Ethosomal Gel for Topical Administration of Dimethyl Fumarate in the Treatment of HSV-1 Infections.

The infections caused by the HSV-1 virus induce lesions on the lips, mouth, face, and eye. In this study, an ethosome gel loaded with dimethyl fumarate was investigated as a possible approach to treat HSV-1 infections. A formulative study was conducted, evaluating the effect of drug concentration on size distribution and dimensional stability of ethosomes by photon correlation spectroscopy. Ethosome morphology was investigated by cryogenic transmission electron microscopy, while the interaction between dimethyl fumarate and vesicles, and the drug entrapment capacity were respectively evaluated by FTIR and HPLC. To favor the topical application of ethosomes on mucosa and skin, different semisolid forms, based on xanthan gum or poloxamer 407, were designed and compared for spreadability and leakage. Dimethyl fumarate release and diffusion kinetics were evaluated in vitro by Franz cells. The antiviral activity against HSV-1 was tested by plaque reduction assay in Vero and HRPE monolayer cells, while skin irritation effect was evaluated by patch test on 20 healthy volunteers. The lower drug concentration was selected, resulting in smaller and longer stable vesicles, mainly characterized by a multilamellar organization. Dimethyl fumarate entrapment in ethosome was 91% w/w, suggesting an almost total recovery of the drug in the lipid phase. Xanthan gum 0.5%, selected to thicken the ethosome dispersion, allowed to control drug release and diffusion. The antiviral effect of dimethyl fumarate loaded in ethosome gel was demonstrated by a reduction in viral growth both 1 h and 4 h post-infection. Moreover, the patch test demonstrated the safety of the ethosomal gel applied on the skin.

Physicochemical Properties, Fatty Acid Composition, Volatile Compounds of Blueberries, Cranberries, Raspberries, and Cuckooflower Seeds Obtained Using Sonication Method.

Every year, thousands of tons of fruit seeds are discarded as agro-industrial by-products around the world. Fruit seeds are an excellent source of oils, monounsaturated fatty acids, and n-6 and n-3 polyunsaturated essential fatty acids. This study aimed to develop a novel technology for extracting active substances from selected seeds that were obtained after pressing fruit juices. The proposed technology involved sonification with the use of ethyl alcohol at a low extraction temperature. Seeds of four species-blueberry (Vaccinium myrtillus L.), raspberry (Rubus idaeus), cranberry (Vaccinium macrocarpon), and cuckooflower (Cardamine pratensis)-were used for extraction. Following alcohol evaporation under nitrogen, the antioxidant activity, chemical composition, and volatile compounds of the obtained extracts were analyzed using chromatographic methods, including gas chromatography (GC)-mass spectrometry (MS) (GC-MS/MS), and high-performance liquid chromatography-MS. We analyzed physicochemical properties, fatty acid, and volatile compounds composition, sterol and tocochromanol content of blueberry, cranberry, raspberry, and cuckooflower seed oils obtained by sonication. This method is safe and effective, and allows for obtaining valuable oils from the seeds.

Influence of phenothiazine molecules on the interactions between positively charged poly-l-lysine and negatively charged DPPC/DPPG membranes.

Phenothiazines are very effective antipsychotic drugs, which also have anticancer and antimicrobial activities. Despite being used in human treatment, the molecular mechanism of the biological actions of these molecules is not yet understood in detail. The role of the interactions between phenothiazines and proteins or lipid membranes has been much discussed. Herein, fourier-transform infrared (FTIR) spectroscopic studies were used to investigate the effect of three phenothiazines: fluphenazine (FPh); chlorpromazine (ChP); and propionylpromazine (PP) on the structures of a positively charged poly-l-lysine (PLL) peptide, a negatively charged dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) membrane, and on the mutual interactions between electrostatically associated PLL molecules and DPPC/DPPG membranes. Phenothiazine-induced alterations in the secondary structure of PLL, the conformational state (trans/gauche) of the hydrocarbon lipid chains, and the hydration of the DPPC/DPPG membrane interface were studied on the basis of amide I' vibrations, antisymmetric and symmetric stretching vibrations of the CH2 groups of the lipid hydrocarbon chains (νsCH2), and stretching vibrations of the lipid C=O groups (νC = O), respectively. It was shown that in the presence of negatively charged DPPC/DPPG membranes, the phenothiazines were able to modify the secondary structure of charged PLL molecules. Additionally, the effect of PLL on the structure of DPPC/DPPG membranes was also altered by the presence of the phenothiazine molecules.

