Plateable cryopreserved human hepatocytes for the assessment of cytochrome P450 inducibility: experimental condition-related variables affecting their response to inducers

Rationale: The aim of the present study was to assess the stability of cryopreserved human hepatocytes over 5 years and to explore experimental condition-related variables such as seeding density, culture matrix and medium, start and duration of treatment that could potentially affect the quality of cultures and their response to cytochrome P450 (CYP) inducers. Results: 63/125 batches of cryopreserved human hepatocytes were plateable after thawing. Of those, 17 batches showed reproducible recovery, viability and plateability (less than 5% intra-batch variability) up to 5 years. When cultured in collagen home-coated 48-well plates at a seeding density allowing 70% confluence, cryopreserved human hepatocytes display activities equivalent to fresh counterparts. Their response to CYP inducers is maximal and equivalent to fresh counterpart for an incubation of 72 h starting at Day 2 or Day 3 after plating when cultured in modified Hepatocyte Maintenance Medium (HMM). The number of cryopreserved human hepatocytes can be further reduced by using a cocktail of CYP substrates for the assessment of their inducibility. Conclusions: Experimental condition-related variables, such as seeding density, culture matrix and medium, start and duration of treatment, affecting the response of plateable thawed cryopreserved human hepatocytes to cytochrome P450 inducers can be reduced by optimizing critical steps of the protocols.     

Prediction of clinical CYP3A4 induction using cryopreserved human hepatocytes

1. The purpose of this study was to construct a method to predict CYP3A4 induction in the clinical setting from in vitro data using cryopreserved human hepatocytes.

2. We recently developed an approach with in vitro assays of HepaRG cell lines for predicting CYP3A4 induction by using a novel value, termed the relative factor (RF), determined from the ratio of the concentration of an inducer to the reference standard. In this study, the applicability of the RF approach was expanded to cryopreserved human hepatocytes.

3. Induction assays were performed in vitro using hepatocytes from four individual donors and eight typical inducers. The obtained RF values were related to the free plasma concentration of each inducer (expressed as Css,u/RF).

4. A good relationship between the Css,u/RF values and the in vivo induction response was found for all donors. Inducers were classified by the Css,u/RF values into three categories for CYP3A4 induction risk (high, medium and low potency), and thereby the degree of CYP3A4 induction in vivo in humans could be predicted from the Css,u/RF values.

5. The RF approach is applicable to human cryopreserved hepatocytes. Thus, a method to predict the potency of CYP3A4 inducers was constructed using cryopreserved human hepatocytes.

     

Species differences in the hepatic effects of inducers of CYP2B and CYP4A subfamily forms: relationship to rodent liver tumour formation

1. Phenobarbitone and related compounds induce hepatic microsomal cytochrome P450 (CYP) 2B forms (mediated by the constitutive androstane receptor), whereas peroxisome proliferators induce CYP4A forms (mediated by the peroxisome proliferator-activated receptor alpha) in rats and mice.

2. A number of non-genotoxic CYP2B and CYP4A inducers have been shown to produce liver tumours in rats and mice.

3. The hepatic effects of CYP2B and CYP4A inducers are reviewed and evaluated with respect to their established modes of action for rodent liver tumour formation and species differences in response. While CYP2B and CYP4A inducers stimulate replicative DNA synthesis in rodent liver, they do not appear to be mitogenic agents in human hepatocytes.

4. Epidemiological studies have demonstrated that phenobarbitone and rodent peroxisome proliferators do not increase the incidence of liver tumours in humans.

5. It is concluded that rodent CYP2B and CYP4A inducers do not pose a hepatocarcinogenic hazard for humans.

     

Generation of proliferating human hepatocytes using upcyte® technology: characterisation and applications in induction and cytotoxicity assays

1. We have developed a novel technique which causes primary human hepatocytes to proliferate by transducing them with genes that upregulate their proliferation.

2. Upcyte^® hepatocytes did not form colonies in soft agar and are not immortalised anchorage-independent cells. Confluent cultures expressed liver-specific proteins, produced urea and stored glycogen.

