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Low-molecular-mass trypsin inhibitors from Arabidopsis thaliana, Brassica napus var. oleifera, and Sinapis alba L. (ATTI, RTI, and MTI, respectively) display more than 69% amino acid sequence identity. Among others, the amino acid sequence Cys-Ala-Pro-Arg-Ile building up the inhibitor reactive site, and the eight Cys residues forming four disulfide bridges are conserved. However, the disulfide bridge connectivity of RTI and MTI (C1-C3, C2-C4, C5-C6, and C7-C8) is different from that of ATTI Cys (C1-C8, C2-C5, C3-C6, and C4-C7). Despite the different disulfide bridge connectivity, the reactive site loop of ATTI, RTI, and MTI is solvent exposed permitting trypsin recognition. Structural considerations here reported suggest that proteins showing high amino acid sequence identity and common functional properties could display different three-dimensional structures. This may reflect high inhibitor plasticity in relation to plant-pathogen interactions, plant tissue development as well as the different redox potential of cell compartments.

The stable, transparent, organogels, which are prepared by adding a minute amount of water to a solution of lecithin in biocompatible oil, are here studied as matrices for solubilization and percutaneous delivery of fenretinide (4 hydroxypropyl phenyl retinamide, 4HPR), a retinoic acid derivative. The influence of different types of oil, content of water and presence of hyaluronic acid was studied on gel properties. Rheology studies were carried out in order to detect the effect of these variables on gel viscosity. 4HPR diffusion from the different organogels was determined by in vitro Franz cell. It was found that diffusion coefficients ( J n ) of 4HPR incorporated in organogels are about five fold lower than J n of 4HPR in organic solution. Stability and shelf life stability studies demonstrate that 4HPR incorporated in organogels does not degrade and that organogels maintain 90% of 4HPR stability for periods up to 4 months.

In this study a formulation suitable to be applied on oral and/or vaginal mucosa has been developed for the treatment of fungal infections. The aim of the research is a comparison between clotrimazole (CLO) containing semisolid formulations based on monoolein aqueous dispersion (MAD) or nanostructured lipid carrier (NLC). MAD and NLC have been characterized in terms of morphology and dimensional distribution by cryogenic Transmission Electron Microscopy (cryo-TEM) and Photon Correlation Spectroscopy (PCS). CLO was encapsulated with high entrapment efficiency both in MAD and in NLC, according to Sedimentation Field Flow Fractionation (SdFFF) combined with HPLC. CLO recovery in MAD and NLC has been investigated by time. In order to obtain formulations with suitable viscosity for mucosal application, MAD was diluted with a carbomer gel, while NLC was directly viscosized by the addition of poloxamer 407 in the dispersion. The rheological properties of MAD and NLC after viscosizing have been investigated. Franz cell has been employed to study CLO diffusion from the different vehicles, evidencing diffusion rates from MAD and NLC superimposable to that obtained using Canesten ® . An anticandidal activity study demonstrated that both CLO-MAD and CLO-NLC were more active against Candida albicans with respect to the pure drug.

The physico-chemical properties and in vivo efficacies of two nanoparticulate systems delivering the antiparkinsonian drug bromocriptine (BC) were compared in the present study. Monoolein Aqueous Dispersions (MADs) and Nanostructured Lipid Carriers (NLCs) were produced and characterized. Cryogenic transmission electron microscopy (cryo-TEM) and X-ray diffraction revealed the morphology of MAD and NLC. Dimensional distribution was determined by Photon Correlation Spectroscopy (PCS) and Sedimentation Field Flow Fractionation (SdFFF). In particular, BC was shown to be encapsulated with high entrapment efficiency both in MAD and in NLC, according to SdFFF combined with HPLC. Two behavioral tests specific for akinesia (bar test) or akinesia/bradykinesia (drag test) were used to compare the effects of the different BC formulations on motor disabilities in 6-hydroxydopamine hemilesioned rats in vivo, a model of Parkinson's disease. Both free BC and BC-NLC reduced the immobility time in the bar test and enhanced the number of steps in the drag test, although the effects of encapsulated BC were longer lasting (5h). Conversely, BC-MAD was ineffective in the bar test and improved stepping activity in the drag test to a much lower degree than those achieved with the other preparations. We conclude that MAD and NLC can encapsulate BC, although only NLC provide long-lasting therapeutic effects possibly extending BC half-life in vivo.

N(6)-cyclopentyladenosine (CPA) has neuronal anti-ischemic properties, but it is not absorbed into the brain from the bloodstream, where it shows poor stability and induces side effects. Microparticulate drug delivery systems designed for CPA nasal administration and constituted by mannitol or chitosan, were prepared by spray-drying and characterized. Mannitol-lecithin microparticles showed high CPA dissolution rate, whereas chitosan microparticles controlled its release rate. In vitro mucoadhesion studies indicated that CPA-loaded chitosan microparticles had higher mucoadhesive properties compared to mannitol particles. Ex vivo studies on sheep nasal mucosa showed that mannitol microparticles promoted CPA permeation across the mucosa, whereas chitosan microparticles controlled CPA permeation rate in comparison with CPA raw material. In vivo studies were carried out on rats. No CPA was detected in rat cerebrospinal fluid (CSF) and brain sections after intravenous administration. In contrast, after nasal administration of loaded microparticles CPA was found in the CSF at concentrations ranging from high nM to microM values and up to two order of magnitude higher than those obtained at systemic level. CPA was also found in the olfactory bulb at concentrations around 0.1 ng/mg of tissue. These results can open new perspectives for the employment of CPA against brain damages following stroke.

This paper describes the synthesis and the physico-chemical characterization of cationic peptides (CPs) for possible application as non-viral gene delivery systems. Particularly, the production of cationic liposomes and micelle solutions was considered. Liposomes were prepared by REV-phase and extrusion presenting an average diameter reflecting the pore size of the membrane used for the extrusion. After DNA complexation the mean diameter of complexes decreased by increasing the number of positive charges. The non-complexed liposome preparations showed a net positive zeta potential comprised between 17.8-30 mV. After adding Defibrotide (DFT) to liposomes (at a 1:4 +/- molar ratio) the zeta potential fell down to a net negative value indicating the formation of the ionic complex. Concerning micelles, before complexation it was not possible to measure their size by PCS. However, after DFT complexation the size of complexes highly increased. In addition, as previously seen for liposomes, before complexation, the five CPs solutions showed a positive zeta potential ranging from 10-17.8 mV, while after addition of DFT the zeta potential fell to negative values. Concerning toxicity studies, in general CP-liposomes displayed a lower toxicity towards K562 cells as compared to the corresponding CP-solution. Taking into account these results, the studied CPs could be efficiently used to obtain both cationic liposomes and micelles. Moreover they are able to complex DNA with different interaction strength, depending on the type of peptide-based cationic molecule used.

