Peg-urate oxidase conjugates, their application, method of tetrameric uricase form separation

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns biopharmaceutics and PEG conjugates of natural or recombinant urate oxidase (uricase). Uricase is bound covalently to poly(ethylene glycol) or poly(ethylene oxide) (both denoted as PEG), with average of 2 to 10 PEG threads are conjugated with each uricase sub-unit, and average molecular weight of PEG is approximately between 5 kDa and 100 kDa.

EFFECT: obtaining almost non-immunogenic PEG uricase conjugates preserving at least 75% of uricolytic activity of non-modified enzyme.

44 cl, 12 ex, 17 dwg

 

The scope of the invention

The present invention relates to chemical modification of proteins to prolong their circulation and reduce their immunogenicity. More specifically, the invention relates to the conjugation of poly(etilenglikola) or poly(ethylenoxide) urotoxicity, as well as to the process of isolating tetramer form uricase from the solution uricase and isolated tetramer uricase, which virtually eliminates the immunogenicity of urotoxicity without compromising its activity in the decomposition of uric acid.

The existing level of technology

Known urotoxicity (uricase; Y.S. 1.7.3.3) are enzymes that catalyze the oxidation of uric acid to a more soluble product, allantoin, purine metabolite that is best allocated. People do not produce the enzyme active uricase as a result of several mutations in the gene for uricase that occurred during the evolution of higher primates. Wu X, et al. (1992) J Mol Evol 34:78-84. Consequently, in susceptible individuals an excessive concentration of uric acid in the blood (hyperuricemia) and in urine (hyperuricuria) can lead to painful arthritis (gout), damage buratovich sediments (gouty nodes) and kidney failure. Some patients available medications such as allopurinol (an inhibitor of the synthesis of uric acid), give the bounding treatment reverse eff the points or not treated state data adequately. Hande, KR, et al. (1984) Am J Med 76:47-56; Fam, AG (1990) Bailliere''s Clin Rheumatol 4:177-192. Injection uricase can reduce hyperuricemia and hyperuricuria, at least temporarily. Because uricase for man is alien protein, even the first injection of unmodified protein from Aspergillus flavus caused anaphylactic reaction in a certain percentage of patients (Pui C-H, et al. (1997) Leukemia 11:1813-1816), and immunological reactions limit its usefulness for chronic or recurrent treatment. Don-adio, D, et al. (1981) Nouv Presse Med 10:711-712; Leaustic, M, et al. (1983) Rev Rhum Mal Osteoartic 50:553-554.

Partially optimal action available treatments hyperuricemia has been known for several decades. Kissel, P, et al. (1968) Nature 217:72-74. Similarly, the possibility that certain groups of patients with severe gout can benefit from safe and effective form of injectable uricase known for many years. Davis, FF, et al. (1978) in GB Broun et al. (ed) Enzyme Engineering, Vol.4 (str-173) New York, Plenum Press; Nishimura, H, et al. (1979) Enzyme 24:261-264; Nishimura, H, et al. (1981) Enzyme 26:49-53; Davis, S, et al. (1981) Lancet 2(8241):281-283; Abuchowski, A, et al. (1981) J Pharmacol Exp Ther 219:352-354; Chen, RH-L, et al. (1981) Biochim Biophys Acta 660:293-298; Chua, CC, et al. (1988) Ann Int Med 109:114-117; Greenberg, ML, et al. (1989) Anal Biochem 176:290-293. Uricase derived from animal organs, almost insoluble in solvents that are compatible with the safe introduction by injection. U.S. patent No. 3616231. Certain uricase derived from plant sludge is from microorganisms, more soluble in medically acceptable solvents. However, injection of microbial enzymes quickly causes an immunological reaction, which can lead to life-threatening allergic reactions or to inactivation and/or accelerated elimination of uricase from circulation. Donadio, et al. (1981); Leaustic, et al. (1983). Enzymes, based on deduced amino acid sequences uricase mammals, including pigs and baboons, or insects, such as Drosophila melanogaster or Drosophila pseudoobscura (Wallrath, LL, et al. (1990) Moll Cell Biol 10:5114-5127), were not suitable candidates for clinical use because of the problems of immunogenicity and solubility at physiological pH.

Covalent modification of proteins with poly(ethylene glycol) or poly(ethylene oxide) (both referred to as PEG) was used to increase the half-life of the protein and reduce immunogenicity. U.S. patent No. 4179337, 4766106 and 4847325; Saifer, MGP, et al. (1994) Adv Exp Med Biol 366:377-387. Linking PEG high molecular weight to obtain conjugates with prolonged circulation time and/or reduced immunogenicity while retaining functional activity was previously demonstrated for another enzyme called superoxide dismutase (Somack, R, et al. (1991) Free Rad Res Commun 12-13:553-562; U.S. Patent No. 5283317 and 5468478) and for other types of proteins, such as cytokines (Saifer, MGP, et al. (1997) Polym Preprints 38:576-577; Sherman, MR, et al. (1997) in M Harris, et al. (EDS), Poly(ethylene glicol) Chemistry and Biological Applications. ACS Symposium Series 680 (page 155-169) Washington, DC: American Chemical Society). The conjugates of uricase with polymers other than PEG, is also described. U.S. patent No. 4460683.

Almost all well-known and the above-mentioned attempts PEG-reroute uricase (i.e. covalently linked PEG with uricase) primary PEG was coupled to the amino groups containing aminoterminal balance and available lysine residues. In the common orikaso the total number of lysine in each of the four identical pojedinici is in the range between 25 (Aspergillus flavus (U.S. Patent No. 5382518)) and 29 (pig (Wu X, et al. (1989) Proc Nati Acad Sci USA 86:9412-9416)). Some of lysine unavailable for PEG-helirovanie inherent in the enzyme conformation. The most common approach to reducing the immunogenicity of uricase was linking a large number of strands of PEG with a low molecular weight. It consistently led to large decreases in enzyme activity of the resulting conjugates.

Previous researchers have used injected to uricase to catalyze the transition of uric acid to allantoin in vivo. Cm. Pui, et al. (1997). This is the basis for use in France and Italy uricase of the fungus Aspergillus flavus (Uricozyme®)to prevent or temporarily adjust the hyperuricemia associated with cytotoxic therapy of hematological disorders, and to temporarily reduce severe Hyper is recebeu in patients with gout. Potaux L, et al. (1975) Nouv Presse Med 4: 1109-1112; Legoux, R, et al. (1992) J Biol Chem 267:8565-8570; U.S. Patent No. 5382518 and 5541098. Due to its short life cycle circulation Uricozyme® requires daily injections. Moreover, it is not very well suited for long-term therapy because of its immunogenicity.

One intravenous injection of the drug uricase from Candida utilis associated with 5 kDa PEG reduced lithate serum to fixed levels of five people, with an average concentration of lithate serum before injection were 6.2 mg/DL, which is within normal limits. Davis, et al. (1981). Subjects were given an additional injection four weeks later, but their reaction was not witnessed. After the second (and last) injection was not detected antibodies to uricase when using a relatively insensitive test gel diffusion. This source does not contain information about chronic or sub-chronic treatment of patients-humans or experimental animals.

Drug uricase from Arthrobacter protoformiae associated with 5 kDa PEG was used for temporary control of hyperuricemia in one patient with lymphoma, in which the concentration of lithate serum before injection was 15 mg/DL. Chua, et al. (1988). Because of the critical condition of the patient and the short duration of treatment (four injections for 14 days) were not evaluated long-term efficacy or safety is the ability of this conjugate.

In this application, the term "immunogenicity" refers to the initiation of immune responses injected drug PEG-modified or unmodified uricase (antigen), and "antigenicity" refers to the reaction of antigen with pre-existing antibodies. Antigenicity and immunogenicity jointly referred to as "immunoreactivity". Previous studies of PEG-uricase immunoreactivity was determined in various ways, including: 1) reaction in vitro PEG-uricase with the previously formed antibodies; 2) measurement of the induced synthesis of antibodies, and 3) increased velocity of blood purification after re-injection.

Previous attempts to eliminate the immunogenicity of uricase according to various sources by linking different amounts of threads PEG through various linkers have had limited success. PEG-uricase first considered FF Davis and Y Inada and their colleagues. Davis, et al. (1978); U.S. Patent No. 4179337; Nishimura, et al. (1979); the Japan Patent No. 55-99189 and 62-55079. The conjugate disclosed in patent '337, was synthesized by reacting uricase from unspecified source with a 2000-fold molar excess of PEG weighing 750 daltons, which shows that a large number of polymer molecules is more likely connected with each polyadenine uricase. Patent '337 discloses the binding of either PEG or poly(propylene glycol) with molecular weights from 50 to 20000 daltons, preferably from 500 to 5000 daltons, to get active, water-soluble, non-immunogenic conjugates of various polypeptide hormones and enzymes containing oxidoreductase, one of the three examples is uricase. In addition, the patent '337 emphasizes linking from 10 to 100 strands of the polymer with each enzyme molecule and save at least 40% of the enzyme activity. However, this was not reported test results on the amount of binding of the PEG with the available amino groups of uricase, the residual specific uricolytic activity or immunoreactivity of the conjugate.

Data from 13 cited sources relative to PEG-helirovanie uricase collected in Table 1. Some of these results are also presented graphically in figa-2B. Seven of these publications describe a significant reduction in uricolytic activity, measured in vitro, caused by linking different number of strands of PEG with uricase from Candida utilis. Linking a large number of strands of PEG weight of 5 kDa with uricase pork liver gave similar results, as described in the publication Chen, and made by the same group of authors report on the Symposium. Chen, et al. (1981); Davis, et al. (1978).

Among the studies collected in Table 1, the reduction of the immunoreactivity uricase noted in seven of them, and the elimination of immunoreactivity is is five. In three of the last five research elimination of immunoreactivity was associated with significant reduction uricolytic activity up to at most 15, 28, or 45% of the initial activity. Nishimura, et al. (1979) (15% activity); Chen, et al., (1981) (28% activity); Nishimura et al. (1981) (45% activity). In the fourth report, it was reported that PEG was associated with 61% of the available lysine residues, but was not installed residual specific activity. Abuchowski et al. (1981). However, the team of researchers, which contained two of these same scientists have used the same techniques in another source reported that the amount of binding was left only 23-28% residual activity. Chen et al. (1981). Publication 1981 Abuchowski et al. and Chen et al. show that for a significant reduction in the immunogenicity of uricase PEG should contact with approximately 60% of the available lysine residues (table 1). The fifth article, which reported the elimination of immunoreactivity in uricase, does not disclose the amount of binding of the PEG, the residual uricolytic activity or the nature of the PEG-protein. Veronese FM, et al. (1997) in Harris JM, et al. (EDS), Poly(ethylene glycol) Chemistry and Biological Applications. ACS Symposium Series 680 (str-192) Washington, DC: American Chemical Society.

The conjugation of PEG to a lower part of the lysine residues in uricase reduces but does not eliminate them immunoreactivity in experimental animals. Tsuji, J, et al. (1985) Int J Immunopharmacol 7:725-730 (28-45% SV is related amino group); Yasuda, Y, et al. (1990) Chem Pharm Bull 38:2053-2056 (38% related amino groups). Residual uricolytic activity of the respective additives are in the range of from <33% (Tsuji, et al.) up to 60% (Yasuda, et al.) from their initial values. Tsuji, et al. synthesized conjugates of PEG-uricase with PEG weighing 7.5-10 kDa in addition to the PEG with a weight of 5 kDa. All of the resulting conjugates were several immunogenic and antigenic, at the same time showing markedly reduced enzyme activity (table 1; figa-1B).

It was reported that PEG-pilirovanny drug uricase from Candida utilis, which safely twice was administered to each of five people, kept only 11% of its initial activity. Davis, et al., (1981). Several years later, a PEG-modified uricase from Arthrobacter protoformiae four was administered to one patient with lymphoma and severe hyperuricemia. Chua, et al. (1988). Although the residual activity of this enzyme preparation was not measured, Chua, et al. demonstrated a lack of antibritish antibodies in the serum of the patient 26 days after the first injection of PEG-uricase using enzyme-linked immunosorbent assay (ELISA).

As shown in Table 1, previous studies of PEG-pilirovanny uricase show that the catalytic activity is markedly inhibited by binding of a significant number of strands of PEG to a significant loss of immunoreactivity. Moreover, Naib is more early preparations of PEG-uricase was synthesized using PEG, activated using cyanoindole chloride, a derivative of triazine (2,4,6-trichloro-1,3,5-triazine), which, as we have seen, introduces new antigenic determinants and causes the formation of antibodies in rabbits. Tsuji, et al. (1985).

Japanese patent No. 3-148298 issued A Sano et al., discloses modified proteins, including uricase, derivateservlet with PEG having a molecular weight from 1 to 12 kDa, which show reduced antigenicity and improved extended action, and methods of obtaining such derivatizing peptides. However, not disclosed the number of threads, enzyme tests, biological tests and the value of the expression "improved extended". Japanese patent No. 55-99189 and 62-55079, both issued by the Inada Y, disclose conjugates uricase obtained using PEG-triazine or bis-REO-triazine (marked PEG2 in Table 1), respectively. Cm. Nishimura et al. (1979 and 1981). In the conjugate of the first type of molecular weight of PEG was 2 and 5 kDa, while in the conjugate of the second type was used only PEG with a weight of 5 kDa. Nishimura et al. (1979) reported the restoration of 15% uricolytic activity after modification 43% of the available lysine using a linear PEG with a weight of 5 kDa, while Nishimura et al. (1981) reported the restoration 31 or 45% urealyticum AK is Yunosti after modification 46 or 36% lysine by PEG2.

The invention

Analysis of prior art shows that when there is a significant reduction in immunogenicity and/or antigenicity of uricase by PEG-helirovanie, it is necessarily associated with a significant loss uricolytic activity. Security, convenience and cost effectiveness of biomedicale have the opposite effect by reducing the capacity of biomedicale and resulting from this the need to increase the input dose. Thus there is a need for a safe and effective alternative tool for reducing elevated levels of uric acid in the body fluids, including blood and urine. One of such means in accordance with the present invention can be practically non-immunogenic PEG-uricase, which retains all or almost all of uricolytic activity of the unmodified enzyme.

Conjugate urotoxicity (uricase) retains at least 75% uricolytic activity of the unconjugated uricase and has significantly reduced immunogenicity. In accordance with the invention provides purified of uricase, in which each polyadenine can covalently to contact in average from 2 to 10 strands of PEG, which can be linear or branched, and each molecule of PEG may have a molecular weight Ciampino international airport is approximately 5 and 100 kDa. Uricase can also be recombinant. Regardless of recombinantly uricase may be a mammal origin. I.e. uricase can be uricase porcine, bovine or Ovine liver. Uricase may be chimerical. Chimerical uricase may contain portions of uricase pork liver and/or liver baboon. For example, the chimerical uricase can be pig-Babuino chimerical uricase (SBH-uricase) or pork uricase containing mutations R291K and T301S (C-KS-uricase) (see sequence figure 6 and the results of physiological and immunological studies 7-12). Alternative uricase can be uricase liver baboon, in which the tyrosine 97 replaced by histidine, due to which the specific activity of uricase can be increased by at least about 60%. Uricase, regardless of origin, may also be a truncated form either from the amino terminal, or from carboxylato terminal or at both terminals. Similarly uricase can be fungal or microbial uricase. Fungal or microbial uricase can be naturally occurring or recombinant form uricase from Aspergillus flavus, Arthrobacter globiformis, or Candida utilis. Alternatively, uricase can be uricase bespozvonochnykh, such as, for example, naturally occurring or recombinant form of uricase the C Drosophila melanogaster or Drosophila pseudoobscura. Uricase can also be vegetable arikati, for example naturally occurring or recombinant form uricase from root nodule of soybean (Glycine max). PEG may have an average molecular weight between about 5 and 100 kDa; preferably PEG may have an average molecular weight of between approximately 10 and 60 kDa; more preferably, the PEG may have an average molecular weight of between approximately 20 and 40 kDa, for example of 30 kDa. The average number of covalently bound threads PEG can range from 2 to 10 threads per polyadenine uricase; preferably the average number of covalently bound threads can be from 3 to 8 on polyadenine; more preferably the average number of strands of PEG can be from 4 to 6 on polyadenine. Uricase can be a tetramer. Strands of PEG can be covalently linked to uricase through urethane (urethane) linkage, a secondary amine communication and/or amide linkages. If uricase is a recombinant form of any implied here uricase, this recombinant form may have substantially the same sequence as a naturally occurring form.

PEG-uricase can be purified uricase of two or more of pojedinici, in which each polyadenine can be covalently linked to an average of 2 to 10 filaments linear or branched PEG, each PEG molecule may and the geta a molecular weight of between approximately 5 and 100 kDa in a pharmaceutically acceptable carrier. Mammal may be a human. The step of introducing can be, for example, intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal injection or inhalation of a drug in aerosol form. Elevated levels of uric acid can exist in the blood, urine and/or other fluids and tissues of the body and can be associated with gout, gouty nodes, renal failure, organ transplantation, or malignant disease.

The essence of the present invention to provide the above biomedicale, as well as the allocation method tetramer form uricase from a solution that contains multiple forms uricase. Initially, the solution may contain the tetramer uricase and collectively uricase. The method can include the following steps: applying the solution to at least one separation column at pH between about 9 and 10.5, for example 10,2; recovery fractions of the eluate and identify those that may contain isolated tetramer to uricase, and faction practically free from complexes uricase; and combining fractions isolated tetramer uricase. The separation column can be based on ion exchange, exclusion or any other effective separation property. The method may also include analysis of the fractions to determine at what ice the tetramer uricase and/or absence of complexes uricase. For example, such analysis may include high-precision liquid chromatography (VTECH), other chromatographic methods, light scattering, centrifugation and/or electrophoresis. In one aspect of this implementation purified tetramer of uricase may contain less than 10% of the complexes uricase.

