A Biomimetic Multiparametric Assay to Characterise Anti-Amyloid Drugs.

Alzheimer's disease (AD) is the most widespread form of senile dementia worldwide and represents a leading socioeconomic problem in healthcare. Although it is widely debated, the aggregation of the amyloid ß peptide (Aß) is linked to the onset and progression of this neurodegenerative disease. Molecules capable of interfering with specific steps in the fibrillation process remain of pharmacological interest. To identify such compounds, we have set up a small molecule screening process combining multiple experimental methods (UV and florescence spectrometry, ITC, and ATR-FTIR) to identify and characterise potential modulators of Aß1-42 fibrillation through the description of the biochemical interactions (molecule-membrane Aß peptide). Three known modulators, namely bexarotene, Chicago sky blue and indomethacin, have been evaluated through this process, and their modulation mechanism in the presence of a biomembrane has been described. Such a well-adapted physico-chemical approach to drug discovery proves to be an undeniable asset for the rapid characterisation of compounds of therapeutic interest for Alzheimer's disease. This strategy could be adapted and transposed to search for modulators of other amyloids such as tau protein.

Relevant Physicochemical Methods to Functionalize, Purify, and Characterize Surface-Decorated Lipid-Based Nanocarriers.

Surface functionalization of lipid-based nanocarriers (LBNCs) with targeting ligands has attracted huge interest in the field of nanomedicines for their ability to overcome some physiological barriers and their potential to deliver an active molecule to a specific target without causing damage to healthy tissues. The principal objective of this review is to summarize the present knowledge on LBNC decoration used for biomedical applications, with an emphasis on the ligands used, the functionalization approaches, and the purification methods after ligand corona formation. The most potent experimental techniques for the LBNC surface characterization are described. The potential of promising methods such as nuclear magnetic resonance spectroscopy and isothermal titration calorimetry to characterize ligand surface corona is also outlined.

Design of donecopride, a dual serotonin subtype 4 receptor agonist/acetylcholinesterase inhibitor with potential interest for Alzheimer's disease treatment.

RS67333 is a partial serotonin subtype 4 receptor (5-HT4R) agonist that has been widely studied for its procognitive effect. More recently, it has been shown that its ability to promote the nonamyloidogenic cleavage of the precursor of the neurotoxic amyloid-ß peptide leads to the secretion of the neurotrophic protein sAPPα. This effect has generated great interest in RS67333 as a potential treatment for Alzheimer's disease (AD). We show herein that RS67333 is also a submicromolar acetylcholinesterase (AChE) inhibitor and therefore, could contribute, through this effect, to the restoration of the cholinergic neurotransmission that becomes altered in AD. We planned to pharmacomodulate RS67333 to enhance its AChE inhibitory activity to take advantage of this pleiotropic pharmacological profile in the design of a novel multitarget-directed ligand that is able to exert not only a symptomatic but also, a disease-modifying effect against AD. These efforts allowed us to select donecopride as a valuable dual (h)5-HT4R partial agonist (Ki = 10.4 nM; 48.3% of control agonist response)/(h)AChEI (IC50 = 16 nM) that further promotes sAPPα release (EC50 = 11.3 nM). Donecopride, as a druggable lead, was assessed for its in vivo procognitive effects (0.1, 0.3, 1, and 3 mg/kg) with an improvement of memory performances observed at 0.3 and 1 mg/kg on the object recognition test. On the basis of these in vitro and in vivo activities, donecopride seems to be a promising drug candidate for AD treatment.

Injectable polyoxazoline grafted hyaluronic acid thermoresponsive hydrogels for biomedical applications.

Injectable thermosensitive hydrogels based on hyaluronic acid (HA) grafted with lower critical solution temperature (LCST) polyoxazoline (copolymers of poly(isopropyl-co-butyl oxazoline)) or P(iPrOx-co-BuOx) have been elaborated with tunable solution/gel temperature transitions and gel state elastic modulus. A suitable HA-g-P(iPrOx-co-BuOx-67/33)-0.10 sample with an iPrOx/BuOx ratio of 67/33, a polymerization degree (DP) of 25, a substitution degree (DS) of 10%, and displaying thermally induced gelling character with elastic (G') and viscous (G'') moduli crossover points at 25 °C and a G' at 37 °C around 80 Pa has been chosen for medical application. Hydrogels obtained with HA-g-P(iPrOx-co-BuOx-67/33)-0.10 exhibited high stability at 37 °C and excellent injectability properties with full and quick reversibility. The incorporation of a secondary network (HA), until 35 wt%, into the thermosensitive hydrogel also demonstrated very good stability and injectability.

MRI-based microthrombi detection in stroke with polydopamine iron oxide.

In acute ischemic stroke, even when successful recanalization is obtained, downstream microcirculation may still be obstructed by microvascular thrombosis, which is associated with compromised brain reperfusion and cognitive decline. Identifying these microthrombi through non-invasive methods remains challenging. We developed the PHySIOMIC (Polydopamine Hybridized Self-assembled Iron Oxide Mussel Inspired Clusters), a MRI-based contrast agent that unmasks these microthrombi. In a mouse model of thromboembolic ischemic stroke, our findings demonstrate that the PHySIOMIC generate a distinct hypointense signal on T2*-weighted MRI in the presence of microthrombi, that correlates with the lesion areas observed 24 hours post-stroke. Our microfluidic studies reveal the role of fibrinogen in the protein corona for the thrombosis targeting properties. Finally, we observe the biodegradation and biocompatibility of these particles. This work demonstrates that the PHySIOMIC particles offer an innovative and valuable tool for non-invasive in vivo diagnosis and monitoring of microthrombi, using MRI during ischemic stroke.

In vitro evaluation of NA1-115-7-loaded nanoemulsions, an MCL-1-specific inhibitor of natural origin, intended to treat B-cell lymphoproliferative disorders after oral administration.

MCL-1, an anti-apoptotic member of the BCL-2 protein family, is overexpressed in many types of cancer and contributes to chemotherapy resistance. The drimane derivative NA1-115-7 is a natural compound isolated from Zygogynum pancheri that can be considered as a very promising lead for treating MCL-1-dependent hematological malignancies. As this drug suffers from low stability in acidic conditions and poor aqueous solubility, we evaluated the potential oral use of NA1-115-7 by encapsulating it in lipid nanoemulsions (NA-NEs) prepared by spontaneous emulsification. NA-NEs showed a particle size of 41.9 ± 2.2 nm, PDI of 0.131 ± 0.016, zeta potential of -5.8 ± 3.4 mV, encapsulation efficiency of approximately 100 % at a concentration of 24 mM. The stability of NA-1-115-7 was sixfold higher than that of the unencapsulated drug in simulated gastric fluid. NA-NEs significantly restored apoptosis and halved the effective doses of NA1-115-7 on BL2, a Burkitt lymphoma cell line, without toxicity in normal cells. Such a drug-delivery system appears to be particularly interesting for the oral administration of NA1-115-7, as it improves its solubility and stability, as well as efficacy, by reducing the therapeutic dose, making it possible to further consider in-vivo studies of this promising drug in BL2 xenografted mice.

New antiplasmodial 4-amino-thieno[3,2-d]pyrimidines with improved intestinal permeability and microsomal stability.

The increasing number of Plasmodium falciparum strains resistant to current treatments justifies the urgent need to discover new compounds active on several stages of the parasite development. Based on the structure of Gamhepathiopine, a 2-tert-butylaminothieno[3,2-d]pyrimidin-4(3H)-one previously identified for its dual activity against the sexual and asexual stages of P. falciparum, 25 new 4-amino-substituted analogues were synthesized and evaluated on the erythrocytic and hepatic stages of Plasmodium. A promising compound, N2-(tert-butyl)-N [4]-(3-(dimethylamino)propyl)-6-(p-tolyl)thieno[3,2-d]pyrimidine-2,4-diamine, showed improved physicochemical properties, intestinal permeability (PAMPA model) and microsomal stability compared to Gamhepathiopine, while maintaining a good antiplasmodial activity on the erythrocytic stage of P. falciparum and on the hepatic stage of P. berghei.

Novel thienopyrimidones targeting hepatic and erythrocytic stages of Plasmodium parasites with increased microsomal stability.

Based on the structure of a previously identified hit, Gamhepathiopine 1, which showed promising antiplasmodial activity, but poor microsomal stability, several strategies were investigated to improve the metabolic stability of the compounds. This included the introduction of fluorine or deuterium atoms, as well as carbocyclic groups. Among the new compounds, the 2-aminocyclobutyl derivative 5g demonstrated enhanced microsomal stability compared to compound 1, while retaining antiplasmodial activity against erythrocytic and hepatic stages of Plasmodium, without significant cytotoxicity against primary hepatocytes.

How nano-engineered delivery systems can help marketed and repurposed drugs in Alzheimer's disease treatment?

