A Double-Blinded,Randomized, Placebo-Controlled Trial to Evaluate Efficacy,Safety, and Tolerability of Single Doses of Tirasemtiv in Patients with Acetylcholine Receptor-Binding Antibody-Positive Myasthenia Gravis

Tirasemtiv is a fast skeletal troponin activator that sensitizes the sarcomere to calcium and increases muscle force following subtetanic nerve input. In an animal model of myasthenia gravis (MG), single oral doses of tirasemtiv improved muscle force and reduced fatigability. The purpose of this study was to determine the effect of single doses of tirasemtiv on skeletal muscle function and fatigability in patients with generalized MG. Thirty-two patients with acetylcholine receptor-antibody positive MG and muscle weakness received single doses of tirasemtiv (250 mg or 500 mg) or placebo in a double-blind, randomized treatment sequence with each treatment separated by at least 1 week. Outcome measures included the Quantitative MG Score (QMG), MG Composite, Manual Muscle Testing, and forced vital capacity. At 6 h after dosing, tirasemtiv produced dose-related improvements from baseline in the QMG score (slope: –0.49 QMG point per 250 mg; p = 0.02) and in percent predicted forced vital capacity (slope: 2.2 % per 250 mg; p = 0.04). QMG improved >3 points in twice as many patients after 500 mg tirasemtiv than after placebo. Both doses of tirasemtiv were well tolerated; there were no premature terminations or serious adverse events. The results of this study suggest that tirasemtiv may improve muscle function in MG and will be used to support further development of tirasemtiv in neuromuscular diseases.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-015-0345-y) contains supplementary material, which is available to authorized users.Key Words: Myasthenia gravis, tirasemtiv, CK-2017357, fast skeletal troponin activator     

Clinical Significance of Repetitive Compound Muscle Action Potentials in Patients with Myasthenia Gravis: A Predictor for Cholinergic Side Effects of Acetylcholinesterase Inhibitors

MethodsWe retrospectively reviewed the clinical records and electrodiagnostic findings of MG patients who underwent electrodiagnostic studies and diagnostic neostigmine testing (NT).ResultsAmong 71 MG patients, 9 could not tolerate oral pyridostigmine bromide (PB) and 17 experienced side effects of PB. R-CMAPs developed in 24 patients after NT. The highest daily dose of PB was lower in the patients with R-CMAPs (240 mg/day vs. 480 mg/day, p<0.001). The frequencies of PB intolerance and side effects were higher in the patients with R-CMAPs than in those without R-CMAPs [37.5% vs. 0% (p<0.001) and 45.8% vs. 12.8% (p=0.002), respectively]. The MG Foundation of America postintervention status did not differ significantly between MG patients with and without R-CMAPs, and the response to immunotherapy was also good in both groups.ConclusionsSide effects of and intolerance to AChEIs are more common in MG patients with R-CMAPs than in those without R-CMAPs. AChEIs should be used carefully in MG patients with R-CMAPs. The presence of R-CMAPs after NT may be a good indicator of the risks of PB side effects and intolerance.     

The daily rhythm of HVA,VMA, (VA) and 5-HIAA in depressionsyndrom

Summary The daily rhythm (8–14 h, 14–20 h, 20–2 h, 2–8 h) of HVA, VMA, (VA) and 5-HIAA was studied in 21 depressed patients and was compared to the values of 13 healthy subjects. In healthy subjects all compounds trend to high values during day-time and significant lower values in the nightphase (2–8 h). Depressed patients did not show a significant rhythm during the four fractions; the values of HVA and VMA were significant lowered in the morning-phase (8–14 h). This demonstrates a good connexion to the clinical feature with the morning listlessness. Normal concentrations were reached in the night-phase (2–8 h). 5-HIAA did not show any significance between healthy subjects and depressed patients during the circadian rhythm. The ratio VMA/HVA in healthy subjects fell significant from morning-phase (8–14 h) to night-phase (20–2 h, 22–8 h). In depressed patients the ratio was approximately equal in all fractions. Whereas the ratio 5-HIAA×100/ VMA>+HVA increased from the morning-phase to the night-phase in healthy persons, this ratio did not show any significance in the four fractions of depressed patients.Also these results suggest some importance in explaining the lost of drive in the activity of depressed patients during morning hours and the remission of these patients often observed in the evening. A very good correlation to the clinical feature is emphasized.Preliminary studies on 6 patients with different depressive diagnosis during depression- and remission-phase gave a different biochemical pattern. Further extended studies have to be carried out to differentiate exactly between nosological different depressions.     

Multiple interactions of anticholinesterases withAplysia acetylcholine responses

The effects of the carbamate anticholinesterases neostigmine and pyridostigmine on the kinetics of densensitization of responses of isolated, voltage-clampedAplysia neurons to microperfused acetylcholine (ACh) was examined. The peak ACh-induced current was potentiated at low carbamate doses and antagonized at higher doses (> 10⁻⁵M); neostigmine was more potent than pyridostigmine in producing both effects. These effects suggest two mechanisms of action of these compounds: (a) inhibition of acetylcholinesterase at low doses, which increases the effective ACh dose, and (b) direct antagonism of the response at higher concentrations, which is associated with a slowing of both the activation and desensitization of the ACh response. These compounds may therefore have direct actions on the excitatory ACh receptor inAplysia neurons which are similar to the effects of these drugs at the vertebrate endplate.     

Presentation of endogenous acetylcholine receptor epitope by an MHC class II-transfected human muscle cell line to a specific CD4 T cell clone from a myasthenia gravis patient

Muscle or thymic myoid cells, if induced to express MHC class II in addition to endogenous acetylcholine receptor (AChR), might present epitopes derived from the AChR to specific CD4⁺ T cells. These T cells could in turn initiate or maintain the anti-AChR response that is responsible for AChR loss in myasthenia gravis (MG). We transfected the AChR⁺ TE671 (rhabdyyosarcomyosarcoma) cells with HLA-DR4 and co-cultured them with the DR4-restricted, CD4⁺ T cell clone (PM-A1; raised from a hyperplastic thymus of an MG patient and previously shown to recognise all forms of the AChR that contain the sequence α144–156). Significant T cell activation, demonstrated both by ³H-thymidine incorporation and by lysis of the TE671 cells, was found in the presence of added α144–156 and, more importantly, in the absence of exogenous antigen. These results show that MHC class II-expressing muscle or other AChR-expressing cells could present endogenous AChR to pathogenic T cells. This process may be important in the aetiology of MG.     

Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels

Recent studies have suggested that anticholinesterases including organophosphates and carbamates act directly on the nicotinic acetylcholine receptor (AChR) channel. We performed whole-cell and single-channel patch-clamp experiments to elucidate the mechanism of action of anticholinesterases on the nicotinic AChR in rat clonal phaeochromocytoma (PC12) cells. Neostigmine and carbaryl showed a biphasic effect; enhancement and suppression of carbachol-induced whole-cell currents. The currents induced by 100 μM carbachol was enhanced by the first co-application with 10 or 100 μM neostigmine, and the current was eventually suppressed below the control level during repeated co-applications. The decay phase of current was accelerated by neostigmine. Carbaryl at 0.1 μM greatly potentiated the carbachol-induced current, and at higher concentrations (0.3–3 μM), current was suppressed. In single-channel experiments, these compounds increased the short closures or gaps during channel opening without changing the single-channel conductance. Mean open time and burst duration were decreased in the presence of neostigmine and carbaryl. These results indicate that neostigmine and carbaryl directly block the nicotinic AChR channel.     

Anti-opioid efficacy of Neuropeptide FF in morphine-tolerant mice

The modulatory effects of 1DMe ( -Tyr-Leu-(NMe)Phe-Gln-Pro-Gln-Arg-Phe-NH₂), an agonist of Neuropeptide FF (NPFF) receptors, on opioid antinociceptive activity have been compared in naive and tolerant mice in the tail-flick and the hot-plate tests. In naive mice, 1DMe alone had no effect on pain threshold but decreased dose-dependently (3–22 nmol) the analgesic activity of morphine in both tests. In tolerant mice, injections of 60-fold lower doses of 1DMe (0.05–0.5 nmol) reverse morphine-induced analgesia in the tail-flick test but this anti-opioid effect was no longer observed with the highest doses of 1DMe tested (3–22 nmol). In the hot-plate test, the anti-opioid action of 1DMe was not detected, whatever doses tested. Neither the NPFF-like immunoreactivity content of spinal cord and of olfactory bulbs, nor the density of NPFF receptors in olfactory bulbs, were altered. These results indicate that a chronic morphine treatment modifies the pharmacological properties of NPFF but the type of pain test is crucial in determining NPFF effects.     

