*2.8. HPLC Analysis*

A 50-µL aliquot of biological sample was mixed with 1 µL hydrocortisone (5 mg/mL); then, 20 µL of 20% ammonia solution was added, mixed with a vortex-mixer (Scientific Industries, Bohemia, NY, USA) for 30 s, and extracted with 750 µL ethyl acetate. The organic layer was collected, evaporated on a thermobath (Eyela, Tokyo, Japan) under a gentle stream of nitrogen gas at 40 ◦C and redissolved by adding 130 µL 20% acetonitrile, and 50 µL of each reconstituted sample was analyzed by HPLC [12,22].

The concentration of tofacitinib in the biological sample was measured using a Prominence LC-20A HPLC system (Shimadzu, Kyoto, Japan). The reconstituted biological samples were filtered through a 0.45-µm filter (Millipore, Billerica, MA, USA) and analyzed with a reversed-phase column (C18; 25 cm × 4.6 mm, 5 µm; Young Jin Biochrom, Seongnam, Korea) using a UV detector at 287 nm. The mobile phase consisted of 10 mM ammonium acetate buffer (pH 5.0) and acetonitrile at a ratio of 69.5:30.5 (*v*/*v*) with a flow rate of 1.0 mL/min. Tofacitinib and the internal standard were separated at approximately 7.21 and 11.3 min, respectively. The lower limits of quantitation of tofacitinib in rat plasma and urine were 0.01 and 0.1 µg/mL, respectively, and the intraday assay precisions (coefficients of variation) were 3.69–5.88% and 4.21–6.18%, respectively. In addition, interday assay precisions in rat plasma and urine were 5.06% and 5.46%, respectively [22].

#### *2.9. Pharmacokinetic Analysis*

To estimate pharmacokinetic parameters such as terminal half-life, the apparent volume of distribution at steady state (*V*ss), area under plasma concentration-time curves from time zero to time infinity (AUC), mean residence time (MRT), and time-averaged total body (CL), and renal (CLR) and nonrenal (CLNR) clearances, standard methods [27] were applied using noncompartmental analysis (WinNonlin, Pharsight Corporation, Mountain View, CA, USA). AUCs were calculated using the trapezoidal rule-extrapolation method [28]. The peak plasma concentration (*C*max) and time that the plasma concentration was peak (*T*max) were directly confirmed from plasma concentration-time curves. To calculate the average values of clearances [29], terminal half-life [30], and *V*ss [31], the harmonic mean method was applied.

#### *2.10. Statistical Analysis*

The *p* values were estimated using Tukey's posttest for comparison among three means after analysis of variance (ANOVA) and were considered significant when less than 0.05. All data are expressed as mean ± standard deviation, and a median (ranges) value is used for *T*max.

#### **3. Results**

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

Renal dysfunction was observed in G-ARF and C-ARF rats. Urea nitrogen (898% and 3449% increase, respectively) and creatinine level (111% and 768% increase, respectively) in the blood showed a significant increase and kidney weight (% of body weight) (58.4% and 59.9% increase, respectively) and urine output (172% and 436% increase, respectively) also showed a significant increase, but CLCR was significantly decreased by 39.7% and 95.3% in G-ARF and C-ARF rats, respectively, than in control rats (Figure 2A). A decrease in renal function was also confirmed by kidney microscopy; severe renal damage including tubular necrosis and inflammation was observed in G-ARF and C-ARF rats (Figure 2B). Liver function also appeared to be impaired in G-ARF and C-ARF rats. GOT was significantly increased by 66.2 and 69.0%, respectively; however, no considerable tissue alterations were found in liver microscopy (Figure 2B). In terms of weight gain changes, the body weight gains significantly decreased in G-ARF (8.24% decrease) and C-ARF (23.0% decrease) rats compared to that in control rats (5.75% increase) (Figure 2A). Comparing the two ARF rat models, the C-ARF model showed more severe renal impairment based on urea nitrogen, creatinine, CLCR, urine output, kidney weight (% of body weight), and kidney microscopy (Figure 2A,B).

**Figure 2.** (**A**) The mean values (± standard deviation) of initial and final body weights, 24-h urine output, plasma concentrations of albumin, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), total protein, SCR, urea nitrogen, CLCR, and relative liver and kidney weights in control, gentamicin (G-ARF) and cisplatin-induced acute renal failure (C-ARF) rats: Bars mean standard deviation. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001; (**B**) Liver and kidney biopsies in control, G-ARF, and C-ARF rats. Black arrows indicate infiltration with immune cells. Blue arrows indicate the tissue damages including tubular necrosis and massive cell death. CLCR: creatinine clearance; SCR: serum creatinine.

