key: cord-251961-g0n85kxz
authors: Li, Guoming; Yuan, Mei; Li, Haihong; Deng, Changsheng; Wang, Qi; Tang, Yexiao; Zhang, Hongying; Yu, Weisheng; Xu, Qin; Zou, Yuanyuan; Yuan, Yueming; Guo, Jiawen; Jin, Chunming; Guan, Xiangdong; Xie, Fengjie; Song, Jianping
title: Safety and efficacy of Artemisinin-Piperaquine for treatment of COVID-19: an open-label, non-randomized, and controlled trial
date: 2020-11-02
journal: Int J Antimicrob Agents
DOI: 10.1016/j.ijantimicag.2020.106216
sha: 
doc_id: 251961
cord_uid: g0n85kxz

BACKGROUND: There are no effective therapies for patients with Coronavirus disease-2019 (COVID-19). METHODS: Forty-one patients with confirmed COVID-19 were enrolled in the study and divided into two groups: artemisinin-piperaquine (AP) group (n=23) and control group (n=18). The primary outcome was the time taken to reach undetectable levels of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) and the percentage of participants with undetectable SARS-CoV-2 on day 7, 10, 14, and 28. The computed tomography (CT) imaging changes within ten days, the corrected QT interval changes, adverse events, and abnormal laboratory parameters were the secondary outcomes. RESULTS: The mean time to reach undetectable viral RNA (mean± standard deviation) was 10.6±1.1 days (95% confidence interval [CI]: 8.4-12.8) for AP group and 19.3±2.1 days (95% CI: 15.1-23.5) for the control group. The percentage of patients with undetectable viral RNA on day 7, 10, 14, 21, and 28 were 26.1%, 43.5%, 78.3%, 100%, and 100%, respectively, in the AP group and 5.6%, 16.7%, 44.4%, 55.6% and 72.2%, respectively, in the control group. The CT imaging within ten days post-treatment showed no significant differences between the two groups (p>0.05). Both groups had mild adverse events. CONCLUSIONS: In patients with mild to moderate COVID-19, the time to reach undetectable SARS-CoV-2 was significantly shorter in the AP group than that in the control group. However, physicians should consider the QT interval changes before using AP.

Coronavirus disease 2019 (COVID- 19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Until April 23, 2020, there were 2.55 million confirmed cases and 179,000 confirmed deaths worldwide. By July 9, 2020, the cumulative number of confirmed cases exceeded 12 million, and deaths exceeded 540,000 [1] .

Several studies have reported the incubation periods in COVID-19 to be 3 ~ 7 days from the first contact [2] [3] . According to the "China's Novel Coronavirus Pneumonia Diagnosis and Treatment Plan (Trial Seventh Edition) ", COVID-19 patients are usually categorized into mild, moderate, severe, and critical based on their symptoms. A large number of clinical trials for potential COVID-19 drugs are underway. Although various treatments such as antiviral therapy, cell therapy, immunotherapy, and Chinese herbal medicine have shown variable efficacy, no drugs or biologics are approved by the FDA for the treatment of COVID-19 at the time of writing this article [4] . Antivirals such as lopinavir and ritonavir did not show better antiviral efficacy than the standard treatment [5] . The use of antimalarialschloroquine and hydroxychloroquine is controversial. A few clinical trials have shown that hydroxychloroquine has failed to treat COVID-19 [6] . The National Institutes of Health has advised discontinuing the clinical trials involving hydroxychloroquine because although harmless, this drug is reported to be ineffective [7] . However, studies from China have demonstrated the efficacy and safety of chloroquine in Chinese patients with COVID-19 [8] . A study from France reported that hydroxychloroquine significantly reduces the viral load in patients with COVID-19, and demonstrates synergistic interactions with azithromycin [9] . Researchers are optimistic that a vaccine will help in getting rid of this epidemic; however, the development cycle of a vaccine is long and time-consuming[10] [11] . Besides fast-tracking the development of COVID-19-specific treatments, we should also test other treatment strategies. One such treatment strategy involves the use of drugs that already in use for different indications, an approach known as drug repurposing. Artemisinin and piperaquine (AP) is a new-generation artemisinin combination therapy (ACT) based antimalarial. The first-line antimalarial drug artemisinin and its derivatives are not only potent antimalarials but also possess considerable antiviral properties [12] [13] [14] .

