Metabolite identification, tissue distribution, excretion and preclinical pharmacokinetic studies of ET-26-HCl, a new analogue of etomidate

ET-26-HCl, a novel anaesthetic agent with promising pharmacological properties, lacks extensive studies on pharmacokinetics and disposition in vitro and in vivo. In this study, we investigated the metabolic stability, metabolite production and plasma protein binding (PPB) of ET-26-HCl along with its tissue distribution, excretion and pharmacokinetics in animals after intravenous administration. Ultra-high performance liquid chromatography–tandem quadrupole time-of-flight mass spectrometry identified a total of eight new metabolites after ET-26-HCl biotransformation in liver microsomes from different species. A hypothetical cytochrome P450-metabolic pathway including dehydrogenation, hydroxylation and demethylation was proposed. The PPB rate was highest in mouse and lowest in human. After intravenous administration, ET-26-HCl distributed rapidly to all tissues in rats and beagle dogs, with the highest concentrations in fat and liver. High concentrations of ET-26-acid, a major hydroxylation metabolite of ET-26-HCl, were found in liver, plasma and kidney. Almost complete clearance of ET-26-HCl from plasma occurred within 4 h after administration. Only a small fraction of the parent compound and its acid form were excreted via the urine and faeces. Taken together, the results added to a better understanding of the metabolic and pharmacokinetic properties of ET-26-HCl, which may contribute to the further development of this drug.

We conducted a comprehensive disposition study and preclinical pharmacokinetics (PK) to support the further development of this new drug. In this study, we evaluated the metabolic stability of ET-26-HCl in liver microsomes from different species and identified CYP metabolites using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). PK of ET-26-HCl was conducted in beagle dogs following intravenous administration. In addition, we investigated biodistribution and excretion profile of ET-26-HCl and ET-26-acid and plasma protein binding (PPB) in vitro.

Materials
ET-26-HCl and its main metabolite ET-26-acid ( purity greater than 99.0% by HPLC) were provided by the Anesthesia and Critical Aid Laboratory, Conversion Neuroscience Center, West China Hospital of Sichuan University. Internal standard (IS), gabapentin (GBP, figure 1d) was obtained from Palm Medicine Chemicals Ltd (Nanyang, Zhejiang, China). HPLC grade methanol was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Formic acid was purchased from Dikma Technologies, Inc. (Lake Forest, CA, USA). Liver microsomes of human, monkey, dog, rat and mouse were purchased from Becton, Dickinson and Company (Franklin Lake, New Jersey, USA).

ET-26-HCl and ET-26-acid analysis in biological samples
Concentrations of ET-26-HCl and ET-26-acid in biological samples were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after deproteinization with methanol. The bioanalytical method for simultaneous quantification of ET-26-HCl and ET-26-acid in the different matrix was fully validated according to FDA guidelines (electronic supplementary material, figure S1 and tables S1 and S2) in our previous study [11,12]. Briefly, a 100 µl sample ( plasma, tissue homogenate, bile, urine and faeces suspension) were spiked with 20 µl of IS solution and vortexed for 30 s. The mixture was then processed for protein precipitation with 300 µl methanol, followed by centrifugation at 12 000 r.p.m. for 5 min. Supernatant was collected and analysed by LC-MS/MS system.
The LC-MS/MS was performed as described previously [11]. Briefly, ET-26-HCl, ET-26-acid and GBP were separated using a CAPCELL PAK C 18 column (50 × 2.00 mm, 5.00 µm) as well as a Security Guard Cartridge (C 18

Metabolic stability in liver microsomes
In vitro metabolic stability of ET-26-HCl was estimated in liver microsomes from human, monkey, dog, rat and mouse. A mixture of 100 mM ET-26-HCl, 2.0 M VIVID, 5.0 M MgCl 2 and liver microsomes (human, monkey, dog, rat and mouse, 0.33 mg microsomes protein/ml) in Tris-HCl phosphate buffer (pH 7.4) was pre-incubated for 10 min at 37°C before the addition of 1.0 mM NADPH and then quenched with 450 µl methanol at 0, 7, 17, 30 and 60 min. Samples were analysed by LC-MS/MS as mentioned above.

