Metabolic Activation of Fluoropyrrolidine Dipeptidyl Peptidase-IV Inhibitors by Rat Liver Microsomes

Abstract The current study evaluated the potential for two dipeptidyl peptidase-IV (DPP-IV) inhibitor analogues (MRL-A and MRL-B), containing a fluoropyrrolidine moiety in the structure, to undergo metabolic activation. The irreversible binding of these tritiumlabeled compounds to rat liver microsomal protein was time-and NADPH-dependent, and was attenuated by the addition of reduced glutathione (GSH) or N-acetylcysteine (NAC) to the incubation, indicating that chemically reactive intermediates were formed and trapped by these nucleophiles. Mass spectrometric analyses and further trapping experiments with semicarbazide indicated that the fluoropyrrolidine ring had undergone sequential oxidation and defluorination events resulting in the formation of GSH or NAC conjugates of the pyrrolidine moiety. The bioactivation of MRL-A was catalyzed primarily by rat recombinant cytochrome (CYP) 3A1 and 3A2. Pretreatment of rats with prototypic CYP3A1 and 3A2 inducers (pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone) enhanced the extent of bioactivation, which in turn, led to a higher degree of in vitro irreversible binding to microsomal proteins (5-and 9-fold increase, respectively). Herein, we describe studies which demonstrate that the fluoropyrrolidine ring is prone to metabolic activation, and that GSH or NAC can trap the reactive intermediates to form adducts that provide insight into the mechanisms of bioactivation

Analysis of benzo(a)pyrene metabolites by mass spectrometry

A continuous-flow fast atom bombardment mass spectrometry (FABMS) method was developed for analysis of benzo(a)pyrene (BP) conjugates. Continuous-flow FAB mass spectra of BP-sulfates and BP-glucuronides acquired in the negative ion mode gave characteristic molecular anions and fragment ions. About fifty times more sample was required to produce spectra with similar signal-to-noise ratio by standard FABMS. A method based on high resolution continuous-flow FABMS was developed for the analysis of BP-sulfates isolated from medium of BP-treated human hepatoma cell cultures (HepG2). A detection limit of less than 1 pg was obtained using high resolution (m/$\Delta$m 5000) single ion monitoring at the molecular anion (m/z 347.0378) of BP-sulfate. HPLC Fractions from BP-treated cells corresponding to the retention time of authentic BP-sulfates gave a mass spectrometric response and fractions from medium of untreated HepG2 cells had UV-absorbing material but gave no mass spectrometric response. The amount of BP-sulfates in the medium was calculated based on a calibration curve and recovery rates obtained by using authentic standards. This continuous-flow FABMS method with some modification was applied to analyses of BP-glucuronides isolated from medium of BP-treated hamster embryo cells, the mouse hepatoma cell line Hepa-1c1c7 and HepG2 cell cultures. A detection limit of 1 pg was obtained by using high resolution (m/$\Delta$m 10,000) single ion monitoring at the molecular anion of BP-glucuronide (m/z 443.1129). An on-line LCMS method, based on high resolution continuous-flow FABMS, was developed for the rapid detection of BP-conjugates from cell cultures. Analysis of medium from BP-treated HepG2, hamster embryo, and Hepa-1c1c7 cell cultures by a reversed phase microbore HPLC system that separates BP-sulfates from BP-glucuronides and also resolves the isomeric 3-BP-glucuronide and 1-BP-glucuronide and showed that the majority of the BP-conjugates in HepG2 cell culture were BP-sulfates and both Hepa-1c1c7 and hamster embryo cell cultures produce at least two isomeric BP-glucuronides. These highly sensitive continuous-flow FABMS methods allow the analysis of polycyclic hydrocarbon metabolites at levels formed in living cells without requiring the use of radioisotope labelled hydrocarbons and should be generally applicable for the direct analysis of hydrocarbon glucuronides and sulfates formed in biological systems

ASSESSMENT OF MARKETING CHALLENGES AND OPPORTUNITIES OF BRANDED AGRICULTURAL PRODUCTS: THE CASE OF NORTH SHOA ZONE

Agricultural commodities play an important role in development. But traditional commodity trading, based on exporting produce in bulk at low prices, limits the profits margin from these products flows to producers in developing countries. Thus, the objective of this study is to assess the marketing opportunities and challenges of branded agricultural products in selected districts of north Shoa Zone. Qualitative and quantitative approaches were used. Measures of central tendency such as mean, standard deviation are employed to interpret the data. The study result proved that, availability of information about market opportunity, inadequacy of benefits obtained from the brand, and packaging practice is highest in Minjar and Arerti, while adequacy of market size is better in Debre-Sina. Problems and challenges associated with market information, warehouse, finance, raw material, expert support, infrastructure, and production and selling space were highest in Mahal-Meda and Debre-Sina, Thus, creating more market linkage strengthening the cooperatives is crucial to help the farmer exploit the benefit of improved price and marketing

Species Difference in Glucuronidation Formation Kinetics with a Selective mTOR Inhibitor

