NO763075L - - Google Patents

Description

Orgotein derivatives

This invention relates to orgotein derivatives and a

procedure for their manufacture.

Organotein is the trivial name established by the Nomenclature Council in the USA. hair. assigned to members of a family of water-soluble protein congeners in substantially pure, injectable form, i.e., substantially free of other proteins intermingled with or associated with them in their sources. U.S. patent document no. 3,758,682 claims pharmaceutical compositions comprising orgotein.

The orgotein metalloproteins are members of a family of protein congeners that have a distinctive combination of physical, chemical, biological and pharmacodynamic properties. Each of these congeners is physically characterized in that they are the isolated, generally substantially pure form of a globular, buffer-acting and water-soluble protein having a highly compact native structure which, although heat-labile, is stable upon heating for several minutes at 65°C at pH 4-10. Chemically, each of them is characterized by containing all but 0-2 of the protein araino acids; a small percentage of carbohydrate, no lipids, 0.1 to 1.0% with metal provided by 1 to 5 gram atoms per moles of one or more chelated divalent metals having an ionic radius of 0.60 to 1.00 Å, and essentially no chelated monovalent metals or such that are cytotoxic in the molecule. The arainoic acid mixtures in the orgotein congeners have a remarkably similar composition regardless of the sources from which they are isolated.

Table 1 contains lists of the distribution of the amino acid residues, calculated for a molecular weight of 32,500, for several orgotein congeners.

It can be seen in Table I that orgotein congeners have from 18-26 and usually 20-23 lysine groups, of which all but 1-3 have titratable (with trinitrobenzenesulfonic acid) £-amino groups. One aspect of this invention is directed to orgotein derivatives in which at least part of the orgotein-lysine groups are alkylated and to a method for their production. Another aspect of this invention is directed to orgotein derivatives in which at least part of the orgotein-lysine groups are carbamylated and to a method for producing these.

One can also see from Table I that orgotein congeners have from 29-37 aspartic acid and 21-38 glutamic acid groups. According to another aspect, this invention is directed to orgotein derivatives in which at least part of these amino acid residues in free acid form are esterified and to a method for their production.

As will be clear to a person skilled in the industry, some esterification reagents and conditions enable a simultaneous alkylation of free amino groups in the orgotein molecule and esterification of carboxylic acid groups to take place. Such N-alkylated and esterified orgoteins and their preparation also form part of this invention.

The natural orgotein proteins have, among other things, anti-inflammatory activity. See U.S. Patent Document No. 3,758,682.

They also have exceptionally high superoxide dismutase activity. See McCord & Fridovich, J. Biol. Chem., 244, 6,176, (1970); ibid.,

246, 2,875 (1971). The anti-inflammatory activity of the natural protein is surprisingly largely unaffected by

esterification, N-alkylation or carbamylation. The orgotein derivatives according to this invention are consequently useful in the same

way as the natural proteins, for example. in the treatment of inflammatory conditions in mammals and other animals, as disclosed in the U.S. patent document no. 3,758,682, and reference is made here to the contents of this patent document. A substantial part of the superoxide dismutase activity of the natural protein is also maintained in the orgotein derivatives according to this invention, for example up to 20-100% of the activity of the natural protein.

N- ALKYLATED ORGOTEINE AND N-CARBAMYLATED ORGOTEINE

As stated above, orgotein congeners contain from 18-26 lysine groups. Since the orgotein molecule consists of two identical peptide chains (under units), half of these lysine groups are present in each chain, and these chains are bound tightly but non-covalently together under moderate temperature and pH conditions. Because of the space structure of the orgotein molecule, usually the £-amino groups of a few lysine groups in each chain are not titratable with trinitrobenzenesulfonic acid (TBNS) and thus not readily available for alkylation or carbamylation. However, it also seems possible with both alkylation and carbamylation of the non-titratable lysine £-amino groups by using a highly active alkylating or carbamylating agent, for example dimethyl sulphate at an alkaline pH value. The extent of the alkylation or carbamylation can be determined by reducing TNBS-reactive amino groups, taking into account that 1-3 of the lysine groups in the natural orgotein protein are not titratable with TNBS. For example, bovine orgotein results-

is for only 18 of its 20 to 22 lysine groups. It must also be borne in mind that monoalkylated lysine groups are still acylatable so that only extensively alkylated orgoteins show a reduction in acylatable lysine groups.

Di- and tri-alkylation or carbamylation of the lysine groups can be followed by counting the charge change shown by electrophoresis of the N-alkylated or N-carbamylated product. For example, orgotein alkylated with dimethyl sulfate at a pH of 10 showed a charge change of -2 after acylation with acetic anhydride compared to -20 for non-alkylated orgotein. Similarly, orgotein N-carbamylated with methyl isothiocyanate at a pH of 9 showed a charge change of -2 after 45 minutes.

As is known, it is the electrophoretic mobility of an ion

a function of the electric field strength, the net charge of the ion (including bound kinetic ions) and the coefficient of friction. See, for example, C. Tanford, "Physical Chemistry of Macromolecules", Wiley, New York (1966). Since the friction coefficient depends on the molecular size and shape and on the composition of the solution,

comparisons of different proteins are not too informative. However, by comparing proteins of similar size and shape,

in this case orgotein molecules that are chemically modified with relatively small groups, under identical electrophoresis conditions, the net charge is the only variable that affects this electrophoretic mobility.

Comparison of the electrophoretic patterns of a number of chemically modified orgotein molecules is consistent with this

conclusion. Natural bovine orgotein electrophoreses mainly as one band (band 1), together with small amounts of off-bands (bands 2, 3, etc.) that move faster and are equally spaced in front of the main band, representing orgotein -molecules with a greater ratio between the groups -COOH and -NH2 than such molecules that form band 1. Treatment of natural bovine orgotein with, for example, successively higher concentrations of acetic anhydride at a pH value of 7, or with methyl isothiocyanate at a pH value of 9, leads to the formation of a series of successively more anodic (migrating towards the electrode) electrophoretic bands as successively more free amino groups on the orgotéine molecules are acetylated or carbamylated. In contrast, treatment with dimethyl sulfate produces a series of bands that are successively more cathodic (shifted from band 1 towards the electrode) as successively more free carbonyl groups on the orgotein molecule are esterified.

A plot of the electrophoretic distance versus band number is relatively linear at low degrees of -COOH or -NH2 modification, but gradually bends at stronger modification, since there is a limit to how fast even the most highly charged species

. can be moved through dissolution. The species that migrate quickly are also more sensitive to salt concentration, and are noticeably retarded by electrophoresis of saline samples. Since

extrapolation of more than approx. two band positions are not always exact, it is therefore necessary for accurate charge counting that the unknown is co-electrophoresed with eh solution containing all bands from 1 up to the position of interest (for example partially acetylated or partially carbamylated orgotein). All the conventional protein modification reactions that have been applied to the orgotein molecule to date have been compatible with this interpretation, i.e. the band shapes correspond to the entire charge changes from the natural orgotein molecule. Both acetylation, carbamylation and N-methylthiocarbamylation give the bands 2, 3, 4, 5, etc, and this shows that 1, 2, 3 and 4 free amino groups respectively have been chemically modified. Similarly, succinylation, which changes >vNH3+ groups to r,-NHCCH2<CH>2<C0>2'"<g>ruppers, gives bands 3, 5, 7, 9, etc, and more extensive carbamylation or 0 s tipcarbamylation, which changes even more /^NHCNH-R and ~NHCNH-R- v groups, gives the bands 6, 7, 8, 9, etc. Esterification with dimethyl sulfate or with ethyl diazoacetate gives the bands -1, -2, -3, -4, etc, and

this shows that respectively 1, 2, 3 and 4 free carboxylic acid groups have been chemically modified.

