Pyridine to aniline: an exceptional biologically driven rearrangement.

During the course of our study on the innovative ligand for nicotinic acetylcholinergic receptors, LNAChR, and in order to assess activity and toxicity profiles of the drug's metabolites, synthesis of the main metabolites was undertaken. This synthesis work was done in parallel by organic chemistry and by biotransformation of LNAChR. Filamentous fungus Aspergillus alliaceus (NRRL 315) neatly afforded three of the main metabolites, one of which arose from a very unexpected and very uncommon rearrangement.

New radioligands for describing the molecular pharmacology of MT1 and MT2 melatonin receptors.

Melatonin receptors have been studied for several decades. The low expression of the receptors in tissues led the scientific community to find a substitute for the natural hormone melatonin, the agonist 2-[125I]-iodomelatonin. Using the agonist, several hundreds of studies were conducted, including the discovery of agonists and antagonists for the receptors and minute details about their molecular behavior. Recently, we attempted to expand the panel of radioligands available for studying the melatonin receptors by using the newly discovered compounds SD6, DIV880, and S70254. These compounds were characterized for their affinities to the hMT1 and hMT2 recombinant receptors and their functionality in the classical GTPS system. SD6 is a full agonist, equilibrated between the receptor isoforms, whereas S70254 and DIV880 are only partial MT2 agonists, with Ki in the low nanomolar range while they have no affinity to MT1 receptors. These new tools will hopefully allow for additions to the current body of information on the native localization of the receptor isoforms in tissues.

An efficient site-selective, dual bioconjugation approach exploiting N-terminal cysteines as minimalistic handles to engineer tailored anti-HER2 affibody conjugates.

Site-selective, dual-conjugation approaches for the incorporation of distinct payloads are key for the development of molecularly targeted biomolecules, such as antibody conjugates, endowed with better properties. Combinations of cytotoxic drugs, imaging probes, or pharmacokinetics modulators enabled for improved outcomes in both molecular imaging, and therapeutic settings. We have developed an efficacious dual-bioconjugation strategy to target the N-terminal cysteine of a chemically-synthesized, third-generation anti-HER2 affibody. Such two-step, one-purification approach can be carried out under mild conditions (without chaotropic agents, neutral pH) by means of a slight excess of commercially available N-hydroxysuccinimidyl esters and maleimido-functionalized payloads, to generate dual conjugates displaying drugs (DM1/MMAE) or probes (sulfo-Cy5/biotin) in high yields and purity. Remarkably, the double drug conjugate exhibited an exacerbated cytoxicity against HER2-expressing cell lines as compared to a combination of two monoconjugates, demonstrating a potent synergistic effect. Consistently, affibody-drug conjugates did not decrease the viability of HER2-negative cells, confirming their specificity for the target.

Novel surfactants with diglutamic acid polar head group: drug solubilization and toxicity studies.

PURPOSE: Novel surfactants made of diglutamic acid (DG) polar head linked to lithocholic, arachidonic, linoleic or stearic acids were designed for drug solubilization. METHODS: Surfactants 3-D conformer and packing parameter were determined by molecular modelling and self-assembling properties by pyrene fluorescence measurements. Cytotoxicity was assessed on Human Umbilical Vein Endothelial Cells (HUVEC) and haemolyitic activity on rat red blood cells. Drug solubilization was quantified and its interaction with hydrophobic moieties was characterized using differential scanning calorimetry and X-ray diffraction. Self organisation of stearoyl-DG was observed by cryogenic transmission electron microscopy. Toxicity after repeated injections of stearoyl-DG was investigated in Wistar rats. RESULTS: DG-based surfactants self-assemble into water and their critical micellar concentrations are comprised between 200 and 920 µg/mL. Cytotoxicity and haemolysis were lower than for polysorbate 80. At best, stearoyl-DG solubilized the drug up to 22% (w/w). Solid-state characterization evidenced drug/lipid interactions leading to the formation of a new complex. Stearoyl-DG formed spherical micelles of 20 nm, as predicted by packing parameter calculation. However, it induced a possible liver toxicity after intravenous administration in rats. CONCLUSIONS: Among the surfactants tested, stearoyl-DG is the more efficient for drug solubilization but its use is limited by its possible liver toxicity.

