Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease caused by deletion and/or mutation of the Survival of Motor Neuron 1 (SMN1) gene. A second gene, SMN2, produces low levels of functional SMN protein that are insufficient to fully compensate for the lack of SMN1. Risdiplam (RG7916; RO7034067) is an orally administered, small‐molecule SMN2 pre‐mRNA splicing modifier that distributes into the central nervous system (CNS) and peripheral tissues. To further explore risdiplam distribution, we assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose‐dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients’ blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues.

The analytical capabilities of liquid chromatography tandem mass spectrometry for sensitive and highly selective determination of target compounds in complex biological samples makes it well suited for high-throughput analysis. We report the fast separation of six benzodiazepines isolated from human urine via selected reaction monitoring liquid chromatography/mass spectrometry using short dwell times to accommodate fast-eluting chromatographic peaks. The analytes were extracted from human urine samples along with their deuterium-labeled internal standards by a simple liquid-liquid extraction in 96-well plates. Using four autosamplers coupled to one chromatographic column and one tandem mass spectrometer operated in the turbo ion spray mode with positive ion detection, 1152 samples (12 96-well plates) were analyzed in less than 12 h. Through an electronic switching box designed and constructed in-house, the autosamplers were synchronized with the mass spectrometer so that injections were made as soon as the mass spectrometer was ready to collect data. Each run required 30 s to complete with another 7-8 s for the data system to load the next data file to be collected. Chromatographic integrity and ion current response remained relatively constant for the duration of the analyses. The results show acceptable precision and accuracy and demonstrate the feasibility of using fast separations with tandem mass spectrometry for high-throughout analysis of biological samples containing multiple analytes.

1. Alectinib is a highly selective, central nervous system-active small molecule anaplastic lymphoma kinase inhibitor. 2. The absolute bioavailability, metabolism, excretion and pharmacokinetics of alectinib were studied in a two-period single-sequence crossover study. A 50 μg radiolabelled intravenous microdose of alectinib was co-administered with a single 600 mg oral dose of alectinib in the first period, and a single 600 mg/67 μCi oral dose of radiolabelled alectinib was administered in the second period to six healthy male subjects. 3. The absolute bioavailability of alectinib was moderate at 36.9%. Geometric mean clearance was 34.5 L/h, volume of distribution was 475 L and the hepatic extraction ratio was low (0.14). 4. Near-complete recovery of administered radioactivity was achieved within 168 h post-dose (98.2%) with excretion predominantly in faeces (97.8%) and negligible excretion in urine (0.456%). Alectinib and its major active metabolite, M4, were the main components in plasma, accounting for 76% of total plasma radioactivity. In faeces, 84% of dose was excreted as unchanged alectinib with metabolites M4, M1a/b and M6 contributing to 5.8%, 7.2% and 0.2% of dose, respectively. 5. This novel study design characterised the full absorption, distribution, metabolism and excretion properties in each subject, providing insight into alectinib absorption and disposition in humans.

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