Biomedical subjects

E Menegatti

Publications and source records attributed to E Menegatti.

136 records · Page 8Linked to original sources

PubMedDate not supplied

Crystal structure of the bovine alpha-chymotrypsin:Kunitz inhibitor complex. An example of multiple protein:protein recognition sites.

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.

Animals↗

PubMedDate not supplied

Binding of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to bovine alpha-chymotrypsin and bovine beta-trypsin. A thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C(p), F-T(p) and F-T(t), respectively) to bovine alpha-chymotrypsin (alpha-chymotrypsin) and bovine beta-trypsin (beta-trypsin) has been investigated. On the basis of Ka values, the proteinase inhibitor affinity can be arranged as follows: alpha-chymotrypsin: BBI approximately beta-trypsin:BBI approximately beta-trypsin:F-T(t) approximately beta-trypsin:F-T(p) much greater than alpha-chymotrypsin:F-C(p). F-C(p), F-T(p) and F-T(t) do not inhibit beta-trypsin and alpha-chymotrypsin action, respectively. On lowering the pH from 9.5 to 4.5, values of Ka for BBI, F-C(p), F-T(p) and/or F-T(t) binding to alpha-chymotrypsin and beta-trypsin decrease, thus reflecting the acid-pK shift of the invariant His57 catalytic residue from 7.0, in the free enzymes, to 5.2, in the proteinase:inhibitor complexes. Considering the known molecular models, the observed binding behaviour of BBI, F-C(p), F-T(p) and F-T(t) was related to the inferred stereochemistry of the proteinase:inhibitor contact regions.

Animals↗

PubMedDate not supplied

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to serine (pro)enzymes: a comparative thermodynamic study.

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.

Animals↗

PubMedDate not supplied

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.

Anti-Asthmatic Agents↗

PubMedDate not supplied

Liposomes containing distamycins: preparation, characterization and antiproliferative activity.

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.

Cell Division↗

PubMedDate not supplied

Kinetics of bromocriptine release from microspheres: comparative analysis between different in vitro models.

This paper describes how the use of different in vitro experimental systems can influence the determination of (a) the drug release profile from microparticles and (b) the interpretation of the release mechanism(s). We employed, as model dosage form, the Parlodel LA, a recently marketed microsphere system especially designed for bromocriptine-controlled delivery. The release kinetics of bromocriptine from microspheres were determined by using two different experimental approaches: a dialysis method and a flow-through cell method. From the comparison of the obtained data it clearly appears that different in vitro experimental models lead to distinct results in terms of drug availability. On the contrary both series of data can be convincingly fitted with the same mathematical equation, giving almost identical results in terms of postulated release mechanism. Taken together these results indicate that different experimental approaches should always be employed to determine drug release kinetics from microparticles in order to obtain more reliable information on the therapeutic dose (bioavailable drug, for in vivo experiments) and on the uniformity of different batches of microspheres.

Bromocriptine↗

PubMedDate not supplied

Production and in vitro evaluation of gelatin microspheres containing an antitumour tetra-amidine.

The preparation and characterization of gelatin microspheres (GMs) containing the antitumour aromatic tetra-amidine TAPP-Br and its release characteristics from microspheres are described. Spherical gelatin microspheres, showing a high percentage of drug encapsulation (over 95 per cent) and an average diameter of 16 microns were obtained by a coacervation method. In addition, the effects of various reaction parameters on the production and chemical hardening of GMs have been investigated. In order to compare the TAPP-Br release profiles from GMs prepared with different procedures, in vitro studies were carried out. The biological effect of TAPP-Br released from gelatin microspheres was determined on 'in vitro' cultured tumour cell lines, showing that, after encapsulation, TAPP-Br still retains antiproliferative activity comparable with that exhibited by the free drug. Taken together, the results obtained suggest that gelatin-based microspheres offer excellent potential as carriers for the controlled release of polyamidines in anticancer chemotherapy.

Antineoplastic Agents↗

PubMedDate not supplied

Biodegradable microparticles for sustained delivery of tetracycline to the periodontal pocket: formulatory and drug release studies.

This paper describes the production and characterization of biodegradable microparticles containing tetracycline, designed for periodontal disease therapy. The influence of production parameters on microparticle characteristics and antibiotic release modality was studied. Microparticles were made by using different preparation procedures and different polyesters, namely poly(L-lactide), [L-PLA] poly(DL-lactide), [DL-PLA] and poly(DL-lactide-co-glycolide) 50:50, [DL-PLG]. A double emulsion preparation method together with a concentrated salt solution as external phase gave the best results in terms of tetracycline incorporation efficacy. In vitro release experiments demonstrated that tetracycline is slowly and appropriately released from microparticles. Release kinetics were found to be influenced by the type of polymer utilized for microparticle production. In vitro experiments, simulating in vivo conditions were carried out for up to 30 days. Only DL-PLG microparticles showed significant changes in their morphology, whereas L-PLA and DL-PLA were found almost intact after the same period of time.

Delayed-Action Preparations↗

PubMedDate not supplied

Production and antiproliferative activity of liposomes containing the antitumour drug chromomycin A3.

In the present paper the production and characterization of liposomes are described as a specialized drug delivery system for chromomycin. Liposomes were prepared by the reverse phase evaporation technique followed by extrusion through polycarbonate filters; afterwards the vesicles were characterized in terms of dimensions, morphology and encapsulation efficacy. The aim of this work was to produce a drug delivery system able to reduce the toxicity problems related to the administration of this drug. The analysis of the in vitro antiproliferative activity on cultured human leukemic K562 cells demonstrated that ionic and neutral liposomes containing chromomycin are 1.5 and 7-fold more effective respectively as compared to the free drug. Based on these results and taking into account the increased solubility of the drug in this system, liposomes could represent a promising drug delivery system for use in the experimental therapy using chromomycin.

Aminoglycosides↗

PubMedDate not supplied

[Hirudin inhibition of human thrombin activity].

Thermodynamics and kinetics for hirudin binding to human alpha-, beta- and gamma-thrombin, under experimental conditions which mimic the in vivo conditions (i.e., pH = 7.35, T = 37.0 degrees C, I = 0.1 M), indicate that the inhibitor specificity for the three species of the enzyme is different. Such a finding agrees with the human thrombin:hirudin binding geometry as revealed by X-ray crystal studies. From the therapeutic viewpoint, thermodynamics and kinetics here reported indicate that the inhibitory activity of hirudin in vivo is more effective than that shown by antithrombin III, which is generally considered the most important plasma thrombin inhibitor.

Hirudins↗