Generally speaking, most, for example all except: 2*-4, of the lysine groups can be alkylated, even with milder alkylating agents, or carbamylated. All but approx. one of the available (TNBS titratable) lysine groups in each of the orgotein peptide subunits can be polyalkylated using stronger alkylation conditions, for example excess dimethyl sulfate,

0.04 M carbonate buffer, pH 10.

As might be expected when not all titratable lysine amino groups are alkylated or carbamylated, the distribution of alkyl or carbamy1 groups on the orgotein compound is probably irregular since none of the titratable lysine amino groups appear abnormally easily alkylated or convertible into carbamylated ones. amino groups. Because the orgoteiri molecule is composed of two identical peptide chains, the alkyl groups on a partially acylated orgotein and the N-carbamylated amino groups on a partially carbamylated orgotein will be distributed more or less randomly along each peptide subunit, but more or less evenly between the two chains. Since

a single alkylation or carbamylation forge 1 is usually used, respectively all the alkyl groups and the carbamylated amino groups will be identical. However, it is possible to

produce alkylated orgoteins and carbamylated orgoteins that have two or more different alkyl groups in the molecule and are identical within each of the molecule's chains.

One way to prepare a mixed alkyl-orgoteine is by alkylating in steps with different alkylating agents. For example, part of the titratable lysine £-amino groups can be alkylated with a moderate concentration of an alkylating agent, for example iodoacetamide, and the rest of the reactive amino groups can be alkylated with a higher concentration of another alkylating agent, for; for example dimethyl sulfate* What constitutes a low or high concentration of alkylating agent will depend on the relative degrees of reaction with protein amino groups and with solvent and will thus depend on the pH during the reaction and on the alkylating agent, and to a lesser extent on buffer medium and temperature. One way to prepare a mixed N-carbamylated orgoteine is to carbamylate in steps with different carbamylating agents.

For example, part of the titratable lysine £-amino groups can be carbamylated with a moderately reactive carbamylation agent1, for

for example KNCO, and the rest of the reactive amino groups can be alkylated with a more reactive carbamylation agent1, for example methyl isocyanate. The reactivity of a carbamylating agent depends on the relative degree of reaction with protein amino groups and with the reaction solvent and thus depends on the pH of the reaction and on the carbamylating agent, and to a lesser extent on the buffer agent and temperature.

Another method of preparing a mixed alkylated or mixed N-carbamylated orgoteine is by hybridization. The term hybridization of orgotein refers to the formation of a mixed orgotein from the peptide chains of two different orgotein molecules, for example A2 and B2, A and B being their respective peptide chains. (A2+ B2^ 2AB). The charge on the heterodimer,

AB, by electrophoresis should be the average of the charges on the homodimers A2 and B2, assuming that the same part of each subunit is involved in the binding in all cases.

£-N-methyl-orgoteine, prepared by alkylation of the natural orgoteine molecule in 0.04 M, pH 10, carbonate buffer with an excess of dimethylsulphate, £-N-ethyl-orgoteine, prepared by

alkylation of orgoteine in the same manner with diethyl sulfate, £-N-propylcarbamyl-orgoteine, prepared by carbamylation of the native orgoteine molecule in 0.1 M, pH 7.6, tris or phosphate buffer with an excess of propyl isocyanate, and £ - N-methylthiocarbamyl-orgotein, prepared by carbamylation of orgoteine in 0.075 M sodium tetraborate with methyl isothiocyanate, can each be hybridized with native orgoteine or with each other by heating together at 50°C for 4 hours.

As will be seen, these semi-alkylated and semi-carbamylated orgotein hybrid molecules can be further alkylated or carbamylated with a different alkylating or carbamylating agent to produce an alkylated or carbamylated orgotein hybrid in which respectively the alkyl groups and the carbamylated amino groups in a peptide chain is distinct from the groups within it

, second chain.

The £-N-alkyl orgoteins and £-N-carbamy1-orgoteins according to this invention appear to have substantially the same spatial structure as the natural orgotein molecule. The content of chelated Cu ++ ' and Zn (gram atom per mole) is approximately the same as in orgotein. In the same way as orgotein, they are very resistant to pronase and other proteolytic enzymatic degradation.

Superoxide dismutase (SOD) enzymatic activity is maintained although, in the case of carbamylated orgo$eins, it is reduced relative to the degree of carbamylation.

v Although the predominantly structural modification of

the natural orgotein molecule which takes place by its alkylation or carbamylation at alkaline pH is mono-, di- and to a lesser extent tri-alkylation of the £-amino groups on lysine-

the groups, when it concerns alkylation, and carbamylation of these £-amino groups, when it concerns carbamylation,

so can also at the same time the free amino groups of the arginine residues thereof and the free carboxyl groups of the aspartic acid and glutamic acid thereof, and also other alkylatable groups present in the molecule, especially -OH bg imidazole nitrogen, and possibly also guanidino nitrogen, - SH, -SCH3, etc, be alkylated or carbamylated, depending on the conditions used and the reactivity of the alkylating or carbamylating agent. While, for example, alkylation at a pH of 10 with iodoacetamide only appears to be £-N-alkylation, the alkylation with dimethyl sulfate at pH 10 is less selective and methyl groups are introduced at the same time anywhere in the molecule. Such accompanying alkylated orgoteins having £-N-alkyl groups are included in the novel compounds of this invention. In the case of carbamylation, however, such co-modified groups are labile and readily hydrolyze in aqueous solutions within a day or less, depending on the pH.

The course of the alkylation, as long as it involves -COOH alkylation, bg of the carbamylation, can be followed directly by a change in total electrophoretic charge and by the appearance of new bands on electrophoresis. Similarly, N-alkylation,

to the extent that it makes an otherwise acylatable -NH2~ group resistant to acylation, is followed by a reduction in the number of acylatable amino groups, in relation to the natural orgotein molecule.

The exact nature of the N-alkyl and N-carbamy1 groups, as well as the number thereof, is not critical as long as the alkyl or carbamy1 radical is physiologically acceptable. Because of the higher molecular weight of the orgotein molecule, even when the orgotein molecule is fully alkylated or carbamylated with alkyl or carbamy1 groups of moderate molecular weight, for example ^100, the estimate of the total chemical composition is relatively small, i.e. less than 10%. Alkylation and carbamylation also have no apparent significant effect on the compact spatial structure of the molecule and the resulting stability, for

example against heating for 1 hour at 60°C, and on attack by proteolytic enzymes.

As will be seen, the alkyl or carbamy1 group must also be one which originates from an alkylating s-r- or carbamylating agent which is capable of alkylating and carbamylating respectively

an amino group in water or a buffer solution, since the reaction is usually carried out in such.

Such alkylating agents include diprimary alkyl sulfates: (RO)2S02 (<R>= = CH3,CH2H5, n-C3H7, n-<C>4Hg), activated alkyl halides: ICH2COX (X = OH, NH2), benzyl- and allyl -bromides, activated vinyl groups CH2= CHX (X = CN, SOCH3, SOC2'H5, COCH3), reductive alkylating agents; RCOR' + BH^ or BH3CN" (R,R = H, CH3, C2H5, CgH5).

