Human Metabolism Studies Research Articles

The role of the Human Metabolome Database in inborn errors of metabolism.

Metabolomics holds considerable promise to advance our understanding of human disease, including our understanding of inborn errors of metabolism (IEM). The application of metabolomics in IEM research has already led to the discovery of several novel IEMs and the identification of novel IEM biomarkers. However, with hundreds of known IEMs and more than 700 associated IEM metabolites, it is becoming increasingly challenging for clinical researchers to keep track of IEMs, their associated metabolites, and their corresponding metabolic mechanisms. Furthermore, when using metabolomics to assist in IEM biomarker discovery or even in IEM diagnosis, it is becoming much more difficult to properly identify metabolites from the complex NMR and MS spectra collected from IEM patients. To that end, comprehensive, open access metabolite databases that provide up-to-date referential information about metabolites, metabolic pathways, normal/abnormal metabolite concentrations, and reference NMR or MS spectra for compound identification are essential. Over the last few years, a number of compound databases, including the Human Metabolome Database (HMDB), have been developed to address these challenges. First described in 2007, the HMDB is now the world's largest and most comprehensive metabolomic resource for human metabolic studies. The latest release of the HMDB contains 114,100 metabolite entries (with 247 being relevant to IEMs), thousands of metabolite concentrations (with 600 being relevant to IEMs), and ~33,000 metabolic and disease-associated pathways (with 202 being relevant to IEMs). Here we provide a summary of the HMDB and offer some guidance on how it can be used in metabolomic studies of IEMs.

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.

Effects of the Artificial Sweetener Neotame on the Gut Microbiome and Fecal Metabolites in Mice.

Although artificial sweeteners are widely used in food industry, their effects on human health remain a controversy. It is known that the gut microbiota plays a key role in human metabolism and recent studies indicated that some artificial sweeteners such as saccharin could perturb gut microbiome and further affect host health, such as inducing glucose intolerance. Neotame is a relatively new low-caloric and high-intensity artificial sweetener, approved by FDA in 2002. However, the specific effects of neotame on gut bacteria are still unknown. In this study, we combined high-throughput sequencing and gas chromatography–mass spectrometry (GC-MS) metabolomics to investigate the effects of neotame on the gut microbiome and fecal metabolite profiles of CD-1 mice. We found that a four-week neotame consumption reduced the alpha-diversity and altered the beta-diversity of the gut microbiome. Firmicutes was largely decreased while Bacteroidetes was significantly increased. The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis also indicated that the control mice and neotame-treated mice have different metabolic patterns and some key genes such as butyrate synthetic genes were decreased. Moreover, neotame consumption also changed the fecal metabolite profiles. Dramatically, the concentrations of multiple fatty acids, lipids as well as cholesterol in the feces of neotame-treated mice were consistently higher than controls. Other metabolites, such as malic acid and glyceric acid, however, were largely decreased. In conclusion, our study first explored the specific effects of neotame on mouse gut microbiota and the results may improve our understanding of the interaction between gut microbiome and neotame and how this interaction could influence the normal metabolism of host bodies.

Clinical Pharmacokinetics and the Impact of Genetic Polymorphism on a CYP2C19 Substrate, BMS-823778, in Healthy Subjects.

