Hepatocyte Suspensions Research Articles

Meta-analysis of expression of hepatic organic anion-transporting polypeptide (OATP) transporters in cellular systems relative to human liver tissue.

Organic anion-transporting polypeptide (OATP)1B1, OATP1B3, and OATP2B1 transporters play an important role in hepatic drug disposition. Recently, an increasing number of studies have reported proteomic expression data for OATP transporters. However, systematic analysis and understanding of the actual differences in OATP expression between liver tissue and commonly used cellular systems is lacking. In the current study, meta-analysis was performed to assess the protein expression of OATP transporters reported in hepatocytes relative to liver tissue and to identify any potential correlations in transporter expression levels in the same individual. OATP1B1 was identified as the most abundant uptake transporter at 5.9 ± 8.3, 5.8 ± 3.3, and 4.2 ± 1.7 fmol/μg protein in liver tissue, sandwich-cultured human hepatocytes (SCHH), and cryopreserved suspended hepatocytes, respectively. The rank order in average expression in liver tissue and cellular systems was OATP1B1 > OATP1B3 ≈ OATP2B1. Abundance levels of the OATP transporters investigated were not significantly different between liver and cellular systems, with the exception of OATP2B1 expression in SCHH relative to liver tissue. Analysis of OATP1B1, OATP1B3, and OATP2B1 liver expression data in the same individuals (n = 86) identified weak (OATP1B1-OATP2B1) to moderately (OATP1B3-OATP2B1) significant correlations. A significant weak correlation was noted between OATP1B1 abundance and age of human donors, whereas expression of the OATPs investigated was independent of sex. Implications of the current analysis on the in vitro-in vivo extrapolation of transporter-mediated drug disposition using physiologically based pharmacokinetic models are discussed.

Metabolism of palmatine by human hepatocytes and recombinant cytochromes P450

In this study, we developed a new liquid chromatography–mass spectrometry (LC–MS) method for analysis of the protoberberine alkaloid palmatine and its metabolites with separation performed on a cyanopropyl-modified stationary phase. Palmatine (10μM) was metabolized using suspensions of human hepatocytes and human recombinant cytochrome P450 (CYP) enzymes. Our analyses using electrospray ionization-quadrupole time-of-flight mass spectrometry revealed that palmatine was relatively resistant to the metabolic activity of human hepatocytes and recombinant CYP enzymes. However, we found that the biotransformation of palmatine in human hepatocytes included O-demethylation or hydroxylation, and that the product of palmatine demethylation was conjugated by glucuronidation or sulfation. Moreover, we found that human recombinant CYP2D6 and, to a lesser extent, CYP1A2 can mediate O-demethylation of palmatine. These results provide fundamental insights into the biotransformation of palmatine in human in vitro models and, together with the LC–MS method, can be applied for further studies on the biotransformation of palmatine and other protoberberine alkaloids.

Regulation of Intracellular Ca2+

Publisher Summary Intracellular Ca 2+ plays an important role in controlling processes such as muscular contraction and secretion. The study of the mechanisms regulating Ca 2+ concentrations in cells has evolved in parallel with studies on the processes regulated by Ca 2+ . The discovery of the relationship among receptor-activated polyphosphoinositide breakdown, inositol 1,4,5-trisphosphate, and mobilization of intracellular Ca 2+ has given great impetus to this area of investigation and has encouraged the study of a wide variety of cell types. The regulation of intracellular Ca 2+ concentration requires interactions between a variety of transport systems in several different membranes. The chapter discusses the Null-point technique for the determination of intracellular Ca 2+ concentration. The technique involves the suspension of hepatocytes in media containing various concentrations of free Ca 2+ and a metallochromic Ca 2+ indicator, Arsenazo III to permeabilize the plasma membranes of the cells with digitonin and to measure whether Ca 2+ has been accumulated or released from intracellular stores.

