In vitro ADME characterization of a very potent 3-acylamino-2-aminopropionic acid-derived GluN2C-NMDA receptor agonist and its ester prodrugs.

The GluN2C subunit exists predominantly, but not exclusively in NMDA receptors within the cerebellum. Antagonists such as UBP1700 and positive allosteric modulators including PYD-106 and 3-acylamino-2-aminopropionic acid derivatives such as UA3-10 ((R)-2-amino-3-{[5-(2-bromophenyl)thiophen-2-yl]carboxamido}propionic acid) represent promising tool compounds to investigate the role of GluN2C-containing NMDA receptors in the signal transduction in the brain. However, due to its high polarity the bioavailability and CNS penetration of the amino acid UA3-10 are expected to be rather low. Herein, three ester prodrugs 12a-c of the NMDA receptor glycine site agonist UA3-10 were prepared and pharmacokinetically characterized. The esters 12a-c showed higher lipophilicity (higher logD 7.4 values) than the acid UA3-10 but almost the same binding at human serum albumin. The acid UA3-10 was rather stable upon incubation with mouse liver microsomes and NADPH, but the esters 12a-c were fast hydrolyzed to afford the acid UA3-10. Incubation with pig liver esterase and mouse serum led to rapid hydrolysis of the esters 12a-c. The isopropyl ester 12c showed a promising logD 7.4 value of 3.57 and the highest stability in the presence of pig liver esterase and mouse serum. These results demonstrate that ester prodrugs of UA3-10 can potentially afford improved bioavailability and CNS penetration.

Diastereoselective synthesis and structure-affinity relationships of σ1 receptor ligands with spirocyclic scaffold.

Spirocyclic scaffolds play an increasing role in drug discovery as they define a rigid three-dimensional space to increase specific interactions with protein binding sites. Herein, a spirocyclic center was introduced into the lead compound 1 to rigidify its flexible benzylaminoethyl side chain. The key step of the synthesis was the reaction of different α,ß-unsaturated amides 6 and 13-16 with methyl acrylate in the presence of TBDMSOTf. DFT calculations explain the mechanism of this transformation as concerted Diels-Alder reaction (functionals B3LYP and TPSS) or double (aza)-Michael addition (functionals PBE and wB97X-D). After separation of the diastereomeric spirocyclic products 8 and 17-20, LiAlH4 reduction provided the spirocyclic hydroxymethyl piperidines 21a,b-25a,b showing low nanomolar σ1 affinity (Ki < 100 nM). trans-Configured ligands (a-series) showed higher or equal σ1 affinity and higher selectivity over σ2 receptors and GluN2B-NMDA receptors than their cis-configured analogs (b-series). The additional hydroxymethyl moiety brings the log D7.4 value in a promising range. The high σ1 affinity (Ki = 3.6 nM) and the low lipophilicity result in the highest lipophilic ligand efficiency for the dispiro compound 23a (LLE = 6.0). The spirocyclic compounds reported herein and in particular the dispiro compound 23a demonstrate that ligands containing a large number of sp3 C-atoms possess favorable pharmacological (σ1 receptor affinity, receptor selectivity) and physicochemical properties (log D7.4 value) resulting in promising LLE.

Negative allosteric modulators of NMDA receptors with GluN2B subunit: synthesis of ß-aminoalcohols by epoxide opening and subsequent rearrangement.

