Human Metabolism Studies Research Articles

P-085 Transcriptomic and proteomic characterization of unexplained male infertility: alterations in energy metabolism

Abstract Study question Which are the transcriptomic and proteomic profiles of spermatozoa belonging to patients with Unexplained Male Infertility (UMI)? Summary answer Transcriptomic and proteomic profiles of UMI patients differ from the fertile men, having the energy metabolism altered. What is known already About 30% of infertile couples suffer from “Unexplained Infertility” (UI). Considering that the male factor contributes to 50% of infertility cases, it is estimated that half of all couple UI cases are due to unexplained male infertility (UMI). Men are diagnosed with UMI when despite having “normal” seminal parameters they are unable to conceive. Due to the lack of UMI classification criteria and the seminogram-only-based infertility diagnosis, little is known about the mechanisms underlying UMI. Study design, size, duration 80 normozoospermic human semen samples with proven fertility and 80 samples from patients with UMI were used for this study. Ejaculates were obtained from Clínica Quirón Bilbao between 2012 and 2018, and after capacitation they were pooled to perform protein and RNA extractions. Participants/materials, setting, methods For transcriptomic analysis, libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v3. Sequencing was performed on NextSeq 2000 system (1x 50 bp, 50 cycles, P3 kit) at Centre de Regulació Genómica (CRG). For the proteomic analysis, Tandem MassTag (TMT) 6-plex isotopic labeling strategy was adopted, and generated peptides were run in a Q Exactive mass spectrometer. To find differentially expressed genes and proteins between both groups EdgeR and Perseus softwares were employed. Main results and the role of chance In the present study, a total of 156.124 transcripts and 1722 proteins were identified in human sperm. Principal Component Analysis (PCA) confirmed the existence of distinguishable differences in the transcriptomic and proteomic profile of control and UMI samples. Specifically, 3112 differentially expressed transcripts and 385 differentially expressed proteins were identified. Among the differentially expressed proteins, 49 were correlated with RNA expression. Coherent RNA-protein signatures were mainly related to axonemal dynein complex assembly and flagellated sperm motility. Proteins involved in energy metabolism were enriched in UMI protein signatures but were not well correlated with the RNA. Furthermore, genes involved in protein transport and chromosome condensation were highly enriched in UMI group but were not detected by proteomics profiling. According to our results, there is a lack of RNA-protein correlation at human sperm. However, differential transcriptomic and proteomic profiles were found when comparing fertile normozoospermic samples to UMI samples. Concretely, our results suggest that altered energy metabolism may be one of the key factors in patients leading with UMI. Thus, the study of human sperm metabolism may be essential to find new targets of male infertility. Limitations, reasons for caution The lack of a consensus on the classification criteria of UMI makes this group of patients very heterogeneous. Further studies are needed to identify and validate specific transcripts and/or proteins that may alter specific metabolic processes and cause infertility. Wider implications of the findings The study of human sperm metabolism may be essential to understand the causes of many UMI cases. In this context, the identification of key protein or transcripts could be very helpful to predict the success of the assisted reproductive treatments. Trial registration number Not applicable

The effect of itraconazole, a strong CYP3A4 inhibitor, on the pharmacokinetics of the first-in-class ACKR3/CXCR7 antagonist, ACT-1004-1239.

