WO2003039487A2 - Cyclo(prolyl-glycine) and methods of use to treat neural disorders - Google Patents

Abstract

Embodiments of pharmaceutical compositions comprising cyclo(Pro-Gly) and methods for use in treating neural degeneration are provided. Cyclic PG substantially prevents toxic neural degeneration and cell death, and promotes neurite outgrowth in neurons, especially cerebellar neurons. The neuroprotective and neurorenenerative effects of cPG are useful to treat behavioural neurological deficits involving motor control pathways.

Description

CYCLO(PROLYL-GLYCINE) AND METHODS OF USE TO TREAT NEURAL DISORDERS

Related Applications

This application claims priority to New Zealand Provisional Application Serial No: 515371 titled "Therapeutic Agent, Composition and Method" filed 9 November 2001, New Zealand Provisional Application Serial No: 515432, titled "Therapeutic Agent, Composition and Method" filed 13 November 2001 and to New Zealand Provisional Application Serial No: 515551, titled "Composition and Methods to Improve Neural Outcome" filed 16 November 2001. Each of these Provisional Applications is herein incoφorated fully by reference.

Field of the Invention

This invention relates to compositions comprising cyclo(Prolyl-Glycine) "cyclo(Pro-Gly)," "cyclic PG" or "cPG") and methods for its use in the treatment or prevention of cell damage in animals, and more particularly relates to compositions and methods of treating injury or disease of neurons in the central nervous system (CNS).

BACKGROUND OF THE INVENTION Related Art

Degeneration and/or death of cells in the nervous system is a substantial medical problem, and results in increased morbidity and mortality among people affected with one or more of a variety of CNS abnormalities. Such abnormalities include traumatic injury, diseases such as Parkinson's Disease, Alzheimer's Disease, stroke, and decreased neural perfusion secondary to cardiac arterial bypass graft surgery ("CABG"). Numerous other causes of damage to nerves or their supporting structures, such as glial cells, myelin sheaths and other cell and tissue types, likewise are major causes of morbidity and mortality. Thus, there are many efforts aimed at decreasing adverse effects of injury and/or disease to nervous system tissues.

Certain compounds have been useful as neuroprotective agents. One such material is Insulin-like growth factor 1 ("IGF-1"). (Scheepens et al, WO 00/13650). Following hypoxic-ischemic brain injury, the brain increases production of IGF-1 and two specific IGF-1 binding proteins, IGF binding protein-2 (IGFBP-2) and IGF binding protein-3 (IGFBP-3) (Gluckman et al Biochem Biophys Res Commun 182:593-599 1992; Klempt et al Brain Res 18:55-61 1992). IGFBP-2 and IGFBP-3 may attract IGF-1 into a region of injury so that concentrations of IGF-1 suitable for promoting neuroprotection were reached. For this reason IGF-1 was anticipated to be more efficacious administered at a site distant from the injury than desu IGF-1, which does not bind well to binding proteins. This was indeed the case. Desι_₃ IGF-1 was not significantly active as a neuronal rescue agent at a dose equivalent to that at which IGF-1 shows neuroprotection.

IGF-1 is a naturally occurring peptide that can decrease binding of glutamate to glutamate receptors of neurons (Bourguinon, U.S. Patent No: 5,804,550, incoφorated herein fully by reference). IGF-1 also can decrease neuronal degeneration caused by damage and/or disease. IGF-1 can be modified by proteolytic cleavage in nervous and other tissues to desι-₃ IGF-1 and a 3 amino acid peptide, glycine-proline-glutamate ("Gly-Pro-Glu" or "GPE"), the amino-terminal tripeptide of IGF-1. GPE is also neuroprotective (Gluckman et al., U.S. Patent No: 6,187,906, incoφorated herein fully by reference).

However, there is a need to identify other therapeutic compositions and methods for treating neural degeneration, damage, and neuronal cell death associated with a variety of CNS conditions. SUMMARY OF THE INVENTION

In view of the prior art described above, one object of the present invention is to provide compositions and new approaches to therapy for injury and disease, particularly CNS injury and disease.

In some aspects of the invention, administering cPG, can be used to restore damaged tissue in mammals. Thus, this invention provides a method of treating an animal to protect neurons otherwise destined to die as a result of an insult from injury or disease, comprising administering to said animal an effective amount of cPG. Certain embodiments of this invention include administering cPG to animals suffering from neuronal or glial cell degeneration. We have unexpectedly found that cPG can substantially reverse or prevent neuronal damage in CNS tissues. Thus, cPG can be used to treat a variety of conditions characterized by neuronal degeneration and/or neuronal cell death. Such conditions include, by way of example only, hypoxic ischemic damage, damage associated with Parkinson's disease, Alzhemier's disease, damage associated with stroke and/or coronary arterial bypass graft (CABG) surgery, or damage caused by neurotoxins. Thus, other embodiments of the invention provide a method of treating a patient with neuronal cell loss as the result of an insult from injury or disease, comprising administering to said patient an amount of cPG effective to stimulate neurite outgrowth.

The effects of cPG are dose-related. At certain doses (about 10 nM), cPG can restore glutamate-induced death of cerebellar cells to values undistinguishable from those found in vehicle-treated controls. Increasing the dose of cPG by about 100 fold also can result in substantial neuroprotection in the light of a neurotoxic insult. Additionally and suφrisingly, at lower doses (e.g., 1 nM), cPG can actually increase neuronal cell numbers compared to vehicle-treated controls. Thus, cPG can also be used to promote neuroregeneration. cPG can also have potent neuroprotective effects in cerebellar neurons exposed to hypoxic or ischemic damage. Thus, cPG can be an effective therapeutic agent to treat neural degeneration, damage or neuronal cell death associated with hypoxia, stroke or CABG.

