key: cord-322503-fynprt6f
authors: Thakur, Aarzoo; Tan, Shawn Pei Feng; Chan, James Chun Yip
title: Physiologically‐Based Pharmacokinetic Modeling to Predict the Clinical Efficacy of the Coadministration of Lopinavir and Ritonavir against SARS‐CoV‐2
date: 2020-08-07
journal: Clin Pharmacol Ther
DOI: 10.1002/cpt.2014
sha: 
doc_id: 322503
cord_uid: fynprt6f

Lopinavir/ritonavir, originally developed for treating the human immunodeficiency virus (HIV), is currently undergoing clinical studies for treating the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Although recent reports suggest that lopinavir exhibits in vitro efficacy against SARS‐CoV‐2, it is a highly protein bound drug and it remains unknown if it reaches adequate in vivo unbound (free) concentrations in lung tissue. We built a physiologically‐based pharmacokinetic (PBPK) model of lopinavir/ritonavir in Caucasian and Chinese populations. Our aim was to perform pharmacokinetic/pharmacodynamic correlations by comparing simulated free plasma and lung concentration values achieved using different dosing regimens of lopinavir/ritonavir with EC(50,unbound) and EC(90,unbound) values of lopinavir against SARS‐CoV‐2. The model was validated against multiple observed clinical datasets for single and repeated dosing of lopinavir/ritonavir. Predicted pharmacokinetic parameters such as the maximum plasma concentration, area under the plasma concentration‐time profile, oral clearance, half‐life and minimum plasma concentration at steady state were within two‐fold of clinical values for both populations. Using the current lopinavir/ritonavir regimen of 400/100 mg twice daily, lopinavir does not achieve sufficient free lung concentrations for efficacy against SARS‐CoV‐2. Although the Chinese population reaches greater plasma and lung concentrations as compared to Caucasians, our simulations suggest that a significant dose increase from the current clinically used dosing regimen is necessary to reach the EC(50,unbound) value for both populations. Based on safety data, higher doses would likely lead to QT prolongation and gastrointestinal disorders (nausea, vomiting and diarrhea), thus, any dose adjustment must be carefully weighed alongside these safety concerns.

On December 31 st , 2019, China notified the World Health Organization (WHO) regarding multiple cases of pneumonia in Wuhan, China. Today, this pneumonia is known as coronavirus disease 2019 (COVID- 19) and has been found to be caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leading to over 11.1 million positive cases and 528,000 deaths worldwide as of 5 th July 2020. 1 Repurposing existing drugs with proven human safety profiles is expected to provide an expedited route to rapidly identify effective pharmacotherapy against SARS-CoV-2. One therapy currently of high interest is lopinavir/ritonavir, a protease inhibitor therapy initially approved for the treatment of human immunodeficiency virus (HIV). 2 This combination has attracted attention because of a long history of use, wide availability and evidence of efficacy against other coronaviruses such as MERS-CoV and SARS-CoV-1. 3, 4 Lopinavir/ritonavir was tested in clinical trials globally through the SOLIDARITY trial conducted by WHO and there are many instances of off-label, compassionate use by physicians to treat COVID-19. 5 Despite widespread use, there is little evidence available in the literature to support its anti-viral activity against SARS-CoV-2. In particular, Cao et al. reported that a recent randomized control trial on 199 critical patients with COVID-19 did not demonstrate significant clinical improvement in those receiving 400/100 mg lopinavir/ritonavir twice daily for 14 days over standard of care. 6 We hypothesized that one possible explanation for the lack of efficacy reported by Cao et al. could be insufficient unbound (free) concentrations of lopinavir in lung tissue (C u,lung ) achieved by the dosing regimen used in the study.

To address this possibility, we utilized physiologically-based pharmacokinetic (PBPK) modeling to simulate the unbound lung concentration of lopinavir achieved by 400/100 mg twice daily dose of lopinavir/ritonavir in both Caucasians and Chinese populations. Lopinavir (victim drug) exhibits complex drug-drug interactions with ritonavir (perpetrator drug), with cytochrome P450 (CYP450) mediated mechanism-based inactivation (MBI) dominating initially, followed by induction. 7 While a minimal PBPK model was previously reported for lopinavir/ritonavir, 8 it did not recapitulate the pharmacokinetic (PK) profile of the single dose administration of 400/100 mg lopinavir/ritonavir, as the model was optimized for a repeated dosing regimen. Furthermore, the use of an empirical additional clearance component for the elimination of ritonavir, which in turn exerts a major influence on the systemic exposure of lopinavir, limited its mechanistic extrapolation to other populations (e.g. ethnic Han Chinese) with lower CYP450 enzyme expression levels. 9, 10 Separately, a population PK model by Aspiroz et al. 11 

This article is protected by copyright. All rights reserved lopinavir clearance in order to simulate the effects of simultaneous inactivation and induction on steady state levels. This top-down approach would similarly limit mechanistic extrapolation to other populations.

Using over 40 sets of in vitro data, we built and rigorously validated a middle-out, full PBPK model for lopinavir alone, and lopinavir/ritonavir using the Simcyp ® Simulator. The model was used to predict C u,lung derived from human lung tissue-to-plasma partition coefficient (K p,lung ) values. As lopinavir is highly protein bound, the free fraction of lopinavir available to interact with pharmacological targets in human tissue and plasma is low. 13 Such considerations influence the free fraction of lopinavir in in vitro assays, which in turn affects potency measurements. 14, 15 Therefore, we derived unbound EC 50 (EC 50,unbound ) values against SARS-CoV-2 from various literature reports and compared it against the predicted C u,lung values to determine if clinically used doses of 400/100 mg twice a day would reach efficacious lung concentrations in Caucasian and Chinese populations. 16, 17 Subsequently, we predicted the optimal dosing regimens required to reach the efficacious lung concentrations in both populations.

This article is protected by copyright. All rights reserved

Middle-out PBPK models of lopinavir and ritonavir were built using population-based Simcyp Simulator version 19 (Certara, Simcyp Division, Sheffield, UK). In particular, a perfusion-limited full PBPK distribution model was implemented to permit prediction of tissue concentrations. For lopinavir, tissue K p values were predicted using the corrected Poulin and Thiel approach (method 1), except for human K p,lung which was calculated from experimental values of unbound lopinavir fraction in lung (f u,lung ) and plasma (f u,p ). 18 The hepatic metabolism of lopinavir by CYP3A4 was accounted for using microsomal kinetic parameters, 2 while the maximal inactivation rate constant (K inact ) for MBI of CYP3A4 by lopinavir was optimized according to Wagner et al. 8 For ritonavir, extensive metabolic kinetic parameters from recombinant CYP enzymes were coupled with intersystem extrapolation factors and incorporated into the model to account for its hepatic metabolism. 19, 20 Subsequently, lopinavir (substrate) and ritonavir (inhibitor) models were linked.

Experimental values for MBI and induction of CYP3A4 by ritonavir were used to define the complex time-dependent inhibition and induction of lopinavir metabolism by ritonavir 21 . Finally, an active hepatic scalar was empirically fitted to achieve an optimal induction of LPV metabolism. All the drug specific parameters of lopinavir and ritonavir are listed in Table S1 .

Simulations were performed for an oral administration of single dose of lopinavir 400 mg, single dose of lopinavir/ritonavir 400/100 mg and repeated dosing of 400/100 mg twice daily regimen for Caucasian and Chinese populations using the Simcyp Healthy Volunteers and Chinese Healthy Volunteers population database, respectively. Simulated results were visually inspected by overlaying clinical plasma concentration-time profiles (extracted using WebPlotDigitizer, San Francisco, California, USA) with model predictions. 2, [22] [23] [24] [25] [26] [27] [28] Thereafter, the PBPK models were validated using a two-fold criterion to compare the clinically observed and model predicted PK parameters such as the maximum plasma concentration (C max ), area under the plasma concentration-time profile (AUC 0-t ), time needed to reach C max (T max ), oral clearance (CL/F), halflife (t 1/2 ) and minimum plasma concentration at steady state (C min ).

