(12) United States Patent (10) Patent No.: US 7,160,899 B2 Peters Et Al

US007160899B2

(12) United States Patent (10) Patent No.: US 7,160,899 B2 Peters et al. (45) Date of Patent: Jan. 9, 2007

(54) ADENOSINE AA RECEPTOR (56) References Cited ANTANGONSTS COMBINED WITH NEUROTROPHIC ACTIVITY COMPOUNDS U.S. PATENT DOCUMENTS IN THE TREATMENT OF PARKINSONS 2001/0027.196 A1 10, 2001 Borroni et al. DISEASE FOREIGN PATENT DOCUMENTS (75) Inventors: Dan Peters, Malmö (SE); Lars Christian B. Ronn, Vekse (DK); Karin DE 43 25 254 A1 2, 1994 Sandager Nielsen, Fredensborg (DK) WO WO 97/40035 * 10/1997 WO WO99/43678 A1 9, 1999 (73) Assignee: Neurosearch A/S, Ballerup (DK) WO WO 99,53909 A2 10, 1999 (*) Notice: Subject to any disclaimer, the term of this WO WO 01/82946 A2 4/2001 patent is extended or adjusted under 35 U.S.C. 154(b) by 342 days. OTHER PUBLICATIONS Beers, M. H. and Berkow, R., Editors-in-chief, The Merck Manual (21) Appl. No.: 10/473,809 of Diagnosis and Therapy, 17th Edition, pp. 1466-1473, 1999.* Hess, Expert Opinion Ther. Patents, vol. 11, No. 10, pp. 1533-1561 (22) PCT Filed: Apr. 4, 2002 (2001). Lee et al., PNAS, vol. 98, No. 6, pp. 3555-3560 (2001). (86). PCT No.: PCT/DKO2/OO228 Kiec-Kononowicz et al., Pure Appl. Chem... vol. 73, No. 9, pp. 1411–1420 (2001). S 371 (c)(1), Ongini et al., Annals NY Academy of Sciences, pp. 30-49, 1997. (2), (4) Date: Oct. 2, 2003 Heese et al., Neuroscience Letters, vol. 231, pp. 83-86 (1997). Bennett, The Neuroscientist, vol. 7, No. 1, pp. 13-17 (2001). (87) PCT Pub. No.: WO02/080957 Morelli et al., Drug Development Research, vol. 52, pp. 387-393 (2001). PCT Pub. Date: Oct. 17, 2002 * cited by examiner (65) Prior Publication Data Primary Examiner Dwayne Jones US 2004/0097540 A1 May 20, 2004 (74) Attorney, Agent, or Firm—Birch, Stewart, Kolasch & Birch, LLP (30) Foreign Application Priority Data Apr. 9, 2001 (DK) ...... 2001 OO583 (57) ABSTRACT (51) Int. Cl. This invention relates to the use of the combined action of A6 IK 3/47 (2006.01) a compound with neurotrophic activity and an adenosine (52) U.S. Cl...... 514/307: 514/2: 514/230.2: A receptor antagonist for the treatment of Parkinson's 514/237 disease. (58) Field of Classification Search ...... None See application file for complete search history. 6 Claims, No Drawings US 7,160,899 B2 1. 2 ADENOSINE AA RECEPTOR The principle combines a fast onset action (the effect of ANTANGONSTS COMBINED WITH the adenosine A receptor antagonist) with a long-term NEUROTROPHIC ACTIVITY COMPOUNDS effective principle (the neurotrophic activity). Thus, the IN THE TREATMENT OF PARKINSONS adenosine A receptor antagonist relieves the symptoms of DISEASE the disease (by increasing the dopaminergic activity), while the neurotrophic activity treats the cause of the disease This application is the national phase under 35 U.S.C. S (degenerating neurons) by slowing or even reversing the 371 of PCT International Application No. PCT/DK02/00228 progression of the disease. which has an International filing date of Apr. 4, 2002, which Other objects of the invention will be apparent to the designated the United States of America. 10 person skilled in the art from the following detailed descrip tion and examples. TECHNICAL FIELD DETAILED DISCLOSURE OF THE INVENTION This invention relates to the use of the combined action of a compound with neurotrophic activity and an adenosine 15 In its first aspect, the invention provides the use of at least A receptor antagonist for the treatment of Parkinson's one compound with neurotrophic activity and at least one disease. adenosine A receptor antagonist for the manufacture of a medicament for the treatment, prevention or alleviation of BACKGROUND ART Parkinson's disease in a Subject. In a second aspect, the invention provides a pharmaceu Parkinson's disease is a neurodegenerative disease char tical composition comprising a therapeutically effective acterised by the progressive deterioration of motor skills, amount of at least one compound with neurotrophic activity affecting about 4 million people worldwide. Parkinson's and at least one adenosine A receptor antagonist, together patients suffer from increasing difficulties in initiating move with at least one pharmaceutically acceptable carrier or ment, rigidity in arms and legs, as well as tremors. Although 25 diluent. the specific cause of Parkinson's disease is unknown, it has In a third aspect, the invention provides a combination of been shown that the disease is associated with the degen at least one compound with neurotrophic activity and at least eration of specific dopamine-containing neurons in a region one adenosine A receptor antagonist for use as a thera of the brain known as the substantia nigra, which is believed peutic agent. to be involved in the coordination of movement. 30 In a further aspect, the invention provides a method of One existing treatment is L-DOPA therapy, alone or treatment, prevention or alleviation of Parkinson's disease in combined with e.g. dopamine agonists. However, after three a subject, which method comprises administering to said to five years of L-DOPA therapy, involuntary motor distur subject a therapeutically effective combination of at least bances (dyskinesia) may appear. one compound with neurotrophic activity and at least one Another treatment is the use of monoamine reuptake 35 adenosine A receptor antagonist. inhibitors (such as dopamine reuptake inhibitors) whereby In a still further aspect, the invention provides a kit of the existing dopamine level in the synaptic cleft is increased. parts comprising at least one compound with neurotrophic A further possible therapy is the use of neurotrophic activity and at least one adenosine A receptor antagonist. compounds which give a neuroregenerative effect on In one embodiment, the adenosine A, antagonist is lesioned and damaged neurons. 40 selected from the group consisting of KW-6002, A still further treatment Suggested is the use of adenosine ZM-241385, 8FB-PTP, SCH-58261, KF-17837, CGS A receptor antagonists, which result in an enhanced 15943, DMPX and pharmaceutically acceptable salts dopaminergic activity. Furthermore, adenosine A recep thereof. tors and their relation to neuroprotection have been dis In a second embodiment, the compound with neu cussed (Ongini, E. et al. (1997) Adenosine A receptors and 45 rotrophic activity is a compound selected from the group neuroprotection, Ann NY Acad Sci 825:30–48). consisting of There is a continued strong interest in the development of 5-(4-Chlorophenyl)-8-methyl-6,7,8,9-tetrahydro-1-H-pyr a more selective and effective therapy with fewer side effects rolo3.2-hisoquinoline-2,3-dione-3-oxime; for the treatment of patients with Parkinson's disease. 