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Background: Although COVID-19 morbidity is significantly lower in pediatrics than in adults, the risk of severe COVID-19 may still pose substantial health care resource burden. This study aimed to describe health care resource utilization (HCRU) and costs associated with COVID-19 in pediatrics 1-17 years old in England., Methods: A population-based retrospective cohort study of pediatrics with COVID-19 using Clinical Practice Research Datalink (CPRD Aurum) primary care data and, where available, linked Hospital Episode Statistics Admitted Patient Care secondary care data. HCRU and associated costs to the National Health Service were stratified by age, risk of severe COVID-19 and immunocompromised status, separately for those with and without hospitalization records (hospitalized cohort: COVID-19 diagnosis August 2020-March 2021; primary care cohort: COVID-19 diagnosis August 2020-January 2022)., Results: This study included 564,644 patients in the primary care cohort and 60 in the hospitalized cohort. Primary care consultations were more common in those 1-4 years of age (face-to-face: 4.3%; telephone: 6.0%) compared with those 5-11 (2.0%; 2.1%) and 12-17 years of age (2.2%; 2.5%). In the hospitalized cohort, mean (SD) length of stay was longer [5.0 (5.8) days] among those 12-17 years old (n = 24) than those 1-4 [n = 15; 1.8 (0.9) days] and 5-11 years old [n = 21; 2.8 (2.1) days]., Conclusions: Most pediatrics diagnosed with COVID-19 were managed in the community. However, hospitalizations were an important driver of HCRU and costs, particularly for those 12-17 years old. Our results may help optimize the management and resource allocation of COVID-19 in this population., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Objective: Management of chronic obstructive pulmonary disease (COPD) with inhaled corticosteroid/long-acting β 2 -agonist (ICS/LABA) improves lung function and health status and reduces COPD exacerbation risk versus monotherapy. This study described treatment use, healthcare resource utilisation (HCRU), healthcare costs and outcomes following initiation of single-device ICS/LABA as initial maintenance therapy (IMT)., Design: Retrospective cohort study., Setting: Primary care, England., Data Sources: Linked data from the Clinical Practice Research Datalink Aurum and Hospital Episode Statistics datasets., Participants: Patients with COPD and ≥1 single-device ICS/LABA prescription between July 2015 and December 2018 were included., Primary and Secondary Outcome Measures: Treatment pathways, COPD-related HCRU and healthcare costs, COPD exacerbations, time to triple therapy, medication adherence (proportion of days covered ≥80%) and indexed treatment time to discontinuation. Data for patients without prior maintenance therapy history (IMT users) and non-triple users were assessed over a 12-month follow-up period., Results: Of 13 451 new ICS/LABA users, 5162 were IMT users (budesonide/formoterol, n=1056; beclomethasone dipropionate/formoterol, n=2427; other ICS/LABA, n=1679), for whom at 3 and 12 months post-index, 45.6% and 39.4% were still receiving any ICS/LABA. At >6 to ≤12 months, the proportion of IMT users with ≥1 outpatient visit (10.1%) and proportion with ≥1 inpatient stay (12.6%) had increased from those at 3 months (9.0% and 7.4%, respectively). Inpatient stays contributed most to total COPD-related healthcare costs. For non-triple IMT users, at 3 and 12 months post-index, 4.5% and 13.7% had ≥1 moderate-to-severe COPD exacerbation. Time to triple therapy initiation and time to discontinuation of index medication ranged from 45.9 to 50.2 months and 2.3 to 2.8 months between treatments. Adherence was low across all time points (21.5-27.6%). Results were similar across indexed therapies., Conclusions: In the year following treatment initiation, ICS/LABA adherence was poor and many patients discontinued or switched therapies, suggesting that more consideration and optimisation of treatment is required in England for patients initiating single-device ICS/LABA therapy., Competing Interests: Competing interests: AC, GR, CC and AI are employees of GSK and hold stock and shares at GSK. AI is also an unpaid part-time Professor at McMaster University. TT, RWood and RWild are employees of Adelphi Real World, which received funding from GSK to conduct the study, but not for manuscript development. VLB was an employee of Adelphi Real World at the time of the study and is currently an employee of Bayer AG UK, and holds stock and shares in Bayer AG UK. Adelphi Real World is a business that provides consulting and other research services to pharmaceutical, device, government and non-government organisations. Adelphi Real World employees work with a variety of companies and organisations and are expressly prohibited from receiving any payment or honoraria directly from these organisations for services rendered., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: To create case definitions for confirmed COVID-19 diagnoses, COVID-19 vaccination status and three separate definitions of high risk of severe COVID-19, as well as to assess whether the implementation of these definitions in a cohort reflected the sociodemographic and clinical characteristics of COVID-19 epidemiology in England., Design: Retrospective cohort study., Setting: Electronic healthcare records from primary care (Clinical Practice Research Datalink, CPRD) linked to secondary care data (Hospital Episode Statistics) data covering 24% of the population in England., Participants: 2 271 072 persons aged 1 year and older diagnosed with COVID-19 in CPRD Aurum between 1 August 2020 and 31 January 2022., Main Outcome Measures: Age, sex and regional distribution of COVID-19 cases and COVID-19 vaccine doses received prior to diagnosis were assessed separately for the cohorts of cases identified in primary care and those hospitalised for COVID-19 (primary diagnosis code of ICD-10 U07.1 'COVID-19'). Smoking status, body mass index and Charlson Comorbidity Index were compared for the two cohorts, as well as for three separate definitions of high risk of severe disease used in the UK (National Health Service Highest Risk, PANORAMIC trial eligibility, UK Health Security Agency Clinical Risk prioritisation for vaccination)., Results: Compared with national estimates, CPRD case estimates under-represented older adults in both the primary care (age 65-84: 6% in CPRD vs 9% nationally) and hospitalised (31% vs 40%) cohorts, and over-represented people living in regions with the highest median wealth areas of England (20% primary care and 20% hospital admitted cases in South East vs 15% nationally). The majority of non-hospitalised cases and all hospitalised cases had not completed primary series vaccination. In primary care, persons meeting high-risk definitions were older, more often smokers, overweight or obese, and had higher Charlson Comorbidity Index score., Conclusions: CPRD primary care data are a robust real-world data source and can be used for some COVID-19 research questions, however, limitations of the data availability should be carefully considered. Included in this publication are supplemental files for a total of over 28 000 codes to define each of three definitions of high risk of severe disease., Competing Interests: Competing interests: KMA, LJM, MR, DM, JLN, CT, DM and JY are employees of Pfizer or Pfizer and may hold Pfizer stock or stock options. KKR and TT are employees of Adelphi Real World which has received consulting fees from Pfizer., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: To quantify direct costs and healthcare resource utilisation (HCRU) associated with acute COVID-19 in adults in England., Design: Population-based retrospective cohort study using Clinical Practice Research Datalink Aurum primary care electronic medical records linked to Hospital Episode Statistics secondary care administrative data., Setting: Patients registered to primary care practices in England., Population: 1 706 368 adults with a positive SARS-CoV-2 PCR or antigen test from August 2020 to January 2022 were included; 13 105 within the hospitalised cohort indexed between August 2020 and March 2021, and 1 693 263 within the primary care cohort indexed between August 2020 and January 2022. Patients with a COVID-19-related hospitalisation within 84 days of a positive test were included in the hospitalised cohort., Main Outcome Measures: Primary and secondary care HCRU and associated costs ≤4 weeks following positive COVID-19 test, stratified by age group, risk of severe COVID-19 and immunocompromised status., Results: Among the hospitalised cohort, average length of stay, including critical care stays, was longer in older adults. Median healthcare cost per hospitalisation was higher in those aged 75-84 (£8942) and ≥85 years (£8835) than in those aged <50 years (£7703). While few (6.0%) patients in critical care required mechanical ventilation, its use was higher in older adults (50-74 years: 8.3%; <50 years: 4.3%). HCRU and associated costs were often greater in those at higher risk of severe COVID-19 than in the overall cohort, although minimal differences in HCRU were found across the three different high-risk definitions. Among the primary care cohort, general practitioner or nurse consultations were more frequent among older adults and the immunocompromised., Conclusions: COVID-19-related hospitalisations in older adults, particularly critical care stays, were the primary drivers of high COVID-19 resource use in England. These findings may inform health policy decisions and resource allocation in the prevention and management of COVID-19., Competing Interests: Competing interests: JY, KMA, MR, LJM, CT, TM, KN, DM, Tamuno Alfred, MA and JLN are employees of Pfizer and may hold stock or stock options. KKR, TT, PP, BEB and RW are employees of Adelphi Real World, which received funds from Pfizer to conduct the study and develop the manuscript., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Osteoarthritis (OA) affects 10% of adults in the UK. Despite over one-third of people with OA experiencing chronic pain, few studies have examined the population-level impact of chronic pain associated with OA. We compared resource-use and epidemiological outcomes in patients with mild, moderate and severe chronic OA-associated pain and matched controls without known OA., Design: Retrospective, longitudinal, observational cohort study (July 2008 to June 2019)., Setting: Electronic records extracted from Clinical Practice Research Datalink GOLD primary care linked to Hospital Episode Statistics (HES)., Participants: Patients (cases; n=23 016) aged ≥18 years with chronic OA-associated pain. Controls (n=23 016) without OA or chronic pain matched on age, sex, comorbidity burden, general practitioner practice and available HES data., Interventions: None., Primary and Secondary Outcome Measures: Total healthcare resource use (HCRU), direct healthcare costs in 0-12, 12-24 and 24-36 months postindex. Secondary outcomes included incidence and prevalence of chronic OA-associated pain and pharmacological management., Results: HCRU was consistently greater in cases versus controls for all resource categories during preindex and postindex periods. Across follow-up periods, resource use was greatest in patients with severe pain. In the first 12 months postindexing, mean total costs incurred by cases were four times higher versus matched controls (£256 vs £62); costs were approximately twice as high in cases vs controls for months 12-24 (£166 vs £86) and 24-36 (£150 vs £81; all p<0.0001). The incidence of new cases of chronic pain associated with OA was 2.64 per 1000 person-years; the prevalence was 1.4%., Conclusions: This study highlights the real-world cost of chronic pain associated with OA in cases versus matched controls. We included patients with mild, moderate and severe pain associated with OA, and showed HCRU in discrete 1-year time frames. The true economic burden of pain associated with OA is likely to be considerably higher when indirect costs are considered., Competing Interests: Competing interests: GC, PC and CL are employees of Pfizer UK, which provided funding for this study; HS was an employee of Pfizer UK during the conduct of the work; RW, LM and TT are employees of Adelphi Real World, which received funding from Pfizer and Eli Lilly & Company in connection with the development of this manuscript and who are paid consultants to Pfizer and Eli Lilly & Company; RK declares a personal financial interest in his employment by the University of Nottingham, which receives funding for his salary in part from the Primary Integrated Community Solutions Ltd. In the past 36 months, RK declares consultancy through his employment with the University of Nottingham to Pfizer and Mibe; AJD declares a personal financial interest in his employment with York Medical Group (YMG), and over the past 36 months with NHS111 and RCP (London), from whom he receives payment for his work as a part-time salaried GP with YMG, and over the past 36 months as a GP on the NHS111 COVID 19 Response and the NICE Gout Committee, respectively, and in his ownership and directorship of AD Outcomes. In the past 36 months, AJD declares the following personal financial interests through medical and health economics consultancy work for AD Outcomes for: Emotive (Agency), Grunenthal (UK), Grunenthal, Pfizer (UK), Pharmacy Magazine, Spink Health, Tilray (Portugal) and RCGP, as a racecourse doctor for various racecourses (Croft, Doncaster, Market Rasen, Newcastle, Pontefract, Redcar, Southwell and Thirsk) and as a locum GP with out-of-hours, NHS111 and urgent care work for Vocare part of Totally Plc. AJD has a non-personal financial interest in his roles as a Trustee of the Primary Care Rheumatology & Musculoskeletal Medicine (PCRMM) Society (Trustee from 2017 2020) and as Trustee of the UK Gout Society (ongoing). The PCRMM Society has received funding from multiple companies for its annual conference including from Pfizer (UK) in 2020. AJD's work as a Trustee of PCRMM Society and UK Gout Society and as GP Clinical Lead for the North of England Low Back Pain Group are voluntary and unpaid; DW declares a personal financial interest in his employment by the University of Nottingham, which receives funding for his salary from the UK Government, Sherwood Forest Hospitals NHS Foundation Trust and UKRI. He is Director UKRI/Versus Arthritis Advanced Pain Discovery Platform (personal pecuniary: paid by University of Nottingham as part of job plan, funded by UKRI), Honorary Consultant Rheumatologist at Sherwood Forest Hospitals NHS Foundation Trust (personal pecuniary; paid by University of Nottingham as part of job plan, funded by Sherwood Forest Hospitals) and Professor of Rheumatology, University of Nottingham (personal pecuniary; paid by University of Nottingham). In the past 36 months, DW declares the following non-personal financial interests: research support to the University of Nottingham from Versus Arthritis and National Institute for Health and care Research; grants from Pfizer, Eli Lilly and UCB Pharma; consultancy through his employment with the University of Nottingham to Pfizer, Eli Lilly, AbbVie, AKL R&D, Galapagos, Reckitt Benckiser Health and GlaxoSmithKline Plc, and responsibilities for investigator-led grants outside the work in this presentation held by the University of Nottingham from Pfizer, Lilly and UCB Pharma, and research grants from Versus Arthritis; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events (to University of Nottingham) from Pfizer and AbbVie., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Despite the high prevalence of chronic low back pain (CLBP) and osteoarthritis (OA), few estimates of the economic cost of these conditions in England have been published. The aim of the present analysis was to characterise the economic burden of moderate-to-severe pain associated with CLBP + OA and CLBP alone compared with general population-matched controls without CLBP or OA. The primary objective was to describe the total healthcare resource use (HCRU) and direct healthcare costs associated with the target patient populations. Secondary objectives were to describe treatment patterns and surgical procedures., Methods: This was a retrospective, observational cohort study of patients receiving healthcare indicative of moderate-to-severe chronic pain associated with CLBP, with or without OA. We used linked longitudinal data from the Clinical Practice Research Datalink GOLD and Hospital Episode Statistics (HES). Patients (cases) were matched 1 : 1 with controls on age, sex, comorbidity burden, GP practice, and HES data availability., Results: The CLBP-alone cohort comprised 13 554 cases with CLBP and 13 554 matched controls; the CLBP + OA cohort comprised 7803 cases with both OA and CLBP and 7803 matched controls. Across all follow-up periods, patients with CLBP alone and those with CLBP + OA had significantly more GP consultations, outpatient attendances, emergency department visits, and inpatient stays than controls (all p  < 0.0001). By 36 months after indexing, the mean (SD) per-patient total direct healthcare cost in the CLBP-alone cohort was £5081 (£5905) for cases and £1809 (£4451) for controls ( p  < 0.0001); in the CLBP + OA cohort, the mean (SD) per-patient total direct healthcare cost was £8819 (£7143) for cases and £2428 (£4280) for controls ( p  < 0.0001)., Conclusion: Moderate-to-severe chronic pain associated with CLBP-with or without OA-has a substantial impact on patients and healthcare providers, leading to higher HCRU and costs versus controls among people with CLBP alone or together with OA., Competing Interests: GC and PC are employees of Pfizer UK. CL was an employee of Pfizer UK at the time of the work and is now employed by Pfizer Aus. HS was an employee of Pfizer UK during the conduct of the work. RW and TT are employees of Adelphi Real World, which was contracted by Pfizer UK to conduct this study. RDK declares a personal financial interest in his employment by the University of Nottingham, which receives funding for his salary in part from the Primary Integrated Community Solutions Ltd. In the past 36 months, RDK declares the following nonpersonal financial interests: consultancy through his employment with the University of Nottingham to Pfizer Ltd. and Mibe Ltd. AJD declares a personal financial interest in his employment with York Medical Group (YMG), and over the past 36 months with NHS111 and RCP (London), from whom he receives payment for his work as a part-time salaried GP with YMG, and over the past 36 months as a GP on the NHS111 COVID-19 Response and the NICE Gout Committee, respectively, and in his ownership and directorship of AD Outcomes Ltd. In the past 36 months, AJD declares the following personal financial interests through medical and health economics consultancy work for AD Outcomes Ltd. for: Emotive (Agency) Ltd., Grunenthal (UK), Grunenthal (GmbH), Pfizer (UK), Pharmacy Magazine, Spink Health, Tilray (Portugal), and RCGP, as a racecourse doctor for various racecourses (Croft, Doncaster, Market Rasen, Newcastle, Pontefract, Redcar, Southwell, and Thirsk), and as a locum GP with out-of-hours, NHS111 and urgent care work for Vocare part of Totally Plc. AJD has a nonpersonal financial interest in his roles as a Trustee of the Primary Care Rheumatology and Musculoskeletal Medicine (PCRMM) Society (Trustee from 2017 to 2020) and as Trustee of the UK Gout Society (ongoing). The PCRMM Society has received funding from multiple companies for its annual conference including from Pfizer (UK) in 2020. AJD's work as a Trustee of the PCRMM Society and the UK Gout Society and as GP Clinical Lead for the North of England Low Back Pain Group are voluntary and unpaid; DAW declares a personal financial interest in his employment by the University of Nottingham, which receives funding for his salary from the UK Government, Sherwood Forest Hospitals NHS Foundation Trust, and UKRI. He is Director of the UKRI/Versus Arthritis Advanced Pain Discovery Platform (personal pecuniary: paid by the University of Nottingham as part of job plan, funded by UKRI), Honorary Consultant Rheumatologist at Sherwood Forest Hospitals NHS Foundation Trust (personal pecuniary; paid by the University of Nottingham as part of job plan, funded by Sherwood Forest Hospitals), and Professor of Rheumatology, University of Nottingham (personal pecuniary; paid by the University of Nottingham). In the past 36 months, DAW declares the following nonpersonal financial interests: research support to the University of Nottingham from Versus Arthritis and National Institute for Health and care Research; grants from Pfizer Ltd., Eli Lilly, and UCB Pharma; consultancy through his employment with the University of Nottingham to Pfizer Ltd., Eli Lilly, AbbVie Ltd., AKL R&D, Galapagos, Reckitt Benckiser Health Ltd., and GlaxoSmithKline Plc, and responsibilities for investigator-led grants outside the work in this presentation held by the University of Nottingham from Pfizer Ltd., Lilly Ltd. and UCB Pharma, and research grants from Versus Arthritis; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events (to University of Nottingham) from Pfizer Ltd. and AbbVie. The authors declare that they have no conflicts of interest., (Copyright © 2023 Greg Coates et al.)

