NL8403782A - METHOD FOR THE PREPARATION OF A HEMINE COMPLEX AND THE THERAPEUTIC USE THEREOF - Google Patents

Description

* * * »-1- 24378 / Vk / mvl

Short designation: Process for the preparation of a hemin complex and its therapeutic application.

The present invention relates to a process for the preparation of a new physiologically active water-soluble hemin compound which can be used as starting material for the preparation of medicines. The invention furthermore relates to the medicaments in the form of tablets, capsules or dry matter for the preparation of injections for the treatment of various types of anemia, in particular anemia associated with metabolic disorders (porphyria). More particularly, the invention also relates to a process for the preparation of a physiologically active water-soluble hemine arginate or hemine lysinate, intended for use as an active agent in medicaments such as tablets or capsules or as a dry matter for the preparation of an injection liquid, namely after mixing with, for example, sterile saline.

Heroin occurs in the organism as a prosthetic group of hemoglobin in most cytochromes and certain enzymes.

Hemoglobin is prepared in the marrow bone. When hemin proteins decompose, hemin is released, but only a small fraction of this is used for the synthesis of new hemin proteins under normal, physiological conditions. Hemine is cleaved by the action of hemine oxygenase to biliverdin, which is further reduced to bilirubin. The internally obtained, intact hemoglobin does not in nature serve as a substrate for hemine oxygenase.

Hemoglobin synthesis abnormalities can be attributed to impaired hemin or globin chain synthesis.

Hemin synthesis can be disrupted by a) lack of some component necessary for synthesis or b) malfunction of an enzyme that catalyzes the synthesis.

a) The lack of iron can be the limiting factor in the synthesis of hemin. The organism obtains the required daily amount of iron (1-2 mg) with the food. A lack of iron can be blamed on the lack of iron in the diet or possibly due to the presence of iron binding compounds in the food. Disturbances in the iron absorption mechanism can also lead to a deficiency of iron 3403782 -2-2A378 / Vk / mvl * ί, * despite an adequate iron content in the food. Irrespective of the cause, a shortage of iron will sooner or later lead to anemia.

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

Iron deficiency anemia is treated by the oral administration of iron preparations (such as iron sulfate or iron gluconate) or optionally by 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, in which iron is bound to hemin, is absorbed by these cells, even in those cases of impaired iron absorption, which are resistant to conventional oral therapy. Thus, hemin iron is the only known effective remedy in the oral treatment of the diseases in which a resistance to the therapy followed occurs. Hemin iron A has been found to be absorbed up to 5 times better than inorganic iron, even in 20 fairly healthy individuals (H. Seppanen and H. Takkunen: Suomen Laakarilehti 36: 2071-2072, 1981).

b) The synthesis of hemin is controlled enzymatically.

Disturbance of the enzymes that catalyze hemin synthesis can be attributed to hereditary factors or 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 most important group of diseases caused by the reduced functioning of the enzymes. Porphyria-30 patients accumulate porphyrins, which are intermediates in the synthesis of hemin and an increased rejection thereof to urine and faeces. Most types of porphyria are manifested by acute attacks that are very difficult to overcome.

Sometimes sideroblastic anemia of different types can be developed instead of porphyria due to malfunction of the enzymes participating in hemin synthesis. Sideroblastic anemia can also be hereditary or develop later.

8403732 -3- 24378 / Vk / mvl

Treatment of porphyria has hitherto been mainly based on the avoidance of certain drugs and the administration of large amounts of carbohydrates during the acute attacks, but the effect has so far been weak. Since the cause of porphyria has been clarified, intravenous treatment with hemin compounds (hematin) has progressed more and more. Hematin has been shown to be effective in treating porphyria attacks, but thrombophlebitis has been caused in more than 50% of patients. Moreover, it is very unstable and therefore unsuitable for industrial scale preparation. Thus, there are very few options for effective treatment for porphyria patients.

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

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 by the hemolysis of 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 containing about 40% by weight of hemin and the remainder is a "blood preparation" of unknown nature. is intended to be used in 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 matter". Since the latter component 35 is not homogeneous, the mixture is not suitable for injection.

Porphyria has been treated in hospitals with a mixture prepared on-site or for immediate use by dissolving hemine at 3403732-4-24378 / Vk / mvl Γr * 41 in sterile sodium carbonate (hematin) solution. Since this solution is not stable, it can be prepared as a large-scale 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 use of the product.

The present invention relates to a process for the preparation of a water-soluble hemine 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, ie the amount of organic solvent relative to water, is surprisingly important for the medical value of the final product. The water content o 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 product formed is separated and dried; the hemin compound is thus obtained in the dry state and is soluble in water, which is important from a medical point of view as well as from a pharmaceutical point of view.

The heminera 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 were dissolved in water and the pH of the solutions was measured and compared at different times with the pH of a regular mixture of hemine and L-arginine dissolved in water. The results are shown in Table A.

30 TABLE A

ph _; 0 min. 60 min. 24 h.

hemin arginate 0.02937 g / 25 ml 8.22 8.22 8.22 35 hemin lysinate 0.02760 g / 25 ml 8.10 7.97 8.13 heroin + 0.01630 g / 25 roll 10 13 9 81 9 33 L-arginine 0.01307 g / 25 ml * 8403782. v ^ -5- 24378 / Vk / mvl

The results of the pH measurements indicate that the pH of the hemin arginate and hemin lysinate is stable (pH about 8) for 24 hours. On the other hand, the pH of the ordinary mixture drops very quickly, probably due to the extremely slow reaction between the 5 carboxylic acid groups in the hemine and the amino group in the L-amino acid.

