Efficacy and Safety of Lopinavir/Ritonavir for Treatment of COVID-19: A Systematic Review and Meta-Analysis

(Background) Lopinavir-ritonavir (LPV/RTV) is a human immunodeficiency virus (HIV) antiviral combination that has been considered for the treatment of COVID-19 disease. (Aim) This systematic review aimed to assess the efficacy and safety of LPV/RTV in COVID-19 patients in the published research. (Methods) A protocol was developed based on the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) statement. Articles were selected for review from 8 electronic databases. This review evaluated the effects of LPV/RTV alone or in combination with standard care ± interferons/antiviral treatments compared to other therapies, regarding duration of hospital stay, risk of progressing to invasive mechanical, time to virological cure and body temperature normalization, cough relief, radiological progression, mortality and safety. (Results) A consensus was reached to select 32 articles for full-text screening; only 14 articles comprising 9036 patients were included in this study; and eight of these were included for meta-analysis. Most of these studies did not report positive clinical outcomes with LPV/RTV treatment. In terms of virological cure, three studies reported less time in days to achieve a virological cure for LPV/RTV arm relative to no antiviral treatment (−0.81 day; 95% confidence interval (CI), −4.44 to 2.81; p = 0.007, I2 = 80%). However, the overall effect was not significant (p = 0.66). When comparing the LPV/RTV arm to umifenovir arm, a favorable affect was observed for umifenovir arm, but not statically significant (p = 0.09). In terms of time to body normalization and cough relief, no favorable effects of LPV/RTV versus umifenovir were observed. The largest trials (RECOVERY and SOLIDARITY) have shown that LPV/RTV failed to reduce mortality, initiation of invasive mechanical ventilation or hospitalization duration. Adverse events were reported most frequently for LPV/RTV (n = 84) relative to other antivirals and no antiviral treatments. (Conclusions) This review did not reveal any significant advantage in efficacy of LPV/RTV for the treatment of COVID-19 over standard care, no antivirals or other antiviral treatments. This result might not reflect the actual evidence.


Exclusion Criteria
Types of articles that were excluded included duplicate articles, editorials, reviews, case reports, and letters to editors.
Any research articles that did not include data on lopinavir/ritonavir use, did not include control patients' group, or reported combined use of lopinavir/ritonavir with other antiviral medications were also excluded. Given the lack of clear benefit and potential for toxicity of hydroxychloroquine [21], studies with evidence on the benefit of LPV/RTV in combination with hydroxychloroquine use in hospitalized COVID-19 patients were excluded in our review.

Data Extraction and Analysis
Two reviewers (SA and MT) independently screened the titles with abstracts using the selection criteria. For relevant articles, full texts were obtained for further evaluation. Disagreements between the two reviewers after full text screening were reconciled via consensus by a third reviewer (AA) [22].
Inclusions and exclusions were recorded following PRISMA guidelines presented in the form of a PRISMA flow diagram and detailed reasons recorded for exclusion. Articles were categorized as clinical trials or cohort studies. The following data were extracted from the selected studies: authors; publication year; study location; study design and setting; sample size, age, and gender; details of study intervention and control therapies in addition to data on adverse events and treatment outcomes; time from symptom onset to treatment initiation; assessment of study risk of bias; and remarks on notable findings.

Risk of Biased Evaluation of Included Studies
The quality assessment of the studies was undertaken based on the revised Cochrane risk of bias tool (RoB 2.0) for randomized controlled studies [23]. The Risk of bias in non-randomized studies-of interventions (ROBINS-I) tool was used to assess non-randomized interventional studies [24], and the Newcastle Ottawa Scale for observational cohort studies [25]. Critical appraisal checklists appropriate to each study design were applied and checked by a third team member.
Three investigators (SA, MT, and AA) separately evaluated the possibility of bias using these tools. Publication bias was not evaluated by funnel plot as there were only three studies that were included in the meta-analysis part of the study.

Assessment of Heterogeneity
Statistical heterogeneity was evaluated using the χ 2 test and I 2 statistics [19]. An I 2 value of 0 to <40% was not considered as significant, 30% to 60% was regarded as moderate heterogeneity, 50% to 90% was considered substantial heterogeneity, and 75% to 100% was considered significant heterogeneity.

