a b s t r a c tInhaled aerosol dose models play critical roles in medicine, the regulation of air pollutants and basic research. The models fall into several categories: traditional, computational fluid dynamical (CFD), physiologically based pharmacokinetic (PBPK), empirical, semi-empirical, and "reference". Each type of model has its strengths and weaknesses, so multiple models are commonly used for practical applications. Aerosol dose models combine information on aerosol behavior and the anatomy and physiology of exposed human and laboratory animal subjects. Similar models are used for in-vitro studies. Several notable advances have been made in aerosol dose modeling in the past 80 years. The pioneers include Walter Findeisen, who in 1935 published the first traditional model and established the structure of modern models. His model combined aerosol behavior with simplified respiratory tract structures. Ewald Weibel established morphometric techniques for the lung in 1963 that are still used to develop data for modeling today. Advances in scanning techniques have similarly contributed to the knowledge of respiratory tract structure and its use in aerosol dose modeling. Several scientists and research groups have developed and advanced traditional, CFD, and PBPK models. Current issues under study include understanding individual and species differences; examining localized particle deposition; modeling non-ideal aerosols and nanoparticle behavior; linking the regions of the respiratory tract airways from nasal-oral to alveolar; and developing sophisticated supporting software. Although a complete history of inhaled aerosol dose modeling is far too extensive to cover here, selected highlights are described in this paper.

The pregnane X receptor (PXR) is an important transcriptional regulator of the expression of xenobiotic metabolism and transporter genes. The receptor is promiscuous, binding many structural classes of molecules that act as agonists at the ligand-binding domain, triggering up-regulation of genes, increasing the metabolism and excretion of therapeutic agents, and causing drug-drug interactions. It has been suggested that human PXR antagonists represent a means to counteract such interactions. Several azoles have been hypothesized to bind the activation function-2 (AF-2) surface on the exterior of PXR when agonists are concurrently bound in the ligand-binding domain. In the present study, we have derived novel computational models for PXR agonists using different series of imidazoles, steroids, and a set of diverse molecules with experimental PXR agonist binding data. We have additionally defined a novel pharmacophore for the steroidal agonist site. All agonist pharmacophores showed that hydrophobic features are predominant. In contrast, a qualitative comparison with the corresponding PXR antagonist pharmacophore models using azoles and biphenyls showed that they are smaller and hydrophobic with increased emphasis on hydrogen bonding features. Azole antagonists were docked into a proposed hydrophobic binding pocket on the outer surface at the AF-2 site and fitted comfortably, making interactions with key amino acids involved in charge clamping. Combining computational and experimental data for different classes of molecules provided strong evidence for agonists and antagonists binding distinct regions on PXR. These observations bear significant implications for future discovery of molecules that are more selective and potent antagonists.

Very few antagonists have been identified for the human pregnane X receptor (PXR). These molecules may be of use for modulating the effects of therapeutic drugs, which are potent agonists for this receptor (e.g., some anticancer compounds and macrolide antibiotics), with subsequent effects on transcriptional regulation of xenobiotic metabolism and transporter genes. A recent novel pharmacophore for PXR antagonists was developed using three azoles and consisted of two hydrogen bond acceptor regions and two hydrophobic features. This pharmacophore also suggested an overall small binding site that was identified on the outer surface of the receptor at the AF-2 site and validated by docking studies. Using computational approaches to search libraries of known drugs or commercially available molecules is preferred over random screening. We have now described several new smaller antagonists of PXR discovered with the antagonist pharmacophore with in vitro activity in the low micromolar range [S-p-tolyl 3Ј,5-dimethyl-3,5Ј-biisoxazole-4Ј-carbothioate (SPB03255) (IC 50 , 6.3 M) and 4-(3-chlorophenyl)-5-(2,4-dichlorobenzylthio)-4H-1,2,4-triazol-3-ol (SPB00574) (IC 50 , 24.8 M)]. We have also used our computational pharmacophore and docking tools to suggest that most of the known PXR antagonists, such as coumestrol and sulforaphane, could also interact on the outer surface of PXR at the AF-2 domain. The involvement of this domain was also suggested by further site-directed mutagenesis work. We have additionally described an FDA approved prodrug, leflunomide (IC 50 , 6.8 M), that seems to be a PXR antagonist in vitro. These observations are important for predicting whether further molecules may interact with PXR as antagonists in vivo with potential therapeutic applications.Our knowledge of ligand-protein interactions for some of the nuclear hormone receptors is in the nascent stages. This has downstream implications for understanding, predicting and modulating the potential xenobiotic and environmental molecule effects on transcription of key genes in human. For example, the pregnane X receptor (PXR; NR1I2; also known as SXR or PAR) regulates multiple genes, including the enzymes CYP3A4 (Bertilsson et al., 1998;Blumberg et al., 1998;Kliewer et al., 1998), CYP2B6 (Goodwin et al., 2001, and CYP2C9 as well as the transporter P-glycoprotein (ABCB1) (Synold et al., 2001) and others. There is a very broad structural diversity in the molecules that bind to human PXR from bile salts (Schuetz and Strom, 2001;Krasowski et al., 2005) to anticancer compounds (Mani et al., 2005;Ekins et al., 2007). Several X-ray crystal structures of the ligand binding domain (LBD) of PXR (Watkins et al., 2001(Watkins et al., , 2002(Watkins et al., , 2003aXue et al., 2007b) have determined that S.E., N.A., V. K., and W.J.W. gratefully acknowledge the support for this work provided by the USEPA-funded Environmental Bioinformatics and Computational Toxicology Center (ebCTC), under STAR Grant number GAD R 832721-010. This work was supported in pa...

