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Research ArticleArticle

Application of a Micropatterned Cocultured Hepatocyte System To Predict Preclinical and Human-Specific Drug Metabolism

T. Eric Ballard, Shuai Wang, Loretta M. Cox, Mark A. Moen, Stacy Krzyzewski, Okechukwu Ukairo and R. Scott Obach
Drug Metabolism and Disposition February 2016, 44 (2) 172-179; DOI: https://doi.org/10.1124/dmd.115.066688

Abstract

Laboratory animal models are the industry standard for preclinical risk assessment of drug candidates. Thus, it is important that these species possess profiles of drug metabolites that are similar to those anticipated in human, since metabolites also could be responsible for biologic activities or unanticipated toxicity. Under most circumstances, preclinical species reflect human in vivo metabolites well; however, there have been several notable exceptions, and understanding and predicting these exceptions with an in vitro system would be very useful. Human micropatterned cocultured (MPCC) hepatocytes have been shown to recapitulate human in vivo qualitative metabolic profiles, but the same demonstration has not been performed yet for laboratory animal species. In this study, we investigated several compounds that are known to produce human-unique metabolites through CYP2C9, UGT1A4, aldehyde oxidase (AO), or N-acetyltransferase that were poorly covered or not detected at all in the selected preclinical species. To perform our investigation we used 24-well MPCC hepatocyte plates having three individual human donors and a single donor each of monkey, dog, and rat to study drug metabolism at four time points per species. Through the use of the multispecies MPCC hepatocyte system, the metabolite profiles of the selected compounds in human donors effectively captured the qualitative in vivo metabolite profile with respect to the human metabolite of interest. Human-unique metabolites that were not detected in vivo in certain preclinical species (normally dog and rat) were also not generated in the corresponding species in vitro, confirming that the MPCC hepatocytes can provide an assessment of preclinical species metabolism. From these results, we conclude that multispecies MPCC hepatocyte plates could be used as an effective in vitro tool for preclinical understanding of species metabolism relative to humans and aid in the choice of appropriate preclinical models.

Introduction

In the research and development of new drug candidates it has become standard practice to evaluate metabolism of candidates using human in vitro hepatic models (Plant, 2004; Pelkonen and Raunio, 2005; Dalvie et al., 2009; Wang et al., 2010; Ballard et al., 2014). The aims of this endeavor are: 1) to provide an understanding of the initial metabolic pathways as a first look at the possible enzymes involved in drug clearance and 2) to provide as comprehensive a picture as possible of the metabolic profile as a forecast of the metabolite profile that will be observed in humans in vivo. To the latter aim, gathering corresponding metabolite profile data in the same in vitro systems derived from common laboratory animal species can strengthen the prediction of the human in vivo metabolic profile if there is agreement between the animal in vitro systems and the metabolite profiles observed in these species in vivo.

In addition to clarifying drug clearance, cross-species comparisons of drug-metabolite profiles are also important from a drug safety standpoint. Laboratory animal species, such as rat, dog, and monkey, among others, are commonly used to test the safety of new drug candidates prior to administration in humans. Risk assessments are derived from the observations of toxicity and the toxicokinetics of the parent drug; however, it is possible that toxicity can be caused not by the parent drug, but by drug metabolite(s). Thus, it is important that laboratory animal species are exposed to the same metabolites to which humans will be exposed. This is highlighted in the Metabolites in Safety Testing guidances that have been issued by government and international drug regulatory authorities over the past decade (FDA, 2010).

The in vitro hepatic-derived systems most commonly used to generate cross-species drug-metabolite profiles include subcellular fractions from liver (microsomes, cytosol, S-9 fraction) and suspensions of primary cryopreserved hepatocytes (Kerns and Di, 2008). Although these systems are mostly successful in generating realistic drug-metabolite profiles (Dalvie et al., 2009), their utility suffers from the relatively short time frame (minutes to a few hours) of their activity (Foti and Fisher, 2004; Gómez-Lechón et al., 2008; Smith et al., 2012), so that when a new drug candidate is slowly but extensively metabolized, the in vitro metabolite profile generated may be an inadequate reflection of the in vivo profile. This problem is not overcome by standard hepatocyte culture systems because of downregulation of drug-metabolizing enzymes in these systems (Gómez-Lechón et al., 2008). Recent advances in tissue engineering have led to the development of in vitro models that more closely mimic the in vivo system and express drug metabolism activity over a period of days; these include liverchip (Dash et al., 2009) and micropatterned coculture (MPCC) systems (Khetani and Bhatia, 2008; Wang et al., 2010; Chan et al., 2013). In our laboratory we have described the use of the human hepatocyte relay method as well as the MPCC system to generate human metabolite profiles that included relevant secondary metabolites that are otherwise challenging to generate using simple in vitro systems (Wang et al., 2010; Ballard et al., 2014). In this study we have extended this effort to include an examination of the MPCC system derived from laboratory animal species with the objective of determining whether cross-species comparisons of metabolite profiles can be generated. We selected test compounds known to have cross-species differences in metabolite profile owing to known differences in enzyme activity [e.g., CYP2C family, UGT1A4, aldehyde oxidase (AO), and N-acetyltransferase]. Thus, such a system can be leveraged effectively to identify instances when one or more laboratory animal species may not yield an important human metabolite.