Sydnone SYD-1 affects the metabolic functions of isolated rat hepatocytes

Previously, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) impairs the mitochondrial functions linked to energy provision and suggested that this effect could be associated with its antitumor activity. Herein, we evaluated the effects of SYD-1 (25 and 50μM) on rat hepatocytes to determine its cytotoxicity on non-tumor cells. SYD-1 (25 and 50μM) did not affect the viability of hepatocytes in suspension after 1–40min of incubation. However, the viability of the cultured hepatocytes was decreased by ∼66% as a consequence of treatment with SYD-1 (50μM) for 18h. Under the same conditions, SYD-1 promoted an increase in the release of LDH by ∼19%. The morphological changes in the cultured cells treated with SYD-1 (50μM) were suggestive of cell distress, which was demonstrated by the presence of rounded hepatocytes, cell fragments and monolayer impairment. Furthermore, fluorescence microscopy showed an increase in the annexin label after treatment with SYD-1 (50μM), suggesting that apoptosis had been induced in these cells. SYD-1 did not affect the states of respiration in the suspended hepatocytes, but the pyruvate levels were decreased by ∼36%, whereas the lactate levels were increased by ∼22% (for the 50μM treatment). The basal and uncoupled states of respiration of the cultured hepatocytes were inhibited by ∼79% and ∼51%, respectively, by SYD-1 (50μM). In these cells, SYD-1 (50μM) increased the pyruvate and lactate levels by ∼84% and ∼16%, respectively. These results show that SYD-1 affects important metabolic functions related to energy provision in hepatocytes and that this effect was more pronounced on cells in culture than those in suspension.

Aggregate culture of human embryonic stem cell-derived hepatocytes in suspension are an improved in vitro model for drug metabolism and toxicity testing.

Early phase drug development relies on primary human hepatocytes for studies of drug metabolism, cytotoxicity, and drug-drug interactions. However, primary human hepatocytes rapidly lose metabolic functions ex vivo and are refractory to expansion in culture and thus are limited in quantity. Hepatocytes derived from human pluripotent stem cells (either embryonic stem (ES) or induced pluripotent stem (iPS) cells), have the potential to overcome many of the limitations of primary human hepatocytes, but to date the use of human pluripotent stem cell-derived hepatocytes has been limited by poor enzyme inducibility and immature metabolic function. Here, we present a simple suspension culture of aggregates of ES cell-derived hepatocytes that compared to conventional monolayer adherent culture significantly increases induction of CYP 1A2 by omeprazole and 3A4 by rifampicin. Using liquid chromatography-tandem mass spectrometry, we further show that ES cell-derived hepatocytes in aggregate culture convert omeprazole and rifampicin to their human-specific metabolites. We also show that these cells convert acetaminophen (APAP) to its cytotoxic metabolite (N-acetyl-p-benzoquinone imine (NAPQI)), although they fail to perform APAP glucuronidation. In summary, we show that human pluripotent stem cell-derived hepatocytes in aggregate culture display improved enzymatic inducibility and metabolic function and is a promising step toward a simple, scalable system, but nonetheless will require further improvements to completely replace primary human hepatocytes in drug development.

Development of a thermal sensor to probe cell viability and concentration in cell suspensions

This paper presents a novel biothermal sensor to probe cell viability and concentration of a cell suspension. The sensing technique exploits the thermophysical properties of the suspension, so no labeling of suspended cells is required. When the sensor is periodically heated, the amplitude and phase of the thermal signal are dependent on the thermal properties of the cell suspension, particularly the thermal conductivity k. We measured k of HeLa, hepatocyte, and NIH-3T3 J2 cell suspensions with various concentrations and viabilities. The results demonstrate that the k of a cell suspension has a strong correlation with its concentration and viability. Accordingly, k can be employed as an index of cell concentration and viability. Furthermore, without data processing to obtain k, the electric signal that reflects the thermal response of the sensor can be used as a tool to probe viability of a cell suspension in real time. The proposed thermal sensing technique offers label-free, non-invasive, long-term, and real-time means to probe the viability and concentration of cells in a suspension.