Purpose: Chronic obstructive pulmonary disease (COPD) exacerbations are associated with significant morbidity and mortality and increased economic healthcare burden for patients with COPD. Long-acting muscarinic antagonist (LAMA)/long-acting β 2 -agonist (LABA) dual therapy is recommended for patients receiving mono-bronchodilator therapy who experience exacerbations or ongoing breathlessness. This study compared two single-inhaler LAMA/LABA dual therapies, umeclidinium/vilanterol (UMEC/VI) and indacaterol/glycopyrronium (IND/GLY), on moderate-to-severe exacerbation rates in patients with COPD in England., Patients and Methods: This retrospective cohort study used linked primary care electronic health record data (Clinical Practice Research Datalink-Aurum) and secondary care data (Hospital Episode Statistics) to assess outcomes for patients with COPD who had a first prescription for single-inhaler UMEC/VI or IND/GLY (index date) between 1 January 2015 and 30 September 2019 (indexing period). Analyses compared UMEC/VI and IND/GLY on moderate-to-severe, moderate, and severe exacerbations, healthcare resource utilization (HCRU), and direct costs at 6, 12, 18, and 24 months, and time-to-first on-treatment exacerbation up to 24 months post-index date. Following inverse probability of treatment weighting (IPTW), non-inferiority and superiority of UMEC/VI versus IND/GLY were assessed., Results: In total, 12,031 patients were included, of whom 8753 (72.8%) were prescribed UMEC/VI and 3278 (27.2%) IND/GLY. After IPTW, for moderate-to-severe exacerbations, weighted rate ratios were <1 at 6, 12, and 18 months and equal to 1 at 24 months for UMEC/VI; around the null value for moderate exacerbations and <1 at all timepoints for severe exacerbations. UMEC/VI showed lower HCRU incidence rates than IND/GLY for all-cause Accident and Emergency visits and COPD-related inpatient stays and associated all-cause costs at 6 months post-indexing. Time-to-triple therapy was similar for both treatments., Conclusion: UMEC/VI demonstrated non-inferiority to IND/GLY in moderate-to-severe exacerbation reduction at 6, 12 and 18 months. These results support previous findings demonstrating similarity between UMEC/VI and IND/GLY on reduction of moderate-to-severe exacerbations., Competing Interests: GR, CCo, KJR, and ASI are employees of GSK and hold stocks and shares at GSK. ASI also holds an unpaid faculty position at McMaster University. AC was an employee of GSK and held stocks and shares at GSK at the time of the study. FJFH is an employee of the Translational Lung Research Center Heidelberg, part of the Germany lung research Foundation (DZL). JKQ holds a position at Imperial College London and has received grants or contracts paid to this institution outside the scope of the submitted work from the Medical Research Council, Health Data Research UK, GSK, Boehringer Ingelheim, Asthma + Lung UK, and AstraZeneca. JKQ has also received payment for advisory board participation, teaching or lectures from GSK, AstraZeneca, and Insmed. CCa, TT, RWo, and RWi are employees of Adelphi Real World. VB and JY were employees of Adelphi Real World at the time of the study. Adelphi Real World is a business that provides consulting and other research services to pharmaceutical, device, government, and non-government organizations which received funding from GSK to conduct the study. Adelphi Real World employees work with a variety of companies and organizations and are expressly prohibited from receiving any payment or honoraria directly from these organizations for services rendered. The authors report no other conflicts of interest in this work., (© 2023 Requena et al.)

