FI57112B - FOER REFRIGERATION FOR ANTI-FLAMMING EPSILON-N-CARBAMYLORGOTEINER - Google Patents

Description

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Patent- och registerstyralMn '' Anastan utlatd odt utUkrlftM pubNcand 29.02.o0 (32) (33) (31) Requested prefix * —Segird priority 09 · 09 · 75 09-09.75 09.12.75 USA 611657 611659 639076 (71) Diagnostic Data, Inc., 518 Logue Avenue, Mountain View, California 92 + 0 ^ 3, USA (72) Wolfgang Huber, Atherton, California, Mark G. Saifer, Berkeley,

California, Lewis D. Williams, Menlow Park, California, USA (US) (7 * 0 Oy Roister Ab (5 * +) Method for the preparation of anti-inflammatory fc-N-carbamylorgotines - Förfarande för framställning av anti-inflammatoriska fc-N-carbamylorgo The present invention relates to a process for the preparation of anti-inflammatory ε-N-carbanyl orgoteins.

The process of the invention is characterized in that an organotein, such as a bovine organotein, is reacted with a carbamylating agent, such as an alkali metal cyanate, alkyl isocyanate, alkyl isothiocyanate, aryl isocyanate or aryl isothiocyanate, to produce an N-carbamylated oncogene, in one of the g-amino groups of its lysine, and preferably having at least 2 to 10 carboxylated amino groups per molecule.

Orgotein is a water-soluble protein conductor in a substantially pure, injectable form, i. substantially free of other proteins, cf. U.S. Patent 3,758,682, as well as Finnish patents 1 * 8,603, 1 * 8 7M *, 1 * 8,881 *, 1 * 8,932, 1 * 9Π0 and 1 * 9,983.

Orgotein has a characteristic combination of physical, chemical, biological, and pharmacodynamic properties. It is a spherical, buffer- and water-soluble protein compound with a very strong native structure, although 2 57112 is thermally labile, it is quite stable in a buffer solution with a pH of U-10 and lasts for several minutes at 65 ° C. Chemically, an organotein is characterized in that it generally contains all the amino acids of the protein, a small amount of carbohydrate, no lipids, 0.1 to 1.0% metal, i.e. 0 to 5 gram atoms (per mole) of one or more chelated divalent metals having the ion beam is 0.60 to 1.00 Å, and essentially no non-chelated monovalent metal or toxic metal.

The amino acid composition of the organotein derivatives is remarkably uniform regardless of the source from which they are isolated.

Table I shows the amino acid composition of the orgotein.

3,57112

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A3 · Η · Η E · Η f] · Η tj 3., 4 fi EH 'rt E -rl o., 1 EEH Ή O' rl ti E -rl ClJ · rl · Η E · Η · Η O i> , "rl · Η E 41 UI" rl EO g EE -rl Cd 43 i> UI 4l H ω -rl Ή H -rl O Ui O · Η E S3 -¾ • HE lo A w 3 t-, ui O 3 M 43 ti O 'ti E: ·> ti H 43 tl rl il w i' »H rl · ,! 01 E E E tl 41 tl tl>, E A3 '3! No; - · <«t i * i o o U3 h pj r-i: ·: (· 1 Ρι ω tu r ι ι · ι: ·> 4 1- a 57112

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It can be seen from Table I that the orgotein has 18-26 and usually 20-23 lysine clusters, all but 1-3 of which have t-amino groups titrated with trinitrobenzenesulfonic acid. These can be alkylated or carbamylated.

Among other things, the natural organotein has anti-inflammatory activity (see U.S. Patent 3,758,682). It also has very high peroxide dismutase activity (see McCord & Fridovich, J. Biol. Chem., 2 months, 6,176 (1970); ibid, 2U6, 2,875 (1971)) · It is surprising that N-carbamylation has virtually no effect. considering at all the anti-inflammatory activity of the organotein derivative prepared according to the invention. Thus, the organotein derivatives prepared according to the present invention are very useful, e.g., for the treatment of inflammatory conditions in mammals or other animals. A significant portion of the peroxide dismutase activity of natural organotein (20-100% *) is also retained.

