CN121729225A - Stabilized lipid and lipid nanoparticle formulations with specific surfactant properties for enhanced pharmaceutical applications - Google Patents

Stabilized lipid and lipid nanoparticle formulations with specific surfactant properties for enhanced pharmaceutical applications

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CN121729225A
CN121729225A CN202480054649.3A CN202480054649A CN121729225A CN 121729225 A CN121729225 A CN 121729225A CN 202480054649 A CN202480054649 A CN 202480054649A CN 121729225 A CN121729225 A CN 121729225A
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surfactant
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linp
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E·贝古因
G·莫里拉
C·多曼
C·普兰克
C·鲁道夫
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Ethris GmbH
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Abstract

本发明提出了用具有特定特征的表面活性剂增强的新型脂质和类脂质纳米颗粒(LNP和LiNP)制剂。这些表面活性剂表现出独特的朗缪尔等温线特性，确保了纳米颗粒的最佳稳定性。所述制剂提高了稳定性，减少了聚集，并缓解了在纯化期间面临的挑战，比如过滤堵塞或结垢。该创新所必需的是基于预定的朗缪尔等温线值和过滤速度确定表面活性剂作为稳定剂的适用性的方法。当与治疗剂一起配制时，稳定的纳米颗粒在医学应用，特别是在mRNA递送、疫苗接种和免疫领域中具有证实的潜力。This invention presents novel lipid and lipid-like nanoparticle (LNP and LiNP) formulations enhanced with surfactants possessing specific characteristics. These surfactants exhibit unique Langmuir isotherm properties, ensuring optimal stability of the nanoparticles. The formulations improve stability, reduce aggregation, and alleviate challenges encountered during purification, such as filter clogging or fouling. This innovation necessitates a method for determining the suitability of surfactants as stabilizers based on predetermined Langmuir isotherm values and filtration rates. When formulated with therapeutic agents, the stabilized nanoparticles demonstrate proven potential in medical applications, particularly in mRNA delivery, vaccination, and immunization.

Description

Stabilized lipid and lipid nanoparticle formulations with specific surfactant properties for enhanced pharmaceutical applications
The present invention relates to stable lipid and/or lipid formulations comprising lipid nanoparticles or lipid nanoparticles and one or more surfactants having specific properties for delivering nucleic acids and enhancing pharmaceutical applications.
Lipid or lipid nanoparticles (LNP or LiNP) are often used to deliver active pharmaceutical ingredients to patients. For example, lipid or lipid formulations of nucleic acids are very useful and effective for introducing nucleic acids into cells. This advantageous property of lipid or lipid formulations of nucleic acids has been used for decades in biological and medical research and therapeutic methods to i) overexpress genes or complement genetic defects in target cells, or ii) down-regulate or up-regulate endogenous gene expression in cells, or iii) repair genetic defects (mutations). Nanoparticle dependent mRNA formulations are now also established as vaccines against COVID-19.
These nanoparticles are central to a range of therapeutic and vaccine applications and often face aggregation and instability problems, particularly under stress conditions such as purification and handling. The present invention provides novel stabilized lipids and lipid formulations with specific surfactants that help overcome problems that occur during such purification, processing and handling.
Disclosure of Invention
In the context of the present invention, it was found that there is a very significant difference between suppliers and within a supplier batch for pharmaceutical grade surfactants such as poloxamers. In the present invention, it was also found that these differences may have a significant impact on the production and purification of LNP and LiNP, etc. The present invention provides a novel Lipid Nanoparticle (LNP) or lipid nanoparticle (LiNP) formulation enhanced with a surfactant having specific characteristics. These surfactants exhibit unique langmuir isotherm characteristics, ensuring optimal stability and efficient membrane purification of the nanoparticles, such as Tangential Flow Filtration (TFF). The formulation improves stability, reduces aggregation, and alleviates challenges faced during purification, such as filtration plugging or fouling.
The present invention proposes novel lipid and lipid nanoparticle (LNP and LiNP) formulations enhanced with surfactants having specific characteristics. These surfactants exhibit unique langmuir isotherm characteristics, ensuring optimal stabilization of the nanoparticles. The formulation improves stability, reduces aggregation, and alleviates challenges faced during purification, such as filtration plugging or fouling. What is essential to this innovation is a method of determining the suitability of a surfactant as a stabilizer based on predetermined langmuir surface pressure/area isotherm (herein simply referred to as "langmuir isotherm") values or filtration rates. When formulated with therapeutic agents, stable nanoparticles have demonstrated potential in medical applications, particularly in the fields of mRNA delivery, vaccination, or immunization.
In this regard, the following aspects of the invention are presented in a non-exclusive manner.
In a first aspect, the present invention provides a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each Lipid Nanoparticle (LNP) or lipid nanoparticle (LiNP) comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanently cationic lipid, and wherein the formulation comprises a surfactant, characterized in that its langerhans surface pressure/area isotherm has a maximum surface pressure (pi max) at a minimum surface area equal to or lower than 4.0 mN/m, the minimum surface area being determined for the lipid mixture or lipid mixture comprised by the nanoparticle.
According to a second aspect, the present invention provides a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanently cationic lipid, wherein the formulation comprises as a stabilizer a surfactant,
And wherein a representative sample bearing on its surface a lipid mixture or lipid mixture comprised by LNP or LiNP comprising said surfactant is characterized by having a langmuir isotherm Δpi equal to or lower than 0.60, preferably equal to or lower than 0.45, at each area point during a langmuir surface pressure/area isotherm cycle comprising a compression phase and an expansion phase and recorded between a maximum surface area and a minimum surface area determined for the lipid mixture or lipid mixture, wherein Δpi is calculated at any area point as:
,
Wherein the method comprises the steps of Is the surface pressure at the area point during the compression phase of the isotherm cycle,
Wherein the method comprises the steps ofIs the surface pressure at the area point during the expansion phase of the isotherm cycle, and
Wherein the method comprises the steps ofIs the maximum surface pressure reached in the isotherm cycle.
In a third aspect, the present invention provides a surfactant for use in a pharmaceutical composition, the surfactant characterized by having a langmuir isotherm having a maximum surface pressure (pi max) at or below 4.0 mN/m at a minimum surface area determined for said pharmaceutical composition.
In a fourth aspect, the present invention provides a method for classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition, the method comprising:
(a) Providing an aqueous solution of surfactant at a concentration (C),
(B) Recording the langmuir surface pressure/area isotherm of the surfactant in the solution to determine a maximum surface pressure pi max of the langmuir surface pressure/area isotherm at a predetermined minimum surface area;
(c) The maximum surface pressure pi max is compared with a threshold value, wherein if the maximum surface pressure pi max is equal to or lower than the threshold value, the surfactant is classified as suitable for use as a stabilizer, and if the maximum surface pressure pi max is greater than the threshold value, the surfactant is classified as unsuitable for use as a stabilizer.
According to this aspect, there is further provided a method of preparing a pharmaceutical composition, the method comprising classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition according to the classification method described above, and incorporating the surfactant into the pharmaceutical composition if the surfactant is classified as suitable for use as a stabilizer for a pharmaceutical composition.
In a fifth aspect, the present invention provides a method for classifying a surfactant as suitable or unsuitable for use as a stabiliser for a pharmaceutical composition comprising a lipid or a lipid-like substance, wherein the method comprises the steps of:
(a) Providing an aqueous solution of surfactant at a concentration (C) of surfactant in the solution;
(b) Recording a langmuir pressure/area isotherm cycle comprising a compression phase and an expansion phase between a maximum surface area and a minimum surface area on a sample comprising an aqueous solution of a surfactant and carrying on its surface a lipid or lipid comprised by the composition:
(c) The langmuir isotherm Δpi for each area point of the langmuir pressure/area isotherm cycle is calculated, wherein Δpi is calculated as follows:
,
Wherein the method comprises the steps of Is the surface pressure at the area point during the compression phase of the isotherm cycle,
Wherein the method comprises the steps ofIs the surface pressure at the area point during the expansion phase of the isotherm cycle, and
Wherein the method comprises the steps ofIs the maximum surface pressure reached in the isotherm cycle, and
(D) Comparing the calculated langmuir isotherm Δpi with a predetermined threshold, wherein if the calculated langmuir isotherm Δpi at each isotherm area point is equal to or below the threshold, the surfactant is classified as suitable for use as a stabilizer, and if the calculated langmuir Miao Er pi at any area point is greater than the threshold, the surfactant is classified as unsuitable for use as a stabilizer.
In the context of this aspect, there is further provided a method of preparing a pharmaceutical composition, the method comprising classifying a surfactant as suitable or unsuitable for use as a stabiliser for a pharmaceutical composition comprising a lipid or lipid according to the above method, and incorporating the surfactant into the pharmaceutical composition if the surfactant is classified as suitable for use as a stabiliser.
In a related sixth aspect, the invention provides a method of reducing or avoiding clogging or scaling of a filtration system during purification of a pharmaceutical composition in the form of a lipid nanoparticle formulation (LNP) or a lipid nanoparticle formulation (LiNP), the method comprising adding a stabilizing surfactant to a first LNP or LiNP formulation to form a second LNP or LiNP formulation, optionally prior to purification, wherein the LNP or LiNP formulation comprises a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of ionizable lipids, ionizable lipids and permanent cationic lipids, and wherein the stabilizing surfactant is a surfactant according to the invention, e.g. a surfactant according to the third aspect discussed above or a surfactant classified as suitable as a stabilizer by a method according to the fourth or fifth aspect.
In another related seventh aspect, the invention provides a method of reducing aggregation of lipid nanoparticles or lipid nanoparticles in a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, preferably in the form of a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, the method comprising adding a stabilizing surfactant to a first LNP or LiNP formulation to form a second LNP or LiNP formulation, optionally prior to purification, wherein the LNP or LiNP formulation comprises a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanent cationic lipid, and the stabilizing surfactant is a surfactant according to the invention, e.g. a surfactant according to the third aspect discussed above, or a surfactant classified as suitable for use as a stabilizing agent by the method of the fourth or fifth aspect.
As a related eighth aspect, the present invention provides a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation obtained by the method according to the fourth to seventh aspects.
Further, in a ninth aspect, the present invention provides a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation according to the first, second or eighth aspects discussed above for use as a medicament.
Also, in a tenth aspect, the present invention provides a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation according to the first, second or eighth aspects discussed above for use in the treatment or prophylaxis of a disease, preferably a disease selected from table a disclosed below, more preferably a disease selected from viral disease, ciliated disease or autoimmune disease, even more preferably a pulmonary disease, a pulmonary viral disease, a pulmonary ciliated disease or an autoimmune disease of the lung.
In another aspect, the invention provides a method of classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition comprising a nucleic acid, optionally as a stabilizer during purification, preferably during TFF purification, the method comprising:
(a) Providing an aqueous solution of a surfactant;
(b) Optionally combining a surfactant with the LNP or LiNP formulation;
(c) Purifying the aqueous solution comprising the surfactant or the LNP or LiNP formulation optionally comprising the surfactant using a membrane purification system (preferably TFF or ultrafiltration, most preferably TFF);
(d) Measuring the filtration time of the aqueous solution or LNP formulation during diafiltration or ultrafiltration;
(e) The measured filtration rate is compared to a predetermined threshold, preferably a time threshold, more preferably a time threshold of 90 minutes, wherein if the filtration rate is equal to or higher than the threshold, the surfactant is classified as unsuitable for use as a stabilizer, and if the filtration rate is lower than the threshold, the surfactant is classified as suitable for use as a stabilizer.
In another aspect, the invention provides a method of reducing or avoiding side effects in treatment with LNP or LiNP carrying at least one therapeutic agent, wherein the method comprises the steps of:
i) Determining whether LNP or LiNP in a pharmaceutical composition comprising LNP or LiNP is aggregated when subjected to mechanical or temperature stress by determining its aggregation level before and after subjecting the pharmaceutical composition to the mechanical or temperature stress,
Ii) if LNP or LiNP shows aggregation after the test of step (i), adding a surfactant, preferably a surfactant according to the third aspect above, to the LNP or LiNP formulation to obtain an LNP or LiNP suspension having a final surfactant concentration of 0.01% w/v up to 10% w/v, preferably between 0.05% w/w surfactant and 5% surfactant, more preferably between 0.33% surfactant and 2.5% surfactant, more preferably between 0.45% and 1.5% surfactant, most preferably between 0.5% and 1.5% surfactant, most preferably about 1% surfactant,
Iii) Recombination is performed using mixing to produce a stable LNP or LiNP suspension.
In a related aspect, the present invention provides the use of a surfactant according to the present invention, for example a surfactant according to the third aspect above, for stabilizing a lipid nanoparticle or suspension of lipid nanoparticles in an aqueous carrier solution under physical stress conditions, preferably shear stress, more preferably shear stress during purification such as TFF, to prevent aggregation of particles, wherein the lipid nanoparticle or lipid nanoparticle comprises the following components (a) and (b):
(a) Therapeutic agent, and
(B) At least one selected from the group consisting of a permanent cationic lipid, an ionizable lipid, and an ionizable lipid.
Without wishing to be bound by theory, the present invention provides stable LNP or LiNP formulations, such as LNP or LiNP suspension formulations, their use and their use in methods of treatment, based on differences in molecular level or differences in quality within pharmaceutical grade surfactant compositions, which are responsible for their suitability or inapplicability for use during the treatment and purification of LNP and LiNP, and further for use to avoid aggregation. The surfactants selected with the method of the present invention also allow surprisingly long shelf life and prolonged stability to shaking. According to the invention, the reduction of aggregation results in a reduction of side effects of the formulations and suspensions of the invention, such as reduction of side effects caused by vaccine formulations or anti-cancer formulations comprising LNP or LiNP.
The following series of clauses provide an overview of the various aspects of the present invention.
Clauses of series
1. A Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each lipid nanoparticle or lipid nanoparticle comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, and a permanently cationic lipid, and wherein the formulation further comprises a surfactant characterized by its langmuir surface pressure/area isotherm (also referred to herein as a "langmuir isotherm") having a maximum surface pressure (pi max) at a minimum surface area of equal to or less than 4.0 mN/m, the minimum surface area being determined for the lipid mixture or lipid mixture comprised by the nanoparticle.
2. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation of clause 1, wherein pi max is equal to or lower than 3.5 mN/m, more preferably equal to or lower than 3.5 and equal to or higher than 0.5 mN/m, even more preferably equal to or lower than 3.0 mN/m and equal to or higher than 0.5 mN/m, even more preferably equal to or lower than 2.5 and equal to or higher than 1.0 mN/m, most preferably equal to or lower than 2.0 and equal to or higher than 1.0 mN/m.
3. A Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each nanoparticle or lipid nanoparticle comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanently cationic lipid, wherein the formulation comprises a surfactant as a stabilizer,
And wherein a representative sample bearing on its surface a lipid mixture or lipid mixture comprised by LNP or LiNP comprising said surfactant is characterized by having a langmuir isotherm Δpi equal to or lower than 0.60, preferably equal to or lower than 0.45, at each area point during a langmuir surface pressure/area isotherm cycle comprising a compression phase and an expansion phase and recorded between a maximum surface area and a minimum surface area determined for the lipid mixture or lipid mixture, wherein Δpi is calculated at any area point as:
,
Wherein the method comprises the steps of Is the surface pressure at the area point during the compression phase of the isotherm cycle,
Wherein the method comprises the steps ofIs the surface pressure at the area point during the expansion phase of the isotherm cycle, and
Wherein the method comprises the steps ofIs the maximum surface pressure reached in the isotherm cycle.
4. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation according to any of clauses 1to 3, wherein the surfactant is a non-ionic surfactant, preferably at least one non-ionic surfactant selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, fatty acid esters of sorbitol, ethoxylated fatty acid esters of sorbitol, fatty acid esters of glycerol, ethoxylated castor oil and ethoxylated vitamin E, e.g. a surfactant selected from poloxamer 124 (P124), poloxamer 188 (P188), poloxamer 338 (P338), poloxamer 407 (P407), tween-20, tween-80, BRIJ35, tyloxapol (tyloxapol), vitE-PEG1000 and Kolliphor EL or a combination thereof.
5. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation of clause 4, wherein the surfactant is a block copolymer of ethylene oxide and propylene oxide, more preferably a poloxamer, even more preferably a poloxamer selected from poloxamer 124, poloxamer 188, poloxamer 338 and poloxamer 407, or a combination thereof, most preferably P188.
6. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation of any of clauses 1 to 5, wherein the lipid nanoparticle or lipid nanoparticle comprises the lipid mixture or lipid mixture and a therapeutic agent.
7. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation of clause 6, wherein the therapeutic agent comprises a nucleic acid, such as RNA, preferably mRNA.
8. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation according to any one of clauses 1 to 7, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanently cationic lipid, and further comprises one or more of the following components (c 1) to (c 6), preferably further comprising components (c 1), (c 2) and (c 3):
(c1) A non-ionizable lipid having a sterol structure;
(c2) A phospholipid;
(c3) PEG conjugated lipids;
(c4) Polysarcosine conjugated lipids
(C5) PAS lipid;
(c6) Ionizable or cationic polymers.
9. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation of clause 8, wherein the lipid mixture or lipid mixture comprises:
i) 30 to 65 mol% of at least one member selected from the group consisting of ionizable lipids, ionizable lipids and permanently cationic lipids,
And further comprises one or more of the following components (c 1) to (c 6):
ii) 10 to 50 mol% of a lipid (c 1) having a sterol structure,
Iii) 4 to 50 mol% of a phospholipid (c 2),
Iv) 0.5 to 10 mol% of one or any combination of PEG conjugated lipid (c 3), poly sarcosine conjugated lipid (c 4) and PAS conjugated lipid (c 5),
V) from 0.5 to 10 mol% of a cationic polymer (c 6),
Such that the sum of the amounts of i) and ii) to v) is 100 mol%,
And more preferably further comprises components (c 1), (c 2) and (c 3) such that the sum of the amounts of i) and ii) to iv) is 100%.
10. A Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation according to any one of clauses 1 to 9, which is a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles, each lipid nanoparticle comprising a lipid mixture, wherein the lipid mixture comprises an ionizable lipid of formula (L-1):
(L-1)
wherein:
a is 1 or 2 and b is an integer from 1 to 4, or a is an integer from 1 to 4 and b is 1 or 2, preferably a is 1 and b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1,
P is either 1 or 2 and the number of the groups,
M is 1 or 2;n is 0 or 1, and m+n≥2, and
R 1A to R 6A are independently of one another selected from the group consisting of hydrogen 、-CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A、-CH2R7A、–C(NH)-NH2、 poly (ethylene glycol) chain and acceptor ligands, wherein R 7A is selected from the group consisting of C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond;
With the proviso that at least two residues from R 1A to R 6A are selected from -CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A and-CH 2R7A, wherein R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond,
Or a protonated form of an ionizable lipid of formula (L-1), wherein one or more of the nitrogen atoms comprised in the compound of formula (L-1) are protonated to provide a positively charged compound.
11. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation of any of clauses 1-10, which is a suspension formulation, and wherein the carrier solution of the suspension formulation is an aqueous carrier solution comprising the surfactant.
12. A surfactant for use in a pharmaceutical composition, said surfactant characterized by having a langmuir surface pressure/area isotherm having a maximum surface pressure (pi max) at a minimum surface area of equal to or lower than 4.0 mN/m, said minimum surface area being determined for said pharmaceutical composition.
13. The surfactant for use according to clause 12, wherein the maximum surface pressure is equal to or lower than 3.5 mN/m, more preferably equal to or lower than 3.5 and equal to or higher than 0.5 mN/m, even more preferably equal to or lower than 3.0 mN/m and equal to or higher than 0.5 mN/m, even more preferably equal to or lower than 2.5 and equal to or higher than 1.0 mN/m, most preferably equal to or lower than 2.0 and equal to or higher than 1.0 mN/m.
14. The surfactant for use according to clause 12 or 13, which is a nonionic surfactant, preferably at least one nonionic surfactant selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, fatty acid esters of sorbitol, ethoxylated fatty acid esters of sorbitol, fatty acid esters of glycerin, ethoxylated castor oil and ethoxylated vitamin E, for example, surfactants selected from poloxamer 124 (P124), poloxamer 188 (P188), poloxamer 338 (P407), tween-20, tween-80, BRIJ35, tyloxapol, vitE-PEG1000 and Kolliphor EL, or a combination thereof.
15. The surfactant for use according to clause 14, which is a block copolymer of ethylene oxide and propylene oxide, more preferably a poloxamer, even more preferably a poloxamer selected from poloxamer 124, poloxamer 188, poloxamer 338 and poloxamer 407 or a combination thereof, most preferably P188.
16. The surfactant for use according to any one of clauses 12 to 15, wherein the pharmaceutical composition is in the form of a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising a plurality of LNPs or LiNP, each comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanent cationic lipid as a component thereof, and wherein the carrier solution of the suspension formulation is an aqueous carrier solution comprising the surfactant.
17. A method for classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition, the method comprising:
(a) Providing an aqueous solution of surfactant at a concentration (C),
(B) Recording the langmuir surface pressure/area isotherm of the surfactant in said solution to determine a maximum surface pressure pi max of the langmuir isotherm at a predetermined minimum surface area;
(c) The maximum surface pressure pi max is compared with a threshold value, wherein if the maximum surface pressure pi max is equal to or lower than the threshold value, the surfactant is classified as suitable for use as a stabilizer, and if the maximum surface pressure pi max is greater than the threshold value, the surfactant is classified as unsuitable for use as a stabilizer.
18. A method for classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition comprising a lipid or a lipid-like substance, optionally during purification of the composition, preferably during tangential flow filtration of the mixture, wherein the method comprises the steps of:
(a) Providing an aqueous solution of surfactant at a concentration (C) of surfactant in the solution;
(b) Recording Langmuir pressure/area isotherm cycle comprising a compression stage and an expansion stage between a maximum surface area and a minimum surface area on a sample comprising an aqueous solution of said surfactant and carrying on its surface the lipid or lipids comprised by said composition,
(C) The langmuir isotherm Δpi for each area point of the langmuir pressure/area isotherm cycle is calculated, wherein Δpi is calculated as follows:
,
Wherein the method comprises the steps of Is the surface pressure at the area point during the compression phase of the isotherm cycle,
Wherein the method comprises the steps ofIs the surface pressure at the area point during the expansion phase of the isotherm cycle, and
Wherein the method comprises the steps ofIs the maximum surface pressure reached in the isotherm cycle, and
(D) Comparing the calculated langmuir isotherm Δpi with a threshold, wherein if the calculated langmuir isotherm Δpi at each isotherm area point is equal to or below the threshold, the surfactant is classified as suitable for use as a stabilizer, and if the calculated langmuir Miao Er pi at any area point is greater than the threshold, the surfactant is classified as unsuitable for use as a stabilizer.
19. A method of preparing a pharmaceutical composition, the method comprising classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition according to the method of clause 17 or 18, and incorporating the surfactant into a pharmaceutical composition if the surfactant is classified as suitable for use as a stabilizer for a pharmaceutical composition.
20. The method according to any of clauses 17 to 19, wherein the surfactant is a nonionic surfactant, preferably at least one nonionic surfactant selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, fatty acid esters of sorbitol, ethoxylated fatty acid esters of sorbitol, fatty acid esters of glycerol, ethoxylated castor oil and ethoxylated vitamin E, e.g., a surfactant selected from the group consisting of poloxamer 124 (P124), poloxamer 188 (P188), poloxamer 338 (P338), poloxamer 407 (P407), tween-20, tween-80, BRIJ35, tyloxapol, vitE-PEG1000 and Kolliphor EL, or a combination thereof.
21. The method of clause 20, wherein the surfactant is a block copolymer of ethylene oxide and propylene oxide, more preferably a poloxamer, even more preferably a poloxamer selected from poloxamer 124, poloxamer 188, poloxamer 338 and poloxamer 407, or a combination thereof, most preferably P188.
22. The method of any one of clauses 17 to 21, wherein the pharmaceutical composition comprises a therapeutic agent comprising a nucleic acid, such as RNA, preferably mRNA.
23. The method of any one of clauses 17 to 22, wherein the pharmaceutical composition is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, and a permanently cationic lipid as a component thereof.
24. The method of clause 23, wherein the pharmaceutical composition is a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation, and wherein the carrier solution of the suspension formulation is an aqueous carrier solution comprising the surfactant.
25. A method of reducing or avoiding clogging or scaling of a filtration system during purification of a pharmaceutical composition in the form of a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, the method comprising adding a stabilizing surfactant to a first LNP or LiNP formulation to form a second LNP or LiNP formulation, optionally prior to purification, wherein the LNP or LiNP formulation comprises a plurality of LNPs or LiNP, each comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises at least one selected from an ionizable lipid, and a permanently cationic lipid as a component thereof, and wherein the stabilizing surfactant is a surfactant according to any one of clauses 12 to 16, or is classified as a surfactant suitable as a stabilizer by the method of any one of clauses 17 to 27.
26. The method of clause 25, wherein the purifying comprises tangential flow filtration.
27. The method of clause 25 or 26, wherein the pharmaceutical composition is a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising an aqueous carrier solution, and wherein the stabilizing surfactant is added to the carrier solution, optionally wherein the surfactant is substantially absent from the LNP or LiNP.
28. The method of any one of clauses 25 to 27, wherein the therapeutic agent is a nucleic acid, such as RNA, more preferably mRNA.
29. A method of mitigating aggregation of a lipid or lipid nanoparticle in a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, the method comprising adding a stabilizing surfactant to a first LNP or LiNP formulation to form a second LNP or LiNP formulation, optionally prior to purification, wherein the LNP or LiNP formulation comprises a plurality of LNPs or LiNP, each comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises at least one selected from an ionizable lipid, and a permanently cationic lipid as a component thereof, and wherein the stabilizing surfactant is a surfactant according to any one of clauses 12 to 16, or is classified as a surfactant suitable as a stabilizer by the method of any one of clauses 17 to 27.
30. The method of clause 29, wherein the formulation is a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising an aqueous carrier solution, and wherein the stabilizing surfactant is added to the carrier solution, optionally wherein the surfactant is substantially absent from the LNP or LiNP.
31. The method of clauses 29 or 30, wherein the lipid nanoparticle or lipid nanoparticle comprises a therapeutic agent, preferably a nucleic acid, such as RNA, more preferably mRNA.
32. The method of clause 31, wherein the lipid nanoparticle or lipid nanoparticle comprises a nucleic acid, such as RNA, and preferably mRNA, and wherein the method comprises the steps of:
i) First, combining the nucleic acid with at least one selected from the group consisting of an ionizable lipid, and a permanently cationic lipid to form an LNP or LiNP,
Ii) second, purifying the LNP or LiNP,
Iii) Thirdly, adding the stabilizing surfactant to the exchange buffer before and during TFF purification, maintaining the surfactant at a stable concentration,
Iv) optionally wherein the stabilizing surfactant is added to the LNP or LiNP formulation after step (i).
33. The method of clause 31 or 32, wherein the method comprises the steps of:
i) The LNP or LiNP preparation is produced by mixing at least one selected from the group consisting of a permanent cationic lipid, an ionizable lipid, and an ionizable lipid dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution,
Ii) diluting the LNP or LiNP preparation with a first solution,
Iii) Concentrating the LNP or LiNP preparation by buffer exchange using ultrafiltration/diafiltration via TFF, wherein the second solution is used for ultrafiltration/diafiltration,
Iv) obtaining a suspension of LNP or LiNP in an aqueous carrier solution,
Wherein the first solution comprises between about 0.01% w/v and 10% stabilizing surfactant, preferably between about 0.01% w/v surfactant and 5% w/v surfactant, more preferably between about 0.01% w/v surfactant and 2.5% w/v surfactant, more preferably between about 0.05% w/v and 1.5% w/v surfactant, even more preferably between about 0.05% w/v and 1.5% w/v surfactant, most preferably about 1% w/v surfactant, and/or
Wherein the second solution comprises between about 0.01% w/v and about 10% stabilizing surfactant, preferably between about 0.01% w/v surfactant and about 5% w/v surfactant, more preferably between about 0.01% w/v surfactant and about 2.5% w/v surfactant, even more preferably between about 0.05% w/v surfactant and 1.5% w/v surfactant, most preferably about 1% w/v;
And wherein the final concentration of stabilizing surfactant from the combined first and second solutions is between 0.01% and 10% surfactant, preferably between 0.01% w/v surfactant and 5% w/v surfactant, more preferably between 0.01% w/v surfactant and 2.5% w/v surfactant, even more preferably between 0.05% w/v and 1.5% w/v surfactant, most preferably about 1% w/v surfactant, relative to the total volume of the suspension of nanoparticles in the aqueous carrier solution.
34. The method of clause 33, wherein:
a) The stabilizing surfactant is not incorporated into the suspension before or during step i),
B) Adding the stabilizing surfactant to the first and second solutions, and/or
C) About half of the stabilizing surfactant is added to the first solution and about half of the surfactant is added to the second solution.
35. The method of any one of clauses 23 to 34, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, and a permanently cationic lipid as a component thereof, and further comprises one or more of the following components (c 1) to (c 6):
(c1) A non-ionizable lipid having a sterol structure;
(c2) A phospholipid;
(c3) PEG conjugated lipids;
(c4) Polysarcosine conjugated lipids
(C5) PAS lipid;
(c6) Ionizable or cationic polymers.
36. The method of clause 35, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, and a permanently cationic lipid as a component thereof, and further comprises components (c 1), (c 2), and (c 3).
37. The method of clause 35, wherein the lipid mixture or lipid mixture comprises:
i) 30 to 65 mol% of at least one selected from the group consisting of the ionizable lipid, and the permanently cationic lipid,
And further comprises one or more of the following components (c 1) to (c 6):
ii) 10 to 50 mol% of a lipid (c 1) having a sterol structure,
Iii) 4 to 50 mol% of a phospholipid (c 2),
Iv) 0.5 to 10 mol% of one or any combination of PEG conjugated lipid (c 3), poly sarcosine conjugated lipid (c 4) and PAS conjugated lipid (c 5),
V) from 0.5 to 10 mol% of a cationic polymer (c 6),
Such that the sum of the amounts of i) and ii) to v) is 100 mol%.
38. The method of clause 37, wherein the lipid mixture or lipid mixture comprises:
i) 30 to 65 mol% of at least one selected from the group consisting of ionizable lipids, and permanently cationic lipids, and further comprising:
ii) 10 to 50 mol% of a lipid (c 1) having a sterol structure,
Iii) 4 to 50 mol% of a phospholipid (c 2),
Iv) 0.5 to 10 mol% of PEG conjugated lipid (c 3),
Such that the sum of the amounts of i) and ii) to iv) is 100%.
39. The method of any of clauses 23-38, wherein the formulation is a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles, each lipid nanoparticle comprising a lipid mixture,
Wherein the lipid mixture comprises an ionizable lipid of formula (L-1):
(L-1)
wherein:
a is 1 or 2 and b is an integer from 1 to 4, or a is an integer from 1 to 4 and b is 1 or 2, preferably a is 1 and b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1,
P is either 1 or 2 and the number of the groups,
M is 1 or 2;n is 0 or 1, and m+n is not less than 2, and
R 1A to R 6A are independently of one another selected from the group consisting of hydrogen ;-CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A、-CH2R7A、–C(NH)-NH2、 poly (ethylene glycol) chain and acceptor ligands, wherein R 7A is selected from the group consisting of C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond;
With the proviso that at least two residues from R 1A to R 6A are selected from -CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A and-CH 2R7A, wherein R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond,
Or a protonated form of an ionizable lipid of formula (L-1), wherein one or more of the nitrogen atoms comprised in the compound of formula (L-1) are protonated to provide a positively charged compound.
40. A lipid nanoparticle formulation or lipid nanoparticle formulation, preferably a lipid nanoparticle suspension formulation or lipid nanoparticle suspension formulation, obtainable by the method of any one of clauses 23 to 39.
41. A lipid nanoparticle formulation or a lipid nanoparticle formulation, preferably a lipid nanoparticle suspension formulation or a lipid nanoparticle suspension formulation according to any one of clauses 1 to 11 or 40, for use as a medicament.
42. A lipid nanoparticle formulation or lipid nanoparticle formulation, preferably a lipid nanoparticle suspension formulation or lipid nanoparticle suspension formulation according to any one of clauses 1 to 11 or 40, for use in the treatment or prophylaxis of a disease, preferably a disease selected from table a, more preferably a disease selected from viral disease, ciliated disease, autoimmune disease and respiratory disease, even more preferably a disease selected from pulmonary disease, airway disease or nasal disease, more preferably a viral disease of the lung, ciliated disease of the lung and autoimmune disease of the lung.
43. The lipid nanoparticle formulation or lipid nanoparticle formulation according to clause 43, wherein the pulmonary disease or pulmonary viral disease is at least one selected from the group consisting of pneumonia and asthma, the airway disease is at least one selected from the group consisting of bronchitis, virus-induced asthma, pulmonary fibrosis and COPD, and/or the nasal disease is at least one selected from the group consisting of rhinitis and sinusitis.
44. A lipid nanoparticle formulation or lipid nanoparticle formulation according to clause 41 for use in vaccination or immunization.
45. A method of avoiding or reducing side effects in treatment with LNP or LiNP carrying at least one therapeutic agent, wherein the method comprises the steps of:
i) Determining whether LNP or LiNP in a pharmaceutical composition comprising LNP or LiNP is aggregated when subjected to mechanical or temperature stress by determining its aggregation level before and after subjecting the pharmaceutical composition to the mechanical or temperature stress,
Ii) if LNP or LiNP shows aggregation after the test of step (i), adding surfactant to the LNP or LiNP formulation to obtain an LNP or LiNP suspension having a final surfactant concentration of 0.01% w/v up to 10% w/v, preferably between 0.05% w/w surfactant and 5% surfactant, more preferably between 0.33% surfactant and 2.5% surfactant, more preferably between 0.45% and 1.5% surfactant, most preferably between 0.5% and 1.5% surfactant, most preferably about 1% w/v surfactant,
Iii) Reconstitution was performed with mixing to produce a stable LNP or LiNP suspension.
46. The method of clause 45, wherein the surfactant is a surfactant according to any of clauses 12 to 16, or is classified as a surfactant suitable as a stabilizer by the method of any of clauses 17 to 24.
47. Use of a surfactant according to any of clauses 12 to 16 or a surfactant classified as suitable as a stabilizer by the method of any of clauses 17 to 24 for stabilizing a lipid nanoparticle or suspension of lipid nanoparticles in an aqueous carrier solution under physical stress conditions, preferably shear stress, more preferably under shear stress during purification such as TFF to prevent aggregation of particles, wherein the lipid nanoparticle or lipid nanoparticle comprises the following components (a) and (b):
(a) Therapeutic agent, and
(B) At least one selected from the group consisting of ionizable lipids, and permanently cationic lipids.
48. The use of the surfactant of clause 47, wherein the physical stress condition is selected from shaking, stirring, vibrating, mixing, tumbling, tapping, or dropping of the suspension, or a combination thereof, or wherein the physical stress condition is caused by pumping the suspension or drawing it into a syringe.
49. The use of a surfactant according to clause 47 or 48, wherein the surfactant is incorporated as an excipient in the aqueous carrier solution.
50. The use of a surfactant of any of clauses 47 to 49, wherein the nanoparticle formulation is not lyophilized.
51. The use of a surfactant of any of clauses 47 to 50, wherein the surfactant is added prior to the lyophilization process.
52. The use of a surfactant of any one of clauses 47 to 51, wherein the presence of the surfactant does not cause a change in the biological activity of the nanoparticle.
53. The use of a surfactant of any of clauses 47 to 52, wherein the presence of the surfactant does not cause a change in the hydrodynamic diameter as nanoparticles and the physical properties measured as a proportion of the therapeutic agent included in the nanoparticles.
54. The use of a surfactant of any of clauses 47 to 53, wherein the lipid nanoparticle or suspension of lipid nanoparticles in an aqueous carrier solution comprises the surfactant at a concentration of 0.01 to 10% (w/v).
55. The use of a surfactant of any of clauses 47 to 54, wherein the therapeutic agent is a nucleic acid.
56. The use of a surfactant of clause 55, wherein the nucleic acid is mRNA.
57. The use of a surfactant of any of clauses 47 to 56, wherein the concentration of the nucleic acid in the suspension formulation is in the range of 0.01 to 10mg/mL based on the total volume of the suspension formulation.
58. The use of a surfactant of any of clauses 47 to 57, wherein the Z-average diameter of the nanoparticle (as determined by dynamic light scattering) is in the range of 10 to 500 nm, preferably about 30 to 100 nm.
