Efficacy and safety of exogenous beta-hydroxybutyrate for preventive treatment in episodic migraine: A single-centred, randomised, placebo-controlled, double-blind crossover trial.

BACKGROUND: Several studies propose that brain energy deficit might be partially involved in the pathophysiology of migraine. Previously, studies demonstrated that ketogenic diet causes a substantial reduction in migraine frequency. Since the ketogenic diet is restricting and its adherence is difficult, we proposed to supplement ketone bodies exogenously to provide a prophylactic effect in migraineurs. AIM: To evaluate the prophylactic effect of exogenous DL-beta-hydroxybutyrate supplementation in episodic migraineurs. METHODS: A double-blind, placebo-controlled, randomised crossover trial was conducted, involving 41 patients with episodic migraine. Patients were randomised 1:1 into placebo or beta-hydroxybutyrate group before entering the first treatment period. Each treatment period was 12 weeks long, followed by four weeks of washout phase and four weeks of run-in phase before entering into the corresponding second treatment period. The primary endpoint was the number of migraine days in the last four weeks of treatment, adjusted for baseline. RESULTS: We observed no clinically significant amelioration of migraine frequency or intensity under DL-beta-hydroxybutyrate treatment as compared to placebo regarding number of migraine days (mean difference [95% CI]: -1.1[-5.07, 2.85]), migraine intensity (0-10 VAS: 1.5[-0.8, 3.7]). CONCLUSION: The selected dose of supplemented exogenous DL-beta-hydroxybutyrate did not demonstrate efficacy in episodic migraineurs.ClinicalTrials.gov Identifier: NCT03132233.

Spinal cord gray matter atrophy is associated with functional decline in post-polio syndrome.

OBJECTIVE: To determine if patients with post-polio syndrome (PPS) show spinal cord gray matter (SCGM) atrophy and to assess associations between SCGM atrophy, muscle strength and patient-reported functional decline. METHODS: Twenty patients diagnosed with PPS (March of Dimes criteria) and 20 age- and sex-matched healthy controls (HC) underwent 3T axial 2D-rAMIRA magnetic resonance imaging at the intervertebral disc levels C2/C3-C6/C7, T9/T10 and the lumbar enlargement level (Tmax ) (0.5 × 0.5 mm2 in-plane resolution). SCGM areas were segmented manually by two independent raters. Muscle strength, self-reported fatigue, depression and pain measures were assessed. RESULTS: Post-polio syndrome patients showed significantly and preferentially reduced SCGM areas at C2/C3 (p = 0.048), C3/C4 (p = 0.001), C4/C5 (p < 0.001), C5/C6 (p = 0.004) and Tmax (p = 0.041) compared to HC. SCGM areas were significantly associated with muscle strength in corresponding myotomes even after adjustment for fatigue, pain and depression. SCGM areaTmax together with age and sex explained 68% of ankle dorsiflexion strength variance. No associations were found with age at or time since infection. Patients reporting PPS-related decline in arm function showed significant cervical SCGM atrophy compared to stable patients adjusted for initial disease severity. CONCLUSIONS: Patients with PPS show significant SCGM atrophy that correlates with muscle strength and is associated with PPS-related functional decline. Our findings suggest a secondary neurodegenerative process underlying SCGM atrophy in PPS that is not explained by aging or residua of the initial infection alone. Confirmation by longitudinal studies is needed. The described imaging methodology is promising for developing novel imaging surrogates for SCGM diseases.

Stable-Isotope Dilution GC-MS Measurement of Metformin in Human Serum and Urine after Derivatization with Pentafluoropropionic Anhydride and Its Application in Becker Muscular Dystrophy Patients Administered with Metformin, l-Citrulline, or Their Combination.

