Safety and Dose Finding Study of NS-065/NCNP-01 in Boys With Duchenne Muscular Dystrophy (DMD)

This is a Phase II, multiple center, 2-period, randomized, placebo-controlled, dose finding study of NS-065/NCNP-01 administered by infusion once weekly for 24 weeks to ambulant boys ages 4-<10 years with DMD. Two dose level cohorts will be enrolled. Period 1 of this study will be conducted in a double-blind fashion. Randomized patients will receive weekly IV infusions of NS-065/NCNP-01 or placebo for the first 4 weeks of their participation (Period 1) and NS-065/NCNP-01 by IV infusion for weeks 5-24 (20 weeks of active treatment - Period 2). Analysis of safety data from Period 1 of the 40mg/kg dose cohort will be completed prior to enrolling patients in the 80mg/kg dose cohort.

Patients completing the 24-week study will be eligible for an open-label extension study.

Clinical efficacy will be assessed at regularly scheduled study visits, including functional tests such as the six-minute walk test (6MWT), time to stand (TTSTAND), time to run/walk 10 meters (TTRW), time to climb 4 stairs (TTCLIMB) and quantitative muscle testing (QMT). All patients will undergo a muscle biopsy of the bicep at baseline and a second muscle biopsy at Week 24.

Safety will be assessed through the collection of adverse events (AEs), blood and urine laboratory tests, electrocardiograms (ECGs), vital signs, and physical examinations throughout the study.

Serial blood samples will be taken at four of the study visits to assess the pharmacokinetics of the study drug.

• NS-065/NCNP-01 (Drug)
• Placebo (Drug)

Primary outcomes

• Incidence of Adverse Events as Assessed by CTCAE v4.0. [24 weeks of treatment]: Treatment emergent adverse events (TEAEs) were summarized for Period 1 by comparing low dose to high dose to placebo and for Period 2 between the low dose cohort and the high dose cohort. TEAEs were summarized both at the patient level for number of TEAEs, highest severity, relationship, action and outcome and at the TEAE level (summarizing events) by organ system and preferred term TEAE as well as severity, relationship, action and outcome. The Medical Dictionary for Regulatory Activities (MedDRA) version 20.1 was used and the Common Terminology Criteria for Adverse Events (CTCAE) grading.
• Dystrophin Production by Western Blot [Baseline and 24 weeks of treatment]: Percentage normal dystrophin production in muscle biopsies from study participants at baseline and after 24 weeks treatment was measured by Western blot. To analyze dystrophin induction, biopsies were taken from a biceps muscle at baseline and the other biceps muscle after 24 weeks of treatment. Muscle samples were snap frozen and delivered to a central laboratory. All laboratory researchers remained blinded to sample identity. Dystrophin protein levels were assessed using standard curves on each gel (range from 0-25% of normal levels) generated by mixing 5 normal control samples with one DMD sample.

