CA1090782A - Orgotein derivatives and their production - Google Patents

Abstract

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

Description

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This invention relates to orgotein derivatives and to a process for their production.
Orgotein is the non-proprietary name assigned by the United States Adopted Name Council to members of a family of water-soluble protein congeners in substantially - pure, injectable form, i.e., substantially free from other proteins which are admixed or associated therewith in the sources thereof. Huber and Schulte U.S. Patent No. 3,758,682, issued September 11, 1973, claims pharmaceutical compositions comprising orgotein.
The orgotein metalloproteins are members of a family of protein congeners 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 heat-ing 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 1.0~ 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 1.00 A., and substantially .
no chelated monovalent metals or those that are cell poisons in the molecule.

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The aminoacid composition of the orgotein congeners is remarkably consistent irrespective of the source from which it is isolated.
Table I lists the distribution of aminoacid residues, S calculated for a molecular weight of 32,500 of several orgotein congeners.

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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 titrable (with trinitrobenzene sulfonic acid) -amino groups. In one aspect, this invention is directed to orgotein derivatives in which at least a portion of the orgotein lysine groups are alkylated and to a process for their ~:
production. In another aspect, this invention is directed to :
orgotein derivatives in which at least a portion of the orgotein lysine groups are carbamylated and to a process for their production.

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It can also be seen from Table I that orgotein congeners have from 29-37 aspartic acid and 21-38 glutamic ;` acid groups. In another aspect, this invention is directed to orgotein derivatives in which at least a portion of these aminoacid residues in free acid form are esterified and to a process for their production.
As will be apparent to those skilled in the art, some - esterification reagents and conditions are capable of simul-taneously alkylating free amino groups in the orgotein mole-cule and esterifying carboxylic acid groups. Such N-alkylated and esterified orgoteins and their production also are within the ambit of aspects of this invention.

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The nati~e orgotein protein possesses, inter alia, ,: .
anti-inflammatory activity. See Huber and Schulte U.S.
Patent 3,758,682, issued September 11, 1973. 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 No. 3,758,682, cited above. A
substantial portion of the superoxide dismutase activity ... . . .
,;s, of the native protein also is retained in:the orgotein derivatives of this invention, e.g., 20-100% of the native protein.

N-ALKYLATED ORGOTEIN AND N-CARBAMYLATED ORGOTEIN
As stat~d 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, which are tightly but non-covalently bound together under moderate conditions of temperature and pH. Because of the spacial conformation of the orgotein '' ' c~
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molecule, usually the -amino groups of a few lysines in each chain are not titrable with trinitrobenzenesulfonic acid (TBNS) and thus not readily accessible for alkylation or carbamylation.
However, both alkylation and carbamylation of the non-titrable lysine E-amino groups also appears possible 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 that 1-3 of the lysines of the native orgotein protein are not titrable with TNBS. 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 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 the electric field strength, net charge of the ion ~including bound conterions), and frictional co-efficient. See, for example, C. Tanford "Physical Chemistry of Macromolecules," Wiley, New York (1966). Since the fric-tional 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 ~ . ~
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groups, under identical electrophoresis conditions, the only variable affecting this electrophoretic mobilit~ is net charge.
Comparison of the electrophoretic patterns of a number of chemically modified orgotein molecules is con-~;
sistent with this conclusion. Native bovine orgotein electro-phoreses mainly as one band (band l), 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 l. Treatment of native bovine orgotein with, e.g., successively higher concentrations of acetic anhydride at pH 7, or with methyl isothiocyanàtë at pH9, leads to the formation of a series of successively more anodic (migrating toward the ~ electrode) electrophoretic bands as successively more free amino groups of the orgotein molecurles are acetylated or carbamy-lated. 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 electrophoresed 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 concentra-tion, and are appreciably retarded when salt-containing samples are electrophoresed. Therefore, since extrapolating more than about two band positions i.s not always precise, accurate `- ~ 1090~;~8Z
charge counting requires that the unknown be co-electro-phoresed with a solution which contains all the bands from 1 through the position of interest (e.g., partially acetylated or partially carbamylated orgotein).
All of the conventional protein modification reactions which have been applied to the orgotein molecule so far have been consistent with this interpretation, viz., the band : positions correspond to integral charge changes from the native orgotein molecule. Acetylation, carbamylation and N-methylthio-carbamylation all give bands 2, 3, 4, 5, etc., indicating that 1, 2, 3 and 4, respectively, free amino groups have been chem-ically modified. Similarly, succinylation, which changes o ~NH3+ groups to ~ NH~CH2CH2C02-groups, gives bands 3, 5, 7, 9, etc. and more extensive carbamylation or thiocarbamylation, which changes even more ~NHCNH-R and ~NHCNH-R groups, gives bands 6, 7, 8, 9, etc. Esterification with dimethyl sulfate or with ethyl diazoacetate gives bands -1, -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 one of the accessible (TNBS
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 amino groups are alkylated or carbamylated, the distri-bution 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 or convertible 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 m~re _g_ ~.09078Z
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or less randomly along each peptide sub-unit but more or less evenly between the two chains. Since a single alkylating or car-bamylating 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 ~-amino groups can be alkylated with a moderate concentration of one alkylating agent, e.g., iodoacetamide, and the remainder of the reactive amino groups alkylated with a high concentration of still another alkylating agent, e.g., dimethyl sulfate.
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>

One way o~ producing a mixed N-carbamylated orgotein is by carbamylating in stages with different carbamylating agents.
For example, a fraction of the titrable lysine ~-amino groups can be carbamylated with a moderately reactive carbamylating agent, e.g., KNCO, and the remainder of the reactive amino groups alkylated with a more reactive 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 plf and on the carbamylating agent, and to a lesser extent on buffer and temperature.

