Chemicals and reagents
Kang-Ai injection samples (lot numbers of 03180805, 03181004, 01180908, 01180909, 02190102, 02190103) were manufactured by Changbaishan Pharmaceutical Co., Ltd. (Jilin, China) with China FDA drug ratification number of GuoYaoZhunZiZ-20026868. Purified matrine, oxymatrine, oxysophocarpine, isoastragaloside IV, and astragaloside III, IV, V, VI were all purchased Guangzhou Fans Biotechnology Co., Ltd (Guangzhou, China). Reference standards of ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2, Rg6, Rh1, Rh4, Rk3, F1, F2, F3, F4, and notoginsenoside R1, R2 were all obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). The purity of these compounds was over 98.0%.
Magnesium chloride (MgCl2), D-saccharic-1, 4-lactone monohydrate, alamethicin, nicotinamide adenine dinucleotide phosphate (NADPH), uridine 5'-diphospho-glucuronosyltransferase (UDPGA) were all obtained from Sigma-Aldrich (St. Louis, MO, USA). 4-hydroxytolbutamide, 4-hydroxymephenytoin, 4-methylumbelliferone, 6α-hydroxy-paclitaxel, 6-hydroxychlorzoxazone, 7-hydroxycoumarin, β-estradiol, bupropion, coumarin, chlorzoxazone, hydroxybupropion, mephenytoin, nifedipine, oxidized nifedipine, phenacetin, paracetamol, paclitaxel, propofol and tolbutamide were all acquired from Aladdin Chemicals (Shanghai, China). 4-MU-glucuronide, β-estradiol-3-O-glucuronide and propofol-O-glucuronide were all obtained from Toronto Research Chemicals (North York, ON, Canada). Recombinant CYP enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4) and UGT isozymes (UGT1A1, 1A9, 2B7) were all provided from Corning Biosciences (Corning, NY, USA).
LC-MS-grade water, methanol and acetonitrile were purchased from Fisher Scientific (Fair Lawn, New Jersey, USA). LC-MS grade formic acid was obtained from Sigma-Aldrich (St. Louis, USA). Other reagents were of analytical or higher grade.
Animals
Specific pathogen free (SPF) grade male Sprague-Dawley rats (250 ± 20) g were provided by the Experimental Animal Center of Zhengzhou University (Zhengzhou, China). These rats were kept in a designated animal room at constant temperature (22 ± 2) ºC and humidity (50 ± 20) % with 12 h of light/dark per day and free access to water and food. All protocols of animal experiments were approved in accordance with the Regulations of Experimental Animal Administration issued by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Prior to the experiments, the rats were fasted for 12 h but with free access to water. After study use, the rats were euthanized with CO2 gas.
Sample preparation
Each plasma sample (50 μL) was transferred to a 1.5-mL polypropylene tube containing 50 μL of internal standard (IS) solution (including 20 nM olanzapine-d3 and 1000 nM mycophenolic acid-d3) and 50 μL of methanol. The mixture was vortex mixed for 30 s, and then 150 μL methanol was precipitated. The tubes were vortex mixed vigorously for 1 min and centrifuged at 13800 g for 10 min at 4 ºC. Finally, 4 μL aliquots of the supernatant was injected into the UHPLC/TQD-MS system. Similarly, rat urine (50 μL) and bile (50 μL) samples were precipitated with methanol at a volumetric sample-to-methanol ratio of 1:3; after centrifugation, the resulting supernatants were applied for analysis. All the biological samples were stored at -80 °C.
Preparation of standard solutions and quality control samples
An appropriate amount of each authentic standard was separately dissolved with 60% methanol-water in a 10 mL volumetric flask to prepare the standard solution. Furthermore, an appropriate volume of each standard solution was transferred into a 10 mL volumetric flask to the desired concentration as the stock solution. The mixed IS solution contained olanzapine-d3 and mycophenolic acid-d3 at final concentration of 20 nM and 1000 nM, respectively. All the solutions were stored at 4 ºC.
