HK40099485A - Use of plinabulin in combination with immune checkpoint inhibitors - Google Patents

Description

Uses of Punabulin in combination with immune checkpoint inhibitors

This application is a divisional application of the invention application filed on February 11, 2016, with application number 201680015268.X and invention title "Use of Punabulin in combination with immune checkpoint inhibitors".

This application claims the benefits of U.S. Provisional Application No. 62/115,468, filed February 12, 2015, and U.S. Provisional Application No. 62/255,259, filed November 13, 2015, the disclosures of which are incorporated herein by reference in their entirety.

Background Technology

field

This invention relates to the fields of chemistry and medicine. More specifically, this invention relates to plinabulin, compositions containing plinabulin, and their therapeutic uses.

Description of related technologies

Human cancers exhibit numerous genetic and epigenetic alterations, generating neoantigens that can potentially be recognized by the immune system (Sjoblom et al., 2006). The adaptive immune system, composed of T lymphocytes and B lymphocytes, possesses potent anti-cancer potential, exhibiting a broad capacity and precise specificity to respond to a wide range of tumor antigens.

Recent research in cancer immunotherapy has focused heavily on enhancing anti-tumor immunity through adoptive transfer of activated effector cells, targeting relevant antigens, providing non-specific immunostimulants such as cytokines, or suppressing anti-cancer effector cells. Efforts to develop specific immune checkpoint inhibitors have begun to offer novel immunotherapies for cancer treatment, including the development of ipilimumab, an antibody that binds to and inhibits cytotoxic T-lymphocyte antigen-4 (CTLA-1), for the treatment of patients with advanced melanoma (Hodi et al., 2010). While cancer remains incurable for most patients, there is a particular need to develop effective therapeutic agents for cancer immunotherapy.

Invention Overview

Some implementations involve pharmaceutical compositions that contain punabulin and one or more immune checkpoint inhibitors.

Some implementation schemes involve methods for treating cancer, which include administering punabrine together with one or more immune checkpoint inhibitors to the recipient.

Attached Figure Description

Figure 1A shows the expression of DC maturation markers CD40, CD80, CD86 and MHCII in dendritic cells treated with different concentrations of purnabulin and LPS; Figure 1B shows the viability of dendritic cells treated with purnabulin and LPS.

Figure 2A shows the expression of CD40 markers in dendritic cells treated with punabulin, paclitaxel, etoposide, or a control; Figure 2B shows the expression of CD80 markers in dendritic cells treated with punabulin, paclitaxel, etoposide, or a control; Figure 2C shows the expression of CD86 markers in dendritic cells treated with punabulin, paclitaxel, etoposide, or a control; Figure 2D shows the expression of MHCII markers in dendritic cells treated with punabulin, paclitaxel, etoposide, or a control.

Figure 3A shows the production of IL-1β in dendritic cells treated with purnabulin, paclitaxel, etoposide, and control; Figure 3B shows the production of IL-6 markers in dendritic cells treated with purnabulin, paclitaxel, etoposide, and control; Figure 3C shows the production of IL-12p40 in dendritic cells treated with purnabulin, paclitaxel, etoposide, and control.

Figures 4A-4C show the increased antitumor activity of plin-induced PD-1 antibody plus CTLA-4 antibody in the MC-38 tumor model of immune-active mice. Figure 4A shows the effect on tumor growth; Figure 4B shows the effect on the average tumor weight at necropsy; Figure 4C shows the time it takes for the tumor to reach 10 times its initial volume.

Figures 5A-5C show the results of fluorescence activated cell sorting (FACS) analysis of tumors during autopsy in the study described in Example 6. Figure 5A shows the effect on Treg cells; Figure 5B shows the ratio of CD8+ cells to Treg cells; and Figure 5C shows the effect on macrophages.

Detailed description of the preferred implementation scheme

Punabulin, namely (3Z,6Z)-3-benzylmethyl-6-{[5-(2-methyl-2-propyl)-1H-imidazol-4-yl]methylene}-2,5-piperazinedione, is a synthetic analog of the natural compound Phenylahistin. Punabulin can be readily prepared according to the methods and steps described in detail in U.S. Patents 7,064,201 and 7,919,497 (which are incorporated herein by reference in their entirety). In some embodiments, punabulin effectively promotes antigen uptake and dendritic cell migration to lymph nodes, where tumor-specific antigens are presented by dendritic cells to initial immune effector cells. Exposure of dendritic cells to punabulin induces dendritic cell maturation and significantly increases their ability to stimulate T cells. In some embodiments, punabulin can mediate tumor size reduction by immunomodulating the tumor microenvironment to enhance antitumor immune response. In some embodiments, significant therapeutic synergy can be achieved when punabulin is combined with immune checkpoint inhibitors.

Some implementations involve the use of punabulin in combination with one or more immune checkpoint inhibitors, such as inhibitors of CTLA4 (cytotoxic T-lymphocyte antigen-4), PD-1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), PD-L3 (programmed cell death ligand 3), PD-L4 (programmed cell death ligand 4), LAG-3 (lymphocyte activation gene-3), and TIM-3 (T-cell immunoglobulin and mucin-3). In some implementations, the immune checkpoint inhibitor is a ligand that binds to PD-1. In some implementations, the immune checkpoint inhibitor is a ligand that binds to CTLA-4.

PD-1 is an important immune checkpoint receptor expressed by activated T cells and B cells and mediates immunosuppression. PD-1 is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isotypes, and species homologs of hPD-1, as well as analogs that share at least one common epitope with hPD-1.

Various cell surface glycoprotein ligands targeting PD-1 have been identified, including PD-L1, PD-L2, PD-L3, and PD-L4, expressed on antigen-presenting cells and in many human cancers. These cell surface glycoprotein ligands have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isotypes, and species homologs of hPD-L1, and analogs sharing at least one common epitope with hPD-L1. As used herein, the term "PD-L2" includes human PD-L2 (hPD-L2), variants, isotypes, and species homologs of hPD-L2, and analogs sharing at least one common epitope with hPD-L2. As used herein, the term "PD-L3" includes human PD-L3 (hPD-L3), variants, isotypes, and species homologs of hPD-L3, and analogs sharing at least one common epitope with hPD-L3. As used herein, the term “PD-L4” includes human PD-L4 (hPD-L4), variants, isotypes and species homologs of hPD-L4, and analogs that share at least one common epitope with hPD-L4.

CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) is a protein receptor that functions as an immune checkpoint, downregulating the immune system. CTLA-4 is present on the surface of T cells and is a member of the immunoglobulin (Ig) superfamily; CTLA-4 contains a single extracellular Ig domain. CTLA-4 transcripts have been found in populations of T cells with cytotoxic activity, suggesting that CTLA-4 may play a role in cell lysis.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All patents, applications, published applications, and other publications are incorporated herein by reference in their entirety. If a term has multiple definitions herein, the definition in this section shall prevail unless otherwise stated.

The terms “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents, etc. The use of such media and reagents in pharmaceutical active substances is well known in the art. Their use in therapeutic compositions is contemplated herein, except where any conventional media or reagent is incompatible with the active ingredient. Furthermore, various adjuvants commonly used in the art may be included, for example. Considerations for including various components in pharmaceutical compositions are described below, e.g., Gilman et al. (Eds.) (1990); Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety. The pharmaceutically acceptable excipient may be a monosaccharide or a monosaccharide derivative.

