Theophylline

Hydroxypropyl methyl cellulose (HPMC) F4M was bought from Colorcon Company, USA. Sodium tauroglycocholate (STGC) and lauric acid were obtained from Camlab Chemicals, England and Applichem GmbH, Germany, respectively. Propylene glycol, sodium sulfite, theophylline, ethylenediamine, monobasic potassium phosphate, and ethanol were bought from Merck, Germany. The snake skin was kindly donated by Razi Institute, Karaj, Iran.

Permeation enhancers (Table 1) were synthesised in the house (see Supplementary Material). Their partition coefficients LogP were calculated by Chemicalize software (ChemAxon, Budapest, Hungary).
Methanol, acetonitrile, theophylline (TH), diclofenac sodium (DF), indomethacin (IND), gentamicin sulphate and phosphate-buffered saline (PBS) tablets were all purchased from Merck life science (Darmstadt, Germany). Azone—N-dodecylazacykloheptan-2-on was purchased from USBiological (Swampscott, MA, USA). Propylene glycol (PG) was purchased from Dr Kulich Pharma (Hradec Králové, Czech Republic). Acetic acid was purchased from Penta (Praha, Czech Republic). All solvents used were of HPLC grade. Water was deionised, distilled and filtered by a Millipore Q purification system.
PBS solution was prepared by dissolving 1 PBS tablet in deionised water (200 mL) at 25 °C to yield a 0.01 M phosphate buffer, pH 7.4.

Theophylline was purchased from Sigma-Aldrich (Warszawa, Poland). The compound PSB-603 (Figure 1) was synthesised at PharmaCenter Bonn, Pharmaceutical Institute, Bonn, Germany, according to a described procedure [28 (link)]. The identity and purity of the final product were assessed by NMR and LC-UV/MS techniques.
For studies, PSB-603 (5 mg/kg b.w. of the mouse or 2 × 5 mg/kg b.w. of the mouse) was suspended in 1% Tween 80 and the volume was adjusted to 10 mL/kg. This dose was chosen because PSB-603 at 5 mg/kg b.w. does not have a sedative effect (locomotor activity study—Section 2.3) and has anti-inflammatory activity [14 (link)]. Theophylline was administered intraperitoneally at a dose of 2 × 50 mg/kg b.w. of the mouse [32 (link)].

Bovine serum albumin (BSA), Tris, HEPES-NaOH, disodium edetate, theophylline, TEP, trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), glucose, fructose and 0.1 M phosphate buffer (PBS) were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). 1-deoxy-1-morpholino-fructose (1-DMF) and nitroblue-tetrazolium (NBT) were purchased from BLD Pharmatech GmbH (Kaiserslautern, Germany). Erythritol was purchased from SHG GROUP (Pustynia, Poland). Xylitol was purchased from Nanga (Złotów, Poland) and collagen was from Hyphen Biomed (Neuville-sur-Oise, France). KCl, Na₂CO₃, NaCl, CaCl₂, KH₂PO₄, MgCl × 6 H₂O, NaHCO₃, FeCl₃ × 6 H₂O, FeSO₄ × 7 H₂, sodium citrate and citric acid were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland).

Forty-eight hours after transfection, HEK293T cells were washed twice with warm DMEM/BSA and then incubated with DMEM/BSA containing 0.5 mM isobutylmethylxanthine (Sigma–Aldrich, St. Louis, MO, USA) at 37 °C for another 15 min. Then, different concentrations of NDP-MSH, ACTH (1–24), α-MSH, or β-MSH were added to make the final concentration of ligands ranging from 10⁻¹² to 10⁻⁶ M for NDP-MSH and ACTH (1–24) or from 10⁻¹¹ to 10⁻⁵ M for α-MSH and β-MSH. After an hour’s incubation at 37 °C, the reaction was terminated on ice and the intracellular cAMP was collected by adding 0.5 M perchloric acid containing 180 μg/mL theophylline (Sigma–Aldrich) and 0.72 M KOH/0.6 M KHCO₃ into each well. The cAMP levels were measured by radioimmunoassay (RIA) [43 (link)].
To investigate the potential effect of caMRAP2 on caMC4R signaling, two ratios of caMC4R and caMRAP2 (1:0 and 1:5) were applied to co-transfect cells, and two ligands, α-MSH and ACTH (1–24), were used. To explore the dose-dependent effect of caMRAP2 on the maximal response (R_max) of cAMP levels to α-MSH and ACTH (1–24) stimulation, cells were co-transfected with caMC4R and caMRAP2 in four ratios (1:0, 1:1, 1:3, and 1:5). To study the constitutive activity of G_s cAMP, cells were transfected with different concentrations caMC4R plasmid (0, 0.007, 0.015, 0.030, 0.060, 0.125, and 0.250 μg/μL).

Gardneramine (purity over 98%, by HPLC, Agilent Technologies, Palo Alto, CA, USA) was isolated from the roots of Gardneria multiflora Makino., and its structure was identified by detailed HR-MS and NMR analysis (Bruker, Karlsruhe, Germany). Theophylline as internal standard was purchased from Sigma-Aldrich (Shanghai, China). HPLC-grade acetonitrile and formic acid were purchased from Fisher Scientific (Tustin, CA, USA). Ultra-pure water, which was prepared by a Milli-Q purification system (Bedford, MA, USA), was used for the mobile phase.