Calibration standard samples were prepared by spiking 50 μL blank plasma (or other biological samples) with 50 μL working solutions and 50 μL IS solution. Therefore, the plasma concentration ranges were 10-1000 nM for matrine (A1), 10-1000 nM for oxysophocarpine (A2), 40-4000 nM for oxymatrine (A3), 40-4000 nM for astragaloside IV (B2), 10-1000 nM for astragaloside III (B3), 10-1000 nM for ginsenoside Rh1 (C1), 10-1000 nM for notoginsenoside R2 (C3), 10-1000 nM for ginsenoside Rg2 (C5), 20-2000 nM for ginsenoside Rg1 (C6), 10-1000 nM for ginsenoside Rf (C7), 10-1000 nM for notoginsenoside R1 (C8), 20-2000 nM for ginsenoside Re (C9), 10-1000 nM for ginsenoside Rd (D2), 10-1000 nM for ginsenoside Rc (D3), 10-1000 nM for ginsenoside Rb2 (D4), 10-1000 nM for ginsenoside Rb1 (D5), respectively. Quality control (QC) samples were independently prepared in the same way at 2, 10, 40 times of LLOQ of each analyte. The calibration standard and QC samples were processed on each analysis day with the same procedures for plasma samples.
Chromatographic and mass spectrometric conditions
Ultra-high-performance liquid chromatography (UHPLC) analysis was performed on a Waters Acquity UHPLC I-Class system (Manchester, UK) for the quantitative analysis of Kang-Ai injection. Chromatographic separation was achieved on an BEH C18 column (2.1 × 50 mm, 1.7 μm) with water (A) and acetonitrile (B) (both including 0.1% formic acid) at a flow rate of 0.45 mL/min with column temperature of 35 ºC. The gradient flow profile was optimized as follows: 10% B from 0 to 0.5 min, 10-20% B from 0.5 to 1.5 min, maintaining 20% B from 1.5 to 4.5 min, 20-30% B from 4.5 to 5.5 min, 30-35% B from 5.5 to 7.5 min, 35-90% B from 7.5 to 9.5 min, keeping 90% B from 9.5 to 10.5 min, 90-10% B from 10.5 to 11.0 min, and maintaining 10% B from 11 to 12 min.
UHPLC system was coupled to a triple quadrupole mass spectrometer (Waters Xevo TQD, Waters, Manchester, UK). The detailed mass spectrometers were adjusted as follows: capillary voltage, 3.5 kV (ESI+) or 1.5 kV (ESI-); cone voltage, 50 V (ESI+) or 50 V (ESI-); source temperature, 350 ºC; desolvation gas flow, 650 L/h; The mass spectrometer was performed in the multiple reaction monitoring mode (MRM) using both positive and negative ionization. The quantitative parameters in MRM modes were same as our previous study. All experimental data were collected and processed using a Quanlynx software in Masslynx 4.1 platform.
Method validation
The developed UHPLC-MS/MS method was validated according to the Guidance for Industry: bioanalytical method validation from the US FDA for specificity, linearity, LLOQ, extraction recovery, matrix effects, precision, accuracy, stability and dilution integrity [23]. The assay validation of each analyte in rat biological samples including plasma, urine and bile were all performed based on the standard specification above.
Specificity was determined by comparing the chromatograms of blank samples (from six individual rats), with dosed samples after an intravenous bolus of Kang-Ai injection, and blank samples spiked with each analyte at LLOQ and IS. The calibration curves were constructed by the peak area ratios of the analytes to IS (Y), versus respective sample concentrations (X) applying a weighted (1/x2) least squares linear regression analysis. LLOQ was determined based on the lowest concentrations for which acceptable linearity, accuracy and precision were demonstrated. The accuracies (the relative error, RE) and inter/intra-day precisions (the relative standard deviation, RSD) of the assay were evaluated by determining six replicates of QC samples (at low, middle and high concentrations) on three consecutive days.
