Innovative oral spray-dried Idebenone systems to improve patient compliance.

Idebenone is a high permeable drug with very slight water solubility that affects the dissolution rate in the biological fluids, causing an irregular and limited in vivo absorption after oral administration. Moreover, it is marketed in Europe as tablets equivalent to 150 mg, with the consequent administration of multiple dose of solid unit to obtain the correct dose, a deterrent for the patients' compliance. According to these considerations, our goal was to develop spray-dried microparticles using a soluble ß-cyclodextrin (CD) polymer and an enhancer of dissolution rate, such as carboxymethyl cellulose, to obtain a formulation easily dosable and soluble in water. The complex in solution was evaluated by phase solubility studies and the Idebenone/CD molar ratio selected was 1:1. According to Higuchi and Connors, adding carboxymethyl cellulose, a Bs-type profile was obtained. This result was due to the presence of carboxymethyl cellulose that competes with the CD in forming Idebenone microsystems, reducing of 10-fold the formulation bulk. UV-Vis absorption, (1)H nuclear magnetic resonance and circular dichroism showed the formation of the CD/Idebenone inclusion complex confirmed also by differential scanning calorimetry, Fourier transform infrared spectroscopy and fluorescence microscope (FM). The water solubility data and the in vitro dissolution tests performed in simulated gastric fluid, showed an increase of the drug water interaction due to the presence of the CD and carboxymethyl cellulose, both able to improve drug wettability, water solubility and dissolution rate. This approach seems to be suitable to produce microsystems which are able to enhance the in vivo absorption of Idebenone after oral administration and to increase the patient compliance.

Polyphenol-enriched fractions from Sicilian grape pomace: HPLC-DAD analysis and antioxidant activity.

On the basis of a preliminary screening of seven different samples of Sicilian grape pomace, the 'Nerello Mascalese' sample NM2 was selected for an ethanol preparative extraction. The defatted NM2 EtOH extract was subjected to DPPH() and GAE assays, showing good radical scavenging activity (SC(50)=9.9 microg/mL) and a GAE value of 397.7 mg/g extract. HPLC-DAD analysis of NM2 extract allowed a quantitative determination of the main anthocyanins (AN) and flavonols/flavonol glycosides (FL/FG). Aliquots of the NM2 extract were subjected to three different fractionation protocols (FP1, FP2 and FP3). The fractions were examined by DPPH() and GAE assays, and subjected to HPLC-DAD analysis for the quantitative determination of the main AN and FL/FG. FP3 allowed obtaining a polyphenol-enriched fraction with SC(50)=14.8 microg/mL and GAE=184.1mg/g of fraction, accounting for only 1.3% in weight of the EtOH extract. Some considerations about the relationship between antioxidant activity and AN/FL/FG HPLC-DAD profiles are also reported.

Hyperhomocysteinemia in acute Plasmodium falciparum malaria: an effect of host-parasite interaction.

BACKGROUND: Plasmodium falciparum utilises the polyamine pathway, essential in proliferation and differentiation, and imposes an oxidative stress on host cell, enhancing the loss of glutathione. METHODS: Standard hematological parameters were determined in 40 black African subjects with acute P. falciparum malaria, 30 aged 5-24 months, 5 aged 4-10 years and 5 aged 19-35 years. Plasma homocysteine, cysteine, glutathione and cysteinylglycine levels were measured by HPLC method. Twenty-eight healthy black children (15 aged 6-24 months and 13 aged 3-10 years) and 20 healthy black adults (aged 20-40 years) were also included as controls. RESULTS: Plasma homocysteine levels were higher in all subjects with P. falciparum malaria and correlated positively with the disease severity and number of parasites, but negatively with Hb levels and patient ages. Cysteine level was found higher in all patients and markedly higher in 4-10 year old patients. Cysteinylglycine level was found lower particularly in 19-35 year old patients. Glutathione level was significantly lower in all patients. CONCLUSIONS: The elevated level of homocysteine during acute P. falciparum infection suggests an imbalance in the folate cycle, which could be a consequence of the reduced availability of NADPH and Vit B12, caused by increased oxidative stress. This may suggest a selection for the C677T MTHFR allele, driven by P. falciparum in sub-Saharan regions. Hence Hcy level could be useful as a predictive parameter of severity, as well as of treatment efficacy.

The photochemistry of flutamide and its inclusion complex with beta-cyclodextrin. Dramatic effect of the microenvironment on the nature and on the efficiency of the photodegradation pathways.

The photochemistry of the anticancer drug flutamide (FM), 2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide, in homogeneous media and in the beta-cyclodextrin (beta-CD) cavity has been investigated. The photoreactivity of the free molecule has been rationalized on the basis of an intramolecular nitro to nitrite rearrangement followed by cleavage of the nitrite intermediate. The twisted geometry of the nitro group with respect to the aromatic plane plays a key role in triggering such a photoprocess. Incorporation of FM in the beta-CD cavity leads to dramatic effects on both the efficiency and the nature of the photochemical deactivation pathways of the guest molecule. A 20-fold increase in the FM photodecomposition quantum yield and the formation of photoproducts originated by both reduction of the nitro group and cleavage of the amide bond were observed in the presence of the macrocycle. Such a behavior cannot be attributed exclusively to the micropolarity of beta-CD and/or to its role as a reactant. The induced circular dichroism spectra and the nature of the photoproducts formed in these experimental conditions provide indications that the photoreactivity in the beta-CD microenvironment could likely be mediated by structural changes of FM upon complexation.

Molecular mechanisms of photosensitization induced by drugs. XII. Photochemistry and photosensitization of rufloxacin: an unusual photodegradation path for the antibacterials containing a fluoroquinolone-like chromophore.

The UVA irradiation of 9-fluoro-2,3-dihydro-10-4'-methyl-1' -piperazinyl-7-oxo-7H-pyrido[1,2,3-de]-1,4-benzo-thiazine-6-carboxylic acid, rufloxacin, a fluoroquinolone antibacterial that shows photosensitizing properties toward biological substrates, leads to formation of two main steady photoproducts characterized by a decarboxylation process and an opening of the piperazinyl ring, respectively. The deprotonation of the 10-piperazinyl group and the dissociation of the 6-carboxyl group of rufloxacin are strictly pH dependent. The photosensitizing activity was tested toward membranes as biological targets. Red blood cell hemolysis and lipid peroxidation were considered as markers of photosensitization. Ultraviolet A-induced damage is strongly influenced by the presence of oxygen, it is triggered by transient species, such as singlet oxygen and free radicals, photogenerated via rufloxacin irradiation, whereas no drug photoproduct is involved in the photosensitization process.

Liposomal delivery of a 30-mer antisense oligodeoxynucleotide to inhibit proopiomelanocortin expression.

An oligodeoxynucleic sequence of 30 bases (30-mer ODN), complementary to a region of beta-endorphin mRNA, was synthesized to have an antisense effect with regard to the expression of this oligopeptide. Following the solid-phase synthesis of the oligodeoxynucleotide, the 30-mer ODN was encapsulated within liposomes to provide a higher resistance against DNases and an improved entrance into cells. The most suitable liposome formulation as a 30-mer ODN carrier consisted of small unilamellar vesicles (50 nm) with an encapsulation capacity of 4.76 microL/micromol. The liposomal formulations containing dipalmitoyl-DL-alpha-phosphatidyl-L-serine presented fusogenic properties, which are of great importance for the delivery of antisense compounds. The antisense activity of 30-mer ODN-loaded liposomes was evaluated by the determination of beta-endorphin levels in AtT-20 cells. The free 30-mer ODN did not provide any lowering of the beta-endorphin production, whereas the liposomally entrapped compound elicited a concentration-dependent inhibition. The inhibition was determined by a sequence-specific binding of the 30-mer ODN with the target mRNA.

