IE43701B1 - Orgotein derivatives and their production - Google Patents

Abstract

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

Description

This invention relates to orgotein derivatives and to a process for their production.

Orgotein is the non-proprietary name assigned hy the United States Adopted Name Council to members of a family of water-soluble protein con5 geners in substantially pure, injectable form, i.e., substantially free from other proteins which are admixed or associated therewith in the sources thereof. U.S. Patent Specification No. 3,758,682 claims pharmaceutical compositions comprising orgotein.

The orgotein metal!oproteins are members of a family of protein con10 geners having a characteristic combination of physical, chemical, biological and pharmacodynamic properties. Each of these congeners is characterized physically by being the isolated, substantially pure form of a globular, buffer and water-soluble protein having a highly compact native conformation which, although heat labile, is stable to heating for several minutes at 65°C. at pH 4-10. Chemically, each is characterized by containing all but 0-2 of the protein aminoacids, a small percentage of carbohydrate, no lipids, 0.1 to 1.02 metal content provided by one to five grams atoms per mole of one or more chelated divalent metals having an ionic radius of 0.60 to l.ool, and substantially no chelated monovalent metals or those that are cell poisons in the molecuTe.

The aminoacid composition of the orgotein congeners is remarkably consistent irrespective of the source from v/hich it is isolated.

Table I lists the distribution of aminoacid residues, calculated for a molecular weight of 32,500 of several orgotein congeners. - 2 QJ rt cn- cm Ci t n?l M3 Q2I O I·, o 00 «sr «3· cm to t— co o AMINO At ID COMPOSITION Of SEVERAL. ORGOTEIlt CONGENERS {Residues per mole, M.W. = 32,5005 o ca Qi ·© o © cr. oo r·*- co i~- CO «rs cn f— i. < < f— r~ cm CS CS X O CM CO CM CM CM CO f— JO © co - 3 dS’j'Oi ο co Οί "3· σ? CM CQ kD Ο Ο σι σι co ν •5J- ι— rCM CM CO Μ Γ— CM CQ CO CM CQ ΟΊ O CQ £= O J3 XI Π3 Οί CQ <3CQ ε sφ •p φ -σ «Ρ c ο ο οί ο Ό Φ Οί 40J ω co ΙΩ ^* <£> r— r— M r** r-~ ·γ- *r" CM Z Z CTi CO CM cn fx. io r- CM CQ C JZ φ •P {/> o. o >> sΛ .>> Ift c. o ’•P i. φ •P Φ Ό υ •Γ s. -p φ .s s_ o *o o •P φ ε o -P o o s-P o Φ a. «ί c (ϋ σ r-l CM - 4 It can be seen from Table I that orgotein congeners have from 18-26 and usually 20-23 lysine groups, of which all but 1-3 have E-amino groups that are titrable with tri nitrobenzene sulphonic acid.

It can also be seen from Table I that orgotein congeners have from 29 to 37 aspartic acid groups and from 21 to 38 glutamic acid groups.

The present invention provides an orgotein having at least one of the following modification: (i) at least one lysine e-amino group is alkylated, (ii) at least one lysine ε-amino group is carbamylated, and (iii) up to 8 free carboxyl groups are esterified.

It is to be understood that references herein to carbamylation and carbamyl groups include thiocarbamylation and the corresponding thiocarbamyl groups.

(The term free carboxyl group is used because of the total of about 50 to 63 glutamic acid and aspartic acid groups only about 20 to 27 have free acid groups, that is to say, carboxyl groups that are not blocked and therefore capable of esterification.) In the orgoteins of the invention, the number of lysine ε-amino groups that are alkylated is preferably at least 10, especially 10 to 18; the number of lysine e-amino groups that are carbamylated is preferably from 6 to 10; and the number of free carboxyl groups that are esterified is preferably at least 4.

The invention also provides a process for the production of an orgotein of the invention, which comprises reacting orgotein with at least one reagent selected from alkylating agents, carbamylating agents and esterifying agents. - 5 βί) ί »» As will be apparent to those skilled in the art, some esterification reagents and conditions are capable of simultaneously alkylating free amino groups in the orgotein molecule and esterifying free carboxylic acid groups. Such N-alkylated and esterified orgoteins and their production also are part of this invention.

The native orgotein protein possesses, inter alia, anti-inflammatory activity. See U.S. Patent Specification No. 3,758,682. It also possesses uniquely high superoxide dismutase activity. See McCord & Fridovich; J. Biol. Chem., 244, 6,176, (1970); ibid, 246, 2,875 (1971). Surprisingly, the anti-inflammatory activity of the native protein is substantially unaffected by esterification, N-alkylation, or carbamylation. Accordingly the orgotein derivatives of this invention are useful in the same manner as the native protein, e.g. for the treatment of inflammatory conditions in mammals and other animals as disclosed in U.S. Patent Specification No. 3,758,682. A substantial portion of the superoxide dismutase activity of the native protein also is retained in the orgotein derivatives of this invention, e.g., 20-100% of that of the native protein.

As stated above, orgotein congeners contain from 18-26 lysine groups. Since the orgotein molecule is made up of two identical peptide chains (sub-units), half of these lysine groups are in each chain. The two chains are tightly but non-covalently bound together under moderate conditions of temperature and pH. Because of the spacial conformation of the orgotein molecule,'the ε-amino groups of a few lysines in each chain are usually non titrable with trinitrobenzenesulfonic acid (TBNS) and are thus not readily accessible for alkylation or carbamylation. However, - 6 both alkylation and carbamylation of the non-titrable lysine e-amino groups also appears possible by employing a highly active alkylating or carbamylating agent, e.g., dimethyl sulfate at an alkaline pH. The extent of alkylation or carbamylation can be determined by the decrease in TNBS-reactive amino groups, taking into account the fact that 1-3 of the lysines of the native orgotein protein are not titrable with TN8S, for example. bovine orgotein assays for only 18 of its 20 to 22 lysines. It should also be borne in mind that monoalkylated lysines are still acylatable so that only extensively alkylated orgoteins show a reduction in acylatable lysine groups Di- and trialkylation or carbamylation of the lysine groups can be followed either directly or after acetylation by counting the charge change shown on electrophoresis of the N-alkylated or N-carbamylated product, for example, orgotein alkylated with dimethyl sulfate at pH 10 showed a charge change of -2 after acylation with acetic anhydride compared to -20 for unalkylated orgotein. Similarly, orgotein N-carbamylated with methyl isothiocyanate at pH 9 showed a charge change of -2 after 45 minutes.

As is known, the electrophoretic mobility of an ion is a function of ihe electric field strength, net charge of the ion (including bound conterions), and frictional coefficient. See, for example, C. Tanford Physical Chemistry of Macromolecules Wiley, Hew York (1966). Since the frictional coefficient is dependent on molecular size and shape, and on the solution composition, comparisons of different proteins are not too informative. However, by comparing proteins of similar size and shape, in this case orgotein molecules chemically modified with relatively small groups, under identical electrophoresis conditions, the only variable - 1 affecting this electrophoretic mobility is net charge.

Comparison of the electrophoretic patterns of a number of chemically modified orgotein molecules is consistent with this conclusion. Native bovine orgotein electrophoreses mainly as one band (band 1), with minor amounts of faster moving bands (bands 2, 3, etc.) equally spaced ahead of the main band, representing orgotein molecules with a higher ratio of -COOH to -NH2 groups than those molecules forming band 1. Treatment of native bovine orgotein with, e.g., successively higher concentrations of acetic anhydride at pH 7, or with methyl isothiocyanate at pH9, leads to che formation of a series of successively more anodic (migrating toward the© electrode) electrophoretic bands as successively more free amino groups of the orgotein molecules are acetylated or carbamylated. Conversely, treatment with dimethyl sulfate gives a series of bands successively more cathodic (displaced from band 1 toward the © electrode) as successively more free carboxylic acid groups of the orgotein molecule are esterified.