59. The use of a surfactant of any of clauses 47 to 58, wherein the nanoparticle further comprises one or more of the following components (c 1) to (c 6):
(c1) A non-ionizable lipid having a sterol structure;
(c2) A phospholipid lipid;
(c3) PEG conjugated lipids;
(c4) Polysarcosine conjugated lipids
(C5) PAS lipid;
(c6) An ionizable or cationic polymer or lipid.
60. The use of a surfactant of any of clauses 47 to 59, wherein the nanoparticle comprises:
a) 30 to 65 mol% of at least one (b) selected from the group consisting of ionizable lipids, and permanently cationic lipids, and one or more of the following components:
ii) 10 to 50 mol% of a lipid (c 1) having a sterol structure,
Iii) 4 to 50 mol% of a phospholipid (c 2),
Iv) 0.5 to 10 mol% of one or any combination of PEG conjugated lipid (c 3), poly sarcosine conjugated lipid (c 4) and PAS conjugated lipid (c 5),
From 0.5 to 10 mol% of a cationic polymer (c 6),
So that the sum of (b) and (c 1) to (c 6) is 100 mol%.
61. The use of a surfactant of any of clauses 47 to 60, wherein the nanoparticle comprises an ionizable lipid (b) of the following formula (a-III):
a-III
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
One of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, and the other of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, or a direct bond;
Each of G 1 and G 2 is independently C 1-C12 alkylene or C 1-C12 alkenylene;
G 3 is C 1-C24 alkylene, C 1-C24 alkenylene, C 3-C8 cycloalkylene, C 3-C8 cycloalkenyl, wherein each of alkylene, alkenylene, cycloalkylene, and cycloalkenyl is optionally substituted;
R a is H or C 1-C12 alkyl, wherein alkyl is optionally substituted;
R 1 and R 2 are each independently C 6-C24 alkyl or C 6-C24 alkenyl, wherein each of the alkyl and alkenyl groups is optionally substituted;
R 3 is H, OR 5、CN、-C(=O)OR4、-OC(=O)R4 or-NR 5C(=O)R4;R4 is C 1-C12 alkyl, wherein alkyl is optionally substituted;
R 5 is H or C 1-C6 alkyl, where the alkyl is optionally substituted, and
X is 0, 1 or 2.
62. The use of a surfactant of any of clauses 47-61, wherein the nanoparticle comprises an ionizable lipid (b) of the formula (L-1),
(L-1)
Wherein:
a is 1 or 2 and b is an integer from 1 to 4, or a is an integer from 1 to 4 and b is 1 or 2, preferably a is 1 and b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1,
P is either 1 or 2 and the number of the groups,
M is 1 or 2;n is 0 or 1, and m+n is not less than 2, and
R 1A to R 6A are each independently selected from the group consisting of hydrogen ;-CH2CH(OH)R7A,-CH(R7A)CH2OH,-CH2CH2(C=O)OR7A,-CH2CH2(C=O)NHR7A,-CH2R7A,–C(NH)-NH2, poly (ethylene glycol) chain, and acceptor ligands, wherein R 7A is selected from the group consisting of C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond,
With the proviso that at least two residues from R 1A to R 6A are selected from -CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A and-CH 2R7A, wherein R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond,
Or a protonated form of an ionizable lipid of formula (L-1), wherein one or more of the nitrogen atoms comprised in the compound of formula (L-1) are protonated to provide a positively charged compound.
63. The use of a surfactant of any one of clauses 47 to 60, wherein the nanoparticle comprises (6 z,9z,28z,31 z) -heptadeca-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate or a protonated form thereof, wherein the nitrogen atom of the compound is protonated, as ionizable lipid (b).
64. The use of a surfactant of any one of clauses 47 to 60, wherein the nanoparticle comprises ((4-hydroxybutyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) or a protonated form thereof (wherein the nitrogen atom of the compound is protonated) and/or (heptadec-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate or a protonated form thereof (wherein the nitrogen atom in the compound is protonated) as ionizable lipid (b).
65. The use of clause 64, wherein the nanoparticle comprises:
((4-hydroxybutyl) azadiyl) bis (hexane-6, 1-dialkyl) bis (2-hexyldecanoate) or a protonated form thereof, wherein the nitrogen atom of the compound is protonated, and optionally further comprising one or more of the following components (d 1) to (d 8):
(d1) 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide (ALC-0159),
(D2) 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC),
(D3) The cholesterol level of the blood is determined by the concentration of cholesterol,
(D4) The potassium chloride is added to the mixture,
(D5) The potassium dihydrogen phosphate is used for preparing the nano-crystalline silicon dioxide,
(D6) The sodium chloride is used for preparing the sodium chloride,
(D7) Disodium phosphate dihydrate is used as a base for the production of the aqueous solution,
(D8) Sucrose.
66. The use of clause 64, wherein the nanoparticle comprises heptadec-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate or a protonated form thereof, wherein the nitrogen atom of the compound is protonated, and further optionally one or more of the following components (e 1) to (e 7):
(e1) 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC),
(E2) The cholesterol level of the blood is determined by the concentration of cholesterol,
(E3) 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (PEG 2000 DMG),
(E4) 2-amino-2- (hydroxymethyl) propane-1, 3-diol (tromethamine) hydrochloride,
(E5) The sodium acetate trihydrate and the sodium acetate trihydrate,
(E6) Acetic acid is used as a solvent for the acetic acid,
(E7) Sucrose.
67. The use according to clause 64, wherein the nanoparticle comprises DLin-MC3-DMA ((6 z,9z,28z,31 z) -seventeen carbon-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butyrate) or a protonated form thereof (wherein the nitrogen atom of the compound is protonated), and optionally further comprising one or more of the following components (e 1) to (e 7):
(e1) 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC),
(E2) The cholesterol level of the blood is determined by the concentration of cholesterol,
(E3) PEG2000-C-DMG (. Alpha. - (3' - { [1, 2-bis (myristoyloxy) propoxy ] carbonylamino } propyl) - ω -methoxy, polyoxyethylene),
(E4) 2-amino-2- (hydroxymethyl) propane-1, 3-diol (tromethamine) hydrochloride,
(E5) Disodium hydrogen phosphate heptahydrate is used for preparing the sodium phosphate,
(E6) The anhydrous potassium dihydrogen phosphate is used for preparing the water-free potassium dihydrogen phosphate,
(E7) Sodium chloride.
It should be understood that the summary of the foregoing clauses forms part of the overall disclosure of the invention so that the information provided in the detailed description below, such as information about other preferred embodiments or optional features, is also applicable to the foregoing clauses and vice versa.
Detailed Description
Unless otherwise indicated in any particular instance, the following explanations, e.g., with respect to a therapeutic agent, lipid nanoparticle or surfactant, apply to all aspects of the invention that include or use any of these components.
As described above, the present invention provides in a first aspect a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, and a permanently cationic lipid, and wherein the formulation further comprises a surfactant characterized by its Langmuir (Langmuir) surface pressure/area isotherm (also referred to herein as a "Langmuir isotherm") having a maximum surface pressure (pi max) of equal to or less than 4.0 mN/m at a minimum surface area determined for the lipid mixture or lipid mixture comprised by the nanoparticle.
Preferably, pi max is equal to or less than 3.5 mN/m, more preferably equal to or less than 3.5 and equal to or greater than 0.5 mN/m, even more preferably equal to or less than 3.0 mN/m and equal to or greater than 0.5 mN/m, even more preferably equal to or less than 2.5 and equal to or greater than 1.0 mN/m, most preferably equal to or less than 2.0 and equal to or greater than 1.0 mN/m.
The maximum surface pressure (pi max) of a surfactant can be determined by recording the langmuir surface pressure/area isotherm of the surfactant in its aqueous solution (e.g., deionized water). Langmuir isotherms were recorded in Langmuir trough (Langmuir trough). Recording the langmuir isotherm of a surfactant involves reducing the available surface area in the langmuir trough at least until a minimum surface area determined for the lipid mixture or lipid mixture is reached. The surface pressure observed at the minimum surface area when recording langmuir isotherms of the surfactant is considered to be the maximum surface pressure (pi max) of the surfactant.
The minimum surface area may be determined by, for example, compressing the lipid mixture or a layer (typically a monolayer) of the lipid mixture (i.e., the lipid mixture or lipid mixture that the nanoparticle comprises) in a langmuir cell until a first phase change is observed in the surface pressure/area isotherm of the lipid mixture or lipid mixture. To determine the minimum surface area, the layer may conveniently be provided on water, for example deionized water. The surface area at the beginning of the phase change is considered to be the minimum surface area determined for the lipid mixture or lipid mixture. Those skilled in the art will appreciate that the phase transition referred to herein in the pressure/area (pi-a) isotherm plot may be objectively determined as the point at which the slope of the curve (dpi/dA) changes significantly, indicating a transition in the molecular organization of the monolayer. The step of determining the minimum surface area may be performed in the langmuir tank, for example as a calibration step, before determining the maximum surface pressure of the surfactant as described above.
As the maximum surface area during recording of the langmuir surface pressure/area isotherms, for example, the maximum area provided by langmuir tanks may be conveniently used.
For example, a monolayer of the aqueous lipid mixture or lipid mixture may be provided in the langmuir trough by applying the lipid mixture or lipid mixture to a surface until a signal change occurs in the langmuir trough detector.
The langmuir surface pressure/area isotherm of the surfactant may, for example, be recorded in the form of a langmuir surface pressure/area isotherm cycle (also referred to herein simply as "isotherm cycle (isotherm cycle)") comprising a compression stage and an expansion stage. For example, the maximum surface pressure (pi max) may be determined by recording a single isotherm cycle, but preferably the result is obtained by sequentially recording a plurality of isotherm cycles, such as three isotherm cycles, for example with a waiting time between cycles of 5 seconds or less, such as 3 seconds. If a single isotherm cycle is recorded, it is preferred that pi max be equal to or less than 3.5 mN/m, more preferably equal to or less than 3.5 and equal to or greater than 0.5 mN/m, even more preferably equal to or less than 3.0 mN/m and equal to or greater than 0.5 mN/m, even more preferably equal to or less than 2.5 and equal to or greater than 1.0 mN/m, and most preferably equal to or less than 2.0 and equal to or greater than 1.0 mN/m, directly in the single cycle. If multiple cycles, e.g., three cycles, are recorded, it is preferred that the surfactant exhibit a Langmuir isotherm having a maximum surface pressure (pi max) as defined above in the last, e.g., third, isotherm cycle.
Typically, the langmuir surface pressure/area isotherm or isotherm cycle referred to herein is recorded at about room temperature, e.g., 22.1±0.2 ℃. Typical concentrations of surfactant in its aqueous solution for maximum surface pressure determination are 1% w/v (corresponding to 1g surfactant in the total volume of 100ml including surfactant, typically measured at room temperature, e.g. 22.1±0.2 ℃).
The calibration procedure that can be used to determine the minimum surface area and the measurement of the maximum surface pressure of the surfactant are further illustrated in the examples section below.
Details of preferred compositions of lipid nanoparticles, lipid mixtures, and lipid mixtures are discussed below.
Details regarding preferred surfactant types, such as poloxamers, are also discussed below.
Preferably, the Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation is a suspension formulation comprising a liquid carrier solution in which LNP or LiNP can be dispersed. The carrier solution is preferably an aqueous carrier solution. Preferably, in such suspension formulations, the surfactant is included in the carrier solution.
For example, in such suspension formulations, the surfactant may advantageously act as a stabilizer to mitigate aggregation of LNP or LiNP or a subset thereof, which may be caused by shaking or shear stress of the suspension during production, purification, handling or transportation, preferably during production or purification, more preferably during purification, most preferably during Tangential Flow Filtration (TFF) purification.
The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation is preferably a formulation in which the LNP or LiNP comprises a lipid mixture or lipid nanoparticle mixture and a therapeutic agent, for example a suspension formulation as described above, i.e. a pharmaceutical formulation. Thus, the first aspect further comprises a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation for use as a medicament comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each lipid nanoparticle or lipid nanoparticle comprising a lipid mixture or lipid mixture and a therapeutic agent. Preferred therapeutic agents are nucleic acids, such as RNA, and particularly preferred are mRNA. Details of therapeutic agents that may be included in LNP or LiNP are also discussed below. The terms "pharmaceutical formulation (pharmaceutical formulation)" and "pharmaceutical composition (pharmaceutical composition)" may be used herein as equivalent terms.
According to the foregoing, the Lipid Nanoparticle (LNP) formulation or the lipid nanoparticle (LiNP) formulation according to the first aspect is particularly preferably a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of ionizable lipids, ionizable lipids and permanently cationic lipids, and wherein the carrier solution of the suspension formulation is an aqueous carrier solution comprising a surfactant characterized in that its langmuir surface pressure/area isotherm has a maximum surface pressure (max) equal to or lower than 4.0 mN/m at a minimum surface area determined for the lipid mixture or lipid mixture comprised by the nanoparticle. Those skilled in the art will appreciate that the preferred values of maximum surface pressure discussed above continue to apply to this preferred embodiment.
According to a second aspect, the present invention provides a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each lipid nanoparticle or lipid nanoparticle comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanently cationic lipid, wherein the formulation comprises a surfactant as a stabilizer,
And wherein a representative sample comprising an aqueous solution of said surfactant bearing on its surface a lipid mixture or lipid mixture comprised by LNP or LiNP is characterized by having a langmuir surface pressure/area isotherm equal to or lower than 0.60, preferably equal to or lower than 0.45, at each area point during the langmuir surface pressure/area isotherm cycle comprising a compression stage and an expansion stage and recorded between a maximum surface area and a minimum surface area determined for the lipid mixture or lipid mixture, wherein Δpi is calculated as:
,
Wherein the method comprises the steps of Is the surface pressure at the area point during the compression phase of the isotherm cycle,
Wherein the method comprises the steps ofIs the surface pressure at the area point during the expansion phase of the isotherm cycle, and
Wherein the method comprises the steps ofIs the maximum surface pressure reached in the isotherm cycle.
Representative samples for determining the langmuir isotherm Δpi value include aqueous solutions of surfactants, such as solutions of deionized water, bearing on their surface the lipid mixture or mixture of lipids, typically a monolayer of the lipid mixture or mixture of lipids, comprised by LNP or LiNP. The lipid mixture or lipid mixture typically has the same composition as the lipid mixture or lipid mixture comprised in the nanoparticle, i.e. it comprises the same lipids and/or lipids in the same proportions. Using this representative sample, langmuir surface pressure/area isotherm cycles comprising a compression phase and an expansion phase can be recorded in a langmuir tank. As described above, the langmuir isotherm Δpi value for each area point is calculated from the surface pressure determined during compression and the surface pressure determined during expansion at each area point of the isotherm. As mentioned above, during the langmuir surface pressure/area isotherm cycle, the langmuir isotherm Δpi value is equal to or lower than 0.60, preferably equal to or lower than 0.45, at each area point. The langmuir isotherm Δpi value calculated as disclosed above is typically above 0.
It is sufficient if one isotherm cycle is recorded and the langmuir isotherm Δpi value of that isotherm cycle is preferably calculated. Multiple isotherm cycles, such as three isotherm cycles, may be recorded sequentially, for example with a latency between cycles of 5 seconds or less, such as 3 seconds. However, if the Δpi value of the first cycle satisfies the above requirement, this is generally also applicable to the subsequent cycles, so that even if a plurality of cycles are recorded, it is preferable to calculate the Δpi value depending on the measurement result of the first cycle.
The minimum surface area may be determined by, for example, compressing the lipid mixture or a layer (typically a monolayer) of the lipid mixture (i.e., the lipid mixture or lipid mixture that the nanoparticle comprises) in a langmuir cell until a first phase change is observed in the surface pressure/area isotherm of the lipid mixture or lipid mixture. To determine the minimum surface area, the layer may conveniently be provided on water, for example deionized water. The surface area at the beginning of the phase change is considered to be the minimum surface area determined for the lipid mixture or lipid mixture. As mentioned above, the phase transition onset referred to herein in the pressure/area (pi-a) isotherm plot can be objectively determined as the point at which the slope of the curve (dpi/dA) changes significantly. The step of determining the minimum surface area may be performed in the langmuir cell, for example as a calibration step, before recording langmuir surface pressure/area isotherm cycles on a representative sample as described above.
As the maximum surface area during recording of the langmuir surface pressure/area isotherm, the surface area before the start of compression in the compression stage, such as the maximum area provided by langmuir tanks, may be conveniently used.
For example, a monolayer of the lipid mixture or lipid mixture may be provided in the langmuir trough on water or an aqueous solution of a surfactant by applying the lipid mixture or lipid mixture to a surface until a signal change occurs in the langmuir trough detector.
Typically, the langmuir surface pressure/area isotherm or isotherm cycle referred to herein is recorded at about room temperature, e.g., 22.1±0.2 ℃. Typical concentrations of surfactant in the aqueous solution of a representative sample are 1% w/v (corresponding to 1 g surfactant in the total volume of 100 ml including surfactant, typically measured at room temperature, e.g., 22.1±0.2 ℃).
The following "examples" section further illustrates calibration steps that may be used to determine the minimum surface area, as well as the recording of langmuir pressure/area isotherms used to calculate the langmuir isotherms Δpi value.
Details regarding preferred surfactant types, such as poloxamers, are discussed below.
Details of the lipid mixture and preferred compositions of the lipid mixture are also discussed below.
Preferably, the Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation is a suspension formulation comprising a liquid carrier solution in which LNP or LiNP can be dispersed. The carrier solution is preferably an aqueous carrier solution. Preferably, in such suspension formulations, the surfactant is included in the carrier solution.
For example, in such suspension formulations, the surfactant may advantageously act as a stabilizer to mitigate aggregation of LNP or LiNP or a subset thereof, which may be caused by shaking or shear stress of the suspension during production, purification, handling or transportation, preferably during production or purification, more preferably during purification, most preferably during Tangential Flow Filtration (TFF) purification.
Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation preferably wherein LNP or LiNP comprises a lipid mixture or a formulation of a lipid nanoparticle mixture and a therapeutic agent, such as a suspension formulation as described above, i.e. a pharmaceutical formulation. Thus, the second aspect also includes a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation for use as a medicament comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each lipid nanoparticle or lipid nanoparticle comprising a lipid mixture or lipid mixture and a therapeutic agent. Preferred therapeutic agents are nucleic acids, such as RNA, and particularly preferred are mRNA. Details of therapeutic agents that may be included in LNP or LiNP are also discussed below. The terms "pharmaceutical formulation" and "pharmaceutical composition" may be used herein as equivalent terms.
According to the above, the Lipid Nanoparticle (LNP) formulation or the lipid nanoparticle (LiNP) formulation according to the second aspect is particularly preferably a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or a lipid mixture and a therapeutic agent, wherein the lipid mixture or the lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanently cationic lipid as a component thereof, and wherein the carrier solution of the suspension formulation is an aqueous carrier solution comprising a surfactant as a stabilizer,
And wherein the representative sample comprising an aqueous solution of a surfactant bearing on its surface a lipid mixture or lipid mixture comprised by LNP or LiNP is characterized by having a langmuir surface pressure/area isotherm equal to or lower than 0.60, preferably equal to or lower than 0.45, at each area point during the langmuir surface pressure/area isotherm cycle comprising a compression stage and an expansion stage and recorded between a maximum surface area and a minimum surface area determined for the lipid mixture or lipid mixture, wherein Δpi is calculated at any area point as described above.
According to a third aspect, the present invention provides a surfactant for use in a pharmaceutical composition, the surfactant being characterized by having a langmuir surface pressure/area isotherm having a maximum surface pressure (pi max) equal to or lower than 4.0 mN/m at a minimum surface area determined for said pharmaceutical composition. Also, this aspect provides the use of a surfactant as a component in a pharmaceutical composition, for example as a stabilizer.
Preferably, in the context of this aspect, pi max is also equal to or lower than 3.5 mN/m, more preferably equal to or lower than 3.5 and equal to or higher than 0.5 mN/m, even more preferably equal to or lower than 3.0 mN/m and equal to or higher than 0.5 mN/m, even more preferably equal to or lower than 2.5 and equal to or higher than 1.0 mN/m, most preferably equal to or lower than 2.0 and equal to or higher than 1.0 mN/m.
For example, the pharmaceutical composition may be a pharmaceutical composition comprising a lipid or a lipid, such as a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP). More preferably, the pharmaceutical composition is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each lipid nanoparticle or lipid nanoparticle comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, and a permanent cationic lipid as a component thereof.
If the pharmaceutical formulation is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, then preferably the formulation is a suspension formulation comprising a liquid carrier solution in which LNP or LiNP may be dispersed. The carrier solution is preferably an aqueous carrier solution. Preferably, in such suspension formulations, the surfactant as defined herein is contained in a carrier solution.
For example, in such suspension formulations, the surfactant may advantageously act as a stabilizer to mitigate aggregation of LNP or LiNP or a subset thereof, which may be caused by shaking or shear stress of the suspension during production, purification, handling or transportation, preferably during production or purification, more preferably during purification, most preferably during Tangential Flow Filtration (TFF) purification.
Those skilled in the art will appreciate that pharmaceutical compositions include therapeutic agents, such as nucleic acids. Preferred therapeutic agents are RNA, and particularly preferred are mRNA. Details regarding the preferred types of therapeutic agents are discussed below. If the pharmaceutical formulation is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, such as a suspension formulation, the therapeutic agent is typically included in LNP or LiNP.
According to the foregoing, the surfactant for use according to the third aspect is particularly preferably a surfactant for use in a pharmaceutical composition in the form of a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises as components thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanent cationic lipid, wherein the carrier solution of the suspension formulation is an aqueous carrier solution and comprises a surfactant, characterized in that the surfactant has a maximum surface pressure (pi max) of equal to or lower than 4.0 mN/m at a minimum surface area determined for the pharmaceutical composition. Those skilled in the art will appreciate that the preferred values of maximum surface pressure discussed above continue to apply to this preferred embodiment. Also, this aspect provides for the use of a surfactant as a component in such a pharmaceutical composition, for example as a stabilizer.
The maximum surface pressure (pi max) of a surfactant can be determined by recording the langmuir surface pressure/area isotherm of the surfactant in its aqueous solution (e.g., deionized water). Langmuir isotherms are recorded in Langmuir cells. Recording the langmuir isotherm of a surfactant involves reducing the available surface area in the langmuir trough at least until a minimum surface area determined for the pharmaceutical composition is reached. The surface pressure observed at the minimum surface area when recording langmuir isotherms of the surfactant is considered to be the maximum surface pressure (pi max) of the surfactant.
The langmuir surface pressure/area isotherm of the surfactant may, for example, be recorded in the form of a langmuir surface pressure/area isotherm cycle (also referred to herein simply as "isotherm cycle") comprising a compression stage and an expansion stage. For example, the maximum surface pressure (pi max) may be determined by recording a single isotherm cycle, but preferably the result is obtained by sequentially recording a plurality of isotherm cycles, such as three isotherm cycles, for example with a waiting time between cycles of 5 seconds or less, such as 3 seconds. If a single isotherm cycle is recorded, it is preferred that pi max be equal to or less than 3.5 mN/m, more preferably equal to or less than 3.5 and equal to or greater than 0.5 mN/m, even more preferably equal to or less than 3.0 mN/m and equal to or greater than 0.5 mN/m, even more preferably equal to or less than 2.5 and equal to or greater than 1.0 mN/m, and most preferably equal to or less than 2.0 and equal to or greater than 1.0 mN/m, directly in the single cycle. If multiple cycles, e.g., three cycles, are recorded, it is preferred that the surfactant exhibit a langmuir isotherm having a maximum surface pressure (pi max) as defined above in the last, e.g., third, isotherm cycle.
Typically, the langmuir surface pressure/area isotherm or isotherm cycle referred to herein is recorded at about room temperature, e.g., 22.1±0.2 ℃. Typical concentrations of surfactant in its aqueous solution for maximum surface pressure determination are 1% w/v (corresponding to 1g surfactant in the total volume of 100ml including surfactant, typically measured at room temperature, e.g. 22.1±0.2 ℃).
Details regarding preferred surfactant types, such as poloxamers, are also discussed below.
For example, if the pharmaceutical composition is a pharmaceutical composition comprising a lipid or lipid, such as a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), the minimum surface area at which the langmuir isotherms of the surfactant are recorded may be determined for the pharmaceutical composition, e.g., by compressing a layer (typically a monolayer) of the lipid or lipid comprised by the pharmaceutical composition in the langmuir trough until a first phase change is observed in the surface pressure/area isotherms of the lipid or lipid. If the pharmaceutical composition, for example as a nanoparticle formulation, comprises a combination of more than one lipid and/or lipid class, the minimum surface area may be determined for the pharmaceutical composition, for example, by compressing a layer (typically a monolayer) of the combination of more than one lipid and/or lipid class comprised by the pharmaceutical composition in a langmuir trough until a first phase change is observed in the combined surface pressure/area isotherm. To determine the minimum surface area, the layer may conveniently be provided on water, for example deionized water.
If, as described above, the pharmaceutical composition is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as its component at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanent cationic lipid, the minimum surface area at which the langmuir isotherm of the surfactant is recorded can be determined for the pharmaceutical composition, for example, by compressing the lipid mixture or layer of lipid mixture (typically a monolayer) comprised by LNP or LiNP in a langmuir trough until a first phase change is observed in the surface pressure/area isotherm of the lipid mixture or lipid mixture. To determine the minimum surface area, the layer may conveniently be provided on water, for example deionized water.
In either case, the surface area at the beginning of the phase change is considered to be the minimum surface area determined for the pharmaceutical composition. As mentioned above, the phase transition onset referred to herein in the pressure/area (pi-a) isotherm plot can be objectively determined as the point at which the slope of the curve (dpi/dA) changes significantly.
The step of determining the minimum surface area may be performed in the langmuir tank, for example as a calibration step, before determining the maximum surface pressure of the surfactant as described above.
As the maximum surface area during recording of the langmuir surface pressure/area isotherm, for example, the maximum area provided by langmuir tanks may be conveniently used.
For example, a monolayer of the water lipids and/or lipids or a water lipid mixture or lipid mixture may be provided in the langmuir trough by applying the lipids and/or lipids or lipid mixture to the surface until a signal change occurs in the langmuir trough detector.
The calibration step that can be used to determine the minimum surface area and the measurement of the maximum surface pressure are further illustrated in the examples section below.
In a fourth aspect, the present invention provides a method for classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition, the method comprising:
(a) Providing an aqueous solution of surfactant at a concentration (C),
(B) Recording the langmuir surface pressure/area isotherm of the surfactant in the solution to determine a maximum surface pressure pi max of the langmuir isotherm at a predetermined minimum surface area;
(c) The maximum surface pressure pi max is compared with a threshold value, wherein if the maximum surface pressure pi max is equal to or lower than the threshold value, the surfactant is classified as suitable for use as a stabilizer, and if the maximum surface pressure pi max is greater than the threshold value, the surfactant is classified as unsuitable for use as a stabilizer.
In the context of this aspect, there is further provided a method for preparing a pharmaceutical composition, the method comprising classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition according to the above method, and incorporating the surfactant into the pharmaceutical composition if the surfactant is classified as suitable for use as a stabilizer for a pharmaceutical composition.
For example, the method may be used to classify surfactants as suitable or unsuitable for use as stabilizers for pharmaceutical compositions comprising lipids or lipids, such as Lipid Nanoparticle (LNP) formulations or lipid nanoparticle (LiNP) formulations comprising a plurality of Lipid Nanoparticles (LNPs) or lipid nanoparticles (LiNP). More preferably, the pharmaceutical composition is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each lipid nanoparticle or lipid nanoparticle comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, and a permanent cationic lipid as a component thereof.
If the pharmaceutical formulation is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, it is preferred that the formulation is a suspension formulation comprising a liquid carrier solution in which the LNP or LiNP can be dispersed. The carrier solution is preferably an aqueous carrier solution. Preferably, in such suspension formulations, surfactants classified as suitable for use as stabilizers are incorporated into the carrier solution.
For example, in such suspension formulations, the surfactant may advantageously act as a stabilizer to mitigate aggregation of LNP or LiNP or a subset thereof, which may be caused by shaking or shear stress of the suspension during production, purification, handling or transportation, preferably during production or purification, more preferably during purification, most preferably during Tangential Flow Filtration (TFF) purification.
Those skilled in the art will appreciate that pharmaceutical compositions include therapeutic agents, such as nucleic acids. Preferred therapeutic agents are RNA, and particularly preferred are mRNA. Details regarding the preferred therapeutic agent types are discussed below. If the pharmaceutical formulation is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, such as a suspension formulation, the therapeutic agent is typically included in LNP or LiNP.
In accordance with the foregoing, it is particularly preferred that the method according to the present aspect is a method for classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition, wherein the pharmaceutical composition is in the form of a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanent cationic lipid as a component thereof, and wherein the carrier solution of the suspension formulation is an aqueous carrier solution.
In step (a) of the method, an aqueous solution of surfactant is provided in concentration (C), for example as a solution in deionized water. Details of preferred surfactant types, such as poloxamers, for performing the method are discussed below. The concentration of the surfactant is not particularly limited, and exemplary concentrations may be in the range of 0.1 to 10.0% w/v (i.e., expressed as weight of surfactant in total volume of 100 ml solution, measured typically at room temperature, e.g., at 22.1±0.2 ℃), preferably in the range of 0.5 to 5.0% w/v, with a concentration of 1% w/v being particularly preferred.
In step (b), the Langmuir surface pressure/area isotherm of the surfactant in the solution provided in step (a) is recorded to determine the maximum surface pressure pi max of the Langmuir surface pressure/area isotherm at a predetermined minimum surface area. Those skilled in the art will appreciate that this may be accomplished using langmuir slots. Recording the langmuir isotherm of a surfactant involves reducing the available surface area in the langmuir tank at least until a predetermined minimum surface area is reached. The surface pressure observed at the minimum surface area when recording the langmuir isotherm of the surfactant is considered to be the maximum surface pressure (pi max) of the surfactant.
As described herein, the inventors have surprisingly found that different langmuir surface pressure/area isotherms can be observed, not only for different types of surfactants, but even for the same type of surfactant, e.g., surfactants obtained from different sources or production lots, and that langmuir isotherms reflect the suitability of the surfactant to act as a stabilizer. The minimum surface area of the langmuir isotherm can be conveniently determined by one skilled in the art as a reference point. The reference point may be used to evaluate the suitability and determine the threshold value that must be complied with. The determination may be based on preliminary stability testing of the desired composition containing the surfactant as a stabilizer, and selecting the minimum surface area and threshold based on the composition having good stability. On this basis, the surfactant may be conveniently selected and/or quality controlled during the actual production of the composition by obtaining langmuir isotherm data as discussed above and comparing it to data previously determined using the stable composition as a template.
This method can be advantageously used, for example, to distinguish between variants of a single surfactant, each variant nominally identified by the same name and meeting the same pharmaceutical quality criteria, to classify each variant as suitable or unsuitable for use as a stabilizer for pharmaceutical compositions.
For example, to classify a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition comprising a lipid or lipid, the minimum surface area may be determined, for example, by compressing a layer (typically a monolayer) of the lipid or lipid in a langmuir trough until a first phase change is observed in the surface pressure/area isotherm of the lipid or lipid. To determine the minimum surface area, the layer may conveniently be provided on water, for example deionized water. If the pharmaceutical composition, for example, comprises a combination of more than one lipid and/or lipid as a nanoparticle formulation, the minimum surface area of the pharmaceutical composition may be determined, for example, by compressing a layer (typically a monolayer) comprising at least one lipid or lipid of the combination in a langmuir trough until a first phase change is observed in the surface pressure/area isotherm of the lipid or lipid. Preferably, the layer comprises at least 100% by weight of the total weight of the combination, of the largest proportion of lipids or lipids in the composition of more than one lipid and/or lipid. More preferably, for pharmaceutical compositions comprising a combination of more than one lipid and/or lipid, the minimum surface area of the pharmaceutical composition may be determined, for example, by compressing a layer (typically a monolayer) comprising the same combination of more than one lipid and/or lipid in the langmuir trough in the same ratio until a first phase change is observed in the combined surface pressure/area isotherm. To determine the minimum surface area, the layer may conveniently be provided on water, for example deionized water.
Similarly, to classify the surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition in the form of a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation as discussed above, the minimum surface area may be determined, for example, by compressing a layer (typically a monolayer) comprising at least one lipid or lipid contained in a lipid mixture or lipid mixture (i.e., the lipid mixture or lipid mixture comprised by the nanoparticle) in a langmuir trough until a first phase change is observed in the surface pressure/area isotherm of the lipid or lipid. Preferably, the layer comprises at least 100% by weight of the lipid mixture or lipid mixture, based on the total weight of the lipid mixture or lipid mixture, the greatest proportion of the lipid or lipid mixture being present in the composition of the lipid mixture or lipid mixture. More preferably, for pharmaceutical compositions in the form of Lipid Nanoparticle (LNP) formulations or lipid nanoparticle (LiNP) formulations as discussed above, the minimum surface area may be determined by compressing a layer (typically a monolayer) of the lipid mixture or lipid mixture (i.e., the lipid mixture or lipid mixture that the nanoparticle comprises) in a langmuir trough until a first phase change is observed in the surface pressure/area isotherm of the lipid mixture or lipid mixture. To determine the minimum surface area, the layer may conveniently be provided on water, for example deionized water.
In either case, the surface area at which the phase change is then initiated may depend on the predetermined minimum surface area in step (b) as the method. As mentioned above, the phase transition onset referred to herein in the pressure/area (pi-a) isotherm plot can be objectively determined as the point at which the slope of the curve (dpi/dA) changes significantly.
Optionally, the step of determining the minimum surface area may be performed in the langmuir tank, for example as a calibration step, before determining the maximum surface pressure of the surfactant as described above.
As the maximum surface area during recording of the langmuir surface pressure/area isotherm, the surface area before the start of compression in the compression stage, for example the maximum area provided by langmuir cells, may be conveniently used.
For example, a monolayer of the water lipids and/or lipids or a water lipid mixture or lipid mixture may be provided in the langmuir trough by applying the lipids and/or lipids or lipid mixture to the surface until a signal change occurs in the langmuir trough detector.
The langmuir surface pressure/area isotherms of the surfactant in the solution in step (b) may, for example, be recorded as langmuir surface pressure/area isotherms cycles comprising a compression stage and an expansion stage. For example, the maximum surface pressure (pi max) may be determined by recording a single isotherm cycle, but preferably the result is obtained by sequentially recording a plurality of isotherm cycles, such as three isotherm cycles, for example with a waiting time between cycles of 5 seconds or less, such as 3 seconds. If a single isotherm cycle is recorded, it is preferred that pi max be equal to or less than 3.5 mN/m. If multiple cycles are recorded, such as three cycles, it is preferred that the surfactant exhibit a Langmuir isotherm with a maximum surface pressure (pi max) equal to or below the threshold in the last, e.g., third, isotherm cycle.
For example, the langmuir surface pressure/area isotherms or isotherm cycles referred to herein may be recorded at room temperature, e.g., 22.1±0.2 ℃.
In step (c), the maximum surface pressure is then compared to a threshold value. Based on this comparison, a determination can be made as to whether the surfactant is suitable for use as a stabilizer.
As mentioned above, the threshold value for the maximum surface pressure pi max for comparison and classification can be conveniently determined, for example, based on surface pressure data prepared in a preliminary test of a stable composition comprising a surfactant. For example, pi max may be equal to or less than 4.0 mN/m, or equal to or less than 3.5 mN/m.
As described above, in the context of this aspect, there is further provided a method for preparing a pharmaceutical composition, the method comprising classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition according to the above method, and incorporating the surfactant into a carrier solution of the pharmaceutical composition, e.g. in the form of a nanoparticle suspension, if the surfactant is classified as suitable for use as a stabilizer for a pharmaceutical composition.
