N1-Alkyl indazoles are a ubiquitous and privileged motif within medicinal chemistry, yet methods to selectively furnish N1-alkyl indazoles with simple alkyl side chains remain sparse. Herein, ...negative data from high-throughput experimentation (HTE) enabled a confident pivot of resource from continued optimisation to the development of an alternative reaction. This workflow culminated in a methodology for the synthesis of N1-alkyl indazoles. The procedure is highly selective for N1-alkylation, practical, and broad in scope, with no N2-alkyl products detected at completion. Mechanistic understandings were consistent with attributing the high selectivity to thermodynamic control. Additional data-driven process development led to this reaction being safely demonstrated on a 100 g scale, with potential for further scale up. This study highlights pragmatic principles followed to develop a necessitated methodology, suitable for large scale manufacture.
The Enduring Consensus Cervical Cancer Screening and Management Guidelines Committee developed recommendations for dual stain (DS) testing with CINtec PLUS Cytology for use of DS to triage high-risk ...human papillomavirus (HPV)-positive results.
Risks of cervical intraepithelial neoplasia grade 3 or worse were calculated according to DS results among individuals testing HPV-positive using data from the Kaiser Permanente Northern California cohort and the STudying Risk to Improve DisparitiES study in Mississippi. Management recommendations were based on clinical action thresholds developed for the 2019 American Society for Colposcopy and Cervical Pathology Risk-Based Management Consensus Guidelines. Resource usage metrics were calculated to support decision-making. Risk estimates in relation to clinical action thresholds were reviewed and used as the basis for draft recommendations. After an open comment period, recommendations were finalized and ratified through a vote by the Consensus Stakeholder Group.
For triage of positive HPV results from screening with primary HPV testing (with or without genotyping) or with cytology cotesting, colposcopy is recommended for individuals testing DS-positive. One-year follow-up with HPV-based testing is recommended for individuals testing DS-negative, except for HPV16- and HPV18-positive results, or high-grade cytology in cotesting, where immediate colposcopy referral is recommended. Risk estimates were similar between the Kaiser Permanente Northern California and STudying Risk to Improve DisparitiES populations. In general, resource usage metrics suggest that compared with cytology, DS requires fewer colposcopies and detects cervical intraepithelial neoplasia grade 3 or worse earlier.
Dual stain testing with CINtec PLUS Cytology is acceptable for triage of HPV-positive test results. Risk estimates are portable across different populations.
Abstract
An increase in the efficiency of clinical trial conduct has been successfully demonstrated in the oncology field, by the use of multi-arm, multi-stage trials allowing the evaluation of ...multiple therapeutic candidates simultaneously, and seamless recruitment to phase 3 for those candidates passing an interim signal of efficacy. Replicating this complex innovative trial design in diseases such as Parkinson’s disease is appealing, but in addition to the challenges associated with any trial assessing a single potentially disease modifying intervention in Parkinson’s disease, a multi-arm platform trial must also specifically consider the heterogeneous nature of the disease, alongside the desire to potentially test multiple treatments with different mechanisms of action.
In a multi-arm trial, there is a need to appropriately stratify treatment arms to ensure each are comparable with a shared placebo/standard of care arm; however, in Parkinson’s disease there may be a preference to enrich an arm with a subgroup of patients that may be most likely to respond to a specific treatment approach. The solution to this conundrum lies in having clearly defined criteria for inclusion in each treatment arm as well as an analysis plan that takes account of predefined subgroups of interest, alongside evaluating the impact of each treatment on the broader population of Parkinson’s disease patients.
Beyond this, there must be robust processes of treatment selection, and consensus derived measures to confirm target engagement and interim assessments of efficacy, as well as consideration of the infrastructure needed to support recruitment, and the long-term funding and sustainability of the platform. This has to incorporate the diverse priorities of clinicians, triallists, regulatory authorities and above all the views of people with Parkinson’s disease.
Multi-arm, multi-stage platform designs have increased the efficiency of clinical trials in oncology. Foltynie et al. consider the issues that would need to be addressed in order to use this approach to assess potential disease-modifying treatments in progressive neurological conditions such as Parkinson's disease.
