CS203992B2 - Method of making the injectable derivates of the ergothein with antiinflammation effect - Google Patents

Abstract

N-alkylated, N-carbamylated and esterified orgoteins, like the native protein, possesses superoxide dismutase and anti-inflammatory activity.

Description

The invention relates to a process for the production of injectable orgotein derivatives having anti-inflammatory activity.

ORGOININ refers to members of the family of water-soluble analogues of proteins in practically pure, injectable form, i.e., virtually free of other proteins that accompany or are associated with them in the sources of their occurrence. U.S. Pat

758,682 describes pharmaceutical compositions comprising an orgotein.

Orgotein methanoproteins are members of a family of protein analogs characterized by a characteristic sum of physical, chemical, biological, and pharmacodynamic properties. Each of these analogs is physically characterized in that it is an isolated, practically pure form of globular, water-soluble and buffer-soluble proteins having a highly compact natural structure which, although heat-sensitive, is stable when heated at 65 ° C. at pH up to 10 for several minutes. Chemically, each of these analogs is characterized in that it contains all protein amino acids in the molecule up to 0 to 2, a small percentage of carbohydrates, no fats, 0.1 to 1.0% of the metal due to the content. 1 to 5 gram-moles / mol of at least one chelated divalent metal having an ion diameter in the range of 0.60 to 1.00 X ¹⁰ m, and virtually no chelated mono-metals or those which are cellular poisons.

The composition of the -orgotein analogs with respect to amino acids is largely unchanged regardless of the source from which they are isolated.

Table I shows the amino acid residue distribution for several orgotein analogues, considered for a molecular weight of 32,500.

It can be seen from Table I that the orgotein analogs have 18 to 26, usually 20 and 23 lysine groups, all of which except for 1 to 3 have titratable (trinitrobenzenesulfonic acid) ε-amino groups. The invention therefore relates to a process for the production of orgotein derivatives in which at least a portion of the lysine groups of the orgotein is alkylated, and further to derivatives of orgotein in which at least a portion of the lysine groups of the orgoteins are carbamoylated.

It is further evident from Table I that the -orgotein analogs have 29-37 aspartic acid groups and 21-38 glutamic acid groups. The invention therefore further relates to a process for the production of orgotein derivatives in which at least a portion of these free acid residues are esterified.

Table I

Amino acid composition of several orgotein analogues (Number of residues in 1 mole, molecular weight = 32,500)

Amino acids of red blood cells

liver, beef cattle beef cattle sheep horse hog dog alanine 19 Dec 19 Dec 18 18 18 16 arginine 8 8 10 6 '8 8 aspartic acid 37 36 35 35 31 29 cystine-1/2 6 6 6 6 6 6 glutamic acid 21 23 22nd 30 28 30 glycine 53 52 52 51 52 53 histidine 16 16 14 20 May 16 15 Dec isoleucine 18 18 18 14 16 18 leucine 17 17 17 18 16 16 lysine 22nd 21 23 26 23 20 May methionine 2 2 2 2 2 6 phenylalanine 8 8 7 9 8 8 proline 12 13 15 Dec 10 10 10 serine 17 17 14 14 13 20 May threonine 26 25 20 May 16 27 Mar: 20 May tryptophan ¹ 0 0 0 0 0 0 tyrosine ^{1 2} 2 2 2 0 4 2 valine 33 32 31 29 29 34 total 317 315 306 304 307 311

Tab. 1 Amino acids rabbit rat red blood cells guinea pig chicken human range alanine 19 Dec 22nd 22nd 23 22nd 16—23 arginine 8 7 8 8 8 6—10 aspartic acid 34 30 34 36 37 29—37 cystine-1/2 6 6 4 10 8 4—10 glutamic acid 25 38 29 26 28 21—3'8 glycine 54 54 53 56 51 51-56 histidine 17 20 May 15 Dec 17 14 14—20 isoleucine 16 16 1’8 15 Dec 17 14—18 leucine 19 Dec 12 17 12 20 May 12-20 lysine 21 18 20 May 21 23 18—26 methionine 3 4 2 3 1 1-6 phenylalanine 9 6 8 8 8 6—9 proline 13 10 12 13 12 10—15 serine 18 18 18 15 Dec 19 Dec 13—20 threonine 21 17 17 18 18 16—27 tryptophan ¹ 0 0 0 1 2 0—2 tyrosine ² 0 2 l? 2 (J 0—4 valine 31 32 32 30 30 29—35 total 315 315 309 317 318 304—318

¹ colorimetric determination ² average of amino acid analysis and spectrophotometric determination

As will be appreciated by those skilled in the art, some eisterification agents and conditions are capable of simultaneously alkylating the free amino groups in the orgotein molecule and esterifying the carboxylic acid groups. The production of such N-alkyl and esterified orgoteins is also part of the invention.

Among other things, the natural orgotein protein has anti-inflammatory activity as disclosed in U.S. Pat. No. 3,758,682. It also has a uniquely high activity of sulphoperoxide dismutase as described by McCord and Fridovich in J. Biol. Chem., 244, 6, 176, (1970) and 246, 2,875, (1971).

Surprisingly, the anti-inflammatory activity of the natural protein is virtually unchanged by esterification, N-alkylation or carbaimylation. Accordingly, the orgotein derivatives of the invention are useful in the same manner as the natural orgotein, for example, for treating inflammatory conditions in mammals and other animals, as described in U.S. Patent 3,758,682.

A substantial portion of the natural protein superoxide dismutase activity is also retained in the orgotein derivatives of the invention, for example, 20-100% of the activity of the natural protein.

as mentioned above, the orgotein analogs contain 1-8 to 26 lysine groups. Since the orgotein molecule consists of two identical peptide chains (subunits), there are half of these lysine groups in each of these chains, which join together firmly but not covalently under mild temperature and pH conditions. Due to the spatial structure of the orgotein molecule, the ε-amino groups of a few lysines in each of these chains are usually not titratable with thiolitolic acid and are therefore not readily available. Available for alkylation or carbamoylation. However, both alkylation and carbamoylation of non-titratable ε-amino lysine groups also appear to be possible using a highly active alkylating or caramoylating agent, for example dimethyl sulfate, at an alkaline pH.

The extent of alkylation or carbamoylation may be determined by reducing the number of amino groups reacting with tinnitrobenzenesulphonic acid, taking into account. the fact that 1 to 3 lysines of the native orgatinine protein are non-titratable with trinitrobenzenesulfonic acid. For example, only 18 of its 20 to 22 lysines are detected in bovine orgotein. It should also be remembered that mylled ^and lysine-containing lysines are still acylated, so that only the largely alkylated orgoteins are characterized by a reduction in the number of acylated lysine groups.

The dialkylation and thialkylation or cation-amylation of the lysine groups can be monitored by the change in charge in electrophoresis of the N-alkylated or N-catoyl-broken product. Also, for example, for an dimethyl sulphate-alkylated orgotein at pH 10, there will be a charge change of -2 upon acylation with acetic anhydride as compared to -20 for unalkylated orgotein. Similarly, the methyl-isothiocyanate N-carbamoylated orgotein at pH 9 exhibits a charge change of -2 after 45 minutes.

As is known, electrophoretic ion mobility is a function of electric field strength, effective ion charge (including coupled counter ions) and friction coefficient, see, for example, C. Tanford, "Physical Chenistry of Maoromolecules," published by Wiley, New York (1969). Since friction coefficients depend on the size and shape of the molecule and the composition of the solution, comparisons of different proteins are not very informative. However, when comparing proteins of similar size and shape, in this case orgotein molecules, chemically modified by relatively small groups, under identical electrophoretic conditions, the only variable affecting this electrophoresis is mobility.

With this in mind, the electroerotic behavior of many chemically modified orgotein molecules is in agreement. The natural bovine orgotein provides predominantly one interface (lane 1) in electrophoresis, with fewer, faster moving interfaces (lane 2,3, etc.), evenly spaced from the main interface, representing orgotein molecules with a larger ratio of -COOH to -NH 2 groups than interface molecules 1.

