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Drug-amino acid co-amorphous systems have become increasingly well-investigated systems to improve dissolution rate of poorly water-soluble drugs. In this study, dipeptides were investigated as co-formers for co-amorphous systems based on the hypothesis that dipeptides might combine the inherent properties of the two included amino acids. Co-amorphization of the model drug mebendazole was investigated with five dipeptides, tryptophan-phenylalanine, phenylalanine-tryptophan, aspartic acid-tyrosine, histidine-glycine and proline-tryptophan. The dipeptides were chosen to investigate whether the side chains (nonpolar, polar, basic and acidic), and the sequence of amino acids (tryptophan-phenylalanine versus phenylalanine-tryptophan) have an influence on the performance of dipeptides as co-formers. All mebendazole-dipeptide systems became amorphous after ball milling for only 30 min, while this generally was not the case for the single amino acids or physical mixtures of the amino acids forming the dipeptides. Dissolution studies showed that the dissolution rate of mebendazole from most co-amorphous systems was increased significantly compared to crystalline and amorphous mebendazole. However, no clear trend for the drug dissolution enhancement was observed within the different co-amorphous drug-dipeptide systems. The stability study revealed that co-amorphous mebendazole-dipeptide systems showed higher physical stability compared to amorphous mebendazole. In conclusion, dipeptides are shown to be promising co-formers for co-amorphous systems.

A series of Gs protein peptidomimetics were designed and synthesised based on the published X-ray crystal structure of the active state β2-Adrenergic receptor (β2AR) in complex with the Gs protein (PDB 3SN6). We hypothesised that such peptidomimetics may function as allosteric modulators that target the intracellular Gs protein binding site of the β2AR. Peptidomimetics were designed to mimic the 15 residue C-Terminal α-helix of the Gs protein and were pre-organised in a helical conformation by (i, i + 4)-stapling using copper catalysed azide alkyne cycloaddition. Linear and stapled peptidomimetics were analysed by circular dichroism (CD) and characterised in a membrane-based cAMP accumulation assay and in a bimane fluorescence assay on purified β2AR. Several peptidomimetics inhibited agonist isoproterenol (ISO) induced cAMP formation by lowering the ISO maximal efficacy up to 61%. Moreover, some peptidomimetics were found to significantly decrease the potency of ISO up to 39-fold. In the bimane fluorescence assay none of the tested peptidomimetics could stabilise an active-like conformation of β2AR. Overall, the obtained pharmacological data suggest that some of the peptidomimetics may be able to compete with the native Gs protein for the intracellular binding site to block ISO-induced cAMP formation, but are unable to stabilise an active-like receptor conformation.

