Metabolite-inactive etomidate analogues alleviating suppression on adrenal function in Beagle dogs

Article abstract of DOI:10.1016/j.ejps.2016.12.041

Owing to rapid generation in body, the metabolites of etomidate softdrug are able to accumulate in either the brain or periphery and subsequently affect the recovery from anaesthesia or cause corticosteroid suppression. This study was designed to investigate the ability of two etomidate analogues (ET-26, ET-42) with inactive metabolites to provide anaesthesia with lesser corticosteroid suppression. The 50% effective dose (ED₅₀) of ET-26, ET-42, Etomidate, MOC-ET (an etomidate softdrug) and CPMM (an improved etomidate softdrug) required to induce anaesthesia intravenously in Beagle dogs were 1.44 mg/kg, 0.72 mg/kg, 0.43 mg/kg 23.12 mg/kg and 0.59 mg/kg, respectively. After adrenocorticotropic hormone (ACTH) stimulation, the serum concentrations of cortisol and corticosterone in the ET-26, ET-42 and CPMM groups were similar to those of controls, and significantly higher than those of the etomidate and MOC-etomidate groups (P < 0.05). Furthermore, no significant differences in serum concentrations of cortisol and corticosterone after ACTH-stimulation between ET-26, ET-42, CPMM, and blank control groups were observed. In this study, anaesthetic potencies of ET-26 (ED₅₀ = 1.44 mg/kg) and ET-42 (ED₅₀ = 0.72 mg/kg) were determined. Both analogues can significantly reduce the corticosteroid suppression in vivo. Metabolite-inactive etomidate derivatives with slow metabolism might provide a novel strategy to improve Etomidate associated corticosteroid suppression.

Full text of DOI:10.1016/j.ejps.2016.12.041

European Journal of Pharmaceutical Sciences 99 (2017) 343–349
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Metabolite-inactive etomidate analogues alleviating suppression on
adrenal function in Beagle dogs
Jun Yang a,1, Yi Kang a,1, Bin Wang a, Linghui Yang a, Jin Liu a,b, Wensheng Zhang a,b,
⁎
a
Laboratory of Anesthesia and Critical Care Medicine, Translational Neuroscience Centre, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
b
Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Owing to rapid generation in body, the metabolites of etomidate softdrug are able to accumulate in either the
brain or periphery and subsequently affect the recovery from anaesthesia or cause corticosteroid suppression.
This study was designed to investigate the ability of two etomidate analogues (ET-26, ET-42) with inactive me-
tabolites to provide anaesthesia with lesser corticosteroid suppression. The 50% effective dose (ED50) of ET-26,
ET-42, Etomidate, MOC-ET (an etomidate softdrug) and CPMM (an improved etomidate softdrug) required to in-
duce anaesthesia intravenously in Beagle dogs were 1.44 mg/kg, 0.72 mg/kg, 0.43 mg/kg 23.12 mg/kg and
0.59 mg/kg, respectively. After adrenocorticotropic hormone (ACTH) stimulation, the serum concentrations of
cortisol and corticosterone in the ET-26, ET-42 and CPMM groups were similar to those of controls, and signifi-
cantly higher than those of the etomidate and MOC-etomidate groups (P b 0.05). Furthermore, no significant dif-
ferences in serum concentrations of cortisol and corticosterone after ACTH-stimulation between ET-26, ET-42,
Received 8 September 2016
Received in revised form 23 December 2016
Accepted 31 December 2016
Available online 03 January 2017
Keywords:
Etomidate analogues
Reduced corticosteroid suppression
Slow metabolism
Inactive metabolite
Intravenous anaesthetic drug
CPMM, and blank control groups were observed. In this study, anaesthetic potencies of ET-26 (ED50
=
1.44 mg/kg) and ET-42 (ED50 = 0.72 mg/kg) were determined. Both analogues can significantly reduce the cor-
ticosteroid suppression in vivo. Metabolite-inactive etomidate derivatives with slow metabolism might provide a
novel strategy to improve Etomidate associated corticosteroid suppression.
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
heart failure and elderly patients (Bovill, 2006). Thus, an unmet need
exists to reduce corticosteroid suppression of Etomidate and therefore
Etomidate is one of the most widely used intravenous anaesthetics.