ABSTRACT The mammalian target of rapamycin (mTOR) is a protein kinase that shows key involvement in age-related disease and promises to be a target for treatment of cancer. In the present study, the elimination of potent ATP-competitive mTOR inhibitor 3-(6-amino-2-methylpyrimidin-4-yl)-N-(1H-pyrazol-3-yl)imidazo[1,2-b]pyridazin-2-amine (compound 1) is studied in bile duct-cannulated rats, and the metabolism of compound 1 in liver microsomes is compared across species. Compound 1 was shown to undergo extensive N-glucuronidation in bile duct-catheterized rats. N-glucuronides were detected on positions N1 (M2) and N2 (M1) of the pyrazole moiety as well as on the primary amine (M3). All three N-glucuronide metabolites were detected in liver microsomes of the rat, dog, and human, while primary amine glucuronidation was not detected in cynomolgus monkey. In addition, N1-and N2-glucuronidation showed strong species selectivity in vitro, with rat, dog, and human favoring N2-glucuronidation and monkey favoring N1-glucuronide formation. Formation of M1 in monkey liver microsomes also followed sigmoidal kinetics, singling out monkey as unique among the species with regard to compound 1 N-glucuronidation. In this respect, monkeys might not always be the best animal model for N-glucuronidation of uridine diphosphate glucuronosyltransferase (UGT) 1A9 or UGT1A1 substrates in humans. The impact of N-glucuronidation of compound 1 could be more pronounced in higher species such as monkey and human, leading to high clearance in these species. While compound 1 shows promise as a candidate for investigating the impact of pan-mTOR inhibition in vivo, opportunities may exist through medicinal chemistry efforts to reduce metabolic liability with the goal of improving systemic exposure

Species difference in glucuronidation formation kinetics with a selective mTOR inhibitor DMD #54809 2 Running Title: Glucuronidation of an mTOR inhibitor

Non-standard abbreviations: CL, clearance; CLint, intrinsic clearance; IV, intravenous; LC-MS/MS, liquid chromatography tandem mass spectrometry; mTOR, mammalian target of rapamycin; PK, pharmacokinetics; UGT, UDP glucuronosyltransferase; NADPH, reduced nicotinamide adenine dinucleotide phosphate; AICc, Akaike information criterion corrected for small sample size DMD #54809 3 ABSTRACT The mammalian target of rapamycin (mTOR) is a protein kinase that shows key involvement in age-related disease and promises to be target for treatment of cancer. In the present study, the elimination of potent ATP-competitive mTOR inhibitor 3-(6-amino-2-methylpyrimidin-4-yl)-N-(1H-pyrazol-3-yl)imidazo[1,2-b]pyridazin-2-amine (compound 1) is studied in bile duct cannulated rats, and the metabolism of compound 1 in liver microsomes is compared across species. Compound 1 was shown to undergo extensive N-glucuronidation in bile duct catheterized (BDC) rats. N-glucuronides were detected on positions N1 (M2) and N2 (M1) of the pyrazole moiety as well as on the primary amine (M3). All three N-glucuronide metabolites were detected in liver microsomes of the rat, dog and human, while primary amine glucuronidation was not detected in cynomolgus monkey. In addition N1 and N2-glucuronidation showed strong species selectivity in vitro, with rat, dog and human favoring N2-glucuronidation and monkey favoring N1-glucuronide formation. Formation of M1 in monkey liver microsomes also followed sigmoidal kinetics, singling out monkey as unique among the species with regard to compound 1 N-glucuronidation. In this respect, monkeys might not always be the best animal model for N-glucuronidation of UGT1A9 or UGT1A1 substrates in humans. The impact of N-glucuronidation of compound 1 could be more pronounced in higher species such as monkey and human leading to high clearance in these species. While compound 1 shows promise as a candidate for investigating the impact of pan-mTOR inhibition in vivo, opportunities may exist through medicinal chemistry effort to reduce metabolic liability with the goal of improving systemic exposure

Localization and Quantification of Drugs in Animal Tissues by Use of Desorption Electrospray Ionization Mass Spectrometry Imaging

Mass spectrometric imaging (MSI) has emerged as a powerful technique to obtain spatial arrangement of individual molecular ions in animal tissues. Ambient desorption electrospray ionization (DESI) technique is uniquely suited for such imaging experiments, as it can be performed on animal tissues in their native environment without prior treatments. Although MSI has become a rapid growing technique for localization of proteins, lipids, drugs, and endogenous compounds in different tissues, quantification of imaged targets has not been explored extensively. Here we present a novel MSI approach for localization and quantification of drugs in animal thin tissue sections. DESI-MSI using an Orbitrap mass analyzer in full scan mode was performed on 6 μm coronal brain sections from rats that were administered 2.5 mg/kg clozapine. Clozapine was localized and quantified in individual brain sections 45 min postdose. External calibration curves were prepared by micropipetting standards with internal standard (IS) on top of the tissues, and average response factors were calculated for the scans in which both clozapine and IS were detected. All response factors were normalized to area units. Quantifications from DESI-MSI revealed 0.2–1.2 ng of clozapine in individual brain sections, results that were further confirmed by extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis