Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington’s disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

Peptide hormones represent an emerging class of potential doping agents. Detection of their misuse is difficult due to their short half-life in plasma and rapid elimination. Therefore, investigating their metabolism can improve detectability. Unfortunately, pharmacokinetic studies with human volunteers are often not allowed because of ethical constraints, and therefore alternative models are needed. This study was performed in order to evaluate in vitro models (human liver microsomes and S9 fraction) for the prediction of the metabolism of peptidic doping agents and to compare them with the established models. The peptides that were investigated include desmopressin, TB-500, GHRP-2, GHRP-6, hexarelin, LHRH and leuprolide. Several metabolites were detected for each peptide after incubation with human liver microsomes, S9 fraction, and serum, which all showed endopeptidase and exopeptidase activity. In vitro models from different organs (liver vs. kidney) were compared, but no significant differences were recorded. Deamidation was not observed in any of the models and was therefore evaluated by incubation with α-chymotrypsin. In conclusion, in vitro models are useful tools for forensic and clinical analysts to detect peptidic metabolic markers in biological fluids.

Cryptophycins are potent tubulin polymerization inhibitors with picomolar antiproliferative potency in vitro and activity against multidrug-resistant (MDR) cancer cells. Because of neurotoxic side effects and limited efficacy in vivo, cryptophycin-52 failed as a clinical candidate in cancer treatment. However, this class of compounds has emerged as attractive payloads for tumor-targeting applications. In this study, cryptophycin was conjugated to the cyclopeptide c(RGDfK), targeting integrin αvβ3, across the protease-cleavable Val-Cit linker and two different self-immolative spacers. Plasma metabolic stability studies in vitro showed that our selected payload displays an improved stability compared to the parent compound, while the stability of the conjugates is strongly influenced by the self-immolative moiety. Cathepsin B cleavage assays revealed that modifications in the linker lead to different drug release profiles. Antiproliferative effects of Arg-Gly-Asp (RGD)–cryptophycin conjugates were evaluated on M21 and M21-L human melanoma cell lines. The low nanomolar in vitro activity of the novel conjugates was associated with inferior selectivity for cell lines with different integrin αvβ3 expression levels. To elucidate the drug delivery process, cryptophycin was replaced by an infrared dye and the obtained conjugates were studied by confocal microscopy.

The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short halflife and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with subnanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).

The metabolite formed by consecutive morpholine cleavage and oxidation of the remaining side chain to a carboxylic group was detected in the highest amounts with the longest detection time. Therefore, it is the best candidate metabolite to detect JWH-200 abuse in urine.

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The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo [4,5]imidazo[1,2a]pyrimidine series that show selective binding to mHTT aggregates over Aβ-and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [ 11 C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.

Qualitative identification of a MGF C-terminal amidated analogue in two black market products was successfully achieved. This report demonstrates that illegal MGF preparations are commercially available for use as doping agent in sports.