**1. Introduction**

Tofacitinib (Figure 1) was developed as a Jak inhibitor for the treatment of rheumatoid arthritis and is particularly effective when methotrexate is poorly treated [1]. Recently, tofacitinib was approved in 2018 for chronic use to treat moderate to severe ulcerative colitis [2], making it the first Food and Drug Administration (FDA)-approved oral Jak inhibitor [3]. Tofacitinib is currently under clinical trials for various diseases, such as psoriasis [4,5], alopecia [6], atopic dermatitis [7], and ankylosing spondylitis [8].

**Figure 1.** Chemical structure of tofacitinib.

Pharmacokinetic analysis following oral administration of tofacitinib (10 mg) to healthy volunteers showed a half-life of 3.2 h and a volume of distribution of 87 L [9–11]. Approximately 40% of the oral dose bound to plasma protein, 30% of the dose was excreted in the urine as an unmetabolized form, and 70% was metabolized and excreted in the urine as metabolized forms [9–11]. Absolute oral bioavailability (*F*) of tofacitinib was found to be approximately 74% [11]. Tofacitinib is primarily metabolized by oxidation and *N*-demethylation in the liver through cytochrome P450 (CYP) 3A4 and CYP2C19 and is further metabolized into glucuronide conjugates [9]. According to a report by Lee and Kim [12], following intravenous, oral, intraportal, intragastric, and intraduodenal administration of 10 mg/kg tofacitinib in male Sprague–Dawley rats, the *F* value was 29.1%, the unabsorbed fraction up to 24 h was 3.16% of the oral dose, the gastric first-pass effect was not significant after intragastric administration of tofacitinib, and 46.1% of the dose administered intraduodenally was metabolized before entering the portal vein. The hepatic first-pass effect was 42% after absorption into the portal vein.

Kidney disease has clinically important significance for patients with rheumatoid arthritis. Kidney disease is correlated with mortality in patients with rheumatoid arthritis [13,14]; therefore, renal impairment can be seen as a predictor of high mortality in patients with rheumatoid arthritis [15]. In addition, rheumatoid arthritis requires long-term treatment, and thus, the possibility of kidney damage is high due to long-term administration of drugs with renal toxicity, such as methotrexate or nonsteroidal anti-inflammatory drugs. Therefore, it is important to accurately measure kidney function in patients with rheumatoid arthritis and necessary to adjust the dose according to the kidney function of the patient. Approximately 36–38% of drugs prescribed in patients with glomerular filtration rate (GFR) < 60 mL/min require dosage adjustment due to pharmacokinetic changes of these drugs [16]. It is known that renal failure occurs in 5–50% of patients with rheumatoid arthritis [17]. Krishnaswami et al. [18] reported that, relative to patients with normal renal function, the mean AUC ratio of tofacitinib for rheumatoid arthritis patients increased progressively with deterioration of renal function. However, no mechanisms for increase of tofacitinib AUC in patients with renal impairment were proposed. There were no reports of tofacitinib showing pharmacokinetic changes associated with hepatic and/or intestinal metabolism, such as CYP protein expression, CYP enzyme activity, or renal function in the acute renal failure model. Since 70% of the dose is metabolized and 30% is excreted in the urine [9–11], renal failure seems to significantly impact the metabolism and excretion of tofacitinib as well as its absorption and distribution.

The aim of this study was to evaluate the effects of renal failure on the pharmacokinetics of tofacitinib using gentamicin (G-ARF) and cisplatin-induced acute renal failure (C-ARF) rat models and to report that the increase in AUC of tofacitinib is attributed to the decreases in renal and nonrenal clearances following intravenous and oral administration of tofacitinib to G-ARF and C-ARF rats.

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

#### *2.1. Chemicals*

Tofacitinib citrate and hydrocortisone (an internal standard) were obtained from Sigma Aldrich (St. Louis, MO, USA), and ethyl acetate for high-performance liquid chromatography (HPLC) analysis was purchased from J.T. Baker (Phillipsburg, NJ, USA). Gentamicin and cisplatin were obtained from Shin Poong Pharmaceutical (Seoul, Korea) and Tokyo Chemical Industry (Tokyo, Japan), respectively. Heparin and 0.9% NaCl-injectable solution were purchased from JW Pharmaceutical Corporation (Seoul, Korea), and β-cyclodextrin is a product of Wako (Osaka, Japan). Primary antibodies to CYP2B1/2, CYP1A1/2, CYP2D1, CYP2C11, CYP2E1, and CYP3A1/2 were produced by Detroit R&D Inc. (Detroit, MI, USA). β-actin was purchased from Cell Signaling Technology (Beverly, MA, USA). Secondary goat, rabbit, and mouse antibodies were purchased from Bio-Rad (Hercules, CA, USA). All other chemicals and reagents were analysis- or HPLC-grade and used without further purification.

#### *2.2. Animals*

Sprague–Dawley rats (male, 7 weeks old, weight 200–230 g) were purchased from OrientBio Korea (Seongnam, Korea) and individually managed in a clean room maintained under 12-h light (07:00–19:00)/12-h dark (19:00–07:00) cycles at 22 ± 1 ◦C with a relative humidity of 50 ± 5% through air purification (Laboratory Animal Research Center of Ajou University Medical Center, Suwon, Korea). All rats were fed with food and water as desired without any restriction. All experimental methods and protocols were carried out according to standard operating procedures with approval by Institutional Animal Care and Use Committee (IACUC No. 2017-0074, 2018) of Laboratory Animal Research Center of Ajou University Medical Center.

#### *2.3. Induction of Acute Renal Failure*

Rats were randomly divided into three groups: control, G-ARF, and C-ARF rats. Acute renal failure was induced in rats by intraperitoneal injection of gentamicin (100 mg/kg, dissolved in 0.9% NaCl-injectable solution) daily for 8 days [19] or by a single intraperitoneal injection of cisplatin (7.5 mg/kg, dissolved in 0.9% NaCl-injectable solution) [20], while control rats were injected with 0.9% NaCl-injectable solution only. The end times of renal failure induction for pharamcokinetic study of tofacitinib were the next day from the last administration of gentamicin and the sixth day from a single intraperitoneal injection of cisplatin. BUN (Blood urea nitrogen) levels were measured in rats on the last day of induction using a BUN detection kit (Asan Pharmaceutical, Seoul, Korea). Rats with a urea nitrogen level of 36 mg/dL or higher were considered to be acute renal failure-induced [21] and were selected for the study.

#### *2.4. Preliminary Study*

For preliminary study, plasma samples were collected from control, G-ARF, and C-ARF rats (*n* = 3 per group) to measure total protein, albumin, creatinine, glutamate pyruvate transaminase (GPT), and glutamate oxaloacetate transaminase (GOT) levels (Green Cross Reference Lab, Seoul, Korea). Urine samples were collected for 24 h, and urine volumes and creatinine levels were also measured to estimate the creatinine clearance (CLCR). CLCR was calculated by dividing the total amount of creatinine excreted in urine for 24 h by area under the plasma concentration-time curve of creatinine from 0 to 24 h (AUC0–24 h), assuming that renal function was stable during the experiment. Whole liver and kidneys were removed from each rat, weighed, partially excised, and soaked in 10% formalin to fix for tissue biopsies.