5-(4-Chlorophenyl)-6,7,8,9-tetrahydro-1-H-pyrrolo3.2-h 50 naphthalene-2,3-dione-3-oxime; SUMMARY OF THE INVENTION GDNF: Neublastin; According to the invention it has now been found that the and pharmaceutically acceptable salts thereof. action of a compound with neurotrophic activity in combi In a special embodiment, the compound with neu nation with an adenosine A receptor antagonist advanta 55 rotrophic activity is GDNF and the adenosine A antagonist geously can be used for the treatment of Parkinson's disease. is SCH-58261. In a further special embodiment, the com Accordingly, in its first aspect, the invention relates to a pound with neurotrophic activity is GDNF and the adenos pharmaceutical composition comprising a therapeutically ine A, antagonist is KF-17837. effective amount of at least one compound with neurotrophic In a further embodiment, the pharmaceutical composition activity and at least one adenosine A receptor antagonist, 60 as described above is for use in the treatment, prevention or together with at least one pharmaceutically acceptable car alleviation of a neurodegenerative condition. In a still further rier or diluent. embodiment, the pharmaceutical composition as described In another aspect, the invention relates to the use of at above is for use in the treatment, prevention or alleviation of least one compound with neurotrophic activity and at least Parkinson's disease in a Subject. one adenosine A receptor antagonist for the manufacture 65 The subject to be treated according to this invention is a of a medicament for the treatment, prevention or alleviation living body, preferably a mammal, most preferably a human, of Parkinson's disease in a subject. in need for Such treatment. US 7,160,899 B2 3 4 Any possible combination of two or more of the embodi CNRS), V-10,367 and V-13,661 (Vertex Pharmaceuticals ments described herein is comprised within the scope of the Inc), ABS-205 (American Biogenic Sciences), Dexanabinol present invention. or HU-211 (Pharmos), or salts, free bases, racemates or enantiomers thereof. Compounds With Neurotrophic Activity The above examples of compounds with neurotrophic activity are not intended to be in any way limiting to the Endogenous neurotrophic factors, such as nerve growth Scope of the invention as claimed. factor (NGF), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), basic fibroblast growth Adenosine A Receptor Antagonists factor (bFGF, or FGF2), NT34, neurturin (NTN), neublastin/ 10 artemin, persephin, and glial cell-line derived neurotrophic In the context of the present invention, adenosine A, factor (GDNF), promote the differentiation, growth and receptor antagonists include Xanthine based analogs and Survival of numerous peripheral and central nervous system non-Xanthine based analogs. neurons during development and adulthood. Examples of adenosine A receptor antagonists include In the context of this invention, compounds with neu 15 KW-6002 (Kyowa Hakko Kogyo Co Ltd), ZM-241385, rotrophic activity are compounds that mimic or enhance the 8FB-PTP, SCH-58261-KF-17837, CGS-15943, DMPX, function of one or more endogenous neurotrophic factors. In 8-(m-chlorostyryl)-DMPX., 8-(m-bromostyryl)-DMPX (or one embodiment, a compound with neurotrophic activity is BS-DMPX), 8-(3,4-dimethoxystyryl)-DMPX. a compound that mimics or enhances the function of NGF, The potential of a given Substance to act as an adenosine BDNF, and/or GDNF. In a further embodiment, a compound A receptor antagonists may be determined using standard with neurotrophic activity is a compound that mimics or in vitro binding assays and/or standard in vivo functionality enhances the function of bfGF and/or EGF. In a special tests, such as those described in "Test methods’. embodiment, a compound with neurotrophic activity is a In one embodiment, the adenosine A receptor antago compound that mimics or enhances the function of NGF. nists show an ICs value of less than 10 uM, preferably less The neurotrophic activity has not been ascribed to a specific 25 than 1 M, and most preferably less than 0.1 uM, when step in the interaction between the growth factor and its tested for In vitro inhibition of H-ZM 241385 binding (test receptor or in the growth factor signal transduction pathway. method 7a). The potential of a given Substance to act as a compound The above examples of adenosine A receptor antago with neurotrophic activity may be determined using standard nists are not intended to be in any way limiting to the scope in vitro binding assays and/or standard in Vivo functional 30 of the invention as claimed. tests, such as those described in “Test methods’. In one embodiment, the compound with neurotrophic Pharmaceutically Acceptable Salts activity at 1 uM shows more than 10% (more preferably more than 20%, and most preferably more than 30%) of the The active compounds for use according to the invention effect of 3 nM NGF when tested in the PC12 cells Survival 35 may be provided in any form suitable for the intended assay (method 2). administration. Suitable forms include pharmaceutically In a second embodiment, the compound with neu (i.e. physiologically) acceptable salts, and pre- or prodrug rotrophic activity at 1 M shows more than 10% (more forms of the chemical compound of the invention. preferably more than 20%, and most preferably more than Examples of pharmaceutically acceptable addition salts 30%) of the effect of 10 ng/ml GDNF when testing the 40 include, without limitation, the non-toxic inorganic and Survival of embryonic rat dopaminergic neurons (method 3). organic acid addition salts such as the hydrochloride derived In a special embodiment, the compound with neu from hydrochloric acid, the hydrobromide derived from rotrophic activity is not an adenosine A receptor antago hydrobromic acid, the nitrate derived from nitric acid, the nist. perchlorate derived from perchloric acid, the phosphate In a further embodiment, the compound with neurotrophic 45 derived from phosphoric acid, the sulphate derived from activity and the adenosine A receptor antagonist are not sulphuric acid, the formate derived from formic acid, the the same compound. acetate derived from acetic acid, the aconate derived from Compounds with neurotrophic activities for use according aconitic acid, the ascorbate derived from ascorbic acid, the to the invention include those substances described in the benzenesulphonate derived from benzensulphonic acid, the patent applications WO 98/07705 (Takeda Chem Ind Ltd), 50 benzoate derived from benzoic acid, the cinnamate derived WO 00/34262 (Takeda Chem Ind Ltd), WO 00/32197 (Al from cinnamic acid, the citrate derived from citric acid, the con Lab Inc), WO 97/40035 (NeuroSearch), WO 00/43397 embonate derived from embonic acid, the enantate derived (NeuroSearch), international application PCT/DK01/00049 from enanthic acid, the fumarate derived from fumaric acid, (NeuroSearch), JP 2000226388-A (Takeda Chem Ind Ltd), the glutamate derived from glutamic acid, the glycolate WO 00/32197 (Alcon Lab), and WO 00/46222 (Schering 55 derived from glycolic acid, the lactate derived from lactic AG). acid, the maleate derived from maleic acid, the malonate Further examples of compounds with neurotrophic activ derived from malonic acid, the mandelate derived from ity according to the invention include 1-(1,3-benzodioxol mandelic acid, the methaneSulphonate derived from meth 5-yl)-7,8,9,10-tetrahydro-1,3-benzodioxola.5-glisoquino ane Sulphonic acid, the naphthalene-2-Sulphonate derived lin-7-one (Takeda), 2-(2,2,4,6,7-Pentamethyl-3-phenyl-2,3- 60 from naphtalene-2-Sulphonic acid, the phthalate derived dihydro-1-benzofuran-5-yl)-isoindoline (Takeda), 4-Aryl-1- from phthalic acid, the salicylate derived from salicylic acid, phenylalkyl-1,2,3,6-tetrahydropyridine (Sanofi the sorbate derived from sorbic acid, the stearate derived Synthelabo), SR-57746A or 1-(2-napht-2-yl)ethyl-4-(3- from Stearic acid, the Succinate derived from Succinic acid, trifluoromethylphenyl)-1,2,5,6-tetrahydropyridine (Sanofi the tartrate derived from tartaric acid, the toluene-p-sulpho Synthelabo), AIT-082 (NeoTherapeutics), NIL-A (Amgen 65 nate derived from p-toluene Sulphonic acid, and the like. Inc), K-252a (Cephalon), CEP-1347, GPI-1046 (Guilford), Such salts may be formed by procedures well known and CTQ3, CTQ5 and CTQ8 (Centre de Neurochimie du described in the art. US 7,160,899 B2 5 6 Other acids such as oxalic acid, which may not be and sterile injectable solutions for parenteral use. Such considered pharmaceutically acceptable, may be useful in pharmaceutical compositions and unit dosage forms thereof the preparation of salts useful as intermediates in obtaining may comprise conventional ingredients in conventional pro an active compound