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: To further assess the ability of PXT3003 to improve peripheral nerve function, we used the in vivo sciatic nerve crush model in mice. The nerve crush model is mostly considered as a model of Wallerian degeneration but demonstrates striking similarities to inherited demyelinating neuropathies with common features of axonal degeneration and dedifferentiation of Schwann cells . In this model, nerve-crush damage results in temporal loss of function (lasting 30 days after nerve crush) accompanied by initial loss and gradual restoration of the amplitude of CMAP (Additional file 5A) as well as by the impairment of axonal morphological parameters (decrease of axonal calibre of the tibial nerve, impaired distribution of myelinated axons and of myelin g-ratio in crushed nerves) in regenerating nerves (Additional file 5B to D). When mice were treated once a day with PXT3003 starting 30 minutes after nerve crush, CMAP amplitudes (Figure 6A) measured at 21 and 30 days were largely normalised. At the end of a 42 day long drug treatment, axonal morphology (Figure 6B), distribution of myelinated fibres (Figure 6C), as well as the distribution g-ratio as an indicator of myelin thickness and/or of the axonal diameter (Figure 6D) were also positively affected in regenerating nerves.Figure 6