We have also unexpectedly found that cPG can promote neurite outgrowth of cerebellar neurons, and can promote fasiculation of those neurites to form bundles of neurites. Therefore, certain embodiments of the invention include a method of treating a patient with neuronal cell loss as the result of an insult from injury or disease, comprising administering to said patient cPG in an amount sufficient to stimulate neural fasiculation.

In still further aspects, this invention provides kits, including, in some embodiments, a kit comprising cPG formulated in a pharmaceutically acceptable buffer, a container for holding said cPG formulated in a pharmaceutically acceptable buffer, and instructions.

Additionally, we have unexpectedly found that treatment with cPG can ameliorate adverse symptoms of neural degeneration in animals. 6-hydroxy dopamine (6-OHDA) is known to produce neural deficits in animals, and those deficits can include motor defects including abnormal rotatory motion. We have unexpectedly found that treatment with cPG can substantially decrease such motor abnormalities in vivo. Therefore, in still further aspects, this invention provides a method of treating an animal having neural degeneration, comprising providing an animal having a functional deficit associated with said neural degeneration; administering a therapeutically effective amount of cPG to said animal; and monitoring a change in said functional deficit in said animal. In yet further embodiments, this invention provides a method for preparing a medicament for treating an animal having a functional deficit associated with neural degeneration, comprising mixing cPG in a pharmaceutically acceptable buffer. BRIEF DESCRIPTION OF DRAWINGS

Aspects of this invention are described with reference to specific embodiments thereof. A better understanding of the invention will be gained from reference to the examples and drawings wherein:

Figure 1 depicts a graph showing effects of cPG on neuronal survival after exposure to glutamate.

Figure 2 depicts a graph showing effects of cPG on neuronal survival and neurite outgrowth.

Figures 3 a and 3b are photomicrographs of cerebellar explants showing effects of cPG on fascicle formation in vitro.

Figure 4 depicts a graph showing effects of cPG on functional recovery of motor behaviour in vivo following a 6-OHDA lesion.

Figure 5 depicts neuronal rescue due to cPG following hypoxic-ischemic brain injury in adult rats.

Figure 6 depicts a graph showing effects of cPG on brain regions following hypoxic-ischemic injury in rats.

DETAILED DESCRIPTION Pharmacology and Utility of Cyclo(Pro-Gly)

Certain aspects of this invention include methods of treatment or prevention of cell damage, degeneration and/or death in mammals in response to injury or disease. Some embodiments comprise delivering a composition comprising cPG to an animal suffering from neural degeneration, and in some cases, conditions involving apoptotic and necrotic cell death. In some embodiments, compositions are desirable to treat in injury or disease of the CNS affecting or liable to affect brain cells. Compositions are provided that can also include one or more other agents that promote neural regeneration, decrease cell degeneration or death, or are neuroprotective. In other aspects of the invention, methods of treatment or prevention of cell damage and death in animals are provided, that comprise providing a composition comprising cPG in response to injury or disease.

In another aspect of the invention, a kit is provided for the method of treatment or prevention of cell damage and death in mammals in response to injury or disease, where the kit comprises a dosage form of cPG formulated in a pharmaceutically acceptable buffer, a container holding the dosage form, and instructions. In a further aspect of the invention, the kit may further comprise one or more other compounds.

Such other compounds may be selected from the group consisting of for example, growth factors and associated derivatives, e.g., insulin-like growth factor-I [IGF-I], insulin-like growth factor-II [IGF-II], the tripeptide GPE, transforming growth factor-βl, activin, growth hormone, nerve growth factor, growth hormone binding protein, and/or IGF-binding proteins. It can be readily appreciated that other compounds may be used along with cPG

Other aspects of the invention include compositions and methods of promoting fasiculation of axons. By promoting formation of nerve bundles, cPG may be useful in treating conditions in which nerve processes (axons and/or dendrites) have become severed, such as in shaφ force injuries, local areas of necrosis or disease, or other localized injuries to nerve processes.

In another aspect of the invention, kit are provided for restoring neural function comprising a dosage form of cPG formulated in a pharmaceutically acceptable buffer, a container holding said dosage form, and instructions. In other embodiments, kits may further comprise cGP along with another neuroprotective compound. Such compounds include IGF-I, GPE, interferon beta lb (Betaseron®) or consensus interferon (Infergen®, interferon alfacon-1).

In yet other embodiments, compositions and methods to treat or prevent cell damage and death in response to injury and disease, including CNS injury and disease, comprise administration of a therapeutic amount of cPG alone or in combination with other agents, after the insult. These embodiments can be particularly desirable in situations of unexpected injury, such as in cardiac arrest, trauma such as head injuries caused by automobile accidents, head wounds and the like.

In still further embodiments, cPG can be used either alone or in combination with other agents to prevent adverse effects of planned brain injury. Such conditions include CABG or other planned surgeries such as brain surgery, vascular surgery or other interventions that may lead to decreased perfusion of the nervous system. By treating an animal, such as a human being, in advance and/or simultaneously and or after the surgery, adverse neurological effects can be ameliorated.

As indicated above, the present invention is broadly based upon the applicant's suφrising finding that cPG can protect cells, particularly nerve cells, against damage, loss of neurites, and/or apoptotic or necrotic cell death. These capabilities of cPG are achieved through increasing the effective concentration or amount of cPG in the affected tissue of a patient.

Increasing Endogenously Produced Cyclo(Pro-Gly)

In addition to direct administration of cPG, the amount of cPG may be increased by providing precursors of cPG. CyclicPG has been isolated from the rat brain tissue and has been shown to improve memory in animal models (US 5,439,930; Gudasheva et al., 1996 FEBS Lett 391: 149-152; Gudasheva et al., 1999 Biull Eksp Biol Med 128: 411-3; Gudasheva et al., 2001, Bull Exp Biol Med 131: 464-6), and cPG has been found in coffee.

