Metabolite ID. Introduction

Transcription

1 Metabolite ID Introduction Drug metabolism plays an increasingly important role in the drug discovery and development process. For the early discovery phase, investigating drug metabolism is intended to identify soft spots and reactive metabolites for optimizing lead compounds. In the preclinical development stage, investigation of species, racial and gender differences of drug metabolism is essential for understanding drug-drug interactions, drug safety profiles and for selecting species for the subsequent toxicology studies. Investigation of drug metabolism is also critical for ensuring the safety of metabolites is adequately tested in animals and for predicting whether toxicological findings observed in animals are likely to link to humans. Identifying drug metabolites in complicated biological matrices continues to be a challenge. The high resolution accurate mass spectrometer has been demonstrated to be one of the most powerful and reliable tools for the structural elucidation of drug metabolites because it provides accurate masses of molecular ions as well as daughter ions to confirm valuable structural-related inion and a semi-empirical formula of the drug metabolites. Meanwhile, data analysis software such as MetaboLynx can automatically generate a list of proposed metabolites by comparing the post-acquisition data of the sample from metabolism studies with those of the blank based on the mass defect filter (MDF) method thereby removing a significant amount of guesswork. WuXi DMPK utilizes a Waters UPLC-QTOF coupled with MetaboLynx software and an AB Sciex 4000 QTrap (for up to MS 3 spectra) for in vitro and in vivo drug Met ID (Figure 1). In addition, radiolabeled ( 14 C and 3 H) in vitro and in vivo MID assays as well as the other in vivo ADME assays are also provided to further support the preclinical development of drug candidates. Figure 1. Waters Acquity UPLC-QTOF Xevo G2 QTOF system for Met ID studies. Assay Description Species Deliverables Fast Met ID Assays Stage LF LO PCC IND TAT (working days) Fast In Vitro Met ID in Liver Microsomes UPLC-UV-Q-TOF MS + MetaboLynx English or Chinese report in word or other Major metabolite (top 3~5 metabolites) Metabolic pathway 5 Fast In Vitro Met ID in S9 Fractions UPLC-UV-Q-TOF MS + MetaboLynx English or Chinese report in word or other Major metabolite (top 3~5 metabolites) Metabolic pathway 5 28 Metabolite ID DMPK LTD.WUXIAPPTEC.COM

2 Assay Description Species Deliverables Fast In Vitro Met ID in Hepatocytes Incubation for 120 min UPLC-UV-Q-TOF MS + MetaboLynx English or Chinese report in word or other Major metabolite (top 3~5 metabolites) Metabolic pathway Stage LF LO PCC IND TAT (working days) 5 Routine Met ID Assays Liver Microsomes Major metabolites (>1% based on UV) Metabolic pathways S9 Fractions Major metabolites (>1% based on UV) Metabolic pathways Hepatocytes Incubation for 120 min Major metabolites (>1% based on UV) Metabolic pathways Plasma Plasma incubation for 60 min Major metabolites (>1% based on UV) Metabolic pathways In Vitro Reactive Metabolite Trapping by GSH Incubation in liver microsomes + GSH GSH adducts and other major metabolites In Vivo Met ID 1~4 matrices (plasma, urine, feces, bile) UPLC-UV-Q-TOF MS + MetaboLynx English or Chinese report in word Structures of major metabolites Metabolic and excretion pathways 10(LO) Table 1. Comprehensive list of WuXi s Met ID studies. LTD.WUXIAPPTEC.COM DMPK Metabolite ID 29

