Terfenadine t-butyl hydroxylation catalyzed by human and marmoset cytochrome P450 3A and 4F enzymes in livers and small intestines

1.?Roles of human cytochrome P450 (P450) 3A4 in oxidation of an antihistaminic drug terfenadine have been previously investigated in association with terfenadine–ketoconazole interaction. Several antihistamine drugs have been recently identified as substrates for multiple P450 enzymes. In this study, overall roles of P450 3A4, 2J2, and 4F12 enzymes in terfenadine t-butyl hydroxylation were investigated in small intestines and livers from humans, marmosets, and/or cynomolgus monkeys.

2.?Human liver microsomes and liver and small intestine microsomes from marmosets and cynomolgus monkeys effectively mediated terfenadine t-butyl hydroxylation. Ketoconazole and N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine (a P450 4A/F inhibitor) almost completely and moderately inhibited these activities, respectively, in human liver microsomes; however, these chemicals did not show substantially suppression in marmoset liver. Anti-human P450 3A and 4F antibodies showed the roughly supportive inhibitory effects.

3.?Recombinant P450 3A4/90 and 4F12 showed high terfenadine t-butyl hydroxylation activities with substrate inhibition constants of 84–144?μM (under 26–76?μM of K_m values), in similar manners to liver and intestine microsomes.

4.?These results suggest that human and marmoset P450 3A4/90 and 4F12 in livers or small intestines played important roles in terfenadine t-butyl hydroxylation. Marmosets could be a model for humans during first pass extraction of terfenadine and related substrates.     

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.     

Caffeine 7-N-demethylation and C-8-oxidation mediated by liver microsomal cytochrome P450 enzymes in common marmosets

1.?3-N-Demethylation of caffeine (1,3,7-trimethylxanthine) is mediated by human cytochrome P450 1A2, whereas 7-N-demethylation and C-8-hydroxylation are reportedly catalyzed by monkey P450 2C9 and rat P450 1A2, respectively.

2.?Roles of marmoset P450 enzymes in caffeine oxidation were investigated using nine marmoset liver microsomes and 14 recombinantly expressed marmoset P450 enzymes.

3.?Predominant caffeine 7-N-demethylation and C-8-hydroxylation activities in marmoset liver microsomes were moderately (r?=?0.78, p?<?0.05) and highly (r?=?0.82, p?<?0.01) correlated with midazolam 1′-hydroxylation activities, respectively, while the former was not strongly affected by ketoconazole or α-naphthoflavone.

4.?Caffeine C-8-hydroxylation in liver microsomes was inhibited by ketoconazole and activated by α-naphthoflavone, suggesting main involvements of P450 3As.

5.?Recombinant marmoset P450 3As had high V_max/K_m values for C-8-hydroxylation, comparable to K_m values for marmoset liver microsomes. Marmoset P450 1As efficiently mediated caffeine 3-N-demethylation and C-8-hydroxylation with apparently lower K_m values than those of liver microsomes.

6.?These results collectively suggest highly active marmoset P450 3A enzymes toward caffeine 8-hydorxylaiton and involvement of multiple P450 isoforms including P450 1A in caffeine 7-N- and 3-N-demethylations in marmoset livers. Marmoset P450s have slightly different properties to human or monkey P450s regarding caffeine metabolic pathways.     

Simultaneous pharmacokinetics evaluation of human cytochrome P450 probes,caffeine, warfarin,omeprazole, metoprolol and midazolam,in common marmosets (Callithrix jacchus)

1.?Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) composite, after single intravenous and oral administrations at doses of 0.20 and 1.0?mg?kg^?1, respectively, to four male common marmosets were investigated.

2.?The plasma concentrations of caffeine and warfarin decreased slowly in a monophasic manner but those of omeprazole, metoprolol and midazolam decreased extensively after intravenous and oral administrations, in a manner that approximated those as reported for pharmacokinetics in humans.

3.?Bioavailabilities were ～100% for caffeine and warfarin, but <25% for omeprazole and metoprolol. Bioavailability of midazolam was 4% in marmosets, presumably because of contribution of marmoset P450 3A4 expressed in small intestine and liver, with a high catalytic efficiency for midazolam 1′-hydroxylation as evident in the recombinant system.

