Long‐term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures

Human primary hepatocytes were cultivated in a microfluidic bioreactor and in Petri dishes for 13 days. mRNA kinetics in biochips showed an increase in the levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6, HNF4a, SULT1A1, UGT1A1 mRNA related genes when compared with post extraction levels. In addition, comparison with Petri dishes showed higher levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6 related genes at the end of culture. Functional assays illustrated a higher urea and albumin production over the period of culture in biochips. Bioreactor drug metabolism (midazolam and phenacetin) was not superior to the Petri dish after 2 days of culture. The CYP3A4 midazolam metabolism was maintained in biochips after 13 days of culture, whereas it was almost undetectable in Petri dishes. This led to a 5000‐fold higher value of the metabolic ratio in the biochips. CYP1A2 phenacetin metabolism was found to be higher in biochips after 5, 9 and 13 days of culture. Thus, a 100‐fold higher metabolic ratio of APAP in biochips was measured after 13 days of perfusion. These results demonstrated functional primary human hepatocyte culture in the bioreactor in a long‐term culture. Copyright © 2016 John Wiley & Sons, Ltd.     

Can a cocktail designed for phenotyping pharmacokinetics and metabolism enzymes in human be used efficiently in rat?

We recently designed the CIME cocktail consisting of 10 drugs to assess the activity of the major human CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A), a phase II enzyme (UGT1A1/6/9), two drug transporters (P-gp and OATP1B1) and a component of the renal function ( Videau et al. 2010 ). The present work aimed at studying the usefulness of the CIME cocktail in the rat.The CIME cocktail was given per os to three male and three female rats, or incubated with rat liver microsomes. Parent substrates and metabolites were quantified by LC-MS/MS in plasma, urine and hepatic microsomal media, and phenotyping index were subsequently calculated.The CIME cocktail could therefore be used in the rat to phenotype rapidly and simultaneously CYP3A1/2 with omeprazole/omeprazole-sulfone, midazolam/1'-hydroxymidazolam or 4-hydroxymidazolam and/or dextromethorphan/3-methoxymorphinan, CYP2C6/11 with tolbutamide/4-hydroxytolbutamide, CYP2D1/2 with omeprazole/5-hydroxyomeprazole or dextromethorphan/dextrorphan, and UGT1A6/7 with acetaminophen/acetaminophen-glucuronide. Our results confirmed also several known gender differences and brought new information on the urinary excretion of rosuvastatin. However, the major rat CYPs, CYP2C11 and CYP2C12, are not specifically assessed. An optimized version of the CIME cocktail should therefore be designed and would be of major importance to more largely phenotype DMPK enzymes in rats to study DMPK variability factors such as disease, age, or to exposure to inductors or inhibitors.     

A comprehensive evaluation of metabolic activity and intrinsic clearance in suspensions and monolayer cultures of cryopreserved primary human hepatocytes

Primary human hepatocytes are widely used for metabolic stability evaluations. However, there are limited data directly comparing phase I and phase II drug‐metabolizing enzymes in fresh and cryopreserved hepatocytes prepared from the same human donor liver. We evaluated the metabolic competency of human hepatocytes prepared from seven donor tissues before and after cryopreservation. Temporal‐dependent enzyme activity in suspension and matched adherent cultures of primary human hepatocytes was also assessed. Cryopreservation of hepatocytes resulted in statistically significant increases in activities of CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A but not CYP2C8, CYP2C19, FMO, UGT, and SULT, relative to fresh hepatocytes. In suspension cultures of hepatocytes, enzyme stabilities were as follows: UGT相似文献   

Potential impact of steatosis on cytochrome P450 enzymes of human hepatocytes isolated from fatty liver grafts.

Liver grafts discarded for transplantation because of macrosteatosis can constitute a valuable source of human hepatocytes for in vitro metabolic and pharmacotoxicological studies or for therapeutic applications. A condition for using hepatocyte suspensions for these purposes is the preservation of their metabolic competence and, particularly, drug-metabolizing enzymes. A reduction in microsomal cytochrome P450 (P450) activities was observed in fatty livers (>40% steatosis) with respect to normal tissue. Similarly, decreased levels of 7-ethoxycoumarin O-deethylation and testosterone metabolism were observed in human hepatocyte cultures prepared from steatotic liver tissue. To clarify the potential impact of lipid accumulation on human hepatic P450 enzymes, we have used an in vitro model of "cellular steatosis" by incubation of cultured hepatocytes with increasing concentrations (0.25-3 mM) of long-chain free fatty acids (FFA). A dose-dependent accumulation of lipids in the cytosol is induced by FFA mixture. Hepatocytes exposed to 1 mM FFA for 14 h showed lower activity values of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 enzymes than nontreated hepatocytes (about 45-65% reduction). This treatment also produced significant decreases in CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 mRNA to about 55 to 75% of mRNA levels in control cells. Our results suggest that although human hepatocytes isolated from steatotic liver show reduced P450 activities, they are metabolically competent and can be used for drug metabolism studies.     

