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The bioactivation of CB 1954 and its use as a prodrug in antibody-directed enzyme prodrug therapy (ADEPT)

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Abstract

Walker cellsin vivo orin vitro are exceptionally sensitive to the monofunctional alkylating agent CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The basis of the sensitivity is that CB 1954 forms DNA interstrand crosslinks in Walker cells but not in insensitive cells. Crosslink formation is due to the aerobic reduction of CB 1954 to form 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide by the enzyme DT diaphorase. The 4-hydroxylamine can not crosslink DNA directly but requires further activation by a non-enzymatic reaction with a thioester (such as acetyl coenzyme A). As predicted from their measured DT diaphorase activities, a number of rat hepatoma and hepatocyte cell lines are also sensitive to CB 1954. However, no CB 1954-sensitive tumours or cell lines of human origin have been found. This is because the rate of reduction of CB 1954 by the human form of DT diaphorase is much lower than that of the Walker enzyme (ratio of kcat= 6.4). To overcome this intrinsic resistance of human cells towards CB 1954 a number of strategies have been developed. First, analogues have been developed that are more rapidly reduced by the human form of CB 1954. Second, the cytotoxicity of CB 1954 can be potentiated by reduced pyridinium compounds. Third, a CB 1954 activating enzyme can be targeted to human tumours by conjugating it to an antibody (ADEPT). A nitroreductase enzyme has been isolated fromE. coli that can bioactivate CB 1954 much more rapidly than Walker DT diaphorase and is very suitable for ADEPT. Thus CB 1954 may have a role in the therapy of human tumours.

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References

  1. Workman P, Morgan JE, Talbot K, Wright KA, Donaldson J, Twentyman PR: CB 1954 Revisited II. Toxicity and antitumour activity. Cancer Chemother Pharmacol 16: 9–14, 1986

    Google Scholar 

  2. Ross WCJ, Mitchley BCV: Nucleotoxic and mutagenic nitrogen mustards, epoxides, ethyleneimines and related substances. Ann Rep Br Empire Cancer Campn 42: 70, 1950

    Google Scholar 

  3. Hendry JA, Homer RF, Rose FL, Walpole AL: Cytotoxic agents: III. Derivatives of ethyleneimine. Br J Pharmacol 6: 357–410, 1951

    Google Scholar 

  4. Khan AH, Ross WCJ: Tumour-growth inhibitory nitrophenylaziridines and related compounds: Structure-activity relationships. Chem Biol Interact 1: 27–47, 1969/70

    Google Scholar 

  5. Cobb LM, Connors TA, Elson LA, Khan AH, Mitchley BCV, Ross WCJ, Whisson ME: 2,4-Dinitro-5-ethyleneiminobenzamide (CB1954): A potent and selective inhibitor of the growth of the Walker carcinoma 256. Biochem Pharmacol 18: 1519–1527, 1969

    Google Scholar 

  6. Connors TA, Melzack DH: Studies on the mechanism of action of 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), a selective inhibitor of the Walker tumour. Int J Cancer 7: 86–92, 1971

    Google Scholar 

  7. Phillips BJ, Ambrose EJ: Cell hybridization study of resistance to alkylating agents. Nature 231: 146–147, 1971

    Google Scholar 

  8. Kavi BR, Bhisey AN, Bapat CV: Effect of standard chemotherapeutic agents on human tumour cell lines ‘in vitro’. Ind J Cancer 12: 295–301, 1975

    Google Scholar 

  9. Bhat AV, Kesava-Rao KV, Bapat CV: Response of human tumours ‘in vitro’ to CB 1954 (5-(aziridinyl-2, 4-nitrobenzamide). Ind J Cancer 14: 132–135, 1977

    Google Scholar 

  10. Cobb LM: Toxicity of the selective antitumor agent 5-aziridino-2, 4-dinitrobenzamide in the rat. Toxicol Appl Pharmacol 17: 231–238, 1970

    Google Scholar 

  11. Sheard CE, Double JA, Berenbaum MC: The sensitivity to chemotherapeutic agents of a rat tumour grown in immunosuppressed mice. Br J Cancer 25: 838–833, 1971

    Google Scholar 

  12. Jarman M, Melzack DH, Ross WCJ: The metabolism of the anti-tumor agent 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954). Biochem Pharmacol 25: 2475–2478, 1976

    Google Scholar 

  13. Connors TA, Mandel HG, Melzack DH: Studies on the reversal of the selective anti-tumour effect of the aziridinyl derivative CB 1954 by 4-amino-5-imidazolecarboxamide. Int J Cancer 9: 126–132, 1972

    Google Scholar 

  14. Khan AH, Ross WCJ: Tumour-growth inhibitory nitrophenylaziridines and related compounds: Structure-activity relationships. II. Chem Biol Interact 4: 11–22, 1971/72

    Google Scholar 

  15. Workman P, White RAS, Talbot K: CB 1954 Revisited I. Disposition kinetics and metabolism. Cancer Chemother Pharmacol 16: 1–8, 1986

    Google Scholar 

  16. Venitt S: The differential response of resistant and sensitive strains ofEscherichia coli to the cytotoxic effects of 5-aziridino-2, 4-dinitrobenzamide (CB 1954). Chem Biol Interact 3: 177–191, 1971

    Google Scholar 

  17. Mandel HG, Connors TA, Melzack DH, Merai K: Studies on the mechanism of action of 5-aziridinyl-2,4-dinitrobenzamide in tumor cells. Cancer Res 34: 275–280, 1974

    Google Scholar 

  18. Hickman JA, Melzack DH: Protection against the effects of the antitumour agent CB 1954 by certain imidazoles and related compounds. Biochem Pharmacol 24: 1947–1952, 1975

    Google Scholar 

  19. Hickman JA, Melzack DH: Studies on the protection by imidazoles against the cytotoxicity of the antitumour alkylating agents melphalan and CB 1954. Biochem Pharmacol 25: 2489–2491, 1976

    Google Scholar 

  20. Tisdale MJ, Habberfield AD: Selective inhibition of ribonucleotide reductase by the monofunctional alkylating agent 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954). Biochem Pharmacol 29: 2845–2853, 1980

    Google Scholar 

  21. Tisdale MJ, Phillips BJ: Inhibition of cyclic 3′,5′-nucleotide phosphodiesterase - A possible mechanism of action of bifunctional alkylating agents. Biochem Pharmacol 24: 211–217, 1975

    Google Scholar 

  22. Tisdale MJ, Phillips BJ: Comparative effects of alkylating agents and other anti-tumour agents on the intracellular level of adenosine 3′,5′-monophosphate in Walker carcinoma. Biochem Pharmacol 24: 1271–1276, 1975

    Google Scholar 

  23. Tisdale MJ, Phillips BJ: The effect of alkylating agents on adenosine 3′,5′-monophosphate metabolism in Walker carcinoma. Biochem Pharmacol 25: 1793–1797, 1976

    Google Scholar 

  24. Tisdale MJ, Phillips BJ: Alterations in adenosine 3′,5′-monophosphate-binding protein in Walker carcinoma cells sensitive or resistant to alkylating agents. Biochem Pharmacol 25: 1831–1836, 1976

    Google Scholar 

  25. Tisdale MJ, Phillips BJ: Cyclic nucleotide metabolism in Walker carcinoma cells resistant to alkylating agents. Biochem Pharmacol 27: 947–952, 1978

    Google Scholar 

  26. Walling JM, Stratford IJ, Stevens MA: Chemopotentiation of CB 1954: the importance of postincubations and the possible involvement of poly(ADP-ribosylation). Int J Radiat Oncol Biol Phys 10: 1661–1664, 1984

    Google Scholar 

  27. Stratford IJ, Williamson C, Hoe S, Adams GE: Radiosensitizing and cytotoxic studies with CB 1954(2,4-dinitro-5-aziridinylbenzamide). Radiat Res 88: 502–509, 1981

    Google Scholar 

  28. Walling JM, Stratford IJ, Adams GE: Radiosensitization by the 2,4-dinitro-5-aziridinylbenzamide CB 1954: a structure/activity study. Int J Radiat Biol 52: 31–41, 1987

    Google Scholar 

  29. Tisdale MJ: Alkylating analogues of the tumour inhibitor 5-aziridinyl-2,4-dinitrobenzamide. Chem Biol Interact 3: 95–107, 1971

    Google Scholar 

  30. Roberts JJ, Friedlos F, Knox RJ: CB 1954 (24-dinitro-5-aziridinyl benzamide) becomes a DNA interstrand crosslinking agent in Walker tumour cells. Biochem Biophys Res Commun 140: 1073–1078, 1986

    Google Scholar 

  31. Knox RJ, Friedlos F, Jarman M, Roberts JJ: A new cytotoxic, DNA interstrand crosslinking agent, 5′(aziridin-1-yl)-4-hydroxylamino-2-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells. Biochem Pharmacol 37: 4661–4669, 1988

    Google Scholar 

  32. Venitt S, Crofton-Sleigh C: The toxicity and mutagenicity of the anti-tumour drug 5-aziridino-2,4-dinitrobenzamide (CB 1954) is greatly reduced in a nitroreductase-deficient strain ofE. coli. Mutagenesis 2: 375–381, 1987

    Google Scholar 

  33. Boland MP, Knox RJ, Roberts JJ: The differences in kinetics of rat and human DT diaphorase result in a differential sensitivity of derived cell lines to CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). Biochem Pharmacol 41: 867–875, 1991

