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Multiple circulating alkaloids and saponins from intravenous Kang-Ai injection inhibit human cytochrome P450 and UDP-glucuronosyltransferase isozymes: potential drug–drug interactions
Chinese Medicine volume 15, Article number: 69 (2020)
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Kang-Ai injection is widely used as an adjuvant therapy drug for many cancers, leukopenia, and chronic hepatitis B. Circulating alkaloids and saponins are believed to be responsible for therapeutic effects. However, their pharmacokinetics (PK) and excretion in vivo and the risk of drug–drug interactions (DDI) through inhibiting human cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes remain unclear.
PK and excretion of circulating compounds were investigated in rats using a validated ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC–MS) method. Further, the inhibitory effects of nine major compounds against eleven CYP and UGT isozymes were assayed using well-accepted specific substrate for each enzyme.
After dosing, 9 alkaloids were found with Cmax and t1/2 values of 0.17–422.70 μmol/L and 1.78–4.33 h, respectively. Additionally, 28 saponins exhibited considerable systemic exposure with t1/2 values of 0.63–7.22 h, whereas other trace saponins could be negligible or undetected. Besides, over 90% of alkaloids were excreted through hepatobiliary and renal excretion. Likewise, astragalosides and protopanaxatriol (PPT) type ginsenosides also involved in hepatobiliary and/or renal excretion. Protopanaxadiol (PPD) type ginsenosides were mainly excreted to urine. Furthermore, PPD-type ginsenosides were extensively bound (fu-plasma approximately 1%), whereas astragalosides and PPT-type ginsenosides displayed fu-plasma values of 12.35% and 60.23–87.36%, respectively. Moreover, matrine, oxymatrine, astragaloside IV, ginsenoside Rg1, ginsenoside Re, ginsenoside Rd, ginsenoside Rc, and ginsenoside Rb1 exhibited no inhibition or weak inhibition against several common CYP and UGT enzymes IC50 values between 8.81 and 92.21 μM. Through kinetic modeling, their inhibition mechanisms towards those CYP and UGT isozymes were explored with obtained Ki values. In vitro-in vivo extrapolation showed the inhibition of systemic clearance for CYP or UGT substrates seemed impossible due to [I]/Ki no more than 0.1.
We summarized the PK behaviors, excretion characteristics and protein binding rates of circulating alkaloids, astragalosides and ginsenosides after intravenous Kang-Ai injection. Furthermore, weak inhibition or no inhibition towards these CYP and UGT activities could not trigger harmful DDI when Kang-Ai injection is co-administered with clinical drugs primarily cleared by these CYP or UGT isozymes.
Herbal medicines are gaining popularity for the treatment of chronic and complex diseases as it works by hitting multiple molecular targets to modulate different signaling pathways [1,2,3]. Major advances have been made regarding their activities related their clinical indications, and more importantly in understanding their pharmacokinetics (PK) and excretion [3]. The PK profiles, just like the pharmacological activities, can be used as a "sieve" to assess the effectiveness of the individual compounds, and identify the chemical basis in an herbal injection [3,4,5]. Therefore, PK behaviors and excretion of circulating compounds should be first implemented in laboratory animals to reveal their systemic exposure. The corresponding PK parameters could show the ability of herbal compounds to be absorbed and transferred from the site of administration to the action targets through multiple biological barriers, and metabolic stability in vivo.
Kang-Ai injection is an herbal medicine approved in 2002 by the China Food and Drug Administration (China FDA). It consists of Astragali Radix (Huangqi), Ginseng Radix et Rhizoma (Renshen) and kushenin (Kushensu), and is indicated for many cancers, leukopenia and chronic hepatitis B [6,7,8]. Previously, we have finished the chemical identification and content determination of the main alkaloids (oxymatrine, matrine), astragalosides (astragaloside IV) and ginseng saponins (ginsenosides Rg1, Re) in Kang-Ai injection [9]. Despite the increasing understanding of Kang-Ai injection in chemical and pharmacological studies, the systemic exposure and excretion of its bioactive compounds after administration remain to be elucidated.
Previous PK researches of individual herb or its characteristic compounds have been carried out, which greatly facilitated the holistic PK properties of Kang-Ai injection. Oxymatrine, the most abundant herbal compound in Kang-Ai injection, occupies over 95.37% of total contents. When intravenously treated with oxymatrine at 30 mg/kg, the AUC0–12 h, Cmax, t1/2 and CL values in rat plasma were 16234.27 ng h/mL, 32847.57 ng/mL, 0.57 h and 3.10 L/h, respectively [10]. Besides, only about 19.9% of oxymatrine was reduced to matrine following intravenous infusion of 600 mg oxymatrine in healthy male volunteers [11]. In addition, systemic exposure of astragaloside IV appeared to be dose proportional over the dose ranged from 200 to 500 mL astragaloside IV (9 mg/100 mL) in healthy volunteers [12]. Furthermore, metabolism of astragaloside IV by intestinal microbiome was a crucial step in its excretion, rather than by hepatic and intestinal enzymes [13,14,15]. Moreover, ginsenosides Rg1, Re, and Rf were not extensively bound in plasma, and also were with substantially shorter t1/2 values and significantly larger systemic clearance (CLtot,p) than other-type ginsenosides [16]. Although several bioanalytical assays have been applied to the PK study of several circulating alkaloids and saponins, PK and excretion information about Kang-Ai injection is still limited. This also brings a huge obstacle in our understanding of the chemical basis responsible for the therapeutic effects of Kang-Ai injection.
In addition, drug–drug interaction (DDI) is significant safety concerns in clinical medication [17,18,19]. Inhibition or induction of human CYP or UGT isozymes could potentially adverse clinical DDI and result in metabolic disorders for endogenous components [20,21,22]. It would be more serious for the clinical drugs with narrow therapeutic indices, such as digoxin, warfarin, SN-38 and so on [17]. Similarly, UGT1A1 is the only one UGT isozyme in the conjugative detoxification of bilirubin [21]. Once UGT1A1 function is inhibited, there is a high risk of liver injury [21]. Considering the intravenous administration of Kang-Ai injection and abundant exposure of its circulating herbal compounds, it should be of particularly clinical significance to investigate the potential risks of DDI.
To this goal, this current report first identified the major circulating alkaloids, astragalosides, ginsenosides in rat samples after an intravenous 30 min-infusion of Kang-Ai injection at 6 mL/kg. In addition, the detailed PK behaviors, and exposure of these herbal substances in plasma were systemically carried out. Meanwhile, we also investigated the plasma binding rate assays of these circulating alkaloids and saponins. Furthermore, the excretion of these compounds in urine and bile were determined by dividing the cumulative amount excreted into urine (Cum.Ae-U) and bile (Cum.Ae-B) by their respect doses. Moreover, we explored the inhibitory effects and mechanism of nine major circulation compounds against several important CYP and UGT enzymes based on their widely recognized probe substrates. Taken together, the current PK and excretion information would facilitate the identification of active herbal compounds responsible for the therapeutic actions of Kang-Ai injection, and would also be helpful to inform its potential DDI in rational clinical use and achieve the optimal herbal therapy.
Materials and methods
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 ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2, Rg6, Rh1, Rh4, Rk3, F1, F2, F3, F4, and notoginsenosides 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 (4-MU), 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 CYP1A2 (Cat. No. 456203), CYP2A6 (Cat. No. 456254), CYP2B6 (Cat. No. 456255), CYP2C8 (Cat. No. 456252), CYP2C9 (Cat. No. 456258), CYP2C19 (Cat. No. 456259), CYP2E1 (Cat. No. 456206), CYP3A4 (Cat. No. 456202), and expressed UGT1A1 (Cat. No. 456411), UGT1A9 (Cat. No. 456419), UGT2B7 (Cat. No. 456427) 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.