Statistical Analysis
Because all of the data were continuous and dichotomous data, either odds ratio (OR) or mean difference were used for estimating the point estimate, along with a 95% confidence interval (CI). In the absence of significant clinical heterogeneity, the meta-analysis using the Mantel Hazel method or inverse variance method for dichotomous data and continuous data were performed, respectively. Employing a conservative approach, a random effects model was used, which produces wider CIs than a fixed effect model. Review Manager (Version 5.3, Oxford, UK; The Cochrane Collaboration, 2014) was used to conduct all statistical analyses and generate forest plots.

Results
A total of 8 literature databases were screened and 76 non-duplicate articles were identified, which were evaluated for possible inclusion using titles and abstracts. Out of these, 32 articles were selected for full-text screening and finally, 14 articles (total participants = 9036) were included in the systematic review, and eight articles were included in the meta-analysis; 18 articles were excluded following full-text screening (reasons: review = 5, study with no relative data = 6, LPV/RTV use data not available = 2, no control patients in the study = 1, combined LPV/RTV use with other antiviral therapies/other medications data = 2, no extractable data = 2). The PRISMA chart for the studies included is displayed in Figure 1. The details of the included studies are depicted in Table 1. Among these, two articles were in preprint versions [26,27].
Trop. Med. Infect. Dis. 2020, 5, x FOR PEER REVIEW 5 of 29 systematic review, and eight articles were included in the meta-analysis; 18 articles were excluded following full-text screening (reasons: review = 5, study with no relative data = 6, LPV/RTV use data not available = 2, no control patients in the study = 1, combined LPV/RTV use with other antiviral therapies/other medications data = 2, no extractable data = 2). The PRISMA chart for the studies included is displayed in Figure 1. The details of the included studies are depicted in Table 1. Among these, two articles were in preprint versions [26,27].

A.2. LPV/RTV vs. No Antiviral Therapy (Conventional)
In terms of CT evidence for radiological progression of pneumonia/lung damage (n = 71 in the LPV/RTV arm vs. n = 75 in conventional arm), treatment with LPV/RTV resulted in no significant decrease in the radiological progression (OR = 0.69; 95% CI, 0.36 to 1.31; p = 0.42, I 2 = 0%; Figure 10) [27,32].   [15,28]. No significant association was observed between the two arms in terms of mortality at 28 days (OR = 1.00; 95% CI, 0.79 to 1.26; p = 0.28, I 2 = 15%; Figure 11). Two trials reported on mortality at 28 days (n = 1715 in LPV/RTV plus standard of care arm vs. n = 3524 in standard of care arm) [15,28]. No significant association was observed between the two arms in terms of mortality at 28 days (OR = 1.00; 95% CI, 0.79 to 1.26; p = 0.28, I 2 = 15%; Figure 11). Two large trials reported on death during treatment at any time (n = 3015 in LPV/RTV plus standard of care arm vs. n = 4796 in standard of care arm) [28,29]. No significant association was observed between the two arms in terms of death during treatment at any time (OR = 1.03; 95% CI, 0.93 to 1.14; p = 0.78, I 2 = 0%; Figure 12).  Two large trials reported on death during treatment at any time (n = 3015 in LPV/RTV plus standard of care arm vs. n = 4796 in standard of care arm) [28,29]. No significant association was observed between the two arms in terms of death during treatment at any time (OR = 1.03; 95% CI, 0.93 to 1.14; p = 0.78, I 2 = 0%; Figure 12). Two trials reported on mortality at 28 days (n = 1715 in LPV/RTV plus standard of care arm vs. n = 3524 in standard of care arm) [15,28]. No significant association was observed between the two arms in terms of mortality at 28 days (OR = 1.00; 95% CI, 0.79 to 1.26; p = 0.28, I 2 = 15%; Figure 11). Two large trials reported on death during treatment at any time (n = 3015 in LPV/RTV plus standard of care arm vs. n = 4796 in standard of care arm) [28,29]. No significant association was observed between the two arms in terms of death during treatment at any time (OR = 1.03; 95% CI, 0.93 to 1.14; p = 0.78, I 2 = 0%; Figure 12).