Cryopreserved rat, dog and monkey hepatocytes: measurement of drug metabolizing enzymes in suspensions and cultures

Metabolism in fresh and cryopreserved (CP) rat, dog and monkey hepatocyte suspensions and cultures was measured using midazolam (CYP3A), tolbutamide (CYP2C), dextromethorphan (CYP2D) and p-nitrophenol (glucuronosyl S-transferases (UGT), sulphotransferases (ST)). CYP3A, CYP2C9, CYP2D6, UGT and ST enzyme functions in fresh and CP rat, dog and monkey hepatocyte suspensions were retained - CP rat hepatocytes lost some CYP2C activity but this was restored by adding NADPH or by placing the cells in culture, suggesting that the enzyme was still functional. Phase 2 activities were equivalent in fresh and CP hepatocyte suspensions. In some cases, incubation conditions increased the rate of metabolism, possibly reflecting de novo cofactor synthesis. However, this effect was substrate and species dependent and was not always the same in fresh and CP cells. CYP3A, CYP2C, CYP2D, UGT and ST activities at 24 hours of culture of rat and monkey hepatocytes were not compromised by cryopreservation. CYP3A, CYP2D but not CYP2C were lower in 24-hour cultures of CP dog hepatocytes than in fresh cells. Despite being lower than fresh cells, UGT activity in dog CP hepatocytes did not decrease from 0 to 24 hours of culture. Species-specific metabolism of p-nitrophenol could be demonstrated in both CP cell suspensions and cultures. In conclusion, these data suggest that the enzyme characteristics of fresh and CP hepatocytes from each species and under specific incubation conditions should be considered when carrying out metabolism studies of new compounds.     

Evaluation of a Liver Microfluidic Biochip to Predict In Vivo Clearances of Seven Drugs in Rats

We investigated metabolic clearances of phenacetin, midazolam, propranolol, paracetamol, tolbutamide, caffeine, and dextromethorphan by primary rat hepatocytes cultivated in microfluidic biochips. The levels of mRNA of the HNF4α, PXR, AHR, CYP3A1, and CYP1A2 genes were enhanced in the biochip cultures when compared with postextraction levels. We measured a high and rapid adsorption on the biochip walls and inside the circuit for dextromethorphan and midazolam, a moderate adsorption for phenacetin and propranolol, and a low adsorption for caffeine, tolbutamide, and paracetamol. Drug biotransformations were demonstrated by the formations of specific metabolites such as paraxanthyne (caffeine), paracetamol (phenacetin), 1-OH midazolam (midazolam), paracetamol sulfate (paracetamol and phenacetin), and dextrorphan (dextromethorphan). We used a pharmacokinetic model to estimate the adsorption and in vitro intrinsic drug clearance values. We calculated in vitro intrinsic clearance values of 0.5, 3, 12.5, 83, 100, 160, and 900 μL/min per 10⁶ cells for the tolbutamide, caffeine, paracetamol, dextromethorphan, phenacetin, midazolam, and propranolol, respectively. A second model describing the liver as a well-stirred compartment predicted in vivo hepatic clearances of 0.1, 13.8, 30, 44.1, 61, 72, 85, and 61 mL/min per kg of body mass for the tolbutamide, caffeine, paracetamol, midazolam, dextromethorphan, phenacetin, and propranolol, respectively. These values appeared consistent with previously reported data. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:706–718, 2014     

Use of a cocktail of probe substrates for drug-metabolizing enzymes for the assessment of the metabolic capacity of hepatocyte preparations

A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation). The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CLint) were calculated from the disappearance of the compounds from the incubations. Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin. The highest CLint estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CLint was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CLint was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively. When the cocktail was incubated together with human hepatocytes and 1 microM ketoconazole, the CLint of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor. It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.     

Liver grafts preserved in Celsior solution as source of hepatocytes for drug metabolism studies: comparison with surgical liver biopsies.

Suitability of human liver grafts preserved in Celsior solution (CS) for preparing metabolically competent hepatocyte cultures has been examined. To this end, basal and induced activity and mRNA levels of major hepatic cytochrome P450 (P450) enzymes have been measured. By 24 h in culture, measurable levels of the 10 P450 mRNAs studied were found in all hepatocyte preparations examined, with CYP2E1, CYP2C9, and CYP3A4 mRNAs being the most abundant. Compared with hepatocytes obtained from surgical liver resections (SLRs), lower content of each P450 mRNA was found in hepatocytes from the CS group; however, the relative distribution of individual P450 mRNAs was similar. Similar results were observed after measuring P450 activities. CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 activities in hepatocytes from CS-flushed grafts were lower than but comparable with those of cultures prepared from SLRs. No differences in the metabolite profile of testosterone were found. Treatment of hepatocytes from CS-preserved grafts with model P450 inducers shows that 2 microM methylcholanthrene only increased CYP1A1 and CYP1A2 mRNAs (>100-fold over control), 1 mM phenobarbital markedly increased CYP2A6, CYP2B6, and CYP3A4 mRNA content (>7-fold), and 50 microM rifampicin highly increased CYP3A4 mRNA levels (>10-fold), whereas minor effects (<3-fold) were observed in CYP2A6, CYP2B6, and CYP2C9 mRNAs. This induction pattern of P450s was similar, in terms of magnitude, reproducibility, and specificity, to that shown in primary hepatocytes from surgical biopsies. Overall, our results indicate that, cold-preserved in CS, liver grafts constitute a valuable source of human hepatocytes for drug metabolism studies.     

Use of a cocktail of probe substrates for drug-metabolizing enzymes for the assessment of the metabolic capacity of hepatocyte preparations

1.?A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation).

2.?The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CL_int) were calculated from the disappearance of the compounds from the incubations.

3.?Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin.

4.?The highest CL_int estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CL_int was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CL_int was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively.

5.?When the cocktail was incubated together with human hepatocytes and 1?μM ketoconazole, the CL_int of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor.

6.?It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.     

CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes

1. In this study, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was evaluated as a substrate to assess the induction of cytochrome P450 (CYP) isoform enzyme activities in rat hepatocytes using a 96-well plate format. 2. BFC was metabolized by both untreated and sodium phenobarbitone (NaPB)-treated rat hepatocytes in a time- and concentration-dependent manner to the highly fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). 3. HFC was extensively conjugated with D-glucuronic acid and/or sulphate in both untreated and NaPB-treated rat hepatocytes, thus necessitating the inclusion of an enzymatic deconjugation step in the assay procedure. 4. The time-course of induction of 7-ethoxyresorufin metabolism by the CYP1A inducer beta-naphthoflavone (BNF), 7-benzyloxyresorufin metabolism by the CYP2B inducer NaPB and BFC metabolism b both BNF and NaPB was studied in rat hepatocytes treated for 24-96 h. The optimal time for induction of metabolism of all three substrates was 72 h, with no medium changes being necessary during this period. 5. The effect of treatment with 0.5-20 microM BNF, 50-2000 microM NaPB, 2-20 microM dexamethasone (DEX), 20-100 microM methylclofenapate (MCP), and 50 and 200 microM isoniazid (ISN) for 72 h on BFC metabolism in cultured rat hepatocytes was studied. BFC metabolism was induced by treatment with BNF, NaPB and MCP, but not with either DEX or ISN. 6. The metabolism of BFC in liver microsomes from the control rat and rat treated with CYP isoform inducers was also studied. BFC metabolism was induced by treatment with NaPB, BNF and DEX. 7. The metabolism of BFC was also studied using microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A, CYP2B, CYP2C and CYP3A isoforms. Whereas BFC was metabolized to some extent by all the rat cDNA-expressed CYP isoforms examined, at a substrate concentration of 2.5 microM the greatest rates of BFC metabolism were observed with the CYP1A1, CYP1A2 and CYP2B1 preparations. 8. In summary, the results demonstrate that BFC is a good substrate for assessing the induction of CYP1A and CYP2B isoforms in rat hepatocytes in a 96-well plate format.     

