Enea Menegatti

The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor): a milestone protein.

The pancreatic Kunitz inhibitor, also known as aprotinin, bovine basic pancreatic trypsin inhibitor (BPTI), and trypsin-kallikrein inhibitor, is one of the most extensively studied globular proteins. It has proved to be a particularly attractive and powerful tool for studying protein conformation as well as molecular bases of protein/protein interaction(s) and (macro)molecular recognition. BPTI has a relatively broad specificity, inhibiting trypsin- as well as chymotrypsin- and elastase-like serine (pro)enzymes endowed with very different primary specificity. BPTI reacts rapidly with serine proteases to form stable complexes, but the enzyme: inhibitor complex formation may involve several intermediates corresponding to discrete reaction steps. Moreover, BPTI inhibits the nitric oxide synthase type-I and -II action and impairs K+ transport by Ca2+-activated K+ channels. Clinically, the use of BPTI in selected surgical interventions, such as cardiopulmonary surgery and orthotopic liver transplantation, is advised, as it significantly reduces hemorrhagic complications and thus blood-transfusion requirements. Here, the structural, inhibition, and bio-medical aspects of BPTI are reported.

Formulation study for the antitumor drug camptothecin: liposomes, micellar solutions and a microemulsion

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.

Effect of cationic liposome composition on in vitro cytotoxicity and protective effect on carried DNA

Positively charged liposomes were prepared by using three different cationic surfactants, namely cetyl-trimethyl-ammonium bromide (CTAB), didodecyl-dimethyl-ammonium bromide (DDAB12) and dioctadecyl-dimethyl-ammonium bromide (DDAB18). A study of the parameters influencing the in vitro toxicity of cationic liposomes on cultured cell lines was performed, demonstrating the lower cytotoxicity of DDAB18-containing liposomes. In addition, the stability of λDNA complexed to cationic liposomes after exposure to serum exo- and endonucleases was evaluated. Our results indicated that DDAB12 and DDAB18 liposomes are able to efficiently protect DNA from degradation, thus representing a potential approach to deliver nucleic acid in vivo.

Comparative analysis of tetracycline-containing dental gels: Poloxamer- and monoglyceride-based formulations

Abstract 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 ( T c ) 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.

Lipid-based supramolecular systems for topical application: A preformulatory study

This article describes the production and characterization of monoglyceride-based supramolecular systems by a simple processing technique, avoiding time-consuming procedures, high energy input, and the use of organic solvents. A preformulatory study was performed to study the influence of the experimental parameters on the production of monoglyceride-based disperse systems. In particular the effects of (1) stirring speed, (2) type and concentration of monoglyceride mixture, and (3) type and concentration of surfactant were investigated on the recovery, fraction of larger particles, mean diameter, and shape of smaller particles (so called nanosomes). Dispersions were first characterized by optical microscopy and freeze-fracture electron microscopy. The mean diameter of standard nanosomes, analyzed by photon correlation spectroscopy (PCS) after elimination of larger particles by filtration, was 193.5 nm. Cryotransmission electron microscopy studies, conducted in order to investigate the structure of dispersions, showed the coexistence of vesicles and particles characterized by a cubic organization. X-ray diffraction data revealed the coexistence of 2 different cubic phases, the first being a bicontinuous cubic phase of spatial symmetry Im3m (Q229) and the second belonging to the Pn3m spatial symmetry. A study on the stability of monoglyceride-based dispersions based on macroscopical analysis of organoleptic properties and dimensional analysis by time was performed after elimination of larger particles by filtration. Organoleptic and morphological features do not change by time, appearing free from phase-separation phenomena for almost 1 year from production. PCS studies showed that nanosomes undergo an initial increase in mean diameter within the first month following production; afterwards they generally maintain their dimensions for the next 4 months.