N-carbamylated orgotein

As discussed above, orgotein derivatives contain 18-26 lysine groups. Because the orgotein molecule consists of two identical peptide chains (subunits), half of the lysine groups are in each chain, which are strongly (albeit non-covalently) bound to each other over a wide range of temperatures and pH. The ε-arino group of only a few lysines is not titratable with trinitrobenzenesulfonic acid (TNBS); these amino groups cannot be carbamylated with other than very strong carbamylating agents, e.g. dimethyl sulfate in alkaline solution. The degree of carbamylation can be determined using TNBS. In this case, it should be noted that not all amino groups are titratable (e.g., when titrating bovine orgotein or 18 of its 20-22 lysine groups react); in addition, it must be borne in mind that monoalkylated lysines are still acylatable.

The carbamylation of lysine groups can be monitored by calculating the change in charge that occurs in the electrophoresis of the N-carbamylated product. For example, an organotein N-carbamylated with methyl isothiocyanate at pH 9 »showed a change in charge after -2 1 * 5 minutes.

As is known, the electrophoretic mobility of an ion is a function of electric field strength, net ion charge (including bound counterions) and frictional action (see, e.g., C. Tanford, Physical Chemistry of Macromolecules Wiley, New York (1966)), because the coefficient of friction depends on molecular size and However, when comparing proteins of similar size and shape, in this case organotein molecules modified with relatively small groups, under identical electrophoretic conditions, the only variable that affects this electrophoretic mobility is the electrophoretic mobility.

A comparison of the electrophoretic analysis results of several chemically modified orgotein molecules is consistent with this conclusion.

Bovine orgotein moves mainly as a single band (band l); only small amounts of substances move faster (lanes 2, 3, etc.), these represent organotein molecules with a higher ratio of -COOH 2 -NH 4 groups than the majority of organotein molecules (lane 1). Treatment of bovine orgotein with increasing concentrations of acetic anhydride at pH 7 »or methyl isothiocyanate at pH 9 results in the formation of a series of more anodic (migrating towards the anode) electrophoretic bands, as do more free amino acids of the organotein molecules. Correspondingly, treatment with dimethyl sulfate 11a gives a series of strips which have moved towards the cathode.

The curve showing the distance traveled by the tape vs. the number of the tape is relatively linear with small degrees of -COOH or -NHg modification, but begins to deviate from the line with larger degrees of modification. Especially when the ionic strength of the solution is high, the velocity of fast ions slows down considerably. Because extrapolating the location of more than about two bands is not accurate, accurate calculation of the charge requires the use of an internal standard.

All conventional protein modification reactions applied to the organotein molecule to date have been consistent with this interpretation, namely, that the positions of the bands correspond to independent charge changes in the natural organotein molecule. Kerb Amy creation and N-methylocarbamylation give bands 2,3,1 +, 5, etc., indicating that 1,2,3 and U free amino groups are chemically modified in the same order. Similarly, succinylation, which converts · ~ ΝΗ® groups to / ^NHCCH.CH groups, gives bands 3,5,7,9, etc., and broader J \\ dd 0 carbamylation or thiocarbamylation, which further converts ^ NHC- NH-R and

II

S o tl ^ NHC-NH-R groups, gives strips 6,7,8,9 etc.

Only the 2-U lysine groups are those that cannot be carbanylated with mild alkylating agents. Only approximately one of the lysine groups titrated with TNBS in each peptide subunit of the organotein is one that cannot be polycarbamylated using more stringent conditions.

When not all of the titratable lysine amino groups are carbamylated, the distribution of the alkyl moieties of the carbamyl groups in the orgotein molecule is likely to be random, as none of the titratable lysine amino groups turn out to be 6,571,122 abnormally easily converted carbamylated amino groups. Because the orgotein molecule consists of two identical peptide chains, the N-carbanylated amino groups of the partially carbamylated orgotein are more or less randomly distributed along each peptide subunit, but more or less evenly distributed between the two chains. Since only one carbaoylating agent is usually used, the carbamylated amino groups are all identical. However, it is possible to prepare carbamylated orgoteins having two or more different alkyl groups in the molecule and even in each of its chains.

