Ethacrynic Acid: A Promising Candidate for Drug Repurposing as an Anticancer Agent.

Ethacrynic acid (ECA) is a diuretic that inhibits Na-K-2Cl cotransporter (NKCC2) present in the thick ascending loop of Henle and muculo dens and is clinically used for the treatment of edema caused by excessive body fluid. However, its clinical use is limited due to its low bioavailability and side effects, such as liver damage and hearing loss at high doses. Despite this, ECA has recently emerged as a potential anticancer agent through the approach of drug repositioning, with a novel mechanism of action. ECA has been shown to regulate cancer hallmark processes such as proliferation, apoptosis, migration and invasion, angiogenesis, inflammation, energy metabolism, and the increase of inhibitory growth factors through various mechanisms. Additionally, ECA has been used as a scaffold for synthesizing a new material, and various derivatives have been synthesized. This review explores the potential of ECA and its derivatives as anticancer agents, both alone and in combination with adjuvants, by examining their effects on ten hallmarks of cancer and neuronal contribution to cancer. Furthermore, we investigated the trend of synthesis research of a series of ECA derivatives to improve the bioavailability of ECA. This review highlights the importance of ECA research and its potential to provide a cost-effective alternative to new drug discovery and development for cancer treatment.

[Research progress of acute kanamycin sulfate-induced deafness in guinea pig].

To present a summary of current knowledge regarding acute kanamycin sulfate-induced deafness in guinea pig, by reviewing the published literature. Animal model of acute deafness induced by a single dose of kanamycin sulfate in combination with ethacrynic acid or furosemide in guinea pig was usually used to investigate the mechanism of cochlear cell degeneration. There were different time sequences of cell degeneration of spiral ganglion cell and hair cell in different studies. The findings may result from different doses, order of two drugs administration or time point chosen. There remains scope for further research in chronic kanamycin-induced deafness, which more replicates the type of exposure to people than acute deafness.

Cellular pharmacokinetic and pharmacodynamic analyses of ethacrynic acid: Implications in topical drug delivery in the eye.

PURPOSE: Ethacrynic acid (ECA) is a potential trabecular meshwork (TM) drug that has shown promising results in preclinical studies for treatment of primary open-angle glaucoma. However, topical application of ECA is currently limited by adverse effects in corneal tissues. To this end, we developed a new theoretical model to evaluate time-dependent toxicity induced by ECA in corneal epithelial cells. METHODS: The model consisted of a cellular pharmacokinetic (PK) module to determine intracellular concentration of ECA, and a pharmacodynamic (PD) module to determine the cytotoxicity of ECA. It was assumed that ECA-induced cytotoxicity depended on drug exposure time and peak concentration of bound ECA in cells. In addition to the model development, we experimentally determined the intracellular concentration of ECA as a function of drug dose and treatment time. RESULTS: The intracellular concentration increased linearly (i.e., no saturation) with increasing the dose of ECA. It also increased initially with time and then reached a steady-state at ~40 min. The percent of cells survived after treatment decreased with increasing the dose of drug or the time of treatment. The experimental data were fit by the new PK and PD models to obtain values of model constants. One of the unique applications of these models was to predict cell survival relative to control when extracellular concentration of ECA varied with time. The prediction showed that the toxicity of ECA might be significantly overestimated by using the traditional LC(50) determined in vitro. CONCLUSIONS: The new PK and PD models developed in this study were capable to fit experimental data and predict time-dependent toxicity of ECA in corneal epithelial cells. The models may be useful for optimizing the dose and schedule in topical application of ECA for glaucoma treatment.

The clinical pharmacology of ethacrynic acid.

Ethacrynic acid (Edecrin) is a loop diuretic that produces a prompt and profound diuresis. The primary action of ethacrynic acid is the inhibition of the activity of the Na⁺-K⁺-2Cl⁻ symporter in the thick ascending limb of the loop of Henle. The onset of action is usually within 30 minutes after an oral dose and within 5 minutes after an intravenous injection. After oral administration, peak diuretic effect occurs in about 2 hours and the effect lasts about 6-8 hours. After intravenous administration, peak diuretic effect occurs within 30 minutes and the diuretic effect is virtually completed in 2-4 hours. The bioavailability of ethacrynic acid approximates 100%, with maximal blood level between 40 and 92 minutes. The elimination half-life has been reported to be less than 1 hour, but highly variable (average 30 minutes with a range of 12-160 minutes). Intravenous ethacrynic acid has a prompt venous dilatory effect and immediately relieves symptoms of pulmonary congestion, before a diuresis can occur. Ethacrynic acid is effective in all types of edema whether there is clinical acidosis, alkalosis, or electrolyte imbalance. Most side effects of ethacrynic acid can be attributed to its effectiveness (volume depletion); however, it may cause metabolic alkalosis that is preventable by KCl replacement. Ethacrynic acid has ototoxic effect that occasionally results in temporally or permanent deafness. Despite limitations, ethacrynic acid has been employed in the treatment of congestive heart failure and other edematous states, especially in patients allergic to sulfa-containing drugs because all the other loop diuretics have a sulfa moiety.

