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From February 20th to February 28th, ten patients with laboratory-confirmed COVID-19 were enrolled in this study to evaluate the efficacy of Leflunomide on SARS-CoV-2 infection. The ten patients randomly assigned into two groups (5 for each) and all received the standard supportive treatments (Arbidol, Lianhua Qingwen Capsule, Magnesium Isoglycyrrhizinate, and Cefoperazone). One group of five patients treated with Leflunomide with a similar dose as the rheumatoid arthritis (RA) treatment, another group of five patients were treated as blank controls without a placebo. There were no significant differences in age (51[49-63] vs 54 [50-59]) and gender (2:3 vs 1:4 [M: F]) between Leflunomide treatment group and blank group, and all the patients are categorized as moderate type according to COVID-19 diagnosis and treatment guide (7th version) issued by the Chinese National Health and Care Commission. All the patients had fevers and respiratory symptoms and clear inflammatory lesions in chest CT before treatment. The patients' information is summarized in Table 1. The Leflunomide treatment group took Leflunomide pills orally for 10 continuous days.
Patient No. Sex Age, y Onset days Anamnesis Patients treated with Leflunomide 1 M 65 10 None 2 F 61 10 Hypertension 3 M 47 9 None 4 F 51 8 Hypertension, Hyperlipidemia 5 F 51 10 Hypertension, Atherosclerosis Patients treated with blank 1 F 54 9 Hypertension, Atherosclerosis, Hysteromyoma 2 F 50 10 Atherosclerosis 3 M 56 10 Hypertension 4 F 63 8 COPD 5 F 51 9 Hypertension P value 1 0.91 Table 1. Clinical characteristics of SARS-CoV-2-infected patients.
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The whole blood routine examination was conducted before and after treatment for all the patients. The data were summarized in Table 2. There were no differences between two groups or between pre- and post-treatment in each group in either the counts of white blood cell (WBC), neutrophilia (N), lymphocytes (L), platelet, or the levels of alkaline, phosphatase, bilirubin, potassium, sodium, urea, creatinine and albumin (P ≥ 0.05). A slight decrease of hemoglobin was observed after treatment in both the Leflunomide treatment group (from 140 to 115 g/L, P > 0.05) and the control group (from 135 to 112 g/L, P < 0.05). Nevertheless, it is considered to be no difference as hemoglobin levels can be affected by multiple factors beyond the drug intervention. The level of creatine kinase also decreased after treatment in both the Leflunomide treatment group (from 50 to 24 mg/L, P > 0.05) and in the control group (from 70 to 48 mg/L, P < 0.05). However, due to samples missing in after-treatment of the Leflunomide treatment group, there is no statistical significance in creatine kinase degrade between two groups (P > 0.05). Nevertheless, the changes in creatine kinase after treatment may suggest that heart muscle damage could occur in SARS-CoV-2 infections.
Parameters Leflunomide, n = 5 Control, n = 5 Before treatment After treatment P value Before treatment After treatment P value WBC, × 109/L (3.5–9.5) 4.67 (3.91–7.61) 5.97 (4.34–9.69) 0.715 5.28 (3.95–6.97) 5.58 (3.27–7.47) 0.893 Differences before and after treatment 1.73 (0.63–5.59) – 0.95 (0.68–2.27) 0.624 N, × 109/L (1.8–6.3) 2.80 (2.00–6.60) 4.56 (2.54–7.52) 0.715 2.78 (1.81–4.62) 2.89 (1.48–4.3) 0.893 Differences before and after treatment 2.04 (0.31–5.08) – 0.49 (0.33–1.90) 0.327 L, × 109/L (1.1–3.2) 0.9 (0.58–1.58) 1.18 (0.95–1.41) 0.465 1.83 (1.13–2.02) 2.21 (1.30–2.36) 0.225 Differences before and after treatment 0.46 (0.30–0.55) – 0.36 (0.17–0.68) 0.624 Platelet count, × 109/L (125–350) 217 (200.5–230.5) 195.5 (152.5–270) 0.465 258 (181–315) 231 (221.5–246.5) 0.893 Differences before and after treatment 40.5 (4.75–86) – 43 (31.5–79.5) 0.624 Alkaline phosphatase, U/L (45–125) 74 (45.5–156) 76 (48–143.75) 0.273 76 (67–83) 73 (65–85.5) 0.893 Differences before and after treatment 15.5 (5.75–54.5) – 4 (2–12) 0.11 Bilirubin, mmol/L (0–23) 15.4 (11.2–18.15) 13.45 (6.7–20.58) 1 8.5 (6.05–10.65) 6.8 (5.95–7.1) 0.225 Differences before and after treatment 4.2 (1.98–10.4) – 1.8 (1.25–4.15) 0.176 Potassium, mmol/L (3.5–5.3) 3.79 (3.45–4.05) 3.75 (3.23–4.07) 0.715 3.9 (3.8–4.16) 4.26 (3.72–4.48) 0.345 Differences before and after treatment 0.19 (0.12–0.35) – 0.57 (0.05–0.7) 0.624 Sodium, mmol/L (137–147) 132 (132–139) 139 (135.75–143.75) 0.068 137 (134.5–141.5) 143 (138.5–147.5) 0.136 Differences before and after treatment 5.5 (3.5–11.25) – 3 (1.5–12.0) 0.387 Urea, mmol/L (2.17–7.14) 3.1 (2.25–4.63) 3.62 (2.1–5.56) 1 4.89 (4.30–5.58) 4.55 (3.66–4.67) 0.138 Differences before and after treatment 0.62 (0.16–2.28) – 0.64 (0.34–1.48) 0.624 Creatinine, μmol/L (57–97) 51 (43.5–78.0) 63 (44.25–78) 0.285 55 (47.5–61.5) 52 (47.5–58) 0.581 Differences before and after treatment 3 (0.5–6.25) – 4 (1.5–8.5) 0.707 Albumin, g/L (40–55) 40.1 (37.0–43.6) 31.7 (26.35–37.43) 