In vitro metabolism of a nitroderivative of acetylsalicylic acid (NCX4016) by rat liver: LC and LC-MS studies

The metabolism of a nitroderivative of acetylsalicylic acid, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX4016), the lead compound of a new class of NO-releasing non steroidal-antiinflammatory drugs has been studied in vitro in rat liver subcellular fractions (S 9000xg, microsomes, cytosol). Samples were extracted with CH3CN (2 vol.) containing 1% H3PO4 (2 M), vortexed for 3 min and then centrifuged for 5 min at 5000 rpm. Supernatants were diluted with 0.02 M phosphoric acid and analysed by reverse-phase LC. Linearity of calibration for NCX4016 and metabolites was observed over the range 0.25-50 microg/ml with coefficients of determination greater than 0.9996. Extraction efficiency from spiked liver samples ranged from 85 to 95% for all the analytes. In the S 9000xg fraction, NCX4016 undergoes rapid metabolization, with the formation of salicylic acid (SA) and [3-(nitrooxymethyl)phenol] (HBN). HBN is then rapidly metabolised to 3-hydroxybenzylalcohol (HBA), and mainly to a new metabolic species, whose formation takes place specifically in the liver cell cytosol. LC-MS analysis (electrospray ionisation) of the cytosol extract in negative and positive-ion modes furnished deprotonated [M-H]- and protonated [M+H]+ molecular ions at m/z 412 and 414, respectively, accompanied by the typical clusters with sodium. MS/MS analysis in negative-ion mode, by selection and collision of the ion at m/z 412, gave a fragmentation pattern characterized by the ions at m/z 272 and 254, which allowed to assign the structure of 1-(glutathion-S-yl)methylene-3-hydroxy-benzene, a conjugated product between GSH and the benzyl carbon atom of HBN. In rat liver cytosol HBN is completely metabolised to this thioether adduct within 30 min incubation; the process is enzymatically mediated by GSH transferase and strictly dependent on GSH availability. The relevance of this new metabolic pathway in NCX4016 detoxification by rat liver is discussed

Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry

The histidine-containing dipeptides carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR), widely distributed in vertebrate organisms, have recently been proposed as endogenous quenchers for highly cytotoxic alpha,beta-unsaturated aldehydes generated by peroxidation. A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination of these peptides in biological matrices in order to establish their plasma/tissue distribution. Samples (plasma or tissue homogenates from male rats) were prepared by protein precipitation with HClO(4) (1 : 1, v/v) containing H-Tyr-His-OH as internal standard. The supernatant was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9 : 1 : 0.01, v/v/v) at a flow-rate of 0.2 ml min(-1), with a run time of 10 min. Detection was effected on an ion trap mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. The acquisitions were in the multiple reaction monitoring mode using the following precursor --> product ion combinations: H-Tyr-His-OH (internal standard) m/z 319 --> 301; CAR m/z 227 --> 210 + 209; ANS m/z 241 --> 224 + 197 + 170; HCAR m/z 241 --> 156. The method was validated over the concentration range 15-1000 nmol g(-1) and the limit of quantification (LOQ) and limit of detection (LOD) were 12.5 and 4.2 pmol injected, respectively. The intra- and inter-day precisions were <10% (< or =17.47% at the LOQ) and the intra- and inter-assay accuracies were within +/-10% for all concentrations. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), followed by the heart, cerebellum and brain (ANS below the LOQ). HCAR was found only in the brain and cerebellum. No histidine-containing dipeptides were detectable in plasma, liver, kidney and lung

Nitric oxide release and distribution following oral and intraperitoneal administration of nitroaspirin (NCX 4016) in the rat