One way to prepare a mixed N-carbamylated orgotein is to step carbamylate with different carbamylating agents. For example, some of the titratable f-amino groups of lysine may be carbamylated with a moderately reactive carbamylating agent, e.g., KNCO, and the remainder of the reactive amino groups may be carbamylated with a more reactive carbamylating agent, e.g., a methyl isocyanate. The reactivity of the carbamylating agent depends on the relative reaction rates with the amino groups of the protein and the reaction solvent, and thus depends on the pH of the reaction and the carbamylating agent and, to a lesser extent, the buffer and temperature.

The t-N-carbaoyl organoteins prepared in accordance with this invention appear to have substantially the same properties as the natural organotein molecule. The chelated Cu and Zn content (gram atoms / mole) is approximately the same as that of the organotein. Like orgoteins, they are highly resistant to pronase and other proteolytic enzyme degradation is required. The enzymatic activity of peroxide dismutase (SOD) decreases with respect to the degree of carbamylation.

Although the predominant structural change in the natural organotein that occurs in its carbanylation at alkaline pH is the alteration of the ε-amino groups of its lysines, other active groups in the molecule, especially -OH and imidateol nitrogen and possibly also guanidino nitrogen, -SH, -SCH 2, etc. can also simultaneously carbamylated, depending on the conditions used and the reactivity of the carbanylating agent.

The exact nature of the N-carbamyl groups, as well as their number, is not critical insofar as the carbamyl radical is physiologically acceptable. Due to the higher molecular weight of the organotein molecule, even when the orgotein molecule is fully carbamylated with carbamyl groups having a reasonable molecular weight, e.g. ssj.00, the effect on the overall chemical composition is relatively small, i. less than 10%. Carbamylation also has no apparent significant effect on the dense spatial structure of the molecule or the stability obtained, e.g. when heated to 60 ° C for one hour, or on proteolytic enzymes.

It will be appreciated that the carbamyl group must also be derived from a carbamylating agent capable of carbamylating the amino group in water or a buffer solution, as the reaction is usually carried out therein.

τ 57112

Examples of suitable carbamylating agents are alkali metal cyanates, e.g. NaNCO, KNCO; alkyl isocyanates and alkyl isothiocyanates, e.g. RNCO and RNCS, where R = CH 2> C 9 H 7, n_C 3 H 7 * n-cl; H 9 »n_G8H17» and aryl isocyanates and isothiocyanates, e.g. phenyl isocyanate and phenyl isothiocyanate and phenyl isothiocyanate.

Although metal cyanates react with many types of side chains, only the reaction with amino groups that converts them to urea groups produces a stable product.

The reaction of alkyl isocyanates and isothiocyanates with orgotein is completely analogous to the reaction between it and cyanates, but much faster. Long chain isocyanates are less reactive than short chain isocyanates.

The reaction of the organotein with isocyanates and isothiocyanates can be detected using a reagent that introduces a fluorescent group into the molecule, e.g., fluorescein isocyanate. This reagent is similar to alkyl isocyanates and reacts with the amino groups of the organotein to produce substituted thioureas. However, even a small amount of substitution significantly reduces the SOD activity of the organotein due to the magnitude of the fluororescein. Mono- and di-substituted orgoteins are not stable in solution and slowly become more heterogeneous due to subunit exchange and hydrolysis.

Carbamylated orgoteins according to the present invention are orgotein derivatives, including bovine, ovine, equine, porcine, rat, canine, rabbit, guinea pig, chicken and human orgotins having at least one of the titratable amino groups, e.g. 1,2,3,5 and even all (about 18-26) are carbamylated, i. contains an unsubstituted or substituted -CONH or -CCSNH group.

In a preferred embodiment, the carbanylated amino groups are those having the formula:

X

-NHCNHR

wherein X is O or S and R is CH2, C8H2, nC2H2, iso-C2H2, n-C8H2 or another alkyl group having up to 12 carbon atoms, Ph or, when X is 0, also a hydrogen atom and Ph is an unsubstituted phenyl group or a phenyl group having 1 to 3 simple substituents, e.g. a methyl, chlorine, bromine, nitro, amido and methoxy group, a carbomethoxy or carboethoxy group, e.g. p-tolyl, sym.- xylyl, p-amidophenyl, m-chlorophenyl and p-methoxyphenyl. Such orgoteins have the formula:

X

(HN) -Org- (NHCNH-R) not m. N 8 57112 wherein n is an integer from 1 to about 26, preferably at least 2 and most preferably from about 6 to 10 and the sum of m + n is the total number of free amino groups to be titrated in the unmodified derivative, X is 0 or S, R is as defined above, preferably an H group or an alkyl group having 1 to 8 carbon atoms, and "Org" is the remainder of the organotein molecule.