BMS-823778 is a potent and selective inhibitor of 11β-HSD1, an enzyme that regulates tissue-specific intracellular glucocorticoid metabolism and is a compelling target for the treatment of metabolic diseases. Metabolism of BMS-823778 was mediated mainly by polymorphic CYP2C19, with minor contributions from CYP3A4/5 and UGT1A4. The clinical pharmacokinetics (PK) of BMS-823778 was first investigated in healthy volunteers after single and multiple ascending doses. BMS-823778 was rapidly absorbed after the oral dose, and systemic exposure at steady state increased proportionally to the dose. Large intersubject variability in BMS-823778 exposure was likely because of the polymorphism of metabolic enzymes. The impact of genetic polymorphism of CYP2C19, UGT1A4, and CYP3A5 on BMS-823778 PK was assessed in healthy Chinese and Japanese subjects, as well as in a human absorption, distribution, metabolism, and excretion study in which all subjects were genotyped either before or after treatment. A clear trend of high exposure and low clearance was seen in poor metabolizers (PMs) of CYP2C19 compared with extensive (EM) and intermediate metabolizer (IM) subjects. The impact of UGT1A4 or CYP3A5 polymorphism on BMS-823778 PK was statistically not significant in CYP2C19 EM and IM subjects; however, in a subject with predicted CYP2C19 PM phenotype, the PK of BMS-823778 was affected significantly by UGT1A4 polymorphism. Overall, BMS-823778 was safe and well tolerated in healthy subjects after single or multiple oral doses. The PK of BMS-823778 was characterized by rapid absorption, and the systemic clearance directly correlated with the genetic polymorphism of CYP2C19.

De-novo identification of specific exposure biomarkers of the alternative plasticizer di(2-ethylhexyl) terephthalate (DEHTP) after low oral dosage to male volunteers by HPLC-Q-Orbitrap-MS

Context: Human exposure biomonitoring relies on the availability of specific, sensitive biomarkers. For emerging chemicals, the identification (prediction, synthesis, verification) of such biomarkers is time and cost intensive.Objective: This study aimed to further elucidate the urinary metabolic profile of the plasticizer di(2-ethylhexyl) terephthalate (DEHTP) in search of probably additional biomarkers of exposure.Materials and methods: Urine samples of an oral low-dose volunteer study were analysed by HPLC-Q-Orbitrap-MS combined with a commercial data mining software. Metabolite identification was based on isotopic pattern, accurate masses of product ions and excretion profiles.Results: Nine phase I metabolites of DEHTP were tentatively identified by HPLC-Q-Orbitrap-MS. Four previously described, side chain oxidized monoester metabolites were confirmed in all samples. In addition, five previously unknown downstream metabolites were tentatively identified.Discussion and conclusion: The excretion profiles obtained by HPLC-Q-Orbitrap-MS were in good agreement with quantitative HPLC-QqQ-MS data. For the newly discovered metabolites, plausible excretion profiles, similar to the ones of the known metabolites, were obtained. The presented approach proved to be successful for metabolite screening in urine samples after low-dose exposure and will be applied in future human metabolism studies for a fast, reliable and cost effective identification of specific biomarkers of exposure.

HMDB 4.0: the human metabolome database for 2018.

The Human Metabolome Database or HMDB (www.hmdb.ca) is a web-enabled metabolomic database containing comprehensive information about human metabolites along with their biological roles, physiological concentrations, disease associations, chemical reactions, metabolic pathways, and reference spectra. First described in 2007, the HMDB is now considered the standard metabolomic resource for human metabolic studies. Over the past decade the HMDB has continued to grow and evolve in response to emerging needs for metabolomics researchers and continuing changes in web standards. This year's update, HMDB 4.0, represents the most significant upgrade to the database in its history. For instance, the number of fully annotated metabolites has increased by nearly threefold, the number of experimental spectra has grown by almost fourfold and the number of illustrated metabolic pathways has grown by a factor of almost 60. Significant improvements have also been made to the HMDB’s chemical taxonomy, chemical ontology, spectral viewing, and spectral/text searching tools. A great deal of brand new data has also been added to HMDB 4.0. This includes large quantities of predicted MS/MS and GC–MS reference spectral data as well as predicted (physiologically feasible) metabolite structures to facilitate novel metabolite identification. Additional information on metabolite-SNP interactions and the influence of drugs on metabolite levels (pharmacometabolomics) has also been added. Many other important improvements in the content, the interface, and the performance of the HMDB website have been made and these should greatly enhance its ease of use and its potential applications in nutrition, biochemistry, clinical chemistry, clinical genetics, medicine, and metabolomics science.

