GB1573730A - Pharmaceutical compositions - Google Patents

Description

(54) PHARMACEUTICAL COMPOSITIONS (71) We, BOEHRINGER MANNHEIM GmbH., of Mannheim-Waldhof, Federal Republic of Germany, a Body Corporate organised under the laws of the Federal Republic of Germany, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention is concerned with new pharmaceutical compositions.

2-Cyanoaziridine is a chemical intermediate which has been known for some time (cf.

Ang. Chem., 84, 108-109/1972). Various derivatives of this compound and the pharmacological properties thereof have been described. However, we have found that this compound itself, when administered orally to rats, brings about a marked increase in the number of leukocytes and lymphocytes, whereas the number of erythrocytes remains substantially unchanged. Furthermore, a considerable increase in the anti-body forming spleen cells has been observed after the administration of 2-cyanoaziridine. These experiments show that, by the administration of 2-cyanoaziridine, the immune defence of animals is strongly stimulated so that this compound can be used therapeutically not only in cases of bacterial and viral infection but also as a cancerostatic.

Furthermore, we have ascertained that numerous derivatives, such as are described, for example, in German Patent Specifications Nos.2,528,460; 2,644,820; 2,656,240 and 2,656,323, break down under physiological conditions with the liberation of 2cyanoaziridine. Insofar as this decomposition takes place quickly, it is to be assumed that at least a part of the effect of these products is due to the action of 2-cyanoaziridine although, of course, a direct action of this compound is not excluded.

Thus according to the present invention, there are provided pharmaceutical compositions which contain 2-cyanoaziridine and/or at least one derivative thereof which, under physiological conditions, liberates 2-cyanoaziridine, together with a solid or liquid pharmaceutical diluent or carrier.

In addition to 2-cyanoaziridine and/or at least one derivative thereof which, under physiological conditions, liberates 2-cyanoaziridine, the new compositions according to the present invention can also contain at least one other known therapeutic substance with bacteriostatic virostatic and/or cancerostatic properties, the action of which is strengthened or supplemented by the immune-stimulating action of the active compound or compounds used according to the present invention.

For the preparation of the pharmaceutical compositions according to the present invention, 2-cyanoaziridine and/or at least one derivative thereof which, under physiological conditions, liberates 2-cyanoaziridine, is mixed in the usual way with appropriate carrier substances and formed, for example, into dosage units, such as tablets or dragees, or, with the addition of appropriate adjuvants, suspended or dissolved in water or in an oil, such as olive oil, and placed into hard gelatine capsules. Since the active material is somewhat acid labile, the composition can, if necessary, be provided with a coating which only dissolves in the alkaline medium of the small intestines or it can be mixed with an appropriate carrier material, for example a high molecular weight fatty acid or carboxymethylcellulose. Solid carrier materials which can be used include, for example, starch, lactose, mannitol, methyl cellulose, talc, highly dispersed silicic acids, high molecular weight fatty acid (such as stearic acid), gelatine, agar-agar, calcium phosphate, magnesium stearate, animal and vegetable fats and solid high molecular weight polymers (such as polyethylene glycol). Compositions which are suitable for oral administration can, if desired, contain flavouring and/or sweetening materials.

As injection medium, which must be sterile, it is preferable to use water which contains the conventional additives for injection solutions, such as stabilising agents, solubilising agents or weakly alkaline buffers. Additives of this kind include, for example, phosphate and carbonate buffers, ethanol, complex-forming agents (such as ethylenediaminetetraacetic acid and the non-toxic salts thereof) and high molecular weight polymers (such as polyethylene oxide) for viscosity regulation.

EXPERIMENTAL RESULTS. a) Rate of hydrolysis of 2-cyanoaziridine derivatives.

In order to ascertain to what extent derivatives of 2-cyanoaziridine are able to liberate 2-cyanoaziridine in solution at physiological pH values, there was determined the rate of hydrolysis at a pH value of 2, which corresponds to a 0.01N hydrochloric acid and which can also be present in the stomach.

High pressure liquid chromatography (HPLC) proved to be the most suitable method of analysis. For this purpose about 1% by weight of the compound to be investigated was dissolved in 0.01N hydrochloric acid and maintained for 16 hours in a thermostatically controlled water-bath at 25"C. Samples of about 100 1ll. were taken from the solutions and then analysed by HPLC.

Separation was carried out in a 0.25 metre long column, the inner diameter of which was 4.6 mm. The column material used was Nucleosil 10-C-18, with a particle size of 10 + 1.5 clam. The mobile phase used was degassed, distilled water with a rate of flow of 1.0 ml./minute at a pressure of about 40 bar and a column temperature of 25"C. The detector used was a Waters differential refractometer 401.

For the determination of the substance-specific factor of 2-cyanoaziridine against n-propanol, 11.66 mg. 2-cyanoaziridine and 92.33 mg. n-propanol were dissolved in 10.0 ml. 0.01 hydrochloric acid. 100 'ill. amounts of this solution were chromatographed under the above-described conditions and, from 5 analyses, there was calculated the average value of the material-specific factor of 2-cyanoaziridine against n-propanol. After introduction of this average value into a calculator, the individual hydrolysis samples were analysed. In the case of 1-carboxamido-2-cyanoaziridine and 1-N-formylglycyl-2-cyanoaziridine, there was also determined, in an analogous manner, a substance-specific factor for the starting materials in order thus to be able to determine the amount of starting material still present.

The results obtained are summarised in the following Table.

Titrimetric examination of the hydrolysis of l-carboxamido-2-cyanoaziridine.

11.35 mg. 1-Carboxamido-2-cyanoaziridine were dissolved in 10.0 ml. 0.01N hydrochloric acid and the solution thermostatically controlled at 25"C. The solution had a pH value of 2.05. This pH value was kept constant with the help of a Radiometer-titration automatic device as pH stat. In the case of complete hydrolysis, a consumption of 1.02 ml. 0.01N hydrochloric acid would have been necessary in order to neutralise the resultant ammonia.

After 15 hours, the pH value was still constant at 2.05. A consumption of 0.01N hydrochloric acid was not noted. b) Determination of leukocytes after a single oral administration to rats In order to ascertain the immune-stimulation action of 2-cyanoaziridine and of its derivatives, the stimulation of the leukocyte formation was investigated in rats. Use was made of female adult Sprague-Dawley rats obtained from the firm WIGA (Gassner, Sulzfeld), which had a body weight of 180 - 220 g. The animals were maintained at a constant temperature (23 i 1"C.) and at a constant atmospheric humidity (55 + 5%) in a 12 hour day/night rhythm. The animals were given rat pellets available under the name "Sniff" from the firm Intermast, Soest and water was available ad libitum. Groups of 10 rats received once only the substance to be investigated (220 mg. thereof dissolved in 10 ml.

0.5% tylose solution/kg. body weight), which was administered with the help of a stomach probe. For control purposes, groups of 10 animals were merely treated with 10 ml. of a 5% tylose solution/kg. body weight. Before the administration, the animals were fasted and blood was collected from the retroorbital vein complex with the help of a he arinised injection capillary needle (type B 3095/2 of the firm Sherwood Med. Inc., St. Louis and the leukocytes determined in known manner with the use of a Coulter counter.

On the fourth day, blood was again collected from the animals from the retroorbital vein complex and the leukocytes counted. From the individual values there were determined the average values with standard deviations. The experimental groups were only evaluated when the control groups showed no physiological variations.

The following Table also shows the leukocyte counts before administration and on the fourth day, as well as the quotients. These data show that, of the substances investigated by oral administration, those bring about a significant increase of the leukocyte count and thus are strongly immune-stimulating which, under physiological conditions, split off 2cyanoaziridine to a significant extent.

TABLE Initial resultant amount of Leukocyte count in Leukocyte amount amount of starting thousands quotient of 2-cyano- material compound aziridine still 0 day 4 day 4/0 (mg) in % of present theory in % 2-[2-Cyanoaziridinyl-(1)]- 107.3 100 # 8.0 13.6 1.7 2-[2carbamoyl-aziridinyl] (1)]-propane 1-N-Formylgycyl-2-cyano- 105.3 54.9+ 7.9 8.5 16.4 1.9 aziridine 1-(N-Methylcarbamoyl)-2- 96.1 # ca. 100 8.3 8.6 1.02 cyanoaziridine 1-(N-p-Sulphamoylphenyl- 102.5 1.5 86.1++ 10.9 11.2 1.02 carbamoyl)-2-cyanoaziridine 1-Carboxamido-2-cyano 104.8 # 90.46+++ 8.9 9.5 1.07 aziridine 2-Cyanoaziridine 103.4 100 ./. 7.6 18.1 2.4 + a part of the initially formed 2-cyanoaziridine has entered into secondary reactions with other hydrolysis products.

++ starting material was not comPletely dissolved in 0.01N HC1 and was measured in potassium phosphate buffer (H2.

+++ since the separation of 2-cyanoaziridine and 1-carboxamido-2-cyanoaziridine is not very good, a titrimetric process was additionally employed.

Claims (7)

WHAT WE CLAIM IS:

1. Pharmaceutical compositions, comprising 2-cyanoaziridine and/or at least one derivative thereof which, under physiological conditions, liberates 2-cyanoaziridine, together with a solid or liquid pharmaceutical diluent or carrier.

2. Composition according to claim 1, wherein the liquid diluent or carrier is sterile.

3. Composition according to claim 1 or 2, wherein the liquid diluent or carrier contains a stabilising agent and/or a solubilising agent and/or a buffer.

4. Composition according to claim 1 or 3, wherein a flavouring agent and/or sweetening agent is present.

5. Composition according to any of the preceding claims, whenever in the form of a dosage unit.

6. Composition according to claim 6, wherein the dosage unit is provided with a coating which only dissolves in the alkaline medium of the small intestines.

7. Pharmaceutical compositions according to claim 1, substantially as hereinbefore described.