Efflux transporters in drug disposition during pregnancy.

Evidence-based dose selection of drugs in pregnant women has been lacking due to challenges in studying maternal-fetal pharmacokinetics. Hence, many drugs are administered off-label during pregnancy based on data obtained from non-pregnant women. During pregnancy, drug transporters play an important role in drug disposition along with known gestational age-dependent changes in physiology and drug-metabolizing enzymes. In this review, as Dr. Qingcheng Mao's former and current lab members, we summarize the collective contributions of Dr. Mao, who lost his life to cancer, focusing on the role of drug transporters in drug disposition during pregnancy. Dr. Mao and his team initiated their research by characterizing the structure of Breast Cancer Resistance Protein [BCRP, ATP-Binding Cassette (ABC) G2]. Subsequently, they have made significant contributions to the understanding of the role of BCRP and other transporters, particularly P-glycoprotein (P-gp/ABCB1), in the exposure of pregnant women and their fetuses to various drugs, including nitrofurantoin, glyburide, buprenorphine, bupropion, tetrahydrocannabinol, and their metabolites. This review also highlights the gestation- and pregnancy-dependent transporter expression at the blood-brain and blood-placenta barriers in mice. Significance Statement Dr. Qingcheng Mao and his team have made significant contributions to the investigation of the role of efflux transporters, especially P-glycoprotein and breast cancer resistance protein, in maternal-fetal exposure to many xenobiotics: nitrofurantoin, glyburide, buprenorphine, bupropion, tetrahydrocannabinol and their metabolites. Studies of individual compounds and the expression of transporters during gestation and pregnancy have improved the understanding of maternal-fetal pharmacokinetics.

Bacterial metabolism rescues the inhibition of intestinal drug absorption by food and drug additives.

Food and drug products contain diverse and abundant small-molecule additives (excipients) with unclear impacts on human physiology, drug safety, and response. Here, we evaluate their potential impact on intestinal drug absorption. By screening 136 unique compounds for inhibition of the key intestinal transporter OATP2B1 we identified and validated 24 potent OATP2B1 inhibitors, characterized by higher molecular weight and hydrophobicity compared to poor or noninhibitors. OATP2B1 inhibitors were also enriched for dyes, including 8 azo (R-N=N-R') dyes. Pharmacokinetic studies in mice confirmed that FD&C Red No. 40, a common azo dye excipient and a potent inhibitor of OATP2B1, decreased the plasma level of the OATP2B1 substrate fexofenadine, suggesting that FD&C Red No. 40 has the potential to block drug absorption through OATP2B1 inhibition in vivo. However, the gut microbiomes of multiple unrelated healthy individuals as well as diverse human gut bacterial isolates were capable of inactivating the identified azo dye excipients, producing metabolites that no longer inhibit OATP2B1 transport. These results support a beneficial role for the microbiome in limiting the unintended effects of food and drug additives in the intestine and provide a framework for the data-driven selection of excipients. Furthermore, the ubiquity and genetic diversity of gut bacterial azoreductases coupled to experiments in conventionally raised and gnotobiotic mice suggest that variations in gut microbial community structure may be less important to consider relative to the high concentrations of azo dyes in food products, which have the potential to saturate gut bacterial enzymatic activity.

Phase Behavior and Crystallization Kinetics of a Poorly Water-Soluble Weakly Basic Drug as a Function of Supersaturation and Media Composition.

