Combined hydroxychloroquine and azithromycin show synergistic effect on SARS-CoV-2 in monkey Vero E6 cells in vitro but may not be effective in human alveoli, pneumocytes and other cells.

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Fig. 2 Effect of hydroxychloroquine and azithromycin association on SARS-CoV 2 replication. 2A. Delta Ct between 0 and 60 h post infection. Ordered axis represents the variation of delta cycle-thresholds obtained by RT-PCR between H0 and H60 for each condition. Each point represents data obtained for one well. Number of replicates was indicated for each conditions are A10H5 n = 16, A10H2 n = 5, A10H1 n = 5, A5H5 n = 15, A5H2 n = 5, A5H1 n = 3, A2H5 n = 3, A2H2 n = 3, A2H1 n = 3 and n = 13 for the positive control. Median and interquartile range were indicated for each condition. *** represent significant results under p < 0,0005. Others are not significant compared to the control. 2B. Percentage of inhibition as compared to control by the combinations of 5 μM of hydroxychloroquine associated with 5 or 10 μM for azithromycin. Data represent the mean ± SD, representing three independent experiments conducted at least in triplicate. Source: Andreani et al., 2020.

Last update and review: August 10, 2020.

Contents hide
A short summary.
After a SARS-CoV-2 has attached to an ACE2, there is another step required for successful infection of the cell: the cleavage of the SARS-CoV-2 “spike protein”.
A combination of hydroxychloroquine with azithromycin had a synergistic effect in limiting viral replication in monkey Vero E6 cells.
What concentrations of hydroxychloroquine were determined as capable to inhibit 50% of viral replication in previous research?
At what concentrations of hydroxychloroquine and azithromycin there was the highest synergistic effect on viral replication?
“The synergy between hydroxychloroquine and azithromycin that we observed herein is at concentrations achieved in vivo.”
Conclusions.
Selected references:

A short summary.

SARS-CoV-2 can infect cells that prominently express angiotensin-converting enzyme 2 (ACE2), a plasma membrane-bound ectoenzyme. The virus uses ACE2 as the attachment receptor. Cells that do not express ACE2 can not be infected. Cells that have only limited expression of ACE2 are not permissive for viral infection and replication.

ACE2 expression in organs and systems most frequently affected in COVID-19.

Hydroxychloroquine and other drugs that limit viral infection and replication by increasing pH in cells endosomes are apparently not effective in many types of cells where SARS-CoV-2 infection is not prevented by higher endosomal pH. The cell types in which hydroxychlorquine is apparently ineffective include epithelial cells lining the nose, trachea, and distal airways, including alveoli and type II pneumocytes.

However, it is useful to know that hydroxychloroquine and azithromycin have a synergistic effect in the cell types where SARS-CoV-2 infection and replication is dependent on endosomal pH.

After a SARS-CoV-2 has attached to an ACE2, there is another step required for successful infection of the cell: the cleavage of the SARS-CoV-2 “spike protein”.

After a SARS-CoV-2 has attached to an ACE2, there is another step required for successful infection of the cell. This step is the cleavage of the SARS-CoV-2 “spike protein”. Cleavage can be pH-dependent and pH-independent. Hydroxychloroquine and other molecules that increase pH in cells’ endosomes can effectively limit viral replication only in some types of cells. The pH-independent cleavage of SARS-CoV-2 spike protein can not be inhibited by hydroxychloroquine.

The pH-independent cleavage of the SARS-CoV-2 spike protein is possible in cells that express the type II transmembrane serine proteases (TTSPs) TMPRSS2 and HAT. TMPRSS2 is expressed in epithelial cells lining the nose, trachea, and distal airways, including alveoli and type II pneumocytes. Given the above, hydroxychloroquine or a combination of hydroxychloroquine with azithromycin, would not be able to inhibit SARS-CoV-2 replication in epithelial cells in the nose, trachea, and distal airways, including alveoli and type II pneumocytes.

RT-PCR analysis of ACE2 and TMPRSS2 gene expression in human primary conjunctival, pterygium, mesenchymal stem cells, and other cell lines. Full-length primary pterygium and conjunctiva tissues (each n = 3) were collected, dissociated and maintained in cell culture. Human ocular cell lines, including retinal pigment epithelial cells ARPE-19, lens epithelial cells B3, primary trabecular meshwork (TM) cells and retinoblastoma cells Y79, human primary mesenchymal stem cells (MSCs), including bone marrow-derived MSCs (BM-MSCs), adipose-derived stem cells (ASC), and periodontal ligament-derived stem cells (PDLSC), as well as other human tissue cell lines, including human umbilical vein endothelial cells (HUVEC), lung epithelial carcinoma cells A549, liver hepatocellular carcinoma cells HepG2 and keratinocytes HaCaT, were also culture and collected. Total RNA was extracted and reverse transcribed (RT). The expression of (a) ACE2 and (b) TMPRSS2 genes was determined by polymerase chain reaction (PCR) with 2 primers for each gene. Housekeeping β-actin gene ACTB was used for normalization. Source: Ma (D) et al., 2020
RT-PCR analysis of ACE2 and TMPRSS2 gene expression in human primary conjunctival, pterygium, mesenchymal stem cells, and other cell lines. Full-length primary pterygium and conjunctiva tissues (each n = 3) were collected, dissociated and maintained in cell culture. Human ocular cell lines, including retinal pigment epithelial cells ARPE-19, lens epithelial cells B3, primary trabecular meshwork (TM) cells and retinoblastoma cells Y79, human primary mesenchymal stem cells (MSCs), including bone marrow-derived MSCs (BM-MSCs), adipose-derived stem cells (ASC), and periodontal ligament-derived stem cells (PDLSC), as well as other human tissue cell lines, including human umbilical vein endothelial cells (HUVEC), lung epithelial carcinoma cells A549, liver hepatocellular carcinoma cells HepG2 and keratinocytes HaCaT, were also culture and collected. Total RNA was extracted and reverse transcribed (RT). The expression of (a) ACE2 and (b) TMPRSS2 genes was determined by polymerase chain reaction (PCR) with 2 primers for each gene. Housekeeping β-actin gene ACTB was used for normalization. Source: Ma (D) et al., 2020.

