Analgesic and anti-inflammatory actions of robenacoxib in acute joint inflammation in dog

The objectives of this study were to establish dose-response and blood concentration-response relationships for robenacoxib, a novel nonsteroidal anti-inflammatory drug with selectivity for inhibition of the cyclooxygenase (COX)-2 isoenzyme, in a canine model of synovitis. Acute synovitis of the stifle joint was induced by intra-articular injection of sodium urate crystals. Robenacoxib (0.25, 0.5, 1.0, 2.0 and 4.0 mg/kg), placebo and meloxicam (0.2 mg/kg) were administered subcutaneously (s.c.) 3 h after the urate crystals. Pharmacodynamic endpoints included data from forceplate analyses, clinical orthopaedic examinations and time course of inhibition of COX-1 and COX-2 in ex vivo whole blood assays. Blood was collected for pharmacokinetics. Robenacoxib produced dose-related improvement in weight-bearing, pain and swelling as assessed objectively by forceplate analysis (estimated ED(50) was 1.23 mg/kg for z peak force) and subjectively by clinical orthopaedic assessments. The analgesic and anti-inflammatory effects of robenacoxib were significantly superior to placebo (0.25-4 mg/kg robenacoxib) and were non-inferior to meloxicam (0.5-4 mg/kg robenacoxib). All dosages of robenacoxib produced significant dose-related inhibition of COX-2 (estimated ED(50) was 0.52 mg/kg) but no inhibition of COX-1. At a dosage of 1-2 mg/kg administered s.c., robenacoxib should be at least as effective as 0.2 mg/kg of meloxicam in suppressing acute joint pain and inflammation in dogs.

Cardiac troponins as indicators of acute myocardial damage in dogs

Cardiac troponin I (cTnI) and T (cTnT) have a high sequence homology across phyla and are sensitive and specific markers of myocardial damage. The purpose of this study was to evaluate the Cardiac Reader, a human point-of-care system for the determination of cTnT and myoglobin, and the Abbott Axsym System for the determination of cTnI and creatine kinase isoenzyme MB (CK-MB) in healthy dogs and in dogs at risk for acute myocardial damage because of gastric dilatation-volvulus (GDV) and blunt chest trauma (BCT). In healthy dogs (n = 56), cTnI was below detection limits (<0.1 microg/L) in 35 of 56 dogs (reference range 0-0.7 microg/L), and cTnT was not measurable (<0.05 ng/mL) in all but 1 dog. At presentation, cTnI, CK-MB, myoglobin, and lactic acid were all significantly higher in dogs with GDV (n = 28) and BCT (n = 8) than in control dogs (P < .001), but cTnT was significantly higher only in dogs with BCT (P = .033). Increased cTnI or cTnT values were found in 26 of 28 (highest values 1.1-369 microg/L) and 16 of 28 dogs (0.1-1.7 ng/mL) with GDV, and in 6 of 8 (2.3-82.4 microg/L) and 3 of 8 dogs (0.1-0.29 ng/mL) with BCT, respectively. In dogs suffering from GDV, cTnI and cTnT increased further within the first 48 hours (P < .001). Increased cardiac troponins suggestive of myocardial damage occurred in 93% of dogs with GDV and 75% with BCT. cTnI appeared more sensitive, but cTnT may be a negative prognostic indicator in GDV. Both systems tested seemed applicable for the measurement of canine cardiac troponins, with the Cardiac Reader particularly suitable for use in emergency settings.

Histamine increases sphingosine kinase-1 expression and activity in the human arterial endothelial cell line EA.hy 926 by a PKC-alpha-dependent mechanism

Sphingosine 1-phosphate (S1P) is a potent mitogenic signal generated from sphingosine by the action of sphingosine kinases (SKs). In this study, we show that in the human arterial endothelial cell line EA.hy 926 histamine induces a time-dependent upregulation of the SK-1 mRNA and protein expression which is followed by increased SK-1 activity. A similar upregulation of SK-1 is also observed with the direct protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, SK-2 activity is not affected by neither histamine nor TPA. The increased SK-1 protein expression is due to stimulated de novo synthesis since cycloheximide inhibited the delayed SK-1 protein upregulation. Moreover, the increased SK-1 mRNA expression results from an increased promoter activation by histamine and TPA. In mechanistic terms, the transcriptional upregulation of SK-1 is dependent on PKC and the extracellular signal-regulated protein kinase (ERK) cascade since staurosporine and the MEK inhibitor U0126 abolish the TPA-induced SK-1 induction. Furthermore, the histamine effect is abolished by the H1-receptor antagonist diphenhydramine, but not by the H2-receptor antagonist cimetidine. Parallel to the induction of SK-1, histamine and TPA stimulate an increased migration of endothelial cells, which is prevented by depletion of the SK-1 by small interfering RNA (siRNA). To appoint this specific cell response to a specific PKC isoenzyme, siRNA of PKC-alpha, -delta, and -epsilon were used to selectively downregulate the respective isoforms. Interestingly, only depletion of PKC-alpha leads to a complete loss of TPA- and histamine-triggered SK-1 induction and cell migration. In summary, these data show that PKC-alpha activation in endothelial cells by histamine-activated H1-receptors, or by direct PKC activators leads to a sustained upregulation of the SK-1 protein expression and activity which, in turn, is critically involved in the mechanism of endothelial cell migration.