With regard to a method for the reductive alkylation of proteins with arbmatics. aldehydes and sodium cyanoborohydride, see Friedman,M. et al., Int. J. Peptide Protein Res., 6, 1974, 183-185, and with respect to alkylation with acrylonitrile, see Means Feeney, "Chemical Modifications of.Proteins", Chapter 6, pages 114-rll7! Fletcher, J.S., Biochem J. 98 34C (1966); Friedman, M. et al., J. Amer. Chem. Soc. 87, 3672 (1965); and Friedman, M. et al. ,: J. Org. Chem, 31, 2888 (1966).

Examples of suitable carbamylating agents include alkali metal cyanates, for example NaNCO, KNCO; alkyl isocyanates and alkyl isothiocyanates, for example RNCO and RNCS where R = CH3,C2H2, n-C3H^, n-C^Hg, n-CgH^^; and aryl isocyanates and isothiocyanates, for example phenyl isocyanate and phenyl isothiocyanate.

Although metal cyanates react with many types of side chains, it is only the reaction with amino groups to

convert them into urea groups which produce a stable product.

The reaction of alkyl isocyanates and -isothiocyanates with orgotheine is completely analogous to the reaction of cyanates with orgoteine, only much faster. The long-chain isocyanates are less reactive than the short-chain isocyanates.

The reaction of orgotein with isocyanates and isothiocyanates can usually be confirmed by using a reagent that introduces a fluorescent group into the molecule, for example fluorescent isothiocyanate. This reagent is similar to alkyl isocyanate and reacts with the amino group on orgotheine to give substituted thioureas. However, even a low degree of substitution markedly reduces the SOD activity of orgotein due to the extent of the fluorescence. Mono- and disubstituted orgoteins are not stable in solution and remain. slowly more heterogeneous due to subunit exchange and hydrolysis.

N-ALKYLATED ORGOTEIN BR

Since the exact chemical nature of the alkyl radical

is not critical, to the extent that it is not physiologically toxic in the orgotein molecule and can be formed on the lysine-£-amino groups, including cyclopentyl, cyclohexyl, menthyl, likewise cycloalkyl, cyclohexylmethyl, /3-cyclopentylpropyl, likewise cycloalkylalkyl, benzyl, p-xylyl and phenethyl and likewise aralkyl as equivalents to the preferred hydrocarbpn-alkyl groups described above, to the extent that they can be prepared. Equivalents also include alkyl with 1-8, preferably 1-4, carbon atoms, and most preferably methyl or ethyl bearing one or more, preferably one, simple substituents, for example carboxy, cyano, carbaloxy and amido, for example carboxymethyl, cyanomethyl, carbethoxymethyl, carbomethoxymethyl and likewise carbo-lower-alkoxymethyl, carbamylmethyl and the corresponding substituted ethyl groups, for example -CH2CH2COOH, -CH2CH2CSN, -CH2CH2CONH2 and -CH2CH2COOR where R is for example methyl or ethyl, -CH2CH2SOCH3,

-CH2CH2SOC2H5 and -CH2CH2COCH3.

The alkylated orgoteins according to this invention are thus orgotein congeners which include those originating from cattle, sheep, horses, pigs, dogs, rabbits, guinea pigs, chickens and humans, where at least one, for example 1, 2, 3, 4 , 5 and up to all (about 18-26) of their titratable amino groups are alkylated, i.e. bear an unsubstituted or substituted alkyl group.

In the preferred embodiment, the alkylated amino groups are those having the formula

where R is H, CH3, C2H5, n-C3H7<iso-C3H7, or other alkyl groups with up to 7 carbon atoms, -COOH, -COO-LA, -CONH-LA, -CON(LA)2,

-C2N, -CH2-C=N, -Ph, -COPh, -CH2OH or -CH(CH3)OH in which LA is lower alkyl with 1-4 carbon atoms and Ph is unsubstituted phenyl or phenyl bearing 1-3 single substituents , for example methyl, chlorine, bromine, nitro, amido and methoxy, carbomethoxy or

carboethoxy, for example p-tolyl, sym.-xylyl, p-amidophenyl, m-chlorophenyl and p-methoxyphenyl. Such orgoteins have the formula

where n is an integer from 1 to approx. 26, preferably at least 10 and more preferably 10-18, and the sum of m and n is the total number of titratable free amino groups in the unmodified congener and R has the meanings indicated above, preferably H or IA, for example methyl or ethyl, and "Org" is the remainder of the orgotein molecule. Some of the alkylating agents used in the method according to this invention will simultaneously alkylate some of the free acid groups on the orgotein molecule. These alkylated orgoteins can be represented by the formula

where "Org", R, m and n have the meanings given above, Y is the number of alkylated free carboxylic acid groups, for example from one up to approx. 8, preferably 2-6, and x are the remaining free carboxylic acid groups in the orgotein molecule.

Reagents useful for introducing carboxymethyl-

and carbamylmethyl groups, are respectively iodoacetic acid and iodoacetamide. Iodoacetic acid can form stable products with cysteine (sulfhydryl), lysine (amino), histidine (imidazole-nitrogen) and methionine (sulfide-sulfur) residues in proteins. Since carboxymethylation of any of these groups, except methionine,

increases the net negative charge on the orgotein protein at pH

8.4, electrophoresis can quantify the total reaction (excluding carboxymethylation of methionine and carboxymethylation of the second imidazole nitrogen).

In natural orgotein, iodoacetic acid and iodoacetamide are converted

predominantly, if not exclusively, with lysine-araino groups„ Even ora free thiol groups are converted several orders of magnitude faster

with iodoacetic acid than the amines do, the former are not reacted

five groups in orgotein that are carboxy-retylated, noticeably faster than the next 10-20 groups do. This is consistent with the turnover of p-mercury benzoate with Orgotein sulfhydrylers, there is no turnover with holo-protein. Even with apo protein,

which gives a p-mercuric-benzoate-sulfhydryl reaction (although it

is slow under non-denaturing conditions), iodoacetic acid in a 10-fold molar excess at pH 7 did not cause any reduction in the sulfhydryl content (determined spectrometrically by p-mercury-benzoate titration) after several weeks at 4°C. The pH dependence of the carboxylation reaction is, as shown by electrophoresis, compatible with turnover of lysine (pK>8) and not of histidine (pK < 6), since the rate of turnover at identical initial concentrations increases steadily as pH approaches 9, and with no appreciable turnover at or under neutral conditions where histidine should still be unprotonated and reactive. On the other hand, ethyl chloroformate titration of histidine showed only 5 histidine groups in carboxyretylated orgoteine under conditions that showed 16 groups in native orgoteine.

Evidence that neither methionine nor histidine is available for turnover in native bovine orgotein was provided by an experiment in which orgotein was incubated with 0.2 M iodoacetamide in 0.5 M phosphate buffer pH 6.5 for 48 hours. Alkylation of methionine or double alkylation of histidine should have produced a more positively charged protein at pH 8.4, but electrophoresis showed no change in SOD^ activity or banding pattern.

However, the lysine-amino groups in orgotein are definitely reacted with iodoacetate. The extensively carboxymethylated orgotein migrates by electrophoresis in a similar manner to acetylated orgotein, but has lower SOD activity (about 20% of the activity of unmodified orgotein protein).

