Influence of treatment of vulvovaginal atrophy with intravaginal prasterone on the male partner.

OBJECTIVE: The aim was to analyze the opinion of the male partner of women treated for vulvovaginal atrophy (VVA) with intravaginal 0.50% DHEA (prasterone), thus providing information on both members of the couple. METHODS: On a voluntary basis, in a prospective, randomized, double-blind and placebo-controlled phase-III clinical trial, the male partner filled a questionnaire at baseline and at 12 weeks stating his observations related to his penis and intercourse before and after VVA treatment. RESULTS: Sixty-six men having a partner treated with intravaginal DHEA and 34 others having a partner treated with placebo answered the questionnaires. Concerning the feeling of vaginal dryness of their female partner, the severity score following DHEA treatment improved by 81% (0.76 units) over placebo (p = 0.0347). Thirty-six percent of men having a partner treated with DHEA did not feel the vaginal dryness of the partner at the end of treatment compared to 7.8% in the placebo group. When analyzing the situation at 12 weeks compared to baseline, an improved score of 1.09 units was the difference found for the DHEA group compared to 0.76 for the placebo group (p = 0.05 vs. placebo). In the DHEA group, 38% of men scored very improved compared to 18% in the placebo group. No adverse event has been reported. CONCLUSION: The male partner had a very positive evaluation of the treatment received by his female partner.

The lipid-lowering effects of lomitapide are unaffected by adjunctive apheresis in patients with homozygous familial hypercholesterolaemia - a post-hoc analysis of a Phase 3, single-arm, open-label trial.

OBJECTIVE: Lomitapide (a microsomal triglyceride transfer protein inhibitor) is an adjunctive treatment for homozygous familial hypercholesterolaemia (HoFH), a rare genetic condition characterised by elevated low-density lipoprotein-cholesterol (LDL-C), and premature, severe, accelerated atherosclerosis. Standard of care for HoFH includes lipid-lowering drugs and lipoprotein apheresis. We conducted a post-hoc analysis using data from a Phase 3 study to assess whether concomitant apheresis affected the lipid-lowering efficacy of lomitapide. METHODS: Existing lipid-lowering therapy, including apheresis, was to remain stable from Week -6 to Week 26. Lomitapide dose was escalated on the basis of individual safety/tolerability from 5 mg to 60 mg a day (maximum). The primary endpoint was mean percent change in LDL-C from baseline to Week 26 (efficacy phase), after which patients remained on lomitapide through Week 78 for safety assessment and further evaluation of efficacy. During this latter period, apheresis could be adjusted. We analysed the impact of apheresis on LDL-C reductions in patients receiving lomitapide. RESULTS: Of the 29 patients that entered the efficacy phase, 18 (62%) were receiving apheresis at baseline. Twenty-three patients (13 receiving apheresis) completed the Week 26 evaluation. Of the six patients who discontinued in the first 26 weeks, five were receiving apheresis. There were no significant differences in percent change from baseline of LDL-C at Week 26 in patients treated (-48%) and not treated (-55%) with apheresis (p = 0.545). Changes in Lp(a) levels were modest and not different between groups (p = 0.436). CONCLUSION: The LDL-C lowering efficacy of lomitapide is unaffected by lipoprotein apheresis.

Utility of ¹8fluoro-deoxyglucose positron emission tomography for prognosis and response assessments in a phase 2 study of romidepsin in patients with relapsed or refractory peripheral T-cell lymphoma.

