Synergistic PXT3003 therapy uncouples neuromuscular function from dysmyelination in male Charcot-Marie-Tooth disease type 1A (CMT1A) rats.

Charcot-Marie-Tooth disease 1 A (CMT1A) is caused by an intrachromosomal duplication of the gene encoding for PMP22 leading to peripheral nerve dysmyelination, axonal loss, and progressive muscle weakness. No therapy is available. PXT3003 is a low-dose combination of baclofen, naltrexone, and sorbitol which has been shown to improve disease symptoms in Pmp22 transgenic rats, a bona fide model of CMT1A disease. However, the superiority of PXT3003 over its single components or dual combinations have not been tested. Here, we show that in a dorsal root ganglion (DRG) co-culture system derived from transgenic rats, PXT3003 induced myelination when compared to its single and dual components. Applying a clinically relevant ("translational") study design in adult male CMT1A rats for 3 months, PXT3003, but not its dual components, resulted in improved performance in behavioral motor and sensory endpoints when compared to placebo. Unexpectedly, we observed only a marginally increased number of myelinated axons in nerves from PXT3003-treated CMT1A rats. However, in electrophysiology, motor latencies correlated with increased grip strength indicating a possible effect of PXT3003 on neuromuscular junctions (NMJs) and muscle fiber pathology. Indeed, PXT3003-treated CMT1A rats displayed an increased perimeter of individual NMJs and a larger number of functional NMJs. Moreover, muscles of PXT3003 CMT1A rats displayed less neurogenic atrophy and a shift toward fast contracting muscle fibers. We suggest that ameliorated motor function in PXT3003-treated CMT1A rats result from restored NMJ function and muscle innervation, independent from myelination.

Highly Variable Pharmacokinetics of Tyramine in Humans and Polymorphisms in OCT1, CYP2D6, and MAO-A.

Tyramine, formed by the decarboxylation of tyrosine, is a natural constituent of numerous food products. As an indirect sympathomimetic, it can have potentially dangerous hypertensive effects. In vitro data indicated that the pharmacokinetics of tyramine possibly depend on the organic cation transporter OCT1 genotype and on the CYP2D6 genotype. Since tyramine is a prototypic substrate of monoamine oxidase A (MAO-A), genetic polymorphisms in MAO-A may also be relevant. The aims of this study were to identify to what extent the interindividual variation in pharmacokinetics and pharmacodynamics of tyramine is determined by genetic polymorphisms in OCT1, CYP2D6, and MAO-A. Beyond that, we wanted to evaluate tyramine as probe drug for the in vivo activity of MAO-A and OCT1. Therefore, the pharmacokinetics, pharmacodynamics, and pharmacogenetics of tyramine were studied in 88 healthy volunteers after oral administration of a 400 mg dose. We observed a strong interindividual variation in systemic tyramine exposure, with a mean AUC of 3.74 min*µg/ml and a high mean CL/F ratio of 107 l/min. On average, as much as 76.8% of the dose was recovered in urine in form of the MAO-catalysed metabolite 4-hydroxyphenylacetic acid (4-HPAA), confirming that oxidative deamination by MAO-A is the quantitatively most relevant metabolic pathway. Systemic exposure of 4-HPAA varied only up to 3-fold, indicating no strong heritable variation in peripheral MAO-A activity. Systolic blood pressure increased by more than 10 mmHg in 71% of the volunteers and correlated strongly with systemic tyramine concentration. In less than 10% of participants, individually variable blood pressure peaks by >40 mmHg above baseline were observed at tyramine concentrations of >60 µg/l. Unexpectedly, the functionally relevant polymorphisms in OCT1 and CYP2D6, including the CYP2D6 poor and ultra-rapid metaboliser genotypes, did not significantly affect tyramine pharmacokinetics or pharmacodynamics. Also, the MOA-A genotypes, which had been associated in several earlier studies with neuropsychiatric phenotypes, had no significant effects on tyramine pharmacokinetics or its metabolism to 4-HPAA. Thus, variation in tyramine pharmacokinetics and pharmacodynamics is not explained by obvious genomic variation, and human tyramine metabolism did not indicate the existence of ultra-low or -high MAO-A activity.

Early short-term PXT3003 combinational therapy delays disease onset in a transgenic rat model of Charcot-Marie-Tooth disease 1A (CMT1A).