In this article we describe the production and characterization of specialized delivery systems for some distamycin derivatives (DD), namely liposomes and micellar dispersions. All the formulations were designed to increase the solubility of DD in an aqueous environment and to reduce the possible toxicity problems related to the administration of these drugs. For instance, liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters, then characterized in terms of dimensions, morphology, and encapsulation efficacy. The analysis of their in vitro antiproliferative activity on cultured human and mouse leukemic cells demonstrated that liposomes and micellar dispersions containing DD exert quite different effects. These effects were compared with those shown by the free drug depending on type of drug and also cell line used.

Low-molecular-mass trypsin inhibitors from Arabidopsis thaliana, Brassica napus var. oleifera, and Sinapis alba L. (ATTI, RTI, and MTI, respectively) display more than 69% amino acid sequence identity. Among others, the amino acid sequence Cys-Ala-Pro-Arg-Ile building up the inhibitor reactive site, and the eight Cys residues forming four disulfide bridges are conserved. However, the disulfide bridge connectivity of RTI and MTI (C1-C3, C2-C4, C5-C6, and C7-C8) is different from that of ATTI Cys (C1-C8, C2-C5, C3-C6, and C4-C7). Despite the different disulfide bridge connectivity, the reactive site loop of ATTI, RTI, and MTI is solvent exposed permitting trypsin recognition. Structural considerations here reported suggest that proteins showing high amino acid sequence identity and common functional properties could display different three-dimensional structures. This may reflect high inhibitor plasticity in relation to plant-pathogen interactions, plant tissue development as well as the different redox potential of cell compartments.

Peroxynitrite (ONOO-) is a transient powerful oxidant produced in vivo as the reaction of nitrogen monoxide (.NO) with superoxide (O2.-). The peroxynitrite reactivity is modulated by carbon dioxide (CO2) which enhances the peroxynitrite-mediated nitration of aromatics and partially impairs the oxidation of thiols. Here, the effect of CO2 on the peroxynitrite-mediated inhibition of human caspase-3, the execution enzyme of the apoptotic cascade, is reported. Peroxynitrite inhibits the catalytic activity of human caspase-3 by oxidizing the Sgamma atom of the Cys catalytic residue. In the absence of CO2, 1.0 equivalent of peroxynitrite inactivates 1.0 equivalent of human caspase-3. In the presence of the physiological concentration of CO2 (=1.3x10(-3) M), 1.0 equivalent of peroxynitrite inactivates only 0.38 equivalents of human caspase-3. Peroxynitrite affects the kcat value of the human caspase-3 catalyzed hydrolysis of N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin, without altering Km. Both in the absence and presence of CO2, the reducing agent dithiothreitol does not prevent human caspase-3 inhibition by peroxynitrite and does not reverse the peroxynitrite-induced inactivation of human caspase-3. These results represent the first evidence for modulation of peroxynitrite-mediated inhibition of cysteine proteinase action by CO2, supporting the role of CO2 in fine tuning of cell processes (e.g., apoptosis).

We have investigated the influence of charge and lipid concentration on the in-vivo percutaneous absorption of a model compound, methyl nicotinate (MN), from liposomal vesicles. MN-loaded liposomes were produced by the reverse-phase evaporation method (REV) using different concentrations of phosphatidyl choline (PC), in association with surfactants such as dioctadecyl dimethyl ammonium bromide (DDAB18) and dicetyl phosphate (DCP), which impart a positive or negative charge to the systems, respectively. The liposomal suspensions were then processed to hydrogels and used to study in-vivo the MN permeation profile. MN was chosen as the model compound since it was capable of causing cutaneous erythema, the intensity and duration of which was proportional to the amount entering the living epidermis over time. The extent of the erythema was monitored by reflectance spectrophotometry, a non-invasive technique. In-vivo findings showed an interesting MN delayed release, which was proportional to the amount of phospholipids in each liposomal formulation. Furthermore, it could be noted that the erythematous effect was more prolonged when MN was delivered from neutral or negatively-charged liposomal forms.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand (e.g., heme and drugs) binding capacity. Here, the binding of chlorpropamide, digitoxin, furosemide, indomethacin, phenylbutazone, sulfisoxazole, and tolbutamide to HSA and ferric heme-HSA is reported. Moreover, ferric heme binding to HSA in the absence and presence of drugs has been investigated. Values of the association equilibrium constant for drug binding to Sudlow's site I of ferric heme-HSA (ranging between 1.7 x 10(3) and 1.6 x 10(5)M(-1)) are lower by one order of magnitude than those for drug binding to ferric heme-free HSA (ranging between 1.9 x 10(4) and 1.8 x 10(6)M(-1)). According to linked functions, the value of the association equilibrium constant for heme binding to HSA decreases from 7.8 x 10(7)M(-1), in the absence of drugs to 7.0 x 10(6)M(-1), in the presence of drugs. These findings represent a clear-cut evidence for the allosteric inhibition of drug binding to HSA Sudlow's site I by the heme. According to linked functions, drugs impair allosterically heme binding to HSA. These results appear to be relevant in the drug therapy and management.

Sulfopropylated dextran microspheres (SP-Ms), (Dm = 80 microm) loaded with a water soluble drug (Tetracycline HCl), were included in cellulose acetate butyrate (CAB) microcapsules. Spherical CAB microcapsules were obtained by oil in water (o/w) solvent evaporation method in the presence of an inert solvent as cyclohexane (CyH) or n-hexane (N-Hex), and different excipients (Phospholipon, Tween, Span, Eudragit RS 100). Chloroform was found to be the best solvent for the preparation of the microcapsules. Also, the sphericity as well as the porosity of the microcapsules was controlled by the presence of an inert solvent. The final concentration of the drug in CAB microparticles was up to 25% (w/w). The key factors for the successful preparation were also the viscosity of the polymer, while the wettability of the resulted microcapsules, the temperature of the preparation, and the porosity have modulated the release of the drug. The higher is the amount of encapsulated microspheres the thinner is the CAB wall between the compartments created by their incorporation. When these microspheres come in contact with the release medium, the pressure created by their swelling breaks the polymer film and the drug starts to be released. The more drug is released in phosphate buffer the higher is the swelling degree of the encapsulated ion exchange resins and the force created by their supplementary swelling will break the more resistants walls. In this way a self-propelled drug release is achieved, until almost all drug was eliberated.