Metformin (N,N-dimethylguanylguanidine) is one of the most prescribed drugs with pleiotropic, exerted in part by not fully elucidated mechanisms of action. We developed and validated a gas chromatography-mass spectrometry (GC-MS) method for the quantitative analysis of metformin (metformin-d0) in 10-µL aliquots of human serum and urine using N,N-[dimethylo-2H6]guanylguanidine (metformin-d6) as the internal standard. The method involves evaporation of the samples to dryness, derivatization with pentafluoropropionic (PFP) anhydride in ethyl acetate (30 min, 65 °C), and extraction into toluene. The negative-ion chemical ionization GC-MS spectra of the PFP derivatives contain a single intense ion with mass-to-charge (m/z) ratios of m/z 383 for metformin-d0 and m/z 389 for metformin-d6. Our results suggest that all amine/imine groups of metformin-d0 and metformin-d6 are converted to their N,N,N-tripentafluoropropionyl derivatives, which cyclize to form a symmetric triazine derivative, of which the non-ring amine group is amidated. Quantification was performed by selected-ion monitoring (SIM) of m/z 383 and m/z 389. Upon validation, the method was applied to determine serum and urine metformin concentrations in 19 patients with Becker muscular dystrophy (BMD). Serum and urine samples were collected at baseline (Visit I), after six weeks of supplementation (Visit II) with metformin (3 × 500 mg/d; metformin group; n = 10) or l-citrulline (3 × 1500 mg/d; citrulline group; n = 9) followed by a six-week supplementation with 3 × 500 mg/d of metformin plus 3 × 1500 mg/d l-citrulline. At Visit I, the metformin concentration in the serum and urine was very low in both groups. The metformin concentrations in the serum and urine of the patients who first took metformin (MET group) were higher at Visit II and Visit III. The metformin concentration in the serum and urine samples of the patients who first took l-citrulline (CITR group) were higher at Visit III. The serum and urine concentrations of metformin were insignificantly lower in the CITR group at Visit III. The mean fractional excretion (FE) rate of metformin was 307% (Visit II) and 322% (Visit III) in the MET group, and 290% in the CITR group (Visit III). This observation suggests the accumulation of metformin in the kidney and its secretion in the urine. The GC-MS is suitable to measure reliably circulating and excretory metformin in clinical settings.

Longitudinal reliability of outcome measures in patients with Duchenne muscular dystrophy.

INTRODUCTION: The definition of reliable outcome measures is of increasing interest in patients with Duchenne muscular dystrophy (DMD). METHODS: In this retrospective study, we analyzed the longitudinal reliability of clinical and radiological endpoints in 29 ambulant patients with DMD. Clinical outcome measures included motor function measure (MFM) and timed function tests, while quantitative MRI data were mean fat fraction (MFF) and T2 relaxation time of thigh muscles. Statistical analysis was based on 3-, 6-, and 12-month follow-up data. RESULTS: Quantitative MRI using the MFF was the most sensitive and powerful marker of disease progression with a sample size of four at 1-year follow-up, followed by the D1 domain of MFM (standing and transfer function) with a sample size of 12. DISCUSSION: Our data support the longitudinal design of clinical trials over at least 12 months and the combinational use of clinical and radiological surrogate outcome measures.

Acquired neuromyotonia in children with CASPR2 and LGI1 antibodies.

Acquired neuromyotonia is a form of peripheral nerve hyperexcitability. In adults, pathogenic antibodies that target the extracellular domains of leucine-rich glioma-inactivated protein 1 (LGI1) and contactin-associated protein-like 2 (CASPR2) have been reported. We describe three paediatric patients with acquired neuromyotonia and CASPR2 and LGI1 serum antibodies. They all presented with acute-onset myokymia and pain in the lower limbs; one patient also had muscle weakness. Electromyography was suggestive of peripheral nerve hyperexcitability. Two patients improved without immunotherapy; one treated patient remained immunotherapy-dependent. Although not fatal, acquired paediatric neuromyotonia can be disabling. It is amenable to symptomatic treatment or may undergo spontaneous recovery. More severe cases may require rational immunotherapy. WHAT THIS PAPER ADDS: The symptoms of neuromyotonia may resolve spontaneously or may require sodium channel blockers. Patients with debilitating symptoms who are refractory to symptomatic therapy may require immunotherapy.