In order to obtain novel antagonists of GluN2B subunit containing NMDA receptors, aryloxiranes were opened with benzylpiperidines. Phenyloxiranes 6 and (indazolyl)oxirane 15 were opened regioselectively at the position bearing the aryl moiety. Reaction of the resulting ß-aminoalcohols 7 and 16 with carboxylic acids under Mitsunobu conditions (DIAD, PPh3) led to rearrangement and after ester hydrolysis to the regioisomeric ß-aminoalcohols 9 and 18. This strategy allows the synthesis of amino-ifenprodil 12 as well using phthalimide in the Mitsunobu reaction. Unexpectedly, the isomeric (indazolyl)oxirane 21 reacted with benzylpiperidines to afford both regioisomeric ß-aminoalcohols 22 and 23. In radioligand receptor binding studies, the indazolyl derivative 18a, which can be regarded as indazole bioisostere of ifenprodil, showed high GluN2B affinity (Ki = 31 nM). Replacement of the benzylic OH moiety of ifenprodil by the NH2 moiety in amino-ifenprodil 12 also resulted in low nanomolar GluN2B affinity (Ki = 72 nM). In TEVC experiments, 18a inhibited the ion flux to the same extent as ifenprodil proving that the phenol of ifenprodil can be replaced bioisosterically by an indazole ring maintaining affinity and inhibitory activity. Whereas 10-fold selectivity was found for the ifenprodil binding site over σ1 receptors, only low preference for the GluN2B receptor over σ2 receptors was detected. The log D7.4 value of 18a (log D7.4 = 2.08) indicates promising bioavailability.

Multifunctional cubic liquid crystalline nanoparticles for chemo- and photodynamic synergistic cancer therapy.

With the aim of engineering multifunctional nanoparticles useful for cancer therapy, a diketopyrrolopyrrole-porphyrin based photosensitizer was here conjugated to a block copolymer (Pluronic F108), and used to stabilize in water lipidic cubic liquid crystalline nanoparticles (cubosomes), also loaded with the antineoplastic agent docetaxel. The physicochemical characterization by SAXS, DLS, and cryo-TEM demonstrated that the formulation consisted of cubosomes, about 150 nm in size, possessing a bicontinuous cubic structure (space group Pn3m). The cellular imaging experiments proved that these nanoparticles localized in lysosomes and mitochondria, while cytotoxicity tests evidenced a slight but significant synergistic effect which, after irradiation, increased the toxicity induced by docetaxel alone, allowing further reduction of cell viability.

Cubosomes for in vivo fluorescence lifetime imaging.

Herein we provided the first proof of principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. This formulation, administered at a non-cytotoxic concentration, was capable of providing both exogenous contrast for NIR fluorescence imaging with very high efficiency and chemospecific information upon lifetime analysis. Time-resolved measurements of fluorescence after the intravenous injection of cubosomes revealed that the dye rapidly accumulated mainly in the liver, while lifetimes profiles obtained in vivo allowed for discriminating between free dye or dye embedded within the cubosome nanostructure after injection.

Dual-Responsive Lipid Nanotubes: Two-Way Morphology Control by pH and Redox Effects.

Lipid nanotubes are the preferred structures for many applications, especially biological ones, and thus have attracted much interest recently. However, there is still a significant need for developing more lipid nanotubes that are reversibly controllable to improve their functionality and usability. Here, we presented a two-way reversible morphology control of the nanotubes formed by the recently designed molecule AQUA (C25H29NO4). Because of its special design, the AQUA has both pH-sensitive and redox-active characters provided by the carboxylic acid and anthraquinone groups. Upon chemical reduction, the nanotubes turned into thinner ribbons, and this structural transformation was significantly reversible. The reduction of the AQUA nanotubes also switched the nanotubes from electrically conductive to insulative. Nanotube morphology can additionally be altered by decreasing the pH below the pKa value of the AQUA, at ∼4.9. Decreasing the pH caused the gradual unfolding of the nanotubes, and the interlayer distance in the nanotube's walls increased. This morphological change was fast and reversible at a wide pH range, including the physiological pH. Thus, the molecular design of the AQUA allowed for an unprecedented two-way and reversible morphology control with both redox and pH effects. These unique features make AQUA a very promising candidate for many applications, ranging from electronics to controlled drug delivery.

Docetaxel-Loaded Fluorescent Liquid-Crystalline Nanoparticles for Cancer Theranostics.