3. CYP activities were low but similar to that in 5-day cultures of primary human hepatocytes. CYP1A2 and CYP3A4 were inducible; moreover, upcyte^® hepatocytes predicted the in vivo induction potencies of known CYP3A4 inducers using the “relative induction score” prediction model. Placing cells into 3D culture increased their basal CYP2B6 and CYP3A4 basal activities and induction responses.

4. Phase 2 activities (UGTs, SULTs and GSTs) were comparable to activities in freshly isolated hepatocytes.

5. Upcyte^® hepatocytes were markedly more sensitive to the hepatotoxin, α-amanitin, than HepG2 cells, indicating functional OATP1B3 uptake. The cytotoxicity of aflatoxin B₁, was decreased in upcyte^® hepatocytes by co-incubation with the CYP3A4 inhibitor, ketoconazole. Upcyte^® hepatocytes also differentiated between ten hepatotoxic and eight non-hepatotoxic compounds.

6. In conclusion, upcyte^® hepatocyte cultures have a differentiated phenotype and exhibit functional phase 1 and 2 activities. These data support the use of upcyte^® hepatocytes for CYP induction and cytotoxicity screening.

     

Comparison of intrinsic metabolic clearance in fresh and cryopreserved human hepatocytes

1. We compared the intrinsic clearance (CL_int) of a number of substrates in suspensions of fresh and cryopreserved human hepatocytes from seven donors.

2. CL_int values for a cocktail incubation of phenacetin, diclofenac, diazepam, bufuralol, midazolam, and hydroxycoumarin were 4.9?±?3.4, 18?±?7.2, 5.1?±?4.9, 6.3?±?3.3, 9.8?±?5.8 and 22?±?14?μl min^?1/10⁶ cells, respectively, and they correlated well with corresponding CL_int values using cryopreserved hepatocytes from 25 different donors.

3. CL_int values of each cocktail substrate and 20 AstraZeneca new chemical entities were compared in fresh and cryopreserved hepatocytes from the same three donors. There was a statistically significant correlation between CL_int in fresh and cryopreserved hepatocytes for each of the three livers (p?int values was 1.03.

4. In conclusion, the results add further support to the use of cryopreserved human hepatocytes as a screening model for the intrinsic clearance of new chemical entities.

     

Hepatic CYP2B6 is altered by genetic,physiologic, and environmental factors but plays little role in nicotine metabolism

1. Human cytochrome P4502B6 (CYP2B6) is predominantly expressed in the liver and it plays a major role in the metabolism of several therapeutically important drugs and environmental toxicants.

2. The objective was twofold: (1) to determine the role of genetic, physiological, and environmental factors in predicting hepatic CYP2B6 protein expression; and (2) to investigate the role of CYP2B6 in nicotine C-oxidation.

3. Human livers (n?=?40) were assessed for CYP2B6 protein and genotype.

4. Linear regression analyses indicated that CYP2B6 genotype (10%), gender (14%), and exposure to inducers (21%), but not age, were predictors of CYP2B6 protein amounts. Livers with at least one CYP2B6*5 or *6 allele were associated with lower CYP2B6. Female livers and livers exposed to inducers (phenobarbital and/or dexamethasone) were associated with higher CYP2B6.

5. A weak correlation between CYP2B6 and nicotine C-oxidation activity was observed, which was abrogated when controlling for CYP2A6 protein levels. CYP2B6*6 was not associated with different nicotine kinetics.

6. In summary, CYP2B6 protein expression was associated with genotype, gender, and exposure to inducers, but not with nicotine C-oxidation activity.

     

Assessment of cryopreserved human hepatocytes as a model system to investigate sulfation and glucuronidation and to evaluate inhibitors of drug conjugation

1. Cultured cryopreserved human hepatocytes are extensively used as a model system for studying drug metabolism, although they remain poorly characterized in respect of the major conjugation reactions glucuronidation and sulfation.

2. Using paracetamol (acetaminophen), we assessed eleven samples of cryopreserved human hepatocytes for their suitability to investigate the simultaneous glucuronidation and sulfation of xenobiotics and evaluated inhibitors of conjugation.

3. Kinetic characterization showed broadly similar values for paracetamol conjugation by hepatocytes (as reported in the literature for in vitro systems), with K_m values of approximately 6 mM and 0.3 mM for glucuronidation and sulfation, respectively. Substantial interindividual differences were observed.