Given the continual increase in the number of patients and the lack of curative treatment, the development of new therapies to treat Alzheimer's disease (AD) is becoming ever more urgent. In this review, we summarize the most promising preclinical studies in, and the significant benefits offered by, nanocarriers to realize the full potential of marketed drugs and identify repurposed drugs. No clinical trials have yet been conducted on nanocarriers for drug repurposing in AD. However, recent preclinical results suggest that nanocarriers could overcome the bioavailability and/or selectivity issues of repurposed drugs, improving their therapeutic efficacy.

NA1-115-7, from Zygogynum pancheri, is a new selective MCL-1 inhibitor inducing the apoptosis of hematological cancer cells but non-toxic to normal blood cells or cardiomyocytes.

The overexpression of antiapoptotic members (BCL-2, BCL-xL, MCL-1, etc.) of the BCL-2 family contributes to tumor development and resistance to chemotherapy or radiotherapy. Synthetic inhibitors targeting these proteins have been developed, and some hematological malignancies are now widely treated with a BCL-2 inhibitor (venetoclax). However, acquired resistance to venetoclax or chemotherapy drugs due to an upregulation of MCL-1 has been observed, rendering MCL-1 an attractive new target for treatment. Six MCL-1 inhibitors (S64315, AZD-5991, AMG-176, AMG-397, ABBV-467 and PRT1419) have been evaluated in clinical trials since 2016, but some were affected by safety issues and none are currently used clinically. There is, therefore, still a need for alternative molecules. We previously described two drimane derivatives as the first covalent BH3 mimetics targeting MCL-1. Here, we described the characterization and biological efficacy of one of these compounds (NA1-115-7), isolated from Zygogynum pancheri, a plant belonging to the Winteraceae family. NA1-115-7 specifically induced the apoptosis of MCL-1-dependent tumor cells, with two hours of treatment sufficient to trigger cell death. The treatment of lymphoma cells with NA1-115-7 stabilized MCL-1, disrupted its interactions with BAK, and rapidly induced apoptosis through a BAK- and BAX-mediated process. Importantly, a similar treatment with NA1-115-7 was not toxic to erythrocytes, peripheral blood mononuclear cells, platelets, or cardiomyocytes. These results highlight the potential of natural products for use as specific BH3 mimetics non-toxic to normal cells, and they suggest that NA1-115-7 may be a promising tool for use in cancer treatment.

Synthesis of antiplasmodial 2-aminothieno[3,2-d]pyrimidin-4(3H)-one analogues using the scaffold hopping strategy.

Gamhepathiopine (also known as M1), is a multi-stage acting antiplasmodial 2-tert-butylaminothieno[3,2-d]pyrimidin-4(3H)-one hydrochloride that was first described in 2015. The development of this compound is limited by poor microsomal stability, insufficient aqueous solubility and low intestinal permeability. In order to obtain new optimized derivatives, we conducted a scaffold hopping strategy from compound M1, resulting in the synthesis of 20 new compounds belonging to six chemical series. All the compounds were tested on the K1 multi-resistant strain of Plasmodium falciparum and the human HepG2 cell-line, to evaluate their antiplasmodial activity and their cytotoxicity. Analogues' biological results also highlighted the mandatory presence of a heteroatom at position 5 of the thieno[3,2-d]pyrimidin-4(3H)-one moeity for the antiplasmodial activity. However, modifications at position 7 were detrimental for the antiplasmodial activity. We identified furane bioisostere 3j as a promising candidate, showing good blood stage antiplasmodial activity, better water solubility and highly improved intestinal permeability in the PAMPA assay.

Improving Aqueous Solubility and In Vitro Pharmacokinetic Properties of the 3-Nitroimidazo[1,2-a]pyridine Antileishmanial Pharmacophore.

An antileishmanial structure−activity relationship (SAR) study focused on positions 2 and 8 of the imidazo[1,2-a]pyridine ring was conducted through the synthesis of 22 new derivatives. After being screened on the promatigote and axenic amastigote stages of Leishmania donovani and L. infantum, the best compounds were tested against the intracellular amastigote stage of L. infantum and evaluated regarding their in vitro physicochemical and pharmacokinetic properties, leading to the discovery of a new antileishmanial6-chloro-3-nitro-8-(pyridin-4-yl)-2-[(3,3,3-trifluoropropylsulfonyl)methyl]imidazo[1,2-a]pyridine hit. It displayed low cytotoxicities on both HepG2 and THP1 cell lines (CC50 > 100 µM) associated with a good activity against the intracellular amastigote stage of L. infantum (EC50 = 3.7 µM versus 0.4 and 15.9 µM for miltefosine and fexinidazole, used as antileishmanial drug references). Moreover, in comparison with previously reported derivatives in the studied series, this compound displayed greatly improved aqueous solubility, good mouse microsomal stability (T1/2 > 40 min) and high gastrointestinal permeability in a PAMPA model, making it an ideal candidate for further in vivo studies on an infectious mouse model.