Modulation of trichloroethylene in vitro metabolism by different drugs in human

Toxicological interactions with drugs have the potential to modulate the toxicity of trichloroethylene (TCE). Our objective is to identify metabolic interactions between TCE and 14 widely used drugs in human suspended hepatocytes and characterize the strongest using microsomal assays. Changes in concentrations of TCE and its metabolites were measured by headspace GC–MS. Results with hepatocytes show that amoxicillin, cimetidine, ibuprofen, mefenamic acid and ranitidine caused no significant interactions. Naproxen and salicylic acid showed to increase both TCE metabolites levels, whereas acetaminophen, carbamazepine and erythromycin rather decreased them. Finally, diclofenac, gliclazide, sulphasalazine and valproic acid had an impact on the levels of only one metabolite. Among the 14 tested drugs, 5 presented the most potent interactions and were selected for confirmation with microsomes, namely naproxen, salicylic acid, acetaminophen, carbamazepine and valproic acid. Characterization in human microsomes confirmed interaction with naproxen by competitively inhibiting trichloroethanol (TCOH) glucuronidation (Ki=2.329mM). Inhibition of TCOH formation was also confirmed for carbamazepine (partial non-competitive with Ki=70μM). Interactions with human microsomes were not observed with salicylic acid and acetaminophen, similar to prior results in rat material. For valproic acid, interactions with microsomes were observed in rat but not in human. Inhibition patterns were shown to be similar in human and rat hepatocytes, but some differences in mechanisms were noted in microsomal material between species. Next research efforts will focus on determining the adequacy between in vitro observations and the in vivo situation.

Generation of Major Human Excretory and Circulating Drug Metabolites Using a Hepatocyte Relay Method

The prediction of human drug metabolites using in vitro experiments containing human-derived reagents is an important approach in modern drug research; however, this can be challenging for drugs that are slowly metabolized. In this report, we describe the use of a recently developed human hepatocyte relay method for the purpose of predicting human drug metabolite profiles. Five compounds for which in vivo human metabolism data were available were selected for the investigation of this method, and the results were compared with data gathered in hepatocyte suspensions as well as previous data from a micropatterned hepatocyte coculture method. The hepatocyte relay method demonstrated an improved performance (generation of 75% of human in vivo metabolites) for those drugs for which previous methods showed a relatively low rate of success (50% of human in vivo metabolites). Metabolites included those arising from both oxidative and conjugative reactions and metabolites that required sequential reactions. Two 4-hour relays were shown to adequately generate metabolites, and no further benefit was derived from more relays. Overall, it can be concluded that the hepatocyte relay assay method can be successfully used in the generation of relevant human metabolites, even for challenging drugs.

Development of 3D Dynamic Flow Model of Human Liver and Its Application to Prediction of Metabolic Clearance of 7-Ethoxycoumarin

Primary and cryopreserved hepatocytes and immortalized hepatic cell lines in static two-dimensional monolayer culture format have been widely used as in vitro liver models for studies of xenobiotic metabolism, enzyme induction, hepatocyte regeneration, and hepatotoxicity. However, the tissue structure and metabolic capacity in these liver models are often ill-defined and are not well preserved compared to in vivo liver-specific architecture and functions. For this reason, we developed a three-dimensional (3D) dynamic flow model with primary human hepatocytes, which was optimized for cell seeding density, medium composition, and extracellular matrix proteins. Human hepatocytes cultured in this system were maintained for up to 7 weeks and reproducibly recapitulated in vivo liver-like structure and important liver-specific functions, such as albumin/total protein production, glucose utilization, lactate production, and cytochrome P450 (CYP) 3A4 activity across multiple tissue donors. The in vitro intrinsic clearance (CLint) of 7-ethoxycoumarin (7-EC) was determined from human hepatocytes cultured in the 3D dynamic flow model and compared to that in hepatocyte suspension. The 7-EC CLint values varied among individual batches and/or the two different in vitro liver models used in this study. The 3D flow model appeared to give more reproducible and stable estimates of clearance that is similar to previously published values. Overall, the results from these studies demonstrate that this culture system could be a valuable tool for making more accurate predictions of the metabolic clearance and long-term effects of chemicals and their metabolites in a complex 3D environment under dynamic flow.