Treatment of the natural bovine orgotein, for example gradually with higher concentrations of acetic anhydride at pH 7 or with methylisothiocyanate at pH 9, results in a series of progressively more anodic (moving towards the positive electrode) electrophoretic bands as more and more free amino groups of orgotein molecules . Conversely, treatment with dimethyl sulfate yields a series of bands progressively more cathodic (distant from band 1 towards the negative electrode) as more and more free carboxylic acid groups of the orgotein molecule are gradually esterified.

Graphical representation of the distance traveled by the interface from the start on the belt number is relatively linear for the low conversion ranges of the -COOH or -NH2 groups, but gradually becomes curved at higher conversion rates because there is a limit to how fast even the most charged components can solution to move. The faster moving components are also more sensitive to salt concentration and are substantially retarded by electrophoresis of salt-containing samples.

For this reason - because the extrapolation of the positions of more than about two bands is not always accurate - for accurate charge determination, the unknown substance needs to be subjected to electrophoresis with a solution containing all bands from 1 to the appropriate position (for example - partially acetylated or partially (an orgyme).

All conventional protein modification reactions that have been applied to an orgotein molecule to date agree with this interpretation, i.e. the position of the bands corresponds to the overall. charge changes over the natural orgotein molecule. Both acetylation and carbamoylation and N-methythiocarbamylation provide bands of 2, 3, 4, 5, etc., indicating that the 1, 2, 3 and 4 free amino groups have been chemically modified. Similarly, the succinylation by which the ~ NH3 ⁺ groups are converted into groups

-NHOCHCHaCOO—,

II o

provides bands 3, 5, 7, 9, etc., and more extensive carbamoylation or thiocarbamoylation, which converts yet other groups into groups

-NHCNH — R a

II.

O ~ NHCNH — R,

II p

provides bands of 6, 7, 8, 9, etc. Esterification with dimethyl sulfate or ethyldiazoacetate affords bands of -1, 2, 3, 4, 4, etc., indicating that 1, 2, 3, or 4 carboxylic acid groups were chemically modified.

Generally speaking, most, for example, all lysines except 2 and 4, can be alkylated, even with milder alkylating agents, or carbamoylated. All but about one of the available (i.e., titratable trinitro-bromosulfonic acid) lysine groups in each of the orgotein peptide subunits may be polyalated using stronger alkylation conditions, for example - excess dimethyl sulfate, 0.04 M carbonate buffer at pH 10.

As can be expected, when less than all of the titratable lysine amino groups are alkylated or carbamoylated, the distribution of the alkyl or carbamoyl groups on the orgotein molecule is likely to be random, since none of the titratable lysine amino groups appears abnormally readily alkylated or convertible to carbamoylated amino groups. Because the orgotein molecule is composed of two identical peptide chains, the alkyl groups of the partially alkylated orgotein and the N-carbamoylated amino group of the partially carbamoylated orgotein will be distributed more or less randomly over each of the two peptide subunits, but more or less equally for both. Since a single alkylating or carbamoylating reagent is usually used, all alkyl or carbamoylated amino groups will be identical. However, it is also possible to prepare alkylated orgoteins and carbamoylated orgoteins having two or more different alkyl groups in the molecule, and even in each of the two chains.

One way to prepare a mixed alkyl orgotein is by step alkylation using various alkylating agents. For example, a portion of the titratable ε-amino lysine groups can be alkylated using a slight concentration of one alkylating agent, for example iodoacetamide, and the remainder of the reactive amino groups can be alkylated using a higher concentration of another alkylating agent, for example dimethylsulfate. What is meant by a low or high concentration of alkylating agent will depend on the relative reaction rates with the protein amino groups and the solvent and thus will depend on the pH of the reaction medium and the alkylating agent and to a lesser extent in ¹ and buffer and temperature.

One way to prepare an orgotein with mixed N-carbamoylated groups is to carbamoylate in stages using various carbamoylating agents. For example, part of the titratable lysine ε-amines may be carbamoylated with a slightly reactive carbamoylating agent, for example KNCO, and the rest of the reactive amino groups may be carbamoylated with a more reactive carbamoylating agent, for example methyl isocyanate. The reactivity of the carbamoylating agent depends on the proportional reaction rates with the protein amino groups and the reaction solvent, and thus on the pH of the reaction medium and the carbamoylating agent, and to a lesser extent buffer and temperature.

Another method to prepare a mixed alkylated or - mixed N-carbamoylated orgotein is by hybridization. The term "orgotein hybridization" refers to the formation of a mixed orgotein from the peptide chains of two different orgotein molecules, for example, A 2 and B 2, wherein A and B represent their respective peptide chains (A 2 + B 2 → 2 AB). The charge of the heterodimer, AB, in electrophoresis should be the average of the charges of the homodimers A2 and B2, provided that in all cases the same part of each of the two subunits is involved.

Both ε-N-methylorgotein, produced by alkylation of a natural orgotein molecule with excess dimethyl sulfate in 0.04 M carbonate buffer at pH-10, and ε-N-ethyllorgotein, prepared by alkylating orgotein in the same manner with diethyl sulfate, and ε-N-propylcarbamoylorgotein, prepared by carbamoylation natural orgotein molecules with an excess of -propylisocyanate in 0.1 M tris or phosphate buffer pH 7.6, or finally ε-N- (1-thiocarbamoylorgotein) produced by carbamoylation of the orgotein with methyl isocyanate in 0.075 M sodium tetraborate solution , they may all be hydrated with natural orgotein or heated together at -50 ° C for 4 hours.

As can be seen, these hybrid semi-alkylated and semi-carbamoylated orgotein molecules may be further alkylated or carbamoylated with a different alkylating or carbamoylating agent to form a hybrid alkylated or carbamoylated orgotein in which the -alkyl groups and optionally carbamoylated -amino groups in a single -peptide chain from these groups in the second chain.

The ε-N-alkyl chloro-proteins and ε-N-carbamoylor-proteins of the invention appear to have virtually the same spatial structure as the natural orgotein molecule. The content of chelated Cu ⁺⁺ and Zn ++ ions (grammatoms per mole) is approximately the same as that of an orgotein. Like an orgotein, they are highly resistant to pronase and degradation by other proteolytic enzymes. is reduced depending on the degree of carbamoylation.

Although the predominant structural modification of the natural orgotein molecule that occurs during its alkylation or carbamoylation at alkaline pH, in the case of alkylation, monoalkylation, dialkylation and, to a lesser extent, trialkylation of the ε-N-amino groups of its lysine residues, and carbamoylation in carbamoylation of these ε-amino groups can. be free amino groups of its arginine residues and free carboxylic acid groups of aspartic acid and glutamic acid residues, as well as other alkylable groups present in the molecule, in particular -OH, and imidazole nitrogen and optionally also guanidine nitrogen, -SH, -SCH3 etc. simultaneously alkylated or carbamoylated according to the conditions employed and the reactivity of the alkylating or carboylating agent. For example, while at pH 10 alkylation with iodoacetamide appears to be merely ε-N-alkylation, alkylation with dimethyl sulfate at pH 10 is less selective, while introducing methyl groups elsewhere into the molecule. Also such concomitantly alkylated orgoteins having ε-N-alkyl groups are included among the novel compounds of the invention. However, in the case of carbamoylation, such concomitantly modified groups are labile and readily hydrolyzable in aqueous solutions within a day or even shorter, depending on the pH.

The course of the alkylation, if it involves the alkylation of -COOH groups, and the course of carbamoylation can be monitored directly by the change in total electrophoretic charge and by the formation of new bands in the electrolyte. Similarly, N-alkylation can be performed because it converts the otherwise acylated -N- -12 per acylation-resistant group, monitored by a reduction in the number of acylated amino groups compared to the natural orgotein molecule.