Once the PBPK model was validated, it was first used to assess lopinavir's efficacy against HIV by comparing its unbound concentrations in white blood cells (C u,WBC ) with EC 50,unbound value against HIV-infected lymphocytes. This was done to verify that the current PBPK model is able to recapitulate the clinical efficacy of lopinavir against HIV. Likewise, the pharmacodynamic (PD)

This article is protected by copyright. All rights reserved assessment against SARS-CoV-2 was performed by predicting if lopinavir reaches therapeutic concentrations in the lung. C u,lung was compared with the EC 50,unbound and EC 90,unbound values of lopinavir against SARS-CoV-2-infected Vero E6/TMPRSS2 cells, respectively. 16, 17 Since lopinavir is a poor substrate of uptake or efflux transporters and its cellular entry occurs passively, 29, 30 it follows the free drug hypothesis. Accordingly, at steady state, free concentration of lopinavir is same on both sides of any biomembrane. 31 Therefore, C u,WBC and C u,lung were calculated using equation 1:

where, C u,tissue represents the unbound tissue concentration; C plasma and C tissue are the total plasma and tissue concentrations, respectively; and f u,tissue is the unbound fraction in tissues. As the Simcyp model does not possess a WBC compartment, C u,WBC was assumed to be the same as C u,plasma . C u,lung was calculated based on simulated total lung concentrations (C lung ) and experimental f u,lung measurements.

The EC 50,unbound value against HIV was obtained from literature. 15 17 Yamamoto et al. used 5% FCS in their culture media. 16 The model is defined by equation 2,

where, K a is the association constant, f u,prot represents the fraction of unoccupied protein sites and P t is the total protein concentration. Keeping the product of K a and f u,prot constant, equation 2 was analyzed using Graphpad Prism 8 (GraphPad Software, La Jolla, CA, USA) to determine the f u,media in 2% FCS. The impact of protein binding on PK/PD assessments were then assessed by comparing the predicted total and unbound lung concentrations of 400/100 mg twice daily lopinavir/ritonavir with EC 50 and EC 50,unbound values of lopinavir against SARS-CoV-2 respectively.

In both Caucasian and Chinese populations, simulations were performed with gradually increasing combinations of loading and maintenance doses in order to reach C u,lung values

This article is protected by copyright. All rights reserved comparable with EC 50,unbound and EC 90,unbound values of lopinavir against SARS-CoV-2. The optimal dose was determined when the steady-state unbound lung C max reached the EC 50,unbound value.

This article is protected by copyright. All rights reserved

Simulated plasma concentration-time profiles for single and repeated twice daily doses of 400/100 mg lopinavir/ritonavir in Caucasian and Chinese populations are shown in Figure 1A -C.

Clinically observed and model predicted PK parameters were compared and the fold difference values were within the two-fold criterion ( 

The predicted lopinavir C u,WBC after twice daily dosing of 400/100 mg lopinavir/ritonavir in both Caucasian and Chinese populations were 100-fold higher than the EC 50,unbound value of lopinavir against HIV (0.00069 ± 0.00006 µg/mL). 15 The concentration-time profiles along with EC 50,unbound values are shown in Figure 1D and E. The f u,media for lopinavir in 2% FCS was determined to be 0.355, which yielded an EC 50,unbound value of 0.386 µg/mL and EC 90,unbound value of 0.806 µg/mL against SARS-CoV-2 from Ohashi et al., 17 while the corresponding f u,media in 5% FCS was reported by Hickman et al. as 0.200, which yielded an EC 50,unbound value of 0.721 µg/mL from Yamamoto et al. 16 Using the same dosing regimen, a comparison of the C u,lung with both EC 50,unbound and EC 90,unbound values of lopinavir against SARS-CoV-2 showed that insufficient unbound lopinavir concentrations were achieved in lung tissue for both Caucasian (unbound lung C max = 0.130 µg/mL) and Chinese populations (unbound lung C max = 0.200 µg/mL) (Figure 2A and B).

As protein binding has a major impact on both unbound tissue concentrations and in vitro EC 50,unbound values, we compared the juxtaposition of both total and unbound values at steady state after twice daily dose of 400/100 mg lopinavir/ritonavir in Caucasian populations (Fig 2C) .

The total lung C min ( 

This article is protected by copyright. All rights reserved values. C u,lung (a function of both plasma and tissue binding) was lower than the EC 50,unbound value of lopinavir (a function of binding to protein within the incubation media).

Different combinations of loading and maintenance doses of lopinavir/ritonavir were trialed to simulate C u,lung values, beginning with the 400/100 mg twice daily regimen currently tested in clinical trials ( Figure 2D ). The predicted unbound lung PK parameters are listed in 

This article is protected by copyright. All rights reserved Lopinavir is a perfusion-limited drug and is neither dependent on transporters for pulmonary entry nor undergoes elimination in the lung. Additionally, it is a neutral compound and does not accumulate in the tissues due to pH-driven partitioning. Therefore, the unbound plasma concentrations can be regarded to be equivalent to unbound tissue concentrations. Based on these observations, we simulated the unbound concentrations of lopinavir in WBCs and lung tissue and juxtaposed these simulations against EC 50,unbound values for inhibition of HIV and

This article is protected by copyright. All rights reserved measurements. 35, 36 Current evidence indicates that SARS-CoV-2 utilises the TMPRSS2 protease for viral spike protein priming, which is necessary for SARS-CoV-2 viral entry. 37 Furthermore, it has been found that the SARS-CoV-2 RNA copies in the Vero E6/TMPRSS2 cell culture supernatants were >100 times greater than those from Vero E6 cells. 38 Thus, the Vero E6/TMPRSS2 cell system is more robust, sensitive and well-suited for performing in vitro efficacy measurements of pharmaceutical compounds against SARS-CoV-2, and here we utilized the EC 50 values obtained from such cell systems.

Understanding the free/unbound drug concentration at the target site is critical as it is the free drug that exerts its pharmacological action. 31 In Caucasian populations, comparison of the predicted total and unbound lung concentration of lopinavir (on administration of twice daily 400/100 mg lopinavir/ritonavir) with its EC 50 and EC 50,unbound against SARS-CoV-2 yield contrasting results. Even though the total lung concentration was greater than the EC 50 , when in vivo and in vitro protein binding were used to correct the corresponding lopinavir concentrations, the opposite trend is observed; where C u,lung is now lower than EC 50,unbound , indicating a lack of efficacy. This highlights the importance of accounting for the free fraction of a drug, both in vivo and in vitro, in performing PK/PD correlation and assessing efficacy particularly for highly protein bound drugs.

Our simulations show that none of the EC 50,unbound values are reached by the C u,lung when 400/100 mg twice daily dosing is administered to either Caucasian or Chinese populations.

This article is protected by copyright. All rights reserved Therefore, it is plausible that the failure of a 400/100 mg twice daily regimen for COVID-19

reported by Cao et al. 6 could be due to insufficient free lung concentrations. During the writing of this manuscript, the WHO announced their decision to discontinue the lopinavir/ritonavir treatment arm in hospitalised COVID-19 patients taking part in the SOLIDARITY trials as well. 39 A dose adjustment may therefore be necessary to increase free lung concentrations of lopinavir.

Our were nausea, vomiting and diarrhoea. 6 Moreover, SARS-CoV-2 can also adversely affect the heart, either directly by infecting cardiac cells and/or indirectly by inducing a cytokine storm. 42

Finally, the elevated doses are highly likely to result in GI adverse effects which may be doselimiting. Therefore, in the absence of efficacy at clinical doses and the presence of safety concerns at higher doses, the utility of lopinavir/ritonavir to treat COVID-19 should be revisited.

Our study has several limitations. Firstly, the f u,lung value used to predict K p,lung was determined using tissue homogenate and may not describe the intracellular drug binding accurately. 18 

This article is protected by copyright. All rights reserved STUDY HIGHLIGHTS (150 words)

Recent reports indicate that lopinavir inhibits the in vitro replication of SARS-CoV-2 but it is uncertain if lopinavir exhibits clinical efficacy. We developed a physiologically-based pharmacokinetic (PBPK) model to predict unbound lung tissue concentrations of lopinavir when co-administered with ritonavir.

We aimed to determine (1) if the recommended dose of 400/100 mg of lopinavir/ritonavir twice daily reaches effective unbound lung concentrations necessary for inhibition of SARS-CoV-2; (2) the optimal dosing regimens required in both Caucasian and Chinese populations; and (3) whether this optimal dosing regimen is clinically safe.

The recommended dose for lopinavir/ritonavir does not reach therapeutic unbound lung concentrations against SARS-CoV-2. A significantly larger loading and maintenance dose is necessary but may pose safety concerns.

Our study exemplifies the utility of PBPK modeling to predict tissue concentrations for building pharmacokinetic/pharmacodynamic (PK/PD) correlations of drugs and the need to account for the free fraction of drugs when making PK/PD assessments. 