In addition to N-methyl "bovine" orgoteins and orgoteins in the following examples, other examples of N-alkyl bovine orgoteins according to this invention are N-ethyl orgoteine, N-propyl orgoteine and N-benzyl -orgotein, where in each case there are 9 such alkyl groups in each of the two subunits of the orgotein molecule, and the corresponding orgoteins where there is an average of respectively 1, 6 or 10 such alkyl groups in each such subunit, and the corresponding congeners from each of these originating from humans, sheep, horses, pigs, dogs, rabbits, guinea pigs and chickens.

N- CARBAMYLATE ORGOTEINES

The carbamylated orgoteins of this invention are orgotein congeners, including those derived from cattle, sheep, horses, pigs, rats, dogs, rabbits, guinea pigs, chickens and humans, wherein at least one, for example 1, 2, 3, 4, 5 and up to all (about 18-26), of their titratable amino groups are carbamylated; i.e. bears an unsubstituted or substituted -CONH or -CSNH group. In a preferred embodiment, the carbamylated amino groups are those having the formula

where X is 0 or S, and R is CH3, C2H5, n-C3H7, iso-C3H7, n-C8H17 or other alkyl groups with up to 12 carbon atoms, Ph, or when X is 0 also a hydrogen atom, and Ph is unsubstituted phenyl or phenyl bearing 1-3 single substituents, for example methyl, chlorine, bromine, nitro, amido and methoxy, carbomethoxy or carboethoxy, for example p-tdlyl, sym.-xylyl, p-amidophenyl, m-chloro-phenyl and p-methoxyphenyl. Such orgoteins have the formula

where n is an integer from 1 to approx. 26, preferably at least 2 and more preferably approx. 6 to 10, and the sum of m and. n is the total number of titratable free amino groups in the unmodified congener, X is 0 or S, R has the above meanings, preferably H or alkyl of 1-8 carbon atoms, and "Org" is the remainder of the orgotein molecule.

Particularly preferred carbamylated orgoteins are alkylcarbamyl and alkylthiocarbamyl orgoteins where the alkyl group is unsubstituted alkyl with 1-8 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, octyl.

Since the exact chemical nature of the carbamy1 group is not critical, to the extent that it is not physiologically toxic in the orgotein molecule and can be formed on the lysine £ -amino groups, those which otherwise correspond to the above formula where R is cyclopentyl, cyclohexyl are included , mehtyl, likewise cycloalkyl, cyclohexylmethyl, /3-cyclopentylpropyl, likewise cycloalkylalkyl, benzyl, p-x<y>lyl and phenethyl and likewise aralkyl as equivalents to the preferred alkylcarbamyl-orgoteins described above, to the extent that they can be prepared. Equivalents also include those where R is alkyl with 1-8, preferably 1-4, carbon atoms, and most preferably methyl or ethyl bearing one or more,

preferably one, other substituents, for example fluoresceinyl.

Other examples of N-carbamyl-bovine-orgoteins according to this invention, in addition to the N-carbamylated "bovine"->orgoteins in the following examples, are correspondingly N-carbamyl-orgotein, N-propylcarbamylorgotein, N-ethylcarbamy1- orgoteine and N-methylthiocarbamy-orgoteine where in each case there are 9 such carbamyl groups in each of the two subunits

on the orgotein molecule, and the corresponding orgoteins where it

in hyer such subunit is one average, of respectively 1, 6 or 10 such carbamyl groups, and the corresponding congeners

for each of these originating from humans, sheep, horses,

pigs, dogs, rabbits, guinea pigs, chickens and rats.

ESTERIFIED ORGOTEINE

As indicated above, orgotein congeners contain approx. 50-68 glutamine and asparagine residues, but only approx. 20-27 of these

has free acid groups. Since the orgotein molecule is formed by two identical or nearly identical peptide chains (subunits), half of these amino acid residues in each chain are firmly but not covalently bound together under moderate temperature-bg pH-xorhpid. Esterification changes the charge on the orgotein molecule, and usually only up to approx. 10 and preferably up to approx. 6 to 8 of these free acid groups are esterified and still maintain the structure of the natural molecule, on which stability and use as a drug depends.

The esterification of the carboxylic acid groups can be quantitated by counting the charge change shown by electrophoresis. If desired, the esterified orgotein can be hybridized with natural orgotein, as described later, thereby reducing the number of esterified carboxylic acid groups in the molecule by half.

Because the orgotein molecule is composed of two identical peptide chains, the ester groups are probably distributed more or less evenly between the two chains. Since it is usually used

a single esterification agent, all the ester groups will be identical. However, it is possible to produce esterified ones

orgoteins that have two or more different ester groups in them

the molecule and even within each chain thereof.

^ .?L A method of preparing a mixed ester of orgotein.

must be esterified step by step with different esterification agents.

For example, part of the free acid groups can be esterified with one esterification agent with a low concentration, for example diethyl sulfate, because another part of the acid groups can be esterified with another esterification agent with a higher concentration, for example diazbmethane. What constitutes a low or high concentration of esterification agent will depend on its relative degree of reaction with protein-acid groups and with dissolving agent1 and will thus depend on the pH of the reaction and of the esterification agent, and to a lesser extent on the buffer agent and temperature.

Another method of preparing a mixed orgotein ester is by hybridization. The term hybridization of orgotein refers to the formation of a mixed orgotein from the subunit chains of two different orgotein molecules, for example A2 and B2, A and B being their respective peptide chains. (a2+

t*2^-A>2AB). The charge of the heterodimer, AB, on electrophoresis should be the average of the charges of the homodimers A2

and B2, assuming that the same part of each subunit is involved in the binding in all cases.

Each of methyl orgoteine and ethyl orgoteine can be hybridized with natural orgoteine or with each other by heating with a small excess of natural orgoteine at 50°C for 4 hours. Electrophoresis of the resulting heterodimers shows a mixture of bands lying between the bands for native orgotein and the bands for the esterified orgotein prior to hybridization.

It is clear that these semi-esterified orgotein hybrid molecules can be further esterified with a different esterification agent to form an esterified orgotein hybrid in which the ester groups in one peptide chain are different from those in the other.

The esterified orgoteins according to this invention appear to have essentially the same spatial structure as the natural orgotein molecule. The content of chelated Cu and Zn (gram atoms per mol) is approximately the same as for orgotein. In the same way as orgotein, they are very resistant to pronase and other proteolytic enzymatic degradation. Superoxide dismutase enzymatic (SOD) activity is not clearly reduced until more than approx. 6-8 carboxylic acid groups are esterified.

The exact nature of the esterified groups, and likewise the exact number of esterified groups, is not critical as long as the esterified group is physiologically acceptable. Due to the high molecular weight of the orgotein molecule, even when the orgotein molecule is esterified with esterifying groups of moderate molecular weight, for example < 160, the estimate of the total chemical composition is relatively small, i.e. less than 5%. Of course, the esterification of the free carboxylic acid groups clearly has a profound effect on the isoelectric point and resulting electrophoretic mobility, but as discussed above, as long as the esterification is limited to approx. 10 or less

with glutamic and aspartic acid groups, it has no apparently significant effect on the compact spatial structure of the molecule and consequently on its stability, for example when heated for 1 hour at 60°C, and on attack by proteolytic enzymes .

It is clear that the performing group must also be

one which is derived from an esterification agent capable of

esterify one carboxylic acid group in water or buffer solution,

since the turnover is usually carried out therein. Such esterification agents include dimethyl and diethyl sulfate, diazomethane and other diazo compounds, for example with the formula N2CH2COX

where X is, for example, 0CH3, O<C>2H5, NH2orNHCH2CONH2, and

other esters bg amides of diazoacetic acid lacking, reactive groups, for example carboxyl or imino.

With respect to methods of preparing such esters,

see Methods in Enzymology, Vol. XI, page 612 (1967); K. T. Fry et al., Biochem. Biophys. Res. Comm., 30,489 (1968); G. R. Delpierre and J.S. Fruton, PNAS, 56 1817 (1966).

More specifically, this invention is directed to -COOH esterified orgoteine where the ester group preferably has up to 4 carbon atoms, for example a monohydric alkanol, for example methyl, ethyl, and most preferably methyl.

Since the exact chemical nature of.the ester radical

is not critical, and as long as it is not physiologically toxic

and can be formed on orgotein's free acid groups, including those bearing one, two or more simple substituents, for example

halogen, alkyloxy, carbo-alkyloxy, carbamido, etc., especially those where the ester group carrying the substituent or substituents,

is methyl, as equivalents to the preferred alkyl esterification groups described above, to the extent that they can be formed.

Equivalents are thus calculated in addition to the ester

orgotein in which they; esterified acid groups are -COOCH^, such where the ester groups are -C00R where R is -CH2C0X where X is OCH^,

OC2H5, NH2, NHCH2CONH2 or CH2CH2-phenyl, or where R is CH(phen<y>l)2.

The esterification of the -COOH groups can be followed by counting the charge bias, shown by electrophoresis, compared to natural orgoteins.

As is known, the electrophoretic mobility of an ion is a function of the electric field strength, the net charge of the ion (including bound counterions) and the coefficient of friction.

See for example C. Tanford, "Physical Chemistry of Macromolecules", Wiley, New York (1966). Since the coefficient of friction depends on molecular size and shape and on the composition of the solution, comparisons of different proteins are not informative* However, when comparing proteins of similar size and shape, in this case orgotein molecules that are chemically modified with relatively small groups, under identical electrophoresis conditions, the net charge is the only variable

which affects this electrophoretic mobility.

Comparison of the electrophoretic patterns of a number of chemically modified ^orgotein molecules is consistent with this conclusion. Natural bovine orgotein electrophoreses mainly as one band (band 1), along with small amounts of bands

(bands 2,3, etc.) which move faster and which are equally spaced for those lt ahead of the main band, and which represent orgotein molecules with a greater ratio between the groups -COOH and -NHj than such molecules which form bands 1. Esterification of successively larger numbers of the free -COOH groups on the natural bovine orgotein leads to the formation of a series of successively more cathodic (migrating towards the negative electrode) electrophoretic bands as successively more carboxylic acid groups on the orgotein molecule are esterified.

A diagram of the electrophoretic distance in relation to band-ta11 is relatively linear at a low degree of -COOH modification, but gradually bends at stronger modification, since there is a limit to how fast even the most highly charged species can move through solution. The species that migrate quickly are also more sensitive to salt concentration, and are noticeably retarded by electrophoresis of salt-containing samples. Since extrapolation of more than approx. two band positions are not always exact, it is therefore necessary for accurate charge division

that the unknown is co-electrophoresed with a solution containing all bands from 1 up to the position of interest (for example partially acetylated orgoteine).

All the conventional protein modification reactions that have been applied to the orgotein molecule to date have been compatible with this interpretation, i.e. that the band positions correspond to the entire charge changes from the natural orgotein molecule. Esterification with dimethyl sulfate or with ethyl diazoacetate gives the bands

-1, -2, ^-3, -4, etc., and this shows that respectively 1, 2, 3 and 4 free carboxylic acid groups have been chemically modified

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

As might be expected when none of the titratable free carboxyl groups seem abnormally easy to esterify, the distribution of the ester-forming ones is. groups on; the orgotein molecule probably irregularly. Because the orgotein molecule is composed of two identical peptide chains, the ester groups on a partially esterified orgotein will be more or less randomly distributed along each peptide subunit#but more or less evenly between the two chains. Since a single esterifying agent is usually used, all the ester groups will be identical. However, it is

possible to produce esterified orgoteins which have two or more different ester groups in the molecule. and even within each of the molecule's chains.

One way to prepare a mixed esterified orgotein is to esterify step by step with different esterification agents. For example, part of the free -COOH groups can be esterified with one esterification agent, for example dimethyl sulfate, and the rest of the reactive -COOH groups can be esterification with a other esterification agent, for example ethyl diazoacetate;

Another method of producing a mixed esterified orgotein is by hybridization. The term hybridization of orgotein refers to the formation of a mixed orgotein from subunit chains of two different orgotein molecules, for example, ^A2 and B2 / where A and B are their respective peptide chains, ( A2 + B2 ^=^- 2AB). The charge on the heterodimer, AB, by electrophoresis should be the average of the charges of the homodimers A2 and B2', assuming that the same part of each subunit is involved in the binding in all cases.

Methyl-esterified orgotein, produced by esterification of approx. 6 free acid groups on the orgotein molecule with dimethyl sulfate, and carboxymethyl-esterified orgotein, produced by esterification of the natural orgotein molecule with ethyl diazoacetate, can each be hybridized with natural orgotein or with each other by heating together at 50°C for 4 hours.

Other examples of orgotein derivatives according to this invention, in addition to bovine orgotein derivatives in

the following examples are corresponding derivatives of other orgotein congeners.

Other examples of esterified bovine orgoteins are . carbomethoxymethyl-orgoteine and carbamymethyl-orgoteine, where in each case there are 3-4 such esterified groups in each of the two subunits of the orgoteine molecule, and the corresponding esterified orgoteins where there are respectively 1 or 2 such ester groups in each subunit, and the corresponding congeners for each of these derived from humans, sheep, horses, pigs, dogs, rabbits, guinea pigs and chickens.

The orgotein derivatives can be isolated from the reaction solution,

preferably after dialysis to remove extraneous ions, by conventional lyophilization, for example in the manner

described in the U.S. Patent Document No. 3,658,682. If desired, orgotein derivatives can first be purified with wood. ion exchange resin chromatography, electrophoresis and/or gel filtration using a polymer that acts as a molecular sieve.

Filtration through a micropore filter with a pore size of 0.01-0.22 micron in an aseptic manner in sterile glasses, possibly after adjusting the ionic strength with NaCl and/or sodium phosphate, for example until it is isotonic, will provide

a bacterially sterile solution suitable for administration by injection. Filtration through a 0.1 micron pore filter will also reduce or eliminate pyrogens in the solution.

The pharmaceutical compositions according to this invention comprise an orgotein ester according to this invention and a pharmaceutically acceptable carrier. The form and character of this carrier is of course determined taking into account the method of administration

The pharmaceutical mixture is preferably in the form of a sterile, injectable preparation, for example as a sterile, injectable aqueous solution. The solution can be formed in accordance with known techniques by using the above-mentioned carriers. The

sterile injectable preparations may also be a sterile injectable solution or suspension in any non-toxic

, parenterally acceptable diluent or solvent, or it may be a lyophilized powder for reconstitution with

such a solvent mite1.

The compositions according to this invention contain an effective unit dose amount of an orgotein derivative according to

to this invention, i.e. that the orgotein derivative is present

at a concentration effective to elicit the desired response when a unit dose of the composition is administered in the manner appropriate for the particular pharmaceutical carrier.

