CYCLIC PEPTIDOMIMETIC FOR THE TREATMENT OF NEUROLOGICAL DISORDERS

The present invention provides to compositions and methods useful in the treatment of neurological disorders including, but not limited to, Angelman syndrome, depression, traumatic brain injury, stroke, and Alzheimer's disease. CN2097, a rationally designed cyclic peptidomimetic drug that has been demonstrated to have effectiveness in preclinical models for the treatment of neurological disorders, is rapidly cleared and has a short half-life. The present invention provides a stable analog of CN2097.

FIELD OF THE INVENTION

The embodiments of the present invention relate to compositions and methods useful in the treatment of neurological disorders including, but not limited to, depression, Angelman syndrome, traumatic brain injury, stroke, and Alzheimer's disease.

BACKGROUND OF THE INVENTION

CN2097 (R7Cs-sCYK[KTE(β-Ala)]V) is a rationally designed cyclic peptidomimetic that has been demonstrated to have effectiveness in preclinical models for the treatment of neurological disorders such as Angelman syndrome, traumatic brain injury (TBI), and stroke.1CN2097 and related compounds have been described in several patents and published patent applications.2CN2097 selectively targets the PDZ binding domain of the PSD-95, a scaffolding protein involved in the development and regulation of neuronal synapses CN 2097 has demonstrated specificity for the NMDA receptor 2B (NR2B) subunit. The association of PSD-95 with NMDA receptors is an element in the propagation of the synaptic changes of LTP and long-term depression (LTD) that induce learning and memory. cPSD-95 is a member of the MAGUK-family of PDZ domain-containing proteins.

CN2097 has all the advantages of peptide-based drugs (high potency and activity, low toxicity), while small enough to remain stable and cross the blood-brain barrier (BBB). Although highly stable, the half-life of CN2097 is drastically reduced (˜1 h in plasma) by the attached poly-arginine transport moiety3required for the transport through the blood brain barrier (BBB). Since CN2097 is cleared in a matter of hours, it's clinical potential is severely limited as it would have to be administered frequently to sustain any beneficial effects.

Because of the tremendous therapeutic potential of CN2097 for the treatment of neurological disorders, there is a need for the development of a more stable CN2097 analog with a longer circulating half-life, enabling the administration of lower doses while maintaining its therapeutic effectiveness.

BRIEF SUMMARY OF THE INVENTION

The embodiments of the present invention provide dR7-2097, a more stable CN2097 analog consisting of all dextrorotary (D)-amino acids, which was shown to be more efficacious than CN2097 in animal models of neurological disorders.

In one embodiment, the present invention provides an analog of CN-2097. CN-2097 has the following structure:

The analog is represented by dR7-2097 and essentially consists of dextrorotatory (D)-amino acids (dR7-NH2).

In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically-effective amount of the analog of CN-2097, dR7-2097, or a pharmaceutically-acceptable salt thereof, and one or more pharmaceutically-acceptable carrier.

In another aspect, the present invention provides a method of preventing or treating a neurological disorder in a subject at risk of, or afflicted with, the neurological disorder comprising administering a composition comprising (a) a therapeutically-effective amount of the analog of CN-2097, dR7-2097, and (b) at least one pharmaceutically-acceptable carrier. The neurological disorders include, but are not limited to, Angelman syndrome, depression, traumatic brain injury, stroke, and Alzheimer's disease.

In one embodiment, the neurological disorder is depression. In another embodiment, the depression is treatment-resistant depression. In yet another embodiment, the depression is major depressive disorder, dysthymic disorder, psychotic depression, postpartum depression, seasonal affective disorder, bipolar disorder, bipolar depression, mood disorder, depressions caused by chronic medical conditions such as cancer or chronic pain, chemotherapy, chronic stress, post-traumatic stress disorders, or manic depressive disorder.

In yet another embodiment, the neurological disorder is Angelman syndrome, traumatic brain injury, spinal cord injury, glaucoma, stroke, cerebrovascular accident (CVA), or Alzheimer's disease.

DETAILED DESCRIPTION OF THE INVENTION

It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention.

Definitions

As used herein, the term “approximately” or “about” in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to “approximately” or “about” a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to “about X” includes description of “X”.

As used herein, the term “subject” refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of either sex.

As used herein, the terms “effective amount” and “therapeutically-effective amount” include an amount sufficient to prevent or ameliorate a manifestation of disease or medical condition, such as Angelman syndrome, depression, traumatic brain injury, and Alzheimer's disease. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds.

As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of a symptom or condition compared to that expected in the absence of treatment.

As used herein, the term “long-term” administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. This includes that the therapeutic agent or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1, 2, 3, 5, 7 or 10 years, or more.

The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, compositions are administered parenterally. The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in THEMERCKMANUAL OFDIAGNOSIS ANDTHERAPY;4THEENCYCLOPEDIA OFMOLECULARCELLBIOLOGY ANDMOLECULARMEDICINE;5MOLECULARBIOLOGY ANDBIOTECHNOLOGY: ACOMPREHENSIVEDESKREFERENCE;6IMMUNOLOGY;7JANEWAY'SIMMUNOBIOLOGY;8LEWIN'SGENESXI;9MOLECULARCLONING: A LABORATORYMANUAL;16BASICMETHODS INMOLECULARBIOLOGY;11LABORATORYMETHODS INENZYMOLOGY;12CURRENTPROTOCOLS INMOLECULARBIOLOGY(CPMB);13CURRENTPROTOCOLS INPROTEINSCIENCE(CPPS);14and CURRENTPROTOCOLS INIMMUNOLOGY(CPI).15

Pharmaceutical Compositions

The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically-acceptable carrier.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art.16

The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; poultry; and pets in general.

In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

As described above, CN2097 (R7Cs-sCYK[KTE(β-Ala)]V) is a rationally designed peptidomimetic that has been demonstrated to have effectiveness in preclinical models for the treatment of neurological disorders such as Angelman syndrome, traumatic brain injury (TBI), and stroke. CN2097 was developed through a rational drug design approach and consists of a cyclic PDZ binding motif through a lactam ring and β-alanine linker based on last five C-terminal residues of the CRIPT scaffolding protein.17Although the PDZ-binding cyclic-ligand of CN2097 is highly stable, the half-life of CN2097 is drastically reduced (˜1 h in plasma) due to the attached polyarginine transport moiety required for the transport through the BBB. Since CN2097 breaks down in a matter of hours, it's clinical potential is severely limited as it would have to be administered frequently to sustain any beneficial effects.

In the present invention, a modified CN2097 analog was developed that exhibits improved affinity and specificity. An all D-polyarginine was synthesized and coupled to the cyclic PDZ-binding motif to generate dR7-2097, a more stable CN2097 analog consisting solely of dextrorotary (D)-amino acids (dR7-NH2).

The synthesis of CN2097 has been described elsewhere.18The analog dR7-2097 was synthesized by Peptides International. For the studies described in the Examples below, highly pure CN2097 and dR7-2097 (>98% purity; 100 mg) were synthesized using our Fmoc/tBu solid phase chemistry protocol.19

As shown in the Examples below, dR7-2097 demonstrated superior efficay compared to CN2097 in various models of neurological disorders.

Antidepressant Effects of CN2097 and dR7-2097

Major Depressive Disorder (MDD) is a debilitating psychiatric disorder that affects ˜7% of U.S. adults in their lifetime. MDD affects over 16 million of the U.S. population and represents a major cause of disability worldwide.20Drugs that increase monoaminergic signaling are the mainstay of depression therapy, however, these drugs have a delayed onset of action and are only effective in about 50% of patients.

