Efficacy of Chloroquine versus Lopinavir/Ritonavir in mild/general COVID-19: a prospective, open-label, multicenter randomized controlled clinical study

Background The outbreak of novel coronavirus pneumonia is very serious, and no effective antiviral treatment has been confirmed. The fresh drug research and development cycle is too long to meet clinical emergency needs, and "old drugs and brand new applications" have a huge therapeutic potential. During our previous treatment, we found that the lopinavir/ritonavir treatment recommended in the Fifth edition of the treatment plan had little effect. Earlier studies have shown that chloroquine can inhibit coronavirus replication through multiple mechanisms. Our previous use of chloroquine to treat patients with SARS-CoV-2(novel coronavirus)-infected pneumonia has a higher negative rate of nucleic acid in throat swabs within 5 days after administration than that using lopinavir/ritonavir. However, the half-life and side effects of chloroquine vary greatly among individuals. Methods/design We plan to conduct a prospective, open-label, multicenter randomized controlled, comprehensive treatment clinical study. The study consisted of three phases: a screening period of 1-110 days, a treatment period of no more than 28 days, and a follow-up period of 1 month. Participants will be assessed at baseline and on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 21, and 28 after the intervention begins. In this study, chloroquine and lopinavir/ritonavir tablets were used to treat patients with eligible novel coronavirus pneumonia diagnosed at various centers between February 12, 2020 and May 31, 2020. The efficacy and safety of chloroquine and lopinavir/ritonavir are to be evaluated. At the same time, explore the correlation between patient genetic polymorphisms and chloroquine steady-state concentration, therapeutic effects and adverse reactions in the body. It is an anti-virus for pneumonitis caused by novel coronavirus. The optimization and update of the antiviral treatment plan provides evidence-based evidence. Disscussion Our study is a prospective, open-label, multicenter randomized controlled, comprehensive treatment clinical study to evaluate the efficacy and safety of chloroquine phosphate and lopinavir/ritonavir in patients with mild/general COVID-2019. The results

cells and interfering with the binding of SARS-CoV and ACE2 receptors 13 . HCoV-229E and SARS-CoV both belong to the α group HCoVs. Chloroquine could inhibit the replication of HCoV-229E on the L132 human embryonic lung cell line by inhibiting p38 mitogen-activated protein kinase (MAPK) activation 14 The latest study found that the S(spike) protein of SARS-CoV-2 is similar in structure with that of SARS-CoV 15 , and can also bind to the ACE2 receptor on the surface of host cell through the S protein, thereby infecting the epithelial cells of the host. At the cellular level, remdesivir  and Chloroquine (Sigma-C6628) can be effective inhibit the infection of SARS-CoV-2 in vitro 16 .
Based on the above evidence, we proposeda prospective, open-label, multicenter randomized controlled clinical study to evaluate the efficacy and safety of anti-viral treatment of chloroquine phosphate compared with lopinavir/ritonavir in patients diagnosed with mild/general type SARS-CoV-2 infection. Previous studies have shown that chloroquine phosphate has a good antiviral effect in the clinic (Data was not shown), and has been highly valued by Guangdong Province and even the National Health Commission. It has been included in the National Health Commission novel coronavirus pneumonia diagnosis and treatment plan (trial version 6, URL: http://www.nhc.gov.cn/yzygj/s7653p/202002/8334a8326dd94d329df351d7da8aefc2/files/b218cfeb1b c54639af227f922bf6b817.pdf ) on February 19, 2020. However, the effectiveness and safety of chloroquine (phosphate) require more evidence-based medical evidence.
Our objective is to initiate antiviral therapy to prevent deterioration to more severe COVID-19, to evaluate the safety of the drug, and to explore the correlation between drug concentration and treatment effect and adverse reactions by measuring the blood concentration of chloroquine phosphate.

Methods/design
Background and design: This study is a prospective, open-label, multicenter randomized controlled, comprehensive treatment clinical study. Eligible participants are randomized assigned to the experimental group (chloroquine phosphate group) or the control group (lopinavir/ritonavir), with 56 patients in each group. Participants arerecruited from 4 hospitals, including the Fifth Hospital Affiliated of Sun Yat-sen University, the Ninth People's Hospital of Dongguan, Zhongshan Second People's Hospital, and the Jiangmen Central Hospital. The study consists of three phases: a screening period of 1-110 days from February 12, 2020 to May 31, 2020, a treatment period of no more than 28 days(outcomes will be evaluated over a period of 28 days from the time of enrolment.), and a followup period of 1 month for each participant. Participants are assessed at baseline on day 0, recorded adverse reactions on each day and clinical and laboratory data on days required after beginning the treatment. This study investigates the use of chloroquine or lopinavir/ritonavir tablets in patients diagnosed with novel coronavirus pneumonia (in line with the inclusion and exclusion criteria) between February 12, 2020 and May 31, 2020, to explore the safety of chloroquine and lopinavir/ritonavir tablets in the treatment of patients with COVID-19, the appropriation of antiviral treatment, the correlation between steady-state concentration of chloroquine phosphate and treatment effects and adverse reactions. The flow chart of the research process is shown in Figure 1.
The schedule of treatment visits and data collection (also known as Clinical Research Flowchart) is shown in Table 1.