Secondary outcomes

• Dystrophin Production by RT-PCR for mRNA - Percentage of Exons Skipped - Molarity [Baseline and 24 weeks of treatment]: The alteration of mRNA splicing was measured by RT-PCR of dystrophin mRNA transcripts from a patient muscle biopsy at baseline and after 24 weeks treatment. To analyze dystrophin induction, biopsies were taken from a biceps muscle at baseline and the other biceps muscle after 24 weeks of treatment. Muscle samples were snap frozen and delivered to a central laboratory. All laboratory researchers remained blinded to sample identity. For RT-PCR, bands corresponding to specific versions of the spliced dystrophin mRNA were visualized by gel electrophoresis, and the amounts of different mRNA isoforms were compared.
• Dystrophin Production by Mass Spectrometry [Baseline and 24 weeks of treatment]: The production of dystrophin protein was measured by stable isotope mass spectrometry methods from a patient muscle biopsy at baseline and after 24 weeks treatment. To analyze dystrophin induction, biopsies were taken from a biceps muscle at baseline and the other biceps muscle after 24 weeks of treatment. Muscle samples were snap frozen and delivered to a central laboratory. All laboratory researchers remained blinded to sample identity. Dystrophin peptides were identified and quantified using reversed-phase nanoflow high-performance liquid chromatography with high resolution Mass Spectrometry.
• Dystrophin Production by Immunofluorescence [Baseline and 24 weeks of treatment]: The production of dystrophin protein was measured by immunofluorescence staining methods from a patient muscle biopsy at baseline and after 24 weeks treatment. To analyze dystrophin induction, biopsies were taken from a biceps muscle at baseline and the other biceps muscle after 24 weeks of treatment. Muscle samples were snap frozen and delivered to a central laboratory. All laboratory researchers remained blinded to sample identity. Immunofluorescence staining for dystrophin was performed on serial muscle biopsy sections in duplicate.
• Change From Baseline in Muscle Strength as Measured by Quantitative Muscle Testing (QMT). [Baseline and 24 weeks of treatment]: A secondary efficacy endpoint was compared to Pre-Infusion Visit: quantitative muscle testing (QMT).
• Change From Baseline in Distance Traveled in the Six-Minute Walk Test (6MWT). [Baseline and 24 weeks of treatment]: A secondary efficacy endpoint was compared to Pre-Infusion Visit: Six-Minute Walk Test (6MWT).
• Change From Baseline in Time to Climb 4 Stairs (TTCLIMB). [Baseline and 24 weeks of treatment]: A secondary efficacy endpoint was compared to Pre-Infusion Visit: Time to Climb 4 Stairs (TTCLIMB).
• Change From Baseline in Time to Climb 4 Stairs (TTCLIMB) Velocity [Baseline and 24 weeks of treatment]: A secondary efficacy endpoint was compared to Pre-Infusion Visit: Time to Run/Walk 10 meters (TTRW). The results were converted into velocity (meter/time).
• Change From Baseline in Time to Run/Walk 10 Meters (TTRW). [Baseline and 24 weeks of treatment]: A secondary efficacy endpoint was compared to Pre-Infusion Visit: Time to Run/Walk 10 meters (TTRW).
• Change From Baseline in Time to Run/Walk 10 Meters (TTRW) Velocity. [Baseline and 24 weeks of treatment]: A secondary efficacy endpoint was compared to Pre-Infusion Visit: Time to Run/Walk 10 meters (TTRW). The results were converted into velocity (meter/time).
• Change From Baseline in Time to Stand (TTSTAND) [Baseline and 24 weeks of treatment]: A secondary efficacy endpoint was compared to Pre-Infusion Visit: Time to Stand (TTSTAND)
• Change From Baseline in Time to Stand (TTSTAND) Velocity [Baseline and 24 weeks of treatment]: A secondary efficacy endpoint was compared to Pre-Infusion Visit: Time to Stand (TTSTAND).The results were converted into velocity (rise/time).
• Change From Baseline in North Star Ambulatory Assessment (NSAA) Score. [Baseline and 24 weeks of treatment]: The NSAA is a functional scale devised for use in ambulant children with Duchenne muscular dystrophy (DMD). It consists of 17 activities graded 0 (unable to perform), 1 (performs with modifications), 2 (normal movement). It assesses abilities necessary to remain ambulant that have been found to progressively deteriorate in untreated DMD patients, as well as in other muscular dystrophies such as Becker Muscular Dystrophy. NSAA Total Score ranges from 0 to 34, with a score of 34 implying normal function.

Sex: Male, ages: 4 Years to 9 Years

Inclusion Criteria:

* Male ≥ 4 years and <10 years of age
* Confirmed DMD mutation(s) in the dystrophin gene that is amenable to skipping of exon 53 to restore the dystrophin mRNA reading frame;
* Able to walk independently without assistive devices;
* Ability to complete the time to stand, time to run/walk and time to climb assessments;
* Stable dose of glucocorticoid for at least 3 months

Exclusion Criteria:

* Acute illness within 4 weeks prior to the first dose of study medication;
* Evidence of symptomatic cardiomyopathy. \[Note: Asymptomatic cardiac abnormality on investigation would not be exclusionary\];
* Severe allergy or hypersensitivity to medications;
* Severe behavioral or cognitive problems that preclude participation in the study, in the opinion of the Investigator;
* Previous or ongoing medical condition, medical history, physical findings or laboratory abnormalities that could affect safety, make it unlikely that treatment and follow-up will be correctly completed or impair the assessment of study results, in the opinion of the Investigator;
* Patient is taking any other investigational drug currently or within 3 months prior to the start of study treatment; or
* Patient has had surgery within the 3 months prior to the first anticipated administration of study medication or surgery is planned for anytime during the duration of the study;
* Patient has previously participated in this study or any other study during which NS-065/NCNP-01 was administered.