Identification of major metabolites in liver microsomes
The major metabolites of ET-26-HCl in liver microsomes from mouse, rat, beagle dog, monkey and human were determined by the UPLC-Q-TOF-MS system. Reaction mixture containing 10 µM ET-26-HCl, 5 M MgCl 2 and 1 mg ml −1 liver microsomes in Tris-HCl phosphate buffer ( pH 7.4) was pre-incubated for 6 min at 37°C, followed by the addition of NADPH. After incubation for 1 h, methanol was added to stop the reaction. Samples were centrifuged royalsocietypublishing.org/journal/rsos R. Soc. open sci. 7: 191666 at 11 000 r.p.m. for 5 min; supernatants were collected and air dried at 40°C. Residues were dissolved in 100 µl acetonitrile/water (1:1, v/v) and analysed by the UPLC-Q-TOF-MS. The UPLC-Q-OF-MS system was equipped with a Waters Synapt G2-Si quadrupole time-of-flight mass spectrometer (Q-TOF-MS), an ESI source and an ACQUITY UPLC H-CLASS liquid chromatography system. The chromatographic separations were achieved on an ACQUITY UPLC BEH C 18 column (2.1 × 100 mm, i.d. 1.7 µm, Waters, USA) kept at 45°C and a flow rate of 0.4 ml min −1 . The mobile phase consisted of 5 mM ammonium acetate with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The optimized gradient elution programme followed was 0 min to 2.1 min, A/B (19/1, v/v); 2.1 min to 7.3 min, A/B (1/4, v/v); 7.3 min to 8.0 min, A/B (1/9, v/v); and 8.0 min to 10 min, A/B (19/1, v/v). The injection volume was 5 µl. The mass experiment was operated in positive ion modes under the following parameters: turbo spray temperature, 120°C; ion spray voltage, 3.0 kV. Nitrogen was used as the nebulizer and the auxiliary gas; meanwhile, the nebulizer gas was set to 650 l h −1 and kept at 350°C. The collision energy was set at 5-10 V and the collision energy spread was 4-6 V. TOF-MS was scanned with the mass range of m/z 50-600 with 200 ms accumulation time. A 400 ng ml −1 leu-enkephalin (m/z 556.2771) was selected as an external standard for mass-charge ratio correction at a flow rate of 5 µl min −1 .

Plasma protein binding of ET-26-HCl
The PPB of ET-26-HCl in mouse, rat, beagle dog, monkey and human plasma was determined according to the equilibrium dialysis method [13,14]. In brief, 1 ml of control plasma samples in dialysis bag was incubated in 10 ml of PBS containing ET-26-HCl at a final concentration of 2.21 × 10 2 , 1.77 × 10 3 and 3.54 × 10 3 ng l −1 at 4°C with an equilibrium time of 9 h to reach the mass balance binding condition. A 100 µl aliquots of the PBS buffer and the plasma in the dialysis bag were transferred to 2 ml EP tubes and diluted to 995 µl with PBS and analysed on the LC-MS/MS system. The PPB (f ppb ) was calculated as follows [15]: where D in is the plasma drug concentration in the dialysis bag and D out is the dialysate drug concentration.

Pharmacokinetics of ET-26-HCl in beagle dogs
Beagle dogs, weighing 9-11 kg, were purchased from Sichuan Industrial of Antibiotics, China National Pharmaceutical Group Corporation (Chengdu, China). A 3 × 3 crossover experimental design with 24 beagles (equal number of male and female dogs) randomly divided into six groups was conducted. Beagle dogs in the six groups received three doses of ET-26-HCl: 1.045 mg kg −1 , 2.090 mg kg −1 and 4.180 mg kg −1 as low (L), medium (M) and high (H) dose. Blood samples (2 ml) were collected from the elbow vein in 4 ml heparinized tubes before dosing, and at 1, 3, 5, 10, 20, 30, 120 and 180 min post-dosing. All blood samples were centrifuged at 8000 r.p.m. for 10 min and plasma collected and stored at −80°C until analysis.