Effects of cytochrome P-450 inducers on the perazine metabolism in a primary culture of human hepatocytes

The metabolism of perazine in a primary culture of human hepatocytes after treatment of cells with TCDD (a CYP1A1/2 inducer) or rifampicin (mainly a CYP3A4 inducer) were studied in vitro. The concentrations of perazine and its main metabolites (perazine 5-sulfoxide, N-desmethylperazine) formed in hepatocytes were assayed in the extracellular medium using the HPLC method. TCDD and rifampicin induced the formation of perazine 5-sulfoxide, however, such an effect was not observed in the case of N-desmethylperazine. The accumulation of perazine 5-sulfoxide in the extracellular medium was enhanced until up to 4 h by rifampicin, and until up to 8 h byTCDD. After 24 h, perazine and perazine 5-sulfoxide were not detected in the extracellular medium of the inducer-treated cultures, except for perazine 5-sulfoxide in the TCDD-treated cultures The obtained results indicate that CYP1A2 and CYP3A4 are involved in the perazine metabolism via 5-sulfoxidation pathway.     

Kupffer cell-mediated differential down-regulation of cytochrome P450 metabolism in rat hepatocytes

Nonparenchymal cells, particularly Kupffer cells, might play an important role in the modulation of xenobiotic metabolism in liver and its pharmacological and toxicological consequences. This intercellular communication via the exchange of soluble factors was investigated in primary rat Kupffer cells and hepatocytes. Freshly isolated rat Kupffer cells were seeded onto cell culture inserts and cocultured with 5 day old serum-free rat hepatocyte monolayer cultures at a ratio of 1:1 for 2 days. Hepatocyte cultures, Kupffer cell cultures or cocultures were treated with 0.1 ng/ml-10 microg/ml lipopolysaccharide (LPS). Within this concentration range, no significant toxicity was observed in either cell type. In LPS-exposed cocultures, tumor necrosis factor alpha (TNFalpha) levels rose up to 5 ng/ml within 5 h; nitric oxide (NO) levels increased up to 70 microM within 48 h of treatment, both in a dose-dependent fashion. The release of negative (albumin) and positive (alpha1-acid-glycoprotein) acute phase proteins from the hepatocytes was strongly down- and up-regulated, respectively. The simultaneous treatment of the cocultures with phenobarbital and LPS (10 ng/ml) or 3-methylcholanthrene and LPS (10 ng/ml) resulted in a strong down-regulation (85%) of the phenobarbital-induced cytochrome P450 (CYP) isoform CYP2B1 in the hepatocytes whereas the 3-methylcholanthrene-induced isoform CYP1A1 was only weakly affected (15%). This specific down-regulation of CYP2B1 was mediated exclusively by TNFalpha, released from the Kupffer cells. It was not linked with NO release from or inducible NO synthase activity in the hepatocytes. The TNFalpha release was not affected by the two xenobiotics. Acetaminophen tested in these cocultures showed no direct interaction with the Kupffer cells. The use of liver cell cocultures is therefore a useful approach to investigate the influence of intercellular communication on xenobiotic metabolism in liver.     

Functionality of cultured human hepatocytes from elective samples, cadaveric grafts and hepatectomies

The major possible sources of human liver for hepatocyte isolation are elective liver biopsies, cadaveric liver grafts and therapeutic liver resections. The suitability in terms of metabolic-competent hepatocyte cultures and risk/benefit of these resources has been comparatively studied. To this end, viability of isolated hepatocytes, yield of isolation procedure, hepatocyte survival during culture and CYP activities were the parameters analysed. The best results were found in hepatocytes prepared from elective biopsies, whereas a marked reduction in viability and functional competence was seen in hepatocytes from hepatectomy samples. Metabolic differences were observed in total CYP oxidative metabolism (7-ethoxycoumarin O-deethylation, total testosterone hydroxylation), as well as in CYP3A4, CYP2C9 or CYP2C19 activities (testosterone oxidations at 6β-, 16β- and 17-positions, respectively). Vascular control during the hepatectomy procedure influenced hepatocyte functionality: higher CYP activities were found in hepatocytes isolated from samples obtained under non-ischemic conditions or continuous vascular clamping than in those obtained under intermittent vascular clamping. In addition to cellular functionality, other criteria such as sample availability or ethical aspects should be considered. Elective biopsies have low, but not absent, surgical risk. However, the better functionality and the higher accessibility of elective liver samples in comparison to the other groups suggest this source of liver tissue as the most appropriate for cell harvesting purposes.     

