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.

Molecular mechanism involved in the transport of a prodrug dopamine glycosyl conjugate.

We have previously demonstrated that dopamine conjugation to glucose allows it to induce therapeutic effects against Parkinson's disease after intravenous administration. In this paper we demonstrate that, unlike dopamine, the prodrug glu-dopamine is a transportable substrate of glucose transporters. Towards this, the effect of glucose-conjugation on the affinity and uptake of dopamine have been assessed in vitro, using human retinal pigment epithelium (HRPE) cells. Glucose transporter-mediated uptake was measured using [(3)H]3-O-methylglucose ([(3)H]3-O-MG) as the tracer. The uptake was found to be rapid and hyperbolically related to its concentrations (K(t)=7.8+/-1.2mM and V(max)=54+/-2 nmol/min mg protein). Inhibition experiments showed that dopamine was able to interact with glucose carriers only when conjugated to glucose (IC(50)=2.6+/-0.6mM). HPLC analysis of HRPE cell extracts showed that both dopamine and the prodrug permeate the cell, but only the uptake of the prodrug is inhibitable by glucose. This confirms that glucose transporters mediate the transport of the prodrug glu-dopamine, but not of dopamine. HRPE cells is therefore proposed as a promising model for in vitro studies involving the glucose transporter-mediated transport of drugs and their conjugates.

Cationic liposomes as potential carriers for ocular administration of peptides with anti-herpetic activity.

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.

Nanoparticle formulation may affect the stabilization of an antiischemic prodrug.

The prodrug 5'-octanoyl-CPA (Oct-CPA) of the antiischemic N6-cyclopentyladenosine (CPA) has been encapsulated by nanoprecipitation in poly(lactic acid) nanoparticles, which have been recovered by gel-filtration, ultra-centrifugation or dialysis. We have analysed how different surfactants and purification methods can influence the nanoparticle characteristics. The particle sizes have been obtained by scanning electron microscope, whereas a SdFFF system was employed to detect their distributions. The Oct-CPA release from nanoparticles and stabilities in human blood of free and encapsulated prodrug have been analysed by HPLC techniques. The effects of nanoparticles on CPA interaction toward adenosine A1 receptor (its action site) have been analysed using radiolabelled drugs. The smallest nanoparticles and the best degree of homogeneity have been obtained using sodium cholate; the best recovery has been achieved by dialysis, whereas gel-filtration and ultra-centrifugation have induced the greatest removal of surfactants. The release of Oct-CPA was better controlled from the nanoparticles obtained using Pluronic F68 and purified by gel-filtration or ultra-centrifugation. Similarly, these nanoparticles better increased the stability of the prodrug in human blood. In particular, the nanoparticles purified by ultra-centrifugation induced a strong stability to a fraction of the encapsulated Oct-CPA. Any interference by unloaded nanoparticles has been registered for CPA-adenosine A1 receptor interaction.

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.

Poly(lactic acid) microspheres for the sustained release of a selective A1 receptor agonist.

A study concerning the feasibility of microsphere use as sustained delivery systems for N(6)-cyclopentyladenosine (CPA) administration has been performed. The release of this drug and the related stability effects in human whole blood have been tested. Moreover, the impact of the delivery system on CPA interaction toward human adenosine A1 receptor and the related cellular responses has been analyzed. The microspheres were prepared by an emulsion-solvent evaporation method using poly(lactic acid). Free and encapsulated CPA was incubated in fresh blood and the drug stability was analyzed with HPLC. The affinity of CPA to human A1 receptor expressed by CHO cells was obtained by binding experiments. Activity was evaluated by measurements of the inhibition of forskolin-stimulated 3',5'-cyclic adenosine monophosphate (c-AMP) performing competitive binding assays. Encapsulated CPA was released within 72 h and its degradation in blood was negligible. Affinity and activity values of CPA obtained in the absence and in the presence of unloaded microspheres were the same. CPA encapsulation in microspheres allows its sustained release and its stabilization in human whole blood to be obtained. The presence of this release system does not interfere with the CPA activity at its action site.

Synthesis and study of 5'-ester prodrugs of N6-cyclopentyladenosine, a selective A1 receptor agonist.

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. 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 candidates for CPA prodrugs.

Binding thermodynamics and intrinsic activity of adenosine A1 receptor ligands.

A thermodynamic analysis of the binding to rat cortex adenosine A1 receptors of 5'-deoxyribose-N6-cyclopentyladenosine (full agonist) and several 8-alkylamino homologues of N6-cyclopentyladenosine (partial agonists) was performed. The intrinsic activity of the compounds was also evaluated by measuring the inhibition of forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (c-AMP) levels in isolated epididymal rat adipocytes. Standard free energy (deltaG), enthalpy (deltaH ) and entropy (deltaS ) of the binding equilibrium were determined by affinity measurements carried out at different temperatures (0, 10, 20, 25, 30 degrees C). Affinity constants of drug-receptor interactions were obtained by displacement experiments in the presence of 1nM [3H]N6-cyclohexyladenosine. Levels of c-AMP were evaluated by performing competitive binding assays. As the affinity of the ligands was found to increase with temperature enhancement, the binding of full and partial agonists is therefore totally entropy-driven. Standard entropy values of a wide series of adenosine derivatives, including the compounds under examination, are strictly correlated to those of intrinsic activity. Similarly, deltaS values appear correlated to the in vivo ability of the adenosine derivatives to inhibit rat heart rate. Thermodymanic data of adenosine A1 receptor ligands are proposed as an indicator of their pharmacodynamics.

Thermodynamic in vitro studies as a method to investigate the pharmacodynamic behavior of adenosine A1 receptor ligands.

PURPOSE: 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 monophosphate (c-AMP) levels in isolated epididymal rat adipocytes. METHODS: The thermodynamic parameters deltaG(o) (standard free energy), deltaH(o) (standard enthalpy), and deltaS(o) (standard entropy) of the binding equilibrium were determined by means of affinity measurements carried out at different temperatures (0, 10, 20, 25, 30 degrees 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 A, full and partial agonists.

Conformational studies on synthetic peptides reproducing the dibasic processing site of pro-ocytocin-neurophysin.

Synthetic peptides reproducing the proteolytic processing site of pro-ocytocin were studied by different spectroscopic techniques, including circular dichroism, Fourier transform infrared absorption, and mono and bidimensional nuclear magnetic resonance, in order to ascertain the possible role of three-dimensional structure in the recognition process by maturation enzymes. Experimental results were compared with energy minimization calculations and suggest that: (i) the region situated on the N-terminus of the Lys-Arg doublet may form a beta-turn; (ii) the sequential organization of the residues participating in the beta-turn determines the privileged relative orientation of the basic amino acid sidechains and the subtype of turn; and (iii) the peptide segment situated on the C-terminal side of the dibasic doublet may assume a helix arrangement. These findings, in spite of the limitations connected to the flexibility of linear peptides, seem to substantiate the hypothesis that structural motifs around the cleavage site could be important for recognition and processing. however, a straightforward correlation between details of the secondary structure and the in vitro reactivity toward a putative convertase is not yet possible.

Structure activity studies on chemically modified homologues of the antibiotic phytotoxic leucinostatin A.

The synthesis and a conformational study of a number of homologues of the well known antibiotic, phytotoxic leucinostatin A are reported. The circular dichroism of all the compounds are discussed. Some conclusions on the SAR of these compounds are drawn. The influence of the alpha-helical conformation and/or the increased lipophile character on their interesting biological activities is emphasized.

Evidence for the presence of a secondary structure at the dibasic processing site of prohormone: the pro-ocytocin model.