In a fifth aspect, the present invention provides a method for classifying a surfactant as suitable or unsuitable for use as a stabiliser for a pharmaceutical composition comprising a lipid or a lipid-like substance, optionally during purification of the composition, preferably during tangential flow filtration of the composition, wherein the method comprises the steps of:
(a) Providing an aqueous solution of surfactant at a concentration (C) of surfactant in the solution;
(b) Recording Langmuir pressure/area isotherm cycles comprising a compression phase and an expansion phase between a maximum surface area and a minimum surface area on a sample comprising an aqueous solution of a surfactant and carrying on its surface a lipid or lipid comprised by the composition,
(C) The langmuir isotherm Δpi for each area point of the langmuir pressure/area isotherm cycle is calculated, wherein Δpi is calculated as follows:
,
Wherein the method comprises the steps of Is the surface pressure at the area point during the compression phase of the isotherm cycle,
Wherein the method comprises the steps ofIs the surface pressure at the area point during the expansion phase of the isotherm cycle, and
Wherein the method comprises the steps ofIs the maximum surface pressure reached in the isotherm cycle, and
(D) The calculated langmuir isotherm Δpi is compared to a threshold value, wherein if the calculated langmuir isotherm Δpi at each isotherm area point is equal to or below the threshold value, the surfactant is classified as suitable for use as a stabilizer and if the calculated langmuir Miao Er pi at any area point is greater than the threshold value, the surfactant is classified as unsuitable for use as a stabilizer.
In the context of this aspect, there is further provided a method of preparing a pharmaceutical composition, the method comprising classifying a surfactant as suitable or unsuitable for use as a stabiliser for a pharmaceutical composition comprising a lipid or lipid according to the above method, and incorporating the surfactant into the pharmaceutical composition if the surfactant is classified as suitable for use as a stabiliser.
For example, the method may be used to classify a surfactant as suitable or unsuitable for use as a stabilizer in a pharmaceutical composition comprising a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP). Preferably, the pharmaceutical composition is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each lipid nanoparticle or lipid nanoparticle comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, and a permanent cationic lipid as a component thereof.
If the pharmaceutical formulation is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, it is preferred that the formulation is a suspension formulation comprising a liquid carrier solution in which LNP or LiNP can be dispersed. The carrier solution is preferably an aqueous carrier solution. Preferably, in such suspension formulations, surfactants classified as suitable for use as stabilizers are incorporated into the carrier solution.
For example, in such suspension formulations, the surfactant may advantageously act as a stabilizer to mitigate aggregation of LNP or LiNP or a subset thereof, which may be caused by shaking or shear stress of the suspension during production, purification, handling or transportation, preferably during production or purification, more preferably during purification, most preferably during Tangential Flow Filtration (TFF) purification.
Those skilled in the art will appreciate that pharmaceutical compositions include therapeutic agents, such as nucleic acids. Preferred therapeutic agents are RNA, and particularly preferred are mRNA. Details regarding the preferred therapeutic agent types are discussed below. If the pharmaceutical formulation is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, such as a suspension formulation, the therapeutic agent is typically included in LNP or LiNP.
In accordance with the foregoing, it is particularly preferred that the method according to the present aspect is a method of classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition, wherein the pharmaceutical composition is in the form of a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein each LNP or LiNP comprises at least one selected from an ionizable lipid, an ionizable lipid and a permanent cationic lipid as a component of the lipid mixture or lipid mixture, and wherein the carrier solution of the suspension formulation is an aqueous carrier solution.
In step (a) of the method, an aqueous solution of surfactant is provided at a concentration (C). Details of preferred surfactant types, such as poloxamers, for performing the method are discussed below. The concentration of the surfactant is not particularly limited, and exemplary concentrations may range from 0.1 to 10.0% w/v (i.e., expressed as the weight of surfactant in the combined volume of surfactant and aqueous solvent of 100ml, expressed in g, typically measured at about room temperature, e.g., at 22.1±0.2 ℃), preferably in the range of 0.5 to 5.0% w/v. Particularly preferred is a concentration of 1% w/v.
In step (b), langmuir pressure/area isotherm cycles comprising a compression stage and an expansion stage are recorded between the maximum surface area and the minimum surface area. Isotherm cycles are recorded for a sample comprising the surfactant in the aqueous solution provided in step (a) and bearing on its surface the lipid or lipids comprised by the pharmaceutical composition. Typically, the sample bears a monolayer of lipid or lipids on its surface. Those skilled in the art will appreciate that the recording of isotherm cycles can be accomplished using langmuir cells containing the sample.
If the pharmaceutical composition, e.g. as a nanoparticle formulation, comprises more than one lipid and/or combination of lipids, the isotherm cycle may be recorded, e.g. on a sample carrying a layer (typically a monolayer) comprising at least one lipid or lipid of the combination. Preferably, the layer comprises at least 100% by weight based on the total weight of the combination of lipids and/or lipids, the largest proportion of lipids or lipids being in the components of the combination of more than one lipid and/or lipid in the pharmaceutical composition. More preferably, for pharmaceutical compositions comprising a combination of more than one lipid and/or lipid, isotherm cycles can be recorded for samples bearing a layer (typically a monolayer) comprising the same combination of more than one lipid and/or lipid in the same proportion as included in the pharmaceutical composition.
According to the preferred embodiment described above, the pharmaceutical composition is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanent cationic lipid. In this case, isotherm cycles can be recorded for example for a sample carrying a layer (typically a monolayer) comprising at least one lipid or lipid contained in a lipid mixture or lipid mixture (i.e. the nanoparticle comprises a lipid mixture or lipid mixture). Preferably, the layer comprises at least 100% by weight of the lipid or lipid based on the total weight of the lipid mixture or lipid mixture, the largest proportion of lipids or lipids being present in the lipid mixture or components of the lipid mixture. More preferably, for a pharmaceutical composition in the form of a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation as described above, the isotherm cycle is recorded on a sample bearing a layer (typically a monolayer) of the lipid mixture or lipid mixture (i.e., the lipid mixture or lipid mixture that the nanoparticle comprises).
The minimum surface area employed for recording an isotherm cycle is typically determined by the lipid or lipid, or by a combination of more than one lipid/lipid contained in the sample used to record the isotherm cycle. In particular, the minimum surface area for recording the isotherm cycles is preferably the surface area at which onset of phase change can be observed when the lipid or a layer of lipid or a combination of more than one lipid/lipid (typically a monolayer) is compressed in a langmuir cell.
Thus, optionally, a step of determining the minimum surface area may be performed in the langmuir trough, for example as a calibration step, prior to recording the isotherm cycles in step (b). For example, the minimum surface area may be determined by compressing a layer (typically a monolayer) of the lipid or lipids or a combination of more than one lipid/lipid contained in the sample used to record the isotherm cycle in step (b) in a langmuir cell until a first phase change is observed in the surface pressure/area isotherm. To determine the minimum surface area, the layer may conveniently be provided on water, for example deionized water. The surface area at the start of the phase change can then be relied upon as the minimum surface area for the isotherm cycle recorded in step (b) of the method. As mentioned above, the phase transition onset referred to herein in the pressure/area (pi-a) isotherm plot can be objectively determined as the point at which the slope of the curve (dpi/dA) changes significantly.
As the maximum surface area during recording of the langmuir surface pressure/area isotherm, the surface area before the start of compression in the compression stage, such as the maximum area provided by langmuir tanks, may be conveniently used.
For example, a monolayer of the water lipids and/or lipids, or a water lipid mixture or lipid mixture, may be provided in the langmuir trough by applying the lipids and/or lipids or lipid mixture to the surface until a signal change occurs in the langmuir trough detector.
It is sufficient if a single isotherm cycle is recorded and the langmuir isotherm Δpi value of that isotherm cycle is preferably calculated in step (c). Multiple isotherm cycles, such as three isotherm cycles, may be recorded sequentially, for example with a latency between cycles of 5 seconds or less, such as 3 seconds. However, if the Δpi value of the first cycle satisfies the above requirement, this is generally applicable to the subsequent cycle as well, and therefore even if a plurality of cycles are recorded, it is preferable to calculate the Δpi value depending on the measurement result of the first cycle.
For example, the langmuir surface pressure/area isotherms or isotherm cycles referred to herein may be recorded at room temperature, e.g., 22.1±0.2 ℃.
In step (c), the langmuir isotherm Δpi for each area point of the langmuir pressure/area isotherm cycle is calculated.
As an aspect closely related to the above-described method for classifying a surfactant, there is provided a method of preparing a pharmaceutical composition, the method comprising classifying the surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition according to the above-described method, and incorporating the surfactant into the pharmaceutical composition, for example into a carrier solution of the pharmaceutical composition in the form of a nanoparticle suspension, if the surfactant is classified as suitable for use as a stabilizer.
In a related sixth aspect, the invention provides a method of reducing or avoiding clogging or scaling of a filtration system during purification of a pharmaceutical composition in the form of a lipid nanoparticle formulation (LNP) or a lipid nanoparticle formulation (LiNP), the method comprising adding a stabilizing surfactant to a first LNP or LiNP formulation to form a second LNP or LiNP formulation, optionally prior to purification, wherein the LNP or LiNP formulation comprises a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises as its components at least one selected from the group consisting of ionizable lipids, ionizable lipids and permanent cationic lipids, and wherein the stabilizing surfactant is a surfactant according to the invention, such as the surfactant described in the third aspect above, or wherein the stabilizing surfactant is a surfactant classified as suitable as a stabilizer by the method of the fourth or fifth aspect above.
LNP formulations or LiNP formulations include therapeutic agents, such as nucleic acids. Preferred therapeutic agents are RNA, and particularly preferred are mRNA. Details regarding the preferred therapeutic agent types are discussed below.
Preferably, the LNP formulation or LiNP formulation is a suspension formulation comprising a liquid carrier solution in which the LNP or LiNP can be dispersed. The carrier solution is preferably an aqueous carrier solution. Preferably, in such suspension formulations, the stabilizing surfactant is added to the carrier solution in the context of the above-described process. Optionally, the LNP or LiNP dispersed in the liquid carrier is substantially free of stabilizing surfactants, e.g., the surfactant is substantially not incorporated into the LNP or LiNP.
Preferably, the purification of the pharmaceutical composition comprises Tangential Flow Filtration (TFF) purification.
In a further related seventh aspect, the invention provides a method of reducing aggregation of lipid nanoparticles or lipid nanoparticles in a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, the method comprising adding a stabilizing surfactant to a first LNP or LiNP formulation to form a second LNP or LiNP formulation, optionally prior to purification, wherein the LNP or LiNP formulation comprises a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanent cationic lipid, and wherein the stabilizing surfactant is a surfactant according to the invention, e.g. a surfactant as discussed in the third aspect above, or wherein the stabilizing surfactant is classified as a surfactant suitable as a stabilizer by a method according to the fourth or fifth aspect discussed above.
Preferably, the method is a method of reducing aggregation of a lipid or lipid nanoparticle in a pharmaceutical formulation in the form of a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture and a therapeutic agent, such as a nucleic acid. Preferred therapeutic agents are RNA, and particularly preferred are mRNA. Details regarding the preferred therapeutic agent types are discussed below.
Also in the context of this method, it is preferred that the LNP formulation or LiNP formulation is a suspension formulation comprising a liquid carrier solution in which the LNP or LiNP can be dispersed. The carrier solution is preferably an aqueous carrier solution. Preferably, in such suspension formulations, the stabilizing surfactant is added to the carrier solution in the context of the above-described process. Optionally, the LNP or LiNP dispersed in the liquid carrier is substantially free of stabilizing surfactants, e.g., the surfactant is substantially not incorporated into the LNP or LiNP.
Thus, it is particularly preferred that the method is a method of reducing aggregation of lipids or lipid nanoparticles in a pharmaceutical formulation in the form of a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising a plurality of LNPs or LiNP, each LNP or LiNP comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanent cationic lipid.
The lipid nanoparticle or lipid nanoparticle may comprise a nucleic acid (e.g. RNA, and preferably mRNA) as therapeutic agent, and the method according to this aspect may comprise, for example, the steps of:
i) First, combining a nucleic acid with at least one selected from the group consisting of an ionizable lipid, and a permanently cationic lipid to form an LNP or LiNP,
Ii) second, purified LNP or LiNP,
Iii) Thirdly, adding stabilizing surfactant before TFF purification and/or during TFF purification in exchange buffer to maintain the surfactant at a stable concentration,
Iv) optionally wherein a stabilizing surfactant is added to the LNP or LiNP formulation after step (i).
In a preferred method, the method according to this aspect may alternatively comprise, for example, the steps of:
i) The LNP or LiNP preparation is produced by mixing at least one selected from the group consisting of a permanent cationic lipid, an ionizable lipid, and an ionizable lipid dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution,
Ii) diluting the LNP or LiNP preparation with the first solution,
Iii) Concentration of LNP or LiNP preparations by buffer exchange using ultrafiltration/diafiltration via TFF, wherein the second solution is used for ultrafiltration/diafiltration,
Iv) obtaining a suspension of LNP or LiNP in an aqueous carrier solution,
Wherein the first solution comprises between about 0.01% w/v and 10% stabilizing surfactant, preferably between about 0.01% w/v surfactant and 5% w/v surfactant, more preferably between about 0.01% w/v surfactant and 2.5% w/v surfactant, more preferably between about 0.05% w/v and 1.5% w/v surfactant, even more preferably between about 0.05% w/v and 1.5% w/v surfactant, most preferably about 1% w/v surfactant, and/or
Wherein the second solution comprises between about 0.01% w/v and about 10% stabilizing surfactant, preferably between about 0.01% w/v surfactant and about 5% w/v surfactant, more preferably between about 0.01% w/v surfactant and about 2.5% w/v surfactant, even more preferably between about 0.05% w/v and 1.5% w/v surfactant, most preferably about 1% w/v surfactant;
And wherein the final concentration of stabilizing surfactant from the combined first and second solutions is between 0.01% and 10% surfactant, preferably between 0.01% w/v surfactant and 5% w/v surfactant, more preferably between 0.01% w/v surfactant and 2.5% w/v surfactant, even more preferably between 0.05% w/v and 1.5% w/v surfactant, most preferably about 1% w/v surfactant, relative to the total volume of the suspension of nanoparticles in the aqueous carrier solution.
In the context of this preferred method, these other optional features can be observed:
a) Incorporation of the stabilizing surfactant into the suspension does not occur before or during step i),
B) Adding a stabilizing surfactant to the first and second solutions, and/or
C) About half of the stabilizing surfactant is added to the first solution and about half of the surfactant is added to the second solution.
As a related eighth aspect, the present invention provides a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation obtained by the method according to the fourth to seventh aspects. For example, the present aspect provides a pharmaceutical composition in the form of a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, the composition comprising a surfactant which has been classified as suitable for use as a stabiliser for a pharmaceutical composition according to the method of the fourth aspect. As another example, an eighth aspect provides a pharmaceutical composition in the form of a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, the composition comprising a surfactant which has been classified as suitable for use as a stabiliser for a pharmaceutical composition comprising a lipid or lipid composition according to the method of the fifth aspect. As another example, an eighth aspect provides a pharmaceutical composition in the form of a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation obtained by a method according to the sixth aspect. As a further example, an eighth aspect provides a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, preferably a pharmaceutical composition in the form of a Lipid Nanoparticle (LNP) or a lipid nanoparticle (LiNP) obtained by a method according to the sixth aspect.
Preferably, the formulation is a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation.
Further, in a ninth aspect, the present invention provides a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation according to the first, second or eighth aspects discussed above for use as a medicament. As will be appreciated, the Lipid Nanoparticle (LNP) formulation or the LNP or LiNP of the lipid nanoparticle (LiNP) formulation used in accordance with this aspect includes a therapeutic agent, for example a nucleic acid, such as RNA, preferably mRNA. They are preferably suspension formulations as discussed in the context of the relevant aspects.
In a related tenth aspect, the present invention provides a Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation according to the first, second or eighth aspects discussed above, for use in the treatment or prophylaxis of a disease. As will be appreciated, the Lipid Nanoparticle (LNP) formulation or the LNP or LiNP of the lipid nanoparticle (LiNP) formulation used in accordance with this aspect includes a therapeutic agent, for example a nucleic acid, such as RNA, preferably mRNA. They are preferably suspension formulations as discussed in the context of the relevant aspects.
The disease may be, for example, a disease selected from table a disclosed below.
Likewise, and preferably, the disease may be a disease selected from viral disease, ciliated disease or autoimmune disease and respiratory disease, even more preferably selected from pulmonary disease, airway disease or nasal disease, more preferably pulmonary viral disease, pulmonary ciliated disease and pulmonary autoimmune disease. Preferably, the pulmonary disease or pulmonary viral disease is at least one selected from the group consisting of pneumonia and asthma, the airway disease is at least one selected from the group consisting of bronchitis, virus-induced asthma, pulmonary fibrosis and COPD, and/or the nasal disease is at least one selected from the group consisting of rhinitis and sinusitis.
In some embodiments, the disease to be treated or prevented is a disease selected from the group consisting of alveolar proteinosis (PAP), interstitial lung diseases (such as pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis), viral infections (such as influenza and COVID-19), acute Respiratory Distress Syndrome (ARDS), nontuberculous mycobacterial (NTM) infections, lung cancer, fungal infections caused by Aspergillus (such as aspergillosis, mycotic sinusitis, otomycosis, keratitis, and A-mycosis, preferably those caused by Aspergillus fumigatus and Aspergillus flavus), infections caused by Mycobacterium tuberculosis, pseudomonas aeruginosa, phanerochaete, plasmodium, cryptococcus, nocardia, and combinations thereof.
In some embodiments, the disease is selected from the group consisting of (autoimmune) alveolar protein deposition (PAP), interstitial lung disease (such as pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis), viral infections (such as influenza and COVID-19), acute Respiratory Distress Syndrome (ARDS), nontuberculous mycobacterial (NTM) infections, lung cancer or fungal infections caused by aspergillus (such as aspergillosis, mycotic sinusitis, otomycosis, keratitis and onychomycosis, preferably those caused by aspergillus fumigatus and aspergillus flavus), infections caused by mycobacterium tuberculosis, pseudomonas aeruginosa, pneumosporium bacteria and plasmodium.
The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation used according to the tenth aspect may be used for vaccination or immunization.
In another aspect, the invention provides a method for classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition comprising a nucleic acid, optionally as a stabilizer during purification, preferably during TFF purification, the method comprising:
(a) Providing an aqueous solution of a surfactant;
(b) Optionally combining a surfactant with the LNP or LiNP formulation;
(c) Purifying the aqueous solution comprising the surfactant or the LNP or LiNP formulation optionally comprising the surfactant using a membrane purification system (preferably TFF or ultrafiltration, most preferably TFF);
(d) Measuring the filtration time of the aqueous solution or LNP formulation during diafiltration or ultrafiltration;
(e) Comparing the measured filtration rate to a predetermined threshold, preferably a time threshold, more preferably a time threshold of 90 minutes, wherein if the filtration rate is equal to or higher than the threshold, the surfactant is classified as unsuitable for use as a stabilizer, and if the filtration rate is less than the threshold, the surfactant is classified as suitable for use as a stabilizer.
In another aspect, the invention provides a method of reducing or avoiding side effects of treatment with LNP or LiNP carrying at least one therapeutic agent, wherein the method comprises the steps of:
i) Determining whether LNP or LiNP in a pharmaceutical composition comprising LNP or LiNP is aggregated when subjected to mechanical or temperature stress by determining its aggregation level before and after subjecting the pharmaceutical composition to the mechanical or temperature stress,
Ii) if LNP or LiNP shows aggregation after the test of step (i), a surfactant is added to the LNP or LiNP formulation to obtain a LNP or LiNP suspension with a final surfactant concentration of 0.01% w/v up to 10% w/v, preferably between 0.05% w/w surfactant and 5% surfactant, more preferably between 0.33% surfactant and 2.5% surfactant, more preferably between 0.45% to 1.5% surfactant, preferably between 0.5% and 1.5% surfactant, most preferably 1% surfactant.
Iii) Reconstitution by mixing to produce a stable LNP or LiNP suspension.
In a related aspect, the present invention provides the use of a surfactant according to the present invention, e.g. a surfactant according to the third aspect above, or a surfactant classified as suitable as a stabilizer in a pharmaceutical composition according to the method of the fourth or fifth aspect above, for stabilizing a lipid nanoparticle or suspension of lipid nanoparticles in an aqueous carrier solution under physical stress conditions, preferably shear stress, more preferably shear stress during purification, e.g. TFF, to prevent aggregation of particles, wherein the lipid nanoparticle or lipid nanoparticle comprises the following components (a) and (b):
(a) Therapeutic agent, and
(B) At least one selected from the group consisting of a permanent cationic lipid, an ionizable lipid, and an ionizable lipid.
Therapeutic agent
When reference is made herein to a therapeutic agent, a pharmaceutical composition comprising a therapeutic agent, and/or a lipid nanoparticle formulation or lipid nanoparticle formulation comprising a therapeutic agent, one or more therapeutic agents may be used in the context of the present invention.
Preferably, the therapeutic agent comprises, consists essentially of, or consists of a nucleic acid, more preferably RNA, still more preferably single stranded RNA, and most preferably mRNA.
In lipid nanoparticle formulations or lipid nanoparticle formulations comprising the therapeutic agents mentioned herein, for example, negatively charged therapeutic agents may be used. Those skilled in the art will appreciate that in such lipid nanoparticle formulations or lipid nanoparticle formulations that include a therapeutic agent, the therapeutic agent is typically included in the nanoparticle. Also in such formulations, it is preferred that the therapeutic agent comprises, consists essentially of, or consists of a nucleic acid, more preferably RNA, still more preferably single stranded RNA, and most preferably mRNA. Thus, in the context of the present invention, a particularly preferred pharmaceutical composition is a lipid nanoparticle formulation or a pharmaceutical composition in the form of a lipid nanoparticle formulation, such as a suspension formulation, wherein the lipid nanoparticle or lipid nanoparticle comprises mRNA as therapeutic agent.
The nature of the nucleic acid is not particularly limited. In principle, any type of nucleic acid may be used in the context of the present invention. Nucleic acids are known to the person skilled in the art and refer to biopolymers or small biomolecules consisting of nucleotides, which are monomers consisting of three components, a 5-carbon sugar, a phosphate group and a nitrogenous base.
The term nucleic acid is a generic term for DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), i.e. members of the above-mentioned family of biopolymers. If fructose is a complex ribose, then the polymer is RNA, and if fructose is deoxyribose derived from ribose, then the polymer is DNA. The term "nucleic acid" includes oligonucleotides or polynucleotides. Since nucleic acids are biopolymers composed of nucleotides, the term "nucleic acid" is also commonly referred to as "nucleotide sequence (sequence of nucleotide)", and thus, one skilled in the art will understand that the terms "nucleic acid" and "nucleic acid sequence (nucleic acid sequence)" are often used interchangeably.
In a preferred embodiment, the nanoparticle comprises ribonucleic acid (RNA), more preferably single stranded RNA, and most preferably mRNA, as the nucleic acid.
The term "nucleic acid" includes all forms of naturally occurring nucleic acid types as well as chemically and/or enzymatically synthesized nucleic acids, and also includes nucleic acid analogs and nucleic acid derivatives. The term specifically includes any backbone-modified, sugar-modified or base-modified single-or double-stranded nucleic acid, such as, for example, locked Nucleic Acids (LNA), peptide Nucleic Acids (PNA), oligonucleotide phosphorothioates and phosphotriesters, morpholino oligonucleotides, cationic oligonucleotides (US 6017700A, WO/2007/069092), substituted ribooligonucleotides or phosphorothioates. Furthermore, the term "nucleic acid" also refers to any molecule comprising a nucleotide or nucleotide analogue. There is no limitation on the sequence or size of the nucleic acid included in the nanoparticle of the present invention. Nucleic acids are primarily defined by the biological effects achieved at the biological target to which the nanoparticles of the present invention are delivered. For example, as will be outlined in more detail below, in the application of a gene or nucleic acid therapy, a nucleic acid or nucleic acid sequence may be defined by the gene or gene fragment to be expressed, or by the intended substitution or repair of a defective gene or any gene target sequence, or by the target sequence of a gene to be suppressed, knocked out, down-regulated or up-regulated.
Nanoparticles may include nucleic acids that are DNA molecules. A preferred embodiment of such a DNA molecule is a DNA molecule which can be transcribed into an mRNA molecule. Transcription is the first step in gene expression in which a specific segment of a DNA molecule is replicated into an mRNA molecule by an RNA polymerase. During transcription, the DNA sequence is read by RNA polymerase, producing complementary antiparallel RNA strands, called primary transcripts.
The DNA molecules may be introduced into the vector, preferably expressed in the vector, by standard molecular biology techniques (see Sambrook et al, molecular Cloning, A laboratory Manual, 2 nd edition, 1989). "vector" in the sense of the present invention, such as an "expression vector" or "cloning vector", is understood as a circular double stranded DNA unit, which is preferably capable of replicating in a cell independently of chromosomal DNA and serves as a vector for transporting genetic material into a cell, in which it can be expressed (i.e. transcribed into RNA and translated into an amino acid sequence). Vectors comprising exogenous DNA are referred to as recombinant DNA. The vector itself is typically a DNA sequence, which typically consists of an insert (e.g., a nucleic acid molecule/DNA molecule of the invention) and a larger sequence that serves as the "backbone" of the vector. Plasmids in the sense of the present invention are most commonly found in bacteria and are used in recombinant DNA studies to transfer genes between cells and are a subset of "vectors" used in the sense of the present invention.
Other regulatory sequences may be added to the DNA molecules of the invention, as will be apparent to those skilled in the art. For example, transcription enhancers and/or sequences that allow for the induction of expression may be used. Suitable induction systems are, for example, the tetracycline regulated gene expression described in Gossen and Bujard, proc. Natl. Acad. Sci. USA89 (1992), 5547-5551 and Gossen, trends Biotech.12 (1994), 58-62, or the dexamethasone inducible gene expression system described, for example, in Crook, EMBO J.8 (1989), 513-519. Vectors comprising DNA molecules, preferably expression vectors, may also be used in the present invention. The vector may be, for example, a plasmid, cosmid, virus, phage, or another vector such as is conventionally used in genetic engineering, and may include other genes, such as marker genes, which allow selection of the vector under appropriate host cells and appropriate conditions.
If the nucleic acid used in the context of the present invention is a DNA molecule, it may be a plasmid DNA (pDNA) molecule.
As described above, the nanoparticle preferably includes ribonucleic acid (RNA), more preferably single-stranded RNA, and most preferably mRNA, as the nucleic acid.
With respect to RNA, in principle, any type of RNA may be used in the context of the present invention. In a preferred embodiment, the RNA is single stranded RNA. The term "single-stranded RNA" refers to a continuous strand of ribonucleotides, in contrast to RNA molecules in which two or more separate strands form a double-stranded molecule as a result of hybridization of the separate strands. The term "single stranded RNA" does not exclude that the single stranded molecule itself forms a double stranded structure, such as a secondary structure (e.g., loop and stem loop) or a tertiary structure. Examples are tRNA and mRNA, but also include any other type of single stranded RNA, such as antisense RNA, siRNA, miRNA, etc.
The term "RNA" encompasses RNA that encodes an amino acid sequence and RNA that does not encode an amino acid sequence. It has been proposed that more than 80% of the genome contains functional DNA elements that do not encode proteins. These non-coding sequences include regulatory DNA elements (binding sites for transcription factors, regulatory factors, co-regulatory factors, etc.) and sequences encoding transcripts that are not translated into proteins. These transcripts are encoded by the genome and transcribed into RNA, but not translated into protein, known as non-coding RNA (ncRNA). Thus, in one embodiment, the RNA is non-coding RNA. Preferably, the non-coding RNA is a single stranded molecule. Studies have shown that ncrnas play a key role in gene regulation, maintenance of genome integrity, cell differentiation and development, and that they are misregulated in various human diseases. There are different types of ncrnas, short (20-50 nt), medium (50-200 nt) and long (> 200 nt) ncrnas. Short ncrnas include microRNA (miRNA), small interfering RNAs (sirnas), piwi-interacting RNAs (pirnas), and transcription initiation RNAs (tirnas). Examples of intermediate ncrnas are small nuclear RNAs (snrnas), small nucleolar RNAs (snornas), transfer RNAs (trnas), transcription initiation site-related RNAs (tssamas), promoter-related small RNAs (PASRs), and promoter upstream transcripts (PROMPT). Long non-coding RNAs (lncRNAs) include long intergenic non-coding RNAs (lincRNAs), antisense lncRNAs, intronic lncRNAs, and transcribed super-conserved RNAs (T-UCR), etc. (Bhan A, mandal SS, chemMedChem., 2014, 3 months 26. Doi: 10.1002/cmdc.201300534). Of the above non-coding RNAs, only siRNA is double-stranded. Thus, since in a preferred embodiment the non-coding RNA is single stranded, it is preferred that the non-coding RNA is not an siRNA. In another embodiment, the RNA is a coding RNA, i.e., an RNA encoding an amino acid sequence. Such RNA molecules are also called mRNA (messenger RNA) and are single stranded RNA molecules. RNA can be prepared by synthetic chemical and enzymatic methods known to those of ordinary skill in the art, or by using recombinant techniques, or can be isolated from natural sources, or by a combination thereof.
Messenger RNAs (mrnas) are copolymers constructed from nucleoside phosphate building blocks with mainly adenosine, cytidine, uridine and guanosine as nucleosides, which act as intermediate vectors, bringing the genetic information of DNA in the nucleus into the cytoplasm where it is translated into a protein. They are therefore suitable as substitutes for gene expression.
In the context of the present invention, mRNA is understood to mean any polyribonucleotide molecule which, if it enters a cell, is suitable for expressing a protein or a fragment thereof or which can be translated into a protein or a fragment thereof. The term "protein" herein includes any kind of amino acid sequence, i.e. a chain of two or more amino acids, each amino acid being linked via a peptide bond, and also peptides and fusion proteins.
MRNA comprises a ribonucleotide sequence that encodes a protein or fragment thereof that is essential or beneficial for its function in or near a cell, e.g., that is deficient or defective in form of a trigger for a disease or illness, that provides a protein that can alleviate or prevent a disease or disorder, or that can promote a process in or near a cell that is beneficial to the body. The mRNA may comprise the sequence of the complete protein or a functional variant thereof. Furthermore, the ribonucleotide sequence may encode a protein acting as a factor, inducer, modulator, stimulator or enzyme, or a functional fragment thereof, wherein the protein is one whose function is essential in order to treat a disease, in particular a metabolic disorder, or in order to initiate an in vivo process, such as the formation of new blood vessels, tissues, etc. Examples of proteins that may be encoded by mRNA include antibodies, cytokines, or chemokines. Functional variants are understood here to mean fragments of a cell which can carry out the function of a protein which is necessary in the cell or whose deficient or defective form is pathogenic. In addition, it is also possible for the mRNA to have other functional regions and/or 3 'or 5' non-coding regions, in particular 3 'and/or 5' UTRs. The 3 'and/or 5' non-coding regions may be naturally flanking regions of the protein coding sequence or of an artificial sequence, for example sequences which aid in RNA stabilization. The person skilled in the art can determine the sequences suitable in each case by routine experimentation.
In a preferred embodiment, the mRNA comprises in particular a 5' -cap consisting of an m7GpppG linked to the mRNA by a 5' to 5' triphosphate bond (five main caps; cap-0), a further methyl group on the penultimate nucleotide at the 5' end of the mRNA (cap-1, anti-reverse cap analogue (ARCA)) and/or an Internal Ribosome Entry Site (IRES) and/or a poly (A) tail at the 3' end in order to improve translation. The mRNA may have other regions that promote translation, such as cap-2 structures or histone stem-loop structures.
RNA that may be present in the nanoparticle may comprise unmodified and modified nucleotides. The term "unmodified nucleotide" as used herein refers to A, C, G and U nucleotides. The term "modified nucleotide" as used herein refers to any naturally occurring or non-naturally occurring isomer of A, C, G and U nucleotides, as well as any naturally occurring or non-naturally occurring analog, substitute or modified nucleotide having, for example, a chemically modified or substituted residue, or an isomer thereof. The modified nucleotide may have a base modification and/or a sugar modification. The modified nucleotides may also have phosphate group modifications, for example, relative to the 5' -main cap of the mRNA molecule. Modified nucleotides also include nucleotides synthesized post-transcriptionally by covalent modification of the nucleotide. Furthermore, any suitable mixture of unmodified and modified nucleotides is possible. Examples of non-limiting numbers of modified nucleotides can be found in the literature (e.g., U.S. 2013/0123481 A1;Cantara et al, nucleic Acids Res,2011,39 (issu supply_1): D195-D201; helm and Alfonzo; chem Biol,2014,21 (2): 174-185; or Carell et al, ANGEW CHEM INT ED ENGL,2012,51 (29): 7110-31), and based on their respective nucleoside residues, some preferred modified nucleotides are exemplified below:
1-methyladenosine, 2-methylsulfanyl-N6-hydroxy-N-valylcarbamoyladenosine, 2-methyladenosine, 2 '-O-ribosyl-phosphate adenosine, N6-methyl-N6-threonyl-carbamoyladenosine, N6-acetyladenosine, N6-glycylcarbamoyladenosine, N6-isopentenyl-adenosine, N6-methyladenosine, N6-threonyl-carbamoyladenosine, N6-dimethyladenosine, N6- (cis-hydroxyisopentenyl) adenosine, N6-hydroxy-N-valylcarbamoyladenosine, 1,2' -O-dimethyladenosine, N6,2 '-O-dimethyladenosine, 2' -O-methyladenosine, N6, N6,2' -O-trimethyladenosine, 2-methylsulfanyl-N6- (cis-hydroxyisopentenyl) adenosine, 2-methylsulfanyl-N6-methyladenosine, 2-methylsulfanyl-N6-isopentenyl adenosine, 2-methylsulfanyl-N6-threonyl carbamoyladenosine, N6-2-methylsulfanyl-N6-threonyl carbamoyladenosine, 2-methylsulfanyl-N6- (cis-hydroxyisopentenyl) adenosine, 7-methyladenosine, 2-methylsulfanyl-adenosine, 2-methoxy-adenosine, 2' -amino-2 ' -deoxyadenosine, 2' -azido-2 ' -deoxyadenosine, 2' -fluoro-2 ' -deoxyadenosine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenosine, 7-deaza-8-aza-adenosine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 2-thiocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 5-hydroxycytidine, lysylcytidine, N4-acetyl-2 '-O-methylcytidine, 5-formyl-2' -O-methylcytidine, 5,2' -O-dimethylcytidine, 2-O-methylcytidine, N4,2' -O-dimethylcytidine, N4,2' -O-trimethylcytidine, isocytidine, pseudocytidine, 2-thio-cytidine, 2' -methyl-2 ' -deoxycytidine, 2' -amino-2 ' -deoxycytidine, 2' -fluoro-2 ' -deoxycytidine, 5-iodocytidine, 5-bromocytidine, 2' -azido-2 ' -deoxycytidine, 2' -amino-2 ' -deoxycytidine, 2' -fluoro-2 ' -deoxycytidine, 5-aza-cytidine, 3-methyl-cytidine, 1-methyl-pseudocytidine, pyrrolo-cytidine, Pyrrolo-pseudoisocytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-1-deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 1-methylguanosine, N2, 7-dimethylguanosine, N2-methylguanosine, 2 '-O-ribosyl-phosphate guanosine, 7-methylguanosine, hydroxy Huai Dinggan (hydroxywybutosine), 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, N2-dimethylguanosine, N2,7,2' -O-trimethylguanosine, N2,2 '-O-dimethylguanosine, 1,2' -O-dimethylguanosine, 2 '-O-methylguanosine, N2,2' -O-trimethylguanosine, N2J-trimethylguanosine, isoguanosine, 4-desmethylguanosine, epoxy quinine (epoxyqueuosine), Modified incomplete hydroxy Huai Dinggan, methylated modified incomplete hydroxy Huai Dinggan, isonicotin, peroxy Huai Dinggan, galactosyl-quinine, mannosyl-quinine, archa-side (archaeosine), huai Dinggan, methyl-bosin, 7-aminopropyl norbosin, 7-aminopropyl bosin, 7-aminopropyl Huai Gan methyl ester, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl inosine, 6-methoxy-guanosine, 1-methyl guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thioguanosine, N2-dimethyl-6-thioguanosine, N1-methyl guanosine, 2 '-amino-3' -deoxyguanosine, 2 '-azido-2' -deoxyguanosine, 2 '-fluoro-2' -deoxyguanosine, 2-thiouridine, 3- (3-amino-3-carboxypropyl) uridine, 3-methyluridine, 4-thiouridine, 5-methyl-2-thiouridine, 5-methylaminomethyluridine, 5-carboxymethyl uridine, 5-carboxymethyl aminomethyluridine, 5-hydroxyuridine, 5-methyluridine, 5-taurinomethyl uridine, 5-carbamoylmethyluridine, 5- (carboxyhydroxymethyl) uridine methyl ester, dihydrouridine, 5-methyldihydrouridine, 5-methyl aminomethyl-2-thiouridine, 5- (carboxyhydroxymethyl) uridine, 5- (carboxyhydroxymethyl) -2' -O-methyluridine methyl ester, 5- (isopentenylaminomethyl) uridine, 5- (isopentenylaminomethyl) -2-thiouridine, 3,2' -O-dimethyluridine, 5-carboxymethyl aminomethyl-2 ' -O-methyluridine, 5-carbamoylhydroxymethyl uridine, 5-carbamoylmethyl-2 ' -O-methyluridine, 5-carbamoylmethyl-2-thiouridine, 5-methoxycarbonylmethyl-2 ' -O-methyluridine, 5- (isopentenylaminomethyl) -2' -O-methyluridine, 5,2' -O-dimethyluridine, 2' -O-methyluridine, 2' -O-methyl-2-thiouridine, 2-thio-2 ' -O-methyluridine, uridine 5-oxyacetic acid, 5-methoxycarbonylmethyluridine, uridine 5-oxyacetic acid methyl ester, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethyl aminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenocysteine, 5-methoxycarbonylmethyl-2-thiouridine, 5-Niu Huangan-methyl-2-thiouridine, pseudouridine, 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine, 1-methyl pseudouridine, 3-methyl pseudouridine, 2 '-O-methyl pseudouridine, 5-formyluridine, 5-aminomethyl-2-geranyluridine, 5-taurolidine, 5-iodouridine, 5-bromouridine, 2' -methyl-2 '-deoxyuridine, 2' -amino-2 '-deoxyuridine, 2' -azido-2 '-deoxyuridine, 2' -fluoro-2 '-deoxyuridine, inosine, 1-methyl inosine, 1,2' -O-dimethylinosine, 2' -O-methyl inosine, 5-aza-uridine, 2-thio-5-aza-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 1-Niu Huangan-methyl-pseudouridine, 5-Niu Huangan methyl-2-thio-uridine, 1-Niu Huangan methyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-1-methyl-l-deaza-pseudouridine, 2-methyl-l-deaza-pseudouridine, Dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-dihydro-pseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 1,2' -O-dimethyl adenosine, 1,2' -O-dimethyl guanosine, 1,2' -O-dimethyl inosine, 2, 8-dimethyl adenosine, 2-methylthio-N6-isopentenyl-adenosine, 2-geranylthiouridine, 2-Lai Bao-glycoside, 2-methylthio-cyclo-N6-threonyl-carbamoyl-adenosine, 2-methylthio-N6- (cis-hydroxyisopentenyl) adenosine, 2-methylsulfanyl-N6-hydroxy-N-valylcarbamoyl adenosine, 2-methylsulfanyl-N6-threonyl carbamoyl adenosine, 2-seleno-uridine, 2-thio-2 '-O-methyluridine, 2' -O-methyladenosine, 2 '-O-methylcytidine, 2' -O-methylguanosine, 2 '-O-methylinosine, 2' -O-methylpseudouridine, 2 '-O-methyluridine 5-oxoacetate methyl ester, 2' -O-ribosyl adenosine phosphate, 2 '-O-ribosyl guanosine phosphate, 3,2' -O-dimethyluridine, 3- (3-amino-3-carboxypropyl) -5, 6-dihydrouridine, 3- (3-amino-3-carboxypropyl) pseudouridine, 5,2' -O-dimethylcytidine, 5,2' -O-dimethyluridine, 5- (carboxyhydroxymethyl) -2' -O-methyluridine methyl ester, 55- (isopentenylaminomethyl) -2' -O-methyluridine, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouride, 5-aminomethyluridine, 5-carbamoylmethyl-2 ' -O-methyluridine, 5-carboxyhydroxymethyl uridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethyl aminomethyl-2-geranylthiouridine, 5-carboxymethyl aminomethyl-2-selenouride, 5-carboxymethyl aminomethyl-2 ' -O-methyluridine, 5-cyanomethyluridine, 5-formyl-2 ' -O-methylcytidine, 5-methoxycarbonylmethyl-2 ' -O-methyluridine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-norxanthosine, 7-methylguanosine, 8-methyladenosine, N2,2' -O-dimethylguanosine, N2,7,2' -O-trimethylguanosine, N2, 7-dimethylguanosine, N2,2' -O-trimethylguanosine, N2, 7-trimethylguanosine, N4,2' -O-dimethylcytidine, N4, N4,2' -O-trimethylcytidine, N4-dimethylcytidine, N4-acetyl-2 ' -O-methylcytidine, N6,2' -O-dimethyladenosine, N6,2' -O-trimethyladenosine, N6-formyladenosine, N6-hydroxymethyladenosine, guanosine, 2-methylthiocyclo-N6-threonyl-carbamoyl adenosine, glutamyl-quinine, guanosine added to any nucleotide, guanylated 5' -end, hydroxy-N6-threonyl-carbamoyl adenosine, most preferably pseudo-uridine, N1-methyl-pseudo-uridine, 2 ́ -fluoro-2 ́ -deoxycytidine, 5-iodocytidine, 5-methylcytidine, 2-thiouridine, 5-iodouridine, and/or 5-methyl-uridine.
