WO1994026272A1

WO1994026272A1 - 3-[n-(2-mercapto-propionyl-amino-acetyl)]-tetrahydro-thiazolyl-4-carboxylic acid as antiinflammatory and immunostimulating agent

Info

Publication number: WO1994026272A1
Application number: PCT/EP1994/001527
Authority: WO
Inventors: Giuseppe Quadro
Original assignee: Yason S.R.L.; Bruno, Graziella
Priority date: 1993-05-14
Filing date: 1994-05-11
Publication date: 1994-11-24
Also published as: JPH07509006A; IT1264446B1; AU6969594A; ITMI930984A0; ITMI930984A1

Abstract

3-[N-(2-mercapto-propionyl-amino-acetyl)]-tetrahydro-thiazolyl-4-carboxylic acid is useful for the preparation of a medicament having antiinflammatory and immunostimulating activity.

Description

3-[N-(2-MERCAPTO-PROPIONYL-AMINO-ACETYL) ]-TETRAHYDRO- THIAZOLYL-4-CARBOXYLIC ACID AS ANTIINFLAMMATORY AND IMMONOSTIMULATING AGENT

This invention is aimed at a new therapeutic use of 3-[N-(2-mercapto-propionyl-amino-acetyl) ]-tetrahy- dro-thiazolyl-4-carboxylic acid.

The compound 3-[N-(2-mercapto-propionyl-amino- acetyl) ]-tetrahydro-thiazolyl-4-carboxylic acid having the following formula (I)

also known as MR 729, is described in the european patent application No. 0 079 464 as a compound endowed with hepatoprotecting, and bronchosecretolytic activity as well as being an inhibitor of the angiotensin converting enzyme.

Surprisingly, it has now been found that MR 729 possesses other pharmacological activities, which make it useful as an antiinflammatory and immuno stimulating agent. The pharmacological characteristics of MR 729 are as follows: Acute Toxicity

It has been evaluated on mice, rats and rabbits, following the Litchfield and Wilcoxon method. , J. Pharm. Exp. Therap. 96, 99, 1949. MR 729 was administered both orally as well as intraperitoneally. The results are reported in table 1, Table 1

SPECIES DL50 (mg/Kg) p.o. i.p.

MOUSE 3748 346

RAT >4000 1020

RABBIT >3000

Primary Antibody Response

The test was carried out on C57B1/6 mice, according to the method described by Jerne N.K., Henry C, Nordin A.A., (Transpl. Rev. 19, 130, 1974). The primary antibody response was assessed by the number of cells which formed plaques in the spleen of mice sensitised with sheep red blood cells (SRBC) , after 4 days of MR729 subcutaneous administration. The controls were not treated. The results are reported in Table No.

Table 2

Effect on the primary antibody response to sheep red blood cells (SRBC) in C57BI/6 mice Mean ± SD (n=5)

Mouse Group n° Treatment Dose Splenocytes PFC/spleen % Strain mg/kg s.c. x 10 6 vs contr .

C57B1/6 1 5 Controls 243 ± 43 14535 ± 4290

2 5 MR-729 100 198 ± 37 30994 ± 15549 * 113,2

C57B1/6 1 5 Controls 198 ± 53 6454 ± 1021 -—

2 5 MR-729 50 295 ± 33 16637 ± 2713 ** 157,8

3 5 MR-729 100 266 ± 39 10037 ± 4632 55.5

PCF - number of antibody-forming cells (plaque-forming cells) in the spleen of mice sensitised with SRBC.

* P < 0,05 ** P< 0,01

IgA Concentration

The effect on the IgA concentration in intestinal secretions of the rat was assessed using the method described by Elson CO. et. al., (J. of Immunol. Method 67, 101, 1984). The IgA concentration was evaluated using the ELISA method. MR 729 was administered daily for 7 days. The results are reported in Table No. 3. Table 3

Effect on IgA concentration in mouse intestinal secretions. Mean ± S.D. (n=10)

Treatment Dose IgA % mg/Kg i •p. μg/ml vs Control

Controls — 30,4 ± 4,52 —

MR 729 40 46,6 ± 3,23 53,3

*

MR 729 70 50,8 ± 3,15 67,1

*

MR 729 100 38,8 ± 2,48 27,6

*

* P < 0,01 vs Controls.

Mitogen stimulation test in human leukocytes

The effect on proliferation of human white blood cells induced by Phytohemagglutinin (PHA) and lipopolysaccharides (LPS) , after treatment with MR 729 was assessed using the method described by Boyum A.,

(J. Clin. Lab. Invest. 21 (97), 1 (1968)) and Hirschhorn K. , Serie Hematologica 9, 26 (1965). 3H- Thymidine was incorporated in human leukocytes. The results are reported in Table No. 4.

