*2.6. Gastric and Intestinal First-Pass E*ff*ect of Tofacitinib*

Rats were fasted overnight with free access to water. Cannulae were inserted into the carotid artery and the cecal vein [13,14]. Using the peristaltic pump, 10 mg/kg tofacitinib was infused over 30 min into the portal vein (*n* = 6), and equal volumes of 0.9% NaCl-injectable solution containing 0.5% β-cyclodextrin were instilled into both the stomach and duodenum using a 23-gauge needle. In addition, 10 mg/kg tofacitinib was instilled into the duodenum (*n* = 5), and equal volumes of 0.9% NaCl-injectable solution containing 0.5% β-cyclodextrin were instilled into the stomach and infused via the portal vein over 30 min. Furthermore, 10 mg/kg tofacitinib was instilled into the stomach (*n* = 5), and equal volumes of 0.9% NaCl-injectable solution containing 0.5% β-cyclodextrin were instilled into the duodenum and infused via the portal vein over 30 min. Blood samples were collected from the carotid artery at times 0, 15, 30, 31, 35, 45, 90, 150, 270, 390, 510, 630, and 750 min after the start of intraportal infusion of tofacitinib, and at times 0, 5, 15, 30, 60, 120, 180, 240, 360, 480, 600, and 720 min after intragastric and intraduodenal instillations of the drug. All sample collection and processing procedures were identical to those described above.

#### *2.7. Tissue Distribution of Tofacitinib*

Rats were handled and processed as described previously [10]. Tofacitinib (10 mg/kg) was administered intravenously to rats for 1 min. After 30 min and 2 h (*n* = 4 each), as much blood as possible was collected from the carotid artery. Blood samples were immediately centrifuged, and plasma was collected. The rats were sacrificed by cervical dislocation, approximately 1 g of each brain, fat, heart, kidney, large intestine, liver, lung, mesentery, muscle, small intestine, spleen, and stomach was removed, rinsed with phosphate-buffered solution (pH 7.4), and blotted dry with paper towels to remove any remaining blood. Each tissue sample was added to four volumes of homogenizing buffer, homogenized using a tissue homogenizer (T25 Ultra-Turrax, IKA Labortechnik, Staufen, Germany) and centrifuged at 8000 × g for 10 min. A 100 µL aliquot of each supernatant was collected and stored at −80 ◦C until HPLC analysis of tofacitinib [12].

#### *2.8. Biliary Excretion of Tofacitinib*

The jugular vein of each rat was cannulated. The abdomen was opened and the bile duct was cannulated with polyethylene tubing [14]. The incision was closed with surgical sutures and each rat was kept warm under an electric light. Each rat was maintained in the supine position during the entire experiment. Tofacitinib (10 mg/kg) was infused for 1 min via the jugular vein (*n* = 3). Bile samples were collected over various time periods, from 0–1, 1–2, 2–4, 4–6, and 6–24 h. The volume of each bile sample was measured, and an aliquot of each was stored at −80 ◦C until HPLC analysis of tofacitinib [12].

#### *2.9. HPLC Analysis of Tofacitinib*

Two microliters of hydrocortisone (5 mg/mL) and 40 µL of 20% ammonia solution were added to 100 µL aliquots of biological samples and vortex-mixed for 30 s using a vortex mixer. Each solution was extracted with 1.5 mL of ethyl acetate by centrifugation at 12,000 rpm for 5 min. The organic layer was collected and dried (Dry Thermobath, Eyela, Tokyo, Japan) under a gentle stream of nitrogen gas at 40 ◦C. The samples were reconstituted in 130 µL of 20% acetonitrile, and 50 µL of resuspended samples were analyzed by HPLC [12].

The concentrations of tofacitinib in the prepared biological samples were determined using a Shimadzu Prominence LC-20A HPLC system (Kyoto, Japan), consisting of a pump (LC-20A), an auto-sampler (SIL-20A), a column oven, and a detector (SPD-20A/20AD), controlled by the CBM-20A system controller. The samples were filtered through 0.45-µm filters (Millipore, Billerica, MA, USA), followed by separation of tofacitinib on a reversed-phase column (AegisPak C18; 25 cm × 4.6 mm, 5 µm; Young Jin Biochrom, Seongnam, Korea). The mobile phase consisted of a 69.5:30.5 (v/v) mixture of 10 mM ammonium acetate buffer (pH 5.0) and acetonitrile, respectively, with a flow rate of 1.0 mL/min. Column effluent was monitored by a UV detector at 287 nm. The retention times of tofacitinib and the internal standard (hydrocortisone) were approximately 7.2 and 11.3 min, respectively. The lower limits of quantitation of tofacitinib in rat plasma, urine, and tissue homogenates were 0.01, 0.1, and 0.1 µg/mL, respectively, with intraday assay precision (coefficients of variation) in these samples being 3.69–5.88%, 4.21–6.18%, and 0.0205–8.74%, respectively. The interday assay precision was 5.06% for rat plasma and 5.46% for rat urine.

#### *2.10. Pharmacokinetics Analysis*

Pharmacokinetic parameters, including AUC, apparent volume of distribution at steady state (*V*ss), mean residence time (MRT), and time-averaged total body (CL), renal (CLR), and nonrenal (CLNR) clearances, were calculated by noncompartmental analysis (WinNonlin, Pharsight Corporation, Mountain View, CA, USA) using standard methods [15]. AUC values were calculated using the trapezoidal rule–extrapolation method [16]. The peak plasma concentration (*C*max) and time to reach *C*max (*T*max) were obtained directly from the experimental data. The mean values of clearance [17], terminal half-life [18], and *V*ss [19] were calculated using the harmonic mean method.
