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

Abstract

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.

Figures

Figure 1

Pharmacology network-based drug repurposing for CMT1A disease. For detailed explanations and abbreviations, see Additional file 1. (A) Three principal pathways regulating expression of PMP22 gene through extracellular GPCR signalling in Schwann cells. The functional interaction between cAMP pathway, neurosteroid-mediated signalling and PI3K-AKT/ERK kinase cascades, activated by receptor tyrosine kinases, control the expression of PMP22 gene in Schwann cells. Individual drugs are shown in green. Blue shading: cAMP pathway; green shading: neurosteroid-mediated signalling; yellow shading: RTK/PI3K-AKT/ERK kinase cascades. (B) Cytoprotective and neuromodulator actions of PXT3003 drug combination in peripheral neurons. Dysfunction of CMT1A Schwann cells could affect membrane excitability of neuronal cells, leading to abnormal processing of neuronal information, cytotoxicity and axonal loss. GPCRs, modulated by PXT3003 drugs, are well-known regulators of neuronal excitability and pain sensation, are able to activate cytoprotective signalling pathways in different experimental settings, and could preserve functional integrity of peripheral neurons in CMT1 patients. Baclofen, naltrexone and sorbitol are shown in green. Red symbols: established or putative functional targets for PTX3003 drugs. Blue arrows: activation links; red lines: inhibition links; dashed lines: functional effect link is receptor-type specific.

Figure 2

BCL, NTX, SRB and their mix (PXT3003) improve myelination in DRG co-cultures. (A) and (B) PXT3003 treatment (Dose 2) of myelinating DRG co-cultures of neurons and Schwann cells derived from CMT1A transgenic rats (TG) demonstrated an increased length of MBP-positive fibres (red staining in (B)) compared to the untreated control (A). Scale bar: 56 μm. Blue staining: DAPI. (C) to (F) After 10–11 days of treatment of TG DRG co-cultures with single compounds BCL (C), NTX (D), SRB (E) or their PXT3003 (F) combination at four doses (Dose 1: 0.32 nM of BCL, 0.32 nM of NTX and 32 nM of SRB; Dose 2: 1.6 nM of BCL, 1.6 nM of NTX and 160 nM of SRB; Dose 3: 8 nM of BCL, 8 nM of NTX and 800 nM of SRB; Dose 4: 40 nM of BCL, 40 nM of NTX and 4 μM of SRB), myelination was significantly improved (featured by an increased length of MBP-stained segments). (G) The synergistic potential of PXT3003 (Dose 2: 1.6 nM of BCL, 1.6 nM of NTX and 160 nM of SRB) was displayed by an isobologram plotted from dose-effect curves of each drug. The calculated Combination Index (CI) of synergy was 0.36; Synergism is characterized by a CI < 1. Greyed surface represents the concentrations of the mixes at which CI = 1. Three independent DRG co-cultures with 6 replicates each were performed and analysed. +, *P < 0.05; ++, **P < 0.01; ***P < 0.001 vs Vehicle; ANOVA with Dunnett’s test (*) and t-test (+). Data are shown as mean + SEM. Vhc: Vehicle.

Figure 3

BCL, NTX, SRB and their mix (PXT3003) down-regulatePmp22mRNA expression in RT4 Schwannoma cells. (A) After a treatment of 8 hours of RT4 Schwannoma cells with single compounds (100 nM of BCL, 100 nM of NTX or 10 μM of SRB) and with PXT3003 combination (100 nM of BCL, 100 nM of NTX and 10 μM of SRB; same drug ratio that was used for all doses in DRG co-cultures), a significant decrease of Pmp22 mRNA expression level was observed when compared to vehicle (Actb and Rps9 used together as reference genes) with a significant observed higher efficacy of PXT3003 over single compounds. (B) The same drugs had no effect on Mpz expression (Actb and Rps9 used as reference genes). (C) The expression of Pmp22 with respect to Mpz was significantly decreased after PXT3003 treatment, unlike single compounds. Three different experiments using 2 cultures each with 3 replicates were performed and analysed. *P < 0.05, ***P < 0.001 vs Vehicle; ANOVA with Dunnett’s test. #P < 0.05 vs the most active compound; post-hoc contrast test. Data are shown as mean + SEM.

