A reference standard, irinotecan hydrochloride, was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). CPT (purity ≥ 98%), which was used as the ISTD, was purchased from Shanghai Yuanye Biological Technology Co. Ltd. (Shanghai, China). SN-38 and SN-38 glucuronide (purity ≥ 98%) were purchased from Toronto Research Chemicals Inc. (Toronto, Canada). Irinotecan hydrochloride for injection was obtained from Hengrui Medicine Co. Ltd. (Jiangsu, China). Sterilized saline for injection was purchased from Shijiazhuang No. 4 Pharmaceutical Co. Ltd. (Hebei, China). D-saccharic acid 1, 4-lactone, alamethicin, and uridine 5′-diphosphoglucuronic acid (UDPGA) triammonium salt were purchased from Sigma-Aldrich (St. Louis, MO, USA).

HPLC-grade acetonitrile and methanol were obtained from Honeywell Burdick & Jackson Company (Mexico City, Mexico). Formic acid (HPLC grade) was purchased from ROE Scientific Inc. (Beijing, China). Deionized water for the HPLC analysis was prepared using a Milli-Q water purification system (Milford, MA, USA). All of the other reagents were of analytical grade.

P. ternata, G. uralensis, C. chinensis, Z. jujuba, Z. officinale, S. baicalensis, C. pilosula, and Z. recens were purchased from the Huamiao Traditional Chinese Medicine Engineering Technology Development Center (Beijing, China). These samples were identified as the dried rhizome of P. ternata (Thunb.) Breit (processed with alumen as adjuvant material), dried root and rhizome of G. uralensis Fisch., dried rhizome of C. chinensis Franch, dried ripe fruit of Z. jujuba Mill., dried rhizome of Z. officinale Rosc., dried root of S. baicalensis Georgi, dried root of C. pilosula (Franch.) Nannf. and rhizome of Z. recens, respectively. A mixture of the eight crude herbal drugs was prepared in a 9:9:3:12:3:9:9:12 ratio on a dry weight basis according to the original record in Shang Han Lun, and the mixture was immersed in distilled water for 30 min. Subsequently, it was added to a 10-fold volume of water and then decocted twice by boiling for 1 h. The decoctions were filtered to remove the herbal residue, combined, and concentrated to generate an extract.

Twenty-four SD rats were randomly divided into four groups with six rats in each group. SXD extract (10 g/kg body weight through oral administration, 10 mL/kg) was given to the rats in groups 1 and 2 twice per day for 5 days, starting from day 0 to 1 h before CPT-11 administration. The animals in groups 3 and 4 received a blank vehicle without SXD extract in a similar fashion. At day 5, CPT-11 diluted with saline was administered intravenously to the rats via their tail veins at a dose of 20 mg/kg (4 mL/kg).

For the pharmacokinetics study, blood samples from groups 1 and 3 were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 10, and 24 h post-dosing from the retro-orbital plexus into heparinized tubes. Plasma was obtained by centrifugation at 3,000 rpm for 10 min. The liver, duodenum, jejunum, ileum, cecum, colon and rectum were removed immediately. The livers were washed with cold physiological saline to remove the blood. The intestinal contents of the corresponding segments were extruded into polypropylene tubes. The intestinal segments were then opened longitudinally, gently rinsed with saline to remove the residual contents and blot-dried with neutral filter paper. All of the obtained samples were stored at −80°C until analysis.

To further measure the concentrations in the liver and intestinal tissues, the animals in groups 2 and 4 were killed by cervical dislocation at 8 h after CPT-11 i.v. administration. Their livers and intestinal segments were removed immediately. The samples were treated in the same way as those obtained at 24 h.

Another 12 SD rats were randomly divided into two groups—an SXD-pre-treated group and a control group—with six rats in each group. SXD extract (10 g/kg, 10 mL/kg) was given to the rats in the pre-treated group twice per day for 5 days. The animals in the control groups received the corresponding volume of blank vehicle. All of the rats were anesthetized with 10% (w/v) chloral hydrate solution (3 mL/kg, i.p.) and placed under a thermo-controlled surgery platform to maintain their body temperatures. Before the CPT-11 administration and bile sample collection, the rats received surgery for the bile cannulation experiments, as described previously (Bansal et al., 2009). Bile was collected at intervals of 0–1, 1–2, 2–3, 3–4, 4–5, 5–6, 6–7, and 7–8 h after CPT-11 i.v. administration at a dose of 20 mg/kg. The samples were stored at −80°C until analysis.