Extracorporal methods for liver failure treatment

A mortality rate in patients with liver failure remains high. Currently the main causes for liver failure are viral hepatitis and alcoholic liver disease. As a result of liver detoxification malfunction a number of complications, including hepatic encephalopathy, hepatorenal syndrome, circulatory disorders develop. Different types of extracorporeal therapies and their combination are used to treat the liver failure for a long time. Figuratively, extracorporeal liver support systems can be divided into two groups: biological and non-biological. Biological methods are based on the use of isolated hepatocytes suspension. Non-biological methods include dialysis, hemofiltration, plasma exchange, hemosorption, Molecular Adsorbent Recirculating System (MARS) and fractionated plasma separation and adsorption (FPSA or Prometheus). Modern technologies allowed to combine liver support systems into a whole and to create an additional method of liver failure treatment — Molecular Adsorbent Recirculating System (MARS). Currently, MARS is a promising trend of liver failure treatment, as it allows support the liver function for a long time until restored or an optimal donor will be found.

Generating an In Vitro–In Vivo Correlation for Metabolism and Liver Enrichment of a Hepatitis C Virus Drug, Faldaprevir, Using a Rat Hepatocyte Model (HepatoPac)

Hepatocytes provide an integrated model to study drug metabolism and disposition. As a result of a loss of polarity or a significant decrease in the expression of enzymes and transporters, suspended and sandwich-cultured hepatocytes have limitations in determining hepatocellular drug concentrations. Underprediction of the extent of glucuronidation is also a concern for these hepatocyte models. Faldaprevir is a hepatitis C virus protease inhibitor in late-stage development that has demonstrated significant liver enrichment in in vivo rat models based on quantitative whole-body autoradiography (QWBA) and liver-to-plasma area under-the-curve ratio. In bile duct cannulated rats, the primary biliary metabolite was a glucuronide. Owing to ethical concerns, it is difficult to assess liver enrichment in humans, and a lack of in vitro and in vivo correlation of glucuronidation has been reported. The current study was conducted to verify whether a hepatocyte model, rat HepatoPac, could overcome some of these limitations and provide validity for follow-up studies with human HepatoPac. With rat HepatoPac, liver enrichment values averaged 34-fold and were consistent with rat QWBA (26.8-fold) and in vivo data (42-fold). In contrast, liver enrichment in suspended hepatocytes was only 2.8-fold. Furthermore, the extent of faldaprevir glucuronidation in HepatoPac studies was in agreement with in vivo results, with glucuronidation as the major pathway (96%). Suspended rat hepatocytes did not generate the glucuronide or two key hydroxylated metabolites that were observed in vivo. Overall, our studies suggest that HepatoPac is a promising in vitro model to predict in vivo liver enrichment and metabolism, especially for glucuronidation, and has demonstrated superiority over suspended hepatocytes.

Mass spectrometric investigation of chelerythrine and dihydrochelerythrine biotransformation patterns in human hepatocytes

The quaternary benzo[c]phenanthridine alkaloids (QBAs) are an important subgroup of plant secondary metabolites. Their main representatives, sanguinarine (SG) and chelerythrine (CHE), have pleiotropic biological effects and a wide spectrum of medicinal applications. The biotransformation of SG and CHE has only been partially studied while subsequent oxidative transformation of their dihydro derivates, the main metabolites, is practically unknown. The aim of this study was to characterize the biotransformation of CHE and dihydrochelerythrine (DHCHE) in detail, with respect to their more extensive biotransformation than SG. Phase I as well as phase II biotransformation of both compounds was examined in human hepatocyte suspensions. Liquid chromatography with electrospray-quadrupole time-of-flight mass spectrometry (LC-ESI-QqTOF MS) was used for analysis of the metabolites. Using the LC-ESI-QqTOF MS method, we analyzed and then suggested the putative structures of 11 phase I and 5 phase II metabolites of CHE, and 11 phase I and 6 phase II metabolites of DHCHE. For the most abundant metabolites of CHE, DHCHE and O-demethylated DHCHE, their cytotoxicity on primary cultures of human hepatocytes was analyzed. Both metabolites were nontoxic up to 50μM concentration and this indicates decreasing toxic effects for CHE biotransformation products, i.e. DHCHE and O-demethylated DHCHE.

