ABSTRACT
Antithrombotic trials in venous thromboembolism treatment and prevention, including those evaluating the new oral anticoagulants, have typically evaluated thromboembolism risk as an efficacy endpoint and bleeding risk as a separate safety endpoint. Findings often occur in opposition (i.e. decreased thromboembolism accompanied by increased bleeding, or vice-versa), leading to variable interpretation of the results, which may ultimately be judged as equivocal. In this paper, we offer an alternative to traditional designs based on the concept of a bivariate primary endpoint that accounts for simultaneous effects on antithrombotic efficacy and harm due to bleeding. We suggest a bivariate endpoint as a general approach to the assessment of 'net clinical benefit' in recently published trials and to the design of future trials. Lastly, we illustrate the bivariate endpoint design using two examples: a recently published superiority trial of rivaroxaban (RECORD1) and an ongoing non-inferiority trial of the duration of anticoagulant therapy in children with venous thrombosis (Kids-DOTT).
Subject(s)
Venous Thromboembolism/therapy , Administration, Oral , Anticoagulants/therapeutic use , Hemorrhage/prevention & control , Humans , Morpholines/therapeutic use , Randomized Controlled Trials as Topic , Research Design , Risk , Rivaroxaban , Thiophenes/therapeutic useABSTRACT
The gene transcript profile responses to metal oxide nanoparticles was studied using human cell lines derived from the colon and skin tumors. Much of the research on nanoparticle toxicology has focused on models of inhalation and intact skin exposure, and effects of ingestion exposure and application to diseased skin are relatively unknown. Powders of nominally nanosized SiO2, TiO2, ZnO and Fe2O3 were chosen because these substances are widely used in consumer products. The four oxides were evaluated using colon-derived cell lines, RKO and CaCo-2, and ZnO and TiO2 were evaluated further using skin-derived cell lines HaCaT and SK Mel-28. ZnO induced the most notable gene transcription changes, even though this material was applied at the lowest concentration. Nano-sized and conventional ZnO induced similar responses suggesting common mechanisms of action. The results showed neither a non-specific response pattern common to all substances nor synergy of the particles with TNF-α cotreatment. The response to ZnO was not consistent with a pronounced proinflammatory signature, but involved changes in metal metabolism, chaperonin proteins, and protein folding genes. This response was observed in all cell lines when ZnO was in contact with the human cells. When the cells were exposed to soluble Zn, the genes involved in metal metabolism were induced but the genes involved in protein refoldling were unaffected. This provides some of the first data on the effects of commercial metal oxide nanoparticles on human colon-derived and skin-derived cells.
Subject(s)
Cell Line/drug effects , Cell Line/physiology , Metal Nanoparticles/chemistry , Transcription, Genetic/drug effects , Zinc Oxide/chemistry , Zinc Oxide/pharmacology , Gene Expression/drug effects , Gene Expression Profiling , Humans , Metal Nanoparticles/ultrastructure , Microarray Analysis , Particle Size , Tumor Necrosis Factor-alpha/pharmacology , Zinc Oxide/metabolismABSTRACT
There is ongoing concern regarding the toxicity of nanoparticles with sizes less than 100 nm as compared to larger particles of the same nominal substance. Two commercial ZnO types, one sold as a 8-10 nm powder and the other described as -325 mesh (<44 mum) powder, were evaluated in human colon-derived RKO cells. The powders had a volume-to-surface area ratio equivalent to 40 and 330 nm spheres, respectively. Both materials formed micrometer-sized agglomerates in cell culture media. The nanosized ZnO was more cytotoxic than the micrometer-sized ZnO with LC(50) values of 15 +/- 1 and 29 +/- 4 mug/cm(2), respectively. Transfer of Zn from the solid phase to the cell culture media in the presence of RKO cells was time- and concentration-dependent. However, direct particle-cell contact was required for RKO cell cytotoxicity, and the toxicity of particles was independent of the amount of soluble Zn in the cell culture media. The mechanism of cell death includes the disruption of mitochondrial function. Robust markers of apoptosis, Annexin V staining, loss of mitochondrial potential, and increased generation of superoxide were observed when cells were treated with ZnO particulate matter but not when treated with comparable concentration of a soluble Zn salt. Both ZnO samples induced similar mechanisms of toxicity, but there was a statistically significant increase in potency per unit mass with the smaller particles.