4.?These results suggest that common marmosets, despite their rapid clearance of some human P450 probe substrates, could be an experimental model for humans and that marmoset P450s have functional characteristics that differ from those of human and/or cynomolgus monkey P450s in some aspects, indicating their importance in modeling in P450-dependent drug metabolism studies in marmosets and of further studies.     

Interaction of metyrapone with adrenal microsomal cytochrome P450 in the guinea pig

Studies were carried out to compare the actions of metyrapone on adrenal mitochondrial and microsomal cytochrome P450-containing enzymes in the guinea pig and rat. As expected. addition of metyrapone to adrenal mitochondria inhibited 11β-hydroxylation in both species. the shape of the type II difference spectrum produced by metyrapone in mitochondria differed somewhat in rat (gDO.D._425?405nm) and guinea pig (gDO.D._425?390nm) and the magnitude of the speetrum was far greater in rat adrenal mitochondria, paralleling species differences in cytochrome P450 concentration (rat > guinea pig). In rat adrenal microsomes, metyrapone produced a small “reverse type I” spectral change (ΔO.D._420-385nm) but did not affect either 21-hydroxylation or the interaction of progesterone with eytochrome P450(as determined spectrally). In guinea pig adrenal microsomes, in contrast, metyrapone produced a large type II spectral change (ΔO.D._423-408nm) and inhibited both 21-hydroxylation and ethylmorphine demethylation, cytochrome P450-dependent reactions. The magnitudes of type I spectra produced by 17α-hydroxyprogesterone and ethylmorphine in guinea pig adrenal microsomes were significantly diminished by prior addition of metyrapone. The results indicate that metyrapone interacts with both microsomal and mitochondrial cytochrome P450 in the guinea pig and that its adrenal sites of action, therefore, are species dependent.     

Intraspecies individuality for the metabolism of steroids

1. A variety of regulatory factors contribute to differences in the rates of 6β-hydroxylation, 16α-hydroxylation and 21-hydroxylation of progesterone as catalysed by liver microsomes prepared from individual rabbits.

2. It is likely that the 6β-hydroxylation of progesterone is catalysed primarily by cytochrome P-450 3c, an enzyme that exhibits allosteric activation by α-napthoflavone, and by a form of P-450 3b, 6β⁺, that is expressed in some rabbits in an autosomal dominant manner.

3. The mechanism of activation for P-450 3c appears to reflect an effector mediated increase of the affinity of the enzyme for substrate as judged by substrate binding studies.

4. A second form of P-450 3b, 6β^-, catalyses a major portion of hepatic progesterone 16α-hydroxylation and exhibits activation by a variety of C21 steroids of which 5β-pregnane-3β,20α-diol is the most efficacious.

5. P-450 1, which catalyses the 21-hydroxylation of progesterone, is expressed at 10-fold higher levels in the 21H phenotype than the 21L phenotype, and the former is inherited as an autosomal dominant characteristic.

6. A cDNA encoding a P-450 1-related gene product exhibits a predicted amino acid sequence that is 95% homologous to that of P-450 1.

7. The P-450 1-related gene product is expressed in liver to a similar degree in both 21H and 21L rabbits.     

Regioselective hydroxylation of steroid hormones by human cytochromes P450

This article reviews in vitro metabolic activities [including Michaelis constants (K_m), maximal velocities (V_max) and V_max/K_m] and drug–steroid interactions [such as induction and cooperativity (activation)] of cytochromes P450 (P450 or CYP) in human tissues, including liver and adrenal gland, for 14 kinds of endogenous steroid compounds, including allopregnanolone, cholesterol, cortisol, cortisone, dehydroepiandrosterone, estradiol, estrone, pregnenolone, progesterone, testosterone and bile acids (cholic acid). First, we considered the drug-metabolizing P450s. 6β-Hydroxylation of many steroids, including cortisol, cortisone, progesterone and testosterone, was catalyzed primarily by CYP3A4. CYP1A2 and CYP3A4, respectively, are likely the major hepatic enzymes responsible for 2-/4-hydroxylation and 16α-hydroxylation of estradiol and estrone, steroids that can contribute to breast cancer risk. In contrast, CYP1A1 and CYP1B1 predominantly metabolized estrone and estradiol to 2- and 4-catechol estrogens, which are endogenous ultimate carcinogens if formed in the breast. Some metabolic activities of CYP3A4, including dehydroepiandrosterone 7β-/16α-hydroxylation, estrone 2-hydroxylation and testosterone 6β-hydroxylation, were higher than those for polymorphically expressed CYP3A5. Next, we considered typical steroidogenic P450s. CYP17A1, CYP19A1 and CYP27A1 catalyzed steroid synthesis, including hydroxylation at 17α, 19 and 27 positions, respectively. However, it was difficult to predict which hepatic drug-metabolizing P450 or steroidogenic P450 will be mainly responsible for metabolizing each steroid hormone in vivo based on these results. Further research is required on the metabolism of steroid hormones by various P450s and on prediction of their relative contributions to in vivo metabolism. The findings collected here provide fundamental and useful information on the metabolism of steroid compounds.     

