Supplementary Appendix

Transcription

1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Goemans NM, Tulinius M, van den Akker JT, et al. Systemic administration of PRO051 in Duchenne s muscular dystrophy. N Engl J Med 2011;364: DOI: /NEJMoa

2 Supplementary Appendix Systemic Administration of PRO051 in Duchenne s Muscular Dystrophy 1. Methods Patient inclusion/exclusion criteria Patients with Duchenne muscular dystrophy aged 5 16 years and with mutations theoretically correctable by exon 51 skipping were recruited through the neuromuscular clinics of the University Hospitals Leuven, Belgium, and the Queen Silvia Children s Hospital, Gothenburg, Sweden. Inclusion and exclusion criteria were similar to the previous study. 1 An estimated life expectancy of 6 months, no other serious pre-existing medical conditions, no assisted ventilation dependency or FEV 1 or FVC < 60% of predicted, and no severe muscle abnormalities (defined as increased signal intensity in >50% of the tibialis anterior muscle at MRI) were required. Concurrent stable glucocorticosteroid treatment was allowed, recent (6 months) or ongoing treatment with anticoagulants, antithrombotics and antiplatelet agents, or investigational products was not allowed. Patients with known presence of dystrophin in 5% of fibers in a pre-study diagnostic muscle biopsy, with aberrant RNA splicing and/or aberrant response to PRO051 (detected by the in vitro PRO051 assay during screening), with elevated creatinine concentration (above 1.5 times the upper limit of age-corrected normal values), with severe mental retardation, or with symptomatic cardiac myopathy, were excluded. Study drug non-clinical safety Non-clinical safety studies showed that PRO051 was not mutagenic or clastogenic and shares class toxicities common to phosphorothioate oligonucleotides: transient prolongation of activated partial thromboplastin time (aptt); increase in complement split products in non-human primates; pro-inflammatory response, and accumulation of compound in liver and kidney. Specific monitoring of these aspects was conducted during both clinical studies. Pharmacokinetic assessments The pharmacokinetic profile of PRO051 was determined during the dose-escalation phase of the study. Plasma levels of PRO051 were determined using a validated hybridization ligation assay adapted from Yu et al., Plasma samples were incubated with a PRO051-specific capture oligonucleotide probe. After separation, a DIG-labeled oligonucleotide was ligated to the

3 complex and detection followed using an anti-dig antibody linked peroxidase. Noncompartmental pharmacokinetic analysis was performed using WINNONLIN software package (model 200, version 5.2, Pharsight, Mountainview, CA). Assessment of RNA and protein Biopsies were taken from the tibialis anterior muscle. 1 Total muscle RNA was isolated from 10 to 15 mg of muscle tissue, and reverse transcription-polymerase chain reaction and sequence analysis performed as described previously. 1, 3-4 Confirmation of correct splicing of the remainder of the DMD gene and immunofluorescence dystrophin analysis were conducted as previously reported. 1 However, for quantitative image analysis, Leica Qwin Software (Leica Microsystems, Rijswijk, The Netherlands) was used. Entire cross-sections were subdivided into series of 3 to 6 non-overlapping images. Fixed exposure settings were used, and pixel saturation was avoided. Absolute fluorescence intensities above saline-vehicle background were determined for each picture (mean pixel intensity of sarcolemma surface) and compared to healthy control muscle intensities. Western blot analysis was performed according to described methods. 1, 4 Between 75 to 150 µl of concentrated lysate was loaded ( µg) for each patient at both time points, depending on the quality of muscle tissue and/or biopsy as determined, and normalized for, by the signal intensity levels of dysferlin reference (NCL- Hamlet1:200, Novocastra Laboratories, Newcastle, UK). Dystrophin and dysferlin intensities were determined using secondary antibody IRDye800 anti-mouse IgG (1:5000) and the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Methods References 1. van Deutekom J, Janson J, Ginjaar H, et al. Local Dystrophin Restoration with Antisense Oligonucleotide PRO051 N Engl J Med 2007;357(26): Yu RZ, Baker B, Chappell A, Geary RS, Cheung E, Levin AA. Development of an ultrasensitive noncompetitive hybridization-ligation enzyme-linked immunosorbent assay for the determination of phosphorothioate oligodeoxynucleotide in plasma. Analytical biochemistry 2002;304(1): Aartsma-Rus A, Bremmer-Bout M, Janson A, den Dunnen J, van Ommen G, van Deutekom J. Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy. Neuromuscul Disord 2002;12 Suppl:S Aartsma-Rus A, Janson AA, Kaman WE, et al. Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum Mol Genet 2003;12(8):

4 2. Tables Table 1. Patient Demographics and Characteristics at Baseline by Dosing Group. Patients 0.5 mg/kg Age (yrs) Deletion Stage Tibialis Anterior Involvement (MRI- T1) 6MWT (m) CK (U/L) Stable minimal Stable minimal Non ambulant minimal NA mg/kg Decline moderate Stable minimal Stable minimal mg/kg Stable mild Decline minimal Stable minimal mg/kg Decline mild Decline moderate Stable minimal MWT=6-minute walk test (distance in meters); CK=Creatine kinase levels (U/L), average of 3 baseline samples.

5 3. Figures Figure 1. Figure 1. Mean PRO051 Plasma Concentrations Over Time. Following the first (A) and fifth (B) subcutaneous injections of 0.5, 2, 4, or 6 mg/kg PRO051, mean plasma levels were quantified up to 24 hours. The shape of the individual plasma concentration time profiles was comparable for Day 1 and Day 29, with slightly higher peak levels on Day 29 at the 4 and 6 mg/kg doses. (C) Trough concentrations were measurable for the majority of patients in the higher dose groups up to 13 weeks post-treatment and increased upon repeated dosing. The mean PRO051 plasma concentrations are presented on log-linear scale.