Materials and Methods

Materials.

Procainamide, tolbutamide, trifluoperazine, and trovafloxacin were purchased from Sigma-Aldrich (St. Louis, MO). Sunitinib was purchased from AK Scientific (Union City, CA). Zoniporide and carbazeran were obtained from Pfizer Global Material Management (Groton, CT). Micropatterned cocultured (MPCC) hepatocyte 24-well plates containing three different human donors, a monkey, a dog, and a rat donor with four wells each were provided by Hepregen Corp. (Medford, MA). The MPCCs were created at Hepregen Corporation and maintained in serum-containing medium for 5–6 days to allow for stabilization of the cocultures prior to shipment. Cryopreserved human hepatocyte donor 1 (HH1023, female) and Cynomolgus monkey hepatocytes (Lot 10106012, female) were purchased from In Vitro ADMET Laboratories (Columbia, MD). Cryopreserved human hepatocyte donors 2 and 3 (TLQ, female, and VNL, female) were purchased from BioreclamationIVT (Westbury, NY). Cryopreserved Sprague-Dawley rat hepatocytes (Rs721, pooled, male) were purchased from Thermo Fisher Scientific (Sunnyvale, CA). Fresh beagle dog hepatocytes (male) were purchased from Triangle Research Laboratories (Research Triangle Park, NC). MPCC hepatocytes are plated with fibroblasts in a 1:3 ratio with ∼25,000 hepatocytes/well in a 24-well plate. For suspension hepatocyte studies a 10-donor mixed-gender pooled cryopreserved human hepatocyte Lot RTH (product no. X008001) was purchased from Celsis IVT (Baltimore, MD), and three-donor male beagle–pooled cryopreserved dog hepatocytes (Lot AAT) were purchased from Celsis IVT (Baltimore, MD) and used in all of the following studies. All other compounds and solvents were purchased from Sigma-Aldrich unless otherwise stated.

Metabolite Profiling Using MPCC Hepatocyte Plates.

Twenty-four-well MPCC plates were shipped to Pfizer; fresh medium was applied, and cultures were kept at 37°C with 90% O2/10% CO2 and 75% relative humidity for 2 days. MPCCs were changed to serum-free medium 2 hours prior to compound treatment. The MPCC hepatocytes were incubated with test compounds (37°C with 90% O2/10% CO2 and 75% relative humidity) at a final concentration of 10 μM [chosen to provide quality UV and mass spectrometry (MS) data] in treatment media (dimethylsulfoxide, final concentration ≈ 0.03%) with a final volume of 0.4 ml (with the exception of rat hepatocytes at 0.3 ml). At 0, 4, 48, and 168 hours, the culture medium was removed from respective wells and transferred to 15-ml vials. The wells were then washed with 2 × 800 μl of acetonitrile (scraping the bottom of the wells with the pipet tips to detach cells) and pooled with the corresponding samples. The samples were mixed on a vortex mixer, centrifuged (1280 × g, 5 minutes), and the supernatants transferred to new 15-ml conical glass tubes. The supernatants were dried in a Genevac (Genevac Inc., Stone Ridge, NY) evaporative centrifuge and the resulting residues were reconstituted in 150 μl of 95% water/5% acetonitrile, centrifuged (1280 × g, 5 minutes), and analyzed by liquid chromatography mass spectrometry. All compounds were incubated in the MPCC hepatocytes with the same time point collections, regardless of known or expected clearance rates (human or preclinical), on the basis of previous studies in Hepregen and hepatocyte relay systems. This generalized collection of time points was implemented to capture metabolite profiles from all compounds regardless of clearance rates that resulted in some compounds producing metabolites of interest at the 2-day time point.

Zoniporide Inhibition in Suspended Hepatocytes.

Williams E medium (WEM) (custom formula number 91-5233EC; Thermo Fisher Scientific, Grand Island, NY) supplemented with 26 mM sodium bicarbonate was warmed to 37°C and bubbled with 95% O2/5% CO2 30 minutes prior to use. Human and dog cryopreserved hepatocyte suspensions (0.75 × 106 cells/ml) in WEM were preincubated with inhibitors (human, 50 μM hydralazine; dog, 1 mM 1-aminobenzotriazole) at 37°C for 30 minutes followed by the addition of zoniporide at a final concentration of 10 μM (dimethylsulfoxide, final concentration 0.1%) in a final volume of 2 ml. Suspensions were incubated for 4 hours (37°C with 90% O2/10% CO2 and 75% relative humidity) taking 0.5-ml time points at 0, 1, and 4 hours. Time points were mixed with 4× volumes of acetonitrile, centrifuged (1280 × g, 5 minutes), and the supernatant decanted into 15-ml conical glass tubes. The supernatants were dried in a Genevac evaporative centrifuge and the resulting residues were reconstituted in 100 μl of 95% water/5% acetonitrile, centrifuged (1280 × g, 5 minutes), and analyzed by liquid chromatography mass spectrometry.

Ultra–High Pressure Liquid Chromatography–Tandem Mass Spectrometry Analysis.

