key: cord-1033089-5pvltcs5
authors: Hu, Fen; Chen, Jiao; Chen, Hao; Zhu, Jin; Wang, Chen; Ni, Haibin; Cheng, Jianming; Hu, Xingxing; Cao, Peng
title: Cinobufacini improves the respiratory function of severe COVID-19 patients
date: 2021-12-31
journal: Pharmacological Research - Modern Chinese Medicine
DOI: 10.1016/j.prmcm.2021.100007
sha: 14d792cddb92bf214c43c8d3cd9aeeac569db772
doc_id: 1033089
cord_uid: 5pvltcs5

Targeted therapeutics for SARS-CoV-2 virus caused COVID-19 are in urgent need. Cinobufacini has been reported to have broad-spectrum antiviral effects and widely used in Southeast Asian countries. This study aims to assess the efficacy of Cinobufacini injection in treating patients with severe COVID-19. A randomized preliminary clinical trial was conducted and eligible patients were allocated to receive general treatment plus Cinobufacini injection or only general treatment as control for 7 days. The primary outcomes of the oxygenation index PaO2/FiO2 and ROX, secondary outcomes of white blood cell count, respiratory support step-down time (RSST), safety indicators, etc were monitored. After 7 days of treatment, the oxygenation index was improved in 95.2% patients in the treatment group compared with 68.4% in the control group. The PaO2/FiO2 and ROX indices in the treatment group (mean, 226.27±67.35 and 14.01±3.99 respectively) were significantly higher than the control group (mean, 143.23±51.29 and 9.64±5.54 respectively). The RSST was 1 day shorter in the treatment group. Multivariate regression analysis suggested that Cinobufacini injection contributed the most to the outcome of PaO2/FiO2. No obvious adverse effects were observed. The preliminary data showed that Cinobufacini injection had apparent efficacy in improving the respiratory function of patients with severe COVID-19.

The current pandemic caused by SARS-CoV-2 represents one of the greatest threats to both human health and socioeconomic development globally. Known as coronavirus disease 2019 , this disease has plagued over 196.5 million cases and claimed about 4.2 million lives as of 30 July, 2021 (data released by the World Health Organization [1] ). Fever, dry cough, and runny nose are common characteristics of COVID-19. Severe cases usually have dyspnea and/or hypoxemia one week after the onset of symptoms, which could quickly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis that is difficult to correct, coagulation dysfunction and multiple organ failure [2] [3] [4] [5] . In severely affected areas, the overall mortality rate is over 10% according to the data released by WHO, while the mortality rate of severe COVID-19 patients is much higher. Therefore, a critical goal of Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATP1A1, Na + /K + -ATPase 1 subunit; BMI, body mass index; CK-MB, cardiac marker creative kinase isoenzyme MB; COVID-19, coronavirus disease 2019; Cr, creatinine; FIPV, feline infectious peritonitis virus; MERS-CoV, Middle East respiratory syndrome; MHV, murine hepatitis virus; PaO 2 /FiO 2 , a ratio of the arterial partial pressure of oxygen to the fraction of inspiration oxygen; PBML, peripheral blood mononuclear lymphocyte; PLT, platelets; ROX, index (ROX = SpO 2 /(FiO 2 * RR)); RR, respiratory rate; RSST, respiratory support step-down time; RSV, respiratory syncytial virus; RT-PCR, reverse-transcriptase-polymerase-chain-reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SpO 2 , pulse oxygen saturation; TB, total bilirubin; VSV, vesicular stomatitis virus; WBC, white blood cell; WHO, World Health Organization. * Crresponding authors. E-mail addresses: huxingxing82@163.com (X. Hu), cao_peng@njucm.edu.cn (P. Cao). 1 F. Hu and J. Chen contributed equally to this work. COVID-19 treatment is reducing the mortality of severe patients. Current treatment recommendation is largely empirical and targeted therapeutics are in urgent need [6] . The Lopinavi-Ritonavir combination has been proved to have little effects on severe COVID-19 patients in clinical trials [7] . However, China's anti-epidemic experience during the outbreak in Wuhan suggested that traditional Chinese medicine might play an important role in the treatment of COVID-19.

