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: We then tested the potency of the three drugs to down-regulate Pmp22 expression in rat RT4 schwannoma when normalised to the levels of both Actb and Rps9 housekeeping genes . Using a drug ratio corresponding to the synergistic effect when combined in DRG co-cultures (Figure 2G, Dose 2), we observed moderately reduced levels of Pmp22 transcript for single drug treatment with NTX or SRB (Figure 3A). Importantly, in the PXT3003 drug combination, this effect was increased, i.e. with significant difference to the single drug action (Figure 3A). We also found that Mpz mRNA, coding for the major protein of peripheral myelin, was not affected by our drugs (Figure 3B), singly or in combination, while the ratio of Pmp22 to Mpz mRNA was significantly lowered (Figure 3C) by the drug combination but not by the single drugs. These results confirmed the importance of combining the chosen drugs and prompted us to use the PXT3003 combination in further testing in animal models.Figure 3