for use according to the invention and portions, with or without additional active compounds or its pharmaceutically acceptable acid addition salt. principles, and Such unit dosage forms may contain any Metal salts of an active compound for use according to the Suitable effective amount of the active ingredient commen invention includes alkali metal salts, such as the Sodium salt Surate with the intended daily dosage range to be employed. of an active compound for use according to the invention The active compound of the present invention can be containing a carboxy group. administered in a wide variety of oral and parenteral dosage In the context of this invention the "onium salts' of 10 forms. It will be obvious to those skilled in the art that the N-containing compounds are also contemplated as pharma following dosage forms may comprise, as the active com ceutically acceptable salts. Preferred “onium salts' include ponent, either a chemical compound of the invention or a the alkyl-onium salts, the cycloalkyl-onium salts, and the pharmaceutically acceptable salt of a chemical compound of cycloalkylalkyl-onium salts. the invention. The active compounds for use according to the invention 15 For preparing pharmaceutical compositions from an may be provided in dissoluble or indissoluble forms together active compound of the present invention, pharmaceutically with a pharmaceutically acceptable solvents such as water, acceptable carriers can be either solid or liquid. Solid form ethanol, and the like. Dissoluble forms may also include preparations include powders, tablets, pills, capsules, hydrated forms such as the monohydrate, the dihydrate, the cachets, Suppositories, and dispersible granules. A solid hemihydrate, the trihydrate, the tetrahydrate, and the like. In carrier can be one or more Substances which may also act as general, the dissoluble forms are considered equivalent to diluents, flavouring agents, solubilizers, lubricants, Suspend indissoluble forms for the purposes of this invention. ing agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Pharmaceutical Compositions In powders, the carrier is a finely divided solid, which is 25 in a mixture with the finely divided active component. While the active compounds for use according to the In tablets, the active component is mixed with the carrier invention in therapy may be administered in the form of the having the necessary binding capacity in Suitable propor raw chemical compounds, it is preferred to introduce the tions and compacted in the shape and size desired. active ingredient, optionally in the form of physiologically The powders and tablets preferably contain from five or acceptable salts, in a pharmaceutical composition together 30 ten to about seventy percent of the active compound. Suit with one or more adjuvants, excipients, carriers, buffers, able carriers are magnesium carbonate, magnesium Stearate, diluents, and/or other customary pharmaceutical auxiliaries. talc, Sugar, lactose, pectin, dextrin, starch, gelatin, traga The active compounds for use according to the invention canth, methylcellulose, Sodium carboxymethylcellulose, a may be administered separately or in combination. Thus the low melting wax, cocoa butter, and the like. The term pharmaceutical compositions for use according to the inven 35 “preparation' is intended to include the formulation of the tion may comprise the active compounds for use separately active compound with encapsulating material as carrier or in combination. providing a capsule in which the active component, with or In one embodiment, the invention provides pharmaceuti without carriers, is surrounded by a carrier, which is thus in cal compositions comprising the active compounds of the association with it. Similarly, cachets and lozenges are invention, or pharmaceutically acceptable salts or derivative 40 included. Tablets, powders, capsules, pills, cachets, and thereof, together with one or more pharmaceutically accept lozenges can be used as Solid forms suitable for oral admin able carriers, and, optionally, other therapeutic and/or pro istration. phylactic ingredients, know and used in the art. The carrier For preparing Suppositories, a low melting wax, such as a (s) must be “acceptable' in the sense of being compatible mixture of fatty acid glyceride or cocoa butter, is first melted with the other ingredients of the formulation and not harmful 45 and the active component is dispersed homogeneously to the recipient thereof. therein, as by stirring. The molten homogenous mixture is Pharmaceutical compositions of the invention may be then poured into convenient sized moulds, allowed to cool, those Suitable for oral, rectal, bronchial, nasal, topical (in and thereby to solidify. cluding buccal and Sub-lingual), transdermal, vaginal or Compositions suitable for vaginal administration may be parenteral (including cutaneous, Subcutaneous, intramuscu 50 presented as pessaries, tampons, creams, gels, pastes, foams lar, intraperitoneal, intravenous, intraarterial, intracerebral, or sprays containing in addition to the active ingredient Such intraocular injection or infusion) administration, or those in carriers as are known in the art to be appropriate. a form suitable for administration by inhalation or insuffla Liquid preparations include Solutions, Suspensions, and tion, including powders and liquid aerosol administration, or emulsions, for example, water or water-propylene glycol by Sustained release systems. Suitable examples of Sustained 55 Solutions. For example, parenteral injection liquid prepara release systems include semipermeable matrices of Solid tions can be formulated as solutions in aqueous polyethylene hydrophobic polymers containing the compound of the glycol Solution. invention, which matrices may be in form of shaped articles, The active compounds for use according to the present e.g. films or microcapsules. invention may thus be formulated for parenteral adminis The active compounds of the invention, together with a 60 tration (e.g. by injection, for example bolus injection or conventional adjuvant, carrier, or diluent, may thus be continuous infusion) and may be presented in unit dose form placed into the form of pharmaceutical compositions and in ampoules, pre-filled Syringes, Small Volume infusion or in unit dosages thereof. Such forms include Solids, and in multi-dose containers with an added preservative. The com particular tablets, filled capsules, powder and pellet forms, positions may take Such forms as Suspensions, Solutions, or and liquids, in particular aqueous or non-aqueous Solutions, 65 emulsions in oily or aqueous vehicles, and may contain Suspensions, emulsions, elixirs, and capsules filled with the formulation agents such as Suspending, stabilising and/or same, all for oral use, Suppositories for rectal administration, dispersing agents. Alternatively, the active ingredient may US 7,160,899 B2 7 8 be in powder form, obtained by aseptic isolation of sterile The pharmaceutical preparations are preferably in unit solid or by lyophilization from solution, for constitution dosage forms. In Such form, the preparation is Subdivided with a Suitable vehicle, e.g. sterile, pyrogen-free water, into unit doses containing appropriate quantities of the before use. active component. The unit dosage form can be a packaged Aqueous solutions suitable for oral use can be prepared by preparation, the package containing discrete quantities of dissolving the active component in water and adding Suit preparation, such as packaged tablets, capsules, and powders able colorants, flavours, stabilising and thickening agents, as in vials or ampoules. Also, the unit dosage form can be a desired. capsule, tablet, cachet, or lozenge