Hydroxychloroquine information sheet V2 2020-04-01

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Intervention

Hydroxchloroquine 2.4g (equivalent to 1.86g base) loading dose in four divided doses over 24 hours, followed by 800 mg (620mg base) daily for a further 9 days or until discharge.

In the RECOVERY Trial we are testing hydroxychloroquine (HCQ). HCQ, a derivative of chloroquine (CQ), has been used for many decades to treat malaria and rheumatological diseases. It has antiviral activity against SARS-CoV-2 in cell culture and preliminary reports from China and France report a beneficial antiviral and clinical effect in COVID-19.

WHO has prioritized the evaluation of chloroquine or hydroxychloroquine in COVID-19 in clinical trials to assess safety and efficacy (see here).

The dose being used in RECOVERY is the same dose as in the WHO-led international clinical trial in COVID-19 and is based on a careful review of the optimal dose in COVID-19, balancing potential risks and benefits (See below and appendix).

Summary of information on chloroquine and its derivatives in betacornavirus infections.

CQ has significant antiviral activity against SARS-CoV-2 in cell culture (EC50 = 1.13

μM; CC50 > 100 μM, SI > 88.50), as it does for the related SARS-CoV-1 1-4. CQ blocks

virus infection by increasing endosomal pH required for virus/ cell fusion, as well as interfering with the glycosylation of cellular receptors of SARS-CoV.3 In SARS-CoV-2 infected Vero cells, HCQ (EC50=0.72 μM) has been reported to be more potent than

CQ (EC50=5.47 μM) 5, although Liu et al reported that CQ was more potent than HCQ.6

These are relatively high levels by comparison with therapeutic exposures in the treatment of malaria but could be achieved with daily oral dosing. Chloroquine has complex pharmacokinetic properties and although the relationship between plasma concentrations and concentrations in respiratory epithelium is not known precisely, in rats the concentration in lung is between 124 and 748-fold that in plasma 7. If active, HCQ concentrations in the human lung would be expected to exceed those required for the EC90 after an initial dose. There are preliminary reports emerging from China and France of clinical benefit in the treatment of COVID-19 infections 8,9.

Potential harm

CQ was discovered in 1934 and introduced generally in 1947. It has been used on a very large scale, with an annual global consumption of hundreds of metric tonnes for over 50 years. It is inexpensive, simple to administer, and, at the appropriate doses, has an excellent safety profile in all age groups and has been the prophylactic drug of choice in pregnancy 10. CQ and HCQ are very similar, with HCQ having a hydroxyl group on the alkyl side chain. In addition to antimalarial uses, CQ and HCQ are used in continuous daily dosing for rheumatoid arthritis, systemic and discoid lupus erythematosus and psoriatic arthritis. HCQ is reported to have better safety profile than CQ, better gastrointestinal tolerability and less retinal toxicity 11.

Hydroxychloroquine information sheet V2 2020-04-01

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Chloroquine and hydroxychloroquine are generally well tolerated. Apart from the bitter taste the most common adverse effect is itching in dark skinned patients. Occasionally there may be transient headache, difficulty focusing, dysphoria, emotional lability decreased appetite; nausea; vomiting. abdominal pain; diarrhoea or skin rash. Chloroquine predictably prolongs the electrocardiograph QT interval but arrhythmias are rare; indeed chloroquine is generally anti-arrhythmic. At high doses, hypotension may occur. Reported retinal and muscle toxicity with HCQ is associated with long term high dose use and it is very unlikely to be encountered in short course regimens.

Choice of intervention and dose

HCQ versus CQ: In animal toxicity assessments HCQ usually has a higher ED50 or LD50 than chloroquine. This toxicity difference in favour of HCQ is greatest in dogs. The elimination kinetics of the two drugs are very similar. In man the two advantages of HCQ most widely quoted are better gastrointestinal tolerability and less retinal toxicity, although the evidence for both is not strong. It is unclear whether the risk of adverse cardiovascular effects (hypotension and QT prolongation) differ in man between the two drugs. From a clinical standpoint there are no downsides to using hydroxychloroquine and there may be a small advantage in tolerability.

