Biomedical subjects

Cornelis E C A Hop

Publications and source records attributed to Cornelis E C A Hop.

At least 19 recordsLinked to original sources

PubMed2006

Method for internal standard introduction for quantitative analysis using on-line solid-phase extraction LC-MS/MS.

A novel approach for on-line introduction of internal standard (IS) for quantitative analysis using LC-MS/MS has been developed. In this approach, analyte and IS are introduced into the sample injection loop in different steps. Analyte is introduced into the injection loop using a conventional autosampler (injector) needle pickup from a sample vial. IS is introduced into the sample injection loop on-line from a microreservoir containing the IS solution using the autosampler. As a result, both analyte and IS are contained in the sample loop prior to the injection into the column. Methodology allowed to reliably introduce IS and demonstrated injection accuracy and precision comparable to those obtained using off-line IS introduction (i.e., IS and analyte are premixed before injection) while maintaining chromatographic parameters (i.e., analyte and IS elution time and peak width). This new technique was applied for direct analysis of model compounds in rat plasma using on-line solid-phase extraction (SPE) LC-MS/MS quantification. In combination with on-line SPE, IS serves as a surrogate IS and compensates for signal variations attributed to sample preparation and instrumentation factors including signal suppression. The assays yielded accuracy (85-119%), precision (2-16%), and analyte recovery comparable to those obtained using off-line IS introduction. Furthermore, on-line IS introduction allows for nonvolumetric sample (plasma) collection and direct analysis without the need of measuring and aliquoting a fixed sample volume prior to the on-line SPE LC-MS/MS analysis. Therefore, this methodology enables direct sample (plasma) analysis without any sample manipulation and preparation.

Animals↗

PubMed2006

A semiquantitative method for the determination of reactive metabolite conjugate levels in vitro utilizing liquid chromatography-tandem mass spectrometry and novel quaternary ammonium glutathione analogues.

An in vitro semiquantitative reactive metabolite detection assay is described that incorporates NADPH-supplemented human liver microsomes, a novel quaternary ammonium glutathione analogue conjugating agent (QA-GSH), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection. The assay was developed to have high sample capacity and the potential for high sample throughput. MS/MS detection is selective and sensitive for the QA-GSH conjugating agent and semiquantitation of QA-GSH-reactive metabolite conjugates is performed using QA-GSH standards added to samples prior to analysis [i.e., internal standards (ISs)]. The reactive metabolite trapping capability of the free thiol group in QA-GSH was assessed using model drugs acetaminophen, clozapine, and flutamide, which are bioactivated to afford reactive metabolites. MS signal responses of equimolar amounts of QA-GSH standards were compared to assess the feasibility of using a QA-GSH IS approach to semiquantify reactive metabolite levels in vitro. The full scan Q1 MS response for each standard was within 3.3-fold of one another even though the "parent" moiety structure of each QA-GSH conjugate standard differed significantly. Standard curve analysis using selected reaction monitoring for each QA-GSH standard gave slope values that differed by only 1.5-fold. The QA-GSH IS semiquantitation method was tested by determining the level of QA-GS-acetaminophen conjugate formation at three different concentrations of acetaminophen and comparing the results to those from linear regression of authentic standards. The calculated levels of conjugate formed compared closely with those calculated from linear regression data of authentic standard curves. These results show that the QA-GSH semiquantitation assay described herein is a viable method for semiquantitatively assessing the bioactivation potential in vitro and is well-suited for use in early drug discovery high throughput screening paradigms.

Acetaminophen↗

PubMed2006

Use of nano-electrospray for metabolite identification and quantitative absorption, distribution, metabolism and excretion studies.

Determination of the pharmacokinetics and metabolite identification have been an integral part of drug discovery and development to ensure that drugs have appropriate absorption, distribution, metabolism and excretion properties. Liquid chromatography interfaced with a mass spectrometer has greatly facilitated these studies. Nano-electrospray has distinct sensitivity advantages and the increased amount of time available to perform mass spectrometric experiments facilitates structural characterization of metabolites. The recently developed silicon chip-based nano-electrospray devices are more practical than pulled capillaries. The use of these devices for the determination of pharmacokinetics and metabolite identification will be described and particular attention will be paid to the distinct advantages and disadvantages these devices offer.

