Amphiphilic Cell-Penetrating Peptides Containing Arginine and Hydrophobic Residues as Protein Delivery Agents.

The entry of proteins through the cell membrane is challenging, thus limiting their use as potential therapeutics. Seven cell-penetrating peptides, designed in our laboratory, were evaluated for the delivery of proteins. Fmoc solid-phase peptide synthesis was utilized for the synthesis of seven cyclic or hybrid cyclic-linear amphiphilic peptides composed of hydrophobic (tryptophan (W) or 3,3-diphenylalanine (Dip) and positively-charged arginine (R) residues, such as [WR]4, [WR]9, [WWRR]4, [WWRR]5, [(RW)5K](RW)5, [R5K]W7, and [DipR]5. Confocal microscopy was used to screen the peptides as a protein delivery system of model cargo proteins, green and red fluorescein proteins (GFP and RFP). Based on the confocal microscopy results, [WR]9 and [DipR]5 were found to be more efficient among all the peptides and were selected for further studies. [WR]9 (1-10 µM) + protein (GFP and RFP) physical mixture did not show high cytotoxicity (>90% viability) in triple-negative breast cancer cells (MDA-MB-231) after 24 h, while [DipR]5 (1-10 µM) physical mixture with GFP exhibited more than 81% cell viability. Confocal microscopy images revealed internalization of GFP and RFP in MDA-MB-231 cells using [WR]9 (2-10 µM) and [DipR]5 (1-10 µM). Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptake of GFP was concentration-dependent in the presence of [WR]9 in MDA-MB-231 cells after 3 h of incubation at 37 °C. The concentration-dependent uptake of GFP and RFP was also observed in the presence of [DipR5] in SK-OV-3 and MDA-MB-231 cells after 3 h of incubation at 37 °C. FACS analysis indicated that the cellular uptake of GFP in the presence of [WR]9 was partially decreased by methyl-ß-cyclodextrin and nystatin as endocytosis inhibitors after 3 h of incubation in MDA-MB-231 cells, whereas nystatin and chlorpromazine as endocytosis inhibitors slightly reduced the uptake of GFP in the presence of [DipR]5 after 3 h of incubation in MDA-MB-231. [WR]9 was able to deliver therapeutically relevant proteins (Histone H2A) at different concentrations. These results provide insight into the use of amphiphilic cyclic peptides in the delivery of protein-related therapeutics.

Cyclic and Linear Peptides Containing Alternate WW and RR Residues as Molecular Cargo Delivery Tools.

Cell-impermeable and negatively charged compounds' cellular uptake across the cell membranes remains challenging. Herein, the synthesis of four linear [(WWRR)2, (WWRR)3, (WWRR)4, and (WWRR)5] and four cyclic ([WWRR]2, [WWRR]3, [WWRR]4, and [WWRR]5) peptides containing alternate two tryptophan (WW) and two arginine (RR) residues and their biological evaluation as molecular transporters are reported. The peptides did not show any significant cytotoxicity in different cell lines (MDA-MB-23, SK-OV-3, and HEK 293) at a concentration of 5 µM and after 3 h of incubation time. The uptake of fluorescence-labeled cargo molecules (F'-GpYEEI, F'-siRNA, and F'-3TC) in the presence of the peptides was monitored in different cell lines (SK-OV-3 and MDA-MB-231) with fluorescence-activated cell sorting. Among all the peptides, [WWRR]5 (C4) showed the highest cellular uptake of cargo molecules, indicating it can act as effective molecular transporter. Confocal microscopy in MDA-MB-231 cells showed the cellular uptake of F'-GpYEEI in the presence of C4 and the intracellular localization of fluorescence-labeled C4 (F'-C4) in the cytosol. The F'-C4 cellular uptake was found to be concentration- and time-dependent, as shown by flow cytometry in MDA-MB-231 cells. Confocal microscopy and flow cytometry of F'-C4 in MDA-MB-231 cells were examined alone and in the presence of different endocytosis inhibitors (chlorpromazine, methyl-ß-cyclodextrin, chloroquine, and nystatin). The data showed that the cellular uptake of F'-C4 in the presence of chlorpromazine, chloroquine, and methyl-ß-cyclodextrin was reduced but not completely eliminated, indicating that both energy-independent and energy-dependent pathways contributed to the cellular uptake of F'-C4. Similar results were obtained using the confocal microscopy of C4 and F'-GpYEEI in the presence of endocytosis inhibitors (chlorpromazine, methyl-ß-cyclodextrin, chloroquine, and nystatin). These data indicate that C4 has the potential to be used as a cell-penetrating peptide and cargo transporter.

The cost-effectiveness of a uniform versus age-based threshold for one-off screening for prevention of cardiovascular disease.

The objective of this article was to assess the cost-effectiveness of screening strategies for cardiovascular diseases (CVD). A decision analytic model was constructed to estimate the costs and benefits of one-off screening strategies differentiated by screening age, sex and the threshold for initiating statin therapy ("uniform" or "age-adjusted") from the Spanish NHS perspective. The age-adjusted thresholds were configured so that the same number of people at high risk would be treated as under the uniform threshold. Health benefit was measured in quality-adjusted life years (QALY). Transition rates were estimated from the European Prospective Investigation into Cancer and Nutrition (EPIC-CVD), a large multicentre nested case-cohort study with 12 years of follow-up. Unit costs of primary care, hospitalizations and CVD care were taken from the Spanish health system. Univariate and probabilistic sensitivity analyses were employed. The comparator was no systematic screening program. The base case model showed that the most efficient one-off strategy is to screen both men and women at 40 years old using a uniform risk threshold for initiating statin treatment (Incremental Cost-Effectiveness Ratio of €3,274/QALY and €6,085/QALY for men and women, respectively). Re-allocating statin treatment towards younger individuals at high risk for their age and sex would not offset the benefit obtained using those same resources to treat older individuals. Results are sensitive to assumptions about CVD incidence rates. To conclude, one-off screening for CVD using a uniform risk threshold appears cost-effective compared with no systematic screening. These results should be evaluated in clinical studies.

Modelling the impact of changes to abdominal aortic aneurysm screening and treatment services in England during the COVID-19 pandemic.

BACKGROUND: The National Health Service (NHS) abdominal aortic aneurysm (AAA) screening programme (NAAASP) in England screens 65-year-old men. The programme monitors those with an aneurysm, and early intervention for large aneurysms reduces ruptures and AAA-related mortality. AAA screening services have been disrupted following COVID-19 but it is not known how this may impact AAA-related mortality, or where efforts should be focussed as services resume. METHODS: We repurposed a previously validated discrete event simulation model to investigate the impact of COVID-19-related service disruption on key outcomes. This model was used to explore the impact of delayed invitation and reduced attendance in men invited to screening. Additionally, we investigated the impact of temporarily suspending scans, increasing the threshold for elective surgery to 7cm and increasing drop-out in the AAA cohort under surveillance, using data from NAAASP to inform the population. FINDINGS: Delaying invitation to primary screening up to two years had little impact on key outcomes whereas a 10% reduction in attendance could lead to a 2% lifetime increase in AAA-related deaths. In surveillance patients, a 1-year suspension of surveillance or increase in the elective threshold resulted in a 0.4% increase in excess AAA-related deaths (8% in those 5-5.4cm at the start). Longer suspensions or a doubling of drop-out from surveillance would have a pronounced impact on outcomes. INTERPRETATION: Efforts should be directed towards encouraging men to attend AAA screening service appointments post-COVID-19. Those with AAAs on surveillance should be prioritised as the screening programme resumes, as changes to these services beyond one year are likely to have a larger impact on surgical burden and AAA-related mortality.

Optimizing Surveillance and Re-intervention Strategy Following Elective Endovascular Repair of Abdominal Aortic Aneurysms.

BACKGROUND: EVAR for abdominal aortic aneurysm has an initial survival advantage over OR, but more frequent complications increase costs and long-term aneurysm-related mortality. Randomized controlled trials of EVAR versus OR have shown EVAR is not cost-effective over a patient's lifetime. However, in the EVAR-1 trial, postoperative surveillance may have been sub-optimal, as the importance of sac growth as a predictor of graft failure was overlooked. METHODS: Real-world data informed a discrete event simulation model of postoperative outcomes following EVAR. Outcomes observed EVAR-1 were compared with those from 5 alternative postoperative surveillance and re-intervention strategies. Key events, quality-adjusted life years and costs were predicted. The impact of using complication and rupture rates from more recent devices, imaging and re-intervention methods was also explored. RESULTS: Compared with observed EVAR-1 outcomes, modeling full adherence to the EVAR-1 scan protocol reduced abdominal aortic aneurysm (AAA) deaths by 3% and increased elective re-interventions by 44%. European Society re-intervention guidelines provided the most clinically effective strategy, with an 8% reduction in AAA deaths, but a 52% increase in elective re-interventions. The cheapest and most cost-effective strategy used lifetime annual ultrasound in primary care with confirmatory computed tomography if necessary, and reduced AAA-related deaths by 5%. Using contemporary rates for complications and rupture did not alter these conclusions. CONCLUSIONS: All alternative strategies improved clinical benefits compared with the EVAR-1 trial. Further work is needed regarding the cost and accuracy of primary care ultrasound, and the potential impact of these strategies in the comparison with OR.