Understanding the supersaturation and precipitation behavior of poorly water-soluble compounds in vivo and the impact on oral absorption is critical to design consistently performing products with optimized bioavailability. Weakly basic compounds are of particular importance in this context since they have an inherent tendency to undergo supersaturation in vivo upon exit from the stomach and entry into the small intestine because of their pH-dependent solubility. To understand and probe potential in vivo variability of supersaturating systems, rigorous understanding of compound physical properties and phase behavior landscape is essential. Herein, we extensively characterize the solution phase behavior of a model, poorly soluble and weakly basic compound, posaconazole. Phase boundaries for crystal-solution and amorphous-solution were established as a function of pH, allowing possible phase transformations, namely, crystallization or liquid-liquid phase separation, to be mapped for different initial doses and fluid volumes. Endogenous surfactants including sodium taurocholate, lecithin, glycerol monooleate, and sodium oleate in biorelevant media significantly extended the phase boundaries due to solubilization, to an extent that was dependent on the concentration of the surface-active agents. The nucleation induction time of posaconazole was much shorter in biorelevant media in comparison to the corresponding buffer solution, with two distinct regions observed in all media that could be attributed to a change in the nucleation mechanism at high and low supersaturation. The presence of undissolved nanocrystals accelerated the desupersaturation. This work enhances our understanding of biorelevant factors impacting precipitation kinetics, which might affect absorption in vivo. It is expected that findings from this study with posaconazole could be broadly applicable to other weakly basic compounds, after taking into consideration differences in pKa, solubility, and molecular structure.

A Bayesian population physiologically based pharmacokinetic absorption modeling approach to support generic drug development: application to bupropion hydrochloride oral dosage forms.

We propose a Bayesian population modeling and virtual bioequivalence assessment approach to establishing dissolution specifications for oral dosage forms. A generalizable semi-physiologically based pharmacokinetic absorption model with six gut segments and liver, connected to a two-compartment model of systemic disposition for bupropion hydrochloride oral dosage forms was developed. Prior information on model parameters for gut physiology, bupropion physicochemical properties, and drug product properties were obtained from the literature. The release of bupropion hydrochloride from immediate-, sustained- and extended-release oral dosage forms was described by a Weibull function. In vitro dissolution data were used to assign priors to the in vivo release properties of the three bupropion formulations. We applied global sensitivity analysis to identify the influential parameters for plasma bupropion concentrations and calibrated them. To quantify inter- and intra-individual variability, plasma concentration profiles in healthy volunteers that received the three dosage forms, each at two doses, were used. The calibrated model was in good agreement with both in vitro dissolution and in vivo exposure data. Markov Chain Monte Carlo samples from the joint posterior parameter distribution were used to simulate virtual crossover clinical trials for each formulation with distinct drug dissolution profiles. For each trial, an allowable range of dissolution parameters ("safe space") in which bioequivalence can be anticipated was established. These findings can be used to assure consistent product performance throughout the drug product life-cycle and to support manufacturing changes. Our framework provides a comprehensive approach to support decision-making in drug product development.

Characterization of iron walnut in different regions of China based on phytochemical composition.

Little is known about the phytochemical composition of iron walnuts. Differences in the geographical origin of iron walnuts associated with economic benefits should also be examined. In this study, the phytochemical composition (fatty acids, Vitamin E, total polyphenols and flavonoids, amino acids, and minerals) of iron walnuts in China was investigated. The results showed that there were significant differences (p < 0.05) in the phytochemical composition of iron walnut oils and flours from different regions. Positive (r > 0.5, p < 0.05) and negative (r < - 0.5, p < 0.05) correlations were found between amino acids/minerals and amino acids/oleic acid, with the highest correlation coefficient (r = 0.742, p < 0.05) between Cu and tyrosine. In addition, based on the 12 phytochemical fingerprints selected by random forest, a geographical-origin identification model for iron walnuts was established, with a corresponding correct classification rate of 96.6%. The top three phytochemical fingerprints for the geographical-origin identification of iron walnut were microelements, macroelements, and antioxidant composition, with contribution rates of 61.7%, 18.1%, and 9.9%, respectively.

Interactions of Oral Molecular Excipients with Breast Cancer Resistance Protein, BCRP.

