Tofacitinib (Fig. 1A), an inhibitor of Janus kinase (JAK) 1 and 3, has been found effective in the treatment of patients with moderate-to-severe rheumatoid arthritis, especially those who are unresponsive to methotrexate (Claxton et al., 2018). Tofacitinib was shown to inhibit the JAK-signal transducer and activator of transcription (STAT) signaling pathway in patients with rheumatoid arthritis (Fukuda et al., 2019). In humans, orally administered tofacitinib has a terminal half-life of 3.2 h, with approximately 70% metabolized in the liver by microsomal cytochrome P450 (CYP) 3A4 and CYP2C19 and the remaining 30% excreted in its unmetabolized form by the kidneys (Bannwarth et al., 2013; Dowty et al., 2014).

Figure 1. Chemical structures of (A) tofacitinib and (B) loganin.

The pharmacokinetics of tofacitinib was shown to be altered in rats with acute kidney injury (AKI) induced by gentamicin and cisplatin (Bae et al., 2020). The time-averaged renal clearance (CL_R) of tofacitinib was markedly reduced due to reduced renal function, as indicated by reduced creatinine clearance (CL_CR). The time-averaged non-renal clearance (CL_NR) of tofacitinib in these rat models was also significantly reduced, a finding attributable to reductions in hepatic and/or intestinal CYP3A1/2 and CYP2C11 activities (Bae et al., 2020).

Renal impairment has been associated with increased mortality in patients with rheumatoid arthritis (Thomas et al., 2003; Koivuniemi et al., 2008), with renal injury being a risk factor for death in these patients (Sihvonen et al., 2004). Because patients with rheumatoid arthritis require long-term treatment, the prolonged administration of drugs with renal toxicity, such as methotrexate (Lee et al., 2020) and non-steroidal anti-inflammatory drugs (Möller et al., 2015), increases the risk of kidney damage. Accurate determination of kidney function in patients with rheumatoid arthritis can enable drug doses to be adjusted accordingly.

Several herbal extracts have shown potential protective effects against gentamicin (Stojiljkovic et al., 2009; Morales et al., 2010; Salem et al., 2010) and cisplatin (Dachuri et al., 2020). Loganin (LGN, Fig. 1B), an iridoid glycoside and the primary constituent of extracts of Corni fructus (Xu et al., 2006; Lee et al., 2009, 2011; Park et al., 2011), has been shown to regenerate the liver and kidneys (Dong et al., 2018). Additionally, LGN has demonstrated beneficial effects in treating various conditions such as acute pancreatitis (Kim et al., 2015), inflammation (Cui et al., 2018), diabetes (Jiang et al., 2012), apoptosis (Kwon et al., 2011), and gastric cancer (Zhou et al., 2020). Moreover, it has been shown to attenuate the severity of acute renal impairment in a mouse model (Kim et al., 2021). Less is known, however, about the ability of LGN to modify the pharmacokinetics of co-administered drugs.

The present study, therefore, investigated the potential of LGN to prevent and/or ameliorate AKI and to modify the pharmacokinetic properties of intravenously and orally administered tofacitinib in rats with cisplatin-induced AKI. In addition, this study assessed the ability of LGN to modify the activities of microsomal CYP isozymes involved in tofacitinib metabolism in the liver and intestines of these rats.

Materials

Tofacitinib citrate (MW: 504.49 daltons) and LGN were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China), respectively. Cisplatin and β-cyclodextrin were obtained from Tokyo Chemical Industry (Tokyo, Japan) and Wako (Osaka, Japan), respectively. Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-generating system was supplied by Corning Inc. (Corning, NY, USA). Primary antibodies against CYP3A1/2, CYP2E1, CYP2C11, CYP2D1, CYP2B1/2 and CYP1A1/2 were provided by Detroit R&D Inc. (Detroit, MI, USA). All other reagents were of high-performance liquid chromatography (HPLC) or analytical grade and were used as received without additional purification.

Animals

Male Sprague-Dawley rats, aged 7 weeks and weighing 200-230 g (OrientBio Korea, Seongnam, Korea) were housed separately at the Laboratory Animal Research Center of Ajou University Medical Center (Suwon, Korea) in a controlled environment, including a temperature of 22 ± 1°C and a relative humidity of 50 ± 5%, a 12-h light cycle (07:00-19:00) with food and water ad libitum. All experimental procedures were in accordance with standard conditions for animal experiments approved by the Institutional Animal Care and Use Committee (IACUC No. 2022-0034) of the Laboratory Animal Research Center.

