Carla Angelozzi

Phenylbutyrate increases SMN expression in vitro: relevance for treatment of spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease, characterized by degeneration of the anterior horn cells of the spinal cord. SMA presents with a highly variable phenotype ranging from very severe to mild (type I–III). No cure for SMA is available at present. All forms of SMA are caused by homozygous loss of the functional survival motor neuron (SMN1) gene. However, all patients have one or more copies of the SMN2 gene, nearly identical to SMN1. Both genes encode the SMN protein but the level produced by SMN2 is insufficient to protect from disease. Increasing SMN2 gene expression could be of considerable therapeutic importance. The aim of this study was to assess whether SMN2 gene expression can be increased by 4-phenylbutyrate (PBA). Fibroblast cell cultures from 16 SMA patients affected by different clinical severities were treated with PBA, and full-length SMN2 transcripts were measured by real-time PCR. In all cell cultures, except one, PBA treatment caused an increase in full-length SMN2 transcripts, ranging from 50 to 160% in type I and from 80 to 400% in type II and III cultures. PBA was found also effective in enhancing SMN protein levels and the number of SMN-containing nuclear structures (gems). These data show that SMN expression is considerably increased by PBA, and suggest that the compound, owing also to its favorable pharmacological properties, could be a good candidate for the treatment of SMA.

Randomized, double-blind, placebo-controlled trial of phenylbutyrate in spinal muscular atrophy

Objective: To assess the efficacy of phenylbutyrate (PB) in patients with spinal muscular atrophy in a randomized, double-blind, placebo-controlled trial involving 10 Italian centers. Methods: One hundred seven children were assigned to receive PB (500 mg/kg/day) or matching placebo on an intermittent regimen (7 days on/7 days off) for 13 weeks. The Hammersmith functional motor scale (primary outcome measure), myometry, and forced vital capacity were assessed at baseline and at weeks 5 and 13. Results: Between January and September 2004, 107 patients aged 30 to 154 months were enrolled. PB was well tolerated, with only one child withdrawing because of adverse events. Mean improvement in functional score was 0.60 in the PB arm and 0.73 in placebo arm (p = 0.70). Changes in the secondary endpoints were also similar in the two study arms. Conclusions: Phenylbutyrate was not effective at the regimen, schedule, and duration used in this study.

Phenylbutyrate increases SMN gene expression in spinal muscular atrophy patients

Spinal muscular atrophy (SMA) is caused by insufficient levels of survival motor neuron (SMN) protein. Recently, we found that sodium 4-phenylbutyrate (PB), a well-tolerated FDA approved drug, enhances SMN gene expression in vitro. We provide here the first evidence that oral administration of PB (triButyrate®) significantly increases SMN expression in leukocytes of SMA patients. This finding provides a strong rationale to further investigate the effects of PB as also supported by preliminary clinical data.

Pilot trial of phenylbutyrate in spinal muscular atrophy

The aim of this study was to evaluate tolerability and efficacy of phenylbutyrate (PB) in patients with spinal muscular atrophy (SMA). Ten patients with SMA type II confirmed by DNA studies (age range 2.6-12.7 years, mean age 6.01) were started on oral PB (triButyrate) in powder or tablets. The dosage was 500 mg/kg per day (maximum dose 19 g/d), divided in five doses (every 4 h, skipping one night-dose) using an intermittent schedule (7 days on and 7 days off). Measures of efficacy were the change in motor function from baseline to 3 and 9 weeks, by means of the Hammersmith functional motor scale. In children older than 5 years, muscle strength, assessed by myometry, and forced vital capacity were also measured. We found a significant increase in the scores of the Hammersmith functional scale between the baseline and both 3-weeks (P < 0.012) and 9-weeks assessments (P < 0.004). Our results indicate that PB might be beneficial to SMA patients without producing any major side effect. Larger prospective randomised, double-blind, placebo controlled trials are needed to confirm these preliminary findings.

Salbutamol increases SMN mRNA and protein levels in spinal muscular atrophy cells

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder caused by homozygous absence of the survival motor neuron gene (SMN1). All patients have at least one, usually two to four copies of the related SMN2 gene which, however, produce insufficient levels of functional SMN protein due to the exclusion of exon 7 in the majority of SMN2 transcripts. Here, we show that salbutamol, a β2-adrenoceptor agonist, determines a rapid and significant increase in SMN2-full length mRNA and SMN protein in SMA fibroblasts, predominantly by promoting exon 7 inclusion in SMN2 transcripts. These data, together with previous clinical findings, provide a strong rationale to investigate further the clinical efficacy of salbutamol in SMA patients.

Salbutamol increases survival motor neuron (SMN) transcript levels in leucocytes of spinal muscular atrophy (SMA) patients: relevance for clinical trial design

Background Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations of the SMN1 gene. Based on severity, three forms of SMA are recognised (types I–III). All patients usually have 2–4 copies of a highly homologous gene (SMN2) which produces insufficient levels of functional survival motor neuron (SMN) protein. Recently, evidence has been provided that SMN2 expression can be enhanced in vitro by salbutamol, a β2-adrenergic agonist. This compound has also been shown to improve motor function of SMA patients in two different pilot trials. Aim To evaluate the in vivo molecular efficacy of salbutamol in SMA patients. Methods Twelve type II–III patients took salbutamol orally for 6 months. SMN2 full length transcript levels were determined in peripheral blood leucocytes by absolute real-time PCR, at baseline and after 3 and 6 months of treatment. Results A significant and constant increase in SMN2 full length transcript levels was detected; the response was directly proportional to SMN2 gene copy number. Conclusions The data strongly support salbutamol as a candidate for treating SMA, and suggest that SMN2 copy number may predict the molecular response to treatment and may be a useful randomisation parameter in a double blind placebo controlled clinical trial design.

The Hammersmith functional score correlates with the SMN2 copy number: A multicentric study

Previous studies showed that SMN2 copy number correlates inversely with the disease severity. Our aim was to evaluate SMN2 copy numbers and the Hammersmith functional motor scale in 87 patients with SMA II in order to establish whether, within SMAII, the number of copies correlates with the severity of functional impairment. Our results showed a relative variability of functional scores, but a significant correlation between the number of SMN2 genes and the level of function.

Clinical and molecular cross-sectional study of a cohort of adult type III spinal muscular atrophy patients: clues from a biomarker study.

Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations of the SMN1 gene. Based on severity, three forms of SMA are recognized (types I–III). All patients usually have 2–4 copies of a highly homologous gene (SMN2), which produces insufficient levels of functional survival motor neuron (SMN) protein due to the alternative splicing of exon 7. The availability of potential candidates to the treatment of SMA has raised a number of issues, including the availability of biomarkers. This study was aimed at evaluating whether the quantification of SMN2 products in peripheral blood is a suitable biomarker for SMA. Forty-five adult type III patients were evaluated by Manual Muscle Testing, North Star Ambulatory Assessment scale, 6-min walk test, myometry, forced vital capacity, and dual X-ray absorptiometry. Molecular assessments included SMN2 copy number, levels of full-length SMN2 (SMN2-fl) transcripts and those lacking exon 7 and SMN protein. Clinical outcome measures strongly correlated to each other. Lean body mass correlated inversely with years from diagnosis and with several aspects of motor performance. SMN2 copy number and SMN protein levels were not associated with motor performance or transcript levels. SMN2-fl levels correlated with motor performance in ambulant patients. Our results indicate that SMN2-fl levels correlate with motor performance only in patients preserving higher levels of motor function, whereas motor performance was strongly influenced by disease duration and lean body mass. If not taken into account, the confounding effect of disease duration may impair the identification of potential SMA biomarkers.

Argl6gly polymorphism of the beta2-adrenoceptor gene (ADRBeta2) as a susceptibility factor for nasal polyposis

Background Nasal polyposis is probably a multifactorial disease, but so far, no genetic susceptibility factor has been identified. The observed associations between the ADRB2 arg16gly polymorphism and asthma-related phenotypes as well as those between nasal polyposis and asthma have prompted us to evaluate the potential involvement of this polymorphism in sinonasal polyposis. Methods We enrolled in our study, 56 patients and 47 sex- and age-matched controls. Genomic DNA from cases and controls was extracted and genotype was assessed by a polymerase chain reaction amplification/Nco I digestion assay. Statistical analysis was performed using JMP software (version 5.1). Results The “number of arg alleles” is significantly higher in cases than in controls (p = 0.0386 at t-test; substantially confirmed by nonparametric tests, p = 0.0396 by Wilcoxon/Kruskal-Wallis tests). Conclusion Although results of this study are preliminary because of the small size of the sample, the arg16 allele seems to be associated with an increased risk of sinonasal polyposis suggesting ADRB2 as a susceptibility gene. This finding, if confirmed, would have a clinical value in helping to assess the genetic risk for sinonasal polyposis thus opening new perspectives for the study of molecular factors underlying the development of nasal polyps.

Polymorphism of Beta2-Adrenoceptor and Regular Use of Formoterol in Asthma: Preliminary Results

Polymorphism at codon 16 of the beta2-adrenoceptor (beta2-AR) affects the responsiveness to salmeterol in asthmatics. Data concerning formoterol are more controversial in the literature. The aim of this study was to verify whether homozygous for arginine-16 (ArgArg16) and homozygous for glycine-16 (GlyGly16) genotypes differently influence the long-term responsiveness to formoterol. Twenty-nine patients with mild-to-moderate asthma, in stable clinical conditions, underwent genotyping at codon 16 of the beta2-AR by RFLP-PCR assay. The effects of a 4-week monotherapy with formoterol (12 μg BID) were tested on the peak expiratory flow (PEF) variability and the forced expiratory volume in 1 sec (FEV1) slope of the dose-response curve to salbutamol. Variability in PEF significantly increased during the 4-week treatment period in 14 patients with GlyGly16, but not in 15 patients with ArgArg16 and ArgGly16 . The FEV1 slope of the dose-response curve to salbutamol decreased after the 4-week treatment period in GlyGly16, but not in pooled ArgArg16 and ArgGly16 patients. This study provides preliminary evidence that tolerance to formoterol develops more frequently in asthmatics with GlyGly16 genotype. If confirmed in a larger population, this finding might be useful in choosing the bronchodilator therapy on the basis of genetic polymorphism of the beta2-AR.