Cryopreservation Of Hepatocytes Research Articles

Use of Mechanistic Modeling to Assess Interindividual Variability and Interspecies Differences in Active Uptake in Human and Rat Hepatocytes

Interindividual variability in activity of uptake transporters is evident in vivo, yet limited data exist in vitro, confounding in vitro-in vivo extrapolation. The uptake kinetics of seven organic anion-transporting polypeptide substrates was investigated over a concentration range in plated cryopreserved human hepatocytes. Active uptake clearance (CL(active, u)), bidirectional passive diffusion (P(diff)), intracellular binding, and metabolism were estimated for bosentan, pitavastatin, pravastatin, repaglinide, rosuvastatin, telmisartan, and valsartan in HU4122 donor using a mechanistic two-compartment model in Matlab. Full uptake kinetics of rosuvastatin and repaglinide were also characterized in two additional donors, whereas for the remaining drugs CL(active, u) was estimated at a single concentration. The unbound affinity constant (K(m, u)) and P(diff) values were consistent across donors, whereas V(max) was on average up to 2.8-fold greater in donor HU4122. Consistency in K(m, u) values allowed extrapolation of single concentration uptake activity data and assessment of interindividual variability in CL(active) across donors. The maximal contribution of active transport to total uptake differed among donors, for example, 85 to 96% and 68 to 87% for rosuvastatin and repaglinide, respectively; however, in all cases the active process was the major contributor. In vitro-in vivo extrapolation indicated a general underprediction of hepatic intrinsic clearance, an average empirical scaling factor of 17.1 was estimated on the basis of seven drugs investigated in three hepatocyte donors, and donor-specific differences in empirical factors are discussed. Uptake K(m, u) and CL(active, u) were on average 4.3- and 7.1-fold lower in human hepatocytes compared with our previously published rat data. A strategy for the use of rat uptake data to facilitate the experimental design in human hepatocytes is discussed.

A Novel Relay Method for Determining Low-Clearance Values

A novel relay method has been developed using cryopreserved human hepatocytes to measure intrinsic clearance of low-clearance compounds. The relay method involved transferring the supernatant from hepatocyte incubations to freshly thawed hepatocytes at the end of the 4-h incubation to prolong the exposure time to active enzymes in hepatocytes. An accumulative incubation time of 20 h or longer in hepatoctyes can be achieved using the method. The relay method was validated using seven commercial drugs (diazepam, disopyramide, theophylline, timolol, tolbutamide, S-warfarin, and zolmitriptan) that were metabolized by various cytochrome P450s with low human in vivo intrinsic clearance at approximately 2 to 15 ml · min⁻¹ · kg⁻¹. The results showed that the relay method produced excellent predictions of human in vivo clearance. The difference between in vitro and in vivo intrinsic clearance was within 2-fold for most compounds, which is similar to the standard prediction accuracy for moderate to high clearance compounds using hepatocytes. The relay method is a straightforward, relatively low cost, and easy-to-use new tool to address the challenges of low clearance in drug discovery and development.

Effect of Spheroid Aggregation on Susceptibility of Primary Pig Hepatocytes to Cryopreservation

Bioartificial liver support systems, which use freshly isolated primary hepatocytes (PH), present severe logistical difficulties. Stored frozen PH that are thawed as required could answer this problem. The aim of this study was to develop a cryopreservation protocol for large-scale preparation of porcine PH. We cryopreserved single and spheroid hepatocytes. Harvested hepatocytes were cryopreserved in various concentrations of dimethyl sulfoxide (DMSO) using hormonally defined medium (HDM) and various presentation solutions, such as University of Wisconsin (UW) and fetal bovine serum. After thawing the hepatocytes, we measured the viability, plating efficiency, ammonia removal, urea synthesis, and albumin secretion. UW solution was most effective for cryopreservation, as evidenced by the viability and liver-specific functions of the thawed hepatocytes. The optimal DMSO concentration for porcine hepatocyte cryopreservation was 15%. After cryopreservation, spheroid hepatocytes maintained greater viability and functional activity compared with single hepatocytes. Moreover, spheroid hepatocytes showed native cell structures and maintained high levels of liver-specific functions. In conclusion, cryopreserved spheroid hepatocytes were superior to cryopreserved single hepatocytes in terms of viability and liver-specific functions.

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.

Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution

To optimize a xeno-free cryopreservation protocol for primary human hepatocytes. The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes. Despite several hepatocyte cryopreservation protocols being available, improvements are urgently needed. We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups. Using the polystyrene box freezing, we compared two xeno-free freezing solutions for freezing of primary human hepatocytes: a new medium (STEM-CELLBANKER, CB), which contains dimethylsulphoxide (DMSO) and anhydrous dextrose, both permeating and non-permeating cryoprotectants, and the frequently used DMSO - University of Wisconsin (DMSO-UW) medium. The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation. The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms (CYPs): CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A7. The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol (P < 0.01). There was no significant difference in viability estimation between both the trypan blue (TB) and the Live-Dead Assay methods. There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols (r(2) = 0.69) using the TB method. However, due to high within-group variability in the activities of the major CYPs, any statistical between-group differences were precluded. Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability. Thus, it may be a better alternative to the standard DMSO-UW protocol. Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples. The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.

Abstract A136: Nonclinical disposition, metabolism, and in vitro drug-drug interaction assessment of DM1, a component of trastuzumab emtansine (T-DM1).

Abstract Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in clinical development for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. T-DM1 is composed of trastuzumab, a stable linker, and the microtubule polymerization inhibitor DM1, a derivative of maytansine. After binding to HER2 on tumor cells, T-DM1 undergoes receptor-mediated internalization and is expected to go through proteolytic degradation and catabolism, resulting in the directed delivery of DM1 to the tumor cells overexpressing HER2. Although catabolism and elimination of T-DM1 have been reported previously, little is known about the metabolism and elimination of the DM1 component. This abstract summarizes aspects of the metabolism and distribution of DM1 to further the understanding of its disposition and potential for T-DM1 drug-drug interactions. In vivo disposition and metabolism in rats: [3H]-DM1 at 91 μCi/kg (200 μg/kg) was administered to rats via a bolus intravenous injection. Blood, various tissues, urine, bile and feces were collected over the following 3 to 5 days and analyzed for radioactivity. DM1 distributed rapidly and extensively to the lungs, liver, kidneys, spleen, heart, gastrointestinal tract, and adrenal glands. The major route of excretion of DM1 was through the bile/feces, with a minimal amount excreted in the urine. A good mass balance (∼100% recovery of the injected dose) was achieved over 5 days. DM1 was extensively metabolized to multiple metabolites through oxidation, hydrolysis, methylation, and glutathione conjugations. In vitro metabolism and potential for drug-drug interactions: To identify the major human cytochrome P450 (CYP) isoforms responsible for the metabolism of DM1 and determine the metabolites formed, DM1 was incubated in human liver microsomes (HLMs) in the presence of selective chemical inhibitors and with human recombinant CYP. The results of these studies demonstrated that DM1 was metabolized mainly by CYP3A4 and, to a lesser extent, by CYP3A5. The oxidative and hydrolysis metabolites of DM1 were detected in the in vitro incubation. The induction and inhibitory properties of DM1 on CYP were evaluated using cryopreserved human hepatocytes and HLMs, respectively. DM1 was found to be neither an inducer nor an inhibitor of major CYP isoforms up to the highest concentrations tested at 1 μM (738 ng/mL) and 0.7 μM (500 ng/mL), respectively. In addition, a monolayer assay using MDCKII-MDR1 cells assessed the potential of DM1 as a substrate and inhibitor of the P-glycoprotein (P-gp) transporter. DM1 was found to be a substrate, but not an inhibitor, of P-gp when tested at 0.5 M (369 ng/mL). Conclusion: Collectively, the in vitro data suggest that the DM1 component of T-DM1 has little potential for drug-drug interactions as a perpetrator, especially considering the low systemic exposure of DM1 (averaging 5 ng/mL or 0.007 μM) achieved in clinical studies and its lack of inhibition of CYP catalytic activities and P-gp activity at clinically relevant concentrations. This is consistent with the lack of evidence of drug-drug interaction observed in phase Ib/II clinical studies of T-DM1 combined with pertuzumab and taxanes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A136.

