Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Transforming Growth Factor Beta (TGF-β) Is a Muscle Biomarker of Disease Progression in ALS and Correlates with Smad Expression.

We recently identified Smads1, 5 and 8 as muscle biomarkers in human ALS. In the ALS mouse, these markers are elevated and track disease progression. Smads are signal transducers and become activated upon receptor engagement of ligands from the TGF-β superfamily. Here, we sought to characterize ligands linked to activation of Smads in ALS muscle and their role as biomarkers of disease progression. RNA sequencing data of ALS muscle samples were mined for TGF-β superfamily ligands. Candidate targets were validated by qRT-PCR in a large cohort of human ALS muscle biopsy samples and in the G93A SOD1 mouse. Protein expression was evaluated by Western blot, ELISA and immunohistochemistry. C2C12 muscle cells were used to assess Smad activation and induction. TGF-β1, 2 and 3 mRNAs were increased in ALS muscle samples compared to controls and correlated with muscle strength and Smads1, 2, 5 and 8. In the G93A SOD1 mouse, the temporal pattern of TGF-β expression paralleled the Smads and increased with disease progression. TGF-β1 immunoreactivity was detected in mononuclear cells surrounding muscle fibers in ALS samples. In muscle cells, TGF-β ligands were capable of activating Smads. In conclusion, TGF-β1, 2 and 3 are novel biomarkers of ALS in skeletal muscle. Their correlation with weakness in human ALS and their progressive increase with advancing disease in the ALS mouse suggest that they, as with the Smads, can track disease progression. These ligands are capable of upregulating and activating Smads and thus may contribute to the Smad signaling pathway in ALS muscle

TGF-β induces Smads1, 5 and 8 in cultured C2C12 muscle cells.

<p>(A) C2C12 muscle cells were treated with TGF-β ligands for the time frames shown and then assessed for Smad1, 5, and 8 mRNA expression by qRT-PCR. Data points were expressed as a fold-increase over vehicle treated cells and represent the mean ± SE of 6–8 independent samples. ** P < 0.005; *** < 0.0005; **** < 0.0001. (B) C2C12 cells were treated with TGF-β ligands for the time frames shown and assessed for p- and t-Smad 1, 5, 8 by Western blot. The experiment was repeated one time with similar results.</p

TGF-β mRNA is increased in muscle samples from ALS patients.

<p>(A) Total RNA from muscle biopsy samples was analyzed by qRT-PCR for TGF-β1, 2, and 3 mRNA expression in patients with ALS (n = 27), myopathy (n = 11), neuropathy (n = 9) or no neuromuscular disease (n = 13). RQ, relative quantity. *, p < 0.05; **, < 0.005; *** < 0.0005; **** < 0.0001. (B) Correlation of TGF-β isoform mRNA levels (expressed as the Ct value from qRT-PCR). (C) Correlation between muscle grade of biopsied ALS muscle samples (as measured by the Medical Research Council scale) and TGF-β mRNA.</p

TGF-β1 is increased in human ALS muscle.

<p><b>(</b>A) Acid activated protein lysates from human muscle biopsy samples were assessed for TGF-β1 by ELISA and compared to a standard curve. Data points are the mean ± SE. Samples include ALS (n = 12); neuropathy (n = 7); myopathy (n = 8); normal (n = 4). *, p < 0.05. (B) Confocal photomicrographs of ALS and control muscle samples labeled with an anti-TGF-β1 antibody. Bx, biopsy specimen; Aut, autopsy specimen; Ctl, normal biopsy. Size marker = 50 microns.</p

TGF-β induces Smads1, 5 and 8 in cultured C2C12 muscle cells.

<p>(A) C2C12 muscle cells were treated with TGF-β ligands for the time frames shown and then assessed for Smad1, 5, and 8 mRNA expression by qRT-PCR. Data points were expressed as a fold-increase over vehicle treated cells and represent the mean ± SE of 6–8 independent samples. ** P < 0.005; *** < 0.0005; **** < 0.0001. (B) C2C12 cells were treated with TGF-β ligands for the time frames shown and assessed for p- and t-Smad 1, 5, 8 by Western blot. The experiment was repeated one time with similar results.</p

TGF-β and Smad mRNA levels correlate in ALS muscle samples.

<p>Smad1, 5 and 8 mRNA levels were determined by qRT-PCR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138425#pone.0138425.ref004" target="_blank">4</a>] and compared with TGF-β mRNA levels from the same ALS muscle biopsy sample (Ct values are shown).</p

TGF-β mRNA levels are increased at early stages of ALS in the G93A SOD1 mouse.

<p>Total RNA was isolated from G93A SOD1 mice and littermate controls (WT) at 40, 60 and 105 d (preclinical stages as previously defined [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138425#pone.0138425.ref004" target="_blank">4</a>]), and early and late clinical stages (125 and 150 d). Samples were analyzed by qRT-PCR for TGF-β1, 2, 3 and BMP4 mRNA expression. Data points represent the mean ± SE of 6–8 mice. * p < 0.05; ** < 0.005; **** < 0.0001. RQ, relative quantity.</p

Smad2 and 3 are increased in ALS muscle.

<p>(A) Total RNA from human muscle biopsy samples was analyzed by qRT-PCR for Smad2 and 3 mRNA expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138425#pone.0138425.g001" target="_blank">Fig 1</a>. (B) Smad2 and3 mRNA levels in the G93A mouse were determined by qRT-PCR. Data points represent the mean ± SE of 3–4 mice. (C) Western blot of C2C12 cells treated with TGF-β ligands for the times shown. Antibodies are shown to the left. RQ, relative quantity. *, P < 0.05; ** < 0.005; ****< 0.0001.</p