Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D203/00—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom
  • C07D203/04—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
  • C07D203/06—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
  • C07D203/08—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring nitrogen atom
  • C07D203/14—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring nitrogen atom with carbocyclic rings directly attached to the ring nitrogen atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
  • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/10—Nitrogen as only ring hetero atom
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
  • C12Y106/05—Oxidoreductases acting on NADH or NADPH (1.6) with a quinone or similar compound as acceptor (1.6.5)
  • C12Y106/05002—NAD(P)H dehydrogenase (quinone) (1.6.5.2)

Definitions

• 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 anti-tumour pro-drugs into anti-tumour agents.
• the alkylating agent 5- (aziridin-1-yl) -2,4- dinitrobenza ide (hereinafter designated CB 1954) has been known, almost for 20 years, as an interesting experimental compound of unique selectivity.
• 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.
• the enzyme does not reduce 2,4-dinitrophenol but will reduce 2,6-dichlorophenolindophenol with either NADH or N?-PH as cofactors.
• the enzyme does not readily reduce the 2-nitro group of CB 1954.
• the fact that the new enzyme can convert CB 1954 into an anti-tumour compound identified as the 4-hydroxylamino derivative has been confirmed by the unambiguous synthesis of 5- (aziridin-1-yl)-4- hydroxylamino-2-nitrobenzamide which was shown to be identical with the product obtained by Walker cell enzyme conversion of CB 1954.
• the enzyme used in the invention can be isolated from the cells of the Walker tumour by extracting the Walker cells and subjecting the extract to sucessive gel filtration and ion exchange high performance liquid chromatography. Given the relatively small size of the enzyme, as an alternative to recovery from natural sources, the enzyme could be prepared synthetically, by conventional peptide synthesis, e.g.
• DNA encoding the enzyme can be obtained by preparing a synthetic polynucleotide encoding the enzyme. Alternatively, conventional techniques could be used using reverse transcriptase with messenger RNA isolated from the Walker cells.
• the enzyme is of particular interest in that it can be utilised to increase the area of applicability of CB 1954 which now has potential clinical application as a pro-drug in view of its relatively low cytotoxicity but, under the influence of the enzyme, is converted into its much more toxic 4-hydroxylamino derivative.
• CB 1954 As a result of our discovery, a further investigation has been made into the activity of CB 1954 against various tumour cells and we found activity (as CB 1954 or as a metabolite ' thereof) against various ovarian, renal, breast and lung tumour cells.
• the availability of the enzyme is of value not only in that it can be used to convert a cytotoxic pro-drug into the cytotoxic drug as a general technique but, by using existing techniques, it is possible to conjugate the enzyme to an antibody, particularly a monoclonal antibody, that will bind with tumour associated antigens. In this way, the enzyme can be directed to the tumour site and bring about a localisation of enzyme in the region of the tumour. Subsequent administration of the cytotoxic pro-drug will ensure that there is a localisation of cytotoxic drug in the region of the tumour with a correspondingly lower concentration of cytotoxic drug and higher concentration of the less toxic pro-drug in other parts of the body.
• the enzyme is also of interest in that antibodies, particularly monoclonal antibodies, to the enzyme can be generated by conventional techniques and assist in the detection of CB 1954 sensitive tumours.
• a new anti-tumour compound which is 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobe ⁇ zamide and its derivatives.
• derivatives we mean derivatives of the hydroxy group e.g., ester and ether derivatives.
