MXPA97006072A - Methods of inhibition of cell-cell adhesion - Google Patents

Abstract

The present invention relates to a method for the inhibition of cell-cell adhesion, comprising administering to a human in need thereof, 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 selected from the group consisting of pyrrolidine, hexamethyleneimino and piperidino, or a pharmaceutically acceptable salt or solvate thereof.

Description

METHODS OF INHIBITION OF CELL-CELL ADHESION DESCRIPTION OF THE INVENTION The vascular endothelium constitutes a main organ that functions as a regulator of blood coagulation, inflammation and exchange of fluids and mediators between the intravascular compartment and the parenchymal tissues. As such, the proper function of the endothelium is critical for complete homeostasis. A poor function of the endothelium results from an alteration in the expression of important surface molecules, which can result in defects in coagulation, local and systemic vascular inflammation, and increased progression and rupture of the atherosclerotic plaque. These effects may also result in conditions that include myocardial infarction, deep vein thrombosis, disseminated intravascular thrombosis, and shock. Certain cell surface proteins are altered in response to vascular damage or destruction, and can be used as markers of a dysfunctional endothelium. A critical class of such proteins is the receptors / ligands that mediate cell-cell adhesion, including integrins, selectins (for example ELAM) and members of the immunoglobulin superfamily, such as ICAM and VCAM. These molecules are increased in response to a variety of stimuli including cytokines, REF: 25225 and besides being important markers of a dysfunctional endothelium, they play an important role in the thrombotic, inflammatory and atherogenic process in the vascular wall. Other activities, such as superficial anticoagulant responses, are also impaired in states of endothelial dysfunction. A compound that could block endothelial dysfunction, as determined by measuring its ability to inhibit cell-cell adhesion or the expression of procoagulant activities, could be useful in the treatment of conditions such as sepsis, damage involving the damage and trauma to the major tissues, systemic inflammatory response syndrome, sepsis syndrome, septic shock and multiple organ dysfunction syndrome (including DIC) as well as rupture of the atherosclerotic plaque and its associated sequelae. Because cell-cell adhesion is a fundamental process of broad biological importance, the ability to specifically modulate adhesive proteins has the potential for many clinical applications outside vascular tissue, including its use as an anti-inflammatory agent. This invention provides methods for inhibiting cell-cell adhesion, comprising administering to a human in need thereof, an effective amount of a compound of formula I (I) wherein R "and R are independently 0 I! hydrogen, -CH3, -C- (alkyl of 1 to 6 carbon atoms), or -C-Ar, wherein Ar is optionally substituted phenyl; R2 is selected from group consisting of pyrrolidino, hexamethyleneimino, and piperidino, and pharmaceutically acceptable salts and solvates thereof The present invention relates to the discovery that a select group of 2-phenyl-3-aroylbenzothiophenes (benzothiophenes), those of the formula, I, are useful for the inhibition of cell-cell adhesion and particularly, cell-cell vascular adhesion. Also, the compounds are useful for inhibiting vascular endothelial dysfunction.

