Marmoset cytochrome P450 2D8 in livers and small intestines metabolizes typical human P450 2D6 substrates,metoprolol, bufuralol and dextromethorphan

Abstract

• 1.?Although the New World non-human primate, the common marmoset (Callithrix jacchus), is a potentially useful animal model, comprehensive understanding of drug metabolizing enzymes is insufficient.

• 2.?A cDNA encoding a novel cytochrome P450 (P450) 2D8 was identified in marmosets. The amino acid sequence deduced from P450 2D8 cDNA showed a high sequence identity (83–86%) with other primate P450 2Ds. Phylogenetic analysis showed that marmoset P450 2D8 was closely clustered with human P450 2D6, unlike P450 2Ds of miniature pig, dog, rabbit, guinea pig, mouse or rat.

• 3.?Marmoset P450 2D8 mRNA was predominantly expressed in the liver and small intestine among the tissues types analyzed, whereas marmoset P450 2D6 mRNA was expressed predominantly in the liver where P450 2D protein was detected by immunoblotting.

• 4.?By metabolic assays using marmoset P450 2D8 protein heterologously expressed in Escherichia coli, although P450 2D8 exhibits lower catalytic efficiency compared to marmoset and human P450 2D6 enzymes, P450 2D8 mediated O-demethylations of metoprolol and dextromethorphan and bufuralol 1′-hydroxylation.

• 5.?These results suggest that marmoset P450 2D8 (also expressed in the extrahepatic tissues) has potential roles in drug metabolism in a similar manner to those of human and marmoset P450 2D6.

     

Marmoset cytochrome P450 2J2 mainly expressed in small intestines and livers effectively metabolizes human P450 2J2 probe substrates,astemizole and terfenadine

1.?Common marmoset (Callithrix jacchus), a New World Monkey, has potential to be a useful animal model in preclinical studies. However, drug metabolizing properties have not been fully understood due to insufficient information on cytochrome P450 (P450), major drug metabolizing enzymes.

2.?Marmoset P450 2J2 cDNA was isolated from marmoset livers. The deduced amino acid sequence showed a high-sequence identity (91%) with cynomolgus monkey and human P450 2J2 enzymes. A phylogenetic tree revealed that marmoset P450 2J2 was evolutionarily closer to cynomolgus monkey and human P450 2J2 enzymes, than P450 2J forms in pigs, rabbits, rats or mice.

3.?Marmoset P450 2J2 mRNA was abundantly expressed in the small intestine and liver, and to a lesser extent in the brain, lung and kidney. Immunoblot analysis also showed expression of marmoset P450 2J2 protein in the small intestine and liver.

4.?Enzyme assays using marmoset P450 2J2 protein heterologously expressed in Escherichia coli indicated that marmoset P450 2J2 effectively catalyzed astemizole O-demethylation and terfenadine t-butyl hydroxylation, similar to human and cynomolgus monkey P450 2J2 enzymes.

5.?These results suggest the functional characteristics of P450 2J2 enzymes are similar among marmosets, cynomolgus monkeys and humans.     

Site-specific oxidation of flavanone and flavone by cytochrome P450 2A6 in human liver microsomes

1. The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes.

2. CYP2A6 was highly active in oxidizing flavanone to form flavone, 2′-hydroxy-, 4′-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5.

3. Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2′-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities.

4. Molecular docking analysis showed that the C2′-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2′ and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively.

5. These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.

     

Deoxyshikonin reversibly inhibits cytochrome P450 2B6

Deoxyshikonin, a natural shikonin derivative, is the major component of Lithospermum erythrorhizon and exhibits various pharmacological effects such as lymphangiogenetic, antibacterial, wound healing, and anticancer effects. To investigate the herb–drug interaction potential associated with deoxyshikonin, the inhibitory effects of deoxyshikonin on eight major cytochrome P450 (CYP) enzymes were examined using cocktail substrate-incubated human liver microsomes. Deoxyshikonin strongly inhibited CYP2B6-catalyzed bupropion hydroxylation, with a K_i value of 3.5 μM, and the inhibition was confirmed using purified human CYP2B6. The inhibition was reversible because the inhibitory effect of deoxyshikonin was not dependent on the preincubation time. The results indicated that deoxyshikonin-induced drug–drug interaction should be considered when any herb containing deoxyshikonin is used for conventional medications.     

人肝脏细胞色素P450 2B6催化药物生物转化研究进展

CYP 2B6是细胞色素P450 2B亚家族中的主要成员,参与催化多种内源、外源化合物,特别是多种临床药物的生物转化.本文综述了CYP 2B6的表达、催化药物生物转化及基因多态性的研究进展.     

