First clinical study using HCV protease inhibitor danoprevir to treat COVID-19 patients

Abstract

1. Introduction

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has spread rapidly since its identification in patients with severe pneumonia in Wuhan, China. The coronavirus disease 2019 (COVID-19), the disease caused by SARS-CoV-2 infection, has been reported more than 118,000 cases in 114 countries, and 4291 people have lost their lives by March 11, 2020. Given its global outbreak and strong human-to-human transmission and high mortality in the elderly or in patients with complications, the World Health Organization (WHO) has made the assessment on March 11, 2020 that COVID-19 can be characterized as a pandemic.^[1]

Investigation into approved drugs for new therapeutic indications, which is also known as drug repurposing, has been proved to be a practical strategy in the situation of an outbreak such as COVID-19.^[2,3] Danoprevir is a potent hepatitis C virus (HCV) protease inhibitor with an IC₅₀ of 0.29 nM against HCV protease ^[4] and was approved and marketed in 2018 in China as an oral direct-acting antiviral agent to treat hepatitis C. The results of a phase 3 clinical trial with 140 patients showed that the triple regimen of ritonavir-boosted danoprevir plus pegylated-interferon α-2a and ribavirin produced a sustained virologic response (SVR12) rate of 97.1% after 12-week treatment in non-cirrhotic HCV genotype 1-infected Chinese patients.^[5] In another phase 2/3 clinical trial, the SVR12 rate is 99% in treatment naïve, non-cirrhotic HCV GT1-infected patients after 12-week all oral treatment of ritonavir-boosted danoprevir combined with ravidasvir (an HCV NS5A inhibitor) and ribavirin.^[6] These results indicated that danoprevir boosted by ritonavir is highly efficacious, safe, and well tolerated in HCV patients.

During SARS-COV-2's life cycle, chymotrypsin-like protease (3CL pro) can cleave viral polyprotein to form the RNA replicase–transcriptase complex, which is essential for both viral transcription and replication.^[7,8] The proteases of HCV and human immunodeficiency virus (HIV) showed similar function as SARS-COV-2, so protease inhibitors are hypothesized to have the therapeutic potential against COVID-19. In vitro and clinical studies in patients with SARS and Middle East respiratory syndrome (MERS) to some extent proved this hypothesis.^[9–11] The homology modeling data indicated that HCV protease inhibitors have the highest binding affinity to SARS-CoV-2 protease among approved drugs.^[12] Molecular docking method analysis showed that danoprevir has the best binding energy for inhibition of main protease of SARS-CoV-2 (−9.3 kcal/mol).^[13] Yet there is still a paucity of clinical data on HCV protease inhibitors in treating COVID-19 patients.

In this study, we for the first time demonstrated the therapeutic efficacy of danoprevir boosted by ritonavir in both treatment naïve and experienced COVID-19 patients.

2. Methods

3. Results

3.1. Baseline demographic and clinical characteristics

A total of 11 moderate COVID-19 patients were enrolled, including 2 naive patients and 9 experienced patients. The baseline demographic characteristics for all enrolled patients are illustrated in Table ​Table1.1. The age range of the patients were 18 to 66 years old, with 4 males in total 11 patients. Two experienced patients, patient 6 and patient 8, had a history of hypertension. The symptoms reported at illness onset were mainly fever, cough, and shortness of breath. Two naive patients were hospitalized for 9 and 7 days, respectively, and the experienced patients were hospitalized for a median duration of 20 days.

Table 1

Baseline demographic characteristics, underlying medical condition, and clinical characteristics of 11 patients receiving danoprevir boosted by ritonavir treatment.

	Naive (N = 2)	Experienced (N = 9)
Median age – yr	44, 18	44 (18–66)∗
Median hospitalization – d	9, 7	20 (7–22)∗
Male	1	3
Underlying medical conditions	No, No	2†
Symptoms reported at illness onset
Fever	No, no	4
Cough	No, yes	3
Shortness of breath	No, yes	6

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The key parameters of 11 patients were presented in Table ​Table22 and illustrated graphically in Figure ​Figure1.1. In general, 9 patients experienced lopinavir/ritonavir along with interferon nebulization treatment were enrolled and switched to danoprevir/ritonavir treatment, including 4 patients with interferon nebulization and 5 patients without interferon nebulization after switching. Two naive patients received danoprevir/ritonavir plus interferon nebulization treatment. After initiation of danoprevir/ritonavir treatment, the first negative RT-PCR test occurred at a median of 2 days, ranging from 1 to 8 days, and the obvious absorption in CT scans occurred at a median 3 days, ranging from 2 to 4 days.

