The preclinical pharmacological study of a novel intravenous anesthetic, ET-26 hydrochloride, in aged rats

Animals and housing

All animal procedures were performed with the approval of the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University, Chengdu, China (2015015A). All animals were housed according to the Guide for the Care and Use of Laboratory Animals by the West China Hospital, Sichuan University. Sprague-Dawley SPF rats (age: 20 months, body weight: 680 ± 23 g for male and 508 ± 59 g for female after 5 days adaptation) were purchased from Dossy Biological Technology Co., Ltd. (Chengdu, China). Rats (n = 5) were housed in polypropylene cages with ad libitum food and water supply, controlled room temperature (22 °C to 26 °C) and humidity (40% to 60%), and under 12 h light-dark cycles (lights on at 7:00 a.m.). Prior to the experiments, animals were acclimated for a minimum of 5 days.

Materials

ET-26HCl was synthesized by Humanwell Pharmaceutical Co., Ltd. (Yichang, China). The solution of ET-26HCl (10 mg/mL) was dissolved in 35% (v/v) propylene glycol and sterile injection water, and this specific solvent was prepared by Humanwell Pharmaceutical Co., Ltd. Propofol (10 mg/mL) and etomidate (10 mg/mL) were purchased from AstraZeneca (Shanghai, China) and Nhwa Pharmaceutical Co., Ltd. (Jiangsu, China), respectively. Isoflurane was purchased from Yipin Pharmaceutical Co., Ltd. (Hebei, China). Dexamethasone sodium phosphate injection and normal saline were purchased from Tianyao Pharmaceutical Co., Ltd. (Hubei, China) and Qingshanlikang Pharmaceutical Co., Ltd. (Chengdu, China), respectively. Adrenocorticotropic hormone (ACTH) fragment 1-24 (human and rat) was purchased from Sigma-Aldrich (MO, USA) and dissolved with dimethyl sulfoxide (20 µg/mL).

Corticosterone and corticosterone-d8 were purchased from the Dr. Ehrenstorfer GmbH (Augsburg, Germany) and C/D/N Isotopes Inc. (Quebec, Canada), respectively. Acetonitrile, formic aid, and dimethyl sulfoxide were purchased from Sigma-Aldrich. Ethyl acetate, ethyl alcohol and methanol were obtained from Thermo Fisher Scientific (NJ, USA). All reagents are HPLC grade solvents. Ultrapure water was generated by the Milli-Q^® water purification system (Merck Millipore, Darmstadt, Germany).

Experimental design

Eighteen aged Sprague-Dawley rats were randomly divided into three groups (three males and three females in each group). According to a previous study (Wang et al., 2017c), the ED₅₀ values for LORR in rats are 2.35 mg/kg, 0.73 mg/kg, and 5.87 mg/kg for ET-26HCl, etomidate and propofol, respectively. Rats were injected intravenously with the two-fold dose of ED₅₀ for LORR via the tail vein in an injection speed of 0.1 mL/s.

LORR measurement

Rats were separately measured for LORR in a transparent box with a thermostatically heated pad (37 °C) to avoid external stimuli. The hypnotic depth was scored continuously until the rat was fully awake according to the following criteria (Salamone et al., 1996; Shimoyama et al., 1999; Wang et al., 2017c): 0 = normal activity; 1 = decreased activity or remained in same position; 2 = could not balance on hind limb; 3 = ataxia; 4 = lay prone and no reaction to mild stimuli; 5 = LORR. Once the score achieved 4, the rat was gently changed to the supine position (recognized as LORR). The time to LORR, time to recovery from LORR, time to stand up, and time to normal activity represented the onset, duration, and recovery of sedative effect. All rats that recovered from LORR were observed closely for mortality and clinical symptoms of toxicity.

Cardiovascular and respiratory function measurement

Rats were anesthetized with 2% isoflurane in oxygen using a mask that was placed in the supine position on a thermostatically heated pad (37 °C). The tail vein was cannulated with a Terumo^® intravenous catheter (24G; Tokyo, Japan) for drug administration. Then, the left femoral artery was isolated and cannulated for monitoring BP. A three-lead electrocardiogram (ECG) was monitored by the subcutaneous placement of an electrode at the left upper limb, right upper limb, and left lower limb. Thoracic movement was measured by a tension sensor for respiratory rate (RR). The ECG, BP and RR were recorded by the BL-420 Biological Signal Acquisition and Analysis System (Techman, Chengdu, China). Rats were acclimated for 20 min prior to the initiation of dosing. In the adaptive phase, the parameters were collected every 4 min and averaged as the baseline. Then, rats were injected with the two-fold dose of the ED₅₀ for LORR as described in 2.3. The biological parameters were used to compare the degree of cardiovascular and respiratory system inhibition by drugs at the following time points: 0.5, 1 min, and every 1 min to 15 min. The mean arterial pressure (MAP) was calculated according to the equation below: ${\displaystyle \text{Mean arterial pressure}=\left(\text{systolic pressure}+2\times \text{diastolic pressure}\right)/3.}$

Serum corticosterone measurement

Data analysis

For pharmacodynamic experiments, data were assessed by one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test (α = 0.025). For comparison of cardiovascular function and adrenal cortical hormones inhibition, data were assessed by two-way ANOVA followed by the Bonferroni post-hoc test (α = 0.025). For respiratory function measurement, data were assessed by Fisher’s exact test (α = 0.025). Data are reported as mean ± SEM, and the level of statistical significance was set at P < 0.05. Data were analyzed using Statistical Package for Social Sciences version 23 (IL, USA).

Pharmacodynamic experiments

After the injection of the drugs at the two-fold dose of ED₅₀ for LORR, there was no significant difference in onset time among those drugs (Fig. 2A, p = 0.053). However, the duration of the sedative-hypnotic effect (score of hypnotic depth = 5, Fig. 2B) for propofol (850.5 ± 77.4 s) was significantly longer than that caused by etomidate (489.8 ± 77.0 s, p = 0.007) and ET-26HCl (347.3 ± 49.0 s, p = 0.0004). Regarding recovery from the state of sedation (Fig. 2C), the time required to stand after administration of propofol, etomidate, and ET-26HCl was 71.3 ± 33.5 s, 140.3 ± 42.6 s, and 129.0 ± 22.3 s, respectively. The time required to normal activity after the administration of propofol, etomidate, and ET-26HCl was 393.0 ± 91.2 s, 382.0 ± 52.6 s, and 378.0 ± 47.8 s, respectively. However, no significant difference was observed in the time to stand (p = 0.330) and normal activity (p = 0.987) among the drugs.

Regarding the clinical symptoms of toxicity, 66.7% (4 of 6) of rats in ET-26HCl group exhibited mild hematuria (abnormal urine color—red), which was considered to be induced by the solvent. All rats recovered from their symptoms of toxicity, no other clinical signs of side-effect were observed, and no rats died.

Cardiovascular and respiratory function measurement

The advantages of etomidate are better cardiovascular stability and milder respiratory depression than propofol. To test cardiovascular and respiratory inhibition in aged rats, BP, ECG, and RR were continuously monitored to explore the effects of the drugs (Fig. 3). In each group, MAP decreased after injection (Fig. 3A). The maximum value of MAP after the administration of etomidate, ET-26HCl and propofol was decreased at 23.8 ± 3.1 mmHg, 30.9 ± 8.1 mmHg, and 44.2 ± 6.7 mmHg, respectively. In etomidate and ET-26HCl groups, the inhibition of BP was temporary and it tended to recovery without any treatment. However, etomidate (p = 0.0001) and ET-26HCl (p = 0.003) had a milder BP inhibition effect than propofol. After drug administration, the etomidate-, ET-26HCl- and propofol-induced decrease in heart rate were not significantly different (Fig. 3B, p = 0.12).

The respiratory depression of the aged rats was compared via the measurement of occurrence rate and duration of apnea (Table 1). Apnea was observed in all rats that were administered propofol (6 of 6, 100%) and the duration of this side effect was 45.0 ± 9.0 s. For etomidate and ET-26HCl, no apnea was observed (p = 0.00016).

Serum corticosterone measurement

In the modified ACTH-stimulation experiment, the concentration of serum corticosterone in aged rats is presented in Fig. 4. The concentration of corticosterone was suppressed by dexamethasone to 37.4 ± 13.6 ng/mL in the ET-26HCl group, 86.7 ± 32.7 ng/mL in the etomidate group, and 39.8 ± 16.5 ng/mL in the solvent group, and there was no significant difference among groups (p = 0.241). No significant difference in the concentration of serum corticosterone was observed between ET-26HCl and solvent groups. However, rats administered etomidate had lower corticosterone concentrations than were administered ET-26HCl at 15, 30, and 60 min after the stimulation of ACTH (p = 0.016, p = 0.004, and p = 0.002, respectively). These results demonstrated that ET-26HCl had no obvious inhibitory effect on the synthesis of adrenal cortical hormones, whereas etomidate significantly decreased the synthesis of corticosterone within 1 h in aged rats.