Penetration of cefuroxime in subcutaneous tissue during coronary artery bypass grafting surgery

A sensitive and rapid HPLC assay for determining cefuroxime penetration in the subcutaneous tissue near to surgical incision of patients submitted to coronary artery bypass grafting (CABC) with or without cardiopulmonary bypass (CPB) was performed. Blood and subcutaneous tissue samples were collected from 14 patients. in four periods during surgery. The analytical method presented linearity from 0.5 to 100 mu g/g. LOQ = 0.50 mu g/g, LOD = 0.25 mu g/g. intra- and interday precision (%CV) ranged from 4.9 to 8.9% and 6.4 to 9.9%, respectively, and intra-and interday accuracy expressed as % of the nominal concentration ranged from 87.1 to 104.6% and 94.8 to 103.8%, respectively (mean of three concentrations). Relative recovery was 98.4%. Tissue/plasma ratios obtained for CPB and non-CPB were, respectively: 14.6% vs 19.0% (0.6 h); 15.7% vs 15.7% (2.1 h); 22.5% vs 19.9% (3.6 h); 15.7% vs 18.8% (4.5 h). Data obtained indicate that tissue/plasma ratio remains unchanged in CPB and non-CPB patients during all period of surgery and the CPB does not affect the penetration of cefuroxime in tissues close to the surgical wound. (C) 2009 Elsevier B.V. All rights reserved.

Effect of Isolated Fractions of Harpagophytum procumbens DC (Devil`s Claw) on COX-1, COX-2 Activity and Nitric Oxide Production on Whole-Blood Assay

The present study evaluates the effect of isolated fractions of Harpagophytum procumbens (devil`s claw) on cyclooxygenase (COX-1 and COX-2) activities and NO production using a whole blood assay. The activity of COX-1 was quantified as platelet thromboxane B(2) production in blood clotting and COX-2 as prostaglandin E(2) production in LPS-stimulated whole blood. Total NO(2)(-)/NO(3)(-) concentration was determined by Griess reaction in LPS stimulated blood. Assays were performed by incubation of isolated fractions obtained by flash chromatography monitored with HPLC, TLC and identified by (1)HNMR, containing different amounts of harpagoside with blood from healthy donors. Indomethacin and etoricoxib were the positive controls of COX-1 and COX-2 Inhibition. Data shows that fraction containing the highest concentration of harpagoside inhibited indistinctively COX-1 and COX-2 (37.2 and 29.5% respectively) activity and greatly inhibited NO production (66%). In contrast the fraction including iridoid pool increased COX-2 and did not alter NO and COX-1 activities. The fraction containing cinnamic acid was able to reduce only NO production (67%). Our results demonstrated that the harpagoside fraction is the main responsible for the effect of devils claw on these enzyme activities. However, other components from devil`s claw crude extract could antagonize or increase the synthesis of inflammatory mediators. Copyright (C) 2010 John Wiley & Sons, Ltd.

Isolated limb perfusion with melphalan for human melanoma xenografts in the hindlimb of nude rats: A surviving animal model

We have established a surviving model of isolated limb perfusion using xenografts of the human melanoma cell line MM 96L injected subcutaneously into the hindlimb of a nude rat, The femoral artery and vein were cannulated via the left renal artery and vein and the hind limb was isolated using tourniquets. The limb was perfused with Krebs Heinseleit buffer at 37 degrees C containing 4.7% bovine serum albumin at a constant flow rate of 4 mi per min for 30-60 min with 100% survival of the animals, Tumour vascularization and blood flow were demonstrated using vascular casts and [Cr-51]-microspheres. Following the addition of melphalan (15 or 100 mu g/ml), drug concentrations in the perfusate, tissues and systemic circulation were determined using high pressure liquid chromatography (HPLC), Systemic leakage, assessed using [I-125]albumin and melphalan and detected by a gamma-counter and HPLC respectively, was <0.5%. The melphalan concentration and tissue flow rate in the tumour deposits were 40 and 30% respectively, when compared with the surrounding subcutaneous tissue, At a dose of 15 mu g/ml, melphalan caused a reduction in tumour growth after 60 min perfusion, and a significant reduction in tumour size was seen when the melphalan dose was 100 mu g/ml. The surviving nude rat model of isolated limb perfusion for recurrent melanoma will allow examination of optimal perfusion conditions, along with the pharmacokinetics, pharmacodynamics and efficacy of melphalan and other drugs.