Aberrant brain-derived neurotrophic factor (BDNF) signaling has been proposed to underlie the pathophysiology of major depressive disorder21and bipolar disorder.22Human studies and animal models support that depression is associated with reduced expression of BDNF.23The hippocampus is one of several limbic structures implicated in the pathophysiology of depression,24,25,26where BDNF and its high-affinity TrkB receptor play a critical role in synaptic plasticity.27Stress, a risk factor for depression, can result in hippocampal neuronal atrophy,28characterized by reduced synaptic connections.29The administration of the selective serotonin-reuptake inhibitor (SSRI) fluoxetine prevents stress-induced atrophy of dendrites and spines,30and reduction of TrkB receptor signaling blocks this effect.31

An advance in treatment was the finding that a single infusion of a sub-anesthetic dose of ketamine, an N-methyl-D-aspartate (NMDA) receptor blocker, produces rapid antidepressant effects in conventional treatment-resistant depression (TRD) patients.32However, ketamine's effects are temporary and limited due to cognitive side effects associated with NMDA receptor inhibition. BDNF is necessary for the rapid antidepressant effects of ketamine33as well as the latent response to conventional antidepressants.34

The discovery of rapidly acting antidepressants, such as ketamine, has transformed our ideas about depression treatment. CN2097 was developed through a rational drug design approach and consists of a cyclic PDZ binding motif through a lactam ring and β-alanine linker based on last five C-terminal residues of the CRIPT scaffolding protein.35

CN2097 selectively targets the PDZ binding domain of the PSD-95, a scaffolding protein involved in the development and regulation of neuronal synapses. The PDZ domain is a common structural domain of 80-90 amino-acids found in the signaling proteins of bacteria, yeast, plants, and animals. PDZ is an acronym combining the first letters of three proteins—post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (zo-1), which were first discovered to share the domain. PDZ domains are also referred to as DHR (Dlg homologous region) or GLGF (glycine-leucine-glycine-phenylalanine) (SEQ ID NO: 1) domains. These domains have been reported as helping to anchor transmembrane proteins to the cytoskeleton and hold together signaling complexes.

CN2097 has demonstrated specificity for the NMDA receptor 2B (NR2B) subunit. The association of PSD-95 with NMDA receptors is an element in the propagation of the synaptic changes of long-term potentiation (LTP) and long-term depression (LTD) that induce learning and memory. cPSD-95 is a member of the MAGUK-family of PDZ domain-containing proteins. Its basic structure includes three PDZ domains, an SH3 domain, and a guanylate kinase-like domain (GK).

A unique effect of CN2097 is that it acts downstream of TrkB to enhance long-lasting increases in synaptic strength in a BDNF-dependent manner. Our studies in Angelman mice,36indicate that the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95) is a TrkB-associated protein essential for Akt-mTORC1 and PLC-CaMKII signaling. We previously designed a soluble cyclic-peptide drug, CN2097 (FIG. 1; MW 2376), that targets the PDZ3-domain of PSD-95 to potentiate TrkB signaling.37As depression is associated with memory impairment,38it was hypothesized that CN2097 could provide a potential new treatment for cognitive deficits.

As described in the Examples below, our preclinical studies show that CN2097 can induce rapid antidepressant effects and dR7-2097 demonstrated superior efficay compared to CN2097.

The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

EXAMPLES

Antidepressant Effects of CN2097

Depressive symptomatology has been reliably characterized using exogenous corticosterone (CORT) administration, e.g., dendritic atrophy in hippocampal pyramidal cells with loss of CA3 and CA1 apical dendrites.39Importantly, CORT administration leads to impaired BDNF-mediated signaling and synaptic plasticity in limbic and forebrain regions. In this model, ketamine, which is known to be effective for the treatment of TRD, significantly reduced depressive-like behavior.40

We hypothesize that antidepressant-like effects of ketamine and CN2097 are mediated via the same signaling pathways, e.g., by the activation of mTOR. The effects of i.v. CN2097 on the depressive-like behavior of repeated corticosterone-injected mice were examined in the Chronic mild stress (CMS) animal model, which comprise both physical and social stressors.41

Mice were divided into two groups, namely, control (Ctrl) and CORT. The mice in the CORT group were injected with CORT (i.p., 20 mg/kg), once per day at random times during the light phase. Control mice were injected with the vehicle.42A schematic of the model of depression is illustrated inFIG. 2A.

As illustrated inFIG. 2, CN2097 reversed depression-like behavior in mice; CN2097 significantly decreased time spent immobile in both the TST and the FST. CN2097 (tail vein injection, 12 hours before TST onset) significantly decreased the time spent immobile in the TST (FIG. 2B) but did not affect the immobility time in the FST (data not shown). CN2097 (tail vein injection, 1 week before TST onset) significantly decreased the time spent immobile in both the TST and the FST (FIG. 2C).

As shown inFIG. 3, a single injection of CN2097 also exhibited anti-depressant efficacy in the CMS model within 2 hours, suggesting a rapid antidepressant response. Day 7 after injection of CN2097, immobility times in the FST and TST were tested as described in Marshall (2018).43The acute injection of CN2097 significantly reduced the duration of immobility time in both the Forced Swim Test (FIG. 3, left panel) and the Tail Suspension Test (FIG. 3, right panel).

As described above, aberrant brain-derived neurotrophic factor (BDNF) signaling has been proposed to underlie the pathophysiology of major depressive disorder and bipolar disorder. An advance in treatment was the finding that a single infusion of a sub-anesthetic dose of ketamine, an NMDA receptor blocker, produces rapid antidepressant effects in conventional treatment-resistant depression (TRD) patients. However, ketamine's effects are temporary and limited due to cognitive side effects associated with NMDA receptor inhibition. BDNF is necessary for the rapid antidepressant effects of ketamine as well as the latent response to conventional antidepressants. With over 5 million people in the U.S. suffering from treatment-resistant depression, there is an urgent need for the development of new antidepressant drugs. CN2097 has been shown to potentiate BDNF signaling pathways.

In the present Example 1, CN2097 was shown to produce rapid antidepressant effects in a chronic mild stress (CMS) mouse model, suggesting that this cyclic peptidomimetic compound is a potential therapeutic for TRD.

CN2097 Reverses Behavioral Alterations Induced in the CMS Model

To further test CN2097, we used the Chronic Mild Stress (CMS) model which has been shown to evoke anxiety and lower sucrose consumption (postulated to reflect anhedonia), symptoms associated with MDD. As described in Marshall (2018),44mice subjected to repeated daily stress for 3 weeks, then received a single injection of CN2097 (10 mg/kg) or vehicle.

The acute effects of CN2097 on anxiety were evaluated by the elevated plus-maze (EPM) and novelty-suppressed feeding (NSF) tests. As shown inFIG. 4(left panel), CN2097 increased the time spent in open arms in the EPM.

In the NSF test, anxiety-induced hypophagia was assessed by measuring the latency of mice to eat a familiar food in an aversive environment. As shown inFIG. 4(right panel), the administration of CN2097 1 hour prior to testing significantly shortened the latency period until feeding. These data suggest that CN2097 effectively reverses behavioral alterations induced in the CMS Model.

The CMS model responds to chronic, but not acute, administration of established antidepressant drugs.45Based on the predictive value of the CMS model,46the above-described data showing that CN2097 caused a reversal of anhedonic and other behavioral effects within 2 hours (FIGS. 3-4) suggests that it provides rapid ketamine-like antidepressant actions.

Antidepressant Effects of CN2097 and dR7-2097

The discovery of rapidly acting antidepressants, such as ketamine, has transformed our ideas about depression treatment. Based on the above-described data (FIGS. 2-4), CN2097 elicits rapid antidepressant effects that merits further testing. Moreover, although dR7-2097 is a stable analog that is not cleared rapidly like CN2097, its functional efficacy remains to be determined and compared to CN2097 in an animal model of depression. Accordingly, the effects of CN2097 and dR7-2097 on memory recall were assessed in the Contextual Fear Conditioning (CFC) test.47

As shown in theFIG. 5, dR7-2097 was more efficacious than CN2097 in significantly increasing memory recall in the CFC test. These results show that it is possible to modify CN2097 with (D)-amino acids and not only maintain the efficacy but enhance it. The suggests that the increased stability will enable the administration of lower doses (10 fold or more) while maintaining anti-depressive properties.

