Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1994-07-27
1997-05-27
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
4352523, 536 232, C12N 902, C12N 120, C07H 2104
Patent
active
056331584
DESCRIPTION:
BRIEF SUMMARY
This APPLN is a 371 of PCT/GB92/01947 filed Oct. 23, 1992. THIS INVENTION relates to the control of neoplastic tissue growth and is particularly concerned with the provision of new anti-tumour agents and with enzymes capable of converting prodrugs into anti-tumour agents.
The alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (hereinafter designated CB 1954) has been known, almost for 20 years, as an interesting experimental compound of unique selectivity. Although CB 1954 is structurally quite closely related to numerous other known alkylating agents which have a relatively broad range of activity, CB 1954 exhibits considerable activity against the Walker tumour cells in vivo or in vitro but was thought to be virtually inactive against other tumours.
It was recently discovered that the selectivity of CB 1954 arose from the fact that it was not an anti-tumour agent per se but was a prodrug for an anti-tumour agent generated from CB 1954 by a nitroreductase enzyme found in the Walker cell. This nitroreductase from the Walker cell was subsequently shown to be an enzyme known from other sources which was an NAD(P)H dehydrogenase (quinone) classified as EC.1.6.99.2, see Robertson et al, J. Biol. Chem. 261, 15794-15799 (1986).
In the course of the previous investigations with CB 1954, it was found that the Walker cell enzyme EC.1.6.99.2 had the ability to reduce the 4-nitro group of CB 1954 to the corresponding hydroxylamine and that it was the resulting 5-(aziridin-1-yl)-2-nitro-4-hydroxylamino-benzamide that was the active anti-tumour agent.
The use of prodrugs represents a clinically very valuable concept in cancer therapy since, particularly where the prodrug is to be converted to an anti-tumour agent under the influence of an enzyme that is linkable to a monoclonal antibody that will bind to a tumour associated antigen, the combination of such a prodrug with such an enzyme monoclonal/antibody conjugate represents a very powerful clinical agent.
We have now discovered new nitroreductases, obtainable from bacterial sources, that are of interest in that not only are they capable of converting CB 1954 into an active anti-tumour agent, but also, unlike EC.1.6.99.2, capable of converting CB 1954 analogues which are also prodrugs into active anti-tumour agents.
DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the results of an experiment in which CB 1954 (100 .mu.M) and reduced cofactor (500 .mu.M) were incubated with enzyme (2 mg/ml) E. coli nitroreductase (A) or 25 .mu.g/ml Walker DT diaphorase (B) in 10 mM sodium phosphate buffer (pH7) in air at 37.degree. C. At various times aliquots (10 .mu.l) were injected onto a Partisil SCX (240.times.4.7 mm) HPLC column and eluted isocratically (2 ml/min) with 100 mM NaH.sub.2 PO.sub.4. The eluate was continuously monitored for absorption at 320, 260 and 360 nm and the concentration of CB 1954 calculated by integration of the peak corresponding to this compound on the HPLC trace.
FIG. 2 shows the formation of actinomycin D (AMD) during incubation of an AMD prodrug with a nitroreductase of the present invention.
FIG. 3 shows the formation of mitomycin C (MC) during incubation of an MC prodrug with a nitroreductase of the present invention.
FIG. 4 shows the binding in vitro of an antibody-enzyme conjugate according to the invention to cells.
The present invention provides a nitroreductase, obtainable from a bacterium having the following characteristics as exemplified by examples isolated from Escherichia coli B and Bacillus amyloliquifaciens: Kilodaltons; analogues thereof to a cytotoxic form e.g. the hydroxylamine.
The nitroreductases of the invention occur naturally within the cells of E. coli B, E. coli C and other E. coli strains e.g. K12 type as well as other gram negative organisms e.g. Thermus aquaticus, and gram positive bacteria such as Bacillus amyloliquifaciens and Bacillus caldotenax. They can be recovered from such cells by disrupting the cells and subjecting the cell contents to chromatographic separation and isolating the nitroreductase.
For example, the ni
REFERENCES:
patent: 4680382 (1987-07-01), Sengupta
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Anlezark Gillian
Connors Thomas
Friedlos Frank
Jarman Michael
Knox Richard
Cancer Research Campaign - Technology Limited
Hobbs Lisa J.
Wax Robert A.
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