Metabolism of a Serotonin-4 Receptor Partial Agonist 4-{4-[4-Tetrahydrofuran-3-yloxy)-Benzo[d]Isoxazol-3-yloxymethyl]-Piperidin-1-ylmethyl}-Tetrahydropyran-4-ol (TBPT): Identification of an Unusual Pharmacologically Active Cyclized Oxazolidine Metabolite in Human

doi:10.1002/jps.23542

Journal of Pharmaceutical Sciences

Volume 102, Issue 9, September 2013, Pages 3277-3293

https://doi.org/10.1002/jps.23542 Get rights and content

ABSTRACT

4-{4-[4-Tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol (PF-4995274, TBPT) is a new agent that is a partial agonist of the human serotonin-4 (5-HT4) receptor and is under investigation for neurological disorders. Metabolism of TBPT was examined in vitro in human liver microsomes and human hepatocytes. Metabolites were also identified in the plasma of healthy human subjects in a phase 1 clinical study. Human-derived metabolite profiles were compared with corresponding profiles obtained in laboratory animal species. There were two major routes of metabolism in vitro: N-dealkylation of the methyltetrahydropyran moiety (M1) and hydroxylation at the seven position of the benzisoxazole moiety (M4). These were also observed in human plasma; however, in that matrix, the major metabolite was an unusual cyclized oxazolidine entity (M2). M2 was proposed to be formed via generation of an intermediate 4° iminium ion on the piperidine ring followed by spontaneous cyclization by attack of the β-hydroxyl substituent of the tetrahydropyran ring to form a cyclized oxazolidine product. An authentic standard of the metabolite was generated using a methylene-blue-sensitized photochemical oxidation reaction as well as microbial transformation. Further investigation of this metabolite showed that it also possessed 5-HT4 agonism activity similar to the parent. The metabolite was 150-fold more highly protein bound in human plasma than TBPT, which is consistent with its presence as a major circulating metabolite while being only a minor metabolite in in vitro systems. Overall, this illustrates the importance of understanding the complex dispositional properties of a pharmacologically active metabolite.

Section snippets

INTRODUCTION

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive decline in cognitive and functional abilities. It is the most common cause of dementia, accounting for 60%–80% of all cases.1 In a study of global prevalence of AD based on the United Nations' global predictions of population growth and other epidemiological studies, it has been estimated that by 2050, this number will be approximately 115 million.² The current standard of pharmacotherapy for AD uses a

Materials

4-{4-[4-Tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol, metabolite M1, and 6-fluoro-3-isopropyl-2-oxo-2,3-dihydro-benzoimidazole-1-carboxylic acid [1(4-hydroxytetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]amide were synthesized at Pfizer, Inc. Metabolite M3 was synthesized using a biomimetic approach under contact from HepatoChem Inc. (Princeton, New Jersey). Liver microsomes from male Wistar rats, male beagle dogs, and humans were

Metabolite Profiles of TBPT in In Vitro Incubations and Plasma from Rat and Dog

The metabolism of TBPT was assessed in rat, dog, and human liver microsomal incubations, as well as an incubation in human hepatocytes. The microsomal incubations were supplemented with both NADPH and UDPGA to support CYP and UGT activities. Chromatograms (UV traces) are shown in Figure 2. A total of 12 metabolites were observed in these incubation extracts and are listed in Table 1 with positive ion mass spectral fragmentation data and proposed structures. Initial metabolic pathways included N

DISCUSSION

Pharmacolgically active drug metabolites are important to identify and understand as early as possible in a drug research program. Knowledge of the intrinsic target receptor potency, free exposure, and target tissue penetrability of a metabolite, relative to the parent drug, is necessary to understand the pharmacokinetic–pharmacodynamic relationship and to select the best dosing regimen that can maximize efficacy and minimize side effects.

The 5-HT4 receptor partial agonist TBPT offers an

CONCLUSIONS

4-{4-[4-Tetrahydrofuran-3-yloxy)-benzo[d]isoxazol-3-yloxymethyl]-piperidin-1-ylmethyl}-tetrahydropyran-4-ol is metabolized to metabolite M2, which also has some activity toward the 5-HT4 receptor. It is an unusual oxazolidine metabolite that arises by oxidation of the piperidine on TBPT to an iminium ion that undergoes cyclization with the β-hydroxy substituent. An “activity-gram” approach, wherein a pooled human plasma extract is fractionated and each fraction is tested for receptor activity,

ACKNOWLEDGEMENTS

The authors extend their appreciation to Jim Cawley and Ming Zeng for the microbial biosynthesis of metabolite M2.

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Metabolism of a Serotonin-4 Receptor Partial Agonist 4-{4-[4-Tetrahydrofuran-3-yloxy)-Benzo[d]Isoxazol-3-yloxymethyl]-Piperidin-1-ylmethyl}-Tetrahydropyran-4-ol (TBPT): Identification of an Unusual Pharmacologically Active Cyclized Oxazolidine Metabolite in Human

ABSTRACT

Section snippets

INTRODUCTION

Materials

Metabolite Profiles of TBPT in In Vitro Incubations and Plasma from Rat and Dog

DISCUSSION

CONCLUSIONS

ACKNOWLEDGEMENTS

Neuropharmacol

Neuropharmacol

Neuropharmacol

J Biol Chem

Exp Gerontol

Tetrahedron

Anti-amyloid-β immunotherapy in Alzheimer's disease: Relevance of transgenic mouse studies to clinical trials

J Alzheimers Dis

The pharmacoeconomics of cognitive enhancers in moderate to severe Alzheimer's disease

CNS Drugs