Effects of CN2097 and dR7-2097 in an Angelman Syndrome Mouse Model

The functional efficacy of dR7-2097 was also assessed and compared to CN2097 in an animal model of Angelman Syndrome (AS).

Disruptions in long-term potentiation (LTP) lead to disruptions in memory and has been associated with several neurological disorders, including autism syndromes and neurodegenerative disorders. LTP failure is also implicated in AS. It is believed that AS is a neurogenetic disorder characterized by intellectual and developmental delay, sleep disturbance, seizures, jerky movements especially hand-flapping, frequent laughter or smiling, and usually a happy demeanor. Some believe that AS is a classic example of genetic imprinting in that it is usually caused by deletion or inactivation of genes on the maternally inherited chromosome 15. The related syndrome, Prader-Willi syndrome, is believed caused by a similar loss of paternally-inherited genes.

AS is a result of loss-of-function mutations or deletions in the maternally inherited allele of UBE3A. Mice that are deficient in maternal Ube3a also show genetically reversible deficits in learning and the induction of hippocampal (LTP),48,49,50a form of synaptic plasticity. Angelman syndrome (AS) is caused by disruptions in function due to mutations and/or deletions in the maternally inherited allele of an E3 ubiquitin ligase, UBE3A, a gene that has also been implicated in the broader spectrum of Autism patients.

A mouse model for AS carries the maternally inherited Ube3a-null mutation and shows profound impairments in hippocampal synaptic plasticity.51It has been reported that UBE3A is also required for experience dependent maturation in the visual cortex. In particular, AS mice show deficits in long-term potentiation (LTP) and long-term depression (LTD), which are the cellular substrates of learning and memory. These forms of synaptic plasticity are highly regulated by activity-dependent changes in the ratio of NMDA subunits, specifically NR2A and NR2B subunits. NMDA receptors gate the influx of calcium ions across the post-synaptic membrane. Depending on the characteristics of the calcium influx generated in response to pre-synaptic activity, either LTP or LTD can be induced. This calcium influx activates calcium-dependent enzymes such as CaMKII, which has been strongly implicated in AS, and triggers its autophosphorylation at Thr286. This autophosphorylation is very important as it allows the enzyme to remain active once the calcium levels return to normal. This pathway requires an interplay between the NMDA receptor, CaMKII and a post-synaptic scaffolding protein (PSD-95 in particular), which brings these biochemical elements into proximity. Thr286 autophosphorylation is instrumental in promoting the association of CaMKII with the PSD by directly binding to the NMDA receptor. Interestingly, there is also a secondary inhibitory autophosphorylation at Thr305/306 that has been suggested to regulate the association of CaMKII to the PSD. Interplay between these states can govern the availability of CaMKII to regulate synaptic plasticity in response to calcium influx during learning and memory activity. The clinical features of AS have been linked to the misregulation of alpha CaMKII function. The role of CaMKII in synaptic plasticity and AS has been investigated at the synapses of the Schaffer-collateral-CA1 pyramidal cells in the adult hippocampus.

CN2097 has previously been shown to restore hippocampal LTP in an Angelman Syndrome mouse model.52After in vivo administration of CN2097, the mouse was sacrificed and CA1 hippocampal LTP measured by extracellular field potential recordings, and the level of potentiation compared to control C57BL/6 mice, which robustly generate long-lasting LTP. In the present study, we tested whether in vivo administration dR7-2097 would reverse the well-established LTP impairment observed in the hippocampus of AS mice.

Typically, 10 mg/kg of CN2097 needs to be injected into AS mice and use of a severe 3×HFS protocol in order to reinstate LTP in AS hippocampal brain slices. With dR7-2097, a lower dose (2.5 mg/kg) and a modest LTP-inducing stimulus of 2×HFS protocol was sufficient to significantly increased the LTP induction rate in AS hippocampal slices prepared 2 hours after dR7-2097 injection. LTP was observed in 67% of AS slices recorded with a mean fEPSP slope of 221 (n=2 of 3). As shown inFIG. 6, dR7-2097 was more efficacious than CN2097 in restoring hippocampal LTP in the AS mouse model.

