Chemicals
Ethanol was purchased from DJ7656 Pharmco (Brookfield, CT); Propylene glycol (two lots used) from VWR (Brisbane, CA) and Avantor Performance Materials, Inc (Center Valley, PA); Carobowax Polyethylene glycol 400 (PEG 400) from Fisher Scientific (Hanover Park, IL); Kleptose HPB (Hydroxypropyl-B-cyclodextrin, Oral Grade (HPBCD) E0110) from Roquette America, Inc (Keokuk, IL); Phosphate buffered saline (PBS) from Invitrogen (Carlsbad, CA); Liver microsomes and NADPH-regenerating system from Xenotech (Kansas City, KS, and Corning (Tewksbury, MA,); Hanks’ balanced salt solution (HBSS) from Thermo Fisher Scientific (Waltham, MA); Caco-2 cells from American Type Culture Collection (Manassas, VA). Other reagents were commercially available and of special reagent grade, liquid chromatography mass spectrometry (MS) grade, or equivalent. (+)-SJ311 and (+)-SJ733 were provided by Dr. David Floyd’s group and synthesized according to the published route (14). The only structural difference is exchange of a single pyridine (SJ733) ring for a pyrazole (SJ311).
Animals.
General procedures for animal care and housing were in accordance with the National Research Council (NRC) Guide for the Care and Use of Laboratory Animals, 8th edition (2011) and the Animal Welfare Standards incorporated in 9 CFR Part 3, 1991.
All murine studies were performed at St Jude Children’s Research Hospital (SJCRH) using C57BL6 mice 8 weeks of age or older (17-23 g). All mice were maintained in a temperature-controlled environment on a fixed 12-hour light/dark cycle with free access to water and food. Studies were performed in strict accordance with the protocol approved by the SJCRH IACUC (Institutional Animal Care and Use Committee).
Rat studies performed at the Centre for Drug Candidate Optimisation (CDCO), Monash University (Australia), used male Sprague Dawley rats 8-9 weeks of age (267- 291 g). Studies were conducted using established procedures in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and the study protocols were reviewed and approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee. Rat studies performed at the SRI International used male Sprague Dawley rats 8-9 weeks of age (220-317 g) with jugular vein catheterization performed by the Charles River Laboratories. Studies were performed in accordance with the protocol approved by the SRI IACUC.
Canine studies carried out at SRI used male beagle dogs 6-7 months of age (6.0-7.7 kg). Studies were performed in accordance with the protocol approved by the SRI IACUC.
Solubility
Kinetic solubility
Solubility assays were carried out on a Biomek FX lab automation workstation (Beckman Coulter, Inc., Fullerton, CA) using µSOL Evolution software (pION Inc., Woburn, MA). Compound stock (10 mM in DMSO, 10 µL) was added to 1-propanol (190 µL) to make a reference stock plate. Reference stock solution (5 µL) was mixed with 1-propanol (70 µL) and citrate phosphate buffered saline (75 µL) to make the reference plate the UV spectrum (250 nm – 500 nm) of the sample plate was read. Test compound stock (10 mM in DMSO, 6 µL) was added to buffer (594 µL) in a 96-well storage plate and mixed. The storage plate was sealed and incubated at room temperature for 18 hours. The suspension was then filtered through a 96-well filter plate (pION Inc., Woburn, MA). Filtrate (75 µL) was mixed with 1-propanol (75 µL) to make the sample plate, and the UV spectrum (250 nm – 500 nm) of the sample plate was read. Calculation was carried out by µSOL Evolution software based on the AUCinf (area under curve) of UV spectrum of the sample plate and the reference plate. All compounds were tested in triplicate.
Thermodynamic solubility
The solubility of (+)-SJ733 was evaluated at 37°C under neutral (isotonic phosphate buffer, ionic strength of 154 mM, pH 7.4) and acidic (0.1 N HCl, pH 1.0) conditions. Solubility was also evaluated in fasted (FaSSIF-V2) or fed (FeSSIF-V2) state simulated intestinal fluids. These media contain lipolysis breakdown products (glycerol monooleate and oleic acid) in addition to bile salt (sodium taurocholate) and phospholipid (lecithin) (15) and were buffered to simulate approximate pH conditions found in the fasted (pH 6.5) or fed (pH 5.8) state small intestine. Control media (blank FaSSIF-V2 and FeSSIF-V2 buffers) lacking bile salt, phospholipid and lipolysis products were also investigated.