Particularly preferred carbanylated orgoteins are alkylcarbamyl and alkylthiocarbanyl orgoteins in which the alkyl group is an unsubstituted alkyl group having 1 to 8 carbon atoms, e.g. a methyl, ethyl, propyl, isopropyl, butyl and octyl group.

Since the exact chemical nature of the carbamyl group is not critical, insofar as it is not a physiologically toxic organotein molecule and can be formed on the ε-amino groups of lysine, conceivable substitutes for the preferred alkylcarbamyl orcoteins described above, insofar as they are similar to those above, are contemplated. R is a cycloalkyl, cyclohexyl, menthyl and the like cycloalkyl group, cyclohexylmethyl-cyclopentylpropyl and the like cycloalkylalkyl group, benzyl, p-xylyl and phenyl and the like aralkyl group. Also substitutable are those in which R is an alkyl group having 1 to 6 and preferably 1 to U carbon atoms, and most preferably a methyl or ethyl group having one or more, preferably one other substituent, e.g. a fluoresinyl group.

In addition to the N-carbanylated "bovine" orcotein of the Examples below, other examples of the N-carbamyl-bovine orgotins of this invention include the corresponding N-carbamyl orcotein, N-propylcarbamyl orcotein, N-ethylcarbamyl orcotein, and N-methylthiocarbamyl orcotein, each having 9 such carbamyl groups. in each of the two orgotein molecules in the subunit, and corresponding orgoteins having an average of 1, 6 or 10 such carbamyl groups in each such subunit and corresponding to each of these human, sheep, equine, porcine, canine, rabbit, guinea pig, chicken and rottaorgoteiini derivatives.

Orgotein derivatives can be isolated from the reaction solution, preferably after dialysis to remove foreign ions, by conventional lyophilization, e.g., as described in U.S. Patent 3,658,682. If desired, the orgotein derivative can first be purified by ion exchange resin chromatography, electrophoresis and / or gel filtration using a polymer that acts as a molecular sieve.

Filtration through a microporous filter with a pore size of 0.01 to 0.22 microns in aseptically sterile vials, optionally after adjusting the ionic strength with NaCl and / or sodium phosphate, e.g. to an isotonic 9,57112 point, provides bacteriophoric or virological control. a solution suitable for injection. Filtration through a 0.1 micron pore filter also reduces or eliminates pyrogens in solution.

Example 1

Unsubstituted carbamylated orgotein

A solution of 3.7 mg of orgotein (bovine) and 10 mg of KNCO in 2 ml of 0.025 M sodium phosphate buffer pH 7.5 was kept at U ° C. Electrophoresis of the subsamples over 5 days showed the appearance of a series of SOD-active bands that were more anodic than the natural organotein. The average change in charge along with the area of the active bands on either side of the mean is shown in the samples below.

Time (days): 0.75 3.8 27 51 *

Reacted lysines 0 0.8 5 + 2 8.5 + 3.5

Example 2

Alkyl carboxylated orgoteins Organotein (from bovine) in 50 silicon fractions in 0.1 M tris or phosphate buffer pH 7.6 at 10 mg / ml was reacted at 1 ° C with 1 silicon in either propyl isocyanate or octyl isocyanate in different periods of time. In two cases, 2 more propyl isocyanate was added after two days. The number of propylcarbamyl and octylcarbamyl groups attached was determined by the change in average charge as determined by electrophoresis as shown below.

Average booking change_

Reagent 2 min. 2 h. 1 day 2 days + 2 f * xsosya- __ _ _ _ __ _ naat______

Propyl isocyanate / tris 8 8 ND x) ND 10

Propyl isocyanate / phosphate 8 8 ND ND> 15

Octyl isocyanate / tris 0.2 1.0 6 6

Octyl isocyanate / phosphate 0 0.2 2.5 3 x) Not done

Example 3

Alkylcarbamylated orgoteins

The procedure of Example 2 was repeated using 10 mg of orgotein in 1 ml of 0.1 M phosphate buffer pH 7.6 and 1 ml of propyl or octyl isocyanate. The solutions were kept at 25 ° C for U hours and examined electrophoretically. An additional 1 ml of isocyanate was then added to each. After standing overnight at H ° C, the solutions were again examined electrophoretically and dialyzed. Carbangrated proteins were less soluble in deionized ίο 57112 water and partially precipitated on dialysis, but were soluble in 0.15 ~ saline. The number of carbamylated lysine groups is shown below.