Reconstituted samples were analyzed by ultra–high pressure liquid chromatography (UHPLC)-UV-MS) operated in positive or negative ion mode using an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). For UHPLC-UV-MS analysis, the capillary temperature was set at 275°C, the source potential was 3500 V, and the source heater was set at 425°C. The mass spectrometer was operated in a data-dependent scanning mode to MS3 with dynamic exclusion enabled (repeat count: 1; repeat duration: 5.0 seconds; list size: 500; exclusion duration: 1.5 seconds). The normalized collision energy for the data-dependent scans was 35%. Other potentials were adjusted as necessary to get optimal ionization and fragmentation of the parent compound. UV absorption spectra were obtained by an inline Accela photodiode array detector. Although each compound required a slightly different gradient system, in general a Kinetex C18 100 Å column (Phenomenex, Torrance, CA) was used (2.1 × 150 mm, 1.7 μm) with a flow rate of 0.4 ml/min at 45°C. Mobile phase A was composed of 0.1% formic acid and mobile phase B was composed of acetonitrile. The gradient system used was: initially, 5% B held for 0.8 minutes followed by a linear gradient to 50% B from 0.8 to 8.25 minutes, a second linear gradient to 95% B at 8.5 to 8.75 minutes, a 0.25-minute wash at 95% B, a third linear gradient to 5% B at 9 to 9.2 minutes, and finally a 0.8-minute re-equilibration period at 5% B. For procainamide, a perfluorophenyl 100-Å column (Phenomenex) was used (2.1 × 150 mm, 1.7 μm) with a flow rate of 0.35 ml/min at 45°C. The gradient system used was: initially, 5% B held for 0.8 minutes followed by a linear gradient to 75% B from 0.8 to 8.25 minutes, a second linear gradient to 95% B at 8.5 to 8.75 minutes, a 0.25-minute wash at 95% B, a third linear gradient to 5% B at 9–9.2 minutes, and finally a 0.8-minute re-equilibration period at 5% B. Injections of 10 μl were made by a CTC PAL autosampler (CTC Analytics).

Results and Discussion

The early detection of human-specific metabolites is of great importance in the research and development of drug candidates. Just as important is the proper choice of preclinical species used to represent the expected human in vivo metabolic drug profile. Incorrect choice of preclinical species, which would provide incorrect drug depletion and metabolic profiles, could negatively affect the success of a drug candidate. A recent example of this was described by Taub et al. (2015), wherein mice predominately generated a toxic metabolite that was not formed in humans. Additionally, metabolites may have inherent biologic activity or toxicity, and tools capable of better predicting these events are sorely needed. To this end, we investigated the ability of micropatterned cocultured hepatocytes of human and preclinical species to recapitulate known in vivo profiles of selected test compounds that possess dissimilar metabolic profiles across species. This is not to say that we were only investigating human-unique metabolites. It was quite the opposite; we were investigating species-unique metabolites that may impact preclinical and clinical pharmacokinetics/pharmacodynamics, safety, and toxicology, including human-unique metabolites. Successful comparison of preclinical species with MPCC hepatocytes can be a useful and efficient tool to evaluate metabolic species differences, as well as to aid preclinical species selection, thus providing higher confidence in a successful transition to human clinical trials.

To investigate the robustness of the MPCC multispecies hepatocyte system, we chose several test compounds with primary major routes of metabolism through CYP2C9 (tolbutamide), aldehyde oxidase (carbazeran and zoniporide), UGT1A4 (trifluoperazine), and N-acetyltransferase (procainamide) (Fig. 1 and Table 1) that also had published in vivo metabolite data. These enzymes have been shown to generate species-unique metabolism, with some species being completely deficient in some activities (e.g., dog does not possess N-acetyltransferase genes). We also investigated two positive control compounds, sunitinib and trovafloxacin, that have in vivo evidence of metabolic profile similarities across species and have been previously studied in human cocultured hepatocytes (Wang et al., 2010). All compounds were incubated at 10 μM to provide in vitro metabolite profiles that are semiquantitatively comparable across species. Although all time points were collected for each compound, metabolite profiles presented in the figures are representative and were selected to illustrate appreciable metabolism. A new design for the MPCC hepatocyte plate was used that allowed for rat, dog, monkey, and human to all be on one plate with human run in triplicate from three individual donors. This new design allowed for facile media handling, drug dosing, and time point collection on one contained multispecies plate per compound.

Fig. 1.
Fig. 1.

Compounds used in the MPCC study and major metabolites of selected compounds.

View this table:
TABLE 1

Major metabolizing enzymes of test compounds and known metabolism differences across species

Sunitinib (1, Fig. 1), an oral tyrosine kinase inhibitor, is primarily metabolized by CYP3A4; it displayed conserved and consistent metabolic profiles across human, monkey, dog, and rat MPCC hepatocytes generating the N-desethyl metabolite (1a) in agreement with known in vivo data (Supplemental Fig. 1) (FDA, 2006; Speed et al., 2012). An N-oxide metabolite (1b), although not major in human in vivo, is also conserved across human, monkey, dog and rat MPCC hepatocytes, and the results were comparable to the in vivo metabolite profiles (Supplemental Fig. 1). Trovafloxacin (2), a broad-spectrum antibacterial, is primarily metabolized by UGT1A1 and also displayed a consistent metabolite profile across the MPCC hepatocyte system for human, dog, and rat in its formation of the acyl glucuronide metabolite (2a), in agreement with available in vivo data (Supplemental Fig. 2) (Dalvie et al., 1997). Monkey metabolism data were not available for trovafloxacin; however, the major metabolite was produced as would be expected on the basis of the known conservation of UGT1A1 activity across species (King et al., 1996; Berry et al., 2014). It was also noted with these initial studies that the replicate human donors all performed satisfactorily with negligible differences between donors. With satisfactory evidence that the MPCC hepatocyte system was recapitulating in vivo data with conserved enzymes across species, we set out to probe the system with our test compounds.

