AZD1979 is a melanin-concentrating hormone receptor 1 (MCHr1) antagonist, which was developed for obesity treatment, but clinical development was halted due to unanticipated liver toxicity. Mitochondrial DNA (mtDNA) variation influences hepatic energy metabolism and may underlie individual susceptibility to drug-induced mitotoxicity. Transmitochondrial cybrids offer a model to assess the impact of mtDNA variation on drug response against a constant nuclear background. This study assessed the mitotoxic potential of AZD1979 in HepG2 wild-type (WT) cells and HepG2 transmitochondrial cybrids representing distinct mtDNA haplogroups from the H and J haplogroup lineages. A 2-hour acute metabolic switch assay was performed in HepG2 WT cells and transmitochondrial cybrids to identify mitochondrial toxicity, with confirmatory Seahorse extracellular flux analysis. AZD1979 was identified as a potent mitotoxicant in HepG2 WT cells, inducing mitochondrial dysfunction prior to cell death. AZD1979 caused reductions in basal respiration, ATP-linked respiration, and spare respiratory capacity, and inhibited complex I-driven respiration. Cybrids displayed haplogroup-specific differences in susceptibility. Haplogroup J2b1g showed the highest sensitivity, while J1c1e, despite lacking spare respiratory capacity, was less susceptible. These findings highlight the role of mtDNA in shaping individual responses to mitochondrial toxicants and that transmitochondrial cybrid models could support early assessment of mitochondrial liability.

Tolvaptan is a vasopressin V2 receptor antagonist which has proven to be an effective and mostly well-tolerated agent for the treatment of autosomal-dominant polycystic kidney disease. However, its administration is associated with rare but serious idiosyncratic liver injury, which has warranted a black box warning on the drug labels and frequent monitoring of liver blood tests in the clinic. This review outlines mechanistic investigations that have been conducted to date and constructs a working narrative as an explanation for the idiosyncratic drug-induced liver injury (IDILI) events that have occurred thus far. Potential risk factors which may contribute to individual susceptibility to DILI reactions are addressed, and key areas for future investigative/clinical development are highlighted.

BACKGROUND:Vancomycin, a glycopeptide antibiotic used for Gram-positive bacterial infections, has been linked with drug reaction with eosinophilia and systemic symptoms (DRESS) in HLA-A*32:01-expressing individuals. This is associated with activation of T lymphocytes, for which glycolysis has been isolated as a fuel pathway following antigenic stimulation. However, the metabolic processes that underpin drug-reactive T-cell activation are currently undefined and may shed light on the energetic conditions needed for the elicitation of drug hypersensitivity or tolerogenic pathways. Here, we sought to characterise the immunological and metabolic pathways involved in drug-specific T-cell activation within the context of DRESS pathogenesis using vancomycin as model compound and drug-reactive T-cell clones (TCCs) generated from healthy donors and vancomycin-hypersensitive patients.METHODS:CD4+ and CD8+ vancomycin-responsive TCCs were generated by serial dilution. The Seahorse XFe96 Analyzer was used to measure the extracellular acidification rate (ECAR) as an indicator of glycolytic function. Additionally, T-cell proliferation and cytokine release (IFN-γ) assay were utilised to correlate the bioenergetic characteristics of T-cell activation with in vitro assays.RESULTS:Model T-cell stimulants induced non-specific T-cell activation, characterised by immediate augmentation of ECAR and rate of ATP production (JATPglyc). There was a dose-dependent and drug-specific glycolytic shift when vancomycin-reactive TCCs were exposed to the drug. Vancomycin-reactive TCCs did not exhibit T-cell cross-reactivity with structurally similar compounds within proliferative and cytokine readouts. However, cross-reactivity was observed when analysing energetic responses; TCCs with prior specificity for vancomycin were also found to exhibit glycolytic switching after exposure to teicoplanin. Glycolytic activation of TCC was HLA restricted, as exposure to HLA blockade attenuated the glycolytic induction.CONCLUSION:These studies describe the glycolytic shift of CD4+ and CD8+ T cells following vancomycin exposure. Since similar glycolytic switching is observed with teicoplanin, which did not activate T cells, it is possible the master switch for T-cell activation is located upstream of metabolic signalling.

