Quantifying the use of the statin antilipemic drugs: Comparisons and contrasts between Nova Scotia, Canada, and Queensland, Australia

Background: jurisdictions are developing public drug insurance systems to improve access to pharmaceuticals, cost-effective prescribing, and patient health and well-being. We compared 2 Jurisdictions with different pharmaceutical policies to determine prescribing patterns for 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (le, statins). Objective: The aim of this work was to investigate the feasibility of using available prescription admimstrative databases to compare the use of statins in Queensland, Australia, and in Nova Scotia, Canada. Methods: Data from the Nova Scotia Pharmacare Program and the Health Insurance Commission in Australia were used to obtain dispensing data. Utilization was compared for the 5-year period from 1997 through 2001, using the World Health Organization anatomic therapeutic chemical/defined daily dose (DDD) system. Results: In the year 2001, there were 177,000 beneficiaries in the public drug plan in Nova Scotia (62% aged ≥ 65 years old) and 960,000 concession beneficiaries (pensioners and social security recipients, 61% aged ≥ 65 years) in Queensland. These 2 groups were comparable. The overall utilization of statin medications increased steadily in both areas over the study period, from 50 to 205 DDD/1000 beneficiaries per day. Comparison of the 2 growth lines showed no statistically significant differences in overall statin use despite differences in brand availabilities and policies about prescribing. In the year 2001, atorvastatin was the most commonly prescribed statin in both areas, comprising 46% of statin use in Nova Scotia and 51% in Queensland. Mean doses of each statin prescribed were slightly above the DDDs. Expenditure on statins per 1000 beneficiaries and per DDD were similar in each jurisdiction, being slightly higher in Nova Scotia. Conclusions: Despite differences in pharmaceutical reimbursement systems, use of the statins was similar in Nova Scotia and Queensland. The feasibility of the methodology was demonstrated. Future studies, including comparisons of drug utilization for other classes of drugs for which drug policies may be divergent (eg, different pricing structures or prior authorization requirements), or for which less evidence for appropriate use is available, may be useful. © 2005 Excerpta Medica, Inc.

Cadmium-induced nephropathy in the development of high blood pressure

In recognition of a central role of the kidney in long-term blood pressure control, we undertook an in-depth analysis of the relationship between blood pressure and kidney damage caused by environmental exposure to the common pollutants cadmium and lead. The subjects were 200 healthy Thais, 16 and 60 years of age (100 female non-smokers, 53 male non-smokers, and 47 male smokers). None of these subjects had been exposed to Cd or Pb in the workplace and their urinary Cd concentrations ranged from 0.4 to 37 nM, whereas their urinary Pb concentrations ranged from 0.1 to 30 nM. The prevalence of high blood pressure was 2%, 8% and 19%, respectively in subjects with low, average and high Cd-burden (linear trend chi(2) = 6.4, P = 0.01). Multiple regression analysis revealed a significant positive association between Cd-burden and blood pressure in male nonsmokers (adjusted beta = 0.31, P = 0.02) and an inverse association between blood pressure and urinary Pb excretion rate in male smokers (adjusted beta = -0.38, P = 0.005). Associations between Cd-burden and nephropathies were evidenced by increases in urinary excretion of beta 2-microglobutin (P = 0.02) and N-acetyl-beta-D-glucosaminidase (P = 0.005) in subjects with high Cd-burden, compared with the subjects with average Cd-burden. In addition, an association between Cd-related nephropathy and high blood pressure was evidenced by a 20% increase in the prevalence of high blood pressure in people with NAG-uria (linear trend chi(2) = 4.3, P = 0.04). Our present study provides first evidence for a possible link between renal tubular damage and dysfunction caused by environmental Cd exposure and increased risk of high blood pressure. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

White blood cell count predicts reduction in coronary heart disease mortality with pravastatin

Background-Elevated serum inflammatory marker levels are associated with a greater long-term risk of cardiovascular events. Because 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors (statins) may have an antiinflammatory action, it has been suggested that patients with elevated inflammatory marker levels may have a greater reduction in cardiovascular risk with statin treatment. Methods and Results-We evaluated the association between the white blood cell count (WBC) and coronary heart disease mortality during a mean follow-up of 6.0 years in the Long-Term Intervention With Pravastatin in Ischemic Disease (LIPID) Study, a clinical trial comparing pravastatin (40 mg/d) with a placebo in 9014 stable patients with previous myocardial infarction or unstable angina. An increase in baseline WBC was associated with greater coronary heart disease mortality in patients randomized to placebo (hazard ratio for 1 X 10(9)/L increase in WBC, 1.18; 95% CI, 1.12 to 1.25; P<0.001) but not pravastatin (hazard ratio, 1.02; 95% CI, 0.96 to 1.09; P=0.56; P for interaction=0.004). The numbers of coronary heart disease deaths prevented per 1000 patients treated with pravastatin were 0, 9, 30, and 38 for baseline WBC quartiles of <5.9, 6.0 to 6.9, 7.0 to 8.1, and >8.2X10(9)/L, respectively. WBC was a stronger predictor of this treatment benefit than the ratio of total to high-density lipoprotein cholesterol and a global measure of cardiac risk. There was also a greater reduction (P=0.052) in the combined incidence of cardiovascular mortality, nonfatal myocardial infarction, and stroke with pravastatin as baseline WBC increased ( by quartile: 3, 41, 61, and 60 events prevented per 1000 patients treated, respectively). Conclusions-These data support the hypothesis that individuals with evidence of inflammation may obtain a greater benefit from statin therapy.

Accumulation and toxicity of monophenyl arsenicals in rat endothelial cells

Clark 1 (diphenylarsine chloride) and Clark 2 ( diphenylarsine cyanide) were used as chemical weapon agents (CWA), and the soil contamination by these CWA and their degraded products, diphenyl and phenyl arsenicals, has been one of the most serious environmental issues. In a series of comparisons in toxicity between trivalent and pentavalent arsenicals we investigated differences in the accumulation and toxicity of phenylarsine oxide (PAO(3+)) and phenylarsonic acid (PAA(5+)) in rat heart microvascular endothelial cells. Both the cellular association and toxicity of PAO(3+) were much higher than those of PAA(5+), and LC50 values of PAO(3+) and PAA(5+) were calculated to be 0.295 muM and 1.93 mM, respectively. Buthionine sulfoximine, a glutathione depleter, enhanced the cytotoxicity of both PAO(3+) and PAA(5+). N-Acetyl-L-cysteine (NAC) reduced the cytotoxicity and induction of heme oxygenase-1 (HO-1) mRNA in PAO(3+)-exposed cells, while NAC affected neither the cytotoxicity nor the HO-1 mRNA level in PAA(5+)-exposed cells. The effect of NAC may be due to a strong affinity of PAO(3+) to thiol groups because both NAC and GSH inhibited the cellular accumulation of PAO(3+), but PAA(3+) increased tyrosine phosphorylation levels of cellular proteins. These results indicate that the inhibition of protein phosphatases as well as the high affinity to cellular components may confer PAO(3+) the high toxicity.

Unidirectional inversion of ibuprofen in Caco-2 cells: Developing a suitable model for presystemic chiral inversion study

Intestinal chiral inversion of ibuprofen is still lacking direct evidence. In a preliminary experiment, ibuprofen was found to undergo inversion in Caco-2 cells. This investigation was thus conducted to determine the characteristics and influence of some biochemical factors on the chiral inversion of ibuprofen in Caco-2 cells. The effects of substrate concentration (2.5-40 mu g/ml), cell density (0.5-2 x 10(6) cells/ well), content of serum (0-20%), coexistence of S ibuprofen (corresponding doses), sodium azide (10mm), exogenous Coenzyme A (CoA) (0.1 - 0.4 mm),. and palmitic acid (5-25 mu m) on inversion were examined. A stereoselective HPLC method based on the Chromasil-CHI-TBB column was developed for quantitative analysis of the drug in cell culture medium. The inversion ratio (F-i) and elimination rate constant were calculated as the indexes of inversion extent. Inversion of ibuprofen in Caeo-2 cells was found to be both dose and cell density dependent, indicating saturable characteristics. Addition of serum significantly inhibited the inversion, to an extent of 2.7 fold decrease at 20% content. Preexistence of S enantiomer exerted a significant inhibitory effect (p < 0.01 for all tests). Sodium azide decreased the inversion ratio from 0.43 to 0.32 (p < 0.01). Exogenous CoA and palmitic acid significantly promoted the inversion at all tested doses (p < 0.01 for all tests). This research provided strong evidence to the capacity and capability of intestinal chiral inversion. Although long incubation times up to 120 h were required, Caco-2 cells should be a suitable model for chiral inversion research of 2-APAs considering the human-resourced and well-defined characteristics from the present study.

Suicide inhibition of acetohydroxyacid synthase by hydroxypyruvate

Acetohydroxyacid synthase (Ec 2.2.1.6) catalyses the thiamine diphosphate-dependent reaction between two molecules of pyruvate yielding 2-acetolactacte and CO2. The enzyme will also utilise hydroxypyruvate with a k(cat) value that is 12% of that observed with pyruvate. When hydroxypyruvate is the substrate, the enzyme undergoes progressive inactivation with kinetics that are characteristic of suicide inhibition. It is proposed that the dihydroxyethyl-thiamine diphosphate intermediate can expel a hydroxide ion forming an enol that rearranges to a bound acetyl group.

Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems

Propionate, a carbon substrate abundant in many prefermenters, has been shown in several previous studies to be a more favorable substrate than acetate for enhanced biological phosphorus removal (EBPR). The anaerobic metabolism of propionate by polyphosphate accumulating organisms (PAOs) is studied in this paper. A metabolic model is proposed to characterize the anaerobic biochemical transformations of propionate uptake by PAOs. The model is demonstrated to predict very well the experimental data from a PAO culture enriched in a laboratory-scale reactor with propionate as the sole carbon source. Quantitative fluorescence in-situ hybridization (FISH) analysis shows that Candidatus Accumulibacter phosphatis, the only identified PAO to date, constitute 63% of the bacterial population in this culture. Unlike the anaerobic metabolism of acetate by PAOs, which induces mainly poly-beta-hydroxybutyrate (PHB) production, the major fractions of poly-beta-hydroxyalkanoate (PHA) produced with propionate as the carbon source are poly-beta-hydroxyvalerate (PHV) and poly-beta-hydroxy-2-methylvalerate (PH2MV). PHA formation correlates very well with a selective (or nonrandom) condensation of acetyl-CoA and propionyl-CoA molecules. The maximum specific propionate uptake rate by PAOs found in this study is 0.18 C-mol/C-mol-biomass h, which is very similar to the maximum specific acetate uptake rate reported in literature. The energy required for transporting 1 carbon-mole of propionate across the PAO cell membrane is also determined to be similar to the transportation of 1 carbon-mole of acetate. Furthermore, the experimental results suggest that PAOs possess a similar preference toward acetate and propionate uptake on a carbon-mole basis. (c) 2005 Wiley Periodicals, Inc.

Cross-talk towards the response regulator NtrC controlling nitrogen metabolism in Rhodobacter capsulatus

Rhodobacter capsulatus NtrB/NtrC two-component regulatory system controls expression of genes involved in nitrogen metabolism including urease and nitrogen fixation genes. The ntrY-ntrX genes, which are located immediately downstream of the nifR3-ntrB-ntrC operon, code for a two-component system of unknown function. Transcription of ntrY starts within the ntrC-ntrY intergenic region as shown by primer extension analysis, but maximal transcription requires, in addition, the promoter of the nifR3-ntrB-ntrC operon. While ntrB and ntrY single mutant strains were able to grow with either urea or N-2 as sole nitrogen source, a ntrB/ntrY double mutant (like a ntrC-deficient strain) was no longer able to use urea or N-2. These findings suggest that the histidine kinases NtrB and NtrY can substitute for each other as phosphodonors towards the response regulator NtrC.

Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 ( unit-cell parameters a = b = 136.83, c = 99.82 angstrom, gamma = 120 degrees). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 angstrom resolution using the laboratory X-ray source and are suitable for crystal structure determination.

Paracetamol [acetaminophen]-induced gastrotoxicity: Revealed by induced hyperacidity in combination with acute or chronic inflammation

Paracetamol is regarded as a relatively safe drug in the gastro-duodenal region of humans but recent epidemiological investigations have suggested that at high doses there may be an increased risk of ulcers and bleeding. To investigate the possibility that inflammatory conditions and gastric acidity may play a role in potentiating development of gastric mucosal injury from paracetamol in rats (as noted previously with various non-steroidal anti-inflammatory drugs) we studied the gastric irritant effects of paracetamol and some phenolic and non-phenolic analgesics and antipyretics in rats with adjuvant or collagen II induced arthritis or zymosan-induced paw inflammation and given 1.0 ml hydrochloric acid (HCl) 0.1 M and/or an i. p. injection of the cholinomimetic, acetyl-β-methyl choline chloride 5.0 mg/kg. Gastric lesions were determined 2 h after oral administration of 100 or 250 mg/kg paracetamol or at therapeutically effective doses of the phenolic or non-phenolic analgesics/antipyretics. The results showed that gastric mucosal injury occurred with all these agents when given to animals that received all treatments so indicating there is an adverse synergy of these three factors, namely: (i) intrinsic disease; (ii) hyperacidity; and (iii) vagal stimulation for rapidly promoting gastric damage, both in the fundic as well as the antral mucosa, for producing gastric damage by paracetamol, as well as the other agents. Removing one of these three predisposing factors effectively blunts/abolishes expression of this paracetamol-induced gastrotoxity in rats. These three factors, without paracetamol, did not cause significant acute gastropathy.

Characterization of tumour necrosis factor alpha release by human granulocytes in response to procainamide challenge

The role of human granulocytes in the promotion of procainamide (PA) toxicity in vitro has been studied and one of the agents responsible for DNA strand scission and cell death in human target cells has been characterized. Crude peripheral blood mononuclear cells (cPBMNs) isolated by density centrifugation, and the lymphocyte cell lines--CCRF-HSB2 and WIL-2NS--were exposed to PA, and DNA strand breaks were quantified by fluorescent analysis of DNA unwinding. Therapeutic plasma concentrations of PA (0-50 microM) caused dose-dependent cytotoxicity, determined by dye exclusion, and strand breaks in cPBMNs incubated for 3 and 1.5 hr at 37 degrees, respectively. Using 50 microM PA a five-fold increase in DNA strand breaks was observed after 1.5 hr, with significant induction of strand breaks also being observed for 10 and 25 microM concentrations. Toxicity was much reduced in lymphocyte cell lines (maximal killing = 3.0% at 50 microM PA compared with 13.2% in cPBMNs). A similar decrease in toxicity was observed where N-acetyl procainamide (NAPA) was substituted for PA (less than 50% of strand breaks at all concentrations). Further investigations showed that the presence of a contaminating granulocyte population in the cPBMN fraction was responsible for the induction of PA toxicity. Incubation of a highly enriched granulocyte population with PA for 1 hr prior to exposure to purified peripheral blood mononuclear cells (pPBMNs) led to the complete restoration of the toxic effects. The resulting cyto- and genotoxicity were not significantly different to levels observed in cPBMNs. Significantly, incubation of granulocytes with NAPA did not induce toxicity in target pPBMNs. Ultrafiltration of granulocyte supernatants led to the identification of two toxic fractions of < 3000 and > 30,000 Da. Temporal studies showed that the toxicity associated with the < 3000 Da fraction appeared during the first 10-15 min incubation with PA whereas the > 30,000 Da fraction did not display significant toxicity until the 40-60 min period. Further assessment of the nature of these agents indicated that the 30,000 Da fraction was a protein. SDS-PAGE analysis showed an inducible 17,800 Da species appearing in granulocyte supernatants after 40 min incubation with PA. Dot blot analysis indicated that tumour necrosis factor alpha (TNF alpha) was present in the > 30,000 Da fraction. Evidence that TNF alpha was the high-molecular weight species responsible for PA-induced toxicity was obtained from neutralization assays employing an anti-TNF alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Ceramide induces a loss in cytosolic peroxide levels in mononuclear cells

Ceramide (a sphingolipid) and reactive oxygen species are each partly responsible for intracellular signal transduction in response to a variety of agents. It has been reported that ceramide and reactive oxygen species are intimately linked and show reciprocal regulation [Liu, Andreieu-Abadie, Levade, Zhang, Obeid and Hannun (1998) J. Biol. Chem. 273, 11313-11320]. Utilizing synthetic, short-chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide formation or using stimulation of CD95 to induce ceramide formation, we found that the principal redox-altering property of ceramide is to lower the [peroxide]cyt (cytosolic peroxide concentration). Apoptosis of Jurkat T-cells, primary resting and phytohaemagglutinin-activated human peripheral blood T-lymphocytes was preceded by a loss in [peroxide]cyt, as measured by the peroxide-sensitive probe 2′,7′-dichlorofluorescein diacetate (also reflected in a lower rate of superoxide dismutase-inhibitable cytochrome c reduction), and this was not associated with a loss of membrane integrity. Where growth arrest of U937 monocytes was observed without a loss of membrane integrity, the decrease in [peroxide]cyt was of a lower magnitude when compared with that preceding the onset of apoptosis in T-cells. Furthermore, decreasing the cytosolic peroxide level in U937 monocytes before the application of synthetic ceramide by pretreatment with either of the antioxidants N-acetyl cysteine or glutathione conferred apoptosis. However, N-acetyl cysteine or glutathione did not affect the kinetics or magnitude of ceramide-induced apoptosis of Jurkat T-cells. Therefore the primary redox effect of cellular ceramide accumulation is to lower the [peroxide]cyt of both primary and immortalized cells, the magnitude of which dictates the cellular response.

Synthetic ceramides inhibit CD36 expression in U937 cells through a reduction in cytosolic peroxide

Ceramide (a sphingolipid) and reactive oxygen species (ROS) are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. It has been reported that synthesis of ceramide and ROS are intimately linked, and show reciprocal regulation. The levels of ceramide are reported to be elevated in atherosclerotic plaques providing circumstantial evidence for a pro-atherogenic role for ceramide. Indeed, LDL may be important sources of ceramide from sphingomyelin, where it promotes LDL aggregation. Using synthetic, short chain ceramides to mimic the cellular responses to fluctuations in natural endogenous ceramides, we have investigated ceramide effects on both intracellular redox state (as glutathione and ROS) and redox-sensitive gene expression, specifically the scavenger receptor CD36 (using RT-PCR and flow cytometry), in U937 monocytes and macrophages. We describe that the principal redox altering properties of ceramide are to lower cytosolic peroxide and to increase mitochondrial ROS formation, where growth arrest of U937 monocytes is also observed. In addition, cellular glutathione was depleted, which was independent of an increase in glutathione peroxidase activity. Examination of the effects of ceramide on stress induced CD36 expression in macrophages, revealed a dose dependent reduction in CD36 mRNA and protein levels, which was mimicked by N-acetyl cysteine. Taken together, these data suggest that ceramides differentially affect ROS within different cellular compartments, and that loss of cytosolic peroxide inhibits expression of the redox sensitive gene, CD36. This may attenuate both the uptake of oxidised LDL and the interaction of HDL with macrophages. The resulting sequelae in vivo remain to be determined.

Modulation of neuronal network activity in the primary motor cortex

In the present study I investigated the mechanisms of modulation of neuronal network activity in rat primary motor cortex using pharmacological manipulations employing the in vitro brain slice technique. Preparation of the brain slice in sucrose-based aCSF produced slices with low viability. Introducing the neuroprotectants N-acetyl-cysteine, taurine and aminoguanidine to the preparatory method saw viability of slices increase significantly. Co-application of low dose kainic acid and carbachol consistently generated beta oscillatory activity in M1. Analyses indicated that network activity in M1 relied on the involvement of GABAA receptors. Dose-response experiments performed in M1 showed that beta activity can be modulated by benzodiazepine site ligands. Low doses of positive allosteric modulators consistently desynchronised beta oscillatory activity, a mechanism that may be driven by a1-subunit containing GABAA receptors. Higher doses increased the power of beta oscillatory activity. Whole-cell recordings in M1 uncovered three interneuronal subtypes regularly encountered in M1; Fast-spiking, regular-spiking non-Pyramidal and low threshold spiking. With the paradoxical effects of positive allosteric modulators in mind, subsequent voltage-clamp recordings in FS cells revealed a constitutively active tonic inhibitory current that could be modulated by zolpidem in two different ways. Low dose zolpidem increased the tonic inhibitory current in FS cells, consistent with the desynchronisation of network oscillatory activity seen at this concentration. High dose zolpidem decreased the inhibitory tonic current seen in FS cells, coinciding with an increase in oscillatory power. These studies indicate a fundamental role for a tonic inhibitory current in the modulation of network activity. Furthermore, desynchronisation of beta activity in M1 decreased as viability of the in vitro brain slice increased, suggesting that the extent of desynchronisation is dependent upon the pathophysiological state of the network. This indicates that low dose zolpidem could be used as a therapeutic agent specifically for the desynchronisation of pathological oscillations in oscillopathies such as Parkinson’s disease.