The cumulative effects of increasing human use of the ocean and coastal zone have contributed to a rapid decline in ocean and coastal resources. As a result, scientists are investigating how ...multiple, overlapping stressors accumulate in the environment and impact ecosystems. These investigations are the foundation for the development of new tools that account for and predict cumulative effects in order to more adequately prevent or mitigate negative effects. Despite scientific advances, legal requirements, and management guidance, those who conduct assessments—including resource managers, agency staff, and consultants—continue to struggle to thoroughly evaluate cumulative effects, particularly as part of the environmental assessment process. Even though 45years have passed since the United States National Environmental Policy Act was enacted, which set a precedent for environmental assessment around the world, defining impacts, baseline, scale, and significance are still major challenges associated with assessing cumulative effects. In addition, we know little about how practitioners tackle these challenges or how assessment aligns with current scientific recommendations. To shed more light on these challenges and gaps, we undertook a comparative study on how cumulative effects assessment (CEA) is conducted by practitioners operating under some of the most well-developed environmental laws around the globe: California, USA; British Columbia, Canada; Queensland, Australia; and New Zealand. We found that practitioners used a broad and varied definition of impact for CEA, which led to differences in how baseline, scale, and significance were determined. We also found that practice and science are not closely aligned and, as such, we highlight opportunities for managers, policy makers, practitioners, and scientists to improve environmental assessment.
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease which presents a grave prognosis for diagnosed patients. Nintedanib (a triple tyrosine kinase inhibitor) and pirfenidone ...(unclear mechanism of action) are the only approved therapies for IPF, but have limited efficacy. The pathogenic mechanisms of this disease are not fully elucidated; however, a role for mast cells (MCs) has been postulated.
The aim of this work was to investigate a role for MCs in IPF and to understand whether nintedanib or pirfenidone could impact MC function.
MCs were significantly elevated in human IPF lung and negatively correlated with baseline lung function (FVC). Importantly, MCs were positively associated with the number of fibroblast foci, which has been linked to increased mortality. Furthermore, MCs were increased in the region immediately surrounding the fibroblast foci, and co-culture studies confirmed a role for MC-fibroblast crosstalk in fibrosis. Nintedanib but not pirfenidone inhibited recombinant stem cell factor (SCF)-induced MC survival. Further evaluation of nintedanib determined that it also inhibited human fibroblast-mediated MC survival. This was likely via a direct effect on ckit (SCF receptor) since nintedanib blocked SCF-stimulated ckit phosphorylation, as well as downstream effects on MC proliferation and cytokine release. In addition, nintedanib ablated the increase in lung MCs and impacted high tissue density frequency (HDFm) in a rat bleomycin model of lung fibrosis.
Nintedanib inhibits MC survival and activation and thus provides a novel additional mechanism by which this drug may exert anti-fibrotic effects in patients with IPF.
Objective To determine the inflammatory effects of time-dependent exposure to the hypobaric environment of simulated aeromedical evacuation following traumatic brain injury (TBI). Methods Mice were ...subjected to a blunt TBI or sham injury. Righting reflex response (RRR) time was assessed as an indicator of neurologic recovery. Three or 24 h (Early and Delayed groups, respectively) after TBI, mice were exposed to hypobaric flight conditions (Fly) or ground-level control (No Fly) for 5 h. Arterial blood gas samples were obtained from all groups during simulated flight. Serum and cortical brain samples were analyzed for inflammatory cytokines after flight. Neuron specific enolase (NSE) was measured as a serum biomarker of TBI severity. Results TBI resulted in prolonged RRR time compared with sham injury. After TBI alone, serum levels of interleukin-6 (IL-6) and keratinocyte-derived chemokine (KC) were increased by 6 h post-injury. Simulated flight significantly reduced arterial oxygen saturation levels in the Fly group. Post-injury altitude exposure increased cerebral levels of IL-6 and macrophage inflammatory protein-1α (MIP-1α), as well as serum NSE in the Early but not Delayed Flight group compared to ground-level controls. Conclusions The hypobaric environment of aeromedical evacuation results in significant hypoxia. Early, but not delayed, exposure to a hypobaric environment following TBI increases the neuroinflammatory response to injury and the severity of secondary brain injury. Optimization of the post-injury time to fly using serum cytokine and biomarker levels may reduce the potential secondary cerebral injury induced by aeromedical evacuation.
Abstract
Background/Aims
The VEDOSS study has recently indicated that more than 50% of patients affected by Raynaud’s phenomenon (RP) and specific SSc anti-nuclear antibodies (ANA) and/or nailfold ...videocapillaroscopy changes (NVC) (sRP) satisfied ACR/EULAR 2013 criteria within 5 years, indicating a window of opportunity for early detection of SSc. Here we aimed to determine whether sera, skin or dermal fibroblasts cultured from VEDOSS patients showed any biological hallmark of SSc.