Participant flow

Outcome results

• Incidence of Adverse Events as Assessed by CTCAE v4.0. (Primary), Count of participants, Participants: Placebo (Period 1) 3; NS-065/NCNP-01 40mg/kg (Period 1) 4; NS-065/NCNP-01 80mg/kg (Period 1) 4; NS-065/NCNP-01 40mg/kg (Period 2) 5; NS-065/NCNP-01 80mg/kg (Period 2) 7; Total 15
• Dystrophin Production by Western Blot (Primary), Mean, % of normal control levels: NS-065/NCNP-01 40mg/kg 5.7 +/- 2.37; NS-065/NCNP-01 80mg/kg 5.9 +/- 4.50; Total NS-065/NCNP-01 Group 5.8 +/- 3.47
• Dystrophin Production by RT-PCR for mRNA - Percentage of Exons Skipped - Molarity (Secondary), Mean, % of normal control levels: NS-065/NCNP-01 40mg/kg 17.4 +/- 7.17; NS-065/NCNP-01 80mg/kg 43.9 +/- 16.68; Total NS-065/NCNP-01 Group 30.6 +/- 18.45
• Dystrophin Production by Mass Spectrometry (Secondary), Mean, % of normal control levels: NS-065/NCNP-01 40mg/kg 2.1 +/- 1.09; NS-065/NCNP-01 80mg/kg 4.2 +/- 3.73; Total NS-065/NCNP-01 Group 3.1 +/- 2.88
• Dystrophin Production by Immunofluorescence (Secondary), Mean, % dystrophin-positive fibers: NS-065/NCNP-01 40mg/kg 14.2 +/- 7.77; NS-065/NCNP-01 80mg/kg 34.8 +/- 20.42; Total NS-065/NCNP-01 Group 24.5 +/- 18.32
• Change From Baseline in Muscle Strength as Measured by Quantitative Muscle Testing (QMT). (Secondary), Mean, lb: NS-065/NCNP-01 40mg/kg -0.2029 +/- 2.98334; NS-065/NCNP-01 80mg/kg -0.8513 +/- 1.85797; Total NS-065/NCNP-01 Group -0.4808 +/- 2.49621
• Change From Baseline in Distance Traveled in the Six-Minute Walk Test (6MWT). (Secondary), Mean, m: NS-065/NCNP-01 40mg/kg 15.6 +/- 26.40; NS-065/NCNP-01 80mg/kg 44.0 +/- 41.98; Total NS-065/NCNP-01 Group 28.9 +/- 36.31
• Change From Baseline in Time to Climb 4 Stairs (TTCLIMB). (Secondary), Mean, second: NS-065/NCNP-01 40mg/kg -0.34 +/- 1.140; NS-065/NCNP-01 80mg/kg 0.00 +/- 0.600; Total NS-065/NCNP-01 Group -0.17 +/- 0.897
• Change From Baseline in Time to Climb 4 Stairs (TTCLIMB) Velocity (Secondary), Mean, m/s: NS-065/NCNP-01 40mg/kg 0.07 +/- 0.105; NS-065/NCNP-01 80mg/kg -0.00 +/- 0.054; Total NS-065/NCNP-01 Group 0.32 +/- 0.088
• Change From Baseline in Time to Run/Walk 10 Meters (TTRW). (Secondary), Mean, second: NS-065/NCNP-01 40mg/kg -0.65 +/- 1.225; NS-065/NCNP-01 80mg/kg -0.66 +/- 0.921; Total NS-065/NCNP-01 Group -0.66 +/- 1.047
• Change From Baseline in Time to Run/Walk 10 Meters (TTRW) Velocity. (Secondary), Mean, m/s: NS-065/NCNP-01 40mg/kg 0.21 +/- 0.291; NS-065/NCNP-01 80mg/kg 0.24 +/- 0.222; Total NS-065/NCNP-01 Group 0.23 +/- 0.251
• Change From Baseline in Time to Stand (TTSTAND) (Secondary), Mean, second: NS-065/NCNP-01 40mg/kg 0.05 +/- 1.446; NS-065/NCNP-01 80mg/kg -0.44 +/- 0.750; Total NS-065/NCNP-01 Group -0.19 +/- 1.141
• Change From Baseline in Time to Stand (TTSTAND) Velocity (Secondary), Mean, rise/time: NS-065/NCNP-01 40mg/kg 0.02 +/- 0.093; NS-065/NCNP-01 80mg/kg 0.02 +/- 0.060; Total NS-065/NCNP-01 Group 0.02 +/- 0.075
• Change From Baseline in North Star Ambulatory Assessment (NSAA) Score. (Secondary), Mean, score on a scale: NS-065/NCNP-01 40mg/kg 0.5 +/- 3.07; NS-065/NCNP-01 80mg/kg 1.1 +/- 2.80; Total NS-065/NCNP-01 Group 0.8 +/- 2.86

Adverse events

Time frame: 24 weeks

Other adverse events by term

• Results publication: Clemens PR, Rao VK, Connolly AM, Harper AD, Mah JK, Smith EC, McDonald CM, Zaidman CM, Morgenroth LP, Osaki H, Satou Y, Yamashita T, Hoffman EP; CINRG DNHS Investigators. Safety, Tolerability, and Efficacy of Viltolarsen in Boys With Duchenne Muscular Dystrophy Amenable to Exon 53 Skipping: A Phase 2 Randomized Clinical Trial. JAMA Neurol. 2020 Aug 1;77(8):982-991. doi: 10.1001/jamaneurol.2020.1264. PMID 32453377

• Study Protocol and Statistical Analysis Plan (PDF, 2017-11-28)

• Paula R. Clemens, MD, Study chair, University of Pittsburgh