The present invention relates to new uses for EP4 receptor agonists in the treatment of neuropathic pain.

The EP4 receptor is a 7-transmembrane receptor and its natural ligand is the prostaglandin PGE2. PIGEA also has affinity for the other EP receptors (types EP1, EP2 and EP3). The EP4 receptor is associated with smooth muscle relaxation, inflammatory activities, lymphocyte differentiation, allergic activities, renal regulation and gastric or enteric mucous secretion.

It is believed that selective EP4 receptor agonists exhibit a number of advantages over current non-steroidal anti-inflammatory (NSAID) and cyclo- oxygenase-2 inhibitor (COX-2i) drugs which act via a number of prostaglandin pathways. By selectively activating the EP4 receptor, the adverse consequences of perturbation of other prostaglandin pathways are avoided. The use according to the instant invention therefore provides greater efficacy and improved gastro-intestinal safety over NSAIDs.

The use of EP4 receptor ligands and antagonists, including [4- (4, 9-diethoxy-1- oxo-1, 3-dihydro-2H-benzo [flisoindol-2-yl) phenyl] acetic acid and pharmaceutical acceptable derivatives thereof, in the treatment of neuropathic pain has been disclosed in PCT/EP/00/07669 (Glaxo Group Limited), unpublished at the priority date of the instant application.

Surprisingly, it has now been found that EP4 receptor agonists are of use in the treatment of neuropathic pain.

Neuropathic pain syndromes can develop following neuronal injury and the resulting pain may persist for months or years, even after the original injury has healed. Neuronal injury may occur in the peripheral nerves, dorsal roots, spinal cord or certain regions in the brain. Neuropathic pain syndromes are traditionally classified according to the disease or event that precipitated them.

Neuropathic pain syndromes include : diabetic neuropathy; sciatica; non-specific

lower back pain; multiple sclerosis pain; fibromyalgia ; HIV-related neuropathy; post-herpetic neuralgia ; trigeminal neuralgia; and pain resulting from physical trauma, amputation, cancer, toxins or chronic inflammatory conditions. These conditions are difficult to treat and although several drugs are known to have limited efficacy, complete pain control is rarely achieved. The symptoms of neuropathic pain are heterogeneous and are often described as spontaneous shooting and lancinating pain, or ongoing, burning pain. In addition, there is pain associated with normally non-painful sensations such as"pins and needles" (paraesthesias and dysesthesias), increased sensitivity to touch (hyperesthesia), painful sensation following innocuous stimulation (dynamic, static or thermal allodynia), increased sensitivity to noxious stimuli (thermal, cold, mechanical hyperalgesia), continuing pain sensation after removal of the stimulation (hyperpathia) or an absence of or deficit in selective sensory pathways (hypoalgesia).

The present invention provides the novel use of an EP4 receptor agonist and pharmaceutical acceptable derivatives thereof for the manufacture of a medicament for the treatment of neuropathic pain.

In a further aspect the present invention provides the novel use of an EP4 receptor agonist and pharmaceutical acceptable derivatives thereof for the manufacture of a medicament for the treatment of neuropathic pain, excluding the use of [4- (4, 9-diethoxy-1-oxo-1, 3-dihydro-2H-benzo [f] isoindol-2- yl) phenyl] acetic acid and pharmaceutical acceptable derivatives thereof.

By pharmaceutical acceptable derivative is meant any pharmaceutical acceptable salt or solvate of an EP4 receptor agonist, or any other compound which upon administration to the recipient is capable of providing (directly or indirectly) an EP4 receptor agonist or an active metabolite or residue thereof.

In a further aspect the invention provides a novel method of treating neuropathic pain in a mammal, including man, comprising administration of an effective amount of an EP4 receptor agonist or a pharmaceutical acceptable derivative thereof.

It is to be understood that reference to treatment as used herein includes treatment of established symptoms and prophylactic treatment, unless explicitly stated otherwise.

The ability of the compounds to agonise EP4 receptors may be demonstrated in the (1251] CAMp Scintillation Proximity Assay (hereafter referred to as'the cAMP assay'). The cAMP assay utilises HEK-293 cells expressing the recombinant human EP4 receptor, obtainable from Receptor Biology, Inc. Beltsville, MD, USA. The cells are cultured in Dulbecco's Modified Eagle Medium-HAM F12 mix (DMEM-F12), containing 10% heat inactivated-foetal bovine serum (FBS) and 2mM L-glutamine. The cells are either passaged into fresh medium or used in an assay once 90% confluency as determined visually had been achieved.

