Metabolite profiling of guanfacine in plasma and urine of healthy Japanese subjects after oral administration of guanfacine extended-release tablets.

Guanfacine is used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), metabolite profiling of guanfacine was performed in plasma and urine collected from healthy Japanese adults following repeated oral administration of guanfacine extended-release formulation. Unchanged guanfacine was the most abundant component in both plasma and urine (from the MS signal intensity). In plasma, the M3 metabolite (a sulfate of hydroxy-guanfacine) was the prominent metabolite; the M2 metabolite (a glucuronide of a metabolite formed by monooxidation of guanfacine), 3-hydroxyguanfacine and several types of glucuronide at different positions on guanfacine were also detected. In urine, the M2 metabolite and 3-hydroxyguanfacine were the principal metabolites. From metabolite analysis, the proposed main metabolic pathway of guanfacine is monooxidation on the dichlorobenzyl moiety, followed by glucuronidation or sulfation. A minor pathway is glucuronidation at different positions on guanfacine. As the prominent metabolites in plasma were glucuronide and sulfate of hydroxyguanfacine, which have no associated toxicity concerns, further toxicity studies of the metabolites, for example in animals, were not deemed necessary.

Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.

Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive "reverse" screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108). Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations.

Absorption, Distribution, Metabolism, and Excretion of the Androgen Receptor Inhibitor Enzalutamide in Rats and Dogs.

BACKGROUND AND OBJECTIVES: Enzalutamide is an androgen receptor inhibitor that has been approved in several countries. Absorption, distribution, metabolism, and excretion (ADME) data in animals would facilitate understanding of the efficacy and safety profiles of enzalutamide, but little information has been reported in public. The purpose of this study was to clarify the missing ADME profile in animals. METHODS: ADME of 14C-enzalutamide after oral administration as Labrasol solution were investigated in non-fasted male Sprague-Dawley rats and beagle dogs. RESULTS: Plasma concentrations of 14C-enzalutamide peaked in rats and dogs at 6-8 h after a single oral administration. In most tissues, radioactivity concentration peaked at 4 h after administration. Excluding the gastrointestinal tract, tissues with the highest concentration of radioactivity were liver, fat, and adrenal glands. The tissue concentrations of radioactivity declined below the limit of quantitation or <0.89 % of maximum concentration by 168 h post-dose. Two known metabolites (M1 and M2) and at least 15 novel possible metabolites were detected in this study. M1 was the most abundant metabolite in both rats and dogs. Unchanged drug was a minor component in excreta. In intact rats, the mean urinary and fecal excretion of radioactivity accounted for 44.20 and 49.80 % of administered radioactivity, respectively. In intact dogs, mean urinary and fecal excretion was 62.00 and 22.30 % of the administered radioactivity, respectively. CONCLUSIONS: Rapid oral absorption was observed in rats and dogs when 14C-enzalutamide was administered as Labrasol solution. Tissue distribution in rats was clarified. The elimination of enzalutamide is mediated primarily by metabolism. Species differences were observed in excretion route.

Formation of the accumulative human metabolite and human-specific glutathione conjugate of diclofenac in TK-NOG chimeric mice with humanized livers.

3'-Hydroxy-4'-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathione-conjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t1/2) of 2.4 hours. 4'-Hydroxydiclofenac (II) and 3'-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (2'-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (4'-hydroxy-3'-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (2'-hydroxy-3'-(glutathion-S-yl)monoclofenac) or M5 (4'-hydroxy-2'-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine-type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans.

New frontiers-accelerator mass spectrometry (AMS): Recommendation for best practices and harmonization from Global Bioanalysis Consortium Harmonization Team.

The technique of accelerator mass spectrometry (AMS) is applicable to the analysis of a wide range of trace elemental isotopes. However, in the context of the pharmaceutical industry, it is invariably used to measure radiocarbon ((14)C). There are two broad modes of application: analysis of total (14)C sometimes termed "direct AMS" and analysis of specific (14)C-labelled analytes in a variety of matrices following some method of isolation. It is the latter application which is within the remit of the GBC team, and the team has made efforts to propose harmonized recommendations for the validation of AMS when used in a regulatory bioanalytical mode, i.e. the quantification of specific analyte(s) using liquid chromatography with off-line detection by AMS now known as "LC + AMS". The GBC team has reached a position where they have agreed to many aspects, but also differ on some aspects of what constitutes a bioanalytical assay validation in support of clinical studies using this technology. The detail of most of this will be covered under separate publication(s), but for the purposes of this paper, we have outlined the points of consensus. The purpose of this article is not to provide a roadmap for validation of LC + AMS assays, but to highlight agreements amongst the industry representative experts and the practitioners, as well as identifying specific areas essential for establishing assay quality but where additional discussion is required to reach agreement.

