Inhibition of Human Cytochrome P450 3A4 by Cholesterol

Abstract

Cholesterol has been shown to be hydroxylated at the 4␤-position by cytochrome P450 3A4, and the reaction occurs in vivo (Bodin, K., Andersson, U., Rystedt, E., Ellis, E., Norlin, M., Pikuleva, I., Eggertsen, G., Björkhem, I., and Diczfalusy, U. (2002) J. Biol. Chem. 277, 31534 -31540). If cholesterol is a substrate of P450 3A4, then it follows that it should also be an inhibitor, particularly in light of the high concentrations found in liver. Heme perturbation spectra indicated a K d value of 8 M for the P450 3A4-cholesterol complex. Cholesterol inhibited the P450 3A4-catalyzed oxidations of nifedipine and quinidine, two prototypic substrates, in liver microsomes and a reconstituted enzyme system with K i ϳ 10 M in an apparently non-competitive manner. The concentration of cholesterol could be elevated 4 -6-fold in cultured human hepatocytes by incubation with cholesterol; the level of P450 3A4 and cell viability were not altered under the conditions used. Nifedipine oxidation was inhibited when the cholesterol level was increased. We conclude that cholesterol is both a substrate and an inhibitor of P450 3A4, and a model is presented to explain the kinetic behavior. We propose that the endogenous cholesterol in hepatocytes should be considered in models of prediction of metabolism of drugs and steroids, even in the absence of changes in the concentrations of free cholesterol.Cytochrome P450 enzymes are nearly ubiquitous in nature. These hemoproteins are involved in the metabolism of numerous steroids, drugs, carcinogens, and other substrates in mammals and, in addition, in many pathways of secondary metabolism and defense in plants and microorganisms (1). Humans have 57 CYP genes potentially coding for P450 enzymes (2). One of these, P450 3A4, is involved in the metabolism of approximately one-half of the drugs marketed today (of those known to be oxidized) (2-4). In addition to drugs, this enzyme also has roles in both the bioactivation and detoxication of chemical carcinogens, e.g. aflatoxin B 1 (2, 5, 6). P450 3A4 also has relatively high catalytic activities in the oxidation of a number of steroids, e.g. testosterone, progesterone, and 17␤-estradiol (7,8). More recently, P450 3A4 has been shown to catalyze the 4␤-hydroxylation of cholesterol (9, 10), and the product has been proposed to be an in vivo biomarker of the activity of P450 3A4 (and the related protein P450 3A5, which is generally expressed at lower levels) (11). P450 3A4 has been described as displaying rather schizophrenic behavior toward some ligands, which has been difficult to understand (2). Evidence for cooperativity (both homotropic and heterotropic) was first described in 1993-1994 (12, 13). Subsequently, considerable work has provided insight into the phenomenon, although not all aspects have been clarified (reviewed in Refs. 2 and 14). A general conclusion is that multiple ligands can be accommodated and that this multiplicity somehow accounts for the cooperative behavior that is seen, e.g. due to forcing substrates into juxtapositions...

4β-Hydroxycholesterol as an Endogenous Biomarker of CYP3A Activity in Cynomolgus Monkeys

“…The K m and V max values were estimated to be 204 mM and 0.600 pg/pmol/min, respectively, for cynomolgus CYP3A8 and 104 mM and 0.310 pg/pmol/min for cynomolgus CYP3A5. The K m values for cynomolgus monkey CYP3A8 and CYP3A5 estimated in this study were higher than reported for human CYP3A4, although similar V max was observed (Shinkyo and Guengerich, 2011). In summary, this study showed that cynomolgus monkeys and humans exhibited similar changes in midazolam exposure and in 4b-hydroxycholesterol concentration after rifampicin treatment at an exposure-comparable dose level, which echoed the reported high homology between cynomolgus monkey CYP3A8 and human CYP3A4.…”

Effect of Cholesterol on the Structure of Membrane-Attached Cytochrome P450 3A4

“…One can hypothesize that the abovediscussed changes might also contribute to the non-competitive inhibition of CYP3A4 by cholesterol observed by Shinkyo and Guengerich. 45 Our results show that cholesterol content significantly influences the structural features of a membrane-anchored enzyme, which in turn may affect the substrate preferences and catalytic efficiency of the respective membranes or membrane domains. The described changes may influence biotransformation processes in different membrane parts and various cellular compartments, which differ in membrane composition and cholesterol content.…”

“…44 On the other hand, cholesterol also inhibits several CYP3A4 reactions in a non-competitive manner. 45 Our MD simulations showed that the presence of cholesterol changes the orientation and rigidifies the membrane-immersed parts of CYP3A4, which could inhibit entry of lipophilic substrates directly from the membrane.…”

The Major Cytochrome P450 Subtype Activities in Diet-Induced Non- Alcoholic Steatohepatitis Mouse Model

“…CYP51 plays a role in cholesterol de novo synthesis in the livers of both humans and mice, whereas the expression of CYP51 is known to be regulated by cholesterol via SREBP [20]. Cholesterol also inhibited CYP3A4 activity in both human liver microsomes and human hepatocytes [21]. However, the livers of patients with NASH typically accumulate TG.…”

“…Our laboratory previously reported that CYP, especially CYP3A, activity was induced more in mice fed a low protein diet (7% (w/w) casein) than in mice fed a control diet (20% (w/w) casein) [20][21][22]. A high-fat diet (P:F:C = 20:60:20) did not cause any changes in the hepatic expression of CYP3A, whereas regular chow (P:F:C = 26:13:61) increased CYP3A11 mRNA and CYP3A protein expression levels in the livers of mice more than the control diet (P:F:C = 20:10:70) [23].…”