Compounds 1, 2, 3 and D were synthesized and purified (over 95%) in our laboratory . The Caco-2 cell line was obtained from the American Type Culture Collection (ATCC HTB-37). Dulbecco's modified Eagle's medium (DMEM), trypsin-EDTA, L-glutamine, non-essential amino acid, penicillin-streptomycin antibiotics and fetal bovine serum (FBS) were obtained from GIBCO-Invitrogen (USA). Transwell (6-well plates) cell culture chambers inserted with 3.0 μm pore size were purchased from Corning Life Sciences (USA). Esterase enzyme was obtained from Sigma (USA). All other chemicals were of cell culture and molecular biology grade from Sigma (USA).
We used a high performance liquid chromatography (HPLC) system consisting of a Knauer pump K-1001 and a Knauer Photometer K-2600 detector (Knauer, Germany) with detection at 254 nm. The separation was performed on a Kromasil 5 μm 100AC18, 250 × 4 mm column (Phenomenex, USA). Flow rate was 0.8 ml per minute and the solvent system was a gradient elution of 1% acetic acid in water and acetonitrile (CH3CN) at 85:15, 70:30, 55:45, 50:50, 30:70, 15:85, 0:100 and 0:100 at 0, 8, 25, 30, 55, 65, 80 and 110 minutes, respectively. Compounds 1, 2 and 3 were separated by non-polar stationary phase (octadecylsilane, ODS) eluted with 100% CH3CN at the last stage of the gradient elution.
As the fatty acid esters were not stable in transported medium, the UV spectroscopy was used to monitor the amounts of compounds 1, 2, 3 and D. Spectrophotometry analysis was performed on a Helios alpha UV-Vis spectrophotometer (Thermo Scientific, USA). The maximum wavelength (λmax) was obtained at 228 nm. Standards of each compound were freshly prepared at the concentration range of 0.62-3.73 μg/mL. Validations were performed by five replicates of intra-day and three replicates of inter-day. The linear correlation coefficients (r) between the UV-absorption and the concentrations of all compounds were in the range of 0.9993-0.9996 (compound 1: Y = 109.67X+0.031, r = 0.9994; compound 2: Y = 109.44X+0.028, r = 0.9995; compound 3: Y = 118.58X+0.049, r = 0.9996; compound D: Y = 169.72X+0.058, r = 0.9993).
Caco-2 cells were maintained in a DMEM at pH7.4, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acid solution and 0.1% penicillin-streptomycin solution in a humidified atmosphere (5% CO2, 95% air, at 37°C). Cells were grown until 60-70% confluence. Cells from passages 20-40 were used for all experiments. Cells were seeded on tissue culture polycarbonate membrane filters (pore size: 3.0 μm) in 6-well Transwell plates (Corning, USA) at a seeding density of 2 × 104cells/cm2. Culture medium was added to both the donor and the acceptor compartments. Medium was changed every two days. Cells were left to differentiate for 15-21 days after seeding with monitoring of trans-epithelial electrical resistance (TEER) values with a Millicell electrical resistance system (Millipore, USA) and the value should be higher than 600Ω.cm2.
Chemical hydrolysis study of the compounds in transport medium
Solution of compound 1, 2 and 3 at 53 μM and 106 μM were prepared in Hank's balanced salt solution (HBSS) at pH7.4. All solutions were kept at 4°C for 96 hours and each solution was then analyzed by HPLC at 12, 24, 48, 72 and 96 hours to determine the hydrolysis product.
Transport experiment across the Caco-2 cell monolayers at pH7.4 was performed. Caco-2 cell monolayers in Transwell (6-well) plates were used for transport studies when they were differentiated and the monolayer was intact, as checked by measuring TEER. Prior to the experiment, the cells were washed twice with phosphate buffered saline (PBS) and pre-equilibrated for one hour with HBSS buffered with 30 mM n-(2-hydroxyethyl) piperazine-n-(2-ethanosulfonic acid) (HEPES) at pH7.4. After medium was removed, the cells were treated with sample solutions (concentrations of 53 μM and 106 μM in HBSS at pH7.4) in an apical compartment. Samples (1 mL) were taken under sink conditions at 0, 5, 20, 40, 60, 80 and 100 minutes from the basolateral side and replaced with an equal volume of fresh HBSS solution. The amount of the compounds from the basolateral side was determined on a UV-spectrophotometer (Thermo Scientific, USA) at 228 nm. Results were expressed as cumulative transport as a function of time. Apparent permeability coefficient was calculated according to the following equation:
where Papp is the apparent permeability coefficient (cm/s), dQ/dt (μg/s) is the rate of appearance of sample on the basolateral side, A is the surface area of the monolayer (cm2) and C0 (μg/mL) is the initial drug concentration in the donor compartment. All rate constants were obtained from the permeation profiles of each compound. Statistical significance was evaluated with one-way analysis of variance (one-way ANOVA). A value of P < 0.05 was considered statistically significant.
Enzyme hydrolysis study of the compounds
Compound 1 (2 mL, 10 μM solution) was pre-incubated at 37°C and 200 μL of esterase enzyme (porcrine liver, 750 units) was added. Samples (200 μL each) were taken at 5, 10, 20, 30 and 60 minutes and added to 200 μL of methanol. Mixtures were vortexed to stop enzymatic activity. Samples were then centrifuged for five minutes at 14,000 × g (Lab Essentials, USA). Supernatant was injected to the HPLC system for the determination of ester pro-drugs and compound D. This procedure was repeated for compounds 2 and 3.