Comparison 2: Efficacy and Safety of LPV/RTV along in Combination with Other Agents versus No Antiviral Therapy (Conventional Therapy) or Control
A total of four studies evaluated the efficacy of LPV/RTV plus interferon (IFN) [30,31,35,37] and three studies [30,31,37] evaluated the safety of the combination. Other studies evaluated the efficacy of LPV/RTV plus standard care [15,28], ribavirin [31], or umifenovir [26,32,37], and evaluated the safety of these combinations.
The combination of LPV/RTV, in addition to standard care, or standard care alone exhibited no difference in the time to clinical improvement (hazard ratio for clinical improvement, 1.31; 95% CI, 0.95 to 1.80) with similar 28-day mortality (19.2% vs. 25.0%; difference, −5.8 percentage points; 95%

Comparison 2: Efficacy and Safety of LPV/RTV along in Combination with Other Agents versus No Antiviral Therapy (Conventional Therapy) or Control
A total of four studies evaluated the efficacy of LPV/RTV plus interferon (IFN) [30,31,35,37] and three studies [30,31,37] evaluated the safety of the combination. Other studies evaluated the efficacy of LPV/RTV plus standard care [15,28], ribavirin [31], or umifenovir [26,32,37], and evaluated the safety of these combinations.
The combination of LPV/RTV, in addition to standard care, or standard care alone exhibited no difference in the time to clinical improvement (hazard ratio for clinical improvement, 1.31; 95% CI, 0.95 to 1.80) with similar 28-day mortality (19.2% vs. 25.0%; difference, −5.8 percentage points; 95% CI, −17.3 to 5.7) [15]. In another recent large study, LPV/RTV combined with standard care was not associated with reductions in 28-day mortality, duration of hospital stay, or risk of progressing to invasive mechanical ventilation or death [28].

