HK40079377A - Oral peptide antagonists of multiple chemokine receptors for reversing loss of synapses and dendritic spines - Google Patents

Description

Oral peptide antagonists of multiple chemokine receptors for reversing synaptic and dendritic spine loss

The present invention broadly relates to compositions and compounds that enter the central nervous system to engage with neuronal chemokine receptors (engage) to promote neuronal remodeling and formation of synapses and dendritic spines. In particular, chemokine receptor antagonists have been found to promote memory and learning in normal animals, and to promote the formation of synapses and dendritic spines to restore cognitive and motor abilities in conditions of brain injury. The present invention promotes the formation of synapses and dendritic spines and is expected to enhance memory and learning in normal individuals and those experiencing loss of synapses, e.g., due to aging or due to a disease condition, wherein the loss of synapses and dendritic spines is accelerated, resulting in impairment of cognitive or motor function.

Background

Improved cognitive and psychological functions are a commonly sought goal. Modulation of protein translation, protein degradation, cytoskeletal dynamics, extracellular matrix interactions, second messenger signaling, and neurotransmitter receptor transport and function are all integral parts of synaptic remodeling essential for cognition. Selective targeting of specific components of these processes can be used as an effective strategy for the treatment of cognitive deficits.

Cognitive enhancement by drug intervention is common and self-administration of psychostimulants such as caffeine, nicotine and amphetamines can improve cognitive performance. However, the use of these stimulants may not be optimal, they have side effects and risks, and they do not improve all cognitive, memory and learning problems. More efficient and specific strategies are needed. Although the causes of cognitive, memory and learning problems are numerous, one strategy to enhance cognitive performance is to target key mechanisms that may have a positive impact on cognitive function, such as promoting the structure, function, formation and regeneration of synapses and dendritic spines.

In the synaptic setting, the actin cytoskeleton is a key element in the synaptic remodeling process and maintains the dendritic spine and neuronal growth cone structures (architecture) driven by synaptic activity. In fact, the shape and synaptic strength of the neuronal growth cones and dendritic spines are precisely controlled to translate short-term changes in synaptic strength into long-term changes caused by stable structural changes. These functional and structural changes are considered to be the biological basis for memory and learning. Synaptic remodeling involves a number of processes that may be considered as potential targets for cognitive therapy. These processes are regulated by signal transduction mechanisms and by a high degree of protein phosphorylation/dephosphorylation, such as the mitogen-actin regulation of cell structures (cytoschikulture). Coordination of these complex processes is critical to optimizing cognitive function.

Cellular stress mediators (inflammation, free radicals, excitotoxic levels of glutamate, mitochondrial inhibitors (e.g., antimycin a), peroxides, NO, a β (amyloid) peptides, and pro-inflammatory cytokines) damage synapses, the major functional units of the brain and negatively affect synapses' ability to store memory and process information. These results have prompted the belief that under normal circumstances, improved cognition can be achieved by promoting synaptic formation. Conditions of memory loss, impaired learning and information processing speeds, and loss of motor function after injury are primarily conditions of synaptic failure, i.e., "synaptopathy". Different stress mediators, even dietary or epigenetic changes, whether significant or not, can induce the rapid formation of transient or persistent rod-like inclusions (rod-like inclusions) containing actin-depolymerizing factor (ADF)/filaggrin and actin in neuronal axons and dendrites, resulting in synaptic loss. The significance of this finding is that the formation of rods provides a target for facilitating synaptic intervention because rod formation is associated with loss of cortical synapses, which is the reason why Mini-mental state cognitive tests score is lower, which becomes more pronounced over time. Generally, cognitive disorders caused by other causes are natural or disease related, such as after brain injury or stroke, or neuropathy, as well as conditions of synaptic failure and failure to establish functional neuronal connectivity. Actin dynamics modulation is an attractive target for memory enhancement strategies due to its many roles in synaptic function and remodeling.

Three basic factors of synaptic degradation are: elevated levels of activated filaggrin, elevated levels of ADP-actin and a highly oxidized environment. Actively stressed neurons usually meet all three requirements, which are not particularly disease-related. Formation of phosphorylated actin/filament cutting protein rods is an early neuronal pathology induced by a number of causes. When activation of the filaggrin persists, it results in loss of synapses prior to neurodegeneration. In cultured neurons, death occurred within a few days after the formation of the silk cutting proteins into rods. The level and extent to which functional synapses may be restored is largely unknown. How the pathway of synaptic loss is related to synaptic reversal is unclear. We have discovered specific peptides that act by selecting chemokine receptors and appear to control the balance between synaptic loss and synaptic regeneration. These peptides modulate the activation of cellular proteins that control neuronal cellular structures that produce growth cone structures that contribute to functional synapses. Reversing synaptic loss to restore function is a clear advantage of the present invention over psychostimulants, which may enhance performance or merely slow or arrest synaptic loss, as this loss may go unnoticed for decades. Loss of functional neurons is considered unrecoverable, although this concept appears to be slowly changing. For the benefit of the patient, it is a more desirable effect to promote synapse formation to improve cognition or to overcome any synaptic loss.