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) °C 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 Ethics Review Committee for Animal Experimentation of Jinan University (Ethical Review NO. 20190301010). 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 13,800g for 10 min at 4 °C. 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 °C.
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 (lower limit of quantification) 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 °C. 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 °C; 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-min 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, notoginsenosides 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 13,800g 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 (13,800g, 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]. After optimization of incubation conditions (Additional file 1: Fig. S1), 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 mixture or herbal compounds (Kang-Ai injection and samples that Kang-Ai injection was diluted 10 times; 1, 10, and 100 μM for matrine and oxymatrine; 0.1, 1, 10 and 100 μM for astragalosides and ginsenosides).
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]. After optimization of incubation conditions (Additional file 1: Fig. S2), the protein concentrations for UGT1A1, 1A9, and 2B7 were 0.125, 0.05, and 0.05 mg/mL, respectively. Kang-Ai injection and samples that Kang-Ai injection was diluted 10 times, were both used to investigate the inhibitory effects against UGT1A1, 1A9 and 2B7. In addition, the concentrations were 1, 10 and 100 μM for matrine and oxymatrine, and 0.1, 1, 10 and 100 μM for astragalosides and ginsenosides 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 Eqs. (1)–(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. Model fitting and parameter estimation were performed using Graphpad Prism V5 software (SanDiego, CA).
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 (∗∗∗).
Xenobiotics detected in rat samples
After dosing Kang-Ai injection to rats (6 mL/kg), a total of 9 circulating alkaloids, 6 astragalosides and 22 ginsenosides were detected and characterized in rat samples (Additional file 1: Fig. S3A and Table S1). Based on their individual content level from Kang-Ai injection [9], these compounds were graded into five dose levels (Additional file 1: Fig. S3B and Table S1): Level I was over 1000 μg/mL for only oxymatrine (A3); Level II was between 100 and 1000 μg/mL for ginsenosides Rg1 (C6) and Re (C9); Level III was ranged from 10 to 100 μg/mL for matrine (A1), astragaloside IV (B2), notoginsenoside R2 (C3), ginsenoside Rg2 (C5), Rf (C7) and Rc (D3); Level IV was from 1 to 10 μg/mL for astragaloside III (B3), VI (B4), V (B5), notoginsenoside R1 (C8), ginsenoside Rh1 (C1), Rd (D2), Rb2 (D4), Rb1 (D5), Rh4 (E2), Rg6 (E3), F4 (E4); Level V was all less than 1 μg/mL for oxysophocarpine (A2), isoastragaloside IV (B1), ginsenoside F1 (C2), F3 (C4), F2 (D1), Rk3 (E1).
Method validation
The typical MRM chromatograms obtained from blank plasma, blank plasma spiked with the analyte (LLOQ), and dosed rat plasma after intravenous Kang-Ai injection (30 min) were presented in Additional file 1: Fig. S4. As seen from MRM chromatograms, there were no endogenous interference for each analyte and two IS compounds, which suggested that the specificity of the quantitative method was satisfactory.
Additional file 1: Table S2 summarized the mean values of linear regression equation of the sixteen compounds. The correlation coefficients (r2) were ranged from 0.9900 to 0.9994, which exhibited good linearity. The LLOQs of each circulation compound were between 5 and 40 nM, with the accuracies and precisions less than 20%, which were enough for the pharmacokinetics study of sixteen analytes following a 30-min infusion of Kang-Ai injection to rats.
The results of the intra- and inter-day precision and accuracy of all analytes and three QC samples were displayed in Additional file 1: Table S3. The intra-day and inter-day precision (RSD) of sixteen analytes were within the range from 2.35 to 12.64%, whereas the accuracy (RE) derived from three QC samples was between − 14.30 and 13.66%, all meeting the requirements. These results proved that the developed method was accurate and reproducible.
Additional file 1: Table S4 exhibited the matrix effect and extraction recovery results of all analytes and two IS. The mean matrix effects of 16 circulation compounds were in the range from 92.06 to 110.45% with RSD values less than 14.14%, and the average absolute matrix effects of olanzapine-d3 (IS-1) and mycophenolic acid-d3 (IS-2) were 100.29% and 109.12% with RSD values of 11.42% and 3.13%, respectively. Similarly, the mean extraction recoveries ranged from 82.85% to 108.98% with RSD values no more than 14.73% at three QC levels, whereas the mean absolute recoveries of IS-1 and IS-2 were 80.54% and 83.60% with RSD values of 2.11% and 5.19%, respectively. These results demonstrated that the recoveries obtained were consistent and reproducible, and the ionization competition (enhancement or suppression effects) between the analytes and the endogenous co-elution was negligible.
The mean stability results (Additional file 1: Table S5) indicated the RE and RSD values of sixteen circulating alkaloids and saponins at three QC levels were in the range from − 14.53% to 14.40% and 13.87%, respectively, indicating that all the analytes were stable under different storage conditions.
In addition, a total of twenty circulating compounds were detected in rat urine. The regression equations, linear ranges and LLOQs for the determination of the analytes in rat urine was shown in Additional file 1: Table S6. Over the considered calibration range, the regression coefficient (r2) was > 0.9906 for the curves of each analyte. Furthermore, twenty-one circulating compounds were obtained in rat bile, and the calibration curves of twenty-one analytes exhibited the good linearity with r2 values over 0.9901 (Additional file 1: Table S7) at a certain concentration range in rat bile. From the data, we can see the developed method showed good linearity and sensibility in rat urine and bile.
Plasma pharmacokinetics of circulating alkaloids and saponins in rats
Table 1 summarized the PK parameters of circulating alkaloids (A1–A3), astragalosides (B2, B3) and ginsenosides (C1, C3, C5–C9, D2–D5) from intravenously dosed Kang-Ai injection. Figure 1a displayed the total plasma concentrations of the alkaloids over time, during and after a 30-min intravenous infusion of Kang-Ai injection in rats. Clearly, the plasma Cmax value of matrine (A1) were determined after stopping the infusion for 30 min. After the 30-min infusion, its plasma concentration increased with mean t1/2 values of 4.33 h. In addition, the Cmax values of oxysophocarpine (A2) and oxymatrine (A3) were both measured just before stopping the infusion, following the declined plasma concentration with shorter t1/2 values of 1.78 and 2.73 h, respectively. The mean VSS values of A1, A2 and A3 were 9.85, 25.20 and 220.11 mL/kg, respectively, suggesting these three alkaloids all had small Vd values. Besides, the CLtot, p values of A3 was 111.34 mL/h/kg and those of A1 and A2 were 1.58 and 12.31, respectively, indicating that A3 and A1, A2 had moderate and low total clearance, respectively. Furthermore, these circulating alkaloids (A1–A3) were not extensively bound with fu-plasma values over 90%.