4. The hepatocytes demonstrated a strong dose-dependent switch from a preponderance of sulfation at low concentrations of paracetamol to glucuronidation at higher doses, consistent with routes of clearance in vivo.

5. A number of drugs, some of which such as probenecid and sulfinpyrazone are known to interact with paracetamol in vivo, were demonstrated to inhibit the sulfation and/or glucuronidation of paracetamol in hepatocytes, demonstrating the potential application of this model system for studying drug–drug interactions involving conjugation.

     

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

1. 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.

2. 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.

3. 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.

4. 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.

     

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.

     

In vitro model for the prediction of clinical CYP3A4 induction using HepaRG cells

1. It is important to predict CYP3A enzyme induction in the drug-discovery process to avoid adverse effects in clinical. In the present study, we constructed a method to correct the variability of in vitro CYP3A induction assays and thereby a method for the prediction of CYP3A induction in the clinical setting.

2. Induction assays were performed in vitro using HepaRG cells and seven typical inducers. An index value was determined for enzyme induction, termed the relative factor (RF), from the ratio of the concentration of the inducers to the reference standards.

3. Using RF as an index, variation among the assays was reduced.

4. A good relationship was obtained between the ratio of the free plasma concentration at steady-state (C_ss,u) to RF (expressed as C_ss,u/RF) and the in vivo induction response. Using rifampicin as a reference standard, compounds with a C_ss,u/RF value greater than 7.31 nmol l^?1 may induce CYP3A in vivo in humans.

     

Metabolism of human cytochrome P450 marker substrates in mouse: a strain and gender comparison

1. The aim was to characterize mouse gender and strain differences in the metabolism of commonly used human cytochrome (CYP) P450 probe substrates.

2. Thirteen human CYP probe substrates (phenacetin, coumarin, 7-ethoxy-4-trifluoromethyl coumarin, amiodarone, paclitaxel, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, chlorzoxazone, p-nitrophenol, testosterone and lauric acid) were used in activity measurements. The metabolism of the probe substrates was compared in liver microsomes from male and female NMRI, CBA, C57bl/6, 129/SvJ and CD1 strains. The expression of proteins identified on Western blots with commonly available antibodies selective for specific human and rat CYP enzymes were compared in the different mouse strains.

3. Males had higher metabolism than corresponding females for phenacetin O-deethylation (human marker for CYP1A2 activity), and a high correlation was found between phenacetin activity and immunoreactivity in Western blots produced with rat CYP1A2 antibodies.

4. Protein detected by antibodies cross-reacting with human CYP2B6 and rat CYP2B1/2 antibodies was female specific except for the 129/SvJ strain, where it was absent in both genders.

5. Females generally had a higher metabolism of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation (human markers for CYP2D activity).

6. Bufuralol 1′-hydroxylation correlated with a female-dominant mouse CYP, which was detected with antibodies against rat CYP2D4.

7. p-Nitrophenol 2-hydroxylation correlated better than chlorzoxazone 6-hydroxylation with the protein detected with antibodies against rat CYP2E1, indicating that p-nitrophenol is a more specific substrate for mouse CYP2E1.

     

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.

     

Pharmacokinetics,disposition and lipid-modulating activity of 5-{2-[4-(3,4-difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid,a potent and subtype-selective peroxisome proliferator-activated receptor α agonist in preclinical species and human

1. 5-{2-[4-(3,4-Difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid (1) is a novel, potent, and selective agonist of the peroxisome proliferator-activated receptor alpha (PPAR-α).

2. In preclinical species, compound 1 demonstrated generally favourable pharmacokinetic properties. Systemic plasma clearance (CL_p) after intravenous administration was low in Sprague–Dawley rats (3.2?±?1.4?ml min^?1 kg^?1) and cynomolgus monkeys (6.1?±?1.6?ml min^?1 kg^?1) resulting in plasma half-lives of 7.1?±?0.7?h and 9.4?±?0.8?h, respectively. Moderate bioavailability in rats (64%) and monkeys (55%) was observed after oral dosing. In rats, oral pharmacokinetics were dose-dependent over the dose range examined (10 and 50?mg kg^?1).