Etomidate has several key pharmacological properties, including rapid
onset of action and recovery from anaesthetic effects, a wide therapeutic
index and a short context-sensitive half-life, in addition to providing
cardiovascular stability (Nauta et al., 1983; Giese et al., 1985; Tomlin
et al., 1998), making this agent an ideal intravenous general anaesthetic.
However, etomidate has also been reported to cause adrenal toxicities
in patients who are critically ill (Wagner and White, 1984; Lundy et
al., 2007), owing to inhibition of corticosteroid synthesis (Duthie et al.,
1985). Data from several studies have demonstrated this risk of adrenal
insufficiency. Whether or not etomidate should be used in clinical prac-
tice, especially in patients who are critically ill, is therefore a matter of
some debate (Cotton et al., 2008). The generally good safety profile
and cardiovascular stability, however, do make etomidate a useful
agent for certain patients, such as those undergoing surgery owing to
expand the potential clinical applications of etomidate.
In the past 7 years, etomidate soft drugs with reduced corticosteroid
suppression, such as MOC-ET (Cotten et al., 2009) and CPMM (Husain et
al., 2012), have been developed. Whether the softdrug design enables
this goal to be achieved will depend not only on the rate of metabolism
of prototype molecule, but also on the activity of metabolite. Etomidate
metabolism in humans depends on hepatic esterase activity, which hy-
drolyzes the drug to etomidate acid and an ethanol-leaving group.
Etomidate acid is excreted mostly in urine and, to a lesser degree, in
bile (Heykants et al., 1975). Etomidate acid does not exhibit any obvious
activity, while the metabolite of MOC-ET (MOC-ECA) shows corticoste-
roid inhibition and central inhibition. MOC-ET can be hydrolyzed rapid-
ly by wide spread hydrolytic enzymes, which are abundant in the brain,
to generate MOC-ECA, which is largely unable to pass through the
blood-brain barrier owing to its increased polarity. As a result, MOC-
ECA accumulates in brain. MOC-ECA has been demonstrated to have a
hypnotic potency that is 350-fold less than that of MOC-ET, resulting
in a significantly increase burst suppression ratio (BSR) after termina-
tion of the infusion in rats (Pejo et al., 2012a). In addition, MOC-ECA
can also produce corticosteroid suppression to a certain extent (about
1/300 of the potency of MOC-etomidate) (Ge et al., 2012a) and probably
⁎
Corresponding author at: Laboratory of Anesthesia and Critical Care Medicine,
Translational Neuroscience Centre, West China Hospital, Sichuan University, Chengdu,
Sichuan 610041, PR China.
E-mail address: zhang_ws@scu.edu.cn (W. Zhang).
Contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejps.2016.12.041
0928-0987/© 2017 Elsevier B.V. All rights reserved.

344
J. Yang et al. / European Journal of Pharmaceutical Sciences 99 (2017) 343–349
produces conspicuous corticosteroid suppression owing to its rapid
generation and accumulation in the periphery. The chemical structure
of metabolite of CPMM, CPMM-ECA, is similar to that of MOC-ECA.
(Fig.1 a.). Experiments have demonstrated that BSR does not increase
significantly after termination of CPMM-ECA infusion in rats (Ge et al.,
2012b), although whether or not CPMM-ECA suppresses serum cortico-
steroid levels remains unknown.
In this study, we used strategy distinct from softdrug design to im-
prove the profile of etomidate. We hypothesized that if the corticoste-
roid suppression of etomidate analogues and its metabolites can be
prevented, even if these analogues have slow metabolic characteristic,
it could not exhibit significant corticosteroid suppression. Based on
this hypothesis, two etomidate analogues ET-26 and ET-42 were stud-
ied in this study.
the test of 50% effective dose. After a washout period of at least
3 weeks, 30 Beagle dogs (half males, half females; 9–12 kg in weight)
were prepared for pharmacodynamic testing procedures. After a wash-
out period of at least 3 weeks, 26 female dogs (10–12 kg) were enrolled
in the adrenal function test. All animals were housed individually and
fed at 4 pm once per day. Water was provided ad libitum. All the exper-
imental procedures were performed in the light phase, and animals
were randomly allocated into each group.
