- Email: [email protected]
G.P.110
828
Abstracts / Neuromuscular Disorders 24 (2014) 791–924
mouse exon 23 appeared to be ineffective after local intra muscular administration (2 times 2.9 nmol) and after 8 weeks of systemic treatment in mdx mice (4 times 50 mg/kg/week). To assess whether this is a generalized effect of 2FPS AON or specific for mouse exon 23 skip, we are currently evaluating a 2FPS AON targeting a human exon in our hDMD mouse model, which contains the complete human DMD gene integrated into the mouse genome. http://dx.doi:10.1016/j.nmd.2014.06.123
G.P.110 Safety and pharmacokinetic profile of eteplirsen, SRP-4045, and SRP-4053, three phosphorodiamidate morpholino oligomers (PMOs) for the treatment of patients with Duchenne muscular dystrophy (DMD) P. Sazani 1, T. Magee 1, J.S. Charleston 1, C. Shanks 1, J. Zhang 1, M. Carver 1, L. Rodino-Klapac 2, Z. Sahenk 2, K. Roush 2, L. Bird 2, L.P. Lowes 2, L. Alfano 2, A.M. Gomez 2, S. Lewis 2, V. Malik 2, K. Shontz 2, K. Flanigan 2, C. Shilling 2, J. Bhalli 3, H. Kaur 4, J. Walisser 4, J. Forget 5, J. Saoud 1, J.R. Mendell 2, E. Kaye 1 1 Sarepta Therapeutics, Cambridge, MA, USA; 2 Nationwide Children’s Hospital, Columbus, OH, USA; 3 Covance Laboratories Inc., Greenfield, IN, USA; 4 Covance Laboratories Inc., Madison, WI, USA; 5 Charles River Laboratories, Montreal, Canada Eteplirsen, SRP-4045 and SRP-4053 are three PMOs developed as treatments for DMD. DMD is mostly caused by mutations in the dystrophin gene that lead to a reading frame shift and premature translation termination. PMOs promote exon skipping during splicing of dystrophin pre-mRNA, restoring the reading frame and translation of internally truncated, but functional dystrophin protein. Eteplirsen, SRP-4045, and SRP-4053 are intended to treat patients with mutations amenable to skipping exon 51, 45 and 53, respectively. IND enabling preclinical studies have been completed for eteplirsen, SRP-4045, and SRP-4053. In vitro metabolism data and PK profiles in animals were similar for all three PMOs. No adverse responses were detected in safety pharmacology studies in non-human primates (NHPs). Genotoxicity studies were negative. NHPs given 12 weekly IV doses up to 320 mg/kg showed no significant sequence-specific toxicities. A clinical study of eteplirsen with doses of 30 and 50 mg/kg/wk reported no clinically significant treatment-related adverse events through 120 weeks of treatment. A few cases of transient, mild urine protein elevation resolved without intervention or drug interruption and one femur fracture was determined to be treatment-unrelated. There were no hospitalizations, discontinuations, or clinically significant treatment-related changes for lab parameters, including liver enzymes, kidney function, coagulation, or platelet counts. Plasma Cmax and AUC increased proportionally with dose, and half-life was approximately 3 h with no plasma accumulation observed. The consistent preclinical profiles and lack of significant, sequence-specific toxicities for eteplirsen, SRP-4045, and SRP-4053 demonstrate PMOs to be a well-tolerated therapeutic class, with potential applications in a wide variety of disease indications. http://dx.doi:10.1016/j.nmd.2014.06.124
G.P.111 Development of an ultrasensitive ELISA method for the determination of phosphorodiamidate morpholino oligonucleotide (PMO) levels in biological samples U. Burki, A. Blain, S. Laval, V. Straub Newcastle University, Newcastle upon Tyne, UK
Antisense oligonucleotide (AON) induced exon skipping is one of the most promising strategies for treating Duchene muscular dystrophy (DMD) and other rare genetic diseases, with the first generation of AONs having been applied in phase 3 clinical trials. Several different chemistries of AONs are available with 2-O-methyl phosphorothioate 0 (2 OMe) and phosphorodiamidate morpholino oligonucleotide (PMO) being the most advanced AONs in development. In addition to determining the efficacy of AON induced dystrophin expression, detecting the levels of AON in blood and tissue is essential for determining the pharmacokinetic/pharmacodynamic (PK/PD) relationship. In comparison with other AON detection methods such as high-performance liquid chromatography or liquid chromatography–mass spectrometry, an ELISA based approach has demonstrated far greater sensitivity with detection levels in the picomolar range. As a result the 0 ELISA is the method of choice for 2 OMe detection in pre-clinical and clinical development. However, to the best of our knowledge, no such assay has been developed for PMO and therefore a novel PMO ELISA assay is currently being developed in our lab with encouraging results. The hybridisation-based assay is currently able to detect PMO levels in buffer with a linear range of detection between 30 and 1000 pM (R2 = 0.99). More importantly the sensitivity is unaffected by various biological samples such as mouse serum, plasma and tissue lysate. The present level of sensitivity should be sufficient for both pre-clinical and clinical analysis. In addition, the assay can be easily adapted for detecting peptide-conjugated PMOs (i.e. PPMOs) which are the next generation of PMO based AONs in pre-clinical development. Here we present the results of studies from in PMO and PPMO treated mdx mice. http://dx.doi:10.1016/j.nmd.2014.06.125
