FR2919184A1 - Use of 5-(4-chlorophennyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxylic acid as a medicament for treating e.g. chronic pain, neuropathic pain, inflammation, benign hypertrophy of the prostate and diabetic neuropathy - Google Patents

Abstract

Use of 5-(4-chlorophennyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxylic acid (I) or its esters, salts, or solvates, as a medicament, is claimed. ACTIVITY : Analgesic; Antiinflammatory; Neuroprotective; Nephrotropic; Antiasthmatic; Antiallergic; Gastrointestinal-Gen. MECHANISM OF ACTION : None given.

Description

The acid

  5- (4-chlorophenyl) -1- (2,4-dichlorophenyl) -4-methylpyrazole-3-carboxylic acid, its esters, pharmaceutically acceptable salts and solvates for use as a medicament.

  The subject of the present invention is 5- (4-chlorophenyl) -1- (2,4-dichlorophenyl) -4-methylpyrazole-3-carboxylic acid, its pharmaceutically acceptable salts and their solvates as well as its esters, in particular with a C1-C6 alkyl or benzyl, for use as a medicament. The subject of the invention is also the pharmaceutical compositions containing 5- (4-chlorophenyl) -1- (2,4-dichlorophenyl) -4-methylpyrazole-3-carboxylic acid, one of its esters, the a pharmaceutically acceptable salt or a solvate thereof as an active ingredient in combination with a pharmaceutically acceptable carrier. Finally, the subject of the invention is also the use of 5- (4-chlorophenyl) -1- (2,4-dichlorophenyl) -4-methylpyrazole-3-carboxylic acid, its esters and its pharmaceutically acceptable salts. and solvates thereof for the preparation of medicaments useful for the treatment of pain, inflammation and benign prostatic hypertrophy. 5- (4-Chlorophenyl) -1- (2,4-dichlorophenyl) -4-methylpyrazole-3-carboxylic acid, hereinafter referred to as compound A, is described in patent EP 656 354. It has now been found that this The compound has pharmacological activity in various therapeutic areas and can be used as a medicament for the treatment of various conditions, particularly for the treatment of pain, inflammation and benign prostatic hypertrophy. More particularly, according to the present invention, compound A, its esters, its pharmaceutically acceptable salts and / or their solvates are useful in the treatment of chronic pain, neuropathic pain, neuropathies including diabetic neuropathy, inflammation, including bronchopulmonary asthmatic inflammation or bronchopulmonary allergic inflammation, irritable bowel syndrome, Crohn's disease, inflammation of the intestines.

  The effects of compound A have been studied in animals in the following experimental models. A - in mice on a model of allergic asthma induced by ovalbumin according to the protocol of K, ps et al., (EurRespirJ 2003, 22, 374-382). Sensitization by intraperitoneal injection of an allergen, ovalbumin, in the presence of alum as an adjuvant recruits and activates subpopulations of T lymphocytes, CD4 + and some Th2, which activate B cells to produce IgE immunoglobulins specific for ovalbumin. These cells also play a memory role. In the second exposure or stimulation, ovalbumin activates mast cells by binding to specific IgE and reactivates T lymphocytes, particularly CD4 + and Th2. T cell and eosinophil infiltration occurs in bronchopulmonary tissues followed by release of mediators that result in tissue damage. This infiltration of inflammatory cells is the consequence of the activation of mononuclear cells (mast cells and macrophages which constitute the first defense barrier at the level of the bronchial epithelium and T lymphocytes) stimulated by antigenic activation during stimulation by ovalbumin. This type of allergic model in mice has made it possible to understand the key roles played by CD4 + T cells and by Th2 and cytokines released by these cells (Kumar & Foster, Am J Resp Ce // Mo / Bi% 27, 267 -272, 2002). B ù in neuropathic rats in which a chronic constriction injury (hereinafter referred to as CCI) was induced according to the protocol described by Bennet et al. Bread, 33 (1988) 87-107. C - on the spinal dorsal ganglia with roots of neuropathic rats in which a chronic constriction lesion (hereinafter referred to as CCI) was induced according to the protocol indicated above, using the protocol of C. Sommer et al. Journal of Neuropathology and Experimental Neurology and R. Lindsay et al. Development Biology, Vol. 112, 30-48 (1985). D - in rats in which a benign prostatic hypertrophy was induced by testosterone (Van Copenol% F. et al., Am J Physio% Endocrino% Metab 280: E120-E129, 2001; Fujimoto N. et al., Cancer Sci: 95: 711-715, 2004). According to another of its aspects, the present invention relates to pharmaceutical compositions comprising, as active ingredient, the compound A one of its esters or a pharmaceutically acceptable salt or a solvate thereof in combination with a pharmaceutically acceptable vehicle. Compound A, its esters, its pharmaceutically acceptable salts or their solvates may be used at daily doses of 0.01 to 100 mg per kilogram of body weight of the mammal to be treated, preferably at daily doses of 0.1 to 50 mg / kg. In humans, the dose may preferably vary from 0.5 to 4000 mg per day, more particularly from 2.5 to 1000 mg depending on the age of the subject to be treated or the type of treatment: prophylactic or curative. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active ingredient can be administered in unit dosage form, in admixture with conventional pharmaceutical carriers. , animals and humans. Suitable unit dosage forms include oral forms, such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual and oral forms of administration, aerosols, implants, forms of subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and forms of rectal administration. In the pharmaceutical compositions of the present invention, the active principle is generally in dosage units containing from 0.5 to 1000 mg, advantageously from 1 to 500 mg, preferably from 2 to 200 mg of said active ingredient per dosage unit for daily administrations. When a solid composition in the form of a tablet is prepared, a wetting agent such as sodium lauryl sulphate may be added to the active ingredient, whether it is micronized or not, and the mixture is mixed with a pharmaceutical vehicle such as silica, starch, lactose, magnesium stearate, talc or the like. The sucrose tablets, various polymers or other suitable materials may be coated or treated in such a way that they have prolonged or delayed activity and continuously release a predetermined amount of active ingredient. A preparation in capsules is obtained by mixing the active ingredient with a diluent such as a glycol or a glycerol ester and incorporating the mixture obtained into soft or hard gelatin capsules.

  A preparation in syrup or elixir form may contain the active ingredient together with a sweetener, preferably calorie-free, methylparaben and propylparaben as antiseptic, as well as a flavoring agent and a suitable colorant.

  The water-dispersible powders or granules may contain the active ingredient in admixture with dispersing agents, wetting agents, or suspending agents, such as polyvinylpyrrolidone, as well as with sweeteners or scavengers. taste. For rectal administration, suppositories are used which are prepared with binders melting at the rectal temperature, for example, cocoa butter or polyethylene glycols. For parenteral administration, aqueous suspensions, isotonic solutions or sterile and injectable solutions which contain dispersing agents and / or pharmaceutically compatible solubilizing agents, for example propylene glycol or polyethylene glycol, are used. Thus, to prepare an aqueous injectable solution intravenously, a cosolvent can be used: an alcohol such as ethanol, a glycol such as polyethylene glycol or propylene glycol and a hydrophilic surfactant such as Tween 80. To prepare an oily solution injectable intramuscularly, the active ingredient can be solubilized by a triglyceride or a glycerol ester. For transdermal administration, patches in multilaminated form or reservoirs in which the active ingredient is in alcoholic solution can be used. The active ingredient may also be formulated in the form of microcapsules or microspheres, optionally with one or more supports or additives.

  Among the sustained-release forms useful in the case of chronic treatment, the implants can be used. These can be prepared as an oily suspension or as a suspension of microspheres in an isotonic medium. The present invention will now be described in more detail by the experimental tests hereinafter. TEST 1: Effect of compound A on cell migration in the bronchoalveolar space after stimulation with ovalbumin.

  Balb / c mice weighing between 28 and 30 g (Charles River) were sensitized on day 1 and day 11 by intraperitoneal injection of a 0.05% solution of ovalbumin (in a 3% AI gel ( OH) 3) in a volume of 0.2 ml / animal.

  At day 19, the mice were stimulated by intratracheal exposure of 10 μl of a 1% solution of ovalbumin (dissolved in sterile isotonic water). 24 hours after ovalbumin injection, the animals were anesthetized with pentobarbital and bronchoalveolar lavage was performed. The washing fluids were recovered, centrifuged and the cells resuspended. The number of cells was counted by differentiating, according to standard morphological criteria, eosinophilic, neutrophilic and mononuclear cells. Intratracheal stimulation with ovalbumin induced a significant increase in the number of mononuclear and eosinophilic cells in the bronchoalveolar space of the mice: compared to the control group, respectively from 26800 to 47700 and from 480 to 11500 cells. The effect of compound A treatment on the ovalbumin-induced recruitment of these cells was studied. Compound A or vehicle (water / Tween 80) was administered to animals 1 hour prior to ovalbumin stimulation at doses ranging from 0.1 to 10 mg / kg / i.p. in a volume of 0.1 ml / 10 g of weight. The results obtained are summarized in Table 1 below.