Furthermore, the term "modified nucleotide" includes nucleotides containing isotopes such as deuterium. The term "isotope" refers to elements having the same number of protons but different numbers of neutrons, which results in different numbers of masses. Thus, isotopes of hydrogen, for example, are not limited to deuterium, but also include tritium. In addition, the polyribonucleotides may also contain isotopes of other elements, including, for example, carbon, oxygen, nitrogen and phosphorus. The modified nucleotide may also be deuterated or contain hydrogen or another isotope of oxygen, carbon, nitrogen or phosphorus.
In U, C, A and G nucleotides, zero, one, two, three or all of them may be modified. Thus, in some embodiments, at least one nucleotide of a nucleotide type, e.g., at least one U nucleotide, can be a modified nucleotide. In some embodiments, at least one nucleotide of the total two nucleotide types, e.g., at least one U nucleotide and at least one C nucleotide, can be a modified nucleotide. In some embodiments, at least one nucleotide of the total three nucleotide types, e.g., at least one G nucleotide, at least one U nucleotide, and at least one C nucleotide, can be a modified nucleotide. In some embodiments, at least one nucleotide of all four nucleotide types may be a modified nucleotide. In all of these embodiments, one or more nucleotides of each nucleotide type may be modified, the percentage of modified nucleotides of each nucleotide type being 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%.
In some embodiments, the total percentage of modified nucleotides included in an mRNA molecule is 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%.
In a preferred embodiment, the mRNA is an mRNA comprising a combination of modified and unmodified nucleotides. Preferably, it is an mRNA comprising a combination of modified and unmodified nucleotides as described in WO 2011/012316. The mRNA described therein is reported to exhibit increased stability and reduced immunogenicity. In a preferred embodiment, 5 to 50% of cytidine nucleotides and 5 to 50% of uridine nucleotides are modified in such modified mRNA. In another preferred embodiment, 5 to 50% of the uridine nucleotides are replaced by N1-methyl-pseudo-uridine. The nucleotides comprising adenosine and guanosine may not be modified. Adenosine and guanosine nucleotides may be unmodified or partially modified, and they are preferably present in unmodified form.
In certain embodiments of any of the above, the percentage of analog of a given nucleotide refers to the percent input (e.g., the percentage of analog in the initial reaction, such as the initial in vitro transcription reaction). In certain embodiments of any of the above, the percentage of a given nucleotide analog refers to the output (e.g., the percentage in a synthetic or transcribed compound). Both options are equally contemplated. RNA (preferably mRNA) molecules can be recombinantly produced in an in vivo system by methods known to those skilled in the art.
Alternatively, the modified RNA (preferably mRNA) molecules may be produced in an in vitro system using, for example, an in vitro transcription system known to those skilled in the art. In vitro transcription systems capable of producing RNA (preferably mRNA) require an input mixture of modified and unmodified nucleoside triphosphates to produce modified RNA. In certain embodiments, 5 to 50% of cytidine in the input mixture is an analog of cytidine, and 5 to 50% of uridine in the input mixture is an analog of uridine. In certain embodiments, 5 to 40% of cytidine in the input mixture is an analog of cytidine, and 5 to 40% of uridine in the input mixture is an analog of uridine. In certain embodiments, 5% to 30% of cytidine in the mixture is an analog of cytidine, and 5% to 30% of uridine in the input mixture is an analog of uridine. In certain embodiments, 5 to 30% of cytidine in such mixtures is an analog of cytidine, and 10 to 30% of uridine in such mixtures is an analog of uridine. In certain embodiments, 5 to 20% of cytidine in the input mixture is an analog of cytidine, and 5 to 20% of uridine in the input mixture is an analog of uridine. In certain embodiments, 5 to 10% of cytidine in the input mixture is an analog of cytidine, and 5 to 10% of uridine in the input mixture is an analog of uridine. In certain embodiments, 25% of cytidine in the input mixture is an analog of cytidine, and 25% of uridine in the input mixture is an analog of uridine. In certain embodiments, the input mixture does not include analogs of adenosine and/or guanosine. In other embodiments, optionally, the input mixture includes one or more analogs (or neither) of adenosine and/or guanosine.
In certain embodiments, the percentage of cytidine in the input mixture that is a cytidine analog is different than the percentage of uridine in the input mixture that is a uridine analog. In certain embodiments, the percentage of cytidine analog in the input mixture is lower than the proportion of uridine analog in the input mixture. As described above, this may be with or without the presence of an analog of adenosine and guanosine in the input mixture, but in some embodiments, with the absence of an analog of adenosine and guanosine in the input mixture.
In certain embodiments, the nucleotide input mixture for an in vitro transcription system for producing an RNA (preferably mRNA) of the invention comprises cytidine analogs and uridine analogs, and 5 to 20% of cytidine in the input mixture is a cytidine analog, and 25 to 45% of uridine is a uridine analog. In other words, the input mixture includes modified and unmodified cytidine and modified and unmodified uridine, and 5 to 20% of cytidine in the input mixture includes analogs of cytidine, and 25 to 45% of uridine in the input mixture includes analogs of uridine. In other embodiments, the input mixture comprises 5 to 10% cytidine analog and 30 to 40% uridine analog, e.g., 7-9% (e.g., 7%, 7.5%, or 8%) cytidine analog and, e.g., 32-38% (e.g., 33%, 34%, 35%, 36%) uridine analog.
In certain embodiments, any of the uridine analogs and cytidine analogs described herein can be used, optionally excluding pseudouridine. In certain embodiments, the cytidine analog comprises or consists of 5-iodocytidine (e.g., it is the single C analog type used), and the uridine analog comprises or consists of 5-iodouridine (e.g., it is the single U analog type used).
Exemplary analogs are described above. It will be appreciated that for modified polyribonucleotides encoding a desired polypeptide, unless otherwise specified, analogs and levels of modification are contemplated throughout the polyribonucleotide encoding the desired polypeptide, including the 5 'and 3' untranslated regions (e.g., the level of modification is based on the rate of entry of the analog in an in vitro transcription reaction such that the analog can be incorporated into the transcribed site).
In addition, the modified RNA (preferably mRNA) molecules can be synthesized chemically, for example, by conventional chemical synthesis on an automated nucleotide sequence synthesizer using solid phase supports and standard techniques, or by chemical synthesis of the corresponding DNA sequence and subsequent transcription in vitro or in vivo.
In another preferred embodiment, mRNA can be bound to the target binding site, target sequence and/or micro-RNA binding site, so as to allow the activity of the desired mRNA only in the relevant cells. In another preferred embodiment, the RNA may bind to micro-RNA or shRNA in the untranslated region.
In general, therapeutic effects can be achieved by the interaction of ribonucleic acids with cellular molecules and organelles. For example, such interactions alone can activate the innate immune system, as is the case with certain CpG oligonucleotides and sequences designed to specifically interact with toll-like and other extracellular or intracellular receptors. Furthermore, uptake or introduction of nucleic acid (preferably nucleic acid, more preferably mRNA) in a cell may be intended for causing expression of a nucleotide sequence, such as a gene (preferably nucleic acid, more preferably mRNA) comprised in the nucleic acid, may be intended for down-regulating, silencing or knocking out endogenous gene expression due to the presence of the introduced exogenous nucleic acid in the cell, or may be intended for modifying an endogenous nucleic acid sequence, such as repairing, excision, insertion or exchange of selected bases or whole endogenous nucleic acid sequence fragments, or may be intended for interfering with almost any cellular process due to the presence and interaction of the introduced exogenous ribonucleic acid (preferably mRNA) in the cell. Overexpression of the introduced exogenous nucleic acid (preferably a nucleic acid, more preferably mRNA) may be aimed at compensating or supplementing endogenous gene expression, in particular in cases where the endogenous gene is defective or silent, resulting in a loss, deficiency or defect or dysfunction of the gene expression product, such as is the case for many metabolic and genetic diseases such as cystic fibrosis, hemophilia or muscular dystrophy, and the like. Overexpression of the introduced exogenous nucleic acid (preferably a nucleic acid, more preferably mRNA) may also be intended to allow the expression product to interact with or interfere with any endogenous cellular process, such as gene expression, signal transduction, and modulation of other cellular processes. Overexpression of the introduced exogenous nucleic acid (preferably a nucleic acid, more preferably mRNA) may also be intended to elicit an immune response in the organism environment in which the cell is transfected or transduced or is made to be. An example is the genetic modification of antigen presenting cells (such as dendritic cells) to render them antigen for vaccination purposes. Other examples are the overexpression of cytokines in tumors to elicit tumor-specific immune responses. In addition, overexpression of the introduced exogenous ribonucleic acid (preferably mRNA) may also be intended to produce in vivo or in vitro transient genetically modified cells for cell therapy, such as modified T cells, NK cells and other lymphocytes or precursor cells or stem cells or other cells for regenerative medicine.
Downregulation, silencing or knockdown of endogenous gene expression for therapeutic purposes can be achieved, for example, by RNA interference (RNAi), with ribozymes, antisense oligonucleotides, trnas, long double-stranded RNAs, where such downregulation can be sequence-specific or non-specific, and can also lead to cell death, as is the case when long double-stranded RNAs are introduced into cells. Downregulation, silencing or knockdown of endogenous or pre-existing gene expression can be used to treat acquired, inherited or spontaneously caused diseases, including viral infections and cancers. It is also contemplated that the introduction of nucleic acids into cells may be used as a prophylactic measure to prevent, for example, viral infections or tumors. Downregulation, silencing or knockout of endogenous gene expression can be performed at both the transcriptional and translational levels. A variety of mechanisms are known to those skilled in the art and include, for example, epigenetic modifications, changes in chromatin structure, selective binding of an introduced nucleic acid to a transcription factor, hybridization of an introduced nucleic acid to complementary sequences in genomic DNA, mRNA, or other RNA species by base pairing, including non-conventional base pairing mechanisms such as triple helix formation. Likewise, gene repair, base or sequence changes may be effected at both genomic and mRNA levels, including exon skipping. For example, base or sequence changes may be achieved by RNA-guided site-specific DNA cleavage, by using trans-splicing, trans-splicing ribozymes, chimeric minibodies (chimeraplast), split-mediated cleavage and attachment mechanisms of RNA trans-splicing, or by using group II or retargeting introns, or by targeted genomic insertion using virus-mediated insertional mutagenesis or using prokaryotic, eukaryotic, or viral integrase systems. Since nucleic acids are vectors of the living system construction program and they participate in many cellular processes in a direct and indirect manner, in theory any cellular process may be affected by the introduction of nucleic acids into the cell from the outside. Notably, such introduction can be performed directly in vivo and in ex vivo cell or organ culture, and then the organ or cell so modified is transplanted into a recipient. Particles with nucleic acids as therapeutically active agents, as used in the context of the present invention, may be used for all of the above purposes.
As described above, RNA (preferably mRNA) may comprise a ribonucleotide sequence encoding a protein or fragment thereof that is essential or beneficial for its function in or near a cell, e.g., a protein whose deficient or defective form is a trigger of a disease or illness, a protein that provides relief or prevention of a disease or illness, or a protein that promotes a process beneficial to the body in or near a cell.
In fact, in recent years, RNA (in particular mRNA) has become increasingly important as a pharmaceutical entity. In contrast to DNA-based gene therapy, mRNA does not need to be transported into the nucleus, but is translated directly into proteins in the cytoplasm (J Control Release, 20111060:238-247 and Eur J Pharm Biopharm, 200971:484-489).
Furthermore, many genetic diseases caused by single gene mutations are known and candidates for RNA (preferably mRNA) treatment methods. Diseases caused by single gene mutations, such as cystic fibrosis, hemophilia, and many others, may be dominant or recessive in the likelihood of a feature appearing in the offspring. Although dominant alleles show phenotype in individuals with only one allelic copy, for recessive alleles, individuals must have two copies, one from each parent. In contrast, polygenic diseases are caused by two or more genes, and the respective diseases are generally smooth in appearance and associated with environmental factors. Examples of polygenic diseases are hypertension, elevated cholesterol levels, cancer, neurodegenerative diseases, psychosis, etc. Also in these cases, therapeutic RNAs (preferably mrnas) representing one or more of these genes may be beneficial to these subjects. Furthermore, the genetic disease must not be transmitted from the parent's gene, but may also be caused by new mutations. Also in these cases, a therapeutic RNA (preferably mRNA) representing the correct gene sequence may be beneficial to the subject.
On-line catalogues of human genes and genetic diseases and their respective genes and descriptions of their phenotypes are provided in ONIM (Mendelian genetic on-line) web page (http:// onim. Org), each of which sequences is available from the Uniprot database (http:// www.uniprot.org). As non-limiting examples, table a below lists some congenital diseases and disorders, as well as the corresponding genes. Mutations in certain genes can lead to a variety of pathogenic symptoms due to the high degree of interaction of cell signaling pathways, only one of which is shown in table a. Thus, the pharmaceutical compositions mentioned herein include compositions suitable for the treatment and prevention of diseases selected from the group listed in table a. Likewise, nanoparticle formulations mentioned herein, such as lipid nanoparticle formulations or lipid nanoparticle formulations comprising RNA (preferably mRNA), may be suitable for the treatment or prevention of diseases selected from those listed in table a.
In some embodiments of the invention, the therapeutic protein encoded by RNA (preferably mRNA) is selected from the cellular proteins listed in table a, which may be present in the suspension formulations and aerosols of the invention. Thus, an RNA (preferably mRNA) molecule may encode a therapeutic cellular protein, wherein the encoded therapeutic protein is a protein listed in table a or a homolog thereof.
In another embodiment of the invention, the therapeutic protein encoded by the RNA (preferably mRNA) is selected from the secreted proteins listed in table a. Thus, the RNA (preferably mRNA) may encode a therapeutic fusion protein, wherein the encoded therapeutic protein or homologue thereof is one of the proteins listed in table a, and the second protein is a signal peptide allowing secretion of the therapeutic protein. The signal peptide is a short sequence (typically 5-30 amino acids long) present at the N-terminus of the therapeutic protein and directs the fusion protein to the secretory pathway of the cell via some cellular organelle (i.e., endoplasmic reticulum, golgi apparatus, or endosome). Thus, such fusion proteins are secreted from the cell or organelle, or inserted into the cell compartment or cell membrane at the cell surface (e.g., multi-transmembrane proteins).
Thus, in embodiments of the invention, the RNA (preferably mRNA) may encode one or more of the following proteins that cause, predispose or prevent a disease, but is not limited thereto. Non-limiting examples of such diseases or conditions that may be treated (or prevented) include those wherein the polypeptide, protein or peptide is selected from the group outlined in table a below.
In some embodiments, the coding sequence of the RNA (preferably mRNA) may be transcribed and translated into a partial or full length protein with cellular activity levels equal to or higher than the native protein. In some embodiments, the RNA (preferably mRNA) encodes a therapeutically or pharmaceutically active polypeptide, protein or peptide having a therapeutic or prophylactic effect, wherein the polypeptide, protein or peptide is selected from those outlined in table a below. RNA (preferably mRNA), and more particularly its coding sequence, can be used to express partial or full-length proteins having cellular activity levels equal to or lower than those of the native protein. This may allow for the therapeutic administration of diseases for which the RNA molecule is suitable.
TABLE A non-limiting examples of human genes and genetic diseases or disorders
Table a above shows an example of a gene, wherein a defect results in a disease that can be treated with RNA (preferably mRNA) that can be present in the pharmaceutical composition or nanoparticle formulation mentioned herein, wherein the RNA (preferably mRNA) comprises a ribonucleotide sequence encoding a complete version of a protein of the defective gene disclosed above or a functional fragment thereof. In particularly preferred embodiments, genetic diseases, for example diseases affecting the lung, such as SPB (surfactant protein B) deficiency, ABCA3 deficiency, cystic fibrosis and alpha 1-antitrypsin deficiency, may be addressed; or diseases affecting plasma proteins (e.g., congenital hemochromatosis (hep-deficiency), thrombotic thrombocytopenic purpura (TPP, ADAMTS 13 deficiency) and leading to coagulation defects (e.g., hemophilia a and B) and complement defects (e.g., protein C deficiency), immune defects such as, for example, SCID (caused by mutations of different genes such as RAG1, RAG2, JAK3, IL7R, CD, CD3 delta, CD3 epsilon) or defects due to the absence of adenosine deaminase (ADA-SCID), septic granulomatosis (caused by mutations of gp-91-phox gene, p47-phox gene, p67-phox or p33-phox gene) and storage diseases such as Gaucher's disease, fabry's disease, MPS I, MPS II (glycogen II) or storage polysaccharide storage diseases.
Other conditions for which RNA (preferably mRNA) may be useful include conditions such as SMN 1-related Spinal Muscular Atrophy (SMA) and the like; amyotrophic Lateral Sclerosis (ALS); GALT-associated galactosylemia; cystic Fibrosis (CF), SLC3A 1-related conditions including cystiuria, COL4A 5-related conditions including Ai Bote (Alport) syndrome, galactosidase deficiency, X-linked adrenoleukodystrophy and adrenomyeloneuropathy, friedreich's ataxia, pelizaeus-Merzbacher disease, TSC1 and TSC 2-related nodular sclerosis, sang Feili wave B syndrome (MPS IIIB), CTNS-related cystitis, FMR 1-related conditions including fragile X syndrome, fragile X-related tremor/ataxia syndrome and fragile X premature senility syndrome, prader-Willi syndrome, hereditary hemorrhagic telangiectasia (AT), niman-pick disease type C1, neuronal ceroid lipofuscin-related conditions including adolescent ceroid cercercerulocerulocerulocerulosis (EI) 62, EIB 24-F2-35, and CNF 2-35F 2-related conditions including the development of the first-aspect of the systems of the invention, and the first-35-type (CNF 2) of the respiratory depression-type CsR 1-related conditions including fragile X syndrome, fragile X-related tremor/ataxia syndrome and fragile X-premature ovarian early syndrome, pradequacy syndrome (Pradequacy-Willi's) type C1-associated with hereditary lymphopenia's' ataxia, neuronal cerulocerulopathy, neuronal ceroid cerulopathy, neuronal ceroid related diseases including neuronal ceroid dyse, and dysceroid dyse, and, type E-E, and, E-type, of, MECP 2-related severe neonatal encephalopathy and PPM-X syndrome, CDKL 5-related atypical Rate syndrome, kennedy's disease (SBMA), notch-3-related autosomal dominant cerebral arterial disease with subcortical infarction and leukoencephalopathy (CADASIL), SCN1A and SCN 1B-related epileptic seizure disorders, polymerase G-related diseases including Alpers-Ha Tengluo Hertz syndrome, POLG-related sensory ataxia neuropathy, dysarthria and oculopathy, and autosomal dominant and recessive progressive exooculopathy with mitochondrial DNA deficiency, X-linked adrenal dysplasia, X-linked agaropectinemia, fabry's disease.
In all these diseases, a protein, such as an enzyme, is defective and can be treated with RNA (preferably mRNA) encoding any of the above-mentioned proteins, which makes available the protein encoded by the defective gene or a functional fragment thereof. Transcriptional replacement therapy/protein replacement therapy does not affect potential genetic defects, but increases the concentration of protein that is absent from the subject. As an example, in Pompe's disease, transcript/enzyme replacement therapy replaces the deficient lysosomal enzyme acid alpha-Glucosidase (GAA).
Thus, non-limiting examples of proteins that may be encoded by mRNA are Erythropoietin (EPO), growth hormone (auxin, hGH), cystic fibrosis transmembrane conductance regulator (CFTR), growth factors (such as GM-SCF, G-CSF), MPS, protein C, hepcidin, ABCA3, and surfactant protein B. Other examples of RNA treatable diseases according to the invention include hemophilia A/B, fabry disease, CGD, ADAMTS13, hull's disease, X-chromosome mediated agaropectinemia, adenosine deaminase-associated immunodeficiency and SP-B associated neonatal respiratory distress syndrome. Particularly preferably, the RNA, preferably mRNA, according to the invention comprises a coding sequence for a surface-active protein B (SP-B) or erythropoietin. Other examples of proteins which can be encoded by the RNA (preferably mRNA) of the invention according to the invention are growth factors such as human growth hormone hGH, BMP-2 or angiogenic factors.
Although the above embodiments are described in the context of RNA (preferably mRNA) molecules that may be present in the pharmaceutical compositions or nanoparticle formulations mentioned herein, as mentioned above, are not limited to the use of RNA (preferably mRNA), but other therapeutic agents, e.g. nucleic acid molecules, such as DNA molecules, may be used.
The DNA molecule may encode the RNA, preferably the mRNA, and thus comprises the genetic information of the corresponding transcribed RNA molecule.
Thus, for the preferred embodiments, as described above and below in the context of RNA molecules (preferably mRNA molecules) in the pharmaceutical compositions or nanoparticle formulations mentioned herein, the same applies, mutatis mutandis, to the DNA molecules of the present invention.
Alternatively, the RNA (preferably mRNA) may comprise a ribonucleotide sequence encoding a full length antibody or a smaller antibody (e.g., both heavy and light chains) that can be used in a therapeutic setting, for example, to confer immunity to a subject. Corresponding antibodies and therapeutic application(s) thereof are known in the art. Antibodies may be encoded by a single mRNA strand or multiple mRNA strands.
In another embodiment, the RNA (preferably mRNA) may encode a functional monoclonal or polyclonal antibody, which may be used to target and/or inactivate a biological target (e.g., a stimulatory cytokine such as tumor necrosis factor). Similarly, the RNA (preferably mRNA) sequence may encode a functional anti-nephrotic factor antibody, for example for the treatment of membranous proliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or alternatively may encode an anti-Vascular Endothelial Growth Factor (VEGF) antibody for the treatment of VEGF mediated diseases such as cancer.
In another embodiment, the RNA (preferably mRNA) may encode a functional monoclonal or polyclonal antibody, which may be used to neutralize or otherwise inhibit the virus or viral replication.
Alternatively, the RNA (preferably mRNA) may comprise a ribonucleotide sequence that encodes an antigen that is preferably useful in a prophylactic or therapeutic environment.
In another embodiment, the mRNA can encode one or more proteins that can induce immunomodulation, such as cytokines, including chemokines, interferons (e.g., interferon lambda), interleukins, lymphokines, and tumor necrosis factors.
In another embodiment, the RNA (preferably mRNA) may comprise a ribonucleotide sequence encoding a polypeptide or protein that can be used in genome editing techniques. Genome editing is a genetic engineering in which nucleases are used to insert, delete or replace DNA in the genome of an organism. These nucleases produce a site-specific cleavage at a desired location in the genome. The induced breaks are repaired by non-homologous end joining or homologous recombination, creating targeted mutations in the genome, thereby "editing" the genome. The break may be a single strand break or a Double Strand Break (DSB), with Double Strand Breaks (DSB) being preferred. Many genome editing systems are known in the art that utilize different polypeptides or proteins, i.e., for example, CRISPR-Cas systems, meganucleases, zinc Finger Nucleases (ZFNs), and nucleases based on transcription activator-like effectors (TALENs). Methods of genome engineering are reviewed in Trends in Biotechnology, 2013, 31 (7), 397-405.
Thus, in a preferred embodiment, the RNA (preferably mRNA) may comprise a ribonucleotide sequence encoding a polypeptide or protein of Cas (CRISPR associated protein) protein family, preferably Cas9 (CRISPR associated protein 9). Proteins of the Cas protein family, preferably Cas9, can be used in CRISPR/Cas 9-based methods and/or CRISPR/Cass9 genome editing techniques. CRISPR-Cas systems for genome editing, regulation and targeting are reviewed in nat. biotechnol, 2014, 32 (4): 347-355.
In another preferred embodiment, the RNA (preferably mRNA) may comprise a ribonucleotide sequence encoding a meganuclease. Meganucleases are endo-deoxyribonucleases that recognize large recognition sites (e.g., 12 to 40 base pair double-stranded DNA sequences) compared to "conventional" endo-deoxyribonucleases. Thus, in any given genome, the corresponding locus occurs only a few times, preferably only once. Thus, meganucleases are considered to be the most specific natural restriction endonucleases and are therefore suitable tools in genome editing technology.
In another preferred embodiment, the RNA, preferably mRNA, comprises a ribonucleotide sequence encoding a Zinc Finger Nuclease (ZFN). ZFNs are artificial restriction enzymes produced by fusing zinc finger DNA binding domains to DNA cleavage domains. The zinc finger domain can be engineered to target a particular desired DNA sequence, and this enables the zinc finger nuclease to target unique sequences in a complex genome. By utilizing endogenous DNA repair mechanisms, ZFNs can be used to precisely alter the genome of higher organisms and are therefore suitable tools in genome editing technology.
In another preferred embodiment, the RNA (preferably mRNA) may comprise a ribonucleotide sequence encoding a transcription activator-like effector nuclease (TALEN). TALENs are restriction enzymes that can be engineered to cleave specific DNA sequences. TALENs are fusion proteins in which the TAL effector DNA binding domain is fused to the DNA cleavage domain of a nuclease. Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any desired DNA sequence. Thus, when bound to a nuclease, the DNA can be cleaved at a specific desired location.
Although the above embodiments are described in the context of RNA (preferably mRNA molecules), as described above, the invention is not limited to the use of RNA (preferably mRNA) but may also use any nucleic acid molecule, such as a DNA molecule.
The DNA molecule may encode the RNA, preferably the mRNA, described above, and thus comprises the genetic information of the corresponding transcribed RNA molecule.
Thus, for the preferred embodiment, as described above and below in the context of RNA molecules (preferably mRNA molecules) that may be present in the nanoparticles used in the present invention, the same applies to DNA molecules, mutatis mutandis.
As an alternative to the above, the RNA comprises a ribonucleotide sequence that is not expressed as a protein or polypeptide. Thus, the term RNA should not be construed as merely any polynucleotide molecule which, if introduced into a cell, can be translated into a polypeptide/protein or fragment thereof. Instead, it is also contemplated that the RNA comprises a ribonucleotide sequence that is not translated into a protein. In this context, it is contemplated that the RNA comprises a ribonucleotide sequence, which preferably provides genetic information for an antisense RNA, siRNA or miRNA sequence or another desired non-coding ribonucleotide sequence.
Thus, the RNA may also be an antisense RNA, siRNA or miRNA sequence. Antisense RNA, siRNA, or miRNA sequences can be used to silence the effect of an RNA molecule at a certain stage. This may be desirable and useful in certain medical settings and in the treatment of certain diseases, and in particular in RNA-based therapies as described above and below.
Silencing of RNA molecules can be achieved by using RNAi (RNA interference) mechanisms through the use of nucleic acid strands that are complementary to a certain RNA sequence. The term "RNA interference" or "inhibitory RNA" (RNAi/iRNA) describes the use of double-stranded RNA to target a particular mRNA for degradation, thereby silencing its translation. Preferred inhibitory RNA molecules may be selected from double-stranded RNA (dsRNA), siRNA, shRNA and stRNA. dsRNA matched to the gene sequence can be synthesized in vitro and introduced into cells. dsRNA can also be introduced into cells in sense and antisense orientation in the form of vectors expressing target gene sequences, for example in the form of hairpin mRNA. The sense and antisense sequences can also be expressed from separate vectors, whereby each antisense and sense molecule forms a double stranded RNA upon its expression. It is known in the art that, in some cases, expression of the sense-oriented sequence or even expression of the promoter sequence is sufficient to produce dsRNA, and subsequently siRNA, due to internal amplification mechanisms in the cell. Thus, according to the present invention, all means and methods will be used which result in a decrease in the activity of the polypeptide or protein encoded by the coding region. For example, sense constructs, antisense constructs, hairpin structures, sense and antisense molecules, and combinations thereof, can be used to produce/introduce these siRNAs. dsRNA enters a natural process, including the highly conserved nuclease Dicer, which cleaves dsRNA precursor molecules into short interfering RNAs (sirnas). Methods for the production and preparation of siRNA(s) and for the inhibition of target gene expression are described in WO02/055693,Wei (2000) Dev. Biol. 15:239-255;La Count (2000) Biochem. Paras. 111:67-76;Baker (2000) Curr. Biol. 10:1071-1074;Svoboda (2000) Development 127:4147-4156 or Marie (2000) Curr. Biol. 10:289-292, et al. These siRNAs then construct the sequence-specific portion of the RNA-induced silencing complex (RISC), a multi-complex nuclease that disrupts messenger RNAs homologous to the silencing trigger. Elbashir (2001) EMBO J.20:6877-6888 shows that a duplex of 21 nucleotide RNAs can be used in cell culture to interfere with gene expression in mammalian cells.
Methods of deriving and constructing siRNA are known in the art and are described in Elbashir (2002) Methods 26:199-213 on the Internet sites of siRNA commercial suppliers, such as Qiagen GmbH, dharmacon, xeragon Inc. and Ambion, or on the sites of Tom Tuschl research group. In addition, the program can be used online to infer siRNA from a given mRNA sequence. Uridine residues in the 2-nt 3 'overhang can be replaced with 2' deoxythymidine without inactivation, which significantly reduces the cost of RNA synthesis and may also enhance siRNA duplex resistance when applied to mammalian cells (Elbashir et al nature 411.6836 (2001): 494-498). siRNA can also be enzymatically synthesized using T7 or other RNA polymerase (Donze (2002) Nucleic Acids Res 30:e46). Short RNA duplex (esiRNA) mediating efficient RNA interference can also be produced by hydrolysis with E.coli RNase III (Yang (2002) PNAS 99:9942-9947). In addition, expression vectors have been developed to express double stranded siRNA joined by small hairpin RNA loops in eukaryotic cells (e.g., brummelkamp (2002) Science 296: 550-553.) all of these constructs can be developed with the aid of the above-described procedure in addition, commercially available sequence prediction tools, e.g., the siRNA design tools provided by www.oligoEngine.com (Seattle, washington), integrated into the sequence analysis procedure or sold separately, can be used for siRNA sequence prediction.
MicroRNA (miRNA) are similar to the small interfering RNAs (siRNAs) described above. microRNA (miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses that plays a role in RNA silencing and post-transcriptional regulation of gene expression. mirnas function via base pairing with complementary sequences within the mRNA molecule. Thus, these mRNA molecules are silenced by one or more of (1) cleavage of the mRNA strand into two parts, (2) destabilization of the mRNA by shortening its poly (A) tail, and (3) less efficient translation of the mRNA into protein by ribosomes. As described above, mirnas are similar to small interfering RNAs (sirnas) of the RNA interference (RNAi) pathway, except that mirnas are derived from RNA transcript regions that reverse fold themselves to form short hairpins, whereas sirnas are derived from longer double stranded RNA regions.
The DNA molecules that may be present in the pharmaceutical compositions or nanoparticle formulations mentioned herein may also be DNA molecules encoding the above-mentioned RNAs, e.g. the above-mentioned sirnas or mirnas, and thus contain the genetic information of the corresponding transcribed RNA molecules. Thus, for the preferred embodiment, as described above in the context of RNA molecules (preferably mRNA molecules) that may be present in the nanoparticles used in the present invention, the same applies to DNA molecules, mutatis mutandis.
It will be appreciated that a pharmaceutical composition, in particular a nanoparticle in the context of the present invention, may comprise a single type of nucleic acid (preferably RNA such as mRNA), but may alternatively comprise a combination of two or more types of nucleic acid (preferably RNA), for example in the form of particles comprising two or more types of nucleic acid (preferably RNA) in a single particle, or in the form of a mixture of particles comprising different types of nucleic acid (preferably RNA such as mRNA) therein.