The liver and jejunum S9 fractions were prepared from rats with or without SXD treatment for 5 days using a method adopted from the literature with minor modifications (Zhu et al., 2010). The livers were perfused with ice-cold 10 mM sodium phosphate buffer (pH 7.4) and chopped into tiny pieces. The jejunums were removed, washed with ice-cold 10 mM sodium phosphate buffer (pH 7.4), blot-dried with neutral filter paper, opened longitudinally and scraped with a glass slide to collect the epithelium at 4°C. The chopped livers and scraped epithelium were homogenized with three-fold volumes of ice-cold homogenization buffer [50 mM potassium phosphate buffer, pH 7.4, 250 mM sucrose, and 1 mM ethylenediaminetetraacetic acid (EDTA)] using an S10 electric tissue homogenizer and centrifuged at 9,357 rpm for 15 min at 4°C. The fat layer was discarded carefully. The supernatant was collected and stored at −80°C until use. The protein concentration was determined using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, Rockford, IL, USA).

For the CES activity assay, incubations of liver or jejunum S9 (protein content: 2 mg/mL) with CPT-11 at a final concentration of 9.47 μM were performed in 50 mM potassium phosphate buffer (pH 7.4) at 37°C in Eppendorf tubes for 2 h.

For the glucuronidation activity assay, SN-38 (38.23 μM), liver or jejunum S9 (final concentration: 2 mg/mL), MgCl₂ (0.88 mM), alamethicin (0.022 mg/mL), and D-saccharic acid 1,4-lactone (4 mM) were mixed in 50 mM potassium phosphate buffer (pH 7.4). The reaction was initiated by adding UDPGA triammonium salt (3.5 mM) and allowed to proceed at 37°C for 2 h.

The hydrolysis and glucuronidation reactions were quenched by adding 400 μL of ice-cold acetonitrile-methanol (1:1, v/v) containing 50 ng/mL CPT (ISTD). After being centrifuged at 12,000 rpm for 10 min, the supernatant was dried and processed further as described for the plasma samples. A 10 μL volume of supernatant was injected into the LC/MS/MS system for SN-38 and SN-38G analyses.

A 100 μL volume of plasma was added with 10 μL of 10% acetic acid before protein precipitation with organic solvent. The liver, intestine and intestinal contents were accurately weighed and homogenized in 3–5 volumes of ice-cold 5% perchloric acid according to the concentration using a S10 electric tissue homogenizer (Ningbo, China). The homogenization was performed in an ice bath. The homogenates were centrifuged at 10,000 rpm for 10 min at 4°C.

A 50 μL volume of ISTD solution containing CPT (200 ng/mL) was evaporated to dryness by a gentle stream of nitrogen in an Eppendorf tube. The plasma, liver, intestine, and intestinal content homogenate samples (100 μL) were added into the abovementioned tubes and then extracted with 500 μL of acetonitrile. The sample was vortexed for 1.0 min and centrifuged (12,000 rpm, 4°C, 10 min). The supernatant was separated and dried under a gentle flow of nitrogen at room temperature. The residue was dissolved with 200 μL of 0.1% aqueous formic acid and loaded onto a pre-conditioned Agela Cleanert® PEP SPE column (Tianjin, China). The SPE column was then eluted with 3 mL of 0.1% aqueous formic acid and 2 mL of methanol in succession. The methanol fraction was collected and dried under a gentle flow of nitrogen at room temperature. The residue was reconstituted with 200 μL of 60% methanol in water and centrifuged again at 12,000 rpm for 10 min. The supernatant was injected into the UHPLC-MS/MS system for analysis.

A 10 μL volume of bile sample was transferred to an Eppendorf tube containing 50 μL of 2.0 μg/mL CPT and spiked with 50 μL of 0.1% acetic acid in acetonitrile. The sample was thoroughly vortexed for 1.0 min and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was separated and dried under a gentle flow of nitrogen. The residue was reconstituted in 2,000 μL of 60% methanol in water before analysis.

The livers and jejunum tissue were homogenized in ice-cold radio immunoprecipitation assay (RIPA) buffer [50 mM Tris HCl, 1 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1.0% Nonidet P-40, and phenylmethane sulfonyl fluoride (PMSF), pH 8.0]. The homogenate was centrifuged at 12,000 rpm for 20 min at 4°C. The supernatants were collected for Western blot analysis, which was performed as described previously (Kong et al., 2015). The antibodies used in the present study included anti-Mrp-2 (1:500; sc-5770, Santa Cruz; California, USA), anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (1:20,000; YM3029, Immunoway; Texas, USA), anti-UGT1A1 (1:4,000; ab194697, Abcam; Cambs, UK), anti-P glycoprotein (1:1,000; ab170904, Abcam; Cambs, UK), anti-CES2 (1:1,000; DF6433, Affinity; Cambs, UK), and anti-Bcrp (1:200; sc-69988, Santa Cruz; California, USA). The gray intensities of the bands were quantified using ImageJ software (National Institute of Health, MD, USA).