Influence of cell culture configuration on the post-cryopreservation viability of primary rat hepatocytes

Cryopreservation has been identified as a necessary barrier to overcome in the production of tissue engineered products for clinical application. Liver engineering and bioartificial liver assisting devices are on the forefront of tissue engineering research due to its high demand and clinical potential. In this study we propose that the cryopreservation of primary mammalian hepatocytes yields better results when these cells are in a tissue-like culture configuration since cell attachment is essential for cell survival in this cell type. We used two different tissue-engineered culture configurations: monolayers and spheroid culture; and two different concepts of cryopreservation, namely vitrification and freezing. Cell suspensions were also cryopreserved using both approaches and results were compared to the engineered cultures. Both engineered configurations and suspension were cryopreserved using both conventional freezing (cooling at 1 °C/minute using 10% DMSO in foetal calf serum) and vitrification (using 40% ethylene glycol 0.6 m sucrose supplemented with 9% Ficoll). These two approaches differ on the degree of mechanical stress they inflict on the material to be cryopreserved. The maintenance of cell-to-cell and the integrity of the actin cytoskeleton were assessed using scanning electron microscopy and immunohistochemistry respectively. Results showed that while there was no significant difference between the degree of integrity shown between vitrified and control engineered cultures, the same did not happen to the frozen engineered constructs. The disruption of the cytoskeletal structure correlated with increased levels of apoptotic markers. With cryopreserved suspensions there was evidence of disruption of the cytoskeletal structure. This study concluded that cell-to-cell contact is beneficial in the maintenance of viability post-cryopreservation and that the vitrification approach was far superior to those of conventional freezing when applied to 2D and 3D hepatocyte based engineered cultures.

Assessment of Compound Hepatotoxicity Using Human Plateable Cryopreserved Hepatocytes in a 1536-Well-Plate Format

Hepatotoxicity is a major concern for both drug development and toxicological evaluation of environmental chemicals. The assessment of compound-induced hepatotoxicity has traditionally relied on in vivo testing; however, it is being replaced by human in vitro models due to an emphasis on the reduction of animal testing and species-specific differences. Since most cell lines and hybridomas lack the full complement of enzymes at physiological levels found in the liver, primary hepatocytes are the gold standard to study liver toxicities in vitro due to the retention of most of their in vivo activities. Here, we optimized a cell viability assay using plateable cryopreserved human hepatocytes in a 1536-well-plate format. The assay was validated by deriving inhibitory concentration at 50% values for 12 known compounds, including tamoxifen, staurosporine, and phenylmercuric acetate, with regard to hepatotoxicity and general cytotoxicity using multiple hepatocyte donors. The assay performed well, and the cytotoxicity of these compounds was confirmed in comparison to HepG2 cells. This is the first study to report the reliability of using plateable cryopreserved human hepatocytes for cytotoxicity studies in a 1536-well-plate format. These results suggest that plateable cryopreserved human hepatocytes can be scaled up for screening a large compound library and may be amenable to other hepatocytic assays such as metabolic or drug safety studies.

Characterization of Aldehyde Oxidase Enzyme Activity in Cryopreserved Human Hepatocytes

Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O⁶-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min⁻¹ · kg⁻¹, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H₂¹⁸O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (∼5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O⁶-benzylguanine was within ∼80% of the observed total clearance in humans after intravenous administration (15 ml · min⁻¹ · kg⁻¹), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.

P63 Ribavirin uptake by primary human hepatocytes corresponds to equilibrative nucleoside transporter 1 expression

IntroductionCombined therapy with pegylated interferon (INF) and ribavirin (RV) is the current standard of care for HCV infection, with a 40% to 80% success rate depending on viral genotype. The...

Influence of Platelet Lysate on the Recovery and Metabolic Performance of Cryopreserved Human Hepatocytes Upon Thawing

Storage of human hepatocytes is essential for their use in research and liver cell transplantation. However, cryopreservation and thawing (C/T) procedures have detrimental effects on the viability and functionality compared with fresh cells. The aim of this study was to upgrade the standard C/T methodology to obtain better quality hepatocytes for cell transplantation to improve the overall clinical outcome. Human hepatocytes isolated from donor livers were cryopreserved in University of Wisconsin solution with 10% dimethyl sulfoxide (standard medium), which was supplemented with 10% or 20% of platelet lysate. Thawing media supplemented with up to 30 mM glucose was also investigated. The effects on cell viability, adhesion proteins (e-cadherin, β-catenin, and β1-integrin) expression, attachment efficiency, apoptotic indicators, Akt signaling, ATP levels, and cytochrome P450 activities have been evaluated. The results indicate that the hepatocytes cryopreserved in a medium supplemented with platelet lysate show better recovery than those preserved in the standard medium: higher expression of adhesion molecules, higher attachment efficiency and cell survival; decreased number of apoptotic nuclei and caspase-3 activation; maintenance of ATP levels; and drug biotransformation capability close to those in fresh hepatocytes. Supplementation of thawing media with glucose led to a significant decrease in caspase-3 activation and to increased adhesion molecules preservation and Akt signal transduction after C/T. Minor nonsignificant changes in cell viability and attachment efficiency were observed. These promising results could lead to a new cryopreservation procedure to improve human hepatocyte cryopreservation outcome.