Bioactivation of pro-proteins by limited proteolysis is a general mechanism in the biosynthesis of hormones, receptors and viral protein precursors. This proceeds by cleavage of peptide bonds at the level of single or pairs of basic residues in the proforms. Examination of a number of cleavage loci in various precursors failed to reveal any consensus primary sequence around the dibasic cleavage sites. Thus it has been proposed, on the basis of secondary structure predictions [Rholam, M., Nicolas, P. and Cohen, P. (1986) FEBS Lett., 207, 1-6], that those basic residues which operate as signal loci for the proteolytic enzyme machinery are situated in, or next to, privileged precursor regions most often constituted by flexible and exposed motifs, e.g. beta-turns and/or loops. Peptides reproducing the N-terminal processing domain of the hormone precursor, pro-ocytocin-neurophysin, were examined by a combination of spectroscopical techniques including circular dichroism, infrared Fourier transform and one- and two-dimensional proton NMR. The results indicate that: (i) the region situated on the N terminus of the Lys-Arg doublet is organized as a beta-turn in solution; (ii) the sequential organization of the residues participating in the beta-turn determines the privileged relative orientation of the basic amino acid side chains and the subtype of turn; (iii) the peptide segment situated on the C-terminal side of the dibasic, corresponding to the N-terminal octapeptide of neurophysin, is organized as an alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for beta-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.

The crystal and molecular structure of the alpha-helical nonapeptide antibiotic leucinostatin A.

The crystal and molecular structure of the nonapeptide antibiotic leucinostatin A, containing some uncommon amino acids and three Aib residues, has been determined by x-ray diffraction analysis. The molecule crystallizes in the orthorhombic space group P2(1)2(1)2(1), a = 10.924, b = 17.810, c = 40.50 A, C62H111N11O13, HCl.H2O, Z = 4. The peptide backbone folds in a regular right-handed alpha-helix conformation, with six intramolecular i----(i + 4) hydrogen bonds, forming C13 rings. The nonapeptide chain includes at the C end an unusual beta-Ala residue, which also adopts the helical structure of the other eight residues. In the crystal the helices are linked head to tail by electrostatic and hydrogen-bond interactions, forming continuous helical rods. The crystal packing is formed by adjacent parallel and antiparallel helical rods. Between adjacent parallel helical columns there are only van der Waals contacts, while between adjacent antiparallel helical columns hydrogen-bond interactions are formed.

Trypsin-pancreatic secretory isoinhibitor A from bovine pancreas (Kazal type). Spectroscopic study on structure and stability.

The secondary and tertiary structure of isoinhibitor A from bovine pancreas secretion (Kazal inhibitor) was investigated by circular dichroism (CD) and fluorescence measurements. The protein shows noteworthy thermal stability as seen by the temperature dependence of the CD spectra and the intensity of emission fluorescence at different pH values.

Conformational studies on sequential polypeptides. Part VII. Structural investigations on (Pro-Phe-Gly)n and (Phe-Pro-Gly)n.

The conformational properties of (Pro-Phe-Gly)n and (Phe-Pro-Gly)n were investigated both in the solid state and in solution. In solid state X-ray diffraction patterns seem to indicate that (Pro-Phe-Gly)n assumes a single chain triple helical structure. In solution, by circular dichroism studies, it was possible to show the existence of beta-bends in the presence of ethylene glycol. Similar studies carried out on (Phe-Pro-Gly)n showed no collagen or polyproline II-like structure in the solid state. In solution the beta-bend formation was apparent in all the solvent systems studied.

Conformational studies on sequential polypeptides. Part V. Synthesis and characterization of (Pro-Leu-Gly)10, (Pro-LeuqGly)n and (Leu-Pro-Gly)n.

Syntheses of (Pro-Leu-Gly)n and (Leu-Pro-Gly)n, two synthetic polytripeptide analogues of the non-polar regions of collagen, via the corresponding tripeptide p-nitrophenyl-esters are described. The sequential polypeptide (Pro-Leu-Gly)10 was also obtained by solid-phase synthesis. In the following paper, conformational investigations on these polymers, both in solution and in solid state, will be described.