The Tao of Anarchy: There is no God. There is no State. They are all superstitions that are established by the power-hunger psychopaths to divide, rule, and enslave us. It's only you and me, we are all true and real existence though in one short life. That is, We all are capable to freely interact with one another without coercion from anyone. We all are capable to take self-responsibility to find ways to live with one another in liberty, equality, harmony, and happiness before leaving this world forever. We all were born free and equal among all beings on this planet. We are not imprisoned in and by a place with a political name just because we were born there by bio-accident and social-chance. We are not chained to a set of indoctrinated beliefs that have been imposed upon us by so-called traditions. This Planet is home to all of us. No one owns it. We share the benefits from and responsibility to this Earth. We pledge no oath, no allegiance to no one; submit to no authority. We are all free and equal. The only obligation we all must undertake constantly with consistency is to respect the same freedoms and rights of others.
Thousands march along the ‘Friedrichstrasse’ during the demonstration against corona measures in Berlin, Germany, Saturday, Aug. 1, 2020. The initiative “Querdenken 711” has called for this. The motto of the demonstration is “The end of the pandemic – Freedom Day”. (Christoph Soeder/dpa via AP)
BERLIN (AP) — Thousands of protesters against German coronavirus restrictions converged Saturday in Berlin for a demonstration proclaiming “the end of the pandemic” has arrived just as authorities voice increasing concern about an upturn in new infections.
Hi there. Great that they are protesting. Here in Portugal there’s only sheeple. Its hard to believe that with all the misery starting to become worse in households, they do nothing. Obey without questioning the nonsense. They are like robots. Few question anything. They are going to let themselves be forced to mandatory masks on public spaces and disastrous misery on a country that already had a fragile economy. Only a few here can see where this going. Portugal is great target market for big pharma.
Hydroxychloroquine
In conclusion, our results show that HCQ can efficiently inhibit SARS-CoV-2 infection in vitro. In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials. We need to point out, although HCQ is less toxic than CQ, prolonged and overdose usage can still cause poisoning. And the relatively low SI of HCQ requires careful designing and conducting of clinical trials to achieve efficient and safe control of the SARS-CoV-2 infection.
• Published: 18 March 2020
Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro
• Jia Liu,
• Ruiyuan Cao,
• Mingyue Xu,
• Xi Wang,
• Huanyu Zhang,
• Hengrui Hu,
• Yufeng Li,
• Zhihong Hu,
• Wu Zhong &
• Manli Wang
Cell Discovery volume 6, Article number: 16 (2020) Cite this article
• 1.56m Accesses
• 271 Citations
• 5188 Altmetric
• Metrics details
Dear Editor,
The outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) poses a serious threat to global public health and local economies. As of March 3, 2020, over 80,000 cases have been confirmed in China, including 2946 deaths as well as over 10,566 confirmed cases in 72 other countries. Such huge numbers of infected and dead people call for an urgent demand of effective, available, and affordable drugs to control and diminish the epidemic.
We have recently reported that two drugs, remdesivir (GS-5734) and chloroquine (CQ) phosphate, efficiently inhibited SARS-CoV-2 infection in vitro1. Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences (USA). A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States2, and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020. However, as an experimental drug, remdesivir is not expected to be largely available for treating a very large number of patients in a timely manner. Therefore, of the two potential drugs, CQ appears to be the drug of choice for large-scale use due to its availability, proven safety record, and a relatively low cost. In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 (sixth edition) published by National Health Commission of the People’s Republic of China.
CQ (N4-(7-Chloro-4-quinolinyl)-N1,N1-diethyl-1,4-pentanediamine) has long been used to treat malaria and amebiasis. However, Plasmodium falciparum developed widespread resistance to it, and with the development of new antimalarials, it has become a choice for the prophylaxis of malaria. In addition, an overdose of CQ can cause acute poisoning and death3. In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine (HCQ) sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less (~40%) toxic than CQ in animals4. More importantly, HCQ is still widely available to treat autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Since CQ and HCQ share similar chemical structures and mechanisms of acting as a weak base and immunomodulator, it is easy to conjure up the idea that HCQ may be a potent candidate to treat infection by SARS-CoV-2. Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry (http://www.chictr.org.cn) for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence.