Dose: The recommended adult dosing of chloroquine for treatment of non-falciparum malaria (BNF) is 620 mg initially, then 310 mg after 6–8 hours, then 310 mg daily for 2 days. This is equivalent to 930mg base in first 24 hours. This is a loading dose to ensure the necessary blood concentrations are achieved rapidly.

Hydroxychloroquine is very similar to chloroquine. It is used mainly to treat rheumatoid arthritis and other related conditions. The adult dose is usually 400-600mg/ day (equivalent to 310 to 465 mg base). Sometimes 800mg/day is given.

The dose in RECOVERY is Hydroxychloroquine (155mg base per 200mg tablet): Initial dose: 4 tablets

6 hours later: 4 tablets 12 hours: 2 tablets 24 hours: 2 tablets Thereafter: 2 tablets every 12 hours for a total of 10 days The first day loading dose in RECOVERY (1.86g base) is twice the first day loading dose for treating malaria. This dose has been selected based on the available data of the IC50 for SARS-CoV-2. The objective is to reach plasma concentrations that are inhibitory to the virus as soon as safely possible. The plasma concentrations that will result are at the higher end of those encountered during steady state treatment of rheumatoid arthritis. Given the significant mortality in patients hospitalised with COVID-19, this dose is felt to be justified. This is the schedule that has been adopted by the World Health Organisation in their SOLIDARITY trial. No dose adjustment is required for weight or kidney function based on the doses defined in this protocol.

The pharmacokinetics of hydroxychloroquine are complex, but are largely determined by distribution rather than excretion. Further details are given in the appendix, but in brief hydroxychloroquine is rapidly absorbed after oral dosing but blood concentrations

Hydroxychloroquine information sheet V2 2020-04-01

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then fall rapidly due to tissue binding which creates an apparent volume of distribution of >100 L. The loading doses included in RECOVERY are intended to ‘fill’ this large volume of distribution rapidly so that blood concentrations can be maintained in the range sufficient to kill the virus. Renal excretion clears about 50% of hydroxychloroquine but does not become an important determinant of blood concentrations for some time, because of the overwhelming effect of distribution, which is why no adjustment for kidney function has been made given the short duration of treatment (compared to chronic administration in rheumatological conditions). The two loading doses are the same as the two loading doses in the WHO-recommended treatment regimen for amoebic liver abscess, but separated by 6 hours rather than 24, and the total dose over 10 days is the same as the standard treatment of amoebic liver abscess, when the same total dose is given over 20 days. The seriousness of COVID-19 (with approximately 20% in-hospital mortality in the UK) justifies testing these higher doses.

Any serious adverse reactions to hydroxychloroquine are to be reported and the independent Data Monitoring Committee will review them alongside other clinical outcome data. If clear evidence of benefit or harm emerge then they would advise the Steering Committee should the trial protocol need to be modified or stopped.

References

1. Wang M, Cao R, Zhang L, et al. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res 2020. 2. Lu R, Zhao X, Li J, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet (London, England) 2020. 3. Vincent MJ, Bergeron E, Benjannet S, et al. Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol J 2005;2:69. 4. Zhou P, Yang XL, Wang XG, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020. 5. Yao X, Ye F, Zhang M, et al. In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Clin Infect Dis 2020. 6. 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 2020;6:16. 7. McChesney EW, Banks WF, Jr., Fabian RJ. Tissue distribution of chloroquine, hydroxychloroquine, and desethylchloroquine in the rat. Toxicol Appl Pharmacol 1967;10:501-13. 8. Gao J, Tian Z, Yang X. Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies. Biosci Trends 2020. 9. GAUTRET P, LAGIER JC, PAROLA P, et al. Hydroxychloroquine and Azithromycin as a treatment of COVID-19: preliminary results of an open-label non-randomized clinical trial. medRxiv 2020:2020.03.16.20037135.

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10. Villegas L, McGready R, Htway M, et al. Chloroquine prophylaxis against vivax malaria in pregnancy: a randomized, double-blind, placebo-controlled trial. Trop Med Int Health 2007;12:209-18. 11. McChesney EW. Animal toxicity and pharmacokinetics of hydroxychloroquine sulfate. Am J Med 1983;75:11-8.

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Near Final Draft

Chloroquine and Hydroxychloroquine Pharmacology

NJ White, J Tarning

Mahidol Oxford Research Unit

Faculty of Tropical Medicine, Mahidol University,

Bangkok, Thailand.

