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\begin{document}

\title{Dosage prediction of chloroquine and ritonavir-boosted lopinavir for
COVID-19 treatment: A physiologically-based pharmacokinetic (PBPK)
modelling}



\author[1]{Teerachat Saeheng}%
\author[2]{Kesara Na-Bangchang}%
\author[3]{Rajith Rajoli}%
\author[3]{Marco Siccardi}%
\author[4]{Juntra Karbwang}%
\affil[1]{Nagasaki University School of Medicine Graduate School of Biomedical Sciences}%
\affil[2]{Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma, Chulabhorn International College, Thammasat University}%
\affil[3]{University of Liverpool Institute of Translational Medicine}%
\affil[4]{NEKKEN}%


\vspace{-1em}



  
  \date{May 19, 2024}


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\selectlanguage{english}
\begin{abstract}
Abstract Background and Purpose: The 2019 novel coronavirus (COVID-19)
has been spread out since December 2019 from China to 29 countries. No
effective treatment is currently available, although the combination
regimen of the antiretroviral drugs-- lopinavir/ritonavir (LPV/r), with
other antiviral drugs have been using, but the evidences are limited. A
recent in vitro study showed that chloroquine could inhibit COVID-19 to
cells, and enhance antiviral efficacy. This study aimed to predict the
optimal dose regimens of LPV/r, and chloroquine in combination as a
potential treatment of COVID-19 infection, using the
physiologically-based pharmacokinetic (PBPK) modelling. Experimental
approach: The whole PBPK models were constructed. The predicted plasma
drug concentrations were compared with the published clinical data. The
validated models were used to predict optimal dosage regimens of LPV/r,
and chloroquine co-administration. The optimal dose regimen was
determined based on the efficacy, and toxicity reported in the published
data. Key Results: The average errors of the predicted values were
within 30\% of the observed data. The proposed optimal dosage regimen is
the once-daily dose of 800/200 mg LPV/r co-administered with chloroquine
at a loading dose of 1,000 mg, followed by twice-daily dose of 500 mg
for 8 doses on the second day, and the twice-daily dose of 400 mg for 18
doses. Conclusion and Implications: PBPK modelling successfully
predicted pharmacokinetic profiles within an acceptable range of errors.
The study provides a focus for clinical studies to confirm the efficacy
of the proposed dosage regimen as a novel treatment for COVID-19
infection.%
\end{abstract}%



\sloppy


\textbf{1. Introduction}

The 2019 novel coronavirus (COVID-19) is becoming a serious global
health catastrophe with 80,248 confirmed cases, 27,754 recovered cases,
and 2,706 deaths reported as of February 25, 2020 (Wolrdometer, 2020).
Currently, there remain 49,788 infected patients, of which 40,573 cases
are with mild symptoms, and 9,215 cases are those with serious
conditions (Wolrdometer, 2020). Since the COVID-19 is a novel
coronavirus, no specific effective treatment is currently available.
Available drug regimens that can potentially represent therapeutic
options for infected patients are the combination of antiretroviral
drugs (lopinavir/ritonavir) as well as other antiviral medications
(ganciclovir, and oseltamivir)(Chen et al., 2020; Huang et al., 2020).
The use of lopinavir/ritonavir (LPV/r) for the treatment of COVID-19 was
based on its efficacy against SARS-CoV(Chu et al., 2004), and the
similarity between the COVID-19 genome with the human SARS-CoV(Zhu et
al., 2020). However, the evidence to support the efficacy, and optimal
dosage regimen of these drugs is limited. Recent data suggest that the
recovery rate of currently used drug combination is less than 20\% (Chen
et al., 2020; Huang et al., 2020).There is an urgent need to look for
other treatment regimens to broad the therapeutic options for COVID-19
to control the emerging epidemic. A recent \emph{in vitro study} showed
that chloroquine (an antimalarial) could inhibit COVID-19 entry, and
post-entry to the infected cells, and enhance antiretroviral drug
efficacy(Wang et al., 2020). Furthermore, chloroquine has been shown to
moderate host immunity, supporting its antiretroviral effect (Wang et
al., 2020). The combination of LPV/r, and chloroquine could represent a
promising drug regimen to treat COVID-19. However, the optimal dosage
regimen(s) of chloroquine in combination with LPV/r that would result in
adequate plasma chloroquine concentrations has not been identified.
Besides, the co-administration of chloroquine with ritonavir may pose
the patients at risk of chloroquine toxicity (\emph{e.g} ., retinopathy)
due to the inhibitory effect of ritonavir on chloroquine metabolism
(Projean et al., 2003; Yeh et al., 2006).