To this end, we evaluated the antiviral effect of HCQ against SARS-CoV-2 infection in comparison to CQ in vitro. First, the cytotoxicity of HCQ and CQ in African green monkey kidney VeroE6 cells (ATCC-1586) was measured by standard CCK8 assay, and the result showed that the 50% cytotoxic concentration (CC50) values of CQ and HCQ were 273.20 and 249.50 μM, respectively, which are not significantly different from each other (Fig. 1a). To better compare the antiviral activity of CQ versus HCQ, the dose–response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection (MOIs) by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection (p.i.). The data summarized in Fig. 1a and Supplementary Table S1 show that, at all MOIs (0.01, 0.02, 0.2, and 0.8), the 50% maximal effective concentration (EC50) for CQ (2.71, 3.81, 7.14, and 7.36 μM) was lower than that of HCQ (4.51, 4.06, 17.31, and 12.96 μM). The differences in EC50 values were statistically significant at an MOI of 0.01 (P < 0.05) and MOI of 0.2 (P 30 cells) was quantified and is shown in b. Representative confocal microscopic images of viral particles (red), EEA1+ EEs (green), or LAMP1+ ELs (green) in each group are displayed in c. The enlarged images in the boxes indicate a single vesicle-containing virion. The arrows indicated the abnormally enlarged vesicles. Bars, 5 μm. Statistical analysis was performed using a one-way analysis of variance (ANOVA) with GraphPad Prism (F = 102.8, df = 5,182, ***P 30 cells for each group). By contrast, in the presence of CQ or HCQ, significantly more virions (35.3% for CQ and 29.2% for HCQ; P < 0.001) were detected in the EEs, while only very few virions (2.4% for CQ and 0.03% for HCQ; P 30 cells) (Fig. 1b, c). This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV7.
Interestingly, we found that CQ and HCQ treatment caused noticeable changes in the number and size/morphology of EEs and ELs (Fig. 1c). In the untreated cells, most EEs were much smaller than ELs (Fig. 1c). In CQ- and HCQ-treated cells, abnormally enlarged EE vesicles were observed (Fig. 1c, arrows in the upper panels), many of which are even larger than ELs in the untreated cells. This is in agreement with previous report that treatment with CQ induced the formation of expanded cytoplasmic vesicles8. Within the EE vesicles, virions (red) were localized around the membrane (green) of the vesicle. CQ treatment did not cause obvious changes in the number and size of ELs; however, the regular vesicle structure seemed to be disrupted, at least partially. By contrast, in HCQ-treated cells, the size and number of ELs increased significantly (Fig. 1c, arrows in the lower panels).
Since acidification is crucial for endosome maturation and function, we surmise that endosome maturation might be blocked at intermediate stages of endocytosis, resulting in failure of further transport of virions to the ultimate releasing site. CQ was reported to elevate the pH of lysosome from about 4.5 to 6.5 at 100 μM9. To our knowledge, there is a lack of studies on the impact of HCQ on the morphology and pH values of endosomes/lysosomes. Our observations suggested that the mode of actions of CQ and HCQ appear to be distinct in certain aspects.
It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200–700 times higher than those in the plasma10. It was reported that safe dosage (6–6.5 mg/kg per day) of HCQ sulfate could generate serum levels of 1.4–1.5 μM in humans11. Therefore, with a safe dosage, HCQ concentration in the above tissues is likely to be achieved to inhibit SARS-CoV-2 infection.
Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity12. Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors. Therefore, in COVID-19 patients, HCQ may also contribute to attenuating the inflammatory response.
In conclusion, our results show that HCQ can efficiently inhibit SARS-CoV-2 infection in vitro. In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials. We need to point out, although HCQ is less toxic than CQ, prolonged and overdose usage can still cause poisoning. And the relatively low SI of HCQ requires careful designing and conducting of clinical trials to achieve efficient and safe control of the SARS-CoV-2 infection.
References
1. 1.
Wang, M. et al. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 30, 269–271 (2020).
Article Google Scholar
2. 2.
Holshue, M. L. et al. First case of 2019 novel coronavirus in the United States. N. Engl. J. Med. https://doi.org/10.1056/NEJMoa2001191 (2020).
