Angelo Scatturin

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.

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.

Complexation of the sunscreen agent, butyl-methoxydibenzoylmethane, with hydroxypropyl-β-cyclodextrin.

Abstract The interaction between the sunscreen, butyl-methoxydibenzoylmethane (BM-DBM), and parent and modified α-, β- or γ-cyclodextrins was investigated in water by phase-solubility analysis. Among the available cyclodextrins, only hydroxypropyl-β-cyclodextrin (HP-β-CD) produced a significant increase in the aqueous solubility of BM-DBM. The complexation of the sunscreen agent with HP-β-CD was studied by circular dichroism, differential scanning calorimetry and X-ray diffractometry. The data from the solubility and the circular dichroism studies suggested the formation of a 1:2 (sunscreen:cyclodextrin) complex. The photodegradation of BM-DBM was reduced by inclusion complexation with HP-β-CD. Therefore the complex can be used to improve the photostability of the sunscreen agent.

Phe-d-Leu-Phe-d-Leu-Phe derivatives as formylpeptide receptor antagonists in human neutrophils: cellular and conformational aspects

We synthesized several Phe-d-Leu-Phe-d-Leu-Phe analogues in which tert-butyloxycarbonyl and four different ureido substituents were included at the N-terminal of the peptides, obtained as free acid and methyl-ester derivatives. Their biological action was analysed on human neutrophil responses induced by N-formyl-Met-Leu-Phe (fMLF). Several in vitro assays were carried out: receptor binding, measurement of Ca2+ intracellular concentration, chemotaxis, superoxide anion production and enzyme release. A conformational investigation, using infrared absorption and circular dichroism, was also performed. Our results demonstrate that the compounds examined prefer an ordered conformation (beta-turn) in amphipathic environment, and are able to antagonize the neutrophil functions evoked by fMLF. Moreover, the extent of inhibition of Ca2+ intracellular enhancement, as well as of superoxide anion production and granule enzyme release, appears related to their affinity toward the formylpeptide receptor. The free acid peptide derivatives appear to be more active antagonists than the methyl-ester ones.

Response of human neutrophils to formyl-peptide modified at the terminal amino and carboxyl groups

Two analogs of chemotactic peptideN-formyl-l-methionyl-l-leucyl-l-phenylalanine were examined for their capacity to activate several functions of human neutrophils. The C-terminus methyl ester derivative of the chemotactic peptide was found to possess strong biological activity and was able to induce levels of chemotaxis, enzyme secretion, and Superoxide generation comparable to those observed with the same concentrations ofN-formyl-l-methionyl-l-leucyl-l-phenylalanine. The analog containing atert-butyloxycarbonyl group at the N-terminus, as well as the C-terrninal methyl ester, was completely devoid of activity towards neutrophils. From these results, it appears that the free carboxyl group is not necessary for biological function. In contrast, the substituent at the N-terminus may play a critical role in the induction of the cellular response.

Evidence for β-turn structure in model peptides reproducing pro-ocytocin/neurophysin proteolytic processing site

The structural organization of small peptides reproducing the amino acid sequence of the common ocytocin/neurophysin precursor around the LysArg cleavage locus was investigated by a combination of spectroscopical techniques. In water both circular dichroism and [1H] NMR spectra indicated that these peptides adopted a random conformation. Evidence for folded structures was obtained when these compounds were placed in a membrane-like environment i.e. 40 mM SDS in phosphate buffer or trifluoroethanol. Whereas the CD spectra indicated the formation of various types of beta-turn in rapid equilibrium, measurements of NH temperature coefficients and Nuclear Overhauser Effects by 400 and 500 MHz NMR revealed the existence of contacts and of a folded conformation. These observations are discussed in relation with previous hypothesis made on the secondary structure organization of the proteolytic processing site of polypeptide hormone precursors.

Solid lipid microparticles for the stability enhancement of the polar drug N6-cyclopentyladenosine

The objective of this study was to prepare solid lipid microparticles (SLMs) loaded with the polar adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA). The microparticles were produced by the conventional hot emulsion technique, using different lipidic carriers (tristearin, glyceryl behenate and stearic acid) and hydrogenated phosphatidylcholine as the surfactant. The controlled release of CPA was achieved only with stearic acid microparticles. This phenomenon has been attributed to direct acid-base interactions between the basic nitrogen atoms of CPA and stearic acid. These SLMs were characterized by release studies, scanning electron microscopy and powder X-ray diffraction analyses. The obtained particles showed proper features in terms of morphology and size distribution (3.2-10.3microm), with a drug loading of 0.15+/-0.04%. The influence of the SLMs carrier system on CPA stability was investigated in vitro using human whole blood. The degradation kinetic of microparticle-entrapped CPA was significantly lower from that measured for the free CPA. The overall results indicate that it was possible to achieve the encapsulation and controlled release of a polar drug, such as CPA, within a lipid matrix without resorting to the complex methods generally used for the preparation of these systems.

Poly(lactic acid) microspheres for the sustained release of antiischemic agents

We report a preliminary study evaluating the encapsulation modalities in microparticles of the antiischemic drug N(6)-cyclopentyladenosine (CPA). The effects of release systems have been evaluated on the stability in human whole blood of CPA and its affinity toward human adenosine A(1) receptors. The microspheres were prepared by an emulsion-solvent evaporation method (different CPA amounts and two stirring rates were employed) using poly(lactic acid). Free and encapsulated CPA was incubated in human blood and the drug stability was analyzed. The affinity of CPA to human A(1) receptor was also obtained in the presence and in the absence of unloaded microspheres. The microspheres obtained using 1200 rpm showed a broad size distribution and a mean diameter value of 21+/-9 microm. Using 1700 rpm the mean diameter decreased to 5+/-2 microm and a more homogeneous size distribution was obtained. The CPA release changed with the particle size and the different amounts of drug employed during the preparation of the microspheres. The degradation in human whole blood of CPA encapsulated in the microspheres was negligible, with respect to that of free CPA. Affinity values of CPA obtained in the absence and in the presence of unloaded microspheres were the same.

Synthesis of a novel series of imidazo[4,5-c]pyrazole derivatives and their evaluation as herbicidal agents

Ever changing problems in agricultural weed control require periodic introduction of new herbicides. Imidazo[4,5‐c]pyrazoles, which were considered of interest as potential herbicides, were synthesized and examined for the pre‐emergence, post‐emergence, and post‐transplant control of weeds in rice against broadleaf and grass weed species. The data obtained suggest that some imidazo[4,5‐c]pyrazoles have potential herbicidal activity against a wide range of weeds, with 5‐methyl, 5‐thiomethyl, and 5‐unsubstituted derivatives being the most effective. No herbicidal activity was observed in the 5‐methylsulfonylimidazo[4,5‐c]pyrazole and imidazo[4,5‐c]pyrazolone series.

Thermodynamic In Vitro Studies as a Method to Investigate the Pharmacodynamic Behavior of Adenosine A1 Receptor Ligands

AbstractPurpose. A thermodynamic analysis of the binding to rat cortex adenosine A1, receptor of N6-substituted (full agonists) and N6-substituted-deoxyribose (partial agonists) adenosine derivatives was performed. The intrinsic activity of the compounds was evaluated by measurements of the inhibition of forskolin stimulated 3′, 5′-cyclic adenosine mono-phosphate (c-AMP) levels in isolated epididymal rat adipocytes. Methods. The thermodynamic parameters ΔG° (standard free energy), ΔH °(standard enthalpy), and ΔS° (standard entropy) of the binding equilibrium were determined by means of affinity measurements carried out at different temperatures (0, 10, 20, 25, 30° C). Levels of c-AMP were evaluated performing competitive protein binding assays. Results. The binding of the ligands increases with temperature enhancement and, as a consequence, is totally entropy driven. Standard entropy values correlate significantly with intrinsic activity ones. Conclusions. It is proposed the data obtained by these in vitro experiments can be used to investigate the in vivo pharmacodynamic of A1, full and partial agonists.