Absolute measurement of species differences in sodium taurocholate cotransporting polypeptide (NTCP/Ntcp) and its modulation in cultured hepatocytes

Species differences among membrane transporters can be remarkable and difficult to properly assess by conventional methods. Herein, we employed the first use of stable isotope labeling in mammals or stable isotope-labeled peptides combined with mass spectrometry to identify species differences in sodium taurocholate cotransporting polypeptide (NTCP/Ntcp) protein expression in liver tissue and to characterize the modulation of protein expression in sandwich-cultured human (SCHH) and rat hepatocytes (SCRH). The lower limit of quantification was established to be 5 fmol on column with a standard curve that was linear up to 2000 fmol. The accuracy and precision were evaluated with three quality control samples and known amounts of synthetic proteotypic peptides that were spiked into the membrane protein extracts. The overall relative error and coefficient of variation were less than 10%. The expression of Ntcp in mouse and rat was significant higher than that in human (five-fold) and monkey (two-fold) and ranked as mouse > rat >> monkey > human. In the cultured hepatocytes, although significant downregulation of Ntcp expression in SCRH at day 5 after the culture was detected, NTCP expression in SCHH was comparable to the suspension hepatocytes. The results suggested that NTCP/Ntcp modulation in cultured hepatocytes is species specific. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3252–3263, 2013

Meeting the Challenge of Predicting Hepatic Clearance of Compounds Slowly Metabolized by Cytochrome P450 Using a Novel Hepatocyte Model, HepatoPac

Generating accurate in vitro intrinsic clearance data is an important aspect of predicting in vivo human clearance. Primary hepatocytes in suspension are routinely used to predict in vivo clearance; however, incubation times have typically been limited to 4-6 hours, which is not long enough to accurately evaluate the metabolic stability of slowly metabolized compounds. HepatoPac is a micropatterened hepatocyte-fibroblast coculture system that can be used for continuous incubations of up to 7 days. This study evaluated the ability of human HepatoPac to predict the in vivo clearance (CL) of 17 commercially available compounds with low to intermediate clearance (<12 ml/min per kg). In vitro half-life for disappearance of each compound was converted to hepatic clearance using the well stirred model, with and without correction for plasma protein binding. Hepatic CL, using three individual donors, was accurately predicted for 10 of 17 compounds (59%; predicted clearance within 2-fold of observed human in vivo clearance values). The accuracy of prediction increased to 76% (13 of 17 compounds) with an acceptance criterion defined as within 3-fold. When considering only low clearance compounds (<5 ml/min per kg), which represented 10 of the 17 compounds, the accuracy of prediction was 60% within 2-fold and 90% within 3-fold. In addition, the turnover of three slowly metabolized compounds (alprazolam, meloxicam, and tolbutamide) in HepatoPac was directly compared with turnover in suspended hepatocytes. The turnover of alprazolam and tolbutamide was approximately 2-fold greater using HepatoPac compared with suspended hepatocytes, which was roughly in line with the extrapolated values (correcting for the longer incubation time and lower cell number with HepatoPac). HepatoPac, but not suspended hepatocytes, demonstrated significant turnover of meloxicam. These results demonstrate the utility of HepatoPac for prediction of in vivo hepatic clearance, particularly with low clearance compounds.

Prediction of fraction metabolized via CYP3A in humans utilizing cryopreserved human hepatocytes from a set of 12 single donors

1. It has previously been demonstrated that metabolism of drugs via a single enzymatic pathway, particularly CYP3A4, is associated with increased risk for drug-drug interactions (DDI). Quantitative experimental systems as well as integrated prediction models to assess such risk during the preclinical phase are highly warranted.2. The present study was designed to systematically investigate the performance of human cryopreserved hepatocytes in suspension to predict fraction metabolized via CYP3A (fmCYP3A) by assessing the ketoconazole sensitive intrinsic clearance (CLint) for five prototypical CYP3A substrates with varying degree of CYP3A dependent CLint in twelve individual hepatocyte batches.3. We demonstrate that in contrast to well predicted mean hepatic metabolic clearance (CLH) and mean fmCYP3A data, the variability in CYP3A contribution for compounds having multiple metabolic pathways cannot be predicted from inhibition experiments using ketoconazole as inhibitor. Instead, data in the present paper indicate that the variability is larger after inhibition of CYP3A for compounds having multiple metabolic pathways.4. It is therefore recommended to estimate the average CLint and fmCYP3A for a given test compound in a series (n = 10) of individual human hepatocyte batches.

