CS247187B2 - Method of production of new rock complex - Google Patents

Abstract

A method for producing a novel water-soluble hemin complex with physiological activity for use in tablets or capsules or as a solid for the preparation of an injection material for the treatment of various types of anemia, especially those associated with porphyria, comprising reacting crystalline hemin with a basic amino acid, preferably L-arginine or L-lysine, at a molar ratio of hemin to L-arginine of 1:1 to 1:3 and hemin to L-lysine of 1:1 to 1:3, in a mixture of an organic solvent and water, preferably acetone and water in a volume ratio of 300:10 to 300:25, preferably 300:20 at room temperature with vigorous stirring for a period of 10 to 15 hours. The product is suitable in the form of tablets, capsules or powder for the preparation of injections for the treatment of anemia, especially those associated with porphyria.

Description

The invention relates to a method for producing a novel hemin complex, physiologically active and water-soluble. It is a hemin arginate or lysinate, which can be used in the form of capsules or tablets or in the form of a solid substance intended for injection after reconstitution with, for example, sterile physiological sodium chloride solution.

Hemin occurs in the body as a prosthetic group of hemoglobin in most cytochromes and in some enzymes. Hemoglobin is formed in the bone marrow. When hemin-containing proteins are broken down, hemin is released, but only a small part of this substance is then used to produce new hemin-containing proteins under normal physiological conditions.

Hemin is cleaved by hemin oxygenase to biliverdin, which is further reduced to bilirubin. Native intact hemoglobin is not a substrate for hemin oxygenase.

Defects in hemoglobin synthesis can be caused by impaired synthesis of the hemin or globin chain. Hemin synthesis can be impaired because a) one of the components necessary for its formation is missing or b) there is a dysfunction of the enzyme that catalyzes the formation of this substance.

a) Iron deficiency is a limiting factor for hemin synthesis. The body receives the necessary daily dose of iron of 1 to 2 mg in the diet. Iron deficiency can occur due to a diet poor in iron or due to the presence of iron-binding substances in the diet. Deficiencies in iron absorption can also lead to iron deficiency in the body, even though the dietary intake is sufficient. Regardless of the cause, iron deficiency sooner or later leads to anemia.

In vitamin B deficiency, which is relatively rare, iron absorption is normal, but the iron is not utilized by the cells. As a result, a type of sideroblastic anemia develops.

Iron deficiency anemia is treated either by oral iron preparations, usually in the form of sulfate or gluconate, or by injection of iron sorbitol. If iron absorption is impaired, there is no point in administering oral preparations. Iron will not even reach the cells of the intestinal mucosa.

Unlike inorganic iron, iron in hemin, where it is directly bound to hemin, is absorbed by cells even in cases of impaired iron absorption where conventional oral treatment is not possible.

This means that hemin-bound iron is the only known oral treatment for cases that are otherwise refractory to treatment. Hemin-bound iron is absorbed

4-5 times better than inorganic iron even in healthy people, as described in the publication Seppánen H and Takkunen H: Suomen Laákárilehti 36: 2 071-2 072, 1981.

b) Hemin synthesis is regulated enzymatically. Impaired function of enzymes catalyzing hemin synthesis can be either congenital or caused by external causes. In both cases, however, there is a reduced formation of hemin, which is manifested by the development of porphyria or some types of sideroblastic anemia or other diseases.

Porphyria is the most important group of diseases caused by impaired enzyme function. Patients experience an accumulation of porphyrins, intermediate products of hemin synthesis, and increased excretion of these substances in the urine and feces.

Most types of this disease manifest as an acute attack that is very difficult to manage.

In some cases, instead of porphyria, sideroblastic anemia of various types develops as a result of improper functioning of the enzymes involved in hemin synthesis. These sideroblastic anemias can also be congenital or acquired.

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The treatment of porphyria has so far been based mainly on the omission of certain substances and the administration of large amounts of carbohydrates during an acute attack, but the results have not been very good. Since the origin of porphyria has been clarified, hemin compounds, such as hematin, have been administered intravenously.

Hematin is very effective in the treatment of seizures, but in more than 50% of patients it causes inflammation of the veins with thrombosis. In addition, it is too unstable and therefore not suitable for industrial production. It is therefore clear that until now only some inadequate means have been available for the treatment of patients with porphyrias.

The invention aims to propose the production of a water-soluble substance containing hemin and iron for the treatment of certain types of anemia, with iron in this case being available directly in the hemin molecule.

This substance should be primarily intended for the treatment of porphyria, in which the normal production of hemoglobin is impaired for some reason. Since the compound is to be intended for oral administration in the form of tablets or capsules, as well as for injection, it must be soluble in water.

Hemin, which is poorly soluble in water, can be obtained in pure form from blood by extraction with hydrochloric or acetic acid from an aqueous solution of disrupted red blood cells.

Another method is based on extraction of hemin with acetone in the presence of, for example, histidylhistidine, pilocarpine or imidazole at pH 7.0 according to the publication Wakid N. W. ot Helou Κ. Y.:

Int. J. Biochem. 4, 259-267, 1973.

