Improvement of Ionization Efficiency and Application of Structural Analysis for MALDI-TOFMS by Derivatization of Polyacrylic Acid.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a suitable method for polymer analysis. MALDI is a soft ionization technique that can generate mainly singly charged ions. Therefore, the polymer's molecular weight distribution is easy to analyze, facilitating the calculation of the number average molecular weight and weight average molecular weight and polydispersity.

Pharmacokinetics and disposition of CS-0777, a sphingosine 1-phosphate receptor modulator, in rats and monkeys.

1. Disposition and metabolism of CS-0777 (1-{5-[(3R)-3-amino-4-hydroxy-3- methylbutyl]-1-methyl-1H-pyrrol-2-yl}-4-(4-methylphenyl) butan-1-one), a selective sphingosine 1-phosphate receptor-1 modulator under development for autoimmune conditions was investigated following oral and/or i.v.

Glutathione S-transferase pi trapping method for generation and characterization of drug-protein adducts in human liver microsomes using liquid chromatography-tandem mass spectrometry.

Covalent binding of reactive metabolites (RMs) to proteins is thought to play an important role in the processes leading to adverse drug reactions. Therefore, there is great interest in methodologies that enable the characterization of covalent binding of drugs to proteins. To facilitate the study of drug-protein adducts, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for characterizing RM-modified proteins formed through drug bioactivation in human liver microsomes (HLMs), which are commonly used for the in vitro drug bioactivation studies.

In vitro metabolism of rivoglitazone, a novel peroxisome proliferator-activated receptor γ agonist, in rat, monkey, and human liver microsomes and freshly isolated hepatocytes.

The in vitro metabolism of rivoglitazone, (RS)-5-{4-[(6-methoxy-1-methyl-1H-benzimidazol-2-yl)methoxy]benzyl}-1,3-thiazolidine-2,4-dione monohydrochloride, a novel thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ selective agonist, was studied in liver microsomes and freshly isolated hepatocytes of rat, monkey, and human as well as cDNA-expressed human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes. Fourteen metabolites were detected, and these structures were elucidated by liquid chromatography-tandem mass spectrometry. Five initial metabolic pathways of rivoglitazone consisting of four oxidation pathways and one N-glucuronidation pathway were predicted in correspondence with those proposed for in vivo studies using rats and monkeys.

Pharmacokinetics, metabolism, and disposition of rivoglitazone, a novel peroxisome proliferator-activated receptor γ agonist, in rats and monkeys.

The pharmacokinetics, metabolism, and excretion of rivoglitazone [(RS)-5-{4-[(6-methoxy-1-methyl-1H-benzimidazol-2-yl)methoxy]benzyl}-1,3-thiazolidine-2,4-dione monohydrochloride], a novel thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ selective agonist, were evaluated in male F344/DuCrlCrlj rats and cynomolgus monkeys. The total body clearance and volume of distribution of rivoglitazone were low in both animals (0.329-0.

Ethylene glycol monomethyl ether-induced toxicity is mediated through the inhibition of flavoprotein dehydrogenase enzyme family.

Ethylene glycol monomethyl ether (EGME) is a widely used industrial solvent known to cause adverse effects to human and other mammals. Organs with high metabolism and rapid cell division, such as testes, are especially sensitive to its actions. In order to gain mechanistic understanding of EGME-induced toxicity, an untargeted metabolomic analysis was performed in rats.

Biotransformation of prasugrel, a novel thienopyridine antiplatelet agent, to the pharmacologically active metabolite.

Prasugrel, a novel thienopyridine antiplatelet agent, undergoes rapid hydrolysis in vivo to a thiolactone, R-95913, which is further converted to its thiol-containing, pharmacologically active metabolite, R-138727, by oxidation via cytochromes P450 (P450). We trapped a sulfenic acid metabolite as a mixed disulfide with 2-nitro-5-thiobenzoic acid in an incubation mixture containing the thiolactone R-95913, expressed CYP3A4, and NADPH. Further experiments investigated one possible mechanism for the conversion of the sulfenic acid to the active thiol metabolite in vitro.

Identification of novel metabolic pathways of pioglitazone in hepatocytes: N-glucuronidation of thiazolidinedione ring and sequential ring-opening pathway.

The metabolism of [(14)C]pioglitazone was studied in vitro in incubations with freshly isolated human, rat, and monkey hepatocytes. Radioactivity detection high-performance liquid chromatography analysis of incubation extracts showed the detection of 13 metabolites (M1-M13) formed in incubations with human hepatocytes. An identical set of metabolites (M1-M13) was also detected in monkey hepatocytes.

CYP2D6-Mediated metabolism of a novel acyl coenzyme A:cholesterol acyltransferase inhibitor, pactimibe, and its unique plasma metabolite, R-125528.

Pactimibe sulfate is a novel acyl coenzyme A:cholesterol acyltransferase inhibitor. We conducted metabolic studies of pactimibe and its plasma metabolite, R-125528. Pactimibe had multiple metabolic pathways including indolin oxidation to form R-125528, omega-1 oxidation, N-dealkylation, and glucuronidation.