Here, we describe a novel monoolein-based cubosome formulation engineered for possible theranostic applications in oncology. The Docetaxel-loaded nanoparticles were stabilized in water by a mixture of commercial Pluronic (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer) F108 (PF108) and rhodamine- and folate-conjugated PF108 so that the nanoparticles possess targeting, therapeutic, and imaging properties. Nanoparticles were investigated by DLS, cryo-TEM, and SAXS to confirm their structural features. The fluorescent emission characterization of the proposed formulation indicated that the rhodamine conjugated to the PF108 experiences an environment less polar than water (similar to chloroform), suggesting that the fluorescent fragment is buried within the poly(ethylene oxide) corona surrounding the nanoparticle. Furthermore, these nanoparticles were successfully used to image living HeLa cells and demonstrated a significant short-term (4 h incubation) cytotoxicity effect against these cancer cells. Furthermore, given their analogy as nanocarriers for molecules of pharmaceutical interest and to better stress the singularities of these bicontinuous cubic nanoparticles, we also quantitatively evaluated the differences between cubosomes and multilamellar liposomes in terms of surface area and hydrophobic volume.

Scanning-tunneling-spectroscopy-directed design of tailored deep-blue emitters.

Frontier molecular orbitals can be visualized and selectively set to achieve blue phosphorescent metal complexes. For this purpose, the HOMOs and LUMOs of tridentate Pt(II) complexes were measured using scanning tunneling microscopy and spectroscopy. The introduction of electron-accepting or -donating moieties enables independent tuning of the frontier orbital energies, and the measured HOMO-LUMO gaps are reproduced by DFT calculations. The energy gaps correlate with the measured and the calculated energies of the emissive triplet states and the experimental luminescence wavelengths. This synergetic interplay between synthesis, microscopy, and spectroscopy enabled the design and realization of a deep-blue triplet emitter. Finding and tuning the electronic "set screws" at molecular level constitutes a useful experimental method towards an in-depth understanding and rational design of optoelectronic materials with tailored excited state energies and defined frontier-orbital properties.

Cancer-cell-targeted theranostic cubosomes.

This work was devoted to the development of a new type of lipid-based (cubosome) theranostic nanoparticle able to simultaneously host camptothecin, a potent anticancer drug, and a squarain-based NIR-emitting fluorescent probe. Furthermore, to confer targeting abilities on these nanoparticles, they were dispersed using mixtures of Pluronic F108 and folate-conjugated Pluronic F108 in appropriate ratios. The physicochemical characterization, performed via SAXS, DLS, and cryo-TEM techniques, proved that aqueous dispersions of such cubosomes can be effectively prepared, while the photophysical characterization demonstrated that these nanoparticles may be used for in vivo imaging purposes. The superior ability of these innovative nanoparticles in targeting cancer cells was emphasized by investigating the lipid droplet alterations induced in HeLa cells upon exposure to targeted and nontargeted cubosomes.

Impact of physicians' attitude to vaccination on local vaccination coverage for pertussis and measles in Germany.

BACKGROUND: Vaccination rates of children in Germany are unsatisfying and regional endemic outbreaks have been reported. Few studies have analysed physicians' attitude towards vaccination. We investigated whether there is an association between physicians' attitude and vaccination coverage on the regional level for Germany. METHODS: In a representative cross-sectional survey, anonymized questionnaires were sent to random samples of all paediatricians (50%) and general practitioners (10%) in private practice in Germany. Attitude towards vaccination was operationalized in three scores. Measles and pertussis vaccination coverage rates were obtained from the 16 Federal States' Health Departments. Geographic methods and linear regression models were used for analysis. RESULTS: A total of 2010 paediatricians (response proportion: 64.1%) and 1712 general practitioners (response proportion 39.1%) were included in the analysis. We found an association of physicians' attitude towards vaccination and vaccination coverage rate (P < 0.0001). There is also an important association between vaccination coverage and the geographic location, with lower coverage rates especially in the States of former Western Germany (compared with our reference State Mecklenburg-Western Pomerania; pertussis: maximum -5.86% in Bavaria, P < 0.0001; measles: maximum -20.20% in Berlin, P = 0.0002). CONCLUSIONS: The regional association between vaccination coverage rates and physicians' attitude towards vaccination seems to be superposed by population-related variables. An increase of vaccination coverage requires better information and training of both, physicians and the general population.

