**1. Introduction**

Tofacitinib (3-[(3*R*,4*R*)-4-methyl-3-[methyl-(7*H*-pyrrolo[2,3-day]pyrimidin-4-l)amino]piperidin-1 yl]-3-oxopropanenitril, Figure 1) potently and selectively inhibits Janus kinases (JAK) 1 and 3 through blocking the signal transducer and activator of transcription 1 (STAT1) signaling pathway, thereby suppressing the production of inflammatory mediators, including interleukins-2, -4, -7, -9, -15 and -21 [1–3]. These findings led to the use of tofacitinib in the treatment of diseases involving the immune system and to its approval for the treatment of rheumatoid arthritis, particularly in patients intolerant to methotrexate therapy [4]. Tofacitinib was also approved by the US Food and Drug Administration in 2018 for the treatment of moderate to severe ulcerative colitis [5], making it the first oral JAK inhibitor for chronic use in patients with ulcerative colitis [6].

Oral administration of 10 mg tofacitinib to healthy volunteers resulted in an absolute oral bioavailability (*F*) of approximately 74% [7]. Pharmacokinetic analysis showed that its volume of distribution was 87 L, and its terminal half-life was 3.2 h [3,7,8]. Studies found that 40% of oral tofacitinib bound to plasma proteins [3,8], and that approximately 70% of eliminated tofacitinib was excreted in urine after being metabolized, with the remaining 30% eliminated unmetabolized through the kidneys [3,7,8]. Tofacitinib is metabolized through hepatic oxidation and *N*-demethylation, primarily by cytochrome P450 (CYP) 3A4, but also by CYP2C19 and glucuronide conjugation [3]. Although the dose-dependent pharmacokinetics of tofacitinib have been described in humans [7,8], the mechanisms underlying its incomplete absorption have not yet been characterized. However, it is difficult to get the information from clinical settings for evaluating the mechanisms of its incomplete absorption. Instead, using a rat model has been considered, but the basic pharmacokinetic characteristics of tofacitinib in rats has not been thoroughly investigated yet.

**Figure 1.** Structure of tofacitinib citrate.

The present study assessed the dose-dependent pharmacokinetics of tofacitinib administered both intravenously and orally in rats by evaluating the total area under the plasma concentration–time curve from time zero to infinity (AUC). This study also investigated the effects of first-pass hepatic, gastric, and intestinal metabolism on tofacitinib administered to rats intravenously, intraportally, intragastrically, and intraduodenally. Furthermore, biliary excretion and tissue distribution of intravenously administered tofacitinib were evaluated in rats.

#### **2. Materials and Methods**

### *2.1. Chemicals*

Tofacitinib citrate and hydrocortisone, the internal standard for high-performance liquid chromatography (HPLC) analysis, were obtained from Sigma-Aldrich (St. Louis, MO, USA), and ethyl acetate was from J.T. Baker (Phillipsburg, NJ, USA). Heparin and 0.9% NaCl-injectable solution were purchased from JW Pharmaceutical Corporation (Seoul, Korea), and β-cyclodextrin was from Wako (Osaka, Japan). All other chemicals were HPLC grade and were used without further purification.

#### *2.2. Animals*

Male Sprague-Dawley rats, aged 7–8 weeks and weighing 240–260 g, were purchased from OrientBio Korea (Seongnam, Korea), housed individually in a clean room, and maintained at a temperature of 22 ± 1 ◦C, with 12-h light (07:00–19:00) and 12-h dark (19:00–07:00) cycles at a relative humidity of 50 ± 5% with air filtration (Laboratory Animal Research Center of Ajou University Medical Center, Suwon, Korea). The rats had access to food (Purina Korea, Pyeongtaek, Korea) and water *ad libitum*. All experimental procedures and protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC No. 2017-0074, 2018) of the Laboratory Animal Research Center of Ajou University Medical Center.

### *2.3. Estimation of the Appropriate Number of Animals*

The appropriate number of animals in each group (*n*) and total number of animals in each experimental setting (*N*) were calculated based on the statistics [9]. For statistical analysis, the degree of freedom should range from 10 to 20. The minimum and maximum number of animals in each group were calculated as:

$$\text{Minimum } n = 10/k + 1 \tag{1}$$

$$\text{Maximum } n = 20\% + 1\tag{2}$$

where *k* is the number of groups in each setting. The minimum and maximum *n* were rounded up and down, respectively. The total minimum and maximum *N* required were minimum *n* multiplied by *k* and maximum *n* multiplied by *k*, respectively.

