Intestinal lymphatic transport for drug delivery.

Intestinal lymphatic transport has been shown to be an absorptive pathway following oral administration of lipids and an increasing number of lipophilic drugs, which once absorbed, diffuse across the intestinal enterocyte and while in transit associate with secretable enterocyte lipoproteins. The chylomicron-associated drug is then secreted from the enterocyte into the lymphatic circulation, rather than the portal circulation, thus avoiding the metabolically-active liver, but still ultimately returning to the systemic circulation. Because of this parallel and potentially alternative absorptive pathway, first-pass metabolism can be reduced while increasing lymphatic drug exposure, which opens the potential for novel therapeutic modalities and allows the implementation of lipid-based drug delivery systems.

Metabolomics analysis reveals elevation of 3-indoxyl sulfate in plasma and brain during chemically-induced acute kidney injury in mice: investigation of nicotinic acid receptor agonists.

An investigative renal toxicity study using metabolomics was conducted with a potent nicotinic acid receptor (NAR) agonist, SCH 900424. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques were used to identify small molecule biomarkers of acute kidney injury (AKI) that could aid in a better mechanistic understanding of SCH 900424-induced AKI in mice. The metabolomics study revealed 3-indoxyl sulfate (3IS) as a more sensitive marker of SCH 900424-induced renal toxicity than creatinine or urea.

Characterization of human liver enzymes involved in the biotransformation of boceprevir, a hepatitis C virus protease inhibitor.

Boceprevir (SCH 503034), a protease inhibitor, is under clinical development for the treatment of human hepatitis C virus infections. In human liver microsomes, formation of oxidative metabolites after incubations with [(14)C]boceprevir was catalyzed by CYP3A4 and CYP3A5. In addition, the highest turnover was observed in recombinant CYP3A4 and CYP3A5.

Quantitation of parent drug and its unstable metabolites by in situ coulometric oxidation and liquid chromatography-tandem mass spectrometry.

Recent FDA and ICH guidances on safety testing of drug metabolites have challenged the way we traditionally think about quantitative bioanalytical methods. Such assays, in general, require a reference standard for each analyte to construct calibration curves and prepare quality control samples. However, early in the drug development process, metabolite standards may not be readily available, and if they are inherently unstable, they are difficult to synthesize or purify.

Identification of human liver cytochrome P450 enzymes involved in the metabolism of SCH 530348 (Vorapaxar), a potent oral thrombin protease-activated receptor 1 antagonist.

Vorapaxar (SCH 530348), a potent oral thrombin protease-activated receptor 1 antagonist, is being developed as an antiplatelet agent for patients with established vascular disease. The objective of this study was to identify the human liver cytochrome P450 (P450) enzyme(s) responsible for the metabolism of SCH 530348. Human liver microsomes metabolized SCH 530348 to M19, an amine metabolite formed via carbamate cleavage, and M20 (monohydroxy-SCH 530348).

Identification of two novel metabolites of SCH 486757, a nociceptin/orphanin FQ peptide receptor agonist, in humans.

The study of human metabolism of endo-8[bis(2-chlorophenyl)methyl]-3-(2-pyrimidinyl)-8-azabicyclo[3.2.1]octan-3-ol (SCH 486757) after a 200-mg oral dose of the drug to healthy volunteers in the first-in-human study is presented.

Metabolism of loratadine and further characterization of its in vitro metabolites.

The present study demonstrated that in addition to CYP3A4 and CYP2D6, the metabolism of loratadine is also catalyzed by CYP1A1, CYP2C19, and to a lesser extent by CYP1A2, CYP2B6, CYP2C8, CYP2C9 and CYP3A5. The biotransformation of loratadine was associated with the formation of desloratadine (DL) and further hydroxylation of both DL and the parent drug (loratadine). Based on the inhibition and correlation studies contribution of CYP2C19 in the formation of the major circulating metabolite DL seems to be minor.

An accurate-mass-based spectral-averaging isotope-pattern-filtering algorithm for extraction of drug metabolites possessing a distinct isotope pattern from LC-MS data.

Detection and identification (ID) of all drug metabolites following liquid chromatography (LC)/mass spectrometry (MS) analysis of complex biological matrixes are not trivial. To facilitate detection of drug-derived materials that possess highly diagnostic isotopic patterns (e.g.

A retention-time-shift-tolerant background subtraction and noise reduction algorithm (BgS-NoRA) for extraction of drug metabolites in liquid chromatography/mass spectrometry data from biological matrices.

A retention-time-shift-tolerant background subtraction and noise reduction algorithm (BgS-NoRA) is implemented using the statistical programming language R to remove non-drug-related ion signals from accurate mass liquid chromatography/mass spectrometry (LC/MS) data. The background-subtraction part of the algorithm is similar to a previously published procedure (Zhang H and Yang Y. J.

Identification of human liver cytochrome P450 enzymes involved in biotransformation of vicriviroc, a CCR5 receptor antagonist.

Vicriviroc (SCH 417690), a CCR5 receptor antagonist, is currently under investigation for the treatment of human immunodeficiency virus infection. The objective of this study was to identify human liver cytochrome P450 enzyme(s) responsible for the metabolism of vicriviroc. Human liver microsomes metabolized vicriviroc via N-oxidation (M2/M3), O-demethylation (M15), N,N-dealkylation (M16), N-dealkylation (M41), and oxidation to a carboxylic acid metabolite (M35b/M37a).

Response normalized liquid chromatography nanospray ionization mass spectrometry.

