WO2005097161A2 - Gpe and g-2mepe, caffeine and alkanol for treatment of cns injury - Google Patents

Gpe and g-2mepe, caffeine and alkanol for treatment of cns injury Download PDF

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WO2005097161A2
WO2005097161A2 PCT/US2005/010816 US2005010816W WO2005097161A2 WO 2005097161 A2 WO2005097161 A2 WO 2005097161A2 US 2005010816 W US2005010816 W US 2005010816W WO 2005097161 A2 WO2005097161 A2 WO 2005097161A2
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alkanol
2mepe
gpe
caffeine
method
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PCT/US2005/010816
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French (fr)
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WO2005097161A3 (en
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James Grotta
Peter D. Gluckman
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Neuren Pharmaceuticals Limited
The University Of Texas
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Priority to US60/557,940 priority
Priority to US57262704P priority
Priority to US60/572,627 priority
Application filed by Neuren Pharmaceuticals Limited, The University Of Texas filed Critical Neuren Pharmaceuticals Limited
Publication of WO2005097161A2 publication Critical patent/WO2005097161A2/en
Publication of WO2005097161A3 publication Critical patent/WO2005097161A3/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/04Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06Tripeptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52Purines, e.g. adenine
    • A61K31/522Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir

Abstract

This invention includes compositions and methods useful for treating CNS injuries or diseases involving neural cell degeneration or cell death or neurological deficits associated with CNS injuries or diseases. Compositions include the tripeptide, Gly-Pro-Glu ('GPE'), the GPE analog Gly-2-methyl Pro-Glu ('G-2MePE') and low doses of caffeine and alkanol ('caffeinol'). Combination therapy with GPE and caffeinol improve neurological function and decrease lesion volume compared to the effects of GPE and caffeinol separately. Unexpectedly, we found that combination therapy with G-2MePE and caffeinol produced a synergistic effect, with the combined effect being greater than the sum of the effects of G-2MePE and caffeinol separately. Thus, combination therapy using GPE, G-2MePE and caffeinol provide an additional therapeutic tool useful for treating a variety of CNS disorders.

Description

GPE AND G-2MePE, CAFFEINE AND ALKANOL FOR TREATMENT OF CNS INJURY

CLAIM OF PRIORITY U.S. Provisional Patent Application No. 60/557,940 entitled GPE AND GPE ANALOGUES/PEPTIDOMIMETICS AND CAFFEINOL IMPROVE SENSORY- MOTOR FUNCTIONAL RECOVERY AFTER STROKE IN RAT, by James Grotta et al, filed March 30, 2004 (Attorney Docket No. NRNZ-01053US0); U.S. Provisional Patent Application No. 60/572,627 entitled GPE AND

G2MePE IN COMBINATION WITH CAFFEINOL IMPROVE SENSORY MOTOR FUNCTIONAL RECOVERY AFTER STROKE, by James Grotta, et al, filed May 19, 2004 (Attorney Docket No. NRNZ-01053US1). Both of the above applications are incorporated herein fully by reference.

FIELD OF INVENTION This invention relates to compositions and methods of treating neurodegenerative conditions, h particular, this invention relates to compositions and methods using an effective amount of Gly-Pro-Glu ("GPE"), Glycyl-2-methyl-Pro-Glu ("G-2MePE"), caffeine and alkanol to treat neurodegenerative conditions.

BACKGROUND Long-term and short-term neurological deficits are common results of injuries to or diseases of the central nervous system ("CNS"). Such neurological deficits can result from degeneration or death of neurons or glial cells in the CNS. Stroke, hypoxia, ischemia, traumatic brain injury, inflammatory conditions and other CNS insults or diseases can decrease neuronal and glial cell numbers in affected individuals. These neuronal deficits can be reflected in cognitive impairment, memory loss, ataxia, abnormalities of gait and/or a variety of other symptoms and clinical findings. Thus, there is a profound need to find ways to decrease the impact of CNS injury or disease, to inhibit or prevent the functional deficits that result from those disorders, and to improve the recovery following a CNS injury or disease. EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acids Gly-Pro- Glu) and its di-peptide derivatives Gly-Pro and Pro-Glu. EP 0 366 638 discloses that GPE is effective as a neuromodulator and is able to alter the release of neurotransmitter from neurons exposed to potassium ions. WO95/172904 and U.S. Patent No. 6,780,848 disclose that GPE has neuroprotective properties and that administration of GPE can reduce damage to the central nervous system (CNS) by the prevention or inhibition of neuronal and glial cell death caused by injury or disease of the CNS . WO02/094856 discloses GPE analogues and peptidomimetics characterised by anti-necrotic and anti-apoptotic activity. One of the peptides disclosed in the application is glycyl-L-2-methylprolyl-L-glutamic acid (G-2MePE). U.S. PatentNumbers 6,500,834, 6,503,915 and 6,503,916 disclose that caffeinol, a combination of low doses of caffeine and the alkanol ethanol, is effective in reducing cerebral infarct damage caused by stroke, with particularly striking effects on the cortex, and improves the chances for complete or substantial recovery. Animal systems have been useful in evaluating potential therapies for CNS damage. In particular, as in humans, rats have cholinergic neurons, dopaminergic neurons, and neurons that use other transmitters, such as glutamate. Further, rats exposed to CNS injury show functional and neurological deficits similar to those found in human beings subjected to similar insults. Thus, rats are a useful system for evaluating efficacy of compounds that have neuroprotective potential. BRIEF SUMMARY Embodiments of this invention include compositions for use in treating neural degeneration or cell death, and neurological behavioral deficits resulting from injury or disease of the CNS. In certain aspects, this invention includes methods for treating CNS injury or disease in a patient suffering from such injury or disease, comprising administering to said patient a pharmaceutically effective composition comprising GPE in combination with a composition comprising an effective amount of alkanol and an effective amount of caffeine ("caffeinol") or G-2MePE in combination with caffeinol or GPE and G-2MePE in combination with caffeinol. In other aspects, this invention includes compositions and methods of treating CNS injury, wherein said CNS injury is hypoxic or ischemic injury. In further aspects, this invention includes compositions and methods for treating

