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Background Headaches are a leading cause of years lived with disability. For some people, headaches become chronic and disabling, with treatment options being primarily pharmaceutical. Non-pharmacological alternative treatment approaches are worthy of exploration. Aim To develop and test an educational and supportive self-management intervention for people with chronic headaches. Objectives To develop and evaluate a brief diagnostic interview to support diagnosis for people with chronic headaches, and then to develop and pilot an education and self-management support intervention for the management of common chronic headache disorders (the CHESS intervention). To select the most appropriate outcome measures for a randomised controlled trial of the CHESS intervention, and then to conduct a randomised controlled trial and economic evaluation of the CHESS intervention with an embedded process evaluation. Design Developmental and feasibility studies followed by a randomised controlled trial. Setting General practice and community settings in the Midlands and London, UK. Participants For our feasibility work, 14 general practices recruited 131 people with chronic headaches (headaches on ≥15 days per month for >3 months). People with chronic headaches and expert clinicians developed a telephone classification interview for chronic headache that we validated with 107 feasibility study participants. We piloted the CHESS intervention with 13 participants and refined the content and structure based on their feedback. People with chronic headaches contributed to the decisions about our primary outcome and a core outcome set for chronic and episodic migraine. For the randomised controlled trial, we recruited adults with chronic migraine or chronic tension-type headache and episodic migraine, with or without medication overuse headache, from general practices and via self-referral. Our main analyses were on people with migraine. Interventions The CHESS intervention consisted of two 1-day group sessions focused on education and self-management to promote behaviour change and support learning strategies to manage chronic headaches. This was followed by a one-to-one nurse consultation and telephone support. The control intervention consisted of feedback from classification interviews, headache management leaflet and a relaxation compact disc. Main outcome measures The primary outcome was headache-related quality of life measured using the Headache Impact Test-6 at 12 months. The secondary outcomes included the Chronic Headache Quality of Life Questionnaire; headache days, duration and severity; EuroQol-5 Dimensions, five-level version; Short Form Questionnaire-12 items; Hospital Anxiety and Depression Scale; and Pain Self-Efficacy Questionnaire scores. We followed up participants at 4, 8 and 12 months. Results Between April 2017 and March 2019, we randomised 736 participants from 164 general practices. Nine participants (1%) had chronic tension-type headache only. Our main analyses were on the remaining 727 participants with migraine (376 in the intervention arm and 351 in the usual-care arm). Baseline characteristics were well matched. For the primary outcome we had analysable data from 579 participants (80%) at 12 months. There was no between-group difference in the Headache Impact Test-6 at 12 months, (adjusted mean difference –0.3, 95% confidence interval –1.23 to 0.67; p = 0.56). The limits of the 95% confidence interval effectively exclude the possibility of the intervention having a worthwhile benefit. At 4 months there was a difference favouring the CHESS self-management programme on the Headache Impact Test-6 (adjusted mean difference –1.0, 95% confidence interval –1.91 to –0.006; p = 0.049). However, the self-management group also reported 1.5 (95% confidence interval 0.48 to 2.56) more headache days in the previous 28 days. Apart from improved pain self-efficacy at 4 and 12 months, there were few other statistically significant between-group differences in the secondary outcomes. The CHESS intervention generated 0.031 (95% confidence interval –0.005 to 0.063) additional quality-adjusted life-years and increased NHS and Personal Social Services costs by £268 (95% confidence interval £176 to £377), on average, generating an incremental cost-effectiveness ratio of £8617 with an 83% chance of being cost-effective at a willingness-to-pay threshold of £20,000 per quality-adjusted life-year. The CHESS intervention was well received and fidelity was good. No process-related issues were identified that would explain why the intervention was ineffective. Limitations Only 288 out of 376 (77%) of those randomised to the CHESS intervention attended one or more of the intervention sessions. Conclusions This short, non-pharmacological, educational self-management intervention is unlikely to be effective for the treatment of people with chronic headaches and migraine. Future work There is a need to develop and test more sustained non-pharmacological interventions for people with chronic headache disorders. Patient and public involvement Substantial patient and public involvement went into the design, conduct and interpretation of the CHESS programme. This helped direct the research and ensured that the patient voice was embedded in our work. Trial registration This trial is registered as ISRCTN79708100. Funding This project was funded by the National Institute for Health and Care Research (NIHR) Programme Grants for Applied Research programme and will be published in full in Programme Grants for Applied Research; Vol. 11, No. 2. See the NIHR Journals Library website for further information. Plain language summary What did we want to find out? We wanted to find out if an education and self-management support programme for people with frequent headaches made these people feel better. What did we do? We first made sure that we could find people with frequent headaches, from general practice, who would want to take part in our study. We then trained nurses to do telephone interviews to find out what sort of headaches people had. We looked at previous research and then, together with people with frequent headaches, designed a group education and self-management programme. It was run by a nurse and another health professional over 2 days, followed by a one-to-one session and telephone support with a nurse. We worked with people with frequent headaches and health professionals specialising in headaches to agree how best to measure how headaches affect people’s quality of life. We then tested our self-management programme. We recruited 736 people with frequent headaches, of whom 727 had migraine. Using a computer, we allocated them at random either to attend the self-management programme or to receive a relaxation compact disc. Everyone was told their headache type. We asked participants to tell us about their headaches and headache quality of life after 4 months, 8 months and 12 months. What did we find? Our main results are for the 727 people with migraine. Our support programme did not help people in our study with frequent migraines to live better. There were also no important differences in the number of headaches people had each month or the amount of prescribed or over-the-counter medication they took for their headaches. What does this mean? Our short 2-day programme did not appear to improve headache-related quality of life or reduce the number of headache days. Other ways of helping people manage their chronic headaches are needed. Scientific summary Background Headaches are second to low back pain as a global cause of years lived with disability. Headaches are the most common neurological disorder treated in primary care. They account for around 3% of general practitioner consultations. Seventy per cent of people with headaches seen by their general practitioner do not get a formal diagnosis. For some people headaches become a chronic disabling disorder. There is a need for more non-pharmacological treatments to help those living with headache disorders. Our overarching aim was to develop and test a supportive education and self-management group intervention, implementable in primary care, for people with chronic headaches. Objectives The objectives of the programme were to: •develop and test strategies for recruiting people with chronic headaches from primary care [work package (WP) 1] •develop and evaluate a brief classification interview to support diagnosis for people with chronic headaches (WP2) •develop and pilot an education and self-management support intervention for the management of common chronic headache disorders [the Chronic Headache Education and Self-management Study (CHESS) intervention] (WP3) •select the most appropriate outcome measures for a randomised controlled trial of the CHESS intervention package (WP4) •run a multicentre randomised controlled trial, including an economic evaluation, of the CHESS intervention package (WP5). Methods and results We used an epidemiological definition of chronic headaches: headaches on ≥15 days per month for >3 months. Phase 1 of our work, WPs 1–4, consisted of interlinked systematic reviews and a feasibility study. Feasibility study (work package 1) Fourteen general practices in the West Midlands recruited 131 people with chronic headaches by writing to people with recorded consultations for headaches and prescriptions for migraine-specific drugs (triptans and pizotifen). Eligibility was confirmed by a telephone call by the study team. This group was our sampling frame for WPs 2–4. Classification interview (work package 2) We wanted to identify the population of interest for the main trial but also to feel confident that those who had other headache types not suitable for our trial were appropriately identified and referred for relevant support. We first reviewed the literature on diagnostic tools and found 38 papers validating 30 tools. We did not find any tools that were suitable for our proposed trial. We therefore organised a consensus meeting to inform our thinking on the content of a new classification tool. This was attended by neurologists with a specialist interest in headaches, general neurologists, headache specialist nurses, general practitioners with a specialist interest in headaches and people with chronic headaches. We established what we needed to know from a person to: •exclude secondary headaches •exclude primary headaches other than chronic migraine and tension-type headache •distinguish between chronic tension-type headache and chronic migraine •identify medication overuse headache. We used this information to develop a classification logic model for use in a nurse-delivered classification interview. A research nurse and a doctor, with expertise in headaches, from the National Migraine Centre then independently interviewed 107 participants. We found a high level of agreement between the nurse and specialist. Over 90% of study participants were classified as having chronic migraine. Intervention development (work package 3) Three systematic reviews informed our intervention development. Using a meta-ethnographic approach our systematic review of the lived experience of chronic headaches (n = 4 studies) we found three overarching themes: •headache as a driver of behaviour •the spectre of headache •strained relationships. In our systematic review of prognostic factors in chronic headache (n = 27 studies), we found moderate evidence for depression and anxiety, poor sleep, stress, medication overuse and poor self-efficacy predicting a poor outcome. We found inconclusive evidence for treatment expectations, age and age at onset, body mass index, employment and headache features predicting a poor outcome. In our systematic review of the effectiveness, style and content of self-management interventions for chronic headache (n = 16 studies) we found beneficial effects of the interventions compared with usual care in pain intensity, headache-related disability and quality of life. Interventions including either education or mindfulness components, and delivered in a group format, showed greater reductions in pain intensity than interventions without these features. A greater beneficial effect on mood was observed in interventions that included a cognitive–behavioural approach component than in those without this. We interviewed seven people living with chronic headaches recruited through our charity partners. We found that participants had tried a range of therapies and interventions, some of which were helpful and others less so. Access to education and peer support was deemed positive, as was learning new skills such as relaxation, mindfulness and stress management. We then presented our findings to 18 people from clinical, academic and lay backgrounds at an intervention development day to agree the structure and content of our new intervention. We agreed on a modular group intervention for 8–10 people delivered by a nurse and a layperson with chronic headaches. It should include educational material, self-management material and medication advice, and include a digital versatile disc (DVD) suitable to share with friends and family. We included a single face-to-face session and up to 8 weeks of telephone support with a specially trained nurse. After piloting with 13 participants, we identified that it was difficult for lay facilitators to commit to the sessions because of the unpredictable nature of their headaches. We therefore changed to using allied health professionals as the second facilitator. The final format was two group days followed by a one-to-one session with a nurse to discuss medication, lifestyle factors and goal-setting, followed by up to 8 weeks of telephone support (individually negotiated). Clinical effectiveness and cost-effectiveness measures (work package 4) In our systematic review of patient-reported outcomes (46 studies evaluating 23 patient measures) we found that for a ‘headache’ population only the Headache Impact Test-6 (HIT-6) had acceptable evidence for its validity and reliability for use in our trial. The Migraine-Specific Quality of Life Questionnaire (MSQ v2.1) had relevance to our population. We modified this measure, changing the focus of each item from ‘migraine’ to ‘headache’ to produce the Chronic Headache Quality of Life Questionnaire (CHQLQ) and did a mixed-methods comparative evaluation of the CHQLQ and HIT-6. Both the CHQLQ and the HIT-6 were well completed, had good psychometric properties and were relevant to the experience of headache. The CHQLQ captured the wide-ranging impact of chronic headache, in particular the emotional impact, to a greater extent than the HIT-6. As this work was not complete before starting the main trial, we set HIT-6 as the primary outcome for the trial and the CHQLQ as a secondary outcome. We developed three questions to capture headache frequency, duration and severity for use in a smartphone application (app) or in a paper diary. Eight feasibility participants tested the app over 11 weeks. Feedback was positive but completion rates varied. We included the app as part of the main trial. From our work on outcome measures we identified the need for a core outcome set for migraine. This work took place after the design of the randomised controlled trial had been finalised. We identified >50 domains from our systematic reviews and our qualitative work. We did a modified, three-round electronic Delphi study with patients and professionals to identify which domains were most important. At a consensus day, when the aim was to ratify the core domains, a two-domain core outcome set was agreed for chronic and episodic migraine: 1.migraine-specific pain – to be assessed with an 11-point numerical pain rating scale, and frequency as the number of headache/migraine days over a specified period 2.migraine-specific quality of life to be assessed with the Migraine Functional Impact Questionnaire (MFIQ). Professor Underwood, the chief investigator for this study, is a director and shareholder of Clinvivo Ltd, who provided the Delphi platform. He recused himself from any discussions related to the choice of Delphi platform for this study. Phase 2: randomised controlled trial, work package 5 Phase 2 of the programme was a randomised controlled trial to evaluate the clinical effectiveness and cost-effectiveness of the CHESS intervention package. We identified people with chronic headaches from general practice records. Self-referral to the trial was also possible. We included adults with migraine or tension-type headache with or without medication overuse headache. People who appeared eligible after an initial telephone call were asked to provide consent and baseline measures. This was followed by a classification interview with a research nurse to confirm eligibility and identify people with suspected non-eligible headaches. After the feasibility study we specified that if at least 85% of our participants had migraine our primary analysis would just be on the population with migraine, with sample size inflated, if necessary, to ensure adequate statistical power for this analysis. The randomisation allocation ratio was 1 : 1.07 in favour of the intervention group to account for clustering in one arm. Randomisation was done using minimisation, stratifying by geographical locality (Midlands and Greater London) and headache type [definite chronic migraine, probable chronic migraine (i.e. episodic migraine plus chronic tension-type headache) and chronic tension-type headache only, with or without medication overuse headache]. Our primary outcome was the HIT-6 score at 1 year. We used the Migraine-Specific Quality of Life Questionnaire as the secondary headache disability outcome. We did follow-ups at 4, 8 and 12 months. The sample size was based on testing the clinical effectiveness in the migraine population excluding participants with just tension-type headache (n = 689 participants: relaxation arm, n = 689; self-management arm, n = 356) provided 90% power to detect a between-group difference of 2 points (standard deviation 6.87 points, from the feasibility study) in HIT-6 score at 12 months for those with migraine using a two-sided test and a 5% significance level with a 20% loss to follow-up. Some over-run on sample size was expected to allow all groups to be adequately populated. We did a within-trial health economic analysis. Between April 2017 and March 2019, staff at 164 general practices in the Midlands and London wrote to 31,026 people and we randomised 736 people, 727 (99%) with migraine: 54% (396/727) had chronic migraine and 56% (407/727) medication overuse headache. Despite reporting chronic headache when eligibility for the study was determined, after receiving informed consent at baseline, 38% (274/727) reported < 15 headache days in the preceding 4 weeks. Unless otherwise stated, analyses were on the 727 participants with migraine. Baseline characteristics were well matched. The first session was attended by 286 out of 376 (76%) intervention participants; 259 (69%) reached the minimum adherence (day 1, and the one-to-one session) and 216 (58%) achieved full adherence to the programme. There was no between-group difference in HIT-6 scores at 12 months [adjusted mean difference –0.3 points, 95% confidence interval (CI) –1.23 to 0.67 points; p = 0.56]. The limits of this 95% CI excluded our target (worthwhile) effect size of 2.0 points and the smaller minimally clinically important difference of 1.5 points suggested by others for studies of episodic migraine. At 4 months there was a difference favouring the CHESS self-management programme (adjusted mean difference –1.0 points; 95% CI –1.91 to –0.006 points; p = 0.049). There were few differences in secondary outcomes. The self-management group had 1.5 (95% CI 0.48 to 2.56; p = 0.004) more headache days over the preceding 28 days at 4 months. They also had improved pain self-efficacy scores at 4 and 12 months. Use of acute drugs, including both prescribed and over-the-counter drugs, and prophylactic drugs was unchanged over time with no between-group differences. Using electronic/paper diary data the difference over 12 months in number of headache days was 0.2 days (95% CI –0.11 to 0.46 days; p = 0.234), difference in duration of each headache was 0.4 hours (95% CI –0.47 to 1.28 hours; p = 0.361) and difference in average headache severity on a 0–10 scale was 0.2 (95% CI –0.08 to 0.46; p = 0.163). We found no subgroup effects. Our complier-average causal effect and sensitivity analyses were not materially different. There were seven adverse events: two in the standard-care arm and five in the self-management arm. The CHESS intervention generated 0.031 (95% CI –0.005 to 0.063) additional quality-adjusted life-years (QALYs) and increased NHS and Personal Social Services costs by £268 (95% CI £176 to £377), generating an incremental cost-effectiveness ratio of £8617 with an 83% chance of being cost-effective at a willingness to pay of £20,000 per QALY gained. Our process evaluation, including all 736 participants, showed that we recruited a nationally representative population including people from practices based in all 10 deciles of the Index of Multiple Deprivation; 18% of participants were from minority ethnic groups. Intervention fidelity was good, with adherence being slightly better than competence [adherence 83% (interquartile range 67–100%); competence 70% (interquartile range 50–90%)]. We carried out semistructured interviews with a purposive sample of 26 study participants. Most participants described gaining some new knowledge or insight about their headaches from the intervention they received, and a few changed medication. Some felt more confident to manage their headaches, but many did not. CHESS was well received by participants, facilitators and general practitioners. Participants enjoyed interacting with others and valued the opportunity to talk, share and discuss their chronic headache experiences with others in a similar situation in a safe knowledgeable space. Patient and public involvement There has been substantial patient and public involvement in the design, conduct and interpretation of the CHESS programme. Throughout the programme we worked closely with three UK migraine charities and a lay advisory group to help direct the research and ensure that the patient voice was embedded in our work. Conclusions Over the duration of the CHESS programme, we have advanced our understanding of the challenges of living with chronic headaches and made some progress in developing the methodology for running randomised controlled trials of complex interventions for people living with chronic headaches. Our data effectively excluded the possibility that this short intervention is effective for the treatment of chronic migraine or chronic tension-type headache and episodic migraine. Although there was no effect on our chosen headache-specific outcomes, we have not excluded the possibility that it produces a worthwhile QALY gain, as measured by the EuroQol-5 Dimensions, five-level version. The health burden of chronic headache disorders, principally chronic migraine, is debilitating. Those living with the condition warrant support to optimise their care planning according to their needs and the latest knowledge about treatment and management. Further advances in this field must be driven by new theoretically and/or biologically informed intervention models. Research recommendations •New work to better understand the health impact of chronic headache disorders and to identify modifiable risk factors for a poor outcome. •Development and testing of new non-pharmacological interventions for a tightly phenotyped group with chronic migraine. •Research is needed to support improved classification of headache disorders in primary care to allow better targeting of the available drug treatments of proven effectiveness, and reduce medication overuse. Trial registration This trial is registered as ISRCTN79708100. Funding This project was funded by the National Institute for Health and Care Research (NIHR) Programme Grants for Applied Research programme and will be published in full in Programme Grants for Applied Research; Vol. 11, No. 2. See the NIHR Journals Library website for further project information.