Recent advances have begun to elucidate a new approach to improving cognition by supporting synapse formation. As explained in detail below, recent findings provide new insights into neuronal growth and regeneration mechanisms, which suggest that conditions of synaptic loss can now be treated by directly promoting nerve regeneration in those neurons by modulating receptors of the innate immune system present on the neurons. This method provides a means to rebalance from the normal process of actually deconstructing and building synapses to support memory consolidation (sometimes called "pruning"), but when excessive or chronic, the deleterious processes into regeneration (means), which is beneficial.

Regenerative therapy is more advantageous than psychostimulants or simple synaptic loss preventives. First, psychostimulants do not bring about a lasting benefit, while prophylactic treatment can only be shown to be effective in preventing the progression of loss of function. It is reasonable to expect that regenerative approaches can improve normal cognition and enhance cognition, memory and learning in the event of synaptic loss, such as following brain injury, neuropathy or dementia.

Neuroinflammation, which involves interactions between neurons and components of the innate immune system (such as cytokines, chemokines, and microglial activity), has been the target of many preclinical and clinical studies, with subsequent clinical trials largely failing to enhance cognition. One reason is the complexity and diversity of the mechanisms of injury, requiring combination therapy or targeting of pathogenic mechanisms common to the early stages. Many effects other than inflammation control the balance between synaptic loss and formation. Blocking inflammation by itself does not seem to be sufficient to bring hopes of clinical benefit. In many cases, loss of synapses occurs early, not necessarily inflammatory, and when unhindered, eventually manifests as cognitive impairment. At present, early intervention seeks the limited goal of preventing disease progression and worsening before loss of function (cognitive or motor being examples) (significantly impairing the quality of life of a person). For this reason, we have attempted to re-concentrate on providing treatments to improve cognition in normal daily life, as well as synaptic disease and subsequent diseases or conditions to enhance cognition or function by promoting the formation of functional synapses to provide greater benefit to the patient.

Since cognitive disorders are usually not evident in the early stages, both normal and pathological processes leading to synaptic loss may persist for years or even decades without diminishing until they become apparent. When cognitive impairment is manifest, the state of the art indicates that effective intervention may be late, resulting in permanent functional loss. There is currently no treatment to reverse synaptic loss, although such regeneration is expected to overcome and reverse existing cognitive deficits and bring people closer to normal function. Thus, while preventing synaptic loss by blocking inflammation or disrupting pathogenic filaggrin rod formation that leads to synaptic loss would have the benefit of slowing or disrupting disease progression, a different and more challenging therapeutic goal is to reverse synaptic damage or loss of initial or already progressing.

The present invention is expected to provide highly inaccessible and long-sought therapeutic benefits for improving memory, learning, cognition and function, manifested in many conditions of synaptic loss, particularly in the hippocampus, which is the site of learning and memory formation. In addition to slowing or stopping cognitive or functional decline, such results are expected to be beneficial to people experiencing loss of synapses, dendritic spines, and reduction of dendritic arborization. The present invention relates to the restoration of synapses, dendrites and neurites to improve cognition and memory, and to restore cognitive function in dementia and motor function in stroke, or to reverse wallerian degeneration in neuropathy of chronic painful conditions caused by injury, surgery, diabetes or chemotherapeutic drugs. The peptides of the invention provide improved benefits by reversing functional decline that is incipient and not very apparent, and has become severe enough to be detected in daily life.

The small peptide chemokine antagonist compounds of the invention provide new opportunities to treat synaptic loss for those in need of synaptic regeneration or experiencing synaptic disease by targeting specific chemokine receptors that control synaptic formation to disrupted balance, and thus can be used to reverse synaptic loss in both mild cognitive impairment and more widespread cognitive impairment conditions.

Numerous studies have shown that Mild Cognitive Impairment (MCI), which may also be the prodromal phase of AD, and early AD are primarily characterized by loss of synapses and connectivity. Synaptic loss of MCI precedes extensive loss of neurons, a characteristic of late stages in AD. May be helpful in neurological aids (hand), COVID19 or other CNS infections, and indeed, in any neurodegenerative condition caused by loss of synapses, and even in non-pathological conditions such as loss of synapses in normal aging. No medical therapy has been developed to facilitate the recovery of many different conditions linked together by synaptic loss (synaptopathy).