Clinical utility and cost modelling of the phi test to triage referrals into image-based diagnostic services for suspected prostate cancer: the PRIM (Phi to RefIne Mri) study.

BACKGROUND: The clinical pathway to detect and diagnose prostate cancer has been revolutionised by the use of multiparametric MRI (mpMRI pre-biopsy). mpMRI however remains a resource-intensive test and is highly operator dependent with variable effectiveness with regard to its negative predictive value. Here we tested the use of the phi assay in standard clinical practice to pre-select men at the highest risk of harbouring significant cancer and hence refine the use of mpMRI and biopsies. METHODS: A prospective five-centre study recruited men being investigated through an mpMRI-based prostate cancer diagnostic pathway. Test statistics for PSA, PSA density (PSAd) and phi were assessed for detecting significant cancers using 2 definitions: ≥ Grade Group (GG2) and ≥ Cambridge Prognostic Groups (CPG) 3. Cost modelling and decision curve analysis (DCA) was simultaneously performed. RESULTS: A total of 545 men were recruited and studied with a median age, PSA and phi of 66 years, 8.0 ng/ml and 44 respectively. Overall, ≥ GG2 and ≥ CPG3 cancer detection rates were 64% (349/545), 47% (256/545) and 32% (174/545) respectively. There was no difference across centres for patient demographics or cancer detection rates. The overall area under the curve (AUC) for predicting ≥ GG2 cancers was 0.70 for PSA and 0.82 for phi. AUCs for ≥ CPG3 cancers were 0.81 and 0.87 for PSA and phi respectively. AUC values for phi did not differ between centres suggesting reliability of the test in different diagnostic settings. Pre-referral phi cut-offs between 20 and 30 had NPVs of 0.85-0.90 for ≥ GG2 cancers and 0.94-1.0 for ≥ CPG3 cancers. A strategy of mpMRI in all and biopsy only positive lesions reduced unnecessary biopsies by 35% but missed 9% of ≥ GG2 and 5% of ≥ CPG3 cancers. Using PH ≥ 30 to rule out referrals missed 8% and 5% of ≥ GG2 and ≥ CPG3 cancers (and reduced unnecessary biopsies by 40%). This was achieved however with 25% fewer mpMRI. Pathways incorporating PSAd missed fewer cancers but necessitated more unnecessary biopsies. The phi strategy had the lowest mean costs with DCA demonstrating net clinical benefit over a range of thresholds. CONCLUSION: phi as a triaging test may be an effective way to reduce mpMRI and biopsies without compromising detection of significant prostate cancers.

Influence of life-style choices on locomotor disability, arthritis and cardiovascular disease in older women: prospective cohort study.

BACKGROUND: many chronic conditions have their roots in modifiable health-related behaviours. METHODS: a total of 4,286 women aged 60-79 in the British Women's Heart and Health Study are followed up for incident cardiovascular disease (CVD), arthritis and locomotor disability over 7 years. Self-reported smoking, alcohol consumption, exercise and fruit intake at baseline is also available. Associations between these and each outcome, plus a composite outcome, are investigated in those without prevalent disease at baseline using logistic regression with multiple imputation. RESULTS: ex-smokers and current smokers showed increased odds of locomotor disability, CVD and the combined outcome. Less regular exercisers had increased odds of all outcomes, particularly locomotor disability. There was no evidence that alcohol or fruit intake was associated with any outcome. Population attributable fractions (PAFs) suggest in addition to the influence of smoking and alcohol, exercise accounts for 9% of incident locomotor disability, 5% of CVD and 4% of arthritis. All four lifestyle factors combined account for 17% of incident locomotor disability and 9% of incident conditions combined. CONCLUSIONS: never smokers and regular exercisers had substantially reduced odds of 7-year disability onset. Low PAFs suggest changes in health-related behaviours in older women would result in only modest reductions in common chronic conditions.

