In situ single-pass intestinal permeability study

Male Sprague–Dawley rats (200–250 g) were supplied by Pasteur Institute of Iran. The animals were kept under 22 ± 2°C with a 12/12 light/dark cycle, and 55 – 60% relative humidity. Prior to experimentation, rats were fasted for 12-16 h (water ad libitum). All procedures were conducted in accordance with the humanity and animal ethic protocols. The animal study protocols were approved by the research ethical committee of Tabriz University of Medical Sciences (Ref No: TBZMED.REC.1394.378).

The animals were anesthetized with an intra-peritoneal injection of thiopantal sodium (60 mg/kg). A midline abdominal incision of 3-4 cm was made and approximately 10 cm of the jejunum segment of the intestine was isolated and cannulated at both ends with polypropylene tubes. The exposed segment was kept moist with body tempered saline. At first, the segment was rinsed with 37℃ normal saline to wash and clear the segment, then PBS (pH = 7.4) containing 50 mg/l phenol red without drug (blank solution) was pumped through the segment at a constant flow rate of 0.2 ml/min (Q_in) through a volumetric infusion pump (Argus Medical AG, Switzerland). Blank perfused solution was collected at the outlet and used to prepare cetirizine and digoxin calibrator solutions and also for stability studies.^15,16 After reaching steady state, perfusates were quantitatively collected for every 10 min (2ml) lasting 90 min (9 samples). Water absorption and secretion and other changes during the perfusion may cause errors in the calculated permeability values, phenol red, therefore, was added at a concentration of 50 mg/L as a non-absorbable marker to correct the results. Digoxin (20 µM in blank solution), Verapamil as a typical P-gp inhibitor (in blank solution with digoxin 20 µM), and different concentrations of cetirizine (0.2, 10, and 100 µM in blank solution with digoxin 20 µM/L) were administrated as described above (n = 3) for each drug and each concentration).^15,16 At the end of procedure, the length of the segment was measured (cm) and the animal was euthanized. Samples were stored at -70°C (ultra-low temperature freezer, Jal Tajhiz Production, Karaj, Iran), until analysis. The concentrations of phenol red in perfused (outlet) samples were measured at 560 nm using an UV-VIS spectrophotometer (Ultra-spec 2000, Pharmacia, Pfizer, New York, NY, USA).¹⁷ Digoxin and cetirizine amount in outlet samples were detected by HPLC method.

The mobile phase for digoxin and cetirizine analysis was 35% (v/v) acetonitrile in water which was filtered through sintered glass filter P5 (1.0-1.6 μ, Winteg, Germany) and degassed in a sonicator. The mobile phase was pumped in isocratic mode at a flow rate of 2 ml/min at ambient temperature. UV detection was accomplished at 218 nm and samples of 20 μl were injected using Hamilton injector syringe (Hamilton, Switzerland) onto the column (Knauer- 15VE081ESJ - 150X4.6 mm with precolumn- Eurospher 100-5 C8, Berlin-Germany).¹⁵ The high performance liquid chromatography (HPLC) system was composed of Smartline manager 5000, Smartline UV detector 2600, and Smartline pump 1000 (Knauer Advanced Scientific Instruments, Berlin, Germany). Figure 2 shows a representative HPLC chromatogram of analyzed samples.

A representative HPLC chromatogram of cetirizine (10 µM) and digoxin (20 µM) in intestinal perfused sample.

P_eff values were calculated from the steady-state concentrations of compounds in the perfusate collected from the outlet tubing. Steady state was confirmed by the ratio of the outlet to inlet concentrations (corrected for water transport) versus time.^15-17 Corrected C_out (outlet concentration of the drug) was calculated from the following equation.¹⁸

Where C_{out (corr)} is corrected outlet concentration of the drug, C_out is outlet concentration of the drug, CPR_in is concentration of phenol red entering the intestinal segment and CPR_out is concentration of phenol red leaving the intestinal segment. Calculations were based on outlet perfusate steady state concentrations achieved after the selected time points. The steady-state intestinal P_eff was calculated according to following equation:

P_eff represents effective permeability (cm/s), Q_in represents the perfusion rate (0.2 ml/min), C_in and C_out represent the concentrations of the test drug entering and leaving the segment respectively, r is the radius of the intestinal segment (0.18 cm), and l is the length of the intestinal segment (cm).