Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
  • A61K31/52—Purines, e.g. adenine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to methods and compositions for treating inflammatory diseases. Discussion of the Background
• cytokines such as tumor necrosis factor- alpha (TNF ⁇ ) by leukocytes
• TNF ⁇ tumor necrosis factor- alpha
• Cytokines stimulate neutrophils to enhance oxidative (e.g., superoxide and secondary products) and nonoxidative (e.g., myeloperoxidase and other enzymes) inflammatory activity.
• Oxidative e.g., superoxide and secondary products
• nonoxidative e.g., myeloperoxidase and other enzymes
• Inappropriate and over-release of cytokines can produce counterproductive exaggerated pathogenic effects through the release of tissue-damaging oxidative and nonoxidative products (Tracey, K. G. et al., I. Exp. Med., vol. 167, pp. 1211- 1227 (1988); and Mannel, D. N. et al, Rev. Tnfect. Dis. : vol. 9 (suppl. 5), pp. S602-S606
• inflammatory cytokines have been shown to be pathogenic in: arthritis (Dinarello, C. A., Semin. Tn ⁇ muno1., vol. 4, pp. 133-45 (1992)); ischemia (Seekamp, A. et al., Agents- Actions-Supp., vol. 41, pp. 137-52 (1993)); septic shock (Mannel, D. N. et al., Rev. Infect. Pis., vol. 9 (suppl. 5), pp. S602- S606 (1987)); asthma (Cembrzynska Nowak M. et al, Am. Rev. Respir. Pis., vol. 147, pp.
• organ transplant rejection Imagawa, P. K. et al, Transplantation, vol. 51, pp. 57-62 (1991)
• multiple sclerosis Hartung, H. P., Ann. Neurol., vol. 33, pp. 591-6 (1993)
• AIPS Matsuyama, T. et al., ATPS. vol. 5, pp. 1405-1417 (1991)
• 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)).
• HIV human immunodeficiency virus
• 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. Tmmunol . . . vol. 135, pp. 1366-1371 (1985)).
• Adenosine can decrease the greatly enhanced oxidative burst of PMN (neutrophil) first primed with TNF- (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)).
• TNF- an inflammatory cytokine
• f-met-leu-phe f-met-leu-phe
• 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)).
• 2-alkylamino substituents increased potency and selectivity, e.g., CV 1808 and CGS21680 (Jarvis, M. F. et al., J. Pharmacol. Exp. Ther. : vol. 251, pp. 888-893 (1989)).
• 2-Alkoxy-substituted adenosine derivatives such as WRC-0090 are even more potent and selective as agonists on the coronary artery A 2A receptor (Ukena, M. et al., I. 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 2A receptor (Niiya, K. et al, I. Med. Chem., vol. 35, pp. 45574561 (1992)).
• 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 2A 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 effective amount of an A 2A adenosine receptor of the following formula:
• .4 -alkyl groups hydroxy groups, amino groups, mono(C j _ 4 -alkyl)amino groups, or di(C, .4 -alkyl)amino groups); C 6.10 -aryl; or C 6.10 -aryl substituted with one or more halogens (fluorine, chlorine, or bromine), hydroxy groups, amino groups, mono(C, .4 -alkyl)amino groups, or di(C, .4 -alkyl)amino groups, or C ].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:
• 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;
• R is -CH 2 OH, -CH 2 H, -CO 2 R 7 , or -C(-O)NR 8 R 9 ; wherein R 7 has the same meaning as R 1 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.
• the Linden et al. invention involves the administration of a Type IV phosphodiesterase (PDE) inhibitor in combination with the A 2A adenosine receptor agonist.
• PDE Type IV phosphodiesterase
• the Type IV phosphodiesterase (PDE) inhibitor can be racemic and optically active 4-(polyalkoxyphenyl)-2— pyrrolidones of the following formula:
• R 18 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 heterocychc 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; 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 structure:
• 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 2A adenosine receptors in combination with rolipram or rolipram derivatives that are Type IV phosphodiesterase or PDE inhibitors.
