In vitro/in silico prediction of drug induced steatosis in relation to oral doses and blood concentrations by the Nile Red assay.

The accumulation of lipid droplets in hepatocytes is a key feature of drug-induced liver injury (DILI) and can be induced by a subset of hepatotoxic compounds. In the present study, we optimized and evaluated an in vitro technique based on the fluorescent dye Nile Red, further named Nile Red assay to quantify lipid droplets induced by the exposure to chemicals. The Nile Red assay and a cytotoxicity test (CTB assay) were then performed on cells exposed concentration-dependently to 60 different compounds. Of these, 31 were known to induce hepatotoxicity in humans, and 13 were reported to also cause steatosis. In order to compare in vivo relevant blood concentrations, pharmacokinetic models were established for all compounds to simulate the maximal blood concentrations (Cmax) at therapeutic doses. The results showed that several hepatotoxic compounds induced an increase in lipid droplets at sub-cytotoxic concentrations. To compare how well (1) the cytotoxicity test alone, (2) the Nile Red assay alone, and (3) the combination of the cytotoxicity test and the Nile Red assay (based on the lower EC10 of both assays) allow the differentiation between hepatotoxic and non-hepatotoxic compounds, a previously established performance metric, the Toxicity Separation Index (TSI) was calculated. In addition, the Toxicity Estimation Index (TEI) was calculated to determine how well blood concentrations that cause an increased DILI risk can be estimated for hepatotoxic compounds. Our findings indicate that the combination of both assays improved the TSI and TEI compared to each assay alone. In conclusion, the study demonstrates that inclusion of the Nile Red assay into in vitro test batteries may improve the prediction of DILI compounds.

HepaChip-MP - a twenty-four chamber microplate for a continuously perfused liver coculture model.

HepaChip microplate (HepaChip-MP) is a microfluidic platform comprised of 24 independent culture chambers with continuous, unidirectional perfusion. In the HepaChip-MP, an automated dielectrophoresis process selectively assembles viable cells into elongated micro tissues. Freshly isolated primary human hepatocytes (PHH) and primary human liver endothelial cells (HuLEC) were successfully assembled as cocultures aiming to mimic the liver sinusoid. Minimal quantities of primary human cells are required to establish micro tissues in the HepaChip-MP. Metabolic function including induction of CYP enzymes in PHH was successfully measured demonstrating a high degree of metabolic activity of cells in HepaChip-MP cultures and sufficient sensitivity of LC-MS analysis even for the relatively small number of cells per chamber. Further, parallelization realized in HepaChip-MP enabled the acquisition of dose-response toxicity data of diclofenac with a single device. Several unique technical features should enable a widespread application of this in vitro model. We have demonstrated fully automated preparation of cell cultures in HepaChip-MP using a pipetting robot. The tubeless unidirectional perfusion system based on gravity-driven flow can be operated within a standard incubator system. Overall, the system readily integrates in workflows common in cell culture labs. Further research will be directed towards optimization of media composition to further extend culture lifetime and study oxygen gradients and their effect on zonation within the sinusoid-like microorgans. In summary, we have established a novel parallelized and scalable microfluidic in vitro liver model showing hepatocyte function and anticipate future in-depth studies of liver biology and applications in pre-clinical drug development.

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

Pharmacokinetics and Pulmonary Distribution of Clarithromycin and Rifampicin after Concomitant and Consecutive Administration in Foals.

