Healthy Chinese volunteers were eligible to participate in this study. Key inclusion criteria were: 1) males and females (not pregnant or lactating) aged 18–45 years; 2) body mass index (BMI) 18–28 kg/m² (body weight: males, ≥ 50 kg, females, 45 kg); and 3) physical examination, medical history, and clinical laboratory test results showed no clinically significant abnormal findings at screening. Key exclusion criteria were: 1) long-term history of smoking or alcohol and/or drug abuse; 2) use of any medication during the 14 days prior to the initial dose of study drug or while participating in the study; 3) blood donation during the 3 months prior to the study; 4) exposure to any CYP3A4, P-gp or Bcrp inducers or inhibitors during the 3 months prior to the initial dose of study drug; or 5) participation in a clinical trial of another investigational drug during the 3 months prior to this study.

This single-center clinical trial was conducted at the Jilin University First Affiliated Hospital-Phase I Clinical Research Center, Changchun City, China between Nov 21, 2018 and Jul 17, 2019. The clinical study protocol was reviewed and approved by the Ethics Committee at the Jilin University First Affiliated Hospital-Clinical Research Institute. The clinical trial was registered at http://www.chinadrugtrials.org.cn/(registration No. CTR20181499, registration date: 22/Nov/2018) and was conducted in accordance with the World Medical Congress Declaration of Helsinki and Good Clinical Practice guidelines. All study subjects provided written informed consent.

This single ascending dose, double-blind, randomized, placebo-controlled clinical trial investigated the safety, tolerability, and PK of HEC30654 in healthy subjects. The study planned to randomize eligible healthy subjects to one of seven treatment groups. In the first treatment group, three sentinel subjects were to receive a single oral dose of 5 mg HEC30654. In the six remaining treatment groups, ten subjects were to receive a single oral dose of 10, 15, 30, 60, 90 or 120 mg HEC30654 (n = 8 per group) or matching placebo (n = 2 per group) after an overnight fast.

Dose escalation was dependant on the safety and tolerability of the previous doses defined according to the National Cancer Institute Common Terminology Criteria for the Classification of Adverse Events (NCI CTCAE) v.4.03. Dose escalation was stopped based on the occurrence of the following drug-related adverse events (AEs): >50% of subjects in the previous treatment group experienced Grade ≥2 drug-related toxicities, or >25% of subjects in the previous treatment group experienced Grade 3–4 drug-related toxicities, or one drug-related serious adverse event (SAE).

The trial was terminated early with subjects in five treatment groups receiving a single dose of 5, 10, 15, 30, or 60 mg HEC30654.

Safety and tolerability, including AEs, vital signs (body temperature, blood pressure at rest, heart rate, and respiratory rate), and findings on electrocardiograms, electroencephalograms, physical examination, and clinical laboratory tests (biochemistry, hematology, urinalysis, coagulation, and immune globulin) were evaluated according to NCI-CTCAE, 4.03. Incidence, severity, and relationship of AEs to study drug were recorded. Tolerability was evaluated on Day 2 and Day 6 for subjects administered 5, 10, 15, or 30 mg HEC30654, and Day 2, Day 6, and Day 10 for subjects administered 60 mg HEC30654.

Blood samples (4 ml each) were collected via an indwelling intravenous angiocatheter into K₂EDTA containing tubes. For subjects administered 5, 10, 15, or 30 mg HEC30654, blood samples were collected at 0 h (pre-dose) and 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, and 120 h after dosing. For subjects administered 60 mg HEC30654, according to the t_1/2 and T_max of previous dosages, the time points were adjusted to 0 h (pre-dose) and 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, 120, 144, 168, 192, and 216 h after dosing. After discarding the first 0.5–1 ml of blood, samples were centrifuged at 1,800 g for 10 min at 4°C, and plasma was stored as two equal aliquots in polypropylene tubes at −80°C until analysis. For subjects administered 60 mg HEC30654, urine and fecal samples were collected during the following time intervals: 0, 0–4, 4–8, 8–12, 12–24, 24–48, 48–72, 72–96, 96–120, 120–144, 144–168, 168–192, 192–216 h (urine) and 0–216 h (feces).

The concentrations of HEC30654 and its metabolite HEC93263 in plasma and feces were determined using an ExionLC HPLC system (AB Sciex, Toronto, Ontario, Canada) equipped with an AB 4000 QTrap MS detector (AB Sciex, Toronto, Ontario, Canada). The concentrations of HEC30654 and its metabolite HEC93263 in urine were determined with a Nexera X2 HPLC system (Shimadzu, Tokyo, Japan) equipped with an API 4000 MS detector (AB Sciex, Toronto, Ontario, Canada). The calibration ranges of the assays for HEC30654 and HEC93263 were 0.500–500 ng/ml in plasma, 2.00–2,000 ng/ml in urine, and 5.00–5,000 ng/ml (HEC30654) and 1.00–1,000 ng/ml (HEC93263) in feces. For HEC30654 and HEC93263 in plasma, urine and feces, accuracies were 3.33–12.8%, –11.93–6.67% and –6.00–8.88%, and precision was within 6.02, 10.5 and 9.10% CV, respectively.

