Lisa D. Marroquin

In Vitro Assessment of Mitochondrial Dysfunction and Cytotoxicity of Nefazodone, Trazodone, and Buspirone

Mitochondrial toxicity is increasingly implicated in a host of drug-induced organ toxicities, including hepatotoxicity. Nefazodone was withdrawn from the U.S. market in 2004 due to hepatotoxicity. Accordingly, we evaluated nefazodone, another triazolopyridine trazodone, plus the azaspirodecanedione buspirone, for cytotoxicity and effects on mitochondrial function. In accord with its clinical disposition, nefazodone was the most toxic compound of the three, trazodone had relatively modest effects, whereas buspirone showed the least toxicity. Nefazodone profoundly inhibited mitochondrial respiration in isolated rat liver mitochondria and in intact HepG2 cells where this was accompanied by simultaneous acceleration of glycolysis. Using immunocaptured oxidative phosphorylation (OXPHOS) complexes, we identified Complex 1, and to a lesser amount Complex IV, as the targets of nefazodone toxicity. No inhibition was found for trazodone, and buspirone showed 3.4-fold less inhibition of OXPHOS Complex 1 than nefazodone. In human hepatocytes that express cytochrome P450, isoform 3A4, after 24 h exposure, nefazodone and trazodone collapsed mitochondrial membrane potential, and imposed oxidative stress, as detected via glutathione depletion, leading to cell death. Our results suggest that the mitochondrial impairment imposed by nefazodone is profound and likely contributes to its hepatotoxicity, especially in patients cotreated with other drugs with mitochondrial liabilities.

Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro

As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.

Effect of the Multitargeted Tyrosine Kinase Inhibitors Imatinib, Dasatinib, Sunitinib, and Sorafenib on Mitochondrial Function in Isolated Rat Heart Mitochondria and H9c2 Cells

Cardiovascular disease has recently been suggested to be a significant complication of cancer treatment with several kinase inhibitors. In some cases, the mechanisms leading to cardiotoxicity are postulated to include mitochondrial dysfunction, either as a primary or secondary effect. Detecting direct effects on mitochondrial function, such as uncoupling of oxidative phosphorylation or inhibition of electron transport chain components, as well as identifying targets within the mitochondrial electron transport chain, can be accomplished in vitro. Here, we examined the effects of the tyrosine kinase inhibitor drugs imatinib, dasatinib, sunitinib, and sorafenib on ATP content in H9c2 cells grown under conditions where cells are either glycolytically or aerobically poised. Furthermore, we measured respiratory capacity of isolated rat heart mitochondria in the presence of the four kinase inhibitors and examined their effect on each of the oxidative phosphorylation complexes. Of the four kinase inhibitors examined, only sorafenib directly impaired mitochondrial function at clinically relevant concentrations, potentially contributing to the cytotoxic effect of the drug. For the other three kinase inhibitors lacking direct mitochondrial effects, altered kinase and other signaling pathways, are a more reasonable explanation for potential toxicity.

Strategies to reduce late-stage drug attrition due to mitochondrial toxicity

Mitochondrial dysfunction is increasingly implicated in the etiology of drug-induced toxicities and negative side-effect profiles. Early identification of mitochondrial liabilities for new chemical entities is therefore crucial for avoiding late-stage attrition during drug development. Limitations of traditional methods for assessing mitochondrial dysfunction have discouraged routine evaluation of mitochondrial liabilities. To circumvent this bottleneck, a high-throughput screen has been developed that measures oxygen consumption; one of the most informative parameters for the assessment of mitochondrial status. This technique has revealed that some, but not all, members of many major drug classes have mitochondrial liabilities. This dichotomy encourages optimism that efficacy can be disassociated from mitochondrial toxicity, resulting in safer drugs in the future.

