Receptor 1 Antagonists

Transcription

1 Supporting Information The Discovery of a Novel Class of Potent and Orally Bioavailable Sphingosine-1-Phosphate Receptor 1 Antagonists Mohamed A. Ibrahim, Henry W. B. Johnson*, Joon Won Jeong, Gary L. Lewis, Xian Shi, Robin T. Noguchi, Matthew Williams, James W. Leahy, John M. Nuss, John Woolfrey, Monica Banica, Frauke Bentzien, Yu-Chien Chou, Anna Gibson, Nathan Heald, Peter Lamb, Larry Mattheakis, David Matthews, Aaron Shipway, Xiang Wu, WenTao Zhang, Sihong Zhou and Geetha Shankar Exelixis, Department of Drug Discovery, 169 Harbor Way, South San Francisco CA, 94083, USA. Table of Contents S2 Pharmacokinetics S5 Efficacy Studies S10 Cytochrome P450 and MLM Assays S 1

2 Pharmacokinetics Experimental Animals The animals were housed at Exelixis vivarium facilities according to the guidelines outlined by the Exelixis Institutional Animal Care and Use Committee (IACUC). Prior to initiation of a study, the animals were allowed to acclimate for a minimum of 48 h. During these studies, animals were provided food and water ad libitum and housed in a room conditioned at F and 60% relative humidity. A 12 h light and 12 h dark cycle was maintained with automatic timers. All animals were examined daily for compound induced or tumor related deaths. Moribund animals were euthanized when necessary and animal carcasses were disposed of according to IACUC guidelines. Bioanalytical Analysis: The concentration of compounds in plasma samples was determined by HPLC/MS/MS analysis using sample preparation and analytical conditions described on page S2. A non-compartmental model was applied to calculate pharmacokinetic (PK) parameters for all routes of administration using WinNonlin 4.2 software Mouse HTS Data: Female Nu/Nu homozygous mice approximately 8 weeks of age and weight approximately grams were purchased from Taconic (Hudson, NY). Compounds were administered orally at a dose of 100 mpk as a solution in 10% EtOH 45% PEG400 / Water plus 1 molar equivalent of HCl, 10% EtOH 40% PEG400 / Water plus 1 molar equivalent of HCl, 10% ethanol / 40%PEG400 / 20% Captisol plus 1 molar equivalent of HCl, or 10% NMP / 90% corn oil at a dose volume of 10 ml/kg. Whole blood was collected at 1 and 4 hours post dose into heparinized tubes, plasma prepared and stored at -80 ºC until analysis. S 2

3 Study of 37 in Female CD Rats: After IV and PO Administration: Female CD rats (Sprague Dawley) approximately 9-10 weeks of age and weighing approximately grams were purchased from Charles River Laboratories (Hollister, CA). The female CD rats were fasted overnight prior to dosing and returned to food 4 h post-dose administration. Compound 37 was administered at 3 mpk orally as a solution in Ethanol / PEG400 / water (EPW, 5:45:50) to groups of 4 female CD Rats. Blood was collected (100 µl) in heparinized tubes via a jugular catheter at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 32, 48, 72, and 144 h post-dosing. The plasma obtained was stored at -80 C and a volume of 50 µl was used for analysis. Study of 37 in Male Beagle Dogs: After PO Administration: The in life portion of the pharmacokinetic studies were conducted by Covance Laboratories (Madison, WI) according to the terms and conditions outlined by Study Number (3 mpk IV and PO). Male Beagle dogs were fasted overnight prior to dosing and returned to food four h post-dose. Compound 37 was administered by oral gavage at 3 mpk as a solution formulated in an EtOH:PEG400:Water (EPW, 5:45:50). Plasma concentrations of 37 were evaluated over a 48-hour period in experimental groups comprised of three animals. Blood (1000 µl) was sampled in potassium EDTA tubes at the following time points: pre-dose, 0.08, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, and 48 h post-dose. Plasma was prepared and the samples were frozen and shipped on dry ice to Exelixis for analysis. Study of 37 in Male Cynomolgus Monkeys: After IV and PO Administration: The in life portion of the pharmacokinetic studies was conducted by Maccine Laboratories (Singapore) according to the terms and conditions outlined by Study Number JPL-004 (3 mpk IV and PO). Male Cynomolgus monkeys were fasted overnight prior to dosing and returned to food four h post-dose. Compound 37 was administered intravenously and by S 3

