Hydrogels containing water soluble conjugates of silver(I) ions with amino acids, metabolites or natural products for non infectious contact lenses.

The poor handling and hygiene practices of contact lenses are the key reasons for their frequent contamination, and are responsible for developing ocular complications, such as microbial keratitis (MK). Thus there is a strong demand for the development of biomaterials of which contact lenses are made, combined with antimicrobial agents. For this purpose, the known water soluble silver(I) covalent polymers of glycine (GlyH), urea (U) and the salicylic acid (SalH2) of formulae [Ag3(Gly)2NO3]n (AGGLY), [Ag(U)NO3]n (AGU), and dimeric [Ag(salH)]2 (AGSAL) were used. Water solutions of AGGLY, AGU and AGSAL were dispersed in polymeric hydrogels using hydroxyethyl-methacrylate (HEMA) to form the biomaterials pHEMA@AGGLY-2, pHEMA@AGU-2, and pHEMA@AGSAL-2. The biomaterials were characterized by X-ray fluorescence (XRF) spectroscopy, thermogravimetric differential thermal analysis (TG-DTA), differential scanning calorimetry (DTG/DSC), attenuated total reflection spectroscopy (FT-IR-ATR) and single crystal diffraction analysis. The antibacterial activity of AGGLY, AGU, AGSAL, pHEMA@AGGLY-2, pHEMA@AGU-2 and pHEMA@AGSAL-2 was evaluated against the Gram negative species Pseudomonas aeruginosa (P. aeruginosa) and Gram positive ones Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus), which mainly colonize in contact lenses. The in vitro toxicity of the biomaterials and their ingredients was evaluated against normal human corneal epithelial cells (HCECs) whereas the in vitro genotoxicity was evaluated by the micronucleus (MN) assay in HCECs. The Artemia salina and Allium cepa models were applied for the evaluation of in vivo toxicity and genotoxicity of the materials. Following our studies, the new biomaterials pHEMA@AGGLY-2, pHEMA@AGU-2, and pHEMA@AGSAL-2 are suggested as efficient candidates for the development of antimicrobial contact lenses.

pHEMA@AGMNA-1: A novel material for the development of antibacterial contact lens.

The Metal Organic Framework (MOF) of formula {[Ag6(µ3-HMNA)4(µ3-MNA)2]2-·[(Et3NH)+]2·(DMSO)2·(H2O)} (AGMNA), a known efficient antimicrobial compound which contains the anti-metabolite, 2-thio-nicotinic acid (H2MNA), was incorporated in polymer hydrogels using, hydroxyethyl-methacrylate (HEMA). The material pHEMA@AGMNA-1 was characterized by X-ray fluorescence (XRF) spectroscopy, X-ray powder diffraction analysis (XRPD), Scanning Electron Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX), Thermogravimetric Differential Thermal Analysis (TG-DTA), Differential Scanning Calorimetry (DTG/DSC), attenuated total reflection spectroscopy (FT-IR-ATR) and Ultrasonic Imaging. The antimicrobial capacity of pHEMA@AGMNA-1 was evaluated against the Gram negative bacterial strain Pseudomonas aeruginosa and the Gram positive ones of the genus of Staphylococcus epidermidis and Staphylococcus aureus, which are the etiology of the microbial keratitis. The % bacterial viability of P. aeruginosa, S. epidermidis and S. aureus upon their incubation with pHEMA@AGMNA-1 discs is significantly low (0.4 ± 0.1%, 1.5 ± 0.4% and 7.7 ± 0.5% respectively). The inhibition zones (IZ) caused by pHEMA@AGMNA-1 discs against P. aeruginosa, S. epidermidis and S. aureus are 14.0 ± 1.1, 11.3 ± 1.3 and 11.8 ± 1.8 mm respectively. Furthermore, pHEMA@AGMNA-1 exhibits low toxicity. Thus, pHEMA@AGMNA-1 might be an efficient candidate for the development of antimicrobial active contact lenses.

An Efficient Disinfectant, Composite Material {SLS@[Zn3(CitH)2]} as Ingredient for Development of Sterilized and Non Infectious Contact Lens.

The [Zn3(CitH)2] (1) (CitH4= citric acid), was dispersed in sodium lauryl sulphate (SLS) to form the micelle of SLS@[Zn3(CitH)2] (2). This material 2 was incorporated in hydrogel made by hydroxyethyl-methacrylate (HEMA), an ingredient of contact lenses, toward the formation of pHEMA@(SLS@[Zn3(CitH)2]) (3). Samples of 1 and 2 were characterized by UV-Vis, 1H-NMR, FT-IR, FT-Raman, single crystal X-ray crystallography, X-ray fluorescence analysis, atomic absorption and TG/DTA/DSC. The antibacterial activity of 1-3 as well as of SLS against Gram-positive (Staphylococcus epidermidis (St. epidermidis) and Staphylococcus aureus (St. aureus)) and Gram-negative (Pseudomonas aeruginosa (PAO1), and Escherichia coli (E. coli)) bacteria was evaluated by the means of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and inhibitory zone (IZ). 2 showed 10 to 20-fold higher activity than 1 against the bacteria tested. Moreover the 3 decreases the abundance of Gram-positive microbes up to 30% (St. aureus) and up to 20% (PAO1) the Gram-negative ones. The noteworthy antimicrobial activity of the obtained composite 3 suggests an effective antimicrobial additive for infection-free contact lenses.