Cytochrome P450 (CYP) 3A4 is an enzyme involved in the metabolism of many drugs that are currently on the market and is therefore a key player in drug-drug interactions (DDIs). ACT-1004-1239 is a potent and selective, first-in-class ACKR3/CXRC7 antagonist being developed as a treatment for demyelinating diseases including multiple sclerosis. Based on the human absorption, distribution, metabolism, and excretion (ADME) study results, ACT-1004-1239 is predominantly metabolized by CYP3A4. This study investigated the effect of the strong CYP3A4 inhibitor, itraconazole, on the pharmacokinetics of single-dose ACT-1004-1239 in healthy male subjects. In the open-label, fixed-sequence DDI study, a total of 16 subjects were treated. Each subject received a single dose of 10 mg ACT-1004-1239 (Treatment A) in the first period followed by concomitant administration of multiple doses of 200 mg itraconazole and a single dose of 10 mg ACT-1004-1239 in the second period. We report a median of difference in tmax (90% confidence interval, CI) of 0.5 h (0.0, 1.0) comparing both treatments. The geometric mean ratio (GMR) (90% CI) of Cmax and AUC0-∞ was 2.16 (1.89, 2.47) and 2.77 (2.55, 3.00), respectively. The GMR (90% CI) of t1/2 was 1.46 (1.26, 1.70). Both treatments were well-tolerated with an identical incidence in subjects reporting treatment-emergent adverse events (TEAE). The most frequently reported TEAEs were headache and nausea. In conclusion, ACT-1004-1239 is classified as a moderately sensitive CYP3A4 substrate (i.e., increase of AUC ≥2- to <5-fold), and this should be considered in further clinical studies if CYP3A4 inhibitors are concomitantly administered.

Potential Role of Pig UCP3 in Modulating Adipocyte Browning via the Beta-Adrenergic Receptor Signaling Pathway.

Adipose tissue plays an important role in regulating body temperature and metabolism, with white adipocytes serving as storage units for energy. Recent research focused on the browning of white adipocytes (beige adipocytes), causing thermogenesis and lipolysis. The process of browning is linked to the activation of uncoupling protein (UCP) expression, which can be mediated by the β3 adrenergic receptor pathway. Transcriptional factors, such as peroxisome proliferator activated receptor γ (PPARγ) and PPARγ coactivator 1 alpha, play vital roles in cell fate determination for fat cells. Beige adipocytes have metabolic therapeutic potential to combat diseases such as obesity, diabetes mellitus, and dyslipidemia, owing to their significant impact on metabolic functions. However, the molecular mechanisms that cause the induction of browning are unclear. Therefore, research using animal models and primary culture is essential to provide an understanding of browning for further application in human metabolic studies. Pigs have physiological similarities to humans; hence, they are valuable models for research on adipose tissue. This study demonstrates the browning potential of pig white adipocytes through primary culture experiments. The results show that upregulation of UCP3 gene expression and fragmentation of lipid droplets into smaller particles occur due to isoproterenol stimulation, which activates beta-adrenergic receptor signaling. Furthermore, PPARγ and PGC-1α were found to activate the UCP3 promoter region, similar to that of UCP1. These findings suggest that pigs undergo metabolic changes that induce browning in white adipocytes, providing a promising approach for metabolic research with potential implications for human health. This study offers valuable insights into the mechanism of adipocyte browning using pig primary culture that can enhance our understanding of human metabolism, leading to cures for commonly occurring diseases.

Exploring mitochondrial metabolism of wild-type and diabetic mice skin explants using the Seahorse technology.

Skin wound healing is a complex mechanism which requires a lot of energy, mainly provided by mitochondrial respiration. However, little is known about the mitochondrial bioenergetics of mice skin. We sought to develop a microplate-based assay to directly measure oxygen consumption in whole mice skin with the goal of identifying mitochondrial dysfunction in diabetic skin using an extracellular flux. Different parameters were optimized to efficiently measure the oxygen consumption rate (OCR). First, the most pertinent skin side of wild-type mice was first determined. Then, concentrations of mitochondrial inhibitors were then optimized to get the best efficacy. Finally, punch sizes were modulated to get the best OCR profile. Dermis had the best metabolic activity side of the skin. Unlike the increased concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and rotenone/antimycin A, which showed no improvement of these drugs' effects, varying the skin punch size was successful. Finally, type II diabetic (T2D) skin produced less ATP through mitochondrial metabolism and had a greater non-mitochondrial oxygen consumption than wild-type or type I diabetic (T1D) skin. Here we designed, for the first time, a reliable protocol to measure mitochondria function in whole mouse skin. Our optimized protocol was valuable in assessing alterations associated with diabetes and could be applied to future studies of pathological human skin metabolism.

