Alessandro Dalpiaz

Progress in Drug Delivery to the Central Nervous System by the Prodrug Approach

This review describes specific strategies for targeting to the central nervous system (CNS). Systemically administered drugs can reach the brain by crossing one of two physiological barriers resistant to free diffusion of most molecules from blood to CNS: the endothelial blood-brain barrier or the epithelial blood-cerebrospinal fluid barrier. These tissues constitute both transport and enzymatic barriers. The most common strategy for designing effective prodrugs relies on the increase of parent drug lipophilicity. However, increasing lipophilicity without a concomitant increase in rate and selectivity of prodrug bioconversion in the brain will result in failure. In these regards, consideration of the enzymes present in brain tissue and in the barriers is essential for a successful approach. Nasal administration of lipophilic prodrugs can be a promising alternative non-invasive route to improve brain targeting of the parent drugs due to fast absorption and rapid onset of drug action. The carrier-mediated absorption of drugs and prodrugs across epithelial and endothelial barriers is emerging as another novel trend in biotherapeutics. Several specific transporters have been identified in boundary tissues between blood and CNS compartments. Some of them are involved in the active supply of nutrients and have been used to explore prodrug approaches with improved brain delivery. The feasibility of CNS uptake of appropriately designed prodrugs via these transporters is described in detail.

Pharmacological and biochemical characterization of purified A2a adenosine receptors in human platelet membranes by [3H]-CGS 21680 binding.

1 The binding properties of human platelet A2a adenosine receptors, assayed with the A2a‐selective agonist, [3H]‐2‐[p‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine ([3H]‐CGS 21680), are masked by a non‐receptorial component, the adenotin site. In order to separate A2a receptors from adenotin sites, human platelet membranes were solubilized with 1% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulphonate (CHAPS). The soluble platelet extract was precipitated with polyethylene glycol (PEG) and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. 2 The present paper describes the binding characteristics of the selective A2a agonist, [3H]‐CGS 21680, to this purified platelet membrane preparation. In addition, receptor affinity and potency of several adenosine agonists and antagonists were determined in binding and adenylyl cyclase studies. 3 Saturation experiments revealed a single class of binding site with Kd and Bmax values of 285 nM and 2.07 pmol mg−1 of protein respectively. Adenosine receptor ligands competed for the binding of 50 nM [3H]‐CGS 21680 to purified protein, showing a rank order of potency consistent with that typically found for interactions with the A2a adenosine receptors. In the adenylyl cyclase assay the compounds examined exhibited a rank order of potency very close to that observed in binding experiments. 4 Thermodynamic data indicated that [3H]‐CGS 21680 binding to the purified receptor is totally entropy‐driven in agreement with results obtained in rat striatal A2a adenosine receptors. 5 It is concluded that in the purified platelet membranes there is a CGS 21680 binding site showing the characteristic properties of the A2a receptor. This makes it possible to use this compound for reliable radioligand binding studies on the A2a adenosine receptor of human platelets.

Binding thermodynamics at A1 and A2a adenosine receptors

Only recently the binding equilibrium of a number of ligands at adenosine A1 and A2a receptors has been analyzed from a thermodynamic point of view. This approach presents the advantage, with respect to usual affinity constant measurements, of a greater capability to give information about the molecular mechanisms underlying the binding process. All available data agree in indicating that, for both A1 and A2a receptors, agonist binding of adenosine derivatives was totally entropy-driven, while xanthine antagonist binding was essentially enthalpy-driven. The differences in thermodynamic behaviour of A1 and A2a agonists and antagonists could be interpreted in terms of a simplified general model of drug-receptor interaction, which accounted for the role played by the ribose moiety and N6-substituents of adenosinic drugs in determining both affinity and intrinsic activity properties. In the frame of this model, measurements of thermodynamic parameters of N6-monosubstituted agonists allowed to hypothesize, for the first time, the existence of partial agonists to adenosine A1 receptors, as now confirmed experimentally. All thermodynamic data concerning the interaction of all ligands studied with A1 and A2a receptors are briefly discussed in terms of the enthalpy-entropy compensation phenomenon which appears to be widely determined by the reorganization of solvent molecules in the binding process.

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.

