Efficacy and safety of the combination of Liushen capsules and Arbidol in the treatment of COVID-19:protocol for a randomized,multi-center pilot study

Yangqing Zhan, Zhengtu Li, Jiayang He, Shaoqiang Li, Ye Lin, Jingyi Liang, Jie Zhou,Yanmei Wu,Xuandan Su,Feng Ye＊,Zifeng Yang,3＊

1 National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health,First Affiliated Hospital of Guangzhou Medical University, State Key Laboratory of Respiratory Disease(Guangzhou Medical University),Guangzhou 510230,China;

2 Guang Zhou Evidence-Based Medicine Technology Co.,Ltd;Guangzhou 510000,China;

3 Macau Institute for Applied Research in Medicine and Health, State Key Laboratory of Quality Research in Chinese Medicine,Macau University of Science and Technology,Macau,999078,China;

Abstract

Keywords:Arbidol,Liushen capsules,COVID-19,Clinical trial

Introduction

2019 novel coronavirus infection (formally defined by WHO as 2019 coronavirus disease, COVID-19)outbroke in Wuhan City, Hubei Province, China in December 2019 [1], with the main clinical manifestations being fever, asthenia, and dry cough.About half of the patients had dyspnea after one week,and severe patients rapidly progressed to acute respiratory distress syndrome (ARDS), refractory metabolic acidosis, disorder coagulation, septic shock,and multiple organ dysfunction syndromes (MODS),etc.[2-4]

From early experience against the COVID-19 epidemic and in vitro experiments, the Chinese research team found that Arbidol Hydrochloride Tablets (Arbidol) [5] and some Traditional Chinese Medicine may have effects on COVID-19.[6]However, there was little definitive clinical research to evaluate it.Due to the lack of specific therapeutic drugs, the control and prevention of disease are still difficult to predict.Besides, studies reported that inflammatory cytokine storms had been observed in patients with novel coronavirus pneumonia, which may be one of the leading causes of sudden multi-organ failure and subsequent death in some patients.Hence, in order to explore the possible therapeutic methods and provide clinical evidence,we designed a pilot study of a combination of Liushen capsules and Arbidol for the treatment of non-critical novel coronavirus infection.The design and consideration of this study will be introduced in detail as follow.

Methods

Objective of the study

The main purpose of this study is to evaluate the efficacy and safety of the combination of Liushen capsules and Arbidol for the treatment of patients with non-critical novel coronavirus infection and to explore the effects of the combination of the two medicines on inflammatory cytokines in patients with novel coronavirus infection.

Overall study design

This is a randomized, blank parallel-controlled,open-label, multi-center,basal treatment add-on study,in which subjects will be allocated randomly into two groups.Subjects in the treatment group will receive Arbidol Hydrochloride Tablets 200 mg/dose, three doses a day orally, and Liushen capsules 0.19 g/dose,three doses a day, on the basis of conventional treatment; subjects in the control group receive standard treatment for COVID-19 based on clinical guidelines issued by the Chinese government, without Liushen capsules and Arbidol.The total duration of treatment is at least seven days (or until the virus becomes negative, which is defined as the virus is negative twice consecutively with an interval of more than 24 hours).

This study has been reviewed and approved by the Independent Ethics Committees (IECs) of the First Affiliated Hospital of Guangzhou Medical University(Medical Research Ethics Committee 2020 No.41).A protocol amendment will be submitted to the IEC for approval if any modifications added.

Study population

The study is planned to be conducted in hospitals in Hubei and Guangdong provinces, etc., and a total of 40 subjects are expected to be enrolled, with 20 subjects in each group, for a total of two groups.The target population of the study consists mainly of isolated patients with confirmed non-critical novel coronavirus infections which were positive in novel coronavirus by real-time PCR for nucleic acid or by gene sequencing according to the Guideline of

Diagnosis and Treatment of Novel Coronavirus Pneumonia(Trial Version 7, the guideline of NCP)by National Health Commission of the People’s Republic of China[7](Hereinafter referred to as theGuideline).

Inclusion criteria: (1) Aged 18 years or older, male or female; (2)Fever, axillary temperature > 37.3℃;(3)Mild, common and severe patients with confirmed novel coronavirus infection according to the guideline(it will be updated accordingly if the guideline of NCP updates;(4)Patients who sign the informed consent.