Liquid mixtures, both topical and injectable, contain

for example usually approx. 0.5 to 20 mg of the orgotein derivative per 0.25. to 10 ml, preferably 0.5 to 5 ml, except for I.V. infusion solutions which can also be more diluted, for example 0.5 to 20 mg of orgotein derivative per 50-1,000 ml, preferably 100-500 ml, with infusion solution. Tablets, capsules and suppositories usually contain 0.1 to 25 mg, preferably 1 to 10 mg, of orgotein derivative per unit.

The orgotein derivative is usually administered by instillation or by injection, for example intramuscularly, subcutaneously, intravenously or intradermally. I. M. is preferred, except in cases such as shock where I.V. sometimes it is preferred because this mode of administration begins to take effect more quickly, and in certain localized diseases, such as radiation or other cystitis, where local injection, infusion and/or instillation are often more effective. Individual doses usually fall within the range of 0.5 to 20 mg. The preferred area for humans is approx. 0.5 to 8 mg, and for horses approx. 5.0 - 10.0 mg. The exact dose is not critical and depends on the type and severity of the disease

The orgotein derivatives according to this invention are, in the same way as orgotein, effective in the treatment of a number of different inflammatory conditions, including those where synthetic anti-inflammatory agents are of limited benefit, for example due to toxic side effects with long-term use.

More particularly, these orgotein derivatives are effective in improving inflammatory conditions and mitigating their effects, for example those related to the urinary organs and

-the joints, in various mammals. They are useful for relief

the symptoms associated with rheumatism, ostep and post-traumatic arthritis, and also bursitis, tendonitis, etc.

With respect to further details regarding how to isolate the starting orgotein congeners and how to

use of the orgotein derivatives 1 according to this invention, including administration methods, dosage forms, dosage regimens and also inflammatory and other conditions amenable to treatment with esterified orgoteins, see U.S. Patent No. 3,758,682, which is incorporated by reference in this description.

It is believed that a person skilled in the industry can use the present invention to its full extent by applying the preceding description, without further extensive work. The following preferred specific embodiments shall therefore only

be considered illustrative and not in any way limiting from the rest of the revelation.

EK5 EMPEL IA

A solution of 5 mg of bovine orgotein in 4 ml of 0.04 M carbonate buffer was treated with 20 µl of dimethyl sulfate and the pH was maintained at 10.0 by the addition of 0.5 M NaOH. The base recording had a half-time of approx. 37 minutes. Electrophoresis showed SOD-active bands 1 to -5 (average -3). After dialysis, lyophilization and re-dissolution in 1 ml of water, the protein retained its SOD activity and unchanged Cu ++ and Zn ++ content compared to the untreated orgotein. Acetylation of an 85 µg/ml solution of the modified protein with a total of 3 µl acetic anhydride in 0.4 ml borate buffer at pH 9 gave an average electrophoretic charge change of only approx. -2, compared to -20 for natural orgotein in the same solution. At pH 10, dlmethylsulphate-treated orgotein is therefore extensively N-methylated, i.e. approx. 18 groups per molecule.

EXAMPLE 2A

The procedure from example IA is followed, and the corresponding congeners originating from humans, sheep, horses, pigs, dogs, rabbits, guinea pigs and chickens are respectively used as starting materials. In each case, all but approx. a lysine group on each subunit of the orgotein molecule, alkylated.

EXAMPLE 3A

The procedure from example IA is followed, but diethyl sulfate is used instead of dimethyl sulfate. The properties of the resulting 6-N-ethylated orgoteine are generally substantially the same as those of the £-N-methylated orgoteine.

EXAMPLE 4A

The α-amino groups on the lysine residues of bovine orgotein were carboxymethylated1 with 0.2 M sodium iodoacetate at ambient temperature under the conditions and with the results indicated below.

From these "electrophoretic patterns it appears that no N-alkylation took place at pH 3.8 and 5.0 and that at pH 9.2 an average of about 3 -CB^COO"" groups were introduced during 2 1/ 2 hours About 5 such groups were introduced in 6 1/2 hours and about 18 such groups were introduced in 72 hours on the £-amino nitrogen atoms.

From the electrophoretic patterns of the thus treated orgotein it appears that at pH 6 less than half of the orgotein molecules were alkylated, at pH 7 after 2 days the orgotein molecules had an average of one £-amino group alkylated with one -CI^ COO" group, and after 5 days an average of two such groups per molecule had been introduced. At pH 10, on average more than three such groups had been introduced after 2 1/2 hours and after 6 1/2 hours there had been an average of more than 10 such groups introduced groups per molecule.

The products of all of the above alkylation methods where

-CH2C00~ groups were introduced, were mixtures of 6-amino-alkylated orgoteins containing varying numbers of such groups, so

as evidenced by the appearance of a plurality of bands with varying electrophoretic mobilities.

EXAMPLE IB Unsubstituted Carbamylated Orgoteine

A solution of 3.7 mg of orgotein (bovine congener) and 14 mg of KNCO in 2 ml of 0.025 M sodium phosphate buffer at pH 7.5 was incubated at 4.C. Electrophoresis of aliquoted samples during

54 days showed the appearance of a series of SOD-active bands

which were more anodic than for natural orgotein. The average charge changes along with the range of active bands

on both sides of the mean for the samples, is indicated below..

EXAMPLE 2B Alky1-carbamylated orgoteins 50 µl portions of orgotein (bovine congener) in 0.1 M tris or phosphate buffer at pH 7.6 and at a concentration of 10 mg/ml were reacted at 4° C with 1 µl of either propyl isocyanate or octyl isocyanate for different time periods. In two cases, 2 µl additional propyl isocyanate was added after two days. The number of introduced propylcarbamy1 and octylcarbamy1 groups was determined by average charge change, as determined by electrophoresis, as shown below. Average charge change

EXAMPLE 3B Alkyl-carbamylated orgotheins

The procedure from example 2B was repeated, using 10 mg of orgotein in 1 ml of 0.1 M phosphate buffer with pH 7.6 and 1 ml of propyl or octyl isocyanate. The solution was kept at 25°C for 4 hours and examined electrophoretically. Then 1 ml of isocyanate was added to each. After incubation at 4°C overnight, the solutions were again examined electrophoretically

and dialyzed. The carbamylated proteins were less soluble in deionized water and were partially precipitated by dialysis, but were soluble in 0.15 M saline. Number of carbamylated lysine-

groups are shown below.

EXAMPLE 4B N-methyl-thiocarbamyl-orgoteine

To a solution of 5 mg of orgotein in 4 ml of 0.075 M Na 2 B 4 O 7 of pH 9 was added 10 µl CH 3 NCS., and the mixture was shaken for one minute until the CH 2 NCS was dissolved. After intervals, samples of 50 µl were withdrawn and these were quenched with 0.4 ml of 0.1 M sodium acetate buffer with pH 5.3. A white precipitate of sulfur (as evidenced by its burning odor and its solubility in CS2) appeared after between 6 and 22 hours. After 22 hours at room temperature, the remaining reaction mixture was quenched with 0.5 ml of 1 M acetate buffer of pH.5.3 and was dialyzed under frequent changes of water for 2 days. The dialyzed solution was briefly centrifuged to remove the white turbidity of the solution.

Electrophoresis of the samples taken from the solution showed the formation of a series of increasingly more anodic SOD-active bands.- Analysis of the samples with cytochrome C experiment at pH 7.8 (McCord & Fridbvich, J. Biol. Chem. 6049- 6055 (1969)) showed a drop in SOD activity with more extensive modification.

