Early detachment of neuromuscular junction proteins in ALS mice with SODG93A mutation

The transgenic animals with mutant copper/zinc superoxide dismutase (SOD1) DNA develop paralytic motor neuron disease resembling human amyotrophic lateral sclerosis (ALS) patients and are commonly used as models for ALS. In the transgenic (Tg) mice with the G93A mutation of the human SOD1 gene SOD1G93A mice), the loss of ventral root axons and the synapses between the muscles and the motor neurons suggested that the motor neuron degeneration might proceed in a dying-back degeneration pattern. To reveal the relationship between axonal degeneration and the progression of the muscle atrophy in the SOD1G93A mice, we investigated the status of the neuromuscular junction along the disease progression. As a presynaptic or postsynaptic marker of neuromuscular junction (NMJ), anti-synaptic vesicle protein 2 (anti-SV2) antibody and α-bungarotoxin (α-BuTX) were chosen in this study and, as a marker of synaptic cleft, anti-agrin antibody was chosen in this study. In the immunohistochemistry of α-BuTX and anti-SV2 antibody, the percentages of double positive NMJs among α-BuTX single positive were decreased in Tg mice through time from ten weeks. The number of postsynaptic acethylcholine receptor (AChR) clusters did not decrease in Tg mice even at the end stage. Immunohistochemistry of α-BuTX and anti-agrin antibody revealed that the increase of immunopositive area of anti-agrin antibody around the muscle fiber in Tg mice from ten weeks of age. In this study, we revealed that the detachment of nerve terminals started at ten weeks in Tg mice. The levels of AChR did not change throughout 5–20 weeks of age in both groups of mice, and AChR remains clustering at NMJs, suggesting that the muscle abnormality is the result of detachment of nerve terminals

Safety and tolerability of bosutinib in patients with amyotrophic lateral sclerosis (iDReAM study) : A multicentre, open-label, dose-escalation phase 1 trial

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the loss of motor neurons, and development of effective medicines is urgently required. Induced pluripotent stem cell (iPSC)-based drug repurposing identified the Src/c-Abl inhibitor bosutinib, which is approved for the treatment of chronic myelogenous leukemia (CML), as a candidate for the molecular targeted therapy of ALS. Methods: An open-label, multicentre, dose-escalation phase 1 study using a 3 + 3 design was conducted in 4 hospitals in Japan to evaluate the safety and tolerability of bosutinib in patients with ALS. Furthermore, the exploratory efficacy was evaluated using Revised ALS Functional Rating Scale (ALSFRS-R), predictive biomarkers including plasma neurofilament light chain (NFL) were explored, and single-cell RNA sequencing of iPSC-derived motor neurons was conducted. Patients, whose total ALSFRS-R scores decreased by 1–3 points during the 12 week, received escalating doses starting from 100 mg quaque die (QD) up to 400 mg QD based on dose-limiting toxicity (DLT) occurrence, and all participants who received one dose of the study drug were included in the primary analysis. This trial is registered with ClinicalTrials.gov, NCT04744532, as Induced pluripotent stem cell-based Drug Repurposing for Amyotrophic Lateral Sclerosis Medicine (iDReAM) study. Findings: Between March 29, 2019 and May 7, 2021, 20 patients were enrolled, 13 of whom received bosutinib treatment and 12 were included in the safety and efficacy analyses. No DLTs were observed up to 300 mg QD, but DLTs were observed in 3/3 patients of the 400 mg QD cohort. In all patients receiving 100 mg–400 mg, the prevalent adverse events (AEs) were gastrointestinal AEs in 12 patients (92.3%), liver function related AEs in 7 patients (53.8%), and rash in 3 patients (23.1%). The safety profile was consistent with that known for CML treatment, and ALS-specific AEs were not observed. A subset of patients (5/9 patients) was found to respond well to bosutinib treatment over the 12-week treatment period. It was found that the treatment-responsive patients could be distinguished by their lower levels of plasma NFL. Furthermore, single-cell RNA sequencing of iPSC-derived motor neurons revealed the pathogenesis related molecular signature in patients with ALS showing responsiveness to bosutinib. Interpretation: This is the first trial of a Src/c-Abl inhibitor, bosutinib, for patients with ALS. The safety and tolerability of bosutinib up to 300 mg, not 400 mg, in ALS were described, and responsiveness of patients on motor function was observed. Since this was an open-label trial within a short period with a limited number of patients, further clinical trials will be required

Induced pluripotent stem cell–based Drug Repurposing for Amyotrophic lateral sclerosis Medicine (iDReAM) study : protocol for a phase I dose escalation study of bosutinib for amyotrophic lateral sclerosis patients

