Novel intranasal treatment for anxiety disorders using amiloride, an acid-sensing ion channel antagonist: Pharmacokinetic modeling and simulation.

OBJECTIVE: To develop a physiologically based pharmacokinetic (PBPK) model for amiloride, an acid-sensing ion channel (ASIC) antagonist, and to simulate its pharmacokinetics in plasma and the central nervous system following intranasal administration in a virtual human population. MATERIALS AND METHODS: We first developed a PBPK model of amiloride after oral administration and optimized the model using data from five clinical studies. Next, we added a nasal compartment to the amiloride oral PBPK model and parameterized using data from previous clinical studies. We simulated amiloride's pharmacokinetics in plasma, brain, and cerebrospinal fluid (CSF) after intranasal administration of amiloride at various doses in a virtual human population. RESULTS: The target amiloride concentration in the central nervous system required for maximal ASIC inhibition was achieved with a 75-mg intranasal amiloride dose. However, this finding is based on simulations performed using a mathematical model and needs to be further validated with appropriate clinical data. CONCLUSION: The nasal PBPK model of amiloride could be used to design future clinical studies and allow for successful clinical translation of intranasal amiloride formulation.

The Effect of Amiloride on Proteinuria in Patients with Proteinuric Kidney Disease.

INTRODUCTION: Proteinuric kidney diseases share an aggressive clinical course of developing end-stage renal disease. However, the treatment is limited. Amiloride, an epithelial sodium channel (ENaC) inhibitor, was reported to reduce proteinuria in animal studies and case reports independent of ENaC inhibition. We hypothesized that amiloride not triamterene (an analog of amiloride) would reduce proteinuria in the patients with proteinuric kidney disease. METHODS: Patients with proteinuria >1.0 g/day and estimated glomerular filtration rate (eGFR) >30 mL/min/1.73 m2 on a maximum tolerable dose of angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers were randomized to receive amiloride 5 mg twice daily or triamterene 50 mg twice daily for 8 weeks, followed by 4 weeks of washout, and then crossed over to the other drug for 8 weeks. The primary outcome was 24-h urine protein reduction. Secondary outcomes were changes in body weight, blood pressure (BP), serum potassium, and eGFR. Data were analyzed by analysis of variance. RESULTS: A total of 12 patients completed the study. Amiloride reduced 24-h urine protein by 38.7% (p = 0.002) and decreased systolic BP by 12.3 mm Hg (p = 0.04). Interestingly, triamterene reduced 24 h urine protein as well, by 32.8% (p = 0.02). Triamterene lowered eGFR by 9.0 mL/min/1.73 m2 (p = 0.007), but it was reversible. The average weight change was insignificant in both groups (p = 0.40 and 0.34 respectively). Three patients withdrew the study due to hyperkalemia. CONCLUSIONS: Both amiloride and triamterene significantly reduced proteinuria in patients with proteinuric kidney disease. The anti-proteinuric effect was additive to renin-angiotensin-aldosterone system (RAAS) blockade, given all patients were on RAAS blockade. Hyperkalemia was a safety concern. Larger trials might be needed to examine the antiproteinuric effects of ENaC inhibitors.

[Antiproteinuric action of amiloride in paediatric patient with corticoresistant nephrotic syndrome]. / Acción antiproteinúrica del amiloride en el paciente pediátrico con síndrome nefrótico corticorresistente.

INTRODUCCION: In nephrotic syndrome, increased podocyturia accompanies pathologic proteinuria. The therapeutic regimen with enalapril, losartan and amiloride could reduce both variables. OBJETIVES: Evaluate the anti-proteinuric effect of 2 non-immunological therapeutic regimens, the quantitative relationship between podocyturia and proteinuria. MATERIAL AND METHODS: We included children aged 4 to 12 years with corticoresistant nephrotic syndrome, using 2 different schemes: group A, enalapril+losartan, and group B, enalapril+losartan+amiloride. RESULTS: In group A, 17 patients completed the study, the initial mean proteinuria was 39mg/m2/h and mean proteinuria at the end was 24mg/m2/h, while in group B 14 patients were treated and the initial average proteinuria was 36mg/m2/h and the end average proteinuria was 13mg/m2/h. The paired T test showed significant differences in the decrease in proteinuria, for patients in group B without variation in podocyturia. The 2 factors associated with an increase in proteinuria were podocyturia and the time elapsed from the diagnosis of cortico-resistant nephrotic syndrome to the start of treatment anti-proteinuric. CONCLUSIONS: The use of amiloride decreased proteinuria, without significantly modifying podocyturia; we did not observe a positive relationship between both variables.