Abstract Background Interventions for preventing falls in older people often involve several components, multidisciplinary teams, and implementation in a variety of settings. We have developed a classification system (taxonomy) to describe interventions used to prevent falls in older people, with the aim of improving the design and reporting of clinical trials of fall-prevention interventions, and synthesis of evidence from these trials. Methods Thirty three international experts in falls prevention and health services research participated in a series of meetings to develop consensus. Robust techniques were used including literature reviews, expert presentations, and structured consensus workshops moderated by experienced facilitators. The taxonomy was refined using an international test panel of five health care practitioners. We assessed the chance corrected agreement of the final version by comparing taxonomy completion for 10 randomly selected published papers describing a variety of fall-prevention interventions. Results The taxonomy consists of four domains, summarized as the "Approach", "Base", "Components" and "Descriptors" of an intervention. Sub-domains include; where participants are identified; the theoretical approach of the intervention; clinical targeting criteria; details on assessments; descriptions of the nature and intensity of interventions. Chance corrected agreement of the final version of the taxonomy was good to excellent for all items. Further independent evaluation of the taxonomy is required. Conclusions The taxonomy is a useful instrument for characterizing a broad range of interventions used in falls prevention. Investigators are encouraged to use the taxonomy to report their interventions.

Abstract Background Depression is common in residents of Residential and Nursing homes (RNHs). It is usually undetected and often undertreated. Depression is associated with poor outcomes including increased morbidity and mortality. Exercise has potential to improve depression, and has been shown in existing trials to improve outcomes among younger and older people. Existing evidence comes from trials that are short, underpowered and not from RNH settings. The aim of the OPERA trial is to establish whether exercise is effective in reducing the prevalence of depression among older RNH residents. Method OPERA is a cluster randomised controlled trial. RNHs are randomised to one of two groups with interventions lasting 12 months Intervention group: a depression awareness and physical activity training session for care home staff, plus a whole home physical activation programme including twice weekly physiotherapist-led exercise groups. The intervention lasts for one year from randomisation, or Control group: a depression awareness training session for care home staff. Participants are people aged 65 or over who are free of severe cognitive impairment and willing to participate in the study. Our primary outcome is the prevalence of depressive symptoms, a GDS-15 score of five or more, in all participants at the end of the one year intervention period. Our secondary depression outcomes include remission of depressive symptoms and change in GDS-15 scores in those with depressive symptoms prior to randomisation. Other secondary outcomes include, fear of falling, mobility, fractures, pain, cognition, costs and health related quality of life. We aimed to randomise 77 RNHs. Discussion Home recruitment was completed in May 2010; 78 homes have been randomised. Follow up will finish in May 2011 and results will be available late 2011. Trial Registration [ISRCTN: ISRCTN43769277]

Abstract Background The National Health Service (NHS) was tasked in 2001 with developing service provision to prevent falls in older people. We carried out a national survey to provide a description of health and social care funded UK fallers services, and to benchmark progress against current practice guidelines. Methods Cascade approach to sampling, followed by telephone survey with senior member of the fall service. Characteristics of the service were assessed using an internationally agreed taxonomy. Reported service provision was compared against benchmarks set by the National Institute for Health and Clinical Excellence (NICE). Results We identified 303 clinics across the UK. 231 (76%) were willing to participate. The majority of services were based in acute or community hospitals, with only a few in primary care or emergency departments. Access to services was, in the majority of cases, by health professional referral. Most services undertook a multi-factorial assessment. The content and quality of these assessments varied substantially. Services varied extensively in the way that interventions were delivered, and particular concern is raised about interventions for vision, home hazard modification, medication review and bone health. Conclusion The most common type of service provision was a multi-factorial assessment and intervention. There were a wide range of service models, but for a substantial number of services, delivery appears to fall below recommended NICE guidance.

Duchenne muscular dystrophy (DMD) is a rare, X-linked neuromuscular disease caused by pathogenic variants in the DMD gene that result in the absence of functional dystrophin, beginning at birth and leading to progressive impaired motor function, loss of ambulation and life-threatening cardiorespiratory complications. Delandistrogene moxeparvovec, an adeno-associated rh74-viral vector-based gene therapy, addresses absent functional dystrophin in DMD. Here the phase 3 EMBARK study aimed to assess the efficacy and safety of delandistrogene moxeparvovec in patients with DMD. Ambulatory males with DMD, ≥4 years to <8 years of age, were randomized and stratified by age group and North Star Ambulatory Assessment (NSAA) score to single-administration intravenous delandistrogene moxeparvovec (1.33 × 10 14 vector genomes per kilogram; n = 63) or placebo (n = 62). At week 52, the primary endpoint, change from baseline in NSAA score, was not met (least squares mean 2.57 (delandistrogene moxeparvovec) versus 1.92 (placebo) points; between-group difference, 0.65; 95% confidence interval (CI), -0.45, 1.74; P = 0.2441). Secondary efficacy endpoints included mean micro-dystrophin expression at week 12: 34.29% (treated) versus 0.00% (placebo). Other secondary efficacy endpoints at week 52 (between-group differences (95% CI)) included: Time to Rise (-0.64 (-1.06, -0.23)), 10-meter Walk/Run (-0.42 (-0.71, -0.13)), stride velocity 95th centile (0.10 (0.00, 0.19)), 100-meter Walk/Run (-3.29 (-8.28, 1.70)), time to ascend 4 steps (-0.36 (-0.71, -0.01)), PROMIS Mobility and Upper Extremity (0.05 (-0.08, 0.19); -0.04 (-0.24, 0.17)) and number of NSAA skills gained/improved (0.19 (-0.67, 1.06)). In total, 674 adverse events were recorded with delandistrogene moxeparvovec and 514 with placebo. There were no deaths, discontinuations or clinically significant complement-mediated adverse events; 7 patients (11.1%) experienced 10 treatment-related serious adverse events. Delandistrogene moxeparvovec did not lead to a significant improvement in NSAA score at week 52. Some of the secondary endpoints numerically favored treatment, although no statistical significance can be claimed. Safety was manageable and consistent with previous delandistrogene moxeparvovec trials. ClinicalTrials.gov: NCT05096221., (© 2024. The Author(s).)

Patients with pre-existing immunity to adeno-associated virus (AAV) are currently unable to receive systemic gene transfer therapies. In this nonhuman primate study, we investigated the impact of immunosuppression strategies on gene transfer therapy safety and efficacy and analyzed plasmapheresis as a potential pretreatment for circumvention of pre-existing immunity or redosing. In part 1, animals received delandistrogene moxeparvovec (SRP-9001), an AAVrh74-based gene transfer therapy for Duchenne muscular dystrophy. Cohort 1 (control, n = 2) received no immunosuppression; cohorts 2-4 (n = 3 per cohort) received prednisone at different time points; and cohort 5 (n = 3) received rituximab, sirolimus, and prednisone before and after dosing. In part 2, cohorts 2-4 underwent plasmapheresis before redosing; cohort 5 was redosed without plasmapheresis. We analyzed safety, immune response (humoral and cell-mediated responses and complement activation), and vector genome distribution. After 2 or 3 plasmapheresis exchanges, circulating anti-AAVrh74 antibodies were reduced, and animals were redosed. Plasmapheresis was well tolerated, with no abnormal clinical or immunological observations. Cohort 5 (redosed with high anti-AAVrh74 antibody titers) had hypersensitivity reactions, which were controlled with treatment. These findings suggest that plasmapheresis is a safe and effective method to reduce anti-AAV antibody levels in nonhuman primates prior to gene transfer therapy. The results may inform human studies involving redosing or circumvention of pre-existing immunity., Competing Interests: R.A.P., E.L.P., D.G., G.C.O., S.L., K.C., and L.R.R.-K. are employees of Sarepta Therapeutics, Inc. J.R.M. has received grants from Parent Project Muscular Dystrophy and personal fees from Sarepta Therapeutics Inc. and Nationwide Children’s Hospital outside the submitted work and holds a pending patent for a micro-dystrophin cassette for gene therapy and an issued patent for rAAV.SGCA delivery isolated limb infusion., (© 2024 The Authors.)