Animals↗

PubMed2005

p38 MAP kinase inhibitors: metabolically stabilized piperidine-substituted quinolinones and naphthyridinones.

Quinolinones and naphthyridinones with C7 N-t-butyl piperidine substituents were found to be potent p38 MAP kinase inhibitors. These compounds significantly suppress TNF-alpha release in both cellular and LPS-stimulated whole blood assays. They also displayed excellent PK profiles across three animal species. Quinolinone at 10 mpk showed comparable oral efficacy to that of dexamethasone at 1 mpk in a murine collagen-induced arthritis model.

Animals↗

PubMed2005

Metabolic activation of the nontricyclic antidepressant trazodone to electrophilic quinone-imine and epoxide intermediates in human liver microsomes and recombinant P4503A4.

Therapy with the antidepressant trazodone has been associated with several cases of idiosyncratic hepatotoxicity. While the mechanism of hepatotoxicity remains unknown, it is possible that reactive metabolites of trazodone play a causative role. Studies were initiated to determine whether trazodone undergoes bioactivation in human liver microsomes to electrophilic intermediates. LC/MS/MS analysis of incubations containing trazodone and NADPH-supplemented microsomes or recombinant P4503A4 in the presence of glutathione revealed the formation of conjugates derived from the addition of the sulfydryl nucleophile to mono-hydroxylated- and hydrated-trazodone metabolites. Product ion spectra suggested that mono-hydroxylation and sulfydryl conjugation occurred on the 3-chlorophenyl-ring, whereas hydration and subsequent sulfydryl conjugation had occurred on the triazolopyridinone ring system. These findings are consistent with bioactivation sequences involving: (1) aromatic hydroxylation of the 3-chlorophenyl-ring in trazodone followed by the two-electron oxidation of this metabolite to a reactive quinone-imine intermediate, which reacts with glutathione in a 1,4-Michael fashion and (2) oxidation of the pyridinone ring to an electrophilic epoxide, ring opening of which, by glutathione or water generates the corresponding hydrated-trazodone-thiol conjugate or the stable diol metabolite, respectively. The pathway involving trazodone bioactivation to the quinone-imine has also been observed with many para-hydroxyanilines including the structurally related antidepressant nefazodone. It is proposed that the quinone-imine and/or the epoxide intermediate(s) may represent a rate-limiting step in the initiation of trazodone-mediated hepatotoxicity.

Antidepressive Agents, Second-Generation↗

PubMed2005

In vitro metabolism of a new oxazolidinedione hypoglycemic agent utilizing liver microsomes and recombinant human cytochrome P450 enzymes.

The compound, 5-{4-[3-(4-cyclohexyl-2-propylphenoxy)propoxy]phenyl}-1,3-oxazolidine-2,4-dione (compound A) is a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist. PPARgamma agonists have proven useful in the treatment of type 2 diabetes, which is characterized by hyperglycemia, insulin resistance and/or abnormal insulin secretion. The metabolism of this oxazolidinedione (OZD) was investigated in male rat, dog, monkey and human liver microsomes, and recombinant human cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in the presence of NADPH. Routes of metabolism included monohydroxylation of the cyclohexane ring at multiple positions, monohydroxylation of the n-propyl side chain or the tether linkage, and OZD ring opening, giving rise to the keto amide and alcohol amide entities. Liver microsomes showed subtle qualitative and quantitative metabolic differences among rat, dog, monkey and human preparations. Further, CYP2C8 and CYP2C19 did not display different regioselectivity for hydroxylation on the cyclohexane ring with both of them giving rise to C-3 and C-4 hydroxy metabolites, but they did display different stereoselectivity with CYP2C8 preferring cyclohexane hydroxylation in equatorial positions and CYP2C19 in axial positions.