Influence of CYP2B6 polymorphism on plasma and intracellular concentrations and toxicity of efavirenz and nevirapine in HIV-infected patients

BACKGROUND: Efavirenz (EFV) and nevirapine (NVP) are metabolized by cytochrome P450 2B6 (CYP2B6). Allele 516 G>T (Gln172His) is associated with diminished activity of this isoenzyme, and may lead to differences in drug exposure. METHODS: We evaluated this allele as a pharmacogenetic marker of EFV and NVP pharmacokinetics and EFV toxicity in 167 participants receiving EFV and 59 receiving NVP recruited within the genetics project of the Swiss HIV Cohort Study. Drug concentrations were measured in plasma and in peripheral blood mononuclear cells (PBMCs) from the same sample. Neuropsychological toxicity of EFV (sleep disorders, mood disorders, fatigue) was assessed using a standardized questionnaire. RESULTS AND CONCLUSIONS: CYP2B6 516TT was associated with greater plasma and intracellular exposure to EFV, and greater plasma exposure to NVP. Intracellular drug concentration, and CYP2B6 genotype were predictors of EFV neuropsychological toxicity. CYP2B6 genotyping may be useful to complement an individualization strategy based on plasma drug determinations to increase the safety and tolerability of EFV.

Tenofovir use is associated with an increase in serum alkaline phosphatase in the Swiss HIV Cohort Study

BACKGROUND: Tenofovir (TDF) use has been associated with proximal renal tubulopathy, reduced calculated glomerular filtration rates (cGFR) and losses in bone mineral density. Bone resorption could result in a compensatory osteoblast activation indicated by an increase in serum alkaline phosphatase (sAP). A few small studies have reported a positive correlation between renal phosphate losses, increased bone turnover and sAP. METHODS: We analysed sAP dynamics in patients initiating (n = 657), reinitiating (n = 361) and discontinuing (n = 73) combined antiretroviral therapy with and without TDF and assessed correlations with clinical and epidemiological parameters. RESULTS: TDF use was associated with a significant increase of sAP from a median of 74 U/I (interquartile range 60-98) to a plateau of 99 U/I (82-123) after 6 months (P < 0.0001), with a prompt return to baseline upon TDF discontinuation. No change occurred in TDF-sparing regimes. Univariable and multivariable linear regression analyses revealed a positive correlation between sAP and TDF use (P < or = 0.003), but no correlation with baseline cGFR, TDF-related cGFR reduction, changes in serum alanine aminotransferase (sALT) or active hepatitis C. CONCLUSIONS: We document a highly significant association between TDF use and increased sAP in a large observational cohort. The lack of correlation between TDF use and sALT suggests that the increase in sAP is because of the bone isoenzyme and indicates stimulated bone turnover. This finding, together with published data on TDF-related renal phosphate losses, this finding raises concerns that TDF use could result in osteomalacia with a loss in bone mineral density at least in a subset of patients. This potentially severe long-term toxicity should be addressed in future studies.

Involvement of the ABC-transporter ABCC1 and the sphingosine 1-phosphate receptor subtype S1P(3) in the cytoprotection of human fibroblasts by the glucocorticoid dexamethasone

Glucocorticoids (GC) represent the most commonly used drugs for the treatment of acute and chronic inflammatory skin diseases. However, the topical long-term therapy of GC is limited by the occurrence of skin atrophy. Most interestingly, although GC inhibit proliferation of human fibroblasts, they exert a pronounced anti-apoptopic action. In the present study, we further elucidated the molecular mechanism of the GC dexamethasone (Dex) to protect human fibroblasts from programmed cell death. Dex not only significantly alters the expression of the cytosolic isoenzyme sphingosine kinase 1 but also initiated an enhanced intracellular formation of the sphingolipid sphingosine 1-phosphate (S1P). Investigations using S1P (3) ((-/-)) -fibroblasts revealed that this S1P-receptor subtype is essential for the Dex-induced cytoprotection. Moreover, we demonstrate that the ATP-binding cassette (ABC)-transporter ABCC1 is upregulated by Dex and may represent a crucial carrier to transport S1P from the cytosol to the S1P(3)-receptor subtype.