Metabolic profile of human coelomic fluid.

AIM: Till now there is very limited knowledge on the molecular content of coelomic fluid and cells. This study presents the first attempt to elucidate the metabolic profile of such samples. METHODOLOGY: Samples were collected via coelocentesis from 41 women during the first trimester of gestation. Metabolic content was assessed using four different analytical platforms. For targeted analysis a hydrophilic interaction chromatography ultra high performance LC-MS/MS method was applied. Holistic analysis performed by GC-MS, NMR spectroscopy and ion cyclotron ultra-high resolution MS (FT-ICR-MS) instrumentation. RESULTS & CONCLUSIONS: Our observations suggest coelomic fluid and cells as promising biosamples, rich in metabolites with potential use in mammalian system biology studies.

The binding of C5-alkynyl and alkylfurano[2,3-d]pyrimidine glucopyranonucleosides to glycogen phosphorylase b: synthesis, biochemical and biological assessment.

C5-alkynyl and alkylfurano[2,3-d]pyrimidine glucopyranonucleosides have been synthesized and studied as inhibitors of glycogen phosphorylase b (GPb). Kinetic experiments have shown that most of these compounds were low micromolar inhibitors of the enzyme. The best inhibitor was 1-(ß-D-glucopyranosyl)-5-ethynyluracil (K(i)=4.7 µM). Crystallographic analysis of these compounds in complex with GPb revealed that inhibitors with a long C5-alkynyl group exploited interactions with ß-pocket of the active site and induced significant conformational changes of the 280s loop compared to GPb in complex with compounds with a short C5-alkynyl group. The results highlight the importance in the length of the aliphatic groups used to enhance inhibitory potency for the exploitation of the hydrophobic ß-pocket. The best of the inhibitors had also a moderate effect on glycogenolysis in the cellular lever with an IC(50) value of 291.4 µM.

Losartan's affinity to fluid bilayers modulates lipid-cholesterol interactions.

Losartan is an angiotensin II receptor antagonist mainly used for the regulation of high blood pressure. Since it was anticipated that losartan reaches the receptor site via membrane diffusion, the impact of losartan on model membranes has been investigated by small angle X-ray scattering. For this purpose 2-20 mol% losartan was incorporated into dimyristoyl-phosphatidylcholine (DMPC) and palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers and into their binary mixtures with cholesterol in the concentration range of 0 to 40 mol%. Effects of losartan on single component bilayers are alike. Partitioning of losartan into the membranes confers a negative charge to the lipid bilayers that causes the formation of unilamellar vesicles and a reduction of the bilayer thickness by 3-4%. Analysis of the structural data resulted in an estimate for the partial area of losartan, A(Los) ≈ 40 Å(2). In the presence of cholesterol, differences between the effects of losartan on POPC and DMPC are striking. Membrane condensation by cholesterol is retarded by losartan in POPC. This contrasts with DMPC, where an increase of the cholesterol content shifts the partitioning equilibrium of losartan towards the aqueous phase, such that losartan gets depleted from the bilayers from 20 mol% cholesterol onwards. This indicates (i) a chain-saturation dependent competition of losartan with lipid-cholesterol interactions, and (ii) the insolubility of losartan in the liquid ordered phase of PCs. Consequently, losartan's action is more likely to take place in fluid plasma membrane patches rather than in domains rich in cholesterol and saturated lipid species such as in membrane rafts.

Synthesis of novel sulfonamide-1,2,4-triazoles, 1,3,4-thiadiazoles and 1,3,4-oxadiazoles, as potential antibacterial and antifungal agents. Biological evaluation and conformational analysis studies.

The significant antifungal activity of a series of sulfonamide-1,2,4-triazole and 1,3,4-thiazole derivatives against a series of micromycetes, compared to the commercial fungicide bifonazole has been reported. These compounds have also shown a comparable bactericidal effect to that of streptomycin and better activity than chloramphenicol against various bacteria. In view of the potential biological activity of members of the 1,2,4-triazole, 1,3,4-thiadiazole and 1,3,4-oxadiazole ring systems and in continuation of our search for bioactive molecules, we designed the synthesis of a series of novel sulfonamide-1,2,4-triazoles, -1,3,4-thiadiazoles and -1,3,4-oxadiazoles emphasizing, in particular, on the strategy of combining two chemically different but pharmacologically compatible molecules (the sulfomamide nucleus and the five member) heterocycles in one frame. Synthesized compounds were tested in vitro for antibacterial and antifungal activity and some analogues exhibited very promising results especially as antifungal agents. In order to explain structure-activity relationships, conformational analysis was performed for active and less active analogues using NMR spectroscopy and molecular modeling techniques. Furthermore, molecular properties which can be further used as descriptors for SAR studies, were predicted for the synthesized analogues. In general, antifungal activity seems to depend more on the triazol-3-thione moiety rather than the different length of the alkyl chain substitutions.

