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

Abstract

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.

Funding: AMED and iPS Cell Research Fund.

Fig. 1

Profile of the clinical trial. A. CONSORT diagram for iDReAM study. B. Design of the study. The study consisted of a 12-week observation period, a 1-week transitional period, a 12-week study treatment period, and a 4-week follow-up period. The patients whose total ALSFRS-R scores decreased by 1–3 points during the 12-week observation period were eligible to enter the study treatment period. C. Dose escalation based on the 3 + 3 design. No DLTs were observed in the 100 mg–300 mg groups. There were 3 DLTs in the 400 mg group. DLT, dose-limiting toxicity; MTD, maximum tolerated dose; RP2D, recommended phase 2 dose.

Fig. 2

Evaluation of exploratory efficacy of bosutinib. A. Change of ALSFRS-R of each patient in the observation and treatment periods. A patient with consistent progression shows a horizontal line. Patients whose progression slowed after treatment with bosutinib show a rise to the right. B. Differences in change of ALSFRS-R score. Δ(change of ALSFRS-R) represents the change in ALSFRS-R during the treatment period minus the change in ALSFRS-R during the observation period. Patients with a positive value indicate that the decrease in ALSFRS-R was suppressed during the treatment period compared with that of the observation period. C. Relevance of plasma NFL levels and Δ(change of ALSFRS-R). Patients with lower NFL levels indicated suppression of ALSFRS-R decline. D. Relevance of plasma pNFH levels and Δ(change of ALSFRS-R). E. Relevance of plasma NFL levels and change of ALSFRS-R during observation period. F. Least-squares mean in lower NFL group (NFL <100 pg/mL, N = 5) and higher NFL group (NFL ≥100 pg/mL, N = 4). Using a linear mixed-effects model with a repeated-measures approach, the change in ALSFRS-R score from baseline was analysed in the lower NFL group and the higher NFL group. Point estimates of group differences in change of ALSFRS-R at 6 month was 4.5 (95% confidence interval, 1.7 to 7.3). Error bars represent 95% confidence interval. ALSFRS-R, Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; NFL, neurofilament light chain; pNFH, phosphorylated neurofilament heavy chain.

Fig. 3

Evaluation of biomarkers. A. Change of biomarkers. For each biomarker, the ratio of the value after the completion of 12 weeks of bosutinib treatment to before the start of bosutinib treatment is shown. B. Relevance between Δ(change of ALSFRS-R) and plasma p-Src/p-c-Abl at baseline. A correlation tendency between Δ(change of ALSFRS-R) and p-c-Abl was observed. C. Bosutinib levels in plasma and CSF. Mean plasma concentrations at 4, 8, and 12 weeks of bosutinib treatment and spinal fluid concentrations at 12 weeks of bosutinib treatment are shown. Bosutinib concentrations were dose-dependent in plasma and CSF. ALSFRS-R, Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; CH3L1, Chitinase 3 Like 1; IL, interleukin; MCP1, monocyte chemotactic protein 1; p-Src, phosphorylated Src; p-c-Abl, phosphorylated c-Abl; OD, optical density; CSF, cerebrospinal fluid.

Fig. 4

Gene expression profiles of motor neurons based on differences in plasma NFL levels. A. Single-cell RNA sequencing of motor neurons derived from iPSCs. Motor neurons from 9 patients with ALS (N = 9) enrolled for 100 mg–300 mg of bosutinib and 6 healthy individuals (N = 6) were analysed. B. A canonical pathway shown by motor neurons in ALS. The top pathways identified by Ingenuity Pathway Analysis are shown. C. Distribution of motor neurons in patients with ALS with higher NFL levels (N = 4) and lower NFL levels (N = 5). D. Pseudotime analysis showed a cell transition trajectory to motor neurons in patients with ALS with higher NFL levels (N = 4) and lower NFL levels (N = 5). E. Among the gene expressions that differ between ALS and healthy individuals, the genes emphasizing the difference between the higher and lower NFL groups were extracted (N = 4, N = 5, respectively, ∗p = 0·0035, ∗∗p < 0·0001, Wilcoxon rank-sum test). iPSCs, induced pluripotent stem cells; NFL, neurofilament light chain; MEG3, maternally expressed 3; NKX6-2, NK6 homeobox2; TEAD1, TEA domain transcription factor 1; TFAP2A, transcription factor AP-2 alpha.