Minimum infusion rate and adrenocortical function after continuous infusion of the novel etomidate analog ET-26-HCl in rats

Introduction

Etomidate, one of the most frequently used intravenous anesthetics, has many favorable properties, such as its ability to maintain hemodynamic stability and generate low incidences of respiratory depression and anaphylaxis. However, the adrenocortical insufficiency caused by etomidate restricts its clinical applications. Several studies have shown that as little as a single administration of etomidate may induce adrenocortical insufficiency, and the increased propensity for this may last 48 h after the administration of etomidate (Hildreth et al., 2008; Tekwani et al., 2008).

In the 1980s, etomidate was infused in a continuous manner in critical patients to maintain sedation; however, studies later showed that this dosage regimen increased mortality (Ledingham & Watt, 1983; Watt & Ledingham, 1984). Pharmacologists have spent years attempting to develop new etomidate analogs in their search for a drug that retains the desirable properties of etomidate but does not cause adrenocortical insufficiency. We recently reported on ET-26-HCl, a promising compound selected from dozens of other etomidate analogs that were designed using our synthesis strategy, showing that ET-26-HCl effectively produces reversible anesthesia, and that a signal administration does not significantly decrease plasma corticosterone levelsin beagle dogs (Yang et al., 2017). The primary aim of the present study was to evaluate the effect of ET-26-HCl on adrenal function after a continuous infusion.

Materials and Methods

Evaluation of adrenocortical function

The infusion protocol for each rat began at 8:30–9:00 a.m. to minimize natural changes in corticosterone levels. After the rats were weighed and the caudal vein was catheterized, dexamethasone (0.2 mg/kg) was administered. Two hours later, the baseline blood sample was drawn. Rats (n = 32) were then randomly divided into the following four groups, with eight rats in each group: etomidate, ET-26-HCl, CPMM, or propylene glycol vehicle control, and the infusion protocol started (see Fig. 1A). The infusion rate in each group was the corresponding predetermined MIR for that compound, and the drug infusion persisted for 60 min. After 30 min of drug infusion, adrenocorticotropic hormone (ACTH) was administered intravenously, and this was repeated every 30 min. The second blood sample was collected at the end of the infusion, and then blood samples were drawn every 30 min for 3.5 h. Blood samples (approximately 0.2 mL each) were kept at room temperature for 10 to 60 min, until coagulation, and then centrifuged at 3,500 gravity (g) for 5 min. The supernatant was transferred to a clean Eppendorf centrifuge tube and subjected to a second centrifugation (3,500 rpm for 5 min). After the second centrifugation, the supernatant was removed again, and a final high-speed centrifugation was performed to remove all red blood cells and particulate. The serum was transferred to a clean tube and immediately placed at −20 °C. The corticosterone concentration in each serum sample obtained at the various time points was quantified within 1–2 days using an enzyme-linked immunosorbent assay (ELISA kit, R & D Systems) and a 96-well plate reader.

During all studies, rats were placed on a warming stage, rectal temperatures were maintained at 36–38 °C, and oxygen was continuously provided at 2 L/min.

Results

Discussion

In the present study, we evaluated the MIRs of etomidate, ET-26-HCl, and CPMM by recording in anesthetized rats either a positive (+) or no (−) reaction to a painful stimulus, an up-and-down design method previously described (Li et al., 2012). Each hypnotic was administered intravenously for 40 min to determine its MIR because our preliminary study showed that the half-life of ET-26-HCl was 6–7 min, the longest of the three hypnotics used in the present study, and because it is generally acknowledged that the in vivo plasma concentration of drugs continuously infused at a constant rate reaches equilibrium at 4–5 half-lives. We determined that the MIR for etomidate was 0.285 mg/kg/min, for ET-26-HCl it was 0.62 mg/kg/min, and for CPMM it was 0.95 mg/kg/min. These results suggested that the anesthetic efficacy of ET-26-HCl was approximately one-half to one-third of that for etomidate, which is consistent with the results of our previous study (Yang et al., 2017). In addition, the MIR of CPMM found in the present study was consistent with the results of Ge and colleagues, which suggested that the immobilizing ED₅₀ (effective dose for 50 percent of the group) of CPMM is 0.89 ± 0.18 mg/kg/min (Ge et al., 2012). These authors also found that the total doses of etomidate and CPMM needed in a 2-h closed-loop infusion protocol to maintain an 80% electroencephalographic burst suppression ratio are 36 mg/kg and 143 mg/kg, respectively, indicating that the average infusion rates for these hypnotics are 0.3 mg/kg/min and 1.19 mg/kg/min, respectively. This infusion rate for etomidate is consistent with the rate found in the present study, while the rate for CPMM in the present study is slightly lower than that observed in the previous study. Thus, we speculate that all rats in the three groups used in the present study were at a similar depth of anesthesia.

