Mechanisms of toxicity associated with six tyrosine kinase inhibitors in human hepatocyte cell lines.

Tyrosine kinase inhibitors have revolutionized the treatment of certain cancers. They are usually well tolerated, but can cause adverse reactions including liver injury. Currently, mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors are only partially clarified. We therefore aimed at investigating the toxicity of regorafenib, sorafenib, ponatinib, crizotinib, dasatinib and pazopanib on HepG2 and partially on HepaRG cells. Regorafenib and sorafenib strongly inhibited oxidative metabolism (measured by the Seahorse-XF24 analyzer) and glycolysis, decreased the mitochondrial membrane potential and induced apoptosis and/or necrosis of HepG2 cells at concentrations similar to steady-state plasma concentrations in humans. In HepaRG cells, pretreatment with rifampicin decreased membrane toxicity (measured as adenylate kinase release) and dissipation of adenosine triphosphate stores, indicating that toxicity was associated mainly with the parent drugs. Ponatinib strongly impaired oxidative metabolism but only weakly glycolysis, and induced apoptosis of HepG2 cells at concentrations higher than steady-state plasma concentrations in humans. Crizotinib and dasatinib did not significantly affect mitochondrial functions and inhibited glycolysis only weakly, but induced apoptosis of HepG2 cells. Pazopanib was associated with a weak increase in mitochondrial reactive oxygen species accumulation and inhibition of glycolysis without being cytotoxic. In conclusion, regorafenib and sorafenib are strong mitochondrial toxicants and inhibitors of glycolysis at clinically relevant concentrations. Ponatinib affects mitochondria and glycolysis at higher concentrations than reached in plasma (but possibly in liver), whereas crizotinib, dasatinib and pazopanib showed no relevant toxicity. Mitochondrial toxicity and inhibition of glycolysis most likely explain hepatotoxicity associated with regorafenib, sorafenib and possibly pazopanib, but not for the other compounds investigated.

Mechanisms of hepatotoxicity associated with the monocyclic ß-lactam antibiotic BAL30072.

BAL30072 is a new monocyclic ß-lactam antibiotic under development which provides a therapeutic option for the treatment of severe infections caused by multi-drug-resistant Gram-negative bacteria. Despite the absence of liver toxicity in preclinical studies in rats and marmosets and in single dose clinical studies in humans, increased transaminase activities were observed in healthy subjects in multiple-dose clinical studies. We, therefore, initiated a comprehensive program to find out the mechanisms leading to hepatocellular injury using HepG2 cells (human hepatocellular carcinoma cell line), HepaRG cells (inducible hepatocytes derived from a human hepatic progenitor cell line), and human liver microtissue preparations. Our investigations demonstrated a concentration- and time-dependent reduction of the ATP content of BAL30072-treated HepG2 cells and liver microtissues. BAL30072 impaired oxygen consumption by HepG2 cells at clinically relevant concentrations, inhibited complexes II and III of the mitochondrial electron transport chain, increased the production of reactive oxygen species (ROS), and reduced the mitochondrial membrane potential. Furthermore, BAL 30072 impaired mitochondrial fatty acid metabolism, inhibited glycolysis, and was associated with hepatocyte apoptosis. Co-administration of N-acetyl-L-cysteine partially protected hepatocytes from BAL30072-mediated toxicity, underscoring the role of oxidative damage in the observed hepatocellular toxicity. In conclusion, BAL30072 is toxic for liver mitochondria and inhibits glycolysis at clinically relevant concentrations. Impaired hepatic mitochondrial function and inhibition of glycolysis can explain liver injury observed in human subjects receiving long-term treatment with this compound.

Mitochondrial oxidative stress plays a critical role in the cardiotoxicity of sunitinib: Running title: Sunitinib and oxidative stress in hearts.

