Methods of Treating Neurodeσenerative Conditions

Cross-Reference of the Application

This case is a CIP of U.S. Serial No. 407,913 filed September 15, 1989.

Background of the Invention

The present invention is directed to methods of treating neurodegenerative conditions by increasing extracellular concentrations of adenosine.

The etiology of major neurodegenerative diseases is not understood. Such diseases, which include Parkinson's Disease, Huntington'ε Disease, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease) and Alzheimer's Disease, have proved difficult to treat; few if any therapies have proved effective in slowing or arresting the degenerative process.

Parkinson's Disease is a prevalent neurodegenerative disease which generally affects older people. As noted above, its specific etiology is not well understood; however, a Parkinson-like syndrome can result from expo¬ sure to certain chemical substances. Two such substances, methamphetamine and l-methyl- -phenyl-1,2,3,6-tetrahy- dropyridine (MPTP) , have been used as models for studying Parkinson's Disease.

Parkinson's Disease is characterized by lesions in the brain, particularly affecting the striatum and results in dopamine depletion, particularly in the striatum (nuclei of the basal ganglia, especially εubstantia nigra, putamen and caudate nucleus) . Attempts to alleviate the dopamine depletion in individuals affected with Parkinson's Disease led to the use of L-dopa, a precursor to dopamine which is better able to cross the blood-brain barrier, as a therapeutic agent to alleviate the symptoms of Parkinson's Disease. In order to better target the global effects of L-dopa, it is often given with

carbidopa, a peripheral decarboxylase inhibitor which decreases the metabolism of L-dopa in the peripheral tissues.

The systemic administration of either methamphetamine or MPTP to experimental animals has been found to produce degenerative changes in nigrostriatal dopaminergic neurons or their axon terminals. Both methamphetamine and MPTP result in decreases in striatal dopamine (DA) and in decreased tyrosine hydroxylase (TH) activity, as well as histochemical indications of nerve terminal degeneration within the neostriatum. It has been postulated that some of those neurodegenerative effects may be associated with overactivity of excitatory amino acid (EAA) neurotrans- mission. Treatment with noncompetitive blockers of one of the EAA receptors, the N-methyl-D-aspartate (NMDA) receptor has been shown to partially antagonize NMDA- mediated decrements in DA content and TH activity produced by administration of methamphetamine or MPTP. It was postulated that those findings implicated EAA's in neurodegenerative conditions such as Parkinson's Disease. (See, Sonsalla, P.K. , et al. "Role for Excitatory Amino Acids in Methamphetamine-Induced Nigrostrial Dopaminergic Toxicity", Science 243: 398-400 (1989)).

Due to their mimicry of effects of Parkinson's Disease, treatment of animals with methamphetamine or MPTP has been used to generate models for Parkinson's Disease. These animal models have been used to evaluate the efficacy of various therapies against Parkinsons'ε Disease. Administration of MPTP to animals provides a useful Parkinsonian model. The end result of MPTP administration is the destruction of the striatum in the brain, an area in the neocortex li bic system in the subcortical area in the center of the brain, an area compromised in Parkinson's Disease. The neurotransmitter dopamine iε concentrated in the striatum. Parkinson's Disease is characterized by lesions in that area of the brain and by

depleted dopamine levels. In some species (primates) the striatal degeneration has been reported to be accompanied by behavioral symptoms that mimic Parkinson's symptoms in humans. Methamphetamine also compromises the striatum, but is somewhat leεs selective than MPTP and may induce strial degeneration by a different mechanism than MPTP.

Adenoεine, 9-,9-D-ribofuranoεyladenine (the nucleoside of the purine adenine) , belongs to the class of bio- chemicals termed purine nucleosides and is a key biochemical cell regulatory molecule, as described by Fox and Kelly in the Annual Reviews of Biochemistry. Vol. 47, p. 635, 1978.

Adenosine interacts with a wide variety of cell types and iε responsible for a myriad of biological effects. Adenosine serves a major role in brain as an inhibitory neuromodulator (see Snyder, S.H., Ann. Rev. Neural Sci. 8: 103-124 1985, Marangos, et al., NeuroSci and Biobehav. Rev. 9:421-430 (1985), Dunwiddie, Int. Rev. Neurobiol.. 21-63-130 (1985)). Thiε action iε mediated by ecto- cellular receptorε (Londos et al.. Regulatory Functions of Adenosine. pp. 17-32 (Berne et al., ed.) (1983)). Among the documented actions of adenosine on nervous tissue are the inhibition of neural firing (Phillis et al., Europ. £. Pharmacol.. 3_Q:125-129 (1975)) and of calcium dependent neurotransmitter release (Dunwiddie, 1985) . Behaviorally, adenoεine and itε etabolically stable analogε have pro¬ found anticonvulεant and sedative effects (Dunwiddie et al., £. Pharmacol, sad Exptl. herapeyt» , 22_QL'' 0-76 (1982); Radulovacki et al., £. Pharmacol. Exptl. Thera.. 228:268-274 (1981)) that are effectively reversed by specific adenosine receptor ^' antagonists. In fact, adenosine has been proposed to serve as a natural anticonvulsant, and agents that alter its extracellular levels are modulators of seizure activity (Dragunow et al., Epilepsia 26:480-487 (1985); Lee et al.. Brain Res.. 21:1650-164 (1984)). In addition, adenosine is a potent