Drug interactions often result from multiple pharmacokinetic changes, such as after rifampicin (RIF) and clarithromycin (CLA) in the treatment of abscessing lung diseases. Comedication of RIF may interact with CLA disposition by either induction of presystemic elimination processes and/or inhibition of uptake mechanisms because it regulates gene transcription and modulates function of various CYP enzymes, multidrug efflux and uptake transporters for which CLA is a substrate. To distinguish the transcriptional changes from the modulating interaction components upon CLA absorption and pulmonary distribution, we initiated a repeated-dose study in 12 healthy foals with CLA (7.5 mg/kg, p.o., b.i.d.) in comedication with RIF (10 mg/kg, p.o., b.i.d.) given either concomitantly with CLA or consecutively 4 h after CLA. Affinity of CLA to human P-gp, MRP2, and MRP3 and to OCT1, OCT3, and PEPT1 was measured using Sf9-derived inside-out membrane vesicles and transfected HEK293 cells, respectively. ABCB1 (P-gp) induction by RIF and affinity of CLA to equine P-gp were studied using primary equine hepatocytes. Absolute bioavailability of CLA was reduced from ∼40% to below 5% after comedication of RIF in both schedules of administration, and Tmax occurred ∼2-3 h earlier. The loss of bioavailability was not associated with increased 14-hydroxyclarithromycin (14-OH-CLA) exposure. After consecutive dosing, absolute bioavailability and pulmonary penetration of CLA increased ∼2-fold compared to concomitant use. In vitro, CLA showed affinity to human and equine P-gp. Expression of ABCB1 mRNA was upregulated by RIF in 7 of 8 duodenal biopsy specimens and in primary equine hepatocytes. In conclusion, the major undesired influence of RIF on oral absorption and pulmonary distribution of CLA is associated with induction of intestinal P-gp. Consecutive administration to avoid competition with its intestinal uptake transport results in significantly, although not clinically relevant, improved systemic exposure.

Long term cultures of primary human hepatocytes as an alternative to drug testing in animals.

Due to species differences, primary human hepatocytes are still the in vitro system of choice to analyse liver specific processes and functions. Human hepatocytes were cultured for several weeks in a serum-free two-dimensional culture system, which was used to study the effects of acetaminophen (APAP) on hepatocellular functions and vitality. Non-invasive determinations of albumin, urea and lactate dehydrogenase concentrations in cell culture supernatants allowed continuous monitoring for at least two weeks. APAP was applied every 4 days for 24 h. Each application reduced urea production by 25% and albumin synthesis by approximately 70% without any effects on cellular viability. After removal of the substance, hepatocellular functions returned to control levels within one (urea) to three (albumin) days. The repetitive analyses of APAP-mediated effects on cellular metabolism led to identical results for up to five cycles. The drug also caused reversible and repetitive ultrastructural modifications, in particular an almost complete replacement of rough endoplasmic reticulum by smooth endoplasmic reticulum and a massive degradation of glycogen stores. The data demonstrate the suitability of the culture system to serve as a model for repetitive testing of drug-mediated changes on hepatocellular functions, thereby reducing animal studies during drug development.

Anticancer drug cis-4-hydroxy-L-proline: Correlation of preclinical toxicology with clinical parameters of liver function.

cis-4-Hydroxy-L-proline (CHP) is being clinically evaluated as an anticancer drug. Since this compound targets the production of L-proline-rich proteins and critical L-proline residues, its impact on long-term cultures of human hepatocytes and toxicity in rats was studied to investigate possible effects on hepatic function, previously reported in rat hepatocytes. In the HEPAC2 human hepatocyte culture system, concentrations of CHP below 3.2 mg/ml had no significant effects on the release of lactate dehydrogenase (LDH), albumin, and urea. In rats, continuous administration of three different doses of CHP were tested for 28 days and resulted in signs of liver damage, as indicated by elevations of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) at a dose of 903 mg/kg, corresponding to a plasma concentration of approximately 200 µg/ml. Data from a clinical study of CHP in bladder and prostate cancer patients showed no adverse effects of administration of 8 g CHP/day, 4 days/week for 3 weeks in liver parameters ALAT, ASAT, γ-glutamyltransferase (γ-GT) and alkaline phosphatase (AP). In conclusion, the HEPAC2 human hepatocyte culture system correlates well with clinical results of a Phase II study of CHP, whereas a previous rat hepatocyte culture system predicted the compound would have toxic effects. The HEPAC2 system therefore constitutes a valuable tool for the preclinical screening of the hepatotoxicity of chemotherapeutic and other drugs, thereby reducing the need for experimental animals.

In vitro system for the prediction of hepatotoxic effects in primary hepatocytes.

Prediction of liver toxicity and compound responses continues to be a major challenge for the pharmaceutical industry. In vitro studies on liver cells have been developed to reduce or replace animal experiments. However, most of the tests in use are based on cell lines which do not necessarily represent normal cell physiology. We compared the response of primary human hepatocytes from two donors with primary rat hepatocytes and the cell line HepG2 to the test compound acetaminophen (AAP) by measuring oxygen consumption, extracellular acidification and cell adhesion as dynamic parameters of cell metabolism. Primary human hepatocytes were cultured on collagen pre-coated sensor chips or in conventional two-dimensional cultures in chemically defined Human Hepatocyte Maintenance Medium. This medium allows cultivation of functionally differentiated hepatocytes for several weeks. Sensor chip based results were compared with conventional assays for hepatocytes like albumin release and urea release. The hepatocytes were exposed to AAP (50-2815 mg/l) for 24 h. Cell respiration was inhibited by AAP concentrations of 500 mg/l and more in all three cell types, whereas only the cellular acidification rates and cell adhesion of the rat hepatocytes and the HepG2 cells were affected by AAP. In conventional cultures of human hepatocytes, AAP had no effect on cellular viability. Whereas high doses of AAP (2815 mg/l) diminished albumin secretion by 70-80%.

Use of a standardised and validated long-term human hepatocyte culture system for repetitive analyses of drugs: repeated administrations of acetaminophen reduces albumin and urea secretion.

Human hepatocytes are the in vitro system of choice to study drug-induced processes in man. Here, we present HEPAC(2): a standardised and validated culture system in which human hepatocytes are maintained in HHMM (Human Hepatocyte Maintenance Medium) with HGF (hepatocyte growth factor) and EGF (epidermal growth factor). Cellular viability and hepatocellular functions were monitored daily. Albumin and urea production remained on a relatively constant level for up to 2-3 weeks. Based on this, a standard protocol was established that allows repeated exposure of hepatocytes to study drug metabolism. We used acetaminophen (AAP) to assay the feasibility of this system. Hepatocytes were exposed to AAP (100-2815 mg/l) for 24 h. Subsequently, the culture medium was replaced by medium without AAP and the same exposure scenario was repeated at intervals of 4 days. High doses of AAP (2815 mg/l) diminished urea production by 15-30% and albumin secretion by 70-80%. These effects were reversible. After removal of AAP, secretion of urea and albumin returned to control levels. AAP hepatotoxicity is caused by its biotransformation to the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) mediated by CYP2E1 and CYP1A2. The AAP activating enzymes were active for at least 21 days and their activity was maintained during at least four repeated cycles of exposure to AAP. In conclusion, these data demonstrate the suitability of our long-term culture system to serve as a tool for repetitive screening of drug-mediated changes of hepatocellular functions. This culture technique may help to overcome the sparse availability of human hepatocytes for testing drugmediated responses in man.

Caspase inhibition in apoptotic T cells triggers necrotic cell death depending on the cell type and the proapoptotic stimulus.

