SR13668 (2,10-Dicarbethoxy-6-methoxy-5,7-dihydro-indolo [2,3-b
]carbazole) was synthesized at SRI International [1
]. The following vehicles were obtained from Sigma–Aldrich (St. Louis, MO): methylcellulose, PEG400, Tween 20, hydroxypropylmethylcellulose, glycerol, corn oil, cremophore EL, Span 85, soybean oil, sesame oil. Gelucire 44/14, Labrasol, Labrafil M1944, Labrafac, and Maisine 35-1 were obtained as gift samples from Gattefosse (Paramus, NJ). DMSO was obtained from Mallinckrodt Baker (Phillipsburg, NJ). PEG300 and propylene glycol were obtained from Spectrum (New Brunswick, NJ); 1-Methyl 2-pyrollidone was obtained from EMD (Gibbstown, NJ). Tween 80, Tween 20, and ethyl alcohol (ethanol) were obtained from Spectrum (Gardena, CA). Solutol HS-15 and Pluronic F127 were obtained from BASF (Mt. Olive, NJ). Miglyol 840 and 812 N were obtained from Creanova (Piscataway, NJ). Cavamax was obtained from EMD (Gibbstown, NJ). Acetonitrile was purchased from Mallinckrodt Baker (Phillipsburg, NJ). Sprague–Dawley rat plasma was purchased from Pelfreez (Rogers, AR). Chromatography C18
column (Luna, 5 μm, 50 × 2 mm) was purchased from Phenomenex (Torrance, CA).
High throughput solubility (HTS) screening
A TECAN FreedomEVO Liquid Handling Instrument (Tecan Systems, San Jose, CA) was used to prepare the excipient combinations. The TECAN dispenser has four tips that can travel in the x, y, and z directions. The FreedomEVOware (software) was programmed to aspirate solvents from the selected positions on the source deck and dispense them into the desired well locations in the flat bottom 96-well plates placed on the dispense deck.
Binary and ternary combinations of excipients were evaluated in n = 3 replicate wells. Baseline turbidity of the excipient combinations was determined using a Tecan GENios Microplate Reader for turbidimetric analysis (Tecan Systems, San Jose, CA) by reading absorbance at 500 nm. A stock solution of the drug was prepared in either absolute ethanol or DMSO and added to 96-well plates containing solvent combinations. The plates were again mixed by shaking at 175–200 rpm for 20–30 min after addition of the drug, and turbidity was measured. Solubility was assessed by evaluating the change in turbidity before and after addition of the drug to the solvent system. Solubility screening was performed at four levels, with the choice of excipients dependent on the behavior of SR13668 at the preceding level. The excipients and the concentrations used at each level of screening are presented in . Based on the data obtained from Level 4 screening, solubility was assessed at 5 mg/ml concentration of SR13668. To verify the results obtained by HTS screening at all four levels, confirmatory manual evaluation of 30 different cosolvent systems was performed, determining SR13668 concentration by UV spectroscopy at 310 nm.
List of solvents with concentrations used for the SR13668 solubility screening
Plasma protein binding
SR13668 was incubated at concentrations of 2.5, 75, 250, and 1,750 ng/ml in rat plasma at 37°C for 2 h. Following incubation, 400 μl samples were transferred in triplicate at each concentration to upper reservoir of the washed Centri-free YM-30 ultrafiltration units (Millipore, Bedford, MA) and then centrifuged for 20 min at 2,000g. Aliquots of the ultrafiltrates and unfiltered incubation mixtures were analyzed for SR13668 by HPLC and the total protein concentration by the bicinchoninic acid assay for protein concentration (BCA assay; Pierce, Rockford, IL).
Rat plasma was incubated at 37°C with 0.05 and 1 μg/ml SR13668 plus and minus sodium fluoride (50 μM) to inhibit plasma esterases for up to 60 min. The reaction was stopped with 4 volumes of acetonitrile, and aliquots were analyzed using HPLC with fluorescence detection, as described above.