itself, or it can be the Aqueous Suspensions suitable for oral use can be made by appropriate number of any of these in packaged form. dispersing the finely divided active component in water with 10 Tablets or capsules for oral administration and liquids for Viscous material. Such as natural or synthetic gums, resins, intravenous administration and continuous infusion are pre methylcellulose, sodium carboxymethylcellulose, or other ferred compositions. well known Suspending agents. Further details on techniques for formulation and admin Also included are solid form preparations, intended for istration may be found in the latest edition of Remington's conversion shortly before use to liquid form preparations for 15 Pharmaceutical Sciences (Maack Publishing Co., Easton, oral administration. Such liquid forms include Solutions, Pa.). Suspensions, and emulsions. In addition to the active com The pharmaceutical composition of the invention prefer ponent Such preparations may comprise colorants, flavours, ably is for use in the treatment, prevention or alleviation of stabilisers, buffers, artificial and natural Sweeteners, dispers Parkinson's disease in a Subject. ants, thickeners, Solubilizing agents, and the like. The actual dosage depend on the nature and severity of the For topical administration to the epidermis the active disease being treated, and is within the discretion of the compound of the invention may be formulated as ointments, physician, and may be varied by titration of the dosage to the creams or lotions, or as a transdermal patch. Ointments and particular circumstances of this invention to produce the creams may, for example, be formulated with an aqueous or desired therapeutic effect. However, it is presently contem oily base with the addition of suitable thickening and/or 25 plated that pharmaceutical compositions containing of from gelling agents. Lotions may be formulated with an aqueous about 0.01 to about 500 mg of active ingredient per indi or oily base and will in general also contain one or more vidual dose, preferably of from about 0.1 to about 100 mg. emulsifying agents, stabilising agents, dispersing agents, most preferred of from about 1 to about 10 mg, are suitable Suspending agents, thickening agents, or colouring agents. for therapeutic treatments. Compositions suitable for topical administration in the 30 The active ingredient may be administered in one or mouth include lozenges comprising the active agent in a several doses per day. A satisfactory result can, in certain flavoured base, usually sucrose and acacia or tragacanth; instances, be obtained at a dosage as low as 0.1 ug/kg i.v. and pastilles comprising the active ingredient in an inert base 1 g/kg p.o. The upper limit of the dosage range is presently Such as gelatin and glycerine or Sucrose and acacia; and considered to be about 10 mg/kg i.v. and 100 mg/kg p.o. mouthwashes comprising the active ingredient in a Suitable 35 Preferred ranges are from about 0.1 ug/kg to about 10 liquid carrier. mg/kg/day i.V., and from about 1 ug/kg to about 100 Solutions or Suspensions are applied directly to the nasal mg/kg/day p.o. cavity by conventional means, for example with a dropper, The pharmaceutical composition according to invention pipette or spray. The compositions may be provided in single may include or may be used or administered in combination or multi-dose form. 40 with one or more additional drugs useful for the treatment, Administration to the respiratory tract may also be prevention or alleviation of Parkinson's disease. Such addi achieved by means of an aerosol formulation in which the tional drugs include L-DODA (optionally in combination active ingredient is provided in a pressurised pack with a with decarboxylase inhibitors (such as carbidopa) or COMT suitable propellant such as a chlorofluorocarbon (CFC) for inhibitors (such as entacapone)), monoamine oxidase inhibi example dichlorodifluoromethane, trichlorofluoromethane, 45 tors, monoamine oxidase B inhibitors (such as selegiline), or dichlorotetrafluoroethane, carbon dioxide, or other suit dopamine agonists (such as bromocriptine, pergolide, cab able gas. The aerosol may conveniently also contain a ergoline, ropinirole, pramipexole, or apomorphine in com Surfactant such as lecithin. The dose of drug may be con bination with domperidone), monoamine reuptake inhibitors trolled by provision of a metered valve. (such as those described in WO 97/16451 (NeuroSearch) Alternatively the active ingredients may be provided in 50 and WO 97/13770 (NeuroSearch), or ALE-26018), dopam the form of a dry powder, for example a powder mix of the ine reuptake inhibitors (such as those described in the compound in a suitable powder base such as lactose, starch, patents U.S. Pat. No. 6,011,070, U.S. Pat. No. 5,821,386, starch derivatives such as hydroxypropylmethyl cellulose U.S. Pat. No. 6,001,330, U.S. Pat. No. 5,795,915, U.S. Pat. and polyvinylpyrrolidone (PVP). Conveniently the powder No. 5,574,060), NA/DA-uptake inhibitors (such as Ven carrier will form a gel in the nasal cavity. The powder 55 lafaxin, Minacipram, Reboxetin), classic tricyclic antide composition may be presented in unit dose form for example pressiva (Such as Imipramin, Amitriptyline, Clomipramine, in capsules or cartridges of, e.g., gelatin, or blister packs Doxepin, Amoxapine, Desipramine, Maprotiline, Nortrip from which the powder may be administered by means of an tyline and Protriptyline), selective dopamine reuptake inhaler. inhibitors (such as GRB-12909, GRB-12935, Indatraline In compositions intended for administration to the respi 60 (Lu-19-005), Bupropion, Amfonelic acid, BTCP, Mazindol, ratory tract, including intranasal compositions, the com Nomifensine, Beta-CFT (WIN 35,428), Beta-CTP (WIN pound will generally have a small particle size for example 35,065-2), Beta-CIT (RTI-55), GYKI 52895, 4',4'-Diflouro of the order of 5 microns or less. Such a particle size may 3-alpha-diphenyl-methoxytropane, 4'-Chloro-3-alpha be obtained by means known in the art, for example by diphenylmethoxytropane, 5-(4-Chlorophenyl)-8-methyl-6, micronization. 65 7,8.9-tetrahydro-1-H-pyrrolo3.2-hisoquinoline-2,3-dione When desired, compositions adapted to give Sustained 3-oxime; and 5-(4-Chlorphenyl)-6,7,8,9-tetrahydro-1-H- release of the active ingredient may be employed. pyrrolo3.2-hlnaphthalene-2,3-dione-3-oxime), or relatively US 7,160,899 B2 10 selective dopamine reuptake inhibitors (such as amineptine, lar Probes, C-7026). Briefly, medium is aspirated, and cells 3,4-dichlorophenyl 4-(3,4-dichlorophenyl)-4-hydroxy-1- are incubated at -80° C. for at least 1 hour. Cells are then methyl-3-piperidyl ketone (Wang, S et al., 1999), 1-2- thawed and incubated in a buffer containing the fluorescent (diphenylmethoxy)ethyl-4-(3-phenylpropyl)-homopipera CyOUANT dye, which exhibits strong fluorescence zine, (LR-1111), 1-2-(diphenylmethoxy)-ethyl4-(3-phenyl enhancement when bound to nucleic acids. Fluorescence 2-propenyl)-homopiperazine, (S)-(-)-1-2 measured with excitation at 480 nm and emission detection (diphenylmethoxy)ethyl-2-N-(3-phenylpropyl)amino af 520 nm can be correlated to the number of living cells in methylpyrrolidine, and (S)-(-)-1-(2-bis(4-fluorophenyl)- the wells. methoxyethyl-2-N-(3-phenylpropyl)aminomethyl Method 3 pyrrolidine). 10 Furthermore, the treatment of the invention may be com Survival of Embryonic Rat Dopaminergic Neurons bined with other known treatments of Parkinson's disease, In this test, the effect of a compound with neurotrophic Such as grafting of dopamine-secreting cells into the stria activity (below: the compound) on the survival of dopam tum or application of neurotrophic growth factors into the inergic neurons in dissociated cultures established from rat lateral ventricle. 