Pharmacokinetics, pharmacodynamics, and efficacy of a small-molecule SMN2 splicing modifier in mouse models of spinal muscular atrophy

Human Molecular Genetics, 2016, Vol. 25, No. 10 1885–1899 doi: 10.1093/hmg/ddw062 Advance Access Publication Date: 29 February 2016 Original Article ORIGINAL ARTICLE Pharmacokinetics, pharmacodynamics, and efficacy of a small-molecule SMN2 splicing modifier in mouse models of spinal muscular atrophy 1 PTC Therapeutics, Inc., South Plainfield, NJ 07080, USA, 2Department of Biological Sciences, Section of Neurobiology, University of Southern California, Los Angeles, CA 90089, USA, 3F. Hoffmann-La Roche, Pharmaceutical Research and Early Development, Roche Innovation Center Basel, Grenzacherstrasse 124, Basel 4070, Switzerland, 4Department of Pathology and Cell Biology, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY 10032, USA and 5SMA Foundation, 888 Seventh Avenue, Suite 400, New York, NY 10019, USA *To whom correspondence should be addressed. Tel: +1 9089129111 (M.W.)/+1 2137409182 (C.P.K.); Fax: +1 9082227231 (M.W.)/+1 2137405687 (C.P.K.); Email: (M.W.)/ (C.P.K.) Abstract Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a ∼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to † The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors. Received: October 27, 2015. Revised: January 25, 2016. Accepted: February 22, 2016 © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 1885 Xin Zhao1,†, Zhihua Feng2,†, Karen K. Y. Ling2,†, Anna Mollin1, Josephine Sheedy1, Shirley Yeh1, Janet Petruska1, Jana Narasimhan1, Amal Dakka1, Ellen M. Welch1, Gary Karp1, Karen S. Chen5, Friedrich Metzger3, Hasane Ratni3, Francesco Lotti4, Sarah Tisdale4, Nikolai A. Naryshkin1, Livio Pellizzoni4, Sergey Paushkin5, Chien-Ping Ko2, * and Marla Weetall1,* 1886 | Human Molecular Genetics, 2016, Vol. 25, No. 10 treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment. Introduction Results Pharmacokinetics of SMN-C1 in neonatal and adult mice We performed a detailed pharmacokinetic evaluation of SMN-C1 in wild-type mice (Fig. 1). SMN-C1 was administered at a dose of 10 mg/kg to neonatal wild-type mice by intraperitoneal injection (IP) or to adult mice by oral gavage (PO). Neonatal mice [ postnatal day 10 (PND10)] were dosed IP due to the difficulty in accurately dosing neonatal mice by oral gavage. As shown in Figure 1B, SMN-C1 plasma concentrations were similar in adult and neonatal mice through ∼8 h, but the compound was eliminated more slowly from the neonatal mice than from the adult mice. Furthermore, concentrations of SMN-C1 in the brain were much higher in the neonatal mice than in the adult mice (Fig. 1C). For that reason, in subsequent survival studies, a lower dose was used in neonatal mice while the dose was increased for adult mice. SMN-C1 increases SMN protein levels in mouse models of SMA To define the pharmacokinetic parameters associated with pharmacodynamic responses as assessed by the increase in SMN protein levels (PK-PD), studies were performed in the C/C-allele mouse using various dosing regimens. This mouse has two SMN genes as a single allele (called the C allele): two copies of a hybrid gene in which murine Smn1 gene is fused to human SMN2 gene with a junction point in intron 6 and two copies of the full human SMN2 gene located immediately downstream of the hybrid gene. The mice have a near-normal life span, but show decreased muscle function, reduced body weight gain, and peripheral necrosis in comparison with normal mice (24). The same total daily dose was administered via oral gavage either as a single dose or in two half doses 6 h apart. The maximal ( peak) plasma concentration (Cmax) was approximately twice as high when the total daily dose was given as a single bolus than when given as two half doses (data not shown). However, the Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease with a spectrum of severity (1,2). Type I is the most severe and frequent form of SMA with disease onset occurring before 6 months of age and death usually by the age of two. Type II is the intermediate form of the disease with onset prior to 18 months of age. Type II patients never gain the ability to walk. Type III is the mild form characterized by onset after 18 months of age. Type III patients typically retain the ability to walk until later in life and have a normal life expectancy. SMA is caused by the mutation or deletion of both copies of the telomeric survival motor neuron 1 (SMN1) gene (3). About 95% of humans carry one or more copies of the paralogous SMN2 gene (4). The protein-coding regions of SMN1 and SMN2 differ only in a translationally synonymous C-to-T transition at nucleotide 840, leading to alternative splicing and preferential exclusion of exon 7 from most of the mature SMN2 transcripts (5,6). The resulting mRNA encodes an unstable SMNΔ7 protein that is rapidly degraded (7,8), while the low levels of full-length (FL) SMN2 transcripts that include exon 7 produce small amounts of fully functional SMN protein. The severity of SMA is inversely correlated with SMN2 gene copy number and SMN protein levels (9,10). SMA is characterized by the loss of proximal spinal alpha motor neurons resulting in neuromuscular junction (NMJ) denervation, (...truncated)