Brief description of drawings

Figa shows the saving activity of PEG-pilirovanny uricase from Candida utilis as a function of the number of strands of PEG-related polyadenine.

Figb shows the saving activity of PEG-pilirovanny uricase from Candida utilis as a function of the total mass of PEG associated with polyadenine.

Figa shows the saving activity of PEG-pilirovanny uricase from pig liver as a function of the number of strands of PEG-related polyadenine.

Figb shows the saving activity of PEG-pilirovanny uricase from pig liver as a function of the total mass of PEG associated with polyadenine.

Figa shows the saving activity of PEG-pilirovanny pig Babuino chimerical (SBH) uricase a function of the number of threads associated with polyadenine.

Figb shows the saving activity of PEG-pilirovanny SBH-uricase a function of the total mass of the PEG associated with polyadenine.

Figa shows the saving activity of PEG-pilirovanny uricase of Aspergillus flavus as a function of the number of strands of PEG associated with what thedenial.

Figb shows the saving activity of PEG-pilirovanny uricase of Aspergillus flavus as a function of the total mass of PEG associated with polyadenine.

Figa shows the saving activity of PEG-pilirovanny recombinant uricase nodules of soybean root as a function of the number of strands of PEG-related polyadenine.

Figb shows the saving activity of PEG-pilirovanny recombinant uricase nodules of soybean root as a function of the total mass of PEG associated with polyadenine.

6 shows the deduced amino acid sequence of pig Babuino the chimerical uricase (SBH-uricase), SPH-uricase, which is truncated from aminoterminal, and carboxylato terminal (SBH-NT-CT), and pork uricase containing mutations R291K and T301S (CKS-uricase), in comparison with the sequences of the pig and baboon.

Fig.7 shows the activity uricase in the serum of mice 24 hours after each of the four or five intraperitoneal injections of PEG-modified SBH-uricase compared with the value obtained 24 hours after the first injection.

Fig shows the inverse relationship between the activity injected PEG-modified SBH-uricase serum pricestability mouse and concentrations of uric acid in serum and urine.

Fig.9 shows the reduction of the severity of the defect concentration of urine from casedefinition (uox-/-) mice, treated with PEG-modified SBH-uricase.

Figure 10 shows the decrease of gravity nephrogenic diabetes insipidus in pricestability (uox-/-) mice that were treated with PEG-modified SBH-uricase.

11 shows the decrease of gravity caused by uric acid nephropathy, according to the testimony of magnetic resonance microscopy, pricestability (uox-/-) mice that were treated with PEG-modified SBH-uricase.

Fig shows rapid purification from blood circulation mouse BALB/c mice injected the octamer SBH-uricase compared with terramara, and they were both associated with 5-6 threads PEG weight of 10 kDa to polyadenine.

A detailed description of the preferred executions

The present invention provides improved conjugates of water-soluble polymers, preferably poly(etilenglikola) or poly(ethylenoxide)urakasumi. These conjugates almost neimenovani and retain at least 75%, preferably 85% or more, preferably 95% or more uricolytic activity of the unmodified enzyme. Uricase suitable for conjugation with water-soluble polymers include naturally occurring of urotoxicity secreted from bacteria, fungi and tissues of plants and animals, both vertebrates and invertebrates, as well as a recombinant form of URI is the basics, including mutant, hybrid and/or truncated enzyme-active variants uricase. Water soluble polymers suitable for use in the present invention include linear or branched poly(etilenglikoli) or poly(ethylenoxide), commonly known as PEG. An example of a forked PEG is the object of U.S. patent No. 5643575. One of the preferred examples of linear PEG is monomethoxy PEG to the General structure of CH3O-(CH2CH2O)nH, where n varies from 100 to 2300.

One of the preferred uricase mammals is recombinant pig Babayeva the chimerical uricase, composed of parts of sequences uricase pork liver and liver baboon, both of which were first defined by Wu et al. (1989). One example of such chimerical uricase contains the first 225 amino acids of the sequence pork uricase (SEQUENCE ID No. 1), and the last 79 amino acids from the sequence uricase baboon (SEQUENCE ID No. 2) (pig Babayeva uricase, or SBH-uricase; see Fig.6). Another example of such a chimerical uricase contains the remains of 7-225 pork sequence (SEQUENCE ID No. 1) and the remains of 226-301 sequence of baboon (SEQUENCE ID No. 2); this is equivalent SBH-uricase, truncated is both aminoterminal, and carboxylato terminal (SBH-NT-CT; see Fig.6). Another example of such a chimerical uricase contains the first 288 amino acids of porcine sequence (SEQUENCE ID No. 1) and the last 16 amino acids of the sequence of baboon (SEQUENCE ID No. 2). Since the latter sequence differs from the sequence of the pig only in two positions, containing lysine (K) instead of arginine at residue 291 and series (S) instead of threonine at residue 301, the mutant is designated as pork-K-S or CKS-uricase. Each of CKS-, SBH and SBH-NT-CT-uricase contains one lysine residue is greater and, therefore, one potential place for PEG-helirovanie more than a sequence of pig or baboon.

cDNA for different uricase mammals, including SBH-uricase, CKS-uricase and recombinant Babuino-like uricase were partially cloned and identified the optimal conditions for expression in E. coli using standard methods. Cm. Erlich, ON, (ed) (1989) PCR Technology. Principles and Applications for DNA Amplification. New York: Stockton Press; Sambrook, J et al. (1989) Molecular Cloning. A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Recombinant uricase was extracted, purified, and their stability and activity was tested using a modification of standard tests. Cm. Fridovich, I, (1965) J Biol Chem 240:2491-2494; Nishimura et al. (1979) and others who measures 1.

Uricase can conjugates through biologically stable, non-toxic, covalent ligament relatively small number of strands of PEG. Such bundles may contain urethane (urethane) chords, secondary amine bond and amide links. Various activated PEG suitable for such conjugation, commercially available from the firm Shearwater Polymers, Huntsville, AL.

For example, urethane links to uricase can be formed by incubating uricase in the presence of succinimidylester (IC) or 4-nitrophenylarsonic (NFC), derived from the PEG. SC-PEG can be synthesized using the procedures described in U.S. patent No. 5612460 included here by reference. NF-PEG can be synthesized by reacting PEG with 4-nitrophenylphosphate in accordance with the methods described in Veronese, FM. et al. (1985) Appl Biochem Biotechnol 11:141-152, and in U.S. patent No. 5286637 included here by reference. The methods described in patent '637, adapted to PEG with high molecular weight by adjusting the concentrations of the reactants to maintain a similar stoichiometry. An alternative method of synthesis of NF-PEG described in Buttner, W. et al., description of GDR patent No. DD 279486 A1.

Amide links to uricase can be obtained by using N-hydroxysuccinimide of ester carboxylato acid derivative of PEG (Shearwater Polymers). Secondary amine ligaments which may be formed by using 2,2,2-cryptgethashparam PEG (trisil PEG; Shearwater Polymers) or by reductive alkylation using PEG-aldehyde (Shearwater Polymers) and laborgerate sodium.

In conjugates containing PEG with molecular weights between 5 and 30 kDa, the maximum number of threads PEG associated with one polyadenine while maintaining at least 75% uricolytic activity of the unmodified enzyme is in the range from 2 threads for uricase soybeans to more than 10 threads for SBH-uricase (see test conditions in Example 1 and the results on figa-5B). The last degree of PEG-helirovanie corresponds to approximately one third of all amino groups. In one embodiment, performing the average number of strands of PEG associated with each polyadenine uricase, is in the range from 2 to 10. In a preferred implementation, the average number of strands of PEG associated with each polyadenine uricase, is in the range from 3 to 8. In a more preferred implementation, the average number of strands of PEG covalently associated with each polyadenine uricase, is in the range from 4 to 6. In yet another implementation of molecular weight of PEG used for binding assays, is in the range from 5 to 100 kDa, preferably from 10 to 60 kDa, and more preferably from 20 to 40 kDa, for example equal to 30 kDa.

As you know, there are several factors which can affect the choice of the optimal molecular weight and the number of strands of PEG to associate with this form uricase. In General, the reduction or elimination of immunogenicity without significant loss uricolytic activity may require the binding of a relatively larger number of strands of PEG lower molecular weight compared with a smaller number of threads larger PEG molecular weight. For example, optimally can be effective or 6 strands of PEG with a weight of 20 kDa to polyadenine or 4 strands of PEG with a weight of 30 kDa to polyadenine. Similarly, each excellent shape uricase may have different optimal values in relation to the size and number of threads. Cm. figa-5B.