-ln-- Another process of ~roducing a mixed alkylated or mixed N-carbamylated orgotein is by hybridization. The term hybridi-zation 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 æ 2A~). 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-init is involved in the binding in all cases.
; 10 -N-methyl orgotein, produced by alkylating the native orgotein molecule in 0.04 pH 10 carbonate buffer with excess dimethyl sulfate, -N-ethyl orgotein, producte~l by alkylating orgotein in the same manner with diethyl sulfate. -N-propyl-carbamyl orgotein, produced by carbamylating the native orgotein molecule in 0.1 M pH 7.6 tris or phosphate buffer with excess propyl isocyanate, and -N-methylthiocarbamyl orgotein, produced by carbamylating orgotein in 0.075 M sodium tetraborate with methyl isothiocyanate, can each be hybridized 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 with 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 ~iffer from those in the other.

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' , ' ' ' ~' ' The E-N-~kyl orgoteins and ~-N-carbamyl orgoteins of aspccts of thls 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 resistan~ to Pronase and other proteolytic enzymatic degradation. Superoxide dismutase (SOD) - enzymatic activity is reta~ned, although, in the case of carbamy-lated orgoteins, lessened in proportion to the degree of carbam-ylation.
Although.the predominant stuctural modification of the native - orgotein molecule which occurs upon alkylation or carbamylation thereof at alkaline pH is the mono-, di- and, to a lesser extent, trialkylation of the ~-amino groups of the lysines thereof, in the case of alkylation, and carbamylation of these ~-amino groups, in the case of carbamylation, the free amino groups of the 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, -SCH3, etc., 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 iodoacetam~de, alkylation appears to be solely ~-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 ~-N-alkyl groups are included in the novel compounds of this invention. However, 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.

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The course of the alkylation, insofar as it involves -COOH
alkylation, and of the carbamylation can be followed directly by a change in overall electrophoretic charge and in the appearance of new bands on electrophoresis. Similarly, N-alkyla-tion, insofar as it renders an otherwise acylatable -NH2 group resistant to acylation, can be followed by a reduction in the number of acylatable amino groups, comparted to the native orgotein molecule.
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. Because of the higher molecular weight o~ the orgotein moleculei even when the orgotein moleucle is fully alkylated or carbamylated with alkyl or carbamyl groups of moderate molecular weight, e.g., C 100, the impact on the overall chemical composition is relatively small, i.e., less than 10%. Alkylation and carbamylation also has no apparent significant effect upon the compact spacial 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 also must be one derived from an alkylating or carbamylating agent capable of alkylating and carbamylating, respectively, an amino group in water of buffer solution, since the reaction is usually conducted therein.
Such alkylating agents include diprimary alkyl suLfates:
(RO)2SO2 (R = CH3, CH2H5, n-C3H7, n-C4Hg, activated alkyl halides:
ICH2COX (X = OH, NH2) benzyl and allyl bromides; activated vinyl groups: CH2 = CHX (X = CN, SOCH3, SOC2H5, COCH3);
reductive alkylating agents: RCOR' + BH4 or BH3CN (R,R' = H, CH3

2 5' 6 5) 1.090782 For a process of reductive alkylation of proteins with aromatic aldehydes 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).
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Examples of suitable carbamylating agents include alakli metal cyanates, e.g., NaNCO, KNCO; alkyl isocyanates and alkyl isothiocyanates, e.g., RNCO and RNCS wherein R = CH3, C2H5, n-C3H7, n-C4Hg, n-C8H17; and aryl isocyanates and iso-thiocyanates, 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 urea groups produces a stable product.
The reaction of alkyl isocyanates and isothiocyanates with orgotein is completely analogous to the reaction of cyanates therewith, only much faster. The long chain iso-., cyanates are less reactive than short chain isocyanates.
The reaction of orgotein with isocyanates and isothio-cyanates 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 appre-ciably because of the 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.