Introduction/aims: Delandistrogene moxeparvovec is indicated in the United States for the treatment of ambulatory pediatric patients aged 4 through 5 years with Duchenne muscular dystrophy (DMD) with a confirmed mutation in the DMD gene. Long-term delandistrogene moxeparvovec microdystrophin protein (a shortened dystrophin that retains key functional domains of the wild-type protein) expression may positively alter disease progression in patients with DMD. We evaluated long-term safety and functional outcomes of delandistrogene moxeparvovec in patients with DMD., Methods: An open-label, phase 1/2a, nonrandomized controlled trial (Study 101; NCT03375164) enrolled ambulatory males, ≥4 to <8 years old, with DMD. Patients received a single intravenous infusion (2.0 × 10 14 vg/kg by supercoiled quantitative polymerase chain reaction) of delandistrogene moxeparvovec and prednisone (1 mg/kg/day) 1 day before to 30 days after treatment. The primary endpoint was safety. Functional outcomes were change from baseline in North Star Ambulatory Assessment (NSAA) and timed function tests., Results: Four patients (mean age, 5.1 years) were enrolled. There were 18 treatment-related adverse events; all occurred within 70 days posttreatment and resolved. Mean NSAA total score increased from 20.5 to 27.5, baseline to year 4, with a mean (standard deviation) change of +7.0 (2.9). Post hoc analysis demonstrated a statistically significant and clinically meaningful 9-point difference in NSAA score, relative to a propensity-score-weighted external control cohort (least-squares mean [standard error] = 9.4 [3.4]; P = .0125)., Discussion: Gene transfer therapy with delandistrogene moxeparvovec treatment is well tolerated, with a favorable safety profile. Functional improvements are sustained through 4 years, suggesting delandistrogene moxeparvovec may positively alter disease progression., (© 2023 Sarepta Therapeutics Inc and The Authors. Muscle & Nerve published by Wiley Periodicals LLC.)

Objective: Delandistrogene moxeparvovec is approved in the USA for the treatment of ambulatory patients (4-5 years) with Duchenne muscular dystrophy. ENDEAVOR (SRP-9001-103; NCT04626674) is a single-arm, open-label study to evaluate delandistrogene moxeparvovec micro-dystrophin expression, safety, and functional outcomes following administration of commercial process delandistrogene moxeparvovec., Methods: In cohort 1 of ENDEAVOR (N = 20), eligible ambulatory males, aged ≥4 to <8 years, received a single intravenous infusion of delandistrogene moxeparvovec (1.33 × 10 14  vg/kg). The primary endpoint was change from baseline (CFBL) to week 12 in delandistrogene moxeparvovec micro-dystrophin by western blot. Additional endpoints evaluated included: safety; vector genome copies; CFBL to week 12 in muscle fiber-localized micro-dystrophin by immunofluorescence; and functional assessments, including North Star Ambulatory Assessment, with comparison with a propensity score-weighted external natural history control., Results: The 1-year safety profile of commercial process delandistrogene moxeparvovec in ENDEAVOR was consistent with safety data reported in other delandistrogene moxeparvovec trials (NCT03375164 and NCT03769116). Delandistrogene moxeparvovec micro-dystrophin expression was robust, with sarcolemmal localization at week 12; mean (SD) CFBL in western blot, 54.2% (42.6); p < 0.0001. At 1 year, patients demonstrated stabilized or improved North Star Ambulatory Assessment total scores; mean (SD) CFBL, +4.0 (3.5). Treatment versus a propensity score-weighted external natural history control demonstrated a statistically significant difference in least squares mean (standard error) CFBL in North Star Ambulatory Assessment, +3.2 (0.6) points; p < 0.0001., Interpretation: Results confirm efficient transduction of muscle by delandistrogene moxeparvovec. One-year post-treatment, delandistrogene moxeparvovec was well tolerated, and demonstrated stabilized or improved motor function, suggesting a clinical benefit for patients with Duchenne muscular dystrophy. ANN NEUROL 2023;94:955-968., (© 2023 Sarepta Therapeutics and The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several cassettes are being tested in clinical trials, this study compared the efficacies of four shortened dystrophin-promoter combinations with implications for outcomes in clinical trials: MHCK7 or MCK promoter with a shortened dystrophin transgene containing the N-terminus and spectrin repeats R1, R2, R3 and R24 (rAAVrh74.MHCK7.micro-dystrophin and rAAVrh74.MCK.micro-dystrophin, respectively); shortened dystrophin construct containing the neuronal nitric oxide (nNOS) binding site (rAAVrh74.MHCK7.DV.mini-dystrophin); and shortened dystrophin containing the C-terminus (rAAVrh74.MHCK7.micro-dystrophin.Cterm). Functional and histological benefit were examined at 4 weeks following intramuscular delivery in mdx mice. rAAVrh74.MHCK7.micro-dystrophin provided the most robust transgene expression and significantly increased specific force output in the tibialis anterior muscle. Muscle environment was normalized (i.e. reductions in central nucleation), indicating functional and histological advantages of rAAVrh74.MHCK7.micro-dystrophin. Thus, promoter choice and transgene design are critical for optimal dystrophin expression/distribution for maximal functional improvement., Competing Interests: Competing interests This study was funded by Sarepta Therapeutics, Inc. and was sponsored by Nationwide Children's Hospital Foundation support to L. R. R. K and J. R. M. All AAVrh74 constructs are investigational and not approved in any country. Rachael Potter is an employee of Sarepta Therapeutics, Inc. Danielle Griffin is an employee of Sarepta Therapeutics, Inc. Kristin Heller has no competing or financial interests. Jerry R. Mendell is the co-inventor of the rAAVrh74.micro-dystrophin technology. This technology has been exclusively licensed to Sarepta Therapeutics, Inc. Louise R. Rodino-Klapac is the co-inventor of the AAVrh74.micro-dystrophin technology and eligible to receive financial consideration as a result, she Rodino-Klapac is an employee of Sarepta Therapeutics, Inc., (© 2023. Published by The Company of Biologists Ltd.)