Exclusion criteria: (1) Critical patients, who fulfill one of the following conditions: I.Respiratory failure and need mechanical ventilation; II.Shock; III.Organ failure which is other than the respiratory system that requires monitor and treatment in an intensive care unit.(2) Patients with respiratory tract infection caused by other virus except novel coronavirus;(3)Patients who have taken Arbidol Hydrochloride Tablets (capsules/granules/granules), Favipiravir,Liushen capsules within one month before screening;(4) Patients known to be allergic to the ingredients of study drugs, or patients with allergic condition; (5)Women in lactation, pregnancy, or with positive urine pregnancy test result or who are disagreed to contraception for three months after participating in the trial; (6)Immunocompromised patients, such as malignant tumor, organ or bone marrow transplant,AIDS, or patients who have taken immunosuppressive drugs within three months before screening examination; (7)Patients with severe hepatic impairment (defined as: alanine aminotransferase(ALT) or aspartate transaminase (AST) > 5 times the upper limit of normal value,or ALT or AST>3 times the upper limit of normal value and bilirubin>3 times the upper limit of normal value); or patients with severe renal impairment (calculated creatinine clearance is less than 50 ml/min according to blood creatinine); (8)Patients who are abandoned rescue;(9)Other patients who are considered unsuitable for this study by the investigators.

Study periods

ScreeningThe investigators will obtain informed consent from Covid-19 patients before the screening.Then subjects who meet the inclusion criteria/do not meet the exclusion will be randomized to the experimental group or the control group.Dedicated nurses will assign daily medication for patients and collect medical data.

RandomizationRandom codes will be generated by Statistics Analysis System(SAS)9.4,the randomization and unblinding will be achieved by investigators with a central randomization system using a multi-center stratified block randomization method.The subjects will be randomized at a 1:1 ratio for the experimental and control group.Subjects'personal information and biological samples will be de-identification, which are processed for randomized code.Since this study was an open-label design, both of the investigators and the subjects are aware of the drug regimen corresponding to the random number.

Follow-upThe subjects will make two visits during the first week after drug administration, and one extra visit if the virus becomes negative.Investigators will evaluate whether the subjects should continue to participate in the research or not.If they continue to participate, the next follow up will be on the day of hospital discharge.All of the subjects will be followed up through telephone calls on day 28.(The study process is shown in Figure 1)

Due to the particularity of the COVID-19,all of the subjects will be isolated and treated in the designated hospitals.Hence the outcome of this study will be collected from the medical records, and entry into the Electronic Data Capture System (EDC).The clinical research associate(CRA)will monitor online.

In addition, the treatment has to be discontinued when subjects had following circumstance: (1) The state of the subjects become critical; (2)Any serious complications, physiological change, and serious adverse events, which lead subject unsuitable for further participation; (3) Non-compliance; (4) The subject who is unwilling to keep participating;(5)The subject who is lost to follow-up.The investigators should take corresponding treatment according to the actual situation of the subjects, and complete the final test as possible for the analysis.The source data of subjects who dropped out should be kept for verification, and the results of the last major efficacy test should be carried forward to the endpoint for statistical analysis.

Study outcome

Primary outcomeTime to major symptomatic remission: The patient’s axillary temperature remains normal for >24 hours (without antipyretic drugs or corticosteroids), and the patient feels no dyspnea (for patients without dyspnea)or relief of dyspnea,oxygen saturation (SpO2) ≥ 94% at rest without oxygen supplementation, and respiratory rate < 24 beats/min(for patients with dyspnea).

Secondary outcome

(1) The time when novel coronavirus nucleic acid of the respiratory tract specimens (such as throat swabs)becomes negative for two consecutive times: Record the test results of the subject’s novel coronavirus nucleic acid, and when two consecutive tests are negative (two tests’ interval ≥24 hours), the time of the last novel coronavirus nucleic acid test shall be recorded.Time to two consecutive negative conversions(unit: days)is defined as the time interval between the last day when two consecutively novel coronavirus nucleic acid test shows negative and the day subject enrolled.

(2)Remission rate and remission time(hours)of other symptoms such as asthenia, dry cough, etc.Calculate the total time from the start of drug administration until the time of remission/complete remission for each and all clinical symptoms.Comparisons of the total time will be made between the two groups.Definition of remission: if symptom score ≠ 0 at baseline and symptom improves with a score ≤1 point after drug administration.Definition of complete remission: if symptom score ≠ 0 at baseline and symptom improves with a score ≤1 point for more than 24 hours after drug administration.The proportion of subjects in the experimental group and the control group who achieve remission or complete remission for each and all clinical symptoms is calculated.Then the remission rate and complete remission rate at Visit 1,Visit 2,Visit 3,and Visit 4 in the experimental group will be compared with those in the control group.

(3) Normalization rate and recover time of inflammatory cytokines: Compared the differences of growth factor, chemokines, T and B cell-related growth factor, innate immune inflammatory factors,and adaptive immune factor between the experimental group and the control group on Day 3 and Day 7 after drug administration,which is including Platelet-derived growth factor-AA/BB(PDGF-AA/BB), Platelet-derived growth factor-AA(PDGG-AA), Eotaxin, Monocyte chemoattractant protein-1(MCP-1),Macrophage Inflammatory Protein 1alpha (MIP-1a), Macrophage Inflammatory Protein 1beta (MIP-1b), Growth-related oncogene (GRO),Regulated upon Activation Normal T Cell Expressed and Presumably Secreted (RANTES), Monocyte chemoattractant protein-3 (MCP-3), Myeloid dendritic cells(MDC),Interleukin 6(IL-6),interferon-γ-inducible protein-10 (IP-10),interleukin-1 receptor antagonist (IL-1ra), Soluble CD40 ligand (sCD40L).And observe the changes in inflammatory cytokines from the baseline.

(4) Time to disease recovery: Record the time from the enrolment to disease recovery which shall refer to criteria of discharge from hospital described in theguidelineas follows: body temperature returns to normal for more than three days (axillary temperature≤37.3℃),respiratory symptoms improve significantly(subjects with all clinical symptoms meeting the definition of the disappearance of symptoms during administration, or feeling remission for all symptoms at follow-up), and two consecutive negative respiratory pathogen nucleic acid tests (at least one day in the time interval of sampling.Time to disease recovery is defined as the time interval between disease recovery and enrollment.

(5) The number of antipyretic drugs used: Compare the number of emergency drugs used in different groups.

(6) Incidence of critical illness on Day 7:Compare the proportion of severe pneumonia between different groups.

(7) Incidence of secondary infection: Calculate the proportion of subjects with secondary infection.

(8)All-cause mortality on Day 28.

(9)Incidence of adverse events.

Basal therapy

The standard therapy in this study was administerated by the investigators according to the updatedGuidelineof NCP to ensure that subjects in each group will receive appropriate treatment in time.The use of anti-viral traditional Chinese medicine and Chinese patent medicine, except for those approved by the protocol,will be prohibited.

The therapeutic scheme of each subject may be different for ethical reasons, therefore the interventions will be recorded in order to stratified analysis.In the event of disease progression to critical illness or the occurrence of other serious complications, the subject will be withdrawn from the study and receive empirical treatment.

Statistical analysis

Statistical hypothesesThe primary endpoint of this study is the time to major symptomatic remission.Suppose H0: AUCt = AUCc, and the time to major symptomatic remission of integrated traditional Chinese medicine and chemical medicine in the treatment of non-critical novel coronavirus infection is the same as that in the control group.H1: AUCt ≠AUCc, the time to major symptomatic remission of integrated traditional Chinese medicine and chemical medicine in the treatment of non-critical novel coronavirus infection is different from that in the control group.If the time to major symptomatic remission in the experimental group is less than that in the control group and has statistical significance, then it can be concluded that the treatment of the experimental group is better than that of the control group.Since this study is a pilot study,no sample size is estimated.However, the results of this study will provide data for the design and sample size estimate of subsequent large-scale clinical trials.

Plan of statistical analysis

(1) Baseline data: Describe demographic data,symptoms, and general conditions.Quantitative data is analyzed by t-test, analysis of variance or nonparametric statistical method according to the distribution of variables; qualitative data is analyzed by chi-square test, Fisher exact probability method,and Wilcoxon rank-sum test.

(2) Endpoint: The primary endpoint is the time to major symptomatic remission, which takes the baseline value and the center as covariates for the superiority test of the time to major symptomatic remission in the experimental group and the control group.One-side for a level of 0.025 is used to test whether the two groups are statistically significant,and 95% confidence interval for the difference value between the experimental group and the control group is calculated.Secondary endpoints are analyzed by group t-test, analysis of variance, χ2/Fisher exact test,or Wilcoxon rank-sum test according to the nature of the endpoints.At the time of statistical analysis,determine the selected analysis time nodes after treatment based on the overall trend.

(3) Safety analysis: The incidence of all adverse events and laboratory abnormalities will be compared between the groups using the χ2 test or Fisher exact probability method.

(4) Sensitivity analysis: Sensitivity analysis is proposed based on different data sets, different missing data estimation methods and concomitant events(if any),etc.

(5) interim analysis and stopping guidelines: The interim analysis is not applicable.The trial will be paused or stopped when this following situation occurs: 1) Any serious safety problems occurred during the trial;2)When it was found that the efficacy of the drug was too poor or even ineffective for clinical during the trial; 3) Significant problems were found during the trial, or any important deviation in the implementation of the protocol, which made it difficult to evaluate the drug efficacy; 4) The requirements for statistical analysis of the primary endpoint have been met; 5) The sponsor requests for suspension (e.g., funding reasons, management reasons, etc.); 6) As the competent administrative department requirement.