Drugs: ET-26, ET-42, MOC-etomidate and CPMM were all prepared
in our laboratory. MOC-etomidate was synthesized using a previously
reported method (Cotten et al., 2009). CPMM was synthesized using a
previously reported method (Husain et al., 2012). ET-26 was synthe-
sized using the indicated synthesis route (Fig.1 b.). ET-42 was synthe-
sized using the indicated synthesis route (Fig.1 c.). Spectral data for
ET-26, ET-42, MOC-etomidate and CPMM are provided as supplementa-
ry data. Also see supplementary data for further details on the prepara-
tion of ET-26 and ET-42. All drugs were dissolved in dimethyl
sulphoxide (DMSO) immediately prior to use.
2. Materials and Methods
2.1. Animal and Drugs
All experiments were approved by the Scientific Research Commit-
tee and the Institutional Animal Experimental Ethics Committee, West
China Hospital, Sichuan University, Chengdu, China. All experimental
animals were cared for in accordance with the Guide for Care and Use
of Laboratory Animals.
Plasma and liver homogenate was sampled from naïve healthy fe-
male Beagle dogs (3 female dogs for plasma sampling, 1 female dog
for liver homogenate sampling). A total of 54 naïve healthy Beagle
dogs (half males, half females; 9–12 kg in weight) were prepared for
2.2. In Vitro Studies: Metabolite Stability in the Plasma and Liver
Homogenate
The whole blood was sampled from one naïve healthy female Beagle
dog and the blood samples were mixed with heparin immediately after
sampling. Liver tissue was taken promptly from a naïve humanely killed
female dog.
A 25% liver homogenate was prepared using a 0.1 mol/L phosphate-
buffered saline solution (pH 7.4). Plasma samples derived from the
Fig. 1. The primary metabolites of (a). MOC-etomidate and CPMM, the synthesis routes of (b). ET-26, and (c) ET-42. MOC-etomidate: methoxycarbonyl etomidate; CPMM: cyclopropyl-
methoxycarbonyl etomidate.

J. Yang et al. / European Journal of Pharmaceutical Sciences 99 (2017) 343–349
345
blood and liver homogenate were divided into five samples, each with a
4-mL volume. Each sample of plasma or liver homogenate was warmed
to 37 °C for 15 min; then, Etomidate or related analogues, including ET-
26, ET-42, MOC-etomidate and CPMM, (10 mg/mL, dissolved in DMSO)
were added to the plasma or liver homogenates to obtain a final concen-
tration of 50 μg/mL. After incubation for 0, 5, 10, 15, 20, 30, 40, 60 and
120 min, 100 μL of the incubated plasma or liver homogenate was re-
moved, and the metabolic reaction was quenched using acetonitrile
(300 μL). All samples were centrifuged at 20,000g for 15 min and ana-
lyzed using high-pressure liquid chromatography (HPLC) (Agilent
1100 series, Agilent Technologies, USA).
Prototype drugs were determined by an isocratic elution chroma-
tography. The ultraviolet (UV) absorbance detector was set at 242 nm
for detection of the prototype drugs. Gradient-elution chromatography
was used to determine the presence of etomidate acid, and the UV ab-
sorbance detector was set at 232 nm. The limits of quantification in plas-
ma for ET-26, ET-42, etomidate, MOC-etomidate, CPMM and etomidate
acid were 1.04 μg/mL, 1.02 μg/mL, 1.06 μg/mL, 1.05 μg/mL, 1.11 μg/mL
and 2.22 μg/mL, respectively. The limits of quantification in liver ho-
mogenate for ET-26, ET-42, etomidate, MOC-etomidate, CPMM and
etomidate acid were 1.06 μg/mL, 1.08 μg/mL, 1.09 μg/mL, 1.07 μg/mL,
0.5 μg/mL and 2.27 μg/mL, respectively. The detailed method of drug
assay was provided as supplementary data.
other groups, resulting in a sample number of 4–6. All drugs were ad-
ministered at a dose of twice the ED50, dissolved in DMSO. DMSO con-
taining no active agent was administered as the control. Cortical
hormone concentrations were measured before administration of
drugs and 60 min after ACTH stimulation.