Purpose: Routinely collected healthcare data on the comparative effectiveness of the long-acting muscarinic antagonist/long-acting β 2 -agonist combination umeclidinium/vilanterol (UMEC/VI) versus tiotropium bromide/olodaterol (TIO/OLO) for chronic obstructive pulmonary disease (COPD) is limited. This study compared rescue medication prescriptions in patients with COPD in England receiving UMEC/VI versus TIO/OLO., Patients and Methods: This retrospective cohort study used primary care data from the Clinical Practice Research Datalink Aurum database linked with secondary care administrative data from Hospital Episode Statistics. Patients with a COPD diagnosis at age ≥35 years were included (indexed) following initiation of single-inhaler UMEC/VI or TIO/OLO between July 1, 2015, and September 30, 2019. Outcomes included the number of rescue medication prescriptions at 12-months (primary), and at 6-, 18- and 24-months (secondary), adherence at 6-, 12-, 18- and 24-months post-index, defined as proportion of days covered ≥80% (secondary), and time-to-initiation of triple therapy (exploratory). Inverse probability of treatment weighting (IPTW) was used to balance potential confounding baseline characteristics. Superiority of UMEC/VI versus TIO/OLO for the primary outcome of rescue medication prescriptions was assessed using an intention-to-treat analysis with a p-value < 0.05., Results: In total, 8603 patients were eligible (UMEC/VI: n = 6536; TIO/OLO: n = 2067). Following IPTW, covariates were well balanced across groups. Patients initiating UMEC/VI had statistically significantly fewer (mean [standard deviation]; p-value) rescue medication prescriptions versus TIO/OLO in both the unweighted (4.84 [4.78] vs 5.68 [5.00]; p < 0.001) and weighted comparison (4.91 [4.81] vs 5.48 [5.02]; p = 0.0032) at 12 months; consistent results were seen at all timepoints. Adherence was numerically higher for TIO/OLO versus UMEC/VI at all timepoints. Time-to-triple therapy was similar between treatment groups., Conclusion: UMEC/VI was superior to TIO/OLO in reducing rescue medication prescriptions at 12 months after treatment initiation in a primary care cohort in England, potentially suggesting improvements in symptom control with UMEC/VI compared with TIO/OLO., Competing Interests: GR, AC, CC, KJR, and ASI are employees of GSK and hold stock and shares at GSK. ASI also holds an unpaid faculty position at McMaster University. FH is an employee of the Translational Lung Research Center Heidelberg, part of the Germany lung research Foundation (DZL). JKQ holds a position at Imperial College London. CMC, TT, RWo, and RWi are employees of Adelphi Real World. VB is currently an employee of Bayer AG UK, and holds stock and shares in Bayer AG UK. VB and JY were employees of Adelphi Real World at the time of the study. Adelphi Real World is a business that provides consulting and other research services to pharmaceutical, device, government, and non-government organizations which received funding from GSK to conduct the study. Adelphi Real World employees work with a variety of companies and organizations and are expressly prohibited from receiving any payment or honoraria directly from these organizations for services rendered. The authors report no other conflicts of interest in this work., (© 2023 Requena et al.)

Objective: Despite the prevalence of osteoarthritis (OA) in England, few studies have examined the health economic impact of chronic pain associated with OA. The aim of this study was to compare outcomes in patients with moderate-to-severe chronic pain associated with OA and matched controls without known OA., Design: Retrospective, longitudinal, observational cohort study., Setting: Electronic records extracted from the Clinical Practice Research Datalink GOLD primary care database linked to Hospital Episode Statistics (HES) data set., Participants: Patients (cases; n=5931) ≥18 years and with existing diagnosis of OA and moderate-to-severe pain associated with their OA, and controls matched on age, sex, comorbidity burden, general practitioner (GP) practice and availability of HES data., Interventions: None., Primary and Secondary Outcome Measures: Total healthcare resource use (HCRU) and direct healthcare costs during 0-6, 0-12, 0-24 and 0-36 months of follow-up. Secondary outcomes measures included pharmacological management and time to total joint replacement., Results: Patients with moderate-to-severe chronic pain associated with OA used significantly more healthcare services versus matched controls, reflected by higher HCRU and significantly higher direct costs. During the first 12 months' follow-up, cases had significantly more GP consultations, outpatient attendances, emergency department visits and inpatient stays than matched controls (all p<0.0001). Total mean costs incurred by cases during 0-12 months' follow-up were five times higher in cases versus controls (mean (SD): £4199 (£3966) vs £781 (£2073), respectively). Extensive cycling through pharmacological therapies was observed; among cases, 2040 (34.4%), 1340 (22.6%), 841 (14.2%), 459 (7.7%) and 706 (11.9%) received 1-5, 6-10, 11-15, 16-20 and >20 lines of therapy, respectively., Conclusions: This wide-ranging, longitudinal, observational study of real-world primary and secondary care data demonstrates the impact of moderate-to-severe chronic pain associated with OA in patients compared with matched controls. Further studies are required to fully quantify the health economic burden of moderate-to-severe pain associated with OA., Competing Interests: Competing interests: GC and PC are employees of Pfizer UK, which provided funding for this study. CL was an employee of Pfizer UK at the time of the work and is now employed by Pfizer Australia. HS was an employee of Pfizer UK during the conduct of the work. RW and TT are employees of Adelphi Real World, which received funding to conduct this study. RK declares a personal financial interest in his employment by the University of Nottingham, which receives funding for his salary in part from the Primary Integrated Community Solutions. In the past 36 months, RK declares consultancy through his employment with the University of Nottingham to Pfizer and Mibe. AJD declares a personal financial interest in his employment with York Medical Group (YMG), and over the past 36 months with NHS111 and RCP (London), from whom he receives payment for his work as a part-time salaried general practitioner (GP) with YMG, and over the past 36 months as a GP on the NHS111 COVID-19 Response and the NICE Gout Committee, respectively, and in his ownership and directorship of AD Outcomes. In the past 36 months, AJD declares the following personal financial interests through medical and health economics consultancy work for AD Outcomes for: Emotive (Agency), Grunenthal (UK), Grunenthal (GmbH), Pfizer (UK), Pharmacy Magazine, Spink Health, Tilray (Portugal) and RCGP, as a racecourse doctor for various racecourses (Croft, Doncaster, Market Rasen, Newcastle, Pontefract, Redcar, Southwell and Thirsk) and as a locum GP with out-of-hours, NHS111 and urgent care work for Vocare part of Totally. AJD has a non-personal financial interest in his roles as a Trustee of the Primary Care Rheumatology & Musculoskeletal Medicine (PCRMM) Society (Trustee from 2017 to 2020) and as Trustee of the UK Gout Society (ongoing). The PCRMM Society has received funding from multiple companies for its annual conference including from Pfizer (UK) in 2020. AJD’s work as a Trustee of PCRMM Society and UK Gout Society and as GP Clinical Lead for the North of England Low Back Pain Group are voluntary and unpaid. DW declares a personal financial interest in his employment by the University of Nottingham, which receives funding for his salary from the UK Government, Sherwood Forest Hospitals NHS Foundation Trust and UKRI. He is Director UKRI/Versus Arthritis Advanced Pain Discovery Platform (personal pecuniary: paid by University of Nottingham as part of job plan, funded by UKRI), Honorary Consultant Rheumatologist at Sherwood Forest Hospitals NHS Foundation Trust (personal pecuniary; paid by University of Nottingham as part of job plan, funded by Sherwood Forest Hospitals) and Professor of Rheumatology, University of Nottingham (personal pecuniary; paid by University of Nottingham). In the past 36 months DW declares the following non-personal financial interests: research support to the University of Nottingham from Versus Arthritis and National Institute for Health and care Research; grants from Pfizer, Eli Lilly, and UCB Pharma; consultancy through his employment with the University of Nottingham to Pfizer, Eli Lilly, AbbVie, AKL R&D, Galapagos, Reckitt Benckiser Health, and GlaxoSmithKline, and responsibilities for investigator-led grants outside the work in this presentation held by the University of Nottingham from Pfizer, Lilly and UCB Pharma, and research grants from Versus Arthritis; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events (to University of Nottingham) from Pfizer and AbbVie., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To compare adherence to once-daily umeclidinium/vilanterol (UMEC/VI), a long-acting muscarinic antagonist/long-acting β 2 -agonist (LAMA/LABA), and twice-daily inhaled corticosteroids (ICS)/LABA single-inhaler dual therapy in patients with chronic obstructive pulmonary disease (COPD) in a primary care cohort in England., Patients and Methods: Active comparator, new-user, retrospective cohort study using CPRD-Aurum primary care data and linked Hospital Episode Statistics secondary care administrative data. Patients without exacerbations in the previous year were indexed on first/earliest prescription date of once-daily UMEC/VI or twice-daily ICS/LABA as initial maintenance therapy between July 2014-September 2019. Primary outcome: medication adherence at 12 months post-index, defined as proportion of days covered (PDC) ≥80%. PDC represented proportion of time over the treatment duration that the patient was theoretically in possession of the medication. Secondary outcomes: adherence at 6, 18, and 24 months post-index, time-to-triple therapy, time-to-first on-treatment COPD exacerbation, COPD-related and all-cause healthcare resource utilization (HCRU), and direct health-care costs. A propensity score was generated and inverse probability of treatment weighting (IPTW) was used to balance potential confounders. Superiority was defined as >0% difference between treatment groups., Results: In total, 6815 eligible patients were included (UMEC/VI:1623; ICS/LABA:5192). At 12 months post-index, weighted odds of a patient being adherent were significantly greater with UMEC/VI versus ICS/LABA (odds ratio [95% CI]: 1.71 [1.09, 2.66]; p=0.0185), demonstrating superiority of UMEC/VI. Patients taking UMEC/VI were statistically significantly more adherent than those taking ICS/LABA at 6, 18, and 24 months post-index (p<0.05). Differences in time-to-triple therapy, time-to-moderate COPD exacerbations, HCRU, and direct medical costs were not statistically significant between treatments after IPTW was applied., Conclusion: At 12 months post-treatment initiation, once-daily UMEC/VI was superior to twice-daily ICS/LABA in medication adherence among patients with COPD without exacerbations in the previous year, newly initiating dual maintenance therapy in England. The finding was consistent at 6, 18, and 24 months., Competing Interests: AC, GR, CC, KJR, and ASI are employees of GSK and hold stock and shares at GSK. ASI also holds an unpaid faculty position at McMaster University in Canada. FH is an employee of the Translational Lung Research Center Heidelberg, part of the Germany lung research Foundation (DZL) and he also reports lecturing and adboard activities for GSK. JKQ holds a position at Imperial College London. CMC, TT, RWo, and RWi are employees of Adelphi Real World. VB is currently an employee of Bayer AG UK and holds stock and shares there. VB and JY were employees of Adelphi Real World at the time of the study. Adelphi Real World is a business that provides consulting and other research services to pharmaceutical, device, government, and non-government organizations, which received funding from GSK to conduct the study. Adelphi Real World employees work with a variety of companies and organizations and are expressly prohibited from receiving any payment or honoraria directly from these organizations for services rendered. The authors report no other conflicts of interest in this work., (© 2023 Czira et al.)

