Efficacy and safety of vamorolone in Duchenne muscular dystrophy: An 18-month interim analysis of a non-randomized open-label extension study.

BACKGROUND: Treatment with corticosteroids is recommended for Duchenne muscular dystrophy (DMD) patients to slow the progression of weakness. However, chronic corticosteroid treatment causes significant morbidities. Vamorolone is a first-in-class anti-inflammatory investigational drug that has shown evidence of efficacy in DMD after 24 weeks of treatment at 2.0 or 6.0 mg/kg/day. Here, open-label efficacy and safety experience of vamorolone was evaluated over a period of 18 months in trial participants with DMD. METHODS AND FINDINGS: A multicenter, open-label, 24-week trial (VBP15-003) with a 24-month long-term extension (VBP15-LTE) was conducted by the Cooperative International Neuromuscular Research Group (CINRG) and evaluated drug-related effects of vamorolone on motor outcomes and corticosteroid-associated safety concerns. The study was carried out in Canada, US, UK, Australia, Sweden, and Israel, from 2016 to 2019. This report covers the initial 24-week trial and the first 12 months of the VBP15-LTE trial (total treatment period 18 months). DMD trial participants (males, 4 to <7 years at entry) treated with 2.0 or 6.0 mg/kg/day vamorolone for the full 18-month period (n = 23) showed clinical improvement of all motor outcomes from baseline to month 18 (time to stand velocity, p = 0.012 [95% CI 0.010, 0.068 event/second]; run/walk 10 meters velocity, p < 0.001 [95% CI 0.220, 0.491 meters/second]; climb 4 stairs velocity, p = 0.001 [95% CI 0.034, 0.105 event/second]; 6-minute walk test, p = 0.001 [95% CI 31.14, 93.38 meters]; North Star Ambulatory Assessment, p < 0.001 [95% CI 2.702, 6.662 points]). Outcomes in vamorolone-treated DMD patients (n = 46) were compared to group-matched participants in the CINRG Duchenne Natural History Study (corticosteroid-naïve, n = 19; corticosteroid-treated, n = 68) over a similar 18-month period. Time to stand was not significantly different between vamorolone-treated and corticosteroid-naïve participants (p = 0.088; least squares [LS] mean 0.042 [95% CI -0.007, 0.091]), but vamorolone-treated participants showed significant improvement compared to group-matched corticosteroid-naïve participants for run/walk 10 meters velocity (p = 0.003; LS mean 0.286 [95% CI 0.104, 0.469]) and climb 4 stairs velocity (p = 0.027; LS mean 0.059 [95% CI 0.007, 0.111]). The vamorolone-related improvements were similar in magnitude to corticosteroid-related improvements. Corticosteroid-treated participants showed stunting of growth, whereas vamorolone-treated trial participants did not (p < 0.001; LS mean 15.86 [95% CI 8.51, 23.22]). Physician-reported incidences of adverse events (AEs) for Cushingoid appearance, hirsutism, weight gain, and behavior change were less for vamorolone than published incidences for prednisone and deflazacort. Key limitations to the study were the open-label design, and use of external comparators. CONCLUSIONS: We observed that vamorolone treatment was associated with improvements in some motor outcomes as compared with corticosteroid-naïve individuals over an 18-month treatment period. We found that fewer physician-reported AEs occurred with vamorolone than have been reported for treatment with prednisone and deflazacort, and that vamorolone treatment did not cause the stunting of growth seen with these corticosteroids. This Phase IIa study provides Class III evidence to support benefit of motor function in young boys with DMD treated with vamorolone 2.0 to 6.0 mg/kg/day, with a favorable safety profile. A Phase III RCT is underway to further investigate safety and efficacy. TRIAL REGISTRATION: Clinical trials were registered at www.clinicaltrials.gov, and the links to each trial are as follows (as provided in manuscript text): VBP15-002 [NCT02760264] VBP15-003 [NCT02760277] VBP15-LTE [NCT03038399].

Cardiac Na+ current regulation by pyridine nucleotides.

RATIONALE: Mutations in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) protein reduce cardiac Na+ current (I(Na)) and cause Brugada Syndrome (BrS). GPD1-L has >80% amino acid homology with glycerol-3-phosphate dehydrogenase, which is involved in NAD-dependent energy metabolism. OBJECTIVE: Therefore, we tested whether NAD(H) could regulate human cardiac sodium channels (Na(v)1.5). METHODS AND RESULTS: HEK293 cells stably expressing Na(v)1.5 and rat neonatal cardiomyocytes were used. The influence of NADH/NAD+ on arrhythmic risk was evaluated in wild-type or SCN5A(+/-) mouse heart. A280V GPD1-L caused a 2.48+/-0.17-fold increase in intracellular NADH level (P<0.001). NADH application or cotransfection with A280V GPD1-L resulted in decreased I(Na) (0.48+/-0.09 or 0.19+/-0.04 of control group, respectively; P<0.01), which was reversed by NAD+, chelerythrine, or superoxide dismutase. NAD+ antagonism of the Na+ channel downregulation by A280V GPD1-L or NADH was prevented by a protein kinase (PK)A inhibitor, PKAI(6-22). The effects of NADH and NAD+ were mimicked by a phorbol ester and forskolin, respectively. Increasing intracellular NADH was associated with an increased risk of ventricular tachycardia in wild-type mouse hearts. Extracellular application of NAD+ to SCN5A(+/-) mouse hearts ameliorated the risk of ventricular tachycardia. CONCLUSIONS: Our results show that Na(v)1.5 is regulated by pyridine nucleotides, suggesting a link between metabolism and I(Na). This effect required protein kinase C activation and was mediated by oxidative stress. NAD+ could prevent this effect by activating PKA. Mutations of GPD1-L may downregulate Na(v)1.5 by altering the oxidized to reduced NAD(H) balance.

Mutation in glycerol-3-phosphate dehydrogenase 1 like gene (GPD1-L) decreases cardiac Na+ current and causes inherited arrhythmias.

BACKGROUND: Brugada syndrome is a rare, autosomal-dominant, male-predominant form of idiopathic ventricular fibrillation characterized by a right bundle-branch block and ST elevation in the right precordial leads of the surface ECG. Mutations in the cardiac Na+ channel SCN5A on chromosome 3p21 cause approximately 20% of the cases of Brugada syndrome; most mutations decrease inward Na+ current, some by preventing trafficking of the channels to the surface membrane. We previously used positional cloning to identify a new locus on chromosome 3p24 in a large family with Brugada syndrome and excluded SCN5A as a candidate gene. METHODS AND RESULTS: We used direct sequencing to identify a mutation (A280V) in a conserved amino acid of the glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) gene. The mutation was present in all affected individuals and absent in >500 control subjects. GPD1-L RNA and protein are abundant in the heart. Compared with wild-type GPD1-L, coexpression of A280V GPD1-L with SCN5A in HEK cells reduced inward Na+ currents by approximately 50% (P<0.005). Wild-type GPD1-L localized near the cell surface to a greater extent than A280V GPD1-L. Coexpression of A280V GPD1-L with SCN5A reduced SCN5A cell surface expression by 31+/-5% (P=0.01). CONCLUSIONS: GPD1-L is a novel gene that may affect trafficking of the cardiac Na+ channel to the cell surface. A GPD1-L mutation decreases SCN5A surface membrane expression, reduces inward Na+ current, and causes Brugada syndrome.

Vascular matrix metalloproteinase-9 mediates the inhibition of myogenic reactivity in small arteries isolated from rats after short-term administration of relaxin.

UNLABELLED: During pregnancy and chronic relaxin administration to nonpregnant rats (for days), vascular MMP (matrix metalloproteinase)-2 is increased and mediates renal vasodilation, hyperfiltration, and inhibition of myogenic reactivity of small renal arteries. However, the renal vasodilatory actions of relaxin also occur after only several hours of hormone administration to nonpregnant rats, and we hypothesized a pivotal role for vascular MMP-2. Accordingly, we used gelatin zymography, which reveals not only vascular MMP-2, but also MMP-9 activity in small renal arteries isolated from rats administered recombinant human relaxin (rhRLX) or vehicle for 4-6 h. Furthermore, we tested whether myogenic reactivity is inhibited, and if so, whether the inhibition is mediated by increased vascular MMP-2. Surprisingly, we detected no significant difference in either pro or active MMP-2 in small renal arteries isolated from rhRLX and vehicle control treatment groups. In contrast, vascular MMP-9 was up-regulated by 70% (P < 0.0005 vs. vehicle). These results were completely unexpected and novel. MMP-9 protein expression was confined to the vascular smooth muscle. MMP-9, but not MMP-2 activity, was also increased in mesenteric arteries after short-term rhRLX administration (P < 0.005 and >0.05 vs. vehicle, respectively). Myogenic reactivity was inhibited in small renal arteries isolated from nonpregnant rats treated with rhRLX for 4-6 h (P < 0.01 vs. vehicle) and was completely restored by incubation with MMP-9, but not MMP-2 neutralizing antibodies in vitro. CONCLUSION: In contrast to chronic rhRLX administration, MMP-9 rather than MMP-2 plays a central role in the vasodilatory effect of short-term relaxin administration.

Evidence for local relaxin ligand-receptor expression and function in arteries.

Relaxin is a 6 kDa protein hormone produced by the corpus luteum and secreted into the blood during pregnancy in rodents and humans. Growing evidence indicates that circulating relaxin causes vasodilatation and increases in arterial compliance, which may be among its most important actions during pregnancy. Here we investigated whether there is local expression and function of relaxin and relaxin receptor in arteries of nonpregnant females and males. Relaxin-1 and its major receptor, Lgr7, mRNA are expressed in thoracic aortas, small renal and mesenteric arteries from mice and rats of both sexes, as well as in small renal arteries from female tammar wallabies (an Australian marsupial). Using available antibodies for rat and mouse Lgr7 receptor and rat relaxin, we also identified protein expression in arteries. Small renal arteries isolated from relaxin-1 gene-deficient mice demonstrate enhanced myogenic reactivity and decreased passive compliance relative to wild-type (WT) and heterozygous mice. Taken together, these findings reveal an arterial-derived, relaxin ligand-receptor system that acts locally to regulate arterial function.

Matrix metalloproteinase-2 activity, protein, mRNA, and tissue inhibitors in small arteries from pregnant and relaxin-treated nonpregnant rats.

Vascular gelatinase activity is essential for pregnancy- and relaxin (Rlx)-induced renal vasodilation and hyperfiltration in rats. The objective of this study was to further elucidate the mechanisms for the increase in vascular matrix metalloproteinase (MMP)-2 activity caused by pregnancy and Rlx. We first corroborated our earlier work by showing that pro- and active forms of MMP-2 were increased in small renal arteries from pregnant compared with virgin rats and Rlx-treated compared with vehicle-treated nonpregnant rats. We next investigated other artery types and showed that MMP-2 activity was upregulated in mesenteric arteries from pregnant rats (pro-MMP-2 by 50% and active MMP-2 by 40%, both P<0.05) and from Rlx-treated nonpregnant rats (pro-MMP-2 by 50% and active MMP-2 by 90%, both P<0.005) compared with their respective controls. To corroborate these results obtained by gelatin zymography, pro-MMP-2 protein was determined by Western analysis in the same small arteries. Pro-MMP-2 protein was increased in small renal arteries from pregnant compared with virgin rats and from Rlx- compared with vehicle-treated nonpregnant rats: pro-MMP-2-to-beta-actin ratio=0.29 vs. 0.21 (P<0.01) and 0.43 vs. 0.32 (P<0.005). Findings were similar for mesenteric arteries. MMP-2 mRNA as measured by real-time PCR was increased in small renal arteries from pregnant and Rlx-treated nonpregnant rats compared with their respective controls. There were no significant differences in tissue inhibitor of metalloproteinase (TIMP-1 or TIMP-2) activity by reverse zymography in small renal arteries. Thus increases in MMP-2 mRNA and protein expression are major factors contributing to increased MMP-2 activity in small arteries from pregnant and Rlx-treated nonpregnant rats.

Essential role for vascular gelatinase activity in relaxin-induced renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small arteries.

During pregnancy, relaxin stimulates nitric oxide (NO)-dependent renal vasodilation, hyperfiltration and reduced myogenic reactivity of small renal arteries via the endothelial ETB receptor subtype. Our objective in this study was to elucidate the mechanisms by which relaxin stimulates the endothelial ETB receptor/NO vasodilatory pathway. Using chronically instrumented conscious rats, we demonstrated that a specific peptide inhibitor of the gelatinases MMP-2 and -9, cyclic CTTHWGFTLC (cyclic CTT), but not the control peptide, STTHWGFTLS (STT), completely reversed renal vasodilation and hyperfiltration in relaxin-treated rats. Comparable findings were observed with a structurally different and well-established, general antagonist of MMPs, GM6001. In contrast, phosphoramidon, an inhibitor of endothelin-converting enzyme, did not significantly change the renal vasodilatory response to relaxin administration. When small renal arteries were incubated with either of the general MMP inhibitors, GM6001 or TIMP-2 (tissue inhibitor of MMP), or with the specific gelatinase inhibitor, cyclic CTT, the reduced myogenic reactivity of these blood vessels from relaxin-treated nonpregnant and midterm pregnant rats was totally abolished. Moreover, a neutralizing antibody specific for MMP-2 completely abrogated the reduced myogenic reactivity of small renal arteries from relaxin-treated nonpregnant and midterm pregnant rats. In contrast, phosphoramidon did not significantly affect the reduction in myogenic reactivity. Using gelatin zymography, we showed increased pro and active MMP-2 activity in small renal arteries from relaxin-treated nonpregnant and midterm pregnant rats relative to the control animals. Thus, inhibitors of MMPs in general and of gelatinases in particular reverse the renal vascular changes induced by pregnancy or relaxin administration to nonpregnant rats. Finally, the typical reduction in myogenic reactivity of small renal arteries from relaxin-treated nonpregnant rats was absent in ETB receptor-deficient rats, despite an increase in vascular MMP-2 activity. These results indicate an essential role for vascular gelatinase, which is in series with, and upstream of, the endothelial ETB receptor/NO signaling pathway in the renal vasodilatory response to relaxin and pregnancy.

Evidence against the hypothesis that endothelial endothelin B receptor expression is regulated by relaxin and pregnancy.

The endothelial endothelin B (ET(B)) receptor subtype is critical for renal vasodilation induced by relaxin in nonpregnant rats and during pregnancy (the latter via endogenous circulating relaxin). Here we tested whether expression of vascular ET(B) receptor protein is regulated by relaxin. Small renal arteries were harvested from virgin and midterm pregnant rats as well as nonpregnant rats that were administered recombinant human relaxin (rhRLX) at 4 mug/h or vehicle for 5 d or 4-6 h. Small renal arteries dissected from additional virgin rats were incubated in vitro with rhRLX or vehicle for 3 h at 37 C. ET(B) expression was also evaluated in cultured human endothelial cells: aortic, coronary, umbilical vein, and dermal microvascular endothelial cells. Cells were incubated for 4, 8, or 24 h with rhRLX (5, 1, or 0.1 ng/ml) or vehicle. ET(B) protein expression in arteries and cells was evaluated by Western analysis. No regulation of ET(B) expression was observed in small renal arteries in any of the experimental protocols, nor was there an increase in the vasorelaxation response to ET-3 in small renal arteries incubated in vitro with rhRLX. rhRLX only sporadically altered ET(B) expression in human coronary artery endothelial cells and human umbilical vein endothelial cells at certain time points or doses, and no regulation was observed in human aortic endothelial cells or human dermal microvascular endothelial cells. These results suggest that regulation of ET(B) receptor protein has little or no role in relaxin stimulation of the endothelial ET(B)/nitric oxide vasodilatory pathway.

Role of relaxin in maternal renal vasodilation of pregnancy.

The remarkable hemodynamic changes of normal pregnancy are briefly reviewed. In addition, new findings and current concepts related to the underlying hormonal and molecular mechanisms are presented. Finally, work that is in progress as well as future directions is briefly discussed.

Renal handling of homocysteine during normal pregnancy and preeclampsia.

OBJECTIVE: Maternal plasma homocysteine decreases in normal pregnancy and is significantly increased in preeclampsia. The goal of this study was to investigate the role of the maternal kidney in the changes of plasma homocysteine during normal pregnancy and preeclampsia. METHODS: Plasma and 24-hour urine samples were collected in the same women before, during (first, second, and third trimesters), and after normal pregnancy; and in a separate cross-sectional study of normal pregnant, preeclamptic and nonpregnant women and homocysteine concentrations were measured. RESULTS: Longitudinally, maternal plasma homocysteine decreased significantly by the first trimester compared with prepregnancy and postpartum levels (5.6 +/- 1.8 versus 6.8 +/- 0.5 and 7.4 +/- 0.4 microM, respectively, P<.05 by analysis of variance) and paralleled a significant increase in the renal clearance of homocysteine (2.9 +/- 0.4 versus 1.8 +/- 0.2 and 1.6 +/- 0.2 L/24 hours, respectively, P<.001). In addition, plasma homocysteine was significantly elevated in preeclampsia compared with normal pregnancy (4.4 +/- 0.6 versus 3.2 +/- 0.2 microM, P<.04); however, renal clearance was not different (1.2 +/- 0.1 versus 1.0 +/- 0.1 L/24 hours, P=.55). CONCLUSION: Increases in renal clearance contribute to the decrease in plasma homocysteine during normal pregnancy. However, changes in renal handling do not appear to contribute to the increase in plasma homocysteine in preeclampsia.

WITHDRAWN: Sex-differences in sodium/calcium exchange expression is a determinant of the arrhythmia phenotype in pre-pubertal rabbit hearts with Long QT type 2.

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