ß-Amyloid peptide interactions with biomimetic membranes: A multiparametric characterization.

Alzheimer's disease is the most common form of senile dementia in the world, and amyloid ß peptide1-42 (Aß1-42) is one of its two principal biological hallmarks. While interactome concept was getting forward the scientific community, we proposed that the study of the molecular interactions of amyloid ß peptide with the biological membranes will allow to highlight underlying mechanisms responsive of AD. We have developed two simple liposomal formulations (phosphatidylcholine, cholesterol, phosphatidylglycerol) mimicking neuronal cell membrane (composition, charge, curvature radius). Interactions with Aß1-42 and mutant oG37C, a stable oligomeric form of the peptide, were characterized according to a simple multiparametric procedure based on ThT fluorescence, liposome leakage assay, ATR-FTIR spectroscopy. Kinetic aggregation, membrane damage and peptide conformation provided our first methodologic bases to develop an original model to describe interactions of Aß peptide and lipids.

Active Targeted Nanoemulsions for Repurposing of Tegaserod in Alzheimer's Disease Treatment.

BACKGROUND AND PURPOSE: The activation of 5-HT4 receptors with agonists has emerged as a valuable therapeutic strategy to treat Alzheimer's disease (AD) by enhancing the nonamyloidogenic pathway. Here, the potential therapeutic effects of tegaserod, an effective agent for irritable bowel syndrome, were assessed for AD treatment. To envisage its efficient repurposing, tegaserod-loaded nanoemulsions were developed and functionalized by a blood-brain barrier shuttle peptide. RESULTS: The butyrylcholinesterase inhibitory activity of tegaserod and its neuroprotective cellular effects were highlighted, confirming the interest of this pleiotropic drug for AD treatment. In regard to its drugability profile, and in order to limit its peripheral distribution after IV administration, its encapsulation into monodisperse lipid nanoemulsions (Tg-NEs) of about 50 nm, and with neutral zeta potential characteristics, was performed. The stability of the formulation in stock conditions at 4 °C and in blood biomimetic medium was established. The adsorption on Tg-NEs of peptide-22 was realized. The functionalized NEs were characterized by chromatographic methods (SEC and C18/HPLC) and isothermal titration calorimetry, attesting the efficiency of the adsorption. From in vitro assays, these nanocarriers appeared suitable for enabling tegaserod controlled release without hemolytic properties. CONCLUSION: The developed peptide-22 functionalized Tg-NEs appear as a valuable tool to allow exploration of the repurposed tegaserod in AD treatment in further preclinical studies.

Drimane Derivatives as the First Examples of Covalent BH3 Mimetics that Target MCL-1.

Drimane sesquiterpenoid dialdehydes are natural compounds with antiproliferative properties. Nevertheless, their mode of action has not yet been discovered. Herein, we demonstrate that various drimanes are potent inhibitors of MCL-1 and BCL-xL, two proteins of the BCL-2 family that are overexpressed in various cancers, including lymphoid malignancies. Subtle changes in their structure significantly modified their activity on the target proteins. The two most active compounds are MCL-1 selective and bind in the BH3 binding groove of the protein. Complementary studies by NMR spectroscopy and mass spectrometry analyses, but also synthesis, showed that they covalently inhibit MCL-1 though the formation of a pyrrole adduct. In addition, cytotoxic assays revealed that these two compounds show a cytotoxic selectivity for BL2, a MCL-1/BCL-xL-dependent cell line and induce apoptosis.

A New Thienopyrimidinone Chemotype Shows Multistage Activity against Plasmodium falciparum, Including Artemisinin-Resistant Parasites.

