AU2108299A - A2A adenosine receptor agonists - Google Patents

Description

WO 99/34804 PCT/US99/00366

A

2 a ADENOSINE RECEPTOR AGONISTS Background of the Invention Field of the Invention 5 The present invention relates to methods and compositions for treating inflammatory diseases. Discussion of the Background The release of inflammatory cytokines such as tumor necrosis factor alpha (TNFo:) by leukocytes is a means by which the immune system combats 10 pathogenic invasions, including infections. Cytokines stimulate neutrophils to enhance oxidative (e.g., superoxide and secondary products) and nonoxidative (e.g., myeloperoxidase and other enzymes) inflammatory activity. Inappropriate and over-release of cytokines can produce counterproductive exaggerated pathogenic effects through the release of tissue-damaging oxidative and 15 nonoxidative products (Tracey, K. G. et al., J. Exp. Med., vol. 167, pp. 1211 1227 (1988); and Minnel, D. N. et al., Rev. Infect. Dis., vol. 9 (suppl. 5), pp. S602-S606 (1987)). For example, inflammatory cytokines have been shown to be pathogenic in: arthritis (Dinarello, C. A., Semin. Immunol., vol. 4, pp. 133-45 (1992)); 20 ischemia (Seekamp, A. et al., Agents-Actions-Supp., vol. 41, pp. 137-52 (1993)); septic shock (Minnel, D. N. et al., Rev. Infect. Dis., vol. 9 (suppl. 5), pp. S602 S606 (1987)); asthma (Cembrzynska Nowak M. et al., Am. Rev. Respir. Dis., vol. 147, pp. 291-5 (1993)); organ transplant rejection (Imagawa, D. K. et al., Transplantation, vol. 51, pp. 57-62 (1991)); multiple sclerosis (Hartung, H. P., 25 Ann Neurol., vol. 33, pp. 591-6 (1993)); and AIDS (Matsuyama, T. et al., AIDS, vol. 5, pp. 1405-1417 (1991)). In addition, superoxide formation in leukocytes has been implicated in promoting replication of the human immunodeficiency virus (HIV) (Legrand-Poels, S. et al., AIDS Res. Hum. Retroviruses, vol. 6, pp. 1389-1397 (1990)). 30 It is well known that adenosine and some relatively nonspecific analogs of adenosine decrease neutrophil production of inflammatory oxidative products WO 99/34804 PCT/US99/00366 2 (Cronstein, B. N. et al., Ann. N.Y. Acad. Sci., vol. 451, pp. 291-314 (1985); Roberts, P. A. et al., Biochem. J., vol. 227, pp. 66.9-674 (19-85); Schrier, D. J. et al., J. Immunol., vol. 137, pp. 3284-3289 (1986); Cronstein, B. N. et al., Clinical Immunol. and Immunopath., vol. 42, pp. 76-85 (1987); lannone, M. A. et al., in 5 Topics and Perspectives in Adenosine Research, E. Gerlach et al., eds., Springer Verlag, Berlin, pp. 286-298 (1987); McGarrity, S. T. et al., J. Leukocyte Biol., vol. 44, pp. 411421 (1988); De La Harpe, J. et al., J. Immunol., vol. 143, pp. 596-602 (1989); McGarrity, S. T. et al., J. Immunol., vol. 142, pp. 1986-1994 (1989); and Nielson, C. P. et al., Br. J. Pharmacol., vol. 97, pp. 882-888 (1989)). 10 For example, adenosine has been shown to inhibit superoxide release from neutrophils stimulated by chemoattractants such as the synthetic mimic of bacterial peptides, f-met-leu-phe (fMLP), and the complement component C 5 a (Cronstein, B. N. et al., J. Immunol., vol. 135, pp. 1366-1371 (1985)). Adenosine can decrease the greatly enhanced oxidative burst of PMN 15 (neutrophil) first primed with TNF-a (an inflammatory cytokine) and then stimulated by a second stimulus such as f-met-leu-phe (Sullivan, G. W. et al., Clin. Res., vol. 41, p. 172A (1993)). There is evidence that in vivo adenosine has anti-inflammatory activity (Firestein, G. S. et al., Clin. Res., vol. 41, p. 170A (1993); and Cronstein, B. N. et al., Clin. Res., vol. 41, p. 244A (1993)). 20 Additionally, it has been reported that adenosine can decrease the rate of HIV replication in a T-cell line (Sipka, S. et al., Acta. Biochim. Biopys. Hung., vol. 23, pp. 75-82 (1988)). It has been suggested that there is more than one subtype of adenosine receptor on neutrophils that have opposite effects on superoxide release 25 (Cronstein, B. N. et al., J. Clin. Invest., vol. 85, pp. 1150-1157 (1990)). The existence of A 2 A receptor on neutrophils was originally demonstrated by Van Calker et al. (Van Calker, D. et al., Eur. J. Pharmacology, vol. 206, pp. 285-290 (1991)). There has been progressive development of compounds that are more and 30 more potent and selective as agonists of A 2 A adenosine receptors based on radioligand binding assays and physiological responses. Initially, compounds with little or no selectivity for A 2 A receptors were used, such as adenosine itself or 5'-carboxamides of adenosine, such as 5'-N-ethylcarboxyamidoadenosine WO 99/34804 PCT/US99/00366 3 (NECA) (Cronstein, B. N. et al., J. Immunol., vol. 135, pp. 1366-1371 (1985)). Later, it was shown that addition of 2-alkylamino substituents increased potency and selectivity, e.g., CV 1808 and CGS21680 (Jarvis, M. F. et al., J. Pharmacol. Exp. There , vol. 251, pp. 888-893 (1989)). 2-Alkoxy-substituted adenosine 5 derivatives such as WRC-0090 are even more potent and selective as agonists on the coronary artery A 2 A receptor (Ukena, M. et al., J. Med. Chem., vol. 34, pp. 1334-1339 (1991)). The 2-alkylhydrazino adenosine derivatives, e.g., SHA 211 (also called WRC-0474) have also been evaluated as agonists at the coronary artery A 2 A receptor (Niiya, K. et al., J. Med. Chem., vol. 35, pp. 10 45574561 (1992)). There is one report of the combination of relatively nonspecific adenosine analogs, R-phenylisopropyladenosine (R-PIA) and 2-chloroadenosine (C1-Ado) with a phosphodiesterase (PDE) inhibitor resulting in a lowering of neutrophil oxidative activity (lannone, M. A. et al., in Topics and Perspectives in 15 Adenosine Research, E. Gerlach et al., Eds., Springer-Verlag, Berlin, pp. 286 298 (1987)). However, R-PIA and C1-Ado analogs are actually more potent activators of A 1 adenosine receptors than of A 2 A adenosine receptors and, thus, are likely to cause side effects due to activation of A 1 receptors on cardiac muscle and other tissues causing effects such as "heart block." 20 Linden et al. SN 08/272,821 is based on the discovery that inflammatory diseases may be effectively treated by the administration of drugs which are selective agonists of A 2 A adenosine receptors, preferably in combination with a phosphodiesterase inhibitor. An embodiment of the Linden et al. invention provides a method for treating inflammatory diseases by administering an 25 effective amount of an A 2 A adenosine receptor of the following formula:

NH

2 N R N N (I) HO OH WO 99/34804 PCT/US99/00366 4 wherein X is a group selected from the group consisting of -OR 1 , -NR 2

R

3 , and -NH-N=R 4 ; wherein R 1 is C 1

-

4 -alkyl; C 1

-

4 -alkyl substituted with one or more

C

14 -alkoxy groups, halogens (fluorine, chlorine, or bromine), hydroxy groups, 5 amino groups, mono(C 1

-

4 -alkyl)amino groups, di(C 1

-

4 -alkyl)amino groups, or

C

6

-

10 o-aryl groups (wherein the aryl groups may be substituted with one or more halogens (fluorine, chlorine, or bromine), C 1

-

4 -alkyl groups, hydroxy groups, amino groups, mono(C 1

-

4 -alkyl)amino groups, or di(Cl- 4 -alkyl)amino groups);

C

6

-

10 -aryl; or C 6

-

1 0 -aryl substituted with one or more halogens (fluorine, chlorine, 10 or bromine), hydroxy groups, amino groups, mono(C 1

-

4 -alkyl)amino groups, or di(Cl- 4 -alkyl)amino groups, or C14-alkyl groups; one of R 2 and R 3 has the same meaning as R' and the other is hydrogen;

R

4 is a group having the formula: R5 __/ R6 wherein each of R 5 and R 6 independently may be hydrogen, C 3 7 -cycloalkyl, or 15 any of the meanings of R', provided that R 5 and R 6 are not both hydrogen; and R is -CH20H, -CH 2 H, -CO 2

R

7 , or -C(=O)NRR 9 ; wherein R 7 has the same meaning as R' and wherein R 8 and R 9 have the same meanings as R 5 and R 6 and

R

8 and R 9 may both be hydrogen. In a preferred embodiment, the Linden et al. invention involves the 20 administration of a Type IV phosphodiesterase (PDE) inhibitor in combination with the A 2 A adenosine receptor agonist. The Type IV phosphodiesterase (PDE) inhibitor can be racemic and optically active 4-(polyalkoxyphenyl)-2pyrrolidones of the following formula: WO 99/34804 PCT/US99/00366 5

OR

18 R19 N X R' (disclosed and described in U.S. Patent Number 4,193,926) wherein R 1 8 and R 19 each are alike or different and are hydrocarbon radicals having up to 18 carbon atoms with at least one being other than methyl, a heterocyclic ring, or alkyl of 5 1-5 carbon atoms which is substituted by one or more of halogen atoms, hydroxy, carboxy, alkoxy, alkoxycarbonyl or an amino group; amino; R' is a hydrogen atom, alkyl, aryl or acyl; and X is an oxygen atom or a sulfur atom. Rolipram is an example of a suitable Type IV phosphodiesterase or PDE inhibitor included within the above formula. Rolipram has the following 10 structure: 0 0

CH

3 / N 0 I H The present invention is based on the inventors' discovery that improved effective treatment of inflammatory disease is achieved by the administration of certain agonists of A 2 A adenosine receptors in combination with rolipram or 15 rolipram derivatives that are Type IV phosphodiesterase or PDE inhibitors.

WO 99/34804 PCT/US99/00366 6 Summary of the Invention Accordingly, one object of the present invention is to provide a novel and improved method for treating inflammatory diseases. It is another object of the present invention to provide novel and 5 improved compositions for the treatment of inflammatory disease. These and other objects, which will become better understood during the course of the following detailed description, have been achieved by the inventors' discovery of improved compositions and methods for effectively treating inflammatory diseases by administration of an agonist of an A 2 A 10 adenosine receptor in combination with rolipram or a rolipram derivative that is a Type IV phosphodiesterase (PDE) inhibitor. Brief Description of the Drawings A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better 15 understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein: Figure 1 illustrates the relative potencies of adenosine analogs to modulate TNF.-primed fMLP-stimulated polymorphonuclear cell (PMN) chemiluminescence as a measure of PMN production of oxidative products (0, 20 no TNFac; , WRC-0474[SHA 211] + TNFa; L, GCS 21680 + TNFa; and A, adenosine + TNFa); Figure 2 illustrates the synergistic effect of WRC-0474[SHA 211] and 4 (3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (rolipram) in inhibiting TNFa-primed (10 U/ml), fMLP-stimulated (100 nM) PMN superoxide 25 production: 0, no 4

-(

3 -cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone; A, 3 nM 4

-(

3 -cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone; l, 30 nM 4-(3 cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone; and, 300 nM 4-(3 cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone; Figure 3 illustrates the synergistic effect of WRC-0474[SHA 211] and 30 rolipram in inhibiting TNFa-stimulated adherent PMN superoxide release; Figure 4 illustrates the effect of WRC-0474[SHA 211] and rolipram on TNFa-stimulated PMN adherence to a fibrinogen-coated surface; WO 99/34804 PCT/US99/00366 7 Figure 5 illustrates synergy between A 2 A adenosine receptor agonists and Rolipram in inhibiting superoxide release from TNFc-stimulated adherent human neutrophils; Figure 6 illustrates the effects of WRC-0470 and rolipram on the 5 oxidative activity of neutrophils in whole blood; Figure 7 illustrates the effects of WRC-0470 and rolipram on the release of TNFa from adherent human monocytes and that this activity is dependent on binding of the adenosine agonist to A 2 A adenosine receptors; Figure 8 illustrates the effect of WRC-0470 on white blood cell 10 pleocytosis in rats; Figure 9 illustrates the effect of WRC-0470 on blood-brain-barrier permeability in rats; Figure 10 illustrates the effect of rolipram on white blood cell pleocytosis in rats; and 15 Figure 11 illustrates the combined effect of WRC-0470 and rolipram on white blood cell pleocytosis in rats. Detailed Description of the Preferred Embodiments Thus, in a first embodiment, the present invention provides a method for treating inflammatory diseases by administering an effective amount of a 20 compound of formula (I):

NH

2 R N NX (I) HO OH wherein X is a group selected from the group consisting of -OR 1 , -NR 2

R

3 , and

-NH-N=R

4 ; wherein R 1 is CI- 4 -alkyl; Cl 14 -alkyl substituted with one or more Cl 4 25 alkoxy groups, halogens, (fluorine, chlorine, or bromine), hydroxy groups, amino groups, mono(Cl- 4 -alkyl)amino groups, di(Cl.