Carbazeran (3) is a phosphodiesterase inhibitor that is primarily metabolized by AO to 4-oxo-carbazeran (3a) in human, whereas this specific oxidation is absent in dogs (Kaye et al., 1984). Studies with freshly prepared livers from multiple preclinical species have also shown that aldehyde oxidase (AO) activities have a wide species variance, with rat and dog displaying low to negligible activity (Beedham et al., 1987). Through multiple studies, it has been confirmed that dog does not possess a functioning AO enzyme, making dog a poor model for drugs metabolized by AO (i.e., carbazeran and zoniporide) (Itoh et al., 2006; O’Connor et al., 2006; Diamond et al., 2010). After only the day 2 time point, carbazeran (3) was nearly fully metabolized by human and monkey MPCC hepatocytes, whereas dog and rat displayed slower rates (Fig. 2). The AO metabolite, 4-oxo-carbazeran (3a), was produced only in human and monkey MPCC hepatocytes and was completely absent in both dog and rat in direct alignment with in vivo data (Fig. 2) (Kaye et al., 1984). A rat-unique oxidative metabolite (C) with a putative hydroxylation on the terminal ethyl carbamate was also detected. A carbazeran glucuronide (3b) was noted to be a major pathway from the MPCC system across all species; however, AO metabolism is the major in vivo pathway in humans. This provides some cautionary context that the MPCC system [like other in vitro systems (Dalvie et al., 2009)] is a qualitative predictive tool of expected pathways, and metabolite ratios should not be taken as evidence of the major in vivo pathway. There are far more precise tools to tease out the fraction metabolized from the parent drug once expected pathways are identified (Youdim et al., 2008).

Fig. 2.
Fig. 2.

Representative UHPLC-UV chromatograms of human, monkey, dog, and rat MPCC hepatocytes incubated with carbazeran (3) after 2 days. A, background; B, unknown conjugative metabolite; C, oxidation (+O) metabolite.

Zoniporide (4) was developed to reduce perioperative myocardial ischemic injury in high-risk surgery patients by inhibition of the sodium/hydrogen exchanger. Zoniporide is another drug that is now known to be extensively metabolized by AO to form 2-oxo-zoniporide (4a) in both humans and rats but not in dogs, and its cross-species metabolism has also been reported (Dalvie et al., 2010). Owing to the low clearance rate of zoniporide (4), the 7-day MPCC hepatocyte system time point displayed formation of the expected AO product in human, monkey (unknown but not unexpected), and rat (Fig. 3). A monkey-unique metabolite (A) was also noted and appeared to be a further oxidation of 4a that introduced a hydroxyl on the phenyl ring. Dog MPCC hepatocytes also appeared to produce a small amount of the AO product (4a), yet dogs are devoid of AO activity (Terao et al., 2006; Kurosaki et al., 2013). Comparison of the dog MPCC metabolite with a synthetic standard of the AO metabolite (4a) definitively confirmed its structure as 2-oxo-zoniporide (4a). We found that this metabolite was also formed in suspended dog hepatocytes (also small), having been overlooked perhaps in previous studies. We followed up these results in suspended human and dog hepatocytes to determine the enzymatic origin of the metabolite, as its production by AO did not appear feasible (Fig. 4, A and B). The zoniporide AO metabolite (4a) was inhibited by hydralazine in human hepatocytes, confirming AO as the human enzyme responsible; however, 1-aminobenzotriazole was required to inhibit metabolite formation in dog hepatocytes, providing evidence for a complementary cytochrome P450-mediated pathway to form 2-oxo-zoniporide (4a) in dog. Qualitatively, the multispecies MPCC hepatocyte system recapitulated the in vivo major metabolite profile producing 2-oxo-zoniporide (4a) in human, monkey, and rat; however, dog hepatocytes appear to have a previously unknown compensatory pathway to formation of 2-oxo-zoniporide.

Fig. 3.
Fig. 3.

Representative UHPLC-UV chromatograms of human, monkey, dog, and rat MPCC hepatocytes incubated with zoniporide (4) after 7 days. A, oxidation (+2O) metabolite.

Fig. 4.
Fig. 4.

Extracted ion chromatograms for 2-oxo-zoniporide (4a) (m/z 337.141) in suspended human and dog hepatocytes after 4 hours with (bottom panels) and without (top panels) the addition of inhibitors: A, human hepatocytes and 50 μM hydralazine; B, dog hepatocytes and 1 mM 1-aminobenzotriazole.

