Current treatment options for the management of depressive episodes in bipolar disorder are often sub-optimal, with some treatments either noted to be only partially effective or to require long durations of treatment prior to a therapeutic response. Therefore, pharmaco-therapeutic options that reduce depressive symptoms in a more rapid manner might provide a viable therapeutic option for some people. Intravenous (IV) scopolamine, a pan muscarinic antagonist, has been demonstrated in a number of studies to confer a rapid antidepressant effect, albeit no study to date has exclusively evaluated its potential therapeutic effect in a cohort consisting solely of individuals with bipolar disorder.Individuals with bipolar disorder who are currently experiencing a depressive episode of at least moderate severity will be included in this study. Eligible participants will undergo a screening and placebo-run in visit and will be randomised at visit 3 to the treatment or placebo group. Participants will receive the three blinded infusions over the course of 2 weeks, with two subsequent follow-up visits, 1 and 3 weeks after the last infusion visit. The total duration of the study will be approximately 6 weeks. Patients will continue their regular treatment regime in addition to study medication. Objective and subjective mood questionnaires, cognitive assessments and other psychometric instruments will be administered and recorded.To our knowledge, this is the first study to investigate the antidepressant effects of IV scopolamine in an exclusively bipolar disorder cohort. Trial findings will contribute to the evidence base regarding the cholinergic hypothesis of mood disorders and specifically might result in an additional safe therapeutic option for the management of depressive episodes in bipolar disorder.NCT04211961. December 26, 2019. EudraCT Number 2017-003112-39ClinicalTrials.gov  Bipolar disorder is a chronic disabling psychiatric disorder characterised by recurrent episodes of mania or hypomania and depression. Individuals with bipolar I disorder experience episodes of mania and depression, while individuals with bipolar II disorder experience episodes of depression with periods of hypomania, but not mania . BipolarCurrent pharmacological treatments for depressive episodes in bipolar disorder remain sub-optimal, with pharmacological strategies employed to date often only partially effective , 4. A nuA rapid-acting antidepressant effect has been demonstrated in some double-blind placebo-controlled trials of intravenous (IV) scopolamine in MDD or bipolar disorder , 9, 10, Scopolamine, also known as “hyoscine”, is most commonly utilised for the management of post-operative nausea, motion sickness and hypersalivation secondary to psychotropic medications such as clozapine –16. ScopThe cholinergic system has long been implicated as a contributory factor in the aetiology of mood disorders. Physostigmine, a cholinesterase inhibitor (opposite effect to scopolamine), has been demonstrated to exacerbate depressive symptomatology , 20 and The aim of this clinical trial is to investigate the efficacy and safety of IV scopolamine, compared to placebo, in reducing the severity of depressive symptoms in individuals with bipolar disorder who are experiencing a depressive episode of at least moderate severity. In addition, the study will investigate if IV scopolamine impacts cognition and improves functioning as measured on a number of objective and subjective psychometric instruments when compared to placebo.This is a single-site, randomised, double-blind, placebo-controlled, parallel, phase IIb clinical trial examining if IV scopolamine exhibits an antidepressant effect in individuals with bipolar disorder. Study visits will take place at the Clinical Research Facility Galway (CRFG), in University Hospital Galway, Galway, Ireland. Patients will be recruited from mental health services in the West of Ireland including a specialised bipolar disorder clinic at University Hospital Galway.Individuals will have a diagnosis of bipolar disorder according to the Diagnostic Statistics Manual (DSM)-V and must be experiencing a depressive episode of at least moderate severity based on a clinical interview by a trained clinician and a Hamilton Depression Rating Scale (HDRS) score ≥ 14. Exclusion criteria include being euthymic or experiencing a manic or hypomanic episode, treatment with any cholinergic medications , a history of a previous allergic reaction or sensitivity to scopolamine, treatment with oral steroids, a history of an alcohol or substance use disorder in the 3 months prior to study entry, an intellectual disability (intelligence quotient < 70), dementia or cognitive impairment, and severity of depressive episode so that participation in a clinical trial is not appropriate.Patients meeting all inclusion criteria will be enrolled into the study . The maximum time between visit 1 (screening) and visit 2 is 14 days. The screening visit and visit 2 can occur on the same day. At visit 2, all participants will receive a placebo run-in . This has been utilised in other IV scopolamine RCTs –7, 9 andn = 25) or 4 μg/kg scopolamine (n = 25) IV in 100 ml of saline over 15 min at:Visit 3 (Day 0),Visit 4 (days 2-6) andVisit 5 (days 6-10)Within 7 days of visit 2, participants will be assessed against the placebo run-in criteria and if these criteria are met (HDRS score ≥ 8 at visit 3), they will be randomised to receive either placebo (Participants will receive scopolamine or placebo in addition to their current treatment regimen. At least 2 days will elapse between IV infusions (visits 2–5). Two follow-up visits (visits 6 and 7) will occur at day 15 (± 5 days) and day 29 (± 7 days) with at least 2 days between visits 5 and the first follow-up visit (visit 6) and at least 3 days between the two follow-up visits (visits 6 and 7). If a trial participant requires medical intervention during or post-infusion, the treating physician must assess if it is appropriate to proceed with the current and/or future infusions. The flow of the study visits is presented in Fig. A 10-item questionnaire to identify harmful use or dependence on alcohol.A rating scale that measures illness severity (CGI-S) (1–7) and global improvement (CGI-I) (1–7) and has an efficacy index (which relates therapeutic effects to adverse effects) (1–4).A 6-item questionnaire examining nicotine dependence.A rating scale from 1 to 100 that measures an individual’s functioning and is observer rated.A 21-item observer rated instrument that measures symptoms of depression.A 10-item observer rated scale that measures symptoms of depression.This 60-item personality inventory measures personality traits in five dimensions .A 9-item self-report instrument measuring adverse effects associated with the gastrointestinal, genitourinary, nervous and cardiac systems, sense organs, skin, sexual functioning, sleep and other general adverse effects.A rating scale where 65 adjectives are rated by participants on a 5-point Likert scale. Six factors are derived from these adjectives .This is a semi-structured interview that evaluates pathology and includes a demographic section followed by nine diagnostic modules, including two that examine mood disorders.A self-report scale used to indicate on a 10-point Likert how participants feel they are on various items, with the VAS utilised for the following terms in this study: (1) happy, (2) restless, (3) sad, (4) anxious, (5) anger, (6) drowsiness and (7) alertness. These items are consistent with those used by Furey et al. [An 11-item observer-rated instrument that measures symptoms of elation.Tests conducted from this battery will entail the following: (1) Emotion Recognition Task (ERT) , (2) Paired Associates Learning (PAL) , (3) One Touch Stockings of Cambridge (OTC) , and (4) Rapid Visual Information Processing (RVP) (assesses attention).Full scale, verbal and performance Intelligence Quotient (IQ) will be estimated using an abbreviated version of this instrument and will comprise the verbal subscales of vocabulary and similarities, and the performance subscales of block-design and matrix reasoning.See Table Clinical interviews and psychometric assessments will be collected by trained clinicians and research associates with a background in psychiatric research. All research staff involved in administration of clinical instruments or data-input will have up-to-date training in Good Clinical Practice (GCP) . InfusioParticipants will be randomly assigned to receive either scopolamine or placebo in a 1:1 ratio. Randomly permuted blocks of sizes 4 and 6 will be used to ensure similar numbers of participants in each arm of the trial. Randomisation will be stratified by the HDRS score at trial entry (a score of < 23 indicating a mild-moderate depressive episode and a score ≥ 23 indicating a severe depressive episode). This will help ensure greater balance between arms in the final trial sample and increase the efficiency of our treatment effect estimates.A validated randomisation system will be used at visit 3 (after the HDRS and YMRS are completed) to randomise patients to either arm. This centralised system will ensure allocation concealment, preventing blinded trial staff from knowing which treatment group will be allocated. Blocks of randomly varying length will also reduce the predictability of the allocation sequence. Randomisation will be carried out by the pharmacy team. In