Therefore, a therapeutically suitable product cannot be obtained by this method. Hemin arginate and hemin lysinate, prepared according to the invention, consists of a complex compound in which L-amino acid has reacted with the heraine carboxylic acid groups.

On the other hand, to determine the optimal molar ratio between the two reagents and, on the other hand, the most suitable composition of the solvent mixture, the following experiments were performed with hemine and arginine.

Crystalline hemine and L-arginine in molar amounts 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 and from about 1.0 g of hemin arginate obtained in each experiment in 50 ml of distilled water.

The solutions were centrifuged (about 3500 rotations / minute) 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.

3403782 -6- 24378 / Vk / mvl 9 '>

TABLE B

hemine: L-arginine ratio tempera in water ____ solvent ml insoluble weight. / ° r \ barable residue (g) m ° lair (%) 6.52: 3.48 1: 2 methanol 300 20 tar 6.52: 3.48 1: 2 ethanol 300 20 * 100 10 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 isopropanol / water 300: 20 20 21.2 6.52 s 5.22 1: 3 isopropanol / 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 20 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 25 6.52: 5.22 1: 3 acetone / water 150: 12.5 20 tar

The tar-like substance that is formed in some experiments can be converted into the powder form. The determined 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 needs a slight excess of L-arginine to react properly *

The local influence of hemin compounds administered intravenously on the surrounding tissue was investigated by infusing 5 mg / kg into the auricular vein of "California White" rabbits. A conventional hemin carbonate solution (hematin) was used as the reference solution.

B 4 0 3 7 8 2 4 Λ * Έ -7- 24378 / Vk / mvl

After infusion of the hemin-arginate solution, the tissue around the vein remained normal, so that 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 administered intravenously.

When a herain decarbonate solution was administered in the same manner, the tissue around the vein became red and irritated, indicating that a manifest sterile inflammation (thrombophlebitis) had developed. Thrombophlebitis was still detectable 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.

The physiological substrate for hemin oxygenase, metheralbumin, is thereby 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 secreted.

The enzyme that limits the reaction rate is thus hemineoxygenase.

In the present enzymatic analyzes performed to firstly find the ability of hemin arginate and hemin lysinate to serve as a substrate for hemin oxygenase, the activity of the comparative substrate methemalbumin was printed at 100. The corresponding value obtained for hemin arginate and hemin lysinate was 106. The activities of other hemine amine derivatives, the amine component being 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 in the organism as normal physiological compounds with respect to hemine oxygenase.

The invention will be further elucidated with reference to the following examples.

Example I

In a beaker equipped with a mechanical stirrer and with a solvent mixture of 300 ml of acetone and 20 ml of water, 6.52 g of «403782 * 7 µg -8- 24378 / Vk / ravl (0.01 mol) of crystalline hemine and 3 , 48 g (0.02 mol) 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 hemin arginate was 9.5 g (95%). The insoluble residue, determined by the above method, was 0.14 g (14%).

Example II

Following the procedure described in Example I, 6.52 g (0.01 mol) of crystalline hemine and 4.36 g (0.025 mol) of crystalline 10 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

According to the procedure described in Example I, 6.52 g (0.01 mol) of crystalline hemine and 5.23 g (0.03 mol) 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 mol) of crystalline hemine and 6.10 g (0.035 mol) 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

Following the procedure described in Example I, 6.52 g (0.01 mol) of crystalline hemine and 4.39 g of crystalline L-lysine (0.03 mol) 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 molar ratio of hemin to arginate is 1: 3 (Example III) because it gave the highest yield to hemin arginate, while the amount of insoluble residue was minimal.

Thus, the present invention relates to a process for the preparation of a novel physiologically active water-soluble complex compound of hemin arginate or hemin lysinate. Crystalline hemine and the amino acid L-arginine or L-lysine in the molar ratio of 1: 3 are reacted with ambient stirring for 10 to 15 hours in a solvent mixture at ambient temperature 8407782 -9- 24378 / Vk / mvl. acetone and water in a volume ratio of 300: 20 · The hemin arginate or hemin lysinate thus formed is a powdery, stable compound suitable for use as an active compound in tablets or capsules or as a dry substance for injection preparations for the treatment of various types of anemia , especially anemia associated with porphyria. Thus, the invention also relates to the drugs thus prepared.

'i 3 v' Y / V L

Claims (9)

Method for the preparation of a new physiologically active water-soluble hemin compound to be used as a starting material for the preparation of tablets or capsules or as a dry substance for the preparation of an injection liquid, characterized in that crystalline hemine is reacted with a basic amino acid, especially L-arginine or L-lysine, in a mixture of an organic solvent and water, such as acetone and water, at ambient temperature under vigorous stirring for 10 to 15 hours, thereby forming a complex compound consisting of hemin arginate or hemin lysinate. A method according to claim 1, characterized in that the molar ratio of hemine to L-arginine is 1: 1-1: 4. 3. Method according to claim 2, characterized in that the ratio is 1: 3. Process according to claim 1, characterized in that the hemin to L-lysine molar ratio is 1: 1-1: 4. Method according to claim 4, characterized in that the ratio is 1: 3. 6. Process according to claims 1-5, characterized in that the solvent mixture consists of acetone and water in a volume ratio of 300: 10-300: 25. Process according to claim 6, characterized in that the acetone-water ratio is 300:20. Process according to claim 1, characterized in that the water-soluble hemine arginate or hemine lysinate formed in the reaction between hemine and L-arginine or L-lysine is used in powder form, in capsules, as a dry substance for injection after formulating with sterile saline or together with carriers into tablets for the treatment of various types of anemia, especially anemia associated with porphyria. Medicament, characterized in that it contains, as active substance, hemin arginate or hemin lysinate, prepared according to a method described in claims 1-8. Eindhoven, December 1984 1403782 35