Physiologically-based pharmacokinetic (PBPK) modelling has been used
successfully for dose optimization in various diseases (Sager, Yu,
Ragueneau-Majlessi, \& Isoherranen, 2015). PBPK modelling is accepted by
regulatory agencies including the United States Food and Drug
Administration (US FDA), and the European Medicine Agencies (EMA) to be
applied as a tool to support drug regulatory submission a prediction of
drug-drug interactions (DDIs) (Shebley et al.,
2018).\textsuperscript{10} The present study aimed to predict optimal
dosage regimens for the co-administration of LPV/r, and chloroquine for
the treatment of COVID-19 using PBPK modelling.

\textbf{2. Methods}

\textbf{2.1 Model construction}

The whole PBPK models for LPV/r, and chloroquine were constructed based
on the previously published articles (Saeheng, Na-Bangchang, Siccardi,
Rajoli, \& Karbwang, 2019; Siccardi, 2015) using Simbiology\selectlanguage{ngerman}® (version
5.8.2), the product of MATLAB® (version 2019a) (MathWorks, Natick, MA,
USA). The physicochemical, and biochemical properties (model parameters)
of each drug were collected from the published articles (Table S1)
(Ernest, Hall, \& Jones, 2005; Koudriakova et al., 1998; Olafuyi \&
Badhan, 2019; Patel, Mandava, Gokulgandhi, Pal, \& Mitra, 2014; Saeheng
et al., 2019; Wagner et al., 2017; Xu, Vela, Shi, Marroum, \& Gao, 2017;
Zhang et al., 2012). Model assumptions included blood-flow limited
model, immediate drug dissolution, absence of drug absorption in the
stomach and large intestine, and absence of enterohepatic recirculation.
The hepatic fraction of metabolism by cytochrome P450 (CYP) 3A4
(f\textsubscript{m, CYP3A4}), and the fraction of metabolism by CYP2D6
(f\textsubscript{m, CYP2D6}) were assumed to be 0.61, and 0.39,
respectively (Projean et al., 2003).

\textbf{2.2 Model verification}

Four published clinical articles were used for model validation (Eron et
al., 2004; Hsu et al., 1997; Mzayek et al., 2007; Na-Bangchang,
Limpaibul, Thanavibul, Tan-Ariya, \& Karbwang, 1994). The absolute
average-folding errors (AAFEs) (the comparison between the predicted
results against clinical published data) were calculated to determine
model accuracy. AAFEs value below 2 is considered as acceptable
following recent publication (Saeheng, Na-Bangchang, \& Karbwang, 2018).
AAFEs can be calculated using the following equation:

\begin{equation}
\text{AAFEs}=10^{\frac{\sum_{i=1}^{n}\left|\log\frac{\text{prediction}}{\text{observation}}\right|}{n}}\nonumber \\
 \end{equation}

Where n is the number of samples; the prediction and observation are
simulated results, and observed clinical data, respectively.

\textbf{2.3 Sensitivity analysis}

The sensitivity analysis (sensitivity coefficient) was performed to
determine the effect of model parameters on the area under the curve
(AUC) following the standard regimen of chloroquine (once-daily dose of
250 mg for 14 days). The model parameters for sensitivity analysis were
absorption rate (K\textsubscript{a}), fraction of unbound drug
(f\textsubscript{u}), liver weight, blood-to-plasma ratio
(R\textsubscript{bp}), f\textsubscript{m, CYP3A4}, and
f\textsubscript{m, CYP2D6}. The mathematical equation is as follow:

Sensitivity coefficient = \%Y/\%X

Where \%Y, and \%X is the percent change of the
AUC\textsubscript{336--360 h}, and the percent change of model
parameter, respectively.