Expression changes of CYP2A and CYP3A in microsomes from pig liver and cultured hepatocytes

The P450 enzymes of the liver are responsible for the metabolism of a wide range of chemical compounds, and hepatocytes are used in pharmacological and toxicological in vitro tests. Thus, it is important to know how stable these enzymes are in culture. We measured the activity of CYP2A and CYP3A in microsomes isolated from both pig liver and primary pig hepatocyte cultures, together with the apoprotein concentration using Western blotting. The CYP2A activity and apoprotein concentration decreased rapidly; only about 5 percent remained after 48 hr incubation, whereas the CYP3A activity and apoprotein concentration was constant. CYP3A was induced 3 times after exposure to rifampicin, whereas neither rifampicin nor pyrazole could induce CYP2A. The hepatocytes were also incubated with varying concentration of FCS and autologous serum, however without effect on the stability of CYP2A, nor did different concentrations of growth hormone and testosterone added to the cultures have any effect.     

Development of a new microfluidic platform integrating co-cultures of intestinal and liver cell lines

We developed a new biological model to mimic the organ–organ interactions between the intestine and the liver. We coupled polycarbonate cell culture inserts and microfluidic biochips in an integrated fluidic platform allowing dynamic co-cultures (called IIDMP for Integrated Insert in a Dynamic Microfluidic Platform). The intestinal compartment was simulated using Caco-2 TC7 cells and the liver one by HepG2 C3A. We showed that Caco-2 TC7 viability, barrier integrity and functionality (assessed by paracellular and active transport), were not altered during co-cultures in the bioreactor in comparison with the conventional insert Petri cultures. In parallel, the viability and metabolism of the HepG2 C3A cells were maintained in the microfluidic biochips. Then, as proof of concept, we used the bioreactor to follow the transport of phenacetin through the intestinal barrier and its metabolism into paracetamol by the CYP1A of the HepG2 C3A cells. Our results demonstrated the performance of this bioreactor with cell co-cultures compared to static co-culture controls in which weak biotransformation into paracetamol was detected. Our study illustrated the interest of such a bioreactor combining the advantages of a cell culture barrier and of liver microfluidic cultures in a common framework for in vitro studies.     

Evaluation of Mutual Drug–Drug Interaction within Geneva Cocktail for Cytochrome P450 Phenotyping using Innovative Dried Blood Sampling Method

Cytochrome P450 (CYP) activity can be assessed using a ‘cocktail’ phenotyping approach. Recently, we have developed a cocktail (Geneva cocktail) which combines the use of low‐dose probes with a low‐invasiveness dried blood spots (DBS) sampling technique and a single analytical method for the phenotyping of six major CYP isoforms. We have previously demonstrated that modulation of CYP activity after pre‐treatment with CYP inhibitors/inducer could be reliably predicted using Geneva cocktail. To further validate this cocktail, in this study, we have verified whether probe drugs contained in the latter cause mutual drug–drug interactions. In a randomized, four‐way, Latin‐square crossover study, 30 healthy volunteers received low‐dose caffeine, flurbiprofen, omeprazole, dextromethorphan and midazolam (a previously validated combination with no mutual drug–drug interactions); fexofenadine alone; bupropion alone; or all seven drugs simultaneously (Geneva cocktail). Pharmacokinetic profiles of the probe drugs and their metabolites were determined in DBS samples using both conventional micropipette sampling and new microfluidic device allowing for self‐sampling. The 90% confidence intervals for the geometric mean ratios of AUC metabolite/AUC probe for CYP probes administered alone or within Geneva cocktail fell within the 0.8–1.25 bioequivalence range indicating the absence of pharmacokinetic interaction. The same result was observed for the chosen phenotyping indices, that is metabolic ratios at 2 hr (CYP1A2, CYP3A) or 3 hr (CYP2B6, CYP2C9, CYP2C19, CYP2D6) post‐cocktail administration. DBS sampling could successfully be performed using a new microfluidic device. In conclusion, Geneva cocktail combined with an innovative DBS sampling device can be used routinely as a test for simultaneous CYP phenotyping.     

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.     

