BackgroundSupport groups for people with Implantable Cardioverter Defibrillators (ICDs) are widely used, however, it is not clear what people with ICDs gain from a support group or what format they should take.ObjectivesThe aim of the present study is to define the perceived benefit of ICD support groups and develop practical recommendations for group format.Methods14 individuals with ICDs were interviewed using a semi-structured interview guide. Reflexive thematic analysis methods were utilised to code and analyse the transcripts before generating themes.ResultsFour themes were defined: confronting mortality, coping through sharing, coping through learning, and providing space. Making connections with other people with ICDs, reassurance, access to information, and advice from health care professionals were important perceived benefits of the support group.ConclusionPeople with ICDs may have to confront their own mortality and adapt to considerable life changes after implant. The findings from the present study have improved understanding of how support groups are perceived and how ICD indication and group format influence the experience. A blended format of in-person community meetings, online forums, HCP-led education and space for person-person interaction is recommended. Importantly, provision of support should not be time-limited to allow people to access it when it most likely to be of benefit to them.

Background Patients with implantable cardioverter defibrillators (ICD) experience anxiety, depression and reduced quality of life (QoL). Objectives This mixed-methods systematic review evaluates whether ICD support groups have a beneficial effect on mental well-being. Methods Literature searches were carried out in MEDLINE, Embase, CINAHL, PsycINFO and Web of Science. Eligible studies investigated patient-led support groups for ICD patients aged 18 years or older, using any quantitative or qualitative design. The Mixed-Methods Assessment Tool was used to assess quality. Meta-analysis of measures of mental well-being was conducted. Thematic synthesis was used to generate analytic themes from the qualitative data. The data were integrated and presented using the Pillar Integration Process. Results Ten studies were included in this review. All studies bar one were non-randomised or had a qualitative design and patients had self-selected to attend a support group. Five contributed to the quantitative data synthesis and seven to the qualitative synthesis. Meta-analysis of anxiety and QoL measures showed no significant impact of support groups on mental well-being, but qualitative data showed that patients perceived benefit from attendance through sharing experiences and acceptance of life with an ICD. Discussion ICD support group attendance improved the patients’ perceived well-being. Attendees value the opportunity to share their experiences which helps to accept their new life with an ICD. Future research could consider outcomes such as patient acceptance and the role of healthcare professionals at support groups.

Nanomaterials have a huge potential to revolutionize the way in which we diagnose and treat disease as tools in biomedical research. This is due to their unique properties and characteristics accredited to the way in which they are fabricated, functionalized, and characterized. However, there is limited focus on the use of silica nanoparticles (SiNPs); in particular, the various methods in which they are used, the assessment of their stability and biosafety, and the strategies used to improve their biocompatibility. This chapter focuses on the potential use of SiNPs as diagnostic tools and therapeutic intervention modalities and provides an overview of the methods used to fabricate and characterize them. The chapter also highlights the strategies used to maximize the biocompatibility of SiNPs to improve their applicability and bioavailability, minimize their off-target localization and effects, and promote their guided delivery.

Nanoparticles are a well-recognized platform for imaging diagnostics and therapeutic intervention where several formulations have gained clinical approval in the treatment of diseases such as cancer. Drug-loaded nanoparticles can be directly administered into the blood stream (usually by intravenous injection), or indirectly by inhalation or ingestion, where the vasculature becomes a key exposure site. Hence, the vascular endothelial layer lining blood vessels have gained attention as a target for the treatment of diseases associated with inflammation and oxidative stress, including cardiovascular disease (CVD). This chapter focuses on current understanding of nanoparticle interactions with cells of the vessel wall, in particular endothelial cells and how these can be targeted by modifying nanoparticle surfaces via conjugation with tagging molecules that recognize surface endothelial determinants. Understanding the cellular interactions will inform the developmental approach of nanoparticles as site-specific delivery modalities toward therapeutic intervention in CVD.