Prior to the test, dexamethasone (0.01 mg/kg) was administered to
each dog to suppress baseline cortisol and corticosterone levels and to
exclude the effects of variable stress responses to handling. Two hours
later, the blood samples were obtained for measurement of the baseline
serum cortisol and corticosterone concentrations. After sample collec-
tion, ET-26, ET-42, etomidate, MOC-ET and CPMM were intravenously
administered at a dose of twice ED50, respectively for each group. A
total of 250 μg of adrenocorticotropic hormone1–24 (ACTH1–24
)
(Sigma-Aldrich Chemical Co., St. Louis, MO) was injected into each
dog 15 min after sampling to stimulate cortisol and corticosterone pro-
duction. The consequent blood sample was collected at 1 h after admin-
istration of ACTH1–24 (Fig. 2). Blood samples (of a 4 mL volume) were
cultured and clotted at 37 °C for approximately 30 min, then centrifuged
at 3500 rpm at 4 °C for 10 min. Serum samples were then transferred
and frozen at −20 °C, followed by measurement of cortisol and cortico-
sterone levels within 1–2 days. After the thawing process, blood sam-
ples were centrifuged at 16,000 rpm, at 4 °C for 10 min, and were
then quantified using an enzyme-linked immunosorbent assay (ELISA
kit; Enzo Life Science) and a 96-well plate reader. A diagnosis of sup-
pression of adrenal function was usually made following the detection
of an impaired cortisol response to corticotrophin (Cotten et al., 2010;
Marik et al., 2008). The methods used to investigate dog adrenal func-
tion were optimized and adapted according to several previously pub-
lished reports (Maze et al., 1991; Foster et al., 2000; Pessina et al., 2009).
2.3. In Vivo Studies
2.3.1. Pharmacodynamic evaluation
54 naïve healthy Beagle dogs (half males, half females; 9–12 kg in
weight) were selected at random. The dogs were catheterized in the ce-
phalic vein with a vein IV catheter (20 gauge, 19 mm). The 50% effective
dose (ED50) for loss of righting reflex (LORR) was established using an
up-and-down sequential allocation design: one Beagle dog was admin-
istered an initial dose of a test drug according to the weight of the dog,
and any incidences of LORR were recorded; if LORR occurred, a lower
dose was then administered to the next dog; if LORR did not occur,
the higher dose was then administered to the next dog; when LORR
happened to a dog and did not happen to the next dog, one crossover
was recorded, the test continued until a total of three crossovers were
obtained, and an ED50 was then calculated based upon these observa-
tions (Dixon, 1991).
The initial doses of all the drugs used were based on the findings of
the preliminary experiment. ET-26, ET-42, etomidate, MOC-etomidate
and CPMM dissolved in DMSO were all administered through the intra-
venous catheter, followed by a 1-mL saline flush. After the injection, the
timing procedure was started. Maintenance of LORR for longer than 30 s
was considered to indicate anaesthesia (Zhang et al., 2014). The onset
time was 0 min in all experiments because all drugs resulted in LORR
immediately after administration.
After ED50 of every drug was obtained, five groups of healthy dogs
(six dogs per group, half males and half females) were used for investi-
gation of the pharmacodynamic characteristics of ET-26, ET-42,
Etomidate, MOC-ET and CPMM. Each agent was intravenously adminis-
tered at a dose of twice the ED50. The corneal reflex and the righting, or
eyelash reflex, were measured to indicate sedation and anaesthesia. The
timing of LORR was regarded as the duration of anaesthesia; the time
from recovery of righting reflex to free walking was regarded as the re-
covery time. The duration of anaesthesia and the recovery time were re-
corded by a blinded observer. Muscle tremor is another major side effect
of etomidate; we thus recorded the incidence of muscle tremor in each
group, using a criterion of a duration of muscle tremor N30 s during an-
aesthesia. Body temperatures of all animals were maintained at 36–
38 °C during anaesthesia.