Discussion

Arbidol is a non-nucleoside broad-spectrum antiviral drug with immunological enhancement.Arbidol is used for treating upper respiratory tract infections caused by influenza A and B viruses and has inhibitory effects on a variety of respiratory viruses,including enveloped and non-enveloped viruses, RNA and DNA viruses since its approval in China in 2006.Arbidol can induce interferon production, activate macrophages, and regulate inflammatory factor levels[8].Several clinical studies have demonstrated that oral administration of Arbidol early in the onset of influenza not only relieves the symptoms but also shortens the course of the illness.A randomized,double-blind, controlled trial of Arbidol for acute influenza found [9] that the median duration of disease was 72 h and 96 h, and median decreases in total symptom scores were 780.0 and 684.0 in the Arbidol group and the placebo group, respectively,indicated that oral administration of Arbidol earlier after the onset of influenza could relieve symptoms and shorten the duration of the disease.A study by Liu Hongbo et al.[10] showed that the symptoms in 23.8% of the patients relieved after 60h of Arbidol administration, compared with 4.2% in the placebo group.Catarrhal symptoms and toxic symptoms also improved significantly after Arbidol administration.

The pharmacodynamic study of Liushen pills showed that Liushen pills had obvious anti-inflammatory and antiviral effects.Liushen pills could significantly inhibit the acetic acid-induced increase of abdominal capillary permeability and carrageenan-induced plantar swelling in mice,showing significant anti-inflammatory effect [11].Liushen pills can activate macrophages, enhance their phagocytic ability, directly kill cells, and inhibit the growth of bacteria, inhibit various pathogenic bacteria to varying degrees, showing antiviral effects.It is commonly used in clinical settings for influenza,mumps, hemorrhagic fever, herpes zoster, rubella,viral hepatitis, and other viral infectious diseases [12].Previous clinical studies of Liushen pills have shown that Liushen Pills could be used in the treatment of various infectious diseases such as throat diseases,bronchitis,herpes zoster,etc.[13]Liushen capsules are comparable with Liushen pills.

Guideline of NCP by National Health Commission of the People’s Republic of China suggested that interferon alfa aerosol inhalation, lopinavir/ritonavir,ribavirin, chloroquine phosphate, and Arbidol can be prescribed for antiviral therapy.However, there were also some alerts in the guideline of NCP that concomitant use of 3 or more antiviral drugs is not recommended and taking care of toxic adverse effects during treatment.Arbidol and Liushen pills all have antiviral effects and have their characteristics, and the combination use may enhance the antiviral effect, but the possible adverse reactions from combination use of the drugs should be noted.The integrated traditional Chinese medicine and chemical medicine has always been a characteristic therapy in China, and the combined use of traditional Chinese medicine and chemical medicine may further increase the therapeutic effect and reduce the occurrence of adverse reactions[14].Therefore, this trial is to study the combined use of the above drugs in order to find the advantages of integrated traditional Chinese medicine and chemical medicine in novel coronavirus infection and to provide a new therapeutic scheme for novel coronavirus infection.The results of this research will be published in the paper.

Acknowledgments

Thanks to all medical staff and investigators for their efforts in this outbreak.Thanks to Shanhu Health Technology Ltd.for donating the central randomization system and providing technical support for this study.We thank CSPC Ouyi Pharmaceutical Co., Ltd., Leiyunshang Pharmaceutical Group Co.,Ltd., and Sunflower Pharmaceutical Group Co., Ltd.for providing the drugs to the patients in this study for free.Thanks to Guangzhou Evidence-based Medicine Tech Co.,Ltd.for providing assistance in the planning and implementation of this study.

Contributors

YQZ, ZTL and JYH contributed equally to this work.FY and ZFY conceived and designed the study and are responsible for the coordination of the study.YQZ,ZTL and JYH developed the study design and revised the protocol.ZTL and ZFY sought funding and ethical approval.All authors contributed to the writing of the manuscript and read and approved the final manuscript.

Protocol version

Version 2.0,Date 28 Feb 2020

Ethics approval

The trial and all substantial amendments have been reviewed and granted approval from the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China(2020 no.54).

Declaration of interests

Researchers do not have any paid consultancies with pharmaceutical companies,and is not a member of the Speaker’s Panel of any company.

Access to data

All Principal Investigators and Government Department representatives will be given access to the cleaned data sets.To ensure confidentiality, data dispersed to project team members will be blinded to any identifying participant information.

Ancillary and post-trial care

If any injury or serious adverse event related to this study occurred during the research, subjects may receive free treatment and/or compensation according to Chinese law.