Hydroxychloroquine can still limit viral replication in some types of cells and this may contribute to the observed positive effect of hydroxychloroquine on the duration of viral shedding in COVID-19 patients.

HCQ+AZ, the pre-therapy workup and the follow-up: electrolytes, electrocardiogram with corrected QT (Bazett’s formula) and more.

A combination of hydroxychloroquine with azithromycin had a synergistic effect in limiting viral replication in monkey Vero E6 cells.

Andreani et al., 2020 (1), found that a combination of hydroxychloroquine with azithromycin had a synergistic effect in limiting viral replication in monkey Vero E6 cells. Monkey Vero E6 cells are often used in virus research. Monkey Vero E6 cells express ACE2 but do not express TMPRSS2.

 

What concentrations of hydroxychloroquine were determined as capable to inhibit 50% of viral replication in previous research?

Andreani et al., 2020 (1):

Against SARS-CoV 2, the IC 50 of hydroxychloroquine was determined to be 4.51, 4.06, 17.31, and 12.96 μM with various MOI of 0.01, 0.02, 0.2, and 0.8, respectively.

At what concentrations of hydroxychloroquine and azithromycin there was the highest synergistic effect on viral replication?

Andreani et al., 2020 (1):

Combination of azithromycin and hydroxychloroquine led to significant inhibition of viral replication for wells containing hydroxychloroquine at 5 μM in combination with azithromycin at 10 and 5 μM (P-values at 0,0003 for A10H5 and at 0,0004 for A5H5).

Fig. 2 Effect of hydroxychloroquine and azithromycin association on SARS-CoV 2 replication. 2A. Delta Ct between 0 and 60 h post infection. Ordered axis represents the variation of delta cycle-thresholds obtained by RT-PCR between H0 and H60 for each condition. Each point represents data obtained for one well. Number of replicates was indicated for each conditions are A10H5 n = 16, A10H2 n = 5, A10H1 n = 5, A5H5 n = 15, A5H2 n = 5, A5H1 n = 3, A2H5 n = 3, A2H2 n = 3, A2H1 n = 3 and n = 13 for the positive control. Median and interquartile range were indicated for each condition. *** represent significant results under p < 0,0005. Others are not significant compared to the control. 2B. Percentage of inhibition as compared to control by the combinations of 5 μM of hydroxychloroquine associated with 5 or 10 μM for azithromycin. Data represent the mean ± SD, representing three independent experiments conducted at least in triplicate. Source: Andreani et al., 2020.
Fig. 2 Effect of hydroxychloroquine and azithromycin association on SARS-CoV 2 replication. 2A. Delta Ct between 0 and 60 h post infection. Ordered axis represents the variation of delta cycle-thresholds obtained by RT-PCR between H0 and H60 for each condition. Each point represents data obtained for one well. Number of replicates was indicated for each conditions are A10H5 n = 16, A10H2 n = 5, A10H1 n = 5, A5H5 n = 15, A5H2 n = 5, A5H1 n = 3, A2H5 n = 3, A2H2 n = 3, A2H1 n = 3 and n = 13 for the positive control. Median and interquartile range were indicated for each condition. *** represent significant results under p < 0,0005. Others are not significant compared to the control. 2B. Percentage of inhibition as compared to control by the combinations of 5 μM of hydroxychloroquine associated with 5 or 10 μM for azithromycin. Data represent the mean ± SD, representing three independent experiments conducted at least in triplicate. Source: Andreani et al., 2020.

“The synergy between hydroxychloroquine and azithromycin that we observed herein is at concentrations achieved in vivo.”

Andreani et al., 2020 (1):

One of the main criticisms of previously published data was that drug concentrations for viral inhibition used in vitro are difficult to translate clinically due to side effects that would occur at those concentrations. The synergy between hydroxychloroquine and azithromycin that we observed herein is at concentrations achieved in vivo and detected in serum and pulmonary tissues respectively.

Conclusions.

The cell types in which hydroxychlorquine is apparently ineffective include epithelial cells lining the nose, trachea, and distal airways, including alveoli and type II pneumocytes.

However, it is useful to know that hydroxychloroquine and azithromycin have a synergistic effect in the cell types where SARS-CoV-2 infection and replication is dependent on endosomal pH.

Selected references:

1. Andreani J, Le Bideau M, Duflot I, et al. In vitro testing of combined hydroxychloroquine and azithromycin on SARS-CoV-2 shows synergistic effect.Microb Pathog. 2020;145:104228.

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