Introduction: Delandistrogene moxeparvovec (SRP-9001) is an investigational gene transfer therapy designed for targeted expression of SRP-9001 dystrophin protein, a shortened dystrophin retaining key functional domains of the wild-type protein. Methods: This Phase 2, double-blind, two-part (48 weeks per part) crossover study (SRP-9001-102 [Study 102]; NCT03769116) evaluated delandistrogene moxeparvovec in patients, aged ≥4 to <8 years with Duchenne muscular dystrophy. Primary endpoints (Part 1) were change from baseline (CFBL) in SRP-9001 dystrophin expression (Week 12), by Western blot, and in North Star Ambulatory Assessment (NSAA) score (Week 48). Safety assessments included treatment-related adverse events (TRAEs). Patients were randomized and stratified by age to placebo (n = 21) or delandistrogene moxeparvovec (n = 20) and crossed over for Part 2. Results: SRP-9001 dystrophin expression was achieved in all patients: mean CFBL to Week 12 was 23.82% and 39.64% normal in Parts 1 and 2, respectively. In Part 1, CFBL to Week 48 in NSAA score (least-squares mean, LSM [standard error]) was +1.7 (0.6) with treatment versus +0.9 (0.6) for placebo; p = 0.37. Disparity in baseline motor function between groups likely confounded these results. In 4- to 5-year-olds with matched baseline motor function, CFBL to Week 48 in NSAA scores was significantly different (+2.5 points; p = 0.0172), but not significantly different in 6-to-7-year-olds with imbalanced baseline motor function (-0.7 points; p = 0.5384). For patients treated with delandistrogene moxeparvovec in Part 2, CFBL to Week 48 in NSAA score was +1.3 (2.7), whereas for those treated in Part 1, NSAA scores were maintained. As all patients in Part 2 were exposed to treatment, results were compared with a propensity-score-weighted external control (EC) cohort. The LSM difference in NSAA score between the Part 2 treated group and EC cohort was statistically significant (+2.0 points; p = 0.0009). The most common TRAEs were vomiting, decreased appetite, and nausea. Most occurred within the first 90 days and all resolved. Discussion: Results indicate robust expression of SRP-9001 dystrophin and overall stabilization in NSAA up to 2 years post-treatment. Differences in NSAA between groups in Part 1 were not significant for the overall population, likely because cohorts were stratified only by age, and other critical prognostic factors were not well matched at baseline., Competing Interests: DG, SL, SW, RP, TS, LRR-K are empolyed by Sarepta Therapeutics Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mendell, Shieh, McDonald, Sahenk, Lehman, Lowes, Reash, Iammarino, Alfano, Sabo, Woods, Skura, Mao, Staudt, Griffin, Lewis, Wang, Potter, Singh and Rodino-Klapac.)

Limb-girdle muscular dystrophy (LGMD) type 2C/R5 results from mutations in the γ-sarcoglycan ( SGCG ) gene and is characterized by muscle weakness and progressive wasting. Loss of functional γ-sarcoglycan protein in the dystrophin-associated protein complex destabilizes the sarcolemma, leading to eventual myofiber death. The SGCG knockout mouse ( SGCG -/- ) has clinical-pathological features that replicate the human disease, making it an ideal model for translational studies. We designed a self-complementary rAAVrh74 vector containing a codon-optimized human SGCG transgene driven by the muscle-specific MHCK7 promoter (SRP-9005) to investigate adeno-associated virus (AAV)-mediated SGCG gene transfer in SGCG -/- mice as proof of principle for LGMD 2C/R5. Gene transfer therapy resulted in widespread transgene expression in skeletal muscle and heart, improvements in muscle histopathology characterized by decreased central nuclei and fibrosis, and normalized fiber size. Histopathologic improvements were accompanied by functional improvements, including increased ambulation and force production and resistance to injury of the tibialis anterior and diaphragm muscles. This study demonstrates successful systemic delivery of the hSGCG transgene in SGCG -/- mice, with functional protein expression, reconstitution of the sarcoglycan complex, and corresponding physiological and functional improvements, which will help establish a minimal effective dose for translation of SRP-9005 gene transfer therapy in patients with LGMD 2C/R5., Competing Interests: The authors are employees of the study sponsor, Sarepta Therapeutics, Inc., and may own stock/options in the company., (© 2023 The Author(s).)

Limb-girdle muscular dystrophy type 2D/R3 (LGMD2D/R3) is a progressive muscular dystrophy that manifests with muscle weakness, respiratory abnormalities, and in rare cases cardiomyopathy. LGMD2D/R3 is caused by mutations in the SGCA gene resulting in loss of protein and concomitant loss of some or all components of the dystrophin-associated glycoprotein complex. The sgca-null ( sgca -/- ) mouse recapitulates the clinical phenotype of patients with LGMD2D/R3, including dystrophic features such as muscle necrosis and fibrosis, elevated serum creatine kinase (CK), and reduction in the generation of absolute muscle force and locomotor activity. Thus, sgca -/- mice provide a relevant model to test the safety and efficacy of gene transfer. We designed a self-complementary AAVrh74 vector containing a codon-optimized full-length human SGCA (hSGCA) transgene driven by a muscle-specific promoter, shortened muscle creatine kinase (tMCK). In this report, we test the efficacy and safety of scAAVrh74.tMCK.hSGCA in sgca -/- mice using a dose-escalation design to evaluate a single systemic injection of 1.0 × 10 12 , 3.0 × 10 12 , and 6.0 × 10 12 vg total dose compared with vehicle-treatment and wild-type mice. In sgca -/- mice, treatment with scAAVrh74.tMCK.hSGCA resulted in robust expression of α-sarcoglycan protein at the sarcolemma membrane in skeletal muscle at all doses tested. In addition, scAAVrh74.tMCK.hSGCA was effective in improving the histopathology of limb and diaphragm muscle of sgca -/- mice, as indicated by reductions in fibrosis, central nucleation, and normalization of myofiber size. These molecular changes were concomitant with significant increases in specific force generation in the diaphragm and tibialis anterior muscle, protection against eccentric force loss, and reduction in serum CK. Locomotor activity was improved at all doses of vector-treated compared with vehicle-treated sgca -/- mice. Lastly, vector toxicity was not detected in a serum chemistry panel and by gross necropsy. Collectively, these findings provide support for a systemic delivery of scAAVrh74.tMCK.hSGCA in a clinical setting for the treatment of LGMD2D/R3.