Subject(s)
Colonic Neoplasms/pathology , Metal Nanoparticles/toxicity , Particulate Matter/toxicity , Zinc Oxide/toxicity , Annexin A5/metabolism , Apoptosis , Colonic Neoplasms/drug therapy , Humans , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Mitochondria/drug effects , Particle Size , Particulate Matter/chemistry , Superoxides/metabolism , Tumor Cells, Cultured , Zinc Oxide/chemistryABSTRACT
OBJECTIVES: The main goal was to develop new z-score reference ranges for common fetal echocardiographic measurements from a large referral population. METHODS: A retrospective cross-sectional study of 2735 fetuses was performed for standard biometry (biparietal diameter (BPD) and femoral diaphysis length (FDL)) and an assessment of menstrual age (MA). Standardized fetal echocardiographic measurements included aortic valve annulus and pulmonary valve annulus diameters at end-systole, right and left ventricular diameters at end-diastole, and cardiac circumference from a four-chamber view of the heart during end-diastole. Normal z-score ranges were developed for these echocardiographic measurements using MA, BPD and FDL as independent variables. This was accomplished by using first standard regression analysis and then weighted regression of absolute residual values for each parameter in order to adjust for inconstant variance. RESULTS: A simple, linear regression model was the best description of the data in each case and correlations between fetal cardiac measurements and the independent variables were excellent. There was significant heteroscedasticity of standard deviation with increasing gestational age, which also could be modeled with simple linear regression. After this adjustment, the residuals conformed to a normal distribution, validating the calculation and interpretation of z-scores. CONCLUSION: Development of reliable z-scores is possible for common fetal echocardiographic parameters by applying statistical methods that are based on a large sample size and weighted regression of absolute residuals in order to minimize the effect of heteroscedasticity. These normative ranges should be especially useful for the detection and monitoring of suspected fetal cardiac size and growth abnormalities.
Subject(s)
Fetal Diseases/diagnostic imaging , Fetal Heart/diagnostic imaging , Ultrasonography, Prenatal/methods , Adolescent , Adult , Biometry/methods , Cross-Sectional Studies , Female , Gestational Age , Humans , Linear Models , Population Groups , Pregnancy , Reference Values , Retrospective Studies , Ultrasonography, Prenatal/classification , Young AdultABSTRACT
Cultured lung cells provide an alternative to animal exposures for comparing the effects of different types of air pollution particles. Studies of particulate matter in vitro have reported proinflammatory cytokine signaling in response to many types of environmental particles, but there have been few studies comparing identical treatments in multiple cell types or identical cells with alternative cell culture protocols. We compared soil-derived, diesel, coal fly ash, titanium dioxide, and kaolin particles along with soluble vanadium and lipopolysaccharide, applied to airway-derived cells grown in submerged culture. Cell types included A549, BEAS-2B, RAW 264.7, and primary macrophages. The cell culture models (specific combinations of cell types and culture conditions) were reproducibly different in the cytokine signaling responses to the suite of treatments. Further, Interleukin-6 (IL-6) response to the treatments changed when the same cells, BEAS-2B, were grown in KGM versus LHC-9 media or in media containing bovine serum. The effect of changing media composition was reversible over multiple changes of media type. Other variables tested included culture well size and degree of confluence. The observation that sensitivity of a cell type to environmental agonists can be manipulated by modifying culture conditions suggests a novel approach for studying biochemical mechanisms of particle toxicity.
Subject(s)
Environmental Pollutants/toxicity , Interleukin-6/metabolism , Particulate Matter/toxicity , Animals , Cell Survival/drug effects , Cells, Cultured , Culture Media , Cytokines/biosynthesis , Dust , Environmental Pollutants/chemistry , Enzyme-Linked Immunosorbent Assay , Humans , Mice , Particulate Matter/chemistry , RatsABSTRACT
Two vital enzymes of the CYP3A subfamily, CYP3A4 and CYP3A5, are differentially expressed in the human lung. However, the molecular mechanisms that regulate tissue-selective expression of the genes are poorly understood. The ability of the 5' upstream promoter region of these two genes to drive luciferase reporter activities in human lung A549 cells was dramatically different. The CYP3A5 promoter region activated luciferase gene expression by 10-fold over the promoterless construct, whereas the CYP3A4 promoter did not drive expression. Sequence comparisons of the promoters identified a 57-base pair insertion in the CYP3A4 promoter region (-71 to -127) that was absent in the CYP3A5 promoter. Deletion of the 57-bp motif from CYP3A4 or insertion into the CYP3A5 promoter, showed that this motif represses CYP3A4 expression in lung. EMSA analysis using nuclear extracts from either A549 cells or human lung tissues showed two specific protein/DNA complexes formed with the (32)P-labeled CYP3A4 57-bp oligonucleotide. EMSA analyses identified two E-box motifs as the minimal specific cis-elements. Supershift assays with antibodies directed against known double- or single-E-box binding factors (TAL1, deltaEF1, E2A, HEB, etc.) failed to identify this factor as a previously characterized trans-acting double E-box binding protein. These results demonstrated that the 5'-upstream region of CYP3A4 contains an active putative double E-box repressor motif, not present in the 5'-upstream region of the CYP3A5 gene, that attenuates CYP3A4 expression in the human lung. We believe that this is the first documented case in which a cytochrome P450 gene is actively repressed in a tissue-specific manner.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , E-Box Elements/genetics , Gene Expression Regulation, Enzymologic , Lung/enzymology , Transcription Factors/metabolism , Base Sequence , Cell Line, Tumor , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Epithelial Cells/enzymology , Genes, Reporter , Humans , Luciferases/metabolism , Lung/cytology , Lung/pathology , Models, Biological , Molecular Sequence Data , Mutagenesis, Site-Directed , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription, Genetic , TransfectionABSTRACT
Lung-selective cytochrome P450 expression is well recognized; however, little is known regarding regulatory mechanisms. To address this knowledge gap, transient expression of CYP4B1/luciferase constructs was used to identify a proximal, positively acting regulatory element, position -139 to -45, that functioned in all cells examined; a negatively acting element, position -457 to -216, that only functioned in HepG2 hepatoblastoma cells; and a distal, positively acting element, position -1087 to -1008, that functioned in A549 or BEAS-2B lung-derived cells but not HepG2 cells or 293 kidney-derived cells. Competitive electrophoretic mobility shift assays further localized specific A549, but not HepG2, nuclear protein binding to two sites within the distal element, CYP4B1 position -1052 to -1042 and -1026 to -1008. Several potential lung-selective transcription factor recognition sequences were identified within these elements. However, attempts to identify specific factor(s) were unsuccessful. In contrast, in vitro DNA/protein binding assays combined with transient expression and mutagenesis studies identified two functional Sephadex protein/Krüppel-like factor families of transcription factor sites within the proximal element (position -118 to -114 and position -77 to -73) that bound both Sephadex protein 1 (Sp1) and Sephadex protein 3 (Sp3) in vitro. Furthermore, Sp1-dependent synergistic regulation was observed in A549 cells involving the proximal and distal regulatory elements. Chromatin immunoprecipitation assays demonstrated binding of neither Sp1 nor Sp3 to the CYP4B1 proximal element in human liver tissue, whereas selective Sp1 binding was observed in human lung tissue. Thus, the composite findings are consistent with both the proximal Sp1 elements and the distal regulatory element acting to synergistically control CYP4B1 lung-selective expression.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Genes, Regulator , Lung/metabolism , Sp1 Transcription Factor/metabolism , Aryl Hydrocarbon Hydroxylases/biosynthesis , Aryl Hydrocarbon Hydroxylases/genetics , Base Sequence , Binding Sites/genetics , Cell Line, Transformed , Cell Line, Tumor , DNA/chemistry , DNA/metabolism , Gene Expression Regulation , Genes, Reporter , Humans , Lung/enzymology , Molecular Sequence Data , Promoter Regions, Genetic , Sp1 Transcription Factor/genetics , TransfectionABSTRACT
Selective transcription of the human CYP2F1 gene in lung tissues may control the susceptibilities of this organ to diverse pneumotoxicants and lung carcinogens. However, the mechanisms responsible for CYP2F1 organ-selective transcription have not been elucidated. The objectives of the current studies were to identify and characterize basal transcription elements within the TATA-less promoter region of CYP2F1. Four putative Sp1-like sites were identified in the CYP2F1 promoter. Competitive electrophoretic mobility shift assay analysis with mutated oligonucleotide probes and lung A549 cell nuclear extract, along with supershift studies using antibodies to either Sp1 or Sp3 proteins, demonstrated that all four sites formed three specific protein-DNA complexes. Mutations in any of the four core Sp1-like motifs abolished protein-DNA binding. Western blot analysis of both human tissues and cells showed that Sp1 was considerably higher in lung than liver and that Sp3 was much higher in liver than lung. Promoter activation of a luciferase reporter construct was sequentially increased by addition of each of the four Sp1-like motifs in lung A549 cells but not in liver HepG2 cells. Cotransfection of a Sp1 expression vector with the reporter construct dramatically increased luciferase activity in either A549 cells or Sp1-deficient Drosophila Schneider line 2 (SL-2) cells. However, similar cotransfections with an Sp3 expression vector failed to increase activity. Cotransfection of both the Sp1 and Sp3 expression vectors considerably decreased Sp1-mediated activity in A549 cells and abolished activity in SL-2 cells. Thus, these studies demonstrated that four Sp1-dependent proximal promoter elements drive organ-selective CYP2F1 gene transcription, and that Sp1 and Sp3 factors interact to modulate constitutive CYP2F1 transcription in lung cells.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , DNA/chemistry , Gene Expression Regulation , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor/metabolism , Transcription, Genetic , Base Sequence , Binding Sites/genetics , Cell Line, Tumor , Cytochrome P-450 Enzyme System/genetics , Cytochrome P450 Family 2 , DNA/metabolism , Genes, Reporter , Humans , Luciferases/genetics , Lung , Molecular Sequence Data , Promoter Regions, Genetic , Repressor Proteins , Sp1 Transcription Factor/genetics , Sp3 Transcription Factor/genetics , Transcriptional Activation , TransfectionABSTRACT
The Tor kinases are the targets of the immunosuppressive drug rapamycin and couple nutrient availability to cell growth. In the budding yeast Saccharomyces cerevisiae, the PP2A-related phosphatase Sit4 together with its regulatory subunit Tap42 mediates several Tor signaling events. Sit4 interacts with other potential regulatory proteins known as the Saps. Deletion of the SAP or SIT4 genes confers increased sensitivity to rapamycin and defects in expression of subsets of Tor-regulated genes. Sap155, Sap185, or Sap190 can restore these responses. Strains lacking Sap185 and Sap190 are hypersensitive to rapamycin, and this sensitivity is Gcn2 dependent and correlated with a defect in translation, constitutive eukaryotic initiation factor 2alpha hyperphosphorylation, induction of GCN4 translation, and hypersensitivity to amino acid starvation. We conclude that Tor signals via Sap-Sit4 complexes to control both transcriptional and translational programs that couple cell growth to amino acid availability.
Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Phosphoprotein Phosphatases/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Biosynthesis/physiology , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction/physiology , Transcription, Genetic/physiology , Adaptor Proteins, Signal Transducing , Gene Expression Regulation/physiology , Genes, Reporter , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Kinases/metabolism , Protein Phosphatase 2 , Protein Serine-Threonine Kinases , Ribonucleoprotein, U2 Small Nuclear/genetics , Ribonucleoprotein, U2 Small Nuclear/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolismABSTRACT
The mechanism whereby cyclooxygenase-2 and its prostaglandin (PG) products are involved in colonic carcinogenesis is not fully understood. Prostacyclin (PGI(2)) is a major PG with antiapoptotic activity and is produced in the gastrointestinal tract. We reported previously that a human colorectal cancer (CRC) cell line, HCA-7, produces significant levels of PGE(2), PGD(2), thromboxane, and PGF(2alpha), but not PGI(2). We now report that human colonic fibroblast cell lines produce significant amounts of PGI(2) and that fibroblast lines derived from normal-appearing colonic mucosa of hereditary nonpolyposis CRC individuals produce 50-fold more PGI(2) than normal fibroblast lines derived from individuals with nonhereditary CRC. Coculture of HCA-7 cells with hereditary nonpolyposis CRC fibroblasts, but not normal fibroblasts, markedly reduced butyrate-induced apoptosis of HCA-7 cells. This antiapoptotic effect was inhibited by the cyclooxygenase-2 inhibitor rofecoxib and was restored by the stable PGI(2) analogue carbaprostacyclin. PGI(2) binds either G protein-coupled cell surface PGI(2) receptor or the nuclear peroxisome proliferator-activated receptor (PPAR) delta. PPAR delta likely mediates this antiapoptotic effect because HCA-7 cells express this receptor, and another PPAR delta agonist, docosahexaenoic acid, mimics the effect. We propose a novel mechanism by which stromal production of PGI(2) promotes survival of colonocytes through PPAR delta activation. This mechanism may have relevance to maintenance of cells in the normal crypt and to clonal expansion of mutant colonocytes during tumorigenesis.