Article PubMed Google Scholar
3. 3.
Weniger, H. Review of side effects and toxicity of chloroquine. Bull. World Health 79, 906 (1979).
Google Scholar
4. 4.
McChesney, E. W. Animal toxicity and pharmacokinetics of hydroxychloroquine sulfate. Am. J. Med. 75, 11–18 (1983).
CAS Article Google Scholar
5. 5.
Mauthe, M. et al. Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy 14, 1435–1455 (2018).
CAS Article Google Scholar
6. 6.
Savarino, A. et al. New insights into the antiviral effects of chloroquine. Lancet Infect. Dis. 6, 67–69 (2006).
Article Google Scholar
7. 7.
Mingo, R. M. et al. Ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to NPC1+ endolysosomes is a rate-defining step. J. Virol. 89, 2931–2943 (2015).
Article Google Scholar
8. 8.
Zheng, N., Zhang, X. & Rosania, G. R. Effect of phospholipidosis on the cellular pharmacokinetics of chloroquine. J. Pharmacol. Exp. Ther. 336, 661–671 (2011).
CAS Article Google Scholar
9. 9.
Ohkuma, S. & Poole, B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Natl Acad. Sci. USA 75, 3327–3331 (1978).
CAS Article Google Scholar
10. 10.
Popert, A. J. Choloroquine: a review. Rheumatology 15, 235–238 (1976).
CAS Article Google Scholar
11. 11.
Laaksonen, A. L., Koskiahde, V. & Juva, K. Dosage of antimalarial drugs for children with juvenile rheumatoid arthritis and systemic lupus erythematosus. A clinical study with determination of serum concentrations of chloroquine and hydroxychloroquine. Scand. J. rheumatol. 3, 103–108 (1974).
CAS Article Google Scholar
12. 12.
Huang, C. et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497–506 (2020).
CAS Article Google Scholar
Download references
Acknowledgements
We thank Professor Zhengli Shi and Dr. Xinglou Yang from Wuhan Institute of Virology and Professor Fei Deng from National Virus Resource Center for providing SARS-CoV-2 strain (nCoV-2019BetaCoV/Wuhan/WIV04/2019); Professor Xiulian Sun for kind help in statistical analysis; Professor Zhenhua Zheng for kindly providing the anti-LAMP1 rabbit polyclonal antibody; Prof. Zhengli Shi for kindly providing the anti-NP polyclonal antibody; Beijing Savant Biotechnology Co., ltd for kindly providing the anti-NP monoclonal antibody; Min Zhou and Xijia Liu for their assistance with this study; Jia Wu, Jun Liu, Hao Tang, and Tao Du from BSL-3 Laboratory and Dr. Ding Gao from the core faculty of Wuhan Institute of Virology for their critical support; Professor Gengfu Xiao, Professor Yanyi Wang and other colleagues of Wuhan Institute of Virology and Wuhan National Biosafety Laboratory for their excellent coordination; and Dr. Basil Arif for scientific editing of the manuscript. This work was supported in part by grants from the National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (2018ZX09711003 to W.Z.), the National Natural Science Foundation of China (31621061 to Z.H.), and the Hubei Science and Technology Project (2020FCA003 to Z.H.).
Author information
Author notes
1. These authors contributed equally: Jia Liu, Ruiyuan Cao, Mingyue Xu
Affiliations
1. State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, 430071, Wuhan, China
Jia Liu, Mingyue Xu, Xi Wang, Huanyu Zhang, Hengrui Hu, Yufeng Li, Zhihong Hu & Manli Wang
2. National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and Toxicology, 100850, Beijing, China
Ruiyuan Cao & Wu Zhong
3. University of the Chinese Academy of Sciences, 100049, Beijing, China
Mingyue Xu, Huanyu Zhang, Hengrui Hu & Yufeng Li
Contributions
Z.H., M.W., and W.Z. conceived and designed the experiments and provided the final approval of the manuscript. J.L., R.C., M.X., X.W., H.Z., H.H., and Y.L. participated in multiple experiments; all the authors analyzed the data. M.W., R.C., J.L., and Z.H. wrote the manuscript.
Corresponding authors
Correspondence to Zhihong Hu or Wu Zhong or Manli Wang.