  Table I: Percent inhibition Rate Total number of eosinophils Number of cells n mg / kg cells Mean and mononuclear deflection Mean and standard deviation Mean and standard deviation standard 0.1 mg / kg 2 18 11 17 0.15 23 6 0.3 mg / kg 35 + 9 19 26 45 7 4 i mg / kg 53 9 31 16 64 10 4 img / kg 64 16 43 17 77 18 9 10 mg / kg 99 18 62 + 14 116 + 21 10 L inhibition of cell migration was greater on mononuclear cells than on eosinophils; thus, at 10 mg / kg, the inhibition of mononuclear migration was complete whereas that of eosinophils reached only 62%.

  The effective dose (ED50) that inhibits the migration of inflammatory cells (total number) was 0.9 mg / kg. In intraperitoneal administration one hour prior to stimulation with an allergenic agent, compound A thus inhibits the migration of inflammatory cells in a dose-dependent manner 24 hours later, the mononuclear cells being more significantly inhibited than eosinophils. Mononuclear cells have not been characterized. Conventionally, the mononuclear cells in the bronchoalveolar fluid of the control mice are predominantly macrophagic while the cells that migrate after stimulation are presumably predominantly lymphocyte but have not been differentiated by flow cytofluorometry. TEST 2: Effect of compound A on the nociceptive response in neuropathic rats.

  The sciatic nerve of each rat was isolated and, near the sciatic nerve trifurcation, four loose ligatures were made around the nerve. The rats thus treated are hereinafter referred to as rats with CCI. Rats in which the sciatic nerve was isolated, but without ligation, were used as control animals (control rats).

  Compound A was administered orally daily and for 8 days at a dose of 30 mg / kg from the 7th day after surgery (ligation) according to the following procedure: - Compound A was dissolved in the vehicle consisting of distilled water (pH 7.5) containing 5% DMSO (dimethylsulfoxide) and 5% Tween 80 and administered by gavage at a rate of 2 ml / kg of body weight. Thermal hyperalgesia and the response of mechanical allodynia were recorded before surgery, then on day 7 and day 14 (24 hours after 7th) after surgery and finally on day 14 (2 hours after the last administration of Compound A.) Thermal hyperalgesia was evaluated by recording the withdrawal latencies of the flaps at a radiation thermal stimulation according to the protocol or plantar test described by Hargreaves et al. (1988) 77-88 using the analgesia meter marketed by the Italian company Ugo Basile, Varese, Italy.Mechanical allodynia was determined according to the method described by Wied et al (The Journal of Pharmaco % ogy and Exper / mental Therapeutics, 306: 804-814,2003) using the instrument for measuring the response to mechanical stimulation (Plantar Aesthesio meter) marketed by the Italian company Ug o Basile The results obtained are collated in Tables 2 and 3 below.

  Table 2: Effect of Compound A on Thermal Hyperalgesia in CCI and Control Rats Latency (s) Day after 0 7 14 14 Intervention 24 h after 7 th - 2 h after the 8 th surgical administration of A Control Rats 10.6 0.8 11.6 0.30 10.4 0.7 10.7 0.4 + Vehicle Control Rats 9.4 0.5 10.6 0.6 10.5 0.2 10 , 3 + 0.2 + Compound A Rats with CCI 9.6 0.3 5.4 0.1 0 5.4 0.2 5.5 0.3 + Vehicle Rats with CCI 10.1 0.3 4, 9 0.300 6.9 0.5 * "7.9 0.4 **" + compounds A 0/0 of 27.7 8.0 48.5 7.2 recovery from controls The results are averages AND for 5 to 8 rats. * P <0.05, ** p <0.001 compared to CCI + vehicle rats, p <0.001 compared to controls (Anovarep 2 passages plus Newman-Keuls test).

