The Role of Breast Cancer Resistance Protein (BCRP) in Disposition of Glucuronide Conjugates

[Purpose] The overall objective is to explore and predict the impact of BCRP on disposition of glucuronides and to change the disposition of glucuronides by manipulating transporters. To approach this goal, three specific aims are: 1) To determine clearance by UGTs and clearance by BCRP for a series of compounds and their glucuronide metabolites; 2) To investigate the role of BCRP in disposition of glucuronide conjugates in vivo; 3) To increase the systemic exposure of glucuronides (and therefore the aglycones) by manipulating efflux transporters. [Methods] Human UGT1A9-overexpressing HeLa cells and MRP2-overexpressing MDCKII-UGT1A1 cells were used as in vitro cellular models to study the excretion of glucuronides by BCRP and MRP2, respectively. Pooled intestinal and hepatic S9 fractions were used to determine the intrinsic glucuronidation clearance. For in vivo studies, pharmacokinetic experiments were performed in wild-type (WT) and Bcrp1 (-/-) mice. A physiologically-based pharmacokinetic (i.e., PBPK) model containing seven organ compartments connected by arterial and venous blood supplies was constructed to predict pharmacokinetic profiles of parent compounds and their glucuronide metabolites. [Results] 1) The clearance by UGTs and the efflux clearance by BCRP were determined for eight compounds (daidzein, chrysin, maackiain, 3,6-DHF, resveratrol, genistein, sorafenib, and MPA) or their glucuronides. More than 4 orders of magnitude difference in glucuronidation clearance existed among these eight compounds. The rank orders of clearance values were almost the same in liver and in intestinal microsomes. Chrysin and maackiain had the highest glucuronidation clearances in mouse S9 fractions. Daidzein glucuronide had the highest clearance by BCRP in human UGT1A9-overexpressing HeLa cells, which was more than 30-fold higher than that of sorafenib glucuronide and maackiain glucuronide. A novel transport-glucuronidation classification system was proposed based on in vitro data. 2a) By using a newly developed and validated LC-MS/MS method, it was found that there was no significant change in systemic exposure of chrysin and its glucuronide, although the Tmax for chrysin glucuronide was significantly shorter (p<0.01) in Bcrp1 deficient mice. 2b) A PBPK model was developed to predict the impact of BCRP on glucuronide disposition for different compounds. For compounds with greater clearance by UGTs and the resulting glucuronides effluxed rapidly and predominantly by BCRP, significant increase in glucuronide AUC in Bcrp deficient mice was predicted and observed (daidzein and genistein). 3) An elevated systemic (i.e., blood) exposure of resveratrol glucuronides was achieved in wild-type and Bcrp1 knockout mice when animals were treated with curcumin, which was an inhibitor of efflux transporters. Curcumin achieved above-stated effects by inhibiting efflux transporters including MRP2 and BCRP, thereby promoting the distribution of resveratrol glucuronides into the systemic circulation (i.e., increased AUC and Cmax). [Conclusion] BCRP plays important role in disposition of glucuronide conjugates. In previous and current study, we observed that the extent of impact from BCRP varied among different compounds. By applying the newly developed transport-glucuronidation classification system and PBPK model, we for the first time demonstrated that the impact of BCRP on disposition of glucuronide can be predicted by knowing if a compound is subjected to fast or slow glucuronidation, with the resulting glucuronides subjected to rapid or slow efflux by BCRP. In addition, we showed that using curcumin as an inhibitor of efflux transporters could result in increased systemic exposure of resveratrol and its glucuronides in vivo, which could be expanded to other co-administered drugs using the similar disposition mechanisms.Pharmacological and Pharmaceutical Sciences, Department o

Determination of Pharmacokinetics of Chrysin and Its Conjugates in Wild-Type FVB and Bcrp1 Knockout Mice Using a Validated LC-MS/MS Method

Chrysin, a flavone found in many plants, is also available as a dietary supplement because of its reported anticancer activities. However, its bioavailability is very poor due to extensive phase II metabolism. The purpose of this study was to develop an UPLC-MS/MS method to simultaneously quantify chrysin and its phase II metabolites, and to determine its pharmacokinetics in FVB wild-type and Bcrp knockout (Bcrp1 −/−) mice. In addition, the role of BCRP in chrysin phase II disposition was further investigated in Caco-2 cells. The results showed that our sensitive and reproducible UPLC-MS/MS method was successfully applied to the pharmacokinetic study of chrysin in wild-type and Bcrp1 (−/−) FVB mice after oral administration (20 mg/kg). Although there was no significant change in systemic exposure of chrysin and its metabolites, it was found that the <i>T</i>_max for chrysin glucuronide was significantly shorter (<i>p</i> < 0.01) in Bcrp1-deficient mice. Furthermore, it was shown that inhibition of BCRP by Ko143 significantly reduced the efflux of chrysin sulfate in Caco-2 cells. In conclusion, BCRP had significant but less than expected impact on pharmacokinetics of chrysin and its conjugates, which were determined using a newly developed and validated LC-MS/MS method

Transport–Glucuronidation Classification System and PBPK Modeling: New Approach To Predict the Impact of Transporters on Disposition of Glucuronides