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. For decades, the research for novel antimalarials focused on the high-throughput screening of molecules that only targeted the asexual blood stages. In a search for new effective compounds presenting a triple action against erythrocytic and liver stages in addition to the ability to block the transmission of the disease via the mosquito vector, 2-amino-thienopyrimidinone derivatives were synthesized and tested for their antimalarial activity. One molecule, named gamhepathiopine (denoted as "M1" herein), was active at submicromolar concentrations against both erythrocytic (50% effective concentration [EC50] = 0.045 µM) and liver (EC50 = 0.45 µM) forms of Plasmodium falciparum. Furthermore, gamhepathiopine efficiently blocked the development of the sporogonic cycle in the mosquito vector by inhibiting the exflagellation step. Moreover, M1 was active against artemisinin-resistant forms (EC50 = 0.227 µM), especially at the quiescent stage. Nevertheless, in mice, M1 showed modest activity due to its rapid metabolization by P450 cytochromes into inactive derivatives, calling for the development of new parent compounds with improved metabolic stability and longer half-lives. These results highlight the thienopyrimidinone scaffold as a novel antiplasmodial chemotype of great interest to search for new drug candidates displaying multistage activity and an original mechanism of action with the potential to be used in combination therapies for malaria elimination in the context of artemisinin resistance. IMPORTANCE This work reports a new chemical structure that (i) displays activity against the human malaria parasite Plasmodium falciparum at 3 stages of the parasitic cycle (blood stage, hepatic stage, and sexual stages), (ii) remains active against parasites that are resistant to the first-line treatment recommended by the World Health Organization (WHO) for the treatment of severe malaria (artemisinins), and (iii) reduces transmission of the parasite to the mosquito vector in a mouse model. This new molecule family could open the way to the conception of novel antimalarial drugs with an original multistage mechanism of action to fight against Plasmodium drug resistance and block interhuman transmission of malaria.

Design of Non-Haemolytic Nanoemulsions for Intravenous Administration of Hydrophobic APIs.

Among advanced formulation strategies, nanoemulsions are considered useful drug-delivery systems allowing to improve the solubility and the bioavailability of lipophilic drugs. To select safe excipients for nanoemulsion formulation and to discard any haemolytic potential, an in vitro miniaturized test was performed on human whole blood. From haemolysis results obtained on eighteen of the most commonly used excipients, a medium chain triglyceride, a surfactant, and a solubilizer were selected for formulation assays. Based on a design of experiments and a ternary diagram, the feasibility of nanoemulsions was determined. The composition was defined to produce monodisperse nanodroplets with a diameter of either 50 or 120 nm, and their physicochemical properties were optimized to be suitable for intravenous administration. These nanoemulsions, stable over 21 days in storage conditions, were shown to be able to encapsulate with high encapsulation efficiency and high drug loading, up to 16% (w/w), two water practically insoluble drug models: ibuprofen and fenofibrate. Both drugs may be released according to a modulable profile in sink conditions. Such nanoemulsions appear as a very promising and attractive strategy for the efficient early preclinical development of hydrophobic drugs.

Pharmacotechnical Development of a Nasal Drug Delivery Composite Nanosystem Intended for Alzheimer's Disease Treatment.

Direct nose-to-brain delivery has been raised as a non-invasive powerful strategy to deliver drugs to the brain bypassing the blood-brain barrier (BBB). This study aimed at preparing and characterizing an innovative composite formulation, associating the liposome and hydrogel approaches, suitable for intranasal administration. Thermosensitive gel formulations were obtained based on a mixture of two hydrophilic polymers (Poloxamer 407, P407 and Poloxamer 188, P188) for a controlled delivery through nasal route via liposomes of an active pharmaceutical ingredient (API) of potential interest for Alzheimer's disease. The osmolarity and the gelation temperature (T° sol-gel) of formulations, defined in a ternary diagram, were investigated by rheometry and visual determination. Regarding the issue of assays, a mixture composed of P407/P188 (15/1%, w/w) was selected for intranasal administration in terms of T° sol-gel and for the compatibility with the olfactory mucosal (280 ± 20 mOsmol, pH 6). Liposomes of API were prepared by the thin film hydration method. Mucoadhesion studies were performed by using mucin disc, and they showed the good natural mucoadhesive characteristics of in situ gel formulations, which increased when liposomes were added. The study demonstrated successful pharmacotechnical development of a promising API-loaded liposomes in a thermosensitive hydrogel intended for nasal Alzheimer's disease treatment.

Synthesis of Pyridoclax Analogues: Insight into Their Druggability by Investigating Their Physicochemical Properties and Interactions with Membranes.

Pyridoclax is considered a promising anticancer drug, acting as a protein-protein interaction disruptor, with potential applications in the treatment of ovarian, lung, and mesothelioma cancers. Eighteen sensibly selected structural analogues of Pyridoclax were synthesized, and their physicochemical properties were systematically assessed and analyzed. Moreover, considering that drug-membrane interactions play an essential role in understanding the mode of action of a given drug and its eventual toxic effects, membrane models were used to investigate such interactions in bulk (liposomes) and at the air-water interface. The measured experimental data on all original oligopyridines allowed the assessment of relative differences in terms of physicochemical properties, which could be determinant for their druggability, and hence for drug development.

New 8-Nitroquinolinone Derivative Displaying Submicromolar in Vitro Activities against Both Trypanosoma brucei and cruzi.

An antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC50 values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 µM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = -0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds.