In Vitro Metabolite Formation in Human Hepatocytes and Cardiomyocytes and Metabolism and Tissue Distribution in Monkeys of the 2'-C-Methylguanosine Prodrug BMS-986094 : Potential Role in Clinical Cardiovascular Toxicity.

BMS-986094, a 2'-C-methylguanosine prodrug for the treatment of chronic hepatitis C virus infection, was withdrawn from phase 2 clinical trials because of unexpected cardiac and renal toxicities. To better understand these toxicities, the in vitro metabolism of BMS-986094 in human hepatocytes (HHs) and human cardiomyocytes (HCMs) and the measurement of BMS-986094 and selected metabolites in monkey plasma and tissues were assessed. BMS-986094 was extensively metabolized by HHs and HCMs, resulting in more efficient formation and accumulation of the active triphosphorylated metabolite, INX-09114, and less efficient efflux of metabolites in HCMs. The predominant metabolism pathway (hydrolysis) in HHs and HCMs was not associated with the formation of reactive metabolites or oxidative stress. In cynomolgus monkeys dosed with BMS-986094 of 15 or 30 mg/kg/d for 3 weeks, the nucleoside metabolite M2 was the major plasma analyte (66%-68% of the combined area under the curve). INX-09114 was the highest drug-related species in the heart and kidney (2,610-4,280 ng/mL [males]; ∼2-420× the concentration of other analytes). Other analytes increased dose dependently, with BMS-986094 highest in diaphragm (≤4,400 ng/mL) followed by M2 in liver and kidney (≤1,360 ng/mL), and M7 and M8 in other tissues (≤124 ng/mL). Three weeks after the last dose, INX-09114 remained high in the heart and kidney (≤1,870 ng/mL), with low M2 (≤37 ng/mL) in plasma and tissues. Persistent high concentrations of INX-09114 in the heart and kidney appeared to correlate with toxicities in these tissues in monkeys.

Thermal sensitisation by lonidamine of human melanoma cells grown at low extracellular pH.

PURPOSE: This study tested the ability of lonidamine (LND), a clinically applicable inhibitor of monocarboxylate transporters (MCT), to thermally sensitise human melanoma cells cultured at a tumour-like extracellular pH (pHe) 6.7. MATERIALS AND METHODS: Human melanoma DB-1 cells cultured at pHe 6.7 and pHe 7.3 were exposed to 150 µM LND for 3 h, beginning 1 h prior to heating at 42 °C (2 h). Intracellular pH (pHi) was determined using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and whole spectrum analysis. Levels of heat shock proteins (HSPs) were determined by immunoblot analysis. Cell survival was determined by colony formation. RESULTS: Treatment with LND at pHe 6.7 reduced pHi to 6.30 ± 0.21, reduced thermal induction of HSPs, and sensitised cells growing at pHe 6.7 to 42 °C. When LND was combined with an acute acidification from pHe 6.7 to pHe 6.5, pHi was reduced to 6.09 ± 0.26, and additional sensitisation was observed. LND had negligible effects on cells cultured at pH 7.3. CONCLUSIONS: The results show that LND can reduce pHi in human melanoma cells cultured at a tumour-like low pHe so that the 42 °C induction of HSPs are abrogated and the cells are sensitised to thermal therapy. Cells cultured at a normal tissue-like pHe 7.3 were not sensitised to 42 °C by LND. These findings support the strategy that human melanoma cells growing in an acidic environment can be sensitised to thermal therapy in vivo by exposure to an MCT inhibitor such as LND.

Intracellular acidification abrogates the heat shock response and compromises survival of human melanoma cells.