vasodilator, an inhibitor of immune cell function, an inhibitor of granulocyte oxygen free radical production, an anti-arrhythmic, and an inhibitory neuromodulator. Considering its broad spectrum of biological activity, considerable effort has been aimed at establishing practical therapeutic uses for adenoεine and its analogs. Since adenoεine iε thought to act at the level of the cell plaεma membrane by binding to receptors anchored in the membrane, past work haε included attemptε to increase extracellular concentrations of adenosine by administering it into the blood stream. Unfortunately, because adeno¬ sine is toxic at concentrationε that have to be administered to a patient to maintain an efficacious extracellular therapeutic concentrations, the administra- tion of adenosine alone iε of limited therapeutic use. Further, adenosine receptors are subject to negative feedback control following exposure to adenoεine, including down-regulation of the receptorε.

Other ways of achieving the effect of a high local extracellular concentration of adenoεine exiεt and have alεo been studied. They include: a) interference with the uptake of adenosine with reagentε that specifically block adenosine transport, as described by Paterson et al., in the Annalε of the New York'Academy of Sciences. Vol. 255, p. 402 (1975); and Deckert et al., in Life Sciences _f Vol. 42, page 1331 to 1345; b) prevention of the degradation of adenoεine, as described by Carson and Seegmiller in The Journal of Clinical Investigation. Vol. 57, p. 274 (1976); and c) the use of analogs of adenosine selectively to bind to adenosine receptors.

Compounds which selectively increase extracellular adenosine would also be useful in the prophylactic protection of cells in the .hippocampus implicated in memory. The hippocampus haε more adenoεine and glutamate receptors than any other area of the brain. Accordingly, as described below, it iε most sensitive to stroke or any condition of low blood flow to the brain. Some recent

studieε εuggest that Alzheimer's disease may result from chronic subclinical cerebral ischemia. Accordingly, compounds which selectively increaεe extracellular adenoεine levels may be used for the treatment and/or prevention of both overt stroke and Alzheimer's disease. It is now established that relatively short periods of brain iεchemia (on the order of 2 to 8 minutes) εet into motion a serieε of eventε that lead to an eventual death of selected neuronal populations in brain. This process is called delayed excitotoxicity and it iε cauεed by the ischemia-induced enhancement of the releaεe of the excitatory amino acid neurotransmitterε, including gluta- mate and aspartate. Within a period of hourε to days post-εtroke, εome neurons in brain are overstimulated by EAA'ε to the point of metabolic exhaustion and death. Because over-released glutamate appears to be the major factor involved *in poεt-εtroke cell damage, the blockade of glutamate receptors in brain could be beneficial in stroke therapy. In animals, glutamate receptor blockers have been shown to be effective in alleviating or pre¬ venting εtroke-associated neural damage. These receptor blockers have, however, been shown to lack specificity and produce many undesirable side effects. Church, et al., "Excitatory Amino Acid Transmission," pp. 115-118 (Alan R. Lisε, Inc. 1^87).

Adenoεine haε been shown to be a potent inhibitor of glutamate release in brain. The CA-I region of brain is selectively sensitive to post-εtroke destruction. In studies where observations were made at one, three and six days poststroke, the CA-I area in the hippocampus was shown to be progresεively destroyed over time. However, where cyclohexyladenosine ("CHA") , a global adenosine agonist, was given shortly after the stroke, the CA-1 area was markedly protected. (Daval et al.. Brain Reε.. 111:212-226 (1989) and Marangos, Med. Hypothesiε 22:45- 49 (1990)). That beneficial effect was also seen in the survival rate of the animals. Because of its global

effect, however, CHA haε non-εpecific εide effectε. For example it undersirably will lower blood preεεure, slow the heart and markedly raise blood glucose.

Hyperglycemia has been reported to be associated with a poor prognosiε for stroke. (Helgaεon, Stroke 1£(8) : 1049-1053 (1988)). In addition, mild hypoglycemia induced by inεulin treatment haε been shown to improve survival and morbidity from experimentally induced infarct. (LeMay et al.. Stroke 19fill: 1411-1419 (1988)). AICA riboεide and the prodrugε of the preεent invention could protect againεt iεchemic injury to the central nervous system (CNS) by their ability to lower blood glucose.

