ArticlePDF Available

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro

DOI:10.1038/s41421-020-0156-0
Authors:
Jia Liu at Nankai University
Jia Liu
Ruiyuan Cao
Ruiyuan Cao
Ruiyuan Cao
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Mingyue Xu
Mingyue Xu
Mingyue Xu
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Xi Wang at Chinese Academy of Sciences
Xi Wang
Show all 10 authorsHide
Download full-text PDF
Read full-text
Download citation

Figures

Figures - available via license: CC BY
Content may be subject to copyright.
Available via license: CC BY
Content may be subject to copyright.
Liu et al. Cell Discovery (2020) 6:16 Cell Discovery
https://doi.org/10.1038/s41421-020-0156-0 www.nature.com/celldisc
CORRESPONDENCE Open Access
Hydroxychloroquine, a less toxic derivative
of chloroquine, is effective in inhibiting
SARS-CoV-2 infection in vitro
Jia Liu
1
,RuiyuanCao
2
, Mingyue Xu
1,3
,XiWang
1
, Huanyu Zhang
1,3
,HengruiHu
1,3
,YufengLi
1,3
, Zhihong Hu
1
,
Wu Zhong
2
and Manli Wang
1
Dear Editor,
The outbreak of coronavirus disease 2019 (COVID-19)
caused by the severe acute respiratory syndrome cor-
onavirus 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 num-
bers 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 vitro
1
. 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 States
2
,
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 pre-
liminary 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 pro-
phylaxis of malaria. In addition, an overdose of CQ can
cause acute poisoning and death
3
. In the past years, due to
infrequent utilization of CQ in clinical practice, its pro-
duction 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 animals
4
. More
importantly, HCQ is still widely available to treat auto-
immune 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
© The Author(s) 2020
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, whi ch permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if
changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a cred it line to the material. If
material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Correspondence: Zhihong Hu (huzh@wh.iov.cn) or Wu Zhong
(zhongwu@bmi.ac.cn) or Manli Wang (wangml@wh.iov.cn)
1
State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety
Mega-Science, Chinese Academy of Sciences, 430071 Wuhan, China
2
National Engineering Research Center for the Emergency Drug, Beijing
Institute of Pharmacology and Toxicology, 100850 Beijing, China
Full list of author information is available at the end of the article.
These authors contributed equally: Jia Liu, Ruiyuan Cao, Mingyue Xu
1234567890():,;
1234567890():,;
1234567890():,;
1234567890():,;
Fig. 1 (See legend on next page.)
Liu et al. Cell Discovery (2020) 6:16 Page 2 of 4
that the 50% cytotoxic concentration (CC
50
) 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 multi-
plicities 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 concentra-
tion (EC
50
) 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 EC
50
values were statistically significant
at an MOI of 0.01 (P< 0.05) and MOI of 0.2 (P< 0.001)
(Supplementary Table S1). It is worth noting that the
EC
50
values of CQ seemed to be a little higher than that in
our previous report (1.13 μM at an MOI of 0.05)
1
, which
is likely due to the adaptation of the virus in cell culture
that significantly increased viral infectivity upon con-
tinuous passaging. Consequently, the selectivity index
(SI =CC
50
/EC
50
) of CQ (100.81, 71.71, 38.26, and 37.12)
was higher than that of HCQ (55.32, 61.45, 14.41, 19.25)
at MOIs of 0.01, 0.02, 0.2, and 0.8, respectively. These
results were corroborated by immunofluorescence
microscopy as evidenced by different expression levels of
virus nucleoprotein (NP) at the indicated drug con-
centrations at 48 h p.i. (Supplementary Fig. S1). Taken
together, the data suggest that the anti-SARS-CoV-2
activity of HCQ seems to be less potent compared to CQ,
at least at certain MOIs.