Standard non-compartmental methods using WinNonlin version 6.4 (Certara United States of America Inc,.) were used to analyze plasma PK data, including peak plasma concentration (C_max), time to peak plasma concentration (T_max), area under the plasma concentration–time curve from time 0 to the last timepoint with a quantifiable concentration (AUC_0-t), AUC from time 0 to infinity (AUC_0-∞), t_1/2, clearance (CL/F), and apparent volume of distribution (Vz/F).

Statistical analysis was performed using SAS software, version 9.4 (SAS Institute Inc., United States). Continuous variables were reported as number, mean with standard deviation, median, maximum, and minimum. Categorical variables, were reported as frequency and percentage. Dose proportionality was explored using a regression power model, relating log-transformed C_max and AUC and log-transformed dose.

This study included a total of 43 healthy subjects enrolled into five treatment groups. Baseline demographic characteristics were well balanced across the treatment groups (Table 1). All subjects completed the study and were included in the safety, tolerability, and PK analyses.

Treatment-emergent AEs (TEAEs) were reported in 33.3, 50.0, 25.0, 12.5, 100, and 37.5% of subjects, and drug-related TEAEs were reported in 33.3, 37.5, 12.5, 12.5, 100, and 25% of subjects administered 5, 10, 15, 30, or 60 mg HEC30654 or placebo, respectively. No dose-dependent TEAEs were observed (Table 2).

Single doses of HEC30654 up to 30 mg were generally safe and well tolerated. 100% of subjects administered 60 mg HEC30654 reported TEAEs, all of which were assessed as related to study drug. TEAEs most frequently reported by subjects administered 60 mg HEC30654 included lymphocytopenia (100%), leukopenia (87.5%), elevated C-reactive protein (75.0%), neutropenia (62.5%), fever (100%), headache (75.0%), and myalgia (75.0%). 100% of subjects in the matching placebo group reported TEAEs (100%), all of which were assessed as related to study drug. TEAEs most frequently reported by subjects administered matching placebo included lymphocytopenia (100%), leukopenia (50.0%), elevated C-reactive protein (100%), neutropenia (50%), fever (100%), headache (100%), and myalgia (50%). Most of the TEAEs reported by subjects administered 60 mg HEC30654 or matching placebo were not reported by subjects in the other treatment groups. There was no clinically significant changes for vital signs, electrocardiograms and electroencephalograms throughout the study for each group.

All the subjects in 60 mg group occurred grade ≥2 drug related AEs, and three of them occurred grade ≥3 drug related AEs (Table 3). As the 60 mg HEC30654 treatment group met the pre-defined stopping rules specified in the protocol, the study was terminated early.

The mean plasma concentration-time profiles of HEC30654 after administration of single escalating doses to healthy subjects under fasted conditions are shown in Figure 1. Mean PK parameters for HEC30654 are summarized in Table 4. Median t_max of HEC30654 was 6 h (range, 4–12 h), t_1/2 of 10–60 mg HEC30654 ranged from 52.1 to 63.8 h, and there was no dose proportional increase (t_1/2 of 5 mg HEC30654 was not included in the analyses due to high variability). The CL/F of HEC30654 was 31.8–63.1 L/h with a large Vz/F 3840–6420 L, suggesting a wide distribution in the body. The AUC_0-∞ and C_max of HEC30654 increased in a dose-dependent manner, but the increases were slightly greater than dose proportional in 60 mg. Dose proportionality over the 5–60 mg HEC30654 dose range was evaluated with a power model. The slope (90%CI) for lnAUC_0-∞ was 1.17 (1.07–1.28) and lnC_max was 0.92 (0.76–1.07). The 90% CI were outside the respective acceptance intervals of 0.86–1.14 and 0.91–1.09; however, as the 90%CI of the slope for lnC_max contained the value of 1, dose proportionality may be claimed for that parameter.

HEC93263 exposure was low. Metabolite PK parameters could only be calculated for 60 mg HEC30654 (Table 4). Median t_max of HEC93263 was 6 h and t_1/2 was 30.3 h. The rate of HEC30654 metabolism to HEC93263 was low, as suggested by C_max (8.9%) and AUC-_0–∞ (3%) values.

HEC30654 and HEC93263 were detected in urine and fecal samples. The percentage cumulative recovery of HEC30654 in the urine and feces from 0 to 216 h was 0.998 and 21.4%, respectively. The percentage cumulative recovery of HEC93263 in the urine and feces from 0 to 216 h was low (<0.1%) (Table 5).