PEG-urate oxidase conjugates and use thereof

Details PEG-urate oxidase conjugates and use thereof PEG-urate oxidase conjugates and use thereof

1999-08-06

2003-06-10

Saidha, Tekchand (Department: 1652)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Oxidoreductases

C435S189000, C435S191000, C435S252300, C435S320100, C536S023200, C530S350000

Reexamination Certificate

active

06576235

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to chemical modification of proteins to prolong their circulating lifetimes and reduce their immunogenicity. More specifically, the invention relates to conjugation of poly(ethylene glycols) or poly(ethylene oxides) to urate oxidases, which substantially eliminates urate oxidase immunogenicity without compromising its uricolytic activity.
BACKGROUND OF THE INVENTION
Statements contained in this background section do not constitute an admission of prior art, but instead reflect the inventors' own subjective comments on and interpretations of the state of the art at the time the invention was made. These interpretations may include personal, heretofore undisclosed, insights of the inventors, which insights were not themselves part of the prior art.
Urate oxidases (uricases; E.C. 1.7.3.3) are enzymes that catalyze the oxidation of uric acid to a more soluble product, allantoin, a purine metabolite that is more readily excreted. Humans do not produce enzymatically active uricase, as a result of several mutations in the gene for uricase acquired during the evolution of higher primates. Wu, X, et al, (1992)
J Mol Evol
34:78-84. As a consequence, in susceptible individuals, excessive concentrations of uric acid in the blood (hyperuricemia) and in the urine (hyperuricosuria) can lead to painful arthritis (gout), disfiguring urate deposits (tophi) and renal failure. In some affected individuals, available drugs such as allopurinol (an inhibitor of uric acid synthesis) produce treatment-limiting adverse effects or do not relieve these conditions adequately. Hande, K R, et al., (1984)
Am J Med
76:47-56; Fam, A G, (1990)
Baillière's Clin Rheumatol
4:177-192. Injections of uricase can decrease hyperuricemia and hyperuricosuria, at least transiently. Since uricase is a foreign protein in humans, however, even the first injection of the unmodified protein from
Aspergillus flavus
has induced anaphylactic reactions in several percent of treated patients (Pui, C-H, et al., (1997)
Leukemia
11:1813-1816), and immunologic responses limit its utility for chronic or intermittent treatment. Donadio, D, et al., (1981)
Nouv Presse Méd
10:711-712; Leaustic, M, et al., (1983)
Rev Rhum Mal Osteoartic
50:553-554.
The sub-optimal performance of available treatments for hyperuricemia has been recognized for several decades. Kissel, P, et al., (1968)
Nature
217:72-74. Similarly, the possibility that certain groups of patients with severe gout might benefit from a safe and effective form of injectable uricase has been recognized for many years. Davis, F F, et al., (1978) in G B Broun, et al., (Eds.)
Enzyme Engineering, Vol
. 4 (pp. 169-173) 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, C C, et al., (1988)
Ann Int Med
109:114-117; Greenberg, M L, et al., (1989)
Anal Biochem
176:290-293. Uricases derived from animal organs are nearly insoluble in solvents that are compatible with safe administration by injection. U.S. Pat. No. 3,616,231. Certain uricases derived from plants or from microorganisms are more soluble in medically acceptable solvents. However, injection of the microbial enzymes quickly induces immunological responses that can lead to life-threatening allergic reactions or to inactivation and/or accelerated clearance of the uricase from the circulation. Donadio, et al., (1981); Leaustic, et al., (1983). Enzymes based on the deduced amino acid sequences of uricases from mammals, including pig and baboon, or from insects, such as, for example,
Drosophila melanogaster
or
Drosophila pseudoobscura
(Wallrath, L L, et al., (1990)
Mol Cell Biol
10:5114-5127), have not been suitable candidates for clinical use, due to problems of immunogenicity and insolubility at physiological pH.
Covalent modification of proteins with poly(ethylene glycol) or poly(ethylene oxide) (both referred to as PEG), has been used to increase protein half-life and reduce immunogericity. U.S. Pat. Nos. 4,179,337, 4,766,106, and 4,847,325; Saifer, M G P, et al., (1994)
Adv Exp Med Biol
366:377-387. The coupling of PEG of high molecular weight to produce conjugates with prolonged circulating lifetimes and/or decreased immunogenicity, while conserving functional activity, was previously demonstrated for another enzyme, superoxide dismutase (Somack, R, et al., (1991)
Free Rad Res Commun
12-13:553-562; U.S. Pat. Nos. 5,283,317 and 5,468,478) and for other types of proteins, e.g., cytokines (Saifer, M G P, et al., (1997)
Polym Preprints
38:576-577; Sherman, M R, et al., (1997) in J M Harris, et al., (Eds.),
Poly
(
ethylene glycol
)
Chemistry and Biological Applications. ACS Symposium Series
680 (pp. 155-169) Washington, D.C.: American Chemical Society). Conjugates of uricase with polymers other than PEG have also been described. U.S. Pat. No. 4,460,683.
In nearly all of the reported attempts to PEGylate uricase (i.e. to covalently couple PEG to uricase), the PEG was attached primarily to amino groups, including the amino-terminal residue and the available lysine residues. In the uricases commonly used, the total number of lysines in each of the four identical subunits is between 25 (
Aspergillus flavus
(U.S. Pat. No. 5,382,518)) and 29 (pig (Wu, X, et al., (1989)
Proc Natl Acad Sci USA
86:9412-9416)). Some of the lysines are unavailable for PEGylation in the native conformation of the enzyme. The most common approach to reducing the immunogenicity of uricase has been to couple large numbers of strands of low molecular weight PEG. This has invariably resulted in large decreases in the enzymatic activity of the resultant conjugates.
Previous investigators have used injected uricase to catalyze the conversion of uric acid to allantoin in vivo. See Pui, et al., (1997). This is the basis for the use in France and Italy of uricase from the fungus
Aspergillus flavus
(Uricozyme®) to prevent or temporarily correct the hyperuricemia associated with cytotoxic therapy for hematologic malignancies and to transiently reduce severe hyperuricemia in patients with gout. Potaux, L, et al., (1975)
Nouv Presse Méd
4:1109-1112; Legoux, R, et al., (1992)
J Biol Chem
267:8565-8570; U.S. Pat. Nos. 5,382,518 and 5,541,098. Because of its short circulating lifetime, Uricozyme® requires daily injections. Furthermore, it is not well suited for long-term therapy because of its immunogenicity.
A single intravenous injection of a preparation of
Candida utilis
uricase coupled to 5 kDa PEG reduced serum urate to undetectable levels in five human subjects whose average pre-injection serum urate concentration was 6.2 mg/dL, which is within the normal range. Davis, et al., (1981). The subjects were given an additional injection four weeks later, but their responses were not reported. No antibodies to uricase were detected following the second (and last) injection, using a relatively insensitive gel diffusion assay. This reference reported no results from chronic or subchronic treatments of human patients or experimental animals.
A preparation of uricase from
Arthrobacter protoformiae
coupled to 5 kDa PEG was used to temporarily control hyperuricemia in a single patient with lymphoma whose pre-injection serum urate concentration was 15 mg/dL. Chua, et al., (1988). Because of the critical condition of the patient and the short duration of treatment (four injections during 14 days), it was not possible to evaluate the long-term efficacy or safety of the conjugate.
In this application, the term “immunogenicity” refers to the induction of an immune response by an injected preparation of PEG-modified or unmodified uricase (the antigen), while “antigenicity” refers to the reaction of an antigen with preexisting antibodies. Collectively, antigenicity and immunogenicity are referred to as “immunoreactivity.” In previou

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