Synthesis, binding studies and in vivo biological evaluation of novel non-peptide antihypertensive analogues.

AT(1) antagonists (SARTANs) constitute the last generation of drugs for the treatment of hypertension, designed and synthesized to mimic the C-terminal segment of the vasoconstrictive hormone angiotensin II (AngII). They exert their action by blocking the binding of AngII on the AT(1) receptor. Up to date eight AT(1) antagonists have been approved for the regulation of high blood pressure. Although these molecules share common structural features and are designed to act under the same mechanism, they have differences in their pharmacological profiles and antihypertensive efficacy. Thus, there is still a need for novel analogues with better pharmacological and financial profiles. An example of a novel synthetic non peptide AT(1) antagonist which devoids the classical template of SARTANs is MM1. In vivo studies showed that MMK molecules, which fall in the same class of MM1, had a significant antihypertensive (40-80% compared to the drug losartan) activity. However, in vitro affinity studies showed that losartan has considerably higher affinity. The theoretical docking studies showed that MM1 acts on the same site of the receptor as losartan. They exert hydrophobic interactions with amino acid Val108 of the third helix of the AT(1) receptor and other hydrophobic amino acids in spatial vicinity. In addition, losartan favours multiple hydrogen bondings between its tetrazole group with Lys199. These additional interactions may in part explain its higher in vitro binding affinity.

Conformation and bioactivity. Design and discovery of novel antihypertensive drugs.

Peptidomimitism is applied to the medicinal chemistry in order to synthesize drugs that devoid of the disadvantages of peptides. AT1 antagonists constitute a new generation of drugs for the treatment of hypertension designed and synthesized to mimic the C-terminal segment of Angiotensin II and to block its binding action on AT1 receptor. An effort was made to understand the molecular basis of hypertension by studying the conformational analysis of Ang II and its derivatives as well as the AT1 antagonists belonging to SARTANs class of molecules. Such studies offer the possibility to reveal the stereoelectronic factors responsible for bioactivity of AT1 antagonists and to design and synthesize new analogs. An example will be given which proves that drugs with better pharmacological and financial profiles may arise based on this rational design.

Efforts to understand the molecular basis of hypertension through drug:membrane interactions.

Biological membranes play an essential role in the drug action. They constitute the first barrier for drugs to exert their biological action. AT1 antagonists are amphiphilic molecules and are hypothesized to act on AT1 receptor through incorporation (first step) and lateral diffusion through membrane bilayers (second step). Various biophysical methods along with Molecular Modelling were applied in order to explore the plausible two step proposed mechanism of action for this class of antihypertensive drugs.

Losartan's molecular basis of interaction with membranes and AT1 receptor.

Physicochemical methods were used to study the thermal and dynamic changes caused by losartan in the membrane bilayers. In addition, molecular modeling was implemented to explore its topography both in membranes and AT(1) receptor. Its incorporation resulted in the modification of thermal profile of dipalmitoyl phosphatidylcholine (DPPC) bilayers in a concentration dependent way up to 20mol% as it is depicted from the combination of differential scanning calorimetry (DSC) and MAS data. In particular, the presence of losartan caused lowering of the phase transition temperature and abolishment of the pretransition. T(1) experiments revealed the location of the drug into the membrane bilayers. The use of a combination of biophysical methods along with docking experiments brought out a possible two-step mechanism which involves incorporation of losartan at the interface of membrane bilayers and diffusion in the upper parts of AT(1) receptor helices IV-VII.

Design and synthesis of novel antihypertensive drugs.

AT1 antagonists constitute a new generation of drugs for the treatment of hypertension and are designed and synthesized to mimic the C-terminal segment of Angiotensin II (Ang II) and to block its binding action on AT1 receptor. For this reason, the conformational analysis of Ang II and its derivatives as well as the AT1 antagonists belonging to SARTANs class of molecules were studied. Such studies offer the possibility to reveal the stereoelectronic factors responsible for bioactivity of AT1 antagonists and to design and synthesize new analogues with better pharmacological and financial profiles. An example of a novel synthetic non-peptide molecule is given which mimics the His(6)-Pro(7)-Phe(8) part of Ang II and is based on the (S)-pyroglutamic acid.

Conformational and biological studies for a pair of novel synthetic AT1 antagonists: stereoelectronic requirements for antihypertensive efficacy.

One of the major systems which interferes with the disease of hypertension, is the Renin Angiotensin Aldosterone System (RAS). The octapeptide hormone angiotensin II is the active product of RAS which causes vasoconstriction when binds to the AT(1) receptor. In the last years, there has been a development of drugs which block the Angiotensin II from binding the AT(1) receptor and are called AT(1) antagonists. In an effort to comprehend their stereoelectronic features, a study has been initiated to compare the conformational properties of drugs already marketed for the treatment of hypertension and others which are designed and synthesized in our laboratory possessing structural characteristics necessary for antihypertensive activity. In this study, two synthetic non-peptide AT(1) antagonists, are structurally elucidated and their conformational properties and bioactivity are compared to the prototype and first approved drug of this category in the market; losartan (trade name: COZAAR).