CA1242713A

CA1242713A - Process for preparation of a new hemin complex compound with medical applications

Info

Publication number: CA1242713A
Application number: CA000473658A
Authority: CA
Inventors: Raimo A. Tenhunen; Reino O. Tokola; Ritva L.A. Penttila; Grels D. Ingberg
Original assignee: Huhtamaki Oyj
Current assignee: Huhtamaki Oyj
Priority date: 1984-12-12
Filing date: 1985-02-06
Publication date: 1988-10-04
Also published as: ATA390784A; SE457958B; SE8406263D0; SU1384188A3; BE901319A; GB2168354A; LU85716A1; GB2168354B; NL192682B; JPH0647541B2; CH666273A5; SE8406263L; NL192682C; DE3446887C2; GB8431398D0; FR2574662B1; DE3446887A1; NL8403782A; AT389308B; FR2574662A1

Abstract

ABSTRACT

The present invention relates to a method for the preparation of a new, physiologically active, water-soluble complex compound of hemin arginate or hemin lysinate. Accord-ing to the invention crystalline hemin and the amino acid L-arginine or L-lysine, in a molar proportion of 1:3, are allowed to react at room temperature under vigorous stirring for 10 to 15 hours in a solvent mixture of acetone and water 300:20 v/v.
The hemin arginate or hemin lysinate thus formed is a powdery, stable compound suitable for use as raw material in tablets or capsules or as dry substance for preparation of injections for treatment of various types of anemia particularly anemias associated with porphyria.

Description

-:L- ~6~6~-2 Process for preparation of a new hemin complex compound with .
medical applications.

The present invention relates to a method for the preparation of a physiologically active water-soluble hemin arginate or hemin lysinate compound intended for use in tablets or capsules or as a dry substance for injection after reconstit-ution e.g. with sterile saline solution.
Hemin occurs 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 pro-teins decompose hemin is released, but only a minor part of it is used in the synthesis of new hemin proteins under normal physiological conditions. Hemin is split by the action of hemin oxygenase into biliverdin, which is further reduced to bilirubin.
Native, intact hemoglobin does not, na-turally, serve as a sub-strate for hemin oxygenase.
DeEects in the hemoglobin synthesis may be due to disturbecl synthqsis of hemin or of globin chains. The hemin synthesis may be ~isturbed because of (a) lack of some component necessary for the synthesis, or (b) dysfunction of an enzym~
catalyzing the synthesis.
a) Iron deficiency is the limiting factor in the hemin synthesis. The organism gets its daily requirement of iron (1-2 mg) with the foodO Iron deficiency may be due to a diet deficien~ in iron ~r~
'~

~ 646~-2.

or possibly to the presence of iron-bindiny com-pounds in the food. Disturbances in the iron ab-sorption mechanism may also lead to iron deficiency despite adecluate iron content in the ~ood. Re~ard-less of the cause, iron deficiency sooner or later leads to anemia.
~n the rarely occurring vitamin B6 deficiency the absorption of iron is normal, but its utilization by the cells is inhibited. As a consequence of this, a certain type of sideroblastic anemia develops.
Iron deficiency anemia is -treated either with oral iron preparations (e.g. iron sulphate or iron glu-conate) or, more rarely, with injections (iron sorbitol).
When the iron absorption mechanism is clisturbed, these conventional oral prepara-tions are useless;
the iron ~oes not even pene-tra-te into the cells of the intestinal mucosa. In contrast -to inor~anic lron, hemln lron, in whlch the iron ls bound to ~0 hemln, is absorbed by these cells even in such cases of dlsturbed lron absorption ~Jhich are resistant to conventional oral therapy~ Thus, hemin iron ls the only known, ef~ective remedy for oral treatment of therapy-resistant cases. Hemln iron has been found to be four to five times better absorbed than .. . .

J~ 3

-2~ 64-2 inorganic iron even in qui-te healthy subjects (Seppanen H & Takkunen H: Suomen La~karilehti 36:
2071-2072, 1981).
b) The synthesis of hemin is enzyma-tically regul~
ated. Impaired function of the enzymes ca-talyzing the hemin synthesis may be either hereditary or due to external factors. It invariably leads to de-creased formation of hemin, manifested by the dev-elopment of porphyria or certain kinds of sidero-blastic anemia or other diseases.

Porphyria i5 the most important group of diseasesresulting from impaired enzyme function. In por-phyria patients there is an accumulation of porphy-rins, intermediary products in -the hemin synthesis, and an increased excretion of these into urine and feces. Mosk kinds of porphyr:ia are manifes-ted by acute attacks which are extremely dlE:icult to ma~ter.
Sometimes, sicleroblastic anemias o cllferent kinds may develop instead of porphyria as a consequence of dysfunction of enzymes participating in the hemin synthesis. Sideroblastic anemias, too, may be either hereditary or acquired.
The treatment of porphyria has until now been based principally on the avoidance oE certain drugs and -2b- 26~64~2 the administrakion of large amounts of carbon hy-drates during the acutR attacks, but the effect has been poorO Since the etiology of porphyria became clarified, intravenous treatment with hemin com-pounds ~hematin) has been continuously gaining ground. Hematin has proved effective in the treat-ment of porphyria attacks, but in more than 50% of the patients it has caused thrombophlebitis. More-over, it is very unstable and therefore unsuitable for production on an industrial scale. There are thus very few possibilities for effective treatment of porphyria patients.

-- . .

The aim oE the presen-t inventlon was to produce a water-soluble hemin iron compound for trea-tmen-t of certain kinds of anemia, with the iron ready at hand, so to speak, in the hemin molecule~ The compound is intended in the first place for treatmen-t of porphyria, where the normal production of hemoglobin is dis-turbed for some reason or other. As the compound is intended for oral adminis-tration in tablets or capsules as well as for injection, it mus-t be wa-ter-soluble.
Hemin, 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 a water solu-tion of hemolyzing erythrocytes. Another method is based on the ex-trac-tion of hemin with acetone in the presence of e.g. histidylhis-tidine, pilocarp-ine, or imidazole a-t pH 7.0 (Wakid ~I.W. & Helou K.Y.: In-t. J.
Biochem. 4 : 259-267, 1973).
'I'he PCT patent application no. 8137~9 (PCT/F'181/00026) describes a method Eor the preparation oE a water-soLuble hern:in Goncentra-te in which about ~Orb w/w is hemin and the rest -is a llbl.oocl r;uhst:rl~cell ol~ unknown nature. rt'he product is interlded Eor 2() uC,e in lyophi:lized form as an iron supplement in food or as an antianemic drug.
The drawback of -this method is that the final product is a mixture of hemin and "blood substance". As the latter component is no-t uniform, the mixture is unsui-table for injection.
Porphyria has been treated in hospitals with a mixture prepared extempore by dissolving hemin in a sterile sodium ~7~ 3 carbonate solution (hematin). As this solution is unstable it cannot be ma~ufactured as a commercial product on a large scale.
Moreover, hematin causes thrombophlebitis at the site of injection in about 50% of the cases, probably because of the high pH of the solution. This is a serious drawback which reduces the usefulness of the product considerably.
The present invention relates to a method of preparing a water-soluble hemin compound by allowing sparingly water-soluble, s-~ a~'~ ClCt~
crystalline hemin to react with a suitable ~ 7-en~r~~~ ~o-~eh~
such as L-lysine or L-arginine, in a solvent mixture containing e.g. water and acetone. The composition of the solvent, i.e. the proportion of organic solvent to water, affects the medical value of the final product. The water content in the solvent mixture is preferably so low (about 7%) that neither hemin nor the fairly readily soluble L-arginine is dissolved. The reaction takes place under vigorous stirring and the pH of the solution is continuously controlled. The product formed is separated and dried; the ,` ' ,~
": :
!:` ``
1` "~

,, "

hemin compound is thus obtained in dry Form and is soluble ;n water~ which i5 essential from the medical as well as the pharmacote(hnical polnt of vlew.
The hemin molecule contains two carboxyl groups which react with the basic amino groups of L-lysine or L-arginine.
Hemin arginate and hemin lysinate prepared according to the inven-t;on were dissolvéd in water, and the pH of the solutions was measured and compared at different time points with the pH of a mechanical mixture of hemin and L-arginine dissolved in waterO The results are seen in Table 1.

Table 1.
.. _ _ ..... . _ pH __ _ _ 0 m;n. 60 min. 24 h Hemin arginate 0.02937 9/25 ml 8.22 8.22 8.22 Hemin lysinate 0.02760 9/25 ml 8.10 7.97 8.13 Hemin ~ 0.01630 9/25 ml 10.13 9.81 9.33 L-arginine 0.01307 .
The results of the pH measurements show tha-t the pM of the hemin arginate and hemin lysinate is stable (pH about 8) -for up to 24 hours. The p~l of the mechanical mixture, on the other hand, decreases very slowly, probably owiny to thc e~tremely slow reaction bekween the carboxyls in the hemin and the arn~no group in the L-amino acid~ A therapeutically use~ul proc!uct cannot therefore be obtained by this method. Hemin arginate and hemin lys~nate prepared according to the invention consist of a complex compound where the L-amino acid has reacted with the hemin carboxyls.
To determine~ on the one hand, the optima7 molar relation bet~een the two reactants and, on the other, the most suitable composition of the solvent mixture, the following tests were performed with hemin and arginine:
Crystalline hemin and L-arginine in molar proportions of 1:2 and 1:3 were allowed to react, under vigorous stirring, in a solvent mixture con-sistiny of an organic solvent and water in varying proportions. The pre-cipitates formed were filtered off~ washed and dried.
The solubility in water was determ;ned by dissolving, under vigorous stirring for about one hour, about 1.0 9 of the hemin arginate obtained in each test in 50 ml of distilled water.

-5~ 2646~~Z
The solutions were centrifuged (about 3500 r/min.
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 consisted mainly of unreacted hemin. The test results are presented in Table 2.
Table 2.
.
hemin : L-arginine water weights molar temp. insoluble (g) proportion solvent ml Cresidue 6.52: 3.48 1:2 methanol 300 20tar 6.52: 3.48 1:2 ethanol 300 20~100%
6.525.22 1:3 " 300 ~020.2%
6.52: 3.48 1:2 isopropanol 300 20~L00%
6.52: 3.48 1:2 isopropanol/water 300:2020 21.2%
6.52: 5.22 1:3 " 300:20 20 9.7%
6.52: 3.48 1:2 acetone/water 300:15 20 16.8%
6.52: 3.4~ 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 ~0 11.4%
6.52: 5.22 1:3 " 300:15 20 8.3%
6.52: 5.22 1:3 " 300:20 20 0.3 6.52: 5.22 1:3 " 300:30 20 tax 6.52: 5.22 1:3 " 150:10 20 4.2 6~52: 5.22 1:3 '' 150:12.5 2,0 tar The tarry substance formed in some of the tes-ts cou:l.d not be kransEerred into powder form.
The practical molar proportion of hemin to arginine L0 was found to be 1:3 and the most suitable solvent mixture 300 ml of acetone and 20 ml of watert because hemin needs a sli.ght ex~
cess of L-arginine to react properly.
The local effect of intravenously infused hemin com-pounds on surrounding tissues was studied by means of infusing '~7 -5a- 2646~-2 5 mg/kg lnto the auricular vein of California White rabbits. A
conven-tional hemin carbonate solution (hematin) was used as reference solution.
After infusion of hemin arginate solution the -tissue surrounding the vein remained normal, i.e. no sterile inf:Lammat-ion (thrombophlebitis) occurred.
A similar resuLt was seen after infusion of a corresponding hemin ;

