An experimental approach to enhance parent ion fragmentation for metabolite identification studies: Application of dual collision cells in an orbital trap

Recent mass spectrometry advancements including data-dependent scanning and high resolution mass spectrometry have aided metabolite profiling for non-radiolabeled xenobiotics. However, narrowing down a site of metabolism is often limited by the quality of collision induced dissociation (CID) based parent ion fragmentation. An alternative dissociation technique, higher energy collisional dissociation (HCD), enriches compound fragmentation and yields “triple quadrupole like fragmentation”. Applying HCD along with CID and data dependent scanning could enhance structural elucidation for small molecules. LC-MSn experiments with CID and HCD fragmentation were run for commercially available compounds on orbital trap, a hybrid linear ion trap-orbitrap mass spectrometer equipped with accurate mass measurement capability. The developed method included stepped normalized collision energy (SNCE) parameters to enhance MS fragmentation without tuning of compounds. All evaluated compounds demonstrated improved fragmentation under HCD as compared to CID. Results suggest that an LC-MSn method that incorporated both SNCE HCD and CID enabled parent ion fragmentations, afforded comprehensive structural information for the compounds under investigation. Such a method was remarkably better than one with only CID MSn in an ion trap. It is evident that such an acquisition method can augment identification of unknown metabolites in drug discovery by improving fragmentation efficiency of both the parent compound and putative metabolite(s)

An in vitro approach to investigate ocular metabolism: Part II - Betaxolol

Topical glaucoma treatments, including multiple β-adrenergic receptor blocking agents, have often been limited by poor ocular absorption and bioavailability. Betaxolol, a selective β1-blocker, has been well studied for its pharmacokinetics and disposition after oral administration. However, limited betaxolol metabolism has been documented after topical dosage despite the growing number of immediate and sustained release, ocular treatments under investigation to improve absorption and retain efficacy. Conversely, plasma, urine and in vitro liver metabolism data has been reported for other topical glaucoma treatments, for example levobunolol. This investigation developed an ocular S9 assay combined with high-resolution mass spectrometry to investigate the role of ocular metabolism for preclinical and human subcellular fractions. Nine metabolites, eight of which were not previously reported, were characterized. Betaxolol’s active metabolite, hydroxybetaxolol, was observed in rat, rabbit and human ocular and liver S9 fractions. To date, hydroxybetaxolol concentrations in vivo or its binding affinity/selectivity have not been reported. Rabbit liver S9 fractions demonstrated extensive glucuronidation that was not observed ocular fractions. For betaxolol and levobunolol, investigated in a companion paper, the complete ocular metabolic profile could not be predicted from the liver S9 assay alone. This is the first comprehensive report of a functional, in vitro ocular metabolism assay, which demonstrates the poor prediction of ocular metabolism using liver as a surrogate model

An in vitro approach to investigate ocular metabolism: Part I - Levobunolol

The number drugs on the market for topical ocular administration in treatment of ocular diseases have been steadily increasing in the recent past. Despite ocular transport and ocular delivery being very well documented, metabolism in the eye is not well-documented. We devised a novel yet simple in vitro approach coupled with high resolution mass spectrometry that may be readily applied to future ocular drug metabolism studies to measure rate and extent of metabolism. The current investigation documents in vitro ocular and liver metabolism of levobunolol, a clinically used, potent non-selective β adrenergic antagonist, in S9 fractions from rat, rabbit and human. In the present study, we identified sixteen metabolites of levobunolol, eight of which were not previously reported. A direct acetyl conjugate of levobunolol that was identified in all ocular and liver fractions was never reported in any prior in vitro or in vivo levobunolol metabolism studies. We identified two human ocular specific conjugative metabolites subsequent to oxidation. Overall, our results for levobunolol metabolism indicate that rat liver S9 and human ocular S9 incubations showed the presence of most metabolites, and that liver was a poor in vitro surrogate for eye across rat, rabbit and human for studying the rate and extent of metabolism. To the best of our knowledge, our investigation is the first documentation of comprehensive in vitro ocular metabolism of levobunolol and betaxolol (see companion paper, Bushee J.L. et. al.) in rat, rabbit and human ocular S9 and its comparison to liver S9 metabolic characterization

Metabolism of bromopride in mouse, rat, rabbit, dog, monkey, and human hepatocytes

Bromopride (BRP) has been utilized clinically for treatment of nausea, vomiting and gastro-intestinal motility disorders. The pharmacokinetics of BRP have been characterized in dogs and humans, however, the metabolic profile of BRP has not been well studied. The present study was aimed at better understanding BRP metabolism across species. We investigated biotransformation of BRP in mouse, rat, rabbit, dog, monkey, and human hepatocytes with the help of LC-MSn and accurate mass measurement. Mouse, rat, dog, and monkey are relevant in drug discovery and development as pre-clinical species to be compared with human, whereas rabbit gastric pacing and post-surgical models have been utilized in historic efficacy studies with BRP. Overall, 20 metabolites of BRP were identified across hepatocytes from six species. Monkey offered the most coverage for human, in terms of number of metabolites identified. Interestingly, M14, an N-sulfate metabolite of BRP was identified as a human-specific metabolite. BRP metabolism had only been reported in dog plasma and urine, historically. Our investigation is the first documentation of in vitro metabolism of BRP in all the species reported here. Metabolites M1, M2, M4-M10, M12, M13, and M15-M20 have not been previously reported. In summary, this report documents seventeen metabolites of BRP for the first time, thus providing a deeper insight into the biotransformation of BRP

