LU85716A1 - PROCESS FOR THE PREPARATION OF A NEW COMPLEX HEMINE COMPOUND WITH MEDICAL APPLICATIONS - Google Patents

Description

BL-3683 / vdw

Process for the preparation of a new complex hemin compound having medical applications.

The present invention relates to a process for the preparation of a physiologically active hemin lysinate or hemin arginate compound, physiologically active and intended for use in tablets or capsules or in the form of a dry substance for injection. after reconstitution, for example, with sterile saline solution.

10 Hemine is present in the body as a prosthetic group of hemoglobin in most cytochromes and in certain enzymes. Hemoglobin is synthesized in the bone marrow. When hemin proteins break down, haem is released, but only a small part of it is used in the synthesis of new hemin proteins under normal physiological conditions. Under the action of hemin oxygenase, hemin is cleaved into biliverdin which is further reduced to bilirubin. Of course, intact natural hemoglobin does not serve as a substrate for hemin oxygenase.

Deficiencies in the synthesis of hemoglobin may be due to the disturbed synthesis of hemin or globin chains. The synthesis of hemin can be disturbed: a) by lack of a component necessary for synthesis, or b) by dysfunction of an enzyme catalyzing synthesis.

a) Iron deficiency is the limiting factor in the synthesis of hemin. The body gets its daily iron requirement (1-2 mg) from food. The iron deficiency can be due to a diet low in iron or possibly to the presence of iron-fixing compounds in nour-.35 riture. Disturbances occurring in the mechanism

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Iron absorption can also lead to iron deficiency even if the food has an adequate iron content. Whatever the cause, iron deficiency sooner or later leads to anemia.

5 In the event of vitamin Bg deficiency, which rarely occurs, iron absorption is normal but its use by cells is inhibited.

As a result, there is a certain type of sideroblastic anemia.

10 Anemia due to iron deficiency is treated either with oral iron preparations (for example, iron sulphate or iron gluconate) or, more rarely, with injections (iron sorbitol). When the iron absorption mechanism is disrupted, these conventional oral preparations are unnecessary; the iron does not even penetrate the cells of the intestinal mucosa. Unlike inorganic iron, hemin iron (iron attached to hemin) is absorbed by these cells even in cases of disturbed iron absorption which resist conventional oral therapy. Hemin iron is therefore the only effective remedy known for the oral treatment of therapy-resistant cases. Hemin iron has been found to be 4 to 5 times better absorbed than inorganic iron even in perfectly healthy subjects (Seppanen H & Takkunen H: Suomen Lääkärilehti 36: 2071-2072, 1981).

b) The synthesis of hemin is regulated enzymatically. The disrupted function of enzymes catalyzing the synthesis of hemin may be inherited or due to external factors. It invariably leads to reduced hemin formation manifested by the development of porphyria or certain types of sideroblastic anemia or other diseases.

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Porphyria is one of the most important group of diseases resulting from the disturbed function of enzymes. In patients with porphyria, there is an accumulation of porphyriins (intermediates in the synthesis of hemin), as well as increased excretion of these porphyrins in the urine and feces. Most types of porphyria manifest themselves as acute attacks which are extremely difficult to control.

Sideroblastic anemias of different types can sometimes develop in place of porphyria due to the dysfunction of the enzymes involved in the synthesis of hemin. Likewise, sideroblastic anemias can be either hereditary or acquired.

Until now, the treatment of porphyria has been based mainly on the withdrawal of certain drugs and on the administration of large quantities of carbohydrates during acute attacks; however, the effects have been poor. As the etiology of porphyria has been clarified, intravenous treatment with hemin compounds (haematin) has steadily gained ground. Hematin has been shown to be effective in treating attacks of porphyria, but in more than 50% of patients it has caused thrombophlebitis. In addition, it is very unstable and therefore it is not suitable for production on an industrial scale. As a result, there are very few possibilities for effective treatment of patients with porphyria.

The object of the present invention has been the production of a water-soluble hemin iron compound “J" ”r“ “

Jk-r 4 iron being, so to speak, available in the hemin molecule. This compound is primarily intended for the treatment of porphyria in which the normal production of hemoglobin is disrupted for one reason or another. Since this compound is intended for oral administration in the form of tablets or capsules, as well as in the form of injection, it must be water-soluble.

Hemin, which is sparingly soluble in water, can be obtained in pure form in the blood by extraction with a mixture of hydrochloric acid or acetic acid from an aqueous solution of hemolyzing erythrocytes. Another method is based on the extraction of hemin with acetone, for example, in the presence of histidylhistidine, pilo-carpine or imidazole at a pH of 7 (V / akid N.W. &

Helou K.Y. : Int. J. Biochem. 4: 259-267, 1973).

