In vitro, cytochrome b 5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b 5 in the liver to create the hepatic microsomal cytochrome b 5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b 5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50 -90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b 5 null mice. These data indicate that microsomal cytochrome b 5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450-and substrate-dependent manner.Microsomal cytochrome b 5 is a 15.2-kDa hemoprotein, located in the endoplasmic reticulum with its obligate electron donor, cytochrome b 5 reductase. This protein is involved in fatty acid desaturation, reactivation of methemoglobin to hemoglobin, and electron transfer into the cytochrome P450 system (1-4).The role of cytochrome b 5 in cytochrome P450 monooxygenase reactions has been controversial for nearly 40 years (5, 6). Depending on the cytochrome P450 involved, the experimental conditions, and the substrate utilized, cytochrome b 5 has been shown to stimulate or inhibit cytochrome P450 reactions (2, 6). Indeed, in the case of cytochrome P450 CYP2B4, activity can be both stimulated or inhibited by the presence of the enzyme, depending on the concentration of cytochrome b 5 (7, 8). Cytochrome b 5 has been shown to affect the in vitro activities of a wide range of human P450s, such as CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, as well as the metabolism of an extensive number of commonly used drugs (9, 10). Although the mechanism(s) of the stimulatory effect of cytochrome b 5 on P450 metabolism appear complex, it is widely accepted that the P450 catalytic cycle involves the sequential transfer of two electrons from NADPH via P450 oxidoreductase (POR).2 Hildebrandt and Estabrook (5) concluded that cytochrome b 5 /cytochrome b 5 reductase was able to supply the second (rate-limiting) electron from NADH and possibly in a more rapid manner. However, recent data from Waskell et al. (8) suggest that cytochrome b 5 and POR are capable of reducing P450s at muc...

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH-cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b 5 , squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b 5 are helping to clarify the P450 isoform-and substrate-specific influences of cytochrome b 5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell-culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism.

We demonstrated recently that cytochrome b 5 plays an important in vivo role in hepatic cytochrome P450 (P450) function [J Biol Chem 283:31385-31393, 2008]. We have now generated a model in which cytochrome b 5 has been deleted in all tissues [cytochrome b 5 complete null (BCN)], which surprisingly results in a viable mouse despite the putative in vivo roles of this protein in lipid and steroid hormone metabolism and the reduction of methemoglobin. In contrast to the liver-specific deletion, complete deletion of cytochrome b 5 leads to a neonatal increase in the expression of many hepatic P450s at both the protein and mRNA level. In extrahepatic tissues, some changes in P450 expression were also observed that were isoformdependent. In vitro cytochrome P450 activities in liver, kidney, lung, and small intestine of BCN mice were determined for a range of model substrates and probe drugs; a profound reduction in the metabolism of some substrates, particularly in lung, kidney, and small intestine, was observed. In vivo, the metabolism of metoprolol was significantly altered in BCN mice, in contrast to the previous finding in the liver-specific cytochrome b 5 deletion, suggesting that extrahepatic cytochrome b 5 plays a significant role in its disposition. Testicular Cyp17 hydroxylase and lyase activities were also significantly reduced by cytochrome b 5 deletion, leading to significantly lower levels of testicular testosterone. The BCN mouse provides an additional model system with which to further investigate the functions of cytochrome b 5 , particularly in extrahepatic tissues.

Mutations in IDH induce epigenetic and transcriptional reprogramming, differentiation bias, and susceptibility to mitochondrial inhibitors in cancer cells. Here, we first show that cell lines, PDXs, and patients with acute myeloid leukemia (AML) harboring an IDH mutation displayed an enhanced mitochondrial oxidative metabolism. Along with an increase in TCA cycle intermediates, this AML-specific metabolic behavior mechanistically occurred through the increase in electron transport chain complex I activity, mitochondrial respiration, and methylation-driven CEBPα-induced fatty acid β-oxidation of IDH1 mutant cells. While IDH1 mutant inhibitor reduced 2-HG oncometabolite and CEBPα methylation, it failed to reverse FAO and OxPHOS. These mitochondrial activities were maintained through the inhibition of Akt and enhanced activation of peroxisome proliferator-activated receptor-γ coactivator-1 PGC1α upon IDH1 mutant inhibitor. Accordingly, OxPHOS inhibitors improved anti-AML efficacy of IDH mutant inhibitors in vivo. This work provides a scientific rationale for combinatory mitochondrial-targeted therapies to treat IDH mutant AML patients, especially those unresponsive to or relapsing from IDH mutant inhibitors.

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Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate–dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate–mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell–autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell–specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ–TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy. Significance: Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies. See related commentary by Zhu and Kwong, p. 604. This article is highlighted in the In This Issue feature, p. 587

ABSTRACT:5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor agent that is also active against autoimmune diseases. The intention of the present studies was to elucidate the role of selected liver enzymes in metabolism of C-1311 and the less active 8-methyl derivative, 5-diethylaminoethylamino-8-methoxyimidazoacridinone (C-1330). Compounds were incubated with rat liver microsomal fraction, with a set of 16 human liver protein samples, and with human recombinant isoenzymes of cytochrome P450, flavin monooxygenases (FMO), and UDPglucuronosyltransferase (UGT). Our results showed that C-1311 and C-1330 were metabolized with human liver microsomal enzymes but not with any tested human recombinant cytochromes P450 (P450s). Two of these, CYP1A2 and CYP3A4, were inhibited by both compounds. In addition, results of C-1311 elimination from hepatic reductase-null mice, in which liver NADPH-P450 oxidoreductase has been deleted indicated that liver P450s were slightly engaged in drug transformation. In contrast, both compounds were good substrates for human recombinant FMO1 and FMO3 but not for FMO5. The product of FMO metabolism, P FMO , which is identified as an N-oxide derivative, was identical to P3 R of liver microsomes. P3 R was observed even in the presence of the P450 inhibitor, 1-aminobenzotriazole, and it disappeared after heating. Therefore, FMO enzymes could be responsible for microsomal metabolism to P3 R ‫؍‬ P FMO . Glucuronidation on the 8-hydroxyl group of C-1311 was observed with liver microsomes supported by UDP-glucuronic acid and with recombinant UGT1A1, but it was not the case with UGT2B7. Summing up, we showed that, whereas liver P450 isoenzymes were involved in the metabolism of C-1311 to a limited extent, FMO plays a significant role in the microsomal transformations of this compound, which is also a specific substrate of UGT1A1.