Longitudinal evaluation of SMN levels as biomarker for spinal muscular atrophy: results of a phase IIb double-blind study of salbutamol.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder, due to the loss of function of the survival motor neuron (SMN1) gene. The first treatment for the condition, recently approved, is based on the reduction of exon 7 skipping in mRNAs produced by a highly homologous gene (SMN2). The primary objective of the present study was to evaluate the applicability of the dosage of SMN gene produts in blood, as biomarker for SMA, and the safety of oral salbutamol, a beta2-adrenergic agonist modulating SMN2 levels. METHODS: We have performed a 1-year multicentre, double-blind, placebo-controlled study with salbutamol in 45 adult patients with SMA. Patients assumed 4 mg of salbutamol or placebo/three times a day. Molecular tests were SMN2 copy number, SMN transcript and protein levels. We have also explored the clinical effect, by the outcome measures available at the time of study design. RESULTS: Thirty-six patients completed the study. Salbutamol was safe and well tolerated. We observed a significant and progressive increase in SMN2 full-length levels in peripheral blood of the salbutamol-treated patients (p<0.00001). The exploratory analysis of motor function showed an improvement in most patients. CONCLUSIONS: Our data demonstrate safety and molecular efficacy of salbutamol. We provide the first longitudinal evaluation of SMN levels (both transcripts and protein) in placebo and in response to a compound modulating the gene expression: SMN transcript dosage in peripheral blood is reliable and may be used as pharmacodynamic marker in clinical trials with systemic compounds modifying SMN2levels. TRIAL REGISTRATION NUMBER: EudraCT no. 2007-001088-32.

Amifampridine phosphate in the treatment of muscle-specific kinase myasthenia gravis: a phase IIb, randomized, double-blind, placebo-controlled, double crossover study.

OBJECTIVE: The aim of this study is to determine the safety and the efficacy of amifampridine phosphate in muscle-specific kinase antibody-positive myasthenia gravis, in a 1:1 randomized, double-blind, placebo-controlled, switchback, double crossover study. METHODS: Eligible patients had muscle-specific kinase myasthenia gravis, >18 years of age, and Myasthenia Gravis Foundation of America class II-IV with a score of ⩾9 on Myasthenia Gravis Composite scale. After the run-in phase, during which amifampridine phosphate was titrated to a tolerable and effective dosage, patients were randomized to receive placebo-amifampridine-placebo sequence or amifampridine-placebo-amifampridine sequence daily for 7 days. Then, patients switched treatment arms twice, for a total of 21 days of double-blind treatment. Safety was determined by serial assessments of adverse events/serious adverse events, physical examinations, and clinical and laboratory tests. The co-primary outcome measures included changes from baseline of Quantitative Myasthenia Gravis score and Myasthenia Gravis-specific Activities of Daily Living Profile score. The secondary outcome measures comprised changes from baseline of Myasthenia Gravis Composite score, Myasthenia Gravis Quality of Life scale-15 questions, Fatigue Severity Scale, and Carlo Besta Neurological Institute-Myasthenia Gravis scale. Statistical analyses were assessed using a switchback model for three-period, two-treatment crossover design. RESULTS: A total of 10 patients were screened, enrolled, and treated. Transient paresthesias (60%) were the only amifampridine phosphate-related adverse events reported. Four patients were randomized to receive placebo-amifampridine-placebo sequence and three patients to receive amifampridine-placebo-amifampridine sequence. The co-primary objectives were statistically met (Quantitative Myasthenia Gravis score: p = 0.0003 and Myasthenia Gravis-specific Activities of Daily Living Profile score: p = 0.0006), as well as all the secondary endpoints (Myasthenia Gravis Composite score: p < 0.0001, Myasthenia Gravis Quality of Life scale-15 questions: p = 0.0025, Fatigue Severity Scale: p = 0.0061, and Carlo Besta Neurological Institute-Myasthenia Gravis scale: p = 0.0014). CONCLUSION: Despite the low number of patients, MuSK-001 study provided evidence that amifampridine phosphate, in the range of 30-60 mg daily dose, was safe and effective in treating muscle-specific kinase myasthenia gravis, suggesting the need for a large multi-center trial to confirm these results.

Usefulness of spinal unenhanced computed tomography and CT-myelography in the age of multidetector CT technology and magnetic resonance imaging - Preliminary considerations.