Another area of medical importance is the treatment of neurological diseases or conditions arising from elevated levels of homocyεteine (e.g., vitamin B12 deficiencies) . The AICA riboside prodrugε may be used for such purposes aε well. .

During εeizureε, certain neural cellε fire abnormally. ATP cataboliεm iε greatly accelerated in the abnormally firing cellε leading to increaεed adenoεine production. Adenoεine haε marked anticonvulεant effects and, thus, haε been termed the brain's natural anticon- vulεant. It appears to play a major role in the brain aε an inhibitory neuromodulator; this action of adenosine is apparently mediated by certain ectocellular receptorε. Adenosine has both post-εynaptic and pre-εynaptic effects. Among the documented effects of adenosine on nervous tissue are the inhibition of neural firing and of calcium dependent neurotransmitter release. Behaviorally, adenosine and its metabolically stable analogs have profound anticonvulsant and sedative effectε.

Aε stated above, adenoεine haε been propoεed to serve aε a natural anticonvulsant with agents that alter its extracellular concentration acting as a modulator of seizure activity. Besides acting as a neuromodulator, adenosine iε a potent vaεodilator, an inhibitor of granulocyte oxygen free radical production, an

antiarrhythmic. In fact, because of the many actionε of adenoεine, it haε been called a "retaliatory molecule" released to protect cellε against certain pathologic aεεaultε. Unfortunately, adenoεine is toxic at concentrations that have to be administered εystemically to a patient to maintain an efficacious extracellular therapeutic concent¬ ration at the target organ, and the administration of adenosine alone so far has been of limited therapeutic use. Likewise, since most cellε in the body carry receptorε for adenoεine, the uεe of techniques that increaεe adenoεine concentrationε generally throughout the body can cauεe unwanted, dramatic changes in normal cellular physiology.

Summary of The Invention

The present invention iε directed to methodε of preventing or decreaεing neural tiεεue damage associated with neurodegenerative diseaseε in an affected individual which compriεes increasing the extracellular concent- rations of adenosine in said neural tissue.

In one aspect of the preεent invention, the extracellular adenoεine concentrationε are increaεed by adminiεtering to the affected individual a therapeutically effective amount of an agent which increases extracellular adenosine. Suitable agents for increasing extracellular adenosine concentrationε include adenosine regulating agentε (e.g. inhibitors of adenosine catabolism and enhancers of adenoεine production), inhibitors of adenoεine transport, and adenosine agonists. One particularly preferred group of agents which act as adenosine regulating agents comprise AICA riboside or AICA riboside prodrugs which compriεe a modified AICA riboεide having an AICA riboεyl moiety and at leaεt one hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety per equivalent weight of AICA riboεyl moiety.

In another aspect, the present invention is directed to preventing neural tissue damage caused by increased release of excitatory amino acids (EAA) by increasing extracellular concentrations of adenoεine. The adenoεine regulating agents, including AICA riboside and itε prodrugε, described herein not only show the beneficial adenosine regulating/EAA inhibiting properties, but also are both site and event specific, avoiding the unwanted global action of known adenosine agonistε.

In an additional aεpect, the preεent invention iε directed to the treatment of Parkinεon'ε Disease in an affected individual and related neurodegenerative diεeaεes by increasing the extracellular concentration of adenosine in the brain of that individual. Such diseaεeε include Alzeheimer'ε Diεeaεe, Amyotropic Laterial Sclerosis (ALS) and Huntington's Disease.

Brief Description of the Drawingε

FIG. 1 depictε the effect of Ketamine and CHA on the development of Methamphetamine-induced Parkinεon Syndrome.

FIG. 2 depictε the effect of Ketamine and CHA (in the preεence and absence of caffeine) on methamphetamine- induced Parkinson'ε Syndrome development.

FIG. 3 depictε the effect of CHA on MPTP-induced decreaεed tyrosine hydroxylase activity.

FIG. 4 depictε a comparison of the effect of CHA and MK-801 on MPTP-induced decreaεe in tyroεine hydroxylaεe activity.

Detailed Description of the Invention The methods of the preεent invention are directed to preventing or decreaεing neural tissue damage by increasing the extracellular concentrations of adenoεine or adenoεine agonists in the neural tissue. One mechanism proposed for this protective effort involves inhibition of excitatory amino acid (EAA) induced neural toxicity.