Comparison of Humanized IgG and FvFc Anti-CD3 Monoclonal Antibodies Expressed in CHO Cells

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

High sucrose intake in rats is associated with increased ACE2 and angiotensin-(1-7) levels in the adipose tissue

Sucrose-fed rats, a model of metabolic syndrome, are characterized by insulin resistance, obesity, hypertension, and high plasma levels of triacylglycerols and angiotensin II (Ang II). However, whether tissue renin-angiotensin system (RAS) is altered in metabolic syndrome is unclear. To study this issue, food ad libitum and water (C) or 20% sucrose solution (SC) were given to adult male Wistar rats, for 30 days. Body weight (BW), blood pressure (BP), epididymal adipose tissue (EPI) mass, rate of in vivo fatty acid (FA) synthesis in EPI, circulating glucose, insulin, leptin, angiotensins I and II, triacylglycerols, and plasma renin (PRA) and angiotensin-converting enzyme (ACE) activities were evaluated. In kidneys and EPI, gene and protein expression of type 1 (AT(1)) and 2 (AT(2)) Ang II receptors, ACE, angiotensinogen (ACT) as well as protein expression of angiotensin-converting enzyme 2 (ACE2) were determined. In both tissues, Ang I, Ang II and Ang-(1-7) contents were also measured by HPLC. In SC rats higher BP, EPI mass, circulating triacylglycerols, insulin, leptin, PRA and, Ang II were found. In EPI, the rate of in vivo FA synthesis was associated with increased Ang-(1-7), protein expression of AT(1) and AT(2) receptors, ACE2, ACT, and gene expression of ACT although a reduction in ACE activity and in adipose Ang I and Ang II contents was observed. In kidneys, AT(1) and AT(2), ACE and ACT gene and protein expression as well as protein expression of ACE2 were unaltered while Ang II, Ang-(1-7) and ACE activity increased. These RAS component changes seem to be tissue specific and possibly are related to enhancement of FA synthesis, EPI mass and hypertension. (C) 2010 Elsevier B.V. All rights reserved.

Biochemical synthesis, purification and preliminary pharmacological evaluation of normorphine-3-glucuronide

Normorphine was synthesised from morphine by thermal decomposition of an N-alpha-chloroethylchloroformate adduct, and purified (> 98% purity) using semipreparative HPLC with ultraviolet detection. Normorphine-3-glucuronide (NM3G) was biochemically synthesised using the substrate normorphine, uridine diphosphoglucuronic acid and Sprague-Dawley rat liver microsomes in a 75% yield (relative to normorphine base). The synthesised NM3G was purified by precipitation and washing with acetonitrile. Determinations of purity using HPLC with electrochemical and ultraviolet detection confirmed that the NM3G produced was of high (> 99%) purity. Mass spectrometry, fourier transform infrared spectrophotometry and nuclear magnetic resonance spectrometry confirmed the structure, especially placement of the glucuronide moiety at the 3-phenolic position and not at the 17-nitrogen. Administration of NM3G by the intracerebroventricular (icy) route to rats in doses of 2.5 and 7.5 mu g resulted in the development of central nervous system (CNS) excitatory behavioural effects including myoclonus, chewing, wet-dog shakes, ataxia and explosive motor behaviour. At an icy dose of 7.5 mu g, NM3G also induced short periods of tonic-clonic convulsive activity. Thus, NM3G elicits CNS excitation following supraspinal administration in a manner analogous to morphine-3-glucuronide (M3G), the major metabolite of morphine (1). Further studies are required to determine whether NM3G attenuates morphine-induced antinociception in se similar manner to M3G.

The effects of perfusion conditions on melphalan distribution in the isolated perfused rat hindlimb bearing a human melanoma xenograft

An isolated rat hindlimb perfusion model carrying xenografts of the human melanoma cell line MM96 was used to study the effects of perfusion conditions on melphalan distribution. Krebs-Henseleit buffer and Hartmann's solution containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 were used as perfusates. Melphalan concentrations in perfusate, tumour nodules and normal tissues were measured using high-performance liquid chromatography (HPLC). Increasing the perfusion flow rates (from 4 to 8 mi min(-1)) resulted in higher tissue blood flow (determined with Cr-51-labelled microspheres) and melphalan uptake by tumour and normal tissues. me distribution of melphalan within tumour nodules and normal tissues was similar for both Krebs-Henseleit buffer and Hartmann's solution; however, tissue concentrations of melphalan were significantly higher for a perfusate containing 2.8% dextran 40 than for one containing 4.7% BSA. The melphalan concentration in the tumour was one-third of that found in the skin if the perfusate contained 4.7% BSA. In conclusion, this study has shown that a high perfusion flow enhances the delivery of melphalan into implanted tumour nodules and normal tissues, and a perfusate with low melphalan binding (no albumin) is preferred for maximum uptake of drug by the tumour.

Bioactivation of Phenytoin by Human Cytochrome P450: Characterization of the Mechanism and Targets of Covalent Adduct Formation

The cytochrome P450-dependent covalent binding of radiolabel derived fi om phenytoin (DPH) and its phenol and catechol metabolites, 5-(4'-hydroxyphenyl)-5-phenylhydantoin (HPPH) and 5-(3',4'-dihydroxyphenyl)-5-phenylhydantoin (CAT), was examined in liver microsomes. Radiolabeled HPPH and CAT and unlabeled CAT were obtained from microsomal incubations and isolated by preparative HPLC. NADPH-dependent covalent binding was demonstrated in incubations of human liver microsomes with HPPH. When CAT was used as substrate, covalent adduct formation was independent of NADPH, was enhanced in the presence of systems generating reactive oxygen species, and was diminished under anaerobic conditions or in the presence of cytoprotective reducing agents. Fluorographic analysis showed that radiolabel derived from DPH and HPPH was selectively associated with proteins migrating with approximate relative molecular weights of 57-59 kDa and at the dye front (molecular weights < 23 kDa) on denaturing gels. Lower levels of radiolabel were distributed throughout the molecular weight range. In contrast, little selectivity was seen in covalent adducts formed from CAT. HPPH was shown to be a mechanism-based inactivator of P450, supporting the contention that a cytochrome P450 is one target of covalent binding. These results suggest that covalent binding of radiolabel derived from DPH in rat and human Liver microsomes occurs via initial P450-dependent catechol formation followed by spontaneous oxidation to quinone and semiquinone derivatives that ultimately react with microsomal protein. Targets for covalent binding may include P450s, though the catechol appears to be sufficiently stable to migrate out of the P450 active site to form adducts with other proteins. In conclusion, we have demonstrated that DPH can be bioactivated in human liver to metabolites capable of covalently binding to proteins. The relationship of adduct formation to DPH-induced hypersensitivity reactions remains to be clarified.

Photodegradation of irinotecan (CPT-11) in aqueous solutions: Identification of fluorescent products and influence of solution composition

The photodegradation of irinotecan (CPT-11), the semisynthetic derivative of the antitumor alkaloid 20(S)-camptothecin, has been investigated. The drug was exposed to laboratory light for up to 5 days in 0.9% saline solution (pH 8.5). Five significant photodegradation products were observed and a high-performance liquid chromatography (HPLC) assay was employed to isolate them from CPT-11 using gradient conditions. The structures were elucidated by nuclear magnetic resonance spectroscopy and tandem mass spectrometry and shown to be the result of extensive modifications of the lactone ring of CPT-11. Three of the compounds were found to belong to the mappicine group of alkaloids. In addition, the effect of light on the stability of CPT-11 in aqueous solutions and biological fluids was also assessed, Potassium phosphate buffers (0.05 M, pH 5.0-8.2) and saline, plasma, urine, and bile solutions containing 20 mu M CPT-11 were equilibrated in the dark for 24 h before being exposed to laboratory light for up to 171 h at ambient temperature. Four of the five identified photodegradation products were observed and quantitated by isocratic HPLC, using a different detection mode (fluorescence) than the one used for gradient elution, In general, CPT-11 was found to be unstable under neutral and alkaline conditions for all solutions investigated, with the exception of bile. We conclude that CPT-11 is photolabile and that care should be taken to protect samples, particularly those intended for the isolation and identification of novel metabolites of CPT-11.