BACKGROUND: For patients with peripheral T-cell lymphoma (PTCL), the value of (18)fluoro-deoxyglucose positron emission tomography (FDG-PET) scans for assessing prognosis and response to treatment remains unclear. The utility of FDG-PET, in addition to conventional radiology, was examined as a planned exploratory end point in the pivotal phase 2 trial of romidepsin for the treatment of relapsed/refractory PTCL. PATIENTS AND METHODS: Patients received romidepsin at a dose of 14 mg/m(2) on days 1, 8, and 15 of 28-day cycles. The primary end point was the rate of confirmed/unconfirmed complete response (CR/CRu) as assessed by International Workshop Criteria (IWC) using conventional radiology. For the exploratory PET end point, patients with at least baseline FDG-PET scans were assessed by IWC + PET criteria. RESULTS: Of 130 patients, 110 had baseline FDG-PET scans, and 105 were PET positive at baseline. The use of IWC + PET criteria increased the objective response rate to 30% compared with 26% by conventional radiology. Durations of response were well differentiated by both conventional radiology response criteria [CR/CRu versus partial response (PR), P = 0.0001] and PET status (negative versus positive, P < 0.0001). Patients who achieved CR/CRu had prolonged progression-free survival (PFS, median 25.9 months) compared with other response groups (P = 0.0007). Patients who achieved PR or stable disease (SD) had similar PFS (median 7.2 and 6.3 months, respectively, P = 0.6427). When grouping PR and SD patients by PET status, patients with PET-negative versus PET-positive disease had a median PFS of 18.2 versus 7.1 months (P = 0.0923). CONCLUSIONS: Routine use of FDG-PET does not obviate conventional staging, but may aid in determining prognosis and refine response assessments for patients with PTCL, particularly for those who do not achieve CR/CRu by conventional staging. The optimal way to incorporate FDG-PET scans for patients with PTCL remains to be determined. TRIAL REGISTRATION: NCT00426764.

Decreased efficacy of twice-weekly intravaginal dehydroepiandrosterone on vulvovaginal atrophy.

OBJECTIVE: While daily intravaginal administration of 0.50% (6.5 mg) dehydroepiandrosterone (DHEA, prasterone) for 12 weeks has shown clinically and statistically significant effects on moderate to severe (MS) dyspareunia as the most bothersome symptom (MBS), the present study analyzes the effect of a reduced dosing regimen on MBS vaginal dryness. METHOD: Daily intravaginal 0.50% prasterone for 2 weeks followed by twice weekly for 10 weeks versus placebo. RESULTS: Maximal beneficial changes in vaginal parabasal and superficial cells and pH were observed at 2 weeks as observed for intravaginal 10 µg estradiol (E2). This was followed by a decrease or lack of efficacy improvement after switching to twice-weekly dosing. The decrease in percentage of parabasal cells, increase in percentage of superficial cells and decrease in vaginal pH were all highly significant (p < 0.0001 to 0.0002 over placebo) at 12 weeks. In parallel, the statistical significance over placebo (p value) on MBS vaginal dryness at 6 weeks was 0.09 followed by an increase to 0.198 at 12 weeks. For MBS dyspareunia, the p value of 0.008 at 6 weeks was followed by a p value of 0.077 at 12 weeks, thus illustrating a decrease of efficacy at the lower dosing regimen. The improvements of vaginal secretions, color, epithelial integrity and epithelial surface thickness were observed at a p value < 0.01 or 0.05 over placebo at 2 weeks, with a similar or loss of statistical difference compared to placebo at later time intervals. No significant adverse event was observed. Vaginal discharge related to the melting of Witepsol was reported in 1.8% of subjects. CONCLUSION: The present data show that daily dosing with 0.50% DHEA for 2 weeks followed by twice-weekly dosing is a suboptimal treatment of the symptoms/signs of vulvovaginal atrophy resulting from a substantial loss of the efficacy achieved at daily dosing.

Intravaginal dehydroepiandrosterone (prasterone), a highly efficient treatment of dyspareunia.

OBJECTIVE: To examine the effect of intravaginal dehydroepiandrosterone (DHEA) on pain at sexual activity (dyspareunia) identified as the most bothersome symptom of vaginal atrophy in postmenopausal women at both screening and day 1. METHODS: This prospective, randomized, double-blind and placebo-controlled phase III clinical trial studied the effect of prasterone (DHEA) applied locally in the vagina on the severity of dyspareunia in 114 postmenopausal women who had identified dyspareunia as their most bothersome symptom of vaginal atrophy, while meeting the criteria for superficial cells ≤ 5% and pH > 5.0 at both screening and day 1. RESULTS: At the standard duration of 12 weeks of treatment, increasing doses of 0.25%, 0.5% and 1.0% DHEA decreased the percentage of parabasal cells by 48.6  ±â€Š 6.78%, 42.4  ±  7.36% and 54.9  ±â€Š 6.60% (p < 0.0001 vs. placebo for all) with no change with placebo (p = 0.769). The effects on superficial cells and pH were also highly significant compared to placebo at all DHEA doses. The severity score of pain at sexual activity decreased by 0.5, 1.4, 1.6 and 1.4 units in the placebo and 0.25%, 0.5% and 1.0% DHEA groups, respectively, with the p value of differences from placebo ranging from 0.0017 to < 0.0001. CONCLUSIONS: Intravaginal DHEA, through local estrogen and androgen formation, causes a rapid and highly efficient effect on pain at sexual activity without systemic exposure of the other tissues, thus avoiding the recently reported systemic effects of estrogens.