\textbf{2.4 Single drug simulation}

One hundred virtual population (100 males, aged 30 to 60 years in the
fasting state) were simulated. The period of simulation time was based
on the maximal admission time of patients in the hospital (14 days)
(Chen et al., 2020).The standard dose regimens of chloroquine for
rheumatoid arthritis treatment (once-daily dose of 250 mg) were
simulated (da Mota et al., 2013).\textsuperscript{19} In addition, the
twice-daily regimen of 400/100 mg LPV/r (based on the regimen used for
COVID-19 treatment) were simulated (Chen et al., 2020).

\textbf{2.5 Estimation of chloroquine toxicity based on the maximum
dosage used in clinical trials}

The dose of 20 mg/kg body weight/day (total dose of 1,200 mg/day for an
average 60 kg body weight) of a once-daily regimen of chloroquine for 6
consecutive weeks (Furst et al., 1999) used for simulations was based on
clinically published data. Maximum plasma concentration
(C\textsubscript{max}) of unbound chloroquine was used as the
pharmacokinetic parameter for chloroquine toxicity.

\textbf{2.6 Simulations of the plasma concentration-time profile of
chloroquine when co-administered with lopinavir (LPV/r)}

Simulation based on standard drug-drug interactions (DDIs) study
approach: multiple doses of the twice-daily regimen of LPV/r (400/100
mg) for 15 consecutive days with the co-administration of a single dose
of 250 mg chloroquine on day 15 were simulated.

Simulation based on actual clinical use approach: multiple doses of the
twice-daily regimen of LPV/r (400/100 mg) with the co-administration of
the once-daily regimen of 250 mg chloroquine given for 14 days were
simulated.

AUC ratios (the comparison of chloroquine AUC administration alone, and
the chloroquine AUC co-administered with LPV/r) were calculated to
determine the effect of LPV/r on chloroquine pharmacokinetics. Dosage
regimens of LPV/r for simulation (total=14 consecutive days) were: (i) a
twice-daily regimen of 400/100 mg, and (ii) a once-daily regimen of
800/100 mg.

\textbf{2.7 Prediction of the optimal dose regimens for chloroquine and
lopinavir}

The optimal dose regimen of lopinavir was determined based on the
previously reported cytopathic effect on SARS-CoV, and maximum dose used
in the published clinical trial, \emph{i.e.,} unbound trough plasma
concentration (C\textsubscript{trough}) \textgreater{} 0\selectlanguage{ngerman}·04 mg
l\textsuperscript{-1} (Chu et al., 2004). The optimal dose regimen of
chloroquine was determined based on the reported 50\% effective
concentration of COVID-19 (EC\textsubscript{50}: 1.13 \selectlanguage{greek}µ\selectlanguage{english}M or 0.36 mg
l\textsuperscript{-1}) (Wang et al., 2020), or unbound
C\textsubscript{trough} at 24 hours, or 12 hours, or 8 hours
\textgreater{} 0.36 mg l\textsuperscript{-1}. The maximum doses of
chloroquine, and LPV/r used for simulations were 20 mg
kg\textsuperscript{-1} body weight day\textsuperscript{-1} (Furst et
al., 1999), and 800/200 mg day\textsuperscript{-1} (Eron et al., 2004),
respectively. The predicted pharmacokinetic parameters were reported as
mean \selectlanguage{ngerman}± standard deviation (SD).