A dose-response curve for the hypoprothrombinemic effect of brodifacoum 3-[-3(4’-bromobiphenyl-4-yl) 1,2,3,4-tetrahydronaphth-1-yl]-4-hydroxycoumarin, was constructed using doses ranging from 0.1 to 0.33 mg/kg. Brodifacoum exhibited a remarkably steep dose-response curve. Brodifacoum failed to exhibit a dose-dependent effect on the degradation rate constant (k_deg) for prothrombin complex activity (PCA) after a PCA-synthesis-blocking dose of warfarin. Both phenobarbital pretreatment and SKF525A treatment altered to anticoagulant response to brodifacoum. Phenobarbital decreased the anticoagulant effect, whereas SKF525A increased it, suggesting that a substantial portion of brodifacoum-induced hypoprothrombinemia is mediated by brodifacoum itself rather than by metabolites. Finally, rats dosed orally with brodifacoum (0.2 mg/kg p.o.) were sacrificed in groups of 3–5 at various times up to 120 h after the dose. Brodifacoum was assayed in serum, small intestine, and liver by an HPLC method. Brodifacoum disappeared slowly from serum with a half-life of 156 h. Disappearance from small intestine was rapid, for 24 h, but intestinal levels began increasing from 24 to 72 h after the dose. Concentrations in liver were rapidly established, and exceeded serum concentrations by 20-fold. Brodifacoum levels in liver remained relatively constant for 96 h. Sustained liver concentrations after a single dose may partially account for brodifacoum’s apparent potency relative to warfarin.

ABSTRACT:We have investigated several in silico and in vitro methods to improve our ability to predict potential drug interactions of antibiotics. Our focus was to identify those antibiotics that activate pregnane X receptor (PXR) and induce CYP3A4 in human hepatocytes and intestinal cells. Human PXR activation was screened using reporter assays in HepG2 cells, kinetic measurements of PXR activation were made in DPX-2 cells, and induction of CYP3A4 expression and activity was verified by quantitative polymerase chain reaction, immunoblotting, and testosterone 6␤-hydroxylation in primary human hepatocytes and LS180 cells. We found that in HepG2 cells CYP3A4 transcription was activated strongly (>10-fold) by rifampin and troleandomycin; moderately (>7-fold) by dicloxacillin, tetracycline, clindamycin, griseofulvin, and (>4-fold) erythromycin; and weakly (>2.4-fold) by nafcillin, cefaclor, sulfisoxazole, and (>2-fold) cefadroxil and penicillin V. Similar although not identical results were obtained in DPX-2 cells. CYP3A4 mRNA and protein expression were induced by these antibiotics to differing extents in both liver and intestinal cells. CYP3A4 activity was significantly increased by rifampin (9.7-fold), nafcillin and dicloxacillin (5.9-fold), and weakly induced (2-fold) by tetracycline, sufisoxazole, troleandomycin, and clindamycin. Multiple pharmacophore models and docking indicated a good fit for dicloxacillin and nafcillin in PXR. These results suggest that in vitro and in silico methods can help to prioritize and identify antibiotics that are most likely to reduce exposures of medications (such as oral contraceptive agents) which interact with enzymes and transporters regulated by PXR. In summary, nafcillin, dicloxacillin, cephradine, tetracycline, sulfixoxazole, erythromycin, clindamycin, and griseofulvin exhibit a clear propensity to induce CYP3A4 and warrant further clinical investigation.