CNS injury, wherein said injury is associated with decreased cerebral blood flow. In still further aspects, this invention includes compositions and methods for CNS injury, wherein said injury is associated with stroke. In yet further aspects, this invention includes compositions and methods for treating decreased cerebral blood flow. In still further aspects, this invention includes compositions and methods for treating decreased cerebral blood flow associated with coronary artery bypass surgery. In yet further aspects, this invention includes compositions and methods for treating CNS injury when the injury is a traumatic brain injury. In other aspects, this invention includes compositions and methods for treating

CNS diseases. In further aspects, this invention includes compositions and methods for treating CNS disease when the disease is associated with damage to glial cells or neurons. In still further aspects, this invention includes compositions and methods for reducing a functional symptom of CNS injury or disease in a patient suffering from such injury or disease, comprising administering to said patient a pharmaceutically effective composition comprising GPE in combination with caffeinol or G-2MePE in combination with caffeinol, or GPE and G-2MePE in combination with caffeinol. In yet further aspects, this invention includes compositions and methods for treating a functional symptom of CNS injury or disease, wherein said functional symptom is selected from the group consisting of paralysis, spasticity, cognitive impairment and/or abnormalities of gait. In still further aspects, this invention includes use of GPE, G-2MePE and caffeinol in the manufacture of a medicament useful for treating a CNS disorder, comprising combining GPE and caffeinol together, combining G-2MePE and caffeinol together or GPE and G-2MePE in combination with caffeinol. We have unexpectedly found that a combination of G-2MePE and caffeinol can have a greater effect than the sum of individual effects of G-2MePE and caffeinol. The finding of this synergy is totally unexpected based on the effects of each agent separately, and provides a new, relatively easy therapeutic regimen for treating functional deficits of CNS damage.

BRIEF DISCRIPTION OF FIGURES This invention is described with reference to specific embodiments thereof. Other details, features and descriptions are found in the Figures, in which: Figure 1 shows the body weight lost in treatment groups (baseline compared to

72h) and rectal temperature measured at 72h after reperfusion. Figure 1A shows that G- 2MePE significantly prevented body weight loss (p<0.05), but all other groups did not differ. Figure IB shows that there was no significant difference in rectal temperature among the groups. Figure 2 shows cortical, striatal, and total lesion volume in each study group. Rats were sacrificed on day 3 after reperfusion and brains sliced into 7 sections and stained with TTC and fixed in 10% formalin for 24h. The data are expressed as Mean ± SD. *, P<0.05; **, P≤0.01; ***, P<0.001. Figure 3 shows changes (baseline compared to 72h) in tests of postural reflex, circling, forward placing and footfault. The data are expressed as Mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001. Figure 4 shows Neurological Deficit Score at 72h after reperfusion. The total NDS (sum of scores from the postural reflex, circling, placing and footfault tests) at 72h after reperfusion expressed as mean + SD. *, P<0.05; **, P≤O.01; ***, P≤O.001.

DETAILED DESCRIPTION OF THE INVENTION Definitions

"GPE" means the tripeptide glycine-proline-glutamate (also referred to as "Gly-Pro-Glu" or "GPE").

"G-2MePE" means Glycyl-L-2-Methylprolyl-L-glutamic Acid, having the formula:

Figure imgf000007_0001

"Caffeine" means 1,3,7-trimethyxantine.

"Caffeinol" means a combination or mixture of caffeine and an alkanol or caffeine plus a combination of alkanols.