GPR139 is an orphan G protein-coupled receptor that is expressed primarily in the brain. Not much is known regarding the function of GPR139. Recently we have shown that GPR139 is activated by the amino acids l-tryptophan and l-phenylalanine (EC50 values of 220 μM and 320 μM, respectively), as well as di-peptides comprised of aromatic amino acids. This led us to hypothesize that GPR139 may be activated by peptides. Sequence alignment of the binding cavities of all class A GPCRs, revealed that the binding pocket of the melanocortin 4 receptor is similar to that of GPR139. Based on the chemogenomics principle "similar targets bind similar ligands”, we tested three known endogenous melanocortin 4 receptor agonists; adrenocorticotropic hormone (ACTH) and α- and β-melanocyte stimulating hormone (α-MSH and β-MSH) on CHO-k1 cells stably expressing the human GPR139 in a Fluo-4 Ca2+-assay. All three peptides, as well as their conserved core motif HFRW, were found to activate GPR139 in the low μM range. Moreover, we found that peptides consisting of nine or ten N-terminal residues of α-MSH activate GPR139 in the submicromolar range. α-MSH1-9 was found to correspond to the product of a predicted cleavage site in the pre-pro-protein pro-opiomelanocortin (POMC). Our results demonstrate that GPR139 is a peptide receptor, activated by ACTH, α-MSH, β-MSH, the conserved core motif HFRW as well as a potential endogenous peptide α-MSH1-9. Further studies are needed to determine the functional relevance of GPR139 mediated signaling by these peptides., GPR139 is an orphan G protein-coupled receptor that is expressed primarily in the brain. Not much is known regarding the function of GPR139. Recently we have shown that GPR139 is activated by the amino acids l-tryptophan and l-phenylalanine (EC50 values of 220 μM and 320 μM, respectively), as well as di-peptides comprised of aromatic amino acids. This led us to hypothesize that GPR139 may be activated by peptides. Sequence alignment of the binding cavities of all class A GPCRs, revealed that the binding pocket of the melanocortin 4 receptor is similar to that of GPR139. Based on the chemogenomics principle "similar targets bind similar ligands", we tested three known endogenous melanocortin 4 receptor agonists; adrenocorticotropic hormone (ACTH) and α- and β-melanocyte stimulating hormone (α-MSH and β-MSH) on CHO-k1 cells stably expressing the human GPR139 in a Fluo-4 Ca(2+)-assay. All three peptides, as well as their conserved core motif HFRW, were found to activate GPR139 in the low μM range. Moreover, we found that peptides consisting of nine or ten N-terminal residues of α-MSH activate GPR139 in the submicromolar range. α-MSH1-9 was found to correspond to the product of a predicted cleavage site in the pre-pro-protein pro-opiomelanocortin (POMC). Our results demonstrate that GPR139 is a peptide receptor, activated by ACTH, α-MSH, β-MSH, the conserved core motif HFRW as well as a potential endogenous peptide α-MSH1-9. Further studies are needed to determine the functional relevance of GPR139 mediated signaling by these peptides.

Aims: Because of its short detection window, uncovering the intake of gammahydroxybutyric acid (GHB) still constitutes a problem. Aim of the experiments was to develop and evaluate new tools for a possible extension of the detection window after the intake of GHB. Methods: Blood, plasma and urine samples (each n=49) of volunteers and of patients (n=3, patient 1 and 2 chronical intake, patient 3 single intake) therapeutically taking up to 4.5 g GHB (Xyrem®) per night were collected at different points in time after the intake up to 72 h. Additionally, hair samples of the patients were taken. Concentration profiles of GHB (high-performance liquid chromatography-tandem mass spectrometry), GHB-β- glucuronide and GHB-4-sulfate (high-performance liquid chromatography-quadrupole timeof- flight-mass spectrometry) in plasma and urine were recorded over time. Hair samples were analyzed by a validated LC-MS/MS method for GHB and GHB-β-glucuronide. Alterations in gene expression of ALDH5A1, AKR7A2, EREG and PEA15 in blood, genes of interest which code for enzymes involved in GHB metabolism, was investigated via quantitative PCR using an empirically derived normalization strategy. Furthermore, possible discrimination of endogenous from exogenous GHB in urine via isotope ratio MS was tested. Results and discussion: The parent compound could be quantified above the usual cut-offs for 4-6 hours both in blood and urine. No discrimination endogenous/exogenous by neither using the phase II metabolites of GHB nor using the expression of the genes of interest was possible. In the hair samples of patients GHB and its glucuronide could not be determined in concentrations higher than the control group. A discrimination endogenous/exogenous GHB was only possible using isotope ratio mass spectrometry, however carbon isotope ratios in urine did not differ longer than GHB was detectable above the cut-off limit.