\textbf{3. Results}

The overall AAFEs was 1.25 (ranged: 1.08--1.59) (Eron et al., 2004; Hsu
et al., 1997; Mzayek et al., 2007; Na-Bangchang et al., 1994). The AAFEs
for ritonavir, lopinavir (LPV/r), and chloroquine were 1.30 (Hsu et al.,
1997), 1.17 (Eron et al., 2004) and 1.27 (Mzayek et al., 2007;
Na-Bangchang et al., 1994), respectively. All AAFEs values were within
the accepted ranges (Table S2). Sensitivity coefficients for
K\textsubscript{a}, f\textsubscript{u}, R\textsubscript{bp},
f\textsubscript{m, CYP3A4}, and f\textsubscript{m, CYP2D6} were +0.13,
-0.90, +0.06, -0.50, and +1.34, respectively. All sensitivity
coefficients except for f\textsubscript{m,CYP2D6} were less than one,
indicating no significant effects of the model parameters on the AUC
values. Although f\textsubscript{m,CYP2D6} was greater than one, this
value was assumed based on the intrinsic clearance of chloroquine from
the \emph{vitro} studies.\textsuperscript{7} All parameters were within
the accepted ranges, indicating model reliability.

\textbf{3.1 Simulations of plasma concentration-time profiles of
chloroquine and lopinavir (LPV/r) when administered alone}

Chloroquine: The average unbound C\textsubscript{trough} at 24 hours
following the standard once-daily regimen of 250 mg was 0.06±0.01 mg
l\textsuperscript{-1}. This dose regimen could not provide drug
concentrations over the EC\textsubscript{50} level. Time to reach
EC\textsubscript{50} was 7 days. Subsequent simulation using the
once-daily dose of 500 mg resulted in the average values of unbound
C\textsubscript{trough} at 24 hours, and the time to reach
EC\textsubscript{50} of 0.13±0.02 mg l\textsuperscript{-1}, and 7 days,
respectively.

Lopinavir (LPV/r): The average unbound C\textsubscript{trough} at 24
hours following the standard twice-daily regimen of LPV/r (400/100 mg)
was 0.0341±0.008 mg l\textsuperscript{-1} (figure 1). Subsequent
simulation using the once-daily dose regimen of 800/100 mg resulted in
the average unbound C\textsubscript{trough} at 24 hours of 0.0468±0.0163
mg l\textsuperscript{-1} (figure 1).

\textbf{3.2 Estimation of chloroquine toxicity based on the maximum dose
regimen used in clinical trials}

The average unbound C\textsubscript{max} as the level of chloroquine
toxicity estimated based on the maximum dose regimen used in clinical
trials was 2.36±0.55 mg l\textsuperscript{-1}.

\textbf{3.3 Simulations of the plasma concentration-time profile of
chloroquine when co-administered with lopinavir (LPV/r)}

Simulation based on standard DDIs study approach: The recommended dosage
of chloroquine for rheumatoid arthritis treatment (250 mg once daily) in
combination with LPV/r (400/100 mg twice-daily dose) resulted in a
1.45-fold increase in the AUC for chloroquine indicating a significant
but weak DDI. The average unbound C\textsubscript{trough} at 24 hours,
and the average unbound C\textsubscript{max} were 0.10±0.01, and
0.30±0.05 mg l\textsuperscript{-1}, respectively.