NEUROMIOTONÍA ADQUIRIDA EN NIÑOS CON ANTICUERPOS CASPR2 Y LGI1: La neuromiotonía adquirida es una forma de hiperexcitabilidad de los nervios periféricos. En algunos adultos, se han notificado anticuerpos patógenos que se dirigen a los dominios extracelulares de la proteína 1 inactivada por glioma rico en leucina (LGI1) y la proteína 2 asociada a contactina (CASPR2). Describimos tres pacientes pediátricos con neuromiotonía adquirida y anticuerpos séricos CASPR2 y LGI1. Todos presentaban mioquimia de inicio agudo y dolor en las extremidades inferiores; un paciente también tenía debilidad muscular. La electromiografía sugirió hiperexcitabilidad del nervio periférico. Dos pacientes mejoraron sin inmunoterapia; un paciente tratado permaneció dependiente de la inmunoterapia. Aunque no es fatal, la neuromiotonía pediátrica adquirida puede ser incapacitante. Es susceptible de tratamiento sintomático o puede sufrir una recuperación espontánea. Los casos más graves pueden requerir inmunoterapia racional.

NEUROMIOTONIA ADQUIRIDA EM CRIANÇAS COM ANTICORPOS PRCAS2 E GIL1: A neuromiotonia adquirida é uma forma de hiperexcitabilidade nervosa periférica. Em alguns adultos, anticorpos patogênicos que visam os domínios extracelulares da proteína glioma-inativada rica em leucina1 (GIL1) e da proteína contactina-associada 2 (PRCAS2) foram reportados. Descrevemos três pacientes pediátricos com neuromiotonia adquirida e anticorpos séricos PRCAS2 e GIL1 CASPR2. Todos apresentaram miocimia de início agudo e dor nos membros inferiores; um paciente também teve fraqueza muscular. A eletromiografia foi sugestiva de hiperexcitabilidade nervosa periférica. Dois pacientes melhoraram sem imunoterapia; um paciente tratado permaneceu imunoterapia-dependente. Embora não seja fatal, a neuromiotomia pediátrica aguda pode ser incapacitante. É responsiva a tratamento sintomático e pode apresentar recuperação espontânea. Casos mais severaos podem requerer imunoterapia racional.

Effects of single and combined metformin and L-citrulline supplementation on L-arginine-related pathways in Becker muscular dystrophy patients: possible biochemical and clinical implications.

The L-arginine/nitric oxide synthase (NOS) pathway is considered to be altered in muscular dystrophy such as Becker muscular dystrophy (BMD). We investigated two pharmacological options aimed to increase nitric oxide (NO) synthesis in 20 male BMD patients (age range 21-44 years): (1) supplementation with L-citrulline (3 × 5 g/d), the precursor of L-arginine which is the substrate of neuronal NO synthase (nNOS); and (2) treatment with the antidiabetic drug metformin (3 × 500 mg/d) which activates nNOS in human skeletal muscle. We also investigated the combined use of L-citrulline (3 × 5 g/d) and metformin (3 × 500 mg/d). Before and after treatment, we measured in serum and urine samples the concentration of amino acids and metabolites of L-arginine-related pathways and the oxidative stress biomarker malondialdehyde (MDA). Compared to healthy subjects, BMD patients have altered NOS, arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) pathways. Metformin treatment resulted in concentration decrease of arginine and MDA in serum, and of homoarginine (hArg) and guanidinoacetate (GAA) in serum and urine. L-Citrulline supplementation resulted in considerable increase of the concentrations of amino acids and creatinine in the serum, and in their urinary excretion rates. Combined use of metformin and L-citrulline attenuated the effects obtained from their single administrations. Metformin, L-citrulline or their combination did not alter serum nitrite and nitrate concentrations and their urinary excretion rates. In conclusion, metformin or L-citrulline supplementation to BMD patients results in remarkable antidromic changes of the AGAT and GAMT pathways. In combination, metformin and L-citrulline at the doses used in the present study seem to abolish the biochemical effects of the single drugs in slight favor of L-citrulline.