The exact nature of the N-alkyl and N-carbamoyl groups, as well as their number, is not critical as long as the alkyl or carbamoyl residue is physiologically suitable. Due to the higher molecular weight of the orgotein molecule, although the orgotein molecule is fully alkylated or carbamoylated with lower molecular weight alkyl or carbamoyl groups, e.g. g 100, the effect on the overall chemical composition is relatively small, i.e. less than. 100 percent. Alkylation and carbamoylation also have no significant effect on the spatial arrangement of the molecule and on the resulting stability, for example, against heating at 60 degrees Celsius for 1 hour and against attack by proteolytic enzymes.

As will be further understood, the alkyl or carbamoyl group must also be derived from an alkylating or carbamoylating agent capable of alkylating or carbamoylating the amino group in water or buffer solution, since the reaction is usually carried out in this medium.

Such alkylating agents include primary dialkyl sulfates: (R 6 SO 2 wherein R = CH 3, C 2 H 5, n-C 5 H 2, n-C 1 -1 H, activated alkyl halides: JCH 2 COX, where X = OH, NH 2, benzyl bromide and allyl bromide activated vinyl groups: CH 2 = CHX) wherein X = CN, SOCH 3 SCC 2 H 5, · CCCH 3 reducing alkylating agents: R.COOR-BH 4 or BHaCN ", wherein R and R '= H = CH, CH 3, C 2 H 5, C 6 H 5.

The method of reductive alkylation of proteins with aromatic aldehydes and sodium cyanoborohydride is described by Friedman M. et al. in Int. J. Peptide Protein Res., 6, 1974, pp. 183-185. alkylation. acrylonitrile then Means and Feeney in the book "Chemical Modifitacion of Proteins", chapter 6, 114-117, by Fletcher J. C., in Biochem. J. 98 34C (1966), Friedman M. et al. in J. Amer. Chem. Soc. 87, p. 3672 (1965) and Friedman M. et al. in J. Org. Chem., 31, 2888 (1966).

Examples of suitable carbamoylating agents include alkali metal cyanates such as NaNCO, KNCO, alkyl isocyanates and alkyl isothiocyanates such as RNCO and RNCS where R = CH3, C2H5, n-C1Hr, n-CdH9, n-CaHiz, and aryl isocyanates and isocyanates such as phenylisocyanate and phenylisothiocyanate.

Although metal cyanates react with many types of side chain, it provides a stable product. only their reaction with amino groups to convert them into the urea group.

The reaction of alkylisothiocyanates and alkylisocyanates with orgotein is quite similar to the reaction of cyanates with orgotein, only much faster. Long chain isocyanates are less reactive than short chain isocyanates.

The reaction of orgoteins with isocyanates and isothiocyanates can usually be monitored using a reagent that introduces a fluorescent moiety such as fluorescein isothiocyanate into the molecule. This reagent is similar to alkyl isocyanates and reacts with amino groups of orgoteins to form substituted thioureas. However, even a low degree of substitution greatly reduces the activity of superoxide orgotein dismutase relative to the amount of fluorescein. Monosubstituted and disubstituted orgoteins are not stable in solution and gradually become more heterogeneous as a result of exchange between subunits and the consequence of hydrolysis.

Since the alkyl moiety in the molecule is not important, unless it is physiologically toxic and can be formed on lysine ε-amino groups, the optional equivalents of the above-described hydrocarbon alkyl groups, if any, can be formed, cyclopentyl, cyclohexyl,. methyl and the like cycloalkyl, cyclohexylmethyl, N-cyclopentylpropyl, and the like cycloalkylalkyl, benzyl, p-xylyl and phenethyl and the like aralkyl groups.

Possible equivalents are alkyl groups of 1 to 8, preferably 1 to 4 carbon atoms, and in particular a methyl or ethyl radical with at least one, preferably one, single substituent such as carboxyl, cyano, carbalkoxy and amido, for example carboxymethyl, cyanomethyl, carbethoxymethyl, carbmethoxymethyl, and the like carb-lower alkoxymethyl, carbamoylmethyl, as well as appropriately substituted ethyl groups, for example -CH2CH2COOH, -CH2CH21CS N, -CH2CH2CONH2 and -CH2CH2COOR, where R is, for example, methyl or ethyl; -CH 2 CH 2 SOCH 3, -CH 2 CH 2 SOC 2 H 5, and -CH 2 CH 2 COCH 3.

Thus, the alkylated orgoteins of the invention are bovine, ovine, equine, swine, canine, rabbit, guinea pig, chicken and human analogues of the bovine, of which at least one, for example 1, 2, 3, 4, 5 and up to all (about 18 to 26] titratable amino groups are alkylated, i.e., have an unsubstituted or substituted alkyl group.

Preferably, alkylated amino groups are —NH — CH2 —R, wherein R is hydrogen, CH3, C2 H5, n -C3 H7, or another alkyl group of up to 7 carbon atoms, -COOH, -COO-LA, -CONH-LA , -CON- (LA) 2, -CN, -CH 2 -CN, -Ph, -COPh, -CH 2 OH or -СН (СНз) OH, where LA is an alkyl radical of 1 to 4 atoms and Ph is an unsubstituted phenyl radical, or a phenyl radical having 1 to 3 simple substituents, for example methyl, chlorine, bromine, nitro, amido and methoxy, carboxy or carboethoxy, such as p-tolyl, symylyl, p-amidophenyl, m-chlorophenyl and p-methoxyphenyl .

Such orgoteins have the general formula (H2N) _m -org- (NHCH2R) n, wherein n- is an integer from 1 to about 26, preferably at least 10, especially 10 to 18, and the sum of m + n represents the total number and R is as defined above, preferably is hydrogen or C1-C4 alkyl, for example methyl, and org is the remainder of the orgotein molecule.

Some of the alkylation. The agents used in the method of the invention will simultaneously alkylate some of the free acid groups of the orgotein molecule. These alkylated orgoteins can be represented by the general formula (HOOC) _X (COOCH2R @ y) / (org) / \ (H2N) m (NHCH2R) n wherein org, R and m are as defined above, y is the number of alkylated free carboxylic acid groups, e.g. to 8, preferably 2 to 6, and x is the remainder of the free carboxylic acid groups in the orgotein molecule.

Suitable reagents for introducing the carboxymethyl and carbamoylmethyl groups are iodoacetic acid and iodoacetamide.

Iodoacetic acid can form stable products with cysteine (sulfhydryl), lysine (amino), histidine (imidazole nitrogen) and methionine (sulfuric) sulfur residues in proteins. *

Since carboxymethylation of any of these groups, with the exception of methionine, increases the effective negative charge in the orgotein protein at pH 8.4, the overall reaction can be quantitated electrophoretically (except for carboxymethylation of methionine and carboxymethylation of the second imidazole nitrogen ').

In natural orgotein, iodoacetic acid and iodoacetamide react predominantly, if not exclusively, with lysine amino groups. Although the free thiols react with iodoacetic acid several times than amines, the first few groups in the carboxymethylated orgotein do not react in any way. noticeably faster than the next 10 to 20 groups react. This is in agreement with the reaction of p-mercuribenzoate with sulfhydryls of orgotein: there is no reaction with holoprotein. Even apoprotein, which provides a p-mercuribezoate sulfhydryl reaction (albeit slow under non-denaturing conditions), does not occur with acid. iodoacetic acid in 10-fold molar excess at pH 7 to no decrease after a few weeks at 4 ° C in the sulfhydryl content (determined spectrophotometrically by titration with p-mercuribenzoate. The pH dependence of the carboxymethylation reaction as determined by electrophoresis is consistent with lysine reaction. ) and not histidine (pK <6), since the extent of reaction at the same initial concentrations is constantly increasing as the pH approaches 9, with no appreciable reaction at or below neutral, where On the other hand, titration with ethyl chloroformate to histidine showed only 5 histidines in the carboxymethylated orgotein under conditions where they showed 16 in the natural orgotein.

Evidence that the natural bovine orgotein did not respond to either methio203992 nin or histidine was the result of an experiment in which the orgotein was incubated for 48 hours with a 0.2 M solution of iodoacetamide in 0.5 M phosphate buffer at pH 6, 5. Alkylation of methionine or double alkylation of histidine should result in a more positively charged protein at pH 8.4, but there was no change in superoxide dismutase activity or change in band system during electrophoresis.