Animals↗

PubMed2005

Strategies for characterization of drug metabolites using liquid chromatography-tandem mass spectrometry in conjunction with chemical derivatization and on-line H/D exchange approaches.

Strategies using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with techniques such as chemical derivatization and on-line hydrogen/deuterium (H/D) exchange for structural elucidation of drug metabolites in crude samples are reviewed. Useful mass spectrometric scan techniques discussed include product ion scan, constant neutral-loss scan, precursor ion scan, multistage MS(n), and accurate mass measurements. In biological systems, xenobiotics are transformed into metabolites, which usually involves introduction of one or more polar functional groups or removal or blockage of such structural moieties. Therefore, chemical derivatization strategies for determination of functional groups and on-line H/D exchange approaches for probing number of exchangeable hydrogens are powerful tools for structural elucidation of drug metabolites in drug metabolism studies. More importantly, these experiments can be carried out on crude samples in microscale, providing sufficient material for LC-MS/MS analysis. Therefore, labor intensive and technically challenging purification of low levels of drug metabolites from complex biological matrices can be avoided. It is the authors' conclusion that strategies such as chemical derivatization and on-line H/D exchange should be used more routinely in drug metabolism studies in order to facilitate metabolite identification.

Chromatography, Liquid↗

PubMed2005

Identification of a novel glutathione adduct of diclofenac, 4'-hydroxy-2'-glutathion-deschloro-diclofenac, upon incubation with human liver microsomes.

Clinical use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with an incidence of idiosyncratic hepatoxicity. The formation of reactive metabolites of DF in vivo has been proposed to be responsible for such toxicity. One type of reactive metabolite, a benzoquinone imine of DF formed through oxidation by cytochromes P450, can be trapped by glutathione in vitro in liver microsomes to form glutathione (GS) adducts. Three GS adducts from DF were reported in the literature, namely, 5-hydroxy (OH)-4-glutathione-DF, 4'-OH-3'-glutathione-DF and 5-OH-6-glutathione-DF, and they all have the same molecular weight of 616. Recently, we developed a sensitive and high throughput method for the detection of GS adducts from liver microsome incubation. This method uses a constant neutral loss scan of m/z 129, a "structure-characteristic" fragment for GS adduct, on an automated chip-based nanoelectrospray (Advion NanoMate 100) attached to a tandem mass spectrometer (Sciex API 3000). The analysis of GS adducts from human liver microsome incubation with DF by the NanoMate 100-API 3000 method unambiguously revealed a new adduct ion with m/z 583 (MH+), in addition to the known adduct peak with m/z 617 (MH+). This new adduct was further confirmed to be 4'-OH-2'-glutathion-deschloro-diclofenac by liquid chromatography (LC) tandem mass spectrometry (MS), LC/MS-NMR, and comparison to a synthetic standard.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2005

Uniformity of ionization response of structurally diverse analytes using a chip-based nanoelectrospray ionization source.

The major drawback of liquid chromatography/mass spectrometry (LC/MS) for the analysis of mixtures is the non-quantitative nature of these studies. The ionization efficiency of the various components in the mixture (e.g., a compound and its metabolites) can vary greatly and, therefore, relative intensities of signals cannot be related to relative abundance. A chip-based nanoelectrospray ionization source was used to compare the ionization efficiencies of compounds with different physical-chemical characteristics. The data indicate that the ionization efficiencies vary much less with the chip-based device than by LC/MS. This was ascribed to the generation of a much higher electric field around the nozzles, which supplies a large excess of protons to the small droplets and reduces/eliminates the differences in the ionization efficiency for the analytes.

Animals↗

PubMed2004

Bioactivation of the nontricyclic antidepressant nefazodone to a reactive quinone-imine species in human liver microsomes and recombinant cytochrome P450 3A4.