Metabolism by human and rat liver microsomes
Human liver microsomes (HLM) were pooled from multiple donors (Gentest, Woburn, MA), and rat liver microsomes (RLM) were prepared at SRI by homogenization and centrifugation to collect the microsomal pellet. Liver microsomes were incubated at a final protein concentration of 0.5 mg/ml with 1 or 10 μM SR13668 (in DMSO, final concentration of DMSO in incubation was 0.1%). Incubations were in phosphate buffer, 100 mM, pH 7.4, and the reaction was started by the addition of 2.5 mM NADPH and 3.3 mM MgCl2. Heat-killed microsomes (100°C for 10 min) were also incubated under the same conditions as a control. Incubations were stopped at 0, 15, 30, and 60 min by the addition of an equal volume of acetonitrile. Samples were centrifuged to pellet the precipitated protein, and the clear supernatant was removed and stored at approximately −70°C until analysis.
Sprague–Dawley rats, male and female, were obtained from Charles River (Hollister, CA) and were 6–8 weeks old at study initiation. Rats were housed either 1 or 3 per cage, with a 12 h light/12 h dark cycle, 68°–72°C temperature, 39–59% humidity. Rats were fed Purina Rodent Chow #5002 and provided water (purified, reverse osmosis) ad libitum.
Protocols for all animal studies were approved by the Institutional Animal Care and Use Committee, and the studies were carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health.
Pharmacokinetics studies in rats
To screen formulations, male Sprague–Dawley rats were administered a single dose of 50 mg/kg SR13668 in one of several formulations, n = 3 rats per treatment group (5 mg/ml). Blood was collected at a limited number of time points, 3 or 6, selected to span the time of maximum blood drug levels (~4 h) based on a pilot study and analyzed for concentration of SR13668. The lungs were also collected and analyzed at 4 h from rats in the first formulation screening study.
For the definitive oral bioavailability and pharmacokinetic study, male and female Sprague–Dawley rats, jugular vein catheterized, n = 3 per sex per treatment group, were administered a single dose of SR13668 by iv (1 mg/kg; 6mg/m2) via the tail vein or oral gavage (1, 10, or 30 mg/kg; 6, 60, 180 mg/m2) routes. The volumes administered were 1 ml/kg for iv and 10 mg/l po. The rats were fasted overnight prior to SR13668 administration. For iv treatment, SR13668 was solubilized in DMSO:PEG300 (15:85) at 1 mg/ml and for oral gavage, it was formulated in PEG400:Labrasol (1:1, v:1) at concentrations of 0.1, 1, and 3 mg/ml. Blood samples (~200 μl) were collected at multiple time points post-dose through 24 h from the jugular canula and transferred to tubes with EDTA to obtain whole anticoagulated blood. Plasma (~200 μl of blood collected at the 2-h time point) was prepared by centrifugation at 2–8°C and stored frozen at approximately −70°C until analysis to determine the concentration of SR13668.
Bioanalytical method for SR13668
Samples were processed by addition of 4 volumes of acetonitrile to 1 volume of plasma or whole blood and well mixed. Lung tissue was processed by adding 80% (v:v) acetonitrile in water to preweighed lung sample. The tissue was homogenized on ice using a polytron tissue grinder, followed by brief sonication. The suspensions (prepared from both blood, plasma or lung homogenates) were then clarified by centrifugation, and the resulting supernatants were transferred to fresh microfuge tubes, and evaporated under vacuum in a centrifugal evaporator without heat application. The dry residues were reconstituted in 200 μl of 80% acetonitrile in water, centrifuged to clarify the samples and the supernatants then transferred to HPLC vials fitted with glass inserts for chromatographic analysis. Study samples were quantitated using a set of SR13668 calibration standards prepared in blank matrix that were processed in parallel (plasma: 1, 5, 25, 50, 100, 500, 1,000, 1,500 and 2,000 ng/ml; whole blood: 0.5, 2.5, 5, 25, 100, 500, 1,000, 1,500 and 2,000 ng/ml). Quality control standards were also processed in parallel and analyzed concurrently with the experimental samples (plasma: 2.5, 75, 250 and 1,750 ng/ml; whole blood: 1, 50, 250 and 1,750 ng/ml). Lung samples were quantitated against a set of neat standard solutions.