Background beta-d-N4-Hydroxycytidine (NHC) is the active metabolite of molnupiravir, a broad-spectrum antiviral approved by the MHRA for COVID-19 treatment. NHC induces lethal mutagenesis of the SARS-CoV-2 virus, undergoing incorporation into the viral genome and arresting viral replication. It has previously been reported that several nucleoside analogues elicit off-target inhibition of mitochondrial DNA (mtDNA) or RNA replication. Although NHC does not exert these effects in HepG2 cells, HepaRG are proven to be advantageous over HepG2 for modelling nucleoside analogue-induced mitochondrial dysfunction. Therefore, the objective of this work was to assess the mitotoxic potential of NHC in HepaRG cells, a model more closely resembling physiological human liver. Methods Differentiated HepaRG cells were exposed to 1-60 mu M NHC for 3-14 days to investigate effects of sub-, supra-, and clinically-relevant exposures (in the UK, molnupiravir for COVID-19 is indicated for 5 days and reported Cmax is 16 mu M). Following drug incubation, cell viability, mtDNA copy number, mitochondrial protein expression, and mitochondrial respiration were assessed. Results NHC induced minor decreases in cell viability at clinically relevant exposures, but did not decrease mitochondrial protein expression. The effects on mtDNA were variable, but typically copy number was increased. At supra-clinical concentrations (60 mu M), NHC reduced mitochondrial respiration, but did not appear to induce direct electron transport chain dysfunction. Conclusions Overall, NHC does not cause direct mitochondrial toxicity in HepaRG cells at clinically relevant concentrations, but may induce minor cellular perturbations. As HepaRG cells have increased physiological relevance, these findings provide additional assurance of the mitochondrial safety profile of NHC.

The United States Food and Drug Administration Adverse Event Reporting System (FAERS) logged 27,140 rhabdomyolysis cases from 2004 to 31 March 2020. We used FAERS to identify 14 drugs frequently reported in 6583 rhabdomyolysis cases and to investigate whether mitochondrial toxicity is a common pathway of drug-induced rhabdomyolysis by these drugs. Preliminary screening for mitochondrial toxicity was performed using the acute metabolic switch assay, which is adapted here for use in murine L6 cells. Fenofibrate, risperidone, pregabalin, propofol, and simvastatin lactone drugs were identified as mitotoxic and underwent further investigation, using real-time respirometry (Seahorse Technology) to provide more detail on the mechanism of mitochondrial-induced toxicity. To confirm the human relevance of the findings, fenofibrate and risperidone were evaluated in primary human skeletal muscle-derived cells (HSKMDC), using the acute metabolic switch assay and real-time respirometry, which confirmed this designation, although the toxic effects on the mitochondria were more pronounced in HSKMDC. Overall, these studies demonstrate that the L6 model of acute modification may find utility as an initial, cost-effective screen for identifying potential myotoxicants with relevance to humans and, importantly, that drug-induced mitochondrial dysfunction may be a common mechanism shared by some drugs that induce myotoxicity.

Introduction Vancomycin is a glycopeptide antibiotic that targets gram-positive bacteria and is the recommended treatment against MRSA infections. However, it is known to cause kidney injury in children. Vancomycin accumulates in proximal tubule cells but relatively little is known regarding the mechanism of this. We conducted a multiparameter assessment of vancomycin toxicity in a novel human renal cell line, Conditionally immortalised proximal tubule epithelial cells (ciPTECs), to determine if these cells recapitulate vancomycin-induced kidney injury (VIKI) in vitro. Methods Three ciPTEC lines were used (parent ciPTEC, and ciPTECs with upregulated expression of organic anion transporters 1 (ciPTEC OAT1) or 3 (ciPTEC OAT3), to assess the respective involvement of these transporters in vancomycin accumulation. ciPTECs were cultured at 33°c and seeded in 96-well plates at 1x104 cells/well. Vancomycin stocks were prepared in distilled water and administered between 1mM and 10mM for 24hr incubations before cell viability (ATP) and cell cytotoxicity (LDH) assays were performed. Results ATP data showed a dose-dependent increase in vancomycin toxicity with 17% cell viability observed at10mM. (EC50=5.6mM) Membrane permeabilization was only observed at 10mM (60% LDH retention), with no change observed at lower concentrations administered. There was no difference in vancomycin toxicity observed between each cell type. Conclusions Our results suggest that ciPTECs recapitulate the nephrotoxicity induced by vancomycin and indicates that OATs are not involved in vancomycin uptake. Here, we established a proof of concept for the use of ciPTECs as a 2D cellular model for VIKI as toxicity was observed after vancomycin administration.