As used in this article, “object” means human or non-human mammal, such as dogs, cats, mice, rats, cattle, sheep, pigs, goats, non-human primates or birds such as chickens, as well as any other vertebrates or invertebrates.

The term "mammal" is used in its usual biological sense. Therefore, it specifically includes, but is not limited to: primates (including apes (chimpanzees, apes, monkeys) and humans), cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, etc.

As used herein, "effective dose" or "therapeutic effective dose" refers to the amount of a therapeutic agent that effectively relieves or reduces the likelihood of one or more symptoms of a disease or condition to a certain extent, and may include curing the disease or condition.

As used herein, “treatment” refers to the administration of a compound or pharmaceutical composition to a subject for preventive and/or therapeutic purposes. The term “preventive treatment” refers to treating a subject who is not yet showing symptoms of a disease or condition but is susceptible to or otherwise has a specific risk of developing such a disease or condition, thereby reducing the likelihood that the patient will develop the disease or condition in the future. The term “therapeutic treatment” refers to treating a subject who already has a disease or condition.

As used herein, the term "chemotherapeutic agent" refers to an agent that can reduce, prevent, mitigate, limit, and/or delay the growth of metastatic tumors or tumors, or directly kill tumor cells through tumor necrosis or apoptosis or any other mechanism, or can otherwise be used in pharmaceutically effective amounts to reduce, prevent, mitigate, limit, and/or delay the growth of metastatic tumors or tumors in a subject of cancerous disease. Chemotherapeutic agents include, but are not limited to, for example, fluoropyrimidines; pyrimidine nucleosides; purine nucleosides; antifolate agents; platinum-based agents; anthracyclines/anthracyclines; epipodophyllotoxin; camptothecin; hormones; hormone complexes; anti-hormones; enzymes, proteins, peptides, and polyclonal and/or monoclonal antibodies; vinca alkaloids; taxanes; epothilone; antimicrotubule agents; alkylating agents; antimetabolites; topoisomerase inhibitors; antiviral agents; and various other cytotoxic agents and cell growth inhibitors.

Dosage and pharmaceutical composition

Some implementations involve pharmaceutical compositions that contain punabulin and one or more immune checkpoint inhibitors.

In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, PD-L2, PD-L3, PD-L4, CTLA-4, LAG3, B7-H3, B7-H4, KIR, or TIM3. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a ligand that binds to PD-L1. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-L2 inhibitor or a combination of PD-L1/PD-L2 inhibitors. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor.

In some embodiments, the compositions described herein comprise a first immune checkpoint inhibitor and a second immune checkpoint inhibitor, wherein the first immune checkpoint inhibitor is different from the second immune checkpoint inhibitor. In some embodiments, the first and second immune checkpoint inhibitors are independently inhibitors of PD-1, PD-L1, PD-L2, PD-L3, PD-L4, CTLA-4, LAG3, B7-H3, B7-H4, KIR, or TIM3. In some embodiments, the first immune checkpoint inhibitor is a PD-1 inhibitor, and the second immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the first immune checkpoint inhibitor is a PD-L1 inhibitor, and the second immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the first immune checkpoint inhibitor is a PD-L2 inhibitor, and the second immune checkpoint inhibitor is a CTLA-4 inhibitor.

In some embodiments, the immune checkpoint inhibitor may be a small peptide agent capable of inhibiting T cell regulation. In some embodiments, the immune checkpoint inhibitor may be a small molecule (e.g., less than 500 Daltons) capable of inhibiting T cell regulation. In some embodiments, the immune checkpoint inhibitor may be a molecule providing co-stimulation for T cell activation. In some embodiments, the immune checkpoint inhibitor may be a molecule providing co-stimulation for natural killer cell activation. In some embodiments, the immune checkpoint inhibitor may be an antibody. In some embodiments, the immune checkpoint inhibitor is a PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L1 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L2 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L3 antibody. In some embodiments, the immune checkpoint inhibitor is a PD-L4 antibody. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antibody. In some embodiments, the immune checkpoint inhibitor is an antibody of CTLA-4, LAG3, B7-H3, B7-H4, KIR, or TIM3.

The antibodies may be selected from: α-CD3-APC, α-CD3-APC-H7, α-CD4-ECD, α-CD4-PB, α-CD8-PE-Cy7, α-CD-8-PerCP-Cy5.5, α-CD11c-APC, α-CD11b-PE-Cy7, α-CD11b-AF700, α-CD14-FITC, α-CD16-PB, α-CD19-AF780, α-CD19-AF700, α-CD20-PO, α-CD25-PE-Cy7, α-CD40-APC, α-CD45-Biotin, Streptavidin-BV605, α-CD62L-ECD, α-CD69 -APC-Cy7, α-CD80-FITC, α-CD83-Biotin, Streptavidin-PE-Cy7, α-CD86-PE-Cy7, α-CD86-PE, α-CD123-PE, α-CD154-PE, α-CD161-PE, α-CTLA4-PE-Cy7, α-FoxP3-AF488 (clone 259D), IgG1-isotype-AF488, α-ICOS(CD278)-PE, α-HLA-A2-PE, α-HLA-DR-PB, α-HLA-DR-PerCPCy5.5, α-PD1-APC, VISTA, co-stimulatory molecules OX40 and CD137.

Various antibodies (Abs) can be used in the compositions described herein, including antibodies that bind to PD-1, PD-L1, PD-L2, PD-L3, or PD-L4 with high affinity. Human mAbs (HuMAbs) that bind specifically to PD-1 with high affinity (e.g., binding to human PD-1 and potentially cross-reactive with PD-1 from other species such as cynomolgus monkeys) are disclosed in U.S. Patent No. 8,008,449, which is incorporated herein by reference in its entirety. HuMAbs that bind specifically to PD-L1 with high affinity are disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference in its entirety. Other anti-PD-1 mAbs have been described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, and 8,168,757, and PCT Publication No. WO 2012/145493, all of which are incorporated herein by reference in their entirety. Anti-PD-Ll mAbs have been described, for example, in U.S. Patent Nos. 7,635,757 and 8,217,149, U.S. Publication No. 2009/0317368, and PCT Publications Nos. WO 2011/066389 and WO2012/14549, all of which are incorporated herein by reference in their entirety.

In some embodiments, anti-PD-1HuMAb may be selected from 17D8, 2D3, 4H1, 5C4 (also referred to herein as nivolumab), 4A11, 7D3, and 5F4, all of which are described in U.S. Patent No. 8,008,449. In some embodiments, anti-PD-1HuMAb may be selected from 3G10, 12A4 (also referred to herein as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, all of which are described in U.S. Patent No. 7,943,743.

In some embodiments, the composition may further comprise one or more pharmaceutically acceptable diluents. In some embodiments, the pharmaceutically acceptable diluent may include Kolliphor (polyethylene glycol (15)-hydroxystearic acid). In some embodiments, the pharmaceutically acceptable diluent may include propylene glycol. In some embodiments, the pharmaceutically acceptable diluent may include Kolliphor and propylene glycol. In some embodiments, the pharmaceutically acceptable diluent may include Kolliphor and propylene glycol, wherein, based on the total weight of the diluent, Kolliphor comprises about 40% by weight and propylene glycol comprises about 60% by weight. In some embodiments, the composition may further comprise one or more other pharmaceutically acceptable excipients.