Is zebrafish (Danio rerio) a tool for human-like metabolism study?

One of the greatest challenges in anti-doping science is the large number of substances available and the difficulty in finding the best analytical targets to detect their misuse. Therefore, metabolism studies involving prohibited substances are fundamental. However, metabolism studies in humans could face an important ethical bottleneck, especially for non-approved substances. An emerging model for metabolism assessment is the zebrafish, due to its genetic similarities with humans. In the present study, the ability of adult zebrafish to produce metabolites of sibutramine and stanozolol, substances with a well-known metabolism that are widely used as doping agents in sports, was evaluated. They represent 2 of the most abused classes of doping agents, namely, stimulants and anabolic steroids. These are classes that have been receiving attention because of the upsurge of synthetic analogues, for which the side effects in humans have not been assessed. The samples collected from the zebrafish tank water were hydrolysed, extracted by solid-phase extraction, and analysed by liquid chromatography with high resolution mass spectrometry (LC-HRMS). Adult zebrafish could produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dehydroxylated, and reduced derivatives, all of which have already been detected in human urine. This study demonstrates that adult zebrafish can absorb, oxidise, and excrete several metabolites in a manner similar to humans. Therefore, adult zebrafish seem to be a very promising tool to study human-like metabolism when aiming to find analytical targets for doping control. Copyright © 2017 John Wiley & Sons, Ltd.

Disposition, profiling and identification of emixustat and its metabolites in humans

1. Emixustat is a small molecule that potently inhibits retinal pigment epithelium 65 isomerohydrolase. Emixustat is in clinical development for the treatment of various retinopathies (i.e. Stargardt disease and diabetic retinopathy).2. A human absorption, distribution, metabolism, and excretion (ADME) study was conducted with a single dose of [14C]-emixustat in healthy male subjects. Total 14C content in plasma, urine, and faeces was determined using accelerator mass spectrometry (AMS), and metabolic profiles in pooled plasma and urine were investigated by both HPLC-AMS and 2D LC-MS/MS.3. After a single, oral 40-mg dose of [14C]-emixustat, recovery of total 14C was nearly complete within 24 h. Urine was the major route of 14C elimination; accounting for > 90% of the administered dose.4. Biotransformation of emixustat occurred primarily at two structural moieties; oxidation of the cyclohexyl moiety and oxidative deamination of the 3R-hydroxypropylamine, both independently and in combination to produce secondary metabolites. Metabolite profiling in pooled plasma samples identified 3 major metabolites: ACU-5124, ACU-5116 and ACU-5149, accounting for 29.0%, 11.5%, and 10.6% of total 14C, respectively. Emixustat was metabolized in human hepatocytes with unchanged emixustat accounting for 33.7% of sample radioactivity and predominantly cyclohexanol metabolites observed.

Direct calorimetry: a brief historical review of its use in the study of human metabolism and thermoregulation.

Direct calorimetry is the gold standard means of measuring human metabolic rate and its use has been fundamental for understanding metabolism in health and disease. While metabolic rate is now more commonly estimated indirectly from measures of the oxygen consumed during respiration, direct calorimetry provides the user with the unique capacity to quantify the heat produced from aerobic and anaerobic metabolism by measuring heat exchange between the body and the environment. This review provides a brief historical overview of the fundamental concepts which underlie direct calorimetry, of pioneer scientists which developed these concepts into functional pieces of equipment and the subsequent use of direct calorimetry to advance our understanding of energy balance, nutrition, and the pathogenesis of metabolic diseases. Attention is directed to seminal studies that successfully employed direct calorimetry to verify that the law of energy conservation also applies to human beings and to establish the validity of indirect calorimetry. Finally, we discuss the more recent use of direct calorimetry for the measurement of whole-body heat exchange and body heat storage in the study of human thermoregulation.

Midostaurin, a Novel Protein Kinase Inhibitor for the Treatment of Acute Myelogenous Leukemia: Insights from Human Absorption, Metabolism, and Excretion Studies of a BDDCS II Drug.

