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Molecular Pharmacology Apr 2015The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and...
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 b5 (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 α-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 (AUC0-8 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 AUC0-8 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.
Topics: Animals; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Cytochromes b5; Debrisoquin; Ethanolamines; Female; Humans; Male; Mice, Knockout; Microsomes, Liver; Nifedipine; Sex Factors; Triazolam
PubMed: 25657337
DOI: 10.1124/mol.114.097394 -
Advances in Experimental Medicine and... 2015Dopamine (DA) is a putative neurotransmitter in the carotid body engaged in the generation of the hypoxic ventilatory response (HVR). However, the action of endogenous... (Review)
Review
Dopamine (DA) is a putative neurotransmitter in the carotid body engaged in the generation of the hypoxic ventilatory response (HVR). However, the action of endogenous DA is unsettled. This study seeks to determine the ventilatory effects of increased availability of endogenous DA caused by inhibition of DA enzymatic breakdown. The peripheral inhibitor of MAO - debrisoquine, or COMT - entacapone, or both combined were injected to conscious rats. Ventilation and its responses to acute 8 % O(2) in N(2) were investigated in a whole body plethysmograph. We found that inhibition of MAO augmented the hyperventilatory response to hypoxia. Inhibition of COMT failed to influence the hypoxic response. However, simultaneous inhibition of both enzymes, the case in which endogenous availability of DA should increase the most, reversed the hypoxic augmentation of ventilation induced by MAO-inhibition. The inference is that when MAO alone is blocked, COMT takes over DA degradation in a compensatory way, which lowers the availability of DA, resulting in a higher intensity of the HVR. We conclude that MAO is the enzyme predominantly engaged in the chemoventilatory effects of DA. Furthermore, the findings imply that endogenous DA is inhibitory, rather than stimulatory, for hypoxic ventilation.
Topics: Adaptation, Physiological; Animals; Blood Pressure; Carotid Body; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Catechols; Debrisoquin; Dopamine; Drug Synergism; Hyperventilation; Hypoxia; Male; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Nitriles; Plethysmography, Whole Body; Rats; Rats, Wistar; Respiration
PubMed: 25310955
DOI: 10.1007/5584_2014_72 -
Drug Metabolism and Disposition: the... Sep 2014Cilengitide is a stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αVβ3 and αVβ5 integrins. The candidate drug showed...
Cilengitide is a stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αVβ3 and αVβ5 integrins. The candidate drug showed unexpected inhibition of cytochrome P450 (P450) 3A4 at high concentrations, that is, a 15-mM concentration caused attenuation of P450 3A4 activity (depending on the probe substrate): 15-19% direct inhibition, 10-23% time-dependent inhibition (30-minute preincubation), and 54-60% metabolism-dependent inhibition (30-minute preincubation). The inactivation efficiency determined with human liver microsomes was 0.003 ± 0.001 min(-1) mM(-1) and was 0.04 ± 0.01 min(-1) mM(-1) with baculovirus-based microsomes containing recombinant P450 3A4. Neither heme loss nor covalent binding to apoprotein could explain the observed reductions in residual activity. Slowly forming type II difference spectra were observed, with maximum spectral changes after 2 hours. Binding to both reduced and oxidized P450 3A4 was observed, with apparent Kd values of 0.66 μM and 6 μM. The significance of the guanidine group in inhibition was demonstrated using ligand binding spectral changes and inactivation assays with guanidine analogs (debrisoquine, N-acetylarginine-O-methyl ester) and the acetylated ornithine derivative of cilengitide. The observed inhibition could be explained by direct inhibition, plus by formation of stable complexes with both ferric and ferrous forms of heme iron and to some extent by the formation of reactive species capable to react to the protein or heme. Formation of the complex required time and NADPH and is attributed to the guanidino group. Thus, the NADPH-dependent inhibition is considered to be mainly due to the formation of a stable complex rather than the formation of reactive species.
Topics: Adult; Cytochrome P-450 CYP3A; Enzyme Inhibitors; Female; Guanidine; Heme; Humans; Male; Microsomes, Liver; NADP; Oxidation-Reduction; Snake Venoms
PubMed: 24985702
DOI: 10.1124/dmd.114.059295