Effectiveness of chlorthalidone/amiloride versus losartan in patients with stage I hypertension and diabetes mellitus: results from the PREVER-treatment randomized controlled trial.

AIMS: To compare the blood pressure (BP)-lowering efficacy of a chlorthalidone/amiloride combination pill with losartan, during initial management of JNC 7 Stage I hypertension in patients with type 2 diabetes mellitus. METHODS: In an a priori subgroup analysis of a randomized, double-blind, controlled trial, volunteers aged 30-70 years, with stage I hypertension and diabetes mellitus, were randomized to 12.5/2.5 mg of chlorthalidone/amiloride (N = 47) or 50 mg of losartan (N = 50), and followed for 18 months in 21 clinical centers. If BP remained uncontrolled after three months, study medication dose was doubled, and if uncontrolled after six months, amlodipine (5 and 10 mg) and propranolol (40 and 80 mg BID) were added as open label drugs in a progressive fashion. RESULTS: Systolic BP decreased to a greater extent in participants allocated to diuretics compared to losartan (P < 0.001). After 18 months of follow-up, systolic BP was 128.4 ± 10.3 mmHg in the diuretic group versus 133.5 ± 8.0 in the losartan group (P < 0.01). In the diuretic group, 36 out of 43 participants (83.7%) had a JNC 7 normal BP, compared to 31/47 (66%) in the losartan group (P = 0.089). Serum cholesterol was higher in the diuretic arm at the end of the trial. Other biochemical parameters and reports of adverse events did not differ by treatment. CONCLUSIONS: Treatment of hypertension based on a combination of chlorthalidone and amiloride is more effective for BP lowering compared to losartan in patients with diabetes mellitus and hypertension. TRIAL REGISTRATION: Clinical trials registration number: NCT00971165.

An Inhibitor of the Sodium-Hydrogen Exchanger-1 (NHE-1), Amiloride, Reduced Zinc Accumulation and Hippocampal Neuronal Death after Ischemia.

Acidosis in the brain plays an important role in neuronal injury and is a common feature of several neurological diseases. It has been reported that the sodium-hydrogen exchanger-1 (NHE-1) is a key mediator of acidosis-induced neuronal injury. It modulates the concentration of intra- and extra-cellular sodium and hydrogen ions. During the ischemic state, excessive sodium ions enter neurons and inappropriately activate the sodium-calcium exchanger (NCX). Zinc can also enter neurons through voltage-gated calcium channels and NCX. Here, we tested the hypothesis that zinc enters the intracellular space through NCX and the subsequent zinc accumulation induces neuronal cell death after global cerebral ischemia (GCI). Thus, we conducted the present study to confirm whether inhibition of NHE-1 by amiloride attenuates zinc accumulation and subsequent hippocampus neuronal death following GCI. Mice were subjected to GCI by bilateral common carotid artery (BCCA) occlusion for 30 min, followed by restoration of blood flow and resuscitation. Amiloride (10 mg/kg, intraperitoneally (i.p.)) was immediately injected, which reduced zinc accumulation and neuronal death after GCI. Therefore, the present study demonstrates that amiloride attenuates GCI-induced neuronal injury, likely via the prevention of intracellular zinc accumulation. Consequently, we suggest that amiloride may have a high therapeutic potential for the prevention of GCI-induced neuronal death.

One-pot porogen free method fabricated porous microsphere-aggregated 3D PCL scaffolds for bone tissue engineering.

