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.

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.

In vivo Metabolism of Nifurtimox and the Drug-Drug Interaction Potential Including its Major Metabolites.

BACKGROUND: Nifurtimox is an effective treatment for patients with Chagas disease, but knowledge of its biotransformation and excretion is limited. OBJECTIVE: This study aimed to better understand the fate of oral nifurtimox in vivo. METHODS: We investigated the exposure and excretion pathways of [14C]-labeled nifurtimox and its metabolites in rats. We then quantified the prominent metabolites and nifurtimox in the urine and plasma of patients receiving nifurtimox using LC-HRMS with reference standards and quantified these compounds in rat plasma after a single, high dose of nifurtimox. We also investigated potential drug-drug interactions (DDIs) of these compounds in vitro. RESULTS: In rats, orally administered nifurtimox was rapidly absorbed (tmax 0.5 h) and eliminated (t½ 1.4 h). Metabolism of nifurtimox yielded six predominant metabolites (M-1 to M-6) in urine and plasma, and the dose was excreted equally via the renal and fecal routes with only traces of unchanged nifurtimox detectable due to its instability in excreta. In patients with Chagas disease, only M-6 and M-4 achieved relevant exposure levels, and the total amount of excreted metabolites in urine was higher in fed versus fasted patients, consistent with the higher systemic exposure. For nifurtimox, M-6, and M-4, no potential perpetrator pharmacokinetic DDIs with the main cytochrome P- 450 enzymes and drug transporters were identified in vitro. CONCLUSION: This contemporary analysis of the complex metabolite profile and associated exposures emerging after oral dosing of nifurtimox in rats and humans, together with the expected low risk for clinically relevant DDIs, expands the understanding of this important anti-trypanosomal drug.

Metabolism and Disposition of the Novel Oral Factor XIa Inhibitor Asundexian in Rats and in Humans.

BACKGROUND AND OBJECTIVES: Current anticoagulants pose an increased risk of bleeding. The development of drugs targeting factor XIa, like asundexian, may provide a safer treatment option. A human mass­balance study was conducted to gain a deeper understanding of the absorption, distribution, metabolism, excretion, and potential for drug-drug interaction of asundexian. Additionally, an overview of the biotransformation and clearance pathways for asundexian in humans and bile-duct cannulated (BDC) rats in vivo, as well as in vitro in hepatocytes of both species, is reported. METHODS: The mass balance, biotransformation, and excretion pathways of asundexian were investigated in six healthy volunteers (single oral dose of 25 mg [14C]asundexian) and in BDC rats (intravenous [14C]asundexian 1 mg/kg). RESULTS: Overall recovery of radioactivity was 101% for humans (samples collected up to 14 days after dosing), and 97.9% for BDC rats (samples collected in the 24 h after dosing). Radioactivity was mainly excreted into feces in humans (80.3%) and into bile/feces in BDC rats (> 94%). The predominant clearance pathways in humans were amide hydrolysis to metabolite M1 (47%) and non-labeled M9 with subsequent N-acetylation to M10; oxidative biotransformation was a minor pathway (13%). In rats, hydrolysis of the terminal amide to M2 was the predominant pathway. In human plasma, asundexian accounted for 61.0% of total drug-related area under the plasma concentration-time curve (AUC); M10 was the major metabolite (16.4% of the total drug-related AUC). Excretion of unmetabolized drug was a significant clearance pathway in both species (human, ~ 37%; BDC rat, ~ 24%). The near-complete bioavailability of asundexian suggests negligible limitations on absorption and first-pass metabolism. Comparison with radiochromatograms from incubations with human or rat hepatocytes indicated consistency across species and a good overall in vitro/in vivo correlation. CONCLUSIONS: Similar to preclinical experiments, total asundexian-derived radioactivity is cleared quantitatively predominantly via feces. Excretion occurs mainly via amide hydrolysis and as the unchanged drug.

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.

Structural and Mechanistic Investigation of the Unusual Metabolism of Nifurtimox.