After determining the MIRs, we next examined the effects of etomidate, ET-26-HCl, and CPMM continuously infused for 1 h and inducing the same anesthesia depth on serum corticosterone concentrations. Compared with those in control rats, ACTH-stimulated serum corticosterone concentrations were significantly decreased by etomidate, while those following ET-26-HCl or CPMM administration were not associated with a significant difference. When ACTH-stimulated serum corticosterone concentrations in rats administered etomidate were compared with those in rats infused with vehicle, ET-26-HCl or CPMM, all time points examined after the drug infusions, except 180 min and 240 min, show significant differences with etomidate. The corticosterone concentrations tended to be reduced for the first hour following ET-26-HCl infusion (as compared to vehicle infusion); however, this reduction did not reach statistical significance. We concluded that ET-26-HCl does not induce obvious inhibition of adrenal function.

The safe dosage range of etomidate has been diminishing owing to its inhibition of adrenocortical function, as the suppression following even a single bolus may last 72 h (Molenaar et al., 2012). This inhibition is mainly the result of the high-affinity binding between the basic nitrogen in the imidazole ring of etomidate and the heme iron on 11β-hydroxylase (Den Brinker et al., 2008; Fellows et al., 1983; Shanmugasundararaj et al., 2013). In the 1980s, etomidate was used as a sedative for critically ill patients; however, in 1983, Watt and colleagues found that the continuous infusion of etomidate may increase mortality, and they speculated that the increased mortality is mainly caused by adrenocortical suppression. A series of studies later verified this speculation and recommended not to blindly administer etomidate to critical patients (Morris & McAllister, 2005). However, no other anesthetic currently possesses the characteristics of etomidate, such as to the ability to maintain stable hemodynamics, especially in aged or critically ill patients. Therefore, researchers have devoted much effort to develop new etomidate analogs that preserve the advantages but reduce the disadvantages of etomidate. There are at least two ways to achieve this goal. The first method involves designing a series of analogs that are rapidly metabolized so that adrenocortical inhibition stops soon after the administration is discontinued. With this in mind, MOC-etomidate and CPMM were designed by researchers at the Massachusetts General Hospital. Among these compounds, CPMM showed the greatest promise for development (Campagna et al., 2014; Cotten et al., 2010; Cotten et al., 2009; Pejo et al., 2012; Santer et al., 2015). The second method involves changing the molecular structure of etomidate to minimize adrenocortical suppression. It is widely acknowledged that the primary mechanism of adrenocortical suppression is through the interaction between the basic nitrogen in the imidazole ring of etomidate and the heme iron of 11β-hydroxylase (Gay et al., 2009; Ouellet, Podust & De Montellano, 2008; Roumen et al., 2007). Several studies have shown that anesthesia efficacy is significantly decreased when the basic nitrogen is replaced with other chemical groups, such as in carboetomidate, which has an anesthesia potency approximately one-seventh of that for etomidate (Cotten et al., 2010). However, Atucha and colleagues suggested that the imidazole carboxylic acid ester side chain of etomidate affects both anesthetic potency and adrenocortical function (Atucha et al., 2009). The design of ET-26-HCl is based on modifications of this side chain, and our previous study showed that ET-26-HCl produces definite and reversible anesthesia in beagle dogs. In the present study, no significant difference was observed in the serum corticosterone concentration after the continuous infusion of ET-26-HCl or vehicle, suggesting that any adrenocortical suppression induced by ET-26-HCl would be lower than that caused by etomidate. These results also indicated that new analogs may be developed by means other than using soft analogs.

Conclusion

The method to evaluate minimum infusion rate of present study is feasible, and it could maintain a similar anesthesia depth of all drugs. The corticosterone concentrations tended to be reduced for the first hour following ET-26-HCl infusion (as compared to vehicle infusion) was mainly because of the molecular structure of ET-26-HCl still have some depression to the release of corticosterone; however, this reduction did not reach statistical significance. Thus, further studies are warranted examining the practicability of using ET-26-HCl as an infused anesthetic.

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