Nanoparticles, lipids and lipids
In various aspects of the invention, a lipid nanoparticle formulation or lipid nanoparticle formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP) is provided. For ease of discussion, lipid nanoparticles and lipid nanoparticles may be collectively referred to herein as nanoparticles. Those skilled in the art will appreciate that nanoparticles comprising at least one lipid, such as an ionizable lipid, are referred to as lipid nanoparticles (LiNP), despite the fact that lipid nanoparticles may additionally comprise one or more lipids. Likewise, the lipid particles referred to herein may comprise a lipid mixture comprising at least one lipid, such as an ionizable lipid, and one or more other components of the lipid mixture may be lipid(s). Furthermore, references to lipid nanoparticle formulations or lipid nanoparticle (LiNP) formulations comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP) are intended to include formulations comprising only lipid or lipid nanoparticles and combinations thereof as nanoparticles unless indicated to the contrary in the specific context.
For example, the lipid nanoparticle or lipid particle, or the lipid mixture or preferred component of the lipid mixture may be a structural lipid. The term "structural lipid (structural lipid)" refers herein to a lipid component that provides structural integrity and shape to the nanoparticle. These lipids may play a fundamental role in forming the primary lipid bilayer or multilamellar structure of LNP or LiNP and may provide a scaffold for particles.
LNP or LiNP are typically included in a lipid or lipid mixture along with at least one selected from the group consisting of ionizable lipids, and permanently cationic lipids, including combinations of different lipid components, each lipid component having a specific effect:
i) Phospholipids these are commonly used as structural lipids, as they are capable of forming bilayers, mimicking the natural cell membrane,
Ii) cholesterol, which helps to regulate the rigidity and permeability of the lipid bilayer,
Iii) Pegylated lipids or poly-sarcosine based lipids which can be added to increase their circulation time in the blood by making the LNP or LiNP more concealed and more difficult for the immune system to recognize.
Among these, phospholipids and cholesterol are generally considered to be the major "structural lipids" because they contribute to the stability of the LNP or LiNP structure. They can be used to maintain the integrity, size and overall morphology of the LNP or LiNP.
LNP and LiNP mentioned in the context of the present invention preferably comprise at least one selected from the group consisting of ionizable lipids, ionizable lipids and permanently cationic lipids. These may be included to facilitate encapsulation of negatively charged molecules (e.g., nucleic acids, such as RNA, preferably mRNA).
The lipid or lipid nanoparticle described herein generally comprises a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, and a permanently cationic lipid as a component thereof. It is to be understood that this includes the possibility that the nanoparticle comprises a combination of different permanent cationic lipids, a combination of different ionizable lipids, or a combination of one or more permanent cationic lipids, one or more ionizable lipids and/or one or more ionizable lipids. Preferred as components are ionizable lipids and ionizable lipids, i.e. it is preferred that the nanoparticle comprises at least one selected from the group consisting of ionizable lipids and ionizable lipids. If the lipid nanoparticle or lipid nanoparticle comprises a lipid mixture or lipid mixture and a therapeutic agent, the lipid mixture or lipid mixture and therapeutic agent are typically included as a mixture in the nanoparticle.
The term "permanently cationic lipid (" PERMANENTLY CATIONIC LIPID) "is used in the lipid nanoparticle art to refer to lipids or lipids containing a permanently cationic charge, for example in the form of a quaternary nitrogen atom.
The terms "ionizable lipid (ionizable lipid)" and "ionizable lipid (ionizable lipidoid)" are used in the lipid nanoparticle and lipid nanoparticle arts to refer to a lipid or lipid that is or may be protonated to carry a cationic charge. Thus, ionizable lipids and lipids are also referred to as "protonatable lipids" and "protonatable lipids", respectively, "ionizable cationic lipids" and "ionizable cationic lipids" or "titratable lipids", respectively. Those skilled in the art will appreciate that reference to an "ionizable lipid" or "ionizable lipid" includes ionizable lipids or lipids in their protonated or non-protonated form. It is further understood that the protonation state or non-protonation state of the lipid or lipid is typically determined by the pH of the medium surrounding the lipid or lipid, for example by the pH of the aqueous carrier solution in which the nanoparticles are suspended. Thus, the terms "ionizable lipid" and "ionizable lipid" also include lipids or lipids that are positively charged at neutral pH.
In the context of the present invention, positively charged permanent cationic lipids, ionizable lipids or positively charged counter ions (anions) of ionizable lipids are typically provided by the anionic moiety contained in the nucleic acid. If the positively charged groups are present in excess compared to the anionic moiety in the nucleic acid, the positive charge may be balanced by other pharmaceutically acceptable anions, such as chloride, bromide or iodide, sulfate, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, carbonate or hydrogen carbonate, or by a polyanionic component other than the nucleic acid, which component may be present as an optional component in the nanoparticle.
Permanent cationic lipids, ionizable lipids, and ionizable lipids are well known lipid nanoparticles or components of lipid nanoparticles. In the context of the present invention, there is no particular limitation on the type of permanent cationic lipid, ionizable lipid or ionizable lipid contained in the nanoparticle.
Typically, the ionizable lipid or lipids include primary, secondary, or tertiary amino groups, respectively, that can act as proton acceptors, and thus can be protonated or non-protonated. The ionizable lipid generally comprises a plurality of such amino groups, such as two or more, preferably three or more.
Preferably, the ionizable lipids that the nanoparticle may comprise are lipids that comprise a protonatable head group comprising one or more (preferably one) primary, secondary, or tertiary amino groups as the protonatable group or protonated groups, and one or more (preferably one or two) hydrophobic moieties attached to the head group.
Examples of these preferred ionizable lipids are:
i) A lipid comprising a protonatable head group and a hydrophobic moiety attached to the head group, the head group comprising one or more (preferably one) primary, secondary or tertiary amino groups as the protonatable group or protonated group;
ii) a lipid comprising one secondary or tertiary amino group as a protonatable or protonatable headgroup, and two hydrophobic moieties attached to the headgroup.
The hydrophobic moiety comprised in these preferred lipids preferably comprises one or more of a linear aliphatic residue, e.g. a linear residue comprising 8 to 18 carbon atoms, a branched aliphatic residue, e.g. a branched residue comprising 8 to 18 carbon atoms, or an alicyclic structure, which may be a fused ring structure, e.g. an alicyclic structure comprising 10 to 18 carbon atoms. Furthermore, the hydrophobic moiety may comprise one or more linking groups which facilitate the attachment of the moiety to the head group or which allow two or more of the above aliphatic residues to bind to each other. Furthermore, the moiety may include one or more substituents provided that its hydrophobic character is maintained.
Preferably, the ionizable lipid that may be included in the nanoparticle is an oligomeric amine, more preferably an oligomeric alkylamine, comprising at least two (preferably at least three) amino groups selected from protonatable or protonated secondary and tertiary amino groups, each of which may have a hydrophobic moiety attached thereto. In addition to amino groups bearing hydrophobic residues, lipids may also include other protonatable or protonated amino groups selected from primary, secondary, and tertiary amino groups. Preferably, the total number of amino groups is 2 to 10, more preferably 3 to 6. Preferably, the total number of hydrophobic moieties attached to the amino groups is from 2 to 6, more preferably from 3 to 6. Preferably, the ratio of the number of hydrophobic moieties attached to the amino groups to the total number of amino groups in the oligoalkylamine is from 0.5 to 2, more preferably from 0.75 to 1.5.
The hydrophobic moiety included in such preferred lipids preferably comprises one or more of linear aliphatic residues, such as linear residues comprising 8 to 18 carbon atoms, and branched aliphatic residues, such as branched residues comprising 8 to 18 carbon atoms. Furthermore, the hydrophobic moiety may comprise one or more linking groups which facilitate the linking of the moiety to the amino group or which allow two or more of the above aliphatic residues to bind to each other. In addition, the moiety may include one or more substituents provided that the hydrophobic character of the moiety is maintained.
For example, suitable lipid fractions of ionized lipids as a group (B) in the Nano-class of the use in the context of the invention or as an ionizable lipid profile are disclosed in WO2006/138380A2、EP2476756A1、US2016/0114042A1、US8,058,069B2、US8,492,359B2、US8,822,668B2、US8,969,535、US9,006,417B2、US9,018,187B2、US9,345,780B2、US9,352,042B2、US9,364,435B2、US9,394,234B2、US9,492,386B2、US9,504,651B2、US9,518,272B2、DE19834683A1、WO2010/053572A2、US9,227,917B2、US9,556,110B2、US8,969,353B2、US10,189,802B2、WO2012/000104A1、WO2010/053572、WO2014/028487、WO2015/095351、US2013/0156849A1(, e.g. claims 13, 33, 34), US9254311B2 (e.g. clause 14), US10501512B2 (e.g. claims 1,6, 9), US2014/0010861A1 (e.g. claims 44 and 78-82), US 2013/0115272 A1 (e.g. clause 12) or Akinc, a. Et al, nature Biotechnology,26 (5), 2008,561-569; sabnis, s. Et al, molecular Therapy, 26 (6), 2018, volume 26, stage 6, 2018, month 6, 1509-1519; kowalski, p.s. Et al, molecular Therapy, 27 (4), 2019, 710-728; kulkarni, j. A. Et al, nucleic Acid Therapeutics, 28 (3), 2018, 146-157; and Li, B. Et al, nano Letters, 15, 2015, 8099-8107.
Preferably, the nanoparticle may comprise a permanently cationic lipid which is a lipid comprising a head group comprising one quaternary nitrogen atom and one or more (preferably one or two) hydrophobic moieties attached to the head group. Preferably, the quaternary nitrogen atom is provided by a group of formula-N (Me) 3 +, wherein Me is methyl.
The hydrophobic moiety comprised in these preferred lipids preferably comprises one or more of a linear aliphatic residue (e.g. a linear residue comprising 8 to 18 carbon atoms) or a branched aliphatic residue (e.g. a branched residue comprising 8 to 18 carbon atoms). Furthermore, the hydrophobic moiety may include one or more linking groups that facilitate the attachment of the moiety to the head group or allow two or more of the above aliphatic residues to bind to each other. In addition, the moiety may include one or more substituents provided that the hydrophobic character of the moiety is maintained. As examples of permanent cationic lipids, reference may be made to DOTMA (dioleoyl-3-trimethylammoniopropane) and DOTAP (dioleoyl-3-trimethylammoniopropane).
In one exemplary embodiment, the nanoparticle may comprise an ionizable lipid or lipid of formula a-I or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof as disclosed in WO2016176330A1, WO2016176330A1 is incorporated herein in its entirety:
a-I
wherein:
One of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, and the other of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、-NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, or a direct bond;
R a is H or C 1-C12 alkyl;
R 1a and R 1b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 1a is H or C 1-C12 alkyl, and R 1b together with the carbon atom to which it is bound form a carbon-carbon double bond with the adjacent R 1b and the carbon atom to which it is bound;
r 2a and R 2b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 2a is H or C 1-C12 alkyl, and R 2b together with the carbon atom to which it is bound form a carbon-carbon double bond with the adjacent R 2b and the carbon atom to which it is bound;
R 3a and R 3b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 3a is H or C 1-C12 alkyl, and R 3b together with the carbon atom to which it is bound form a carbon-carbon double bond with the adjacent R 3b and the carbon atom to which it is bound;
R 4a and R 4b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 4a is H or C 1-C12 alkyl, and R 4b together with the carbon atom to which it is bound form a carbon-carbon double bond with the adjacent R 4b and the carbon atom to which it is bound;
R 5 and R 6 are each independently methyl or cycloalkyl;
r 7 is independently at each occurrence H or C 1-C12 alkyl;
r 8 and R 9 are each independently unsubstituted C 1-C12 alkyl, or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5, 6 or 7 membered heterocyclic ring comprising one nitrogen atom, a and d are each independently integers from 0 to 24;
b and c are each independently integers from 1 to 24;
e is 1 or 2, and
X is 0, 1 or 2.
In some embodiments, the ionizable lipid has a structure of formula a-II or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof:
a-II
wherein:
One of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, and the other of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, or a direct bond;
G 1 is C 1-C2 alkylene, - (c=o) -, -O (c=o) -, -SC (=o) -, -NR a C (=o) -or a direct bond;
G 2 is-C (=o) -, - (c=o) O-, -C (=o) S-, -C (=o) NR a -, or a direct bond;
g 3 is C 1-C6 alkylene;
R a is H or C 1-C12 alkyl;
R 1a and R 1b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 1a is H or C 1-C12 alkyl, and R 1b together with the carbon atom to which it is attached form a carbon-carbon double bond with the adjacent R 1b and the carbon atom to which it is attached;
R 2a and R 2b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 2a is H or C 1-C12 alkyl, and R 2b together with the carbon atom to which it is attached form a carbon-carbon double bond with the adjacent R 2b and the carbon atom to which it is attached;
R 3a and R 3b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 3a is H or C 1-C12 alkyl, and R 3b together with the carbon atom to which it is bound form a carbon-carbon double bond with the adjacent R 3b and the carbon atom to which it is bound;
r 4a and R 4b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 4a is H or C 1-C12 alkyl, and R 4b together with the carbon atom to which it is attached form a carbon-carbon double bond with the adjacent R 4b and the carbon atom to which it is attached;
r 5 and R 6 are each independently H or methyl;
R 7 is C 4-C20 alkyl;
R 8 and R 9 are each independently C 1-C12 alkyl, or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-or 7-membered heterocyclic ring;
a. b, c and d are each independently integers from 1 to 24, and
X is 0, 1 or 2.
In some embodiments, the ionizable lipid has a structure of formula a-III or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof:
a-III
wherein:
One of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, and the other of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, or a direct bond;
each of G 1 and G 2 is independently unsubstituted C 1-C12 alkylene or C 1-C12 alkenylene;
G 3 is C 1-C24 alkylene, C 1-C24 alkenylene, C 3-C8 cycloalkylene, C 3-C8 cycloalkenyl;
R a is H or C 1-C12 alkyl;
R 1 and R 2 are each independently C 6-C24 alkyl or C 6-C24 alkenyl;
R 3 is H, OR 5、CN、-C(=O)OR4、-OC(=O)R4 or-NR 5C(=O)R4;R4 is C 1-C12 alkyl;
r 5 is H or C 1-C6 alkyl, and
X is 0, 1 or 2.
In some embodiments, the ionizable lipid has the following formulas a-IV or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof:
(a-IV)
wherein:
One of G 1 or G 2 is at each occurrence –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)y-、-S-S-、-C(=O)S-、SC(=O)-、-N(Ra)C(=O)-、-C(=O)N(Ra)-、-N(Ra)C(=O)N(Ra)-、-OC(=O)N(Ra)- or-N (R a) C (=o) O-, and the other of G 1 or G 2 is at each occurrence -O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)y-、-S-S-、-C(=O)S-、-SC(=O)-、-N(Ra)C(=O)-、-C(=O)N(Ra)-、-N(Ra)C(=O)N(Ra)-、-O(C=O) N(Ra)- or-NR a C (=o) O-, or a direct bond;
l is at each occurrence-O (C=O) -, where-represents a covalent bond with X;
X is CR a;
Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1, or is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
R a is independently at each occurrence H, C 1-C12 alkyl, C 1-C12 hydroxyalkyl, C 1-C12 aminoalkyl, C 1-C12 alkylaminoalkyl, C 1-C12 alkoxyalkyl, C 1-C12 alkoxycarbonyl, C 1C12 alkylcarbonyloxy, C 1-C12 alkylcarbonyloxy alkyl or C 1-C12 alkylcarbonyl;
R is independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R and the carbon atom to which it is attached;
Each occurrence of R 1 and R 2 has the following structure:
And
R 1、R2、a1 and a 2 are independently at each occurrence an integer from 3 to 12, b 1 and b 2 are independently at each occurrence 0 or 1;
c 1 and c 2 are independently at each occurrence an integer from 5 to 10, d 1 and d 2 are independently at each occurrence an integer from 5 to 10, y is independently at each occurrence an integer from 0 to 2, and n is an integer from 1 to 6,
Wherein each alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy alkyl, and alkylcarbonyl is optionally substituted with one or more substituents.
In some embodiments, the ionizable lipid has the following formula (a-V) or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof:
(a-V)
wherein:
One of G 1 or G 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)y-、-S-S-、-C(=O)S-、SC(=O)-、-N(Ra)C(=O)-、-C(=O)N(Ra)-、-N(Ra)C(=O)N(Ra)-、-OC(=O)N(Ra)- or-N (R a) C (=o) O-at each occurrence, and the other of G 1 or G 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)y-、-S-S-、-C(=O)S-、-SC(=O)-、-N(Ra)C(=O)-、-C(=O)N(Ra)-、-N(Ra)C(=O)N(Ra)-、-OC(=O)N(Ra)- or-N (R a) C (=o) O-or a direct bond at each occurrence;
L is at each occurrence-O (C=O) -, where-represents a covalent bond with X;
X is CR a;
Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1, or is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
R a is independently at each occurrence H, C 1-C12 alkyl, C 1-C12 hydroxyalkyl, C 1C12 aminoalkyl, C 1-C12 alkylaminoalkyl, C 1-C12 alkoxyalkyl, C 1-C12 alkoxycarbonyl, C 1C12 alkylcarbonyloxy, C 1-C12 alkylcarbonyloxy alkyl or C 1-C12 alkylcarbonyl;
R is independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R and the carbon atom to which it is attached;
Each occurrence of R 1 and R 2 has the following structure:
R' is independently at each occurrence H or C 1-C12 alkyl, a 1 and a 2 are independently at each occurrence integers from 3 to 12, b 1 and b 2 are independently at each occurrence 0 or 1;
c 1 and c 2 are independently at each occurrence an integer from 2 to 12, d 1 and d 2 are independently at each occurrence an integer from 2 to 12, y is independently at each occurrence an integer from 0 to 2, and n is an integer from 1 to 6,
Wherein a 1、a2、c1、c2、d1 and d 2 are selected such that the sum of a 1+c1+d1 is an integer from 18 to 30 and the sum of a 2+c2+d2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy alkyl, and alkylcarbonyl is optionally substituted with one or more substituents.
In some embodiments, the ionizable lipid is selected from the group consisting of the lipids in table 1, table 2, table 3, or table 4.
In some embodiments, the ionizable lipid has one of the following structures:
。
In another exemplary embodiment, the ionizable lipid has the following structure or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof:
,
wherein:
Each occurrence of R 1 and R 2 is independently an optionally substituted C 10-C30 alkyl, an optionally substituted C 10-C30 alkenyl, an optionally substituted C 10-C30 alkynyl or an optionally substituted C 10-C30 acyl;
R 3 is H, optionally substituted C 10-C10 alkyl, optionally substituted C 2-C10 alkenyl, optionally substituted C 2-C10 alkynyl, alkyl heterocycle, alkyl phosphate, alkyl thiophosphate, alkyl dithiophosphate, alkyl phosphonate, alkylamine, hydroxyalkyl, omega-aminoalkyl, omega (substituted) aminoalkyl, omega-alkyl phosphate, omega-alkyl thiophosphate, optionally substituted polyethylene glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl or heterocycle, or linker-ligand, and
E is O、S、N(Q)、C(O)、N(Q)C(O)、C(O)N(Q)、(Q)N(CO)O、O(CO)N(Q)、S(O)、NS(O)2N(Q)、S(O)2、N(Q)S(O)2、SS、O═N、 aryl, heteroaryl, ring or heterocycle, and
Q is H, alkyl, omega-aminoalkyl, omega- (substituted) aminoalkyl, omega-alkyl phosphate or omega alkyl thiophosphate.
In some embodiments, the ionizable lipid has one of the following structures:
。
In a preferred embodiment, the lipid nanoparticle referred to herein comprises the ionizable lipid [ (4-hydroxybutyl) azadiyl ] bis (hexane-6, 1-diyl) bis (2-hexyl decanoate), also known as ALC-0315, and is represented by the formula:
。
In some embodiments, the lipid nanoparticle may further comprise a neutral lipid and/or a polymer conjugated lipid, preferably both a neutral lipid and a polymer conjugated lipid. The molar ratio of the ionizable lipid to neutral lipid described above is preferably in the range of about 4.1:1.0 to about 4.9:1.0, 4.5:1.0 to about 4.8:1.0, or 4.7:1.0 to about 4.8:1.0. In some embodiments, the molar ratio of ionizable lipid to neutral lipid ranges from about 2:1 to about 8:1, preferably from 5:1 to 1:1.
The molar ratio of ionizable lipid to polymer conjugated lipid ranges from about 35:1 to about 25:1, or from 100:1 to about 20:1.
In another exemplary embodiment, the ionizable lipid has the following structure:
. 。
according to another exemplary embodiment, the lipid nanoparticle mentioned herein comprises:
i) A first cationic lipid as ionizable lipid (a) having a first effective pKa;
ii) a second cationic lipid as ionizable lipid (a) having a second effective pKa that is greater than the first effective pKa;
iii) Neutral lipids;
iv) a steroid;
v) polymer conjugated lipids;
vi) a therapeutic agent or pharmaceutically acceptable salt or prodrug thereof encapsulated within or associated with the lipid nanoparticle, and
Vii) a surfactant, which is a surfactant,
Wherein the effective pKa of the lipid nanoparticle is between the first effective pKa and the second effective pKa.
In some embodiments, the first effective pKa is less than 5.75. In some embodiments, the second effective pKa is greater than 6.25. In some embodiments, the lipid nanoparticle of any one of claims 48-50, wherein the effective pKa of the lipid nanoparticle is in the range of 5.90 to 6.35. In some embodiments, the molar ratio of the first cationic lipid to the second cationic lipid is in the range of 1:20 to 1:2.
In some embodiments, LNP or LiNP of the present invention comprises a first cationic lipid that is an ionizable lipid (a), or a second cationic lipid that is an ionizable lipid (a), and one or both have the structure of formula a-I or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof:
a-I
wherein:
One of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, and the other of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, or a direct bond;
R a is H or C 1-C12 alkyl;
R 1a and R 1b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 1a is H or C 1-C12 alkane, and R 1b together with the carbon atom to which it is bound form a carbon-carbon double bond with the adjacent R 1b and the carbon atom to which it is bound;
r 2a and R 2b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 2a is H or C 1-C12 alkyl, and R 2b together with the carbon atom to which it is bound form a carbon-carbon double bond with the adjacent R 2b and the carbon atom to which it is bound;
R 3a and R 3b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 3a is H or C 1-C12 alkyl, and R 3b together with the carbon atom to which it is bound forms a carbon-carbon double bond with the adjacent R 3b and the carbon atom to which it is bound, R 4a and R 4b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 4a is H or C 1-C12 alkyl, and R 4b together with the carbon atom to which it is bound forms a carbon-carbon double bond with the adjacent R 4b and the carbon atom to which it is bound;
R 5 and R 6 are each independently methyl or cycloalkyl;
r 7 is independently at each occurrence H or C 1-C12 alkyl;
R 8 and R 9 are each independently unsubstituted C 1-C12 alkyl, or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-or 7-membered heterocyclic ring comprising one nitrogen atom;
a and d are each independently integers from 0 to 24;
b and c are each independently integers from 1 to 24;
e is 1 or 2, and
X is 0, 1 or 2.
In some embodiments, the first and second cationic lipids as ionizable lipid (a) are each independently selected from lipids of formulas a-I. In some embodiments, the first cationic lipid or the second cationic lipid or both have the structure of formula a-II or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof:
a-II
wherein:
one of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, and the other of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, or a direct bond;
G 1 is C 1-C2 alkylene, - (c=o) -, -O (c=o) -, -SC (=o) -, -NR a C (=o) -or a direct bond;
G 2 is-C (=o) -, - (c=o) O-, -C (=o) S-, -C (=o) NR a -, or a direct bond; G 3 is C 1-C6 alkylene;
R a is H or C 1-C12 alkyl;
R 1a and R 1b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 1a is H or C 1-C12 alkyl, and R 1b together with the carbon atom to which it is attached form a carbon-carbon double bond with the adjacent R 1b and the carbon atom to which it is attached;
R 2a and R 2b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 2a is H or C 1-C12 alkyl, and R 2b together with the carbon atom to which it is attached form a carbon-carbon double bond with the adjacent R 2b and the carbon atom to which it is attached;
R 3a and R 3b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 3a is H or C 1-C12 alkyl, and R 3b together with the carbon atom to which it is bound form a carbon-carbon double bond with the adjacent R 3b and the carbon atom to which it is bound;
r 4a and R 4b are independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R 4a is H or C 1-C12 alkyl, and R 4b together with the carbon atom to which it is attached form a carbon-carbon double bond with the adjacent R 4b and the carbon atom to which it is attached;
r 5 and R 6 are each independently H or methyl;
R 7 is C 4-C20 alkyl;
R 8 and R 9 are each independently C 1-C12 alkyl, or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-or 7-membered heterocyclic ring;
a. b, c and d are each independently integers from 1 to 24, and
X is 0, 1 or 2.
In some embodiments, the first and second cationic lipids as ionizable lipid (a) are each independently selected from lipids of formulas a-II.
In some embodiments, the first cationic lipid that is the ionizable lipid (a), or the second cationic lipid that is the ionizable lipid (a), or both, has the structure of formulas a-III or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof:
a-III
wherein:
One of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, and the other of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, or a direct bond;
each of G 1 and G 2 is independently unsubstituted C 1-C12 alkylene or C 1-C12 alkenylene;
G 3 is C 1-C24 alkylene, C 1-C24 alkenylene, C 3-C8 cycloalkylene, C 3-C8 cycloalkenyl;
R a is H or C 1-C12 alkyl;
R 1 and R 2 are each independently C 6-C24 alkyl or C 6-C24 alkenyl;
R 3 is H, OR 5、CN、-C(=O)OR4、-OC(=O)R4 or-NR 5C(=O)R4;R4 is C 1-C12 alkyl;
r 5 is H or C 1-C6 alkyl, and
X is 0, 1 or 2.
In some embodiments, the first and second cationic lipids as ionizable lipid (a) are each independently selected from lipids of formulas a-III.
In some embodiments, the first cationic lipid that is the ionizable lipid (a), or the second cationic lipid that is the ionizable lipid (a), or both, has the structure of formulas a-IV or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof:
(a-IV)
wherein:
One of G 1 or G 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)y-、-S-S-、-C(=O)S-、SC(=O)-、-N(Ra)C(=O)-、-C(=O)N(Ra)-、-N(Ra)C(=O)N(Ra)-、-OC(=O)N(Ra)- or-N (R a) C (=o) O-at each occurrence and the other of G 1 or G 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)y-、-S-S-、-C(=O)S-、-SC(=O)-、-N(Ra)C(=O)-、-C(=O)N(Ra)-、-N(Ra)C(=O)N(Ra)-、-OC(=O)N(Ra)- or-N (R a) C (=o) O-or a direct bond at each occurrence;
X is CR a;
Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1, or is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
R a is independently at each occurrence H, C 1-C12 alkyl, C 1-C12 hydroxyalkyl, C 1C12 aminoalkyl, C 1-C12 alkylaminoalkyl, C 1-C12 alkoxyalkyl, C 1-C12 alkoxycarbonyl, C 1C12 alkylcarbonyloxy, C 1-C12 alkylcarbonyloxy alkyl or C 1-C12 alkylcarbonyl;
R is independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R and the carbon atom to which it is attached;
Each occurrence of R 1 and R 2 has the following structure:
a 1 and a 2 are independently at each occurrence integers from 3 to 12, b 1 and b 2 are independently at each occurrence 0 or 1;
c 1 and c 2 are independently at each occurrence an integer from 5 to 10, d 1 and d 2 are independently at each occurrence an integer from 5 to 10, y is independently at each occurrence an integer from 0 to 2, and n is an integer from 1 to 6,
Wherein each alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy alkyl, and alkylcarbonyl is optionally substituted with one or more substituents.
In some embodiments, the first and second cationic lipids as ionizable lipid (a) are each independently selected from lipids of formulas a-IV.
In some embodiments, the first cationic lipid that is the ionizable lipid (a), or the second cationic lipid that is the ionizable lipid (a), or both, has the structure of formulas a-V or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof:
(a-V)
wherein:
One of G 1 or G 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)y-、-S-S-、-C(=O)S-、SC(=O)-、-N(Ra)C(=O)-、-C(=O)N(Ra)-、-N(Ra)C(=O)N(Ra)-、-OC(=O)N(Ra)- or-N (R a) C (=o) O-at each occurrence, and the other of G 1 or G 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)y-、-S-S-、-C(=O)S-、-SC(=O)-、-N(Ra)C(=O)-、-C(=O)N(Ra)-、-N(Ra)C(=O)N(Ra)-、-OC(=O)N(Ra)- or-N (R a) C (=o) O-or a direct bond at each occurrence;
l is at each occurrence-O (C=O) -, where-represents a covalent bond with X;
X is CR a;
Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1, or is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
R a is independently at each occurrence H, C 1-C12 alkyl, C 1-C12 hydroxyalkyl, C 1C12 aminoalkyl, C 1-C12 alkylaminoalkyl, C 1-C12 alkoxyalkyl, C 1-C12 alkoxycarbonyl, C 1C12 alkylcarbonyloxy, C 1-C12 alkylcarbonyloxy alkyl or C 1-C12 alkylcarbonyl;
R is independently at each occurrence (a) H or C 1-C12 alkyl, or (b) R together with the carbon atom to which it is attached forms a carbon-carbon double bond with the adjacent R and the carbon atom to which it is attached;
Each occurrence of R 1 and R 2 has the following structure:
R' is independently at each occurrence H or C 1-C12 alkyl, a 1 and a 2 are independently at each occurrence integers from 3 to 12, b 1 and b 2 are independently at each occurrence 0 or 1;
c 1 and c 2 are independently at each occurrence an integer from 2 to 12, d 1 and d 2 are independently at each occurrence an integer from 2 to 12, y is independently at each occurrence an integer from 0 to 2, and n is an integer from 1 to 6,
Wherein a 1、a2、c1、c2、d1 and d 2 are selected such that the sum of a 1+c1+d1 is an integer from 18 to 30 and the sum of a 2+c2+d2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy alkyl, and alkylcarbonyl is optionally substituted with one or more substituents.
In some embodiments, the first and second cationic lipids as ionizable lipid (a) are each independently selected from lipids of formulas a-V.
In some embodiments, the first and second cationic lipids as ionizable lipid (a) each have the following structure:
。
in some embodiments, the first cationic lipid that is the ionizable lipid (a), the second cationic lipid that is the ionizable lipid (a), or both have one of the following structures:
In some embodiments, when using the compounds of formulas a-II, the total mole percent of cationic lipid as ionizable lipid (a) within the lipid nanoparticle ranges from 40 to 55 mole percent, based on the total lipid present in the lipid nanoparticle. In some embodiments, the lipid nanoparticle may further comprise a neutral lipid, a steroid (such as a sterol), and/or a polymer conjugated lipid, preferably all of the neutral lipid, the steroid, and the polymer conjugated lipid, and the molar ratio of total cationic lipid to neutral lipid ranges from about 2:1 to about 8:1. In some embodiments, the molar ratio of total cationic lipid to steroid ranges from 5:1 to 1:1. In some embodiments, the molar ratio of total cationic lipid to polymer conjugated lipid ranges from about 100:1 to about 20:1.
In some embodiments, the neutral lipid is distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1 carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE) or 1, 2-di-trans-oleoyl-sn-glycero-3-phosphoethanolamine (DOPE), preferably the neutral lipid is DSPC, DPPC, DMPC, DOPC, POPC, DOPE or SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol.
In some embodiments, the polymer conjugated lipid is present at a concentration ranging from 1.0 to 2.5 mol%, preferably about 1.7 mol%, wherein the polymer conjugated lipid is present at a concentration of about 1.5 mol%.
In some embodiments, the polymer conjugated lipid is a pegylated lipid. In some embodiments, the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxypropyl carbamate. In some embodiments, the pegylated lipid has the following formula (a-VI) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:
wherein:
R 12 and R 13 are each independently a linear or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, where the alkyl chain is optionally interrupted by one or more ester bonds, and
The average value of w is in the range of 30 to 60.
Optionally, R 12 and R 13 are each independently a straight saturated alkyl chain containing from 12 to 16 carbon atoms. Optionally, the w-means ranges from 42 to 55, preferably the w-means is about 49. In some embodiments, the pegylated lipid has the following formula (VIa):
Wherein w means about 49.
In some embodiments, the lipid nanoparticle forms a plurality of nanoparticles having a polydispersity of less than 0.12. Preferably, the polydispersity is less than 0.08.
In some embodiments, the average diameter ranges from 50nm to 100 nm, preferably the diameter ranges from 60 nm to 85 nm.
According to a preferred embodiment, the nanoparticles referred to herein may comprise or consist of an ionizable lipid of the following formula (L-1) or a protonated form thereof. It is particularly preferred that the Lipid Nanoparticle (LNP) formulation or the lipid nanoparticle (LiNP) formulation is a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles (LiNP), each comprising a lipid mixture, wherein the lipid mixture comprises an ionizable lipid of the following formula (L-1) or a protonated form thereof. Ionizable lipids of the following formula (L-1) or protonated forms thereof that may be used as preferred ionizable lipids in the context of the present invention are described in detail in PCT application WO2014/207231A 1.
(L-1),
Wherein variables a, b, p, m, n and R 1A to R 6A are defined as follows:
a is 1 or 2 and b is an integer from 1 to 4, or a is an integer from 1 to 4 and b is 1 or 2,
Preferably a is 1 and b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1,
P is either 1 or 2 and is preferably chosen,
M is 1 or 2;n is 0 or 1 and m+n≥2, and
R 1A to R 6A are each independently selected from the group consisting of hydrogen 、-CH2CH(OH)R7A、-CH(R7A)-CH2-OH、-CH2-CH2-(C=O)-O-R7A、-CH2CH2(C=O)NHR7A、-CH2R7A、-C(NH)NH2、 poly (ethylene glycol) chain and acceptor ligands, wherein R 7A is selected from the group consisting of C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond,
Provided that at least two residues of R 1A to R 6A are selected from -CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A and-CH 2R7A, wherein R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond.
In the protonated form of the compound of formula (L-1), one or more of the nitrogen atoms contained in the compound of formula (L-1) are protonated to provide a positively charged compound.
Preferably, R 1A to R 6A are independently selected from hydrogen, the radicals -CH2CH(OH)R7A、-CH(R7A)-CH2-OH、-CH2-CH2-(C=O)-O-R7A、-CH2CH2(C=O)NHR7A; and-CH 2R7A, wherein R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond,
Provided that at least two residues of R 1A to R 6A, more preferably at least three residues of R 1A to R 6A and still more preferably at least four residues of R 1A to R 6A are groups selected from -CH2CH(OH)R7A、-CH(R7A)-CH2-OH、-CH2CH2-(C=O)-O-R7A、-CH2CH2(C=O)NHR7A and-CH 2R7A, wherein R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond.
More preferably, R 1A to R 6A are independently selected from hydrogen and the group-CH 2CH(OH)R7A, wherein R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond, with the proviso that at least two residues of R 1A to R 6A, more preferably at least three residues of R 1A to R 6A and still more preferably at least four residues of R 1A to R 6A are groups-CH 2CH(OH)R7A, wherein R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond.
Preferably, R 7A is selected from the group consisting of C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, and more preferably from the group consisting of C8-C12 alkyl and C8-C12 alkenyl having one C-C double bond. Generally, alkyl is more preferred over alkenyl as R 7A.
As long as any of the groups R 1A to R 6A is a protecting group for an amino group, such as described for example in WO2006/138380, a preferred embodiment thereof is tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) or benzyloxycarbonyl (carbobenzyloxy) (Cbz).
To the extent that any of the groups R 1A to R 6A is a receptor ligand, philipp and Wagner give useful examples in "GENE AND CELL THERAPY-Therapeutic MECHANISMS AND STRATEGY", 3 rd edition, chapter 15, CRC Press, taylor & Francis Group LLC, boca Raton 2009. Preferred receptor ligands for lung tissue are described in Pfeifer et al, 2010, ther Deliv.1 (1): 133-48. Preferred receptor ligands include synthetic cyclic or linear peptides, such as peptides derived from a library of screening peptides to bind to a particular cell surface structure or a particular cell type, cyclic or linear RGD peptides, synthetic or natural carbohydrates, such as sialic acid, galactose or mannose, or synthetic ligands derived from, for example, the reaction of a carbohydrate with a peptide, antibodies that specifically recognize a cell surface structure, folic acid, epidermal growth factor and its derivatives, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, or approved drugs that bind to known cell surface molecules.
As long as any of the groups R 1A to R 6A is a poly (ethylene glycol) chain, the preferred molecular weight of the poly (ethylene glycol) chain is from 100 to 20,000 g/mol, more preferably from 1,000 to 10,000 g/mol and most preferably from 1,000 to 5,000 g/mol.
The variable p in formula (L-1) is preferably 1.