Mechanistic Investigation of Imatinib-Induced Cardiac Toxicity and the Involvement of c-Abl Kinase

The Bcr-abl tyrosine kinase inhibitor imatinib mesylate is the frontline therapy for chronic myeloid leukemia. Imatinib has been reported to cause congestive heart failure and left ventricular contractile dysfunction in patients and cardiomyopathy in rodents, findings proposed to be associated with its pharmacological activity. To investigate the specific role of Abelson oncogene 1 (c-Abl) in imatinib-induced cardiac toxicity, we performed targeted gene inhibition of c-Abl by RNA interference in neonatal cardiomyocytes (NCMs). Suppression of c-Abl did not lead to cytotoxicity or induction of endoplasmic reticulum (ER) stress. To further dis associate c-Abl from imatinib-induced cardiac toxicity, we designed imatinib structural analogs that do not have appreciable c-Abl inhibition in NCMs. The c-Abl inactive analogs induced cytotoxicity and ER stress, at similar or greater potencies and magnitudes as imatinib. Furthermore, combining c-Abl gene silencing with imatinib and analogs treatment did not significantly shift the cytotoxicity dose response curves. Imatinib and analogs were shown to accumulate in lysosomes, likely due to their physicochemical properties, and disrupt autophagy. The toxicity induced by imatinib and analogs can be rescued by bafilomycin A pretreatment, demonstrating the involvement of lysosomal accumulation in cardiac toxicity. The results from our studies strongly suggest that imatinib induces cardiomyocyte dysfunction through disruption of autophagy and induction of ER stress, independent of c-Abl inhibition.

Identifying Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore in Isolated Rat Liver Mitochondria

The mitochondrial permeability transition pore (MPTP) is a protein pore that forms in the inner mitochondrial membrane and allows the membrane to be permeable to all molecules of less than 1500 Da. Ca2+, numerous reactive chemicals, and oxidative stress induce MPTP opening, whereas cyclosporin A (CsA) or bongkrekic acid block it. In addition, several drugs have been shown to induce MPTP opening, leading to the loss of mitochondrial membrane potential, swelling of the matrix because of water accumulation, rupture of the outer mitochondrial membrane, and release of intermembrane space proteins into the cytosol. This ultimately leads to the rupture of the outer mitochondrial membrane and cell demise. Here, we describe an assay using isolated rat liver mitochondria that can detect Ca2+‐dependent drug‐induced opening of the MPTP, providing protocols for screening in both cuvette and 96‐well format. Curr. Protoc. Toxicol. 60:25.4.1:‐25.4.17.

Inhibition of Hepatobiliary Transport Activity by the Antibacterial Agent Fusidic Acid: Insights into Factors Contributing to Conjugated Hyperbilirubinemia/Cholestasis

Conjugated hyperbilirubinemia accompanied by cholestasis is a frequent side effect during chronic treatment with the antimicrobial agent fusidic acid. Previous studies from our laboratory, addressing mechanisms of musculoskeletal toxicity arising from coadministration of fusidic acid with statins, demonstrated the ability of fusidic acid to potently inhibit human organic anion transporting polypeptides OATP1B1 (IC50 = 1.6 μM) and OATP1B3 (IC50 = 2.5 μM), which are responsible for the uptake-limited clearance of statins as well as bilirubin glucuronide conjugates. In the present work, inhibitory effects of fusidic acid were characterized against additional human hepatobiliary transporters [Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), and multidrug resistance-associated proteins MRP2 and MRP3] as well as uridine glucuronosyl transferase (UGT1A1), which mediate the disposition of bile acids and bilirubin (and its conjugated metabolites). Fusidic acid demonstrated concentration-dependent inhibition of human NTCP- and BSEP-mediated taurocholic acid transport with IC50 values of 44 and 3.8 μM, respectively. Inhibition of BSEP activity by fusidic acid was also consistent with the potent disruption of cellular biliary flux (AC50 = 11 μM) in the hepatocyte imaging assay technology assay, with minimal impact on other toxicity end points (e.g., cytotoxicity, mitochondrial membrane potential, reactive oxygen species generation, glutathione depletion, etc.). Fusidic acid also inhibited UGT1A1-catalyzed β-estradiol glucuronidation activity in human liver microsomes with an IC50 value of 16 μM. Fusidic acid did not demonstrate any significant inhibition of ATP-dependent LTC4 transport (IC50s > 300 μM) in human MRP2 or MRP3 vesicles. R values, which reflect maximal in vivo inhibition, were estimated from a static mathematical model by taking into consideration the IC50 values generated in the various in vitro assays and clinically efficacious unbound fusidic acid concentrations. The magnitudes of in vivo interaction (R values) resulting from the inhibition of OATP1B1, UGT1A1, NTCP, and BSEP transport were ∼1.9-2.6, 1.1-1.2, 1.0-1.1, and 1.4-1.7, respectively, which are indicative of some degree of inherent toxicity risk, particularly via inhibition of OATP and BSEP. Collectively, these observations indicate that inhibition of human BSEP by fusidic acid could affect bile acid homeostasis, resulting in cholestatic hepatotoxicity in the clinic. Lack of direct inhibitory effects on MRP2 transport by fusidic acid suggests that conjugated hyperbilirubinemia does not arise via interference in MRP2-mediated biliary disposition of bilirubin glucuronides. Instead, it is possible that elevation in the level of bilirubin conjugates in blood is mediated through inhibition of hepatic OATPs, which are responsible for their reuptake and/or downregulation of MRP2 transporter as a consequence of cholestatic injury.

