In vitro assessment of metabolic drug–drug interaction potential of AZD2624, neurokinin-3 receptor antagonist,through cytochrome P450 enzyme identification,inhibition, and induction studies

1. AZD2624 was pharmacologically characterized as a NK3 receptor antagonist intended for treatment of schizophrenia. The metabolic drug–drug interaction potential of AZD2624 was evaluated in in vitro studies.

2. CYP3A4 and CYP3A5 appeared to be the primary enzymes mediating the formation of pharmacologically active ketone metabolite (M1), whereas CYP3A4, CYP3A5, and CYP2C9 appeared to be the enzymes responsible for the formation of the hydroxylated metabolite (M2). The apparent K_m values were 1.5 and 6.3 µM for the formation of M1 and M2 in human liver microsomes, respectively.

3. AZD2624 exhibited an inhibitory effect on microsomal CYP3A4/5 activities with apparent IC₅₀ values of 7.1 and 19.8 µM for midazolam and testosterone assays, respectively. No time-dependent inactivation of CYP3A4/5 activity (midazolam 1′-hydroxylation) by AZD2624 was observed. AZD2624 demonstrated weak to no inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6.

4. AZD2624 was not an inducer of CYP1A2 or CYP2B6. Although AZD2624-induced CYP3A4 activity in hepatocytes, the potential of AZD2624 to cause inductive drug interactions of this enzyme was low at relevant exposure concentration.

5. Together with targeted low efficacious concentration, the results of this study demonstrated AZD2624 has a relatively low metabolic drug–drug interaction potential towards co-administered drugs. However, metabolism of AZD2624 might be inhibited when co-administrated with potent CYP3A4/5 inhibitors.

     

In vitro inhibition and induction of human cytochrome P450 enzymes by mirabegron,a potent and selective β3-adrenoceptor agonist

1. The potential for mirabegron, a β₃-adrenoceptor agonist for the treatment of overactive bladder, to cause drug–drug interactions via inhibition or induction of cytochrome P450 (CYP) enzymes was investigated in vitro.

2. Mirabegron was shown to be a time-dependent inhibitor of CYP2D6 in the presence of NADPH as the IC₅₀ value in human liver microsomes decreased from 13 to 4.3 μM after 30-min pre-incubation. Further evaluation indicated that mirabegron may act partly as an irreversible or quasi-irreversible metabolism-dependent inhibitor of CYP2D6. Therefore, the potential of mirabegron to inhibit the metabolism of CYP2D6 substrates in vivo cannot be excluded. Mirabegron was predicted not to cause clinically significant metabolic drug–drug interactions via inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5 because the IC₅₀ values for these enzymes both with and without pre-incubation were >100 μM (370 times maximum human plasma concentration [C_max]).

3. Whereas positive controls (100 µM omeprazole and 10 µM rifampin) caused the anticipated CYP induction, the highest concentration of mirabegron (10 µM; 37 times plasma C_max) had minimal effect on CYP1A2 and CYP3A4/5 activity, and CYP1A2 and CYP3A4 mRNA levels in freshly isolated human hepatocytes, suggesting that mirabegron is not an inducer of these enzymes.

     

Drug interaction potential of toremifene and N-desmethyltoremifene with multiple cytochrome P450 isoforms

Toremifene is an effective agent for the treatment of breast cancer in postmenopausal women and is being evaluated for its ability to prevent bone fractures in men with prostate cancer taking androgen deprivation therapy. Due to the potential for drug-drug interactions, the ability of toremifene and its primary circulating metabolite N-desmethyltoremifene (NDMT) to inhibit nine human cytochrome P450 (CYP) enzymes was determined using human liver microsomes. Induction of CYP1A2 and 3A4 by toremifene was also investigated in human hepatocytes. Toremifene did not significantly inhibit CYP1A2 or 2D6. However, toremifene is a competitive inhibitor of CYP3A4, non-competitive inhibitor of CYP2A6, 2C8, 2C9, 2C19 and 2E1 and mixed-type inhibitor of CYP2B6. CYP inhibition by NDMT was similar in magnitude to toremifene. Toremifene did not induce CYP1A2 but increased CYP3A4 monooxygenase activity and gene expression in drug-exposed human primary hepatocytes. Although clinical doses of toremifene produce steady state exposures to toremifene and NDMT that may be sufficient to cause pharmacokinetic drug-drug interactions with other drugs metabolised by CYP2B6, CYP2C8, CYP3A4, CYP2C9 and CYP2C19, these data indicate that toremifene is unlikely to play a role in clinical drug-drug interactions with substrate drugs of CYP1A2 and CYP2D6.     

