Olea Europea-derived phenolic products attenuate antinociceptive morphine tolerance: an innovative strategic approach to treat cancer pain.

Morphine and related opioid drugs are currently the major drugs for severe pain. Their clinical utility is limited in the management of severe cancer pain due to the rapid development of tolerance. Restoring opioid efficacy is therefore of great clinical importance. A great body of evidence suggests the key role of free radicals and posttranslational modulation in the development of tolerance to the analgesic activity of morphine. Epidemiological studies have shown a relationship between the Mediterranean diet and a reduced incidence of pathologies such as coronary heart disease and cancer. A central hallmark of this diet is the high consumption of virgin olive oil as the main source of fat which contains antioxidant components in the non-saponifiable fraction, including phenolic compounds absent in seed oils. Here, we show that in a rodent model of opiate tolerance, removal of the free radicals with phenolic compounds of olive oil such as hydroxytyrosol and oleuropein reinstates the analgesic action of morphine. Chronic injection of morphine in mice led to the development of tolerance and this was associated with increased nitrotyrosin and malondialdehyde (MDA) formation together with nitration and deactivation of MnSOD in the spinal cord. Removal of free radicals by hydroxytyrosol and oleuropein blocked morphine tolerance by inhibiting nitration and MDA formation and replacing the MnSOD activity. The phenolic fraction of virgin olive oil exerts antioxidant activities in vivo and free radicals generation occurring during chronic morphine administration play a crucial role in the development of opioid tolerance. Our data suggest novel therapeutic approach in the management of chronic cancer pain, in particular for those patients who require long-term opioid treatment for pain relief without development of tolerance.

Effect of MN (III) tetrakis (4-benzoic acid) porphyrin by photodynamically generated free radicals on SODs keratinocytes.

Superoxide, a reactive form of oxygen, can be produced in vivo either in normal and under pathophysiologic conditions or by photosensitizing chemicals, as during photodynamic treatment. Photodynamic therapies (PDT), widely adopted in Dermatology and Oncology, are known to generate reactive oxygen species (ROS) and may contribute to structural alterations and oxidatively generated modifications of cellular antioxidants. We hypothesized that over-production of free radicals would decrease the enzymatic activities of endogenous cellular antioxidants. To test this hypothesis, keratinocytes were treated with the photosensitizer Photofrin plus visible light to produce free radicals and CuZnSOD and MnSOD activities were measured. Photodynamic treatment of keratinocytes increases malonylaldehyde production, nitrotyrosine staining and superoxide production. The enzymatic activities of CuZnSOD and MnSOD were significantly decreased after Photofrin plus visible light treatment. Our results suggest that the main cellular antioxidant system can be inactivated by photodynamically generated ROS. Pretreatment of keratinocytes with free radicals scavenger such as Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) was able to restore the endogenous antioxidant system activities, inhibiting the MDA formation, nitrotyrosine staining and superoxide formation. Antioxidant therapy could therefore be a useful tool in protecting healthy epidermal cells against common side effects induced by antitumor targeted therapies.

Pharmacokinetic study of a new angiotensin-AT1 antagonist by HPLC.

Recently an innovative novel class angiotensin-AT1 antagonist has been developed by Rottapharm. In this study, we present a validated method for detecting CR 3834 in biological matrices using high-performance liquid chromatography (HPLC) with diode array detection. After oral administration (30mg/kg) to Wistar rats, the plasma and urine concentrations of CR 3834 and its potential metabolic products were determined. Moreover, the plasmatic time course in rats has been determined after intravenous (IV) administration of CR 3834 (5mg/kg). Biological samples (0.5ml of plasma and 1ml of urine) were purified using solid-phase extraction (SPE) of analytes and the internal standard Idebenone, 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1-4-benzoquinone. A chromatographic separation was performed on an Adsorboshere C18 at 25 degrees C, with a pre-column of the same matrix; the eluent was made up of acetonitrile/acidified water with CF3COOH (pH 2.01) in ratio of 75:25 (v/v); the flow rate was 1.0ml/min and a 100microl loop. The lower limit of detection (LOD) was taken as 25ng/ml in plasma and 50ng/ml in urine samples. The lower limit of quantification (LOQ) was taken as 0.1 and 0.2microg/ml in plasma and urine samples, respectively. The procedures were validated according to international standards with a good reproducibility and linear response (r=0.9916 in plasma; r=0.9997 in urine). The coefficients of variation inter assay ranged between 2.579 and 4.951% in plasma, and between 0.813 and 2.460% in urine. Mean recovery for CR 3834 was 79% in plasma and 97% in urine samples. The experiments performed demonstrated that the method presented was suitable for determining this new angiotensin-AT1 antagonist in rat plasma and urine.