The dialyzed 22-hour turnover product showed a fast-moving anodic band by electrophoresis, retained 18% of the SOD activity of the natural orgotein protein, and contained 2.06 GAPM Zn++ and 1.76 GAPM Cu<++> (compared to 2.26 GAPM Zn++ and 2.08 GAPM Cu<++>in unmodified orgotein). All products were soluble and maintained chelated Cu and Zn and at least part of the superoxide dismutase activity of the unmodified protein.

EXAMPLE 5B N-Fluoresceylcarbamyl-orgotein

A* solution of 100 mg of orgotein (bovine congener) and 6 mg of fluorescein isothiocyanate in 10 ml of 0.14 M phosphate buffer of pH 8.5 was kept at 4°C for 18 hours. The reaction mixture was added to a microporous cross-linked chromatographic column

dextran ("Sephadex G-50", Pharmacia, Upsala, Sweden) and eluted therefrom with bbrate-buffered saline at pH 8 (0.15 M). The eluted fractions with yellow protein were dialyzed against water. The

dialyzed protein that was precipitated (20 mg), was intensely yellow and fluorescent and was easily soluble in borate-buffered saline with pH 8. Electrophoresis showed that the 96 mg of soluble protein consisted of approx. half with mono-carbamylated orgoteine (-3 charge change) along with some double and triple carbamylated orgoteine (-6 and -9 charge changes) and unreacted orgoteine. The redissolved smear appears to be somewhat more extensively modified protein, since electrophoresis showed a fast anodic fat.

The soluble protein was chromatographed on a weakly basic (DEAE-cellulose) ion-exchange coionhe of pH 6 with a 0.01 to 0.2 M linear gradient of phosphate buffer. The mono-carbamylated and di-carbamylated orgoteins were isolated, dialyzed and lyophilized.

The mono-carbamylated orgotein was SOD active by electrophoresis and was stained with NBT-riboflavin. According to the cytochrome C assay at pH 7.5, it had 48% of the SOD activity of native orgotein. In the Ungar-bio test, the protein showed approx.

50% of the activity of natural orgotein.

A solution of the mono-carbamylated orgotein stored at 4°C for 22 weeks was changed to a mixture of orgotein, mono-carbamylated orgotein and di-carbamylated orgotein (apparent result of hybridization), and some of the non-protein fluorescent -compoundIse (apparently the result of hydrolysis of

the thiourea group on carbamylated orgotein to give free aminofluorescein). V

By following the procedure in the examples above, but by using as starting material the respective orgotein congeners derived from humans, sheep, horses, pigs, dogs,

rabbits, guinea pigs, rats and chickens, the corresponding carbamylated derivatives of these congeners are formed.

EXAMPLE 1C Methyl esterification

A solution (125 µg/ml) of 0.5 mg bovine orgotein and 0.5% v/v dimethyl sulfate in 4 ml of 0.05 M acetate buffer is maintained at pH 5 for 100 minutes. Electrophoretic analysis of the reaction product shows SOD-active bands from +1 to - 6

(average -4). The product is orgotein with an average of 4 -COOCH^ groups.

EXAMPLE 2C Methyl Esterification

A solution of 0.5 mg bovine orgotein and 10 µl dimethyl sulfate in 4 mL water was kept at constant pH by the addition of 0.1 M NaOH. The degree of base uptake in the pH range 7-10 was clearly independent of pH and of the addition of 0.25 mol sodium phosphate buffer. Electrophoresis showed the formation of more cathodically SOD-active bands with an onset rate of approx. -0.5 charge/hour. After 21 hours, mainly at pH 7-8, the solution was analyzed

with respect to N-methylation and esterification, as described below. N-methylation could not be detected. (a) Acetylation of a 0.5 mL aliquot of the esterified orgoteine with 1 µl acetic anhydride + 3 µl 6 M NaOH at 4°C gave a solution whose electrophoresis pattern was a fast-moving anodic band similar to that of acetylated natural orgotein, and this proves that the free amino groups were unaffected during the esterification. (b) A 1 ml aliquot of the esterified orgotein solution was adjusted to a pH of 10.5 and stored enclosed in a desiccator along with NaOH pellets. Although the cathodic band patterns of bands 1 to -4 were stable at pH 7-9, the cathodic bands at pH 10.5 gradually disappeared over a period of 4 days as the electrophoretic patterns of native orgotheine appeared new. Protein methyl esters are usually readily hydrolyzed at alkaline pH values, and this confirms that the more cathodic bands that appeared after esterification were orgotheine esters.

From the foregoing, it is clear that dimethyl sulfate at pH 7-10 after two hours esterifies on average one -COOH group per molecule, after four hours approx. 2 per molecule and continues there-after to increase the number of esterified -COOH groups up to a maximum of approx. 6 per molecule.

By following the procedure from examples 1C and 2C, and by using in each case the corresponding orgotein congeners originating respectively from humans, sheep, horses, pigs, dogs, rabbits, guinea pigs and chickens as starting materials,

will average approx. four carboxylic acid groups on the orgotein molecule converted to methyl esters thereof.

By following the procedure from examples 1C and 2C, and using diethyl sulfate instead of dimethyl sulfate,

orgotein esters with from one to six free carboxylic acid groups converted to ethyl esters thereof. The properties of the resulting esterified orgoteins are essentially the same as for methyl-esterified orgoteins.

If desired, the esterified orgoteins can be further purified by ion-exchange chromatography to separate arteries with different net charges, and consequently different degrees of esterification. For example, by elution of 200 mg of orgotein through a DEAE "Sephadex" column of 2.5 x 40 om with 4 liters of eri 0.01 M to 0.2 M linear gradient of tris buffer with pH 8.5, orgotein is separated -bands from each other, as the electrophoretically more cathodic bands are eluted first. With such a method, the mixture of methyl-esterified orgoteins produced by the method in example 1C can be separated into fractions

which mainly contain 1, 2,3, 4, 5 or 6 methyl ester groups per orgotein molecule. Such fractionation of partially modified orgotheine by ion-exchange chromatography can be applied to any modified orgoteine whose molecular charge depends on the degree of modification,

for example methyl-esterified orgoteine, carbethoxymethyl-esterified orgoteine and N-acetylated orgoteine.

EXAMPLE 3C Carbethoxymethyl Esterification

Ethyl diazoacetate is prepared by reacting glycine ethyl ester with nitric acid, as in "Organic Synthesis", Coll. Vol. IV, p. 424, but using CCl^ instead of CH 2 Cl 2 to extract the ethyl diazoacetate from aqueous solution.

2 ml of approximately 1 M ethyl diazoacetate/CCl 2 solution

placed in a flask on. 25 ml and almost all CCl^ is evaporated under aspirator pressure. One solution of 9 mg bovine orgotein/3 ml water is added and this is then rotated to disperse the organic phase. The solution is stored at 4°C and rotated for a few days. The reaction mixture remains heterogeneous throughout. Electrophoresis shows gradual formation of more cathodic SOD-active protein bands -1 to -6. The average charge change is 1.2 after 5 days and 3 after 12 days. The product

is one mixture of carbethoxymethyl-orgoteine esters which after 5

° days have either one or two slikéV ester groups per molecule, and after 12 days from 1 to 6 such ester groups.

The aqueous phase is then filtered and desalted by chromatographic fractionation using a 10 cm 3's

"Sephadex G-25" column. The protein fractions are lyophilized

and redissolved in 2 ml of water, and the pH is raised from 3.7 to 5.6 with

2<y>ul of 1 M NaOH. Electrophoresis shows no change from

before desalting and lyophilization. Re-reaction with this solution for one month under the same conditions as above gives fat-like protein on electrophoresis with less SOD activity and no material more cathodic than band -6.