In the formula (L-1), m is 1 or 2;n is 0 or 1, and m+n is not less than 2. In other words, if m is 1, n must also be 1, and if m is 2, n may be 0 or 1. If n is 0, then m must be 2. If n is 1, then m may be 1 or 2.
The variable n in formula (L-1) is preferably 1. More preferably, m is 1 and n is 1.
Thus, a combination of p=1, m=1, and n=1 is also preferable.
For the variables a and b in formula (L-1), a is 1 or 2 and b is an integer of 1 to 4, or a is an integer of 1 to 4 and b is 1 or 2. Thus, for example, a may be 1 or 2, and b may also be independently 1 or 2. Preferably, a is 1 and b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1, and more preferably one of a and b is 1 and the other is 2 or 3. Still more preferably a is 1 and b is 2, or a is 2 and b is 1. Most preferably, a is 1 and b is 2.
In view of the above, it is further preferred that the compound of formula (L-1) is a compound of formula (L-1 a), and that the ionizable lipids in the lipid nanoparticles referred to herein comprise or more preferably consist of the ionizable lipids of formula (L-1 a) below or a protonated form thereof:
R1A-NR2A-CH2-(CH2)a-NR3A-CH2-(CH2)b-NR4A-CH2-(CH2)a-NR5A-R6A(L-1a),
Wherein a, b and R 1A to R 6A are as defined in formula (L-1), including exemplary and preferred embodiments thereof.
In the protonated form of the compound of formula (L-1 a), one or more of the nitrogen atoms contained in the compound of formula (L-1 a) are protonated to provide a positively charged compound.
According to another preferred embodiment, the ionizable lipids in the lipid nanoparticles described herein comprise or more preferably consist of an ionizable lipid of the following formula (L-1 b):
(L-1b)
Wherein R 1A to R 6A are as defined for formula (L-1), including preferred embodiments thereof;
In the protonated form of the compound of formula (L-1 b), one or more of the nitrogen atoms contained in the compound of formula (L-1 b) are protonated to provide a positively charged compound.
Thus, according to a particularly preferred embodiment, the ionizable lipids in the lipid profile mentioned herein comprise or more preferably consist of an ionizable lipid of formula (L-1 b) or a protonated form thereof, wherein R 7A is selected from the group consisting of C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, provided that at least two residues of R 1A to R 6A are-CH 2-CH(OH)-R7A, more preferably at least three residues of R 1A to R 6A, still more preferably at least four residues of R 1A to R 6A are-CH 2-CH(OH)-R7A, wherein R 7A is selected from the group consisting of C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond.
As examples of suitable lipid compounds that can be used as ionizable lipids in the context of the present invention, reference may be made to lipid dl_05 (R) or protonated forms thereof having the structure wherein one or more of the nitrogen atoms comprised in the compound are protonated:
。
According to another exemplary embodiment, the ionizable lipids that may be comprised by the nanoparticles referred to herein include or consist of an ionizable lipid of formula (L-2) or a protonated form thereof,
(L-2)
Wherein R 1B is an organic group comprising one or more primary, secondary or tertiary amino groups,
Wherein one or more of the nitrogen atoms contained in the primary, secondary or tertiary amino groups included in R 1B are protonated to provide a positively charged compound.
Preferably, the compound of formula (L-2) has the following structure:
。
according to another exemplary embodiment, the ionizable lipids that may be comprised by the nanoparticles mentioned herein include or consist of an ionizable lipid of formula (L-3) or a protonated form thereof
(L-3)
Wherein the method comprises the steps of
R 1C and R 2C are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably from C12-C18 alkyl and C12-C18 alkenyl,
R 3C is C1-C6 alkanediyl, preferably C2 or C3 alkanediyl, and
R 4C and R 5C are independently hydrogen or C1-C3 alkyl, and preferably methyl;
wherein one or more nitrogen atoms contained in the compound of formula (L-3) are protonated to provide a positively charged compound.
As an example of ionizable lipids of formula (L-3), reference may be made to DLin-MC3-DMA (6Z, 9Z,28Z, 31Z) -seventeen carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate.
According to another exemplary embodiment, the ionizable lipids that may be comprised by the nanoparticles referred to herein include or consist of the ionizable lipids of formula (L-4) or a protonated form thereof,
(L-4)
Wherein the method comprises the steps of
R 1D and R 2D are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably from C12-C18 alkyl and C12-C18 alkenyl,
R 3D is C1-C6 alkanediyl, preferably C2 alkanediyl, and
R 4D and R 5D are independently hydrogen or C1-C3 alkyl, and preferably methyl;
Wherein one or more nitrogen atoms contained in the compound of formula (L-4) are protonated to provide a positively charged compound.
According to another exemplary embodiment, the ionizable lipids that may be comprised by the nanoparticles referred to herein include or consist of the ionizable lipids of formula (b-5) or a protonated form thereof,
(L-5)
Wherein R 1E to R 5E are independently of one another selected from hydrogen ,-CH2CH(OH)-R7E、-CH(R7E)-CH2-OH、-CH2-CH2-(C=O)-O-R7E、-CH2CH2(C=O)NHR7E and-CH 2R7E, wherein R 7E is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond, with the proviso that at least two residues in R 1E to R 5E are selected from -CH2-CH(OH)-R7E、-CH(R7E)-CH2-OH、-CH2-CH2-(C=O)-O-R7E、-CH2-CH2-(C=O)-NH-R7E and-CH 2-R7E, wherein R 7E is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond;
wherein one or more nitrogen atoms contained in the compound of formula (L-5) are protonated to provide a positively charged compound.
In formula (L-5), R 1E to R 5E are preferably independently-CH 2CH(OH)R7E, wherein R 7E is selected from C8-C18 alkyl or C8-C18 alkenyl having one C-C double bond.
Still another exemplary ionizable lipid that may be included in or consist of the herein mentioned nanoparticles as ionizable lipid suitable for use in the present invention is an ionizable lipid disclosed as "cationic lipid of formula I" in PCT application WO2012/000104A1, beginning at page 104 of this document, and including all the specific embodiments discussed in this document.
Other exemplary ionizable lipids suitable for use in the present invention that may be included in or consist of the nanoparticles referred to herein are those disclosed and claimed as "aminoalcohol lipids" in PCT application WO2010/053572A2, including all compounds of the general formula shown in page 4 of the invention of that document, and further defined in the remainder of that application.
Yet another exemplary ionizable lipid suitable for use in the present invention that may be included in or may consist of the nanoparticles referred to herein is the ionizable lipid disclosed in PCT application WO2014/028487A1 as amine-containing lipids of formulas I-V, including specific embodiments thereof.
Another preferred example of an ionizable lipid that may be included in or consist of the herein mentioned nanoparticles as ionizable lipid suitable for use in the present invention is the ionizable lipid ((4-hydroxybutyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), or a protonated form thereof, wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.
Another preferred example of an ionizable lipid that may be included in or consist of the herein mentioned nanoparticles as an ionizable lipid suitable for use in the present invention is the ionizable lipid (6 z,9z,28z,31 z) -thirty-heptadec-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate, or a protonated form thereof, wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.
Another preferred ionizable lipid example as an ionizable lipid in the nanoparticles mentioned herein or which may be comprised in component (b) or which component (b) may consist of is the ionizable lipid heptadec-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate (SM-102), or a protonated form thereof, wherein the nitrogen atom of the compound is protonated to provide a positively charged compound.
As a preferred optional component in addition to at least one selected from the group consisting of a permanent cationic lipid, an ionizable lipid and an ionizable lipid, the nanoparticle may generally comprise one or more of the following components (c 1) to (c 6) as a lipid mixture or components of a lipid mixture:
(c1) A non-ionizable lipid having a sterol structure;
(c2) A phosphoglyceride lipid;
(c3) PEG conjugated lipids;
(c4) A polyglutamic acid conjugated lipid;
(c5) PAS lipid, and
(C6) Cationic polymers.
Those skilled in the art will appreciate that the possibility of nanoparticles comprising one or more of components (c 1) to (c 6) includes not only combinations between (c 1) - (c 6), but also combinations of one type of different components, e.g. two components (c 2), or combinations of one type of different components with other components of (c 1) to (c 6).
Component (c 1) is a lipid having a sterol structure. Thus, suitable lipids are compounds having a steroid core structure with a hydroxyl group at the 3-position of the a-ring.
Exemplary non-ionizable lipids having a sterol structure that may be included in component (c 1) or that constitute component (c 1) have a structure of formula (c 1-1),
(c1-1)
Wherein R 1L is C3-C12 alkyl.
Other exemplary non-ionizable lipids having sterol structures that may be included in component (c 1) or from which component (c 1) is composed include those disclosed in s.patel et al ,Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA, Nature Communications, 2020, 11:983, particularly those shown in fig. 2 of the publication.
Preferably, component (c 1) comprises or consists of cholesterol.
Component (c 2) is a phosphoglyceride.
Preferably, component (c 2) comprises or consists of a phospholipid selected from the group consisting of a compound of formula (c 2-1) or a pharmaceutically acceptable salt thereof and a phospholipid of formula (c 2-2) or a pharmaceutically acceptable salt thereof,
(c2-1)
Wherein the method comprises the steps of
R 1F and R 2F are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably from C12-C18 alkyl and C12-C18 alkenyl;
(c2-2)
Wherein the method comprises the steps of
R 1G and R 2G are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably from C12-C18 alkyl and C12-C18 alkenyl.
More preferably, component (c 2) comprises or consists of 1, 2-dipalmitoyl-sn-glycerol-3-phosphorylcholine (DPPC) or a pharmaceutically acceptable salt thereof or 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine (DSPC) or a pharmaceutically acceptable salt thereof.
Exemplary salt forms of the compounds of formula (c 2-1) include salts formed from acidic OH groups with bases, or salts formed from amino groups with acids. As salts with bases, mention may be made of alkali metal salts, such as sodium or potassium salts, alkaline earth metal salts, such as calcium or magnesium salts and ammonium salts. As exemplary salts with acids, salts with acidic groups of nucleic acids may be mentioned, but other salts are not excluded, and inorganic acid salts such as chloride, bromide or iodide, sulfate, nitrate, phosphate, hydrogen phosphate or dihydrogen phosphate, carbonate and bicarbonate may be mentioned as examples.
Exemplary salt forms of the compounds of formula (c 2-2) include salts formed from acidic-OH groups attached to the P atom with bases, or salts formed from quaternary amino groups with anions. As salts with bases, mention may be made of alkali metal salts, such as sodium or potassium salts, alkaline earth metal salts, such as calcium or magnesium salts and ammonium salts. As exemplary salts with anions, salts with acidic groups of nucleic acids may be mentioned, but other salts are not excluded, and inorganic acid salts such as chloride, bromide or iodide, sulfate, nitrate, phosphate, hydrogen phosphate or dihydrogen phosphate, carbonate and bicarbonate may be mentioned as examples.
Component (c 3) is a PEG conjugated lipid, i.e. a lipid covalently linked to a polyethylene glycol chain.
Preferably, component (c 3) comprises or consists of a PEG conjugated lipid selected from the group consisting of a compound of formula (c 3-1), a compound of formula (c 3-2) or a pharmaceutically acceptable salt thereof or a compound of formula (c 3-3),
(c3-1)
Wherein:
R 1H and R 2H are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably from C12-C18 alkyl or C12-C18 alkenyl, and p is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60;
(c3-2)
wherein:
r 1J and R 2J are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably from C12-C18 alkyl or C12-C18 alkenyl, and q is an integer from 5 to 200, preferably from 10 to 100, more preferably from 20 to 60,
(c3-3)
Wherein:
R 1K and R 2K are independently C8-C18 alkyl or C8-C18 alkenyl, preferably C12-C18 alkyl or C12-C18 alkenyl, and q is an integer from 5 to 200, preferably from 10 to 100, and more preferably from 20 to 60.
Exemplary salt forms of the compounds of formula (c 3-2) include salts formed from acidic-OH groups attached to the P atom with bases. As salts with bases, mention may be made of alkali metal salts, such as sodium or potassium salts, alkaline earth metal salts, such as calcium or magnesium salts and ammonium salts.
More preferably, component (c 3) comprises or consists of 1, 2-dimyristoyl-sn-glycerylethoxy (polyethylene glycol) (DMG-PEG), and still more preferably component d) comprises or consists of 1, 2-dimyristoyl-sn-glycerylethoxy (polyethylene glycol) -2000 (DMG-PEG 2 k) or 2- [ (polyethylene glycol) -2000] -N, N-dimyristoyl acetamide (ALC-0159).
Component (c 4) is a polygluc-acid conjugated lipid, i.e. a lipid covalently linked to a polymer moiety of formula (c 4-1):
-[C(O)-CH2-N(CH3)]r-(c4-1)
Wherein r represents the number of repeating units, and is preferably 10 to 100. An example of a poly-sarcosine based lipid is N-TETAMINE-pSar.
Component (c 5) is a PAS lipid, e.g., a lipid covalently linked to a polymer moiety formed by proline (pro)/alanine (ala)/serine (ser) repeat residues.
The contents of WO2017/109087A1 and EP3394266B1 are incorporated herein by reference with respect to the PAS lipids used herein. In particular, the definitions and embodiments described below, which specifically enumerate nucleic acids encoding PAS polypeptides, are incorporated herein by reference. The PAS lipids may include, for example, a polypeptide consisting of at least 100 amino acid residues of proline, alanine, and optionally serine, wherein the polypeptide forms a random coil.
Component (c 6) is a cationic polymer. Such polymers suitable for forming nanoparticles comprising nucleic acids are known in the art. Exemplary suitable cationic polymers are discussed in A.C. Silva et al, current Drug Metabolism, 16, 2015, 3-16 and references cited therein, J.C. Kasper et al, J.Contr. Rel 151 (2011), 246-255, WO 2014/207231 and references cited therein, and WO2016/097377 and references cited therein.
Suitable cationic oligomers or polymers include in particular cationic polymers comprising a plurality of units comprising amino groups therein. The amino groups may be protonated to provide a cationic charge to the polymer.
Polymers comprising a plurality of units independently selected from the following (1), (2), (3) and (4) are preferred:
-CH2-CH2-NH-(1)
(2)
-CH2-CH2-CH2-NH-(3)
(4),
Wherein one or more of the nitrogen atoms of repeating units (1), (2), (3) and/or (4) may be protonated to provide the cationic charge of the polymer.
Particularly preferred cationic polymers are the following four classes of polymers comprising a plurality of units (containing amino groups therein).
As a first preferred class, mention is made of poly (ethyleneimine) ("PEI"), including branched polyethyleneimines ("brPEI").
A second preferred class of cationic polymers are polymers comprising a plurality of groups of formula (c 6-1) as side chains and/or end groups, as disclosed in WO 2014/207231 (which is incorporated herein in its entirety by reference, applicant's Ethris GmbH) as groups of formula (II):
(c6-1)
Wherein, for each of a plurality of such groups of formula (c 6-1), variables a, b, p, m, n and R 2 to R 6 are independently defined as follows:
a is 1 or 2 and b is an integer from 1 to 4, or a is an integer from 1 to 4 and b is 1 or 2,
Preferably a is 1 and b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1,
P is either 1 or 2 and is preferably chosen,
M is 1 or 2;n is 0 or 1 and m+n≥2, and
R 2 to R 5 are independently of one another selected from hydrogen, a protecting group for the amino group of the radical -CH2-CH(OH)-R7、-CH(R7)-CH2-OH、-CH2-CH2-(C=O)-O-R7、-CH2-CH2-(C=O)-NH-R7、-CH2-R7、 and a poly (ethylene glycol) chain, wherein R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond;
R 6 is selected from hydrogen, a protecting group for the group -CH2-CH(OH)-R7,-CH(R7)-CH-OH、-CH2-CH2-(C=O)-O-R7、-CH2-CH2-(C=O)-NH-R7、-CH2-R7 、 amino, -C (NH) -NH 2, a poly (ethylene glycol) chain, and a receptor ligand, wherein R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond;
And wherein one or more of the nitrogen atoms shown in formula (c 6-1) may be protonated to provide a cationic group of formula (c 6-1).
With regard to further preferred definitions of these polymers and of the variables contained in the above-mentioned formula (c 6-1), the corresponding disclosure in WO2014/207231 regarding the groups of formula (II) disclosed in this document also applies to the invention described herein.
A third class of preferred cationic polymers are polymers comprising a plurality of groups of the following formula (c 6-2) as repeat units, as disclosed in WO 2014/207231 (which is incorporated herein in its entirety by reference, applicant's Ethris GmbH) as groups of the formula (III):
(c6-2)
Wherein, for each of a plurality of such groups of formula (c 6-2), variables a, b, p, m, n and R 2 to R 5 are independently defined as follows:
a is 1 or 2 and b is an integer from 1 to 4, or a is an integer from 1 to 4 and b is 1 or 2,
Preferably a is 1 and b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1,
P is either 1 or 2 and the number of times,
M is 1 or 2;n is 0 or 1 and m+n≥2, and
R 2 to R 5 are independently of one another selected from the group consisting of hydrogen, protecting groups for the amino group -CH2-CH(OH)-R7、-CH(R7)-CH2-OH、-CH2-CH2-(C=O)-O-R7、-CH2-CH2-(C=O)-NH-R7、-CH2-R7、, -C (NH) -NH 2 and poly (ethylene glycol) chains;
And wherein one or more of the nitrogen atoms shown in formula (c 6-2) may be protonated to provide a cationic group of formula (c 6-2).
With regard to further preferred definitions of these polymers and of the variables contained in the above-mentioned formula (c 6-2), the corresponding disclosure in WO2014/207231 regarding the repeat units of formula (III) disclosed in this document also applies to the invention described herein.
A fourth preferred class of cationic polymers is provided by statistical copolymers, as disclosed in WO2016/097377 (incorporated herein in its entirety by reference, applicant's Ethris GmbH). Comprising a plurality of repeating units (a) independently selected from repeating units of the following formulas (a 1) and (a 2) and a plurality of repeating units (b) independently selected from repeating units of the following formulas (b 1) to (b 4):
-CH2-CH2-NH-(a1),
(a2),
-CH2-CH2-CH2-NH-(b1),
(b2),
-CH2-CH2-CH2-CH2-NH-(b3),
(b4),
And the molar ratio of the sum of the repeating units (a) to the sum of the repeating units (b) is in the range of 0.7/1.0 to 1.0/0.7, and one or more nitrogen atoms of the repeating units (a) and/or (b) contained in the copolymer may be protonated to provide a cationic copolymer.
With regard to a further preferred definition of the copolymer, the corresponding disclosure in WO 2016/097377 also applies to the invention described herein. As described therein, particularly preferred copolymers are linear copolymers comprising repeating units (a 1) and (b 1), or linear copolymers consisting of repeating units (a 1) and (b 1).
As an optional component of the nanoparticle, if the nanoparticle comprises a nucleic acid as a preferred therapeutic agent, it may also comprise a polyanionic component other than the nucleic acid, in particular in addition to the nucleic acid. Examples of such polyanions are polyglutamic acid and chondroitin sulfate. If such a polyanionic component other than a nucleic acid is used in the nanoparticle, the amount thereof is preferably limited so that the amount of anionic charge provided by the polyanionic component is not higher than the amount of anionic charge provided by the nucleic acid.
Preferably, the nanoparticles mentioned herein comprise, more preferably consist of:
i) Optionally, the above therapeutic agent as component (a), preferably a nucleic acid, more preferably an RNA, still more preferably an mRNA,
Ii) a lipid or lipid mixture comprising or consisting of:
At least one selected from the group consisting of permanent cationic lipids, ionizable lipids and ionizable lipids, more preferably ionizable lipids or ionizable lipids as component (b), and one or more of the following as preferred other components of the lipid or lipid mixture:
-a non-ionizable lipid (c 1) having a sterol structure;
-phosphoglyceride lipid (c 2);
-PEG conjugated lipid (c 3);
Poly sarcosine conjugated lipid (c 4);
PAS-formed lipid (c 5);
Cationic polymer (c 6).
Exemplary suspensions comprising nanoparticles formed from the above-listed components are also suitable for use as nanoparticle formulations in the context of the present invention, including those disclosed in s.patel et al ,Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA, Nature Communications, 2020, 11:983.
It will be appreciated that the components of the nanoparticle, particularly components (a) and (b), and optionally one or more of (c 1) to (c 6), are typically included as a mixture in the nanoparticle.
It is particularly preferred that the lipid or lipid mixture comprises components (c 1), (c 2) and (c 3).
With respect to the amounts of these components, it is further preferred that the nanoparticle comprises or consists of:
a therapeutic agent optionally as component (a), and
A lipid or lipid mixture comprising or consisting of:
i) 30 to 65 mol% of at least one selected from the group consisting of permanent cationic lipids, ionizable lipids and ionizable lipids, preferably ionizable lipids or ionizable lipids as component (b),
And one or more of the following components as a lipid or other component of a lipid mixture:
ii) 10 to 50 mol% of a lipid (c 1) having a sterol structure,
Iii) 4 to 50 mol% of phosphoglyceride lipid (c 2),
Iv) 0.5 to 10 mol% of one of PEG conjugated lipid (c 3), poly sarcosine conjugated lipid (c 4) and PAS conjugated lipid (c 5), or any combination thereof,
V) from 0.5 to 10 mol% of a cationic polymer (c 6),
Such that the sum of i) and ii) to v) is 100 mol%.
With respect to 30 to 65 mol% of at least one selected from the group consisting of permanent cationic lipids, ionizable lipids and ionizable lipids as component (b), it is understood that if a cationic lipid or two or more of a lipid, an ionizable lipid and an ionizable lipid are present as component (b), the indicated mole percentages refer to the total amount of these components in the nanoparticle. Also, it is understood that the molar percentages given for components (c 1) to (c 6) are such that not all of these components need to be present in the nanoparticle. Thus, for example, in the context of this preferred embodiment, the cationic polymer may be present or absent, but if present, is used in an amount of from 0.5 to 10 mole%. As described above, in the context of the present preferred embodiment, the amounts of components (c 1), (c 2), (c 3), (c 4), (c 5) and/or (c 6) are such that the sum of (b) and (c 1) to (c 6) is 100mol%.
It is further preferred that the nanoparticle comprises or consists of:
optionally, as a therapeutic agent of component (a), more preferably an mRNA, and
A lipid or a mixture of lipids comprising or consisting of at least one selected from the group consisting of a permanently cationic lipid, an ionizable lipid and an ionizable lipid as component (b), preferably an ionizable lipid or an ionizable lipid,
A non-ionizable lipid (c 1) having a sterol structure,
Phosphoglyceride lipid (c 2), and
PEG conjugated lipid (c 3).
With respect to the amounts of these components, more preferably, the nanoparticle comprises, more preferably consists of:
a therapeutic agent optionally as component (a), and
A lipid or lipid mixture comprising or consisting of:
30 to 65 mol% of at least one (b) selected from the group consisting of a permanent cationic lipid, an ionizable lipid and an ionizable lipid, preferably an ionizable lipid or an ionizable lipid,
10 To 50 mol% of a lipid (c 1) having a sterol structure,
4 To 50 mol% of phosphoglyceride lipid (c 2), and
From 0.5 to 10 mol% of PEG conjugated lipid (c 3),
So that the sum of (b) and (c 1) to (c 3) is 100 mol%.
Based on the above information regarding preferred therapeutic agents, in particular nucleic acids, and information regarding preferred components of lipid compositions other than therapeutic agents, lipid nanoparticles in the context of the present invention preferably comprise:
(a) mRNA as therapeutic agent, and
A lipid mixture comprising:
(b) An ionizable lipid of formula (L-1 b), or a protonated form thereof, wherein one or more of the nitrogen atoms shown in formula (L-1 b) are protonated to provide a cationic lipid,
(L-1b)
Wherein R 1A to R 6A are independently selected from hydrogen and-CH 2CH(OH)R7A, wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, provided that at least two residues of R 1A to R 6A are-CH 2-CH (OH) -R 7A, more preferably at least four residues of R 1A to R 6A are-CH 2-CH(OH)-R7A, wherein R 7A is selected from C8-C18 alkyl and C8C18 alkenyl having one C-C double bond, (C1) a non-ionizable lipid of the formula (C1-1) having a sterol structure,
(c1-1)
Wherein R 1L is C3-C12 alkyl;
(c2) A phosphoglyceride of formula (c 2-2) or a pharmaceutically acceptable salt thereof,
(c2-2)
Wherein R 1G and R 2G are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably from C12-C18 alkyl and C12-C18 alkenyl, and
(C3) PEG conjugated lipids of formula (c 3-1),
(c3-1)
Wherein R 1H and R 2H are independently selected from C8-C18 alkyl and C8-C18 alkenyl, preferably from C12-C18 alkyl or C12-C18 alkenyl, and p is an integer from 5 to 200, preferably from 10 to 100, more preferably from 20 to 60. In such lipid particle compositions, a lipid dL_05 (R) having the formula shown above would be a particularly preferred variant of an ionizable lipid.
Another preferred exemplary composition of lipid nanoparticles suitable for use in the context of the present invention comprises a nucleic acid, more preferably an mRNA, as a therapeutic agent, and a lipid mixture as an ionizable lipid comprising ((4-hydroxybutyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), or a protonated form thereof, wherein the nitrogen atom of the compound is protonated to provide a positively charged compound, and optionally further comprising one or more of the following components (d 1) to (d 8):
(d1) 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide (ALC-0159),
(D2) 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC),
(D3) The cholesterol level of the blood is determined by the concentration of cholesterol,
(D4) The potassium chloride is added to the mixture,
(D5) The potassium dihydrogen phosphate is used for preparing the nano-crystalline silicon dioxide,
(D6) The sodium chloride is used for preparing the sodium chloride,
(D7) Disodium phosphate dihydrate is used as a base for the production of the aqueous solution,
(D8) Sucrose.
More preferably, they further comprise at least (d 1), (d 2) and (d 3), and still more preferably they comprise all of (d 1) to (d 8).
Another preferred exemplary composition of lipid nanoparticles suitable for use in the context of the present invention comprises a nucleic acid, more preferably an mRNA, as a therapeutic agent, and a lipid mixture as an ionizable lipid comprising heptadec-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate (SM-102), or a protonated form thereof, wherein the nitrogen atom of the compound is protonated to provide a positively charged compound, and optionally one or more of the following components (e 1) to (e 7):
(e1) 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC),
(E2) The cholesterol level of the blood is determined by the concentration of cholesterol,
(E3) 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (PEG 2000 DMG),
(E4) The ammonia butanetriol hydrochloride is used for preparing the medicine,
(E5) The sodium acetate trihydrate and the sodium acetate trihydrate,
(E6) Acetic acid is used as a solvent for the acetic acid,
(E7) Sucrose.
More preferably, they also include at least (e 1), (e 2) and (e 3), and still more preferably they include all of (e 1) to (e 7).
Another preferred exemplary composition of lipid nanoparticles suitable for use in the context of the present invention comprises a nucleic acid, more preferably an mRNA, as a therapeutic agent, and a lipid mixture comprising DLin-MC3-DMA ((6 z,9z,28z,31 z) -heptadeca-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butyrate), or a protonated form thereof, wherein the nitrogen atom of the compound is protonated, and optionally one or more of the following components (e 1) to (e 7):
(e1) 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC),
(E2) The cholesterol level of the blood is determined by the concentration of cholesterol,
(E3) PEG2000-C-DMG (. Alpha. - (3' - { [1, 2-bis (myristoyloxy) propoxy ] carbonylamino } propyl) - ω -methoxy, polyoxyethylene),
(E4) 2-amino-2- (hydroxymethyl) propane-1, 3-diol (tromethamine) hydrochloride,
(E5) Disodium hydrogen phosphate heptahydrate is used for preparing the sodium phosphate,
(E6) The anhydrous potassium dihydrogen phosphate is used for preparing the water-free potassium dihydrogen phosphate,
(E7) Sodium chloride.
More preferably, components (e 1), (e 2) and (e 3) are present, and more preferably they comprise all of (e 1) to (e 6).
The nanoparticle preferably comprises a therapeutic agent, preferably a nucleic acid, more preferably RNA, and still more preferably mRNA, in which case the composition of the nanoparticle is preferably such that the weight ratio of the sum of the weights of the components in the nanoparticle other than the therapeutic agent to the weight of the therapeutic agent is in the range of 50:1 to 1:1, more preferably 40:1 to 2:1, and most preferably 30:1 to 3:1.
The N/P ratio, i.e. the ratio of the number of amine nitrogen atoms provided by the ionizable lipid, and/or the permanently cationic lipid to the number of phosphate groups provided by any nucleic acid of the nanoparticle (if the nucleic acid is included as a therapeutic agent), is preferably in the range of 0.5 to 20, more preferably in the range of 0.5 to 10.
The lipid or lipid nanoparticles, for example in suspension formulations, preferably have a Z-average diameter in the range of 10 to 500nm, more preferably in the range of 10 to 250 nm, still more preferably 20 to 200 nm. The indicated particle size is the hydrodynamic diameter of the particle as determined by Dynamic Light Scattering (DLS). The measurement is typically performed at 25 ℃.
The polydispersity index of the nanoparticles, e.g. of the lipids or lipid nanoparticles in the suspension formulation, is preferably in the range of 0.02 to 0.4, more preferably in the range of 0.03 to 0.2. The polydispersity index may be determined by Dynamic Light Scattering (DLS). The measurement is typically performed at 25 ℃.
A formulation, such as a suspension formulation, comprising different lipids or lipid-like nanoparticles (i.e., particles of different compositions) as described above may be provided. Preferably, however, the nanoparticles comprised in the formulation consist of the same components.
Nanoparticles comprising an active agent, such as a nucleic acid, may be conveniently prepared by mixing solutions comprising the active agent, and optionally a salt, such as sodium chloride, in an aqueous solvent, for example comprising a buffer, such as a citrate buffer at a pH of 4.5, and solutions comprising an ionizable lipid or ionizable lipid in an organic solvent, for example ethanol. For example, other optional components may be incorporated by adding them to one of the two solutions. Nanoparticles produced in this way may be further processed by chromatography and/or dialysis and/or Tangential Flow Filtration (TFF) to obtain the desired nanoparticles in the liquid composition. Preferably, they are further processed using TFF.
To provide a nanoparticle suspension, the lyophilized nanoparticles prepared according to the procedure described above, followed by lyophilization, may also be relied upon, followed by re-suspension in an aqueous carrier solution.
The lipid nanoparticle formulation or lipid nanoparticle formulation referred to herein is preferably a suspension formulation in which the nanoparticles are contained in a liquid carrier solution. The liquid carrier solution is preferably an aqueous carrier solution.
Those skilled in the art will appreciate that an aqueous carrier solution is one in which the primary solvent is water, preferably a solution comprising more than 70% water, more preferably more than 90% water as solvent, expressed as a volume percentage of water (at a temperature of 25 ℃) of the total volume of solvent(s) contained in the carrier solution, based on the total volume of solvent(s). Most preferably, water is the only solvent in the carrier solution. Thus, the carrier solution is a liquid at room temperature (e.g., 25 ℃). Likewise, the aqueous solvent referred to herein is water or a mixture of solvents, wherein water represents the primary solvent based on the total volume of solvent(s). Typically, the aqueous solvent comprises more than 70% water, more preferably more than 90% water as solvent, expressed as a volume percentage of water (at a temperature of 25 ℃) of the total volume of solvent(s) contained in the aqueous solvent. Most preferably, water is the only solvent in the aqueous solvent.
The weight/volume ratio of nanoparticles in the carrier solution of the suspension formulation is preferably in the range of 0.1g/L to 300g/L, more preferably 0.2g/L to 300 mg/L, still more preferably 0.5g/L to 250g/L, and most preferably 0.5g/L to 125g/L (measured at 25 ℃).
If the nanoparticle comprises nucleic acid as therapeutic agent and is provided as a suspension formulation, the concentration of nucleic acid provided by the lipid or lipid nanoparticle in suspension is preferably in the range of 0.01 to 10mg/ml, more preferably 0.02 to 10mg/ml, still more preferably 0.05 to 5mg/ml, and most preferably 0.05 to 2.5 mg/ml, based on the total volume of the suspension (measured at 25 ℃).
As mentioned above, the lipid or lipid nanoparticles comprised in the suspension preferably have a Z-average diameter in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, still more preferably 20 to 200 nm. The indicated particle size is the hydrodynamic diameter of the particle as determined by Dynamic Light Scattering (DLS). The measurement is typically performed at 25 ℃.
The polydispersity index of the nanoparticles contained in the suspension is preferably in the range of 0.02 to 0.4, more preferably in the range of 0.03 to 0.2. The polydispersity index may be determined by Dynamic Light Scattering (DLS). The measurement is typically performed at 25 ℃.
Surface active agent
Different types of surfactants may be used in the context of the present invention. Preferably, the surfactant comprises (or consists of) a nonionic surfactant, more preferably it is a nonionic surfactant. Examples of suitable nonionic surfactants include fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, fatty acid esters of sorbitol, ethoxylated fatty acid esters of sorbitol, fatty acid esters of glycerin, ethoxylated castor oil, and ethoxylated vitamin E.
Thus, the surfactant used in the context of the present invention, e.g. as a component of a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, preferably comprises, or consists of, at least one surfactant selected from the group consisting of, yet more preferably, a fatty alcohol ethoxylate, a fatty acid ethoxylate, a block copolymer of ethylene oxide and propylene oxide, an alkylphenol ethoxylate or an oligomer of an alkylphenol ethoxylate, a fatty acid ester of sorbitol, an ethoxylated fatty acid ester of sorbitol, a fatty acid ester of glycerin, an ethoxylated castor oil and an ethoxylated vitamin E.
Preferably, the block copolymer of ethylene oxide and propylene oxide is a poloxamer. The poloxamer is preferably one comprising one poly (propylene oxide) block B of formula (p-1) and two poly (ethylene oxide) blocks a of formula (p-2):
(p-1)
Wherein s is an integer of 15 to 60,
(p-2)
Wherein r is independently an integer from 8 to 150, preferably from 10 to 150, for each block.
Preferably, the surfactant used in the context of the present invention comprises, or preferably consists of, at least one selected from the group consisting of poloxamer 124, poloxamer 188, poloxamer 338, poloxamer 407, polysorbate 20 or Tween-20, polysorbate 80 or Tween-80, polyoxyethylene lauryl ether, polyoxyethylene-35 castor oil, D-alpha-tocopheryl polyethylene glycol 1000 succinate and tyloxapol.
Particularly preferred surfactants include, more preferably are (or consist of) poloxamers, such as the preferred poloxamers discussed above, still more preferably are poloxamers selected from poloxamer 124, poloxamer 188, poloxamer 338 and poloxamer 407 or selected from combinations thereof, most preferably P188.
It will be appreciated that the surfactants described herein are generally surfactants meeting the requirements set out in the aspects of the invention (e.g. first, second or third aspects) as described above, or are surfactants classified as suitable as stabilisers by the method according to the fourth or fifth aspects.
In the case of Lipid Nanoparticle (LNP) formulations or lipid nanoparticle (LiNP) formulations in suspension formulations, such as aqueous suspension formulations, it is preferred that the carrier solution in which the nanoparticles are suspended include a surfactant dissolved therein. Those skilled in the art will appreciate that this does not exclude the possibility of a certain amount of surfactant molecules adsorbing onto the lipid or lipid nanoparticles contained in the suspension.
In the context of the present invention, it has been found that the beneficial effect of the surfactant can already be achieved by a relatively low concentration of surfactant, for example 0.01% (w/v) in suspension. Thus, typically, the surfactant content in the suspension is 0.01% (w/v) or more relative to the total volume of nanoparticle suspension in the carrier solution (typically measured at 25 ℃).
For example, the present invention relates to incorporating surfactants into suspension formulations, preferably into carrier solutions, more preferably into aqueous carrier solutions, in an amount of 0.01 to 10% (w/v), preferably 0.1 to 10% (w/v), more preferably 0.25 to 5% (w/v), still more preferably 0.33 to 2.5% (w/v), even more preferably 0.45 to 1.5% (w/v) and most preferably 0.5 to 1.5% (w/v), relative to the total volume of the suspension of nanoparticles in the carrier solution. It will be appreciated that the concentration of the substance in% (w/v) or (weight/volume) corresponds to the amount of substance in g in 100 mL volumes, typically measured at 25 ℃, so that 1% (w/v) corresponds to 1 g surfactant per 100 mL total volumes of suspension.