Mechanistic-understanding-based selection of excipients may improve formulation development strategies for generic drug products and potentially accelerate their approval. Our study aimed at investigating the effects of molecular excipients present in orally administered FDA-approved drug products on the intestinal efflux transporter, BCRP (ABCG2), which plays a critical role in drug absorption with potential implications on drug safety and efficacy. We determined the interactions of 136 oral molecular excipients with BCRP in isolated membrane vesicles and identified 26 excipients as BCRP inhibitors with IC50 values less than 5 µM using 3H-cholecystokinin octapeptide (3H-CCK8). These BCRP inhibitors belonged to three functional categories of excipients: dyes, surfactants, and flavoring agents. Compared with noninhibitors, BCRP inhibitors had significantly higher molecular weights and SLogP values. The inhibitory effects of excipients identified in membrane vesicles were also evaluated in BCRP-overexpressing HEK293 cells at similar concentrations. Only 1 of the 26 inhibitors of BCRP identified in vesicles inhibited BCRP-mediated 3H-oxypurinol uptake by more than 50%, consistent with the notion that BCRP inhibition depends on transmembrane or intracellular availability of the inhibitors. Collectively, the results of this study provide new information on excipient selection during the development of drug products with active pharmaceutical ingredients that are BCRP substrates.

Evaluation of risk levels of trace elements in walnuts from China and their influence factors: Planting area and cultivar.

The potential health risk of trace elements in nut foods has been widely concerned. The accumulations of trace elements by plants in the environment are disturbed by multiple factors. The objective of this work was to investigate the risk levels of trace elements in walnuts and their influence factors (planting area and cultivar). A total of 228 walnut samples were collected from four major walnut producing areas of China. The contents of essential elements for Fe, Cu, Zn, Mo and Se were 35.8, 21.9, 14.8, 0.3 and 0.04 mg/kg, respectively. The contribution of Cu for dietary reference intake was as high as 82.22%. The risk levels of potential toxicological elements and toxic elements within the acceptable limits. Significantly, the hazard quotients (HQs) of Ba and Co were up to 26.14% and 25.31%, respectively. The effect of planting area on trace elements was determined from the aspects of regional distribution and urbanization. Significant differences of essential elements were found between northeast and northwest areas. The urbanization directly influenced toxic elements, which could cause variation up to 85.0% (Pb) and 42.9% (As). Finally, cultivar effect was confirmed that all walnut cultivars were divided into four categories with different trace element characteristics.

Risk assessment and prediction for toxic heavy metals in chestnut and growth soil from China.

BACKGROUND: Toxic heavy metals (THMs) cause severe environmental hazards and threaten human health through various consumption of food stuff. However, little is known of THMs in chestnuts. In this study, the risk assessment and prediction of THMs [lead (Pb), arsenic (As), chromium (Cr), cadmium (Cd) and mercury (Hg)] in chestnuts and growth soils from China were investigated. RESULTS: The main detected THMs in chestnuts and growth soils were As and Cd. The total pollution levels of the five THMs (Nemerow pollution indexes, NPIs) were 0.062 and 1.06, respectively. The dietary risks for children were higher than those of adults, especially short-term non-carcinogenic risk. The main combined risks from the relationships between THMs were Pb-Cr (r = 0.85, P < 0.01) in chestnuts and Pb-As (r = 0.59, P < 0.01) in growth soils. The risk source was found to be the uptake effect of THMs from soil to chestnut, with the highest bioaccumulation factors (BCFs) of Cd (0.254). Several comprehensive risk models were established with the highest coefficient of determination (R2 ) of 0.79. In addition, the main contribution rates of different soil parameters to comprehensive risk of THMs uptake were 49.8% (Cd), 23.4% (pH), 13.8% (Cr) and 13.0% (organic carbon). CONCLUSION: The total pollution levels of THMs fell outside of the safety domain in growth soils. Furthermore, more attention needs to be paid to Cd pollution owing to its low environment background value and high accumulation ability. Three main soil parameters (Cr content, pH, organic carbon) played important roles in the formations and accumulations of THMs in chestnuts. © 2019 Society of Chemical Industry.

Environmental behaviors of phoxim with two formulations in bamboo forest under soil surface mulching.

