Butyrylcholinesterase (BChE) inhibitors are or could be used for the treatment of Alzheimer's disease, canine cognitive dysfunction, depression, multiple sclerosis, heroin abuse and metabolic disorders. Monoamine oxidase (MAO) inhibitors are or could be used for the treatment of depression, anxiety, Alzheimer's disease, Parkinson's disease, cancer, cardiovascular disease and chronic inflammatory diseases. We have designed, synthesized, and evaluated ten new N-propargylpyrrolidine-based inhibitors of these enzymes. Sulfonamide 10 is the most potent human (h)BChE (IC50 = 0.203 μM) of the series, and secondary carboxamide 1 is a time-dependent and irreversible inhibitor of hMAO-A (IC50 = 6.42 μM) and hMAO-B (IC50 = 7.83 μM). The X-ray crystal structures of carboxamide 4 [IC50(hBChE) = 3.89 μM] and sulfonamide 10 with hBChE confirmed our previous observation that carboxamides and sulfonamides have distinct binding poses in the active site of hBChE. The X-ray crystal structure of the complex of pyrrolidine 4 with hBChE also revealed a distinct binding pose compared to its direct piperidine analogue (PDB code 5LKR). Furthermore, compounds 1 and 10 should be able to cross the blood-brain barrier, exhibit low cytotoxicity (>50 μM) in two cell lines and protect against amyloid β1-42-induced neuronal cell death.

Butyrylcholinesterase (BChE) is a promising drug target for alleviating the symptoms of canine cognitive dysfunction (CCD) and Alzheimer's disease (AD). We have recently developed lead compound 2, a racemic, nanomolar BChE inhibitor with procognitive effects in mice with scopolamine-induced AD-like symptoms and dogs suffering from CCD. To overcome its modest brain exposure, we developed compound (R)-(-)-3, a more potent BChE inhibitor with a 7-fold higher in vivo brain exposure. It has procognitive effects in mice with scopolamine-induced AD-like symptoms and, superior to compound 2, also in mice with Aβ1-42-induced AD-like symptoms. Compound (R)-(-)-3 produces no cholinergic adverse effects or motor deficits and has no acute toxic effects in mice. This makes sulfonamide (R)-(-)-3 an optimized lead compound for alleviating the symptoms of AD.

BACKGROUND AND PURPOSE:Major depressive disorder (MDD) is often treatment resistant, particularly in addressing anhedonia and cognitive deficits. Novel pharmacological strategies are needed. While butyrylcholinesterase, ghrelin, and dopamine (DA) have been well studied in the context of stress and MDD, their interaction remains unclear. EXPERIMENTAL APPROACH:The dose-dependent antidepressant effects of a novel butyrylcholinesterase inhibitor (BChEI) were evaluated in the Flinders Sensitive Line (FSL) rat model of MDD. Behavioural assessments included the forced swim test (despair), sucrose preference test (reward-related), and novel object recognition test (cognition). Brain-derived neurotrophic factor (BDNF), acetylcholine (ACh), and brain monoamines were analysed, as well as serum growth hormone and acyl- and desacyl-ghrelin. To confirm the role of ghrelin, pharmacological exploration was undertaken using the ghrelin receptor antagonist, D-Lys-3-GHRP-6. KEY RESULTS:FSL rats had significantly lower ghrelin ratios, BDNF, ACh, DA and growth hormone levels. In FSL rats, both BChEI and escitalopram significantly reduced despair. BChEI significantly outperformed escitalopram in enhancing reward-related and cognitive behaviours. Biochemically, BChEI treatment significantly increased ghrelin ratios and brain DA levels without altering brain 5-HT, ACh or BDNF. D-Lys-3-GHRP-6 significantly reversed the antidepressant-like, rewarding, and pro-cognitive effects of BChEI, accompanied by significant reductions in BDNF and DA. CONCLUSIONS AND IMPLICATIONS:FSL rats display impaired ghrelin, DA, serotonin, growth hormone, and BDNF signalling, akin to MDD. BChEI exerts antidepressant-like effects across despair, reward, and cognitive domains, most likely via the BChE-ghrelin-DA cascade. Reversal of these effects by ghrelin antagonism underscores the critical role of ghrelin, specifically via growth hormone secretagogue receptor-ghrelin interaction. These findings suggest a potentially novel multimodal neurobiological target for the treatment of MDD.