Three-dimensional (3D) scaffolds with interconnected, hierarchically structured pores, and biomimetic nanostructures are desirable for tissue engineering, where preparation with a facile and biocompatible strategy remains challenging. In the present work, an innovative porous microspheres-aggregated 3D PCL scaffold with macropores, micropores, and nanofibrous-like structures was fabricated through a one-pot thermally induced phase separation (TIPS) method without the use of any porogen or specific instruments. Importantly, the porosity, pore size, and mechanical properties of our scaffolds were tailorable through tuning of the polymer concentration. Interestingly, the bioactivity of our 3D PCL scaffolds was significantly improved, as abundant apatite-like layers were formed on the 3D porous scaffolds, while no obvious apatite was observed on the 2D flat PCL film. Moreover, the high surface area attributed to the hierarchical macro/micro/nanostructure enabled our 3D porous scaffold to serve as a drug delivery depot for sustained release of both small molecule drug (phenamil) and protein (BMP2). In addition to sustained drug release, the hierarchical structure and high mechanical properties also contribute to significantly improving BMP2-induced osteogenic differentiation. In summary, we developed a novel PCL porous scaffold through a facile, one-pot TIPS method and demonstrated its promising potential application in large bone defect repair.

A Randomized Trial of Distal Diuretics versus Dietary Sodium Restriction for Hypertension in Chronic Kidney Disease.

BACKGROUND: Distal diuretics are considered less effective than loop diuretics in CKD. However, data to support this perception are limited. METHODS: To investigate whether distal diuretics are noninferior to dietary sodium restriction in reducing BP in patients with CKD stage G3 or G4 and hypertension, we conducted a 6-week, randomized, open-label crossover trial comparing amiloride/hydrochlorothiazide (5 mg/50 mg daily) with dietary sodium restriction (60 mmol per day). Antihypertension medication was discontinued for a 2-week period before randomization. We analyzed effects on BP, kidney function, and fluid balance and related this to renal clearance of diuretics. RESULTS: A total of 26 patients (with a mean eGFR of 39 ml/min per 1.73 m2) completed both treatments. Dietary sodium restriction reduced sodium excretion from 160 to 64 mmol per day. Diuretics produced a greater reduction in 24-hour systolic BP (SBP; from 138 to 124 mm Hg) compared with sodium restriction (from 134 to 129 mm Hg), as well as a significantly greater effect on extracellular water, eGFR, plasma renin, and aldosterone. Both interventions resulted in a similar decrease in body weight and NT-proBNP. Neither approaches decreased albuminuria significantly, whereas diuretics did significantly reduce urinary angiotensinogen and ß2-microglobulin excretion. Although lower eGFR and higher plasma indoxyl sulfate correlated with lower diuretic clearance, the diuretic effects on body weight and BP at lower eGFR were maintained. During diuretic treatment, higher PGE2 excretion correlated with lower free water clearance, and four patients developed mild hyponatremia. CONCLUSIONS: Distal diuretics are noninferior to dietary sodium restriction in reducing BP and extracellular volume in CKD. Diuretic sensitivity in CKD is maintained despite lower diuretic clearance. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: DD-study: Diet or Diuretics for Salt-sensitivity in Chronic Kidney Disease (DD), NCT02875886.

Efficacy of chlorthalidone and hydrochlorothiazide in combination with amiloride in multiple doses on blood pressure in patients with primary hypertension: a protocol for a factorial randomized controlled trial.

BACKGROUND: Thiazide diuretics have demonstrated favorable blood pressure lowering efficacy, but the equivalent doses of their more common agents, chlorthalidone and hydrochlorothiazide, are still unclear. Further, concerns exist regarding adverse metabolic effects, which may be attenuated with the concomitant administration of a potassium-sparing diuretic, such as amiloride. This trial aims to investigate the efficacy of chlorthalidone and hydrochlorothiazide, in combination with amiloride at different doses, for initial management of patients with primary hypertension. METHODS/DESIGN: This is a factorial (2 × 2) randomized double-blinded clinical trial comparing the association of a thiazide diuretic (chlorthalidone 25 mg/day or hydrochlorothiazide 50 mg/day) with a potassium-sparing diuretic (amiloride 10 mg/day or amiloride 20 mg/day) in patients with primary hypertension. The primary outcome will be the mean change from baseline in 24-h systolic and diastolic blood pressure measured by ambulatory blood pressure monitoring. The secondary outcomes will be the mean change from baseline in daytime and nighttime systolic and diastolic blood pressure measured by ambulatory blood pressure monitoring, mean change from baseline in systolic and diastolic blood pressure measured by office blood pressure, incidence of adverse events, variation of laboratory parameters, and proportion of patients who achieved blood pressure control. The follow-up will last 12 weeks. For a P alpha of 0.05, power of 80%, standard deviation of 9 mmHg, and absolute difference of 6 mmHg on systolic blood pressure on 24-h ambulatory blood pressure monitoring, it will be necessary to study a total of 76 patients. The sample size will be increased by 10% to compensate for losses, resulting in 84 patients being randomized. DISCUSSION: Diuretics are pivotal drugs for the treatment of hypertension. Chlorthalidone and hydrochlorothiazide, in combination with amiloride in multiple doses, will be tested in terms of blood pressure lowering efficacy and safety. Since the intensity of blood pressure reduction is the major determinant of reduction in cardiovascular risk in hypertensive patients, this study will help to determine which combination of diuretics represents the most appropriate treatment for this population. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03928145. Registered on 25 April 2019. Last update on 29 April 2019.