0.068 41.8 (38.45–44.45) 40.8 (39.6–41.65) 0.686 Differences before and after treatment 6.35 (4.48–16.18) – 3.8 (1.05–4.5) 0.05 Haemoglobin, g/L (130–175) 140 (117–157.5) 115 (101–134) 0.08 135 (125–178.5) 112 (108.5–127) 0.043 Differences before and after treatment 26 (4–38) – 27 (8–58) 0.465 Creatine kinase, U/L (50–310) 50 (34.5–79.5) 24 (19– N.T.) 0.109 70 (41.5–96) 48 (30.5–63) 0.043 Differences before and after treatment 31 (25– N.T.) – 11 (3.5–46) 0.368 CRP, mg/L (0–10) 37.4 (7.8–120.6) 5 (5–5) 0.109 5 (5–14.75) 5 (5–5.7) 0.18 Differences before and after treatment 32 (5.6–N.T.) – 0 (0–9.05) 0.047 ALT, U/L (9–50) 26 (19.5–80.5) 123.5 (61.25–251.75) 0.068 18 (17–34.5) 23 (15–42) 0.684 Differences before and after treatment 58.5 (36.75–186) – 8 (6.5–29.5) 0.049 AST, U/L (15–40) 23 (17.5–47.5) 83(37.25–96.5) 0.068 18 (17–23.5) 16 (14.5–35) 0.893 Differences before and after treatment 48.5 (13.5–55) – 6 (4–20) 0.176 Tmax (℃) 38 (38–38.85) 38 (37.95–38.65) 0.738 Antipyretic time (d) 2 (1–3) 3 (2.5–3.5) 0.131 The data are presented as the medians and interquartile ranges. P values comparing cases are from Mann-Whitney U-test.
WBC white blood cell, N neutrophil, L lymphocyte, CRP C-reactive protein, ALT alanine aminotransferase, AST aspartate aminotransferase, Tmax maximum body temperature, N.T. not tested.Table 2. Clinical parameters between the Leflunomide group and the control group.
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There was a significant decrease in the level of the inflammatory biomarker C-reactive protein (CRP) in the Leflunomide treatment group (from 37.4 to 5 mg/L) when compared with the control group (from 5 to 5 mg/L) (P = 0.047) (Table 2), indicating that Leflunomide could repress harmful inflammations in COVID-19 as it does in RA treatment. However, due to patient recruitment difficulties, all five patients in the control group exhibited low levels of CRP before treatment as compared to the Leflunomide treatment group.
Considering the clinical features, although there were no differences in body maximum temperature (Tmax) and antipyretic time, the Leflunomide treatment group showed significantly shorter duration of viral shedding time in 3 patients (5 days [median]) as compared to the control group (n = 3) (11 days [median]) with P = 0.046 (Table 3). Two patients in each group (Patient No. 2 and 5 in the Leflunomide treatment group and Patient No. 2 and 3 in the control group) showed negative in viral shedding immediately on the day of drug administration but still with obvious lung inflammatory opacities. We, therefore, excluded these four patients in viral shedding time analysis but continued with treatment until they met the discharge criteria.
Patient No. Patients treated with Leflunomide Patients treated with blank P value 1 3 4 1 4 5 Viral shedding time after treatment (d) 4 8 5 11 9 11 0.046 The data are presented as medians. P values comparing cases are from Mann-Whitney U-test. Table 3. Viral shedding time after drug treatment of indicated patients.
Because viral nucleic acid detection by RT-PCR kit may have false-negative results due to the limit of detection, the chest CT is more reliable for pneumonia diagnosis to visualize the lung damage lesions. The chest CT imaging from a representative patient in the Leflunomide treatment group showed patchy ground-glass opacity in both left and right lobes before treatment, which are the common CT features for COVID-19 patients (Fig. 1A). However, at 7 days after Leflunomide treatment, the areas of ground-glass opacity became much smaller and there was obvious absorption of lesions in the bilateral lung as compared to before-treatment (Fig. 1B). Moreover, this patient (No. 4) has a disease history of both hypertension and hyperlipidemia, but she responded positively to Leflunomide treatment by clearing the virus within 5 days after-treatment and showed obvious absorption of lung inflammatory opacities.
Figure 1. CT image of a patient in the Leflunomide treatment group. A 51-year-old female was admitted to the hospital for 8 days due to fever, fatigue, and myalgia, with a maximum body temperature of 38 ℃. A comparison of CT imaging was shown before and at day 7 after treatment with Leflunomide. Arrows show the ground-glass opacity in the lung.
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The levels of liver enzymes Alanine Aminotransferase (ALT) and Aspartate transaminase (AST) both increased in the Leflunomide treatment group as compared to the control group (P = 0.049 and P = 0.176 respectively) (Table 2). The elevations of these two enzymes reflected the commonly observed adverse effects of Leflunomide and other drugs that need to be metabolized through the liver. However, such increases are normally reversible after stopping drug administration and can be cleaned by standard clearance protocols. Besides this, no other obvious adverse effects in the Leflunomide treatment group were observed.