Enhanced drug loading in polymerized micellar cargo.

A new drug carrier system based on self-assembly and polymerization of polydiacetylenic amphiphiles is described. Although classical amphiphiles can help in solubilizing hydrophobic molecules upon self-arrangement into a variety of nanometric structures, a greater effect on drug loading was observed for our polymerized micelles as compared to the non-polymerized analogues. This permitted higher aqueous solubilization of lipophilic drugs with low micelle concentration. (14)C labeling of a model drug on one side and of the amphiphile on the other side permitted assessment, after intravenous injection, of biodistribution and excretion profiles of the drug cargo.

Context-Dependent Pharmacological Effects of Metformin on the Immune System.

Metformin is used for the treatment of type 2 diabetes mellitus and has shown therapeutic effects in preclinical models of other pathologies, such as cancer and autoimmune diseases. The antitumor activity of metformin is due, in part, to immunostimulatory effects. In the context of other pathologies, such as autoimmune or inflammatory diseases, metformin has immunosuppressive effects. There is evidence that the immunostimulatory effects of metformin are indirect. The immunosuppressive effects of metformin in other pathologies appear to be a direct consequence of its action on immune cells. Based on these observations we opine that the pharmacology of metformin is dependent on the pathological context which, to our knowledge, is a novel concept in pharmacology.

Leuconostoc mesenteroides glucansucrase synthesis of flavonoid glucosides by acceptor reactions in aqueous-organic solvents.

The enzymatic glucosylation of luteolin was attempted using two glucansucrases: the dextransucrase from Leuconostoc mesenteroides NRRL B-512F and the alternansucrase from L. mesenteroides NRRL B-23192. Reactions were carried out in aqueous-organic solvents to improve luteolin solubility. A molar conversion of 44% was achieved after 24h of reaction catalysed by dextransucrase from L. mesenteroides NRRL B-512F in a mixture of acetate buffer (70%)/bis(2-methoxyethyl) ether (30%). Two products were characterised by nuclear magnetic resonance (NMR) spectroscopy: luteolin-3'-O-alpha-d-glucopyranoside and luteolin-4'-O-alpha-d-glucopyranoside. In the presence of alternansucrase from L. mesenteroides NRRL B-23192, three additional products were obtained with a luteolin conversion of 8%. Both enzymes were also able to glucosylate quercetin and myricetin with conversion of 4% and 49%, respectively.

Anti-Cancer Stem-like Cell Compounds in Clinical Development - An Overview and Critical Appraisal.

Cancer stem-like cells (CSC) represent a subpopulation of tumor cells with elevated tumor-initiating potential. Upon differentiation, they replenish the bulk of the tumor cell population. Enhanced tumor-forming capacity, resistance to antitumor drugs, and metastasis-forming potential are the hallmark traits of CSCs. Given these properties, it is not surprising that CSCs have become a therapeutic target of prime interest in drug discovery. In fact, over the last few years, an enormous number of articles describing compounds endowed with anti-CSC activities have been published. In the meanwhile, several of these compounds and also approaches that are not based on the use of pharmacologically active compounds (e.g., vaccination, radiotherapy) have progressed into clinical studies. This article gives an overview of these compounds, proposes a tentative classification, and describes their biological properties and their developmental stage. Eventually, we discuss the optimal clinical setting for these compounds, the need for biomarkers allowing patient selection, the redundancy of CSC signaling pathways and the utility of employing combinations of anti-CSC compounds and the therapeutic limitations posed by the plasticity of CSCs.

Active targeting with particulate drug carriers in tumor therapy: fundamentals and recent progress.

