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 C or H, is critical to obtain relevant information. Sometimes, a biotransformation reaction may lead to cleavage of a part of the molecule.

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.

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.

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 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.

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.

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 H 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).

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.

Improving and Maintaining On-Time Start Times for Nonelective Cases in a Major Academic Medical Center.

Background: As health care expenditures continue to increase, thoughtful use of perioperative resources is important. Efforts to improve operating room (OR) efficiency often focus on increasing on-time first case starts to improve OR utilization, reduce subsequent delays, and reduce adverse events. One institution, with severely limited inpatient hospital capacity and an extensive daily add-on list of surgical cases, focused efforts to improve OR efficiency by improving on-time first case starts for unscheduled, nonemergent surgeries.

Improving surgical efficiency of immediate implant-based breast reconstruction following mastectomy.

Purpose: Traditionally, during a mastectomy with implant-based reconstruction, the surgical oncologist completes their operative procedure prior to the reconstructive surgeon entering the room. In this scenario, two separate instruments kits and tables are utilized. In our institution, we created a combined instrument kit for use by both surgical teams.

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.

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

1. Unbound brain drug concentration (C), a valid surrogate of interstitial fluid drug concentration (C), cannot be directly determined in humans, which limits accurately defining the human C:C of investigational molecules. 2.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Concordance between trifluoroacetic acid and hepatic protein trifluoroacetylation after disulfiram inhibition of halothane metabolism in rats.

Background: Cytochrome P4502E1(CYP2E1)-mediated oxidation of halothane to a reactive intermediate (trifluoroacyl chloride) that covalently binds to hepatic proteins forming trifluoroacetylated neoantigens is believed to be the initiating event in a complex immunologic cascade culminating in antibody formation and severe hepatic necrosis ('halothane hepatitis') in susceptible patients. Trifluoroacyl chloride may also hydrolyze to the stable metabolite trifluoroacetic acid (TFA). CYP2E1 inactivation by disulfiram or its primary metabolite, diethyldithiocarbamate, inhibits human halothane oxidation to TFA in vitro and in vivo.

Cutaneous mucinosis and mastocytosis in a shar-pei.

A 7-year-old shar-pei was presented because of a recurrent dermatologic condition. Skin biopsies revealed an idiopathic (primary) cutaneous mucinosis that initially responded to corticosteroids. The condition reappeared 2 years later and subsequent biopsies revealed a mast cell tumor in some of the skin sites previously diagnosed with mucinosis.

Dose-dependent metabolism of fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A), an anesthetic degradation product, to mercapturic acids and 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid in rats.

The volatile anesthetic sevoflurane is degraded in anesthesia machines to fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE), to which humans are exposed. FDVE is metabolized in rats and humans to two alkane and two alkene glutathione S-conjugates that are hydrolyzed to the corresponding cysteine S-conjugates. The latter are N-acetylated to mercapturic acids, or bioactivated by renal cysteine conjugate beta-lyase to metabolites which may react with cellular macromolecules or hydrolyze to 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid.