San Jose State University SJSU ScholarWorks Master's Theses Master's Theses and Graduate Research Fall 2015 An Evaluation of the Disposition of R941000, a TetrazoloneTelmisartan Analog: A Case Study of the Suitability of Tetrazolone As a Carboxylic Acid Bioisostere Ryan Brant Murray San Jose State University Follow this and additional works at: https://scholarworks.sjsu.edu/etd_theses Recommended Citation Murray, Ryan Brant, "An Evaluation of the Disposition of R941000, a Tetrazolone-Telmisartan Analog: A Case Study of the Suitability of Tetrazolone As a Carboxylic Acid Bioisostere" (2015) Master's Theses 4655 DOI: https://doi.org/10.31979/etd.c45w-e8ma https://scholarworks.sjsu.edu/etd_theses/4655 This Thesis is brought to you for free and open access by the Master's Theses and Graduate Research at SJSU ScholarWorks It has been accepted for inclusion in Master's Theses by an authorized administrator of SJSU ScholarWorks For more information, please contact scholarworks@sjsu.edu AN EVALUATION OF THE DISPOSITION OF R941000, A TETRAZOLONETELMISARTAN ANALOG IN RATS: A CASE STUDY ON THE SUITABILITY OF TETRAZOLONE AS A CARBOXYLIC ACID BIOISOSTERE A Thesis Presented to The Faculty of the Department of Chemistry San José State University In Partial Fulfilment of the Requirements for the Degree Master of Science By Ryan Murray December 2015 © 2015 Ryan Murray ALL RIGHTS RESERVED The Designated Thesis Committee Approves the Thesis Titled AN EVALUATION OF THE DISPOSITION OF R941000, A TETRAZOLONETELMISARTAN ANALOG IN RATS: A CASE STUDY ON THE SUITABILITY OF TETRAZOLONE AS A CARBOXYLIC ACID BIOISOSTERE by Ryan Murray APPROVED FOR THE DEPARTMENT OF CHEMISTRY SAN JOSÉ STATE UNIVERSITY December 2015 Dr Joseph Pesek Department of Chemistry Dr Roger Terrill Department of Chemistry Dr Christophe Colas Department of Pharmacokinetics, Rigel Pharmaceuticals Abstract Carboxylic acids are ubiquitous in medicinal compounds, such as nonsteroidal antiinflammatories, statins, hypertensives, and anticoagulants Despite their prolific use, unfavorable characteristics such as metabolic instability, poor membrane permeability, and toxicity have been associated with this moiety in some instances Bioisosteres have been employed to attenuate these issues However, bioisostere use can alter drug potency and disposition Recently, our company demonstrated the feasibility of the tetrazolone moiety as a carboxylic acid bioisostere for the angiotensin II antagonist telmisartan R941000 (telmisartan-tetrazolone analog) was a potent in vitro inhibitor of angiotensin II and possessed a similar disposition to telmisartan To the best of our knowledge, no studies of the changes in disposition caused by bioisosteric replacement of a carboxylic acid with a tetrazolone have been published In this work, the disposition of R941000 was evaluated in Sprague Dawley rats, and in vitro metabolism was conducted using human and rat hepatocytes and supplemented microsomes Results indicated comparable PK parameters for R941000 relative to telmisartan, respectively, bioavailability (64.7% vs 59.2%), exposure (2610 ngL/h vs 1850 ngL/h) Clpred (4.51 ml/min vs 7.23 ml/min) t1/2 (5.37h vs 3.64 h) and Vss (1.67L/kg vs 1.59L/kg) Both compounds underwent biliary excretion, and glucuronide metabolites were found in rat bile; however, no significant glucuronidation was observed in in vitro assays Additional studies utilizing tetrazolone bioisosteres in other species and classes of compounds are needed to further characterize their utility as a carboxylic acid substitute ACKNOWLEDGEMENTS I would like to express my gratitude to San Jose State University for allowing me to pursue my research in both academia and the private sector I would like to thank the department of chemistry for the opportunity to write and present my thesis work Additionally, I would like to thank Rigel Pharmaceuticals and the DMPK department for their support of me in my graduate studies To my committee members, Dr Pesek, Dr Terrill, and Dr Colas, I am very grateful for your insight, suggestions, guidance, and time To my research advisor, Dr