Tolbutamide (5) is a low clearance potassium channel blocker that is used to treat type 2 diabetes. The major excretory metabolite in human and rat is 4-hydroxytolbutamide (5a) catalyzed mainly by CYP2C9, with some production of 4-carboxytolbutamide (5b) as well (Thomas and Ikeda, 1966). In our study with multispecies MPCC hepatocytes, human donors were able to produce 4-hydroxy tolbutamide (5a) as the major metabolite with smaller amounts of carboxytolbutamide (5b) formed. Monkey and rat were able to produce 4-hydroxytolbutamide (5a), whereas dog failed to produce it at all (Fig. 5). It was anticipated that dog would not form the 4-hydroxy metabolite (5a), owing to the polymorphic and semihomologous nature of the dog CYP2C family (Blaisdell et al., 1998; Graham et al., 2003). As an example of nonhuman specific metabolites, an urea N-dealkylation product (5c) is also known to be formed in dog and rat but not in human. Using an authentic standard for comparison, the N-dealkylation product was not detected in human but was formed in dog and rat consistent with known literature (Supplemental Fig. 3) (Gee et al., 1984; Back and Orme, 1989). The multispecies MPCC hepatocyte system accurately recapitulated the human and known preclinical species’ metabolism profiles of tolbutamide providing evidence for the usefulness of such a system in early-stage research and development efforts.

Fig. 5.
Fig. 5.

Representative UHPLC-UV chromatograms of human, monkey, dog, and rat MPCC hepatocytes incubated with tolbutamide (5) after 7 days. A, background.

Procainamide (6) is a low clearance antiarrhythmic used to treat abnormal heart rhythms. N-acetylprocainamide (6a) is the major human metabolite and it also possesses potency similar to the parent drug (Minchin et al., 1978). N-Acetyltransferase (NAT) catalyzes the acetylation of many aniline-containing drugs, including procainamide, in humans and other species; however, the NAT gene is completely absent in dogs (Trepanier et al., 1997). After a 7-day incubation in the multispecies MPCC hepatocyte system, human, monkey, and rat produced N-acetylprocainamide (6a) as the major metabolite; however, dog also generated a small amount of the acetylated product (Fig. 6). As the production of this metabolite in dog by NAT was completely impossible, we followed up these results in suspended human and dog hepatocytes (data not shown). In suspended hepatocytes, dog did not produce the metabolite, but human did, suggesting the MPCC system is producing a false-positive for NAT activity. The MPCC system uses human fibroblasts for dog matrix coculture, whereas all other species are cocultured with mouse fibroblasts. We hypothesized that the human fibroblasts were providing NAT activity to the dog MPCC system, generating a false-positive metabolite. Upon incubations of procainamide (6) in human fibroblast cells, the N-acetylprocainamide metabolite (6a) was produced in qualitatively similar amounts after a 7-day incubation period, as compared with the dog MPCC hepatocytes, confirming our hypothesis (data not shown). The other compounds in our study were not tested in fibroblasts, and we cannot exclude that fibroblasts may form some of the minor metabolites; however, fibroblasts are not known to possess significant phase 1 (i.e., cytochrome P450, AO) or phase II (i.e., NAT, UGT) enzymatic activity (Cui et al., 2000; Moriwaki et al., 2001; Du et al., 2014). Overall, the MPCC hepatocyte system recapitulated in vivo metabolism data when controlled for the stromal cell component, which should be monitored in dog specifically.

Fig. 6.
Fig. 6.

Representative UHPLC-UV chromatograms of human, monkey, dog, and rat MPCC hepatocytes incubated with procainamide (6) after 7 days. Trp, tryptophan; A, background.

Trifluoperazine (7) is a moderate clearance antipsychotic drug used in the treatment of schizophrenia. Trifluoperazine is an old drug, and in vivo human data has not been published (it may never have been collected), but its major metabolite from human in vitro systems was an N-glucuronide (7a), later shown to be solely mediated by human UGT1A4 (Uchaipichat et al., 2006). Both in vitro and in vivo studies in rats failed to produce the same N-glucuronide (7a) product, although rapid N-demethylation (7b) was observed (Gaertner et al., 1974; Breyer and Schmalzing, 1977). The multispecies MPCC hepatocyte system formed the N-glucuronide (7a) in both human and monkey, whereas dog and rat did not produce the metabolite (Fig. 7). There is no in vivo data to support or refute formation in monkey or dog, but the system’s ability to recapitulate human and rat profiles was exceptional. In vitro studies with trifluoperazine and other substrates have highlighted human and preclinical species UGT1A4 differences (Kaku et al., 2004; Xiao et al., 2014; Troberg et al., 2015). Additionally, N-desmethyl-trifluoperazine (7b) was formed across all species as an example of a conserved cross-species metabolite and provides evidence that the system is functioning appropriately.

Fig. 7.
Fig. 7.

Representative UHPLC-UV chromatograms of human, monkey, dog, and rat MPCC hepatocytes incubated with trifluoperazine (7) after 2 days. A, oxidation (+O) metabolite.