Long-term BP control and vascular health in patients with hyperaldosteronism treated with low-dose, amiloride-based therapy.

Whether aldosterone itself contributes directly to macro- or microcirculatory disease in man or to adverse cardiovascular outcomes is not fully known. We report our long-term single-practice experience in 5 patients with chronic hyperaldosteronism (HA, including 3 with glucocorticoid remediable aldosteronism, GRA) treated with low-dose amiloride (a specific epithelial sodium channel [ENaC] blocker) 5-10 (mean 7) mg daily for 14-28 (mean 20) years. Except for 1 GRA diagnosed in infancy, all had severe resistant hypertension. In each case, BP was normal or near-normal within 1-4 weeks after starting amiloride and office BP's were well controlled for 20 years thereafter. Vascular studies and 24-hour ambulatory BP monitoring with pulse wave analysis (cardiac output, vascular resistance, augmentation index, and reflection magnitude) were assessed after a mean of 18 years as were regional pulse wave velocities, pulse stiffening ratio, ankle-brachial index, serum creatinine, estimated glomerular filtration rate, and spot urinary albumin:creatinine ratio. All indicators were completely normal in all patients after 18 years of amiloride, and none had a cardiovascular event during the 20-year mean follow-up. We conclude that long-term ENaC blockade can normalize BP and protect macro- and microvascular function in patients with HA. This suggests that (a) any vasculopathic effects of aldosterone are mediated via ENaC, not MR activation itself, and are fully preventable or reversible with ENaC blockade or (b) aldosterone may not play a major BP-independent role in human macro- and microcirculatory diseases. These and other widely divergent results in the literature underscore the need for additional studies regarding aldosterone, ENaC, and vascular disease.

Urokinase-type plasminogen activator (uPA) is not essential for epithelial sodium channel (ENaC)-mediated sodium retention in experimental nephrotic syndrome.

AIM: In nephrotic syndrome, aberrantly filtered plasminogen (plg) is converted to active plasmin by tubular urokinase-type plasminogen activator (uPA) and thought to lead to sodium retention by proteolytic activation of the epithelial sodium channel (ENaC). This concept predicts that uPA is an important factor for sodium retention and that inhibition of uPA might be protective in nephrotic syndrome. METHODS: Activation of amiloride-sensitive currents by uPA and plg were studied in Xenopus laevis oocytes expressing murine ENaC. In doxorubicin-induced nephrotic mice, uPA was inhibited pharmacologically by amiloride and genetically by the use of uPA-deficient mice (uPA-/- ). RESULTS: Experiments in Xenopus laevis oocytes expressing murine ENaC confirmed proteolytic ENaC activation by a combination of plg and uPA which stimulated amiloride-sensitive currents with concomitant cleavage of the ENaC γ-subunit at the cell surface. Treatment of nephrotic wild-type mice with amiloride inhibited urinary uPA activity, prevented urinary plasmin formation and sodium retention. In nephrotic mice lacking uPA (uPA-/- ), urinary plasmin formation from plg was suppressed and urinary uPA activity absent. However, in nephrotic uPA-/- mice, sodium retention was not reduced compared to nephrotic uPA+/+ mice. Amiloride prevented sodium retention in nephrotic uPA-/- mice which confirmed the critical role of ENaC in sodium retention. CONCLUSION: uPA is responsible for the conversion of aberrantly filtered plasminogen to plasmin in the tubular lumen in vivo. However, uPA-dependent plasmin generation is not essential for ENaC-mediated sodium retention in experimental nephrotic syndrome.