We have demonstrated that cPG exhibits neuroprotection in both cell culture and in animal models of neurodegenerative disease and can therefore be an effective addition or alternative to conventional therapies for neural degeneration. Although the mechanism of cPG's protective effects are not known, one possible mechanism involves protecting cells from apoptotic and necrotic cell death. However, regardless of the mechanism of action, cPG can be used as an effective therapy for a variety of neurological diseases, including hypoxia, ischemia and neurotoxin-induced nerve damage. Moreover, cPG can be used in the absence of any particular neurological deficit to promote neurite outgrowth and fasiculation of nerves. Thus, in situations in which cell death is not necessarily associated with the neurological disorder (e.g., axonal damage such as caused by spinal cord injury), cPG may be an effective way of promoting neurite regeneration.

Therapeutic Applications of Cyclo(Pro-Gly)

Compositions and methods of the invention find use in the treatment of animals, such as human patients, suffering from neural injury or disease. Still more generally, the compositions and methods of the invention find use in the treatment of mammals, such as human patients, suffering from nerve damage or potential apoptotic and/or necrotic cell death, due to injuries and diseases.

Specific conditions and diseases include septic shock, ischemia, administration of cytokines, overexpression of cytokines, ulcers, gastritis, ulcerative colitis, Crohn's disease, diabetes, rheumatoid arthritis, asthma, Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke, cirrhosis, allograft rejection, transplant rejection, encephalomyelitis, meningitis, pancreatitis, peritonitis, vasculitis, lymphocytic choriomeningitis, glomerulonephritis, uveitis, glaucoma, blepharitis, chalazion, allergic eye disease, corneal ulcer, keratitis, cataract, retinal disorders, age-related macular degeneration, optic neuritis ileitis, inflammation induced by oveφroduction of inflammatory cytokines, hemorrhagic shock, anaphylactic shock, burn, infection leading to the oveφroduction of inflammatory cytokines induced by bacteria, virus, fungus, and parasites, hemodialysis, chronic fatigue syndrome, stroke, cancers, cardiovascular diseases associated with oveφroduction of inflammatory cytokines, heart disease, cardiopulmonary bypass, ischemic/reperfusion injury, ischemic/reperfusion associated with oveφroduction of inflammatory cytokines, toxic shock syndrome, adult respiratory distress syndrome, cachexia, myocarditis, autoimmune disorders, eczema, psoriasis, heart failure, dermatitis, urticaria, cerebral ischemia, systemic lupus erythematosis, AIDS, AIDS dementia, chronic neurodegenerative disease, chronic pain, priapism, cystic fibrosis, amyotrophic lateral sclerosis, schizophrenia, depression, premenstrual syndrome, anxiety, addiction, migraine, Huntington's disease, epilepsy, gastrointestinal motility disorders, obesity, hypeφhagia, neuroblastoma, malaria, hematologic cancers, myelofϊbrosis, lung injury, graft-versus-host disease, head injury, CNS trauma, hepatitis, renal failure, e.g., chronic hepatitis C, paraquat poisoning, transplant rejection and preservation, fertility enhancement, bacterial translocation, circulatory shock, traumatic shock, hemodialysis, hangover, and combinations of two or more thereof.

Still more generally, the invention has application in the induction of nerve bundle formation following insult in the form of trauma, toxin exposure, asphyxia or hypoxia-ischemia. And has application in the treatment or prevention of apoptosis in response to injury or disease in the form of cancers, viral infections, autoimmune diseases, neurological diseases and injuries and cardiovascular diseases.

Injuries to and diseases of the cerebellum include cerebellar infarction, cerebellar hemorrhage, adult onset hereditary ataxias including SCA1, SCA2, SCA3/MJD, SCA4, SCA5, SCA6, SCA7, dominantly inherited olivopontocerebellar atrophy, recessively inherited olivopontocerebellar atrophy; sporadic cerebellar degeneration including sporadic olivopontocerebellar atrophy, multiple system atrophy; drug induced, metabolic and endocrine disorders affecting the cerebellum including cerebellar dysfunction and loss of Purkinje cells due to the administration of the chemotherapeutic agents 5-fluorouracil and cytosine arabinoside, cerebellar atrophy in patients suffering from epilepsy exposed to phenytoin, alcoholic cerebellar degeneration and Wemicke's encephalopathy. cPG treatment may be given before an injury, for example, before elective surgery. Examples of relevant elective procedures include neural surgery, in which retraction of lobes of the brain may lead to cerebral oedema, or heart operations, such as valve replacement, in which inevitable small emboli are said to lead to detectable impairment of brain function in some 75% of cases.

Determining Efficacy of Cyclo(Pro-Gly) cPG's anti-apoptotic and anti-necrotic activity can be measured by in vivo using cell counts by methods such as those discussed in Klempt et al, 1992. cPG can also be measured in vitro using mass spectroscopy, immunological, or chromatographic methods known in the art.

CNS damage may for example be measured clinically by the degree of permanent neurological deficit cognitive function, and/or propensity to seizure disorders. We herein disclose histological techniques suitable for measuring cPG effects and behavioural tests for motor deficits in vivo.

The therapeutic ratio of a compound can be determined, for example, by comparing the dose that gives effective anti-apoptotic and anti-necrotic activity in a suitable in vivo model such as experimental immune encephalomyelitis (Mendel et al., 1995 Ewr. J. Immunol. 25: 1951-1959) in a suitable animal species such as the mouse, with the dose that gives significant weight change (or other observable side-effects) in the test animal species.

As a general proposition, the total pharmaceutically effective amount of the cPG agonist compound administered parenterally per dose will be in a range that can be measured by a dose response curve. One can administer increasing amounts of the cPG agonist compound to the patient and check the serum levels of the patient for cPG. The amount of cPG agonist to be employed can be calculated on a molar basis based on these serum levels of cPG.

Specifically, one method for determining appropriate dosing of the compound entails measuring cPG levels in a biological fluid such as a body or blood fluid. Measuring such levels can be done by any means, including RIA and ELISA. After measuring cPG levels, the fluid is contacted with the compound using single or multiple doses. After this contacting step, the cPG levels are re-measured in the fluid. If the fluid cPG levels have fallen by an amount sufficient to produce the desired efficacy for which the molecule is to be administered, then the dose of the molecule can be adjusted to produce maximal efficacy. This method can be carried out in vitro or in vivo. Preferably, this method is carried out in vivo, i.e. after the fluid is extracted from a mammal and the cPG levels measured, the compound herein is administered to the mammal using single or multiple doses (that is, the contacting step is achieved by administration to a mammal) and then the cPG levels are remeasured from fluid extracted from the mammal.

Pharmaceutical Compositions and Routes of Administration cPG may be administered using any suitable means. For example, to administer cPG to the CNS, a shunt into a ventricle of the animal may be used. Alternatively, peripheral administration via a blood vessel, such as a vein may be used. Alternatively, direct injection into the site of therapy may be desirable. Using stereotactic devices and accurate maps of an animals CNS, cPG may be injected directly into a site of neural damage. Such routes of administration may be especially desired in situations in which perfusion of that location is compromised either by decreased vascular perfusion or by decreased cerebral spinal fluid (CSF) flow to that area. However, there is no intention on the part of the applicants to exclude administration of other forms of cPG. By way of example, the effective amount of cPG in the CNS can be increased by administration of a pro-drug form of cPG which comprises cPG and a carrier, cPG and the carrier being joined by a linkage which is susceptible to cleavage or digestion within the patient. Any suitable linkage can be employed which will be cleaved or digested to release cPG following administration. Additionally, analogs of cPG, naturally occurring precursors of cPG may be administered and converted to cPG by endogenous means.

Another option is for cPG levels to be increased through an implant which is or includes a cell line which is capable of expressing cPG in an active form within the CNS of the patient. cPG can be administered as part of a medicament or pharmaceutical preparation. This can involve combining cPG with any pharmaceutically appropriate carrier, adjuvant or excipient. The selection of the carrier, adjuvant or excipient will of course usually be dependent upon the route of administration to be employed.

The administration route can vary widely. An advantage of cPG is that it can be administered peripherally. This means that it need not be administered directly to the CNS of the patient in order to have effect in the CNS.

Any peripheral route known in the art can be employed. These can include parenteral routes for example injection into the peripheral circulation, subcutaneous, intraorbital, ophthalmic, intraspinal, intracisternal, topical, infusion (using eg. slow release devices or minipumps such as osmotic pumps or skin patches), implant, aerosol, inhalation, scarification, intraperitoneal, intracapsular, intramuscular, intranasal, oral, buccal, pulmonary, rectal or vaginal. The compositions can be formulated for parenteral administration to humans or other mammals in therapeutically effective amounts (eg. amounts which eliminate or reduce the patient's pathological condition) to provide therapy for the neurological diseases described above.

Convenient administration routes include subcutaneous injection (e.g. dissolved in a physiologically compatible carrier such as 0.9% sodium chloride) or orally (in a capsule).

It will also be appreciated that it can be desirable to directly administer cPG compounds to the CNS of the patient. Cyclic PG can be administered using any convenient route. Examples include administration by lateral cerebroventricular injection or through a surgically inserted shunt into the lateral cerebroventricle of the brain of the patient, intraveneously, direct injection into the desired location or other routes..

The calculation of the effective amount of cPG compounds to be administered is within the skill of one of ordinary skill in the art, and will be routine to those persons skilled in the art. Needless to say, the final amount to be administered will be dependent upon the route of administration and upon the nature of the neurological disorder or condition to be treated. A suitable dose range may for example be between about lμg to lOOmg of cPG per lOOg of body weight where the dose is administered centrally.

In still further embodiments of the invention, the amount of cPG can be from about lμg per Kg body weight to about lOOmg of cPG per kg of body weight of the animal.

In further embodiments of the invention, restoring nerve function in an animal can comprise administering a therapeutic amount of cPG in combination with another neuroprotective agent, such as IGF-1. IGF-1 can be administered in a dose range of about 0.1 to lOOOμg of IGF-1 per lOOg body weight of the mammal or an interferon from about 0.1 to 1000/xg of interferon per lOOg of body weight of the mammal. In certain embodiments, the interferon is interferon beta. In another embodiment, the interferon is interferon beta lb (Betaseron®). In a further embodiment, the interferon comprises consensus interferon (Infergen®, interferon alfacon-1).

For inclusion in a medicament, cPG compounds can be obtained from a suitable commercial source. Alternatively, cPG can be directly synthesized by conventional methods such as the stepwise solid phase synthesis method of Merryfield et al, 1963. Alternatively synthesis can involve the use of commercially available peptide synthesizers such as the Applied Biosystems model 430 A.

The compound is suitably administered by a sustained-release system. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al, 1983), poly(2-hydroxyethyl methacrylate) (Langer et al, 1981), ethylene vinyl acetate (Langer et al, supra), or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also include a liposomally entrapped compound. Liposomes containing the compound are prepared by methods known per se: DE 3,218,121; Epstein et al, 1985; Hwang et al, 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (from or about 200 to 800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the most efficacious therapy.

PEGylated peptides having a longer life can also be employed, based on, e.g., the conjugate technology described in WO 95/32003 published November 30, 1995.

For parenteral administration, in one embodiment, the compound is formulated generally by mixing each at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically, or parenterally, acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the compound uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, a buffered solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine; amino acids such as glutamic acid, aspartic acid, histidine, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, trehalose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counter-ions such as sodium; non-ionic surfactants such as polysorbates, poloxamers, or polyethylene glycol (PEG); and/or neutral salts, e.g., NaCl, KC1, MgCl₂, CaCl₂, etc. The compound is typically formulated in such vehicles at a pH of from or about 4.5 to 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of salts of the compound. The final preparation may be a stable liquid or lyophilized solid.

Formulations of the peptide in pharmaceutical compositions can also include adjuvants. Typical adjuvants which may be incoφorated into tablets, capsules, and the like are a binder such as acacia, com starch, or gelatin; an excipient such as microcrystalline cellulose; a disintegrating agent like corn starch or alginic acid; a lubricant such as magnesium stearate; a sweetening agent such as sucrose or lactose; a flavoring agent such as peppermint, wintergreen, or cherry. When the dosage form is a capsule, in addition to the above materials, it may also contain a liquid carrier such as a fatty oil. Other materials of various types may be used as coatings or as modifiers of the physical form of the dosage unit. A syrup or elixir may contain the active compound, a sweetener such as sucrose, preservatives like propyl paraben, a coloring agent, and a flavoring agent such as cherry. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice. For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants, and the like can be incoφorated according to accepted pharmaceutical practice.

For injection, intraventrucilar administration, and other invasive routes of administration, the compounds used must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes) although other methods are known in the art and are included herein. Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Kits are also contemplated for this invention. A typical kit would comprise a container, preferably a vial, for the cPG formulation comprising cPG agonist compound in a pharmaceutically acceptable buffer, and instructions, such as a product insert or label, directing the user to utilize the pharmaceutical formulation.

The pharmaceutical formulation ordinarily will be stored in unit or multi-dose containers, for example, in sealed ampules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-mL vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution of compound, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound using bacteriostatic Water-for-Injection. It can be readily appreciated that other dosage forms and types of preparations can be used, and all are considered to be part of this invention.

Combination therapy with the cPG agonist compound herein and one or more other appropriate reagents that increase total cPG in the blood or enhance the effect of the cPG agonist is also part of this invention. These reagents generally allow the cPG agonist compound herein to release the generated cPG.

The present invention is further illustrated by the following examples. These examples are offered by way of illustration only and are not intended to limit the invention in any manner. All patent and literature references cited throughout the specification are expressly incoφorated by reference in their entirety.

EXAMPLES

The following Examples are provided to demonstrate features of this invention. They are not intended to be limiting, and other compositions and methods of this invention can be developed without undue experimentation. All of those compositions and methods are considered to be part of this invention. All the following experiments were carried out using protocols developed under guidelines approved by the University of Auckland Animal Ethics Committee.

Example 1: Effects of CyclofPro-Gly) on Cerebellar Cell Explants

To determine the effects of cPG on neuronal cells in vitro, we carried out a series of studies using cerebellar explants from adult rats. In vitro systems are suitable for studying neuronal proliferation, neurite growth, formation of nerve bundles and effects of toxins on neural cells. Thus, in vitro cerebellar explants are predictive of effects of interventions in vivo.

In a first series of studies, we determined the effects of glutamate on cerebellar explants. At physiological concentrations, glutamate is a neurotransmitter in the CNS of mammals, including humans. However, at sufficiently high concentrations, glutamate is neurotoxic, resulting in neuronal cell death. Because glutamate is a naturally occurring neurotransmitter in the CNS of mammals, including humans, it is a valuable predictive tool useful for identifying and characterizing agents used to treat neurotoxicity.

Materials and Methods

Ten coverslips were placed into a large Petri dish and washed in 70% alcohol for 5 minutes, then washed with Millipore H₂O. The coverslips were air dried, and coated with Poly-D-Lysine (1 mg/ml stock solution in PBS, 90- lOOμl) for 2 hours at 34°C.

Extraction of Cerebellar Tissue

Postnatal day 4 Wistar rats were used for the study. The rats were placed in ice for 1 minute, then decapitated and the cerebellum removed and placed on ice. Cerebellum tissue was placed in 1 ml of 0.65% glucose- supplemented PBS (10 μl 65% stock D (+)glucose/lml PBS) in a large Petri dish, chopped up into smaller sections and triturated with a 1ml insulin syringe via a 23 G (0.4mm) needle, and then squirted back into the glucose solution in the large Petri dish. The tissue was sieved through (125μm pore size gauze) and centrifuged (2 minutes at 60g) twice to exchange the medium into serum-free BSA-supplemented START V medium (Biochrom). The second centrifugation step was done with 1ml of START V medium. The microexplants were reconstituted into 500μl of START V medium and put on ice.

Cultivation of Cerebellar Cells

Two hours after PDL-coating, the slides were washed with Millipore H₂O and air dried. Each slide was placed into a small Petri dish (diameter: 35 mm) and 40μl of START V/cell suspension was added. The tissue was incubated for 2 hours at 34°C (settlement period). START V-medium (1ml) was then added to the Petri dish and cultivated at 34° C in the presence of 5% CO₂ in air at 100% humidity for 48 hours.

Drug Application

For each of the two studies, certain explant cultures were exposed to vehicle only. In the first study (Study 1) ten (10) μl of toxin (L-glutamate-100 mM in Millipore water; final concentration: 1 mM) was applied simultaneously with cGP (10 mM stock solution prepared in PBS and diluted to final concentrations between 1-100 nM). In a second study (Study 2), explants were exposed to glutamate for 6 hours prior to administration of cPG. In each case, the drugs were left in contact with the explants for the duration of the study (45 hours for Study 1 and 48 hours for study 2).

Methods for Determining Drug Effects

After explants were exposed to drugs for the study period, cells were then rinsed in PBS and then fixed in increasing concentrations of paraformaldehyde (500μl of 0.4% PFA was applied; then 1.2% PFA; then 3% PFA and finally 4% PFA (each fixation step: 2-3 minutes. All fixation solutions contain 0.2% glutaraldehyde). Finally, the microexplants were rinsed in PBS. Neurons in the explants were then evaluated for moφhology (presence of neurites) and presence of necrosis/apoptosis. Because glutamate is neurotoxic, we considered the presence of neurites to be indicative of neuronal survival. Numbers of cells having neurite outgrowth in each culture dish (18 mm x 18 mm or 324 mm²) were counted, and the data presented as mean ± standard error of the mean (SEM); n= 4 each. Results were compared using one-way analysis of variance.

Results

Study 1

The results of Study 1 are shown in Figure 1. Glutamate treatment (1 mM; filled bar) resulted in about an 85% loss of cerebellar neurons having neurites compared to vehicle-treated controls (open bar). In contrast, cyclic PG significantly increased the numbers of cells having neurites in a dose-dependent manner when administered simultaneously with glutamate (hatched bars). Treatment with low doses of cPG (10-100nM) showed significant recovery from glutamate-induced neurotoxicity.

Study 2

The results of Study 2 are shown in Figure 2. As with Study 1, glutamate (1 mM) caused substantial decrease in cells having neurites. Six hours after addition of glutamate, addition of cPG substantially increased the appearance of cells with neurites in a dose range of 10-lOOnM. Suφrisingly, at a concentration of 1 nM, cPG significantly increased neurite outgrowth compared to vehicle-treated controls. Thus, cPG promoted neuronal survival in the presence of neurotoxic amounts of glutamate. Another suφrising result of this study is shown in Figure 3. The top panel (Figure 3 a) shows fascicle formation in a cerebellar explant in the presence of 1 mM glutamate for 6 hours, followed by 42 hours incubation with 1 nM cPG. A bundle of nerve processes (a fascicle) can be seen in the middle of Figure 3a, extending toward the right from a group of surviving neurons.

In contrast, cells exposed to glutamate and the vehicle for cPG (bottom panel; Figure 3b) shows no fasiculation. In fact, the cells appeared to be disorganized, and few had neurites at all. These results indicate that cPG can promote neuronal cell survival, can promote neurite outgrowth and can result in reformation of nerve bundles.

Conclusions

Cyclic PG prevented (Study 1) and reversed (Study 2) glutamate- induced neurotoxicity, indicating that cPG is neuroprotective of neurons exposed to a highly toxic dose of glutamate. Moreover, in the presence of glutamate, cPG significantly increased neurite outgrowth compared to explants treated with glutamate plus the vehicle for cPG, indicating that cPG treatment substantially improves neurite outgrowth or neural maturation. Moreover, the finding that cerebellar explants formed fasciles after exposure to cPG indicates that cPG can be useful for promoting neurite and nerve regeneration. In particular, cPG can be desirably used to promote neurite regeneration after, for example, axonal damage. Many conditions are characterized by loss of neural function. By promoting neurite and nerve regeneration, cPG can be a useful therapeutic agent to treat spinal cord injuries, and other conditions in which neural processes are lost, due either to cell death or to injury to peripheral nerve processes. Example 2: Effects of CycIo(Pro-Gly) on 6-Hydroxy Dopamine-Induced Motor Deficits

To determine whether the in vitro effects of cPG have functional correlation in vivo, we carried out a series of studies in rats that had been pre- treated to produce motor deficits. It is known that 6-hydroxydopamine (6- OHDA) can cause neuronal damage in animals. Further, in 6-OHDA-treated animals, further exposure to apomoφhine can result in motor dysfunctions characterized by the animal's exhibiting rotatory walking behaviour. This system is predictive of motor dysfunction in other animals, including humans. Therefore, studies of effects of cPG on rats treated in this fashion is predictive of effects of cPG in human beings experiencing motor deficits resulting from neural damage.

Materials and Methods

Twenty male Wistar rats (280-3 lOg) were used. After exposing the skull, 6-OHDA (8μg in a base of 2μl 0.9% saline containing 1% ascorbic acid) was administered into the right medial forebrain bundle (MFB) using coordinates AP +4.7mm, R 1.6mm, v -8mm (Guan, J., Krishnamurthi, R., Waldvogel, H.J., Faull, R.L.M., Clark, R. and Gluckman, P. N-terminal tripeptide of IGF-1 (GPE) prevents the loss of TH positive neurons after 6- OHDA induced nigral lesion in rats. Brain Research 859/2. 286-292, 2000) under 3% halothane anaesthesia.

6-OHDA was injected through a 25G needle connected via a polyethylene catheter to a lOOμl Hamilton syringe. The 6-OHDA was infused by a microdialysis infusion pump at a rate of 0.5μl/min. The needle was left in the brain for a further 3 minutes before being slowly withdrawn. The skin was sutured with 2.0 silk and the rats were allowed to recover from anaesthesia. The rats were housed in a holding room with free access to food and water at all times except during behavioural testing. Cyclic PG was dissolved in saline. Four different doses of cPG (0, 0.1 0.5 lmg/kg, Bachem) were administered intraperitoneally 2h after injecting the 6-OHDA.

Seven days after administering the 6-OHDA, rats were injected with 0. lmg/kg apomoφhine, and the number of contralateral rotations/hour was recorded and calculated using a computerised Rotameter (St Diego Instruments). The experimenters were blinded from the treatment groups.

Results

The group treated with lmg cPG (n=5, 154±64.1) showed a trend toward a reduction in the number of rotations compared to the vehicle treated group (n=5, 290.08±18.9) indicating a role for cPG in improving functional recovery in 6-OHDA induced nigrostriatal injury (Figure 4).

Conclusions

Cyclic PG improved functional recovery after a 6-OHDA induced nigral-striatal lesions in a dose-dependent manner. The highest dose tested (lmg/kg) reduced the functional deficit (apomoφhine induced rotations) by 47%. This data indicates cPG has potential value as a treatment for neuromotor deficits, such as those characteristic of Parkinson's disease. Moreover, because the cerebellum is responsible for promoting and maintaining smoothness of motion by inhibiting overactivity of motoneuron pathways in the cerebrum and spinal cord, these results indicate an important therapeutic role of cPG in treatment of a variety of conditions involving the cerebellum and associated structures.

