Clopidogrel

Two osteotropic metastatic human breast cancer cell lines were used in this study: MDA-MB-231/B02 (MDA-B02) and a subclone expressing the three markers GFP, luciferase, and a beta-galactosidase referred to as MDA-B02-Luc for simplification and which was described previously [46 (link)], as well as their parental breast cancer cell line MDA-MB-231 (MDA-231) and the murine 4T1 breast cancer cells obtained from the American Type Culture Collection (ATCC^®). The 4T1 cells derive from a BALB/c spontaneous mammary carcinoma and are naturally resistant to 6-thioguanine [47 (link)]. They were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Zafirlukast and montelukast, the CysLT1R antagonists, everolimus, the mTORC1 inhibitor, and clopidogrel, the P2Y₁₂ inhibitor, were purchased from Sigma. HAMI3379, the CysLT2R antagonist, and MK-886, the FLAP inhibitor, were from Cayman. LTD₄ was from Bertin.

DH was provided from Jai Radhe Sales (Gujarat, India). Chitosan (103.2 g/mol, degree of deacetylation 76.6%), 2-imino thiolane hydrochloride, 2-mercaptoethanol, triethanolamin reagent grade, formic acid (ultra-performance liquid chromatography MS/MS (UPLC MS/MS) grade), clopidogrel (as internal standard (IS)), ammonium formate, and acetonitrile were purchased from Sigma Aldrich Company (St. Luis, MO, USA). Cholesterol (CHOL) and dipalmitoylphosphocholine (DPPC) were purchased from Fisher Scientific Company (Fair Lawn, NJ, USA) and Avanti Polar Lipid Inc., (Birmingham, AL, USA), respectively. Polyvinyl pyrolidone (PVP), known as Luviskol® K-9, was purchased from BASF (Aktien gesellschaft Ludwig Shafen, Germany). Polyethylene glycol 600 (PEG600) was obtained from Merck Schuchardt (Hohenbrunn, Germany). Lactic acid glutaraldehyde 50% solution and chloroform were purchased from BDHL laboratory Supplies (PooleBH151TD, England). Mono basic potassium dihydrogen phosphate and cellophane membrane (Spectra/Por® Dialysis Tubing from regenerated cellulose) with cut-off 12–14000 Da were purchased from panreac Quimica, SA, (Barcelona, Espana) and Spectrum Laboratories (Houston, TX), respectively. All chemicals used were of analytical grade and were used as received.

The experimental protocol for allergen sensitization and challenge was modified from previously described procedures 19. Briefly, BALB/c mice were sensitized intraperitoneally with 10 μg of OVA (Fisher Scientific, Pittsburgh, PA, USA) emulsified in 1 mg of alum (Imject Alum; Pierce, Rockford, IL, USA) on days 0 and 14. For the allergen challenge, mice were subjected to airway allergen challenges by exposure to 1% OVA aerosols for 20 min. on days 28, 29 and 30 with an ultrasonic nebulizer (NE‐U22; Omron, Kyoto, Japan). To treat P2Y12 antagonist, mice were administered by oral gavage with 30 mg/kg of clopidogrel (Sigma‐Aldrich, St. Louis, MO, USA) in PBS for 30 min. before each OVA challenge. PBS was given as a vehicle. Each experiment in our study was repeated 3 times with 5 mice per group.

The human colon cancer cell lines HCT116 and LoVo and the human liver cancer cell line SNU398 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI medium (Capricorn Scientific, Ebsdorfergrund, Germany) containing 10% FBS (Capricorn Scientific, Ebsdorfergrund, Germany) and penicillin/streptomycin (Capricorn Scientific, Ebsdorfergrund, Germany). The cells were maintained at 37ºC in a humidified incubator with an atmosphere of 5% CO₂. Antiplatelet agents, aspirin (Cat #A3160), clopidogrel (Cat #C0614), prasugrel (Cat #SML0331), and ticagrelor (Cat #CDS023238) were purchased from Sigma-Aldrich (St.Louis, MO, USA). TM5275 sodium salt, which is known to inhibit SERPINE1, was purchased from Tocris Bioscience (Cat #5769, Bristol, Avon, UK).

AMB (99.8% purity), clopidogrel, the internal standard (IS), GA and formic acid were purchased from Sigma–Aldrich (St. Louis, MO).
PEGylated Poly (D,L-lactide-co-glycolide) copolymer: RGPd 50155 Diblock with molecular weight 6000 Da (Lactic to glycolic acid molar ratio of 1:1) with 15% polyethylene glycol and Poly(D,L-lactide): Poly(D,L-lactide) named R 203 H Monoblock were supplied by Boehringer Ingelheim (Ingelheim, Germany). Fungizone® (AMB micelle dispersion) was obtained from Bristol-Myers Squibb (Montreal, Canada). RPMI 1640 medium was purchased from Gibco/BRL (Grand Island, NY). Sabouraud Dextrose Agar plates (RODAC™) were obtained from BD Diagnostics (Becton, Dickinson and Company, NJ). All other reagents and chemicals were of HPLC analytical grade, and were used as received. Water was deionized and purified by a Milli-Q Reagent Grade water system (Millipore Corporation, Bedford, MA).

Sauchinone (over 98% purity) was provided by Professor Chin Y.-W. (Pharmacognosy Laboratory of Dongguk University, Seoul, Korea) according to the modification of previously reported method [48 (link)]. Hydroxybupropion, 4-hydroxymephenytoin, 6-hydroxychlorzoxazone, S-mephenytoin, and pooled human liver microsomes from a mixed pool of 50 donors (HLMs; BD Ultra Pool HLM 50, cat. 452156) were purchased from BD Gentest Co. (Woburn, MA, USA). Acetaminophen, chlorzoxazone, clopidogrel, coumarin, furafylline, 6α-hydroxytestosterone, 7-hydroxycoumarin, ketoconazole, 4-methylpyrazole, omeprazole, paroxetine, phenacetin, quinidine, sibutramine, sulfaphenazole, testosterone, tetraethylthiuram disulfide, ticlopidine, tienilic acid, tolbutamide, tranylcypromine, verapamil, and reduced form of NADPH (as a tetrasodium salt) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dextrorphan and 4-hydroxytolbutamide were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). Bupropion, carbamazepine (internal standard, IS), dextromethorphan, fluvoxamine, fluoxetine, and 8-methoxypsoralen were purchased Wako Co. (Tokyo, Japan). All other chemicals and reagents used were of analytical grade.