Effects of the potassium-sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells.

BACKGROUND: Osteosarcoma (OSA) is a common bone tumor of mesenchymal origin in dogs. Chemotherapy delays metastasis, yet most dogs die of this disease within 1 year of diagnosis. The high metabolic demand of cancer cells promotes proton pump upregulation, leading to acidification of the tumor microenvironment and chemoresistance. The potassium-sparing diuretic amiloride is among a class of proton pump inhibitors prescribed for refractory heart failure treatment in dogs. OBJECTIVE: We hypothesized that amiloride treatment improves chemotherapy response by reducing acidification in canine OSA cells. Our objective was to assess the in vitro effects of amiloride on cell viability, apoptosis, and metabolism. METHODS: In vitro study. Assessments of cell viability and apoptosis were performed after single agent or combination treatment, along with calculations of pharmacological synergism using the combination index. Protein signaling during apoptosis was evaluated by Western blotting. Metabolic profiling was performed using a Seahorse bioanalyzer. RESULTS: Amiloride strongly synergized with doxorubicin in combination treatment and exhibited additive or antagonistic effects with carboplatin in canine OSA cells. Combination treatment with doxorubicin significantly upregulated p53-mitochondrial signaling to activate apoptosis and downregulate Akt phosphorylation. Amiloride-treated cells further exhibited metabolic switching with reductions in glycolytic capacity and maximal respiration. CONCLUSION AND CLINICAL IMPORTANCE: Amiloride synergized with doxorubicin to potentiate apoptosis in canine OSA cells. These results justify further investigation into repurposing of amiloride as an oncology drug for the treatment of OSA in dogs.

Characterization of mouse chorda tympani responses evoked by stimulation of anterior or posterior fungiform taste papillae.

Different gustatory papilla types vary in their locations on the tongue. Distinctions have often made between types, but variation within fungiform papillae has seldom been explored. Here, regional differences in fungiform papillae were investigated by flowing solutions selectively over either an anterior fungiform (AF, tongue tip) or a posterior fungiform (PF, middle third) region as taste-evoked activity was measured in the chorda tympani nerve of C57BL/6J (B6) mice. Significantly larger responses were evoked by NaCl applied to the AF than PF region, and the ENaC blocker amiloride reduced the NaCl response size only for the former. Umami synergy, based on co-presenting MSG and IMP, was larger for the AF than PF region. The regions did not differ in response size to sour chemicals, but responses to l-lysine, l-arginine, sucrose, and tetrasodium pyrophosphate were larger for the AF than PF region. Thus, fungiform papillae on the tongue tip differed from those found further back in their transduction mechanisms for salty and umami compounds. Gustatory sensitivity also showed regional variation, albeit with a complex relationship to palatability and taste quality. Overall, the data support a regional organization for the mouse tongue, with different functional zones for the anterior, middle, and posterior thirds.

Effects of systemic inhibitors of acid-sensing ion channels 1 (ASIC1) against acute and chronic mechanical allodynia in a rodent model of migraine.

BACKGROUND AND PURPOSE: Acid-sensing ion channels (ASICs) are neuronal proton sensors emerging as potential therapeutic targets in pain of the orofacial region. Amiloride, a non-specific ASIC blocker, has been shown to exert beneficial effects in animal models of migraine and in patients. We explored the involvement of the ASIC1-subtype in cutaneous allodynia, a hallmark of migraine affecting cephalic and extra-cephalic regions in about 70% of migrainers. EXPERIMENTAL APPROACH: We investigated the effects of systemic injections of amiloride and mambalgin-1, a specific inhibitor of ASIC1a- and ASIC1b-containing channels, on cephalic and extra-cephalic mechanical sensitivity in a rodent model of acute and chronic migraine induced by i.p. injections of isosorbide dinitrate. KEY RESULTS: I.v. injections of these inhibitors reversed cephalic and extra-cephalic acute cutaneous mechanical allodynia in rats, a single injection inducing a delay in the subsequent establishment of chronic allodynia. Both mambalgin-1 and amiloride also reversed established chronic allodynia. The anti-allodynic effects of mambalgin-1 were not altered in ASIC1a-knockout mice, showing the ASIC1a subtype is not involved in these effects which were comparable to those of the anti-migraine drug sumatriptan and of the preventive drug topiramate on acute and chronic allodynia respectively. A single daily injection of mambalgin-1 also had a significant preventive effect on allodynia chronification. CONCLUSIONS AND IMPLICATIONS: These pharmacological data support the involvement of peripheral ASIC1-containing channels in migraine cutaneous allodynia as well as in its chronification. They highlight the therapeutic potential of ASIC1 inhibitors as both an acute and prophylactic treatment for migraine.