Both CQ and HCQ are weak bases that are known to
elevate the pH of acidic intracellular organelles, such as
endosomes/lysosomes, essential for membrane fusion
5
.In
addition, CQ could inhibit SARS-CoV entry through
changing the glycosylation of ACE2 receptor and spike
protein
6
. Time-of-addition experiment confirmed that
HCQ effectively inhibited the entry step, as well as the
post-entry stages of SARS-CoV-2, which was also found
upon CQ treatment (Supplementary Fig. S2). To further
explore the detailed mechanism of action of CQ and HCQ
in inhibiting virus entry, co-localization of virions with
early endosomes (EEs) or endolysosomes (ELs) was ana-
lyzed by immunofluorescence analysis (IFA) and confocal
microscopy. Quantification analysis showed that, at
90 min p.i. in untreated cells, 16.2% of internalized virions
(anti-NP, red) were observed in early endosome antigen 1
(EEA1)-positive EEs (green), while more virions (34.3%)
were transported into the late endosomal–lysosomal
protein LAMP1
+
ELs (green) (n> 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 vir-
ions (2.4% for CQ and 0.03% for HCQ; P< 0.001) were
found to be co-localized with LAMP1
+
ELs (n> 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-CoV
7
.
Interestingly, we found that CQ and HCQ treatment
caused noticeable changes in the number and size/mor-
phology 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
vesicles
8
. Within the EE vesicles, virions (red) were loca-
lized 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
(see figure on previous page)
Fig. 1 Comparative antiviral efficacy and mechanism of action of CQ and HCQ against SARS-CoV-2 infection in vitro. a Cytotoxicity and
antiviral activities of CQ and HCQ. The cytotoxicity of the two drugs in Vero E6 cells was determined by CCK-8 assays. Vero E6 cells were treated with
different doses of either compound or with PBS in the controls for 1 h and then infected with SARS-CoV-2 at MOIs of 0.01, 0.02, 0.2, and 0.8. The virus
yield in the cell supernatant was quantified by qRT-PCR at 48 h p.i. Y-axis represents the mean of percent inhibition normalized to the PBS group. The
experiments were repeated twice. b,cMechanism of CQ and HCQ in inhibiting virus entry. Vero E6 cells were treated with CQ or HCQ (50 μM) for 1 h,
followed by virus binding (MOI =10) at 4 °C for 1 h. Then the unbound virions were removed, and the cells were further supplemented with fresh
drug-containing medium at 37 °C for 90 min before being fixed and stained with IFA using anti-NP antibody for virions (red) and antibodies against
EEA1 for EEs (green) or LAMP1 for ELs (green). The nuclei (blue) were stained with Hoechst dye. The portion of virions that co-localized with EEs or ELs
in each group (n> 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< 0.001).
Liu et al. Cell Discovery (2020) 6:16 Page 3 of 4
of lysosome from about 4.5 to 6.5 at 100 μM
9
. 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 con-
centrations in the liver, spleen, kidney, and lung reaching
levels of 200–700 times higher than those in the plasma
10
.
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 μMin
humans
11
. Therefore, with a safe dosage, HCQ con-
centration 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 severity
12
.
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 func-
tion, 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.
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 details
1
State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety
Mega-Science, Chinese Academy of Sciences, 430071 Wuhan, China.
2
National
Engineering Research Center for the Emergency Drug, Beijing Institute of
Pharmacology and Toxicology, 100850 Beijing, China.
3
University of the
Chinese Academy of Sciences, 100049 Beijing, China
Author 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.
Conflict of interest
The authors declare that they have no conflict of interest.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary Information accompanies the paper at (https://doi.org/
10.1038/s41421-020-0156-0).
Received: 24 February 2020 Accepted: 4 March 2020
References
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).
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).
3. Weniger, H. Review of side effects and toxicity of chloroquine. Bull. World
Health 79, 906 (1979).
4. McChesney, E. W. Animal toxicity and pharmacokinetics of hydroxy-
chloroquine sulfate. Am. J. Med. 75,11–18 (1983).
5. Mauthe,M.etal.Chloroquine inhibits autophagic flux by decreasing
autophagosome-lysosome fusion. Autophagy 14,1435–1455 (2018).
6. Savarino, A. et al. New insights into the antiviral effects of chloroquine. Lancet
Infect. Dis. 6,67–69 (2006).
7. Mingo, R. M. et al. Ebola virus and severe acute respiratory syndrome cor-
onavirus display late cell entry kinetics: evidence that transport to NPC1+
endolysosomes is a rate-defining step. J. Virol. 89,2931–2943 (2015).