The cells are harvested by treatment with Versene, re-suspended in fresh culture medium and plated out to yield approximately 10,000 cells per well of a 96-well plate for overnight culture in culture medium additionally supplemented with 3pM indomethacin. For assay, the culture medium is replaced with assay medium (DMEM-F12 containing 300pM isobutylmethylxanthine (IBMX) and elm indomethacin) and incubated for 30 minutes. Subsequently the cells are challenged with varying concentrations of test compounds or known control agonists for 15 minutes in order to construct agonist concentration-effect curves.

The reaction is stopped by the aspiration of the assay medium and the addition of ice-cold ethanol. All incubations are carried out at 37°C in a 5% carbon dioxide atmosphere. Care should be taken to ensure the constancy of IBMX, indomethacin and vehicle (DMSO) concentrations throughout. The amount of cAMP in each well is then determined by l1251] cAMP scintillation proximity assay using a proprietary kit, obtainable from Amersham, Buckinghamshire, UK, and according to the manufacturer's instructions.

Data from cAMP assays are expressed as pmol cAMP per well. A four- parameter logistic equation of the form: E = ( (Em. [A]) AnH)/((EC50^nH) + ( [A]"nH)) is then fitted to E/ [A] curve data in order to estimate maximum effect (Em), curve mid-point (EC50), and Hill slope (nH); other terms in the equation are effect (E) and concentration ( [A]). Individual estimates of curve parameters are obtained from each curve and then averaged to provide mean data. Quoted values are

therefore the mean standard deviation (s. d.) of n separate experiments, each derived from a separate cAMP assay.

EP4 receptor ligands, including EP4 receptor agonists, have been described in WOOO/03980, WO00/15608, WO00/21542, W099102164, WO01/37877 and Bioorganic and Medicinal Chemistry Letters (2001), 11 (15) pages 2029-2031 and 2033-2035, all incorporated herein by reference. These applications and publications also describe suitable methods for the preparation of EP4 receptor ligands and doses for their administration.

Compounds ONO-AE1-329, ONO-AE1-437 and its prodrug ON04819 are described in WO01/37877 and WO00/03980.

Compounds described in WO00/03980 are 5-thia-co-substituted phenyl- prostaglandin E derivatives of formula (I) wherein each symbol is as defined in the specification, incorporated herein by reference.

Compounds described in WO00/15608 are o-substituted phenyl-prostaglandin E derivatives of formula (IA)

wherein each symbol is as defined in the specification, incorporated herein by reference.

Compounds described in WO00/21542 are prostaglandin Ei, prostaglandin E2, misoprostal, 19-hydroxy prostaglandin E2,9-oxo-8-phenyl-8- (5- phenylpentyl) decanoic acid, 8-acetyl-8-phenyl-13-phenoxytridecanoic acid and the pharmaceutically acceptable salts thereof, and mixtures thereof.

Compounds described in W099/02164 are prostaglandin derivatives of the formula (IB) wherein R is hydrogen, a salt moiety (e. g. alkali or ammonium), a straight or branched, saturated or unsaturated alkyl cahin, preferably with 1-10 carbon atoms, an alicyclic ring, preferably with 3-8 carbons, arylalkyl, preferably-C2-5 alkyly, or an aryl ring; X is a straight saturated or unsaturated alkyl chain, preferably containing 2-5 carbon atoms, optionally interrupted by a heteroatom, selected from oxygen, nitrogen and sulfur, and optionally containing a cycloalkyl, aryl, or a heteroaryl group; R1 and R2, which are identical or different, are hydrogen, hydroxy, halogen, oxygen (keto or alkoxy) or an alkyl group with 1-3 carbons or an ester OCOR3, where R3 is a straight or branched, saturated or unsaturated alkyl group, preferably containing 1-10, especially 1-6 carbons, or a cycloalkyl, preferably containing 3-7 carbons, or an arylalkyl group, especially aryl C2-5 alkyl (e. g. benzyl) ; Z is an alkyl chain of 1-8 carbons, saturated or unsaturated, optionally interrupted by one or more heteroatoms (O, N, S), straight or branched

containing alkyl substituents, or containing an alicyclic ring or an aryl or heteroaryl ring, and containing one or more, preferably one substituent Y, selected form hydroxy, oxygen (keto), hydroxy, sulfhydryl, amino, methylamino, dimethylamino and Ci-satkoxy.

Compounds described in Bioorganic and Medicinal Chemistry Letters (2001), 11 (15) pages 2029-2031 are 3,7-dithiaprostaglandin E1 analogues of the formula (IC) wherein X is as defined in the publication, incorporated herein by reference.