Phoxim (emulsifiable concentrate (EC) and granules (G)) has been widely used in bamboo forests. The persistence and magnitude of phoxim residues in the crop and soil must be investigated to ensure human and environmental safety. The environmental behaviors of the two formulations were investigated in a bamboo forest under soil surface mulching conditions (CP) and non-covered cultivation conditions (NCP). The half-lives of phoxim in soil under the two conditions in soil were 4.1-6.2days (EC) and 31.5-49.5days (G), respectively. Phoxim in EC could be leached from the topsoil into the subsoil. A minimized leaching effect was observed for G under NCP. Inversely, an enhanced leaching effect was observed for G under CP. The G formulation resulted in more parent compound (in bamboo shoots) and metabolite (in soil) residues of phoxim than in the case of EC, especially under CP conditions. In addition, the intensity and duration of the formulation effect on soil pH adjustment from G were more obvious than that from EC. Results showed that the environmental behaviors (distribution, degradation, residue) of phoxim in the bamboo forest were significantly influenced by the type of formulation. The prolongation effect from phoxim G might cause persistence and long-term environmental risk. However, bamboo shoot consumption could be considered relatively safe after applying the recommended dose of the two phoxim formulations.

Quantitative methods and modeling to assess COVID-19-interrupted in vivo pharmacokinetic bioequivalence studies with two reference batches.

The coronavirus disease 2019 (COVID-19) has presented unprecedented challenges to the generic drug development, including interruptions in bioequivalence (BE) studies. Per guidance published by the US Food and Drug Administration (FDA) during the COVID-19 public health emergency, any protocol changes or alternative statistical analysis plan for COVID-19-interrupted BE study should be accompanied with adequate justifications and not lead to biased equivalence determination. In this study, we used a modeling and simulation approach to assess the potential impact of study outcomes when two different batches of a Reference Standard (RS) were to be used in an in vivo pharmacokinetic BE study due to the RS expiration during the COVID-19 pandemic. Simulations were performed with hypothetical drugs under two scenarios: (1) uninterrupted study using a single batch of an RS, and (2) interrupted study using two batches of an RS. The acceptability of BE outcomes was evaluated by comparing the results obtained from interrupted studies with those from uninterrupted studies. The simulation results demonstrated that using a conventional statistical approach to evaluate BE for COVID-19-interrupted studies may be acceptable based on the pooled data from two batches. An alternative statistical method which includes a "batch" effect to the mixed effects model may be used when a significant "batch" effect was found in interrupted four-way crossover studies. However, such alternative method is not applicable for interrupted two-way crossover studies. Overall, the simulated scenarios are only for demonstration purpose, the acceptability of BE outcomes for the COVID19-interrupted studies could be case-specific.

Percutaneous absorption of steroids from finite doses: Predicting urinary excretion from in vitro skin permeation testing.

Pharmacokinetic (PK) models are widely used to describe drug permeation across the epidermal membrane barrier, the stratum corneum (SC). Here, we extend our previously reported diffusion and compartment-in-series models to describe plasma concentrations, urinary excretion-time profiles and exposure estimates after topically applied finite doses of solvent deposited solids. In vivo models were derived by convolution of a skin absorption input function for finite dosing with that for in vivo disposition PK. In vitro skin permeation test (IVPT) and in vivo urinary excretion data for cortisone, desoxycorticosterone, and testosterone were extracted from literature for model validation and establishment of in vitro - in vivo relationships (IVIVR). Both SC diffusion and SC 3-compartment-in-series PK models adequately described experimental in vitro and in vivo permeation data, with similar model parameter estimates for SC diffusion time and bioavailability. A satisfactory IVIVR was generated for cortisone, whereas testosterone and desoxycorticosterone showed higher bioavailability in vitro compared to in vivo. In recognising that future prospective studies need to both have an adequate sampling schedule and be harmonized for robust IVIVRs, we developed expressions for predicting extent of absorption and time for peak absorption for both in vitro and in vivo studies. Other study parameters, such as application site, applied dose, and application techniques, can also affect drug permeability through skin during dosage form metamorphosis after finite dose application, and a lack of correlation may result if these are poorly matched.

Identification of proline residues in or near the transmembrane helices of the human breast cancer resistance protein (BCRP/ABCG2) that are important for transport activity and substrate specificity.