Effect of buffer conditions on CYP2C8-mediated paclitaxel 6α-hydroxylation and CYP3A4-mediated triazolam α- and 4-hydroxylation by human liver microsomes

Abstract

1.?Buffer conditions in in vitro metabolism studies using human liver microsomes (HLM) have been reported to affect the metabolic activities of several cytochrome P450 (CYP) isozymes in different ways, although there are no reports about the dependence of CYP2C8 activity on buffer conditions.

2.?The present study investigated the effect of buffer components (phosphate or Tris-HCl) and their concentration (10–200?mM) on the CYP2C8 and CYP3A4 activities of HLM, using paclitaxel and triazolam, respectively, as marker substrates.

3.?The K_m (or S₅₀) and V_max values for both paclitaxel 6α-hydroxylation and triazolam α- and 4-hydroxylation, estimated by fitting analyses based on the Michaelis–Menten or Hill equation, greatly depended on the buffer components and their concentration.

4.?The CL_int values in phosphate buffer were 1.2–3.0-fold (paclitaxel) or 3.1–6.4-fold (triazolam) higher than in Tris-HCl buffer at 50–100?mM. These values also depended on the buffer concentration, with a maximum 2.3-fold difference observed between 50 and 100?mM which are both commonly used in drug metabolism studies.

5.?These findings suggest the necessity for optimization of the buffer conditions in the quantitative evaluation of metabolic clearances, such as in vitro–in vivo extrapolation and also estimating the contribution of a particular enzyme in drug metabolism.     

“Detoxication,a most Stimulating Concept of Toxicology”

1. Large, independent variations occur among New Zealand White rabbits in the 21-and 6β-hydroxylation of progesterone as catalysed by liver microsomes.

2. These reactions are catalysed respectively by two electrophoretically distinct types of rabbit-liver microsomal cytochrome P-450, 1 and 3b, as judged by their catalytic efficiency and the capacity of specific monoclonal antibodies to extensively inhibit the respective microsomal hydroxylases.

3. The relatively large variations in progesterone 6β-hydroxylase activity do not appear to be associated with differences in microsomal content of cytochrome P-450 3b, whereas differences in the microsomal concentration of cytochrome P-450 1 may underlie variations in 21-hydroxylase activity.

4. Preparations of cytochrome P-450 3b contain at least two catalytically distinct subforms, one of which catalyses both 6β- and 16α-hydroxylation of progesterone with a low K_m while the other subform catalyses predominantly 16α-hydroxylation with a significantly greater K_m.

5. The two catalytic subforms of cytochrome P-450 3b can be independently modulated in vitro by positive and negative effectors that can arise in vivo from the metabolism of progesterone.

6. The 6β-hydroxylase subform of cytochrome P-450 3b is not expressed in a genetically defined strain of rabbits, IIIVO/J, indicating a heritable basis for the differential expression of the two subforms of cytochrome P-450 3b.

7. These results indicate that the extent of cytochrome P-450 multiplicity may be greater than is evident from the isolation of electrophoretically distinct forms of cytochrome P-450, and that small differences in structure may underlie large differences in catalytic properties.

8. It is not known whether the differences among outbred New Zealand White rabbits in the expression of either cytochrome P-450 1 or the subforms of cytochrome P-450 3b reflect regulatory phenomena or genetic polymorphism.     

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.     

Molecular modelling of CYP2A enzymes: application to metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

1.?Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in a variety of animal models and a putative human lung carcinogen. Its tumorigenic potential is unmasked via cytochrome P450 (CYP)-mediated hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e.?α-hydroxylation). Therefore, elucidation of enzyme–substrate interactions that facilitate?α-hydroxylation is important to gain insight into the tumorigenic mechanism of NNK and to develop potent inhibitors of this detrimental reaction.