Introduction Amyotrophic lateral sclerosis (ALS) is a progressive and severe neurodegenerative disease caused by motor neuron death. There have as yet been no fundamental curative medicines, and the development of a medicine for ALS is urgently required. Induced pluripotent stem cell (iPSC)-based drug repurposing identified an Src/c-Abl inhibitor, bosutinib, as a candidate molecular targeted therapy for ALS. The objectives of this study are to evaluate the safety and tolerability of bosutinib for the treatment of patients with ALS and to explore the efficacy of bosutinib on ALS. This study is the first clinical trial of administered bosutinib for patients with ALS. Methods and analysis An open-label, multicentre phase I dose escalation study has been designed. The study consists of a 12-week observation period, a 1-week transitional period, a 12-week study treatment period and a 4-week follow-up period. After completion of the transitional period, subjects whose total ALS Functional Rating Scale-Revised (ALSFRS-R) score decreased by 1–3 points during the 12-week observation period receive bosutinib for 12 weeks. Three to six patients with ALS are enrolled in each of the four bosutinib dose levels (100, 200, 300 or 400 mg/day) to evaluate the safety and tolerability under a 3+3 dose escalation study design. Dose escalation and maximum tolerated dose are determined by the safety assessment committee comprising oncologists/haematologists and neurologists based on the incidence of dose-limiting toxicity in the first 4 weeks of the treatment at each dose level. A recommended phase II dose is determined by the safety assessment committee on completion of the 12-week study treatment in all subjects at all dose levels. The efficacy of bosutinib is also evaluated exploratorily using ALS clinical scores and biomarkers. Ethics and dissemination This study received full ethical approval from the institutional review board of each participating site. The findings of the study will be disseminated in peer-reviewed journals and at scientific conferences

Safety and tolerability of bosutinib in patients with amyotrophic lateral sclerosis (iDReAM study): A multicentre, open-label, dose-escalation phase 1 trial

筋萎縮性側索硬化症（ALS）患者さんを対象とした ボスチニブ第1相試験；iDReAM試験の成果報告 （論文発表）. 京都大学プレスリリース. 2022-10-26.Phase I clinical trial of bosutinib for amyotrophic lateral sclerosis (ALS); iDReAM study. 京都大学プレスリリース. 2022-11-28.[Background] Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the loss of motor neurons, and development of effective medicines is urgently required. Induced pluripotent stem cell (iPSC)-based drug repurposing identified the Src/c-Abl inhibitor bosutinib, which is approved for the treatment of chronic myelogenous leukemia (CML), as a candidate for the molecular targeted therapy of ALS. [Methods] An open-label, multicentre, dose-escalation phase 1 study using a 3 + 3 design was conducted in 4 hospitals in Japan to evaluate the safety and tolerability of bosutinib in patients with ALS. Furthermore, the exploratory efficacy was evaluated using Revised ALS Functional Rating Scale (ALSFRS-R), predictive biomarkers including plasma neurofilament light chain (NFL) were explored, and single-cell RNA sequencing of iPSC-derived motor neurons was conducted. Patients, whose total ALSFRS-R scores decreased by 1–3 points during the 12-week, received escalating doses starting from 100 mg quaque die (QD) up to 400 mg QD based on dose-limiting toxicity (DLT) occurrence, and all participants who received one dose of the study drug were included in the primary analysis. This trial is registered with ClinicalTrials.gov, NCT04744532, as Induced pluripotent stem cell-based Drug Repurposing for Amyotrophic Lateral Sclerosis Medicine (iDReAM) study. [Findings] Between March 29, 2019 and May 7, 2021, 20 patients were enrolled, 13 of whom received bosutinib treatment and 12 were included in the safety and efficacy analyses. No DLTs were observed up to 300 mg QD, but DLTs were observed in 3/3 patients of the 400 mg QD cohort. In all patients receiving 100 mg–400 mg, the prevalent adverse events (AEs) were gastrointestinal AEs in 12 patients (92.3%), liver function related AEs in 7 patients (53.8%), and rash in 3 patients (23.1%). The safety profile was consistent with that known for CML treatment, and ALS-specific AEs were not observed. A subset of patients (5/9 patients) was found to respond well to bosutinib treatment over the 12-week treatment period. It was found that the treatment-responsive patients could be distinguished by their lower levels of plasma NFL. Furthermore, single-cell RNA sequencing of iPSC-derived motor neurons revealed the pathogenesis related molecular signature in patients with ALS showing responsiveness to bosutinib. [Interpretation] This is the first trial of a Src/c-Abl inhibitor, bosutinib, for patients with ALS. The safety and tolerability of bosutinib up to 300 mg, not 400 mg, in ALS were described, and responsiveness of patients on motor function was observed. Since this was an open-label trial within a short period with a limited number of patients, further clinical trials will be required