Induction of AKI

The rats were randomly assigned to four groups: the AKI, LGN, AKI-LGN, and control (CON) groups. AKI was induced by a single intraperitoneal administration of cisplatin (6.5 mg/kg in saline), as described previously (Abd El-Kader and Taha, 2020). LGN rats were orally administered LGN (20 mg/kg in saline). LGN-AKI rats were orally administered 20 mg/kg LGN 1 h prior to the intraperitoneal administration of cisplatin (Kim et al., 2021; Szentmihályi et al., 2014). CON rats received saline. Blood urea nitrogen (BUN) levels >36 mg/dL 6 days after cisplatin administration were regarded as confirmation of AKI induction (Feng et al., 2013).

Biochemical profiles and tissue microscopy

Plasma and 24-h urine samples were collected from three rats in each of the four groups. The plasma concentrations of glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT), total protein, albumin, creatinine and urine volume were quantified, and CL_CR was calculated by dividing the total creatinine excreted in the urine over 24 h by the area under the plasma concentration-time curve of creatinine from 0 to 24 h (AUC_{0-24 h}), with renal function assumed to be stable throughout the experimental period. In addition, liver and kidney weights were measured. For histological examination, a segment of each tissue specimen was fixed in 10% formalin.

Rat plasma protein binding of tofacitinib

The binding of plasma proteins to tofacitinib was evaluated using freshly obtained rat plasma (n=3 per group) using an ultrafiltration method (Barre et al., 1985). Briefly, rat plasma containing 5 µg/mL tofacitinib was introduced into a Nanosep 10 K centrifugal filter device (Pall Co., Ann Arbor, MI, USA) and centrifuged at 1,500 g for 30 min, and a 100-µL aliquot of each ultrafiltrate containing unbound tofacitinib was stored at -80°C (Kim et al., 2020). The nonspecific binding of tofacitinib to the ultrafiltration device was determined by loading phosphate buffer (pH 7.4) containing 5 µg/mL tofacitinib onto the device and centrifuging it under the same conditions (n=3). The plasma protein binding percentage (%) of tofacitinib was calculated using the formula:

where C_F is the concentration of unbound tofacitinib measured in the ultrafiltrate and C_T is the concentration of tofacitinib loaded onto the ultrafiltration device prior to centrifugation (Barre et al., 1985).

Intravenous and oral administration of tofacitinib to rats

All experimental procedures, including rat pretreatment, were performed as previously described (Lee and Kim, 2019; Bae et al., 2022). The carotid artery and jugular vein were cannulated for blood collection and intravenous drug administration, respectively. The experiments were started approximately 3-4 h after the surgical cannulation procedure, allowing the rats to recover and move freely.

Tofacitinib (10 mg/kg) dissolved in saline with β-cyclodextrin (0.5%, w/v) was infused intravenously for 1 min into CON (n=6), LGN (n=7), AKI (n=7), and AKI-LGN (n=6) rats through the jugular vein. An aliquot of blood samples was withdrawn from the carotid artery before administration of the drug (0 min) and 1, 5, 15, 30, 45, 60, 90, 120, 180, 240, and 360 min after drug administration. Alternatively, tofacitinib (20 mg/kg) was administered orally to CON (n=5), LGN (n=5), AKI (n=6), and AKI-LGN (n=7) rats, and an aliqot of blood samples was collected at before administration of the drug (0 min), and 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 600, and 720 min after drug administration. Plasma sample (50 μL) was collected after centrifugation and was kept at -80°C until quantification of tofacitinib (Kim et al., 2020). The gastrointestinal track and urine samples over 24 h were also collected as described (Lee and Kim, 2019; Bae et al., 2022) and the concentration of tofacitinib was measured (Kim et al., 2020).

Measurement of V_max, K_m, and CL_int

In vitro metabolic ability in hepatic and intestinal microsomes obtained from three rats in each of the four groups were determined as described (Duggleby, 1995; Bae et al., 2022). The in vitro metabolic system included 1 mg of microsomal protein, 1 μL of tofacitinib (5, 10, 20, 50, 100, 200, or 500 μM), and NADPH-generating system. The system was adjusted to a total volume of 1 mL by 0.1 M phosphate-buffered saline (pH 7.4). The samples were incubated at 37°C for 15 min at a rate of 50 rpm. After adding two volumes of methanol, the reaction was terminated.