Conjugation with PEG makes all protestirovany uricase soluble and stable in buffers at physiological pH without adding similar substrate or inhibitor, such as an 8-asasantin, which is used as a stabilizer in mushroom uricase (Uricozyme®). Two different conjugate SBH-uricase is one that contains 6 strands of PEG weight of 10 kDa to polyadenine, and the other containing 2 strands of PEG weight of 19 kDa on polyadenine, retained significant activity after incubation in mouse serum for more than one month at 37°C. in Addition, several conjugates had in mice circulation pologize longer than two days as opposed to polurotny 8 hours or 24 hours, reported previously in respect of PEG-Modific the level of uricase mammalian and microbial origin. Chen et al. (1981); Fuertges, F. et al. (1990) J Contr Release 11:139-148; Fujita, T, et al., (1991) J Pharmacobiodyn 14:623-629. Longer half-life of injectable protein drugs make them more economical and can lead to improved perception of the patient. Longer pologize also shows that these products are better tolerated by the body.

When PEG-conjugates SBH-uricase were prepared from purified tetramer form of the protein (four pojedynczy 35 kDa), they showed a strongly reduced immunogenicity in mice (7) in contrast to the moderate immunogenicity of PEG-conjugates with large forms of the enzyme (for example, oktamery 35 kDa on polyadenine; see Fig) and very high immunogenicity unmodified enzyme. Repeated injections of PEG-uricase of the present invention pricestability mice were removed from them hyperuricemia more than 2 months and protect the structure and function of their kidneys from damage associated with uric acid (Fig-11).

Injection fully active conjugates SBH-uricase with PEG weight of 10 kDa (figa-3B) strongly reduced hyperuricemia in homozygous pricestability mice (Fig). Uric acid levels in the urine were also strongly reduced in all pricestability mice who were treated with PEG-modified SBH-uricase. Pricestability mice received a series of injections with the drug PEG-uricase similar used to obtain the data Fig. This treatment reduced the severity of the defect concentration of the urine, as shown by measurement of urine osmolality in normal conditions and after a 12-hour period of water deprivation (Fig.9) and their water consumption and urine output (figure 10) compared with the corresponding measurements for genetically similar mice not receiving treatment. It was also demonstrated that ten weeks of treatment initiated within the first ten days of life, homozygous pricestability (uox-/-) "knocked out" mice with PEG-uricase of the present invention reduced the severity caused by the lithate of renal architecture, as shown by magnetic resonance microscopy (11). Cm. methods microscopy in Hedlund, LW, et al. (1991) FundAppl Toxicol 16:787-797; Johnson, GA. et al. (1992) in Gore JC (ed), Reviews of Magnetic Resonance in Medicine, Vol.4 (str-219) New York: Pergamon Press.

Purified preparations of naturally occurring and recombinant uricase usually contain a mixture of complexes of the enzyme in addition to the tetramer (140 kDa) form. The percentage of tetramer forms in each uricase the drug is usually varies from 20 to 90%. Despite the evidence of things not PEG-lirovannye a combination of several other proteins are highly immunogenic (see, for example, Moore, WV. et al. (1980) J Clin Endocrinol Metab 51:691-697), previous studies of PEG-uricase not describe any efforts to restrict the content of sosaku the values, assuming that the potential immunogenicity of PEG-modified aggregates were not taken into account. Based on observations it seems likely that such a combination was present in the enzyme preparations used for previous syntheses of PEG-uricase. Their presence could make the task of cooking non-immunogenic conjugates more complex. It also seems that large losses uricolytic activity observed in previous studies on PEG-helirovanie uricase, were associated with a large number of associated threads PEG of low molecular weight. On the other hand, methods of purification and PEG-helirovanie uricase described here provide covalent joining of the 10 strands of PEG on polyadenine when saving more than 75% uricolytic activity, at least for certain uricase, for example pig Babuino the chimerical uricase the enzyme from A. flavus (see figa and 4A).

In yet another preferred implementation of almost all together tetramer form of the enzyme can be removed by ion exchange or rozmirovskaya chromatography at pH ranging from 9 to 10.5, preferably 10,2, up to conjugation with PEG result almost entirely of tetramer drug uricase. Molecular weight uricase in each fraction, leaving the preparation of the column, which may be controlled by any analytical technology based on size, including, for example, WPGH (high performance liquid chromatography), traditional rozmirovskaya chromatography, centrifugation, light scattering, capillary electrophoresis or gel electrophoresis sedentarism buffer. For the tetramer uricase allocated by using rozmirovskaya chromatography, the fractions containing only the shape of the enzyme with a weight of 140 kDa, can be combined and used for conjugation with PEG. For the tetramer uricase allocated by using ion-exchange chromatography, fractions emerging from the ion exchange column can be analyzed in relation to the size to determine which fractions contain significant amounts of tetramer forms without detectable aggregates. From the United thus uricase at least 90% may be in the tetramer form; junk together, thus, approximately 10%, 5%, 2% or less of all the selected uricase.

Results presented here show that even with a strong PEG-Yerevani form SBH, bógreater than the tetramer, are highly immunogenic for mice (Fig). Moreover, in mice that were injected with PEG-conjugates complexes uricase, prikaitinta activity in subsequent injections or PEG-Yerevani tetramers, or PEG-Ilir the bathrooms populations were rapidly eliminated from the circulation. In contrast, conjugates prepared from uricase containing less than 5% of the populations, could repeatedly re-inetservices without any acceleration of their purification (Fig.7) and without detectable formation of antibodies, which showed a sensitive enzyme-linked immune test. The use of highly purified tetramer uricase advanced features superior conjugates of the present invention as previously described preparations of PEG-uricase. On the contrary, the presence of a significant proportion (e.g., >10%) aggregates in uricani drugs used by some previous researchers, led them to bind a large number of strands of PEG in an attempt to suppress the immunogenicity. Consequently, the enzymatic activity of the obtained conjugates was significantly reduced. The invention involves PEG-lirovannomu uricase in nethermere form, such as, for example, dimers of uricase, to the extent until such conjugate of uricase kept at least 75% of its uricolytic activity was almost non-immunogenic.

The following examples, which in no way should be construed as limiting the invention, illustrate various disclosed above aspects. These examples describe PEG-uricase prepared by binding activated (i.e., the electrophile is different) derivative of PEG of different sizes and compositions found in nature pork, fungal or bacterial urakasumi or recombinant soybean, pork or pig Babuino chimerical urakasumi. The results of the research activity, solubility, stability, pharmacokinetics, pharmacodynamics, and immunologic studies are contained in the examples. Data on Fig-11 make clear the ability of PEG-modified SBH-uricase of the present invention to adjust the hyperuricemia and hyperuricosuria and to protect the structure and function of kidney in an animal model with hyperuricemia and hyperuricuria, causing serious destruction of the kidneys. Wu, X. et al. (1994) Proc Natl Acad Sci USA 91:742-746. These examples provide experts guidance for the preparation of almost non-immunogenic conjugates of uricase that retains at least 75% uricolytic activity of the unmodified enzyme.

EXAMPLE 1

Cleaning tetramer form uricase

Tetramer form uricase (molecular weight of about 140 kDa) was obtained in pure form from a solution of uricase pork liver by preparative rozmirovskaya or ion-exchange chromatography with subsequent analytical rozmirovskaya chromatography. Uricase pork liver was purchased from the company Sigms-Aldrich, St. Louis, MO, catalog number U2350 or U3377 or the company Boehringer Mannheim, Indianapolis, IN.

Preparative and analytical rozmirovskaya x is matography was carried out at pH 10-10,5, preferably 10,2 10 mmol sodium buffer containing 0.1 mol of NaCl, in columns Superdex 200, pre-calibrated with proteins of known molecular weight. Superdex were purchased from Amersham Pharmacia, Piscataway, NJ. You can use any buffer capable of maintaining the desired pH and compatible with the chemistry, which is intended for subsequent binding to PEG. Such buffers are well known in the prior art. UV absorption of the eluate from the preparative column was observed at 280 nm, and containing uricase part of the eluate corresponding to the molecular weight desired tetramer forms, but free from compounds with high molecular weights were collected for use in the synthesis of essentially non-immunogenic PEG-uricase as described in Example 2. Alternative tetramer form uricase can be selected using another razminayuschego tools, such as Superose 12 (Amersham Pharmacia) or any other means compatible with a mild alkaline solution and having a range of fractionation of a suitable size. Such tools are available and well known in the prior art.

Ion exchange chromatography was performed at pH 10-10,5, preferably of 10.2, column Mono Q (Amersham Pharmacia, Piscataway, NJ), which were balanced with a 0.1 molar sodium carbonate buffer. Any who Ufer, compatible with the chemistry of binding to PEG and capable of maintaining the desired pH may be used at a sufficiently low ionic concentration, which provides the adsorption of uricase to the column. Such buffers are well known in the prior art. UV absorption of the eluate was observed at 280 nm during elution of uricase of the ion exchange resin by increasing the ionic concentration of the supplied buffer solution, for example, by a linear gradient from 0 to 0.5 mol NaCl in sodium carbonate buffer. Then used rozmirovskaya WPGH to identify fractions of the eluate containing the desired tetramer form uricase without detectable aggregates, for the synthesis of essentially non-immunogenic, uricase. Alternative tetramer form uricase can be isolated using other ion-exchange means, such as Q-Sepharose (Amersham Pharmacia) or any other means that is compatible with a mild alkaline solutions. Such tools are available and well known in the prior art.