Table 4

Effect on the proliferation induced by phytohemagglutinin (PHA and lipopolysaccharides (LPS) . Mean ± S.D.

Substance Concentration PHA LPS meg/ml cpm % cpm %

Controls 149998 ± 24461 1187 ± 156 σi

MR 729 20 126651 ± 10426 -15,6 1795 ± 310 * 51,2

MR 729 40 123094 + 16483 -17,9 1884 ± 376 * 58,7

P < 0 , 01 Vs . control s

Immunorestoration test

The test was carried out according to the method described by Farrant et al. , (J. Immunol. Methods, 1980), in order to evaluate the effect of mitogens on splenocyte proliferation in mice treated on day 0 with azathioprine. The splenocytes were obtained on day +3. The results are reported in table 5. MR 729 was administered on days 0, +1, +2.

Immunorestoration test

Table 5

Effect on PWA (2 μg/ml) or LPS (60 μg/ml) induced proliferation.

Each group represents pool from 3 animals - Means 1 S.D.

PWM LPS

Treatment Dose dpm % dpm % of controls of controls

Controls - 1342.01235.6 100.0 3813.51421.0 100.0 oo

Azathioprine 250 mg/kg p.o. 1269.51194.5 94.6 3634.81762.0 95.3

Azathioprine+MR 729 50 mg/kg s.c. 870.81110.4 64.9 2848.91416.0 74.7

Azathioprine+MR 729 100 mg/kg s.c. 1012.01150.3 75.4 6790.51796.5* 178.1

Azathioprine+MR 729 50 mg/kg p.o. 1529.11165.6 113.9 5751.81957.6* 150.8

Azathioprine+MR 729 100 mg/kg p.o. 3159.01514.7* 235.4 7475.811306.2* 196.0

* P<0.01 vs controls

Subacute antiinflammatory activity Carrageenin abscess in the rat

The test was carried out according to the procedure described by Benitz K.F. et al . , (Arch. Int. Pharmacodyn. 144, 185, 1963). The drugs were administered 30 minutes and 6 hours after the carrageenin injection. After 24 hours the subcutaneous pouch containing the abscess was removed and weighed. The results appear in Table No. 6. (Mean 1 S.D.; n= 9) Table 6

Treatment Dose Weight of P % mg/kg po abscess (mg) vs Controls

Controls 1470,6 1 40,6

MR 729 100 929,60 1 24,3 < 0,01 -36,8 MR 729 200 813,8 1 17,7 < 0,01 -44,7

Phenylbutazone 200 911,8 1 17,3 < 0,01 -38,0

Carrageenin Granuloma in the rat

The test was carried out as described by Boris A. et al., (Arch. int. Pharmacodyn. 153, 205, 1965). The drugs were administered for 4 days. On the 5th day the pouch containing the exudate was removed. The results are reported in table No. 7. (Mean ± S.E.; n=10 ). Table 7

Treatment Dose Exudate % P mg/kg po volume (ml) vs Controls

Controls 5,23 1 0,14

Phenylbutazone 100 2,83 1 0,1 -45,9 <0,01

MR 729 100 3,18 1 0,12 ■39,2 <0,01

The above reported-pharmacological tests are generally recognized as being valid and predictive experimental models of clinical utilities in humans. Therefore, this invention also concerns pharmaceutical formulations containing MR 729 as active ingredient possessing immunostimulating and antiinflammatory activity, in particular for the prevention of recurring respiratory infections against which the concomitant presence of the above reported activities exerts particulartly beneficial effects.

Examples of pharmaceutical formulations are, for instance, tablets, sachets, capsules, small bottles, containing from 100 to 500 mg of active ingredient. Administration routes different from the oral one can also be used. The posology will depend on several factors, such as patient's weight and conditions but it will usually range from 100 mg to 2 g per day, optionally divided in more administrations.

Claims

1. Pharmaceutical formulations with immunostimulating and antiinf lammatory activities containing 3- [N- ( 2- mercapto-propionyl-amino-acetyl ) ] -tetrahydro-thiazolyl- 4-carboxylic acid as active ingredient , having the following formula :

2. Pharmaceutical formulations according to claim 1, in tablet, sachet, capsule, small bottle form containing 100 to 500 mg of active ingredient.

3. Use of 3-[N-(2-mercapto-propionyl-amino-acetyl) ]- tetrahydro-thiazolyl-4-carboxylic acid for the preparation of a medicament with immunostimulating and antiinflammatory activity.

4. Use of 3-[N-(2-mercapto-propionyl-amino-acetyl) ]- tetrahydro-thiazolyl-4-carboxylic acid for the preparation of a medicament for the prevention of recurring respiratory infections.