Progesterone hydroxylation by cytochromes P450 2C and 3A enzymes in marmoset liver microsomes

1.?Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical drug metabolism studies. However, the roles of marmoset cytochrome P450 (P450) isoforms in the oxidation of endobiotic progesterone have not been fully investigated. In this study, the roles of marmoset P450 isoforms in progesterone hydroxylation were extensively determined.

2.?The activities of liver microsomes from individual marmosets with respect to progesterone 21/17α- and 16α/6β-hydroxylation were significantly correlated with those for flurbiprofen 4-hydroxylation and midazolam 1′-hydroxylation, respectively, as similar correlations have been found in humans. Anti-P450 2?C and 3?A antibodies suppressed progesterone 21/17α- and 16α/6β-hydroxylation, respectively, in marmoset liver microsomes.

3.?Recombinant marmoset P450 2C58 and 2C19 catalyzed progesterone to form 21-hydroxyprogesterone and 16α-hydroxyprogesterone, respectively, as major products with high maximum velocity/K_m values of 0.53 and 0.089?mL/min/nmol, respectively. Recombinant marmoset P450 3A4/90 oxidized progesterone to form 6β-hydroxyprogesterone as a major product with homotropic cooperativity (>1 of Hill coefficients).

4.?These results indicate that the overall activities and roles of liver microsomal P450 enzymes in marmoset livers are similar to those in humans, especially for progesterone 21/17α- and 16α/6β-hydroxylation by marmoset P450 2?C and 3?A enzymes, respectively, suggesting important roles for these P450 enzymes in the metabolism of endobiotics in marmosets.     

Regioselective hydroxylation of an antiarrhythmic drug,propafenone, mediated by rat liver cytochrome P450 2D2 differs from that catalyzed by human P450 2D6

1. Propafenone, an antiarrhythmic drug, is a typical human cytochrome P450 (P450) 2D6 substrate used in preclinical studies. Here, propafenone oxidation by mammalian liver microsomes was investigated in vitro.

2. Liver microsomes from humans and marmosets preferentially mediated propafenone 5-hydroxylation, minipig, rat and mouse livers primarily mediated 4′-hydroxylation, but cynomolgus monkey and dog liver microsomes differently mediated N-despropylation.

3. Quinine, ketoconazole or anti-P450 2D antibodies suppressed propafenone 4′/5-hydroxylation in human and rat liver microsomes. Pretreatments with β-naphthoflavone or dexamethasone increased N-despropylation in rat livers.

4. Recombinant rat P450 2D2 efficiently catalysed propafenone 4′-hydroxylation in a substrate inhibition manner, comparable to rat liver microsomes, while human P450 2D6 displayed propafenone 5-hydroxylation. Human and rat P450 1A, 2C and 3A enzymes mediated propafenone N-despropylation with high capacities.

5. Carbon-4′ of propafenone docked favourably into the active site of P450 2D2 based on an in silico model; in contrast, carbon-5 of propafenone docked into human P450 2D6.

6. These results suggest that the major roles of individual P450 2D enzymes in regioselective hydroxylations of propafenone differ between human and rat livers, while the minor roles of P450 1A, 2C and 3A enzymes for propafenone N-despropylation are similar in livers of both species.     

Mechanism-based inactivation of cytochrome P450 2B6 by isopsoralen

1.?Isopsoralen (IPRN) is a major component in many traditional medicinal herbs widely used in Asian countries. The objective of the present study was to investigate the inhibitory effect of IPRN on cytochrome P450 2B6 (CYP2B6) and the mechanism involved in the enzyme inactivation.

2.?Pre-incubation of CYP2B6 with IPRN resulted in a time- and concentration-dependent enzyme activity loss. The values of K_I and k_inact were found to be 7.89?μM and 0.067?min^?1, respectively. Ticlopidine exhibited protective effect on the IPRN-induced enzyme inactivation. The estimated partition ratio of the inactivation was 122. The GSH trapping experiments indicate that an epoxide and/or γ-ketoenal intermediate were/was generated in IPRN-fortified microsomal incubations. The synthetic work verified the formation of the reactive intermediate(s). Additionally, CYPs2E1, 2C19, 2B6 and 1A2 were found to be the major enzymes participating in the bioactivation of IPRN.

3.?IPRN was characterized as a mechanism-based inactivator of CYP2B6. An IPRN-derived furanoepoxide and/or γ-ketoenal intermediate(s) were/was generated and may be responsible for the inactivation of CYP2B6.     

Involvement of CYP2B6 in the biotransformation of propofol by human liver microsomes

Objective To determine whether the cytochrome P4502B6(CYP2B6)is involved in the oxidation of propofol by human liver microsomes.Methods The change of propofol concentration in an incubation mixture with human liver microsomes was monitored by the high performance liquid chromatography(HPLC),in order to calculate the rate constants of metabolism of propofol.The correlation between the rate constants and the rate of metabolism of CYP2B6 selective substrate bupropion,and the effect of two different CYP2B6 specific inhibitors on the propofol metabolism were examined.Results The mean rate constant of propofol metabolism by liver microsomes obtained from twelve individuals was 3.9(95% confidence intervals 3.3,4.5)nmol·min-1·mg-1 protein.The rate constants of propofol metabolism by liver microsomes were significantly correlated with bupropion hydroxylation(r=0.888,P<0.001).Both selective chemical inhibitors of CYP2B6,orphenadrine and N,N',N″-triethylenethiophosphoramide(thioTEPA),reduced the rate constants of propofol metabolism by 37.5%(P<0.001)and 42.7%(P<0.001)in liver microsomes,respectively.Conclusions CYP2B6 is predominantly involved in the oxidation of propofol by human liver microsomes.     

Inhibition of cytochrome P450 2E1 by propofol in human and porcine liver microsomes

While almost anesthetics are metabolized by the cytochrome P450 (CYP) 3A4, some major volatile ones such as halothane and sevoflurane are metabolized by CYP2E1 in humans. To determine whether 2,6-diisopropylphenol (propofol), a widely used intravenous anesthetic agent, known to inhibit CYP3A4 and CYP1A2, also inhibits CYP2E1, 6-OH hydroxylation of chlorzoxazone, a prototypical CYP2E1 substrate, was estimated using two pools of human microsomes and one pool of porcine microsomes from seven livers. Basal human enzyme activities were characterized by a V(max) of 1426+/-230 and 288+/-29 pmol min(-1)mg(-1) protein and a K(m) of 122+/-47 and 149+/-42 microM, while the corresponding porcine activities were associated with a V(max) of 352+/-42 pmol min(-1)mg(-1) protein and a K(m) of 167+/-38 microM. A competitive inhibition of CYP2E1 by propofol was observed with low inhibition constants in the therapeutic range in both porcine (19 microM) and human (48 microM) liver microsomes. These in vitro results suggest that propofol could have a protective effect on toxic metabolite activation of compounds catalyzed by CYP2E1.     

In vitro inhibitory effects of dihydromyricetin on human liver cytochrome P450 enzymes

Context: Dihydromyricetin (DHM) is the most abundant and active flavonoid component isolated from Ampelopsis grossedentata (Hand-Mazz) W.T. Wang (Vitaceae) and it possesses numerous pharmacological activities. However, whether DHM affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.

Materials and methods: The inhibitory effects of DHM on eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro using human liver microsomes (HLMs).

Results: The results showed that DHM could inhibit the activity of CYP3A4, CYP2E1 and CYP2D6, with IC₅₀ values of 14.75, 25.74 and 22.69?μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that DHM was not only a non-competitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2E1 and CYP2D6, with Ki values of 6.06, 9.24 and 10.52?μM, respectively. In addition, DHM is a time-dependent inhibitor for CYP3A4 with K_I/K_inact value of 12.17/0.057?min^?1?μM^?1.

Discussion and conclusion: The in vitro studies of DHM with CYP isoforms indicate that DHM has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4, CYP2E1 and CYP2D6. Further clinical studies are needed to evaluate the significance of this interaction.     

Deactivation of anti-cancer drug letrozole to a carbinol metabolite by polymorphic cytochrome P450 2A6 in human liver microsomes

1. Cytochromes P450 (P450) involved in letrozole metabolism were investigated. Among 13 recombinant P450 forms examined, only P450 2A6 and 3A4 showed activities in transforming letrozole to its carbinol metabolite with small K_m and high V_max values yielding apparent V_max/K_m values of 0.48 and 0.24 nl min^?1 nmol^?1 P450, respectively.

2. The metabolic activities of individual human liver microsomes showed a significant correlation with coumarin 7-hydroxylase activities (P450 2A6 marker) at a letrozole concentration of 0.5 μM, while a good correlation was also seen with testosterone 6β-hydroxylase activities (P450 3A4 marker) at 5 μM substrate concentration with different inhibition by 8-methoxypsolaren.

3. Significantly low carbinol-forming activities were seen in human liver microsomes from individuals possessing CYP2A6*4/*4 (whole CYP2A6 gene deletion) at a letrozole concentration of 0.5 μM. A V_max/K_m value measured for CYP2A6.7 (amino acid substitution type) in human liver microsomes, in the presence of anti-P450 3A4 antibodies, was approximately seven-fold smaller than that for CYP2A6.1 (wild-type).

4. These results demonstrate that P450 2A6 and 3A4 catalyse the conversion of letrozole to its carbinol metabolite in vitro at low and high concentrations of letrozole. Polymorphic variation of CYP2A6 is considered to be relevant to inter-subject variation in therapeutic exposure of letrozole.

     

Effects of nicotine on cytochrome P450 2A6 and 2E1 activities

AIMS

Smoking slows the metabolism of nicotine and accelerates the metabolism of chlorzoxazone, which are probe reactions for cytochrome P450 2A6 (CYP2A6) and CYP2E1 activities, respectively. We aimed to determine the role of nicotine in these metabolic effects of cigarette smoking.

METHODS

The study had a single-blind, randomized, crossover two-arm design. Twelve healthy smokers were given two transdermal patches with 42-mg nicotine a day or placebo patches, each for 10 days. The subjects abstained from smoking during the study arms. Oral chlorzoxazone was given on day 7 and deuterium-labelled nicotine-d₂ and cotinine-d₄ infusion on day 8.

RESULTS

There was no significant influence of transdermal nicotine administration on pharmacokinetic parameters of nicotine-d₂ or on the formation of cotinine-d₂. Nicotine decreased the volume of distribution (62.6 vs. 67.7 l, 95% confidence interval of the difference −9.7, −0.6, P= 0.047) of infused cotinine-d₄. There were no significant differences in disposition kinetics of chlorzoxazone between the treatments.

CONCLUSIONS

CYP2A6 and CYP2E1 activities are not affected by nicotine. The tobacco smoke constituents responsible for the reduced CYP2A6 and increased CYP2E1 activities remain unknown.     

Inhibitory effects of antiparasitic drugs on cytochrome P450 2D6

The interaction of antiparasitic drugs with the polymorphic cytochrome P450 2D6 was studied in human liver microsomes.Of ten different drugs tested, three quinolines, oxamniquine, primaquine and chloroquine inhibited microsomal CYP2D6-catalysed formation of 1hydroxybufuralol at concentrations that might have clinical consequences in drug use. These drugs inhibited competitively bufuralol metabolism with K _i values of 22, 23 and 15 M, respectively, indicative of high affinity for the CYP2D6-active site. The results imply that oxamniquine, primaquine and chloroquine could be substrates of cytochrome P4502 D6 or that they are potent non-substrate inhibitors of the enzyme similar to quinidine.In either case, the inhibition of CYP2D6 by these agents could lead to interference with in vivo population-phenotyping procedures in the tropical regions where treatment with the drugs is common.     

细胞色素P4502D6的研究进展

ＣＹＰ２Ｄ６在临床重要药代谢中具有重要作用。本文综述了ＣＹＰ２Ｄ６的结构特点,底物,探针药物,影响其活性的因素,基因多态性以及它与疾病的关系。     

己烯雌酚对人肝微粒体内CYP3A4和CYP2C9酶的抑制作用

目的:体外实验考察己烯雌酚(DES)对细胞色素P450 3A4(CYP3A4)和细胞色素P450 2C9(CYP2C9)活性的抑制作用,以评佑DES通过抑制这两个重要的细胞色素P450(CYP)亚型而引发药物-药物相互作用的可能性.方法:混合人肝微粒体与不同浓度的DES(或阳性抑制剂),CYP3A4或CYP2C9的探针...     

Involvement of human liver cytochrome P4502B6 in the metabolism of propofol

AIMS: To determine the cytochrome P450 (CYP) isoforms involved in the oxidation of propofol by human liver microsomes. METHODS: The rate constant calculated from the disappearance of propofol in an incubation mixture with human liver microsomes and recombinant human CYP isoforms was used as a measure of the rate of metabolism of propofol. The correlation of these rate constants with rates of metabolism of CYP isoform-selective substrates by liver microsomes, the effect of CYP isoform-selective chemical inhibitors and monoclonal antibodies on propofol metabolism by liver microsomes, and its metabolism by recombinant human CYP isoforms were examined. RESULTS: The mean rate constant of propofol metabolism by liver microsomes obtained from six individuals was 4.2 (95% confidence intervals 2.7, 5.7) nmol min(-1) mg(-1) protein. The rate constants of propofol by microsomes were significantly correlated with S-mephenytoin N-demethylation, a marker of CYP2B6 (r = 0.93, P < 0.0001), but not with the metabolic activities of other CYP isoform-selective substrates. Of the chemical inhibitors of CYP isoforms tested, orphenadrine, a CYP2B6 inhibitor, reduced the rate constant of propofol by liver microsomes by 38% (P < 0.05), while other CYP isoform-selective inhibitors had no effects. Of the recombinant CYP isoforms screened, CYP2B6 produced the highest rate constant for propofol metabolism (197 nmol min-1 nmol P450-1). An antibody against CYP2B6 inhibited the disappearance of propofol in liver microsomes by 74%. Antibodies raised against other CYP isoforms had no effect on the metabolism of propofol. CONCLUSIONS: CYP2B6 is predominantly involved in the oxidation of propofol by human liver microsomes.