Fluorenone 1,4-dihydropyridine derivatives with cardiodepressant activity: enantiomeric separation by chiral HPLC and conformational aspects.

The direct HPLC enantiomeric separation of five fluorenone-1,4-dihydropyridine-3,5-dicarboxylic diesters has been achieved using a Chiralpak AD stationary phase obtaining simultaneously good enantioselectivities, resolution factors, and elution times. CD spectra of the individual enantiomers for two compounds were measured. Thermodynamic parameters associated with the adsorption equilibria of the enantiomers with the chiral stationary phase were obtained from HLPC runs at various temperatures. The conformational preferences of the synperiplanar fluorenone group and of the cis/cis ester groups were obtained by 1H NMR spectra, including NOE experiments.

Molecular mechanism of drug photosensitization. Part 6. Photohaemolysis sensitized by tolmetin.

The photosensitizing properties of tolmetin, 5-(p-toluoyl)-1-methyl-2-pyrrolyacetic acid (TLM), have been studied in vitro following the lysis of erythrocytes in phosphate buffer suspensions irradiated with UVA light in the presence of the drug. It was found that the phototoxic properties of the drug are negligible in nitrogen and significant in aerated medium, but that they decrease in oxygen-saturated solution. The investigation of the drug photolysis showed that TLM undergoes photodecarboxylation to p-tolyl 1,2-dimethyl-5-pyrrolyl ketone in nitrogen and to p-tolyl 1-methyl-2-hydroxymethyl-5-pyrrolyl ketone and 5-(p-toluoyl)-1-methyl-2-pyrrole carbaldehyde in air. These photoproducts also undergo photodegradation. The comparison between the photohaemolysis and photolysis results and the effect of suitable additives such as sodium azide, mannitol, butylated hydroxy-anisole, reduced glutathione, superoxide dismutase and copper (II) suggest that the phototoxicity of TLM can be attributed essentially to singlet oxygen in the first step and to its photoproducts when they accumulate and compete with the starting drug in light absorption. Their phototoxic effect is much higher with respect to that of TLM, as shown by comparison of the doses needed to attain 50% photohaemolysis.

Molecular mechanism of drug photosensitization. 5. Photohemolysis sensitized by Suprofen.

Red blood cell lysis photosensitized by Suprofen (SPF) and the photolysis of the drug were investigated. The photohemolysis process occurs at a higher rate in anaerobic than aerobic conditions. The effect of additives demonstrates the involvement of free radicals and, to a lesser extent, singlet oxygen and hydroxyl radicals in the process. Photolysis of the drug at 310-390 nm in deaerated buffered solutions (pH 7.4) leads to a decarboxylation process with the formation of p-ethylphenyl 2-thienyl ketone (I), whereas in aerated solutions formation of photoproduct I and of the photoproducts p-acetylphenyl 2-thienyl ketone (II) and p-(1-hydroxyethyl)phenyl-2-thienyl ketone (III) occurs. The photodegradation products, which were separated and characterized, show a moderate lytic and photolytic activity. The rate of SPF photodegradation decreases in the presence of oxygen and increases in the presence of hydrogen donors. The overall results lead us to propose a mechanism of SPF photodegradation and a hemolysis scheme in which cell damage is provoked principally by the direct attack of drug radicals and secondarily by singlet oxygen and hydroxyl radicals.

Molecular mechanism of drug photosensitization. 4. Photohemolysis sensitized by carprofen.

Red blood cell lysis photosensitized by carprofen (CPF) was investigated. The photohemolysis process was observed in both aerobic and anaerobic conditions. Irradiation (310-390 nm) of buffered CPF solutions at pH 7.4 in deaerated conditions leads to a dehalogenation process via intermediate radicals with formation of the compound 2-(2-carbazolyl)propanoic acid (I) in the presence of hydrogen donors. Irradiation of I produces decarboxylation via free radicals and the formation of a stable decarboxylated compound, 2-ethylcarbazole (II). The photodegradation products I and II do not show lytic activity. The dechlorinated product I shows photosensitizing ability which was studied in the presence of the red blood cells in both aerated and deaerated solution. When carried out in the presence of additives, the observed photohemolysis suggests the involvement of free radicals and singlet oxygen in the membrane damage induced by both CPF and photoproduct I irradiation, whereas there is no evidence of any role for hydroxyl radicals. Superoxide anion is involved only in the photosensitization process induced by photoproduct I.