Subject(s)
Apoptosis/physiology , Colon/cytology , Colon/metabolism , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Epoprostenol/biosynthesis , Coculture Techniques , Colorectal Neoplasms, Hereditary Nonpolyposis/metabolism , Colorectal Neoplasms, Hereditary Nonpolyposis/pathology , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Epithelial Cells/metabolism , Epoprostenol/analogs & derivatives , Humans , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Lactones/pharmacology , Membrane Proteins , Prostaglandin-Endoperoxide Synthases/metabolism , Stromal Cells/metabolism , Sulfones , Tumor Cells, CulturedABSTRACT
Cholinesterase (ChE) inhibition represents the most efficacious treatment approach for Alzheimer's disease (AD) to date. This multiple-dose study has examined the relationship between inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in the cerebrospinal fluid (CSF) and cognitive change (measured by the Computerised Neuropsychological Test Battery [CNTB]) following administration of the ChE inhibitor, rivastigmine (Exelon). In 18 patients with mild to moderate AD, CNTB scores, activities of AChE and BuChE in the CSF, and plasma BuChE activity were determined prior to treatment with rivastigmine. Doses of rivastigmine were then titrated (1 mg b.i.d./week) to final doses of 1, 2, 3, 4, 5 or 6 mg b.i.d. (n = 3 per dose). Following treatment with the target dose of rivastigmine for at least 3 days, CNTB scores were re-determined. CSF samples were continuously collected together with plasma samples prior to and for 12 hours after the final dose of rivastigmine, and AChE and BuChE activities determined.AChE in CSF and BuChE in plasma were dose-dependently inhibited by rivastigmine treatment. The inhibition of BuChE in CSF was not clearly dose-dependent. A statistically significant correlation was observed between the change in CNTB summary score and inhibition of AChE activity (r = -0.56, p < 0.05) and BuChE activity (r = -0.65, p < 0.01) in CSF. Improvement in speed-, attention- and memory-related subtests of the CNTB correlated significantly with inhibition of BuChE but not AChE activity in CSF. Weak or absent correlation with change in cognitive performance was noted for inhibition of plasma BuChE. These results indicate that cognitive improvement with rivastigmine in AD is associated with central inhibition of ChEs and support a role for central BuChE in addition to AChE inhibition in modulating cholinergic function in AD.
Subject(s)
Acetylcholinesterase/cerebrospinal fluid , Alzheimer Disease/cerebrospinal fluid , Alzheimer Disease/drug therapy , Butyrylcholinesterase/cerebrospinal fluid , Carbamates/therapeutic use , Cholinesterase Inhibitors/therapeutic use , Cognition/drug effects , Phenylcarbamates , Acetylcholinesterase/blood , Aged , Aged, 80 and over , Alzheimer Disease/blood , Alzheimer Disease/psychology , Butyrylcholinesterase/blood , Cholinesterase Inhibitors/administration & dosage , Dose-Response Relationship, Drug , Female , Humans , Male , Middle Aged , Neuropsychological Tests , RivastigmineABSTRACT
Sibutramine is a serotonin-noradrenaline reuptake inhibitor that is effective for long-term weight reduction and maintenance in obese patients when used as an adjunct to dietary and behavioural measures. Because the inhibition of noradrenaline reuptake may be expected to increase systolic and diastolic blood pressure (SBP and DBP) and pulse rate (PR), a 12-week multi-centre, placebo-controlled, double-blind study was designed to evaluate the efficacy and tolerability of sibutramine for weight loss in obese patients whose hypertension was well controlled (DBP < or = 95 mm Hg) by beta-adrenergic blocking agents (beta-blockers), with or without concomitant thiazide diuretics. Of the 61 patients randomised to sibutramine 20 mg once daily or placebo, 55 patients (90%) completed the study. After 12 weeks, sibutramine-treated patients lost significantly more weight than placebo-treated patients: mean weight reductions were 4.2 kg (4.5%) in the sibutramine group vs 0.3 kg (0.4%) in the placebo group (P<0.001). Greater weight reduction on sibutramine was accompanied by trends for greater mean reductions in serum triglycerides and very low density lipoprotein cholesterol. Sibutramine was well tolerated, and most adverse events were mild or moderate in severity. No sibutramine patient discontinued treatment because of an adverse event. Mean supine and standing DBP and SBP were not statistically significantly different between the sibutramine group and the placebo group at any post-baseline visit during the 12-week trial. At week 12, mean increases from baseline supine SBP and DBP, respectively, were 1.6 and 1.7 mm Hg for the sibutramine group vs increases of 0.4 and 1.3 mm Hg for the placebo group. At week 12, mean increases from baseline standing SBP and DBP, respectively, were 1.5 and 1.8 mm Hg for the sibutramine group vs an increase of 0.3 and a decrease of 0.8 mm Hg for the placebo group (P > 0.05 for treatment comparison). A statistically significant mean increase of 5.6 bpm (+/-8.25, s.d.) in supine PR from a baseline of 62 bpm was reported in sibutramine-treated patients at week 12, whereas placebo-treated patients had a mean supine PR decrease of 2.2 bpm (+/-6.43) (P < 0.001). In summary, sibutramine was well tolerated and effective in weight reduction. The addition of sibutramine did not result in an increase in BP in obese patients whose hypertension was well controlled by a beta-blocker. However, based on the potential for changes in BP and PR, obese patients being treated with sibutramine should be monitored periodically for changes in BP and PR and managed appropriately.
Subject(s)
Adrenergic beta-Antagonists/therapeutic use , Appetite Depressants/adverse effects , Appetite Depressants/therapeutic use , Blood Pressure/drug effects , Blood Pressure/physiology , Cyclobutanes/adverse effects , Cyclobutanes/therapeutic use , Heart Rate/drug effects , Heart Rate/physiology , Hypertension/drug therapy , Hypertension/physiopathology , Obesity/drug therapy , Obesity/physiopathology , Weight Loss/drug effects , Weight Loss/physiology , Adult , Aged , Benzothiadiazines , Diuretics , Double-Blind Method , Drug Therapy, Combination , Female , Humans , Hypertension/complications , Male , Middle Aged , Obesity/complications , Sodium Chloride Symporter Inhibitors/therapeutic useABSTRACT
Rapamycin binds and inhibits the Tor protein kinases, which function in a nutrient-sensing signal transduction pathway that has been conserved from the yeast Saccharomyces cerevisiae to humans. In yeast cells, the Tor pathway has been implicated in regulating cellular responses to nutrients, including proliferation, translation, transcription, autophagy, and ribosome biogenesis. We report here that rapamycin inhibits pseudohyphal filamentous differentiation of S. cerevisiae in response to nitrogen limitation. Overexpression of Tap42, a protein phosphatase regulatory subunit, restored pseudohyphal growth in cells exposed to rapamycin. The tap42-11 mutation compromised pseudohyphal differentiation and rendered it resistant to rapamycin. Cells lacking the Tap42-regulated protein phosphatase Sit4 exhibited a pseudohyphal growth defect and were markedly hypersensitive to rapamycin. Mutations in other Tap42-regulated phosphatases had no effect on pseudohyphal differentiation. Our findings support a model in which pseudohyphal differentiation is controlled by a nutrient-sensing pathway involving the Tor protein kinases and the Tap42-Sit4 protein phosphatase. Activation of the MAP kinase or cAMP pathways, or mutation of the Sok2 repressor, restored filamentation in rapamycin treated cells, supporting models in which the Tor pathway acts in parallel with these known pathways. Filamentous differentiation of diverse fungi was also blocked by rapamycin, demonstrating that the Tor signaling cascade plays a conserved role in regulating filamentous differentiation in response to nutrients.