The methods of use provided by this invention are practiced by administering to a human in need thereof, a dose of the compound of formula I or a pharmaceutically acceptable salt or solvate thereof, which is effective to inhibit cell-cell adhesion. or its effects. The term "inhibit" includes its generally accepted meaning, which includes prohibition, prevention, restriction, and decrease, detention or reversal. As such, the present method includes medical therapeutic and / or prophylactic administration, as appropriate. Raloxifene, a compound of this invention, wherein this is the hydrochloride salt of a compound of formula 1, R1 and R3 are hydrogen and R2 is 1-piperidinyl, is a nuclear regulatory molecule. Raloxifene has been shown to bind to the estrogen receptor, and was originally thought to be a molecule whose function and pharmacology was that of an antiestrogen, and which blocked the ability of estrogen to activate uterine tissue, and dependent breast cancers of estrogen. Of course, raloxifene does block the estrogen action of some cells, but in other cell types raloxifene activates the same genes as estrogen, and shows the same pharmacology, for example, osteoporosis, hyperlipidemia. As a result, raloxifene has been termed as an anti-estrogen with mixed agonist-antagonist properties. The unique profile that shows raloxifene and differs from that of estrogen, is now thought to be due to the single activation and / or deletion of several functions of genes by the receptor complex or raloxifene-estrogen, as opposed to activation and / or deletion of genes by the estrogen-estrogen receptor complex. Therefore, although raloxifene and estrogen use and compete for the same receptor, the pharmacological receptor of the gene regulation of the two is not easily predicted and is unique in each. In general, the compound is formulated with common excipients, diluents or carriers, and compressed in the form of tablets, or formulated as elixirs or solutions for convenient oral administration, or administered via intramuscular or intravenous routes. The compounds can be administered transdermally, and can be formulated as sustained release dosage forms, and the like. The compounds used in the methods of the present invention can be made according to established procedures, such as those detailed in US Patent Nos. 4,133,814, 4,418,068, and 4,380,635 all of which are incorporated by reference herein. In general, the process begins with a benzo [b] thiophene having a 6-hydroxyl group and a 2- (4-hydroxyphenyl) group. The starting compound is protected, acylated and deprotected to form the compounds of formula I. Examples of the preparation of such compounds are given in the US Patents described above. Optionally substituted phenyl includes phenyl, and phenyl substituted one or two times with alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 4 carbon atoms, hydroxyl, nitro, chloro, fluoro, or tri (chloro) or fluoro) methyl. The compounds used in the methods of this invention form the pharmaceutically acceptable acid and base addition salts with a wide variety of organic and inorganic acids and bases, and include the physiologically acceptable salts which are frequently used in pharmaceutical chemistry. Such salts are also part of this invention. Typical inorganic acids are used to form such salts, including hydrochloric, hydrobromic, iohydric, nitric, sulfuric, phosphoric, hypophosphoric acids and the like. Salts derived from organic acids, such as mono- and dicarboxylic aliphatic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic and hydroxyalkanedioic acids, aromatic acids, aliphatic acids and aromatic sulfonic acids, may also be used. Such pharmaceutically acceptable salts include the salts of acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, β-hydroxybutyrate. , butin-1,4-dioate, hexin-1,4-dioate, caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate, glycolate, heptanoate, hippurate, lactate, maleate malate, Hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, teraphthalate, phosphate, onoácido phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate , pyrosulfate, sulfite, bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartarate, and the like. A preferred salt is the hydrochloride salt. The pharmaceutically acceptable acid addition salts are typically formed by the reaction of a compound of the formula I with an equimolar or excess acid amount. The reagents are generally combined in a mutual solvent such as diethyl ether or benzene. The salt normally precipitates from the solution within about one hour to 10 days and can be isolated by filtration, or the solvent can be removed by conventional means. The bases commonly used for the formation of salts include ammonium hydroxide and hydroxides of alkali and alkaline earth metals, carbonates, as well as aliphatic and primary, secondary and tertiary amines, aliphatic diamines, etc. Bases especially useful in the preparation of addition salts include ammonium hydroxide, potassium carbonate, methylamine, diethylamine, ethylenediamine and cyclohexyl ina. The pharmaceutically acceptable salts generally have improved solubility characteristics, as compared to the compound from which they are derived, and thus are frequently 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 formed 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 extenders 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 calcium carbonate and sodium bicarbonate; agents for delaying dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds, surface active agents 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 by intramuscular, subcutaneous or intravenous routes. In addition, the compounds are well suited for formulation as controlled release dosage forms, and the like. The formulations can also be constructed so that they release the active ingredient uniquely or preferably at a particular site of the intestinal tract, possibly over a period of time. The coatings, envelopes, and protective matrices can be made, for example, of polymeric substances or waxes. The particular dosage of the compound of formula I required to inhibit cell-cell adhesion or its effects, or any other use described herein, and 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. In general, accepted and effective daily doses will be from about 0.1 to about 1000 mg / day, and more typically from about 50 to about 200 mg / day. Such doses will be administered to a subject in need thereof, from one to about three times a day, or more frequently as necessary to effectively inhibit cell-cell adhesion, or its effects, or any other use described in I presented. It is generally preferred to administer a compound of the formula I in the form of a salt by the addition of acid, as is customary in the administration of pharmaceutical products having a basic group, such as the piperidino ring. It is also advantageous to administer such compound by the oral route. For these purposes the following oral dosage forms are available.

Formulations In the following formulations, "Active ingredient" means a compound of the formula Y.