#### *2.4. Intravenus and Oral Administration of Tofacitinib*

The pretreatment and surgical procedures for oral and intravenous administration were similar to those described previously [10,11]. For oral administration, the rats were fasted overnight with free access to water. The rats were anesthetized with ketamine (200 mg/kg), and their carotid arteries were cannulated using polyethylene tubing (Clay Adams, Parsippany, NJ, USA) for blood sampling. For intravenous administration, the rats were anesthetized with ketamine (200 mg/kg), and their jugular veins and carotid arteries were cannulated for drug administration and blood sampling, respectively. Rats were allowed to recover for 4–5 h after surgical procedures. The rats were not restrained during the experimental period and had free access to water and food.

For intravenous administration, tofacitinib, dissolved in 0.9% NaCl-injectable solution containing 0.5% β-cyclodextrin, was injected via the jugular vein for 1 min at doses of 5 (*n* = 9), 10 (*n* = 8), 20 (*n* = 7), and 50 (*n* = 7) mg/kg. Blood samples (110–220 µL) were collected via the carotid artery at times 0 (prior to drug administration), 1 (at the end of drug infusion), 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, and 600 min. The total amount of blood collected from each rat did not exceed 10% of the total blood volume during the entire experimental period so as not to alter the pharmacokinetics and physiological functions. These blood samples were immediately centrifuged at 8000× *g* for 10 min, and plasma was collected and stored at −80 ◦C until HPLC analysis of tofacitinib [12]. To prevent blood clotting, 0.3 mL of heparinized 0.9% NaCl-injectable solution (20 IU/mL) was immediately injected into the carotid artery after each blood sampling. Urine samples were collected over 24 h; in addition, each metabolic cage was rinsed with 20 mL of distilled water 24 h after drug administration, and the rinses were combined with their corresponding 24-h urine samples. The volumes of the combined urine samples were measured, and two 100 µL aliquots of each were stored at −80 ◦C until HPLC analysis of tofacitinib [12]. At 24-h, each rat was exsanguinated, followed by cervical dislocation. The abdomen of each rat was opened and the entire gastrointestinal tract, including its contents and feces, was removed, transferred to a beaker containing 50 mL methanol, and cut into small pieces using scissors. The contents of each beaker were stirred manually with a glass rod for 1 min, and two 100 µL aliquots of each supernatant were collected and stored at −80 ◦C until HPLC analysis of tofacitinib [12].

For oral administration, approximately 1.0 mL tofacitinib was administered to rats at doses of 10 (*n* = 7), 20 (*n* = 8), 50 (*n* = 9), and 100 (*n* = 7) mg/kg. Blood samples (110–220 µL) were collected via the carotid artery at times 0 (prior to drug administration), 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 600, and 720 min. Urine and gastrointestinal tract samples were also obtained over 24 h were processed as described above for the corresponding samples collected after intravenous administration.

#### *2.5. Hepatic First-Pass E*ff*ects of Tofacitinib*

The carotid artery and jugular vein were handled as described previously [10,11]. In addition, the vein from the cecum was cannulated and the cannula was pushed forward about 4 cm toward the liver through the portal vein to minimize the damage from blood flowing into the portal vein [13,14]. Using a peristaltic pump (BT-300CA, JIH Pump, Chongqing, China), 10 mg/kg tofacitinib was infused over 30 min into the jugular vein (*n* = 7) and an equal volume of 0.9% NaCl-injectable solution containing 0.5% β-cyclodextrin was infused simultaneously over 30-min into the portal vein. In another group of rats, 10 mg/kg tofacitinib was infused over 30 min into the portal vein (*n* = 7) and an equal volume of 0.9% NaCl-injectable solution containing 0.5% β-cyclodextrin was infused simultaneously over 30 min into the jugular vein. Blood samples were collected from the carotid artery at times 0, 15, 30 (at the end of the infusion), 31, 35, 45, 60, 90, 150, 210, 270, 390, 510, and 630 min, with all sample collection and processing procedures identical to those described above.
