Cytochromeb₅Is a Major Determinant of Human Cytochrome P450 CYP2D6 and CYP3A4 Activity In Vivo

Abstract

The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and .80% of therapeutic drugs. Cytochrome P450 (P450) activity is regulated transcriptionally and by the rate of electron transfer from P450 reductase. In vitro studies have demonstrated that cytochrome b 5 (Cyb5) also modulates P450 function. We recently showed that hepatic deletion of Cyb5 in the mouse (HBN) markedly alters in vivo drug pharmacokinetics; a key outstanding question is whether Cyb5 modulates the activity of the major human P450s in drug disposition in vivo. To address this, we crossed mice humanized for CYP2D6 or CYP3A4 with mice carrying a hepatic Cyb5 deletion. In vitro triazolam 4-hydroxylation (probe reaction for CYP3A4) was reduced by .50% in hepatic microsomes from CYP3A4-HBN mice compared with controls. Similar reductions in debrisoquine 4-hydroxylation and metoprolol a-hydroxylation were observed using CYP2D6-HBN microsomes, indicating a significant role for Cyb5 in the activity of both enzymes. This effect was confirmed by the concentration-dependent restoration of CYP3A4-mediated triazolam turnover and CYP2D6-mediated bufuralol and debrisoquine turnover on addition of Escherichia coli membranes containing recombinant Cyb5. In vivo, the peak plasma concentration and area under the concentration time curve from 0 to 8 hours (AUC 0-8 h ) of triazolam were increased 4-and 5.7-fold, respectively, in CYP3A4-HBN mice. Similarly, the pharmacokinetics of bufuralol and debrisoquine were significantly altered in CYP2D6-HBN mice, the AUC 0-8 h being increased ∼1.5-fold and clearance decreased by 40-60%. These data demonstrate that Cyb5 can be a major determinant of CYP3A4 and CYP2D6 activity in vivo, with a potential impact on the metabolism, efficacy, and side effects of numerous therapeutic drugs.

The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

“…5) pharmacokinetics were determined in vivo in CYP3A4-HBN and CYP2D6-HBN mice, respectively, metabolism was again shown to be profoundly reduced in mice lacking hepatic b 5 , with significant increases in C max and AUC and decreased clearance relative to control animals. The minor perturbations in hepatic lipids reported in the CYP3A4-HBN and CYP2D6-HBN mice (Henderson et al, 2015) strongly support the view that the effects observed on drug disposition in vivo are not a result of changes to the lipid composition of the ER but rather reflect the consequences of a lack of b 5 . It is thus possible that the expression of b 5 , and its functionality, may make a significant contribution to the observed heterogeneity in plasma levels of many commonly prescribed drugs.…”

“…Using substrates for human CYP3A4 (triazolam) and CYP2D6 (debrisoquine), it was shown in vitro in hepatic microsomes that the metabolism of these drugs was significantly reduced in the absence of b 5 . Moreover, in both cases, addition of exogenous b 5 to reaction mixtures restored activity in a dose-dependent manner (Henderson et al, 2015). When triazolam (Fig.…”

“…To this end, we crossed the hepatic b 5 null (HBN) (Finn et al, 2008) mouse with two mouse lines in which key human P450s-CYP2D6 or CYP3A4-are expressed on a Cyp2d or Cyp3a gene cluster null background (Hasegawa et al, 2011;Scheer et al, 2012). CYP2D6-HBN and CYP3A4-HBN mice (Henderson et al, 2015) were fertile and phenotypically normal and had only minor changes in hepatic lipids, perhaps surprising given the known involvement of b 5 in lipid desaturation (Jeffcoat et al, 1977;Finn et al, 2011). Using substrates for human CYP3A4 (triazolam) and CYP2D6 (debrisoquine), it was shown in vitro in hepatic microsomes that the metabolism of these drugs was significantly reduced in the absence of b 5 .…”

Membrane Associated Progesterone Receptors: Promiscuous Proteins with Pleiotropic Functions – Focus on Interactions with Cytochromes P450

“…Since POR is present in the endoplasmic reticulum (ER) at sub-stoichiometric amounts compared to CYP, it has been proposed that their interactions involve transient oligomeric complexes (Backes and Kelley, 2003). In contrast to POR, the small hemoprotein cytochrome b5 (CYB5), which is located together with CYB5-reductase and the other components on the cytoplasmic side of the ER, is not an obligatory electron donor for CYPs but acts as modulator of enzyme activity in CYP- and sometimes substrate-dependent way (Henderson et al, 2015). For a long time, POR and CYB5 were the only proteins known to functionally interact with CYPs during the microsomal monooxygenase reaction.…”

Cholic Acid Feeding Leads to Increased CYP2D6 Expression in CYP2D6-Humanized Mice

“…4B) but exhibited large variability. Considering that CYP2D6 activity can be regulated by the rate of electron transfer from CPR and Cyb5 (Henderson et al, 2015), we measured the activity of CPR and the content of Cyb5 in S9 fractions. Neither CPR activity nor Cyb5 content in hepatic S9 fractions differed between the control and CA-fed groups (Fig.…”