Compounds were weighed into individual screw cap polypropylene tubes and aqueous buffer, 0.1 N HCl, or simulated intestinal fluid added to provide a compound concentration of between 700 and 5000 μg/mL. Samples were vortexed, placed in a 37 °C incubator and mixed on an orbital shaker (IKA® VXR basic Vibrax® orbital shaker) set at 600 rpm. Samples were regularly examined to ensure excess solid was present. Sampling was conducted after 1, 4, 6, and 24 h. by centrifuging each sample at 10000 rpm for 3 minutes, transferring a 200 µL aliquot into fresh Eppendorf tubes, and centrifuging again at 10000 rpm for 3 min. Duplicate aliquots of the final supernatant were removed and diluted to an appropriate analytical concentration in 50% aqueous methanol prior to analysis by HPLC. HPLC analysis was conducted on a Waters 2695 HPLC system coupled to a Waters 2487 dual absorbance wavelength detector, analyzing at 254 nm. A Phenomenex Luna C18(2) column (3 µm, 50 x 2.0 mm i.d.) was used for analysis, with the column temperature maintained at 40°C. Concentrations were quantified by comparison to a calibration curve prepared over the concentration range in 50% aqueous methanol. The mobile phase consisted of water, methanol, and 1% aqueous formic acid. Separations were conducted using a flow rate of 0.4 mL/min and an injection volume of 5 µL. Processed samples were maintained in the autosampler at a temperature of 10°C.
Permeability
Parallel Artificial membrane Permeability Assay (PAMPA)
The assay was conducted using a Biomek FX lab automation workstation (Beckman Coulter, Inc., Fullerton, CA) with PAMPA evolution 96 command software (pION Inc., Woburn, MA). Test compound stock (10 mM in DMSO, 3 µL) was mixed with citrate phosphate buffered saline (597 µL) to make diluted test compound. Diluted test compound (150 µL) was transferred to a UV plate (pION Inc., Woburn, MA) and the UV spectrum (250 nm – 500nm) was read as the reference plate. Each well of the donor plate in a PAMPA sandwich plate (pION Inc., Woburn, MA) contained a filter that was painted on one side with 4 µL GIT lipid (pION Inc., Woburn, MA) to form a membrane. Each well in the acceptor plate in a PAMPA sandwich, preloaded with magnetic stir bars, was filled with acceptor solution buffer (200 µL, pION Inc., Woburn, MA). The donor plate was filled with diluted test compound (180 µL). The combined PAMPA plate was placed on a pIon Gut-boxTM and stirred for 30 minutes. The UV spectrum (250-500 nm) of the donor and the acceptor were read. The permeability coefficient and recovery were calculated using PAMPA evolution 96 command software (pION Inc., Woburn, MA) based on the whole spectrum measured from the reference plate, the donor plate, and the acceptor plate. All compounds were tested in triplicate.
Caco-2 Permeability
Caco-2 cells were maintained at 37 °C in a humidified incubator with an atmosphere of 5% CO2. The cells were cultured in Eagle's Minimum Essential Medium (EMEM) containing 20% fetal bovine serum (FBS) in 75 cm2 flasks, supplemented with 100 units/ml of penicillin and 100 µg/ml of streptomycin. The Caco-2 cells were seeded onto inserts of a 96-well plate (HTS-Transwell inserts, surface area: 0.143 cm2, Corning) at a cell density of 0.5 × 105 cells/insert. The culture medium was replaced every 2 days, and the cells were maintained for 7 days at 37°C and 5% CO2. Caco-2 cell monolayers with trans epithelial electrical resistance (TEER) values greater than 400 ohm•cm2 were used for the subsequent assays. The permeability assay was initiated by adding an appropriate volume of HBSS/HEPES containing test compounds to either the apical ( A to B) or basolateral (B to A) side of the monolayer, then adding the blank HBSS/HEPES buffer in the receiving compartment, the basolateral or apical side of the monolayer. The Caco-2 cell monolayers were then incubated for 2 h at 37 °C. To make a sample plate, fractions were collected from the basolateral side or apical side and quenched by adding 1-fold volume of acetonitrile with internal standard (2 µM warfarin) to each well. In a reference plate, the above HBSS/HEPES buffer containing test compounds were diluted with quenching solvent the same as that in the sample plate. 10 µL of supernatants were injected and analysis by UPLC/MS (Waters; Milford, MA). The test compound concentrations were quantified by comparing the sample well to the reference well via peak areas. The A→B (or B→A) apparent permeability coefficients (Papp, 10-6 cm/s) of each compound were calculated using the equation, Papp=dQ/dt×1/AC0. The flux of a drug across the monolayer was dQ/dt (µmol/s). The initial drug concentration on the apical or basolateral side was C0 (µM). The surface area of the monolayer was A (cm2). The efflux ratio is the ratio of apparent permeability for a test compound in the basolateral to apical (B→A) direction to that in the A→B direction. All compounds were tested in triplicate.