[18F]Fluspidine-A PET Tracer for Imaging of σ1 Receptors in the Central Nervous System.

σ1 receptors play a crucial role in various neurological and neurodegenerative diseases including pain, psychosis, Alzheimer's disease, and depression. Spirocyclic piperidines represent a promising class of potent σ1 receptor ligands. The relationship between structural modifications and σ1 receptor affinity and selectivity over σ2 receptors led to the 2-fluoroethyl derivative fluspidine (2, Ki = 0.59 nM). Enantiomerically pure (S)-configured fluspidine ((S)-2) was prepared by the enantioselective reduction of the α,ß-unsaturated ester 23 with NaBH4 and the enantiomerically pure co-catalyst (S,S)-24. The pharmacokinetic properties of both fluspidine enantiomers (R)-2 and (S)-2 were analyzed in vitro. Molecular dynamics simulations revealed very similar interactions of both fluspidine enantiomers with the σ1 receptor protein, with a strong ionic interaction between the protonated amino moiety of the piperidine ring and the COO- moiety of glutamate 172. The 18F-labeled radiotracers (S)-[18F]2 and (R)-[18F]2 were synthesized in automated syntheses using a TRACERlab FX FN synthesis module. High radiochemical yields and radiochemical purity were achieved. Radiometabolites were not found in the brains of mice, piglets, and rhesus monkeys. While both enantiomers revealed similar initial brain uptake, the slow washout of (R)-[18F]2 indicated a kind of irreversible binding. In the first clinical trial, (S)-[18F]2 was used to visualize σ1 receptors in the brains of patients with major depressive disorder (MDD). This study revealed an increased density of σ1 receptors in cortico-striato-(para)limbic brain regions of MDD patients. The increased density of σ1 receptors correlated with the severity of the depressive symptoms. In an occupancy study with the PET tracer (S)-[18F]2, the selective binding of pridopidine at σ1 receptors in the brain of healthy volunteers and HD patients was shown.

Drug-loaded fluorescent cubosomes: versatile nanoparticles for potential theranostic applications.

In this work, monoolein-based cubosomes were doped with two fluorescent probes, namely, fluorescein and dansyl, properly modified with a hydrocarbon chain to increase their encapsulation efficiency within the monoolein palisade. The same nanocarriers were also loaded with quercetin, a hydrophobic molecule with potential anticancer activity. Particularly, the cubosomes doped with the modified fluorescein probe were successfully exploited for single living cell imaging. The physicochemical and photophysical characterizations reported here, along with the well-known ability of cubosomes in hosting molecules with pharmaceutical interest, strongly encourage the use of these innovative fluorescent nanocarriers for theranostic purposes.

An OFF-ON chemosensor for biological and environmental applications: sensing Cd2+ in water using catanionic vesicles and in living cells.

A new OFF-ON fluorescent chemosensor (L(1)) for Cd(2+) recognition based on a 5-chloro-8-hydroxyquinoline pendant arm derivative of 1,4,7-triazacyclononane ([9]aneN3) will be presented and its photochemical features in an MeCN-H2O 1 : 1 (v/v) mixture, in pure water, after inclusion within catanionic vesicles, and in living cells will be discussed. The coordination properties of L(1) both in solution and in the solid state were preliminarily studied and its selectivity towards Cd(2+)versus a set of different metal ions (Cu(2+), Zn(2+), Cd(2+), Pb(2+), Al(3+), Hg(2+), Co(2+), Ni(2+), Mn(2+), Mg(2+), K(+), Ca(2+), Ag(+), and Na(+)) was verified in MeCN-H2O 1 : 1 (v/v). In water, upon addition of increasing amounts of Cd(2+) to L(1) an enhancement of the fluorescence emission was detected. To overcome this serious drawback, L(1) was dissolved in an innovative catanionic vesicular solution based on sodium bis(2-ethylhexyl) sulfosuccinate, a traditional surfactant, and 1-dodecyl-3-methylimidazolium bromide, an ionic liquid. When enclosed within the vesicle bilayers in water, L(1) restored its fluorescence emission property upon addition of Cd(2+). Remarkably, L(1) enters the cellular membrane of living cells thus allowing the detection of intracellular Cd(2+). These findings encourage the application of this new fluorescent chemosensor in real samples for histological and environmental analyses.

