Optimizing Caco-2 cell monolayers to increase throughput in drug intestinal absorption analysis.

Introduction: The aim of this investigation was to evaluate methods for increasing Caco-2 cell throughput for assessing drug intestinal absorption. The use of 6-, 12-, and 24-well membranes and the effect of membrane size on permeability and the integrity of the Caco-2 cell monolayer were assessed. In an effort to optimize the assessment of drug permeability, increased throughput was investigated by testing compounds singly or as mixtures of analytes.

An improved resazurin-based cytotoxicity assay for hepatic cells.

A simple resazurin-based cytotoxicity assay is presented for screening of cytotoxicity in hepatocytes and liver cell lines. Human hepatoma (HepG2) cells in 96-well culture plates were exposed to known toxic (cisplatin, 5-fluorouracil, ethionine, flufenamic acid, and diflunisal) and control (transplatin, 5-chlorouracil, methionine, and acetylsalicylic acid) compounds for 1-3 days, and resazurin (5 micromol/L) was added. A conventional short-term (1 h) assay was first performed, where cytotoxicity is indicated by decreased reduction of resazurin to its fluorescent product resorufin.

MAP-2 immunolabeling can distinguish diffuse from dense-core amyloid plaques in brains with Alzheimer's disease.

Alzheimer's disease (AD) neuropathology is characterized by the presence of diffuse and dense-core (neuritic) amyloid plaques in specific areas of the brain. The origin of these plaques and the relationship between them is poorly understood. Current methods to identify clearly these types of plaques in the AD brains are largely dependent upon morphological characteristics.

Metabolism of the analgesic drug ULTRAM (tramadol hydrochloride) in humans: API-MS and MS/MS characterization of metabolites.

1. Metabolism of the analgesic agent tramadol hydrochloride has been investigated after a single oral administration of tramadol to three male volunteers (100 mg/subject), and a urine pool (4-12h) was obtained. 2.

A HPLC assay for coating integrity of topiramate sprinkle formulation.

A HPLC method has been developed for the determination of coating integrity of topiramate sprinkle formulation. This method determines the completeness of the sprinkle coating and, indirectly, the completeness of taste masking of the product. This method utilizes a sample preparation where the sprinkles are placed in a specially designed stainless steel basket equipped with a screen, 25-mesh size, at the bottom.

Evaluation of the excretion and metabolism of the new analgesic agent RWJ-22757 in male and female CR Wistar rats.

The excretion and metabolism of (+/-)-trans-3-(2-bromophenyl)octahydroindolizine hydrochloride (RWJ-22757) have been investigated in male and female CR Wistar rats. Radiolabeled [14C] RWJ-22757 was administered orally to each of the rats as a single 60 mg/kg suspension dose. Plasma (0-48 h), urine (0-168 h) and fecal (0-168 h) samples were collected and analyzed.

Lipofuscin and Abeta42 exhibit distinct distribution patterns in normal and Alzheimer's disease brains.

Our recent study has provided evidence that Abeta42, a 42 amino acid fragment of the amyloid precursor protein, accumulates intracellularly in vulnerable neurons. This study appears to show that neurons lyse and form dense-core amyloid plaques in Alzheimer's disease (AD) entorhinal cortex. Previous studies have suggested that intracellular Abeta42 co-localizes with lipofuscin in neurons and those increased levels of lipofuscin and Abeta42 are associated with AD.

Effects of transdermal estradiol delivered by a matrix patch on bone density in hysterectomized, postmenopausal women: a 2-year placebo-controlled trial.

This 2-year, double-masked, randomized, placebo-controlled trial was designed to evaluate the safety and efficacy in preventing bone loss in postmenopausal women of two doses of transdermal 17betaestradiol (estradiol) delivered by a matrix patch, compared with placebo. One hundred and sixty healthy, hysterectomized postmenopausal volunteers aged 40-60 years with serum estradiol levels < 20 pg/ml were started on treatment at four centers in The Netherlands. Every 6 months, bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) at the lumbar spine, non-dominant wrist and left hip, and markers of bone turnover were assessed in urine and serum.

The use of on-line and off-line chromatographic extraction techniques coupled with mass spectrometry for support of in vivo and in vitro assays in drug discovery.

We have investigated various sample chromatographic extraction and sample preparation methods for liquid chromatography mass spectrometry analysis in order to increase the throughput of various in vivo and in vitro assays in support of drug discovery. The results indicated that direct plasma injection, although certainly faster than conventional protein precipitation for sample preparation, had problems associated with column longevity and overall robustness. Frequently a single study could not be completed without column replacement.

Metabolism profiling, and cytochrome P450 inhibition & induction in drug discovery.

To reduce the high attrition rates of NCEs in preclinical and clinical development uncovering pharmacokinetics, toxicokinetics, drug metabolism, and drug-drug interactions early in drug discovery would be highly valuable. There have been many in vitro screens developed for these areas that have higher sample throughput, which is consistent with the iterative cycle of a typical drug discovery research project. We have presented the present status and given detailed descriptions of biotransformation, metabolic stability assays, identification of drug metabolizing P450 enzymes, prediction of pharmacokinetic parameters from in vitro metabolism data, structure elucidation of metabolites, CYP450 inhibition assays and CYP450 induction assays from a drug discovery perspective.

The new pre-preclinical paradigm: compound optimization in early and late phase drug discovery.

The attrition rates of new chemical entities (NCEs) in preclinical and clinical development are staggeringly high. NCEs are abandoned due to insufficient efficacy, safety issues, and economic reasons. Uncovering drug defects that produce these failures as early as possible in drug discovery would be highly effective in lowing the cost and time of developing therapeutically useful drugs.

Pharmacokinetics of a contraceptive patch (Evra/Ortho Evra) containing norelgestromin and ethinyloestradiol at four application sites.

Aims: To determine the pharmacokinetic profile of norelgestromin (NGMN) and ethinyloestradiol (EE) following application of the contraceptive patch, Evra/Ortho Evra, at each of four anatomic sites (abdomen, buttock, arm, and torso).

Methods: Thirty-seven healthy, nonpregnant women aged 20-45 years participated in this open-label, four-period crossover study. Subjects were randomized to one of four treatment (site of application) sequences.

Nile Red binding to HepG2 cells: an improved assay for in vitro studies of hepatosteatosis.

Nile Red is a fluorescent dye used extensively to study fat accumulation in many types of cells; unfortunately protocols that work well for most cells are not effective for studying drug-induced lipid accumulation in cultured liver cells and hepatocyte-derived cell lines. Using human hepatoma (HepG2) cells, we have developed a simple Nile Red binding assay as a screen for steatosis-inducing compounds. Increases in Nile Red binding in response to known hepatotoxic compounds were observed after incubating treated cells with 1 microM Nile Red for several hours, washing away free Nile Red, and then allowing redistribution, and/or clearance of the lipid-indicator dye.

Thrombin regulation of cell function through protease-activated receptors: implications for therapeutic intervention.

The serine protease thrombin is well recognized as being pivotal to the maintenance of hemostasis under both normal and pathological conditions. Its cellular actions are mediated through a unique family of protease-activated receptors (PARs). These receptors represent a novel family of G protein-coupled receptors that undergo proteolytic cleavage of their amino terminus and subsequent autoactivation by a tethered peptide ligand.

Determination of the absolute configuration of a key tricyclic component of a novel vasopressin receptor antagonist by use of vibrational circular dichroism.

We present the results of a study using vibrational circular dichroism (VCD) for (+)-1, which furnished an unambiguous determination of its absolute configuration as S. The most abundant conformation of (+)-1 in CDCl(3) solution was also established.

Functional regulation of immunoproteasomes and transporter associated with antigen processing.

The central event in the cellular immune response to invading pathogens is the presentation of non-self antigenic peptides by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes (CTLs). As peptide binding and transport proteins, MHC class I molecules have evolved distinct biochemical and cellular strategies for acquiring antigenic peptides, providing CTLs an extracellular representation of the intracellular antigen content. Whereas efficient generation of MHC class I binding peptides depends on the intracellular, immunoproteasome-mediated proteolysis machinery, translocation of peptides into the lumen of the endoplasmic reticulum requires the endoplasmic reticulum-resident, adenosine 5'-triphosphate (ATP) binding cassette transporter associated with antigen processing (TAP).