The therapeutic benefits of the antidepressant nefazodone have been hampered by several cases of acute hepatotoxicity/liver failure. Although the mechanism of hepatotoxicity remains unknown, it is possible that reactive metabolites of nefazodone play a causative role. Studies were initiated to determine whether nefazodone undergoes bioactivation in human liver microsomes to electrophilic intermediates. Following incubation of nefazodone with microsomes or recombinant P4503A4 in the presence of sulfydryl nucleophiles, conjugates derived from the addition of thiol to a monohydroxylated nefazodone metabolite were observed. Product ion spectra suggested that hydroxylation and sulfydryl conjugation occurred on the 3-chlorophenylpiperazine-ring, consistent with a bioactivation pathway involving initial formation of p-hydroxynefazodone, followed by its two-electron oxidation to the reactive quinone-imine intermediate. The formation of novel N-dearylated nefazodone metabolites was also discernible in these incubations, and 2-chloro-1,4-benzoquinone, a by-product of N-dearylation, was trapped with glutathione to afford the corresponding hydroquinone-sulfydryl adduct. Nefazodone also displayed NADPH-, time-, and concentration-dependent inactivation of P4503A4 activity, suggesting that reactive metabolites derived from nefazodone bioactivation are capable of covalently modifying P4503A4. A causative role for 2-chloro-1,4-benzoquinone and/or the quinone-imine intermediate(s) in nefazodone hepatotoxicity is speculated. Although the antianxiety agent buspirone, which contains a pyrimidine ring in place of the 3-chlorophenyl-ring, also generated p-hydroxybuspirone in liver microsomes, no sulfydryl conjugates of this metabolite were observed. This finding is consistent with the proposal that two-electron oxidation of p-hydroxybuspirone to the corresponding quinone-imine is less favorable due to differences in the protonation state at physiological pH and due to weaker resonance stabilization of the oxidation products as predicted from ab initio measurements.

Antidepressive Agents, Second-Generation↗

PubMed2004

The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: evaluation using the MDR1A/1B knockout mouse model.

Thirty-two structurally diverse drugs used for the treatment of various conditions of the central nervous system (CNS), along with two active metabolites, and eight non-CNS drugs were measured in brain, plasma, and cerebrospinal fluid in the P-glycoprotein (P-gp) knockout mouse model after subcutaneous administration, and the data were compared with corresponding data obtained in wild-type mice. Total brain-to-plasma (B/P) ratios for the CNS agents ranged from 0.060 to 24. Of the 34 CNS-active agents, only 7 demonstrated B/P area under the plasma concentration curve ratios between P-gp knockout and wild-type mice that did not differ significantly from unity. Most of the remaining drugs demonstrated 1.1- to 2.6-fold greater B/P ratios in P-gp knockout mice versus wild-type mice. Three, risperidone, its active metabolite 9-hydroxyrisperidone, and metoclopramide, showed marked differences in B/P ratios between knockout and wild-type mice (6.6- to 17-fold). Differences in B/P ratios and cerebrospinal fluid/plasma ratios between wild-type and knockout animals were correlated. Through the use of this model, it appears that most CNS-active agents demonstrate at least some P-gp-mediated transport that can affect brain concentrations. However, the impact for the majority of agents is probably minor. The example of risperidone illustrates that even good P-gp substrates can still be clinically useful CNS-active agents. However, for such agents, unbound plasma concentrations may need to be greater than values projected using receptor affinity data to achieve adequate receptor occupancy for effect.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2004

In vitro metabolism of MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl) phenyl]methyl]benzamide], a peroxisome proliferator-activated receptor alpha/gamma agonist. I. Role of cytochrome P450, methyltransferases, flavin monooxygenases, and esterases.