Determination of a potent non-competitive AMPA receptor antagonist in rat brain.

N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivative (PS3Ac) has been determined in brain tissues by high performance liquid chromatography (HPLC) coupled with a diode array detection. In a previous paper we presented a validation method for detecting PS3Ac and its metabolites in plasma samples after intraperitoneal administration to Wistar rats. In the present paper, we report the results of the determination of PS3Ac and its N-deacetyl (PS3) and O-demethyl (PS3OH) metabolites, in the brain after extraction based on a polymeric matrix with a high hydrophilic-lipophilic balance, using Oasis cartridges. The chromatographic separation was performed in an octadecylsilica stationary phase at 25 degrees C using a mixture of 10 mM potassium dihydrogen orthophosphate (pH 2.24) and acetonitrile in ratio of 30:70 (v/v) as mobile phase, with a flow rate of 0.8 ml/min. The method exhibited a large linear range from 0.05 to 2 microg/ml for all studied compounds (n=6). In the within-day assay (n=4), the accuracy ranged from 87.5% determined with 0.05 microg/ml of PS3 to 110.1% determined with 0.2 microg/ml of PS3OH. In the between-day assay the coefficient of variation ranged from 2.4 determined with 0.05 microg/ml of PS3 to 9.7 determined with 0.2 microg/ml of PS3OH. The extraction efficiency ranged from 77.8% for PS3OH at 0.2 microg/ml to 94.3 for PS3Ac at 0.5 microg/ml. The limit of detection for all the tetrahydroisoquinoline derivatives ranged around 50 ng/ml. The method proved to be highly sensitive and specific to determinate PS3Ac and its metabolites and has been successfully applied to value their concentrations in brain matrix over the time.

HPLC determination of phenolics adsorbed on yeasts.

An analytical HPLC method was developed to determine monomeric anthocyans as catechin (CA), flavonols as rutin (RU) and phenol acids as gallic acid (GA) adsorbed on the cell wall of 23 Saccharomyces cerevisiae strains grown on two media containing high levels of phenolic compounds, from grape seeds and grape skins, and on one control medium. Microbial biomass purification by liquid-liquid extraction of the phenolics is followed by reversed-phase chromatographic separation and CA, RU and GA detection by ultraviolet detector. The method was linear over the studied range of concentrations: GA at 0.12-0.96 microg/ml, CA at 0.25-20.00 microg/ml and RU at 0.02-0.20 microg/ml. The correlation coefficient for each analyte was greater than 0.9983. The recovery was greater than 85% for both GA and RU, and greater than 94% for CA. The detection limits for GA, CA and RU were determined to be 0.015, 0.025 and 0.029 microg/mg of biomass, respectively. The proposed method is highly responsive for the determination of different phenolics, and seems to be useful to evaluate their adsorption profile on yeasts.

SPE-HPLC determination of new tetrahydroisoquinoline derivatives in rat plasma.

Recently a novel class of non-competitive AMPA receptor (AMPAR) antagonists, such as, N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (PS3Ac) have been developed using molecular modeling studies. In this study we present a validated method for detecting PS3Ac in biological matrices by high performance liquid chromatography with ultraviolet detection. In this study PS3Ac was administered to Wistar rats. After intraperitoneal administration, the plasma concentrations of PS3Ac and its potential metabolic products, i.e., PS3OH, PS3 and PS3OHAc were determined. Serum samples (0.5 ml) were purified by solid-phase extraction of analytes using Oasis cartridges. The chromatographic separation was performed on a LiChrosorb RP-1 at 30 degrees C. The eluent was made of potassium dihydrogen phosphate/acetonitrile in ratio of 50:50 (v/v); the flow rate was 1 ml/min. The detection was performed at 220 nm. The method exhibited a large linear range from 0.05 to 5 microg/ml for all studied compounds. The intra-assay accuracy ranged from 92% determined at 0.1 microg/ml of PS3OH, to 108% determined at 0.05 microg/ml of PS3OHAc. The average coefficient of variation of inter-assay was 6.27%. The average recovery from plasma was 78.5%. The limits of quantification for all the tetrahydroisoquinoline derivatives was 20 ng. The method proved to be highly sensitive and specific for the determination of the studied compounds in rat plasma and has been successfully applied to the evaluation of the pharmacokinetic profile of the inoculated compound.