The widely different LC-MS response observed for many structurally different compounds limits the use of LC-MS in full scan detection mode for quantitative determination of drugs and metabolites without using reference standard. The recently introduced nanospray ionization (NSI) technique shows comparable MS response for some compounds under non-LC-MS conditions. However, in the presence of numerous endogenous compounds commonly associated with biological samples such as urine, plasma, and bile, LC-MS is required to separate, detect, identify, and measure individual analytes.

Metabolism-based identification of a potent thrombin receptor antagonist.

The metabolism of our prototypical thrombin receptor antagonist 1, Ki = 2.7 nM, was studied and three major metabolites (2, 4, and 5) were found. The structures of the metabolites were verified independently by synthesis.

Identification of unstable metabolites of Lonafarnib using liquid chromatography-quadrupole time-of-flight mass spectrometry, stable isotope incorporation and ion source temperature alteration.

Structural characterization of unstable metabolites and other drug-derived entities poses a serious challenge to the analytical chemist using instrumentation such as LC-MS and LC-MS/MS, and may lead to inaccurate identification of metabolite structures. The task of structural elucidation becomes even more difficult when an analyte is unstable in the ion source of the mass spectrometer. However, a judicious selection of the experimental conditions and the advanced features of new generation mass spectrometers can often overcome these difficulties.

Application of mass spectrometry for metabolite identification.

Metabolism studies play a pivotal role in drug discovery and development. Characterization of metabolic "hot-spots" as well as reactive and pharmacologically active metabolites is critical to designing new drug candidates with improved metabolic stability, toxicological profile and efficacy. Metabolite identification in the preclinical species used for safety evaluation is required in order to determine whether human metabolites have been adequately tested during non-clinical safety assessment.

Thermally induced N-to-O rearrangement of tert-N-oxides in atmospheric pressure chemical ionization and atmospheric pressure photoionization mass spectrometry: differentiation of N-oxidation from hydroxylation and potential determination of N-oxidation site.

N-Oxides are known to undergo deoxygenation during atmospheric pressure chemical ionization (Ramanathan, R.; Su, A.-D.

Ezetimibe: a review of its metabolism, pharmacokinetics and drug interactions.

Ezetimibe is the first lipid-lowering drug that inhibits intestinal uptake of dietary and biliary cholesterol without affecting the absorption of fat-soluble nutrients. Following oral administration, ezetimibe is rapidly absorbed and extensively metabolised (>80%) to the pharmacologically active ezetimibe-glucuronide. Total ezetimibe (sum of 'parent' ezetimibe plus ezetimibe-glucuronide) concentrations reach a maximum 1-2 hours post-administration, followed by enterohepatic recycling and slow elimination.

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of 3-hydroxydesloratadine.

Desloratadine is a non-sedating antihistamine recently approved for the treatment of seasonal allergic rhinitis. The major metabolite of desloratadine in human plasma and urine is the glucuronide conjugate of 3-hydroxydesloratadine. 3-Hydroxydesloratadine-glucuronide is also the major in vitro metabolite of 3-hydroxydesloratadine formed by incubation of 3-hydroxydesloratadine with human liver microsomes supplemented with uridine 5'-diphosphate-glucuronic acid (UDPGA).

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of ezetimibe (Zetia).

Ezetimibe [1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone] (Zetia; Schering-Plough, Kenilworth, NJ) is the first in a new class of cholesterol-lowering agents known as cholesterol absorption inhibitors. The objective of this study was to identify the isoform(s) of human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for the glucuronidation of ezetimibe. The main circulating metabolite of this drug in human plasma is SCH 60663, the phenolic glucuronide conjugate of ezetimibe.

Identification of human UDP-glucuronosyltransferase enzyme(s) responsible for the glucuronidation of posaconazole (Noxafil).

Posaconazole (Noxafil, SCH 56592), an orally available broad-spectrum triazole antifungal, is currently in phase III clinical studies for treating serious opportunistic fungal infections. The major in vitro metabolite of posaconazole formed by human liver microsomes supplemented with uridine 5'-diphosphate-glucuronic acid was a glucuronide of posaconazole (m/z877). Screening of 10 cDNA-expressed recombinant human UDP-glucuronosyltransferase (UGT) enzymes showed that only UGT1A4 exhibited catalytic activity with respect to the formation of the glucuronide of posaconazole.

Assessment of temporal biochemical and gene transcription changes in rat liver cytochrome P450: utility of real-time quantitative RT-PCR.

Purpose: A conventional approach to assess cytochrome P450 (CYP) induction in preclinical animal models involves daily dosing for a least a week followed by Western blot and/or enzyme activity analysis. To evaluate the potential benefit of a third more specific and sensitive assay, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), with the objective of reducing the duration of the conventional 1-week study, we simultaneously assessed gene expression by qRT-PCR along with Western blots and enzyme activity assays as a time course in an in vivo model.

Methods: Rats were dosed daily for 8 days with model inducers of CYP1A, CYP2B, CYP3A, or CYP4A.

High-performance liquid chromatographic method for the quantification of unbound evernimicin in human plasma ultrafiltrate.

A rapid HPLC method was developed for quantification of unbound evernimicin in human plasma. Protein-free samples prepared by ultrafiltration were injected directly onto a polymeric reversed-phase column and the eluent monitored at 302 nm. Evernimicin that eluted within 3.

Disposition of the selective cholesterol absorption inhibitor ezetimibe in healthy male subjects.

Ezetimibe [SCH 58235; 1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone], a selective cholesterol absorption inhibitor, is being developed for the treatment of primary hypercholesterolemia. The absorption, metabolism, and excretion of ezetimibe were characterized in eight healthy male volunteers in this single-center, single-dose, open-label study. Subjects received a single oral 20-mg dose of [14C]ezetimibe (approximately 100 microCi) with 200 ml of noncarbonated water after a 10-h fast.