N-ALKYLATED ORGOTEINS

Since the exact chemical nature of the alkyl radical - is not critical, so long as it is not pllysioloyically toxic in the orgotein molecule and can be formed on the lysine ~-amino groups, contemplated equivalents of the preferred hydrocarbon alkyl groups described above, insofar as they can be formed, are cyclopentyl, cyclohexyl, menthyl, and like cycloalkyl, cyclohexylmethyl, ~-cyclopentylpropyl, and like cycloalkylalkyl, benzyl, p-xylyl and phenethyl and like aralkyl. Also contemplated as equivalents are alkyl oE 1-8, preferably 1-4, and most preferably methyl or ethyl bearing one or more, preferably one, simple substituents, e.g., carbbxy, cyano, carbalkoxy, and amido, e.g., carboxymetl-yl, . -' . ' ` --`` ``" ~10'~0~78~
' cyanomethyl, carbethoxymethyl, carbomethoxymethyl and like carbo-lower-alkoxymethyl, carbamylmethyl, and the correspond-ing substituted ethyl groups, e.g., -CH2CH2COOH, -CH2CH2C -N, -CH2CH2CONH2 and -CH2CH2COOR, wherein R is, e.g., methyl or ethyl, -CH2CH2SOCH3, -CH2CH2SOC2H5 and -CH2CH2COCH3.
Thus, the alkylated orgoteins of aspects of this inven-tion 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 (18-26) of whose titrat-able amino groups are alkylated, i.e., bearing an unsubstituted or substituted alkyl group.
In the preferred embodiment, the alkylated amino groups ` are those of the formula ` -NH-CH2-R
wherein R is H, CH3, C2H5, n-C3H7, iso-C3H7, or other alkyl - of up to 7 carbon atoms, -COOH, -COO-LA, -CONH-LA, -CON(LA) -C - N, -CH2-C_N, -Ph, -COPh, -CH2OH or -CH(CH3)OH, in which LA is lower alkyl of 1-4 carbon atoms and Ph is unsubstituted phenyl or phenyl bearing 1-3 simple substituents, e.g., methyl, chloro, bromo, nitro, amido and methoxy, carbomethoxy or carboethoxy, e.g., p-tolyl, sym.-xylyl, p-amidophenyl, m-chlorophenyl and p-methoxyphenyl. Such orgoteins have the formula (H2N)m-Org- (NHCH2R)n wherein n is an integer from 1 to 26, preferably at least 10, more preferably 10-18, and the sum of m and n is the total number of titratable free amino groups in the unmodified con-gener and R has the values given above, preferably H or LA, e.g., methyl or ethyl, and "Org" is the remainder of the orgotein molecule.
Some of the alkylating agents employed in the process of an aspect of this invention will simNltaneously alkylate some of the - ^ 10~0782 free acid groups of the orgoteln molecule. These alkyl~ted orgoteins can be represented by the formula (HOOC)X ~COOCH2R)y ;~ (Org) (H2N) . \ (NHCH2R)n wherein "Org", R, m and n have the values given above, y is the number of alkylated free carboxylic acid groups, e.g., from one up to 8, preferably 2-6, and x is the remain- ~
~; der of free carboxylic acid groups in the orgotein molecule.
Reagents useful for introducing carboxymethyl and carb-~? amylmethyl groups, respectively, are iodoacetic acid and iodoacetamide. Iodoacetic acid can form stable ~roducts ; 10 with cysteine (sulfhydryl), ~ysine (amino), histidine (imidazole nitrogens), and methionine (sulfide sulfur) re-sidues in proteins. Since carboxymethylation of any of these groups except methionine increases the net negative charge into the orgotein protein at pH ~.4, electrophoresis can quantitate the total xeac~i~n: (except carboxymethylation of `
methionine and carboxymethylation of the second imidazole r~ nitrogen).
In native orgotein, iodoacetic acid and iodoacetamide react predominantly, if not exclusively, with lysine amino groups. Although free thiols react with iodoacetic acid several orders of magnitude faster than do amines, the first few groups in orgotein which are carboxymethylated do not react appreciably faster than do the next 10-20 groups. This is consistent with the reaction of p-mercuribenzoate with , . , ~ .
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orgotein sulfhydryls: there is no reaction with holo-protein.
Even with apo-protein, which gives a p-mercuribenzoate-sulfhydryl reaction (although slowly under non-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 p-mercuribenzoate titration). The pH dependence of the carboxymethylation reaction as shown by electrophoresis is consistent with reac-tion of lysine (pK>8) and not of histidine (pK~6); , since the extent of reaction at identical initial concentrations increases 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, ethylchloro-formate 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 un-modified orgotein protein).
In addition to the N-methyl "bovine" orgoteins and orgoteins of the examples hereinafter, other examples o l()sot7s~

N-alkyl bovine orgoteins of this invention are N-ethyl orgotein, N-propyl orgotein and N-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 corre-sponding 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.
N-CARBAMYLATED ORGOTEINS
The carbamylated orgoteins of aspects of this inven-tion are orgotein congeners, including bovine, sheep, horse, por~, rat, dog, rabbit, guinea pig, chicken and human, at least one, e.g., 1,2,3,4,5 and up to all (18-26), of whose titratable 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 of the formula X
-NHCNHR
wherein X is O or S and R is CH3, C2H5, n-C3H7, iso-C3H7, ; 20 n-C8H17 or other alkyl of up to 12 carbon atoms, Ph, or when X is O, also a hydrogen atom, and Ph is unsubstituted phenyl or phenyl bearing 1-3 simple substituents, e.g., methyl, chloro, bromo, nitro, amido and methoxy, carbomethoxy or carboethoxy, e.g., p-tolyl, sym.-xylyl, p-amidophenyl, m-chlorophenyl and p-methoxyphenyl. Such orgoteins have the .

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formula X
(H2N)m-Org-(NH~NH-R)n wherein n is an integer from 1 to about 26, preferably at least 2 , more preferably about 6 to 10 and the sum of m and n is the total number of titratable free amino groups in the unmod~
ified congener, X is O or S, R-has the values given above, preferably 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 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 and can be formed on the lysine ~ -amino groups, contemplated equivalents of the preferred alkyl carbamyl orgoteins described above, insofar as they can be formed, are those otherwise corresponding to the above formula wherein R is cyclopentyl, cyclohexyl, menthyl, and like cycloalkyl, cyclohexylmethyl, ~-cyclopentylpropyl, and like cycloalkylalkyl, benzyl, p-xylyl and phenethyl and like aralkyi. Also contemplated as equivalents are those wherein R is alkyl of 1-8, preferably 1-4, and most preferably methyl or ethyl,bearing one or more, preferably one, other substitu-ents, e.g., fluoresceinyl.
.

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In addition to the N-carbamylated "bovine"
orgotein of the examples hereinafter, other examples of N-carbamyl bovine orgoteins of aspects of this invention are the corresponding N-carbamyl orgotein, N-propylcarbamyl orgotein, N-ethylcarbamyl orgotein, and N-methylthio-carbamyl 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, Ç 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.
ESTERIFIED ORGOTEIN
As stated above, orgotein congeners contain 50-68 . 15 glutamic and aspartic residues but only 20-27 of these have free acid groups. Since the orgotein molecule is made up of two identical or almost identical peptide chains . (sub-units), half of these aminoacid residues are in each chain, which are tightly but non-covalently bound together under moderate conditions of temperature and pH. Esterification changes the . .