A significant number of patients with severe cardiovascular disease, undergoing coronary artery bypass grafting (CABG), present with hypertension. While internal mammary arteries (IMAs) may be a better alternative to vein grafts, their impaired vasodilator function affects their patency. Our objectives were to (1) determine if inhibition of the cytochrome P450 enzyme CYP1B1, using liposome-encapsulated 2,3′,4,5′-tetramethoxystilbene (TMS), can potentiate vasodilation of IMAs from CABG patients, and (2) assess mechanisms involved using coronary arteries from normal rats, in an ex vivo model of hypertension. PEGylated liposomes were synthesized and loaded with TMS (mean diameter 141 ± 0.9 nm). Liposomal delivery of TMS improved its bioavailability Compared to TMS solution (0.129 ± 0.02 ng/mL vs. 0.086 ± 0.01 ng/mL at 4 h; p < 0.05). TMS-loaded liposomes alleviated attenuated endothelial-dependent acetylcholine (ACh)-induced dilation in diseased IMAs (@ACh 10−4 M: 56.9 ± 5.1%; n = 8 vs. 12.7 ± 7.8%; n = 6; p < 0.01) for TMS-loaded liposomes vs. blank liposomes, respectively. The alleviation in dilation may be due to the potent inhibition of CYP1B1 by TMS, and subsequent reduction in reactive oxygen species (ROS) moieties and stimulation of nitric oxide synthesis. In isolated rat coronary arteries exposed to a hypertensive environment, TMS-loaded liposomes potentiated nitric oxide and endothelium-derived hyperpolarization pathways via AMPK. Our findings are promising for the future development of TMS-loaded liposomes as a promising therapeutic strategy to enhance TMS bioavailability and potentiate vasodilator function in hypertension, with relevance for early and long-term treatment of CABG patients, via the sustained and localized TMS release within IMAs.

Nanostructured lipid carriers (NLCs) are an emerging drug delivery platform for improved drug stability and the bioavailability of antihypertensive drugs and vasoprotective nutraceutical compounds, such as resveratrol (RV). The objective of this study was to ascertain NLCs’ potential to deliver RV and restore attenuated dilator function, using an ex vivo model of acute hypertension. Trimyristin–triolein NLCs were synthesized and loaded with RV. The uptake of RV-NLCs by human coronary artery endothelial cells (HCAECs) maintained their viability and reduced both mitochondrial and cytosolic superoxide levels. Acute pressure elevation in isolated coronary arteries significantly attenuated endothelial-dependent dilator responses, which were reversed following incubation in RV-NLCs, superoxide dismutase or apocynin (p < 0.0001). RV-NLCs demonstrated a five-fold increase in potency in comparison to RV solution. At elevated pressure, in the presence of RV-NLCs, incubation with Nω-nitro-l-arginine (L-NNA) or indomethacin resulted in a significant reduction in the restored dilator component (p < 0.0001), whereas apamin and TRAM-34 had no overall effect. Incubation with the adenosine monophosphate-activated protein kinase (AMPK) inhibitor dorsomorphin significantly attenuated dilator responses (p < 0.001), whereas the SIRT-1 inhibitor EX-527 had no effect. RV-NLCs improved the impaired endothelial-dependent dilation of small coronary arteries, following acute pressure elevation, via NO and downstream COX elements, mediated by AMPK. We suggest that RV-NLCs are an effective delivery modality for improved potency and sustained drug release into the vasculature. Our findings have important implications for the future design and implementation of antihypertensive treatment strategies.

BACKGROUND:Home-based interventions might facilitate the lifelong uptake of a physically active lifestyle following completion of a supervised phase II exercise-based cardiac rehabilitation. Yet, data on the long-term effectiveness of home-based exercise training on physical activity and exercise capacity are scarce.OBJECTIVE:The purpose of the TeleRehabilitation in Coronary Heart disease (TRiCH) study was to compare the long-term effects of a short home-based phase III exercise programme with telemonitoring guidance to a prolonged centre-based phase III programme in coronary artery disease patients. The primary outcome was exercise capacity. Secondary outcomes included physical activity behaviour, cardiovascular risk profile and health-related quality of life.METHODS:Ninety coronary artery disease patients (80 men) were randomly assigned to 3 months of home-based (30), centre-based (30) or a control group (30) on a 1:1:1 basis after completion of their phase II ambulatory cardiac rehabilitation programme. Outcome measures were assessed at discharge of the phase II programme and after one year.RESULTS:Eighty patients (72 (91%) men; mean age 62.6 years) completed the one-year follow-up measurements. Exercise capacity and secondary outcomes were preserved in all three groups (Ptime > 0.05 for all), irrespective of the intervention (Pinteraction > 0.05 for all). Eighty-five per cent of patients met the international guidelines for physical activity (Ptime < 0.05). No interaction effect was found for physical activity.CONCLUSION:Overall, exercise capacity remained stable during one year following phase II cardiac rehabilitation. Our home-based exercise intervention was as effective as centre-based and did not result in higher levels of exercise capacity and physical activity compared to the other two interventions.TRIAL REGISTRATION:ClinicalTrials.gov NCT02047942. https://clinicaltrials.gov/ct2/show/NCT02047942.

