MXPA97009189A - Methods to inhibit the migration of cells of the smooth muscle vascu - Google Patents

Abstract

Methods are provided for inhibiting the migration of vascular smooth muscle cells, which comprises administering to a human or other mammal in need of treatment an effective amount of a compound having the formula (I) wherein R1 and R3 are independently hydrogen. CH3, (a) or (b), wherein Ar is optionally substituted phenyl, R2 is pyrrolidino, hexamethyleneimino or piperidino, and pharmaceutically acceptable salts and solvates thereof.

Description

METHODS TO INHIBIT MASSAGE MUSCLE SMOOTH VASCULAR Deactivation gives the Invention ftnMedmet flffi a Invention Cell migration plays an important role in wound healing, inflammation, in adult respiratory disease syndrome and in the invasion of malignant cancers (Savani et al., J, Clin, Invest. 95: 1158-1168, nineteen ninety five; Kullmann et al. , Am J. Respir. Cell. Mol. Biol. 8: 83-88, 1993; Brooks et al. , Cell: 79, 1157-1164, 1994). Migration of vascular smooth muscle cells from the media to the intima plays a critical role in neointima formation leading to the pathogenesis of vascular diseases such as atherosclerosis, post-PTCA restenosis and atherosclerosis by vein shunting (Jackson et al. ., Arteriesclerosis and Thrombosis 13: 1218-1226, 1993; Brown et al., Cardiovascular Res. 28: 1815-1820, 1995; Bell and Madri, Am. J. Pathol. 137: 7-12, 1990). It has been found that the use of antibodies to growth factors that stimulate the migration of smooth muscle cells or peptides that block cell migration mediated REF: 26043 per integrin inhibit the formation of neointima and animal models of vascular damage (Ferns et al., Science 253: 1129-1132, 1991., Choi et al., J. Vasc. Surg. 19: 125-135, 1994 ). Vascular restenosis after percutaneous transluminal coronary angioplasty (PTCA) has been shown to be a tissue response characterized by an early and a delayed phase. Thrombosis and / or vasospasm may contribute to the early phase occurring from hours to days after PTCA. The late phase seems to be dominated by SMC migration, vascular proliferation and remodeling. In this disease, the increased accumulation of SMC by migration of the intima media contributes significantly to the pathogenesis of the disease. Excessive proliferation and migration of vascular smooth muscle cells may be the main mechanism for coronary artery reocclusion after PTCA, atherectomy, angioplasty. laser and graft surgery for arterial bypass. See "Intimal Proliferation of Smooth Muscle Cells as an Explanation for Recurring Coronary Artery Stenosis after Percutaneous Transluminal Coronary Angioplasty," Austin et al. , Journal of the American College of Cardiology 8: 369-375 (August 1985). Vascular restenosis remains a major long-term complication after coronary artery surgery by percutaneous transluminal coronary angioplasty (PTCA), atherectomy, laser angioplasty, and graft surgery for arterial bypass. In approximately 35% of patients who undergo PTCA, reocclusion occurs within three to six months after the procedure. Current strategies for treating vascular restenosis include mechanical intervention by devices such as stents or pharmacological therapies including heparin, low molecular weight heparin, cuolin, aspirin, fish oil, calcium antagonists, steroids and prostacyclin. These strategies have not eliminated the reocclusion rate and have been ineffective for the treatment and prevention of vascular restenosis. See "Prevention of Restenosis after Percutaneous Transluminal Coronary Angioplasty: The Search for a 'Magic Bullet', "Hermans et al., American Heart Journal 122: 171-187 (July 1991) In the pathogenesis of restenosis there is an excessive proliferation and migration of cells as a result of factors of growth mediated by cellular constituents in the blood and in the wall of the damaged arterial vessel which mediates the proliferation of smooth muscle cells in vascular restenosis Agents that inhibit the migration of smooth muscle cells are useful in the treatment and prevention of The present invention provides the use of compounds as inhibitors for the migration of smooth muscle cells.

BRIEF DESCRIPTION OF THE INVENTION The invention provides a method for inhibiting the migration of vascular smooth muscle cells in a human or other mammalian subject comprising administering to the subject a pharmaceutically effective dose of a compound of the formula wherein R1 and R3 are independently hydrogen, -CH3, or (Alkyl of C-C) 1 6 Ar wherein Ar is optionally substituted phenyl; R2 is pyrrolidino, hexamethyleneimino or piperidino; and pharmaceutically acceptable salts and solvates thereof.

DESCRIPTION PETFTT.T.ftPft OF THE IMVENCIQN The present invention relates to the discovery that a select group of compounds, those of formula I, are useful for inhibiting the migration of vascular smooth muscle cells. The methods of treatment provided by this invention are practiced by administering to the human or other animal in need of a dose of a compound of formula I or II, or a pharmaceutically acceptable salt or solvate thereof, which is effective to inhibit migration of vascular smooth muscle cells. The term "inhibit" is defined to include its generally accepted meaning, which includes prophylactically treating a human subject to prevent migration of smooth muscle cells, and maintaining verification and / or treatment of smooth muscle cell migration. existing. As such, the present method includes both medical therapeutic and prophylactic treatment, as appropriate. Generally, the compound is formulated with common excipients, diluents or carriers, and compressed into tablets or formulated as elixirs or solutions for convenient oral administration, or administered intramuscularly or intravenously. The compounds can be administered transdermally and can be formulated as sustained release dosage forms and the like. The compounds of formula I used in the methods of the present invention can be manufactured according to established procedures, such as those detailed in US Pat. Nos. 4,133,814, 4,418,068 and 4,380,635, all of which are incorporated herein by reference. reference. In general, processes begin with benzo [b] thiophene having a β-hydroxyl group and a 2- (4-hydroxyphenyl) group. The starting compound is protected, alkylated and deprotected to form the compounds of formula I. Examples of the preparation of such compounds are provided in the North American patents described above. The use of the following compounds is included in the invention: (IA) (IB) Substituted phenyl includes phenyl substituted once or twice with C 1 -C 6 alkyl, C 1 -C 4 alkoxy, hydroxy, nitro, chloro, fluoro or tri (chloro or fluoro) methyl. The compounds used in the methods of this invention form pharmaceutically acceptable base acid addition salts with a wide variety of organic and inorganic acids and bases and include the physiologically acceptable salts which are often used in pharmaceutical chemistry. Such salts are also part of this invention. Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric and the like. Salts derived from organic acids, such as monoaliphatic and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkane and hydroxyalkanedioic acids, aromatic acids, aliphatics and aromatic sulfonic acids can also be used. Such pharmaceutically acceptable salts, therefore, include acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, β- hydroxybutyrate, butyl-1,4-dioate, hexin-1,4-dioate, caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate, glycolate, heptanoate, hippurate, lactate, malate, maleate, hydroxyalate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethane sulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2 sulfonate, p-toluenesulfonate, xylene sulfonate, tartrate and the like. A preferred salt is a hydrochloride salt. The pharmaceutically acceptable acid addition salts are typically produced by reacting a compound of formula I with an equimolar or excess amount of acid. The reagents are generally combined in a mutual solvent such as diethyl ether or benzene. The salt is usually separated by precipitation from the solution in about 1 hour to 10 days and can be isolated by filtration, or the solvent can be removed by distillation by conventional means. Bases commonly used for the formation of salts include ammonium hydroxide and alkali metal or alkaline earth metal hydroxide, carbonates and bicarbonates, as well as aliphatic and aromatic amines, aliphatic diamines and hydroxyalkylamines. Bases especially useful in the preparation of addition salts include ammonium hydroxide, potassium carbonate, sodium bicarbonate, calcium hydroxide, methylamine, diethylamine, ethylenediamine, cyclohexylamine and ethanolamine. The pharmaceutically acceptable salts generally have improved solubility characteristics as compared to the compound from which they are derived, and therefore are often more susceptible to formulation as liquids or emulsions. The pharmaceutical formulations can be prepared by methods known in the art. For example, the compounds can be formulated with common excipients, diluents or carriers and can be constituted into tablets, capsules, suspensions, powders and the like. Examples of excipients, diluents and carriers that are suitable for such formulations include the following: fillers and solvents such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethylcellulose and other cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; agents for delaying dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surfactants such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate and solid polyethylene glycols. The compounds can also be formulated as elixirs or solutions for convenient oral administration or as solutions suitable for parenteral administration, for example, intramuscularly, subcutaneously or intravenously. Additionally, the compounds are suitable for formulation as sustained release dosage forms and the like. The formulations may be constituted so as to release the active ingredient uniquely or preferably in a defined part of the intestinal tract, possibly over a period of time. The coatings, envelopes and protective matrices can be made, for example, from polymeric or silk materials. The particular dosage of a compound of formula I required to inhibit the migration of smooth muscle cells according to this invention will depend on the severity of the condition, the route of administration and related factors that will be decided by the attending physician. Generally, effective daily accepted dosages will be between about 0.1 to about 1000 mg / day, and more typically from about 50 to about 200 mg / day. Such dosages will be administered to a subject in need of treatment from once to approximately three times each day, or more frequently as necessary to effectively inhibit the migration of smooth muscle cells. The local delivery of inhibitory amounts of the active compound for the treatment of vascular smooth muscle cell migration or restenosis can be by various techniques with which the compound is administered at or near the affected site. The examples of local supply techniques are not considered as limiting but as illustrative of the available techniques. Examples include local delivery catheters, site specific carriers, implants, direct injection or direct applications. The local supply by a catheter allows the administration of a pharmaceutical agent directly in the affected lesion. Examples of local delivery using a balloon catheter are described in EPO 383 492 A2 and U.S. Patent No. 4,636,195 (January 13, 1987).