lysinate solution. Thus, it can be concluded that -the compounds do not cause thrombophlebitis when infused intravenously.
When a hemin carbonate solution was adminis-tered in the same manner, the tissue surrounding the vein became red and irritated; i.e., a manifest sterile inflammation (thrombophlebitis~
developed. Three days after the infusion of the hemin carbonate solution the thrombophlebitis was still manifest.
The physiological character of the different water-soluble hemin compounds was assessed by testing the ability of hemin oxygenase to split the compounds. The physiological sub-strate for hemin oxygenase, methemalbumin, is split by this into biliverdin, which is fur-ther reduced to bilirubin by biliverdin reductase. Thus, the excess hemin which the organism cannot utilize is decomposed, in the Eirst place, by hemin oxygenase into bilirubin and other, closely related substances, which a.re then normally excre-ted. The reaction rate l:imit:ing erlzyme is thus hemln oxygenase.
Irl ou:r enzymat:i.c ana:Lyses, performed :in order to ~Eind out, .Eor one thing, the ability of hemin arginate and hemin lysinate to serve as substrates for hemin oxygenase, the activity of the reference substrate methemalbumin was expressed by 100.
The corresponding value obtained for hemin arginate and hemin lysi.nate was 106. The activities of other hemin amine der.ivati.ves, where the amine component was diethanol amine, ethyl amine, cyclohexyl amine or piperidine, were found to be 13, 21, 31 and /8 respectively. The tests show that hemin arginate and hemin -6a-lysinate behave in the organism like normal physiological cornpounds with regard to hemin oxygenase.
The invention is illustrated by the following examples:
Example 1.
6.52 g of crystalline hemin (0.01-M) and 3.48 g of crystalline L-arginine (0.02 M) in a beaker provided with a mechanical stirrer and containing a solvent mixture of 300 ml of acetone and 20 ml of water were vigorously stirred 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 method men-tioned above:
0.14 g (14%).

'~ 3 Example 2.
6.52 9 of crystalline hernin (0.01 M) and 4.36 g of cr~s-talline L-arginine (0.025 M) were treated as described in example ~.
Yield of hemin arginate: 11.1 y (about 100%). Insoluble residue:
0.042 g (4~2%).

Example 3.
6.52 g of crystalline hemin (0.01 M) and 5.23 9 of crystalline 1.-arginine (0.03 M) were treated as described in example 1.
Yield of hemin arginate: 12.0 9 (about 102%). Insoluble residue:
~.001 g (0~ 1%)o Example 4.
6.52 y of crystalline hem-in ~0.01 M) and 6.10 g o-F crystalline L-arginine (0.035 M) were treated as described in example 1.
Yield of hemin arginate: 12.0 y (95%). Insoluble residue: 0.0005 9 (O~D5%).

Example 5.
6.52 g of crystalline hemin ~0.01 M) and 4.39 9 oF crystalline L~lysine (0.03 M) were treated as described in example 1.
Yield of he~in lysinate: 10.8 y (99%). Insoluble residue: 0~020 9 . . (2~8%).
.
It appears that the optimal molar proportion of hemin ~o aryinate is 1:3 (example 3), because this gave the hiyhest yield of hemin arginate, while the amount of insoluble residue was minimal,

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for preparing a physiologically active, water-soluble hemin compound comprising reacting crystalline hemin with a basic amino acid, in a mixture of an organic solvent and water, at room temperature with vigorous stirring for 10 to 15 hours, whereby a complex of hemin arginate or hemin lysinate, is formed.

2. A method according to claim 1, wherein the basic amino acid is L-arginine and the molar proportion of hemin to L-arginine is 1:1 - 1:4.

3. A method according to claim 1, wherein the basic amino acid is L-lysine and the molar proportion of hemin to L-lysine is 1:1 - 1:4.

4. A method according to claim 1, wherein the solvent is a mixture of acetone and water in a proportion of 300:10 - 300:25 v/v.

5. A method according to claim 1, wherein the basic amino acid is L-arginine or L-lysine and the water-soluble hemin arginate or hemin lysinate formed in the reaction between hemin and L-arginine or L-lysine is used in powder form in capsules, as dry substance for injection after reconstitution with sterile saline solution, or together with a vehicle in tablets, for treat-ment of anemia.

6. A method according to claim 1, wherein the basic amino acid is L-arginine or L-lysine.

7. A method according to claim 1, wherein the organic solvent is acetone.

8. A method according to claim 2 wherein the molar pro-portion of hemin to L-arginine is 1:3.

9. A method according to claim 3, wherein the molar pro-portion of hemin to L-lysine is 1:3.

10. A method according to claim 4, wherein the acetone and water is in a proportion of 300:20.