IntroductionThe methylated analogue of the polyphenol Resveratrol (RV), 2, 3’, 4, 5’-Tetramethoxystilbene (TMS), displays significantly more antioxidant effects than RV and is a potent inhibitor of CYP1B1, shown to contribute to the development of hypertension. While TMS bioavailability is low1, liposomes are a promising modality for TMS encapsulation and delivery to improve uptake into tissues2. The objective of this study was to determine the effect of TMS, delivered via liposomes, on endothelial cell viability and vasodilator responses of isolated coronary arteries, after acute pressure elevation, ex vivo, and assess mechanisms involved.MethodsLiposomes were synthesised using a thin-lipid film process and characterised using UV-Vis and fluorescence spectroscopy, Dynamic Light Scattering and Fourier-transform infrared spectroscopy. The effect of TMS-loaded liposomes on human coronary artery endothelial cell viability was determined in vitro using Alamar Blue assay. Small coronary arteries were isolated from male Wistar rats (in accordance with Home office guidelines and institutional ethics approval) and their function assessed at 60mmHg and following acute pressure elevation (150 mmHg, 30 minutes) to mimic a hypertensive environment. Endothelial-dependent (acetylcholine, ACh 1.0 nM – 1.0 mM) and independent (Sodium nitroprusside -SNP, 100 μM, Papaverine –PAPA, 100 μM) responses were measured in the presence/absence of TMS and TMS-loaded liposomes, using pressure myography. Data are expressed as mean percent dilation ± SEM.ResultsTMS-loaded liposomes (157 ± 6 nm diameter; zeta potential -13.13 ± 0.67 mV) maintained cell viability without toxicity, following 48h incubation. Acute pressure elevation significantly reduced endothelial-dependent dilator responses but did not affect endothelial-independent vasodilation. Co-incubation with TMS liposomes significantly improved endothelial-dependent vasodilation (@ ACh 100 μM: 86.06 ± 5.63% and 89.84 ± 3.05% for TMS liposomes and TMS solution respectively, compared to control PSS 38.52 ± 6.34; n = 5; p ≤ 0.01). The potentiated dilator response was sustained over a longer period (4h) with TMS liposomes, when compared to TMS solution (@ ACh 100 μM: 77.32 ± 8.70% vs 41.70 ± 8.70%; n = 4; p ≤ 0.05).ConclusionTMS-loaded liposomes have the potential to restore attenuated coronary endothelial-dependent dilator responses in an acute hypertensive environment. Our findings will help establish whether TMS-loaded liposomes are a valid therapeutic drug-delivery strategy in hypertension.ReferencesNawaz et al., 2017. Nutrients, 9(11), p.1188. Hu et al., 2018. Molecular Pharmaceutics, 15(12), pp.5493-5500.Conflict of InterestNone

Resveratrol is a plant-derived phytoalexin with antioxidant, anti-inflammatory and cardio-protective properties and may be a promising therapeutic intervention strategy in cardiovascular disease. Here, we investigated the acute direct effects of trans-resveratrol (RV), on acetylcholine (ACh)-induced and flow-mediated dilation (FMD) of isolated pressurized femoral arteries of young (4-month-old) and old (26-month-old) mice. Vessel exposure to RV enhanced ACh (0.01-1.0 mM)-induced dilation (p < 0.05), but not FMD (@ 5-10 mu L.min(-1)) (p < 0.05) in both young and old mice. After RV incubation, acute nitric oxide (NO) production by cultured endothelial cells was increased in response to 0.01 mM ACh, but reduced by flow (5-10 mu L.min(-1); p < 0.05). In isolated femoral arteries from endothelial nitric oxide synthase knockout (eNOS(-/-)) mice, RV had no overall effect on FMD, but potentiated ACh induced dilation, that was completely abolished by potassium channel blockers, Apamin and Tram 34 (p < 0.01). We demonstrate that the non-metabolised form of RV stimulates ACh-induced dilation via the NO and EDHF pathways, but not FMD by interaction with the cyclo-oxygenase pathway. Our findings have important implications in the use of RV (for both young and aged) under 'normal' non-diseased physiological states.