the case of an emergency, when knowledge of the treatment assignment is essential for the clinical management of the participant, a treating physician may request to unblind a single participant.Participants randomised to the scopolamine group will receive a 15-min infusion of IV scopolamine in 100 ml saline, repeated over 3 visits .Participants randomised to the placebo group will receive a 15-min infusion of IV saline, repeated over 3 visits .Non-adherence will be extremely unlikely given the mode of administration, with participants monitored by a team researcher throughout the infusion period.t-test with a conservative standard deviation of 13 units and a significance level of 0.05. Allocating 25 participants to each arm will allow a loss to follow-up of approximately 12% post-randomisation (6 participants missing at visit 6 follow-up). It is suggested that approximately 60 participants will need to be recruited to ensure 50 participants (83.3%) are eligible for randomisation (visit 3). All participants will attain a placebo infusion at visit 2.Based on previous data , the allAdverse events (AEs) and serious adverse events (SAEs) will be recorded on AE case report forms for all participants from the time of consent for the duration of their participation in the study. A full AE review will be conducted at each study visit to ensure that a complete evaluation of the safety and tolerability of the investigational medical product (placebo or scopolamine) is conducted. The site investigator will follow-up all AEs reported during the treatment period until resolved, considered stable, including completion of patient participation in the trial . All SAEs will be followed-up until resolution or until they are clearly determined to be due to a participant’s stable or chronic condition or intercurrent illness(es) including after trial completion if required. SAEs will be reported to the sponsor and principal investigator within 24 h after site awareness of the SAE to find a reasonable solution. Any suspected unexpected serious adverse reaction (SUSAR) that potentially occur will be reported by the sponsor to the competent authority (HPRA and/or EudraVigilance), the approving ethics committee and PI. Indemnity is in place pertaining to any adverse effects experienced by study participants.The mean scores at visit 6 will be compared across the study arms using an analysis of co-variance (ANCOVA) model. The response variable will be the change in HDRS score (primary outcome measure) from visit 3 to visit 6. For this effect analysis of the primary outcome, HDRS score at visit 3 is added as a co-variate as it is expected that pre-randomisation measurements of the HDRS score will be correlated with scores obtained at visit 6. The inclusion of stratifying variables (i.e. severe v. mild or moderate depressive symptoms at baseline) and other variables as covariates (i.e. bipolar I or II disorder) will be considered as appropriate to increase the power to detect significant differences between the groups. Inverse probability weighting will be applied to the primary outcome analysis. These weights will be derived based on the inverse of the probability of a patient’s data being missing given their pre-randomisation measurements. This will ensure the estimate and inference is more representative of all patients randomised, reducing bias in the estimation of the treatment effect due to participants being lost to follow-up and missing data. Inference regarding the treatment effectiveness will focus on the point estimate, confidence interval and p-value for hypothesis confirmation.Secondary outcomes will be analysed as appropriate (according to the distribution of each outcome), comparing the difference between groups at the specified follow-up visit and relative to baseline values (where specified/measured). Baseline variables predictive of each outcome (the measure at baseline where available), and stratifying variables will be included in ANCOVA or generalised linear models of outcome variables. For secondary outcomes (i.e. acute and long-term effects on cognition (CANTAB), change in other mood rating scales including the MADRS and POMS, changes in functioning (GAF), treatment changes), the focus will be on the point estimates and confidence intervals for hypothesis generation.Additional analysis of the primary outcome will compare the time course of HDRS under placebo and treatment using a mixed-effects model including fixed-effects terms for time since randomisation/study visit, trial arm and their interactions and a random effect to account for correlation of multiple observations per participant.Required documents including the study protocol , informed consent form, participant information leaflet and any other required documents (i.e. psychometric instruments) have been submitted and approved by the Galway University Hospital's Research Ethics Committee and the Health Products Regulatory Authority (HPRA). The sponsor  will submit and obtain approval from the above parties for substantial amendments to the original approved documents. The sponsor is responsible for implementing and maintaining quality assurance and quality control systems in line with written standard operating procedures (SOPS), ensuring data are recorded and reported in compliance with the protocol, Good Clinical Practice and regulatory requirements.All study information will be stored in Microsoft Excel 365 by a data manager using an electronic database. The database will record all subject data to include the baseline characteristics, pre- and post-assessments, and possible adverse events. The computer and database will use password protection with the database only accessible to the study researchers. To ensure the confidentiality of the data, all subjects will be provided with identification numbers. All researchers will have access to the final trial data. Electronic data and paper documents will be kept for 5 years, after which they will be destroyed.A Data Monitoring Committee (DMC), which is independent from the sponsor and competing interests, comprises clinical experts, trial experts, investigators and a statistician and will conduct regular monitoring tests and interim analyses (approximately every 4 months) to ensure the authenticity of the data. A trial steering committee, comprising the PI, clinicians and researchers with experience in this area, will meet every 6 months to review study progress and address any concerns subsequent to DMC reports. The study will be monitored on an approximate 6 monthly basis by the sponsor, entailing a blinded and unblinded study monitor of extensive experience. These monitoring visits will ensure appropriate patient eligibility for the study, staff training, accurate recording of data including source data verification and protocol deviations. The study will be open to external auditing by the HPRA (a process independent from investigators and the sponsor), throughout the time-frame of this study to ensure adherence to GCP.The end of trial will be the date of the last visit of the last participant (50th randomised participant). The trial may be terminated prematurely if (1) new information about safety or efficacy becomes available , (2) there is unsatisfactory progress of the study, including unsatisfactory progress in relation to participant recruitment or (3) deemed necessary by the sponsor, principal investigator, DMC, trial steering committee and/or the funder .This trial may be subject to internal or external auditing or inspection procedures to ensure adherence to GCP. Access to all trial-related documents will be given at that time.The results of this study will be published in peer-reviewed journals and be presented at domestic or international academic congresses. Individualised results will be reported by the researcher to the participants of this study, after data publication in peer-reviewed journals. Data will be available for public access via the National Institute for Mental Health Data Archive 1 year after submission of all participant data.The purpose of this study is to examine the efficacy and safety of IV scopolamine compared to placebo in individuals with bipolar disorder who are experiencing a depressive episode. In addition, this study will also assess whether IV scopolamine, compared to placebo, impacts cognition and functioning in individuals with bipolar disorder experiencing a depressive episode of at least moderate severity. This is the first study to our knowledge to investigate the antidepressant effects of IV scopolamine in an exclusively bipolar disorder cohort. This study can help further research investigate the potential antidepressant effects of anticholinergic medications.Some commonly documented adverse effects of IV scopolamine include drowsiness, blurred vision, dry mouth, thirst and urinary retention. No serious adverse events are expected from the IV scopolamine infusion, and no serious adverse events were witnessed in previous studies using IV scopolamine in depressed patients.In relation to ethical considerations and patient confidentiality, the vulnerability of this study group is fully appreciated, and every effort will be undertaken to protect their safety and well-being. During their time on the trial, participants will continue usual care, as recommended by their responsible clinical team.In line with the applicable regulatory requirements, consenting processes will be standardised and a robust standard operating procedure (SOP) for consenting participants will be adhered to. Patients will be given an information leaflet prior to participating in the study. The consenting process will be performed by trained clinical staff  and will include providing a detailed explanation of the study, along with ample time for the research participants to ask questions about the study. Once agreeable, both the research participant and clinical staff taking consent will sign the consent form. A copy will be given to the participant and another copy will be stored in their medical file. In addition to having a full awareness of all study procedures prior to entering the study, participants will understand that research participation is completely voluntary and that they can withdraw from the study at any time.n = 50) has been attained. It is consequently anticipated that participant recruitment will finish in March 2024.Recruitment has commenced in March 2021. The duration of this study is 3 years, or until the number of necessary participants who have completed randomisation (Vice-President for Research, National University of Ireland GalwayE-Mail: vpresearch@nuigalway.ieTel: +353-91-495678