• the ester derivatives of the present invention are essentially esters of the hydroxylamine with lower alkanoic acids, e.g. those containing from 1 to 6 carbon atoms and in this category, attention focusses on the saturated alkanoic acids, particularly those containing a straight chain of 2 to 4 carbon atoms such as the acetate and propionate ester. Esters with other acids, e.g., phosphate and sulphate esters are also within the scope of the invention.
• Ether derivatives of the hydroxylamine are preferably those containing a hydrocarbyl ether group of 1 to 6 carbon atoms, particularly where the hydrocarbyl group is a C,-C. al'kyl group such as methyl or ethyl.
• the new anti-tumour compounds of the present invention are compounds of the formula:
• R is H or an acyl group, e.g., a carboxylic acyl group of up to 6 carbon atoms particularly an acetyl group or R is a phosphate or sulphate group.
• the hydroxylamines of the present invention can be prepared by reduction of the corresponding 4-nitro compound, that is CB 1954. We have found that the most satisfactory method of selective reduction of the 4-nitro group is to use zinc dust and ammonium acetate in acetone in accordance with the method of Jarman e_t a_l, Biomed. Mass. Spectrom. 1983, 10, 1983.
• the 4-hydroxylamine obtained by the selective reduction method mentioned above can then be converted into its ester or ether derivatives by methods known per se using the appropriate esterifying or etherifying agent.
• An alternative method of preparing the hydroxylamines of the present invention is, of course, by selective reduction of CB 1954 using the enzyme EC 1.6.99.2 and isolating the resulting 4-hydroxylamino compound.
• Further aspects of the present invention comprise methods of controlling neoplastic tissue growth in humans or animals by converting cytotoxic pro-drugs _in vivo into cytotoxic drugs by the use of the enzyme EC
• a cytotoxic pro-drug e.g. CB 1954
• intravenous injection at a rate of about 0.2-1.6 mg/Kg body weight corresponding to a clinical dosage of about 12.5 to 100 mg.
• a specific dosage regime involves injection of 25 mg followed by a further 12.5 mg every third day.
• compositions comprising the hydroxylamines of the present invention, or their ether and ester derivatives, in a formulation suitable for parenteral administration.
• Such compositions will normally be prepared in a form suitable for intravenous administration.
• the invention also extends to methods of treatment for controlling neoplastic tissue growth by administering to a human in need of such treatment effective amounts e.g. by the intravenous route, of CB 1954 and EC 1.6.99.2.
• the invention provides a prodrug/enzyme system for use in controlling neoplastic tissue growth in humans comprising (1) an amount of 5-(aziridin-1-yl)-2,4-dinitrobenzamide in a form suitable for parenteral administration in association with (2) an amount of the enzyme EC 1.6.99.2 such that the 5-(aziridin-l-yl)-2,4-dinitrobenzamide is converted in vivo into 5-(aziridin-l-yl)-4-hydroxylamino-2- nitrobenzamide.
• Figure 1 illustrates the results of cytotoxicity tests described in Example 2.
• Figure 2A and 2B illustrate the results of comparative tests described in Examples 3 and 4.
• Figures 3 and 4 illustrate the results of cytotoxicity tests described in Example 6.
• the present invention is further illustrated by the following Examples.
• Protein purity was confirmed by PAGE as a single band of 33.5 Kd. Yield was about 10 mg per 10 cells.
• the protein was quantified by its absorbance at 450 nm given that 1 mg per ml gives an absorbance of 0.125 (10 mm pathlength) .
• the yellow enzyme was heated at 56° for 20 minutes and the separated flavin cofactor removed from the pure protein by ultrafiltration.
• the pure protein having a molecular weight of about 33.5 Kd was then subjected to amino acid composition analysis when the following results were obtained. Cone, nmoles/ml

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• 1988
  • 1988-02-22 GB GB888804068A patent/GB8804068D0/en active Pending
• 1989
  • 1989-02-22 EP EP89301678A patent/EP0330432A1/de not_active Withdrawn
  • 1989-02-22 AU AU34398/89A patent/AU3439889A/en not_active Abandoned
  • 1989-02-22 WO PCT/GB1989/000179 patent/WO1989007592A1/en not_active Application Discontinuation
  • 1989-02-22 ZA ZA891363A patent/ZA891363B/xx unknown
  • 1989-02-22 EP EP89903769A patent/EP0429454A1/de active Pending
  • 1989-02-22 JP JP1503762A patent/JPH03503761A/ja active Pending

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