• Summary of the Invention Accordingly, one object of the present invention is to provide a novel and improved method for treating inflammatory diseases.
• compositions and methods for effectively treating inflammatory diseases by administration of an agonist of an A 2A adenosine receptor in combination with rolipram or a rolipram derivative that is a Type IV phosphodiesterase (PDE) inhibitor.
• PDE Type IV phosphodiesterase
• 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, no TNF ⁇ ; ⁇ , WRC-0474[SHA 211] + TNF ⁇ ; D, GCS 21680 + TNF ⁇ ; and A, adenosine + TNF ⁇ );
• PMN polymorphonuclear cell
• Figure 2 illustrates the synergistic effect of WRC-0474[SHA 211] and 4- (3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (rolipram) in inhibiting TNF ⁇ -primed (10 U/ml), fMLP-stimulated (100 nM) PMN superoxide production: 0, no 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone; A, 3 nM 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone; D, 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 rolipram in inhibiting TNF ⁇ -stimulated adherent PMN superoxide release;
• Figure 4 illustrates the effect of WRC-0474[SHA 211] and rolipram on TNF ⁇ -stimulated PMN adherence to a fibrinogen-coated surface
• Figure 5 illustrates synergy between A 2A adenosine receptor agonists and Rolipram in inhibiting superoxide release from TNF ⁇ -stimulated adherent human neutrophils;
• Figure 6 illustrates the effects of WRC-0470 and rolipram on the oxidative activity of neutrophils in whole blood
• Figure 7 illustrates the effects of WRC-0470 and rolipram on the release of TNF ⁇ from adherent human monocytes and that this activity is dependent on binding of the adenosine agonist to A 2A adenosine receptors;
• Figure 8 illustrates the effect of WRC-0470 on white blood cell 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
• Figure 11 illustrates the combined effect of WRC-0470 and rolipram on white blood cell pleocytosis in rats.
• the present invention provides a method for treating inflammatory diseases by administering an effective amount of a compound of formula (I):
• 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)
• 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;
• C 6.10 -aryl groups examples include phenyl and naphthyl.
• the compound of formula (I) has X being a group of the formula (III)
• the compound of formula (IV) has X being a group of the formula (I)
• Cy is a C 3 . 7 -cycloalkyl group, preferably cyclohexyl or a C -alkyl group, preferably isopropyl.
• WRC-0474[SHA 211] and WRC-0470 are particularly preferred.
• Such compounds may be synthesized as described in: Hutchinson, A. J. et al., I. Pharmacol. KxD. Ther., vol. 251, pp. 47-55 (1989); Olsson, R. A. et al., I. Med. Chem. r vol. 29, pp. 1683-1689 (1986); Bridges, A. J. et al, I. Med. 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, I. Med. Che ⁇ n. r vol. 34, pp. 1334-1339 (1991); Francis, J. E. et al, J. Med. Chem, vol. 34, pp. 2570-
• the present method includes the administration of a Type IV phosphodiesterase (PDE) inhibitor in combination with the compound of formula (I).
• PDE Type IV phosphodiesterase
• Examples of Type IV phosphodiesterase inhibitors include those disclosed in U.S. Patent No. 4,193,926, WO 92-079778, and Molnar-Kimber,
• PDE Type IV phosphodiesterase
• R 18 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 heterocychc 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.
• hydrocarbon R 18 and R 19 groups are saturated and unsaturated, straight-chain and branched alkyl of 1-18, preferably 1-5, carbon atoms, cycloalkyl and cycloalkylalkyl, preferably of 3-7 carbon atoms, and aryl and aralkyl, preferably of 6-10 carbon atoms, especially monocyclic.
• alkyl examples include 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 18 and R 19 is methyl, the other is a value other than methyl.
• unsaturated alkyl groups are alkenyl and alkynyl, e.g., vinyl, 1-propenyl, 2-propenyl, 2-propynyl and 3-methyl-2-propenyl.
• cycloalkyl and cycloalkylalkyl which preferably contain a total of 3-7 carbon atoms are cyclopropyl, cyclopropylmethyl, cyclopentyl and cyclohexyl.
• aryl and aralkyl are phenyl and benzyl, which are preferred, and tolyl, xylyl, naphthyl, phenethyl and 3phenylpropyl.
• heterocychc R 18 and R 19 groups are those wherein the heterocychc 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- 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.
• heterocychc 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 N-alkylpiperazino.
• substituted alkyl R 18 and R 19 groups are those mono- or polysubstituted, for example, by halogen, especially fluorine, chlorine and bromine.
• halogen- substituted alkyl are 2-chloroethyl, 3-chloropropyl, 4-bromobutyl, difluoromethyl, trifluoromethyl, l,l,2-trifluoro-2-chloroethyl, 3,3,3- trifluoropropyl, 2,2,3,3,3-pentafluoropropyl and l,l,l,3,3,3-hexafluoro-2-propyl.
• alkyl groups examples include hydroxy groups, e.g., 2-hydroxyethyl or 3-hydroxypropyl; carboxy groups, e.g., carboxymethyl or carboxyethyl; alkoxy groups, wherein each alkoxy group contains 1-5 carbon atoms, e.g., ethoxymethyl, isopropoxymethyl, 2- methoxyethyl, 2-isopropoxyethyl, 2-butoxyethyl, 2-isobutyoxyethyl, and 3- pentoxypropyl.
• alkoxycarbonyl of 1-5 carbon atoms in the alkoxy group are alkoxycarbonyl of 1-5 carbon atoms in the alkoxy group.
• alkoxycarbonyl substituted alkyl groups are ethoxycarbonylmethyl and 2-butoxycarbonylethyl.
• Alkyl groups of 1-5 carbon atoms can also be substituted, e.g., in the ⁇ , 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 phosphodiesterase inhibitors.
• 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 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), TNF ⁇ -enhanced HIV replication and TNF ⁇ inhibition of reverse transcriptase inhibitor activity, wasting diseases (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 from
• 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.
• a suitable dosage of the compound of formula (I) is 0.5 to 100 ⁇ g/kg of body weight, preferably 1 to 10 ⁇ g/kg of body weight.
• 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, the mode of administration will be oral, topical, intravenous, parenteral, subcutaneous, or intramuscular injection.
• the compound of formula (I) may be administered in the form of a pharmaceutically acceptable salt.
• salts include acid addition salts.
• 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, such as carboxysuccinic acid, citric acid, and the like.
• 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.
• 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.
• the compounds of formula (I) can be administered orally, for example, with an inert diluent with an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
• the compounds can be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, waters, 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 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 ⁇ g and about 5 mg, preferably between 50 to 500 ⁇ g, of active compound.
• Tablets, pills, capsules, troches, and the like can contain the following 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 peppermint, methyl sahcylate, or orange flavoring.
• a liquid carrier such as a fatty oil.
• compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 30 ⁇ g to 5 mg, preferably between 50 to 500 ⁇ g, 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, 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 or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose.
• a sterile diluent such as water for injection, 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
• Effective amounts of the Type IV phosphodiesterase inhibitor can be administered to a subject by any one of various methods, for example, orally as 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.
• 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, such as carboxysuccinic acid, citric acid, and the like.
• 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
• salts of tribasic carboxylic acids such as carboxysuccinic acid, citric acid, and the like.
• 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).
• Type IV phosphodiesterase inhibitor is administered to a subject requiring such treatment as an effective oral, parenteral or intravenous dose as described below.
• 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- 50 mg, preferably 1-30 mg p.o.
• 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.
• 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.
• 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 those described above in the context of the compound of formula (I).
• the dosage of the A 2A 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.
• 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).
• 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 action also synergistic with rolipram. Further, the present studies establish that activation of A 2A receptors on human monocytes strongly inhibits TNF ⁇ (an inflammatory cytokine) release. This mechanism further contributes to the anti- inflammatory action of the A 2A adenosine receptor agonists of the present invention.
• 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 Whittaker Bioproducts (WalkersviUe, 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.
