Fabrizio Bortolotti

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.

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.

Brain uptake of an anti-ischemic agent by nasal administration of microparticles

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.

Purification, inhibitory properties, amino acid sequence and identification of the reactive site of a new serine proteinase inhibitor from oil‐rape (itBrassica napus) seed

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 β‐trypsin and bovine α‐chymotrypsin with apparent dissociation constants of 3.0 × 10−10 M and 4.1 × 10−7 M, at pH 8.0 and 21°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‐pi, 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.

Pullulan–cyclodextrin microspheres: A chromatographic approach for the evaluation of the drug–cyclodextrin interactions and the determination of the drug release profiles

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 CyDs. 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 CyDs 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.

Synthesis and Study of 5′-Ester Prodrugs of N6-Cyclopentyladenosine, a Selective A1 Receptor Agonist

AbstractPurpose. 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. Methods. An appropriate synthetic procedure was adopted to avoid concomitant deamination at position 6. Log P values were obtained by the Mixxor system. The stability of CPA and its 5′-ester was evaluated in human plasma and whole blood and analyzed with high-performance liquid chromatography. The affinities to human A1 receptor expressed by N6-cyclohexyladenosine cells were obtained by binding experiments. The activities were evaluated by measurements of the inhibition of forskolin stimulated 3′-5′-cyclic adenosine monophosphate, performing competitive binding assays. Results. All prodrugs were more lipophilic than CPA, and their hydrolysis, in whole blood and in plasma, was found related, respectively, to the length and hindrance of 5′-substituents. Affinity and activity values indicated a very weak interaction toward adenosine A1 receptor of the intact prodrugs. Conclusions. We propose 5′-esters of CPA, characterized by suitable lipophilicity and elevated degree of stability in physiological fluids, as possible canditates for CPA prodrugs.

Multicomponent antibiotic substances produced by fermentation: Implications for regulatory authorities, critically ill patients and generics

Teicoplanin and polymyxin E (colistin) are antibiotics consisting of multiple, closely related subcomponents, produced by fermentation. The principal components comprise a complex mixture of chemically related, active substances (teicoplanin A(2-1)-A(2-5) and polymyxin E(1-2), respectively), which might be required to be present in specific ratios to ensure optimal antibacterial and clinical efficacy. These subcomponents differ in their fatty acid and amino acid composition and, as such, the lipophilic and protein binding characteristics differ between components. This has therapeutic implications for critically ill patients, as the volume of distribution of the teicoplanin A2 and polymyxin E analogues at the onset of an intravenous infusion may impact on expected pharmacokinetics and influence outcome.

Reversed-phase HPLC study on the in vitro enzymic degradation of dermorphin

A high-performance liquid chromatographic (HPLC) method for the separation of the opioid heptapeptide dermorphin and related fragments has been developed. The chromatographic system was applied in the study of the kinetics of degradation of dermorphin (Der) in various tissues. Der was found to be extremely resistant to human and rat plasma (T 1/2 greater than 180 min). Upon incubation with homogenates of rat brains and kidneys, Der was cleaved with a half-life of 20.8 +/- 2.2 min and 2.4 +/- 0.3 min respectively. The catabolite formed was identified, in both tissues, as the N-terminal tetrapeptide H-Tyr-D-Ala-Phe-Gly-OH. The stability to rat kidney and brain of the N-terminal hexa- and pentapeptides and of the [4 psi 5, NHCO] Der analogue was also investigated. The nature of the enzyme systems involved in the in vitro degradations is discussed.

Amphiphilic association systems for Amphotericin B delivery.

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.

Isolation and characterization of a new form of the porcine pancreatic secretory trypsin inhibitor Biochemical studies and high-resulution 1H-NMR

Abstract A new active form of porcine PSTI (pancreatic secretory trypsin inhibitor) was isolated during the fractionation by ion-exchange chromatography of the already known forms PSTI I and II. Biochemical and 1H-NMR techniques were used to characterize the new inhibitor, which is referred to as PSTI III. The amino acid composition, the nature of the N-terminal residue and data obtained from the tryptic peptides all indicate that PSTI III lacks the N-terminal octapeptide of PSTI I; hence, it starts and ends with disulfide bridges. The conclusion is supported by the 1H-NMR spectrum of the protein at 270 MHz. The biological activity and the most prominent conformational and dynamic features of forms I and II are retained in inhibitor IIL However, PSTI III appears to be less compact than its parent forms I and II, suggesting that in the latter inhibitors an interaction between the N-terminal tail and the bulk of the protein may contribute to the overall stability. The genetic origin of PSTI III is discussed.