Diphenhydramine hydrochloride

Pharmaceutical grade standards of diphenhydramine hydrochloride (DIPH), azelastine hydrochloride (AZE) and bepotastine besylate (BEPO), papaverine and xylomethazoline hydrochlorides (the internal standards for HPLC methods) from Sigma-Aldrich (St. Louis, MO, USA), acetonitrile, methanol and water for chromatography from Merck (Darmstad, Germany), glacial acetic acid, sodium acetate, hydrochloric acid, sodium chloride, sodium tetraborate, phosphoric acid, sodium hydrogen phosphate, sodium dihydrogen phosphate, kalium dihydrogen phosphate and kalium hydroxide from POCh (Gliwice, Poland) were used. The buffer solutions, i.e., acetate buffer of pH 4, phosphate buffer of 7 and borate buffer of pH 10, were used as degradation media. Phosphate buffer of pH 3 was used for preparing the mobile phases in our HPLC methods. All buffers were prepared according to European Pharmacopoeia [18 ].

Clonidine hydrochloride (C7897; Sigma), Amitriptyline hydrochloride (A8404; Sigma), HC-30031 (TRPA1 antagonist; H4415; Sigma), Gabapentin (G154; Sigma), A-803467 (Nav1.8 blocker; 2976; Tocris), TC-N1752 (Nav1.7 blocker; 4435; Tocris), Atenolol (A7655; Sigma), Ibuprofen (I4883;Sigma), Naloxone (0599; R&D Systems) Atenolol (A7655; Sigma), Lamotrigine (L3791; Sigma), Enalipril (CDS020548; Aldrich), Ethyl 3-aminobenzoate methanesulfonate (Tricaine; E10521; Sigma), Omeprazole (O104; Sigma), Promethazine hydrochloride (P4651: Sigma), Diphenhydramine hydrochloride (D3630; Sigma), and Pancuronium bromide (P1918; Sigma) were dissolved in DMSO to a 1000x concentration. Buprenorphine hydrochloride (12496–0757; Reckitt Benckiser Pharmaceuticals) was obtained as a 3mg/mL injection solution from UW Medicine Drug Services. Candidate analgesics were further diluted in a solution of EM and 2% DMSO for the behavioral assay.

All chemicals were used as received from the supplier. Diphenhydramine hydrochloride (20 mg kg⁻¹ or 50 mg kg⁻¹ Sigma Aldrich, St. Louis, MO, USA), zolantidine dimaleate (10 mg kg⁻¹; Tocris, Minneapolis, MN, USA), immepip dihydrobromide (5 mg kg⁻¹; Sigma Aldrich, St. Louis, MO, USA), thioperamide maleate (20 mg kg⁻¹; Sigma Aldrich and Tocris), tacrine hydrochloride (2 mg kg⁻¹; Tocris), and α-fluoromethylhistidine (20 mg kg⁻¹; Toronto Research Chemicals, North York, ON, CAN) were all dissolved in sterile saline (0.9% NaCl solution, Mountainside Medical Equipment, NY, USA) at 5 mL kg⁻¹. Reserpine (10 mg kg⁻¹; Sigma Aldrich, St. Louis, MO, USA) was dissolved in 0.1% acetic acid (Sigma Aldrich, St. Louis, MO, USA) in sterile saline at 5 mL kg⁻¹. Tetrabenazine (Sigma Aldrich) was dissolved in 10% DMSO (Sigma Aldrich, St. Louis, MO, USA) in sterile saline with 1 M HCl (10 µL mL⁻¹ injection volume). All solutions were made fresh at the time of injection and all injections were given via intraperitoneal (i.p.) injection. Urethane (Sigma Aldrich, St. Louis, MO, USA) was dissolved in sterile saline as a 25% w/v solution and administered at 7 µL g⁻¹.