Cinobufacini (Chansu), aqueous extracts of venoms from the parotid and skin glands of the toad Bufo bufo gargarizans Cantor , has been widely used in China, Japan and other Southeast Asian countries as an cardiotonic, antimicrobial, anodyne, antineoplastic, and local anesthetic agent for thousands of years, and it has been used to treat plague in history [ 8 , 9 ] . Recently, Cinobufacini, in dosage forms of injection, capsules, oral solution and tablets, has been approved by the Chinese State injection (the ethanol extract of dry secretions from the parotid and skin glands of Bufo bufo gargarizans Cantor processed into injection) is used to treat severe upper and lower respiratory tract infections, chronic hepatitis B therapy and cancer, and the incidence of adverse reactions is "occasionally", with good clinical safety [10] [11] [12] [13] . The main active constituents of Cinobufacini are bufadienolides, including bufalin, resibufogenin, and cinobufagin etc. Our preliminary in vitro studies have shown that Cinobufacini or its main constituents have anti-H1N1, HIV and other RNA viruses activities (data have not been published). Reported studies also showed that, bufalin, the major active constituent of Cinobufacini, can inhibit the infection of cells by murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), Middle East respiratory syndrome coronavirus (MERS-CoV), and vesicular stomatitis virus (VSV) via targeting the Na + /K + -ATPase 1 subunit (ATP1A1)-mediated Src signaling pathway [ 14 , 16 ] , which also plays a critical role in the cell entry of Ebola virus and respiratory syncytial virus (RSV) [ 15 , 21 ] . ATP1A1 was shown to be required for macropinocytic entry and replication of a series of coronaviruses [ 15 , 16 ] . In addition, cardiac glycosides targeting ATP1A1 have been reported to be effective against both DNA and RNA viruses, emerging as potential broad-spectrum antiviral drugs [17] [18] [19] [20] .

SARS-CoV-2 is a positive-sense RNA virus. Recently, using affinitypurification mass spectrometry and CRISPR methods, researchers identified ATPase as one of the targets of SARS-CoV-2 [ 22 , 23 ] . Based on these findings, Cinobufacini injection was empirically applied to patients with severe COVID-19. The HPLC fingerprint of Cinobufacini injection and the chemical structures of its main constituents (bufalin, cinobufagin and resibufogenin) are presented in Supplementary Figure S1 and Fig. 1 .

Of note, adequate and well-controlled clinical studies are difficult to perform in the condition of such a life threatening epidemic. Herein, we present the results of a randomized, preliminary clinical study that evaluated the effect of Cinobufacini injection plus the general treatment in improving the respiratory function of patients with severe COVID-19, using the general treatment group as control.

From February 5, 2020 to March 5, 2020, a total of 50 patients with severe COVID-19 admitted to the Department of Respiratory and Critical Care Medicine of the First People's Hospital of Jiangxia District, Wuhan, China were enrolled in the trial after approval by the ethics committees. All patients were confirmed as COVID-19 positive by either a reversetranscriptase-polymerase-chain-reaction (RT-PCR) assay or clinical diagnosis according to the Guidelines for the Prevention, Diagnosis, and Treatment of Pneumonia Caused by COVID-19 (version 5) issued by the National Health Commission of the People's Republic of China [24] . Patients meeting any of the following diagnostic criteria were considered as severe patients: respiratory rate (RR) ≥ 30 bpm in calm state, pulse oxygen saturation (SpO 2 ) ≤ 93% while breathing ambient air in resting state, oxygenation index (a ratio of the arterial partial pressure of oxygen to the fraction of inspiration oxygen, PaO 2 /FiO 2 ) ≤ 300 mmHg, or respiratory failure (PaO 2 ≤ 60 mmHg with or without carbon dioxide retention under standard conditions).

Main exclusion criteria included age < 18 years old, pregnancy, history of arrhythmia, chronic respiratory failure caused by other diseases such as heart failure, thoracic deformity, structural lung disease, hemodynamic instability, severe immunodeficiency, recent use of immunosuppressants, allergies, estimated survival time < 3 days, withdrawal from research or return visits. Severe COVID-19 patients admitted to the hospital mainly presented with hypoxemia, and very few were hemodynamically instable. Hemodynamic instability in patients with end-stage COVID-19 is mostly caused by factors such as high-dose sedation, muscle relaxation, and high-strength mechanical ventilation etc. Good stability of the blood pressure of the enrolled patients was observed in this study. Survival time was estimated by two experienced clinicians. Patients with persistent severe hypoxemia that was difficult to correct, recurring malignant arrhythmia, coma, severe gastrointestinal dysfunction, and uncontrollable gastrointestinal bleeding were excluded to avoid difficulty in the observation of drug efficacy. Written informed consent was obtained from all patients or their legal representatives. The trial was registered at ChiCTR.org.cn (registration number: ChiCTR2000030704) and conducted in accordance with the principles of the Declaration of Helsinki and the Good Clinical Practice guidelines of the International Conference on Harmonisation.

Using a random-numbers table, the enrolled patients were randomly assigned in a 1:1 ratio to receive general treatment (the control group) and general treatment plus Cinobufacini injection (20 mL/day, containing about 60 g dry extract of Cinobufacini, produced by Jiangsu Pujin Pharmaceutical Co., Ltd., Nanjing, China. Batch number: 191002, SFDA approval number: Z32020694). General treatment includes, as necessary, empirical antiviral therapy with peramivir, arbidol and interferon , -globulin, supplemental oxygen and standard glucocorticoid therapy, nutritional support, etc (Supplementary Table S1 ). Besides general treatment, the treatment group was also given Cinobufacini injection (20 mL formulated into 250 mL 0.9% physiological saline) intravenously at a rate of 125 mL/h every day, while the control group was given 250 mL 0.9% physiological saline. According to the instructions of Cinobu- 

Baseline characteristics include gender, age, body mass index (BMI), time from onset to treatment, basic diseases, and vital signs including body temperature, RR, heart rate, and SpO 2 etc. at the time of enrollment.

Primary outcomes include PaO 2 /FiO 2 and ROX index (ROX = SpO 2 /(FiO 2 * RR)). Secondary outcomes include the white blood cell (WBC) count, peripheral blood mononuclear lymphocyte (PBML) count and respiratory support step-down time (RSST). The supporting intensity of the four respiratory supports of non-invasive mechanical ventilation, high-flow oxygen therapy, oxygen mask, and nasal cannula oxygen therapy, gradually weakens. Therefore, RSST is defined as the transition time from advanced respiratory support to low respiratory support after enrollment, or the time required for FiO 2 to decrease by 50%. The respiratory support transition was deemed successful for patients when their SpO 2 stayed above 94% and the increase of their respiratory rates was less than 20%.

Safety outcomes include liver function indicators of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin (TB), kidney function indicator of creatinine (Cr), cardiac marker creative kinase isoenzyme MB (CK-MB), platelets (PLT) and adverse events such as systemic or local rash without other explanations, gastrointestinal symptoms such as nausea, vomiting, abdominal pain and diarrhea, new arrhythmia, etc. Adverse events were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0.

SPSS software version 22 (SPSS Inc., Chicago, IL, USA) was used to perform data analyses. Continuous variables were summarized as either medians and ranges or arithmetic means with standard deviations. Student's t test was used for assessing statistical significance of continuous variables between the control group and treatment group. Categorical variables were summarized as percentages (%), with their statistical significance between groups assessed by the 2 test. Multiple linear regression analysis was performed to evaluate the effect of the variables on the primary outcome by adjustment of imbalance for important baseline variables due to the withdrawal of some patients. Univariate linear regression analysis was performed to identify variables with regression coefficients statistically significant ( p < 0.05), which were further included in the multivariate linear regression to evaluate their effects on the primary outcome.

This prospective study preliminarily evaluated the efficacy and safety of Cinobufacini injection for severe COVID-19 patients infected by SARS-CoV-2. Following eligibility assessment, a cohort of 50 patients were randomly allocated into general treatment group (the control group) and general treatment plus Cinobufacini injection group (the treatment group) in a 1:1 ratio. Ten patients were excluded from the final analysis, including 3 patients in the treatment group and 6 patients in the control group transferring to another hospital for further treatment, and 1 patient in the treatment group failed to undergo re-examination on schedule. The remaining 40 patients, including 21 in the treatment group and 19 in the control group, were included for statistical analysis ( Fig. 2 ) .

Of the 40 participants, 24 (60%) were male. The median age was 61.5 years old (interquartile range [IQR], 51.0 to 71.0 years) and most patients (82.5%) were > 50 years old. At least one third of the patients had one or more basic diseases. The median time from onset to treatment was 6 days (IQR, 4.0 to 7.8 days). 36 patients (90%) had a body temperature > 37°C at enrollment. The mean SpO 2 was 88.73%. No significant differences were found between the treatment group and control group in gender, age, BMI, time from onset to treatment, basic diseases and vital signs at enrollment ( p > 0.05, Table 1 ).

General treatments included peramivir, arbidol and interferon , glucocorticoid therapy, -globulin and respiratory support as necessary, which had no significant difference between the two groups ( p > 0.05, Supplementary Table S1). Compared with CT imaging characteristics and various biochemical indicators, the patients' respiratory function indicators such as PaO 2 /FiO 2 , SpO 2 and RR related ROX index are more direct indicators of disease outcome. Therefore, PaO 2 /FiO 2 and ROX index were used as the primary outcomes in this study. No significant difference was found in the initial PaO 2 /FiO 2 and ROX index on Day 1 between the treatment group and control group ( p = 0.118, 95% CI, -6.46 to 55.18 for PaO 2 /FiO 2 ; p = 0.716, 95% CI, -1.89 to 2.73 for ROX) Table 2 ). After 7 days of treatment, patients receiving general treatment plus Cinobufacini injection (20 mL/day) improved significantly in both PaO 2 /FiO 2 and ROX index ( p < 0.001, 95% CI, -111.30 to -35.90 for PaO 2 /FiO 2 ; p < 0.001, 95% CI, -7.56 to -2.94 for ROX), while no significant differences were found between Day 1 and Day 7 in the control group receiving only general treatment ( p > 0.05). The PaO 2 /FiO 2 and ROX index were improved in 20 patients (95.2%) in the treatment group, as compared with 13 (68.4%) and 14 (73.7%) patients in the control group, while the mean values were significantly higher in the treatment group than in the control group on Day 7 ( p < 0.001, 95% confidence interval [CI], 44.39 to 121.70 for PaO 2 /FiO 2 and p = 0.006, 95% CI, 1.31 to 7.44 for ROX) ( Table 2 ). These results indicated that most severe COVID-19 patients benefited from Cinobufacini injection, while general treatment with paramivir, arbidol and interferon , standard glucocorticoid therapy or other symptom relievers and oxygen therapy had little effect on improving the respiratory function of severe COVID-19 patients.

COVID-19 patients usually have normal or decreased WBC and decreased PBML. For the secondary outcomes of WBC and PBML, no obvious improvement was observed after 7 days treatment in both groups ( p > 0.05). RSST is the time needed for the patient to transform from ad-vanced respiratory support to low respiratory support. The shorter RSST is associated with the faster prognosis of patients' respiratory function. The median RSST was 5.0 (IQR, 4.0 to 6.0) days in the treatment group, 1 day shorter than that in the control group (median: 6.0 days, IQR: 5.0 to 8.0 days) ( Table 2 ).

Univariate linear regression analyses were performed on the treatment interventions, patients' baseline characteristics (including age, gender and basic diseases), PaO 2 /FiO 2 , ROX index, WBC and PBML on Day 1 to screen out variables with regression coefficients statistically significant. As a result, four characteristics, the PaO 2 /FiO 2 and PBML on Day 1, the initial respiratory support mode and Cinobufacini injection affected the primary outcome of PaO 2 /FiO 2 on Day 7 statistically ( p < 0.05). To determine the main variables affecting the primary outcome of PaO 2 /FiO 2 on Day 7, multivariate regression analysis was applied. The PaO 2 /FiO 2 on Day 1, the initial respiratory support mode and Cinobufacini injection were further confirmed to affect the primary outcome of PaO 2 /FiO 2 on Day 7 statistically ( p < 0.05). Among them, Cinobufacini injection contributed the most to the regression model (Beta = 0.486) ( Table 3 ) .

No obvious adverse events, such as systemic and local rash, gastrointestinal symptoms, abdominal pain, diarrhea, and new arrhythmia, occurred during the treatment with Cinobufacini injection. No significant difference in other safety outcomes including liver function indicators (AST, ALT, TB), kidney function indicators (Cr), cardiac marker CK-MB and PLT was found after treatment with Cinobufacini injection ( p > 0.05). There was also no significant difference between the treatment group and control group in these safety indicators ( Table 4 ). All these indicated that the dose of Cinobufacini injection used in this study is safe for severe COVID-19 patients.