Sunitinib is cardiotoxic, but the mechanisms are not entirely clear. We aimed to enlarge our knowledge about the role of mitochondria in cardiac toxicity of sunitinib in vitro and in vivo. For this reason, we studied the toxicity of sunitinib on cardiac H9c2 cells exposed for 24 h, permeabilized rat cardiac fibers exposed for 15 min and in mice treated orally with sunitinib for 2 weeks (7.5 mg/kg/day). In H9c2 cells exposed for 24 h, sunitinib was more cytotoxic under galactose (favoring mitochondrial metabolism) compared to glucose conditions (favoring glycolysis). Sunitinib dissipated the mitochondrial membrane potential starting at 10 µM under glucose and at 5 µM under galactose conditions. Sunitinib reduced activities of mitochondrial enzyme complexes of the electron transport chain (ETC), increased mitochondrial ROS accumulation and decreased the cellular GSH pool. Electron microscopy revealed swollen mitochondria with loss of cristae. Accordingly, sunitinib caused caspase 3 activation and DNA fragmentation in H9c2 cells. Co-exposure with mito-TEMPO (mitochondrial-specific ROS scavenger) for 24 h prevented ATP and GSH depletion, as well as the increases in H2O2 and caspase 3/7 activity observed with sunitinib. In mice, treatment with sunitinib for two weeks increased plasma concentrations of troponin I and creatine kinase MB, indicating cardiomyocyte damage. The activity of enzyme complexes of the ETCwas decreased, mitochondrial ROS were increased and cleavage of caspase 3 was increased, suggesting cardiomyocyte apoptosis. In conclusion, mitochondrial damage with ROS accumulation appears to be an important mechanism of cardiotoxicity associated with sunitinib, eventually leading to apoptotic cell death.

Sunitinib induces hepatocyte mitochondrial damage and apoptosis in mice.

Reports concerning hepatic mitochondrial toxicity of sunitinib are conflicting. We therefore decided to conduct a toxicological study in mice. After having determined the highest dose that did not affect nutrient ingestion and body weight, we treated mice orally with sunitinib (7.5 mg/kg/day) for 2 weeks. At the end of treatment, peak sunitinib plasma concentrations were comparable to those achieved in humans and liver concentrations were approximately 25-fold higher than in plasma. Sunitinib did not affect body weight, but increased plasma ALT activity 6-fold. The activity of enzyme complexes of the electron transport chain (ETC) was decreased numerically in freshly isolated and complex III activity significantly in previously frozen liver mitochondria. In previously frozen mitochondria, sunitinib decreased NADH oxidase activity concentration-dependently in both treatment groups. The hepatic mitochondrial reactive oxygen species (ROS) content and superoxide dismutase 2 expression were increased in sunitinib-treated mice. Protein and mRNA expression of several subunits of mitochondrial enzyme complexes were decreased in mitochondria from sunitinib-treated mice. Protein expression of PGC-1α, citrate synthase activity and mtDNA copy number were all decreased in livers of sunitinib-treated mice, indicating impaired mitochondrial proliferation. Caspase 3 activation and TUNEL-positive hepatocytes were increased in livers of sunitinib-treated mice, indicating hepatocyte apoptosis. In conclusion, sunitinib caused concentration-dependent toxicity in isolated mitochondria at concentrations reached in livers in vivo and inhibited hepatic mitochondrial proliferation. Daily mitochondrial insults and impaired mitochondrial proliferation most likely explain hepatocellular injury observed in mice treated with sunitinib.

Mechanisms of mitochondrial toxicity of the kinase inhibitors ponatinib, regorafenib and sorafenib in human hepatic HepG2 cells.

Previous studies have shown that certain kinase inhibitors are mitochondrial toxicants. In the current investigation, we determined the mechanisms of mitochondrial impairment by the kinase inhibitors ponatinib, regorafenib, and sorafenib in more detail. In HepG2 cells cultured in galactose and exposed for 24 h, all three kinase inhibitors investigated depleted the cellular ATP pools at lower concentrations than cytotoxicity occurred, compatible with mitochondrial toxicity. The kinase inhibitors impaired the activity of different complexes of the respiratory chain in HepG2 cells exposed to the toxicants for 24 h and in isolated mouse liver mitochondria exposed acutely. As a consequence, they increased mitochondrial production of ROS in HepG2 cells in a time- and concentration-dependent fashion and decreased the mitochondrial membrane potential concentration-dependently. In HepG2 cells exposed for 24 h, they induced mitochondrial fragmentation, lysosome content and mitophagy as well as mitochondrial release of cytochrome c, leading to apoptosis and/or necrosis. In conclusion, the kinase inhibitors ponatinib, regorafenib, and sorafenib impaired the function of the respiratory chain, which was associated with increased ROS production and a drop in the mitochondrial membrane potential. Despite activation of defense measures such as mitochondrial fission and mitophagy, some cells were liquidated concentration-dependently by apoptosis or necrosis. Mitochondrial dysfunction may represent a toxicological mechanism of hepatotoxicity associated with certain kinase inhibitors.

Induction of mitophagy in the HEI-OC1 auditory cell line and activation of the Atg12/LC3 pathway in the organ of Corti.