4 -alkyl)amino groups, or WO 99/34804 PCT/US99/00366 8

C

6

-

1 0 -aryl groups (wherein the aryl groups may be substituted with one or more halogens (fluorine, chlorine, or bromine), Cl- 4 -alkyl groups, hydroxy groups, amino groups, mono(C 1 -4-alkyl)amino groups, or di(C 1

-

4 -alkyl)amino groups);

C

6

-

1 0 -aryl; or C 6

-

10 -aryl substituted with one or more halogens (fluorine, chlorine, 5 or bromine), hydroxy groups, amino groups, mono(Cl- 4 -alkyl)amino groups, or di(Cl- 4 -alkyl)amino groups, or Cl- 1 4 -alkyl groups; one of R 2 and R 3 has the same meaning as R 1 and the other is hydrogen;

R

4 is a group having the formula (II) R5 C (II) R6 wherein each of R 5 and R 6 independently may be hydrogen, C 3

-

7 -cycloalkyl, or 10 any of the meanings of R 1 , provided that R 5 and R 6 are not both hydrogen; Examples of suitable C6-10-aryl groups include phenyl and naphthyl. Preferably, the compound of formula (I) has X being a group of the formula (III) --- CH2 - - A r (III) wherein n is an integer from 1-4, preferably 2, and Ar is a phenyl group, tolyl 15 group, naphthyl group, xylyl group, or mesityl group. Most preferably, Ar is a para-tolyl group and n = 2. Even more preferably, the compound of formula (IV) has X being a group of the formula (I) -NH-N=CHCy (IV) 20 wherein Cy is a C 3

-

7 -cycloalkyl group, preferably cyclohexyl or a C 1

-

4 -alkyl group, preferably isopropyl. Specific examples of such compounds of formula (I) include WRC-0470, 25 WRC-0474 [SHA 211], WRC-0090 and WRC-0018, shown below: WO 99/34804 PCT/US99/00366 9

NH

2 N N N N NH I OH N O1f OH

NH

2 N N ON N NH OH N OH OH NH2 N N OH Of OH and

NH

2 N~N 0 0 N N 0 OH Of OH Of these specific examples, WRC-0474[SHA 211] and WRC-0470 are particularly preferred. Such compounds may be synthesized as described in: Hutchinson, A. J. 5 et al., J. Pharmacol. ExD. Ther., vol. 251, pp. 47-55 (1989); Olsson, R. A. et al., J. Med. Chem., vol. 29, pp. 1683-1689 (1986); Bridges, A. J. et al., J. Med.

WO 99/34804 PCT/US99/00366 10 Chem., vol. 31, pp. 1282-1285 (1988); Hutchinson, A. J. et al., J. Med. Chem., vol. 33, pp. 1919-1924 (1990); Ukena, M. et al., J. Med Chem., vol. 34, pp. 1334-1339 (1991); Francis, J. E. et al., J. Med. Chem., vol. 34, pp. 2570 2579 (1991); Yoneyama, F. et al., Eur. J. Pharmacol., vol. 213, pp. 199-204 5 (1992); Peet, N. P. et al., J. Med. Chem., vol. 35, pp. 3263-3269 (1992); and Cristalli, G. et al., J. Med. Chem., vol. 35, pp. 2363-2368 (1992); all of which are incorporated herein by reference. The present method includes the administration of a Type IV phosphodiesterase (PDE) inhibitor in combination with the compound of 10 formula (I). Examples of Type IV phosphodiesterase inhibitors include those disclosed in U.S. Patent No. 4,193,926, WO 92-079778, and Molnar-Kimber, K. L. et al., J. Immunol., vol. 150, p. 295A (1993), all of which are incorporated herein by reference. Specifically, the suitable Type IV phosphodiesterase (PDE) inhibitors 15 include racemic and optically active 4-(polyalkoxyphenyl)-2-pyrrolidones of the general formula (V) OR18 R190 RO (V) N 0 H (disclosed and described in U.S. Patent No. 4,193,926) wherein R' 8 and R 19 each are alike or different and are hydrocarbon radicals having up to 18 carbon atoms 20 with at least one being other than methyl, a heterocyclic ring, or alkyl of 1-5 carbon atoms which is substituted by one or more of halogen atoms, hydroxy, carboxy, alkoxy, alkoxycarbonyl or an amino group or amino. Examples of hydrocarbon R" 8 and R" 9 groups are saturated and unsaturated, straight-chain and branched alkyl of 1-18, preferably 1-5, carbon 25 atoms, cycloalkyl and cycloalkylalkyl, preferably of 3-7 carbon atoms, and aryl and aralkyl, preferably of 6-10 carbon atoms, especially monocyclic.

WO 99/34804 PCT/US99/00366 11 Examples of alkyl are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, 2-methylbutyl, 2,2-dimethylpropyl, hexyl, heptyl, octyl, nonyl, 1,2-dimethylheptyl, decyl, undecyl, dodecyl and stearyl, with the proviso that when one of R' 8 and R 19 is methyl, the other is a value other than methyl. 5 Examples of unsaturated alkyl groups are alkenyl and alkynyl, e.g., vinyl, 1-propenyl, 2-propenyl, 2-propynyl and 3-methyl-2-propenyl. Examples of cycloalkyl and cycloalkylalkyl which preferably contain a total of 3-7 carbon atoms are cyclopropyl, cyclopropylmethyl, cyclopentyl and cyclohexyl. 10 Examples of aryl and aralkyl are phenyl and benzyl, which are preferred, and tolyl, xylyl, naphthyl, phenethyl and 3phenylpropyl. Examples of heterocyclic R 18 and R' 9 groups are those wherein the heterocyclic ring is saturated with 5 or 6 ring members and has a single 0, S or N atom as the hetero atom, e.g., 2- and 3-tetrahydrofuryl, 2- and 3 15 tetrahydropyranyl, 2- and 3-tetrahydrothiophenyl, pyrrolidino, 2- and 3 pyrrolidyl, piperidino, 2-, 3- and 4-piperidyl, and the corresponding N-alkyl pyrrolidyl and piperidyl wherein alkyl is of 1-4 carbon atoms. Equivalents are heterocyclic rings having fewer or more, e.g., 4 and 7, ring members, and one or more additional hetero atoms as ring members, e.g., morpholino, piperazino and 20 N-alkylpiperazino. Examples of substituted alkyl R" 8 and R' 9 groups, preferably of 1-5 carbon atoms, are those mono- or polysubstituted, for example, by halogen, especially fluorine, chlorine and bromine. Specific examples of such halogen substituted alkyl are 2-chloroethyl, 3-chloropropyl, 4-bromobutyl, 25 difluoromethyl, trifluoromethyl, 1,1,2-trifluoro-2-chloroethyl, 3,3,3 trifluoropropyl, 2,2,3,3,3-pentafluoropropyl and 1,1,1,3,3,3-hexafluoro-2-propyl. Examples of other suitable substituents for such alkyl groups are hydroxy groups, e.g., 2-hydroxyethyl or 3-hydroxypropyl; carboxy groups, e.g., carboxymethyl or carboxyethyl; alkoxy groups, wherein each alkoxy group 30 contains 1-5 carbon atoms, e.g., ethoxymethyl, isopropoxymethyl, 2 methoxyethyl, 2-isopropoxyethyl, 2-butoxyethyl, 2-isobutyoxyethyl, and 3 pentoxypropyl.