The oral antiparasitic drug nifurtimox has been used to treat Chagas disease for more than 50 years. Historical studies determined that very little nifurtimox is excreted unchanged, but contemporaneous preclinical studies of radiolabeled nifurtimox found almost all of the radiolabel was rapidly excreted, suggesting that metabolism is extensive. Attempts to study nifurtimox metabolism have had limited success, yet this knowledge is fundamental to characterizing the pharmacokinetics and pharmacodynamics of the drug. We conducted in vitro studies using hepatic and renal sources with 14C-labeled nifurtimox as substrate and obtained samples of urine, plasma, and feces from rats administered 2.5 mg/kg [14C]-nifurtimox, and samples of human urine and plasma from phase 1 clinical studies in which participants received a single dose of 120 mg nifurtimox. Analysis of metabolites was done by high-performance liquid chromatography (HPLC)-high-resolution mass spectrometry (HRMS) and HRMS/MS with offline liquid scintillation counting of radiolabeled samples. Surprisingly, only traces of a few metabolites were identified from in vitro incubations with hepatocytes and subcellular fractions, but more than 30 metabolites were identified in rat urine, mostly with atypical mass changes. We developed an HRMS scouting method for the analysis of human samples based on the sulfur atom in nifurtimox and the natural abundance of 34S, as well as a characteristic tandem mass spectrometry (MS/MS) fragmentation of nifurtimox and metabolites. Fragmentation patterns on HRMS/MS were used to propose structures for 18 metabolites (22 including stereoisomers), and based on these structures, the six most abundant products were synthesized and the structures of the synthetic forms were confirmed by HRMS and two-dimensional nuclear magnetic resonance (2D NMR). Overall, we determined that the metabolism of nifurtimox is almost certainly not mediated by typical hepatic and renal drug-metabolizing enzymes, and instead is rapidly metabolized mainly by reduction or nucleophilic attack, with some evidence of oxidation. Knowledge of the most abundant metabolites of nifurtimox affords the possibility of future studies to investigate levels of exposure and possible drug-drug interactions.

Drug-drug interaction of atazanavir on UGT1A1-mediated glucuronidation of molidustat in human.

Molidustat is an oral inhibitor of hypoxia-inducible factor (HIF) prolyl-hydroxylase enhancing the erythropoietin (EPO) response to HIF; it is in clinical development for the treatment of anaemia related to chronic kidney disease. The predominant role of glucuronidation for molidustat clearance (formation of N-glucuronide metabolite M1) and subsequent renal excretion was confirmed in a human mass balance study, with about 85% of the drug being excreted as M1 in urine. The inhibitory effects of 176 drugs and xenobiotics from various compound classes on the UGT-mediated glucuronidation of molidustat in human liver microsomes (HLMs) were investigated. Based on preclinical findings, glucuronidation of molidustat was predominantly mediated by the 5'-diphospho-glucuronosyltransferase (UGT) isoform UGT1A1. Therefore, atazanavir, which is a potent inhibitor of UGT1A1, was chosen for the evaluation of pharmacokinetics and EPO release following a single oral dose of 25 mg molidustat. Molidustat exposure increased about twofold upon coadministration with atazanavir when considering area under plasma concentration-time curve from zero to infinity (AUC) and maximum plasma concentration (Cmax ). Baseline-corrected increase of EPO was 14% and 34% for Cmax and AUC (calculated over 24 hours), respectively. Coadministration of molidustat and atazanavir was well tolerated.

Design, Synthesis, and Pharmacological Characterization of a Neutral, Non-Prodrug Thrombin Inhibitor with Good Oral Pharmacokinetics.

Despite extensive research on small molecule thrombin inhibitors for oral application in the past decades, only a single double prodrug with very modest oral bioavailability has reached human therapy as a marketed drug. We have undertaken major efforts to identify neutral, non-prodrug inhibitors. Using a holistic analysis of all available internal data, we were able to build computational models and apply these for the selection of a lead series with the highest possibility of achieving oral bioavailability. In our design, we relied on protein structure knowledge to address potency and identified a small window of favorable physicochemical properties to balance absorption and metabolic stability. Protein structure information on the pregnane X receptor helped in overcoming a persistent cytochrome P450 3A4 induction problem. The selected compound series was optimized to a highly potent, neutral, non-prodrug thrombin inhibitor by designing, synthesizing, and testing derivatives. The resulting optimized compound, BAY1217224, has reached first clinical trials, which have confirmed the desired pharmacokinetic properties.

Discovery and Characterization of the Potent and Selective P2X4 Inhibitor N-[4-(3-Chlorophenoxy)-3-sulfamoylphenyl]-2-phenylacetamide (BAY-1797) and Structure-Guided Amelioration of Its CYP3A4 Induction Profile.

The P2X4 receptor is a ligand-gated ion channel that is expressed on a variety of cell types, especially those involved in inflammatory and immune processes. High-throughput screening led to a new class of P2X4 inhibitors with substantial CYP 3A4 induction in human hepatocytes. A structure-guided optimization with respect to decreased pregnane X receptor (PXR) binding was started. It was found that the introduction of larger and more polar substituents on the ether linker led to less PXR binding while maintaining the P2X4 inhibitory potency. This translated into significantly reduced CYP 3A4 induction for compounds 71 and 73. Unfortunately, the in vivo pharmacokinetic (PK) profiles of these compounds were insufficient for the desired profile in humans. However, BAY-1797 (10) was identified and characterized as a potent and selective P2X4 antagonist. This compound is suitable for in vivo studies in rodents, and the anti-inflammatory and anti-nociceptive effects of BAY-1797 were demonstrated in a mouse complete Freund's adjuvant (CFA) inflammatory pain model.