The absorption, metabolism, and excretion of midostaurin, a potent class III tyrosine protein kinase inhibitor for acute myelogenous leukemia, were evaluated in healthy subjects. A microemulsion formulation was chosen to optimize absorption. After a 50-mg [14C]midostaurin dose, oral absorption was high (>90%) and relatively rapid. In plasma, the major circulating components were midostaurin (22%), CGP52421 (32.7%), and CGP62221 (27.7%). Long plasma half-lives were observed for midostaurin (20.3 hours), CGP52421 (495 hours), and CGP62221 (33.4 hours). Through careful mass-balance study design, the recovery achieved was good (81.6%), despite the long radioactivity half-lives. Most of the radioactive dose was recovered in feces (77.6%) mainly as metabolites, because only 3.43% was unchanged, suggesting mainly hepatic metabolism. Renal elimination was minor (4%). Midostaurin metabolism pathways involved hydroxylation, O-demethylation, amide hydrolysis, and N-demethylation. High plasma CGP52421 and CGP62221 exposures in humans, along with relatively potent cell-based IC50 for FMS-like tyrosine kinase 3-internal tandem duplications inhibition, suggested that the antileukemic activity in AML patients may also be maintained by the metabolites. Very high plasma protein binding (>99%) required equilibrium gel filtration to identify differences between humans and animals. Because midostaurin, CGP52421, and CGP62221 are metabolized mainly by CYP3A4 and are inhibitors/inducers for CYP3A, potential drug-drug interactions with mainly CYP3A4 modulators/CYP3A substrates could be expected. Given its low aqueous solubility, high oral absorption and extensive metabolism (>90%), midostaurin is a Biopharmaceutics Classification System/Biopharmaceutics Drug Disposition Classification System (BDDCS) class II drug in human, consistent with rat BDDCS in vivo data showing high absorption (>90%) and extensive metabolism (>90%).

Differentiation and Metabolic Interrogation of Human Adipocytes.

Adipocytes differentiated from preadipocytes provide a valuable model for the study of human adipocyte metabolism. We describe methods for isolation of human stromal vascular cells, expansion of preadipocytes, differentiation into mature adipocytes, and in vitro metabolic interrogation of adipocytes.

An evaluation of human ADME and mass balance studies using regular or low doses of radiocarbon.

There has been increased interest in conducting human absorption, distribution, metabolism, and excretion (ADME) studies with low doses (up to 0.1MBq) as opposed to regular doses (1.85-3.7MBq) of radiocarbon (14 C). This is due to the fact that low-dose human ADME studies may be conducted without dosimetry calculations and will lead to lower human radiation exposure. Here, we sought to compare the outcomes of low-dose versus regular-dose human ADME studies in healthy volunteers. Forty oral human ADME studies conducted at PRA were surveyed, among which 12 were low-dose studies. The fraction of drug material absorbed was 67%±7% in the regular-dose studies (data for 13 studies) versus 39%±16% in the low-dose studies (data for 5 studies). The average total recovery of 14 C in excreta was 93%±5% for regular-dose studies, and 21 of 28 such studies showed recoveries more than 90%. For low-dose studies, average total recovery was 89%±9%, and 6 of 12 studies showed recoveries more than 90%. Metabolite profiling was successful in all cases reported (13 regular-dose studies and 5 low-dose studies). There was no obvious relationship between the total recoveries of 14 C in excreta and the proportion of 14 C excreted in feces, or between the total recoveries and the plasma elimination half-lives for parent or total 14 C, neither in the low-dose nor the regular-dose studies. A significant correlation was found between the fraction absorbed and the recovery in feces in the low-dose but not in the regular-dose studies, and no correlation was found between the fractions absorbed and the total recoveries in both types of studies. Low-dose studies were more often conducted on drugs that had a plasma elimination half-life of parent drug more than 100hours (5 of 12 studies) than regular-dose studies (1 of 26 studies). We conclude that both low-dose as well as regular-dose human ADME studies provide adequate data to support decision making for further drug development.

Assessment of human energy exchange: historical overview.

Energy exchange is fundamental to life and is a cornerstone in the study of human physiology, metabolism and nutrition. A global effort is underway to further our understanding of human energy exchange and its components as a means of establishing the mechanistic underpinnings of the evolving obesity and chronic disease epidemics. The current report establishes a conceptual historical framework for examining the evolution of energy exchange concepts and measurement methods. We review developments taking place over more than 2000 years during which humans endeavored to establish the source of body heat, the 'fire of life'. Major conceptual and methodological advances over the past three centuries have incrementally advanced the field and created the energy exchange paradigm within which we now work. As in the past, innovative experimental ideas and measurement methods are now needed to answer important questions brought to light by the obesity and chronic disease epidemics. Nevertheless, older classical measurement methods based on calorimetry techniques still hold a strong position in the field as many diet and weight-related questions remain unanswered.

Editorial introductions

Editorial introductions

Disrupted Murine Gut-to-Human Liver Signaling Alters Bile Acid Homeostasis in Humanized Mouse Liver Models.

The humanized liver mouse model is being exploited increasingly for human drug metabolism studies. However, its model stability, intercommunication between human hepatocytes and mouse nonparenchymal cells in liver and murine intestine, and changes in extrahepatic transporter and enzyme expressions have not been investigated. We examined these issues in FRGN [fumarylacetoacetate hydrolase (Fah-/-), recombination activating gene 2 (Rag2-/-), and interleukin 2 receptor subunit gamma (IL-2rg -/-) triple knockout] on nonobese diabetic (NOD) background] and chimeric mice: mFRGN and hFRGN (repopulated with mouse or human hepatocytes, respectively). hFRGN mice showed markedly higher levels of liver cholesterol, biliary bilirubin, and bile acids (liver, bile, and plasma; mainly human forms, but also murine bile acids) but lower transforming growth factor beta receptor 2 (TGFBR2) mRNA expression levels (10%) in human hepatocytes and other proliferative markers in mouse nonparenchymal cells (Tgf-β1) and cholangiocytes [plasma membrane-bound, G protein-coupled receptor for bile acids (Tgr5)], suggestive of irregular regeneration processes in hFRGN livers. Changes in gene expression in murine intestine, kidney, and brain of hFRGN mice, in particular, induction of intestinal farnesoid X receptor (Fxr) genes: fibroblast growth factor 15 (Fgf15), mouse ileal bile acid binding protein (Ibabp), small heterodimer partner (Shp), and the organic solute transporter alpha (Ostα), were observed. Proteomics revealed persistence of remnant murine proteins (cyotchrome P450 7α-hydroxylase (Cyp7a1) and other enzymes and transporters) in hFRGN livers and suggest the likelihood of mouse activity. When compared with normal human liver tissue, hFRGN livers showed lower SHP mRNA and higher CYP7A1 (300%) protein expression, consequences of tβ- and tα-muricholic acid-mediated inhibition of the FXR-SHP cascade and miscommunication between intestinal Fgf15 and human liver fibroblast growth factor receptor 4 (FGFR4), as confirmed by the unchanged hepatic pERK/total ERK ratio. Dysregulation of hepatocyte proliferation and bile acid homeostasis in hFRGN livers led to hepatotoxicity, gallbladder distension, liver deformity, and other extrahepatic changes, making questionable the use of the preparation for drug metabolism studies.

The synthesis and analysis of [phenyl-14 C(U)]BMS-770767 and [13 C6 ]BMS-770767 for use in discovery biotransformation, human ADME and bioanalytical studies.

Type 2 diabetes is a significant worldwide health problem. To support the development of BMS-770767 as an inhibitor of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) for type 2 diabetes was required the synthesis of carbon-14-labelled material for use in metabolic profiling and for the human adsorption, distribution, metabolism and excretion (ADME) study. Initially, [phenyl-14 C(U)]BMS-770767 was synthesized in two steps from a late-stage intermediate and [14 C(U)]2-chlorophenol to give the desired final product in 18% yield. Later, the synthesis was completed for the human ADME clinical study using a combination of the discovery and process chemistry routes under cGMP to prepare [phenyl-14 C(U)]BMS-770767. The radiochemical purity of the synthesized [phenyl-14 C(U)]BMS-770767 after dilution with unlabelled clinical grade BMS-770767 was 99.1% having a specific activity of 1.61 μCi/mg. In addition, to support the quantification of BMS-770767 in LC/MS analyses, [13 C6 ]BMT-770767 was prepared in two steps from a late-stage intermediate and [13 C6 ]2-chlorophenol.

Abstract CT153: Pharmacokinetic drug interactions between abemaciclib and CYP3A inducers and inhibitors

Abstract Abemaciclib is a selective and potent small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) being investigated for treatment of refractory hormone-receptor positive (HR+) advanced or metastatic breast cancer. In vitro, CYP3A is responsible for >99% of the CYP-mediated microsomal metabolism of abemaciclib and its active metabolites. Three clinical studies evaluated the disposition and metabolism and drug interaction potential of abemaciclib in the presence of a strong CYP3A-inducer, rifampin, or a strong CYP3A-inhibitor, clarithromycin. Abemaciclib disposition and metabolism were determined following a single oral 150 mg dose of [14C]-abemaciclib in healthy subjects (N = 6). In the rifampin interaction study, abemaciclib was administered as a single oral 200 mg dose in healthy subjects (N = 24) on 2 occasions: alone on Day 1 of Period 1 and in combination with 600 mg rifampin on Day 7 of Period 2, after 6 days of rifampin once daily (QD) dosing; rifampin continued QD for 7 days after abemaciclib. In the clarithromycin interaction study, abemaciclib was administered as a single oral 50 mg dose in patients with advanced cancer (N = 26) on 2 occasions: alone in Period 1 and on Day 5 of clarithromycin dosing (500 mg BID) in Period 2 followed by an additional 7 days of clarithromycin. Abemaciclib was extensively metabolized, with less than 10% of parent drug recovered unchanged in feces. Parent drug and 3 active metabolites; (LSN2839567 [M2], LSN3106729 [M18], and LSN3106726 [M20]) were detected in plasma. The mean t1/2 in healthy subjects was 29.0, 104.0, 55.9, and 43.1 hours for abemaciclib, M2, M18, and M20, respectively. Coadministration with rifampin compared to abemaciclib alone decreased abemaciclib AUC(0-?) and Cmax by 95% and 92%, respectively, and decreased AUC(0-?) and Cmax of total active species (abemaciclib + M2 + M18+ M20) by 77% and 45%, respectively. Coadministration with clarithromycin compared to abemaciclib alone increased abemaciclib AUC(0-?) and Cmax by 237% and 30%, respectively; and increased the total active species AUC(0-?) by 119% and decreased Cmax by 7%. The mean abemaciclib t1/2 was prolonged from 28.8 to 63.6 hours. No clinically significant safety concerns were observed following single doses of abemaciclib in healthy subjects or in patients with advanced cancer based on vital signs, clinical laboratory evaluations, and electrocardiogram data. The human absorption, distribution, metabolism and excretion study indicated that abemaciclib was cleared primarily by hepatic metabolism, and the clinical drug-drug interaction studies with strong CYP3A inducer and inhibitor substantiated the major role of CYP3A in the metabolism of abemaciclib. Due to significant changes in abemaciclib and active-metabolite exposure in the presence of strong CYP3A inducers and inhibitors, concomitant use with abemaciclib should be avoided, or abemaciclib dose may require adjustment. Citation Format: Palaniappan Kulanthaivel, Daruka Mahadevan, P. Kellie Turner, Jane Royalty, Wee Teck Ng, Ping Yi, Jessica Rehmel, Kenneth Cassidy, Jill Chappell. Pharmacokinetic drug interactions between abemaciclib and CYP3A inducers and inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT153.