Pesek, thank you very much for your flexibility and helping me find a project I could pursue while working full time To my beautiful family Toni and Ruby, thank you for all your support and patience while I have been pursuing my degree I am very grateful for all the motivation and encouragement both of you have given me during this time v Table of Contents 1.0 Introduction 1.1 Drug Disposition: Principles of ADME 1.2 Basic Pharmacokinetic Principles 1.3 Drug Metabolism 1.4 In vitro Tools: Cryopreserved Hepatocytes and Microsomes 14 1.5 Principles behind LC/MS 16 1.5.1 Chromatography Theory and HPLC and UPLC Applications 16 1.5.2 Principles of Mass Spectroscopy in Metabolism and PK Studies 18 2.0 Experimental 26 2.1 Chemicals & Biological Materials 26 2.2 Formulation Preparation 27 2.3 Pharmacokinetic Studies 27 2.4 Elimination Route Studies 28 2.5 Hepatic Extraction Studies 29 2.6 Rat Bile Metabolite Identification Studies 30 2.6.1 β-Glucuronidase 31 2.7 Microsomal Stability Studies 31 2.7.1 UDPGA and Alamethicin Supplemented Human and Rat Liver and Intestinal Microsomes 32 2.8 Metabolite Identification through Cryopreserved Human and Rat Hepatocytes 33 2.9 Plasma Protein Binding 34 3.0 Results 34 3.1.0 Pharmacokinetic Studies 35 3.1.1 Hepatic Extraction 40 3.1.2 Elimination Studies 42 3.2.0 Rat In vivo Metabolism: Searching for Metabolites in Bile 43 3.2.2 Rat and Human Cryopreserved Hepatocyte and Microsomal Studies 56 4.0 Discussion 69 Pharmacokinetics 69 vi Metabolism 70 Conclusion 74 5.0 Future Studies 74 References 77 vii List of Figures Figure Chemical Structure of R941000 Figure An illustration of ADME principles Figure The elimination phase of a drug Figure Volume of distribution Figure Phase I and II metabolism Figure Cytochrome P450 mechanism 10 Figure UGT catalytic cycle 11 Figure Reactivity mechanism of acyl glucuronides 13 Figure Location of UGT and P450 on the ER 15 Figure 10 A schematic of the ESI process 19 Figure 11 A schematic for MRM 20 Figure 12 A hypothetical fragmentation pattern of two isobaric ions 22 Figure 13 Use of MRM scanning mode for metabolite identification 24 Figure 14 R941000 IV PK data 35 Figure 15 Telmisartan IV PK data 36 Figure 16 R941000 PO PK data 37 Figure 17 Telmisartan PO PK data 38 Figure 18 Telmisartan and telmisartan-O-acyl glucuronide chromatograms 40 Figure 19 Hepatic extraction ratios for R941000 and telmisartan 41 Figure 20 XIC chromatograms of R941000 and telmisartan 45 Figure 21 TIC and XIC chromatograms in bile of rats dosed with R941000 45 Figure 22 XICs of R941000-glucuronide 47 viii Figure 23 Mass fragmentation spectra of observed parent and metabolite peaks 48 Figure 24 Potential tetrazolone glucuronidation sites 49 Figure 25 Incubation of rat bile samples with β-glucuronidase 50 Figure 26 UV chromatogram of rat bile samples 51 Figure 27 Oxidized metabolites of R941000 in rat bile samples 53 Figure 28 Potential oxidation sites of R941000 54 Figure 29 Incubation of telmisartan in HCH 57 Figure 30 Incubation of R941000 in HCH 59 Figure 31 Incubation of telmisartan in alamethicin HLM and RLM 61 Figure 32 Incubation of R941000 in alamethicin treated HLM and RLM 0h 62 Figure 33 Incubation of R941000 in alamethicin treated HLM and RLM 2h 63 Figure 34 Incubation of R941000 in alamethicin treated RLM 2h 64 Figure 35 Incubation of R941000 in alamethicin treated RIM at 2h 65 Figure 36 The stability of R941000 in HLM 66 Figure 37 The stability of R941000 in RLM 67 Figure 38 Incubation of telmisartan and R941000 in alamethicin treated RLM with tris buffer system 68 Figure 39 Potential reactivity of N-glucuronidated tetrazolones 71 Figure 40 Potential O-glucuronide tetrazolone reactivity 72 Figure 41 Potential reactivity of O-glucuronide tetrazolone towards nucleophiles 73 ix Figure 35 10 µM R941000 incubated in alamethicin treated RIM for h The top chromatogram shows the parent R941000 channel, followed by the glucuronide (middle) and +16 metabolite (bottom) channels From the microsomal experiments, oxidation mechanisms appeared to be more relevant than glucuronidation activity, contrary to