Tissue distribution of ET-26-HCl in rats
Sprague Dawley (SD) rats (200-240 g) were provided by the Animal Facility at Sichuan University (Chengdu, China). All rats received a single 4.2 mg kg −1 dose of ET-26-HCl intravenously. Rats were sacrificed and tissues, including heart, spleen, lung, intestine, colon, kidney, brain, fat, stomach, liver, muscle, testis and ovary were collected at 0, 1, 3, 6 and 10 h post-dosing (six rats/time-point). Tissue samples were rinsed thrice in ice cold saline, and blotted dry with filter papers. Weighed amount of tissues were homogenized in normal saline (thrice the tissue weight, w/v) and homogenates were stored at −80°C until analysis. Bile fistulas of SD rats (four male and four female) were cannulated with polyethylene tube (Instech Laboratories, Inc., USA) and bile was collected at the following time-points: 0-3, 3-6, 6-9, 9-12 and 12-24 h post-dosing. The volume of the collected bile was measured and stored at −80°C until analysis.

Pharmacokinetics, tissue distribution, excretion and plasma protein binding
All calculations were performed on Microsoft Excel 2016 (Microsoft Co., USA). The Drug and Statistics 3.2.7 (DAS 3.2.7) software package (Mathematical Pharmacology Professional Committee of China, China) was used to acquire the main pharmacokinetic parameters of ET-26-HCl. All data were shown as mean ± standard deviation (s.d.). Statistical significances were calculated by one-way analysis of variance (ANOVA).

Metabolic stability of ET-26-HCl
ET-26-HCl was not metabolically stable in liver microsomes from monkey, dog, rat and mouse, but was relatively stable in human liver microsomes (figure 2), as assessed by the disappearance of ET-26-HCl (table 1). After incubation under the given condition, ET-26-HCl completely disappeared within 15 min in the monkey liver microsomes, followed by rat, mouse and dog. Human liver microsomes did not wholly transform ET-26-HCl at 1 h. The CL int, in vitro of ET-26-HCl, was 50.0, 2216, 466, 984 and 629 ml min g −1 protein in liver microsomes from human, monkey, dog, rat and mouse, respectively.

Identification of major metabolites of ET-26-HCL in liver microsomes
The parent compound ET-26-HCl was detected only in liver microsomes samples from human and dog. The fragmentation behaviour was similar to that of ET-26-HCl with ion at m/z 139.0569. As shown in figure 4b, characteristic product ion at m/z 157.0613 was CH 2 less than the fragment ion of ET-26-HCl with ion at m/z 171.0773, suggesting that the methyl linked with the side chain oxygen was demethylation. Dehydrogenation might have occurred on the left side of the imidazole ring. Therefore, M1 was speculated to be the oxygen-demethylated and desaturated metabolites of ET-26-HCl.

Metabolite M2
Metabolite M2 was detected in every liver microsomal preparation. It was eluted at 5.25 min, with a protonated molecular ion at m/z 261.1230, indicating a molecular composition of C 14 H 16 N 2 O 3 , CH 2 less than that of the parent compound ET-26-HCl. The characteristic ions at m/z 95.0225 and 139.0494, were similar to that of ET-26-HCl. Similar to M1, the ion at m/z 157.0596 was 14 Da lower than that of m/z 171.0773, suggesting that M2 was an oxygen-demethylated metabolite of ET-26-HCl (figures 3 and 4c).