Several decades ago, Hill et al (1) suggested the value of chemokine antagonists as interventions capable of reversing synaptopathy, showing the protection of dendritic spine morphology, the expansion of dendritic tree structure and the reversal of functional defects by the non-oral analogue peptide Dala 1-peptide T-amide ("DAPTA") of the peptide of the invention in animal models of HIV cognitive decline. This finding was not well understood at the time, as chemokine receptors have not been discovered. DAPTA is able to reverse cognitive deficits in HIV-associated neurocognitive disease (HAND) phase 2 human trials and brain scans, and its action was later understood to be mediated by a panel of HIV entry chemokine receptors (CCR2/CCR5/CCR8/CXCR4) that are targets for DAPTA action. Further focus on the role of CCR5 as an inhibitor of cortical plasticity and hippocampal memory and learning comes from studies by Zhou,2016(2) which show that overexpression of CCR5 results in memory deficits in normal mice, which can be prevented by a "knockout" or deletion of CCR 5. Joy et al (3) in 2019 unexpectedly found CCR5 as a therapeutic target for recovery after stroke and traumatic brain injury. Knock-down of CCR5, or drug blockade with the antagonist maraviroc (maraviroc), was associated with retention of dendritic spines and a new neurite cortical projection pattern. HIV chemokine entry into the receptor CXCR4 was also identified as having a negative impact on recovery following traumatic brain injury, as its antagonist AMD3100 improved recovery.

Both long-term hippocampal slice cultures from adult rodent brains and isolated neuronal cultures from mice have shown that CXCR4 and CCR5 are two important chemokine receptors that when activated lead to synaptic loss and cognitive impairment. Additional results implicate CCR2 and CCR8 in connection with memory impairment, identifying a group of HIV entry receptors targeted by the peptides of the present invention as therapeutic targets.

Collectively, these studies have encouraged us to investigate the benefit of a multi-chemokine receptor antagonist peptide (multi-chemokine receptor antagonist peptide) to reverse synaptic loss by promoting the formation of neuronal growth cones, the organizational structure of neurons that produce functional synapses. Since activation of multiple chemokine pathways is implicitly associated with improved recovery from brain injury, better results can generally be expected by agents that antagonize multiple chemokine receptors to reverse the loss of synapses and dendritic spines.

Brief Description of Drawings

FIG. 1A is a graphical representation of the activity of a class of four representative peptides to block chemokine receptor 2(CCR 2).

Figure 1B is a graph depicting the activity of several all D pentapeptides blocking chemokine receptor 2(CCR 2).

FIG. 2A is a graph depicting direct neuronal activation of chemokine receptors CXCR4 or CCR5 by the HIV envelope protein gp120, with synaptic loss resulting from mitogen rod formation by the HIV envelope protein gp 120.

Figure 2B illustrates that filaggrin rod formation (an early pathology leading to synaptic loss) is caused by the AD-associated peptide β -amyloid (a β -trimer/dimer) and can be prevented by blocking neuronal CCR5 with the specific antagonist maraviroc.

FIG. 3 is a graph depicting the dose response of a multi-chemokine receptor antagonist peptide (RAP-103 (all-D-TTNYT) or RAP-310 (all-D-ASTTTNYT) to prevent A β -trimer/dimer pathology (leading to synaptic loss).

FIG. 4 is a graph depicting the effect of the multi-chemokine receptor antagonist RAP-103(R103) in blocking synaptic injury caused by preformed fibrils of amyloid beta (A β) and alpha-synuclein (PPF).

Figure 5 illustrates the ability of amyloid β (a β) trimer to cause pathology leading to synaptic loss (rod formation) (left, arrow). Addition of RAP-103(R103) inhibits activation of filaggrin into rods and into growth cones for synaptic regeneration.

In particular embodiments, the invention relates to the treatment of various synaptic diseases and their associated neurodegenerative conditions, including aging, alzheimer's disease, huntington's disease, and other conditions, such as parkinson's disease, type 2 diabetes, HIV-associated neurocognitive disorder (HAND), as well as functional deficits following injury, stroke, or trauma, or viral encephalopathy and other encephalopathies associated with loss of synapses. The invention also relates to pharmaceutical compositions useful for such treatment and/or reversal of loss of synapses and dendritic spines, as well as to certain orally active peptides themselves.

Continued exposure of neurons to soluble β -amyloid peptide dimers/trimers (Α β d/t) (AD), α -synuclein fibrils (PFF), HIV envelope gp120 protein (HAND), or proinflammatory cytokines (TNF α, IL-1 β, IL-6) disrupts synaptic function and leads to synaptic loss. Synaptic loss, whether caused by neuroinflammation, oxidative and energetic stress, injury, trauma, bacterial/viral or other infectious agents, cytotoxic drug therapy, or radiation therapy, underlies many neurodegenerative conditions that the peptides of the invention can reverse.