    Google Scholar 

  34. Knox RJ, Friedlos F, Sherwood RF, Melton RG, Anlezark GM: The bioactivation of CB 1954. II. A comparison of anEscherichia coli nitroreductase and Walker DT diaphorase. Biochem Pharmacol 44: 2297–2301, 1992

    Google Scholar 

  35. Knox RJ, Friedlos F, Marchbank T, Roberts JJ: Bioactivation of CB 1954: reaction of the active 4-hydroxylamino derivative with thioesters to form the ultimate DNA-DNA interstrand crosslinking species. Biochem Pharmacol 42: 1691–1697, 1991

    Google Scholar 

  36. Knox RJ, Lydall DA, Friedlos F, Basham C, Rawlings CJ, Roberts JJ: The Walker 256 carcinoma: a cell type inherently sensitive only to those difunctional agents that can form DNA interstrand crosslinks. Mutation Res 255: 227–240, 1991

    Google Scholar 

  37. Knox RJ, Lydall DA, Friedlos F, Basham C, Roberts JJ: The effect of monofunctional or difunctional platinum adducts and of various other associated DNA damage on the expression of transfected DNA in a pair of mammalian cell lines sensitive or resistant to difunctional agents. Biochim Biophys Acta 908: 214–223, 1987

    Google Scholar 

  38. Friedlos F, Quinn J, Knox RJ, Roberts JJ: The properties of total adducts and interstrand crosslinks in the DNA of cells treated with CB 1954. Exceptional frequency and stability of the crosslink. Biochem Pharmacol 43: 1249–1254, 1992

    Google Scholar 

  39. Roberts JJ, Friedlos F: Quantitative estimation of cisplatininduced DNA interstrand cross-links and their repair in mammalian cells: Relationship to toxicity. Pharmac Ther 34: 215–246, 1987

    Google Scholar 

  40. Knox RJ, Boland M, Friedlos F, Coles B, Southan C, Roberts JJ: The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2). Biochem Pharmacol 37: 4671–4677, 1988

    Google Scholar 

  41. Ernster L: DT diaphorase: A historical review. Chemica Scripta 27A: 1–13, 1987

    Google Scholar 

  42. Riley RJ, Workman P: DT-diaphorase and cancer chemotherapy. Biochem Pharmacol 43: 1657–1669, 1992

    Google Scholar 

  43. Friedlos F, Jarman M, Davies LC, Boland MP, Knox RJ: Identification of novel reduced pyridinium derivatives as synthetic co-factors for the enzyme DT diaphorase (NAD (P)H dehydrogenase (quinone) (EC 1.6.99.2). Biochem Pharmacol 44: 25–31, 1992

    Google Scholar 

  44. Roberts JJ, Marchbank T, Kotsaki-Kovatsi VP, Boland MP, Friedlos F, Knox RJ: Caffeine, aminoimidazolecarboxamide and dicoumarol, inhibitors of NAD(P)H dehydrogenase (quinone) (DT diaphorase), prevent both the cytotoxicity and DNA interstrand crosslinking produced by 5-(aziridin-1-yl)-2, 4-dinitrobenzamide (CB 1954) in Walker cells. Biochem Pharmacol 38: 4137–4143, 1989

    Google Scholar 

  45. Schor NA, Ogawa K, Lee G, Farber E: The use of the D-T diaphorase for the detection of foci of early neoplastic transformation in rat liver. Cancer Letters 5: 167–171, 1978

    Google Scholar 

  46. Pickett CB, Williams JB, Lu AYH, Cameron RG: Regulation of glutathione transferase and DT-diaphorase mRNAs in persistent hepatocyte nodules during chemical hepatocarcinogenesis. Proc Natl Acad Sci 81: 5091–5095, 1984

    Google Scholar 

  47. Beyer RE, Segura-Aguilar JE, Ernster L: The anticancer enzyme DT diaphorase is induced selectively in liver during ascites hepatoma growth. Anticancer Res 8: 233–238, 1988

    Google Scholar 

  48. Martin LF, Patrick SD, Wallin R: DT-diaphorase in morbidly obese patients. Cancer Lett 36: 341–347, 1987

    Google Scholar 

  49. Smith D, Martin LF, Wallin R: Human DT-diaphorase, a potential cancer protecting enzyme. Its purification from abdominal adipose tissue. Cancer Lett 42: 103–112, 1988

    Google Scholar 

  50. Berger MS, Talcott RE, Rosenblum ML, Silva M, Ali-Osman F, Smith MT: Use of quinones in brain-tumor therapy: Preliminary results of preclinical laboratory investigations. J Toxicol Environ Health 16: 713–719, 1985