Jet dispensing of multi-layered films for the co-delivery of three antihypertensive agents.

Three-layer thin films comprising of two polymers as substrate (ethyl cellulose and, copovidone K28) and three antihypertensive agents (hydrochlorothiazide, amiloride HCl, and carvedilol) were printed using jet dispensing technology. Two film formulations with different ethyl cellulose to copovidone K28 ratio (i.e., 90/10 and 50/50 w/w) were prepared using a three-course dispensing. The films were characterized regarding surface morphology, solid-state properties, polymer-drug interactions, drug distribution in each layer, and in vitro drug release. All the components of the films were found to be in the amorphous state apart from hydrochlorothiazide which retained its crystallinity. FT-IR spectroscopy revealed hydrogen bond interactions between carvedilol and copovidone K28. Combinations of ethyl cellulose and copovidone K28 provide suitable polymeric film substrates with the ability to modify drug release. Particularly, decreased ethyl cellulose to copovidone K28 weight ratio was found to suppress the crystallization of hydrochlorothiazide and to increase the release rate of the dispensed drugs. Jet dispensing was found to be a rapid technology for the preparation of multi-layered films that can be used as personalized formulations for the delivery of combinations of drugs.

Effectiveness of low-dose diuretics for blood pressure reduction to optimal values in prehypertension: a randomized clinical trial.

BACKGROUND: To determine the effectiveness of low-dose diuretic therapy to achieve an optimal level of blood pressure (BP) in adults with prehypertension. METHODS: The PREVER-prevention trial was a randomized, parallel, double-blinded, placebo-controlled trial, with 18 months of follow-up, conducted at 21 academic medical centers in Brazil. Of 1772 individuals evaluated for eligibility, 730 volunteers with prehypertension who were aged 30-70 years, and who did not reach optimal blood pressure after 3 months of lifestyle intervention, were randomized to a fixed association of chlorthalidone 12.5 mg and amiloride 2.5 mg or placebo once a day. The main outcomes were the percentage of participants who achieved an optimal level of BP. RESULTS: A total of 372 participants were randomly allocated to diuretics and 358 to placebo. After 18 months of treatment, optimal BP was noted in 25.6% of the diuretic group and 19.3% in the placebo group (P < 0.05). The mean net reduction in SBP and DBP for the diuretic group compared with placebo was 2.8 mmHg (95% CI 1.1 to 4.5) and 1.1 mmHg (95% CI -0.09 to 2.4), respectively. Most participants in the active treatment group (74.5%) and in the placebo group (80.7%) continued to have BP in the prehypertension range or progressed to hypertension. CONCLUSION: Low-dose diuretic therapy increased the probability of individuals with prehypertension to achieve optimal BP but most of those treated continued to have a BP in the prehypertension range or progressed to having overt hypertension.

Influence of Inhaled Amiloride on Lung Fluid Clearance in Response to Normobaric Hypoxia in Healthy Individuals.

Wheatley, Courtney M., Sarah E. Baker, Bryan J. Taylor, Manda L. Keller-Ross, Steven C. Chase, Alex R. Carlson, Robert J. Wentz, Eric M. Snyder, and Bruce D. Johnson. Influence of inhaled amiloride on lung fluid clearance in response to normobaric hypoxia in healthy individuals. High Alt Med Biol 18:343-354, 2017. AIM: To investigate the role of epithelial sodium channels (ENaC) on lung fluid clearance in response to normobaric hypoxia, 20 healthy subjects were exposed to 15 hours of hypoxia (fraction of inspired oxygen [FiO2] = 12.5%) on two randomized occasions: (1) inhaled amiloride (A) (1.5 mg/5 mL saline); and (2) inhaled saline placebo (P). Changes in lung fluid were assessed through chest computed tomography (CT) for lung tissue volume (TV), and the diffusion capacity of the lungs for carbon monoxide (DLCO) and nitric oxide (DLNO) for pulmonary capillary blood volume (VC). Extravascular lung water (EVLW) was derived as TV-VC and changes in the CT attenuation distribution histograms were reviewed. RESULTS: Normobaric hypoxia caused (1) a reduction in EVLW (change from baseline for A vs. P, -8.5% ± 3.8% vs. -7.9% ± 5.2%, p < 0.05), (2) an increase in VC (53.6% ± 28.9% vs. 53.9% ± 52.3%, p < 0.05), (3) a small increase in DLCO (9.6% ± 29.3% vs. 9.9% ± 23.9%, p > 0.05), and (4) CT attenuation distribution became more negative, leftward skewed, and kurtotic (p < 0.05). CONCLUSION: Acute normobaric hypoxia caused a reduction in lung fluid that was unaffected by ENaC inhibition through inhaled amiloride. Although possible amiloride-sensitive ENaC may not be necessary to maintain lung fluid balance in response to hypoxia, it is more probable that normobaric hypoxia promotes lung fluid clearance rather than accumulation for the majority of healthy individuals. The observed reduction in interstitial lung fluid means alveolar fluid clearance may not have been challenged.

Amiloride, An Old Diuretic Drug, Is a Potential Therapeutic Agent for Multiple Myeloma.

Purpose: The search for new drugs that control the continuous relapses of multiple myeloma is still required. Here, we report for the first time the potent antimyeloma activity of amiloride, an old potassium-sparing diuretic approved for the treatment of hypertension and edema due to heart failure.Experimental Design: Myeloma cell lines and primary samples were used to evaluate cytotoxicity of amiloride. In vivo studies were carried out in a xenograft mouse model. The mechanisms of action were investigated using RNA-Seq experiments, qRT-PCR, immunoblotting, and immunofluorescence assays.Results: Amiloride-induced apoptosis was observed in a broad panel of multiple myeloma cell lines and in a xenograft mouse model. Moreover, amiloride also had a synergistic effect when combined with dexamethasone, melphalan, lenalidomide, and pomalidomide. RNA-Seq experiments showed that amiloride not only significantly altered the level of transcript isoforms and alternative splicing events, but also deregulated the spliceosomal machinery. In addition, disruption of the splicing machinery in immunofluorescence studies was associated with the inhibition of myeloma cell viability after amiloride exposure. Although amiloride was able to induce apoptosis in myeloma cells lacking p53 expression, activation of p53 signaling was observed in wild-type and mutated TP53 cells after amiloride exposure. On the other hand, we did not find a significant systemic toxicity in mice treated with amiloride.Conclusions: Overall, our results demonstrate the antimyeloma activity of amiloride and provide a mechanistic rationale for its use as an alternative treatment option for relapsed multiple myeloma patients, especially those with 17p deletion or TP53 mutations that are resistant to current therapies. Clin Cancer Res; 23(21); 6602-15. ©2017 AACR.

Phenamil, an amiloride derivative, restricts long bone growth and alters keeled-sternum bone architecture in growing chickens.

"Broiler-type" chickens are fast-grow-ing, heavy-bodied birds with high demands on bone quality. Phenamil increased mineralization in cultured murine mesenchymal stem cells. Phenamil effects were tested in 2 groups of weight and gender matched day-old broiler chickens (n = 13). Oral administration of 30 mg phenamil/kg body weight d 1 to 13 reduced growth of chicks d 5 to 14 (P = 0.002); with phenamil-treated (PT) chick body weight being 84% of vehicle-treated (VT) chicks' body weight on d 14. Tissues collected on d 15 showed that femur lengths and widths did not differ, but tibias from PT chicks were 6% shorter (P = 0.002) and 13% narrower (P = 0.012) with 18% thinner tibial cross-sections (P < 0.008) than in VT chicks. Angles of the caudal aspect of the anterior surface of keeled-sternums were 166° in PT chicks, flatter than the 148° found in VT chicks (P = 0.000). Total mineral content of both tibia and femur were lower in PT chicks (P = 0.005 for both). Bone Ca, P, and Mg (ppm) in ash were similar, but Ca:P was lower (1.70 vs 1.75) in PT versus VT chicks (P < 0.05). Osteocalcin was ∼20% lower (P = 0.020), PINP was ∼45% higher (P = 0.000) in PT chicks. Carboxy-terminal telopeptide type I collagen (ICTP) and cross-linked N-telopeptide of type I collagen (NTX1) were similar in the 2 groups. Phenamil had unexpected and detrimental effects on bone formation in growing broiler chicks, reducing linear skeletal growth and markedly changing bone architecture.