Table 2

Key parameters of 11 patients receiving danopreivr boosted by ritonavir treatment.

	Days of hospitalization	Antiviral experience before danoprevir/ritonavir treatment	Days of lopinavir/ritonavir plus IFN nebulization treatment prior to the study	Days of danoprevir/ritonavir plus IFN nebulization treatment during the study	Days of danoprevir/ritonavir treatment without IFN nebulization during the study	Days of first negative RT-PCR test after initiation of danoprevir/ritonavir treatment∗	Days of obvious absorption in CT scans after initiation of danoprevir/ritonavir treatment
Patient 1	9	Naive	NA	9	NA	2	4
Patient 2	7	Naive	NA	7	NA	4	2
Patient 3	7	Experienced	3	4	NA	1	2
Patient 4	10	Experienced	2	8	NA	1	4
Patient 5	17	Experienced	5	12	NA	2	3
Patient 6	9	Experienced	3	6	NA	3	3
Patient 7	22	Experienced	13	NA	9	2	3
Patient 8	20	Experienced	11	NA	5	2	3
Patient 9	21	Experienced	13	NA	8	2	4
Patient 10	21	Experienced	13	NA	8	4	†
Patient 11	22	Experienced	10	NA	9	8	3

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Figure 1

Timeline of RT-PCR tests, CT scans, and antiviral treatments of the enrolled patients. ^#Patient 10's lung imaging did not show obvious lesion at admission and during the hospitalization.

3.2. Naïve patients receiving danoprevir/ritonavir plus interferon nebulization

Two treatment naïve patients received danoprevir/ritonavir plus interferon nebulization upon hospitalization. No patient had composite adverse outcomes during this study. The medical history, treatment, and various examinations for 1 of 2 patients, patient 1, were described in details below.

On February 19, 2020, a woman aged 44, who had a close contact with a COVID-19 patient, was tested positive SARS-COV-2 in nasal swab with RT-PCR and thus admitted to the hospital. The vital sign of the patient showed body temperature (BT) of 36.4 °C and oxygen saturation (SPO₂) of 99% under ambient air. The breathing sound was normal initially. She had no other underlying disease before this onset. In the morning of February 20, she was given the first dose of danoprevir boosted by ritonavir, along with interferon nebulization. On February 20, both lower lungs showed multiple diffuse patch-shape areas and ground-glass opacity (GGO), especially obvious in the left lower lung, based on chest CT scans (Fig. ​(Fig.2A).2A). The laboratory results revealed lymphopenia (0.62 × 10⁹/L) and elevated platelet (322 × 10⁹/L) (Table ​(Table3).3). On February 22, 3 days after receiving danoprevir boosted by ritonavir treatment, the patient did not show abnormal symptoms or vital signs. The lower lobe of bilateral lung showed decreased GGO and patch-shaped areas compared to the previous image on February 20, indicating partial absorption (Fig. ​(Fig.2B).2B). The RT-PCR results of nasal swab became negative. These results indicated the patient had a quick response to the treatment of danoprevir boosted by ritonavir, along with interferon nebulization. On February 24, 5 days after receiving danoprevir boosted by ritonavir treatment, the GGO and patch-shaped areas in both lungs significantly decreased in density and scale (Fig. ​(Fig.2C)2C) compared to the previous image on February 22. And the RT-PCR results of nasal swab were repeatedly negative on February 24 and February 26. On February 28, patient #1 was given the last dose of danoprevir boosted by ritonavir, along with interferon nebulization. Since she met all 4 discharge standards described in Section 2, the patient was discharged from the hospital on the same day.

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Figure 2

Naïve patient receiving danoprevir/ritonavir plus interferon nebulization (patient 1). GGO, ground-glass opacity. Red arrows indicate the possible lesions.

Table 3

Laboratory tests and underlying disease of 3 representative patients before and after receiving danopreivr boosted by ritonavir treatment.

	Patient 1 (naïve)		Patient 6 (lopinavir/ritonavir experienced)		Patient 8 (lopinavir/ritonavir experienced)
Age (yr)	44		47		42
Sex	Female		Male		Female
Underlying disease	No		Hypertension		Hypertension
	Hospital day 1, before danoprevir/ritonavir treatment	5 d after danoprevir/ritonavir treatment	Hospital day 1, before danoprevir/ritonavir treatment	4 d after switching to danoprevir/ritonavir treatment	Hospital day 1, before danoprevir/ritonavir treatment	4 d after switching to danoprevir/ritonavir treatment
White-cell count (109/L)	4–10	8.26	–	6.5	6.34	5.58
Red-cell count (109/L)	3.5–5.5	3.81	–	5.69↑	3.95	4.77
Neutrophil count (109/L)	2–7.7	7.3	–	3.66	4.42	4.46
Lymphocyte count (109/L)	0.8–4	0.62↓	–	2.23	1.36	0.65↓
Platelet count (109/L)	100–300	322↑	–	170	211	145
Hemoglobin (g/L)	110–160	120	–	112	101	147
Hematocrit (%)	36–50	35.2↓	–	36	27.3↓	43.0
Sodium (mmol/L)	136–144	142	139.5	142.3	141.41	139.8
Potassium (mmol/L)	3.6–5.5	3.52↓	4.47	3.54↓	4.18	3.21↓
Chloride (mmol/L)	96–108	93.7↓	96.0	96.76	97.96	98.7
Calcium (mmol/L)	2.2–3.2	2.2	–	2.22	2.19↓	2.31
Glucose (mmol/L)	3.88–6.11	6.58↑	4.76	10.2↑		6.06
Urea (mmol/L)	2.9–6.42	2.49↓	3.07	4.82	4.55	2.83↓
Creatinine (mmol/L)	56–97	62	67	85	93	69
Total protein (g/L)	60–87	73.9	68	71	63.7	74.1
Albumin (g/L)	35–55	39.4	37.7	42.8	37.5	47.9
Total bilirubin (μmol/L)	3.42–20.5	7.8	10.5	9.6	9	12.9
Procalcitonin (ng/ml)	0–0.5	0.12	–	<0.1	<0.1	–
ALT (U/L)	0–40	11.8	10.4	24	18.8	26.4
AST (UL)	0–40	15.5	14.2	22.6	15.5	21.0
ALP (U/L)	0–150	56	53	79	75	72
Fibrinogen (g/L)	2–4	2.92	–	4.38↑	–	3.86
LDH (U/L)	109–245	151	–	177	150	219
PT (s)	10–14	11	–	12.3	–	12.1

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3.3. Experienced patients treated with lopinavir/ritonavir plus interferon nebulization and switched to danoprevir/ritonavir with or without interferon nebulization

Nine treatment experienced patients were on lopinavir/ritonavir plus interferon nebulization treatment ranging from 2 to 13 days. Four patients experienced lopinavir/ritonavir plus interferon nebulization treatment for 2 to 5 days with CT scans worsened and switched to danoprevir/ritonavir with interferon nebulization. No patient had composite adverse outcomes during this study. The medical history, treatment, and various examinations for one of the patients, patient 6, were described in details below.

On February 14, 2020, a man aged 47 was admitted in to the hospital for positive RT-PCR nucleic acid test of SARS-CoV-2 in nasal swab. His family member was diagnosed with COVID-19 on February 11. Based on chest CT scans, subpleural area of the right lower lung showed GGO (Fig. ​(Fig.3A).3A). On admission, the patient reported a 3-day history of chest distress and fatigue. He showed no signs of fever, shortness of breath, nausea, and diarrhea. He had a history of hypertension. In the evening of February 14, he was given the first dose of Kaletra (lopinavir (200 mg)/ritonavir (50 mg), per tablet), 2 tablets at a time, twice per day, along with interferon nebulization. On February 15, the patient reported he still had chest distress. Soft thin stool was still reported and occasional cough and white sputum appeared. The patient continued lopinavir/ritonavir treatment and received rehydration supportive care. On February 17, 3 days after receiving lopinavir/ritonavir treatment, the lower lung showed further consolidation, along with GGO and patch-shape areas based on CT scans (Fig. ​(Fig.3B).3B). Therefore, in the evening of February 17, lopinavir/ritonavir treatment was stopped and the patient was switched to danoprevir boosted by ritonavir, along with interferon nebulization. On February 18, patient had a weak positive RT-PCR test for viral RNA in nasal swab. On February 20, 4 days after receiving danoprevir boosted by ritonavir treatment, the right lower lung showed shrunken patch-shape areas and decreased density of GGO (Fig. ​(Fig.3C).3C). On the same day, RT-PCR test with the nasal swab became negative and remained negative at the next day RT-PCR test. In the morning of February 23, the patient received the last dose of danoprevir boosted by ritonavir treatment. Since he met all 4 discharge standards described in Section 2, the patient was discharged from the hospital on the same day.