Functional Integrity of the Chimeric (Humanized) Mouse Liver: Enzyme Zonation, Physiologic Spaces, and Hepatic Enzymes and Transporters

Chimeric mouse liver models are useful in vivo tools for human drug metabolism studies; however, liver integrity and the microcirculation remain largely uninvestigated. Hence, we conducted liver perfusion studies to examine these attributes in FRGN [Fah(-/-), Rag2(-/-), and Il2rg(-/-), NOD strain] livers (control) and chimeric livers repopulated with mouse (mFRGN) or human (hFRGN) hepatocytes. In single-pass perfusion studies (2.5 ml/min), outflow dilution profiles of noneliminated reference indicators ((51)Cr-RBC, (125)I-albumin, (14)C-sucrose, and (3)H-water) revealed preservation of flow-limited distribution and reduced water and albumin spaces in hFRGN livers compared with FRGN livers, a view supported microscopically by tightly packed sinusoids. With prograde and retrograde perfusion of harmol (50 µM) in FRGN livers, an anterior sulfation (Sult1a1) over the posterior distribution of glucuronidation (Ugt1a1) activity was preserved, evidenced by the 42% lower sulfation-to-glucuronidation ratio (HS/HG) and 14% higher harmol extraction ratio (E) upon switching from prograde to retrograde flow. By contrast, zonation was lost in mFRGN and hFRGN livers, with HS/HG and E for both flows remaining unchanged. Remnant mouse genes persisted in hFRGN livers (10%-300% those of FRGN). When hFRGN livers were compared with human liver tissue, higher UGT1A1 and MRP2, lower MRP3, and unchanged SULT1A1 and MRP4 mRNA expression were observed. Total Sult1a1/SULT1A1 protein expression in hFRGN livers was higher than that of FRGN livers, consistent with higher harmol sulfate formation. The composite data on humanized livers suggest a loss of zonation, lack of complete liver humanization, and persistence of murine hepatocyte activities leading to higher sulfation.

(13)C MRS and LC-MS Flux Analysis of Tumor Intermediary Metabolism.

We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate–malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to 13C magnetic resonance spectroscopy (13C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable 13C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of mammalian target of rapamycin (mTOR) signaling produced changes in flux through the glycolytic, pentose shunt, and TCA cycle pathways that were evident within 8 h of treatment and increased at 24 and 48 h. Lactate was demonstrated to be a suitable biomarker of mTOR inhibition that could readily be monitored by 1H MRS and perhaps also by FDG-PET and hyperpolarized 13C MRS methods.

Conception, realization and qualification of a radioactive clean room lab facility dedicated to the synthesis of radiolabeled API for human ADME studies.

The human absorption, distribution, metabolism and elimination study administering radiolabeled drugs to human volunteers is an important clinical study in the development program of new drug candidates. The manufacture of radiolabeled Active Pharmaceutical Ingredients is covered by national drug laws and may come within the scope of regulatory GMP requirements. Additionally, authorities may request an appropriate environmental zoning to minimize the risk of microbiological contaminations particularly during the synthesis of radiolabeled Active Pharmaceutical Ingredients intended for parenteral application. Thus, a radioactive clean room lab facility in line with both GMP and radiation safety regulations was installed and the environmental zoning validated by appropriate testing of technical parameters and microbial and particle monitoring. The considerations detailed in this paper cover only GMP aspects related to the synthesis of radioactive drug substance. The subsequent, final formulation step in the overall process for manufacturing of radioactive drug product for any kind of administration is not within the scope of this paper. Under these qualified and controlled environmental conditions, we are now in a position to provide radiolabeled drug substances for all kinds of drug administration including both po and iv.