Extraction recoveries (ER) and matrix effects (ME) were evaluated by a published experimental protocol [24]. The response of extracted QC sample was defined as A1. In addition, A2 referred to the corresponding standard solutions added into the post-extracted supernatant from biological matrix, while A3 corresponded to the responses of each analyte at three QC levels. The ER and ME values were calculated as follows: ER% = A1/A2 × 100%, and ME% = A2/A3 × 100%.
Stability tests were investigated at three QC levels under different conditions, including short-term stability (8 h exposure at room temperature), autosampler storage stability of the methanol-treated samples (at 8 °C for 18 h), freeze/thaw stability (three -80 °C ↔ 23 °C cycles), long-term stability (14 days storage at -80 °C). The dilution integrity was investigated by analyzing six replicate samples with each analyte, and each analyte were diluted 10-fold with blank rat matrix. Furthermore, the diluted samples were analyzed using freshly prepared calibration curve to calculate RE and RSD, which should be no more than ±15% as criterion.
In vivo rat studies
The rats in this study all received a single 30-minute intravenous infusion of Kang-Ai injection via the tail veins using TYD01-02 infusion pumps (Lead Fluid, Baoding, China). The dose was derived from the label daily dose of Kang-Ai injection (60 mL/person, once daily) according to dose normalization by body surface area [25]. For plasma pharmacokinetic studies, the rats were randomly received a single intravenous 30 min-bolus dose at 6 mL/kg. Blood samples (100 μL) were collected from the rats’ external jugular veins into heparinized polypropylene tubes at 5, 15, 30, 45 min, and 1, 1.5, 2, 3, 4, 6, 8, 12 h). After gently shaking for 10 s, the blood samples were centrifuged at 13000 g for 10 min to yield plasma samples, which were kept frozen at -80 °C until analysis.
For urine sampling, the rats were housed singly in metabolic cages. And then, the urine samples were collected at 0-4, 4-8 and 8-12 h after starting infusion and were weighed. Likewise, bile samples were also collected at 0-3, 3-6, 6-9 and 9-12 h after starting infusion and were weighed. The urine and bile collection tubes were all frozen at -80 °C.
Protein binding assay
Binding of circulating alkaloids and saponins to rat plasma was measured by equilibrium dialysis using Spectra/Por 2 dialysis membranes (molecular weight cutoff, 12-14 kDa; Rancho Dominguez, CA, USA) as described [26]. After equilibrating for 24 h at 55 rpm and 37 °C, the dialysate and the plasma were sampled for analysis. In brief, the concentrations of each analyte in dialysate and rat plasma samples were determined for analysis after equilibrating at 55 rpm and 37 °C for 24 h. The concentrations of tested matrine, oxysophocarpine and oxymatrine were 2 μmol/L, while those of astragaloside IV, III, and ginsenosides Rh1, Rg2, Rg1, Rf, Re, Rd, Rc, Rb1, Rb2, notoginsenoside R1, R2, were 4 μmol/L. The unbound fraction (fu-plasma) in rat plasma was calculated as follow: fu-plasma = Cd/Cp × 100%, where the Cd and Cp values were the concentrations of each analyte in the dialysate sample and in the post-dialysis plasma sample, respectively.
Incubation systems
Phase I incubation system (100 μL) contained Tris-HCl buffer (50 mM, pH = 7.4), MgCl2 (5 mM), each CYP isozyme, specific substrates, circulating compounds and NADPH (1 mM) as prescribed previously [18]. After incubation (37 °C, 60 min), the reaction was terminated by ice-cold acetonitrile (100 μL), following by centrifugation at 13800 g for 10 min. The supernatant (8 μL) was acquired for ultra-high-performance liquid chromatography (UHPLC) system (Waters, Manchester, UK) analysis.
For glucuronidation assays, the incubation system (100 μL) contained Tris-HCl buffer (50 mM, pH = 7.4), alamethicin (22 μg/mL), D-saccharic-1, 4-lactone (4.4 mM), each UGT enzyme, specific substrates, circulating compounds and UDPGA (3.5 mM) as described recently [18]. The reaction was terminated by ice-cold acetonitrile (100 μL) after incubation at 37 °C for 60 min. After centrifugation (13800 g, 10 min), the supernatant (8 μL) was obtained for determination by UHPLC system.
