LT3589B - Using of known combination for treatment of rejection reaction by transplantation of organs - Google Patents

Abstract

Known compounds are described and their formulas are the following:<IMAGE><IMAGE>physically tolerated compound 2 saline usage, during the production of medicine, designed for treating recipient rejection reaction in the aspect of transplanted organs.

Description

The present invention relates to the use of N- (4-trifluoromethyl) -5-methylisoxazole-4-carboxanilide and N- (trifluoromethylphenyl) -2-cyano-3-crotonamide and / or their physiologically tolerated salts for the preparation of a medicament for treating a recipient organ transplant rejection.

From European patent application no. 13376 is known as N- (4-trifluoromethyl) -5-methylisoxazole-4-carboxanilide (Compound 1) as a substance having anti-inflammatory properties. The process for preparing this compound is also described there.

In addition, Compound 1 and its metabolite N- (4-trifluoromethylphenyl) -2-cyano-3-hydroxycrotonamide (Compound 2) are known to have immune modulatory properties and can therefore be used in the treatment of chronic homologous diseases (graft versus host). ) as well as autoimmune diseases such as systemic lupus erythematosus (EP-A-217206).

U.S. Pat. No. 4061767 discloses the use of 2-hydroxyethylidene-cyanoacetanilide derivatives for the preparation of medicaments having anti-inflammatory and analgesic activity.

In the United States in 1990, 15,000 organ transplants were performed. The majority of them were incisional transplants, but recently the number of heart, skin, lung, liver and pancreas transplants has increased. Many patients experience rejection of an organ transplanted from another person. In these cases, there are three forms of rejection reactions: extremely acute, acute and chronic rejection.

Particularly acute rejection occurs mainly due to the presence of antibodies circulating in the blood, which affect the tissues of the transplanted organ (graft), which can lead to graft necrosis within a very short time, sometimes within minutes.

In acute graft rejection, the rejection reaction is slightly delayed. In addition, chronic rejection can lead to illness. Transplants that have successfully survived. the first year of transplant, the following year may be rejected. It is also known that host-graft dependency goes beyond mere rejection by the host organism. In some cases, an immune response from the graft side to the host tissues may occur (EP-A-217 206). Therefore, it is accepted to distinguish between rejection reactions between the graft and the host and between the host and the graft.

In addition, rejection reactions are known to occur when organ transplantation is performed from different types of organisms, such as the rat rat (Roitt et al., Immunology, Gower, Medical Publishing Ltd. 1985).

To date there is no known drug that can provide effective protection against extremely acute rejection reactions. To date, donor-recipient compatibility studies have been conducted in clinics. 20-40% of patients are unable to receive an organ transplant. Acute rejection reactions can be treated, but medication often has side effects of nephrotoxicity. To date, there is no known drug whose action is directed to the cause of rejection.

Allophilic and kseLT 3589 B nophilic antibodies may be the major pathogenic tissue destruction factor in transplantation (Auchincloss H., Transplantation, 4 6, 1, 1988). These antibodies essentially determine the rejection of transplanted organs between animals of the same species (alo) and between two different species (xenon).

Unexpectedly, Compound 1 and its metabolite, Compound 2 above, have been identified as potential inhibitors of allophilic and xenophilic antibody formation. Therefore, it is possible to have an effective effect on the extremely acute, acute and chronic rejection reactions of the recipient in relation to the transplanted organ.

The present invention is therefore directed to the use of N- (4-trifluoromethyl) -5-methylisoxazole-4-carboxanilide and N- (4-trifluoromethylphenyl) -2-cyano-3-hydroxycrotonamide and / or its physiologically tolerated salts for the manufacture of a medicament for use in for organ transplant reactions.

Suitable physiologically tolerated salts of Compound 2 are, for example, alkali metal, alkaline earth metal salts or ammonium salts, including salts of physiologically tolerated organic ammonium bases.

The term organs should be understood to refer to all mammalian organs, including the human, such as kidneys, heart, skin, liver, pancreas, muscle, bone, stomach or intestine, as well as blood and hair.

Rejection reactions are protective reactions in the recipient organism which ultimately lead to the remission or destruction of cells or tissues of the transplanted organ or affect its viability.

Compounds 1 and 2 can be obtained as follows:

Compound of Formula I:

O

(D where X is a halogen atom such as chlorine or bromine exposed to an amine of formula II:

and Compound 1 is obtained and this compound can be further treated with a basic agent and converted to Compound 2.