Standard pharmaceutical formulation techniques can be used to prepare the pharmaceutical compositions described herein, such as those disclosed in Remington's *The Science and Practice of Pharmacy*, 21st Ed., Lippincott Williams & Wilkins (2005), which are incorporated herein by reference in their entirety. Therefore, some embodiments include pharmaceutical compositions comprising: (a) a safe and therapeutically effective amount of punabulin or a pharmaceutically acceptable salt thereof, (b) an immune checkpoint inhibitor, and (c) a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

Other embodiments include co-administering punabulin and one or more immune checkpoint inhibitors in separate compositions. Thus, some embodiments include a first pharmaceutical composition comprising: (a) a safe and therapeutically effective amount of punabulin or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof; and a second pharmaceutical composition comprising: (a) one or more immune checkpoint inhibitors, and (b) a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

The pharmaceutical compositions described herein can be administered via any acceptable mode of administration suitable for similar purposes, including but not limited to: oral, sublingual, oral, subcutaneous, intravenous, intranasal, topical, percutaneous, intradermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, or intraocular administration. Oral and parenteral administration are commonly used for the therapeutic indications (which are the objects of preferred embodiments).

The terms “pharmaceutically acceptable carrier” or “pharmaceuticalally acceptable excipient” include any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents, etc. The use of such media and reagents in pharmaceutical active substances is well known in the art. Except where any conventional media or reagents are incompatible with the active ingredient, their use in therapeutic compositions is contemplated herein. Furthermore, various adjuvants commonly used in the art may be included, for example. Considerations for including various components in pharmaceutical compositions are described below, e.g., Gilman et al. (Eds.) (1990); Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety.

Some examples of substances that can serve as pharmaceutically acceptable carriers or components of other substances include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth gum; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as TWEENS; wetting agents such as sodium dodecyl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline and phosphate buffer solutions.

The compositions described herein are preferably provided in unit dosage forms. As used herein, a “unit dosage form” is a composition according to good medical practice that contains compounds or compositions in amounts suitable for administration to an animal, preferably a mammalian, at a single dose. However, a single or unit dosage form does not imply that the dosage form is administered once daily or once per course of treatment. Such a dosage form is intended to be administered once, twice, three or more times daily, and over an infusion period (e.g., about 30 minutes to about 2-6 hours), or as a continuous infusion, and may be administered more than once during treatment, although single administration is not specifically excluded. Those skilled in the art will recognize that the dosage form is not specifically intended for the entire course of treatment, and these decisions are left to those skilled in the art of treatment rather than the art of formulation.

The useful compositions described above can be any of a variety of suitable forms for use in a variety of routes of administration, such as oral, sublingual, oral, nasal, rectal, topical (including transdermal and intradermal), ocular, intracranial, intrathecal, intrathecal, intra-arterial, intravenous, intramuscular, or other parenteral administration routes. Those skilled in the art will understand that oral and nasal compositions include compositions prepared by inhalation and using available methods. Depending on the specific route of administration desired, a variety of pharmaceutically acceptable carriers well known in the art can be used. Pharmaceutically acceptable carriers include, for example, solid or liquid fillers, diluents, water-soluble agents, surfactants, and encapsulating substances. Optional pharmaceutically active substances may be included that do not materially interfere with the inhibitory activity of the compound or composition. The amount of carrier used in conjunction with the compound or composition is sufficient to provide the actual amount of material for administration per unit dose of the compound. Techniques and compositions for preparing dosage forms that can be used in the methods described herein are described in the following references (all of which are incorporated herein by reference): Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

Various oral dosage forms can be used, including solid forms such as tablets, capsules (e.g., solid gel capsules and liquid gel capsules), granules, and bulk powders. Tablets can be compressed, ground, enteric-coated, sugar-coated, film-coated, or repeatedly compressed, containing suitable binders, lubricants, diluents, disintegrants, colorants, flavorings, flow inducers, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent particles, and effervescent formulations reconstituted from effervescent particles, containing suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, colorants, and flavorings.

Pharmaceutically acceptable carriers suitable for preparing unit dosage forms for oral administration are well known in the art. Tablets typically contain conventional pharmaceutically compatible adjuvants such as calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose as inert diluents; binders such as starch, gelatin, and sucrose; disintegrants such as starch, alginate, and croscarmellose; and lubricants such as magnesium stearate, stearic acid, and talc. Flow aids such as silica can be used to improve the flow properties of powder mixtures. For appearance, colorants such as FD&C dyes can be added. Sweeteners and flavorings, such as aspartame, saccharin, menthol, peppermint, and fruit flavorings, are useful adjuvants for chewable tablets. Capsules typically contain one or more of the solid diluents disclosed above. The selection of carrier components depends on secondary considerations such as taste, cost, and storage stability, which are not critical and can be readily performed by those skilled in the art.

Oral compositions may also comprise liquid solutions, emulsions, suspensions, etc. Pharmaceutically acceptable carriers suitable for preparing such compositions are well known in the art. Typical components of carriers used in syrups, elixirs, emulsions, and suspensions include ethanol, glycerin, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol, and water. For suspensions, typical suspending agents include methylcellulose, sodium carboxymethylcellulose, AVICEL RC-591, tragali gum, and sodium alginate; typical wetting agents include lecithin and polysorbate 80; typical preservatives include methylparaben and sodium benzoate. Oral liquid compositions may also contain one or more components such as sweeteners, flavoring agents, and coloring agents disclosed above.

This composition can also be coated using conventional methods, typically pH- or time-dependent coatings, to release the subject composition in the gastrointestinal tract near the desired topical application, or at different times to prolong the desired effect. Such dosage forms typically include, but are not limited to, one or more of the following: cellulose acetate, polyvinyl acetate, hydroxypropyl methylcellulose phthalate, ethyl cellulose, Eudragit coating, waxes, and shellac.

The compositions described herein may optionally include other pharmaceutically active substances.

Other compositions for achieving systemic delivery of the subject compound include sublingual, oral, and nasal dosage forms. Such compositions typically comprise one or more soluble filler substances such as sucrose, sorbitol, and mannitol; and binders such as gum arabic, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methyl cellulose. They may also include the flow aids, lubricants, sweeteners, colorants, antioxidants, and flavoring agents disclosed above.

Liquid compositions are formulated (for topical ophthalmic application) so that they can be applied topically to the eye. Comfort should be maximized as much as possible, but sometimes, due to formulation considerations (e.g., drug stability), optimal comfort may not be achievable. Where maximizing comfort cannot be achieved, the composition should be formulated as a liquid so that the liquid is tolerable for patients applying it topically. Furthermore, ophthalmologically acceptable liquids should be packaged for single use or contain preservatives to prevent contamination after repeated use.

For ophthalmic applications, solutions or drugs are typically prepared using physiological saline solution as the primary medium. It is preferable to use an appropriate buffering system to maintain a comfortable pH for the ophthalmic solution. The formulation may also contain conventional, pharmaceutically acceptable preservatives, stabilizers, and surfactants.

Preservatives that can be used in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, PHMB, chlorobutanol, thimerosal, phenylmercuric acetate, and phenylmercuric nitrate. Useful surfactants include, for example, Tween 80. Similarly, a variety of useful media can be used in the ophthalmic preparations disclosed herein. These media include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamer, carboxymethyl cellulose, hydroxyethyl cellulose, and purified water.