3 In Vitro Metabolite ID WuXi offer in vitro metabolite ID studies in mice, rats, beagles, primates and humans liver microsomes, hepatocytes, S9 fraction and plasma incubation samples. WuXi also provides the reactive metabolite trapping studies via GSH in liver microsomes. Samples are all analyzed using UPLC-PDA-QTOF system, detected by both UV and MS. Case Studies Using Cold Compound Met ID of Midazolam in Liver Microsomes Liver microsomes that contain most of the CYP450s are commonly used for in vitro drug metabolism. WuXi DMPK has established phase I metabolism systems in mouse, rat, beagle, monkey and human liver microsomes using midazolam as a positive control. A typical incubation system consists of 1 mg/ml microsomal proteins and 10 µm test article. A typical Met ID study for midazolam is presented in the following Table 3 and figures 2-3. The measured and calculated molecular masses were in agreement within 5 ppm indicating a high level of confidence in the proposed elemental compositions of the metabolites. Metabolite [M + H] + (m/z) RT (min) Mouse Rat Dog NHP Human Metabolic %(UV) %(UV) %(UV) %(UV) %(UV) Pathway MA Di-oxygenation MA Oxygenation Midazolam (parent) N/A Table 2. Observed metabolic profile of midazolam in liver microsomes of various species as detected by LC-MS. MA 1 MA 2 midazolam Figure 2. LC-UV chromatograms of midazolam and its metabolites in mouse, rat, dog, monkey and human liver microsomes after incubation for 60 min at 37 C. Figure 3. MS and MS 2 spectra of MA2 and its proposed fragmentation pathways. Met ID of 7-ethoxycoumarin in Hepatocytes Although liver microsomes are the most commonly used medium for in vitro metabolism studies, hepatocytes are also commonly used in drug metabolism studies as they provide more metabolic inion including phase II metabolism. Thus WuXi DMPK offers Met ID studies in mouse, rat, dog, monkey and human hepatocytes. 7-ethoxycoumarin (7EC) is used as positive control for Met ID in hepatocytes, typically at 10 µm incubated with 1x10 6 hepatocytes per ml. The result for a typical 7EC Met ID study is presented in Table 4 and figures 4-5. The measured and calculated molecular masses were in agreement within 5 ppm indicating a high level of confidence in the proposed elemental compositions of the metabolites. 30 Metabolite ID DMPK LTD.WUXIAPPTEC.COM

4 Metabolite [M + H] + (m/z ) RT (min) Mouse Rat Dog NHP Human %(UV) %(UV) %(UV) %(UV) %(UV) MA Metabolic Pathway Deethylation and glucuronidation 7-EC (parent) N/A Table 3. Observed metabolites of 7-ethoxycoumarin in mouse, rat, dog, monkey and human hepatocytes. MA 1 7-EC Figure 4. LC-UV chromatograms of 7-ethoxycoumarin and its metabolites in mouse, rat, dog, monkey and human hepatocytes after incubation for 120 min at 37 C. Figure 5. MS and MS 2 spectra of MA1 and its proposed fragmentation pathways. Reactive Metabolite Trapping via GSH in Liver Microsomes Reactive metabolites might bind to proteins and nucleic acids potentially resulting in idiosyncratic toxicities. Identification of these reactive species is an essential process in the optimization of drug candidates. At WuXi DMPK, GSH is used to trap reactive metabolites by spiking matrices of human liver microsomes. A typical incubation system consists of 1 mg/ml microsomal proteins, 5 mm GSH and 10 µm test article. Acetaminophen is used as positive control and the results of a typical Met ID study is presented in Table 5 and figures 6-7. The measured and calculated molecular masses are in agreement within 5 ppm indicating a high level of confidence in the proposed elemental compositions of the metabolites. Metabolites RT (min) %(UV) [M + H] + (nominal m/z) [M + H] + (observed m/z) PPM Formula Metabolic Pathway Acetaminophen (parent) [C 8 H 9 NO 2 +H] + N/A MA [C 18 H 24 N 4 O 8 S+H] + S-glutathione adduct Table 4. Observed GSH conjugated metabolites of acetaminophen in human liver microsomes. acetaminophen MA 1 Figure 6. LC-UV chromatograms of acetaminophen and the GSH adducts of its metabolites in human liver microsomes. Figure 7. MS and MS 2 spectra of MA1 and its proposed fragmentation pathways. LTD.WUXIAPPTEC.COM DMPK Metabolite ID 31

5 In Vivo Met ID Complementary to in vitro Met ID, WuXi offers in vivo studies in mice, rats, beagles, primates and humans. Samples of urine, feces, plasma and bile are all analyzed using UPLC-PDA-QTOF system. Presented in figure 8 is an in vivo study of the drug rosglitazone where the determined mass errors between the measured and calculated values are < 5 ppm indicating a high level of confidence in the proposed elemental compositions of the metabolites. This particular study was conducted in SD rats. Based on the MS data, a proposed metabolic profile for rosglitazone is presented in scheme 1. Figure 8. MS and MS 2 spectra for rosglitazone and its proposed fragmentation pathways in SD rats. Scheme 1. Proposed metabolic pathways of rosiglitazone in SD rats. 14 C Radiolabeled Assays for Mass Balance, Tissue Distribution and Met ID Studies WuXi DMPK can perform studies with radiolabled compounds provided by our clients or synthesize them in-house to lower costs for our clients. WuXi studies use two low energy β-emitters, 14 C and 3 H for Met ID and distribution studies; 14 C is preferred due to its higher scintillation efficiency and stability. There are two major advantages to using radiolabeled versus unlabeled compounds for metabolic and distribution studies. First, the metabolites can be easily identified and separated by specific detection of the radiolabel. Second, the total radioactivity of all metabolites can be specifically quantified with only minor biological matrix effects, a common issue in LC-MS methods. In addition, the higher sensitivity of radioanalysis leads to much higher recoveries compared to those from non-radiolabeled compounds by LC-MS. Table 6 shows the available assays for radiolabeled compounds, and figure 9 shows the analytical instruments for quantifying these compounds. LSC Counter SSC Counter Biological Oxidizer Figure 9. Instruments used for preparing and quantifying radioactive samples in mass balance studies. 32 Metabolite ID DMPK LTD.WUXIAPPTEC.COM

6 Assay Description Species Deliverables Stage LF LO PCC IND TAT (working Liver Microsomes LC-FSA-MS for metabolite profiling Major Metabolites Quantitative and qualitative metabolite profiling inion 20 In Vitro ID in Hepatocytes Incubation for 120 min LC-FSA-MS for metabolite profiling Major Metabolites Quantitative and qualitative metabolite profiling inion 20 In Vivo Met ID Met ID (plasma, bile, urine, feces). Mouse LC-FSA-MS for metabolite Rat profiling Non-rodents as needed Major Metabolites Quantitative and qualitative metabolite profiling inion 20 Mass Balance Mass balance (oxidizer and SC for Mouse sample measurement) Rat Non-rodents as needed %Recovery Excretion pathways 15 Tissue Distribution Tissue distribution (oxidizer and Mouse LSC for sample measurement) Rat Non-rodents as needed Tissue BA data PK data 20 Table 5. Met ID and distribution studies that incorporate 14 C- or 3 H-labeled compounds. LTD.WUXIAPPTEC.COM DMPK Metabolite ID 33

7 Case Studies Using Radiolabeled Compound Met ID Study of Compound X Met ID, mass balance and tissue distribution studies are all commonly conducted as part of an IND package. While radiolabled compounds are not required for any of these studies, they do simplify the process and increase the sensitivity of quantitation. Distribution studies provide support for the toxicology portion of an IND and facilitate translation into the clinical phase. The following data are derived from a study in mice (Figure 10) and rats (Figures 11 and 12) following the dose of 14 C-labled compound X to examine the metabolic profile, mass balance and tissue distribution which provides a very broad context along with the other PK data acquired earlier for this compound. Figure 10. Radiochromatograms of plasma, bile, urine and feces of 14 C-compound X following a single oral dose in mice. Mass Balance Figure 11. The accumulated excretion of 14 C-compound X in rats as analyzed by SC. 34 Metabolite ID DMPK LTD.WUXIAPPTEC.COM

8 Tissue Distribution Figure 12. The comprehensive tissue distribution of 14 C-compound X in rats over 24 h following a single oral dose. References 1. Zhang, D. L., Zhu, M. S. & Humphreys, W. G. Drug Metabolism in Drug Design and Development: Basic Concepts and Practice, 1 st ed. (Wiley, 2008). 2. Food Drug Administration Center for Drug Evaluation and Research (2008). Guidance for Industry: Safety Testing of Drug Metabolites. (FDA Maryland). LTD.WUXIAPPTEC.COM DMPK Metabolite ID 35