Interferon beta in relapsing–remitting multiple sclerosis

Background and objective Long–term observational studies may provide additional information about the behaviour of different drugs in the post–marketing period. We present the data of our cohort of relapsing–remitting multiple sclerosis (RRMS) patients treated with interferon beta (IFNβ). Methods We analysed RRMS patients followed for at least 2 years. From 1995, we initiated therapy with IFNβ.As they became available, patients were allocated to one of the IFNs at standard doses (IFNβ–1b, IFNβ–1a i. m. or IFNβ–1a s. c.). Each patient was included in a follow–up protocol containing demographic and baseline clinical data. Results Between 1995 and 2004, 382 patients have completed at least 2 years of follow–up. Significant differences at entry were observed. Patients on IFNβ–1b had a higher disease activity and disability at baseline than those on IFNβ–1a i. m. or IFNβ–1a s. c. A significant reduction in the relapse rate was observed for the three drugs (70 % for IFNβ–1b, 64% for IFNβ–1a i. m. and 74 % for IFNβ–1a s. c.). We observed a sustained progression of disability in 11% of patients on IFNβ–1b, 17% on IFNβ–1a i. m. and 19% on IFNβ–1a s. c.; and at four years of follow–up in 24% of patients on IFNβ–1b, 23% on IFNβ–1a i. m. and 35% on IFNβ–1a s. c. No unexpected major adverse events were observed with any of the drugs. Conclusions Interferon beta is safe and well tolerated. The various registered interferon beta drugs provide a comparable efficacy in a large non–selected cohort of RRMS patients.     

Clinical and Electrophysiologic Responses to Acetylcholinesterase Inhibitors in MuSK-Antibody-Positive Myasthenia Gravis: Evidence for Cholinergic Neuromuscular Hyperactivity

Background and Purpose

Patients with muscle-specific tyrosine kinase (MuSK) antibody (MuSK-Ab)-positive myasthenia gravis (MG) show distinct responses to acetylcholinesterase inhibitors (AChEIs). Although clinical responses to AChEIs in MuSK-Ab MG are reasonably well known, little is known about the electrophysiologic responses to AChEIs. We therefore investigated the clinical and electrophysiologic responses to AChEIs in MuSK-Ab-positive MG patients.

Methods

We retrospectively reviewed the medical records and electrodiagnostic findings of 17 MG patients (10 MuSK-Ab-positive and 7 MuSK-Ab-negative patients) who underwent electrodiagnostic testing before and after a neostigmine test (NT).

Results

The frequency of intolerance to pyridostigmine bromide (PB) was higher in MuSK-Ab-positive patients than in MuSK-Ab-negative patients (50% vs. 0%, respectively; p=0.044), while the maximum tolerable dose of PB was lower in the former (90 mg/day vs. 480 mg/day, p=0.023). The frequency of positive NT results was significantly lower in MuSK-Ab-positive patients than in MuSK-Ab-negative patients (40% vs. 100%, p=0.035), while the nicotinic side effects of neostigmine were more frequent in the former (80% vs. 14.3%, p=0.015). Repetitive compound muscle action potentials (R-CMAPs) developed more frequently after NT in MuSK-Ab-positive patients than in MuSK-Ab-negative patients (90% vs. 14.3%, p=0.004). The frequency of a high-frequency-stimulation-induced decrement-increment pattern (DIP) was higher in MuSK-Ab-positive patients than in MuSK-Ab-negative patients (100% vs. 17.7%, p=0.003).

Conclusions

These results suggest that MuSK-Ab-positive MG patients exhibit unique and hyperactive responses to AChEIs. Furthermore, R-CMAP and DIP development on a standard AChEI dose may be a distinct neurophysiologic feature indicative of MuSK-Ab-positive MG.     