Selective gamma-ketoaldehyde scavengers protect Nav1.5 from oxidant-induced inactivation.

The cardiac sodium channel (SCN5A, Na(V)1.5) is a key determinant of electrical impulse conduction in cardiac tissue. Acute myocardial infarction leads to diminished sodium channel availability, both because of decreased channel expression and because of greater inactivation of channels already present. Myocardial infarction leads to significant increases in reactive oxygen species and their downstream effectors including lipoxidation products. The effects of reactive oxygen species on Na(V)1.5 function in whole hearts can be modeled in cultured myocytes, where oxidants shift the availability curve of I(Na) to hyperpolarized potentials, decreasing cardiac sodium current at the normal activation threshold. We recently examined potential mediators of the oxidant-induced inactivation and found that one specific lipoxidation product, the isoketals, recapitulated the effects of oxidant on sodium currents. Isoketals are highly reactive gamma-ketoaldehydes formed by the peroxidation of arachidonic acid that covalently modify the lysine residues of proteins. We now confirm that exposure to oxidants induces lipoxidative modification of Na(V)1.5 and that the selective isoketal scavengers block voltage-dependent changes in sodium current by the oxidant tert-butylhydroperoxide, both in cells heterologously expressing Na(V)1.5 and in a mouse cardiac myocyte cell line (HL-1). Thus, inhibition of this lipoxidative modification pathway is sufficient to protect the sodium channel from oxidant induced inactivation and suggests the potential use of isoketal scavengers as novel therapeutics to prevent arrhythmogenesis during myocardial infarction.

Development of a large-scale de-identified DNA biobank to enable personalized medicine.

Our objective was to develop a DNA biobank linked to phenotypic data derived from an electronic medical record (EMR) system. An "opt-out" model was implemented after significant review and revision. The plan included (i) development and maintenance of a de-identified mirror image of the EMR, namely, the "synthetic derivative" (SD) and (ii) DNA extracted from discarded blood samples and linked to the SD. Surveys of patients indicated general acceptance of the concept, with only a minority ( approximately 5%) opposing it. As a result, mechanisms to facilitate opt-out included publicity and revision of a standard "consent to treatment" form. Algorithms for sample handling and procedures for de-identification were developed and validated in order to ensure acceptable error rates (<0.3 and <0.1%, respectively). The rate of sample accrual is 700-900 samples/week. The advantages of this approach are the rate of sample acquisition and the diversity of phenotypes based on EMRs.

Molecular basis of isolated cardiac conduction disease.

Cardiac conduction disorders are among the most common rhythm disturbances causing disability in millions of people worldwide and necessitating pacemaker implantation. Isolated cardiac conduction disease (ICCD) can affect various regions within the heart, and therefore the clinical features also vary from case to case. Typically, it is characterized by progressive alteration of cardiac conduction through the atrioventricular node, His-Purkinje system, with right or left bundle branch block and QRS widening. In some instances, the disorder may progress to complete atrioventricular block, with syncope and even death. While the role of genetic factors in conduction disease has been suggested as early as the 1970s, it was only recently that specific genetic loci have been reported. Multiple mutations in the gene encoding for the cardiac voltage-gated sodium channel (SCN5A), which plays a fundamental role in the initiation, propagation, and maintenance of normal cardiac rhythm, have been linked to conduction disease, allowing for genotype-phenotype correlation. The electrophysiological characterization of heterologously expressed mutant Na+ channels has revealed gating defects that consistently lead to a loss of channel function. However, studies have also revealed significant overlap between aberrant rhythm phenotypes, and single mutations have been identified that evoke multiple distinct rhythm disorders with common gating lesions. These new insights highlight the complexities involved in linking single mutations, ion-channel behavior, and cardiac rhythm but suggest that interplay between multiple factors could underlie the manifestation of the disease phenotype.