The Changing Landscape for Human Absorption, Metabolism, and Excretion: Practical Experiences From a Data Analysis of 500 Studies.

Coincidental with the intensified regulatory and industry focus on the design and conduct of human absorption, metabolism, and excretion (hAME) studies in the past 12 months, we have recently completed our 500th cohort involving radiolabeled test item administration to humans. Here, we build upon a recent industry white paper in this journal1 and share some of our own experiences as a Contract Research Organization based upon collaborations with numerous pharma companies and their differing approaches to design and timing, to add further context to the discussion regarding hAME studies and the pivotal role that drug metabolism and pharmacokinetics plays. In this article, we explore how both changing relationships within the industry and shifting regulatory guidelines are impacting strategies, and compare EU and US pre-study approval requirements, before evaluating the trends from over 500 studies conducted at our global facilities conducted over more than 30 years. We conclude with a review of how improved technical capabilities and strategies are influencing the design and conduct of hAME studies, before speculating on some of the driving factors which may shape the direction they take in the future.

Assessment of virgin coconut oil in a balanced diet on indicators of cardiovascular health in non-obese volunteers: A human metabolic study

Background and aimsConsumption of coconut oil is implicated in cardiovascular disease risk. On the contrary, virgin coconut oil (VCO) is believed to offer better health benefits, however, the evidence to support such claims is lacking, particularly in humans. Therefore, this study aimed at assessing the impact of VCO in a balanced diet on HDL-C and some of the anthropometric and biochemical parameters associated with human cardiovascular health before and after the feeding experiment. MethodsIn a crossover observational study, apparently healthy non-obese male volunteers (n = 22) aged between 28 and 50years with a mean body weight of 67.5 kg were inducted into a two-arm controlled feeding experiment one after another for eight weeks with a six-week washout period. In the first arm, the diets were prepared with VCO, whereas peanut oil was used in the second arm (∼35g/day) as the control. ResultsCompared to baseline, the consumption of VCO did not affect HDL-C and anthropometric measures at the end of the 8th week, whereas plasma total cholesterol (TC) and LDL-C levels (Means±standard error; 172 ± 5.6 mg/dL versus 186 ± 5.9 mg/dL and 113 ± 4.29 mg/dL versus 126 ± 4.17 mg/dL respectively) increased significantly. However, plasma triglycerides and some of the cardiovascular risk markers (namely, vascular cell-adhesion molecules, serum amyloid proteins and C-reactive protein) remained unaltered. Further, most of the changes in the VCO arm were comparable to the peanut oil regimen. ConclusionThe consumption of VCO in a balanced diet displayed neutral effects on most parameters related to cardiovascular risk. However, the rise in TC and LDL-C must be tested in a larger sample size over longer periods.

Spatial metabolomics in head and neck tumors: a review.

The joint analysis of single-cell transcriptomics, proteomics, lipidomics, metabolomics and spatial metabolomics is continually transforming our understanding of the mechanisms of metabolic reprogramming in tumor cells. Since head and neck tumor is the sixth most common tumor in the world, the study of the metabolic mechanism of its occurrence, development and prognosis is still undeveloped. In the past decade, this field has witnessed tremendous technological revolutions and considerable development that enables major breakthroughs to be made in the study of human tumor metabolism. In this review, a comprehensive comparison of traditional metabolomics and spatial metabolomics has been concluded, and the recent progress and challenges of the application of spatial metabolomics combined multi-omics in the research of metabolic reprogramming in tumors are reviewed. Furthermore, we also highlight the advances of spatial metabolomics in the study of metabolic mechanisms of head and neck tumors, and provide an outlook of its application prospects.

Editorial introductions.

Editorial introductions.