Artemisinin is reported to reduce the proliferation of the hepatitis B virus [15] , hepatitis C virus [16] [17] , and human immunodeficiency virus [18] [19] . Piperaquine is a bis-quinoline antimalarial drug, similar to chloroquine. Piperaquine was first synthesized in China in the 1960s and is widely used in China and other countries ever since. Chloroquine is not only welltolerated but also has similar potency against P. falciparum and P. vivax [20] . Chloroquine is reported to treat Middle-east respiratory syndrome and atypical respiratory syndrome (SARS) [21] . Moreover, in vitro experiments have shown that chloroquine has an inhibitory effect on SARS-CoV-2 [22] .

AP is the fourth-generation ACT, which plays an essential role in the prevention and treatment of malaria. If AP also treats COVID-19, many countries will benefit, especially the developing countries affected by malaria.

The study was approved by the ethical committee of Hongqi Hospital affiliated with Mudanjiang Medical University (registration number: 202014). The trial protocol and the application documents were submitted to the China Clinical Trial Registration Center for online registration (registration number: ChiCTR2000033049). Initially, this trial was an open-label randomized parallel-group controlled trial intended to compare the efficacy and safety of AP tablets in comparison with hydroxychloroquine to treat patients with mild to moderate COVID-19. Due to the rapid control of this pandemic in China, the number of patients failed to meet the trial requirements. Therefore, the trial was modified to a controlled clinical trial and conducted during the same period. Patients with confirmed SARS-CoV-2 infection were divided into two groups.

Patients of the first group were treated with AP tablets as antiviral and symptomatic treatments, and the second group's patients acted as controls and received hydroxychloroquine/abidol as antiviral and symptomatic treatments.

Inclusion criteria: 1) Age ≥18 years; 2) Confirmed SARS-CoV-2 infection in upper respiratory tract specimens by real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR); 3)

Signature on the informed consent form.

Exclusion criteria: 1) Age <18 years; 2) Pregnancy; 3) Severe malignancy, heart/liver/kidney disease or poorly controlled metabolic diseases; 4) Allergy to 4aminoquinolines; 5) Blood system diseases; 6) Arrhythmia or chronic heart disease; 7) Retinal disease or hearing loss; 8) Mental illnesses or skin diseases (including rash, dermatitis, psoriasis).

The COVID-19 patients were categorized according to the "China's Novel Coronavirus Pneumonia Diagnosis and Treatment Plan (Trial Seventh Edition) " as follows:

Mild: mild clinical symptoms with no pneumonia manifestation in CT imaging; Moderate: symptoms such as fever, cough, and respiratory symptoms, and pneumonia manifestation on CT imaging.

AP group: AP (ARTEPHARM Co., Ltd) was used as an antiviral and symptomatic treatment. AP was orally administrated with a loading dose of two tablets (artemisinin 125mg and piperaquine 750mg) for the first day and followed by a maintenance dose of one tablet/day (artemisinin 62.5mg and piperaquine 375mg) for the next six days. The total dose was eight tablets in 7 days.

Control group: Hydroxychloroquine/Arbidol, according to the "China's Novel Coronavirus Pneumonia Diagnosis and Treatment Plan (Trial Seventh Edition) ", was mainly used as an antiviral and symptomatic treatment. Hydroxychloroquine sulfate (Shanghai Zhongxi Pharmaceutical Co., Ltd.) was orally administered as a loading dose of 800mg/day for the first three days, followed by a maintenance dose of 400mg daily for the next five days. Arbidol hydrochloride (CSPC Ouyi Pharmaceutical Co., Ltd.) was orally administrated 600 mg/day for eight days, divided into three doses daily.

When drug doses completed, positive patients would be continued to receive symptomatic treatment, and meet the discharge conditions until two consecutive tests for nucleic acid turn negative. All the patient should be quarantined for 14-day observation after discharge. The quarantine restriction could be lifted if the tests remain negative.

Critical inspection indicators: Upper respiratory tract specimens were daily obtained from each patient, and RT-PCR for SARS-CoV-2 was conducted in the local Center for Disease Control and Prevention or the hospital. The RT-PCR test kits (BioGerm) [23] could detect the ORF1ab/N gene for SARS-CoV-2 nucleic acid and the reference mean Ct value for detecting the genes was 38. For the FAM channel, the HEX/VIC channel, and the ROX channel of RT-PCR, if the Ct value of the two channels was less than or equal to 38, the result was positive, otherwise was negative. The epidemiological characteristics, clinical symptoms, signs, and adverse events were recorded with case report forms. The clinical features, laboratory findings, chest CT scan data were recorded on case report forms and the hospital case report system. The patients who were negative for SARS-CoV-2 RNA two consecutive times were placed in quarantine for 14 days.

