Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Transferases
Reexamination Certificate
1996-01-11
2001-09-18
Wax, Robert A. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Transferases
C424S178100
Reexamination Certificate
active
06290953
ABSTRACT:
The present invention relates to the medical use of thymidine phosphorylase in the modulation of cellular proliferation.
BACKGROUND OF THE INVENTION
Thymidine phosphorylase (thymine: orthophosphate deoxyribosyl transferase, EC 2.4.2.4) is a cytosolic enzyme which catalyses the reversible phosphorolysis of thymidine and other pyrimidine 2′-deoxyribosides, except for 4-amino substituted compounds such as 2′-deoxycytidine, as follows:
PyrdR+P
i
←→Pyr+dR−1−P
The phosphorolytic and synthetic reactions may also be used to transfer a deoxyribose moiety of one deoxynucleoside to form a second deoxynucleoside in a nucleoside deoxyribosyl transferase reaction (Schwartz, M. (1971)
Eur. J. Biochem.
21, 191-198).
Thymidine phosphorylase has been purified and characterized from a number of micro-organisms (Schwartz, M. (1971)
Eur. J. Biochem.
21, 191-198, Schwartz, M. (1978)
Methods Enzymol.
51, 442-445; Avraham, Y. et al (1990)
Biochim. Biophys. Acta
1040, 287-293; Hoffee, P. A. et al (1978)
Methods Enzymol.
51, 437-442) and from human tissues (Desgranges, C. et al (1981)
Biochim. Biophys. Acta.
654, 211-218; Kubilus, J. et al (1978)
Biochim. Biophys. Acta
527, 221-228; Yoshimura et al (1990)
Biochim. Biophys. Acta
1034, 107-113).
Escherichia coli
thymidine phosphorylase is a dimer of 90 kD composed of two identical subunits with a molecular weight of 45 kD (Schwartz, M. (1978)
Methods Enzymol.
51, 442-445; Walter, M. et al (1990)
J. Biol. Chem.
265, 14016-14022). The three-dimensional crystal structure of
Escherichia coli
has been determined to resolution of 2.8 Å (Walter, M. et al (1990)
J. Biol. Chem.
265, 14016-14022). The monomer subunit consists of a small &agr;-helical domain and a large &agr;/&bgr; domain. The active site, which binds both thymidine and phosphate, has been located in a cleft between the two domains.
Human thymidine phosphorylase has been identified in many tissues including lymphocytes, heart, spleen, lung and placenta (Yoshimura, A. et al (1990)
Biochim. Biophys. Acta.
1034, 107-113) and purified from both placenta (Yoshimura, A. et al (1990)
Biochim. Biophys. Acta.
1034, 107-113; Kubilus, J. (1978)
Biochem. Biophys. Acta.
527, 221-228) and platelets (Desgranges, C. (1981)
Biochim. Biophys. Acta
654, 211-218). It has been suggested that thymidine phosphorylase plays an essential role in maintaining intracellular thymidine homeostasis (Shaw, T. et al (1988)
Mutant Res.
200, 99-116). The thymidine salvage pathway requires the action of a permease to transport thymidine across the lipid bilayer. Intracellular thymidine is then phosphorylated by thymidine kinase, generating thymidine monophosphate (TMP) which is further phosphorylated to generate thymidine triphosphate.
Thymidine triphosphate not only regulates the thymidine salvage pathway by inhibition of thymidine kinase, but also inhibits the production of other deoxyribonucleotides by allosteric effects on ribonucleotide diphosphate reductase. The action of thymidine phosphorylase may therefore be to regulate the size of the intracellular thymidine nucleotide pool and hence the size of the other nucleotide pools via ribonucleotide diphosphate reductase as suggested by Shaw, T. et al,
Mutant Res.
200, 99-116 (1988), and hence regulate DNA synthesis.
There has been no suggestion that thymidine phosphorylase could be used as an extracellular growth factor in medicine.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a method of modulating cellular proliferation by the application of a thymidine phosphorylase to an organism. In a further aspect of the subject method, the thymidine phosphorylase is a conjugate which includes a targeting portion adapted to target the conjugate to a specific cell type or anatomical location.
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patent: 4178212 (1979-12-01), Krenitsky et al.
patent: 4347315 (1982-08-01), Krenitsky et al.
patent: 5227302 (1993-07-01), Heldin et al.
patent: 5314995 (1994-05-01), Fell et al.
patent: 5756686 (1998-05-01), Heldin et al.
patent: 0 289 229 (1988-11-01), None
Callard et al., The Cytokine Fact Book, Academic Press Limited, pp. 119, 122, 123 and 133, 1994.*
Yoshimura, A. et al. (1990) “Purification and tissue distribution of human thymidine phosphorylase; high in lymphocytes, reticulocytes and tumors” Biochim. Biophys. Acta. 1034(1):107-113, Apr. 1990.*
Usuki, K. et al. (1992) “Platelet-derived endothelial cell growth factor has thymidine phosphorylase activity” Biochem. Biophys. Res. Comm. 184(3):1311-1316, May 1992.*
Hammerberg, C. et al. (1991) “Elevated thymidine phosphorylase activity in psoriatic lesions accounts for the apparent presence of an epidermal ‘growth inhibitor,’ but is not itself growth inhibitory” J. Invest. Dermatol. 97:286-290, Aug. 1991.
Ballance David J.
Courtney Michael G.
Finnis Christopher J. A.
Sleep Darrell
Biswas Naomi S.
Delta Biotechnology Ltd.
Wax Robert A.
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