Kinetic constants, maximum velocity (V_max), and the apparent Michaelis–Menten constant (K_m; the concentration of tofacitinib at one-half of V_max) were determined by nonlinear regression analysis (Duggleby, 1995; Bae et al., 2022). The intrinsic clearance (CL_int) of tofacitinib in the liver and intestine was estimated by dividing V_max by K_m (Duggleby, 1995; Bae et al., 2022).

Immunoblot analysis

Hepatic and intestinal microsomal proteins (20-40 μg per lane) were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to membranes. The membranes were incubated overnight at 4°C with primary antibodies (1:2,000 dilution) against CYP1A1/2, CYP2B1/2, CYP2C11, CYP2D1, CYP2E1, CYP3A1/2, and β-actin, followed by incubation with secondary antibodies (1:10,000 dilution). Bands were visualized by enhanced chemiluminescence and their densities were measured using ImageJ 1.45s software (NIH, Bethesda, MD, USA), with β-actin serving as a loading control (Bae et al., 2022).

HPLC analysis

Biological samples for HPLC analysis of tofacitinib were prepared as described (Kim et al., 2020). Briefly, a 50 μL aliquot of each biological sample was mixed with 20% ammonia solution (20 μL) containing hydrocortisone (10 μg/mL) as an internal standard and was extracted with ethyl acetate (750 μL). The organic layer was evaporated under nitrogen gas. The residue was reconstituted and the supernatant (50 μL) was injected onto a reversed-phase column (C₁₈; 250×4.6 mm, 5 μm; Young Jin Biochrom, Seongnam, Korea) and analyzed using a Prominence LC-20A HPLC system (Shimadzu, Kyoto, Japan) equipped with a UV detector set at 287 nm. The mobile phase consisted of 10 mM ammonium acetate buffer (pH 5.0) and acetonitrile (69.5:30.5, v/v) and a flow rate was 1 mL/min. The retention times for tofacitinib and hydrocortisone were approximately 7.2 and 11.3 min, respectively.

The lower limits of quantitation for tofacitinib in rat plasma and urine were 0.01 and 0.1 μg/mL, respectively. Intra-day assay precisions (coefficients of variation) in rat plasma and urine ranged from 3.69% to 5.88% and from 4.21% to 6.18%, respectively, and the corresponding inter-day assay precisions in rat plasma and urine were 5.06% and 5.46%, respectively (Kim et al., 2020).

Pharmacokinetic analysis

Standard method (Gibaldi and Perrier, 1982) was applied to estimate pharmacokinetic parameters by non-compartmental analysis (WinNonlin; Pharsight Co., Mountain View, CA, USA). AUCs were calculated using the trapezoidal rule extrapolation method (Chiou, 1978). The peak plasma concentration (C_max) and time to reach C_max (T_max) were measured directly from the plasma concentration–time curves. The absolute oral bioavailability (F) of tofacitinib was calculated by dividing the dose-normalized AUC_oral by the dose-normalized AUC_intravenous, with AUC_oral and AUC_intravenous indicating the AUCs after oral and intravenous administration, respectively.

Statistical analysis

Results are represented as mean ± standard deviation (SD). The comparison of four groups was performed by analysis of variance (ANOVA), followed by Tukey’s post-test, with p-values <0.05 defined as statistically significant.

Preliminary data and tissue microscopy

The effects of LGN on rats with cisplatin-induced AKI and on the pharmacokinetics of tofacitinib were assessed by measuring body weights, biochemical profiles, plasma protein binding of tofacitinib, CL_CR, and relative liver and kidney weights in the four groups of rats (Fig. 2A). Kidney function was impaired in AKI rats, as shown by significantly higher BUN levels (1310%) and relative kidney weights (53.6%) and significantly lower CL_CR (87.3%) compared with CON rats (Stojiljkovic et al., 2009; Bae et al., 2020). LGN administration significantly improved kidney function in AKI rats, resulting in a 65.4% reduction in BUN levels, a 16.8% reduction in relative kidney weights, and a 490% increase in CL_CR compared with AKI rats (Fig. 2A). Hepatic function was not altered in AKI rats, with plasma concentrations of total proteins, albumin, GOT, and GPT, and relative liver weights being within normal ranges (Mitruka and Rawnsley, 1981) and comparable in the four rat groups. Tissue microscopy confirmed severe renal tissue damage in AKI rats, including necrosis and massive cell death (Fig. 2B). Liver microscopy of AKI rats showed the infiltration of some immune cells, but little or no alterations in tissue (Fig. 2B). In contrast to AKI rats, AKI-LGN rats showed no evidence of serious abnormalities in liver or kidney tissue (Fig. 2B).