Activity uricase was investigated using a modification of standard methods. See, for example, Fridovich (1965); Nishimura et al. (1979). Solutions of uric acid were prepared daily in 50-Molina sodium borate buffer, pH of 9.2 to ensure that the final concentrations in the test from 6 to 150 Ám. Drugs arikasumatikris in borate buffer, containing bovine serum albumin (Sigma-Aldrich, St. Louis, MO, catalog number A-7030), so that the final concentration of albumin in this test was 0.1 mg/ml After mixing of the different solutions of the enzyme with the substrate in the cell plate microtitre in microplastics the reader the rate of disappearance of uric acid at 25°was monitored at 292 nm every 4 seconds for 3 minutes. Of samples in which from 10 to 40% of the substrate was consumed within 3 minutes, at least 20 data points were used to calculate the maximum speed decrease absorption per minute. One international unit (ME) activity uricase is defined as the amount of enzyme that consumes one micromoles of uric acid per minute; specific activity expressed in IU/mg protein. Some data for the relative activities of uricase on figa-5B were obtained using 100 micromole of uric acid in the test. Other results for speed at 100 micromole of uric acid (V100) were calculated from the values of Michaelis constant (Km) and maximum velocity (Vmax) for the respective enzyme preparations using the formula:

V100=100×Vmax/(Km+100),

where Km is expressed in micromol.

EXAMPLE 2

Linking PEG with tetramer pork uricase

To a solution of the tetramer uricase in 0.1-molar sodium carbonate buffet is e, pH 10.2, pray for each of polyadenine uricase (molecular weight 35 kDa) was added 10-200 mol activated derivative of monomethoxy PEG, for example 4-nitrophenyl carbonate (NF-PEG)of various sizes. These and other suitable activated PEG and supplied by Shearwater Polymers. Instructions for linking these PEG to proteins are given in the directory Shearwater Polymers on the Internet at www.swpolymers.com and JM Harris et al. (ed) (1997) Poly(ethylene glycol) Chemistry and Biological Applications. ACS Symposium Series 680, Washington, DC: American Chemical Society. The binding reaction proceeds at 0-8°as long as the volume of the binding of PEG does not cease to significantly change over time. Unreacted PEG was then removed from the reaction product by chromatography and/or ultrafiltration.

The number of strands of PEG associated with each polyadenine uricase was determined by adaptation of the methods described in Kunitani, M. et al. (1991) J Chromatogr 588:125-137; Saifer et al. (1997) and Sherman et al. (1997). Aliquot the number of mixtures or fractions of the reaction of PEG-helirovanie from preparative ion-exchange or rozmirovskaya columns was determined by analytical rozmirovskaya WPGH on TSK 5000PWXLat room temperature in 10-Molina sodium carbonate buffer, pH 10.2, containing 0.1 mol of NaCl. As columns WPGH column was used, manufactured by TosoHaas, Montgomeryville, PA. Proteins and PEG were monitored by ultraviolet detectors is about the absorption and index of refraction. The amount of protein in the conjugate was calculated by the peak area of the index of refraction, adjusted for the contribution of protein in the index of refraction, relative to the peak area of the index of refraction of a suitable standard PEG.

Figa shows the saving activity of PEG-pilirovanny uricase pork liver as a function of the number of strands of PEG associated with each polyadenine. The data obtained in accordance with the present invention (▴, □) are compared with those obtained by Chen et al. (1981). Data point in a large circle denotes the conjugate, which reimmunization according to Chen et al. (1981). As shown in figa, conjugates tetramer pork uricase containing up to 6 strands of PEG weight of 30 kDa for each polyadenine or containing up to 7 thread weight PEG 5 kDa for each polyadenine kept at least 75% of the activity of the unmodified enzyme. The apparent increase in specific activity when the number of strands of PEG weighing 5 or 30 kDa (up to 4 threads per polyadenine) can affect the relative insolubility or instability unmodified enzyme compared with conjugates. As shown in figb, conjugates pork uricase with on average more than 3 threads PEG weight of 30 kDa for each polyadenine contain a large mass of the PEG than was found sufficient to eliminate immunoreactivity in Chen et al. (981).

EXAMPLE 3

Properties of PEG-conjugates tetramer recombinant CBH-uricase

Recombinant pig Babayeva chimerical (SBH) uricase was subcloned in the vector expresii pET3d (Novagen, Madison, WI), and the resulting plasmid construct was transformed into a culture of Escherichia coli BL21(DE3)pLysS (Novagen) and expressed in it. These procedures were carried out using methods well known from the prior art in molecular biology. Cm. Erlich (1989); Sambrook et al. (1989); Ausubel, F. et al. (ed) (1997) Short Protocols in Molecular Biology. New York: John Wiley & Sons.

6 shows the amino acid sequence set SBH-uricase (amino acids 1-225 of SEQUENCE ID No. 1 and amino acids 226-304 of SEQUENCE ID No. 2) in comparison with the sequences of the pig (SEQUENCE ID No. 1) and baboon (SEQUENCE ID No. 2). Residues in the sequence of baboon, other than residues in the sequence pigs in bold. The first sequence of the pig and baboon were identified Wu et al. (1989) and confirmed by the present inventors. The SEQUENCE ID No. 1 is identical to the Access Number R GenBank except for the lack of initial meteorologi residue in the sequence of GenBank. The SEQUENCE ID No. 2 is identical to the Access Number R GenBank except for the lack of initial meteorologi residue and replacement of histidine threonine at residue 153 in the GenBank sequence (residue 154 figure 6).

Tetramer form SBH-uricase isolated and contacted with PEG of different molecular weight, as described in Examples 1 and 2. Conjugates prepared with PEG weighing 5, 10, 19 or 30 kDa, contained up to 10 strands of PEG per polyadenine. Those that were prepared using PEG weight of at least 10 kDa, retained more than 95% of the initial specific activity of recombinant uricase (figa-3B).

The following properties of the conjugate tetramer SBH-uricase with approximately 6 threads weight PEG 10 kDa each polyadenine illustrated in the following drawings: lack of immunogenicity (Fig.7) and efficiency at uricase-deficient mice at 1) the correction of hyperuricemia and hyperuricosuria (Fig); 2) reduce the severity of the defect concentration of urine (Fig.9) and (3) reducing the severity of nephrogenic diabetes insipidus (figure 10). In addition, this PEG-uricase reduced the severity associated with uric acid kidney damage, according to magnetic resonance microscopy (11).

Fig.7 shows the activity SBH-uricase in the serum of mice 24 hours after each of the four or five intraperitoneal injections of PEG-uricase in relation to the value of 24 hours after the first injection. PEG-conjugates were prepared from three different preparations SBH-uricase using two different activation technologies PEG. One drug ● was tested on uricase-deficient (uox-/-) mice; the other two (Δ, ▪) tested on normal BALB/c mice. The most immunoreactive product (Δ) was prepared from purified CBH-uricase containing an unknown number of complexes uricase associated with an average of 7 threads weight PEG 5 kDa for each polyadenine, using Succinimidyl-carbonate derivative of PEG (CK-PEG). Zalipsky, U.S. patent No. 5612460 included here by reference. Moderately immunoreactive product (▪) was prepared by linking the drug SBH-uricase containing 11% of aggregates with an average of 2 threads PEG weight of 19 kDa for each polyadenine, using 4-nitrophenylacetonitrile derivative of PEG (NF-PEG). Sherman et al. (1997). Least immunoreactive product (●) was prepared by linking an average of 6 threads NF-weight PEG 10 kDa each polyadenine with drug SBH-uricase containing <5% of the aggregate uricase.

Fig shows the inverse relationship between concentrations of uric acid in serum and urine and activity injected PEG-uricase serum uricase-deficient (uox-/-) mouse. Injection at time 0 and after 72 hours contained 0,43 IU SBH-uricase, conjugated with an average of 6 threads weight PEG 10 kDa each polyadenine enzyme.

Fig.9 shows that the treatment uricase-deficient mice using PG-modified SBH-uricase reduced the severity of the defect concentration of the urine. The mean and standard deviation data for the urine osmolality is shown for two mice containing one copy of the normal mouse gene uricase (uox+/-), six not treated homozygous uricase-deficient mice (uox-/-) and six homozygous uricase-deficient mice, which were injected ten times between the third and seventy-second day of life through either 95 or 190 mIU PEG-uricase. Mouse each genetic group either received water ad libitum (shaded rectangles), or were deprived of water for 12 hours (hatched rectangles) to collect their urine.

Figure 10 shows that treatment uricase-deficient mice with PEG-modified SBH-uricase reduced the severity of nephrogenic diabetes insipidus, characterized by abnormally high water consumption and abnormally high urine output. The genetic basis of mice and treatment Protocol were the same as in Fig.9. The mean and standard deviation of daily consumption of water (solid rectangles) and urine output (hatched rectangles) are shown for three groups of six mice.