LogP, LogD, and pKa
LogD and pKa
Octanol/pH 7.4 buffer partitioning experiments were conducted using a shake flask method, and pKa was assessed by potentiometric titration. Both methods have been described previously (16).
LogP
LogP was measured using a Gemini Profiler instrument (pION Inc., Billerica, MA). 1-2 mg of compound was dissolved in octanol (0.5 mL) in a test tube. The test tube was purged with argon and a magnetic stir bar was added. The solution was treated with aqueous KCl (2.5 mL, 0.15 M) and stirred for 10 min. The pH was adjusted to 2 by addition of aqueous HCl (0.5 M). The resulting solution was titrated by adding aqueous KOH (0.5 M) in small aliquots (controlled by the Gemini Profiler software), until the pH reached 12. The volume of each addition and the corresponding pH of the test solution were recorded. Data were processed using pS software. The data points were fitted to a Bjerrum plot to achieve the best GOF (goodness of fitness) and a logP value was obtained. All measurements were conducted in triplicate.
Stability in SGF (simulated gastric fluid) and CPBS
Compound stocks (10 mM in DMSO) were diluted to 2 mM in DMSO. The positive control was chlorambucil (10 mM in DMSO) and the internal standard was warfarin (2 µM in methanol). Freshly prepared simulated gastric fluid (0.4 g NaCl, 0.64 g pepsin, 1.4 ml concentrated HCl, 198 ml DI water) and citrate phosphate buffered saline (CPBS, pH 3, 5, and 7.4) (1.9 mL) were added to the wells of a master plate (2 mL 96-well deep well plate, pION Inc., MA, #110023). Chlorambucil (3.8 µL) or diluted compound solutions (3.8 µL, 2 mM) were added to each well and mixed. 600 µL of mixed solution was then removed from each well into two new wells to make triplicates. From the master plate, 65 µL of each sample was transferred into each of 8 storage plates (pION Inc., MA) allowing for eight time points. The storage plates were then incubated at 37 ºC while shaking at 60 rpm. Stability was assessed at 0 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h by quenching the reaction with 195 µL of chilled methanol containing the internal standard, centrifuging at 4000 rpm for 15 min, and analyzing the supernatant by UPLC-MS. The compound and internal standard were detected by selected ion recording (SIR). Quantification of compound degradation was measured as a ratio to the internal standard and log peak area ratio was plotted as a function of time (h). Using the slope from the linear portion of this curve, the degradation rate constant was calculated. The rate constant was then used to calculate the half-life in SGF or CPBS.
Plasma Stability
Plasma stability assays were conducted in the same way as those of SGF/CPBS, except that three concentrations of compounds were prepared in DMSO:acetonitrile (1:4, v:v): high (2 mM), medium (0.4 mM) and low (0.08 mM). 1.9 mL each of mouse (Fisher Scientific, catalog #: NC9050370), rat (Fisher Scientific, catalog #: 50-415-345), dog (Fisher Scientific, catalog #: 50-415-573) or human plasma (Innovative Research Inc., catalog # IPLA-1) were added to wells, transferred and analyzed the same way as those in the SGF and CPBS stability assay. The degradation rate constant and half-life in plasma was also calculated accordingly.
Protein Binding
A Rapid Equilibrium Dialysis (RED) Plate (Thermo Scientific, catalog #, PI-90007) was used to determine the percentage of compound binding to plasma proteins. The positive control for this experiment was propranolol-HCl (10 mM in DMSO) and the internal standard was warfarin (2 µM in methanol). 10 mM stocks of compound in DMSO were diluted with DMSO and acetonitrile to three different intermediate concentrations: high (2 mM), medium (0.4 mM) and low (0.08 mM) in DMSO:acetonitrile (1:4, v:v). A 10 mM stock of propranolol in DMSO was diluted to 0.4 mM concentration in DMSO:acetonitrile (1:4 v:v). In 16 Eppendorf tubes, the control (10 µL) or each of three compound dilutions (10 µL) were each added to separate aliquots of mouse, rat, dog, and human plasma (990 µL). In the RED plate, potassium phosphate buffer (500 µL, 0.1 M, pH 7.4, 37°C) was placed in every white well and each plasma/compound mixture was added to each of 3 red wells. The RED plate holds triplicate samples of one control (final concentration 0.4 µM) and one compound (final concentrations: 20 µM, 4 µM, 0.8 µM). The RED Plate was sealed and incubated at 37 ºC with shaking at 60 rpm for 4 h. The changes of pH value over the course of incubation is less than 0.1. After incubation, aliquots (50 µL) from each well in the RED plate were transferred to an assay plate. In order to create a uniform matrix in every well of the assay plate, plasma (50 µL) was added to each of the wells that already contained buffer and potassium phosphate buffer (50 µL) was added to each of the wells that already contained plasma/compound. Pre-cooled internal standard (300 µL) was added to the assay plate to quench the reaction. The compounds and internal standard were detected by selected ion recording (SIR). Using the peak area ratio of compound to warfarin from the SIR spectra, we calculated percentage of free compound [1] and bound compound [2] using the following equations: 1) % free = (concentration buffer chamber/concentration plasma chamber)*100, and 2) % bound = 100 - % free.
Whole blood-plasma partitioning
Human whole blood was procured from the Volunteer Blood Donor Registry (Walter and Eliza Hall Institute of Medical Research) and used on the day of collection. Blood was collected using heparin as anticoagulant, and the hematocrit (Hct) determined by centrifugation (13000 x g for 3 min using a Clemets® Microhematocrit centrifuge and Safecap® Plain Self-sealing Mylar Wrapped capillary tubes) was 42%. Blood to plasma partitioning was determined as previously described (16).
Microsomal stability
The metabolic stability assay was performed by incubating compounds individually (0.8, 4, 20 µM) with mouse, rat, dog and human liver microsomes (Fisher) at 37°C and 0.5 mg/mL protein concentration. The metabolic reaction was initiated by the addition of a NADPH-regenerating system and quenched at various time points by the addition of acetonitrile according to a published method (14).
The remaining concentration of each compound was measured as a ratio of peak area to the internal standard. The log peak area ratio was plotted vs. time (h), and the slope was determined to calculate the elimination rate constant [k = (-2.303) * slope]. If deviation from first order kinetics was evident, only the initial linear portion of the plot was used to determine the rate constate, k. The half-life (h) was calculated as t1/2 = 0.693/k. Intrinsic clearance in vitro was calculated as CLintin vitro = (1000)*(0.693/t1/2*60)/0.5, where microsomal concentration in the reaction solution is 0.5 mg/mL;1000 and 60 are scaling factors for volume (µL) and time (min), respectively. The intrinsic in vitro clearance was scaled to the intrinsic in vivo clearance using physiology based scaling factor (PBSF): CLint in vivo= CLint in vitro *PBSF : (microsome protein/gram liver) * (gram liver/kg b.w.) (14, 17), with PBSF: 47 (mouse), 47(rat), 58 (dog), 32 (human); and liver weight proportions: 54.9 (mouse), 36.6 (rat), 32.9 (dog) 25.7 (human)
Hepatocyte Stability
SJ733 (1 µM; n=2 replicates) was incubated at 37°C with suspensions of human, dog, rat and mouse cryopreserved hepatocytes (XenoTech, Lenexa, KS). The average viable cell concentration over the incubation period was determined by the Trypan Blue exclusion method (in the absence of test compound). At various time points over the 60 min incubation period, the incubation mixtures were quenched by addition of ice-cold acetonitrile containing 0.52 µM of diazepam as an internal standard. The relatively short incubation time of 60 min was used to ensure hepatocyte viability over the incubation period. The relative loss of parent compound was quantified by LC-MS using a Waters Micromass Xevo G2QTOF mass spectrometer against calibration standards prepared in pre-quenched (to inactivate enzymes) blank hepatocyte mixture. The lower limit of quantitation value for the assay was 0.039 µM.
Test compound concentration versus time data were fitted to an exponential decay function to determine the apparent first-order rate constant for substrate depletion (k) that was then used to calculate the degradation half-life and the in vitro intrinsic clearance [3]: CLint = (k/hepatocyte cell number (106 viable cells / mL).
Each value for CLint, in vitro was multiplied by a PBSF to obtain the predicted in vivo intrinsic hepatic clearance, CLint, in vivo (17, 18). The predicted in vivo blood clearance (predicted CLblood) was then obtained by application of the well-stirred model of hepatic elimination [4]: Predicted Blood CL = (Q*CLint vivo/Q + CLint vivo), where Q is the nominal hepatic blood flow. Binding to hepatocytes and plasma protein were not taken into account.
Recombinant human cytochrome P450 (rhCYP) enzyme assays
SJ733 was first pre-incubated with BactosomesTM (Cypex Ltd, final P450 concentration: CYP1A1 25 pmol/mL, CYP1A2 100 pmol/mL,CYP1B1 100 pmol/mL, CYP2B6 100 pmol/mL, CYP2C8 50 pmol/mL, CYP2C9, 25 pmol/mL, CYP2C19 100 pmol/mL, CYP2D6 50 pmol/mL and CYP3A4 25 pmol/mL, 0.1 M phosphate buffer pH 7.4) at 37 °C prior to the addition of NADPH (final concentration 1 mM) to initiate the reaction with a final incubation volume of 50 μL. Incubations were also performed using control BactosomesTM (no P450 enzymes present) to reveal any non-enzymatic degradation. Control compounds known to be metabolized specifically by each P450 isoform were included individually. Test articles and controls were incubated with each isoform for 0, 5, 15, 30 and 45 min. The reactions were stopped by transferring 20 μL of incubate to 60 μL methanol at the appropriate timepoints.
In an alternate approach SJ311 was studied using a validated cocktail probe substrate method. SJ311 was incubated (500 μL, 10 μM) at 37 °C with a cocktail of two or three probe substrates at concentrations equal to their approximate Km values for human CYP enzymes (0.2 mg/mL human liver microsomes, 10 mM MgCl2, and 100 mM potassium phosphate buffer (pH 7.4). SJ311 was pre-incubated for 5 min in with the addition of NADPH regenerating system, followed by incubation for 10 min and terminated by addition of 0.5 mL acetonitrile containing 0.2 μM dextrorphan as an internal standard. The termination plates were centrifuged at 3400 rpm for 10 min at room temperature to precipitate the protein. All samples were analyzed using LC MS/MS, with either positive atmospheric pressure chemical ionization (APCI) mode (SJ311) or ESI (SJ733) mode utilizing multiple reaction monitoring (MRM) scans.
In vivo PK studies
Studies were undertaken to determine the plasma pharmacokinetics of SJ733 and SJ311. These studies included: i) single oral gavage (PO) or intravenous (IV) dose administration to female C57BL/6 mice; ii) single PO or IV dose administration to male Sprague Dawley (SD) rats; iii) single PO or IV dose administration to male beagle dogs; iv) toxicokinetic (TK) study following single PO administration to male SD rats.