Aryloxoalcanoic compounds induce resistance to antibiotic therapy in urinary tract infection caused by Escherichia coli.

Clofibric acid (CL) is a compound used to control hypertriglyceridemia, and ethacrynic acid (ET) is administered to enhance diuresis. These compounds are structurally analogous to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), as they have a chlorinated phenoxy moiety. As these agents are mainly excreted by the renal route, they could potentially coexist with Escherichia coli in the urinary tract of infected patients. Induction of the in vitro resistance of E. coli to hydrophilic antibiotics was determined by increasing the values of the minimum inhibitory concentration (2-40-fold). These results correlated with drastically inhibited expression of the hydrophilic bacterial channel OmpF. In vivo assays were performed in ascending urinary tract infection in female BALB/c mice. Treatment with the hydrophilic antibiotic cephalexin 25 mg kg(-1) day(-1) by the oral route diminished renal infection. The CFU mean values in the kidneys were between 75% and 89% lower than those in animals without treatment. Simultaneous exposure to CL (at a therapeutic dose, 28.6 mg kg(-1) day(-1)) did not change the effect of the treatment. In contrast, ET at 2.9 mg kg(-1) day(-1) or 2,4-D at 70 mg kg(-1) day(-1) inhibited the antibiotic therapeutic effect. Moreover, 2,4-D dramatically increased bacterial infection after 9 days of exposure.

Ototoxic drugs and sensorineural hearing loss following severe neonatal respiratory failure.

AIM: To determine relationships between ototoxic drugs and 4-y sensorineural hearing loss (SNHL) in near-term and term survivors of severe neonatal respiratory failure. METHODS: All 81 survivors of the Canadian arm of the Neonatal Inhaled Nitric Oxide Study (mortality 32, loss to follow-up 9) received loop diuretics, aminoglycosides, and neuromuscular blockers (NMB), and 50 received vancomycin as neonates. Prospective, longitudinal secondary outcome using audiological tests diagnosed late-onset, progressive SNHL in 43 (53%); not flat (sloping) in 29, flat (severe to profound) in 14. Risk for SNHL was determined. RESULTS: A combination of duration of diuretic use of >14 d and average NMB dose of >0.96 mg/kg/d contributed to SNHL among survivors (odds ratio 5.2; 95% CI 1.6, 16.7). Markers of illness severity did not contribute. Dosage or duration of aminoglycosides use did not relate to SNHL. Cumulative dosages and duration of use of diuretics; NMB; use of vancomycin; and overlap of diuretics with NMB, aminoglycosides, and vancomycin individually linked to SNHL (p<0.001). CONCLUSION: Overuse of loop diuretics and/or NMB contributes to SNHL after neonatal respiratory failure; markers of illness severity or the appropriate administration of aminoglycosides do not.

Novel antiglaucoma prodrugs and codrugs of ethacrynic acid.

The purpose of this study was to synthesize a novel prodrug of ethacrynic acid (ECA) with short chain polyethylene glycols (PEGs) and codrugs of ECA with the beta-adrenergic blocking agent atenolol (ATL) or timolol (TML) to overcome the adverse effects of ECA and to enhance its physicochemical properties.

Glial cell line-derived neurotrophic factor. Potential for otoprotection.

Sensorineural hearing loss results from the degeneration of hair cells and/or auditory neurons in the cochlea of the inner ear. BDNF and NT-3 were shown to support survival of auditory neurons both in vitro and in vivo. Cochlea from P3-P4 rats were cultured as floating explants and hair cells in the organ of Corti were identified by phalloidin-FITC immunostaining. Treatment with cisplatin (35 micrograms/mL) or neomycin (0.6 mM) resulted in 21.2 +/- 6.0% and 7.4 +/- 4.7% surviving hair cells, respectively, after 3 days in culture. GDNF, added together with the ototoxins, increased their number to 46.7% and 37.4%, respectively. In cultures of dissociated cochlea from 4-week-old rat, cisplatin (5 mg/mL) added 24 h after seeding resulted in only 6.1 +/- 1.2% surviving neurons. However, when cisplatin was added together with GDNF (10 ng/mL), 32.8 +/- 1.0% of the neurons survived. The efficacy of GDNF in animal models of ototoxicity was tested next. Guinea pigs were pretreated with GDNF in one ear, delivered either by infusion into the inner ear (scala tympani) with Alzet minipumps (50 ng/mL at a 0.5 microL/h), or injected into the middle ear (120 microL at 1 mg/mL) through the tympanic membrane. The ear that did not receive GDNF always served as control. Ototoxicity was induced systemically either by intraperitoneal cisplatin injections (1 mg/kg/day for 15 days or two injections of 7.5 mg/kg at a 5-day interval or by a combination of kanamycin (200-300 mg/kg, administered subcutaneously) and ethacrinic acid (40 mg/kg, intravenous). It was found that the number of surviving hair cells in GDNF-treated ears was about twice that of control ears in animals exposed to the ototoxins. The transducing GDNF receptor (ret) is expressed in the inner ear.