Conclusions

The MPCC hepatocyte system successfully recapitulated the human and preclinical species’ metabolites catalyzed by both phase I and phase II enzymes with compounds representing diverse clearance rates and metabolic liabilities. Whereas pooled human liver microsomes, liver S-9 fraction, and hepatocytes have been shown to be effective at predicting primary human metabolites in vivo (Dalvie et al., 2009), human MPCC hepatocytes have shown success at predicting both primary and secondary major human excretory and circulating metabolites, especially for low turnover compounds (Wang et al., 2010). With the development of a multispecies MPCC hepatocyte system encompassing human, monkey, dog, and rat cocultured hepatocytes on one plate, preclinical in vitro metabolism studies could be conducted simultaneously to better predict both human and preclinical major circulating and excretory metabolites. Evaluation of seven diverse test compounds yielded good correlation between in vivo metabolite profiles and in vitro MPCC hepatocyte profiles across human and preclinical species (Table 2). The qualitative cross-species MPCC metabolite profiles of the investigational compounds also generally agreed with reported clearance data displaying parent depletion in close agreement with in vivo half-life data across species. Dog MPCC hepatocytes did produce some discrepancies, which could be mitigated and contextualized by comparison with incubations in the control fibroblasts and with targeted inhibition studies. Although our test compounds provided good in vitro–in vivo correlation, there is still a need to better investigate the system with compounds displaying sequential and multiple routes of metabolism in preclinical species. Overall, the multispecies MPCC hepatocyte system is a predictive preclinical tool to investigate the qualitative cross-species in vitro metabolite profiles of investigational new drugs in preparation for in vivo preclinical and clinical toxicology and efficacy trials.

View this table:
TABLE 2

Multispecies MPCC formation of species-specific in vivo metabolites

Acknowledgments

The authors thank Jack McGeehan for assistance in preparing this manuscript.

Authorship Contributions

Participated in research design: Ballard, Wang, Cox, Moen, Krzyzewski, Ukairo, Obach.

Conducted experiments: Ballard, Wang, Cox, Moen, Krzyzewski.

Performed data analysis: Ballard, Wang.

Wrote or contributed to the writing of the manuscript: Ballard, Wang, Cox, Moen, Krzyzewski, Ukairo, Obach.

Footnotes

    • Received August 17, 2015.
    • Accepted November 19, 2015.

  • ↵1 Current affiliation: University of Illinois at Chicago, Chicago, Illinois.

  • ↵2 Current affiliation: Ipsen Biosciences, Inc., Cambridge, Massachusetts.

  • dx.doi.org/10.1124/dmd.115.066688.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

AO
aldehyde oxidase
MPCC
micropatterned cocultured
MS
mass spectrometry
NAT
N-acetyltransferase
UHPLC
ultra–high pressure liquid chromatography