Conformationally Restricted σ1 Receptor Antagonists from (-)-Isopulegol.

Antagonists at σ1 receptors have great potential for the treatment of neuropathic pain. Starting from monoterpene (-)-isopulegol (1), aminodiols 8-11 were obtained and transformed into bicyclic 13-16 and tricyclic ligands 19-22. Aminodiols 8-11 showed higher σ1 affinity than the corresponding bicyclic 13-16 and tricyclic derivatives 19-22. (R)-configuration in the side chain of aminodiols (8 and 10) led to higher σ1 affinity than (S)-configuration (9 and 11). 4-Benzylpiperidines (b-series) revealed higher σ1 affinity than 4-phenylbutylamines (a-series). Aminodiol 8b showed very high σ1 affinity (Ki = 1.2 nM), excellent selectivity over σ2 receptors, and promising logD7.4 (3.05) and lipophilic ligand efficiency (5.87) values. Molecular dynamics simulations were conducted to analyze the σ1 affinity and selectivity on an atomistic level. In the capsaicin assay, 8b exhibited similar antiallodynic activity to the prototypical σ1 antagonist S1RA. The antiallodynic activity of 8b was removed by co-application of the σ1 agonist PRE-084, proving σ1 antagonism being involved in the antiallodynic effect.

Indazole as a Phenol Bioisostere: Structure-Affinity Relationships of GluN2B-Selective NMDA Receptor Antagonists.

Negative allosteric modulation of GluN2B subunit-containing NMDA receptors prevents overstimulation, resulting in neuroprotective effects. Since the phenol of prominent negative allosteric modulators is prone to rapid glucuronidation, its bioisosteric replacement by an indazole was envisaged. The key step in the synthesis was a Sonogashira reaction of non-protected iodoindazoles with propargylpiperidine derivatives. Modification of the alkynyl moiety allowed the introduction of several functional groups. The synthesized indazoles showed very high GluN2B affinity but limited selectivity over σ receptors. Molecular dynamics simulations revealed the same molecular interactions with the ifenprodil binding site as the analogous phenols. In two-electrode voltage-clamp experiments, enantiomeric 3-(4-benzylpiperidin-1-yl)-1-(1H-indazol-5-yl)propan-1-ols (S)-10a and (R)-10a displayed higher inhibitory activity than ifenprodil. In contrast to phenolic GluN2B antagonists, the indazoles were not conjugated with glucuronic acid. It can be concluded that the phenol of potent GluN2B antagonists can be replaced bioisosterically by an indazole, retaining the high GluN2B affinity and activity but inhibiting glucuronidation.

Grey parrots use inferential reasoning based on acoustic cues alone.

Our ability to make logical inferences is considered as one of the cornerstones of human intelligence, fuelling investigations of reasoning abilities in non-human animals. Yet, the evidence to date is equivocal, with apes as the prime candidates to possess these skills. For instance, in a two-choice task, apes can identify the location of hidden food if it is indicated by a rattling noise caused by the shaking of a baited container. More importantly, they also use the absence of noise during the shaking of the empty container to infer that this container is not baited. However, since the inaugural report of apes solving this task, to the best of our knowledge, no comparable evidence could be found in any other tested species such as monkeys and dogs. Here, we report the first successful and instantaneous solution of the shaking task through logical inference by a non-ape species, the African grey parrot. Surprisingly, the performance of the birds was sensitive to the shaking movement: they were successful with containers shaken horizontally, but not with vertical shaking resembling parrot head-bobbing. Thus, grey parrots seem to possess ape-like cross-modal reasoning skills, but their reliance on these abilities is influenced by low-level interferences.