Estimation of life-years gained and cost effectiveness based on cause-specific mortality.

Cost-effectiveness analysis is usually based on life-years gained estimated from all-cause mortality. When an intervention affects only a few causes of death accounting for a small fraction of all deaths, this approach may lack precision. We develop a novel technique for cost-effectiveness analysis when life-years gained are estimated from cause-specific mortality, allowing for competing causes of death. In the context of randomised trial data, we adjust for other-cause mortality combined across randomised groups. This method yields a greater precision than analysis based on total mortality, and we show application to life-years gained, quality-adjusted life-years gained, incremental costs, and cost effectiveness. In multi-state health economic models, however, mortality from competing causes is commonly derived from national statistics and is assumed to be known and equal across intervention groups. In such models, our method based on cause-specific mortality and standard methods using total mortality give essentially identical estimates and precision. The methods are applied to a randomised trial and a health economic model, both of screening for abdominal aortic aneurysm. A gain in precision for cost-effectiveness estimates is clearly helpful for decision making, but it is important to ensure that 'cause-specific mortality' is defined to include all causes of death potentially affected by the intervention.

Uncertainty and validation of health economic decision models.

Health economic decision models are based on specific assumptions relating to model structure and parameter estimation. Validation of these models is recommended as an indicator of reliability, but is not commonly reported. Furthermore, models derived from different data and employing different assumptions may produce a variety of results.A Markov model for evaluating the long-term cost-effectiveness of screening for abdominal aortic aneurysm is described. Internal, prospective and external validations are carried out using individual participant data from two randomised trials. Validation is assessed in terms of total numbers and timings of key events, and total costs and life-years. Since the initial model validates well only internally, two further models are developed that better fit the prospective and external validation data. All three models are then extrapolated to a life-time horizon, producing cost-effectiveness estimates ranging from pound1600 to pound4200 per life-year gained.Parameter uncertainty is now commonly addressed in health economic decision modelling. However, the derivation of models from different data sources adds another level of uncertainty. This extra uncertainty should be recognised in practical decision-making and, where possible, specifically investigated through independent model validation.

A sustained mortality benefit from screening for abdominal aortic aneurysm.

BACKGROUND: Longer-term mortality benefit and cost-effectiveness for abdominal aortic aneurysm (AAA) screening are uncertain. OBJECTIVE: To estimate the benefits, in terms of AAA-related and all-cause mortality, and cost-effectiveness of ultrasonography screening for AAA in a group that was invited to screening compared with a group that was not invited at a mean 7-year follow-up. DESIGN: Randomized trial. SETTING: 4 centers in the United Kingdom. PATIENTS: Population-based sample of 67,770 men age 65 to 74 years. INTERVENTION: Patients with an AAA detected at screening had surveillance and were offered surgery after predefined criteria were met. MEASUREMENTS: Mortality data were obtained after flagging on the national database. Unit costs obtained from large samples were applied to individual event data for the cost analysis. RESULTS: The hazard ratio was 0.53 (95% CI, 0.42 to 0.68) for AAA-related mortality in the group invited for screening. The rupture rate in men with normal results on initial ultrasonography has remained low: 0.54 rupture (CI, 0.25 to 1.02 ruptures) per 10 000 person-years. In terms of all-cause mortality, the observed hazard ratio was 0.96 (CI, 0.93 to 1.00). At the 7-year follow-up, cost-effectiveness was estimated at $19 500 (CI, $12,400 to $39,800) per life-year gained based on AAA-related mortality and $7600 (CI, $3300 to infinity) per life-year gained based on all-cause death. (All values are reported in U.S. dollars [U.K. 1 pound sterling = U.S. $1.58]). LIMITATION: Inclusion of deaths from aortic aneurysm at an unspecified site, which may include some thoracic aortic aneurysms, may have underestimated the treatment effect. CONCLUSIONS: These results from a large, pragmatic randomized trial show that the early mortality benefit of screening ultrasonography for AAA is maintained in the longer term and that the cost-effectiveness of screening improves over time. International Standard Randomized Controlled Trial registration number: ISRCTN37381646.

The credibility of health economic models for health policy decision-making: the case of population screening for abdominal aortic aneurysm.

OBJECTIVES: To review health economic models of population screening for abdominal aortic aneurysm (AAA) among elderly males and assess their credibility for informing decision-making. METHODS: A literature review identified health economic models of ultrasound screening for AAA. For each model focussing on population screening in elderly males, model structure and input parameter values were critically appraised using published good practice guidelines for decision analytic models. RESULTS: Twelve models published between 1989 and 2003 were identified. Converting costs to a common currency and base year, substantial variability in cost-effectiveness results were revealed. Appraisals carried out for the nine models focusing on population screening showed differences in their complexity, with the simpler models generating results most favourable to screening. Eight of the nine models incorporated two or more simplifying structural assumptions favouring screening; uncertainty surrounding these assumptions was not investigated by any model. Quality assessments on a small number of parameters revealed input values varied between models, methods used to identify and incorporate input data were often not described, and few sensitivity analyses were reported. CONCLUSIONS: Large variation exists in the cost-effectiveness results generated by AAA screening models. The substantial number of factors potentially contributing to such disparities means that reconciliation of model results is impossible. In addition, poor reporting of methods makes it difficult to identify the most plausible and thus most useful model of those developed.

Assessment of the criteria for elective surgery in screen-detected abdominal aortic aneurysms.

OBJECTIVES: Apart from aortic diameter, two other widely used criteria for considering surgery in screen-detected abdominal aortic aneurysms (AAAs)--annual aortic expansion > or =1.0 cm and presence of symptoms attributable to the AAA--are based on accepted practice and AAA expansion rates, rather than direct evidence. The Multi-centre Aneurysm Screening Study (MASS) enables assessment of their contribution to this risk reduction. METHODS: MASS employs three criteria for referral for considering elective open surgery: maximum aortic diameter > or =5.5 cm, rapid aortic expansion (> or =1.0 cm/year), and/or the presence of symptoms attributable to the AAA. Data from MASS are used to examine the value of these criteria in practice. RESULTS: No patients were referred for symptoms alone. Of those referred for rapid expansion, 88% were returned to surveillance, compared with only 12% of those referred for diameter > or =5.5 cm at initial scan, and 34% of those referred for diameter > or =5.5 cm at a follow-up scan. Return to surveillance following referral for rapid expansion was strongly associated with aortic diameter (age-adjusted odds ratio for return 0.89 per mm, 95% confidence interval 0.79-1.00). Of those 5.0-5.4 cm at the time of referral for rapid expansion who were returned, 31% reached 5.5 cm during a median post-referral follow-up of 0.9 years. Among those referred for expansion, the rupture rate was only 8 per 1000 person-years of follow-up prior to reaching 5.5 cm. CONCLUSIONS: A single criterion for considering elective surgery is recommended in screen-detected AAA, based on a maximum aortic diameter of > or =5.5 cm. This criterion detects the majority of those at risk from rupture, and is simple to assess.

Poorer self assessed health in a prospective study of men with screen detected abdominal aortic aneurysm: a predictor or a consequence of screening outcome?

STUDY OBJECTIVES: To assess the extent to which poorer self assessed health in men in whom an abdominal aortic aneurysm (AAA) is detected at screening is a consequence or a predictor of screening outcome. DESIGN: Prospective study. SETTING: Community based screening. PARTICIPANTS: 23 654 men who attended for AAA screening as part of the UK multicentre aneurysm screening study completed a measure of self assessed health before screening. A total of 1156 had an aneurysm detected. A sub-sample of screened men (571 with an aneurysm and 609 with a normal aorta) also completed the measure of self assessed health six weeks after screening. MAIN RESULTS: Men in whom an aneurysm was detected at screening perceived their health to be poorer before screening than those with a normal aorta. Adjusting for risk factors for AAA made no difference to this RESULT: self assessed health remained a strong predictor of having an aneurysm (odds ratio 1.7 comparing the extreme quartiles of self assessed health, 95% confidence intervals: 1.4 to 2.0). Men with an aneurysm also perceived their health to be poorer after screening had detected their aneurysms, but only to an extent in line with their pre-screening perceptions. CONCLUSIONS: Self assessed health seems to predict having an aortic aneurysm, independently of known risk factors. This emphasises the importance of assessing baseline perceptions of health to prevent erroneously inferring that poorer self assessed health in those who screen positive is a consequence as compared with a predictor of screening outcome.