Subject(s)
Cell Differentiation , Drosophila Proteins , Nitrogen/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Signal Transduction , Actin Cytoskeleton/metabolism , Adaptor Proteins, Signal Transducing , Cell Differentiation/drug effects , Cell Division , Cyclic AMP-Dependent Protein Kinases/metabolism , Fungal Proteins/metabolism , Hyphae/drug effects , Hyphae/genetics , Hyphae/growth & development , Mitogen-Activated Protein Kinases/metabolism , Models, Biological , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 2 , Repressor Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/metabolism , Sirolimus/pharmacologyABSTRACT
Rivastigmine is a cholinersterase inhibitor approved recently for the treatment of Alzheimer's disease (AD). The objective of this study is to characterize the pharmacokinetics-pharmacodynamics of rivastigmine in patients with AD. Eighteen AD patients received doses ranging from 1 to 6 mg bid for about 11 weeks. Rivastigmine and its active (major) metabolite (ZNS 114-666, also called NAP 226-90), plasma, and cerebrospinal fluid (CSF) concentrations were determined together with the AChE activity and computerized neuropsychological test battery (CNTB) scores. Nonlinear mixed-effects modeling of pharmacokinetic and pharmacodynamic data was conducted using NONMEM. Rivastigmine and its metabolite exhibited dose-disproportional pharmacokinetics. The apparent clearance and volume of distribution (plasma) of rivastigmine were estimated to be 120 L/h and 236 L, respectively. The relative bioavailability at the 6 mg dose was about 140%. The metabolite had a clearance of about 100 L/h and a volume of distribution of 256 L. The kinetics of the parent and metabolite in CSF showed an equilibration half-life of about 0.2 and 0.5 hours, respectively. The metabolite levels in CSF correlated very well with the acetylcholinesterase inhibition, with a ZNS 114-666 concentration of about 5.4 microg/L required for half-maximal inhibition of acetylcholinesterase activity. No statistically significant correlation of the CNTB scores with enzyme inhibition, parent or metabolite concentration (plasma/CSF), or rivastigmine dose could be established. The PK-PD model presented in this study can provide valuable information to optimize the drug development of rivastigmine and other related compounds and also in rationalizing dosing recommendations.
Subject(s)
Alzheimer Disease/metabolism , Carbamates/pharmacokinetics , Cholinesterase Inhibitors/pharmacokinetics , Models, Biological , Phenylcarbamates , Acetylcholinesterase/cerebrospinal fluid , Aged , Alzheimer Disease/blood , Alzheimer Disease/cerebrospinal fluid , Alzheimer Disease/enzymology , Carbamates/blood , Carbamates/cerebrospinal fluid , Cholinesterase Inhibitors/blood , Cholinesterase Inhibitors/cerebrospinal fluid , Female , Humans , Male , Middle Aged , Neuropsychological Tests/statistics & numerical data , Patients/statistics & numerical data , RivastigmineABSTRACT
In this randomized, double-blind, placebo-controlled, parallel-group study, patients received a single 50-mg oral dose of a 5-HT(1D) agonist, PNU-142633 (n = 34), or matching placebo (n = 35) during an acute migraine attack. No statistically significant treatment effects were observed at 1 and 2 h after dosing, even after stratifying by baseline headache intensity. At 1 and 2 h post-dose, 8.8% and 29.4% of the PNU-142633 group, respectively, and 8.6% and 40.0% of the placebo group, respectively, experienced headache relief; 2.9% and 8.8% of the PNU-142633 group and 0% and 5.7% of the placebo group were free of headache pain. Adverse events associated with PNU-142633 treatment included chest pain (two patients) and QTc prolongation (three patients). Results from this study suggest that anti-migraine efficacy is not mediated solely through the 5-HT(1D) receptor subtype, although this receptor may contribute, at least in part, to the adverse cardiovascular effects observed with 5-HT agonist medications.
