Efficacy of Chloroquine and Lopinavir/Ritonavir in mild/general COVID-2019: 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,

There are currently no clinically specific drugs for these 7 HCoVs. An effective treatment is urgently needed to control the COVID-2019 epidemic. The National Health Commission of China announced a new coronavirus-infected pneumonia diagnosis and treatment program (the Fifth edition), which proposed the trial of lopinavir/ritonavir(CYP3A inhibitor, also metabolized by CYP3A). However, our previous use found that the effcet of lopinavir/ritonavir on COVID-2019 was unsatisfactory (data was not shown).
Two independent research teams found that chloroquine has anti-SARS-CoV activity at the cellular level. Chloroquine phosphate can inhibit virus replication in Vero E6 cell line induced by SARS-CoV, with a 50% inhibitory concentration (IC50) of 8.8 ± 1.2 µm, which is close to the chloroquine plasma concentration achieved during the treatment of acute malaria 2 . It is significantly lower than the 50% cytostatic concentration(CC50 = 261.3 ± 14.5 µm), suggesting the safety of chloroquine for this cell line. At the same time, the antiviral activity of chloroquine can be extended to 5 hours after infection without a significant decrease. A study by the United States Centers for Disease Control and Prevention further clarified that chloroquine inhibits virus replication by reducing terminal glycosylation of angiotensin-converting enzyme 2(ACE2) receptors on Vero E6 cells and interfering with the binding of SARS-CoV and ACE2 receptors 3 . 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 4 . The latest study found that the S(spike) protein of SARS-CoV-2 is similar in structure with that of SARS-CoV 5 , 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 (GS-5734) and Chloroquine (Sigma-C6628) can be effective inhibit the infection of SARS-CoV-2 in vitro 6 .
Based on the above evidence, we first use a prospective, open-label, multicenter randomized controlled clinical study to evaluate the efficacy 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) on February 19, 2020. However, the effectiveness and safety of chloroquine (phosphate) require more evidence-based medical evidence.
Our objective is 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 will be randomly assigned to the experimental group (chloroquine phosphate group) and the control group The schedule of treatment visits and data collection (also known as Clinical Research Flowchart) is shown in Table 1. Table 1 The schedule of treatment visits and data collection.  Participants:

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 and Treatment of pneumonitis caused by novel coronavirus (trial version 5)): ① Epidemiological history; ② Clinical manifestations (in accordance with any 2 of the following): fever; normal or decreased white blood cell counts in the early stages of disease, or decreased lymphocyte counts; multiple small patchy shadows and interstitial changes in the early stage of chest imaging, which are evident with the extrapulmonary zones. Furthermore, it develops multiple ground glass infiltrations and infiltrates throughout both lungs. In severe cases, pulmonary consolidation may occur, and pleural effusion is rare.
③ Confirmed: The suspected case has one of the following pathogenic evidence: respiratory specimens, blood specimens, or stool specimens are detected by real-time fluorescent RT-PCR(Reverse Transcription PCR) to detect positive of novel coronavirus nucleic acid; the above-mentioned specimens are genetically sequenced and highly homologous to known novel coronavirus.
④ mild or general patients; ⑤ Those who have not used antiviral drugs.

Exclusion criteria
The exclusion criteria are as follows: (If the subject meets any of the following conditions, they cannot enter the study)

Sample size
In this study, Logrank method was used to compare the difference in clinical recovery time between the two groups of patients. According to the existing research, the median clinical recovery time of the control group is 8 days, and the experimental group is 4 days.
According to the type I error α = 0.05, the test power is 0.85, and the ratio between the test group and the control group is 1: 1. Considering the 5% shedding rate, the number of statistical cases is no less than 56 respectively in the two groups (the experimental group and the control group), and a total of 112 subjects are included in the study.

Recruitment
Participants will be recruited from SARS-CoV-2 infected inpatients. The volunteers 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) . All participants can withdraw their consent at any time during the trial.

Randomization allocation and blinding
Grouping was performed using a block randomization method. Before the experiment, a statistical expert randomly assigns 1 to 112 numbers according to the statistical software to generate a random allocation table. The selected block length and random seed number are stored together with the statistical expert. According to the random allocation table in advance, the statistical expert gives random numbers (1-112) in ascending order. Each random number corresponds to an envelope. The envelope contains the corresponding random number. The envelope is sealed and given to the researchers responsible for screening. The qualified subjects are selected, and the envelopes are received in the order of enrollment. After the envelopes are opened, the random number is taken out and given to the researcher responsible for treatment and observation. The researcher will contact the statistician. The statistician informs the group corresponding to the random number according to the random allocation table, 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 remains the same throughout the trial.

Interventions
The study subjects were divided into experimental group and control group for corresponding treatment regimens.  Outcomes:

Primary outcome
The clinical recovery time (not more than 28 days), that is, the time (in hours) from the start of study drug intervention to normalization of body temperature, respiratory symptoms, respiratory frequency, and blood oxygen saturation. Specifically meet the following criteria at the same time: ⑧Frequency of serious adverse events.

Safety assessment and adverse events
Test safety is monitored 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 assessed by monitoring adverse events(AEs) and vital signs.

Quality control
This study will be conducted in four hospitals and the following five measures will be taken to ensure its rigor and quality. Quality control must be used at each stage of data processing to ensure that all data is reliable and processed correctly.

(1) Balance analysis of baseline values
For demographic data and other baseline values such as vital signs, disease history, and basic treatment, in order to measure the balance of each group, continuous comparison test was used to compare the count data between groups. When the theoretical frequency in the fourfold table is less than 5, Fisher's exact probability method is used; Measurement data normally distributed are compared using group t-tests, otherwise, comparisons between groups are tested using Wilcoxon Rank Sum. Baseline evaluations were performed on FAS(full analysis set).
(2) Effectiveness analysis The comparison of the main efficacy indicators' clinical recovery time was a log-rank test.
If the influence of confounding factors needs to be considered, Cox proportional hazard model can be used. 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 compare the rate differences between groups.
(3) The negative rate and adverse reaction rate were the dependent variables on the 7th, 14th, and 21st days in the two groups. Its steady-state valley concentration is taken as the independent variable, Logistic regression analysis and receiver operating characteristic (ROC) curve analysis after assignment. Investigate the correlation between blood concentration and clinical efficacy and adverse reactions.

(4) 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 test, and the relationship with the test drug when abnormal changes occur, apply a statistical test when necessary.

Clinical trial registration
The trial was registered under the registration number

Discussion
Chloroquine is a known 4-aminoquinoline that has been used clinically since 1944. In addition to being used as an antimalarial drug, chloroquine is also used for the treatment of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, etc.
due to its immunomodulatory activity 7  The results of this study will provide meaningful information and evidence for clinical practice and will help design a proven and reasonable RCT (Randomized Controlled Trial) soon.
This study will confirm the safety and effectiveness of chloroquine in the diagnosis of mild/general patients with COVID-2019, and evaluate whether chloroquine is antiviral. At the same time, investigate the correlation between drug concentration and treatment effect and adverse reactions by measuring blood concentration of chloroquine phosphate.

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