Marina Zacchigna

Bi-layered self-emulsifying pellets prepared by co-extrusion and spheronization: Influence of formulation variables and preliminary study on the in vivo absorption

The aim of this work was to produce by co-extrusion-spheronization pellets with two cohesive layers, one of them containing a self-emulsifying system for vinpocetine, a poorly water soluble model drug. Two layers were prepared: an inert layer of microcrystalline cellulose, lactose and water and a second one wetted with the self-emulsifying system. Different formulations of both layers were tested, evaluating the effects of formulation variables with an experimental design. The screening amongst formulations was performed preparing rod extrudates and using the extrusion profiles to assess their suitability for extrusion and to anticipate quality of the spheronized extrudates. Tubular extrudates and co-extrudates/spheronized pellets were then produced. Two types of bi-layered pellets were prepared: type I with the self-emulsifying system internally and the inert matrix externally, whereas type II vice versa. The pellets were characterized for sizing and shape, density, hardness, in vitro dissolution and disintegration and released droplets size and in vivo tests. Although both types of pellets demonstrated adequate morphological and technological characteristics, pellets type II revealed an improved drug solubility and in vivo bioavailability. These preliminary technological and pharmacokinetic data demonstrated that co-extrusion/spheronization is a viable technology to produce bi-layered cohesive self-emulsifying pellets of good quality and improved in vivo bioavailability.

Nitric oxide-mediated activity in anti-cancer photodynamic therapy.

Cell recurrence in cancer photodynamic therapy (PDT) is an important issue that is poorly understood. It is becoming clear that nitric oxide (NO) is a modulator of PDT. By acting on the NF-κB/Snail/RKIP survival/anti-apoptotic loop, NO can either stimulate or inhibit apoptosis. We found that pheophorbide a/PDT (Pba/PDT) induces the release of NO in B78-H1 murine amelanotic melanoma cells in a concentration-dependent manner. Low-dose PDT induces low NO levels by stimulating the anti-apoptotic nature of the above loop, whereas high-dose PDT stimulates high NO levels inhibiting the loop and activating apoptosis. When B78-H1 cells are treated with low-dose Pba/PDT and DETA/NO, an NO-donor, intracellular NO increases and cell growth is inhibited according to scratch-wound and clonogenic assays. Western blot analyses showed that the combined treatment reduces the expression of the anti-apoptotic NF-κB and Snail gene products and increases the expression of the pro-apoptotic RKIP gene product. The combined effect of Pba and DETA/NO was also tested in C57BL/6 mice bearing a syngeneic B78-H1 melanoma. We used pegylated Pba (mPEG-Pba) due to its better pharmacokinetics compared to free Pba. mPEG-Pba (30 mg/Kg) and DETA/NO (0.4 mg/Kg) were i.p. injected either as a single molecule or in combination. After photoactivation at 660 nM (fluence of 193 J/cm(2)), the combined treatment delays tumor growth more efficiently than each individual treatment (p<0.05). Taken together, our results showed that the efficacy of PDT is strengthened when the photosensitizer is used in combination with an NO donor.

Syntheses, chemical and enzymatic stability of new poly(ethylene glycol)–acyclovir prodrugs

Two known antiherpetic agents, acyclovir and valaciclovir, were coupled with activated poly(ethylene glycol). In vitro drug release studies demonstrated the conjugates to be stable in buffer solutions at pH 7.4 and 5.5, while only PEG-valacyclovir2 was stable in a buffer solution at pH 1.2. The ability of the macromolecular conjugate to release the free drug was also evaluated in plasma, in which the most stable prodrug also proved to be PEG-valacyclovir2. The derivatives are degraded in the presence of proteolytic enzyme. The rate of hydrolysis was monitored by HPLC-analysis.

Chemical composition and functional characterisation of commercial pumpkin seed oil

BACKGROUND Pumpkin (Cucurbita pepo L.) seed oil is a common product in Slovenia, Hungary and Austria and is considered a preventive agent for various pathologies, particularly prostate diseases. These properties are related to its high content of carotenoids and liposoluble vitamins. In this study the carotenoid (lutein and zeaxanthin), vitamin E (α- and γ-tocopherol) and fatty acid contents of 12 samples of commercial pumpkin seed oil were investigated together with the composition of the volatile fraction resulting from the roasting process. RESULTS The aromatic profile obtained from the commercial samples was directly related to the intensity of the roasting process of the crushed pumpkin seeds. The roasting temperature played a crucial role in the concentrations of volatile substances originating from Strecker degradation, lipid peroxidation and Maillard reaction. CONCLUSION The findings suggest that high-temperature roasting leads to the production of an oil with intense aromatic characteristics, while mild conditions, generally employed to obtain an oil with professed therapeutic characteristics, lead to a product with minor characteristic pumpkin seed oil aroma. The nutraceutical properties of the product are confirmed by the high content of α- and γ-tocopherol and carotenoids.

p-nitrophenyllaurate: a substrate for the high-performance liquid chromatographic determination of lipase activity

Many assay procedures have been devised to measure lipolytic activity, but none is without problems. It is for this reason that new methods are still being proposed. In this work we have investigated the use of two esters of p-nitrophenol, the palmitic acid and lauric acid esters, as substrates for a highly sensitive high-performance liquid chromatographic method. Data on recovery, specific activity and reproducibility are reported only for the lauric ester, because the palmitic ester turned out to be a very poor substrate.