The Behaviors of Long‐Term Cryopreserved Human Hepatocytes on Different Biomaterials

Isolated human hepatocytes have been extensively investigated due to clinical demand and scientific interest. With a limited supply of available liver tissue and the rapid loss of liver-specific functions in vitro, cryopreservation of human hepatocytes serves as an alternative way to maintain availability of hepatocytes. The purpose of this study was to evaluate the biological behaviors of long-term (more than 4 years) cryopreserved human hepatocytes on biomaterials, including polyvinyl alcohol (PVA), poly(ethylene-co-vinyl alcohol), polyvinylidene fluoride, commercial tissue culture polystyrene (TCPS), and collagen-coated TCPS. Cell attachment was observed by scanning electron microscopy and quantified by lactate dehydrogenase assay. Cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction activity. Cell functions were determined by albumin secretion and urea synthesis. Results indicated that human hepatocytes could be cryopreserved for more than 4 years without losing liver-specific functions. Also, PVA was proposed to serve as an appropriate and promising substrate for culturing long-term cryopreserved hepatocytes, maintaining high cell attachment and high level of liver-specific functions.

Effect of cryopreservation on the activity of OATP1B1/3 and OCT1 in isolated human hepatocytes

Drug metabolism in liver is the major pathway for xenobiotic elimination from the body. Access to intracellular metabolising enzymes is possible through passive diffusion of lipophilic drugs through cell membrane or active uptake of more polar drugs by specific uptake transporters. Organic Anion Transporting Polypeptides (OATP/SLCO) and Organic Cation Transporters (OCT/SLC22A) are among the most important transporters involved in xenobiotic transport into hepatocytes. Isolated hepatocytes are the model of choice for drug metabolism and drug transport investigations. These primary cells are used either as fresh directly after isolation from liver biopsies, or after subsequent cryopreservation in liquid nitrogen. While cryopreserved hepatocytes are a more convenient and flexible tool for in vitro investigations, information on the functionality of transporter activity after cryopreservation is still sparse. The present study investigated the effect of cryopreservation of human hepatocytes on the uptake of [ 3H]-estradiol-17β-glucuronide (E 217βG, substrate of OATP1B1/3/SLCO1B1/3) and [ 3H]-1-methyl-4-phenylpyridinium (MPP+, substrate of OCT1/SLC22A1) into hepatocytes from 6 and 5 human donors, respectively. The results showed that cryopreserved human hepatocytes display carrier-mediated uptake of E 217βG and MPP+. While the affinity of E 217βG for OATP1B1/3/SLCO1B1/3 was not affected by cryopreservation (Km unchanged, the Wilcoxon signed pair t test gave p = 1), V max and CL uptake values decreased in average by 47% ( p = 0.06). The passive diffusion of E 217βG decreased significantly after cryopreservation ( p = 0.03). Cryopreservation did not affect Km, V max or the passive diffusion of MPP+ in human hepatocytes. In conclusion, the present study showed that cryopreserved human hepatocytes are useful tool to investigate hepatic uptake mediated by OATP1B1/3/SLCO1B1/3 or OCT1/SLC22A1, two of the most important hepatic uptake transporters.

Isolation, culture and cryopreservation of adult human hepatocytes

Isolation, culture and cryopreservation of adult human hepatocytes

Prediction of clinical CYP3A4 induction using cryopreserved human hepatocytes

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.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.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).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.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.

Step Double Barrier Options

We report here the application of a novel hepatocyte system, the cofactor-supplemented permeabilized cryopreserved human hepatocytes (MetMax human hepatocytes (MMHHs)) in a higher throughput 384-well plate assay for the evaluation of P450 inhibition. The assay was developed to develop physiologically relevant P450 inhibition information, taking advantage of the complete organelle composition and their associated drug metabolizing enzymes of the MMHH, but with the ease of use of human liver microsomes including storage at -80 deg C instead of liquid nitrogen, and thaw and use without centrifugation and microscopic evaluation as required for intact hepatocytes. Nine key cytochrome P450 (CYP) isoforms for drug metabolism: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, were evaluated using multiple isoform-selective inhibitors. Results with MMHH were found to be comparable to that obtained with intact cryopreserved human hepatocytes (CHHs). Isoform-selective drug metabolizing enzyme pathways evaluated were phenacetin O-deethylation (CYP1A2), coumarin hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), amodiaquine N-deethylation (CYP2C8), diclofenac hydroxylation (CYP2C9), mephenytoin hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone hydroxylation (CYP2E1), midazolam and testosterone hydroxylation (CYP3A4). The Km values obtained with MMHHs were comparable with those reported in the literature for CHHs. Using substrate concentrations at or near Km values, the IC50 values for the standard inhibitors against the P450 activities were found to be comparable between MMHHs and CHHs, with 73% and 84% of values falling within two-fold and three-fold, respectively, from the line of unity. The results indicate that MMHHs can be an efficient experimental system for the evaluation of P450 inhibition in hepatocytes. <h3>SIGNIFICANCE STATEMENT</h3> We report here the development of a higher throughput hepatocyte-based P450 inhibition assay using a novel human hepatocyte system, the cofactor-supplemented permeabilized human hepatocytes (MetMax Human Hepatocytes; MMHH) (Li et al., 2018). MMHHs have the desirable properties of both intact hepatocytes and HLMs - the completeness of the DME’s in hepatocytes and the robustness and ease of handling of HLMs including storage at -80 deg. C freezer instead of liquid nitrogen, and use directly after thawing without a need for centrifugation and microscopic examination as required for CHHs. Our results show that MMHH can be used in a 384-well plate format for the evaluation of P450 inhibition, yielding results similar to that obtained with intact human hepatocytes. The MMHH 384-well plate P450 inhibition assay may represent an effective approach for the early screening of DDI potential of NCEs.

Cytochrome P450 3A4 mRNA Is a More Reliable Marker than CYP3A4 Activity for Detecting Pregnane X Receptor-Activated Induction of Drug-Metabolizing Enzymes

Induction of cytochrome P450 (P450) activity in the clinic can result in therapeutic failure such as tissue rejection in transplant patients or unwanted pregnancy, among others. CYP3A4 is by far the most abundant isoform and is responsible for the majority of P450-related metabolism of all marketed drugs. However, it is of importance to understand the significance of induction mediated through other P450 enzymes. The objective of this investigation was to evaluate several known inducers in vitro using cryopreserved human hepatocytes, with the aim of assessing the relevant induction of CYP3A4, CYP2B6, CYP2C9, CYP2C19, and CYP3A5, based on mRNA expression. CYP3A4 induction was also assessed based on enzymatic activity in three different lots to investigate whether mRNA expression data have any advantages over enzymatic activity. In general, the mRNA fold-induction data results were more sensitive compared with activity data, and more informative in cases in which the drug is also a P450 inhibitor. The induction of transcription of other drug-metabolizing enzymes including CYP2B6 and CYP2C enzymes occurred every time that CYP3A4 mRNA levels increased, but to a lesser extent, indicating that measurement of CYP3A4 mRNA is a sensitive marker for the induction of these other enzymes. This was the case even for enzymes and inducers that are known to also act via the constitutive androstane receptor pathway. Finally, the utility of in vitro induction measurements in the identification of clinically meaningful inducers was tested by using two simple binary classification approaches: 1) fold-induction versus vehicle control and 2) induction response relative to rifampin. The best classification was observed when the cutoff criteria based on fold induction relative to the vehicle control, using mRNA data are used.

