Simultaneous determination of forsythin and its major metabolites in human plasma via liquid chromatography-tandem mass spectrometry.

Forsythiae suspensa is widely used in China as a traditional Chinese medicine. Forsythin is extracted from Forsythiae Fructus and has undergone phase II clinical trials as an antipyretic drug in China. The main metabolites of forsythin in human plasma are aglycone sulfate (KD-2-SO3H) and aglycone glucuronide (KD-2-Glc). In the present study, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the simultaneous analysis of forsythin, KD-2-Glc, and KD-2-SO3H, in human plasma. After precipitating proteins with methanol, these three analytes were separated on a Gemini-C18 column along with teniposide as an internal standard. Mass spectrometry detection, under multiple reaction monitoring, was then carried out in negative mode using the Triple Quad™ 6500+ LC-MS/MS system coupled with an electrospray ionization (ESI) ion source. The transitions of m/z 371.1→356.1 for forsythin, m/z 547.2→356.0 for KD-2-Glc and m/z 451.2→356.2 for KD-2-SO3H were chosen to effectively maintain the balance between selectivity and sensitivity. The developed method was linear over the following concentrations in human plasma samples: 1.00-1000 ng/mL for forsythin, 2.50-2500 ng/mL for KD-2-Glc, and 5.00-5000 ng/mL for KD-2-SO3H. Assays were validated and satisfied the acceptance criteria recommended by the CFDA guidance. Furthermore, this LC-MS/MS method was successfully implemented in a Phase I, first-in-human, dose-escalation pharmacokinetic study among Chinese healthy participants after single oral administration of forsythin tablets.

Pretreatment with broad-spectrum antibiotics alters the pharmacokinetics of major constituents of Shaoyao-Gancao decoction in rats after oral administration.

The influence of broad-spectrum antibiotics on the pharmacokinetics and biotransformation of major constituents of Shaoyao-Gancao decoction (SGD) in rats was investigated. The pharmacokinetic behaviors of paeoniflorin (PF), albiflorin (AF), liquiritin (LT), isoliquiritin (ILT), liquiritin apioside (LA), isoliquiritin apioside (ILA), and glycyrrhizic acid (GL), seven major constituents of SGD, as well as glycyrrhetinic acid (GA), a major metabolite of GL, were analyzed. A 1-week pretreatment with broad-spectrum antibiotics (ampicillin, metronidazole, neomycin, 1 g L-1; and vancomycin, 0.5 g L-1) via drinking water reduced plasma exposure of the major constituents. The AUC0-24 h of PF and LT was significantly decreased by 28.7% and 33.8% (P < 0.05 and P < 0.005), respectively. Although the differences were not statistically significant, the AUC0-24 h of AF, ILT, LA, ILA, and GL was decreased by 31.4%, 50.9%, 16.9%, 44.1%, and 37.0%, respectively, compared with the control group. In addition, the plasma GA exposure in the antibiotic-pretreated group was significantly lower (P < 0.005) than the control group. The in vitro stability of the major constituents of SGD in the rat intestinal contents with or without broad-spectrum antibiotics was also investigated. The major constituents were comparatively stable in the rat duodenum contents, and the biotransformation of GL mainly occurred in the rat colon contents. In summary, broad-spectrum antibiotics suppressed the absorption of the major constituents of SGD and significantly inhibited the biotransformation of GL to GA by suppressing the colon microbiota. The results indicated a potential clinical drug-drug interaction (DDI) when SGD was administered with broad-spectrum antibiotics.

CYP3A4 inducer and inhibitor strongly affect the pharmacokinetics of triptolide and its derivative in rats.

Triptolide is the most active ingredient of Tripterygium wilfordii Hook F, which is used to treat rheumatoid arthritis. (5R)-5-Hydroxytriptolide is a hydroxylation derivative of triptolide with a reduced toxicity. To investigate the metabolic enzymes of the two compounds and the drug-drug interactions with enzyme inducers or inhibitors, a series of in vitro and in vivo experiments were conducted. In vitro studies using recombinant human cytochrome P450 enzyme demonstrated that cytochrome P450 3A4 (CYP3A4) was predominant in the metabolism of triptolide and (5R)-5-hydroxytriptolide, accounting for 94.2% and 64.2% of the metabolism, respectively. Pharmacokinetics studies were conducted in male SD rats following administration of triptolide or (5R)-5-hydroxytriptolide (0.4 mg/kg, po). The plasma exposure to triptolide and (5R)-5-hydroxytriptolide in the rats was significantly increased when co-administered with the CYP3a inhibitor ritonavir (30 mg/kg, po) with the values of AUC0-∞ (area under the plasma concentration-time curve from time zero extrapolated to infinity) being increased by 6.84 and 1.83 times, respectively. When pretreated with the CYP3a inducer dexamethasone (50 mg·kg-1·d-1, for 3 d), the AUC0-∞ values of triptolide and (5R)-5-hydroxytriptolide were decreased by 85.4% and 91.4%, respectively. These results suggest that both triptolide and (5R)-5-hydroxytriptolide are sensitive substrates of CYP3a. Because of their narrow therapeutic windows, clinical drug-drug interaction studies should be carried out to ensure their clinical medication safety and efficacy.