The methylated analogue of the polyphenol resveratrol (RV), 2,3′,4,5′-tetramethoxystilbene (TMS) displays potent antioxidant properties and is an effective cytochrome P450 (CYP) 1B1 inhibitor. The bioavailability of TMS is low. Therefore, the use of liposomes for the encapsulation of TMS is a promising delivery modality for enhanced uptake into tissues. We examined the effect of delivery of TMS in liposomes on the restoration of vasodilator responses of isolated aortic vessels after acute tension elevation ex vivo. Aortic vessels from young male Wistar rats were isolated, and endothelial-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside, SNP) responses assessed. Acute tension elevation (1 h) significantly reduced ACh dilator responses, which were restored following incubation with superoxide dismutase or apocynin (an NADPH oxidase inhibitor). Incubation with TMS-loaded liposomes (mean diameter 157 ± 6 nm; PDI 0.097) significantly improved the attenuated dilator responses following tension elevation, which was sustained over a longer period (4 h) when compared to TMS solution. Endothelial denudation or co-incubation with L-NNA (Nω-nitro-l-arginine; nitric oxide synthase inhibitor) resulted in loss of dilator function. Our findings suggest that TMS-loaded liposomes can restore attenuated endothelial-dependent dilator responses induced by an oxidative environment by reducing NADPH-oxidase-derived ROS and potentiating the release of the vasodilator nitric oxide. TMS-loaded liposomes may be a promising therapeutic strategy to restore vasodilator function in vascular disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Blood vessel disease is a major contributor to cardiovascular morbidity and mortality and is hallmarked by dysfunction of the lining endothelial cells (ECs). These cells play a significant role in vascular homeostasis, through the release of mediators to control vessel diameter, hence tissue perfusion. Mesoporous silica nanoparticles (MSNs) can be used as potential drug delivery platforms for vasodilator drugs. Here, using an ex vivo model of vascular function, we examine the use of titania coating for improved biocompatibility and release dynamics of MSN loaded sodium nitroprusside (SNP). MSNs (95 ± 23 nm diameter; pore size 2.7 nm) were synthesised and fully characterised. They were loaded with SNP and coated with titania (TiO 2 ), using the magnetron sputtering technique. Pre-constricted aortic vessels were exposed to drug loaded MSNs (at 1.96 x 10 12 MSN mL -1 ) and the time course of vessel dilation observed, in real time. Exposure of viable vessels to MSNs lead to their internalization into the cytoplasm of ECs, while TiMSNs were also observed in the elastic lamina and smooth muscle cell layers. We demonstrate that titania coating of MSNs significantly improves their biocompatibility and alters the dynamics of drug release. A slow and more sustained relaxation was evident after uptake of TiMSN-SNP, in comparison to uncoated MSN-SNP (rate of dilation was 0.08% per min over a 2.5 h period). The use of titania coated MSNs for drug delivery to the vasculature may be an attractive strategy for therapeutic clinical intervention in cardiovascular disease. that titania coating of MSNs significantly improves their biocompatibility and uptake within aortic blood vessels and furthermore, enables a slower and more sustained release of the vasodilator drug, sodium nitroprusside within the vessel, thus making them an attractive strategy for the treatment of vascular disease.

Objective: Resveratrol (RV) is a polyphenol with antioxidant, anti-inflammatory and cardio-protective properties. Our objective was to investigate whether acute supplementation with high doses of RV would improve flow-mediated dilation (FMD) and oxygen consumption (VO2) kinetics in older coronary artery disease (CAD) patients. Design: We employed a placebo-controlled, single-blind, crossover design in which ten participants (aged 66.6 +/- 7.8 years) received either RV or placebo (330 mg, 3x day(-1)) during three consecutive days plus additional 330 mg in the morning of the fourth day with a seven-day wash-out period in-between. On the fourth day, FMD of the brachial artery and VO2 on-kinetics were determined. Results: RV improved FMD in patients who had undergone coronary artery bypass grafting (CABG; -1.4 vs. 5.0%; p = .004), but not in those who had undergone percutaneous coronary intervention (PCI; 4.2 vs. -0.2%; NS). Conclusion: Acute high dose supplementation with RV improved FMD in patients after CABG surgery but impaired FMD in patients who underwent PCI. The revascularization method-related differential effects of RV may be due to its direct effects on endothelial-dependent dilator responses. Our findings have important implications for personalized treatment and stratification of older CAD patients.