Simulation based on actual clinical use approach: The recommended
once-daily dose of 250 mg chloroquine co-administered with LPV/r
(400/100 mg twice-daily dose) provided a 1.50-fold increase in the AUC
of chloroquine (AUCR=1.50). The average unbound
C\textsubscript{trough}at 24 hours, and the average unbound
C\textsubscript{max} were 0.11±0.03 mg l\textsuperscript{-1}, and
1.82±0.15 mg l\textsuperscript{-1}, respectively. The time to reach
EC\textsubscript{50} was 5 days. The weak DDI (AUCR \textgreater{} 1.25
to \textless{}2 fold) resulted in the lower average unbound
C\textsubscript{trough} at 24 hours than the EC\textsubscript{50}, and
the lower unbound C\textsubscript{max} than the toxicity level, and thus
significant dose reduction is probably not requiring. Subsequent
simulation of the once-daily regimen of 250 mg chloroquine
co-administered with LPV/r (800/100 mg once-daily dose) resulted in the
average unbound C\textsubscript{trough} at 24 hours, and unbound
C\textsubscript{max} of 0.11±0.02 mg l\textsuperscript{-1}, and
0.80±0.15 mg l\textsuperscript{-1}, respectively. A 500 mg once-daily
dose of chloroquine co-administered with LPV/r (400/100 mg once-daily
dose was simulated. The average unbound C\textsubscript{trough} at 24
hours, and unbound C\textsubscript{max} were 0.22±0.04 mg
l\textsuperscript{-1}, 1.62±0.27 mg l\textsuperscript{-1}, respectively.
Besides, the average unbound C\textsubscript{trough} at 24 hours, and
unbound C\textsubscript{max} following the once-daily regimen of 500 mg
chloroquine co-administered with LPV/r (800/100 mg once-daily dose) were
0.22±0.05 mg l\textsuperscript{-1}, and 1.62±0.30 mg
l\textsuperscript{-1}, respectively. None of the current clinically used
dosage regimens of chloroquine provided unbound
C\textsubscript{trough}above the EC\textsubscript{50} level.

Subsequent simulations were performed to identify the potential dose
regimen(s) of chloroquine that could result in chloroquine plasma
concentrations above the EC\textsubscript{50} level. Chloroquine at the
twice-daily regimen of 500 mg co-administered with 400/100 mg
twice-daily dose, or 800/200 mg once-daily dose LPV/r provided adequate
unbound C\textsubscript{max}, but insufficient unbound
C\textsubscript{trough} at 24 hours (figure 2 (regimen A), and 3
(regimen A)). Time to reach EC\textsubscript{50} for the 400/100 mg
twice-daily dose, and 800/100 mg once-daily dose LPV/r dose regimen was
48 hours. Chloroquine at the dose of 400 mg given three times a day in
combination with 400/100 mg twice-daily dose (figure 2 (regimen B)) or
800/200 mg once-daily dose LPV/r (figure 3 (regimen B)) provided toxic
level of unbound C\textsubscript{max}, and inadequate unbound
C\textsubscript{trough} at 8 hours. This regimen, however, reduced the
time to reach EC\textsubscript{50} to 24 hours. The pharmacokinetic
parameters of chloroquine following other dose regimens (\emph{i.e.} , a
once-daily dose of 1000 mg, a loading dose of 1,000 mg, followed by
twice-daily dose of 500 mg, and a loading dose of 1,000 mg, followed by
twice-daily dose of 500 mg for 8 doses, and twice-daily dose of 400 mg
for 18 doses) are shown in figure 2 (co-administered with 400/100 mg
LPV/r regimen C,D, E, respectively), and figure 3 (co-administered with
800/200 mg LPV/r regimen C, D, E, respectively). All three doses of
chloroquine regimens provided unbound C\textsubscript{trough} above the
EC\textsubscript{50} level within 24 hours.

\textbf{4. Discussion and conclusion}

Results of the current study suggest that PBPK modelling successfully
predicted plasma concentration-time profiles of chloroquine and
lopinavir (LPV/r) when given alone, or in combination. The results
suggest that there is a weak but potentially significant DDIs between
chloroquine, and LPV/r. Using PBPK modelling can guide the selection of
appropriate dose regimens in terms of both efficacy, and safety.
Simulations of several dosage regimens of chloroquine administration
alone, and when co-administered with lopinavir (LPV/r) offer potential
dose optimization strategies for the treatment regimen for COVID-19
infected patients.

\textbf{4.1 LPV/r alone for the treatment of COVID-19 infection}

The simulation of LPV/r at the dose of 400/100 mg twice-daily dose
(currently used to treat COVID-19) may not be an optimal dose regimen
due to insufficient unbound C\textsubscript{trough} at 24 hours (figure
1(regimen A)). The 800/200 mg LPV/r once-daily dose may be a better
option to ensure the unbound C\textsubscript{trough} at 24 hours above
0·04 mg l\textsuperscript{-1} (figure 1 (regimen
B)).\textsuperscript{4}This may explain the recovery rate of less than
20\% of the currently used 400/100 mg twice-daily dose LPV/r even when
used in combination with other antiretroviral drugs (Chen et al., 2020;
Huang et al., 2020). The successful treatment reported in some patients
(Chen et al., 2020) treated with 400/100 mg twice-daily dose LPV/r could
be explained by a relatively higher sensitivity of the COVID-19 to
lopinavir compared with SARS-CoV.