The human breast cancer resistance protein (BCRP/ABCG2) confers multidrug resistance and mediates the active efflux of drugs and xenobiotics. BCRP contains one nucleotide-binding domain (NBD) followed by one membrane-spanning domain (MSD). We investigated whether prolines in or near the transmembrane helices are essential for BCRP function. Six proline residues were substituted with alanine individually, and the mutants were stably expressed in Flp-In(TM)-293 cells at levels comparable to that of wild-type BCRP and predominantly localized on the plasma membrane of the cells. While P392A showed a significant reduction (35-50%) in the efflux activity of mitoxantrone, BODIPY-prazosin, and Hoechst 33342, P485A exhibited a significant decrease of approximately 70% in the efflux activity of only BODIPY-prazosin. Other mutants had no significant changes in the efflux activities of these substrates. Drug resistance profiles of the cells expressing the mutants correlated well with the efflux data. ATPase activity was not substantially affected for P392A or P485A compared to that of wild-type BCRP. These results strongly suggest Pro(392) and Pro(485) are important in determining the overall transport activity and substrate selectivity of BCRP, respectively. Prazosin differentially affected the binding of 5D3, a conformation-sensitive antibody, to wild-type BCRP, P392A, or P485A in a concentration-dependent manner. In contrast, mitoxantrone had no significant effect on 5D3 binding. Homology modeling indicates that Pro(392) may play an important role in the communication between the MSD and NBD as it is predicted to be located at the interface between the two functional domains, and Pro(485) induces flexible hinges that may be essential for the broad substrate specificity of BCRP.

Heavy metals in bamboo shoots from Southeastern China and risk assessment.

Bamboo shoot is an indispensable vegetable in Southeastern China, so a survey of heavy metals in bamboo shoots is a relevant topic of interest. The present study sought to analyse the content of seven heavy metals (As, Cd, Cr, Cu, Mn, Pb and Zn) in six bamboo shoot species from Southeastern China. Percentages of 3.8% (Cd) and 8.9% (Pb) of the samples exceeded the maximum limits as established by the Chinese legislation. Further health risk assessment exhibited that the Cd intake contribution of these samples could not be negligible. The hazard index (HI) at average exposure was less than one. The average HI values in moso bamboo shoot were significantly higher than those of other bamboo shoot species. Overall, the results indicated that the intake of bamboo shoots is safe for consumers in Southeastern China.

Physiologically Based Pharmacokinetic Modeling Approach to Identify the Drug-Drug Interaction Mechanism of Nifedipine and a Proton Pump Inhibitor, Omeprazole.

BACKGROUND AND OBJECTIVES: Proton pump inhibitors (PPIs) can affect the intragastric release of other drugs from their dosage forms by elevating the gastric pH. They may also influence drug absorption and metabolism by interacting with P-glycoprotein or with the cytochrome P450 (CYP) enzyme system. Nifedipine is a Biopharmaceutics Classification System (BCS) class II drug with low solubility across physiologic pH and high permeability. Previous studies have demonstrated that drug-drug interaction (DDI) existed between omeprazole and nifedipine with significantly increased systemic exposure of nifedipine in subjects after pre-treatment for 7 days with omeprazole compared to the subjects without omeprazole treatment. It was shown that omeprazole not only induced an increase in intragastric pH, but also inhibited the CYP3A4 activity, while CYP3A4-mediated oxidation is the main metabolic pathway of nifedipine. The purpose of this study is to apply a physiologically based pharmacokinetic (PBPK) modeling approach to investigate the DDI mechanism for an immediate release formulation of nifedipine with omeprazole. METHODS: A previously published model for omeprazole was modified to integrate metabolites and to update CYP inhibition based on the most updated published in vitro data. We simulated the nifedipine pharmacokinetics in healthy subjects with or without the multiple-dose pretreatment of omeprazole (20 mg) following oral administrations of immediate-release (IR) (10 mg) nifedipine. Nifedipine solubility at different pHs was used to simulate the nifedipine pharmacokinetics for both clinical arms. Multiple sensitivity analyses were performed to understand the impact of gastric pH and the CYP3A4-mediated gut and liver first pass metabolism on the overall nifedipine pharmacokinetics. RESULTS: The developed PBPK model properly described the pharmacokinetics of nifedipine and predicted the inhibitory effect of multiple-dose omeprazole on CYP3A4 activity. With the incorporation of the physiologic effect of omeprazole on both gastric pH and CYP3A4 to the PBPK model, the verified PBPK model allows evaluating the impact of the increase in gastric pH and/or CYP3A4 inhibition. The simulated results show that the nifedipine metabolic inhibition by omeprazole may play an important role in the DDI between nifedipine and omeprazole for IR nifedipine formulation. CONCLUSION: The developed full PBPK model with the capability to simulate DDI by considering gastric pH change and metabolic inhibition provides a mechanistic understanding of the observed DDI of nifedipine with a PPI, omeprazole.