The conclusion was drawn by the professional imaging doctors comparing the before and after images. The main analysis criteria were the number of affected lobes, presence of groundglass nodules, patchy/punctate ground-glass opacities, patchy consolidation, fibrous stripes, and irregular solid nodules in each CT image [24] .

'The time to undetectable viral RNA' was defined as the time between the first dose of drug administered and the time when the RT-PCR for SARS-CoV-2 was negative for the first time.

And the rate of patients to undetected SARS-CoV-2 by RT-PCR at day 7, 10, 14, 21, and 28 during drug administration, the CT images results within ten days, the abnormal laboratory index and adverse events would be compared between the two treatments. To assess the safety of AP, we used the ECG (Japanese photoelectric electrocardiograph ECG-1250C six-channel automatic analysis of 12 leads) to monitor the QT change before and 3-8 days after treatment in the AP group. The Bazetts algorithm corrects the QTc algorithm, and as per the formula QTc = QT / (RR^0.5), and RR= 60 / Heart Rate.

Qualitative indicators were described by percentage or composition ratio for descriptive statistical analysis; the mean and standard deviation describe the quantitative indicators. For qualitative data, we used the chi-square test, Fisher's exact probability method, and the Wilcoxon rank-sum test. The quantitative data conformed to the normal distribution with the t-test and did not conform to the normal distribution with the Wilcoxon rank-sum test for the two groups. The hypothesis test uses a two-sided test uniformly. A p-value of less than 0.05 is considered statistically significant. The overall negative conversion probability was estimated by analyzing the time taken by the patient to reach undetectable viral RNA levels. For this, we used the Kaplan-Meier method and compared the findings with a log-rank test. SPSS19.0 was used to perform statistical analyses. As there was no treatment to prove that the virus has a specific effect, based on ethical requirements, other drugs may interfere with the antiviral effect that has been used clinically, as shown in the list ( Table 2 ). The enumeration of other antiviral drugs was based on facts and might affect the inevitable factors in evaluating the efficacy. Interferon α-1b, ribavirin, lopinavir, oseltamivir, and carrimycin might have certain effects on COVID-19 patients. However, there was no significant statistical difference between the two groups(p＞0.05). In this study, Herbal medicine was used for symptomatic treatment, and the results showed that there was a significant difference between the two groups (p=0.030＜0.05).

The average time to achieve undetectable SARS-CoV-2 RNA in the AP group was significantly less than that in the control group (AP: 10.6±1.1 days (95% CI: 8.4-12.8), control: 19.3±2.1 days (95% CI: 15.1-23.5)) (p=0.001<0.005) ( Figure 2 ). The percentage of the patients to achieve undetectable SARS-CoV-2 at the day 7, 10, 14, 21, and 28 during drug administration in the AP group were 26.1%, 43.5%, 78.3%, 100%, and 100%, respectively, while that in the control group were 5.6%, 16.7%, 44.4%, 55.6% and 72.2%, respectively ( Table 3) . Analysis of these data indicated that the elimination rate of SARS-CoV-2 RNA in the AP group was significantly higher than that in the control group (RD=0.28; 95%CI 0.07-0.49) (Figure 3 ). The length of the patient's hospital stay for AP group was 13.3±4.8 days, and 21.3±9.1 days for control group (Table 3) . No patients had been transferred to severe cases.

Before treatment, the lungs of 82.6% (19/23) of the patients in the AP group and that of 83.3% (15/18) of the patients in the control group had visible inflammation. The CT imaging changes of the patients were analyzed within the next ten days. Improvements in CT imaging were detected in 36.8% (7/19) of the AP group patients and 46.7% (7/15) of the control group patients. 21.1% (4/19) and 6.7% (1/15) of the patients of AP and the control group, respectively, had no significant change. Exacerbations occurred in 10.5% (2/19) of the AP group patients and 13.3%

(2/15) of the control group patients. 31.6% (6/19) of the AP group patients and 33.3% (5/15) of the control group patients were not reexamined within ten days (Table 4) .