2.4. Statistical Analysis
Data were analyzed using the Dixon–Mood method to derive the
median effective doses [ED50 = x0 + i(A/N 0.5)] with a 95% confi-
dence interval (CI). In vitro data on the stability of ET-26, ET-42,
etomidate, MOC-etomidate and CPMM, and the concentrations of corti-
cal hormones are presented as mean standard deviation (SD), and the
N denotes the number of dogs in each group. Data on rates of metabo-
lism were analyzed using a one-way analysis of variance (ANOVA)
followed by Bonferroni's post-hoc test. A Kruskal–Wallis test, followed
by a Mann–Whitney U test was applied to assess differences in the du-
ration and recovery time observed in the in vivo tests. Plasma cortisol
and corticosterone concentrations after compound or vehicle adminis-
tration were linearized, log-transformed and compared using a one-
way ANOVA followed by a least-significant difference (LSD) or Tamhane
multiple-comparison test. A P-value b 0.05 was considered statistically
significant. All analysis was performed using Statistical Package for So-
cial Sciences (SPSS™) software for Windows, version 21 (SPSS, Chicago,
IL, USA). Figure preparation and curve fitting was performed using Ori-
gin 8.0 (Origin Lab Corp., Northampton, MA, USA).
3. Results
3.1. Metabolite Stability in Plasma and Liver Homogenate
ET-26, ET-42 and etomidate were stable in plasma samples, even
after a 2-h incubation. MOC-ET can be metabolized 25.23 1.78% and
CPMM can be metabolized 19.83
0.79% in plasma in 2 h (Fig.3 a).
The primary metabolites of MOC-ET and CPMM were MOC-ECA (Ge et
al., 2012a, b) and CPMM-ECA (Campagna et al., 2014), no etomidate
acid was detected at any point during the 2-h incubation period.
ET-42 was stable in liver homogenate samples, even after a 2-h incu-
bation period. The percentages of metabolized ET-26 and etomidate
were 19.02 0.06% and 10.74 1.71% in liver homogenate after a 2-
h incubation, and the rate of decomposition of ET-26 was significantly
greater than that of etomidate (P b 0.05). CPMM was undetectable,
2.3.2. Adrenocortical Function Test
A total of 26 adult female dogs were randomly divided into six
groups, with 6 dogs in the CPMM treatment group and 4 dogs in all

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J. Yang et al. / European Journal of Pharmaceutical Sciences 99 (2017) 343–349
Fig. 2. The experimental protocol of the cortical hormone stimulation test.
and MOC-etomidate was barely detectable (1.54 0.06% of the concen-
tration at the start of the incubation) after a 10-min incubation in liver
homogenate (Fig.3 b.), indicating that CPMM and MOC-etomidate are
much more rapidly degraded than the other agents tested (Vs. ET-26,
P b 0.05). When incubated in plasma, no etomidate acid was detected
after a 2-h incubation of MOC-etomidate or CPMM in liver homogenate.
Etomidate acid was found to be the primary metabolite of both ET-
26 and etomidate: the concentrations of etomidate acid were 8.79
0.37 μg/mL and 3.24 0.16 μg/mL in the ET-26 and etomidate groups,
respectively. The initial concentration of both ET-26 and etomidate in
liver homogenate was 50 μg/mL. Theoretically, 19.02% of this concentra-
tion of ET-26 and 10.74% of this concentration of etomidate would result
in etomidate acid concentrations of 7.50 μg/mL and 4.75 μg/mL, respec-
tively. The measured concentrations of etomidate acid were found to
conform approximately with these calculated values. Therefore, we
are able to confirm that our measurements of etomidate acid produc-
tion are consistent with our measurements of ET-26 and etomidate
degradation.