Wedelolactone metabolism in rats through regioselective glucuronidation catalyzed by uridine diphosphate-glucuronosyltransferases 1As (UGT1As).

BACKGROUND: Wedelolactone (WEL), a medicinal plant-derived coumestan, has been reported to exhibit a diverse range of pharmacological activities. However, the metabolism and disposition of WEL remain unexplored. PURPOSE: The present study aims to investigate the metabolism of WEL in rats and identify the enzymes responsible for forming major WEL metabolites. METHODS: Plasma, urine, feces, and bile samples were collected before and after 50 mg/kg WEL was orally administered to rats. Metabolites were profiled by ultrahigh performance liquid chromatography/quadrupole time-of-flight mass spectrometry and identified by high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy. The in vitro WEL glucuronidation activities of human liver microsomes, human kidney microsomes, human intestine microsomes, and 12 recombinant human uridine diphosphate-glucuronosyltransferase (UGT) isoforms were screened. Molecular docking simulation of the interaction between WEL and UGT1A9 was conducted. RESULTS: WEL underwent extensive metabolism, and 17 metabolites were identified. The major metabolic pathways observed were glucuronidation and methylation. Glucuronic acid was preferentially introduced into 5-OH, whereas no obvious regioselectivity was observed in the methylation of 11-OH and 12-OH. Multiple UGTs, including UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10, were involved in forming WEL glucuronides and O-methylated WEL glucuronides. CONCLUSION: The extensive glucuronidation and methylation is responsible for the low oral bioavailability of WEL in rats. UGT1A1 and UGT1A9 were the major enzymes involved in the glucuronidation of WEL and O-methylated WEL. Molecular docking studies revealed that 5-OH was accessible to the catalytic domain of UGT1As; therefore, 5-OH exhibited a high probability of glucuronidation.

Isomer-selective distribution of 3-n-butylphthalide (NBP) hydroxylated metabolites, 3-hydroxy-NBP and 10-hydroxy-NBP, across the rat blood-brain barrier.

AIM: To investigate the mechanisms underlying the isomer-selective distribution of 3-n-butylphthalide (NBP) hydroxylated metabolites, 3-hydroxy-NBP (3-OH-NBP) and 10-hydroxy-NBP (10-OH-NBP), across the blood brain barrier (BBB). METHODS: After oral administration of NBP (20 mg/kg) to rats, the pharmacokinetics of two major hydroxylated metabolites, 3-OH-NBP and 10-OH-NBP, in plasma and brains were investigated. Plasma and brain protein binding of 3-OH-NBP and 10-OH-NBP was also assessed. To evaluate the influences of major efflux transporters, rats were pretreated with the P-gp inhibitor tariquidar (10 mg/kg, iv) and BCRP inhibitor pantoprazole (40 mg/kg, iv), then received 3-OH-NBP (12 mg/kg, iv) or 10-OH-NBP (3 mg/kg, iv). The metabolic profile of NBP was investigated in rat brain homogenate. RESULTS: After NBP administration, the plasma exposure of 3-OH-NBP was 4.64 times that of 10-OH-NBP, whereas the brain exposure of 3-OH-NBP was only 11.8% of 10-OH-NBP. In the rat plasma, 60%±5.2% of 10-OH-NBP was unbound to proteins versus only 22%±2.3% of 3-OH-NBP being unbound, whereas in the rat brain, free fractions of 3-OH-NBP and 10-OH-NBP were 100%±9.7% and 49.9%±14.1%, respectively. In the rats pretreated with tariquidar and pantoprazole, the unbound partition coefficient Kp,uu of 3-OH-NBP was significantly increased, while that of 10-OH-NBP showed a slight but not statistically significant increase. Incubation of rat brain homogenate with NBP yielded 3-OH-NBP but not 10-OH-NBP. CONCLUSION: The isomer-selective distribution of 10-OH-NBP and 3-OH-NBP across the BBB of rats is mainly attributed to the differences in plasma and brain protein binding and the efflux transport of 3-OH-NBP. The abundant 10-OH-NBP is not generated in rat brains.