"Alkanol" means an alkyl alcohol, and includes ethanol. The term alkanol also includes combinations of two or more alkyl alcohols.

The term "treat" when used herein refers to at least attempting to effect a reduction in the severity of the CNS damage, by reducing neuronal loss, and loss of glial cells and other cells, suffered after a CNS injury or due to CNS disease. It encompasses the process of minimizing such damage following a CNS injury or disease.

Technical Details of the Invention GPE or the GPE analog G-2MePE when used in combination with caffeinol show improved efficacy compared to the effects of the individual compounds. Beneficial effects include decreased lesion volume and improved neurological function. We have unexpectedly found that low concentrations of G-2MePE and caffeinol can act synergistically to provide greater effects when used together than the sum of the individual effects. Thus, the use of the compounds and compositions of this invention can be a useful alternative for therapy of numerous CNS disorders, including stroke, hypoxia/ischemia, and other conditions resulting in neurological deficits. We measured the effects of GPE, and its analogue G-2MePE, alone or combined with caffeinol, on functional performance, CNS lesion volume, and physiological variables in a well accepted animal system for studying stroke (middle cerebral artery suture occlusion, MCAo). We found that GPE was neuroprotective alone at the doses used, that G-2MePE was also effective, but the effect was less at the dose used than GPE, and that G-2MePE in combination with caffeinol, was even more neuroprotective than GPE alone, G-2MePE alone or caffeinol alone, even when administration was delayed until 75-min after MCAo. Both GPE and caffeinol decreased cortical lesion volume and improved neurological functional recovery, findings consistent with previous reports from different stroke models (Smith. IDrugs. 2003;6:1173-1177; Strong et al. Neuropharmacology. 2000;39:515-522). The combination of GPE plus caffeinol had a greater effect than either GPE or caffeinol alone, although the effect was not statistically significant. G-2MePE alone did not have a significant protective effect on lesion volume or NDS at the dose used, but had substantial and statistically significant additive effect when combined with caffeinol. The relatively weaker effect of G-2MePE alone to reduce damage may be related to the low dose (0.9 mg/kg). Multiple deleterious factors have been implicated in the pathology of ischemic damage, including neurotransmitter release, ion imbalance, free-radical formation, mitochondrial dysfunction, gene expression, protein synthesis impairment, inflammation and programmed cell death. The treatments tested in these experiments may target one or several of these pathways. Special attention during the search for an effective stroke treatment has been devoted to the role of NMD A, GABA and adenosine receptors, which are directly implicated in modulation and execution of ischemia-evoked excitotoxic damage. Adenosine modulates neuronal excitability as well as the release of many neurotransmitters. These include the excitatory amino acids (EAA) (Fastbom et al. Acta Physiol Scand. 1985; 125 : 121-123; Dunwiddie. Int Rev Neurobiol. 1985;27:63-139; Dunwiddie et al. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1985;19:259-272), which, through activation of specific ionotropic receptors (primarily NMDA but also certain AMPA and kainate receptors), play a role in ischemia-induced Ca2+-mediated excitotoxic damage (Corsi etα/. Neurobiol Aging. 1997;18:243-250; Goda etal. JNuτr. 1998;128:2028-2031; O'Regan et al. Brain Res. 1992;582:22-26). In vivo, both caffeine and ethanol are readily absorbed and distributed to all body fluids, including the cerebrospinal fluid ("CSF"). The protective mechanism of caffeinol, or the biological activities of caffeine and ethanol in stroke are not completely known. Biological activity of caffeine is dose-dependent and can be expressed through its ability to: (1) induce the intracellular release of C?X (possibly via an interaction at the level of IP3 and ryanodine receptor), (2) inhibit phosphodiesterase ("PDE"), and (3) block gamma amino butyric acid ("GABA") receptors and (4) adenosine receptors (Fredholm. Lakartidningen. 1995;92:4079-4080; Daly etα/. Drug Alcohol Depend. 1998;51:199-206; Daly et al. Lakartidningen. 1998;95:5878-5883). The biological activities of ethanol include inhibition of excitatory NMDA receptors and activation of inhibitory GABA receptors (Faingold et al. Prog Neurobiol. 1998;55:509-535). However, it should be understood that the above mechanisms are not necessarily the only ones through which caffeine or ethanol exert neuroprotective effects. Insulin-Like Growth Factor (IGF-1) has significant neuroprotective properties but does not cross the blood brain barrier ("BBB") because of its relatively large size. GPE, an active peptide component cleaved from IGF-1, can across the BBB with excellent brain penetration. However, the mechanism of its neuroprotective effect is still unknown. At relatively high doses (uM range), GPE binds to the NMDA receptor (most probably at the glutamate binding site) at a concentration >10 uM, where it may act as a partial agonist, mimicking (with lower efficacy) the effects of glutamate at 10 to 300 uM, and antagonizing glutamate's effects at concentrations above about 300 uM. In situations of excessive glutamate release, such as occurs during stroke and brain injury, GPE may be acting as an NMDA receptor antagonist, and might inhibit the neurotoxic effects of glutamate overload. However, NMDA receptor activity of GPE probably does not explain the neuroprotective effect seen in our study because only nM concentrations of GPE are achieved in CSF following infusion of neuroprotective doses. The cytoprotective effect of GPE may be mediated through its own unique receptor, but this novel binding site is still undetermined. Regardless of the exact mechanism of action, the finding that GPE, G-2MePE and caffeinol exhibit desirable physiological and clinical effects is nonetheless, a significant new finding. Moreover, as other mechanisms of action of GPE, G-2MePE and caffeinol may account for at least some of the observed effects, we do not intend to limit the scope of this invention to any particular mechanism of action. Rather, all mechanisms of action are considered to be part of this invention.

Administration of Compounds of the Invention In cases of acute neural injuries or insults, GPE, G-2MePE and caffeinol can be administered from 0.1-lOOh after said neuronal injury or insult. Preferably the compounds of the invention are administered between 1 hours to about 24 hours from the injury to the CNS. We have previously shown in PCT International Application No : PCT/US04/35165 that due to its rapid plasma clearance, GPE can be desirably administered as an intravenous infusion with or without a prior bolus injection. When GPE is administered by intravenous infusion, it can be administered over a duration of from about 1 to about 4 hours. In certain embodiments, a bolus dose of GPE can be from about 0.03 mg/kg to about 30 mg/kg, and in other embodiments, about 3 mg/kg. When GPE is infused intravenously, it can be desirable to use an infusion rate from about 0.03 mg/kg/h to about 30 mg/kg/h and in other embodiments, at the rate of 3 mg/kg/hr. GPE can be administered directly to the CNS, either by lateral cerebroventricular injection or through a surgically inserted shunt into the lateral cerebral ventricle of the brain. Another way of administering GPE directly to CNS is through inj ection into cerebral parenchyma. When GPE is administered for infusion in artificial CSF into the lateral ventricle or other perfusion sites, a dose rate of about lOμg/kg can be effective. G-2MePE can be administered according to the description of patent application

WO02/094856. G-2MePE can be administered in therapeutically effective amounts by any of the usual modes known in the art. Therapeutically effective amounts of G-2MePE may range from 0.001 to 100 milligrams per kilogram (mg/kg) mass of the patient, for example, 0.1 to 10 mg/kg, with lower doses such as 0.001 to 0.1 mg/kg, e.g. about 0.01 mg/kg, being appropriate for administration through the cerebrospinal fluid, such as by intracerebroventricular administration, and higher doses such as 1 to 100 mg/kg, e.g. about 10 mg/kg, being appropriate for administration by methods such as oral, systemic (e.g. transdermal, intranasal, or by suppository), or parenteral administration (e.g. intramuscular, subcutaneous, or intravenous injection). In some embodiments, G-2MePE can be administered once daily when given orally, and 2-3 times per day when administered subcutaneously. If G-2MePE is administered by intravenous infusion, an effective dosage is between about 0.1 to 3mg/kg/h given for the duration of 1-4 hours. A person of ordinary skill in the art will be able without undue experimentation, having regard to that skill and this disclosure, to determine a therapeutically effective amount of a compound of this invention for a given disease or injury. Pharmaceutical compositions including G-2MePE may take the form of tablets, pills, capsules, semisolids, powders, sustained release formulation, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions; and comprise at least one compound of this invention in combination with at least one pharmaceutically acceptable excipient. Suitable excipients are well known to persons of ordinary skill in the art, and they, and the methods of formulating the compositions, may be found in such standard references as Gennaro AR: Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins, 2000. Suitable liquid carriers, especially for injectable solutions, include water, aqueous saline solution, aqueous dextrose solution and glycols. Desirably, if possible, when administered as anti-apoptotic agent, anti-necrotic agent, compounds of this invention will be administered orally. The amount of a compound of this invention in the composition may vary widely depending on the type of composition, size of a unit dosage, kind of excipients, and other factors well known to those of ordinary skill in the art. In some embodiments, the final composition comprises from 0.0001 percent by weight (% w) to 10% w of the compound of this invention, preferably 0.001% w to 1% w, with the remainder being the excipient or excipients. An effective amount of caffeine and alkanol (such as ethanol) can be administered separately from each other or in a mixture. Both can be administered to the subject or patient in an oral, intravenous, or other form that provides an effective amount of the caffeine and alkanol (e.g., ethanol). The caffeine and alkanol can be co-administered from a common source or to a common site or be provided separately through distinct delivery sites and modes. Caffeine can be administered in doses from 1 to 50mg/kg of patient's weight. In some embodiments, the amount is between 1-lOmg/kg. In some embodiments, an alkanol is ethanol or a mixture of ethanol and another alkanol. Preferably ethanol is dissolved at about