Analysis of inhibitory effects
In this study, phenacetin (100 μM), coumarin (100 μM), bupropion (100 μM), paclitaxel (60 μM), tolbutamide (200 μM), mephenytoin (100 μM), chlorzoxazone (200 μM) and nifedipine (40 μM) have been used as the probe substrates for CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4, respectively [27]. The substrates were incubated with each CYP isozyme at different protein concentrations (0.05 mg/mL for CYP1A2 and 3A4; 0.1 mg/mL for CYP2A6, 2B6, 2C8, 2C9, 2C19 and 2E1) in the absence (control) and presence of different herbal compounds (1, 10, 100 and 1000 μM for matrine and oxymatrine; 0.1, 1, 10 and 100 μM for astragaloside and ginsenoside).
Similarly, β-estradiol (60 μM), propofol (40 μM) and 4-MU (350 μM) were typically used as the substrates for UGT1A1, 1A9 and 2B7, respectively [28]. The protein concentrations for UGT1A1, 1A9, and 2B7 were 0.125, 0.05, and 0.05 mg/mL, respectively. The concentrations were 1, 10, 100 and 1000 μM for matrine and oxymatrine, and 0.1, 1, 10 and 100 μM for astragaloside and ginsenoside based on the detailed plasma concentration after intravenous Kang-Ai injection.
The half-inhibition concentration (IC50) values were determined by non-linear regression analysis. The inhibitory effects towards each CYP or UGT enzyme were divided into four categories as follows, potent (IC50 < 1 μM), moderate (1 μM < IC50 < 10 μM), weak (10 μM < IC50 < 100 μM), or no inhibition (IC50 > 100 μM) [18]. The inhibition mechanism towards corresponding CYP and UGT isoforms were further explored.
Inhibition kinetic analysis
The inhibition constant (Ki) values were obtained by multiple concentrations of substrates in the absence or presence of multiple concentrations of herbal compounds as described previously [18, 27, 28]. Competitive inhibition, noncompetitive inhibition, and mixed-type inhibition models were used to determine the Ki values by nonlinear regression analysis using the equation (1), equation (2) and equation (3), respectively. The model with the smallest Akaike information criterion (AIC) and Schwartz information criterion (SC) values was considered as the best model, following the obtained appropriate Ki values.
The detailed parameters for three equations were as follow. V is the velocity of the reaction. The [S] and [I] are the concentrations of substrate and herbal compounds, respectively. Ki is the constant describing the affinity between herbal compounds and the enzyme. Km is the substrate concentration at half of the maximum velocity (Vmax) of the reaction. The αKi describes the affinity of herbal compounds to the complex of enzyme and substrate; When α is very large (α >> 1), the binding of inhibitor would prevent the binding of substrate, and the mixed inhibition model becomes identical to competitive inhibition.
Data processing
Pharmacokinetic parameters were estimated by non-compartmental analysis using WinNonlin 6.3 software (Pharsight, NC, US). Data are presented as the mean ± SD (n = 3). The area under the concentration-time curve up to the last measured point in time (AUC0-t) was calculated using the trapezoidal rule. The total plasma clearance (CLtot, p) was calculated by dividing the compound dose by the AUC0-∞, and the distribution volume at steady state (VSS) was estimated by multiplying the CLtot, p by the mean residence time (MRT). The renal excretory clearance (CLR) and hepatobiliary (CLB) was determined by dividing the cumulative amount excreted into urine (Cum.Ae-U) and bile (Cum.Ae-B) by the plasma AUC0-∞, respectively. The fractions of dose excreted into urine (fe-U) and the fractions of dose excreted into bile (fe-B) were established using the relationship Cum.Ae-U/Dose and Cum.Ae-B/Dose, respectively. Mean differences between treatment and control groups were analyzed by two-tailed unpaired Student’s t test by Graphpad Prism V5 software (SanDiego, CA). The level of significance was set at p < 0.05 (∗), p < 0.01 (∗∗) or p < 0.001 (∗∗∗).