Molecular mechanism of drug photosensitization. Part 3. Photohemolysis sensitized by diflunisal.

The lysis of red blood cells photosensitized by diflunisal (DFN) was investigated. Photohemolysis is inhibited by butylated hydroxyanisole and reduced glutathione, but is unaffected by mannitol and enhanced by sodium azide; the presence of oxygen markedly reduces the lysis which is accelerated in anaerobic conditions. These results contrast with those expected for a photodynamic mechanism. High lytic activity is observed for pre-irradiated solutions, mainly under anaerobic conditions. Direct irradiation of DFN in buffer solution at pH 7.4 leads to the formation, under anaerobic conditions, of compound 2'-(2''',4'''-difluoro-3''-carboxy-[1'',1'''-biphenyl]-4''-oxy)-4'- fluoro-4-hydroxy-[1,1'-biphenyl]-3-carboxylic acid (PhP), whereas under aerobic conditions formation of PhP is accompanied by unidentified photo-oxidation products; only compound PhP displays strong lytic activity. The overall results for DFN-photosensitized hemolysis suggest a mechanism involving a concerted action of free radicals, superoxide anion, singlet oxygen and sensitizer photoproducts.

Melanosomes from liver and skin of Rana esculenta L. A comparative chemical study.

1. Melanosomes from skin and liver of Rana esculenta L. have been isolated and some chemical properties of the relevant melanin and protein components were compared. 2. In both cases the pigments show spectroscopic (ESR) and chemical characteristics similar to those of eumelanins. The melanin content in skin melanosomes is higher than in the liver counterparts. 3. Amino acid patterns of the two protein components are different in their quantitative composition and both are characterized by high levels of glycine and proline. 4. The results as a whole indicate that skin and liver melanosomes from the same animal markedly differ in their chemical composition.

Simultaneous high-performance liquid chromatographic determination of antazoline phosphate and tetrahydrozoline hydrochloride in ophthalmic solution.

A method for the determination of 2-[(N-phenyl)benzylaminomethyl]-2-imidazoline X H3PO4 (antazoline phosphate) and 2-(1,2,3,4-tetrahydro-1-naphthyl)-2-imidazoline X HCl (tetrahydrozoline hydrochloride) in ophthalmic solution is described. The pharmaceutical preparation is analysed directly by reversed-phase ion-pair high-performance liquid chromatography and the method is very rapid, selective and simple.

Urinary steroid profiles in gestosis syndrome.

Urinary steroid profiles were studied using gas chromatography in eighteen pregnant women with EPH-gestosis. A drastic and probably aspecific decrease in urinary steroid concentrations was observed in all metabolic profiles when compared with physiological counterparts. The phenomenon seems to be strictly dependent upon the syndrome and disappears after effective therapy.

Gibberellin- and cytokinin-like activities in marine algae from central Mediterranean.

The distribution of gibberellin- and cytokinin-like activities in twenty-one species of mediterranean algae belonging to Phaeophyta and Rhodophyta has been investigated. Fractions containing each of these activities have been obtained by partitioning aqueous-methanolic extracts of algae against ethyl acetate and butanol successively at different pH values. The stimulation or inhibition of amaranthin synthesis in Amaranthus sp. seedlings have been used a biological tests to assay the cytokinin- or gibberellin-like activities respectively. One or both these two hormonal activities have been detected in all species examined. While cytokinin-like activity has been found with the same frequency in the two phyla, the gibberellin-like activity was less frequent in Phaeophyta.