Composition and properties of complexes between spherical polycationic brushes and anionic liposomes.

A spherical polycationic brush (SPB) is made by graft-polymerizing a cationic monomer onto the surface of a 100 nm polystyrene bead. It is possible to adsorb anionic liposomes (40-60 nm diameter) onto the SPBs while maintaining the liposome integrity. The liposomes were constructed with phosphatidyl choline (PC) admixed with 0.05-0.4 mol fraction of an dianionic lipid, cardiolipin (CL(2-)). As shown by electrophoretic mobility measurements, SPB-to-liposome complexation leads to a conversion from the initial positive charge of the copolymer to a negative charge. The higher the CL(2-) content of the liposomes, the lower the concentration needed for charge neutralization. Dynamic light scattering (DLS) revealed that multicomplex aggregates are formed with a maximum size at the SPB/liposome charge-equivalence point. Experiments with fluorescent-labeled liposomes show that at low CL(2-) content about 80 liposomes are adsorbed per SPB. As the mole fraction of CL(2-) increases from 0.05 to 0.4, fewer liposomes adsorb owing to electrostatic repulsion among neighboring liposomes. The effect of added NaCl also depends upon the CL(2-) content. With 0.05 mol fraction CL(2-), the SPB/liposome complex dissociates into its components at 0.15 M NaCl. With a mole fraction of >0.1, complexes fail to dissociate even at 1.2 M NaCl. Additional information about the SPB/liposome morphology was obtained from cryo-TEM. For example, cryo-TEM data confirm liposome integrity upon complexation, a behavior that contrasts with the liposome destruction as found with adsorption to many other types of surfaces.

When trade law meets public health evidence: the World Trade Organization and clove cigarettes.

A recent trade dispute between the USA and Indonesia, overseen by the World Trade Organization, challenges piecemeal approaches to tobacco regulation.

The effect of intercalants on the host liposome.

When phospholipids are vigorously dispersed in water, liposomes are formed. In the present study, we have explored the effect of intercalant concentration on various properties of unilamellar liposomes. Liposomes were sonically intercalated with vitamin E acetate (VitEAc) and hypericin (Hy) until no difference in light transmission was observed, which reflects the formation of liposomes of minimal diameter. Our studies indicate that the intercalant structure and concentration have an influence on the liposome diameter, which could be directly measured by cryogenic transmittance electronic microscopy. Thus, intercalated VitEAc substantially decreased the diameter of unilamellar dimyristoylphosphatidylcholine liposomes, whereas Hy did not. In addition, we followed peak intensities in the absorbance and fluorescence spectra of Hy as a function of intercalant concentration in the liposomal solution. Initially, the fluorescence intensity increased linearly with concentration; however, the curve then arched asymptotically, followed by a decrease in fluorescence at yet higher concentrations. Because the Hy monomer is the only species that emits fluorescence, we believe that the decrease of fluorescence intensity is the result of Hy aggregation.

Liposome fusion rates depend upon the conformation of polycation catalysts.

Cryo-TEM and NaCl-leakage experiments demonstrated that the cationic polymer polylysine induces fusion of anionic liposomes but that the cationic polymer poly(N-ethyl-4-vinylpyridinium bromide) (PEVP) does not, although both polymers bind strongly to the liposomes. The difference was traced to the thickness of the coatings at constant charge coverage. Polylysine is believed to form planar ß-sheets that are sufficiently thin to allow membrane fusion. In contrast, looping and disorganization among adsorbed PEVP molecules physically prevent fusion. A similar effect is likely to be applicable to important polycation-induced fusion of cell membranes.