PCT Patent Application No. 813,749 describes a process for the preparation of a water-soluble concentrate of hemin comprising about 40% w / w hemin, the remainder being a "blood substance" of unknown nature. This product is intended to be used in lyophilized form as an iron supplement for food or as an anti-anemic drug.

The disadvantage of this process is that the end product is a mixture of hemin and a "blood substance". Since the latter component is not uniform, the mixture 30 is unsuitable for injection.

In hospitals, porphyria was treated with an extemporaneous mixture prepared by dissolving hemin in a sterile solution of sodium carbonate (haematin). Since this solution is insurable, it cannot be made as a suitable product.

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5 large-scale mercial. 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 disadvantage ’5 considerably reducing the usefulness of the product.

The present invention relates to a process for the preparation of a water-soluble hemin compound by reacting crystalline hemin poorly soluble in water with an appropriate base, for example, an amino acid such as L-lysine or L-arginine, in a mixture of solvents containing, for example, water and acetone. The composition of the solvent, i.e. the proportion of the 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 (around 7%) that neither hemin nor fairly readily soluble L-arginine is dissolved. The reaction takes place 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 from the medical point of view and from the pharmacotechnical point of view.

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

The hemin arginate and the hemin lysinate prepared according to the invention were dissolved in; water, the pH of the solutions was measured and compared at various times with the pH of a mechanical mixture of hemin and L-arginine dissolved in "water." The results are shown in Table 1.

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1 · TABLE 1 | R I | 0 mid | 60 mi- | 24 hours 111 - 5 _ nute nutes res mk l Hemin arginate 0.02937 g / 25 ml | 8.22 | 8.22 | 8,22 B Hemine lysinate 0.02760 g / 25 ml 8.10 7.97 8.13 | Hemlock + 0.01630 g / 25 ml | | | K j L-arginine 0.01307 g / 25 ml j 10.13 d 9.81 d 9.33 BB The results of the pH measurement reveal BK that the pH of hemline arginate and HS lysinate hemin is stable (pH: about 8) during a SK „· * period of up to 24 hours. On the other hand, the

At this point the pH of the mechanical mixture decreases very slowly, probably due to the extremely slow reaction between the carboxy groups of hemin and the group ^^^ BraB; friend no L-amino acid. Consequently, a therapeutically useful product cannot be obtained by this method. The hemine arginate and the hemine lysinate prepared according to the invention consist of a complex compound in which the L-amino acid reacted with the carboxy groups of the hemine.

In order to determine, on the one hand, the optimum molar ratio between the two reactants and, on the other hand, the most suitable composition of the mixture of solvents, the tests described below were carried out with hemin and l 'arginine.

Hemin and L-arginine were reacted in the crystalline state in molar proportions of 1: 2 and 1: 3 while stirring vigorously in a mixture of solvents consisting of an organic solvent and water in varying proportions. By filtration, the precipitates formed were separated, they were washed at Λ 7 * *

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

The solutions were subjected to centrifugation (about 3,500 rpm) and the residue was washed with 10 ml of distilled water and 10 ml of acetone, after which it was dried and weighed. The insoluble residue 20 consisted mainly of unreacted hemin. The results of the tests are given in table 2. = ·, TABLE 2 25 Hemin / L-arginine Solvent ml Tempera- Residue

Weight Proportion ture (° C) insoluble (g) _molar ___ in water 6.52: 3.48 1: 2 Methanol 300 20 Tar 6.52: 3.48 1: 2 Ethanol 300 20 - 100% 20 6.52: 5.22 1: 3 "300 40 20.2% 6.52: 3.48 1: 2 Isopropanol 300 20 - 100% 6.52: 3.48 1: 2 Isopropanol / 300: 20 20 21.2% water 6.52: 5.22 1: 3 "300: 20 20 9.7% 25 6.52: 3.48 1: 2 Acetone / ean 300: 15 20 16.8% 6.52: 3.48 1: 2 "300: 20 20 12.4% 6.52: 5.22 1: 3" 300: 10 20 ~ 100% 6.52: 5.22 1: 3 "300: 10 40 11.4%.

6.52: 5.22 1: 3 "300: 15 20 - 8.3% 30 6.52: 5.22 1: 3" 300: 20 20 0.3% 6.52: 5.22 1: 3 "300: 30 20 Tar 6.52: 5.22 1: 3" 150: 10 20 4.2% 6.52: 5.22 1: 3 * "150: 12.5 20 Tar i- * 8

In some tests, the tarry substance could not be transformed into powder.

It has been found that the practical molar ratio between hemin and arginine is 1: 3 and that the. The most suitable solvent mixture was 300 ml of acetone and 20 ml of water, since hemin requires a slight excess of L-arginine to react properly.

The local effect of intravenous infused hemin compounds on peripheral tissue was studied by infusing 5 mg / kg into the ear vein of California white rabbits. As a reference solution, a conventional solution of hemin carbonate (hematin) was used.

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

A similar result was observed after infusion of a corresponding solution of hemin lysinate. It can therefore be concluded that the compounds do not cause thrombophlebitis when infused intravenously.

When a solution of hemin carbo-nate was administered in the same way, the tissue surrounding the vein became red and irritated, i.e. manifest sterile inflammation developed ( thrombophlebitis). Three days after the infusion of the hemin carbonate solution, the thrombophle-30 bite was still evident.

The physiological character of the various water-soluble hemin compounds was determined by testing the ability of hemin oxygenase to cleave. the compounds. The physiological substrate for the hemin oxy-35 I genase, namely methemalbumin, is cleaved

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k 9 by the latter into biliverdin which is further reduced to bilirubin by biliverdin reductase. Therefore, the excess hemin that the body cannot use is broken down, first, by the hemin oxy-5 genase into bilirubin and other closely related substances which are then normally excreted. Consequently, the enzyme limiting the reaction rate is hemin oxygenase. In the enzymatic analyzes carried out by Applicant 10 to discover the ability of hemin arginate and hemin lysinate to act as substrates for hemin oxygenase, the activity of the reference substrate, namely methemalbumin, was expressed by 100. The corresponding value obtained for hemin argi-15 nate and hemin lysinate was 106.

It was found that the activities of the other amino derivatives of hemin in which the amino component was diethanolamine, ethylamine, cyclohexylamine or piperidine, were 13, 21, 31 and 78, respectively. The tests demonstrate that the hemin arginate and hemin lysinate have behaved in the body similar to that of normal physiological compounds with respect to hemin oxygenase.

The invention is further illustrated by the following examples.

Example 1

For 10 to 15 hours, in a beaker comprising a mechanical stirrer and containing a mixture of solvents consisting of 300 ml of acetone and 20 ml of water, 6.52 g of crystalline hemin were vigorously stirred (0, 01M) and 3.48 g of crystalline L-arginine (0.02M). By filtration, the product formed was separated, washed with acetone and dried. Hemin arginate yield: 9.5 g (95%). 35 insol insoluble residue determined by the above process: 0.14 g, 10 Γ (14%).

Example 2

6.52 g of crystalline hemin (Ο, ΟΙΜ) and 4.36 g of crystalline L-arginine (0.025M) were treated as described in Example 1. Yield of hemin arginate: 11.1 g (around 100%). Insoluble residue: 0.042 g (4.2%).

Example 3

6.52 g of crystalline hemin 10 (Ο, ΟΙΜ) and 5.23 g of crystalline L-arginine (0.03M) were treated as described in Example 1. Yield of hemin arginate: 12 g ( about 102%). Insoluble residue: 0.001 g (0.1%).

EXAMPLE 4 6.52 g of crystalline hemin (Ο, ΟΙΜ) and 6.10 g of crystalline L-arginine (0.035 M) were treated as described in Example 1. Yield of hemin arginate: 12 g (95%). Insoluble residue: 0.0005 g (0.05%).

20 Example 5

6.52 g of crystalline hemin (Ο, ΟΙΜ) and 4.39 g of crystalline L-lysine (0.03M) were treated as described in Example 1. Yield of hemin lysinate: 10.8 g (99%). Insoluble residue: 0.020 g 25 (2.8%).

It appears that the optimal molar ratio between hemin and arginate is 1: 3 (Example 3), because this ratio gave the maximum yield of hemin arginate, while the amount of residue inso-30/1 fad was minimal.

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Claims (5)

11 1. Process for the preparation of a new water-soluble and physiologically active hemin compound intended for use as raw material * 5 in tablets or capsules or in the form of a dry substance for the preparation of injections intended for the treatment of different types of anemia, in particular, anemia associated with porphyria, characterized in that hemisinis 10 talline is reacted with a basic amino acid, preferably L-arginine or L- lysine, in a mixture of an organic solvent and water, preferably acetone and water, at room temperature, while stirring vigorously for 10 to 15 hours, to form <15 thus a complex compound consisting hemin arginate or hemin lysinate respectively. 2. Method according to claim 1, characterized in that the molar ratio between hemin and L-arginine is 1: 1 - 1: 4, preferably 1: 3. 3. Method according to claim 1, characterized in that the molar ratio between hemin and L-lysine is 1: 1-1: 4, preferably 1: 3. 4. Method according to claim 1, charac-25 terized in that the solvent mixture consists of acetone and water in a ratio of 300: 10-300: 25 (volume / volume), preferably 300 : 20. 5. Method according to claim 1, characterized in that the hemin arginate or the water-soluble hemin lysinate formed during the reaction between * hemin and L-arginine or L-lysine is used in powder in capsules, in the form of a dry substance for injection after reconstitution with sterile saline, or together with a 35/1 vehicle in tablets, for the treatment of f 12 different types of anemias, in particular , the anemias associated with porphyria. ^