Inherited sodium channelopathies: novel therapeutic and proarrhythmic molecular mechanisms.

Voltage-gated sodium (Na) channels, transmembrane proteins that produce the ionic current responsible for the rapid upstroke of the cardiac action potential, are key elements required for rapid conduction through the myocardium and maintenance of the cardiac rhythm. The exquisite sensitivity of the cardiac rhythm to Na channel function is manifest in the proarrhythmic complications of "antiarrhythmic" Na channel blockade in patients with myocardial ischemia. More recently, studies of inherited single amino acid substitutions in Na channels have unveiled a remarkable array of cardiac rhythm disturbances, as well as surprising pharmacologic sensitivities. Hence, the sodium channelopathies are providing new molecular insights into mechanisms whereby altered ion channel behavior precipitates cardiac arrhythmias.

Gating-dependent mechanisms for flecainide action in SCN5A-linked arrhythmia syndromes.

BACKGROUND: Mutations in the cardiac sodium (Na) channel gene (SCN5A) give rise to the congenital long-QT syndrome (LQT3) and the Brugada syndrome. Na channel blockade by antiarrhythmic drugs improves the QT interval prolongation in LQT3 but worsens the Brugada syndrome ST-segment elevation. Although Na channel blockade has been proposed as a treatment for LQT3, flecainide also evokes "Brugada-like" ST-segment elevation in LQT3 patients. Here, we examine how Na channel inactivation gating defects in LQT3 and Brugada syndrome elicit proarrhythmic sensitivity to flecainide. METHODS AND RESULTS: We measured whole-cell Na current (I(Na)) from tsA-201 cells transfected with DeltaKPQ, a LQT3 mutation, and 1795insD, a mutation that provokes both the LQT3 and Brugada syndromes. The 1795insD and DeltaKPQ channels both exhibited modified inactivation gating (from the closed state), thus potentiating tonic I(Na) block. Flecainide (1 micromol/L) tonic block was only 16.8+/-3.0% for wild type but was 58.0+/-6.0% for 1795insD (P<0.01) and 39.4+/-8.0% (P<0.05) for DeltaKPQ. In addition, the 1795insD mutation delayed recovery from inactivation by enhancing intermediate inactivation, with a 4-fold delay in recovery from use-dependent flecainide block. CONCLUSIONS: We have linked 2 inactivation gating defects ("closed-state" fast inactivation and intermediate inactivation) to flecainide sensitivity in patients carrying LQT3 and Brugada syndrome mutations. These results provide a mechanistic rationale for predicting proarrhythmic sensitivity to flecainide based on the identification of specific SCN5A inactivation gating defects.

A novel extracellular calcium sensing mechanism in voltage-gated potassium ion channels.

Potassium (K(+)) channels influence neurotransmitter release, burst firing rate activity, pacing, and critical dampening of neuronal circuits. Internal and external factors that further modify K(+) channel function permit fine-tuning of neuronal circuits. Human ether-à-go-go-related gene (HERG) K(+) channels are unusually sensitive to external calcium concentration ([Ca(2+)](o)). Small changes in [Ca(2+)](o) shift the voltage dependence of channel activation to more positive membrane potentials, an effect that cannot be explained by nonspecific surface charge screening or channel pore block. The HERG-calcium concentration-response relationship spans the physiological range for [Ca(2+)](o). The modulatory actions of calcium are attributable to differences in the Ca(2+) affinity between rested and activated channels. Adjacent extracellular, negatively charged amino acids (E518 and E519) near the S4 voltage sensor influence both channel gating and Ca(2+) dependence. Neutralization of these charges had distinct effects on channel gating and calcium sensitivity. A change in the degree of energetic coupling between these amino acids on transition from closed to activated channel states reveals movement in this region during channel gating and defines a molecular mechanism for protein state-dependent ligand interactions. The results suggest a novel extracellular [Ca(2+)](o) sensing mechanism coupled to allosteric changes in channel gating and a mechanism for fine-tuning cell repolarization.

A sodium-channel mutation causes isolated cardiac conduction disease.