Treatment Oof Neural Injuries

A stroke, or cerebrovascular accident (CVA), is the rapid loss of brain function due to disturbance in the blood supply to the brain. This can be due to ischemia (lack of blood flow) caused by blockage (thrombosis, arterial embolism), or a hemorrhage (broadly termed “neurological insult”). As a result, and often worsening over time, brain injury becomes permanent dysfunction/damage. Typically, affected areas of the brain have diminished or complete loss of function. In particular instances this is exhibited as an inability or reduced ability to move one or more limbs on one side of the body, inability to or difficulty in understanding or formulating speech, or an inability to see one side of the visual field. In the case of stroke, secondary neuronal damage following the initial insult is a significant factor in patient decline and eventual death. This decline manifests itself weeks to months following the initial stoke and, without effective therapeutic intervention, leads to a slow but continuous decline in the patients physical and mental health.

In the case of TBI, the progression of damage is typically slower than that in stroke. Recent reports, however, establish TBI as equally destructive in outcome in the more severe cases. Current reports find that about 40% of people with TBI exhibit a continuing decline in both physical and mental capacity. This represents a significantly higher incidence of dementia, in some instances is an aggravating factor in death.

An important consideration in therapeutic treatment and improved outcomes in patients afflicted with neuronal injury (including either Stroke or TBI) is to block secondary injury of surrounding neurons that were not, in the first instance, directly affected by the primary insult. Literature reports that secondary neuronal damage occurs after a variety of brain insults including subarachnoid hemorrhage, stroke, and traumatic brain injury and involves metabolic cascades. Noted metabolic aspects include prolonged secondary ischemia, cerebral hypoxia, hypotension, cerebral edema, changes in the blood flow to the brain; and raised intracranial pressure. Other secondary insults include hypercapnia, acidosis, meningitis, and brain abscess. In addition, alterations in the release of neurotransmitters particularly glutamate can cause excitotoxic neuronal cell death by causing free radicals generation and oxidative stress within these neurons that leads to neurodegeneration.

CN2097 and dR7-2097 are both capable of neuroprotection. Without being bound by any particular theory, reference is made to the strong positive charge of the polyarginine compositions, e.g., over all charge of the peptide is +7 with 7 arginine residues. Notably, substituting lysine (also positively charged) for arginine in the presence or absence of a terminal cysteine did not provide neuroprotection. Further, a peptide with at least 5 or more arginine residues can be flanked by 1 or 2 neutral amino acids and still be therapeutically active.

Because it is cleared less rapidly, dR7-2097 is more effective than CN2097 for neuroprotection. Reference is made to the chiral nature of arginine and cysteine, e.g., d-based compositions (e.g., d-R) had superior neuroprotective properties. The terminal cysteine(s) are contemplated as attached at either/both the N-terminus or C-terminus via peptide bond and to reduce the reactivity of the exposed SH group, will have the option of being protected by way of a disulfide linkage to a second cysteine (designated here as C-s-s-C).

Dosing is stated for dR7-2097 compositions but is similar for all compositions disclosed herein. Broadly, therapeutically effective dosages intraventricular or intrathecal administrations range from about 1,000 μg/kg to about 1 μg/kg. For i.v administrations, therapeutically effective dosages from about 0.01 mg/kg to about 20 mg/kg. All forms of administration are contemplated. Dosing should be instituted is soon as possible after the subject presents with a neuronal injury, e.g., stroke-like indicia or traumatic brain injury.

In a test model, the retina was protected from NMDA-induced chemical stroke. Notably d-amino acid peptides are resistant to most protease digestion and, in some embodiments, exhibit longer half-life in vivo. The compositions of this invention are useful in a variety of clinical situations, most of which are characterized by neurological insult or cellular insult typified by mitochondrial damage. Without being bound by any particular theory, untreated mitochondrial damage is believed to lead to generation of free radical species. The compositions of this invention appear to avert or curtail oxidative stress in the retina following NMDA-insult by reducing mitochondrial originated free radical generation. Without being bound by any particular theory, such activity is believed to be significant in neuroprotection arising from the compositions of this invention.