Doxorubicin‐induced cardiomyopathy is a clinically prevalent pathology, occurring as a sequelae following chemotherapy for cancer patients. In particular, the “first dose” effect has been particularly challenging, given the heterogeneous and multifactorial nature of this pathophysiology. Here, we describe the development of a physiologically relevant in vitro model for cardiotoxicity testing, using human cells. Primary cardiomyocytes, endothelial, and smooth muscle cells were tri‐cultured in 2D, or within nano‐fibrous scaffolds in a 3D environment, under dynamic nutrient flow, using the Quasi Vivo® system. State‐of‐the‐art sensor chips were used to detect troponin I levels, 2 h after acute exposure to doxorubicin. We demonstrate a significant improvement in cardiomyocyte viability when grown in a 3D tri‐culture environment over a 5‐day period and a 10‐fold reduction in doxorubicin‐induced toxicity. Our tri‐culture model can be used as a valuable tool for physiologically relevant assessment of drug‐induced cardiotoxicity in vitro.

Background: Cardiac rehabilitation (CR) is an essential part of coronary heart disease (CHD) management. However, patients exiting a center-based CR program have difficulty retaining its benefits. Objective: The purpose of the TRiCH study is to evaluate the added benefit of a home-based (HB) CR program with telemonitoring guidance on physical fitness in patients with coronary artery disease (CAD) who are being discharged from a phase II ambulatory CR program, and to compare its effectiveness to a prolonged center-based (CB) CR intervention. Methods: Between February 2014 and August 2016, 90 CAD patients (61.2±7.6yrs, 89% males, 1.73±0.7m, 82.9±13kg, 27.5±3.4kg/m2) who successfully completed a three month ambulatory CR program were randomly allocated to one of three groups: HB (=30), CB (=30) or a control group (CG) (=30) on a 1:1:1 basis. HB patients received a home-based exercise intervention with telemonitoring guidance consisting of weekly emails or phone calls, CB patients continued the ambulatory-hospital CR and CG patients received usual care including the recommendation to remain physically active. All patients underwent cardiopulmonary exercise testing to assess peak oxygen uptake (VO2P) at baseline and after the 12 week intervention period. Secondary outcomes included physical activity behavior, anthropometric characteristics, cardiovascular risk factors and quality of life. Results: The increase in VO2P was larger following 12 weeks of intervention in the CB group (P = .03) and HB group (P = .04) compared to the control group. In addition, oxygen uptake at the first ventilatory threshold (P-interaction = .03) and the second ventilatory threshold (P-interaction = .03) increased significantly more in the HB group compared to CB. No significant changes were found in the secondary outcomes. Conclusions: Adding a HB exercise program with telemonitoring guidance following completion of a phase II ambulatory CR program results in further improvement of physical fitness and is as equally effective as prolonging a CB CR in patients with coronary artery disease.

There is an acute clinical need for small-diameter vascular grafts as a treatment option for cardiovascular disease. Here, we used an intelligent design system to recreate the natural structure and hemodynamics of small arteries. Nano-fibrous tubular scaffolds were fabricated from blends of polyvinyl alcohol and gelatin with inner helices to allow a near physiological spiral flow profile, using the electrospinning technique. Human coronary artery endothelial cells (ECs) were seeded on the inner surface and their viability, distribution, gene expression of mechanosensitive and adhesion molecules compared to that in conventional scaffolds, under static and flow conditions. We show significant improvement in cell distribution in helical vs. conventional scaffolds (94% ± 9% vs. 82% ± 7.2%; P < 0.05) with improved responsiveness to shear stress and better ability to withhold physiological pressures. Our helical vascular scaffold provides an improved niche for EC growth and may be attractive as a potential small diameter vascular graft.