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).
9. Ohkuma, S. & Poole, B. Fluorescence probe measurement of the intralyso-
somal pH in living cells and the perturbation of pH by various agents. Proc.
NatlAcad.Sci.USA75, 3327–3331 (1978).
10. Popert, A. J. Choloroquine: a review. Rheumatology 15,235–238 (1976).
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).
12. Huang, C. et al. Clinical features of patients infected with 2019 novel cor-
onavirus in Wuhan, China. Lancet 395,497–506 (2020).
Liu et al. Cell Discovery (2020) 6:16 Page 4 of 4
... The accumulation of hydroxychloroquine and chloroquine in lysosomes causes an increase in pH level. Thanks to this elevation, this elevation limits the functionality of lysosomal enzymes (29,30). mechanisms with low affinity. ...
... mechanisms with low affinity. These changes can also affect the function of autophagy by blocking the fusion occurred between autophagosomes and lysosomes (29)(30)(31)(32). In addition to molecular-level studies, activities of hydroxychloroquine and chloroquine drugs are related to the activation of immunity. ...
... In addition, the production of cytokines, such as IL-1 and IL-6, has been observed to decrease as a cellular effect of this treatment. The main reason for this is that the TLR signal plays a significant role in cytokine production, and the decrease in the affinity of Toll-like receptors with the utilization of these two drugs triggers the decrease in the production of inflammatory cytokines (29)(30)(31)(32). According to some crucial research, hydroxychloroquine and chloroquine drugs have an essential role in the immune response. ...
CLINICAL AND RADIOLOGICAL CHARACTERISTICS OF TESTICULAR TORSION CASES IN CHILDREN AND ADOLESCENTS AND POSTOPERATIVELY FOLLOW-UP RESULTS
Article
  • Oct 2023
Introduction: Testicular torsion is a surgical acute condition characterised by loss of blood supply to the testicle due to abnormal bending of the testicle. Early diagnosis and treatment are vitally important for the prevention of testicular loss. In this study, we aimed to present the clinical and radiological features and postoperative follow-up results of testicular torsion cases in children and adolescents. Methods: In this cross-sectional study, the data of patients aged 17 years and younger who were operated for testicular torsion between January 2014 and June 2023 were evaluated retrospectively. Patients' age, symptoms, hospital admission time, physical examination findings, scrotal colour Doppler ultrasound results, surgical techniques (manual detorsion or orchiectomy) and late follow-up results were statistically analysed. Results: The mean age of 66 patients operated with the diagnosis of testicular torsion was calculated as 6.55 years (14 days-17 years). Left testicular torsion was detected in 48 (72.7%) and right testicular torsion in 18 (27.2%) patients. There were no cases of bilateral torsion. Four patients had a history of scrotal trauma and three patients had undescended testis. A history of sudden scrotal pain on awakening from sleep in the morning was reported by 31 patients (46.9%). When the time from the onset of symptoms to admission to the emergency department was evaluated, it was found that 50% (n=33) of the patients were admitted within 0-12 hours, 36.3% (n=24) within 12-24 hours and 13.6% (n=9) more than 24 hours. A total of 34 patients underwent orchiopexy and 32 patients underwent orchiectomy. Orchiectomy was performed in 15.1% of the patients with a presentation time of less than 12 hours, 79.1% of the patients with a presentation time of 12-24 hours and 88.9% of the patients with a presentation time of more than 24 hours. None of the patients with 360 degrees of torsion during surgery required orchiectomy. 64.5% of patients with 540 degrees of torsion and 92.3% of patients with 720 degrees of torsion underwent orchiectomy. Late orchiectomy was performed in 7 patients who developed testicular atrophy as a result of clinical follow-up. Conclusion: In children presenting with acute scrotum, it is vital that the differential diagnosis of testicular torsion is made and treated as soon as possible. However, raising public awareness for early presentation is as important as the surgical procedure
View
... Chloroquine or hydroxychloroquine, a typical anti-malarial treatment, is an alternative to these antiviral medications because it prevents viral entrance by altering endosomes, modulating inflammatory mediators, and changing ACE2 (113). However, there is inconsistent evidence about the effectiveness of these drugs in treating SARS-CoV-2, in addition to legitimate concerns about chloroquine toxicity (114). Ammonium chloride, an acidotic drug that suppresses SARS-CoV virus multiplication in vitro, is one more treatment that may work in a manner that is similar to that of this one. ...