Figure 2. (A) Body weight, plasma chemistry data, creatinine clearance (CLCR), plasma protein binding of tofacitinib, and relative liver and kidney weights in the four groups of rats (control (CON), loganin (LGN), acute kidney injury (AKI) and acute kidney injury-loganin (AKI-LGN); n=3 each). Data are shown as means ± standard deviations. (B) Liver and kidney biopsy samples from rats in the four groups. Black arrows indicate immune cell infiltration and stars indicate tissue damage, including massive cell death. BUN, blood urea nitrogen; BW, body weight; GOT, glutamate oxaloacetate transaminase; GPT, glutamate pyruvate transaminase; SCR, serum creatinine concentration. *p<0.05, **p<0.01, ***p<0.001.

Plasma protein binding of tofacitinib

The binding of tofacitinib to proteins in fresh plasma obtained from rats in the CON (58.8%), LGN (56.8%), AKI (62.6%), and AKI-LGN (62.2%) groups was comparable (Fig. 2A). The mean recovery of tofacitinib after ultrafiltration of phosphate buffer containing tofacitinib (5 μg/mL) was 101 ± 0.596% (n=3), indicating that the non-specific binding of tofacitinib to the ultrafiltration device was negligible.

Pharmacokinetics of tofacitinib after its intravenous and oral administration to rats

The effects of AKI and LGN on the pharmacokinetics of intravenously administered tofacitinib (10 mg/kg) were evaluated by plotting the mean arterial plasma concentration–time curves of tofacitinib (Fig. 3A) and by analyzing its pharmacokinetic parameters (Table 1) in the four groups of rats. Compared with CON rats, AKI rats exhibited notable changes in the pharmacokinetics of tofacitinib, including a significantly increased AUC (224%), a significantly prolonged terminal half-life (235%), significantly slower CL (68.6%), CL_NR (67.2%), and CL_R (96.6%), and an 89.8% reduction in the amount of tofacitinib excreted in the urine over 24 h (Ae_{0-24 h}). Compared with AKI rats, however, AKI-LGN rats showed significant improvements in all pharmacokinetic parameters of tofacitinib, reaching levels comparable to CON rats (Table 1), including a significantly decreased AUC (68.4%), a substantially reduced terminal half-life (60.3%), significantly faster CL (227%), CL_NR (223%), and CL_R (1816%), and a 394% increase in Ae_{0-24 h}. Comparisons of the CON and LGN groups showed no significant differences in pharmacokinetic parameters. Considering the half-life of LGN (approximately 50-140 min, Park et al., 2021), LGN was completely eliminated from the body when pharmacokinetic study of tofacitinib started at 6th day after LGN administration. Therefore, LGN did not seem to affect the pharmacokinetics of tofacitinib in LGN rats.

Table 1 Mean pharmacokinetic parameters (± standard deviation) of tofacitinib after its intravenous administration at a dose of 10 mg/kg to CON, LGN, AKI, and AKI-LGN rats

Parameters	CON (n=6)	LGN (n=6)	AKI (n=7)	AKI-LGN (n=6)
Body weight (g)	292 ± 12.2	268 ± 10.9	181 ± 3.91*	243 ± 17.0
Terminal half-life (min)	46.0 ± 12.0	28.0 ± 11.7	154 ± 65.8*	61.1 ± 16.0
AUC (µg∙min/mL)	243 ± 22.0	257 ± 13.1	788 ± 128*	249 ± 36.6
CL (mL/min/kg)	41.4 ± 3.64	39.0 ± 1.99	13.0 ± 1.93*	42.5 ± 4.18
CLR (mL/min/kg)	2.17 ± 0.284	2.25 ± 0.892	0.0741 ± 0.0629*	1.42 ± 0.358
CLNR (mL/min/kg)	39.3 ± 3.45	36.8 ± 2.23	12.9 ± 1.89*	41.7 ± 4.45
Vss (mL/kg)	880 ± 405	490 ± 84.0	889 ± 475	1585 ± 346
Ae0-24 h (% of dose)	5.24 ± 0.524	5.79 ± 2.37	0.536 ± 0.424	2.65 ± 1.61
GI24 h (% of dose)	0.0393 ± 0.0241	0.165 ± 0.139	0.419 ± 0.296	0.0303 ± 0.00232