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, impacts of advances in technology and instrumentation on 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 ADME studies versus a "human-first, human-only strategy" will be presented. Along with the information above, this manuscript will highlight how 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 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.

CMC development of [14C]-labeled sotorasib for the conduct of microtracer human ADME study

Human absorption, distribution, metabolism, and excretion (hADME) studies of new drugs are required for global regulatory filings. Recent advances in high sensitivity analytical technologies have enabled microtracer hADME studies wherein very low radioactive doses can be administered to healthy volunteers to study drug pharmacokinetic profile. Microtracer hADME studies are advantageous to accelerate study timelines during drug development. However, there are limited examples in peer-reviewed journals that highlight the key chemistry, manufacturing, and control (CMC) development challenges and requirements for enabling these clinical microtracer hADME studies. The current manuscript summarizes the CMC activities, risk assessment, and mitigation strategies that were put in place and executed to enable and accelerate the microtracer hADME study of [14C]-labeled sotorasib (AMG 510, compound 1). Sotorasib is a first in class KRASG12C inhibitor used to treat non-small cell lung cancer (NSCLC) in patients with a KRASG12Cmutation (Canon et al., Nature 575:217–223, 2019). The key CMC activities included the synthesis of low nanocurie [14C]-labeled AMG 510 drug substance, development of a drug-in-bottle (DIB) formulation, use of simulation software to predict absorption profiles, associated drug substance and drug product analytical control strategies development, and the utilization of accelerator mass spectrometry (AMS) as a CMC tool enabling low radioactive strength formulation analysis.

Tiered approach to evaluate the CYP3A victim and perpetrator drug-drug interaction potential for vonoprazan using PBPK modeling and clinical data to inform labeling.

Vonoprazan is metabolized extensively through CYP3A and is an in vitro time-dependent inhibitor of CYP3A. A tiered approach was applied to understand the CYP3A victim and perpetrator drug-drug interaction (DDI) potential for vonoprazan. Mechanistic static modeling suggested vonoprazan is a potential clinically relevant CYP3A inhibitor. Thus, a clinical study was conducted to evaluate the impact of vonoprazan on the exposure of oral midazolam, an index substrate for CYP3A. A physiologically-based pharmacokinetic (PBPK) model for vonoprazan was also developed using in vitro data, drug- and system-specific parameters, and clinical data and observations from a [14 C] human absorption, distribution, metabolism, and excretion study. The PBPK model was refined and verified using data from a clinical DDI study with the strong CYP3A inhibitor, clarithromycin, to confirm the fraction metabolized by CYP3A, and the oral midazolam clinical DDI data assessing vonoprazan as a time-dependent inhibitor of CYP3A. The verified PBPK model was applied to simulate the anticipated changes in vonoprazan exposure due to moderate and strong CYP3A inducers (efavirenz and rifampin, respectively). The clinical midazolam DDI study indicated weak inhibition of CYP3A, with a less than twofold increase in midazolam exposure. PBPK simulations projected a 50% to 80% reduction in vonoprazan exposure when administered concomitantly with moderate or strong CYP3A inducers. Based on these results, the vonoprazan label was revised and states that lower doses of sensitive CYP3A substrates with a narrow therapeutic index should be used when administered concomitantly with vonoprazan, and co-administration with moderate and strong CYP3A inducers should be avoided.

Production of Large Amounts of [13C]Leucine-Enriched Milk Proteins by Lactating Cows

During protein metabolism kinetic studies, oral tracers are administered as labeled free amino acids and do not necessarily represent the metabolic fate of amino acids ingested as proteins. However, sufficient quantities of 13C-labeled proteins are not currently available. We here present a new methodology for producing large amounts of milk proteins intrinsically labeled with [13C]leucine. After surgical preparation, two lactating cows were infused with 80–90 g of L-[1-13C]leucine for 24–32 h, and milk was collected during and after the infusion. Casein and whey protein fractions were purified by membrane separation techniques. Arteriovenous balance across the udder indicated a very efficient extraction of leucine by the mammary gland. Five batches of pure casein and whey proteins, totaling 3854 g of protein of excellent bacteriological quality, were obtained. Two thirds of these proteins had [13C]leucine enrichments ranging from 10.5 to 19.4% ([13C]leucine atom percent excess). The overall tracer recoveries were 22 and 27% (cows 1 and 2, respectively). Thus, pure milk proteins were produced in large amounts with sufficient 13C enrichment to be used in human protein metabolism studies.

Peptidomic Characterization and Amino Acid Availability after Intake of Casein vs. a Casein Hydrolysate in a Pig Model.

It is known that casein hydrolysis accelerates gastrointestinal transit in comparison to intact casein, although the effect of the protein hydrolysis on the composition of the digests is not fully understood. The aim of this work is to characterize, at the peptidome level, duodenal digests from pigs, as a model of human digestion, fed with micellar casein and a previously described casein hydrolysate. In addition, in parallel experiments, plasma amino acid levels were quantified. A slower transit of nitrogen to the duodenum was found when the animals received micellar casein. Duodenal digests from casein contained a wider range of peptide sizes and a higher number of peptides above five amino acids long in comparison with the digests from the hydrolysate. The peptide profile was markedly different, and although β-casomorphin-7 precursors were also found in hydrolysate samples, other opioid sequences were more abundant in the casein digests. Within the same substrate, the evolution of the peptide pattern at different time points showed minimal changes, suggesting that the protein degradation rate relies more on the gastrointestinal location than on digestion time. Higher plasma concentrations of methionine, valine, lysine and amino acid metabolites were found in animals fed with the hydrolysate at short times (<200 min). The duodenal peptide profiles were evaluated with discriminant analysis tools specific for peptidomics to identify sequence differences between both substrates that can be used for future human physiological and metabolic studies.

Generation and analysis of context-specific genome-scale metabolic models derived from single-cell RNA-Seq data

Single-cell RNA sequencing combined with genome-scale metabolic models (GEMs) has the potential to unravel the differences in metabolism across both cell types and cell states but requires new computational methods. Here, we present a method for generating cell-type-specific genome-scale models from clusters of single-cell RNA-Seq profiles. Specifically, we developed a method to estimate the minimum number of cells required to pool to obtain stable models, a bootstrapping strategy for estimating statistical inference, and a faster version of the task-driven integrative network inference for tissuesalgorithm for generating context-specific GEMs. In addition, we evaluated the effect of different RNA-Seq normalization methods on model topology and differences in models generated from single-cell and bulk RNA-Seq data. We applied our methods on data from mouse cortex neurons and cells from the tumor microenvironment of lung cancer and in both cases found that almost every cell subtype had a unique metabolic profile. In addition, our approach was able to detect cancer-associated metabolic differences between cancer cells and healthy cells, showcasing its utility. We also contextualized models from 202 single-cell clusters across 19 human organs using data from Human Protein Atlas and made these available in the web portal Metabolic Atlas, thereby providing a valuable resource to the scientific community. With the ever-increasing availability of single-cell RNA-Seq datasets and continuously improved GEMs, their combination holds promise to become an important approach in the study of human metabolism.

Application of Accelerator Mass Spectrometry to Characterize the Mass Balance Recovery and Disposition of AZD4831, a Novel Myeloperoxidase Inhibitor, following Administration of an Oral Radiolabeled Microtracer Dose in Humans.