Particular reference is made to neuronal injuries such as stroke and TBIs (including closed head injuries, e.g., sports-related injuries, accidental injuries involving a concussion, blast injuries (combat or other). In one embodiment, football players and boxers are dosed prophylactically prior to a game, practice, or bout. Additionally, dosing may be immediately after first contact in a scheduled game or bout. Dosing is based on an assumption of neurological insult or incipient damage without regard to actual (detected) presenting symptoms.

PROPHETIC EXAMPLES

Stroke Patient

A 57 year old male enters the emergency room and is diagnosed with an ischemic event involving the middle cerebral artery, with symptomatology onset being under 1 hour. Immediately, the patient was administered an i.v. supplemented with dR7-2097 while blood supply to the restricted area is restored.

Daily testing of cognitive and motor systems show no deficits in physical or mental capacities. In addition, CT/MRI imaging show no signs of lesioning in the affected region, immediately following recovery and that this was maintained and confirmed by follow-up imaging and behavior testing.

A 64 year old woman is revived following stoke in her home by EMS and is rushed to the nearest hospital 80 miles away. Upon arriving at the hospital, it is determined that the patient is showing signs of weakness on her left side. In addition, she complains of having blurred vision and exhibits difficulty in speaking clearly. She is immediately started an i.v. and administered dR7-2097 (4 mg/kg/hr over a period of 5 days) to arrest post-stroke neuronal damage.

The patient is monitored for recovery signs. Improvement in her speech pattern is noted over the next 30 days as is increased muscle tone on the left side and muscle recovery strength and usage of her effected limbs.

TBI Patient

A 17 year old high school football player is rushed to the emergency room of a local hospital suffering from a severe concussion following a helmet to helmet impact with an opposing player. The patient is initially unconscious on the field for over 20 min. EMS personnel arrived to find the patient responding to stimuli, but completely disoriented and unable to coordinate muscle movement, articulate speech or focus eye movements.

Upon arriving at the ER, the patient is administered an i.v. containing dR7-2097 at 0.05 mg/kg. Delivery is continued until brain swelling resulting from the impact returns to normal state (3-5 days). Without being bound by any particular theory, the therapeutic is to arrest neuronal cell death resulting from these documented post-traumatic effects.

Monitoring the patient though this period (and beyond) shows that he remains symptom free of TBI and post-concussion effects (i.e., avoiding short-term memory loss or exhibiting longer-term learning deficits).

TBI Patient

The same scenario as above is noted here, except the dR7-2097 dosage is 20 mg/kg.

Spinal Cord Injury

A 30 year old female presents with a spinal cord injury following an automobile accident. The subject is conscious but unable to move her legs. Multi-slice spiral computed tomography (MSCT) demonstrates a foreign body in spinal canal at the level of the Th11-Th12. A Th(11)-L(2) laminectomy is performed along with retrieval of foreign bodies and dura repair. Upon admission, the patient is administered an i.v. containing dR7-2097 at 0.15 mg/kg. Delivery of the peptide is continued every other day for 30 days when substantial ability to move her legs is returned.

Glaucoma

A 60 year old female presents with a complaint of blurry vision in her left eye for 1 week. Her intraocular pressure (IOP) is above 21 mmHg. A diagnosis of glaucoma is made.

Effort is made to surgically reduce the IOP. In post-surgical monitoring, IOP levels remain above normal accepted levels. To prevent retinal damage resulting from the prolonged exposure to elevated IOP, the patient is administered dR7-2097 in the form of eye drops suspended in a corneal penetrating lipophilic suspension. Each 20 μL drop contains 1.25 μg of dR7-2097. The total applied/treatment is 2.5 μg dR7-2097; two drops are administered morning and two drops at night.

Treatment is continued chronically, and no progression of neuronal cell death is detected.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the present aspects and embodiments. The present aspects and embodiments are not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect and other functionally equivalent embodiments are within the scope of the disclosure. Various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects described herein are not necessarily encompassed by each embodiment. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

REFERENCES