Effect of phenothiazine compounds on the secondary structure and fibrillogenesis of poly-l-lysine.

Phenothiazine molecules are effective and commonly used antipsychotic drugs, especially in the treatment of schizophrenia. However, they produce strong extrapyramidal side-effects manifested by drug-induced parkinsonism. Because Parkinson's disease as a neurodegenerative illness is associated with the formation of amyloid fibrils in neuronal cells, it is postulated that the development of phenothiazine-induced parkinsonism may be related to the phenothiazine-induced formation of fibrillar aggregates. The effect of phenothiazine compounds (fluphenazine (FPh), chlorpromazine (ChP) and propionylpromazine (PP)) on the fibrillogenesis of poly-l-lysine (PLL) was studied using Fourier-transform infrared (FTIR) spectroscopy supported by principal component analysis (PCA), vibrational circular dichroism (VCD), transmission electron microscopy (TEM) and Congo red binding assay. The fibrillogenesis of PLL is accompanied by fibril formation with charged or uncharged polypeptides with PPII (polyproline-like extended helix), α-helix or ß-sheet conformations. All of the phenothiazine molecules investigated effectively reduced the temperature required to induce the formation of ß-sheet-rich fibrils from α-helix-rich fibrils of PLL.

Resveratrol modulates fibrillogenesis in a poly­l­lysine peptide.

Resveratrol (Res) is an effective inhibitor of amyloid fibril formation and reduces neuron cell toxicity. The effect of Res on the fibrillogenesis of a polar polypeptide, poly­l­lysine (PLL), was studied by Fourier-transform infrared spectroscopy, vibrational circular dichroism and transmission electron microscopy. Res molecules exhibited strong and specific inhibition of ß-sheet-rich fibrils formed by PLL. Side chain-side chain hydrophobic interactions of Lys side chains in aggregated ß-sheet structures of PLL stabilize this aggregated state, and this interaction is targeted by Res via hydrophobic interactions. The effect of Res on PLL and other previously reported proteins/peptides sequences with fewer polar amino acids reveals that Res shows rather low sequence specificity. Instead, Res targets sequences that support strong hydrophobic interactions. The fibril-inhibition activity of Res, which is specific toward ß-sheet-rich fibrils of proteins/peptides and rather non-specific to the amino acid sequence, indicates that Res is a very promising drug candidate for effective treatment of amyloid-related diseases.

Influence of resveratrol on interactions between negatively charged DPPC/DPPG membranes and positively charged poly-l-lysine.

Resveratrol (Res), a natural polyphenol present in different plants and vegetables, exhibits potential therapeutic activity with cardioprotective, antineurodegenerative, antioxidant, and antitumor action. In this study, the effect of Res on the mutual interactions between positively charged poly-l-lysine (PLL) and negatively charged dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) membranes was studied using Fourier-transform infrared (FTIR) spectroscopy supported by principal component analysis (PCA). The interactions between PLL and DPPC/DPPG membranes were strongly affected by the presence of Res molecules. Depending on the Res concentration and method of its supply (through the water or lipid phase) to the studied peptide-membrane systems, the membrane-induced transition of PLL from an α-helix to an extended left-handed polyproline II helix (PPII) occurred at different temperatures, with different cooperativity, or was even completely suppressed. The influence of PLL on the conformational (trans/gauche) state of the hydrocarbon chain region of the lipid membranes and the hydration state of the polar/apolar membrane interface was also modulated by Res, depending on the membrane phase state.

Interaction of anesthetic molecules with α-helix and polyproline II extended helix of long-chain poly-l-lysine.