Cytochrome P450 mono-oxygenase gene expression and protein activity in cultures of adult cardiomyocytes of the rat

There are a substantial number of drugs acting either directly or indirectly on the heart, but surprisingly, little is known about the metabolic capacity of heart muscle cells. We therefore investigated the gene expression and protein activity of cytochrome P450 isozymes in cultures of adult cardiomyocytes of the rat. Semi-quantitative CYP gene expression pattern suggests CYP1A1 and CYP2B1/2 to be key players in cardiomyocytes and upon treatment with Aroclor 1254 approximate 4 fold inductions could be observed for both gene families, when compared with appropriate controls. The mRNA expression of most genes was sustained for prolonged periods of time, e.g. up to 120 h in culture and in the case of the CYP3A1 gene an approximate 10 fold induction was observed at the higher Aroclor 1254 dose level (10 microM) in 24 h old cultures. The constitutively expressed genes, e.g. CYP2C11 and CYP2E1 are expressed throughout the entire culture period (5 days) and did not respond to Aroclor 1254 treatment. CYP4A1 was mainly expressed in freshly isolated cardiomyocytes of control animals and its expression declined rapidly in culture. There was good agreement between gene expression and translated protein activity using 7-ethoxyresorufin and testosterone as substrates. The data reported herein should foster the routine use of freshly isolated and cultivated cardiomyocytes for drug profiling and toxicity studies.     

Characterization of in vitro oxidative and conjugative metabolic pathways for brevetoxin (PbTx-2).

Brevetoxins are potent marine toxins produced by the dinoflagellate Karenia brevis, the causative organism of Florida red tides. An in vitro metabolism of PbTx-2 was performed using purified cDNA-expressed rat liver cytochrome P-450 (CYP) enzymes and freshly isolated rat hepatocytes. The metabolic activities of six CYP enzymes, CYP1A2, CYP2A2, CYP2C11, CYP2D1, CYP2E1, and CYP3A1, were examined by incubation with PbTx-2 for up to 4 h in the presence of a NADPH-generating system. Further identification of the metabolites produced by CYP1A2 and CYP3A1 was preformed using high performance liquid chromatography-mass spectrometry (LC/MS). Both CYP1A2 and CYP3A1 metabolized PbTx-2 to PbTx-3 (MH+: m/z 897), PbTx-9 (MH+: m/z 899), and a newly recorded diol brevetoxin-2 metabolite (MH+: m/z 929). CYP3A1 also produced a considerably higher amount of BTX-B5 (MH+: m/z 911). Subsequent incubation of PbTx-2 with rat hepatocytes produced additional phase 1 metabolites of MH+: m/z 911, 913, 915, 917, and 931, indicating a CYP-catalyzed epoxidation at H-ring (C27,C28-double bond) and a subsequent A-ring hydrolysis of PbTx-2 metabolic products. A conjugation metabolism was identified by the production of a glutathione-brevetoxin conjugate (MH+: m/z 1222) and a cysteine-brevetoxin conjugate (MH+: m/z 1018). Structures of the new metabolites are postulated, and a likely CYP-catalyzed metabolism pathway of PbTx-2 metabolism are discussed.     

The olivacine derivative s 16020 (9-hydroxy-5,6-dimethyl-N-[2-(dimethylamino)ethyl)-6H-pyrido(4,3-B)-carbazole-1-carboxamide) induces CYP1A and its own metabolism in human hepatocytes in primary culture.

The olivacine derivative 9-hydroxy-5,6-dimethyl-N-[2-(dimethylamino)ethyl)-6H-pyrido(4,3-b)-carbazole-1-carboxamide (S 16020) exhibits a potent antitumor activity. However, when administered in cancer patients, its blood clearance increases after repeated administrations, whereas the volume of distribution remains constant, suggesting that the drug is able to induce its own metabolism. The aim of this work was to identify the enzymes involved in S 16020 metabolism and determine whether this molecule is an enzyme inducer in human hepatocytes in primary cultures. Among a battery of cDNA-expressed cytochromes P450 (P450s) and flavin monooxygenase (FMO), only CYP1A1, CYP1A2, and FMO3 were able to generate detectable amounts of metabolites of S 16020. In primary hepatocytes, S 16020 behaved as a CYP1A inducer, producing an increase in CYP1A2 protein, acetanilide 4-hydroxylation, ethoxyresorufin O-deethylation, and chlorzoxazone 6-hydroxylation to an extent similar to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical CYP1A inducer. The levels of other P450 proteins, including CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1, and CYP3A4, and related activities were not affected by S 16020. In primary hepatocytes, pretreatment of cells with S 16020 or TCDD produced a significant and similar increase of S 16020 metabolism, consistent with the previous indications on the role of CYP1As. We conclude that CYP1As and FMO3 are the major phase I enzymes involved in the metabolism of S 16020 and that this molecule is a potent hydrocarbon-like inducer able to stimulate its own metabolism in primary human hepatocytes and liver.