The diagnostic significance of autoantibodies to the acetylcholine receptor

The predictive value of the assay for antibodies to the acetylcholine receptor (anti-AChR) is dependent upon the reference range used and the question being asked by the clinician. A reference range has been established after assaying sera from 200 healthy individuals, 314 patients with diseases often considered in the differential diagnosis of myasthenia gravis (MG) or found in association with MG, and 72 patients with active adult onset MG. If the assay is to be used to screen at unselected population for MG a conservative cut off point (2 units) should be used. After establishment of a differential diagnosis more significant may be attributed to a lower result (1 unit or greater). A negative result does not exclude MG. In patients with Systemic Lupus Erythematosus, Graves' disease or thymoma anti-AChR has been demonstrated in the absence of signs of MG. Such patients may have latent or subclinical MG. Two such patients subsequently developed clinically evident MG concomitant with a rise in anti AChR titre above their particular ‘biological threshold’.     

No evidence for an association of AChR beta-subunit gene (CHRNB1) with myasthenia gravis

Using a polymorphic dinucleotide repeat, we have investigated the contribution of the gene encoding the beta-subunit of the muscle acetylcholine receptor (CHRNB1), the target autoantigen, to the susceptibility to myasthenia gravis (MG). We have combined a case-control study (comparing 143 patients and 162 controls) and a transmission–disequilibrium test bearing on 35 simplex families with heterozygous parents. There was no evidence for an association of CHRNB1 with MG, even after subgrouping patients according to thymus histology, or other clinical criteria. Interestingly however, the shortest four variants of the CHRNB1 microsatellite were seen only in patients with thymus hyperplasia and in none of the control subjects (P<0.0025).     

Dysphonia as a primary manifestation in myasthenia gravis (MG): a retrospective review of 7 cases among 1520 MG patients

Myasthenia gravis (MG) is an autoimmune disorder of neuromuscular transmission, usually recognized with ocular complaints or generalized muscle weakness. However, among the 1520 MG cases that had been diagnosed and treated in our hospital in the last 15 years (1990–2005), we have identified 7 MG patients whose initial and prominent complaint was dysphonia and all had been misdiagnosed elsewhere. The diagnoses were confirmed with fibrolaryngoscope and voice analysis employed before and after a positive neostigmine (anticholinesterase) test. Electromyography with repetitive stimulations, single-fiber electromyography, and laboratory and radiographic evaluations were also conducted for diagnosis. A surprisingly low seropositivity rate of anti-acetylcholine-receptor antibodies (1/7) and anti-MuSK (Muscle Specific Kinase) antibodies (0/6) were found in these dysphonia MG patients. A cholinesterase inhibitor (ChEI) and immunosuppressive therapy were applied for treatment. Extended thymectomy was applied to MG patients with thymus hyperplasia or thymic tumor. Significant improvement was found in all 7 cases after these treatments. We have developed a sere of diagnostic protocol for this rare type of laryngeal MG, and discussed the clinical implication of our data. In summary, dysphonia or laryngeal disorder can be the only prominent manifestation of MG in rare cases, which should be taken into consideration during the diagnosis to patients with exclusive laryngeal complaints.     

Involvement of M3 muscarinic receptors of the spinal cord in formalin-induced nociception in mice