Samples were analyzed by HPLC with fluorescence and UV detection using a Waters 2690 Separation Module (Waters, Milford, MA), autosampler at 4°C, Luna C18 [2
] column (5 μm, 250 × 4.6 mm) at ambient temperature. The mobile phase consisted of 50 mM sodium acetate buffer, pH 5.0 (A) and acetonitrile (B). Flow rate was 1 ml/min and the elution mode used 65% A: 35% B from 0 to 6 min, 25% A: 75% B from 6.2 to 25 min, 65% A: 35% B from 25.2 to 30 min. Detection by UV absorbance was 307 nm, and by fluorescence, the excitation wavelength was 345 nm with emission wavelength of 480 nm. The retention time of SR13668 was ~16.9 min. Calibration standards and quality control, QC, standards were analyzed in triplicate with each day of study sample analysis and processed according to the extraction protocol. Lower limit of quantitation was 1.0 ng/ml plasma, whole blood or lung homogenate. Mean percent accuracies for whole blood and plasma were 107.87 and 107.45%, respectively. Relative standard deviations (RSD) for whole blood and plasma were 10.50 and 4.08%, respectively. QC results show that accuracy and precision values were well within ±15% of the corresponding nominal QC concentrations.
Plasma was mixed with an equal volume of acetonitrile to precipitate plasma proteins, and then centrifuged (18,000g, 10 min) to pellet the precipitate. Twenty microliters of the resulting supernatants were injected directly onto the LC–MS/MS system described below. Chromatography (Waters 2795 separation module, Phenomenex Synergi Fusion 4 μ, 150 × 3 mm column, autosampler and column temperature 20°C, mobile phase water with 0.1% formic acid and acetonitrile with 0.1% formic acid) was monitored in tandem with a photodiode array detector and Micromass Ultima triple quadrupole mass spectrometer (Waters, Milford, MA) set to monitor in the full scan mode over a range of m/z values of 60–1,000. The mass spectrometer was configured as follows in the characterization of metabolites in the blood samples. The HPLC eluant was examined by full scan mass spectrometry in the negative ion mode set for unit mass resolution. The capillary voltage was 2.5 kV, the cone voltage was 40 v, the ion source temperature was 115°C, and the desolvation temperature was 350°C with desolvation gas at a sufficient flow rate to give an even spray at 0.4 ml/min HPLC flow. The chromatograms shown are the extracted ion chromatograms obtained from the total ion chromatograms at the expected m/z values for SR13668 (m/z 429) and a monohydroxylated metabolite of SR13668 (m/z 445).
Pharmacokinetics data evaluation
Pharmacokinetic analysis was performed on whole blood drug concentration data for individual animals using non-compartmental methods and WinNonlin® Professional (Version 5.2, Pharsight, Mountain View, CA). Pharmacokinetic parameters, calculated for each individual animal, included observed maximal plasma concentration (Cp), extrapolated plasma concentration at time 0 (C0; iv group only), area under the plasma concentration–time curve from time 0 to the last time point (AUClast) and extrapolated to infinity (AUCinf), terminal elimination half-life (t1/2), apparent volume of distribution (V), and total clearance (Cl). Mean residence time (MRT) was calculated by dividing area under the first moment curve (AUMC) by the AUC. Bioavailabilty (F) was estimated using the following formula: F = AUCinf po × Doseiv/AUCinf iv × Dosepo. In the oral treatment groups, V and CL are presented as V/F and CL/F. Terminal phase parameters (t1/2, AUCinf,V, Cl, and MRT) were not determined in animals with fewer than 3 non-zero points in the terminal phase.
Student’s t test was used to determine statistically significant differences in Cmax, AUC, CL for male and female rats.