1-10% in water. An effective amount of alkanol is between 0.1 and 0.5 mg/kg of patient's weight. GPE, G-2MePE, caffeine and alkanol maybe administered in a pharmaceutically acceptable formulation. This can involve combining the compounds with any pharmaceutically appropriate carrier, adjuvant or excipient. In some embodiments, formulations can be prepared by contacting a compound uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if desired, the product can be shaped into the desired formulation. In some embodiments, the carrier is a parenteral carrier, alternatively, 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 desirably non-toxic to recipients at the dosages and concentrations employed, and include, by way of example only, 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, MgC12, CaC12, etc. 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. In other embodiments, adjuvants can be used. Typical adjuvants which may be incorporated into tablets, capsules, and the like are a binder such as acacia, corn 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. 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 incorporated according to accepted pharmaceutical practice. Desirably, GPE or G-2MePE compound to be used for therapeutic administration may be sterile. Sterility can be readily accomplished by filtration through sterile filtration membranes (e.g., membranes having pore size of about 0.2 micron). Therapeutic compositions generally can be 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. In other embodiments, GPE or G-2MePE compound can be stored in unit or multi- dose containers, for example, 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 0.01% (w/v) aqueous solution of compound, and the resulting mixture is lyophilized. The infusion solution can be prepared by reconstituting lyophilized compounds using bacteriostatic water or other suitable solvent. EXAMPLES

Example 1 : Experimental Methods Subjects Adult Long-Evan's rats, weighing 325 ± 25g (Harlan) at the time of order, were used. The rats were kept on a 12:12 hour ligh dark cycle and allowed food and water ad libitum, and the rats were tamed by gentle handling twice daily for one week before inclusion in the experiment. The rats were pre-selected and pre-trained on the behavior tests including footfault, and forelimb placing (Whisker, Forward, and Lateral-Tactile Placing). These tests are well known in the art and need not be described in detail herein. Behavior tests and rectal temperature were measured before surgery to exclude abnormal rats. Only rats with less than 20% footfault, normal forelimb placing and rectal temperature at 37.7 ± 0.5°C were included and subjected to MCAo.

Statistical Analysis Investigators blinded to treatment assignment did all behavioral testing and subsequent histological analyses. Statistical analyses were performed using GraphPad Prism version 3.00 for Windows and GraphPad InStat. Behavioral tests and body weight changes were analyzed using One-way analyses of variance ("ANOVA") followed by Student- Newman-Keuls multiple comparison post hoc tests with correction for multiple comparisons. A separate ANOVA was performed for each of the time points to see at what time point the NDS scores became significantly different. Pearson correlation coefficients were calculated between the infarct volume and behavioral tests at the 72-h time point. All data were expressed as mean±SD. The above statistical tests were two-tailed and considered significant at thejp<0.05 level. Mortality was analyzed by Fisher's exact test.

Example 2. Induction of Middle Cerebral Artery Occlusion (MCAO) Focal brain ischemia was induced by the MCAo intraluminal suture method as previously described by Longa (Longa et al. Stroke. 1989;20: 84-91). Briefly, the rats were anesthetized with 2% isofluorine in a mixture of 30% oxygen and 70% nitrous oxide delivered by tracheal intubation. A rectal probe was inserted 4-cm into rectum and the probe was temporarily fixed to the tail. Micro-Renathane tubing (type MRE-040, Braintree Scientific Inc.) was cannulated into the jugular vein for IV drug delivery before the MCAo. The right common carotid artery ("CCA"), internal carotid artery ("ICA"), and external carotid artery ("ECA") were exposed through a midline neck incision. A 4-0 poly-

L-Lysine-coated nylon suture was inserted through a stump of the ECA, and the CCA was kept open and intact. The suture occluder was advanced into the ICA 19 to 21 mm beyond the carotid bifurcation. Mild resistance indicated that the occluder was properly lodged in the anterior cerebral artery and thus blocked blood flow to the middle cerebral artery (MCA). The rat was allowed to awaken right after finishing the operation and the Neurological Deficit Score (NDS) was measured 60-65 min after initiation of the MCAo. Rats with satisfactory deficits on NDS (NDS score 11 to 15) were re-anesthetized with isofluorine by a facemask. 75 min after starting MCAo, reperfusion was started by withdrawing the suture. The CCA and ICA were inspected to ensure the return of good pulsation, and the neck incision closed with silk suture. Drug treatments were started immediately after the start of reperfusion by connecting the pre-cannulated tubing to a perfusion pump to infuse a 3-ml solution into the jugular vein at a dose rate of 1 ml/hr. The rats freely moved around in a cylinder during the drug infusion by using a swivel tether system. After finishing the infusion, the rats were returned to their homecage. MCA suture occlusion for 75 minutes caused a relative high mortality (12%); 11 rats died out of 92 subjected to MCAo mainly related to cerebral edema, hyperthermia, and hemorrhagic conversion of the infarct (Fagan et al. Pharmacotherapy. 1999;19:139-142; Fagan et al. Neurol Res. 2003;25:377-382; Li etal. Stroke. 1999;30:2464-2470; discussion 2470-2461; Schmid-Elsaesser et al. Stroke. 1998;29:2162-2170). This mortality rate and its causes, in fact, are similar to those observed in human stroke patients with MCA occlusions. There was no significant difference in mortality between any of the study groups.