Discussion
This systematic review included 14 articles relating to the efficacy and safety of LPV/RTV in COVID-19 patients, with a total of 9036 patients included, and only eight articles, that comprised 8438 patients had findings on the efficacy and safety of LPV/RTV alone or in combination with standard care ± interferons/antiviral treatments compared to other therapies in the treatment of COVID-19, were deemed legible for quantitative synthesis (meta-analysis) [26][27][28][29][32][33][34]36].
Two studies reported no significant effect on time to temperature normalization for LPV/RTV arm (n = 93) versus umifenovir arm (n = 71) (OR = 0.87 day; 95% CI, 0.42 to 1.78; p = 0.70, I 2 = 0%); or alleviation of cough duration (p = 0.69) [27,32]. The total number of cough days was found to be lower in the LPV/RTV arm compared with the umifenovir arm or no antiviral therapy (conventional) arm after 7 days of treatment; however, the overall effect was found to be not significant [27,32]. Although a favorable therapeutic effect for umifenovir was observed in a small cohort study when the drug was combined with LPV/RTV treatment in (n = 16) COVID-19 patients rather than LPV/RTV alone (n = 17) [38]; it should be noted that the treatment of LPV/RTV alone groups (n = 127) versus umifenovir plus LPV/RTV groups (n = 69) did not reveal any significant mean difference between the two groups in terms of virological cure at day seven [26,32,37]. In another study that involved 81 COVID-19 patients, the umifenovir treatment group had a longer hospital stay than patients in the control group (13 days (IQR 9-17) vs. 11 days (IQR 9-14), p = 0.04) [39]. Of note, umifenovir, which is branded as Arbidol ® , has a wide antiviral activity against RNA and DNA viruses, is licensed in Russia and China for the treatment and prophylaxis of influenza and recommended for treatment of MERS-CoV, was investigated in SARS-CoV, and is currently being trialed in COVID-19 patients [40].
In terms of CT evidence for radiological progression of pneumonia/lung damage, fewer patients exhibited radiological progression in the LPV/RTV arm compared with the umifenovir arm or no antiviral therapy (conventional) arm after 7 days of treatment, this effect was not significant (p = 0.59), and similarly, with LPV/RTV (n = 71) versus no antiviral therapy [27,32].
It is worth mentioning that initiating therapy earlier is known to be more effective [41], since systemic hyperinflammation rather than viral pathogenicity dominates later stages of SARS-CoV-2 infection. Although patients in five of the studies [15,27,30,31,34] included in our review were administered LPV/RTV early in the infection (median of <7 days); LPV/RTV therapy was not found to be totally effective.
In terms of safety, this study found greater adverse events reported in the LPV/RTV arm versus no antiviral treatment (conventional) or umifenovir, respectively. Adverse events associated with LPV/RTV alone or in combination with other medicines were reported in COVID-19 patients, and were typically gastrointestinal (GIT) in nature, including nausea, vomiting, and diarrhea [32]; nevertheless, serious GIT adverse drug reactions such as acute gastritis and GIT bleeding and acute kidney injury (n = 3) were also reported [32]. It was reported that most ADRs associated with LPV/RTV in combined groups of medicines are resolved within three days of drug initiation [30].
To address the efficacy and safety of LPV/RTV combined with other drugs in patients with COVID-9, LPV/RTV plus IFN combination in addition to ribavirin was found to be superior and safer than LPV/RTV alone by shortening the time to negative nasopharyngeal swab compared to the LPV/RTV arm alone [31]. Additionally, a combined treatment regimen of LPV/RTV plus IFN and umifenovir resulted in a shorter time by normalizing body temperature and turning PCRs negative compared to the umifenovir plus IFN arm with reasonable safety profile [37]. However, the use of LPV/RTV plus IFN combination resulted in less therapeutic responses on COVID-19 in terms of viral clearance and chest CT changes compared to the favipiravir plus IFN combination. Favipiravir arm patients had fewer AEs than patients in the LPV/RTV arm [30]. Additionally, there was no significant difference in average PCR negative conversion times among IFN plus LPV/RTV or IFN plus LPV/RTV plus ribavirin treatment arms [35]. The combination of LPV/RTV, in addition to standard care, or standard care alone revealed no difference in the time to clinical improvement, duration of hospitalization, initiation of invasive mechanical ventilation and death [15,28,29]. A serious case of elevated alanine aminotransferase (ALT) was reported [28], GI AEs were more common in the LPV/RTV group and treatment was stopped early in 13.8% because of AEs [15].
In a recent systematic review that included 41 studies which considered therapeutics for COVID-19, LPV/RTV was found to be the third therapy associated with positive outcomes (54.9%) with less negative outcomes (12.3%) compared to systemic corticosteroids (21.3%), remdesivir (16.9%), moxifloxacin (13.4%) and oseltamivir (12.5%) [2]; however, further controlled studies were needed to draw a valid conclusion. Antiviral ineffectiveness of LPV/RTV against SARS-CoV-2 in the studies included in our review was justified by the necessity to give the drug at a daily amount higher than 800 mg/200 mg; as an in vitro analysis identified antiviral activity of LPV/RTV against SARS-CoV-2 with a half-maximal effective concentration (EC 50 ) of 16.4 µg/mL [42]. However, there is a potential to intoxicate the patient, because high doses of LPV/RTV may lead to delayed ventricular repolarisation (QT prolongation) [7]. Thus, it might be logical to argue that there is a need to determine the effective and safe dose of LPV/RTV against the SARS-CoV-2 virus for better clinical benefit [13].
It is important to consider drug concentrations at the site of infection, and currently, the lack of robust lung penetration data is an important gap that exists for many agents being considered for repurposing. In the case of LPV/RTV, lung penetration is complex and not well understood; however, typically it is the plasma-free fraction that is available to penetrate into tissues. Therefore, given its potency, lung penetration of LPV/RTV would have to be high to provide concentrations in the therapeutic range [43]. The antiviral activity in vivo is estimated by calculating the ratio of unbound drug concentrations achieved in the lung at the administered dose to the in vitro EC50 (R LTEC ) [44]. Even though the majority of the observed total LPV/RTV plasma concentrations in COVID-19 patients were above the published EC50 for SARS-CoV-2 [42], boosted LPV/RTV is unlikely to attain sufficient effective levels in the lung to inhibit the virus. Indeed, the largest trials of RECOVERY [28] and SOLIDARITY [29] found LPV/RTV had little or no effect on overall mortality, initiation of ventilation and duration of hospital stay in hospitalized patients.
There is uncertainty about the optimal approach to treat hospitalized COVID-19 patients. Management approaches are based on limited data and evolves rapidly as clinical data emerge. For patients with non-severe disease, care is primarily supportive, with close monitoring for disease progression. Remdesivir is suggested in hospitalized patients with severe disease (i.e., they have hypoxia) but who are not yet on oxygen [45,46]. For patients who are receiving supplemental oxygen (including those who are on high-flow oxygen and noninvasive ventilation), low-dose dexamethasone and, if available, remdesivir is/are suggested [47,48]. However, the optimal role of remdesivir remains uncertain, and some guidelines panels (including the WHO) suggest not using it in hospitalized patients because there is no clear evidence that it improves patient-important outcomes for hospitalized patients (e.g., mortality, need for mechanical ventilation). In general, use of LPV/RTV for treatment of SARS-CoV-2 in hospitalized patients is not suggested as several trials have failed to prove efficacy [15,28,29]. Evidence as to whether LPV/RTV is beneficial in outpatients with mild or moderate severity COVID-19 infection is lacking; therefore, use of LPV/RTV is suggested in outpatients only in the context of a clinical trial.
Vaccines to prevent COVID-19 infection are considered the most promising approach for controlling the pandemic. COVID-19 vaccine development is occurring at an unprecedented pace. Several different platforms are being utilized to develop COVID-19 vaccines such as: inactivated virus or live-attenuated virus platforms (traditional methods); recombinant proteins and vector vaccines (newer methods); and RNA and DNA vaccines (methods never previously employed in a licensed vaccine) [49]. Several vaccine candidates have demonstrated immunogenicity without major safety concerns in early-phase human trials [50]. Two mRNA vaccine candidates have also been reported to have approximately 95% vaccine efficacy [51,52]. AstraZeneca's Oxford coronavirus vaccine is 70% effective on average, data shows, with no safety concerns [53]. Importantly, the AstraZeneca vaccine can be distributed and administered within existing healthcare systems, as it can be stored, transported and handled in normal refrigerated conditions for at least six months, the company said. The vaccine will also be cheaper than rival coronavirus vaccines from makers Pfizer and Moderna [53].
Since disease resulting from SARS-CoV infection is driven by both virus and host immune response factors, depending on the stage of the disease progression, early initiation of antiviral therapy, and/or holistic combination therapies will likely be needed to diminish virus replication, immunopathology, and/or promote repair and restoration of pulmonary homeostasis [54]. Until sufficient evidence is available, the WHO has warned against physicians and medical associations recommending or administering unproven treatments to patients with SARS-CoV-2 or people self-medicating with them.
The key limitations of this study were the limited number of clinical studies investigating the efficacy and safety of LPV/RTV in combination with a limited number of participants. Another limitation is the inability to perform any type of meta-analysis specifically for the results of efficacy and safety of using LPV/RTV in combination with other agents versus no antiviral therapy (conventional therapy) or control because of the large methodological differences. Despite these limitations, this systematic review provided valuable insight into the efficacy, safety, and clinical outcomes of LPV/RTV alone or with other antiviral medications.

Conclusions
The small number of studies included in this systematic review and meta-analysis study did not reveal any statistically significant advantage in the efficacy of LPV/RTV in COVID-19 patients, over no antiviral or other antiviral treatments. In terms of safety, this study found a greater number of adverse events reported in LPV/RTV arm versus no antiviral treatment (conventional) or umifenovir arms, respectively. There is a general understanding of the need to conduct large randomized clinical trials to determine the efficacy and safety of LPV/RTV in the treatment of COVID-19. Ideally, these studies should be double-blinded and conducted in a wide range of settings.  Abbreviations  coronavirus disease 2019 SARS-CoV-2 severe acute respiratory syndrome coronavirus 2 MERS-CoV Middle East respiratory syndrome coronavirus LPV/RTV lopinavir/ritonavir PRISMA Preferred Reporting Items for Systematic reviews and Meta-Analysis RoB 2 Version 2 of the Cochrane risk-of-bias tool for randomized trials ROBINS-I Risk of bias in non-randomized studies-of interventions RT-PCR real-time reverse transcription-polymerase chain reaction