Recommendations on the Use of Multiple Labels in Human Mass Balance Studies.

The administration of radiolabeled drug candidates is considered the gold standard in absorption, distribution, metabolism, and excretion studies for small-molecule drugs since it allows facile and accurate quantification of parent drug, metabolites, and total drug-related material independent of the compound structure. The choice of the position of the radiolabel, typically 14C or 3H, is critical to obtain relevant information. Sometimes, a biotransformation reaction may lead to cleavage of a part of the molecule. As a result, only the radiolabeled portion can be followed, and information on the fate of the nonlabeled metabolite may be lost. Synthesis and administration of two or more radiolabeled versions of the parent drug as a mixture or in separate studies may resolve this issue but comes with additional challenges. In this paper, we address the questions that may be considered to help make the right choice whether to use a single or multiple radiolabel approach and discuss the pros and cons of different multiple-labeling strategies that can be taken as well as alternative methods that allow the nonlabeled part of the molecule to be followed. SIGNIFICANCE STATEMENT: Radiolabeled studies are the gold standard in drug metabolism research, but molecules can undergo cleavage with loss of the label. This often results in discussions around potential use of multiple labels, which seem to be occurring with increased frequency since an increasing proportion of the small-molecule drugs are tending towards larger molecular weights. This review provides insight and decision criteria in considering a multiple-label approach as well as pros and cons of different strategies that can be followed.

Metabolite Bioanalysis in Drug Development: Recommendations from the IQ Consortium Metabolite Bioanalysis Working Group.

The intent of this perspective is to share the recommendations of the International Consortium for Innovation and Quality in Pharmaceutical Development Metabolite Bioanalysis Working Group on the fit-for-purpose metabolite bioanalysis in support of drug development and registration. This report summarizes the considerations for the trigger, timing, and rigor of bioanalysis in the various assessments to address unique challenges due to metabolites, with respect to efficacy and safety, which may arise during drug development from investigational new drug (IND) enabling studies, and phase I, phase II, and phase III clinical trials to regulatory submission. The recommended approaches ensure that important drug metabolites are identified in a timely manner and properly characterized for efficient drug development.

Non-Labeled, Stable Labeled, or Radiolabelled Approaches for Provision of Intravenous Pharmacokinetics in Humans: A Discussion Piece.

A review of the use of microdoses and isotopic microtracers for clinical intravenous pharmacokinetic (i.v. PK) data provision is presented. The extent of application of the varied approaches available and the relative merits of each are highlighted with the aim of assisting practitioners in making informed decisions on the most scientifically appropriate design to adopt for any given new drug in development. It is envisaged that significant efficiencies will be realized as i.v. PK data in humans becomes more routinely available for suitable assets in early development, than has been the case prior to the last decade.

Human Absorption, Distribution, Metabolism, and Excretion Studies: Origins, Innovations, and Importance.

Human absorption, distribution, metabolism, and excretion (hADME) studies represent one of the most important clinical studies in terms of obtaining a comprehensive and quantitative overview of the total disposition of a drug. This article will provide background on the origins of hADME studies as well as provide an overview of technological innovations that have impacted how hADME studies are carried out and analyzed. An overview of the current state of the art for hADME studies will be provided, the impacts of advances in technology and instrumentation on the timing of and approaches to hADME studies will be discussed, and a summary of the parameters and information obtained from these studies will be offered. Additionally, aspects of the ongoing debate over the importance of animal absorption, distribution, metabolism, and excretion studies versus a "human-first, human-only strategy" will be presented. Along with the information above, this manuscript will highlight how, for over 50 years, Drug Metabolism and Disposition has served as an important outlet for the reporting of hADME studies. SIGNIFICANCE STATEMENT: Human absorption, distribution, metabolism, and excretion (hADME) studies have and will continue to be important to the understanding and development of drugs. This manuscript provides a historical perspective on the origins of hADME studies as well as advancements resulting in the current-state-of the art practice for these studies.

Considerations for Human ADME Strategy and Design Paradigm Shift(s) - An Industry White Paper.

