SAD: A total of 62 subjects were involved in this part. Subjects were randomly assigned to one of the seven groups receiving placebo or HSK16149 at doses of 5, 10, 20, 40, 60, 90, and 120 mg. 4 (3 for HSK16149 and 1 for placebo) subjects were assigned to the starting dose of 5 mg group, 8 (6 for HSK16149 and 2 for placebo) for the highest dose group of 120 mg, and 10 (8 for HSK16149 and 2 for placebo) for the remaining dose groups (Figure 1).

Eligible subjects were admitted to the Phase I unit 1 day prior to drug administration. On the morning of Day 1, after a period of fasting for ≥10 h, subjects were administered HSK16149 or placebo at the respective dose level with 240 mL of water. All subjects stayed at the Phase I unit until 48 h after dosing with close medical monitoring. A follow-up visit by telephone or WeChat was performed by investigators on Day 7.

MAD: A total of 60 subjects were involved in this part. Subjects were randomly assigned to one of the six groups receiving HSK16149 (n = 8 per dose group) or placebo (n = 2 per dose group)at doses of 15, 30, 45, 60, and 80 mg, twice a day (BID), and of 90 mg once a day (QD) (Figure 1). The dose group 45 mg BID and 90 mg QD are parallel dose groups, that means when the tolerance assessment of the previous dose group is completed and the results show good tolerance, they can be carried out at the same time. Eligible subjects were admitted to the Phase I unit one day prior to the first administration. On the morning and evening (excluding the 90 mg QD group) of Day 1–8, HSK16149 capsule or placebo at the respective dose level was administered with 240 mL of water under fasting conditions. All subjects stayed at the Phase I unit until 48 h after the last dose. A follow-up visit by telephone or WeChat was performed by investigators on Day 15. This study initiated from the lowest dose group, and the decision to proceed to the next higher dose group was made by both investigator and sponsor according to the safety and tolerability of the preceding dose group within 7 days after the last administration or terminate according to the dose-escalation termination criteria specified in the protocol.

Randomization in this study was carried out using a stratified block randomization method. The double-blind study uses identical placebos corresponding to different specifications of HSK16149, with identical labels and outer packaging, ensuring that both subjects and researchers are completely unaware of the distribution of the experimental drug.

Healthy Chinese males or females aged between 18 and 45 years were erolled if they met the following inclusion criteria, but did not meet the exclusion criteria.

Inclusion criteria: bodyweight ≥50 kg for males, or ≥45 kg for females, and body mass index (BMI) between 19 and 26 kg/m² (included); vital signs, physical examination, 12-lead ECG, chest X-ray and results of laboratory tests was normal or abnormality with no clinical significance; willing to use effective contraceptive measures during the study and within 3 months after the administration of investigational products.

Exclusion criteria: history of any systemic disorders or diseases, orthostatic hypotension, allergy, angioedema or any disease history or current disease that may affect the safety evaluation of the subject or the process of the study drug in vivo; clinically significant dizziness or vertigo; people with insomnia, anxiety disorder, depression disorder, or other mental disorders; any drug that inhibits or induces liver drug-metabolizing enzymes used within 30 days before the first administration of the study drug; blood donation or blood loss ≥400 mL within 3 months before dosing; any concomitant medication within 2 weeks before screening; participation in any clinical trials within 3 months before dosing; drug or alcohol addicts, or heavy smokers; positive test for hepatitis B, hepatitis C, HIV, or syphilis; positive pregnancy test for females.

The safety of the study was evaluated through vital signs (blood pressure, pulse rate, respiratory rate, and body temperature), physical examinations, clinical laboratory tests, 12-lead ECGs, and adverse events (AEs) monitoring throughout the study. AEs were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE, version 5.0), and were processed and recorded promptly by qualified investigators according to relevant regulations. All AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA, version 22.0). The incidence of AEs was calculated by dividing the number of subjects who have experienced at least one AE by the total number of subjects in the corresponding part.