The plasma concentration–time curves and PK parameters of astragaloside IV (B2) and III (B3) were shown in Fig. 1b and Table 1, respectively. The plasma Cmax (2363.99 nmol/L) and AUC0-∞ (4523.65 nmol/L*h) values of B2 were far higher than those of B3 with Cmax and AUC0-∞ values of (54.75 nmol/L) and AUC0-∞ (34.29 nmol/L*h), due to the differences in the compounds' doses from the injection. Besides, the t1/2 values of these two astragalosides were both small and less than 2 h. Similarly, B2 and B3 both exhibited the small Vd values (< 100 mL/kg), whereas B2 (28.39 mL/h/kg) and B3 (206.96 mL/h/kg) had low and moderate total clearance, respectively. Protein binding assay demonstrated that B2 and B3 were extensively bound in rat plasma (nearly 90%).
These circulating ginsenosides in plasma could be classified into two categories, protopanaxatriol (PPT) type ginsenosides (C1, C3, C5–C9) and protopanaxadiol (PPD) type ginsenosides (D2–D5). Fig. 1c, d summarized the plasma concentrations of the PPT-type ginsenosides and PPD-type ginsenosides over time, respectively. It was notably shown that the Tmax values of PPT-type ginsenosides were 30 min, whereas those of PPD-type ginsenosides were measured after stopping infusion for 15 min. In addition, the C4h values of the PPT-type ginsenosides were very low, while those of the PPD-type ginsenosides still maintained a moderate plasma concentration. These suggested that the PPT-type ginsenosides possessed the longer MRT, and were eliminated more quickly than PPD-type ginsenosides. Another strong evidence was that CLtot, p values of PPT-type ginsenosides (72.70–255.80 mL/h/kg) were approximately 10 times of those of PPT-type ginsenosides (5.60–7.80 mL/h/kg). Besides, except for ginsenoside Rg2 (C5) about the t1/2 value of 9.77 h, the PPT-type ginsenosides exhibited the shorter elimination half-lives values of i.e., 0.29–1.20 h for ginsenoside Rh1 (C1), Rg1 (C6), Rf (C7), Re (C9) and notoginsenoside R1 (C8), R2 (C3) than those of 4.12–7.22 h for ginsenosides Rd (D2), Rc (D3), Rb2 (D4) and Rb1 (D5). The VSS values of PPT-type ginsenosides and PPD-type ginsenosides were 50.05–791.61 and 35.69–46.96 mL/kg, respectively, indicating that the PPT-type ginsenosides were more easily distributed in various body fluids and tissues than the PPD-type ginsenosides. Additionally, the PPT-type ginsenosides (C1, C3, C5–C9) were not significantly bound, whereas the PPD-type ginsenosides (D2–D5) were obviously bound (around 99%) in rat plasma.
Moreover, due to lack of the available authentic standards, virtual quantification of other main circulating herbal compounds in rat plasma, including baptifoline (A5), 9α-hydroxy-sophocarpine (A6), leontalbinine N-oxide (A7), mamanine (A8), sophoranol (A9), were achieved using the calibration curve of an available reference standard of an analog that bore close structural similarity to the analyte. Figure 1e, Table 1 showed their mean plasma concentration–time curves and calculated relative PK parameters, respectively. The Cmax values ranged from 202.37 to 421.27 nmol/L, and the Tmax values were all determined just before stopping the infusion. Besides, A7 exhibited the longest MRT (7.94 h) and t1/2 (5.73 h) values among these five alkaloids. The small VSS (16.89–150.70 mL/kg) and CLtot, p (7.93–56.05 mL/h/kg) values indicated that these alkaloids were slowly distributed in various body fluids and tissues, and slowly cleared from the systemic circulation.
To better demonstrate the systemic exposure in rat plasma, the comparison of the AUC0–12h values of circulation alkaloids and saponins were displayed in Fig. 2a. Obviously, oxymatrine (A3) exhibited the most abundant exposure with 502.71 μmol/L*h, due to the most abundant dose level from Kang-Ai injection. Additionally, A1, B2, D3, D2, D5, C6, C9, A7, D4, C7, A8, A6, A9, A5, A2, C5, C8, in descending order, exhibited substantially higher total levels of plasma exposure with the AUC0–12h values over 100 nmol/L*h, whereas the levels of plasma exposure to other compounds (B3, C1, C3) were quite low, due to their low compound dose from Kang-Ai injection.
Urinary and biliary excretion of circulating alkaloids and saponins in rats
Table 1 summarized the urinary and biliary excretion parameters of circulating alkaloids and saponins. Meanwhile, Fig. 2b, c showed their cumulative excretion amounts in urine and bile, respectively. For the circulating alkaloids (A1–A3), the renal and hepatobiliary excretion of the unchanged compounds represented the major routes of elimination (Table 1). This was attributed that A1 (57.40%), A2 (38.60%) and A3 (63.83%) exhibited an extraordinarily high fe-U,0–12h values, the sum of which with the fe-B,0–12h values (36.55%, 51.92%, 28.96%, respectively) exceeded 90% of individual dose levels.
Astragaloside III (B3) was eliminated more extensively via hepatobiliary excretion (fe-B, 67.06%) than renal excretion (fe-U, 5.25%) (Table 1). Unlike for B3, renal excretion was a minor elimination pathway for astragaloside IV (B2) (fe-U, 4.84%), and the same fact was that the fraction of dosed B2 was excreted into bile (fe-B) about 2.49%.
The PPT-type ginsenosides (C1–C9), except ginsenoside Rg2 (C5), all exhibited the large fe-U values (9.59–72.17%) (Table 1), suggesting that the renal excretion was the predominant elimination route responsible for their systemic clearance. Similarly, the PPD-type ginsenosides (D2–D5), exhibited moderate fe-U values (6.41–27.8%), which was around a half of the values of PPT-type ginsenosides. In addition, in rat bile, the PPT-type ginsenosides were the most abundant herbal compounds with fe-B values of 9.71–67.93%, whereas only two PPD-type ginsenosides, ginsenoside F2 (D1) and Rd (D2) could be detected and displayed the small fe-B values of 7.46% and 5.55%, respectively. Consistent with the inter-compound differences in CLtot, p values, the CLR and CLB values for these PPT-type ginsenosides (C1–C9) were significantly greater than those respect values for the PPD-type ginsenosides (D1–D5).
Moreover, other alkaloids (A4–A9) were eliminated more extensively unchanged via the renal excretion (fe-U, 12.92–61.05%) than the hepatobiliary excretion (fe-B, 1.27–34.12%) (Table 1). The CLR and CLB values for the alkaloids (A4–A9) kept in line with their CLtot, p values. Another astragaloside, astragaloside VII (B6) was excreted 74.96% of the dose derived from Kang-Ai injection to urine. Another four PPT-type ginsenosides, ginsenosides R1 (C10), Re4 (C11), notoginsenoside R3 (C12) and 20-gluco-ginsenoside Rf (C13) were all subjected to be eliminated by the renal excretion with fe-U values of 36.16–48.30%, whereas their hepatobiliary excretion appeared to be negligible.
Inhibitory effects of Kang-Ai injection and herbal compounds towards each CYP and UGT isozyme
Kang-Ai injection showed inhibitory effects towards CYP2A6, 2E1 and 3A4 with residual activity of 73.63%, 70.28% and 56.92%, respectively (Fig. 3a). Meanwhile, Kang-Ai injection almost exhibited no inhibitory effects against other CYP and UGT enzymes (Fig. 3a). Based on Table 1, matrine (A1), oxymatrine (A3), astragaloside IV (B2), ginsenosides Rg1 (C6), Rf (C7), Re (C9), Rd (D2), Rc (D3), Rb1 (D5) exhibited the Cmax values about or over 0.5 μmol/L after intravenous Kang-Ai injection. Therefore, these nine major circulating compounds were selected to evaluate the inhibitory effects toward several common CYP and UGT enzymes. A1 (100 μM) displayed inhibitory effects against CYP3A4 (66.63%) (Additional file 1: Fig. S5A), whereas A3 (100 μM) could inhibit the metabolic activity of CYP2A6 (77.63%), 2E1 (80.28%), and 3A4 (64.92%) (Fig. 3b). In addition, B2 (100 μM) exhibited inhibitory activity towards CYP2C9 (52.36%) (Fig. 3c). Besides, PPT-type ginsenosides, C6 (100 μM) (Fig. 3d) and C9 (100 μM) (Additional file 1: Fig. S5C) mainly inhibited the function of UGT1A1 (53.26%) and CYP2C8 (30.25%), respectively, while almost no changes were observed for C7 (100 μM) against these CYP and UGT enzymes (Additional file 1: Fig. S5B). Furthermore, D2 (100 μM) exhibited inhibition on the metabolic activity of CYP2B6 (30.36%) and 3A4 (42.36%) (Additional file 1: Fig. S5D), whereas D3 (100 μM) (Fig. 3e) and D5 (100 μM) (Additional file 1: Fig. S5E) showed inhibitory effects on UGT1A9 (30.26%) and CYP2C9 (15.36%), respectively.
Moreover, the inhibition data (Fig. 4) were fit to log (I) and normalized response equations to obtain the IC50 values. The inhibition trend of the abundant compounds towards these CYP and UGT enzymes showed in a dose dependent manner, and their IC50 values were 65.00 (B2 towards CYP2C9), 92.21 (C6 against UGT1A1), 24.03 (C9 towards CYP2C8), 20.19 (D2 against CYP2B6), 40.96 (D2 towards CYP3A4), 28.96 (D3 against UGT1A9) and 8.81 μM (D5 towards CYP2C9), respectively (Additional file 1: Table S8). The inhibition mechanism was further evaluated based on three inhibition kinetic models.
Analysis of inhibition mechanism
The AIC and SC values (Table 2) were obtained after the inhibition data were modeled by three conventional inhibition equation (i.e., competitive, noncompetitive and mixed-type). Based on the smallest AIC and SC values principle, C6 displayed competitive inhibition against UGT1A1, while C9 exhibited mixed-type inhibition against CYP2C8. Noncompetitive inhibition kinetics were observed for B2 against CYP2C9, D2 towards CYP2B6 and 3A4, D3 against UGT1A9 and D5 towards CYP2C9.
Furthermore, the Dixon plots for B2 against CYP2C9 (Fig. 5a), C6 towards UGT1A1 (Fig. 5b), C9 against CYP2C8 (Fig. 5c), D2 towards CYP2B6 (Fig. 5d) and 3A4 (Fig. 5e), D3 against UGT1A9 (Fig. 5f) and D5 towards CYP2C9 (Fig. 5g) also provided strong evidences to support this judgment about the inhibition mechanism. Their respective Ki values were 55.8, 140.6, 13.3, 37.2, 32.8, 22.3 and 16.1 μM (Table 2). These findings suggested that the major circulating compounds have weak or no inhibition on the metabolic activity of common CYP and UGT enzymes with Ki values all over 10 μM.
Potential DDI risks
Based on the [I]/Ki values in vitro, potential DDI risks in vivo could be predicted [27, 28]. Traditionally, [I]/Ki < 0.1, low possibility DDI; 1 > [I]/Ki > 0.1, medium possibility DDI; [I]/Ki > 1, high possibility DDI [18]. In this study, [I]/Ki values for these herbal compounds were all no more than 0.1 (Table 2). Therefore, it seemed impossible to trigger DDI when Kang-Ai injection were co-administrated with clinical drugs which were metabolized by these enzymes.
Bioactive compounds with adequate abundance in herbal injection and favorable PK properties are mostly believed to be responsible for the pharmacological effects and therapeutic efficacy [4]. In the current study, alkaloids and saponins derived from Kang-Ai injection are two classes of bioactive compounds which are likely responsible for the adjuvant therapy of many cancers (Additional file 1: Table S1) [6, 8]. After dosing, oxymatrine (A3), matrine (A1), astragaloside IV (B2), ginsenosides Rc (D3), Rd (D2), Rb1 (D5), Rg1 (C6) and Re (C9), all exhibited considerably high levels of systemic exposure (over 1000 nmol/L*h) (Fig. 2a), indicating that these circulating compounds are worth special consideration in the pharmacological research. Besides, the abundant exposure of A1 may be attributed the metabolism of A3 by CYP3As [29]. In addition, the considerable systemic exposure for A3, A1, D3, D2 and D5, seemed to be correlated with the significantly longer t1/2 values of 2.73–7.22 h than those of B2, C6 and C9 (1.07–1.64 h) (Table 1). This phenomenon that long elimination half-life could significantly counterbalance influence on systemic exposure to herbal compounds with poor bioavailability has been proved previously [30].
Except the systemic exposure of major circulating compounds, we also investigate their elimination in the current study (Table 1). After dosing Kang-Ai injection, oxymatrine (A3) and matrine (A1) mainly involved in the renal and hepatobiliary excretion of the parent compounds (over 90%), of which parts of circulated A1 was originated from the metabolism of A3 by CYP3A4 [11]. Only about 25% of dosed astragaloside IV (B2) were detected as parent compound in urine and bile, and this was probably attributed to the de-glycosylation of B2 to its sapogenin [15]. Additionally, ginsenosides Rg1 (C6) and Re (C9) were eliminated mainly to urine and bile (nearly 60%), whereas it proved that approximately 25% of ginsenoside Rd (D2) was excreted via renal and hepatobiliary elimination. Ginsenosides Rc (D3) and Rb1 (D5) were not detected in bile, and mainly were eliminated through renal excretion. Except the in vivo metabolism, i.e., oxidation and de-glycosylation [16], the delivery to the rat tissues of circulating herbal compounds through blood flow were also an important factor to determine the systemic exposure and excretion of circulating compounds [31]. The VSS values of dosed A3, C6 and C9 were 100.55–220.11 mL/kg, which were near the rat extracellular volume (300 mL/kg) [32], indicating that these herbal compounds could be distributed evenly in various body tissues, except several barriered tissues. However, this elimination route warrant future investigation.
In addition, the obtained PK parameters (Table 1) also could enable us to compare the plasma concentrations of the alkaloids and saponins with the reported effective concentrations (IC50 or EC50 values) for pharmacological activities. Oxymatrine (A3) exhibited the most abundant concentration (422.70 μmol/L) after dosing Kang-Ai injection, which is still obviously below the reported effective concentrations (473.48–3787.84 μmol/L) for several tumor cell lines, including HepG2, MCF-7, HeLa, A549 cells [33]. However, for SW-620, BGC823, A375 cells, A3 is sufficiently effective to inhibit the tumor cell viability (0.10–40.00 μmol/L) [33]. Similar results were obtained for matrine (A1) and its derivatives [34], astragaloside IV (B2) [35], and several main ginsenosides [36]. Although the low plasma concentrations do not result in the ineffectiveness of Kang-Ai injection for the therapeutic effects, they may limit the contribution of bioactive compounds to the current adjuvant therapy. How to achieve the higher plasma concentrations is an important idea to develop new and superior adjuvant agents derived from the current Kang-Ai injection.