Mitochondria are the main energy producers of the cell, providing energy in the form of adenosine triphosphate. Drug-induced toxicity may directly target mitochondria in various organs including the liver, heart, and kidney, as well as in organ systems such as the auditory, nervous, and muscular, which can result in clinical consequences. Understanding the causes and mechanisms of drug-induced mitochondrial dysfunction (DIMD) can enable the better prediction of mitochondrial toxicity in early stages of drug development. In this chapter, we will introduce the organs and organ systems most commonly affected by mitochondrial intoxication, specifically focussing on drugs and will describe the role of DIMD in the pathogenesis toxicity. We will also consider the influence of interindividual variation on the risk of DIMD and discuss the importance of appropriate and thorough preclinical testing in order to mitigate the risk of drugs carrying unidentified mitochondrial liabilities.

Background:Evidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here, we describe the generation of HepG2-derived transmitochondrial cybrids, to investigate the impact of mtDNA variation on mitochondrial (dys)function and susceptibility to iDILI. This study created 10 cybrid cell lines, each containing distinct mitochondrial genotypes of haplogroup H or haplogroup J backgrounds. Methods:HepG2 cells were depleted of mtDNA to make rho zero cells, before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide, and tolcapone, and their less toxic counterparts bicalutamide and entacapone utilizing ATP assays and extracellular flux analysis. Results:Whilst only slight variations in basal mitochondrial function were observed between haplogroups H and J, haplogroup-specific responses were observed to the mitotoxic drugs. Haplogroup J showed increased susceptibility to inhibition by flutamide, 2-hydroxyflutamide, and tolcapone, via effects on selected mitochondrial complexes (I and II), and an uncoupling of the respiratory chain. Conclusions:This study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest. This provides a practical and reproducible system to investigate the cellular consequences of variation in the mitochondrial genome, against a constant nuclear background. Additionally, the results show that inter-individual variation in mitochondrial haplogroup may be a factor in determining sensitivity to mitochondrial toxicants. Funding:This work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).

The clinical course of chronic liver diseases is significantly dependent on the progression rate of fibrosis which is the unstructured replacement of injured parenchyma by extracellular matrix. Despite intensive studies, the clinical opportunities for patients with fibrosing liver diseases have not improved. This will be changed by increasing knowledge of new pathogenetic mechanisms, which complement the "canonical principle" of fibrogenesis. The latter is based on the activation of hepatic stellate cells and their transdifferentiation to myofibroblasts induced by hepatocellular injury and consecutive inflammatory mediators such as TGF-β. Stellate cells express a broad spectrum of matrix components. New mechanisms indicate that the heterogeneous pool of (myo-)fibroblasts can be supplemented by epithelial–mesenchymal transition (EMT) from cholangiocytes and potentially also from hepatocytes to fibroblasts, by influx of bone marrow-derived fibrocytes in the damaged liver tissue and by differentiation of a subgroup of monocytes to fibroblasts after homing in the damaged tissue. These processes are regulated by the cytokines TGF-β and BMP-7, chemokines, colony-stimulating factors, metalloproteinases and numerous trapping proteins. They offer innovative diagnostic and therapeutic options. As an example, modulation of TGF-β/BMP-7 ratio changes the rate of EMT, and so the simultaneous determination of these parameters and of the connective tissue growth factor (CTGF) in serum might provide information on fibrogenic activity. Also, proteomic and glycomic approaches of serum are under investigation to set up specific protein profiles in patients with liver fibrosis. The aim of this article is to present the current pathogenetic concepts of liver fibrosis and to discuss established and novel diagnostic approaches to reflect the process of hepatic fibrogenesis in the medical laboratory.

