Two branched polar groups and polar linker moieties of thiophene amide derivatives are essential for MRP2/ABCC2 recognition.

Previously we demonstrated that the torsion angle between two biphenyl rings forming a three-dimensional conformation is the determinant factor for multi-drug resistance protein 2 (Mrp2/Abcc2) interaction [1]. More recently, we reported a heterocyclic compound, 1-(1-(4-bromophenyl)-3-carbamoyl-1H-pyrazol-4-yl) urea that shares the polar head groups with the biphenyl-substituted heterocycles, is highly secreted from bile by Mrp2/Abcc2, [2]. Collectively we hypothesized that the two branched polar groups and linkers might be essential with proposed Mrp2/Abcc2 recognition fitting in two primarily positive regions deep in the binding site. To test the hypothesis, a discovery lead compound (Compound 1) was examined to confirm the Mrp2/Abcc2 involvement resulting in hepatobiliary secretion in rats. The structural requirement of Mrp2/Abcc2 recognition was further explored in a series of thiophene amides derivatives divided into eight structural classes, with structural changes focused on the amide linker orientation or substitution from amide and sulfonamide to alkene, alkane, or alkyne linkers. In Caco-2 cell bidirectional transport assays and Mrp2/Abcc2 membrane vesicle uptake assays, the involvement of Mrp2/Abcc2 mediated transport was confirmed in structural classes 1 - 5, which contains polar amide or sulfonamide linker, but not in classes 6 - 8 with non-polar aliphatic linker. The Mrp2/Abcc2 recognition showed strong correlation with structural descriptors in predictive Bayesian model, as well as with polar surface area and lipophilicity (LogP). The result provided valuable information for predicting transporter recognition in silico, for improved predictions of transporter involved ADME in early drug discovery.

Novel metabolic bioactivation mechanism for a series of anti-inflammatory agents (2,5-diaminothiophene derivatives) mediated by cytochrome p450 enzymes.

The thiophene moiety is considered a structural alert in molecular design in drug discovery, largely because several thiophene-containing drugs, including tienilic acid and suprofen, have been withdrawn from the market because of toxicities. Reactive thiophene intermediates, activated via sulfur oxidation or ring epoxidation, are possible culprits for these adverse side effects. In this work, the metabolic activation of an anti-inflammatory agent, 1-(3-carbamoyl-5-(2,3,5-trichlorobenzamido)thiophen-2-yl)urea), containing a 2,5-diaminothiophene structure, was studied in liver microsomes in the presence of glutathione or N-acetylcysteine as trapping agents. In addition, the glutathione conjugate was detected in bile from a bile duct-cannulated rat study. The structure of the glutathione conjugate was identified by mass spectrometry and (1)H NMR. The glutathione molecule was attached to the thiophene ring, replacing the existing proton. Metabolic phenotyping experiments, using chemical inhibitors or recombinant cytochromes P450 (P450), demonstrated that CYP3A4 was the major P450 enzyme responsible for the metabolic activation, followed by CYP1A2, 2Cs, and 2D6. A novel metabolic activation mechanism is proposed whereby the 2,5-diaminothiophene moiety undergoes oxidation to a 2,5-diimine thiophene reactive intermediate. This mechanism was used to support efforts to eliminate reactive metabolite generation via structural modification of ring substituents using structure-activity relationships. The disruption of formation of the 2,5-diimine reactive intermediate resulted in the elimination of glutathione conjugate formation both in vitro and in vivo and provided a rational approach to mitigating potential safety risks associated with this class of thiophenes in drug research and development.

Euthanasia method for mice in rapid time-course pulmonary pharmacokinetic studies.