... Collaboration efforts should use both traditional and innovative methods to provide a fast and efficient worldwide reaction to the COVID-19 epidemic. (110,113,114) ...
FUTURE PROGNOSIS FOR CARDIOVASCULAR COMPLICATIONS ASSOCIATED WITH SARS-COV-2: A PHARMACOLOGICAL AND MOLECULAR PERSPECTIVE
Preprint
  • Nov 2023
Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19), which is associated with cardiovascular problems and serious lung damage. COVID-19 patients with comorbid conditions are at a significantly elevated risk of increased morbidity and mortality. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) are the two key host contributing factors for the severity and pathogenesis of COVID-19. The principal SARS-CoV-2 entrance receptor, ACE2, is expressed equally in most organs and produces cardio-protective vasodilators by physically degrading angiotensin II, the main controller of the Renin-Angiotensin Aldosterone System. However, treatment for cardiovascular disease (CVD) commonly involves RAAS inhibitors, which may increase ACE2 expression. Objective: To summarize the pharmacological molecular discoveries into the processes of viral infection and its consequences for cardiovascular disease and to offer suggestions for the practical management and treatment of COVID-19-related cardiovascular injury. Methods: This review focuses on the important considerations related to the cardiovascular manifestations of COVID-19 and discusses the various mechanisms of COVID-19 that contribute to its molecular and pharmacological presentation of cardiovascular injury. Results: The host-pathogen relationship began with ACE2’s attachment to the S-protein and proceeded with TMPRSS2’s proteolytic cleavage of the viral spike (S)-protein and ACE2. Currently discovered protein-protein interactions explain the uniqueness of SARS-CoV-2 infection. Conclusion: COVID-19 is associated with cardiovascular problems and serious lung damage. ACE2 and TMPRSS2 are key host contributing factors for the severity and pathogenesis of COVID-19. The molecular discoveries into the processes of viral infection and its consequences for cardiovascular disease provide important considerations for the management and treatment of COVID-19-related cardiovascular injury.
View
... When patients passed away, IL-6 levels increased dramatically and consistently. It serves as a reminder that if the IL-6 level is observed to continue rising during primary care, even above 1,000, it means that a cytokine storm has broken out, the patient's condition is deteriorating, and the patient may eventually pass away [114]. The survival showed no significant difference in measurements before and after TCZ application. ...
... It also agrees with published data that ivermectin treatment during early illness did not prevent progression to severe disease and with the author who called for the discontinuation of ineffective ivermectin as a COVID-19 drug [109,110]. Actually, there are both pros [111][112][113][114][115] and cons [116,117] to the clinical use of HCQ in COVID-19 patients. Actually, we have two sides of the coin; the first is the immune-modulatory, and the second is the antiviral effect of HCQ. ...
Even one dose of tocilizumab could hinder bad prognosis of cytokines storm in COVID-19 patients
Article
Severe COVID-19 pneumonia is a principal cause of death due to cascade of hyper inflammatory condition that leading to lung damage. Therefore, an effective therapy to countercurrent the surge of uncontrolled inflammation is mandatory to propose. Anti-interlukin-6 receptor antagonist monoclonal therapy, tocilizumab (TCZ) showed potential results in COVID-19 patients. This study aimed to emphasize the factors associated with mortality in COVID-19 patients that treated with tocilizumab and may influence the level of serum IL-6. A retrospective cohort study included all patients with clinical parameters that pointed to presence of cytokines storm and treated with one or more doses of TCZ beside the regular protocol of COVID-19 pneumonia. The factors that influence the mortality in addition to the level of serum IL-6 were analyzed. A total of 377 patients were included, 69.5 % of them received only one dose of TCZ which started mainly at the third day of admission. The mortality rate was 29.44 %. Regardless the time of starting TCZ, just one dose was fair enough to prevent bad consequence; OR = 0.04, P = 0.001.However, in spite of protective action of TCZ, older age and female sex were significant risk factors for mortality, P = 0.001 and 0.01 respectively, as well heart disease. Moreover, increasing the level of neutrophil, AST and IL-6 were associated with bad prognosis. In the same line, treatment with ivermectin, chloroquine and remdesivir inversely affect the level of IL-6. Early treatments of COVID-19 pneumonia with at least one dose of tocilizumab minimized the fatality rate.