Breast carcinoma is the second largest cancer in the world among women. Early detection of breast cancer has been shown to increase the survival rate, thereby significantly increasing patients’ lifespan. Mammography, a noninvasive imaging tool with low cost, is widely used to diagnose breast disease at an early stage due to its high sensitivity. Although some public mammography datasets are useful, there is still a lack of open access datasets that expand beyond the white population as well as missing biopsy confirmation or with unknown molecular subtypes. To fill this gap, we build a database containing two online breast mammographies. The dataset named by Chinese Mammography Database (CMMD) contains 3712 mammographies involved 1775 patients, which is divided into two branches. The first dataset CMMD1 contains 1026 cases (2214 mammographies) with biopsy confirmed type of benign or malignant tumors. The second dataset CMMD2 includes 1498 mammographies for 749 patients with known molecular subtypes. Our database is constructed to enrich the diversity of mammography data and promote the development of relevant fields. The popularity of mammography uptake in breast carcinoma treatment has dramatically improved the 5-year survival rate of breast carcinoma since the 1980s2. Due to the sensitivity of mammography and the heterogeneity of breast cancer lesions, invasive methods such as biopsy, surgery is critical to confirm the benign and malignant tumors, and the molecular subtypes to optimize the type of treatment3.Breast carcinoma is one of the most commonly diagnosed cancer and the second leading cause of death from cancer in women9 aims at locating suspect lesions such as mass and microcalcification, leaving the classification to the radiologist; and (2) Computer-aided diagnosis (CADx)14 aims to characterize the suspicious region of lesion and/or estimate its probability of onset; and (3) Findings of predictive image-based biomarkers18 by applying the computational methods to mine the potential relationships between image representation and molecular subtype, including luminal A, luminal B, HER2 positive, and Triple-negative. Although mammography imaging is rapidly growing in the three areas, the promising results of radiomics approaches have not been widely used in daily clinical practice. Limited data sharing is an essential reason for reducing the development of radiomics strategies.Advances in both imaging and computer have synergistically lead to a rapid rise of the artificial intelligence (AI) for breast imaging in the following three tasks: (1) Computer-aided detection (CADe)23 that are publicly and freely available to authorized investigators. The datasets involve the Digital Database for Screening Mammography (DDSM), the Mammographic Imaging Analysis Society (MIAS) database, the Image Retrieval in Medical Application (IRMA) project, and the Curated Breast Imaging Subset of DDSM (CBIS-DDSM). Notwithstanding these public datasets are useful, there is still a lack of open access datasets that expand beyond the white population, which will enable researchers to verify previous findings and make the dataset more diverse. Furthermore, the biopsy confirmed results, such as immunohistochemical or molecular subtype, for most of the current datasets are missing. Therefore, an open-access database consisting of large samples with immunohistochemical type is valuable for researchers who are interested in this domain or who require an independent database for cross-validation. In this study, we built a database that contained two branches labeled by Chinese Mammography Database  for allowing researchers to investigate the relationships among image features, pathological assessment, and tumor molecular subtypes. Specifically, CMMD1 including 1026 cases diagnosed with benign or malignant tumors were collated to promote the development of the CADx and CADe. While the CMMD2 included 749 cases, its purpose is to investigate the relationship between image features of invasive carcinoma and molecular subtypes. Note, the cases in CMMD2 have more complete immunohistochemical markers than CMMD1. Both datasets involved mammography images and clinical data such as age, and benign or malignant tumor. Currently, it is available for research through the International Data-sharing Initiative. Our free data sharing can hasten the clinical application of radiomics approaches. Table In investigating the CADe and CADx, there are several datasetsEthical approval was acquired for this retrospective analysis, and the requirement to obtain informed consent was waived. Our study was conducted on 1775 patients  with benign or malignant breast who underwent mammography examination between July 2012 and January 2016. CMMD1 involves 1026 patients , which have the mammography data and complete clinical data. CMMD2 includes 749 patients  with complete immunohistochemical markers. Figure Image data were acquired on a GE Senographe DS mammography system and a Siemens Mammomat Inspiration mammography system in the SunYat-sen University Cancer Center in Guangzhou, and the Nanhai Affiliated Hospital of Southern Medical University in Fushan, China. The scans were processed by the operator with a fixed operating procedure. For each subject, craniocaudal (CC) projection images and mediolateral oblique (MLO) projections images were obtained. In the released database, the raw images were stored as 8-bit grayscale in the Digital Imaging and Communications in Medicine format. All images were digitized at a resolution of 2294 × 1914 pixels.24. By referring to commonly used X-ray classification methods, the images are divided into three types of masses, calcifications, and both. Note, the two radiologists independently reviewed the mammography in our study. When the results of the two doctors are inconsistent, they will combine the pathology report to further determine the type of abnormality.Two radiologists with at least five years of experience performed mammography interpretation and guidance before surgery to determine which patients should be treated surgically. It was asked to refer to the standard readings of the breast imaging report and data system, established by the American College of RadiologyIn this study, biopsy samples were collected from all patients by core needle biopsy. The sample tissues were routinely stored as formalin-fixed and paraffin-embedded tissue blocks. The pathologist stained the section of biopsy tissue with hematoxylin and eosin (HE), analyzed the tissue morphology under the microscope. If necessary, surgery was performed to extract the suspicious lesion specimen. The immunohistochemistry test is conducted to determine the pathological result.25. The surgical specimens were fixed with 4% neutral buffer formaldehyde solution. The monoclonal antibodies were adopted for nuclear staining to evaluate the status of ER and PR. A negative test was defined as staining less than 1% (<1%) of tumor cells, while a positive test was defined as staining of greater than or equal to 1% (≥1%) of tumor cells. In assessing the expression of HER2, the specimen was first graded by IHC and scored by 0 to 3+, according to the recommendations of the American Society of Clinical Oncology/College of American Pathologists26. If there is no observed staining or faintly/barely perceptible membrane staining in less than 10% (<10%) of tumor cells, the score was set as 0. If there are greater than or equal to 10% (≥10%) of tumor cell membrane staining or the cell membrane staining faintly/barely noticeable, the score was marked as 1+. If there is weakly to moderately complete membrane staining observed in more than 10% (>10%) of tumor cells, the score was marked as 2+. In this case, the tissue was further evaluated by fluorescence in situ hybridization (FISH) analysis for HER2 gene amplification. In assessing the expression of Ki-67, immunostaining was performed by the monoclonal antibody Ki-67. The Ki-67 expression is divided between 0% and 100%. A cutoff value of 20% was used to classify the sample into low or high expression27.According to the different expressions for immunohistochemistry including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67, invasive breast carcinoma