The present paper describes the production of biodegradable microparticles using different hyaluronan polymers, such as native hyaluronan, the esterified derivative of hyaluronan Hyaff 11p50 (where 50% of the carboxy groups of hyaluronic acid are esterified with benzyl alcohol) and the autocross-linked polymer (ACP) internally esterified derivative of hyaluronan, by solvent evaporation and spray-drying methods. As model drugs cromolyn sodium salt, metronidazole and prednisolone hemisuccinate sodium salt were employed. The influence of polymer and preparation procedure has been evaluated on microparticle characteristics (i.e. morphology and encapsulation yield) and on the drug release profiles. The use of solvent evaporation method, a polymeric matrix constituted of Hyaff 11p50 3% (w/v), a dispersing phase constituted of 80 g of mineral oil (w/o ratio: 0.1), Span 85 0.1% (w/w) as stabilizer, and a stirring speed of 700 rpm resulted in the production of microspheres characterized by spherical shape, absence of aggregates, a mean diameter of 6.4 μm and a recovery of 90% (w/w). The production of drug containing microspheres led to an increase of mean diameter of microspheres and to high encapsulation yields. Moreover in vitro models have demonstrated that in all cases drugs were released from Hyaff 11p50 microspheres in a controlled fashion. Finally mathematical analysis of the drug release modalities has evidenced that drug release from Hyaff 11p50 microspheres is more consistent with kinetics of the diffusion rather than of the dissolution type.

This article describes the production and characterization of two liposome formulations containing antitumor drugs, namely distamycin A (Dist) and a new alkyl derivative of distamycin A (C16-Dist). Egg-PC/cholesterol liposomes (4:1 mol/mol) were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. The encapsulation efficiency was found to be almost complete for C16-Dist (99.8%), while native distamycin A showed a lower yield (19.0%). The in vitro antiproliferative activity of the distamycins-containing liposomes determined on human leukaemic K562 cells, was 11-fold and 8-fold higher for native and alkyl derivative distamycin A, respectively, compared with that of the corresponding free drugs. Liposomal formulations show an increase in the activity and specificity of distamycins in experimental antitumor therapy.

The present study describes the production and characterization of amphiphilic association systems for Amphotericin B (AMB). In particular, three different classes of microemulsions and different monoglyceride–water systems were produced. Formulations were characterized for macroscopic aspect, pH, rheology, mean size and size distribution, both in the absence and in the presence of AMB. AMB solubility was investigated in the different formulations by HPLC studies. The formulations increased AMB solubility up to 20-fold with respect to the single oil and aqueous phases employed for microemulsion production. AMB diffusion studies from two microemulsions taken as models were performed in a Franz cell system using a nylon membrane. The physical and chemical stability of AMB-containing amphiphilic association systems were investigated for three months after production. For physical stability studies both the macroscopic aspect, droplet mean size and dimensional distribution were analysed. For chemical stability studies, the AMB content of the formulations was quantified by HPLC analysis. Microemulsions and monoglyceride–water systems were free from phase separation for up to three months and in some cases the AMB content was unchanged even after three months.

Pullulan microspheres containing cyclodextrin (CyD) were obtained by chemical crosslinking with epichlorohydrin of an alkaline solution of pullulan (Pul) and alpha-, beta- or gamma-CyD. The amount of alpha-, beta- and gamma-CyD in microspheres was 120, 156, and 138 micromol/g, respectively, as determined from the percentage of iodine incorporated in the hydrophobic cavity of CyD's. Microspheres were packed in a glass column and the liquid chromatographic behaviour by isocratic elution of different drugs or typical organic compounds (TOC), taken as model drugs, was investigated. The increase of the retention volume (V(R)) of each compound, depending on the interaction(s) between CyD's cavity and the considered molecule, is characterized by a broadening of the peaks. The interaction coefficient K, corresponding to the ratio between the V(R) value of each tested molecule on Pul-alpha-, Pul-beta- and Pul-gamma-CyD active stationary phase and the V(R) value of benzoic acid on St/maltodextrin neutral stationary phase, was determined. According to K values, the accurate prediction can be done on the potential drugs to be conditioned in suitable CyD cavity. Values of K allow to anticipate the release profiles of drugs considered.

The aim of the present paper was to study production of methacrylate microparticles for the delivery (administration) of ascorbic acid via the oral route. Vitamin C is an important antioxidant that may be involved in the reduction of the risk of certain types of cancer, such as colorectal cancer. As polymers different acrylic compounds were considered, namely Eudragit® RL, L and RS. Spray-drying was used as preparation method of vitamin C/Eudragit® microspheres. Microspheres were first characterized by size and morphology by scanning electron microscopy, then in vitro release kinetics by mean of dialysis method were studied. Although the produced microparticles were unable to slow down the release of the drug with respect to the free form of ascorbic acid, these microspheres showed a good morphology and size distribution that permit to propose them as candidate for the delivery of vitamin C as associated therapy in the treatment of colorectal cancer by oral route.

Purpose. A series of 5′-esters of N6-cyclopentyladenosine (CPA) were prepared with the aim to improve stability and bioavailability of selective A1 agonists. Log P values, stability, affinity, and activity toward human adenosine A1 receptors were evaluated.