Kang-Ai injection are the combinations of two herbs and Kushensu, of which complex chemical compositions increased the DDI risks. Usually, the induction and inhibition of CYP and UGT enzymes can facilitate the metabolism and elimination of their drug substrates and caused unexpected adverse effect. Previously, oxymatrine (A3) was subjected to be metabolized to matrine (A1) rapidly in pooled HLM (Km = 220.78 μM) and HIM (Km = 150.22 μM), and CYP3A4 (Km = 341.35 μM) isozyme greatly contributed to this transformation [37]. However, A1 could not be extensively metabolized by CYPs and UGTs [38]. In addition, considering that most of clinical drugs or endogenous compounds are metabolized or eliminated by CYP1A2 (9%), 2A6, 2B6 (2%), 2C8, 2C9 (16%), 2C19 (12%), 2E1 (2%), 3A4 (46%), and UGT1A1 (15%), 1A9, 2B7 (35%) [39, 40], we focused on the inhibitory effects of herbal compounds derived from Kang-Ai injection against these enzymes above.
Our results showed that Kang-Ai injection demonstrated weak inhibitory effects against CYP2A6, 2E1 and 3A4 (Fig. 3a), which could be mainly attributed to the contribution of oxymatrine. This is because the content level of oxymatrine in Kang-Ai injection (37731.41 μM) is far higher than those of other herbal compounds (0.23–158.95 μM) (Additional file 1: Table S1) [9]. Furthermore, it was notable that the de-glycosylation of astragaloside IV (B2) to sapogenin-cycloastragenol strongly increased the inhibitory effects towards UGT1A8 (Ki = 0.034 µM) and 2B7 (Ki = 20.98 µM) [15]. Interestingly, naturally occurring alkaloids and ginsenosides, including A1, A3, ginsenosides Rg1 (C6), Rf (C7), Re (C9), Rd (D2), Rc (D3) and Rb1 (D5), were unlikely to cause clinically relevant DDI mediated via the induction or inhibition of most CYPs or UGTs involving in drug metabolism in vivo, which agreed with previous study [41].
Identification of the induction or inhibition of the abundant circulating alkaloids and saponins towards uptake and efflux transporters was another significant risk factor for the DDI. In clinics, oxymatrine (A3) could interfere with hOCT1-mediated hepatic uptake and renal elimination, and intestinal absorption by hOCT3, whereas matrine (A1) only had the potential to decrease hOCT3 expressed in enterocytes [42]. Besides, A1 and A3 did not demonstrate the inhibition of P-gp in vitro and in vivo [43]. Additionally, it was reported that astragaloside IV (B2) could induce the protein levels of P-gp, MRP1, MRP3, but produced the inhibitory effects on BCRP [44]. In contrast, in MDR1-MDCKII cells, B2 only exhibited weak inhibition activity of 8.22% when treated with 100 μM [43]. Recently, several scholars got the fact that B2 could induce the upregulation of P-gp and BCRP, and might be a potential substrate of P-gp [45]. For ginsenosides, ginsenoside Rg1 (C6) was potential inhibitor of NTCP (IC50 = 50.49 μM), whereas ginsenoside Re (C9) was potential substrates of NTCP [46]. Further, DDI between ginsenosides Rb1 (D5), Rc (D3), Rd (D2) and methotrexate (a probe substrate for Mrp2) might occur owing to the decrease in the mRNA and protein levels of Mrp2 [47]. In addition, D5, D3, D2 and D9 were predicted to possess the high potential for human OATP1B3-mediated drug interactions [16]. Moreover, PPT and PPD, the aglycones of PPT-type ginsenosides and PPD-type ginsenosides, respectively, were potent P-gp inhibitors, suggesting the unpredictable interactions when ginsenosides were co-administered with the P-gp substrate drugs [48]. The interactions of complex circulating compounds and clinical drugs based on transporters may be one of the important compatibility mechanisms for Kang-Ai injection.
In clinical consumption, the adverse drug reactions (ADR) derived from herbal injections, should be gaining more attention. It was essential to evaluate the objectivity of ADR, develop a reasonable application strategy, and ensure the security and validity of Kang-Ai injection. Data and information obtained from PK studies on the circulating alkaloids and saponins could help understand the complex action of herbal injections, and predict a variety of events related to the efficacy and toxicity of herbal injections. In Kang-Ai injection, the most abundant compounds, oxymatrine (A3) and matrine (A1) both could cause the hepatic toxicities, of which the toxicity of A1 was greater than that of A3 [49]. The elevated metabolism of A3 by CYP3A4 may result in more severe liver damage. Therefore, the clarification of the PK studies could be helpful in deciphering the ADR and hepatorenal toxicity of herbal injections.
Herein, nine alkaloids, six astragalosides and twenty-two ginsenosides were the main circulating compounds after dosing Kang-Ai injection. Oxymatrine (A3), matrine (A1), astragaloside IV (B2), ginsenosides Rc (D3), Rd (D2), Rb1 (D5), Rg1 (C6), Re (C9), Rf (C7), and Rb2 (D4) exhibited high exposure in plasma. Renal and hepatobiliary excretion were their major elimination pathways. In addition, PPD-type ginsenosides (D2–D5) were extensively bound with fu-plasma values approximately 1%, whereas alkaloids and PPT-type ginsenosides (C6, C7, C9) displayed higher fu-plasma values (60.23–93.24%). Furthermore, A1, A3, B2, C6, C7, C9, D2, D3 and D5 displayed weak inhibition or no inhibition towards several common CYP and UGT enzymes with IC50 values ranging from 8.81 to 92.21 μM, suggesting negligible DDI risk. Taken together, the current PK and excretion information gained here could serve as a crucial step in understanding the roles of circulating alkaloids, astragalosides and ginsenosides in therapeutic actions of Kang-Ai injection. These results here will also inform the negligible DDI risks in clinical use of Kang-Ai injection.
Availability of data and materials
Most of the data in the present study are included in this article and its additional information.
Akaike information criterion
Breast cancer resistance protein
Cytochrome P450
Drug–drug interactions
- hOCT:
Human organic cation transporter
Human liver microsomes
Internal standard
Lower limit of quantification
- MDR1/P-gp:
Multidrug resistance protein 1
- MRPs:
Multidrug resistance-associated proteins
Multiple reactions monitoring
Nicotinamide adenine dinucleotide phosphate
Organic anion transporting polypeptide
Pharmacokinetics (PK)
Quality control
Schwartz information criterion
Uridine 5'-diphosphoglucuronosyltransferase
Ultra high‑performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry
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The authors would like to thank Miss Panpan Yang for the UHPLC-TQD-MS tests.
This work was supported by the National Natural Science Foundation of China (81903704, 81803638), Foundation of He'nan Educational Committee (20A350012), and National Major Scientific and Program of Introducing Talents of Discipline to Universities (B13038).
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The animal studies were in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). The study protocol was approved by the Ethics Review Committee for Animal Experimentation of Jinan University (Ethical Review NO. 20190301010).
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The authors declare that they have no competing interests
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Supplementary information
Additional file 1: Table S1.