2.?Molecular models of CYP2A enzymes from mice, rats and humans that are catalysts of NNK bioactivation were constructed and used, in conjunction with docking experiments, to identify active-site residues that make important substrate contacts.

3.?Docking studies revealed that hydrophobic residues at positions 117, 209, 365 and 481, among others, play critical roles in orienting NNK in the active site to effect?α-hydroxylation. These molecular models were then used to rationalize the stereo- and regioselectivity, as well as the efficiency, of CYP2A-mediated NNK metabolism.     

Drug interactions of diclofenac and its oxidative metabolite with human liver microsomal cytochrome P450 1A2-dependent drug oxidation

Abstract

1.?The purpose of this study was to investigate the inhibitory effects of diclofenac on human cytochrome P450 1A2-, 2C19- and 3A4-mediated drug oxidations and to evaluate the drug interaction potential of diclofenac and 4′-hydroxydiclofenac.

2.?Diclofenac was converted to 4′-hydroxydiclofenac by recombinantly expressed human P450 1A2 with K_m and V_max values of 33?µM and 0.20?min^?1, respectively. Diclofenac and 4′-hydroxydiclofenac suppressed flurbiprofen 4′-hydroxylation by P450 2C9 strongly and moderately, respectively; however, they did not affect P450 2C19-dependent S-mephenytoin hydroxylation or P450 3A4-dependent midazolam hydroxylation.

3.?Although the caffeine 3-N-demethylation activity of liver microsomal P450 1A2 was inhibited by simultaneous incubation with diclofenac, the riluzole N-hydroxylation activities of recombinant P450 1A2 and human liver microsomes were inhibited after preincubation with diclofenac or 4′-hydroxydiclofenac for 20?min in the presence of NADPH. Using the inhibition constant (37?µM) of diclofenac on caffeine 3-N-demethylation and the reported 95th percentiles of maximum plasma concentration (10.5?µM) after an oral dose of diclofenac, the in vivo estimated increase in area under the plasma concentration–time curve was 29%.

4.?These results suggest that diclofenac could inhibit drug clearance to a clinically important degree that depends on P450 1A2. Clinically relevant drug interactions in vivo with diclofenac are likely to be invoked via human P450 1A2 function in addition to those caused by the effect of diclofenac on P450 2C9.     

Effects of aging and rifampicin pretreatment on the pharmacokinetics of human cytochrome P450 probes caffeine,warfarin, omeprazole,metoprolol and midazolam in common marmosets genotyped for cytochrome P450 2C19

1. The pharmacokinetics were investigated for human cytochrome P450 probes after single intravenous and oral administrations of 0.20 and 1.0?mg/kg, respectively, of caffeine, warfarin, omeprazole, metoprolol and midazolam to aged (10–14?years old, n?=?4) or rifampicin-treated/young (3?years old, n?=?3) male common marmosets all genotyped as heterozygous for a cytochrome P450 2C19 variant.

2. Slopes of the plasma concentration–time curves after intravenous administration of warfarin and midazolam were slightly, but significantly (two-way analysis of variance), decreased in aged marmosets compared with young marmosets. The mean hepatic clearances determined by in silico fitting for individual pharmacokinetic models of warfarin and midazolam in the aged group were, respectively, 23% and 56% smaller than those for the young group.

3. Significantly enhanced plasma clearances of caffeine, warfarin, omeprazole and midazolam were evident in young marmosets pretreated with rifampicin (25?mg/kg daily for 4?days). Two- to three-fold increases in hepatic intrinsic clearance values were observed in the individual pharmacokinetic models.

4. The in vivo dispositions of multiple simultaneously administered drugs in old, young and P450-enzyme-induced marmosets were elucidated. The results suggest that common marmosets could be experimental models for aged, induced or polymorphic P450 enzymes in P450-dependent drug metabolism studies.     

Potential of pranlukast and zafirlukast in the inhibition of human liver cytochrome P450 enzymes

1.?The potential of zafirlukast to inhibit several human cytochrome P450 enzymes is well known. However, pranlukast, a structural analogue of zafirlukast, has not been studied. Accordingly, the inhibitory potential of pranlukast was evaluated and compared with that of zafirlukast, a known CYP2C9 inhibitor, in in vitro microsomal incubation studies.