Purpose: Selection of treatments for patients with chronic obstructive pulmonary disease (COPD) may impact clinical outcomes, healthcare resource use (HCRU) and direct healthcare costs. We aimed to characterize these outcomes along with treatment patterns, for patients with COPD following initiation of single-inhaler long-acting muscarinic antagonist/long-acting β 2 -agonist (LAMA/LABA) dual therapy in the primary care setting in England., Patients and Methods: This retrospective cohort study used linked primary care electronic medical record data (Clinical Practice Research Datalink-Aurum) and secondary care administrative data (Hospital Episode Statistics) in England to assess outcomes for patients with COPD who had a prescription for one of four single-inhaler LAMA/LABA dual therapies between 1st June 2015-31st December 2018 (indexing period). Outcomes were assessed during a 12-month follow-up period from the index date (date of earliest prescription of a single-inhaler LAMA/LABA within the indexing period). Incident users were those without previous LAMA/LABA dual therapy prescriptions prior to index; this manuscript focuses on a subset of incident users: non-triple therapy users (patients without concomitant inhaled corticosteroid use at index)., Results: Of 10,991 incident users included, 9888 (90.0%) were non-triple therapy users, indexed on umeclidinium/vilanterol (n=4805), aclidinium/formoterol (n=2109), indacaterol/glycopyrronium (n=1785) and tiotropium/olodaterol (n=1189). At 3 months post-index, 63.3% of non-triple therapy users remained on a single-inhaler LAMA/LABA, and 22.1% had discontinued inhaled therapy. Most patients (86.9%) required general practitioner consultations in the first 3 months post-index. Inpatient stays were the biggest contributor to healthcare costs. Acute exacerbations of COPD (AECOPDs), adherence, time-to-triple therapy, time-to-first on-treatment moderate-to-severe AECOPD, time-to-index treatment discontinuation, HCRU and healthcare costs were similar across indexed therapies., Conclusion: Patients initiating treatment with single-inhaler LAMA/LABA in primary care in England were unlikely to switch treatments in the first three months following initiation, but some may discontinue respiratory medication. Outcomes were similar across indexed treatments., Competing Interests: AC, GR, CC, and ASI are employees of GSK and hold stock and shares at GSK. ASI is also a part-time member of the McMaster university faculty. TT, RWo and RWi are employees of Adelphi Real World, which received funding from GSK to conduct the study, but not for manuscript development. VB was an employee of Adelphi Real World at the time of the study, and is currently an employee of Bayer AG UK, and holds stock and shares in Bayer AG UK. Adelphi Real World is a business that provides consulting and other research services to pharmaceutical, device, government, and non-government organizations. Adelphi Real World employees work with a variety of companies and organizations and are expressly prohibited from receiving any payment or honoraria directly from these organizations for services rendered. The authors report no other conflicts of interest in this work., (© 2023 Requena et al.)

Purpose: Triple therapy comprising a long-acting muscarinic antagonist, long-acting β 2 -agonist and inhaled corticosteroid is recommended for patients with chronic obstructive pulmonary disease (COPD) who continue to experience frequent exacerbations or symptoms whilst receiving dual therapy. Adherence and persistence to multiple-inhaler triple therapy (MITT) is known to be poor. This study assessed comparative adherence to single-inhaler triple therapy (SITT) versus MITT in a real-world setting in England., Patients and Methods: This was a retrospective cohort study using linked primary care (Clinical Practice Research Datalink Aurum) and secondary care (Hospital Episode Statistics [HES] Admitted Patient Care) data to identify patients with COPD who were newly initiated on SITT or MITT between November 2017 and June 2019. Eligible patients were aged ≥35 years and had a forced expiratory volume in 1 second/forced vital capacity <0.7, linkage to HES and continuous registration with a general practitioner for 12 months pre- and 6 months post-initiation. Inverse probability of treatment weighting was used to balance baseline characteristics between cohorts. Adherence was measured using the proportion of days covered by days' supply of SITT or MITT prescriptions. Persistence was measured with a gap of >30 days between the end of a prescription and the following refill used to determine non-persistence., Results: Overall, 4080 SITT and 6579 MITT users comprised the study cohort. After weighting, the baseline characteristics between the cohorts were comparable (absolute standardized mean difference <10%). SITT users had significantly higher adherence than MITT users at 6, 12, and 18 months post-initiation (p<0.001 for all comparisons). Median persistence was higher among SITT users than MITT users (5.09 months vs 0.99 months)., Conclusion: Patients with COPD in England initiating SITT had significantly better adherence and persistence compared with MITT initiators. These improvements continued at least 18 months following treatment initiation., Competing Interests: Kieran J Rothnie, Alexandrosz Czira, Chris Compton, Kirill Nikitin, Raj Sharma, and Afisi S Ismaila are employees of and/or hold stocks/shares in GSK. Neil Snowise is a previous employee of GSK and holds GSK stocks/shares. Victoria Banks, Robert Wood, Theo Tritton, Olivia Massey, and Rosie Wild are employees of Adelphi Real World. Adelphi Real World received funding from GSK to conduct the study. David MG Halpin reports personal fees from AstraZeneca, personal fees and non-financial support from Boehringer Ingelheim, personal fees from Aerogen, Chiesi, CSL Behring, and GSK, personal fees and non-financial support from Novartis, and personal fees from Pfizer and Sanofi. Claus F Vogelmeier has received research grants from AstraZeneca, Boehringer Ingelheim, CSL Behring, Chiesi, GSK, and Grifols, and has given presentations at symposia and/or served on scientific advisory boards sponsored by Aerogen, AstraZeneca, Boehringer Ingelheim, CSL Behring, Chiesi, GSK, Grifols, Menarini, Novartis, and Nuvaira. The authors report no other conflicts of interest in this work., (© 2022 Halpin et al.)