Local delivery by an implant describes the surgical placement of a matrix containing the pharmaceutical agent in the lesion. The implanted matrix releases the pharmaceutical agent by diffusion, chemical reaction or solvent activators. Lange, Science 249: 1527-1533 (September 1990). An example of local delivery by an implant is the use of a stent. The stents are designed to mechanically avoid the collapse or reocclusion of the coronary arteries. The incorporation of a pharmaceutical agent in the stent delivers a medication directly to the affected site. Local supply by this technique is described in Kohn, Pharmaceutical Technology (October 1990). Another example is a delivery system in which a polymer containing the pharmaceutical agent is injected into the lesion in liquid form. The polymer is then cured to form an implant in situ. This technique is described in PCT WO 90/03768 (Donn, April 19, 1990). Another example is the delivery of a pharmaceutical agent by the polymeric endoluminal seal. This technique uses a catheter to apply a polymer implant to the inner surface of the lumen. The pharmaceutical agent incorporated in the biodegradable polymer implant in this manner is released at the surgical site. This method is described in PCT WO 90/01969 (Schindler, August 23, 1989). A final example of local delivery by an implant is the direct injection of vesicles or microparticles into the affected site. These microparticles can be constituted by substances such as proteins, lipids, carbohydrates or synthetic polymers. These microparticles have the pharmaceutical agent incorporated through the microparticle or the microparticle as a coating. Delivery systems that incorporate microparticles are described in Lange, Science 249: 1527-1533 (September 1990) and Mathiowitz, et al. , J. App. Poly. Sci. , 26: 809 (1981). Local delivery by site-specific carriers describes the binding of the pharmaceutical agent to a carrier which will direct the drug to the lesion. Examples of the delivery technique include the use of carriers such as a protein ligand or a monoclonal antibody. Lange, Science 249: 1527-1533 (September). Local supply by direct administration includes the use of topical applications. An example of a local delivery by direct application is the application of the pharmaceutical agent directly to the arterial bypass graft during the surgical procedure.

It is usually preferred to administer a compound of formula I in the form of an acid addition salt, as is usual in the administration of pharmaceutical substances having a basic group, such as the piperidino ring. It is also advantageous to administer such a compound orally to an elderly human (e.g., a post-menopausal woman). For these purposes the following oral dosage forms are available.

Foraulacianes In the formulations that follow, "active ingredient" means a compound of formula I.

Formulation 1: Gelatin capsules Hard gelatin capsules are prepared using the following: The ingredients are mixed, passed through a No. 45 mesh North American sieve and filled into hard gelatin capsules. Examples of capsule formulations specific to the compound of formula I in which the compound is raloxifene, include those shown below.

Formulation 2; Raloxifene capsule Formulation; Raloxifene capsule Formulation 4; Raloxifene capsule Formulation 5: Raloxifene Capsule The above specific formulations may change in accordance with the reasonable variations provided. A tablet formulation is prepared using the following ingredients: Formulation 6 Tablets Acid stearate 15 The components are mixed and compressed to form tablets. Alternatively, tablets are made as follows, each containing 0.1 - 1000 mg of active ingredient: Formulation 7: Tablets The active ingredient, starch and cellulose are passed through an American mesh screen No. 45 and mix carefully. The polyvinylpyrrolidone solution is mixed with the resulting powders which are then passed through a No. 14 mesh North American sieve. The granules produced in this way are dried at 50 ° -60 ° C and passed through the a North American sieve of mesh No. 18. Sodium carboxymethyl starch, magnesium stearate and talcum, which are previously passed through a North American sieve No. 60, then add the granules, which, after mixing, compress in a tabletting machine to produce tablets. Suspensions are prepared as indicated below, each containing 0.1 - 1000 mg of medication per 5 ml dose: Formulation 8: Suspensions The medicament is passed through a No. 45 mesh North American sieve and mixed with sodium carboxymethyl cellulose and syrup to form a smooth or regular paste. The benzoic acid solution, flavor and color are diluted with a little water, and added with agitation. Subsequently enough water is added to produce the required volume.

PROOF PROCEDURE The compounds of the invention have the ability to inhibit the migration of vascular smooth cells, as evidenced by the following.

Smooth muscle cells of porcine aorta.

Porcine aorta is obtained from castrated male pigs freshly slaughtered in a local trail. Smooth muscle cells are prepared using a procedure similar to that previously described (Bonin et al., 1989). Briefly, the aorta is cut longitudinally, and the endothelium is removed by gently scraping the surface of the lumen with a razor. The aorta is then washed several times in sterile growth medium consisting of Eagle's medium modified by Dubecco (DMEM), 10% fetal bovine serum, L-glutamine (2 mM), penicillin (100 U / ml) and streptomycin ( 100 μg / ml). The strips of medium smooth muscle cells are then detached from the adventitia and cut into 1 - 2 mm pieces. The explants are placed in 24-well culture vessels containing the above growth medium. The growth of the cells of the explants is observed in the following 5-7 days. After 10-14 days, the explants are removed, the cells are trypsinized and subcultured in T75 flasks containing 15 ml of the growth medium.

Human smooth muscle cells Human coronary and aortic smooth muscle cells are purchased from Clonetics Corporation (San Diego, CA). Both cell types are grown in growth medium as described for porcine smooth muscle cells.

Smooth muscle cell migration assay The directed migration of smooth muscle cells, derived from porcine and human arteries, to a gradient of platelet-derived growth factor is carried out using a modified Boyden chamber using a 96-well piercing system and polycarbonate filter with pores of 8 μm (Neuro Probé, Inc., Cabin John, NJ). Smooth muscle cells growing in T75 flasks are transferred to Dubecco-modified Eagle medium free of phenol / F12 medium (DMEM / F-12), containing 2% fetal bovine serum, L-glutamine (2 mM) , penicillin (100 U / ml) and streptomycin (100 μg / ml). After 24 hours, the cells are trypsinized using phenol-free red / EDTA trypsin (Gibco, BRL). The cells (2.5 x 10"6 cells / ml) are suspended in DMEM / F12 free of phenol red containing 1% platelet poor plasma, and various concentrations of the compounds of formula I. 100 μl of cell suspension is added. The upper wells of the modified Boyden chamber The lower chamber wells are filled with 43 μl of DMEM / F12 containing 1% platelet-poor plasma, 5 ng / ml PDGF and varying concentrations of the compounds. they are incubated at 37 ° C in 5% C02 for 5 hours.The migration membrane is removed from the chamber and the cells on the upper side of the membrane are separated with a cotton swab.The cells migrate to the lower side of the membrane, they are fixed in methanol and stained with Diff-Quick staining solution (Baxter). Cell migration is quantified spectrophotometrically using a microtiter plate reader (ThermoMax, Molecular Dynamics, Inc.) or by counting cells in a 40X high power field (HPF) using an inverted microscope (Nikon, Inc.). For experiments involving the re-incubation of cells with the compound, the drug is placed in the pretreatment medium at the indicated concentrations, in separate flasks, and incubated for 18 hours. The test conditions of the cells in these pretreatment experiments are exactly the same as those used in the experiments described for acute drug effects.

Table I Stimulation of Migration of Smooth Muscle Cells Porcine Aortic (SMC) by Growth Factor Platelet Derivatives (PDGF) Table II Inhibition of Swine Atrial SMC Migration Induced by PDGF by Compound A * and Migration of β-Estradiol SMC (D.O. 650 nm) * Compound A is of the formula I, wherein Rx and R3 are hydrogen and R2 pyrrolidino. The activity in the above indicates that the compounds of the invention have potential in the inhibition of vascular smooth muscle cell migration and its effects. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (4)

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1. Use of a compound having the formula (I) for the manufacture of a medicament for inhibiting the migration of vascular smooth muscle cells comprising administering to a human or other mammal in need of treatment an effective amount of said compound wherein R1 and R3 are independently hydrogen, -CH3, (Alkyl of C - Ar wherein Ar is optionally substituted phenyl; R2 is pyrrolidino, hexamethyleneimino or piperidino; and pharmaceutically acceptable salts and solvates thereof.

2. The use according to claim 1, characterized in that the compound is the hydrochloride salt thereof.

3. The use according to claim 1, characterized in that the compound is or its hydrochloride salt.

4. The use according to claim 1, characterized in that the administration is for inhibition of atherosclerosis, restenosis, inflammation or invasion by malignant cancer cells. THE INVENTION Methods for inhibiting the migration of vascular smooth muscle cells are provided, which comprises administering to a human or other mammal in need of treatment an effective amount of a compound having the formula (I) wherein R x and R 3 are independently hydrogen, -CH3, (a) or (b), wherein Ar is optionally substituted phenyl; R2 is pyrrolidino, hexamethyleneimino or piperidino; and pharmaceutically acceptable salts and solvates thereof.