15 E14 ventral mesencephali (VM) is assessed. The invention is further illustrated with reference to the following test methods and examples which are not intended Method to be in any way limiting to the scope of the invention as Embryonic rat brains (Wistar, E14) are isolated under claimed. sterile conditions placed in chilled Gey's balanced salt solution (GIBCO) with glucose (6.5 mg/ml). Test Methods The ventral mesencephali are dissected out, cut into Small Method 1 tissue pieces, placed in Neurobasal medium with B27 Supplement and gently pressed through a 80 um Nitex filter. Stimulation of Neurite Outgrowth in PC12 Cells The cells are counted using a hemocytometer and plated in 25 a 6 well multi-dish at a density of approximately 2.0x10° In this test, the ability of a compound with neurotrophic cells/well. Culture dishes are pre-coated with poly-D-lysine. activity (below the compound) to potentiate NGF-induced After 1 hour, the medium is removed and fresh medium neurite outgrowth in PC12 cells is assessed. added with or without the compound to be tested (1.5 Method ml/well). (untreated cultures serve as controls). The medium PC12 cells are seeded in tissue culture plates coated with 30 is changed every other day and antimitotics and antibiotics collagen at a cell density of 15,000/cm in DMEM with are not used at any stage. 7.5% FCS and 7.5% DHS. Next day the medium is changed After 7 days in culture, cultures are immunostained for to medium Supplemented with the compound in the absence tyrosine hydroxylase (TH). Briefly, the cells are washed in or presence of NGF. 0.05M tris-buffered saline (TBS, pH 7.4) containing 1% Two days after the medium change, cells are fixed in 4% 35 Triton X-100 for 3X15 minutes and incubated with 10% paraformaldehyde and stained for neurofilament. Cells are foetal bovine serum (FBS, Life Technologies) in TBS for 30 fixed by in tissue culture plates by incubation in 4% minutes. The cells are then incubated for 24 hours at 4° C. paraformaldehyde in PBS, followed by permeabilization in with monoclonal mouse anti-TH antibody (Boehringer Man 0.05% Triton-X100 in the presence of 10% DHS to block nheim) diluted 1:600 in TBS with 10% FBS. After rinsing in non-specific binding sites. After washing, the plates are 40 TBS with 1% Triton X-100 for 3X15 minutes, cells are incubated with anti-neurofilament (NF) antibody (clone incubated for 60 minutes with biotinylated anti-mouse IgG RT97, Boehringer) diluted 1:200 in 0.05% Triton-X100/ antibody (Amersham) diluted 1:200 in TBS with 10% FBS. 10% DHS followed by incubation with biotinylated anti The cells are then washed in TBS with 1% Triton X-100 mouse immunoglobulin RPN1001 (Amersham) diluted (3x15 minutes) and incubated for 60 minutes with strepta 1:200. NF-immunoreactive cells are stained using the ABC 45 vidine-peroxidase (Dako) diluted 1:200 in TBS with 10% complexHRP kit K0355 (DAKO) and 3.3-diaminobenzidine FBS. After washing in TBS (3x15 minutes), bound antibody tetrahydrochloride (DAB) as substrate. is visualised by treatment with 0.05% 3.3-diaminobenzidine Estimation of total cell number per well, as well as the (Sigma) in TBS containing 0.01% HO. TH-immunoreac total neurite length are done using unbiased 2D stereology tive (ir) cells are counted manually. (CAST-grid system connected to a Olympus BH-2 micro 50 Scope). Method 4 Survival of Dopaminergic Neurons from E28 Pig Ventral Method 2 Mesencephali PC12 Cells Survival Assay In this test, the effect of a compound with neurotrophic In this test, the effect of a compound with neurotrophic 55 activity (below: the compound) on the survival of dopam activity (below: the compound) on the survival of PC12 cells inergic neurons in organotypic slice cultures established is assessed. from pig E28 Ventral mesencephali is assessed. Method Method PC12 cells are seeded in collagen-coated 96 well plates in 60 Ventral mesencephali (VM) are isolated from porcine growth medium supplemented with 2 nM mouse 7S NGF embryos (E28) under sterile conditions, chopped into 400 (Alomone Labs Ltd., Jerusalem, Israel) and cultured for 6 um slices and placed in chilled Gey's balanced salt Solution days. The medium is then changed to serum-free DMEM (GIBCO) with glucose (6.5 mg/ml). The tissue slices are supplemented with the compound. NGF (3 nM) is included cultured by the interface culture method, originally devel as a positive control. After 4 days of incubation, cell viability 65 oped by Stoppini et al. L. Stoppini, P. A. Buchs, D. Muller: is evaluated by using the CyOUANT Cell Proliferation A simple method for organotypic cultures of nervous tissue; assay according to the manufacturers instructions (Molecu J. Neurosci. Methods 1991 37 173–182. US 7,160,899 B2 11 12 In brief, slices are placed on semiporous membranes Phosphorylated CREB is immunodetected by using rabbit (Millipore, 0.3 um; 4 slices/membrane) placed as inserts in anti-Phospho-CREB (Upstate Biotechnology #06-519) fol 6-well plates (CoStar) with serum containing medium lowed by HRP-linked anti-rabbit antibody (Amersham Life (Gibco BRL). Each well contained 1 ml medium (50% Science #NA 934). Bands are detected by chemilumines Optimem, 25% horse serum, 25% Hank's balanced salt cence using the ECL system (Amersham). solution (all GIBCO)) supplemented with D-glucose to a Method 7a final concentration of 25 mM. At day 3, the medium is replaced by defined serum-free In Vitro Inhibition of H-ZM 241385 Binding medium (Neurobasal medium with B27 supplement, Life In this method the ability of an adenosine A receptor Technologies). The cultures are grown in an incubator with 10 antagonist to inhibit the specific binding of the selective and 5% CO at 36° C. for 21 days after which the sections are potent adenosine A receptor antagonist H-ZM 241385 in immunostained for TH as described in Test 2. One group of striatal tissue is assessed. slice cultures are treated chronically with the compound at Tissue Preparation a concentration of 1 uM. Untreated cultures serves as Preparations are performed at 0–4° C. unless otherwise controls. The medium is changed twice a week and antimi 15 indicated. Striatal tissue from male Wistar rats (150–250 g) totics and antibiotics are not used at any stage. is homogenised using an Ultra-Turrax homogeniser for Quantification of TH-ir neurons is performed on coded 10–20 sec in 20 vol. of Tris, HCl (50 mM, pH 7.4). This slides (to allow analysis by experiments “blinded to sample membrane homogenate is then centrifuged at 48,000xg for identity) using an Olympus C.A.S.T. Grid system (version 10 min. The supernatant is discarded and the pellet is 1.10: Olympus, Albertslund, Denmark) composed of an resuspended in buffer containing 2 IU/ml of adenosine Olympus BX50 microscope and a computer controlled x-y-Z deaminase to 10 mg/ml of original tissue weight and incu step motor stage. The area of the culture slice is delineated bated at 37°C. for 30 minto remove endogenous adenosine. and a counting frame is randomly placed to mark the first This membrane homogenate is recentrifuged and the final area to be sampled. The frame is then systematically moved pellet is resuspended in 50 vol. of buffer and frozen at -80° through the sections and the TH-ir cells counted. 