Tension modulators may be added as needed or for convenience. These include, but are not limited to, salts (especially sodium chloride and potassium chloride), mannitol and glycerol, or any other suitable ophthalmologically acceptable tension modulators.

A variety of buffers and methods can be used to adjust the pH, provided the resulting formulation is ophthalmologically acceptable. For many compositions, the pH ranges from 4 to 9. Therefore, buffers include acetate buffers, citrate buffers, phosphate buffers, and borate buffers. The pH of these formulations can be adjusted with acids or bases as needed.

Ophthalmologically acceptable antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene.

Other excipients that may be included in ophthalmic preparations are chelating agents. Disodium edetate is a useful chelating agent, but other chelating agents can be used in place of it or in combination with it.

For topical applications, creams, ointments, gels, solutions, or suspensions comprising the compositions disclosed herein may be used. Topical formulations may typically consist of a drug carrier, a cosolvent, an emulsifier, a penetration enhancer, a preservative system, and an emulsifier.

For intravenous administration, the compositions described herein may be dissolved or dispersed in a pharmaceutically acceptable diluent (e.g., saline or glucose solution). Suitable excipients may also be included to achieve a desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HCl, and citric acid. In many embodiments, the pH of the final composition is 2 to 8, or preferably 4 to 7. Antioxidant excipients may include sodium bisulfite, sodium acetone-sodium bisulfite, sodium formaldehyde sulfoxylate, thiourea, and EDTA. Other non-limiting examples of suitable excipients that may be found in the final intravenous composition may include sodium or potassium phosphate, citric acid, tartaric acid, gelatin, and carbohydrates such as glucose, mannitol, and dextran. Other acceptable excipients are described in: Powell et al., Compendium of Excipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998, 52 238-311 and Nema et al., Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions, PDA J Pharm Sci and Tech 2011, 65 287-332, both of which are incorporated herein by reference in their entirety. Antimicrobial agents may also be included to obtain solutions that inhibit bacteria or fungi, including but not limited to phenylmercuric nitrate, thimerosal, benzyl chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.

The composition for intravenous administration may be provided to caregivers in one or more solid forms and may be rehydrated immediately prior to administration with a suitable diluent such as sterile water, saline, or glucose solution. In other embodiments, the composition is provided as a prepared solution for parenteral administration. In other embodiments, the composition is provided as a solution that requires further dilution prior to administration. In embodiments that include administration of the compound described herein in combination with other reagents, the combination is provided to caregivers as a mixture, or the caregivers mix the two reagents prior to administration, or the two reagents may be administered separately.

The actual dosage of the active compound described herein depends on the specific compound and the condition to be treated; the selection of an appropriate dosage is well known to those skilled in the art. In some embodiments, the daily dose of pannabulin may be from about 0.25 mg/kg body weight to about 120 mg/kg body weight or more, from about 0.5 mg/kg body weight or less to about 70 mg/kg body weight, from about 1.0 mg/kg body weight to about 50 mg/kg body weight, or from about 1.5 mg/kg body weight to about 10 mg/kg body weight. Thus, for a person weighing 70 kg, the dosage range may be: from about 17 mg/day to about 8000 mg/day, from about 35 mg/day or less to about 7000 mg/day or more, from about 70 mg/day to about 6000 mg/day, from about 100 mg/day to about 5000 mg/day, or from about 200 mg to about 3000 mg/day.

In some embodiments, the compositions described herein may be used in combination with other therapeutic agents. In some embodiments, the compositions described herein may be administered or applied in combination with treatments such as chemotherapy, radiotherapy, and biological therapy.

Treatment

Some embodiments relate to methods of treating cancer by applying the pharmaceutical composition described herein to a subject in need. Some embodiments relate to methods of treating cancer that include co-administering pannabulin with one or more immune checkpoint inhibitors to a subject in need. In some embodiments, the subject may be an animal, such as a mammal or a human. In some embodiments, the subject is a human.

Some implementation methods involve providing co-stimulation of cancer-targeting T-cell activation by co-administering prazolam and one or more immune checkpoint inhibitors. Other implementation methods involve providing co-stimulation of cancer-targeting natural killer cells by co-administering prazolam and one or more immune checkpoint inhibitors.

In some embodiments, the cancer comprises cancer cells expressing a ligand that binds to PD-1. In some embodiments, the ligand that binds to PD-1 is PD-L1. In some embodiments, the ligand that binds to PD-1 is PD-L2.

In some embodiments, the methods for treating cancer described herein further include identifying cancer cells expressing a ligand that binds to PD-1. In some embodiments, the methods for treating cancer described herein further include identifying cancer cells expressing PD-L1. In some embodiments, the methods for treating cancer described herein further include identifying cancer cells expressing PD-L2. In some embodiments, the methods for treating cancer described herein further include identifying cancer cells expressing PD-L3 or PD-L4.

In some implementations, identifying cancer cells expressing ligands that bind to PD-1 includes using analytical assays to detect the presence of the binding ligands. Examples of suitable analytical assays include, but are not limited to, the PD-L1IHC22C3pharmDx kit and the PD-L1IHC 28-8pharmDx kit from Dako Pharmaceuticals.

In some embodiments, the cancer comprises cancer cells expressing a ligand that binds to CTLA-4. In some embodiments, the ligand that binds to CTLA-4 is B7.1 or B7.2.

In some embodiments, the methods for treating cancer described herein further include identifying cancer cells expressing a ligand that binds to CTLA-4. In some embodiments, the methods for treating cancer described herein further include identifying cancer cells expressing B7.1 or B7.2.

In some embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, ipilimumab, dacarbazine, BMS 936559, atezolizumab, durvalimumab, or any combination of the above inhibitors.

In some embodiments, the cancer is head and neck cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer, prostate cancer, breast cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, glioblastoma, myeloma, lymphoma, or leukemia. In some embodiments, the cancer is renal cell carcinoma, malignant melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, Hodgkin's lymphoma, or squamous cell carcinoma. In some embodiments, the cancer is selected from: breast cancer, colon cancer, rectal cancer, lung cancer, prostate cancer, melanoma, leukemia, ovarian cancer, stomach cancer, renal cell carcinoma, liver cancer, pancreatic cancer, lymphoma, and myeloma. In some embodiments, the cancer is a solid tumor or a hematologic malignancy.

In some implementations, the cancer does not have any cells expressing PD-1, PD-L1, or PD-L2 at detectable levels.

In some embodiments, the cancer is selected from: breast cancer, colon cancer, rectal cancer, lung cancer, prostate cancer, melanoma, leukemia, ovarian cancer, stomach cancer, renal cell carcinoma, liver cancer, pancreatic cancer, lymphoma, and myeloma. In some embodiments, the cancer is a solid tumor or a hematologic malignancy.

Some implementation methods involve methods for inducing dendritic cell maturation in cancer patients, which include administering a composition containing punabulin to the cancer patient.

Some implementation methods involve disrupting the cancer-associated tumor vascular system of a subject, including co-administering a mixture of punabulin and one or more immune checkpoint inhibitors to the subject.