Inclusion criteria
The study inclusion criteria are as follows:(All the following criteria are met before being selected) (1) Age ≥18 years; (2) Meet all the following criteria (refer to confirmed cases in the Diagnosis 3. Multiple small patchy shadows and interstitial changes in the early stage of chest imaging, which are evident with the extrapulmonary zones it develops multiple ground glass infiltrations and infiltrates throughout both lungs. In severe cases, pulmonary consolidation may occur, and pleural effusion is rare.  ⑦The time for the improvement of chest imaging (chest CT), the improvement of imaging is determined by the professional doctor of radiology based on the reduction of the scope of the lesion and the decrease in density; ⑧Frequency of serious adverse events.

Sample size
The main therapeutic index of this study is the clinical recovery time (not more than 28 days), which is from the beginning of the study drug intervention treatment to the normalization of body temperature, respiratory symptoms, respiratory rate and blood oxygen saturation, In the later analysis, the Logrank method is used to compare the differences in clinical recovery time between the two groups of patients. The sample size of this study is calculated based on the Logrank method by using the Logrank Tests (Lakatos) (Median Survival Time) module in the PASS11.0 statistical software.
Based on clinical experience 17 18 , the median clinical recovery time of the patients in the control group is expected to be 8 days, and the median clinical recovery time of patients in the experimental group can be shortened to 4 days (corresponding HR = 2.0) 112 patients (56 in each group) will be required to detect this difference with a significant level of α = 0.05 (both sides) with 85% confidence.
The trial is planned to be enrolled for 90 days, followed up for 28 days, and final analysis is performed after 78 clinical recovery events occurr. It is estimated the drop-out rate of the experimental group and the control group is 5%.

Recruitment
Participants will be recruited from SARS-CoV-2 infected inpatients according to the Inclusion criteria and the Exclusion criteria. The potential study candidate will be screened to determine if they meet the basic criteria. Once volunteered participants have been included or excluded from the criteria assessment, researchers will explain the research procedures in detail and require them to sign a written informed consent form (written informed consent form signed by the subject or his legal representative, these are available from the corresponding author on request) . All participants can withdraw their consent at any time during the trial.

Randomization allocation and blinding
Grouping is carried outusing a central stratification, randomization block method. Before the experiment, a statistical expert uses SAS software to set the number of centers to be 4, the length of the block to be 4, the number of blocks to be 28, a 1: 1 ratio between the test group and the control group, to generate 112 random numbers and corresponding grouping information. According to the haphazard allocation table in advance, the statistical expert gives random numbers (1-112) in ascending order. Each random number and grouping information corresponds to an envelope. The envelope is sealed and given to the researchers responsible for screening.Qualified subjects are selected, and the envelopes are received in the order of enrollment. After the envelopes are opened, the random number and grouping information is taken out, so that the subjects will be randomly assigned to the experimental group or the control group, and the corresponding treatment and observation were performed. Each subject's random number is unique and is the same throughout the trial.

Interventions
The study subjects were divided into experimental group and control group for corresponding treatment regimens. Researchers fill out the inpatient medical records at the same time when the subjects are being treated, ensuring that the data records are timely, complete, accurate and true. At the same time, the researcher fills out the case report form after the diagnosis and treatment of the subjects in time to ensure that the content of the case report form is consistent with the content on the outpatient or inpatient medical records., the researcher should fill out the relevant data on the case report form in time, and submit it to the main researcher at the center for review and signature confirmation.

Office of Clinical Research Center and the Medical Ethics Committee of the Fifth Affiliated Hospital of
Sun Yat-sen University monitorthe safety throughout the test. Typical laboratory safety tests include routine tests for blood, urine, liver function, eg: ALT(Alanine aminotransferase) and AST(Aspartate aminotransferase), and renal function, eg: blood urea nitrogen(BUN) and creatinine(Cr) will be performed during the treatment period. Along with treatment, safety will also be evaluated by monitoring adverse events(AEs) and vital signs.
1.Adverse events (AE) refer to adverse medical events that occur after a patient or clinical trial subject receives a drug, but they are not necessarily causally related to treatment.
2 Severe adverse events (SAE) are adverse events that occur during medication and obvious abnormalities in hematology or other laboratory tests, which need to take targeted medical measures to return to normal. Record of adverse events: The researcher should strictly follow protocol requirements, and record the adverse events/ serious adverse events in the original medical record and case report form(CRF) in a true, accurate, complete, timely and legal manner, sign and date them.
The record includes at least the name of the adverse event and a description of all symptoms, the start time, the end time, the severity and the frequency of attacks, the relevance to the test drug, the examination due to the adverse event, the treatment measures and the outcome.

Data collection and verification
In our research, professional researchers specialize in recording. At the same time, we appoint qualified supervisors to conduct on-site comprehensive inspection visits to the research center. In order to ensure the accuracy of numerical data, Epidata 3.1 is used for data double-checking. The data is entered and proofread. For the questions in the case report form, the data administrator will fill them in the Data Rating Questionnaire(DRQ) and send an inquiry to the researcher through the clinical monitor. The data administrator will modify the data according to the researcher's response, Test termination criteria: (1) Due to lack of efficacy, no improvement in results or worsening of symptoms, the investigator believes it is not suitable to continue the trial.
(2) The subject have an adverse event, and after taking appropriate treatment measures, the investigators consider it inappropriate to continue the trial.
(3) Other than the above reasons, those who cannot follow the plan.
(4) After using the test drugs, it is found that the subjects did not meet the inclusion criteria.
(5) Subjects or family members request to withdraw from the trial.
(6) Other reasons that the researcher thinks it is necessary to terminate the experiment.

Quality control
This study will be conducted in four hospitals to ensure its rigor and quality.
All observations and findings in clinical research should be verified to ensure the reliability of the data and to ensure that the conclusions in the clinical research are derived from the original data. Quality control must be used at each stage of data processing to ensure that all data is reliable and processed correctly.
Before the formal start of clinical research, the head of the research centertrain the researchers on the research plan, to help them to unify the knowledge, be familiar with the collection methods and procedures, and understand the special requirements of the research project, to improve the internal observational consistency and inter-observer consistency of clinical research data collectors, to ensure the reliability of clinical research conclusions.
Investigators conscientiously implement an informed consent so that subjects fully understand the research requirements and cooperate with the research.
A qualified auditor is appointed, and regular on-site inspections of the research center are conducted to ensure that all the contents and requirements of the research plan are strictly observed, and the original data is checked to ensure consistency in the content on the CRF(Clinical Research Flowchart).
The clinical research management department and the project responsible unit may entrust the inspectors to conduct a systematic inspection of the clinical research to determine whether the research execution is consistent with the research plan, and whether the reported data is consistent with the records of the clinical participating units, that is, whether the data recorded in the case report form is the same as that of the medical record or other original records.

Statistical analysis (1) Full analysis set (FAS)
According to the principle of intention-to-treat (ITT), all the cases that are randomized and use the study drug at least once and have post-medication evaluation data constitute the FAS of this study.
The missing data of the relevant part of the efficacy in FAS will be supplemented by the method of the last observation carryied forward.
(2) Per-protocol set (PPS) The standards of the PPS set and its population will be finalized during the blind data verification, including at least the following standards: Meet the inclusion criteria specified in the test plan; Complete all planned visits; No drugs or treatments that may affect the evaluation of efficacy are used and received during the trial.
(3) Safety analysis data set (safety set, SS) All cases that are randomized into groups and have used the study drug at least once and have postmedication safety evaluation data constitute the safety population in this study.

2.Effectiveness analysis
The comparison of the main efficacy indicators' clinical recovery time is a log-rank test. Cox proportional hazard model can be used to provide hazard ratios and 95% confidence intervals(CIs).
For the comparison of other efficacy indicators, t-test or Wilcoxon rank sum test was used for comparison of measurement data, and test or Fisher's exact probability method was used to test differences in a binary outcome and provide 95% CI of relevant indicators Take the viral nucleic acid negative or not and the occurrence of adverse events as the dependent variables on the 7th, 14th, ,21st and 28th days in the two groups. Its steady-state valley concentration is taken as the independent variable, Logistic regression analysis is used to investigate the correlation between blood concentration and clinical efficacy and adverse reactions.

3.The safety analysis
Use the test or Fisher's exact probability method to compare the incidence of adverse events in each group, and list the adverse events occurred in this study; the normal/abnormal changes in laboratory test results before and after the trialand the relationship with the test drug when abnormal changes occur.

Clinical trial registration
The trial was registered under the registration number

Discussion
To the 10 April 2020, the total number of COVID-19 diagnosed in the world is more than 1. Gautret P 20 concluded that chloroquine is significantly associated with viral load reduction/disappearance in COVID-19 patients.
In the seventh edition of the Chinese version of the COVID-19 Diagnosis and Treatment Plan

Limitations
Randomized controlled studies still have some design limitations. First, the sample size is relatively small and the 28-day treatment period is shorter. We will not be able to estimate the possible relapse of pneumonia after long-term treatment. Second, the pathophysiology of novel coronavirus pneumonia has not been elucidated. Only clinician assessment (including lung CT results and viral accounting load), there is no objective indicator to judge the effect of treatment on COVID-19. Finally, the follow-up period in this study was relatively short. In light of these limitations, we will develop a more reasonable treatment cycle and follow-up period to explore the efficacy of chloroquine in patients with COVID-19. We also know there will be many biases in the open trial, and have taken a number of measures to control the possible bias in the trial, as follows: (1) Strict exclusion criteria are formulated to effectively control other confounding factors that may affect the efficacy; (2) The trial uses random grouping to ensure that the two groups of patients are comparable; (3) Before the patient signs the informed consent form, the researchers and the patients make full communication to ensure the patients understand the entire trial content, and try to eliminate the impact of the patient's psychological state on the trial effect. The flow chart of the research process