Acetaldehyde as an underestimated risk factor for cancer development: role of genetics in ethanol metabolism

Chronic ethanol consumption is a strong risk factor for the development of certain types of cancer including those of the upper aerodigestive tract, the liver, the large intestine and the female breast. Multiple mechanisms are involved in alcohol-mediated carcinogenesis. Among those the action of acetaldehyde (AA), the first metabolite of ethanol oxidation is of particular interest. AA is toxic, mutagenic and carcinogenic in animal experiments. AA binds to DNA and forms carcinogenic adducts. Direct evidence of the role of AA in alcohol-associated carcinogenesis derived from genetic linkage studies in alcoholics. Polymorphisms or mutations of genes coding for AA generation or detoxifying enzymes resulting in elevated AA concentrations are associated with increased cancer risk. Approximately 40% of Japanese, Koreans or Chinese carry the AA dehydrogenase 2*2 (ALDH2*2) allele in its heterozygous form. This allele codes for an ALDH2 enzyme with little activity leading to high AA concentrations after the consumption of even small amounts of alcohol. When individuals with this allele consume ethanol chronically, a significant increased risk for upper alimentary tract and colorectal cancer is noted. In Caucasians, alcohol dehydrogenase 1C*1 (ADH1C*1) allele encodes for an ADH isoenzyme which produces 2.5 times more AA than the corresponding allele ADH1C*2. In studies with moderate to high alcohol intake, ADH1C*1 allele frequency and rate of homozygosity was found to be significantly associated with an increased risk for cancer of the upper aerodigestive tract, the liver, the colon and the female breast. These studies underline the important role of acetaldehyde in ethanol-mediated carcinogenesis.

In vivo analysis of efavirenz metabolism in individuals with impaired CYP2A6 function

INTRODUCTION: The antiretroviral drug efavirenz (EFV) is extensively metabolized into three primary metabolites: 8-hydroxy-EFV, 7-hydroxy-EFV and N-glucuronide-EFV. There is a wide interindividual variability in EFV plasma exposure, explained to a great extent by cytochrome P450 2B6 (CYP2B6), the main isoenzyme responsible for EFV metabolism and involved in the major metabolic pathway (8-hydroxylation) and to a lesser extent in 7-hydroxylation. When CYP2B6 function is impaired, the relevance of CYP2A6, the main isoenzyme responsible for 7-hydroxylation may increase. We hypothesize that genetic variability in this gene may contribute to the particularly high, unexplained variability in EFV exposure in individuals with limited CYP2B6 function. METHODS: This study characterized CYP2A6 variation (14 alleles) in individuals (N=169) previously characterized for functional variants in CYP2B6 (18 alleles). Plasma concentrations of EFV and its primary metabolites (8-hydroxy-EFV, 7-hydroxy-EFV and N-glucuronide-EFV) were measured in different genetic backgrounds in vivo. RESULTS: The accessory metabolic pathway CYP2A6 has a critical role in limiting drug accumulation in individuals characterized as CYP2B6 slow metabolizers. CONCLUSION: Dual CYP2B6 and CYP2A6 slow metabolism occurs at significant frequency in various human populations, leading to extremely high EFV exposure.

Molecular genetic analysis of Dichelobacter nodosus proteases AprV2/B2, AprV5/B5 and BprV/B in clinical material from European sheep flocks

Dichelobacter nodosus, the etiological agent of ovine footrot, exists both as virulent and as benign strains, which differ in virulence mainly due to subtle differences in the three subtilisin-like proteases AprV2, AprV5 and BprV found in virulent, and AprB2, AprB5 and BprB in benign strains of D. nodosus. Our objective was a molecular genetic epidemiological analysis of the genes of these proteases by direct sequence analysis from clinical material of sheep from herds with and without history of footrot from 4 different European countries. The data reveal the two proteases known as virulent AprV2 and benign AprB2 to correlate fully to the clinical status of the individuals or the footrot history of the herd. In samples taken from affected herds, the aprV2 gene was found as a single allele whereas in samples from unaffected herds several alleles with minor modifications of the aprB2 gene were detected. The different alleles of aprB2 were related to the herds. The aprV5 and aprB5 genes were found in the form of several alleles scattered without distinction between affected and non-affected herds. However, all different alleles of aprV5 and aprB5 encode the same amino acid sequences, indicating the existence of a single protease isoenzyme 5 in both benign and virulent strains. The genes of the basic proteases BprV and BprB also exist as various alleles. However, differences found in samples from affected versus non-affected herds do not reflect the currently known epitopes that are attributed to differences in biochemical activity. The data of the study confirm the prominent role of AprV2 in the virulence of D. nodosus and shed a new light on the presence of the other protease genes and their allelic variants in clinical samples.