#### *3.2. Pharmacokinetics of Tofacitinib After Intravenous Administration*

After intravenous administration of 10 mg/kg tofacitinib to control, G-ARF, and C-ARF rats, the mean arterial plasma concentration-time curves of tofacitinib declined in a polyexponential fashion for the three groups, with significantly higher plasma levels in rats with G-ARF and C-ARF than in

control rats (Figure 3). This resulted in a significantly higher AUC of tofacitinib (64.0 and 163% increase, respectively) than that in control rats (Table 1). The higher AUC of tofacitinib could be due to the significantly lower CL of tofacitinib by 37.7 and 62.3% in rats with G-ARF and C-ARF, respectively (Table 1). A significantly longer terminal half-life (78.7 and 240% increase, respectively) and MRT (96.7 and 154% increase, respectively) of tofacitinib in rats with G-ARF and C-ARF also supports the lower CL of tofacitinib in rats with G-ARF and C-ARF. Lower CL of tofacitinib was due to the slower metabolism of tofacitinib; CLNRs of tofacitinib in rats with G-ARF and C-ARF were significantly lower by 33.2 and 57.4%, respectively (Table 1). Tofacitinib excreted in urine as unchanged for 24 h (*Ae*0–24 h) was significantly lower by 37.7 and 95.2% in rats with G-ARF and C-ARF, respectively, than that in control rats (Table 1), perhaps due to significantly impaired kidney function in rats with G-ARF and C-ARF. Thus, CLRs of tofacitinib were significantly lower by 69.5 and 98.6% in rats with G-ARF and C-ARF, respectively, compared to that in control rats (Table 1). The percentage of dose remaining in the gastrointestinal tract at 24 h (GI24 h) was 0.00919–0.195% of the intravenous dose and did not significantly differ among the three groups of rats, suggesting that the contribution of gastrointestinal excretion (including biliary excretion) of tofacitinib to CLNR of tofacitinib was not significant. The *V*ss values were comparable among the three groups (Table 1). Therefore, the significantly lower CL of tofacitinib in rats with G-ARF and C-ARF may be due to slower metabolism and lower renal excretion of tofacitinib than control rats. Plasma concentration and pharmacokinetic parameters of tofacitinib in C-ARF rats were significantly different from those in G-ARF rats (Table 1 and Figure 3) due to more severe renal impairment in C-ARF rats (Figure 2).

**Figure 3.** Mean arterial plasma concentration-time curves of tofacitinib after 1-min intravenous infusion at a dose of 10 mg/kg to control (black; *n* = 6), G-ARF (blue; *n* = 8) and C-ARF (red; *n* = 7) rats: Bars represent standard deviation. G-ARF: gentamicin-induced acute renal failure; C-ARF: cisplatin-induced acute renal failure.


**Table 1.** Mean (±standard deviation) pharmacokinetic parameters of tofacitinib after 1-min intravenous infusion at a dose of 10 mg/kg to control, G-ARF, and C-ARF rats.

*Ae*0–24 h: percentage of the dose excreted in the 24-h urine; AUC: total area under the plasma concentration–time curve from time zero to time infinity; C-ARF: cisplatin-induced acute renal failure; CL: time-averaged total body clearance; CLNR: time-averaged nonrenal clearance; CLR: time-averaged renal clearance; G-ARF: gentamicin-induced acute renal failure; GI24 h: percentage of the dose remaining in the gastrointestinal tract (including its contents and feces) at 24 h; MRT: mean residence time; *V*ss: apparent volume of distribution at steady state. <sup>a</sup> Control is significantly different (*p* < 0.01) from C-ARF and G-ARF. <sup>b</sup> Control is significantly different from C-ARF (*p* < 0.001) and G-ARF (*p* < 0.01). <sup>c</sup> Control is significantly different from G-ARF (*p* < 0.05). <sup>d</sup> Control is significantly different from G-ARF and C-ARF (*p* < 0.001). G-ARF and C-ARF were significantly different (*p* < 0.05). <sup>e</sup> Control is significantly different from G-ARF and C-ARF (*p* < 0.001). <sup>f</sup> Control is significantly different from G-ARF (*p* < 0.01) and C-ARF (*p* < 0.001). G-ARF and C-ARF were significantly different (*p* < 0.01).

#### *3.3. Pharmacokinetics of Tofacitinib After Oral Administration*

After oral administration of 20 mg/kg tofacitinib to control, G-ARF, and C-ARF rats, the mean arterial plasma concentration-time profiles of tofacitinib were created and are shown in Figure 4. Relevant pharmacokinetic parameters of tofacitinib were summarized in Table 2.