Centella asiatica water extract inhibits iPLA(2) and cPLA(2) activities in rat cerebellum

Centella asiatica (L.) Urb an is distributed widely in South America and Asia and is known as a therapeutic agent in folk medicine, capable of improving memory and treating several neurological disorders. Asiaticoside is one of the compounds found in C asiatica leaves that is suggested to be responsible for its pharmacological potential. Phospholipase A(2) (PLA(2)) is a group of enzymes that has abnormal activity in the central nervous system in some neuropsychiatric diseases. In this work, the asiaticoside present in C asiatica water extract was quantified by HPLC analysis. We also evaluated the activity of subtypes of PLA(2) in cerebellar samples from rats after C asiatica water extract treatment using a radioenzymatic assay. Asiaticoside was the major compound (84%) found in Centella water extract. We found a dose-dependent inhibitory effect of C asiatica water extract on the activity of Ca(2+)-independent PLA(2) (iPLA(2)) and cytosolic PLA(2) (cPLA(2)). The inhibition of these enzymes in the brain suggests that C asiatica may be useful to treat conditions associated with increased PLA(2) activity in the brain, such as epilepsy, stroke, multiple sclerosis and other neuropsychiatric disorders. (C) 2008 Elsevier GmbH. All rights reserved.

PM(2.5) and PM(10): The influence of sugarcane burning on potential cancer risk

In Brazil, sugarcane fields are often burned to facilitate manual harvesting, and this burning causes environmental pollution from the large amounts of soot released into the atmosphere. This material contains numerous organic compounds such as PAHs. In this study, the concentrations of PAHs in two particulate-matter fractions (PM(2.5) and PM(10)) in the city of Araraquara (SE Brazil, with around 200,000 inhabitants and surrounded by sugarcane plantations) were determined during the sugarcane harvest (HV) and non-harvest (NHV) seasons in 2008 and 2009. The sampling strategy included four campaigns, with 60 samples in the NHV season and 220 samples in the HV season. The PM(2.5) and PM(10) fractions were collected using a dichotomous sampler (10 L min(-1), 24 h) with Teflon (TM) filters. The filter sets were extracted (ultrasonic bath with hexane/acetone (1:1 v/v)) and analyzed by HPLC/Fluorescence. The median concentration for total PAHs (PM(2.5) in 2009) was 0.99 ng m(-3) (NHV) and 3.3 ng m(-3) (HV). In the HV season, the total concentration of carcinogenic PAHs (benz(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, and benzo(a)pyrene) was 5 times higher than in the NHV season. B(a)P median concentrations were 0.017 ng m(-3) and 0.12 ng m(-3) for the NHV and HV seasons, respectively. The potential cancer risk associated with exposure through inhalation of these compounds was estimated based on the benzo[a]pyrene toxic equivalence (BaP(eq)), where the overall toxicity of a PAR mixture is defined by the concentration of each compound multiplied by its relative toxic equivalence factor (TEF). BaP(eq) median (2008 and 2009 years) ranged between 0.65 and 1.0 ng m(-3) and 1.2-1.4 ng m(-3) for the NHV and HV seasons, respectively. Considering that the maximum permissible BaPeq in ambient air is 1 ng m(-3), related to the increased carcinogenic risk, our data suggest that the level of human exposure to PAHs in cities surrounded by sugarcane crops where the burning process is used is cause for concern. (C) 2010 Published by Elsevier Ltd.

Synthesis of Fructooligosaccharides in Banana `Prata` and Its Relation to Invertase Activity and Sucrose Accumulation

Levels of sucrose and total fructool igosaccha rides (FOS) were quantified in different phases of banana `Prata` ripening during storage at ambient (similar to 19 degrees C) and low (similar to 10 degrees C) temperature. Total FOS levels were detected in the first days after harvest, whereas 1-kestose remained undetectable until the sucrose levels reached approximately 200 mg/g (dry weight) in both groups. Sucrose levels increased slowly but constantly at low temperature, but they elevated rapidly when the temperature was raised to 19 degrees C. Total FOS and sucrose levels were higher in bananas stored at low temperature than in the control group. In both samples, total FOS levels were higher than those of 1-kestose. The carbohydrate profiles obtained by HPLC and TLC suggest the presence of neokestose, 6-kestose, and bifurcose. The enzymes putatively involved in banana fructosyltransferase activity were also evaluated. Results obtained indicate that the banana enzyme responsible for the synthesis of FOS by transfructosylation is an invertase rather than a sucrose-sucrosyl transferase-like enzyme.