4 oral gavage at 3 mpk as a solution formulated in an EtOH:PEG400:Water (EPW, 5:45:50). Plasma concentrations of 37 were evaluated over a 48-hour period in experimental groups comprised of three animals. Blood (1000 µl) was sampled in potassium EDTA tubes at the following time points: pre-dose, 0.08, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, and 48 h post-dose. Plasma was prepared and the samples were frozen and shipped on dry ice to Exelixis for analysis. S 4

5 Efficacy Studies Experimental Animals The animals were housed at Exelixis vivarium facilities according to the guidelines outlined by the Exelixis Institutional Animal Care and Use Committee (IACUC). Prior to initiation of a study, the animals were allowed to acclimate for a minimum of 48 h. During these studies, animals were provided food and water ad libitum and housed in a room conditioned at F and 60% relative humidity. A 12 h light and 12 h dark cycle was maintained with automatic timers. All animals were examined daily for compound induced or tumor related deaths. Moribund animals were euthanized when necessary and animal carcasses were disposed of according to IACUC guidelines. Female athymic nude mice (NCr) 5-8 weeks of age and weighing approximately 20 g were purchased from Taconic (Germantown, NY). Cancer Cell lines All cell lines were tested by the University of Missouri Research Animal Diagnostic Animal Laboratory for all known species of mycoplasma and for a panel of murine viruses, which included: sendai virus, parvovirus, murine hepatitis virus, Theiler s murine encephalomyelitis virus, Reovirus3, Rotavirus, Extramelia, Lymphocytic choriomeningitis virus, Polyoma virus, K virus, adenovirus, cytomeglavirus, lactate dehydrogenase elevating virus, thymic virus, and Hanta virus. Solid Tumor Models - Tumor Cell Implantation and Measurement The MDA-MB-231T human breast cancer cell line was chosen as a highly vascularized orthotopic xenograft model, with an average of 90 tumor vessels per mm 2 in control tumors at the end of a 14 dosing period (Exelixis in-house data). The MDA-MB-231T cell line harbors a homozygous activating mutation in B-RafG464V, and a heterozygous S 5

6 activating mutation in K-RasG13D. MDA-MB-231T cells also harbor homozygous mutations in tumor suppressor genes TP53 and CDKN2A known to regulate cell cycle progression. MDA-MB-231T cells were cultured in vitro in DMEM (Mediatech) supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and non-essential amino acids at 37 C in a humidified, 5% CO 2 atmosphere. On day 0, cells were harvested by trypsinization, and 1 x 10 6 cells (passage #8-12, > 98% viability) in 0.1 ml ice-cold Hank s balanced salt solution were implanted subcutaneously into the mammary fat pad of 5-8 week old female athymic nude mice. Analysis of Efficacy Data In mammary fat pad implanted tumors, the mean tumor weight of each animal in the respective control and treatment groups was determined twice-weekly during the duration of the study. Tumor weight (TW) was determined by measuring perpendicular diameters with a caliper, using the following formula: tumor weight (mg) = [tumor volume = length (mm) x width 2 (mm 2 )]/2 These data were recorded and plotted on a tumor weight vs. days post-implantation line graph and presented graphically as an indication of tumor growth rates. Percent inhibition of tumor growth (TGI) is determined with the following formula: 1 ( X f X 0 ) * 100 ( Y f X 0 ) where X 0 = average TW of all tumors on staging day X f = TW of treated group on Day f Y f = TW of vehicle control group on Day f S 6

7 If tumors regress below their starting sizes, then the percent tumor regression is determined with the following formula: ( X 0 X f ) * 100 X 0 Tumor size is calculated individually for each tumor to obtain a mean ± SEM value for each experimental group. Statistical significance is determined using the 2-tailed Student s t-test (significance defined as p < 0.05). In addition to tumor weight determination, body weights of all animals on study were recorded daily. A transponder was implanted in each mouse for identification, and animals were monitored daily for clinical symptoms and survival. Formulation The compound was formulated for oral (PO) administration in efficacy studies by dissolution of the dry powder in 10% EtOH 40% PEG400 / Water + 1:1 HCl (12 and 15), Water (25, ZD6474), 0.75% CMC 0.1% Tween80 / Water (26, Merck Receptor Agonist), 10% NMP / corn oil (37 and 38). Merck Receptor Agonist was synthesized in-house. All dosing solutions were prepared fresh daily and administered at 10 ml/kg of body weight. S 7