11 shows that treatment uricase-deficient mice with PEG-modified SBH-uricase reduced the severity caused by uric acid nephropathy, as shown by magnetic resonance microscopy. The genetic basis of tre the groups of mice and the treatment Protocol were the same as figures 9 and 10.

In addition to the results shown Fig-11, it was demonstrated that the levels of uric acid in the urine of all uricase-deficient mice was strongly reduced after treatment with PEG-modified uricase. Finally, Fig shows that in contrast to PEG-modified tetramer forms SBH-uricase octamine form (molecular weight =280 kDa) even when a strong PEG-Yerevani is immunogenic for mice. This property is reflected in the accelerated purification of PEG-modified octamer within 5 days after a single intraperitoneal injection. The same mouse was re-inheritability the same dose of the same drugs PEG-uricase at 8 and 15 days. Twenty-four hours after the second and third injections prikaitinta activity was neonatorium in the sera of mice injected with PEG-lirovannomu of the octamer, but detectable in the sera of those mice that were injected with PEG-lirovannomu tetramer. These results in combination with the rapid purification of PEG-lirovannomu of octamer observed after the first injection (Fig)support the usefulness of the elimination of all forms of uricase larger than the tetramer, to PEG-helirovanie enzyme.

EXAMPLE 4

PEG-conjugation uricase from Candida utilis

Uricase from Candida utilis were purchased either from Sigma-Aldrich (St. Louis, MO; catalog number U1878), or from whom the assured Worthington Biochemical Corporation (Freehold, NJ; catalog number URYW). Acting on the descriptions of Examples 1 and 2, tetramer form isolated and synthesized PEG-conjugates with PEG weighing 5, 10 or 30 kDa (figa-1B). Figa shows the saving activity of PEG-pilirovanny uricase from Candida utilis as a function of the number of strands of PEG associated with each polyadenine. The data obtained in accordance with the present invention (▴, ●, □) are compared with the data of Nishimura et al. (1979); Nishimura et al. (1981); Chen et al. (1981); Davis et al. (1981); Tsuji et al. (1985); Yasuda et al. (1990) and Fujita et al. (1991). Data points in large circles denote the conjugates described as pantagonia in Nishimura et al. (1979 or 1981) or reimmunization in Chen et al. (1981).

Figb shows the saving activity of PEG-pilirovanny uricase from Candida utilis as a function of the total mass of PEG associated with each polyadenine. The data obtained in accordance with the present invention (▴, ●, □) are compared with the data of the same works that figa. Data points in large circles have the same meaning as in figa.

As shown in figa and 1B, compared with the average up to 6 strands of PEG weighing 5 or 30 kDa or 9 threads weight PEG 10 kDa each polyadenine retain at least 75% of the activity of the unmodified enzyme. The apparent increase in specific activity when attaching the increased number of strands of PEG weight of 30 kDa (up to 5 or 6 threads on the AC is blowing polyadenine) may reflect the relative insolubility or instability unmodified enzyme compared with conjugates.

EXAMPLE 5

PEG-conjugation uricase from Aspergillus flavus

Uricase of Aspergillus flavus were purchased from Sanofi Whinthrop (Gentilly Cedex, France). Operating as described in Example 2, was synthesized conjugates with PEG of different molecular weight (figa-4B). Conjugates prepared by binding the enzyme from A. flavus with on average up to 12 threads weight PEG 5 kDa, or up to 7 strands of PEG weight of 30 kDa for each polyadenine kept 75% of the initial specific activity of this mushroom uricase.

EXAMPLE 6

PEG-conjugation uricase soy

Recombinant uricase from nodules of soybean root (also called nodulin 35) prigotovlyalos and were purified as described by Kahn and Tipton (Kahn, K. et al. (1997) Biochemistry 36:4731-4738), and was provided by Dr. Tipton (University of Missouri, Columbia, MO). Acting according to the descriptions given in Examples 1 and 2, isolated tetramer form and to prepare conjugates with PEG of different molecular weight (figa-5B). In contrast uricase from Candida utilis (figa), pork uricase (figa), pig-Babuino the chimerical uricase (figa) and uricase from Aspergillus flavus (figa) soybean enzyme endured binding only about 2 threads PEG weighing 5 or 30 kDa each polyadenine with preservation of at least 75% of the initial uricolytic activity.

EXAMPLE 7

PEG-conjugation uricase from Arthrobacter slobiformis

Uricase from Arthrobacter globiformis were purchased from comp the Institute Sigma-Aldrich (catalog number U7128). Cm. the Japan patent No. 9-154581. Acting according to the descriptions given in Examples 1 and 2, isolated tetramer form and to prepare conjugates with PEG weight 5 and 30 kDa. Although compared with an average of more than 3 threads weight PEG 5 kDa for each polyadenine kept less than 60% of the initial specific activity, compared with the average about 2 threads PEG weight of 30 kDa for each polyadenine maintained at least 85% of the initial specific activity.

EXAMPLE 8

PEG-conjugation aminosidine pork and SBH-uricase

Recombinant porcine and SBH-uricase, which destroyed the first six amino acids from aminoterminal, expressed in E. coli and purified from it by standard methods, as described in Example 3. Acting according to the descriptions given in Examples 1 and 2, was synthesized PEG-conjugates aminosuccinic uricase to obtain non-immunogenic conjugates that retain at least 75% of the initial specific activity.

EXAMPLE 9

PEG-conjugation pork and SBH-uricase, truncated by carboxylato terminal either side aminoterminal, and carboxylato terminal

Recombinant porcine and SBH-uricase, which destroyed the last three amino acids from carboxylato terminal, expressed in E. coli and purified from it by standard methods, as described in point is the iMER 3. This carboxylato-terminal destruction can improve the solubility of unmodified enzyme, because it removes peroxisome-target signal. Cm. Miura et al. (1994). Acting according to the description given in Examples 1 and 2, was synthesized PEG-conjugates carboxyl-truncated uricase to get virtually non-immunogenic conjugates that retain at least 75% of the initial specific activity. The sequence of recombinant CBH-uricase, truncated to six residues from aminoterminal and three residue from carboxylato terminal (SBH-NT-CT), is shown in Fig.6. This uricase expressed, purified and PEG-yiruma, as described in Examples 1, 2 and 3, to obtain a practically non-immunogenic conjugates that retain at least 75% of the initial specific activity.

EXAMPLE 10

PEG-conjugation mutants pork uricase containing an increased number of seats attaching PEG

Recombinant swine uricase, in which the potential number of seats attaching PEG increased by replacing one or more arginine residues lysine, prepared as described in Example 3. Cm. Hershfield MS, et al. (1991) Proc Natl Acad Sci USA 88:7185-7189. Amino acid sequence of one of the examples of such mutant (CKS-uricase), in which arginine at residue 291 replaced by lysine and threonine at residue 301 is replaced by serine, the azan figure 6. Acting according to the descriptions given in Examples 1 and 2, PEG conjugatively with this uricase to get virtually non-immunogenic conjugates that retain at least 75% of the initial specific activity of recombinant uricase.

EXAMPLE 11

PEG-conjugation mutant recombinant uricase baboon

Using standard methods of molecular biology, as in Example 3, is constructed of uricase baboon with amino acid substitution (histidine instead of tyrosine) at position 97 (see sequence of baboon figure 6). Acting according to the descriptions given in Examples 1 and 2, was synthesized PEG-conjugates tetramer forms of mutant recombinant uricase baboon to obtain conjugates with significantly reduced immunogenicity, retaining at least 75% of the initial specific activity of recombinant uricase.

EXAMPLE 12

The immunogenicity of PEG-conjugates of Candida utilis, Aspergillus flavus and Arthrobacter globiformis

Uricase from Candida utilis, Aspergillus flavus and Arthrobacter globiformis obtained as described in Examples 4, 5 and 7, respectively. Acting according to the descriptions given in Examples 1 and 2, was synthesized PEG-conjugates with PEG weighing 5, 10, 20 or 30 kDa. The immunogenicity of these conjugates greatly reduced or eliminated.

1. Conjugate comprising at least 90% of the tetramer uricase, conjugated to PEG, and the PEG has an average molecular weight of from about 5 is about 100 kDa, and these conjugate uricase retains at least about 75% uricolytic activity of the unconjugated uricase, and where immunogenicity mentioned conjugate uricase significantly reduced compared with unconjugated uricase.

2. The conjugate according to claim 1, in which the aforementioned PEG is polyethylene glycol.

3. The conjugate according to claim 2, in which the said polyethylene glycol has an average molecular weight of about 20 kDa.

4. The conjugate according to claim 2, in which the said polyethylene glycol has an average molecular weight of about 10 kDa.

5. The conjugate according to claim 2, in which the said polyethylene glycol has an average molecular weight of about 30 kDa.

6. The conjugate according to claim 1, in which the above-mentioned uricase contains less than 5% of the units uricase that are more than tetramera.

7. Pharmaceutical composition for reducing the levels of uric acid in the fluid or tissue of the body containing the conjugate according to claim 1 and a pharmaceutically acceptable carrier.

8. The conjugate according to claim 1, in which each polyadenine uricase covalently linked, on average, with 2-10 threads PEG.