The metabolic fate of nitric oxide (NO) released from nitroaspirin, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX 4016), the lead compound of a new class of NO-releasing non steroidal anti-inflammatory drugs (NO-NSAIDs), has been studied in the rat following p.o. and i.p. administration of 100 mg/kg, by monitoring in plasma the bioactive storage forms of NO (S-nitrosothiols, RS-NO) and its oxidation products (nitrites/nitrates, NOx) by a chemiluminescent assay. In parallel, plasma was analyzed for unchanged drug and metabolites by reverse-phase HPLC. In orally treated rats, no unchanged drug is observed in the 0-24 h interval post-dosing, but only salicylic acid (SA), NOx and RS-NO. The time-course of SA formation parallels that of plasma NOx (plateau after 6 h). Nitrosothiols in plasma are detectable at 1 h, peak at 4 h post-administration, and decline thereafter. The results relative to i.p. administration show a more pronounced and rapid NO delivery (peak of both NOx and RS-NO at 1 h and plateau between 1 and 2 h), still coincident with the peak of SA, and the presence in plasma of NCX 4015 (a metabolite of NCX 4016 which still bears the nitrate function). In myocardial tissue from p.o. treated rats, no drug or metabolites were ever detected, and the NOx levels were always in the range of the controls. Conversely, following i.p. treatment, we observed a rapid compartmentalization within the heart of the unchanged drug, which rapidly disappears in favour of its breakdown products NCX 4015 and SA, with a concomitant rise in myocardial NOx levels up to 2 h. To check the stability of NCX 4016 in the acidic gastric milieu and to explain the different distribution of the drug following p.o. or i.p. administration, the gastric content of the orally-treated animals at different post-dosing times was analysed by HPLC. The unchanged drug was detected up to 8 h post-dosing (levels slowly decreased with time), and the only metabolite to be detected was the O-deacetylated derivative (NCX 4023), which was present in low concentrations up to 4 h post-dosing. This indicates that NCX 4016 does not undergo biotransformation in the upper part of gastrointestinal tract (no direct release of NO in this district) and that the stomach acts as a reservoir for the drug

Pharmacological profiles of animal- and nonanimal-derived sulfated polysaccharides – comparison of unfractionated heparin, the semisynthetic glucan sulfate PS3, and the sulfated polysaccharide fraction isolated from Delesseria sanguinea

Sulfated polysaccharides (SP) such as heparin are known to exhibit a wide range of biological activities, e.g., anticoagulant, anti-inflammatory, and antimetastastic effects. However, since the anticoagulant activity of heparin is dominating, its therapeutic use for other medical indications is limited due to an associated risk of bleeding. Further disadvantages of heparin are its animal origin, the shortage of resources, and its complex and variable composition. However, SP without these limitations may represent a substance class with good prospects for applications other than anticoagulation. In this study, the in vitro pharmacological profiles of two nonanimal-derived SP were investigated in comparison with unfractionated heparin. One is the natural SP fraction from the red algae Delesseria sanguinea (D.s.-SP). The other one is the chemically defined PS3, a semisynthetic β-1,3-glucan sulfate with proven in vivo anti-inflammatory and antimetastatic activities. All three polysaccharides were examined in vitro for their inhibitory effects on the coagulation and complement system, polymorphonuclear neutrophil elastase, hyaluronidase, matrix metalloproteinase-1, heparanase, and p-selectin-mediated cell adhesion. Compared with heparin, the nonanimal-derived polysaccharides have a four times weaker anticoagulant activity, but mostly exhibit stronger (1.4–224 times) effects on test systems investigating targets of inflammation or metastasis. According to their different structures, PS3 and D.s.-SP differ in their pharmacological profile with PS3 being the strongest inhibitor of heparanase and cell adhesion and D.s.-SP being the strongest inhibitor of hyaluronidase and complement activation. Considering both pharmacological profile and pharmaceutical quality parameters, PS3 represents a candidate for further development as an anti-inflammatory or antimetastatic drug whereas D.s.-SP might have perspectives for cosmetic applications

Oxidative Modifications of Rat Liver Cell Components During Fasciola hepatica Infection

The aim of this paper was to assess the influence of Fasciola hepatica infection on oxidative modifications of rat liver cell components such as proteins and lipids. Wistar rats were infected per os with 30 metacercariae of F. hepatica. Activities and concentrations of liver damage markers were determined in the 4th, 7th, and 10th week postinfection (wpi). A decrease in antioxidant capacity of the host liver, manifested by a decrease in total antioxidant status (TAS), was observed. Diminution of antioxidant abilities resulted in enhanced oxidative modifications of lipids and proteins. F. hepatica infection enhanced lipid peroxidation, which was visible in the statistically significant increase in the level of different lipid peroxidation products such as conjugated dienes (CDs), lipid hydroperoxides (LOOHs), malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). The level of protein modification markers in the rat liver was also significantly changed and the most intensified changes were observed at seventh week postinfection. Concentration of carbonyl groups and dityrosine was significantly increased, whereas the level of tryptophan and sulfhydryl and amino groups was decreased. Changes in the antioxidant abilities of the liver and in the lipid and protein structure of the cell components resulted in destruction of the function of the liver. F. hepatica infection was accompanied by raising serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as markers of liver damage. A significant decrease in lysosomal as well as in the total activity of cathepsin B during fasciolosis was also observed