Rhabdomyolysis in association with simvastatin and amiodarone

OBJECTIVE To report a case of severe myopathy associated with concomitant simvastatin and amiodarone therapy. CASE SUMMARY A 63-year-old white man with underlying insulin-dependent diabetes, recent coronary artery bypass surgery, and postoperative hemiplegia was treated with aspirin, metoprolol, furosemide, nitroglycerin, and simvastatin. Due to recurrent atrial fibrillation, oral anticoagulation with phenprocoumon and antiarrhythmic treatment with amiodarone were initiated. Four weeks after starting simvastatin 40 mg/day and 2 weeks after initiating amiodarone 1 g/day for 10 days, then 200 mg/day, he developed diffuse muscle pain with generalized muscular weakness. Laboratory investigations revealed a significant increase of creatine kinase (CK) peaking at 40 392 U/L. Due to a suspected drug interaction of simvastatin with amiodarone, both drugs were stopped. CK normalized over the following 8 days, and the patient made an uneventful recovery. An objective causality assessment revealed that the myopathy was probably related to simvastatin. DISCUSSION Myopathy is a rare but potentially severe adverse reaction associated with statins. Besides high statin doses, concomitant use of fibrates, defined comorbidities, and concurrent use of inhibitors of cytochrome P450 are important additional risk factors. This is especially relevant if statins predominantly metabolized by CYP3A4 are combined with inhibitors of this isoenzyme. Amiodarone is a potent inhibitor of several different CYP isoenzymes, including CYP3A4. CONCLUSIONS Avoiding the concomitant use of drugs with the potential to inhibit CYP-dependent metabolism (eg, amiodarone) or elimination of statins may decrease the risk of statin-associated myopathy. Alternatively, if drug therapy with a potent CYP inhibitor is inevitable, choosing a statin without relevant CYP metabolism (eg, pravastatin) should be considered.

Biochemical changes in barley plants after excessive supply of copper and manganese

The present study was undertaken to identify changes in some important proteins involved in CO2 fixation (Rubisco, Rubisco activase (RA), Rubisco binding protein (RBP)), NH4+ assimilation (glutamine synthetase (GS) and glutamate synthase (GOGAT)), using immunoblotting, and in the antioxidative defense as a result of Cu or Mn excess in barley leaves (Hordeum vulgare L. cv. Obzor). Activities and isoenzyme patterns of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase (CAT), as well as the levels of ascorbate (ASC), non-protein sulfhydryl groups, hydrogen peroxide and oxidative damage to proteins were determined. Data were correlated to the accumulation of Cu or Mn in the leaves after 5 days supply of heavy metal (HM) excess in the nutrient solution. In the highest Cu excess (1500 μM), Rubisco LS and SS were reduced considerably whereas under the highest Mn concentrations (18,300 μM) only minor changes in Rubisco subunits were detected. The RBP was diminished under the highest concentrations of both Cu or Mn. The bands of RA changed differently comparing Cu and Mn toxicity. GS decreased and GOGAT was absent under the highest concentration of Cu. At Mn excess Fd-GOGAT diminished whereas GS was not apparently changed. The development of toxicity symptoms corresponded to an accumulation of Cu or Mn in the leaves and to a gradual increase in protein carbonylation, a lower SOD activity and elevated CAT and GPX activities. APX activity was diminished under Mn toxicity and was not changed under Cu excess. Generally, changes in the isoenzyme profiles were similar under both toxicities. An accumulation of H2O2 was observed only at Mn excess. Contrasting changes in the low-molecular antioxidants were detected when comparing both toxicities. Cu excess affected mainly the non-protein SH groups, while Mn influenced the ASC content. Oxidative stress under Cu or Mn toxicity was most probably the consequence of depletion in low-molecular antioxidants as a result of their involvement in detoxification processes and disbalance in antioxidative enzymes. The link between heavy metal accumulation in leaves, leading to different display of oxidative stress, and changes in individual chloroplast proteins is discussed in the article.