Essentials In this crossover study the anticoagulant effects of rivaroxaban and apixaban were compared. Healthy volunteers received rivaroxaban 20 mg once daily or apixaban 5 mg twice daily. Rivaroxaban was associated with more prolonged inhibition of thrombin generation than apixaban. Rivaroxaban induced a clear prolongation of prothrombin time and activated partial thromboplastin time.Background The anticoagulant actions of the oral direct activated factor Xa inhibitors, rivaroxaban and apixaban, have not previously been directly compared. Objectives To compare directly the steady-state pharmacokinetics and anticoagulant effects of rivaroxaban and apixaban at doses approved for stroke prevention in patients with non-valvular atrial fibrillation. Methods Twenty-four healthy Caucasian male volunteers were included in this open-label, two-period crossover, phase 1 study (EudraCT number: 2015-002612-32). Volunteers were randomized to receive rivaroxaban 20 mg once daily or apixaban 5 mg twice daily for 7 days, followed by a washout period of at least 7 days before they received the other treatment. Plasma concentrations and anticoagulant effects were measured at steady state and after drug discontinuation. Results Overall exposure was similar for both drugs: the geometric mean area under the plasma concentration-time curve for the 0-24-h interval was 1830 μg h L-1 for rivaroxaban and 1860 μg h L-1 for apixaban. Rivaroxaban was associated with greater inhibition of endogenous thrombin potential (geometric mean area under the curve relative to baseline during the 0-24-h interval: 15.5 h versus 17.5 h) and a more pronounced maximal prolongation relative to baseline of prothrombin time (PT) (1.66-fold versus 1.14-fold) and activated partial thromboplastin time (APTT) (1.43-fold versus 1.16-fold) at steady state than apixaban. Conclusions Despite similar exposure to both drugs, rivaroxaban 20 mg once daily was associated with greater and more sustained inhibition of thrombin generation than apixaban 5 mg twice daily. Sensitive PT and APTT assays can be used to estimate the anticoagulant effects of rivaroxaban.
On a global scale, there is an increasing tendency for a more aggressive treatment of hypercholesterolemia. Minor effects of statins on brain cholesterol metabolism have been reported in some in vivo animal studies, and it seems that this is due to a local effect of the drug. We treated male mice of the inbred strain C57/BL6 with a high daily dose of lipophilic simvastatin (100 mg/kg b.wt.) or hydrophilic pravastatin (200 mg/kg b.wt.) or vehicle (controls) by oral gavage for 3 days. To compare the impact of both statins on brain cholesterol synthesis and degradation, levels of cholesterol, its precursor lathosterol, and its brain metabolite 24(S)-hydroxycholesterol as well as statin concentrations were determined in whole-brain lipid extracts using mass spectrometry. The expression of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase mRNA and of other target genes were evaluated using real-time reverse transcription-polymerase chain reaction. In addition, analysis of liver and serum samples was performed. Similar levels of simvastatin and pravastatin were detected in whole-brain homogenates. Cholesterol contents in the brain, liver, and serum were not affected by high-dose statin treatment. Whereas brain cholesterol precursor levels were reduced in simvastatin-treated animals only, no effect was observed on the formation of the brain cholesterol metabolite, 24(S)-hydroxycholesterol. Polymerase chain reaction analysis revealed that mRNA expression of HMG-CoA reductase and ATP-binding cassette transporter A1 in the brain was significantly up-regulated in simvastatin-treated animals compared with pravastatin-treated or control animals. We conclude that, under the present experimental conditions, brain cholesterol synthesis is significantly affected by short-term treatment with high doses of lipophilic simvastatin, whereas whole-brain cholesterol turnover is not disturbed.
A fast and sensitive method for the specific detection of E. coli and coliform bacteria in water samples based on the Fluorescence in situ Hybridization (FISH) technology was developed by transferring the standard slide-based FISH protocol to a filter membrane-based method. For detection and quantification two different strategies were implemented and tested: (1) the direct detection of single E. coli and coliform bacterial cells on the filter membranes (single cell protocol) and (2) the detection of micro-colonies after incubation of the filter membranes on a nutrient agar plate (micro-colony protocol). Both protocols were validated using drinking water samples spiked with pure cultures as well as naturally contaminated water samples including heat treated and disinfected water samples (chlorine, UV).
The single cell approach was shown to be very fast. Yet especially for low cell numbers the microscopic evaluation can become very labour-intensive. For routine application, an automated cell detection and quantification would be necessary which so far could not be implemented. Moreover, the approach showed some limitations with disinfected samples
Due to the incubation step the micro-colony approach is more time consuming but it yielded very good results for all different samples and conditions tested. This approach has the potential of practical use as only a low magnification factor is needed for microscopic evaluation, making even manual quantification feasible. In addition automated quantification can be implemented. Consequently this approach can be recommended as an alternative method to detect E. coli and coliform bacteria in water samples.
Presenilins (PS) are essential components of the γ-secretase complex and critically involved in the generation of the Alzheimer-associated amyloid β-peptide (Aβ). In addition to the β-amyloid precursor protein (βAPP), more than 30 type I membrane proteins are known to be cleaved by γ-secretase. Accordingly, γ-secretase is involved in the regulation of distinct biological functions, including signal transduction, protein trafficking, and cellular metabolism. Notably, γ-secretase has also been linked to membrane lipid metabolism. Levels of cholesterol and its precursors were determined by gas-liquid chromatography mass spectrometry (GC-MS). The expression of enzymes involved in biosynthesis of cholesterol was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Consistent with previous results, we also demonstrate that MEFs of PS deficient mice have elevated cholesterol concentrations as compared to that of wt mice. The pharmacologic inhibition of γ-secretase activity in wt MEFs by treatment with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) also led to increased cholesterol levels. On the other hand, expression of human PS1 in PS-deficient cells normalized cellular cholesterol levels, indicating that the observed alterations in cholesterol levels are dependent on PS-mediated γ-secretase activity. GC-MS analysis of lipid extracts from cultured cells revealed that levels of desmosterol, the immediate precursor of cholesterol, as well as that of several other precursors are also increased in PSdKO MEFs as compared to wt cells. The analysis of mRNA expression by RT-PCR revealed a selective up-regulation of critical enzymes involved in cholesterol biosynthesis. Our data indicate that PS proteins are involved in the cellular homeostasis of cholesterol levels by transcriptional regulation of enzymes involved in the biosynthetic.
Myopathy, probably caused by 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition in skeletal muscle, rarely occurs in patients taking statins. This study was designed to assess the effect of high-dose statin treatment on cholesterol and ubiquinone metabolism and mitochondrial function in human skeletal muscle.Forty-eight patients with hypercholesterolemia (33 men and 15 women) were randomly assigned to receive 80 mg/d of simvastatin (n = 16), 40 mg/d of atorvastatin (n = 16), or placebo (n = 16) for 8 weeks. Plasma samples and muscle biopsy specimens were obtained at baseline and at the end of the follow-up.The ratio of plasma lathosterol to cholesterol, a marker of endogenous cholesterol synthesis, decreased significantly by 66% in both statin groups. Muscle campesterol concentrations increased from 21.1 +/- 7.1 nmol/g to 41.2 +/- 27.0 nmol/g in the simvastatin group and from 22.6 +/- 8.6 nmol/g to 40.0 +/- 18.7 nmol/g in the atorvastatin group (P = .005, repeated-measurements ANOVA). The muscle ubiquinone concentration was reduced significantly from 39.7 +/- 13.6 nmol/g to 26.4 +/- 7.9 nmol/g (P = .031, repeated-measurements ANOVA) in the simvastatin group, but no reduction was observed in the atorvastatin or placebo group. Respiratory chain enzyme activities were assessed in 6 patients taking simvastatin with markedly reduced muscle ubiquinone and in matched subjects selected from the atorvastatin (n = 6) and placebo (n = 6) groups. Respiratory chain enzyme and citrate synthase activities were reduced in the patients taking simvastatin.High-dose statin treatment leads to changes in the skeletal muscle sterol metabolism. Furthermore, aggressive statin treatment may affect mitochondrial volume.
During the last years, several clinical studies have been published trying to elucidate the effect of statin treatment on amyloid precursor protein (APP) processing and metabolism of brain cholesterol in Alzheimer's disease (AD) in humans. We present an open biochemical study where 19 patients with AD have been treated with simvastatin (20 mg/day) for 12 months. The aim was to further investigate the effect of simvastatin treatment on cerebrospinal fluid (CSF) biomarkers of APP processing, AD biomarkers as total tau and tau phosphorylated at threonine 181, brain cholesterol metabolism as well as on cognitive decline in patients with AD. Despite biochemical data suggesting that treatment with 20 mg/day of simvastatin for 12 months does affect the brain cholesterol metabolism, we did not find any change in CSF or plasma levels of beta-amyloid (Abeta)(1-42). However, by analysis of APP isoforms, we found that statin treatment may favor the nonamyloidogenic pathway of APP processing. The relevance and mechanism between statin treatment and AD has to be further elucidated by using statins of different lipophility in different dosages over a longer period of time.
Presenilins (PS) are essential components of the γ–secretase complex and critically involved in the generation of the Alzheimer–associated amyloid β–peptide (Aβ). Aβ has recently been shown to inhibit hydroxymethylglutaryl–CoA reductase activity [Grimm et al., 2005, Nat Cell Biol. 11; 1118–23]. Accordingly, PS–deficient cells showed increased levels of cholesterol, providing a link between PS–dependent γ–secretase activity and cellular cholesterol biosynthesis. To investigate the role of PS proteins in cholesterol metabolism further, we analyzed the levels of cholesterol and its precursors as well as mRNA levels of enzymes involved in the biosynthetic pathway of cholesterol in mouse embryonic fibroblasts (MEFs) derived from wild–type (wt) and PS double knock–out (PSdKO) mice. Levels of cholesterol and its precursors in MEFs from wt and PSdKO mice were determined by gas–liquid chromatography mass spectrometry (GC–MS). The expression of enzymes involved in biosynthesis of cholesterol was analyzed by reverse transcription–polymerase chain reaction (RT–PCR). Consistent with previous results, we also demonstrate that MEFs of PS deficient mice have elevated cholesterol concentrations as compared to that of wt mice. The pharmacologic inhibition of γ–secretase activity in wt MEFs by treatment with N–[N–(3,5–difluorophenacetyl)–l–alanyl]–S–phenylglycine t–butyl ester (DAPT) also led to increased cholesterol levels. On the other hand, expression of human PS1 in PS–deficient cells normalized cellular cholesterol levels, indicating that the observed alterations in cholesterol levels are dependent on PS–mediated γ–secretase activity. GC–MS analysis of lipid extracts from cultured cells revealed that levels of desmosterol, the immediate precursor of cholesterol, as well as that of several other precursors are also increased in PSdKO MEFs as compared to wt cells. The analysis of mRNA expression by RT–PCR revealed a selective up–regulation of critical enzymes involved in cholesterol biosynthesis. Our data indicate that PS proteins are involved in the cellular homeostasis of cholesterol levels by transcriptional regulation of enzymes involved in the biosynthetic pathway. The molecular mechanisms that might be involved in the PS–dependent control of cholesterol biosynthesis will be discussed. This work is supported by the Deutsche Forschungsgemeinschaft
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