Information of unchanged compounds in rats after a 30 min-infusion of Kang-Ai injection (6 mL/kg) by UHPLC/Q-TOF–MS. Table S2. Linear correlation parameters, limits of detection (LODs) and lower limits of quantification (LLOQs) of each circulating compound in rat plasma after a 30 min-infusion of Kang-Ai injection (6 mL/kg). Table S3. Relative standard deviation (RSD) values of intra-day and inter-day precisions, and of sixteen compounds in rat plasma after a 30 min-infusion of Kang-Ai injection (6 mL/kg). Table S4. Matrix effect and extraction recovery of each compound and two internal standard (IS) in rat plasma after a 30 min-infusion of Kang-Ai injection (6 mL/kg). Table S5. Stability results of each compound and internal standard in rat plasma after a 30 min-infusion of Kang-Ai injection (6 mL/kg). Table S6. Linear correlation parameters, LLOQs of twenty analytes in rat urine after a 30 min-infusion of Kang-Ai injection (6 mL/kg). Table S7. Linear correlation parameters, LLOQs of twenty-one analytes in rat bile after a 30 min-infusion of Kang-Ai injection (6 mL/kg). Table S8. The IC50 values of six circulating alkaloids and saponins towards several recombinant CYP and UGT isozymes. Data represent the mean ± standard deviation of triplicate. Fig S1. Effects of different incubation conditions on the metabolism of mephenytoin by human CYP2C19 (mean ± SD, n = 3). (A) Tris buffer concentration; (B) pH values; (C) MgCl2 concentration; (D) NADPH concentration; (E) incubation time; (F) protein concentration. Fig S2. Effects of different incubation conditions on the metabolism of estradiol by UGT1A1. (A) Tris buffer concentration; (B) pH values; (C) MgCl2 concentration; (D) UDPGA concentration; (E) incubation time; (F) protein concentration. Data represent the mean ± standard deviation of triplicate. Fig S3. Chemical structures (A) and content levels (B) of circulating alkaloids, astragalosides and ginsenosides in rats after intravenous Kang-Ai injection (6 mL/kg). See Additional file 1: Table S1 for compounds identification and names. Fig S4. Representative MRM chromatograms of sixteen circulating compounds in plasma. (A) blank plasma, (B) blank plasma spiked with sixteen analytes at LLOQs, (C) rat plasma samples at 0.5 h after an intravenous 30 min-infusion of Kang-Ai injection. Fig. S5 Effects of several circulating compounds towards eight expressed CYP isozymes and three recombinant UGT enzymes. (A) matrine, (B) ginsenoside Rf, (C) ginsenoside Re, (D) ginsenoside Rd, (E) ginsenoside Rb1; The probe substrates were incubated at 37 °C in the absence (control, 0 μM) and presence of tested compounds (1, 10, and 100 μM for matrine; 0.1, 1, 10 and 100 μM for astragalosides and ginsenosides). Data represent the mean ± standard deviation of triplicate, (* compared with those of control, * p < 0.05).
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Cite this article
Qin, Z., Jia, M., Yang, J. et al. Multiple circulating alkaloids and saponins from intravenous Kang-Ai injection inhibit human cytochrome P450 and UDP-glucuronosyltransferase isozymes: potential drug–drug interactions. Chin Med 15, 69 (2020). https://doi.org/10.1186/s13020-020-00349-3
DOI: https://doi.org/10.1186/s13020-020-00349-3
- Kang-Ai injection
- Pharmacokinetics
- Excretion
- Plasma protein binding rate
- Cytochrome P450
- UDP-glucuronosyltransferase
- Drug–drug interactions


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Art Nouveau German Silver Plate Deer Theme Garland Wall Plaque Stamped 331. Stunning silver plate over pewter wall plaque originating from Germany and possibly made by. Kayser Sohn, though it is not stamped with a makers mark.The back of the plaque is stamped with 331. The design is very typical of the art nouveau period with a high relief deer or doe amongst foliage and around the outside an oval shaped pretty garland feature.
The rear of the plaque has a ring to attach it to a wall. The overall condition is good without any plate loss.The german'Kayserzinn Pewter' was exhibited all over the world has been exhibited in numerous art galleries due to the fine workmanship and almost handmade looking technique used to hand press the pewter moulds. Length 22 cm x Width 15.5 cm x Thickness 2 mm. The item "Art Nouveau German Silver Plate Deer Theme & Garland Wall Plaque Stamped 331" is in sale since Tuesday, March 10, 2020.
This item is in the category "Antiques\Art Nouveau". The seller is "hnco" and is located in brisbane. This item can be shipped worldwide.

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text: homepage Urbana Civilian Police Review Board Votes to End Mandatory Taser Camera Reviews of All Taser Displays Image from Taser Presentation to Urbana City Council On September 25, 2019, the Urbana Civilian Police Review Board (CPRB) met (only audio recording is made available to the public) to continue the discussion of the Urbana Police Department (UPD) Taser Utilization and Taser Policy. Specifically, UPD had decided independently without consultation with CPRB to phase out the use of Taser Cameras. In the last CPRB meeting on July 24, 2019 (article here), board members had concerns regarding the decision not continue using Taser Cams, and board member Scott Dossett called for this special meeting to look into this issue. Said Dossett last meeting, "I made a promise to myself. I was going to be as transparent and thorough about this with the citizens of Urbana. We had 12-year olds crying in their testimony about how their City of Urbana was going to be damaged because the police would abuse Tasers. That happened in this room for hours. So, I don't take any change to the implementation of the Tasers or the policies that we created lightly." However, Dossett was not present during this special meeting. Other board members – Megan McGinty, Grace Mitchell, Mikhail Lyubansky, Ricardo Diaz, Darius White, and Darrell Price were present for the quorum, with Urbana Police Department Criminal Investigation Division Commander and Deputy Chief, Richard Surles representing UPD. CPRB Chairperson Grace Mitchell set the tone for the meeting stating, "I must say thus far the officers have done a good job with Taser usage, a couple of things I'm probably going to suggest, that we could be more involved. We don't have to see all or so many, maybe we could randomly select videos to look at and process, randomly, so we won't be looking at details of all videos, cause some videos we are only looking at the ground, or looking at the tree, I see that as something we don't need to do." The main issue discussed was how much time Surles takes to put together the Taser reports and videos, as well as the time spent by the board reviewing the videos. According to Surles, in the past 4 years since UPD started utilizing Tasers, there have been about 50 cases requiring Taser Utilization Reports. Out of those cases, there were 3 Taser discharges, the rest were Taser displays. In the past 2 weeks, Surles had processed 7-8 cases, spending 25-30 hours on the task. "It is a lot, especially when I have a division to run that's responsible for lots of important things," says Surles. Surles goes through the steps he takes to select pertinent footage to show the board and redact the footage, telling the board "You don't want to hear all the back and forth about what's going on, you just want to say, OK, he pointed the taser and it looks reasonable." Image from Taser Presentation to Urbana City Council Board member Ricardo Diaz commented that when UPD first started using Tasers, the Taser cam was the only available footage, so it was great. The good thing about the Taser cam is that the recording starts automatically when the weapon's safety mechanism is deactivated, where as officers have to make the decision to turn on their body worn cameras. He agreed that more information is usually helpful, but stated that every piece of info comes at a cost, in this case, the cost is valuable time. Says Diaz, "I do agree that time and investment, the resource investment, the capture, ought not to be as much, so maybe we can think through how to take that up." He added that in the beginning, the board agreed that they wanted to review everything recorded because board members were in the process of training themselves. Moving forward though, it makes no sense to continue, since the board is already "trained" in performing reviews. Diaz went on to say when Tasers usage was first implemented, it was the agreement that there would be no Tasers without cameras. At the time however, there were no other types of recordings. He asked, "Is there ever a situation in which there is a Taser without a camera, does a Taser come on without a camera?" This segued into a discussion of body worn cameras and their associated policies. Surles explained that Tasers are carried by patrol officers and all patrol officers are required to wear body cams. However, it was not possible to guarantee that there would never be a case of an officer without a camera. Surles gave an example where an officer might be in the middle of transferring footage off his camera, when he gets a call and forgets, and leaves