Formulations
The PO and IV formulation used for mouse studies at SJCRH was 1% hydroxypropyl-beta-cyclodextrin (w/v), 10% ethanol (v/v), 10% propylene glycol (v/v), 40% PEG-400 (v/v) and 39% PBS (pH 7.4) isotonic (v/v). Compounds were dosed orally as suspensions, and intravenously as filtered solutions. Compound concentrations were confirmed post filtration using UV spectroscopy. The IV formulation used for rat studies (Monash University) was the same as that used for mice. For PO dosing to rats (Monash University) at 2 mg/kg, a suspension formulation was used containing 0.5% (w/v) hydroxypropyl methylcellulose, 0.5% (v/v) benzyl alcohol and 0.4% (v/v) Tween 80. The formulation used for rat studies at SRI (PO, high dose) was the same as that used in mouse PK studies. The formulation for the high dose TK study at SRI was 0.5% methylcellulose in sterile water. The formulation used for dog studies was the same as that used in mouse PK studies.
i) Mouse
The PO and IV PK of SJ733 and SJ311 were studied in female C57BL/6 mice. Mice had access to water and food ad libitum throughout the pre- and post-dose sampling period. Doses were administered at 15 mg/kg for IV and 10-200 mg/kg for PO with 20 mice in each dosage group. Two samples were taken from each mouse, with the first sample being a retro-orbital bleed (~200 µL) at the indicated time point (5, 15, 30 min, 1, 4, 24 h) and the second being terminal cardiac puncture (~500 µL) at the indicated time point (usually 48 h). EDTA disodium was used as anticoagulant and added to whole blood (10% volume of EDTA for 1% w/v final concentration) followed by centrifugation at 13000 rpm for 2 min. Plasma was collected and stored frozen at -20°C until analysis.
ii) Rat
The PO and IV PK of SJ733 and SJ311 were studied in overnight-fasted male Sprague Dawley rats. Rats had access to water ad libitum throughout the pre- and post-dose sampling period, and access to food was re-instated 4 h post-dose. Each compound was independently administered as a10 min constant rate IV infusion (4.5-5.1 mg/kg, 1.0 mL per rat, n = 2-3 rats) through a cannula surgically implanted in the jugular vein on the day prior to dosing. Oral doses (1.9-21.3 mg/kg) were administered via gavage. Samples of arterial blood and total urine were collected up to 48 h post-dose. Once collected, blood samples were centrifuged, supernatant plasma was removed and stored frozen (-20°C) until LC-MS analysis.
The high dose levels of both compounds (50, 100, 200 mg/kg, PO, (+)- SJ733/311) were also independently tested by SRI, with a single oral gavage administration. Blood (through jugular vein) and urine were collected at time points up to 72 and 24 h post dose, respectively. Supernatant plasma was removed following centrifugation and stored frozen (-20°C) until LC-MS analysis. For toxicology studies, male Sprague Dawley rats were administered 50, 100, 250, 500 or 750 mg/kg of (r)-SJ733 or (r)-SJ311 by oral gavage (one dose per animal). Body weights were recorded on Day 1 prior to dose administration and on Day 4. Blood samples (400 µL) were collected at 4 and 24 h post drug administration from the retro-orbital sinus. Potassium EDTA treated plasma was collected and kept frozen at -70°C for bioanalytical analysis.
iii) Dog
The plasma PK of SJ733 and SJ311 following a single PO gavage or IV dose (via saphenous vein) to male beagle dogs was determined at an IV dose of 3 mg/kg and PO doses of 3 and 30 mg/kg (n=3 for each). Briefly, this PK study was carried out in three sessions with one week washout period in between to allow for complete clearance of compounds. In the first session, 6 male dogs were administered a single 3 mg/kg IV dose of SJ311 or SJ733. In the subsequent second and third sessions, the same 6 male dogs were given a single 3 mg/kg or 30 mg/kg PO dose of SJ311 or SJ733, respectively. Plasma (0 -72 h) and urine (0 - 48 h) samples were collected for further analysis of SJ311 or SJ733 concentrations.
Bioanalytical Methods
For studies at SJCRH, all blood samples were kept on wet ice after collection and processed to plasma within 30 min of collection. Plasma samples were kept on dry ice and transferred to ≤ -20ºC until analysis. Mouse plasma samples were extracted via protein precipitation with cold acetonitrile. The detection of the SJ733, SJ311, and warfarin (IS) was conducted by LC-MS with SIR or LC-MS/MS with MRM detection. Aliquots (3 µL) were injected onto a Waters Acquity UPLC equipped with an ABI Sciex 6500 Qtrap MS/MS and separated using an Acquity BEH C18 column (50 x 2.1 mm, 1.7 µm) with a methanol-water gradient containing 0.1% formic acid.
