Pre-clinical drug trial in SOD1G93A mice

The pre-clinical trial with JNJ-47965567 in SOD1^G93A mice was designed in accordance with recommendations of the ALS Therapy Development Institute (Cambridge, USA) [30] and the European ALS/MND Group [31]. Equal numbers of female and male mice were used in experiments and mice were matched for date of birth and sex. Each treatment group consisted of 24 mice per group, but 1 male control-treated mouse died (day 93) from causes unrelated to ALS and was excluded from all analysis. Mice were scored and weighed three times a week in a blinded fashion from day 60 using the criteria outlined by the ALS Therapy Development Institute [32]. Motor coordination of mice was assessed by rotarod performance once per week as described [25]. From disease onset (days 97–104) until end stage disease, mice were injected i.p. with 30 mg/kg JNJ-47965567 or an equivalent volume of β-CD (vehicle control) three times per week. The dose of 30 mg/kg for JNJ-47965567 was based on previous studies demonstrating efficacy of this drug at this same dose in rodent models of neuropathic pain [12] or epileptic seizure [13, 14]. Commencement and dosing frequency of JNJ-47965567 was based on our previous study using BBG which displayed efficacy in SOD1^G93A mice when injected i.p. from disease onset [25]. Disease onset was defined as when mice in either group had a clinical score of 1 over two consecutive measurements. Disease end stage was defined as when a mouse lost 20% body weight compared to the initial pre-treatment maximum body weight or when a mouse was unable to right itself within 10 s of being placed on either of its sides. End stage mice were euthanised by CO₂, and whole blood (via cardiac puncture), spleens and CNS draining (cervical) and non-draining (inguinal) lymph nodes were collected. Mice were then perfused transcardially with PBS, and spinal cords (lumbar region) stored in RNAlater at − 20 °C until required.