Duchenne muscular dystrophy (DMD) is a rare, X-linked, fatal, degenerative neuromuscular disease caused by mutations in the DMD gene. More than 2,000 mutations of the DMD gene are responsible for progressive loss of muscle strength, loss of ambulation, and generally respiratory and cardiac failure by age 30. Recently, gene transfer therapy has received widespread interest as a disease-modifying treatment for all patients with DMD. We designed an adeno-associated virus vector (rAAVrh74) containing a codon-optimized human micro-dystrophin transgene driven by a skeletal and cardiac muscle-specific promoter, MHCK7. To test the efficacy of rAAVrh74.MHCK7.micro-dystrophin, we evaluated systemic injections in mdx (dystrophin-null) mice at low (2 × 10 12 vector genome [vg] total dose, 8 × 10 13 vg/kg), intermediate (6 × 10 12 vg total dose, 2 × 10 14 vg/kg), and high doses (1.2 × 10 13 vg total dose, 6 × 10 14 vg/kg). Three months posttreatment, specific force increased in the diaphragm (DIA) and tibialis anterior muscle, with intermediate and high doses eliciting force outputs at wild-type (WT) levels. Histological improvement included reductions in fibrosis and normalization of myofiber size, specifically in the DIA, where results for low and intermediate doses were not significantly different from the WT. Significant reduction in central nucleation was also observed, although complete normalization to WT was not seen. No vector-associated toxicity was reported either by clinical or organ-specific laboratory assessments or following formal histopathology. The findings in this preclinical study provided proof of principle for safety and efficacy of systemic delivery of rAAVrh74.MHCK7.micro-dystrophin at high vector titers, supporting initiation of a Phase I/II safety study in boys with DMD.

Importance: Micro-dystrophin gene transfer shows promise for treating patients with Duchenne muscular dystrophy (DMD) using recombinant adeno-associated virus serotype rh74 (rAAVrh74) and codon-optimized human micro-dystrophin driven by a skeletal and cardiac muscle-specific promoter with enhanced cardiac expression (MHCK7)., Objective: To identify the 1-year safety and tolerability of intravenous rAAVrh74.MHCK7.micro-dystrophin in patients with DMD., Design, Setting, and Participants: This open-label, phase 1/2a nonrandomized controlled trial was conducted at the Nationwide Children's Hospital in Columbus, Ohio. It began on November 2, 2017, with a planned duration of follow-up of 3 years, ending in March 2021. The first 4 patients who met eligibility criteria were enrolled, consisting of ambulatory male children with DMD without preexisting AAVrh74 antibodies and a stable corticosteroid dose (≥12 weeks)., Interventions: A single dose of 2.0 × 1014 vg/kg rAAVrh74.MHCK7.micro-dystrophin was infused through a peripheral limb vein. Daily prednisolone, 1 mg/kg, started 1 day before gene delivery (30-day taper after infusion)., Main Outcomes and Measures: Safety was the primary outcome. Secondary outcomes included micro-dystrophin expression by Western blot and immunohistochemistry. Functional outcomes measured by North Star Ambulatory Assessment (NSAA) and serum creatine kinase were exploratory outcomes., Results: Four patients were included (mean [SD] age at enrollment, 4.8 [1.0] years). All adverse events (n = 53) were considered mild (33 [62%]) or moderate (20 [38%]), and no serious adverse events occurred. Eighteen adverse events were considered treatment related, the most common of which was vomiting (9 of 18 events [50%]). Three patients had transiently elevated γ-glutamyltransferase, which resolved with corticosteroids. At 12 weeks, immunohistochemistry of gastrocnemius muscle biopsy specimens revealed robust transgene expression in all patients, with a mean of 81.2% of muscle fibers expressing micro-dystrophin with a mean intensity of 96% at the sarcolemma. Western blot showed a mean expression of 74.3% without fat or fibrosis adjustment and 95.8% with adjustment. All patients had confirmed vector transduction and showed functional improvement of NSAA scores and reduced creatine kinase levels (posttreatment vs baseline) that were maintained for 1 year., Conclusions and Relevance: This trial showed rAAVrh74.MHCK7.micro-dystrophin to be well tolerated and have minimal adverse events; the safe delivery of micro-dystrophin transgene; the robust expression and correct localization of micro-dystrophin protein; and improvements in creatine kinase levels and NSAA scores. These findings suggest that rAAVrh74.MHCK7.micro-dystrophin can provide functional improvement that is greater than that observed under standard of care., Trial Registration: ClinicalTrials.gov Identifier: NCT03375164.

Introduction : The development of adeno-associated virus (AAV) vectors as safe vehicles for in vivo delivery of therapeutic genes has been a major milestone in the advancement of gene therapy, enabling a promising strategy for ameliorating a wide range of diseases, including Duchenne muscular dystrophy (DMD). Areas covered : Based on experience with the development of a gene transfer therapy agent for DMD, we discuss ways in which gene therapy for rare disease challenges traditional clinical development paradigms, and recommend a step-wise approach for design and evaluation to support broader applicability of gene therapy. Expert opinion : The gene therapy development approach should intentionally design the therapeutic construct and the clinical study to systematically evaluate agent delivery, safety, and efficacy. Rigorous preclinical work is essential for establishing an effective gene delivery platform and determining the efficacious dose. Clinical studies should thoroughly evaluate transduction, on-target transgene expression at the tissue and cellular level, and functional efficacy.

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene, leading to a progressive dystrophy characterized by chronic muscle fiber loss, fat replacement, and fibrosis. To correct the underlying histopathology and function, expression of full-length DYSF is required. Dual adeno-associated virus vectors have been developed, defined by a region of homology, to serve as a substrate for reconstitution of the full 6.5 kb dysferlin cDNA. Previous work studied the efficacy of this treatment through intramuscular and regional delivery routes. To maximize clinical efficacy, dysferlin-deficient mice were treated systemically to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at multiple time points between 4 and 13 months post treatment for dysferlin expression and functional improvement using magnetic resonance imaging and magnetic resonance spectroscopy and membrane repair. A systemic dose of 6 × 10 12 vector genomes resulted in widespread gene expression in the muscles. Treated muscles showed a significant decrease in central nucleation, collagen deposition, and improvement of membrane repair to wild-type levels. Treated gluteus muscles were significantly improved compared to placebo-treated muscles and were equivalent to wild type in volume, intra- and extramyocellular lipid accumulation, and fat percentage using magnetic resonance imaging and magnetic resonance spectroscopy. Dual-vector treatment allows for production of full-length functional dysferlin with no toxicity. This confirms previous safety data and validates translation of systemic gene delivery for dysferlinopathy patients.

A method using reverse-phase ultra-high-performance liquid chromatography coupled with tandem mass spectrometry is described for eight classes of therapeutants that are used in marine aquaculture products. Validation studies to evaluate recovery, precision, method detection limits, and measurement uncertainty were performed at three levels, using three representative matrices [salmon (fatty fish), tilapia (lean fish), and shrimp (crustaceans)] to assess the method performance for use as a screening or determinative (quantitative and confirmatory) method. A total of 16 sulfonamides (plus 2 potentiators), 2 tetracyclines, 11 (fluoro)quinolones, 7 nitroimidazoles, 3 amphenicols, 5 steroids, and 3 stilbenes met the quantitative criteria for method validation. An additional 5 triphenylmethane dyes, 2 sulfonamides, 2 tetracyclines, and 1 amphenicol met the required performance for use as a screening method. Limits of detection (LODs) for the compounds that met the quantitative criteria ranged from 0.1 to 5 μg/kg, while LODs for compounds from the screening group ranged from 0.1 to 30 μg/kg. This method provides a comprehensive approach to the determination of different classes of compounds in aquaculture products.