Effectiveness of Chlorthalidone Plus Amiloride for the Prevention of Hypertension: The PREVER-Prevention Randomized Clinical Trial.

BACKGROUND: Prehypertension is associated with higher cardiovascular risk, target organ damage, and incidence of hypertension. The Prevention of Hypertension in Patients with PreHypertension (PREVER-Prevention) trial aimed to evaluate the efficacy and safety of a low-dose diuretic for the prevention of hypertension and end-organ damage. METHODS AND RESULTS: This randomized, parallel, double-blind, placebo-controlled trial was conducted in 21 Brazilian academic medical centers. Participants with prehypertension who were aged 30 to 70 years and who did not reach optimal blood pressure after 3 months of lifestyle intervention were randomized to a chlorthalidone/amiloride combination pill or placebo and were evaluated every 3 months during 18 months of treatment. The primary outcome was incidence of hypertension. Development or worsening of microalbuminuria, new-onset diabetes mellitus, and reduction of left ventricular mass were secondary outcomes. Participant characteristics were evenly distributed by trial arms. The incidence of hypertension was significantly lower in 372 study participants allocated to diuretics compared with 358 allocated to placebo (hazard ratio 0.56, 95% CI 0.38-0.82), resulting in a cumulative incidence of 11.7% in the diuretic arm versus 19.5% in the placebo arm (P=0.004). Adverse events; levels of blood glucose, glycosylated hemoglobin, creatinine, and microalbuminuria; and incidence of diabetes mellitus were no different between the 2 arms. Left ventricular mass assessed through Sokolow-Lyon voltage and voltage-duration product decreased to a greater extent in participants allocated to diuretic therapy compared with placebo (P=0.02). CONCLUSIONS: A combination of low-dose chlorthalidone and amiloride effectively reduces the risk of incident hypertension and beneficially affects left ventricular mass in patients with prehypertension. CLINICAL TRIAL REGISTRATION: URL: http://www.ClinicalTrials.gov, www.ensaiosclinicos.gov. Unique identifiers: NCT00970931, RBR-74rr6s.

Effects of aerobic dance training on blood pressure in individuals with uncontrolled hypertension on two antihypertensive drugs: a randomized clinical trial.

There is a dearth of reports on possible additive blood pressure (BP)-reducing effect of aerobic exercise on antihypertensive drug in humans. This study investigated the additive BP-reducing effect of aerobic exercise on BP in individuals with uncontrolled hypertension. In this 12-week double-blind study, 120 new-diagnosed individuals with mild-to-moderate hypertension were randomized to receive coamilozide + 5/10 mg of amlodipine + aerobic dance or coamilozide + 5/10 mg of amlodipine alone. Forty-five and 43 participants in exercise and control groups, respectively, completed the 12-week intervention. Addition of aerobic exercise to antihypertensive drug therapy significantly reduced systolic BP (7.1 mm Hg [95% confidence interval: 5.0, 9.3]; P < .001) and diastolic BP (1.7 mm Hg [95% confidence interval: 0.4, 3.0]; P = .009) at 12 weeks. BP control rate differed significantly between exercise (53.9%) and control (35.3%) groups, P < .001. Postintervention, proportion of participants in exercise group who had their number of antihypertensive drug reduced to one (20.3%) differed from that in control group (11.1%); (χ(2) = 11.0; P = .001). Combination of aerobic dance and antihypertensive drugs reduces number of antihypertensive drugs needed to achieve BP control and enhances BP control in individuals with hypertension on two antihypertensive drugs.