Longitudinal 2-point dixon muscle magnetic resonance imaging in becker muscular dystrophy.

INTRODUCTION: Quantitative MRI techniques detect disease progression in myopathies more sensitively than muscle function measures or conventional MRI. To date, only conventional MRI data using visual rating scales are available for measurement of disease progression in Becker muscular dystrophy (BMD). METHODS: In 3 patients with BMD (mean age 36.8 years), the mean fat fraction (MFF) of the thigh muscles was assessed by MRI at baseline and at 1-year follow-up using a 2-point Dixon approach (2PD). The motor function measurement scale (MFM) was used for clinical assessment. RESULTS: The mean MFF of all muscles at baseline was 61.6% (SD 7.6). It increased by 3.7% to 65.3% (SD 4.7) at follow-up. The severity of muscle involvement varied between various muscle groups. CONCLUSIONS: As in other myopathies, 2PD can quantify fatty muscle degeneration in BMD and can detect disease progression in a small sample size and at relatively short imaging intervals.

Metabolic markers of short and long-term exogenous DL-beta-hydroxybutyrate supplementation in episodic migraine patients: an exploratory analysis of a randomized-controlled-trial.

Background: Emerging findings propose that the pathophysiology of migraine may be associated with dysfunctional metabolic mechanisms. Recent findings suggest that migraine attacks are a response to the cerebral energy deficit, and ingestion of ketone bodies stabilizes the generation of a migraine attack. Based on these findings, ketone body supplementation is postulated as a prophylactic treatment approach to restore cerebral metabolism deficiency. Metabolic markers are unexplored after exogenous ketone body supplementation in episodic migraineurs. Therefore, the present single-arm uncontrolled explorative analysis evaluated blood ketone body and glucose concentration after short and long-term 6 g exogenous DL-Mg-Ca-beta-hydroxybutyrate (DL-ßHB) supplementation. Methods: The presented data are part of the MigraKet randomized-control cross-over clinical trial of 41 episodic migraineurs (Number NCT03132233). Patients were given a single dose of 6 g DL-ßHB. Ketone body and glucose blood concentration were assessed before intake, 20, and 40 min after DL-ßHB intake. Ketone body, glucose concentration and glycated hemoglobin values were evaluated after 12 weeks of 18 g DL-ßHB ingestion (total dose), taken three times daily (6g/dose; 3x/day). Linear models explored the association between the ketone body and glucose levels. Results: Ketone body concentration increased within-group to a mean of 0.46 (0.30) mmol/L after 40 min post- DL-ßHB supplementation [estimate = 0.24 mmol/L, CI = (0.20.0.27), p < 0.01]. This within-group increase of ketone body concentration did not change after repeated daily intake of DL-ßHB supplementation over 12 weeks [estimate = 0.00 mmol/L, CI = (-0.03.0.04), p = 0.794]. DL-ßHB intake significantly reduced blood glucose concentration within-group from a mean baseline of 4.91 (0.42) mmol/L to 4.75 (0.47) mmol/L 40 min post-DL-ßHB supplementation [estimate = -0.16 mmol/L, CI = (-0.15, 0.03), p < 0.01]. Repeated DL-ßHB supplementation for 12 weeks showed no change within-group in acute ketone bodies concentration [estimate = 0.00 mmol/L, CI = (-0.03.0.04), p = 0.794] and in the HbA1c value [estimate = 0.02, CI = (-0.07.0.11), p = 0.69]. Conclusion: A single dose of 6 g DL-ßHB significantly elevated blood ketone bodies and decreased blood glucose concentration within-group in episodic migraineurs. Long-term DL-ßHB supplementation for 12 weeks showed no effect within-group on acute ketone body concentration and had not impact on HbA1c. The elevation of the ketone body concentration was moderate, indicating that nutritional ketosis was not reached. Therefore, a dose higher than 6 g of DL-ßHB is required to reach the nutritional level of ketosis. ClinicalTrials.gov Identifier: NCT03132233.