Methods
VEDOSS patients were enrolled in the national inception cohort for patients at risk of Scleroderma (Kennedy Cohort, n = 64) . Sera were tested for IFN-inducible chemokines and biomarker of extracellular matrix turnover (ELF test) and compared to Healthy control (HC) and SSc sera (n = 56, 64, respectively). 3mm skin punch biopsies were taken from the forearms of 12 VEDOSS patients, and 5 healthy controls and 6 SSc patients. Biopsies were analysed for haematoxylin and eosin (H&E), masson trichrome staining (MT) and immunohistological (IHC) staining for CD45. A dermatopathologist blinded to clinical features undertook the analysis. A probabilistic image analysis model was used to detect areas of immunopositivity (CD45) and collagen staining (MT) within the samples, using HeteroGenius Medical Imaging Manager colour analysis. In addition, 7 biopsies along with 2 healthy control and 3 SSc, were used to explant fibroblasts cultures. mRNA and protein were isolated from primary fibroblasts and processed for RT-qPCR and western blotting analyses.
Results
Skin biopsies from VEDOSS patients showed evidence of fibrosis and increased collagen bundles within the dermis evidenced in H&E and MT staining, as usually seen in SSc, compared to HC. IHC showed increased number of CD45+ cells in VEDOSS. Semiquantitative analysis of histopathological samples indicated a significant correlation between MT and CD45 staining for VEDOSS (R = 0.8828 P = 0.0016). In vitro, fibroblasts from VEDOSS patients showed an average 5-fold higher collagen mRNA levels compared to HC fibroblasts, at a similar range to that seen in SSc fibroblasts, confirmed at protein level via western blotting. Sera from VEDOSS patients showed significantly higher IFN-inducible chemokines compared to HC and an intermediate levels compared to SSc Mean (STDV) HC = 4.69(0.29) vs. VEDOSS= 4.93 (0.39); SSc= 5.30 (0.54). ELF score was within normal range for most patients with SRP and showed no statistical difference to HC.
Conclusion
Although pilot in nature, this study suggests that patients with no clinical signs of skin fibrosis in their forearms already show biomarker signs of SSc both in their sera, skin biopsies and fibroblast level. Our data indicate that skin thickening is a late manifestation of SSc pathogenesis and early window of opportunity of patients with VEDOSS could be targeted for immune intervention and antifibrotic intervention.
Disclosure
R.L. ross: None. E. Clarke: None. W. Merchant: None. I. Georgiou: None. P. Mulipa: None. A. Carriero: None. G. Abignano: None. A. herrick: None. C. Denton: None. N. Riobo-Del Galdo: None. F. Del Galdo: Consultancies; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.
ObjectivesTo assess antibody and T cell responses to SARS-CoV-2 vaccination in patients with rheumatoid arthritis (RA) on disease-modifying antirheumatic drugs (DMARDs).MethodsThis prospective study ...recruited 100 patients with RA on a variety of DMARDs for antibody and T cell analysis, pre-vaccination and 4 weeks post-vaccination. Positive antibody response was defined as sera IgG binding to ≥1 antigen. Those that remained seronegative after first vaccination were retested 4 weeks after second vaccination; and if still seronegative after vaccination three. A T cell response was defined an ELISpot count of ≥7 interferon (IFN)γ-positive cells when exposed to spike antigens. Type I IFN activity was determined using the luminex multiplex assay IFN score.ResultsAfter vaccine one, in patients without prior SARS-CoV-2 exposure, 37/83 (45%) developed vaccine-specific antibody responses, 44/83 (53%) vaccine-specific T cell responses and 64/83 (77%) developed either antibody or T cell responses. Reduced seroconversion was seen with abatacept, rituximab (RTX) and those on concomitant methotrexate (MTX) compared to 100% for healthy controls (p<0.001). Better seroconversion occurred with anti-tumour necrosis factor (TNF) versus RTX (p=0.012) and with age ≤50 (p=0.012). Pre-vaccine SARS-CoV-2 exposure was associated with higher quantitative seroconversion (≥3 antibodies) (p<0.001). In the subgroup of non-seroconverters, a second vaccination produced seroconversion in 54% (19/35), and after a third in 20% (2/10). IFN score analysis showed no change post-vaccine.ConclusionPatients with RA on DMARDs have reduced vaccine responses, particularly on certain DMARDs, with improvement on subsequent vaccinations but with approximately 10% still seronegative after three doses.