Subcutaneous injection of formalin into a paw of mice caused two distinct phases of licking and biting, first phase (1–5 min) and the second phase (7–30 min) after the injection. The muscarinic antagonist atropine (0.1–10 ng, i.t.) and the M₃ receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (0.1–20 ng, i.t.) inhibited the second phase of this response, whereas higher doses of atropine (20–100 ng, i.t.) did not cause inhibition. The M₁ muscarinic receptor antagonist pirenzepine (10–100 ng, i.t.) did not inhibit either the first or the second phase response, but a high dose of pirenzepine (1000 ng, i.t.) tended to inhibit the second phase response. On the other hand, the M₂ muscarinic receptor antagonist 11-{(2-[(diethylamino)methyl]-1-piperidinyl}acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one (AF-DX116; 10–1000 ng, i.t.) had no effect on either the first or the second phase of response. The opioid receptor antagonist naloxone did not affect the 4-DAMP-induced anti-nociceptive response. The i.t. injection of the acetylcholinesterase inhibitor neostigmine (25 ng) significantly inhibited only the second phase. The acetylcholine (ACh) depletor hemicholinium-3 (HC-3) (1 μg, i.t.) completely abolished the 4-DAMP-induced anti-nociceptive response. The ACh content of the spinal cord was significantly increased 14 min after formalin injection. This significant increase in the ACh content was inhibited by pretreatment with 4-DAMP (10 ng, i.t.). These results suggest that endogenous ACh in the spinal cord acts as a transmitter anti-nociception, and that ACh release regulated by presynaptic M₃ muscarinic receptors in the spinal cord is involved in the second phase of nociception induced by formalin.     

Involvement of the bed nucleus of the stria terminalis in hippocampal cholinergic system-mediated activation of the hypothalamo–pituitary–adrenocortical axis in rats

This study was designed to determine the effect of the bed nucleus of the stria terminalis (BNST) in hippocampal cholinergic system-mediated activation of the hypothalamo–pituitary–adrenocortical (HPA) axis in the rat. Neurons in the BNST were lesioned by bilateral injection of the cell-selective neurotoxin, ibotenic acid (1.5 μg/μl of solution per side). Two weeks later, neostigmine was microinjected into the rats’ hippocampus. Rats in which ibotenic acid had been injected into the BNST showed attenuated expression of c-Fos in the hypothalamic paraventricular nucleus (PVN) and blunted elevation of plasma adrenocorticotropic hormone (ACTH) after microinjection of neostigmine into the hippocampus compared with rats in which saline had been injected into the BNST. The results of this study indicate that the BNST relays signals of hippocampal cholinergic system-mediated activation of the HPA axis in rats.     

Cardiac involvement in myasthenia gravis

Non-specific ECG changes and histological changes in the myocardium in patients with myasthenia gravis (MG) have been described. In this study, the left ventricular function using systolic time intervals (STI) was assessed in patients with MG. 4 patients out of the 10 studied showed abnormalities of the STI which reverted towards normal after injection of neostigmine. This finding suggests that myocardial involvement in MG may be more common than clinically suspected.     

Fatal or severely disabling cerebral infarction during hospitalization for stroke or transient ischemic attack

Summary Six (1%) of 578 patients admitted for cerebral infarction or transient ischemic attack (TIA) suffered a fatal or severely disabling in-hospital cerebral infarction following a period of stabilization or improvement lasting more than 1 day. These infarctions were characterized by the sudden onset of stupor or coma and subsequent development of transtentorial herniation due to carotid or middle cerebral artery territory infarction, or widespread brain-stem infarction due to basilar occlusion. Only one patient survived. Four patients had largevessel disease documented by Doppler, angiography, or at autopsy. Each of these six infarcts occurred during the morning hours, 4–9 days after the initial event, 3–8 days after initiation of intravenous heparin, and within 4–8 h after intravenous heparin had been discontinued. No coagulation abnormalities were documented. We believe that these cases indicate that among patients admitted for cerebral infarction or TIA, fatal or severely disabling in-hospital cerebral infarction after a period of stabilization or improvement may occur in patients having an initially mild to moderate clinical deficit, that those suffering large artery disease may be at greater risk, and that there may be a relationship between heparin withdrawal and cerebral infarction in some patients.Supported in part by the Sunny von Bulow Coma and Head Trauma Research Foundation     

Synthesis of anti-acetylcholine receptor antibodies by CD5- B cells from peripheral blood of myasthenia gravis patients