Cynomolgus monkey CYPs: a comparison with human CYPs

1. Characteristics of twelve cytochromes P450 (CYPs) from cynomolgus monkeys were compared with those of human CYPs that play an important role in drug metabolism.

2. Eleven members of CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A subfamilies from cynomolgus monkeys exhibited a high degree of homologies (more than 90%) in cDNA and amino acid sequences with corresponding human CYPs, and catalysed typical reactions of corresponding human CYPs.

3. One member of the cynomolgus monkey CYP2C subfamily, CYP2C76, exhibited a lower homology (around 70%) in amino acid sequences with other cynomolgus monkey and human CYP2C subfamilies. CYP2C76 catalysed typical CYP2C substrates with low activities, and has not been found in humans.

4. CYPs identified in cynomolgus monkeys were similar to CYP1A1, CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 in humans.

5. These results indicate that cynomolgus monkeys express CYPs similar to human CYPs that are important in drug metabolism.

     

Evaluation of the effects of 18 non-synonymous single-nucleotide polymorphisms of CYP450 2C19 on in vitro drug inhibition potential by a fluorescence-based high-throughput assay

1. To comprehensively understand the effects of CYP2C19 genetic polymorphisms on inhibition-based drug–drug interactions (DDIs), 18 human CYP2C19 non-synonymous single-nucleotide polymorphic variants and the wild-type isoform (CYP2C19.1A) were expressed in yeast cells. Using a fluorescence-based high-throughput method, the kinetic constants of these variants, as well as the inhibition constants for 10 drugs, were determined.

2. CYP2C19.5B and CYP2C19.6 showed no activity towards CEC (3-cyano-7-ethoxycoumarin) O-deethylation. CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.16, CYP2C19.19, E122A and A161P* (an allele containing both A161P and I331V) exhibited significantly reduced catalytic activities compared with CYP2C19.1A. The inhibition assay showed that the CYP2C19 genotype significantly affected the in vitro drug inhibition potential. Although the effect on drug inhibition potential is genotype- and inhibitor-dependent, there was an obvious trend: drugs tended to exhibit higher IC₅₀ values (i.e. decreased inhibition potential) towards variants with reduced activity compared with variants with normal activity. This indicated that patients with reduced-function alleles may be less susceptible to CYP2C19-related DDIs.

3. In this study, we provided the first in vitro evidence of CYP2C19 genotype-dependent effects on drug inhibition potential. This work greatly extends our understanding of the functional consequences of CYP2C19 genetic polymorphisms, and thus should prove valuable for CYP2C19 genotype-based therapy.

     

A novel CYP2A26 identified in cynomolgus monkey liver metabolizes coumarin

1. A novel cytochrome P450 (CYP), CYP2A26, was identified and characterized in cynomolgus monkey, one of the animal species used in preclinical studies.

2. Deduced amino acid sequences of CYP2A26 cDNA showed high sequence identities (91–95%) with cynomolgus monkey CYP2A23 and CYP2A24, and human CYP2A6 and CYP2A13.

3. Phylogenetic analysis showed that macaque CYP2As (CYP2A26, CYP2A23, and CYP2A24) were most closely clustered with human CYP2As, unlike CYP2As of dog, rat, and mouse (other species also used in drug metabolism).

4. Quantitative polymerase chain reaction analysis showed that CYP2A26 mRNA, along with CYP2A23 and CYP2A24 mRNAs, was expressed predominantly in the liver, where CYP2A proteins were also detected by immunoblotting.

5. Drug-metabolizing assays using the CYP2A26 protein heterologously expressed in Escherichia coli indicated that CYP2A26 catalyzed coumarin 7-hydroxylation with its apparent K_m lower than that of CYP2A24, but similar to those of CYP2A6 and CYP2A23.

6. These results suggest an evolutionary closeness and functional similarity of cynomolgus monkey CYP2A26 (together with CYP2A23 and CYP2A24) to human CYP2A6, and its functional role as a drug-metabolizing enzyme in the liver.

     

Ilaprazole,a new proton pump inhibitor,is primarily metabolized to ilaprazole sulfone by CYP3A4 and 3A5

1. Ilaprazole is a new proton pump inhibitor, designed for treatment of gastric ulcers, and developed by Il-Yang Pharmaceutical Co (Seoul, Korea). It is extensively metabolised to the major metabolite ilaprazole sulfone.

2. In the present study, several in vitro approaches were used to identify the cytochrome P450 (CYP) enzymes responsible for ilaprazole sulfone formation. Concentrations of ilaprazole sulfone were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

3. Incubation of ilaprazole with cDNA-expressed recombinant CYPs indicated that CYP3A was the major enzyme that catalyses ilaprozole to ilaprazole sulfone. This reaction was inhibited significantly by ketoconazole, a CYP3A inhibitor, and azamulin, a mechanism-based inhibitor of CYP3A, while no substantial effect was observed using selective inhibitors for eight other P450s (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1).

4. In addition, the formation of ilaprazole sulfone correlated well with CYP3A-catalysed testosterone 6β-hydroxylation and midazolam 1′-hydroxylation in 20 different human liver microsome panels. The intrinsic clearance of the formation of ilaprazole sulfone by CYP3A4 was 16-fold higher than that by CYP3A5.

5. Collectively, these results indicate that the formation of the major metabolite of ilaprazole, ilaprazole sulfone, is predominantly catalysed by CYP3A4/5.

     

Identification of the human liver cytochrome P450 isoenzymes responsible for the 5-methylhydroxylation of the novel anti-fibrotic drug AKF-PD

1. Identification of cytochrome P450 isoforms (CYPs) involved in flourofenidone (5-methyl-1-(3-fluorophenyl)-2-[1H]-pyridone, AKF-PD) 5-methylhydroxylation was carried out using human liver microsomes and cDNA-expressed human CYPs (supersomes). The experiments were performed in the following in vitro models: (A) a study of AKF-PD metabolism in liver microsomes: (a) correlations study between the rate of AKF-PD 5-methylhydroxylation and activity of CYPs; (b) the effect of specific CYPs inhibitors on the rate of AKF-PD 5-methylhydroxylation; (B) AKF-PD biotransformation by cDNA-expressed human CYPs (1A2, 2D6, 2C9, 2C19, 2E1, 3A4).

2. In human liver microsomes, the formation of AKF-PD 5-methylhydroxylation metabolite significantly correlated with the caffeine N3-demethylase (CYP1A2), chlorzoxazone 6-hydroxylase (CYP2E1), midazolam 1’- hydroxylase (CYP3A4), tolbutamide 4-hydroxylase (CYP2C9), and debrisoquin 4-hydroxylase (CYP2D6) activities. The production of AKF-PD 5-methylhydroxylation metabolite was completely inhibited by a-naphthoflavone (a CYP1A2 inhibitor) with the IC50 value of 0.12 μM in human liver microsomes. The cDNA-expressed human CYPs generated different amounts of AKF-PD 5-methylhydroxylation metabolites, but the preference of CYP isoforms to catalyze AKF-PD metabolism was as follows: 2D6?>?2C19?>?1A2?>?2E1?>?2C9?>?3A4.

3. The results demonstrated that CYP1A2 is the main isoform catalyzing AKF-PD 5-methylhydroxylation while CYP3A4, CYP2C9, CYP2E1, CYP2C19, and CYP2D6 are engaged to a lesser degree. Potential drug–drug interactions involving CYP1A2 may be noticed when AKF-PD is used combined with CYP1A2 inducers or inhibitors.

     

In vitro metabolism of nobiletin,a polymethoxy-flavonoid,by human liver microsomes and cytochrome P450

1. Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs.

2. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4′-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4′-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL.

3. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4′-OH-NBL.

4. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

     

Inhibition and induction of human cytochrome P450 enzymes in vitro by capsaicin

1. Widespread exposure to capsaicin occurs through food and topical medicines. To investigate potential food-drug or drug–drug interactions, capsaicin was evaluated in vitro against seven human drug-metabolizing cytochrome P450 (CYP) enzymes.

2. At concentrations occurring after ingestion of chili peppers or topical administration of a high-concentration patch, capsaicin did not cause direct inhibition of any CYP enzyme. Direct inhibition was only observed at much higher concentrations; the lowest IC₅₀ value was 2.0 μM. For CYP2E1, the IC₅₀ value was too high to calculate. With pre-incubation, inhibition decreased for CYP1A2, 2C9, 2C19 and 3A4/5, whereas inhibition of CYP2B6 increased and moderately increased for CYP2D6.

3. Induction of CYP activity was evaluated in microsomes from hepatocyte primary cultures. Capsaicin did not induce CYP1A2, 2B6, 2C9, 2C19, 2E1 or 3A4/5. 10 μM capsaicin caused a statistically significant increase in CYP1A2 activity (8.6% of the positive control).

4. Inhibition of drug metabolism by capsaicin should be minimal, as the ratio of [I]/K_i for direct inhibition is?<?0.1. Although pre-incubation did enhance the potency for CYP2B6 inhibition to 5.1 μM, given that exposure to capsaicin from either food or a topical medicine is very low (≤58?nM) and transient, effects on CYPs appear unlikely.

     

Effects of Phyllanthus amarus on the pharmacokinetics of midazolam and cytochrome P450 activities in rats

1. Phyllanthus amarus, a commonly used medicinal herb, was investigated for possible herb–drug interactions. The effect on CYP3A-mediated drug metabolism in rats after single dose administration of P. amarus extract was investigated using midazolam (MDZ) as a probe substrate. The effect of multiple dose administration of P. amarus extract on activity and expression of various CYP isoforms were studied.

2. Oral administration of P. amarus extract (800?mg/kg) 1?h before oral MDZ increased the C_max and AUC_0–-∞ of MDZ by 3.9- and 9.6-fold and decreased the clearance by 12%, but did not alter the pharmacokinetics of intravenous MDZ.

3. Daily administration of P. amarus extract (200 or 800?mg/kg/day) for 15 days in rats increased the activity and expression of CYP3A and CYP2B1/2. In contrast, the activities and expressions of CYP1A, CYP2C and CYP2E1 were not significantly changed.

4. The dual effects of P. amarus extract on CYP enzymes were demonstrated. Single dose administration of the extract increased oral bioavailability of MDZ through inhibition of intestinal CYP3A whereas repeated administration of the extract slightly induced hepatic CYP3A and CYP2B1/2 in rats, which suggested that herb–drug interactions by P. amarus may potentially occur via CYP3A and 2B.

     

Role for protein kinase C delta in the functional activity of human UGT1A6: implications for drug–drug interactions between PKC inhibitors and UGT1A6

1. Many UDP-glucuronosyltransferases (UGTs) require phosphorylation by protein kinase C (PKC) for glucuronidation activity. Inhibition of UGT phosphorylation by PKC inhibitor drugs may represent a novel mechanism for drug–drug interactions.

2. The potential for PKC-mediated inhibition of human UGT1A6, an isoform involved in the glucuronidation of drugs such as acetaminophen (paracetamol) and endogenous substrates including serotonin, was evaluated using various cell model systems.

3. Of ten different PKC inhibitors screened for their effects on acetaminophen glucuronidation by human LS180 colon cells, only rottlerin (PKC δ selective inhibitor; IC₅₀?=?9.0?±?1.2 μM) and the non-selective PKC inhibitors (calphostin-C, curcumin and hypericin) decreased glucuronidation by more than 50%.

4. Using UGT1A6-infected Sf9 insect cells, calphostin-C and hypericin showed three times more potent inhibition of serotonin glucuronidation in treated whole cells versus cell lysates. However, both curcumin and rottlerin showed significant direct inhibition and so (indirect) PKC effects could not be differentiated in this model system.

5. Of nine PKC isoforms co-expressed with UGT1A6 in human embryonic kidney 293T cells only PKC δ increased protein-normalized UGT1A6-mediated serotonin glucuronidation significantly (by 63% ± 4%).

6. These results identify an important role for PKC δ in UGT1A6-mediated glucuronidation and suggest that PKC δ inhibitors could interfere with glucuronidation of UGT1A6 substrates.

     

Assessment of the in vitro cytochrome P450 (CYP) inhibition potential of ZYTP1, a novel poly (ADP-ribose) polymerase inhibitor

Abstract

1. ZYTP1 is a novel Poly (ADP-ribose) polymerase protein inhibitor being developed for cancer indications.

2. The focus of the work was to determine if ZYTP1 had a perpetrator role in the in vitro inhibition of cytochrome P450 (CYP) enzymes to aid dosing decisions during the clinical development of ZYTP1.

3. ZYTP1 IC₅₀ for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4/5 was determined using human liver microsomes and LC-MS/MS detection. CYP3A4/5 IC₅₀ of depropylated metabolite of ZYTP1 was also determined. Time dependent inhibition of CYP3A4/5 by ZYTP1 was also assessed using substrates, testosterone and midazolam.

4. The mean IC₅₀ values of ZYTP1 were >100 µM for CYP1A2, 2B6 and 2D6, while 56.1, 24.5, 39.5 and 23.3–58.7 µM for CYP2C8, 2C9, 2C19 and 3A4/5, respectively. The CYP3A4/5 IC₅₀ of depropylated metabolite was 11.95–24.51 µM. Time dependent CYP3A4/5 inhibition was noted for testosterone and midazolam with IC₅₀ shift of 10.9- and 39.9-fold, respectively. With midazolam, the kinact and KI values of ZYTP1 were 0.075?min^?1 and 4.47 µM for the CYP3A4/5 time dependent inhibition, respectively.

5. Because of potent inhibition of CYP3A4/5, drugs that undergo metabolism via CYP3A4/5 pathway should be avoided during ZYTP1 therapy.

     

CYP2D plays a major role in berberine metabolism in liver of mice and humans

1. Berberine is a widely used plant extract for gastrointestinal infections, and is reported to have potential benefits in treatment for diabetes and hypercholesterolemia. It has been suggested that interactions between berberine-containing products and cytochromes P450 (CYPs) exist, but little is known about which CYPs mediate the metabolism of berberine in vivo.

2. In this study, berberine metabolites in urine and feces of mice were analyzed, and the role that CYPs play in producing these metabolites were characterized in liver microsomes from mice (MLM) and humans (HLM), as well as recombinant human CYPs. Eleven berberine metabolites were identified in mice, including 5 unconjugated metabolites, mainly in feces, and 6 glucuronide and sulfate conjugates, predominantly in urine. Three novel berberine metabolites were observed. Three unconjugated metabolites of berberine were produced by MLM, HLM, and recombinant human CYPs. CYP2D6 was the primary recombinant human CYP producing these metabolites, followed by CYP1A2, 3A4, 2E1 and CYP2C19. The metabolism of berberine in MLM and HLM was decreased the most by a CYP2D inhibitor, and moderately by inhibitors of CYP1A and 3A.

3. CYP2D plays a major role in berberine biotransformation, therefore, CYP2D6 pharmacogenetics and potential drug-drug interactions should be considered when berberine is used.

     

Effect of a traditional Chinese medicine Liu Wei Di Huang Wan on the activities of CYP2C19, CYP2D6 and CYP3A4 in healthy volunteers

1. Liu Wei Di Huang Wan (LDW), a well-known traditional Chinese medicine, is widely used for the treatment of various diseases in China. This study was designed to investigate the potential herb–drug interactions of LDW in healthy volunteers and attempted to ascertain whether the interaction might be affected by genotypes.

2. We assessed the effect of LDW on the activities of CYP2C19, CYP2D6 and CYP3A4 in 12 Chinese healthy subjects in a single-center, controlled, non-blinded, two-way crossover clinical trial. The subject pool consisted of six extensive metabolizers with CYP2C19*1/*1 and six poor metabolizers with CYP2C19*2/*2. Placebo or 4.8?g LDW (12 pills, 0.2?g/pill, twice daily) was given to each participant for 14 continuous days with a wash-out period of 2 weeks after an oral administration of 30?mg omeprazole, 30?mg dextromethorphan hydrobromide and 7.5?mg midazolam. The activities of CYP2C19, CYP2D6 and CYP3A4 were ascertained by their respective plasma or urinary metabolic ratios on day 14 post-treatment.

3. There is no difference in the activities of the three tested enzymes before or after a 14-day administration of LDW. LDW had no effect on the pharmacokinetic parameters of the substrates and their metabolites.

4. A 14-day administration of LDW did not affect the activities of CYP2C19, CYP2D6 and CYP3A4. LDW is unlikely to cause pharmacokinetic interaction when it is combined with other medications predominantly metabolized by these enzymes.

     

Functional characterization of five CYP2C8 variants and prediction of CYP2C8 genotype-dependent effects on in vitro and in vivo drug–drug interactions

1. To analyze the polymorphic activities of CYP2C8 and evaluate their impact on drug inhibitory potential, three CYP2C8 allelic variants (CYP2C8.2, CYP2C8.3, and CYP2C8.4), two non-synonymous single nucleotide polymorphic variants (R139K and K399R, carried by CYP2C8.3), and wild-type CYP2C8 (CYP2C8.1) were heterologously expressed in yeast, and their enzymatic activities were characterized. CYP2C8 inhibition-based in vitro and in vivo drug–drug interactions (DDIs) in wild-type and variant CYP2C8s were then predicted.

2. Functional characterization of five CYP2C8 variants revealed similar enzymatic activity in R139K and low activity in CYP2C8.2, CYP2C8.3, CYP2C8.4, and K399R compared with CYP2C8.1. The systematic analysis of these CYP2C8 variants can provide more homogeneous data for predicting CYP2C8 phenotypes and could be applied to personalized drug therapy.

3. Prediction of DDIs indicated that CYP2C8.4, R139K, and K399R dramatically alter the IC₅₀ values of nifedipine, troglitazone, and raloxifene, and R139K qualitatively and quantitatively reduces the risk of in vivo paclitaxel–raloxifene and paclitaxel–troglitazone interactions. The results provide the first evidence that CYP2C8 inhibition-based DDIs may be influenced by CYP2C8 genetic polymorphisms. These inhibition data can be used by pharmacologists in the design of in vivo studies to further assess and address the potential role of CYP2C8 genotype-dependent inhibition in clinical DDIs.

     

In vitro metabolism of the specific endothelin-A receptor antagonist ZD4054 and clinical drug interactions between ZD4054 and rifampicin or itraconazole in healthy male volunteers

1. ZD4054 is an oral specific endothelin-A receptor antagonist in development for the treatment of hormone-resistant prostate cancer. Both renal and metabolic processes contribute to its overall clearance.

2. Two preclinical in vitro studies investigated the metabolism of ZD4054 using human liver microsomes, individual cytochrome P450 (CYP) isozymes, and flavin-containing monooxygenase isoforms. Two Phase I open-label crossover volunteer studies subsequently investigated in vivo drug interactions between ZD4054 and the CYP450 inducer rifampicin or CYP3A4 inhibitor itraconazole.

3. The most abundant metabolite produced in in vitro incubations accounted for 12.8% of radioactivity after ZD4054 was incubated with CYP3A4. No significant flavin-containing monooxygenase metabolism of ZD4054 was observed. In the in vivo studies, rifampicin co-administration reduced the area under the concentration–time curve and maximum plasma concentration of ZD4054 by 68% and 29%, respectively, whilst co-administration with itraconazole was associated with an increase in ZD4054 area under the curve of approximately 28%.

4. While co-administration of CYP450 inducers might be associated with reduced efficacy of ZD4054, dose reduction is unlikely to be required with concomitant administration of CYP3A4 inhibitors.

     

Method for predicting the risk of drug–drug interactions involving inhibition of intestinal CYP3A4 and P-glycoprotein

1. To develop a method to predict the risk of drug–drug interactions involving the inhibition of intestinal CYP3A4 or P-glycoprotein, data from clinical drug–drug interaction studies of CYP3A4 and/or P-glycoprotein substrates were analysed. The ratio of inhibitor dose (Dose_i) to inhibition constant (K_i), termed the drug-interaction number, was used to index intestinal drug–drug interaction.

2. From the analysis, it was found that (1) CYP3A4 inhibitors with a drug-interaction number below 2.8?L have a low risk of interacting with substrates which exhibit intestinal first-pass metabolism and those with a drug-interaction number above 9.4?L have a high risk; (2) P-glycoprotein inhibitors with a drug-interaction number below 10.8?L have a low risk of interacting with P-glycoprotein substrates and those with a drug-interaction number above 27.9?L have a high risk; and (3) the drug-interaction number indexes, 2.8?L and 9.4?L for CYP3A4 and 10.8?L and 27.9?L for P-glycoprotein were validated by data from dual CYP3A4/P-glycoprotein substrates.

3. In conclusion, the drug-interaction number is useful for classifying the risk of drug–drug interactions involving the inhibition of intestinal CYP3A4 and P-glycoprotein. This drug-interaction number-based approach is similar to the method that the US Food and Drug Administration (USFDA) recently proposed in the draft guidance for predicting P-glycoprotein-mediated drug–drug interaction.

     

Identification of the novel canine CYP1A2 1117 C>T SNP causing protein deletion

1. The pharmacokinetics of YM-64227 (4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1H)-one), a novel and selective phosphodiesterase type 4 inhibitor, was characterized in beagle dogs. Based on the plasma parent drug to major hydroxylated metabolite ratio, 21 dogs were phenotyped as 16 extensive metabolizers (EM) and five poor metabolizers (PM).

2. Nucleotide sequences of CYPs 1A2, 2B11, 2C21, 2D15, 2E1 and 3A12 were investigated in the EM and PM dogs. A CYP1A2 1117 C>T single nucleotide polymorphism was found, which resulted in an amino acid change from an Arg codon to a stop codon at position 373. All dogs phenotyped as PM were T/T homozygous, whereas EMs were C/C homozygous and C/T heterozygous.

3. In Western blotting of liver microsomes, CYP1A protein expression was detected in the C/C and C/T types, but not in the T/T type.

4. Of 65 dogs genotyped using genome DNA, the frequencies of the C and T alleles were 0.61 and 0.39, respectively, suggesting approximately 15% of the dogs would not express the CYP1A2 protein.

5. The findings provide a coherent explanation for the inter-individual variability in the pharmacokinetics of CYP1A2 substrate drugs in dogs.

     

Flavin monooxygenases,FMO1 and FMO3, not cytochrome P450 isoenzymes,contribute to metabolism of anti-tumour triazoloacridinone,C-1305, in liver microsomes and HepG2 cells

1. 5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, being the close structural analogue of the clinically tested imidazoacridinone anti-tumour agent, C-1311, expressed high activity against experimental tumours and is expected to have more advantageous pharmacological properties than C-1311.

2. The aim of this study was to elucidate the role of selected liver enzymes in the metabolism of C-1305.

3. We demonstrated that the studied triazoloacridinone was transformed with rat and human liver microsomes, HepG2 hepatoma cells and with human recombinant flavin-containing monooxygenases FMO1, FMO3 but not with CYPs. Furthermore, this compound was an effective inhibitor of CYP1A2 and CYP3A4. The product of FMO catalysed metabolism was shown to be identical to the main metabolite from liver microsomes and HepG2 cells. It was identified as an N-oxide derivative and, under hypoxia, it underwent retroreduction back to C-1305, what was extremely effective with participation of CYP3A4.

4. In summary, this work revealed that the involvement of the P450 enzymatic system in microsomal and cellular metabolism of C-1305 was negligible, whereas this agent was an inhibitor of CYP1A2 and CYP3A4. In contrast, FMO1 and FMO3 were crucial for metabolism of C-1305 by liver microsomes and in HepG2 cells, which makes C-1305 an attractive potent anti-tumour agent.