Over the last decade, magnetic resonance imaging (MRI) and multidetector computed tomography (MDCT) have revolutionized diagnostic potential in small animal practice, providing adequate assessment of spinal diseases at levels comparable to that achieved in human radiology. T2-weighted MRI images are extremely sensitive to intramedullary parenchymal disorders, while balanced steady-state free precession sequences provide high-quality myelographic images of the spine without the need of intrathecal contrast medium administration. Multidetector computed tomography, with its near-isotropic spatial resolution and multiplanar reformatting of the acquired datasets, provides sufficient stratigraphic details of the spinal cord and the epidural space, facilitating the detection of compressive pathologies without the need of subarachnoid opacification. Nowadays, MDCT and low-field (LF) MRI have become fairly standard and available in academic institutions and private veterinary facilities, appearing to be valuable, complementary, and non-invasive diagnostic tools for imaging the spine. In this scenario, this clinical communication provides a series of preliminary observations that may help to reconsider the usefulness of CT-myelography in the light of its invasiveness and actual diagnostic advantages compared to MRI and unenhanced MDCT for the assessment of compressive and non-compressive spinal diseases in small animals.

Doxycycline in Creutzfeldt-Jakob disease: a phase 2, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Creutzfeldt-Jakob disease (CJD) is a fatal, untreatable prion encephalopathy. Previous studies showed that doxycycline is effective in in-vitro and in-vivo models of disease, and patients with CJD who received compassionate treatment with doxycycline showed increased survival time compared with historical series. We therefore did a randomised, double-blind study of doxycycline versus placebo in CJD. METHODS: We recruited patients older than 18 years old who had a diagnosis of definite or probable sporadic CJD or genetic forms of the disease via Italian reference centres and the French national referral system. Patients were randomly assigned (ratio 1:1) to receive oral doxycycline (100 mg daily) or placebo under double-blind conditions from the day of randomisation to death. Centralised randomisation was done independently of enrolment or evaluation of patients using a minimisation method in Italy and a simple randomisation in France. Participants, caregivers, and clinicians were masked to group assignment. The primary efficacy variable was the survival time from randomisation. Interim analyses were planned to detect a significant effect of treatment as early as possible. This trial is registered with EudraCT, 2006-001858-27 for the Italian study and 2007-005553-34 for the French study. FINDINGS: From April 12, 2007, to Aug 19, 2010, in Italy, and from Jan 30, 2009, to Jan 10, 2012, in France, 121 patients with CJD were enrolled in the study, 62 of whom were randomly assigned to the treatment group and 59 to the placebo group. The first interim analysis showed absence of superiority of doxycycline compared with placebo, and the trial was stopped for futility. Efficacy analyses did not show significant differences between patients treated with doxycycline and placebo with regard to survival times (HR 1.1, 95% CI 0.8-1.7, p=0.50). Serious adverse events were judged not to be related to treatment, whereas a relation was deemed probable or possible for five non-serious adverse events that occurred in each treatment group. INTERPRETATION: Doxycycline at a dose of 100 mg per day was well tolerated but did not significantly affect the course of CJD, at variance with the results of previous observational studies. Our experience could be useful in the design of large multinational controlled trials of potential anti-prion molecules in this rare disease. FUNDING: Agenzia Italiana Farmaco, Italian Ministry of Health, AIEnP, and French Ministry of Health.

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.

Electroacupuncture is not effective in chronic painful neuropathies.

OBJECTIVE: To investigate the analgesic efficacy of electroacupuncture (EA) in patients with chronic painful neuropathy. DESIGN: Double-blind, placebo-controlled, cross-over study. Inclusion criteria were diagnosis of peripheral neuropathy, neuropathic pain (visual analog scale > 4) for at least 6 months, and stable analgesic medications for at least 3 months. PATIENTS: Sixteen patients were randomized into two arms to be treated with EA or pseudo-EA (placebo). INTERVENTIONS: The protocol included 6 weeks of treatment, 12 weeks free of treatment, and then further 6 weeks of treatment. EA or pseudo-EA was performed weekly during each treatment period. OUTCOME MEASURES: The primary outcome was the number of patients treated with EA achieving at least 50% of pain relief at the end of each treatment compared with pain intensity at baseline. Secondary outcomes were modification in patient's global impression of change, depression and anxiety, and quality of life. RESULTS: Eleven patients were randomized to EA and five patients to pseudo-EA as the first treatment. Only one patient per group (EA and pseudo-EA) reported 50% of pain relief at the end of each treatment compared with pain intensity at baseline. Pain intensity did not differ between EA (5.7 ± 2.3 at baseline and 4.97 ± 3.23 after treatment) and pseudo-EA (4.9 ± 1.9 at baseline and 4.18 ± 2.69 after treatment). There was no difference between patients who received EA as the first treatment and patients initially treated with placebo. There was no change in the secondary outcomes. CONCLUSIONS: Our results do not support the use of EA in this population of painful neuropathy patients. Further studies in larger groups of patients are warranted to confirm our observation.

Erythropoietin in amyotrophic lateral sclerosis: a pilot, randomized, double-blind, placebo-controlled study of safety and tolerability.