Example 3: Effects of Cyclo(Pro-Gly) on Hypoxic-ischemic Injury Materials and Methods

To determine whether cPG might promote neural regeneration or inhibit neuronal degeneration in response to stroke, cardiac arterial bypass graft surgery (CABG) or other hypoxic insults, we carried out a series of studies in rats that had been exposed to hypoxic-ischemic injury (HI). Adult rats (Wistar, 280-3 lOg, male) were used. The modified Levine model preparation and experimental procedures have been previously described. Briefly, HI injury was induced by unilateral carotid artery ligation followed by inhalational asphyxia in the animals with an implanted lateral ventricular cannula. A guide cannula was stereotaxically placed on the top of the dura 1.5mm to the right of the mid- line and 7.5mm anterior to the interaural zero plane under halothane anaesthesia. The right carotid artery was double ligated one day after the cannulation. After 1 hour recovery from the anaesthesia, each of the rats were placed in an incubator where the humidity (90±5%) and temperature (31°±0.5°C) were controlled for another hour, then exposed to hypoxia (6% oxygen) for lOmin. The animals were kept in the incubator for an additional 2 hours before treatment.

In a first study, twenty two pairs of rats were treated intracerebral ventricularly (icv) with either cPG (0.2μg and 2μg) or its vehicle (normal saline) alone. In a second study, nine pairs of rats were treated with either cPG (20ng; icv) or the vehicle 2 hours after hypoxic-ischemic insult. Rats in each group were simultaneously infused with cPG or its vehicle under light anaesthesia (1.5% halothane) 2 hours after the insult. A total volume of 20μl was infused (icv) over 20 minutes by a micro-infusion pump.

Histological examination was performed on rats 5 days after the hypoxic-ischemic injury. The rats were killed with an overdose of sodium pentobarbital and were perfused transcardially with normal saline followed by 10% formalin. The brains were kept in the same fixative for a minimum of 2 days before being processed using a standard paraffin imbedding procedure.

Coronal sections 8μm in thickness were cut from the striatum, cerebral cortex and hippocampus and were stained with thionin and acid fuchsin. The histological outcome was assessed at three levels: (1) the mid level of the striatum, (2) where the completed hippocampus first appeared and (3) the level where the ventral horn of the hippocampus just appears. The severity of tissue damage was scored in the striatum, cortex and the CAl-2, CA3, CA4 and dentate gyrus of the hippocampus. Tissue damage was identified as neuronal loss (acidophilic (red) cytoplasm and contracted nuclei), pan-necrosis and cellular reactions. Tissue damage was scored using the following scoring system: 0: tissue showed no tissue damage, 1: <5% tissue was damaged, 2: <50% tissue was damaged, 3: >50% tissue was damaged and 4: >95% tissue was damaged.

Results and Conclusion

Results of these studies are shown in Figures 5 and 6. Figure 5 shows that hypoxic-ischemic injury (left bars of each set) resulted in significant damage scores in each of the areas of the brain studied. Figure 5 also shows that central administration of a relatively low dose of cPG (middle bars of each set; 0.2μg) significantly reduced the tissue damage in each brain region examined compared to the vehicle treated group (p<0.001). Figure 5 also shows that in some regions of the brain, a higher dose of cPG (2 μg; right bars of each set) decreased brain damage.

Figure 6 shows that hypoxia/ischemia produces tissue damage (left bars of each set), and that at a dose of 20 ng (right bars of each set), cPG selectively reduced tissue damage in the striatum CA 1-2 and CA4, and by more than 50% in the dentate gyrus and the cortex.

We conclude that cPG can be neuroprotective against neural damage caused by hypoxic-ischemic injury, even when administered after hypoxic- ischemic injury. This suφrising finding indicates that cPG can be a useful agent to treat a variety of conditions characterized by neural degeneration or cell death, including stroke and cardiac arterial bypass graft surgery, and can stimulate neural regeneration though increased neurite outgrowth and increased formation of nerve bundles.

The descriptions and examples provided herein are for puφoses of illustration only. We do not intend the scope of this invention to be limited to the described embodiments. Other embodiments incoφorating elements of the invention can be practiced without undue experimentation by persons of ordinary skill in the art. All such embodiments are therefore considered to be part of this invention. All references cited herein are incoφorated fully by reference.

INDUSTRIAL APPLICABILITY Compositions and methods of this invention are useful for preparing medicaments useful for treating neural degeneration and reversing functional neurological deficits in animals afflicted with neurodegenerative conditions including hypoxic-ischemic injury, diseases such as Parkinson's Disease and Alzheimer's Disease.

Claims

We Claim:

1. A method of treating an animal to protect neurons otherwise destined to die as a result of an insult from injury or disease, comprising administering to said animal an effective amount of cPG.

2. A method as claimed in claim 1, wherein said disease is characterized by apoptotic cell death.

3. A method as claimed in claim 1, wherein said disease is characterized by necrotic cell death.

4. The method as claimed in claim 1, wherein said disease is selected from Alzheimer's disease, Huntington's disease and Parkinson's disease.

5. The method as claimed in claim 1, wherein said injury or disease is a result of one or more conditions selected from the group consisting of traumatic brain injury, stroke, cardiac artery bypass graft surgery, a toxin and asphyxia.

6. The method of claim 1, wherein the peptide administered is cPG.

7. The method of claim 1 , further comprising administering IGF- 1.

8. The method as claimed in claim 1, wherein said disease is selected from the group consisting of acute or chronic encephalomyelitis, optic neuritis, transverse myelitis, Devic's disease, a leucodystrophy, progressive multifocal leukoencephalopathy, central pontine myelinolysis, neuromyelitis optica, diffuse cerebral sclerosis of Schilder, and acute or subacute necrotizing haemorrhagic encephalitis.

9. The method of claim 1, wherein said cPG is administered simultaneously with said injury.

10. The method of claim 1, wherein said cPG is administered after said injury or disease.

11. The method of claim 1, wherein said cPG is administered before said injury or disease.

12. The method as claimed in claims 1, wherein said cPG is administered in a pharmaceutical composition including a pharmaceutically acceptable carrier.