Figure 4

Daily oral treatment of CMT1A rats with PXT3003 down-regulatesPmp22expression and reduces signs of motor and sensory neuropathy. (A) A 9-week treatment with PXT3003 (BCL 30 μg/kg, NTX 3.5 μg/kg and SRB 1.05 mg/kg) decreased Pmp22 to Mpz mRNA ratio in the sciatic nerve. n = 18, 20 and 18 animals for respectively WT vehicle, TG vehicle and TG PXT3003 groups. (B) Bar test. The latency to fall for transgenic rats was significantly improved after 9 weeks of treatment with PXT3003. (C) Kaplan-Meier representation of the data set in (B) demonstrated a positive effect of PXT3003. (D) After 9 weeks of treatment, the inclined plane score was significantly improved for transgenic rats. (B) to (D)n = 36, 38 and 36 animals for respectively WT vehicle, TG vehicle and TG PXT3003 groups. (E) A 4-month long PXT3003 treatment normalised the impaired thermal sensitivity of transgenic rats in the hot plate test. n = 12, 10 and 7 animals respectively for WT, vehicle, TG vehicle and TG PXT3003 groups. *P < 0.05; **P < 0.01; ***P < 0.001 vs TG vehicle; ANOVA with Dunnett’s test (except in (C), logrank). Data are shown as mean + SEM.

Figure 5

Daily oral treatment of CMT1A rats with PXT3003 improves myelination and electrophysiology. A 4-month PXT3003 treatment (BCL 30 μg/kg, NTX 3.5 μg/kg and SRB 1.05 mg/kg) of TG rats significantly increased the number of myelinated axons in sciatic nerve cross sections (A) mostly in the small to medium sized axon class of myelinated axons (< 4 μm) (B). n = 12, 12 and 15 animals for respectively WT vehicle, TG vehicle and TG PXT3003 groups. (C) 8-month PXT3003 treatment increased the motor nerve conduction velocity of TG rats in sciatic nerve. n = 11, 9 and 7 animals for respectively WT vehicle, TG vehicle and TG PXT3003 groups. (D) Representative images of toluidine blue-stained sciatic nerve cross sections for each group. Scale bar: 25 μm. *,+ P < 0.05; ** P < 0.01; ***P < 0.001 vs TG vehicle; ANOVA with Dunnett’s test (except in (C), + t-test). Data are shown as mean + SEM.

Figure 6

Daily oral PXT3003 treatment of nerve-crushed mice restores nerve physiology, improves axonal and myelin integrity and functional behaviour. (A) A 21 and 30 day-treatment with PXT3003 (BCL 60 μg/kg, NTX 7 μg/kg and SRB 2.1 mg/kg) increased the amplitude of CMAP (measured in the gastrocnemius) of crushed nerves in male mice. n = 10 animals in each group. (B) to (D) A 42 day-treatment with PXT3003 significantly improved axonal calibre size (B), normalised the distribution of the number of myelinated axons in terms of axonal diameter (C) and improved the distribution of myelin g-ratio in tibial nerve cross sections (D). n = 6 for each group. (E) After 13 days of treatment, PXT3003 (BCL 600 μg/kg, NTX 70 μg/kg and SRB 21 mg/kg) improved significantly the functional activity of crushed mice as assessed by the paw surface pressure against the floor (paw surface bearing) of the affected paw normalised to the contralateral non-affected one. (F) to (H) In contrast to PXT3003, single drugs had no effect on surface bearing ratio. n = 10 for each group. *P < 0.05, **P < 0.01, ***P < 0.001 vs Crush Vehicle; t–test. Data are shown as mean ± SEM.