Introduction: Treatment pathways of patients with chronic obstructive pulmonary disease (COPD) receiving single-device dual therapies in England remain unclear. This study describes the characteristics of patients with COPD before initiating treatment with a single-device inhaled corticosteroid/long-acting β 2 -agonist (ICS/LABA) in primary care in England., Methods: This is a retrospective, descriptive study of linked primary and secondary healthcare data (Clinical Practice Research Datalink Aurum, Hospital Episode Statistics). Patients with COPD were indexed on first prescription of fixed-dose, single-device ICS/LABA (June 2015-December 2018). Demographics, clinical characteristics, prescribed treatments, healthcare resource use (HCRU) and direct healthcare costs were assessed over 12 months pre-index. Incident users (indexed on first ever prescription) could be non-triple users (no concomitant long-acting muscarinic antagonist at index); a subset were initial maintenance therapy (IMT) users (no history of pre-index maintenance therapy)., Results: Overall, 13 451 incident users (non-triple users: 7448, 55.4%; IMT users: 5162, 38.4%) were indexed on beclomethasone dipropionate/formoterol (6122, 45.5%), budesonide/formoterol (2703, 20.1%) or Other ICS/LABA combinations (4626, 34.4%). Overall, 20.8% of incident users had comorbid asthma and 42.6% had ≥1 moderate-to-severe acute exacerbation of COPD pre-index. Baseline characteristics were similar across indexed therapies. At 3 months pre-index, 45.3% and 35.4% of non-triple and IMT users were receiving maintenance treatment. HCRU and direct healthcare costs were similar across indexed treatments. Prescribing patterns varied regionally., Conclusion: Patient characteristics, prior treatments, prior COPD-related HCRU and direct healthcare costs were similar across single-device ICS/LABAs in primary care in England. A high proportion of patients were not receiving any respiratory medication pre-index, indicating that prescribing in primary care in England is more closely aligned with national guidelines than global treatment strategies. Comorbid asthma may have influenced prescribing decisions. Less than half of users had preindex exacerbations, suggesting that ICS/LABA is not being prescribed principally based on exacerbation history., Competing Interests: Competing interests: AC, GR, CC, MD and ASI are employees of GSK and hold stock and shares at GSK. ASI is also a part-time member of the McMaster University faculty. VB, TT, RWo and RWi are employees of Adelphi Real World (or were at the time of the study), which received funding from GSK to conduct the study. Adelphi Real World is a business that provides consulting and other research services to pharmaceutical, device, government and non-government organisations. Adelphi Real World employees work with a variety of companies and organisations and are expressly prohibited from receiving any payment or honoraria directly from these organisations for services rendered., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Treatment pathways of patients with chronic obstructive pulmonary disease (COPD) receiving single-inhaler dual therapies remain unclear. We aimed to describe characteristics, prescribed treatments, healthcare resource use (HCRU) and costs of patients with COPD who initiated single-inhaler long-acting muscarinic antagonist/long-acting β 2 -agonist (LAMA/LABA) dual therapy in primary care in England., Patients and Methods: Retrospective study using linked data from Clinical Practice Research Datalink Aurum and Hospital Episode Statistics datasets. Patients with COPD with ≥1 single-inhaler LAMA/LABA prescription between June 2015 and December 2018 (index) were included. Demographic and clinical characteristics, prescribed treatments, HCRU and costs were evaluated in the 12 months pre-index. Data are presented for patients not receiving concomitant inhaled corticosteroids at index (non-triple users)., Results: Of 10,991 patients initiating LAMA/LABA, 9888 were non-triple users, of whom 21.3% (n=2109) received aclidinium bromide/formoterol, 18.1% (n=1785) received indacaterol/glycopyrronium, 12.0% (n=1189) received tiotropium bromide/olodaterol and 48.6% (n=4805) received umeclidinium/vilanterol. Demographic and clinical characteristics were similar across indexed therapies. LAMA monotherapy was the most frequently prescribed respiratory therapy at 12 (18.4-25.8% of patients) and 3 months (23.9-33.7% of patients) pre-index across indexed therapies; 42.5-59.0% of patients were prescribed no respiratory therapy at these time points. COPD-related HCRU during the 12 months pre-index was similar across indexed therapies (general practitioner consultations: 62.0-68.6% patients; inpatient stays: 19.3-26.1% patients). Pre-index COPD-related costs were similar across indexed therapies, with inpatient stays representing the highest contribution. Mean total direct annual COPD-related costs ranged from £805-£1187., Conclusion: Characteristics of patients newly initiating single-inhaler LAMA/LABA dual therapy were highly consistent across indexed therapies. As half of non-triple users were not receiving respiratory therapy one year prior to LAMA/LABA initiation, there may be an opportunity for early optimization of treatment to relieve clinical burden versus current prescribing patterns in primary care in England., Competing Interests: AC, GR, CC, MD and ASI are employees of GSK and hold stock and shares at GSK. ASI is also a part-time member of the McMaster university faculty. VB, TT, RWo and RWi are employees of Adelphi Real World (or were at the time of the study), which received funding from GSK to conduct the study. Adelphi Real World is a business that provides consulting and other research services to pharmaceutical, device, government, and non-government organizations. Adelphi Real World employees work with a variety of companies and organizations and are expressly prohibited from receiving any payment or honoraria directly from these organizations for services rendered. Since concluding the study, VB left employment with Adelphi Real World and started full time employment with Bayer PLC., (© 2022 Requena et al.)

Aims: To determine the proportion of UK patients with type 2 diabetes (T2D) who meet the cardiovascular (CV) or combined CV/core eligibility criteria used for the CV outcome trials (CVOTs) of UK-marketed glucagon-like peptide-1 receptor agonists (GLP-1RAs) showing CV benefit (dulaglutide in REWIND, liraglutide in LEADER and injectable semaglutide in SUSTAIN-6)., Materials and Methods: Adults with T2D on/before June 2018 were identified from the UK Clinical Practice Research Datalink GOLD primary care database and linked to Hospital Episode Statistics data (Protocol 19&#95;262). Patient CV and clinical data were evaluated against the CVOT eligibility criteria. Data were analysed descriptively., Results: The study cohort (N = 33 118 patients with T2D) had a mean (standard deviation) age of 66.0 (13.3) years and 56.6% were male. Almost two-thirds (64.5%) of the study cohort met the CV criteria for REWIND, versus 43.0% for both LEADER and SUSTAIN-6. The proportions of the study cohort who met the CVOT criteria of "established CV disease" and "CV risk factors only" for REWIND were 22.4% and 42.1%, respectively, versus 38.7% and 4.3%, respectively, for both LEADER and SUSTAIN-6. The proportions of patients satisfying both CV and core criteria were 44.4% for REWIND, 13.3% for LEADER and 13.5% for SUSTAIN-6. Study findings remained consistent when restricted to GLP-1RA users., Conclusions: REWIND captured a trial population more representative of the real-world T2D population in the United Kingdom than LEADER or SUSTAIN-6 with regard to both CV and combined CV/core eligibility criteria., (© 2021 Eli Lilly and Company. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Objective: The aim of the study was to improve understanding of adherence and persistence to biologics, and their association with health-care resource utilization (HCRU), in Japanese patients with moderate to severe ulcerative colitis (UC)., Methods: Data were from Medical Data Vision, a secondary care administrative database. A retrospective, longitudinal cohort analysis was conducted of data from UC patients initiating biologic therapy between August 2013 and July 2016. Data collected for 2 years prior (baseline) and 2 years after (follow-up) the index date were evaluated. Patients completing biologic induction were identified, and adherence/persistence to biologic therapy calculated. HCRU, steroid, and immunosuppressant use during baseline and follow-up were assessed. Biologic switching during the follow-up was evaluated. Descriptive statistics (e.g., means and proportions) were obtained and inferential analyses (from Student's t tests, Fisher's exact tests, χ 2 tests, the Cox proportional hazard model, and negative binomial regression) were performed., Results: The analysis included 649 patients (adalimumab: 265; infliximab: 384). Biologic induction was completed by 80% of patients. Adherence to adalimumab was higher than that to infliximab ( p < 0.001). Persistence at 6, 12, 18, and 24 months was higher with infliximab than with adalimumab ( p < 0.05). Overall, gastroenterology outpatient visits increased, and hospitalization frequency and duration decreased, from baseline to follow-up. UC-related hospitalizations were fewer and shorter, and endoscopies fewer, in persistent than in nonpersistent patients, although persistent patients made more outpatient visits than nonpersistent patients. Hospitalization duration was lower in persistent than nonpersistent patients. Approximately 50% of patients received an immunosuppressant during biologic therapy; 5% received a concomitant steroid during biologic therapy. Overall, 17% and 3% of patients, respectively, received 2nd line and 3rd line biologics., Conclusions: Poor biologic persistence was associated with increased non-medication-associated HCRU. Effective treatments with high persistence levels and limited associated HCRU are needed in UC., Competing Interests: Joseph C. Cappelleri, Irene Modesto, Marco DiBonaventura, Timothy W. Smith, Sean Gardiner, and Irene Modesto are stockholders and employees of Pfizer Inc (who funded this study). At the time of conducting the study, Danielle Bargo and Takanori Tsuchiya were employees of Pfizer Inc. Theo Tritton and Tim Holbrook are employed by Adelphi Real World, which received funding from Pfizer in connection with the development of this manuscript. At the time of conducting the study, Daniel Bluff was an employee of Adelphi Real World., (Copyright © 2021 by S. Karger AG, Basel.)

Background: Major depressive disorder (MDD) is a chronic recurrent or episodic psychiatric illness that can be successfully treated with oral antidepressants, yet one-in-three patients do not respond to currently-available treatments. According to the FDA and EMA, patients are considered to have treatment-resistant depression (TRD) when their MDD fails to respond adequately to ≥2 successive antidepressants in a single episode., Aims: To describe current clinical management of patients with MDD and TRD in England, including treatment strategies and referral to secondary mental healthcare., Method: A retrospective cohort study of adult patients identified in primary care with diagnosed MDD, including a TRD subgroup (≥2 treatment failures as determined by treatment dynamics) was conducted using the Clinical Practice Research Datalink GOLD primary care database linked to Hospital Episode Statistics and Mental Health Services Data Set data (Protocol 19&#95;019R)., Results: 41,375 patients with MDD (mean age 44yrs, 62% female, median follow-up 29mths); and 1,051 (3%) patients with TRD were identified. Mean time-to-TRD was 18 months. Most patients (>99%) received first-line antidepressant monotherapy. Following TRD criteria being met, antidepressant monotherapy use remained most frequent from TRD first-line (70%) to fifth-line (48%). Dual/triple antidepressant use remained constant (range:24%-26%), while augmented antidepressant use increased from TRD first-line (7%) to third-line (17%). Minimal non-pharmacological therapies were observed., Conclusions: Despite current clinical guidelines recommending a stepwise approach, many patients frequently cycle through numerous antidepressants with similar mechanisms of action and efficacy. These findings indicate a high unmet need for new treatments that improve outcomes in these patient populations., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Limited real-world data exist on healthcare resource utilization (HCRU) and associated costs of patients with heart failure (HF) with reduced ejection fraction (HFrEF) and preserved EF (HFpEF), including urgent HF visits, which are assumed to be less burdensome than HF hospitalizations (hHFs) HYPOTHESIS: This study aimed to quantify the economic burden of HFrEF and HFpEF, via a retrospective, longitudinal cohort study, using IBM® linked claims/electronic health records (Commercial and Medicare Supplemental data only)., Methods: Adult patients, indexed on HF diagnosis (ICD-10-CM: I50.x) from July 2012 through June 2018, with 6-month minimum baseline period and varying follow-up, were classified as HFrEF (I50.2x) or HFpEF (I50.3x) according to last-observed EF-specific diagnosis. HCRU/costs were assessed during follow-up., Results: About 109 721 HF patients (22% HFrEF, 31% HFpEF, 47% unclassified EF; median 18 months' follow-up) were identified. There were 3.2 all-cause outpatient visits per patient-month (HFrEF, 3.3; HFpEF, 3.6); 69% of patients required inpatient stays (HFrEF, 80%; HFpEF, 78%). Overall, 11% of patients experienced hHFs (HFrEF, 23%; HFpEF, 16%), 9% experienced urgent HF visits (HFrEF, 15%; HFpEF, 12%); 26% were hospitalized less than 30 days after first urgent HF visit versus 11% after first hHF. Mean monthly total direct healthcare cost per patient was $9290 (HFrEF, $11 053; HFpEF, $7482)., Conclusions: HF-related HCRU is substantial among contemporary real-world HF patients in US Commercial or Medicare supplemental health plans. Patients managed in urgent HF settings were over twice as likely to be hospitalized within 30 days versus those initially hospitalized, suggesting urgent HF visits are important clinical events and quality improvement targets., (© 2021 The Authors. Clinical Cardiology published by Wiley Periodicals LLC.)