The dose-escalation termination criteria in the whole study were determined by AEs. Dose escalation should be terminated when 1) AEs related to the study drug of CTCAE Grade 2 and above occur in ≥50% of the subjects in a certain dose group or AEs related to the study drug of CTCAE Grade 3 and above occur in ≥33.3% of the subjects in a certain dose group; 2) Serious adverse events (SAEs) related to the study drug occur in at least one subject; 3) The drug exposure level shows a saturation trend and is approaching a plateau.

Blood samples (approximately 3 mL each) were collected from a suitable forearm vein by an indwelling catheter or by immediate venipuncture. In the 5 mg group of SAD, blood samples were collected at the following 16-time points: 0 (pre-dose), 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h post-dose. Subsequently, the time points were slightly adjusted depending on the pharmacokinetic profile of the 5 mg group of SAD. In other dose groups of SAD, blood samples were collected at the following time points: 0 (pre-dose), 0.33, 0.66, 1, 1.33, 1.66, 2, 3, 4, 6, 8, 10, 12, 24, 36, and 48 h post-dose. Urine samples were collected within 2 h before dosing and during the intervals 0-4 h, 4-8 h, 8-12 h, 12-24 h and 24-48 h after dosing.

In MAD, blood samples were collected at the following time points: 0 (pre-dose), 0.33, 0.66, 1, 1.33, 1.66, 2, 3, 4, 6, 8, 10, 12 h post-dose at Day 1 and 0 (pre-dose), 0.33, 0.66, 1, 1.33, 1.66, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48 h post-dose at Day 8, in addition, 0 h (pre-dose) at Day 5–7 for determining the steady-state. In the 90 mg QD group, 24 h post-dose at Day 1 should be collected.

The blood samples were separated by centrifugation at 4°C, 1,500 g for 10 min. The separated plasma was divided into two centrifuge tubes (each with at least 0.5 mL of plasma), and stored frozen under −80°C until analysis.

The plasma and urine concentrations of HSK16149 were analyzed by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in Shanghai Xuhui Central Hospital. The LC-MS/MS system includes a Triple Quad 6500+ mass spectrometer (AB SCIEX, USA) and a liquid chromatograph waters I class system (Waters, USA). HSK16149-D₄ was chosen as the internal standard. For plasma concentration detection, the lower limit of quantification (LLOQ) was 3 ng/mL, the linear detection range was 3 ng/mL to 3,000 ng/mL. For urine concentration detection, the LLOQ was 0.2 μg/mL, the linear detection range was 0.2 μg/mL to 200 μg/mL.

Pharmacokinetic parameters were calculated using PhoenixWinNonlin 8.0 software (Pharsight, Cary, NC, United States). Plasma and urine concentration-time data were analyzed by noncompartmental methods. PK parameters mainly included: maximum observed plasma concentration (C_max), the maximum plasma concentration at steady-state (C_max,ss) after multi-dose, the area under the concentration-time curve (AUC), AUC from time zero (pre-dose) to the time of the last measurable concentration (AUC_0-t), AUC from time zero (pre-dose) to infinity (AUC_0-inf), the AUC over the dosing interval at steady-state (AUC_ss), time to maximum plasma concentration (T_max), time to C_max,ss (T_max), terminal elimination half-life (t_1/2), apparent distribution volume (Vz/F), clearance rate (CL/F), renal clearance (CL_R); average retention time from time zero to infinity (MRT_0-inf), accumulation ratio at steady state (R_ac), degree of fluctuation (DF) and the cumulative fraction of HSK16149 excreted in urine during each collection interval (Fe). For urine PK parameters, we calculated Ae_0–48h, which was the cumulative amount of drug excreted into the urine over the 48-h collection interval. CLR(L/h) was the renal clearance index, which was calculated from Ae (mg) ×1,000/AUC_0-t (h*ng/mL) and Fe_0–48h, which was the cumulative fraction of the dose excreted as unchanged parent in urine over the 48-h collection interval.

Statistical analysis was performed using SAS Software version 9.4 or above (SAS Institute, Cary, NC, United States). Continuous variables were statistically described using the number of cases, mean, median, standard deviation (SD), minimum values, and maximum values; categorical variables were statistically described using the frequency and percentage of each category. Geometric mean and coefficient of variation (%CV) were also used for applicable plasma concentrations and pharmacokinetic parameters. p ≤ 0.05 was considered to be statistically significant. A power model was used to investigate the correlation between PK parameters (AUC, C_max) and doses. ANOVA model was used to calculate the accumulation index of PK parameters after 8 days of continuous administration.

A total of 62 eligible subjects were enrolled in SAD and all of them completed the study per protocol. A total of 60 eligible subjects were enrolled in MAD, 1 subject withdrew from the study after administration due to an AE (Grade 2 of urticaria, this subject was proved to be in placebo group after unblinding), 1 subject withdrew from the study after administration due to protocol deviation (The collected serum and urine samples were included in PK analysis group), and the remaining 58 subjects completed the study. The distribution of subjects is displayed in Figure 1. The demographic profile of all enrolled subjects is demonstrated in Table 1.

In SAD, 49 subjects taking HSK16149, of which 28 subjects (57.1%) had TEAEs (Table 2) and 23 subjects (46.9%) (Table 2) had TEAEs judged to be related to the study drug. 13 subjects taking placebo, of which 5 subjects (38.5%) (Table 2) had TEAEs. Among the reported TEAEs after HSK16149 administration, 2 cases with CTCAE Grade 3 in the 120 mg group were considered to be related to the study drug, 1 case in the 60 mg group, and 1 case in the 90 mg group with CTCAE Grade 2 were considered to be related to the study drug. The rest are grade 1 AEs. The most common drug-related AEs were somnolence and dizziness. No SAEs occurred throughout the study, and no subject withdrew from the study due to AE.

In MAD, 48 subjects taking HSK16149, of which 44 subjects (91.7%) had TEAEs (Table 3), and 43 subjects (89.6%) (Table 3) had TEAEs judged to be related to the study drug; 12 subjects taking placebo, of which 11 subjects (91.7%) had TEAEs (Table 3). Among the reported TEAEs after HSK16149 administration, 1 case in the 30 mg BID group and 1 case in the 45 mg BID group with CTCAE Grade 2 were considered to be related to the study drug, 1 case in the 90 mg QD group with CTCAE Grade 2 was not considered to be related to the study drug, 1 subject in the placebo group withdrew from the study due to urticaria, the rest are grade 1 AEs. The most common drug-related AEs were dizziness, elevated blood triglycerides, and diarrhea. No AEs of grade 3 or higher occurred throughout the study.

For SAD: The main plasma PK parameters in each dose group after a single dose of HSK16149 are summarized in Table 4, and the mean plasma drug concentration-time curves are shown in Figure 2.

The Vz/F, Cl/F, and MRT_0-inf of the 7 dose groups were similar (p > 0.05), while the parameters AUC and C_max were dose proportional. HSK16149 was rapidly absorbed, T_max occurred at 1.5-1.7 h and t_1/2 ranged from 3.7 to 6.4 h. The PK parameters of HSK16149 in urine are shown in Table 4. The cumulative amount of HSK16149 Ae_0–48 h increased with the dosage increment, the average renal clearance was consistent in different dose groups. The range of Fe_0–48 h in different dose groups was 70.84%-83.53% which means HSK16149 was mainly eliminated via urinary excretion.

For MAD: The main PK parameters of HSK16149 for MAD are presented in Table 5. The mean plasma drug concentration-time curves at D1 and D8 are demonstrated in Figure 3.

The AUC and C_max were almost dose-proportional. The average clearance rate and distribution volume of HSK16149 at all dose levels were comparable. The mean t_1/2 of HSK16149 ranged from 3.7 to 7.1 h, and R_ac ranged from 1.04 to 1.35, indicating a slight accumulation on day 8.