View
... However, it was found that treatment with chloroquine caused acute toxicity cases as reported by the United States [77] and Nigeria [78] media. Hydroxychloroquine showed inhibitory effect to SARS-CoV-2 infection in vitro and less toxicity effect as well but receiving an overdose may result in poisoning [79]. Accordingly, investigating of other natural products that could be more effective and safer inhibitors is needed. ...
Network pharmacology, molecular docking, and dynamics analyses to predict the antiviral activityof ginger constituents against coronavirus infection
Preprint
Full-text available
  • Nov 2023
Background: COVID-19 is a global pandemic that caused a dramatic loss of human life worldwide, leading to accelerated research for antiviral drug discovery. Herbal medicine is one of the most commonly used for of alternative medicine for prevention and treatment of many conditions including respiratory system diseases. Methods: In this study, a computational pipeline was employed, including network pharmacology, molecular docking simulations, and molecular dynamics simulations, to analyze the common phytochemicals of ginger rhizomes and identify candidate constituents as viral inhibitors. Furthermore, experimental assays were performed to analyze the volatile and non-volatile compounds of ginger and to assess the antiviral activity of ginger oil and hydro-alcoholic extract. Results: Network pharmacology analysis showed that ginger compounds target human genes that are involved in related cellular processes to the viral infection. Docking analysis highlighted five pungent compounds and zingiberenol as potential inhibitors for the main protease (Mpro), spike receptor-binding domain (RBD), and human angiotensin-converting enzyme 2 (ACE2). Finally, (6)-gingerdiacetate was selected for molecular dynamics (MD) simulations as it exhibited the best binding interactions and free energies over the three target proteins. Trajectories analysis of the three complexes showed that RBD and ACE2 complexes with the ligand preserved similar patterns of root mean square deviation (RMSD) and radius of gyration (Rg) values to their respective native structures. These pungent compounds contribute to the higher antiviral activity of ginger extract than HD oil with CC50 of 3.480 μg/ml and IC50 of 2.727 μg/ml. Conclusion: Our study provides predictive insights that could be useful for development of anti-coronavirus drugs especially (6)-gingerdiacetate that may enhance the host immune response and block RBD binding to ACE2, thereby, inhibiting SARS-CoV-2 infection.
View
... Compounds with a toxicity rate of over 50% were eliminated, while others were subjected to infection using a VPP assay to assess their ability to reduce infection rates ( Figure 2B). To further examine the efficiency of our VPP assay, we also used well-known inhibitors, hydroxychloroquine (HCQ) and oligomeric proanthocyanidins (OPCs), as positive controls (Supplementary Figure S1A) [31][32][33]. Several studies have reported that high expression levels of ACE2 are correlated with severe infection and COVID-19-related complications [34,35]. We therefore sought to understand whether ACE2 is indeed critical for SARS-CoV-2 infection. ...
Herbal Compounds Dauricine and Isoliensinine Impede SARS-CoV-2 Viral Entry
Article
Full-text available
  • Oct 2023
Targeting viral entry has been the focal point for the last 3 years due to the continued threat posed by SARS-CoV-2. SARS-CoV-2’s entry is highly dependent on the interaction between the virus’s Spike protein and host receptors. The virus’s Spike protein is a key modulator of viral entry, allowing sequential cleavage of ACE2 at the S1/S2 and S2 sites, resulting in the amalgamation of membranes and subsequent entry of the virus. A Polybasic insertion (PRRAR) conveniently located at the S1/S2 site can also be cleaved by furin or by serine protease, TMPRSS2, at the cell surface. Since ACE2 and TMPRSS2 are conveniently located on the surface of host cells, targeting one or both receptors may inhibit receptor-ligand interaction. Here, we show that Dauricine and Isoliensinine, two commonly used herbal compounds, were capable of inhibiting SARS-CoV-2 viral entry by reducing Spike-ACE2 interaction but not suppressing TMPRSS2 protease activity. Further, our biological assays using pseudoviruses engineered to express Spike proteins of different variants revealed a reduction in infection rates following treatment with these compounds. The molecular modeling revealed an interconnection between R403 of Spike protein and both two compounds. Spike mutations at residue R403 are critical, and often utilized by ACE2 to gain cell access. Overall, our findings strongly suggest that Dauricine and Isoliensinine are effective in blocking Spike-ACE2 interaction and may serve as effective therapeutic agents for targeting SARS-CoV-2′s viral entry.