CD95 (Fas/Apo-1) triggers apoptotic cell death via a caspase-dependent pathway. Inhibition of caspase activation blocks proapoptotic signaling and thus, prevents execution of apoptosis. Besides induction of apoptotic cell death, CD95 has been reported to trigger necrotic cell death in susceptible cells. In this study, we investigated the interplay between apoptotic and necrotic cell death signaling in T cells. Using the agonistic CD95 antibody, 7C11, we found that caspase inhibition mediated by the pancaspase inhibitor, zVAD-fmk, prevented CD95-triggered cell death in Jurkat T cells but not in A3.01 T cells, although typical hallmarks of apoptosis, such as DNA fragmentation or caspase activation were blocked. Moreover, the caspase-independent cell death in A3.01 cells exhibited typical signs of necrosis as detected by a rapid loss of cell membrane integrity and could be prevented by treatment with the radical scavenger butylated hydroxyanisole (BHA). Similar to CD95-induced cell death, apoptosis triggered by the DNA topoisomerase inhibitors, camptothecin or etoposide was shifted to necrosis when capsase activation was inhibited. In contrast to this, ZVAD was fully protective when apoptosis was triggered by the serpase inhibitor, Nalpha-tosyl-phenyl-chloromethyl ketone (TPCK). TPCK was not protective when administered to anti-CD95/ZVAD-treated A3.01 cells, indicating that TPCK does not possess anti-necrotic activity but fails to activate the necrotic death pathway. Our findings show (a) that caspase inhibition does not always protect apoptotic T cells from dying but merely activates a caspase-independent mode of cell death that results in necrosis and (b) that the caspase-inhibitor-induced shift from apoptotic to necrotic cell death is dependent on the cell type and the proapoptotic stimulus.

Dual activity of phosphorothioate CpG oligodeoxynucleotides on HIV: reactivation of latent provirus and inhibition of productive infection in human T cells.

CpG oligodeoxynucleotides (CpG ODNs) bind to toll-like receptor-9 (TLR-9) and activate immune cells with antigen-presenting activity, including B cells and dendritic cells. Here we show that treatment of the latently human immunodeficiency virus (HIV)-infected T cell line ACH-2 with the CpG ODNs 2006 or 2040 triggers activation of viral gene expression, demonstrating that CpG-signaling activity can also be found in T cells. The CpG ODNs g12AAC and g12GTC had no effect on virus reactivation. In contrast to the stimulating effects on viral gene expression in latently infected cells, CpG ODNs potently suppressed HIV replication in productively infected MT4 T cells or PBLs. Inhibition of virus replication was not related to the CpG motif but similarly occurred with non-CpG phosphorothioate (PTO)-ODNs. Thus, virus inhibition was likely caused by the PTO backbone of the CpG ODNs, probably by interfering with events prior to integration of the viral cDNA into the host genome. The ability of CpG PTO-ODNs to trigger reactivation of latent HIV in combination with their antiviral activity on productive infection makes this substance class an interesting candidate for further test to asses their potential as supplements in HIV therapy.

CpG oligodeoxynucleotides activate HIV replication in latently infected human T cells.

CpG oligodeoxynucleotides (CpG ODNs) stimulate immune cells via the Toll-like receptor 9 (TLR9). In this study, we have investigated the effects of CpG ODNs on latent human immunodeficiency virus (HIV) infection in human T cells. Treatment of the latently infected T cell line ACH-2 with CpG ODNs 2006 or 2040 stimulated HIV replication, whereas no effects were evident when ODNs without the CpG motif were used. CpG-induced virus reactivation was blocked by chloroquine, indicating the involvement of TLR9. In contrast to the responsiveness of ACH-2 cells, CpG ODNs failed to activate HIV provirus in the latently infected Jurkat clone J1.1. We also studied the effects of CpG ODNs on productive HIV infection and found enhancement of viral replication in A3.01 T cells, whereas again no stimulating effects were observed in Jurkat T cells. CpG ODN treatment activated NF-kappaB in ACH-2 cells, which was similarly triggered in uninfected A3.01 T cells following exposure to CpG ODNs, indicating that TLR9-induced signal transduction was not dependent on proviral infection. Our study demonstrates that CpG ODNs directly trigger the activation of NF-kappaB and reactivation of latent HIV in human T cells. Our results point to a novel role for CpG ODNs as stimulators of HIV replication and open new avenues to eradicate the latent viral reservoirs in HIV-infected patients treated with antiretroviral therapy.