Subject(s)
Chromans/adverse effects , Chromans/therapeutic use , Migraine Disorders/drug therapy , Receptors, Serotonin , Serotonin Receptor Agonists/adverse effects , Serotonin Receptor Agonists/therapeutic use , Acute Disease , Adolescent , Adult , Double-Blind Method , Female , Humans , Male , Middle Aged , Receptor, Serotonin, 5-HT1D , Receptors, Serotonin/physiologyABSTRACT
The current approach to antimigraine therapy comprises potent serotonin 5-HT1B/1D receptor agonists collectively termed triptans. Sumatriptan was the first of these compounds to be developed, and offered improved efficacy and tolerability over ergot-derived compounds. The development of sumatriptan was quickly followed by a number of 'second generation' triptan compounds, characterised by improved pharmacokinetic properties and/or tolerability profiles. Triptans are believed to effect migraine relief by binding to serotonin (5-hydroxy-tryptamine) receptors in the brain, where they act to induce vasoconstriction of extracerebral blood vessels and also reduce neurogenic inflammation. Although the pharmacological mechanism of the triptans is similar, their pharmacokinetic properties are distinct. For example, bioavailability of oral formulations ranges between 14% (sumatriptan) and 74% (naratriptan), and their elimination half-life ranges from 2 hours (sumatriptan and rizatriptan) to 25 hours (frovatriptan). Clearly, such diverse pharmacokinetic properties will influence the effectiveness of the compounds and favour the prescription of one over another in different patient populations. This article reviews the pharmacological properties of the triptans (time to peak plasma concentration, half-life, bioavailability and receptor binding) and relates these properties to efficacy and time of onset. It also considers the effects of concomitant medication, food, age and disease on the pharmacokinetics of the compounds. In addition, the relative merits, such as headache recurrence, tolerability and route of administration, are discussed. Finally, the performance of the triptans is considered in the context of direct head-to-head comparative trials that have assessed the efficacy profile of the compounds.
Subject(s)
Migraine Disorders/metabolism , Serotonin Receptor Agonists/pharmacology , Serotonin Receptor Agonists/pharmacokinetics , Animals , Humans , Migraine Disorders/drug therapy , Serotonin Receptor Agonists/therapeutic useABSTRACT
Mild cognitive impairment (MCI) is a term used to describe memory decline or other specific cognitive impairment in individuals who do not have dementia or significant impairment of other cognitive functions beyond that expected for their age or education. It has been suggested that as much as 38% of the elderly population would meet criteria for MCI and although the associated memory deficits are mild, the fact that up to 15% of MCI patients, particularly those with a particular type of memory impairment, convert to Alzheimer's disease (AD) annually has prompted serious attention. Despite the high conversion rate, MCI cannot be used synonymously with early or mild AD, as patients with AD are impaired not only in memory performance but in other cognitive domains as well; they meet diagnostic criteria for dementia. However, since there is a high conversion rate from MCI to AD, it is likely many with MCI have the underlying neuropathology of AD, though they do not yet meet clinical diagnostic criteria. Therefore, treatment strategies developed for AD, specifically acetylcholinesterase inhibitors and Cox-2 inhibitors, have been among the first employed to treat MCI. It is hoped that by impeding the progression of MCI in this manner, fewer patients will convert to AD. This article will give a brief overview of the condition of mild cognitive impairment and an account of trial methodology and current treatment strategies being employed for MCI.
Subject(s)
Cognition Disorders/drug therapy , Anti-Inflammatory Agents/therapeutic use , Antioxidants/therapeutic use , Cholinesterase Inhibitors/therapeutic use , Clinical Trials as Topic , Free Radical Scavengers/therapeutic use , Humans , Research DesignABSTRACT
In clinical trials of dopamine-blocking antipsychotics, significant adverse events may occur in healthy volunteers at dose levels that are well tolerated by schizophrenic patients. Because of these differences in tolerability, bioequivalence and pharmacokinetic studies of antipsychotics should be performed in schizophrenic patients rather than in healthy volunteers. When clozapine is the drug being investigated, pharmacokinetic and bioequivalence studies should be carried out in real-life dosage conditions because the half-life of clozapine increases with multiple doses. Under real-life conditions, the evaluation of multiple doses of clozapine in a population of schizophrenic patients can provide direct therapeutic relevance to bioavailability findings. This article discusses patient recruitment and informed consent in pharmacokinetic trials of schizophrenia, issues in studying antipsychotic agents in healthy volunteers versus schizophrenic patients, and a bioequivalency study of Clozaril (Novartis Pharmaceuticals) and generic clozapine (Creighton [Sandoz]) in schizophrenic patients.