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: Increased PMP22 copy number in CMT1A patients causes peripheral nerve dysmyelination through dysbalance in the expression of genes for myelin proteins and consecutive downstream signalling events . It can be studied in neuron-Schwann cell co-cultures derived from dorsal root ganglia (DRG) of CMT1A transgenic (TG) rats, a model of human CMT1A [8,40]. In this model, axons show reduced amounts of myelinated segments when compared to the wild-type . We treated DRG co-cultures from CMT1A rats during 10–11 days with single drugs and their combination PXT3003. Myelination was measured by quantifying myelin basic protein (MBP)-stained segments of neuronal fibres (Figure 2A and B). We observed that, acting alone, each of the three drugs improved myelination in a dose-dependent fashion (Figure 2C to E). Then, we tested their combination PXT3003 and also demonstrated a dose-related effect on axonal myelination (Figure 2F) with a synergistic interaction between the 3 drugs (dose 2) as calculated through Combination Index analysis (CI = 0.36) and 3D isobologram obtained from dose-effect curves  (Figure 2G), supporting the concept of an increased drug potency when used in combination.Figure 2