Our therapeutic approach seeks to promote synaptic formation, which is advantageous for reversing synaptic loss by blocking neuronal chemokine receptor activation, a previously unidentified activity of the subject peptides. Targeting neuronal chemokine receptors with the peptides of the invention to promote the formation of synapses and growth cones, thereby improving memory and cognition is a previously unknown and unexpected therapeutic advance and in contrast to the prior art, which focuses on blocking innate immune inflammation via microglia and their secreted cytokines to prevent killing of neurons or the formation of filaggrin rods that disrupt the loss of synapses just prior to neuronal death. To distinguish between the different effects of chemokine receptor blockade, we performed experiments on pure cultures of neurons in the absence of immune cells or cytokines, compared to brain tissue slices or whole animals. Unlike reports showing microglia and cytokine-activated chemokine blockade through the NKkB pathway in whole animals or CNS tissue sections, our results point to a previously unrecognized direct effect of blocking neuronal chemokine receptors. We have observed that chemokine receptor blockade is one such means: this approach shifts neurons away from the stress response that leads to synaptic loss and stimulates their regeneration through growth cone formation, which drives neurite extension, restoring synapses, their connectivity, and function in patients suffering from synaptic loss.

The chemokine antagonist peptides of the invention can block multiple neuronal chemokine receptors at doses of pM, which is an advantage over single receptor antagonists, especially when multiple chemokine receptors appear to be associated with synaptic restoration. They enter the brain efficiently and rapidly by oral and parenteral routes of administration, and, because they are protected from proteolysis, persist in the CNS at therapeutic doses for a sufficient period of time to establish benefit. All of these different features combine to provide one result: enabling treatment for conditions of synaptic loss, i.e., synapsis, restoring synaptic connectivity to reverse defects and promote functional recovery. The present invention relates to pharmaceutical compositions useful in such treatment and/or reversal of synaptic loss and neurodegeneration, as well as to certain active peptides themselves.

The peptides can be used in pharmaceutical and composition of matter for the treatment and prevention of any disease or condition caused by an organism, compound or immune dysfunction that results in the formation of persistent or pathological filaggrin-actin sticks associated with synaptic loss.

The peptides may be administered orally, buccally, parenterally, topically, rectally, vaginally, by intranasal inhalation spray, by intrapulmonary inhalation, or otherwise. In particular, the peptides according to the invention may be formulated for oral use, for inhalation in a spray or powder, for injection (e.g. subcutaneous, intramuscular, intravenous, intra-articular or intracisternal) or for infusion and may be presented in unit dosage form in ampoules or tablets, or in multi-dose vials or other containers with an added preservative.

The compositions may take such forms as suspensions, solutions or emulsions, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder and/or lyophilized form for direct administration or for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water, physiological saline, or 5% dextrose) before use. The pharmaceutical composition containing one or more peptides may also contain other active ingredients, such as antibacterial agents or preservatives.

The composition may comprise 0.001% to 99% (w/v, or preferably w/w) of the active substance. Preferred administration is oral pills or capsules. The composition is administered in a therapeutically or prophylactically effective dose, i.e., 0.1mg peptide/day to 500mg peptide/day, particularly 5-50 mg/day. Even larger doses may be used, since the peptides according to the invention are non-toxic. However, this is not generally necessary.

For administration by injection or infusion of the composition, the daily dose for treating an adult human of about 70kg body weight generally ranges from 0.2mg to 20mg of active substance, and can be administered in the form of 1 to 4 doses per day. The invention is useful for preventing or treating diseases or medical conditions, particularly those involving loss of synapses, such as mild cognitive impairment, dementia, frontal dementia, lewy body disease, alzheimer's disease, parkinson's disease, HAND, stroke recovery, or normal aging.

According to a first aspect of the invention there is provided the use of a linear peptide of formula 1 wherein all amino acids are in the D-stereoisomeric configuration, except when Gly is present and the Gly is achiral:

A-B-C-D-E-F-G-H. General formula 1)

Wherein

A is Ala or is absent,

b is Ser, Thr or absent,

c is Ser, Thr or absent,

d is Ser, Thr, Asn, Glu, Arg, Ile, Leu,

e is Ser, Thr, Asp or Asn,

f is Thr, Ser, Asn, Arg, Gln, Lys, Trp,

g is a group of Tyr, wherein,

h is Thr, Ser, Arg, Gly.

All amino acids mentioned in formula 1 are in the D-stereoisomeric configuration and the H candidate may be esterified or amidated. The peptide comprises at least 5 amino acids. In previous studies, the all-L amino acid form of the linear peptide of formula 1 was designated peptide T, and its modified analog was designated DAPTA.