in vivo observations Relative abundance by mass spectroscopy can be deceptive due to ionization efficiencies; additionally, other microsomal products may be forming that were not caught with the monitored transitions To capture the importance of total phase I contribution to R941000 metabolism, microsomal stability assays were employed For microsomal stability assays, a compound’s disappearance over time is recorded and a half-life is established (see Equation 11) In this way, all contributing factors can be accounted for, and the importance of phase I activity can be established 65 R941000 was incubated with HLM or RLM at µM (reduced concentration to avoid saturation of enzymes) in mg/ml protein over 45 min, with quenching times of 0, 5, 15, 30, and 45 Standards midazolam and propranolol were used for short and long stability controls (propranolol was metabolized quickly in rat; therefore, RLM lacked a long positive control) Results are recorded in Figures 36 and 37 (HLM, RLM) where midazolam had a half-life of around 3.5 in HLM and RLM and propranolol 38 in HLM and nearly in RLM R941000 appeared stable with a half-life exceeding 45 for both human and rat liver microsomes Figure 36 The stability of R941000 in HLM 66 Figure 37 The stability of R941000 in RLM In vitro data had thus far failed to significantly correlate with in vivo results; however, a modified supplemented microsome protocol was attempted Several methods report improved UGT1A4 and UGT1A9 using 100 mM tris buffer pH 7.5-7.7 in human microsomes and expressed enzymes, while having no effect on other isoforms.17 Both of these enzymes are capable of N-glucuronidation and may thus provide a more vigorous metabolism of R941000 While UGT1A4 and 1A9 are human UGTs (rodent homologs are inactive), it was hoped a change in buffer would result in improved R941000 glucuronidation Results are shown in Figure 38 for the h time points Telmisartan acyl glucuronide formation had peak intensities of 106 cps, while no glucuronidation was observed for R941000 67 Figure 38 Incubation of R941000 in alamethicin treated RLM with tris-buffer The two top chromatograms are the MRM transitions for telmisartan and its acyl glucuronide metabolite while the bottom two are of R941000 and its glucuronide metabolite 68 4.0 Discussion Recently, our lab demonstrated the feasibility of using a tetrazolone group as a bioisostere of a carboxylic acid for a number of compounds including telmisartan.5 It was uncertain how replacement of this moiety with a tetrazolone would alter compound DMPK attributes Thus, PK and metabolism studies were performed on the telmisartantetrazolone analog R941000 as an example of how disposition may be altered with this novel bioisostere Pharmacokinetics R941000 total exposure levels and half-life were approximately 1.5 fold greater than telmisartan, while clearance was reduced about 1.5 fold Volume of distribution for the two compounds was nearly identical, and bioavailability values were comparable at 65% and 59% for R941000 and telmisartan, respectively No significant hepatic first pass effect of R941000 was observed (HE 45 73 Conclusion PK and metabolism analysis confirmed R941000 maintained similar to slightly improved PK profile in rat, while differing in metabolism with the formation of two major glucuronide metabolites It is hypothesized that these glucuronides are an Nglucuronide and O-glucuronide In vitro assays were unable to mimic in vivo results, and it is unclear where and what UGT isozyme is responsible for R941000 glucuronidation in vivo While only one compound and one preclinical species were assessed in this study, analysis of R941000 does provide a useful first step in demonstrating the feasibility of replacement of carboxylic acids with tetrazolones 5.0 Future Studies Currently, development of an in vitro assay mimicking in vivo results of R941000 glucuronidation is needed Future studies using UGT expressed enzymes are planned to address this issue UGT expressed enzymes may provide more robust UGT activity and will allow for identification of individual UGT isozymes