SLC2A1 mediates glucose cellular uptake; key to appropriate immune function. Our previous work has shown efavirenz and lopinavir exposure inhibits T cell and macrophage responses, to known agonists, likely via interactions with glucose transporters. Using human cell lines as a model, we assessed glucose uptake and subsequent bioenergetic profiles, linked to immunological responses. Glucose uptake was measured using 2-deoxyglucose as a surrogate for endogenous glucose, using commercially available reagents. mRNA expression of SLC transporters was investigated using qPCR TaqMan™ gene expression assay. Bioenergetic assessment, on THP-1 cells, utilised the Agilent Seahorse XF Mito Stress test. In silico analysis of potential interactions between SLC2A1 and antiretrovirals was investigated using bioinformatic techniques. Efavirenz and lopinavir exposure was associated with significantly lower glucose accumulation, most notably in THP-1 cells (up to 90% lower and 70% lower with efavirenz and lopinavir, respectively). Bioenergetic assessment showed differences in the rate of ATP production (JATP); efavirenz (4 μg/mL), was shown to reduce JATP by 87% whereas lopinavir (10 μg/mL), was shown to increase the overall JATP by 77%. Putative in silico analysis indicated the antiretrovirals, apart from efavirenz, associated with the binding site of highest binding affinity to SLC2A1, similar to that of glucose. Our data suggest a role for efavirenz and lopinavir in the alteration of glucose accumulation with subsequent alteration of bioenergetic profiles, supporting our hypothesis for their inhibitory effect on immune cell activation. Clarification of the implications of this data, for in vivo immunological responses, is now warranted to define possible consequences for these, and similar, therapeutics.

The RENOWNED study investigated the treatment patterns, real-world effectiveness and safety of ranibizumab in Taiwanese patients with neovascular age-related macular degeneration (nAMD) treated with ranibizumab 0.5 mg in accordance with the first reimbursement scheme effective from 2012 to 2014.This study was a Phase IV, 12-month, open-label, prospective, observational study conducted in Taiwan. Patients with visual impairment due to nAMD initiating treatment with ranibizumab 0.5 mg were included. The primary endpoint was mean change in best-corrected visual acuity (BCVA) from Baseline at Month 3.Overall, 202 patients with nAMD were included. Mean (standard deviation [SD]) BCVA Early Treatment Diabetic Retinopathy Study letters improved from Baseline (49.6 [21.5] letters) at Month 3 (+4.9 [11.8], P < 0.0001), and at Month 12 (+3.5 [14.1], P = 0.0043). The proportion of patients with nAMD who lost ≥5 letters at Months 3 and 12 was 13.6% (n = 27) and 26.6% (n = 37), respectively. Mean (SD) central retinal thickness decreased from Baseline (320.1 [127.2] μm) with a mean reduction of 49.1 (107.3) μm at Month 3 (P < 0.0001), but was not significant at Month 12 with a mean reduction of 11.6 (115.6) μm (P = 0.2861). Mean (SD) number of ranibizumab injections over 12 months was 3.1 (1.0). A mean treatment interval of 109.5 days was observed between the third and fourth injections. After limited reimbursed ranibizumab injections, 43.8% patients received other treatments. The safety findings are consistent with previous studies.Ranibizumab 0.5 mg treatment for 12 months under real-world settings improved visual outcomes in Taiwanese patients with nAMD.

Inhibition of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzymatic step in de novo pyrimidine synthesis, has broad immunosuppressive effects in vivo and shows promise as a therapeutic target for the treatment of malignancies, viral infections and auto-immune diseases. Whilst there are numerous DHODH inhibitors under development, leflunomide and teriflunomide are the only FDA approved compounds on the market, each of which have been issued with black-box warnings for hepatotoxicity. Mitochondrial dysfunction is a putative mechanism by which teriflunomide and leflunomide elicit their hepatotoxic effects, however it is as yet unclear whether this is shared by other nascent DHODH inhibitors. The present study aimed to evaluate the propensity for DHODH inhibitors to mediate mitochondrial dysfunction in two hepatic in vitro models. Initial comparisons of cytotoxicity and ATP content in HepaRG® cells primed for oxidative metabolism, in tandem with mechanistic evaluations by extracellular flux analysis identified multifactorial toxicity and moderate indications of respiratory chain dysfunction or uncoupling. Further investigations using HepG2 cells, a hepatic line with limited capability for phase I xenobiotic metabolism, identified leflunomide and brequinar as positive mitochondrial toxicants. Taken together, biotransformation of some DHODH inhibitor species may play a role in mediating or masking hepatic mitochondrial liabilities.