Sensitive SPE-HPLC method to determine a novel angiotensin-AT1 antagonist in biological samples.

A high-performance liquid chromatography (HPLC)-method after solid-phase extraction (SPE) has been developed in order to determine a new angiotensin-AT1 antagonist, i.e. CR 3210 (C27H24N8; MW = 460.54), 4-[4-[(2-ethyl-5,7-dimethylimidazo[4,5-b]pyridin-3-yl)methyl]phenyl]-3-(2H-tetrazol-5-yl)quinoline in rat plasma and urine after oral administration to Sprague-Dawley rats. CR 3210 and the internal standard (IS) CR 1505 (loxiglumide), i.e. 4-[(3,4-dichlorobenzoyl)amino]-5-[(3-methoxypropyl)pentylamino]-5-oxopentanoic acid, were isolated from rat urine and plasma by solid-phase extraction. The procedure was optimized regarding the sorbent extraction material, the pH in the conditioning solution, the washing step, the dry time and the type of elution solvent. The separation was performed by reversed-phase high-performance liquid chromatography with ultraviolet detection. The samples were injected onto the analytical column (Tracer Extrasil ODS1) and detected at 238 nm, giving a capacity factor of 1.87 for CR 3210 and 1.10 for the internal standard. The selectivity of the method was satisfactory. The mean recovery of CR 3210 from spiked rat plasma was 68.5 at 75 ng/ml and 80.9 at 3000 ng/ml; the mean recovery of CR 3210 from spiked rat urine was 69.9 at 75 ng/ml and 78.6 at 3000 ng/ml. The lower limit of detection (LOD) was 14 ng/ml in plasma and 22 ng/ml in urine samples. The lower limit of quantification (LOQ) was taken as 30 ng/ml, the lowest calibration standard using 500 microl rat plasma and urine. The procedures were validated according to international standards with a good reproducibility and linear response from 30 to 3000 ng/ml, for either plasma or urine. The sensitivity of the method allowed for its application to pharmacokinetic studies.

Determination of a novel angiotensin-AT1 antagonist CR 3210 in biological samples by HPLC.

A simple and sensitive method for the determination of a new angiotensin-AT(1) antagonist i.e. CR 3210, 4-[4-[(2-ethyl-5,7-dimethylimidazo[4,5-b]pyridin-3-yl)methyl]phenyl]-3-(2H-tetrazol-5-yl)quinoline, is described. The assay was utilised to describe the pharmacokinetic profile of the title compound after intravenous and intraperitoneal administration to Sprague Dawley rats. CR 3210 and the internal standard CR 1505 (loxiglumide, 4-[(3,4-dichlorobenzoyl)amino-5-[(3-methoxypropyl)pentylamino]-5-oxopentanoic acid) were isolated from rat plasma by solid-phase extraction. The sorbent extraction material along with the pH in the conditioning solution and the washing volume were considered pivotal parameters for the optimisation of the procedure. The separations were performed by reversed-phase high-performance liquid chromatography with ultraviolet detection. The samples were injected onto the analytical column (Tracer Extrasil ODS1) and detected at 238 nm, giving a retention time of 6.19 min for CR 3210 and 4.39 min for the internal standard, respectively. The selectivity of the method showed to be satisfactory. The mean recovery of CR 3210 from spiked rat plasma was 80.3 at 1 microg/ml and 79.9 at 2 microg/ml. The lower limit of detection (LOD) was taken as 0.014 microg/ml in plasma samples. The lower limit of quantification (LOQ) was taken as 0.02 microg/ml, the lowest calibration standard using 500 microg rat plasma. The procedures were validated according to international standards with a good reproducibility and linear response from 0.02 to 2 microg/ml. The sensitivity of the method allowed for its application to pharmacokinetic studies. The maximal concentration was detected 5' after the IV administration, whereas no significant absorption was evident after IP administration of CR 3210 to Sprague-Dawley rats. Our study suggests the absence of extensive bio-transformation of the drug in vivo, supported by the evidence that no metabolites were detected in plasma samples.