Experiments for a systematic comparison between stable-isotope-(deuterium) labeling and radio-(14C) labeling for the elucidation of the in vitro metabolic pattern of pharmaceutical drugs (original) (raw)
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Evaluation of isotope ratio (IR) mass spectrometry for the study of drug metabolism
Biomedical Mass Spectrometry, 1985
Isotope ratio (IR) mass spectrometry was evaluated for the study of drug metabolism and balance using 13C,1sNZlabelled antipyrine (AP) as a test drug. Rats were given 40 mg kg-' (13C,'SN,)AP intraperitoneally. Breath, urine, faeces and blood were collected. Except for breath, samples were combusted in sealed quartz tubes. The resulting CO, and N, were analysed for excess 13C and "N, relative to pre-dose samples, by IR mass spectrometry. In addition, blood levels of AP and cumulative excretion of urinary AP metabolites were determined by gas chromatographyhass spectrometry/selected ion monitoring (GC/MS/SIM) and high-performance liquid chromatography (HPLC) respectively. Excess I3C and 15N levels in blood were comparable with observed levels of AP, and urinary recoveries of 13C (42%) were in good agreement with those calculated from HPLC data (45%). N-Demethylation, one of the important pathways of AP metabolism, was most rapidly determined by excess I3CO2 excretion in breath (S0/o). The IR mass spectral analysis complemented gas chromatographichass spectrum and HPLC analyses, and was less complex.
Chemical Research in Toxicology, 2012
Absorption, distribution, metabolism, and excretion (ADME) studies are an integral part of the comprehensive safety evaluation of a new molecular entity, and they represent a standard suite of studies included in the registration package for all new small molecule drugs. In vivo studies in preclinical toxicology species and humans using radiolabeled (3 H or 14 C) compound provide quantitative assessments of overall routes of excretion of drug-related material, pharmacokinetics of total drug-derived radioactivity in circulation, relative to parent compound and quantitation, and characterization of metabolites in excreta and circulation. These data serve as the starting point for metabolite in safety testing (MIST). These studies involve the administration of a radiolabeled drug to laboratory animals and humans followed by a quantitative collection of excreta and blood. Using appropriate plasma-pooling strategies, these studies could allow for modeling the metabolite exposure at the steady state. Information from the radiolabeled human study is used to design clinical drug−drug interaction (DDI) studies and to obtain a waiver for bioequivalence studies. This article describes the various aspects of conducting ADME studies and the use of radiolabeled analogues of drug candidates to investigate their metabolism and how to compare the exposures of metabolites in humans and toxicology species. ■ CONTENTS 1. Introduction 513 2. Conduct of Radiolabeled Absorption, Distribution, Metabolism, and Excretion (ADME) Studies 514 2.1. Objectives of Radiolabeled ADME Studies 514 2.2. Choice of Radioisotopes and Their Position in the Drug Molecule 515 2.3. Standard or Typical Studies with Radiolabeled Compounds 515 2.4. Sample Analysis 517 3. Utilities of Radiolabeled Studies in Development 517 3.1. Characterization of Absorption and Routes of Excretion 517 3.2. Metabolism/Clearance Mechanism 518 3.3. Characterization of Unusual and Unexpected Metabolites 519 3.4. Covalent Protein Binding (CPB)/Reactive Metabolites 519 3.5. Distribution/Tissue Targeting 521 3.6. Human MIST 522 3.7. Microdosing/Microtracer Studies 524 3.8. Bioequivalence Waiver 525 4. Limitations of Radiolabeled ADME Studies 525 5. Regulatory Requirements and Their Effect on the Timing of Radiolabeled Studies 525 6. Summary 527 Author Information 527 Corresponding Author 527 Notes 527 Acknowledgments 527 Abbreviations 527 References 527
Applications of stable isotopes in clinical pharmacology
British Journal of Clinical Pharmacology, 2011
This review aims to present an overview of the application of stable isotope technology in clinical pharmacology. Three main categories of stable isotope technology can be distinguished in clinical pharmacology. Firstly, it is applied in the assessment of drug pharmacology to determine the pharmacokinetic profile or mode of action of a drug substance. Secondly, stable isotopes may be used for the assessment of drug products or drug delivery systems by determination of parameters such as the bioavailability or the release profile. Thirdly, patients may be assessed in relation to patient-specific drug treatment; this concept is often called personalized medicine. In this article, the application of stable isotope technology in the aforementioned three areas is reviewed, with emphasis on developments over the past 25 years. The applications are illustrated with examples from clinical studies in humans.