2.?Both pranlukast and zafirlukast showed moderate inhibition of CYP2C9-catalysed tolbutamide 4-methylhydroxylation, competitively inhibiting tolbutamide 4-methylhydroxylation with estimated mean K_i values of 3.82 ± 0.50 and 5.86 ± 0.08?μM, respectively.

3.?Pranlukast had no effect on CYP2C19-catalysed S-mephenytoin 4′-hydroxylation or CYP3A4-catalysed midazolam 1-hydroxylation. However, zafirlukast showed minor inhibition of these reactions. Neither pranlukast nor zafirlukast inhibited CYP1A2-catalysed phenacetin O-deethylation, CYP2D6-catalysed dextromethorphan O-demethylation or CYP2E1-catalysed chlorzoxazone 6-hydroxylation.

4.?The results suggest that like zafirlukast, pranlukast also has the potential moderately to inhibit CYP2C9-catalysed tolbutamide 4-methylhydroxylation. Therefore, the inhibitory potential of pranlukast should be considered when it is co-administered with CYP2C9 substrates with narrow therapeutic ranges (e.g. S-warfarin, phenytoin).     

Oxidation of 1-chloropyrene by human CYP1 family and CYP2A subfamily cytochrome P450 enzymes: catalytic roles of two CYP1B1 and five CYP2A13 allelic variants

1.?1-Chloropyrene, one of the major chlorinated polycyclic aromatic hydrocarbon contaminants, was incubated with human cytochrome P450 (P450 or CYP) enzymes including CYP1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C9, 2D6, 2E1, 3A4 and 3A5. Catalytic differences in 1-chloropyrene oxidation by polymorphic two CYP1B1 and five CYP2A13 allelic variants were also examined.

2.?CYP1A1 oxidized 1-chloropyrene at the 6- and 8-positions more actively than at the 3-position, while both CYP1B1.1 and 1B1.3 preferentially catalyzed 6-hydroxylation.

3.?Five CYP2A13 allelic variants oxidized 8-hydroxylation much more than 6- and 3-hydroxylation, and the variant CYP2A13.3 was found to slowly catalyze these reactions with a lower k_cat value than other CYP2A13.1 variants.

4.?CYP2A6 catalyzed 1-chloropyrene 6-hydroxylation at a higher rate than the CYP2A13 enzymes, but the rate was lower than the CYP1A1 and 1B1 variants. Other human P450 enzymes had low activities towards 1-chloropyrene.

5.?Molecular docking analysis suggested differences in the interaction of 1-chloropyrene with active sites of CYP1 and 2?A enzymes. In addition, a naturally occurring Thr134 insertion in CYP2A13.3 was found to affect the orientation of Asn297 in the I-helix in interacting with 1-chloropyrene (and also 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and caused changes in the active site of CYP2A13.3 as compared with CYP2A13.1.     

Mechanism-based inactivation of human cytochrome P450 1A2 and 3A4 isoenzymes by anti-tumor triazoloacridinone C-1305

1.?5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, is a promising anti-tumor therapeutic agent with high activity against several experimental tumors.

2.?It was determined to be a potent and selective inhibitor of liver microsomal and human recombinant cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1305 might modulate the effectiveness of other drugs used in multidrug therapy.

3.?The objective of this study was to investigate the mechanism of the observed C-1305-mediated inactivation of CYP1A2 and CYP3A4.

4.?Our findings indicated that C-1305 produced a time- and concentration-dependent decrease in 7-ethoxycoumarin O-deethylation (CYP1A2, K_I?=?10.8?±?2.14?μM) and testosterone 6β-hydroxylation (CYP3A4, K_I = 9.1?±?2.82?μM). The inactivation required the presence of NADPH, was unaffected by a nucleophilic trapping agent (glutathione) and a reactive oxygen species scavenger (catalase), attenuated by a CYP-specific substrate (7-ethoxycoumarin or testosterone), and was not reversed by potassium ferricyanide. The estimated partition ratios of 1086 and 197 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively.

5.?In conclusion, C-1305 inhibited human recombinant CYP1A2 and CYP3A4 isoenzymes by mechanism-based inactivation. The obtained knowledge about specific interactions between C-1305 and/or its metabolites, and CYP isoforms would be useful for predicting the possible drug–drug interactions in potent multidrug therapy.