Tianyan Zhou

Genistein Induction of Human Sulfotransferases in HepG2 and Caco‐2 Cells

Sulfotransferases are phase II drug-metabolizing enzymes. While the induction of sulfotransferases by hormones and endogenous molecules is relatively well known, induction by xenobiotics is not well studied. Isoflavones are naturally occurring phyto-oestrogens, mainly existing in soy food products. They have been described as health-promoting, disease-preventing dietary supplements and as agents with cancer-preventive activities. Recently, isoflavones have been reported to interact with nuclear receptors, including those that are known to mediate the induction of drug-metabolizing enzymes. In the present investigation, the isoflavone genistein was shown to be a xenobiotic inducer of human sulfotransferases in transformed human liver cells (HepG2) and colon carcinoma cells (Caco-2). Enzymatic activity assay, Western blot, and real-time reverse transcription-polymerase chain reaction (RT-PCR) results demonstrated that genistein significantly induced protein and mRNA expression of human simple phenol sulfotransferase (hSULT1A1) and human dehydroepiandrosterone sulfotransferase (hSULT2A1) in HepG2 and Caco-2 cells. The induction was time-dependent and dose-dependent. Western blot results agreed well with real-time RT-PCR results, suggesting that induction occurred at the gene transcription level. This isoflavone is the first nutritionally related phyto-oestrogen shown to induce human sulfotransferases in HepG2 and Caco-2 cells.

Sulfation of dietary flavonoids by human sulfotransferases.

Dietary flavonoids catechin, epicatechin, eriodictyol, and hesperetin were investigated as substrates and inhibitors of human sulfotransferases (hSULTs). Purified recombinant proteins and human intestine cytosol were used as enzyme sources. hSULT1A1 and hSULT1A3 as well as human intestine cytosol can catalyse the sulfation of the investigated flavonoids. Sulfation of catechin, epicatechin, eriodictyol, and hesperetin by recombinant hSULTs showed substrate inhibition at high flavonoid concentrations. Hesperetin and eriodictyol are potent inhibitors of purified hSULT1A1, hSULT1A3, hSULT1E1, and hSULT2A1. Catechin and epicatechin inhibited hSULT1A1 and hSULT1A3, but not hSULT1E1 and hSULT2A1. The sulfation efficacy and potency of inhibition is related to the C-ring structure of flavonoids. These results suggest that dietary flavonoids may regulate human SULT activity and, therefore, affect the regulation of hormones and neurotransmitters, detoxification of drugs, and the bioactivation of pro- carcinogens and pro-mutagens.

Biochanin A induction of sulfotransferases in rats.

Biochanin A (BCA) is a dietary isoflavone present in red clover (Trifoliumn pretense) and many herbal products. BCA has been reported to have chemopreventive actions against various cancers including prostate, breast, colon cancer, and so on. Sulfotransferases are a family of phase II drug‐metabolizing enzymes, which are important for xenobiotic detoxification and regulation of biological signaling molecule biological activities. Sulfotransferase gene expressions are regulated by different hormones and xenobiotics. Improper regulation of sulfotransferases leads to improper functions of biological signaling molecules, which in turn can cause cancer or other diseases. BCA inhibits the enzyme activities of the phase I drug‐metabolizing enzymes CYP1A1 and CYP1B1 in Chinese hamster ovary cells and induces the phase II drug‐metabolizing enzymes UDP‐glucuronosyltransferases in human prostate cancer cells. BCA induction of sulfotransferases has not been studied. This investigation evaluates the in vivo regulation of sulfotransferases at protein and mRNA levels in the liver and intestine of Sprague‐Dawley rats treated with BCA (0, 2, 10, and 50 mg/kg/day) for 7 days. Our experimental results demonstrate for the first time that chronic BCA treatment can significantly induce the expression of rat sulfotransferase 1A1 (rSULT1A1, AST‐IV), sulfotransferase 2A1 (rSULT2A1, STa), and rat estrogen sulfotransferase (rSULT1E1, EST) in rat liver and intestine. Our Western blot results are in good agreement with real‐time RT‐PCR data, suggesting that BCA induction of sulfotransferases occurs at the transcriptional level.