For studies at Monash University, rat plasma and urine samples were extracted utilizing protein precipitation with a 2-fold volume ratio of acetonitrile. SJ733, SJ311, and diazepam (IS) were detected using LC-MS/MS instrumentation. Aliquots (3 µL) were injected onto a Waters Acquity UPLC equipped with a Waters Micromass Xevo TQ MS/MS and separated using a Supelco Ascentis Express RP Amide column (50 x 2.1 mm, 2.7 µm) with a methanol-water gradient containing 0.05% formic acid. Calibration standards were prepared by spiking blank matrix (plasma or urine) and the calibration range was from 1 to 10,000 ng/mL for plasma or 2.5 to 5,000 ng/mL for urine.
Rat samples from the toxicokinetic study at SRI were analyzed at SJCRH using the method described above. The calibration range was from 47 to 26,000 ng/mL for plasma.
Plasma and urine samples from the high dose rat PK studies were analyzed by SRI. In both matrices, the sample volume was 50 μL, and assay entailed the addition of 50 μL of internal standard solution to the standards and study samples. The compounds were used as reciprocal standards for one another (427 nM SJ733 in Milli-Q-Water for SJ571311 and 437 nM SJ311 in Milli-Q-Water for SJ733). These mixtures were then extracted with 1000 μL of ethyl acetate by vortexing for 10 min on a multi-tube vortex mixer at maximum speed followed by separation of the organic and aqueous phases by centrifugation (18000 × g, 5 min). Eight hundred microliters of the ethyl acetate (upper) layer of each sample were transferred to a clean tube and evaporated in a centrifugal evaporator without the application of heat. The dried samples were reconstituted with 100 µl of 10/90 (v/v) acetonitrile/Milli-Q-Water solution containing 0.1% formic acid. The reconstituted samples were then vortexed for 5 min on a multi-tube vortex mixer at one quarter speed and clarified by centrifugation (18000 × g, 3 min), and then transferred to HPLC vials fitted with glass inserts for LC-MS/MS analysis. Aliquots (10 µL) were injected using a Waters 2795 Alliance LC and Waters Micromass Quattro Ultima MS/MS and separated using a Phenomenex Luna C18 column (30 × 3 mm, 5 µm) with 2-propanol-water gradient containing 20 mM acetic acid. Study samples were quantitated using a set of calibration standards prepared in blank matrix that were processed in parallel.
Dog plasma was extracted utilizing protein precipitation using a 2-fold volume ratio of acetonitrile, while urine samples were extracted utilizing liquid-liquid extraction with ethyl acetate. SJ733, SJ311 and verapamil (IS) were quantitated by LC-MS/MS as described above.
SJ733 and SJ311 were shown to be stable (± 15% variance) when stored at -80ºC for more than 14 days. All plasma samples that were shipped elsewhere from the testing facility were all analyzed within the validated stability time period.
Pharmacokinetic analysis
Plasma concentration time (Ct) data for SJ733 and SJ311 were grouped by nominal time point, and the mean Ct values were subjected to noncompartmental analysis (NCA) using Phoenix WinNonlin 8.1 (Certara USA, Inc., Princeton, NJ). For all administrations, the area under the Ct and first moment curves (AUC, AUMC) were estimated using the “linear up log down” method. The terminal phase was defined as at least three time points at the end of the Ct profile, and the elimination rate constant (Kel) was estimated using an unweighted log-linear regression of the terminal phase. The terminal elimination half-life (T1/2) was estimated as 0.693/Kel, and the AUC from time 0 to infinity (AUCinf) was estimated as the AUC to the last time point (AUClast) + Clast (predicted)/Kel with the AUCinf similarly calculated. Additional parameters estimated included observed maximum concentration (Cmax), time of Cmax (Tmax), concentration at the last observed time point (Clast), time of Clast (Tlast), and the apparent oral terminal volume of distribution (V/F). The apparent oral clearance (CL/F), systemic clearance (CL) and volume of distribution at steady state (Vss) were all estimated using standard formulae (19) .