Conjugated PDT drug: Photosensitizing activity and tissue distribution of PEGylated pheophorbide a

The design of new photosensitizers with enhanced phototoxicity and pharmacokinetic properties remains a central challenge for cancer photodynamic therapy (PDT). In this study, Pheophorbide a (Pba) has been pegylated to methoxypolyethylene glycol (mPEG-Pba) to produce a soluble photosensitizer that exhibits a higher tissue distribution than free Pba. In vitro studies have shown that mPEG-Pba promotes a fairly strong photosensitizing effect in cancer cells, as previously observed for the unpegylated molecule. mPEG-Pba targets the mitochondria where, following photoactivation, ROS are produced which cause a cellular injury by lipid peroxidation. The effect of pegylation on the photosensitizer biodistribution has been examined in different selected organs of female mice, at different time points after intraperitoneal administration of the drug (50 µmol/Kg body weight). Other than free Pba, which showed a low tissue accumulation, mPEG-Pba has been detected in significant amounts (8 to 16 μg/ml) in liver, spleen, duodenum and kidney and, 3-5 hours after intraperitoneal injection, in moderate amounts (3 to 8 μg/ml) in brain and lung. In vivo optical imaging performed on living female C57/BL6 mice bearing a subcutaneous melanoma mass, showed that injected mPEG-Pba distributes all over the body, with an higher uptake in the tumor respect to free Pba. Our results indicate that although pegylation somewhat decreases the phototoxicity, it significantly increases the drug solubility and tissue distribution and tumor uptake of mPEG-Pba, making the conjugate an interesting photosensitizer for PDT.

Improvement of physicochemical and biopharmaceutical properties of theophylline by poly(ethylene glycol) conjugates.

In the present paper two theophylline esters with poly (ethylene glycol) (PEG) and methoxy poly (ethylene glycol) (mPEG) were prepared. Quantitative yields of the pure products were obtained. Unlike the free drug, the drug-polymer conjugates are freely water-soluble at room temperature. In vitro release experiments in aqueous buffer demonstrate that both conjugates are stable in buffer of pH 7.4 and 1.2. In vivo release studies after oral administration of theophylline conjugates demonstrate a good release of parent drug.

Synthesis of 4-thiophen-2'-yl-1,4-dihydropyridines as potentiators of the CFTR chloride channel.

The gating of the CFTR chloride channel is altered by a group of mutations that cause cystic fibrosis. This gating defect may be corrected by small molecules called potentiators. Some 1,4-dihydropyridine (DHP) derivatives, bearing a thiophen-2-yl and a furanyl ring at the 4-position of the nucleus, were prepared and tested as CFTR potentiators. In particular, we evaluated the ability of novel DHPs to enhance the activity of the rescued DeltaF508-CFTR as measured with a functional assay based on the halide-sensitive yellow fluorescent protein. Most DHPs showed an effect comparable to or better than that of the reference compound genistein. The potency was instead significantly improved, with some compounds, such as 3g, 3h, 3n, 4a, 4b, and 4d, having a half effective concentration in the submicromolar range. CoMFA analysis gave helpful suggestions to improve the activity of DHPs.

Melt extruded helical waxy matrices as a new sustained drug delivery system

The aim of this research was to prepare helical and cylindrical extrudates by melt extrusion and to evaluate their potential as sustained release dosage form. The systems contained theophylline as water-soluble model drug and microcrystalline wax as thermoplastic binder. The temperature suitable to ensure a successful extrusion process of formulations containing the wax in three different percentages was found to be below the melting point of the excipient. After the production of the extrudates in three different helical shapes (having 2, 3 and 4 blades) and a classical cylindrical shape, the systems were studied by means of X-ray powder diffraction and differential scanning calorimetry to check possible variations of the solid state of the drug during the thermal process. The morphology and chemical composition of the surface of the extrudates were examined by Scanning Electron Microscopy/Energy Dispersive X-ray Microanalysis to evaluate the presence of the drug on the surface of the extrudates and to monitor changes on the aspect of the waxy matrix during dissolution. Then, the different systems were analysed from the in vitro dissolution point of view to study the influence of the shape and of the composition on the drug release. An in vivo pilot study on the best performing system (helix with 3 blades) was carried out on five healthy volunteers and monitoring the intestinal transit by X-ray images. The resulting plasma profiles were analysed by means of a suitable pharmacokinetic analysis. Finally, an ad hoc mathematical model was developed to perform an accurate description of the in vitro release and in vivo performance of the 3-blades helical system.

Cerebrosides with antiproliferative activity from Euphorbia peplis L.

Two new cerebrosides have been isolated from the whole plants of Euphorbia peplis L. The structures were established by FT-IR spectroscopy, FAB MS, EI-MS, ESI-MS, 1D and 2D NMR spectroscopy. The structures of the cerebrosides were characterized as 1-O-beta-d-glycosides of phytosphingosines, which comprised a common long-chain base, (2S, 3S, 4R, 8Z)-2-amino-8 (Z)-octadecene-1,3,4-triol with 2-hydroxy fatty acids of varying chain lengths (C25, C22) linked to the amino group. The isolated compounds were shown to possess significant antiproliferative properties against cultured human tumor cell lines KB and IMR-32.