Preferred D peptides are (except when Gly is present and Gly is achiral):

Thr Thr Asn Tyr Thr,(SEQ ID NO:1)

Ser Ser Thr Tyr Arg,(SEQ ID NO:2)

Thr Thr Ser Tyr Thr,(SEQ ID NO:3)

Asn Thr Arg Tyr Arg,(SEQ ID NO:4)

Ile Asp Asn Tyr Thr,(SEQ ID NO:5)

Asn Thr Ser Tyr Arg,(SEQ ID NO:6)

Ile Asn Asn Tyr Thr,(SEQ ID NO:7)

asn Thr Ser Tyr Gly, (SEQ ID NO:8) and

Glu Thr Trp Tyr Ser.(SEQ ID NO:9)。

Method

isolation of a β d/t from the culture medium of cultured 7PA2 cells, 7PA2 cells are CHO cell lines expressing a form of human APP found in familial AD. The amount of Α β d/t was quantified by western blotting with a standard of synthetic Α β monomers. Neuron culture, fixation, permeabilization, blocking and immunostaining were performed by widely used common methods starting with E16.5 mouse hippocampal neurons. Assays were performed on 7 and 21 DIV cultures, with or without drug treatment at 6 or 20 DIVs (10) ^-14 M to 10 ^-10 Peptides RAP-103 and RAP-310 and 10 of M ^-11 M to 10 ^-7 M Malavirroseo/AMD 3100, with or without A β d/t-1 nM). GP120 protein was obtained from CCR5 tropic strain CM, CXCR4 tropic strain MN and amphotropic strain MN. After treatment, cells were fixed, as published, immunostaining for the silk-cutting protein and growth cones with 2G13 antibody, and counterstaining with DAPI, and three coverslips also stained for the neuronal nuclear antigen NeuN. Analysis gave the percentage of neurons (NeuN) in total nuclei (DAPI) to obtain rods/neurons.

Images of fixed neurons immunostained across silk-cutting proteins, growth cones and nuclei captured throughout the coverslip were exported to ImageJ. Applying a macro-function sets local thresholds and shape/size segmentation (segmentation) for both the silk-cutting protein and the growth cone antibody. The material stained with the silk-cutting protein that is not within the neurite is removed. Results are reported as rods/nucleus, number of rods/area, percent of total area of rods, or rods/neurons if NeuN is stained or morphologically identified.

The invention will now be illustrated by the following non-limiting examples. The embodiments refer to the figures.

Example 1

FIG. 1A is a graph depicting the activity of the all-D form of the peptide compared to the predominantly L-form of the peptide of formula I. It is shown that three related peptides of formula I, which differ only in enantiomeric form, having the same primary sequence or share a partial sequence in common, block CCL2 chemotaxis. The peptides function as chemokine receptor antagonists for both CCR2 and CCR5 (4). Figure 1B shows the general nature of this finding, in which eight additional all-D-amino acid pentapeptides also blocked the CCL1/CCR2 interaction.

Example 2

Neurons expressing CCR5 and CXCR4 receptors that control synaptic regeneration are illustrated. Figure 2A shows that the chemokine receptors CXCR4 or CCR5 are activated by the HIV envelope protein gp120 in cultured mouse hippocampal neurons without addition of immune cells, microglia, cytokines or other inflammatory mediators, confirming the relevance of direct neuronal chemokine receptors to synaptic loss and cognitive impairment. These chemokine receptor-mediated effects of HIV gp120 lead to the cognitive performance of neuro-aids (hand) through synaptic loss, as shown by Hill et al (1), which can therefore be reversed by the specific chemokine receptor antagonists maraviroc (Mar, CCR5) or AMD3100(AMD, CXC 4).

Figure 2A also shows that neurons treated with gp120 form a filament-cutting protein rod via CXCR4 or CCR5 receptors, an early transition to synaptic disruption, as the different gp120 tropic (CCR5vs CXCR4) forms are inhibited by their receptor-specific antagonists maraviroc (CCR5) and AMD3100(CXCR 4).

Fig. 2B shows that the early pathology of synaptic loss induced by the AD-associated peptide β -amyloid (a β -trimer/dimer) can be reversed and that the realization of the synaptic regeneration pathway by blocking neuronal CCR5 with maraviroc indicates that neuronal chemokine receptors are important under different (HIV of fig. 2A compared to AD of fig. 2B) conditions where synaptic regeneration is required.

Example 3

The ability of the multi-chemokine receptor (CCR2/CCR5/CCR8/CXCR4) antagonists RAP-103 (all-D-TTNYT) and RAP-310 (all-D-ASTTTNYT) to block the formation of mitotic protein rods in cultured mouse hippocampal neurons at early pathological A β and TNF (not shown) at pM and lower concentrations. Synaptic regeneration cannot occur with activated filaggrin-actin rods, and the results indicate that the multi-chemokine receptor antagonistic peptide shifts the neuronal state towards regeneration by decreasing the percentage of neurons expressing the filaggrin rods. Thus, blocking chemokine receptors CCR2/CCR5/CCR8 or CXCR4 with the peptides of the invention is a specific approach to block early AD pathology and reverse synaptic loss. Blocking multiple chemokine receptors, implicitly associated with synaptic loss, is an unexpected advantage of the present all-D peptides of the invention, as they can provide better protection than targeting a single chemokine receptor.

