DD249028A5 - METHOD FOR PRODUCING A NEW HAEMIN COMPLEX COMPOUND WITH MEDICAL AVAILABILITY - Google Patents

Abstract

The invention relates to a process for the preparation of a novel, physiologically active, water-soluble complex compound of hemaringinate or hememin lysinate. According to the invention, crystalline haemin and the amino acids L-arginine or L-lysine react at a molar ratio of 1: 3 at room temperature with vigorous stirring over a period of 10 to 15 hours in a solvent mixture of acetone and water 300: 20 vol / vol. The hemisphere material or haeminylsinate formed is a powdery, stable compound and suitable for use as a starting material in tablets or capsules or as a dry substance for the preparation of injection solutions for the treatment of various types of anemia, in particular porphyria-associated anemia.

Description

Field of application of the invention

The present invention relates to a process for the preparation of a physiologically active, water-soluble Häminarginat- or Häminlysinatverbindung that in tablets or capsules or as a dry substance for injection after re-dissolution z. B. can be used with sterile saline.

Well-known technical solutions

Hemin is found in the organism as a prosthetic group of hemoglobin in most cytochromes and in certain enzymes. Hemoglobin is synthesized in the bone marrow. When hemin proteins are decomposed, hemin is released but only a minor portion thereof is used for the synthesis of new hemin proteins under normal physiological conditions. Hemin is cleaved by heme oxygenase in biliverdin, which is further reduced to bilirubin. Natural, intact hemoglobin, of course, does not serve as a substrate for hemin oxygenase.

Defects in hemoglobin synthesis can lead to impaired synthesis of hemin or globin chains. The hemin synthesis can be disturbed a) by the lack of components necessary for the synthesis or b) by malfunction of a synthesis catalyzing enzyme.

a) Iron deficiency is the limiting factor of hemin synthesis.

The organism needs 1-2 mg of iron daily through the diet. Iron deficiency may be due to a dietary deficiency of iron or possibly the presence of iron-binding compounds in the diet. Disruptions in the iron absorption mechanism can also lead to iron deficiency, regardless of the corresponding iron content of the food. Irrespective of the cause, iron deficiency sooner or later leads to anemia.

In the rare case of vitamin B ₆ deficiency, iron absorption is normal, but cell utilization is inhibited. As a result, a certain type of sideroblastic anemia develops.

Iron deficiency anemia is treated with either oral iron supplements (eg iron sulfate or iron gluconate) or, more rarely, injections (iron sorbitol). When the iron absorption mechanism is disturbed, these conventional preparations are unusable; The iron does not penetrate easily into the cells of the intestinal mucosa. In contrast to inorganic iron, the hemin iron, in which the iron is bound to hemin, is absorbed by these cells, especially in cases of disturbed iron absorption, which are not countered by conventional oral therapy. Thus, hemin-iron is the only known effective remedy for the oral treatment of drug-resistant cases. It has been found that hemin iron is absorbed four to five times better by the same test subjects than inorganic iron (Seppänen, H. and Takkunen, H .; Suomen Lääkärilehti 36: 2071-2072, 1981).

b) The hemin synthesis is enzymatically regulated. A damaging function of the enzymes that catalyze the synthesis of hemin may be either hereditary or caused by external factors. This inevitably leads to degraded hemin forms, as evidenced by the occurrence of porphyria or certain types of sideroblastic anemia or other diseases.

Porphyria is one of the most important disease group, which results from a damaged enzyme function. In porphyria patients, circulation of porphyrins, intermediates of hemin synthesis and increased secretion of these into the urine and stool occurs. Most types of porphyria are characterized by acute seizures that are extremely difficult to control.

In part, sideroblastic anemias of various types may develop in place of porphyria as a result of the dysfunction of enzymes involved in hemin synthesis. Sideroboastic anemias may also be either hereditary or acquired.

The treatment of prophylaxis to this day is based in principle on the prevention of certain drugs and the administration of large amounts of carbohydrates during acute seizures, but the effect is low. Since the etiology of porphyria has been elucidated, intravenous treatment with hemin compounds (hematin) has been steadily increasing. Hematin has been shown to be effective in the treatment of porphyria attacks, but more than 50% of patients have experienced thrombophlebitis. In addition, it is very unstable and therefore unsuitable for industrial scale production. Thus, there are only limited possibilities for the effective treatment of porphyria patients. The object of the present invention is to produce a water-soluble hemin-iron compound for the treatment of various anemia types in which the iron is present in the hemin molecule. The compound is intended primarily for prophylactic treatment where normal hemoglobin production is disturbed for one reason or another. Since the compound should be suitable both for oral administration in tablets or capsules and for injection, it must be water-soluble.

