The Effect of Metformin and Hydrochlorothiazide on Cytochrome P450 3A4 Metabolism of Ivermectin: Insights from In Silico Experimentation

Abstract

The spread of SARS-CoV-2 has led to an interest in using ivermectin (a potent antiparasitic agent) as an antiviral agent despite the lack of convincing in vivo clinical data for its use against COVID-19. The off-target prophylactic use of ivermectin adds a substantial risk of drug-drug interactions with pharmaceutical medications used to treat chronic conditions like diabetes and hypertension (metformin and hydrochlorothiazide, respectively). Therefore, this study aims to evaluate the potential drug-drug interactions between ivermectin with either metformin or hydrochlorothiazide. In silico experiments and high-throughput screening assays for CYP3A4 were conducted to understand how metformin and hydrochlorothiazide might affect CYP3A4's role in metabolizing ivermectin. The study findings indicated that hydrochlorothiazide is more stable than both ivermectin and metformin. This conclusion was further supported by root mean square fluctuation analysis, which showed that hydrochlorothiazide is more flexible. The variation in the principal component analysis scatter plot across the first three normal modes suggests hydrochlorothiazide has a more mobile conformation than ivermectin and metformin. Additionally, a strong inhibition of CYP3A4 by hydrochlorothiazide was observed, suggesting that hydrochlorothiazide's regulatory effects could significantly impede CYP3A4 activity, potentially leading to a reduced metabolism and clearance of ivermectin in the body. Concurrent administration of these drugs may result in drug-drug interactions and hinder the hepatic metabolism of ivermectin.

Keywords: COVID-19; SARS-CoV-2; cytochrome P450 3A4; diabetes; drug–drug interactions; hepatic metabolism; hydrochlorothiazide; hypertension; ivermectin; metformin.

Figure 1

Illustration of the front view of CYP3A4 [11] in complex with the heme. The CYP3A4 chain is composed of helix (green), sheet (purple), and loop (orange) structures, with the heme depicted in black. The heme is positioned between the flexible loop regions of the protein, which are believed to play a crucial role in the mechanism of ligand dissociation and association [12].

Figure 2

Illustration of ivermectin, metformin, and hydrochlorothiazide in terms of their binding orientation. Metformin (Cyan) displays a known binding site located at the center of the CYP3A4 protein (light gray) next to the Heme (dark khaki). On the other hand, ivermectin (green) and hydrochlorothiazide (orange) display unknown binding sites. Hence, the results suggest that the CYP3A4 protein has more than one binding site.

Figure 3

Ligand–residue interactions of (A) CYP3A4-IVM, (B) CYP3A4-MET, and (C) CYP3A4-HCTZ complexes. The red eyelashes represent the hydrophobic interactions; green dotted lines (H-bonds); black (carbon); red (oxygen); and blue (nitrogen).

Figure 4

RMSD of CYP3A4-HCTZ, CYP3A4-IVM, and CYP3A4-MET.

Figure 5

RMSF of CYP3A4–hydrochlorothiazide, CYP3A4–ivermectin, and CYP3A4–metformin.

Figure 6

RoG comparison across the 500 ns molecular dynamic simulation of CYP3A4 + hydrochlorothiazide (black), CYP3A4 + ivermectin (red), and CYP3A4 + metformin (green).

Figure 7

Inhibition of BOMC transformation by CYP3A4 in the presence of ivermectin (A). The rate of enzyme inhibition over 60 min of incubation (B) and the remaining enzyme activity (C). The results are from three independent experiments conducted in triplicates (n = 3) and are expressed as mean ± SD. One-way ANOVA was used for the statistical analysis, where **** = p $<0.0001,$** = p$<$ 0.006, and * = p $=$ 0.05, respectively.

Figure 8

Inhibition of BOMC transformation by CYP3A4 in the presence of metformin (A). The rate of enzyme inhibition over this incubation period is shown in (B). The residual enzyme activity (C). The results are based on three independent experiments conducted in triplicate (n = 6) and are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA, with significance levels indicated as **** = p < 0.0001.

Figure 9

Inhibition of BOMC transformation by CYP3A4 in the presence of hydrochlorothiazide (A) The rate of CYP3A4 inhibition over 60-minute incubation (B) and the remaining enzyme activity (C). The results are presented as the mean SD of three independent tests performed in duplicates (n = 3), and CYP inhibition is shown as a percentage of the vehicle control with a value of 100 per cent. One-way ANOVA was used for the statistical analysis, where *** p $<$ 0.001.