Example 4

The ability of the multi-chemokine receptor antagonist RAP-103 to block early neuronal pathology leading to synaptic damage or loss in Alzheimer's disease and Parkinson's disease caused by preformed fibrils of amyloid beta (A β) and alpha-synuclein (PPF), mediators of synaptic loss, respectively, is demonstrated. RAP-103 functions to transform neuronal state into synaptic regeneration by reducing the area within the neuron occupied by the silk-cutting protein rod.

Example 5

The following experiment illustrates the ability of RAP-103 to promote growth cone formation, a synaptic regeneration event. Amyloid beta (Α β) trimer was added to pure cultures of mouse hippocampal neurons (lacking immune cells, microglia and inflammatory mediators they secrete) to evaluate actin dynamics and the formation of mitogen bars. In the left panel of fig. 5, (no RAP-103) synaptic pathology (arrows indicate immunostained bars) shows AD-associated systems. Almost all dendrites, especially their extensions, show rod formation. These neurons died on day 3. Related is that bar pathology leads to loss of synapses in people associated with dementia. This example further shows (fig. 5, right) the unexpected effect of RAP-103 in switching neurons from a synaptic degeneration state (stick) to a synaptic regeneration state (no stick, but now expressing growth cones, stained with 2G13 antibody). Note that in the right panel of fig. 5, the silk cutting protein rod-like structures at the ends of the dendrites are eliminated and instead are a multiplicity of growth cone-like structures distributed along the entire length of the dendritic stalk (dendritic arbor) as a dominant phenotype. RAP-103 transforms neurons from a state of synaptic collapse (synapse collapse) and loss to growth cone expansion and synaptic regeneration.

It is relevant that the growth cone comprises the growing tip of an axon seeking and forming a synaptic contact, and that such a structure is one aspect of dendritic spine formation, the site of synapse formation. Blocking chemokine receptors appears to control the bifurcation of a complex series of pathways in actin-filaggrin dynamics from synaptic destruction and loss to synaptic preservation, reconstruction and regeneration. This result is consistent with other reports (3) on CCR5 blockade as a means to promote synapse formation. Importantly, synaptic loss is the basis for all neurodegeneration and leads to a number of conditions and diseases that have little or no therapeutic approach. The present invention provides a new, important and unique therapeutic intervention for a wide range of uses, including nonpathological conditions of synaptic disease, such as normal aging. Although the inventors do not claim exact knowledge of the mechanism of action, we provide our current model to help explain our data showing two effects caused by the peptides of the invention. The first is to block the formation of the silk-cleaved protein rod, which has an additional commensurate effect of stimulating growth cone formation. Both of these characteristics are superior to either alone and may represent a unique neuronal regeneration phenotype.

Various compositions and methods are disclosed herein. Specific exemplary embodiments of these compositions and methods are disclosed below:

embodiment 1. a composition comprising a D-peptide multi-chemokine receptor antagonist in a pharmaceutically acceptable carrier for use in a method of improving cognitive function in a human by enhancing the formation of synapses and dendritic spines, wherein further the D-peptide comprises the general structure: A-B-C-D-E-F-G-H, wherein:

a is Ala or is absent,

b is Ser, Thr or absent,

c is Ser, Thr or absent,

d is Ser, Thr, Asn, Glu, Arg, Ile, Leu,

e is Ser, Thr, Asp or Asn,

f is Thr, Ser, Asn, Arg, Gln, Lys, Trp,

g is Tyr, and

h is Thr, Ser, Arg, Gly,

wherein said D peptide further comprises the case where all amino acids are in D stereoisomeric configuration, except when Gly is present and is achiral, and wherein

The composition is useful for preventing the formation of actin/silk-cutting protein rods, which enhances the formation of synapses and dendritic spines, and thereby improves cognitive function in said human.

Embodiment 2. the composition for use according to embodiment 1, wherein said human experiences symptoms of loss of synapses and dendritic spines.

Embodiment 3. the composition for use according to embodiment 2, wherein said symptoms of synapse and dendritic spine loss are selected from the group consisting of: cognitive loss associated with aging, mild cognitive impairment, cognitive loss associated with dementia, cognitive loss associated with brain trauma, neuropathy.

Embodiment 4. the composition for use according to embodiment 2, wherein said symptoms of synapse and dendritic spine loss are selected from the group consisting of: alzheimer's disease, frontal dementia, Huntington's disease, synucleinopathy, Parkinson's disease, Lewy body disease, neuro-AIDS/HAND, demyelinating diseases, HTLV-1-associated myelopathy (HAM), Multiple Sclerosis (MS), amyotrophic lateral sclerosis, diabetic cognitive decline, normal aging, or pathological neurological symptoms and manifestations that occur after brain injury, stroke, diabetes, neuropathy, encephalopathy, and viral encephalopathy.