time for the effects of all agents was 0 min. The duration of anaesthesia
was 3.21 0.90, 7.94 0.41, 5.36 1.91, 2.43 0.35 and 2.21
0.42 min in the ET-26, ET-42, etomidate, MOC-etomidate and CPMM
groups, respectively. The duration of the recovery time was 1.84
0.82, 9.67 0.50, 1.78 0.93, 5.08 1.09 and 1.21 0.53 min in the
ET-26, ET-42, etomidate, MOC-etomidate and CPMM groups, respec-
tively (Table 1). No significant differences were observed in the dura-
tion of anaesthesia in the ET-26 group compared with that of the
MOC-etomidate and CPMM groups (P = 0.655 versus MOC-etomidate,
and P = 0.426 versus CPMM). The mean duration of anaesthesia in the
ET-26, CPMM and MOC-etomidate groups was significantly shorter
than that of the ET-42 group (P b 0.05). No significant differences in
the mean duration of recovery time were observed in the ET-26 group
compared with that of the etomidate and CPMM groups (P = 1.000 ver-
sus etomidate, P = 0.394 versus CPMM). The mean duration of recovery
time in the MOC-etomidate and ET-42 groups was significantly longer
than that of the ET-26, etomidate and CPMM groups (P b 0.05). In addi-
tion, the incidence of muscle tremor in the ET-26 group was lower than
that of the other four groups (Table 1).
3.2. LORR Test
3.4. Adrenocortical Function Test
DMSO, the solvent control, had no anaesthetic or adverse effects. The
ED50 of ET-26, ET-42, etomidate, MOC-etomidate and CPMM for LORR in
dogs was 1.44, 0.72, 0.43, 23.12, and 0.59 mg/kg, respectively. Pharma-
codynamic characteristics of ET-26, ET-42, etomidate, MOC-etomidate
and CPMM were also investigated (Table 1).
The effects of ET-26, ET-42, etomidate, MOC-etomidate and CPMM
on adrenocortical function were assessed using measurements of
serum cortisol and corticosterone concentrations.
The baseline of serum cortisol concentrations was 347.42
83.19 pg/mL, 368.66
204.38 pg/mL, 251.55
75.30 pg/mL,
3.3. Equivalent Study
269.76 97.26 pg/mL, 248.28
44.73 pg/mL and 308.12
290.86 pg/mL in the ET-26, ET-42, etomidate, MOC-etomidate, CPMM
and cntrol groups, respectively. After ACTH stimulation, serum cortisol
Doses corresponding to twice the ED50 were given to induce LORR.
All the dogs were anaesthetized during injection; therefore, the onset
concentrations was 3493.84
1558.09 pg/mL, 3657.97
Fig. 3. In vitro 2-h decomposition curves of the five test drugs in (a). plasma and (b). liver homogenate.

J. Yang et al. / European Journal of Pharmaceutical Sciences 99 (2017) 343–349
347
Table 1
Pharmacodynamic characteristic of the 5 drugs in Beagle dogs (N = 6).
Drug
ED50
Dose
Onset time (Bovill, Duration time (Campagna et Recovery time (Cotton et
Incidence of muscle tremor (Cotten
(with 95% CI, mg/kg) (Atucha
et al., 2009)
(2ED50
mg/kg
)
2006)
min
al., 2014)
min
al., 2008)
min
et al., 2009)
ET-26
ET-42
1.44 (1.63–1.27)
0.72 (0.75–0.69)
2.88
1.44
0.86
46.24
1.18
0
0
0
0
0
3.21
7.94
5.36
2.43
2.21
0.90
1.84
9.67
1.78
5.08
1.21
0.82
2/6
3/6
4/6
6/6
6/6
0.41
1.91
0.35
0.42
0.50
0.93
1.09
0.53
Etomidate 0.43 (0.48–0.39)
MOC-ET
CPMM
23.12 (26.19–20.40)
0.59 (0.60–0.57)
1: The ED50 was determined by the up-and-down method (Dixon, 1991).
2: Onset time denotes the time from injection of the drug to disappearance of the righting reflex.
3: Duration time denotes the time from disappearance of the righting reflex to recovery of the righting reflex.
4: Recovery time denotes the time from recovery of the righting reflex to free walking.
5: Criterion of muscle tremor was that the tremor time of extremities or trunk was N30 s during anaesthesia.
1533.68 pg/mL, 436.79
5450.39 1947.61 pg/mL and 8596.66
26, ET-42, etomidate, MOC-etomidate, CPMM and cntrol groups,
respectively.
109.52 pg/mL, 357.29
104.36 pg/mL,
compared with those of the control group (P = 0.674 versus CPMM,
P = 0.389 versus ET-26, P = 0.199 versus ET-42) (Fig.4 b.). These results
indicate that, unlike etomidate and MOC-etomidate, ET-26, ET-42 and
CPMM could relieve cortex suppression in Beagle dogs.
3315.59 pg/mL in the ET-
The baseline of serum corticosterone concentrations was 478.60
195.37 pg/mL, 247.10
165.10 pg/mL, 464.13
293.78 pg/mL,
4. Discussion
193.84 176.60 pg/mL, 241.38
185.08 pg/mL and 150.80
49.52 pg/mL in the ET-26, ET-42, etomidate, MOC-etomidate, CPMM
and cntrol groups, respectively. After ACTH stimulation, serum cortico-
Etomidate has a potent inhibitory effect on 11-β hydroxylase en-
zymes; however, its metabolite, etomidate acid, has an inhibitory po-
tency approximately 1/120,000 of that of etomidate (Zolle et al.,
2008), indicating almost no inhibitory effect. Intravenous etomidate re-
sults in obvious inhibition of cortical hormone synthesis, owing to slow
in vivo metabolism. To overcome this adverse effect of etomidate, a
number of promising etomidate softdrug have been developed
(Cotten et al., 2009; Husain et al., 2012; Pejo et al., 2012b). These mole-
cules are rapidly metabolized in vivo; therefore, the prototype drug can
be rapidly eliminated from the body. These prototype drugs are unable
to suppress cortical hormone synthesis for a long period of time after
drug withdrawal.
However, these softdrug are generally not metabolized primarily to
etomidate acid. Etomidate softdrug are rapidly metabolized in vivo by
hydrolytic enzymes located throughout the body. Owing to their con-
siderable polarity, the primary metabolites generated by the hydrolytic
enzymes cannot easily cross the blood–brain barrier, and can accumu-
late in both the brain and the periphery. If these primary metabolites
have some inhibitory effects on the CNS, they will affect the quality of
the patient's postoperative recovery; if the primary metabolites have
some cortical hormone inhibitory activity, they may produce obvious
sterone concentrations was 3088.19
884.99 pg/mL, 609.50
3335.25 1943.27 pg/mL and 4380.09
1886.67 pg/mL, 2197.99
208.30 pg/mL,
1651.06 pg/mL in the ET-
73.34 pg/mL, 468.21
26, ET-42, etomidate, MOC-etomidate, CPMM and cntrol groups,
respectively.
No significant differences in baseline cortisol and corticosterone
concentrations were observed across all groups (cortisol: P = 0.731;
corticosterone: P = 0.431). Serum cortisol concentrations in the
CPMM, ET-26 and ET-42 groups were significantly higher than those
of the etomidate and MOC-etomidate groups at 1 h after ACTH stimula-
tion (P b 0.05). No significant differences in serum cortisol concentra-
tions at 1 h after ACTH stimulation were observed between the
control group and the ET-26, ET-42 and CPMM groups (P = 0.755 versus
CPMM, P = 0.115 versus ET-26, P = 0.136 versus ET-42) (Fig.4 a.).
Serum corticosterone concentrations in the ET-26, ET-42 and CPMM
groups at 1 h after ACTH stimulation were significantly higher than
those of the etomidate and MOC-etomidate groups (P b 0.05). No signif-
icant differences were observed in serum corticosterone concentrations
at 1 h after ACTH stimulation in the ET-26, ET-42 and CPMM groups
Fig. 4. Comparisons of (a). ACTH-stimulated serum cortisol concentrations, and (b). ACTH-stimulated serum corticosterone concentrations. ACTH; adrenocorticotropic hormone.

348
J. Yang et al. / European Journal of Pharmaceutical Sciences 99 (2017) 343–349
cortical hormone suppression after drug withdrawal. For example,
MOC-ECA has been confirmed to have an inhibitory effect on both the
CNS and on cortical hormone (Ge et al., 2012a, b).
therapeutic index and cardiovascular stability of etomidate. These unre-
solved concerns will form the basis of subsequent research.
In this study, MOC-etomidate and CPMM were rapidly metabolized
in both plasma and liver homogenate samples. After incubation in
liver homogenates for 15 min, MOC-etomidate and CPMM are almost
completely metabolized. Interestingly, no etomidate acid was detected
even after a 2-h incubation of MOC-etomidate or CPMM in either plas-
ma or liver homogenates. MOC-etomidate can be hydrolyzed in vivo to
MOC-ET-ECA (Cotten et al., 2009), indicating that MOC-ET-ECA cannot
be rapidly hydrolyzed to etomidate acid. Despite MOC-etomidate
being rapidly metabolized, ACTH stimulation did not result in an in-
crease in cortical hormone levels 75 min after administration of MOC-
etomidate, indicating that cortical hormone synthesis continued to be
inhibited. We speculate that this prolonged inhibition is probably
caused by accumulation of MOC-ET-ECA. Similar to MOC-etomidate,
CPMM can also be rapidly hydrolyzed during in vitro incubation in
liver homogenate for 10 min. CPMM-ECA, as a primary metabolite of
CPMM, could not be rapidly metabolized to etomidate acid. However,
no significant suppression of corticosteroid levels was observed, sug-
gesting that CPMM-ECA does not suppress circulating cortical hormone
levels. These results demonstrate that knowledge of the major primary
metabolites is a very important consideration in the design and testing
of etomidate analogues.
5. 5. Conclusions
In this study, ET-26 and ET-42 were demonstrated to be highly po-
tent anaesthetics. ET-26, similar to etomidate, is slowly metabolized to
etomidate acid in liver homogenates. ET-42 is more slowly metabolized
than both ET-26 and etomidate. Despite this slow metabolism, both ET-
26 and ET-42 have no significant effects on adrenal function. Our results
indicate that etomidate derivatives that are not rapidly metabolized and
that have pharmacologically inactive metabolites (such as etomidate
acid) might provide an alternative to etomidate, with a reduced risk of
adverse effects on corticosteroid levels.
Abbreviations
ACTH
ET-26
ET-42
adrenocorticotropic hormone
methoxyethyl etomidate
methoxy-2-methylpropan etomidate
MOC-ET methoxycarbonyl etomidate
MOC-ET-ECA methoxycarbonyl etomidate acid
CPMM
cyclopropyl-methoxycarbonyl etomidate
CPMM-ECA cyclopropyl-methoxycarbonyl etomidate acid
The anaesthetic effects of etomidate originate from its ability to acti-
vate GABAA receptors (Siegwart et al., 2002), while the suppression of
cortical hormone levels is a result of inhibition of 11-β hydroxylase en-
zymes (Vanden Bossche et al., 1984). Investigations of the structure–ac-
tivity relationships of etomidate indicate that the structure of the
imidazole carboxylic acid ester side chain is important for both of
these effects (Atucha et al., 2009). Therefore, specific modifications of
these ester side chains might enable the anaesthetic effects of etomidate
to be retained, with a reduction in the extent of suppression of cortical
hormone levels. If etomidate acid is the primary metabolite of such mol-
ecules, this would suggest that neither the prototype agents, nor its me-
tabolite, are likely to inhibit cortical hormone biosynthesis.
Conflicts of Interest
The authors declare that they have no competing interests.
Funding
This work was supported by National Natural Science Foundation of
China 81302640, National Science and Technology Major Project
2014ZX09101001003 and 2014ZX09101001004.
With these previous findings in mind, the properties of ET-26 and
ET-42, which are etomidate analogues with different ester side chains,
were investigated in this study. The results of this study show that ET-
26 and ET-42 produce definite and reversible anaesthetic effects, and
that the anaesthetic efficacy of ET-26 is close to one-third that of
etomidate, and the efficacy of ET-42 is close to half that of etomidate.
In addition, we observed that the incidence of muscle tremor in the
ET-26 group is significantly lower than that of the other four groups.
Etomidate acid, as observed in liver homogenates, was found to be a
major metabolite of ET-26. Furthermore, ET-26 and ET-42, similar to
etomidate, was found to be much more slowly metabolized than both
MOC-etomidate and CPMM.
Acknowledgements
The authors thank Bowen Ke, Ph.D., for his helpful suggestion on the
study design.
Appendix A. Supplementary Data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ejps.2016.12.041.
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