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

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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 conrmed in
China, including 2946 deaths as well as over 10,566
conrmed 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, efciently
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 rst
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 Peoples 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 rst 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 efcacious
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
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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
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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 signicantly different from each other (Fig. 1a). To
better compare the antiviral activity of CQ versus HCQ,
the doseresponse curves of the two compounds against
SARS-CoV-2 were determined at four different multi-
plicities of infection (MOIs) by quantication 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 signicant
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 signicantly 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 immunouorescence
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 conrmed 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 immunouorescence analysis (IFA) and confocal
microscopy. Quantication 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 endosomallysosomal
protein LAMP1
+
ELs (green) (n> 30 cells for each group).
By contrast, in the presence of CQ or HCQ, signicantly
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
signicantly (Fig. 1c, arrows in the lower panels).
Since acidication 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 gure on previous page)
Fig. 1 Comparative antiviral efcacy 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 quantied 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 xed 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 quantied 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 efcient. In animals, both drugs
share similar tissue distribution patterns, with high con-
centrations in the liver, spleen, kidney, and lung reaching
levels of 200700 times higher than those in the plasma
10
.
It was reported that safe dosage (66.5 mg/kg per day) of
HCQ sulfate could generate serum levels of 1.41.5 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-inammatory agent that has been used
extensively in autoimmune diseases and can signicantly
decrease the production of cytokines and, in particular,
pro-inammatory factors. Therefore, in COVID-19
patients, HCQ may also contribute to attenuating the
inammatory response. In conclusion, our results show
that HCQ can efciently inhibit SARS-CoV-2 infection
in vitro. In combination with its anti-inammatory func-
tion, we predict that the drug has a good potential to
combat the disease. This possibility awaits conrmation 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
efcient 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 scientic 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 nal 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.
Conict of interest
The authors declare that they have no conict of interest.
Publishers note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional afliations.
Supplementary Information accompanies the paper at (https://doi.org/
10.1038/s41421-020-0156-0).
Received: 24 February 2020 Accepted: 4 March 2020
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... In addition, they could impede SARS-CoV-2 entry by restricting the glycosylation of ACE2 and S protein 292 and inhibiting acidification-dependent endocytosis. 293 Several in vitro studies showed that CQ and HCQ made inhibitory effects on SARS-CoV-2-infected Vero E6 cells. [293][294][295] A prospective observational study indicated that CQ provided positive effects on reduced viral load and time to clinical recovery (TTCR) in Fig. 6 The complicated mechanisms of SARS-CoV-2 manipulating cell autophagy. ...
... 293 Several in vitro studies showed that CQ and HCQ made inhibitory effects on SARS-CoV-2-infected Vero E6 cells. [293][294][295] A prospective observational study indicated that CQ provided positive effects on reduced viral load and time to clinical recovery (TTCR) in Fig. 6 The complicated mechanisms of SARS-CoV-2 manipulating cell autophagy. The phagophore derived from subcellular membranes expands and elongates to form the autophagosome, further fusing with lysosome to form the autophagolysosome for degradation, whose processes are closely regulated by ATGs. ...
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Objectives: We evaluated the 6-week mortality of SARS-CoV-2 hospitalized patients treated using a standardized protocol in 2020 in Marseille, France. Methods: A retrospective monocentric cohort study was conducted in the standard hospital wards at the Institut Hospitalo-Universitaire Méditerranée Infection, between March and December 2020 in adults with SARS-CoV-2 PCR-proven infection. Results: Of the 2111 hospitalized patients (median age, 67 [IQR 55-79] years; 1154 [54.7%] men), 271 were transferred to the intensive care unit (12.8%) and 239 died (11.3%; the mean age of patients who died was 81.2 (±9.9)). Treatment with hydroxychloroquine plus azithromycin (HCQ-AZ), used in 1270 patients, was an independent protective factor against death (0.68 [0.52 - 0.88]). This effect was consistent for all subgroups of age, comorbidities, severity of the disease and comedications with zinc or corticosteroids. Zinc was independently protective against death (0.39 [0.23 - 0.67]), in a subgroup analysis of patients treated with HCQ-AZ without dexamethasone. The use of high-flow oxygen therapy in elderly patients who were not eligible for intensive care unit transfer saved 19 patients (33.9%). Conclusions: In our 2020 cohort, treating COVID-19 with HCQ-AZ was associated with lower mortality. These results need to be analyzed in the context of academic discussions about observational studies versus randomized clinical trials. More data will deserve to be analyzed in the SARS-Cov 2 variants, vaccination and post-vaccination era.
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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.
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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.
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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.
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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.
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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.
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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.
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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)
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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.
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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.