EXAMPLE 4C Methyl Esterification

By following the procedure in Examples 1 and 2, bovine orgotein was alkylated under the conditions indicated in the table below.

The product from example (a) had an average of 2 esterified -COOH groups per molecule, and the product from example (b) an average of 4 such groups. The presence of a plurality of cathodic bands proves that the esterified products consist of., a plurality of esterified orgoteins containing from one and up to 6 ester groups per molecule.

EXAMPLE 5C Methyl ester/N-acetyl hybrid

To a solution (125 µg/ml) of 0.5 mg of methyl-esterified orgoteine, prepared by the method of Example 1, was added 0.5 mg of fully N-acetylated orgoteine. Electrophoresis of the mixture showed only one band corresponding to methyl-esterified orgoteine plus the anodic band corresponding to N-acetyl-orgoteine. However, after the mixture had been heated at 50°C for 4 hours, electrophoresis showed that several new species had formed (at band positions +7 to +11) and that there was over a 50% reduction of the original N-acetyl and methyl -ester orgotein bond.

The new species formed by heating the mixture of modified orgoteins are hybrids (heterodimers) containing a subunit each of N-acetyl-orgotein (containing 10 N-acetyl-lysine groups per subunit) and methyl-esterified orgotein (containing 0 to 3 -COOMe groups per subunit).

The hybrids can be isolated from their equilibrium mixture with N-acetyl and -COOMe orgoteins (homodimers) by ion-exchange chromatography at low temperature, as described in Example 2C.

The isolated heterodimers can, upon storage, continue to re-hybridize to restore a mixture that also contains both homodimers. The degree of re-hybridization is temperature dependent and is low at low temperatures.

The previous examples can be repeated with similar ones

success by substituting the generally or specifically described reactants and/or operating conditions according to this invention instead of those used in the preceding examples.

From the foregoing description, one skilled in the industry can easily ascertain the essential features of this invention, and can, without departing from its spirit and scope, make various changes and modifications to the invention to adapt it to different applications and conditions.

Claims (33)

1. Process for the production of injectable orgotein derivatives having anti-inflammatory activity, characterized by reacting orgotein with (a) an alkylating agent or a carbamylating agent to produce, respectively, an N-alkylated orgotein with an alkyl group or an N-carbamylated orgotein with a carbamyl group on at least one lysine- £-amino group thereof, or (b) an esterification agent to produce an esterified orgotein where up to 10 of the free carboxylic acid groups on the orgotein protein molecule are esterified. 2. Method according to claim 1, characterized in that an orgotein originating from cattle is used. 3. Method according to claim 1, characterized in that orgotein is reacted with an alkylating agent using such alkylation conditions. that an alkylated orgotein is produced which has at least 10 alkylated £-amino groups per molecule. 4. Method according to claim 3, characterized in that such alkylation conditions are used that an alkylated orgoteine is produced with at least 18 alkylated ^-amino groups per molecule. 5. Method according to claim 3, characterized in that a di-primary alkyl sulphate, an activated alkyl halide, an activated vinyl compound or a reductive alkylating agent is used as alkylating agent. 6. Method according to claim 3, characterized in that a dialkyl sulfate is used as alkylating agent, where each alkyl is alkyl with 1-4 carbon atoms. 7. Method according to claim 3, whereby dimethyl sulfate is used as alkylating agent. 8. Procedure according to claim 1. characterized in that orgotein is reacted with a carbamylating agent using such reaction conditions that a carbamylated orgotein is produced with at least two carbamylated £-amino groups per molecule. 9. Procedure in accordance with claim 8, vessel aft in that such reaction conditions are used that the a carbamylated orgotein with approx. 6 to 10 carbamylated £-amino groups per molecule. 10. Method according to claim 8, characterized in that the carbamylating agent used is a , alkyl metal cyanate, alkyl isocyanate, alkyl isothiocyanate <,> ar <y>l isocyanate or aryl isothiocyanate. 11. Method according to claim 8, characterized in that the carbamylating agent used is that alkyl metal cyanate; alkyl isocyanate or alkyl isothiocyanate where the alkyl group contains up to 12 carbon atoms. 12. Method according to claim 8, characterized in that an alkyl isocyanate is used as carbamylation agent where the alkyl group contains 1-8 carbon atoms or an alkyl metal cyanate. 13. Method according to claim 8, characterized in that an alkyl isothiocyanate is used as carbamylating agent, where the alkyl group consists of 1-8 carbon atoms. 14. Method according to claim 1, characterized in that orgotein is reacted in an aqueous solution with an esterification agent which esterifies carboxylic acids in aqueous solution or buffer solution. 15. Process according to claim 14, characterized in that dimethyl or diethyl sulfate, an azide with the formula N2 CH2 COX where X is 0C <H>3, OC2H5, NH2 or NHCH2 CQN <H>2 or another ester or amide of diazoacetic acid lacking a reactive group. 16. Forward <g> mode of action according to claim 14, character cert in that the esterification agent used is one that forms alkyl esters with 1 to 4 carbon atoms. 17. Method according to claim 14, characterized in that the esterification agent is used which \ form methyl esters. 18. Method according to claim 14, characterized in that dimethyl sulfate is used as esterification agent. • ! 19. An orgotein derivative selected from the group consisting of an alkylated orgotein in which an alkyl group with up to 8 carbon atoms is present on at least one lysine-£ -amino group thereof? an N- carbaphthylated orgoteine in which a carbamyl group is present on at least one lysine-t-amino group; and an esterified orgotein in which up to 10 of the five carboxylic acid groups on the orgotein protein molecule are esterified. 20. Orgotein derivative according to claim 19, characterized in that the orgotein originates from cattle. 21. Alkylated. orgotein according to claim 19, characterized in that it contains at least 10 alkylated £-amino groups per molecule. 22. Alkylated orgotein according to claim 19, characterized in that it contains at least 18 alkylated £-amino groups per molecule. 23. Alkylated orgoteine according to claim ^. characterized in that alkyl contains 1-4 carbon atoms. 24. Alkylated orgotein according to claim 19, characterized by. that alkyl is methyl. 25. Carbamylated orgotein according to claim 19, characterized in that it contains at least two carbamylated E-amino groups per molecule. 26. Carbamylated orgotein according to claim 19, characterized in that it contains approx. 6 to 10 carbamylated ^-amino groups per molecule. 27. Carbamylated orgotein according to claim 19, characterized in that the carbamylated amino group has "X IC the formula -NHCNHR where X is 0 or S and R is alkyl with up to 12 carbon atoms or, when X is 0, also a hydrogen atom. 28. Carbamylated orgotein according to claim 27, characterized in that X is 0 and R is alkyl with 1-8 carbon atoms or a hydrogen atom. 29. Carbamylated orgotein according to claim 27, characterized in that X is S and R is alkyl with 1-8 carbon atoms. 30. Esterified orgotein according to claim 19, k a r a k It is characterized by the fact that it contains up to 8 esterified carboxylic acid groups per molecule. 31. Esterified orgotein according to claim 19, characterized in that the ester groups are alkyl esters with 1 to 14 carbon atoms.. 32* Esterified orgotein according to claim 19, characterized in that the ester groups are methyl esters. 33. Pharmaceutical composition which, mixed with a pharmaceutically acceptable carrier, comprises an anti-inflammatory effective unit dose amount of an orgotein derivative according to claim 19 i