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Figure 3

Experienced patient treated with lopinavir/ritonavir plus interferon nebulization for 3 d and switched to danoprevir/ritonavir plus interferon nebulization (patient 6). GGO, ground-glass opacity. Red arrows indicate the possible lesions.

Five patients failed lopinavir/ritonavir plus interferon nebulization and switched to danoprevir/ritonavir without interferon nebulization. The medical history, treatment, and various examinations for 1 of patients, patient 8, were described in details below.

On February 9, a woman aged 42, who was a permanent resident in Wuhan City, was admitted to the hospital due to maximum BT 38 °C and RT-PCR positive SARS-CoV-2 in nasal swab. She had occasional coughs with white sputum. The vital sign of the patient showed BT of 37.4 °C and SPO₂ of 99% under ambient air. The patient had half-year history of hypertension. The patient was given the first dose of lopinavir/ritonavir, 2 tablets at a time, twice per day, along with interferon nebulization in the evening of February 9. On February 10, based on CT scans, subpleural areas of both lower lungs and the tongue-like segment of the left lung showed GGO and patch-shape areas (Fig. ​(Fig.4A).4A). From February 10 to February 13, the patient showed slightly fluctuating BT (maximum 37.3 °C to minimum 36.4 °C), coughs with white sputum, anorexia, and diarrhea with yellow thin stool. The laboratory results revealed leukopenia (3.40 × 10⁹/L) and lymphopenia (0.7 × 10⁹/L) (Table ​(Table3).3). On February 13, 4 days after receiving lopinavir/ritonavir treatment, GGO and patch-shape areas showed a slightly higher density (Fig. ​(Fig.4B)4B) compared to previous CT image on February 10. On February 17, 8 days after receiving lopinavir/ritonavir treatment, the patient reported improved appetite and sleeping while coughing with white sputum remained. CT scan showed that no significant changes were observed compared to previous CT image on February 13 and the opacities at tongue-like segment increased (Fig. ​(Fig.4C).4C). On February 19, the patient received the last dose of lopinavir/ritonavir and interferon nebulization. The treatment duration of both lopinavir/ritonavir and interferon nebulization exceeded the recommendation by China National Standards.^[14] As a result, both lopinavir/ritonavir and interferon nebulization were stopped. On February 20, she was switched to danoprevir boosted by ritonavir without interferon nebulization. On February 22, 3 days after receiving danoprevir/ritonavir treatment, RT-PCR test was negative in both nasal and phlegm swabs. On February 23, 4 days after switching to danoprevir boosted by ritonavir, the patient showed improved white blood cell count (4.31 × 10⁹/L) (Table ​(Table3).3). CT scans showed GGO and patch-shape areas decreased in density compared to previous CT images on February 17, indicating absorption in both lungs (Fig. ​(Fig.4D).4D). On February 25, the patient received the last dose of danoprevir boosted by ritonavir. On February 26, CT scans showed the lesions in tongue-like segment of the lung were completely absorbed. Opacities in both lower lungs decreased in density compared to previous CT images (Fig. ​(Fig.4E).4E). RT-PCR tests were repeatedly negative in both nasal and phlegm swabs on February 26 and February 27. On February 29, the patient was discharged from the hospital since she met all 4 discharge standards as described in Section 2.

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Figure 4

Patient failed lopinavir/ritonavir plus interferon nebulization and switched to danoprevir/ritonavir without interferon nebulization (patient 8). GGO, ground-glass opacity. Red arrows indicate the possible lesions. ^∗Viral RNA was undetectable in both nasal and phlegm swabs.

4. Discussion

Our study for the first time demonstrated the therapeutic efficacy in moderate COVID-19 patients with the treatment of danoprevir, an HCV NS3/4A protease inhibitor marketed in China, boosted by ritonavir, a pharmacokinetic enhancer of danoprevir and an HIV protease inhibitor. As a marketed drug, danoprevir has been demonstrated safe and well tolerated in thousands of HCV patients in China. Ritonavir is approved HIV drug and pharmacokinetic booster with more than 15-year clinical use.

