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Oxidative Medicine and Cellular... 2023Patients with severe asthma respond poorly to corticosteroids, and their care accounts for more than 60% of the total costs attributed to asthma. Neutrophils form...
OBJECTIVE
Patients with severe asthma respond poorly to corticosteroids, and their care accounts for more than 60% of the total costs attributed to asthma. Neutrophils form neutrophil extracellular traps (NETs), which play a crucial role in severe asthma. Statins have shown anti-inflammatory effects by reducing NETosis. In this study, we investigate if simvastatin can attenuate severe asthma by reducing NETosis and the underlying mechanism.
METHODS
Mice were concomitantly sensitized with ovalbumin (OVA), house dust mite (HDM), and lipopolysaccharide (LPS) during sensitization to establish a mouse model of severe asthma with neutrophil predominant inflammation (OVA+LPS mice) and treated with or without simvastatin. In inflammatory response, proportions of Th2, Th17, and Treg cells in lung tissue were detected by flow cytometry, and the levels of cytokines, dsDNA, and MPO-DNA in bronchoalveolar lavage fluid (BALF) were analyzed by ELISA. Citrullinated histone H3 (CitH3) and peptidyl arginine deiminase 4 (PAD4) in lung tissue were determined by Western blot and immunofluorescence imaging. PAD4 mRNA was determined by quantitative PCR (qPCR). HL-60 cells were differentiated into neutrophil-like cells by 1.25% DMSO. The neutrophil-like cells were treated with or without LPS, and simvastatin was then stimulated with PMA. CitH3 and PAD4 expressions were determined.
RESULTS
Sensitization with OVA, HDM, and LPS resulted in neutrophilic inflammation and the formation of NETs in the lungs. Simvastatin treatment reduced the inflammation score, cytokine levels, total cells, and neutrophil counts in the BALF and reduced proportions of Th2 and Th17 but increased Treg cells in lungs of OVA+LPS mice. Simvastatin-treated OVA+LPS mice show reduced NET formation in BALF and lung tissue compared to control mice. Adoptive transfer of neutrophils was sufficient to restore NETosis and neutrophilic inflammation in simvastatin-treated OVA+LPS mice. Simvastatin reduced PAD4 mRNA and protein expression in lung tissues and neutrophils isolated from lungs of OVA+LPS mice and consequent NET formation. In vitro, simvastatin reduced LPS-induced PAD4 upregulation and NETosis in HL-60-differentiated neutrophil-like cells. Furthermore, PAD4-overexpressed lentiviral transduction was sufficient to restore PAD4 protein expression and NETosis in simvastatin-treated HL-60-differentiated neutrophil-like cells.
CONCLUSIONS
Simvastatin reduces Th17-mediated neutrophilic inflammation and airway hyperreactivity by reducing PAD4 expression and inhibiting NETosis in a mouse model of severe asthma. Severe asthmatic patients with high levels of circulating NETs or sputum NETs may show improved responses to statin treatment.
Topics: Animals; Mice; Asthma; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; DNA; Histones; Inflammation; Lipopolysaccharides; Lung; Neutrophils; Ovalbumin; RNA, Messenger; Simvastatin; Extracellular Traps
PubMed: 36778209
DOI: 10.1155/2023/1493684 -
Journal of Nanobiotechnology Oct 2021Triple-negative breast cancer (TNBC), a management of aggressive breast cancer, remains an unmet medical challenge. Although a wave of efforts had spurred to design...
