Polytherapy with a combination of three repurposed drugs (PXT3003) down-regulates Pmp22 over-expression and improves myelination, axonal and functional parameters in models of CMT1A neuropathy.
Bottom Line: Combination of (RS)-baclofen, naltrexone hydrochloride and D-sorbitol, termed PXT3003, improved myelination in the Pmp22 transgenic co-culture cellular model, and moderately down-regulated Pmp22 mRNA expression in Schwannoma cells.In both in vitro systems, the combination of drugs was revealed to possess synergistic effects, which provided the rationale for in vivo clinical testing of rodent models.PXT3003 also improved axonal regeneration and remyelination in the murine nerve crush model.
Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited sensory and motor peripheral neuropathy. It is caused by PMP22 overexpression which leads to defects of peripheral myelination, loss of long axons, and progressive impairment then disability. There is no treatment available despite observations that monotherapeutic interventions slow progression in rodent models. We thus hypothesized that a polytherapeutic approach using several drugs, previously approved for other diseases, could be beneficial by simultaneously targeting PMP22 and pathways important for myelination and axonal integrity. A combination of drugs for CMT1A polytherapy was chosen from a group of authorised drugs for unrelated diseases using a systems biology approach, followed by pharmacological safety considerations. Testing and proof of synergism of these drugs were performed in a co-culture model of DRG neurons and Schwann cells derived from a Pmp22 transgenic rat model of CMT1A. Their ability to lower Pmp22 mRNA in Schwann cells relative to house-keeping genes or to a second myelin transcript (Mpz) was assessed in a clonal cell line expressing these genes. Finally in vivo efficacy of the combination was tested in two models: CMT1A transgenic rats, and mice that recover from a nerve crush injury, a model to assess neuroprotection and regeneration. Combination of (RS)-baclofen, naltrexone hydrochloride and D-sorbitol, termed PXT3003, improved myelination in the Pmp22 transgenic co-culture cellular model, and moderately down-regulated Pmp22 mRNA expression in Schwannoma cells. In both in vitro systems, the combination of drugs was revealed to possess synergistic effects, which provided the rationale for in vivo clinical testing of rodent models. In Pmp22 transgenic CMT1A rats, PXT3003 down-regulated the Pmp22 to Mpz mRNA ratio, improved myelination of small fibres, increased nerve conduction and ameliorated the clinical phenotype. PXT3003 also improved axonal regeneration and remyelination in the murine nerve crush model. Based on these observations in preclinical models, a clinical trial of PTX3003 in CMT1A, a neglected orphan disease, is warranted. If the efficacy of PTX3003 is confirmed, rational polytherapy based on novel combinations of existing non-toxic drugs with pleiotropic effects may represent a promising approach for rapid drug development.
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Mentions: PXT3003 was assessed in CMT1A rats [5,9]. These rats carry additional copies of the murine Pmp22 gene leading to 1.6-fold overexpression of Pmp22 mRNA in peripheral nerves. This model is characterised by dysmyelination, reduced MNCV, diminished muscle strength and sensory nerve involvement, mimicking the clinical phenotype of human CMT1A patients. CMT1A rats have been used previously to assess the efficacy of progesterone antagonists and recombinant Neuregulin 1 (NRG1) as single drugs [3,9,13,42]. Young adult male Pmp22 TG rats and their WT littermates were treated during 9 weeks by daily oral treatment starting at 4 weeks of age. We confirmed by RTqPCR analysis the ~1.6-fold up-regulation of normalised Pmp22 mRNA relative to Mpz mRNA in the TG group when compared to WT littermates (Figure 4A). In order to examine the ability of PXT3003 to lower Pmp22 gene levels in vivo, we determined the expression of this gene in sciatic nerves at the end of 9 weeks of PXT3003 treatment. Importantly, we observed its significant but limited (−12%) down-regulation (Figure 4A) in line with the in vitro data obtained with Schwannoma cells (Figure 3C).Figure 4