Five new low-molecular-mass trypsin inhibitors belonging to the RTI/MTI-2 family were identified from white mustard (Sinapis alba L. ; MTI-2) seed. Purified MTI-2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C-terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI-2 inhibitors originated from different genes. The presence of 5-oxo-proline (pyroglutamic acid; 5-oxoPro1) and Kyn44 reflected post-translational processing of the serine proteinase inhibitor. MTI-2 showed approximately 70% amino-acid identity with low-molecular-mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI-III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI-2 was homologous to brazzein, the sweet-tasting protein from Pentadiplandra brazzeana Baillon fruit ( approximately 30% amino-acid identity). Although snake-venom toxins showed a low amino-acid identity (< 20%) with MTI-2, RTI-III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI-2 (Cys5-Cys27, Cys18-Cys31, Cys42-Cys52, and Cys54-Cys57) corresponded to that of RTI-III and of snake-venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three-dimensional arrangement rather than to the amino-acid sequence. Values of Ka for MTI-2 binding to bovine beta-trypsin (trypsin) and bovine alpha-chymotrypsin were 6.3 x 109 M-1 and 2.0 x 106 M-1, respectively, at pH 8.0 and 21.0 degrees C. Moreover, values of kon for MTI-2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 x 105 M-1.s-1 and 8.9 x 10-5 M-1.s-1, respectively, at pH 8.0 and 21.0 degrees C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI-2 inhibitors were indistinguishable.

Gabexate mesylate (GM; commercialized under the brand name FOY) is a nonantigenic synthetic inhibitor of plasmatic and pancreatic serine proteinases that is used therapeutically in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. The inhibitory effect of GM on nitric oxide synthase as well as serine proteinases and swine kidney copper amine oxidase, all acting on cationic substrates, has been investigated. On the basis of the available X-ray crystal structures of the enzymes considered, the possible binding mode(s) of GM has(have) been analyzed. The enzyme cross-inhibition by GM suggests that the use of this drug should be under careful control. With the aim to improve the scarce plasma stability of GM, the positively charged drug has been complexed to the surface of preformed anionic liposomes. The liposome-complexed GM half-life increases about five-fold, indicating the protective effect of liposomes on GM degradation. Moreover, the GM complexation with liposomes does not alter its inhibitory activity on NOS-I and porcine pancreatic trypsin.

In the present paper we describe the production and characterization of specialized delivery systems for camptothecin, namely liposomes, micellar solutions and microemulsion. For instance, liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. Liposomes were characterized in term of dimensions, morphology and encapsulation efficacy. All the formulations were designed firstly to increase the solubility of camptothecin in aqueous environment and secondly to reduce the toxicity problems related to the administration of this drug. The analysis of their in vitro antiproliferative activity on cultured human leukemic K562 cells demonstrated that liposomes, micellar solutions and microemulsion containing camptothecin exert similar or slightly enhanced effect as compared to that shown by the free drug. Based on these results, the specialized delivery systems here proposed typify an interesting starting point for a future use in experimental therapy.

Amiloride, a diuretic and antihypertensive drug, inhibits Na+ transporting systems, diamine oxidases and the human urinary plasminogen activator. Present results indicate that amiloride is a competitive inhibitor of mouse brain nitric oxide synthase (NOS; Ki = 4.5 x 10(-4) M, at pH 7.5 and 37.0 degrees C), but not a nitric oxide (NO) precursor, representing a case for enzyme cross-inhibition. The decreased levels of NO, as a consequence of NOS inhibition, might induce some clinically-adverse amiloride reactions, such as an unexpected reduced antihypertensive effect.

The crystal structure of bovine alpha-chymotrypsin (alpha-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 A resolution (R-factor = 0.18). The proteinase:inhibitor complex forms a compact dimer (two alpha-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between alpha-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P1 residue of BPTI, however, does not occupy the alpha-CHT S1 specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217.

The aim of the paper was to develop tetracycline-containing formulations for the treatment of periodontitis by direct periodontal intrapocket administration. Two different semi-solid formulations were prepared, based on poly(oxyethylene)poly(oxypropylene) block copolymer (poloxamer) and monoglycerides, respectively. Both formulations possess interesting properties as delivery systems. They are easily administered by syringe equipped with needles appropriate for intrapocket delivery, they are characterized by a sol-gel transition, becoming semi-solid once in the periodontal pocket and, finally, they represent biocompatible formulations eliminated from the body by normal routes. A rheological characterization of both formulations was performed in the presence or in the absence of tetracycline, determining the sol-gel transition temperature (Tc) by ‘time cure tests’ and the z coefficient by ‘frequency sweep test’. In addition, the in vitro tetracycline release from formulations was determined. Comparative in vivo studies were conducted, in order (a) to compare the persistence of the gels on the gum and (b) to evaluate the clinical performances of the gels. These latest studies indicated that both poloxamer and monoglycerides gels, when applied subgingivally, produce a significantly improved outcome in moderate to deep periodontal pockets.

The effects of glucosinolates (GLs) (sinigrin, gluconapin, progoitrin, epi-progoitrin, sinalbin, glucotropaeolin, glucoerucin, glucocheirolin, and glucoraphenin) and their enzymatic hydrolysis-derived products (EHDPs) have been studied in controlling the proliferation of cancer cell lines. The results of this study indicate the following: (i) neither myrosinase nor intact GLs have any effect on tumor cell growth when used up to 36 U/mL and 500 μM, respectively; (ii) all EHDPs show a clear inhibition of human erythroleukemic K562 cell proliferative growth, which is particularly evident for EHDPs from sinigrin, glucotropaeolin, glucoerucin, and glucocheirolin (IC50 < 20 μM); (iii) the EHDP production by in situ or pre-mix procedures gives rather similar antiproliferative effects; and finally, (iv) the EHDPs from glucoraphenin are active toward several other tumor cells, viz. FL (murine erythroleukemic cells), Jurkat (human T-lymphoid cells), HeLa (human cervix carcinoma cells), H9 (human T-lymphoid cells),...

The purpose of this study was to investigate the potential of intranasal immunization with non-ionic surfactant vesicles (NISV) containing either the secretory recombinant form of glycoprotein B (gBs) of herpes simplex virus type 1 or a related polylysine reach peptides (DTK) for induction of protective immunity against genital herpes infection in mice. NISV were prepared by lipid film hydration method. The mean diameter of vesicles was around 390 nm for DTK-containing NISV (DTK-NISV) and 320 nm for gB1s-containing NISV (gB1s-NISV). The encapsulation efficiency of the molecules was comprised between 57% and 70%. After 7-14 day NISV maintained stable dimensions and a drug encapsulation higher than 48%. We showed that intranasal immunization with gB1s-NISV induces gB-specific IgG antibody and lymphoproliferative responses, whereas vaccination with DTK-NISV was not able to generate a gB-specific immune response. Our results indicate that vaccination of BALB/c mice with gB1s-NISV induced Th1 responses, as characterized by increased titre of IG2a in plasma and IFN-production in CD4+ splenic cells. Intranasal immunization with gB1s-NISV could elicit 90% (almost complete) protection against a heterologous lethal vaginal challenge with herpes simplex virus type 2. These data may have implications for the development of a mucosal vaccine against genital herpes.

A new serine proteinase inhibitor, rapeseed trypsin inhibitor (RTI), has been isolated from rapeseed (Brassica napus var. oleifera) seed. The protein inhibits the catalytic activity of bovine beta-trypsin and bovine alpha-chymotrypsin with apparent dissociation constants of 3.0 x 10(-10) M and 4.1 x 10(-7) M, at pH 8.0 and 21 degrees C, respectively. The stoichiometry of both proteinase-inhibitor complexes is 1:1. The amino acid sequence of RTI consists of 60 amino acid residues, corresponding to an M(r) of about 6.7 kDa. The P1-P1' reactive site bond has been tentatively identified at position Arg20-Ile21. RTI shows no similarity to other serine proteinase inhibitors except the low molecular weight mustard trypsin inhibitor (MTI-2). RTI and MTI-2 could be members of a new class of plant serine proteinase inhibitors.

This paper describes a comparative study on the performances of ethosomes and solid lipid nanoparticle as delivery systems for acyclovir. Ethosomes were spontaneously produced by dissolution of phosphatidylcholine and acyclovir in ethanol followed by addition of an aqueous buffer while solid lipid nanoparticle were produced by homogenization and ultrasonication. Both colloidal systems were morphologically characterized by cryo-transmission electron microscopy. The encapsulation efficiency was 94.2±2.8% for ethosomes and 53.2±0.2% for solid lipid nanoparticle. Concerning Z potential, both formulations are close to neutrality. The diffusion coefficients of the drug from ethosomes and solid lipid nanoparticle, determined by a Franz cell method, were 9.4 and 1.2-fold lower as compared to the free acyclovir in solution, thus evidencing the ability of both colloidal systems in enhancing the diffusion of the drug. The antiviral activity against HSV-1 of both systems was tested by plaque reduction assay in monolayer cultures of Vero cells. Data showed that no significant differences in the antiviral activity were observed by acyclovir in the free or loaded forms. Taken together these results, colloidal systems could be interesting to mediate the penetration of acyclovir within Vero cells.

Catalytic and inhibitor binding properties of bovine alpha-chymotrypsin, in which the Met-192 residue has been converted by treatment with chloramine T to the sulfoxide derivative (Met(O)192 alpha-chymotrypsin), have been examined relative to the native enzyme (alpha-chymotrypsin), between pH 4.5 and 8.0 (mu = 0.1), and/or 5.0 degrees C and 40.0 degrees C. Values of kcat, k+2 and/or k+3 for the hydrolysis of all the substrates examined (i.e., tMetAcONp, ZAlaONp, ZLeuONp, ZLysONp and ZTyrONp) catalyzed by native and Met(O)192 alpha-chymotrypsin are similar, as well as values of Km for the hydrolysis of ZLeuONp, ZLysONp and ZTyrONp. On the other hand, Ks and Km values for the hydrolysis of ZAlaONp and tMetAcONp are decreased by about 5-fold. Met-192 oxidation does not affect the kinetic and thermodynamic parameters for the (de)stabilization of the complex formed between the proteinase and the bovine basic pancreatic trypsin inhibitor. On the other hand, the recognition process between between alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis is influenced by the oxidation event. Considering known molecular models, the observed catalytic and inhibitor binding properties of native and Met(O)192 alpha-chymotrypsin were related to the inferred stereochemistry of the proteinase-substrate and proteinase-inhibitor contact region(s).

The present article describes a comparative study of the performances of liposomes and ethosomes as specialized delivery systems for distamycin A (DA) and two of its derivatives. Liposomes and ethosomes were prepared by classical methods, extruded through polycarbonate filters, and characterized in terms of dimensions, morphology, and encapsulation efficiency. It was found that DA was associated with vesicles (either liposomes or ethosomes) by around 16.0%, while both derivatives of DA showed a percentage of association around 80% in the case of liposomes and around 50% in the case of ethosomes. In vitro antiproliferative activity experiments performed on cultured human and mouse leukemic cells demonstrated that vesicles were able to increase the activity of both derivatives of DA. In addition, it was demonstrated that the aging of both liposomes- and ethosomes-associated distamycin suspensions did not heavily influence the vesicle size, while all samples showed a relevant drug leakage with time. Moreover, according to the different physicochemical characteristics of DA and its derivatives (i.e., log P), vesicle-associated DA showed the highest loss of drug with respect to both its derivatives. In conclusion, the enhancement of drug activity expressed by these specialized delivery systems-associated DD could be interesting to obtain an efficient therapeutic effect aimed at reducing or minimizing toxic effects occurring with distamycins administration.

In the present study are reported the differential DNA binding activity of the anti-tumor polyamidine tetra-p-amidinophenoxyneopentane (TAPP-H) and its 2'-halo derivative (TAPP-Br), and their effects on the binding of the recombinant Epstein-Barr virus (EBV) nuclear antigen to a synthetic oligonucleotide mimicking the target DNA sequence present in the EBV genome. In addition, the proliferation kinetics and cell cycle analysis of human leukemia K562 cells treated with TAPP-H and TAPP-Br are reported. The possible in vivo relationship between DNA binding affinity and cytotoxicity is also discussed.

The effect of pH and temperature on kinetic and thermodynamic parameters (i.e., k(on),k(off),Ka,delta G0, delta H0 and delta S0 values) for the binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds (ETI) to bovine beta-trypsin, bovine alpha-chymotrypsin, the human tissue plasminogen activator, human alpha-, beta- and gamma-thrombin, as well as the M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator (also named urokinase) has been investigated. At pH 8.0 and 21.0 degrees C: (i) values of the second-order rate constant (K(on)) for the proteinase:ETI complex formation vary between 8.7 x 10(5) and 1.4 x 10(7)/M/s; (ii) values of the dissociation rate constant (k(off)) for the proteinase: ETI complex destabilization range from 3.7 x 10(-5) to 1.4 x 10(-1)/s; and (iii) values of the association equilibrium constant (Ka) for the proteinase:ETI complexation change from1.0 x 10(4) to 3.8 x 10(11)/M. Thus, differences in k(off) values account mostly for the large changes in Ka values for ETI binding. The affinity of ETI for the serine proteinases considered can be arranged as follows: bovine beta-trypsinhuman tissue plasminogen activatorbovine alpha-chymotrypsinhuman alpha-, beta- and gamma-thrombin approximately M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator. Moreover, the serine proteinase:ETI complex formation is an endothermic, entropy-driven, process.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamic parameters for the binding of hirudin to human α, β and γ-thrombin have been determined between pH 5.0 and 9.0, and from 10 °C to 40 °C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 °C. These results have been analysed in parallel with the inhibitor-binding properties of human α, β and γ-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human γ-thrombin have been determined between pH 5.0 and 9.0, at 21 °C. The different binding behaviour of hirudin and BPTI with respect to human α, β and γ-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the β and γ-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system.

A new serine proteinase inhibitor, mustard trypsin inhibitor 2 (MTI-2), has been isolated from white mustard ( Sinapis alba L.) seed by affinity chromatography and reverse phase HPLC. The protein inhibits the catalytic activity of bovine β-trypsin and bovine α-chymotrypsin, with dissociation constants ( K d ) of 1.6 × 10 −10 M and 5.0 × 10 −7 M, respectively, at pH 8.0 and 21°C, the stiochiometry of both proteinase-inhibitor complexes being 1:1. The amino acid sequence of MTI-2, which was determined following S -pyridylethylation, is comprised of 63 residues, corresponding to a molecular weight of about 7 kDa, and shows only extremely limited homology to other serine proteinase inhibitors.

The binding of the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis to serine (pro)enzymes belonging to the chymotrypsin and subtilisin families has been investigated from the thermodynamic viewpoint, between pH 4.5 and 9.5 and from 10 degrees C to 40 degrees C. The affinity of eglin c for the serine (pro)enzymes considered shows the following trend: Leu-proteinase [the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves] greater than human leucocyte elastase congruent to human cathepsin G congruent to subtilisin Carlsberg congruent to bovine alpha-chymotrypsin greater than bovine alpha-chymotrypsinogen A congruent to porcine pancreatic elastase congruent to bovine beta-trypsin. The serine (pro)enzyme-inhibitor complex formation is an entropy-driven process. On increasing the pH from 4.5 to 9.5, the affinity of eglin c for the serine (pro)enzymes considered increases thus reflecting the acid pK shift of the invariant hystidyl catalytic residue from approximately to 6.9 in the free serine proteinases and bovine alpha-chymotrypsinogen A to congruent to 5.1 in the serine (pro)enzyme-inhibitor complexes. Considering the known molecular models, the observed binding behaviour of eglin c was related to the inferred stereochemistry of the serine (pro)enzyme-inhibitor contact regions.

The N -α- l -isoleucyl- l -valine (Ile-Val) activating dipeptide, sequentially homologous to the Ile 16-Val 17 N-terminus of bovine β-trypsin, displays an activating effect on equilibria involved in the binding of strong ligands (i.e., n -butylamine and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen. This property has been investigated between pH 3.0 and 9.0 ( I = 0.1 M at 21.0°C. The thermodynamics for the interaction of strong ligands with bovine β-trypsin has also been studied under the same experimental conditions. The equilibria involved in the binding of the Ile-Val activating dipeptide and/or inhibitors to bovine β-trypsin and its zymogen are described according to linkage relationships, wherefore interaction(s) between different functional and structural domains of the (pro)enzyme (i.e., the so-called Ile-Val pocket and the primary and/or secondary recognition subsite(s)), possibly revolved in the bovine trypsinogen-to-β-trypsin activation pathway, are considered.

The crystal structure of the molecular complex formed by bovine alpha-chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 A resolution were employed to determine the structure by molecular replacement techniques. Bovine alpha-chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 A resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine alpha-chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine alpha-chymotrypsin between subsites P3 and P'2; Leu 451 acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic- and secretory-inhibitors are found around the scissile peptide bond.

The effect of activating dipeptides, sequentially homologous to the Ile16-Val17N-terminus of bovine beta-trypsin (beta-trypsin), on equilibria involved in the binding of strong ligands (i.e., n-butylamine, the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI) and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen (trypsinogen) was investigated at pH 5.51 (I = 0.1 M) and T = 21.0 +/- 0.5 degrees C; under the same experimental conditions, thermodynamics for the binding of strong ligands to beta-trypsin was also obtained. The equilibria involved in the binding of activating dipeptides and/or inhibitors to beta-trypsin and to its zymogen are described according to an induced-fit formalism, taking into account ligand-linked interaction(s) between different functional and structural domains of the (pro)enzyme possibly involved in the trypsinogen-to-beta-trypsin activation pathway. The analysis of data is focussed on parameters describing interactions between the so-called Ile-Val pocket (where the Ile16-Val17 N-terminus of beta-trypsin or activating dipeptides bind) and the primary and/or secondary recognition subsite(s) (where strong ligands associate) present in the (pro)enzyme. Such an analysis allows to dissect the contributions due to the primary recognition subsite, where small mono-functional ligands (e.g., n-butylamine) bind, from those of the secondary subsite(s), which are additional recognition clefts for macromolecular inhibitors (e.g., BPTI and PSTI).

This paper describes the production and characterization of polyacrylic polymer (Eudragit RL, RS and NE) microparticles by spray drying method. Microparticles were designed for ophthalmic administration of acyclovir. Microparticle morphology was characterized by optical and electron microscopy. The release kinetics of the drug from microspheres were determined by a dialysis method. The spray drying method described allows the production of microparticles with acceptable encapsulation efficiency and appropriate dimensional characteristics for ophthalmic administration. Release profile data indicate that acyclovir is released from microparticles in a controlled manner. In addition the release pattern of the drug is influenced by the type of Eudragit used for microparticle production. Moreover the plaque reduction efficiency of acyclovir containing microparticles (except for RS/NE microspheres) is comparable to that displayed by the free drug. Finally our results suggest that acyclovir containing microparticles could represent an interesting system for the release of this antiviral drug at the eye site.

