ABSTRACT
AIMS: The role of CYP pharmacogenetics in the bioactivation of cyclophosphamide is still controversial. Recent clinical studies have suggested a role for either CYP2C19 or CYP2B6. The aim of this study was to clarify the role of these pharmacogenes. METHODS: We used a combined in vitro-in vivo approach to determine the role of these pharmacogenes in the bioactivation of the prodrug to 4-hydroxy cyclophosphamide (4-OHCP). Cyclophosphamide metabolism was determined in a human liver biobank (n= 14) and in patients receiving the drug for treatment of lupus nephritis (n= 16) RESULTS: In livers of known CYP2C19 and CYP2B6 genotype and protein expression we observed that there was a combined role for both CYP2C19 and CYP2B6 in the bioactivation of cyclophosphamide in vitro. The presence of at least one loss of function (LoF) allele at either CYP2C19 or CYP2B6 resulted in a significant decrease in both V(max) (P= 0.028) and CL(int) (P= 0.0017) compared with livers with no LoF alleles. This dual genotype relationship was also observed in a preliminary clinical study, with patients who had ≥1 LoF allele at either CYP2C19 or CYP2B6 also displaying significantly (P= 0.0316) lower bioactivation of cyclophosphamide. The mean 4-OHCP : CP bioactivation ratio was 0.0014 (95% CI 0.0007, 0.002) compared with 0.0071 (95% CI 0.0001, 0.014) in patients with no LoF alleles at either of these genes. CONCLUSIONS: The presence of ≥1 LoF allele(s) at either CYP2B6 or CYP2C19 appeared to result in decreased bioactivation of cyclophosphamide both in vitro and in patients. Further clinical studies to confirm this relationship are warranted.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Cyclophosphamide/pharmacokinetics , Immunosuppressive Agents/pharmacokinetics , Oxidoreductases, N-Demethylating/genetics , Adult , Aged , Alleles , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP2B6 , Cytochrome P-450 CYP2C19 , Female , Genotype , Humans , Lupus Nephritis/blood , Lupus Nephritis/genetics , Male , Microsomes, Liver/metabolism , Middle Aged , Oxidoreductases, N-Demethylating/metabolism , Tissue BanksABSTRACT
The dinitrobenzamide mustards are a class of bioreductive nitro-aromatic anticancer prodrugs, of which a phosphorylated analog (PR-104) is currently in clinical development. They are bioactivated by tumor reductases to form DNA cross-linking cytotoxins. However, their biotransformation in normal tissues has not been examined. Here we report the aerobic in vitro metabolism of three N-(2 hydroxyethyl)-3,5-dinitrobenzamide 2-mustards and the corresponding nonmustard analog in human, mouse, rat, and dog hepatic S9 preparations. These compounds have a range of mustard structures (-N(CH(2)CH(2)X)(2) where X = H, Cl, Br, or OSO(2)Me). Four metabolic routes were identified: reduction of either nitro group, N-dealkylation of the mustard, plus O-acetylation, and O-glucuronidation of the hydroxyethyl side chain. Reduction of the nitro group ortho to the mustard resulted in intramolecular alkylation and is considered to be an inactivation pathway, whereas reduction of the nitro group para to the mustard generated potential DNA cross-linking cytotoxins. N-Dealkylation inactivated the mustard moiety but may result in the formation of toxic acetaldehyde derivatives. Increasing the size of the nitrogen mustard leaving group abrogated the ortho-nitroreduction and N-dealkylation routes and thereby improved overall metabolic stability but had little effect on aerobic para-nitroreduction. All four compounds underwent O-glucuronidation of the hydroxyethyl side chain and further studies to elucidate the relative importance of this pathway in vivo are in progress.
Subject(s)
Antineoplastic Agents/metabolism , Mustard Compounds/metabolism , Prodrugs/metabolism , Animals , Antineoplastic Agents/chemistry , Dogs , Female , Glucuronides/metabolism , Humans , Male , Mice , Mice, Nude , Microsomes, Liver/metabolism , Molecular Structure , Mustard Compounds/chemistry , Prodrugs/chemistry , Rats , Rats, Sprague-DawleyABSTRACT
5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954), a promising anti-tumour compound, is associated with clinical hepatotoxicity. We have previously demonstrated that human liver preparations are capable of endogenous 2- and 4-nitroreduction of CB 1954 to generate highly potent cytotoxins. The present study initially examined the in vitro metabolism of CB 1954 in S9 preparations of several non-clinical species and strains. The CD-1 nu/nu mouse and Sprague-Dawley rat were subsequently chosen for further assessment of in vivo metabolism and hepatotoxicity of CB 1954, as well as the mechanisms that may be involved. Animals were administered the maximum tolerated dose (MTD). At 562 micromol/kg, the mouse exhibited transaminase elevation and centrilobular hepatocyte injury. Moreover, thiol adducts as well as hepatic glutathione depletion paralleled temporally by maximal nitroreduction were observed. The rat had a much lower MTD of 40 micromol/kg and showed signs of gastro-intestinal disturbances. In contrast to mouse, peri-portal damage and biliary changes were observed in rat without any alterations in plasma biomarkers or hepatic glutathione levels. Immunohistochemical analysis did not reveal any correlation between the location of injury and expression of cytochrome P450 reductase and NAD(P)H quinone oxidoreductase 1, two enzymes implicated in the bioactivation of this drug. In conclusion, the present study showed that following administration of CB 1954 at the respective MTDs, hepatotoxicity was observed in both mouse and rat. However, the degree of sensitivity to the drug and the mechanisms of toxicity involved appear to be widely different between CD-1 nu/nu mice and Sprague-Dawley rats.