^Ae_{0-24 h}, the amount of tofacitinib excreted in the urine over 24 h; AKI, acute kidney injury; AKI-LGN, acute kedney injury-logaini; AUC, area under plasma concentration-time curves from time zero to time infinity; CL, time-averaged total body clearance; CL_NR, time-averaged non-renal clearance; CL_R, time-averaged renal clearance; CON, control; GI_{24 h}, the percentage of drug remaining in the gastrointestinal tract at 24 h; LGN, loganin; ^V_ss, the apparent volume of distribution at steady state. *AKI is significantly different (p<0.05) from CON, LGN and AKI-LGN.

Figure 3. Mean arterial plasma concentration-time profiles of tofacitinib after (A) 1-min intravenous infusion (10 mg/kg) into control (CON, n=6), loganin (LGN, n=6), acute kidney injury (AKI, n=7) and acute kidney injury-loganin (AKI-LGN, n=6) rats, and (B) oral administration (20 mg/kg) to CON (n=5), LGN (n=5), AKI (n=6) and AKI-LGN (n=7) rats. Bar represent standard deviations.

The effects of AKI and LGN on the mean arterial plasma concentration–time curves (Fig. 3B) and pharmacokinetics (Table 2) of orally administered tofacitinib (20 mg/kg) were similarly evaluated in the four groups of rats. Compared with CON rats, AKI rats exhibited a significant increase in AUC (166%) and significant reductions in CL_R (94.8%) and Ae_{0-24 h} (86.6%). All pharmacokinetic parameters in AKI-LGN rats, however, were comparable to those in CON rats. Compared with AKI rats, AKI-LGN rats showed a significant reduction in AUC (49.4%) and significant increases in CL_R (769%) and Ae_{0-24 h} (363%). In contrast, the pharmacokinetic parameters of tofacitinib were comparable in the CON and LGN groups due to the same reason as the intravenous study.

Table 2 Mean pharmacokinetic parameters (± standard deviation) of tofacitinib after its oral administration at a dose of 20 mg/kg to CON, LGN, AKI, and AKI-LGN rats

Parameters	CON (n=5)	LGN (n=5)	AKI (n=6)	AKI-LGN (n=7)
Body weight (g)	257 ± 10.1	251 ± 5.29	195 ± 2.40*	223 ± 14.7
AUC (µg∙min/mL)	215 ± 27.4	277 ± 33.1	571 ± 124*	289 ± 33.3
Cmax (µg/mL)	1.98 ± 0.712	4.03 ± 2.06	2.09 ± 0.525	2.52 ± 0.886
Tmax (min)	30.0 ± 10.6	16.0 ± 8.94	75.0 ± 48.4	39.3 ± 28.8
CLR (mL/min/kg)	11.7 ± 1.40	7.21 ± 0.950	0.610 ± 0.361*	5.30 ± 0.733
Ae0-24 h (% of dose)	12.4 ± 0.833	9.95 ± 1.36	1.66 ± 0.836*	7.69 ± 1.55
GI24 h (% of dose)	0.256 ± 0.162	0.120 ± 0.0821	0.859 ± 0.497	0.463 ± 0.142
F (%)	44.2	45.7	43.7	58.0

Ae_{0-24 h}, the drug excreted as an unchanged form in the urine for 24 h; AKI, acute kidney injury; AKI-LGN, acute kedney injury-logaini; AUC, area under plasma concentration-time curves from time zero to time infinity; C_max, maximum plasma concentration; CL_R, time-averaged renal clearance; CON, control; F, absolute oral bioavailability; GI_{24 h}, the percentage of drug remaining in the gastrointestinal tract at 24 h; LGN, loganin; T_max, time to reach C_max. *AKI is significantly different (p<0.05) from CON, LGN and AKI-LGN.

In vitro metabolism of tofacitinib

Tofacitinib metabolism in hepatic microsomes obtained from the four groups of rats was evaluated by measuring V_max, K_m, and CL_int values (Fig. 4A). V_max was significantly slower (59.8%) in AKI than in CON rats, but was partially restored by administration of LGN, with V_max in AKI-LGN rats being 80.4% of that in CON rats. K_m values did not differ significantly among the groups, suggesting that the affinity of the enzyme for tofacitinib was unaltered by AKI or LGN plus AKI. CL_int in AKI rats was 38.1% of that in CON rats, but was restored to 93.1% of that in CON rats in the AKI-LGN group. These findings indicate that hepatic metabolism of tofacitinib was reduced in AKI rats, but was restored in AKI-LGN rats after LGN administration.

Figure 4. Mean Vmax, Km and CLint values for tofacitinib metabolism in (A) hepatic and (B) intestinal microsomes from control (CON), loganin (LGN), acute kidney injury (AKI) and acute kidney injury-loganin (AKI-LGN) rats (n=3 per group). Data are shown as means ± standard deviations of three independent experiments. Vmax, maximum velocity; Km, apparent Michaelis–Menten constant; CLint, intrinsic clearance. *p<0.05; **p<0.01; ***p<0.001.

Tofacitinib metabolism, including V_max, K_m, and CL_int values, was also analyzed in intestinal microsomes obtained from the four groups of rats (Fig. 4B). V_max was significantly slower (69.6%) in AKI rats than in CON rats, but was partially restored by LGN administration, with V_max in AKI-LGN rats being 64.8% of that in CON rats. Similar to findings with hepatic microsomes, K_m values did not differ significantly in the four groups. CL_int was significantly slower (51.6%) in AKI rats than in CON rats, but was restored to 80.8% of that in CON rats by the administration of LGN in AKI-LGN rats. These results suggest that intestinal metabolism of tofacitinib was lower in AKI rats than in CON rats, but was restored to CON levels in AKI-LGN rats by LGN administration.

Expression of CYP isozymes

CYP3A4 and CYP2C19 are expressed in human and mainly involved in the metabolism of tofacitinib in human. CYP3A1/2 and CYP2C11 play same roles in rats as CYP3A4 and CYP2C19 do in human. Human CYP3A4 and rat CYP3A1 proteins have 73% homology (Lewis, 1996). Human CYP2C19 and rat CYP2C11 share a high degree of amino acid homology (Banerjee et al., 2015). In this study, the expression of rat CYP proteins was measured instead of human CYP proteins since rat models were used in the experiment.

Analysis of the expression of CYP1A1/2, CYP2C11, and CYP3A1/2 in hepatic microsomes showed that the levels of these enzymes were 56.5%, 47.7%, and 63.8% lower, respectively, in AKI rats than in CON rats, but were 40.5%, 95.0%, and 94.2% higher, respectively, in AKI-LGN rats than in AKI rats (Fig. 5). Interestingly, the expression levels of intestinal CYP2C11 and CYP3A1/2 were 160% and 2930% higher, respectively, in AKI rats than in CON rats, but were 64.0% and 39.6% lower, respectively, in AKI-LGN rats than in AKI rats. These findings suggest that AKI altered the expression of CYP isozymes in the liver and/or intestine, which may have been responsible for the reduced metabolism of tofacitinib, and that these effects were restored by administration of LGN.

Figure 5. Immunoblot analyses of cytochrome P450 (CYP) isozymes in (A) hepatic and (B) intestinal microsomes from control (CON), loganin (LGN), acute kidney injury (AKI) and acute kidney injury-loganin (AKI-LGN) rats. β-Actin was used as a loading control. Results are representative of three independent experiments. Band density was measured using ImageJ 1.45s software (NIH).

Cisplatin induces irreversible and severe acute renal impairment, including tubular necrosis and/or apoptosis, through the generation of reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide, and hydroxyl radicals (Siddik et al., 1987), which accumulate in renal tubules (Martínez-Salgado et al., 2007; Arjumand et al., 2011; Malik et al., 2016). Patients taking drugs with the potential for severe nephrotoxicity, such as cisplatin, are therefore advised to take dietary supplements that can protect kidneys from acute renal impairment. The iridoid glycoside LGN has been shown to alleviate diabetic nephropathy (Jiang et al., 2012) and mitigate the severity of AKI (Kim et al., 2021). Moreover, the Chinese extract, QiongYu-Gao, which contains iridoid glycosides, has been shown to prevent cisplatin-induced nephrotoxicity (Teng et al., 2015).

Analyses of plasma, urine, kidney, and liver in the present study showed that the administration of LGN to AKI rats resulted in the recovery of renal function, with findings in AKI-LGN rats being similar to those in CON rats. These findings suggest that LGN suppresses renal damage to a certain extent. Other studies have indicated that some natural products, including LGN, exhibit nephroprotective effects by preventing the accumulation of nephrotoxic drugs in the kidneys (Lee et al., 2013; Teng et al., 2015; Kim et al., 2021), suggesting that drug accumulation in the kidneys is associated with nephrotoxicity (Navarro and Senior, 2006; Lee et al., 2013). The results of the present study therefore indicate that LGN prevents nephrotoxicity by reducing the renal accumulation of cisplatin.

Because the AUCs of both intravenously and orally administered tofacitinib have been reported to be dose-independent under 50 mg/kg (Lee and Kim, 2019), rats were administered intravenous and oral tofacitinib at doses of 10 mg/kg and 20 mg/kg, respectively. The AUC of intravenously administered tofacitinib was significantly greater in AKI rats than in CON rats, possibly because CL was significantly slower in AKI rats. Because tofacitinib is primarily metabolized in the liver, the decreased CL of tofacitinib in AKI rats was likely due to its reduced CL_NR (Dowty et al., 2014; Lee and Kim, 2019). The CL_NR of tofacitinib represents its metabolic clearance because tofacitinib showed negligible contribution of gastrointestinal and biliary excretion of unchanged tofacitinib to CL_NR (Lee and Kim, 2019) and the absence of chemical and enzymatic degradation of tofacitinib in rat gastric fluid (Kim et al., 2020).

Tofacitinib is primarily metabolized by hepatic CYP2C11 and CYP3A1/2 in rats (Dowty et al., 2014; Lee and Kim, 2019). The hepatic metabolism of tofacitinib is markedly lower in AKI rats than in CON rats, resulting in a significantly slower CL_NR. Because tofacitinib is an intermediate hepatic extraction ratio drug (42.0%) in rats (Lee and Kim, 2019), its hepatic clearance depends more on its CL_int and the percentage unbound to plasma proteins than on hepatic blood flow rate (Wilkinson and Shand, 1975). Thus, the slower CL_NR of tofacitinib in AKI rats than in CON rats was likely due to its significantly reduced in vitro CL_int, as evidenced by the reduced levels of expression of CYP2C11 and CYP3A1/2 in hepatic microsomes. In contrast, renal dysfunction and changes in the pharmacokinetic parameters of tofacitinib, such as AUC, CL, CL_R, and CL_NR, were alleviated in AKI-LGN compared with AKI rats and were comparable to findings in CON rats. The Ae_{0-24 h} of tofacitinib was substantially lower in AKI rats than in CON rats, but was significantly higher in AKI-LGN rats than in AKI rats, indicating that renal function was less impaired in AKI-LGN rats than in AKI rats by the administration of LGN. Taken together, these results suggest that LGN may prevent cisplatin-induced AKI and reverse the associated alterations in pharmacokinetics of tofacitinib to a status similar to that in CON rats.

The decreased CL of tofacitinib in AKI rats was also due to its reduced CL_R, a reduction primarily attributed to a markedly smaller Ae_{0-24 h} resulting from impaired kidney function, and a greater AUC. The CL_R of tofacitinib was based on the fraction unbound to plasma proteins, with rats in the CON, LGN, AKI, and AKI-LGN groups having tofacitinib CL_R values of 5.27, 5.21, 0.198, and 3.76 mL/min/kg, respectively, rates markedly higher than the CL_CR values in these groups. These findings indicated that tofacitinib is actively secreted through renal tubules in all four groups of rats (Lee and Kim, 2019) and that cisplatin-induced kidney damage reduced the excretion of tofacitinib (Bae et al., 2020). The Ae_{0-24 h} of tofacitinib, however, was significantly higher in AKI-LGN rats than in AKI rats, suggesting that LGN mitigated the impairment of renal function and increased the urinary excretion of tofacitinib in AKI-LGN rats.