Introduction: Prophylactic treatment of severe hemophilia A is burdensome, requiring frequent intravenous injections. Extended half-life (EHL) factor VIII replacement therapies offer longer intervals between infusions while still meeting efficacy and safety outcomes; however, patient perspectives following long-term use of such products in the real-world remain unknown., Objective: We aimed to explore the importance of infusion frequency and the potential benefits of reduced infusion frequency among patients receiving prophylactic treatment with an EHL product (BAY 94-9027)., Methods: Patients with severe hemophilia A participating in the PROTECT VIII extension study were invited to participate in a semi-structured, concept elicitation 'exit' interview to discuss their experiences. Participants were recruited from Israel, The Netherlands, and the US. Interview transcripts were translated into English and analyzed using thematic analysis methods., Results: Sixteen participants (29-68 years of age) infusing with BAY 94-9027 once every 7 days, once every 5 days, or twice weekly were interviewed. Participants reported infusion frequency (alongside efficacy) as the most important treatment attribute influencing their satisfaction with therapy. Patient-reported benefits of reduced infusion frequency and longer duration of factor coverage included greater ability to participate in physical activities; better vein health; less time to schedule and administer factor VIII; reduced impact on work; and improved emotional well-being., Conclusions: This study provides rich insights into the experiences of patients with EHL products and the value of reduced infusion frequency. Such data could be of value to a range of stakeholders (e.g. regulators, payers) and facilitate patient-clinician discussions to promote tailored treatment decisions.

Objectives: Individuals with tuberous sclerosis complex (TSC) experience a wide range of health impacts, including epileptic seizures, negatively impacting their health-related quality of life (HRQoL). Health state utility values (HSUVs) are index values representing HRQoL and are used as key inputs for health economic analyses. Such data are currently very limited in the TSC population. The objective of this study was to generate HSUVs for TSC health states, defined by the number and type of seizures experienced in the previous week, and to compare with UK normative values., Methods: This cross-sectional study involved 186 participants (individuals with TSC = 61, caregivers reporting for individuals with TSC = 125) from Europe and North America who completed a web-based survey. Participants completed the [EuroQol - 5 dimensions - 3 levels] (self-report version for individuals with TSC or proxy version 1 for caregivers)., Results: The mean age of individuals with TSC was 27.3 years (self-reported: 41.3 years, caregiver-reported: 20.5 years); 56% were males. Most individuals with TSC (71%) reported experiencing between one and ten seizures in the week prior to participating in the study. The most frequently reported type of seizure was focal: simple partial (50%). Across all participants (combined self-report and caregiver-report), the mean HSUV was 0.474 (95% confidence interval [CI]: 0.424-0.524), significantly lower than the UK norm (0.856, 95%CI: 0.848-0.864) [1]. Mean HSUV and HRQoL scores were consistently lower when reported by caregivers than when self-reported by individuals with TSC (HSUV = 0.351 vs. 0.727). This is in part because caregivers reported for individuals with TSC who experienced more frequent and severe seizures than those who were able to self-report. HSUVs incrementally decreased with the experience of more frequent (1-5 per week: HSUV = 0.666 vs. >20: HSUV = 0.290) and more severe seizures (focal: simple partial: HSUV = 0.450 vs. generalized: convulsive: HSUV = 0.194)., Conclusions: The HRQoL and HSUV index scores indicate substantial impairment among individuals with TSC; HSUVs were shown to decrease considerably with increases in seizure frequency or seizure severity, indicating that more burdensome seizure health states are associated with poorer HRQoL., (Copyright © 2018 Elsevier Inc. All rights reserved.)

On the surface, higher education has served the nation well. An increasing percentage of adults have attended college. Information is widely and freely available to most citizens; the economy is steady; and technological developments appear to offer ever-increasing improvements in the quality of life. Looking just beneath the surface, though, one realizes that knowledge is fragmented, access to knowledge is not universal, and there are no guarantees that past successes will sustain the future. Thus, it is worth examining how higher learning has contributed to the general prosperity and how education might evolve to address society's evident problems. This essay discusses the core approach of liberal arts in the context of the value--and obstacles--of integration across the traditional disciplines. Critical to the natural sciences is a firm grounding in the central importance of rigorous evidence, while the humanities keep us rooted in the importance of human values. Seeking linkages and connections between these realms is the lively challenge of this conference. The details of curricular design will be of less interest at this juncture than locating the critical issues and discussing how education might serve the goal of unifying knowledge and learning.

Anthracyclines are, above all, DNA intercalators, which induce genetic damage leading to cell death. However, increasing evidence firmly suggests that the underlying mechanism for anthracycline cytotoxicity is the induction of apoptosis through intracellular-mediated signaling pathways. Whether drug/DNA interaction is necessary for such apoptosis signaling is unknown. We investigated the cellular effects of the anthracyclines daunorubicin (DNR) and doxorubicin (DOX) using the myeloid leukemia cell line U937. By comparing free drug against agarose bead-immobilized drug iDNR and iDOX (which cannot accumulate within the cell), we observed that whereas both free and immobilized anthracyclines were cytotoxic, only the former induced apoptosis; the latter induced necrosis. Indeed, we did not observe ceramide generation, neutral sphingomyelinase activation, poly (ADP-ribose) polymerase cleavage, or other apoptotic events with iDNR or iDOX. However, both free and immobilized drug were similarly capable of triggering nuclear factor kappaB activation. These observations demonstrate that whereas activation of certain cellular signaling pathways can be achieved solely through membrane interaction, apoptosis signaling requires anthracycline internalization. These results also show that the initiation of cell survival pathways (illustrated by nuclear factor kappaB activation) is independent of intracellular drug/target interaction.

We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c-fos/c-jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR (P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c-fos but not of c-jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c-fos and the development of apoptosis. The ability to block asbestos-induced elevations in c-fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.

Resistance to anticancer drugs is a major cause of failure of many therapeutic protocols. A variety of mechanisms have been proposed to explain this phenomenon. The exact mechanism depends upon the drug of interest as well as the tumor type treated. While studying a cell line selected for its resistance to cisplatin we noted that the cells expressed a > 25,000-fold collateral resistance to methotrexate. Given the magnitude of this resistance we elected to investigate this intriguing collateral resistance. From a series of investigations we have identified an alteration in a membrane protein of the resistant cell as compared to the sensitive cells that could be the primary mechanism of resistance. Our studies reviewed here indicate decreased tyrosine phosphorylation of a protein (molecular mass = 66) in the resistant cells, which results in little or no transfer of methotrexate from the medium into the cell. Since this is a relatively novel function for tyrosine phosphorylation, this information may provide insight into possible pharmacological approaches to modify therapeutic regimens by analyzing the status of this protein in tumor samples for a better survival of the cancer patients.

It is demonstrated that methotrexate/cisplatin-sensitive L1210 cells express low levels of major histocompatibility complex (MHC) class II relative to the high levels expressed on methotrexate (MTX)/cisplatin-resistant L1210/DDP cells. L1210 cells express cell-surface Fas, while the L1210/DDP cells express no cell-surface Fas. Expression of costimulatory molecules B7-1/B7-2 and Fas is increased on L1210 cells, but not L1210/DDP, in the presence of methotrexate or trimetrexate (TMTX). Therefore, a component of the mechanism of action of some anti-cancer agents may be to facilitate immune recognition and T cell-directed, Fas-induced cell death. Loss of cell-surface Fas expression and failure of Fas (CD95)-dependent apoptotic death has been observed when cells develop drug resistance. The defect in apoptosis can be overcome by anti-cancer agents or experimental manipulation that induce Fas expression on the drug-resistant cells.

We have previously shown that methotrexate (MTX) transport is impaired in murine L1210 cells selected for cisplatin (DDP) resistance (L1210/DDP) and that the decreased MTX uptake may be due to an altered 66 kDa membrane protein. We have further hypothesized that tyrosine phosphorylation is necessary for the function of this protein. To determine the importance of tyrosine phosphorylation we studied the effect of genistein, a tyrosine kinase inhibitor, on methotrexate sensitivity, uptake and tyrosine phosphorylation of the 66 kDa protein in L1210/0 cells. After 5 h of treatment with 50 microM genistein, methotrexate uptake was decreased by nearly 50% and the cells were protected from methotrexate cytotoxicity. Immunoblotting of whole cell lysates with a phosphotyrosine monoclonal antibody demonstrated that genistein treatment decreased phosphorylation of the 66 kDa membrane protein. We concluded that phosphorylation of a 66 kDa protein may be critical for methotrexate transport and that genistein protects L1210/0 cells from methotrexate toxicity.

Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival in part because of the ability of individual cells to migrate significant distances into brain tissue. Invasion is a difficult process to model, because many such human tumors do not invade immunologically competent animal tissue, tumors grown in animals do not invade human tissue, and relevant human tissue substrates are not easily reproduced. We discuss models for examining invasion in vitro, and in particular review work using the tumor spheroid--fetal rat brain aggregate co-culture model, assessed with confocal microscopy and four-dimensional imaging. Quantitation of invasion in this model is discussed, as well as the invasion-inhibitory properties of tyrosine kinase (TK) inhibitors. The effects of receptor-specific tyrphostins strongly support a dominant role for Epidermal Growth Factor Receptor activation in this process and show that invasion can be effectively inhibited at much lower concentrations of TK inhibitors than is necessary for growth suppression. Inhibition of activation of the purported growth factor receptor second messenger phospholipase C- gamma 1, by pharmacological means and gene transfection, also profoundly inhibits the invasive properties of human glioblastoma and rat C6 glioma cells. We have assessed invasiveness in several human tumor specimens, which may provide information relative to prognosis and recurrence risk. Our data supports the concept of differential control of invasion and proliferation, and points to possible strategies for anti-invasive therapy for glioblastoma multiforme.