The effect of halothane, enflurane, sevoflurane, and isoflurane molecules, as volatile anesthetics, on the α-helices and polyproline II extended helices (PPII) of long-chain poly-l-lysine (PLL) were studied using Fourier-transform infrared and vibrational circular dichroism spectroscopy. Uncharged and charged α-helices, as well as charged extended PPII helices, were subjected to anesthetic actions in solvents with different pD values or methanol to water ratios. A crucial factor responsible for hindering the anesthetic-PLL interactions is shown to be the ionization of amino groups of the PLL side chains. The α-helix to ß-sheet transition was triggered only for the uncharged α-helical structures of PLL by the nonpolar anesthetics under study.

Ethanol- and trifluoroethanol-induced changes in phase states of DPPC membranes. Prodan emission-excitation fluorescence spectroscopy supported by PARAFAC analysis.

It has been shown that Prodan emission-excitation fluorescence spectroscopy supported by Parallel Factor (PARAFAC) analysis is a fast, simple and sensitive method used in the study of the phase transition from the noninterdigitated gel (Lß') state to the interdigitated gel (LßI) phase, triggered by ethanol and 2,2,2-trifluoroethanol (TFE) molecules in dipalmitoylphosphatidylcholines (DPPC) membranes. The relative contribution of lipid phases with spectral characteristics of each pure phase component has been presented as a function of an increase in alcohol concentration. It has been stated that both alcohol molecules can induce a formation of the LßI phase, but TFE is over six times stronger inducer of the interdigitated phase in DPPC membranes than ethanol molecules. Moreover, in the TFE-mixed DPPC membranes, the transition from the Lß' to LßI phase is accompanied by a formation of the fluid phase, which most probably serves as a boundary phase between the Lß' and LßI regions. Contrary to the three phase-state model of TFE-mixed DPPC membranes, in ethanol-mixed DPPC membranes only the two phase-state model has been detected.

The PPII-to-α-helix transition of poly-l-lysine in methanol/water solvent mixtures accompanied by fibrillar self-aggregation: An influence of fluphenazine molecules.

Fourier-transform infrared, vibrational circular dichroism spectroscopy and transmission electron microscopy are used to follow the structural changes of pure and fluphenazine (FPh)-mixed poly-l-lysine (PLL) triggered by variations of the methanol to water ratio in solvent mixtures. FPh molecules are used as an effective psychotic drug but with a strong Parkinson's-related side effect. To answer the question whether FPh molecules can modify the fibril development, the PLL polypeptide was used as a model of α-helix- and PPII-rich fibrils. It was stated that the presence of FPh molecules did not inhibit the creation of both types of PLL fibrils with clustering features. The methanol-poor aqueous solutions promote the formation of extended polyproline II (PPII) helices; however, the methanol-rich aqueous solutions induce the development of α-helices of both pure and FPh-mixed PLL. Unpredicted and interesting features of PLL fibrillogenesis are evidenced by the formation of uncommon fibrillar aggregates, which are developed in methanol/water solvents from PLL molecules rich in either α-helix or PPII structures. Possibility of PLL molecules to form ß-sheet-, α-helix- and PPII-rich fibrils demonstrating that fibrillogenesis is a common phenomenon, and fibrillar aggregates can be based on all of the basic protein secondary structures.

Anesthetic-dependent changes in the chain-melting phase transition of DPPG liposomes studied using near-infrared spectroscopy supported by PCA.

The effect of inhalation anesthetics (enflurane, isoflurane, sevoflurane or halothane) on the lipid chain-melting phase transition of negatively charged phospholipid membranes was studied using near-infrared (NIR) spectroscopy supported by Principal Component Analysis (PCA). NIR spectra of anesthetics-mixed dipalmitoylphosphatidylglycerol (DPPG) membranes were recorded in a range of the first overtone of the symmetric and antisymmetric stretching vibrations of CH2 groups of lipid aliphatic chains as a function of increasing temperature. Anesthetic-dependent changes in the trans to gauche conformers ratio of CH2 groups in the hydrocarbon lipid chains were characterized in detail and compared with the zwitterionic lipid membranes, which were built of dipalmitoylphosphatidylcholine (DPPC) molecules.

Alpha-helix to beta-sheet transition in long-chain poly-l-lysine: Formation of alpha-helical fibrils by poly-l-lysine.

The temperature-induced α-helix to ß-sheet transition in long-chain poly-l-lysine (PLL), accompanied by the gauche-to-trans isomerization of CH2 groups in the hydrocarbon side chains of Lys amino acid residues, and formation of ß-sheet as well as α-helix fibrillar aggregates of PLL have been studied using Fourier-transform infrared (FT-IR) and vibrational circular dichroism (VCD) spectroscopy, and transmission electron microscopy (TEM). In a low-temperature alkaline water solution or in a methanol-rich water mixture, the secondary structure of PLL is represented by α-helical conformations with unordered and gauche-rich hydrocarbon side chains. Under these conditions, PLL molecules aggregate into α-helical fibrils. PLLs dominated by extended antiparallel ß-sheet structures with highly ordered trans-rich hydrocarbon side chains are formed in a high-temperature range at alkaline pD and aggregate into fibrillar, protofibrillar, and spherical forms. Presented data support the idea that fibrillar aggregation is a varied phenomenon possible in repetitive structural elements with not only a ß-sheet-rich conformation, but also an α-helical-rich conformation.

Laurdan emission study of the cholesterol-like effect of long-chain alkylresorcinols on the structure of dipalmitoylphosphocholine and sphingomyelin membranes.

Long-chain alkylresorcinols (ARs) are commonly found in plant and bacteria cells, and they exhibit a wide variety of biological effects, including antifungal, antitumor, and antiphrastic activities. The cholesterol (Chol)-like effect of ARs with hydrocarbon side-chain lengths ranging from C15 to C25 on the structure of pure and Chol-doped dipalmitoylphosphocholine (DPPC) and sphingomyelin (SM) membranes was investigated by Laurdan fluorescence spectroscopy. The Laurdan emission generalized polarization parameter was analyzed as a function of the temperature and excitation wavelength in DPPC (or SM)/Chol, DPPC (or SM)/AR, and DPPC/Chol/AR systems. It was found that AR incorporation into both DPPC and SM bilayers induces an increase in the temperature of the main lipid phase transition, similar to the effect of Chol molecule incorporation. The phase separation, lipid-chain ordering, and membrane hydration are discussed for the AR-mixed membranes and compared with DPPC (or SM)/Chol membranes.

Fluphenazine: from an isolated molecule to its interaction with lipid bilayers.

Fluphenazine (FPh) belongs to the phenothiazine family of compounds and exhibits a wide variety of biological effects, including antimutagenic, proapoptotic, antiproliferative and anti-multidrug resistance (MDR) activities. The ability of FPh to interact with lipid membranes can have a significant impact on its biological activities. However, the mechanisms involved in the interaction of FPh with lipid membranes are poorly understood. FTIR-ATR spectroscopy has been used in this study to visualize the interactions between FPh and a model lipid bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Subsequent interpretation of the temperature-dependent FTIR spectra obtained for FPh:DPPC systems containing different concentrations of FPh was efficiently supported by principal component analysis.

Phase separation in phosphatidylcholine membrane caused by the presence of a pyrimidine analogue of fluphenazine with high anti-multidrug-resistance activity.

Phenothiazine compounds are known as effective inhibitors of a multidrug resistance (MDR) of tumor cells to chemotherapeutic agents. This group consists of many important substances used in human medicine such as antipsychotic drugs in the case of fluphenazine (FPh) or chlorpromazine (CPZ). Fluphenazine was on the World Health Organization (WHO) list of Essential Medicines of 2009, and its new pyrimidine analog (FPh-prm) presented in this work has been documented to have a high anti-MDR activity. In order to discover the character of alterations of the lipid bilayer structure caused by the presence of FPh-prm inside the lipid membrane, which is responsible for the essential increase of an anti-MDR activity of FPh-prm, microcalorimetric (differential scanning calorimetry), Laurdan fluorescence, (31)P nuclear magnetic resonance spectroscopy (NMR), and attenuated total reflectance Fourier transfer infrared spectroscopy (FTIR-ATR) were used for dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes mixed with a different concentration of amine analogue. It was stated that the formation of domains with different content of FPh-prm/DPPC can be a reason for the membrane-related mechanism of chemoprevention associated with the inhibition of the outward transport of anticancer drugs by the glycoprotein P (Pgp) in cancer cells by the pyrimidine analog of FPh. To our best knowledge, this report is the first to show the bilayer structure of domains formed by incomplete miscibility of fluphenazine-related compounds and phospholipid molecules. Our results provide a sound basis for the design of future modifications of anti-MDR drugs by providing very effective inhibitors of the pump activity of Pgp.

Interaction of piperidin derivative of Mannich base with DPPC liposomes.

The long chain Mannich bases, especially with the piperidine and morpholine groups, display very promising antimicrobial activity. In order to extend our knowledge on their impact on biological systems, we examined the interactions of the 5-pentadecyl-2-((piperidin-1-yl)methyl)phenol (PPDP) with model lipid membrane by means of differential scanning calorimetry (DSC) and fluorescence measurements. The small unilamellar vesicles of dipalmitoylophosphatidylcholine (DPPC) with different piperidine Mannich base concentration were investigated as a function of the increase of temperature. The phase separation accompanied by the rise of the transition enthalpy of both subcomponents, the increase of the function of the GP values of Laurdan versus the wavelength of excitation in the gel phase of PPDP/DPPC systems, and no remarkable differences in the fluorescence anisotropy of PPDP molecules in lipid environment for different mixtures of PPDP/DPPC was observed. Additionally, it was shown that PPDP itself interdigitated in solid state.

Chemical structure of phenothiazines and their biological activity.

Phenothiazines belong to the oldest, synthetic antipsychotic drugs, which do not have their precursor in the world of natural compounds. Apart from their fundamental neuroleptic action connected with the dopaminergic receptors blockade, phenothiazine derivatives also exert diverse biological activities, which account for their cancer chemopreventive-effect, as: calmodulin- and protein kinase C inhibitory-actions, anti-proliferative effect, inhibition of P-glycoprotein transport function and reversion of multidrug resistance. According to literature data on relations between chemical structure of phenothiazines and their biological effects, the main directions for further chemical modifications have been established. They are provided and discussed in this review paper.

New fluphenazine analogues as inhibitors of P-glycoprotein in human lymphocyte cultures.

AIM OF THE STUDY: To evaluate the inhibitory effect of 17 new analogues of FPh on the Pgp transport function, by estimation of the rhodamine 123 (Rod-123) accumulation inside cultured lymphocytes. MATERIAL AND METHODS: Lymphocyte were cultured in the presence of a lectin (PHA; 2%, v/v), incubated with benzo[α]pyrene (B[α]P; 7.5 µM, 48 h) to induce genotoxic damage and to increase Pgp expression in the cells. Lymphocytes cultured without the tested compounds were considered as controls. RESULTS: It was established that 10 analogues of FPh, among 17 tested, significantly increased Rod-123 accumulation in lymphocytes at the concentration of 10 µM. As compared to the control cultures the Pgp transport function was the most strongly inhibited by 1a, 1b, 1d, 3f, 3h and 3i analogues (approximately by 25%). CONCLUSIONS: FPh analogues 1a, 1b, 1d, 3f, 3h and 3i should be further studied as promising candidates for adjuvant cancer chemotherapeutics.

Generalized solvent scales as a tool for investigating solvent dependence of spectroscopic and kinetic parameters. Application to fluorescent BODIPY dyes.

Two difluoroboron dipyrromethene (BODIPY) based fluorescent dyes - 4,4-difluoro-3-{2-[4-(dimethylamino)phenyl]ethenyl}-8-[4-(methoxycarbonyl)phenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (1) and 4,4-difluoro-3-[2-(4-fluoro-3-hydroxyphenyl)ethenyl]-8-[4-(methoxycarbonyl)phenyl]-1,5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (3) - have been synthesized via condensation of p-N,N-dimethylaminobenzaldehyde and 4-fluoro-3-hydroxybenzaldehyde, respectively, with 4,4-difluoro-8-[4-(methoxycarbonyl)phenyl]-1,3,5,7-tetramethyl-3a,4a-diaza-4-bora-s-indacene (2). UV-vis spectrophotometry and steady-state and time-resolved fluorometry have been used to study the spectroscopic and photophysical characteristics of in various solvents. The multi-parameter Kamlet-Taft {pi*, alpha, beta} solvent scales and a new, generalized treatment of the solvent effect, proposed by Catalán (J. Phys. Chem. B, 2009, 113, 5951-5960), have been used in the analysis of the solvatochromic shifts of the UV-vis absorption and fluorescence emission maxima of 1-3, and the rate constants of excited-state deactivation via fluorescence (k(f)) and radiationless decay (k(nr)). The four Catalán solvent scales (dipolarity, polarizability, acidity and basicity of the medium) are the most appropriate for describing the solvatochromic effects. Solvent dipolarity and polarizability are the important causes for the solvatochromism of 1. Conversely, the absorption and emission maxima of 2 and 3 are hardly dependent on the solvent: the small changes reflect primarily the polarizability of the solvent surrounding the dye. Fluorescence decay profiles of 1 can be described by a single-exponential function in aprotic solvents, whereas two decay times are found in alcohols. The fluorescence decays of 2 (lifetimes tau in 1.9-2.9 ns range) and 3 (tau between 3.5 and 4.0 ns) are mono-exponential in all solvents studied. The fluorescence properties of dye are very sensitive to the solvent: upon increasing solvent dipolarity, the fluorescence quantum yields and k(f) values decrease and the emission maxima become more red-shifted. The k(f) values of 2 [(1.6 +/- 0.3) x 10(8) s(-1)] and 3 [(1.5 +/- 0.2) x 10(8) s(-1)] are practically independent of the solvent properties. The crystal structure of reveals that the BODIPY core is nearly planar with the boron atom moved out of the plane. The angle between the phenyl group at the meso-position and the BODIPY plane equals 80 degrees.

Interactions of dihydrochloride fluphenazine with DPPC liposomes: ATR-IR and 31P NMR studies.

The influence of dihydrochloride fluphenazine (FPh) on the dipalmitoylphosphatidylcholine (DPPC) bilayer structure was investigated using ATR-IR and (31)P NMR methods. The ATR-IR results indicate an increase in conformational disorder in the hydrophobic part compared with pure DPPC liposomes and a decrease in temperature of the chain-melting phase transition in FPh/DPPC liposomes. These effects depended on the concentration of the drug in the DPPC bilayer. The dihydrochloride fluphenazine molecules form H-bonds with the proton-acceptor carbonyl groups of DPPC molecules. At a higher concentration of the drug, the lipid bilayer structure is destroyed, and an isotropic phase is observed using (31)P NMR spectroscopy. The interactions between FPh and the lipid bilayer have a crucial role in MDR (multidrug-resistant) activity of this drug. These results improve one possible strategy of cancer chemoprevention with FPh accompanied by fluidization and destabilization of the model lipid bilayer structure.

Photophysics and stability of cyano-substituted boradiazaindacene dyes.

The solvatochromic photophysical properties of two fluorescent, cyano-substituted BODIPY dyes-8-(4-bromophenyl)-3,4,4,5-tetracyano-4-bora-3a,4a-diaza-s-indacene (4CN) and 8-(4-bromophenyl)-3,5-dicyano-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (2CN)-have been studied in various solvents by UV-vis spectrophotometry and steady-state and time-resolved fluorometry. These two BODIPY analogues have comparable photophysical properties, implying that displacement of F by CN at boron has a negligible effect. Both compounds have high fluorescence quantum yields Phi(f) (0.65-0.90 for 4CN and 0.63-0.88 for 2CN) in the solvents studied and display mono-exponential fluorescence decay profiles in nonprotic solvents. A new, generalized treatment of the solvent effect based on four mutually independent, empirical solvent scales (dipolarity, polarizability, acidity, and basicity of the medium) indicates that solvent polarizability and, to a lesser degree, solvent (di)polarity are crucial factors causing the solvent-dependent shifts of the UV-vis absorption and fluorescence emission. The rate constants of radiative deactivation (k(f)) are nearly independent of the nonprotic solvent [k(f) = (1.4 +/- 0.1) x 10(8) s(-1) for 4CN and (1.5 +/- 0.2) x 10(8) s(-1) for 2CN]. Both compounds undergo a color change in polar aprotic solvents (acetone, acetonitrile, and N,N-dimethylformamide), which can be stopped by addition of HClO(4). The kinetics of this color change indicates that the decomposition of these cyano-substituted BODIPY compounds is complex.