The pharmacokinetic parameters, including area under the concentration time curve to the respective sampling point (AUC_0−t), area under the concentration time curve to infinity (AUC_{0 → ∞}), initial plasma concentration (C₀), maximum plasma concentration (C_max), mean residence time (MRT), clearance rate (CL_obs), volume of distribution (V_d), and half-life (t_1/2), were evaluated using Phoenix WinNonlin 6.0 (Certara, USA). Statistical comparisons were performed using Student's unpaired t-tests (SPSS 16.0 software). The data were expressed as the means ± standard error of the mean (SEM). The statistical significance was considered at p < 0.05.

ESI was performed in positive ionization mode for CPT-11, SN-38, and SN-38G. All of the analyzed components were quantified in multiple reaction monitoring (MRM) mode. CPT was selected as the internal standard (ISTD) because it showed a strong MS response in positive ion mode and exhibited chromatographic behavior similar to those of CPT-11, SN-38, and SN-38G. The precursor and product ion pairs for MRM detection and their corresponding declustering potential (DP), collision energy (CE), entrance potential (EP), and collision cell exit potential (CXP) values were optimized to obtain the maximum sensitivity and response. The results are presented in Figure 1 and Table 1. A gradient elution with 0.1% formic acid in water and acetonitrile was adopted for the LC to achieve satisfactory sensitivity and good peak shapes (Figure 2).

The method was validated for its selectivity, calibration curve, sensitivity, precision, accuracy, recovery, matrix effect, dilution integrity, and stability (as shown in Supplementary Information), in compliance with the guidelines on bioanalytical method validation from the European Medicines Agency (European Medicine Agency, 2011) and the US Food and Drug Administration (FDA) [Center for Drug Evaluation and Research (CDER) and US Food and Drug Administration, 2001].The data obtained from method validation are shown in Figure S1 and Tables S1–S8. All of the results were acceptable, indicating that the method can reliably determine the CPT-11, SN-38, and SN-38G concentrations in the tested matrixes.

The developed method was successfully applied to a pharmacokinetics study of CPT-11 in SD rats after the i.v. administration of CPT-11. The mean plasma concentration-time profiles of CPT-11 and its metabolites (SN-38 and SN-38G) are shown in Figure 3. The primary pharmacokinetics parameters of CPT-11, SN-38, and SN-38G are listed in Table 2. In the SXD-pre-treated rats, the corresponding AUC and C₀ of CPT-11 were significantly higher than those in the control group (p < 0.05 and p < 0.001, respectively), whereas the AUC and C_max of SN-38 were clearly reduced (p < 0.01 and p < 0.001, respectively). Moreover, the pretreatment of rats with SXD significantly decreased the elimination CL of CPT-11. The V_d of CPT-11 also decreased, although this difference did not reach statistical significance. SN-38G exhibited a significant (p < 0.01) increase in its C_max after pretreatment with SXD. However, no significant variations were found for MRT and t_(1/2) between the control group and the SXD-pre-treated group. The decreased AUC ratio of SN-38 to CPT-11 in the SXD group may suggest the inhibition of the metabolic transformation of CPT-11 to SN-38, while the significantly increased AUC ratio of SN-38G to SN-38 indicated that the glucuronidation of SN-38 was accelerated (Paoluzzi et al., 2004; Jong et al., 2007).

Figure 4 shows the cumulative biliary excretions of CPT-11, SN-38, and SN-38G for 8 h following i.v. administration in control and SXD-pre-treated rats. In the SXD-pre-treated group, the cumulative biliary excretions of CPT-11 and SN-38 decreased significantly from 3 to 8 h, whereas that of SN-38G decreased from 5 to 8 h. The pretreatment of rats with SXD decreased the cumulative biliary excretion of CPT-11 over 8 h from 1873.77 ± 28.76 to 1580.39 ± 54.93 μg (p < 0.01). Similarly, the pretreatment decreased the cumulative biliary excretions of SN-38 and SN-38G significantly from 130.44 ± 7.99 to 95.67 ± 6.23 (p < 0.01) and from 192.54 ± 12.97 to 152.36 ± 4.67 μg (p < 0.01), respectively. The biliary excretions of CPT-11, SN-38, and SN-38G over 8 h, expressed as percentages of the CPT-11 dose, are shown in Figure 4. Following the i.v. administration of CPT-11, approximately 32% of the CPT-11 dose was excreted into bile over 8 h, whereas this value was reduced to 28% in the SXD-pre-treated group (p < 0.05). The biliary excretions of both SN-38 and SN-38G were also reduced by SXD pretreatment.