Example 3. Treatment Groups and Doses Rats with moderate level injury (NDS of 11 to 15 right before reperfusion) were randomly assigned to six groups: 1. Buffer group (n=l 1): 3 ml of saline or 3 ml of succinate buffer (pH 6.0) at lml/h for 3 hours. 2. GPE group (n=9): 9 mg/kg of GPE in 3 ml of succinate buffer at 1 ml/hr for 3 hours. 3. G-2MePE group (n=9): 0.9 mg/kg of G-2MePE in 3 ml of saline at lml/h for 3 hours. 4. Caffeinol group (n=12): 10 mg/kg of caffeine (1,3,7-trimethylxanthine, Acros Organics) and 0.32 g/kg of ethanol (Quantum, IL) in 3 ml of saline at 1 ml/h for 3 hours. 5. GPE + caffeinol (n=12): Combination of groups 2 and 4 in 3 ml saline at 1 ml/h for 3 hours. 6. G-2MePE + caffeinol group (n=9): Combination of groups 3 and 4 in 3 ml saline at 1 ml/h for 3 hours.

Example 4. Physiological Signs There were no significant differences in baseline body weight between the groups

(p=0.71). G-2MePE combined with caffeinol significantly prevented body weight loss at 72 h (p<0.05) compared to the buffer group, but there were no significant differences among all the other groups (Figure 1A). Rectal temperature did not differ between the groups

(Figure IB). Example 5. Measurement of Lesion Volume On day 3 after MCAo, the rats were deeply anesthetized by injection of 0.6 mg/kg of chloral hydrate, perfused intracardially with 150 to 200 ml of ice-cold PBS, then decapitated. The brain was removed within 30 seconds, rinsed in PBS, and sliced into 2- rnm sections. The sections were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC). After TTC staining, each section was scanned into a Macintosh computer and analyzed by a computer-interfaced BRAIN imaging system as previously described (Aronowski et al. J Cereb Blood Flow Metab. 1999;19:652-660). Lesion volume was calculated as the sum of 7 slices. Figure 2 shows that by itself, GPE significantly reduced the lesion volume in the cortex (125.7 mm3; ? <0.01), but not in the striatum (89.5, p=0.14) compared to the buffer group (221.1 and 110.3). Caffeinol alone also significantly reduced cortical lesion volume (134.2; p <0.01), but not in striatum (87.0; p=0.06). Although G-2MePE did reduce lesion volume in both cortex and striatum (cortex 192.5, p= .25; striatum 92.0, p=0.0$), these effects were not statistically significant. GPE combined and caffeinol together reduced cortical infarct volume to 100.50 (p < 0.01) and striatal volume to 82.13 (p<0.05), but the additive effect of the combination was not significantly different from that of GPE alone. G-2MePE combined with caffeinol displayed an additive effect compared to either treatment alone. The protective effect of the combination on lesion volume was shown in both the cortex (94.8,/j O.001) and also in the striatum (74.0, p O.001), see Figure 2.

Example 6. Behavioral Measurements All behavioral tests took place in a quiet and low light room by an experimenter blinded with respect to the treatment groups. The footfault and forelimb placing tests were done according to previously published methods by Bland (Bland et al. Behav Brain Res. 2001;126:33-41). The postural reflex and circling tests were done as described by Bederson (Bederson et al. Stroke. 1986;17:472-476). NDS was measured pre-occlusion, pre-reperfusion (60 to 65 min after initiation of MCAo), and 72 hours after MCAo. NDS (0 to 18) was calculated by combining the score on the following 4 tests. Postural Reflex Test The degree of abnormal posture was estimated by suspending rats with their tails

20 cm above a tabletop. Intact rats extended both forelimbs toward the table surface. Rats displaying this behavior were recorded as score 0. Rats with only flexing of the contralateral limb toward the body were recorded as 2. Rats only rotating the contralateral shoulder toward the tail were graded as 4.

Circling or Sidewalk Rats that circled or sidewalked toward tire paretic side on 10 trials were recorded. A score of 2 or 4 was given to each rat according to the severity of their deficits.