Application of Model-Based Methods to Characterize Exenatide-Loaded Double-Walled Microspheres: In Vivo Release, Pharmacokinetic/Pharmacodynamic Model, and In Vitro and In Vivo Correlation

The objective of this study was to characterize exenatide double-walled microspheres (DWMS) using model-based methods. Exenatide DWMS were prepared using oil-in-oil-in-water method, and physicochemical characterization and in vitro release and degradation of DWMS were evaluated. The pharmacokinetics (PK) and pharmacodynamics (PD) were investigated after subcutaneous injection to diabetic rats. Transit compartment model was used to describe the in vivo release of exenatide from DWMS successfully. On the basis of the insulinotropic effects of exenatide and hypoglycemic effects of insulin, PK/PD model was developed and nicely described the concentration-effect relationship of exenatide. Moreover, on the basis of the transit compartment model, a simulation method was applied to predict in vivo release, and in vitro and in vivo correlation was established. In conclusion, DWMS was a promising vehicle for delivery of exenatide, and the proposed PK/PD model allowed a better understanding of the pharmacological properties of exenatide DWMS. Transit compartment model-based modeling and simulation methods provided more options for the description and prediction of the in vivo exenatide release from DWMS.

Preclinical Pharmacokinetic/Pharmacodynamic Models to Predict Schedule-Dependent Interaction Between Erlotinib and Gemcitabine

PurposeTo investigate the pharmacological effects of different erlotinib (ER) and gemcitabine (GM) combination schedules by in vitro and in vivo experiments and PK/PD models in non-small cell lung cancer cells.MethodsH1299 cells were exposed to different ER combined with GM schedules. Cell growth inhibition was analyzed to evaluate these schedules. A preclinical in vivo study was then conducted to compare tumor suppression effects of different schedules in H1299 xenografts. PK/PD models were developed to quantify the anti-tumor interaction of ER and GM.ResultsSynergism was observed when ER preceded GM, but other sequences showed antagonism. The optimal in vitro schedule, or interval schedule, was applied to the animal study, which showed greater anti-tumor effect than simultaneous group. PK/PD models implied that interaction of the two drugs was additive in simultaneous treatment but synergistic in interval schedule. The simulation results showed that interval schedule can delay tumor growth for a longer time, and demonstrated more evident anti-tumor effect compared with simultaneous group if the treatment duration was longer.ConclusionsInterval schedule of the two drugs can achieve synergistic anti-tumor effect, and is superior to simultaneous treatment.

Simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat plasma using HPLC with on-line solid-phase extraction

A HPLC method with on-line solid phase extraction (SPE) and DAD detection was developed for the simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat (SHR) plasma. Plasma samples (100 μL) were injected directly onto a CAPCELL MF C(8) SPE column. High-abundance proteins and most matrixes in plasma were removed by on-line SPE technology, while nitrendipine and hydrochlorothiazide trapped on the SPE column were effectively separated on a C(18) analytical column. The column temperature was maintained at 20°C. The optimal detection wavelength was 237 nm for NTDP and 271 nm for HCTZ. The total analytical run time was 34 min. The proposed method was linear over the range 5-500 ng mL(-1) for nitrendipine and 10-1000 ng mL(-1) for hydrochlorothiazide. The lower limit of detection (LLOD) was 0.5 and 0.6 ng mL(-1) for nitrendipine and hydrochlorothiazide, respectively. The sensitivity and precision of the method were within acceptable limits during validation period. The method was successfully used to investigate the pharmacokinetic characteristics of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rats.

A mechanism-based pharmacokinetic/pharmacodynamic model for CYP3A1/2 induction by dexamethasone in rats

Aim:To develop a pharmacokinetic/pharmacodynamic (PK/PD) model describing the receptor/gene-mediated induction of CYP3A1/2 by dexamethasone (DEX) in rats.Methods:A group of male Sprague-Dawley rats receiving DEX (100 mg/kg, ip) were sacrificed at various time points up to 60 h post-treatment. Their blood sample and liver were collected. The plasma concentration of DEX was determined with a reverse phase HPLC method. CYP3A1/2 mRNA, protein levels and enzyme activity were measured using RT-PCR, ELISA and the testosterone substrate assay, respectively. Data analyses were performed using a first-order conditional estimate (FOCE) with INTERACTION method in NONMEM version 7.1.2.Results:A two-compartment model with zero-order absorption was applied to describe the pharmacokinetic characteristics of DEX. Systemic clearance, the apparent volume of distribution and the duration of zero-order absorption were calculated to be 172.7 mL·kg−1·h−1, 657.4 mL/kg and 10.47 h, respectively. An indirect response model with a series of transit compartments was developed to describe the induction of CYP3A1/2 via PXR transactivation by DEX. The maximum induction of CYP3A1 and CYP3A2 mRNA levels was achieved, showing nearly 21.29- and 8.67-fold increases relative to the basal levels, respectively. The CYP3A1 and CYP3A2 protein levels were increased by 8.02-fold and 2.49-fold, respectively. The total enzyme activities of CYP3A1/2 were shown to increase by up to 2.79-fold, with a lag time of 40 h from the Tmax of the DEX plasma concentration. The final PK/PD model was able to recapitulate the delayed induction of CYP3A1/2 mRNA, protein and enzyme activity by DEX.Conclusion:A mechanism-based PK/PD model was developed to characterize the complex concentration-induction response relationship between DEX and CYP3A1/2 and to resolve the drug- and system-specific PK/PD parameters for the course of induction.