A graph of distance elsctrophoresed versus band number is relatively linear at low extents of -COOH or -NH2 modification, but curves gradually at higher modification, since there is a limit to how fast even the most highly charged species can move through solution. The faster migrating species are also more sensitive to salt concentration, and are appreciably retarded when salt-containing samples are electrophoresed. Therefore, since extrapolating more than about two band positions is not always precise, accurate charge counting requires that the unknown be co-electrophoresed with a solution which contains all the bands from 1 through the position of interest (e.g., partially acetylated or partially carbamylated orgotein). - 8 All Of the conventional protein modification reactions which have been applied to the orgotein molecule so far have been consistent with the interpretation, viz., the band positions correspond to integral charge changes from the native orgotein molecule. Acetylation, carbamylation, and N-methylthiocarbamylation all give bands 2, 3, 4, 5, etc., indicating that 1, 2, 3, and 4, respectively, free amino groups have been chemically modified. Similarly, succinyl ation, which changes ''-N^ + li groups to /'zNHCCH2CH2C02-groups, gives bands 3, 5, 7, 9, etc. and more extensive carbamylation or thiocarbamylation, which creates even more S II li (--NHCNH-R and~NHCNH-R groups, gives bands 6, 7, 8, 9, etc. Esterification with dimethyl sulfate or with ethyl diazoacetate gives bands-!, -2, -3, -4, etc., indicating the 1, 2, 3, and 4, respectively, free carboxylic acid groups have been chemically modified.

Generally speaking, most e.g., all except 2-4, of the lysines can be alkylated, even with the milder alkylating agents, or carbamylated. All but about one of the accessible (TN5S titrable) lysine groups in each of the orgotein peptide sub-units can be polyalkylated using stronger alkylating conditions, e.g., excess dimethyl sulfate 0.04 M carbonate buffer, pH 10.

As would be expected, when less than all of the titrable lysine ami no groups are alkylated or carbamylated, the distribution of the alkyl of carbamyl groups on the orgotein molecule probably is random since none of the titrable lysine amino groups appear abnormally readily alkylatable - 9 O?0i or convertable to carbamylated amino groups. Because the orgotein molecule is composed of two identical peptide chains, the alkyl groups of a partially alkylated orgotein and the N-carbamylated amino groups of a partially carbamylated orgotein will be distributed more or less randomly along each peptide sub-unit but more or less evenly between the two chains. Since a single alkylating or carbamylating agent is ordinarily employed, the alkyl groups and carbamylated amino groups, respectively, will all be identical. However, it is possible to produce alkylated orgoteins and carbamylated orgoteins having two or more different alkyl groups in the molecule and even within each chain thereof.

One way of producing a mixed alkyl orgotein is by alkylating in stages with different alkylating agents, for example, a fraction of the titrable lysine ε-amino groups can be alkylated with a moderate concentration of one alkylating agent, e.g., iodoacetamide, and the remainder of the re15 active amino groups alkylated with a high concentration of another alkylating agent, e.g., dimethyl sulphate. What constitutes a low, or high, concentration of alkylating agent will depend on the relative rates of reaction with protein amino groups and with solvent and will thus depend on the reaction pH and on the alkylating agent, and to a lesser extent on buffer and temperature.

In an analogous manner, one way of producing a mixed N-carbamylated orgotein is by carbamylating in stages with different carbamylating agents, for example, a fraction of the titrable lysine e-amino groups can be carbamylated with a moderately reactive carbamylating agent, e.g., KNCQ, and the remainder of the reactive amino groups carbamylated with a more active - 10 carbamylating agent, e.g., methyl isocyanate. The reactivity of a carbamylating agent depends on the relative rates of reaction with protein amino groups and with the reaction solvent and thus depends on the reaction pH and on the carbamylating agent, and to a lesser extent on buffer and temperature.

It is also possible, of course, to alkylate some of the lysine eamino groups and then to carbamylate all or some of the remaining amino groups, or vice versa, to obtain an orgotein containing e-amino groups and e-carbamyl groups in both chains.

Another method of producing a mixed alkylated or mixed N-carbamylated orgotein 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, e.g., A2 and B2 , A and B being their respective peptide chains. (A2 + B2 2AB). The charge of the heterodimer, AB, on electrophoresis should be the average of that of the homodimers A2 and B2, assuming that the same portion of each sub-unit is involved in the binding in all cases.

It is possible to hybridize a modified orgotein with another modified orgotein or with native orgotein, for example, an ε-N-alkylated orgotein may be hybridized with a different eS N-alkyl orgotein, with an e-N-carbamylated orgotein or with native orgotein. e-N-methyl orgotein, for example, produced by alkylating the native orgotein molecule in 0.04 pH 10 carbonate buffer with excess dimethyl sulphate, e-N-ethyl orgotein, for example, produced by alkylating orgotein in the same manner with diethyl sulphate, ε-Ν-propylcarbamyl orgotein, for - 11 example, produced by carbamylating the native orgotein molecule in 0.1 pH 7.6 tris or phosphate buffer with excess propyl isocyanate, and ε-N-methyl thiocarbamyl orgotein, for example, produced by carbamylating orgotein in 0.075 M sodium tetraborate with methyl isothiocyanate, may each be hybrid5 ized with native orgotein or with each other by heating together at 50°C for 4 hours.

As will be apparent, these hybrid semi-alkylated and semi-carbamylated orgotein molecules can be further alkylated or carbamylated v/ith a different alkylating or carbamylating agent to produce a hybrid alkylated or carbamylated orgotein in which the alkyl groups and carbamylated amino groups, respectively, in one peptide chain differ from those in the other. Alternatively, a hybrid semi-alkylated orgotein molecule can be e-N-carbamylated, and a hybrid semi-carbamylated orgotein molecule can be alkylated. Furthermore, a mixed hybrid orgotein molecule (in which the two chains contain lysine ε-amino groups bearing different substituents) can be further alkylated or carbamylated, thus making it possible to produce an orgotein molecule containing three different types of groups on the lysine ε-amino groups.

The ε-Ν-alkyl orgoteins and e-N-carbamyl orgoteins of this invention appear to have essentially the same spatial conformation as the native orgotein molecule. Chelated Cu++ and Zn++ contents (Gram Atoms Per Mole) are about the same as that of orgotein. Like orgotein, they are highly resistant to Pronase and other proteolytic enzymatic degradation. Superoxide dismutase (SOD) enzymatic activity is retained, although, in the case of carbamylated orgoteins, lessened in proportion to the degree of carb- 12 <23763 amylation.

Although the predominant structural modification of the native orgotei molecule which occurs upon alkylation or carbamylation thereof at alkaline pH is the mono-, di- and, to a lesser extent, trialkylation of the ε-amino groups of the lysines thereof, in the case of alkylation, and carbamylation of these ε-asiino groups, in the case of carbamylation, the free amino groups of tha arginine residues thereof and the free carboxylic acid groups of the aspartic acid and glutamic acid groups thereof, as well as other alkylatable groups present in the molecule, especially -OH, and imidazole nitrogen, and possibly also guanidino nitrogen, -SH, and -SCH3, can also be concurrently alkylated or carbamylated, depending on the conditions employed and the reactivity of the alkylating agent or carbamylating agent, for example, whereas at pH 10 with iodoacetamide, alkylation appears to be solely e-N-alkylation, alkylation with dimethyl sulfate at pH 10 is less selective and concurrently introduces methyl groups elsewhere in the molecule. Such concomittantly alkylated orgoteins having e-N-alkyl groups are compounds of this invention. In the case of carbamylation, such concurrently modified groups are labile and readily hydrolyzible in aqueous solutions within a day or less, depending on the pH.

The course of alkylation, insofar as it involves -COOH alkylation, and of carbamylation can be followed directly by a change in overall electrophoretic charge and in the appearance of new bands on electrophoresis Similarly, N-alkylation, insofar as it renders an otherwise acylatable -NH;, group resistant to acylation, can be followed by a reduction in the number of acylatable amino groups, comparted to the native orgotein molecule - 13 6 S ( u i The exact nature of the N-alkyl and N-carbamyl groups, like the number thereof, is not critical as long as the alkyl or carbamyl radical is physiologically acceptable if the orgotein is intended for administration to an animal. Because of the relatively high molecular weight of the orgotein molecule, even when the orgotein molecule is fully alkylated or carbamylated with alkyl or carbamyl groups of moderate molecular weight, e.g. <100, the impact on the overall chemical composition is relatively small, i.e., less than 10$. Alkylation and carbamylation also have no apparent significant effect upon the compact spatial conformation of the molecule and resultant stability, e.g., to heating for one hour at 60°C. and to attack by proteolytic enzymes.