Study on Multicellular Systems Using a Phase Field Model

A model of multicellular systems with several types of cells is developed from the phase field model. The model is presented as a set of partial differential equations of the field variables, each of which expresses the shape of one cell. The dynamics of each cell is based on the criteria for minimizing the surface area and retaining a certain volume. The effects of cell adhesion and excluded volume are also taken into account. The proposed model can be used to find the position of the membrane and/or the cortex of each cell without the need to adopt extra variables. This model is suitable for numerical simulations of a system having a large number of cells. The two-dimensional results of cell adhesion, rearrangement of a cell cluster, and chemotaxis as well as the three-dimensional results of cell clusters on the substrate are presented.Comment: 13 pages, 7 figure

Successful management of refractory pleural effusion due to systemic immunoglobulin light chain amyloidosis by vincristine adriamycin dexamethasone chemotherapy: a case report

<p>Abstract</p> <p>Introduction</p> <p>Refractory pleural effusion in systemic immunoglobulin light chain amyloidosis without cardiac decompensation is rarely reported and has a poor prognosis in general (a median survival of 1.6 months). Moreover, the optimum treatment for this condition is still undecided. This is the first report on the successful use of vincristine, adriamycin and dexamethasone chemotherapy for refractory pleural effusion due to systemic immunoglobulin light chain amyloidosis without cardiac decompensation.</p> <p>Case presentation</p> <p>We report the case of a 68-year old Japanese male with systemic immunoglobulin light chain amyloidosis presenting with bilateral pleural effusion (more severe on the right side) in the absence of cardiac decompensation that was refractory to diuretic therapy. The patient was admitted for fatigue, exertional dyspnea, and bilateral lower extremity edema. He had been receiving intermittent melphalan and prednisone chemotherapy for seven years. One month before admission, his dyspnea had got worse, and his chest radiograph showed bilateral pleural effusion; the pleural effusion was ascertained to be a transudate. The conventionally used therapeutic measures, including diuretics and thoracocentesis, failed to control pleural effusion. Administration of vincristine, adriamycin, and dexamethasone chemotherapy led to successful resolution of the effusion.</p> <p>Conclusion</p> <p>Treatment with vincristine, adriamycin, and dexamethasone chemotherapy was effective for the refractory pleural effusion in systemic immunoglobulin light chain amyloidosis without cardiac decompensation and appears to be associated with improvement in our patient's prognosis.</p

発症早期ALS患者に対する超高用量メチルコバラミンの有効性・安全性について : ランダム化比較試験

Importance: Post hoc analysis in a phase 2/3 trial indicated ultra-high dose methylcobalamin slowed decline of the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) total score at week 16 as well as at week 182, without increase of adverse events, in patients with amyotrophic lateral sclerosis (ALS) who were enrolled within 1 year from onset. Objective: To validate the efficacy and safety of ultra-high dose methylcobalamin for patients with ALS enrolled within 1 year of onset. Design: A multicenter, placebo-controlled, double-blind, randomized phase 3 trial with 12-week observation and 16-week randomized period, conducted from October 2017 to September 2019. Setting: Twenty-five neurology centers in Japan. Participants: Patients with ALS diagnosed within 1 year of onset by the updated Awaji criteria were initially enrolled. Of those, patients fulfilling the following criteria after 12-week observation were eligible for randomization: 1- or 2-point decrease in ALSFRS-R total score, a percent forced vital capacity over 60%, no history of noninvasive respiratory support and tracheostomy, and being ambulant. The target number was 64 in both methylcobalamin and placebo groups. Of 203 patients enrolled in the observation, 130 patients (age, 61.0 ± 11.7 years; female, 56) met the criteria and were randomly assigned through an electronic web-response system to methylcobalamin or placebo (65 for each). Of these, 129 patients were eligible for the full analysis set, and 126 completed the double-blind stage. Interventions: Intramuscular injection of methylcobalamin 50 mg or placebo twice weekly for 16 weeks. Main outcomes and measures: The primary endpoint was change in ALSFRS-R total score from baseline to week 16 in the full analysis set. Results: The least-squares mean difference in ALSFRS-R total score at week 16 of the randomized period was 1.97 points greater with methylcobalamin than placebo (−2.66 versus −4.63; 95% CI, 0.44–3.50; P = 0.012). The incidence of adverse events was similar between the two groups. Conclusions and relevance: Ultra-high dose methylcobalamin was efficacious in slowing functional decline and safe in the 16-week treatment period in ALS patients in the early stage and with moderate progression rate. Trial registration: UMIN-CTR Identifier: UMIN000029588 (umin.ac.jp/ctr); ClinicalTrials.gov Identifier: NCT03548311 (clinicaltrials.gov

Development of a Multi-Step Leukemogenesis Model of MLL-Rearranged Leukemia Using Humanized Mice

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ−/− (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia

灌流指標に対する大和当帰葉茶・当帰葉湯手浴の急性効果の評価

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