Evaluation of the excretion, and metabolism of the cardiotonic agent bemoradan in male rats and female beagle dogs.

The excretion and metabolism of (+/-) [6-(3,4-dihydro-3-oxo-1,4[2H]-benzoxazine-yl)-2,3,4,5-tetrahydro-5-methylpyridazin-3-one] (bemoradan; RWJ-22867) have been investigated in male Long-Evans rats and female beagle dogs. Radiolabeled [14C] bemoradan was administered to rats as a singkle 1 mg/kg suspension dose while the dogs received 0.1 mg/kg suspension dose.

RWJ-270201 (BCX-1812): a novel neuraminidase inhibitor for influenza.

The influenza virus neuraminidase (NA) is important in the pathogenesis of infection and, thus, is an attractive target for agents used in the treatment and prophylaxis of influenza. This article describes preclinical and early clinical data related to RWJ-270201 (BCX-1812), a novel, orally active NA inhibitor that was rationally designed for having potent and selective activity against influenza A and B viruses. RWJ-270201 is a unique NA inhibitor with a cyclopentane ring structure and high selectivity for the influenza NA.

Multiple-dose pharmacokinetics of a contraceptive patch in healthy women participants.

This open-label, randomized study evaluated the pharmacokinetics of norelgestromin (NGMN) and ethinyl estradiol (EE) following the application of a contraceptive patch (1/week) for three cycles (3 weeks/cycle). Healthy women (n = 24) wore a 20-cm(2) patch (ORTHO EVRA/EVRA) on either their abdomen or buttock during blood sampling weeks and on any of four approved sites at other times. Serum was analyzed for NGMN and EE from samples taken during Week 1 of Cycle 1 and Weeks 1-3 of Cycle 3.

Pharmacokinetics of norelgestromin and ethinyl estradiol delivered by a contraceptive patch (Ortho Evra/Evra) under conditions of heat, humidity, and exercise.

The objectives of this randomized, open-label, three-period, incomplete block design study were to evaluate the pharmacokinetics of norelgestromin (NGMN) and ethinyl estradiol (EE) delivered by the contraceptive patch, Ortho Evra/Evra, and to evaluate patch adhesion under conditions of heat, humidity, and exercise. During each treatment period, 30 healthy women wore Ortho Evra on the abdomen for 7 days under one of six conditions (normal activity, sauna, whirlpool, treadmill, cool water immersion, or a combination of activities). Blood samples were collected before and several times to 240 hours after patch application.

Diversity and coverage of structural sublibraries selected using the SAGE and SCA algorithms.

It is often impractical to synthesize and test all compounds in a large exhaustive chemical library. Herein, we discuss rational approaches to selecting representative subsets of virtual libraries that help direct experimental synthetic efforts for diverse library design. We compare the performance of two stochastic sampling algorithms, Simulating Annealing Guided Evaluation (SAGE; Zheng, W.

Transdermal contraception.

This review summarizes the clinical studies involving the once-weekly Ortho Evra/Evra contraceptive patch. The patch delivers norelgestromin (NGMN), 150 microg, and ethinyl estradiol (EE), 20 microg, daily to the systemic circulation. The contraceptive patch provided ovulation suppression and cycle control similar to that of oral norgestimate 250 microg/EE 35 microg, significantly decreased mean maximum follicular diameter following a 3-day intentional delayed dosing phase when compared with oral levonorgestrel (LNG) 50/75/125 microg/EE 30/40/30 micorg and oral LNG 100 microg/EE 20 microg, and was as effective as oral LNG 50/75/125 microg/EE 30/40/30 microg and oral desogestrel 150 microg/EE 20 microg in altering cervical mucus composition (i.

Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase A degrades collagen IV but not fibronectin.

Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin.

The use of selective adsorbents in capillary electrophoresis-mass spectrometry for analyte preconcentration and microreactions: a powerful three-dimensional tool for multiple chemical and biological applications.

Much attention has recently been directed to the development and application of online sample preconcentration and microreactions in capillary electrophoresis using selective adsorbents based on chemical or biological specificity. The basic principle involves two interacting chemical or biological systems with high selectivity and affinity for each other. These molecular interactions in nature usually involve noncovalent and reversible chemical processes.