  TABLE 3 Effect of Compound A on Mechanistic Mechanism in CCI and Control Rats Mechanical Threshold (g) Day after 0 7 14 14 Intervention 24h after 7th day 2h after 8th Surgical administration of A Administration of A Control rats 37.6 + 2.5 33.7 1, 6 37.4 0.9 38.2 1.5 + Vehicle Control rats 37.3 2.7 36.7 1.2 37, 3 2.7 36.7 2.1 + Compound A Rats with CCI 37.5 2.2 10.6 0.5 O 11.5 0.3 12.1 0.4 + Vehicle ù Rats with CCI 38 , 1 2.6 11 0.700 16.9 + 0.8 "15.4 + 1.0 **" + Compound A% of 20.2 2.9 14.1 3.7 recovery from controls The results are + AND means for 5 to 8 rats. * p <0.05, ** p <0.001 compared to CCI + vehicle rats, p <0.001 compared to controls (Anovarep 2 passages plus Newman-Keuls test).

  TEST 3: Effect of Compound on Inflammation Back L4, L5 and L6 dorsal root ganglia with attached roots (DRG) were removed by micro-dissection in naïve rats (controls without CCI) and rats rendered neuropathic (rats with CCI) according to the protocol described in Example 2 above after anesthesia with pentobarbital sodium (100 mg / kg body weight ip). In both cases, the rats were adult Charles River Cree: CD BR rats weighing 400-500 g. The removed ganglia were incubated in DULBECCO medium-modified EAGLE medium (DMEM medium) containing 0.5 mg / ml of trypsin, 1 mg / ml of collagenase and 0.1 mg / ml of DNAase for 90 minutes. C. A soy trypsin inhibitor was then added to neutralize the trypsin and the resulting suspension was centrifuged at 1000 g for one minute. The neurons contained in the centrifuge cake were suspended in DMEM medium containing 10% calf serum, 10% horse serum, 2 mM glutamine, 0.1 mg / ml penicillin / streptomycin and 2, 5 mg / ml of DNAase and distributed in Petri dishes (diameter 35 mm) coated with Matrigel. The neurons were then incubated at 37 ° C. in the presence of 95% CO 2 and 5% O 2 in a base medium under different experimental conditions: in the presence of compound A (0.1 μM and 1 NM); in the presence of nerve growth factor (NGF) (1 ng / ml and 50 ng / ml); - in the absence of any agent (control rats). After incubation for 48 h under the above conditions, the cultured neurons were fixed in 4% paraformaldehyde for 5-10 minutes, washed with phosphate buffer (PBS) and immediately incubated with blocking buffer ( 1% Tween 20 + 1.5% lean milk powder in PBS at pH 4) for 30 minutes. The cultures were then incubated overnight at 4 ° C. with a first antibody solution (mouse monoclonal antibodies anti-neurofilament H and M sold under the name Chemicon by CHEMICON International Inc., California, USA) in the US. blocking buffer. The next day cultures were washed and incubated with a second universal peroxidase-conjugated antibody (according to the ABC technique using an avidin-biotin enzyme complex with the kit sold under the name Vectastain by VECTOR Laboratories in Burlingame, California. USA) and stained with a solution of 3,3'-diaminobenzidine (DAB). The neurons were observed under a ZEISS microscope and pictures were taken with a Canon Power Shot G (4.0 megapixel) digital camera. The DRG explants were measured on each photograph in a random manner using SCION Image NIH software (National Institute of Health, Bethesda, Maryland USA). A minimum of 15 separate DRG explants were measured for each box. In each case, the maximum growth of radial neurites was measured and converted to metric units (elongation) as compared to a calibration grid established under the same experimental conditions. The NGF-induced DRF elongation of neurites (expressed in {gym) under the different experimental conditions is shown in FIG. 1: control rats (without CCI); control rats (without CCI) + NGF at 20 ng / ml; rats with CCI; rats with CCI + NGF at 20 ng / ml; - at day 0 (t = 0 days of the creation of the chronic lesion by constriction); - at day 3 and day 7 (t = 3 and t = 7, respectively the 3rd and 7th days after the creation of the chronic lesion by constriction). In a deleterious condition, the exogenous NGF causes an increase in DRG neurite elongation that is maximal on the 3rd day after the lesion. On day 7, the exogenous NGF is unable to induce a further increase in DRG neurite elongation, probably due to activation of Schwann cells (regeneration process). The percent neurite elongation was then measured after administration of Compound A at the concentration of 10-6 M or 10 "M. The percentages of effect are calculated with respect to the effect due to NGF. summarized in Table 4 below.

