Oxyntomodulin (OXM), an enteroendocrine hormone, causes appetite suppression, increased energy expenditure, and weight loss in obese humans via activation of GLP-1 and glucagon receptors. However, the effects of OXM on glucose homeostasis remain ill defined. To address this gap, we evaluated the effects of an i.v. infusion of native OXM on insulin secretion rates (ISRs) and glycemic excursion in a graded glucose infusion (GGI) procedure in two separate randomized, placebo (PBO)-controlled, single-dose crossover trials in 12 overweight and obese subjects without diabetes and in 12 obese subjects with type 2 diabetes mellitus (T2DM), using the GLP-1 analog liraglutide (LIRA) as a comparator in T2DM. In both groups, in the GGI, 3.0 pmol/kg/min of OXM significantly increased ISR and blunted glycemic excursion relative to PBO. In T2DM, the effects of OXM were comparable to those of LIRA, including restoration of β-cell glucose responsiveness to that of nonobese subjects without diabetes. Our findings indicate that native OXM significantly augments glucose-dependent insulin secretion acutely in obese subjects with and without diabetes, with effects comparable to pharmacologic GLP-1 receptor activation and independent of weight loss. Native OXM has potential to improve hyperglycemia via complementary and independent induction of insulin secretion and weight loss.

To address questions relating to the safety assessment of circulating drug metabolites in humans, the US Food and Drug Administration (FDA) and the International Conference on Harmonisation (ICH) have recently issued regulatory guidances(1,2) that effectively require metabolite profiling in humans during early clinical development of a candidate drug. The clinical metabolite profiling results may require separate safety assessment studies in humans of stable circulating metabolites if such metabolites are not formed in sufficient concentrations in the plasma during nonclinical safety assessment of the parent drug.

Species differences in drug metabolism present two challenges that may confound the nonclinical safety assessment of candidate drugs. The first challenge is encountered when metabolites are formed uniquely or disproportionately in humans. Another challenge is understanding the human relevance of toxicities associated with metabolites formed uniquely or disproportionately in a nonclinical species. One potential approach to minimize the impact of metabolite related challenges is to consider genetically engineered mouse models that express human P450 enzymes. Human P450 expressing mouse models may have the ability to generate major human metabolites and eliminate or reduce the formation of mouse specific metabolites. Prior to determining the utility of any particular model, it is important to qualify by characterizing protein expression, establishing whether the model generates an in vivo metabolite profile more closely related to that of humans than the wild-type mouse, verifying genetic stability, and evaluating animal health. When compared to the current strategy for handling metabolite challenges (i.e., direct administration of metabolite), identifying an appropriate human P450 expressing model could provide a number of benefits. Such benefits include improved scientific relevance of the evaluation, decreased resource needs, and a possible reduction in the number of animals used. These benefits may ultimately improve the quality and speed by which promising new drug candidates are developed and delivered to patients.

After oral treatment (once daily) for 4 weeks with the potent bradykinin B 1 receptor antagonist methyl 3-chloro-3 (cid:1) -fluoro-4 (cid:1) -{(1 R )-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)- directed radiochemical Radiochemical detection was performed using a cell spectrometry using Quantum equipped ESI operated in a mode, a of (GenBank accession no. NM_001040211) were designed using Primer Express software version 1.0 (Applied Biosystems). The binding position, length, and specificity of each primer and probe were optimized using the BLAST program on the National Center for Bio-technology Information homepage (http://www.ncbi.nlm.nih.gov/blast). PCR-amplified cDNAs were detected by real-time fluorescence on an ABI Prism 7700 Sequence Detection System (PerkinElmer Life and Analytical Sciences). The relevant change of the target cDNA in treated samples versus DMSO control samples was calculated using the 2 (cid:5)(cid:8)(cid:8) Ct method, following normalization of the 18S ribosomal RNA in each sample (Livak and Schmittgen, 2001).

After oral treatment (once daily) for 4 weeks with the potent bradykinin B1 receptor antagonist methyl 3-chloro-3′-fluoro-4′-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1′-biphenyl-2-carboxylate (MK-0686), rhesus monkeys (Macaca mulatta) exhibited significantly reduced systemic exposure of the compound in a dose-dependent manner, suggesting an occurrence of autoinduction of MK-0686 metabolism. This possibility is supported by two observations. 1) MK-0686 was primarily eliminated via biotransformation in rhesus monkeys, with oxidation on the chlorophenyl ring as one of the major metabolic pathways. This reaction led to appreciable formation of a dihydrodiol (M11) and a hydroxyl (M13) product in rhesus liver microsomes supplemented with NADPH. 2) The formation rate of these two metabolites determined in liver microsomes from MK-0686-treated groups was ≥2-fold greater than the value for a control group. Studies with recombinant rhesus P450s and monoclonal antibodies against human P450 enzymes suggested that CYP2C75 played an important role in the formation of M11 and M13. The induction of this enzyme by MK-0686 was further confirmed by a concentration-dependent increase of its mRNA in rhesus hepatocytes, and, more convincingly, the enhanced CYP2C proteins and catalytic activities toward CYP2C75 probe substrates in liver microsomes from MK-0686-treated animals. Furthermore, a good correlation was observed between the rates of M11 and M13 formation and hydroxylase activities toward probe substrates determined in a panel of liver microsomal preparations from control and MK-0686-treated animals. Therefore, MK-0686, both a substrate and inducer for CYP2C75, caused autoinduction of its own metabolism in rhesus monkeys by increasing the expression of this enzyme.

Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, West Point, Pennsylvania (C.T., B.A.C., B.M., S.L.P., Y.K., K.S., A.N., K.R., R.E., E.J.C., N.X.Y., C.E.R., T.R.,T.P.); Laboratories Merck Sharp & Dohme-Chibret, Merck Research Laboratories, Route de Marsat, Riom, 63963 Clermont-Ferrand Cedex 9, France (F.P.); Department of Medicinal Chemistry, Merck Research Laboratories, West Point, Pennsylvania (C.N.D.M., S.D.K., M.G.B.); and Department of Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania (C.B.F.) JPET Fast Forward. Published on February 29, 2008 as DOI:10.1124/jpet.107.136044

The metabolic A/-oxidation of the carcinogen 2-aminofluorene was examined in vitro using fortified hepatic microsomes from a variety of species. Rat, dog, human, and pig liver microsomes catalyzed the formation of N-hydroxy-2-aminofluorene (N-OH-AF) from AF at rates of 1.6, 1.0, 1.2, and 3.5 nmol/min/mg protein, respectively. The involvement of both cytochrome P-450 and the flavin-containing monooxygenase was demonstrated with hepatic microsomes and with purified enzymes by using specific enzyme inhibitors. 2-[(2,4-Dichloro6-phenyl)phenoxy]ethylamine, a potent cytochrome P-450 in hibitor, decreased microsomal N-OH-AF formation by 96, 83, 70, and 46% in the rat, dog, human, and pig, respectively; and further addition of methimazole, a high-affinity flavin-containing monooxygenase substrate, abolished the residual A/-hydroxylating activity. Using the purified porcine flavin-containing monooxygenase, metabolic formation of N-OH-AF occurred at a rate of 4.9 nmol/min/nmol flavin adenine dinucleotide and was insensitive to 2-[(2,4-dichloro-6-phenyl)phenoxy]ethylamine inhibition. In addition, purified rat liver cytochrome P-450 (isolated from 5,6-naphthoflavone-induced animals) N-hydroxylated AF (1.1 nmol/min/nmol P-450) and was completely inhibited by 2-[(2,4-dichloro-6-phenyl)-phenoxy]ethylamine, but the reaction was insensitive to methimazole. To determine whether or not the metabolic formation of NOH-AF could lead directly to covalently bound adduct(s) with DNA under these incubation conditions (30 min, pH 7.5), the binding of synthetic and metabolically formed [3H]-N-OH-AF to added calf thymus DNA and to DNA in isolated rat liver nuclei was investigated. In all cases, the amount of DNA-bound car cinogen accounted for 0.08 to 0.15% of the N-OH-AF present in the incubation mixtures. These data, when compared to the levels of AF bound to hepatic nuclear DNA reported in vivo, suggest that the nonenzymatic reaction of N-OH-AF with nu clear DNA may be sufficient to account for a substantial portion of the observed in vivo binding of this carcinogen.

A workshop convened to define research needs in toxicology identified several deficiencies in data and methods currently applied in risk assessment. The workshop panel noted that improving the link between chemical exposure and toxicological response requires a better understanding of the biological basis for inter- and intra-human variability and susceptibility. This understanding will not be complete unless all life stages are taken into consideration. Because animal studies serve as a foundation for toxicological assessment, proper accounting for cross-species extrapolation is essential. To achieve this, adjustments for dose-rate effects must be improved, which will aid in extrapolating toxicological responses to low doses and from short-term exposures. Success depends on greater use of validated biologically based dose-response models that include pharmacokinetic and pharmacodynamic data. Research in these areas will help define uncertainty factors and reduce reliance on underlying default assumptions. Throughout the workshop the panel recognized that biomedical science and toxicology in particular is on the verge of a revolution because of advances in genomics and proteomics. Data from these high-output technologies are anticipated to greatly improve risk assessment by enabling scientists to better define and model the elements of the relationship between exposure to biological hazards and health risks in populations with differing susceptibilities.

