Patients
The median age of the patients was 62 (0.1 mg/kg NuSepin), 59 (0.2 mg/kg NuSepin), and 63 (placebo) years-old (Fig. 1, Supplementary table 1). 45% (0.1 mg/kg NuSepin), 55% (0.2 mg/kg NuSepin), and 50% (placebo) were male. 100% of patients were Caucasian. The patients had comorbidities at enrollment, most commonly hypertension [n= 35 (55%)], obesity [n=19 (30%)], and diabetes mellitus [n=16 (25%)].
17 patients (27%) were mild (with baseline NEWS2 < 5), 11 patients (17%) were moderate (with baseline NEWS2 = 5~6), and 36 patients (56%) were severe (with baseline NEWS2 ≥ 7) at randomization day (Supplementary Table 2). No patients were with baseline OS = 5, and one patient in each group had a baseline OS = 2 (Supplementary Table 3). 38 patients (59.4%) received anti-viral drugs, such as Lopinavir-Ritonavir, Remdesvir, Favipiravir, etc. (Supplementary Table 4). 16 patients received immune modulators, such as tocilizumab, hydroxychloroquine, or anakinra, respectively (Supplementary Table 5).
Primary outcome
There were no significant differences in TTCI_P between groups. In this report, data for the efficacy of 0.1 mg/kg NuSepin were not included because it did not show statistically significant differences with placebo in outcomes tested. In the mITT population, none of the patients recovered until day 4 when assessed by TTCI_P. At day 9, 38.1% and 15.8% of patients recovered in the NuSepin group and the placebo group, respectively. The RR was 1.34 (95% C.I., 0.7 ~2.6, p > 0.05), which favored improved recovery in the NuSepin group numerically. In the PP population, 42.1% and 17.6% of patients recovered at day 9 in the NuSepin group and the placebo group, respectively. The RR was 1.9 (95% C.I., 1.0 ~3.9, p > 0.05), which favored improved recovery in the NuSepin group numerically. When moderate-to-severe patients of the PP population were analyzed, the mean % decrease of OS at day 9 in the patients of the NuSepin group (41.7 ± 3.7) was significantly higher than the placebo group (24.3 ± 5.9, p = 0.028, Supplementary Fig. 1).
Secondary outcome
First, the effects of covariates on TTCI_S were evaluated by Cox proportional hazard regression analysis (Fig. 2). The baseline NEWS2 and use of anti-viral drugs significantly affected TTCI_S. In the patient stratum with baseline NEWS2 ≥ 5, RR = 2.7 (95% CI: 1.2-6.4, p = 0.02). In the patient stratum who received anti-viral drugs, RR = 3.7 (95% CI: 1.4-10.2, p = 0.01). When the significant covariates (baseline NEWS2 ≥5 and use of anti-viral drugs) were adjusted, overall RR between NuSepin and placebo was 3.4 (1.5-7.4, p = 0.0026), which significantly favors the recovery rate of the NuSepin group in the PP population.
We analyzed covariate effects of baseline NEWS2 (Supplementary Fig. 2, Supplementary Table 6). In the ITT population, clinical remission of patients with baseline NEWS2 ≥ 5 was significantly delayed compared to patients with baseline NEWS2 < 5. For this reason, we analyzed TTCI_S after stratifying the ITT population with baseline NEWS2.
When PP population was analyzed, mean % decrease of NEWS2 at day 9 in the moderate-to-severe patients of the NuSepin group (89.4 ± 3.0) was significantly higher than the placebo group (64.3 ± 10.2, p = 0.041, Supplementary Fig. 3).
When clinical recovery of moderate-to-severe patients in the ITT population was analyzed, the median TTCI_S was 9.0 d [95% C.I. 7.0~12.0] in the NuSepin group and 12.5 d [95% C.I. 8.0~25.0] in the placebo group (p = 0.086) (Fig. 3A). The median TTCI_S was 9.0 d [95% C.I. 7.0~11.0] in the NuSepin group and 12.5 d [95% C.I. 8.0~25.0] in the placebo group (p = 0.016), when moderate-to-severe patients of the PP population were analyzed (Fig. 3B). In this subgroup of patients, RR was 2.7 (95% C.I., 1.2 ~ 6.4, p = 0.02), which favors improved recovery in the NuSepin group significantly faster than the placebo group.
We next analyzed covariate effects of anti-viral drugs. No significant differences of TTCI_S between the groups were observed in patients of the ITT population who did not receive anti-viral drugs (Supplementary Table 7). When NuSepin was combined with anti-viral drugs (Lopinavir–Ritonavir or Favipiravir), median TTCI_S was 9.0 d [7.0~12.0], which is comparable with the group of placebo + anti-viral drugs of 13.0 d [4.0~ND] d (p = 0.013), when the ITT population was analyzed (Fig. 3C). The RR in this subgroup was 3.5 [1.2~10.0], which favors the group of NuSepin + anti-viral drugs compared with the group of placebo + anti-viral drugs (p = 0.02). When NuSepin was combined with anti-viral drugs, median TTCI_S was 9.5 d [7.0~12.0], which is comparable to the group of placebo + anti-viral drugs 13.0 [10.0~ND] (p = 0.007), when moderate-to-severe patients in the ITT population were analyzed (Fig. 3D). The RR in this subgroup was 4.2 [1.3~13.6], which favors the group of NuSepin + anti-viral drugs compared with the group of placebo + anti-viral drugs (p = 0.01).
When NuSepin was combined with Lopinavir–Ritonavir, median TTCI_S was 8.0 d [5.0~11.0], which is comparable to the placebo + Lopinavir–Ritonavir group of 12.5 d [4.0~ND] (p = 0.08), when the patients in the ITT population were analyzed (Supplementary Fig. 4A). The RR in this subgroup was 7.7 [1.1~54.0], which significantly favors the group of NuSepin + Lopinavir–Ritonavir compared with the group of placebo + Lopinavir–Ritonavir (p = 0.04). When NuSepin was combined with Favipiravir, median TTCI_S was 10.0 d [7.0~13.0], which is comparable with the placebo + Favipiravir group of 19.0 d [10.0~ND] (p = 0.07), when the patients in the ITT population were analyzed (Supplementary Fig. 4B). The RR in this subgroup was 3.8 [0.78~18.83], which favors the group of NuSepin + Favipiravir compared with the group of placebo + Favipiravir, numerically (p = 0.10).
We hypothesized that anti-viral drugs might be given more frequently to more severe patients than to less severe patients. Therefore, we analyzed the baseline NEWS2 of patients and compared given prognoses of these patients. As expected, baseline NEWS2 was significantly higher in patients who were given anti-viral drugs (Supplementary Fig. 5). The mean baseline NEWS2 of patients who received anti-viral drugs was 7.6 ± 0.71 (not given with NuSepin) and 8.4 ± 0.62 (given with NuSepin together). In comparison, the mean baseline NEWS2 of patients who did not receive anti-viral drugs was 4.5 ± 0.75 (not given with NuSepin) and 5.2 ± 0.95 (given with NuSepin together) (Supplementary Fig. 5A). These findings indicate that anti-viral drugs were given more often to more severe patients. Moreover, clinical remission of patients who received anti-viral drugs without NuSepin was delayed compared to patients who did not receive anti-viral drugs nor NuSepin (Supplementary Fig. 5B), suggesting that anti-viral drugs were given to patients with higher baseline NEWS2 whose clinical improvement was significantly delayed compared to patients who did not receive anti-viral drugs.
Next, we compared supplemental oxygen-free days between the groups (Supplementary Fig. 6). When patients in the PP population were analyzed after adjusting the effects of baseline NEWS2, the RR was 2.2 (95% C.I., 1.0~ 4.6, p = 0.04), which favors longer supplementary oxygen-free days in the NuSepin group than in the placebo group.
CRP levels decreased after randomization both in the NuSepin group and the placebo group (Fig. 4). However, the CRP level in the NuSepin group was normalized faster than that in the placebo group. At day 4, CRP levels in 52% of patients in the NuSepin group returned within normal range, which is comparable to 20% of patients in the placebo group. When patients who received anti-viral drugs were analyzed, CRP levels in 64.3% of patients in the NuSepin group returned within the normal range at day 4, which is comparable to the 11.1% of the placebo group (Supplementary Fig. 7).
At EoS, blood pro-inflammatory cytokines levels were significantly returned within normal range in the NuSepin group, and the percentage of patients with pro-inflammatory cytokines within normal ranges was higher in patients in the NuSepin group compared with those in the placebo group (Fig. 4). For example, IL-8 levels in patients of the NuSepin group (12.8 ± 2.2) at EoS were significantly lower than those of the randomization day (22.1 ± 4.0, p <0.05). In comparison, IL-8 levels in patients of the placebo group were not significantly decreased at EoS day (259.4 ± 167.8) compared with those of randomization day (22.0 ± 3.2). IL-8 levels returned within normal ranges in 55% of patients in the NuSepin group and in 33% of patients in the placebo group. At EoS day, the levels of TNF-α in patients of the NuSepin group were (7.4 ± 0.7) significantly lower than those of patients in the placebo group (24.8 ± 12.3, p = 0.007), and TNF- α levels returned within normal ranges in 35% of patients in the NuSepin group and in 11% of patients in the placebo group. Furthermore, IL-6 levels returned within normal ranges in 60% of patients in the NuSepin group and in 33% of patients in the placebo group. IL-6 levels in patients in the NuSepin group at EoS (9.7 ± 5.1) were significantly lower than those of randomization day (19.0 ± 3.9, p < 0.05). Because no deceased cases were observed in either the NuSepin or placebo groups, we were unable to compare 28-d mortality.
Safety Outcomes
In the SAS population, which includes all 64 patients, 25% of patients experienced more than one adverse event (AE) (Table 1). The total number of events was 31. 27.3% (six patients, 12 events) of the 0.1 mg/kg NuSepin group, 27.3% (six patients, 12 events) of the 0.2 mg/kg NuSepin group, and 20.0% (four patients, seven events) of the placebo group reported at least one treatment-emergent AE. AEs were mild and recovered by EoS. The relationship between 13% AEs and 0.2 mg/kg NuSepin was determined to be ‘possible’ when graded as “probable, possible, or definite”. There was one serious AE of dyspnea in the 0.2 mg/kg NuSepin arm which was “not related” to NuSepin. The most common AE was increases in ALT, i.e., one case (5%), two cases (9%), and two cases (10%) in the 0.1 mg/kg NuSepin, 0.2 mg/kg NuSepin, and placebo arm, respectively.