The human absorption, distribution, metabolism, and excretion (hADME) study is the cornerstone of the clinical pharmacology package for small molecule drugs, providing comprehensive information on the rates and routes of disposition and elimination of drug-related material in humans through the use of 14 C-labeled drug. Significant changes have already been made in the design of the hADME study for many companies, but opportunity exists to continue to re-think both the design and timing of the hADME study in light of the potential offered by newer technologies, that enable flexibility in particular to reducing the magnitude of the radioactive dose used. This paper provides considerations on the variety of current strategies that exist across a number of pharmaceutical companies and on some of the ongoing debates around a potential move to the so called "human first/human only" approach, already adopted by at least one company. The paper also provides a framework for continuing the discussion in the application of further shifts in the paradigm.

Mini-Review: Comprehensive Drug Disposition Knowledge Generated in the Modern Human Radiolabeled ADME Study.

The human radiolabeled absorption, distribution, metabolism, and excretion (ADME) study offers a quantitative and comprehensive overall picture of the disposition of a drug, including excretion pattern and metabolite profiles in circulation and excreta. The data gathered from the ADME study are highly informative for developing a cohesive strategy for clinical pharmacology studies. Elements of standard ADME study designs are described. An exciting new development in human ADME studies is the application of accelerator mass spectrometry (AMS) as the detection technique for carbon-14, in replacement of radioactivity measurements. This technology permits administration of 100-fold to 1,000-fold lower amounts of carbon-14, and thus opens the door to the application of new study designs. A new ADME study design, termed the AMS-Enabled Human ADME study, is described. In this design, both oral and intravenous administration are assessed in a single clinical study with a two-period crossover. In addition to all of the standard ADME study end points (e.g., mass balance and quantitative metabolite profiles), the AMS-Enabled ADME study can provide the fundamental pharmacokinetic parameters of clearance, volume of distribution, absolute oral bioavailability, and even estimates of the fraction of the dose absorbed. Thus, we have entered a new era of human ADME study design that can yield vastly more informative and complete data sets enabling a superior understanding of overall drug disposition.

Late-Stage Lead Diversification Coupled with Quantitative Nuclear Magnetic Resonance Spectroscopy to Identify New Structure-Activity Relationship Vectors at Nanomole-Scale Synthesis: Application to Loratadine, a Human Histamine H1 Receptor Inverse Agonist.

An experimental approach is described for late-stage lead diversification of frontrunner drug candidates using nanomole-scale amounts of lead compounds for structure-activity relationship development. The process utilizes C-H bond activation methods to explore chemical space by transforming candidates into newly functionalized leads. A key to success is the utilization of microcryoprobe nuclear magnetic resonance (NMR) spectroscopy, which permits the use of low amounts of lead compounds (1-5 µmol). The approach delivers multiple analogues from a single lead at nanomole-scale amounts as DMSO-d6 stock solutions with a known structure and concentration for in vitro pharmacology and absorption, distribution, metabolism, and excretion testing. To demonstrate the feasibility of this approach, we have used the antihistamine agent loratadine (1). Twenty-six analogues of loratadine were isolated and fully characterized by NMR. Informative SAR analogues were identified, which display potent affinity for the human histamine H1 receptor and improved metabolic stability.

Effective Application of Metabolite Profiling in Drug Design and Discovery.

At one time, biotransformation was a descriptive activity in pharmaceutical development, viewed simply as structural elucidation of drug metabolites, completed only once compounds entered clinical development. Herein, we present our strategic approach using structural elucidation to enable chemistry design/SAR development. The approach considers four questions that often present themselves to medicinal chemists optimizing their compounds for candidate selection: (1) What are the important clearance mechanisms that mediate the disposition of my molecule? (2) Can metabolic liabilities be modulated in a favorable way? (3) Does my compound undergo bioactivation to a reactive metabolite? (4) Do any of the metabolites possess activity, either on- or off-target? An additional question necessary to support compound development relates to metabolites in safety testing (MIST) and our approach also addresses this question. The value in structural elucidation is derived from its application to better design molecules, guide their clinical development, and underwrite patient safety.