is divided into four molecular subtypes, including Luminal A , Luminal B (ER+ and/or PR+ and Her2+ or Ki-67 > 20%), HER2-enriched , and triple-negative To sum up, we list the clear and transparent about each step in the generation of the dataset, ultimately presenting a fully reproducible dataset, as shown in Fig. A unique identifier for each subject was identical in all two public datasets in this database. Subject IDs were 4-digit numbers in the form of D1-xxxx or D2-xxxx.28 contains breast mammography images and corresponding clinical data. Imaging, clinical data for all subjects are stored in The Cancer Imaging Archive https://www.cancerimagingarchive.net/ under 10.7937/tcia.eqde-4b16. Imaging data for all subjects are were store in the folder CMMD. All image data were processed using standard TCIA curation workflows. TCIA uses a standards-based approach for de-identification of images stored in the Digital Imaging and Communications in Medicine format. One comma-delimited file  contains clinical data for all subjects with unique subject identifiers. Table The CMMD collectionOur data has some notable limitations. First, the sample size is not very large. Second, the ROI is not marked. We will add more available information and increase the amount of data in the future.All data were collected by the hospital and used as part of the diagnosis, therefore all quality assurances were performed by the institution that collected the data.29 are analyzed on CMMD1, while CMMD2 with molecular subtypes is our newly added data. All data are raw data without any preprocessing. We also welcome any cooperation with us to fully explore our dataset.The data of our previous publications


ACSCentral Science, Fiedler and co-workers report a new approachto elucidate the structures of myo-inositol polyphosphates(InsPs) and quantify them as part of complex mixtures, which was previouslypossible only to a very limited extent.1 An NMR-based method was developed to characterize compounds arisingfrom the metabolism of InsPs. With structural information on the metabolitesin hand, the authors then studied the actions of an enzymatic modulator,the phosphatase MINPP1, on different substances in detail.1 Their investigations reveal two distinct metabolicpathways as well as potential mechanisms of regulation and make significantprogress toward a fully assigned network of the different inositolphosphate metabolites.In this issue of myo-inositol and can act as signaling molecules, among other biologicalroles.2 The study of these molecules ischallenging due to the presence of multiple isomers, and many currentmethods rely on radioactive labeling or require separation of thecompounds prior to analysis.3 Frequently,isolation is based on exploiting the highly charged nature of InsPs,which creates a bias against less charged metabolites such as InsP2. Additionally, some InsPs are chiral, and both enantiomersbehave identically in conventional purification methods. To overcomethese limitations, Nguyen Trung et al. combine 13C-labelingof myo-inositol with an NMR method that suppressesany signals from protons bound to 12C, that is unlabeledmaterial.4 This strategy allows for selectivemonitoring of 13C-labeled myo-inositolmetabolites in a complex sample such as cellular extracts and therebycircumvents the need for isolation of the metabolites of interest.In this study, the 2D HMQC signals of 19 different InsPs and inositol pyrophosphates (PP-InsPs) were assigned and collated in order to identify the metabolites bytheir NMR signals. This “map” of NMR signals shows clusteringdependent on the position of the group and its phosphorylation status,and importantly provides a reference for the assignment of observedsignals in samples. As a limitation, unknown products resulting fromother pathways may be observed but cannot be assigned yet withouta reference spectrum, as was the case for the labeled metabolic extractsof Schizosaccharomyces pombe. To resolve the identityof chiral InsPs asymmetrically labeled myo-inositol was added to cells, resulting in NMR signals only fromthe proton attached to the labeled carbon atom(s)  1a. Using13C atom and that there are no other modifications that resultin a highly similar signal. For this to be the case, the metabolicmodification needs to be close to the CH group of interest. Additionally,a reference library of known metabolites is necessary to assign chemicalshifts. If these requirements can be met, this strategy is a highlypromising avenue for fields that are facing similar challenges asthe study of InsPs.Excitingly, this method is theoretically applicable for studyingthe metabolism of other types of compounds. A prerequisite for successfulassignment is that metabolism of the labeled molecule results in anobservable change in chemical shift of a proton attached to a 13C-labeledmetabolites was then applied in this study for the detailed characterizationof the only human enzyme known to dephosphorylate InsP6, MINPP1.1 Previously unassigned metaboliteswere identified in MINPP1-deficient cells, and InsPs resulting from MINPP1 acting on two different substrates, InsP5[2OH] and InsP6, were quantified in invitro assays. A potential limitation is the sensitivity ofthe NMR method, which precludes the assignment of less abundant species.Addressing this issue, experiments requiring higher sensitivity such as kinetic analysis or metabolic flux were carried out by combiningthe metabolic labeling with capillary electrophoresis coupled to massspectrometry (CE-MS),5 which also distinguishesdifferently labeled metabolites. By adding differently labeled myo-inositol at different time points, the sequence of (de)phosphorylationevents in cells could be determined.The established NMR analysis methodology of 2 was shown to be dependent on MINPP1 and appearednot to be an intermediate in the biosynthesis of higher InsPs. Theyalso discovered that InsP6 acts as a competitive inhibitor,alluding to a potential regulatory mechanism. Regulation of thesepathways is of particular interest, since, at least in vitro, MINPP1is capable of acting on a minimum of 12 substrates, although not allmetabolites observed in vitro were observed in cellular extracts.In addition, results from this work point toward the existence ofan additional unknown 3-phosphatase that acts on InsP6.It is therefore likely that not all of these reactions are catalyzedby MINPP1 in cells. Nonetheless, the multitude of potential reactionscatalyzed by MINPP1 and their potential signaling roles indicate thatregulation is required. The newly discovered substrate inhibitionby InsP6, possibly in conjunction with spatial separation,may be one mechanism of directing flux toward certain metabolites.Maintaining the delicate balance between different InsPs is importantfor cellular function, as an imbalance of InsPs has been linked tomutations of MINPP1 in a subtype of pontocerebellar hypoplasia, agroup of early onset neurodegenerative disorders.6 Interestingly, the involvement in disease appears to relateto the type of mutations, as different mutations in MINPP1 were suggestedto play a role in a subset of malignant follicular thyroid tumors,7 while the complete absence of MINPP1 in micedid not lead to a disease phenotype.8 Theherein established methodologies in combination with CE-MS open upthe possibility to identify InsP metabolites with high sensitivityin the presence of different mutations in disease settings, whichpromises to advance our understanding of the effects of MINPP1 mutationsin the disease mechanisms.The powerof the newly developed methodology primarily lies in the detailedstructural information it can provide. Importantly, in this work theauthors demonstrate how this information can be harnessed to uncovertwo completely distinct metabolic pathways that both involve MINPP11b. The gOverall, the newly developed methodologyenables mapping out the mammalian InsP metabolitenetwork 1b. A sim


For locally advanced non-small cell lung cancer (NSCLC), the neoadjuvant therapy strategy of preoperative nivolumab combined with chemotherapy has great potential, especially for locally advanced NSCLC which are initially unresectable. They may be cured after neoadjuvant immunotherapy, and this may become a new direction of treatment. We hope that this representative medical record and literature review can provide some assistance for clinicians using immune checkpoint inhibitors to treat lung cancer.A 50-year-old male patient was admitted to Zhongshan Hospital of Traditional Chinese Medicine on April 27, 2020 due to “coughing for more than one month.”. The patient had nothing of note in either his medical history or that of his family, and no history of smoking.The diagnosis was cT4N2M0IIIB stage right lower lung NSCLC with right hilar and mediastinal lymph node metastasis. The stage was inoperable stage IIIB NSCLC, but the patient had a strong willingness for doing surgery.The patient received 3 rounds of the neoadjuvant nivolumab therapy combined with TP (paclitaxel plus cisplatin) regimen, on 5-14-21, 06-07-21 and 07-07-21.The tumor's area shrunk. Then the patient underwent thoracoscopic radical resection of the cancer in the right upper lung and postoperative pathology achieved pathological complete response (pCR)In this case, combined with the wishes of the patient and the latest research results, we confirmed pCR by radical surgery after 3 rounds of the neoadjuvant nivolumab therapy combined with chemotherapy. This may be a modality to cure more lung cancer patients in the future. In recent years, immunotherapy has gradually become one of the 3 most important means of cancer treatment. Some programmed death-1 (PD-1)/programmed death receptor ligand-1 (PDL-1) inhibitors can activate the autoimmune system and inhibit tumor progression. As such, an increasing number of cancer patients have benefitted from immunotherapy.,3 In recent years, great achievements have been made regarding immune checkpoint inhibitors in research on neoadjuvant immunotherapy. Among them, nivolumab's performance has been particularly eye-catching, bringing significant clinical benefits to some patients with early and middle stage tumors, and becoming a hot topic in the field of cancer treatment.