WO 99/34804 PCT/US99/00366 12 Also suitable as preferably terminal-positioned substituents on alkyl groups of 1-5 carbon atoms are alkoxycarbonyl of 1-5 carbon atoms in the alkoxy group. Examples of such alkoxycarbonyl substituted alkyl groups are ethoxycarbonylmethyl and 2-butoxycarbonylethyl. 5 Alkyl groups of 1-5 carbon atoms can also be substituted, e.g., in the 3, T and preferably terminal position with amino groups wherein the nitrogen atom optionally is mono- or disubstituted by alkyl, preferably of 1-5 carbon atoms, or is part of a 4- to 7-membered ring. Rolipram and its analogues are specific examples of preferred Type IV 10 phosphodiesterase inhibitors. Examples of inflammatory diseases which may be treated according to the present invention include: autoimmune diseases such as lupus erythematosus, multiple sclerosis, type I diabetes mellitis, Crohn's disease, ulcerative colitis, inflammatory bowel 15 disease, osteoporosis, arthritis, allergic diseases such as asthma, infectious diseases such as sepsis, septic shock, infectious arthritis, endotoxic shock, gram negative shock, toxic shock, cerebral malaria, bacterial meningitis, adult respiratory distress syndrome (ARDS), TNFa-enhanced HIV replication and TNFx inhibition of reverse transcriptase inhibitor activity, wasting diseases 20 (cachexia secondary to cancer and HIV), skin diseases like psoriasis, contact dermatitis, eczema, infectious skin ulcers, cellulitis, organ transplant rejection (including bone marrow, kidney, liver, lung, heart, skin rejection), graft versus host disease, adverse effects from amphotericin B treatment, adverse effects from interleukin-2 treatment, adverse effects from OKT3 treatment, adverse effects 25 from GM-CSF treatment, adverse effects of cyclosporine treatment and adverse effects of aminoglycoside treatment, ischemia, mucositis, infertility from endometriosis, circulatory diseases induced or exacerbated by an inflammatory response such as atherosclerosis, peripheral vascular disease, restenosis following angioplasty, inflammatory aortic aneurysm, ischemia/reperfusion 30 damage, vasculitis, stroke, congestive heart failure, hemorrhagic shock, vasospasm following subarachnoid hemorrhage, vasospasm following cerebrovascular accident, pleuritis, pericarditis, and encephalitis.

WO 99/34804 PCT/US99/00366 13 The exact dosage of the compound of formula (I) to be administered will, of course, depend on the size and condition of the patient being treated, the exact condition being treated, and the identity of the particular compound of formula (I) being administered. However, a suitable dosage of the compound of 5 formula (I) is 0.5 to 100 gg/kg of body weight, preferably 1 to 10 gg/kg of body weight. Typically, the compound of formula (I) will be administered from 1 to 8, preferably 1 to 4, times per day. The preferred mode of administration of the compound of formula (I) may also depend on the exact condition being treated. However, most typically, 10 the mode of administration will be oral, topical, intravenous, parenteral, subcutaneous, or intramuscular injection. Of course, it is to be understood that the compound of formula (I) may be administered in the form of a pharmaceutically acceptable salt. Examples of such salts include acid addition salts. Preferred pharmaceutically acceptable 15 addition salts include salts of mineral acids, for example, hydrochloric acid, sulfuric acid, nitric acid, and the like; salts of monobasic carboxylic acids, such as, for example, acetic acid, propionic acid, and the like; salts of dibasic carboxylic acids, such as maleic acid, fumaric acid, oxalic acid, and the like; and salts of tribasic carboxylic acids, such as carboxysuccinic acid, citric acid, and 20 the like. In the compounds of formula (I) in which R is -CO2H, the salt may be derived by replacing the acidic proton of the -CO 2 H group with a cation such as Na', K , NH'4 mono-, di-, tri- or tetra(C 1

-

4 -alkyl)ammonium, or mono-, di-, tri-, or tetra(C 2

-

4 -alkanol)ammonium. It is also to be understood that many of the compounds of formula (I) 25 may exist as various isomers, enantiomers, and diastereomers and that the present invention encompasses the administration of a single isomer, enantiomer, or diastereomer in addition to the administration of mixtures of isomers, enantiomers, or diastereomers. The compounds of formula (I) can be administered orally, for example, 30 with an inert diluent with an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the compounds can be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, waters, WO 99/34804 PCT/US99/00366 14 chewing gums, and the like. These preparations should contain at least 0.5% by weight of the compound of formula (I), but the amount can be varied depending upon the particular form and can conveniently be between 4.0% to about 70% by weight of the unit dosage. The amount of the compound of formula (I) in such 5 compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention are prepared so that an oral dosage unit form contains between about 30 tg and about 5 mg, preferably between 50 to 500 Rg, of active compound. Tablets, pills, capsules, troches, and the like can contain the following 10 ingredients: a binder, such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient, such as starch or lactose; a disintegrating agent, such as alginic acid, Primogel, corn starch, and the like; a lubricant, such as magnesium stearate or Sterotes; a glidant, such as colloidal silicon dioxide; a sweetening agent, such as sucrose, saccharin or aspartame; or flavoring agent, such as 15 peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule it can contain, in addition to the compound of formula (I), a liquid carrier, such as a fatty oil. Other dosage unit forms can contain other materials that modify the physical form of the dosage unit, for example, as coatings. Thus, tablets or pills 20 can be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and preservatives, dyes, colorings, and flavors. Materials used in preparing these compositions should be pharmaceutically pure and nontoxic in the amounts used. For purposes of parenteral therapeutic administration, the compounds of 25 formula (I) can be incorporated into a solution or suspension. These preparations should contain at least 0.1% of the aforesaid compound, but may be varied between 0.5% and about 50% of the weight thereof. The amount of active compound in such compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention are 30 prepared so that a parenteral dosage unit contains between 30 gg to 5 mg, preferably between 50 to 500 gg, of the compound of formula (I). Solutions or suspensions of the compounds of formula (I) can also include the following components: a sterile diluent, such as water for injection, WO 99/34804 PCT/US99/00366 15 saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents: antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates 5 or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Effective amounts of the Type IV phosphodiesterase inhibitor can be administered to a subject by any one of various methods, for example, orally as 10 in a capsule or tablets, topically, or parenterally in the form of sterile solutions. The Type IV phosphodiesterase inhibitors, while effective themselves, can be formulated and administered in the form of their pharmaceutically acceptable addition salts for purposes of stability, convenience of crystallization, increased solubility, and the like. 15 Preferred pharmaceutically acceptable addition salts include salts of mineral acids, for example, hydrochloric acid, sulfuric acid, nitric acid, and the like; salts of monobasic carboxylic acids, such as, for example, acetic acid, propionic acid, and the like; salts of dibasic carboxylic acids, such as maleic acid, fumaric acid, oxalic acid, and the like; and salts of tribasic carboxylic acids, 20 such as carboxysuccinic acid, citric acid, and the like. The Type IV phosphodiesterase may be administered in the form of a pharmaceutical composition similar to those described above in the context of the compound of formula (I). While dosage values will vary with the specific disease condition to be 25 alleviated, good results are achieved when the Type IV phosphodiesterase inhibitor is administered to a subject requiring such treatment as an effective oral, parenteral or intravenous dose as described below. For oral administration, the amount of active agent per oral dosage unit usually is 0.1-20 mg, preferably 0.5-10 mg. The daily dosage is usually 0.1 30 50 mg, preferably 1-30 mg p.o. For parenteral application, the amount of active agent per dosage unit is usually 0.005-10 mg, preferably 0.01-5 mg. The daily dosage is usually 0.01-20 mg, preferably 0.02-5 mg i.v. or i.m.

WO 99/34804 PCT/US99/00366 16 With topical administration, dosage levels and their related procedures would be consistent with those known in the art, such as those dosage levels and procedures described in U.S. Patent No. 5,565,462 to Eitan et al., which is incorporated herein by reference. 5 It is to be understood, however, that for any particular subject, specific dosage regimens should be adjusted to the individual need and the professional judgment of the person administering or supervising the administration of the Type IV phosphodiesterase inhibitor. It is to be further understood that the dosages set forth herein are exemplary only and that they do not, to any extent, 10 limit the scope or practice of the present invention. In a particularly preferred embodiment, the compound of formula (I) and the Type IV phosphodiesterase inhibitor are coadministered together in a single dosage unit. The compound of formula (I) and the Type IV phosphodiesterase inhibitor may be administered in the same type of pharmaceutical composition as 15 those described above in the context of the compound of formula (I). By coadministering a Type IV phosphodiesterase inhibitor with the agonist of the A 2 A adenosine receptor, it is possible to dramatically lower the dosage of the A 2 A adenosine receptor agonist and the Type IV phosphodiesterase inhibitor due to a synergistic effect of the two agents. Thus, in the embodiment 20 involving coadministration of the A 2 A adenosine receptor agonist with the Type IV phosphodiesterase inhibitor, the dosage of the A 2 A adenosine receptor agonist may be reduced by a factor of 5 to 10 from the dosage used when no Type IV phosphodiesterase inhibitor is administered. This reduces the possibility of side effects. 25 The present invention will now be described in more detail in the context of the coadministration of WRC-0470, WRC-0474[SHA 211], WRC-0090 or WRC-0018 and rolipram. However, it is to be understood that the present invention may be practiced with other compounds of formula (I) and other Type IV phosphodiesterase inhibitors of formula (V). 30 The present studies establish that anti-inflammatory doses have no toxic effects in animals; the effect of WRC-0470 to inhibit neutrophil activation is synergistic with rolipram; and intravenous infusion of WRC-0470 profoundly inhibits extravasation of neutrophils in an animal model of inflammation, an WO 99/34804 PCT/US99/00366 17 action also synergistic with rolipram. Further, the present studies establish that activation of A 2 A receptors on human monocytes strongly inhibits TNFa (an inflammatory cytokine) release. This mechanism further contributes to the anti inflammatory action of the A 2 A adenosine receptor agonists of the present 5 invention. Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof. Examples 10 Materials and Methods Materials. f-Met-Leu-Phe(fMLP), luminol, and trypan blue were from Sigma Chemical. Ficoll-hypaque was purchased from Flow Laboratories (McLean, VA) and Los Alamos Diagnostics (Los Alamos, NM). Hanks balanced salt solution (HBSS), and limulus amebocyte lysate assay kit were from 15 Whittaker Bioproducts (Walkersville, MD). Human serum albumin (HSA) was from Cutter Biological (Elkhart, IN). Recombinant human tumor necrosis factor-alpha was supplied by Dianippon Pharmaceutical Co. Ltd. (Osaka, Japan). ZM241385 was a gift of Dr. Simon Poucher, Zeneca Pharmaceuticals (Cheshire, England). 20 Leukocyte Preparation. Purified PMN (-98% PMN and >95% viable by trypan blue exclusion) containing <1 platelet per 5 PMN and <50 pg/ml endotoxin (limulus amebocyte lysate assay) were obtained from normal heparinized (10 Units/ml) venous blood by a one step Ficoll-hypaque separation procedure (Ferrante, A. et al., J. Immunol. Meth., vol. 36, p. 109 (1980)). 25 Residual RBC were lysed by hypotonic lysis with iced 3 ml 0.22% sodium chloride solution for 45 seconds followed by 0.88 ml of 3% sodium chloride solution. Chemiluminescence. Luminol-enhanced chemiluminescence, a measure of neutrophil oxidative activity, is dependent upon both superoxide production 30 and mobilization of the granule enzyme myeloperoxidase. The light is emitted from unstable high-energy oxygen species generated by activated neutrophils. Purified PMN (5 x 10 5 /ml) were incubated in HBSS containing 0.1% human serum albumin (1 ml) with or without adenosine, adenosine analogs, and TNFa WO 99/34804 PCT/US99/00366 18 (1 U/mL) for 30 minutes at 37oC in a shaking water bath. Then luminol (1 x 10-4 M) enhanced f-met-leu-phe (1 [M) stimulated chemiluminescence was read with a Chronolog Photometer (Chrono-log Corp., Havertown, PA) at 37'C for 8 minutes. Chemiluminescence is reported as relative peak light emitted 5 (= height of the curve) compared to samples with TNF and without adenosine or adenosine analogs. WRC-0474[SHA 211] was 10 times more potent than either adenosine (ADO) or CGS21680 in decrease TNFa-primed f-met-leu-phe stimulated PMN chemiluminescence (see Figure 1). Synergy of A 2 A Adenosine Receptor Agonist and Phosphodiesterase 10 Inhibitors. The synergy between WRC-0474[SHA 211] and 4-(3 cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (rolipram) was examined by measuring the effect of combined WRC-0474[SHA 211] and rolipram on TNF primed f-met-leu-phe-stimulated suspended neutrophil superoxide release and on the oxidative burst of neutrophils adhering to matrix proteins (in this model the 15 PMN oxidative burst is enhanced by small concentrations of TNFu [e.g., 1 U/ml] when added prior to the addition of a second stimulus such as the peptide f-met leu-phe). Suspended PMN Superoxide Release. Human PMN (1 x 10 6 /ml) from Ficoll-hypaque separation were primed for 30 minutes (37 0 C) with or without 20 rhTNF (10 U/ml), with adenosine deaminase (1 U/ml), and with or without 4-(3 cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone and SHA 211. Cytochrome c (120 RM), catalase (0.062 mg/ml) and fMLP (100 nM) were added and the samples incubated for 10 minutes more at 37 0 C. SOD (200 U/ml) was added to matched samples. The samples were iced and centrifuged (2000 g x 25 10 minutes). The optical density of the supernatant were read at 550 nm against the matched SOD samples, and the nmoles of SOD-inhibitable superoxide released in 10 minutes were calculated. A synergistic effect of WRC-0474[SHA 211] and rolipram in decreasing the TNFa-primed fMLP-stimulated PMN oxidative burst was observed (see 30 Figure 2). TNFT-stimulated superoxide release of PMN adherent to a matrix protein (fibrinogen) coated surface. Human PMN (1 x 10 6 /ml) from Ficoll-hypaque separation were incubated for 90 minutes in 1 ml of Hanks balanced salt solution WO 99/34804 PCT/US99/00366 19 containing 0.1% human serum albumin, cytochrome c (120 ptM), and catalase (0.062 mg/ml) in the presence and absence of rhTNF (1 U/ml), WRC-0474[SHA 211] (10 nM) and rolipram (100 nM) in a tissue culture well which had been coated overnight with human fibrinogen. SOD (200 U/ml) was added to 5 matched samples. The supernatants were iced and centrifuged (2000 g x 10 minutes) to remove any remaining suspended cells, and the optical density of the supernatants were read at 550 nm against the matched SOD samples, and the nmoles of SOD-inhibitable superoxide released in 90 minutes were calculated. A synergistic effect of WRC-0474[SHA 211] and rolipram in decreasing 10 the TNFo-stimulated release of superoxide from PMN adherent to fibrinogen was observed (see Figure 3). Effect of WRC-0474[SHA 211] with and without rolipram on TNF Stimulated PMN Adherence to a Fibrinogen-Coated Surface. Cronstein et al., . Immunol., vol. 148, p. 2201 (1992) reported that adenosine binding to A 1 15 receptors increases PMN adherence to endothelium and matrix proteins and binding to A 2 receptors decreases adherence to these surfaces when the PMN are stimulated with fMLP. Despite this, others have failed to see much of an effect of adenosine (10 gM) on TNFaT-stimulated PMN adherence to matrix proteins. In contrast, adenosine dramatically decreases the oxidative burst of TNFa 20 stimulated PMN adhering to matrix proteins (DeLa Harpe, J., J. Immunol., vol. 143, p. 596 (1989)). The experiments described above establish that WRC 0474[SHA 211] decreases TNF-stimulated oxidative activity of PMN adhering to fibrinogen, especially when combined with rolipram. PMN adherence to fibrinogen was measured as follows as adapted from 25 Hanlon, J. Leukocte Biol., vol. 50, p. 43 (1991). Twenty-four well flat bottomed tissue culture plates were incubated (37 0 C) overnight with 0.5 ml of fibrinogen (5 mg/ml) dissolved in 1.5% NaHCO 3 . The plates were emptied and each well washed 2x with 1 ml of normal saline. The wells were then filled with 1 ml of HBSS-0.1% human serum albumin containing PMN (1 x 10 6 /ml) with 30 and without rhTNFa (1 U/ml), adenosine deaminase (ADA) (1 U/ML), WRC 0474[SHA 211] (10 nM), CGS21680 (30 nM), adenosine (100 nM) and rolipram (100 nM). The plates were incubated for 90 minutes at 37 0 C in 5% CO 2 . Following incubation the tissue culture wells were washed free of non-adherent WO 99/34804 PCT/US99/00366 20 cells with normal saline. The adherent monolayer of PMN was lysed with 0.1% triton-X, the amount of lactic dehydrogenase (LDH) released from the monolayer assayed (LDH kit, Sigma Co., St. Louis, MO), and compared to a standard curve relating the LDH content ot PMN numbers. The results are 5 shown in Figure 4. As a comparison to WRC-0474[SHA 211] (at only 10 nM), CGS21680 (30 nM) decreased TNF-stimulated adherence in the presence of ADA from 38% to 30% adhered (p = .004) (see Fig. 4), and ten times as much adenosine (100 nM) decreased adherence to 28% adhered (p = .009 compared to TNF in the 10 presence of ADA). Additional effects of adenosine A 2 A agonists on adherent human neutrophil oxidative activity. The bioactivity of test compounds WRC 0474[SHA 211], WRC-0470, WRC-0090 and WRC-0018 were evaluated according to the following method modified from Sullivan, G. W. et al., Intl J. 15 Immunopharmacol., 17, 793-803 (1995). Neutrophils (1 x 10 6 /ml) from Ficoll hypaque separation were incubated for 90 minutes in 1 ml of Hanks balanced salt solution containing 0.1% human serum albumin, cytochrome c (120 [M) and catalase (0.062 mg/ml) in the presence and absence of rhTNFa (1 U/ml), WRC 0474[SHA 211], WRC-0470, WRC-0090 and WRC-0018 (3-300 nM), and 20 rolipram (100 nM) in a tissue culture well which had been coated overnight with human fibrinogen. The supernatants were iced and centrifuged (200 g x 10 minutes) to remove any remaining suspended cells, and the optical densities of the supernatants were read at 550 nm against matched superoxide dismutase (SOD) (200 U/ml) samples. The nmoles of SOD = inhibitable superoxide 25 released in 90 minutes were calculated. Figure 5 shows synergy between A 2 A adenosine agonists and rolipram in inhibiting TNFca-stimulated adherent PMN oxidative activity (p < 0.05). WRC 0474[SHA 211] (30-300 nM), WRC-0470 (300 nM), WRC-0090 (300 nM) and WRC-0018 (300 nM) combined with rolipram synergistically decreased 30 superoxide release (p < 0.05). All four compounds had some activity in the presence of rolipram. WRC-0474[SHA 211] and WRC-0470 were the most active. Nanomolar concentrations of WRC-0474[SHA 211] resulted in biphasic WO 99/34804 PCT/US99/00366 21 activity. All compounds were synergistic with rolipram to decrease TNFa stimulated adherent PMN oxidative activity. PMN degranulation (adherent cells). The following methods were adapted from Sullivan, G. W. and G. L. Mandell, Infect. Inumun., 30, 272-280 5 (1980). Neutrophils (3.1 x 10 6 /ml) from Ficoll-hypaque separation were incubated for 120 minutes in 1 ml of Hanks balanced salt solution containing 0.1% human serum albumin, ± rh TNFce (10 U/ml), ± WRC-0470 (3-300 nM) and ± rolipram (300 nM) in a tissue culture well which had been coated overnight with human fibrinogen. The supernatant fluids with any suspended 10 neutrophils were harvested following incubation, centrifuged (2000 x g for 10 minutes) to remove any suspended cells and the cell-free supernatants frozen. Release of lysozyme, a component of neutrophil primary and secondary granules was assayed. Lysis of a suspension of Micrococcus lysodeikticus by the "cell free supernatant" was measured by spectrophotometric analysis (540 mm) to 15 determine the amount of release of granule contents to the surrounding medium. Results showed that WRC-0470 (300 nM) with rolipram (300 nM) significantly decreased TNFa-stimulated adherent neutrophil degranulation 67%; P = 0.027. The data indicate that in addition to decreasing TNFa stimulated PMN adherent and the oxidative burst of these adherent neutrophils, 20 WRC-0470 also decreases degranulation-activated PMN adhering to a biological surface. PMN oxidative activity in whole blood. The following methods were adapted from Rothe, G. A. et al., J. Immunol. Meth., 138, 133-135 (1991). Heparinized whole blood (0.8 ml) was incubated (370; 30 minutes) with 25 adenosine deaminase (ADA, 1 U/ml), catalase (14,000 U/ml), ± dihydrorhodamine 123, ± WRC-0470 (3-300 nM), ± rolipram (300 nM) and ± TNFa (10 U/ml). The primed blood samples were stimulated with fMLP (15 minutes), then iced, the red blood cells lysed with FACS lysing solution (Becton-Dickinson, San Jose, CA), washed and the leukocytes resuspended in 30 phosphate buffered saline (PBS). These samples containing mixed leukocytes were gated for neutrophils by forward and side scatter and the fluorescence of 10,000 neutrophils measured in the FL1 channel of a FACScan (Becton Dickinson) fluorescence-activated cell sorter.

WO 99/34804 PCT/US99/00366 22 The results are reported as relative mean fluorescence intensity in Figure 6 of the drawings. WRC-0470 decreased oxidative activity of TNFa primed fMLP-stimulated neutrophils in whole blood and acted synergistically with rolipram. WRC-0470 (30-300 nM) decreased neutrophil oxidative activity 5 synergistically with rolipram (300 nM) in samples stimulated with fMLP and in blood samples primed with TNFh and then stimulated with fMLP. Production of TNFe by purified human adherent monocytes. A monocyte-rich monolayer (>95% monocytes) was prepared by incubating 1 ml of the mononuclear leukocyte fraction (5 x 10 5 /ml) from a Ficoll-hypaque 10 separation in wells of a 24 well tissue culture plate (1 hour; 37 0 C; 5% CO 2 ). The non-adherent leukocytes were removed by washing and culture medium added to the wells (1 ml RMPI 1640 containing 1.5 mM HEPES-1% autologous serum with penicillin and streptomycin (250 U/ml and 250 gg/ml, respectively) and ADA (1 U/ml) + WRC-0470 (30-100 nM), ± endotoxin (10 ng/ml), ± rolipram 15 (300 nM) and ± the adenosine A 2 A selective antagonist 4-(2-[7-amino-2-(2 furyl)[ 1,2,4]-triazolo[2,3a]-[1,3,5]trazinyl-amino]ethyl)-phenol (ZM241385) (50 nM). The samples were incubated for 4 hours (37 0 C; 5% CO 2 ) and the supernatants harvested. Any suspended cells were removed by centrifugation and the cell-free samples frozen (-70 0 C). TNFa was assayed in the cell-free 20 supernatants by an ELISA kit (Cistron Biotechnology, Pine Brook, NJ). As shown in Figures 7A and 7B, WRC-0470 + rolipram decreased endotoxin-stimulated adherent monocyte production of TNFV (P < 0.050). As illustrated in Figure 7B, the A 2 A selective antagonist SM241385 significantly inhibited the effect of WRC-0470 (300 nM) combined with rolipram (300 nM) 25 (p= 0.020) on TNFu release from monocytes. Hence, WRC-0470 affects TNFa stimulated neutrophil activity and decreases endotoxin-stimulated TNFa production by monocytes. Effects of WRC-0470 and rolipram on the extravasation of white blood cells in a rat model of inflammation. Adult wistar rats (approximately 200 g) 30 were anesthetized with intermuscular injections of ketamine and xylazine. Bacteria meningitis (BM) was induced via intracisternal inoculation of either E. coli strain 026:B6LPS (200 ng), cytokines (IL-1 and TNFa, or LPS plus cytokines). The animals were then infused with rolipram and/or WRC-1470 WO 99/34804 PCT/US99/00366 23 over the duration of the experiment using a Harvard pump. CSF (cerebrospinal fluid) and blood was then sampled at 4 hours postinoculation and alterations in BBBP (blood-brain-barrier permeability) and WBC (white blood cell) counts were determined. CSF and WBC concentrations were determined with standard 5 hemacytometer methods. For assessment of % BBBP, rats were given an intravenous injection of 5 tCi 1 25 I-labeled bovine serum albumin concomitant with intracisternal inoculation. Equal samples of CSF and blood were read simultaneously in a gamma counter and after subtraction of background radioactivity, % BBBP was calculated by the following formula: % BBBP = 10 (cpm CSF/cpm blood) x 100. All statistical tests were performed using Instat biostatistical software to compare the post-inoculation samples of experimental rats with the control rats. The statistical tests used to generate p-values were Student's t -test and ANOVA. Results of the tests are reported in Figures 8 and 9. Infusion of WRC 15 0470 at a rate of 0.005-1.2 gg/kg/hour inhibited pleocytosis (p < 0.05 as compared to control). The effect of WRC-0470 on BBBP is shown in Figure 9. A significant response is seen with a range of 0.01-0.015 gg/kg/hour (p < 0.05 as compared to control). A rebound effect is noted with the administration of 1.2 gg/kg/hour where % BBBP returned to control. Figure 10 shows the effect 20 of rolipram on CSF pleocytosis in a range of 0-0.01 gg/kg/hour with 0.01 pg/kg/hour inhibiting 99% of the pleocytosis (p < 0.05). Rolipram at either 0.01 or 0.005 gg/kg/hour showed significant inhibition of alterations of BBBP (p <0.05), while a dose of 0.002 gg/kg/hour had no significant effect. The effect of a combination of rolipram and WRC-0470 on CSF WBC 25 pleocytosis is illustrated in Figure 11. Rolipram (0.001 pg/kg/hour) in combination with WRC-0470 (0.1 gg/kg/hour) inhibited migration of WBC's (200 ± 70 WBC/gl) into the sub-arachnoid space (SAS) to a greater extent than did either rolipram (1,670 ± 1,273 WBC/gl, p < 0.050) or WRC-0470 (600 ± 308 WBCs/il, p < 0.050) alone. The data show a powerful inhibiting 30 effect of WRC-0470 and a synergy with rolipram to prevent inflammation in an animal model. Application of AA adenosine receptors with or without rolipram on balloon angioplasty and gene therapy. Balloon angioplasty is commonly used to WO 99/34804 PCT/US99/00366 24 treat coronary artery stenosis. Restenosis following balloon angioplasty (BA) occurs in up to 40% of coronary interventions. Holmes et al., American Journal of Cardiology, 53, 77C-81C (1984). (40%). Restenosis results from a complex interaction of biologic processes, including (i) formation of platelet-rich 5 thrombus; (ii) release of vasoactive and mitogenic factors causing migration and proliferation of smooth muscle cells (SMC); (iii) macrophage and other inflammatory cell accumulation and foam cell (FC) formation; (iv) production of extracellular matrix; and (v) geometric remodeling. Recently the use of coronary stents and pharmacologic intervention using a chimeric antibody to block the 10 integrin on platelets have been partially successful in limiting restenosis after percutaneous coronary interventions in man. Topol et al., Lencet, 343, 881-886 (1994). Since inflammatory cell infiltration might be central to the response to injury, and restenotic processes, and adenosine, active via A 2 A adenosine receptors, inhibits tissues' inflammatory cell accumulation, we hypothesize that 15 agonists of A 2 A adenosine receptors ± Type IV PDE inhibitors will reduce the incidence of restenosis following balloon angioplasty. In addition, recent advances in local delivery catheters and gene delivery techniques raise the interesting and exciting possibility of administering genes locally into the vessel wall. Nabel et al., Science, 249, 1285-1288 (1990); 20 Leclerc et al., Journal of Clinical Investigation, 90, 936-944 (1992). Adenoviral mediated gene transfer affords several advantages over other techniques. However, gene expression is only transient, and has been observed for 7-14 days with diminution or loss of expression by 28 days. Lack of persistence may result form host immune cytolytic responses directed against infected cells. The 25 inflammatory response generated by the present generation of adenovirus results in neointimal lesion formation and may thus offset the benefit of a therapeutic gene. Newman et al., Journal of Clinical Investigation, 96, 2955-1965 (1995). An A 2 A adenosine receptor agonist ± a Type IV phosphodiesterase inhibitor in combination with adenovirus may improve the efficiency of gene transfer. 30 Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims (11)

1. The use of an agonist of an A 2 A adenosine receptor in combination with a compound that is a Type IV phosphodiesterase inhibitor, preferably rolipram, a 5 rolipram derivative, to treat inflammatory disease.

2. A pharmaceutical composition comprising an effective amount of an agonist of an A 2 A adenosine receptor in combination with a Type IV phosphodiesterase inhibitor, preferably rolipram or a rolipram derivative or a 10 rolipram analogue.

3. The pharmaceutical composition of Claim 2, wherein said agonist of an A 2 A adenosine receptor has the formula (I) NH 2 N NNX HO OH wherein X is a group selected from the group consisting of -OR 1 , -NR 2 R 3 , 15 and -NH-N=R 4 ; wherein R 1 is C,- 4 -alkyl; C 14 -alkyl substituted with one or more C 1 . 4 alkoxy groups, halogen (preferably fluorine, chlorine, or bromine), hydroxy groups, amino groups, mono(Cl- 4 -alkyl)amino groups, di(Cl- 4 -alkyl)amino groups, or C 6 - 1 0 -aryl groups (wherein the aryl groups may be substituted with one 20 or more halogens (preferably fluorine, chlorine, or bromine), Cl- 4 -alkyl groups, hydroxy groups, amino groups, mono(C 1 - 4 -alkyl)amino groups, or di(C 1 - 4 alkyl)amino groups; C 6 o 10 -aryl; or C 6 - 1 0 -aryl substituted with one or more halogens (fluorine, chlorine, or bromine), hydroxy groups, amino groups, mono(Cl- 4 -alkyl)amino groups, or di(Cl- 4 -alkyl)amino groups, or Cl 4 -alkyl 25 groups; one of R 2 and R 3 has the same meaning as R 1 and the other is hydrogen; WO 99/34804 PCT/US99/00366 26 R 4 is a group having the formula R5 c/ (II) R6 wherein each of R 5 and R 6 independently may be hydrogen, C 3 7 cycloalkyl, or any of the meanings of R 1 , provided that R 5 and R 6 are not both hydrogen; 5 or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition of Claim 2, wherein said agonist of an A 2 A adenosine receptor is selected from the group consisting of NH 2 N N O N NH OH NOH NH 2 N N N:N OH OH NH 2 O N 0 OH Off OH WO 99/34804 PCT/US99/00366 27 and NH 2 0N 0 OH OH OH

5. The pharmaceutical composition of Claim 2, wherein said Type IV 5 phosphodiesterase inhibitor is a compound having formula (V): OR18 R 19 0 (V) N 0 I H wherein R 1 8 and R' 9 each are alike or different and are hydrocarbon radicals having up to 18 carbon atoms with at least one being other than methyl, a heterocyclic ring, or alkyl of 1-5 carbon atoms which is substituted by one or more of halogen atoms, hydroxy, carboxy, alkoxy, alkoxycarbonyl or an amino 10 group; amino; R' is a hydrogen atom, alkyl, aryl or acyl; and X is an oxygen atom or a sulfur atom.

6. The pharmaceutical composition of Claim 2, wherein said Type IV phosphodiesterase inhibitor is rolipram. 15

7. The pharmaceutical composition of Claim 2, wherein said agonist of an A 2 A adenosine receptor is WO 99/34804 PCT/US99/00366 28 NH 2 NA / O N NH OH N Off OH and said Type IV phosphosterase inhibitor is rolipram.

8. The pharmaceutical composition of Claim 2, wherein said agonist of an A 2 A adenosine receptor is NH 2 N N NO N NH OH-- N' OH NOH Off OH 5 and said Type IV phosphosterase inhibitor is rolipram.

9. The use of an agonist of an A 2 A adenosine receptor in combination with a Type IV phosphodiesterase inhibitor, preferably rolipram or a rolipram derivative, during and for a limited time after balloon angioplasty to reduce 10 frequency and extent of restenosis.

10. The use of an agonist of an A 2 A adenosine receptor in combination with a Type IV phosphodiesterase inhibitor, preferably rolipram or a rolipram derivative, in conjunction with a gene delivery modality to limit inflammation 15 and thereby improve efficiency and stability of gene therapy. WO 99/34804 PCT/US99/00366 29

11. The use of claim 10, wherein said gene delivery modality is viruses and lipid vesicles.