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The authors declare that they have no conflict of interest.
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Liu, J., Cao, R., Xu, M. et al. Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discov 6, 16 (2020). https://doi.org/10.1038/s41421-020-0156-0
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• Received24 February 2020
• Accepted04 March 2020
• Published18 March 2020
• DOIhttps://doi.org/10.1038/s41421-020-0156-0
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• Autophagy
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Hi there. Great that they are protesting. Here in Portugal there’s only sheeple. Its hard to believe that with all the misery starting to become worse in households, they do nothing. Obey without questioning the nonsense. They are like robots. Few question anything. They are going to let themselves be forced to mandatory masks on public spaces and disastrous misery on a country that already had a fragile economy. Only a few here can see where this going. Portugal is great target market for big pharma.
LikeLike
Hydroxychloroquine
In conclusion, our results show that HCQ can efficiently inhibit SARS-CoV-2 infection in vitro. In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials. We need to point out, although HCQ is less toxic than CQ, prolonged and overdose usage can still cause poisoning. And the relatively low SI of HCQ requires careful designing and conducting of clinical trials to achieve efficient and safe control of the SARS-CoV-2 infection.
• Published: 18 March 2020
Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro
• Jia Liu,
• Ruiyuan Cao,
• Mingyue Xu,
• Xi Wang,
• Huanyu Zhang,
• Hengrui Hu,
• Yufeng Li,
• Zhihong Hu,
• Wu Zhong &
• Manli Wang
Cell Discovery volume 6, Article number: 16 (2020) Cite this article
• 1.56m Accesses
• 271 Citations
• 5188 Altmetric
• Metrics details
Dear Editor,
The outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) poses a serious threat to global public health and local economies. As of March 3, 2020, over 80,000 cases have been confirmed in China, including 2946 deaths as well as over 10,566 confirmed cases in 72 other countries. Such huge numbers of infected and dead people call for an urgent demand of effective, available, and affordable drugs to control and diminish the epidemic.
We have recently reported that two drugs, remdesivir (GS-5734) and chloroquine (CQ) phosphate, efficiently inhibited SARS-CoV-2 infection in vitro1. Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences (USA). A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States2, and a phase III clinical trial of remdesivir against SARS-CoV-2 was launched in Wuhan on February 4, 2020. However, as an experimental drug, remdesivir is not expected to be largely available for treating a very large number of patients in a timely manner. Therefore, of the two potential drugs, CQ appears to be the drug of choice for large-scale use due to its availability, proven safety record, and a relatively low cost. In light of the preliminary clinical data, CQ has been added to the list of trial drugs in the Guidelines for the Diagnosis and Treatment of COVID-19 (sixth edition) published by National Health Commission of the People’s Republic of China.
CQ (N4-(7-Chloro-4-quinolinyl)-N1,N1-diethyl-1,4-pentanediamine) has long been used to treat malaria and amebiasis. However, Plasmodium falciparum developed widespread resistance to it, and with the development of new antimalarials, it has become a choice for the prophylaxis of malaria. In addition, an overdose of CQ can cause acute poisoning and death3. In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China. Hydroxychloroquine (HCQ) sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less (~40%) toxic than CQ in animals4. More importantly, HCQ is still widely available to treat autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Since CQ and HCQ share similar chemical structures and mechanisms of acting as a weak base and immunomodulator, it is easy to conjure up the idea that HCQ may be a potent candidate to treat infection by SARS-CoV-2. Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry (http://www.chictr.org.cn) for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence.
To this end, we evaluated the antiviral effect of HCQ against SARS-CoV-2 infection in comparison to CQ in vitro. First, the cytotoxicity of HCQ and CQ in African green monkey kidney VeroE6 cells (ATCC-1586) was measured by standard CCK8 assay, and the result showed that the 50% cytotoxic concentration (CC50) values of CQ and HCQ were 273.20 and 249.50 μM, respectively, which are not significantly different from each other (Fig. 1a). To better compare the antiviral activity of CQ versus HCQ, the dose–response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection (MOIs) by quantification of viral RNA copy numbers in the cell supernatant at 48 h post infection (p.i.). The data summarized in Fig. 1a and Supplementary Table S1 show that, at all MOIs (0.01, 0.02, 0.2, and 0.8), the 50% maximal effective concentration (EC50) for CQ (2.71, 3.81, 7.14, and 7.36 μM) was lower than that of HCQ (4.51, 4.06, 17.31, and 12.96 μM). The differences in EC50 values were statistically significant at an MOI of 0.01 (P < 0.05) and MOI of 0.2 (P 30 cells) was quantified and is shown in b. Representative confocal microscopic images of viral particles (red), EEA1+ EEs (green), or LAMP1+ ELs (green) in each group are displayed in c. The enlarged images in the boxes indicate a single vesicle-containing virion. The arrows indicated the abnormally enlarged vesicles. Bars, 5 μm. Statistical analysis was performed using a one-way analysis of variance (ANOVA) with GraphPad Prism (F = 102.8, df = 5,182, ***P 30 cells for each group). By contrast, in the presence of CQ or HCQ, significantly more virions (35.3% for CQ and 29.2% for HCQ; P < 0.001) were detected in the EEs, while only very few virions (2.4% for CQ and 0.03% for HCQ; P 30 cells) (Fig. 1b, c). This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV7.
Interestingly, we found that CQ and HCQ treatment caused noticeable changes in the number and size/morphology of EEs and ELs (Fig. 1c). In the untreated cells, most EEs were much smaller than ELs (Fig. 1c). In CQ- and HCQ-treated cells, abnormally enlarged EE vesicles were observed (Fig. 1c, arrows in the upper panels), many of which are even larger than ELs in the untreated cells. This is in agreement with previous report that treatment with CQ induced the formation of expanded cytoplasmic vesicles8. Within the EE vesicles, virions (red) were localized around the membrane (green) of the vesicle. CQ treatment did not cause obvious changes in the number and size of ELs; however, the regular vesicle structure seemed to be disrupted, at least partially. By contrast, in HCQ-treated cells, the size and number of ELs increased significantly (Fig. 1c, arrows in the lower panels).
Since acidification is crucial for endosome maturation and function, we surmise that endosome maturation might be blocked at intermediate stages of endocytosis, resulting in failure of further transport of virions to the ultimate releasing site. CQ was reported to elevate the pH of lysosome from about 4.5 to 6.5 at 100 μM9. To our knowledge, there is a lack of studies on the impact of HCQ on the morphology and pH values of endosomes/lysosomes. Our observations suggested that the mode of actions of CQ and HCQ appear to be distinct in certain aspects.
It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200–700 times higher than those in the plasma10. It was reported that safe dosage (6–6.5 mg/kg per day) of HCQ sulfate could generate serum levels of 1.4–1.5 μM in humans11. Therefore, with a safe dosage, HCQ concentration in the above tissues is likely to be achieved to inhibit SARS-CoV-2 infection.
Clinical investigation found that high concentration of cytokines were detected in the plasma of critically ill patients infected with SARS-CoV-2, suggesting that cytokine storm was associated with disease severity12. Other than its direct antiviral activity, HCQ is a safe and successful anti-inflammatory agent that has been used extensively in autoimmune diseases and can significantly decrease the production of cytokines and, in particular, pro-inflammatory factors. Therefore, in COVID-19 patients, HCQ may also contribute to attenuating the inflammatory response.
In conclusion, our results show that HCQ can efficiently inhibit SARS-CoV-2 infection in vitro. In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials. We need to point out, although HCQ is less toxic than CQ, prolonged and overdose usage can still cause poisoning. And the relatively low SI of HCQ requires careful designing and conducting of clinical trials to achieve efficient and safe control of the SARS-CoV-2 infection.
References
1. 1.
Wang, M. et al. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 30, 269–271 (2020).
Article Google Scholar
2. 2.
Holshue, M. L. et al. First case of 2019 novel coronavirus in the United States. N. Engl. J. Med. https://doi.org/10.1056/NEJMoa2001191 (2020).
Article PubMed Google Scholar
3. 3.
Weniger, H. Review of side effects and toxicity of chloroquine. Bull. World Health 79, 906 (1979).
Google Scholar
4. 4.
McChesney, E. W. Animal toxicity and pharmacokinetics of hydroxychloroquine sulfate. Am. J. Med. 75, 11–18 (1983).