GAPM

Isocyanate Reacted lysines Me ££ Lipid content% of initial K h overnight Cu Zn SOD activity

Propyl 10 + U> 15 2.2 1.5 approximately 50%

Octyl 3 + .3 6 + 5 2.1 1.8 approximately 25%

Example U

N-metyylitiokarbamyyliorgoteiini

To a solution of 5 mg of organotein in U ml of 0.075 M Na 2 B 2 O 2 at pH 9 was added 10 μl of CH 2 Cl 2 and the mixture was shaken for one minute until the CH 2 NCS dissolved. At intervals, 50 μl samples were removed and 0.1 ml of 0.1 M sodium acetate buffer pH 5.3 was added. A white sulfur precipitate (indicated by its odor on combustion and its solubility in CS2) appeared between 6 and 22 hours. After 22 hours at room temperature, the remaining reaction mixture was treated with 0.5 ml of 1-M acetate buffer pH and dialyzed against several changes of water for 2 days. The dialyzed solution was centrifuged briefly to remove the white turbidity of the solution.

Electrophoresis of samples taken from the solution increasingly showed the formation of a series of more anodic SOD active bands. Analysis of the samples by the cytochrome c assay at pH 7-8 (McCord & Fridovich, J. Biol. Chem. 60U9-6055 (1969)) showed a decrease in SOD activity with greater organ protein modification.

Time (h) Average SOD activity% change in charge o (0) (100%) 0.08 0.5 Hl 0.25 1 118 0.75 2 91 1.9 U 86 6 9.5 ^ 3 22 16 20

The reaction product, dialyzed for 22 hours, electrophoresed as a fast-moving anodic band, retained 18% of the SOD activity of the native organotein protein, and contained 2.06 GAPM Zn ** and 1.76 GAPM Cu ** (compared to 2.26 in the unmodified organotein). GAPM Zn and 2.08 Cu). All + Φ ft products are soluble, retaining coils of fused Cu and Zn and at least a portion of the unmodified protein peroxide disructase activity.

11 5711 2

Example 5 N-fluoresynylcarbamyl orcotein

A solution of 100 mg of orgotein (bovine) and 6 mg of fluorescein isothiocyanate in 10 ml of 0.1U-M phosphate buffer pH 8.5 was kept at U ° C for 18 hours. The reaction mixture was applied to a chromatography column containing microporous crosslinked dextran (Sephadex G-50, Pharmacia, Uppsala, Sweden) and eluted with borate buffered saline (0.15 μM) pH 8. The eluted fractions of the yellow protein were dialyzed against water. The dialyzed protein that precipitated (20 mg) was strongly yellow and fluorescent and was readily soluble in borate-buffered saline at pH 8. Electrophoresis showed that 96 mg of soluble protein was approximately half monocarbamylated orgotein (-3 charge change) with some double and triple carbamylated orgotein (-6 and -9 charge change) and unreacted orgotein. The reconstituted precipitate turns out to be a more strongly modified protein.

The soluble protein was chromatographed on a weakly basic (DEAE-cellulose) ion exchange column at pH 6 using a linear gradient of phosphate buffer from 0.01 to 0.2 M. Monocarbanylated and dicarbanylated orgotein was isolated, dialyzed, and lyophilized.

The monocarbanylated orgotein was SOD active in electrophoresis and NBT-riboflavin stained according to the pH 7 * 5 cytochrome c assay showed its SOD activity to be H8% of the native organotein. In the Ungar bioassay, this protein showed approximately 50% of the activity of a natural organotein.

The monocarbamylated orgotein solution stored at U ° C for 2 weeks was converted to a mixture of orgotein, monocarbamylated orgotein and dicarbamylated orgotein (apparently the result of hybridization) and some non-proteinaceous hydrolyzate, apparently carbamylated gives free aminofluorescein).