9. The conjugate according to claim 1, in which uricase is uricase mammal.

10. The conjugate according to claim 1, in which uricase is uricase pork liver, uricase bovine liver or sheep's liver.

11. The conjugate according to claim 1, in which uricase is recombinant.

1. The conjugate according to claim 11, in which uricase has essentially the sequence uricase pork liver, bovine liver, goat liver or liver baboon.

13. The conjugate according to claim 11, in which uricase is chimerical.

14. The conjugate according to claim 11, in which the chimerical uricase contains part of uricase pork liver and uricase liver baboon.

15. The conjugate according to 14, in which the chimerical uricase is SBH-uricase.

16. The conjugate according to 14, in which the chimerical uricase is CKS-uricase.

17. The conjugate according to claim 11, in which uricase has essentially the sequence uricase liver baboons, in which the tyrosine 97 replaced by histidine.

18. The conjugate according to claim 11, in which uricase contains aminoterminal and carboxylates terminal, and in which uricase truncated from one terminal or both terminals.

19. The conjugate according to claim 1, in which uricase is fungal or microbial uricase.

20. The conjugate according to claim 19, in which fungal or microbial uricase stands out from Aspergillus flavus, Arthrobacter globiformis, or Candida utilis, or is a recombinant enzyme having essentially the sequence of one of these uricase.

21. The conjugate according to claim 1, in which uricase is uricase bespozvonochnykh.

22. The conjugate according to item 21, wherein uricase bespozvonochnykh stands out from Drosophila melanogaster or Drosophila pseudoobscura, or is a recombinant enzyme, have them essentially the sequence of one of these uricase.

23. The conjugate according to claim 1, in which uricase is a plant uricase.

24. The conjugate according to item 23, in which vegetable uricase stands out from the root nodules of Glycine max, or is a recombinant enzyme having essentially the sequence of this uricase.

25. The conjugate of claim 1, wherein the PEG has an average molecular weight of between approximately 10 and 60 kDa.

26. Conjugate on A.25, in which the PEG has an average molecular weight of between approximately 20 and 40 kDa.

27. The conjugate of claim 8, in which the average number of covalently bound threads PEG is 3 to 8 threads per polyadenine uricase.

28. The conjugate according to item 27, in which the average number of covalently bound threads PEG is from 4 to 6 threads per polyadenine uricase.

29. The conjugate according to claim 1, in which the threads PEG covalently linked to uricase through chords, selected from the group consisting of urethane chords, secondary amine ligaments and amide ligaments.

30. The conjugate according to claim 1, in which the PEG is linear.

31. The conjugate according to claim 1, in which the PEG is branched.

32. The pharmaceutical composition according to claim 7 in which the said composition is stabilized by lyophilization and dissolved after restoring to provide solutions suitable for parenteral administration.

33. The use of conjugate uricase according to claim 1 in the preparation of medications to reduce the Oia elevated levels of uric acid in the fluid or body tissue of a mammal.

34. The use of conjugate uricase on p, in which the aforementioned mammal is man.

35. The use of conjugate uricase on p in which the drug is administered to a mammal by intravenous, intradermal, subcutaneous, intramuscular and intraperitoneal injection or inhalation aerosol formula.

36. The use of conjugate uricase on p, which referred to elevated levels of uric acid are associated with a condition selected from the group consisting of gout, gouty nodes, kidney failure, organ transplantation and malignant diseases.

37. The use of conjugate uricase on p, in which PEG is linear.

38. The use of conjugate uricase on p, in which PEG is branched.

39. The conjugate according to claim 1, in which said conjugate uricase prepared from uricase containing not more than about 10% neutralinos aggregated uricase.

40. The conjugate according to claim 1, in which uricase contains less than 10% of the complexes uricase.

41. The conjugate of claim 1, wherein the PEG has a molecular weight of more than 5 kDa and less than 100 kDa.

42. The conjugate of claim 1, wherein the PEG has a molecular weight between about 10 kDa and about 100 kDa.

43. The conjugate according to paragraph 41, in which each polyadenine uricase covalently linked, on average, from 2 to 10 strands of PEG.

44. The conjugate according to claim 1, in which the EO PEG has a molecular weight of more than about 10 kDa, less than about 100 kDa.



 

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4 cl, 3 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: agent contains Rifabutin sorbated in polymeric nanoparticles matrix, potassium cholesterylphosphate, or sodium glycocholate, or hexadecyl dihydrogen phosphate, or a-tocopheryl succinate, water-soluble polymeric stabiliser and bulking agents. Polymeric nanoparticles sized 100-800 nm include lactic acid polymer/polymers and/or lactic and glycolic acid copolymer/copolymers at glycolic acid content in specified copolymers up to 50 mole %. Molecular weight of specified polymers and copolymers is 5 to 300 kDa. Molecular weight of water-soluble polymeric stabiliser is no more than 70 kDa and is selected from the group including polyvinyl alcohol, polyvinylpyrrolidone, polysorbate and seralbumin.

EFFECT: new agent provides durable action of Rifabutin; higher bioavailability of Rifabutin and efficiency of bacterial infection treatment.

3 dwg, 1 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention concerns coordination complex of platinum (II) diaminocyclohexane with block copolymer containing structure of the general formula PEG-block-poly(carbo), where PEG is a poly(ethyleneglycol) segment, and carbo is a repeating chain containing carboxylic group in the side chain, and platinum (II) diaminocyclohexane is immobilised by block copolymer due to linkage between carboxylic carbo residue anion and platinum; as well as method of obtaining the complex and anticancer composition including effective anticancer quantity of coordination complex and pharmaceutically acceptable carrier. In addition, invention concerns coordination complex of platinum (II) diaminocyclohexane and block copolymer with structure of the general formula (1-a) or (2-a) , where R1 is a hydrogen atom or unsubstituted or substituted serial or furcated C1-C12 alkyl group, L1 and L2 are linkage group, R3 is a hydrogen atom, protective group of aminogroup, hydrophobic group or polymerisation-capable group, R4 is hydroxylic group or initiator residue, each of R5 radicals is independently a hydrogen atom, alkali metal ion or protective group of carboxylic group, m is an integer from 5 to 20000, n is an integer from 2 to 5000 if alkali metal ion comprises 50% or more of the number of R5 groups which is n, with platinum (II) diaminocyclohexane immobilised by the said block copolymer due to linkage between carboxylic carbo residue anion and platinum, and equivalent ratio of diaminocyclohexane platinum (Pt) to carboxylic groups of the said block copolymer (Pt/COO-) is 0.3-1. The invention also concerns the method of obtaining this coordination complex and method of tumour treatment involving introduction of effective quantity of combined coordination complex of platinum (II) diaminocyclohexane and coordination complex of cis-platinum to a patient.

EFFECT: increased composition efficiency.

20 cl, 2 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention concerns aldehyde derivatives and conjugates of di-, oligo- or polysaccharide, of the general formula (I), methods of obtaining them, and pharmaceutical composition based on them and capable of staying in blood flow for prolonged time. , where R is -CH(CHO)CH2OH, -CH2CHO, -CH(CH2NHR1)CH2OH, -CH(CH2NHNHR1)CH2OH, -CH(CH=NNHR1)CH2OH, -CH2CH2NHR1, -CH2CH=N-NHR1, -CH2CH2NHNHR1; R1 is polypeptide or albumen; GlyO is a sialic acid bond; R3 is H; R4 is OH; n is 2 or more.

EFFECT: obtaining pharmaceutical composition based on aldehyde derivatives of sialic acid capable of staying in blood flow for prolonged time.

20 cl, 7 tbl, 22 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: present invention refers to medicine and concerns microparticles containing sphere mainly consisting of cross-linked agarose carbohydrate and allergen covalently bonded with sphere, applied for immune system disturbance treatment. Used allergen is produced of plant pollen, specifically of timothy grass pollen. Application of specified microparticles provides effective treatment for patients suffering from allergy, as well as reduces by-effects of parenteral introduction.

EFFECT: development of effective method of allergy treatment and prevention.

9 cl, 4 dwg, 1 tbl, 5 ex

FIELD: medicine; pharmacology.

SUBSTANCE: medicinal agent is mixed with preaerated and irradiated with accelerated electron current or pulse UV-laser radiation 5.0-50.0% polyethylene oxide solution of molecular weight 0.4-20 kilodaltons in presence of 0.01-0.1 M phosphate buffer, pH 6.0-8.0 and containing 0.05-0.3 M sodium chloride. Offered method is easy and multipurpose, and can be applied to increase enteral bioavailability of medicinal agents introduced mainly or solely parenterally due to low resorption by intestine walls.

EFFECT: increased enteral bioavailability of medicinal agents.

4 cl, 3 tbl, 10 ex, 1 dwg

FIELD: chemistry; pharmacology.