8 25.0 Mean Body Weight (g) Vehicle, 10 ml/kg, PO qd 32, mpk, PO qd 33, mpk, PO qd 16, mpk, PO qd 19, mpk, PO qd Dose Day Figure 1: Mean Body Weights in the MDA-MB-231T Xenograft Model. S 8

9 25.0 Mean Body Weight (g) 20.0 Vehicle:1, 10 ml/kg + Vehicle:1, 10 ml/kg 38 53, 100 mpk, PO qd 37 52, 100 mpk, PO qd Dose Day Figure 2: Mean Body Weights in the MDA-MB-231T Xenograft Model. S 9

10 Cytochrome P450 Assay CYP450 inhibition profiling was evaluated by quantification of drug substrate metabolism to specific products known to be catalyzed by CYP450 isozymes present as pooled human liver microsomal fractions. Tolbutamide methyl-hydroxylation is catalyzed by CYP2C9 and bufuralol-1-hydroxylation is catalyzed by CYP2D6. The assays were performed in 96-well microtiter plates using a final volume of 500 µl in 100 mm potassium phosphate buffer (50 mm potassium phosphate buffer for CYP2C8), ph 7.4 at 37 C. Reaction and analysis conditions were optimized for all CYP450 isozymes, and are summarized Table 1. At the end of the incubation period, reactions were quenched by addition of 100 µl acetonitrile to 100 µl of reaction samples, cooled to 4 C, and clarified by centrifugation at 2500 xg for 15 min. The supernatant was transferred to a fresh 96-well microtiter plate, sealed, and quantified by LC/MS/MS analysis. Inhibition potential was evaluated at six concentrations ( µm). Enzyme activity was calculated based on quantification of metabolite signal. IC 50 values were calculated by nonlinear regression using a 4-parameter sigmoidal curve fit equation (XLFit). Table 1. CYP450 LC/MS/MS Assay Conditions Isozyme Substrate Substrate (µm) Metabolite NADPH (µm) HLM (mg/ml) Incubation Time CYP2C9 Tolbutamide 100 Hydroxytolbutamide C CYP2D6*1 Bufuralol 5 Hydroxybufuralol C CYP3A4 Testosterone 3 Hydroxytestosterone C LC/MS/MS analysis was performed using an API 3000 instrument, coupled to a Shimadzu SCL-10AVP LC Pump system. Analytical samples were separated using a Waters Atlantis dc 18 reverse phase HPLC column (50 mm x 4.6 mm) at a flow rate of S 10

11 1-2 ml/min. The mobile phase consisted of 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in water (solvent B). Elution conditions are detailed in Table 2. Table 2: Gradient Conditions for CYP450 Assay Time (min) Flow (µl/min) %A %B MLM Assay Microsomal oxidation of test compounds in the presence of mouse liver microsomal fractions was conducted in 96-well microtiter plates. Liver microsomal preparations were purchased from BD Gentest (Bedford, MA). Test compounds were incubated in duplicate at 37 C for 30 min in the presence of liver microsomes and NADPH. Reaction mixtures (75 µl) contained a final concentration of 15 µm test compound, 0.15% DMSO, 0.5 mg/ml microsomal protein and 1 mm NADPH in 100 mm potassium phosphate, ph 7.4 buffer. Control incubations contained the full complement of enzyme and substrate but no NADPH added. All test-sets included reference CYP450 substrates to verify assay performance. Reactions were terminated by the addition of 150 µl acetonitrile containing 0.1% formic acid. The concentration of the test compound remaining was determined by LC/MS/MS analysis on Sciex API-3000 instrument. The extent of metabolism was calculated as a disappearance of the test compound, compared to the no-nadph control reaction incubations. Chromatographic separation of analytes was achieved using a Phenomenex Synergi Hydro-RP column (Torrance, CA) and a mobile phase consisting of S 11

12 0.1% formic acid in acetonitrile and 0.1% formic acid in water. A linear gradient from 2-98% acetonitrile was used to elute compounds from the HPLC column. S 12

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