    Google Scholar 

  51. Beyer RE, Seugra-Aguilar J, Lind C, Castro VM: DT diaphorase activity in various cells in culture with emphasis on induction in ascites hepatoma cells. Chemica Scripta 27A: 145–150, 1987

    Google Scholar 

  52. Schor NA, Cornelisse CJ: Biochemical and quantitative histochemical study of reduced pyridine nucleotide dehydrogenation by human colonic carcinomas. Cancer Res 43: 4850–4855, 1983

    Google Scholar 

  53. Jaiswal AK, McBride OW, Adesnik M, Nebert DW: Human dioxin-inducible cytosolic (NAD(P)H: menadione oxidoreductase: cDNA sequence and localization of gene to chromosome 16. J Biol Chem 263: 13572–13578, 1988

    Google Scholar 

  54. Boland MP: Studies on the bioactivation of 5-(aziridin-1-yl)-2-4-dinitrobenzamide (CB 1954). Ph.D. Thesis, University of London, 1991

  55. Lambert AJ, Friedlos F, Boland MP, Knox RJ: A CB 1954 analogue that is highly cytotoxic in human tumour cell lines (Abstract). Br J Cancer 65 (Suppl. XVI): 59, 1992

    Google Scholar 

  56. Friedlos F, Biggs PJ, Abrahamson JA, Knox RJ: Potentiation of CB 1954 cytotoxicity by reduced pyridine nucleotides in human tumour cells by stimulation of DT diaphorase activity. Biochem Pharmacol 44: 1739–1743, 1992

    Google Scholar 

  57. Friedlos F, Knox RJ: Metabolism of NAD(P)H by blood components: Relevance to bioreductively activated prodrugs in a targeted enzyme therapy system. Biochem Pharmacol 44: 631–635, 1992

    Google Scholar 

  58. Bagshawe KD: Antibody directed enzymes revive anti-cancer prodrugs concept. Br J Cancer 56: 531–532, 1987

    Google Scholar 

  59. Bagshawe KD: Towards generating cytotocix agents at cancer sites. Br J Cancer 60: 275–281, 1989

    Google Scholar 

  60. Bagshawe KD: Antibody-directed enzyme/prodrug therapy (ADEPT). Biochem Soc Transactions 18: 750–751, 1990

    Google Scholar 

  61. Bagshawe KD, Springer CJ, Searle F, Antoniw P, Sharma SK, Melton RG, Sherwood RF: A cytotoxic agent can be generated selectively at cancer sites. Br J Cancer 58: 700–703, 1988

    Google Scholar 

  62. Senter PD, Saulnier MG, Schreiber GJ, Hirschberg DL, Brown JP, Hellstrom I, Hellstrom KE: Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate. Immunol 85: 4842–4846, 1988

    Google Scholar 

  63. Shepherd TA, Jungheim LN, Meyer DM, Starling JJ: A novel targeted delivery system utilizing a cephalosporin-oncolytic prodrug activated by an antibody-lactamase conjugate for the treatment of cancer. Bioorg Med Chem Lett 1: 21–26, 1991

    Google Scholar 

  64. Alexander RP, Beeley NRA, O'Driscoll M, O'Neill FP, Millican TA, Pratt AJ, Willenbrock FW: Cephalosporin nitrogen mustard carbamate prodrugs for ‘ADEPT’. Tetrahedron Lett 32: 3269–3272, 1991

    Google Scholar 

  65. Kerr DE, Senter PD, Burnett WV, Hirschberg DL, Hellstrom I, Hellstrom KE: Antibody-penicillin-V-amidase conjugates kill antigen-positive tumor cells when combined with doxorubicin phenoxyacetamide. Cancer Immunol Immunother 31: 202–206, 1990

    Google Scholar 

  66. Hellstrom KE, Senter PD: Activation of prodrugs by targeted enzymes. Eur J Cancer 27: 1342–1343, 1991

    Google Scholar 

  67. Anlezark GM, Melton RG, Sherwood RF, Coles B, Friedlos F, Knox RJ: The bioactivation of CB 1954. I. Purification and properties of a nitroreductase enzyme forEscherichia colia potential enzyme for antibody directed enzyme prodrug therapy (ADEPT). Biochem Pharmacol 44: 2289–2295, 1992

    Google Scholar 

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  1. Molecular Pharmacology Unit, Section of Drug Development, Block F, Institute of Cancer Research, Cotswold Rd., SM2 5NG, Sutton, Surrey, United Kingdom

    Richard J. Knox, Frank Friedlos & Marion P. Boland

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  1. Richard J. Knox
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Knox, R.J., Friedlos, F. & Boland, M.P. The bioactivation of CB 1954 and its use as a prodrug in antibody-directed enzyme prodrug therapy (ADEPT). Cancer Metast Rev 12, 195–212 (1993). https://doi.org/10.1007/BF00689810

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