without his camera. Surles assured the board though that "they (the officers) love the cameras, they want the cameras". Board members wanted to know more about UPD's body cam policy. Surles explained that UPD's body cam policy falls in line with the Illinois Law Enforcement Officer-Worn Body Camera Act and that officers should turn on their camera if they are doing "cop stuff". In addition, UPD body cameras and squad car cameras are activated automatically if they are in the vicinity of their squad car and the squad car lights are turned on. According to the Illinois Law Enforcement Officer-Worn Body Camera Act, all recordings made with an officer-worn body camera must be destroyed after 90 days unless any encounter captured on the recording has been flagged. Board members asked if this time frame could mean that footage is deleted before they are able to review it. Surles said that it was unlikely because if there was anything suspect, a complaint would probably be made and complaints need to be filed within 45 days, so a supervisor would have flagged the footage. However, there could be cases where the complaint filing countdown is frozen, for example if the subject is in jail. If 90 days have elapsed by the time the subject is released and files a complaint, the video footage would have been deleted. The board also wanted to know how much of the body cam footage was available through the Freedom of Information Act. "Actually, the exemptions for FOIA are pretty extensive on body cams", says Surles. A request will be usually be denied if the requestor is not a party to the recording. City staff clarified that "For the most part, body cam videos are not FOIA-able unless there's a complaint filed, it's very similar to the flags – complaint filed, discharge of a firearm, use of force, arrest or detention, or bodily harm." Wrapping up, board members debated the best way to move forward. Chairperson Mitchell was in favor of randomly selecting 3 or 4 videos to review each time. One board member suggested just getting rid of all video from the Tasers. Another board member was concerned that if they randomly selected videos to watch, what happens if the miss something? How would it reflect on the board with the responsibility placed by the citizens? Surles spoke from the police department's viewpoint, saying that from a staff allocation perspective UPD would "greatly appreciate it", especially since he was being promoted to Deputy Chief that week and did not know how it would fit into his new workload, and if someone else could be trained to process the videos. Surles then suggested only looking at every Taser discharge and did not want the responsibility of selecting which video CPRB should review. To date, there have been 3 Taser discharges – 2 have been reviewed by CPRB, 1 is pending. "In terms of (Taser) displays, listen to me, you've seen the displays they are usually kind of the same thing, um, I would say something like every 5th video, or random number generator, or something like that. Don't let me decide", says Surles. Board member Megan McGinty proposed that the board look at the Taser reports as a group and decide which ones should be further examined. But Chairperson Mitchell quickly shot down the proposal, saying "Then that puts us in a position of having to look at them all." McGinty responded that it does not take that long to review the reports. Surles also assured the board that if a Taser incident "goes sideways", the board will probably hear about it through the complaint process. To date, there has not been a single complaint about Taser usage. Diaz asked to confirm that all body cam footage would be available to CPRB via the complaint process. However, it is unclear from the dialogue if this is indeed the case, as city staff chimed in to say that the body cam footage will be available if the complaint goes through the appeals process. In conclusion, the CPRB members present at this meeting voted unanimously to approve the following motions: 1) Eliminate the mandatory Taser camera review of all displays unless there is a discharge. 2) Require a review of Taser discharges on human beings, regardless of the effect of the discharge. The board also wanted to make sure all body cam footage related to a Taser discharge (not just the footage that Surles chooses to show) is flagged for non-erasure, even if a complaint is not made. Moving forward, Surles will review the UPD Taser policy to see if anything needs to be changed based on the two motions approved at this meeting. One or two board members will be assigned to work with him on this task. Share This September 27, 2019


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"It's not the mic, it's the podcaster." There's no way around it. We're humongous life-long fans of the original "Top Gun" and haven't loved a movie like this in a long time. We gush and thoroughly cover what we feel is one of the best legacy sequels of all-time, as well as talk some about the career of Tom Cruise and how this film speaks about both his future and the film industry. Enjoy.
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Understanding "Wage Theft": Evasion and Avoidance Responses to Minimum Wage Increases
A holistic assessment of the labor market effects of minimum wage regulation requires understanding employer compliance. We investigate how minimum wage increases and the strength of enforcement regimes affect the prevalence of subminimum wage payment. Using the Current Population Survey (CPS), we find strong evidence that higher minimum wages lead to a greater prevalence of subminimum wage payment. We estimate that increases in measured underpayment following minimum wage increases average between 14 and 22 percent of realized wage gains. Furthermore, we provide evidence that these estimates are unlikely to be driven by measurement error in the CPS's wage data, which are self-reported. Taken together, we interpret these findings as evidence that minimum wage noncompliance is an important reality in the low-wage labor market. We find some evidence that enforcement regimes mediate both baseline rates of subminimum wage payment and the response of subminimum wage payment to increases in minimum wages.
We thank Duncan Hobbs for excellent research assistance. We thank Julie Cullen, Gaurav Khanna, Diane Schanzenbach, and Ian Schmutte for comments. The views expressed herein are those of the authors and do not necessarily reflect the views of the National Bureau of Economic Research.
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Jeffrey Clemens & Michael R. Strain, 2022. "Understanding "Wage Theft": Evasion and Avoidance Responses to Minimum Wage Increases," Labour Economics, . citation courtesy of


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text:
Stu Versus San Francisco, Part 17
Page 82
Stu Versus San Francisco, Part 17
Original Release Date: October 14, 2008
So...some of this dialogue is a little bit cringe-worthy for me at this point, particularly that between Renee and Tiffany, but I love what's going on here. Mike and Renee's plan has obviously gone awry, so they're testy with one another, and Tiffany and Gary continue to needle each other to the point where Mike and Renee forget about their argument because they're too fascinated by the other one that's going on in the same vehicle. Maybe it seems unrealistic because Tiffany has a gun and Mike and Renee wouldn't be arguing in this situation because they should be afraid of the gun, but they've been in enough tight spots at this point that maybe it just doesn't phase them. And, again, I like the idea of Tiffany and Gary bickering over work-related stuff and embarrassing social foibles. I like it because it just feels real. Sure, they could be all-business, but they've obviously got stuff on their minds.


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Speaker Profile
M.S., MBA, Co-founder and CEO of Xcell Biosciences
Prior to Xcell, Brian worked with Strategyn Equity Partners, a mid-market private equity firm and with L.E.K. Consulting, a business strategy management consulting firm where he focused on advising life sciences clients in corporate strategy, product strategy and planning, and transaction support. Brian has also lived and worked in several countries in Africa with the non-profit TechnoServe, where he wrote a grant for and led the launch of a $13M program funded by the Gates Foundation and Coca-Cola to double the incomes of 54,000 farmers. Brian earned his Masters of Business Administration from UC Berkeley's Haas School of Business, his Masters in Bioscience from the Keck Graduate Institute of Applied Sciences, and Bachelor of Science degrees with honors in molecular biology and in chemistry from Purdue University.
Presentation Title and Company Description
Immunotherapy Showcase: Controlling Cell State for Immunotherapy Development
Xcell Bio has developed a proprietary cell control system for use with tumor, stem, and immune cells. This novel platform enables researchers to easily grow primary human cells, flexibly modulate cellular function, and improves the delivery of materials into cells.