As will be apparent, the alkyl or carbamyl group is generally one derived from an alkylating or carbamylating agent capable of alkylating and carbamylating, respectively, an amino group in water or a buffer solu15 tion, since the reaction is usually conducted therein.

Such alkylating agents include dipriraary alkyl sulfates: e.g. a dialkyl sulphate in which each alkyl group has up to 4 carbon atoms e.g.(RO)2SO2(R= CH3, C2H5, n-C3H7, N-CijHg); activated alkyl halides: ICH2C0X(X = OH, NH2) benzyl and allyl bromides; activated vinyl compounds: CH2 = CHX (X = CN, S0CH3, SOC2Hs, C0CH3); reductive alkylating agents: RCOR1 + BHi, or BH3CN (R, R‘ = H, CH3C2H5, C6H5). An activated alkyl halide or vinyl compound is one which contains a group which increases its reactivity. A reductive alkylating agent is an alkylating agent used in conjunction with a reducing agent.

For a method of reductive alkylating of proteins with aromatic alde- 14 43701 hydes and sodium cyanoborohydride, see Friedman, M. et al., Int. J. Peptide Protein Res., 6, 1974, 183-185; alkylation with acrylonitrile, see Means & Feeney, Chemical Modifications of Proteins Chapter 6, pages 114-117; Fletcher, J.C., Biochem J. 98 34C (1966); Friedman, M. et al., J. Amer.

Chem. Soc. 87, 3672 (1965); Friedman, M. et al., J. Org. Chem, 31, 2888 (1966).

Examples of suitable carbamylating agents include alkali metal cyanates, e.g., NaNCO, KNCO; alkyl isocyanates and alkyl isothiocyanates, e.g. wherein the alkyl moiety has up to 12 carbon atoms, especially up to 8 carbon atoms, e.g. RNCO and RNCS wherein R = CH3, C2H6, n-C3H7, n-C^Hg, n-CgHj7; and aryl isocyanates and isothiocyanates, e.g., phenyl isocyanate and phenyl isothiocyanate.

Although metal cyanates react with many types of side chain, only the reaction with amino groups to convert them to yrea groups produces a product stable in aqueous solution.

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

The reaction of orgotein with isocyanates and isothiocyanates can be confirmed usually by employing a reagent which introduces a fluorescent group into the molecule, e.g., fluorescein isothiocyanate. This reagent is similar to the alkyl isocyanates, and reacts with the amino groups of orgotein to give substituted thioureas. However, even a low level of substitution reduces the SOD activity of orgotein appreciably because of the - 15 bulk of the fluorescein. The mono- and disubstituted orgoteins are not stable in solution and slowly become more heterogeneous due to subunit interchange and hydrolysis.

The exact chemical nature of the N-alkyl group is not critical. Preferred groups are straight and branched chain alkyl groups, for example, methyl, ethyl, n-propyl and n-butyl groups; cycloalkyl groups, for example, cyclopentyl, cyclohexyl, and menthyl groups; cycloalkyl-alkyl groups, for example cyclohexy!methyl and β-cyclopentylpropyl groups; and aralkyl groups, for example, benzyl, p-xylyl and phenethyl groups. Also preferred are alkyl groups having 1 to 8, preferably 1-4 carbon atoms, and most preferably a methyl or ethyl group, bearing one or more, preferably one substituent, e.g. carboxy, substituted carboxy (e.g. -C00CH3 or -COOC2H5), cyano, cyanoalkyl, amido, hydroxya!kyl, phenyl and phenyl carbonyl groups, e.g. carboxymethyl , cyanomethyl, carbethoxymethyl, carbomethoxymethyl, carbamylmethyl, and the correspondingly substituted ethyl groups, e.g., -CH2CH2COOH, -CH2CH2CaN, -CH2CH2CONH2 and -CH2CH2COOR, (wherein R is, e.g., methyl or ethyl,) -CH2CH2S0CH3, -CH2CH2SOC2H5 and -CH2CH2COCH3.

Thus, the alkylated orgoteins of this invention are orgotein congeners including bovine, sheep, horse, pork, dog, rabbit, guinea pig, chicken and human, at least one, e.g,, 1, 2, 3, 4, 5 and up to all (about 18-26) of whose amino groups are alkylated, i.e., bearing an unsubstituted or substituted alkyl group.

The alkylated amino groups are especially those of the formula -NH-C^-R1 wherein R1 is H, CH3, C2H5, n-C3H7, iso-C3H7, or other alkyl of up to 7 - 16 £3701 carbon atoms, -COOH, -COO-LA, -CONH-LA, -CON(LA)2, -C=N, -CH?Cs!i, -Ph, -COPh, -CH2-0H or -CH(CH3)0H, in which LA is an alkyl group having 1 to 4 carbon atoms and Ph represents an unsubstituted or substituted phenyl group, a substituted phenyl group preferably having from 1 to 3 substituents selected from alkyl groups, halogen atoms, nitro and amido groups, alkoxy and carboalkoxy groups, for example, chlorine and bromine atoms, methyl, methoxy, carbomethoxy and carboetnoxy groups, for example, £-tolyl, sym.xylyl, £-amidophenyl, m-chlorophenyl and jj-methoxypbenyl. Such preferred orgoteins have the formula (H.,N)ra-Org- (NHCH2RI)n wherein £ is an integer from 1 to about 26, preferably at least 10, more preferably 10-18, and the sum of m and £ is the total number of free amino groups in the unmodified congener and R1 has the meanings given above, and preferably represents a hydrogen atom or an alkyl group having from 1 to 8 carbon atoms, e.g., methyl or ethyl, and Org is the remainder of the orgotein molecule.

Some of the alkylating agents employed in the process of this invention will simultaneously alkylate some of the free acid groups of the orgotein molecule. These alkylated orgoteins are especially those represented by the formula (HOOC)X ^^.(COOC^Rjy (Org)^ (NHCH2Ri)n wherein Org. R1, m and n have tne values given above, y is the number of alkylated free carboxylic acid groups, e.g., from one up to 8, preferably - 17 O?0i 2-6, and χ is the remainder of free carboxylic acid groups in the orgotein molecule.

Reagents useful for introducing carhoxymethyl and carbamylmethyl groups, respectively,’are iodoacetic acid and iodoacetamide. Iodoacetic acid can form stable products with cysteine (sulfhydryl), lysine (amino), histidine (imidazole nitrogens), and methionine (sulfide sulfur) residues in proteins. Since carboxymethylation of any of these groups except methionine increases the net negative charge into the orgotein protein at pH 8.4, electrophoresis can quantitate the total reaction (except carboxy10 methylation of methionine and carboxymethylation of the second imidazole nitrogen).

In native orgotein, iodoacetic acid and iodoacetamide- react predominantly, if not exclusively, with lysine amino groups. Although free thiols react with iodacetic acid several orders of magnitude faster than do amines, the first few groups in orgotein v/hich are carboxymethyl ated do not react appreciably faster than do the next 10-20 groups. This is consistent with the reaction of £-mercuribenzoate with orgotein sulfhydryls: there is no reaction with hoio-protein. Even with apo-protein, which gives a ]3-mercuribenzoate-sulfbydryl reaction (although slowly under non20 denaturing conditions), iodoacetic acid in 10-fold molar excess at pH 7 gave no decrease after several weeks at 4°C. in the sulfhydryl content (determined spectrophotometrically by £-mercuribenzoate titration). The pH dependence of the carboxymethylation reaction as shown by electrophoresis is consistent with reaction of lysine (pK>8) and not of histidine (pK<6); since the extent of reaction at identical initial concentrations increases - 18 steadily as the pH approaches 9, with no appreciable reaction at or below neutrality where histidine should still be unprotonated and reactive. On the other hand, ethylehloroformate titration for histidine showed only 5 histidines in carboxymethylated orgotein under conditions which showed 16 in native orgotein.

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

The lysine amino groups in orgotein definitely react with iodoacetate, however. The extensively carboxymethylated orgotein migrates on electrophoresis similarly to acetylated orgotein, but has lower SOD activity (about 20% of the unmodified orgotein protein). in addition to the N-methyl bovine orgoteins and orgoteins of the examples hereinafter, other examples of N-alkyl bovine orgoteins of this invention are N-ethyl orgotein, N-propyl orgotein and Ν-benzyl orgotein, wherein in each instance there are 9 such alkyl groups in each of the two sub-units of the orgotein molecule and the corresponding orgoteins wherein there are an average of 1, 6 or 10 such alkyl groups in each such sub-unit, respectively, and the corresponding human, sheep, horse, pork, dog, rabbit, guinea pig and chicken congeners of each of these.

The carbamylated orgoteins of this invention are orgotein congeners, including bovine, sheep, horse, pork, rat, dog, rabbit, guinea pig, chicken - 19 and human, at least one, e.g., 1, 2, 3, 4, 5 and up to all (about 18-26), of whose amino groups are carbamylated, i.e., bear an unsubstituted or substituted -CONH- or -CSNH- group.

In a preferred embodiment, the carbamylated amino groups are those 5 of the formula X1 ll , -NHCNHR2 wherein X1 is 0 or S and R2 is CH3, C2H5, n-C3H7, iso-C3H7, ji-C8H17 or another alkyl group of up to 12 carbon atoms, Ph, or, when X is 0, also a hydrogen atom, and wherein Ph represents an unsubstituted or substituted phenyl group, a substituted phenyl group preferably having from 1 to 3 substituents selected from alkyl groups, halogen atoms, nitro and amido groups, alkoxy and carboalkoxy groups, for example, chlorine and bromine atoms, methyl, methoxy, carbomethoxy and carboethoxy groups, for example, £-tolyl, sym.-xylyl, £-amidophenyl, m-chlorophenyl and p-methoxyphenyl.

Such orgoteins have the formula X (H2N)ra,-Org-(NHCNH-R2)n, wherein n’ is an integer from 1 to about 26, preferably at least 2, more preferably 6 to 10 and the sum of m’ and jr is the total number of free amino groups in the unmodified congener, X is 0 or S, R2 has meanings given above, and preferably represents H or alkyl of 1-8 carbon atoms, and Org is the remainder of the orgotein molecule.

Especially preferred carbamylated orgoteins are alkylcarbamyl and alkylthiocarbamyl orgoteins wherein the alkyl group is unsubstituted alkyl - 20 4370% of 1-8 carbon atoms, e.g., methyl, ethyl, propyl, isopropyl, butyl, octyl.

Since the exact chemical nature of the carbamyl group is not critical, so long as it is not physiologically toxic in the orgotein molecule if the molecule is to be administered to ail animal - and can be formed on the lysine ε-amino groups, in the above formula R2 may represent cyeloalkyl e.g. cyclopentyl, cyclohexyl, and menthyl; cycloalkylalkyl e.g. cyclohexylmethyl, and g-cyclopentylpropyl; or aralkyl e.g. benzyl, p-xylyl or phenethyl. Rz may also represent alkyl of 1-8, preferably 1-4 carbon atoms, and most preferably methyl or ethyl, bearing one or more, preferably one, other substituents, e.g. those described above as substituents for an N-alkyl group, e.g. a fluoresceinyl group.

In addition to the N-carbamylated bovine orgotein of the examples hereinafter, other examples of N-carbamyl bovine orgoteins of this invention are the corresponding N-carbamyl orgotein, N-propylcarbamyl orgotein, Nethylcarbamyl orgotein, and N-methylthiocarbamyl orgotein wherein in each instance there are 9 such carbamyl groups in each of the two sub-units of the orgotein molecule and the corresponding orgoteins wherein there are an average of 1, 6 or 10 such carbamyl groups in each such sub-unit, respectively, and the corresponding human, sheep, horse, pork, dog, rabbit, guinea pig, chicken and rat congeners of each of these.

As stated above, orgotein congeners contain a total of about 50-68 glutamic and aspartic residues but only about 20-27 of these have free acid groups, the rest being already annidated as glutamine and asparagine residues. Since the orgotein molecule is made up of two identical or almost identical peptide chains (sub-units), half of these aminoacid resi- 21 4370ft dues are in each chain, whieh are tightly out non-convalently bound together under moderate conditions of temperature and pH. Esterification changes the charge of the orgotein molecule and usually only up to about 10 and preferably up to about 6 of these iree acid groups can be esterified and still retain that conformation of the native molecule, upon which stability and drug utility is dependent.

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

Because the orgotein molecule is composed of two identical peptide chains, the ester groups probably are distributed more or less evenly between the two chains.. Since a single esterifying agent is ordinarily employed, the ester groups will all be identical. However, it is possible to produce esterified orgoteins having two or more different ester groups in the molecule and even within each chain thereof.

One way of producing a mixed ester of orgotein is by esterifying in stages with different esterifying agents, for example, a fraction of the free acid groups can be esterified with a low concentration of one esterify· ing agent, e.g. diethyl sulphate, and another fraction of the acid groups esterified with a higher concentration of another esterifying agent, e.g. diazomethane. What constitutes a low or high concentration of esterifying agent will depend on its relative rates of reaction with protein acid groups and with solvent and will thus depend on the reaction pH and on the - 22 43701 esterifying agent, and to a lesser extent on buffer and temperature.

Another method of producing a mixed orgotein ester is by hybridization, the principle of which has been discussed above. An esterified orgotein molecule may be hybridized with native orgotein or with any other modified orgotein, that is to say, with another esterified orgotein, with an Ν-εalkyl orgotein or with an N-e-carbamyl orgotein, for example, methyl orgotein and ethyl orgotein may each be hybridized with native orgotein or with each other by heating with a slight excess of native orgotein at 50°C. for 4 hours. The resulting heterodimers electrophorese as a mixture of bands intermediate between native orgotein and the bands of the esterified orgotein prior to hybridization.

As will be apparent, a hybrid semi-esterified orgotein molecule can be further esterified with a different esterifying agent to produce a hybrid esterified orgotein in which the ester groups in one peptide chain differ from those in the other, or it can be alkylated or carbamylated.

A mixed hybrid may be further modified e.g. by esterification, alkylation or carbamylation as appropriate.

The esterified orgoteins of this invention appear to have essentially the same spatial conformation as the native orgotein molecule. Chelated Cu++ and Zn+ (Gram Atoms Per Mole) contents are about the same as that of orgotein. Like orgotein, they are highly resistant to degradation by Pronase and other proteolytic enzymes.

Superoxide dismutase enzymatic (SOD) activity is not markedly reduced until more than about 6-8 carboxylic acid groups are esterified.

The exact nature of the esterifying groups, like the exact number of - 23 esterified groups is not critical as long as the esterifying group is physiologically acceptable if the orgotein is to be administered to an animal. Because of the relatively high molecular weight of the orgotein molecule, even when the orgotein molecule is esterified with esterifying groups of moderate molecular weight, e.g. < 160, the impact on the overall chemical composition is relatively small, i.e., less than 5%. Of course, the esterification of the free carboxylic acid groups obviously has a profound impact upon the isoelectric point and resulting electrophoretic mobility but, as discussed above, as long as esterification is limited to about 10 or less glutamic and aspartic acid groups, it has no apparent significant effect upon the compact spatial conformation of the molecule and resultant stability, e.g., to heating for one hour at 60°C. and to attack by proteolytic enzymes.

As will be apparent, the esterifying group generally must be one derived from an esterifying agent capable of esterifying a carboxylic acid group in water or buffer solution, since the reaction is usually conducted therein. Such esterifying agents include a dialkyl sulphate e.g., dimethyl and diethyl sulphate, diazomethane and other diazo compounds, e.g. of the formula N2CH2COX wherein X is, e.g., OCH3, OC2H5, NH2 or NHCH2CONH2, and other esters and amides of diazoacetic acid which lack reactive groups, e.g., carboxyl or imino.

For 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 particularly, this invention is directed to -COOH esterified - 24 0701 orgotein wherein the ester group preferably is of up to 4 carbon atoms, e.g., derived from a monohydric alkanol, e.g., methyl or ethyl, and most preferably methyl.

Since the exact chemical nature of the ester radical is not critical, as long as it is not physiologically toxic if it is to be administered to an animal, and it can be formed on orgotein's free acid groups, the alkyl esterifying group may be one bearing one, two or more substituents, e.g., those described above as substituents of alkyl groups. Especially preferred are those wherein the ester group bearing the substituent or sub10 stituents is methyl, e.g. the esterified carboxy group has the formula -COO-CHji-R1 wherein R1 is as defined above. Thus, in addition to esterified orgotein in which the esterified acid groups are -C00CH3, also preferred are those wherein the ester groups are -COOR3 wherein R3 is -CHjCOX in which X is OCH:i, 0C2H5, NH2, NHCH2C0NH2 or CH2CH2-Ph or wherein R3 is CH(Ph)2, wherein Ph represents an unsubstituted or substituted phenyl radical as defined above.

Esterification of the -COOH groups can be followed by counting the charge change shown on electrophoresis compared to native orgoteins, as described earlier. Esterification with dimethyl sul fate or with ethyl diazoacetate gives bands -1, -2, -3, -4 etc., indicating that 1, 2, 3, and 4, respectively, 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 Z5 than 6-8 -COOH groups, i.e. to esterify more than four -COOH groups of each - 25 of the two orgotein peptide sub-units, usually leads to denaturation and loss of superoxide dismutase activity.

As would be expected, the distribution of the esterifying alkyl groups on the orgotein molecule probably is random since none of the titrable free carboxy groups appear abnormally readily esterifiable. Because the orgotein molecule is composed of two identical peptide chains, the ester groups of a partially esterified orgotein will be distributed more or less randomly along each peptide sub-unit but more or less evenly betweeen the two chains. Since a single esterifying agent is ordinarily employed, the ester groups will all be identical. However, it is possible to produce esterified orgoteins having two or more different ester groups in the molecule and even within each chain thereof.

One way of producing a mixed esterified orgotein is by esterifying in stages with different esterifying agents, as mentioned above, for example, a portion of the free -COOH groups can be esterified with one esterifying agent, e.g. dimethyl sulfate, and the remainder of the reactive -COOH groups esterified v/ith another esterifying agent, e.g., ethyl diazoacetate, Another method of producing a mixed esterified orgotein is by hybridization, as mentioned above, for example, methyl esterified orgotein, e.g., produced by esterifying about 6 free acid groups of the orgotein molecule with dimethyl sulfate and carboxymethyl esterified orgotein, e.g. produced by esterifying the native orgotein molecule with ethyl diazoacetate, can each be hybridized with native orgotein or with each other by heating together at 50°C. for 4 hours. - 26 43Τ0ί In addition to ths esterified bovine orgoteins of the examples hereinafter, other examples of orgoteins derivatives of this invention are the corresponding derivatives of other orgotein congeners.

Other examples of esterified bovine orgoteins are carbomethoxymethyl orgotein and carbamylmethyl orgotein, wherein in each instance there are 3-4 such esterified groups in each of the two sub-units of the orgotein molecule and the corresponding esterified orgoteins wherein there are 1 or 2 such ester groups in each such sub-unit, respectively, and the corresponding human, sheep, horse, pork, dog, rabbit, guinea pig, and chicken congeners of each of these.

As disclosed above, the present invention provides an orgotein having at least one of the following modifications: (i) at least one lysine e-amino group is alkylated, (ii) at least one lysine e-amino group is carbamylated, and (iii) up to 8 free carboxyl groups are esterified.

At least two of the modification (i) to (i'ii) an; present for example.

It will be appreciated that it is possible to produce a large variety of orgotein molecules containing more than one modification and/or one modification carried out with different groups of the same type by using one or more of the techniques of successively modifying one molecule with two reagents (of the same or different types), by hybridizing two different molecules and by further modifying a hybrid molecule.

The invention accordingly also provides an orgotein having at least one cf the following modifications: (iv) at least two lysine ε-amino groups are alkylated, - 27 (v) at least two lysine ε-amino groups are carbamylated, and (vi) at least two carboxyl groups are esterified, there being present at least two different alkyl, carbamyl and ester groups respectively.

The orgotein derivatives may be isolated from the reaction solution, preferably after dialysis to remove extraneous ions, by conventional lyophilization, e.g., in the manner described in U.S. Patent Specification No. 3,658,682. If desired, the orgotein derivative may first be purified by ion exchange resin chromatography, electrophoresis and/or gel filtration employing a polymer which acts as a molecular sieve.

Filtration through a micropore filter of pore size 0.01 to 0.22 micron in an aseptic manner into sterile vials, optionally after adjusting ionic strength with NaCl and/or sodium phosphate, e.g., to isotonicity, will provide a bacterial,ly and virally 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 of this invention comprise an orgotein derivative of this invention in admixture or conjunction with a pharmaceutically suitable carrier. The form and character which this carrier takes is, of course, dictated by the mode of administration.

The pharmaceutical composition preferably is in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous solution. The solution can be formulated according to the known art.

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 a lyophilized powder for reconstitution with such solvent. - 28 43701 The compositions of this invention preferably comprise an effective unit dosage amount of an orgotein derivative of this invention, i.e., the orgotein derivative is present at a concentration effective to evoke the desired response when a unit dose of the composition is administered by the route appropriate for the particular pharmaceutical carrier, for example, liquid compositions, both topical and injectable, usually contain 0.5 to 20 mg. of the orgotein derivative per 0.25 to 10 ml., preferably 0.5 to 5 ml., except I.V. infusion solutions, v/hich may also be more dilute, e.g., 0.5 to 20 mg. orgotein derivatives per 50-1,000 ml., preferably 100500 ml. of infusion solution. The preparation may be in a form suitable for enteral administration, and 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, e.g. intramuscularly, subcutaneously, intravenously or intradermally. I.M. is preferred, except in case of shock, where I.V. is sometimes preferred for more rapid onset of effect, and in certain localized disorders, e.g., radiation and other cystitis, where local injection, infusion and/or instillation is often more effective. Individual doses usually fall within the range of 0.5 to 20 mg. The preferred range for humans is 0.5 to 8 mg.: for horses, 5.0 - 10.0 mg. The exact dosage is not critical and depends on the type and the severity of the disease.

The orgotein derivatives of this invention, like orgotein itself, is effective in treating a wide variety of inflammatory conditions, including those in which synthetic anti-inflammatory agents have limited utility, - 29 e .j ( v i e.g., because of toxic side effects upon prolonged use.

More specifically, these orgotein derivatives are efficacious in ameliorating inflammatory conditions and mitigating the effects thereof, for instance those involving the urinary tract and the joints, in various mammals. It is useful in alleviating the symptoms associated with, for example, rheumatoid, osteo and post-traumatic arthritis, as well as bursitis and tendonitis.

For further details relating to how to isolate the starting orgotein congeners and how to use the orgotein derivatives of this invention, in10 eluding modes of administration, dosage forms, dosage regimen and inflammatory and other conditions susceptible to treatment with esterified orgotein, see U.S. Patent Specification No. 3,758,682.

Without further elaboration, it is believed that one skilled in the art can, using preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

EXAMPLE IA A solution of 5 mg. bovine orgotein in 4 ml. of 0.04 M carbonate buffer was treated with 20 μΐ dimethyl sulfate and the pH kept at 10.0 by addition of 0.5 M NaOH. The uptake of base had a half-time of about 37 minutes. Electrophoresis showed SOD-active bands 1 through -5 (average -3). After dialysis, lyophilization and re-solution in 1 ml. water, the protein still had its SOD activity and Cu44 and Zn++ content unchanged from the untreated orgotein. Acetylation of an 85 yg./ml. solution of the - 30 «370 j. modified protein with a total of 3 ul acetic anhydride in 0.4 ml. borate buffer at pH 9 gave an average electrophoretic charge change of only about -2, compared to -20 for native orgotein in the same solution. The pH 10 dimethyl sulfate-treated orgotein is therefore extensively N-methylated, i.e., about 18 per molecule.

EXAMPLE 2A Follow the procedure of Example IA, employing, respectively, the corresponding human, sheep, horse, pig, dog, rabbit, guinea pig and chicken orgotein congeners as starting materials. In each case, all except about one lysine of each sub-unit of the orgotein molecule is alkylated.

EXAMPLE 3A Follow the procedure of Example IA, employing diethyl sulfate instead of dimethyl sulfate. The properties of the resulting ε-Ν-ethylated orgotein are essentially the same as the ε-N-methylated orgotein.

EXAMPLE 4A The ε-amino groups of the lysine residues of bovine orgotein were carboxymethyiated with 0.2 M sodium iodoacetate at ambient temperatures under the conditions and with the results set forth below.