In Vitro Characterization of Axitinib Interactions with Human Efflux and Hepatic Uptake Transporters: Implications for Disposition and Drug Interactions

Axitinib is an inhibitor of tyrosine kinase vascular endothelin growth factor receptors 1, 2, and 3. The ATP-binding cassette (ABC) and solute carrier (SLC) transport properties of axitinib were determined in selected cellular systems. Axitinib exhibited high passive permeability in all cell lines evaluated (Papp ≥ 6 × 10(-6) cm/s). Active efflux was observed in Caco-2 cells, and further evaluation in multidrug resistance gene 1 (MDR1) or breast cancer resistance protein (BCRP) transfected Madin-Darby canine kidney cells type 2 (MDCK) cells indicated that axitinib is at most only a weak substrate for P-glycoprotein (P-gp) but not BCRP. Axitinib showed incomplete inhibition of P-gp-mediated transport of digoxin in Caco-2 cells and BCRP transport of topotecan in BCRP-transfected MDCK cells with IC50 values of 3 μM and 4.4 μM, respectively. Axitinib (10 mg) did not pose a risk for systemic drug interactions with P-gp or BCRP per regulatory guidance. A potential risk for drug interactions through inhibition of P-gp and BCRP in the gastrointestinal tract was identified because an axitinib dose of 10 mg divided by 250 mL was greater than 10-fold the IC50 for each transporter. However, a GastroPlus simulation that considered the low solubility of axitinib resulted in lower intestinal concentrations and suggested a low potential for gastrointestinal interactions with P-gp and BCRP substrates. Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 transfected human embryonic kidney 293 (HEK293) cells transported axitinib to a minor extent but uptake into suspended hepatocytes was not inhibited by rifamycin SV suggesting that high passive permeability predominates. Mouse whole-body autoradiography revealed that [(14)C]axitinib-equivalents showed rapid absorption and distribution to all tissues except the brain. This suggests that efflux transport of axitinib may occur at the mouse blood-brain barrier.

In vitro pharmacokinetic profile of a benzopyridooxathiazepine derivative using rat microsomes and hepatocytes: Identification of phases I and II metabolites

In the present study, the in vitro metabolic behavior of a benzopyridooxathiazepine (BZN), a potent tubulin polymerization inhibitor, was investigated by liquid chromatography-UV detection (LC-UV). First, simple and fast LC-UV methods have been optimized and validated to evaluate the pharmacokinetic profile of BZN using rat liver microsomes or hepatocytes primary cultures suspensions. Whatever the medium investigated, baseline resolution between the internal standard and BZN was achieved in a run time less than 15min using a Symmetry ODS column (150mm×4.6mm i.d., 5μm) and a mobile phase consisting of acetonitrile/water/formic acid 60:40:0.1 (v/v/v). Linearity was assessed in the 0.1–50μM and in the 0.05–5μM concentration ranges, respectively, in microsomal and hepatocyte matrix. According to the novel strategy based on the build of the accuracy profile, total error of the developed methods was included within the ±10% limits of acceptance. Then, from incubation of BZN with both liver microsomes and or hepatocytes, structural informations on phase I and phase II metabolites were acquired using liquid chromatography coupled to electrospray orbitrap mass spectrometer (LC–MS). Mass spectrum, double bond equivalent and elemental composition were useful data to access to the chemical structure of each metabolite. In microsomal suspension, four main metabolites were observed including monohydroxylation and dihydroxylation of the benzopyridooxathiazepine core, demethylation of the methoxyphenyl moiety, as well as their combinations. The phase II metabolites detected in hepatocytes suspension were the glucuronide adducts of both demethylated BZN and mono-oxygenated BZN. Based on the structural elucidation of the metabolites detected, we proposed an in vitro metabolic pathway of BZN, a new tubulin polymerization inhibitor.

Evaluation of Hepatic Disposition of Paroxetine Using Sandwich-Cultured Rat and Human Hepatocytes

Paroxetine, a selective serotonin reuptake inhibitor, is metabolized in the liver and excreted into bile and urine as metabolites, but species differences have been observed in hepatic disposition between rats and humans. A major metabolite in rats is M1-glucuronide, whereas M1-glucuronide and M1-sulfate are found in humans. The primary excretion route of paroxetine-derived radioactivity in rats and humans is bile and urine, respectively. The aim of this study was to examine the usefulness of sandwich-cultured hepatocytes (SCH) to evaluate in vivo species differences of the hepatic disposition of paroxetine between rats and humans. The metabolite profile of [(3)H]paroxetine in SCH was similar to that in hepatocytes in suspension, and the in vitro metabolite profiles were similar to the published in vivo metabolic pathways for both species. Furthermore, the biliary excretion index (BEI) of formed M1-glucuronide in rat SCH (25.8-50.9%) was higher than that in human SCH (15.1-16.7%). The BEI of formed M1-sulfate (16.4-29.1%) was comparable to that of M1-glucuronide in human SCH, whereas the BEIs of paroxetine were negligible in SCH of both species. Moreover, M1-glucuronide was demonstrated to be a multidrug resistance-associated protein 2 substrate in both species, as determined by its uptake into ATP-binding cassette transporter-expressing membrane vesicles. SCH should prove to be useful to evaluate the processes of hepatic uptake and metabolism of parent drugs and the simultaneous examination of the biliary excretion of both parent drug and liver-derived metabolites.

In UteroHepatocellular Transplantation in Rats

This work represents a step forward in the experimental design of an in utero hepatocellular transplantation model in rats. We focused on the enrichment optimization of isolated fetal hepatocytes suspension, arranging the surgery methodology of in utero transplantation, monitoring the biodistribution of the transplanted hepatocytes, and assessing the success of the transplants. Rat fetuses have been transplanted at the 17th embryonic day (ED17) with fetal hepatocytes isolated from rats at the end of pregnancy (ED21). We assessed possible differences between lymphocyte population, CD4 positive, CD8 positive, double-positive T-cells, and anti-inflammatory cytokines interleukins 4 and 10 (IL4 and IL10) as well. Cellular viability reached the rates of 90–95%. Transplanted groups had a limited success. Transplanted hepatocytes were not able to pass through the hematoplacental barrier. The hepatocytes injected were primarily located in the liver. There was an upward trend in the whole amount of T CD4 and T CD8 cells. There was an increased IL4 in the transplanted groups observed in the pregnant rats. The possibility to induce tolerance in fetuses with a hepatocyte transplant in utero could be a key point to avoid the immunosuppression treatments which must be undergone by transplanted patients.

The effects of autologous hepatocyte transplantation on acute hepatic failure caused by 90% hepatectomy in rats.

This study aims to build rat acute hepatic failure models by 90% hepatectomy, perform autologous hepatocyte transplantation with the cells separated from the resected liver tissues, and observe the effectiveness of the transplantation on acute hepatic failure. Ninety percent hepatectomy was performed on twenty male Sprague-Dawley rats. No other treatment was given in the control group, and autologous hepatocyte transplantation was given in the experimental group. The outcomes of two groups were compared. The survival rate of the rats of the experimental group was evidently higher than that of the control group (60% vs. 20%). The TB, ALT and AST levels of the experimental group were obviously smaller than the control group on the first postoperative day. Two days later the TB, AST and ALT levels of the control group were larger than the experimental group. Three days after the operation, the quantities of TB and ALT of the experimental group were evidently smaller than those of the control group. The quantity of AST of the experimental group was smaller than the control group. Autologous hepatocyte transplantation with multi-point injection of 0.6mL hepatocyte suspension into the spleen effectively eased the symptoms of AHF caused by 90% hepatectomy.