13. The method as claimed in claim 12, wherein said cPG is administered in a pharmaceutical composition including dextran or gelatin.

14. The method as claimed in claim 12, wherein said cPG is administered in a pharmaceutical composition containing mannitol.

15. The method as claimed in claim 1, wherein said cPG is administered directly to the brain or cerebrospinal fluid by cerebro-ventricular injection, by injection into the cerebral parenchyma or through a surgically inserted shunt into the lateral cerebral ventricle of the brain.

16. The method as claimed in claim 1, wherein said cPG is administered in combination with artificial cerebrospinal fluid.

17. A method as claimed in claim 1, wherein said cPG is administered via at least one route selected from the group consisting of intravenous, oral, rectal, nasal, subcutaneous, inhalation, intraperitoneal and intramuscular.

18. The method as claimed in claim 1, wherein the dose of said cPG administered is in the range of about 1 μg to about 100 mg of cPG per kg of body weight.

19. The method as claimed in claim 1, further comprising the step of administering to said patient a neuroprotective amount of at least one other neuroprotective agent.

20. The method as claimed in claim 19, wherein said cPG is administered in combination with another neuroprotective agent selected from the group consisting of insulin-like growth factor-I [IGF-I], insulin-like growth factor-II [IGF-II], transforming growth factor-βl, activin, growth hormone, nerve growth factor, growth hormone binding protein, an IGF-binding protein, basic fibroblast growth factor, acidic fibroblast growth factor, the hst/Kfgk gene product, FGF-3, FGF-4, FGF-6, keratinocyte growth factor, androgen-induced growth factor, int-2, fibroblast growth factor homologous factor-1 (FHF-1), FHF-2, FHF-3 and FHF-4, keratinocyte growth factor 2, glial-activating factor, FGF-10 and FGF-16, ciliary neurotrophic factor, brain derived growth factor, neurotrophin 3, neurotrophin 4, bone moφhogenetic protein 2 [BMP-2], glial- cell line derived neurotrophic factor, activity-dependant neurotrophic factor, cytokine leukaemia inhibiting factor, oncostatin M, interleukin, β-interferon, α- interferon, χ-interferon, consensus interferon, TNF-α, clomethiazole; kynurenic acid, Semax, FK506 [tacrolimus], L-threo-l-pheyl-2-decanoylamino-3- moφholino-1-propanol, andrenocorticotropin-(4-9-analogue [ORG 2766], dizolcipine [MK-801], selegiline; a glutamate antagonist selected from the group consisting of NPS1506, GV1505260, MK-801, GV150526; an AMPA antagonist selected from the group consisting of 2,3-dihydroxy-6-nitro-7- sulfamoylbenzo(f)quinoxaline (NBQX), LY303070 and LY300164, an anti- inflammatory agent directed against MAdCAM-1 and/or integrin α4 receptors (α4βl and α4β7), and anti-MAdCAM-lmAb MECA-367 (ATCC accession no. HB-9478).

21. The method as claimed in claim 19, wherein said at least one other neuroprotective agent is a glutamate receptor antagonist.

22. The method as claimed in claim 19, wherein said said at least one other neuroprotective agent is a peptide that modifies glycine binding.

23. The method as claimed in claim 1, further comprising the step of administering to said animal an anti-inflammatory agent.

24. The method as claimed in claim 23, wherein said anti-inflammatory agent is selected from the group consisting of anti-integrin alpha 4 subunit reagents, anti-integrin beta 7 subunit reagents, anti-integrin beta 2 subunit reagents, anti-integrin alpha L subunit reagents, andti-MAdCAM-1, anti- VCAM-1 reagents and anti-ICAM reagents.

25. A method of treating a patient with neuronal cell loss as the result of an insult from injury or disease, comprising administering to said patient an amount of cPG effective to stimulate neurite outgrowth.

26. A method of treating a patient with neuronal cell loss as the result of an insult from injury or disease, comprising administering to said patient cPG in an amount sufficient to stimulate neural fasciculation.

27. A kit comprising: cPG formulated in a pharmaceutically acceptable buffer, a cpntainer for holding said cPG formulated in a pharmaceutically acceptable buffer, and instructions.

28. A method for promoting neurite outgrowth in a neuron subjected to neural degeneration, comprising exposing said neuron to an effective amount of cPG.

29. The method of claim 28, wherein said neuron is a cerebellar neuron.

30. The method of claim 28, wherein said neural degeneration is associated with hypoxia or ischemia.

31. The method of claim 28, wherein said neural degeneration is associated with Parkinson's Disease, Alzheimer's Disease, or Huntington's Disease.

32. A method of treating an animal, comprising:

(a) providing an animal having a functional deficit associated with said neural degeneration;

(b) administering a therapeutically effective amount of cPG to said animal; and

(c) monitoring a change in said functional deficit in said animal.

33. The method of claim 32, wherein said neural degeneration is associated with damage to said animal's cerebellum.

34. The method of claim 33, wherein said damage to said animal's cerebellum is associated with at least one condition selected from the group consisting of cerebellar infarction, cerebellar hemorrhage, SCA1, SCA2, SCA3/MJD, SCA4, SCA5, SCA6, SCA7, dominantly inherited olivopontocerebellar atrophy, recessively inherited olivopontocerebellar atrophy; sporadic olivopontocerebellar atrophy, multiple system atrophy; drug induced disorders of the cerebellum, metabolic disorders of the cerebellum, endocrine disorders of the cerebellum, 5-fluorouracil-induced loss of Purkinje cells, cytosine arabinoside-induced loss of Purkinje cells, phenytoin-induced cerebellar atrophy, alcoholic cerebellar degeneration and Wernicke's encephalopathy.

35. A method for preparing a medicament for use in treating an animal having a neurological injury or disease, comprising mixing a therapeutically effective amount of cPG in a pharmaceutically acceptable buffer and providing instructions for use of said medicament to treat said injury or disease associated with neuronal degeneration.