This study evaluated the mass balance and disposition of AZD4831, a novel myeloperoxidase inhibitor, in six healthy participants using a 14C-labeled microtracer coupled with analysis by accelerator mass spectrometry (AMS). A single oral dose of 10 mg 14C-AZD4831 (14.8 kBq) was administered as a solution, and 14C levels were quantified by AMS in blood, urine, and feces over 336 hours postdose. AZD4831 was rapidly absorbed, and AZD4831 plasma concentrations declined in a biphasic manner, with a long half-life of 52 hours. AZD4831 was eliminated via metabolism and renal excretion. An N-carbamoyl glucuronide metabolite of AZD4831 (M7), formed primarily via UGT1A1, was the predominant circulating metabolite. Presumably, M7 contributed to the long half-life of AZD4831 via biliary elimination and hydrolysis/enterohepatic recirculation of AZD4831. On average, ∼84% of administered 14C-AZD4831 was recovered by 336 hours postdose (urine, 51.2%; feces, 32.4%). Between 32%-44% of the dose was excreted as unchanged AZD4831 in urine, indicating renal elimination as the major excretory route. Only 9.7% of overall fecal recovery was recorded in the first 48 hours, with the remainder excreted over 48%-336 hours, suggesting that most fecal recovery was due to biliary elimination. Furthermore, only 6% of unchanged AZD4831 was recovered in feces. Overall, the fraction of the administered AZD4831 dose absorbed was high. 14C-AZD4831 was well tolerated. These findings contribute to increasing evidence that human absorption, distribution, metabolism, and excretion studies can be performed with acceptable mass balance recovery at therapeutically relevant doses and low radiolabel-specific activity using an AMS-14C microtracer approach. SIGNIFICANCE STATEMENT: In this study, the human absorption, distribution, metabolism, and excretion (hADME) of the novel myeloperoxidase inhibitor AZD4831 was assessed following oral administration. This included investigation of the disposition of M7, the N-carbamoyl glucuronide metabolite. Resolution of challenges highlighted in this study contributes to increasing evidence that hADME objectives can be achieved in a single study for compounds with therapeutically relevant doses and low radiolabel-specific activity by using an AMS-14C microtracer approach, thus reducing the need for preclinical radiolabeled studies.

A "dilute-and-shoot" column-switching UHPLC-MS/MS procedure for the rapid determination of branched nonylphenol in human urine: method optimisation and some fundamental aspects of nonylphenol analysis.

Technical grade branched nonylphenol (NP) was determined in human urine by online solid phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry (SPE-UHPLC-MS/MS). Prior to analysis, urine specimens were simply diluted and enzymatically deconjugated. The run time of the chromatography, including SPE and re-equilibration, was 9min per injection. The enzymatic cleavage of NP conjugates was optimised with incurred sample material from a human metabolism study: the highest recoveries were obtained with β-glucuronidase from E. coli K 12 in 0.1M ammonium acetate at pH 6.5, within a minimal hydrolysis time of 30 to 60min. Using sodium acetate instead of ammonium acetate led to systematically decreased recovery rates. The analytical method was validated regarding its precision (coefficients of variation: 2.9-7.4%), accuracy (relative recovery rates: 93-105%), robustness (relative recovery rates in individual urine matrices: 92-117%), selectivity, and limit of quantification (1.0μgL-1). Fundamental aspects in the analysis of technical product mixtures such as NP, comprising various isomers and homologues, were considered. Validation results, an exposure scenario and the application of the procedure to real samples, show that it enables a rugged monitoring of NP exposures above, at, and significantly below health-based guidance values, corresponding to daily NP intakes in the low μg kg-1 d-1 range. On the other hand, background levels in non-specifically exposed populations cannot be detected with this method. Hence, while alternative approaches should be pursued for NP analysis at environmental trace level, the speed and simplicity of our method are ideal for high-throughput human biomonitoring in occupational medicine.