3. In vitro metabolism studies on 1 in cryopreserved rat, monkey, and human hepatocytes revealed that 1 was metabolized via oxidation and phase II glucuronidation pathways. In rats, a percentage of the dose (approximately 19%) was eliminated via biliary excretion in the unchanged form.

4. Studies using recombinant human CYP isozymes established that the rate-limiting step in the oxidative metabolism of 1 to the major primary alcohol metabolite M1 was catalysed by CYP3A4.

5. Compound 1 was greater than 99% bound to plasma proteins in rat, monkey, mouse, and human.

6. No competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, P4502C9, P4502C19, P4502D6 and P4503A4 (IC₅₀’s?>?30 μM) was discerned with 1.

7. Because of insignificant turnover of 1 in human liver microsomes and hepatocytes, human clearance was predicted using rat single-species allometric scaling from in vivo data. The steady-state volume was also scaled from rat volume after normalization for protein-binding differences. As such, these estimates were used to predict an efficacious human dose required for 30% lowering of triglycerides.

8. In order to aid human dose projections, pharmacokinetic/pharmacodynamic relationships for triglyceride lowering by 1 were first established in mice, which allowed an insight into the efficacious concentrations required for maximal triglyceride lowering. Assuming that the pharmacology translated in a quantitative fashion from mouse to human, dose projections were made for humans using mouse pharmacodynamic parameters and the predicted human pharmacokinetic estimates.

9. First-in-human clinical studies on 1 following oral administration suggested that the human pharmacokinetics/dose predictions were in the range that yielded a favourable pharmacodynamic response.

     

In vitro metabolism of magnolin and characterization of cytochrome P450 enzymes responsible for its metabolism in human liver microsomes

1. Magnolin is a major bioactive component found in Shin-i, the dried flower buds of Magnolia fargesii; it has anti-inflammatory and anti-histaminic activities. Incubation of magnolin in human liver microsomes with an nicotinamide adenine dinucleotide phosphate-generating system resulted in the formation of five metabolites, namely, O-desmethyl magnolin (M1 and M2), didesmethylmagnolin (M3), and hydroxymagnolin (M4 and M5).

2. In this study, we characterized the human liver cytochrome P450 (CYP) enzymes responsible for the biotransformation of three major metabolites—M1, M2, and M4—of magnolin. CYP2C8, CYP2C9, CYP2C19, and CYP3A4 were identified as the major enzymes responsible for the formation of the two O-desmethyl magnolins (M1 and M2), on the basis of a combination of correlation analysis and experiments, including immunoinhibition of magnolin in human liver microsomes and metabolism of magnolin by human cDNA-expressed CYP enzymes. CYP2C8 played a predominant role in the formation of hydroxymagnolin (M4).

3. These results suggest that the pharmacokinetics of magnolin may not be affected by CYP2C8, CYP2C9, CYP2C19, and CYP3A4 responsible for the metabolism of magnolin or by the co-administration of appropriate CYP2C8, CYP2C9, CYP2C19, and CYP3A4 inhibitors or inducers due to the involvement of multiple CYP enzymes in the metabolism of magnolin.

     

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.

     

Role of individual human cytochrome P450 enzymes in the in vitro metabolism of hydromorphone

1. The aim was to identify the individual human cytochrome P450 (CYP) enzymes responsible for the in vitro N-demethylation of hydromorphone and to determine the potential effect of the inhibition of this metabolic pathway on the formation of other hydromorphone metabolites.

2. Hydromorphone was metabolized to norhydromorphone (apparent K_m = 206?? 822?μM, V_max = 104 ? 834?pmol?min^?1?mg^?1 protein) and dihydroisomorphine (apparent K_m = 62 ? 557?μM, V_max = 17 ? 122?pmol?min^?1?mg^?1 protein) by human liver microsomes.

3. In pooled human liver microsomes, troleandomycin, ketoconazole and sulfaphenazole reduced norhydromorphone formation by an average of 45, 50 and 25%, respectively, whereas furafylline, quinidine and omeprazole had no effect. In an individual liver microsome sample with a high CYP3A protein content, troleandomycin and ketoconazole inhibited norhydromorphone formation by 80%.