Stable Isotopes Labeling of Drugs in Pediatric Clinical Pharmacology
Pediatrics, 1999
S table isotope labeling (SIL) still has not been used very much in pediatric and perinatal clinical pharmacology. However, this method has numerous advantages. It allows one to determine the concentration of drugs with great sensitivity, thereby allowing quantification in small amounts of biologic samples. It also allows quantification with great specificity, eliminating interference by drug metabolites found in some radioactive tracer methods. SIL is safe because stable isotopes are not radioactive. The only possible toxicity that may be related to an isotope effect is a slowing of biochemical reactions because of the greater mass of the stable isotope. Because of the great difference between deuterium and hydrogen, a significant isotope effect occurs only with deuterium. However, toxicity related to the isotope effect of deuterium can only be produced by very high levels of deuteration (Ͼ15% of body water), far higher than the amount of deuterium in a typical tracer dose of drug. 1 SIL also allows noninvasive in vivo studies such as the co 2 breath test (CBT). All of these advantages explain the great potential interest in SIL in pediatric pharmacology. The costs of stable isotopes and limited facilities available for sample analysis are undoubtedly the greatest limitations to their increased use.
Journal of Chromatography A, 2009
The in vitro metabolic profile of BAL30630, an antifungal piperazine propanol derivative, which inhibits the 1,3-beta-d-glucansynthase, was investigated by incubation with microsomes of several species and with rat hepatocytes. For the spotting of the metabolites, mixtures of BAL30630 with a stable isotope (deuterium) labeled analogue were incubated. The metabolic pattern comprises several oxidized metabolites. Based on isotope exchange experiments, their structures could be assigned to epoxide-and hydroxylated metabolites. In hepatocyte incubations, several glucuronides formed from these oxidized metabolites could be observed. From the analysis of the metabolic pattern in microsomes, products of carbamate hydrolysis were characterized. This hydrolysis was highly species dependent. In activated incubations and in rat hepatocytes, those metabolites were further oxidized. In incubations without NADPH activation, the resulting hydrolytic metabolites could be enriched without the subsequent oxidation. Final structural elucidation of the metabolites was performed using accurate mass determination and isotope exchange experiments, in which incubations were analyzed by deuterium exchange and capillary HPLC-QTof-MS and MS/MS. The use of non-radioactive, stabile isotope labeled drug analogues in combination with isotope exchange studies was essential in particular for a defined assignment of the functional groups in the structures of the investigated metabolites.
Drug Metabolism and Disposition, 2012
The exposure of a drug candidate and its metabolites in humans and preclinical species during drug development needs to be determined to ensure that the safety of drug-related components in humans is adequately assessed in the standard toxicology studies. The in vivo radiolabeled studies in preclinical species and human volunteers provide total fate of the drug-derived radioactivity including the relative abundance of metabolites. Here we describe how the single dose radiolabeled human studies could provide the exposure of circulating metabolites at steady state using a case study of an extensively metabolized drug, lixivaptan. Following an oral dose of [ 14 C]lixivaptan to humans, a total of nine metabolites were detected in systemic circulation; eight of them exceed 10% of the parent exposure (2 to 41% of total radioactivity). The plasma samples were profiled for all subjects at each time point by HPLC and metabolites were quantified using a radioactive detector. Based on single dose AUC values, exposure of six human metabolites was greater at least in one preclinical species used in toxicology evaluation. Based on T 1/2 of lixivaptan and two major metabolites from single dose in humans, their AUC and C max values were simulated at the steady state. The simulated exposures (C max and AUC) values of parent drug and the two most abundant metabolites were similar to those of from a 7 day clinical study obtained using a validated LC-MS/MS assay, suggesting that a well-designed single dose radiolabeled human study can help in addressing the metabolites in safety testing-related issues.
Synthesis of stable isotope labelled internal standards for drug–drug interaction (DDI) studies
Bioorganic & Medicinal Chemistry, 2012
The syntheses of stable isotope labelled internal standards of important CYP-isoform selective probes, like testosterone 1, diclofenac 3, midazolam 5, and dextromethorphan 7, as well as their corresponding hydroxylated metabolites 6b-hydroxytestosterone 2, 4 0-hydroxydiclofenac 4, 1 0-hydroxymidazolam 6 and dextrorphan 8 are reported. Microwave-enhanced H/D-exchange reactions applying either acid, base, or homogeneous and heterogeneous transition metal catalysis, or combinations thereof proved to be highly efficient for direct deuterium labelling of the above mentioned probes. Compared to conventional stepwise synthetic approaches, the combination of H/D exchange and biotransformation provides the potential for considerable time-and cost savings, in particular for the synthesis of the stable isotope labelled internal standards of 4 0-hydroxydiclofenac 4 and 1 0-hydroxymidazolam 6.
Drug Metabolism and Disposition, 2012
The pharmacokinetic properties of drugs may be altered by kinetic deuterium isotope effects. With specifically deuterated model substrates and drugs metabolized by aldehyde oxidase, we demonstrate how knowledge of the enzyme's reaction mechanism, species differences in the role played by other enzymes in a drug's metabolic clearance, and differences in systemic clearance mechanisms are critically important for the pharmacokinetic application of deuterium isotope effects. Ex vivo methods to project the in vivo outcome using deuterated carbazeran and zoniporide with hepatic systems demonstrate the importance of establishing the extent to which other metabolic enzymes contribute to the metabolic clear-ance mechanism. Differences in pharmacokinetic outcomes in guinea pig and rat, with the same metabolic clearance mechanism, show how species differences in the systemic clearance mechanism can affect the in vivo outcome. Overall, to gain from the application of deuteration as a strategy to alter drug pharmacokinetics, these studies demonstrate the importance of understanding the systemic clearance mechanism and knowing the identity of the metabolic enzymes involved, the extent to which they contribute to metabolic clearance, and the extent to which metabolism contributes to the systemic clearance.
Drug Metabolism and Disposition, 2009
The primary objective of this study was to demonstrate the use of stable isotopes (SI) as an approach for pharmacokinetic analysis such as fraction absorbed, hepatic extraction ratio and fraction metabolized from parent drug to a metabolite. (S,S)-3-[3-(methylsulfonyl)phenyl]-1propylpiperidine hydrochloride (PNU96391) was selected as the model compound because of its simple biotransformation pathway, i.e., the predominant metabolic pathway to N-despropyl metabolite (M1), which makes it a suitable candidate. The second objective was to fully characterize pharmacokinetics of PNU96391 in rats using the SI coadministration approach with quantitative analysis by liquid-chromatography tandem mass spectrometry. Overall the present study showed that 1) absorption of PNU96391 from the gastrointestinal tract was near-complete (>90% of the dose), 2) PNU96391 was predominantly metabolized to M1 (approximately 70% of the dose) and 3) M1 was exclusively eliminated into urine with negligible biotransformation (the ratio of renal clearance to plasma clearance ≈ 0.9). Therefore, the present study demonstrated the utility of the SI methodology for characterizing the pharmacokinetics of a compound within the drug discovery and development process. Furthermore, the compartmental pharmacokinetic modeling provided insights into the disposition and biotransformation rates of PNU96391 and M1, suggesting that the modeling could add further advantages to the SI coadministration approach. Despite the greater availability of SI-labeled compounds, ADME scientists have yet to take full advantage of the potential use of these analogues for mechanistic ADME studies. These SI-labeled compounds can be used more widely to gain a better understanding of ADME properties in drug discovery and development.