Rapid quantification of seven major neonicotinoids and neonicotinoid-like compounds and their key metabolites in human urine

Neonicotinoids and neonicotinoid-like compounds (NNIs) are frequently used insecticides worldwide and exposure scenarios can vary widely between countries and continents. We have developed a specific and robust analytical method based on liquid chromatography-electrospray tandem mass spectrometry coupled to online-SPE (online-SPE-LC-ESI-MS-MS) to analyze the seven most important NNIs from a global perspective together with nine of their key metabolites in human urine. The method also includes the neonicotinoid-like flupyradifurone (FLUP), an important future substitute for classical neonicotinoids, and two of its major human metabolites, 5-hydroxy– and N-desfluoroethyl-FLUP. Validation of the method was carried out using pooled urine samples from low-dose human metabolism studies and spiked urine samples with a wide range of creatinine concentrations. Depending on the analyte, the limits of quantitation were between 0.06 and 2.1 µg L−1, the inter-day and intra-day imprecisions ≤6%, and the mean relative recoveries between 89% and 112%. The method enabled us to successfully quantify NNIs and their metabolites at current environmental exposures in 34 individuals of the German general population and 43 pregnant women from Brazil with no known occupational exposures to NNIs.

RF34 | PMON202 The Human Metabolic Profile of Estetrol

Abstract Objectives Estetrol (E4), a native estrogen produced by the fetal human liver, has four hydroxyl-groups, making it structurally distinct from other estrogens. We determined the human metabolic profile of E4, which is recently marketed combined with drospirenone as a novel contraceptive and is currently in development for the relief of menopausal symptoms. Method: We studied the in vitro metabolism of E4 using human hepatocytes, human liver microsomes and recombinant CYP enzymes in reaction phenotyping studies to evaluate the role of human cytochrome P450 enzymes, UDP-glucuronosyltransferases (UGT) and sulfotransferases (SULT). Furthermore, we evaluated the in vivo metabolism of E4 in an open-label, non-randomised phase 1 human absorption, distribution, metabolism, and excretion study, in which six healthy postmenopausal participants received a single oral solution containing 15 mg [14C]-E4. We analysed blood (up to 240h), urine and faecal samples (up to 312h). Metabolite profiling and identification was performed using liquid chromatography with tandem mass spectrometry. Results The in vitro studies showed that E4 undergoes extensive phase 2 metabolism to form glucuronide and sulphate conjugates. UGT2B7 is the key enzyme catalysing the direct glucuronidation of E4 and SULT1E1 is the dominant sulfotransferase for sulphate conjugation in vitro. Unlike for other estrogens, oxidative phase 1 metabolism by P450 enzymes does not play a major role in the metabolism of E4. Following oral administration of E4 to humans, the major metabolites observed in human plasma were E4-16-glucuronide, E4-3-glucuronide and E4-glucuronide-sulfate (respectively 62%, 17% and 9% of radioactivity at the time of maximum plasma concentration [Tmax]). At Tmax, unchanged E4 accounts for only 7.5% of plasma radioactivity. Main metabolites quantified in urine during the first 6 hours after administration were E4-16-glucuronide and E4-3-glucuronide (respectively 77.4% and 16.5% of urine radioactivity). Approximately 69% of the total administered radioactivity was recovered in urine and 22% in faeces. Mass spectrometry showed that E4 was not present in urine but was detected in faeces and that E4 is not converted back into one of the other natural estrogens (estriol, estradiol, estrone). Conclusions Altogether, these data suggest that E4 has a unique metabolic profile. E4 is a terminal end-product of estrogen metabolism, which is not converted back, in vivo, into active metabolites like estriol, estradiol or estrone. Unlike for other estrogens, CYP450 enzymes do not play a major role in the metabolic pathway of E4. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 12:48 p.m. - 12:53 p.m.

Pipeline Impact of Radiolabeled Compounds in Drug Discovery and Development.

The long-lived radionuclides tritium and carbon-14 have been used for many years in pharmaceutical research and development for making key efficacy and toxicological decisions. Early discovery utilizes radiolabels for compound selection through radioligand binding assays and autoradiography. In preclinical safety evaluation, the use of labeled compounds for adsorption, distribution, metabolism, and excretion studies is often preferred for the added detection sensitivity. As the drug substance proceeds to the clinic, human metabolism studies are reliant on the use of labeled materials to fulfill required regulatory applications.

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.