Dimethyl fumarate modulates the dystrophic disease program following short-term treatment.

New medicines are urgently required to treat the fatal neuromuscular disease Duchenne muscular dystrophy (DMD). Dimethyl fumarate (DMF) is a potent immunomodulatory small molecule nuclear erythroid 2-related factor 2 activator with current clinical utility in the treatment of multiple sclerosis and psoriasis that could be effective for DMD and rapidly translatable. Here, we tested 2 weeks of daily 100 mg/kg DMF versus 5 mg/kg standard-care prednisone (PRED) treatment in juvenile mdx mice with early symptomatic DMD. Both drugs modulated seed genes driving the DMD disease program and improved force production in fast-twitch muscle. However, only DMF showed pro-mitochondrial effects, protected contracting muscles from fatigue, improved histopathology, and augmented clinically compatible muscle function tests. DMF may be a more selective modulator of the DMD disease program than PRED, warranting follow-up longitudinal studies to evaluate disease-modifying impact.

Safety and efficacy of tamoxifen in boys with Duchenne muscular dystrophy (TAMDMD): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Drug repurposing could provide novel treatment options for Duchenne muscular dystrophy. Because tamoxifen-an oestrogen receptor regulator-reduced signs of muscular pathology in a Duchenne muscular dystrophy mouse model, we aimed to assess the safety and efficacy of tamoxifen in humans as an adjunct to corticosteroid therapy over a period of 48 weeks. METHODS: We did a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial at 12 study centres in seven European countries. We enrolled ambulant boys aged 6·5-12·0 years with a genetically confirmed diagnosis of Duchenne muscular dystrophy and who were on stable corticosteroid treatment for more than 6 months. Exclusion criteria included ophthalmological disorders, including cataracts, and haematological disorders. We randomly assigned (1:1) participants using an online randomisation tool to either 20 mg tamoxifen orally per day or matched placebo, stratified by centre and corticosteroid intake. Participants, caregivers, and clinical investigators were masked to treatment assignments. Tamoxifen was taken in addition to standard care with corticosteroids, and participants attended study visits for examinations every 12 weeks. The primary efficacy outcome was the change from baseline to week 48 in scores on the D1 domain of the Motor Function Measure in the intention-to-treat population (defined as all patients who fulfilled the inclusion criteria and began treatment). This study is registered with ClinicalTrials.gov (NCT03354039) and is completed. FINDINGS: Between May 24, 2018, and Oct 14, 2020, 95 boys were screened for inclusion, and 82 met inclusion criteria and were initially enrolled into the study. Three boys were excluded after initial screening due to cataract diagnosis or revoked consent directly after screening, but before randomisation. A further boy assigned to the placebo group did not begin treatment. Therefore, 40 individuals assigned tamoxifen and 38 allocated placebo were included in the intention-to-treat population. The primary efficacy outcome did not differ significantly between tamoxifen (-3·05%, 95% CI -7·02 to 0·91) and placebo (-6·15%, -9·19 to -3·11; 2·90% difference, -3·02 to 8·82, p=0·33). Severe adverse events occurred in two participants: one participant who received tamoxifen had a fall, and one who received placebo suffered a panic attack. No deaths or life-threatening serious adverse events occurred. Viral infections were the most common adverse events. INTERPRETATION: Tamoxifen was safe and well tolerated, but no difference between groups was reported for the primary efficacy endpoint. Slower disease progression, defined by loss of motor function over time, was indicated in the tamoxifen group compared with the placebo group, but differences in outcome measures were neither clinically nor statistically significant. Currently, we cannot recommend the use of tamoxifen in daily clinical practice as a treatment option for boys with Duchenne muscular dystrophy due to insufficient clinical evidence. FUNDING: Thomi Hopf Foundation, ERA-Net, Swiss National Science Foundation, Duchenne UK, Joining Jack, Duchenne Parent Project, Duchenne Parent Project Spain, Fondation Suisse de Recherche sur les Maladies Musculaires, Association Monegasque contre les Myopathies.