Novel in vitro dynamic metabolic system for predicting the human pharmacokinetics of tolbutamide.

Liver metabolism is commonly considered the major determinant in drug discovery and development. Many in vitro drug metabolic studies have been developed and applied to understand biotransformation. However, these methods have disadvantages, resulting in inconsistencies between in vivo and in vitro experiments. A major factor is that they are static systems that do not consider the transport process in the liver. Here we developed an in vitro dynamic metabolic system (Bio-PK metabolic system) to mimic the human pharmacokinetics of tolbutamide. Human liver microsomes (HLMs) encapsulated in a F127'-Acr-Bis hydrogel (FAB hydrogel) were placed in the incubation system. A microdialysis sampling technique was used to monitor the metabolic behavior of tolbutamide in hydrogels. The measured results in the system were used to fit the in vitro intrinsic clearance of tolbutamide with a mathematical model. Then, a PBPK model that integrated the corresponding in vitro intrinsic clearance was developed to verify the system. Compared to the traditional incubation method, reasonable PK profiles and the in vivo clearance of tolbutamide could be predicted by integrating the intrinsic clearance of tolbutamide obtained from the Bio-PK metabolic system into the PBPK model. The predicted maximum concentration (Cmax), area under the concentration-time curve (AUC), time to reach the maximum plasma concentration (Tmax) and in vivo clearance were consistent with the clinically observed data. This novel in vitro dynamic metabolic system can compensate for some limitations of traditional incubation methods; it may provide a new method for screening compounds and predicting pharmacokinetics in the early stages, supporting the development of compounds.

Establishment and assessment of a novel in vitro bio-PK/PD system in predicting the in vivo pharmacokinetics and pharmacodynamics of cyclophosphamide.

1. A novel bio-pharmacokinetic/pharmacodynamic (PK/PD) system was established and assessed in predicting the PK parameters and PD effects of the model drug cyclophosphamide (CP) considering the interrelationships between drug metabolism, pharmacological effects and dynamic blood circulation processes in vitro. 2. The system contains a peristaltic pump, a reaction chamber with rat liver microsomes (RLMs) encapsulated in pluronic F127-acrylamide-bisacrylamide (FAB) hydrogels, an effector cell chamber and a recirculating pipeline. The metabolism and pharmacological effects of CP (5, 10 and 20 mM) were measured by HPLC and MTT assay. A mathematical model based on mass balance was used to predict the in vitro clearance of CP. In vivo clearance of CP was estimated by in vitro to in vivo extrapolations (IVIVE) and simulations using Simcyp® software. 3. The predicted in vivo clearance of CP at concentrations of 5, 10 and 20 mM was 11.36, 10.12 and 10.68 mL/min/kg, respectively, within two-fold differences compared with the reported 11.1 mL/min/kg. The survival ratio of effector cells during the metabolism and circulation of CP was significantly enhanced. 4. This system may serve as an alternative approach to predict in vivo metabolism, pharmacological effects and toxicity of drugs, ensuring an efficient drug screening process.

[Absorption of flavonoids from Abelmoschus manihot extract by in situ intestinal perfusion].

To explore the mechanism of the absorption of flavonoids from Abelmoschus manihot flowers, in situ intestinal recirculation was performed to study the effect of the absorption at different concentrations and different intestinal regions. To evaluate the conditions of the absorption of six flavonoids from Abelmoschus manihot flowers, the concentrations of Abelmoschus manihot in the perfusion solution were determined by HPLC at predesigned time. And we have investigated the inhibitory effect of six flavonoids from Abelmoschus manihot flowers on P-glycoprotein (P-gp) drug efflux pump. The results demonstrated that the absorption rates of flavonoids from Abelmoschus manihot flowers are not significantly different (P > 0.05) at various drug concentrations, the absorption of flavonoids from Abelmoschus manihot flowers is a first-order process with the passive diffusion mechanism. The absorption rates of each of flavonoids are significantly different. The absorption rate of flavonoid glycoside was lower than that of aglycone; the flavonoids from Abelmoschus manihot flowers could be absorbed in all of the intestinal segments. The best parts of intestine to absorb hyperoside and myricetin are jejunum and duodenum, separately. Verapamil could enhance the absorption of isoquercitrin, hyperoside, myricetin and quercetin-3'-O-glucoside by inhibiting P-glycoprotein (P-gp) drug efflux pump.

[Identification of the metabolites of Sinisan extract in rat plasma, urine, feces and bile after intragastric administration].