The adverse reactions in both the groups were mild and disappeared quickly after treatment. 3 (13%) patients in the AP group and 3 (17%) patients in the control group encountered adverse reactions. The adverse reactions in the AP group patients included nausea, muscle aches, and fatigue while that in the control group patients included nausea, rash, and itching (Table 5 ). In the AP group, post-treatment, three patients had alanine aminotransferase (ALT) levels higher than 80U/L, and one patient had aspartate aminotransferase (AST) levels higher than 60U/L post-treatment. In the control group, post-treatment, two patients had ALT levels higher than 80U/L, and one patient had AST levels higher than 60U/L. showed varying degrees of prolongation, 6 (35.29%) showed mild prolongation (<30ms), 4 (23.53%) demonstrated moderate prolongation (30-60ms), and 2 (11.76%) demonstrated severe prolongation (>60ms). Furthermore, the paired sample t-test showed significant differences between the two groups (p<0.05) (Figure 4 ). AP treatment did not cause TdP and other arrhythmias in the patients. The patients with prolonged QT interval returned to normal after the treatment was discontinued. We have not collected and recorded ECG changes for the control group patients.

Although our study has various limitations, including that of insufficient sample size and trial design, we have decided to publicly share the research data to help in meeting the urgent treatment needs.

In the AP treatment, artemisinin has fast and complete oral absorption, wide distribution, fast metabolism and excretion, and a short half-life (1.93 hours) [25] . And piperaquine disappears slowly in the body, with a long half-life (11.7 days) [26] . Continuous administration has the risk of accumulating toxicity and AP may cause heart rate prolongation in the QT phase [27] .

Therefore, after the first dose of 2 tablets was administered, one tablet a day was chosen to minimize the side effects and maximize the efficacy of artemisinin.

Herbal medicine, including Lianhua Qingwen and Huoxiang Zhengqi in treatment of COVID-19, could significantly improve the patient's symptoms, improve patient prognosis and without serious adverse reactions, but did not significantly contribute to viral assay findings [28] [29] [30] . Based on this, the focus of this study was that AP could significantly shorten the time to reach undetectable SARS-CoV-2. Due to the use of herbal medicine and other symptomatic treatment measures, no further analysis and inferences were made on the improvement of clinical symptoms.

COVID-19 spreads from human to human through droplets, contaminated hands, or surfaces. SARS-CoV-2 has an incubation time of 2-14 days. The spread index of this virus, as estimated by most studies, is between 2.24 and 3.58, which is slightly higher than SARS [31] .

COVID-19 can quickly progress from mild to severe [32] . Classical public health measures, including isolation, quarantine, social distancing, and community containment, are effective to curb this pandemic [33] .

We found that AP shortens the time the virus remains in the body. CT imaging results within ten days of taking AP showed a similar effect on lung improvement as the control group.

As there are no effective antiviral drugs, we recommend AP to be used as eight tablets in seven days for patients with mild to moderate COVID-19. For regions lacking medical facilities, we also recommend AP during isolation and quarantine periods, if the condition of the patient worsens. For the patients with suspected close contacts of COVID-19, we recommend AP (eight tablets within seven days) as a precautionary treatment.

Besides, SARS-CoV-2 directly infects endothelium and causes immune cell recruitment that results in extensive endothelial dysfunction and apoptosis. These manifestations make the blood of the patient more viscous and result in thrombus formation [34] . As per studies, the adult dose of AP for malaria consists of four tablets (artemisinin 250 mg and piperaquine 1500 mg), which showed prolonged QT-interval in some people [35] . The total recommended AP adult dose for the treatment of COVID-19 consists of eight tablets (artemisinin 500 mg and piperaquine 3000 mg). We found severe prolongation in two patients (11.76%) and significant differences between the two groups. Although AP treatment can cause the QT interval prolongation in some patients, it did not cause TdP and other arrhythmias. Moreover, patients with prolonged QT intervals return to normal after the drug is discontinued. However, considering the effect of SARS-CoV-2 on blood vessels and the side effects of AP, we still recommend close monitoring of QT segment changes during AP treatment.

Moreover, we will also investigate the mechanism of how AP is improving the health of patients with COVID-19. 

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We would like to thank all the patients who participated in this study and their families. We would like to thank all the health care workers at the field sites who are fighting this pandemic.The authors would like to thank all the reviewers who participated in the review and MJEditor (www.mjeditor.com) for its linguistic assistance during the preparation of this manuscript.

Funding: This work was supported by the Natural Science Foundation of China [Grant Number