To develop a means of euthanasia to support rapid time-course pharmacokinetic studies in mice, we compared retroorbital and intravenous lateral tail vein injection of ketamine-xylazine with regard to preparation time, utility, tissue distribution, and time to onset of euthanasia. Tissue distribution and time to onset of euthanasia did not differ between administration methods. However, retroorbital injection could be performed more rapidly than intravenous injection and was considered to be a technically simple and superior alternative for mouse euthanasia. Retroorbital ketamine-xylazine, CO(2) gas, and intraperitoneal pentobarbital then were compared as euthanasia agents in a rapid time-point pharmacokinetic study. Retroorbital ketamine-xylazine was the most efficient and consistent of the 3 methods, with an average time to death of approximately 5 s after injection. In addition, euthanasia by retroorbital ketamine-xylazine enabled accurate sample collection at closely spaced time points and satisfied established criteria for acceptable euthanasia technique.

Saturation of multidrug-resistant protein 2 (mrp2/abcc2)-mediated hepatobiliary secretion: nonlinear pharmacokinetics of a heterocyclic compound in rats after intravenous bolus administration.

Multidrug-resistant protein 2 (MRP2/ABCC2), expressed on the canalicular membrane of hepatocytes, mediates the secretion of conjugated or nonconjugated compounds into bile and plays an important role in physiology and drug elimination. A heterocyclic compound, BPCPU [1-(1-(4-bromophenyl)-3-carbamoyl-1H-pyrazol-4-yl) urea], which was metabolically stable in vitro in rat liver microsomes and freshly isolated rat hepatocytes, demonstrated a saturable nonlinear pharmacokinetic profile in the rat. Polarized efflux was observed for this compound in Caco-2 cells, with a low K(m) = 1.06 +/- 0.06 microM. The Caco-2 efflux was dose-dependent and saturable. Coadministration of 25 microM MK571 ([3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]), an MRP inhibitor, blocked the polarized efflux in Caco-2 cells. In contrast, this compound did not inhibit calcein efflux in MRP2 gene-transfected Madin-Darby canine kidney cells, suggesting that it is a substrate, not an inhibitor, of the MRP2/ABCC2 transporter. To investigate the mechanism for the nonlinear pharmacokinetics, bile duct-cannulated rats were used to obtain time profiles of plasma concentration, biliary, and urinary excretion after intravenous administration at various doses. The plasma clearance increased remarkably with decreased dose, from 1.5 ml/min/kg at 5 mg/kg to 14.9 ml/min/kg at 0.05 mg/kg. A dose-dependent biliary excretion also was observed. The results revealed that saturation of hepatobiliary secretion played a role in the dose-dependent changes in total body clearance and biliary clearance. Saturating concentrations of the Mrp2/Abcc2 substrate, BPCPU, causing decreased hepatobiliary clearance could be the major cause for the nonlinear pharmacokinetics observed in rats.

Evaluation of Aerosol Delivery of Nanosuspension for Pre-clinical Pulmonary Drug Delivery.

Asthma and chronic obstructive pulmonary disease (COPD) are pulmonary diseases that are characterized by inflammatory cell infiltration, cytokine production, and airway hyper-reactivity. Most of the effector cells responsible for these pathologies reside in the lungs. One of the most direct ways to deliver drugs to the target cells is via the trachea. In a pre-clinical setting, this can be achieved via intratracheal (IT), intranasal (IN), or aerosol delivery in the desired animal model. In this study, we pioneered the aerosol delivery of a nanosuspension formulation in a rodent model. The efficiency of different dosing techniques and formulations to target the lungs were compared, and fluticasone was used as the model compound. For the aerosol particle size determination, a ten-stage cascade impactor was used. The mass median aerodynamic diameter (MMAD) was calculated based on the percent cumulative accumulation at each stage. Formulations with different particle size of fluticasone were made for evaluation. The compatibility of regular fluticasone suspension and nanosuspension for aerosol delivery was also investigated. The in vivo studies were conducted on mice with optimized setting. It was found that the aerosol delivery of fluticasone with nanosuspension was as efficient as intranasal (IN) dosing, and was able to achieve dose dependent lung deposition.