In laboratory studies of rodents, the inhalation of organic vapors often results in preferential damage to olfactory epithelium. Such focal lesion formation may be due either wholly or in part to a corresponding nonuniformity in the spatial distribution of vapor uptake within the nasal cavities. As a tool for determining this dose distribution, a mathematical model based on a combination of computational fluid dynamics (CFD) and physiologically based pharmacokinetic (PBPK) modeling was developed for simulating toxicant vapor uptake in the rat nose. The nasal airways were subdivided into four distinct meatuses selected such that each contained a major air flow stream. Each meatus was further divided into four serial regions attached to separate tissue stacks containing mucus, epithelial, and subepithelial compartments. Values for the gas-phase mass transfer coefficients and gas flows in the 16 airway regions were determined by a solution of the Navier–Stokes and convection–diffusion equations using commercially available CFD software. These values were then input to a PBPK simulation of toxicant transport through the 16 tissue stacks. The model was validated by using overall uptake data from rodent inhalation studies for three “unreactive” vapors that were either completely inert (i.e., acetone), reversibly ionized in aqueous media (i.e., acrylic acid), or prevented from being metabolized by an enzyme inhibitor (i.e., isoamyl alcohol). A sensitivity analysis revealed that accurate values of the mass transfer coefficient were not necessary to simulate regional concentrations and uptake of unreactive vapors in the rat nose, but reliable estimates of diffusion coefficients in tissue were crucial for accurate simulations.

Analysis of the default cancer risk assessment methodology suggests that the confidence interval usually associated with the prediction of an upper bound on risk underestimates the uncertainty in the risk estimate. This underestimate of uncertainty is based on the use of a large number of policy decisions or professional judgements that are incorporated into the methodology as exact values with no estimate of error. An alternative approach is to develop a comprehensive biologically based risk assessment that provides scientific data to substitute for many of the policy decisions of the default methodology.

Gavage dosing of the irritant, ethyl acrylate (EA), has been found to induce hyperplasia in the rat forestomach, but no signs of toxicity in the glandular stomach or in organs remote from the site of dosing. To quantitatively describe this effect as a background for subsequent modeling studies, pulse measurements of the number of S-phase cells were made following a single gavage dose of EA. The time-course of the S-phase response in the forestomach epithelium following a high dose (200 mg/kg or a 4% solution in corn oil) indicated that the number of S-phase nuclei was decreased relative to control animals immediately following gavage dosing with a minimum at 6 hr, but that the number of S-phase nuclei increased significantly above control values by 20 hr and remained significantly elevated until at least 48 hr following the gavage dose. A single-dose dose-response study with gavage doses of 0, 2, 10, 20, 50, 100, or 200 mg/kg EA and S-phase analysis at 24 hr following gavage dosing indicated that a significant increase in S-phase nuclei was evident at doses of 20 mg/kg or higher. Dosing with EA for 2 weeks at dose levels of 0, 10, 50, or 200 mg/kg caused a prolonged elevation of S-phase nuclei only at the 200 mg/kg dose level during the 24 hr following the last gavage dose. Lower doses did not induce a significant increase in the S-phase nuclei. In contrast to the forestomach, the S-phase response of the glandular stomach was transient following a single 200 mg/kg gavage dose, and only a marginal response was observed following multiple 200 mg/kg doses. No effects were observed at lower doses. Comparison of these results to prior determinations of the effect of EA on the concentration of nonprotein sulfhydryls (primarily glutathione) in the forestomach and glandular stomach indicate a correlation of the stimulation in S-phase activity in the forestomach with the repletion and overshoot of tissue nonprotein sulfhydryl levels.

Risk AnalysisVolume 11, Issue 4 p. 581-582 Comments on Incorporating Mechanistic Data into Quantitative Risk Assessment1 Clay B. Frederick, Clay B. Frederick American Industrial Health Council, 1330 Connecticut Avenue NW, Suite 300, Washington, D.C. 20036-1702.Search for more papers by this authorAlan G. E. Wilson, Alan G. E. Wilson American Industrial Health Council, 1330 Connecticut Avenue NW, Suite 300, Washington, D.C. 20036-1702.Search for more papers by this author Clay B. Frederick, Clay B. Frederick American Industrial Health Council, 1330 Connecticut Avenue NW, Suite 300, Washington, D.C. 20036-1702.Search for more papers by this authorAlan G. E. Wilson, Alan G. E. Wilson American Industrial Health Council, 1330 Connecticut Avenue NW, Suite 300, Washington, D.C. 20036-1702.Search for more papers by this author First published: December 1991 https://doi.org/10.1111/j.1539-6924.1991.tb00647.xCitations: 2 1 Manuscript received Jury 3, 1991. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume11, Issue4December 1991Pages 581-582 RelatedInformation

A siderophore isolated from Epicoccus purpurascens has been found to be an isomer of triornicin (I) which differs from I only in interchange of the positions of acetyl and (E)-5-hydroxy-3-methyl-2-pentenoyl hydroxamate groups. The structure of the new compound, isotriornicin (II), was determined by spectroscopic techniques and by cleavage with methanol-ammonia to a new dihydroxamic acid (III) and N-acetyl-trans-fusarinine methyl ester (IV).