Glucuronide metabolites require the action of efflux transporters to exit cells due to their hydrophilic properties. In this study, we proposed a transport–glucuronidation classification system and developed a PBPK model to predict the impact of BCRP on systemic exposure of glucuronides. The clearance by UGTs in S9 fractions and the efflux clearance of glucuronides by BCRP in human UGT1A9-overexpressing HeLa cells were incorporated in the classification system and PBPK model. Based on simulations for glucuronide AUC for theoretical compounds in the classification system, it was indicated that BCRP was more important for compounds with greater efflux clearance of their glucuronides by BCRP regardless of differences in clearance by UGTs. Pharmacokinetic studies were performed in WT and Bcrp1 (−/−) mice for 8 compounds to verify our predictions. Among eight compounds, the glucuronide AUC of daidzein and genistein increased significantly in Bcrp1 (−/−) mice, while only slight increases in systemic exposure were observed for other glucuronides. The results from pharmacokinetic studies were in agreement with the predictions except for resveratrol, which was effluxed predominantly by transporters other than BCRP. Therefore, for glucuronides that were predominantly mediated by BCRP, this study provided a useful approach in predicting the impact of BCRP on its disposition and the potential DDIs involving BCRP

Role of Filler Shape and Connectivity on the Viscoelastic Behavior in Polymer Nanocomposites

We compare the rheological behavior of three classes of polymer nanocomposites (PNCs) to understand the role of particle shape and interactions on mechanical reinforcement. The first two correspond to favorably interacting composites formed by mixing poly­(2-vinylpyridine) with either fumed silica nanoparticles (NPs) or colloidal spherical silica NPs. We show that fumed silica NPs readily form a percolated network at low NP volume fractions. We deduce that the NPs act as network junctions with the effectively irreversibly bound polymer chains serving as the connecting bridges. By comparing with colloidal spherical silica, which has a significantly higher percolation threshold, we conclude that the fractal shape of the fumed silica is responsible for its unusually low percolation threshold. The third system corresponds to polystyrene grafted colloidal silica nanoparticles (PGNPs) in a polystyrene matrix. These PNCs have an even lower percolation threshold probably because the grafted chains increase the effective volume fraction of the NPs. When we take these different thickness of the polymer layers in the two cases into account (i.e., grafted layer vs adsorbed layer thickness), the percolation threshold for the fumed and the grafted system occurs at similar effective loadings, but the NP network with fumed silica has a higher low-frequency plateau modulus than that formed with the PGNPs. These findings can be reconciled by the fact that the fumed silica NPs are composed of fused entities, thus ensuring that they have a higher modulus than the PGNPs where the modulus is largely attributed to interactions between the grafts. Our results systematically stress the important role of the nanofiller shape and connectivity on the mechanical reinforcement of PNCs

Population Pharmacokinetics of Metoclopramide in Infants, Children, and Adolescents.

Metoclopramide is commonly used for gastroesophageal reflux. The aims of the present study were to develop a pediatric population pharmacokinetic (PopPK) model, which was applied to simulate the metoclopramide exposure following dosing used in clinical practice. Opportunistic pharmacokinetic data were collected from pediatric patients receiving enteral or parenteral metoclopramide per standard of care and these data were simultaneously fitted using NONMEM. Allometric scaling with body weight was included a priori in the model. Using the final model, the steady-state maximum concentrations (Css,max ) and the area under the metoclopramide plasma concentration-time curve at steady state from 0 to 6 hours (AUCss,0-6h ) were simulated following 0.1 or 0.15 mg/kg orally every 6 hours in virtual patients, and compared with previously reported ranges associated with toxicity or the efficacy for gastroesophageal reflux in infants. A two-compartment model with first-order absorption best characterized 87 concentration measurements from 50 patients (median [range] postnatal age of 8.89 years [0.01-19.13]). There were 20 infants (≤ 2 years), 9 children (2 years to age ≤ 12 years), and 21 adolescents (&gt; 12 years). Body weight was the only covariate included in the final model. For &gt; 75% of virtual patients, simulated Css,max and AUCss,0-6h estimates were within the range associated with efficacy for gastroesophageal reflux in infants; however, slightly lower exposures were predicted in virtual patients &lt; 2 years. Our study suggests that a metoclopramide enteral dose of 0.1 mg/kg every 6 hours, which was previously recommended for pediatric patients, results in simulated exposure generally within suggested ranges for the treatment of gastroesophageal reflux

Population Pharmacokinetics of Metoclopramide in Infants, Children, and Adolescents.

Metoclopramide is commonly used for gastroesophageal reflux. The aims of the present study were to develop a pediatric population pharmacokinetic (PopPK) model, which was applied to simulate the metoclopramide exposure following dosing used in clinical practice. Opportunistic pharmacokinetic data were collected from pediatric patients receiving enteral or parenteral metoclopramide per standard of care and these data were simultaneously fitted using NONMEM. Allometric scaling with body weight was included a priori in the model. Using the final model, the steady-state maximum concentrations (Css,max ) and the area under the metoclopramide plasma concentration-time curve at steady state from 0 to 6 hours (AUCss,0-6h ) were simulated following 0.1 or 0.15 mg/kg orally every 6 hours in virtual patients, and compared with previously reported ranges associated with toxicity or the efficacy for gastroesophageal reflux in infants. A two-compartment model with first-order absorption best characterized 87 concentration measurements from 50 patients (median [range] postnatal age of 8.89 years [0.01-19.13]). There were 20 infants (≤ 2 years), 9 children (2 years to age ≤ 12 years), and 21 adolescents (&gt; 12 years). Body weight was the only covariate included in the final model. For &gt; 75% of virtual patients, simulated Css,max and AUCss,0-6h estimates were within the range associated with efficacy for gastroesophageal reflux in infants; however, slightly lower exposures were predicted in virtual patients &lt; 2 years. Our study suggests that a metoclopramide enteral dose of 0.1 mg/kg every 6 hours, which was previously recommended for pediatric patients, results in simulated exposure generally within suggested ranges for the treatment of gastroesophageal reflux