PCT Patent Application No. 813,749 (PCT/Fl81/00026) describes a process for the production of a water-soluble hemin concentrate in which approximately 40% by weight is hemin and the remainder is a blood substance of unknown nature. The product is intended for use in lyophilized form as an iron-containing food supplement or as a drug for anemia. '

The disadvantage is that the resulting product is a mixture of hemin and blood substance. Because the latter component is not uniform, the mixture is unsuitable for injection.

Porphyria has been treated until now with a hospital-prepared mixture obtained by dissolving hemin in a sterile sodium carbonate solution (hematin). Because this solution is unstable, it cannot be produced industrially on a large scale.

Furthermore, as mentioned above, hematin causes phlebitis with thrombosis at the injection site in approximately 50% of cases, probably due to the high pH of the solution. This represents a serious disadvantage which considerably reduces the usefulness of the substance.

The subject of the invention is a method for producing a new hemin complex, water-soluble, with physiological effect for use in tablets or capsules or as a solid for the preparation of injection material for the treatment of various types of anemia, especially those associated with porphyria, characterized in that crystalline hemin is reacted with a basic amino acid, namely L-arginine or L-lysine, at a molar ratio of hemin to L-arginine of 1:1 to 1:4 and hemin to L-lysine of 1:1 to 1:4, in a mixture of an organic solvent and water, preferably acetone and water in a volume ratio of 300:10 to 300:25, preferably 300:20 at room temperature with vigorous stirring for a period of 10 to 15 hours.

The composition of the solvent, especially the proportion of organic solvent to water, is very important for the use of the resulting product, which is surprising. The water content in the solvent mixture is so low, approximately 7%, that neither hemin nor the more soluble L-arginine dissolves in it. The reaction proceeds under control of the pH of the reaction solution. The resulting solution is separated and sent.

The hemin-containing compound is obtained in dry form and is soluble, which is essential from a medical and also from a pharmaceutical point of view.

The heroin molecule contains two carboxyl groups that react with the basic amino groups of L-lysine or L-arginine.

Heminarginate and hemin lysinate, prepared by the method of the invention, were dissolved in water and the pH of these solutions was measured and compared at different time intervals with the pH of the mechanical mixture of hemin and L-arginine/dissolved in water. The results are shown in Table 1.

Table 1

PH

0 minutes 60 minutes 24 hours Heminarginate 0.02937 g/25 ml 8.22 8.22 8.22 Hemin lysinate 0.02760 g/25 ml 8.10 7.97 8.13 Hemin + 0.01630 g/25 ml 10.13 9.81 9.33 L-arginine 0.01307 g/25 ml

The pH measurement results show that the pH of heminarginate and heminlysinate is constant, i.e. approximately 8 throughout 24 hours. On the other hand, the pH of the mechanical mixture slowly decreases, probably due to the slow reaction between the carboxyl groups of hemin and the amino group of the L-amino acid.

For this reason, it is not possible to obtain a product suitable for treatment by simply mixing it. Heminarginate and hemin lysinate, prepared by the method of the invention, are complex compounds in which the L-amino acid has reacted with the carboxyl groups of hemin.

In order to determine the optimal molar ratio between the two components and the most suitable solvent composition, the following experiments were performed with hemin and arginine:

Crystalline hemin and L-arginine were added to the reaction in molar ratios of 1:2 and 1:3 under vigorous stirring in a solvent consisting of organic solvent and water in various ratios. The precipitates formed were filtered off, washed and dried.

Water solubility was determined by dissolving and stirring for 1 hour a total of 1.0 g of heminarginate obtained in each experiment in 50 ml of distilled water.

The solutions were centrifuged at approximately 3500 rpm, the pellet was washed with 10 ml of distilled water and 10 ml of acetone, and then dried and weighed. The insoluble residue consisted mainly of unreacted hemin. The results of these experiments are shown in the following table 2

Table 2

Hemin: L-arginine

Weight (g) Molar fraction Solvent ml Temperature °C The rest 6.52:3.48 1:2 methanol 300 20 tar 6.52:3.48 1:2 ethanol 300 20 2^.100% 6.52:5.22 1:3 ethanol 300 40 20.2¾ 6.52:3.48 1:2 isopropanol 300 20 ~ 100% 6.52:3.48 1:2 isopropanol/water 300:20 20 21.2% 6.52:5.22 1 : 3 isopropanol/water 300:20 20 9.7% 6.52:3.48 1:3 acetone/water 300/15 20 16.8% 6.52:3.48 ] : 2 acetone/water 300/20 20 12.4% 6.52:5.22 1:3 acetone/water 300:10 20 •^100% 6.52:5.22 1:3 acetone/water 300:10 40 11.4% 6.52:5.22 1 : 3 acetone/water 300:15 20 8.3% 6.52:5.22 1:3 acetone/water 300:20 20 0.3%

continuation of table 2

Hemin: L-arginine

Weight (g) Molar fraction Solvent ml Temperature °C The rest 6.52:5.22 1:3 acetone/water 300:30 20 tar 6.52:5.22 1:3 acetone/water 150:10 20 4.2% 6.52:5.22 1:3 acetone/water 150:12.5 20 tar

The tar-like substance that formed in some experiments could not be converted into a powder form.