Background: Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2., Results: In this study, PKCθ knockdown (PKCθshRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 and extracellular signal-regulated kinase1/2 (ERK1/2) was increased in PKCθshRNA cells, with no change in ERK5 phosphorylation, highlighting a PKCθ-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCθshRNA cells. Thus, with reduced PKCθ, differentiation and fusion occur in the absence of PI3-kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCθshRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCθ regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation., Conclusion: Cellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCθ regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCθ had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCθ regulated signaling may reveal important interactions regulating skeletal muscle health in an insulin resistant state.

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of fluoroquinolones-ciprofloxacin (CIPRO), danofloxacin (DANO), enrofloxacin (ENRO) and sarafloxacin (SARA)-in aquaculture products, specifically salmon, shrimp and tilapia. After initial sample extraction with an acidic acetonitrile solution, the extract was diluted with dichloromethane and centrifuged, then an aliquot was concentrated and applied to a C18 solid-phase extraction cartridge and concentrated for a second time. The resultant residue was dissolved in acetonitrile, diluted with water, and then further defatted with hexane. The fluoroquinolone residues were determined by UPLC with an HSS T3 C18 reverse-phase column using an ammonium hydroxide-formic acid buffer in an acetonitrile gradient with MS/MS detection using multiple reaction monitoring. Average recoveries for salmon tissue ranged from 73% for DANO to 95% for SARA, for shrimp from 71% for DANO to 109% for SARA, and from 62% for DANO to 111% for SARA in tilapia, fortified at the 1.0 ng g(-1) level. Standard curves were linear between 0.002 and 0.5 ng injected for all compounds. Detection limits of 0.2 ng g(-1) for CIPRO, DANO, ENRO, and SARA were easily obtainable. The operational errors, interferences, and recoveries for fortified samples demonstrate that this described method is suitable for routine use in a regulatory programme. The recommended method is simple, rapid, specific and reliable for the routine monitoring of fluoroquinolone residues in aquatic species such as salmon, tilapia and shrimp.

A simple, robust method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 17 sulfonamides [sulfanilamide (SNL), sulfacetamide (SAA), sulfaguanidine (SGD), sulfapyridine (SPY), sulfadiazine (SDZ), sulfathiazole (STZ), sulfamerazine (SMR), sulfamethoxazole (SOZ), sulfamoxole (SXL), sulfisoxazole (SXZ), sulfamethizole (SML), sulfamethazine (SMZ), sulfamethoxypyridazine (SMP), sulfamonomethoxine (SMM), sulfachloropyridazine (SCP), sulfaquinoxaline (SQX), and sulfadimethoxine (SDM)] and 2 potentiators [ormetoprim (OMP) and trimethoprim (TMP)] in fish tissue has been developed. The analytes were extracted from homogenized fish tissue with water-acetonitrile (50 + 50). The extract was clarified by centrifugation and a portion defatted with hexane. The analytes were partitioned into chloroform and evaporated to dryness. The redissolved residue was applied to a C18 reversed-phase column with a water-acetonitrile (0.1% acetic acid) gradient. All of the compounds were completely separated and detected in <10 min at 30 degrees C using LC/MS/MS. Standard curves were linear over the range of 0.02 to 5 ng injected. The limit of detection varied from 0.1 ng/g for SMZ and OMP to 0.9 ng/g for SXL and SOZ. Recoveries varied from 100% for SDM, SOZ, and SQX and 85% for SMR, OMP, and TMP to approximately 30% for SAA. Relative standard deviations for repeat analysis varied from 4% for SMZ and SCP to 23% for SAA.

The air temperatures inside uninsulated and insulated huts were recorded on an outdoor pig unit in the south of England between September 1997 and September 1998, and the herd's production parameters were also recorded. During the summer the temperatures inside some of the uninsulated huts exceeded 45 degrees C, but the temperatures inside the insulated huts were lower and fluctuated less. Despite the high temperatures, the weaning weight of piglets reared in the uninsulated huts were often higher than those of the piglets reared in the insulated huts, possibly as a result of the higher mortality of small piglets in the uninsulated huts, especially during the winter. The weaning weights of the piglets were higher during the summer.

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for determining the residues of malachite green (MG) and leucomalachite green (LMG) in a number of aquatic species. MG and its metabolite were extracted from homogenized tissues with a perchloric acid-acetonitrile solution; the extract was centrifuged; and an aliquot was taken, concentrated, and passed through a chemically bonded octadecyl C18 solid-phase extraction column. The compounds of interest were eluted with acetonitrile, and the eluate was evaporated to dryness. The residue was dissolved in acetonitrile and diluted with water in preparation for analysis by LC/MS/MS. MG and its metabolite were determined by reversed-phase LC using a Luna C18 column with an ammonium hydroxide-formic acid buffer in acetonitrile gradient and MS/MS detection using multiple reaction monitoring. Calibration curves were linear for all analyses between 5 and 500 pg injected for both analytes, with recoveries ranging from 81% for LMG to 98% for MG in salmon spiked at the 1 ng/g level. Detection limits of 0.1 ng/g for both MG and LMG were easily obtainable using the recommended method. The operational errors, interferences, and recoveries for spiked samples compared favorably with those obtained by established methodology. The recommended method is simple, rapid, and specific for monitoring residues of MG and LMG in a number of aquatic species.

A liquid chromatographic (LC)/mass spectrometric (MS) method was developed for determining the residues of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in a number of aquatic species. The phenicols are extracted with acetone, the extracts are partitioned with dichloromethane, the aqueous layer is removed, and the organic layer is evaporated to dryness. The residue is dissolved in dilute acid and defatted with hexane, and the aqueous layer is prepared for analysis by LC. The phenicols are determined by reversed-phase LC by using a Hypersil C18-BD column with a water-acetonitrile gradient and MS detection using selected-ion recording. Calibration curves were linear for all analytes between 0.015 and 0.425 ng injected. The relative standard deviations for measurements by the proposed method were < 10% for all of the analytes studied, with recoveries ranging from 71% for florfenicol amine to 107% for florfenicol in salmon tissue spiked at the 2 ng/g level. Detection limits of 0.1 ng/g for florfenicol and chloramphenicol, 0.3 ng/g for thiamphenicol, and 1.0 ng/g for florfenicol amine are easily obtainable. The operational errors, interferences, and recoveries for spiked samples compare favorably with those obtained by established LC methodology. The proposed method is simple, rapid, and specific for monitoring residues of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in a number of aquatic species.