Metabolite M3
Metabolite M3 was detected at the retention time of 6.12 min only in liver microsomes from human and dog ( figure 3). The formula was conjectured as C 15

Determination of plasma protein binding rate
PPB rates of ET-26-HCl at 654, 2.62 × 10 3 and 5.23 × 10 3 ng ml −1 in mouse, rat, beagle dog, monkey and human plasmas are shown in table 2. Binding rates in the plasma of mouse, rat, beagle dog, monkey and human were 94.35 ± 1.15%, 83.03 ± 1.76%, 66.02 ± 4.10%, 62.72 ± 3.56% and 68.31 ± 5.16%, respectively. A one-way single factor ANOVA and post hoc LSD t-test manifested that only the PPB rates of ET-26-HCl in mouse, rats and humans were significantly different ( p < 0.05).

Pharmacokinetics in beagle dogs
Plasma concentrations of ET-26-HCl in beagles after i.v. administration of three doses are shown in figure 5, and the major PK parameters are shown in table 3. The plasma concentration ET-26-acid was almost non-detectable in the beagles. Moreover, the halflife (t 1/2 ) and volume of distribution (V d ) of ET-26-HCl increased while the clearance (CL) deceased at higher doses.

Tissue distribution in Sprague Dawley rats
In rats, both ET-26-HCl and ET-26-acid widely and rapidly distributed to several tissues ( figure 6). CL of ET-26-HCl and ET-26-acid from tissues took around 3 h indicating that ET-26-HCl was a short-acting anaesthetic drug, consistent with the trend in plasma concentrations. Adipose tissue and liver had the highest concentration of ET-26-HCl followed by kidney, stomach, plasma, colon, spleen, intestines,  royalsocietypublishing.org/journal/rsos R. Soc. open sci. 7: 191666 brain, heart, ovary, lung, muscle and testis. Meanwhile, the highest concentration of ET-26-acid was found in plasma and liver, followed by kidney, fat, lung, spleen, testis, stomach, intestines, colon, ovary, heart, muscle and brain.

Excretion in Sprague Dawley rats
The accumulative excretion results of rats injected intravenously with ET-26-HCl