Adenylyl cyclases as innovative therapeutic goals

Pharmacological modulation of intracellular cyclic AMP (cAMP) signalling could provide new therapeutic and experimental tools. Although drugs interfering with this pathway have traditionally targeted membrane receptors, the effector enzyme adenylyl cyclase (AC), which functions as a signalling catalyst, also presents an interesting target. Thus, development of isoform-selective stimulator and/or inhibitor compounds for AC could lead to organ-specific pharmacotherapeutics for treating heart failure, cancer and neurodegenerative diseases. In this review, the potential of AC as the object of drug therapy is discussed.

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.

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]‐SCH 58261 binding

1 The present study describes for the first time the characterization of the adenosine A2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [3H]‐5‐amino‐7‐(2‐phenylethyl)‐2‐(2‐furyl)‐pyrazolo [4,3‐e]‐1,2,4 triazolo [1,5‐c] pyrimidine, ([3H]‐SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies. 2 Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 0.85 nM and 35 fmol mg−1 protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [3H]‐SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A2A‐adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments. 3 Thermodynamic data indicate that [3H]‐SCH 58261 binding to human lymphocytes is entropy and enthalpy‐driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A2A‐adenosine receptors. 4 It is concluded that in human lymphocyte membranes [3H]‐SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A2A‐receptor. The presence of A2A‐receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.

Solid microparticles based on chitosan or methyl-β-cyclodextrin: a first formulative approach to increase the nose-to-brain transport of deferoxamine mesylate

We propose the formulation and characterization of solid microparticles as nasal drug delivery systems able to increase the nose-to-brain transport of deferoxamine mesylate (DFO), a neuroprotector unable to cross the blood brain barrier and inducing negative peripheral impacts. Spherical chitosan chloride and methyl-β-cyclodextrin microparticles loaded with DFO (DCH and MCD, respectively) were obtained by spray drying. Their volume-surface diameters ranged from 1.77 ± 0.06 μm (DCH) to 3.47 ± 0.05 μm (MCD); the aerodynamic diameters were about 1.1 μm and their drug content was about 30%. In comparison with DCH, MCD enhanced the in vitro DFO permeation across lipophilic membranes, similarly as shown by ex vivo permeation studies across porcine nasal mucosa. Moreover, MCD were able to promote the DFO permeation across monolayers of PC 12 cells (neuron-like), but like DCH, it did not modify the DFO permeation pattern across Caco-2 monolayers (epithelial-like). Nasal administration to rats of 200 μg DFO encapsulated in the microparticles resulted in its uptake into the cerebrospinal fluid (CSF) with peak values ranging from 3.83 ± 0.68 μg/mL (DCH) to 14.37 ± 1.69 μg/mL (MCD) 30 min after insufflation of microparticles. No drug CSF uptake was detected after nasal administration of a DFO water solution. The DFO systemic absolute bioavailabilities obtained by DCH and MCD nasal administration were 6% and 15%, respectively. Chitosan chloride and methyl-β-cyclodextrins appear therefore suitable to formulate solid microparticles able to promote the nose to brain uptake of DFO and to limit its systemic exposure.

Studies on human neutrophil biological functions by means of formyl-peptide receptor agonists and antagonists.

Phagocytes are activated by several extracellular signals, including formyl-peptides derived from bacterial proteins or disrupted cells. The most intensely studied member of the formylpeptide family is the synthetic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), whose specific receptors have been identified on neutrophil plasma membrane and subsequently cloned. The fMLP-receptor interaction activates multiple transduction pathways responsible for various neutrophil functions such as adhesion, chemotaxis, exocytosis of secretory granules and superoxide anion production, which represent the physiological response to bacterial infection and tissue damage. An unresolved question is whether signaling requirements are identical or specific for each physiological function. The development of fMLP receptor agonists and antagonists has led to an improvement of our knowledge about the above issue. Of particular interest is the possibility that receptorial antagonists, able to transiently inhibit neutrophil responses to formylpeptides, could be therapeutic agents in the treatment of inflammation-related diseases. Aim of this review is, i) to summarise the current understanding of the series of events that begins at the level of formylpeptide-receptor interaction and is responsible for the activation of transduction pathways, which finally determine neutrophil response; ii) to define the state of art regarding the synthesis as well as the biological actions of fMLP receptor agonists and antagonists.

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.