Forelimb Placing (Whisker, Forward Tactile and Lateral Tactile) Animals were held by their torsos with forelimbs hanging freely. Contralateral and ipsilateral forelimb Whisker placing responses were induced by gently brushing the respective vibrissae on the edge of a tabletop for 10 trials. A score of one was given each time the rat placed its forelimb on the edge of the tabletop in response to the vibrissae stimulation. Percent successful placing responses were determined (number correct x 10). The lateral tactile placing is similar to the Whisker placing, except the placing response was induced by gently contacting the lateral side of the forelimb to the edge of the tabletop, while forward tactile placing was induced by contacting the frontal side of the forelimb to the edge of a tabletop. The scale was scored as: 0, immediate and complete placing 8 or more out of 10 trials; 1, delayed and/or incomplete placing >2 out of 10 trials; 2, no placing. Footfault Animals were placed on an elevated grid, with openings of 2.3 cm2. As the animals traversed the grid, a footfault was scored each time the contralateral forepaw slipped through an opening in the grid. The total number of steps was also counted. The percent footfault was calculated as the number of footfaults/total steps X 100. A score of 0 to 4 was given to each rat according to the severity of the deficit by calculating the percent footfaults x 0.04. Behavioral Test Results Sensory-motor deficits were improved in parallel with the changes seen in cortical lesion volume. NDS changes from before reperfusion to 72 hours for the individual tests are shown in Figure 3. Figure 3 A shows results of studies of footfault change. G-2MePE alone had a very small effect, caffeinol alone had a small effect, but the effect of G-2MePE and caffeinol together produced an effect that was greater than the sum of the individual effects. This finding of synergy was completely unexpected given the effects of G-2MePE and caffeinol alone. Figure 3B shows effects of GPE, G-2MePE and caffeinol on circling change. As with Figure 3A, G-2MePE alone had a relatively weak effect at the dose studied, as did caffeinol alone. However, the combination of G-2MePE and caffeinol together had an effect greater than the sum of the separate effects of G-2MePE and caffeinol. This finding of synergy was completely unexpected based on the results obtained for G-2MePE and caffeinol alone. Figure 3C shows effects of GPE, G-2MePE and caffeinol on reflexing change. As with Figures 3 A and 3B, G-2MePE alone had a relatively weak effect at the dose used, as did caffeinol alone. However, the combination of G-2MePE and caffeinol together had an effect greater than the sum of the separate effects. This finding of synergy was completely unexpected based on the results obtained for G-2MePE and caffeinol alone. Figure 3D shows effects of GPE, G-2MePE and caffeinol on forelimb placing change. As with Figures 3A, 3B and 3C, G-2MePE alone had a relatively weak effect at the dose studied, as did caffeinol alone. However, the combination of G-2MePE and caffeinol together had an effect greater than the sum of the separate effects. This finding of synergy was completely unexpected based on the results obtained for G-2MePE and caffeinol alone. The total NDS at 72h after reperfusion is shown in Figure 4. As with Figures 3 A- 3D, G-2MePE had a relatively weak effect by itself, as did caffeinol. However, the combination of G-2MePE and caffeinol together had an effect greater than the sum of the individual effects. This finding of synergy was completely unexpected based on the results obtained with G-2MePE and caffeinol alone. The correlation of lesion volume to changes on the individual tests and the total NDS are given in Table 1 below. Table 1 Correlation of Lesion Volume (LV) and Neurological Deficit Score (NDS)

Figure imgf000019_0001
GPE significantly decreased the NDS by 33.8% (8.35 vs.12.6, ρ< 0.01) (Figure 4).

Caffeinol reduced the NDS by 35.5% (8.13 vs. 12.6, pO.OT). G-2MePE did not significantly decrease the NDS (10.1 vs. I2.6,p = 0.05), but when combined with caffeinol, had a very significant effect on reducing NDS by 63.5% (4.6 vs. 12.6,p<0.001). Similarly, GPE produced significant changes in neurological deficit score, and further addition of caffeinol further increased the beneficial effect in Figures 3B and 3D. It can be appreciated that the above descriptions and Examples are not intended to limit the scope of the invention. Rather, other embodiments and uses of the compounds and compositions of this invention are within the ordinary skill in the art and are considered to be part of this invention. All references cited in here are fully and in their entirety incorporated herein by reference.

INDUSTRIAL APPLICABILITY Embodiments of this invention are useful for treating disorders involving CNS injury or disease. Other embodiments of this invention are useful in the medical and pharmaceutical arts for the manufacture of medicaments useful for treating neurological deficits associated with CNS injury or disease.