text: Mother's Day is this weekend. What a better way to say "Thank You" to all the mothers out there than cooking dinner for them with Shot in the Dark Rubs & Seasonings. Stop by and pick your gifts up while you can. Contact us or order on our website www.shot-in-the-dark-bbq-staging.jaszunq5-liquidwebsites.com We will be around some this weekend for local pick ups.


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Modern coaching theory is becoming increasingly complex. New sport science revelations and practices by elite sporting nations indicate that coaching programs have to be up-to-date to be effective. This workshop describes the basic principles which underlie coaching excellence, the features of a complete coaching program, and the pitfalls that often occur in coaching endeavors. Participants should gain an understanding of what needs to be done to produce maximized performances in athletes.
Prospective Audience
Senior athletes, coaches, sports administrators, and parents.
A master copy of a 30-page workbook which needs to be reproduced and distributed to participants will be supplied.
Return to List of Workshops.


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Your Huntsville business is your passion and livelihood. That's why your business should get a professionally installed Huntsville business security system to keep your business and employees protected and secure.
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ECB Comes to Greece's Aid by Waiving Collateral Rules; ECB Plays With Fire; Europe's Web of Debt
Mish Moved to MishTalk.Com Click to Visit.
In a move that is supposed to stop contagion and inspire confidence, the ECB Comes to Greece's Aid by Waiving Collateral Rules
The European Central Bank joined the international rescue of Greece, saying it would indefinitely accept the country's debt as collateral regardless of its country's credit rating, underpinning gains in the bond market.ECB Plays With Fire
The decision came less than a day after Greece agreed to a 110 billion-euro ($145 billion) package of emergency loans from the International Monetary Fund and its euro-region allies. Under the plan backed by the ECB, Greece pledged 30 billion euros in budget cuts to bring a deficit of 13.6 percent of gross domestic product within the EU limit of 3 percent in 2014.
Further downgrades from credit-rating companies had threatened to render Greek bonds ineligible for collateral for ECB loans after Standard & Poor's last week cut the nation to junk status. Had Moody's Investors Service and Fitch Ratings followed suit, Greece's debt would have no longer been accepted under the previous rules, threatening to inflict further pain on the economy and its banks.
Today's decision was a reversal for ECB President Jean-Claude Trichet, who began the year saying the ECB would not change its "collateral policy for the sake of any particular country."
The ECB is foolish. Greece may default a couple years down the road. In the meantime, banks can swap Greek risk straight on to the ECB in exchange for Euros.
What the ECB does for Greece, it may now feel obliged to do for Portugal and Spain.
This is a dangerous precedent that challenges the credibility of both the ECB and Jean-Claude Trichet.
Intermediate-term, the ECB's actions add more tinder to the woodpile. Spain and Portugal are the matches.
Europe's Web of Debt
The New York times has a nice European Web of Debt graphic worth a good look.
click on graph for sharper image
For the related story please see In and Out of Each Other's European Wallets
The chart and story show some interesting facts
- Italy owes France $511 Billion (20% of French GDP)
- Portugal owes Spain a net a net of $58 billion
- Spain holds nearly a third of Portugal's debt
- Spain owes France $220 Billion
- Spain owes Germany $238 billion
- Spain owes the UK $114 billion
Does anyone seriously think this debt can be paid back? If so, how? Instead, I suggest this is another clear sign of just how insolvent the global banking system is.
Misguided US-Centric hyperinflationists who tend to only see US problems clearly have something else to think about.
Mike "Mish" Shedlock
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BIDMC's Dan Barouch, MD, PhD, Director of Vaccine and Virology Research, elected to the National Academy of Medicine
Boston – Dan Barouch, MD, PhD, virologist and immunologist and Director of Vaccine and Virology Research at Beth Israel Deaconess Medical Center (BIDMC), has been elected to the prestigious National Academy of Medicine (NAM). Election to the Academy is considered one of the highest honors in the fields of health and medicine and recognizes individuals who have demonstrated outstanding professional achievement and commitment to service.
Barouch, who is also a William Bosworth Castle Professor of Medicine at Harvard Medical School, was elected for being an international leader in virology and immunology and developing novel vaccines and cure strategies for viruses of global importance, including one of the first COVID-19 vaccine candidates, the first Zika virus vaccine, and the first global mosaic HIV-1 vaccine, as well as defining immunotherapeutic HIV-1 cure strategies.
"This distinguished and diverse class of new members is a truly exceptional group of scholars and leaders whose expertise in science, medicine, health, and policy will be integral to helping the NAM address today's most pressing health challenges and inform the future of health and health care for the benefit of everyone around the globe," said National Academy of Medicine President Victor J. Dzau. "It is my privilege to welcome these esteemed individuals to the National Academy of Medicine."
New members are elected by current members through a process that recognizes individuals who have made major contributions to the advancement of the medical sciences, health care and public health. A diversity of talent among NAM's membership is assured by its Articles of Organization, which stipulate that at least one-quarter of the membership is selected from fields outside the health professions — for example, from such fields as law, engineering, social sciences, and the humanities.
"Dr. Barouch's election to the National Academy of Medicine is a well-deserved recognition of his remarkable scientific discoveries and leadership in developing novel vaccines to fight existing and emerging pathogens that threaten global public health," said Gyongyi Szabo, MD, PhD, Chief Academic Officer at BIDMC. "BIDMC congratulates Dr. Barouch on this milestone accomplishment. At the top of his impressive list of accomplishments in vaccine development, Dr. Barouch and his team developed one of the leading COVID-19 vaccine candidates, now in a phase 3 clinical trial. We look forward to Dr. Barouch's continued success and scientific discoveries here at BIDMC."
Established originally as the Institute of Medicine in 1970 by the National Academy of Sciences, the National Academy of Medicine addresses critical issues in health, science, medicine, and related policy and inspires positive actions across sectors. NAM works alongside the National Academy of Sciences and National Academy of Engineering to provide independent, objective analysis and advice to the nation and conduct other activities to solve complex problems and inform public policy decisions. The National Academies of Sciences, Engineering, and Medicine also encourage education and research, recognize outstanding contributions to knowledge, and increase public understanding of STEMM. With their election, NAM members make a commitment to volunteer their service in National Academies activities.
"Dr. Barouch has performed elegant, innovative science to develop strong vaccine candidates for HIV, and applied what he has learned in HIV to develop an effective Zika vaccine and what we hope will be an effective COVID-19 vaccine," said Mark Zeidel, MD, Chairman of the Department of Medicine at BIDMC. "His innovations include the development of novel and highly effective adenoviral vectors, and the development and application of highly relevant animal models to test the efficacy of vaccine candidates. Dr. Barouch is also an outstanding citizen-scientist who performs rigorous scientific work and keeps the public conversation focused on the need for careful scientific work as we develop effective COVID-19 vaccines."
About Beth Israel Deaconess Medical Center
Beth Israel Deaconess Medical Center is a patient care, teaching and research affiliate of Harvard Medical School and consistently ranks as a national leader among independent hospitals in National Institutes of Health funding. BIDMC is the official hospital of the Boston Red Sox.
Beth Israel Deaconess Medical Center is a part of Beth Israel Lahey Health, a health care system that brings together academic medical centers and teaching hospitals, community and specialty hospitals, more than 4,800 physicians and 36,000 employees in a shared mission to expand access to great care and advance the science and practice of medicine through groundbreaking research and education.