SUBSTANCE: polysaccharide, in form of polysialic acid, with at least, two sialic acid links, joined to each other at positions 2,8 and/or 2,9, and with a lateral part, linked to at least, one end link, obtained from a sialic acid link, which consists of a functional group chosen from N-maleimide groups, vinylsufonyl groups, N-iodoacetamide groups and orthopyridyl disulphide groups. Polysaccharide is reacted with a hetero bi-functional reagent, with a first functional group, chosen from N-maleimide groups, vinylsufonyl groups, N-iodoacetamide groups and orthopyridyl disulphide groups, and a second functional group, different from the first group, where the stated second functional group reacts with the stated end link, derivative of sialic acid, with formation of a covalent bond and functional polysaccharide, suitable for selective pairing with a thiol group.

EFFECT: reaction with a hetero bi-functional reagent allows for introduction of a side functional group for site-specific bonding to sulfhydryl groups, for example, to side chains of cysteic links in medicines, medication delivery systems, proteins or peptides.

29 cl, 3 dwg, 1 tbl, 4 ex

FIELD: chemistry; medicine.

SUBSTANCE: description is given of a conjugate of hydroxyalkyl starch and a low-molecular substance, in which the binding interaction between the molecular hydroxyalkyl starch and the low-molecular substance is based on a covalent bond, which forms from a bonding reaction between (i) an end aldehyde group or carboxyl group, formed due to selective oxidation of the end aldehyde group, or activated carboxyl group, obtained from conversion of the carboxyl group of hydroxyalkyl starch molecules and (ii) a functional group of a low-molecular substance, chosen from a group, consisting of an amino group, carboxyl group, thiol group and a hydroxyl group, which is reactive, relative the given aldehyde group or carboxyl group or an activated carboxyl group of hydroxyalkyl starch molecules. The bond directly formed from the bonding reaction can be modified for further reaction until formation of the above mentioned covalent bond, if necessary.

EFFECT: use of the conjugate allows for increasing duration of the presence of the low-molecular substance in blood plasma.

35 cl, 29 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention group refers to compositions containing hapten-carrier conjugate within arranged and repeating matrix, and method of related composition production. Offered hapten-carrier conjugate used for induction of agent-specified immune reaction in case of addiction or abuse, contains cortex particle including at least one first apposition site, where specified cortex particle is virus-like particle of RNA-phage, and at least one nicotine hapten with at least one second apposition site, where specified second apposition site is associated by at least one covalent non-peptide bond with specified first apposition site, thus forming arranged and repeating hapten-carrier conjugate. Offered conjugates and compositions under this invention can include virus-like particles connected to various haptens including hormones, toxins and agent, especially agents causing addiction, as nicotine and can be applied for induction of hapten immune reaction for therapeutic, preventive and diagnostic purposes.

EFFECT: vaccines can induce stable immune reactions for nicotine and fast reduce nicotine availability for brain absorbing.

31 cl, 6 dwg

FIELD: medicine, cosmetology.

SUBSTANCE: invention consists in creating a preparation, containing efficient amount of superoxidedismutase.

EFFECT: invention allows strengthening nails, reducing their fragility, improving nail content, enhancing their elasticity, reducing nail splitting, as well as restoring healthy appearance of nails and accelerating their growth.

5 cl, 3 ex

FIELD: medicine.

SUBSTANCE: operative intervention follows intravenous single dosing of 0.03% sodium hypochlorhyd solution in amount 1:40 to amount of circulating blood. During operation cavity is sanitised with 0.06 % sodium hypochlorhyd solution. Operative intervention is followed with daily intravenous introduction of 0.03% sodium hypochlorhyd solution within two days once a day in the same amount and continued sanitation of cavity with 0.06 % sodium hypochlorhyd solution. Within the second phase of wound process wound is applied with ointment Soderm once a day before secondary suturing. The third phase includes removal of sutures and gel Contractubex infriction in a cicatricial tissue.

EFFECT: increased efficiency of treatment and prevention of inflammatory process progressing owing to antioxidant and detoxicative properties in various phases of wound process.

1 ex

FIELD: medicine, oral surgery.

SUBSTANCE: before surgical interference one should intravenously once inject by drops 0.03%-sodium hypochlorite solution at 1:40 against the volume of a patient's circulating blood. In 2 h one should intravenously once by drops inject rexod preparation at 16 mg. Then comes surgical interference in the course of which one should additionally carry out sanitation with 0.06%-sodium hypochlorite solution. In post-surgical period for 2 d after interference it is necessary to inject the same quantity of 0.03%-sodium hypochlorite solution. Rexod should be injected during the 1st d after operation at the same dosage, and during the next 3 d - per 8 mg. Additionally, locally during the 1st phase of wound process one should treat purulent wound with 0.06%-sodium hypochlorite solution, During the 2nd phase of wound process till applying early secondary sutures one should apply "Soderm" ointment onto the wound, and in the 3d phase - "Contractubex" gel onto cicatricial tissue. The innovation enables to avoid the progress of inflammatory process and shorten terms of therapy due to combined general and local impact of preparations being of detoxication and antioxidant action.

EFFECT: higher efficiency of therapy.

1 ex

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to preparations used in treatment of inflammatory diseases. The claimed composition comprises a nonsteroid anti-inflammatory drug (NSAID) and the preparation "REKSOD" representing [Cu,Zn]-superoxide dismutase as a substance enhancing its effectiveness. The ratio of nonsteroid anti-inflammatory drug (NSAID) and the preparation "REKSOD" in the composition is, wt.-% from 50:1 to 400:1 usually. The claimed composition enhances effectiveness of NSAID by about two-fold, it provides decreasing the effective dose and to prolong their effect on body also.

EFFECT: improved and valuable properties of composition.

1 tbl, 9 ex

FIELD: medicine, biochemistry, pharmaceutical industry, pharmacy.

SUBSTANCE: method involves separation of the mammalian enzyme uricase purified preparation for fractions followed by combination of fractions showing the absence of mammalian uricase aggregates exceeding octamer size based on data obtained in measurement of light scattering and ultraviolet absorption of these fractions. The prepared preparation is conjugated with poly-(ethylene glycol) or poly-(ethylene oxide) and used as a component of the pharmaceutical composition used for reducing of the concentration of uric acid in liquid or body tissue. Use of invention provides decreasing the immunogenic effect of uricase being without reducing its uricolytic effect.

EFFECT: improved preparing method, valuable medicinal and pharmaceutical properties of enzyme and pharmaceutical composition.

19 cl, 6 dwg, 6 ex

FIELD: medicine, biochemistry.

SUBSTANCE: claimed method includes administration of uricase solution into at least one separation column at pH approximately 9-10,5 and reduction from the said column of one or more fractions containing isolated tetra-dimensional urecase aggregate-free uricase, wherein tetra-dimensional urecase aggregates are larger than tetra-dimensional urecase.

EFFECT: 9 cl, 12 ex, 13 dwg.

FIELD: medicine, veterinary science.

SUBSTANCE: the present innovation deals with treating malignant tumors. For this purpose, its is necessary to provide a blood supply of an agent that destroys extra-cellular blood DNA. This agent should be introduced in dosages providing alteration of electrophoretic profile of extra-cellular blood DNA. Agent, also, should be introduced at the dosages and modes that provide the level of DNA-hydrolytic activity of blood plasma measured in blood plasma being above 150 Kunz units/l plasma during totally above 12 h daily. Therapy may last without intervals for 2 d, not less. As an agent destroying extra-cellular blood DNA one may apply DNAse, in peculiar case, bovine pancreatic DNAse or recombinant human DNAse. The innovation suggests, also, to apply and agent that binds extra-cellular blood DNA, for example, anti-DNA antibodies. The method provides low-toxic and efficient treatment of tumors, particularly at prolonged, even one's life-long therapy with preparations mentioned.

EFFECT: higher efficiency of therapy.

10 cl, 7 ex, 6 tbl

FIELD: biochemistry, pharmaceutical chemistry.

SUBSTANCE: invention relates to preparing conjugate of naturally occurring or recombinant urate oxidase (uricase) bound covalently with poly-(ethylene glycol) or poly-(ethylene oxide) (both are designated as PEG) wherein in average from 4 to 10 PEG strands are conjugated with each subunit of uricase and molecular mass of PEG is about between 20 and 40 kDa. Prepared PEG-uricase conjugates are nonimmunogenic practically and retain at least 75% of uricolytic activity of nonmodified enzyme.

EFFECT: improved preparing method, valuable properties of conjugates.

22 cl, 17 dwg, 12 ex

Medical-cosmetic // 2236843
The invention relates to the field of cosmetology and concerns of cosmetic products using algae

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, in particular to hepatic cells production, and may be used in medical science. From the whole liver or resected part thereof, a cell population enriched with living cells of human liver, including hepatic stem cells/precursor cells, is obtained. Cell population contains functional hepatocytes and biliary cells expressing cytokeratin 19 (CK19), but not expressing albumin, as well as hepatic stem cells/precursor cells 9 to 13 mcm in diameter and expressing EP-CAM, CD 133 markers. Resulting cell population is used for hepatotherapy.

EFFECT: production of living population of hepatic cells sufficiently efficient for regeneration.

60 cl, 16 dwg

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