A major limitation of cancer chemotherapy is development of resistance. In this study, we analyzed KB-3-1 cells and adriamycin-selected multidrug resistant sublines KB-A1 and KB-A10 cells for mechanisms of resistance. KB-A10 cells are 10-fold more resistant than KB-A1 cells but have lower P-glycoprotein. Of the known mechanisms of multidrug resistance, topoisomerase II and lung-resistance-related protein were altered between the resistant cell lines. Glutathione-S-transferase activity and multidrug-resistance-related protein levels were higher in the resistant cell lines compared to the sensitive cells but were similar in KB-A1 and KB-A10 cells. Results indicate differential regulation of mechanisms of resistance with stepwise selection.

Objective: Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival despite aggressive treatment, in part because of its propensity to aggressively infiltrate into brain tissue. Glioblastoma multiforme is also unique because it is the only nonepithelial human tumor for which excessive activation of epidermal growth factor receptor (EGFR) has been consistently linked to tumor growth and patient survival, and EGFR activation promotes glioblastoma multiforme infiltration in vitro., Methods: Cocultures of human glioblastoma spheroids (derived from three separate patients) and fetal rat brain aggregates were examined for infiltration using confocal microscopy, in the presence of 0 to 100 mumol/L genistein, a tyrosine kinase (TK) inhibitor, and 3 mumol/L tyrphostin A25, a specific EGFR-TK inhibitor., Results: Infiltration (not attachment) was completely inhibited by genistein at 10 mumol/L, the IC20 for monolayer growth inhibition in two cell lines. Tyrphostin A25 at 3 mumol/L (the IC20 for monolayers) reduced invasion in a third cell line from 38.8 +/- 6.1% invasion-hour per hour (n = 5) to 2.9 +/- 1.2% invasion-hour per hour (n = 6) (P = 0.0002, two-tailed t test, 93% inhibition), and from 0.54 +/- 0.065% per hour (slope) to 0.028 +/- 0.018% per hour (P = 0.00001, 95% inhibition). Maximal percent invasion was reduced from 100 +/- 0 to 7.4 +/- 5.6% of the fetal rat brain aggregate. No change was detected in EGFR-associated tyrosine phosphorylation at those doses in monolayers by 32P immunolabeling, consistent with the known effects of low concentrations of TK inhibitors. An increase in expression of wild-type and truncated EGFR was demonstrated by Western blotting. Invasion was equally well inhibited by a monoclonal antibody to the high-affinity ligand binding domain of EGFR and not by antibody to an inactive domain., Conclusion: Our observations support the role of EGFR activation as a determinant by which glioblastoma invades normal brain tissue, and we show that invasion can be effectively inhibited at much lower concentrations of TK inhibitors than are necessary for growth suppression.

Asbestos fibers are human carcinogens with undefined mechanisms of action. In studies here, we examined signal transduction events induced by asbestos in target cells of mesothelioma and potential cell surface origins for these cascades. Asbestos fibers, but not their nonfibrous analogues, induced protracted phosphorylation of the mitogen-activated protein (MAP) kinases and extracellular signal-regulated kinases (ERK) 1 and 2, and increased kinase activity of ERK2. ERK1 and ERK2 phosphorylation and activity were initiated by addition of exogenous epidermal growth factor (EGF) and transforming growth factor-alpha, but not by isoforms of platelet-derived growth factor or insulin-like growth factor-1 in mesothelial cells. MAP kinase activation by asbestos was attenuated by suramin, which inhibits growth factor receptor interactions, or tyrphostin AG 1478, a specific inhibitor of EGF receptor tyrosine kinase activity (IC50 = 3 nM). Moreover, asbestos caused autophosphorylation of the EGF receptor, an event triggering the ERK cascade. These studies are the first to establish that a MAP kinase signal transduction pathway is initiated after phosphorylation of a peptide growth factor receptor following exposure to asbestos fibers.

Expression of murine P-glycoprotein (P-gp), encoded by mdrl or mdri3, confers a multidrug resistance phenotype. Higher expression of c-fos and c-jun has also been demonstrated in multidrug resistant human and murine cells. We detected increased expression of c-fos early in the derivation of two series of murine erythroleukemia sublines selected for resistance to vincristine or adriamycin which eventually overexpress mdr3. We speculate that early expression of c-fos prepares cells for overexpression of other genes, such as mdr3, that contribute to the multidrug resistance phenotype.

We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-beta 1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-beta was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-beta.

Collateral resistance to cisplatin and methotrexate has been reported in several cell lines. A murine leukemia cell line (L1210/DDP) selected for cisplatin resistance also has been shown to be highly resistant to methotrexate. Of the mechanisms proposed for methotrexate resistance, only changes in methotrexate transport into the cells were found in an earlier report. Methotrexate enters mammalian cells via an active transport system. In the present study, we demonstrated that the transport into the cell may be impaired in the resistant cells due to altered tyrosine phosphorylation of a membrane protein with a molecular mass of 66 kDa. This alteration was manifested by altered tyrosine phosphorylation of the 66 kDa protein and may be an underlying modification that renders the cells resistant to methotrexate. These results suggest involvement of tyrosine phosphorylation in folate transport and methotrexate resistant in L1210/DDP cells.

We report a murine leukemia cell variant (L1210/DDP), selected for cisplatin (DDP) resistance, to be cross-resistant to methotrexate (MTX). Cross-resistance of L1210 cells to DDP and MTX has been observed by others, and has also been recorded in P388 murine leukemia and SSC-25 human squamous carcinoma cells. We demonstrated that MTX resistance is not due to dihydrofolate reductase (DHFR) gene amplification, increased DHFR enzyme activity or decreased MTX binding to the target enzyme. Of the mechanisms commonly proposed for MTX resistance, only differences in transport were observed when comparing sensitive (L1210/0) and resistant (L1210/DDP) cells. Our results suggest that MTX resistance in L1210/DDP cells is due to altered methotrexate uptake.

We present studies which suggest that the cytotoxic action of Adriamycin (ADR) may involve intracellular pathways of vesicular transport. The movement of proteins or lipids from the endoplasmic reticulum to the plasma membrane via the Golgi organelle and associated compartments exhibits several temperature-sensitive steps between 15 degrees C and 20 degrees C. In this same temperature range, ADR loses its cytotoxic capacity. Using the inhibitor brefeldin A (BFA), we have investigated the possible role of intravesicular trafficking in the loss of ADR activity and the induction of protection from cytotoxicity at low temperature in L1210 cells. We show here that cells pretreated at 37 degrees C for 2 h with BFA could be protected from a subsequent exposure to ADR. The concentration causing 50% inhibition, determined by cloning in soft agar, was increased approximately 3.5 fold. L1210 cells could also be protected from the topoisomerase II inhibitors etoposide and amsacrine, but to a lesser extent; the concentration causing 50% inhibition for the latter inhibitors were increased 2-fold. Spectrofluorometric analysis of intracellular ADR accumulation revealed that there was no significant difference in the level of ADR in cells with or without BFA pretreatment. In addition, examination of ADR-induced cleavable complex formation by alkaline elution showed no significant difference in the level of DNA strand breaks in cells which had been pretreated with BFA even though there was a large difference in survival. Further examination of the persistence of DNA damage after a period of up to 6 h of repair revealed that cells which were pretreated with BFA removed DNA strand breaks at rates equivalent to those of cells which had received ADR directly. These results suggest that the protective effect induced by brefeldin A does not involve uptake, DNA damage, or repair but instead implicates protein or lipid interactions which may be independent of DNA damage and which may influence critical events that take place after the topoisomerase II-DNA complex has been formed.

We have examined the activities of two novel aza-anthracene-9,10-diones (aza), 1-aza and 2-aza, in HL-60 human leukemia cell lines containing type II topoisomerases with different sensitivities to inhibition by other intercalating agents. The sensitive line, HL-60, was sensitive to 2-aza but not to 1-aza, whereas the resistant HL-60/AMSA was sensitive to neither agent. Measurements of 1- and 2-aza-induced, topoisomerase II-mediated DNA cross-linking in the cells revealed patterns of resistance and sensitivity that paralleled the results in the cytotoxicity assays. However, measurements of drug-induced topoisomerase II-mediated DNA cross-linking using purified HL-60 and HL-60/AMSA topoisomerase II indicated that both agents could stabilize a covalent complex between DNA and the HL-60 enzyme. HL-60/AMSA topoisomerase II resisted stabilization by either agent. This suggests that the resistance of HL-60 cells to 1-aza is not due to the inability of this drug to inhibit topoisomerase II but rather to another, undefined mechanism.

KB-A1 and KB-A10 are 2 multi-drug-resistant cell lines which are 100- and 1,000-fold resistant to Adriamycin, respectively. We have examined the expression of P-glycoprotein at the molecular and cellular levels in these human carcinoma cells. Both MDR cell lines, when compared to the parental KB-3-1, show characteristic increases in mdr 1 gene copy number, an increase in mdr 1 mRNA expression, a corresponding increase in transcription rate and a consequent over-expression of P-glycoprotein. However, the more highly resistant KB-A10 cells have a lower gene copy number, express less mdr 1 mRNA and contain less P-glycoprotein than the A1 cell line. To determine whether higher levels of cellular resistance were attributable to enhanced efficacy of P-glycoprotein or to other cellular regulatory mechanisms, we examined other major cellular properties known to be associated with the mdr phenotype. Both the KB-A1 and KB-A10 lines exhibit similar increases in protein kinase C activity as compared to the drug-sensitive parent. In addition, neither glutathione-S-transferase nor topoisomerase II activities account for enhanced resistance of the KB-A10 cells. The above observations are contrary to the premise that the level of drug resistance is necessarily proportional to expression of P-glycoprotein or to other common factors thought to participate in drug insensitivity; consequently, new mechanisms of resistance must be in operation in these cells.

Adriamycin is a cytotoxic drug which has enjoyed considerable success in the treatment of cancer. This agent has a bewildering variety of biological effects both within and on the surface of cells exposed to drug, and it has proved difficult to unambiguously assign a single mechanism of action. In this report we are able to separate intracellular and extracellular actions by taking advantage of the complete lack of Adriamycin-induced cytotoxicity at low temperature. For example, cells exposed to 100 microM Adriamycin at 0 degree C are not killed by the drug, even though this concentration is orders of magnitude higher than the concentration needed to cause 100% cell death at 37 degrees C. If cells exposed to 100 microM Adriamycin at 0 degree C are shifted to fresh drug-free medium at 37 degrees C, there is a time-dependent decrease in survival. However, if the drug-free medium contains calf thymus DNA (1.5 mg/ml) to act as a reservoir for Adriamycin binding of effluxed drug, there is no ensuing cytotoxicity. Thus, the results show that no matter how much drug is present inside the cell, there must also be extracellular drug available for membrane interaction in order to initiate nuclear DNA damage and the cytotoxic cascade.

