Ibuprofen, Flurbiprofen, Etoricoxib or Paracetamol Do Not Influence ACE2 Expression and Activity In Vitro or in Mice and Do Not Exacerbate In-Vitro SARS-CoV-2 Infection

Abstract

SARS-CoV-2 uses the human cell surface protein angiotensin converting enzyme 2 (ACE2) as the receptor by which it gains access into lung and other tissue. Early in the pandemic, there was speculation that a number of commonly used medications-including ibuprofen and other non-steroidal anti-inflammatory drugs (NSAIDs)-have the potential to upregulate ACE2, thereby possibly facilitating viral entry and increasing the severity of COVID-19. We investigated the influence of the NSAIDS with a range of cyclooxygenase (COX)1 and COX2 selectivity (ibuprofen, flurbiprofen, etoricoxib) and paracetamol on the level of ACE2 mRNA/protein expression and activity as well as their influence on SARS-CoV-2 infection levels in a Caco-2 cell model. We also analysed the ACE2 mRNA/protein levels and activity in lung, heart and aorta in ibuprofen treated mice. The drugs had no effect on ACE2 mRNA/protein expression and activity in the Caco-2 cell model. There was no up-regulation of ACE2 mRNA/protein expression and activity in lung, heart and aorta tissue in ibuprofen-treated mice in comparison to untreated mice. Viral load was significantly reduced by both flurbiprofen and ibuprofen at high concentrations. Ibuprofen, flurbiprofen, etoricoxib and paracetamol demonstrated no effects on ACE2 expression or activity in vitro or in vivo. Higher concentrations of ibuprofen and flurbiprofen reduced SARS-CoV-2 replication in vitro.

Keywords: ACE protein expression; ACE2 activity; ACE2 mRNA expression; NSAIDs; SARS-CoV-2 infection; ibuprofen.

Figure 1

Effect of NSAIDs and paracetamol on angiotensin converting enzyme 2 (ACE2) mRNA and protein expression in Caco-2 cells. Caco-2 cells were incubated with test substances or vehicle in the indicated concentrations for 24 h or 48 h. (A) The mRNA expression was determined by quantitative polymerase chain reaction (PCR) and normalized to β-actin. (B) Protein expression determined by western blot technology was analysed by the optical densitometric analysis achieved with the Image Lab software version 6.0 (Bio-Rad Laboratories, Hercules, CA, USA). The protein expression was normalized to β-actin. To obtain the fold induction the mRNA or protein expression of drug treated samples were related to the vehicle treated samples (control). The experiment was achieved in three biological and three technical replicates. The mean of three technical replicates are shown and used for statistical analysis (two-way ANOVA with Dunnett’s multiple comparisons test). * p < 0.05, ** p < 0.01 and *** p < 0.001 show statistically significant difference between drug treated and vehicle treated samples.

Figure 2

Effect of NSAIDs and paracetamol on soluble and membrane-bound angiotensin converting enzyme 2 (ACE2) activity in Caco-2 cells. For the soluble ACE activity Caco-2 supernatant and for the membrane-bound ACE2 activity Caco-2 cells were incubated with test substances or positive control (MLN-4760) for 18 h and the activity was determined with a fluorogenic substrate. To calculate the relative ACE2 activity, the fluorescence intensity (FI) values of the samples were related to untreated samples. The untreated samples were defined as 100% activity. Two-way ANOVA with multiple comparisons test was used to analyse statistical difference between drug treated and vehicle treated samples.

Figure 3

Effect of NSAIDs and paracetamol on SARS-CoV-2 virus replication. Caco-2 cells were preincubated with test substances or vehicle in the indicated concentrations for 1 h and subsequently infected with SARS-CoV-2 at an MOI of 0.01 for 24 h in the presence of compound dilutions. Afterwards, the cells were fixed and stained via immunohistochemistry against SARS-CoV-2 spike protein. The percentage of spike positive area per well was quantified and the values of the compound treated samples were normalized to the virus control without compounds (=100%). Values lower or higher than 100% represent virus inhibition or promotion, respectively. The experiment was achieved in three biological replicates. For statistical analysis, one-way ANOVA with multiple comparisons test was used. * p < 0.01, ** p < 0.01, **** p < 0.0001 show statistically significant differences between drug treated and vehicle treated samples.

Figure 4

ACE2 mRNA/protein expression and activity in ibuprofen treated mice. Male C57Bl/6J mice were treated with 50, 100, 200 mg/kg ibuprofen or vehicle for 7 days. ACE2 mRNA (A) or protein (B) levels of heart, lung and aorta were determined by quantitative PCR or western blot technology, respectively. To obtain the relative values the mRNA and protein levels of treated mice were related to untreated mice. (C) Angiotensin 1–7 plasma levels determined with ELISA. Each treatment group consists of 8 mice.