Discussion
ET-26-HCl, a promising short-acting anaesthetic agent, was investigated with regard to its distribution, metabolism, excretion and PK in animals in this study according to Product Development Under the Animal Rule: Guidance for Industry from FDA [16]. Several CYP-mediated metabolites were identified. Metabolically, ET-26-HCl was significantly more stable in human liver microsomes compared with microsomes from other species. A significant amount of parent compound was found in the human microsome, whereas there was none or little residual amount of ET-26-HCl in liver microsomes from other species, which were mainly O-demethylated and dehydrogenated O-demethylated metabolites. No human-specific metabolite was identified in this study. Relevant information on ET-26-HCl and its metabolites are summarized in table 4. Because of space limitation, only the most likely fragment ions are shown. The presumed metabolic pathways of ET-26-HCl in liver microsomes of all species are shown in figure 8. Based on the results of hepatic microsomal metabolic stability, possible metabolic pathway and metabolites, only dogs and rats were selected for PK analysis. In our previous study, we had successfully established an LC-MS/MS method for quantification of ET-26-HCl and ET-26-acid in rat plasma and used it for a pharmacokinetic study [11]. In the present study, this simple, sensitive and reliable method was successfully used to evaluate metabolic stability, PK in beagle dogs, tissue distribution and excretion in SD rats, and PPB of ET-26-HCl. We observed rapid metabolism of ET-26-HCl with a half-life of less than 5 min in the liver microsomes from monkey, rat, mouse and dog. However, it was relatively stable with a half-life of 42.0 min in human liver microsomes. In beagles, the t 1/2 was about 1 h and V d was about 4 l kg −1 (2.09 mg kg −1 ), which suggested short residence time and rapid clearance of ET-26-HCl from the body. It is worth mentioning that there is a species difference for ET-26-HCl pharmacokinetics, since a large gap was observed in PK parameters, i.e. t 1/2 , CL and V d of ET-26-HCl between dogs and rats (figure 5 and table 3; electronic supplementary material, figure S2 and table S4) [11]. ET-26-acid was almost undetectable in the plasma of dogs, most likely due to the difference in the type and expression of carboxylesterases between dogs and rats [11,17]. A greater than dose proportional increase in AUC and deceased clearance at higher doses were observed, which indicated the possible nonlinear PK of ET-26-HCl in dogs. Because hepatic clearance is the major pathway of elimination, the saturation of metabolism is considered to be the main reason.
The extrapolated hepatic clearances (CL hep, in vivo in table 1) are 19.1 and 296 ml min −1 for rat and dog, respectively, which can be normalized to while the observed total body clearances are 1.78 and    (table 3; electronic supplementary material, table S4). The results indicate that the prediction is reasonable and hepatic clearance is the major pathway for drug elimination.
As per Kleiber's law for allometric scaling between species, the animal metabolic rate can be scaled to around 0.75 power of the animal's size [18]. The relations in terms of clearance can be examined using the equation of CL = a × BW b , where a is the typical value of clearance, BW is the average body weight of various species and b is the power which should close to 0.75. Log-linear regression of the predicted clearance versus the average BW of mouse, rat, dog, monkey and human resolved that a = 40, b = 0.74 and R 2 = 0.999. Our results suggested a good fit using Kleiber's equation for allometric scaling (electronic supplementary material, figure S3).
In rats, ET-26-HCl was rapidly metabolized and distributed to blood-rich tissues and organs. The overall high concentrations of ET-26-HCl and ET-26-acid was observed in liver, kidney and plasma. However, the distribution into fat and brain was significantly higher for ET-26-HCl rather than ET-26acid. Because ET-26-acid is more hydrophilic than ET-26-HCl, which may result in reduced passive permeability across cell membrane. Considerable amounts of ET-26-HCl in the brain (concentration 2.57 × 10 3 ng ml −1 at the onset) suggest that it can effectively penetrate across BBB. The rapid elimination of ET-26-HCl from the brain within 3 h and complete excretion from kidney within 24 h, supporting the use of ET-26-HCl as a short-acting anaesthetic with rapid clearance. We believe that ET-26-HCl with its minimal adrenal suppression and potent sedative action in preclinical studies could be a promising short-acting anaesthetic drug candidate [6,7].
PPB of beagle dog, monkey and human were much lower than that of mouse and rat (table 2) indicating the inter-species variation between rodent and non-rodent. As a weak basic compound, albumin could be the major binding protein in the blood, similar to etomidate [19,20]. However, the exact binding sites need to be further identified. The pharmacokinetics of drugs with strong PPB (e.g. 13 phenytoin and warfarin) may be influenced by co-administration of ET-26-HCl due to its moderate binding rate in human plasma (68%) [21,22]. Less than 30% of ET-26-HCl was excreted through urine and faeces as parent drug or ET-26-acid, indicating that other important metabolic pathways should be considered. However, as the reference standards of proposed metabolites were not available, the proportion of each metabolite in the urine and faeces remains undetermined. Also, measurement of the kinetics of metabolite formation was not possible at this stage. Besides, pharmacological activity of the metabolites and their relative contribution to the total efficacy of ET-26-HCl warrant further clarification.

Conclusion
In this study, simple and sensitive UPLC-Q-OF-MS and LC-MS/MS methods were developed and successfully used to examine metabolic stability, metabolic products, PK, tissue distribution, excretion and PPB of ET-26-HCl, a novel etomidate analogue. We identified ET-26-HCl as a promising shortacting anaesthetic drug candidate based on its PK and distribution properties.
Ethics. All experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals and approved by the Committee of Scientific Research and the Committee of Animal Care of the West China Hospital, Sichuan University ( protocol no. 2015012311). Liver microsomes from human, monkey, dog, rat and mouse were purchased from Becton, Dickinson and Company. We were not required to complete an ethical assessment prior to conducting the researches involving liver microsomes. Human plasma was provided by Chengdu Fan Microanalysis Pharmaceutical Technology Co., Ltd and approved by the Ethical Committee of The Phase I Clinical Trial Research Office of the 416 Hospital of the Nuclear Industry, China ( protocol no. LSD2018001).