G.P.112 Pulmonary function is stable through week 120 in patients with Duchenne muscular dystrophy (DMD) treated with exon-skipping drug eteplirsen in phase 2b study J.R. Mendell 1, L.P. Lowes 1, L. Alfano 1, J. Saoud 2, P. Duda 2, E. Kaye 2 1 Nationwide Children’s Hospital, Columbus, OH, USA; 2 Sarepta Therapeutics, Cambridge, MA, USA DMD is a rare, degenerative, genetic disease that results in progressive muscle loss and premature death and affects 1:5000 male births. Clinically manifest pulmonary dysfunction often occurs when DMD patients become non-ambulant and is preceded by subclinical deterioration of pulmonary function tests (PFTs). Specifically, MIP and MEP % predicted deteriorate by approximately 4 % per year between the ages of 8–19. Eteplirsen is an investigational drug designed to enable functional dystrophin production in boys who are amenable to skipping exon 51. When dosed for up to 120 weeks, stabilization of 6 min walk distance was demonstrated in a Phase 2b study. 12 boys were randomized 1:1:1 to 30/50 mg/kg or placebo. Upon completion of a 24-week double-blind, placebo-controlled phase, all patients were enrolled in an open-label extension and the placebo-treated patients initiated eteplirsen treatment. FVC, FVC % predicted, MIP, MEP and MIP and MEP % predicted were assessed every 12 to 24 weeks. For all 12 patients changes in function at Week 120 were examined from Week 1, and from last assessment pre-eteplirsen administration (Week 1 for 8 patients and Week 24 for 4 patients). One-sample t-test was used for statistical analysis. Reported here are the results for all 12 patients, including two patients who became non ambulant by Week 24. Median age at Week 120 was 12 years. The 120 week data for 5 of the 6 PFT parameters were not statistically different from baseline, with the exception of a statistically significant increase in MEP. Furthermore, individual patient values for all 12 patients continue to be in the age-adjusted normal ranges indicative of continued normal pulmonary function without the
Abstracts / Neuromuscular Disorders 24 (2014) 791–924
mouse exon 23 appeared to be ineffective after local intra muscular administration (2 times 2.9 nmol) and after 8 weeks of systemic treatment in mdx mice (4 times 50 mg/kg/week). To assess whether this is a generalized effect of 2FPS AON or specific for mouse exon 23 skip, we are currently evaluating a 2FPS AON targeting a human exon in our hDMD mouse model, which contains the complete human DMD gene integrated into the mouse genome. http://dx.doi:10.1016/j.nmd.2014.06.123
G.P.110 Safety and pharmacokinetic profile of eteplirsen, SRP-4045, and SRP-4053, three phosphorodiamidate morpholino oligomers (PMOs) for the treatment of patients with Duchenne muscular dystrophy (DMD) P. Sazani 1, T. Magee 1, J.S. Charleston 1, C. Shanks 1, J. Zhang 1, M. Carver 1, L. Rodino-Klapac 2, Z. Sahenk 2, K. Roush 2, L. Bird 2, L.P. Lowes 2, L. Alfano 2, A.M. Gomez 2, S. Lewis 2, V. Malik 2, K. Shontz 2, K. Flanigan 2, C. Shilling 2, J. Bhalli 3, H. Kaur 4, J. Walisser 4, J. Forget 5, J. Saoud 1, J.R. Mendell 2, E. Kaye 1 1 Sarepta Therapeutics, Cambridge, MA, USA; 2 Nationwide Children’s Hospital, Columbus, OH, USA; 3 Covance Laboratories Inc., Greenfield, IN, USA; 4 Covance Laboratories Inc., Madison, WI, USA; 5 Charles River Laboratories, Montreal, Canada Eteplirsen, SRP-4045 and SRP-4053 are three PMOs developed as treatments for DMD. DMD is mostly caused by mutations in the dystrophin gene that lead to a reading frame shift and premature translation termination. PMOs promote exon skipping during splicing of dystrophin pre-mRNA, restoring the reading frame and translation of internally truncated, but functional dystrophin protein. Eteplirsen, SRP-4045, and SRP-4053 are intended to treat patients with mutations amenable to skipping exon 51, 45 and 53, respectively. IND enabling preclinical studies have been completed for eteplirsen, SRP-4045, and SRP-4053. In vitro metabolism data and PK profiles in animals were similar for all three PMOs. No adverse responses were detected in safety pharmacology studies in non-human primates (NHPs). Genotoxicity studies were negative. NHPs given 12 weekly IV doses up to 320 mg/kg showed no significant sequence-specific toxicities. A clinical study of eteplirsen with doses of 30 and 50 mg/kg/wk reported no clinically significant treatment-related adverse events through 120 weeks of treatment. A few cases of transient, mild urine