Application of ultrasound‐guided cholecystocentesis to the evaluation of the metabolite profiling in bile of dogs and cynomolgus monkeys

Abstract In this study, we describe a novel approach for collecting bile from dogs and cynomolgus monkeys for metabolite profiling, ultrasound‐guided cholecystocentesis (UCC). Sampling bile by UCC twice within 24 hours was well tolerated by dogs and monkeys. In studies with atorvastatin (ATV) the metabolite profiles were similar in bile obtained through UCC and from bile duct‐cannulated (BDC) dogs. Similar results were observed in UCC and BDC monkeys as well. In both monkey and dog, the primary metabolic pathway observed for ATV was oxidative metabolism. The 2‐hydroxy‐ and 4‐hydroxyatorvastatin metabolites were the major oxidation products, which is consistent with previously published metabolite profiles. S‐cysteine and glucuronide conjugates were also observed. UCC offers a viable alternative to bile duct cannulation for collection of bile for metabolite profiling of compounds that undergo biliary excretion, given the similar metabolite profiles in bile obtained via each method. Use of UCC for metabolite profiling may reduce the need for studies using BDC animals, a resource‐intensive model.


| ATV dose solution preparation for monkey
Monkeys were dosed with ATV in a suspension formulation.
Suspension formulations were prepared for the study on the morning of dosing. Briefly, ATV tablets totaling 1 gram active were placed in a mortar and pestle and crushed into a fine powder. Suspension vehicle (0.5% methylcellulose E4M/0.02% Docusate Sodium) was slowly added to the powder to form a suspension at the desired concentration. The formulation was triturated until a homogenous suspension was formed.

| Dogs
Beagle dogs (n = 3, males) were orally dosed ATV tablets at 40 mg/ kg 1 hour after a morning meal in order to prevent gallbladder emptying after compound dosing and to allow for bile collection at an optimal time. Plasma samples were collected at 0.5, 1, 2, 4, 6, and 24 hours postdose via cephalic vein or indwelling cephalic catheter.
At the 6-and 24-hours postdose time points, dogs were sedated with propofol at 6 mg/kg through an indwelling cephalic catheter.
The dogs were then positioned in a dorsal-recumbency with head elevated. Heart rate, rhythm, and arterial oxygen saturation were monitored throughout the procedure. After shaving and aseptically preparing the abdominal skin immediately caudal to the xiphoid process, an ultrasound exam of the gallbladder and cranial abdomen was done using a Mylab30cv ultrasound system with 9-3 MHz micro-convex transducer (Esoate, Indianapolis, IN). A 21-gauge, 1.5-inch needle, attached to a 5-mL syringe, was inserted percutaneously through the abdominal wall into the gallbladder lumen.
Effort was made to completely empty the gallbladder by aspiration (typically, 2-5 mL bile was obtained). The needle and syringe were withdrawn and the bile samples were immediately saved on dry ice for later analysis. Dogs were allowed to recover in their home cages.
Both plasma and bile samples were stored at −20°C until analysis.
For bile collection monkeys were sedated with ketamine (8 mg/kg) and dexmedetomidine (0.05 mg/kg) intramuscularly. At the conclusion of the procedure, sedation was reversed with atipamezole (0.15 mg/kg) intramuscularly. All other procedures were the same as for the dog.

| Quantification of ATV and its metabolites by LC-MS/MS analysis
Quantification of ATV and its metabolites in dog and monkey plasma and bile was performed using LC-MS/MS-based analysis. Standard curves and quality control (QC) samples defining the dynamic range of the bioanalytical method were prepared in commercially available control plasma and processed in the same fashion as the test samples. When dilutions were required, an aliquot of the sample was diluted into control plasma. Aliquots (50 µL) of plasma or bile diluted into plasma from in vivo study and standard/QC samples were treated with acetonitrile (150 µL) containing internal standard SIL-ATV (0.2 µmol/L), followed by vortex mixing for 2 minutes.
The supernatant was then separated from the precipitated proteins after a 10-minute centrifugation at 3500 rpm (2109× g) at 10°C and transferred to an autosampler 96-well plate. An aliquot (10 µL) was injected onto the ultrahigh performance liquid chromatography (UHPLC) column for LC-MS/MS-based analysis. Samples from individual animals were not pooled prior to quantitative analysis.
The UHPLC system Nexera (Shimadzu, Japan) consisted of two pumps (LC30AD), a column heater (CTO-30A), and an autosampler (SIL-30AC) equipped with a cooling compartment that maintained samples at 15°C during analysis. The analytical column used was a BEH C18 2.1 mm × 50 mm, 1.7 µm (Waters Corporation, Milford, MA) at 50°C. The mobile phase, which consisted of 0.1% formic acid in water (A) and acetonitrile (B), was delivered at a flow rate of 0.6 mL/min. The initial conditions were 100% aqueous followed by 1-minute gradient to 100% acetonitrile then hold for 0.4 minutes and back to initial conditions. The retention times of each compound were as follows: ATV and SIL-ATV at 1.28 minutes, 2-OH ATV at The analysis of ATV and its metabolites was conducted against a standard curve ranging from 1 to 10 000 nmol/L. The standard curve was fitted with a linear regression weighted by reciprocal concentration squared (1/x 2 ). QC samples were prepared in control rat plasma.
The predicted concentrations of more than 2/3 of the QCs were within 20% of nominal values, indicating acceptable assay performance.

| Plasma pharmacokinetic analysis
The pharmacokinetic parameters of ATV were determined by noncompartmental analysis of plasma concentration vs time data (KINETICA ™ software, Version 5.0, Thermo Fisher Scientific Corporation, Philadelphia, PA). The peak concentration (C max ) and time for C max (T max ) were recorded directly from experimental observations. The area under the curve from time 0 to 24 hours (AUC 0-24h ) was calculated using a combination of linear and log trapezoidal summations.

| Quantitative analysis of ATV and Major metabolites in plasma and bile in dog and monkey
In general, the plasma concentrations of ATV were similar in the BDC and UCC dogs. The mean ATV AUC 0-24h of 2346 nmol/L × h for the UCC dogs was higher than the mean AUC 0-24h of 1072 nmol/L × h observed in the BDC dogs. The mean AUC 0-24h for the metabolites 2-OH ATV and 4-OH ATV were similar between the UCC and BDC dogs as well (Table 1). Table 2  In the UCC monkeys the mean ATV AUC 0-24h of 501 nmol/L × h was lower than the mean AUC 0-24h of 1077 nmol/L × h observed in the BDC monkeys. Similarly, the mean AUC 0-24h for the metabolites 2-OH ATV and 2-OH ATV-L were higher in the plasma samples from the BDC monkeys (Table 3). The concentrations of 4-OH ATV and 4-OH ATV-L were below the limits of quantitation in both the UCC and BDC monkey plasma samples. Table 4 shows the concentrations of ATV and the four metabolites in monkey bile obtained by UCC and BDC. ATV and metabolite concentrations were broadly similar across the 24-hour collection period in bile collected via both methods. In the UCC bile samples, the mean concentrations of the ATV and the metabolites ranged from 9 μmol/L to 594 μmol/L. In BDC bile samples, the mean concentrations of the parent drug and metabolites ranged from 2 μmol/L to 412 μmol/L during the 24-hour collection period. It is important to note here than some bile samples from the BDC monkeys were not collected/lost due to blocked or displaced cannulas.

| Metabolite profile of ATV in bile obtained by cholecystocentesis and bile duct-cannulation in dogs
Representative dog bile UV chromatograms from 6 and 24 hours UCC samples and 0-24 hours pooled BDC samples are shown in

| D ISCUSS I ON
ATV was chosen as a probe compound to evaluate the use of UCC for metabolite profiling due to its great extent of metabolism and biliary excretion. Biliary excretion is reported to be a major route of elimination for ATV and its metabolites in dog, accounting for 33% of the oral dose, with peak biliary excretion at 4-8 hours. 17 In the    Abbreviations: ATV, atorvastatin; BDC, bile duct-cannulated; UCC, ultrasound-guided cholecystocentesis. a n = 2 due to blocked/displaced cannulas. b n = 1; other samples below the lower limit of quantitation.  In conclusion, the current results demonstrate the utility of UCC for collecting bile samples from dogs and monkeys for metabolite profiling of compounds that undergo biliary elimination.
Bile samples obtained through UCC may be considered similar to pooled bile samples from BDC animals in that they represent an accumulation of metabolites between dosing and sample time.
Thus, UCC is a viable alternative methodology to help investigate drug candidate metabolism and disposition. This technique may provide sufficient metabolism data to obviate the need for investigations using surgically prepared dogs or monkeys, particularly in drug discovery. Alternatively, it could be used as a complement to a BDC study to provide a more complete picture of the metabolic pathways and excretion in intact animals. Additionally UCC avoids possible sample contamination that can occur with other alternative sampling methods and may be translational to clinical ADME studies.