Charge Change on Electrophoresis, Reaction Buffer average ί range orgotein (1.4 mg./ml.) + ICH2C0®Na® (0.2 M) (a) 0.3 M pH 3.8 acetate (b) 0.4 M pH 5.0 acetate (C) 0.23 M pH 9,2 carbonate (only changes are those due to low pH) no change up to 72 hours 0 (15 min), 3 + 2 (2j hrs.), ± 3 (63, hrs.) >18 (72 hrs.) - 31 01 From these electrophoretic patterns, it appears that no N-alkylation occurred at pH 3.8 and 5.0 and that at pH 9.2 an average of about 3 -CH2COO' groups were introduced in 2ί hours; about 5 such groups were introduced in 6i hours; and about 18 such groups were introduced in 72 hours on the e-amino nitrogen atoms. orgotein (2.8 mg./ml.) + ICH2CO2Na® (0.43 M) (d) pH 6.0 0.4 after 5 days at room temperature (e) pH 7.0 0.6 (1 day), 1.0 (2 days), 1.5 (5 days) (f) pH 10 0 (10 min.), >3 (2. hrs.), >10 (6| hrs.) From the electrophoretic patterns of the thus-treated orgotein it appears that at pH 6 less than half the orgotein molecules were alkylated; at pH 7, in 2 days the orgotein molecules had an average of one e-amino θ group alkylated with a -CH2C00 group and in 5 days an average of two such groups per molecule were introduced. At pH 10, an average of more than three such groups were introduced by 2J hours and by 6£ hours an average of more than 10 such groups per molecule were introduced. g The products of all the above alkylations in which -CH2COO groups were introduced were a mixture of ε-amino alkylated orgoteins containing varying numbers of such groups as evidenced by the appearance of a plurality of bands with varying electrophoretic mobilities.

EXAMPLE IB Unsubstituted Carbamylated Orgotein A solution of 3.7 mg. of orgotein (bovine congener) and 14 mg. KNCO in 2 ml. 0.025 M pH 7.5 sodium phosphate buffer was incubated at 4°C. Electrophoresis of aliquot samples over a period of 54 days showed the appearance - 32 43701 of a series of SOD active bands more anodic than native orgotein. The average charge change along with the range of active bands on either side of the average for the samples is given below. Time (days): 0.75 3.8 27 54 Lysines reacted: 0 0.8 5 ± 2 8.5 ± 3.5 EXAMPLE 2B Alkyl Carbamylated Orgoteins ul portions of orgotein (bovine congener) in 0.1 M pH 7.6 tris or phosphate buffer at a concentration of 10 mg/ml were reacted at 4°C with either 1 ul of propyl isocyanate or octyl isocyanate for various times. In two instances, 2 μΐ additional propyl isocyanate was added after two days. The number of propylcarbamyl and octylcarbamyl groups introduced was determined by the average charge change as determined by electrophoresis, as shown below.

Average Charge Change + 2 ul isocy- REAGENT 2 min. 2 hr. 1 day 2 days anate propylisocyanate/tris 8 8 ND* ND 10 propylisocyanate/phosphate 8 8 ND ND >15 octyli socyanate/tri s 0.2 1.0 6 6 octyli socyanate/phosphate 0 0.2 2.5 3 *Not done EXAMPLE 3B Alkyl Carbamylated Orgoteins The procedure of Example 2B was repeated employing 10 mg. of orgotein in 1 ml. 0.1 M pH 7.6 phosphate buffer and 1 ml. of propyl or octyl iso- 33 cyanate. The solutions were maintained at 25°C. for 4 hours and examined electrophoretically. Then an additional 1 ml. of the 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 partially precipitated on dialysis, but were soluble in 0.15 M saline solution. The number of carbamylated lysine groups are shown below.

GAPM Isocyanate Lysines 4 Hours Reacted Overnight Metal Cu++ Content Zn++ % of Original SOO Activity propyl 10+4 > 15 2.2 1.5 approx. 50% octyl 3+3 6 + 5 2.1 1.8 approx. 25% EXAMPLE 4B N-Methyl Thiocarbamyl Orgotein To a solution of 5 mg. orgotein in 4 ml. 0.075 M pH 9 Na2Bi,O7 was added 0 μΐ CH3NCS, and the mixture shaken for a minute until the CH3NCS dissolved. At intervals, 50 pi samples were withdrawn and quenched with 0.4 ml. 0.1 M pH 5.3 sodium acetate buffer. A white precipitate of sulfur (as shown by its odor on burning and its solubility in CS2) appeared between 6 and 22 hours. After 22 hours at room temperature, the remaining reaction mixture was quenched with 0.5 ml. 1 M pH 5.3 acetate buffer and dialyzed vs. frequent changes of water for 2 days.

The dialyzed solution was centrifuged briefly to remove the white cloudiness of the solution.

Electrophoresis of samples taken from the solution showed production of a series of increasingly more anodic SOD-active bands. Analysis of - 34 43701 the samples by the pH 7.8 cytochrome c assay (McCord & Fridovich, 2· Biol. Chem. 5049-6055 (1969) showed a drop in SOD activity with more extensive modification. Average SOD Activity Time (Hours) Charge Change % 0 (0) (100%) 0.08 0.5 m 0.25 1 118 0.75 2 91 1.9 4 86 6 9.5 43 22 16 20 The dialyzed 22 hour reaction product electrophoresed as a fast-moving anodic band, retained 18% of the SOD activity of the native orgotein protein and contained 2.06 GAPM Zn++ and 1.76 GAPM Cu++ (compared with 2.26 GAPM Zn and 2.08 GAPM Cu++ in unmodified orgotein). Ail of the products are soluble retain chelated Cu++ and ZnT+ and at least a portion of the superoxide dismutase activity of the unmodified protein.

EXAMPLE 5B N-Fluoresceinylcarbamyl Orgotein A solution of 100 mg. orgotein (bovine congener) and 6 mg. fluorescin isothiocyanate in 10 mi. of 0.14 M pH 8.5 phosphate buffer was maintained at 4°C. for 18 hours. The reaction mixture was applied to a chromatographic column of microporous cross-linked dextran (Sephadex G-50, Pharmacia, Upsala, Sweden (Sephadex being a Trade Mark) and eluted therefrom with pH 8 borate buffered saline (0.15 M). The eluted fractions containing the - 35 yellow protein were dialyzed against water. The dialyzed protein which precipitated (20 mg.) was intensely yellow and fluorescent and was readily soluble in pH 8 borate buffered saline. Electrophoresis showed the 96 mg of soluble protein to be about half mono-carbamylated orgotein (-3 charge change) along with some doubly and triply carbamylated orgotein (-6 and -9) charge changes) and unreacted orgotein. The redissolved precipitate appears to be more extensively modified protein, since electrophoresis showed a fast anodic smear.

The soluble protein was chromatographed on a weakly basic (DEAE10 cellulose) ion exchange column at pH 6 with 0.01 to 0.2 M linear gradient of phosphate buffer. The moho-carbamylated and the di-carbamylated orgoteins were isolated, dialyzed and lyophylized.

The mono-carbamylated orgotein was SOD active on electrophoresis and NBT-riboflavin staining. According to the pH 7.5 cytochrome c assay, it has 48» of the SOD activity of native orgotein. In the Ungar bioassay, that protein showed about 50% of the activity of native orgotein.

A solution of the mono-carbamylated orgotein stored at 4°C. for 2 weeks changed to a mixture of orgotein, monocarbamylated orgotein and dicarbamylated Orgotein, (apparently the result of hybridization) and some non-protein fluorescent compound (apparently the result of hydrolysis of the thiourea group of carbamylated orgotein to give free aminofluorescein).

Following the procedure of the above examples but employing, respectively, the corresponding human, sheep, horse, pig, dog, rabbit, guinea pig, rat and chicken orgotein congeners as starting materials, the corresponding carbamylated derivatives of these congeners are produced. - 36 43?0i EXAMPLE JC METHYL ESTERIFICATION A solution (125 ug/ml) of 0.5 mg bovine orgotein and 0.5% vol/vol of dimethyl sulfate in 4 ml. of 0.05 M acetate buffer is maintained at pH 5 for 100 min. Electrophoretic analysis of the reaction product showed SOD active bands from +1 through -6 (average -4). The product is orgotein having an average of 4 -GOOCH, groups.

EXAMPLE 2C METHYL ESTERIFICATION A solution of 0.5 mg. bovine orgotein and 10 μΐ dimethyl sulfate in 4 ml. water was kept at constant pH by addition of 0.1 M NaOH. The rate of base uptake in the pH range 7-10 was fairly independent of pH and of the condition of 0.25 mole sodium phosphate buffer. Electrophoresis showed the formation of more cathodic SOD-active bands at an initial rate of about -0.5 charge/hour. After 21 hours, predominantly at pH 7-8, the solution was analyzed for N-methylation and for esterification, as described below. N-Methylation could not be detected. (a) Acetylation of a 0.5 ml. aliquot, of the esterified orgotein with μΐ acetic anhydride +3 ul 6M NaOH at 4°C., gave a solution whose electrophoresis pattern was a fast-moving anodic band similar to that of acetylated native orgotein, thus establishing that the free amino groups were unaffected during the esterification. (b) A 1 ml. aliquot of the esterified orgotein solution was adjusted to pH 10.5 and stored covered in a dessicator with NaOH pellets. Although the cathodic band pattern of bands 1 through -4 was stable at pH 7-9, at pH 10.5 the cathodic bands gradually disappeared over a period of 4 days as the electrophoretic pattern of native orgotein reappeared. Protein - 37 asvoi methyl esters commonly hydrolyze readily at alkaline pH's, thus confirming the more cathodic bands appearing after the esterification were orgotein esters.

From the foregoing it is apparent that after two hours, dimethyl 5 sulfate at pH 7-10 esterified an average of one -COOH group per molecule; after four hours, about 2 per molecule; and continues thereafter to increase the number of esterified -COOH groups to a maximum of about 6 per molecule.

Following the procedure of Examples 1C and 2C, employing, respectively, 10 the corresponding human, sheep, horse, pig, dog, rabbit, guinea pig and chicken orgotein congeners as starting materials, in each case, an average of about four carboxylic acid groups of the orgotein molecule are converted to methyl esters thereof.

Following the procedure of Example 1C and 2C, employing diethyl sul15 fate instead of dimethyl sulfate, orgotein esters having from one to six free carboxylic acid groups converted to ethyl esters thereof are produced. The properties of the resulting esterified orgoteins are essentially the same as the methyl esterified orgotein.

The esterified orgoteins can be further purified, if desired, by 20 ion exchange chromatography to separate from each other the species of different net charge and, hence, different extents of esterification.

For example, elution of 200 mg orgotein through a 2.5 x 40 cm DEAE Sephadex column with 4 liter of a 0.01 M to 0.2 M linear gradient of tris pH 8.5 buffer separates the orgotein bands from each other, the electrophoretically more cathodic bands eluting first. By such a procedure, the mixture of - 38 methyl esterified orgoteins produced by the procedure of Example 1C can be separated into fractions containing predominantly 1, 2, 3, 4, 5 or 6 methyl ester groups per orgotein molecule.

Such fractionation of a partially modified orgotein by ion-exchange chromatography is applicable to any modified orgotein whose molecular charge depends upon the extent of modification; e.g. methyl esterified orgotein, carbethoxymethyl esterified orgotein, and N-acetylated orgotein. EXAMPLE 3C: CARBETHOXYMETHYL ESTERIFICATION Ethyl diazoacetate is prepared by reaction of glycine ethyl ester with nitrous acid, as in Organic Synthesis Coll. Vol. IV, p.424, but using CCb, rather than CH2C12 to extract the ethyl diazoacetate from aqueous solution.

Two mi. of the approximately IM ethyl diazoacetate/CCl,, solution are placed in a 25 ml. flask and most of the CC14 is evaporated under aspirator pressure. A solution of 9 mg. bovine orgotein/3 ml. water is added and swirled to disperse the organic phase. The solution is stored at 4°C. and swirled every few days. The reaction mixture remains heterogeneous throughout. Electrophoresis shows the gradual formation of more cathodic SOD active protein bands -1 through -6. The average charge change is 1.2 after 5 days ana 3 after 12 days. The product is a mixture of carbethoxymethyl orgotein esters having after 5 days either one or two such ester groups per molecule, and after 12 days, from 1 through 6 such ester groups.

The aqueous phase is then filtered and desalted by chromatographic fractionation employing a 10 cc Sephadex G-25 column. The protein fractions - 39 are lyophylized and redissolved in 2 ml. of water, and the pH raised from 3.7 to 5.6 with 2 μΐ IM NaOH. Electrophoresis shows no change from before desalting and lyophylization. Re-action of this solution for a month under the same conditions as above gives a smear of protein on electrophoresis with less SOD activity and no material more cathodic than band -6. EXAMPLE 4C: METHYL ESTERIFICATION Following the procedure of Examples i and 2, bovine orgotein was alkylated under the conditions set forth n the table below.

PH Buffer Orgotein Dimethy. Sulfate % by vol. Hydrolysis Half-time* SOD Active Bands on Electrophoresis (a) 5.0 0.05 M 125 ug./ml . 0.25 54 min. +1 through -4 (b) 5.0 acetate 125 ug./ml . 0.5 54 min (avg. -2) -1 through -6 (c) 7.0 0.016 M 80 It 0.65 1 hr. (avg. -4) 12 through -6 (d) 7.0 phosphate 80 tl 1.3 1 hr. faint streak of (e)11.2- 0.05 M 125 II 0.5 J hr. SOD from +4 to -6 peaking at -6 position +2 to -3 9.3 *Half-time carbonate for NaOH uptake needed to maintain (at pH 10) constant pH.

The product of Example (a) has an average of 2 esterified -COOH groups per molecule arid Example (b), an average of 4 such groups. The presence of’a plurality of cathodic bands establishes that the esterified products consist of a plurality of esterifed orgoteins containing from one up to about 6 ester groups per molecule. - 40 427«! EXAMPLE 5C: METHYL ESTER/N-ACETYL HYBRID To a solution of (125 ug/ml) of 0.5 mg methyl esterified orgotein, produced by the procedure of Example 1C, was added 0.5 mg completely Nacetylated orgotein. Electrophoresis of the mixture showed only the bands corresponding to methyl esterified orgotein plus the anodic band corresponding to N-acetyl orgotein. After the mixture was heated at 50°C for four hours, however, electrophoresis showed the formation of several new species (at band positions +7 to +11) and over 50% diminution of the original N-acetyl and methyl ester orgotein bands.

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

The hybrids can be isolated from their equilibrium mixture with the N-acetyl and -COOMe orgoteins (homodimers) by ion exchange chromatography at low temperature, as described in Example 2C. The isolated heterodimers on storage can continue to re-hybridize to reform a mixture containing both homodimers as well. The rate of re-hybridization is dependent on temperature and is low at low temperatures.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without de25 parting from the spirit and scope thereof, can make various changes and - 41 modifications of the invention to adapt it to various usages and conditions The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

Claims (51)

1. An orgotein having at least one of the following modifications: (i) at least one lysine ε-amino group is alkylated, (ii) at least one lysine ε-amino group is carbamylated, and 5 (iii) up to 8 free carboxyl groups are esterified.

2. An orgotein as claimed in claim 1, having at least one of the following modifications: (iv) at least two lysine ε-amino groups are alkylated, (v) at least two lysine ε-amino groups are carbamylated, and 10 (vi) at least two free carboxyl groups are esterified, there being present at least two different alkyl, carbamyl and ester groups respectively.

3. An orgotein as claimed in claim 1, wherein at least two of the modifications (i), (ii) and (iii) are present.

4. An orgotein as claimed in claim 1, wherein 15 (a) one peptide sub-unit of the orgotein molecule is unmodified and the other has at least one of the modifications, (i), (ii) and (iii). (b) one peptide sub-unit has one of the modifications (i), (ii) and (iii), and the other has a different one of said modifications, or (c) one peptide sub-unit has one of the modifications (i), (ii) and (iii) 20 and the other has the same modification but with a different group.

5. An orgotein as claimed in claim 4, wherein any modified peptide sub-unit has another of the modifications (i) to (iii) or has one modification (iv), (v) or (vi) as defined in claim 2.

6. An orgotein as claimed in any one of claims 1 to 5, wherein at 25 least 10 lysine ε-amino groups are alkylated. - 43 'iivt

7. An orgotein as claimed in claim 6, wherein at least 18 lysine e-amino groups are alkylated.

8. An orgotein as claimed in any one of claims 1 to 7, wherein at least 6 free carboxyl groups are esterified. 5

9. An orgotein as claimed in any one of claims 1 to 3, wherein from 6 to 10 lysine e-amino groups are carbamylated.

10. An orgotein as claimed in any one of claims 1 to 7, wherein the alkyl group in an alkylated lysine ε-amino group is a straight or branched chain alkyl group, a cycloaikyl or cycloaikyl-alkyl group, or an aralkyl 10 group. IT. An orgotein as claimed in claim 10, wherein the alkyl group is one having 1 to 8 carbon atoms and which may be substituted.

11. 12. An orgotein as claimed in claim 11, wherein the alkyl group is substituted by one or more substituents selected from carboxy, substituted 15 carboxy, cyano, cyanoalkyl, carbalkoxy, amido, hydroxyalkyl, phenyl and phenyl carbonyl groups.

12. 13. An orgotein as claimed in claim 12, wherein the substituted carboxy substituent has the formula -COOR wherein R represents a methyl or ethyl · group. 20

13. 14. An orgotein as claimed in claim 11, wherein the alkylated amino group has the formula -NH-CH2-R 1 wherein R 1 represents a hydrogen atom, a (C 1 -C 7 )-alkyl group, or a group of the formula -COOH, -COO-LA, -CONH(LA) -C0N(LA) 2 , -CsN, -CH Z -C=N, -Ph, 25 -COPh, -CH 2 OH or -CH(CH 3 )0H in which LA represents a (C^Ci,)-alkyl group - 44 and Ph is an unsubstituted or substituted phenyl group.

14. 15. An orgotein as claimed in claim 14, wherein the phenyl radical Ph has 1 to 3 substituents selected from alkyl groups, halogen atoms, nitro and amido groups, alkoxy and carboalkoxy groups. 5

15. 16. An orgotein as claimed in any one of claims 1 to 3 or claim 8, wherein the esterified carboxy group has the formula -COO-CH^R 1 wherein R 1 is as defined in claim 14 or claim 15.

16. 17. An Grgotein as claimed in any one of claims 1 to 3 or claim 8, wherein the carboxyl group is esterified by an alkyl group having up to 4 10 carbon atoms which may be substituted.

17. 18. An orgotein as claimed in claim 17, wherein the alkyl group is substituted as claimed in claim 12 or claim 13.

18. 19. An orgotein as claimed in any one of claims 1 to 3, or claim 8, wherein the esterified carboxy group has the formula -COOR 3 wherein R 3 15 represents -CH.XOX in which X represents -OCH.,, -OC Z H.,, -NH 2 , -NHCH 2 C0NH 2 or -CH 2 CH2ph, or wherein R 3 represents -CH(Ph) 2 wherein Ph is defined as in claim 14 or claim 15.

19. 20. An orgotein as claimed in any one of claims 1 to 3, or claim 9, wherein the carbamylated/amino group has the formula X' -NHCH N HR 2 wherein X' is an oxygen or sulphur atom and R 2 represents an alkyl group of up to 12 carbon atoms, Ph is as defined in claim 14 or claim 15 and, when X' is oxygen, a hydrogen atom.

20. 21. An orgotein as claimed in claim 20, wherein R ? represents an unsub- 45 43701 stituted (Cx-CgJ-alkyl group.

21. 22. An orgotein as claimed in any one of claims 11, 14, 17, 20 and 21, wherein an alkyl group is a methyl group.

22. 23. An orgotein as claimed in any one of claims 1 to 22, which is 5 bovine orgotein.

23. 24. An orgotein as claimed in claim 1 and which is substantially as described in any one of the Examples herein.

24. 25. A process for the production of an orgotein as claimed in claim 1, which comprises reacting orgotein with at least one reagent selected from 10 alkylating agents, carbamylating agents and esterifying agents.

25. 26. A process as claimed in claim 25, wherein the alkylating agent is a di(primary alkyl)sulphate, an activated alkyl halide, an activated vinyl compound or a reductive alkylating agent.

26. 27. A process as claimed in claim 26, wherein the alkylating agent 15 is a dialkyl sulphate wherein each alkyl group has up to 4 carbon atoms.

27. 28. A process as claimed in claim 27, wherein the alkylating agent is dimethylsulphate.

28. 29. A process as claimed in claim 25, wherein the carbamylating agent is an alkyl isocyanate, alkyl isothiocyanate or aryl isocyanate or aryl 20 isothiocyanate,

29. 30. A.process as claimed in claim 29, wherein the carbamylating agent is an alkyl isocyanate or isothiocyanate having up to 12 carbon atoms in the alkyl moiety.

30. 31. A process as claimed in claim 30, wherein the carbamylating agent 25 is (Οχ-Cg)-alkyl isocyanate or isothiocyanate.

31. 32. A process as claimed in claim 26, wherein the esterifying agent - 46 4370ft is a dialkyl sulphate, diazomethane or a diazo compound of formula N 2 CH 2 COX wherein X is -OCH 3 , -OC 2 H 3 . -NH 2 , or -NHCH 2 CONH 2 .

32. 33. A process as claimed in claim 32, wherein the esterifying agent is dimethyl sulphate. 5

33. 34. A process as claimed in any one of claims 25 to 33, for the production of an orgotein as claimed in claim 2, wherein the orgotein or at least one peptide sub-unit thereof is reacted successively with at least two alkylating, carbamylating or esterifying agents, each successive agent being more reactive and/or used at a higher concentration than the previous 10 agent.

34. 35. A process as claimed in any one of claims 25 to 33 for the production of an orgotein as claimed in claim 4, wherein two orgoteins are modified separately and the resulting orgoteins are hybridized, or one orgotein is modified and is hybridized with native orgotein. 15 35, A process as claimed in claim 35 for the production of an orgotein as claimed in claim 5 wherein the, each or an orgotein is modified as claimed in claim 34 before hybridization, or the resulting hybrid molecule is reacted with a reagent as defined in claim 25.

35. 37. A process as claimed in any one of claims 26 to 36, wherein the 20 orgotein is bovine orgotein.

36. 38. A process as claimed in claim 26, carried out substantially as described in any one of the Examples herein.

37. 39. An orgotein as claimed in claim 1, whenever produced by a process as claimed in any one of claims 28 to 38. 25

38. 40. A pharmaceutical preparation which comprises an orgotein as claimed - 47 43702. in any one of claims! to 25 or claim 39 in admixture or conjunction with a pharmaceutically suitable carrier.

39. 41. A pharmaceutical preparation as claimed in claim 40, in the form of a sterile injectable solution or suspension. 5

40. 42. A pharmaceutical preparation as claimed in claim 41, which comprises from 0.5 to 20 mg of the orgotein per 0.25 to 10 ml.

41. 43. A pharmaceutical preparation as claimed in claim 42, which comprises from 0.5 to 20 mg of the orgotein per 0.5 to 5 ml.

42. 44. A pharmaceutical preparation as claimed in claim 41, in a form 10 suitable for administration by infusion.

43. 45. A pharmaceutical preparation as claimed in claim 44, v/hich comprises from 0.5 to 20 mg of the orgotein per 50 to 1,000 ml.

44. 46. A pharmaceutical preparation as claimed in claim 45, which comprises from 0.5 to 20 mg of the orgotein per 100 to 500 ml. 15

45. 47. A pharmaceutical preparation as claimed in claim 40, in a form suitable for topical administration.

46. 48. A pharmaceutical preparation as claimed in claim 47, which comprises from 0.5 to 20 mg of the orgotein per 0.25 to 10 ml.

47. 49. A pharmaceutical preparation as claimed in claim 48, which comprises 20 from 0.5 to 20 mg of the orgotein per 0.5 to 10 cc.

48. 50. A pharmaceutical preparation as claimed in claim 40, in a form suitable for enteral administration.

49. 51. A pharmaceutical preparation as claimed in claim 50, in unit dosage form. 25

50. 52. A pharmaceutical preparation as claimed in claim 51, which comprises - 48 4370 ί from 0.1 to 25 mg of the orgotein per dosage unit.

51. 53. A pharmaceutical preparation as claimed in claim 52, which comprises from 1 to 10 mg of the orgotein per dosage unit.