Sinisan is a widely used traditional Chinese medicine (TCM) in treating various diseases; however, the in vivo metabolic profile of its multiple components remains unknown. In this paper, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was applied to identify the metabolites of Sinisan extract in rat plasma, urine, feces and bile after intragastric administration. Using MS(E) and mass defect filter techniques, 41 metabolites of 10 parent compounds (naringin, naringenin, hesperidin, neohesperidin, liquiritin, liquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, saikosaponin a and saikosaponin d) were detected and tentatively identified. It was shown by our results that these compounds was metabolized to the forms of hydroxylation, glucuronidation, sulfation, glucuronidation with sulfation and glucuronidation with hydroxylation in vivo.

Establishment of rat liver microsome-hydrogel system for in vitro phase II metabolism and its application to study pharmacological effects of UGT substrates.

Studies on the efficacy evaluation of UDP-glucuronosyltransferases (UGTs) substrates often ignore the existence of active metabolites. However, the present study aims to establish an in-vitro Phase II metabolism system to predict their pharmacological effects after metabolism. Rat liver microsomes (RLMs) encapsulated in a F127'-Acr-Bis (FAB) hydrogel were placed in the incubation system. Baicalein (BA) was chosen as a model drug and the metabolic activity was investigated by quantitating the metabolite Baicalin (BG). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to measure the cell viability in Traditional cell culture system (TCCS) and Microsome-hydrogel added to cell culture system for Phase II metabolism (MHCCS-II). Finally, MHCCS-II was applied to predict the metabolic effects of Oroxylin A (OA) and Wogonin (W). Compared to TCCS group, for HepG2 and MCF-7 cells, BA in MHCCS-II led to lower survival ratios of cells (P < 0.05), while for PC12 cells it led to higher survival ratios of cells (P < 0.01). For HepG2 cells, OA and W showed obviously enhanced tumor inhibition after metabolism with the IC50 of 32.7 ± 2.9 µM and 76.1 ± 5.1 µM, respectively (P < 0.01). In conclusion, the MHCCS-II could be a useful tool for studying the pharmacokinetics and pharmacodynamics of UGTs substrates.

Prediction of Drug-Drug Interactions with Bupropion and Its Metabolites as CYP2D6 Inhibitors Using a Physiologically-Based Pharmacokinetic Model.

The potential of inhibitory metabolites of perpetrator drugs to contribute to drug-drug interactions (DDIs) is uncommon and underestimated. However, the occurrence of unexpected DDI suggests the potential contribution of metabolites to the observed DDI. The aim of this study was to develop a physiologically-based pharmacokinetic (PBPK) model for bupropion and its three primary metabolites-hydroxybupropion, threohydrobupropion and erythrohydrobupropion-based on a mixed "bottom-up" and "top-down" approach and to contribute to the understanding of the involvement and impact of inhibitory metabolites for DDIs observed in the clinic. PK profiles from clinical researches of different dosages were used to verify the bupropion model. Reasonable PK profiles of bupropion and its metabolites were captured in the PBPK model. Confidence in the DDI prediction involving bupropion and co-administered CYP2D6 substrates could be maximized. The predicted maximum concentration (Cmax) area under the concentration-time curve (AUC) values and Cmax and AUC ratios were consistent with clinically observed data. The addition of the inhibitory metabolites into the PBPK model resulted in a more accurate prediction of DDIs (AUC and Cmax ratio) than that which only considered parent drug (bupropion) P450 inhibition. The simulation suggests that bupropion and its metabolites contribute to the DDI between bupropion and CYP2D6 substrates. The inhibitory potency from strong to weak is hydroxybupropion, threohydrobupropion, erythrohydrobupropion, and bupropion, respectively. The present bupropion PBPK model can be useful for predicting inhibition from bupropion in other clinical studies. This study highlights the need for caution and dosage adjustment when combining bupropion with medications metabolized by CYP2D6. It also demonstrates the feasibility of applying the PBPK approach to predict the DDI potential of drugs undergoing complex metabolism, especially in the DDI involving inhibitory metabolites.

Anticonvulsant, antidepressant-like activity of Abelmoschus manihot ethanol extract and its potential active components in vivo.

Depression is the most common psychiatric comorbidity in patients with epilepsy. Searching for antiepileptic (anticonvulsant) and antidepressant-like medicines from natural products is very important for the treatment of this disease. The flower of Abelmoschus manihot (Linn.) Medicus has been reported to have neuroprotective effect against cerebral ischemia injury. In order to further explore the activity of Abelmoschus manihot on the central nervous system, the anticonvulsant and antidepressant-like effects of Abelmoschus manihot ethanol extract (AMEE) as well as its potential active components in vivo was investigated in the present study. It was found that AMEE could protect mice against PTZ-induced clonic convulsions and mortality. AMEE could also decrease immobility time in the FST in mice. Furthermore, the potential active components of AMEE in rat brain were identified by ultra performance liquid chromatography-mass spectrometer (UPLC-MS). Five parent components including isoquercitrin, hyperoside, hibifolin, quercetin-3'-O-glucoside, quercetin and three metabolites were detected in rat brain after administration of AMEE. In conclusion, eight flavonoids were identified in rat brain after administration of AMEE; meanwhile, these flavonoids might represent the potential bioactive components of AMEE and contribute to its anticonvulsant and antidepressant-like activity in vivo.

Screening for in vitro metabolites of Abelmoschus manihot extract in intestinal bacteria by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Abelmoschus manihot has drawn much attention recently due to its potential beneficial health effects after oral administration. However, the metabolic fate of A. manihot in intestinal flora is not well understood. In this paper, we describe a strategy using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) with automated data analysis software (MetaboLynx™) for fast analysis of the metabolic profile of flavonoids from A. manihot in intestinal flora. The human and rat incubated samples collected 72 h in the anaerobic incubator were analyzed by UPLC-Q-TOF MS within 10 min. A total of 14 metabolites were identified in human and rat incubated solution compared with blank samples. The results indicated that hydrolysis, hydroxylation and acetylation were the major metabolic pathways of flavonoids in A. manihot extract in vitro. MS(E) was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of metabolites. This work demonstrated the potential of the UPLC-Q-TOF MS approach using Metabolynx for fast and automated identification of metabolites of natural product in intestinal flora.

Identification of the potential active components of Abelmoschus manihot in rat blood and kidney tissue by microdialysis combined with ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

In this paper, microdialysis combining with ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was applied to simultaneously identify components in blood and kidney dialysis after oral administration of Abelmoschus manihot extract. Microdialysis probe was implanted in the jugular vein and the kidney medulla, respectively; microdialysis samples were collected continuously, transferred to microtubes and analyzed by UPLC-Q-TOF/MS. The components in microdialysis samples were separated by an UPLC HSS T3 column and eluted with acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.4 mL/min. The results showed that unbound constituents in blood circulation of the rat include hyperoside, isoquercitrin, quercetin monoglucuronide, quercetin-3'-O-glucoside, quercetin, myricetin, and hibifolin while unbound constituents in kidney are hyperoside, isoquercitrin, quercetin monoglucuronide, which might be the potential active components in vivo. The developed method was simple and reliable, and could be adopted to rapidly screen and identify potential active components contributing to pharmacological effects of TCM and to better clarify its action mechanism.

Identification of hyperoside metabolites in rat using ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

In this paper, ultra performance liquid chromatography (UPLC)/quadrupole-time-of-flight mass spectrometry (QTOF) with automated data analysis software (Metabolynx™) were applied for fast analysis of hyperoside metabolites in rat after intravenous administration. MS(E) was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of 12 metabolites in rat plasma, urine and bile. The results indicated that methylation, sulfation and glucuronidation were the major metabolic pathways of hyperoside in vivo, and among them, 3'-O-methyl-hyperoside was confirmed by matching its fragmentation patterns with standard compound. The present study provided important information about the metabolism of hyperoside which will be helpful for fully understanding the mechanism of this compound's action. Furthermore, this work demonstrated the potential of the UPLC/QTOFMS approach using Metabolynx for fast and automated identification of metabolites of natural product.

UPLC-QTOF/MS-based screening and identification of the constituents and their metabolites in rat plasma and urine after oral administration of Glechoma longituba extract.

Glechoma longituba is a widely used traditional Chinese medicine (TCM) in treating various diseases; however, the in vivo integrated metabolism of its multiple bioactive components remains unknown. In this paper, ultra-performance liquid chromatography (UPLC) coupled to quadrupole time-of-flight (QTOF) and the MetaboLynx™ software combined with mass defect filtering (MDF) together provide unique high throughput capabilities for drug metabolism study, with excellent MS mass accuracy and enhanced MS(E) data acquisition. This rapid automated analysis method was successfully applied for screening and identification of the constituents absorbed and metabolized studies of G. longituba extract after oral administration to rats. The results showed that 21 parent components of G. longituba extract were absorbed into the blood circulation of the rats and a total of 80 metabolites of 9 parent compounds were tentatively detected in vivo by their MS spectra obtained at low or high collision energy scan with the comparison of the authentic standards and literature data. The developed method was simple and reliable, revealing that it could be used to rapid screen and identify the structures of active components responsible for pharmacological effects of G. longituba and to better clarify its action mechanism. This work suggests that the integrative metabolism approach makes a useful template for drug metabolism research of TCM.