Autophagy is a highly evolutionary conserved quality control defense mechanism within cells, which has also been implicated in cell death processes. In the mammalian inner ear, autophagy has been shown to play a role during early morphogenesis as well as in adult cochlear hair cells exposed to ototoxic insults. Mitophagy, a selective autophagic cell process targeting mitochondria, hasn't been studied in the inner ear so far. On this work, we searched for molecular indicators of mitophagy within House Ear Institute-Organ of Corti-1 (HEI-OC1) cells as well as in the organ of Corti (OC). We first tested for the expression of Pink1/Park2 mRNA in 5-day-old C57BL/6 mice's cochleae using RT-PCR. We focused on the induction of mitophagy in HEI-OC1 cells as well as in the OC and investigated a possible mitophagic potential of the aminoglycoside agent gentamicin. The induction of mitophagy in HEI-OC1 cells was detected by objectivizing the translocation of fluorescence-tagged LC3 to mitochondria using confocal microscopy after a 6-h incubation with a well-described mitochondrial uncoupler and mitophagy-inducing agent: carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Incubation with gentamicin generated no mitochondrial translocation of LC3. Protein levels of COXIV, Atg5/12 and LC3 were evaluated by an immunoblot analysis after a 24-h CCCP treatment as well as gentamicin. We demonstrated mitophagy after CCCP exposure in HEI-OC1 cells by showing a downregulation of COXIV. A downregulation of COXIV could also be visualized in the OC after CCCP. A significant oxygen consumption rate (OCR) changed in cells treated with CCCP as well as significant morphological changes of mitochondria by electron microscopy (EM) strengthen this assumption. Gentamicin exposure generated no impact on OCR or mitochondrial morphological changes by EM. Finally, we demonstrated changes in the expression of Atg12 and LC3 proteins in both the OC and HEI-OC1 cells after CCCP exposure but not after gentamicin. Our data indicate that gentamicin had no impact in the activation of mitophagy-neither in the HEI-OC1 cell line nor in the OC. Therefore, we speculate that mitophagic-independent mechanisms may underly aminoglycoside ototoxicity.

Prediction of Safety Margin and Optimization of Dosing Protocol for a Novel Antibiotic using Quantitative Systems Pharmacology Modeling.

Elevations of liver enzymes have been observed in clinical trials with BAL30072, a novel antibiotic. In vitro assays have identified potential mechanisms for the observed hepatotoxicity, including electron transport chain (ETC) inhibition and reactive oxygen species (ROS) generation. DILIsym, a quantitative systems pharmacology (QSP) model of drug-induced liver injury, has been used to predict the likelihood that each mechanism explains the observed toxicity. DILIsym was also used to predict the safety margin for a novel BAL30072 dosing scheme; it was predicted to be low. DILIsym was then used to recommend potential modifications to this dosing scheme; weight-adjusted dosing and a requirement to assay plasma alanine aminotransferase (ALT) daily and stop dosing as soon as ALT increases were observed improved the predicted safety margin of BAL30072 and decreased the predicted likelihood of severe injury. This research demonstrates a potential application for QSP modeling in improving the safety profile of candidate drugs.

Hepatocellular Toxicity Associated with Tyrosine Kinase Inhibitors: Mitochondrial Damage and Inhibition of Glycolysis.

Tyrosine kinase inhibitors (TKIs) are anticancer drugs with a lesser toxicity than classical chemotherapeutic agents but still with a narrow therapeutic window. While hepatotoxicity is known for most TKIs, underlying mechanisms remain mostly unclear. We therefore aimed at investigating mechanisms of hepatotoxicity for imatinib, sunitinib, lapatinib and erlotinib in vitro. We treated HepG2 cells, HepaRG cells and mouse liver mitochondria with TKIs (concentrations 1-100 µM) for different periods of time and assessed toxicity. In HepG2 cells maintained with glucose (favoring glycolysis), all TKIs showed a time- and concentration-dependent cytotoxicity and, except erlotinib, a drop in intracellular ATP. In the presence of galactose (favoring mitochondrial metabolism), imatinib, sunitinib and erlotinib showed a similar toxicity profile as for glucose whereas lapatinib was less toxic. For imatinib, lapatinib and sunitinib, cytotoxicity increased in HepaRG cells induced with rifampicin, suggesting formation of toxic metabolites. In contrast, erlotinib was more toxic in HepaRG cells under basal than CYP-induced conditions. Imatinib, sunitinib and lapatinib reduced the mitochondrial membrane potential in HepG2 cells and in mouse liver mitochondria. In HepG2 cells, these compounds increased reactive oxygen species production, impaired glycolysis, and induced apoptosis. In addition, imatinib and sunitinib impaired oxygen consumption and activities of complex I and III (only imatinib), and reduced the cellular GSH pool. In conclusion, imatinib and sunitinib are mitochondrial toxicants after acute and long-term exposure and inhibit glycolysis. Lapatinib affected mitochondria only weakly and inhibited glycolysis, whereas the cytotoxicity of erlotinib could not be explained by a mitochondrial mechanism.