A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development under the "Metabolites in Safety Testing" Regulatory Guidance.

Since the introduction of metabolites in safety testing (MIST) guidance by the Food and Drug Administration in 2008, major changes have occurred in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and nonclinical studies to generate this information. In this cross-industry review, we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radio chromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated, which has led to more frequent determination of human metabolite profiles from multiple ascending-dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. Together with technological advances, these events have led to a general shift of focus toward earlier human metabolism studies using high-resolution mass spectrometry and to a reduction in animal radiolabel absorption/distribution/metabolism/excretion studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industry.

Quantitative projection of human brain penetration of the H3 antagonist PF-03654746 by integrating rat-derived brain partitioning and PET receptor occupancy.

1. Unbound brain drug concentration (Cb,u), a valid surrogate of interstitial fluid drug concentration (CISF), cannot be directly determined in humans, which limits accurately defining the human Cb,u:Cp,u of investigational molecules. 2. For the H3R antagonist (1R,3R)-N-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-lmethyl)phenyl]cyclobutane-1-carboxamide (PF-03654746), we interrogated Cb,u:Cp,u in humans and nonhuman primate (NHP). 3. In rat, PF-03654746 achieved net blood-brain barrier (BBB) equilibrium (Cb,u:Cp,u of 2.11). 4. In NHP and humans, the PET receptor occupancy-based Cp,u IC50 of PF-03654746 was 0.99 nM and 0.31 nM, respectively, which were 2.1- and 7.4-fold lower than its in vitro human H3 Ki (2.3 nM). 5. In an attempt to understand this higher-than-expected potency in humans and NHP, rat-derived Cb,u:Cp,u of PF-03654746 was integrated with Cp,u IC50 to identify unbound (neuro) potency of PF-03654746, nIC50. 6. The nIC50 of PF-03654746 was 2.1 nM in NHP and 0.66 nM in human which better correlated (1.1- and 3.49-fold lower) with in vitro human H3 Ki (2.3 nM). 7. This correlation of the nIC50 and in vitro hH3 Ki suggested the translation of net BBB equilibrium of PF-03654746 from rat to NHP and humans, and confirmed the use of Cp,u as a reliable surrogate of Cb,u. 8. Thus, nIC50 quantitatively informed the human Cb,u:Cp,u of PF-03654746.

Biosynthesis of drug metabolites and quantitation using NMR spectroscopy for use in pharmacologic and drug metabolism studies.

The contribution of drug metabolites to the pharmacologic and toxicologic activity of a drug can be important; however, for a variety of reasons metabolites can frequently be difficult to synthesize. To meet the need of having samples of drug metabolites for further study, we have developed biosynthetic methods coupled with quantitative NMR spectroscopy (qNMR) to generate solutions of metabolites of known structure and concentration. These quantitative samples can be used in a variety of ways when a synthetic sample is unavailable, including pharmacologic assays, standards for in vitro work to help establish clearance pathways, and/or as analytical standards for bioanalytical work to ascertain exposure, among others. We illustrate five examples of metabolite biosynthesis and qNMR. The types of metabolites include one glucuronide and four oxidative products. Concentrations of the isolated metabolite stock solutions ranged from 0.048 to 8.3 mM, with volumes from approximately 0.04 to 0.150 ml in hexadeutarated dimethylsulfoxide. These specific quantified isolates were used as standards in the drug discovery setting as substrates in pharmacology assays, for bioanalytical assays to establish exposure, and in variety of routine absorption, distribution, metabolism, and excretion assays, such as protein binding and determining blood-to-plasma ratios. The methods used to generate these materials are described in detail with the objective that these methods can be generally used for metabolite biosynthesis and isolation.

Deuterium isotope effects on drug pharmacokinetics. I. System-dependent effects of specific deuteration with aldehyde oxidase cleared drugs.

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 clearance 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.

Discovery of two clinical histamine H(3) receptor antagonists: trans-N-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidinylmethyl)phenyl]cyclobutanecarboxamide (PF-03654746) and trans-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-ylmethyl)phenyl]-N-(2-methylpropyl)cyclobutanecarboxamide (PF-03654764).