–9 We report a case of a patient with IIIB lung cancer. After 3 rounds of neoadjuvant nivolumab therapy combined with chemotherapy, we performed radical surgery to achieve pCR.Lung cancer is one of the most common malignant tumors in the world, and it is also the leading cause of death among malignant tumors.22.1A 50-year-old male patient was admitted to Zhongshan Hospital of Traditional Chinese Medicine on April 27, 2020 due to “coughing for more than one month.” He had with no special medical history or family medical history, and no smoking history. Physical examination: there was no palpable enlargement of the lymph nodes anywhere in his body. Cardiopulmonary physical examination showed nothing out of the ordinary. Imaging examination: 04–20–2021) enhanced CT conducted in Zhongshan Hospital of Traditional Chinese Medicine showed a lumpy soft tissue density shadow in the dorsal segment of the lower lobe of the right lung, with an area of about 69 mm × 56 mm. The edges were not smooth, the internal density was uneven, and the corresponding bronchus was occluded. Additionally, there were nodules in the middle lobe of the right lung, with a diameter of about 3 mm. The mediastinal shadow had no deviation, and there were many enlarged lymph nodes in the mediastinum, the length and diameter of which were about 19 mm. There was no pleural effusion, no pleural thickening and no destruction of bony thorax. We also found multiple cysts in the liver, the diameter of which were about 28 mm  NSCLC patients were enrolled in this clinical trial. We administered 2 doses of PD-1 inhibitor nivolumab (3 mg/kg) intravenously every 2 weeks before surgery, and surgery was scheduled for about 4 weeks after the first dose. The main endpoints of this study were safety and feasibility, with case remission, PD-L1 expression level, tumor mutation load and mutation-related new antigen-specific T cell response as secondary and exploratory endpoints. The results showed that of the 21 patients, 2 (10%) had partial remission, 18 (85%) had stable disease, and only 1 (5%) had progression. Long-term follow-up showed that the 20 patients who had received radical surgery had a disease-free recurrence rate of 73% after 18 months, and the overall survival rate reached as high as 95%.The Checkmate159 study was the first clinical study to use immunotherapy in the field of neoadjuvant therapy for NSCLC.Lancet Oncology on September 24, 2020. The data showed that all patients were removed by R0, and the 12-month progress free survival (PFS) rate was 95.7%. The PFS rate at 18 months was 87%, and the PFS rate at 24 months was 77.1%. In the revised intended treatment group (ITT) population, the overall survival (OS) rates of 12 months, 18 months and 24 months were 97.8%, 93.5%, and 89.9%, respectively. At the 2021 WCLC meeting, the researchers presented data from the NADIM trial with a median follow-up of 37.9 months. The updated data showed that in the ITT, the PFS rate for neoadjuvant therapy with nivolumab combined with chemotherapy for 36 months and 42 months was the same, both 69.6%. This suggested that there had been no disease progression over these 6 months. In the population treated according to the scheme (PP), the PFS rates of 36 months and 42 months were the same, both 81.8%. The OS rates were 91.0% and 87.3%, respectively, almost three times the previous survival figures.The NADIM study is an open-label, multicenter, single-arm, 2-phase clinical study that includes histopathologically/cytopathologically confirmed, untreated and potentially resectable patients with stage IIIA NSCLC. The subjects in this study were resectable IIIA stage , EGFR/ALK negative NSCLC patients. A total of 46 patients were enrolled in the group, and all patients received at least 1 cycle of neoadjuvant therapy. The preliminary data were published at the 19th World Lung Cancer Congress, and the data were updated at many major conferences such as ASCO and WCLC. According to the preliminary data, 83% of the patients with major pathologic response (MPR) achieved complete pathological remission (defined as no tumor cells being found), and 90% of the patients had decreased staging. The updated follow-up data were published in the journal  Its primary endpoints were pCR and event-free survival. The secondary endpoints were OS, primary pathological remission  and time to death or distant metastasis. Exploratory endpoints included outcomes such as surgical feasibility and perioperative related adverse events. The results showed that the pCR of Nivolumab combined with chemotherapy in the ITT population was 24%, while the pCR of the patients treated with chemotherapy alone was only 2.2%, an increase of about 10 fold . Moreover, regardless of the stage of the disease, the pCR rate and the depth of pathological remission improved significantly. In terms of secondary end points, the MPR of patients who had received Nivolumab combined with chemotherapy before surgery was 4 times higher than that of patients treated with chemotherapy alone . Exploratory analysis showed that the ctDNA clearance rate in the nivolumab combined with chemotherapy group exceeded that of the chemotherapy group (56% vs 34%). At the same time, the higher ctDNA clearance rate also suggested a higher pCR rate. This indicated that there was a strong correlation between the pCR clearance rate and pCR. Combined with the clear predictive effect of pCR and ctDNA clearance on long-term survival in the NADIM study, the pCR benefits of neoadjuvant therapy with immunotherapy plus chemotherapy compared with chemotherapy alone are to be expected, as is the higher proportion of ctDNA eventually translating into long-term survival benefits.CheckMate816 was the first 3-phase study to confirm the benefit of neoadjuvant therapy with immunotherapy plus chemotherapy for resectable NSCLC.4.2,20 The main adverse reactions of nivolumab combined with chemotherapy in neoadjuvant therapy for non-small cell lung cancer are as follows.Although nivolumab has produced numerous positive results in clinical trials, we should also pay attention to the adverse effects of immunotherapy, which can affect multiple organs in the body, such as the liver, lungs, skin, eyes, endocrine system and gastrointestinal tract.In terms of safety in the Checkmate159 study, treatment-related adverse reactions happened in only 23% of the cases, and only one case was more than grade 3. The study revealed that after immunotherapy, the use of PD-1 blockers before surgery not only improves the response of anti-tumor T cells, but also stimulates the peripheral blood's production of CD8+T cells with memory characteristics, and continuously terminates cancer cells through the T cells’ anti-tumor activity. This reduces the tumor cells’ viability in micrometastases, potentially reducing the likelihood of tumor micrometastasis after surgery, and mitigating the risk of recurrence. In the NADIM study, the incidence of adverse reactions of degree 3 or above was 30%. No adverse reactions resulted in discontinuation of treatment, delayed surgery or death during the neoadjuvant therapy stage. During the postoperative adjuvant therapy stage, 14% of the patients terminated the treatment due to adverse reactions. In the CheckMate816 study, the patients tolerated the neoadjuvant immunotherapy, and there was no significant increase in surgical complications. The proportions of surgery-related adverse event reported in the nivolumab combined with chemotherapy group and the chemotherapy group were 41% and 47%, respectively, and the surgery-related adverse event for grades 3 to 4 were 11% and 15%, respectively.Several existing studies have shown that the safety of neoadjuvant immunotherapy is similar to that of previous neoadjuvant chemotherapy and radiotherapy. Most of the treatment-related adverse events during the nivolumab combined with chemotherapy neoadjuvant therapy were grade 1–2, and there was no death caused by the adverse events. Neoadjuvant nivolumab therapy combined with chemotherapy maintains tolerable safety features and does not hinder surgery feasibility. In exploratory subgroup analysis, the nivolumab plus chemotherapy group's ctDNA clearance rate was higher than that of the single drug chemotherapy group, indicating that it may have been related to pCR. Data from several clinical studies have shown that immunoadjuvant therapy does not affect surgery timing. We expect this to change the traditional lung cancer treatment mode.5Lung cancer is the leading cause of cancer death throughout the world, and cases are on the rise due to a variety of factors. A growing number of clinical trials have demonstrated that immunotherapy is essential