Ocular Non-P450 oxidative, hydrolytic and conjugative drug metabolizing enzymes

Metabolism in the eye for any species, preclinical or human, is gaining rapid interest as pharmaceutical scientists aim to treat a wide range of so-called incurable ocular diseases. Over a period of decades, reports of metabolic activity toward various drugs and biochemical markers in select ocular tissues of in animals and humans have emerged. Ocular P450 enzymes and transporters have been recently reviewed. However, there is a dearth of collated information on non-P450 drug metabolizing enzymes in eyes of various preclinical species and humans in health and disease. In an effort to complement ocular P450s and transporters, which have been well reviewed in the literature, this review is aimed at presenting collective information on non-P450 oxidative, hydrolytic and conjugative ocular drug metabolizing enzymes. We also present a list of xenobiotics or drugs that have been reported to be metabolized in the eye

Identification of saturated and unsaturated fatty acids as microsomal degradation by-products by direct high resolution -ESI-MS/MS

In vitro clearance in liver microsomes is routinely measured in drug discovery and development for understanding the disposition properties of new chemical entities. Literature reports indicate that long chain fatty acids such as arachidonic, linoleic and oleic acids may be released over a period of time during a microsomal incubation. These fatty acids are known to be substrates for conjugating enzymes like Uridine diphosphoglucuronosyl transferases (UGTs), thus potentially inhibiting microsomal clearance of UGT substrates. The present study was aimed at deciphering the fatty acids that may be released as by-products of microsomal degradation. LC-MS analytical methods were developed to characterize and qualitatively assess the fatty acids without chemical derivatization in rat, monkey and human liver microsomes. Additionally, incubations with UDPGA were utilized to trap the released fatty acids in their glucuronate ester form, also characterized and confirmed by high resolution LC-MS/MS. Our results indicate for the first time that eicosapentanoic, trans- and cis- eicosenoic, docosanoic, and nervonic acid were released as microsomal by-products in incubation conditions. Our results corroborate that palmitic, stearic, linoleic, and arachidonic acid were also formed as previously reported. Additionally, α- and γ-linolenic, eicosapentaenoic, palmitoleic, linoleic, arachidonic, palmitic, oleic, and stearic acid were identified as their corresponding acyl-glucuronides in rat, monkey and human liver microsomes. Our investigation renders a deeper understanding about the fatty acids which may be released by degradation of liver microsomes

Evaluation of pharmaceutical excipients as cosolvents in 4-methyl umbelliferone glucuronidation in human liver microsomes: An application for compounds with low solubility

Introduction: In order to minimize the potential inhibitory effects of organic solvents on metabolic activity, standard incubation procedures for carrying out microsomal assays involve the use of less than 1% w/v organic solvents. Often, solvents needed to dissolve the substrate add-up nearly to this concentration. This presents a practical limitation for poorly soluble xenobiotics, which cannot be incubated at concentrations high enough to obtain a Vmax, and therefore subsequent values for Km and Clint cannot be calculated. Our goal was to study the application of a variety of pharmaceutical excipients to aid the solubilization of compounds in vitro in glucuronidation incubations, without affecting the reaction kinetics. Methods: In vitro glucuronidation incubations were carried out in human liver microsomes with 4-methylumbelliferone (4-MU) and the kinetics of 4-MU glucuronidation in the presence of excipients were compared to that in control incubations without any excipients. In addition, IC75 values were calculated for each excipient. Results and Summary: We observed that HPBCD may be employed in in vitro glucuronidation incubations up to 0.5% w/v without affecting the Clint of 4-MU. Although NMP and DMA showed low IC75 values approximately 0.1% w/v each, neither excipients altered the Clint of 4-MUG formation. Our studies point toward a possible application of pharmaceutical excipients to carry out in vitro glucuronidation of substrates with poor aqueous solubility, in order to estimate Clint and subsequently scaled organ clearance values

Identifying and Attracting the "right" Investors: Evidence on the Behavior of Institutional Investors

This article summarizes the findings of research the author has conducted over the past seven years that aims to answer a number of questions about institutional investors: Are there significant differences among institutional investors in time horizon and other trading practices that would enable such investors to be classified into types on the basis of their observable behavior? Assuming the answer to the first is yes, do corporate managers respond differently to the pressures created by different types of investors- and, by implication, are certain kinds of investors more desirable from corporate management's point of view? What kinds of companies tend to attract each type of investor, and how does a company's disclosure policy affect that process? 2004 Morgan Stanley.

Oxidative ipso Substitution of 2,4-Difluoro-benzylphthalazines: Identification of a Rare Stable Quinone Methide and Subsequent GSH Conjugate

ABSTRACT: In vitro metabolite identification and GSH trapping studies in human liver microsomes were conducted to understand the bioactivation potential of compound 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol], an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a GSH adduct (M2). The stability of this metabolite arises from extensive resonance-stabilized conjugation of the substituted benzylphthalazine moiety. Cytochrome P450 (P450) phenotyping studies revealed that the formation of M1 and M2 were NADPH-dependent and primarily catalyzed by CYP3A4 among the studied P450 isoforms. In summary, an unusual and stable quinone methide metabolite of compound 1 was identified, and a mechanism was proposed for its formation via an oxidative ipso substitution