However, the lysine amino groups in the orgotein definitely react with iodoacetate. Extensively carboxymethylated orgotein moves similarly to acetylated orgotein in electrophoresis but has a lower superoxide dismutase activity (approximately 20% of the unmodified orgotein protein).

In addition to the bovine N-methyl-organogoteins and the orgoteins of the examples below, other examples of the bovine N-alkyl chlorgoteins of the invention are N-ethylorgotein, N-propylorgotein and N-benzylorgotein, wherein in each case there are 9 such alkyl groups in each both subunits of the orgotein molecule, or the respective orgoteins, on average 1, 6 or 10 such alkyl groups in each subunit, and the respective analogues of these orgoteins from humans, sheep, horses, pigs, dogs, rabbits, guinea pigs and chickens.

The carbamoylated orgoteins of the invention are bovine, ovine, equine, pig, rat, dog, rabbit, guinea pig, chicken and human orgotein analogues in which at least one, for example, 1, 2, 3, 4, 5 to all (about 18 to 18). 26) titratable amino groups are carbamoylated, i.e., have an unsubstituted or substituted "CONH" or "CSNH" group.

Preferably, the carbamoylated amino groups are amino groups of the general formula

X

II — NHCNHR where

X is oxygen or sulfur; and

R is CH 3, C 2 H 5, n-C 5 H 6, n-C 5 H 17 or another alkyl group of up to 12 carbon atoms, Ph or, when X is oxygen, also hydrogen, and Ph is unsubstituted phenyl or phenyl having 1 up to 3 simple substituents such as methyl, chloro, bromo, nitro, amido and methoxy, carbomethoxy or carboethoxy, for example p-tolyl, s-xylyl, p-amidophenyl, m-chlorophenyl and p-methoxyphenyl. Such orgoteins have the formula

X

II (H2N) _m -org- (NHCNH-R) _n , wherein n is an integer from 1 to about 26, preferably at least 2, in particular 6 to 10, and the sum of n + m represents the total number of washable free amino groups in an unmodified analog,

X is oxygen or sulfur,

R is as defined above, preferably hydrogen or alkyl of 1 to 8 carbon atoms, and org is the remainder of the orgotein molecule.

Particularly preferred carbamoylated orgoteins are alkylcarbamoylorgotein and alkylthiocarbamoylorgotein wherein the alkyl group is an unsubstituted C 1 -C 8 alkyl radical, for example a methyl, ethyl, propyl, isopropyl, butyl or octyl radical.

Since the carbamoyl group in the orgotein molecule does not matter if it is not only physiologically toxic and can be formed on the lysine ε-amino groups, the possible equivalents of the preferred alkylcarbamoylorogoteins described above, if they can be formed, are those which otherwise correspond to the above of the above formula wherein R represents a cyclopentyl, cyclohexyl, methyl and the like cycloalkyl, cyclohexylmethyl, n-cyclopentylpropyl and the like cycloalkyl, benzyl, cycloalkyl and phenethyl and the like aralkyl radical.

Possible equivalents are those orgoteins of the above formula wherein R is an alkyl radical of 1 to 8, preferably 1 to 4 carbon atoms, especially a methyl or ethyl radical optionally substituted by at least one, preferably one other, substituent, for example a fluoresceinyl radical .

In addition to the N-carbamoylated bovine orgotein of the following examples, other examples of the bovine N-carbamoylorgotein of the present invention are the corresponding N-carbamoylorgotein, N-propylcarbamoylorgotein, N-thecoccarbamoylglorine and N-methylthiocarbamoylgotein groups, both of which are both carbamoylgotein groups. , and the respective orgoteins having an average of 1, 6, or 10 such carbamoyl groups in each of the two subunits, and the respective human, sheep, horse, pork, canine, rabbit, guinea pig, chicken and rat analogs of each compound.

As mentioned above, the orgotein analogs contain about 50-68 glutamic and aspartic acid residues, but only about 20-27 of these residues have free acid groups. Since the orgotein molecule consists of two identical or nearly identical peptide chains (subunits), half of these amino acid residues in each of the two chains are rigid but not covalently bound to each other under mild temperature and pH conditions. Esterification changes the charge of the orgotein molecule and usually only about 10 to 10, preferably only about 6 to 8 of these free acid groups can be esterified while still retaining the structure of the natural molecule upon which the stability and usefulness of the substance depend.

The esterification of carboxylic acid groups can be quantitatively monitored by a change in charge during electrophoresis. If desired, the esterified orgotein can be hybridized with a natural orgotein, as described below, thereby reducing by half the number of esterified carboxylic acid groups per molecule.

Because the molecule. The orgotein consists of two identical peptide chains, the ester groups are probably more or less evenly distributed between the two chains. Since a single esterifying agent is usually used, all ester groups will be identical. However, it is possible to prepare esterified orgoteins having two or more different ester groups per molecule and even in each of its chains.

One way to prepare a mixed orgotein ester is to sequentially esterify with different esterifying agents. For example, a portion of the free acid groups can be esterified with one low concentration esterifying agent, such as diethyl sulfate, and another portion of the free acid groups can be esterified with another high concentration esterifying agent, such as diazomethane. What is meant by a low or high concentration of esterifying agent will depend on its relative rates of reaction with the acid groups of the protein and the solvent, and thus will depend on the pH of the reaction medium and the esterifying agent, and to a lesser extent buffer and temperature.

Another method of obtaining a mixed orgotein ester is by hybridization. The term hybridization of orgotein expresses the formation of a mixed orgotein from the subunit chains of two different orgotein molecules, for example. Up to and Bž, where A and B are their respective peptide chains (Α2 + Β22ΎΑΒ). In electrophoresis, the charge of the heterodimer AB should be the average of the charges of the homodimers A 1 and B 2, provided that in all cases the same portion of each of the two subunits is involved.

Both the methyl chloro-protein and the ethyl chloro-protein can be hybridized with natural orgotein or by heating with each other with a small excess of natural orgotein at 50 ^; ° C for 4 hours. The resulting heterodimers appear in electrophoresis as a mixture of bands lying between the natural orgotein band and the esterified orgotein band prior to hybridization.

As can be seen, these hybrid molecules of the semi-esterified orgotein can be further esterified with another esterifying agent to form a hybrid. an esterified orgotein in which the ester groups in one peptide chain differ from the ester groups in the other chain.

The esterified orgoteins of the invention appear to have virtually the same spatial structure as the natural orgotein molecule. The contents of chelated Cu ++ and Zn + ⁺ ions (number of grammatomes per mole) are approximately the same as those of an orgotein. Like orgotein, they are highly resistant to pronase and degradation by other proteolytic enzymes. The enzymatic activity of the superoxide dismutase is not appreciably reduced until more than about 6-8 carboxylic acid groups are esterified.

The nature of the esterifying groups, like the number of esterified groups, is not critical as long as the esterifying group is physiologically appropriate. Due to the higher molecular weight of the orgotein molecule, although the orgotein molecule is esterified by low molecular weight esterifying groups, such as 60, the effect on the overall chemical composition is relatively small, i.e. less than 5%. The esterification of the free carboxylic acid groups has, of course, a large effect on the isoelectric point and the resulting electrophoretic mobility, but - as mentioned above - if esterification is limited to about 10 or less glutamic and aspartic acid groups, it has no significant effect on the spatial arrangement of the molecule and the resulting stability, for example against heating at. at 60 ° C for 1 hour and against proteolytic enzyme attack.

As will be further appreciated, the esterifying groups must also be derived from an esterifying agent capable of esterifying the carboxylic acid group in an aqueous solution or a buffer solution, since the reaction is usually carried out in these environments. Such esterifying agents include dimethyl sulfate and diethyl sulfate, diazomethane and other diazo compounds, for example

N2CH2COX, where

X is, for example, OCH 3, OC 2 H 5, NH 2 or NHCH 2 CONH 2, and other diazoacetic acid esters and amides that do not have reactive groups such as a carboxyl group or an imino group.

Methods for preparing such esters are described in "Methods in Enzymology," Vol. XI, p. 612 (1967), and further in Biochem. Biohys, Ress. Comm., 30, p. 489 (196'8) by K. T. Fry et al., And in PNAS, 56, p. 1817 (1966), G. R. Delpierr and J. S. Fruton.

The invention further relates to an COOH esterified orgotein in which the ester group preferably has up to 4 carbon atoms, for example from a monovalent alkanol such as methyl or ethyl, preferably methyl.

Since the ester moiety does not matter if it is not only physiologically toxic and can be formed on the free acid groups in the orgotein, the possible equivalents of the preferred alkyl esterifying groups described above, if they can be formed, are those having one two or more simple substituents, for example halogen, alkoxy, carbal17, alkoxy, carbamino, etc., especially those in which the ester group bearing the substituent or substituents is a methyl group. For example, except for esterified orgoteins in which the esterified acid groups are —СООСНз, the possible equivalents are those in which the ester groups are —COOR, where R is —GHzCOX, where X is ОСНз, OCžils, N112, NHCH 2 CONH 2 or CH 2 CH 2 -phenyl, or wherein R represents C 1 (phenyl) 2.

Esterification of the COOH groups can be followed by determining the charge change detected electrophoretically by comparison with natural orgoteins.

As is known, the electrophoretic mobility of an ion is a function of the electric field strength, the effective charge of the ion (including bonded ions) and the coefficient of friction, see, for example, C. Tanford's "Physical. Chemistry of Macromolecules', Wilsy Publishing House, New York (1966). Since the coefficient of friction depends on the size and shape of the molecule and the composition of the solution, comparisons of the different proteins are not very informative. However, when compared to proteins of similar size and shape, in this case the orgotein molecules chemically modified by relatively small groups, under identical electrophoretic conditions, the only variable affecting this electrophoretic mobility is the effective charge.

The electrophoretic behavior of many chemically modified orgotein molecules is in agreement with this conclusion. The natural bovine orgotein in ecotrophoresis provides predominantly one interface (lane 1) with fewer, faster moving interfaces (lanes 2, 3, etc.) evenly spaced from the main interface, representing orgotein molecules with a higher group ratio —COOH к - NH? 1. Esterification of progressively higher numbers of free groups — COOH of natural bovine orgoteins leads to the formation of a series of progressively more cathodic (moving towards the negative electrode) electrophoretic bands as more and more free carboxylic acid groups of the orgotein molecule progressively esterifuje.

Graphical representation of the distance traveled by the interface from start to belt number is relatively linear at low conversion ranges —COOH, but gradually curves at higher conversion rates as there is a limit to how fast even the most charged components can move through the solution . The faster moving components are also more sensitive to salt concentration and are substantially retarded by the salt-containing ions electrophoresis. For this reason - because the extrapolation of positions of more than about 2 bands is not always accurate -..... it is necessary to accurately determine the charge of an unknown substance to be subjected to common eciekophoresis with a solution containing all bands from 1 to the appropriate position ( for example partially acetylated orgotein).

All conventional protein modification reactions previously applied to an orgotein molecule agree with this interpretation, i.e., the position of the bands corresponds to the overall charge changes compared to the natural orgotein molecule. Esterification with dimethyl sulfate or ethyldiazoacetate affords bands of -1, -2, -3, -4, etc., indicating that 1, 2, 3 and 4 free carboxylic acid groups have been chemically modified.

Generally speaking, at most only about eight of the free COOH groups can be esterified without deleterious effects. Attempts to esterify more than 6-8 COOH groups, i.e., esterify more than three COOH groups of each of the two orgotein peptide subunits, usually lead to denaturation and loss of superoxide dismutase activity.

As expected, the distribution of the esterifying alkoxy groups on the orgotein molecule is likely to be random, since none of the titratable free carboxyl groups appears to be extremely easily esterifiable. Because the orgotein molecule consists of two identical peptide chains, the ester groups of the partially esterified orgotein will be distributed more or less randomly along each peptide subunit, but more or less equally between the two. Since a single esterifying agent is usually used, all ester groups will be identical. However, it is also possible to prepare esterified orgoteins having two or more different ester groups per molecule and even in each of the two chains.

One way to prepare a mixed esterified orgotein is to carry out the esterification stepwise using various esterifying agents. For example, a portion of the -COOH free groups can be esterified with one esterifying agent, for example dimethyl sulfate, and the rest of the COOH reactive groups can be esterified with another esterifying agent, for example ethyldiazoacetate.

Another method to prepare a mixed esterified orgotein is by hybridization. The term "hybridization of orgoteins" refers to the formation of mixed orgoteins from the subunit chains of two different orgotein molecules, for example Аг а B2, where А а В represent their respective peptide chains (Аг + Вз * -2AB) . The charge of heterodimer AB before electrophoresis should be the diameter of the charge of homodimers A? and B2, provided that in all cases the same portion of each of the two subunits participates in the concentration.

Both the methyl esterified orgotein, prepared by esterifying about 6 free acid groups of the orgotein molecule with dimethyl sulfate, and the carboxymethyl esterified orgotein, prepared by esterifying the natural orgotein molecule with ethyldiazoacetate, can hybridize to natural orgotein or together with each other at 50 ° C for 4 ° C. hours. In addition to the bovine orgotein derivatives shown in the following examples, other examples of the orgotein derivatives of the invention are the corresponding derivatives of other orgotein analogues.

Other examples of esterified bovine orgoteins are carbomethoxymethylorgotein and carbamoyimethylorgotein, each of which contains 3 to 4 such esterified groups in each of the two subunits of the orgotein molecule, and the corresponding esterified orgoteins in which there are 1 to 2 such ester groups in each such subunit, and furthermore, the respective human, sheep, equine, pork, canine, rabbit, guinea pig and chicken analogues of each of these compounds.

The orgotein derivatives can be isolated from the reaction solution, preferably after dialysis to remove foreign ions, by conventional lyophilization, for example as described in U.S. Pat. No. 3,658,682. If desired, the orgotein derivative can first be purified by ion exchange chromatography. resin, electrophoretically, and / or gel filtration using a polymer that acts as a molecular sieve.

Filtration with a microporous filter having a pore size of from 0.01 to 0.22 μ / m under aseptic conditions into sterile vials, optionally after adjusting the ionic strength with sodium chloride and / or sodium phosphate, for example up to isotonicity, is obtained bacterially and virally, or bacterial sterile solution suitable for injection. Filtration through a 0.1 µm filter also reduces the number of pyrogens or removes them from the solution.

The method for producing injectable orgotein derivatives having anti-inflammatory activity is characterized in that the aqueous solution of the natural orgotein, in particular from bovine, is reacted in an alkaline medium with an alkylating and / or carbamoylating agent, such as dialkyl sulfate C 3 -C 8 alkyl moiety, C 2 -C 8 activated alkyl halide, vinyl-activated alkylating agent, reducing alkylating agent, - or alkali metal cyanate, C 3 -C 8 alkyl isocyanate an alkyl moiety, an aryl isocyanate, an isothiocyanate, in an amount sufficient to convert at least one of the titratable ε-amino groups of the orgotein into an N-alkyl or N-carbamoyl group, or in an acidic environment with an esterifying agent, such as dialkyl sulfate, diazomethane, in an amount sufficient to convert up to 10 of the free % of the carboxyl groups in the orgotein molecule to the esterified carboxyl group at a temperature ranging from the freezing point of the reaction mixture to 80 ° C.

The pharmaceutical compositions of the invention include an orgotein ester of the invention and a pharmaceutically acceptable carrier. The form and character of this carrier are determined by the method of application.

The pharmaceutical composition of the invention is preferably in the form of a sterile injectable preparation, for example a sterile injectable aqueous solution. The solution may be formulated in a known manner using the above carriers. The sterile injectable preparation may also be a sterile injectable solution or suspension in any non-toxic parenterally-acceptable diluent or solvent, or may be an lyophilized powder reconstitutable with such a solvent.

The compositions of the invention contain an effective amount of a dosage unit of an orgotein derivative according to the invention, i.e. the orgotein derivative is present in a concentration effective to elicit the desired response when the unitary agent of the composition is administered in a manner suitable for the pharmaceutical carrier used. For example, liquid preparations, both topical and injectable, usually contain about 0.5 to 20 mg of the orgotein derivative in 0.25 to 10 ml, preferably -0.5 to 5 ml, in addition to intravenous infusion solutions, which may also be more dilute and may contain, for example, 0.5 to 20 mg of the orgotein derivative per 50 to 1000 ml, preferably 100 to 500 ml of infusion solution. Tablets, capsules and suppositories generally contain 0.1 to 25 mg, preferably 1 to 10 mg, of an orgotein derivative per unit.

The orgotein derivative is usually administered by instillation or injection, for example, intramuscularly, subcutaneously, intravenously or intradermally. Intramuscular administration is preferred, except in the case of shock, where intravenous administration is sometimes preferred due to the more rapid effect, and in some localized disorders such as radiation and some cystitis where local injections, infusions and / or dripping. - Individual doses usually range from 0.5 to 20 mg. A preferred range in humans is about 0.5 to 8 mg; in horses, about 5.0 to 10.0 mg. The exact dose is not a determining factor and depends on the type of disease and the severity of the case.

The orgotein derivatives of the invention, like the orgotein, are effective for treating a wide variety of inflammatory processes, including those in which synthetic anti-inflammatory agents have limited utility, for example, to toxic side effects with prolonged use.

In particular, these orgotein derivatives are effective for improving inflammatory processes and mitigating their effects, for example in those cases where the urinary tract and joints in mammals are affected. They are useful in alleviating the symptoms associated with rheumatoid arthritis, osteoarthritis and post-traumatic arthritis as well as bursitis, tendonitis, etc.

The anti-inflammatory activity of the injectable ororgotein derivatives produced by the method of the invention was investigated with n-karage21 ninema cream tests performed on female Swiss Webster mice weighing between 20 and 23 g, compared to that of orgotein alone. Groups of 10 test animals were injected subcutaneously into the hind paw with 0.03 ml of saline containing 1.0% carrageenan. The solutions of the tocotable orgotein derivative produced by the process of the invention and the comparative ergot were sprayed subcutaneously every 3 hours before and 2 hours after application of the carrageenin solution, at a dose of 0.1 ml. 24 hours after carrageenin administration, the animals were sacrificed with chloroform, their hind paws amputated in the joint and weighed. The results of inhibition of carrageenin-induced edema are shown in the following table:

number of animals average swelling increase --S.D. in 24 h application 10 57.1+ 8.6 physiological saline solution 10 44.0 + 10.5 0.1 mg / ml ORG 10 43.7+ 5.6 0.1 mg / ml DMS-ORG 10 29.6 - + 5.7 1.0 mg / ml ORG 10 35.0- + 11, -8 1.0 mg / ml DMS-ORG

inhibition t 2p < 22.9% 3,057 0.01 23.5% 4,122 0.001 48.2% 8,423 0.001 38.7% 4,786 0.001

ORG + orgotein 740706/740705 DMS-ORG = N-methylorgotein having 18 N-methyl-substituted lysine groups

The results in the table show that N-methylorgotein (DMS-ORG) has approximately the same degree of efficacy as orgotein (ORG). To determine toxicity, one group of 10 mice received a subcutaneous dose of 4.5 mg / kg twice, and another group of mice received a subcutaneous dose of 0.45 mg / kg twice. No signs of toxicity were observed within 24 hours after administration. This is consistent with the total non-toxicity of the orgotein, even at high doses for extended periods of time.

Further details regarding the isolation of parent orgotein analogs and the use of orgotein derivatives made by the method of the invention, including administration methods, dosage forms, dosing regimen and inflammatory and other processes that can be treated with sterified orgotein, are disclosed in U.S. Patent 3,758,682.

Based on the foregoing description, one of ordinary skill in the art will be able to utilize the invention to the fullest extent without further elaboration. The following examples are intended to illustrate the invention and are not to be construed as being limited thereto.

Example IA

To a solution of 5 mg of bovine orgotein in 4 ml of 0.04 M carbonate buffer is added. The reaction mixture was maintained at 10.0 by addition of 0.5 M sodium hydroxide solution. Half of the base is consumed in about 37 minutes. During electrophoresis, bands 1-5 appear, characterized by superoxide dismutase activity. After dialysis, lyonization and redissolution of 1 ml of water, the superoxide dismutase activity of the protein and its content of Cu ⁺⁺ and Zn ++ ions are still unchanged compared to the untreated orgotein. Acetylation of a 85 µg / ml solution of a 3 µl acetic anhydride solution in a medium of 0.4 µg of a britanite buffer at pH 9 achieves an average electrrfretic charge change of only about -2 compared to -20 for a natural orgotein in the same solution. . The dimethyl sulfate-treated orgotein at pH 10 is therefore extensively N-methylated, i.e. it contains about 18 methyl groups per molecule.

Example 2

The procedure of Example IA is followed, but using, respectively, the respective human, sheep, horse, pig, dog, rabbit, turkey and chicken orgotein analogs as starting material. In each case, all of the up to about one lysine of each of the subunits of the orgotein molecule are alkylated.

Example 3 A

The procedure of Example IA, but using diethyl sulfate instead of dimethyl sulfate, yields ε-N-ethylated orgotein, the properties of which are substantially the same as those of ε-N-methylated orgotein.

EXAMPLE 4 The ε-amino groups of bovine orgotein lysine residues are cationized with a 0.2 M sodium iodoate solution at ambient temperature under the conditions given below and with the results given below. *

Reaction buffer charge change during electrophoresis, mean 4- range orgotein (1.4 mg / ml) + JCH2COONa ⁺ (0.2 M)

a) 0.3 M acetate, pH 3.8 (the only changes are those due to low pH)

b) 0.4 m acetate, pH 5.0 no change in hours

c) 0.23 M carbonate, 0 (15 min), pH 9.2 3 + 2 (2.5 h),

5 + 3 (6.5 hours)> 18 (72 hours).

From these electrophoretic. the images show that at pH 3, 8 and 5.0, there is no N-alkylation and that at pH 9.2 on average about 3 groups - CH 2 COO - are introduced in 2.5 hours; about 5 such groups are introduced in 6.5 hours and about 18 such groups are introduced in 72 hours on the nitrogen atoms of the ε-amino groups.

orgotein (2.8 mg / ml) + JCH ² COO'Na ⁺ (0.43 M)

d) pH 6.0

e) pH 7.0

f) pH 10

0.4 after 5 days at room temperature,

0.6 (1 day), 1.0 (2 days), 1.5 (5 days), 0 (10 minutes,> 3 (2.5 hours),> 10 (6.5 hours)

Time (days): 0.75

Number of lysine reacted: 0

The electrophoretic patterns of the thus processed orgotein show that at pH 6 less than half of the orgotein molecules are alkylated, at pH 7 the orgotein molecules had on average one ε-amino group in the alkylated group - CH2COO in 2 days and two such groups in 1 day. molecules. Example 10 was introduced

2.5 hours an average of more than 3 such groups and 6.5 hours an average of more than 10 such groups per molecule.

The products in all of the above-described alkylations wherein the -CH 2 COO groups are introduced are mixtures of ε-aminoalkylated orgoteins containing a different number of such groups, as evidenced by the formation of multiple bands with different electrophoretic motions.

Example 1 В

Unsubstituted catamoylated orgoitein

A solution of 3.7 mg of orgotein (bovine analog) and 14 mg of KNCO in 2 ml of 0.025 M sodium phosphate pH 7.5 is left at 4 ° C. Upon electrophoresis of aliquots for 54 days, a number of bands with superoxide dismutase activity appear more anodic than natural orgotein. Below is the average charge change along with the range of active bands on either side of the average u

samples: 54 3.8 27 Mar: 0.8 5 + 2 8.5 + 3.5

Example 2B

Alkylcarbamoylated orgoteins μΐ aliquots of orgotein (bovine analogue) in 0,1 M tris solution or phosphate buffer pH 7,6 and a concentration of 10 milligrams / ml are allowed to react at min2 h.

Isopropyl cyanate / tris 88 reagent propyl isocyanate / phosphate 88 octyl isocyanate / tris 0.21.0 octyl isocyanate / phosphate 00.2 ° C with 1 μΐ of either propyl isocyanate or octyl isocyanate for various times. In two cases, an additional 2 μΐ of propyl isocyanate is added after 2 days. The number of propylcarbamoyl and octylcarbamoyl groups introduced is determined from the average charge change determined electrophoretically as shown below.

Average charge change day 2 days 4- 2 μϊ isocyanate.

not determined10 not determined> 15

2.53 cyanate or octyl isocyanate. The solutions were kept at 25 ° C for 4 hours and electrophoresed. Then an additional 1 ml of isocyanate is added to each of them. After leaving overnight at 4 ° C, the solutions are again electrophoretically examined and dialyzed.

Carbamoylated proteins are less resolved

P г í к 1 and cl 3 В

Alkylcarbamoylated orgoteins

The procedure of Example 2B was repeated using 10 mg of orgotein in 1 ml of 0.1 M phosphate buffer pH 7.6 and 1 ml of g-at / mol propyl isolate.

Cu ⁺⁺ Zn ++% of the original superoxide dismutase activity is released in deionized water and is partially excreted after dialysis but is soluble in

0.15 M saline. The number of carbamoylated lysine groups is given below:

Isocyanate number of reacting lysines after 4 hours overnight propyloctyl10 + 4> 15 2.2

3 + 3 6 + 5 2.1

1.5

1.8 about 50 about 25

Example 4B

N-im (R) -ocrine-rhocarbamoyl orgotein

To a solution of 5 mg of orgotein in 4 ml of a 0.075 M sodium tetraborate solution, pH 9, was added 10 µl of CH 3 NCS, and the mixture was shaken for one minute until the CH 3 NCS dissolved. 50 µl samples were taken at intervals and 0.4 ml of 0.1 M sodium acetate pH 5.3 was added as a buffer. After 6 to 22 hours, a white sulfur precipitate appears (as determined by the burning odor and the carbon disulfide solubility). After 22 hours at room temperature, 0.5 ml of 1 M pH acetate buffer solution is added to the rest of the reaction mixture.

5.3 and the mixture was dialyzed for 2 days with frequent water changes. The dialyzed solution is briefly centrifuged to remove white. turbidity of the solution.

The electrophoresis of the samples taken from the solution gives rise to a series of increasingly anodic bands with superfluous diatomic activity.

From the analysis of samples by the cychochrome assay at pH 7.0 [McCord and Fridovich, J. Biol. Chem. pp. 6049-6055 (1969)] shows a decrease in superoxide dismutase activity with more intensive modification.

Time (hours) average charge time superoxide dismutase activity (%) 0 (0) (100 ·%) 0.08 0.5 111 0.25 1 118 0.75 2 91 1.9 4 86 6 9.5 43 22nd 16 20 May

The dialyzed reaction product becomes apparent as a fast moving anodic band after 22 hours in ecophoresis; retains 18% of the superoxide dismutase activity of the natural orgotein protein and contains 2.06 g-at / mol Zn ++ ions and 1.76 g-at / mol Cu ++ ions (compared to 2.26 g-at / mol Zn + + ions and 2.08 g-at / mol Cu + + ions for unmodified orgotein). All of these products are soluble, retaining chelated Cu ⁺⁺ and Zn ++ ions and at least a portion of the superoxide dismutase activity of the unmodified protein.

Example 5 B

NIfluoretcemylcarbamoyl orgotein

A solution of 100 mg of orgotein (bovine analog) and 6 mg of flouretecithiothiocyanate in 10 ml of a 0.14 M phosphate solution at pH 8.5 is kept at 4 ° C for 18 hours. The reaction mixture was then loaded onto a chromatographic column containing microporous crosslinked dextran (Sephadex G-50) and eluted with 0.15 M borate buffered saline to pH 8. The eluted yellow protein fractions were dialyzed against water. The dialyzed protein that is reported (20 milligrams) is intensely yellow and fluorescent and readily dissolves in borate buffered saline to pH 8.

Ecotoxophoresis reveals that about 96 mg of soluble protein is about half of the monocarbamylated orgotein (charge change of -3), along with a smaller amount of double and triple carbamoylated orgotene (charge changes of -6 and -9) and. unreacted orgotein. The redissolved precipitate appears to be more extensively modified. protein, since it provides a rapid anodic spot in ecotrophoresis.

The soluble protein is chromatographed on a weakly basic ion exchange column at pH 6 with a phosphate buffer having a linearly increasing concentration from 0.01 to 0.2 M. The monocarbamoylated and dicarbamoylated orgoterins are isolated, dialyzed and lyophilized.

The monocaprbamoylated orgotein is characterized by superoxide dimitutase activity and riboflavin assay activity in electrophoresis. According to the cytochrome assay at pH 7.5, 4'8 '% has superoxide ditmutasse activity of natural orgotein. According to Ungar's bioassay, this protein is characterized by approximately 50% natural orgotein activity.

Monocarbamoylated orgotein solution,

203882, stored at 4 ° C for two weeks, turns into a mixture of orgotein, monocarbamoylated orgotein and dicarbamoylated orgotein [apparently the result of hybridization] and non-protein fluorescent compounds [apparently the result of hydrolysis of the thiourea group of the carbainoyl orgotein to give free arainofluorescein).

Using the procedure of the above examples, but using the appropriate human, sheep, equine, canine, rabbit, guinea pig, rat and chicken orgotein analogs as starting materials, the corresponding carbamoylated derivatives of these analogs are obtained.

Example 1C

Methyl esterification

Solution [125 (Ug / ml) 0.5 mg of bovine orgotein and 0.5% v / v a solution of dimethyl sulfate in 4 ml of 0.05 M acetate buffer was maintained at pH 5 for 100 minutes. Electrophoretic analysis of the reaction product yields bands of superoxide dismutase activity from +1 to -6 (mean -4). The product is orgotein, with an average content of 4 groups —СООСНз.

Example 2C

Methyl esterification

A solution of 0.5 mg. Of beef cattle and 10 μϊ of dimethyl sulfate in 4 ml of water is kept constant by the addition of 0.1 M sodium hydroxide solution. The rate at which the base reacts in the pH range of 7 to 10 is largely independent of pH and the addition of 0.25 moles of sodium phosphate buffer. In electrophoretic analysis, multiple cathode bands with superoxide dismutase activity are formed at an initial rate of approximately 00.5 charges / h. After 21 hours, predominantly at pH 7-8, the solution is analyzed for N-methylation and esterification as described below. No N-methylation can be detected in this analysis.

a] Acetylation of a 0.5 ml aliquot of esterified orgotein using 1 µl of acetic anhydride + 3 µΐ of 6 M sodium hydroxide solution at 4 ° C gives a solution whose electrophoretic analysis reveals a fast moving anodic strip, similar to the one electrophoresis of the acetylated natural orgotein, demonstrating that the free amino groups are unaffected by esterification.

b) Adjust 1 ml of the esterified orgoteln solution to pH 10.5 and place in a sodium hydroxide grain desiccator. Although the pattern of cathodic bands 1 to 4 is stable at pH 7 to 9, at pH 10.5 the cathodic bands gradually disappear within 4 days, and electrophoretic patterns of the natural orgotein reappear. Protein methyl esters usually readily hydrolyze at alkaline pH, demonstrating that the multicode bands occurring after esterification are orgotein esters.

It is apparent from the foregoing that, after two hours, an average of one -COOH group per molecule is esterified with dimethyl sulfate at pH 7-10; after 4 hours about two groups in 1 molecule; further esterification increases the number of esterified groups —COOH · to a maximum of about 6 in 1 molecule.

Using the procedures of Examples 1C, 2C and using the appropriate human, sheep, equine, pig, canine, rabbit, guinea pig and chicken analogues of orgotein as starting materials, an average of about 4 carboxylic acid groups of the orgotein molecule are each converted to methyl ester groups.

Following the procedure of Examples 1C, 2C using diethyl sulfate instead of dimethyl sulfate, orgotein esters having from 1 to 6 free carboxylic acid groups converted to ethyl ester groups were obtained. The properties of the resulting esterified orgoteins are practically the same as those of the methyl esterified orgoteins.

Alternatively, the esterified orgoteins may be further purified by ion exchange chromatography to separate species with different effective charges and thus esterified to a different degree. For example, when eluting with 200 mg in a 2.5 X 40 cm ion exchange column (SEPHADEX) using 4 liters of a tris pH 8.5 solution with a linear increasing concentration of 0.01 M to 0.2 M, they were separated orgotein bands, wherein the electrophoretically more cathodic bands elute first. In this way, the mixture of methyl esterified orgoteins produced by Example 1C can be separated into fractions containing predominantly 1, 2, 3, 4, 5 or 6 methyl ester groups per 1 orgotein molecule.

Such fractionation of a partially modified orgotein by ion exchange chromatography is applicable to any modified orgotein whose molecular charge depends on the extent of the modification, for example, the esterified orgotein and the N-acetyilofused orgotein by methyl or carboethoxymethyl groups.

Example 3C

Carbethoxymethyl esterification

Ethyldiazoacetate is prepared by reacting glycine ethyl ester with nitrous acid as described in "Orgnic Synthesis" Coli. St. IV, p. 424, but using carbon tetrachloride instead of dichloromethane to extract ethyldiazoacetate from the aqueous solution.

ml of an approximately 1 molar solution of ethyl dichloroacetate in carbon tetrachloride was introduced into a 25 ml flask and most of the carbon tetrachloride was evaporated under reduced pressure by a water pump. A solution of 9 milligrams of bovine orgotein in 3 milliliters of water is added and the organic phase is dispersed by swirling. The solution is left at 4 ° C and shaken every few days. The reaction mixture remains heterogeneous at all times. Electrophoretic analysis revealed the gradual formation of multiple cathodic protein bands -1 through 6 with superoxide dismutase activity. The average charge change is 1.2 after 5 days and 3 after 12 days. The product is a mixture of esters karbethoxymethylových orgoteins having after 5 days either ^r one or two of such ester groups in one molecule, and after 12 days 1 to 6 such ester groups.

The aqueous phase is then filtered and freed by chromatographic fractionation in a column consisting of 10 ml of Sephadex G-25. Protein fractions are lyophilized and redissolved in 2 ml of water and the pH is raised from 3.7 to 5.6 by adding 2 μΐ of 1 M sodium hydroxide solution. According to electrophoretic analysis, there was no change compared to the state prior to salt removal and lyophilization.

After a further reaction of this solution for 1 month under the same conditions as above, an electrophoretic analysis stained a protein with a lower superoxide dismutase activity and no material more cathodic than band-6.

Example 4 C

Methyl esterification

Following the procedure of Examples 1 and 2, the bovine orgotein is alkylated under the conditions in the following table:

pH buffer orgotein dimethylsulphate by volume hydrolysis half-life ⁺ bands with superoxide dismutase activity in electrophoresis (a) 5.0 0.05 M acetate 3.25 .mu.g / ml 0.25 54 min. + 1 to —4 (average —2) (b) 5.0 0.05 M acetate 125, ug / m! 0.5 54 min. —1 to —6 (average —4) (c) 7.0 0.016 M phosphate 80 µg / ml 0.65 1 hour —2 to –6 (d) 7.0 0.016 M phosphate 80 /.ig/ml 1.3 1 hour weak spot SOD from +4 to —6, peaking at —6 (e) 11,7- -9.3 0.05 M carbonate 125 µg / ml 0.5 ¥ 2 hours (at pH 10) +2 to —3

⁺ Half ^- life for NaOH uptake required to maintain a constant pH.

The product of Example [a] has an average of 2 esterified —COOH groups per molecule and the product of Example (b) has an average of 4 such groups. The presence of multiple cathodic bands demonstrates that the esterified products consist of a plurality of esterified orgoteins containing 1 to about 6 ester groups per molecule.

Example 5C

Hybrid containing methyl ether and N-acetyl groups

0.5 mg of totally N-acetylated orgotein was added to the solution (125 µg / ml) of 0.5 mg methyl esterified orgotein produced according to Example 1. In the electrophoretic analysis of the mixture, only bands corresponding to methyl esterified orgotein and an anodic band corresponding to N-acetylorogotein appear.

However, when the mixture is heated at 50 ° C for 4 hours, electrophoresis reveals the formation of several new constituents (at band positions -1-7 to +11) and a reduction of more than 50% of the original band corresponding to N-acetylorgotein and orgotein methyl ester .

The new components resulting from the heating of a mixture of modified orgoteins are hybrids (heterodimers) containing each subunit of N-acetylorgotein (with 10 N-acetyl-lysines in 1 subunit) and a subunit of esterified esterified orgotein (with 0 to 3 groups) ——СООСНз in 1 subunit).

The hybrids can be isolated from their equilibrium mixture with N-acetylorgotein and an orgotein containing groups of —СООСНз (homodlimers) by low-temperature ion exchange chriomatography as described in Example C 2. On standing of isolated heterodimers, rehybridization can continue to form a mixture containing both homodimers . The rate of rehybridization is temperature dependent and is low at low temperatures.

The foregoing examples may be repeated with a similar result by replacing the reagents and conditions used therein with the generic and specifically described reagents and / or reaction conditions of the invention.

From the foregoing description, one of ordinary skill in the art can readily determine the features of the invention and can do so without departing from the scope of

Claims (3)

A process for the preparation of injectable orgotein derivatives having anti-inflammatory activity, characterized in that an aqueous solution of natural orgotein, in particular of bovine, is reacted in an alkaline medium with an alkylating and / or carbamoylating agent, such as dialkyl sulfate with C 3 -C 8 alkyl moiety, C 2 -C 8 activated alkyl halide, vinyl-activated alkylating agent, reducing alkylating agent, or alkali metal cyanate, C 3 -C 8 alkyl isocyanate, aryl isocyanate, isothiocyanate , in an amount sufficient to convert at least one of the titratable ε-amino groups of the orgotein into an N-alkyl or N-carbamoyl group, or in an acid medium with an esterifying agent such as dialkyl sulfate, diazomethane in an amount sufficient to convert up to 10 of the free carboxyl groups in the orgotein molecule to an esterified carboxyl group, at a temperature ranging from the freezing point of the reaction mixture to 80 ° C. 2. A method according to claim 1 for the production of injectable orgotein derivatives having anti-inflammatory activity, characterized in that, to obtain N-alkylated or N-carbamoylated orgoteins, the aqueous scope of the invention, various changes and modifications of the invention are allowed to adapt it to different applications and different conditions. OF THE INVENTION A solution of a natural orgotein, in particular of bovine, is reacted in an alkaline medium with an alkylating and / or carbamoylating agent, such as a C 3 -C 8 dialkyl sulfate, an activated C 2 -C 8 alkyl halide, an alkylating agent having activated vinyl group, reducing alkylating agent, or with alkali metal cyanate, C 3 -C 8 alkyl isocyanate, aryl isocyanate, isothiocyanate, in an amount sufficient to convert at least one of the titratable ε-amino groups of the orgotein into N-alkyl or N-carbamoyl group, at a temperature ranging from the freezing point of the reaction mixture to 80 ° C. 3. A composition according to claim 1 for the production of injectable orgotein derivatives having anti-inflammatory activity, characterized in that an aqueous solution of a natural orgotein, in particular from bovine, is reacted in an acidic medium with an esterification agent to obtain ester derivatives of orgotein. an agent such as a C 3 -C 8 dialkyl sulfate, diazomethane, in an amount sufficient to convert up to 10 of the free carboxyl groups in the orgotein molecule to an esterified carboxylic acid group, at a temperature ranging from the freezing point of the reaction mixture to 80 ° C.