We describe the development of quinolylnitrones（QNs） as multifunctional ligands inhibiting cholinesterases（ChEs: acetylcholinesterase and butyrylcholinesterase—h BChE） and monoamine oxidases（hMAO-A/B） for the therapy of neurodegenerative diseases. We identified QN 19, a simple, low molecular weight nitrone, that is readily synthesized from commercially available 8-hydroxyquinoline-2-carbaldehyde. Quinolylnitrone 19 has no typical pharmacophoric element to suggest ChE or MAO inhibition, yet unexpectedly showed potent inhibition of h BChE（IC50= 1.06 ± 0.31 nmol/L） and h MAO-B(IC 50 =4.46±0.18 μmol/L). The crystal structures of 19 with hBChE and hMAO-B provided the structural basis for potent binding, which was further studied by enzyme kinetics. Compound 19 acted as a free radical scavenger and biometal chelator, crossed the blood—brain barrier, was not cytotoxic, and showed neuroprotective properties in a 6-hydroxydopamine cell model of Parkinson’s disease. In addition, in vivo studies showed the anti-amnesic effect of 19 in the scopolamine-induced mouse model of AD without adverse effects on motoric function and coordination. Importantly, chronic treatment of double transgenic APPswe-PS1δE9 mice with 19 reduced amyloid plaque load in the hippocampus and cortex of female mice, underscoring the disease-modifying effect of QN 19.

Hydrazoic acid (HN 3 ) and its deprotonated form azide ion (N 3 − ) (AHA) are toxic because they inhibit the cytochrome c oxidase complex IV (CoX IV) embedded in the inner mitochondrial membrane that forms part of the enzyme complexes involved in cellular respiration. Critical to its toxicity is the inhibition of CoX IV in the central nervous system and cardiovascular system. Hydrazoic acid is an ionizable species and its affinity for membranes, and the associated permeabilities, depend on the pH values of aqueous media on both sides of the membranes. In this article, we address the permeability of AHA through the biological membrane. In order to understand the affinity of the membrane for the neutral and ionized form of azide, we measured the octanol/water partition coefficients at pH values of 2.0 and 8.0, which are 2.01 and 0.00034, respectively. Using a Parallel Artificial Membrane Permeability Assay (PAMPA) experiment, we measured the effective permeability through the membrane, which is logP e − 4.97 and − 5.26 for pH values of 7.4 and pH 8.0, respectively. Experimental permeability was used to validate theoretical permeability, which was estimated by numerically solving a Smoluchowski equation for AHA diffusion through the membrane. We demonstrated that the rate of permeation through the cell membrane of 8.46·10 4 s −1 is much higher than the rate of the chemical step of CoX IV inhibition by azide of 200 s −1 . The results of this study show that transport through the membrane does not represent the rate-limiting step and therefore does not control the rate of CoX IV inhibition in the mitochondria. However, the observed dynamics of azide poisoning is controlled by circulatory transport that takes place on a time scale of minutes.

The RPMI 2650 and Calu-3 cell lines have been previously evaluated as models of the nasal and airway epithelial barrier, and they have demonstrated the potential to be used in drug permeation studies. However, limited data exist on the utilization of these two cell models for the assessment of nasal formulations. In our study, we tested these cell lines for the evaluation of in vitro permeation of intranasally administered drugs having a local and systemic effect from different solution- and suspension-based formulations to observe how the effects of formulations reflect on the measured in vitro drug permeability. Both models were shown to be sufficiently discriminative and able to reveal the effect of formulation compositions on drug permeability, as they demonstrated differences in the in vitro drug permeation comparable to the in vivo bioavailability. Good correlation with the available bioavailability data was also established for a limited number of drugs formulated as intranasal solutions. The investigated cell lines can be applied to the evaluation of in vitro permeation of intranasally administered drugs with a local and systemic effect from solution- and suspension-based formulations.