Also, the method according to the invention may involve incorporating the surfactant into the nanoparticle suspension formulation in an amount of, for example, 0.01 to 10% (w/v), preferably 0.1 to 10% (w/v), more preferably 0.25 to 5% (w/v), still more preferably 0.33 to 2.5% (w/v), even more preferably 0.45 to 1.5% (w/v), and most preferably 0.5 to 1.5% (w/v), relative to the total volume of the suspension of the nanoparticles in the aqueous carrier solution (typically measured at 25 ℃).
Although a concentration of 0.5 to 1.5% (w/v) is particularly preferred as described above, the invention also provides and relates in its various aspects to suspension formulations wherein the concentration of surfactant is lower, for example in the range of 0.01 to 0.45% (w/v), or 0.1 to 0.40% (w/v).
In the context of the various aspects of the present invention, in lipid nanoparticles or suspension formulations of lipid nanoparticles in a carrier solution, preferably an aqueous carrier solution, it is generally preferred that the surfactant is substantially non-adhering to the nanoparticles, e.g., it is substantially not comprised in the nanoparticles and is substantially non-adhering to the nanoparticles. For example, in the context of the various aspects of the present invention, more than 90 wt%, preferably more than 95 wt%, of the total amount of surfactant contained in or incorporated into the lipid nanoparticles or suspension of lipid nanoparticles in an aqueous carrier solution is present in the aqueous carrier solvent without adhering to the nanoparticles.
In addition to the surfactant, other excipients may be present in the carrier solution. Preferably, the carrier solution further comprises at least one of sugar and salt, more preferably sucrose and NaCl.
The surfactant may conveniently be incorporated into the nanoparticle suspension formulation, for example by a method comprising adding the surfactant to a suspension comprising an aqueous carrier solution and lipid or lipid nanoparticles or a method comprising adding the lipid or lipid nanoparticles to an aqueous carrier fluid comprising the surfactant. For example, as described above, if the nanoparticles are provided in lyophilized form, they may be resuspended in an aqueous carrier solution comprising a surfactant.
In this regard, a method of preparing a lipid nanoparticle or suspension formulation of lipid nanoparticles as defined herein may comprise producing a lipid nanoparticle or preparation of lipid nanoparticles by mixing at least one selected from the group consisting of a permanently cationic lipid, an ionizable lipid, and an ionizable lipid dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution, and
The nanoparticles are combined with a surfactant to obtain a suspension of nanoparticles in an aqueous carrier solution.
Preferably, the method comprises the steps of:
i) Lipid nanoparticles or preparations of lipid nanoparticles are produced by mixing at least one selected from the group consisting of a permanent cationic lipid, an ionizable lipid, and an ionizable lipid dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution,
Ii) diluting the lipid nanoparticle or the preparation of lipid nanoparticles by dilution with the first solution,
Iii) Concentrating the lipid nanoparticles or diluted preparation of lipid nanoparticles by buffer exchange using ultrafiltration/diafiltration of TFF, wherein the second solution is used for ultrafiltration/diafiltration,
Iv) obtaining lipid nanoparticles or a suspension of lipid nanoparticles in an aqueous carrier solution comprising a surfactant,
Wherein the first solution comprises between 0.01% w/v and 10% w/v surfactant, preferably between 0.1% w/v and 10% surfactant, more preferably between 0.25% w/v surfactant and 5% w/v surfactant, still more preferably between 0.33% w/v surfactant and 2.5% w/v surfactant, even more preferably between 0.45% w/v surfactant and 1.5% w/v surfactant, most preferably between 0.5% w/v and 1.5% w/v surfactant, and/or
Wherein the second solution comprises between 0.01% w/v and 10% w/v surfactant, preferably between 0.1% w/v and 10% surfactant, more preferably between 0.25% w/v surfactant and 5% w/v surfactant, still more preferably between 0.33% w/v surfactant and 2.5% w/v surfactant, even more preferably between 0.45% w/v surfactant and 1.5% w/v surfactant, most preferably between 0.5% w/v and 1.5% w/v surfactant,
And wherein the final concentration of surfactant from the combined first and second solutions is between 0.01% w/v and 10% w/v surfactant, preferably between 0.1% w/v and 10% surfactant, more preferably between 0.25% w/v surfactant and 5% w/v surfactant, still more preferably between 0.33% w/v surfactant and 2.5% w/v surfactant, even more preferably between 0.45% w/v surfactant and 1.5% w/v surfactant, most preferably between 0.5% w/v and 1.5% w/v surfactant, relative to the total volume of the suspension of nanoparticles in the aqueous carrier solution.
In the above method, the surfactant is preferably not incorporated into the suspension before or during step i).
In addition, it is preferable to add the surfactant together with the first and second solutions. For example, wherein 30 to 70 wt% of the surfactant, preferably 40 to 60 wt%, and more preferably 45 to 55 wt% of the surfactant may be added with the first solution based on the total weight of the surfactant in the suspension obtained in step iv), and 70 to 30 wt% of the surfactant, preferably 60 to 40 wt%, and more preferably 55 to 45 wt% of the surfactant may be added with the second solution based on the total weight of the surfactant in the suspension obtained in step iv), such that the sum of the amounts of the surfactants added with the first and second solutions is 100 wt%. In general, it is preferable to add about half of the surfactant with the first solution and about half of the surfactant with the second solution.
In the context of the present invention, lipid nanoparticle formulations or lipid nanoparticle formulations are provided that are stabilized by the use of surfactants. As used herein, the term "stabilized" refers to the state of a Lipid Nanoparticle (LNP) formulation, wherein its physical and chemical integrity is maintained over time under specific conditions. This includes preferably maintaining particle size, structural integrity and/or uniformity, and preventing aggregation or fusion. Stabilization may also protect the encapsulated therapeutic agent from degradation, ensuring sustained efficacy and controlled release. Surfactants, such as poloxamers, help stabilize by reducing surface tension and preventing caking, increasing the overall stability of the lipid nanoparticle dispersion. For example, stability can be assessed by particle size, encapsulation efficiency, mRNA integrity, and potency measurements. Particle size can be measured using Dynamic Light Scattering (DLS) in combination with an instrument such as Malvern Zetasizer. Encapsulation efficiency can be quantified using an assay such as the RiboGreen assay. The integrity of the mRNA can be assessed using a fragment analyzer. Efficacy can be measured using an enzyme-linked immunosorbent assay (ELISA). For example, a stable lipid or lipid formulation may have a Z-average particle size of 25 to 150 nm over a prolonged period of time, e.g., weeks or months.
For example, by using the surfactants (preferably nonionic surfactants) described herein, nanoparticle suspensions may be stabilized in the context of the present invention, e.g., to prevent aggregation of particles under physical stress conditions. To achieve this effect, the surfactant may be incorporated into a suspension, preferably as an excipient into an aqueous carrier solution. In preferred embodiments, membrane fouling can be avoided by using the surfactants described herein.
In some embodiments, the LNP and/or LiNP have not been lyophilized. In some embodiments, the surfactant is added prior to the lyophilization process. In some embodiments, the surfactant is not present in the carrier solution during the lyophilization process.
In general, the presence of the surfactant does not result in a change in the biological activity of the nanoparticle. Biological activity refers to the expression level of a therapeutic nucleic acid in a target cell(s). The biological activity can be quantified, for example, by transfecting a cell line (e.g., HEK-293) with nanoparticles in vitro, followed by quantification of the nucleic acids produced by Southern/northern blotting or quantification of the proteins by ELISA. When the same assay is performed with the same LNP or LiNP in the absence of surfactant, the detected protein levels calculated as the average of three measurements at each concentration must not differ by more than 10%, preferably not more than 5%, more preferably statistically no difference.
The presence of the surfactant generally does not result in a change in the physical properties of the nanoparticle, as measured by the hydrodynamic diameter of the nanoparticle and the proportion of encapsulated therapeutic agent (preferably nucleic acid).
The hydrodynamic diameter of the nanoparticle may be measured, for example, by dynamic light scattering (also known as photon correlation spectroscopy). Optionally, the average of three measurements of the hydrodynamic diameter of the nanoparticle in the presence of the surfactant must not differ from the same nanoparticle in the absence of the surfactant by more than 5%, preferably not more than 1%, more preferably no statistical difference. The viscosity change of the surfactant must be taken into account during the measurement. The percentage of encapsulated nucleic acid can be determined, for example, by measuring the fluorescence intensity in a RiboGreen assay. Nanoparticles were analyzed under two different conditions, untreated samples were used for external nucleic acid analysis, and samples treated with Triton X-100 were used for total mRNA analysis. The percentage content of encapsulated nucleic acid was calculated. Optionally, the value calculated from the average of three measurements of the nanoparticle in the absence of surfactant should not differ by more than 5%, optionally not more than 3%, preferably not statistically different from the same nanoparticle in the presence of surfactant.
Those skilled in the art will appreciate that the action taken to stabilize the nanoparticle suspension against particle aggregation may prevent aggregation of the nanoparticles, or may reduce the extent of aggregation of the nanoparticles, as compared to the case where no relevant action is applied. Preferably, stabilization of the nanoparticle suspension is demonstrated by an increase in Z-average particle size of the suspended particles under physical stress conditions of less than 50%, more preferably less than 20%, still more preferably less than 10% and most preferably no such increase.
Also, it is understood that stabilization of the nanoparticle suspension to particle aggregation under physical stress conditions means that aggregation of the nanoparticles is prevented or reduced, which would be observed if the nanoparticle suspension was not stabilized when exposed to physical stress.
The physical stress conditions to which the nanoparticle suspension may be exposed are typically those encountered during handling or transportation of the suspension. For example, they involve a rapid movement of the volume of the suspension, which can lead to collisions of the nanoparticles contained in the unstable suspension. As examples of physical stress conditions, mention may be made of shaking, stirring, vibrating, mixing, tumbling, tapping or dropping of the nanoparticle suspension, or physical stress conditions caused by, for example, pumping the nanoparticle suspension or withdrawing it from a syringe. Those skilled in the art will appreciate that physical stress conditions include not only conditions under which the nanoparticle suspension is exposed during conventional processing, but also conditions under which the suspension may be abnormally exposed (such as in difficult conditions for transportation) or unintentionally exposed (such as by dropping a suspension sample).
Other pharmaceutical aspects
The pharmaceutical compositions mentioned herein, such as the Lipid Nanoparticle (LNP) formulations or lipid nanoparticle (LiNP) formulations mentioned herein, including therapeutic agents, are particularly useful in the medical environment and in the treatment or prevention of diseases and conditions, preferably in the treatment or prevention of diseases or conditions that rely on nucleic acids, such as RNA, preferably mRNA, as an active agent. Thus, such compositions are typically provided or used as medicaments or pharmaceutical compositions.
In particular, the pharmaceutical compositions mentioned herein, such as Lipid Nanoparticle (LNP) formulations or lipid nanoparticle (LiNP) formulations mentioned herein that include a therapeutic agent, are suitable for administration to a subject. In this way, the therapeutic agent, preferably a nucleic acid such as RNA (preferably mRNA), contained in the nanoparticle may also be administered to the subject.
The therapeutic agent, preferably a lipid or nucleic acid contained in lipid nanoparticle particles, may be delivered to the target cells via administration to a subject. The term "delivering to a target cell" preferably refers to transferring nucleic acid into a cell. Administration may be accomplished in a variety of ways known to those skilled in the art, including administration to or via the respiratory tract, such as by aerosolization of nanoparticles, or intramuscular or intravenous administration.
Diseases or conditions may be treated or prevented by administering to a subject a pharmaceutical composition, such as a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation as referred to herein, including a therapeutic agent. The term "disease" refers to any possible pathological condition that can be treated, prevented or vaccinated by using a suspension. The disease may be, for example, genetic, acquired, infectious or non-infectious, age-related, cardiovascular, metabolic, intestinal, oncological (in particular cancer) or genotypic. For example, a disease may be based on irregularities in physiological processes, molecular processes, biochemical reactions within an organism, which in turn may be based on, for example, genetic equipment, behavior, social or environmental factors of the organism (such as exposure to chemicals or radiation).
The pharmaceutical compositions mentioned herein, such as nanoparticle (LNP) formulations or lipid nanoparticle (LiNP) formulations mentioned herein comprising a therapeutic agent, are generally suitable for treating or preventing a disease selected from table a disclosed above.
Likewise, pharmaceutical compositions, such as nanoparticle (LNP) formulations or lipid nanoparticle (LiNP) formulations including therapeutic agents as referred to herein, are generally suitable for treating or preventing a disease selected from the group consisting of viral, ciliated or autoimmune diseases and respiratory diseases, even more preferably from the group consisting of pulmonary, airway or nasal diseases, more preferably from the group consisting of pulmonary viral, pulmonary ciliated and autoimmune diseases. Preferably, the pulmonary disease or pulmonary viral disease is at least one selected from the group consisting of pneumonia and asthma, the airway disease is at least one selected from the group consisting of bronchitis, virus-induced asthma, pulmonary fibrosis and COPD, and/or the nasal disease is at least one selected from the group consisting of rhinitis and sinusitis.
In some embodiments, the disease to be treated or prevented is a disease selected from the group consisting of alveolar proteinosis (PAP), interstitial lung diseases (such as pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis), viral infections (such as influenza and COVID-19), acute Respiratory Distress Syndrome (ARDS), nontuberculous mycobacterial (NTM) infections, lung cancer, fungal infections caused by Aspergillus (such as those caused by aspergillosis, fungal sinusitis, otomycosis, keratitis, and A-mycosis, preferably by Aspergillus fumigatus and Aspergillus flavus), infections caused by Mycobacterium tuberculosis, pseudomonas aeruginosa, phlebsiella, plasmodium protozoa, cryptococcus, nocardia, and combinations thereof.
In some embodiments, the disease is selected from the group consisting of (autoimmune) alveolar protein deposition (PAP), interstitial lung disease (such as pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis), viral infections (such as influenza and COVID-19), acute Respiratory Distress Syndrome (ARDS), nontuberculous mycobacterial (NTM) infections, lung cancer or fungal infections caused by aspergillus (such as those caused by aspergillosis, mycotic sinusitis, otomycosis, keratitis and onychomycosis, preferably by aspergillus fumigatus and aspergillus flavus), infections caused by mycobacterium tuberculosis, pseudomonas aeruginosa, pneumosporium bacteria and plasmodium.
In this regard, the invention also provides a lipid nanoparticle or lipid nanoparticle formulation comprising a therapeutic agent as described herein for use in the treatment or prevention of a disease. Also, the lipid nanoparticle or lipid nanoparticle formulation according to the present disclosure may be used in a method of treating or preventing a disease, the method comprising administering a suspension or formulation to a subject in need thereof.
In a related aspect, the invention also provides a lipid nanoparticle or a formulation of lipid nanoparticles comprising a therapeutic agent as described herein for use as a medicament.
For example, the invention provides a lipid nanoparticle or lipid nanoparticle formulation comprising a therapeutic agent as described herein for use in vaccination or immunization. Likewise, the lipid nanoparticles or lipid nanoparticle formulations comprising a therapeutic agent described herein can be used in a method of vaccination or immunization comprising administering a suspension or formulation to a subject in need thereof.
According to another aspect, the present invention provides a method of inducing an immune response against a target pathogen in a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle or lipid nanoparticle formulation comprising a therapeutic agent as described herein.
In another example, the invention provides a lipid nanoparticle or lipid nanoparticle formulation comprising a therapeutic agent as described herein for use in the treatment of cancer. Likewise, the lipid nanoparticles or lipid nanoparticle formulations comprising a therapeutic agent described herein can be used in a method of treating cancer, the method comprising administering the formulation to a subject in need thereof.
In another aspect, the present invention provides a method of avoiding or reducing side effects in therapy using a lipid nanoparticle or lipid nanoparticle comprising at least one therapeutic agent as described herein, wherein the method comprises the steps of:
i) Determining whether lipid nanoparticles or lipid nanoparticles in a pharmaceutical composition comprising the lipid nanoparticles or lipid nanoparticles are aggregated when subjected to mechanical or temperature stress by determining their aggregation level before and after subjecting the pharmaceutical composition to the mechanical or temperature stress,
Ii) if the lipid nanoparticle or lipid nanoparticle shows aggregation after the test of step (i), adding a surfactant as defined herein to the lipid nanoparticle or lipid nanoparticle formulation to obtain a LNP or LiNP suspension with a final surfactant concentration in the range of between 0.01% w/v and 10% w/v surfactant, preferably between 0.1% w/v and 10% w/v surfactant, more preferably 0.25% w/v surfactant to 5% w/v surfactant, still more preferably 0.33% w/v surfactant to 2.5% w/v surfactant, even more preferably 0.45% w/v surfactant to 1.5% w/v surfactant, most preferably 0.5% w/v to 1.5% w/v surfactant,
Iii) Reconstitution is performed by mixing to produce lipid nanoparticles or a stable suspension of lipid nanoparticles.
In a related aspect, the invention further provides a method of reducing one or more side effects associated with a vaccine formulation or an anti-cancer formulation comprising lipid nanoparticles or lipid nanoparticles carrying a therapeutic agent (such as a nucleic acid, preferably RNA, more preferably mRNA), as described herein, the method comprising modifying the vaccine formulation or anti-cancer formulation by adding a surfactant as described herein to the vaccine formulation or anti-cancer formulation comprising a suspension of lipid nanoparticles or lipid nanoparticles. Preferably, the surfactant represents between 0.01% w/v and 10% w/v surfactant, preferably between 0.1% w/v and 10% surfactant, more preferably 0.25% w/v surfactant to 5% w/v surfactant, still more preferably 0.33% w/v surfactant to 2.5% w/v surfactant, even more preferably 0.45% w/v surfactant to 1.5% w/v surfactant, most preferably 0.5% w/v to 1.5% w/v surfactant.
In a related aspect, the invention provides a method of reducing the occurrence or severity of one or more side effects associated with an LNP/LiNP-based vaccine in a subject, the method comprising administering to the subject a vaccine formulation or an anti-cancer formulation comprising a lipid nanoparticle or lipid nanoparticle formulation comprising a therapeutic agent as described herein.
The reduction in the occurrence or severity of one or more side effects may be caused by a reduction in LNP/LiNP aggregation, which may be measured by determining the hydrodynamic diameter of the nanoparticle, for example, by dynamic light scattering or photon correlation spectroscopy.
It will be appreciated that if the pharmaceutical composition, such as a lipid or a formulation of lipid nanoparticles, includes a therapeutic agent, the therapeutic agent is included in the composition or formulation in an effective amount. The term "effective amount (EFFECTIVE AMOUNT)" refers to an amount sufficient to induce a detectable therapeutic response or prophylactic effect in a subject to whom the pharmaceutical composition or formulation is administered. According to the above, the content of the therapeutic agent is not limited as long as it is useful for the above-mentioned treatment or prevention. For example, as described above, the suspension formulation in which the nucleic acid-containing nanoparticles are included preferably includes a quantity of particles to provide a concentration thereof of from 0.01 to 10mg/ml, more preferably from 0.02 to 10mg/ml, still more preferably from 0.05 to 5mg/ml, and most preferably from 0.05 to 2.5 mg/ml of the nucleic acid contained in the particles, based on the total volume of the composition. Also, it is understood that in the case where a pharmaceutical composition, such as a lipid nanoparticle or lipid nanoparticle formulation comprising a therapeutic agent as described herein, is administered to a subject, it will be administered in an effective amount.
Exemplary subjects include mammals, such as dogs, cats, pigs, cattle, sheep, horses, rodents, e.g., rats, mice, and guinea pigs, or primates, e.g., gorillas, chimpanzees, and humans. In a most preferred embodiment, the subject is a human.
Langmuir-Brookfield (Langmuir-Blodgett) trough
Langmuir-Brookfield (LB) technology is a method for precisely preparing ordered molecular assemblies and controlling molecular orientation and layer thickness. The method is based on the principle that a stable monolayer can be formed at the air-water interface when the amphiphilic molecules are spread on an aqueous subphase. By adjusting the surface pressure, the organization and bulk density of these molecules can be finely manipulated.
Langmuir surface pressure/area (pi-A) isotherms are a fundamental concept in the field of surface science, for example, when studying amphiphilic molecular monolayers at the air-water interface.
In a typical langmuir device, amphiphilic molecules (molecules having both hydrophilic and hydrophobic components) are spread into an aqueous subphase, forming a monolayer at the air-water interface. The area available for these molecules can be changed by using a moving barrier, which in turn changes the surface pressure (pi) of the monolayer. By plotting the change in surface pressure (pi) as a function of the area (a) of each molecule in the monolayer, a pi-a isotherm can be derived.
The main characteristics of the pi-A isotherm are:
Monolayer phase-different regions on an isotherm correspond to different molecular arrangements or phases. These may include a gas phase, a liquid expanded phase, and a liquid condensed phase.
Collapse pressure-the pressure at which a monolayer undergoes phase change and collapses can be determined on an isotherm. This may be a relevant parameter as it provides insight into the stability and strength of the monolayer.
Molecular area the area occupied by a single molecule in a monolayer can be determined from the isotherms. This is particularly useful for understanding the geometry and orientation of the molecules.
Phase transition-A change or abrupt change in the slope of the isotherm indicates a phase transition within a single layer. These can be used to infer molecular interactions and behavior under different conditions.
Intermolecular interactions the shape and characteristics of the isotherms allow for a deep understanding of the nature and strength of intermolecular forces in a monolayer.
In the context of the present invention, it has surprisingly been found that compression-expansion isotherms can be used to test the suitability of surfactants in lipid or lipid nanoparticle purification and processing.
*.*.*
In this specification, a number of documents including patent application and manufacturer manuals are cited. The disclosures of these documents, while deemed irrelevant to the patentability of the present invention, are incorporated herein by reference in their entirety. More specifically, all cited documents are incorporated by reference as if each individual document were specifically and individually indicated to be incorporated by reference.
Drawings
FIG. 1 shows a time chart of TFF processing of different P188 solutions.
Fig. 2 shows a histogram of refractive indices of different P188 solutions before and after TFF treatment.
Fig. 3 shows langmuir pressure-area diagrams of four representative poloxamer 188 samples in aqueous solution. Two distinct groups of poloxamers can be distinguished by considering the isotherm maximum surface pressure. S8 and S9 with maximum surface pressures above 4mN/m correspond to poloxamer P188 samples leading to fouling of the tangential flow filtration membranes, whereas samples S2 and S7 are suitable for purification and treatment of lipid nanoparticles.
Fig. 4 shows langmuir pressure-area diagrams of representative poloxamer 188 samples S1 to S11 in aqueous solution for three consecutive compression-expansion cycles. Samples S8 and S9 were clearly prominent with respect to isotherm maximum surface pressure, which was above 4 mN/m, confirming the poloxamer P188 sample that resulted in fouling of the tangential flow through the filter.
Figure 5 shows langmuir pressure-area diagrams of four representative poloxamer 188 samples in aqueous solutions containing lipid mixtures. The large hysteresis broadening observed in samples S8 and S9 can be used to identify poloxamer P188 samples that resulted in fouling of the tangential flow filtration membranes, while samples S2 and S7 were suitable for purification and processing of lipid nanoparticles.
Fig. 6 shows langmuir pressure-area diagrams of representative poloxamer 188 samples S1 to S11 in aqueous solution and lipid mixtures for three consecutive compression-expansion cycles. Samples S8 and S9 were clearly prominent with broad hysteresis, with the recognition that poloxamer P188 samples resulted in fouling of the tangential flow filter membranes, taking into account isotherm hysteresis.
Fig. 7 shows each calculated Δpi data point obtained for a lipid mixture representing lipid nanoparticle formulations deposited on 11 different poloxamer 188 (S1 to S11) aqueous subphases in three consecutive compression-expansion langmuir trough cycles. The superimposed Δpi data points are displayed as a wider distribution to visualize them. Thus, an isotherm with a generally tight hysteresis and a broader hysteresis for only a limited area will be represented herein by a pear-shaped distribution (e.g., fig. 6, s 7). Thus, the wider hysteresis feature (e.g., fig. 6, S8, and S9) results in a higher Δpi value and a narrower Δpi data point distribution. Two distinct sets of poloxamers can be distinguished and the narrow figure corresponds to poloxamer P188 samples that lead to fouling of the tangential flow through the filter membrane.
Fig. 8 shows a comparison of langmuir pressure-area diagrams of three consecutive compression-expansion cycles in an aqueous solution containing a lipid mixture for a representative poloxamer 188 sample S3 using two different LT devices (device 1 and device 2). The device 2 is characterized by a larger surface area than the device 1. The map shape and surface pressure values of both devices are repeatable.
Fig. 9 shows langmuir pressure-area diagrams of three consecutive compression-expansion cycles of poloxamer 188 test sample in an aqueous solution containing the lipid mixture of test sample S23. Considering isotherm hysteresis, the samples are characterized by low Δpi values and are therefore classified as suitable for TFF.
Fig. 10 shows a TFF treatment time diagram for different P188 solutions (including test sample S23). It was shown that sample S9 showed a non-linear relationship and three samples (S2, S3 and test sample S23) showed a linear relationship between permeate volume and time, confirming that the method is suitable for predicting surfactant behavior during TFF purification.
Figure 11 shows a comparison of langmuir pressure-area diagrams of three successive compression-expansion cycles of a test sample of poloxamer 124 in an aqueous solution containing a lipid mixture using device 1. The graph shape and surface pressure values indicate suitability for use as stabilizers.
Fig. 12 shows TFF treatment time charts for two different P188 solutions (S3 and S9) and poloxamer P124 test samples. Sample S9 is shown to exhibit a non-linear relationship. The P188 sample S2 and P124 test samples showed a linear relationship between permeation volume and time, confirming that this method was suitable for predicting the applicability of the surfactant during TFF purification.
Examples
Example 1-some P188 caused membrane fouling during TFF purification
During LiNP purification, it was observed that some components of the nanoparticle preparation can in some cases lead to blockage or fouling of the TFF membrane, leading to a substantial delay in filtration time or complete loss of LiNP preparation.
To test the source of the fouling causing substances, different batches of poloxamer P188 were tested during LiNP purification using TFF.
Materials and methods
Material
Poloxamer P188 is a pharmaceutical grade product obtained from a number of suppliers. The test mRNA was used at a concentration of 1 mg/mL.
TABLE 1
Method of
LiNP formulations and TFF
Lipid nanoparticles were produced in NanoAssemblr TM BT using the nanoprecipitation method, which included ionizable lipids (dl_05 (R), scheme 1), helper lipids DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, avanti Polar Lipids) and cholesterol (Avanti Polar Lipids) and PEG lipids DMG-PEG2k (1, 2-dimyristoyl-sn-glycero-methoxy (polyethylene glycol) -2000,Avanti Polar lipids) in a molar ratio of 8.00/5.29/4.41/0.88, respectively. Poloxamer P188 was added to a final concentration of 1% w/v prior to purification by Tangential Flow Filtration (TFF). TFF uses a C02-E100-05-N filter unit from Repligen. These devices are prepared according to manufacturer's manuals. A flow rate of 54 mL/min (main pump) was selected for the system to achieve the desired maximum shear rate of 12000 s -1. TFF was performed for 10 volumes in diafiltration mode, followed by ultrafiltration to reduce the volume by a factor of two.
Quality Control (QC)
Time measurements of TFF were recorded. Samples in which the TFF duration exceeded 75 minutes were considered to fail the test and were unsuitable for TFF purification.
Results
For some LiNP/P188 samples, fouling or plugging of the TFF column can be observed. The summary is shown in Table 2.
TABLE 2 summary of TFF blocking for certain pharmaceutical grade poloxamer 188 samples
Conclusion(s)
Scaling or clogging can be observed for different poloxamer P188 used at the same concentration. Surfactants such as poloxamers, particularly p188, may be considered suitable or unsuitable for membrane purification by purifying a composition comprising nanoparticles and poloxamers using TFF. However, this method is both expensive and time consuming, as in the case of poloxamers that fail the test, the TFF column needs to be discarded, and LiNP or LiNP, which are expensive to produce, need to be discarded.
Example 2-TFF purification with surfactant alone may indicate the applicability of TFF.
The following experiments were aimed at developing a test method for identifying P188 materials suitable for use in LiNP formulation processes, such as during TFF filtration. To develop this test, TFF was used to purify a solution containing only surfactant and NaCl.
The poloxamer samples S1-S11 discussed in example 1 were tested, either for the duration of TFF purification or for the Refractive Index (RI) of the poloxamer solution:
Materials and methods
Material
TABLE 3 materials
TABLE 4 apparatus
Sample preparation
The P188 solutions were prepared using P188 provided by different suppliers and different batches listed in table 2.
The final composition was 0.5% (w/v) P188,50 mM NaCl.
The solution was visually inspected for aggregates. Unless otherwise indicated, the solution did not appear to aggregate or cloudy.
TFF processing
All poloxamer 188 solutions were tested at a concentration of 0.5% (w/v). The TFF system was rinsed with the corresponding poloxamer solution and diafiltered 100 mL with the automatic back pressure valve fully opened. The feed flow rate was set at 54 mL/min and the maximum TMP was 690 mbar.
Before and after TFF treatment, 100 μl samples were drawn from each solution for Refractive Index (RI) measurements.
Refractive index measurement
The refractometer (DR 201-95, kru ss OptronicTM, germany) was calibrated with 100. Mu.L HPLC grade H 2 O. The RI was determined using a sample volume of 100 μl before and after treatment of poloxamer solution by TFF.
TABLE 5 Table data of FIG. 2
The experiment was intended to evaluate the processing time of P188 materials from different suppliers. Fig. 1 shows that the solution containing some samples, in particular sample S8, has to be treated for more than three times longer than all other samples. Furthermore, for most samples, there is indeed a slight increase in RI, whereas for sample S8, the RI of the treatment solution is increased to the highest extent, see fig. 2.
Discussion and conclusion
The experiment aims at developing a test method of different poloxamer materials as commodity entering control. It provides information as to whether the P188 lot or P188 vendor is suitable for formulation and/or available for standard production and/or purification.
Experiments have shown that surprisingly the blocking phenomenon observed when purifying nanoparticles in the presence of poloxamers can be reproduced in the absence of said nanoparticles. Thus, it was found that there was a difference in the handling properties of the poloxamers tested when tested without nanoparticles. From the results generated, the limit defining the processing time during the material test was 75 minutes. Materials with processing times exceeding 75 minutes are considered unsuitable for formulation LiNP. Poloxamer P188 featuring shorter treatment times was given a better suitability score.
In summary, it was found that when TFF was run with poloxamer dissolved in the aqueous phase, the quality of poloxamer could be determined. Of the 5 drug-grade poloxamer samples tested, one contained drug-grade poloxamer that resulted in a delay or blockage of filtration, which resulted in an increase in the reflectivity of the sample.
Example 3 preparation of surfactant solution and lipid mixture
Preparation of aqueous surfactant solutions
A total of 11 aqueous solutions were prepared by dissolving 4g of 11 different poloxamer P188 (1% w/v) and 584.4mg NaCl (25 mM) in 400mL purified deionized water in a glass vial.
Preparation of lipid mixtures
Lipid mixtures representing lipid nanoparticle formulations were prepared with ionizable lipids (dl_05 (R), scheme 1), helper lipids DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, avanti Polar Lipids) and cholesterol (Avanti Polar Lipids) and PEG lipid DMG-PEG2k (1, 2-dimyristoyl-sn-glycero-methoxy (polyethylene glycol) -2000,Avanti Polar lipids) in a molar ratio of 8.00/5.29/4.41/0.88, respectively. These were resuspended together in 15mg/mL chloroform and vortexed for 1 minute to achieve complete dissolution.
Scheme 1 chemical structure of dL_05 (R)
The 15mg/mL stock was then transferred to a 10mL glass vial and stored in a-20℃refrigerator. The lipids were then further diluted to 1mg/mL in chloroform before use on langmuir tanks.
Example 4 Langmuir trough analysis of poloxamer P188
Exemplary protocol for Langmuir surface pressure/area isotherm measurements of aqueous solutions of test surfactants
1. An aqueous solution of the surfactant to be tested was prepared at a concentration of 1% w/v, hereinafter referred to as "aqueous test solution",
2. A lipid mixture solution having a concentration of 1mg/mL, hereinafter referred to as "lipid mixture" was prepared in chloroform,
3. According to manufacturer's recommendations, ensure that langmuir trough (e.g., model MicrotroughX from Kibron Inc or model 112D from Nima Technologies) is ready for use.
4. Calibration control run:
a) Deionized water was added to the tank at the manufacturer's recommended volume on the cleaned langmuir tank, to properly contact the tank sensor,
B) A glass syringe is used to add sufficient lipid mixture to the deionized water surface to form a monolayer (e.g., 30 μl),
C) The samples were allowed to equilibrate at 22.1 + 0.2C for 5 minutes,
D) The tank barrier was compressed starting from the maximum allowed area at a speed of 20 cm 2 a/min until the first phase change was observed on the surface-pressure diagram. Compression is stopped when the first phase transition has passed, and the area reached when the first phase transition starts, hereinafter referred to as the "minimum target area",
E) The slot is inflated back to the maximum area allowed.
4. Test sample:
a) On the cleaned langmuir tank, an aqueous test solution was added to the tank in the volume recommended by the manufacturer, so that it properly contacted the tank sensor,
B) The samples were allowed to equilibrate at 22.1 + 0.2C for 5 minutes,
C) Starting from the maximum area allowed, compressing the tank barrier at a speed of 20 cm 2/min, until the "minimum target area" is reached,
D) The slot is inflated back to the maximum area allowed,
E) After waiting for a period of three seconds between compression-expansion cycles, steps 4. C) through d) are repeated, if necessary, to obtain a plurality of surface-pressure hysteresis cycles (e.g., three),
F) The tank was cleaned as recommended by the manufacturer.
Langmuir trough hardware
Langmuir-Brookfield tank model 112D manufactured by Nima Technologies (UK) was used in all experiments of example 4. Before use, the well ventilated area was fitted with a polyethylene glove and the tank was thoroughly cleaned with a Kimwipe cube wipe (model 7105, kimtech 75512 or EX-L WIPES 34256) impregnated with chloroform. The chloroform was removed and the tank was rinsed several times with purified deionized water.
Langmuir trough arrangement
Isotherm cycle set up:
compression/expansion speed 20cm/min
Total number of cycles of 3 to 4 times
Maximum area 79 cm 2
Minimum area of 20cm 2
Waiting 3 seconds when fully open,
During all experiments, the cells were covered and the temperature was maintained at 22.1±0.2 ℃.
Langmuir trough experiment
About 80mL of the poloxamer P188 aqueous solution was added to the langmuir trough and allowed to stand for five minutes to reach equilibrium. The compression-expansion isotherm was run starting from a maximum area of 79cm 2 and decreasing the compression rate of 20cm/min to a minimum area of 20cm 2. The tank is then inflated at the same speed to again reach maximum area. After waiting three seconds, the compression-expansion cycle is repeated. This procedure was performed three to four times in total for each poloxamer aqueous solution. After each set of experiments, the tank was rinsed with five times the tank volume of deionized water.
Results
An isotherm plot of the aqueous surfactant solution is shown in fig. 4, with the legend in the plot indicating which poloxamer P188 is present in the aqueous subphase filling the langmuir trough and the heading indicating which compression-expansion cycle is shown.
Surprisingly, it was found that compression-expansion isotherms can be used to test the applicability of poloxamer P188 in lipid nanoparticle purification and processing. Particularly significant results were obtained in the third compression-expansion cycle. It was further found that unsuitable poloxamers can be characterized by isotherms reaching a maximum surface pressure above 4 mN/m.
Figure 3 shows a summary of isotherms for representative stabilized and unstable poloxamers.
EXAMPLE 5 Langmuir trough analysis of poloxamer P188 and lipid mixtures
Exemplary Langmuir surface pressure/area isotherm measurement protocol for aqueous solutions of test surfactants with lipid blend layers added to the surface
1. An aqueous solution of the surfactant to be tested was prepared at a concentration of 1% w/v, hereinafter referred to as "aqueous test solution",
2. A lipid mixture solution having a concentration of 1mg/mL, hereinafter referred to as "lipid mixture" was prepared in chloroform,
3. According to manufacturer's recommendations, it is ensured that Langmuir trough (e.g., model MicrotroughX from Kibron Inc or model 112D from Nima Technologies) is ready for use.
4. Calibration control run:
a) Deionized water was added to the tank at the manufacturer's recommended volume on the cleaned langmuir tank, to properly contact the tank sensor,
B) A glass syringe is used to add sufficient lipid mixture to the deionized water surface to form a monolayer (e.g., 30 μl),
C) The samples were allowed to equilibrate at 22.1 + 0.2C for 5 minutes,
D) The tank barrier was compressed at a rate of 20 cm 2 a/min starting from the maximum area allowed until the first phase change was observed on the surface-pressure diagram. Compression is stopped when the first phase transition has passed, and the area reached when the first phase transition starts, hereinafter referred to as the "minimum target area",
E) The tank is inflated to the maximum area allowed.