Novel non-xanthine antagonist of the A2B adenosine receptor: From HTS hit to lead structure.

The A2B adenosine receptor is a G protein-coupled receptor that belongs to the four member family of adenosine receptors: A1, A2A, A2B, A3. While adenosine-mediated A2B receptor signaling attenuates acute inflammation, facilitates tissue adaptation to hypoxia, and induces increased ischemia tolerance under conditions of an acute insult, persistently elevated adenosine levels and A2B receptor signaling are characteristics of a number of chronic disease states. In this report we describe the discovery of certain thienouracils (thieno[2,3-d]pyrimidine-2,4(1H,3H)-diones) as antagonists of the A2B adenosine receptor by high-throughput screening from our corporate substance collection. The structure optimization of the initial screening hits led to BAY-545, an A2B receptor antagonist that was suitable for in vivo testing. The structure optimization work, SAR that was derived from there, as well as the properties of BAY-545 are also described. In vivo efficacy of BAY-545 was demonstrated in two models of lung fibrosis and data is presented.

Skeletal effects in Angle Class II/1 patients treated with the functional regulator type II : Cephalometric and tensor analysis.

OBJECTIVES: The purpose of this work was to employ both cephalometric and tensor analysis in characterizing the skeletal changes experienced by patients with Angle Class II/1 malocclusion during functional orthodontic treatment with the functional regulator type II. METHODS: A total of 23 patients with Class II/1 malocclusion based on lateral cephalograms obtained before and after treatment with the functional regulator type II were analyzed. Another 23 patients with Angle Class II/1 malocclusion who had not undergone treatment were included as controls. RESULTS: Our cephalometric data attest to significant therapeutic effects of the functional regulator type II on the skeletal mandibular system, including significant advancement of the mandible, increases in effective mandibular length with enhancement of the chin profile, and reduction of growth-related bite deepening. No treatment-related effects were observed at the cranial-base and midface levels. In addition, tensor analysis revealed significant stimulation of mandibular growth in sagittal directions, without indications of growth effects on the maxilla. Its growth-pattern findings differed from those of cephalometric analysis by indicating that the appliance did promote horizontal development, which supports the functional orthodontic treatment effect in Angle Class II/1 cases. CONCLUSIONS: Tensor analysis yielded additional insights into sagittal and vertical growth changes not identifiable by strictly cephalometric means. The functional regulator type II was an effective treatment modality for Angle Class II/1 malocclusion and influenced the skeletal development of these patients in favorable ways.

No evidence for effects of negative emotions on eating behaviour in overweight children.

Negative emotions have been hypothesised to trigger a short-term eating pattern in the obese that leads to a high calorie intake. The present study induced emotions of different quality (negative, neutral, positive) and compared laboratory eating behaviour in overweight and normal weight children. No significant emotion related differences in cumulative food intake curves during a laboratory meal were found. Results point to an adequate regulation of hunger and satiety in children, which possibly cannot be maintained during adulthood.

Curcumin uptake and metabolism.

Curcumin (CUR) is the major orange pigment of turmeric and believed to exert beneficial health effects in the gastrointestinal tract and numerous other organs after oral intake. However, an increasing number of animal and clinical studies show that the concentrations of CUR in blood plasma, urine, and peripheral tissues, if at all detectable, are extremely low even after large doses. The evidence and possible reasons for the very poor systemic bioavailablity of CUR after oral administration are discussed in this brief review. Major factors are the chemical instability of CUR at neutral and slightly alkaline pH, its susceptibility to autoxidation, its avid reductive and conjugative metabolism, and its poor permeation from the intestinal lumen to the portal blood. In view of the very low intestinal bioavailablity, it is difficult to attribute the putative effects observed in peripheral organs to CUR. Therefore, metabolites and/or degradation products of CUR should be taken into consideration as mediators of the pharmacological activity.

Stress-induced laboratory eating behavior in obese women with binge eating disorder.

OBJECTIVE: To compare the microstructural eating behavior of obese patients with and without binge eating disorder (BED) after stress induction in laboratory. METHOD: Forty-eight obese women were investigated. Seventeen were assigned to the group of BED. Group (BED vs. non-BED) by condition (stress vs. no stress) interaction effect on feeding variables, measured by a universal eating monitor, was tested. Stress was induced by the trier social stress test (TSST) and chocolate pudding served as laboratory food. RESULTS: From the nonstress to the stress condition, patients with BED, when compared with non-BED had a greater increase in average eating rate (p < .01) and a corresponding greater increase in the frequency of spoonfuls (p < .02). The BED group also showed a different change in acceleration/deceleration from the nonstress to the stress condition compared to the non-BED group (p < .04). DISCUSSION: Obese individuals with BED appear to exhibit a different response to stress than obese non-BED individuals and individuals with bulimia nervosa.