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charge of the orgotein molecule and usually only up to 10 and preferably up to 6 to 8 of these free 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 electro-phoresis. 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. ~owever, 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 .~ .
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-' 1.09U7~3~
with a low concentratlon of one esterifying acJent, e.g., diethyl sulfate, and another fraction of the ~cid groups esterified with a higher concentration of another esterify-ing agent, e.g., diazomethane. What constitutes a low or S high concentration of esterifying agent will depend on its relative r~tes of reaction with protein acid groups and with solvent and will thus depend on the reaction pl~ and on the esterifying agent, and to a lesser extent on buffer and temperature.
Another process of producing a mixed orgotein ester is by hybridization. The term hybridization of orgotein refers to the formation of a mixed orgotein from the sub~it 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.
Methyl orgotein and ethyl orgotein can each be hybridized with native orgotein or with each other by heating with a slight excess of native orgotein at 50C. for 4 hours. The resulting heterodimers electrophorese as a mixture of bands intermediate between native orgotein and the bands of the esterified orgo-tein prior to hybridization.

,, .

78'~

As will be apparent, these hybrid semi-esterified orgotein molecules 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.
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 Pronase and other pro-teolytic enzymatic degradation. 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 esterified groups is not critical as long as the esterifying group is physiologically acceptable. Because of the 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 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 also must be one derived from an esterifying agent capable of esterifying '7l3 ~' a carboxylic acid group in water or buffer solution, since the reaction is usually conducted therein. Such esterifying agents include dimethyl and diethyl sulfate, diazomethane ~: and other diazo compounds, e.g., of the formula N2CH2COX
wherein X is, e.g., OCH3, OC2H5, NH2 or NHCH2COHN2, and other esters and amides of diazoacetic acid which lack reactive groups, e.g., carboxyl or imino.
For processes 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 in various aspects is directed to -COOH esterified orgotein wherein the ester group preferably is of up to 4 carbon atoms, e.g., of a mono-hydric alkanol, e.g., methyl, 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 and ' it can be formed on orgotein's free acid groùps, contemplated equivalents of the preferred alkyl esterifying groups described above, insofar as they can be formed, are those bearing one, two or more simple substituents, e.g., halo, alkoxy, carb-alkoxy, carbamido, stc., especially those wherein the ester group bearing the substituent or substituents is methyl. Thus, in addition to esterified orogtein in which the esterified acid groups are -COOCH3, contemplated equiva-lents are those wherein the ester groups are -COOR wherein R is -CH2COX in which X is OCH3, OC2H5, NH2, NHCH2CONH2 or CH2CH2-phenyl, or wherein R is CH(phenyl)2.

-` :1090782 Esterification of the -COO~I groups can be followed by counting the charge change shown on electrophoresis com-pared to native orgoteins.
As is known, the electrophoretic mobility of an ion is a function of the electric field strength, net charge of the ion ( including bound conterions), and frictional coefficient. See, for example, C. Tanford "Physical Chemistry of Macromolecules" Wiley, New York (1966). Since the frictional coefficient is dependent on molecular size and shape, and on the solution composition, comparisons of different proteins are not informative. However, by com--paring proteins of similar size and shape, in this case orgotein molecules chemically modified with relatively small groups, under identical electrophoresis conditions, the only variable affecting this electrophoretic mobility is net charge.
Comparison of the electrophoretic patterns of a number of chemically modified orgotein molecules is con-sistent with this conclusion. Native bovine orgotein elec-trophoreses 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 orming band 1. Esterification of successively higher nun~ers of the free -COOH groups of the native bovine orgotein leads to the formation of a series of successively more cathodic (miyrating toward the negative electrode) electrophoretlc bands as successively more free carboxy~ic acid groups of the orgotein molecule are esterified.
A graph of distance electrophoresed versus band number 109~78'~
is relatively linear at low extents of -COO~I 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 elec-trophoresed. Therefore, since extrapolating more than , about two band positions is not always precise, accurate charge counting requires that the unknown be co-electrophoresed ` 10 with a solution which contains all the bands from 1 through the position of interest (e.g., partially acetylated orgotein).
All the conventional protein modification reactions which have been applied to the orgotein molecule so far have i, .
been consistent with this interpretation, viz., the band positions correspond to integral charge changes from the native orgotein molecule. Esterification with dimethyl-sulfate 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 esterlfied without deleterious effects. AttemptS to esterify more than 6-8 -COOH groups, i.e., to esterify more than three -COOH groups of each of ;
the two orgotein peptide sub-units,usually leads to denat-uration and loss of superoxide dismutase activity.
~s would be expected, the distribution of the esterify-ing alkyl groups on the orgotein molecule probably is random since none of the titrable frëe carboxy groups appear abnormally readily esteriEi~ble. Because the orgotein mole-cule 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 - 27 ~

IC~78Z

less evenly between the two chains. Since a single esteri-fying 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.
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 with another esterifying agent, e.g., ethyl diazoacetate.
Another process of producing a mixed esterified orgotein is by hybridization. The term hybridization of orgotein refers to the formation of a mixed orogtein from the sub-unit 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.
Methyl esterified orgotein, produced by esterifying about 6 free acid groups of the orgotein molecule with diemthyl sulfate and carboxymethyl esterified orgotein, produced by esterifying the native orgotein molecule with ethyl diazo-acetate, can each be hybridized with native orgotein orwith each other by heating together at 50~ C. for 4 hours.
In addition to the bovine orgotein derivati~e of the examples hereinafter, other examples of orgotein derivatives of this invention are the corresponding derivatives of other orgotein congeners.