References

  1. ↵
    1. Back DJ and
    2. Orme MLE
    (1989) Genetic factors influencing the metabolism of tolbutamide. Pharmacol Ther 44:147–155.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Ballard TE,
    2. Orozco CC, and
    3. Obach RS
    (2014) Generation of major human excretory and circulating drug metabolites using a hepatocyte relay method. Drug Metab Dispos 42:899–902.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Beedham C,
    2. Bruce SE,
    3. Critchley DJ,
    4. al-Tayib Y, and
    5. Rance DJ
    (1987) Species variation in hepatic aldehyde oxidase activity. Eur J Drug Metab Pharmacokinet 12:307–310.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Berry LM,
    2. Liu J,
    3. Colletti A,
    4. Krolikowski P,
    5. Zhao Z, and
    6. Teffera Y
    (2014) Species difference in glucuronidation formation kinetics with a selective mTOR inhibitor. Drug Metab Dispos 42:707–717.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Blaisdell J,
    2. Goldstein JA, and
    3. Bai SA
    (1998) Isolation of a new canine cytochrome P450 CDNA from the cytochrome P450 2C subfamily (CYP2C41) and evidence for polymorphic differences in its expression. Drug Metab Dispos 26:278–283.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Breyer U and
    2. Schmalzing G
    (1977) Metabolism and disposition of trifluoperazine in the rat. I. A thin-layer chromatographic method for the measurement of trifluoperazine and its metabolites in rat tissues. Drug Metab Dispos 5:97–103.
    OpenUrlAbstract
  7. ↵
    1. Chan TS,
    2. Yu H,
    3. Moore A,
    4. Khetani SR, and
    5. Tweedie D
    (2013) Meeting the challenge of predicting hepatic clearance of compounds slowly metabolized by cytochrome P450 using a novel hepatocyte model, HepatoPac. Drug Metab Dispos 41:2024–2032.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Cui L,
    2. Takahashi S,
    3. Tada M,
    4. Kato K,
    5. Yamada Y,
    6. Kohri K, and
    7. Shirai T
    (2000) Immunohistochemical detection of carcinogen-DNA adducts in normal human prostate tissues transplanted into the subcutis of athymic nude mice: results with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 3,2′-dimethyl-4-aminobiphenyl (DMAB) and relation to cytochrome P450s and N-acetyltransferase activity. Jpn J Cancer Res 91:52–58.
    OpenUrlCrossRef
  9. ↵
    1. Dalvie DK,
    2. Khosla N, and
    3. Vincent J
    (1997) Excretion and metabolism of trovafloxacin in humans. Drug Metab Dispos 25:423–427.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Dalvie D,
    2. Obach RS,
    3. Kang P,
    4. Prakash C,
    5. Loi CM,
    6. Hurst S,
    7. Nedderman A,
    8. Goulet L,
    9. Smith E,
    10. Bu HZ,
    11. et al.
    (2009) Assessment of three human in vitro systems in the generation of major human excretory and circulating metabolites. Chem Res Toxicol 22:357–368.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Dalvie D,
    2. Zhang C,
    3. Chen W,
    4. Smolarek T,
    5. Obach RS, and
    6. Loi CM
    (2010) Cross-species comparison of the metabolism and excretion of zoniporide: contribution of aldehyde oxidase to interspecies differences. Drug Metab Dispos 38:641–654.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Dash A,
    2. Inman W,
    3. Hoffmaster K,
    4. Sevidal S,
    5. Kelly J,
    6. Obach RS,
    7. Griffith LG, and
    8. Tannenbaum SR
    (2009) Liver tissue engineering in the evaluation of drug safety. Expert Opin Drug Metab Toxicol 5:1159–1174.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Diamond S,
    2. Boer J,
    3. Maduskuie TP Jr.,
    4. Falahatpisheh N,
    5. Li Y, and
    6. Yeleswaram S
    (2010) Species-specific metabolism of SGX523 by aldehyde oxidase and the toxicological implications. Drug Metab Dispos 38:1277–1285.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Du Y,
    2. Wang J,
    3. Jia J,
    4. Song N,
    5. Xiang C,
    6. Xu J,
    7. Hou Z,
    8. Su X,
    9. Liu B,
    10. Jiang T,
    11. et al.
    (2014) Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming. Cell Stem Cell 14:394–403.
    OpenUrlCrossRefPubMed
  15. ↵
    FDA (2006) Sutent (Sunitinib Malate) Drug Approval Package, http://www.accessdata.fda.gov/drugsatfda_docs/nda/2006/021938s000_021968s000_Stutent.cfm, accessed 21 November 2015.
  16. ↵
    FDA (2010) Guidance for Industry M3(R2). Nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals, http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm073246.pdf, accessed 21 November 2015.
  17. ↵
    1. Foti RS and
    2. Fisher MB
    (2004) Impact of incubation conditions on bufuralol human clearance predictions: enzyme lability and nonspecific binding. Drug Metab Dispos 32:295–304.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Gaertner HJ,
    2. Breyer U, and
    3. Liomin G
    (1974) Metabolism of trifluoperazine, fluphenazine, prochlorperazine and perphenazine in rats: in vitro and urinary metabolites. Biochem Pharmacol 23:303–311.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Gee SJ,
    2. Green CE, and
    3. Tyson CA
    (1984) Comparative metabolism of tolbutamide by isolated hepatocytes from rat, rabbit, dog, and squirrel monkey. Drug Metab Dispos 12:174–178.
    OpenUrlAbstract
  20. ↵
    1. Gómez-Lechón MJ,
    2. Castell JV, and
    3. Donato MT
    (2008) An update on metabolism studies using human hepatocytes in primary culture. Expert Opin Drug Metab Toxicol 4:837–854.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Graham MJ,
    2. Bell AR,
    3. Crewe HK,
    4. Moorcraft CL,
    5. Walker L,
    6. Whittaker EF, and
    7. Lennard MS
    (2003) mRNA and protein expression of dog liver cytochromes P450 in relation to the metabolism of human CYP2C substrates. Xenobiotica 33:225–237.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Itoh K,
    2. Yamamura M,
    3. Takasaki W,
    4. Sasaki T,
    5. Masubuchi A, and
    6. Tanaka Y
    (2006) Species differences in enantioselective 2-oxidations of RS-8359, a selective and reversible MAO-A inhibitor, and cinchona alkaloids by aldehyde oxidase. Biopharm Drug Dispos 27:133–139.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Kaku T,
    2. Ogura K,
    3. Nishiyama T,
    4. Ohnuma T,
    5. Muro K, and
    6. Hiratsuka A
    (2004) Quaternary ammonium-linked glucuronidation of tamoxifen by human liver microsomes and UDP-glucuronosyltransferase 1A4. Biochem Pharmacol 67:2093–2102.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Kaye B,
    2. Offerman JL,
    3. Reid JL,
    4. Elliott HL, and
    5. Hillis WS
    (1984) A species difference in the presystemic metabolism of carbazeran in dog and man. Xenobiotica 14:935–945.
    OpenUrlCrossRefPubMed
  25. ↵
    Kerns EH and Di L (2008) Drug-Like Properties: Concepts, Structure Design, and Methods: From ADME to Toxicity Optimization, pp 329–347, Elsevier, Inc., Waltham, MA.
  26. ↵
    1. Khetani SR and
    2. Bhatia SN
    (2008) Microscale culture of human liver cells for drug development. Nat Biotechnol 26:120–126.
    OpenUrlCrossRefPubMed
  27. ↵
    1. King CD,
    2. Green MD,
    3. Rios GR,
    4. Coffman BL,
    5. Owens IS,
    6. Bishop WP, and
    7. Tephly TR
    (1996) The glucuronidation of exogenous and endogenous compounds by stably expressed rat and human UDP-glucuronosyltransferase 1.1. Arch Biochem Biophys 332:92–100.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Kurosaki M,
    2. Bolis M,
    3. Fratelli M,
    4. Barzago MM,
    5. Pattini L,
    6. Perretta G,
    7. Terao M, and
    8. Garattini E
    (2013) Structure and evolution of vertebrate aldehyde oxidases: from gene duplication to gene suppression. Cell Mol Life Sci 70:1807–1830.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Minchin RF,
    2. Ilett KF, and
    3. Paterson JW
    (1978) Antiarrhythmic potency of procainamide and N-acetylprocainamide in rabbits. Eur J Pharmacol 47:51–56.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Moriwaki Y,
    2. Yamamoto T,
    3. Takahashi S,
    4. Tsutsumi Z, and
    5. Hada T
    (2001) Widespread cellular distribution of aldehyde oxidase in human tissues found by immunohistochemistry staining. Histol Histopathol 16:745–753.
    OpenUrlPubMed
  31. ↵
    1. O’Connor D,
    2. Jones P,
    3. Chambers MS,
    4. Maxey R,
    5. Szekeres HJ,
    6. Szeto N,
    7. Scott-Stevens P,
    8. Macleod AM,
    9. Braun M, and
    10. Cato B
    (2006) Aldehyde oxidase and its contribution to the metabolism of a structurally novel, functionally selective GABAA alpha5-subtype inverse agonist. Xenobiotica 36:315–330.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Pelkonen O and
    2. Raunio H
    (2005) In vitro screening of drug metabolism during drug development: can we trust the predictions? Expert Opin Drug Metab Toxicol 1:49–59.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Plant N
    (2004) Strategies for using in vitro screens in drug metabolism. Drug Discov Today 9:328–336.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Smith CM,
    2. Nolan CK,
    3. Edwards MA,
    4. Hatfield JB,
    5. Stewart TW,
    6. Ferguson SS,
    7. Lecluyse EL, and
    8. Sahi J
    (2012) A comprehensive evaluation of metabolic activity and intrinsic clearance in suspensions and monolayer cultures of cryopreserved primary human hepatocytes. J Pharm Sci 101:3989–4002.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Speed B,
    2. Bu HZ,
    3. Pool WF,
    4. Peng GW,
    5. Wu EY,
    6. Patyna S,
    7. Bello C, and
    8. Kang P
    (2012) Pharmacokinetics, distribution, and metabolism of [14C]sunitinib in rats, monkeys, and humans. Drug Metab Dispos 40:539–555.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Taub ME, Ludwig-Schwellinger E, Ishiguro N, Kishimoto W, Yu H, Wagner K, and Tweedie D (2015) Sex-, species-, and tissue-specific metabolism of empagliflozin in male mouse kidney forms an unstable hemiacetal metabolite (m466/2) that degrades to 4-hydroxycrotonaldehyde, a reactive and cytotoxic species. Chem Res Toxicol PMID: 25489797 [published ahead of print].
  37. ↵
    1. Terao M,
    2. Kurosaki M,
    3. Barzago MM,
    4. Varasano E,
    5. Boldetti A,
    6. Bastone A,
    7. Fratelli M, and
    8. Garattini E
    (2006) Avian and canine aldehyde oxidases. Novel insights into the biology and evolution of molybdo-flavoenzymes. J Biol Chem 281:19748–19761.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Thomas RC and
    2. Ikeda GJ
    (1966) The metabolic fate of tolbutamide in man and in the rat. J Med Chem 9:507–510.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Trepanier LA,
    2. Ray K,
    3. Winand NJ,
    4. Spielberg SP, and
    5. Cribb AE
    (1997) Cytosolic arylamine N-acetyltransferase (NAT) deficiency in the dog and other canids due to an absence of NAT genes. Biochem Pharmacol 54:73–80.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Troberg J,
    2. Järvinen E,
    3. Muniz M,
    4. Sneitz N,
    5. Mosorin J,
    6. Hagström M, and
    7. Finel M
    (2015) Dog UDP-glucuronosyltransferase enzymes of subfamily 1A: cloning, expression, and activity. Drug Metab Dispos 43:107–118.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Uchaipichat V,
    2. Mackenzie PI,
    3. Elliot DJ, and
    4. Miners JO
    (2006) Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone) “probes” for human udp-glucuronosyltransferases. Drug Metab Dispos 34:449–456.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Wang WW,
    2. Khetani SR,
    3. Krzyzewski S,
    4. Duignan DB, and
    5. Obach RS
    (2010) Assessment of a micropatterned hepatocyte coculture system to generate major human excretory and circulating drug metabolites. Drug Metab Dispos 38:1900–1905.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Xiao JF,
    2. Liu XJ,
    3. Liu GW,
    4. Yang XY,
    5. Xiao P,
    6. Hou XM,
    7. Wang HT,
    8. Tang JJ,
    9. Zhang YT,
    10. Zhen C,
    11. et al.
    (2014) The metabolism of trifluoperazine (TFP) exhibits atypical kinetic behavior in both human liver microsomes (HLMs) and monkey liver microsomes (MyLM). Eur J Drug Metab Pharmacokinet 39:335–337.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Youdim KA,
    2. Zayed A,
    3. Dickins M,
    4. Phipps A,
    5. Griffiths M,
    6. Darekar A,
    7. Hyland R,
    8. Fahmi O,
    9. Hurst S,
    10. Plowchalk DR,
    11. et al.
    (2008) Application of CYP3A4 in vitro data to predict clinical drug-drug interactions; predictions of compounds as objects of interaction. Br J Clin Pharmacol 65:680–692.
    OpenUrlCrossRefPubMed
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Multispecies MPCC Metabolite Profiling

T. Eric Ballard, Shuai Wang, Loretta M. Cox, Mark A. Moen, Stacy Krzyzewski, Okechukwu Ukairo and R. Scott Obach
Drug Metabolism and Disposition February 1, 2016, 44 (2) 172-179; DOI: https://doi.org/10.1124/dmd.115.066688
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