responsible for glucuronidation if observed, thus potentially determining what enzyme(s) are responsible for the N- or Oglucuronide Additionally, identification of the human recombinant UGT enzyme(s) may provide for a better understanding of potential human metabolism as well as potential drug-drug interactions While an N- and O-glucuronide are the suspected metabolites, further characterization of these species is needed to definitively confirm their identity Chemical synthesis of the proposed metabolites is being attempted and isolation of the 74 observed metabolites in sufficient quantities in bile for NMR analysis are currently underway Determining potential reactivity mechanisms of a compound or particular molecular moiety can be a long and arduous process, and one that sometimes may only become evident after a compound has been on the market for a number of years.10-12 Indeed, much time, energy, and research have gone into developing the current hypothesis of acyl glucuronide toxicity.10 However, there are a few assays that may be employed to test if tetrazolone glucuronides may be prone to interact with endogenous proteins in the same manner as acyl glucuronides One method, as previously suggested, would be to incubate tetrazolone glucuronides with nucleophiles to assess their susceptibility to nucleophilic attack A second method, developed by Sawamura and coworkers, correlates reactivity of acyl glucuronides with its stability in a buffer solution and its tendency towards acyl migration The shorter the half-life, the more prone an acyl glucuronide is to migrate and react with electron rich amino acids.12 In this paper, Sawamura tested several O-acyl glucuronide metabolites for compounds such as zomepirac (short half-life), diclofenac (intermediate half-life) and telmisartan (long halflife) Compounds forming acyl glucuronides with short half-lives were strongly correlated with toxicity and subsequent market withdrawal; those with intermediate halflives were associated with warnings; those with good stability were considered “safe.”12 Synthesizing O- and N-glucuronide equivalents to compounds such as zomepirac, diclofenac, and telmisartan acyl glucuronides and testing their stability in buffer may provide an informative method of assessing potential reactivity and ranking of 75 tetrazolone glucuronides relative to each other and their acyl glucuronide counterparts.12 Additionally, the stability of N-glucuronides may help confirm its inability for tetrazolone migration Finally, animal studies comparing toxicity between tetrazolones and carboxylic compounds may provide a general yet informative study on the relative toxicity of this bioisostere to its carboxylic acid counter parts While the R941000 tetrazolone analog of telmisartan has thus far behaved as a carboxylic acid bioisostere, analysis of additional tetrazolone analogs is needed to better characterize this moiety as a carboxylic acid replacement since other tetrazolone analogs may have a different disposition and potency relative to their carboxylic acid counterparts A larger set of examples would provide greater context into the range of disposition alterations caused by this bioisostere 76 References Ballatore, C.; Huryn, D., M.; Smith III, A B Carboxylic Acid (Bio)isosteres in Drug Design ChemMedChem 2013, 8, 385 – 396 Williams, D A.; Lemke, T L.; Foye’s Principles of Medicinal Chemistry, 5th ed.; Lippincott Williams & Wilkins: Massachusetts, 2002; chapter Papastavrou, N.; Chatzopoulou, M.; Kyriaki, P.; Nicolaou, L 1-Hydroxypyrazole, as a Bioisostere of the Acetic Acid Moiety in a 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Table IV PK parameters of R941000 and telmisartan... by tracking the disappearance of the TA over time by a linear plot of TA/IS peak area ratio From the linear plot, the elimination constant, k, was estimated and the half-life of the TA was determined.. .AN EVALUATION OF THE DISPOSITION OF R941000, A TETRAZOLONETELMISARTAN ANALOG IN RATS: A CASE STUDY ON THE SUITABILITY OF TETRAZOLONE AS A CARBOXYLIC ACID BIOISOSTERE A Thesis Presented to The