` ` 109078Z

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-unlts of the orgotein molecule S and the corresponding esterified orgoteins wherein there are 1 or 2 such ester groups in each such sub-unit, respective ly, and the corresponding human, sheep, horse, pork, dog, rabbit, guinea pig, and chicken congeners of each of these.
, - 2~ -:` ` `
lO9V7~
The orgotein derivatives can 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 No. 3,758,682, cited above. If desired, the orgotein derivative can 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 - 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 bacterially and virally or bacterially sterile solution ; suitable for administration by injection. Filtration through an 0.1 micron pore filter will also reduce or eliminate pyrogens in the solution.
The pharmaceutical compositions containing the compo-sitions of aspects of this invention comprise the orgotein ester and a pharmaceutically acceptable 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 f form of a-sterile injectable preparation, for example, as a ~A~ sterile injectable aqueous solution. The solution can be formulated according to the known art using those carriers mentioned above. The sterile injectable preparation can also be sterile injectable solution or suspension in any non-toxic parenterally acceptable diluent or solvent, or can be a lyophilized powder for reconstitution with such solvent.
-30 The pharmaceutical compositions containing the compo-sitions of this invention combine an effective unit dosage : B amount of the orgotein derivative, i.e., the orgotein ' ' - ' ' . , :
. . . . . . . . . .

. , . ' . .

iO9078~

derivative is present at a concentration effective to evoke the desired response when a unit dose of the compo-sition is administered by the route appropriate for the par-ticular pharmaceutical carrier. For example, liquid compo-sitions, both topical and injectable, usually contain 0.5to 20 mg. of the orgotein derivative per 0.25 to 10 ml., preferably 0.5 to 5 ml., except I.V. infusion solutions, which can also be more dilute, e.g., 0.5 to 20 mg. orgotein derivative per 50 - 1,000 ml., preferably 100 - 500 ml. of 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, e.g., intramuscularly, sub-cutaneously, intravenously or intradermally. I.M. is pre-ferred, 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 aspects of this invention like orgotein, is effective in treating a wide variety of inflammatory conditions, including those in which synthetic anti-inflammatory agents have limited utility, e.g., because of toxic side effects upon prolonged use.

.: . . . .
. ' , ,: ' ' "
.

` ~ iO9078~
~``'` ' ' .
More specifically, these orgotein derivatives are efficacious in ameliorating inflammatory conditions and . mitigating the effects thereof, for instance those in-. volvlng the urinary tract and the joints, in various mammals. It is useful in alleviating the symptoms associated with rheumatoid, osteo and post-traumatic arthritis, as well as bursitis, tendonitis, etc.
For further details relating to how to isolate the starting orgotein congeners and how to use the orgotein derivatives of this invention, including modes of adminis-tration, dosage forms, dosage regimen and inflammatory and other conditions susceptible to treatment with esterified orgotein, see U.S. Patent No. 3,758,682, cited above.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative.

`
~, 1~-' ., ~, ~ -32--, ' ' , ' . . .
. ' '; ' . `. .

10~078'~

EXI~MPLI~ lA
~ solution of 5 mg. bovine orgotein in 4 ml. of 0.04 M
carbonate buffer was treated with 20/ll dimethyl sulfate and the p~l kept at 10.0 by addition of 0.5 M NaOII. The uptake of base had a half-time of about 37 minutes. ~lectrophoresis showed SOD-active bands l through -5 (average -3). After dialysis, lyophilization and re-solution in 1 ml. water, the protein still had its SOD activity and Cu and Zn content unchanged from the untreated orgotein. ~cetylation of an , 85 /lg./ml. solution of the modified protein with a total of :; 3 ~ 1 acetic anhydride in 0.4 ml. borate buffer at pH 9 gavean average electrophoretic charge change of only about -2, compared to -20 for native orgotein in the same solution.
The p~l 10 dimethyl sulfate-treated orgotein is therefore - 15 extensively N-methylated, i.e., about 18 per molecule.

.~ .
, EXAMPLE 2A
Follow the procedure of Example lA, employing, respec-tively, the corresponding human, sheep, horse, pig, dog, rabbit, guinea pig and chlcken orgotein conyellers as start-ing materials. In each case, all except about one lysine of each sub-unit of the orgotein molecule is alkylated.
.
j- EXAMPLE 3 Follow the procedure of Example lA, employing diethyl-.
sulfate instead of dimethyl sulfate. The properties of the resulting ~-N-ethylated orgotein are essentially tlle same as the ~-N-methylated orgotein.

EX~MPLE 4A
The ~-amino groups of the lysine residues of bovine , . . .

10907~

orgotein were carboxymethylated with 0.2 M sodium iodoacetate at ambient temperature under the conditions and with the results set forth below.
Charge Chanye on Electrophoresis, l~eaction BufEer average + range oryotein (1.4 mg./ml.) + ICl12C02Na (0.2 M) ~a) 0.3 M pH 3.8 acetate (only changes are those due to low p~
(b) 0.4 M p~l 5.0 acetate no change up to 72 hours (c) 0.23 M pH 9.2 carbonate 0 (15 min), 3-+2 (2 1/2 hrs.), 5-+3 (6 1/2 hrs.) ~ 181(72 hrs.) From these electrophoretic patterns, it appears that no N-alkylation occurred at pH 3.8 and 5.0 and that at pll 9.2 an average of about 3 -CH2COO groups were introduced in 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.
orgotein (2.8 mg./ml.) + ICH2CO2Na (0.43 M) ... .
(d) p~l 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 da~
(f) pH 10 0 (10 min.), ~3 (2 1/2 hrs.), > 10 (6 1/2 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 ~-amino yroup alkylated wit:h a -C112COO
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 2 1/2 hours and by 6 1/2 hours an ,, ,,, , . . . ~

;' average of more than 10 such groups per molecule were intro-duced.
The products of all the above alkylations in which -C~I2COO groups were introduced were a mixture of ~-amino alkylated orgoteins containing varying numbers of such groups as evidenced by the appearance of a plurality of bands with varying electrophoretic mobilities.

i~, ' ' . ' :.
, . .
.

., EXAMPLE lB 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 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 50~1 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 ~1 of propylisocyanate or octylisocyanate for various times. In two instances, 2 ~l 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 + 2~1 2 min. 2 hr. 1 da~ 2 days ana~Ye~
.REAGENT
propylisocyanate/tris 8 8 ND* ND 10 propylisocyanate/phosphate 8 8 ND ND > 15 octylisocyanate/tris0.2 1.0 6 6 octylisocyanate/phosphate 0 0.2 2.5 3 *Not done .. . . . . . . .

