Aim:Although regulatory guidances require human metabolism information of drug candidates early in the development process, the human mass balance study (or hADME study), is performed relatively late. hADME studies typically involve the administration of a 14 C-radiolabelled drug where biological samples are measured by conventional scintillation counting analysis. Another approach is the administration of therapeutic doses containing a 14 C-microtracer followed by accelerator mass spectrometry (AMS) analysis, enabling hADME studies completion much earlier. Consequently, there is an opportunity to change the current drug development paradigm. Materials & methods: To evaluate the applicability of the MICADAScAMS method, we successfully performed: the validation of MICADAS-cAMS for radioactivity quantification in biomatrices and, a rat ADME study, where the conventional methodology was assessed against a microtracer MICADAS-cAMS approach. Results & discussion: Combustion AMS (cAMS) technology is applicable to microtracer studies. A favorable opinion from EMA to complete the hADME in a Phase I setting was received, opening the possibilities to change drug development. Keywords:In the scope of drug development, regulatory guidances encourage the early identification of relevant human metabolites [1][2][3]. A human ADME study that used radiolabeled drugs allows the identification of metabolites and the elucidation of key biotransformation pathways and clearance mechanisms in humans. However, due to the high study cost, the needs for a dosimetry assessment (which include a rat distribution investigation) and for a GMP manufacturing as well as the high attrition rates encountered in drug development, the conventional hADME is generally performed late in drug development (Phase II, see Supplementary Figure 1). The administered dose in a conventional hADME study can contain up to 3.7 mBq of radioactivity which is analysed by scintillation counting. To address the regulatory demands earlier, a first investigation of pharmacokinetics (PK) and excretion routes is typically performed with a radiolabeled drug in rodents. Subsequently, exploratory analysis of potentially relevant metabolites is investigated in early clinical samples (Phase I) without the use of radiolabel [4,5] (Supplementary Figure 1). However, the applied LC-MS methods can fail to identify unknown metabolites. If human-specific metabolites are generated in early clinical samples, these metabolites will not be detected in preclinical species using radiolabeled drug, and will not be discovered in humans until the radiolabeled human mass balance ADME study is completed. To address this gap, we propose to dose a normal, therapeutically relevant dose spiked with very low amounts of a radiotracer to healthy volunteers or patients in a Phase I setting. Thereafter, the mass balance, the excretion routes and levels of circulating metabolites in humans are determined by accelerator mass spectrometry (AMS) as suggested by Lappin and Garner [6,7].AMS is a highly sensit...

Polycomb Repressive Complex 2 (PRC2) plays an important role in transcriptional regulation during animal development and in cell differentiation, and alteration of PRC2 activity has been associated with cancer. On a molecular level, PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), resulting in mono-, di-, or trimethylated forms of H3K27, of which the trimethylated form H3K27me3 leads to transcriptional repression of polycomb target genes. Previously, we have shown that binding of the lowmolecular-weight compound EED226 to the H3K27me3 binding pocket of the regulatory subunit EED can effectively inhibit PRC2 activity in cells and reduce tumor growth in mouse xenograft models. Here, we report the stepwise optimization of the tool compound EED226 toward the potent and selective EED inhibitor MAK683 (compound 22) and its subsequent preclinical characterization. Based on a balanced PK/PD profile, efficacy, and mitigated risk of forming reactive metabolites, MAK683 has been selected for clinical development.

Fevipiprant is a novel oral prostaglandin D receptor 2 (DP; also known as CRTh2) antagonist, which is currently in development for the treatment of severe asthma and atopic dermatitis. We investigated the absorption, distribution, metabolism, and excretion properties of fevipiprant in healthy subjects after a single 200-mg oral dose of [C]-radiolabeled fevipiprant. Fevipiprant and metabolites were analyzed by liquid chromatography coupled to tandem mass spectrometry and radioactivity measurements, and mechanistic in vitro studies were performed to investigate clearance pathways and covalent plasma protein binding. Biotransformation of fevipiprant involved predominantly an inactive acyl glucuronide (AG) metabolite, which was detected in plasma and excreta, representing 28% of excreted drug-related material. The AG metabolite was found to covalently bind to human plasma proteins, likely albumin; however, in vitro covalent binding to liver protein was negligible. Excretion was predominantly as unchanged fevipiprant in urine and feces, indicating clearance by renal and possibly biliary excretion. Fevipiprant was found to be a substrate of transporters organic anion transporter 3 (OAT3; renal uptake), multidrug resistance gene 1 (MDR1; possible biliary excretion), and organic anion-transporting polypeptide 1B3 (OATP1B3; hepatic uptake). Elimination of fevipiprant occurs via glucuronidation by several uridine 5'-diphospho glucuronosyltransferase (UGT) enzymes as well as direct excretion. These parallel elimination pathways result in a low risk of major drug-drug interactions or pharmacogenetic/ethnic variability for this compound.

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Neonatal dopamine (DA) depletion produces learning impairments both during development and throughout adulthood in the rat. The present experiment further investigated the memory capabilities of the dopamine-depleted rat by assessing performance in the radial arm maze. Results showed that, following neonatal injection of 6-hydroxydopamine and desmethylimipramine, lesioned rats per-performed more accurately than controls. In this paradigm, DA-depleted rats tended to enter each arm to obtain a food pellet and not enter unbaited, incorrect arms. The difference in performance of control and treated rats could not be accounted for by differences in locomotor activity, body weights, or motivational factors. A computer analysis of the data revealed that DA-depleted animals adopted a strategy of choosing adjacent arms consecutively, which probably accounted for their superior performance. Results are discussed in terms using algorithms versus extra-maze cues to complete the maze following early brain injury.