Although many clinical trials involving many different drugs are ongoing in China and globally, including protease inhibitors, RNA-dependent RNA polymerase (RDRP) inhibitors, none of them has been approved for COVID-19 indication by regulatory authorities of major counties. In a randomized, controlled, open-label trial involving hospitalized adult patients with severe illness caused by SARS-CoV-2 infection (SaO₂ of 94% or less while they were breathing ambient air or PaO₂/FiO₂ ≤ 300 mm Hg), there were no significant differences between lopinavir–ritonavir treatment and standard care in terms of time to clinical improvement, mortality at 28 days, and percentages of patients with detectable viral RNA at various time points.^[15] A retrospective study of COVID-19 patients demonstrated that no significant differences were observed between lopinavir/ritonavir group (all 52 patients were moderate) and control group (without receiving any antiviral drugs; 91.67% of 48 patients were moderate) in terms of median time to temperature normalization, median time to PCR negative, PCR negative rates at day 7 after admission and radiological worsening at day 7.^[16]

Around 5% of COVID-19 cases were critical with a mortality of over 50%,^[17] so it is essential to treat and prevent the mild or moderate cases progressing into severe or critical ones. Our report demonstrated that danoprevir boosted by ritonavir can suppress viral replication in less than 1 week and effectively reduce GGO and patch-shape areas. Viral nucleic acids in nasal swabs turned negative at a median of 2 days and the absorption occurred at a median of 3 days after the initiation of danoprevir/ritonavir treatment.

The mortality rate of COVID-19 patients with hypertension, cardiovascular diseases, and with preexisting comorbid conditions was much higher than normal patients (6.0–10.5% vs 2.3%).^[18] Two patients involved in this report had a history of hypertension, but they showed a quick response with negative PCR tests in 2 days and normalized lung imaging in 3 days after the initiation of danoprevir/ritonavir treatment, respectively. However, we still need large-scale study to investigate the efficacy of danoprevir/ritonavir in COVID-19 patients with hypertension.

Of 11 patients enrolled, 8 patients showed significant clinical manifestations such as fever, cough, and dry coughing, while 3 patients did not show significant clinical manifestations. Therefore, the most reliable indicators for treatment efficacy should be improved lung imaging via CT scans and dramatically reduced virus load through RT-PCR tests.

This study highlights the first-time successful treatment of 3 COVID-19 patients by HCV protease inhibitor. Nucleic acid testing results suggested 4 to 12-day treatment of danoprevir/ritonavir can effectively suppress viral replication and improve patient's health conditions, especially for those patients with moderate COVID-19. This report also highlights the therapeutic effect of danoprevir/ritonavir treatment on both antiviral naive and experienced patients. SARS-COV-2 is an RNA virus and thus prone to mutate under the antiviral drug pressure. In the real world of clinic practice, patients usually experience various kinds of treatments, which could lead to drug-resistant mutations. Combination of agents that target different phases of viral life cycle should minimize the incidents of drug-resistance. However, there were several limitations in this study. First, lack of a control group limited the comparability to other studies since failure in therapy may be due to the factors otherwise the investigational regimen. Second, due to the adequate control of SARS-COV-2 pandemic in China, the sample size is small. Thus, large-scale study with control is needed to investigate the efficacy and safety of danoprevir on the COVID-19.

5. Conclusions

In conclusion, repurposing danoprevir, a marketed HCV protease inhibitor, represents a promising therapeutic option for COVID19 patients.

Acknowledgments

We thank all the patients involved in this study; the nurses and clinical staff who are providing care for the patients; staff at the clinical laboratory of the Ninth hospital of Nanchang.

Author contributions

HC and JW conceived and designed the study. ZZ, FG, ZH, and LW contributed and performed data collection and analysis. HC, XL, and JW contributed to the literature search and manuscript drafting. YC, HC, and JW contributed to the planning and operation of the clinical study. All authors provided critical review of the manuscript and approved the final draft for publication.

Conceptualization: Hongyi Chen, Jinzi Wu.

Data curation: Zhicheng Zhang, Li Wang, Zhihua Huang, Fanghua Gong.

Investigation: Hongyi Chen, Xiaodong Li, Jinzi Wu.

Project administration: Hongyi Chen, Yahong Chen, Jinzi Wu.

Writing – original draft: Hongyi Chen, Xiaodong Li, Jinzi Wu.

Writing – review & editing: Hongyi Chen, Zhicheng Zhang, Li Wang, Zhihua Huang, Fanghua Gong, Xiaodong Li, Yahong Chen, Jinzi Wu.

Footnotes

References