Cardiac conduction disorders slow the heart rhythm and cause disability in millions of people worldwide. Inherited mutations in SCN5A, the gene encoding the human cardiac sodium (Na+) channel, have been associated with rapid heart rhythms that occur suddenly and are life-threatening; however, a chief function of the Na+ channel is to initiate cardiac impulse conduction. Here we provide the first functional characterization of an SCN5A mutation that causes a sustained, isolated conduction defect with pathological slowing of the cardiac rhythm. By analysing the SCN5A coding region, we have identified a single mutation in five affected family members; this mutation results in the substitution of cysteine 514 for glycine (G514C) in the channel protein. Biophysical characterization of the mutant channel shows that there are abnormalities in voltage-dependent 'gating' behaviour that can be partially corrected by dexamethasone, consistent with the salutary effects of glucocorticoids on the clinical phenotype. Computational analysis predicts that the gating defects of G514C selectively slow myocardial conduction, but do not provoke the rapid cardiac arrhythmias associated previously with SCN5A mutations.

The cardiac sodium channel: gating function and molecular pharmacology.

Cardiac sodium (Na) channels are dynamic molecules that undergo rapid structural changes in response to the changing electrical field in the myocardium. Inherited mutations in SCN5A, the gene encoding the cardiac Na channel, provoke life-threatening cardiac arrhythmias, often by modifying these voltage-dependent conformational changes. These disorders (i.e. the long QT syndrome and Brugada syndrome) may serve as valuable models for understanding the mechanistic linkages between Na channel dysfunction and cardiac arrhythmias in more common, acquired conditions such as cardiac ischemia. In addition, the balance between therapeutic and adverse effects from Na channel blockade by antiarrhythmic compounds may be shifted by subtle alterations in Na channel function. This review examines recent studies that tie key loci in the Na channel primary sequence to its dynamic function, while examining the emerging themes linking Na channel structure, function, and pharmacology to inherited and acquired disorders of cardiac excitability.

Probing the interaction between inactivation gating and Dd-sotalol block of HERG.

Potassium channels encoded by HERG underlie I:(Kr), a sensitive target for most class III antiarrhythmic drugs, including methanesulfonanilides such as Dd-sotalol. Recently it was shown that these drugs are trapped in the channel as it closes during hyperpolarization. At the same time, HERG channels rapidly open and inactivate when depolarized, and methanesulfonanilide block is known to develop in a use-dependent manner, suggesting a potential role for inactivation in drug binding. However, the role of HERG inactivation in class III drug action is uncertain: pore mutations that remove inactivation reduce block, yet many of these mutations also modify the channel permeation properties and could alter drug affinity through gating-independent mechanisms. In the present study, we identify a definitive role for inactivation gating in Dd-sotalol block of HERG, using interventions complementary to mutagenesis. These interventions (addition of extracellular Cd(2+), removal of extracellular Na(+)) modify the voltage dependence of inactivation but not activation. In normal extracellular solutions, block of HERG current by 300 micromol/L Dd-sotalol reached 80% after a 10-minute period of repetitive depolarization to +20 mV. Maneuvers that impeded steady-state inactivation also reduced Dd-sotalol block of HERG: 100 micromol/L Cd(2+) reduced steady-state block to 55% at +20 mV (P:<0.05); removing extracellular Na(+) reduced block to 44% (P:<0.05). An inactivation-disabling mutation (G628C-S631C) reduced Dd-sotalol block to only 11% (P:<0.05 versus wild type). However, increasing the rate of channel inactivation by depolarizing to +60 mV reduced Dd-sotalol block to 49% (P:<0.05 versus +20 mV), suggesting that the drug does not primarily bind to the inactivated state. Coexpression of MiRP1 with HERG had no effect on inactivation gating and did not modify Dd-sotalol block. We postulate that Dd-sotalol accesses its receptor in the open pore, and the drug-receptor interaction is then stabilized by inactivation. Whereas deactivation traps the bound methanesulfonanilide during hyperpolarization, we propose that HERG inactivation stabilizes the drug-receptor interaction during membrane depolarization.

A structural rearrangement in the sodium channel pore linked to slow inactivation and use dependence.