\textbf{4.2 Chloroquine alone for the treatment of COVID-19 infection}

The initial once-daily dose of 250 mg chloroquine provided adequate
plasma concentration-time profiles to achieve the therapeutic effect
(EC\textsubscript{50}) for COVID-19 on day 7. Nevertheless, the unbound
C\textsubscript{trough} at 24 hours was not sufficient to exert
inhibitory effect (EC\textsubscript{50}) on the virus. This regimen is,
therefore, not appropriate for the treatment of COVID-19 infection. This
finding is in accordance with that was reported by Wang and
colleagues\textsuperscript{6}, of which the EC\textsubscript{50} could
not be achieved with the drug concentration delivered following the
once-daily dose regimen of 250 mg chloroquine (for 28 days) in
rheumatoid patients. Simulation of chloroquine 500 mg once-daily dose
also did not provide adequate the unbound C\textsubscript{trough} at 24
hours to exert inhibitory effect (EC\textsubscript{50}) on the virus.
This finding is in contrast with Wang and colleagues' proposal that the
concentration achieved following the once-daily dose regimen of 500 mg
chloroquine could be sufficient to inhibit the entry of the virus into
host cells (Wang et al., 2020). This could be due to the fact that the
reported 500 mg-plasma concentration profiles were not actual observed
clinical data, but the extrapolation from the once-daily dose regimen of
250 mg in rheumatoid patients.

\textbf{4.3 Chloroquine and LPV/r combination for the treatment of
COVID-19 infection}

Based on the results of the simulation, co-administration of LPV/r
(400/100 mg twice-daily dose, or 800/200 mg once-daily dose), and
once-daily regimen of 250 mg, and 500 mg are not optimal due to
inadequate concentration-time profiles of chloroquine above the
EC\textsubscript{50} level (C\textsubscript{trough}). Increasing
chloroquine dose to reach EC\textsubscript{50} should offer better
clinical benefits in term of treatment efficacy. Based on the
simulations, only one chloroquine dosage regimen provided adequate
plasma drug concentrations for preventing viral entry, and below the
toxic levels \emph{i.e.,} a 1,000 mg loading dose, followed by the
twice-daily dose of 500 mg for 8 doses on the second day, and the
twice-daily dose of 400 mg for 18 doses, co-administered with once-daily
dose of 400/100 mg, and 800/200 mg LPV/r (figure 2 (regimen E), and
figure 3 (regimen E)). The regimen offered chloroquine unbound
C\textsubscript{trough} of higher than EC\textsubscript{50}. Although
plasma chloroquine concentrations following both regimens of LPV/r were
similar, the desirable dose of LPV/r should be 800/200 mg once-daily
dose to ensure sufficient cytopathic effect at 24 hours (Chu et al.,
2004). The results of this study provide a focus for further clinical
studies to confirm this proposed regimen.

Although other two regimens also provided adequate plasma
concentration-time profiles for COVID-19 infection, the unbound
C\textsubscript{max} was over toxic level (figure 2 (regimen C\&D), and
figure 3 (regimen C\&D)), therefore, is an inappropriate dosage.

The simulated once-daily dose regimen of chloroquine (\emph{i.e.,} 1000
mg once-daily dose) may be desirable when considering the reduction of
frequency of drug administration; this regimen however, provided unbound
C\textsubscript{max} of chloroquine over the toxic level (figure 3
(regimen C)).