Preclinical studies demonstrated that erythropoietin is neuroprotective in different models of peripheral and central nervous system diseases. We investigated safety and tolerability of recombinant human erythropoietin (rhEPO) in amyotrophic lateral sclerosis (ALS). We performed a phase II double-blind, randomized, placebo-controlled study. After screening, 23 patients were randomly assigned to rhEPO or placebo arm. Patients were examined during a six-month lead-in period, and then they received fortnightly either 40,000 units of rhEPO or placebo for 24 months. Primary outcomes were adverse events, safety, and death or tracheotomy. Treatment was safe and well tolerated. One patient in the rhEPO arm dropped out for a superficial phlebitis. Median values of haematocrit, haemoglobin, red cells, and reticulocytes were non-significantly higher in rhEPO than placebo arm. Haemoglobin did not increase >1 g/dl between subsequent doses. Anti-rhEPO antibodies were not detected. Survival and slope of ALSFRS-R curves did not significantly differ between treatment groups. RhEPO treatment was safe and well tolerated in ALS patients. Our results suggest that larger studies are warranted to confirm safety of treatment and to investigate different dose schedule and efficacy.

A novel method for species identification in milk and milk-based products.

This study describes a method for species-specific detection of animal DNA from different species (cattle, sheep, goat, water buffalo) in milk and dairy products. A primer set was designed in conserved region on the basis of the alignment of the sequence codifying the genomic kappa-casein gene in order to amplify all four species with a single primer pair. Polymorphisms were detected via minisequencing with extension primers designed in conserved sequences for haplotype determination that allow unambiguous assignment to each species. The method was successfully applied to the detection of raw and pasteurized milk from the four different species considered as well as to cheese products from the retail trade. Estimation of the limit of detection was carried out using a progression of dilutions of genomic DNA as well as DNA isolated from milk of a known number of somatic cells from different species in order to be able to achieve detection rates as low as 0.1% bovine milk mixed with buffalo milk.

Short- and intermediate-term efficacy of buprenorphine TDS in chronic painful neuropathies.

Buprenorphine is a potent opioid available as a transdermal delivery system (TDS) formulation. This open-label study investigated its safety, tolerability, and efficacy in 30 patients with chronic painful neuropathy. Subjects with visual analogue scale (VAS) score > or = 5 under stable analgesic treatment were entered. The starting dosage of 35 microg/h was increased up to 70.0 microg/h in case of unsatisfactory pain control as assessed by fortnightly visits. The primary endpoint was the number of patients achieving at least 30% pain relief at day 42 visit. Treatment was safe over the study period. Nine patients dropped out for side effects, mostly nausea and daily sleepiness. Buprenorphine TDS was well tolerated in 21 patients. Thirteen patients achieved > 30% of pain relief at day 42 visit. Five patients needed to increase the dosage to 52.5 microg/h. Eight patients did not meet the primary outcome, but none allowed increasing the dosage to 70 microg/h, and four patients withdrew consent to continue the study before day 42 visit because of a 'fear to become addicted,' although 40% had obtained VAS reduction. In our study, which needs to be confirmed by a controlled trial, buprenorphine TDS induced clinically meaningful pain relief in about 40% of patients with chronic painful neuropathy, suggesting its use as a third-line treatment.

Myasthenia gravis (MG): epidemiological data and prognostic factors.

Data from 756 myasthenic patients were analyzed for diagnostic criteria, clinical aspects, and therapeutic approaches. The patients were followed up at our institution from 1981 to 2001. Clinical evaluation was performed according to the myasthenia gravis score adopted at our clinic. Clinical features of each patient (comprising demographic, clinical, neurophysiological, immunological, radiological, and surgical data, as well as serial myasthenia gravis scores) were filed in a relational database containing more than 7000 records. Clinical efficacy and variables influencing outcome were assessed by life-table methods and Cox proportional hazards regression analysis. Complete stable remission, as defined by the Task Force of the Medical Scientific Advisory Board of the Myasthenia Gravis Foundation of America, was the end point for good prognosis. Four hundred and ninety-nine patients (66%) were female and 257 (34%) were male. Mean follow-up was 55.1 +/- 48.1 months. Onset of symptoms peaked in the third decade in females, whereas the male distribution was bimodal with peaks in the third and sixth decades. Modality of myasthenia gravis presentation was as follows: ocular, 39.3%; generalized, 28.5%; bulbar, 31.3%; and respiratory, 0.8%. Thymectomy was carried out on 63.7% of our patients by different approaches: (1) transcervical; (2) transsternal; (3) video-thoracoscopic mini-invasive surgery. The last approach has been preferentially used in more recent years and accounted for 62.4% of the thymectomized myasthenia gravis population. Univariate analysis and Kaplan-Meier analysis showed that variables such as sex (female), age at onset (below 40 years), thymectomy, and histological diagnosis of thymic hyperplasia were significantly associated with complete stable remission, whereas on multivariate analysis only age at onset below 40 years and thymectomy were confirmed.