  Table 4: Elongation of neurites NEURITES COMPOUND COMPOUND AT ELONGATION 10- 'M 10-6 M effect compared to the maximum effect due 52 + 10% to NGF 45 + 17% ù ** (no neuropathy) effect compared at maximum effect due 34 5% 45 5 NGF * (neuropathy - rats with CCI) * p <0.05, ** p <0.01 ANOVA Duncan test / controls n = 3-5

  TEST 4: Effect of Compound A on Benign Prostatic Hypertrophy Benign Prostatic Hypertrophy (BPH) was induced in 10 male CD rats by administration of 18.75 mg testosterone per day subcutaneously for 24 days.

  Compound A was then administered orally in combination with 10% NaOH + 5% Tween 80 + 1N HCl 9.8% + H2O as carrier (5 or 10 mg of compound A per ml of vehicle) at 10 or 30 mg / kg body weight and daily for 24 days. The volume and weight of the total prostate and ventral prostate were then measured. The results obtained are shown in FIGS. 2a and 2b (weight) and FIGS. 3a and 3b (volume) as well as those obtained with male control rats in which a benign prostatic hypertrophy was provoked according to the above procedure. but to which only the vehicle has been administered.

  TEST 5: Isometric measurement The following ex vivo test was performed on the ventral lobe of the prostate of rats in which benign hypertrophy was induced according to the procedure described in test 4.

  The prostate was rapidly excised in male rats CrI: CD Charles River) weighing 300-350 g and placed in Krebs bicarbonate buffer (118.4 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 1.2 mM KH 2 PO 4, 25 mM NaHCO 3, 11.7 mM glucose). The prostate was cleared of surrounding tissue and the ventral portion was isolated and cut into two lobes. Each lobe was mounted longitudinally in a 20 ml isolated organ chamber for recording the isometric tension, put into Krebs bicarbonate buffer at 37 C, oxygenated with 95% O2 - 5% CO2 and stretched to at a passive voltage of 250 mg. After an equilibration period during which the lobe was washed with Krebs buffer as defined above 4 times for 10 minutes, the norepinephrine response curve (1 nM -0.1 mM) was obtained in the presence 0.5 μM DMI (desmethylimipramine, Ciba) and 30 μM hydrocortisone hemisuccinate (Flebocortid (D, Lepetit) .Isometric force changes were recorded using the PowerLab data acquisition system (AD Instruments Pty Ltd, Castle Hill, Australia).

  The generation of isometric force was measured as the percentage of contraction induced by 40 mM KCI (100% maximal tissue contraction) added at the end of the experiment after the highest concentration of norepinephrine.

  The results are expressed in EC50 (concentration producing 50% of the maximum effect). The norepinephrine response curves are shown in Figure 4.

  In rats with benign testosterone-induced hypertension (control + BPH rats), the norepinephrine response curve showed a shift to the right compared with the control rats. This shift is indicative of a decrease in tissue contraction capacity. Compound A administered at 10 or 30 mg / kg body weight showed a normal contraction capacity to norepinephrine. Compound A thus restores the contractibility of the tested prostate sample.

Claims (8)

1. Use of 5- (4-chlorophenyl) -1- (2,4-dichlorophenyl) -4-methylpyrazole-3-carboxylic acid, its esters, pharmaceutically acceptable salts and solvates thereof as a medicament.   2. Pharmaceutical composition containing 5- (4-chlorophenyl) -1- (2,4-dichlorophenyl) -4-methylpyrazole-3-carboxylic acid, its esters, its pharmaceutically acceptable salts and their solvates as active ingredient in combination with an acceptable pharmaceutical vehicle.   3. Pharmaceutical composition according to claim 2 containing 0.5 to 1000 mg of active ingredient. 4. Use of the acid   5- (4-chlorophenyl) -1- (2,4-dichlorophenyl) -4-methylpyrazole-3-carboxylic acid, its esters, pharmaceutically acceptable salts and solvates for the preparation of medicaments useful for the treatment of pain. 5. Use according to claim 4 for the preparation of medicaments useful for the treatment of chronic pain.   6. Use according to claim 4 for the preparation of medicaments useful for the treatment of neuropathic pain. 7. Use of 5- (4-chlorophenyl) -1- (2,4-dichlorophenyl) -4-methylpyrazole-3-carboxylic acid, its esters, pharmaceutically acceptable salts and solvates for the preparation of medicaments useful for the treatment of inflammation. 8. Use of 5- (4-chlorophenyl) -1- (2,4-dichlorophenyl) -4-methylpyrazole-3-carboxylic acid, its esters, pharmaceutically acceptable salts and solvates for the preparation of medicaments useful for treatment of benign prostatic hypertrophy.