The present study was a single-center, randomized, open-label, placebo-controlled trial conducted on healthy Chinese subjects to evaluate the effect of the ET-26 blood concentration on the QT interval using the C-QTc model. This study aimed to investigate the pharmacokinetic characteristics and safety profiles of ET-26 and its primary metabolite etomidate acid (ETA) (Yu et al., 2020). The study strictly adhered to the established protocol and complied with relevant laws and regulations governing drug clinical trial quality management, as well as the guidelines set forth by the ethics committee. It has been registered under the identification number CTR20233230 at the dedicated platform for registering clinical trials (http://www.chinadrugtrials.org.cn). Informed consent was obtained from all subjects before they participated in the clinical trial.

The study enrolled healthy male and female subjects. The exclusion criteria included high-risk factors for torsades de pointes such as hypokalemia, hypomagnesemia, bradycardia, heart failure, and recent myocardial infarction. Subjects with abnormal 12-lead ECG results and a baseline QT interval corrected by Fridericia formula (QTcF) ≥ 450 ms, PR interval ≥200 ms, and QRS wave duration ≥120 ms were also excluded. Additionally, subjects with potentially difficult airways (Mallampati grade III–IV) or those who had taken medications within 30 days prior to the trial were excluded.

In this trial, eighteen healthy subjects were assigned to two dosage groups: the low-dose group (0.8 mg/kg, the recommended Phase III dose) and the high-dose group (2.8 mg/kg, the highest well-tolerated dose in Phase I), with nine subjects in each group. Additionally, a placebo control group was established at a 2:1 ratio from both dosage groups, comprising three subjects from each group, totaling six subjects in the placebo group. Throughout the trial, all participants received intravenous injections of either 0.8 mg/kg or 2.8 mg/kg ET-26-HCl or placebo (physiological saline) in ascending order. Blood samples and ECG data were collected.

The subjects were instructed to fast for at least 10 h before receiving the experimental drug. The low-dose group received the injection over a duration of 60 ± 5 s, while the high-dose group received it at a rate of 1.2 mg/kg/min for approximately 140 ± 10 s. Subjects strictly adhered to fasting conditions for 4 h following drug administration, with no water intake permitted for 2 h before or after drug administration. A standardized diet was provided, starting 4 h after drug administration.

In the low-dose group, venous blood samples were collected before dosing (within 60 min before administration) and at various time intervals after administration, including 0.0167, 0.0333, 0.05, 0.0833, 0.117, 0.167, 0.25, 0.417, 0.667, 1, 1.5, 2, 3, 4, 6, 8, and 24 h. An additional venous blood sample was collected immediately after dosing in the high-dose group. The collected blood samples were centrifuged for 10 minutes (1,700 g, 4 °C), and the plasma was subsequently stored at -70 °C for further analysis. Dynamic ECG machines were utilized in this study to acquire consistent ECG data parameters, such as heart rate, PR interval, QRS duration, QT interval, QTcF, and QTcB. The ECG data were collected simultaneously with the blood samples at various time intervals. Furthermore, baseline ECG data were obtained 2 days prior to dosing. Modified observer’s assessment of alertness/sedation (MOAA/S) score evaluations were performed every 120 ± 30 s after administration until three consecutive scores ≥5 were achieved. Eyelash reflexes were assessed concurrently with the MOAA/S scoring. Moreover, continuous ECG monitoring was conducted using a cardiac monitor from the start of dosing until three consecutive MOAA/S scores of ≥5 were achieved. One set of 12-lead ECG was obtained at 2, 4, 8, and 24 h post-administration and on the day before dosing. The placebo group was not subjected to MOAA/S scoring, eyelash reflex evaluation, or continuous ECG monitoring.

A validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was employed to determine the concentrations of ET-26 and ETA in plasma samples collected at various time points. The analysis was performed using an AB SCIEX API 4000 mass spectrometer equipped with a C18 column (Phenomenex Luna, 2.0 mm ID × 50 mm). The mobile phase consisted of (A) a salt solution prepared by mixing ultrapure water (1:1,000, v/v) with (B) methanol. ET-26 and ETA were analyzed using positive ion mode electrospray ionization with multiple reaction monitoring. The standard curve ranges for ET-26 were 10.0–4,000 ng/mL, and for ETA, they were 2.0–800 ng/mL. For ET-26, intra-day accuracy ranged from -6.37% to 3.30%, while for ETA, the range was -3.48% to -0.91%. The maximum precision for ET-26 and ETA was 5.08% and 6.73%, respectively. The pharmacokinetic parameters of ET-26/ETA, including the peak concentration (C _max), time to reach peak concentration (T _max), area under the curve from 0 to the last measurable concentration (AUC _0-t ) and from 0 to infinity (AUC _0-∞ ), elimination half-life (t _1/2 ), apparent clearance rate (CL), apparent volume of distribution during terminal phase (V _z ), and mean residence time (MRT), were estimated using Phoenix WinNonlin software version 8.1 or above with a non-compartmental model.

The safety assessment in this trial encompassed various aspects, including the frequency and incidence rate of AEs. It also involves physical examinations, pre- and post-medication vital sign assessments, MOAA/S score evaluations, eyelash reflex examinations, 12-lead ECG analysis, and laboratory tests, such as complete blood count, blood biochemistry analysis, coagulation function test, and urinalysis. These safety evaluation indicators are widely acknowledged as reliable and accurate.

This experiment utilized a linear mixed-effects C-QTc model to investigate the relationship between ΔQTcF and blood concentrations of ET-26 and ETA. ΔQTcF refers to QTcF corrected by baseline, which is calculated as the average of QTcF values at different time points minus their respective baseline values. The model considered post-dose ΔQTcF as the dependent variable and examined the effects of medication (experimental drug, placebo), blood concentrations of ET-26/ETA, sampling time points, and centered the QTcF baseline as independent variables. The centered QTcF baseline for each participant was calculated by subtracting the individual baseline QTcF values from the mean baseline QTcF values of all participants within their respective medication groups. Additionally, random effects were incorporated in conjunction with intercepts to account for variations in the blood concentration levels of ET-26/ETA. Detailed information can be found in Formula 1. ∆ Q T c F i j k = θ 0 + η 0 , i + θ 1 T R T j + θ 2 + η 2 , i C i j k + θ 3 T I M E k + θ 4 Q T c F i j * − Q T c F j * ¯ (1)

ΔQTcF _ijk represents the change in QTcF for subject i under treatment j at time point k. θ ₀ and η _0,i are the fixed and random effects of the intercept term respectively. TRTj indicates treatment (1 for experimental drug, 2 for placebo), and θ ₁ is the fixed effect of treatment. C _ijk represents the blood concentration of ET-26/ETA at time point k for subject i under treatment j, while θ ₂ and η _2,i represent the fixed and random effects of blood concentration of ET-26/ETA respectively. TIME refers to the time points where PK blood tests and QTcF measurements were conducted; θ ₃ is the fixed effect of measurement time. θ ₄ is the fixed effect of baseline, where QTcF_ij represents the mean value of QTcF at baseline for subject i under treatment j. Q T c F j * ¯ is the mean value across all baseline values.

The C-QTc model analysis was employed using R software (version 4.3.2) and SAS software (version 9.4) to simulate the correlation between ∆QTcF and blood drug concentration. Utilizing the C-QTc model, we assessed the upper limit of the 90% two-sided confidence interval (CI) for QTcF interval corrected by baseline and placebo (ΔΔQTcF) values corresponding to the geometric mean of ET-26 and ETA C _max at clinically relevant doses.

The study enrolled 18 healthy Chinese participants, with an equal distribution of six individuals each in the low-dose, high-dose, and placebo groups. Demographic details are presented in Table 1. Overall, there were 14 males and four females, with an average age of 31.2 years. The participants had a mean height of 170.2 cm, a mean weight of 67.6 kg, and a mean body mass index (BMI) of 23.3 kg/m². No statistically significant differences in these characteristics were observed between groups when analyzed using a t-test.

All participants in the low and high-dose groups completed the experiments. Figure 1 illustrates the drug–time curve of ET-26 and its metabolite ETA, and Table 2 shows the PK parameters. With increasing dosage, the C _max of ET-26 in the high-dose group showed a roughly 2.7-fold increase, and the AUC demonstrated an approximately 4.3-fold rise. Regarding ETA, the C _max in the high-dose group was 5.5 times higher than that in the low-dose group, with a corresponding AUC elevation of approximately 7.7-fold. Both ET-26 and ETA showed an approximately 1.5-fold increase in the MRT in the high-dose group.

A linear mixed-effects C-QTc model was employed in this study to investigate the correlation between ΔQTcF and the ET-26 and ETA concentration. The analysis encompassed the baseline period and post-administration ECG data, along with the corresponding concentration data of the placebo, low-dose, and high-dose groups. When summarizing QTcF and ΔQTcF data, it was observed that expect for one subject in the placebo group who exhibited a ΔQTcF >30 ms and ≤60 ms at 1, 2, and 3 h post-administration, all other subjects had QTcF values ≤450 ms at various time points, and ΔQTcF values ≤30 ms, as described in Table 3. The establishment of a C-QTc model was based on the evaluation of key assumptions regarding the C-QTc relationship, as recommended in the scientific white paper on modeling C-QTc (Garnett et al., 2017; Garnett et al., 2018), and the assumptions of this experimental model were evaluated through graphics.

The four hypotheses mentioned above were not contradicted by any apparent evidence found in this experiment, indicating the continued validity of the assumptions. A linear mixed-effects model was employed to establish a C-QTc model, with the ET-26/ETA blood drug concentration, sampling time points, and centralized QTcF baseline as the independent variables and ΔQTcF as the dependent variable. The established model parameters are listed in Table 4.

The final models produced estimated slopes of 0.001 ng/mL (90% CI: −0.001–0.003 ng/mL) for ET-26 and 0.013 ng/mL (90% CI: −0.001–0.027 ng/mL) for ETA, indicating a limited correlation between the concentrations of ET-26 and ETA with ΔQTcF. Importantly, all parameters exhibited insignificant standard errors, implying that the estimates closely approximated their true values with minimal variability among the data points.

The mean ΔΔQTcF value and upper limit of the 90% CI for C _max, predicted using a linear mixed-effects model, are presented in Figure 6; Table 5. The geometric mean (GM) of ET-26 C _max was 2,640 ng/mL, resulting in a mean ΔΔQTcF value of −2.305 ms with an upper limit of the 90% CI at 4.204 ms, which did not exceed the predefined threshold value of 10 ms. In contrast, ETA exhibited C _max levels at 238 ng/mL, corresponding to a mean ΔΔQTcF value of −1.913 ms with an upper limit below the predetermined threshold value set at 10 ms (0.936 ms). However, both ET-26 and ETA showed maximum C _max values that exceeded the predefined threshold values.

The clinical efficacy of the experimental drug was evaluated using the MOAA/S score and eyelash reflex assessment. The changes in the MOAA/S scores for each subject are shown in Figure 7. In the low-dose group, the median duration of loss of consciousness (MOAA/S score ≤1) was 2.30 min, while it was observed to be 2.20 min in the high-dose group. Regarding the eyelash reflex, 83.3% of the subjects (5/6) consistently maintained their eyelash reflex throughout the experiment in the low-dose group. Conversely, all six subjects in the high-dose group experienced temporary disappearance and subsequent recovery of their eyelash reflex after drug administration, with the disappearance time ranging from 2 to 4 min and recovery time ranging from 11 to 22 min. ET-26 exhibited a rapid onset of action and quick recovery, while also demonstrating dose-dependent sedative and hypnotic effects.

During the experiment, 10 subjects in the ET-26-HCl group experienced 19 treatment-emergent AEs (TEAEs), all of which were deemed related to the drug. Details of the TEAEs are listed in Table 6. The overall incidence rate of TEAEs was 55.6% (10/18). In the low-dose group, four subjects experienced six instances of TEAEs, with a severity level distribution of 83.3% grade 1 (5/6) and 16.7% grade 2 (1/6). In the high-dose group, six subjects experienced a total of 13 instances of TEAEs, all at the grade 1 severity level. Myoclonus was the most frequently observed TEAE, which occurred in nine subjects (9/12, 75%). Most TEAEs did not necessitate interventions except for one patient who received non-pharmacological treatment.