Embodiment 5. the composition for use according to embodiment 1, wherein the administration of the composition to the patient is selected from the group consisting of: oral, buccal, parenteral, topical, rectal, vaginal, by intranasal inhalation spray, by intrapulmonary inhalation.

Embodiment 6. the composition for use according to embodiment 1, further comprising,

the D peptide is up to twenty (20) D amino acid residues in length, except when Gly is present and the Gly is achiral, and comprises five consecutive D amino acid residues having a sequence selected from the group consisting of:

Thr Thr Asn Tyr Thr,(SEQ ID NO:1)

Ser Ser Thr Tyr Arg,(SEQ ID NO:2)

Thr Thr Ser Tyr Thr,(SEQ ID NO:3)

Asn Thr Arg Tyr Arg,(SEQ ID NO:4)

Ile Asp Asn Tyr Thr,(SEQ ID NO:5)

Asn Thr Ser Tyr Arg,(SEQ ID NO:6)

Ile Asn Asn Tyr Thr,(SEQ ID NO:7)

asn Thr Ser Tyr Gly, (SEQ ID NO:8) and

Glu Thr Trp Tyr Ser.(SEQ ID NO:9)。

embodiment 7. the composition for use according to embodiment 6, further comprising,

derivatives of the D-peptide are up to twelve (12) D amino acid residues in length.

Embodiment 8. the composition for use according to embodiment 6, further comprising,

derivatives of the D-peptide are up to eight (8) D amino acid residues in length.

Embodiment 9. the composition for use of embodiment 6, further comprising that said D peptide is five (5) D amino acid residues in length.

Embodiment 10. a composition comprising a peptide analog and a pharmaceutically acceptable carrier for use in a method of treating synapses and dendritic spine loss in a human experiencing one or more symptoms of synapses and dendritic spine loss, wherein

The peptide analog is [ D-Ala1] -Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH2

Wherein the first amino acid is a D stereoisomer and the remaining amino acids are L stereoisomers, and the N-terminal amino acid is an amide, and wherein the composition functions to treat synapse and dendritic spine loss in the patient.

Embodiment 11 the composition for use according to embodiment 10, wherein the symptoms of synapse and dendritic spine loss are selected from the group consisting of: alzheimer's disease, Huntington's disease, synucleinopathies, Parkinson's disease, demyelinating diseases, HTLV-1-associated myelopathy (HAM), Multiple Sclerosis (MS), amyotrophic lateral sclerosis, pathological neurological symptoms after injury or trauma, encephalopathy and viral encephalopathy.

Cited documents

1.Hill J,Mervis R,Avidor R,Moody T,Brenneman D.HIV envelope protein-induced neuronal damage and retardation of behavioral development in rat neonates.Brain Res.1993；603(2):222-33.

2.Zhou M,Greenhill S,Huang S,Silva TK,Sano Y,Wu S,Cai Y,Nagaoka Y,Sehgal M,Cai DJ,Lee YS,Fox K,Silva AJ.CCR5 is a suppressor for cortical plasticity and hippocampal learning and memory.Elife.2016；5.Epub 2016/12/21.doi:10.7554/eLife.20985.PubMed PMID:27996938；PMCID:PMC5213777.

3.Joy MT,Ben Assayag E,Shabashov-Stone D,Liraz-Zaltsman S,Mazzitelli J,Arenas M,Abduljawad N,Kliper E,Korczyn AD,Thareja NS,Kesner EL,Zhou M,Huang S,Silva TK,Katz N,Bornstein NM,Silva AJ,Shohami E,Carmichael ST.CCR5 Is a Therapeutic Target for Recovery after Stroke and Traumatic Brain Injury.Cell.2019；176(5):1143-57e13.Epub 2019/02/23.doi:10.1016/j.cell.2019.01.044.PubMed PMID:30794775.

4.Padi S,Shi X,Zhao Y,Ruff M,Baichoo N,Pert C,Zhang J.Attenuation of rodent neuropathic pain by an orally active peptide,RAP-103,which potently blocks CCR2-and CCR5-mediated monocyte chemotaxis and inflammation.Pain.2012；153(1):95-106.doi:S0304-3959(11)00579-3[pii]10.1016/j.pain.2011.09.022.PubMed PMID:22033364；PMCID:22033364。

Sequence listing

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<120> oral peptide antagonists of multiple chemokine receptors for reversing synaptic and dendritic spine loss

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<210> 6

<211> 5

<212> PRT

<213> Human immunodeficiency virus (Human immunodeficiency virus)

<400> 6

Asn Thr Ser Tyr Arg

1 5

<210> 7

<211> 5

<212> PRT

<213> Human immunodeficiency virus (Human immunodeficiency virus)