4. The reduction in norhydromorphone formation by troleandomycin and ketoconazole was accompanied by a stimulation in dihydroisomorphine production.

5. Recombinant CYP3A4, CYP3A5, CYP2C9 and CYP2D6, but not CYP1A2, catalysed norhydromorphone formation, whereas none of these enzymes was active in dihydroisomorphine formation.

6. In summary, CYP3A and, to a lesser extent, CYP2C9 catalysed hydromorphone N-demethylation in human liver microsomes. The inhibition of norhydromorphone formation by troleandomycin and ketoconazole resulted in a stimulation of microsomal dihydroisomorphine formation.

     

The in vitro metabolism of bupropion revisited: concentration dependent involvement of cytochrome P450 2C19

1. The involvement of cytochrome P450 2B6 (CYP2B6) to the in vitro and in vivo metabolism of bupropion has been well studied. In these investigations we performed a detailed in vitro phenotyping study to characterize isoforms other than CYP2B6.

2. A total of nine metabolites were identified (M1–M9) in the incubations with cDNA-expressed P450s (rhCYP) and human liver microsomes (HLM).

3. Incubations in rhCYP identified CYP2B6 as the isoform responsible for the formation of hydroxybupropion (M3). CYP2C19 was involved in bupropion metabolism primarily through alternate hydroxylation pathways (M4–M6) with higher activity at lower substrate concentrations, near 1 µM.

4. The results from HLM inhibition studies using CYP2B6 and CYP2C19 inhibitory antibodies indicated that CYP2B6 contributed to approximately 90% of M3 formation, and CYP2C19 contributed to approximately 70–90% of M4, M5, and M6 formation.

5. Studies using single donor HLM with varying degrees of CYP2B6 and CYP2C19 activities showed a good relationship between M3 formation and CYP2B6 activity and M4/M5 formation and CYP2C19 activity.

6. These results confirmed the principle role of CYP2B6 in hydroxybupropion formation, as a selective CYP2B6 probe. In addition, the new findings revealed that CYP2C19 also contributes to bupropion metabolism through alternate hydroxylation pathways.

     

Evaluation of cytochrome P450 inductions by anti-epileptic drug oxcarbazepine, 10-hydroxyoxcarbazepine,and carbamazepine using human hepatocytes and HepaRG cells

1. Anti-epileptic drug oxcarbazepine is structurally related to carbamazepine, but has reportedly different metabolic pathway. Auto-induction potentials of oxcarbazepine, its pharmacologically active metabolite 10-hydroxyoxcarbazepine and carbamazepine were evaluated by cytochrome P450 (CYP) 1A2, CYP2B6 and CYP3A4 mRNA levels and primary metabolic rates using human hepatocytes and HepaRG cells.

2. For the CYP1A2 the induction potential determined as the fold change in mRNA levels was 7.2 (range: 2.3–11.5) and 10.0 (6.2–13.7) for oxcarbazepine and carbamazepine, respectively, while 10-hydroxyoxcarbazepine did not induce. The fold change in mRNA levels for CYP2B6 was 11.5 (3.2–19.3), 7.0 (2.5–10.8) and 14.8 (3.1–29.1) for oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine, respectively. The fold change for CYP3A4 induction level by oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine was 3.5 (1.2–7.4), 2.7 (0.8–5.7) and 8.3 (3.5–14.5), respectively. The data suggest lower induction potential of oxcarbazepine and 10-hydroxyoxcarbazepine relative to carbamazepine. The results in HepaRG cells showed similar trend as the human hepatocytes.

3. After incubation for 72?h in hepatocytes and HepaRG cells, auto-induction was evident for only carbamazepine metabolism. The 10-keto group instead of double bond at C10 position is evidently a determinant factor for limited auto-induction of P450 enzymes by oxcarbazepine.

     

Activation of CYP3A-mediated testosterone 6β-hydroxylation by tanshinone IIA and midazolam 1-hydroxylation by cryptotanshinone in human liver microsomes

1. This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone.

2. The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed.

3. Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not.

4. The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.