Excitotoxicity haε been implicated in the methamphetamine animal model of Parkinson's Diεeaεe (Sonεalla et al.. Science 243:398-400 (1989)). According to the methamphetamine model, a neurodegenerative mechanism involves the release of newly synthesized dopamine oxidation products which exhibit neurotoxicity. Another neurodegenerative diεeaεe model involveε methylphenyl- tetrahydropyridine (MPTP) which iε thought to cause damage by glial conversion of MPTP to l-methyl-4-phenylpyridi- niu ion ("MPP ^* ") and which is then thought to be selectively taken up by dopaminergic neurons and which produces εuperoxide in mitochondria by redox cycling uεing neuromelanin as an electron source. (See, Markey, et al., Medicinal Research Reviews £(4):389-429 (1986)). The methods of the preεent invention have shown efficacy in protecting against neurodegenerative effects in both the methamphetamine and MPTP models.

Preferred Adenosinergic Compounds

One aspect of the present invention iε directed to the uεe of agentε which enhance extracellular adenoεine levels to protect against neurodegenerative damage. As noted, these agents include adenosine regulating agents εuch aε AICA riboεide and, analogε and prodrugε thereof.

Agents which enhance extracellular • adenoεine concentrations include inhibitors of adenosine transport or adenosine regulating agents, e.g. inhibitors of adenosine catabolism or enhancers of adenosine production.

Agents that can inhibit the cellular transport of adenosine include those that do so specifically, and are essentially competitive inhibitors of adenosine uptake, and others that inhibit nonspecifically. P-nitrobenzyl- thioinosine and dipyradamole appear to be competitive inhibitors, while a variety of other chemicals, including colchicine, phenethyalcohol and papaverine inhibit uptake nonspecifically.

Alternatively, extracellular concentrations of adenosine can be increased by the uεe of chemicalε that inhibit enzymatic degradation of adenoεine. One group includes inhibitors of adenosine deaminase, an enzyme which participates in the conversion of adenosine to inosine. Inhibitors of adenosine deaminase activity include coformycin, 2'-deoxycoformycin, and erythro-9-(2- hydroxy-3-nonyl) adenine hydrochloride.

Adenosine receptor agonists and antagonists include adenosine analogs which have εtructural modificationε in the purine ring, alterationε in εubεtituent groupε attached to the purine ring, and modificationε or alterationε in the εite of attachment of the carbohydrate moiety. Although inhibitors of adenoεine transport, inhibitors of adenoεine deaminase and adenosine agoniεtε provide advantages in increaεing extracellular adenoεine over the uεe of adenoεine alone, they may exhibit dis¬ advantages which include adverse side effects, primarily due to the fact that they must be administered in doseε that are toxic, and have nonεelective effects on most cell types. Since moεt cellε in the body have functional receptorε for adenoεine, techniques that increase adenosine concentrations generally throughout the body can cauεe unwanted, dramatic changeε in normal cellular phyεiology. (See, Purine Metabolism in Man, (eds. De Baryn, Sim onds and Muller) , Plenum Press, New York (1984)). In addition, adenosine deaminase inhibitors prevent the degradation of deoxyadenosine which is a potent im unotoxin. (see Gruber et al. Ann. New York Acad. ≤si. 451:315-318 (1985)).

Accordingly, adenosinergic agents which selectively increase extracellular adenosine levels in the areas of the brain affected by a neurodegenerative condition, such as Parkinson's Diseaεe, are preferred. Thuε, a preferred aspect of the methods of the preεent invention iε directed to the uεe of adenoεine regulating agentε which selec-

tively enhance extracellular adenoεine concentration in neural tissue to protect against neurodegenerative damage. Methods for enhancing extracellular adenosine concentrations utilize the administration of compounds which are believed to alter one or more of the biochemical pathways of adenoεine etaboliεm (e.g., adenoεine kinaεe, AMP deaminase, AMP nucleotidase) , so that the net result is an enhanced extracellular concentration of adenosine. This may result from one or more processes, including enhanced intracellular production and/or decreased catabolism of adenoεine) . Examples of compounds useful in the methods of the preεent invention include compounds broadly classified aε purine nucleosides and related analogs, such as AICA riboside, AICA ribotide, 1-3-D- ribofuranosyl-lB-1,2,4-triazole-3-carboxamide (ribavirin) , ribavirin monophosphate, and variouε pro-formε of the above compounds. The compounds can be taken up by cellε and, if necessary, are believed to be converted to their monophosphate and, to a lesser extent, their diphosphate and triphoεphate formε. Also included are (1) agents that can enhance endogenouε synthesis of AICA riboside or metabolites, such as purine intermediary metaboliteε or compoundε that can form theεe metaboliteε, e.g.. succinylaminoi idazole carboxamide (SAICA) riboside, (2) agents that cause a buildup of AICA-riboside or its metabolites, including ethotrexate, and (3) agentε that cauεe bacterial flora to increase AICA riboεide production, such as εulfonamideε. Theεe compoundε can be administered to a patient either prophylactically, in some caseε, and/or in direct reεponεe to a bodily condition in otherε. Purine nucleosides or analogs that enhance the extracellular concentration of adenosine and/or adenosine analogs may be administered to a living system over the concentration range of 0.05 millimolar to 0.5 millimolar and, for AICA riboside typically, are administered in concentrations up to 0.5 millimolar.