Triple-negative breast cancer (TNBC), a management of aggressive breast cancer, remains an unmet medical challenge. Although a wave of efforts had spurred to design novel therapeutic method of TNBC, unpredictable prognosis with lacking effective therapeutic targets along with the resistance to apoptosis seriously limited survival benefits. Ferroptosis is a non-apoptotic form of cell death that is induced by excessive lipid peroxidation, which provide an innovative way to combat cancer. Emerging evidence suggests that ferroptosis plays an important role in the treatment of TNBC cells. Herein, a novel ferroptosis nanomedicine was prepared by loading simvastatin (SIM), a ferroptosis drug, into zwitterionic polymer coated magnetic nanoparticles (FeO@PCBMA) to improve the therapeutic effect of TNBC. The as-obtained FeO@PCBMA-SIM nanoparticles demonstrated more cytotoxicity against MDA-MB-231 than MCF-7 due to the higher expression of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), which demonstrated that statins could effectively kill TNBC. Further experiments showed that SIM could inhibit the expression of HMGCR to downregulate the mevalonate (MVA) pathway and glutathione peroxidase 4 (GPX4), thereby inducing cancer cell ferroptosis. What's more, PCBMA endows FeO@PCBMA longer blood circulation performance to enhance their accumulation at tumor sites. Given that FeO have proven for clinical applications by the U.S. Food and Drug Administration (FDA) and SIM could induce cancer cell ferroptosis, the developed FeO@PCBMA-SIM nanosystem would have great potential in clinics for overcoming the drug resistance brought about by apoptotic drugs to cancer cells.
Topics: Animals; Cell Line, Tumor; Delayed-Action Preparations; Drug Carriers; Female; Ferroptosis; Humans; MCF-7 Cells; Magnetite Nanoparticles; Male; Mice, Nude; Signal Transduction; Simvastatin; Triple Negative Breast Neoplasms; Mice
PubMed: 34627266
DOI: 10.1186/s12951-021-01058-1 -
Hepatology Communications May 2022Patients with decompensated cirrhosis, particularly those with acute-on-chronic liver failure (ACLF), show profound alterations in plasma metabolomics. The aim of this... (Randomized Controlled Trial)
Randomized Controlled Trial
Patients with decompensated cirrhosis, particularly those with acute-on-chronic liver failure (ACLF), show profound alterations in plasma metabolomics. The aim of this study was to investigate the effect of treatment with simvastatin and rifaximin on plasma metabolites of patients with decompensated cirrhosis, specifically on compounds characteristic of the ACLF plasma metabolomic profile. Two cohorts of patients were investigated. The first was a descriptive cohort of patients with decompensated cirrhosis (n = 42), with and without ACLF. The second was an intervention cohort from the LIVERHOPE-SAFETY randomized, double-blind, placebo-controlled trial treated with simvastatin 20 mg/day plus rifaximin 1,200 mg/day (n = 12) or matching placebo (n = 13) for 3 months. Plasma samples were analyzed using ultrahigh performance liquid chromatography-tandem mass spectroscopy for plasma metabolomics characterization. ACLF was characterized by intense proteolysis and lipid alterations, specifically in pathways associated with inflammation and mitochondrial dysfunction, such as the tryptophan-kynurenine and carnitine beta-oxidation pathways. An ACLF-specific signature was identified. Treatment with simvastatin and rifaximin was associated with changes in 161 of 985 metabolites in comparison to treatment with placebo. A remarkable reduction in levels of metabolites from the tryptophan-kynurenine and carnitine pathways was found. Notably, 18 of the 32 metabolites of the ACLF signature were affected by the treatment. Conclusion: Treatment with simvastatin and rifaximin modulates some of the pathways that appear to be key in ACLF development. This study unveils some of the mechanisms involved in the effects of treatment with simvastatin and rifaximin in decompensated cirrhosis and sets the stage for the use of metabolomics to investigate new targeted therapies in cirrhosis to prevent ACLF development.
Topics: Acute-On-Chronic Liver Failure; Carnitine; Humans; Kynurenine; Liver Cirrhosis; Metabolomics; Rifaximin; Simvastatin; Tryptophan
PubMed: 34964311
DOI: 10.1002/hep4.1881 -
EBioMedicine Apr 2023Glioblastoma is one of the most devastating and incurable cancers due to its aggressive behaviour and lack of available therapies, being its overall-survival from...
BACKGROUND
Glioblastoma is one of the most devastating and incurable cancers due to its aggressive behaviour and lack of available therapies, being its overall-survival from diagnosis ∼14-months. Thus, identification of new therapeutic tools is urgently needed. Interestingly, metabolism-related drugs (e.g., metformin/statins) are emerging as efficient antitumour agents for several cancers. Herein, we evaluated the in vitro/in vivo effects of metformin and/or statins on key clinical/functional/molecular/signalling parameters in glioblastoma patients/cells.