The discovery of two histamine H(3) antagonist clinical candidates is disclosed. The pathway to identification of the two clinical candidates, 6 (PF-03654746) and 7 (PF-03654764) required five hypothesis driven design cycles. The key to success in identifying these clinical candidates was the development of a compound design strategy that leveraged medicinal chemistry knowledge and traditional assays in conjunction with computational and in vitro safety tools. Overall, clinical compounds 6 and 7 exceeded conservative safety margins and possessed optimal pharmacological and pharmacokinetic profiles, thus achieving our initial goal of identifying compounds with fully aligned oral drug attributes, "best-in-class" molecules.

Discovery of 4-(5-methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane (CP-810,123), a novel alpha 7 nicotinic acetylcholine receptor agonist for the treatment of cognitive disorders in schizophrenia: synthesis, SAR development, and in vivo efficacy in cognition models.

A novel alpha 7 nAChR agonist, 4-(5-methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane (24, CP-810,123), has been identified as a potential treatment for cognitive deficits associated with psychiatric or neurological conditions including schizophrenia and Alzheimer's disease. Compound 24 is a potent and selective compound with excellent pharmaceutical properties. In rodent, the compound displays high oral bioavailability and excellent brain penetration affording high levels of receptor occupancy and in vivo efficacy in auditory sensory gating and novel object recognition. The structural diversity of this compound and its preclinical in vitro and in vivo package support the hypothesis that alpha 7 nAChR agonists may have potential as a pharmacotherapy for the treatment of cognitive deficits in schizophrenia.

Differential effects of various typical and atypical antipsychotics on plasma glucose and insulin levels in the mouse: evidence for the involvement of sympathetic regulation.

Atypical antipsychotic treatment has been associated with serious metabolic adverse events, such as glucose dysregulation and development of type 2 diabetes. As part of our studies on possible underlying mechanisms, we investigated the acute effects of various typical and atypical antipsychotics on plasma glucose and insulin in FVB/N mice, a strain that showed a more pronounced hyperglycemic response to clozapine than C57BL/6 and CD-1 mice. Acute administration of high doses of clozapine, olanzapine, quetiapine, perphenazine, or chlorpromazine significantly increased plasma glucose by 100%-140% above basal levels without significant effects on insulin levels. In contrast, risperidone reduced plasma glucose (-30%) and markedly enhanced plasma insulin levels. Doses of ziprasidone that gave 50-fold higher free plasma concentrations than therapeutic plasma levels, as well as high doses of aripiprazole and haloperidol, did not significantly alter either glucose or insulin levels. Clozapine- and olanzapine-induced hyperglycemia occurred at free plasma concentrations that were within, or one order of magnitude above, the range of therapeutic plasma levels. Pretreatment with either the ganglionic blocker hexamethonium, or the alpha(2) adrenergic receptor antagonist yohimbine, blocked the clozapine- and chlorpromazine-induced increase in glucose levels. Taken together, these results suggest that typical and atypical antipsychotics with known metabolic liability produce acute hyperglycemia in mice and that this effect is likely driven by activation of the sympathetic autonomic nervous system via a central mechanism.

Synthesis and SAR studies of 1,4-diazabicyclo[3.2.2]nonane phenyl carbamates--subtype selective, high affinity alpha7 nicotinic acetylcholine receptor agonists.

The synthesis and SAR studies about the bicyclic amine, carbamate linker and aromatic ring of a 1,4-diazabicyclo[3.2.2]nonane phenyl carbamate series of alpha7 nAChR agonists is described. The development of the medicinal chemistry strategy and SAR which led to the identification of 5 and 7aa as subtype selective, high affinity alpha7 agonists as excellent leads for further evaluation is discussed, along with key physicochemical and pharmacokinetic data highlighting their lead potential.

3-Benzyl-1,3-oxazolidin-2-ones as mGluR2 positive allosteric modulators: Hit-to lead and lead optimization.