[Metabolites of injected chlorogenic acid in rats].

Chlorogenic acid (5-CQA) is one of the major components in some Chinese herbal injections. However, the metabolism of 5-CQA in rats after intravenous injection has not been determined. An ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) method was applied to identify the metabolites in bile, urine, feces and plasma after a single intravenous administration of 10 mg x kg(-1) 5-CQA to rats. Using MSE and mass defect filter techniques, a total of 35 metabolites were detected in bile, urine, feces and plasma. The predominant metabolites in bile were glutathione conjugates of O-methyl-5-CQA, accounting for approximately 80% of the metabolites excreted in bile. The major components in urine were parent drug, O-methyl-5-CQA, hydrolyzed metabolites and glucuronide conjugates. The major components in feces were O-methyl-5-CQA and its cysteine conjugates. The major component in plasma was the parent drug. The urinary and fecal excretion pathways were equally important to 5-CQA in rats. These results demonstrate that 5-CQA undergoes extensively metabolism in rats and are highly reactive to nucleophiles such as GSH. This finding indicates that attention should be paid on the injections containing 5-CQA, which may covalently bind to proteins, leading to allergenic drug reactions.

Separation and identification of Aconitum alkaloids and their metabolites in human urine.

To study the safety of Aconitum medicinal herbs in clinic and identify Aconitum alkaloids poisoning in forensic medicine, Aconitum alkaloids and their metabolites were separated and identified in human urine by liquid chromatography-electrospray ionization-multi-stage mass spectrometry (LC-ESI-MS(n)) and chemical pathway of metabolism was investigated. The alkaloids and their metabolites in the urine sample were extracted with solid-phase cartridges and separated by HPLC with acetonitrile-water-formic acid (40:60:0.5) mobile phase. Structures of five metabolites and three parent Aconitum alkaloids were identified with multi-stage mass spectrometry data through comparison with authentic substances as aconitine (M(1)), mesaconitine (M(2)), hypaconitine (M(3)), benzoylaconine (M(4)), benzoylmesaconine (M(5)), benzoylhypaconine (M(6)), 16-O-demethylaconitine (M(7)) and 16-O-demethylhypaconitine (M(8)), respectively. Among them, M(8) was identified and reported for the first time. Metabolic pathways of Aconitum alkaloids in human body were proposed.

[Determination of wogonin in rat plasma by liquid chromatography-tandem mass spectrometry].

AIM: To develop a sensitive and rapid LC/MS/MS method for the determination of wogonin, an active flavonoid shown to have an inhibitory effect on the proliferation of a carcinoma cell line, in rat plasma after an oral administration. METHODS: Wogonin and daidzein (internal standard) were extracted from plasma directly with n-hexane-diethyl ether (1:4). After liquid-liquid extraction, the analytes of interest were separated on a Diamonsil C18 column. The mobile phase was acetonitrile-water-formic acid (80:20:1) with a flow rate of 0.8 mL.min-1. A Finnigan TSQ (triple stage quadruple) tandem mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source was used as detector and was operated in positive ion mode. Selected reaction monitoring (SRM) was used and transitions selected for quantitation were: m/z 284.8-->269.5 for wogonin and m/z 254.7-->198.5 for daidzein. The mass spectrometric conditions were as follows: The temperatures of the vaporizer and heated capillary were 450 degrees C and 250 degrees C, respectively. The corona discharge current was 4.00 microA. Nitrogen was used as the sheath and auxiliary gas, whose settings were 0.6 MPa and 3 mL.min-1, respectively. Argon was used as the collision gas at a pressure of 1.4 Pa. The collision energy of 35 V was chosen for both wogonin and daidzein. RESULTS: The calibration curve was linear over the concentration range of 0.25 to 20.0 ng.mL-1. The limit of quantitation was 0.25 ng.mL-1. Within-day and between-day precision expressed by relative standard deviation (RSD) was 2.2% to 13.1% and 5.9% to 7.3%, respectively, and the accuracy expressed by RE was -0.3% to 1.3%. CONCLUSION: This method proved to be specific, accurate and sensitive enough to be applied to the pharmacokinetic studies of wogonin in rats after a single dose of 5 mg.kg-1 by oral administration.