METHODS
An exploratory-observational-randomized retrospective glioblastoma patient cohort (n = 85), human glioblastoma/non-tumour brain human cells (cell lines/patient-derived cell cultures), mouse astrocytes progenitor cell cultures, and a preclinical xenograft glioblastoma mouse model were used to measure key functional parameters, signalling-pathways and/or antitumour progression in response to metformin and/or simvastatin.
FINDINGS
Metformin and simvastatin exerted strong antitumour actions in glioblastoma cell cultures (i.e., proliferation/migration/tumoursphere/colony-formation/VEGF-secretion inhibition and apoptosis/senescence induction). Notably, their combination additively altered these functional parameters vs. individual treatments. These actions were mediated by the modulation of key oncogenic signalling-pathways (i.e., AKT/JAK-STAT/NF-κB/TGFβ-pathways). Interestingly, an enrichment analysis uncovered a TGFβ-pathway activation, together with AKT inactivation, in response to metformin + simvastatin combination, which might be linked to an induction of the senescence-state, the associated secretory-phenotype, and to the dysregulation of spliceosome components. Remarkably, the antitumour actions of metformin + simvastatin combination were also observed in vivo [i.e., association with longer overall-survival in human, and reduction in tumour-progression in a mouse model (reduced tumour-size/weight/mitosis-number, and increased apoptosis)].
INTERPRETATION
Altogether, metformin and simvastatin reduce aggressiveness features in glioblastomas, being this effect significantly more effective (in vitro/in vivo) when both drugs are combined, offering a clinically relevant opportunity that should be tested for their use in humans.
FUNDING
Spanish Ministry of Science, Innovation and Universities; Junta de Andalucía; CIBERobn (CIBER is an initiative of Instituto de Salud Carlos III, Spanish Ministry of Health, Social Services and Equality).
Topics: Humans; Mice; Animals; Metformin; Glioblastoma; Proto-Oncogene Proteins c-akt; Simvastatin; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Retrospective Studies; Transforming Growth Factor beta; Cell Line, Tumor; Cell Proliferation
PubMed: 36907105
DOI: 10.1016/j.ebiom.2023.104484 -
Acta Pharmaceutica (Zagreb, Croatia) Jun 2018Simvastatin is poorly bioavailable because it is practically insoluble in water and shows dissolution rate-limited absorption. Solubilizing effects of several... (Comparative Study)
Comparative Study
Simvastatin is poorly bioavailable because it is practically insoluble in water and shows dissolution rate-limited absorption. Solubilizing effects of several β-cyclodextrin (βCD) derivatives such as HPβCD, SBEβCD and DMβCD on simvastatin in aqueous solution were investigated using the phase solubility technique. The solubility diagram of simvastatin with each βCD derivative could be classified as AL-type, indicating soluble complex formation of 1:1 stoichiometry. Among the above βCD derivatives DMβCD was found to be the ideal complexing agent for improving drug solubility. The simvastatin complex with DMβCD was prepared using the co-evaporation method and was then characterized by differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FT-IR) and in vitro dissolution. Dissolution and pharmacokinetic studies indicated that the simvastatin/DMβCD complex exhibited an increased dissolution rate, rapid absorption, and improved bioavailability in rats compared to free drug. Maximum plasma concentration (cmax) and the time to reach it (tmax) were 21.86 μg mL-1 and 1.4 h for the drug complex, 8.25 μg mL-1 and 3.0 h for free drug, respectively. Main pharmacokinetic parameters such as tmax, cmax were significantly different (p < 0.01) between the simvastatin complex and free drug. Bioavailability of the simvastatin complex relative to free drug was up to 167.0 %.
Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; Biological Availability; Calorimetry, Differential Scanning; Chemistry, Pharmaceutical; Excipients; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Male; Rats; Rats, Wistar; Simvastatin; Solubility; Spectroscopy, Fourier Transform Infrared; beta-Cyclodextrins
PubMed: 29702479
DOI: 10.2478/acph-2018-0016 -
Cancer Science Feb 2023Multiple cancers have been reported to be associated with angiogenesis and are sensitive to anti-angiogenic therapies. Vascular normalization, by restoring proper tumor...
Multiple cancers have been reported to be associated with angiogenesis and are sensitive to anti-angiogenic therapies. Vascular normalization, by restoring proper tumor perfusion and oxygenation, could limit tumor cell invasiveness and improve the effectiveness of anticancer treatments. However, the underlying anticancer mechanisms of antiangiogenic drugs are still unknown. Metformin (MET) and simvastatin (SVA), two metabolic-related drugs, have been shown to play important roles in modulating the hypoxic tumor microenvironment and angiogenesis. Whether the combination of MET and SVA could exert a more effective antitumor effect than individual treatments has not been examined. The antitumor effect of the synergism of SVA and MET was detected in mouse models, breast cancer patient-derived organoids, and multiple tumor cell lines compared with untreated, SVA, or MET alone. RNA sequencing revealed that the combination of MET and SVA (but not MET or SVA alone) inhibited the expression of endothelin 1 (ET-1), an important regulator of angiogenesis and the hypoxia-related pathway. We demonstrate that the MET and SVA combination showed synergistic effects on inhibiting tumor cell proliferation, promoting apoptosis, alleviating hypoxia, decreasing angiogenesis, and increasing vessel normalization compared with the use of a single agent alone. The MET and SVA combination suppressed ET-1-induced hypoxia-inducible factor 1α expression by increasing prolyl hydroxylase 2 (PHD2) expression. Furthermore, the MET and SVA combination showed a more potent anticancer effect compared with bosentan. Together, our findings suggest the potential application of the MET and SVA combination in antitumor therapy.
Topics: Animals; Mice; Simvastatin; Metformin; Endothelin-1; Neovascularization, Pathologic; Cell Line, Tumor; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Neoplasms
PubMed: 36156330
DOI: 10.1111/cas.15602 -
British Journal of Clinical Pharmacology Sep 2004
Topics: Fees, Pharmaceutical; Humans; Nonprescription Drugs; Patient Participation; Safety; Simvastatin
PubMed: 15327581
DOI: 10.1111/j..2004.02191.x -
The New England Journal of Medicine Jun 2014Retrospective studies have shown that statins decrease the rate and severity of exacerbations, the rate of hospitalization, and mortality in chronic obstructive... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
Retrospective studies have shown that statins decrease the rate and severity of exacerbations, the rate of hospitalization, and mortality in chronic obstructive pulmonary disease (COPD). We prospectively studied the efficacy of simvastatin in preventing exacerbations in a large, multicenter, randomized trial.
METHODS
We designed the Prospective Randomized Placebo-Controlled Trial of Simvastatin in the Prevention of COPD Exacerbations (STATCOPE) as a randomized, controlled trial of simvastatin (at a daily dose of 40 mg) versus placebo, with annual exacerbation rates as the primary outcome. Patients were eligible if they were 40 to 80 years of age, had COPD (defined by a forced expiratory volume in 1 second [FEV1] of less than 80% and a ratio of FEV1 to forced vital capacity of less than 70%), and had a smoking history of 10 or more pack-years, were receiving supplemental oxygen or treatment with glucocorticoids or antibiotic agents, or had had an emergency department visit or hospitalization for COPD within the past year. Patients with diabetes or cardiovascular disease and those who were taking statins or who required statins on the basis of Adult Treatment Panel III criteria were excluded. Participants were treated from 12 to 36 months at 45 centers.
