This single-center, non-randomized, open-label Phase I clinical trial was designed to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of ET-26 in healthy subjects, comprising both non-elderly and elderly populations. A total of 16 healthy subjects were enrolled, including 8 non-elderly subjects aged 18–64 years and 8 elderly subjects aged ≥65 years, with at least 3 subjects being ≥75 years old. To ensure group comparability, the non-elderly group was matched to the elderly group in terms of gender, and the mean body weight of non-elderly subjects was maintained within ±10 kg of the elderly group’s mean. All participants provided informed consent before participation in the trial. This study had appropriate inclusion/exclusion criteria and was approved by the Ethics Committee of the Shandong Provincial Qianfoshan Hospital before implementation (Supplementary Material S1).

Plasma concentrations of ET-26 at various time points were measured using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Pharmacokinetic samples were collected according to the planned schedule (Supplementary Table S1), and the bioanalytical methods are provided in the Supplementary Material S1.

Data from both the elderly and non-elderly groups were analyzed to generate individual as well as mean (Mean ± SD) plasma concentration-time (c-t) curves and semi-logarithmic c-t curves. The key PK parameters of ET-26 include Peak concentration (C_max), Time to peak concentration (T_max), Area under the concentration-time curve (AUC_0-t, AUC 0 − ∞ ), Percentage extrapolated AUC (AUC_{_%Extrap}), Elimination half-life (T_1/2), Apparent clearance (CL, CL/F), Apparent volume of distribution (Vz, Vz/F), and Mean residence time (MRT) were calculated via a non-compartmental model using WinNonlin software (version 8.1), based on the actual sampling times and administered dose. For each group, the arithmetic mean, standard deviation, and coefficient of variation were computed. Descriptive statistical summaries of ET-26 plasma protein binding data (including mean ± standard deviation) were generated using SAS software.

After the log transformation of the primary PK parameters (C_max, AUC_0-t, and AUC 0 − ∞ ), analysis of variance and two-sided tests were performed to calculate the geometric mean ratios and 90% confidence intervals. If the 90% confidence intervals fall within the 80.0%–125.0% range, it is concluded that there is no significant difference in the PK profiles between the non-elderly and elderly groups; otherwise, a comprehensive evaluation, incorporating exposure-response relationships, will be conducted to guide clinical dosing in the elderly population.

Following the Technical Guidelines for Clinical Evaluation of Intravenous General Anesthetics (NMPA, 2022), the pharmacodynamic evaluation in this study focused on two primary endpoints: the time to loss of consciousness and the area under the curve (AUC) of the Bispectral Index (BIS). Loss of consciousness was assessed by continuously monitoring the Modified Observer’s Assessment of Alertness/Sedation (MOAA/S) scale, with the endpoint defined as the interval from administration to the first instance when the MOAA/S score reached ≤1. Simultaneously, BIS values were recorded via electroencephalography to determine the minimum BIS level. The resulting BIS curves were subsequently fitted, and the AUC was calculated using WinNonlin or SAS 9.4 software. Descriptive statistics, including mean, median, standard deviation, maximum, and minimum values, were then used to comprehensively characterize the anesthetic effect.

Descriptive statistical methods were employed to evaluate laboratory test results, vital signs, 12-lead electrocardiograms (ECG), physical examination findings, and additional safety parameters. Adverse events (AEs) were coded per the International Council for Harmonisation (ICH) Medical Dictionary for Regulatory Activities (MedDRA, version 25.1) and their severity was graded according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE, version 5.0) (see Supplementary Table S2). Treatment-emergent adverse events (TEAEs), serious adverse events (SAEs), and TEAEs leading to study discontinuation were systematically assessed. These events were further categorized based on participant subgroup (elderly vs. non-elderly), severity, and their causality relative to the investigational drug.

A total of 16 subjects were enrolled in the study, with 8 allocated to the healthy elderly group and 8 to the healthy non-elderly group. The median age was 57.5 years (range: 24–81 years). All subjects were male and of Han ethnicity. The median height was 164.75 cm (range: 153.0–173.5 cm), the median weight was 63.8 kg (range: 56.1–81.5 kg), and the median body mass index (BMI) was 23.15 kg/m² (range: 19.3–27.7 kg/m²) (Table 1).

After intravenous administration of 0.8 mg/kg ET-26, plasma protein binding of ET-26 and the free fraction showed no significant time-dependent changes in healthy elderly subjects (n = 8) and healthy non-elderly subjects (n = 8). In the elderly group, plasma protein binding was 56.3% (free fraction 0.437) at 1 min post - dose, showing a slight increase to 60.3% (free fraction 0.397) after 4 h. In the non-elderly group, the binding rate was 57.3% (free fraction 0.427) at 1 min and 61.0% (free fraction 0.390) at 4 h. The binding rate fluctuated slightly within 4 h in both groups (4.0% in the elderly, 3.7% in the non-elderly), with no regular upward or downward trend. The inter-group comparison showed that the plasma protein binding difference was within 1.0% (1.0% at 1 min, 0.7% at 4 h), and the free fraction difference was less than 0.04 units at corresponding time points.

Eight subjects in the healthy elderly group received a single intravenous injection of ET-26 at a dose of 0.8 mg/kg. The median T_max was 1.94 min. Geometric mean PK parameters were as follows: C_max of 2,500 ng/mL, AUC_0-t of 804 h·ng/mL, AUC 0 − ∞ of 865 h·ng/mL. Eight subjects in the healthy non-elderly group received the identical dosing regimen. The median T_max was 4.00 min. Their geometric mean PK parameters were: C_max of 1,260 ng/mL, AUC_0-t of 641 h·ng/mL, and AUC 0 − ∞ of 700 h·ng/mL. Comparing the two groups (n = 16), the geometric mean ratio for C_max (elderly vs. non-elderly) was 198.81% (90% CI: 126.51–312.45). The corresponding ratios for AUC_0-t and AUC 0 − ∞ were 125.43% (90% CI: 107.56–146.27) and 123.54% (90% CI: 107.57–141.87), respectively. Since these confidence intervals do not entirely fall within the predefined equivalence range of 80%–125% (with overlap at the boundaries), further evaluation incorporating the exposure-response relationship is warranted (Table 2; Figure 1).

In the healthy elderly group, the median time to loss of consciousness post-dose was 1.933 min (range: 0.93–2.93 min). The area under the BIS curve was 855.55 (range: 711.89–1,345.71) (Table 3).

In the healthy non-elderly group, one subject exhibited a MOAA/S score >1 due to a response to repeated loud calls of his name. Excluding this subject, the remaining 7 subjects showed a median loss of consciousness time of 1.933 min (range: 1.92–1.97 min), with a BIS AUC of 648.68 (range: 240.67–816.13).

In this study, 9 TEAE events were reported in 6 subjects. Among these, 7 events were deemed related to the study drug, resulting in an overall incidence of 37.5% (6/16 subjects). In the healthy elderly group, drug-related TEAEs comprised Grade 1 injection site pain (12.5%, 1/8) and Grade 2 cough (12.5%, 1/8). The cough was managed with non-pharmacological treatment (suctioning), and no treatment was required for the other events; all recovered completely. In contrast, in the healthy non-elderly group, drug-related TEAEs included Grade 1 injection site pain (50.0%, 4/8) and myoclonus (12.5%, 1/8), none of which required any pharmacological or non-pharmacological intervention, and all recovered (Table 4).