Gamma-hydroxybutyric acid (GHB) is used as a so-called knock-out drug in drug-facilitated sexual assault. Due to GHB's short detection window in blood and urine, crime verification remains problematic in many cases. Phase II metabolites can enhance the detectability and prolong the detection window of some lower-molecular-mass molecules, as exemplified by ethanol. GHB-beta-O-glucuronide (GHB-Gluc) was recently introduced as one phase II metabolite, and GHB-4-sulfate (GHB-Sulf) was also postulated to represent a urinary metabolite of exogenous GHB. To unambiguously verify the presence of GHB-Sulf in urine, the reference standard material of GHB-Sulf was synthesized, together with its deuterated analogue, GHB-Sulf-d (6). This reference standard material, together with GHB-Gluc and GHB-Gluc-d (4), was employed to develop and validate a quantitative method for determining both target analytes in plasma and urine samples. Compounds were separated by high-performance liquid chromatography on a Hypercarb column and detected by electrospray ionization time-of-flight-mass spectrometry. Calibration curves were linear over the entire calibration range up to 20 mg/L, and quantification limits of < 0.01 mg/L were achieved. Accuracy and interday and intraday precision fulfilled all forensic guidelines criteria. In plasma, only GHB-Gluc was detected, while both metabolites were clearly identified in urine, even in real human specimens. Stability was investigated under different temperature conditions for up to 4 weeks, and both phase II metabolites were found to be stable under refrigerated conditions. This method proved to be suitable for this purpose, and allowed for the first time the analysis and quantification of two different GHB metabolites from plasma and urine samples.

INTRODUCTION: The label-free dynamic mass redistribution-based assay (DMR) is a powerful method for studying signalling pathways of G protein-coupled receptors (GPCRs). Herein we present the label-free DMR assay as a robust readout for pharmacological characterization of formyl peptide receptors (FPRs) in human neutrophils.METHODS: Neutrophils were isolated from fresh human blood and their responses to FPR1 and FPR2 agonists, i.e. compound 43, fMLF and WKYMVm were measured in a label-free DMR assay using Epic Benchtop System from Corning®. Obtained DMR traces were used to calculate agonist potencies.RESULTS: The potencies (pEC50) of fMLF, WKYMVm and compound 43, determined on human neutrophils using the label-free DMR assay were 8.63, 7.76 and 5.92, respectively. The DMR response to fMLF, but not WKYMVm and compound 43 could be blocked by the FPR1-specific antagonist cyclosporin H.DISCUSSION: We conclude that the DMR assay can be used, and complements more traditional methods, to study the signalling and pharmacology of endogenous FPR receptors in human neutrophils.

Gamma-hydroxybutyrate (GHB) is a drug of abuse, an approved therapeutic for narcolepsy, an agent employed for facilitation of sexual assault, as well as a biomarker of succinic semialdehyde dehydrogenase deficiency (SSADHD). Our laboratory seeks to identify surrogate biomarkers in SSADHD that can shed light on the developmental course of this neurometabolic disease. Since GHB may be quantified in hair as a potential surrogate to identify victims of drug-related assault, we have opted to examine its level in SSADHD. We quantified GHB in hair derived from ten patients with SSADHD, and documented a significant negative age correlation. These findings are consistent with recent results in patient biological fluids, including plasma and red blood cells. These findings may provide additional insight into the developmental course of SSADHD (Jansen et al., J Inherit Metab Dis 39:795-800, 2016).

The introduction of macrocyclic constraints in peptides (peptide stapling) is an important tool within peptide medicinal chemistry for stabilising and pre-organising peptides in a desired conformation. In recent years, the copper-catalysed azide-alkyne cycloaddition (CuAAC) has emerged as a powerful method for peptide stapling. However, to date CuAAC stapling has not provided a simple method for obtaining peptides that are easily diversified further. In the present study, we report a new diversity-oriented peptide stapling (DOPS) methodology based on CuAAC chemistry. Stapling of peptides incorporating two azide-modified amino acids with 1,3,5-triethynylbenzene efficiently provides (i, i+7)- and (i, i+9)-stapled peptides with a single free alkyne positioned on the staple, that can be further conjugated or dimerised. A unique feature of the present method is that it provides easy access to radiolabelled stapled peptides by catalytic tritiation of the alkyne positioned on the staple.

Among the new psychoactive substances (NPS) that have recently emerged on the market, many of the new synthetic opioids have shown to be particularly harmful. A new synthetic analogue of fentanyl, N-phenyl-N-[1-(2-phenethyl)piperidin-4-yl]prop-2-enamide (acrylfentanyl), was identified in powder from a seized capsule found at a forensic psychiatric ward in Denmark. Gas chromatography with mass spectrometry (GC-MS) identified a precursor to synthetic fentanyls, N-phenyl-1-(2-phenylethyl)piperidin-4-amine; however, the precursor 1-(2-phenethyl)piperidin-4-on, was not detected. Analysis of the electron impact mass spectrum of the main, unknown chromatographic peak (GC) tentatively identified an acryloyl analogue of fentanyl. Further analyses by quadrupole time-of-flight high resolution mass spectrometry (QTOF-MS), matrix-assisted laser ionisation orbitrap mass spectrometry (MALDI-Orbitrap-MS), nuclear magnetic resonance spectroscopy (NMR) and infra-red spectroscopy (IR) confirmed the presence of acrylfentanyl (also known as acryloylfentanyl). Quantitative analysis with liquid chromatography and triple quadrupole mass spectrometry (LC-MS/MS) determined the content of acrylfentanyl in the powder, equal to 88.3 mass-% acrylfentanyl hydrochloride. An impurity observed by NMR was identified as triethylamine hydrochloride. Acrylfentanyl is sold on the Internet as a 'research chemical'. Like other synthetic fentanyls, such as acetylfentanyl, it poses a serious risk of fatal intoxication.

Compound 1 is a selective and potent agonist of the G protein-coupled receptor GPR139 (EC50 = 39 nM). In this study, we describe the synthesis, radiolabelling and in vitro evaluation of [3H]-1 for the characterisation of GPR139 and its spatial expression in the brain using autoradiography. Two different synthesis routes for the radiolabelling of 1 based on a reductive debromination strategy were investigated using deuterium (D2, g). The route based on reductive debromination of the bromonaphthyl precursor 5 proved superior over arylbromide 4 and was employed for the radiolabelling experiments. Reductive debromination of precursor 5 was accomplished using 3H2, Pd/C and triethylamine in DMF at ambient temperature to give target molecule [3H]-1 with a specific activity of 19.3 Ci mmol-1 and a radiochemical purity of ≥95%. By application of autoradiography and binding studies, it was not possible to discriminate [3H]-1 binding to wildtype mice brains from GPR139 knockout mice brains and total binding from non-specific binding in CHO-k1 cells stably expressing human GPR139 receptor. Based on these experiments we conclude that [3H]-1 is not a suitable radioligand for the characterisation of GPR139.

GPR132 is an orphan class A G protein-coupled receptor. It has been proposed to be activated by protons and to regulate apoptosis, atherosclerosis and inflammation, but these results are still preliminary. In the current work, we designed and screened a focused compound library using a β-arrestin recruitment assay, and thereby identified the first disclosed surrogate GPR132 agonist 1 with a potency of 3.4 μM. This constitutes the first available pharmacological tool for the in vitro characterization of the orphan receptor GPR132. The testing of 32 analogs furthermore identified a number of compounds with lower activity – of which six were agonists and two were antagonists – that were used to construct preliminary structure–activity relationships. Docking followed by a molecular dynamics simulation of compound 1 in a structural model of GPR132 displayed the putative interactions for the key ligand functionalities.

Privileged structures such as 2-arylindoles are recurrent molecular scaffolds in bioactive molecules. We here present an operationally simple, high yielding and scalable method for regioselective 3-acylation of 2-substituted indoles under basic conditions using functionalized acid chlorides. The method shows good tolerance to both electron-withdrawing and donating substituents on the indole scaffold and gives ready access to a variety of functionalized 3-acylindole building blocks suited for further derivatization.