NL192682C - Method for preparing a hemin complex, and drug. - Google Patents

Description

1 192682

Method for preparing a hemin complex, and drug

The present invention relates to a method of preparing a physiologically active water-soluble hemin compound and to a medicament comprising the compound thus prepared.

It is well known that the water-insoluble hermine exists in the organism as a prosthetic group of hemoglobin in most cytochromes and in certain enzymes. Hemoglobin is synthesized in the marrow bone. When hemin proteins decompose, hemin is released, but under normal physiological conditions only a small part of it is used for the synthesis of new hemin proteins. By the action of hemin-oxygenase, hemin is cleaved to biliverdin, which is further reduced to bilirubin. Naturally, intact hemoglobin does not serve as a substrate for hemin oxygenase, of course.

The object of the present invention is to prepare a water-soluble hemin iron compound for treating certain types of anemia with iron readily available in the hemin molecule. The compound is primarily intended for the treatment of poryphyria in which the normal production of hemoglobin is disrupted for some reason. Since the compound is intended for oral administration, in tablets or capsules, as well as for injection, the compound must be soluble in water.

The process of the present invention is characterized in that crystalline hemine is reacted with L-arginine or 20 L-lysine, in an aqueous organic solvent selected from the group consisting of 10-15 hours at ambient temperature and with vigorous stirring. acetone, isopropyl alcohol, methanol and ethanol, containing more than 3.2% by volume of water, but not more than 7% by volume of water, with a molar ratio of hemine to L-arginine or L-lysine not exceeding 0.5 but is at least 0.25 is used.

Defects of hemoglobin synthesis may be due to impaired synthesis of hemin or globin chains. Hemin synthesis may be disrupted by (a) a lack of a component required for synthesis, or (b) dysfunction of an enzyme that catalyzes the synthesis.

Ad (a) Iron deficiency can be the limiting factor in hemin synthesis. The organism gets the necessary amount of iron (1-2 mg) with the food every day. Iron deficiency may be due to dietary iron deficiency or possibly the presence of iron binding compounds in the food. Disturbances of the iron absorption mechanism, despite an adequate iron content in the food, can also lead to iron deficiency. Regardless of the cause, iron deficiency sooner or later leads to anemia.

In the rare vitamin B6 deficiency, the absorption of iron is normal, but its use is inhibited by the cells. As a result, a certain type of sideroblastic anemia develops.

Iron deficiency anemia is treated with oral iron preparations (such as iron sulfate or iron gluconate) or, more rarely, with injections (iron sorbitol). When the mechanism of iron absorption is disturbed, these conventional oral preparations are of no use; the iron will not even enter the cells or intestinal tissue. Unlike inorganic iron, hemin iron, where iron is bound to hemin, is absorbed by these cells even in the cases of impaired iron absorption that are resistant to conventional oral therapy. Thus, hemin iron is the only known effective remedy in the oral treatment of cases resistant to therapy. Hemin iron has been found to be absorbed 4 to 5 times better than inorganic iron, even in fairly healthy individuals (H. Seppanen and H. Takkunen: Suomen Laakarilehti 36: 2071-2072, 1981).

45 Ad (b) The synthesis of hemine is controlled enzymatically. An impaired function of the enzymes that catalyze hemin synthesis can be hereditary or due to external factors. It inevitably leads to reduced hemin formation, which is reflected by the development of porphyria or certain types of sideroblastic anemia or other diseases.

Porphyria is the main group of diseases caused by the diminished action of enzymes.

In porphyria patients, an accumulation of porpyrins, which are intermediates in the synthesis of hemin, occurs and an increased excretion thereof in urine and faeces. Most types of porphyria are manifested by current attacks that are very difficult to control.

Sometimes sideroblastic anemias of different types can develop instead of porphyria due to the dysfunction of the enzymes participating in hemin synthesis. Sideroblastic 55 anemias can also be hereditary or acquired.

The treatment of porphyria has hitherto been mainly based on the avoidance of certain drugs and on the administration of large amounts of carbohydrates during the acute replenishment, 192682 2, but the activity has hitherto been limited. Since the etiology of porphyria has been clarified, intravenous treatment with hemin compounds (hematin) has progressed. Hematin has been shown to be effective in treating porphyria attacks, but thrombophlebitis has been caused in more than 50% of patients. In addition, it is very unstable and therefore unsuitable for industrial scale preparation. Therefore, there are very few options for effective treatment for porphyria patients.

Hemine, which is sparingly soluble in water, can be obtained in pure form from blood by extraction with a mixture of hydrochloric or acetic acid from an aqueous solution of hemolysing erythrocytes.

Another method is based on the extraction of hemine with acetone in the presence of, for example, histidylhistidine, pilocarpine or imidazole, at a pH of 7.0 (N.W. Wakid & K.Y. Helou: Int. J.

Biochem. 4, pp. 259-267, 1973).

PCT patent application 813749 (PCT / FI81 / 00026) describes a process for the preparation of a water-soluble hemine concentrate in which about 40% by weight of hemin is present and the remainder is a "blood substitution" of unknown nature. The product is intended to be used in a lyophilized form as a dietary iron supplement or as an anti-anemic drug.

The drawback of this method is that the final product is a mixture of hemin and "blood substance". Since the latter component is not uniform, the mixture is not suitable for injection.

Porphyria has been treated in hospitals with a mixture prepared ex tempore by dissolving hemin in a sterile solution of sodium carbonate (hematin). Since this solution is not stable, it cannot be widely prepared as a commercial product. In addition, hematin causes thrombophlebitis at the injection site in about 50% of cases, probably due to the high pH of the solution. This is a serious drawback, which significantly reduces the applicability of the product.

The present invention relates to a process for the preparation of a water-soluble hemin compound by reacting the sparingly water-soluble crystalline hemine with a suitable base such as an amino acid, in particular L-lysine or L-arginine in a solvent mixture which contains, for example, water and acetone. The composition of the solvent, i.e. the proportion of organic solvent to water, is surprisingly important for the medical value of the final product. The water content in the solvent mixture is so low (about 7%) that neither hemine nor the fairly easily soluble L-arginine is dissolved. The reaction takes place under strong stirring and the pH of the solution is monitored continuously. The formed product is separated and dried; the hemin compound is thus obtained in a dry state and is soluble in water, which is important from a medical point of view as well as from a pharmacotechnical point of view.

The hemin molecule contains two carboxylic acid groups that react with the basic amino groups of L-lysine or L-arginine.

Hemin arginate and hemin lysinate prepared according to the invention are dissolved in water and the pH of the solutions was measured at different times and compared to the pH of a regular mixture of hemine and L-arginine dissolved in water. The results are shown in Table A.

40 TABLE A

PH

0 min. 60 min. 24 h.

45 hemin arginate 0.02937 g / 25 ml 8.22 8.22 8.22 hemine lysinate 0.02760 g / 25 ml 8.10 7.97 8.13 hemine + 0.01630 g / 25 ml 10, 13 9.81 9.33 L-arginine 0.01307 g / 25 ml 50

The results of the pH measurements indicate that the pH of the hemin arginate and hemin lysinate is stable for 24 hours (pH about 8). In contrast, the pH of the regular mixture drops very quickly, probably due to the extremely slow reaction between the carboxyl groups in the hemine and the amino group in the L-amino acid. Therefore, a therapeutically suitable product cannot be obtained by this method 55. Hemin arginate and hemin lysinate prepared according to the invention each consist of a complex compound in which the L-amino acid has reacted with the hemin carboxylic acid groups.

In order to determine the optimum molar ratio between the two reagents on the one hand, and the most suitable composition of the solvent mixture on the other hand, the following experiments were carried out with hemine and arginine.

Crystalline hemine and L-arginine in molar ratios of 1: 2 and 1: 3 were reacted with vigorous stirring in a solvent mixture consisting of an organic solvent and water in various amounts. The precipitates formed were filtered off, washed and dried.

Water solubility was determined by dissolving, with vigorous stirring, for about 1 hour about 1.0 g of the hemin arginate obtained in each experiment in 50 ml of distilled water.

The solutions were centrifuged (about 3500 rpm) and the residue was washed with 10 ml of distilled water and 10 ml of acetone, then dried and weighed. The insoluble residue mainly consisted of unreacted hemine. The results of the experiments are shown in Table B. The percentage of the water-insoluble residue listed in this Table is based on the weight of the residue obtained by centrifugation.

TABLE B

15 -

Test hemin-L-arginine ratio solvent ml temperature in water - (° C) insoluble weight (g) molar residue (%) 20 1 * 6.52: 3.48 1: 2 methanol 300 20 tar 2 * 6.52 : 3.48 1: 2 ethanol 300 20 = 100 3 * 6.52: 5.22 1: 3 ethanol 300 40 20.2 4 * 6.52: 3.48 1: 2 isopropyl alcohol 300 20 = 100 5 6, 52: 3.48 1: 2 isopropyl alcohol / 300: 20 20 21.2 25 water 6 6.52: 5.22 1: 3 isopropyl alcohol / 300: 20 20 9.7 water 7 6.52: 3.48 1: 2 acetone / water 300: 15 20 16.8 8 6.52: 3.48 1: 2 acetone / water 300: 20 20 12.4 30 9 6.52: 5.22 1: 3 acetone / water 300:10 20 = 100 10 6.52: 5.22 1: 3 acetone / water 300: 10 40 11.4 11 6.52: 5.22 1: 3 acetone / water 300: 15 20 8.3 12 6.52: 5.22 1: 3 acetone / water 300: 20 20 0.3 13 * 6.52: 5.22 1: 3 acetone / water 300: 30 20 tar 35 14 6.52: 5.22 1: 3 acetone / water 150: 10 20 4.2 15 * 6.52: 5.22 1: 3 acetone / water 150: 12.5 20 tar *) Not according to the invention 40 The tar-like substance formed in some experiments could not be converted to the powder form. The practical molar ratio of hemine to arginine was found to be 1: 3 and the most suitable solvent mixture was 300 ml of acetone and 20 ml of water because hemine requires a slight excess of L-arginine to react properly.

The local effect of intravenously infused hemin compounds on surrounding tissues 45 was investigated by infusing 5 mg / kg into the ear vein of "California White" rabbits. A conventional hemin carbonate solution (hematin) was used as the reference solution.

After infusion of the hemin-arginate solution, the tissue around the vein remained normal, i.e. no sterile inflammation (thrombophlebitis) occurred.

A similar result was found after infusion of a corresponding hemin lysinate solution. Thus, it can be concluded that the compounds do not cause thromophlebitis when infused intravenously.

When a hemin carbonate solution was administered in the same manner, the tissue around the vein became red and irritated, indicating that a manifest sterile inflammation (thromophlebitis) developed. Thrombophlebitis was still evident three days after the infusion of the hemin carbonate solution.

The physiological nature of the various water-soluble hemin compounds was determined by examining the ability of hemin oxygenase to cleave the compounds. It

Claims (2)

192682 4 physiological substrate on heminoxigenase, methemalbumin, is cleaved to biliverdin, which is further reduced to bilirubin by biliverdin reductase. Thus, the excess hemin, which cannot be used by the organism, is decomposed primarily by hemine oxygenase to bilirubin and other closely related substances, which are normally excreted. The enzyme that limits the reaction rate is thus hemine oxygenase. In the present enzymatic analyzes, conducted first to determine the ability of hemin arginate and hemin lysinate to serve as a substrate for hemin oxygenase, the activity of the comparative substrate methemalbumin was expressed as 100. The corresponding value obtained for hemin arginate and hemin lysinate was 106. The activities of other hemineamine derivatives, where the amine component was diethanolamine, ethylamine, cyclohexylamine or piperidine, were found to be 13, 21, 31 and 78, respectively. The experiments indicated that hemin arginate and hemin lysinate behaved as normal physiological compounds in the organism to hemine oxygenase. The invention will be further elucidated by means of the following examples. Example I In a beaker equipped with a mechanical stirrer and with a solvent mixture of 300 ml acetone and 20 ml water, 6.52 g (0.01 m) of crystalline hemine and 3.48 g (0.02 m) of crystalline L- arginine and stirred vigorously for 10 to 15 hours. The product formed was filtered off, washed with acetone and dried. The yield of hemine arginate was 9.5 g (95%). The insoluble residue, determined by the above method, was 0.14 g (14%). EXAMPLE 2 Following the procedure described in Example I, 6.52 g (0.01 m) of crystalline hemine and 4.36 g (0.025 m) of crystalline L-arginine were reacted with each other. The yield of hemin arginate was 11.1 g (about 100%). The insoluble residue was 0.042 g (4.2%). EXAMPLE III Following the procedure described in Example I, 6.52 g (0.01 m) of crystalline hemine and 5.23 g (0.03 m) of crystalline L-arginine were reacted with each other. The yield of hemin arginate was 12.0 g (about 102%). The insoluble residue was 0.001 g (0.1%). EXAMPLE IV Following the procedure described in Example I, 6.52 g (0.01 m) of crystalline hemine and 6.10 g (0.035 m) of crystalline L-arginine were reacted with each other. The yield of hemine arginate was 12.0 g (95%). The insoluble residue was 0.0005 g (0.05%). Example V 40 Following the procedure described in Example I, 6.52 g (0.01 m) of crystalline hemine and 4.39 g of crystalline L-lysine (0.03 m) were reacted with each other. The yield of hemin lysinate was 10.8 g (99%) and the insoluble residue was 0.020 g (2.8%). This shows that the optimal hemin to arginate molar ratio is 1: 3 (Example III) because it gave the highest yield of hemin arginate, while the amount of insoluble residue was minimal. 45 1. A method of preparing a physiologically active water-soluble hemine compound, characterized in that crystalline hemine is reacted with L-arginine or L-lysine at ambient temperature and with vigorous stirring for 10 to 15 hours, in an aqueous organic solvent selected from the group consisting of acetone, isopropyl alcohol, methanol and ethanol, containing more than 3.2% by volume of water, but not more than 7% by volume of water, with a molar ratio of hemine to L arginine or L-lysine which is at most 0.5 but at least 0.25 is used. A medicament comprising as the active ingredient hemin arginate or hemin lysinate prepared by the method of claim 1.