Formulation 1; Gelatin Capsules Hard gelatin capsules are prepared using the following: Ingredient Quantity (mg / capsule) Active ingredient 0.1 - 1000 Starch NF 0 - 650 Flowable powder starch or - 650 Silicone fluid of 350 or 15 centistokes The ingredients are mixed, passed through a No. 45 mesh North American sieve, and filled into hard gelatin capsules. The examples of capsule formulations, specific for raloxifene that have been elaborated, include those shown below: Formulation 2: raloxifene capsule Ingredient Quantity (mg / capsule) Raloxifene 1 Starch, NF 112 Fluid starch powder 225.3 Silicone fluid of 350 1.7 centistokes Formulation 3: Raloxifene capsule Ingredient Quantity (mg / capsule) Raloxifene 5 Starch, NF 108 Fluid starch powder 225.3 Silicone fluid 350 1.7 centistokes Formulation 4: raloxifene capsule Ingredient Quantity (mg / capsule) Raloxifene 10 Starch NF 103 Fluid starch powder 225.3 Silicone fluid 350 1.7 centistokes Formulation 5 Ingredient Qty: (mg / <- Raloxifene capsule 50 Starch NF 150 Fluid starch powder 397 Silicone fluid of 350 3.0 centistokes The above specific formulations may be changed in compliance with the reasonable variations provided. A tablet formulation is prepared using the following ingredients: Formulation 6: Tablets Ingredient Cantidaci (mg / capsule) Active ingredient 0.1 - 1000 Microcrystalline cellulose 0 - 650 Silicon dioxide, smoked 0 - 650 Stearic acid 0 15 The components are mixed and compressed to form tablets. Alternatively, the tablets each containing 0.1-1000 mg of active ingredient, are constituted as follows: Formulation 7: Tablets Ingredient Quantity (mg / capsule) Active ingredient 0.1 - 1000 Starch 45 Cellulose, microcrystalline 35 Polyvinylpyrrolidone 4 (as a 10% solution in water) Sodium carboxymethylcellulose 4.5 Magnesium stearate 0.5 Talcum 1 The active ingredient, starch and cellulose are made pass through a North American mesh screen No. 45, and they blend perfectly. The solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a No. 14 mesh North American sieve. The granules thus produced are dried at 50 ° -60 ° C and passed through a North American mesh screen No. 18. Sodium carboxymethyl starch, magnesium stearate and talcum, previously passed through a No. 60 mesh American sieve, are then added to the granules which, after mixing, they are compressed in a tabletting machine to produce tablets. The suspensions that each contain 0.1 - 1000 mg of medication per 5 ml of dose, are elaborated as follows: Formulation 8: Suspensions Ingredient Quantity (mg / capsule) Active ingredient 0.1 - 1000 mg Sodium carboxymethylcellulose 50 mg Syrup 1.25 mg Benzoic acid solution 0.10 ml Taste css Color css Purified water cbp 5 ml The medicament is passed through a No. 45 mesh North American sieve and mixed with the sodium carboxymethyl cellulose and the syrup to form a smooth paste. The benzoic acid solution, the flavor and the color are diluted with some of the water, and added, with agitation. Sufficient water is then added to produce the required volume.

In vitro cell adhesion assays Human Umbilical Vein Endothelial Cells (HUVEC) or Human Aortic Endothelium (HAE) were obtained from Clonetics (San Diego), and were developed in EBM medium provided by Clonetics. The cells were plated in 96-well plates at a density to obtain confluent monolayers after overnight incubation at 37 degrees C. The test compound was added and incubated in serum-free medium for 8-20 hours. The monolayers were then incubated with or without 2 ng / ml of IL-1 or with 20 nanograms of Tumor Necrosis Factor (TNF) for 4 to 24 hours before the binding assay in a total volume of 75 to 100 microliters in the presence of the test compound After the incubations, tritium-labeled U937 cells were added in 50 microlitre volumes from 1 to 3 x 10 cells per well. U937 cells were labeled with tritium by the addition of 3H-thymidine to a final concentration of 1 microcurie per milliliter, followed by 18 to 20 hours of incubation. The cells were washed with PBS before use to remove the excess marker. After a 20 minute incubation period of the labeled U937 cells, with the endothelial cells, the wells were aspirated and washed four times with PBS containing calcium. The monolayer and adherent U937 cells were solubilized by the addition of 0.25% SDS / 0.1 N NaOH for 5 minutes, with agitation. The level of binding was determined by scintillation counting of the solubilized cells.

Anticoagulant activity assay Confluent cultures of human endothelial cells treated with IL1 (2 ng / ml) or untreated, in 96-well plates, were washed once with HBSS to remove the serum proteins and incubated with serum-free medium (DMEM / F medium). -12, 20 mM-HEPES, pH 7.5, 50 mg / ml gentamicin, 1 mg / ml human transferrin and 1 mg / ml bovine insulin) containing 400 nM recombinant human protein C, and 10 nM human thrombin. The cells were incubated at 37 ° C, and at various times the medium was removed and added to an equal volume of a solution of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mg / ml BSA and 10 U / ml of Hirudina. The samples were incubated in the buffer containing hirudin for 5 minutes to inhibit thrombin activity. The amount of activated protein C generated was determined by the addition of chromogenic substrate (S-2366) to a final concentration of 0.75 mM, and measuring the change in absorbance units / minute at 405 mM in a microtiter plate reader TermoMax kinetic (Molecular Devices). In all experiments, samples of the protein C / thrombin solution were incubated in wells without cells to determine basal levels of protein C activation triggered by thrombin. The amount of activated protein C, generated, is expressed as the absorbance (mOD) per minute, per microgram of cellular protein.

