A systematic review of lopinavir therapy for SARS coronavirus and MERS coronavirus—A possible reference for coronavirus disease‐19 treatment option

Abstract In the past few decades, coronaviruses have risen as a global threat to public health. Currently, the outbreak of coronavirus disease‐19 (COVID‐19) from Wuhan caused a worldwide panic. There are no specific antiviral therapies for COVID‐19. However, there are agents that were used during the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) epidemics. We could learn from SARS and MERS. Lopinavir (LPV) is an effective agent that inhibits the protease activity of coronavirus. In this review, we discuss the literature on the efficacy of LPV in vitro and in vivo, especially in patients with SARS and MERS, so that we might clarify the potential for the use of LPV in patients with COVID‐19.

Currently, there is no specific antiviral treatment for COVID-19.
Therefore, identifying drug treatment options as soon as possible is critical for the response to the COVID-19 outbreak.

SARS-CoV, MERS-CoV, and COVID-19 belong to the same genera of
CoV and all are beta-CoV. COVID-19 shares 79.5% sequence identity with SARS-CoV. 4 Therefore, the existing treatment LPV for SARS and MERS may be helpful for developing COVID-19 therapeutics.
Proteinase is a key enzyme in CoV polyprotein processing. In recent years, research on SARS-CoV and MERS-CoV protease inhibitors has been carried out in vitro and in vivo. Lopinavir (LPV) is a proteinase inhibitor. Both peak (9.6 µg/mL) and trough (5.5 µg/mL) serum concentrations of LPV inhibit SARS-CoV. 5 LPV also blocks a post-entry step in the MERS-CoV replication cycle. 6 Ritonavir (RTV) inhibits the CYP3A-mediated metabolism of LPV, thereby increasing the serum concentration of LPV.
The antiviral activity of LPV/r is similar to that of LPV alone, suggesting that the effect is largely driven by LPV. 7,8 In this review, we analyze the efficacy of LPV or LPV/r in patients with SARS-CoV and MERS-CoV, which can be a useful reference for COVID-19 treatment option.

| In vitro studies of SARS
An analysis of molecular dynamics simulations showed that the SARS-CoV 3CLpro enzyme could be inhibited by the combination of lopinavir and ritonavir. 9 A binding analysis of the main SARS coronavirus proteinase with LPV showed that half of lopinavir is left outside the catalytic site, and the efficacy of lopinavir may be poor. 10 Another study showed that neither lopinavir nor ritonavir has an effect on the replication of SARS-CoV. 11 However, studies have revealed that lopinavir has antiviral activity. The 50% effective inhibitory concentration (EC 50 ) of LPV for the plaque reduction assay is 6 µg/mL in the Vero cell line. The selectivity index (SI) of LPV is 8 to 32. 12 In vitro activity against SARS-CoV has been demonstrated for lopinavir at 4 µg/mL after 48 hours of incubation. Cytopathic inhibition has been achieved down to a concentration of lopinavir 1 µg/mL combined with ribavirin at 6.25 µg/mL and data suggested that this combination may be synergistic against SARS-CoV in vivo. 13

| Animal studies of SARS
There have been some animal studies of SARS, 14 however, no study of lopinavir or ritonavir has been performed.

| In vitro studies of MERS
In an in vitro study, LPV inhibited MERS-CoV-induced cytopathic effect (CPE) with an EC 50 of 8.0 μM (SI = 3.1), and a maximal protective effect (89% inhibition) was observed at a dose of 12 μM. 6 However, an in vitro study showed that LPV was not effective. LPV showed a suboptimal EC 50 in the initial cytopathic effect inhibition assay and was therefore not evaluated further. 15 Another in vivo study of MERS showed that EC 50 values generated for lopinavir and ritonavir were 11.6 and 24.9 μM with CC 50 values > 50 μM, the SI for LPV and RTV was >4.3 and >2, respectively. 7 Compared with remdesivir and interferon-β (IFN-β), LPV has inferior in vitro antiviral activity. RTV does not significantly enhance the antiviral activity of LPV in vitro. 7

| Animal studies of MERS
For the MERS-CoV mouse model, prophylactic LPV/r combined with IFN-β slightly reduced the viral loads. 7 However, therapeutic LPV/r and IFN-β improved pulmonary function, but failed to reduce viral replication and lung hemorrhaging. This in vivo evidence is suggestive of the potential for LPV/r to treat MERS-CoV infections. When LPV/r was combined with IFN-β, the antiviral activity (EC 50 = 160 IU/mL) was indistinguishable from that of IFN-β alone (EC 50 = 175 IU/mL, P = .62). This suggests that the observed in vitro antiviral activity of the LPV/r-IFN-β combination against MERS-CoV is dominated by IFN-β when LPV/r is used at clinically relevant concentrations.
Chan et al 16 explored the therapeutic potential of LPV/r and/or IFN-β in common marmosets. Animals treated with LPV/r alone or in combination with interferonβ1b had better clinical scores, less weight reduction, and less pulmonary infiltrate than untreated animals. Furthermore, necropsied lung and extrapulmonary tissues from the treated group had lower mean viral loads than those from the control group.
The in vitro and animal studies of SARS and MERS are summarized in Table 1.