View
... It also inhibits glycosylation of the ACE2 receptor, which interferes with virus binding [81]. In vitro studies suggest that chloroquine and hydroxychloroquine block the transport of SARS-CoV-2 from endosomes to endolysosomes, thus possibly preventing the release of viral genetic material [82]. Also, hydroxychloroquine inhibits the cytokine storm induced by SARS-CoV-2 via suppressing T-cell activation [83]. ...
What are the current anti-COVID-19 drugs? From traditional to smart molecular mechanisms
Article
Full-text available
Background Coronavirus disease 19 (COVID-19) is the disease caused by SARS-CoV-2, a highly infectious member of the coronavirus family, which emerged in December 2019 in “Wuhan, China”. It induces respiratory illness ranging from mild symptoms to severe disease. It was declared a “pandemic” by the World Health Organization (WHO) in March 2020. Since then, a vast number of clinical and experimental studies have been conducted to identify effective approaches for its prevention and treatment. Main body The pathophysiology of COVID-19 represents an unprecedented challenge; it triggers a strong immune response, which may be exacerbated by “a cytokine storm syndrome”. It also induces thrombogenesis and may trigger multi-organ injury. Therefore, different drug classes have been proposed for its treatment and prevention, such as antivirals, anti-SARS-CoV-2 antibody agents (monoclonal antibodies, convalescent plasma, and immunoglobulins), anti-inflammatory drugs, immunomodulators, and anticoagulant drugs. To the best of our knowledge, this review is the first to present, discuss, and summarize the current knowledge about the different drug classes used for the treatment of COVID-19, with special emphasis on their targets, mechanisms of action, and important adverse effects and drug interactions. Additionally, we spotlight the latest “October 2023” important guidelines (NIH, IDSA, and NICE) and FDA approval or authorization regarding the use of these agents in the management of COVID-19. Conclusion Despite the wide array of therapeutic strategies introduced for the treatment of COVID-19, one of the most prominent therapeutic challenges is SARS-CoV-2 mutations and emerging new variants and subvariants. Currently, the anti-COVID-19 drug pipeline is continuously affording novel treatments to face this growing challenge.
View
View
View
Autophagy of the ER : the secretome finds the lysosome
Article
  • Nov 2023
Lysosomal degradation of the endoplasmic reticulum (ER) and its components through the autophagy pathway has emerged as a major regulator of ER proteostasis. Commonly referred to as ER‐phagy and ER‐to‐lysosome‐associated degradation (ERLAD), how the ER is targeted to the lysosome has been recently clarified by a growing number of studies. Here, we summarize the discoveries of the molecular components required for lysosomal degradation of the ER and their proposed mechanisms of action. Additionally, we discuss how cells employ these machineries to create the different routes of ER‐lysosome‐associated degradation. Further, we review the role of ER‐phagy in viral infection pathways, as well as the implication of ER‐phagy in human disease. In sum, we provide a comprehensive overview of the current field of ER‐phagy.
View
Phytochemicals for mitigating the COVID-19 crisis: evidence from pre-clinical and clinical studies
Article
  • Oct 2023
The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in December 2019 quickly escalated to pandemic levels and had a severe impact on public health. There are 761 million confirmed coronavirus disease 2019 (COVID-19) cases, with over 6.88 million deaths worldwide till March 2023. Severe cases of the disease caused critical respiratory failure followed by multiorgan involvement. Clinical escalation of COVID-19 has been correlated with markedly increased plasma inflammatory markers [e.g., C-reactive protein (CRP)] and pro-inflammatory cytokine levels [e.g., interleukin (IL)-6, tumor necrosis factor-α (TNF-α)]. Therapeutic options have mostly utilized corticosteroids, antivirals (e.g., remdesivir), and monoclonal antibody-based immunomodulation (e.g., tocilizumab). These existing treatments have adverse side effects, inadequate efficacy, and limitations in administering to patients with comorbidities and other underlying diseases. Monoclonal antibody-based therapies and some of the antivirals are very costly. Many phytochemicals have previously reported anti-inflammatory, antiviral, and antioxidant properties. Studying the effectiveness of such phytochemicals against COVID-19 and identifying new plant-derived molecules with antiviral properties have been a focus since the SARS-CoV-2 outbreak. This review article has documented in vitro, in vivo, and clinical studies encompassing 28 different phytochemicals belonging to various chemical groups (e.g., polyphenols, alkaloids, terpenes) that show anti-COVID-19 activity. These findings suggest that multiple phytochemicals can interfere with virus entry and replication inside the host cell. Many of them can protect from cytokine storm by acting on intracellular signalling pathways in addition to inhibiting virus multiplication. Phytochemicals may prove useful in alleviating post-COVID complications associated with kidney injury, and central nervous system complications, as well. Plant-derived compounds are usually cheaper and have fewer side effects. But, developing new formulations with better absorption and bioavailability remains a priority. This review informs the readers of the current status and indicates the ongoing research in this highly relevant field.
View
View
Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China
Article
Full-text available
  • Jan 2020
Background: A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of these patients. Methods: All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analysed data on patients with laboratory-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. Findings: By Jan 2, 2020, 41 admitted hospital patients had been identified as having laboratory-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0-58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0-13·0]). 26 (63%) of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. Interpretation: The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was associated with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiology, duration of human transmission, and clinical spectrum of disease need fulfilment by future studies. Funding: Ministry of Science and Technology, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technology Commission.
View
Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion
Article
Full-text available
  • Jun 2018
Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A1 (BafA1), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA1. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.
View
Ebola and SARS Display Late Cell Entry Kinetics: Evidence that Transport to NPC1+ Endolysosomes is a Rate-Defining Step.
Article
Full-text available
Unlabelled: Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1(+) LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity. Importance: Ebola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1(+) LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1(+) LE/Lys, as a therapeutic target for SARS and EBOV.
View
First Case of 2019 Novel Coronavirus in the United States
Article
  • Jan 2020
An outbreak of novel coronavirus (2019-nCoV) that began in Wuhan, China, has spread rapidly, with cases now confirmed in multiple countries. We report the first case of 2019-nCoV infection confirmed in the United States and describe the identification, diagnosis, clinical course, and management of the case, including the patient's initial mild symptoms at presentation with progression to pneumonia on day 9 of illness. This case highlights the importance of close coordination between clinicians and public health authorities at the local, state, and federal levels, as well as the need for rapid dissemination of clinical information related to the care of patients with this emerging infection.
View
Effect of Phospholipidosis on the Cellular Pharmacokinetics of Chloroquine
Article
In vivo, the weakly basic, lipophilic drug chloroquine (CQ) accumulates in the kidney to concentrations more than a thousand-fold greater than those in plasma. To study the cellular pharmacokinetics of chloroquine in cells derived from the distal tubule, Madin-Darby canine kidney cells were incubated with CQ under various conditions. CQ progressively accumulated without exhibiting steady-state behavior. Experiments failed to yield evidence that known active transport mechanisms mediated CQ uptake at the plasma membrane. CQ induced a phospholipidosis-like phenotype, characterized by the appearance of numerous multivesicular and multilamellar bodies (MLBs/MVBs) within the lumen of expanded cytoplasmic vesicles. Other induced phenotypic changes including changes in the volume and pH of acidic organelles were measured, and the integrated effects of all these changes were computationally modeled to establish their impact on intracellular CQ mass accumulation. Based on the passive transport behavior of CQ, the measured phenotypic changes fully accounted for the continuous, nonsteady-state CQ accumulation kinetics. Consistent with the simulation results, Raman confocal microscopy of live cells confirmed that CQ became highly concentrated within induced, expanded cytoplasmic vesicles that contained multiple MLBs/MVBs. Progressive CQ accumulation was increased by sucrose, a compound that stimulated the phospholipidosis-like phenotype, and was decreased by bafilomycin A1, a compound that inhibited this phenotype. Thus, phospholipidosis-associated changes in organelle structure and intracellular membrane content can exert a major influence on the local bioaccumulation and biodistribution of drugs.
View
Fluorescence Probe Measurement of the Intralysosomal pH in Living Cells and the Perturbation of pH by Various Agents
Article
  • Aug 1978
A quantitative method is described for the measurement of intralysosomal pH in living cells. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength, or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.7-4.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic potassium ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (2-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping.
View
Chloroquine: A review
Article
There is general agreement that chloroquine produces improvement in mild and moderate rheumatoid arthritis similar to that of gold but relapse is common within 3 mth after stopping treatment. In 1 long-term controlled trial of 134 cases no serious toxic effects were observed. The treated group showed a significant fall in sheep-cell agglutination titer which was correlated with clinical improvement. There was no definite improvement in the radiological status in the treated group whilst there was some deterioration in the control group. Both gold and chloroquine are relatively weak and slow to act but may induce remission in the less severe cases. Retinopathy is the most serious potential hazard but is very rare. It seems to be related to high individual doses rather than length of treatment with chloroquine. The author saw only 1 doubtful case in about 1000 cases over a 10-yr period and this low incidence appears to be the general experience. (Csonka - Radlett)
View
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
Article
  • Feb 1974
Treatment of juvenile rheumatoid arthritis with antimalarial drugs, chloroquine and hydroxychloroquine, is widely accepted, but the dosages are arbitrary, and serum concentrations are not usually determined. In this study, 123 child patients, 119 with juvenile rheumatoid arthritis (JRA) and 4 with systemic lupus erythematosus (SLE) were treated with antimalarial drugs, 60 patients with chloroquine (CQ) diphosphate and 63 patients with hydroxychloroquine (HCQ) sulphate. Side effects were found in 36 patients in the CQ group and in 23 patients in the HCQ group. No correlation between the occurrence of side effects and duration of treatment or total drug dose was observed. The influence of the daily dose of CQ or HCQ on the appearance of the side effects was demonstrable. The maximum safe dose of CQ diphosphate is considered to be 4 mg/kg/day (100 mg/m2/ day) and that of HCQ sulphate 5 to 7 mg/kg/day (120 to 150 mg/rrr/day) in the treatment of children with JRA. With a dosage of 4 mg/kg/day of either CQ or HCQ, the frequencies of side effects are about equal, but at higher dosages, CQ is apparently more toxic. The maxima of safe serum concentrations are correspondingly considered to be 250 to 280 μg/1 (0.8 to 0.9 /μmol/1) during CQ therapy and 370 to 470 μg/l (1.4 to 1.5 /imol/1) during HCQ therapy. The appearance of keratopathy correlated to high CQ and HCQ serum concentrations and is considered to be a sign of overdosage of these drugs. Despite the same dose in mg/kg of body weight or in mg/m2 of body surface area, significant individual variations in serum concentrations of CQ and HCQ were noted. Large variations were seen even when the drug had been administered for months. The control of serum concentrations is considered necessary during antimalarial therapy, particularly when child patients are in question.
View
Animal toxicity and pharmacokinetics of hydroxychloroquine sulfate
Article
Chloroquine is two to three times as toxic in animals as hydroxychloroquine, even though various single and repeated oral dosage regimens in man have given nearly identical plasma level curves. Tissue distributions are qualitatively similar for both drugs in albino rats--namely, bone, fat, and brain less than muscle less than eye less than heart less than kidney less than liver less than lung less than spleen less than adrenal--but the absolute distribution values are about 2.5 times higher for chloroquine. The metabolism of chloroquine and hydroxychloroquine differs only in that the latter drug gives two first-stage metabolites, whereas chloroquine gives one. Oral absorption of both drugs in man is nearly complete. However, three times as much chloroquine as hydroxychloroquine appears in the urine, and three times as much hydroxychloroquine as chloroquine appears in the feces.
View