Blood vessel disease is a major contributor to cardiovascular morbidity and mortality and is hallmarked by dysfunction of the lining endothelial cells (ECs). These cells play a significant role in vascular homeostasis, through the release of mediators to control vessel diameter, hence tissue perfusion. Mesoporous silica nanoparticles (MSNs) can be used as potential drug delivery platforms for vasodilator drugs. Here, using an ex vivo model of vascular function, we examine the use of titania coating for improved biocompatibility and release dynamics of MSN loaded sodium nitroprusside (SNP). MSNs (95 +/- 23 nm diameter; pore size 2.7 nm) were synthesised and fully characterised. They were loaded with SNP and coated with titania (TiO2), using the magnetron sputtering technique. Pre-constricted aortic vessels were exposed to drug loaded MSNs (at 1.96 x 10(12) MSN mL(-1)) and the time course of vessel dilation observed, in real time. Exposure of viable vessels to MSNs lead to their internalization into the cytoplasm of ECs, while TiMSNs were also observed in the elastic lamina and smooth muscle cell layers. We demonstrate that titania coating of MSNs significantly improves their biocompatibility and alters the dynamics of drug release. A slow and more sustained relaxation was evident after uptake of TiMSN-SNP, in comparison to uncoated MSN-SNP (rate of dilation was 0.08% per min over a 2.5 h period). The use of titania coated MSNs for drug delivery to the vasculature may be an attractive strategy for therapeutic clinical intervention in cardiovascular disease. Statement of Significance Cardiovascular disease is a major cause of mortality and morbidity worldwide, with a total global cost of over $918 billion, by 2030. Mesoporous silica nanoparticles (MSNs) have great potential for the delivery of drugs that can treat vessel disease. This paper provides the first description for the use of titania coated MSNs with increased vascular penetration, for the delivery of vasodilator drugs, without compromising overall vessel function. We demonstrate that titania coating of MSNs significantly improves their biocompatibility and uptake within aortic blood vessels and furthermore, enables a slower and more sustained release of the vasodilator drug, sodium nitroprusside within the vessel, thus making them an attractive strategy for the treatment of vascular disease. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

BACKGROUND:Cardiac rehabilitation (CR) is an essential part of contemporary coronary heart disease management. However, patients exiting a center-based CR program have difficulty retaining its benefits. OBJECTIVE:We aimed to evaluate the added benefit of a home-based CR program with telemonitoring guidance on physical fitness in patients with coronary artery disease (CAD) completing a phase II ambulatory CR program and to compare the effectiveness of this program in a prolonged center-based CR intervention by means of a randomized controlled trial. METHODS:Between February 2014 and August 2016, 90 CAD patients (unblinded, mean age 61.2 years, SD 7.6; 80/90, 89.0% males; mean height 1.73 m, SD 0.7; mean weight 82.9 kg, SD 13; mean body mass index 27.5 kg/m2, SD 3.4) who successfully completed a 3-month ambulatory CR program were randomly allocated to one of three groups: home-based (30), center-based (30), or control group (30) on a 1:1:1 basis. Home-based patients received a home-based exercise intervention with telemonitoring guidance consisting of weekly emails or phone calls; center-based patients continued the standard in-hospital CR, and control group patients received the usual care including the advice to remain physically active. All the patients underwent cardiopulmonary exercise testing for assessment of their peak oxygen uptake (VO2 P) at baseline and after a 12-week intervention period. Secondary outcomes included physical activity behavior, anthropometric characteristics, traditional cardiovascular risk factors, and quality of life. RESULTS:Following 12 weeks of intervention, the increase in VO2 P was larger in the center-based (P=.03) and home-based (P=.04) groups than in the control group. In addition, oxygen uptake at the first (P-interaction=.03) and second (P-interaction=.03) ventilatory thresholds increased significantly more in the home-based group than in the center-based group. No significant changes were observed in the secondary outcomes. CONCLUSIONS:Adding a home-based exercise program with telemonitoring guidance following completion of a phase II ambulatory CR program results in further improvement of physical fitness and is equally as effective as prolonging a center-based CR in patients with CAD. TRIAL REGISTRATION:ClinicalTrials.gov NCT02047942; https://clinicaltrials.gov/ct2/show/NCT02047942 (Archived by WebCite at http://www.webcitation.org/70CBkSURj).