
Antitumour prodrug development using cytochrome P450 (CYP) mediated activation
No Abstract
Keywords: AQ4 (T50/80 Km = 26.7 μM; AQ4N metabolism should be restricted to hypoxid tumour cells. The isoform selectivity of AQ4N reduction; An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme; Vmax = 0.42 μM/mg protein/min) via AQM; Vmax = 0.43 μM/mg protein/min; RIF-1 Km = 33.5 μM; Vmax = 3.5 pmol/mg protein/min) and normal kidney Km = 4 μM; Vmax = 4.0 pmol/mg protein/min); a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite; a mono-N-oxide intermediate (T50/80 Km Km = 37.5 μM; Vmax = 1.4 μM/mg protein/min; RIF Km = 37.5 μM; Vmax = 1.2 μM/mg protein/min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3 fold over a 12 h period; after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely; are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed; both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species; breast; but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (Km = 4 μM; cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air; despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4; in addition to its air sensitivity; including colon; including identification of prodrugs activated by tumour-specific polymorphic CYPs; indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active site bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.bioreduction; CYP polymorphism; cytochrome P450; gene therapy; hypoxia; prodrugs; kidney and prostate; liver; lung; or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers; the process requires low oxygen conditions. Hence; use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxid tumour regions. The last approach is best exemplified by AQ4N
Document Type: Research Article
Affiliations: 1: School of Biomedical Sciences, University of Ulster at Jordanstown, Jordanstown, UK 3: School of Pharmacy and Pharmaceutical Sciences, De Montfort University, Leicester LE1 9BH, UK
2:Publication date: December 1, 1999
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