Lignan Derivatives from Krameria lappacea Roots Inhibit Acute Inflammation in Vivo and Pro-inflammatory Mediators in Vitro

The roots of Krameria lappacea are used traditionally against oropharyngeal inflammation. So far, the astringent and antimicrobial properties of its proanthocyanidin constituents are considered to account for the anti-inflammatory effect. The aim of the present study was to characterize pharmacologically a lipophilic extract of K. lappacea roots and several isolated lignan derivatives (111) in terms of their putative anti-inflammatory activity. The dichloromethane extract (ID50 77 \u3bcg/cm2) as well compounds 111 (ID50 0.310.60 \u3bcmol/cm2) exhibited topical antiedematous properties comparable to those of indomethacin (ID50 0.29 \u3bcmol/cm2) in a mouse ear in vivo model. Two of the most potent compounds, 2-(2-hydroxy-4-methoxyphenyl)-5-(3-hydroxypropyl)benzofuran (5) and (+)-conocarpan (7), were studied regarding their time-dependent edema development and leukocyte infiltration up to 48 h after croton oil-induced dermatitis induction, and they showed activity profiles similar to that of hydrocortisone. In vitro studies of the isolated lignan derivatives demonstrated the inhibition of NFkB, cyclooxygenase-1 and -2, 5-lipoxygenase, and microsomal prostaglandin E2 synthase-1 as well as antioxidant properties, as mechanisms possibly contributing to the observed in vivo effects. The present findings not only support the ethnopharmacological use of K. lappacea roots but also reveal that the isolated lignan derivatives contribute strongly to the anti-inflammatory activity of this herbal drug

The Antinociceptive and Anti-Inflammatory Activities of Caulerpin, a Bisindole Alkaloid Isolated from Seaweeds of the Genus Caulerpa

The antinociceptive and anti-inflammatory activity of caulerpin was investigated. This bisindole alkaloid was isolated from the lipoid extract of Caulerpa racemosa and its structure was identified by spectroscopic methods, including IR and NMR techniques. The pharmacological assays used were the writhing and the hot plate tests, the formalin-induced pain, the capsaicin-induced ear edema and the carrageenan-induced peritonitis. Caulerpin was given orally at a concentration of 100 μmol/kg. In the abdominal constriction test caulerpin showed reduction in the acetic acid-induced nociception at 0.0945 μmol (0.0103–1.0984) and for dypirone it was 0.0426 μmol (0.0092–0.1972). In the hot plate test in vivo the inhibition of nociception by caulerpin (100 μmol/kg, p.o.) was also favorable. This result suggests that this compound exhibits a central activity, without changing the motor activity (seen in the rotarod test). Caulerpin (100 μmol/kg, p.o.) reduced the formalin effects in both phases by 35.4% and 45.6%, respectively. The possible anti-inflammatory activity observed in the second phase in the formalin test of caulerpin (100 μmol/kg, p.o.) was confirmed on the capsaicin-induced ear edema model, where an inhibition of 55.8% was presented. Indeed, it was also observed in the carrageenan-induced peritonitis that caulerpin (100 μmol/kg, p.o.) exhibited anti-inflammatory activity, reducing significantly the number of recruit cells by 48.3%. Pharmacological studies are continuing in order to characterize the mechanism(s) responsible for the antinociceptive and anti-inflammatory actions and also to identify other active principles present in Caulerpa racemosa

Strategy for the management of diabetic macular edema: the European Vitreo-Retinal Society macular edema study

Objective. To compare the efficacy of different therapies in the treatment of diabetic macular edema (DME). Design. Nonrandomized, multicenter clinical study. Participants. 86 retina specialists from 29 countries provided clinical information on 2,603 patients with macular edema including 870 patients with DME. Methods. Reported data included the type and number of treatment(s) performed, the pre-and posttreatment visual acuities, and other clinical findings.The results were analyzed by the French INSEE (National Institute of Statistics and Economic Studies). Main Outcome Measures. Mean change of visual acuity and mean number of treatments performed. Results.The change in visual acuity over time in response to each treatment was plotted in second order polynomial regression trend lines. Intravitreal triamcinolone monotherapy resulted in some improvement in vision. Treatmentwith threshold or subthreshold grid laser also resulted in minimal vision gain. Anti-VEGF therapy resulted in more significant visual improvement. Treatment with pars plana vitrectomy and internal limiting membrane (ILM) peeling alone resulted in an improvement in vision greater than that observed with anti-VEGF injection alone. In our DME study, treatment with vitrectomy and ILM peeling alone resulted in the better visual improvement compared to other therapies