Executive summary

Enormous clinical experience with chloroquine and hydroxychloroquine has been gained

over the past 60 years in the treatment of malaria, amoebic liver abscess and several

rheumatological conditions. COVID19 represents a new potential indication. Dosing based

on previous therapeutic guidelines has therefore to be adapted, based on the well

characterised pharmacokinetic properties of both drugs and the likely pharmacodynamic

effects, to increase the chance of overall benefit in this potentially lethal infection. In

general chloroquine and hydroxychloroquine are safe and reasonably well tolerated,

although they are dangerous in overdose and parenteral administration needs careful

control. This is because of their unusual pharmacokinetic properties. Chloroquine and

hydroxychloroquine have enormous apparent volumes of distribution

(chloroquine>hydroxychloroquine) because of extensive tissue binding and they are both

eliminated very slowly. Thus the free plasma concentrations which drive potentially serious

adverse effects (hypotension and delayed ventricular repolarization) are determined

largely by distribution processes. Hydroxychloroquine was slightly safer than chloroquine

in preclinical testing and is considered better tolerated over the long term. Taking into

account the concentrations of both drugs that have been documented in the acute

treatment of malaria (over 3 days) and the chronic treatment of rheumatological conditions

(over years), and their likely weak activity against the SARS-2-CoV virus, clinical trials are

using the highest doses of these drugs that are considered to be reasonably safe. The two

loading doses proposed are the same as the two loading doses in the WHO-recommended

treatment regimen for amoebic liver abscess, but they are separated by 6 hours instead of

24 hours, and the total treatment dose, given over 10 days is the same as the total

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treatment dose given over 20 days in the amoebic liver abscess regimen. Using lower doses

risks failing to identify the potential benefit in a potentially lethal infection. Thus from the

standpoint that both drugs might be beneficial in COVID19, the risks of overdosing have

been balanced against the risks of underdosing.

Background

Chloroquine [7-chloro-4-[[4-(diethylamino)-1-methylbutyl]amino] quinoline is a 4-

aminoquinoline compound discovered in Germany in 1934 as part of a research programme

to develop new antimalarial drugs, but it was not evaluated fully until the end of the Second

World War (Shannon et al 1948). Hydroxychloroquine, in which one of the ethyl groups in the

alkyl side chain is hydroxylated, was synthesized in 1946. Early clinical pharmacology

assessments in the USA characterized the

safety, tolerability and antimalarial efficacy of

chloroquine – and it was selected from a very

long list of potential antimalarials as the most

promising. By the early 1950s chloroquine had

become the treatment of choice for all malaria throughout the world and hundreds of metric

tonnes (corresponding to nearly 100 million malaria treatment doses) were dispensed

annually. Thus over 5 billion treatments have been dispensed. Chloroquine was used both in

prevention and treatment, and it remains today a first line treatment for non-falciparum

malaria. It was, and it still is, used to prevent malaria in pregnancy. In the 1950s chloroquine

was even added in large quantities to salt in some regions to provide mass antimalarial

prophylaxis. At the same time the other potential benefits from this drug class were being

explored, notably their anti-inflammatory properties. Hydroxychloroquine was shown to have

some safety advantages in experimental animals (McChesney 1983), and was developed

more for its use in rheumatological conditions. It is generally considered to be slightly safer

than chloroquine (Table shows animal data) although the evidence to support this in man is

not strong. Chloroquine has also been used at high doses (10mg base/kg daily for two days

followed by 5mg base/kg daily for 2-3 weeks) for the treatment of hepatic amoebiasis.

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From McChesney et al 1983

Chemistry

Chloroquine and hydroxychloroquine are racemic mixtures. The enantiomers have different

pharmacokinetic (see below) and pharmacodynamic properties. Hydroxychloroquine is more

hydrophilic compared to chloroquine.