Herein, we report the design of a focused library of novel bacterial topoisomerase inhibitors (NBTIs) based on innovative mainly monocyclic right-hand side fragments active against DNA gyrase and Topo IV. They exhibit a very potent and wide range of antibacterial activity, even against some of the most concerning hard-to-treat pathogens for which new antibacterials are urgently needed, as reported by the WHO and CDC. NBTIs enzyme activity and whole cell potency seems to depend on the fine-tuned lipophilicity/hydrophilicity ratio that governs the permeability of those compounds through the bacterial membranes. Lipophilicity of NBTIs is apparently optimal for passing through the membrane of Gram-positive bacteria, but the higher, although not excessive lipophilicity and suitable hydrophilicity seems to determine the passage through Gram-negative bacterial membranes. However, due to the considerable hERG inhibition, which is still at least two orders of magnitude away from MICs, continued optimization is required to realize their full potential.

The complex nature of neurodegenerative diseases (NDDs), such as Alzheimer's disease (AD) and Parkinson's disease (PD) calls for multidirectional treatment. Restoring neurotransmitter levels by combined inhibition of cholinesterases (ChEs) and monoamine oxidases (MAOs, MAO-A and MAO-B), in conjunction with strategies to counteract amyloid 13 (A13) aggregation, may constitute a therapeutically strong multi-target approach for the treatment of NDDs. Chalcones are a subgroup of flavonoids with a broad spectrum of biological activity. We report here the synthesis of 2 '-hydroxychalcones as MAO-A and MAO-B inhibitors. Compounds 5c (IC50 = 0.031 +/- 0.001 mu M), 5a (IC50 = 0.084 +/- 0.003 mu M), 2c (IC50 = 0.095 +/- 0.019 mu M) and 2a (IC50 = 0.111 +/- 0.006 mu M) were the most potent, selective and reversible inhibitors of human (h)MAO-B isoform. hMAO-B inhibitors 1a, 2a and 5a also inhibited murine MAO-B in vivo in mouse brain homogenates. Molecular modelling rationalised the binding mode of 2 '-hydroxychalcones in the active site of hMAO-B. Additionally, several derivatives inhibited murine acetylcholinesterase (mAChE) (IC50 values from 4.37 +/- 0.83 mu M to 15.17 +/- 6.03 mu M) and reduced the aggregation propensity of A13. Moreover, some derivatives bound to the benzodiazepine binding site (BDZ-bs) of the gamma-aminobutyric acid A (GABAA) receptors (1a and 2a with Ki = 4.9 +/- 1.1 mu M and 5.0 +/- 1.1 mu M, respectively), and exerted sedative and/or anxiolytic like effects on mice. The biological results reported here on 2 '-hydroxychalcones provide an extension to previous studies on chalcone scaffold and show them as a potential treatment strategy for NDDs and their associated comorbidities.

The therapeutic indications for monoamine oxidases A and B (MAO-A and MAO-B) inhibitors that have emerged from biological studies on animal and cellular models of neurological and oncological diseases have focused drug discovery projects upon identifying reversible MAO inhibitors. Screening of our in-house academic compound library identified two hit compounds that inhibit MAO-B with IC50 values in micromolar range. Two series of indole (23 analogues) and 3-(benzyloxy)benzyl)piperazine (16 analogues) MAO-B inhibitors were derived from hits, and screened for their structure-activity relationships. Both series yielded low micromolar selective inhibitors of human MAO-B, namely indole 2 (IC50 = 12.63 ± 1.21 µM) and piperazine 39 (IC50 = 19.25 ± 4.89 µM), which is comparable to selective MAO-B inhibitor isatin (IC50 = 6.10 ± 2.81 µM), yet less potent in comparison to safinamide (IC50 = 0.029 ± 0.002 µM). Selective MAO-B inhibitors 2, 14, 38 and 39 exhibited favourable permeation of the blood-brain barrier and low cytotoxicity in the human neuroblastoma cell line SH-SY5Y.

Dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamide (tetraene) is the main component of Echinacea angustifolia DC. lipophilic extract, the bioavailability and immunomodulatory effect after oral administration in soft gel capsules in healthy volunteers of which we have already demonstrated. In the present work, we assessed the transdermal administration as an alternative route of administration of such an alkamide. The first step, therefore, encompassed the preparation of a drug-in-adhesive patch with an area of 868 mm2 and containing a dose of 0.64 mg of tetraene. In vitro skin permeation studies in Franz-type diffusion chambers resulted in a tetraene flux of (103 ± 10) ng × cm−2 × h−1 with a very good linearity (r = 0.99). The relatively low lag time of just 13 min indicates low binding and the accumulation of tetraene in the skin. Finally, the patch was administered to six healthy volunteers, and the pharmacokinetic analysis was performed by nonlinear mixed effects modelling with soft gel oral capsules serving as the reference formulation. The in vivo results correlated well with the in vitro permeation and indicated an initial burst tetraene absorption from the patch that was in parallel with the zero-order kinetics of absorption. The rate of the latter process was in good agreement with the one estimated in vitro. The tetraene absorption rate was therefore slow and prolonged with time, resulting in a bioavailability of 39% relative to the soft gel capsules and a very flat plasma concentration profile.

The Calu-3 cell line has been largely investigated as a physiological and pharmacological model of the airway epithelial barrier. Its suitability for prediction of drug permeability across the airway epithelia, however, has not been yet evaluated by using large enough set of model drugs. We evaluated two Calu-3 cell models (air-liquid and liquid-liquid) for drug permeability prediction based on the recent regulatory guidelines on showing suitability of in vitro permeability methods for drug permeability classification. Bidirectional permeability assays using 22 model drugs and several zero permeability markers, as well as using ABC transporter substrates were conducted. Functional activity of P-gp, but not of BCRP was revealed. The potential of the Calu-3 cells to be used as a model of the nasal epithelial barrier, despite their different anatomical origin, has been demonstrated by the obtained excellent correlation with the fully differentiated 3D human nasal epithelial model (MucilAir™) for 11 model drugs, as well as by the good correlation obtained with the human nasal epithelial cell line RPMI 2650. In addition, the permeability values determined in the two Calu-3 models correlated well with the intestinal permeability model Caco-2.

Compounds capable of interacting with single or multiple targets involved in Alzheimer's disease (AD) pathogenesis are potential anti-Alzheimer's agents. In our aim to develop new anti-Alzheimer's agents, a series of 36 new N-alkylpiperidine carbamates was designed, synthesized and evaluated for the inhibition of cholinesterases [acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)] and monoamine oxidases [monoamine oxidase A (MAO-A and monoamine oxidase B (MAO-B)]. Four compounds are very promising: multiple AChE (IC50 = 7.31 mu M), BChE (IC50 = 0.56 mu M) and MAO-B (IC50 = 26.1 mu M) inhibitor 10, dual AChE (IC50 = 2.25 mu M) and BChE (IC50 = 0.81 mu M) inhibitor 22, selective BChE (IC50 = 0.06 mu M) inhibitor 13, and selective MAO-B (IC50 = 0.18 mu M) inhibitor 16. Results of enzyme kinetics experiments showed that despite the carbamate group in the structure, compounds 10, 13, and 22 are reversible and non-time-dependent inhibitors of AChE and/or BChE. The resolved crystal structure of the complex of BChE with compound 13 confirmed the non-covalent mechanism of inhibition. Additionally, N-propargylpiperidine 16 is an irreversible and time-dependent inhibitor of MAO-B, while N-benzylpiperidine 10 is reversible. Additionally, compounds 10, 13, 16, and 22 should be able to cross the blood-brain barrier and are not cytotoxic to human neuronal-like SH-SY5Y and liver HepG2 cells. Finally, compounds 10 and 16 also prevent amyloid beta(1-42) (A beta(1-42))-induced neuronal cell death. The neuroprotective effects of compound 16 could be the result of its A beta(1-42) anti-aggregation effects. (C) 2020 Elsevier Masson SAS. All rights reserved.

The resurgence of interest in monoamine oxidases (MAOs) has been fueled by recent correlations of this enzymatic activity with cardiovascular, neurological, and oncological disorders. This has promoted increased research into selective MAO-A and MAO-B inhibitors. Here, we shed light on how selective inhibition of MAO-A and MAO-B can be achieved by geometric isomers of cis- and trans-1-propargyl-4-styrylpiperidines. While the cis isomers are potent human MAO-A inhibitors, the trans analogues selectively target only the MAO-B isoform. The inhibition was studied by kinetic analysis, UV-vis spectrum measurements, and X-ray crystallography. The selective inhibition of the MAO-A and MAO-B isoforms was confirmed ex vivo in mouse brain homogenates, and additional in vivo studies in mice show the therapeutic potential of 1-propargyl-4-styrylpiperidines for central nervous system disorders. This study represents a unique case of stereoselective activity of cis/trans isomers that can discriminate between structurally related enzyme isoforms.

The biopharmaceutical classification of drugs was designed as a basis for bio-waivers - a mechanism with the double ethical benefit of delivering new drug formulations to the market with less human testing and lower cost. However, many drugs defy simple classification because in vitro permeability and stability assessment can be challenging as shown in this study for desloratadine. Literature shows that desloratadine is highly soluble, while data on luminal stability and permeability are circumstantial. Combined with borderline bioavailability and not really known fraction of absorbed dose, desloratadine was found to be a good example for showing the innovative in vitro approaches necessary to unambiguously classify desloratadine according to Biopharmaceutical Classification System (BCS) guideline. Presented study undoubtedly confirmed that desloratadine solubility is high and dissolution is very rapid for immediate release reference tablets. We have demonstrated deslorata-dine stability under legally required conditions and also in more physiologically relevant media. High in vitro desloratadine permeability was confirmed using Caco-2 and Parallel Artificial Membrane Permeability Assay (PAMPA). Well-established in vitro model with rat intestinal tissue could not be used due to reasons elaborated in this paper.

Canine cognitive dysfunction (CCD) is common in aged dogs and has many similarities with Alzheimer’s disease. Unfortunately, like Alzheimer’s disease, CCD cannot be cured. In the present study, we treated dogs with CCD with our newly developed and characterized butyrylcholinesterase inhibitor (BChEi). Seventeen dogs were randomized into two groups (treated with BChEi and untreated) and followed for 6 months at regular check-ups. The dogs’ cognitive status was determined by a Canine Dementia Scale (CADES) questionnaire and two cognitive tests. In dogs with moderate cognitive impairment, treatment caused significant improvement in the clinical rating of cognitive abilities and the performance-based tests of cognitive functioning when compared to the untreated group (p < 0.001). Dogs treated with BChEi showed markedly improved cognitive function with enhanced quality of life. No side effects were observed in the treated dogs with moderate cognitive impairment. According to the results of this preliminary study, there is an indication that novel BChEi may be a promising drug for the treatment of CCD in dogs and may be an interesting candidate for the treatment of Alzheimer's disease in humans. However, further clinical studies are needed to confirm this.

Prediction of the effect of food on drug’s pharmacokinetics using modeling and simulation could cause difficulties due to complex in vivo processes. A generic formulation with amorphous form of BCS 2 class drug substance was developed and compared in vitro and in vivo to the reference drug product with drug substance in crystalline form. In order to approve generic formulation, some regulatory agencies are requesting to perform bioequivalence (BE) studies also in fed state. Food can have various effects on drug dissolution and absorption, depending also on drug’s properties. A physiologically based pharmacokinetic (PBPK) absorption model was built in GastroPlus™ to predict the food effect on generic and reference formulation and to predict the fed BE study outcome. During model development, we were searching for model inputs that impact and describe in vivo behavior of amorphous and crystalline forms of active pharmaceutical ingredient (API) in fast and fed conditions. The developed model was able to predict the food effect with up to 10% prediction error (PE). Performed virtual BE trials confirmed the BE of drug products in fed state. Our model was able to capture the difference between the two drug products containing different forms of API (amorphous and crystalline) and predict the food effect on both formulations.

The significance of thermodynamic solubility in biopharmaceutical compound or drug characterization as well as the importance of having methods that accurately establish it have been extensively addressed. Nonetheless, its precise determination continues to remain a challenging task to accomplish. Even more so when the number of compounds to evaluate is high and the available amount of each compound is low, both of which are inevitable for the compound characterization during the drug development process. Except for the shake-flask method which is still considered as the ‘gold standard’ in obtaining thermodynamic data, it is currently difficult to say that another satisfactory model which is routinely used to determine thermodynamic solubility is being applied. Therefore, this review summarizes the various experimental approaches which are based on the classical shake flask method but have yet attempted to speed up the experimental process of obtaining such data more conveniently. The most important experimental features of these approaches are provided to the reader. Some advantages and disadvantages associated with each approach are also highlighted, consequently offering a resource to those looking for the most appropriate of the approaches that have already fared well at determining the biopharmaceutically relevant drug solubility.

The RPMI 2650 cell line has been a subject of evaluation as a physiological and pharmacological model of the nasal epithelial barrier. However, its suitability for drug permeability assays has not yet been established on a sufficiently large set of model drugs. We investigated two RPMI 2650 cell models (air-liquid and liquid-liquid) for nasal drug permeability determination by adopting the most recent regulatory guidelines on showing suitability of in vitro permeability methods for drug permeability classification. The permeability of 23 model drugs and several zero permeability markers across the cell models was assessed. The functional expression of two efflux transporters P-glycoprotein (P-gp) and Breast Cancer Resistant Protein (BCRP) was shown to be negligible by bidirectional transport studies using appropriate transporter substrates and inhibitors. The model drug permeability determined in the two RPMI 2650 cell models was correlated with the fully differentiated nasal epithelial model (MucilAir™). Additionally, correlations between the drug permeability in the investigated cell models and the ones determined in the Caco-2 cells and isolated rat jejunum were established. In conclusion, the air-liquid RPMI 2650 cell model is a promising pharmacological model of the nasal epithelial barrier and is much more suitable than the liquid-liquid model for nasal drug permeability prediction.

The paper presents the effect of polymer solution composition on the morphology, mechanical properties and drug permeability of the asymmetric polyamide 6 (PA6) membranes prepared by immersion precipitation. The effect of polymer solution composition on morphology, mechanical properties and permeability of the produced membrane is considered, since these properties are of relevance for drug delivery applications. PA6-formic acid-deionized water solutions were used to prepare membranes for further characterization with differential scanning calorimetry and scanning electron microscopy for morphology analysis, tensile testing and drug permeability tests. The results show that the amount of PA6 does not significantly affect morphology of the membrane, while having pronounced effect on tensile elastic modulus (50% increase). On the other hand, the concentration of formic acid in solution (dissolution intensity) influences crystallization dynamics and significantly changes the morphology of membrane (in the range of approximately 75-100 wt% of formic acid concentrations), consequently having effect on drug permeability.

The limited clinical efficacy of current symptomatic treatment and minute effect on progression of Alzheimer's disease has shifted the research focus from single targets towards multi-target-directed ligands. Here, a potent selective inhibitor of human butyrylcholinesterase was used as the starting point to develop a new series of multifunctional ligands. A focused library of derivatives was designed and synthesised that showed both butyrylcholinesterase inhibition and good antioxidant activity as determined by the DPPH assay. The crystal structure of compound 11 in complex with butyrylcholinesterase revealed the molecular basis for its low nanomolar inhibition of butyrylcholinesterase (Ki = 1.09 +/- 0.12 nM). In addition, compounds 8 and 11 show metal-chelating properties, and reduce the redox activity of chelated Cu2+ ions in a Cu-ascorbate redox system. Compounds 8 and 11 decrease intracellular levels of reactive oxygen species, and are not substrates of the active efflux transport system, as determined in Caco2 cells. Compound 11 also protects neuroblastoma SH-SY5Y cells from toxic A beta(1-42) species. These data indicate that compounds 8 and 11 are promising multifunctional lead ligands for treatment of Alzheimer's disease. (C) 2018 Elsevier Masson SAS. All rights reserved.