**Figure 4.** Mean arterial plasma concentration–time curves of tofacitinib after oral administration at a dose of 20 mg/kg to control (black; *n* = 8), G-ARF (blue; *n* = 6), and C-ARF (red; *n* = 8) rats: Bars represent standard deviation. G-ARF: gentamicin-induced acute renal failure; C-ARF: cisplatin-induced acute renal failure.


**Table 2.** Mean (±standard deviation) pharmacokinetic parameters of tofacitinib after oral administration at a dose of 20 mg/kg to control, G-ARF, and C-ARF rats.

*Ae*0–24 h: percentage of the dose excreted in the 24-h urine; AUC: total area under the plasma concentration–time curve from time zero to last time; C-ARF: cisplatin-induced acute renal failure; CLR: time-averaged renal clearance; *C*max: peak plasma concentration; G-ARF: gentamicin-induced acute renal failure; GI24 h: percentage of the dose remaining in the gastrointestinal tract (including its contents and feces) at 24 h; *T*max: time that the plasma concentration was peak. <sup>a</sup> Control is significantly different from G-ARF and C-ARF (*p* < 0.001). G-ARF and C-ARF were significantly different (*p* < 0.05). <sup>b</sup> Control is significantly different from C-ARF (*p* < 0.01). <sup>c</sup> Control is significantly different from G-ARF (*p* < 0.05). <sup>d</sup> C-ARF is significantly different from control (*p* < 0.001) and G-ARF (*p* < 0.05).

Absorption of tofacitinib from the gastrointestinal tract occurred rapidly; the plasma concentration of tofacitinib was found at 5 min, the first blood collection time after oral administration in all three groups. Compared to the control rats, G-ARF and C-ARF rats showed higher mean arterial plasma concentration of tofacitinib, resulting in a significant increase in AUC (142 and 247% increase, respectively) and *C*max (198 and 141% increase, respectively) (Table 2). However, the CL<sup>R</sup> values significantly decreased by 69.8 and 94.0% in G-ARF and C-ARF rats, respectively, due to the significant increase in AUC and a significant decrease in *Ae*0–24 h (22.4 and 81.3% decrease, respectively) in G-ARF and C-ARF rats (Table 2). GI24 h values were 0.231, 1.27, and 0.505% of the oral dose in control, G-ARF, and C-ARF rats, respectively, indicating that absorption of tofacitinib from the gastrointestinal tract was almost complete with no significant difference among the three groups (Table 2). The *T*max values were likewise not significantly different among the three groups. After oral administration, *F* values of tofacitinib were 41.3, 60.7, and 54.3% in control, G-ARF, and C-ARF rats, respectively (Table 2). Plasma concentration and pharmacokinetic parameters of tofacitinib in C-ARF rats, such as AUC, CLR, and *Ae*0–24 h, were significantly different from those in G-ARF rats due to severe renal impairment in C-ARF rats (Table 2 and Figure 4); this was similar to the results produced by the intravenous study.

#### *3.4. E*ff*ect of Acute Renal Failure on CYP Enzyme Expression*

In rats with G-ARF and C-ARF, hepatic and intestinal expression of CYP2B1/2, CYP1A1/2, CYP2D1, CYP2C11, CYP2E1, and CYP3A1/2 were monitored (Figure 5). Immunoblot analysis showed that hepatic expression of CYP2C11 in rats with G-ARF and C-ARF decreased to 53.1 and 49.2% of the level in control rats, respectively, and CYP3A1/2 expression in rats with G-ARF and C-ARF also decreased by 14.6 and 60.8%, respectively, compared to that in control rats. However, CYP2E1 expression in rats with G-ARF and C-ARF increased by 1.33 and 1.73 times, respectively, compared to that in control rats. Expression of CYP1A1/2 and CYP2D1 was comparable among the three groups of rats. Interestingly, protein expression in the intestine showed the opposite trend (Figure 5). The intestinal expression of CYP3A1/2 in rats with G-ARF and C-ARF increased 5.30 and 7.97 times, respectively, and CYP2C11 expression also increased by 3.30 and 3.27 times, respectively, compared to those in control rats. Other CYP protein expressions except CYP2E1 also increased in the intestine of G-ARF and C-ARF rats.

**Figure 5.** Protein expression of CYP450 isozymes in hepatic and intestinal microsomes in control, G-ARF, and C-ARF rats by immunoblot analyses: ß-actin was used as a loading control. This experiment was performed three times. Band density was measured using ImageJ1.45s software (NIH). G-ARF: gentamicin-induced acute renal failure; C-ARF: cisplatin-induced acute renal failure.