25 C. until the time of assay. Method 5 Assay Potentiation of NGF Signal Transduction in PC12 Cells The membrane preparation is thawed and centrifuged at In this test the effect of a compound with neurotrophic 2 C. for 10 min at 27,000xg, and the pellet is resuspended activity (below: the compound) on NGF-induced phospho 30 in 50 mM Tris, HCl, pH 7.4, containing 10 mM MgCl, (500 rylation of the ERKs and the Akt kinase is assessed. ml buffer per g of original tissue), and then used for binding Method assays. Aliquots of 0.5 ml homogenate are added to 0.025 ml Approximately 200,000 PC12 cells are plated in a 24 well of test solution and 0.025 ml of H-ZM241385 (1 nM, final plate in DMEM with 7.5% FCS and 7.5% DHS and incu concentration), mixed and incubated for 30 min at room bated ON. The next day NGF and the compound are added 35 temperature. Non-specific binding is determined using to the cells and they are incubated for 24 hours after which NECA (100 uM, final concentration). After incubation the the cells are harvested in 2x Laemmli sample buffer. samples are poured directly onto Whatman GF/C glass fibre Total cell lysate is electrophoresed on 8–18% gradient filters under suction and immediately washed with 2x5 ml SDS gels which are electroblotted to PVDF membranes. ice-cold buffer. The amount of radioactivity on the filters is Phosphorylated ERK1 and ERK2 are immunodetected by 40 determined by conventional liquid Scintillation counting. using mouse anti-Phospho-p44/p42 MAP kinase E 10 mAb Specific binding is total binding minus non-specific binding. (New England Biolabs #9106) and HRP-linked anti-mouse Results antibody. Phosphorylated Akt kinase is immunodetected by The test value is given as an ICso (the concentration (LM) using rabbit phospho-specific Akt (Ser473) antibody (New of the test substance which inhibits the specific binding of England Biolabs #9271) and HRP-linked anti-rabbit anti 45 H-ZM 241385 by 50%). body. Bands are detected by chemiluminescence using the ECL system (Amersham). Method 7b Method 6 In Vitro Inhibition of H-CGS 21680 Binding 50 In this method the ability of an adenosine A receptor Stimulation of CREB Phosphorylation in Undifferentiated antagonist to inhibit the specific binding of the adenosine PC12 Cells A agonist H-CGS 21680 in striatal tissue is assessed. In this method the effect of a compound with neurotrophic activity (below: the compound) on CREB (cyclic AMP Tissue Preparation responsive element binding protein) phosphorylation is As described in method 7a above. 55 assessed. Assay Method The membrane preparation is thawed and centrifuged at Approximately 7.5x10 PC12 cells per well are plated in 2 C. for 10 min at 27,000xg, and the pellet is resuspended collagen coated 6-well plates in DMEM with 0.75% FCS in 50 mM Tris, HCl, pH 7.4, containing 10 mM MgCl, (200 and 0.75% DHS and incubated for 48 hours. Cells are then 60 ml buffer per g of original tissue), and then used for binding further starved for 2 hours in serum free DMEM before assays. Aliquots of 0.5 ml homogenate are added to 0.025 ml stimulation with the indicated compounds for 5, 10 or 20 of test solution and 0.0250 ml of H-CGS 21680 (5 nM, final minutes. Cells are harvested in 1x heated sample buffer (2% concentration), mixed and incubated for 2 hr at room tem SDS, 400 mM Tris, pH 8.0, 10 mM DTT and 0.25 mM perature. Non-specific binding is determined using NECA NaVO) and the cell lysates are electrophoresed on 8-18% 65 (100 uM, final concentration). After incubation the samples gradient SDS gels, which are electroblotted to PVDF mem are poured directly onto Whatman GF/C glass fibre filters branes. under suction and immediately washed with 2x5 ml ice-cold US 7,160,899 B2 13 14 buffer. The amount of radioactivity on the filters is deter injected into the HPLC system. The concentration of dopam mined by conventional liquid Scintillation counting. Specific ine (DA), dihydroxy phenyl acetic acid (DOPAC), homo binding is total binding minus non-specific binding. vanillic acid (HVA) and 5-hydroxy indolacetic acid (5-HIM) Results are determined by high-performance liquid chromatography 5 with electrochemical detection (HPLC-ED). The column is The test value is given as an ICso (the concentration (LM) a reverse-phase liquid chromatography Catecholamine 3 um of the test substance which inhibits the specific binding of ESA column at 23°C., the mobile phase consisting of 0.055 H-CGS 21680 by 50%). MSodium acetate with 0.1 nMoctanesulfonic acid, 0.01 mM The ICs values of method 7a and 7b are determined from Na EDTA, and 10% methanol pH 3.7 adjusted with glacial the inhibition curve. If a full curve is not available a 25-75% 10 acetic acid). The mobil phase is delivered by a HPLC pump inhibition of specific binding must be obtained, before (ESA) at 0.55 ml/min. Electrochemical detection is accom calculation of an ICso. plished using an amperometric detector (Antec) with a glassy carbon electrode (0.8 V an Ag/AgCl reference) or a 1 coloumetric detector (Choulochem II model ESA; with a Cso = (applied test substance concentration, uM) X 15 high sensitivity analytical cell (5011). (0.4V an Ag/AgCl 2-1 reference). Chromatograrns are recorded by an integrator. (-1) The data are calculated as percent change of the basal concentration, the 100% value being defined as the average of the last 3 pretreatment values for each rat. The mean where C is specific binding in control assays and C is the percentage values are then calculated for each 20 min specific binding in the test assay. (The calculations assume sample for the rats in each group of treatment. normal mass-action kinetics). Method 9 Method 8 Effect of a Compound on Degeneration of Nigral Dopam Effect of a Compound on Extracellular Dopamine Measured 25 inergic Neurons After a 6-OHDA Lesion of the Medial by Microdialysis Forebrain Bundle and the Ventral Tegmental Area In this test, the ability of a compound with neurotrophic In this test, the ability of a compound with neurotrophic activity to increase dopamine in various brain regions is activity to increase the number of Surviving dopamine assessed. neurons in the substantia nigra after a striatal 6-OHDA Male SPF Mol Wistar rats weighing 300–350 g are 30 lesion is assessed. obtained from Mollegaard Breeding and Research Centre FluoroGold (0.2% solution in 0.9% NaCl, 0.2 ul/side) is and housed in standard Macrolon cages sized 24x36x18 cm injected bilaterally in the striatum of halothane anaesthetised for at least 5 days understandard conditions at a temperature female Sprague Dawiey rats weighing approximately of 23+2° C. and a humidity of 60%+10%, and a 12 h light 200–250 g with a 10 ul Hamilton syringe. The following and dark cycle. The rats are housed in groups of two with 35 food and water freely available ad libitum. For microdialy coordinates are used: AP=+1.0 mm, ML=+/-3.0 mm, DV=- sis, the rat is placed in a stereotaxic instrument under 5.0 mm, tooth bar=0.0. After 1 week, 6-OHDA (20 ug free halothane anesthesia using 1/2% halothane, 20% oxygen base dissolved in 0.9% NaCl supplemented with 0.02% and 80% nitrous oxide. The rectal temperature is monitored ascorbic acid) is injected unilaterally in the medial forebrain and maintained at 37.0+1° C. during the experimental period 40 bundle (MFB) and the ventral tegmental area (VTA) with a using a heating pad (CMA 150 Carnegie Medicin). A small glass capillary using the following coordinates: AP -4.4 hole is drilled to allow a vertical probe (CMA/123), to be mm, ML=-1.2 mm, DV=-7.8 mm, tooth bar -2.3 (MFB) Stereotaxically implanted into the right striatum, using the and AP=-4.0 mm, ML=-0.8.mm, DV=-8.0 mm, tooth following coordinates relative to bregma: AP + 1 mm; L 3 bar +3.4 (VTA). mm, DV -6 mm. The probes for the nucleus accumbens 45 Test compound or vehicle is administered i.p. p.o., s.c. or (CMA 122) is implanted vertical at the following coordi i.v. either daily or at specified time points starting after the nates: AP +2.4, L1.4 and DV -8 mm. Similar experiments 6-OHDA injection. Three to four weeks after the 6-OHDA are performed with probes implanted into the nucleus injection, the rats are deeply anaesthetised and transcardially accumbens in non anaesthetised freely moving animals. perfused with 0.9% NaCl for 1 min followed by 4% These experiments are performed 48 h after Surgery during 50 paraformaldehyde in 0.1 M phosphate buffer for 6 min. the daylight period in animals housed individually in plastic Brains are dissected out and postfixed for three to six hours cages with food and water available ad libitum. In all cases, in formalin and then transferred to 25% sucrose in 0.1 M the injection sites are confirmed histologically according to phosphate buffer for 48 hours. Series of 40 um sections are the atlas of Paxinos and Watson. obtained by freezing microtomy through the striatum and the After an initial 2 h period, samples of dialysate are 55 Substantia nigra. Sections are stained for tyrosine hydroxy collected from halothane anaesthetised rats. The dosing of a lase (TH). Surviving dopaminergic neurons in the 6-OHDA test compound to these rats are usually initiated after the lesioned and intact sides are quantified blindly by stereo collection of 3 base line analyses. Dopamine and its metabo logically counting the number of retrogradely labelled neu lites are rapidly frozen to -18°C. and then analyzed as soon rons in the Substantia nigra displaying fluorogold fluores as possible thereafter. The dialysis probe is perfused at a rate 60 cence and by counting the number of neurons displaying TH of 2 ul/min (by a CMA/100 microperfusion pump) with immunoreactivity. Ringer's solution (147 mM NaCl, 4 mM KC1, 2.3 mM CaCl) Method 10 i.e. Ringer's solution (NaCl 4.3 g. KCl 150 mg, CaCl 110.3 mg ad 500 ml) adjusted to pH 6.5 with 2 mM sodium Effect of a Compound on Turning Behaviour After 6-OHDA phosphate buffer. The Ringer solution is filtered before use 65 Lesion through Millipore glass filters (0.22 um). The dialysate In this test, the ability of an adenosine A receptor fractions (40 ul) are collected at 20 min intervals and then antagonist and/or a compound with neurotrophic activity to US 7,160,899 B2 15 16 influence the turning behaviour after a striatal or medial 1–5 days prior to MPTP treatment. The mice are sacrificed forebrain bundle and ventral tegmental area 6-OHDA lesion 48 hr after the MPTP treatment. The effect of the separate is assessed. administration of the neurotrophic compound is examined 6-OHDA (20 ug free base dissolved in 0.9% NaCl supple after daily administration of the compound in various doses mented with 0.02% ascorbic acid) is injected unilaterally in S.c., p.o., i.p. or i.c. v. 1–5 days succeeding MPTP treatment. the striatum or in the medial forebrain bundle and the ventral The mice are sacrificed 48 hr after the last treatment with the tegmental area of halothane anaesthetised female Sprague neurotrophic compound. The effect of the combined admin Dawley rats weighing approximately 200–250 g with a glass istration of the two compounds are examined by adminis capillary. Test compound or vehicle is administered i.p. p.o. tering the adenosine A receptor antagonist daily in various S.c. or i.v. either daily or at specified time points starting 10 doses s.c., p.o., i.p. or i.c.V for 1–5 days prior to MPTP after the 6-OHDA injection. treatment and in the same animals, administering the neu At different time points after the 6-OHDA injection, the rotrophic compound daily in various doses s.c., p.o., i.p. or rotational behaviour of the 6-OHDA lesioned animals after i.c. v. for 1–5 days post MPTP treatment. The mice are administration of amphetamine (2.5 mg/kg i.p.), apomor sacrificed 48 hr after the last treatment with the neurotrophic phine (0.25 mg/kg. s.c.), or L-dopa (2–10 mg/kg i.p.) is 15 compound. The brains are rapidly removed and the striatum monitored in automated rotometer bowls. of the mice are dissected, frozen and stored at -80° C. until biochemical analysis of dopamine and its metabolites HVA Method 11 and DOPAC. On the day of analysis, one striatum per mouse Effect of a Compound on Catalepsy (weighing 5-7 mg) is homogenised in 1 ml of 0.1N per In this test, the ability of an adenosine A receptor chloric acid containing 5% EDTA. After centrifugation antagonist and/or a compound with neurotrophic activity to 14,000xG for 30 min. 200 uL of the supernatant is filtered influence catalepsy induced by haloperidol is assessed. through a glass 0.22 um filter. 20 LL is then injected into the Male wistar rats weighing 200–250 g are housed in cages ESA Coulochem II HPLC equipment with the following of four rats with food and water ad lib and with a 12 hour column (Catecholamine HR-80 4.6 mmx80 mm 3 um light cucle. Test compound or vehicle is administered i.p. 25 Nucleosil C. 18). The eluent is 10.25 g NaH2PO, 185 mg p.o., s.c. or i.v. at specified time points before haloperidole EDTA, 100 mg octansulphonic acid, 9% methanol, pH 3.7, administration (0.1 mg/kg, s.c.). For each dose levels 6 rats add 500 ml MilliO water, filtered through 0.22 um filter. The are tested. Testing for catalepsy is performed at 15 min Colochem ESA analytical cell is 5014A and the ESA detec intervals including 4 tests performed consecutively, in each tor has the following settings: E2–175 mV. run time 16 min. test evaluating the intensity of catalepsy for 10 sec. 30 for the elution of dopamine, DOPAC and HVA (DOPAC: 4.3 1) A vertical wire netting (40x40 cm high). The meshes min; dopamine=6.4 min and HVA=12.7 min). The autoin (openings) of the netting are approximately 1x2 cm. jector SHIMADZY sil-10A has the following settings: injec 2) A horizontal bar 9 cm above the floor tion vol. 20 uL. 16min. analysis, temp. 4°C. Flow rate from 3) A 9 cm high block (bar) the pump is 0.80 ml/min. The analyses are calibrated with 35 standards of 3 pM of dopamine, HVA and DOPAC for each 4) A3 cm high block (cork) 12-analysis run and are compared with the standard curves. The rat is placed in the middle of the vertical wire netting, then on the horizontal bar in an extended position Supporting Method 13 the forelegs on the bar. The intensity of catalepsy is evalu Effect of the Separate or Combined Administration of an ated according to a criterion of 10 sec of total immobility for 40 Adenosine Receptor A. Antagonist and a Compound with a score of 2. Minor movements of the head or the body give Neurotrphic Activity on Degeneration of Nigral Dopamin the score of 1 and a score of 0 is given, if the rat shows no ergic Neurons After Striatal 6-OHDA Lesion syndrome. The rats are then tested after the bar test, whether In this test, the ability of the separate or combined or not they were willing to sit with the left or right foreleg administration of an adenosine Areceptor antagonist and a placed first on the 9 and then on the 3 cm block for a duration 45 compound with neurotrophic activity to increase the number of 10 sec. The maximum score for all 4 tests is thus a total of Surviving dopamine neurons in the Substantia nigra after of 8. a striatal 6-OHDA lesion is assessed. FluorCold (0.2% Method 12 solution in 0.9% NaCl, 0.2 LL/side) is injected bilaterally in the striatum of halothane anaesthetised female Sprague Effect of the Separate or Combined Administration of an 50 Dawley or Wistar rats weighing approximately 200–250 Adenosine Receptor A. Antagonist and a Compound with grams with a 10 uL Hamilton Syringe. The following coor Neurotrophic Activity on Striatal Dopamine in Mice Treated dinates are used: AP: +1.0 mm, ML=+/-3.0 mm, DV=-5.0 With MPTP mm, tooth bar 0.0 mm. After 1 week, 6-OHDA (20 ug free In this test, the ability of the separate or combined base dissolved in 0.9% NaCl supplemented with 0.02% administration of an adenosine A receptor antagonist and 55 ascorbic acid) is injected unilaterally in the striatum with a a compound with neurotrophic activity to increase striatal glass capillary using the