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: As a first step of our discovery we performed a systematic analysis of available data to define a group of signalling pathways important for peripheral nerve structure and function affected in CMT1A disease (Figure 1 and Additional file 1). Among them, modular pathways known to affect myelin gene expression such as cAMP-dependent mechanisms, neurosteroid signalling and the Akt/Erk pathway were of particular interest [24-28] (Figure 1A and Additional file 1A). We hypothesised that these modules are integrated as a unified system that is influenced by G protein coupled receptors (GPCRs) leading to the differential regulation of genes for peripheral myelin proteins. Since PMP22 is not only a structural component of myelin, but may have signalling functions in Schwann cells, its transcriptional control could be different from “classical” myelin genes such as MPZ. The topology of these putative regulatory networks permitted us to suggest that drugs acting on different GPCRs could cause a more potent and robust influence when combined. Thus, we have focused on the drugs able to modify relevant branches of GPCR signalling. This class of compounds is functionally pleiotropic, acting on multiple pathways and is well represented in approved pharmacopeia. This fact permitted us to apply additional safety criteria for their selection. We also preferred drugs that could be important for other aspects of peripheral nerve physiology (Figure 1B and Additional file 1B) that are affected in CMT1A, particularly drugs potentially promoting neuronal protection. Eventually, three drugs – (RS)-baclofen, naltrexone and D-sorbitol – were chosen for testing in the relevant cellular and animal models of CMT1A.Figure 1