Claims

CLAIMS What is claimed is:
1. A method for treating CNS injury or disease in a mammal, comprising administering to said mammal a pharmaceutically effective composition comprising an effective amount of:
(a) Gly-Pro-Glu, caffeine and an alkanol; or
(b) G-2MePE, caffeine and an alkanol; or
(c) GPE, G-2MePE, caffeine and an alkanol.
2. The method of claim 1 , wherein said CNS injury is hypoxic or ischemic injury.
3. The method of claim 2, wherein said injury is associated with decreased cerebral blood flow.
4. The method of claim 2, wherein said injury is associated with stroke.
5. The method of claim 2, wherein said injury is associated with coronary artery bypass surgery.
6. The method of claim 2, wherein said injury is associated with a traumatic brain injury.
7. The method of claim 1 , wherein said disease is associated with damage to glial cells or neurons.
8. A method for reducing a functional symptom of CNS injury or disease in a mammal suffering from such injury or disease, comprising administering to said mammal a pharmaceutically effective composition comprising:
(a) Gly-Pro-Glu, caffeine and an alkanol; or
(b) G-2MePE, caffeine and an alkanol; or
(c) GPE, G-2MePE, caffeine and an alkanol.
9. The method of any of claims 1 to 8, wherein said functional symptom is selected from the group consisting of paralysis and spasticity, cognitive impairment, and abnormalities of gait.
10. The method of any of claims 1 to 8, wherein said alkanol is ethanol.
11. The method of any of claims 1 to 9, wherein said alkanol is ethanol, or a mixture of ethanol and another alkanol.
12. The method of claim 11 , wherein the alkanol is between 1 and 10 percent ethanol in water.
13. The method of any of claims 1 to 8, wherein the effective amount of alkanol is between 0.1 and 0.5mg/kg of said patient's weight.
14. The method of any of claims 1 to 13, wherein the effective amount caffeine is between about 1 and 50mg/kg of patient's weight.
15. The method of any of claims 1 to 14, wherein the effective amount of GPE is from about 0.03 mg/kg/h to about 30 mg/kg h.
16. The method of any of claims 1 top 14, wherein the effective amount of G-2MePE is from lμg to lOOmg/kg.
17. Use of GPE, G-2MePE, caffeine and alkanol in the manufacture of a medicament useful for treating a CNS disorder, comprising combining together:
(a) Gly-Pro-Glu, caffeine and an alkanol; or
(b) G-2MePE( caffeine and an alkanol; or
(c) GPE, G-2MePE, caffeine and an alkanol.
18. The use of claim 17, wherein said alkanol is ethanol.
19. The use of any of claims 17 or 18, wherein said CNS disorder is selected from the group consisting of hypoxia/ischemia, stroke, traumatic brain damage, decreased cerebral blood flow and coronary artery bypass surgery.
20. A composition for treating a CNS injury or disease, comprising: Gly-Pro-Glu, caffeine and an alkanol; G-2MePE, caffeine and an alkanol; or GPE, G-2MePE, caffeine and an alkanol.
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WO2007106555A2 (en) 2006-03-14 2007-09-20 Neuren Pharmaceuticals Limited Oral formulations of glycyl-2-methylprolyl-glutamate
WO2009033806A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of gly-pro-glu-oh (gpe) as a therapeutic agent
WO2009033805A3 (en) * 2007-09-11 2009-09-03 Mondobiotech Laboratories Ag Use of somatostatin-14 as a therapeutic agent

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US6780848B2 (en) * 1993-12-23 2004-08-24 NeuronZ, Ltd. Use of GPE to protect glial cells or non-dopaminergic cells from death from neural injury or disease

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007106555A2 (en) 2006-03-14 2007-09-20 Neuren Pharmaceuticals Limited Oral formulations of glycyl-2-methylprolyl-glutamate
EP2001400A2 (en) * 2006-03-14 2008-12-17 Neuren Pharmaceuticals Limited Oral formulations of glycyl-2-methylprolyl-glutamate
JP2009538269A (en) * 2006-03-14 2009-11-05 ジンユアン ウェン Oral formulation of glycyl-2-methylprolyl glutamate
EP2001400A4 (en) * 2006-03-14 2013-01-23 Neuren Pharmaceuticals Ltd Oral formulations of glycyl-2-methylprolyl-glutamate
WO2009033806A2 (en) * 2007-09-11 2009-03-19 Mondobiotech Laboratories Ag Use of gly-pro-glu-oh (gpe) as a therapeutic agent
WO2009033806A3 (en) * 2007-09-11 2009-07-09 Gerald Bacher Use of gly-pro-glu-oh (gpe) as a therapeutic agent
WO2009033805A3 (en) * 2007-09-11 2009-09-03 Mondobiotech Laboratories Ag Use of somatostatin-14 as a therapeutic agent
US8211856B2 (en) 2007-09-11 2012-07-03 Mondobiotech Laboratories Ag Use of somatostatin-14 as a therapeutic agent

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