Isolation and identification of diglucuronides of some endogenous steroids in dogs.

Diglucuronidation is a novel glucuronidation reaction where the second glucuronosyl moiety is attached at the C2' position of the first glucuronosyl moiety. To examine whether diglucuronidation takes place in endogenous substrates in vivo, control urine and bile samples were collected from male Crl:CD(SD) IGS rats, beagle dogs, and cynomolgus monkeys and analyzed by liquid chromatography-mass spectrometry (LC-MS) after solid phase extraction. Several diglucuronides of C(19) steroids, including M1 (C(31)H(46)O(14)) and M2 (C(31)H(44)O(14)), were detected in the urine and bile of the dogs but not in the excreta of the rats and monkeys.

Characterization of phenotypes in Gstm1-null mice by cytosolic and in vivo metabolic studies using 1,2-dichloro-4-nitrobenzene.

Glutathione S-transferase Mu 1 (GSTM1) has been regarded as one of the key enzymes involved in phase II reactions in the liver, because of its high expression level. In this study, we generated mice with disrupted glutathione S-transferase Mu 1 gene (Gstm1-null mice) by gene targeting, and characterized the phenotypes by cytosolic and in vivo studies. The resulting Gstm1-null mice appeared to be normal and were fertile.

Human UDP-glucuronosyltransferase, UGT1A8, glucuronidates dihydrotestosterone to a monoglucuronide and further to a structurally novel diglucuronide.

We identified human UDP-glucuronosyltransferase (UGT) isoforms responsible for producing dihydrotestosterone (DHT) diglucuronide, a novel glucuronide in which the second glucuronosyl moiety is attached at the C2' position of the first glucuronosyl moiety, leading to diglucuronosyl conjugation of a single hydroxyl group of DHT at the C17 position. Incubation of the DHT monoglucuronide with 12 cDNA-expressed recombinant human UGT isoforms and uridine 5'-diphosphoglucuronic acid resulted in a low but measurable DHT diglucuronidation activity primarily with UGT1A8, a gastrointestinal UGT, and to a lesser extent with UGT1A1 and UGT1A9. In contrast, the activity of DHT monoglucuronidation was high and was found in UGT2B17, UGT2B15, UGT1A8, and UGT1A4 in descending order.

Repeated glucuronidation at one hydroxyl group leads to structurally novel diglucuronides of steroid sex hormones.

Androgens (androsterone, dihydrotestosterone and testosterone) and estrogens (estradiol, estriol and estrone) were incubated with liver microsomes from rats, dogs, monkeys and humans in the presence of uridine diphosphoglucuronic acid (UDPGA), and the glucuronides produced were structurally characterized by liquid chromatography-tandem mass spectrometry. After 2-h incubation with dog liver microsomes, all substrates tested were converted (approximately 2-10%) to structurally novel diglucuronides, where two glucuronosyl groups are bound to a single hydroxyl group in tandem. Two-dimensional nuclear magnetic resonance spectroscopy unambiguously elucidated the chemical structures of the 3-O-diglucuronide of estrone and the 17-O-diglucuronide of testosterone isolated from the incubation mixture.

Formation of a structurally novel, serial diglucuronide of 4-hydroxybiphenyl by further glucuronidation of a monoglucuronide in dog liver microsomes.

Incubation of 4-hydroxybiphenyl (p-phenylphenol) in the presence of UDP-glucuronic acid (UDPGA) with liver microsomes from male and female dogs produced a more polar metabolite peak than a simultaneously produced peak of 4-hydroxybiphenyl monoglucuronide in the high performance liquid chromatography (HPLC) chromatogram. Tandem mass spectrometry (MS/MS) and two-dimensional nuclear magnetic resonance (NMR) analyses revealed this polar metabolite as a 4-hydroxybiphenyl diglucuronide having a beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronopyranosyl moiety, where the two glucuronic acids are connected directly at the 1''-->2' position. Liver microsomes from Sprague-Dawley rat, cynomolgus monkey and human, converted 4-hydroxybiphenyl only to the monoglucuronide, suggesting that there is a dog UDP-glucuronosyltransferase (UGT), with a wider substrate specificity capable of glucuronidating 4-hydroxybiphenyl monoglucuronide to the diglucuronide.

Formation of a novel quinone epoxide metabolite of troglitazone with cytotoxicity to HepG2 cells.

Troglitazone, an oral antidiabetic drug, was reported to cause adverse hepatic effects in certain individuals, leading to its withdrawal from the market. After incubation of troglitazone (100 microM) with the human hepatoma cell line, HepG2 cells, and human primary hepatocytes for 48 to 72 h, an unknown peak was detected in the cell culture. The formation of this peak from troglitazone (100 microM) was also catalyzed by expressed CYP3A4, and further HPLC analysis revealed that there were three metabolites (metabolite A, B, and C) in the peak.