In both mouse sarcoma 180 and human KB cells selected for the multiple drug resistance (MDR) phenotype, there is an elevation in the steady state mRNA level of c-fos. There is no detectable gene amplification for c-fos, nor is there any significant change in the rate of mRNA transcription or degradation, suggesting that other factors are responsible for the increased expression level in resistance. Cells selected for resistance to methotrexate, a drug not in the MDR group, do not have an increase in c-fos mRNA expression. When drug-sensitive cells are exposed for 30 min to an ED50 concentration of vinblastine, Adriamycin, colchicine, or VP-16, but not to methotrexate or cisplatin, there is a 3-6-fold induction in the level of c-fos message. Because the former drugs are members of the MDR class and the latter are not, the results are consistent with the hypothesis that induction of c-fos by low levels of cytotoxic drugs may be an early event in the acquisition of the MDR phenotype. If this were the case, then c-fos would be expected to act in concert with c-jun to control transcription by binding to a specific DNA regulatory site. Consistent with this explanation is the existence of an AP-1 sequence in the promotor region for the P-glycoprotein gene (mdr1), as well as the fact that c-jun is also overexpressed in MDR cells.

Adriamycin has a vast range of reported actions on the structural and functional properties of cells. This review summarizes the literature on the ability of the drug to modulate the cell surface membrane and attempts to address the question of how such actions could be linked to cytotoxicity. In addition, we consider the use of polymer immobilization of adriamycin to separate intracellular from plasma membrane effects of the drug, and show how this approach has been helpful in interpreting the pharmacology of adriamycin. Finally, a range of biophysical and spectroscopic approaches to defining the molecular details of adriamycin-bilayer interactions is surveyed, and the results used to discuss a model for how this antineoplastic agent binds to membranes.

Quenching of anthracycline fluorescence by a series of spin-labeled fatty acids was used to probe the transverse location of the drug in phosphatidylcholine bilayers in the form of small unilamellar vesicles. Stern-Volmer plots of the quenching data indicate that the fluorophore moiety of the anthracycline is intercalated into the hydrocarbon region of the bilayer, with deeper penetration observed in fluid-phase than in solid-phase vesicles. 31P-NMR parameters (T1 and nuclear Overhauser enhancement (NOE] are unaffected by the presence of drug, consistent with a binding site removed from the interfacial region. Comparison of intensity (F0/F) plots with lifetime (tau 0/tau) data shows that the predominant mechanism of anthracycline quenching by membrane-bound nitroxides is static. Since the membrane-bound drug is also accessible to quenching by I-, the binding site in the membrane must create a channel which is accessible to solvent. Two other fluorescent probes, 12-(9-anthroyloxy)stearate (12-AS) and diphenylhexatriene (DPH), were employed to confirm the results obtained with the anthracyclines, giving quenching data representative of their location in the bilayer.

Most approaches to cancer chemotherapy have centered around the idea that cytotoxic drugs can be used to eradicate proliferating neoplastic cells. Cytotoxicity is generally thought to evolve from the presence of drug-induced damage to the genetic material, and DNA has served admirably as a primary focus for drug development. Other cellular targets should also be vulnerable, however, and over the past decade the plasma membrane in particular has received considerable attention as a therapeutic locus. In the early stages of this work there were only vague notions as to how membrane disruption could lead to cell death, but recent thinking has coalesced around the idea that cell-surface signal transduction and growth control pathways represent an ideal target for the rational development of new cancer therapies. In this review, we discuss three aspects of signal transduction-phosphoinositide turnover, phosphorylation by protein kinase C, and phosphorylation by protein tyrosine kinases--and summarize the existing evidence that these vital processes can be specifically disrupted, and that such pharmacology offers rich prospects for future therapeutic design.

We report an assessment of the applicability of the Bolton-Hunter method to the radioiodination of epidermal growth factor (EGF). Recombinant human EGF (hEGF) could be radioiodinated successfully by this method, whereas murine EGF could not. Bolton-Hunter 125I-labeled hEGF was compared with commercial 125I-labeled hEGF prepared by the chloramine-T radioiodination method. Neither radioligand was sufficiently pure for a detailed characterization of the purportedly heterogeneous pattern of binding of EGF to its receptors. A procedure based on receptor adsorption was thus developed for repurification of the Bolton-Hunter 125I-labeled hEGF. This provided a much purer radioligand suitable for detailed studies of receptor-binding heterogeneity.

Vero cells treated for 24 h with a concentration of 2-deoxy-D-glucose (2dGlc) that inhibited the production of infectious herpes simplex virus type 1 grew at the same rate as untreated cells. Longer exposures to 2dGlc inhibited the growth of Vero cells in a dose-dependent manner, but without any loss of viability, and could be reversed by replating the cells in the absence of drug. To exhibit antiviral activity, 2dGlc had to be present during the replication cycle of herpes simplex virus type 1. Treatment of Vero cells, even with a cytotoxic dose of 2dGlc, was without effect on the yield of infectious virus, provided the drug was removed before infection. Thus the antiviral effects of 2dGlc were not the result of, and appeared to be independent of, persistent host cell toxicity.

The complex between ribosomal protein L24 and its RNA binding site (that region of the 23S RNA which the protein protects from ribonuclease digestion) has been studied by various physicochemical methods. The RNA is composed of two fragments of about 160 and 140 nucleotides which interact with each other to form the L24 binding site. Circular dichroism spectroscopy suggests that the two interacting fragments have a unique region of secondary structure which is not present in either of the two components alone; hence there are important structural interactions between regions of the RNA which are separated in the primary sequence. Addition of the L24 protein to the RNA site promotes a structural change associated with base unstacking, but with little or no change in the hydrogen-bonded base pairing. Heat activation is not required for complex formation. Thermal denaturation studies reveal a broad featureless transition and the amount of hypochromic change indicates that the RNA site contains less secondary structure than other RNAs such as tRNA and total rRNA. Temperature-jump relaxation measurements on the mechanism of unfolding of the RNA show a concerted melting of the entire secondary and tertiary structure, which is altered upon addition of the protein. A structrual basis for this RNA-protein complex is discussed.

Pharmacologic agents may exert their biological activity at the level of the cell membrane. Of particular interest is the anticancer agent adriamycin. This drug has previously been considered to act by intercalation with nuclear DNA, but recent evidence suggests the possibility that the cell surface membrane may represent an alternative target. To test this hypothesis, adriamycin was attached to insoluble supports, and conditions suggesting that the drug was actively cytotoxic without entering cells were demonstrated.

The effects of anthracyclines on the stimulation of oxygen consumption in the presence of HL-60 cell sonicates, beef heart mitochondria and NADPH cytochrome c reductase were determined as a measure of oxygen radical production. Drug-induced oxygen radical formation in each of these systems was modulated by structural changes in the aglycone as well as in the amino sugar portion of the anthracycline molecule. Cytotoxic potency was not correlated with anthracycline-induced oxygen consumption, suggesting that net oxygen radical production was not the primary factor in tumor cell killing by anthracyclines. In contrast, available data on anthracycline cardiotoxicity appeared to correlate with the drug-induced stimulation of oxygen consumption by beef heart mitochondria, providing support for the premise that drug-induced oxygen radicals formed in the presence of mitochondrial flavoproteins are involved in the adverse effects of anthracyclines on the heart. Cyanomorpholinoadriamycin, an analogue which is 100 to 1000 times more potent than adriamycin (doxorubicin) as an antineoplastic agent, has been shown here and elsewhere to be equivalent to adriamycin in stimulating oxygen radical production by beef heart mitochondria and to produce similar cardiotoxicity at equimolar concentrations. Thus, it appears possible to separate the favorable antitumor activity of adriamycin from its unwanted cardiotoxicity by structural changes such as substitution of the antibiotic by a cyanomorpholino moiety.

We have demonstrated a drug-dependent increase in the capacity of HeLa and 3T3 cells, grown in the presence of lethal and sublethal concentrations of adriamycin, to bind epidermal growth factor (EGF). Scatchard analysis ascribes this effect to an increase in the number of binding sites, with little change in affinity. The time course of binding of 125I-EGF is unchanged by adriamycin treatment, in both 3T3 and HeLa cells, at both 0 and 37 degrees C. This increase appears gradually over 3 or 4 days' exposure to the drug and is reversible over a similar period. Although in HeLa cells the increase reaches a maximum of about 4-fold, regardless of cell density, the maximum observed in 3T3 cells, over 100-fold, is seen only at low cell densities. This could be related to the density-dependent growth regulation seen in 3T3 cells, but not in HeLa cells. We suggest that the ability of the anticancer agent adriamycin to alter the cellular response to a growth-regulatory substance may be related to the mechanism of its cytotoxic action.

The equilibrium-binding affinities of six adriamycin analogues and four daunomycin derivatives for negatively charged dimyristoyl phosphatidylcholine/dimyristoyl phosphatidic acid (DMPC/DMPA) small unilamellar vesicles are compared with values for electroneutral DMPC liposomes. Binding of the daunomycin series to negatively charged dimyristoyl phosphatidyl glycerol (DMPG) vesicles was also examined. Under physiological conditions of pH and ionic strength, substitution of the amino group of adriamycin or daunomycin resulted in a reduced affinity for negatively charged bilayers, even if the substituent enhanced the degree of ionization of the amine. Decreasing the ionic strength increases the binding affinity for acidic membranes but decreases the drug affinity for neutral membranes. We propose that the electrostatic bond of the phosphate-amino group that has been shown to exist between anthracyclines and phosphatidic acid is sterically destabilized by substitution of the amino group. The results are consistent with a mode of anthracycline binding to negatively charged membranes which is driven by hydrophobic and electrostatic considerations but is destabilized by steric bulk at the amino group. The data also provide insight into the design of new anthracyclines with high membrane affinities and reduced uptake; such directed interaction with plasma membranes may enhance antineoplastic potential while reducing cardiac toxicity.