The above reactions may be carried out under standard conditions in a known manner (EP-B-13 376, US 4,061,767).

The starting materials for the reactions are known or can be readily obtained by methods described in the literature.

The invention is also directed to medicaments containing an effective amount of Compound 1 or Compound 2 and / or physiologically acceptable salts of John 2, together with suitable pharmaceutical additives, carriers, diluents and / or other active ingredients and excipients.

The invention also relates to a process for the preparation of a medicament for treating rejection reactions of a recipient in a transplanted organ which comprises converting Compound 1 or 2 and / or a physiologically tolerated salt of Compound 2 together with a pharmaceutically acceptable, physiologically acceptable carrier. and other suitable active substances, additives and excipients in a suitable pharmaceutical form.

The pharmaceutical compositions of this invention may be administered orally, topically, rectally, intravenously or parenterally. They are used by the recipient and / or the donor before, during and after organ transplantation.

Suitable solid or liquid galenical formulations may be, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops or solutions for injection, as well as sustained release formulations, which may be formulated in conventional pharmaceutical excipients such as carriers, disintegrants, binders, astringents, creams, lotions, as well as flavoring, sweetening or dissolving agents. The most commonly used excipients are, for example, magnesium carbonate, titanium dioxide, lactose, mannitol or other sugars, talc, milk protein, gelatin, starch, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents such as sterile water or mono- or polyhydric alcohols, such as glycerol.

It is recommended that preparations be prepared and administered in a uniform dosage form, each of which must contain, as an active component, a quantity of the active ingredient 1 / ·

Ό either dose of 2 and / or physiologically tolerated salts of compound 2. For solid dosage forms such as tablets, capsules or suppositories, this dosage may be up to 300 mg, preferably 10 mg to 200 mg.

For the treatment of an organ transplant patient (70 kg), an intravenous infusion is performed in the early stages of transplantation, with a maximum active compound dose of 1,200 mg per day, followed by oral administration of 300 mg of compound 1 or 2 3 times daily. and / or the corresponding salts of compound 2.

However, in certain circumstances, it may be necessary to administer higher or lower doses. The dose may be taken as a single unit dose or in a number of smaller doses, or it may be divided into a number of smaller doses and given over time.

Finally, Compound 1 or 2 and / or its corresponding salts may be combined with other active ingredients, for example, for use in the treatment of urinary organs, platelet aggregation inhibitors, analgesics and steroidal and non-steroidal anti-inflammatory agents, in the manufacture of the above dosage forms.

The following examples illustrate the invention without limiting it.

Example 1

Pharmacological tests and results

Rats (LEW) at 2 to 3 months of age were sensibilized by intraperitoneal injection (i.p.) of 2x10 'human peripheral blood lymphocytes. Administration of Compound 2 to the abdominal cavity was initiated 4 days prior to sensitization and completed 10 days after rat sensitization. Serum samples were taken from the vertebral vein and stored at −80 ° C. Thermal decontamination avoids complement function.

Naturally occurring xenophilic antibodies (ΝΧΑ) and sensitization-induced xenophilic antibodies (SXA) are titrated and live human peripheral blood lymphocytes are added (45 min at 20 ° C). After intensive rinsing, FITC-labeled goat-rat IgG or IgM antibody was added and binding to SXA or ΝΧΑ current cytometry (FACScan, Becton Dickinson) was determined.

A) Non-sensitized rats

Male (LEW) male serum contains ΝΧΑ, which binds to viable human peripheral blood lymphocytes, more typically with a mean IgM titer of 1: 4 and a mean IgG titer of less than 1: 1.

Rats (n = 8) treated daily with 10 mg / kg compound on day 2 and 11 show a 30% decrease in IgG titer and a 50% decrease in IgM titer.

B) Sensitized rats

Sensitized rats have significantly elevated levels of SXA. The IgG titer is 1: 1024 to 1: 16384 and the IgM titer is 1: 4 to 1: 256. IgG titer remains stable for more than 50 days while IgM titer decreases slowly after day 10.

Sensitized rats (n = 5) were exposed to 3 or 10 mg / kg of compound 2. On day 11, the following SXA titer values were obtained:

Compound (mg / kg) IgG IgM 0 1: 2048 1:90 3 1:25 1:16 10th 1: 1 1: 1.5 Effect of Compound 2 allophilic antibodies formation.

Male Brown-Norway rats weighing 200 to 250 g were used as cardiac donors. Their hearts were transplanted into Lewis male males of the same weight. All rats were subjected to the standard diet and water regimen. Rats were housed in a 12-h light / dark cycle.

Recipient rats (Lewis rats) were harvested from the vertebral vein on the day of transplantation and then every 4 days with 1 ml of blood. The titre of the allophilic antibody in the recipient rat blood is determined by incubation of the recipient at varying dilution with spleen cells from the donor rat (Brown-Norway rat) and rabbit complement. Cytotoxicity was determined by the Trypan Blue displacement method. Antibody p-values were determined using a standard t-test and compared to untreated control animals.

Compound 2 was started on day 4 after transplantation. In each case, 10 mg / kg of compound 2 is injected once daily into the abdomen. The number of animals tested at a time is 5 (n = 5). Operation ends on the 17th. The table below shows the mean cytotoxicity values (%) over the course of the assay.

Serum p-value of the day after transplantation

dilution 0 4 7th 11th 14th 17th 17th day 1: 5 3 63 36 19th 22nd 11th 0.001 1:25 3 58 31st 16th 11th 6th 0.001 1: 125 4 34 20th 9th 6th 5 0.050

Example 2

Preparation of N- (4-trifluoromethyl) -5-methylisoxazole-4-carboxanilide (Compound 1)

A solution of 0.05 mol of 5-methylisoxazole-4-carbonyl chloride (7.3 g) in 20 ml of acetonitrile was added dropwise at room temperature to a solution of 0.1 mol of 4-trifluoromethylaniline (16.1 g) in 150 ml of acetonitrile. After 20 minutes the precipitate of 4-trifluoromethylaniline hydrochloride which is stirred under suction is suctioned off and washed twice with 20 ml of acetonitrile. The combined filtrate is concentrated under reduced pressure. 12.8 g of a white crystalline product are obtained, which is N- (4-trifluoromethyl) -5-methylisoxazole-4-carboxanilide (compound 1).

io

Example 3

N- (4-Trifluoromethylphenyl) -2-cyano-3-hydroxycrotonamide (Compound 2)

Dissolve 0.1 mol of N- (4-trifluoromethyl) -5-methylisoxazole-4-carboxanilide in 100 ml of methanol and treat at 10 ° C with 0.11 mol (4.4 g) of sodium hydroxide in 100 ml of water. The mixture is stirred for 30 minutes, diluted with water and acidified with concentrated hydrochloric acid. The precipitated crystal mass is suctioned off, washed with water and air dried. The yield of N- (4-trifluoromethylphenyl) -2-cyano-3-hydroxycrotonamide (Compound 2) is 24.4 g. Lyd. mp 205-206 ° C (from methanol).

Example 4

Acute toxicity By ingestion

Acute toxicity was determined in NMR mice (20-25 g) and SD rats (120-195 g) after intraperitoneal administration. The test substance was suspended in 1% sodium carboxymethylcellulose solution. Different doses of test substance were administered in mice at a volume of 10 ml / kg and in rats at a volume of 5 ml / kg. For each dosing, 10 animals were administered. After 3 weeks, u was determined

acute toxicity according to Litchfield and Wiicoxon. The results are shown in the table.

Table

Compound 1 Acute Abdominal Toxicity LD ₅₀ (mg / kg)

Compound 2 Acute Abdominal Toxicity LD ₅₀ (mg / kg)

Mouse NMRI

In the rat SD

185 (163-210) 150 (100-200)

170 (153-189)

Claims (4)

DEFINITION OF INVENTION CF, (2) wherein Compound 2 is formulated in the form of a salt for the treatment of a very acute reaction to a transplanted or physiologically acceptable compound in the treatment of a rejection of an organ of the recipient. 2. The use according to claim 1, wherein the compounds are used for the treatment of rejection by transplantation of organs from one type of organism to another. Use according to claim 1 or 2, characterized in that the compounds are used for the treatment of rejection reactions in mammals, including humans. Use according to one or more of claims 1 to 3, characterized in that other active substances such as anti-uricopathics, platelet aggregation inhibitors, analgesics and steroidal or non-steroidal anti-inflammatory agents can be used.