protein elevation resolved without intervention or drug interruption and one femur fracture was determined to be treatment-unrelated. There were no hospitalizations, discontinuations, or clinically significant treatment-related changes for lab parameters, including liver enzymes, kidney function, coagulation, or platelet counts. Plasma Cmax and AUC increased proportionally with dose, and half-life was approximately 3 h with no plasma accumulation observed. The consistent preclinical profiles and lack of significant, sequence-specific toxicities for eteplirsen, SRP-4045, and SRP-4053 demonstrate PMOs to be a well-tolerated therapeutic class, with potential applications in a wide variety of disease indications. http://dx.doi:10.1016/j.nmd.2014.06.124
G.P.111 Development of an ultrasensitive ELISA method for the determination of phosphorodiamidate morpholino oligonucleotide (PMO) levels in biological samples U. Burki, A. Blain, S. Laval, V. Straub Newcastle University, Newcastle upon Tyne, UK
Antisense oligonucleotide (AON) induced exon skipping is one of the most promising strategies for treating Duchene muscular dystrophy (DMD) and other rare genetic diseases, with the first generation of AONs having been applied in phase 3 clinical trials. Several different chemistries of AONs are available with 2-O-methyl phosphorothioate 0 (2 OMe) and phosphorodiamidate morpholino oligonucleotide (PMO) being the most advanced AONs in development. In addition to determining the efficacy of AON induced dystrophin expression, detecting the levels of AON in blood and tissue is essential for determining the pharmacokinetic/pharmacodynamic (PK/PD) relationship. In comparison with other AON detection methods such as high-performance liquid chromatography or liquid chromatography–mass spectrometry, an ELISA based approach has demonstrated far greater sensitivity with detection levels in the picomolar range. As a result the 0 ELISA is the method of choice for 2 OMe detection in pre-clinical and clinical development. However, to the best of our knowledge, no such assay has been developed for PMO and therefore a novel PMO ELISA assay is currently being developed in our lab with encouraging results. The hybridisation-based assay is currently able to detect PMO levels in buffer with a linear range of detection between 30 and 1000 pM (R2 = 0.99). More importantly the sensitivity is unaffected by various biological samples such as mouse serum, plasma and tissue lysate. The present level of sensitivity should be sufficient for both pre-clinical and clinical analysis. In addition, the assay can be easily adapted for detecting peptide-conjugated PMOs (i.e. PPMOs) which are the next generation of PMO based AONs in pre-clinical development. Here we present the results of studies from in PMO and PPMO treated mdx mice. http://dx.doi:10.1016/j.nmd.2014.06.125
G.P.112 Pulmonary function is stable through week 120 in patients with Duchenne muscular dystrophy (DMD) treated with exon-skipping drug eteplirsen in phase 2b study J.R. Mendell 1, L.P. Lowes 1, L. Alfano 1, J. Saoud 2, P. Duda 2, E. Kaye 2 1 Nationwide Children’s Hospital, Columbus, OH, USA; 2 Sarepta Therapeutics, Cambridge, MA, USA DMD is a rare, degenerative, genetic disease that results in progressive muscle loss and premature death and affects 1:5000 male births. Clinically manifest pulmonary dysfunction often occurs when DMD patients become non-ambulant and is preceded by subclinical deterioration of pulmonary function tests (PFTs). Specifically, MIP and MEP % predicted deteriorate by approximately 4 % per year between the ages of 8–19. Eteplirsen is an investigational drug designed to enable functional dystrophin production in boys who are amenable to skipping exon 51. When dosed for up to 120 weeks, stabilization of 6 min walk distance was demonstrated in a Phase 2b study. 12 boys were randomized 1:1:1 to 30/50 mg/kg or placebo. Upon completion of a 24-week double-blind, placebo-controlled phase, all patients were enrolled in an open-label extension and the placebo-treated patients initiated eteplirsen treatment. FVC, FVC % predicted, MIP, MEP and MIP and MEP % predicted were assessed every 12 to 24 weeks. For all 12 patients changes in function at Week 120 were examined from Week 1, and from last assessment pre-eteplirsen administration (Week 1 for 8 patients and Week 24 for 4 patients). One-sample t-test was used for statistical analysis. Reported here are the results for all 12 patients, including two patients who became non ambulant by Week 24. Median age at Week 120 was 12 years. The 120 week data for 5 of the 6 PFT parameters were not statistically different from baseline, with the exception of a statistically significant increase in MEP. Furthermore, individual patient values for all 12 patients continue to be in the age-adjusted normal ranges indicative of continued normal pulmonary function without the