Clinical Pharmacokinetics

Even in the 1940s, when drug measurement was in its infancy, it was clear that the 4-

aminoquinolines had unusual pharmacokinetic properties. Absorption after oral

administration was rapid and generally reliable but the total apparent volume of distribution

was enormous (>100 L/kg) reflecting extensive tissue binding. Estimates for the terminal

elimination half-life lengthened as assays improved (i.e. with increasing sensitivity a greater

proportion of the terminal

phase could be

characterized). The true

terminal elimination half-life

of chloroquine approximates

one month. The quoted average values for terminal phase elimination half-lives (chloroquine

~38 days and hydroxychloroquine ~54 days) may not represent significant differences. Thus,

the initial plasma or whole blood concentration profile in the treatment of acute illness is

determined mainly by distribution processes and not by elimination. This is critical to

understanding dosing and concentration profiles and associated risks with short course

treatments. In the treatment of malaria (or COVID19) the initial (loading) doses are designed

The plasma or blood concentration profile of chloroquine or hydroxychloroquine in acute treatment is determined primarily by distribution not elimination

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to “fill” the body so that concentrations that would take weeks to achieve without a loading

dose, are achieved as soon as safely possible. The dosing and spacing of the loading dose aims

to provide sufficient time for chloroquine or hydroxychloroquine to diffuse out from the small

central “compartment” and thereby avoid transient very high concentrations that are

potentially toxic.

Chloroquine is well absorbed by mouth. It is very rapidly absorbed following subcutaneous or

intramuscular injection, such that absorption may outpace distribution, and transiently toxic

concentrations may occur. To circumvent this, intravenous chloroquine is administered by

constant-rate infusion, and subcutaneous or intramuscular chloroquine is given in small (2.5-

3.5mg base/kg) frequent injections. The principal metabolite of chloroquine,

desethylchloroquine, has approximately equivalent antimalarial and other biological

activities.

Enantiomer binding and elimination kinetics

Using HPLC assays (which are less sensitive than currently used LC-MS/MS methods) the

estimated mean terminal elimination half-life was longer for (R)-chloroquine (12.3 days) than

for (S)-chloroquine (9.8 days). The estimated mean total body clearance was lower and

distribution volume was smaller for the (R)-enantiomer (136 ml/min and 3410 L, respectively)

than for the (S)-enantiomer (237 ml/min and 720 L, respectively). Protein binding was also

different for the chloroquine stereoisomers, with opposite preferential binding to human

albumin and the “acute phase protein” alpha 1-acid glycoprotein. Total

human plasma protein binding was 67% for (S)-chloroquine and 43% for the (R)-

enantiomer. The plasma

protein binding estimates

for hydroxychloroquine are

very similar. The (S)-

enantiomer

of hydroxychloroquine was

64% bound in plasma, while

(R)-hydroxychloroquine was

The metabolism of hydroxychloroquine

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37% bound. The S enantiomers showed relatively

greater binding to albumin and lower binding to alpha

1-acid glycoprotein. Thus in contrast to avid tissue

binding chloroquine and hydroxychloroquine are not

highly protein bound to plasma proteins.

Both drugs are metabolized slowly. Renal elimination

accounts for approximately 50% of total clearance.

Chloroquine and hydroxychloroquine are both

desethylated, and the hydroxyl group is removed from

hydroxychloroquine so that desethylchloroquine and

chloroquine are all formed (Figure). As described above,

drug metabolism and clearance have little effect on the

plasma or whole blood concentrations in the first few

days of treatment but they are relevant to chronic

dosing. Accumulation does occur slowly in patients with

renal failure.

Plasma chloroquine levels in the treatment of acute malaria after intravenous infusion (White

et al 1987)

From Gustafsson et al 1983

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Antiviral and antimalarial activities.

The 4-aminoquinolines inhibit the pH-dependent steps of replication of a broad range of

viruses (including flaviviruses, retroviruses, and coronaviruses). The exact mechanism of

antiviral action is unclear as these drugs may interfere with nearly every step of cellular

infection and replication, i.e. viral fusion, viral penetration, nucleic acid replication, viral

protein glycosylation, virus assembly, new virus particle transport, and virus release. Activities

(EC50s) against the SAR-CoV-2 virus are in the low micromolar range which represents the

upper end of the free plasma concentration range encountered in clinical practice. The

antimalarial mode of action of the quinoline antimalarials has been a source of controversy

for years. These drugs are weak bases, and they concentrate in the acid food vacuole of the

parasite, but this in itself does not explain their antimalarial activity. Chloroquine intercalates

DNA, but only at concentrations (1-2mM) much higher than required to kill parasites (10-

20nM). Chloroquine binds to ferriprotoporphyrin IX, a product of haemoglobin degradation,

and thereby chemically inhibits haem dimerization. This is an essential defence mechanism

for the malaria parasite to detoxify haem, and inhibition of this process provides a plausible

explanation for the selective antimalarial action of these drugs. Chloroquine also

competitively inhibits glutathione mediated haem degradation, another parasite

detoxification pathway.