Certain purine prodrugs and analogε which exhibit and, in some caseε improve upon, the positive biological effectε of AICA riboεide and other adenoεine regulating compoundε without the negative effects of adenosine are diεcloεed in the commonly assigned pending patent application "AICA Riboside Prodrugε," USSN 301,222, filed January 24, 1989, the commonly assigned patent application "Method and Compounds for AICA Riboside Delivery and for Lowering Blood Glucose" USSN 408,107 filed September 15, 1989, and "Methods and Compoundε for AICA Riboεide Delivery and for Lowering Blood Glucoεe, USSN 466,979, filed January 18, 1990, the disclosures of which are incorporated herein by reference. The compounds therein defined may be used aε prodrugε. The novel compoundε typically exhibit one or more of the following improvementε over AICA riboεide: 1) more potent adenoεine regulating effectε; 2) increased half-lives; 3) increaεed brain penetration; 4) increaεed oral bioavailability; 5) increaεed myocardial targeting; 6) in some caseε synergism with AICA riboεide itself. The AICA riboside prodrugs may be used in methods of the preεent invention.

Adenoεine or inoεine are generated from adenoεine triphoεphate in the course of rapid cellular energy utilization, such as during seizure activity, arrhythmias, or a condition resulting in decreased blood flow (ischemia) , such as a stroke, heart attack, or angina. Normally, during such an event, the production of inosine iε greater than that of adenoεine. In the area of low flow during coronary occluεion, for example, the ratio of inoεine to adenoεine iε approximately 100 to 1. A certain percentage of inoεine and adenosine subsequently exit the cell and are present in the immediate extracellular environment. These adenoεine regulating agentε are useful in the methods described herein and have been shown to enhance the extracellular concentration of adenosine, and the production of inosine haε been shown to be decreaεed in some settings. Adenosine levels are not altered

εignificantly throughout the patient becauεe alterations in adenosine production only occur in areas of, and at the time of, net ATP use and because adenoεine iε rapidly degraded. Thuε, the uεe of theεe adenoεine regulating agents according to the methods of the preεent invention will cauεe a localized increased concentration of extracellular adenosine instead of a systemic or generalized adenosine enhancement.

Because the purine nucleoside analog AICA riboside can be metabolized to uric acid, this agent may be used with allopurinol or other drugs that prevent uric acid synthesis, or with a uricosuric agent such as probenecid. Certain agentε, εuch aε methotrexate and ribavirin, whoεe metaboliteε inhibit AICA riboεide transfor ylaεe, may cauεe an elevation of endogenouεly synthesized AICA ribotide and create effects similar to administering the purine nucleoside. Concomitant administration of AICA riboside or AICA riboεide with an inhibitor of AICA riboside transformylaεe should have at least additive effects. In addition, any one of the de novo purine nucleotide synthesis intermediates (after the first committed step for purine synthesis) or their nucleosideε or bases can be assumed to be rapidly converted to AICA riboside. An example is SAIGA riboside or its nucleotide or base.

Upon contact with cells, it is believed that the adenosine regulating purine nucleosideε and analogs useful in the methods of the present invention enter the cell where, if necessary, they, may be phosphorylated by adenoεine kinase or, in the case of administration of base, they may be converted to a nucleotide by a phosphoribosyl transferase enzyme to yield a purine nucleotide monophosphate, and eventually also the nucleoside diphosphate or triphoεphate. The diphoεphate or triphosphate form may comprise a pool for breakdown to the monophosphate form.

In one aεpect of the preεent invention, preferred AICA riboεide prodrug compoundε are uεed which include those having the following formula:

wherein X ₂ , and X ₃ are independently (a) hydrogen, (b) -

CR,, O

8 or -C0R ₂ wherein R, iε independently hydrocarbyl preferably of from 1 to about 24 carbon atomε, or independently mono- or di-hydrocarbylamino and R _g is independently hydrocarbyl, preferably of 1 to 24 carbon atoms, or (c) two of X. , X ₂ and X ₃ taken together form a cyclic carbonate group, provided that at least one of X. , X ₂ and X ₃ is not hydrogen. Preferred R, and ΕL*, groups include lower alkyl groups. One preferred class of lower alkyl groups are those having at least one secondary or tertiary carbon

atom. Another preferred class of lower alkyl groups are those having up to about 6 carbon atomε. Hydrocarbyl groups having more than 24 carbon atoms may be used.