Apart from efficacy, chloroquine-induced irreversible retinopathy is
also of great concern when used in combination with LPV/r. The reported
threshold dose of chloroquine-induced retinopathy was 5·1 mg/kg/day (300
mg for the average body weight of 60 kg) (Mackenzie, 1983). However, the
retinopathy from chloroquine is due to the accumulation of chloroquine
after long-term use for at least 5 years (Mackenzie, 1983). Based on the
previous clinical study, the high dose of chloroquine (20 mg
kg\textsuperscript{-1} body weight day\textsuperscript{-1}, or total
dose of 1,200 day\textsuperscript{-1} for the average body weight of 60
kg) was safe for short-term treatment (6 weeks) (Furst et al., 1999).
The chloroquine dose regimen used in our study is therefore, likely to
be safe as the total duration of chloroquine administration for COVID-19
treatment is only 14 days.

One of the limitations of this study was that the contributions of
P-glycoprotein transporter in the disposition of ritonavir, lopinavir,
and chloroquine were not included in the model construction.
Nevertheless, the inhibitory effect of ritonavir on P-glycoprotein may
have a less significant impact on chloroquine disposition as chloroquine
is a weak substrate of this transporter (Crowe, Ilett, Karunajeewa,
Batty, \& Davis, 2006).

In summary, the developed PBPK models successfully predicted a
potentially suitable regimen of chloroquine and LPV/r for COVID-19
infected patients. The proposed dosage regimen is the combination of the
once-daily dose of 800/200 mg LPV/r, in combination with chloroquine at
a loading dose of 1,000 mg (16.67 mg kg\textsuperscript{-1} body weight
day\textsuperscript{-1}), followed by twice-daily 500 mg for 8 doses on
the second day, and twice-daily 400 mg for 18 doses. The total duration
of treatment is 14 days. The simulated results in this study represent
an advanced framework to rationalize the selection of dose regimens to
fight against this COVID-19 infection outbreak. The study provides a
focus for further clinical studies to confirm the efficacy of the
proposed dosage regimen as a novel treatment for COVID-19 infection.

\textbf{ACKNOWLEDGEMENTS}

T.S. is a recipient of the Nagasaki University Special Research Student
Scholarship. J.K. is the recipient of Bualuang ASEAN Chair Professors
and Center of Excellence in Pharmacology and Molecular Biology of
Malaria and Cholangiocarcinoma.

\textbf{AUTHOR CONTRIBUTIONS}

J.K., K.N., and T.S. designed the study. M.S., R.K.R., and T.S.
collected the data. M.S., R.K.R., and T.S. performed research and
analysis. T.S. drafted the manuscript. M.S., and R.K.R. revised
manuscript. J.K., and K.N. were responsible for the critical revision of
the manuscript. All authors approved the final version.

\textbf{CONFLICT OF INTEREST}

All authors declare no competing interests.

\textbf{What is already known}

\begin{itemize}
\tightlist
\item
  Chloroquine exhibited anti-COVID-19 activity \emph{in vitro} study.
\item
  Lopinavir (ritonavir-boosted lopinavir or LPV/r) showed treatment
  efficacy for COVID-19.
\end{itemize}

\textbf{What this study adds}

\begin{itemize}
\tightlist
\item
  The proposed dosage regimen of LPV/r was 800/100 mg once-daily regimen
  for 14 consecutive days.
\item
  Chloroquine dose (mg) was 1000 loading dose, 500 twice-daily (8
  doses), 400 twice-daily (18 doses).
\end{itemize}

\textbf{What is the clinical significance}

This study provided an alternative promising dosage regimen to fight
against COVID-19 in current outbreak.

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\textbf{Figure legends}

Figure 1 The prediction of lopinavir (LPV/r) concentration-time profiles
given twice daily (LPV/r: 400/100 mg), or once daily (LPV/r: 800/200
mg). Data present as mean±SD.

Figure 2 The prediction of chloroquine concentration-time profiles in
combination with 400/100 mg LPV/r given twice daily for 14 consecutive
days. Data present as mean±SD.

Figure 3 The prediction of chloroquine concentration-time profiles in
combination with 800/200 mg LPV/r given once daily for 14 consecutive
days. Data present as mean±SD.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

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