Drug therapy for the treatment of tumors is often limited by a narrow therapeutic index. One approach that overcomes this limitation is the active targeting of tumors with particulate drug carriers. The derivatization of particulate drug carriers with a ligand leads to the selective targeting of the particulate to selected cells, thereby focusing drug delivery. In addition, particulate drug carriers have a high loading capacity, do not need covalent conjugation of the drug and the formulation protects the entrapped drug from enzymatic inactivation. Despite these favorable properties, their therapeutic efficacy in animal models has been reported only in recent years. The use of internalizing ligands and the targeting of intravascular tumor cells and endothelial cells of tumor blood vessels have been instrumental in demonstrating the clinical effectiveness of particulate drug carriers in animal models. As a result, several actively targeted particulate carriers have now entered, or are about to enter, clinical investigation. Recent findings, for example, the identification of cell-penetrating peptides with restricted cell selectivity, suggest that further improvements in this approach are likely in the near future.

Molecular pharmacology of the ovine melatonin receptor: comparison with recombinant human MT1 and MT2 receptors.

The variations of the pharmacological properties of melatonin receptors between different mammalian species in transfected cell lines have been poorly investigated. In the present study, melatonin analogues have been used to characterize the pharmacology of the recombinant ovine melatonin receptor (oMT1) expressed in CHO cell lines and the native oMT1 from the pars tuberalis (PT). Studies with selective ligands on native and transfected oMT1 showed similar properties for binding affinities [r2(PT/CHO) = 0.85]. The affinities and the functional activities of these ligands were compared with the human receptors (hMT1 or hMT2) expressed in CHO cells as well. The oMT1 and hMT1 receptors had similar pharmacological profiles (r2=0.82). Nevertheless, some of the selective compounds at the human receptor presented a reduced affinity at the ovine receptor. Furthermore, some compounds showed marked different functional activities at oMT1 vs. hMT1 receptors. Our findings demonstrated differences in the pharmacological properties of melatonin receptors in ovine and human species.

New selective ligands of human cloned melatonin MT1 and MT2 receptors.

Melatonin has a key role in the circadian rhythm relay to periphery organs. Melatonin exerts its multiple roles mainly through two seven transmembrane domain, G-coupled receptors, namely MT1 or MT2 receptors. A pharmacological characterization of these human cloned melatonin hMT1 and hMT2 receptors stably expressed in HEK-293 or CHO cells is presented using a 2-[125I]-iodo-melatonin binding assay and a [35S]-GTPgammaS functional assay. Both reference compounds and new chemically diverse ligands were evaluated. Binding affinities at each receptor were found to be comparable on either HEK-293 or CHO cell membranes. Novel non-selective or selective hMT1 and hMT2 ligands are described. The [35S]-GTPgammaS functional assay was used to define the functional activity of these compounds which included partial, full agonist and/or antagonist activity. None of the compounds acted as an inverse agonist. We report new types of selective antagonists, such as S 25567 and S 26131 for MT1 and S 24601 for MT2. These studies brought other new molecular tools such as the selective MT1 agonist, S 24268, as well as the non-selective antagonist, S 22153. Finally, we also discovered S 25150, the most potent melatonin receptor agonist, so far reported in the literature.

Original Design of Fluorescent Ligands by Fusing BODIPY and Melatonin Neurohormone.

An original design and synthesis of fluorescent ligands for melatonin receptor studies is presented and consists in the fusion of the endogenous ligand with the fluorescent BODIPY core. Probes I-IV show high affinities for MT1 and MT2 melatonin receptors and exhibit fluorescence properties compatible with cell observation.

Branching pattern of gluco-oligosaccharides and 1.5kDa dextran grafted by the α-1,2 branching sucrase GBD-CD2.

