Conjugates of superoxide dismutase coupled to high molecular weight polyalkylene glycols (US5006333)


	Parent Case:			This is a Continuation-In-Part application of parent application Ser. No. 081,009 filed Aug. 3, 1987 now abandoned. The present invention is directed to superoxide dismutase (SOD) conjugates or adducts in which at least a portion of the SOD amino, carboxyl, or sulfhydryl groups are coupled to a polyalkylene glycol (PAG), such as a polyethylene glycol (PEG) or a polyethylene-polypropylene glycol copolymer, wherein the PAG has a molecular weight greater than 20,000 and desirably an average molecular weight of about 35,000 to 1,000,000 daltons. Unless otherwise indicated, molecular weights for PAG are those determined by high performance, size exclusion liquid chromatography (HPLC) using PEG as standard. Preferred is a PAG with an average molecular weight of about 40,000 or greater but no greater than 200,000 and especially preferred is an average molecular weight for the PAG of about 40,000-150,000. Such PAG can be linear or branched and be unsubstituted or substituted by straight-chained or branched C_1-4 -alkyl groups to form a C_1-4 -alkoxy group. Of special value to prevent linkage to two SOD molecules are PAG molecules in which one terminal group is a C_1-4 -alkyl ether group such as an isopropoxy group. PAG, which is a mixture of terminally substituted and unsubstituted molecules (e.g., partially isopropylated), can also be used. BACKGROUND OF THE INVENTION Previous workers have utilized PEG or methoxy-PEG of low molecular weight (350 to 20,000, and typically about 5,000) attached to superoxide dismutase (SOD) and other proteins to obtain adducts demonstrating varying degrees of (a) increased serum persistence and (b) reduced immunogenicity. However, the extent of modification of protein groups with low molecular weight PEG or methoxy-PEG required to adequately attain both objectives (a) as well as (b), often leads to substantial losses in enzyme activity or biological activity. For example, Pyatak et al. (Res. Commun. Chem. Pathol. Pharmacol. 29, 113-127, 1980) showed that attaching 7 to 18 methoxy-PEG strands (MW 5000, each strand) to SOD produced adducts demonstrating a serum half life of about 25 hours in mice but which retained only 50-60% of the native enzyme activity. Attaching fewer PEG strands of MW 5000 gave adducts of higher enzyme activity but reduced the serum persistence (to about 10 hours.) Yoshimoto et al. (Jpn. J. Cancer Res. 77, 1264-1270, 1986) have attempted to solve the reduced activity problem observed with L-asparaginase, by modifying fewer protein groups using a chloro-triazine reagent substituted with two strands of methoxy-PEG, each strand having a molecular weight in the same range (5000 or less) employed by others. However, cyanuric chloride is toxic to animals and many workers have cautioned against introducing a triazine ring into an adduct used for pharmaceutical purposes, since this chemical moiety would be anticipated to be immunogenic. Yabuki and Iwashita (European Patent Application #86303058.1, filed 4/23/86) substituted PEG for methoxy-PEG, but in the same NW range employed by other workers, to prepare adducts which claim to be composed of SOd copolymers in which both ends of the PEG are attached to different SOD molecules. They report serum half-lives for these adducts of 4 to 8 hours. Other workers, including Lee and Sehon (Int. Archs. Allergy Appl. Immun., 56 159-170, 1978), Lee and Sehon (ibid., 56, 193-206, 1978) and Mueller et al., (ibid, 68, 312-319, 1982 and ibid., 68, 320-325, 1982) had previously used PEG in the same MW range (6,000-20,000) as Yabuki and Iwashita to produce adducts which probably contained similar copolymer structures. Wie et al. (Int. Archs. Allergy Appl. Immun., 64, 84-99, 1981) and Lee et al. (ibid., 64, 100-114, 1981) specifically replaced PEG with methoxy-PEG in the 2000-20,000 MW range to avoid possible inter- and intramolecular linkages DETAILS OF THE INVENTION Superoxide dismutase (SOD) is an intracellular enzyme responsible for catalyzing the conversion of the superoxide radical to oxygen and hydrogen peroxide Native orgotein protein has been shown to possess uniquely high SOD activity. Amongst the fields of application studied for orgotein have been inflammatory conditions such as arthritis and related conditions, cystitis, epicondylitis, tendovaginitis, bursitis, uses in cardiology including reperfusion injury following ischemia associated with myocardial infarction, congestive failure, arteriosclerotic cardiovascular disease, pulmonary conditions, oxygen toxicity, reperfusion injury following ischemia associated with organ transplantation, immune complex disease, dermatological conditions and in avoiding side-effects of radiotherapy. Preferred methods of administration include injection into joint spaces or musculoskeletal junctions. Such administration is commonly preferred to delay systemic inactivation, which occurs especially in the kidneys. It is an object of the invention to provide a product which is more persistent than the native SOD protein in vivo and to delay inactivation by the kidneys. It is especially important that the product retain enzymatic activity for a prolonged time while exhibiting a low level of immunogenicity. It is also a special object of this invention to provide a conjugate with anti-inflammatory activity, as shown, e.g., in the conventional carrageenan-induced paw edema test. This activity makes the product especially promising for treatment of rheumatoid conditions The novel concept embodied in the present invention utilizes high molecular weight PAG strands, greater than 20,000. The PAG used in the present invention can be any water soluble polymer of alkylene oxide, for example, poly(ethylene oxide) or certain copolymers of ethylene oxide and propylene oxide. The PAG can be linear or branched and can be substituted at one or more hydroxyl positions (but not at all terminal hydroxy groups) with a C_1-4 -alkyl group or other chemical group. However, the molecular weight of the PAG polymer used for the preparation of the conjugate in the present invention is greater than 20,000 and preferably in the 35,000 to 200,000 MW range and especially about 40,000 to 150,000. Use of polymers larger than 200,000 is also possible but they are not preferred due to their higher viscosity and susceptibility to cleavage by shearing. The PAG-SOD adducts of the present invention have molecular weights (relative to PEG standards of known molecular weight) ranging from about 40,000 to about 2,000,000 daltons, and preferably about 90,000 to 1,000,000 daltons. Furthermore, the PAG-SOD adducts of the present invention usually retain most of the enzymatic activity of the native protein. (It is to be noted that molecular weights herein are based on PEG standards of known molecular weight. For the purpose of HPLC calibration, the protein equivalent molecular weights, i.e., MW based on protein standards of known MW, appear to be approximately 5 to 8 times larger). The conjugates of the present invention are an improvement over previous products in that by attaching fewer PAG strands, less chemical modification of the active parent molecule results, so that more of the original character of the parent molecule is preserved. Thus, by using fewer strands of high molecular weight PAG, as in the present invention, the adducts retain most, if not substantially all of the activity of the parent molecule, while also demonstrating increased persistence in the bloodstream. Another advantage of the adducts of the present invention is that by using high molecular weight PAG, larger adducts can be made with the same degree of modification attained by other workers. Furthermore, in some applications, larger PAG adducts are clearly advantageous. For example, PEG-SOD adducts of this invention, which were prepared using PEG-strands in the 40,000-130,000 MW range by the methods of this invention and which illustrate the principle of this invention, had serum half-lives in mice of about 36 hours and greater, longer than the PEG-SOD adducts described by previous workers. The PAG-SOD adducts preferably contain from 1 to 5 chains of attached PAG per protein molecule, and more preferably 2 to 4 chains of PAG per molecule. The number of chains needed to achieve satisfactory serum persistence decreases when longer chains are used. The SOD preparations of this invention are typically the mammalian copper- and zinc-containing superoxide dismutase forms of the enzyme derived from bovine, other animal (e.g., sheep, horse, pig, dog, rabbit, chicken) or human cells. Also available are SOD preparations containing other metal ions such as iron or manganese. Also useful is the enzyme with congeneric structures derived from microbial cultures in which such structures have been cloned and expressed. The SOD may also be non-identical to the naturally occurring proteins as a result of infidelity of translation in such microbial cultures, since the products of this invention have reduced immunogenicity. It has also been found that when the SOD preparation contains traces of non-SOD proteins which would otherwise make such preparation immunologically unsafe for repeated parenteral administration, coupling such SOD by the methods of this invention can render the product reasonably useful, since the impurity proteins are rendered substantially less immunogenic. In the coupling process, a number of conventional reactions can be used. A preferred reaction proceeds by way of formation of a reactive carbonate half ester, PAG-O-CO-X, wherein X is a good leaving group, using reagents such as carbonyl diimidazole, p-nitrophenyl chloroformate or bis-N-succinimidyl carbonate. The activated PAG, PAG-0-CO-X, is then reacted with the protein under conditions which do not destroy its enzymatic activity, leading predominantly to urethane linkages PAG--O--CO--NH-protein attached through protein amino- groups such as the epsilon-NH₂ group of lysine. For example, carbonyl diimidazole, can be reacted with the terminal hydroxyl groups of the PAG. The reaction mixture is quenched in aqueous solution at neutral pH and the activated PAG (polyalkylene glycol-carbonyl-imidazole) is isolated by dialysis and/or size exclusion chromatography. Reaction of the [Figure] with SOD is carried out in solution, with an excess of activated PAG. In a variant of this reaction, a solution of SOD and activated PAG is freeze-dried. The coupled products are conveniently isolated by size exclusion chromatography. Other purification processes can be employed including ion exchange chromatography. In an alternate coupling reaction, the polyalkylene glycol is dissolved in an inert organic solvent, the reaction mixture is rendered weakly alkaline and reacted with cyanuric chloride. The unreacted cyanuric chloride is removed by precipitating the PAG with petroleum ether. The residual solvent is evaporated to yield a 2-PAG-6-chloro-1,3,5-triazine. The resulting activated polymers are then reacted with SOD in a suitable buffer, e.g., a borate solution. The unreacted activated PAG is removed and the product isolated by chromatography. There is thus obtained a 4-hydroxy-1,3,5-triazine to which there is attached at the 2-position the polyalkylene glycol group PAG--O-- while at the 6-position there is attachment to the epsilon-amino group of a reactive lysine group of SOD. A terminal hydroxy group of PAG can also be converted to a carboxyl group, e.g., by reaction with succinic anhydride or with ethyl bromoacetate and alkali, or by oxidation of the terminal --OCH₂ CH₂ OH with alkaline permanganate to form the PAG acetic acid ethers, PAG--O--CH₂ --COOH. The carboxyl groups are then activated by methods commonly known to be useful for protein modification, e.g., formation of the N-hydroxy succinimide ester by reaction with a carbodiimide and N-hydroxysuccinimide, or formation of the acyl azide by nitrosation of the acyl hydrazide. The activated PAG is then reacted with the protein under conditions which do not destroy the enzymatic activity of the protein, leading predominantly to amide linkages (PAG--C(=O)NH-protein) via protein amino groups (such as amino terminal NH₂ and lysine epsilon amino groups). A terminal PAG hydroxyl group can also be converted to an amino group, e.g., by reaction first with thionyl bromide to form PAG-Br, followed by aminolysis with excess ammonia to form PAG-NH₂. The amino-PAG can then be coupled through amide bonds directly to the protein carboxyl groups by use of reagents such as water-soluble carbodiimide or Woodward's Reagent K. Alternatively, the amino function can be converted to a carboxylic acid function, e.g., by reaction with succinic anhydride, which is then activated and reacted with the protein in the manner described above. The PAG terminal --CH₂ OH can also be converted to an aldehyde group, --CH(=O) e.g., by oxidation with MnO₂. The aldehyde group can then be reductively alkylated onto the protein via the latter's free amino groups, e.g., with cyanoborohydride, to give a linkage predominantly via secondary amine groups, forming a PAG--OCH₂ CH₂ NH-protein bridge. In addition to protein amino groups, protein carboxyl and sulfhydryl groups can also be used for coupling to the PAG. As stated above, in selecting coupling reactions, those are preferred which leave only non-aromatic groups composed of carbon, oxygen, sulfur, nitrogen and hydrogen as part of the bridge linking the PAG to the protein. The conjugated SOD can be isolated from the reaction solution, preferably after dialysis to remove extraneous ions, by conventional lyophilization. If desired or necessary, the conjugate can be further purified by ion exchange chromatography, electrophoresis, and/or gel filtration. Filtration through a micropore filter in a conventional manner into sterile vials, optionally after adjusting ionic strength, e.g., with sodium chloride and/or sodium phosphate to isotonicity, provides a sterile solution suitable for administration by injection. The pharmaceutical compositions of this invention comprise PAG-SOD conjugates of this invention and a pharmaceutically acceptable carrier. The pharmaceutical composition preferably is in the form of a sterile injection preparation, for example a sterile injectable aqueous solution. The solution can be formulated according to the known art using pharmaceutically acceptable carriers mentioned above. The sterile injectable preparation can also be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent. The compositions of this invention combine an effective unit dosage amount of SOD conjugate at a concentration effective to evoke the desired response when a unit dose of the compositions is administered by the route appropriate for the particular pharmaceutical carrier. For example, liquid compositions usually contain about 0.5 to 40 mg of conjugate protein per 0.25 to 10 ml, preferably about 0.5 to 5 ml, except I.V. infusion solutions, which can also be more dilute, e.g., 0.5 to 200 mg SOD conjugate protein 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 conjugate protein per dosage unit. The SOD conjugates of this invention, like the established product orgotein, are 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. More specifically, the SOD conjugates are efficacious in ameliorating damage to the lungs from oxygen toxicity (e.g., bronchopulmonary dysplasia of the newborn and adult respiratory distress syndrome), reperfusion injury (e.g., following ischemia associated with myocardial infarction, open heart surgery, organ transplantation and shock), and inflammatory conditions and mitigating the effects thereof, for instance those involving the urinary tract and the joints, in various mammals. They are useful in alleviating the symptoms of and the structural deformities associated with post-traumatic arthritis, and rheumatoid diseases, such as bursitis, tendinitis, and osteoarthritis. The invention is further illustrated by the following examples.
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	First Claim: Show all 13 claims			What is claimed is: 1. A an anti-imflammatory water-soluble substantially non-immunogenic conjugate of a superoxide dismutase coupled with a coupling agent to 1 to 5 strands of a polyalkylene glycol which is a polyethylene glycol or polyethylene-polypropylene glycol copolymer residue, wherein all of polyalkylene glycol so coupled has an average molecular weight as determined by HPLC using PEG as standard of about 35,000-200,000 and is unsubstituted or substituted at one terminal by a C_1-4 -alkyl group.
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Buy PDF	Patent	Pub.Date	Inventor	Assignee	Title
	US4179337	1979-12	Davis et al.		Non-immunogenic polypeptides
	US4563349	1986-01	Miyata et al.	Takeda Chemical Industries, Ltd.	Superoxide dismutase, its immobilized form, and their production and use