to lung cancer treatment. The patient in our study was initially diagnosed as being at the locally advanced T4N2M0IIIB stage, with a TPS of only 2%. Before the era of immunotherapy, previous evidence for patients with stage NSCLC showed that without considering radical surgery, the highest level of evidence for NSCLC patients with IIIB was simultaneous radiotherapy and chemotherapy, but the effects have not been satisfactory, especially for patients in China. In this case, combined with the wishes of the patient and the latest research results, we confirmed pCR by radical surgery after 3 rounds of neoadjuvant therapy consisting of nivolumab combined with chemotherapy. This has revealed a potential cure for more patients with lung cancer. Of course, determining whether patients with locally advanced NSCLC with low TPS expression can achieve success in neoadjuvant therapy necessitates further exploration of possible mechanisms and more accurate biomarkers. Whether each of these patients needs 3 rounds of neoadjuvant therapy as immunotherapy combined with chemotherapy is also worthy of further discussion. For example, can patients who receive 2 cycles of neoadjuvant therapy also receive 4 rounds of neoadjuvant therapy? If patients have poor response to 2 cycles of neoadjuvant therapy, should we be more flexible to adjusting the original neoadjuvant therapy regimen? Should we consider changing multiple disciplinary team treatment strategies? These questions could be addressed with more evidence from randomized controlled trial studies.Although the neoadjuvant therapy model of immunotherapy plus chemotherapy has produced a qualitative leap in the proportion of pCR in non-advanced NSCLC patients, we still need to continue exploring the following issues: Does pCR show long-term benefits in prospective studies on larger populations? Long-term survival is the most important outcome, and we look forward to the CM816 long-term follow-up); Is it necessary for pCR/MPR patients to adjust their surgical plans and methods? For a more accurate population distinction, which population is most suitable for quantifying postoperative assistance with individualized regimens ? Are there specific patients who are exempt from surgery? Is overtreatment a concern? Is the same scheme is used in different types, different stages and different PD-L1 expressions? For each patient, we need to combine the evidence from randomized controlled trial research with patients’ wishes to arrive at a comprehensive diagnosis and treatment strategy so that the patient can be an active participant in their treatment. Only in this way can patients maximize benefits. Based on this case report and related studies, we have reason to believe that the future will bring greater progress in immunotherapy, such that more lung cancer patients will be cured .We would like to thank the Department of Radiology, Pathology, and Pharmacy at the Zhongshan Hospital of Traditional Chinese Medicine.Data curation: Ting Chen.Investigation: Xianhui Zhao.Methodology: Jiaming Wu.Resources: Ao Zhang, Huisi Hong, Lexia Wu, Sihong Lin.Resources: Huatang Zhang.Supervision: Cantu Fang.Validation: Kexin Wang.Writing – original draft: Luzhen Li.Writing – review & editing: Huiqin Lai.


Data from case series suggest that clozapine may benefit inpatients with borderline personality disorder (BPD), but randomised trials have not been conducted.Multicentre, double-blind, placebo-controlled trial. We aimed to recruit 222 inpatients with severe BPD aged 18 or over, who had failed to respond to other antipsychotic medications. We randomly allocated participants on a 1:1 ratio to receive up to 400 mg of clozapine per day or an inert placebo using a remote web-based randomisation service. The primary outcome was total score on the Zanarini Rating scale for Borderline Personality Disorder (ZAN-BPD) at 6 months. Secondary outcomes included self-harm, aggression, resource use and costs, side effects and adverse events. We used a modified intention to treat analysis (mITT) restricted to those who took one or more dose of trial medication, using a general linear model fitted at 6 months adjusted for baseline score, allocation group and site.The study closed early due to poor recruitment and the impact of the COVID-19 pandemic. Of 29 study participants, 24 (83%) were followed up at 6 months, of whom 21 (72%) were included in the mITT analysis. At 6 months, 11 (73%) participants assigned to clozapine and 6 (43%) of those assigned to placebo were still taking trial medication. Adjusted difference in mean total ZAN-BPD score at 6 months was -3.86 . There were 14 serious adverse events; 6 in the clozapine arm and 8 in the placebo arm of the trial. There was little difference in the cost of care between groups.We recruited insufficient participants to test the primary hypothesis. The study findings highlight problems in conducting placebo-controlled trials of clozapine and in using clozapine for people with BPD, outside specialist inpatient mental health units.https://doi.org/10.1186/ISRCTN18352058ISRCTN18352058.  Some people with severe BPD have very high levels of contact with inpatient mental health services. It is estimated that more than a fifth of inpatients on general adult wards,4 more than 40% of women on Psychiatric Intensive Care Units, and more than 60% of women treated on medium secure units have BPD.Borderline personality disorder (BPD) is a common mental health condition that is characterised by instability in emotions, identity, and interpersonal relationships. people with the most severe problems are less likely to engage in them.9 No medication is licenced for the treatment of BPD, but despite this, people with severe BPD are often prescribed multiple medications.11 Clozapine is used to treat people with schizophrenia who have not responded to other antipsychotic medications. As well as improving the mental health of people with psychosis, clozapine appears to reduce the incidence of aggressive and impulsive behaviour and it is licenced in the United States for reducing the risk of recurrent suicidal behaviour among people with schizophrenia.While psychological treatments improve the mental health of people with BPD,16 In an observational study of 102 Danish patients with BPD who had ever been prescribed clozapine, levels of self-harm were found to be lower when people took this medication compared to when they did not. Qualitative data collected from women with borderline personality disorder treated with clozapine on secure wards also highlight potential benefit, with many describing marked improvements in their mental health and relationships with others. While it is possible that clozapine improves the mental health of people with borderline personality disorder, it has serious side effects including weight gain and associated metabolic complications, constipation, seizures and potentially fatal agranulocytosis, pneumonia, myocarditis and paralytic ileus.1921 People prescribed clozapine are therefore required to have regular checks on their physical health including weekly blood tests for the first 18 weeks of treatment. Previous placebo-controlled trials of medication for people with borderline personality disorder have shown clear evidence of improved outcomes for those prescribed placebos. We do not know to what extent the benefits of clozapine seen in open-label studies can be explained by non-specific factors including instillation of hope and the structured care that people taking clozapine receive through additional monitoring of their physical health.Open label studies of clozapine for people with borderline personality disorder have reported improved mental health, reductions in aggressive and self-harming behaviour, and lower costs of care.We therefore designed a placebo-controlled trial to test the clinical effectiveness and cost-effectiveness of clozapine for inpatients with borderline personality disorder. We followed up participants 3 and 6 months after randomisation to examine the clinical effects, costs and safety of this treatment.DSM-IV diagnostic criteria for borderline personality disorder using the Structured Clinical Interview for Axis II Personality Disorders. Severity of personality disorder was based on service use and risk. To take part in the study potential participants had to have been an inpatient on a mental health ward for more than 28 days in the last 12 months, or to have had two or more admissions to hospital/periods of care provided by Home Treatment over the last 12 months, plus a lifetime history of two or more incidents of harm to self or others which resulted in permanent damage, or would have done so had services not intervened. Potential participants had to have not shown an adequate clinical response despite taking antipsychotic medication other than clozapine for at least 3 months. They also needed to have their current body weight and blood glucose level recorded in their clinical records and have a satisfactory pre-treatment full blood count (white blood cell count > = 3.5 x109/L and absolute neutrophil count > = 2.0 x109/L). We excluded those who had a current coexisting clinical diagnosis of schizophrenia, or bipolar I disorder, and those who had been prescribed clozapine within the last 2 weeks, as well as those women known to be pregnant, trying to conceive, breastfeeding, or who were of childbearing potential but not using a highly effective birth control. We also excluded any potential participant who was due to be discharged within the following 2 weeks unless arrangements for necessary monitoring of physical health as an outpatient could be made. We excluded those unable to speak sufficient English to complete the baseline assessment, those unable to undergo regular blood tests, those with contraindications for clozapine and those unwilling or unable to provide written informed consent to take part in the study.The CALMED  study is a two-arm, parallel group, blinded, randomised trial of clozapine versus placebo for inpatients with a diagnosis of borderline personality disorder. We recruited inpatients aged 18 years or over who had severe borderline personality disorder and were being treated by one of seven NHS and independent sector organisations in England. Potential participants had to meet https://doi.org/10.1186/ISRCTN18352058The study was approved by the Wales Research Ethics Committee 1 Research Ethics Committee (Ref: 217828). The Medicines and Healthcare Products Regulatory Agency (MHRA) gave Clinical Trial Authorisation and the Research and Development departments of the participating provider organisations approved the trial prior to the start of recruitment. The participants provided written informed consent. This trial was prospectively registered at ISRCTN with the ID #18352058:  and the International Personality Disorder Examination (IPDE) Screening Questionnaire and all were enrolled with the Clozaril Patient Monitoring Service. We used this service to ensure that no study participant could be administered study medication if they had a significantly reduced white blood cell count during the previous 7 days during the first 18 weeks of treatment and for 14 days thereafter (white blood cell count  < 3.0 x109/L and absolute neutrophil count  < 1.5 x109/L).Eligible participants were asked to complete the Psychosis Screening Questionnaire A trial prescription form was then completed by the responsible clinician and sent to a local pharmacy for dispensing to the ward where the participant was being treated.We then randomly allocated participants on a 1:1 ratio to either clozapine or placebo via a secure and automated randomisation service operated by NWORTH, University of Bangor. We used a sequentially randomised, dynamic adaptive algorithm, stratified by centre, ward type and gender.All study researchers, aside from the trial manager, remained masked to allocation status until after an initial discussion of the trial findings was completed. We asked participants and researchers to guess allocation status after they had collected 6-month follow-up data.All those taking part in the study continued to receive usual treatment including inpatient psychosocial care prior to transfer to another ward, or discharge to the community contingent upon their mental health and risk assessment. We imposed no restrictions on the use of other treatments, except that the participants who remained in the trial could not be prescribed clozapine. Those in the active arm of the trial were prescribed up to 300 mg of clozapine titrated over a 15-day period. The dose could then be increased to 400 mg of clozapine daily, depending on clinical response, patient preference and side effects. Those randomised to control treatment were prescribed an inert placebo capsules that were identical in appearance but contained a microcrystalline cellulose backfill. The ZAN-BPD is a widely used outcome in trials for BPD and provides a reliable and valid assessment of core features of the condition and is sensitive to change.29 A higher score on the measure indicates poorer mental health.The primary outcome was total score on the Zanarini rating scale for Borderline Personality Disorder (ZAN-BPD) at 6 months.The secondary outcomes were;i. Total score on the Zanarini rating scale for Borderline Personality Disorder at 3 months. The BPRS has been used in previous open-label studies of clozapine in people with borderline personality disorder.16ii. General mental health using the Brief Psychiatric Rating Scale (BPRS) at 3 and 6 months.iii. Incidence and severity of suicidal behaviour using the Acts of Deliberate Self-Harm Inventory.iv. Level of aggressive behaviour using the Modified Overt Aggression Scale (MOAS).v. Health related quality of life using the EuroQuol-5 Dimensions (EQ-5D-5 L). and motor and extrapyramidal side effects using the Extrapyramidal Side Effects Scale.vi. Side effects of medication using the Antipsychotic Non-Neurological Side Effects Scale (ANNSERS)vii. Incidence of withdrawal of trial medication due to adverse effects.viii. Medication adherence at 3 and 6 months using the Brief Adherence Rating Scale (BARS).ix. Indication of the severity of personality problem collected at baseline and 6 months using the self-report Standardised Assessment of Severity of Personality Disorder (SAS-PD). This includes detailed information about length of inpatient treatment and type of ward, contacts with community mental health services and emergency medical services, and the type and dose of psychotropic medication that people were prescribed.x. Resource use collected using a modified version of the Adult Service Use Schedule.Using PASS software  we calculated that we would need to analyse data from 166 participants (83 receiving clozapine and 83 receiving placebo) to have 90% power to detect a four-point clinically important difference in ZAN-BPD score at 6 months, with an assumed standard deviation of 7.89 and a significance level of 0.05 using a two-sided two sample t-test. To take account of 25% loss to follow-up we planned to recruit 222 subjects.The analysis of both the primary and secondary outcomes used a linear mixed model adjusted for baseline score along with site as a random effect. For the primary analysis we used a modified intention to treat analysis (mITT), using data from all randomised participants who took at least one dose of trial medication. Where the number of missing items on an outcome measure was 20% or less, we substituted the individual’s mean score for the remaining items on the scale. If there were more than 20% missing items on the scale, the outcome measure was not calculated for the participant at that time point and multiple imputation methods were used where appropriate. on the primary outcome by including this as a factor in the model. We also conducted a sensitivity analysis of trial arm perception of both the participant and the researcher; the primary analysis model was run including allocation perception to assess any impacts on the scores. Finally, we examined differences in the primary outcome in different treatment settings: general adult wards, low, medium and high secure units. All statistical tests were two-sided using a 5% significance level and we calculated 95% confidence intervals for estimated effect sizes.We undertook secondary exploratory sensitivity analyses. A per protocol analysis was conducted in which we analysed data from a subset of participants who took trial medication until the first follow-up interview at three months, in order to explore the effect of clozapine among people who took it. We undertook sensitivity analysis of patient-reported lethargy, derived from the ANNSERS, We collected service use data using a modified version of the Adult Service Use Schedule (AD-SUS), which was based on our previous research in this group and was piloted and adapted in collaboration with clinical and service user members of the research team. Total costs were calculated by matching each service use item with a relevant unit cost, which were sourced from routine UK sources.41 Primary economic analysis was at 6-month follow-up. Differences in service use were explored descriptively and differences in costs were calculated using standard t-tests adjusted for baseline costs and with bootstrapped confidence intervals, as recommended for cost data.The economic analysis took a health and personal social services perspective, which is relevant to UK decision makers and to this patient group.Recruitment took place from September 2019 to March 2020 and was then suspended due to the COVID-19 pandemic. We attempted to restart recruitment in autumn 2020, but researchers were no longer permitted to visit participating sites due to infection control policies. Continuing pressures on clinical staff resulting from the pandemic and concerns about the off-label use of clozapine during the crisis that further limited recruitment, which subsequently closed in February 2021. We assessed the records of 474 inpatients for eligibility see . Of thesn = 23, 79.3%) were being treated on a compulsory basis and half  were recruited from medium secure units. In addition to meeting DSM-IV criteria for borderline personality disorder, self-report data from the IPDE, which was completed by 27 participants, indicated that most participants met criteria for probable avoidant , schizotypal , compulsive , dependent , schizoid , histrionic , and paranoid 19 (66%) personality disorder. At baseline, 26 participants (90%) had a score of 10 or more on the SASPD, the threshold for moderate/ severe personality disorder (12 out of 14 (86%) in the placebo arm and 14 out of 15 (93%) in the clozapine arm of the trial).Three-quarters of the study sample were female and the mean age was 30 years (range = 18 to 53). Most of the sample (n = 4), non-compliance (n = 2), adverse event (n = 1), physician decision (n = 1) and problems encountered with blood monitoring when a patient was discharged to a community setting. Levels of adherence among those who continued to take trial medication were very high, with a mean percentage score on the Brief Adherence Rating Scale of 98.2 (SD = 2.51) at 3 months and 99.0 (SD = 1.56) at 6 months.Among 12 participants in the clozapine arm of the trial who guessed their allocation status, 8 thought they had been taking clozapine and 4 did not know what they were taking. Of six participants in the placebo arm of the trial who guessed their allocation status, five believed they were taking a placebo and one stated that they did not know. Researchers correctly guessed that 11 of 12 participants in the active arm of the trial were allocated to clozapine and five of the six participants in the control arm of the trial were allocated to placebo.p = 0.22) at 6 months. For the secondary per protocol analysis, the adjusted mean ZAN-BPD was 7.61 (SD = 2.30) in the clozapine arm and 12.77 (SD = 3.16) in the placebo arm  at 6 months. When lethargy was added to the model, the adjusted difference in mean ZAN-BPD score = -8.54, 95%, C.I = -13.31 to -3.75, p < 0.01). Among participants recruited from medium secure units, ZAN-BPD scores changed from 19.1 at baseline to 10.1 at 6 months among those in the clozapine arm, and from 19.2 (SD = 7.7) to 14.5 (SD = 7.6) among those in the placebo arm of the trial.There were no statistically significant differences in study outcomes between those in the clozapine and placebo arm of the trial see . ZAN-BPDRegarding patient safety, 68 adverse events and 49 adverse reactions were reported among participants in the clozapine arm of the trial and 31 adverse events and 8 adverse reactions were reported among those in the placebo arm of the trial. Adverse events were predominantly mental health-related, including deterioration in mental health and self-harm. The main adverse reactions were gastrointestinal (including constipation), sedation and cardiac (including tachycardia). There were also 14 serious adverse events; six among those in the clozapine arm of the trial and eight among those in the placebo arm of the trial.The use of services over the 6-month follow-up period is summarised in We obtained data on 25 (86.2%) participants after they completed the 6-month follow-up interview to see if they were prescribed clozapine after the trial period. Of the 15 participants in the clozapine arm of the trial, 9 (60%) continued to be prescribed it. This included all eight of those in the clozapine arm who were being treated on inpatient units and one patient who had been treated on a general adult ward. Among 10 participants in the placebo arm of the trial, four were subsequently prescribed clozapine; all four participants were being treated as inpatients on specialist wards.p = 0.22). This finding is consistent with the improved outcomes associated with clozapine treatment reported in open-label studies.16 However, the magnitude of the difference between arms was small relative to the size of the placebo response; during the 6-month follow-up period, the general mental health of study participants in both arms of the study improved, and levels of aggression and self-harm halved among those in both the clozapine and the placebo arm of the trial. There were no major tolerability issues identified with clozapine, but again this finding must be interpreted in the context of our small sample size. Fourteen serious adverse events were reported during the trial . None of the 15 participants in the clozapine arm of the trial experienced sustained falls in white blood cell count nor experienced a serious adverse event that related to trial medication. However, one participant experienced a number of non-serious adverse events  that resulted in them stopping taking clozapine.The CALMED study is the first randomised trial of clozapine for people with borderline personality disorder. Problems with recruitment, which were greatly compounded by the COVID-19 pandemic, meant that we fell well short of the sample size required for a fully powered study. Among the 26 participants who took at least one dose of trial medication and completed a 6-month follow-up assessment, there was a numerically lower ZAN-BPD score in the clozapine arm of the trial compared to those in the placebo arm, but this was not statistically significant .47 Adherence to clozapine is particularly important because people who miss more than 48 h of treatment need to be re-titrated and medication has to be stopped if people do not have regular blood monitoring. Problems with adherence are an important obstacle to the use of clozapine for people with borderline personality disorder.Another limitation of the study was adherence to trial medication. Only 15 (52%) of our 29 study participants were taking trial medication at six months. Three people did not take a first dose of study medication. Among the 26 that did take at least one dose, 6 (55%) of those taking placebo and 4 (27%) of those in the clozapine arm of the trial were no longer taking it 6 months later. The reasons why people did not start trial medication or stopped it before the end of the follow-up period varied. In some instances, patients withdrew consent. In other instances, patients who had started to take trial medication were discharged from hospital before arrangements for monitoring their physical health in the community were agreed. Aware of the potential side effects of clozapine, some of the participants who started trial medication and experienced no side effects, judged that they were being given a placebo and withdrew from the study. Levels of adherence with pharmacological and other treatments have been reported to be low among people with borderline personality disorder.49While we were able to keep researchers masked to the allocation status of study participants, the high side-effect profile of clozapine, which includes sedation and hypersalivation, meant that many patients, researchers and clinicians believed they knew which arm of the trial they were in. Among 18 participants who guessed their allocation status after completing their 6-month follow-up assessment, 13 (72%) guessed correctly. Similarly, researchers correctly guessed the allocation status of 89% of study participants. These data suggest that most participants and researchers in placebo-controlled trials of clozapine are able to correctly guess their allocation status, threatening the internal validity of such trials.In addition to halting recruitment, the COVID-19 pandemic meant that researchers were no longer able to conduct face-to-face follow-up assessments. Instead, we arranged remote assessments by telephone or videoconferencing. Pairs of researchers completed all remote follow-up assessments, which helped ensure they were reliable. However, it was not possible to complete the Extrapyramidal Side Effects Scale as this requires a physical examination of the patient.The data from this trial do not form a sound basis for changing current clinical practice. Clinicians working on specialist inpatient units who have seen the mental health of people with borderline personality disorder improve with clozapine are likely to continue to prescribe it. The high cost of care and the relatively low cost of clozapine suggest that, if it was effective in reducing the amount of time people with BPD spend in hospital, clozapine would be a cost-effective treatment. But the logistical challenges we encountered on acute inpatient units when trying to ensure that clozapine was safely prescribed add to concerns about the use of clozapine for people with borderline personality disorder in this setting and in the community. The value placed on clozapine by clinicians working in specialist units is illustrated by the decision to start clozapine in two-thirds of the participants in the placebo arm of the trial who had been recruited in this setting. In contrast, none of the participants in the placebo arm who had been recruited from general adult mental health services were prescribed clozapine after the end of the study. Differences in the beliefs of clinicians working on specialist units and in general adult teams about the value of clozapine for people with severe BPD have implications for patient management. It is important to check that the general adult mental health team that will oversee the care of a person with severe BPD after they have been discharged from hospital is willing and able to provide ongoing physical health monitoring for people on clozapine before a decision is made to initiate this treatment in hospital.Future studies of clozapine for inpatients with severe borderline personality disorder would need to identify units where clinicians supported the need for better information on the costs and benefits of this treatment. Consideration should be given to the use of an alternative antipsychotic medication, such as olanzapine, for those in the control arm of the trial, in order to make it less easy for study participants to correctly guess which arm of the trial they are in.In conclusion, uncertainty remains about the role of clozapine in the treatment of inpatients with severe borderline personality disorder. However, this sense of uncertainty is not shared by many clinicians working on inpatient units in England and this may render randomised trials of clozapine unfeasible in this setting.