CAS Article Google Scholar
5. 5.
Mauthe, M. et al. Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy 14, 1435–1455 (2018).
CAS Article Google Scholar
6. 6.
Savarino, A. et al. New insights into the antiviral effects of chloroquine. Lancet Infect. Dis. 6, 67–69 (2006).
Article Google Scholar
7. 7.
Mingo, R. M. et al. Ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to NPC1+ endolysosomes is a rate-defining step. J. Virol. 89, 2931–2943 (2015).
Article Google Scholar
8. 8.
Zheng, N., Zhang, X. & Rosania, G. R. Effect of phospholipidosis on the cellular pharmacokinetics of chloroquine. J. Pharmacol. Exp. Ther. 336, 661–671 (2011).
CAS Article Google Scholar
9. 9.
Ohkuma, S. & Poole, B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Natl Acad. Sci. USA 75, 3327–3331 (1978).
CAS Article Google Scholar
10. 10.
Popert, A. J. Choloroquine: a review. Rheumatology 15, 235–238 (1976).
CAS Article Google Scholar
11. 11.
Laaksonen, A. L., Koskiahde, V. & Juva, K. Dosage of antimalarial drugs for children with juvenile rheumatoid arthritis and systemic lupus erythematosus. A clinical study with determination of serum concentrations of chloroquine and hydroxychloroquine. Scand. J. rheumatol. 3, 103–108 (1974).
CAS Article Google Scholar
12. 12.
Huang, C. et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497–506 (2020).
CAS Article Google Scholar
Download references
Acknowledgements
We thank Professor Zhengli Shi and Dr. Xinglou Yang from Wuhan Institute of Virology and Professor Fei Deng from National Virus Resource Center for providing SARS-CoV-2 strain (nCoV-2019BetaCoV/Wuhan/WIV04/2019); Professor Xiulian Sun for kind help in statistical analysis; Professor Zhenhua Zheng for kindly providing the anti-LAMP1 rabbit polyclonal antibody; Prof. Zhengli Shi for kindly providing the anti-NP polyclonal antibody; Beijing Savant Biotechnology Co., ltd for kindly providing the anti-NP monoclonal antibody; Min Zhou and Xijia Liu for their assistance with this study; Jia Wu, Jun Liu, Hao Tang, and Tao Du from BSL-3 Laboratory and Dr. Ding Gao from the core faculty of Wuhan Institute of Virology for their critical support; Professor Gengfu Xiao, Professor Yanyi Wang and other colleagues of Wuhan Institute of Virology and Wuhan National Biosafety Laboratory for their excellent coordination; and Dr. Basil Arif for scientific editing of the manuscript. This work was supported in part by grants from the National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (2018ZX09711003 to W.Z.), the National Natural Science Foundation of China (31621061 to Z.H.), and the Hubei Science and Technology Project (2020FCA003 to Z.H.).
Author information
Author notes
1. These authors contributed equally: Jia Liu, Ruiyuan Cao, Mingyue Xu
Affiliations
1. State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, 430071, Wuhan, China
Jia Liu, Mingyue Xu, Xi Wang, Huanyu Zhang, Hengrui Hu, Yufeng Li, Zhihong Hu & Manli Wang
2. National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and Toxicology, 100850, Beijing, China
Ruiyuan Cao & Wu Zhong
3. University of the Chinese Academy of Sciences, 100049, Beijing, China
Mingyue Xu, Huanyu Zhang, Hengrui Hu & Yufeng Li
Contributions
Z.H., M.W., and W.Z. conceived and designed the experiments and provided the final approval of the manuscript. J.L., R.C., M.X., X.W., H.Z., H.H., and Y.L. participated in multiple experiments; all the authors analyzed the data. M.W., R.C., J.L., and Z.H. wrote the manuscript.
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Correspondence to Zhihong Hu or Wu Zhong or Manli Wang.
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Liu, J., Cao, R., Xu, M. et al. Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discov 6, 16 (2020). https://doi.org/10.1038/s41421-020-0156-0
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• Received24 February 2020
• Accepted04 March 2020
• Published18 March 2020
• DOIhttps://doi.org/10.1038/s41421-020-0156-0
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• Autophagy
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