Voltage-gated sodium (Na(+)) channels are a fundamental target for modulating excitability in neuronal and muscle cells. When depolarized, Na(+) channels may gradually enter long-lived, slow-inactivated conformational states, causing a cumulative loss of function. Although the structural motifs that underlie transient, depolarization-induced Na(+) channel conformational states are increasingly recognized, the conformational changes responsible for more sustained forms of inactivation are unresolved. Recent studies have shown that slow inactivation components exhibiting a range of kinetic behavior (from tens of milliseconds to seconds) are modified by mutations in the outer pore P-segments. We examined the state-dependent accessibility of an engineered cysteine in the domain III, P-segment (F1236C; rat skeletal muscle) to methanethiosulfonate-ethylammonium (MTSEA) using whole-cell current recordings in HEK 293 cells. F1236C was reactive with MTSEA applied from outside, but not inside the cell, and modification was markedly increased by depolarization. Depolarized F1236C channels exhibited both intermediate (I(M); tau approximately 30 ms) and slower (I(S); tau approximately 2 s) kinetic components of slow inactivation. Trains of brief, 5-ms depolarizations, which did not induce slow inactivation, produced more rapid modification than did longer (100 ms or 6 s) pulse widths, suggesting both the I(M) and I(S) kinetic components inhibit depolarization-induced MTSEA accessibility of the cysteine side chain. Lidocaine inhibited the depolarization-dependent sulfhydryl modification induced by sustained (100 ms) depolarizations, but not by brief (5 ms) depolarizations. We conclude that competing forces influence the depolarization-dependent modification of the cysteine side chain: conformational changes associated with brief periods of depolarization enhance accessibility, whereas slow inactivation tends to inhibit the side chain accessibility. The findings suggest that slow Na(+) channel inactivation and use-dependent lidocaine action are linked to a structural rearrangement in the outer pore.

Enhanced Na(+) channel intermediate inactivation in Brugada syndrome.

Brugada syndrome is an inherited cardiac disease that causes sudden death related to idiopathic ventricular fibrillation in a structurally normal heart. The disease is characterized by ST-segment elevation in the right precordial ECG leads and is frequently accompanied by an apparent right bundle-branch block. The biophysical properties of the SCN5A mutation T1620M associated with Brugada syndrome were examined for defects in intermediate inactivation (I:(M)), a gating process in Na(+) channels with kinetic features intermediate between fast and slow inactivation. Cultured mammalian cells expressing T1620M Na(+) channels in the presence of the human beta(1) subunit exhibit enhanced intermediate inactivation at both 22 degrees C and 32 degrees C compared with wild-type recombinant human heart Na(+) channels (WT-hH1). Our findings support the hypothesis that Brugada syndrome is caused, in part, by functionally reduced Na(+) current in the myocardium due to an increased proportion of Na(+) channels that enter the I:(M) state. This phenomenon may contribute significantly to arrhythmogenesis in patients with Brugada syndrome. The full text of this article is available at http://www.circresaha.org.

Two distinct congenital arrhythmias evoked by a multidysfunctional Na(+) channel.

The congenital long-QT syndrome (LQT3) and the Brugada syndrome are distinct, life-threatening rhythm disorders linked to autosomal dominant mutations in SCN5A, the gene encoding the human cardiac Na(+) channel. It is believed that these two syndromes result from opposite molecular effects: LQT3 mutations induce a gain of function, whereas Brugada syndrome mutations reduce Na(+) channel function. Paradoxically, an inherited C-terminal SCN5A mutation causes affected individuals to manifest electrocardiographic features of both syndromes: QT-interval prolongation (LQT3) at slow heart rates and distinctive ST-segment elevations (Brugada syndrome) with exercise. In the present study, we show that the insertion of the amino acid 1795insD has opposite effects on two distinct kinetic components of Na(+) channel gating (fast and slow inactivation) that render unique, simultaneous effects on cardiac excitability. The mutation disrupts fast inactivation, causing sustained Na(+) current throughout the action potential plateau and prolonging cardiac repolarization at slow heart rates. At the same time, 1795insD augments slow inactivation, delaying recovery of Na(+) channel availability between stimuli and reducing the Na(+) current at rapid heart rates. Our findings reveal a novel molecular mechanism for the Brugada syndrome and identify a new dual mechanism whereby single SCN5A mutations may evoke multiple cardiac arrhythmia syndromes by influencing diverse components of Na(+) channel gating function. The full text of this article is available at http://www.circresaha.org.