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

Abstract

Walker cells in vivo or in 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 from E. 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.

Nitroreductase-mediated cell ablation is very rapid and mediated by a p53-independent apoptotic pathway

“…10,11 This system is based on the E. coli enzyme nitroreductase (NTR) which activates the aziridine compound CB1954 (5-aziridin-1-yl-2,4-dinitrobenzamide), to a potent cytotoxic agent. The activated prodrug is thought to induce DNA interstrand cross-linking at a high frequency leading to cell death 12 which is not dependent on cell proliferation.…”

Esporos bacterianos para el tratamiento del cáncer.

“…Uno de los genes que ha sido probado es el gen codA que codifica para la síntesis de la enzima bacteriana Citosina Deaminasa (CD-asa) que convierte el compuesto aplicado por vía sistémica 5-fluorocitosina (5FC) en otro compuesto tóxico antitumoral, el 5-fluoracilo (5FU), que inhibe la síntesis de ADN y ARN resultando por lo tanto la disrupción de la síntesis proteica en las células tumorales, Fox et al, (1996). También se ha estudiado la inserción del gen que codifica para la Nitroreductasa (NTR), Minton et al, (1995), Lemmon et al, (1997), que convierte la prodroga CB1954 en un derivado 10.000 veces más tóxico, la 4-hidroxilamina (4HX) que causa el entrecruzamiento del ADN e induce apoptosis, Knox et al, (1988Knox et al, ( , 1993.…”

Enhanced efficacy of Escherichia coli nitroreductase/CB1954 prodrug activation gene therapy using an E1B-55K-deleted oncolytic adenovirus vector

“…In contrast, activation of CB1954 generates a potent DNA-crosslinking agent whose cytotoxicity is independent of ongoing DNA replication. 12,19,20 Treatment of mice with CB1954 can lead to complete regression of tumour xenografts that stably express NTR following in vitro retroviral transduction 21 or transfection. 22 Tumour regression and prolonged survival have also been observed following in vivo delivery of replication-defective adenovirus expressing NTR and treatment with CB1954.…”