The most common type of Charcot-Marie-Tooth disease is caused by a duplication of PMP22 leading to dysmyelination, axonal loss and progressive muscle weakness (CMT1A). Currently, no approved therapy is available for CMT1A patients. A novel polytherapeutic proof-of-principle approach using PXT3003, a low-dose combination of baclofen, naltrexone and sorbitol, slowed disease progression after long-term dosing in adult Pmp22 transgenic rats, a known animal model of CMT1A. Here, we report an early postnatal, short-term treatment with PXT3003 in CMT1A rats that delays disease onset into adulthood. CMT1A rats were treated from postnatal day 6 to 18 with PXT3003. Behavioural, electrophysiological, histological and molecular analyses were performed until 12 weeks of age. Daily oral treatment for approximately 2 weeks ameliorated motor deficits of CMT1A rats reaching wildtype levels. Histologically, PXT3003 corrected the disturbed axon calibre distribution with a shift towards large motor axons. Despite dramatic clinical amelioration, only distal motor latencies were improved and correlated with phenotype performance. On the molecular level, PXT3003 reduced Pmp22 mRNA overexpression and improved the misbalanced downstream PI3K-AKT / MEK-ERK signalling pathway. The improved differentiation status of Schwann cells may have enabled better long-term axonal support function. We conclude that short-term treatment with PXT3003 during early development may partially prevent the clinical and molecular manifestations of CMT1A. Since PXT3003 has a strong safety profile and is currently undergoing a phase III trial in CMT1A patients, our results suggest that PXT3003 therapy may be a bona fide translatable therapy option for children and young adolescent patients suffering from CMT1A.

OCT1 Deficiency Affects Hepatocellular Concentrations and Pharmacokinetics of Cycloguanil, the Active Metabolite of the Antimalarial Drug Proguanil.

Cycloguanil, the active metabolite of proguanil, acts on malaria schizonts in erythrocytes and hepatocytes. We analyzed the impact of the organic cation transporter OCT1 on hepatocellular uptake and pharmacokinetics of proguanil and cycloguanil. OCT1 transported both proguanil and cycloguanil. Common variants OCT1*3 and OCT1*4 caused a substantial decrease and OCT1*5 and OCT1*6 complete abolishment of proguanil uptake. In 39 healthy subjects, low-activity variants OCT1*3 and OCT1*4 had only minor effects on proguanil pharmacokinetics. However, both, cycloguanil area under the time-concentration curve and the cycloguanil-to-proguanil ratio were significantly dependent on number of these low-functional alleles (P = 0.02 for both). Together, CYP2C19, CYP3A5, OCT1 polymorphisms, and sex accounted for 61% of the variation in the cycloguanil-to-proguanil ratio. Most importantly, in vitro OCT1 inhibition caused a fivefold decrease of intracellular cycloguanil concentrations in primary human hepatocytes. In conclusion, OCT1-mediated uptake is a limiting step in bioactivation of proguanil, and OCT1 polymorphisms may affect proguanil efficacy against hepatic malaria schizonts.

Increased Systemic Exposure and Stronger Cardiovascular and Metabolic Adverse Reactions to Fenoterol in Individuals with Heritable OCT1 Deficiency.

Fenoterol is a widely used anti-asthmatic and tocolytic agent, but high plasma concentrations of fenoterol may lead to severe and even fatal adverse reactions. We studied whether heritable deficiency of the liver organic cation transporter 1 (OCT1), a trait observed in 3% of Europeans and white Americans, affects fenoterol plasma concentrations and toxicity. OCT1 transported fenoterol with high affinity, and OCT1 inhibition in human hepatocytes reduced fenoterol uptake threefold. After administration of 180 µg of fenoterol to 39 healthy individuals, the OCT1-deficient individuals (zero active OCT1 alleles; n = 5) showed 1.9-fold greater systemic fenoterol exposure (P = 4.0 × 10-5 ) and 1.7-fold lower volume of distribution (P = 8.0 × 10-5 ). Correspondingly, the OCT1-deficient individuals had a 1.5-fold stronger increase in heart rate (P = 0.002), a 3.4-fold greater increase in blood glucose (P = 3.0 × 10-5 ), and significantly lower serum potassium levels. In conclusion, heritable OCT1 deficiency significantly increases plasma concentrations of fenoterol and may be an important factor underlying the excess mortality associated with fenoterol.

Biomarkers predict outcome in Charcot-Marie-Tooth disease 1A.

BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited neuropathy, a debilitating disease without known cure. Among patients with CMT1A, disease manifestation, progression and severity are strikingly variable, which poses major challenges for the development of new therapies. Hence, there is a strong need for sensitive outcome measures such as disease and progression biomarkers, which would add powerful tools to monitor therapeutic effects in CMT1A. METHODS: We established a pan-European and American consortium comprising nine clinical centres including 311 patients with CMT1A in total. From all patients, the CMT neuropathy score and secondary outcome measures were obtained and a skin biopsy collected. In order to assess and validate disease severity and progression biomarkers, we performed qPCR on a set of 16 animal model-derived potential biomarkers in skin biopsy mRNA extracts. RESULTS: In 266 patients with CMT1A, a cluster of eight cutaneous transcripts differentiates disease severity with a sensitivity and specificity of 90% and 76.1%, respectively. In an additional cohort of 45 patients with CMT1A, from whom a second skin biopsy was taken after 2-3 years, the cutaneous mRNA expression of GSTT2, CTSA, PPARG, CDA, ENPP1 and NRG1-Iis changing over time and correlates with disease progression. CONCLUSIONS: In summary, we provide evidence that cutaneous transcripts in patients with CMT1A serve as disease severity and progression biomarkers and, if implemented into clinical trials, they could markedly accelerate the development of a therapy for CMT1A.

Tolerability and efficacy study of P2X7 inhibition in experimental Charcot-Marie-Tooth type 1A (CMT1A) neuropathy.

Charcot-Marie-Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy for which pharmacological treatments are not yet available. An abnormally high intracellular Ca(2+) concentration was observed in Schwann cells (SC) from CMT1A rats, caused by the PMP22-mediated overexpression of the P2X7 purinoceptor. The purpose of this study was to investigate the tolerability and therapeutic potential of a pharmacological antagonist of the P2X7 receptor (A438079) in CMT1A. A438079 ameliorated in vitro myelination of organotypic DRG cultures from CMT1A rats. Furthermore, we performed an experimental therapeutic trial in PMP22 transgenic and in wild-type rats. A preliminary dose-escalation trial showed that 3mg/kg A438079 administered via intraperitoneal injection every 24h for four weeks was well tolerated by wild type and CMT1A rats. Affected rats treated with 3mg/kg A438079 revealed a significant improvement of the muscle strength, when compared to placebo controls. Importantly, histologic analysis revealed a significant increase of the total number of myelinated axons in tibial nerves. Moreover, a significant decrease of the hypermyelination of small caliber axons and a significant increase of the frequency and diameter of large caliber myelinated axons was highlighted. An improved distal motor latencies was recorded, whereas compound muscle action potentials (CMAP) remained unaltered. A438079 reduced the SC differentiation defect in CMT1A rats. These results show that pharmacological inhibition of the P2X7 receptor is well tolerated in CMT1A rats and represents a proof-of-principle that antagonizing this pathway may correct the molecular derangements and improve the clinical phenotype in the CMT1A neuropathy.

Curcumin therapy in a Plp1 transgenic mouse model of Pelizaeus-Merzbacher disease.

OBJECTIVE: Pelizaeus-Merzbacher disease (PMD) is a progressive and lethal leukodystrophy caused by mutations affecting the proteolipid protein (PLP1) gene. The most common cause of PMD is a duplication of PLP1 and at present there is no curative therapy available. METHODS: By using transgenic mice carrying additional copies of Plp1, we investigated whether curcumin diet ameliorates PMD symptoms. The diet of Plp1 transgenic mice was supplemented with curcumin for 10 consecutive weeks followed by phenotypical, histological and immunohistochemical analyses of the central nervous system. Plp1 transgenic and wild-type mice fed with normal chow served as controls. RESULTS: Curcumin improved the motor phenotype performance of Plp1 transgenic mice by 50% toward wild-type level and preserved myelinated axons by 35% when compared to Plp1 transgenic controls. Furthermore, curcumin reduced astrocytosis, microgliosis and lymphocyte infiltration in Plp1 transgenic mice. Curcumin diet did not affect the pathologically increased Plp1 mRNA abundance. However, high glutathione levels indicating an oxidative misbalance in the white matter of Plp1 transgenic mice were restored by curcumin treatment. INTERPRETATION: Curcumin may potentially serve as an antioxidant therapy of PMD caused by PLP1 gene duplication.

Soluble neuregulin-1 modulates disease pathogenesis in rodent models of Charcot-Marie-Tooth disease 1A.

Duplication of the gene encoding the peripheral myelin protein of 22 kDa (PMP22) underlies the most common inherited neuropathy, Charcot-Marie-Tooth 1A (CMT1A), a disease without a known cure. Although demyelination represents a characteristic feature, the clinical phenotype of CMT1A is determined by the degree of axonal loss, and patients suffer from progressive muscle weakness and impaired sensation. CMT1A disease manifests within the first two decades of life, and walking disabilities, foot deformities and electrophysiological abnormalities are already present in childhood. Here, we show in Pmp22-transgenic rodent models of CMT1A that Schwann cells acquire a persistent differentiation defect during early postnatal development, caused by imbalanced activity of the PI3K-Akt and the Mek-Erk signaling pathways. We demonstrate that enhanced PI3K-Akt signaling by axonally overexpressed neuregulin-1 (NRG1) type I drives diseased Schwann cells toward differentiation and preserves peripheral nerve axons. Notably, in a preclinical experimental therapy using a CMT1A rat model, when treatment is restricted to early postnatal development, soluble NRG1 effectively overcomes impaired peripheral nerve development and restores axon survival into adulthood. Our findings suggest a model in which Schwann cell differentiation within a limited time window is crucial for the long-term maintenance of axonal support.