~ 109~)78Z

EXAMPLE 3B Alkyl Carbamylated Orgoteins The procedure of Example 2~ was repeated employing 10 mg. of orgotein in 1 ml 0.l M pH 7.6 phosphate buffer and 1 ml of propyl or octyl isocyanate. The solutions were maintained at 25 C. for 4 hours and examined electrophoretically. Then an additional l 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 Reacted Metal Content ~ of Origina~
-` 4 llours Overniqht Cu~t Z~ SOD Activity ,: .
propyl 10_4' > 15 2.2 1.5 approx. 50~

. .
` octyl 3+3 6~5 2.1 1.8 approx. 25~

~X~MPL~ 4B N-Methyl Thiocarbamyl Orgotein To a solution of 5 mg. orgotein in 4 ml. 0.075 M p~l 9 Na2B4O7 was added 10~1` CH3NCS, and the mixture shaken for a minute until the C~13NCS dissolved. At intervals, 50 ~1 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. Af~er 22 hours at room tcm~eraturc, tllc rcmainin~
reaction mixture was quenched with 0.5,ml. 1_ pll 5.3 acetate buffer and dialyzed vs. frequent changes of water for 2 days.

~09~)78Z

. The dialyzed solution was centrifuged briefly to remove the whlte ~` 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 the samples by the pH 7.8 cytochrome c assay (McCord & Fridovich, J. 13iol. Chem. 6049-6055 (1969)) " showed a drop in SOD activity with more extensive modification.
;; . .
Time (Hours)Average SOD ~ctivity Charge Change %
~,,. 10 o (O) (100~) ^ 0.08 0.5 111 0.25 1 118 0.75 2 91 ` 1.9 4 86 6 9.5 43 , . . .
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 Cutt (compare~d with 2.26 GAPM Zn and 2.08 GAPM Cuttin unmodified orgotein). All of the products are soluble, retain chelated Cu ++ and Zn + and at least a portion of the superoxlde dismutase activity of the u~Lmodified protein.
, EXAMPLE 5B N-Fluoresceinylcarbamyl Orgotein A solution of 100 mg. orgotein (bovine congener) and 6 mg. fluorescein isothiocynate in 10 ml. of 0.14 M pll 8.5 phosphate buffer was maintained at 4 C. for 1~ hours. The reaction mixture was applied to a chromatographic column of

- 3~ --- ; ~
109()78Z

microporous cross-linked dextran (Sephadex G-50, Pharmacia, Upsala, Sweden) and eluted therefrom with pl~ 8 borate buffered saline (0.15 M). The yellow protein eluted fractions were dialyzed against water. The dialyzed protein which precipitated (20 mg.) was intensely yellow and fluorescent and was readily sol~ble in pl~ 8 borate buffered saline. Elec~rophoresis 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 mo~ extensively modified pro-tein, since electrophoresis showed a fast anodic smear.
The soluble protein was chromatographed on a weakly basic (DEAE-cellulose) ion exchange column at pH 6 with 0.01 to 0.2 M linear gradient of phosphate buffer. The mono-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, mono-carbamylated orgotein and dicarbamylated orgotein, (apparently the result of hybridization) and some non-protein fluorescen~t compound (apparently the result of hydrolysis of the thiourea group of carbamylated orgotein to give free aminofluorescein).

- 3~ -` ` 1090782 : Pollowing the procedu.re 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 S of these congeners are produced.

-~', ~'`

-- ~0 --. .

EXAMPLE lC : METHYL ESTERIFICATION
:
solution (125 lug/ml) of 0.5 mg bovine orgotein and 0.5~ vol/vol of dimethyl sulfate in 4 ml. of 0.05 M acetate )u~rcrlimairltainccl at p)l 5 for ]00 min. ~lectrophoretic ana~ysls of the reaction product showed SOD active bands from +1 through -6 (average -4). The product is orgotein having an average of 4 -COOCH3 groups.
EXAMPLE 2C : METHYL ESTERIFICATION ~
A solution of 0.5 mg. bovine orgotein an lO \11 dimethyl sulfate in 4 ml. water was kept at constant pH by addition of O.l M NaOII. The rate of base uptake in the pll range 7-10 was fairly independent of pH and of the addition of 0.25 mole sodium phosphate buffer. Electrophoresis showed the formation of more cathodic SOD-active bands at :.; .
15 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.
ta) Acetylation of a 0.5 ml. aliquotof the esterified org~tein with 1 111 acetic anhydride +3 ~1 6M NaOH at 4 C., gave a solu-tion whose electrophoresis pattern was a fast-moving anodic band similar to that of acetylated native orqotein, thus establishin~ that the free amino groups were unaffected during the esterification.
2S (b) A l ml. aliquot of the esterified orgotein solution was adjusted to pl~ lO.5 and stored covered in a dessicator with NaOH pellets. Although the cathodic band pattern of bands 1 through -4 was stable at pll 7-9, at pH 10.5 the cathodic bands gradually disappeared over a period of 4 days as the electrophoretic Fattern of native orgotein reappeared.
~' .
~ ' .
- 4l -~09078~
Protein methyl esters commonly hydrolyze readily at alkaline pH's, thus confirmlng the more cathodic bands appearing after the esterification were orgotein esters.
From the foregoing it is apparent that after two hours, dimethyl sùlfate at pH 7-10 esterified an averaqe 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 lC and 2C, employing, respectively, 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 lC and 2C, employing diethyl sulfate instead of dimethyl sulfate, orgotein esters haying from one to six free carboxylic acid groups converted to ethyl esters thereof are produced. The properties of the re-s~lting esterified orgoteins are essentially the same as the methyl esterified orgotein.
The esterified orgoteins can be further purified, if de-sired, by 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 orgo-tein through a 2.5 x 40 cm DEA SEPHADEX (the trade mark for a group of synthetic organic compounds derived from the polysac-charide dextrans, of Pharmacia Fine Chemicals) column with 4 liter of a 9.91 M to 0.2 M linear gradient of tris pH 8.5 buffer sep-arates the orgotein bands from each other, the electrophoretic-ally more cathodic bands eluting first. By such a procedure, the mixture of .

' .' ' : ' '' :, - . ; . , :

" ,''' ' '' ' ' ' ~ '.: .

109078Z ~
.

methyl esterified orgoteins produced by the procedure of Example lC
, 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 esterifled orgotein, carbethoxymethyl esterified orgotein, and N-acetylated orgotein.

.

. .

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- ~3 -.. .

1.090'7~Z
.
EXAMPLE 3C: CARBETHOXYMETHYL ESTERIFICATION

Ethyl diazoacetate is prepared by reaction of glycine ethyl ester with nitrpus acid, as in "Organic Synthesis" Coll.
Vol. IV, p. 424, but using CC14 rather than CH2C12 to extract the ethyl diazoacetate from aqueous solution.
Two ml. of the approximately lM ethyl diazoacetate/CC14 solution are placed in a 25 ml. flast 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 and 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 filetered and desalted by chromatographic fractionation employing a 10 cc SEpHADEx G-25 column. The protein fractions are lyophylized and redissolved in 2 ml. of water, and the pH raised from 3.7 to 5.6 with , 2 ,ul lM NaOH. Electrophoresis shows no change from before de-salting and lyophylization. Re-reaction 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 mat-erial more cathodic than band -6.

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.

` 1090'78Z

EXAMPLE 4C : METHYL ESTERIFICATION

Following the procedure of Examples 1 and 2, bovine orgotein was alkylated under the conditions set forth in the table below SOD Active Dimethyl Hydrolysis Bands on Sulfate Half-time* Electrophoresis ~H Buffer Or~otein ~ bv vol (a) 5.0 0.05 M 125 ~g/ml 0.25 54 min. +1 through -4 (Av. -2) (b) 5.0 acetate 125 yg/ml 0.5 54 min. -1 through -6 (Av. -4) (c) 7.0 0.016 M 80 "0.65 1 hr. -2 through -6 (d) 7.0 phosphate 80 "1.3 1 hr. raint streak of SOD
from +4 to -6, peak-ing at -6 position (e) 11.2- 0.05 M 125 " 0.5 1/2 hr. +2 to -3 9.3 carbonate (@ pH 10) Half-time for NaOH uptake needed to maintain constant pH.

The product of Example (a) has an average of 2 esterified -COOH groups per molecule and 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 esterified orgoteins containing from one up to about 6 ester groups per molecule.

` - -`" 109078Z

:
~` EXAMPLE 5C: METHYL ESTER/N-ACETYL HYBRID
To a solution of (125Jug/ml) of 0.5 mg methyl esteri-fied orgotein, produced by the procedure of Example l, was added 0.5 mg completely N-acetylated orgotein. Electro-phoresis of the mixture showed only the bands correspondingto methyl esterified orgotein plus the anodic band correspond-` ing 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) ' 10 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 !,.'' 15 lysines per subunit) and of methyl esterified orgotein (con-- taining 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 con-tinue 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.

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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.
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Claims (34)

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A process for the production of injectable orgotein derivatives having anti-inflammatory activity, which comprises reacting orgotein with (a) an alkylating agent or a carbamylat-ing agent to produce an N-alkylated orgotein or an N-carbamylated orgotein having an alkyl group or carbamyl group, respectively, on at least one lysine .epsilon.-amino group thereof, or (b) an esterifying agent to produce an esterified orgotein wherein up to 10 of the free carboxylic acid groups of the orgotein protein molecule are esterified.

2. A process according to Claim 1, wherein the orgotein is bovine.

3. A process according to Claim 1, wherein orgotein is reacted with an alkylating agent employing alkylation conditions which produce an alkylated orgotein having at least 10 alkylated .epsilon.-amino groups per molecule.

4. A process according to Claim 1, wherein orgotein is reacted with an alkylating agent employing alkylation conditions which produce an alkylated orgotein having at least 18 alkylated .epsilon.-amino groups per molecule.

5. A process according to Claim 1, wherein orgotein is reacted with an alkylating agent employing alkylation conditions which produce an alkylated orgotein having at least 10 N-alkyl-.epsilon.-amino groups per molecule in which alkyl is of 1-4 carbon atoms.

6. A process according to Claim 1, wherein orgotein is reacted with an alkylating agent employing alkylation conditions which produce an alkylated orgotein having at least 10 N-methyl-.epsilon.-amino groups per molecule.

7. A process according to Claim 3, wherein the alkylating agent is a diprimary alkyl sulfate, an activated alkyl halide, an activated vinyl compound or a reductive alkylating agent.

8. A process according to Claim 3, wherein the alkylating agent is a dialkyl sulfate wherein each alkyl is alkyl of 1-4 carbon atoms.

9. A process according to Claim 3, wherein the alkylating agent is dimethyl sulfate.

10. A process according to Claim 1, wherein orgotein is reacted with a carbamylating agent employing reaction conditions which produce a carbamylated orgotein having at least two carbamylated .epsilon.-amino groups per molecule.

11. A process according to Claim 1, wherein orgotein is reacted with a carbamylating agent employing reaction conditions which produce a carbamylated orgotein having 6-10 carbamylated .epsilon.-amino groups per molecule.

12. A process according to Claim 10, wherein the carbamylating agent is an alkali metal cyanate, alkyl iso-cyanate, alkyl isothiocyanate, aryl isocyanate or aryl iso-thiocyanate.

13. A process according to Claim 1, wherein orgotein is reacted with a carbamylating agent selected from an alkali metal cyanate or an alkyl isocyanate or alkyl iso-thiocyanate wherein alkyl is up to 12 carbon atoms, under conditions which produce a carbamylated orgotein having at least two carbamylated .epsilon.-amino groups per molecule, each being of the formula wherein X is O or S and R is alkyl of up to 12 carbon atoms or, when X is O, also a hydrogen atom.

14. A process according to Claim 1, wherein orgotein is reacted with a carbamylating agent selected from an alkali metal cyanate or an alkyl isocyanate wherein alkyl is up to 8 carbon atoms, under conditions which produce a carbamylated orgotein having at least two carbamylated .epsilon.-amino groups per molecule, each being of the formula wherein X is O
and R is alkyl of up to 8 carbon atoms or a hydrogen atom.

15. A process according to Claim 1, wherein orgotein is reacted with a carbamylating agent selected from an alkyl isothiocyanate wherein alkyl is up to 8 carbon atoms, under conditions which produce a carbamylated orgotein having at least two carbamylated .epsilon.-amino groups per molecule, each being of the formula wherein X is S and R is alkyl of up to 8 carbon atoms.

16. A process according to Claim 10, wherein the alkylating agent is an alkyl isothiocyanate wherein alkyl is 1-8 carbon atoms.

17. A process according to Claim l, wherein orgotein is reacted in an aqueous solution with an esterifying agent which esterifies carboxylic acids in aqueous or buffer solu-tion, under conditions which esterify up to 8 carboxylic acid groups per molecule.

18. A process according to Claim 1, wherein orgotein is reacted in an aqueous solution with an esterifying agent which esterifies carboxylic acids in aqueous or buffer solution, selected from dimethyl or diethyl sulfate, an azide of the formula N2CH2COX wherein X is OCH3, OC2H5, NH2 or NHCH2-CONH2 or other ester or amide of diazoacetic acid which lacks a reactive group, under conditions which esterify up to 8 carboxylic acid groups per molecule.

19. A process according to Claim 1, wherein orgotein is reacted in an aqueous solution with an esterifying agent is one which forms alkyl esters of 1 to 4 carbon atoms of up to 8 carboxylic acid groups per molecule.

20. A process according to Claim 1, wherein orgotein is reacted in an aqueous solution with an esterifying agent is one which forms methyl esters of up to 8 carboxylic acid groups per molecule.

21. An orgotein derivative selected from the group consisting of an alkylated orgotein wherein an alkyl group of up to 8 carbon atoms is present on at least one lysine .epsilon.-amino group thereof; an N-carbamylated orgotein wherein a carbamyl group is present on at least one lysine .epsilon.-amino group; and an esterified orgotein wherein up to 10 of the free carboxylic acid groups of the orgotein protein molecule are esterified, whenever produced according to the process of Claim 1 or an obvious chemical equivalent thereof.

22. An orgotein derivative of Claim 21, wherein the orgotein is bovine, whenever produced according to the process of Claim 2 or an obvious chemical equivalent thereof.

23. An alkylated orgotein of Claim 21, having at least 10 alkylated .epsilon.-amino groups per molecule, whenever produced according to the process of Claim 3 or an obvious chemical equivalent thereof.

24. An alkylated orgotein of Claim 21, having at least 18 alkylated .epsilon.-amino groups per molecule, whenever produced according to the process of Claim 4 or an obvious chemical equivalent thereof.

25. An alkylated orgotein of Claim 21, having at least 10 N-alkyl-.epsilon.-amino groups per molecule, wherein alkyl is alkyl of 1-4 carbon atoms, whenever produced according to the process of Claim 5 or an obvious chemical equivalent thereof.

26. An alkylated orgotein of Claim 21, having at least 10 N-methyl-.epsilon.-amino groups per molecule, whenever produced according to the process of Claim 6 or an obvious chemical equivalent thereof.

27. A carbamylated orgotein of Claim 21, having at least two carbamylated .epsilon.-amino groups per molecule, whenever produced according to the process of Claim 10 or an obvious chemical equivalent thereof.

28. A carbamylated orgotein of Claim 21, having about 6 to 10 carbamylated .epsilon.-amino groups per molecule, whenever produced according to the process of Claim 11 or an obvious chemical equivalent thereof.

29, A carbamylated orgotein of Claim 21, wherein the carbamylated amino group is of the formula wherein X is O or S and R is alkyl of up to 12 carbon atoms or, when X is O, also a hydrogen atom, whenever produced according to the process of Claim 13 or an obvious chemical equivalent thereof.

30. A carbamylated orgotein of Claim 21, wherein the carbamylated amino group is of the formula wherein X is O and R is alkyl of up to 8 carbon atoms or, a hydrogen atom, whenever produced according to the process of Claim 14 or an obvious chemical equivalent thereof.

31. A carbamylated orgotein of Claim 21, wherein the carbamylated amino group is of the formula wherein X is S and R is alkyl of up to 8 carbon atoms, whenever produced according to the process of Claim 15 or an obvious chemical equivalent thereof.

32. An esterified orgotein of Claim 21, having up to 8 esterified carboxylic acid groups per molecule, whenever produced according to the process of Claim 17 or an obvious chemical equivalent thereof.

33. An esterified orgotein of Claim 21, having up to 8 esterified carboxylic acid groups per molecule, wherein the ester groups are alkyl esters of 1 to 4 carbon atoms, whenever produced according to the process of Claim 19 or an obvious chemical equivalent thereof.

34. An-esterified orgotein of Claim 21, having up to 8 esterified carboxylic acid groups per molecule, wherein the ester groups are methyl esters, whenever produced according to the process of Claim 20 or an obvious chemical equivalent thereof.