<400> 7

Ile Asn Asn Tyr Thr

1 5

<210> 8

<211> 5

<212> PRT

<213> Human immunodeficiency virus (Human immunodeficiency virus)

<400> 8

Asn Thr Ser Tyr Gly

1 5

<210> 9

<211> 5

<212> PRT

<213> Human immunodeficiency virus (Human immunodeficiency virus)

<400> 9

Glu Thr Trp Tyr Ser

1 5

Claims (11)

1. A method of improving cognitive function in a human by enhancing the formation of synapses and dendritic spines, the method comprising:

administering a composition comprising a D-peptide multi-chemokine receptor antagonist in a pharmaceutically acceptable carrier,

further, the D peptide comprises the following general structure: A-B-C-D-E-F-G-H, wherein:

a is Ala or is absent,

b is Ser, Thr or absent,

c is Ser, Thr or absent,

d is Ser, Thr, Asn, Glu, Arg, Ile, Leu,

e is Ser, Thr, Asp or Asn,

f is Thr, Ser, Asn, Arg, Gln, Lys, Trp,

g is Tyr, and

h is Thr, Ser, Arg, Gly,

wherein the D peptide further comprises all amino acids in a D stereoisomeric configuration, except when Gly is present and Gly is achiral, and wherein said administering comprises administering to the human said composition in a therapeutically effective dose, wherein said composition is for preventing formation of actin/silk cutting protein rods, which enhances formation of synapses and dendritic spines, and thereby improves cognitive function in the human.

2. The method of claim 1, wherein the human further comprises a human experiencing symptoms of loss of synapses and dendritic spines.

3. The method of claim 2, wherein the symptom of synapse and dendritic spine loss is selected from the group consisting of: cognitive loss associated with aging, mild cognitive impairment, cognitive loss associated with dementia, cognitive loss associated with brain trauma, neuropathy.

4. The method of claim 2, wherein the symptom of synapse and dendritic spine loss is selected from the group consisting of: alzheimer's disease, frontal dementia, Huntington's disease, synucleinopathy, Parkinson's disease, Lewy body disease, neuro-AIDS/HAND, demyelinating diseases, HTLV-1-associated myelopathy (HAM), Multiple Sclerosis (MS), amyotrophic lateral sclerosis, diabetic cognitive decline, normal aging, or pathological neurological symptoms and manifestations that occur after brain injury, stroke, diabetes, neuropathy, encephalopathy, and viral encephalopathy.

5. The method of claim 1, wherein administering the composition to the patient is selected from the group consisting of: oral, buccal, parenteral, topical, rectal, vaginal, by intranasal inhalation spray, by intrapulmonary inhalation.

6. The method of claim 1, further comprising,

the D peptide is up to twenty (20) D amino acid residues in length, except when Gly is present and the Gly is achiral, and comprises five consecutive D amino acid residues having a sequence selected from the group consisting of:

Thr Thr Asn Tyr Thr,(SEQ ID NO:1)

Ser Ser Thr Tyr Arg,(SEQ ID NO:2)

Thr Thr Ser Tyr Thr,(SEQ ID NO:3)

Asn Thr Arg Tyr Arg,(SEQ ID NO:4)

Ile Asp Asn Tyr Thr,(SEQ ID NO:5)

Asn Thr Ser Tyr Arg,(SEQ ID NO:6)

Ile Asn Asn Tyr Thr,(SEQ ID NO:7)

asn Thr Ser Tyr Gly, (SEQ ID NO:8) and

Glu Thr Trp Tyr Ser.(SEQ ID NO:9)。

7. the method of claim 6, further comprising,

derivatives of the D-peptide are up to twelve (12) D amino acid residues in length.

8. The method of claim 6, further comprising,

derivatives of the D-peptide are up to eight (8) D amino acid residues in length.

9. The method of claim 6, further comprising the D peptide is five (5) D amino acid residues in length.

10. A method of treating synapse and dendritic spine loss in a human experiencing one or more symptoms of synapse and dendritic spine loss, said method comprising the steps of:

administering a composition comprising the peptide analog and a pharmaceutically acceptable carrier,

the peptide analog is [ D-Ala1] -Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH2

Wherein the first amino acid is the D stereoisomer and the remaining amino acids are the L stereoisomer, and the N-terminal amino acid is an amide, and

wherein said administering comprises administering a therapeutically effective dose of said peptide analog to said patient, wherein said composition acts to treat synapse and dendritic spine loss in said patient.

11. The method of claim 10, wherein the symptoms of synapse and dendritic spine loss are selected from the group consisting of: alzheimer's disease, Huntington's disease, synucleinopathies, Parkinson's disease, demyelinating diseases, HTLV-1-associated myelopathy (HAM), Multiple Sclerosis (MS), amyotrophic lateral sclerosis, pathological neurological symptoms after injury or trauma, encephalopathy and viral encephalopathy.