Spinal muscular atrophy is a disorder of lower motor neurons, most commonly caused by recessive mutations in SMN1 on chromosome 5q. Cases without SMN1 mutations are subclassified according to ...phenotype. Spinal muscular atrophy, lower extremity-predominant, is characterized by lower limb muscle weakness and wasting, associated with reduced numbers of lumbar motor neurons and is caused by mutations in DYNC1H1, which encodes a microtubule motor protein in the dynein-dynactin complex and one of its cargo adaptors, BICD2. We have now identified 32 patients with BICD2 mutations from nine different families, providing detailed insights into the clinical phenotype and natural history of BICD2 disease. BICD2 spinal muscular atrophy, lower extremity predominant most commonly presents with delayed motor milestones and ankle contractures. Additional features at presentation include arthrogryposis and congenital dislocation of the hips. In all affected individuals, weakness and wasting is lower-limb predominant, and typically involves both proximal and distal muscle groups. There is no evidence of sensory nerve involvement. Upper motor neuron signs are a prominent feature in a subset of individuals, including one family with exclusively adult-onset upper motor neuron features, consistent with a diagnosis of hereditary spastic paraplegia. In all cohort members, lower motor neuron features were static or only slowly progressive, and the majority remained ambulant throughout life. Muscle MRI in six individuals showed a common pattern of muscle involvement with fat deposition in most thigh muscles, but sparing of the adductors and semitendinosus. Muscle pathology findings were highly variable and included pseudomyopathic features, neuropathic features, and minimal change. The six causative mutations, including one not previously reported, result in amino acid changes within all three coiled-coil domains of the BICD2 protein, and include a possible 'hot spot' mutation, p.Ser107Leu present in four families. We used the recently solved crystal structure of a highly conserved region of the Drosophila orthologue of BICD2 to further-explore how the p.Glu774Gly substitution inhibits the binding of BICD2 to Rab6. Overall, the features of BICD2 spinal muscular atrophy, lower extremity predominant are consistent with a pathological process that preferentially affects lumbar lower motor neurons, with or without additional upper motor neuron involvement. Defining the phenotypic features in this, the largest BICD2 disease cohort reported to date, will facilitate focused genetic testing and filtering of next generation sequencing-derived variants in cases with similar features.
Tubal ectopic pregnancies can cause substantial morbidity or even death. Current treatment is with methotrexate or surgery. Methotrexate treatment fails in approximately 30% of women who subsequently ...require rescue surgery. Gefitinib, an epidermal growth factor receptor inhibitor, might improve the effects of methotrexate. We assessed the efficacy of oral gefitinib with methotrexate, versus methotrexate alone, to treat tubal ectopic pregnancy.
We performed a multicentre, randomised, double-blind, placebo-controlled trial across 50 UK hospitals. Participants diagnosed with tubal ectopic pregnancy were administered a single dose of intramuscular methotrexate (50 mg/m2) and randomised (1:1 ratio) to 7 days of additional oral gefitinib (250 mg daily) or placebo. The primary outcome, analysed by intention to treat, was surgical intervention to resolve the ectopic pregnancy. Secondary outcomes included time to resolution of ectopic pregnancy and serious adverse events. This trial is registered at the ISRCTN registry, ISCRTN 67795930.
Between Nov 2, 2016, and Oct 6, 2021, 328 participants were allocated to methotrexate and gefitinib (n=165) or methotrexate and placebo (n=163). Three participants in the placebo group withdrew. Surgical intervention occurred in 50 (30%) of 165 participants in the gefitinib group and in 47 (29%) of 160 participants in the placebo group (adjusted risk ratio 1·15, 95% CI 0·85 to 1·58; adjusted risk difference –0·01, 95% CI –0·10 to 0·09; p=0·37). Without surgical intervention, median time to resolution was 28·0 days in the gefitinib group and 28·0 days in the placebo group (subdistribution hazard ratio 1·03, 95% CI 0·75 to 1·40). Serious adverse events occurred in five (3%) of 165 participants in the gefitinib group and in six (4%) of 162 participants in the placebo group. Diarrhoea and rash were more common in the gefitinib group.
In women with a tubal ectopic pregnancy, adding oral gefitinib to parenteral methotrexate does not offer clinical benefit over methotrexate and increases minor adverse reactions.
National Institute of Health Research.
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