The discovery, synthesis and SAR of a novel series of 3-benzyl-1,3-oxazolidin-2-ones as positive allosteric modulators (PAMs) of mGluR2 is described. Expedient hit-to-lead work on a single HTS hit led to the identification of a ligand-efficient and structurally attractive series of mGluR2 PAMs. Human microsomal clearance and suboptimal physicochemical properties of the initial lead were improved to give potent, metabolically stable and orally available mGluR2 PAMs.

Concentration-dependent modulation of amyloid-beta in vivo and in vitro using the gamma-secretase inhibitor, LY-450139.

LY-450139 is a gamma-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-beta (Abeta) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Abeta responses to LY-450139 in the guinea pig, a nontransgenic model that has an Abeta sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma Abeta levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Abeta levels at early time points, with return to baseline within hours. Higher doses inhibited Abeta levels in all compartments at early time points, but elevated plasma Abeta levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma Abeta was significantly inhibited at 10-30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Abeta elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted Abeta were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the gamma-secretase complex, eliciting Abeta lowering at high concentrations but Abeta elevation at low concentrations.

The role of muscarinic receptor antagonism in antipsychotic-induced hippocampal acetylcholine release.

Olanzapine and clozapine produce robust increases in hippocampal acetylcholine release during acetylcholinesterase inhibition, while other antipsychotics, including thioridazine, have only small effects. Since thioridazine binds with similar high affinities to muscarinic receptors as olanzapine and clozapine, muscarinic autoreceptor blockade was ruled out as a primary mechanism [Neuropsychopharmacology 26 (2002) 583]. This study compared in vitro binding affinities and functional activities of olanzapine, clozapine, thioridazine, ziprasidone, risperidone, chlorpromazine and scopolamine at muscarinic M2 receptors with their in vivo potencies to increase acetylcholine release in the rat hippocampus. We found that scopolamine, olanzapine and clozapine, but also high doses of thioridazine and chlorpromazine, markedly increase acetylcholine release. The reduced in vivo potencies of thioridazine and chlorpromazine are consistent with their significantly weaker functional antagonist activity at human muscarinic M2 receptors, while thioridazine's reduced binding affinity for rat muscarinic M2 receptors and lower brain exposure, may further contribute to its weak in vivo potency compared to olanzapine. The excellent correlation between in vitro antagonist activities of antipsychotics at muscarinic M2 receptors and their in vivo potencies to increase acetylcholine release, suggests that olanzapine, clozapine, as well as thioridazine and chlorpromazine, increase acetylcholine release via blockade of terminal muscarinic M2 autoreceptors.

The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: evaluation using the MDR1A/1B knockout mouse model.

Thirty-two structurally diverse drugs used for the treatment of various conditions of the central nervous system (CNS), along with two active metabolites, and eight non-CNS drugs were measured in brain, plasma, and cerebrospinal fluid in the P-glycoprotein (P-gp) knockout mouse model after subcutaneous administration, and the data were compared with corresponding data obtained in wild-type mice. Total brain-to-plasma (B/P) ratios for the CNS agents ranged from 0.060 to 24. Of the 34 CNS-active agents, only 7 demonstrated B/P area under the plasma concentration curve ratios between P-gp knockout and wild-type mice that did not differ significantly from unity. Most of the remaining drugs demonstrated 1.1- to 2.6-fold greater B/P ratios in P-gp knockout mice versus wild-type mice. Three, risperidone, its active metabolite 9-hydroxyrisperidone, and metoclopramide, showed marked differences in B/P ratios between knockout and wild-type mice (6.6- to 17-fold). Differences in B/P ratios and cerebrospinal fluid/plasma ratios between wild-type and knockout animals were correlated. Through the use of this model, it appears that most CNS-active agents demonstrate at least some P-gp-mediated transport that can affect brain concentrations. However, the impact for the majority of agents is probably minor. The example of risperidone illustrates that even good P-gp substrates can still be clinically useful CNS-active agents. However, for such agents, unbound plasma concentrations may need to be greater than values projected using receptor affinity data to achieve adequate receptor occupancy for effect.