Preferred compounds include thoεe having one or two ester groupε. Especially preferred are compounds having an ester group at either the 3'- or 5' - position or both positionε of the ribosyl ring.

Since for many indications, it would be advantageous and preferred to administer these prodrugs orally, those prodrugs which exhibit enhanced oral bioavailability would offer a therapeutic advantage. Accordingly, prodrugs where one or more of X_,, X ₂ and X ₃ compriseε a short chain hydrocarbylcarbonyl group are preferred. In view of their enhanced bioavailability when given orally in either a liquid or solid (e.g., capsule) form, particularly preferred are those prodrugs where X ¹ is isobutyryl or pivaloyl and X ₂ and X ₃ are both hydrogen, and where X,, X ₂ and X ₃ are both acetyl. Alεo preferred are thoεe prodrugε where X, iε n-butyryl and X ₂ and X ₃ are both hydrogen, and where X ₁ and X ₃ are both acetyl and X ₂ iε hydrogen. Especially preferred are certain prodrug compoundε which have been iεolated in an advantageous crystalline form, in particular 2*,3•,5'-triacetyl AICA riboside 3',5'-diacetyl AICA riboside and 3*-neopentoxycarbonyl. Moreover, in the acetyl-substituted prodrug compounds, the leaving groupε comprise acetate which is advantageously relatively pharmacologically silent.

Preparation of AICA Riboside Prodrug Compounds

The AICA riboside prodrug compounds may be conveniently prepared according to the following reaction scheme:

II III

I wherein X ^k ,1 ' 2 1 "3 > ~*Λ i and R ₂ , are as defined m conjunction with formula (I) . Reaction (1) is carried out by combining II, AICA riboεide, and III, the appropriate acid chloride, acid anhydride or chloroformate, in solvent. The acid chloride may be conveniently prepared by conventional procedures such as reaction of the corresponding acid with thionyl chloride. Some acid chlorides and acid anhydrides are commercially available. Many chloroformates are commercially available; also, the chloroformates may be conveniently prepared by conventional procedures known to thoεe skilled in the art by the reaction of phosgene with the appropriate alcohol. Reaction (1) is conducted at a temperature of from about -1O*C to about 5'C, preferably from about-5'C to about 0*C and is generally complete within about 2 to about 4 hours. For ease of handling, the reaction is carried out in solvent. Suitable solvents include dimethylformamide (DMF) , pyridine, methylene chloride and the like. For convenience, the reaction is carried out at ambient pressure. The reaction product(s) are isolated by conventional procedures as column chromatography, crystallization and the like. As may be appreciated, the reaction may result in a mixture of products, mono, di, and tri-esterε at the 2*-, 3'- and/or

5'- positions of the riboεyl moiety. The product eεterε may be εeparated by conventional procedureε such aε thin layer chromatography (TLC) , high preεsure liquid chromatography (HPLC) , column chromatography, crystallization, and the like which are well known to thoεe skilled in the art.

The 5•-monoesters may be conveniently prepared according to the following reaction scheme to give an intermediate blocked at the 2' and 3' positions:

O O

I I wherein X _1a iε -CR-, or -COΕL-, and DbAg iε a deblocking agent. Reaction (2) iε conducted by combining II, IV, V and VI. Although the reactantε may be combined in any order, it may be preferred to add II to a mixture of IV, V and VI. The reaction iε carried out at a temperature of about 10'C to about 25*C, preferably from about 15 ^* C to about

25*C and is generally complete within about 45 minutes. Intermediate VI is isolated by conventional procedures.

Reaction (3) is the reaction of intermediate VII with the appropriate acid chloride, acid anhydride or chloroformate and iε carried out aε described in connection with Reaction (1) .

Reaction (4) is an optional step to remove, if desired, the cyclic blocking group from the 2* and 3 ^» positionε. It iε carried out by reacting with IX, the appropriate deblocking agent. Suitable deblocking agents include H ^* resin in water/ acetone, tetraethyl-ammonium fluoride/THF, acetic acid/water, formic acid/water and the like. Such deblocking reactions are conventional and well known to those skilled in the art. Mixed ester compounds may be conveniently prepared by first reacting AICA riboside with the appropriate acid chloride or acid anhydride according to Reaction (1) to add the acyl ester group and then reacting the acyl ester- substituted compound with the appropriate chloroformate according to Reaction (1) to obtain the mixed ester. Alternatively, mixed ester compoundε may be prepared by firεt converting AICA riboεide to a monoacyl ester according to Reaction (1) , Reaction (2) and then reacting the purified monoacylated product with the appropriate chloroformate according to Reaction (1) . In addition, some mixed esters are prepared by first converting AICA riboside to a mono-alkoxycarbonate according to Reaction (1) or (2) and then reacting the purified carbonate ester with an appropriate acid . chloride or acid anhydride according to Reaction (1) .

utility

The methods of the present invention involve treatment of neurodegenerative diseaεe using agentε which enhance extracellular adenoεine concentrationε. Aε noted, adenosine regulating agents including AICA riboside and the AICA riboside prodrug compounds described herein are

useful in treating such conditions where increased extracellular concentrationε of adenoεine are beneficial.