Interaction of Commonly Used Oral Molecular Excipients with P-glycoprotein.

P-glycoprotein (P-gp) plays a critical role in drug oral bioavailability, and modulation of this transporter can alter the safety and/or efficacy profile of substrate drugs. Individual oral molecular excipients that inhibit P-gp function have been considered a mechanism for improving drug absorption, but a systematic evaluation of the interaction of excipients with P-gp is critical for informed selection of optimal formulations of proprietary and generic drug products. A library of 123 oral molecular excipients was screened for their ability to inhibit P-gp in two orthogonal cell-based assays. ß-Cyclodextrin and light green SF yellowish were identified as modest inhibitors of P-gp with IC50 values of 168 µM (95% CI, 118-251 µM) and 204 µM (95% CI, 5.9-1745 µM), respectively. The lack of effect of most of the tested excipients on P-gp transport provides a wide selection of excipients for inclusion in oral formulations with minimal risk of influencing the oral bioavailability of P-gp substrates.

Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport.

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane α-helices 1 and 6 of BCRP in drug transport. We substituted Asn(387), Gln(398), Asn(629), and Thr(642) with Ala, Thr(402) with Ala and Arg, and Tyr(645) with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that Thr(402) within transmembrane 1 may be important for helical interactions, and Asn(629) may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity.

Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport.

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics out of cells. In this study, we investigated the role of five basic residues within or near transmembrane (TM) 2 of BCRP in transport activity. Lys(452), Lys(453), His(457), Arg(465), and Lys(473) were replaced with Ala or Asp. K452A, K453D, H457A, R465A, and K473A were stably expressed in human embryonic kidney (HEK) cells, and their plasma membrane expression and transport activities were examined. All of the mutants were expressed predominantly on the plasma membrane of HEK cells. After normalization to BCRP levels, the activities of K452A and H457A in effluxing mitoxantrone, boron-dipyrromethene-prazosin, and Hoechst33342 were increased approximately 2- to 6-fold compared with those of wild-type BCRP, whereas the activities of K453D and R465A were decreased by 40 to 60%. Likewise, K452A and H457A conferred increased resistance to mitoxantrone and 7-ethyl-10-hydroxy-camptothecin (SN-38), and K453D and R465A exhibited lower resistance. The transport activities and drug-resistance profiles of K473A were not changed. These mutations also differentially affected BCRP ATPase activities with a 2- to 4-fold increase in V(max)/K(m) for K452A and H457A and a 40 to 70% decrease for K453D and R465A. These mutations may induce conformational changes as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin and altered trypsin digestion. Molecular modeling and docking calculations indicated that His(457) and Arg(465) might be directly involved in substrate binding. In conclusion, we have identified several basic residues within or near TM2 that may be important for interaction of substrates with BCRP.

Diffusion modelling of percutaneous absorption kinetics. Predicting urinary excretion from in vitro skin permeation tests (IVPT) for an infinite dose.

