(T1430-11-64) SARS-CoV-2 Infection Dysregulates Drug Metabolism and Transport

Purpose: We proposed that COVID-19-associated inflammatory response may be limiting the safety and efficacy profile of promising COVID-19 drugs by dysregulating the expression of drug metabolizing enzymes and membrane-associated drug transporters through regulatory proteins including nuclear factor kappa B (NF-kβ), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and phosphorylated signal transducer and activator of transcription 3 (pSTAT3), tissue damages, and/or activation of immune cells such as macrophages and leukocytes. The overall goal of this novel research was to investigate the interactions between SARS-CoV-2 infection and clinically relevant drug metabolizing enzymes and membrane-associated drug transporters in Vero E6 cells and postmortem COVID-19 lung tissues.

Methods: We performed in vitro studies in Vero E6 cells, an African monkey kidney epithelial cell line that has been used to characterize SARS-CoV-2 virus<[1]. Vero E6 cells were infected with SARS-CoV-2 virus containing the spike protein D614G mutation, and cell pellets were collected at 0, 6, 24, and 48 hours post-infection. mRNA expression levels of selected COVID-19-associated inflammatory markers (10), drug metabolizing enzymes (8), and membrane-associated drug transporters (7) were measured using qPCR. To allow for the clinical translation of our in vitro findings in Vero E6 cells, we assessed the changes at the protein level, as well as the localization of COVID-19-associated inflammatory markers (2), regulatory proteins (4), drug metabolizing enzymes (7), and membrane-associated drug transporters (6) in postmortem human lung tissues obtained from 10 infected COVID-19 patients and 5 age/sex-matched non-infected controls using chromogenic immunohistochemistry analyses.

Results: mRNA expression levels of IL-6, IL-1β, TNF-α, CCL2, and CXCL10 were significantly upregulated in SARS-CoV-2 infected Vero E6 cells compared to the control (Fig.1). CYP3A4 mRNA expression was significantly upregulated while UGT1A1 was significantly downregulated in the infected Vero E6 cell (Fig.1). However, none of the investigated membrane-associated drug transporters showed significant dysregulation in infected Vero E6 cells (Fig.1). In postmortem human lung tissues, PXR was localized in smooth muscle cells of pulmonary arterioles and bronchioles, intravascular platelets, and alveolar macrophages. CAR, NF-kβ, CYP2C9, and CYP3A4 were constitutively expressed in all the cells of the lung tissues. CYP1A2 was detected in leukocytes while CYP2B6 was primarily expressed in macrophages, partly in smooth muscle cells, and pneumocytes. Localization of CYP2C8 was observed in the cytoplasm and nucleus of pneumocytes, endothelial cells, macrophages, and some smooth muscle cells. CYP2D6 was localized in pneumocytes, and subsets of macrophages while CYP2C19 and MRP1 were primarily expressed in bronchial epithelium and slightly expressed in smooth muscles of bronchi (Fig.2). P-gp was predominantly localized in respiratory epithelium and pneumocytes (Fig.2) while BCRP was primarily expressed in endothelial cells of larger pulmonary arteries. MRP2 was expressed in endothelial cells of alveolar capillaries and a subset of pneumocyte membranes. ENT1 was infrequently found in hematopoietic elements while ENT2 was localized in the membrane and cytoplasm of alveolar walls. IL-6 and IL-1β were localized in alveolar walls and bronchial epithelium while pSTAT3 was found in alveoli, bronchial epithelium, and endothelial cells. In the infected postmortem human lung tissues, P-gp was downregulated and MRP1 was upregulated but there was no significant dysregulation in the other investigated drug metabolizing enzymes and membrane-associated drug transporters (Fig.3).

Conclusion: To the best of our knowledge, this is the first work to demonstrate that some inflammatory processes seen in SARS-CoV-2 infection in patients are recapitulated in SARS-CoV-2-infected Vero E6 cells. COVID-19-associated inflammatory response dysregulated the mRNA expression of CYP3A4 and UGT1A1 in SARS-CoV-2-infected Vero E6 cells; this may present efficacy and safety concerns for COVID-19 patient subpopulations on polypharmacy. The lack of observable significant differences in the investigated membrane-associated drug transporters in Vero E6 cells may be due to subphysiological inflammatory responses, species differences in transporter expression between humans and monkeys, and less quantitative expression of selected transporters in kidney epithelial cells. In postmortem COVID-19 human lung tissues, only P-gp and MRP1 were significantly dysregulated, and this could affect the safety and efficacy of promising COVID-19 drugs such as remdesivir and nirmatrelvir as both drugs are substrates of P-gp. No significant dysregulation of the other investigated drug metabolizing enzymes and membrane-associated drug transporters, as well as inflammatory markers and regulatory proteins were observed in postmortem COVID-19 human lung tissues; and this could be attributed to differences in COVID-19 disease stages, and genetic variation in enzyme and transporter expression amongst subjects. Nonetheless, the localization of these clinically relevant drug metabolizing enzymes and transporters in pulmonary cells such as alveoli, macrophages, and blood vessels that are affected by COVID-19 pathogenesis may alter drug metabolism and transport.

References: [1] Rosa RB, Dantas WM, Do Nascimento JCF, da Silva MV, de Oliveira RN, Pena LJ. In vitro and in vivo models for studying sars-cov-2, the etiological agent responsible for covid-19 pandemic. Viruses 2021;13. https://doi.org/10.3390/v13030379.

Acknowledgments: This work is supported by grants from the Ontario HIV Treatment Network (fund #506657) and Canadian Institutes of Health Research (fund # 51794) awarded to Dr. Reina Bendayan. Chukwunonso K. Nwabufo is a recipient of the Canadian Institutes of Health Research Doctoral Scholarship, Ontario Graduate Scholarship, and Leslie Dan Faculty of Pharmacy Dean’s Fellowship.

Figure 1. Comparison of mRNA Expression Levels by qPCR Analyses of Vero E6 Cells



Figure 2. Localization of Pgp and MRP1 in Postmortem Human Lung Tissues



Figure 3. Comparison of Protein Expression Levels by Chromogenic Immunohistochemistry Analyses of Postmortem Human Lung Tissues