The present study also found that AUC values of orally administered tofacitinib were significantly higher in AKI rats than in CON rats. Almost 100% of orally administered tofacitinib was absorbed from the gastrointestinal tract in both CON and AKI rats, with GI24 h values accounting for less than 0.859% of the oral dose for both groups. Therefore, absorption alone does not explain the increased AUCs in AKI rats. Despite the intestinal expression of CYP3A1/2 and CYP2C11 being markedly higher in AKI rats than in CON rats, the AUC of tofacitinib was significantly higher in AKI rats (Bae et al., 2020), a finding that may be attributed to the significantly lower CL_int for tofacitinib metabolism in the intestinal microsomes of AKI rats.

Intestinal CYP3A activity and/or expression has been found to differ in animal models of renal failure induced by various treatments, such as cisplatin, gentamicin, glycerol, bilateral ligation, or nephrectomy (Okabe et al., 2003; Bae et al., 2020). Therefore, the increased tofacitinib AUC in AKI rats may be associated with a reduction in its active secretion caused by cisplatin-induced tubular necrosis, increasing tofacitinib accumulation. This resulted in the Ae_{0-24 h} of orally administered tofacitinib being significantly lower and its CL_R being significantly slower in AKI rats than in CON rats, with AUCs being significantly greater in the former. The finding that approximately 21.3% of orally administered tofacitinib was metabolized in the livers of CON rats (Lee and Kim, 2019) suggested that the hepatic first-pass effect was lower in AKI rats than in CON rats following absorption into the portal vein. This decrease may have been due to the lower hepatic activity and protein expression of CYP3A1/2 and CYP2C11 in AKI rats, further contributing to the increased AUC of tofacitinib after its oral administration.

The AUC of tofacitinib after its oral administration was found to be significantly lower in AKI-LGN rats than in AKI rats. This reduction may have been associated with the restoration of enzyme activity in the intestine and liver resulting from pretreatment with LGN. Iridoid glycosides are main constituents of Rehmanniae radix (Lee et al., 2011) that have been found to protect against cisplatin-induced renal toxicity by, for example, amelioration of renal tubular lesions, reduced apoptosis, and accelerated tubular cell regeneration (Teng et al., 2015). These protective effects have been shown to involve the modulation of expression in the kidneys of pro-inflammatory factors such as tumor necrosis factor-alpha (TNF-α) mRNA, interleukin-1 beta (IL-1β) mRNA, and cyclooxygenase-2 (COX-2) protein (Teng et al., 2015). Furthermore, iridoid glycosides have been shown to reduce platinum accumulation in the kidney by reducing the expression of copper transporter 1 and organic cation transporter 2 (Teng et al., 2015). Because LGN is a major iridoid glycoside, it may protect the kidneys by similar mechanisms.

Consistent with the present study in rat models of AKI, the expression of CYP3A proteins was found to be lower in patients with end-stage renal failure than in healthy control subjects (Dowling et al., 2003). The plasma concentration of tofacitinib was significantly higher in patients with severe renal impairment, with an AUC more than two-fold higher, than in healthy control subjects. These findings suggest that tofacitinib dosage should be lower in patients with than without severe renal failure (Krishnaswami et al., 2014). Although the renal excretion of tofacitinib is greater in humans (approximately 30%, Dowty et al., 2014) than in rats (12.4%), complicating the ability to draw clear clinical conclusions from rat results, the higher AUC in rats with than without renal failure was likely due to the slower rates of hepatic and intestinal metabolism of tofacitinib and its reduced urinary excretion in rats with renal failure.

The administration of LGN to rats was found to restore CYP3A and CYP2C activities by attenuating nephrotoxicity and promoting the regeneration of renal tubules, thereby reducing cisplatin accumulation. These findings could have implications for interactions between drugs and natural components in clinical practice, especially when treating patients with renal failure with the combination of a drug and a natural extract containing LGN or another iridoid glycoside. The co-administration of an iridoid glycoside may alter the plasma concentrations of drugs by affecting both renal and nonrenal elimination pathways.

This work was partly supported by the Basic Science Research Program through a grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT) (NRF-2021R1A2C1011142) and by the GRRC program of Gyeonggi province (GRRCAjou2023-B04), Korea.

The authors declare no conflicts of interest.