Results The human umbilical vein endothelial cells (HUVEC) were treated with the compound A doncc R: and R3 are hydrogen and R3 is pyrrolidino, concurrent to the induction of the expression of the adhesion molecule by TNF. As shown in Table I, the presence of 100 nM of compound A resulted in an approximate reduction of 40% in the cell-cell adhesion level in this assay. When cells were previously treated with only 10 nM of compound A for approximately 20 hours before induction with TNF, an approximate 65% reduction in adhesion was observed (Table 2). Both HUVECs and human aortic endothelial cells (HAEC) were also treated with IL1, another mediator of inflammation to induce the expression of adhesion molecules in the presence of the compound at 10 nM. As shown in Table 3, compound A effectively inhibited the induction of IL1 adhesion in both cell lines. In this way compound A can block the induction of the expression of adhesion molecules, mediated by two independent media and in the venous and arterial cells. As additional evidence for the ability of this compound to modulate the functional properties of the endothelium, the ability of endothelial cells to activate human protein C, a natural regulatory function that is deregulated during endothelial dysfunction states, was measured. As shown in Table 4, treatment with IL1 of the cells significantly reduced the ability of the endothelium to support the generation of protein C. However, after the treatment of the cells with compound A, the suppression of this was essentially eliminated. function by IL1. The above data indicate that compound A protects cells from the activation of inflammatory and procoagulant activities.

Table 1. Effect of compound A on the adhesion of U937 cells to human umbilical vein endothelial cells activated by TNF (HUVEC) Condition% link activity to untreated control 0 ± 7.5 Treated with TNF 100 ± 9.5 TNF plus compound A (100 62 ± 17 nM) aThe level of binding is expressed as the percent of the number of U937 cells linked to the endothelium, before and after induction with TNF.

Table 2. Effect of pretreatment with compound A on the adhesion of U937 cells to endothelial cells of the human umbilical vein activated with TNF (HUVEC) Condition% link activity to untreated control 0 ± 20 Treated with TNF 100 ± 16 TNF plus compound A (10 34 ± 8 nM) aThe level of binding is expressed as the percent of the number of U937 cells linked to the endothelium before and after induction with TNF.

Table 3. Effect of pretreatment with compound A on the adhesion of U937 cells to human aortic endothelial cells (HAEC) activated with IL1 and human umbilical vein endothelial cells (HUVEC) Condition Percent binding activity * for HUVEC HAEC Treated with IL1 100 ± 14 100 ± 8 IL1 plus compound A (10 18 ± 13 54 ± 7 nM) aThe link level is expressed as the percent number of linked U937 cells. to the endothelium before and after induction with IL1.

Table 4. Effect of compound A on the thrombin-catalyzed activation of human protein C on endothelial cells treated with IL-1 Condition Level of protein C produced (mOD / min / μg) Control untreated 8.8 ± 1.4 Treated with IL1 3.7 ± 0.4 111 plus compound A 7.6 ± .6 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 (6)

1. A method for the inhibition of cell-cell adhesion, characterized in that it comprises administering to a human in need thereof an effective amount of a compound of the formula wherein R * and R ° are independently O II hydrogen, -CH3, -C- (alkyl of 1 to 8 carbon atoms), or -C-Ar, wherein Ar is optionally substituted phenyl; R2 is selected from the group consisting of pyrrolidino, hexamethyleneimino, and piperidino; and pharmaceutically acceptable salts and solvates thereof.

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

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

4. A method for inhibiting the normal inflammatory and coagulation process interrupted in patients with vascular endothelial disorders, characterized in that it comprises administering to a patient in need thereof an effective amount of a compound having the formula (I) wherein R1 and R3 are independently O hydrogen, -CH3, -C- (alkyl of 1 to 6 O atoms) carbon), or -C-Ar, wherein Ar is optionally substituted phenyl; R is selected from the group consisting of pyrrolidino, hexamethyleneimino, and piperidino; or a pharmaceutically acceptable salt or solvate thereof.

5. The method according to claim 4, characterized in that the compound is the hydrochloride salt thereof.

6. The method according to claim 4, characterized in that the compound is (I) or its hydrochloride salt.