| SARS
In a preliminary report, there were no deaths at 30 days after the onset of symptoms among 34 patients treated with LPV/r (400 mg ritonavir and 100 mg lopinavir) in combination with ribavirin initially, compared to 10% mortality in 690 patients taking only ribavirin.
Twenty-one percent of 33 patients who received LPV/r as a rescue therapy died, whereas 42% of 77 patients who received ribavirin alone died. 17 However, these results were given only as a presentation, and no formal paper was published. Thus, this evidence is not credible.
A retrospective matched cohort study including 1052 SARS patients (75 treated patients and 977 control patients) showed that the addition of LPV/r as an initial treatment was associated with a reduced death rate (2.3%) and intubation rate (0%) compared with that in a matched cohort who received standard treatment (11.0% and 15.6%, respectively, P < .05). 18 In addition, the rate and dose of pulsed methylprednisolone were decreased. These SARS patients were retrospectively matched with control subject. Matching was performed with respect to age, sex, the presence of comorbidities, lactate dehydrogenase level, and the use of pulsed steroid therapy.
However, the mortality, oxygen desaturation, and intubation rates of the subgroup of patients who received lopinavir-ritonavir as rescue therapy were not different from those in the matched cohort and patients who received an increased dose of pulsed methylprednisolone. This result suggests that the combination of lopinavir and ribavirin has a synergistic effect for the treatment of SARS; it may play an essential role in the early phase of the infection. The viral replication phase peaks around day 10. 19 LPV/r use within this replication window decreases the peak viral load and the subsequent immune response.
Another retrospective matched cohort study of SARS patient also revealed that the rate of acute respiratory distress syndrome YAO ET AL.    Table 2.

| MERS
A MERS patient who received LPV/r, ribavirin, and interferon had a resolution of viremia after 2 days of treatment. 20 However, the patient eventually died from septic shock 2 months and 19 days after the initial diagnosis. Another 64-year-old MERS patient from Korea was also treated with LPV/r, ribavirin, and interferon. After 6 days of antiviral therapy, negative PCR result in the serum sample, sputum samples, and swab samples were achieved. 21 The patient was discharged on day 13 of admission after achieving complete recovery.
These two simple cases may show that LPV is effective against MERS.
However, they do not exclude the possibility of other combination therapies being effective or spontaneous improvement occurring.
The treatment effect of LPV/r against MERS is still controversial.
A retrospective study enrolled healthcare workers (HCWs) with high-risk exposure to MERS-CoV pre-isolation pneumonia and revealed that an effective post-exposure prophylaxis (PEP) strategy including LPV/r may limit the spread of infection. 22 Table 3.

| COVID-19
There are no reported in vitro studies of COVID-19. Four patients with COVID-19 were given antiviral treatment including LPV/r. After treatment, three patients showed significant improvement in pneumonia-associated symptoms, two of whom were confirmed to be COVID-19 negative and discharged, and one of whom was negative for the virus at the first test. 25 This study shows the positive effects of LPV/r therapy. Two reviews, including a Chinese review and communication showed that LPV may be drug treatment option for COVID-19. 26,27 However, a retrospective study enrolled 134 NCP patients revealed that there is no significant difference between LPV/r-treated group (n = 52), Abidol-treated group (n = 34), and control group (n = 48) in improving symptom or in reducing viral loads. 28 The negative rate of COVID-19 nucleic acid on the 7 day was 71.8%, 82.6%, and 77.1%, respectively (P = .79). The efficacy of LPV/r antiviral treatment warrants further verification in future studies.
Nine randomized controlled trials of LPV/r in patients with COVID- 19 have been registered in China up to February 22 (Table 4) Additional studies are needed to gain further insights into the origin, tropism, and pathogenesis of COVID-19.

ACKNOWLEDGMENTS
This study was supported by the Scientific Research Seed Fund of Peking University First Hospital (#2018SF049 to JD Qian) and the National Natural Science Foundation of China (#81870417 to YW).