Many cancers are associated with the formation of tumor vascular systems. In some implementation schemes, the cancers are selected from: melanoma, pancreatic cancer, colorectal adenocarcinoma, brain tumors, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hormone-refractory metastatic prostate cancer, metastatic breast cancer, non-small cell lung cancer, renal cell carcinoma, head and neck cancer, prostate cancer, colon cancer, and anaplastic thyroid cancer.

Some embodiments include co-administering the compositions and/or pharmaceutical compositions described herein with other drugs. For example, as described above, some embodiments include co-administering punabulin with one or more immune checkpoint inhibitors. "Co-administration" means that two or more agents are administered in such a manner that the administration of one or more agents affects the efficacy and/or safety of one or more other agents, regardless of when or how they are actually administered. In one embodiment, the agents are administered simultaneously. In one such embodiment, combined administration is achieved by combining the agents in one dosage form. In another embodiment, the agents are administered sequentially. In one embodiment, the agents are administered via the same route, such as oral or intravenous. In another embodiment, the agents are administered via different routes, such as one agent administered orally and another agent administered intravenously. In some implementations, the time interval between the application of one or more reagents and the application of one or more reagents applied together can be: approximately 1 hour, 2 hours, 3 hours, 5 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 28 days, or 30 days.

In some implementations, the treatment cycle may include co-administration of pramabulin and one or more immune checkpoint inhibitors, in combination with pramabulin alone or one or more immune checkpoint inhibitors alone. In some implementations, pramabulin and one or more immune checkpoint inhibitors are co-administered on day 1, followed by pramabulin alone after 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, or 3 weeks, and then co-administered pramabulin and one or more immune checkpoint inhibitors again after 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, or 3 weeks. In some implementations, pramabulin and one or more immune checkpoint inhibitors are co-administered on day 1, followed by pramabulin or one or more immune checkpoint inhibitors alone on a selected day from day 2 to day 31, and then co-administered pramabulin and one or more immune checkpoint inhibitors on a selected day from day 3 to day 31. In some embodiments, pranabuline and one or more immune checkpoint inhibitors are co-administered on day 1, followed by pranabuline alone on day 8, and then pranabuline and one or more immune checkpoint inhibitors co-administered on day 15. In some embodiments, the treatment cycle may be repeated two or more times.

Examples of other drugs include other chemotherapeutic agents.

In some embodiments, the chemotherapeutic agents may be selected from: abiraterone acetate, methotrexate, abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (berentuzumab-vitoline), ADE, Ado-trastuzumab-emetansine, doxorubicin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Ak ynzeo (netupirant and palonosetron hydrochloride), Aldara (imiquimod), adeleukin, Alecensa (alectinib), alectinib, alenzab, pemetrexed disodium, palonosetron hydrochloride, chlorambucil (chlorambucil), aminolevulinic acid, anastrozole, aprepitant, Aredia (pamir phosphate disodium), anastrozole (anastrozole), and exemestane (exemestane). Arranon (nerapine), arsenic trioxide, Arzerra (orfamumab), Erwinia chrysogenum asparaginase, Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, Becenum (carmustine), Beleodaq (belistat), belistat, bendamustine hydrochloride, BEP, bevacizumab, besalodine, Bexxar (tosimomumab and iodine-131 tosimomumab), bicalutamide, BiCN U (carmustine), bleomycin, bonnetumab, Blincyto (bonnetumab), bortezomib, Bosulif (bosutinib), bosutinib, belentoumab-vitoltin, busulfan, cabazitaxel, cabozantinib-S-malate, CAF, Camppath (alemumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Carac (fluorouracil - topical), carboplatin, carboplatin-paclitaxel, carfilzomib Carmubris (carmustine), Carmustine, Carmustine implants, Casodex (bicalutamide), CeeNU (lomustine), Ceritinib, Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), Cetuximab, Leukonazole, Leukonazole-prednisone, CHOP, Cisplatin, Clafen (cyclophosphamide), Clofarex (clofarex), Clolar (clofarex), CMF, Cobitinib, Cometriq (cazozidinib-S-malate), COPDAC, COPP, COPP-ABV, Cosmegen (daunorubicin), Cotellic (cobitinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), Cytarabine, Cytarabine liposomes, Cytosar-U (cytarabine) Cytosine, Cytoxan (cyclophosphamide), Darafenib, Dacarbazine, Dacogen (decitabine), Dermasomycin, Darazalex (daratumumab), Dasatinib, Dornoximycin Hydrochloride, Decitabine, Degarelix, Dernexin-Diphtheria Toxin Conjugate, Dernoxim, DepoCyt (cytarabine liposome), Dexamethasone, Dexamethasone Hydrochloride, Detoximumab (Din Doxulimab, Docetaxel, Doxil (doxorubicin hydrochloride liposome), Doxorubicin hydrochloride, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), Efudex (fluorouracil - topical), Elitek (raburicase), Ellence (epirarubicin hydrochloride), Erotozumab, Eloxatin (oxaliplatin), Eltrombopag ethanolamine, Emen d (Aprepitant), Empliciti (Erotocilizumab), Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vimidege), Erlotinib Hydrochloride, Erwinaze (Erwinia chrysanthemum asparaginase), Etopophos (Etoposide phosphate), Etoposide, Etoposide phosphate, Evacet (Doxorubicin Hydrochloride Liposome) Everolimus, Evista (raloxifene hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil for topical use), Fareston (toremifene), Farydak (pabistat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoro plex (fluorouracil - topical), fluorouracil injection, fluorouracil - topical, flutamide, Foles (methotrexate), Foles PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pratroxa), FU-LV, fulvestrant, Gardasil (recombinant quadrivalent HPV vaccine) Gardasil 9 (recombinant HPV 9-valent vaccine), Gazyva (atuzumab), Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gemcitabine-Ozomicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel Round Tablet (Carmustine implant), Carboxypeptidase, Goserelin acetate, Halaven (Eribulin mesylate), Herceptin (Trastuzumab), Bivalent HPV vaccine, Recombinant HPV nine-valent vaccine, Recombinant HPV quadrivalent vaccine, Recombinant Hycamtin (Topotecan hydrochloride), Hyper-CVAD, Ibrance (Pabocenib), Tiuxetan (Ilimumab), Ibrutinib, ICE, Iclusig (Panatinib hydrochloride), Idamycin (Idarubicin hydrochloride), Adelalipid, Ifex (Ifosfamide), Ifosfamide, IL-2 (Imatinib mesylate), Imbruvica (Ibrutinib), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Interferon α-2b, Recombinant Interleukin-2 (Imatinib), In Tron A (recombinant interferon α-2b), iodine-131 tosimomumab and tosimomumab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposomes, Istodax (romidesin), ixaprilone, ixazomib citrate, Ixempra (ixaprilone), Jakafi (ruxotinib phosphate), Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab Emtansi) ne), Keoxifene (raloxifene hydrochloride), Kepivance (palivmin), Keytruda (pembrolizumab), Kyprolis (carfilzomib), lanreotide acetate, lapatinib dimethylbenzenesulfonate, lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (leucovorin), leuprolide acetate, Levulan (aminolevulinic acid), Linfoliz In (LipoDox), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluorouridine and Telbipyrimidine Hydrochloride), Lupron (Leupron Acetate), Lupron Depot (Leupron Acetate), Lupron-Ped (Leupron Acetate), Lupron-3 Months (Leupron Acetate), Lupron-4 Months (Leupron Acetate), Lynparza (Olaparib), Marqibo (vincristine sulfate liposome), Matulane (methylbenzylhydrazine hydrochloride), nitrogen mustard hydrochloride, Megace (medroxyprogesterone acetate), medroxyprogesterone acetate, Mekinist (trametinib), mercaptopurine, mesna, Mesnex (mesna), Methazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), Mexate-AQ ( Methotrexate, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plesafox), Mustargen (Nitrogen Mustard Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Germuzumab-Ozomicin), Paclitaxel Nanoparticles (Paclitaxel Albumin-Stable Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Nexituzumab, Nerapine, Neosar (Cyclophosphamide), Netupitan and Palonosetron Hydrochloride, Neupogen (Figrex), Nexavar (Sorafenib Tosylate), Nilotinib, Ninlaro (Ixazomib Citrate), Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romistachytin), Atouzumab, O Domzo (Sonigibr), OEPA, Ofatumumab, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar (pegaspargase), Ondansetron Hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ontak (dinitroleum oleoresin-diphtheria toxin conjugate), Opdivo (nivolumab), OPPA, Ostinib, Oxaliplatin Paclitaxel, Paclitaxel Albumin-Stable Nanoparticle Formulation, PAD, Palbociclib, Palivmin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitan, Pamir Disodium Phosphate, Panitumumab, Pabistat, Paraplatin, Paraplatin, Pazopanib Hydrochloride, PCV, Pegaspargase, Pegylated Interferon Alpha-2b, PEG-Intron (Pegylated Interferon Alpha-2b), Pemetrexed Disodium Per jeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), prasalifos, pomalidomide, pomalyst (pomalidomide), panatinib hydrochloride, Portrazza (nexituzumab), prastrexar, prednisone, procarbazine hydrochloride, Proleukin (aldeleukin), Prolia (denomab), Promacta (eltrombopag ethanolamine), Provenge (Sipuleucel-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, raburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nine-valent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, R-EPOCH, Revlimi d (lenalidomide), Rheumatrex (methotrexate), rituximab, lorapidan hydrochloride, romidisin, romistamine, erythromycin (danomycin hydrochloride), ruxotetinib phosphate, slanso intrapleural aerosol (Talc), sutuximab, Sipuleucel-T, semaduline reservoir type (lanreotide acetate), sonogilb, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, sterile talc. (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Pegylated Interferon Alpha-2b), Sylvant (Stetuximab), Synovir (Salidomide), Synribo (Homoharringtonine), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Ostinib), Talc, Talimogene Laherparepvec, Temoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Besalotin), Tasigna (Nillotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar (Temozolomide) Temozolomide, Tamromoxetine, Salidone, Thioguanine, Thiotepa, Tolak (fluorouracil - topical), Toposar (etoposide), Topotecan hydrochloride, Toremifene, Torisel (tamromoxetine), Tosimomab and Iodine-131 Tosimomab, Totect (dexrazoxen hydrochloride), TPF, Trabectin, Trametinib, Trastuzumab, Treanda (bendamustine hydrochloride), Trifluorouridine and Telbipyrimidine hydrochloride, Trisen ox (arsenic trioxide), Tykerb (lapatinib dimethylbenzenesulfonate), Unituxin (detoximab), uridine triacetate, VAC, vandetanib, VAMP, Varubi (lorapidan hydrochloride), Vectibix (panitumumab), VeIP, Velban (vincrine sulfate), Velcade (bortezomib), Velsar (vincrine sulfate), vemurafenib, VePesid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), vincrine sulfate, VincasarPFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomes, vinorelbine tartrate, VIP, vemodigine, Vistogard (uridine triacetate), Voraxaze (carboxypeptidase), vorinostat, Votrient (pazopanib hydrochloride), Wellcovorin (leucovorin), X Alkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denomab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectedine), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zelboraf (vemurafenib), Zevalin (tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (ederalipix), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