This study tests the hypothesis that lowering intracellular pH (pHi) in melanoma cells grown at low extracellular pH (pHe) selectively abrogates 42 degrees C-induced heat shock protein (HSP) expression and reduces survival. Cells were acidified by a combination of a 0.2-pH-unit decrease in pHe coupled with the lactate/H+ transport inhibitor alpha-cyano-4-hydroxy-cinnamic acid (CNCn). A mild acute extracellular acidification was used to mimic the acute extracellular acidification observed in tumors that can be induced in vivo by oral glucose administration. CNCn blocks the activity of H(+)-linked monocarboxylate transporters (MCTs), particularly MCT isoform 1 (MCT-1). This transporter removes lactic acid from cells and has a high activity in DB-1 melanoma cells grown at low pHe. The effect of extracellular acidification combined with CNCn on pHi was measured in cells grown at pHe 6.7 and pHe 7.3. Cells grown at pHe 6.7 serve as an in vitro model for cells in an acidic tumor microenvironment. When cells were grown at pHe 6.7 and incubated with CNCn at pHe 6.5, the pHi decreased from 6.9 to below 6.5, and the 42 degrees C induction of HSP70 and HSP27 was blocked. The abrogation of HSP induction correlated positively with decreased clonogenic survival. In contrast, when cells growing at pHe 7.3 were acidified by a 0.2-pH unit to pHe 7.1, the inhibitor had less effect on pHi, which remained above 7.0. Under these conditions, the 42 degrees C-induction of HSPs was not inhibited, and cytotoxicity was not enhanced. These results indicate that a significant decrease in the pHi of melanoma cells can selectively sensitize the cells to 42 degrees C hyperthermia, possibly through the inhibition of HSP expression. This strategy could result in a therapeutic gain, because normal tissues, existing at a pHe above 7.0, would not be sensitized.

Accurate prediction of drug-induced liver injury using stem cell-derived populations.

Despite major progress in the knowledge and management of human liver injury, there are millions of people suffering from chronic liver disease. Currently, the only cure for end-stage liver disease is orthotopic liver transplantation; however, this approach is severely limited by organ donation. Alternative approaches to restoring liver function have therefore been pursued, including the use of somatic and stem cell populations. Although such approaches are essential in developing scalable treatments, there is also an imperative to develop predictive human systems that more effectively study and/or prevent the onset of liver disease and decompensated organ function. We used a renewable human stem cell resource, from defined genetic backgrounds, and drove them through developmental intermediates to yield highly active, drug-inducible, and predictive human hepatocyte populations. Most importantly, stem cell-derived hepatocytes displayed equivalence to primary adult hepatocytes, following incubation with known hepatotoxins. In summary, we have developed a serum-free, scalable, and shippable cell-based model that faithfully predicts the potential for human liver injury. Such a resource has direct application in human modeling and, in the future, could play an important role in developing renewable cell-based therapies.

Developing high-fidelity hepatotoxicity models from pluripotent stem cells.

Faithfully recapitulating human physiology "in a dish" from a renewable source remains a holy grail for medicine and pharma. Many procedures have been described that, to a limited extent, exhibit human tissue-specific function in vitro. In particular, incomplete cellular differentiation and/or the loss of cell phenotype postdifferentiation play a major part in this void. We have developed an interdisciplinary approach to address this problem, using skill sets in cell biology, materials chemistry, and pharmacology. Pluripotent stem cells were differentiated to hepatocytes before being replated onto a synthetic surface. Our approach yielded metabolically active hepatocyte populations that displayed stable function for more than 2 weeks in vitro. Although metabolic activity was an important indication of cell utility, the accurate prediction of cellular toxicity in response to specific pharmacological compounds represented our goal. Therefore, detailed analysis of hepatocellular toxicity was performed in response to a custom-built and well-defined compound set and compared with primary human hepatocytes. Importantly, stem cell-derived hepatocytes displayed equivalence to primary human material. Moreover, we demonstrated that our approach was capable of modeling metabolic differences observed in the population. In conclusion, we report that pluripotent stem cell-derived hepatocytes will model toxicity predictably and in a manner comparable to current gold standard assays, representing a major advance in the field.