Transporter-Mediated Disposition, Clinical Pharmacokinetics and Cholestatic Potential of Glyburide and Its Primary Active Metabolites

Glyburide is widely used for the treatment of type 2 diabetes. We studied the mechanisms involved in the disposition of glyburide and its pharmacologically active hydroxy metabolites M1 and M2b and evaluated their clinical pharmacokinetics and the potential role in glyburide-induced cholestasis employing physiologically based pharmacokinetic (PBPK) modeling. Transport studies of parent and metabolites in human hepatocytes and transfected cell systems imply hepatic uptake mediated by organic anion–transporting polypeptides. Metabolites are also subjected to basolateral and biliary efflux by P-glycoprotein, breast cancer resistance protein, and multidrug resistance–associated proteins, and are substrates to renal organic anion transporter 3. A PBPK model in combination with a Bayesian approach was developed considering the identified disposition mechanisms. The model reasonably described plasma concentration time profiles and urinary recoveries of glyburide and the metabolites, implying the role of multiple transport processes in their pharmacokinetics. Predicted free liver concentrations of the parent (∼30-fold) and metabolites (∼4-fold) were higher than their free plasma concentrations. Finally, all three compounds showed bile salt export pump inhibition in vitro; however, significant in vivo inhibition was not apparent for any compound on the basis of a predicted unbound liver exposure-response effect model using measured in vitro IC50 values. In conclusion, this study demonstrates the important role of multiple drug transporters in the disposition of glyburide and its active metabolites, suggesting that variability in the function of these processes may lead to pharmacokinetic variability in the parent and the metabolites, potentially translating to pharmacodynamic variability.

Assessment of Bile Salt Export Pump (BSEP) Inhibition in Membrane Vesicles Using Radioactive and LC/MS‐Based Detection Methods

The bile salt export pump (BSEP, ABCB11) belongs to the ATP‐binding‐cassette superfamily of transporters and is predominately found in the liver. BSEP is an efflux transporter that plays a critical role in the secretion of bile salts into the bile. Inhibition of BSEP function by drugs can result in the buildup of bile salts in the liver and eventually leads to cholestasis and drug‐induced liver injury (DILI). DILI is a major cause of withdrawal of drugs from the pharmaceutical market and accounts for >50% of acute liver failures. Therefore, early detection of BSEP inhibition by drugs can help to mitigate the possibility of BSEP‐associated liver injury. This unit describes two assays that investigate the relationship between drug interference with BSEP function and liver injury using membrane vesicles prepared from Hi5 insect cells transfected with human BSEP. Comprehensive protocols for assessing BSEP inhibition in a 384‐well format using radiolabeled and liquid chromatography/mass spectrometry (LC/MS)–based detection methods are described.