The only slightly soluble in water hemin can be obtained in pure form from blood by extraction with a mixture of hydrochloric acid or acetic acid from an aqueous solution of hemolyzed erythrocytes. Another method is based on the extraction of hemin with acetone in the presence of e.g. Histidy I histidine, pilocarpine or imidazole at pH 7.0 (Wa kid, N.W. and Helon, K.Y., Int J. Biochem 4: 259-267, 1973).

PCT Patent Application No. 813749 (PCT / FI 81/00026) describes a process for producing a water-soluble heme concentrate in which about 40% (mass / mass) is hemin and the remainder is a "blood substance" of unknown type. The product is intended The disadvantage of this method is that the final product is a mixture of hemin and the "blood substance". Since the latter component is not consistent, the mixture is unsuitable for injection.

Porphyria was treated in clinics with a mixture prepared immediately before by dissolving hemin in a sterile solution of sodium carbonate (hematin). Since this solution is unstable, it can not be manufactured on a commercial scale as a commercial product. In addition, hematin induces thrombophlebitis in the injection site in about 50% of cases, presumably because of the high pH of the solution. This is a serious drawback that significantly reduces the usefulness of the product.

Object of the invention

It is an object of the invention to substantially eliminate these disadvantages.

Essence of the invention

The invention has for its object to produce a water-soluble hemin-iron compound for the treatment of various anemia types, in which the iron is present in the hemin molecule.

According to the invention, the process for producing a water-soluble hemin compound in a minor reaction of water-soluble, crystalline hemin with a suitable base, for. As an amino acid such as L-Ly si η or L-arginine, in a solvent mixture containing water and acetone. The composition of the solvent, i. the proportion of organic solvent and water is surprisingly important for the medicinal value of the final product. The water content in the solvent mixture is so low (about 7%) that neither hemin nor the readily soluble L-arginine is dissolved.

The reaction is carried out with vigorous stirring and the pH of the solution is continuously monitored. The product formed is separated and dried; the hemin compound is thus obtained in dry form and is soluble in water, which is essential in both medical and pharmacotechnical terms.

The urinary molecule contains two carboxy groups which react with the basic amino groups of L-lysine or L-arginine.

Häminarginat and Häminlysinat prepared in accordance with the invention were dissolved in water and the pH of the solutions was measured and compared at different times with the pH of a mechanical mixture of hemin and L-arginine dissolved in water.

The results are shown in Table 1.

Table 1

pH OMin. 60 min. 24 h

Heminine 0.09937 g / 25 ml 8.22 8.22 8.22

Hemin Lysine 0.02760g / 25ml 8.10 7.97 8.13

Hemin + 0.01630g / 25ml 10.13 9.81 9.33

L-arginine 0.01307 g / 25 ml 10.13 9.81 9.33

The results of the pH measurements show that the pH of hemin arginate and hemin lysinate is stable (pH about 8) for up to 24 hours. In contrast, the pH of the mechanical mixture decreases very slowly, probably because of the extremely slow reaction between the carboxy groups in the hemin and the amino group in the L-amino acid. Therefore, no therapeutically useful product can be obtained by this method. Heminicginate and hemin lysinate prepared according to the invention consist of a complex compound in which the L-amino acid has reacted with the heminic carboxy groups.

In order to determine both the optimal molar relationship between the two reactants and the most suitable composition of the solvent mixture, the following tests were performed with hemin and arginine:

Crystalline hemin and L-arginine were allowed to react in molar ratios of 1: 2 and 1: 3 with vigorous stirring and a solvent mixture consisting of an organic solvent and water in different proportions was used. The precipitates formed were filtered off, washed and dried.

Solubility in water was determined by dissolving about 1.0 g of hemin arginate obtained in each experiment in 50 mL of distilled water with vigorous stirring for about one hour.

The solutions were centrifuged (at about 3500 rpm) and the residue washed with 10 ml of distilled water and 10 ml of acetone, then dried and weighed. The insoluble residue consisted essentially of unreacted hemin. The results are included in Table 2.

Hemin: L-arginine

Dimensions molar ratio solvent ml Temp. ⁰ C water-insoluble rest 6.52: 3.48 1: 2 methanol 300 20 tar 6.52: 3.48 1: 2 ethanol 300 20 = 100% 6.52: 5.22 1: 3 ethanol 300 40 20.2% 6.52: 3.48 1: 2 isopropanol »300 20 = 100% 6.52: 3.48 1: 2 i-propanol / water 300: 20 20 21.2% 6.52: 5.22 1: 3 i-propanol / water 300: 20 20 9.7% 6.52: 3.48 1: 2 Acetone / water 300: 15 20 16.8% 6.52: 3.48 1: 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% 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 tarry substance obtained in some experiments could not be converted into a powder form.

The substantially different ratio of hemin to arginine was found to be 1: 3, and the most suitable solvent mixture was 300 ml of acetone and 20 ml of water, which hemin requires a slight L-arginine excess for the good reaction.

The local effect of intravenously infused hemin compounds on the surrounding tissue was investigated by infusing 5mg / kg into the auricular vein of California White, using a standard heme carbonate solution (hematin) as the reference solution and then infusing the hemarinargin solution into the surrounding tissue The vein normal, ie no sterile inflammation (thrombophlebitis) occurred.

A similar result was seen after infusion of a corresponding hemin lysinate solution. It can be concluded that the compounds cause no thrombophlebitis on intravenous infusion.

When a hemocyanin solution is administered in the same way, the tissue around the vein becomes red and irritated, i. H.

it develops a sterile inflammation (thrombophlebitis). Three days after infusion of the hemicarbonate solution, thrombophlebitis was still present.

The physiological character of the various water soluble hemin compounds was assessed by testing the ability of hemin oxygenase to cleave the compounds. The physiological substrate for hemin oxygenase, methaemalbumin, was cleaved into biliverdin, which was further reduced to bilirubin by biliverdin reductase. Thus, the excess hemin, which the organism does not need, is decomposed, primarily by heme oxygenase in bilirubin and other related substances, which are then excreted normally. The reaction rate limiting enzyme is thus hemin oxygenase.

In enzymatic analyzes carried out with the aim of finding, on the one hand, the ability of hemin arginate and hemin lysinate to serve as substrates for hemin oxygenase, the activity of the reference substrate methaemum albumin was expressed as 100. The corresponding value obtained for hemoglobin and hemin lysine was 106. The activities of other hemin derivatives in which the amine component was diethanolamine, ethylamine, cyclohexylamine or piperidine were respectively 13, 21, 31 and 78. The tests show that hemin arginate and Häminlysinat behave like normal physiological compounds with respect to Häminoxygenase in the organism.

The invention is best illustrated by the following examples:

Step Example! 1

6.52 g of crystalline hemin (0.01 mol) and 3.48 g of crystalline L-arginine (0.02 mol) were placed in a beaker provided with a mechanical stirrer and containing a mixed solvent of 300 ml of acetone and 20 ml of water. stirred vigorously for 10 to 15 hours. The product formed was filtered off, washed with acetone and dried. Yield of hemin arginate: 9.5 g (95%). Insoluble residue determined by the above method: 0.14 g (14%).

Example 2

6.52g of crystalline hemin (0.01 mol) and 4.36g of crystalline L-arginine (0.025 mol) were as described in Example 1. Yield of hemin arginate: 11.1 g (about 100%); insoluble residue: 0.042g (4.2%).

Example 3

6,52g crystalline hemin (0.01 mol) and 5,23g crystalline L-arginine (0.03 mol) of _(as described in Example 1 _(treated Häminarginat yield. 12.0 g (about 102%); insoluble residue : 0.001 g (0.1%).

Example 4

6.52g of crystalline hemin (0.01 mol) and 6.10g of crystalline L-arginine (0.035 mol) were treated as described in Example 1. Yield of hemin arginate: 12.0 g (95%); insoluble residue: 0.0005g (0.05%).

Example 5

6.52 g of crystalline hemin (0.01 mol) and 4.39 g of crystalline L-lysine (0.03 mol) were treated as described in Example 1. Yield of hemin lysinate: 10.8 g (99%); insoluble residue: 0.020g (2.8%).

It appears that the optimum molar ratio of hemin to arginate is 1: 3 (Example 3), since the highest yield of hemin arginate was obtained while the amount of insoluble residue was minimal.

Claims (4)

1. A process for the preparation of a novel, physiologically active, water-soluble hemin compound for use as a starting material for tables or capsules or as a dry substance for the preparation of injections for the treatment of various types of anemia, in particular associated with porphyria anemias, characterized in that crystalline hemin with a basic amino acid , preferably L-arginine or L-lysine, in a mixture before organic solvent and water, preferably acetone and water, at room temperature with vigorous stirring for 10 to 15 hours with each other to form a complex compound was formed from hemin arginate or Häminlysinat consists. 2. The method according to item 1, characterized in that the molar ratio of hemin to L-arginine 1: 1 to 1: 4, preferably 1: 3. 3. The method according to item 1, characterized in that the molar ratio of hemin to L-lysine 1: 1 to 1: 4, preferably 1: 3. 4. The method according to item 1, characterized in that the solvent mixture of acetone and water in a ratio of 300: 10 to 300: 25 Vof / vol is, preferably 300: 20.