GBD-CD2, an engineered sucrose-acting enzyme of glycoside hydrolase family 70, transfers D-glucopyranosyl (D-Glcp) units from sucrose onto dextrans or gluco-oligosaccharides (GOS) through the formation of α-(1→2) linkages leading to branched products of interest for health, food and cosmetic applications. Structural characterization of the branched products obtained from sucrose and pure GOS of degree of polymerization (DP) 4 or DP 5 revealed that highly α-(1→2) branched and new molecular structures can be synthesized by GBD-CD2. The formation of α-(1→2) branching is kinetically controlled and can occur onto vicinal α-(1→6)-linked D-Glcp residues. To investigate the mode of branching of 1.5 kDa dextran, simulations of various branching scenarios and resistance to glucoamylase degradation were performed. Analysis of the simulation results suggests that the branching process is stochastic and indicates that the enzyme acceptor site can accommodate both linear and poly-branched acceptors. This opens the way to the design of novel enzyme-based processes yielding carbohydrate structures varying in size and resistance to hydrolytic enzymes.

Drug solubilization and in vitro toxicity evaluation of lipoamino acid surfactants.

To improve solubilization of a water insoluble anticancer drug, novel surfactants were synthesized. All surfactants derived from lysine, with a so-called nitrilo triacetic acid (NTA) polar head, and differed from the length and saturation degree of their hydrophobic moieties: C19:0-NTA, C20:4-NTA, C25:0-NTA and C25:4-NTA. Self-assembling properties and critical micellar concentration (CMC) values were determined using pyrene fluorescence and cytotoxicity using MTT and LDH assays on endothelial cells. Surfactant haemolytic activity and drug solubilization capacity were also evaluated. All surfactants self-assemble with low CMC values from 0.012 to 0.430 mg/mL. Cytotoxicity assays showed that C20:4-NTA and C25:0-NTA were less cytotoxic than polysorbate 80. Unsaturations and alkane chain length have a marked influence on toxicity. Saturated surfactants had a similar haemolytic activity, explained by their low CMC values and the linear configuration of their hydrophobic tail. C20:4-NTA and C25:4-NTA were less haemolytic than polysorbate 80. Furthermore, C19:0-NTA, C25:0-NTA and C25:4-NTA increased drug solubility from <0.15 µg/mL up to 7 mg/mL, with 46% (w/w) drug loading, due to their linear and flexible hydrophobic chain configuration, as evidenced by molecular modelling. Although these solubilizers are promising, a compromise between drug solubilization and toxicity remains to be found.

Physicochemical characterization and toxicity evaluation of steroid-based surfactants designed for solubilization of poorly soluble drugs.

To overcome poor water-solubility of new drug candidates, four innovative surfactants based on naturally-occuring hydrophilic and hydrophobic moities were designed and synthesized: cholesteryl-glutamic acid, cholesteryl-poly[N-2-hydroxyethyl-l-glutamine] (PHEG), ursodeoxycholanyl-PHEG (UDCA-PHEG) and ursodeoxycholanyl-poly-l-glutamic acid (UDCA-PGA). Their self-assembling capacity was evaluated using pyrene fluorescence measurements which allow to determine their critical aggregation concentration (CAC). Size measurements were carried out using dynamic light scattering (DLS). Surfactant cytotoxicity was investigated on human umbilical vein endothelial cells (HUVEC) by determining tetrazolium salt (MTT) activity and lactate dehydrogenase (LDH) release. In addition, surfactant haemolytic activity was assessed using rat red blood cells (RBCs). Finally, the ability of these surfactants to solubilize a model poorly soluble drug was quantified. Surfactant self-assembly, cytotoxicity and solubilization properties were compared to those obtained with polysorbate 80, a model solubilizer. Except for cholesteryl-glutamic acid, surfactants were water-soluble. UDCA-PGA was not able to self-assemble or to increase significantly drug solubility. Results showed that cholesteryl-PHEG and UDCA-PHEG were self-assembling with low CAC values (17 and 120µg/ml) into nano-structures with mean diameters of 13 and 250nm, respectively. Cholesteryl-PHEG was the most efficient surfactant in increasing drug solubility (2mg/ml) but exhibited a similar or higher toxicity than polysorbate 80. UDCA-PHEG did not present any cytotoxicity but was far less efficient to solubilize the drug (0.2mg/ml). These results evidence interesting properties of cholesteryl-PHEG and UDCA-PHEG as novel solubilizers.