The present investigation describes a formulative study for the development of innovative drug delivery systems for bromocriptine. Solid lipid nanoparticles (SLN) based on different lipidic components have been produced and characterized. Morphology and dimensional distribution have been investigated by electron microscopy and Photon Correlation Spectroscopy. The antiparkinsonian activities of free bromocriptine and bromocriptine encapsulated in nanostructured lipid carriers were evaluated in 6-hydroxydopamine hemilesioned rats, a model of Parkinson’s disease. Tristearin/tricaprin mixture resulted in nanostructured lipid carriers with stable mean diameter up to 6 months from production. Bromocriptine was encapsulated with high entrapment efficiency in all of the SLN samples, particularly in the case of tristearin/tricaprin mixture. Bromocriptine encapsulation did not change nanoparticle dimensions. In vitro release kinetics based on a dialysis method demonstrated that bromocriptine was released in a prolonged fashion for 48 h. Tristearin/tricaprin nanoparticles better controlled bromocriptine release. Both free and encapsulated bromocriptine reduced the time spent on the blocks (i.e. attenuated akinesia) in the bar test, although the action of encapsulated bromocriptine was more rapid in onset and prolonged. It can be concluded that nanostructured lipid carriers encapsulation may represent an effective strategy to prolong the half-life of bromocriptine.

Poly[(N-isopropylacrylamide-co-acrylamide-co-(hydroxyethylmethacrylate))] [poly(NIPAAm-co-AAm-co-HEMA)] copolymer was synthesized as a new thermoresponsive material possessing a lower critical solution temperature (LCST) around 37 °C in phosphate buffer, pH 7.4, at a solution concentration of 1%, w/v. The influence of polymer concentration on LCST was determined by cloud point measurements and by microcalorimetric analysis. The copolymer was transformed in hydrogel microspheres by suspension reticulation of OH groups with glutaraldehyde. The volume phase transition temperature (VPTT) of microspheres was determined by a new approach, which involves measurement of the increase in concentration of a blue dextran (BD) solution at different temperatures in the presence of dry microspheres. The minimum BD concentration that gives reliable and reproducible results was determined to be 1 mg/ml. However, the higher is the concentration of BD in solution the smaller is the error. Contrary to solution of the linear polymer which displays a sharp phase transition temperature, the dependence of water regain of the hydrogel with temperature lasts from 4 °C to 50 °C.

The aim of this work is to safely transport bioadhesive microspheres loaded with DNA to intestine and to test their bioadhesive properties. Poly(vinyl alcohol) (PVA) microspheres were prepared by dispersion reticulation with glutaraldehyde and further aminated. These microspheres were firstly loaded with plasmid DNA by electrostatic interactions and then entrapped in cellulose acetate butyrate (CAB) microcapsules for gastric protection. The entrapped PVA microspheres do not have enough force by swelling to produce the rupture of CAB shell, therefore the resistance of microcapsules was weakened by incorporating different amount of the pH/thermosensitive polymer (SP) based on poly(N-isopropylacrylamide-co-methyl methacrylate-co-methacrylic acid) (NIPAAm-co-MM-co-MA). This polymer is insoluble in gastric juice at pH 1.2 and 37 degrees C, but quickly solubilized in intestinal fluids (pH 6.8 and pH 7.4). Therefore, DNA loaded PVA microspheres were not expelled in acidic media but were almost entirely discharged in small intestine or colon. The integrity of DNA after entrapment was tested by agarose gel electrophoresis indicating that no DNA degradation occurs during encapsulation. The percentage of adhered microspheres on the mucus surface of everted intestinal tissue was 65+/-18% for aminated PVA microspheres without DNA and almost 50+/-15% for those loaded with DNA. Non-aminated PVA microspheres display the lowest adhesive properties (33+/-12%). In conclusion DNA loaded microspheres were progressively discharged in intestine. The integrity of DNA was not modified after entrapment and release, as proved by agarose gel electrophoresis. Both loaded and un-loaded aminated microspheres display good bioadhesive properties.

Poly(vinyl alcohol) (PVA) microspheres were prepared by dispersion reticulation with glutaraldehyde and further aminated. These microspheres were firstly loaded with diclofenac (DF) and then entrapped in cellulose acetate butyrate (CAB) microcapsules by an o/w solvent evaporation technique for intestinal delivery of drug. The encapsulated PVA microspheres due to their low swelling degree in intestinal fluids, do not have enough force to produce the disruption of CAB shell, therefore different amounts of succinoylated pullulan microspheres (SP-Ms) (exchange capacity up to 5.2 meq/g) were co-encapsulated. The SP-Ms do not swell in acidic pH, but swell up to 20-times in intestinal fluids causing the rupture of CAB shell and facilitating the escape of loaded PVA microspheres.

In the present study the preparation, characterization and activity of cationic liposomes containing the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) or two related polylysine rich peptides, namely DTK1 and DTK2, were described. The immunotherapeutic potential of these HSV antigens containing liposomes was examined with a rabbit ocular model of HSV-1 infection. Our study indicates that the liposomes (i) are able to encapsulate quantitatively gB1s and around 30% the DTK peptides, (ii) are characterized by dimensions compatible with ocular applications and (iii) can release the peptide comparably to the free solution. In addition, neutralization studies demonstrated that an anti-DTK specific polyclonal antiserum can inhibit HSV-1 infection, indicating that such peptides could be a good immunogen/antigen in an anti-HSV vaccine formulation. Although the vaccination protocol did not induce protection against the eye disease, a significative protection against a lethal ocular challenge was detectable together with the absence of reactivation episodes from latency on the survived animals. In this respect, the use of cationic liposomes coupled to gB1s and DTK peptides, as a local ocular vaccine, could represent an interesting approach in order to obtain a possible efficacy in protecting animals against a subsequent HSV-1 ocular challenge.