following coordinates: APO+1.0 dopamine in mice treated with MPTP is assessed. mm, ML=-3.0 mm, DV=-5.0 mm, toothbar-0.0 mm. The Female C57B1/6J mice weighing 20–25 grams (M&E effect of separate administration of the adenosine A recep breeding centre, Ltd. Ejby, Denmark) are adapted to the tor antagonist is examined after administration of the com laboratory for 5–7 days before the experiments with food 60 pound in various doses s.c., p.o., i.p. or i.c. v. either daily or and water freely available, room temperature 22–24° C. at specified time points prior to the 6-OHDA injection. The Light is on/off at 7 am and 6 pm respectively. At least 5–8 effect of separate administration of the neurotrophic com mice are used per group. MPTP (RBI) is dissolved in saline pound is examined after administration of the compound in just before the experiments and is tested in various doses. various doses s.c., p.o., i.p. or i.c. v. either daily or at The effect of the separate administration of the adenosine 65 specified time points after the 6-OHDA injection. The effect A receptor antagonist is examined after daily administra of the combined administration of the two compounds are tion of the compound in various doses s.c., p.o., i.p. or i.c.V. examined by administering the adenosine A receptor US 7,160,899 B2 17 18 antagonist daily or at specified time points in various doses 2. The method according to claim 1, wherein the com S.c., p.o., i.p. or i.c.V prior to the 6-OHDA injection, and in pound having a neurotrophic activity is GDNF and the the same animals, administering the neuro-trophic com compound having adenosine A antagonist activity is SCH pound daily or at specified time points in various doses s.c., 58.261 or KF-17837. p.o., i.p. or i.c.V. after the 6-OHDA lesion. Three to four 3. A pharmaceutical composition comprising a therapeu weeks after the 6-OHDA injectio, the rats are deeply anaes tically effective amount of at least one compound having a thetised and transcardially perfused with 0.9% NaCl for 1 neurotrophic activity which mimics or enhances the function min. followed by 4% paraformaldehyde in 0.1M phosphate of NGF, BDNF, and/or GDNF and at least one compound buffer for 6 min. having adenosine A receptor antagonist activity, wherein Brains are dissected out and postfixed for three to six 10 the compound having a neurotrophic activity is at least one hours in formalin and then transferred to 25% sucrose in 0.1 compound selected from the group consisting of 5-(4- M phosphate buffer for 48 hours. Series of 40 um sections Chlorophenyl)-8-methyl-6,7,8,9-tetrahydro-1-H-pyrrolo are obtained by freezing microtomy through the striatum and 3.2-hisoquinoline-2,3-dione-3-oxime; 5-(4-chlorophenyl)- the Substanba nigra. Sections are stained for tyrosine 6,7,8,9-tetrahydro-1-H-pyrrolo3.2-hlnaphthalene-2,3- hydroxylase (TH) immuno activity using mouse-anti-TH 15 dione-3-oxime; GDNF: and neublastin; and (Chemicon, #MAB 318). Sections are rinsed in KPBS and pharmaceutically acceptable salts thereof, and the com thereafter quenched using 10% methanol-3% hydrogenper pound having adenosine A receptor antagonist activity is oxide in KPBS. Preincubation for one hour in 2% normal at least one compound selected from the group consisting of horse serum (NHS)+0.3% Triton X-100 in KPBS. Thereaf KW-6002: ZM-241385; 8FB-PTP:SCH-58261; KF-17837; ter, sections are incubated inmouse-anti-TH (Chemicon, CGS-15943; DMPX; and pharmaceutically acceptable salts #MAB 318) 1:2000+2% NHS+0.3% Triton X-100 in KPBS thereof, together with at least one pharmaceutically-accept over night. After rinsing in KPBS, sections are incubated in able carrier or diluent, wherein the therapeutically effective biotinylated horse-anti-mouse (Vector) 1:200 in 0.3% Triton amount of the combination of compounds administered is an in KPBS for 2 hours. After rinsing in KPBS, immunoreativ amount that is effective for treating or alleviating Parkin ity is visualised by the ABC reaction (Vector Kit) followed 25 son's disease. by DAB staining. Surviving dopaminergic neurons in the 4. The pharmaceutical composition of claim 3, for use in 6-OHDA lesioned and intact sides are quantified blindly by the treatment or alleviation of Parkinson's disease in a Stereologically counting the number of retrogradely labelled Subject. neurons in the Substantia nigra displaying fluorogold fluo rescence and by counting the number of neurons displaying 30 5. A combination of at least one compound having a TH immunoreactivity. In some cases, the-degree of neuronal neurotrophic activity which mimics or enhances the function survival is estimated by assigning a score from one to five of NGF, BDNF, and/or GDNF and at least one compound to each section depending on the fraction of Surviving having adenosine A receptor antagonist activity, wherein dopaminergic cells as estimated blindly by observing sec the compound having a neurotrophic activity is at least one tions processed for fluorogold flourescence and/or TH 35 compound selected from the group consisting of 5-(4- immunohistochemistry. The score “1” is assigned to sections chlorophenyl)-8-methyl-6,7,8,9-tetrahydro-1-H-pyrrolo in which all neurons Survive and are morphologically indis 3.2-hisoquinoline-2,3-dione-3-oxime; 5-(4-chlorophenyl)- tinguishable from non-lesioned neurons whereas the score 6,7,8,9-tetrahydro-1-H-pyrrolo3.2-hlnaphthalene-2,3- “5” is assigned to sections in which no neurons Survive in dione-3-oxime; GDNF: and neublastin; and the 6-OHDA lesioned side. 40 pharmaceutically acceptable salts thereof, and the com The invention claimed: pound having adenosine A receptor antagonist activity is 1. A method of treatment or alleviation of Parkinson's at least one compound selected from the group consisting of disease in a Subject, which method comprises administering KW-6002: ZM-241385; 8FB-PTP:SCH-58261; KF-17837; to said Subject a combination of at least one compound CGS-15943; DMPX; and pharmaceutically acceptable salts having a neurotrophic activity which mimics or enhances the 45 thereof, for use as a therapeutic agent. function of NGF, BDNF, and/or GDNF and at least one 6. A kit of parts comprising at least one compound having compound having adenosine A receptor antagonist activ a neurotrophic activity which mimics or enhances the func ity, tion of NGF, BDNF, and/or GDNF and at least one com wherein the compound having a neurotrophic activity is at pound having adenosine A receptor antagonist activity, least one compound selected from the group consisting 50 wherein the compound having a neurotrophic activity is at of 5-(4-Chlorophenyl)-8-methyl-6,7,8,9-tetrahydro-1- least one compound selected from the group consisting of H-pyrrolo3.2-hisoquinoline-2,3-dione-3-oxime; 5-(4- 5-(4-chlorophenyl)-8-methyl-6,7,8,9-tetrahydro-1-H-pyr Chlorophenyl)-6,7,8,9-tetrahydro-1-H-pyrrolo3.2-h rolo3.2-hisoquinoline-2,3-dione-3-oxime; 5-(4-chlorophe naphthalene-2,3-dione-3-oxime; GDNF: and nyl)-6,7,8,9-tetrahydro-1-H-pyrrolo3.2-hlnaphthalene-2,3- neublastin; and pharmaceutically acceptable salts 55 dione-3-oxime; GDNF: and neublastin; and thereof; and the compound having adenosine A pharmaceutically acceptable salts thereof, and the com receptor antagonist activity is at least one compound pound having adenosine A receptor antagonist activity is selected from the group consisting of: KW-6002: at least one compound selected from the group consisting of ZM-241385; 8FB-PTP:SCH-58261; KF-17837; CGS KW-6002: ZM-241385; 8FB-PTP:SCH-58261; KF-17837; 15943; DMPX; and pharmaceutically acceptable salts 60 CGS-15943; DMPX; and pharmaceutically acceptable salts thereof, and thereof. wherein the combination of compounds administered is effective for treating or alleviating Parkinson's disease.