Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1998-06-09
2001-06-26
Saidha, Tekchand (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S229000, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06251388
ABSTRACT:
FIELD OF INVENTION
The present invention relates to methodologies for the production of microbial enzymes, particularly native and recombinant
Wolinella succinogenes
asparaginase and its analogs, which possesses potent in vitro and in vivo activity against diseases correlated with asparagine dependence. In addition, the present invention also relates to methods for the utilization of recombinant microbial enzymes in the treatment of diseases which respond to asparagine depletion, including various hematologic, autoimmune, and infectious diseases.
BACKGROUND OF INVENTION
The references cited below are not admitted to be prior art to the inventions described herein.
Asparaginases are enzymes which catalyze the deamidation of L-asparagine (asparaginase activity) and L-glutamine (glutaminase activity). See Cantor, P. S. & Schimmell, M. R.,
Enzyme Catalysis,
2nd ed., (T. Pettersonn & Y. Tacashi, eds.) Sanders Scientific Press, New York pp. 219-23. (1990). L-glutamine serves as the amide donor in purine biosynthesis, as well as other transamination reactions, and hence plays a role in DNA and cyclic nucleotide metabolism.
In vivo biochemical activity of asparaginase was first documented to be present in guinea pig serum in 1922 (see Clementi, A., La desamidation enzmatique de l'asparagine chez les differentes especes-animals et la signification physiologique de sa presence dass l'organisme, 19
Arch. Intern. Physiol.
369 (1922)). The subsequent discovery that asparaginase isolated from guinea pig serum was the active agent which inhibited the in vivo growth of certain asparagine-dependent mammalian tumors without concomitant deleterious effects on normal tissue (see Broome, J. D., Evidence that the asparaginase activity of guinea pig serum is responsible for its anti-lymphoma effects, 191
Nature
1114 (1961)) suggested that this enzyme could be utilized as an anti-neoplastic agent. Because L-asparagine is a non-essential amino acid, asparaginase was initially thought to represent a unique prototype of selective chemotherapy in which treatment could be directed specifically and selectively against asparagine-dependent cells. However, the low levels of asparaginase in guinea pig serum necessitated the development of a more practical source of this anti-neoplastic enzyme.
Subsequently, microbial asparaginase isolated from
Escherichia coli
and
Erwinia carotovora
were shown to act as potent anti-leukemic agents (see Howard, J. B. & Carpenter, F. H., L-asparaginase from
Erwinia carotovora:
substrate specificity and enzymatic properties, 247
J. Biol. Chem.
1020 (1972); Campbell, H. A., et al., Two asparaginases from
Escherichia coli
B: their separation, purification, and anti-tumor activity, 6
Biochemistry
721 (1967)), and when one of these enzymes was utilized in combination with the chemotherapeutic agent vincristine and the corticosteroid prednisone for the treatment of acute lymphoblastic or acute undifferentiated human leukemia, an overall remission rate of 93% was reported (see Ortega, J. A., et al., L-asparaginase, vincristine, and prednisone for the induction of first remission in acute lymphocytic leukemia, 37
Cancer Res.
535 (1977)).
While these asparaginases possess potent anti-leukemic activity, clinical utilization of the aforementioned microbial asparaginases resulted in a wide range of host toxicity (e.g., hepatic, renal, splenic, pancreatic dysfunction and blood coagulation) and pronounced immunosuppression (see Ohno, R. & Hersh, E. M., Immunosuppressive effects of L-asparaginase, 30
Cancer Res.
1605 (1970)), unlike asparaginase isolated from guinea pig serum (see Cooney, D. A., et al., L-asparaginase and L-asparagine metabolism, 10
Ann. Rev. Pharmacol.
421 (1970)).