1. Table S1 Accepted Article

This article is protected by copyright. All rights reserved

This article is protected by copyright. All rights reserved 

This article is protected by copyright. All rights reserved 

This article is protected by copyright. All rights reserved 

World Health Organization Coronavirus disease

Centre for Drug Evaluation and Research FDA Kaletra Clinical Pharmacology and Biopharmaceutical Review

Role of lopinavir/ritonavir in the treatment of SARS: Initial virological and clinical findings

Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture

Solidarity" clinical trial for COVID-19 treatments

A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19

Complex drug interactions of HIV protease inhibitors 2: In vivo induction and in vitro to in vivo correlation of induction of cytochrome P450 1A2, 2B6, and 2C9 by ritonavir or nelfinavir

Physiologically Based Pharmacokinetic Modeling for Predicting the Effect of Intrinsic and Extrinsic Factors on Darunavir or Lopinavir Exposure Coadministered With Ritonavir

Differences in cytochrome P450-mediated pharmacokinetics between Chinese and Caucasian populations predicted by mechanistic physiologically based pharmacokinetic modelling

A Review of Clinical Pharmacokinetic and Pharmacodynamic Profiles of Select Antiretrovirals: Focus on Differences among Chinese Patients

Population pharmacokinetics of lopinavir/ritonavir (Kaletra) in HIVinfected patients

Dosing will be a key success factor in repurposing antivirals for COVID-19

Metabolism and disposition of the HIV-1 protease inhibitor lopinavir (ABT-378) given in combination with ritonavir in rats, dogs, and humans

ABT-378, a highly potent inhibitor of the human immunodeficiency virus protease

Estimation of serum-free 50-percent inhibitory concentrations for human immunodeficiency virus protease inhibitors lopinavir and ritonavir

Shutoku Matsuyama; Tyuji Hoshino; Naoki Yamamoto Nelfinavir inhibits replication of severe acute respiratory syndrome coronavirus 2 in vitro

Multidrug treatment with nelfinavir and cepharanthine against COVID-19

Evaluation of Fraction Unbound Across 7 Tissues of 5 Species

Cytochrome P450-mediated metabolism of the HIV-1 protease inhibitor ritonavir (ABT-538) in human liver microsomes

Investigating the contribution of CYP2J2 to ritonavir metabolism in vitro and in vivo

Time-Dependent Interaction of Ritonavir in Chronic Use: The Power Balance Between Inhibition and Induction of P-Glycoprotein and Cytochrome P450 3A

Pharmacokinetics of Adjusted-Dose Lopinavir-Ritonavir Combined with Rifampin in Healthy Volunteers

Steady-state pharmacokinetics of etravirine and lopinavir/ritonavir melt extrusion formulation, alone and in combination, in healthy HIV-negative volunteers

Pharmacokinetics of plasma lopinavir/ritonavir following the administration of 400/100 mg, 200/150 mg and 200/50 mg twice daily in HIV-negative volunteers

Evaluation of pharmacokinetic interactions between long-acting HIV-1 fusion inhibitor albuvirtide and lopinavir/ritonavir, in HIV-infected subjects, combined with clinical study and simulation results

Echinacea purpurea significantly induces cytochrome P450 3A activity but does not alter Lopinavir-Ritonavir exposure in healthy subjects

Once-Daily versus Twice-Daily Lopinavir/Ritonavir in Antiretroviral-Naive HIV-Positive Patients: A 48-Week Randomized Clinical Trial

Pharmacokinetics of two generic co-formulations of lopinavir/ritonavir for HIV-infected children: A pilot study of paediatric Lopimune versus the branded product in healthy adult volunteers

Protease inhibitors atazanavir, lopinavir and ritonavir are potent blockers, but poor substrates, of ABC transporters in a broad panel of ABC transporteroverexpressing cell lines

Interaction between HIV protease inhibitors (PIs) and hepatic transporters in sandwich cultured human hepatocytes: implication for PI-based DDIs

The effect of plasma protein binding on in vivo efficacy: misconceptions in drug discovery

Measurement of antiretroviral drugs in the lungs of HIV-infected patients

Detection of intrapulmonary concentration of lopinavir in an HIV-infected patient

Intracellular drug concentrations and transporters: Measurement, modeling, and implications for the liver

Identification of antiviral drug candidates against SARS-CoV-2 from FDAapproved drugs

Remdesivir, lopinavir, emetine, and homoharringtonine inhibit SARS-CoV-2 replication in vitro

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

World Health Organization (WHO) WHO discontinues hydroxychloroquine and

The authors thank Drs Venkateshan Srirangam Prativadibhayankara and Andrea Kwa for helpful discussions on the clinical pharmacology of lopinavir/ritonavir. 

This article is protected by copyright. All rights reserved

This article is protected by copyright. All rights reserved