An increased frequency of CD5⁺ B cells (or, according to a new nomenclature, B 1 cells) has been detected in the peripheral blood of a proportion of patients with myasthenia gravis (MG), as in some other autoimmune diseases. To elucidate the pathogenic significance of this B-cell subset in myasthenia gravis, mononuclear cells from the peripheral blood of six MG patients were separated into T and B lymphocytes by a magnetic cell separation procedure employing superparamagnetic microbeads (MACS). Subsequently, the B-cell fraction was depleted of CD5⁺ B cells in a second separation. The resulting purified CD5^– B-cell fraction was cultured alone or with the addition of autologous T cells. Anti-acetylcholine receptor (AChR) synthesis by CD5^– B cells in cultures with T cells was significantly increased by pokeweed mitogen (176 ±130 fmol/ml per week/2 × 10⁵ B cells) compared with unfractionated cells (75 ± 101) or CD5^– B cells alone (19 ± 4). These results demonstrate that in MG anti-AChR are synthesized, at least in part, by CD5^– B cells which are dependent on T cells. Although this does not exclude the existence of AChR-specific CD5⁺ B cells, it provides evidence against a pivotal role of this B-cell subset in anti-AChR synthesis.     

Aβ25–35 induces rapid lysis of red blood cells: contrast with Aβ1–42 and examination of underlying mechanisms

Amyloid β-peptide (Aβ) is produced by many different cell types and circulates in blood and cerebrospinal fluid in a soluble form. In Alzheimer's disease (AD), Aβ forms insoluble fibrillar aggregates that accumulate in association with cells of the brain parenchyma and vasculature. Both full-length Aβ (Aβ1–40/42) and the Aβ25–35 fragment can damage and kill neurons by a mechanism that may involve oxidative stress and disruption of calcium homeostasis. Circulating blood cells are exposed to soluble Aβ1–40/42 and may also be exposed to Aβ aggregates associated with the luminal surfaces of cerebral microvessels. We therefore examined the effects of Aβ25–35 and Aβ1–42 on human red blood cells (RBCs) and report that Aβ25–35, in contrast to Aβ1–42, induces rapid (10–60 min) lysis of RBCs. The mechanism of RBC lysis by Aβ25–35 involved ion channel formation and calcium influx, but did not involve oxidative stress because antioxidants did not prevent cell lysis. In contrast, Aβ1–42 induced a delayed (4–24 h) damage to RBCs which was attenuated by antioxidants. The damaging effects of both Aβ25–35 and Aβ1–42 towards RBCs were completely prevented by Congo red indicating a requirement for peptide fibril formation. Aβ1–42 induced membrane lipid peroxidation in RBC, and basal levels of lipid peroxidation in RBCs from AD patients were significantly greater than in age-matched controls, suggesting a possible role for Aβ1–42 in previously reported alterations in RBCs from AD patients.     

The effects of naloxone on the neural control of the urinary bladder of the cat

Naloxone in doses ranging from 0.5 to 512μg/kg i.v., enhanced reflex contractions of the urinary bladder of the cat. At the lowest doses (threshold, 0.5–5 μg/kg) the drug increased the frequency of spontaneous bladder contractions. In large doses (10–100 μg/kg) the drug produced an initial tonic contraction of bladder lasting 15–40 min followed by a period of high frequency rhythmic activity. Multiunit firing in parasympathetic postganglionic nervs on the surface of the urinary bladder was also enhanced. Bursts of firing which in untreated animals occured during large bladder contractions continously during the entire sustained contraction of the bladder following large doses of naloxone. Various evidence indicates that the site of action of naloxone is in the central nervous system. These findings suggest that the parasympathetic reflex pathway to the urinary bladder may be subject to tonic enkephalinergic inhibitory control.     

Heterogeneity of antibodies directed against the α-bungarotoxin binding site on human acetylcholine receptor and severity of myasthenia gravis

A rapid and sensitive radioimmunological method is described, using decamethonium (DC), which revealed antibodies which blocked α-bungarotoxin (α-Bgt) binding to human acetylcholine receptor (AChR) in 98% of myasthenia gravis (MG) patients' sera tested. These sera had anti-AChR antibody titres by the conventional assay. The titre of blocking antibodies (1 to 110 nM) could be measured and was found to produce from 1 to 54% inhibition of α-Bgt binding. No relationship was found between these titres and anti-AChR antibody titres. MG sera were divided into 2 major groups on the basis of their blocking effects, with and without DC, but there was no correlation between these and the clinical status, as defined by Osserman's classification. However, no sera from asymptomatic or ocular MG patients had the dual capacities of blocking α-Bgt binding, directly and in the presence of DC.