Molecular Networking for Drug Toxicities Studies: The Case of Hydroxychloroquine in COVID-19 Patients

Using drugs to treat COVID-19 symptoms may induce adverse effects and modify patient outcomes. These adverse events may be further aggravated in obese patients, who often present different illnesses such as metabolic-associated fatty liver disease. In Rennes University Hospital, several drug such as hydroxychloroquine (HCQ) have been used in the clinical trial HARMONICOV to treat COVID-19 patients, including obese patients. The aim of this study is to determine whether HCQ metabolism and hepatotoxicity are worsened in obese patients using an in vivo/in vitro approach. Liquid chromatography high resolution mass spectrometry in combination with untargeted screening and molecular networking were employed to study drug metabolism in vivo (patient’s plasma) and in vitro (HepaRG cells and RPTEC cells). In addition, HepaRG cells model were used to reproduce pathophysiological features of obese patient metabolism, i.e., in the condition of hepatic steatosis. The metabolic signature of HCQ was modified in HepaRG cells cultured under a steatosis condition and a new metabolite was detected (carboxychloroquine). The RPTEC model was found to produce only one metabolite. A higher cytotoxicity of HCQ was observed in HepaRG cells exposed to exogenous fatty acids, while neutral lipid accumulation (steatosis) was further enhanced in these cells. These in vitro data were compared with the biological parameters of 17 COVID-19 patients treated with HCQ included in the HARMONICOV cohort. Overall, our data suggest that steatosis may be a risk factor for altered drug metabolism and possibly toxicity of HCQ.

PXR Suppresses PPARα-Dependent HMGCS2 Gene Transcription by Inhibiting the Interaction between PPARα and PGC1α

Background: PXR is a xenobiotic-responsive nuclear receptor that controls the expression of drug-metabolizing enzymes. Drug-induced activation of PXR sometimes causes drug–drug interactions due to the induced metabolism of co-administered drugs. Our group recently reported a possible drug–drug interaction mechanism via an interaction between the nuclear receptors CAR and PPARα. As CAR and PXR are structurally and functionally related receptors, we investigated possible crosstalk between PXR and PPARα. Methods: Human hepatocyte-like HepaRG cells were treated with various PXR ligands, and mRNA levels were determined by quantitative reverse transcription PCR. Reporter assays using the HMGCS2 promoter containing a PPARα-binding motif and mammalian two-hybrid assays were performed in HepG2 or COS-1 cells. Results: Treatment with PXR activators reduced the mRNA levels of PPARα target genes in HepaRG cells. In reporter assays, PXR suppressed PPARα-dependent gene expression in HepG2 cells. In COS-1 cells, co-expression of PGC1α, a common coactivator of PPARα and PXR, enhanced PPARα-dependent gene transcription, which was clearly suppressed by PXR. Consistently, in mammalian two-hybrid assays, the interaction between PGC1α and PPARα was attenuated by ligand-activated PXR. Conclusion: The present results suggest that ligand-activated PXR suppresses PPARα-dependent gene expression by inhibiting PGC1α recruitment.

Phenobarbital Induces SLC13A5 Expression through Activation of PXR but Not CAR in Human Primary Hepatocytes

Phenobarbital (PB), a widely used antiepileptic drug, is known to upregulate the expression of numerous drug-metabolizing enzymes and transporters in the liver primarily via activation of the constitutive androstane receptor (CAR, NR1I3). The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays an important role in intracellular citrate homeostasis that is associated with a number of metabolic syndromes and neurological disorders. Here, we show that PB markedly elevates the expression of SLC13A5 through a pregnane X receptor (PXR)-dependent but CAR-independent signaling pathway. In human primary hepatocytes, the mRNA and protein expression of SLC13A5 was robustly induced by PB treatment, while genetic knockdown or pharmacological inhibition of PXR significantly attenuated this induction. Utilizing genetically modified HepaRG cells, we found that PB induces SLC13A5 expression in both wild type and CAR-knockout HepaRG cells, whereas such induction was fully abolished in the PXR-knockout HepaRG cells. Mechanistically, we identified and functionally characterized three enhancer modules located upstream from the transcription start site or introns of the SLC13A5 gene that are associated with the regulation of PXR-mediated SLC13A5 induction. Moreover, metformin, a deactivator of PXR, dramatically suppressed PB-mediated induction of hepatic SLC13A5 as well as its activation of the SLC13A5 luciferase reporter activity via PXR. Collectively, these data reveal PB as a potent inducer of SLC13A5 through the activation of PXR but not CAR in human primary hepatocytes.

Drug-Drug Interaction Potential, Cytotoxicity, and Reactive Oxygen Species Production of Salix Cortex Extracts Using Human Hepatocyte-Like HepaRG Cells

Herbal preparations of willow bark (Salix cortex) are available in many countries as non-prescription medicines for pain and inflammation, and also as dietary supplements. Currently only little information on toxicity and drug interaction potential of the extracts is available. This study now evaluated the effects of two Salix cortex extracts on human hepatocyte-like HepaRG cells, in view of clinically relevant CYP450 enzyme activity modulation, cytotoxicity and production of reactive oxygen species (ROS). Drug metabolism via the CYP450 enzyme system is considered an important parameter for the occurrence of drug-drug interactions, which can lead to toxicity, decreased pharmacological activity, and adverse drug reactions. We evaluated two different bark extracts standardized to 10 mg/ml phenolic content. Herein, extract S6 (S. pentandra, containing 8.15 mg/ml total salicylates and 0.08 mg/ml salicin) and extract B (industrial reference, containing 5.35 mg/ml total salicylates and 2.26 mg/ml salicin) were tested. Both Salix cortex extracts showed no relevant reduction in cell viability or increase in ROS production in hepatocyte-like HepaRG cells. However, they reduced CYP1A2 and CYP3A4 enzyme activity after 48 h at ≥25 μg/ml, this was statistically significant only for S6. CYP2C19 activity inhibition (0.5 h) was also observed at ≥25 μg/ml, mRNA expression inhibition by 48 h treatment with S6 at 25 μg/ml. In conclusion, at higher concentrations, the tested Salix cortex extracts showed a drug interaction potential, but with different potency. Given the high prevalence of polypharmacy, particularly in the elderly with chronic pain, further systematic studies of Salix species of medical interest should be conducted in the future to more accurately determine the risk of potential drug interactions.