To further illustrate the invention, the following embodiments are included. These embodiments should not be construed as specifically limiting the invention. Variations of these embodiments within the scope of the claims are within the skill of the art and are considered to fall within the scope of the invention described and claimed herein. The reader will recognize that, with the aid of this disclosure, those skilled in the art can prepare and use the invention without exhaustive examples.

Example

Example 1. Effects of Punabulin on Dendritic Cell Maturation

Cell lines: Immature mouse DC cell line SP37A3 (provided by Merck KGaA) was cultured in Iscove modified Duchenne medley medium (IMDM; Sigma) supplemented with the following components: 10% heat-inactivated and endotoxin-tested FBS (PAA), sodium pyruvate (Gibco), penicillin/streptomycin L-glutamine mixture (Gibco), Eagle minimum essential medium (MEM) non-essential amino acids (Sigma), Ciproxin (Bayer), and 0.05 mmol/L 2-mercaptoethanol (Gibco). IMDM complete medium was supplemented with 20 ng/mL recombinant mouse GM-CSF and 20 ng/mL recombinant mouse M-CSF (both from Peprotech). Mouse tumor cell lines EG7 and 3LL-OVA were obtained from ATCC or provided by Douglas T. Fearon (Cancer Research UK Cambridge Institute, Li Ka Shing Center, University of Cambridge, Cambridge, UK). All cell lines were tested and verified to be mycoplasma-free. OVA expression in EG7 and 3LL-OVA and Thy1.1 expression in RMAThy1.1 were confirmed; no genome identification was performed.

SP37A3DC (mouse DC strain, Merck) were plated in 180 μL of IMDM complete medium supplemented with the following components: 10% heat-inactivated and endotoxin-tested FBS (PAA), sodium pyruvate (Gibco), penicillin/streptomycin L-glutamine mixture (Gibco), MEM non-essential amino acids (Sigma), and 0.05 mM ² -mercaptoethanol (Gibco)]. The IMDM complete medium was supplemented with 20 ng/mL recombinant mouse GM-CSF. DCs were allowed to adhere for 2 hours, after which punabulin, medium, or LPS was added as controls, 10x concentrated in 20 μL. DCs were incubated for 20 hours with different concentrations of punabulin (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM), medium, and LPS, respectively. The supernatant from these cultures was collected and used to detect cytokine production via ELISA (using a kit from BD). Cells were stained with LD-IR cell viability identification dye (Invitrogen) and monoclonal antibodies labeled with fluorescent dyes targeting CD80, CD86, CD40, and MHCII for flow cytometry analysis. Cells were analyzed using a BDFortessa cell counter equipped with DIVA software. The mean fluorescence intensity (MFI) of DC maturation markers CD40, CD80, CD86, and MHCII in live cells was normalized to the MFI of those markers detected in untreated (cultured) DCs. As shown in Figure 1A, purnabulin significantly increased the expression of all four DC maturation markers CD40, CD80, CD86, and MHCII. As shown in Figure 1B, DC viability did not change significantly at any of the drug concentrations tested, as determined using SytoxGreen staining.