Effects of phospholipid based formulations on in vitro and in vivo percutaneous absorption of methyl nicotinate

In this paper we evaluate the influence of phospholipid based formulations (PBFs) on skin absorption. In particular we describe the production and characterization of different PBFs, namely liposomes and w/o microemulsion gels, and their influence on in vitro and in vivo absorption of methyl nicotinate (MN) used as model compound. In order to compare the influence of various vehicles on skin absorption, Franz cell and MN induced erythema were used as in vitro and in vivo experimental models respectively. The formulations tested were: (a) unilamellar liposomes consisting of soybean lecithin/ cholesterol (9:1 w/w) suspended in water or incorporated into hydrophilic gels (Carbomer and carboxymethyl cellulose based gels) and (b) soybean lecithin based gels. The results indicate that vehicles containing phospholipids in liposomal form provided enhanced in vivo MN skin permeation compared to the corresponding vehicles without phospholipids. Lecithin gel showed a different behaviour characterized by a short and intense persistence of MN induced erythema.

Dextran cross-linked gelatin microspheres as a drug delivery system

This paper describes the use of oxidized dextran as a cross-linker for the preparation of gelatin microspheres. Microspheres were obtained by a thermal gelation method and their dissolution kinetic was examined. In order to find evidence of sugar mediated cross-linking, swelling tests and gelatin microspheres dissolution experiments were performed. The obtained results indicated that oxidized dextran can form a cross-linked gelatin network which can reduce the dissolution of gelatin. More interestingly, gelatin microspheres treated by both native and oxidized dextran slow down, even if to a different extent, the release of the antitumor drug TAPP-Br used as a model compound. Taken together, our results suggest that oxidized dextran could be an interesting means to cross-link gelatin microspheres allowing the use of this delivery formulation for controlled release of drugs.

Preparation and characterization of starch/cyclodextrin bioadhesive microspheres as platform for nasal administration of Gabexate Mesylate (Foy®) in allergic rhinitis treatment

Bioadhesive and biodegradable microspheres were obtained by chemical cross-linking with epichlorohydrin of an alkaline solution of a mixture of starch and alpha-, beta-, or gamma-cyclodextrin (CyD). Microspheres were characterized by scanning electron microscopy, swelling degree, and water retention. The percentage of the effective CyD in microspheres was estimated by measuring the amount of iodine and typical organic compounds (TOCs) retained in the hydrophobic cavity of CyD. Gabexate Mesylate (trade name Foy); GM), an antiallergic drug, was included in microspheres by soaking in an aqueous solution containing the drug, followed by solvent evaporation or lyophilization. UV, IR, and DSC data indicated that despite the fact that GM is a hydrophilic drug, its hydrophobic moiety close to the benzene ring is able to penetrate the CyD cavity and to form stable inclusion complexes. Values of the association equilibrium constant for GM binding to CyD, obtained by UV differential spectroscopy, indicated that the affinity of the drug for alpha- and gamma-CyD is higher than that for beta-CyD. In vitro, GM was gradually released during 1h. Even if the release rate of the drug is relatively fast, the microspheres might actually provide the best platform since the material adheres to the nasal mucosa which was proved by adhesion tests. The GM integrity was checked by comparing its anti-trypsin activity before and after release.

Preparation of liposomes by reverse-phase evaporation using alternative organic solvents

The organic solvents employed in liposome preparation, such as chlorinated solvents, diethyl ether or methanol, although usually removed by evaporation, may remain as traces in the final formulation representing a possible risk for human health and influencing the stability of the vesicles. In order to solve the above mentioned disadvantages, this paper describes the use of different organic solvents, namely ethanol, ethyl acetate and two mixtures of ethanol/ethyl acetate, for the production of liposomes by the reverse phase evaporation technique. After preparation, liposomes were extruded through polycarbonate filters and then characterized by size and encapsulation efficacy. As model drugs retinyl acetate and sodium cromoglycate have been used. The association yield for the hydrophilic drug and the encapsulation yield for the hydrophilic model drug were found, in all the liposome preparations, to be satisfactory. In particular, the use of more polar organic solvents (with respect to diethyl ether) appear to enhance the encapsulation of the hydrophilic drug, sodium cromoglycate. The use of alternative organic solvents to diethyl ether can be proposed with the aim of reducing the toxic problems associated with the presence of residual traces of organic solvents in the final liposome formulation.

Purification, inhibitory properties and amino acid sequence of a new serine proteinase inhibitor from white mustard (Sinapis alba L.) seed

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.