Maternal alcohol consumption may influence cord blood ascorbic acid concentration: findings from a study of Brazilian mothers and their newborns

Studies that have investigated ascorbic acid (AA) concentrations in cord blood have pointed to significant associations with maternal blood AA concentrations. smoking, age, diet, type of delivery, duration of gestation, fetal distress and birth weight. The aim of the present study was to determine the relationship between cord blood AA concentrations in newborns and maternal characteristics. A total of 117 Brazilian healthy parturients were included in this cross-sectional study. The concentrations of AA in blood were determined by the HPLC method. Data concerning socio-economic, demographic, obstetric, nutritional and health characteristics of the parturients, including alcohol consumption and smoking habit, were assessed by a standardised questionnaire. A FFQ was used to investigate the intake of foods rich in vitamin C. Cord blood AA concentration was significantly correlated with per capita income (r 0.26; P=0.005), maternal blood AA concentration (r 0.48; P<0.001) and maternal vitamin C-rich food intake score (r 0.36; P<0.001). The linear regression model including maternal AA concentration, alcohol consumption, smoking, parity, vitamin C-rich food intake score and per capita income explained 31.13% of the variation in cord blood AA concentrations in newborns. We recommend further experimental studies to assess the effects of ethanol on placental AA uptake, and epidemiological cohort studies to evaluate in detail the influence of maternal alcohol consumption on cord blood AA concentrations.

The role of polar phytocomplexes on anticonvulsant effects of leaf extracts of Lippia alba (Mill.) NE Brown chemotypes

Objectives The purpose of the present work was to characterize file pharmacological profile of different L. alba chemotypes and to correlate the obtained data to the presence of chemical constituents detected by phytochemical analysis. Methods Essential oils from each L. alba chemotype (LP1-LP7) were characterized by gas chromatography-mass spectrometry (GC-MS) and extracted non-volatile compounds were analysed by HPLC and GC-MS. The anticonvulsant actions of file extracted compounds were studied in pentylenetetrazole-induced clonic seizures in mice and then effect oil motor coordination was studied using the rota-rod test in rats. The synaptosomes and synaptic membranes of the rats were examined for the influence of LP3 chemotype extract oil GABA uptake and binding experiments. Key findings Behavioural parameters encompassed by the pentylenetetrazole test indicated that 80% ethanolic extracts of LP1, LP3 and LP6 L. alba chemotypes were more effective as anticonvulsant agents. Neurochemical assays using synaptosomes and synaptic membranes showed that L. alba LP3 chemotype 80% ethanolic extract inhibited GABA uptake and GABA binding ill a dose-dependent manner. HPLC analysis showed that LP1, LP3 and LP6 80% ethanolic extracts presented a similar profile of constituents, differing from those seen in LP2, LP4, LP5 and LP7 80% ethanolic extracts, which exhibited no anticonvulsant effect. GC-MS analysis indicated the Occurrence of phenylpropanoids in methanolic fractions obtained from LP1, LP3 and LP6 80% ethanolic extracts and also the accumulation of inositol and flavonoids in hydroalcoholic fractions. Conclusions Our results suggest that the anticonvulsant properties shown by L. alba might be correlated to the presence of it complex of non-volatile Substances (phenylpropanoids, flavonoids and/or inositols), and also to the volatile terpenoids (beta-myrcene, citral, limonene and carvone), which have been previously Validated as anticonvulsants.

Influence of Quinidine, Cimetidine, and Ketoconazole on the Enantioselective Pharmacokinetics and Metabolism of Metoprolol in Rats

Metoprolol is a beta-blocker and its racemic mixture is used for the treatment of hypertension. In the present study we investigated the influence of CYP2D and CYP3A on the stereoselective metabolism of metoprolol in rats. Male Wistar rats (n = 6 per group) received racemic metoprolol (15 mg/kg) orally, with or without pretreatment with the CYP inhibitor ketoconazole (50 mg/kg), cimetidine (150 mg/kg), or quinidine (80 mg/kg). Blood samples were collected up to 48 h after metoprolol administration. The plasma concentrations of the stereoisomers of metoprolol, O-demethylmetoprolol (ODM), alpha-hydroxymetoprolol (OHM) (Chiralpak(R) AD column), and metoprolol acidic metabolite (AODM) (Chiralcel(R) OD-R column) were determined by HPLC using fluorescence detection (lambda(exc) = 229 nm; lambda(em) = 298 nm). CYP3A inhibition by ketoconazole reduced the plasma concentrations of ODM and AODM and favored the formation of OHM. CYP2D and CYP3A inhibition by cimetidine reduced the plasma concentrations of OHM and AODM and favored the formation of ODM. The inhibition of CYP2D by quinidine reduced the plasma concentrations of OHM and favored the formation of ODM. In conclusion, the results suggest that CYP3A is involved in the formation of ODM and CYP2D is involved in the formation of AODM. Chirality 21:886-893, 2009. (C) 2009 Wiley-Liss, Inc.