An Improvement in the Attaching Capability of Cryopreserved Human Hepatocytes by a Proteinaceous High Molecule, Sericin, in the Serum-Free Solution

The methodology of cryopreservation of human hepatocytes remains unsatisfactory. Even when the viability of thawed cells is tolerable, the cells often lose the attaching capability to a culture dish, resulting in the cells' inability to survive. Previously, we described the effectiveness of maltose on the attachment of hepatocytes. This article demonstrates that a silk-derived high molecular protein, sericin, improves the cell-attaching capability in the serum-free freezing medium. When human hepatocytes [initial viability: 60.9 ± 3.1% (mean ± SD, n = 3)] were frozen with serum-free Dulbecco's modified Eagle medium (DMEM) containing 10% dimethyl sulfoxide (DMSO), the viability was 29.4 ± 3.2% and the cell-attaching capability 20.4 ± 4.1%. On the other hand, DMEM containing 10% DMSO and 1% sericin increased the values to 45.0 ± 0.8% and 26.2 ± 3.2%. Moreover, the addition of 0.1 mol/L maltose to the sericin-containing medium improved to 42.2 ± 3.2% and 51.1 ± 1.0%, as we demonstrated in a previous report. The present results indicated that sericin combined with maltose is a novel additive in the serum-free freezing medium for human hepatocytes.

Screening of OATP1B1/3 and OCT1 inhibitors in cryopreserved hepatocytes in suspension

Drug–drug interactions involving hepatic drug transporters may have clinical consequences and jeopardize development of promising drug candidates. Organic anion transporting polypeptides (OATP/Oatp) and the organic cation transporters (OCT/Oct) are among the most important transporters involved in xenobiotic uptake in the liver. In the present study, 179 molecules have been tested as inhibitors of the uptake of estradiol-17βD-glucuronide (E 217βG), substrate of OATP1B1/3 (rOatp), or 1-methyl-4-phenylpyridinium (MPP+), substrate of OCT1 (rOct1), into suspended cryopreserved hepatocytes from humans and rats. Uptake was assessed in 96-well plates by measuring intracellular accumulation of radioactive substrate in hepatocytes in presence or absence of inhibitor. In rat hepatocytes 140 compounds were identified as inhibitors (inhibition at 20 μM ≥ 30%) of E 217βG uptake and 77 compounds inhibitors of MPP+ uptake. The most potent inhibitors of rOatp and rOct1 were dantrolene sodium ( K i = 2 ± 9 μM) and bepridil ( K i = 14 ± 2 μM), respectively. In human hepatocytes, the most potent inhibitors of E 217βG and MPP+ uptake were capsazepine ( K i = 14 ± 5 μM) and cyproheptadine ( K i = 19 ± 3 μM), respectively. Structure–activity relationship (SAR) analysis of all tested compounds suggested that lipophilicity, polarity, p K a and the number of hydrogen bond donors and acceptors play a role in their interaction with the transporters investigated. The method used here is a simple tool to screen large number of compounds as inhibitors of the uptake of substrates into suspended hepatocytes.

Effects of Selective Serotonin Reuptake Inhibitors on Timolol Metabolism in Human Liver Microsomes and Cryo‐Preserved Hepatocytes

Timolol has been widely used in the treatment of glaucoma. Topically applied, timolol may cause adverse cardiovascular effects due to systemic absorption through the nasolacrimal duct. Timolol is mainly metabolized by cytochrome P450 2D6 (CYP2D6) in the liver. The aim of the present study was to characterize further the metabolism of timolol in vitro. Especially the effect of several drugs such as selective serotonin reuptake inhibitors on the metabolism of timolol was evaluated. In human liver microsomes, four timolol metabolites were identified, in cryo-preserved hepatocytes nine. In both in vitro experiments, the hydroxy metabolite M1 was the main metabolite. The in vivo half-life predicted for timolol was 3.7 hr. in cryo-preserved hepatocytes, corresponding to the half-life of timolol in humans in vivo. Fluoxetine, paroxetine, sertraline, citalopram and fluvoxamine inhibited the formation of M1 in microsomes with IC(50) values of 1.4, 2.0, 3.5, 21 and 20 microM, respectively. In human cryo-preserved hepatocytes, the IC(50) values for fluoxetine, paroxetine and fluvoxamine were 0.7, 0.5 and 5.9 microM, respectively. In conclusion, compounds known to be potent CYP2D6 inhibitors inhibited timolol metabolism in in vitro experiments. The present results strongly suggest that fluoxetine and paroxetine may significantly affect the metabolism of timolol also in vivo and may thus potentiate the adverse cardiovascular effects of topically administered timolol.