Background: The triad of drug efficacy, toxicity and resistance underpins the risk-benefit balance of all therapeutics. The application of pharmacogenomics has the potential to improve the risk-benefit balance of a given therapeutic via the stratification of patient populations based on DNA variants. A growth in the understanding of the particulars of the mitochondrial genome, alongside the availability of techniques for its interrogation has resulted in a growing body of literature examining the impact of mitochondrial DNA (mtDNA) variation upon drug response. Objective: To critically evaluate and summarize the available literature, across a defined period, in a systematic fashion in order to map out the current landscape of the subject area and identify how the field may continue to advance. Methods: A systematic review of the literature published between January 2009 and December 2020 was conducted using the PubMed database with the following key inclusion criteria: reference to specific mtDNA polymorphisms or haplogroups, a core objective to examine associations between mtDNA variants and drug response, and research performed using human subjects or human in vitro models. Results: Review of the literature identified 24 articles reporting an investigation of the association between mtDNA variant(s) and drug efficacy, toxicity or resistance that met the key inclusion criteria. This included 10 articles examining mtDNA variations associated with antiretroviral therapy response, 4 articles examining mtDNA variants associated with anticancer agent response and 4 articles examining mtDNA variants associated with antimicrobial agent response. The remaining articles covered a wide breadth of medications and were therefore grouped together and referred to as “other.” Conclusions: Investigation of the impact of mtDNA variation upon drug response has been sporadic to-date. Collective assessment of the associations identified in the articles was inconclusive due to heterogeneous methods and outcomes, limited racial/ethnic groups, lack of replication and inadequate statistical power. There remains a high degree of idiosyncrasy in drug response and this area has the potential to explain variation in drug response in a clinical setting, therefore further research is likely to be of clinical benefit.

Mitochondrial DNA (mtDNA) is highly polymorphic and encodes 13 proteins which are critical to the production of ATP via oxidative phosphorylation. As mtDNA is maternally inherited and undergoes negligible recombination, acquired mutations have subdivided the human population into several discrete haplogroups. Mitochondrial haplogroup has been found to significantly alter mitochondrial function and impact susceptibility to adverse drug reactions. Despite these findings, there are currently limited models to assess the effect of mtDNA variation upon susceptibility to adverse drug reactions. Platelets offer a potential personalised model of this variation, as their anucleate nature offers a source of mtDNA without interference from the nuclear genome. This study, therefore, aimed to determine the effect of mtDNA variation upon mitochondrial function and drug-induced mitochondrial dysfunction in a platelet model. The mtDNA haplogroup of 383 healthy volunteers was determined using next-generation mtDNA sequencing (Illumina MiSeq). Subsequently, 30 of these volunteers from mitochondrial haplogroups H, J, T and U were recalled to donate fresh, whole blood from which platelets were isolated. Platelet mitochondrial function was tested at basal state and upon treatment with compounds associated with both mitochondrial dysfunction and adverse drug reactions, flutamide, 2-hydroxyflutamide and tolcapone (10–250 μM) using extracellular flux analysis. This study has demonstrated that freshly-isolated platelets are a practical, primary cell model, which is amenable to the study of drug-induced mitochondrial dysfunction. Specifically, platelets from donors of haplogroup J have been found to have increased susceptibility to the inhibition of complex I-driven respiration by 2-hydroxyflutamide. At a time when individual susceptibility to adverse drug reactions is not fully understood, this study provides evidence that inter-individual variation in mitochondrial genotype could be a factor in determining sensitivity to mitochondrial toxicants associated with costly adverse drug reactions.