The reference standard for ephedrine (purity ≥ 99%; CAS Number: 299-42-3) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The reference standards for cinnamic acid (purity ≥ 98%; CAS Number: 140-10-3) and paeoniflorin (purity ≥ 98%; CAS Number: 23180-57-6) were obtained from the National Development Institute of Korean Medicine (Jangheung-gun, Jeollanam-do, Republic of Korea). Diphenhydramine hydrochloride (purity ≥ 99%; CAS Number: 147-24-0) and geniposide (purity ≥ 98%; CAS Number: 24512-63-8) as an internal standard (IS) were purchased from Sigma-Aldrich. Figure 1 shows the structure of cinnamic acid, paeoniflorin, ephedrine, and the IS. Liquid chromatography–mass spectroscopy (LC-MS)-grade acetonitrile, methanol, water (18.2 mΩ), and high-performance liquid chromatography (HPLC)-grade ethyl acetate were purchased from Fisher Scientific (Hampton, NH, USA). LC-MS-grade formic acid and acetic acid were supplied from Sigma-Aldrich. All other reagents were in the highest HPLC grades or quality available. Heparin sodium (25,000 IU/5 mL) was purchased from JW Pharmaceutical Corporation (Seoul, Republic of Korea). The SCRT tablets (manufacturing date: 10.01.2017; manufacturing number: H71501502) used in this study were supplied from Kyung-bang Pharmaceutical Company (Incheon, Republic of Korea).

Mepyramine maleate was obtained from Tocris Bioscience (Bristol, UK) and Sigma-Aldrich (St. Louis, MO, USA). Cetirizine dihydrochloride, azelastine hydrochloride, diphenhydramine hydrochloride, amoxicillin, sulfadiazine sodium salt, trimethoprim, enrofloxacin, colistin sulfate salt, gentamicin sulfate and kanamycin sulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetracycline hydrochloride was obtained from Carl Roth (Karlsruhe, Germany) and florfenicol from Cayman Chemical Company (Ann Arbor, MI, USA). All agents were directly diluted in M9 minimal medium (described below), except florfenicol, which was first dissolved in 10 μl dimethyl sulfoxide (DMSO), and enrofloxacin by adding 5% of 1 N sodium hydroxide solution. The trimethoprim-potentiated sulfonamides were used at a ratio of 19:1 (sulfadiazine:trimethoprim) stock solution dissolved with 0.5 ml of DMSO in 20 ml M9 minimal medium.

Four hours post-infection, larvae were transferred to wells containing E3 + PTU supplemented with the compound being tested. For most studies, larvae were kept in six-well plates (COSTAR 3736) containing E3 + PTU supplemented with either 0.5% DMSO (MP Bio CAS 67-68-5) or 5 μM clemastine fumarate (Sigma-Aldrich SML0445) dissolved in 100% DMSO to a final concentration of 0.5% DMSO in each treatment group. Diphenhydramine hydrochloride (Sigma-Aldrich D3630) was dissolved in 100% DMSO and diluted to a final concentration of 5 μM. All drugs were added directly to E3 + PTU. Whenever agarose was used to mount fish, the same concentration of drug was added to the agarose prior to solidification.

To represent acidic, basic, and neutral pharmaceuticals diclofenac sodium, loratadine, acetaminophen, carbamazepine, citalopram hydrobromide, propranolol hydrochloride, fluoxetine hydrochloride, diphenhydramine hydrochloride, and erythromycin were purchased from Sigma–Aldrich (Poole, UK) and assigned according to their predicted state at pH 7 (see Supporting Information). For the amphiphilic (surfactant) class, quaternary ammonium compounds (QACs) were purchased from Sigma–Aldrich (Poole, UK) and included benzyldimethyldodecylammonium chloride (BAC‐C12), benzyldimethyltetradecylammonium chloride (BAC‐C14), and an aliphatic surfactant hexadecyltrimethylammonium chloride (HDTMA). While as ISs acetaminophen‐(methyl‐d₃) and 10,11‐dihydrocarbamazpine, pronethalol hydrochloride, and talopram hydrochloride were purchased from Tocris (Abingdon, England) and benzyldimethyltetradecylammonium chloride‐d₇ (BAC‐C14‐d₇) from Toronto Research Chemicals (Ontario, Canada). For sample preparation, modified QuEChERS extraction kits were obtained from Biotage (Uppsala, Sweden) with formic acid, acetonitrile, and water (HPLC‐grade) from Fisher Scientific (Loughborough, UK). As complex matrices for proof‐of‐concept testing, garden soil was collected from an undisclosed location in West Wales and blood plasma sourced from Seralab (West Sussex, UK).