5. Test sample:
a) On the cleaned langmuir tank, an aqueous test solution was added to the tank in the volume recommended by the manufacturer, so that it properly contacted the tank sensor,
B) A glass syringe is used to add sufficient lipid mixture to the deionized water surface to form a monolayer (e.g., 30 μl),
C) The samples were allowed to equilibrate at 22.1 + 0.2C for 5 minutes,
D) The tank barrier is compressed at a rate of 20 cm 2/min, starting from the maximum area allowed, until the "minimum target area" is reached,
E) The tank is inflated to the maximum area allowed,
F) After waiting three seconds between compression-expansion cycles, steps 5.D-e are repeated, if necessary, to obtain multiple surface-pressure hysteresis cycles (e.g., three),
G) The tank was cleaned as recommended by the manufacturer.
Langmuir trough hardware
As described in example 4, the langmuir-blodgett cell manufactured by Nima Technologies (uk) in form 112D was used in all experiments of example 5.
Langmuir trough arrangement
As described in example 4, the following langmuir trough settings were used:
compression/expansion speed 20cm/min
Total number of cycles of 3 to 4 times
Maximum area 79 cm 2
Minimum area 20 cm 2
Wait 3 seconds when fully open.
During all experiments, the cells were covered and the temperature was maintained at 22.1±0.2 ℃.
Langmuir trough experiment
About 80mL of the poloxamer P188 aqueous solution was added to the langmuir trough and allowed to stand for five minutes to reach equilibrium. A control compression-expansion isotherm of the poloxamer-only aqueous solution was recorded. After control runs, 4. Mu.L of lipid mixture (1 mg/mL) was added using a 10. Mu.L glass syringe. The sample was allowed to stand for five minutes starting at a maximum area of 79 cm 2 and a compression rate of 20 cm/min and then reduced to a minimum area of 20 cm 2 before starting the compression-expansion cycle. The tank is then inflated at the same rate, again to a maximum area. After waiting three seconds, the compression-expansion cycle is repeated. This procedure was performed three to four times in total for each poloxamer aqueous solution and lipid mixture. After each set of experiments, the tank was rinsed with five times the tank volume of deionized water.
Results
An isotherm plot of the aqueous surfactant solution is shown in fig. 6, with the legend indicating which stabilizer is present in the aqueous subphase filling the langmuir trough, and the use of lipid mixtures in the experiment. The heading indicates which compression-expansion cycle is displayed. Figure 5 shows a summary of the isotherms of representative stabilized and unstable poloxamers.
Surprisingly, it was found that analysis of langmuir isotherms of lipid mixtures in the poloxamer Sha Mshui sub-phase could be used to test the suitability of poloxamer P188 in lipid nanoparticle purification and processing.
To define a threshold between suitable and unsuitable poloxamers P188, a parameter Δpi of the isotherm is defined, which is calculated at any area point within the three compression-expansion cycles.
For each cycle, the hysteresis Δpi is calculated asWherein:
Pi comp is the surface pressure of the compression stage,
Pi exp is the surface pressure of the expansion phase,
Pi max is the maximum surface pressure reached in the isotherm cycle
The calculation is performed for a match of pi comp and pi exp in area. Δpi is calculated for any area point of the langmuir trough experiment and is plotted in fig. 7. If up to three isotherm cycles are considered in the case of any Δpi higher than 0.6, poloxamer P188 is identified which is unsuitable for lipid nanoparticle purification and processing. As shown in fig. 7, during the first isotherm cycle, a consistently lower Δpi value is observed.
Example 6-Langmuir trough analysis of poloxamer P188 with lipid mixtures in LT hardware with larger test area.
Langmuir trough hardware
To demonstrate the reproducibility of the process of the present invention, apparatus 2, langmuir-Brookfield tank model MicrotroughX from Kibron Inc, was used in all experiments of example 6. Before use, the tank was thoroughly cleaned with ethanol. The ethanol was removed and the tank was rinsed several times with purified deionized water.
Langmuir trough arrangement
The following langmuir trough settings were used:
Compression/expansion speed 34 mm/min
Total number of cycles 3 times
Maximum area 224 cm 2
Minimum area 110 cm 2
Wait 3 seconds when fully open.
During all experiments, the cells were covered and the temperature was maintained at 22.1±0.2 ℃.
Langmuir trough experiment
About 250mL of poloxamer P188 aqueous solution (sample S3) was added to the langmuir trough and allowed to stand for five minutes to reach equilibrium. mu.L of the lipid mixture (1 mg/mL) was added using a 50. Mu.L glass syringe. The sample was allowed to stand for five minutes to reach equilibrium and then the compression-expansion cycle was started starting at maximum area 224 cm 2 and dropping at a compression rate of 34 m/min to a minimum area of 110cm 2. The tank is then inflated at the same rate, again to a maximum area. After waiting three seconds, the compression-expansion cycle is repeated. This procedure was performed three times in total for each poloxamer aqueous solution and lipid mixture. After each set of experiments, the tank was rinsed with five times the tank volume of deionized water.
TFF processing
The same tangential flow filtration process as described in example 2 was also used herein.
Results
Isothermal diagrams of the aqueous surfactant solutions are shown in fig. 8 and 9, with the legends indicating which stabilizer is present in the aqueous subphase filling the langmuir trough, and the use of lipid mixtures in the experiments. The heading indicates which compression-expansion cycle is displayed.
The langmuir isotherm of the lipid mixture on the poloxamer aqueous subphase was found to be reproducible between device 1 (112D,Nima Technologies) and device 2 (MicrotroughX, kibron Inc) (fig. 8). This demonstrates the properties of suitable poloxamer P188, showing a thin hysteresis and Δpi <0.6. The suitability of poloxamer sample S3 and test sample S23 was further confirmed by reprocessing the same sample via TFF (fig. 10).
Considering a new batch of poloxamer P188 and the methods described herein, the langmuir isotherm of the lipid mixture in the poloxamer Sha Mshui subphase (fig. 9) suggests that the new batch is suitable for purification and treatment of lipid nanoparticles. These results were further confirmed by TFF treatment of new poloxamer P188 batches in aqueous solution (fig. 10), which supports the surprising suitability of the test method of the present invention for distinguishing and determining the suitability of poloxamer P188 in TFF purification. The method of the present invention provides a method that requires significantly less time, less test material and less consumables than testing surfactants in a practical production environment. Multiple samples can be tested quickly and economically using the method of the present invention to effectively determine the suitability of a surfactant as a stabilizer.
Example 7-Langmuir trough analysis and TFF treatment of poloxamer P124.
The analysis was extended to different poloxamers and this example examined the performance of the method of the invention on poloxamer P124.
Langmuir trough hardware, setup and experimental procedure
The same langmuir cell hardware, setup, experimental setup and procedure as described in example 5 was used.
TFF processing
The same experimental setup and procedure as described in example 2 was followed.
Results
Fig. 11 shows an isotherm plot of an aqueous surfactant solution comprising P124 with a lipid mixture added to the surface in three compression-expansion cycles. The thin hysteresis (Δpi < 0.6) observed in these samples suggests that the excipient is suitable for purification and processing of lipid nanoparticles. These results are further confirmed by TFF treatment of the poloxamer P124 in aqueous solution shown in fig. 12 as a permeation/time graph. The P124 sample showed a linear relationship between permeation and time, confirming that poloxamer P124 is suitable for TFF purification, particularly for this sample. The results show that the method of the present invention requires significantly less time, less test material and less consumables than testing surfactants in a practical production environment.

Claims (67)

1. A Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each lipid nanoparticle or lipid nanoparticle comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanently cationic lipid, and wherein the formulation further comprises a surfactant characterized by having a langmuir surface pressure/area isotherm at a minimum surface area determined for the lipid mixture or lipid mixture comprised by the nanoparticle of equal to or less than 4.0 mN/m.
2. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation according to claim 1, wherein pi max is equal to or lower than 3.5 mN/m, more preferably equal to or lower than 3.5 and equal to or higher than 0.5 mN/m, even more preferably equal to or lower than 3.0 mN/m and equal to or higher than 0.5 mN/m, even more preferably equal to or lower than 2.5 and equal to or higher than 1.0 mN/m, most preferably equal to or lower than 2.0 and equal to or higher than 1.0 mN/m.
3. A Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation comprising a plurality of Lipid Nanoparticles (LNP) or lipid nanoparticles (LiNP), each nanoparticle or lipid nanoparticle comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises as a component thereof at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanently cationic lipid, wherein the formulation comprises a surfactant as a stabilizer,
And wherein a representative sample bearing on its surface a lipid mixture or lipid mixture comprised by LNP or LiNP comprising said surfactant is characterized by having a langmuir isotherm Δpi equal to or lower than 0.60, preferably equal to or lower than 0.45, at each area point during a langmuir surface pressure/area isotherm cycle comprising a compression phase and an expansion phase and recorded between a maximum surface area and a minimum surface area determined for the lipid mixture or lipid mixture, wherein Δpi is calculated at any area point as:
,
Wherein the method comprises the steps of Is the surface pressure at the area point during the compression phase of the isotherm cycle,
Wherein the method comprises the steps ofIs the surface pressure at the area point during the expansion phase of the isotherm cycle, and
Wherein the method comprises the steps ofIs the maximum surface pressure reached in the isotherm cycle.
4. A Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation according to any one of claims 1 to 3, wherein the surfactant is a nonionic surfactant, preferably at least one nonionic surfactant selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, fatty acid esters of sorbitol, ethoxylated fatty acid esters of sorbitol, fatty acid esters of glycerin, ethoxylated castor oil and ethoxylated vitamin E.
5. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation of claim 4, wherein the surfactant is a block copolymer of ethylene oxide and propylene oxide.
6. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation of any one of claims 1 to 5, wherein the lipid nanoparticle or lipid nanoparticle comprises the lipid mixture or lipid mixture and a therapeutic agent.
7. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation according to claim 6, wherein the therapeutic agent comprises a nucleic acid, preferably mRNA.
8. Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation according to any one of claims 1 to 7, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of ionizable lipids, ionizable lipids and permanently cationic lipids as a component thereof, and further comprises one or more of the following components (c 1) to (c 6), preferably further comprises components (c 1), (c 2) and (c 3):
(c1) A non-ionizable lipid having a sterol structure;
(c2) A phospholipid;
(c3) PEG conjugated lipids;
(c4) Polysarcosine conjugated lipids
(C5) PAS lipid;
(c6) Ionizable or cationic polymers.
9. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation of claim 8, wherein the lipid mixture or lipid mixture comprises:
i) 30 to 65 mol% of at least one selected from the group consisting of the ionizable lipid, and the permanently cationic lipid,
And further comprises one or more of the following components (c 1) to (c 6):
ii) 10 to 50 mol% of a lipid (c 1) having a sterol structure,
Iii) 4 to 50 mol% of a phospholipid (c 2),
Iv) 0.5 to 10 mol% of one or any combination of PEG conjugated lipid (c 3), poly sarcosine conjugated lipid (c 4) and PAS conjugated lipid (c 5),
V) from 0.5 to 10 mol% of a cationic polymer (c 6),
Such that the sum of the amounts of i) and ii) to v) is 100 mol%,
And more preferably further comprises components (c 1), (c 2) and (c 3) such that the sum of the amounts of i) and ii) to iv) is 100%.
10. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation according to any one of claims 1 to 9, which is a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles, each lipid nanoparticle comprising a lipid mixture, wherein the lipid mixture comprises an ionizable lipid of formula (L-1):
(L-1)
wherein:
a is 1 or 2 and b is an integer from 1 to 4, or a is an integer from 1 to 4 and b is 1 or 2,
P is either 1 or 2 and the number of the groups,
M is 1 or 2;n is 0 or 1, and m+n≥2, and
R 1A to R 6A are independently of one another selected from the group consisting of hydrogen 、-CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A、-CH2R7A、–C(NH)-NH2、 poly (ethylene glycol) chain and acceptor ligands, wherein R 7A is selected from the group consisting of C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond;
With the proviso that at least two residues from R 1A to R 6A are selected from -CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A and-CH 2R7A, wherein R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond,
Or a protonated form of an ionizable lipid of formula (L-1), wherein one or more of the nitrogen atoms comprised in the compound of formula (L-1) are protonated to provide a positively charged compound.
11. The Lipid Nanoparticle (LNP) formulation or lipid nanoparticle (LiNP) formulation according to any one of claims 1 to 10, which is a suspension formulation, and wherein the carrier solution of the suspension formulation is an aqueous carrier solution comprising the surfactant.
12. A surfactant for use in a pharmaceutical composition, said surfactant characterized by having a langmuir surface pressure/area isotherm having a maximum surface pressure (pi max) at a minimum surface area of equal to or lower than 4.0 mN/m, said minimum surface area being determined for said pharmaceutical composition.
13. The surfactant for use according to claim 12, wherein the maximum surface pressure is equal to or lower than 3.5 mN/m, more preferably equal to or lower than 3.5 and equal to or higher than 0.5 mN/m, even more preferably equal to or lower than 3.0 mN/m and equal to or higher than 0.5 mN/m, even more preferably equal to or lower than 2.5 and equal to or higher than 1.0 mN/m, most preferably equal to or lower than 2.0 and equal to or higher than 1.0 mN/m.
14. The surfactant for use according to claim 12 or 13, which is a nonionic surfactant, preferably at least one nonionic surfactant selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, fatty acid esters of sorbitol, ethoxylated fatty acid esters of sorbitol, fatty acid esters of glycerin, ethoxylated castor oil and ethoxylated vitamin E.
15. A surfactant for use according to claim 14, which is a block copolymer of ethylene oxide and propylene oxide, more preferably.
16. The surfactant for use according to any one of claims 12 to 15, wherein the pharmaceutical composition is in the form of a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising a plurality of LNPs or LiNP, each comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, an ionizable lipid and a permanent cationic lipid as a component thereof, and wherein the carrier solution of the suspension formulation is an aqueous carrier solution comprising the surfactant.
17. A method for classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition, the method comprising:
(a) Providing an aqueous solution of surfactant at a concentration (C),
(B) Recording the langmuir surface pressure/area isotherm of the surfactant in said solution to determine a maximum surface pressure pi max of the langmuir isotherm at a predetermined minimum surface area;
(c) The maximum surface pressure pi max is compared with a threshold value, wherein if the maximum surface pressure pi max is equal to or lower than the threshold value, the surfactant is classified as suitable for use as a stabilizer, and if the maximum surface pressure pi max is greater than the threshold value, the surfactant is classified as unsuitable for use as a stabilizer.
18. A method for classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition comprising a lipid or a lipid-like substance, optionally during purification of the composition, preferably during tangential flow filtration of the mixture, wherein the method comprises the steps of:
(a) Providing an aqueous solution of surfactant at a concentration (C) of surfactant in the solution;
(b) Recording langmuir pressure/area isotherm cycles comprising a compression phase and an expansion phase between a maximum surface area and a minimum surface area on a sample comprising an aqueous solution of said surfactant and bearing on its surface a lipid or lipid comprised in said composition:
(c) The langmuir isotherm Δpi for each area point of the langmuir pressure/area isotherm cycle is calculated, wherein Δpi is calculated as follows:
,
Wherein the method comprises the steps of Is the surface pressure at the area point during the compression phase of the isotherm cycle,
Wherein the method comprises the steps ofIs the surface pressure at the area point during the expansion phase of the isotherm cycle, and
Wherein the method comprises the steps ofIs the maximum surface pressure reached in the isotherm cycle, and
(D) Comparing the calculated langmuir isotherm Δpi with a threshold, wherein if the calculated langmuir isotherm Δpi at each isotherm area point is equal to or below the threshold, the surfactant is classified as suitable for use as a stabilizer, and if the calculated langmuir Miao Er pi at any area point is greater than the threshold, the surfactant is classified as unsuitable for use as a stabilizer.
19. A method of preparing a pharmaceutical composition, the method comprising classifying a surfactant as suitable or unsuitable for use as a stabilizer for a pharmaceutical composition according to the method of claim 17 or 18, and incorporating the surfactant into a pharmaceutical composition if the surfactant is classified as suitable for use as a stabilizer for a pharmaceutical composition.
20. The method according to any one of claims 17 to 19, wherein the surfactant is a nonionic surfactant, preferably at least one nonionic surfactant selected from the group consisting of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, fatty acid esters of sorbitol, ethoxylated fatty acid esters of sorbitol, fatty acid esters of glycerol, ethoxylated castor oil and ethoxylated vitamin E.
21. The method of claim 20, wherein the surfactant is a block copolymer of ethylene oxide and propylene oxide, more preferably a poloxamer, even more preferably a poloxamer selected from poloxamer 124, poloxamer 188, poloxamer 338 and poloxamer 407, or a combination thereof, most preferably P188.
22. The method according to any one of claims 17 to 21, wherein the pharmaceutical composition comprises a therapeutic agent comprising a nucleic acid, such as RNA, preferably mRNA.
23. The method of any one of claims 17 to 22, wherein the pharmaceutical composition is a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation comprising a plurality of LNPs or LiNP, each comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of an ionizable lipid, and a permanent cationic lipid as a component thereof.
24. The method of claim 23, wherein the pharmaceutical composition is a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation, and wherein the carrier solution of the suspension formulation is an aqueous carrier solution comprising the surfactant.
25. A method of reducing or avoiding clogging or scaling of a filtration system during purification of a pharmaceutical composition in the form of a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, the method comprising adding a stabilizing surfactant to a first LNP or LiNP formulation to form a second LNP or LiNP formulation, optionally prior to purification, wherein the LNP or LiNP formulation comprises a plurality of LNPs or LiNP, each comprising a lipid mixture or lipid mixture and a therapeutic agent, wherein the lipid mixture or lipid mixture comprises at least one selected from an ionizable lipid, and a permanently cationic lipid as a component thereof, and wherein the stabilizing surfactant is a surfactant according to any one of claims 12 to 16, or is classified as a surfactant suitable as a stabilizer by the method of any one of claims 17 to 27.
26. The method of claim 25, wherein the purifying comprises tangential flow filtration.
27. The method of claim 25 or 26, wherein the pharmaceutical composition is a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising an aqueous carrier solution, and wherein the stabilizing surfactant is added to the carrier solution, optionally wherein the surfactant is substantially absent from the LNP or LiNP.
28. The method according to any one of claims 25 to 27, wherein the therapeutic agent is a nucleic acid, such as RNA, more preferably mRNA.
29. A method of mitigating aggregation of a lipid or lipid nanoparticle in a Lipid Nanoparticle (LNP) formulation or a lipid nanoparticle (LiNP) formulation, the method comprising adding a stabilizing surfactant to a first LNP or LiNP formulation to form a second LNP or LiNP formulation, optionally prior to purification, wherein the LNP or LiNP formulation comprises a plurality of LNPs or LiNP, each comprising a lipid mixture or lipid mixture, wherein the lipid mixture or lipid mixture comprises at least one selected from an ionizable lipid, and a permanently cationic lipid as a component thereof, and wherein the stabilizing surfactant is a surfactant according to any one of claims 12 to 16, or is classified as suitable as a stabilizer by the method of any one of claims 17 to 27.
30. The method of claim 29, wherein the formulation is a Lipid Nanoparticle (LNP) suspension formulation or a lipid nanoparticle (LiNP) suspension formulation comprising an aqueous carrier solution, and wherein the stabilizing surfactant is added to the carrier solution, optionally wherein the surfactant is substantially absent from the LNP or LiNP.
31. The method of claim 29 or 30, wherein the lipid nanoparticle or lipid nanoparticle comprises a therapeutic agent, preferably a nucleic acid, such as RNA, more preferably mRNA.
32. The method of claim 31, wherein the lipid nanoparticle or lipid nanoparticle comprises a nucleic acid, such as RNA, and preferably mRNA, and wherein the method comprises the steps of:
i) First, combining the nucleic acid with at least one selected from the group consisting of an ionizable lipid, and a permanently cationic lipid to form an LNP or LiNP,
Ii) second, purifying the LNP or LiNP,
Iii) Thirdly, adding the stabilizing surfactant to the exchange buffer before and during TFF purification, maintaining the surfactant at a stable concentration,
Iv) optionally wherein the stabilizing surfactant is added to the LNP or LiNP formulation after step (i).
33. The method according to claim 31 or 32, wherein the method comprises the steps of:
i) The LNP or LiNP preparation is produced by mixing at least one selected from the group consisting of a permanent cationic lipid, an ionizable lipid, and an ionizable lipid dissolved in an organic phase with a therapeutic agent dissolved in an aqueous solution,
Ii) diluting the LNP or LiNP preparation with a first solution,
Iii) Concentrating the LNP or LiNP preparation by buffer exchange using ultrafiltration/diafiltration via TFF, wherein the second solution is used for ultrafiltration/diafiltration,
Iv) obtaining a suspension of LNP or LiNP in an aqueous carrier solution,
Wherein the first solution comprises between about 0.01% w/v and 10% stabilizing surfactant, preferably between about 0.01% w/v surfactant and 5% w/v surfactant, more preferably between about 0.01% w/v surfactant and 2.5% w/v surfactant, more preferably between about 0.05% w/v and 1.5% w/v surfactant, even more preferably between about 0.05% w/v and 1.5% w/v surfactant, most preferably about 1% w/v surfactant, and/or
Wherein the second solution comprises between about 0.01% w/v and about 10% stabilizing surfactant, preferably between about 0.01% w/v surfactant and about 5% w/v surfactant, more preferably between about 0.01% w/v surfactant and about 2.5% w/v surfactant, even more preferably between about 0.05% w/v surfactant and 1.5% w/v surfactant, most preferably about 1% w/v;
And wherein the final concentration of stabilizing surfactant from the combined first and second solutions is between 0.01% and 10% surfactant, preferably between 0.01% w/v surfactant and 5% w/v surfactant, more preferably between 0.01% w/v surfactant and 2.5% w/v surfactant, even more preferably between 0.05% w/v and 1.5% w/v surfactant, most preferably about 1% w/v surfactant, relative to the total volume of the suspension of nanoparticles in the aqueous carrier solution.
34. The method according to claim 33, wherein:
a) The stabilizing surfactant is not incorporated into the suspension before or during step i),
B) Adding the stabilizing surfactant to the first and second solutions, and/or
C) About half of the stabilizing surfactant is added to the first solution and about half of the surfactant is added to the second solution.
35. The method according to any one of claims 23 to 34, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of ionizable lipids, ionizable lipids and permanently cationic lipids as a component thereof, and further comprises one or more of the following components (c 1) to (c 6):
(c1) A non-ionizable lipid having a sterol structure;
(c2) A phospholipid;
(c3) PEG conjugated lipids;
(c4) Polysarcosine conjugated lipids
(C5) PAS lipid;
(c6) Ionizable or cationic polymers.
36. The method of claim 35, wherein the lipid mixture or lipid mixture comprises at least one selected from the group consisting of ionizable lipids, and permanently cationic lipids as a component thereof, and further comprises components (c 1), (c 2), and (c 3).
37. The method of claim 35, wherein the lipid mixture or lipid mixture comprises:
i) 30 to 65 mol% of at least one selected from the group consisting of the ionizable lipid, and the permanently cationic lipid,
And further comprises one or more of the following components (c 1) to (c 6):
ii) 10 to 50 mol% of a lipid (c 1) having a sterol structure,
Iii) 4 to 50 mol% of a phospholipid (c 2),
Iv) 0.5 to 10 mol% of one or any combination of PEG conjugated lipid (c 3), poly sarcosine conjugated lipid (c 4) and PAS conjugated lipid (c 5),
V) from 0.5 to 10 mol% of a cationic polymer (c 6),
Such that the sum of the amounts of i) and ii) to v) is 100 mol%.
38. The method of claim 37, wherein the lipid mixture or lipid mixture comprises:
i) 30 to 65 mol% of at least one selected from the group consisting of ionizable lipids, and permanently cationic lipids, and further comprising:
ii) 10 to 50 mol% of a lipid (c 1) having a sterol structure,
Iii) 4 to 50 mol% of a phospholipid (c 2),
Iv) 0.5 to 10 mol% of PEG conjugated lipid (c 3),
Such that the sum of the amounts of i) and ii) to iv) is 100%.
39. The method of any one of claim 23 to 38, wherein the formulation is a lipid nanoparticle formulation comprising a plurality of lipid nanoparticles, each lipid nanoparticle comprising a lipid mixture,
Wherein the lipid mixture comprises an ionizable lipid of formula (L-1):
(L-1)
wherein:
a is 1 or 2 and b is an integer from 1 to 4, or a is an integer from 1 to 4 and b is 1 or 2,
P is either 1 or 2 and the number of the groups,
M is 1 or 2;n is 0 or 1, and m+n is not less than 2, and
R 1A to R 6A are independently of one another selected from the group consisting of hydrogen ;-CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A、-CH2R7A、–C(NH)-NH2、 poly (ethylene glycol) chain and acceptor ligands, wherein R 7A is selected from the group consisting of C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond;
With the proviso that at least two residues from R 1A to R 6A are selected from -CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A and-CH 2R7A, wherein R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond,
Or a protonated form of an ionizable lipid of formula (L-1), wherein one or more of the nitrogen atoms comprised in the compound of formula (L-1) are protonated to provide a positively charged compound.
40. A lipid nanoparticle formulation or lipid nanoparticle formulation, preferably a lipid nanoparticle suspension formulation or lipid nanoparticle suspension formulation, obtained by the method of any one of claims 23 to 39.
41. Lipid nanoparticle formulation or lipid nanoparticle formulation, preferably lipid nanoparticle suspension formulation or lipid nanoparticle suspension formulation according to any one of claims 1 to 11 or 40, for use as a medicament.
42. Lipid nanoparticle formulation or lipid nanoparticle formulation, preferably lipid nanoparticle suspension formulation or lipid nanoparticle suspension formulation, according to any one of claims 1 to 11 or 40, for use in the treatment or prophylaxis of a disease, preferably a disease selected from table a, more preferably a disease selected from viral disease, ciliated disease, autoimmune disease and respiratory disease, even more preferably a disease selected from pulmonary disease, airway disease or nasal disease, more preferably a viral disease of the lung, ciliated disease of the lung and autoimmune disease of the lung.
43. The lipid nanoparticle formulation or lipid nanoparticle formulation of claim 43, wherein the pulmonary disease or pulmonary viral disease is at least one selected from the group consisting of pneumonia and asthma, the airway disease is at least one selected from the group consisting of bronchitis, virus-induced asthma, pulmonary fibrosis and COPD, and/or the nasal disease is at least one selected from the group consisting of rhinitis and sinusitis.
44. The lipid nanoparticle formulation or lipid nanoparticle formulation of claim 41 for use in vaccination or immunization.
45. A method of avoiding or reducing side effects in treatment with LNP or LiNP carrying at least one therapeutic agent, wherein the method comprises the steps of:
i) Determining whether LNP or LiNP in a pharmaceutical composition comprising LNP or LiNP is aggregated when subjected to mechanical or temperature stress by determining its aggregation level before and after subjecting the pharmaceutical composition to the mechanical or temperature stress,
Ii) if LNP or LiNP shows aggregation after the test of step (i), adding surfactant to the LNP or LiNP formulation to obtain an LNP or LiNP suspension having a final surfactant concentration of 0.01% w/v up to 10% w/v, preferably between 0.05% w/w surfactant and 5% surfactant, more preferably between 0.33% surfactant and 2.5% surfactant, more preferably between 0.45% and 1.5% surfactant, most preferably between 0.5% and 1.5% surfactant, most preferably about 1% w/v surfactant,
Iii) Reconstitution was performed with mixing to produce a stable LNP or LiNP suspension.
46. The method of claim 45, wherein the surfactant is a surfactant according to any one of claims 12 to 16, or is classified as suitable as a stabilizer by the method of any one of claims 17 to 24.
47. Use of a surfactant according to any one of claims 12 to 16 or a surfactant classified as suitable as a stabilizer by the method of any one of claims 17 to 24 for stabilizing a lipid nanoparticle or suspension of lipid nanoparticles in an aqueous carrier solution under physical stress conditions, preferably shear stress, more preferably under shear stress during purification such as TFF to prevent aggregation of particles, wherein the lipid nanoparticle or lipid nanoparticle comprises the following components (a) and (b):
(a) Therapeutic agent, and
(B) At least one selected from the group consisting of ionizable lipids, and permanently cationic lipids.
48. The use of a surfactant according to claim 47, wherein the physical stress condition is selected from shaking, stirring, vibrating, mixing, tumbling, tapping or dropping of a suspension, or a combination thereof, or wherein the physical stress condition is caused by pumping the suspension or drawing it into a syringe.
49. The use of a surfactant according to claim 47 or 48, wherein the surfactant is incorporated as an excipient in the aqueous carrier solution.
50. The use of a surfactant of any one of claims 47-49, wherein the nanoparticle formulation is not lyophilized.
51. The use of a surfactant according to any one of claims 47 to 50, wherein the surfactant is added prior to the lyophilization process.
52. The use of a surfactant according to any one of claims 47 to 51, wherein the presence of the surfactant does not cause a change in the biological activity of the nanoparticle.
53. The use of a surfactant according to any one of claims 47 to 52, wherein the presence of the surfactant does not cause a change in the hydrodynamic diameter as nanoparticles and the physical properties measured as a proportion of the therapeutic agent included in the nanoparticles.
54. The use of a surfactant according to any one of claims 47 to 53, wherein the lipid nanoparticle or suspension of lipid nanoparticles in an aqueous carrier solution comprises the surfactant in a concentration of 0.01 to 10% (w/v).
55. The surfactant for use according to any one of claims 47 to 54, wherein the therapeutic agent is a nucleic acid.
56. The use of a surfactant according to claim 55, wherein the nucleic acid is mRNA.
57. The use of a surfactant of any one of claims 47-56, wherein the concentration of nucleic acid in the suspension formulation is in the range of 0.01 to 10mg/mL based on the total volume of the suspension formulation.
58. The use of a surfactant according to any one of claims 47 to 57, wherein the Z-average diameter of the nanoparticles, as determined by dynamic light scattering, is in the range of 10 to 500 nm, preferably about 30 to 100 nm.
59. The use of a surfactant of any one of claims 47 to 58, wherein the nanoparticle further comprises one or more of the following components (c 1) to (c 6):
(c1) A non-ionizable lipid having a sterol structure;
(c2) A phospholipid lipid;
(c3) PEG conjugated lipids;
(c4) Polysarcosine conjugated lipids
(C5) PAS lipid;
(c6) An ionizable or cationic polymer or lipid.
60. The use of a surfactant of any one of claims 47-59, wherein the nanoparticle comprises:
a) 30 to 65 mol% of at least one (b) selected from the group consisting of ionizable lipids, and permanently cationic lipids, and one or more of the following components:
ii) 10 to 50 mol% of a lipid (c 1) having a sterol structure,
Iii) 4 to 50 mol% of a phospholipid (c 2),
Iv) 0.5 to 10 mol% of one or any combination of PEG conjugated lipid (c 3), poly sarcosine conjugated lipid (c 4) and PAS conjugated lipid (c 5),
From 0.5 to 10 mol% of a cationic polymer (c 6),
So that the sum of (b) and (c 1) to (c 6) is 100 mol%.
61. The use of a surfactant of any one of claims 47-60, wherein the nanoparticle comprises an ionizable lipid (b) of the following formula (a-III):
a-III
or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:
One of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, and the other of L 1 or L 2 is –O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NR a C (=o) O-, or a direct bond;
Each of G 1 and G 2 is independently C 1-C12 alkylene or C 1-C12 alkenylene;
G 3 is C 1-C24 alkylene, C 1-C24 alkenylene, C 3-C8 cycloalkylene, C 3-C8 cycloalkenyl, wherein each of alkylene, alkenylene, cycloalkylene, and cycloalkenyl is optionally substituted;
R a is H or C 1-C12 alkyl, wherein alkyl is optionally substituted;
R 1 and R 2 are each independently C 6-C24 alkyl or C 6-C24 alkenyl, wherein each of the alkyl and alkenyl groups is optionally substituted;
R 3 is H, OR 5、CN、-C(=O)OR4、-OC(=O)R4 or-NR 5C(=O)R4;R4 is C 1-C12 alkyl, wherein alkyl is optionally substituted;
R 5 is H or C 1-C6 alkyl, where the alkyl is optionally substituted, and
X is 0, 1 or 2.
62. The use of a surfactant according to any one of claims 47 to 61, wherein the nanoparticle comprises an ionizable lipid (b) of formula (L-1),
(L-1)
Wherein:
a is 1 or 2 and b is an integer from 1 to 4, or a is an integer from 1 to 4 and b is 1 or 2,
P is either 1 or 2 and the number of the groups,
M is 1 or 2;n is 0 or 1, and m+n is not less than 2, and
R 1A to R 6A are each independently selected from the group consisting of hydrogen ;-CH2CH(OH)R7A,-CH(R7A)CH2OH,-CH2CH2(C=O)OR7A,-CH2CH2(C=O)NHR7A,-CH2R7A,–C(NH)-NH2, poly (ethylene glycol) chain, and acceptor ligands, wherein R 7A is selected from the group consisting of C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond,
With the proviso that at least two residues from R 1A to R 6A are selected from -CH2CH(OH)R7A、-CH(R7A)CH2OH、-CH2CH2(C=O)OR7A、-CH2CH2(C=O)NHR7A and-CH 2R7A, wherein R 7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond,
Or a protonated form of an ionizable lipid of formula (L-1), wherein one or more of the nitrogen atoms comprised in the compound of formula (L-1) are protonated to provide a positively charged compound.
63. The use of a surfactant of any one of claims 47-60, wherein the nanoparticle comprises (6 z,9z,28z,31 z) -seventeen carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate or a protonated form thereof wherein the nitrogen atom of the compound is protonated as ionizable lipid (b).
64. Use of a surfactant according to any one of claims 47 to 60, wherein the nanoparticle comprises ((4-hydroxybutyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) or a protonated form thereof wherein the nitrogen atom of the compound is protonated and/or (heptadec-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate or a protonated form thereof wherein the nitrogen atom of the compound is protonated as ionizable lipid (b).
65. The use of claim 64, wherein the nanoparticle comprises:
((4-hydroxybutyl) azadiyl) bis (hexane-6, 1-dialkyl) bis (2-hexyldecanoate) or a protonated form thereof in which the nitrogen atom of the compound is protonated, and optionally further comprising one or more of the following components (d 1) to (d 8):
(d1) 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide (ALC-0159),
(D2) 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC),
(D3) The cholesterol level of the blood is determined by the concentration of cholesterol,
(D4) The potassium chloride is added to the mixture,
(D5) The potassium dihydrogen phosphate is used for preparing the nano-crystalline silicon dioxide,
(D6) The sodium chloride is used for preparing the sodium chloride,
(D7) Disodium phosphate dihydrate is used as a base for the production of the aqueous solution,
(D8) Sucrose.
66. The use according to claim 64, wherein the nanoparticle comprises heptadec-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate or a protonated form thereof wherein the nitrogen atom of the compound is protonated, and further optionally one or more of the following components (e 1) to (e 7):
(e1) 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC),
(E2) The cholesterol level of the blood is determined by the concentration of cholesterol,
(E3) 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (PEG 2000 DMG),
(E4) 2-amino-2- (hydroxymethyl) propane-1, 3-diol (tromethamine) hydrochloride,
(E5) The sodium acetate trihydrate and the sodium acetate trihydrate,
(E6) Acetic acid is used as a solvent for the acetic acid,
(E7) Sucrose.
67. The use of claim 64, wherein the nanoparticle comprises DLin-MC3-DMA ((6 z,9z,28z,31 z) -seventeen carbon-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butyrate) or a protonated form thereof in which the nitrogen atom of the compound is protonated, and optionally further comprising one or more of the following components (e 1) to (e 7):
(e1) 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC),
(E2) The cholesterol level of the blood is determined by the concentration of cholesterol,
(E3) PEG2000-C-DMG (. Alpha. - (3' - { [1, 2-bis (myristoyloxy) propoxy ] carbonylamino } propyl) - ω -methoxy, polyoxyethylene),
(E4) 2-amino-2- (hydroxymethyl) propane-1, 3-diol (tromethamine) hydrochloride,
(E5) Disodium hydrogen phosphate heptahydrate is used for preparing the sodium phosphate,
(E6) The anhydrous potassium dihydrogen phosphate is used for preparing the water-free potassium dihydrogen phosphate,
(E7) Sodium chloride.
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