Caffeine induction of sulfotransferases in rat liver and intestine

Sulfotransferases (SULTs) are important phase II drug‐metabolizing enzymes. Regulation of SULTs by hormones and other endogenous molecules is relatively well understood, while xenobiotic induction of SULTs is not well studied. Caffeine is one of the most widely consumed psychoactive substances. However, SULT regulation by caffeine has not been reported. In this report, male and female rats were treated with different oral doses of caffeine (2, 10, 50 mg kg−1 per day) for 7 days. Western blot and real‐time RT‐PCR were used to investigate the changes in SULT protein and mRNA expression following the caffeine treatment. Caffeine induced both rat aryl sulfotransferase (rSULT1A1, AST‐IV) and rat hydroxysteroid sulfotransferase (rSULT2A1, STa) in the liver and intestine of female rats in a dose‐dependent manner. Caffeine induction of rSULT1A1 and rSULT2A1 in the female rat intestine was much stronger than that in the liver. Although caffeine induced rSULT1A1 significantly in the male rat liver, it did not significantly induce rSULT2A1. In male rat intestine, caffeine significantly induced rSULT2A1. The different SULTs induction patterns in male and female rats suggest that the regulation of rat SULTs by caffeine may be affected by different hormone secretion patterns and levels. Our results suggest that consumption of caffeine can induce drug metabolizing SULTs in drug detoxification tissues. Copyright

Pharmacokinetic/pharmacodynamic studies on exenatide in diabetic rats.

Aim:To quantitatively evaluate the blood glucose-lowering effect of exenatide in diabetic rats.Methods:Male Harlan-Sprague-Dawley rats were treated with high-fat diet/streptozotocin to induce type 2 diabetes. After subcutaneous administration of a single dose of exenatide (4.2, 42, or 210 μg/kg), serum exenatide, insulin concentration and blood glucose were measured. The pharmacokinetics of exenatide was characterized by a two-compartment model with first-order absorption. Insulin turnover was characterized by an effect compartment and indirect response combined model. Glucose turnover was described using an indirect response model with insulin (in effect compartment) stimulating glucose disposition and insulin (in insulin compartment) inhibiting glucose production simultaneously. The model parameters were estimated using nonlinear mixed-effects model program. Visual predictive check and model evaluation were used to make assessments.Results:Exenatide exhibited rapid absorption with ka=4.45 h-1, and the two-compartment model well described its pharmacokinetic profile. For the pharmacodynamic model, exenatide increased insulin release with the estimated Sm1 of 0.822 and SC50 of 4.02 μg/L. It was demonstrated that insulin stimulated glucose dissipation (Sm2=0.0513) and inhibited the production of glucose (Im=0.0381). Visual predictive check and model evaluation study indicated that a credible model was developed.Conclusion:The glucose-lowering effect of exenatide in diabetic rats is reliably described and predicted by the combined effect compartment/indirect response model.

Initial dosage regimens of vancomycin for Chinese adult patients based on population pharmacokinetic analysis.

OBJECTIVE To build a population pharmacokinetic model for Chinese adult patients and develop initial dosage regimens for patients with different degrees of renal function to achieve target steady-state trough concentrations in the range of 10 - 15 and 15 - 20 mg/l. METHOD Data on serum vancomycin concentration was collected from a retrospective study including 72 Chinese adult patients. NONMEM was used to build the population pharmacokinetic model, and a one-compartment model was chosen to describe the vancomycin concentration-time profile. Internal evaluation by bootstrap and visual predictive check (VPC) was performed to evaluate the robustness and prediction of the final model. Monte Carlo simulations were conducted to develop initial dosage regimens to achieve target trough concentrations. RESULTS A one-compartment model was built with creatinine clearance (CLcr) as the key covariate influencing drug clearance. The estimated drug clearance for patients with normal renal function (CLcr ≥ 80 ml/min) was 4.90 l/h, and 0.0654 × CLcr if CLcr was < 80 ml/min. The apparent volume of the central compartment was 47.76 l and no covariate was found to affect it. The results of bootstrap analysis were in agreement with the original parameters of the final model, and VPC of the final model demonstrated good predictability. Initial dosage regimens were developed based on the simulations of the population pharmacokinetic model. CONCLUSION A one-compartment model fitted the retrospective data and CLcr had a significant effect on drug clearance. Initial dosage regimens for vancomycin were proposed to provide some help to individual therapy for Chinese adult patients with different renal functions.