Example 2. Effects of Punabulin on Dendritic Cell Maturation Compared to Paclitaxel and Etoposide

Two other cancer drugs, paclitaxel and etoposide, were also tested to compare their effects on DC maturation with that of punabulin. SP37A3 DCs (mouse DC strain, Merck) were seeded in 180 μL of IMDM complete medium [IMDM medium (Sigma) supplemented with: 10% heat-inactivated and endotoxin-tested FBS (PAA), sodium pyruvate (Gibco), penicillin/streptomycin L-glutamine mixture (Gibco), MEM non-essential amino acids (Sigma), and 0.05 mM 2-mercaptoethanol (Gibco)] (8 x ^10⁴ cells/well, 96-well flat bottom, tissue culture treated). The IMDM complete medium was supplemented with 20 ng/mL recombinant mouse GM-CSF. DCs were allowed to attach for 2 hours, after which punabulin, paclitaxel, etoposide, medium, or LPS (positive control) were added, 10x concentrated in 20 μL. DCs were incubated for 20 h each with purnabulin (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM), paclitaxel (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM), etoposide (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM), culture medium, and LPS (positive control). The supernatant from these cultures was collected and used to detect cytokine production by ELISA (using a kit from BD). Cells were also stained with LD-IR cell viability identification dye (Invitrogen) and monoclonal antibodies labeled with fluorescent dyes targeting CD80, CD86, CD40, and MHCII for flow cytometry analysis. Cells were analyzed using a BD Fortessa cell counter equipped with DIVA software. The mean fluorescence intensity (MFI) of the DC maturation markers CD40 (Fig. 2A), CD80 (Fig. 2B), CD86 (Fig. 2C), and MHCII (Fig. 2D) in live cells was normalized to the MFI of those markers detected in untreated (culture medium) DCs. The production of pro-inflammatory cytokines IL-1β (Fig. 3A), IL-6 (Fig. 3B), and IL-12p40 (Fig. 3C) was also measured by ELISA. These pro-inflammatory cytokines from DC culture supernatant were analyzed; they have been shown to play a crucial role in regulating T cell function and anti-tumor immune responses.

It was noted that punabulin was the most potent inducer of DC maturation among all three drugs. Compared to paclitaxel and etoposide, punabulin showed higher expression of all four DC maturation markers (CD40, CD80, MHCII, and CD86). Compared to the positive control LPS, punabulin also showed a significant increase in the expression of all four markers. Compared to paclitaxel, etoposide, and LPS, punabulin induced increased production of IL1β, IL6, and IL12. Therefore, punabulin increased the upregulation of maturation markers and the production of pro-inflammatory cytokines, thereby producing enhanced T cell stimulation capacity.

Example 3. Synergistic effect of punebulin and immune checkpoint inhibitors (PD-1 antibodies)

The combination therapy of punabulin and a PD-1 checkpoint inhibitor was compared with treatment with punabulin alone and treatment with a PD-1 antibody alone. The experiment was conducted using 7-10 week old mice subcutaneously injected with MC-38 tumor cells. Five experimental groups were prepared, each containing 9 mice.

Group 1 received saline; Group 2 received pranabulin diluent (without pranabulin); Group 3 received pranabulin diluted with diluent at a concentration of 7.5 mg/kg; Group 4 received PD-1 antibody; and Group 5 received a combination of pranabulin and PD-1 antibody. For the pranabulin/PD-1 antibody combination therapy (Group 5), mice were administered pranabulin diluted with diluent (7.5 mg/kg) twice weekly (days 1 and 4), followed by PD-1 antibody administration 1 hour after each pranabulin administration. For pranabulin-only treatment (Group 3) or antibody-only treatment (Group 4), mice were administered pranabulin (7.5 mg/kg, diluted with diluent) or antibody alone twice weekly (days 1 and 4). For Groups 1 and 2, mice were administered saline or pranabulin diluent alone twice weekly.

Each treatment began at a tumor size of approximately 125 ^mm³ and continued until the tumor size reached 1500 ^mm³ . If the average tumor size in any group did not reach 1500 ^mm³ by day 45 of the experiment, treatment was discontinued and tumor size was reassessed. To determine the efficacy of each treatment, the following data were collected: mortality before the tumor size reached 1500 ^mm³ ; mouse body weight assessed twice weekly before treatment; tumor growth rate determined by tumor size measurements (twice weekly); tumor growth index; overall survival; and time required for the tumor size to double. Results from the combined treatment of punebulin and PD-1 antibody indicated that punebulin and PD-1 antibody had a synergistic effect in inhibiting tumor growth.

Example 4. In vivo stimulation of OVA-specific OT-I and OT-II T cells

SP37A3 cells or BMDC cells from day 7 were pulsed for 1 hour with full-length OVA protein (0.1 mg/mL) or OVA257-264 peptide (T4)/OVA323-339 peptide (500 ng/mL; after activation) before activation with purnabulin, and then CD8 ⁺ /CD4 ⁺ T cells purified from OT-I/OT-II transgenic mice ( ² x 10⁵ total cells/well, 96-well round-bottom plate) were added at the specified ratio. CD4 ⁺ T cells were loaded with the proliferation dye eFluor670 before co-culture. Proliferation was assessed by flow cytometry after 3 days.

Example 5. In vivo stimulation of antigen-specific CD4 and CD8 T cells

Langerhans cells (LC) and spleen cells from naïve OT-I and OT-II transgenic mice (Ly5.2) were labeled with eFluor670 and adoptively transferred to C57BL/6-Ly5.1 mice. Twenty-four hours later, mice were immunized by tail injection with either OVA257-264 peptide (T4: SIINFEKL; a low-affinity variant of SIINFEKL) or OVA323-339 peptide along with punabulin or LPS. Four days after adoptive transfer by flow cytometry, the proliferation of OT-1CD8 ⁺ and OT-IICD4 ⁺ T cells was assessed.

Example 6. Analysis of DC homing to tumor drainage lymph nodes

To detect DC homing upon injection of punabulin, mice with subcutaneous EG7 tumors were intratumorally injected with FITC-conjugated dextran (100 mg/mouse; Sigma) and either punabulin or PBS/carrier (dummy control). Forty-eight hours after punabulin injection, single-cell suspensions from tumor-drained and non-drained lymph nodes were prepared and analyzed by flow cytometry.

Example 7. Synergistic effect of punebulin and immune checkpoint inhibitors (PD-1 antibody and CTLA-4 antibody)

The combination therapy of punabulin and a PD-1 checkpoint inhibitor with a CTLA-4 checkpoint inhibitor was compared with punabulin alone, PD-1 antibody alone, or a combination of PD-1 and CTLA-4 antibodies. The experiment was conducted using 7-10 week old mice subcutaneously injected with MC-38 tumor cells. Six experimental groups were prepared, each containing 10 mice.

Group 1 was administered IgG2a and punabulin vector; Group 2 was administered punabulin at a concentration of 7.5 mg/kg diluted with a diluent; Group 3 was administered a PD-1 antibody; Group 4 was administered a combination of punabulin and PD-1 antibody; Group 5 was administered a combination of PD-1 and CTLA-4 antibodies; and Group 6 was administered a combination of PD-1 antibody/CTLA-4 antibody/punabulin. For the punabulin/PD-1 antibody combination therapy (Group 4) and the punabulin/PD-1/CTLA-4 antibody therapy (Group 6), mice were administered punabulin diluted with a diluent (7.5 mg/kg) twice a week (on day 1 and day 4 of each week), followed by administration of the antibody one hour after each punabulin administration. For mice treated with only punabulin (Group 2) or only with antibody (Groups 3 and 5), punabulin (7.5 mg/kg, dissolved in diluent) or antibody was administered twice a week (on the 1st and 4th of each week).