Progesterone antagonist therapy in a Pelizaeus-Merzbacher mouse model.

Pelizaeus-Merzbacher disease (PMD) is a severe hypomyelinating disease, characterized by ataxia, intellectual disability, epilepsy, and premature death. In the majority of cases, PMD is caused by duplication of PLP1 that is expressed in myelinating oligodendrocytes. Despite detailed knowledge of PLP1, there is presently no curative therapy for PMD. We used a Plp1 transgenic PMD mouse model to test the therapeutic effect of Lonaprisan, an antagonist of the nuclear progesterone receptor, in lowering Plp1 mRNA overexpression. We applied placebo-controlled Lonaprisan therapy to PMD mice for 10 weeks and performed the grid slip analysis to assess the clinical phenotype. Additionally, mRNA expression and protein accumulation as well as histological analysis of the central nervous system were performed. Although Plp1 mRNA levels are increased 1.8-fold in PMD mice compared to wild-type controls, daily Lonaprisan treatment reduced overexpression at the RNA level to about 1.5-fold, which was sufficient to significantly improve the poor motor phenotype. Electron microscopy confirmed a 25% increase in the number of myelinated axons in the corticospinal tract when compared to untreated PMD mice. Microarray analysis revealed the upregulation of proapoptotic genes in PMD mice that could be partially rescued by Lonaprisan treatment, which also reduced microgliosis, astrogliosis, and lymphocyte infiltration.

Antiprogesterone therapy uncouples axonal loss from demyelination in a transgenic rat model of CMT1A neuropathy.

OBJECTIVE: Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy, and a duplication of the Pmp22 gene causes the most frequent subform CMT1A. Using a transgenic rat model of CMT1A, we tested the hypothesis that long-term treatment with anti-progesterone (Onapristone) reduces Pmp22 overexpression and improves CMT disease phenotype of older animals, thereby extending a previous proof-of-concept observation in a more clinically relevant setting. METHODS: We applied placebo-controlled progesterone-antagonist therapy to CMT rats for 5 months and performed grip-strength analysis to assess the motor phenotype. Quantitative Pmp22 RT-PCR and complete histological analysis of peripheral nerves and skin biopsies were performed. RESULTS: Anti-progesterone therapy significantly increased muscle strength and muscle mass of CMT rats and reduced the performance difference to wildtype rats by about 50%. Physical improvements can be explained by the prevention of axon loss. Surprisingly, the effects of anti-progesterone were not reflected by improved myelin sheath thickness. Electrophysiology confirmed unaltered NCV, but less reduced CMAP recordings in the treatment group. Moreover, the reduction of Pmp22 mRNA, as quantified in cutaneous nerves, correlated with the clinical phenotype at later stages. INTERPRETATION: Progesterone-antagonist long-term therapy reduces [corrected] Pmp22 overexpression to a degree at which the axonal support function of Schwann cells is better maintained than myelination. This suggests that axonal loss in CMT1A is not caused by demyelination, but rather by a Schwann cell defect that has been partially uncoupled by anti-progesterone treatment. Pmp22 expression analysis in skin may provide a prognostic marker for disease severity and for monitoring future clinical trials.

Myelin disorders: Causes and perspectives of Charcot-Marie-Tooth neuropathy.

Charcot-Marie-Tooth (CMT) disease is a common hereditary neuropathy that causes progressive distally pronounced muscle weakness and can lead to life-long disability in patients. In most cases, the disorder has been associated with a partial duplication of human chromosome 17 (CMT1A), causing 1.5-fold overexpression of the peripheral myelin protein 22 kDa (PMP22). Increased PMP22 gene dosage results in demyelination, secondary axonal loss, and neurogenic muscle atrophy. Experimental therapeutic approaches based on the role of progesterone and ascorbic acid in myelin formation recently have reached preclinical proof-of-principle trials in rodents. It was shown that progesterone receptor antagonists can reduce PMP22 overexpression and clinical severity in a CMT1A rat model. Furthermore, ascorbic acid treatment reduced premature death and demyelination in a CMT1A mouse model. Thus, basic research has opened up new vistas for the understanding and treatment of hereditary neuropathies.