In vivo biotransformation studies

In vitro studies can only give a limited, mechanistic picture of biotransformation in animals or humans. The quantitative importance of each individual metabolite can only be assessed in vivo. Also, samples collected from in vivo studies give rise to comprehensive metabolite identification work (Watt et al. 2003 Clarke et al. 2001) which is also required from a regulatory point of view (Baillie et al. 2002). Due to the labour-intense nature of these studies and the need of applying radiolabeled compounds in order to get a complete picture of biotransformation these studies are performed at a later stage of development during preclinical and clinical phase. 

Urine and feces homogenates might serve as a basis for structure elucidation of metabolites by LC-MS/MS directly or - if necessary - by more detailed NMR/MS investigation after isolation of the metabolites by preparative chromatography. 

Samples are analyzed by a suitable chromatographic system, typically HPLC with on-line radiodetection. Metabolite profiles in plasma and tissues are given as percent of radioactivity present in total. Profiles in urine, bile and feces are given as percent of the dose administered. 

In most instances classical approaches in isolation of metabolites followed by MS and NMR analyses are still necessary to obtain the definitive structural information. This is due for complex structures of metabolites as they are drugs in development in most instances with the need of multiple NMR experiments in addition to an initial one dimensional1H NMR experiment. 

Techniques applying test compounds labeled with stable isotopes are applied as well (Browne et al. 1993). However, they have not found broad acceptance in substituting radioisotopes in this field due to their restrictions in quantification of unknown metabolites. 

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This book chapter refers to rat in vivo biotransformation studies performed by LC-ESI MS/MS [5,40],... 

Numerous studies have been published on the in vivo metabolism of peptides. However, these studies are concerned mainly with assessment of pharmacokinetic parameters such as half-life and clearance. Only seldom is the in vivo biotransformation of peptides that contain only common amino acids investigated in any detail, due to the difficulty of monitoring products of proteolysis that are identical to endogenous peptides and amino acids. More importantly, such studies fail to yield mechanistic and biochemical insights. For this reason, we begin here with a discussion of the metabolism of just a few peptides in some selected tissues, namely portals of entry (mouth, gastro-intestinal tract, nose, and skin), plasma, organs of elimination (liver, kidney), and pharmacodynamic sites (brain and cerebrospinal fluid). These examples serve as introduction for the presentation in Sect. 6.4.2 of the involvement of individual peptidases in peptide metabolism. 

In vitro biotransformation studies in pig, rat, and dog liver revealed that hydroxyl mebendazole and amino mebendazole are the main metabolites of mebendazole (24). To verify the formation in vivo of the expected metabolites in eel, wild European eels were subjected to 6, 24, and 48 h treatments (25). The results clearly indicated that mebendazole given to eels via the water was absorbed and metabolized into the hydroxyl and amino metabolites, as was also tlie case with... 

A Generalized Scheme of Pathways for the Biotransformation of CDDs Based on Information from In Vivo Mammalian Studies... 

Studies on in vivo biotransformation of benztropine in rats were supported by LC-ESI MS analysis and are referred herein [59],... 

Accordingly, Chen et al. performed a serious number of charming in vivo and in vitro biotransformation studies for diverse TTA (anisodamine, anisodine, atropine, scopolamine) after oral administration of the drugs to rat [5-7,51, 52,87], Urine was analysed by LC-MS/MS methods that provided the following operation modes ... 

Meteor can be interfaced with the MetaboLynx mass spectrometer software from Waters Corporation to integrate mass spectrometry data from metabolism studies directly. MetaboLynx is part of the Waters MassLynx Application Managers, a suite of mass spectrometry instrument software [16]. It is designed for automated metabolism studies with data from LC/MS or LC/MS/MS time-of-flight (TOP) experiments. MetaboLynx is able to detect peaks in an LC/MS data file resulting from in vitro or in vivo biotransformation and provides a list of elemental formulae for unidentified components in a mass spectrum. Meteor uses these data to filter the list of predicted metabolites. System requirements are similar to DEREK for Windows. 

Limited information is available on the biotransformation of carbon disulfide in humans, and the metabolic products of carbon disulfide are not completely known. In animals and humans the proposed metabolic pathways involved in the metabolism of carbon disulfide (Beauchamp et al. 1983) are depicted in Figure 2-3, reactions i-x. Reaction i has been demonstrated in in vivo animal studies and in in vitro assays. Reactions ii-v are proven by in vitro studies, while products of reactions vi-ix are the results of proposed metabolic pathways of carbon disulfide in animals and humans. Carbon disulfide is metabolized by cytochrome P-450 to an unstable oxygen intermediate (reaction i). The intermediate may either spontaneously degrade to atomic sulfur and carbonyl sulfide (reaction ii) or hydrolyze to form atomic sulfur and monothiocarbonate (reaction iii). The atomic sulfur generated in these reactions may either covalently bind to macromolecules (reaction iv) or be oxidized to products such as sulfate (reaction v). 

For example, van Heek and coworkers observed a lead candidate that underwent extensive first-pass metabolism and yet elicited a significant level of pharmacological activity (van Heek et al., 1997). To evaluate the biological activity of the in vivo biotransformation products, they collected samples of bile from rats dosed with a lead compound and directly administered the samples to a bile duct cannulated rats via an intraduodenal cannula. As a control study, the parent compound prepared in a blank bile was dosed in a similar fashion to the recipient rats. The results indicated that the in vivo activity elicited by the bile samples was higher than the parent control sample, clearly indicating the presence of an active metabolite(s) that was more potent than the parent compound. To identify the active component, the bile sample was then fractionated and each fraction tested for biological activity. The structure of the metabolite was then established following the detection of the active fraction. As mentioned before, further modification of the active metabolite led to the discovery of ezetimibe. 

Cineole is a avor constituent of Citrus aurantifolia and Piper cubeba (Bornscheuer et al., 2014). In vitro and in vivo animal studies demonstrated extensive biotransformation of this mono-terpene strongly suggesting biotransformation in the human body too. After oral application to rabbits, four neutral and one acidic metabolite could be isolated from urine (Asakawa et al., 1988). Using rat and human liver microsomes, however, only 2-hydroxylation could be observed indicating species related differences in 1,4 cineole metabolism (Miyazawa et al., 2001a) (Figure 9.8). 

Prokai, L., Kim, H.-S., Zharikova, A., Roboz, J., Ma, L., Deng, L. and Simonsick Jr., W.J., Electrospray ionization mass spectrometric and LC/MS studies on the metabolism of synthetic dynorphin A peptides in brain tissue in vitro and in vivo. J. Chromatogr. A, 800, 59-68 (1998). Klintenberg, R. and Andren, P.E., Altered extracellular striatal in vivo biotransformation of the opioid neuropeptide dynorphin A(l-17) in the unilateral 6-OHDA rat model of Parkinson s disease. J. Mass Spectrom., 40, 261-270 (2005). 

Kassahun, K. Pearson, P. G. Tang, W. McIntosh, I. Leung, K. Elmore, C. Dean, D. Wang, R. Doss, G. Baillie, T. A. Studies on the metabolism of troglitazone to reactive intermediates in vitro and in vivo. Evidence for novel biotransformation pathways involving quinone methide formation and thiazolidinedione ring scission. Chem. Res. Toxicol. 2001, 14, 62-70. 

Studies on the metabolism of troglitazone to reactive intermediates in vitro and in vivo. Evidence for novel biotransformation pathways involving quinone methide formation and thiazolidinedione ring scission. Chemical Research in Toxicology, 14 (1), 62—70. 

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