RESULTS
A total of 885 participants with COPD were enrolled for approximately 641 days; 44% of the patients were women. The patients had a mean (±SD) age of 62.2±8.4 years, an FEV1 that was 41.6±17.7% of the predicted value, and a smoking history of 50.6±27.4 pack-years. At the time of study closeout, the low-density lipoprotein cholesterol levels were lower in the simvastatin-treated patients than in those who received placebo. The mean number of exacerbations per person-year was similar in the simvastatin and placebo groups: 1.36±1.61 exacerbations and 1.39±1.73 exacerbations, respectively (P=0.54). The median number of days to the first exacerbation was also similar: 223 days (95% confidence interval [CI], 195 to 275) and 231 days (95% CI, 193 to 303), respectively (P=0.34). The number of nonfatal serious adverse events per person-year was similar, as well: 0.63 events with simvastatin and 0.62 events with placebo. There were 30 deaths in the placebo group and 28 in the simvastatin group (P=0.89).
CONCLUSIONS
Simvastatin at a daily dose of 40 mg did not affect exacerbation rates or the time to a first exacerbation in patients with COPD who were at high risk for exacerbations. (Funded by the National Heart, Lung, and Blood Institute and the Canadian Institutes of Health Research; STATCOPE ClinicalTrials.gov number, NCT01061671.).
Topics: Adult; Aged; Female; Forced Expiratory Volume; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lipids; Male; Middle Aged; Prospective Studies; Pulmonary Disease, Chronic Obstructive; Quality of Life; Severity of Illness Index; Simvastatin; Treatment Failure; Vital Capacity
PubMed: 24836125
DOI: 10.1056/NEJMoa1403086 -
The Cochrane Database of Systematic... Mar 2012Age-related macular degeneration (AMD) is a progressive late onset disorder of the macula affecting central vision. Age-related macular degeneration is the leading cause... (Review)
Review
BACKGROUND
Age-related macular degeneration (AMD) is a progressive late onset disorder of the macula affecting central vision. Age-related macular degeneration is the leading cause of blindness in people over 65 years in industrialized countries (Congdon 2003). Recent epidemiologic, genetic and pathological evidence has shown AMD shares a number of risk factors with atherosclerosis, leading to the hypothesis that statins may exert protective effects in AMD.
OBJECTIVES
To examine the effectiveness of statins compared with other treatments, no treatment, or placebo in delaying the onset and/or progression of AMD.
SEARCH METHODS
We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2011, Issue 9), MEDLINE (January 1950 to September 2011), EMBASE (January 1980 to September 2011), Latin American and Caribbean Health Sciences Literature Database (LILACS) (January 1982 to September 2011), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com), ClinicalTrials.gov (www.clinicaltrials.gov) and the WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). There were no date or language restrictions in the electronic searches for trials. The electronic databases were last searched on 16 September 2011.
SELECTION CRITERIA
We included randomized controlled trials (RCTs) that compared statins with other treatments, no treatment, or placebo in participants who were either susceptible to or diagnosed as having early stages of AMD.
DATA COLLECTION AND ANALYSIS
Two authors independently evaluated the search results against the selection criteria. Two Italian speaking colleagues extracted data. One author entered data. We did not perform a meta-analysis because only one completed RCT was identified.
MAIN RESULTS
Two studies met the selection criteria. One trial reported insufficient details to assess the risk of bias; the other trial is ongoing.Of the completed trial, the analyses of 30 participants did not show a statistically significant difference between the simvastatin and the placebo arm in visual acuity at three months of treatment (decimal visual acuity 0.21± 0.56 in simvastatin and 0.19± 0.40 in placebo arm) or 45 days after the completion of treatment (decimal visual acuity 0.20± 0.50 in simvastatin and 0.19± 0.48 in placebo arm). The lens and retina status were unchanged during and after the treatment period for both groups.Of the ongoing trial, the preliminary analyses of 42 participants who completed 12 months follow-up did not show a statistically significant difference between the simvastatin and the placebo arm in visual acuity, drusen score or visual function (effect estimates and confidence intervals were not available). We contacted the investigators and will update the review as data become available.
AUTHORS' CONCLUSIONS
Evidence from currently available RCTs was insufficient to conclude that statins have any role in preventing or delaying the onset or progression of AMD.
Topics: Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Macular Degeneration; Randomized Controlled Trials as Topic; Simvastatin
PubMed: 22419318
DOI: 10.1002/14651858.CD006927.pub3 -
The Cochrane Database of Systematic... Aug 2016Age-related macular degeneration (AMD) is a progressive, late-onset disorder of the macula affecting central vision. It is the leading cause of blindness in people over... (Review)
Review
BACKGROUND
Age-related macular degeneration (AMD) is a progressive, late-onset disorder of the macula affecting central vision. It is the leading cause of blindness in people over 65 years in industrialized countries. Recent epidemiologic, genetic, and pathological evidence has shown that AMD shares a number of risk factors with atherosclerosis, leading to the hypothesis that statins may exert protective effects in AMD.
OBJECTIVES
The objective of this review was to examine the effectiveness of statins compared with other treatments, no treatment, or placebo in delaying the onset and progression of AMD.
SEARCH METHODS
We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (which contains the Cochrane Eyes and Vision Trials Register) (2016, Issue 3), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to March 2016), EMBASE (January 1980 to March 2016), Latin American and Caribbean Health Sciences Literature Database (LILACS) (January 1982 to March 2016), PubMed (January 1946 to March 2016), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com) (last searched 5 June 2014), ClinicalTrials.gov (www.clinicaltrials.gov), and the WHO International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 31 March 2016.
SELECTION CRITERIA
We included randomized controlled trials (RCTs) and quasi-randomized trials that compared statins with other treatments, no treatment, or placebo in people who were diagnosed as having the early stages of AMD.
DATA COLLECTION AND ANALYSIS
We used standard methodological procedures expected by Cochrane. Two review authors independently evaluated the search results against the selection criteria, abstracted data, and assessed risk of bias. We did not perform meta-analysis due to heterogeneity in the interventions and outcomes between the included studies.
MAIN RESULTS
Two RCTs with a total of 144 participants met the selection criteria. Both trials compared simvastatin versus placebo in older people (older than 50 or 60 years) with high risk of developing AMD (drusen present on examination). Overall, we judged the quality of the evidence to be low, as we downgraded all outcomes due to limitations in the designs of the trials and insufficient outcome reporting. The larger trial, with 114 participants, was conducted in Australia and used a higher dose (40 mg daily) of simvastatin for three years. Participants and study personnel in this trial were adequately masked, however data were missing for 30% of participants at three years' follow-up. The smaller trial, with 30 participants, was conducted in Italy and used a lower dose (20 mg) of simvastatin for three months. This trial reported insufficient details to assess the risk of bias.Neither trial reported data for change in visual acuity. Low-quality evidence from the smaller trial, with 30 participants, did not show a statistically significant difference between the simvastatin and placebo groups in visual acuity values at three months of treatment (decimal visual acuity 0.21 ± 0.56 in simvastatin group and 0.19 ± 0.40 in placebo group) or 45 days after the completion of treatment (decimal visual acuity 0.20 ± 0.50 in simvastatin group and 0.19 ± 0.48 in placebo group). The lack of a difference in visual acuity was not explained by lens or retina status, which remained unchanged during and after the treatment period for both groups.Preliminary analyses of 42 participants who had completed 12 months' follow-up in the larger trial did not show a statistically significant difference between simvastatin and the placebo groups for visual acuity, drusen score, or visual function (effect estimates and confidence intervals were not available). Complete data for these outcomes at three years' follow-up were not reported. At three years, low-quality evidence showed an effect of simvastatin in slowing progression of AMD compared with placebo to be uncertain (odds ratio 0.51, 95% confidence interval 0.23 to 1.09).One trial did not report adverse outcomes. The second trial reported no difference between groups in terms of adverse events such as death, muscle aches, and acute hepatitis.
AUTHORS' CONCLUSIONS
Evidence from currently available RCTs is insufficient to conclude that statins have a role in preventing or delaying the onset or progression of AMD.
Topics: Female; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Macular Degeneration; Male; Middle Aged; Randomized Controlled Trials as Topic; Simvastatin; Visual Acuity
PubMed: 27490232
DOI: 10.1002/14651858.CD006927.pub5