Except for the vaccines, only remdesivir is approved by FDA as targeted therapeutics for COVID-19 at present [25] , whereas other treatment recommendations such as chloroquine, hydroxychloroquine, favipiravir, corticosteroids and convalescent plasma are largely empirical [26] . Cinobufacini is a type of traditional Chinese medicine extracted from the skin and parotid glands of the toad, and it has been widely used for antiviral therapies in China and other Southeast Asian countries. To evaluate the effect of Cinobufacini plus general treatment in patients with severe COVID-19, we conducted a randomized, controlled clinical trial in this study.

From clinical experience, the patients' respiratory function status such as SpO 2 , PaO 2 /FiO 2 , and ROX index were found to be more direct indicators to evaluate pulmonary function [ 27 , 28 ] . This preliminary trial found that, compared with the general treatment alone, Cinobufacini injection combined with general treatment significantly improved the primary outcomes of PaO 2 /FiO 2 and ROX index in patients with severe COVID-19 after 7 days of treatment. General treatment with empirical antiviral therapy such as paramivir, arbidol and interferon , standard glucocorticoid therapy or other symptom relievers and oxygen therapy had little effect on improving the respiratory function of severe COVID-19 patients. In addition, the median RSST needed for the patients in the treatment group is 5.0 days, about one day shorter than the control group. Multivariate regression analysis suggested that, among various variables, Cinobufacini injection contributed the most to the primary outcomes of the patients in the treatment group.

The main active ingredients of Cinobufacini include bufalin, resibufogenin and cinobufagin, etc. Researchers have found that Cinobufacini has various activities including anti-inflammatory, anti-infection and immunomodulatory [29] [30] [31] . Previous studies reported that Cinobufacini or its active ingredient bufalin shows obvious anti-HBV activities and can inhibit infection of cells with MERS-CoV, Ebola, RSV, MHV, FIPV, and VSV [ 11-12 , 15 ] . The mechanism of Cinobufacini inhibiting coronavirus entry into host cells might be related to ATP1A1-mediated Src signaling pathway. A recent study identified ATPases as one of the Known side effects of Cinobufacini are mainly related to its cardiac glycoside property [33] . However, Cinobufacini injection shows good clinical safety at the treatment dosage [ 34 , 35 ] . In this clinical trial, no obvious adverse events occurred in the patients receiving Cinobufacini injection. Our preliminary results suggests that the dose of Cinobufacini injection (20 mL/day, containing about 60 g dry extract of Cinobufacini) used in this study is safe and effective in treating patients with severe COVID-19. Such results are promising, offering an alternative strategy to treat the emerging SARS-CoV-2 infection in real time. Given the urgent need for effective drugs against SARS-CoV-2 in the current pandemic, we recommend treatment consisting of Cinobufacini for severe COVID-19 patients to improve their respiratory function.

This clinical trial conducted in such hastiness has several limitations. Firstly, the trial was not blind, which could influence clinical decisionmaking and expose the study to observer bias. Secondly, owing to limited conditions, virologic clearance was not assessed after 7 days of treatment. In addition, the effect of different interventions on the final outcome of patients was difficult to evaluate, as some patients were transferred to other hospitals for follow-up treatment. Herein, we use the outcomes of respiratory function such as PaO 2 /FiO 2 and ROX index on the 7 th day after treatment to evaluate the efficacy of Cinobufacini injection. Thirdly, some patients had a history of out-of-hospital treatment, leading to inconsistencies of disease course. Interventions were not carried out at the same time after the patients transformed from mild symptoms to severe symptoms, which may lead to biased results. Other limitations include a small sample size and limited follow-up assessment of long-term outcome.

In conclusion, we found that Cinobufacini might have a therapeutic effect on severe COVID-19 in this preliminary study. It can improve the patients' respiratory function significantly. These promising early data provide a reference for the treatment of severe COVID-19, although the effects need further confirmation by clinical trials with larger sample sizes.

This work was supported by the National Natural Science Foundation of China ( 81973527 ) and Priority Academic Program Development of Jiangsu Higher Education Institutions ( Integration of Chinese and Western Medicine).

X. Hu and P. Cao conceived the presented idea, F. Hu, J. Chen, X. Hu and P. Cao designed the research; F. Hu, H. Chen, J. Zhu, C. Wang, and H. Ni performed research; F. Hu, J. Chen, J. Cheng, and X. Hu analyzed data; and J. Chen, P. Cao and X. Hu wrote the paper. F. Hu and J. Chen contributed equally to this work.

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

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We thank Yun Shi for providing language help.

Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.prmcm.2021.100007 .