• Luminol-enhanced chemiluminescence a measure of neutrophil oxidative activity, is dependent upon both superoxide production and mobilization of the granule enzyme myeloperoxidase. The light is emitted from unstable high-energy oxygen species generated by activated neutrophils.
• Purified PMN (5 ⁇ l0 5 /ml) were incubated in HBSS containing 0.1% human serum albumin (1 ml) with or without adenosine, adenosine analogs, and TNF ⁇ (1 U/mL) for 30 minutes at 37°C in a shaking water bath.
• the samples were iced and centrifuged (2000 g x 10 minutes).
• the optical density of the supematants were read at 550 nm against the matched SOD samples, and the nmoles of SOD-inhibitable superoxide released in 10 minutes were calculated.
• PMN adherence to fibrinogen was measured as follows as adapted from Hanlon, J. Leukocyte Biol, vol. 50, p. 43 (1991). Twenty-four well flat- bottomed tissue culture plates were incubated (37°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 2 with 1 ml of normal saline.
• the wells were then filled with 1 ml of HBSS-0.1% human serum albumin containing PMN (1 lOVml) with and without rhTNF ⁇ (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°C in 5% CO 2 . Following incubation the tissue culture wells were washed free of non- adherent cells with normal saline.
• 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., Int. J. Tmmunopharmaco 12, 793-803 (1995).
• Neutrophils (1 x lOVml) 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 rhTNF ⁇ (1 U/ml), WRC- 0474[SHA 211], WRC-0470, WRC-0090 and WRC-0018 (3-300 nM), and rolipram (100 nM) in a tissue culture well which had been coated ovemight with human fibrinogen.
• SOD superoxide dismutase
• Figure 5 shows synergy between A 2A adenosine agonists and rolipram in inhibiting TNF ⁇ -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 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 activity. All compounds were synergistic with rolipram to decrease TNF ⁇ - stimulated adherent PMN oxidative activity.
• 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 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. The results are reported as relative mean fluorescence intensity in Figure 6 of the drawings.
• WRC-0470 decreased oxidative activity of TNF ⁇ - primed fMLP-stimulated neutrophils in whole blood and acted synergistically with rolipram.
• WRC-0470 (30-300 nM) decreased neutrophil oxidative activity synergistically with rolipram (300 nM) in samples stimulated with fMLP and in blood samples primed with TNF ⁇ and then stimulated with fMLP.
• a monocyte-rich monolayer (>95% monocytes) was prepared by incubating 1 ml of the mononuclear leukocyte fraction (5 x lOVml) from a Ficoll-hypaque separation in wells of a 24 well tissue culture plate (1 hour; 37°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 ⁇ g/ml, respectively) and ADA (1 U/ml) ⁇ WRC-0470 (30-100 nM), ⁇ endotoxin (10 ng/ml), ⁇ rolipram (300 nM) and ⁇ the adenosine A 2A 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).
• TNF ⁇ was assayed in the cell-free supematants 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 TNF ⁇ (P ⁇ 0.050).
• WRC-0470 affects TNF ⁇ - stimulated neutrophil activity and decreases endotoxin-stimulated TNF ⁇ production by monocytes.
• BM Bacteria meningitis
• E. coli strain 026:B6LPS 200 ng
• cytokines IL-1 and TNF ⁇
• LPS plus cytokines The animals were then infused with rolipram and/or WRC-1470 over the duration of the experiment using a Harvard pump.
• CSF cerebrospinal fluid
• BBBP blood-brain-barrier permeability
• WBC white blood cell
• % BBBP (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 ANONA.
• Balloon angioplasty is commonly used to 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-81 C (1 84). (40%).
• Restenosis results from a complex interaction of biologic processes, including (i) formation of platelet-rich 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.
• SMC smooth muscle cells
• FC macrophage and other inflammatory cell accumulation and foam cell
• Adenoviral- mediated gene transfer affords several advantages over other techniques.
• 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 inflammatory response generated by the present generation of adenovims results in neointimal lesion formation and may thus offset the benefit of a therapeutic gene.

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