The present study concerns the production and characterization of monooleine (MO) dispersions as drug delivery systems for indomethacin, taken as model anti-inflammatory drug. Dispersions were produced by emulsification and homogenization of MO and poloxamer in water. Morphology and dimensional distribution of the disperse phase have been characterized by cryo-transmission electron microscopy and photon correlation spectroscopy, respectively. X-ray diffraction has been performed to determine the structural organization of the disperse phase. Sedimentation field flow fractionation (SdFFF) has been performed to investigate drug distribution in the dispersion. An in vitro diffusion study was conducted by Franz cell associated to stratum corneum epidermis membrane on cubosome dispersions viscosized by carbomer. In vivo studies based on skin reflectance spectrophotometry and tape stripping were performed to better investigate the performance of cubosome as indomethacin delivery system. Microscopy studies showed the coexistence of vesicles and cubosomes. X-ray diffraction revealed the presence of a bicontinuous cubic phase of spatial symmetry Im3m (Q229). SdFFF demonstrated that no free drug was present in the dispersion. Indomethacin incorporated in viscosized MO dispersions exhibited a lower flux with respect to the analogous formulation containing the free drug in the aqueous phase and to the control formulation based on carbomer gel. Reflectance spectroscopy demonstrated that indomethacin incorporated into MO dispersions can be released in a prolonged fashion. Tape-stripping experiments corroborated this finding. MO dispersions can be proposed as nanoparticulate systems able to control the percutaneous absorption of indomethacin.

Despite the large number of publications and patents concerning pH/thermoresponsive polymers, few data are available concerning the preparation of thermoresponsive cross-linked microspheres from preformed polymers. Therefore, N-isopropylacrylamide-co-acrylamide-co-(2-hydroxyethyl acrylate) copolymers were obtained as a new thermoresponsive material with a lower critical solution temperature (LCST) around 36 degrees C, in phosphate buffer at pH 7.4, and with a cross-linkable OH group in their structure. The LCST value was determined both by UV spectroscopy and microcalorimetric analysis. These copolymers were solubilised in acidified aqueous solution below their LCST, dispersed in mineral oil, and transformed into stable microspheres by cross-linking with glutaraldehyde. The thermoresponsive microspheres were characterised by optical and scanning electron microscopy, degree of swelling, and water retention. The pore dimensions of the microspheres and the retention volumes of some drugs and typical compounds were evaluated at different temperatures by liquid chromatography. Indomethacin, as a model drug, was included in the microspheres by the solvent evaporation method. Finally, the influence of temperature and of temperature cycling on drug release was investigated.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional capacity to bind ligands (e.g. heme and drugs). Here, binding of the anti-HIV drugs abacavir, atazanavir, didanosine, efavirenz, emtricitabine, lamivudine, nelfinavir, nevirapine, ritonavir, saquinavir, stavudine, and zidovudine to HSA and ferric heme-HSA is reported. Ferric heme binding to HSA in the absence and presence of anti-HIV drugs was also investigated. The association equilibrium constant and second-order rate constant for the binding of anti-HIV drugs to Sudlow's site I of ferric heme-HSA are lower by one order of magnitude than those for the binding of anti-HIV drugs to HSA. Accordingly, the association equilibrium constant and the second-order rate constant for heme binding to HSA are decreased by one order of magnitude in the presence of anti-HIV drugs. In contrast, the first-order rate constant for ligand dissociation from HSA is insensitive to anti-HIV drugs and ferric heme. These findings represent clear-cut evidence for the allosteric inhibition of anti-HIV drug binding to HSA by the heme. In turn, anti-HIV drugs allosterically impair heme binding to HSA. Therefore, Sudlow's site I and the heme cleft must be functionally linked.

The basic properties and the in vitro release rate kinetics of azelaic acid (AA), alternatively vehiculated in different phospholipid-based vesicles such as ethosomes or liposomes, were investigated. Ethosomes were produced by a simple method based on addition of an aqueous phase to an ethanol solution (comprised between 20\% and 45%, v/v) of soy phosphatidyl choline (5%, w/w) and AA (0.2%, w/w) under mechanical stirring. Liposomes were obtained by the same composition in the absence of ethanol with the reverse-phase evaporation method. Vesicle size was measured by photon correlation spectroscopy (PCS), evidencing smaller mean diameters and narrower dimensional distributions in the case of ethosomes with respect to liposomes. In order to obtain homogeneously sized vesicles, both ethosomal and liposomal dispersions were extruded through polycarbonate membranes with pores of calibrated diameter (400 nm and 200 nm). Vesicle morphology was characterized by freeze-fracture scanning electron microscopy (SEM) showing the presence of unilamellar vesicles both in liposome- and in ethosome-based dispersions. Free energy measurements of the vesicle bilayers were conducted by differential scanning calorimetry (DSC). AA diffusion from ethosomal or liposomal dispersions and from ethosomes and liposomes incorporated in a viscous gel was investigated by a Franz cell assembled with synthetic membranes. The release rate was more rapid from ethosomal systems than from liposomal systems. In particular, ethosomes produced by the highest ethanol concentration released AA more rapidly, and the same trend was found using viscous forms.

NO-donors block Plasmodium, Trypanosoma, and Leishmania life cycle inactivating parasite cysteine proteinases. In this study, the inactivation of falcipain, cruzipain, and Leishmania infantum cysteine proteinase by S-nitroso-5-dimethylaminonaphthalene-1-sulphonyl (dansyl-SNO), S-nitrosoglutathione (GSNO), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), and S-nitrosoacetylpenicillamine (SNAP) is reported. With NO-donors in excess over the parasite cysteine proteinase, the time course of enzyme inactivation corresponds to a pseudo-first-order reaction for more than 90% of its course. The concentration dependence of the pseudo-first-order rate constant is second-order at low NO-donor concentrations but tends to first-order at high NO-donor concentrations. This behavior may be explained by a relatively fast pre-equilibrium followed by a limiting pseudo-first-order process. Kinetic parameters of cruzipain inactivation by GSNO were affected by the acidic pK shift of one ionizing group (from pKunl = 5.7 to pKlig = 4.8) upon GSNO-induced enzyme inactivation. Falcipain, cruzipain, and L. infantum cysteine proteinase inactivation by dansyl-SNO, GSNO, NOR-3, and SNAP is prevented and reversed by dithionite and l-ascorbic acid. However, the incubation of L. infantum cysteine proteinase with dansyl-SNO does not result in the appearance of fluorescence of the enzyme. More than 90% of the S-transnitrosylation product GSH existed in the inactivation reaction, suggesting that S-transnitrosylation is the favorite process for parasite cysteine proteinase inactivation. Furthermore, the fluorogenic substrate N-alpha-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) protects L. infantum cysteine proteinase from inactivation by SNAP. These results indicate that parasite cysteine proteinase inactivation by NO-donors occurs via NO-mediated S-nitrosylation of the Cys25 catalytic residue.