In practice, the most useful molar ratio of hemin to arginine is probably 1:3 and the most preferred solvent mixture is 300 ml of acetone and 20 ml of water, because hemin needs a small excess of L-arginine for the reaction to proceed properly.

The local effect of intravenously administered hemin compounds on surrounding tissues was monitored by injecting 5 ml/kg into the ear vein of rabbits (California White). A standard hemin carbonate solution (hematin) was used as a reference solution.

After heminarginate infusion, the surrounding tissue remained normal, meaning that sterile inflammation (thrombophlebitis) did not occur.

Similar results were observed after infusion of the corresponding hemin lysinate solution.

It can therefore be concluded that these substances do not cause thrombophlebitis when administered intravenously.

When hemin carbonate was administered in the same way, the skin around the vein became red and irritated, causing sterile inflammation (thrombophlebitis). This condition persisted three days after the injection of the hemin carbonate solution.

The physiological properties of various water-soluble hemin compounds have been investigated by examining the ability of hemin oxygenase to cleave these substances. The physiological substrate for the enzyme is methemalbumin, which is cleaved to biliverdin, which is further reduced to bilirubin by biliverdin reductase.

This means that excess hemin that the body cannot use is broken down primarily by heminoxygenase into bilirubin and other related substances, which are then excreted normally. Heminoxygenase is therefore the limiting enzyme.

In our enzymatic assays, the efficiency of methemalbumin was expressed as 100 to determine the ability of heminarginate and heminlysinate to serve as substrates for heminoxygenate. The corresponding value for heminarginate and heminlysinate was 106. The efficiencies of other hemin amine derivatives in which the amine moiety was diethanolamine, ethylamine, cyclohexylamine, or piperidine were 13, 21, 31, and 78, respectively.

It is therefore clear that both of the above-mentioned substances, i.e. heminarginate and heminlysinate, behave in the organism with respect to heminoxygenase as normal physiological compounds.

The invention will be illustrated by the following examples.

Example

6.52 g (0.01 ml) of crystalline hemin and 3.48 g (0.02 mol) of crystalline L-arginine are stirred vigorously for 10 to 15 hours in a beaker equipped with a mechanical stirrer and containing 300 ml of acetone and 20 ml of water. The resulting product is filtered off, washed with acetone and dried.

In this way, 9.5 g of heminarginate are obtained as a solid in a yield of 95%. The insoluble fraction, determined by the above method, is 0.13 g (14%).

Example 2

6.52 g (0.01 mol) of crystalline hemin and 4.35 g (0.025 mol) of crystalline L-arginine are processed according to the method of Example 1.

. In this way, 11.1 g of heminarginate is obtained in a yield of approximately 100%. The insoluble fraction is 0.042 g (4.2%). ^

Example 3

6.52 g (0.01 mol) of crystalline hemin and 5.23 g (0.03 mol) of crystalline L-arginine are processed as described in Example 1.

In this way, 12.0 g of heminarginate is obtained in a yield of approximately 102%. The insoluble fraction is 0.001 g (0.1%).

Example 4

6.52 g (0.01 mol) of crystalline hemin and 6.10 g (0.035 mol) of crystalline L-arginine are treated as described above in Example 1.

In this way, a total of 12.0 g of the resulting heminarginate is obtained in a yield of 95%. In addition, 0.0005 g (1.05%) of the resulting insoluble fraction is obtained.

Example 5

6.52 g (0.01 mol) of crystalline hemin and 4.39 g (0.03 mol) of crystalline L-lysine are processed according to the method of Example 1.

In this way, 10.8 g of hemin lysinate is obtained in a yield of 99%. The insoluble fraction is 0.020 g (2.8%).

It is clear that the optimal molar ratio of hemin to arginate is 1:3 as in Example 3, because in this case the highest yield of heminarginate and the lowest amount of insoluble fraction are obtained.

Claims (3)

A process for the production of a novel water-soluble hemin complex having a physiological effect for use in tablets or capsules or as a solid for the preparation of injectable material for the treatment of various types of anemia, in particular those associated with porphyria, characterized in that the crystalline hemin is reacted with a basic amino acid, L-arginine or L-lysine at a molar ratio of hemin to L-arginine of 1: 1 to 1: 4 and hemin to L-lysine of 1: 1 to 1: 4, in a mixture organic solvent and water, preferably acetone and water in a volume ratio of 300: 10 to 300: 25, preferably 300: 20 at room temperature with vigorous stirring for 10 to 15 hours. 2. The method according to claim 1, wherein the molar proportion of hemin to L-arginine is in the range 1: 2 to 1: 4, preferably 1: 3. 3. The method according to claim 1, wherein the molar proportion of hemin to L-lysine is 1: 3 to 1: 4, preferably 4: 3.