Microdose pharmacogenetic study of ¹4C-tolbutamide in healthy subjects with accelerator mass spectrometry to examine the effects of CYP2C9∗3 on its pharmacokinetics and metabolism.

Microdose study enables us to understand the pharmacokinetic profiles of drugs in humans prior to the conventional clinical trials. The advantage of microdose study is that the unexpected pharmacological/toxicological effects of drugs caused by drug interactions or genetic polymorphisms of metabolic enzymes/transporters can be avoided due to the limited dose. With a combination use of accelerator mass spectrometry (AMS) and (14)C-labaled compounds, the pharmacokinetics of both parent drug and its metabolites can be sensitively monitored. Thus, to demonstrate the usability of microdose study with AMS for the prediction of the impact of genetic polymorphisms of CYP enzyme on the pharmacokinetics of unchanged drugs and metabolites, we performed microdose pharmacogenetic study using tolbutamide as a CYP2C9 probe drug. A microdose of (14)C-tolbutamide (100 µg) was administered orally to healthy volunteers with the CYP2C9(∗)1/(∗)1 or CYP2C9(∗)1/(∗)3 diplotype. Area under the plasma concentration-time curve for the (14)C-radioactivity, determined by AMS, or that for the parent drug, determined by liquid chromatography/mass spectrometry, was about 1.6 times or 1.7 times greater in the CYP2C9(∗)1/(∗)3 than in the CYP2C9(∗)1/(∗)1 group, which was comparable to the previous reports at therapeutic dose. In the plasma and urine, tolbutamide, carboxytolbutamide, and 4-hydroxytolbutamide were detected and practically no other metabolites could be found in both diplotype groups. The fraction of metabolites in plasma radioactivity was slightly lower in the CYP2C9(∗)1/(∗)3 group. Microdose study can be used for the prediction of the effects of genetic polymorphisms of enzymes on the pharmacokinetics and metabolic profiles of drugs with minimal care of their pharmacological/toxicological effects.

Comprehensive quantitative and qualitative liquid chromatography-radioisotope-mass spectrometry analysis for safety testing of tolbutamide metabolites without standard samples.

Liquid chromatography-radioisotope-mass spectrometry (LC-RI-MS) analysis was used to determine the structures of 12 (four previously unknown) (14) C-tolbutamide (TB) metabolites in rat biological samples (plasma, urine, bile, feces, and microsomes). The four novel metabolites are ω-carboxy TB, hydroxyl TB (HTB)-O-glucuronide, TB-ortho or meta-glutathion, and tolylsulphoaminocarbo-glutathion. In rat plasma, after oral administration of (14) C-TB at therapeutic dose (1 mg/kg) and microdose (1.67 µg/kg), the total RI and six metabolites [HTB, carboxy TB (CTB), M1: desbutyl TB, M2: ω-hydroxyl TB, M3: α-hydroxyl TB, and M4: ω-1-hydroxyl TB] were quantified by LC-RI-MS. The plasma concentrations were calculated using their response factors (MS-RI intensity ratio) without standard samples, and the area under the curve (AUC) of plasma concentration per time for evaluation of Safety Testing of Drug Metabolites (MIST) was calculated using the ratio of TB metabolites AUC/total RI AUC. The ratios were as follows: TB 94.5% and HTB 2.5% for the microdose (1.67 µg/kg) and TB 95.6%, HTB 0.96%, CTB 0.065%, M1 0.62%, M2 0.0035%, M3 0.077%, and M4 0.015% for the therapeutic dose (1 mg/kg). These values were less than 10% of the MIST criteria.

Absolute stereochemistry and antitumor activity of iejimalides.

The absolute configurations at five chiral centers, except for C-32(S) reported previously, in iejimalides A, C, and D, potent cytotoxic 24-membered macrolides isolated from a tunicate Eudistoma cf. rigida, were assigned as 4R, 9S, 17S, 22S, and 23S on the basis of detailed analysis of NMR data and chemical means. Furthermore, the structures proposed for iejimalides A, C, and D were revised to their 13Z-isomers. Iejimalides A-D (1-4) exhibited antitumor activity in vivo.

Solution conformations of amphidinolide H.

Solution conformations of amphidinolide H (1), a 26-membered macrolide exhibiting potent cytotoxic and antitumor activity, in CDCl3 and DMSO-d6 were investigated on the basis of NMR data, distance geometry calculation, and restrained energy minimization. Three-dimensional conformations in CDCl3 were suggested to be close to the X-ray structure of 1, while those in DMSO-d6 were indicated to be different from both those in CDCl3 and the X-ray structure.

Rigidins B-D, new pyrrolopyrimidine alkaloids from a tunicate Cystodytes species.

Three new pyrrolopyrimidine alkaloids, rigidins B-D (1-3), have been isolated from an Okinawan marine tunicate Cystodytes sp., and the structures were elucidated on the basis of spectroscopic data.