Kinetics of lipid bilayer permeation of a series of ionisable drugs and their correlation with human transporter-independent intestinal permeability.

For low molecular weight drugs, lipid bilayer permeation is considered the major route for in vivo cell barrier passage. We recently introduced a fluorescence assay with liposomes to determine permeation kinetics of ionisable compounds across the lipid bilayer by monitoring drug-induced pH changes inside the liposomes. Here, we determined the permeability coefficients (PFLipP, FLipP for "Fluorescence Liposomal Permeability") across 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers of 35 ionisable drugs at pH6.0 and compared them to available in vivo human jejunal permeability (Peff) data. PFLipP values were furthermore compared with published Caco-2 cell permeability coefficients (PCaco-2), permeability coefficients determined with the parallel artificial membrane permeability assay (PAMPA) and with log D (pH6.0). The log PFLipP, corrected for predicted para-cellular diffusion, and log PCaco-2 correlated best with log Peff, with similar adjusted R2 (0.75 and 0.74, n=12). Our results suggest that transporter-independent intestinal drug absorption is predictable from liposomal permeability.

Mechanistic insights into selective killing of OXPHOS-dependent cancer cells by arctigenin.

Arctigenin has previously been identified as a potential anti-tumor treatment for advanced pancreatic cancer. However, the mechanism of how arctigenin kills cancer cells is not fully understood. In the present work we studied the mechanism of toxicity by arctigenin in the human pancreatic cell line, Panc-1, with special emphasis on the mitochondria. A comparison of Panc-1 cells cultured in glucose versus galactose medium was applied, allowing assessments of effects in glycolytic versus oxidative phosphorylation (OXPHOS)-dependent Panc-1 cells. For control purposes, the mitochondrial toxic response to treatment with arctigenin was compared to the anti-cancer drug, sorafenib, which is a tyrosine kinase inhibitor known for mitochondrial toxic off-target effects (Will et al., 2008). In both Panc-1 OXPHOS-dependent and glycolytic cells, arctigenin dissipated the mitochondrial membrane potential, which was demonstrated to be due to inhibition of the mitochondrial complexes II and IV. However, arctigenin selectively killed only the OXPHOS-dependent Panc-1 cells. This selective killing of OXPHOS-dependent Panc-1 cells was accompanied by generation of ER stress, mitochondrial membrane permeabilization and caspase activation leading to apoptosis and aponecrosis.

A liposomal fluorescence assay to study permeation kinetics of drug-like weak bases across the lipid bilayer.

Lipid bilayer permeation is considered the major route for in vivo barrier passage of drugs. Despite this fact, no technique is currently available to measure the kinetics of permeation across a single lipid bilayer of structurally unrelated drug-like solutes. We developed a liposomal fluorescence assay capable to determine permeation kinetics of basic drug-like solutes across lipid bilayers. The assay is based on the hypothesis that permeation of a weak base along a concentration gradient results in net proton release at the cis-side and net proton capture at the trans-side of the bilayer. The resulting pH changes were monitored with pH-sensitive fluorophores: Test compounds were incubated with liposomes containing a pH-sensitive fluorophore at the bilayer surfaces or in the aqueous lumen and fluorescence changes were monitored with a stopped-flow apparatus in solution or by total internal reflection fluorescence microscopy with surface-captured liposomes on a microfluidic platform. Incubation with lipophilic basic drugs resulted in the expected fluorescence changes while incubation with compounds without basic functionality or high polarity did not affect fluorescence. Kinetics of fluorescence changes followed bi-exponential functions. Logarithmic permeation coefficients (logPermapp) determined in solution and by microfluidics technology showed a good correlation (r(2)=0.94, n=7) and logPermapp increased with increasing lipophilicity. Neither diffusion in the aqueous phase nor partitioning into the bilayer was rate-limiting. PEGylation of 2% of the liposomal lipids reduced Permapp by a factor ~300. In conclusion, the presented liposomal fluorescence assay is capable to determine permeation kinetics of weak basic drug-like solutes across lipid bilayers. The method is adaptable to microfluidics technology for high-throughput measurements and can potentially be modified to work for weak acid solutes.