Examination of the effects of
E. coli
asparaginase treatment on spleen histology and lymphocyte populations revealed a marked reduction in both the size and reactivity of the splenic germinal centers which was concomitantly associated with a marked reduction in the cytoplasmic immunoglobulin-containing cells (B-cell immunoblasts; see Distasio, J. A., et al., Alteration in spleen lymphoid populations associated with specific amino acid depletion during L-asparaginase treatment, 42
Cancer Res.
252 (1982)). Additionally, examination of the lymphocyte sub-population within the spleen revealed that there was a 40% reduction in the percentage of surface immunoglobulin-expressing cells (B-cells) accompanied by an increase in the ratio of Thy-1.2-expressing cells (T-cells), whereas the ratio of Lyt-2 to Lyt-1 cells remained unchanged in comparison to the control animal group. These results supported the hypothesis that glutamine, or glutamine combined with asparagine depletion initially resulting from administration of
E. coli
asparaginase, caused a marked decrease in spleen lymphocytic cells of the B-cell lineage.
Another important adverse clinical effect associated with traditional microbial asparaginase treatment is hepatic dysfunction (see Schein, P. S., et al., The toxicity of
E. coli
asparaginase, 29
Cancer Res.
426 (1969)). Patients treated with
E. coli
asparaginase generally exhibit decreased plasma levels of albumin, antithrombin III, cholesterol, phospholipids, and triglycerides. Other indications of asparaginase-induced hepatic dysfunction and pathology include fatty degenerative changes, delayed bromosulfophthalein clearance, and increased levels of serum glutamic-oxaloacetic transaminase and alkaline phosphatase. Although some investigators have reported that low dosages of
E. coli
asparaginase result in limited hepatotoxic complications, sensitive indicators of hepatic function in some patients receiving low dosages, however, still reveals significant hepatic disease which may result in life-threatening coagulopathy (see Crowther, D., Asparaginase and human malignant disease, 229
Nature
168 (1971)).
The hepatotoxic effects of microbial asparaginases may be a result of their capability to hydrolyze both asparagine and glutamine. One biochemical difference between
E. coli
and
E. carotovora
asparaginases and the enzyme derived from guinea pig is the non-specific amidohydrolase activity associated with the microbial enzymes (see Howard, J. B. & Carpenter, F. H., (1972) supra; Campbell, H. A., et al., (1967) supra). For example,
E. coli
asparaginase has been shown to possess a 130-fold greater level of glutaminase activity as compared to the activity of
Wolinella succinogenes
(previously classified as
Vibrio succinogenes
) asparaginase. As a result, patients treated with the conventional microbial asparaginases show a marked reduction in serum levels of both glutamine and asparagine (see Schrek, R., et al., Effect of L-glutaminase on transformation and DNA synthesis of normal lymphocytes, 48
Acta Haematol.
12 (1972)), which may demonstrate a possible correlation between glutamine deprivation and asparaginase-induced clinical toxicity (see Spiers, A. D. S., et al., L-glutaminase/L-asparaginase: human pharmacology, toxicology, and activity in acute leukemia, 63
Cancer Treat. Rep.
1019 (1979)).
The relative importance of L-glutamine in mammalian intermediary metabolism served to stimulate further research into the possible role of glutamine deprivation in asparaginase-induced immunosuppression. Lymphoid tissue has been shown to have relatively low levels of glutamine synthetase activity (see El-Asmar, F. A. & Greenberg, D. H., Studies on the mechanism of inhibition of tumor growth by glutaminase, 26
Cancer Res.
116 (1966); Hersh, E. M., L-glutaminase: suppression of lymphocyte blastogenic responses in vitro, 172
Science
139 (1971)), suggesting that these tissues may be particularly sensitive to the depletion of exogenous glutamine. In contrast, some investigators have proposed that asparagine depletion alone may be responsible for asparagine-induced immunosuppression (see Baechtel, F. S., et al., The influence of glutamine, its decomposition products, and glutaminase on the transformation of human lymphocytes, 421
Biochem. Biophys. Acta
33 (1976
Children's Hospital Los Angeles
Lyon & Lyon LLP
Saidha Tekchand
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