Compounds described in Bioorganic and Medicinal Chemistry Letters (2001), 11 (15) pages 2033-2035 are 5-thiaprostaglandin E1 and 9ß-chloroprostaglandin F2 analogues of the formula (ID) (ID)

wherein W, X, Y and Z are as defined in the publication, incorporated herein by reference. In addition, when W is oxygen, X is CH20Me, Y is H and Z is CH2S, this publication also discloses the methyl ester analogue.

The precise amount of the compounds administered to a host, particularly a human patient, will be the responsibility of the attendant physician. However, the dose employed will depend upon a number of factors including the age and

sex of the patient, the precise condition being treated and its severity, and the route of administration.

In general, the compounds for use in the invention may be administered orally at a dose of from 0.005 to 10 mg/kg body weight per day, such as from 0.01 to 5 mg/kg body weight per day, more particularly from 0.1 to 3 mg/kg body weight per day, calculated as the free base, which may be administered in single or divided doses, for example one to four doses.

The compounds and their pharmaceutically acceptable derivatives are conveniently administered in the form of pharmaceutical compositions. Such compositions may conveniently be presented for use in conventional manner in admixture with one or more physiologically acceptable carriers or excipients.

Thus, in another aspect of the invention, we provide a pharmaceutical composition comprising an EP4 receptor agonist or a pharmaceutically acceptable derivative thereof for use in the treatment of neuropathic pain.

While it is possible for the compounds to be administered as the raw chemical, it is preferable to present it as a pharmaceutical formulation. The formulations comprise the compounds together with one or more acceptable carriers or diluents therefor and optionally other therapeutic ingredients. The carrier (s) must be"acceptable"in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The formulations include those suitable for oral, parenteral (including subcutaneous e. g. by injection or by depot tablet, intradermal, intrathecal, intramuscular e. g. by depot and intravenous), rectal and topical (including dermal, buccal and sublingual) administration although the most suitable route may depend upon for example the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the compounds ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into

association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Formulations suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets (e. g. chewable tablets in particular for paediatric administration) each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in- oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of a sterile liquid carrier, for example, water-for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter, hard fat or polyethylene glycol.

Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.

The compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

In addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

The EP4 receptor agonist compounds for use in the instant invention may be used in combination with other therapeutic agents, for example COX-2 inhibitors, such as celecoxib, rofecoxib, valdecoxib or parecoxib; 5-lipoxygenase inhibitors; NSAID's, such as diclofenac, indomethacin, nabumetone or ibuprofen; leukotriene receptor antagonists; DMARD's such as methotrexate; adenosine A1 receptor agonists; sodium channel blockers, such as lamotrigine ; NMDA receptor modulators, such as glycine receptor antagonists; gabapentin and related compounds; tricyclic antidepressants such as amitriptyline ; neurone stabilising antiepileptic drugs; mono-aminergic uptake inhibitors such as venlafaxine ; opioid analgesic ; local anaesthetics; and 5HT1 agonists, such as triptans, for example sumatriptan, naratriptan, zolmitriptan, eletriptan, frovatriptan, almotriptan or rizatriptan. When the compounds are used in combination with other therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route.

The invention thus provides, in a further aspect, the use of a combination comprising an EP4 receptor agonist with one or more further therapeutic agents in the treatment of neuropathic pain.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutical acceptable carrier or excipient comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

When an EP4 receptor agonist is used in combination with a second therapeutic agent active against the same disease, the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

Reference Example 1 Testing of [4- (4, 9-diethoxy-1-oxo-1, 3-dihydro-2H-benzo [f] isoindol-2- yl) phenyl] acetic acid in the [125cAMP Scintillation Proximity Assay hereinbefore described showed the molecule to be an EP4 receptor agonist with a pEC50 of 7.7 0.2.

The chronic constriction injury (CCI) model was used to induce neuropathic hypersensitivity (Bennett & Xie, 1988) in mate random hooded rats. Under isoflurane anaesthesia, the common left sciatic nerve was exposed at mid thigh level and four loose ligatures of Chromic gut tied around it. The wound was then closed and secured using suture clips. The surgical procedure was identical for the sham operated animals except the sciatic nerve was not ligated. The rats were allowed a period of seven days to recover from the surgery before behavioural testing began.

[4- (4, 9-Diethoxy-1-oxo-1, 3-dihydro-2H-benzo [fjisoindol-2-yf) phenyllacetic acid (10mgkg-1 b. i. d. PO) was dosed chronically for 14 days (days 20-33 post- operative). A reversal of the CCI-induced decrease in paw withdrawal threshold became apparent following 3 days of chronic dosing which was maximal after 1 week. This reversal was maintained throughout the remainder of the dosing period. Following cessation of the drug treatment the paw withdrawal threshold returned to that of the vehicle treated CCI-operated animals.