In this work, we developed a number of generalised skin diffusion based pharmacokinetic models to relate published in vivo urinary excretion data to matching experimentally generated in vitro human skin permeation test (IVPT) data for a series of topically applied salicylate esters. A simplified linear in vivo model was found to inadequately describe the time course of urinary excretion over the entire sampling period. We represented the skin barrier as both a one layer (stratum corneum) and a two-layer (stratum corneum with viable epidermis) diffusion model and convoluted their Laplace solutions with that for a single exponential disposition phase to describe the urinary excretion profiles in the Laplace domain. We also derived asymptotic approximations for the model and estimated the conditions under which they could be used. We then sought to develop in vitro - in vivo relationships (IVIVR) for topically applied methyl, ethyl and glycol salicylates using our experimental IVPT data and the literature urinary excretion data. Good linear IVIVRs for ethyl and glycol salicylates were obtained, but the IVIVR for methyl salicylate was poor, perhaps because of topical stimulation of local skin blood flow by methyl salicylate. The ratio of the hydrated to dehydrated skin permeation for all salicylate esters was the same in both the IVPT and in vivo studies. A diffusion based one compartment pharmacokinetic model was also developed to describe the urinary excretion of solutes after removal of topical products and to compare the methyl salicylate skin permeation for five different body sites. The work presented here is consistent with the development of skin IVIVRs, but suggests that different skin conditions, application sites and local skin effects may affect model predictions.

Membrane topology of the human breast cancer resistance protein (BCRP/ABCG2) determined by epitope insertion and immunofluorescence.

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and organic anions across the plasma membrane. Hydropathy analysis suggests that BCRP consists of a nucleotide-binding domain (residues approximately 1-395) and a membrane-spanning domain (MSD) (residues approximately 396-655); however, its exact topology structure remains unknown. In this study, we determined the topology structure of BCRP by inserting hemagglutinin (HA) tags in its predicted hydrophilic regions of the MSD. HA-tagged BCRP mutants were expressed in HEK cells and tested for their ability to efflux mitoxantrone and BODIPY-prazosin. Polarity of the inserted tags with respect to the plasma membrane was determined by immunofluorescence. All of the mutants were expressed at levels comparable to wild-type BCRP as revealed by immunoblotting with specific antibodies against BCRP and the HA tag. Insertions at residues 423, 454, 462, 499, 529, 532, and 651 produced functional mutants, whereas insertions at residues 560, 594, and 623 resulted in mutants with significantly reduced activity and insertions at residues 387, 420, 474, and 502 completely abrogated the activity. HA tags inserted at residues 387, 474, 529, 532, 560, and 651 were localized intracellularly, whereas those inserted at residues 420, 423, 454, 499, 502, 594, and 623 revealed an extracellular location. Residue 462 was localized in a transmembrane (TM) segment. These results provide the first direct experimental evidence in support of a 6-TM model for BCRP with the amino and carboxyl termini of the MSD located intracellularly. These data may have important implications for understanding the transport mechanism of BCRP.

Uptake effects of toxic heavy metals from growth soils into jujube and persimmon of China.

Compared with other agricultural plants, information about uptake effects of toxic heavy metals from growth soils into persimmon and jujube are scarce. In this study, the single and comprehensive uptake effects of five toxic heavy metals (Pb, As, Cd, Cr, and Hg) between the two fruits and their growth soils were investigated. The results showed that the average concentrations of heavy metals in the two fruits were found to be 30 (Pb), 6.6 (As), 2.3 (Cd), 38 (Cr), and 0.33 (Hg) µg/kg, respectively. The average concentrations of heavy metals in their growth soils were 26.31 (Pb), 9.63 (As), 0.12 (Cd), 57.6 (Cr), and 0.049 (Hg) mg/kg, respectively. An uptake effect was observed for the two fruits. The values of Nemerow pollution index (NPI) in the two fruits and their growth soils were 0.10 and 0.55, respectively. The average bioconcentration factor (BCF) values of Pb, As, Cd, Cr, and Hg in the two fruits were 0.0012, 0.00075, 0.021, 0.00077, and 0.012, respectively. Based on the residue levels of toxic heavy metals in the growth soils and soil parameters, the prediction models for NPI and BCF were established, with the adjusted regression coefficients of 0.65 (NPI) and 0.81 (BCF). The contribution rates of different soil parameters to NPI were 21.7% (OC), 16.1% (Pb), 17.1% (Cr), 19.8% (Cd), and 25.4% (As), respectively. The contribution rates of different soil parameters to BCF were 10.2% (OC), 9.4% (Cr), and 80.4% (Cd), respectively.