A Mouse Model with Liver-Specific Deletion and Global Suppression of the NADPH-Cytochrome P450 Reductase Gene: Characterization and Utility for in Vivo Studies of Cyclophosphamide Disposition

Abstract

A mouse model combining liver-specific deletion with global suppression of the NADPH-cytochrome P450 reductase gene (Cpr) has been developed and characterized. These mice (designated "Cpr-low and liver-Cpr-null" or CL-LCN) retain the respective phenotypes of the previously reported Cpr-low (CL) and liver-Cpr-null (LCN) mouse strains, but hepatic deletion of the Cpr gene occurs at an earlier age in the CL-LCN mouse than in the LCN mouse. Residual hepatic microsomal CPR activities are very low in both CL-LCN and LCN mice (at 1.5 and 2.5% of wild-type levels, respectively). The utility of CL-LCN mice for in vivo drug metabolism studies was explored using the cytochrome P450 (P450) prodrug cyclophosphamide (CPA). After i.p. injection of CPA at 100 mg/kg, the t 1/2 and the area under the concentration-time curve for plasma CPA were significantly increased in mice deficient in liver CPR compared with wild-type controls, indicating a lower rate of metabolism, with the effects greater in CL-LCN mice than in LCN mice. Correspondingly, substantial decreases in C max , and increases in T max , and t 1/2 , of 4-hydroxycyclophosphamide (4-OH-CPA) formation were observed in both LCN and CL-LCN mice relative to wild-type controls. In contrast, CPA and 4-OH-CPA pharmacokinetic parameters were essentially unchanged in CL mice, relative to wild-type controls. The slower elimination of CPA in CL-LCN mice compared with LCN mice suggests a role for extrahepatic P450 in the in vivo metabolism of CPA and demonstrates the utility of the CL-LCN model in determining the role of extrahepatic P450 enzymes in drug metabolism and chemical toxicity.

Enhancement of intratumoral cyclophosphamide pharmacokinetics and antitumor activity in a P450 2B11-based cancer gene therapy model

“…4-OH-CPA was dose-dependent, without reaching saturation at 140 mg CPA kg À1 BW (Figure 3c). The inability to saturate the active site of P450 2B11 at this dose, which corresponds to a C max (CPA) of B200 mM, 32 suggests there is restricted uptake of circulating CPA by the tumor cells, reducing the availability of CPA for i.t. metabolism.…”

Role of Small Intestinal Cytochromes P450 in the Bioavailability of Oral Nifedipine

“…C57BL/6 (B6) mice, which were used as the wild-type control strain for the CL mice, were obtained from breeding stocks maintained at the Wadsworth Center. LCN (B6/N10) and WT control littermates (Cpr lox/lox ), CL (B6/N1), and CL-LCN (B6/N1.5) mouse models have been described recently (Gu et al, , 2007Wu et al, 2003Wu et al, , 2005. All studies with mice were approved by the Wadsworth Center Institutional Animal Care and Use Committee.…”

“…Hepatic CPR/P450 was also found to play significant roles in systemic disposition of cyclophosphamide (Pass et al, 2005) but apparently not in the clearance of diclofenac and doxorubicin (Henderson et al, 2006). A role for extrahepatic tissue P450 enzymes in the in vivo disposition of cyclophosphamide was subsequently demonstrated through comparison of the pharmacokinetics of cyclophosphamide metabolism between the LCN and CL-LCN mice (Gu et al, 2007). It should be noted that none of these studies compared the impact of CPR deficiency on the disposition of intraperitoneally administered drugs with the impact on orally administered drugs; for oral drugs, the small intestine is expected to play a greater role in first-pass metabolism than for drugs given intraperitoneally (Grundy et al, 1997).…”

“…In the LCN mouse, Cpr expression is abolished in essentially all hepatocytes, but it is normal in other tissues . In the CL-LCN mouse, which combines the phenotypes of the CL and LCN mice, Cpr expression is absent in hepatocytes of adult mice, whereas CPR levels are substantially decreased in other tissues (Gu et al, 2007). These mouse models have made it possible to determine the relative contributions of liver and extrahepatic tissue P450 enzymes in the disposition and bioactivation of xenobiotic compounds.…”

“…Several mouse models targeting the Cpr locus have been developed, including the "Cpr-low" (CL) mouse (Wu et al, 2005), the "liver-Cpr-null" (LCN) mouse Henderson et al, 2003), and the "Cpr-low and liver-Cpr-null" (CL-LCN) mouse (Gu et al, 2007). In the CL mice, CPR expression is decreased by Ͼ70% in all tissues examined, including olfactory mucosa, adrenal gland, brain, testis, ovary, lung, kidney, liver, and heart (Wu et al, 2005).…”