The CPT-11, SN-38, and SN-38G concentrations in the duodenal, jejunal, ileal, cecal, colonic, and rectal contents from the SXD-pre-treated and control groups are shown in Figure 5. The CPT-11 and SN-38 concentrations in the large intestinal (cecum, colon, and rectum) contents were higher than those in the contents from the small intestinal (duodenum, jejunum, and ileum) lumens. However, SN-38G was hardly detected in the large intestinal contents of the two groups. Compared with the control group, SXD pretreatment decreased the concentrations of CPT-11 and SN-38 in all of the intestinal contents to some extent. The CPT-11 concentrations in the jejunal and ileal contents of the SXD-pre-treated rats were only 34.21 and 23.76%, respectively, of those in the control group. The concentrations of SN-38 in the ileal contents and SN-38G in the jejunal contents were reduced to 30.55 and 37.92%, respectively. Notably, the SN-38G concentration in the ileal content increased.

Figure 6 shows the tissue levels of CPT-11 and its metabolites in the liver, duodenum, jejunum, ileum, cecum, colon, and rectum at 8 and 24 h after the i.v. administration of CPT-11 to the control and SXD-pre-treated rats. Regarding the concentrations of CPT-11 and its metabolites in the liver, the effects of SXD on the hepatic concentrations of SN-38G were insignificant, whereas SXD increased the hepatic CPT-11 concentration to 143.42% and reduced the SN-38 concentration to 27.66% relative to the values in the control group at 8 h after i.v. CPT-11 administration. Although, the SN-38 concentration in the liver remained lower than that in the control group (p < 0.01) at 24 h, no differences were found in the hepatic distributions of CPT-11 and SN-38G between the two groups.

In the control rats, the concentrations of CPT-11 and its metabolite SN-38 in the cecum tissue were higher than those in other intestinal segments. At 8 h after CPT-11 administration, SXD significantly decreased the concentrations of CPT-11 and SN-38 in the cecum to 31.26 and 22.36% (p < 0.05), respectively. Although, SXD reduced the concentrations of CPT-11 and SN-38 in the ileum and colon, these differences were not significant. The SN-38G concentration in the small intestinal tissue was slightly increased. At 24 h after i.v. administration, significant reductions in the concentrations of CPT-11, SN-38, and SN-38G were observed in the duodenum. Compared with those in the control rats, the SN-38 concentrations in the jejunum and ileum tissues and that of SN-38G in the jejunum from SXD-pre-treated rats were decreased to 43.14, 61.87, and 40.76 (p < 0.05), respectively. SXD also decreased the CPT-11 concentrations in the jejunum and ileum tissues but increased the tissue accumulation of SN-38G in the ileum; however, these differences were not significant.

The production of SN-38 from CPT-11 over a 2-h incubation period with the hepatic and jejunal S9 fractions from control and SXD-pre-treated rats is shown in Figure 7. The SN-38 level in the hepatic S9 fraction of SXD-pre-treated rats was only 84.81% of that of the control rats (p < 0.05), which suggested that the CES-mediated hydrolysis of CPT-11 was lower in the liver S9 fractions from SXD-pre-treated rats. This finding implied that the conversion from CPT-11 to SN-38 in the liver was hindered by SXD. Similarly, the higher SN-38G concentration produced from SN-38 in the jejunal S9 fraction of SXD-pre-treated rats indicated that the SXD pre-treated rats had a greater SN-38 glucuronidation capacity than the control rats. Differences were observed in the CPT-11 hydrolysis in the jejunal S9 fraction and SN-38 glucuronidation in the hepatic S9 fraction between the two groups, although these differences were not significant.

Potential changes in the expression of CES2, UGT1A1, Mrp-2, Bcrp, and P-gp in rat liver and UGT1A1 in jejunal segment from the control and SXD-pre-treated groups were explored by Western blot analyses (Figure 8, Figures S2–S7). The expression of Mrp-2, P-gp, and CES2 in the liver was significantly decreased in the SXD-pre-treated rats (p < 0.01, p < 0.001), whereas the expression of UGT1A1 in the liver and jejunum was increased (p < 0.05, p < 0.01). No significant difference in Bcrp protein expression was observed.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.