In the studies described herein, we have uεed two agents, MPTP and methamphetamine, which induce neurodegenerative and Parkinson's-like conditions in animals. Neurodegeneration was induced in male Swiεs Webster mice by four systemic injections of either 20 mg/kg of MPTP or 5 g/kg of methamphetamine, two hours between each. CHA was administered in three injections of 0.5 mg/kg at zero, three and six hours relative to the first toxin injection. Ketamine was coadminiεtered with the toxins at 100 mg/kg per injection. Animals were permitted to survive for seven days before sacrifice, upon which the brains were removed and the εtriata diεεected out. The tissue was homogenized and analyzed for tyrosine hydroxylaεe activity by the method of Reinhard et al. rLife Sciences 39:2185-2189).

Both Meth and MPTP induced a 40-45% reduction in striatal tyrosine hydroxylase activity. Treatment with either CHA (p<.01) or Ketamine (a glutamine receptor antagonist) (p<.05) was effective in completely reversing the Meth induced decrement. Ketamine, while not tried by us, waε not reported to be effective by Sonεalla et al. against MPTP. CHA, on the other hand, completely prevented the decrement in this model as well (p<.01). In both models, it was posεible to partially reverse the protection afforded by CHA by coadministration of the adenosine antagonist, caffeine (30-50mg/kg) .

CHA protected against the development of both MPTP and methamphetamine induced Parkinsonian syndrome. This is a superior protection than that reported for the EAA blockers which have only been shown to be significantly effective by the tyrosine hydroxylaεe diεcriminator, in the methamphetamine model. The ineffectiveneεε of the NMDA-specific EAA receptor blocker MK-801 is shown in Figure 4. Additionally, the doses of CHA required are not highly sedative unlike those of the EAA blockers. This

protection iε adenoεine receptor mediated since it can be reversed by an adenosine receptor antagonist. This data suggests that adenoεine may be an endogenous neuroprotec- tive agent in Parkinson's neurodegeneration, and demonstrates the potential use of adenosinergic strategieε for the treatment of Parkinson's disease.

According to one aspect of the present invention, we have found that adenosine receptor agonists can afford significant protection against experimentally induced Parkinson's diseaεe in animals. This diseaεe iε one that progresses over a long period (yearε) and can generally be recognized initially by a mild tremor. Current treatmentε involve symptom abatement (L-DOPA) but do not block the progression of the diεeaεe. According to methods of the present invention, we propoεe to utilize agentε which act via an adenoεine mechaniεm (adenosinergic) to inhibit this progresεive deterioration in Parkinson's patients. By "adenosinergic agents" are meant either one or a combination of the following: 1) adenosine receptor agoniεtε; 2) adenoεine tranεport inhibitorε; or 3) enzyme inhibitorε or activatorε that raise extracellular adenosine concentrations. One or a combination of the above agents may be administered chronically on probably a daily basis. Due to the chronic, long-term aspect of the treatment it is envisioned that agents of category 2 or 3 will most probably be employed since their actions are more subtle in nature and their side effect profile much more manageable.

The adenosine receptor agonist, cyclohexyladenosine (CHA), has been shown to inhibit the development of the Parkinson'ε syndrome in rats in both the MPTP and methamphetamine models. Decrease in striatal (an area of the limbic syεtem rich in dopamine which selectively degenerates in Parkinson's Disease) tyrosine hydroxylase in response to either MPTP or methamphetamine was monitored. Administration of CHA was shown to accord εignificant protection in both the MPTP and

methamphetamine models. (See Examples 1 to 4 and Figures 1 to 4.)