Each treatment began with a tumor size of approximately 125 ^mm³ and continued until the tumor size reached 3000 ^mm³ . The experiment ended when the average tumor size in Group 1 reached 3000 ^mm³ . To determine the efficacy of each treatment, the following data were collected: mortality before the tumor size reached 3000 ^mm³ ; mouse body weight assessed twice weekly before treatment; tumor growth rate determined by tumor size measurements (twice weekly); tumor growth index; overall survival; tumor weight at necropsy; and time required for the tumor size to increase tenfold. Tissues were weighed and FACS analyzed at necropsy.

Results of studies on the combined treatment of punebulin and PD-1 antibody with CTLA-4 antibody showed that punebulin and the antibody had a synergistic effect in inhibiting tumor growth, and the longest time to achieve a 10-fold increase in tumor weight was observed in all six experimental groups. Figure 4A shows the effects of groups 1, 5, and 6 on tumor growth. As shown in Figure 4A, the combined treatment of punebulin, PD-1 antibody, and CTLA-4 antibody in group 6 had a better tumor growth inhibition effect than the combination of PD-1 antibody and CTLA-4 antibody in group 5, and both groups 5 and 6 showed inhibitory effects on tumor growth compared to the control group 1. Figure 4B shows the effects of the six treatment groups on the mean tumor weight at autopsy. As shown in Figure 4B, the combined treatment with punebulin, PD-1 antibody, and CTLA-4 antibody produced the lowest mean tumor weight at autopsy, followed by the punebulin and PD-1 antibody treatment group. Figure 4C shows the time to reach a 10-fold increase in tumor volume in the six treatment groups. As shown in Figure 4C, the treatment group using a combination of pomabulin, PD-1 antibody, and CTLA-4 antibody experienced the longest time to reach 10 times their initial volume. Therefore, pomabulin alone, or the combination of pomabulin with PD-1 antibody or PD-1 plus CTLA-4 antibody, resulted in reduced tumor weight at autopsy. The combination of pomabulin, PD-1 antibody, and CTLA-4 antibody showed better tumor-suppressive activity than pomabulin and PD-1 antibody alone, while pomabulin and PD-1 antibody alone demonstrated better tumor-suppressive activity than pomabulin alone.

Figure 5 shows the FACS analysis results of tumors at autopsy in the MC-38CRC tumor model described above, including changes in the percentage of Treg cells, the CD8 ⁺ /Treg ratio, and the percentage of macrophages among CD45+ lymphocytes. Figure 5A shows the effect of the six treatment groups on the percentage of Treg cells. As shown in Figure 5A, compared with the control group without punabulin, treatment with punabulin, PD-1 antibody and CTLA-4- antibody, treatment with punabulin and PD-1 antibody, and treatment with punabulin alone all showed a reduction in %Treg cells. Figure 5B shows the ratio of CD8+ cells to Treg cells. As shown in Figure 5B, treatment with punabulin, PD-1 antibody and CTLA-4- antibody showed the highest CD8+/Treg cell ratio. Figure 5C shows the effect of the six treatment groups on macrophages. As shown in Figure 5C, compared with the control groups, the treatment groups of ponabulin, PD-1 antibody and CTLA-4 antibody, ponabulin treatment group, and PD-1 antibody and CTLA-4 antibody treatment group all showed a reduced percentage of macrophages.

Therefore, FACS analysis of tumor tissues showed that treatment with punebulin alone, and treatment with punebulin combined with immune checkpoint inhibitors (e.g., punebulin with PD-1 antibody, punebulin with PD-1 antibody and CTLA-4 antibody), were all associated with a decrease in the percentage of regulatory T cells (Treg cells), a decrease in the percentage of macrophage-stained cells, and a concomitant increase in the CD8+/Treg cell ratio. The decrease in the percentage of Treg cells and macrophage-stained cells, and the increase in the CD8+/Treg cell ratio, were more significant in the punebulin combined with immune checkpoint inhibitors compared to the punebulin alone or antibody alone groups. These data demonstrate the synergistic immuno-oncological properties of combination therapy with punebulin and immune checkpoint inhibitors (e.g., PD-1 antibody and CTLA-4 antibody).

Claims (21)

1. Use of punabulin in the preparation of a medicament for the treatment of cancer in combination with one or more immune checkpoint inhibitors, wherein the one or more immune checkpoint inhibitors are PD1 antibodies. 2. The use as described in claim 1, wherein the combination comprises punabulin, PD1 antibody, and CTLA-4 antibody. 3. The use as described in claim 1, wherein punabulin is administered in combination with one or more other chemotherapeutic agents. 4. The use as described in claim 2, wherein punabulin is administered in combination with one or more other chemotherapeutic agents. 5. The use as described in any one of claims 1-3, wherein the cancer comprises cancer cells expressing a ligand that binds to PD-1. 6. The use as claimed in claim 5, wherein the ligand binding PD-1 is PD-L1 or PD-L2. 7. The use as described in claim 5, wherein the cancer is head and neck cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer, prostate cancer, breast cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, glioblastoma, myeloma, lymphoma, or leukemia. 8. The use as described in claim 5, wherein the cancer is renal cell carcinoma, malignant melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, Hodgkin's lymphoma, or squamous cell carcinoma. 9. The use as claimed in claim 1, wherein the cancer comprises cancer cells expressing a ligand that binds to CTLA-4. 10. The use as claimed in claim 9, wherein the ligand binding CTLA-4 is B7.1 or B7.2. 11. The use as described in claim 1, wherein the one or more checkpoint inhibitors are PD1 antibody and CTLA-4 antibody. 12. The use as described in any one of claims 1-3, wherein the one or more immune checkpoint inhibitors are nivolumab, pembrolizumab, pidilizumab, ipilimumab, BMS 936559, atezolizumab, durvalimumab, or any combination of the above antibodies. 13. The use as described in any one of claims 1-3, wherein the cancer is selected from: breast cancer, colon cancer, rectal cancer, lung cancer, prostate cancer, melanoma, leukemia, ovarian cancer, gastric cancer, renal cell carcinoma, liver cancer, pancreatic cancer, lymphoma, and myeloma. 14. The use as claimed in any one of claims 1-3, wherein the cancer is a solid tumor or a blood cancer. 15. The use as described in any one of claims 1-3, wherein the cancer does not have any cells expressing PD-1, PD-L1 or PD-L2. 16. A pharmaceutical composition comprising punabulin and one or more immune checkpoint inhibitors, wherein the one or more immune checkpoint inhibitors are PD1 antibodies or PD1 antibodies and CTLA-4 antibodies. 17. The composition of claim 16, wherein the pharmaceutical composition comprises punabulin, a PD1 antibody, and a CTLA-4 antibody. 18. The composition of claim 16, further comprising one or more pharmaceutically acceptable excipients. 19. The composition of claim 16, further comprising one or more additional chemotherapeutic agents. 20. The composition of claim 16, wherein the one or more immune checkpoint inhibitors are nivolumab, pembrolizumab, piperizumab, ipilimumab, BMS 936559, atezolizumab, durvalimumab, or any combination of the above antibodies. 21. Use of the pharmaceutical composition of any one of claims 16-20 in the preparation of a medicament for treating cancer.