The metabolism of MK-0767, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl) phenyl]methyl]benzamide, a thiazolidinedione (TZD)-containing peroxisome proliferator-activated receptor alpha/gamma agonist, was studied in liver microsomes and hepatocytes from humans and rat, dog, and rhesus monkey, to characterize the enzyme(s) involved in its metabolism. The major site of metabolism is the TZD ring, which underwent opening catalyzed by CYP3A4 to give the mercapto derivative, M22. Other metabolites formed in NADPH-fortified liver microsomes included the TZD-5-OH derivative (M24), also catalyzed by CYP3A4, and the O-desmethyl derivative (M28), whose formation was catalyzed by CYP2C9 and CYP2C19. Metabolite profiles from hepatocyte incubations were different from those generated with NADPH-fortified microsomal incubations. In addition to M22, M24, and M28, hepatocytes generated several S-methylated metabolites, including the methyl mercapto (M25), the methyl sulfoxide amide (M16), and the methyl sulfone amide (M20) metabolites. Addition of the methyl donor, S-adenosyl methionine, in addition to NADPH, to microsomal incubations enhanced the turnover and resulted in metabolite profiles similar to those in hepatocyte incubations. Collectively, these results indicated that methyltransferases played a major role in the metabolism of MK-0767. Using enzyme-specific inhibitors, it was concluded that microsomal thiol methyltransferases play a more important role than the cytosolic thiopurine methyltransferase. Baculovirus-expressed human flavin-containing monooxygenase 3, as well as CYP3A4, oxidized M25 to M16, whereas further oxidation of M16 to M20 was catalyzed mainly by CYP3A4. Esterases were involved in the formation of the methyl sulfone carboxylic acids, minor metabolites detected in hepatocytes.

Animals↗

PubMed2004

In vitro metabolism of MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)-phenyl] methyl]benzamide], a peroxisome proliferator-activated receptor alpha/gamma agonist. II. Identification of metabolites by liquid chromatography-tandem mass spectrometry.

The in vitro metabolism of MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl) methyl]-2-methoxy-N-[[(4-trifluoromethyl)-phenyl] methyl]benzamide], a novel 2,4-thiazolidinedione (TZD)-containing peroxisome proliferator-activated receptor alpha/gamma agonist, was studied in rat, dog, monkey, and human liver microsomes and hepatocytes, as well as in recombinant human CYP3A4-containing microsomes. Twenty-two metabolites (some at trace levels) were detected by liquid chromatography-tandem mass spectrometry analysis. All appeared to be phase I metabolites except for a glucuronide conjugate of a hydroxylated metabolite that was detected at trace levels. A constant neutral loss scan experiment performed on a triple quadrupole mass spectrometer proved to be very useful for resolving the metabolites from endogenous compounds. It was observed that the initial site of metabolism of MK-0767 was at the TZD ring leading to two major metabolites, namely the 5-hydroxy-TZD metabolite (M24) and the mercapto metabolite (M22). The latter was formed via the cleavage of the TZD ring with the elimination of the carbonyl adjacent to the sulfur atom. The structure of M24 was established by accurate mass measurements and NMR analysis. This hydroxy-TZD metabolite might represent an important precursor for a group of metabolites formed by TZD ring opening and subsequent loss of the sulfur moiety. The mercapto metabolite, on the other hand, is probably the key precursor for the TZD ring-opened metabolites with retention of the sulfur, even though the detailed mechanism of the ring scission remains to be characterized. From these studies, it was concluded that the TZD ring was the major site of metabolism of MK-0767. All the metabolites produced in vitro from human preparations were detected in the corresponding preparations from the nonclinical species.

Animals↗

PubMed2004

The metabolic disposition of aprepitant, a substance P receptor antagonist, in rats and dogs.

The absorption, metabolism, and excretion of [14C]aprepitant, a potent and selective human substance P receptor antagonist for the treatment of chemotherapy-induced nausea and vomiting, was evaluated in rats and dogs. Aprepitant was metabolized extensively and no parent drug was detected in the urine of either species. The elimination of drug-related radioactivity, after i.v. or p.o. administration of [14C]aprepitant, was mainly via biliary excretion in rats and by way of both biliary and urinary excretion in dogs. Aprepitant was the major component in the plasma at the early time points (up to 8 h), and plasma metabolite profiles of aprepitant were qualitatively similar in rats and dogs. Several oxidative metabolites of aprepitant, derived from N-dealkylation, oxidation, and opening of the morpholine ring, were detected in the plasma. Glucuronidation represented an important pathway in the metabolism and excretion of aprepitant in rats and dogs. An acid-labile glucuronide of [14C]aprepitant accounted for approximately 18% of the oral dose in rat bile. The instability of this glucuronide, coupled with its presence in bile but absence in feces, suggested the potential for enterohepatic circulation of aprepitant via this conjugate. In dogs, the glucuronide of [14C]aprepitant, together with four glucuronides derived from phase I metabolites, were present as major metabolites in the bile, accounting collectively for approximately 14% of the radioactive dose over a 4- to 24-h period after i.v. dosing. Two very polar carboxylic acids, namely, 4-fluoro-alpha-hydroxybenzeneacetic acid and 4-fluoro-alpha-oxobenzeneacetic acid, were the predominant drug-related entities in rat and dog urine.

Administration, Oral↗

PubMed2003

p38 Inhibitors: piperidine- and 4-aminopiperidine-substituted naphthyridinones, quinolinones, and dihydroquinazolinones.

We have synthesized a series of C7-piperidine- and 4-aminopiperidine-substituted naphthyridinones, quinolinones, and dihydroquinazolinones that are highly potent inhibitors of both p38MAP kinase activity and TNF-alpha release. The 4-aminopentamethylpiperidine naphthyridinone 5, which was designed to block metabolism at major 'hot spots', combined excellent inhibitory potency with good oral bioavailability in the rat.

Animals↗

PubMed2003

Design and synthesis of potent, orally bioavailable dihydroquinazolinone inhibitors of p38 MAP kinase.

The development of potent, orally bioavailable (in rat) and selective dihydroquinazolinone inhibitors of p38alpha MAP kinase is described. These analogues are hybrids of a pyridinylimidazole p38alpha inhibitor reported by Merck Research Laboratories and VX-745. Optimization of the C-5 phenyl and the C-7 piperidinyl substituents led to the identification of 15i which gave excellent suppression of TNF-alpha production in LPS-stimulated whole blood (IC(50)=10nM) and good oral exposure in rats (F=68%, AUCn PO=0.58 microM h).

Animals↗

PubMed2003

p38MAP kinase inhibitors. Part 1: design and development of a new class of potent and highly selective inhibitors based on 3,4-dihydropyrido[3,2-d]pyrimidone scaffold.

A new class of p38 antagonists based on 3,4-dihydropyrido[3,2,-d]pyrimidine scaffold has been developed. These inhibitors exhibit unprecedented selectivity towards p38 over other very closely related kinases. Compounds 25, 33, and 34 were identified as benchmark analogues for follow-up studies. They show good potency for enzyme inhibition and excellent functional activity.

Drug Design↗

PubMed2003

The unanticipated loss of SO2 from sulfonamides in collision-induced dissociation.

A potent and selective sulfonamide beta3 agonist with an excellent pharmacokinetic profile has recently been synthesized. During the analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of metabolites of the sulfonamide N-[4-[2-(2-hydroxy-2-pyridin-3-ylethylamino)ethyl]phenyl]-4-[4-(4-trifluoromethylphenyl)thiazol-2-yl]benzulfonamide (compound A), we observed loss of 64 Da for a few of the metabolites in the negative ion mode. Accurate mass measurements performed with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and quadrupole time-of-flight (Q-TOF) mass spectrometry suggested that the loss of 64 Da corresponded to the loss of SO(2). The same phenomenon was observed for a group of structurally related and commercially available compounds that also contain a sulfonamide moiety. MS/MS analysis of the fragment ions that had lost SO(2) in the ion source suggested that these ions were covalently bound rather than ion-molecule complexes. The neutral loss involving the cleavage of two bonds was unanticipated and suggested a complex rearrangement process. A mechanism for the loss of SO(2) has been proposed.

Animals↗