Hydroxychloroquine PKs from Lim et al 2009 400mg given to 6 healthy adults

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Formulations

Chloroquine has been formulated as

sulphate, phosphate and hydrochloride

salts, and is prescribed for malaria in

weights of base content. Various liquid

formulations are available for

paediatric use. Chloroquine can be

given by intravenous infusion,

intramuscular or subcutaneous

injection, orally, or by suppository.

Hydroxychloroquine has been made in a parenteral formulation but the usual form is a tablet

of the sulphate salt.

Toxicity

Chloroquine and hydroxychloroquine are generally well tolerated. The most common adverse

reactions reported are dyspepsia, nausea, occasionally vomiting visual disturbances

(particularly transient accommodation difficulties) and headache. These adverse effects can

often be lessened by taking chloroquine with food. Orthostatic hypotension may be

accentuated in febrile patients. Chloroquine and hydroxychloroquine (and the structurally

related 4-aminoquinoline amodiaquine and the bisquinoline piperaquine) block several

different cation channels. Blockade of the inwardly rectifying Kir2.1 potassium (hERG) channel

delays ventricular repolarization and results in electrocardiographic QT prolongation. This is

a risk factor for polymorphic ventricular tachycardia (TdP: torsade de pointes) although there

is much debate about the determinants of the risk relationship and the potential ameliorating

effect of multichannel blockade. It is unclear whether the risk of TdP is increased with

chloroquine or hydroxychloroquine. TdP has been reported in chronic dosing in systemic

lupus erythematosus (SLE), where myocarditis may be a confounder, but there is very little

evidence for significant risk in acute treatment. Sudden unexplained death has not been

associated with chloroquine but there have not been very large prospective studies to assess

this in malaria. Recent prospective studies with data from 300,000 patients with the related

antimalarial compound piperaquine, which has very similar hERG blocking properties, found

There are different salts, each with a different base equivalent. This has led to confusion in dosing. Malaria treatment is usually recommended in terms of base. Chloroquine diphosphate 250 mg salt contains 155 mg base. Hydroxychloroquine sulphate 200 mg salt contains 155 mg base.

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no increased risk of TdP. In contrast, chloroquine clearly does have beneficial anti-arrhythmic

properties. This is particularly useful in SLE where arrhythmias are common. Pruritus is

particularly troublesome in dark-skinned patients and may be dose limiting. Itching is

described as a widespread prickling sensation mostly affecting the palms, soles and scalp

which starts within 6 to 24 hours and may last for several days. It can be very distressing.

Antihistamine treatment is not usually very effective. Hydroxychloroquine may be associated

with less itching. Very rarely chloroquine may cause an acute and self-limiting

neuropsychiatric reaction. In prophylaxis, cumulative doses over 100g (>5 years prophylaxis)

are associated with an increased risk of retinopathy. Retinal signs include a pale optic disc,

arteriolar narrowing, peripheral retinal depigmentation, macular oedema, retinal granularity

and oedema, and retinal pigmentary changes consisting of a circle of pigmentation and

central pallor; the so called 'doughnut' or 'bull's eye' macula. Reversible corneal opacities can

be seen in 30-70% of rheumatology patients within a few weeks of high dose treatment. Half

are asymptomatic but others may complain of photophobia, visual halos around lights, and

blurred vision. Myopathy is rare at the doses used in antimalarial prophylaxis. Less common

cutaneous side effects include lightening of skin colour, various rashes (photoallergic

dermatitis, exacerbation of psoriasis (severe psoriasis is probably a contraindication), bullous

pemphigoid, exfoliative dermatitis, pustular rash), skin depigmentation (with long term use),

and hair loss. Parenteral chloroquine causes hypotension if administered too rapidly or a large

dose (>3.5mg base/kg) is given by intramuscular or subcutaneous injection. In self-poisoning,

chloroquine produces hypotension, arrhythmias, and coma, and is commonly lethal. It has

been suggested that diazepam is a specific antidote, but more recent studies do not support

a specific role for this drug above good haemodynamic and ventilatory support. Chloroquine

must be kept in child resistant containers out of reach of children. Chloroquine should not be

prescribed to patients with a history of suicide or those who have suicidal ideas.