It is anticipated that adenosinergic compounds uεeful in the methods of the present invention will be effectively administered in amounts ranging from about 0.01 mg/kg/day to about 500 mg/kg/day, preferably from about 15 mg/kg/day to about 200 mg/kg/day. That range of dosageε should be eεpecially suitable for compoundε useful in the invention as prophylacticε for the prevention of neurodegenerative damage aεεociated with undeεired reεtricted or decreased blood flow. The use of at least about 0.1 mg/kg/day of AICA riboεide or AICA ribotide, preferably from about 1.0 mg/kg/day to about 500 mg/kg/day for εaid prophylaxiε and, more preferably, from about 20 mg/kg/day to about 100 mg/kg/day, iε further anticipated. For some of the adenosinergic compounds, a dosage of more than 200-500 mg/kg/day may be needed becauεe of the blood/brain barrier. The uεe of brain-directed prodrugε may, however, enable a lower dosage. To deliver these adenoεinergic compoundε to patientε, it iε anticipated that they will moεt often be administered orally, since these compounds (such as prodrugε of AICA ribotide) are not readily degraded by extracellular enzymes in the body or by exposure to low pH present in the stomach. These compounds can also be administrated intravenously, by direct intramuscular injection, subcutaneously, topically to skin or mucous membranes, rectally, or by inhalation. Compoεitions acceptable for pharmaceutical use are well known. Prodrugs may also be utilized, i.e.. those which, when introduced into the body, metabolize to the active forms of the adenoεinergic agents.

To aεsist in understanding the present invention, the following examples are described which include the results of a series of experiments. The following examples relating to this invention are illustrative and should not, of courεe, be conεtrued aε εpecifically limiting the

invention. Moreover, such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are to be considered to fall within the scope of the present invention hereinafter claimed.

Example 1

Striatal decrement was induced in male Swiss Webster mice by four εyεtemic adminiεtrationε of 5 mg/kg of (+) methamphetamine, two hourε between each. CHA waε adminiεtered in three doεeε of 1 mg/kg, the first preceding the first methamphetamine injection by 15 minuteε, and the others three and six hours following the first. Ketamine was administered in four doses of 100 mg/kg. These injectionε coincided with the methamphetamine^injections, as did the injections of 0.9% saline. All of the compoundε were delivered aε an intraperitoneal boluε in a εaline vehicle at a volume of 1 ml/lOOg animal body weight.

Seven days later, the animalε were sacrificed and the brainε quick frozen on dry ice. Brainε were later thawed in 50 mM potassium phosphate buffer (pH 6.1) and the striata dissected out. The tissue from the two hemispheres of each brain waε combined and homogenized in 100 μl of the same buffer containing 0.2% triton x-100, and centrifuged for ten minutes at 17,000xg. The supernatant waε analyzed for tyrosine hydroxylase activity by the method of J.F. Reinhard, et al. [Life Sci. 39, 2185 (1986)] in 50 mM phosphate buffer (pH 6.1) with 6,7- Dimethyl tetrahydropterin as a cofactor. Results are shown in Figure 1.

Example 2

Striatal decrement was induced in mice using the same protocol as that described in Example 1. Ketamine was again administered in four injections of 100 mg/kg coincident with the methamphetamine, but in this

experiment, the saline injections were at zero, three and εix hourε after the first methamphetamine injection as were the adminiεtrationε of the other drugε. CHA was given as three injections of either 1 mg/kg, 0.5 mg/kg, or 0.5 mg/kg plus 30 mg/kg of caffeine.

Seven days later, animals were sacrificed and their striata dissected out. The striata from the two hemispheres of each brain were combined, weighed and homogenized in ten volumes of 50 mM potasεium phosphate buffer with 0.2% triton x-100. The homogenates were spun at 17,000xg and the supernatantε analyzed for tyrosine hydroxylase activity as described in figure 1.

Results are shown in Figure 2.

Example 3 MPTP was used to induce the Parkinson's syndrome in mice by four systemic administrations of 20 mg/kg with two hours between each. CHA at 0.5 mg/kg/injection or thiε pluε 50 mg/kg/injection of caffeine waε adminiεtered in three injections, zero, three and six hours from the first MPTP injection as were injectionε of 0.9% εaline. All injections were given as an intraperitoneal bolus injection in a volume of 1 ml/lOOg body weight.

Seven days later, animals were sacrificed, and their brains removed. Their εtriata were dissected out, the two hemispheres combined, weighed and homogenized in one volume of 50 mM potasεium phosphate buffer (pH 6.1). These homogenates were then assayed for tyrosine hydroxylase activity as deεcribed in connection with Example 1. Results are shown in Figure 3.

Example 4

MPTP was uεed to induce the Parkinson's syndrome in mice according to the procedure described in Example 3.

CHA (0.5 mg/kg) and MK-801 (3.5 mg/kg) were administered in three injections, zero, three and six hours from the

first MPTP injection, as were injections of 0.9% saline. All injections were given aε an intraperitoneal boluε injection in a volume of 1 ml/lOOg body weight.

Strial tyroεine hydroxylane activity waε aεεayed aε described in Example 3.

Results are shown in Figure 4.