Why use these large doses in COVID109 infections?

The inhibitory activity of chloroquine and hydroxychloroquine against the SAR-CoV-2 virus is

seen only at high concentrations in-vitro so if there is any benefit from the drug it is likely to

require high concentrations of free drug in blood to drive high concentrations in the infected

respiratory epithelium. We assume that the cytosolic concentrations of the drugs in the

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respiratory epithelium will be in dynamic equilibrium with the blood. We know from the

treatment of life-threatening infections that the earlier in the evolving disease process that

pathogen multiplication is inhibited the better is the outcome. This argues for achieving high

blood concentrations of chloroquine or hydroxychloroquine as soon as possible, but this has

to be balanced against toxicity. These doses are certainly high, but the disease is serious. The

concentrations that will occur in the first few hours of treatment are similar to those which

will occur after about one week’s twice daily treatment (See modelled profiles below).

Although there is little experience to date in the treatment of COVID19 the extensive previous

experience in acute malaria suggests that serious toxicity (hypotension, arrhythmias,

convulsions) should be unlikely at these doses. Paradoxically by analogy with malaria, sicker

bed-bound patients generally tolerate these drugs relatively well whereas ambulant febrile

patients may feel nauseated and temporarily dysphoric. It is hoped that COVID19 patients

would respond in a similar way to malaria patients but this will need to be determined.

Simulated blood concentration time profiles of chloroquine (n=1,000).

0 10 20 30 400.1

1

10

Time (days)

Chl

oroq

uine

(µM

)

0 1 2 3 4 5 6 7 8 9 100.1

1

10

Time (days)

Chl

oroq

uine

(µM

)

0 10 20 30 400.1

1

10

Time (days)

Chl

oroq

uine

(µM

)

0 1 2 3 4 5 6 7 8 9 10 11 12 130.1

1

10

Time (days)

Chl

oroq

uine

(µM

)

7 days of chloroquine 7 days of chloroquine

10 days of chloroquine 10 days of chloroquine

Putative IC50

Putative IC50

Putative IC50

Putative IC50

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Loading dose of 4 tablets of chloroquine diphosphate (250 mg salt, equivalent to 155 mg base per tablet) at time 0 and 6 hours, followed by a maintenance dose of 2 tablets of chloroquine diphosphate at time 12 hours, and then twice daily for a total of 7 or 10 days of treatment. Population pharmacokinetic model based on Hoglund et al, Malaria Journal (2016). Red dashed line indicates a putative in vitro IC50-value for SARS-CoV-2 (1.13 µM; Wang et al, Cell Research (2020)).

Simulated blood concentration time profiles of hydroxychloroquine (n=1,000). Loading dose of 4 tablets of hydroxychloroquine sulfate (200 mg salt, equivalent to 155 mg base per tablet) at time 0 and 6 hours, followed by a maintenance dose of 2 tablets of hydroxychloroquine sulfate at time 12 hours, and then twice daily for a total of 7 or 10 days of treatment. Population pharmacokinetic model based on Rosenfeld et al, Autophagy (2014). Red dashed line indicates a putative in vitro IC50-value for SARS-CoV-2 (0.72 µM; Yao et al CID (2020)).

0 10 20 30 400.1

1

10

Time (days)

Hyd

roxy

chlo

roqu

ine

(µM

)

0 1 2 3 4 5 6 7 8 9 100.1

1

10

Time (days)

Hyd

roxy

chlo

roqu

ine

(µM

)

0 10 20 30 400.1

1

10

Time (days)

Hyd

roxy

chlo

roqu

ine

(µM

)

0 1 2 3 4 5 6 7 8 9 10 11 12 130.1

1

10

Time (days)

Hyd

roxy

chlo

roqu

ine

(µM

)

7 days of hydroxychloroquine 7 days of hydroxychloroquine

10 days of hydroxychloroquine 10 days of hydroxychloroquine

Putative IC50

Putative IC50

Putative IC50

Putative IC50

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