Topical gentamicin and ethacrynic acid: effects on cochlear function.

OBJECTIVE: To determine whether concurrent intravenous administration of the loop diuretic ethacrynic acid potentiates the toxicity of the aminoglycoside antibiotic gentamicin applied topically on the round window. STUDY DESIGN: The authors studied the effects on cochlear sensitivity of co-administered intracardiac ethacrynic acid (40 mg/kg) and high-dose topical gentamicin solution (100%) applied to the round window. Comparisons were made with animals receiving ethacrynic acid plus systemic gentamicin (100 mg/kg); topical gentamicin alone; systemic gentamicin alone; and intravenous ethacrynic acid alone. METHODS: Experiments were carried out on pigmented guinea pigs weighing 400 to 500 g. Changes in cochlear function were characterized by monitoring shifts in compound action potential (CAP) thresholds by use of chronic indwelling electrodes implanted at the round window, vertex, and contralateral mastoid. RESULTS: After 20 days animals receiving ethacrynic acid in combination with topical gentamicin to the round window failed to demonstrate a significant deterioration in cochlear sensitivity, whereas all animals receiving systemic gentamicin plus ethacrynic acid experienced profound increases in CAP thresholds. CONCLUSIONS: This study supports the contention that ethacrynic acid potentiates aminoglycoside ototoxicity by facilitating the entry of the antibiotics from the systemic circulation into the endolymph. In addition, this study answers important clinical concerns regarding the safety of the use of topical aminoglycoside agents in combination with loop diuretics.

Inhibición del cotransporte de NA/K/2CI por ácido etacrínico en células aisladas del epitelio / Inhibition of the cotransport of NA/K/2C1 by ethacrynic acid in insolated cell of the ciliary epithelium of the rabbits eyes

La formación de humor acuoso es debida principalmente al transporte activo de iones específicos desde el espacio extracelular del estroma a través de la doble capa de células del epitelio ciliar hacia la cámara posterior, y este proceso conduce al flujo osmótico de agua. Estudios recientes demuestran que el ácido etacrínico (AE), una droga utilizada durante muchos años como diurético produce una disminución de la presión intraocular en ojos de monos glaucomatoso e incrementa el flujo de salida de humor acuoso en ojos de monos, orangutanes y humanos. El propósito de este trabajo fue evaluar el efecto del AE sobre los mecanismos de transporte activo de K+ en células del epitelio ciliar de ojos de conejo usando Rb como trazador. Esta droga produjo una inhibición significativa del cotransporte de Na/K/2CI (De 180 ñ 8 a 35 ñ2, P<0.05), obteniéndose un efecto inhibitorio máximo a una concentración de 10 elevado a la cinco M. No observamos efecto significativo sobre la actividad de la ATPasa de Na/K. Nuestros resultados indican que la reducción de la presión intraocular producida por el AE en animales y humanos podría deberse en gran parte a la inhibición de la actividad del Cotransporte Na/K/C1 presente en el epitelio ciliar

Ototoxicity of loop diuretics.

The loop diuretics are drugs that increase the excretion of water and electrolytes in the urine by their action on the cells in the loop of Henle. Clinical reports of ototoxicity of these agents are reviewed, and the results of a number of studies in experimental animals are discussed. These drugs can cause either a temporary, or in some cases, a permanent loss of hearing in patients. Animal experiments show that these drugs act on the stria vascularis, producing edema of these tissues and a temporary loss of function, resulting in a decrease of the endocochlear potential. This can result in secondary effects on sound-evoked measures of hearing. As new information unfolds about protective agents, it may be possible to preserve hearing and maintain the desired therapeutic effect.

The effect of intracameral ethacrynic acid on the intraocular pressure of living monkeys.

Previous studies have shown that the sulfhydryl-reactive ethacrynic acid increases outflow facility in living monkeys when perfused via the anterior chamber. To study its potential clinical use further, living monkeys were intracamerally injected with 10 microliters of ethacrynic acid, with concentrations ranging from 0.5 to 7.5 mmol/l. The fellow control eye was injected with 10 microliters of diluent. The status of the anterior segment was monitored by slit-lamp biomicroscopy and the intraocular pressure was measured by pneumatonometry with the monkeys anesthetized with ketamine. The anterior segment of living monkeys tolerated injections up to 3.0-mmol/l ethacrynic acid without marked adverse effects. One of 13 monkey eyes injected with 3.0-mmol/l ethacrynic acid demonstrated mild reversible segmental corneal edema. The greatest mean intraocular pressure reduction in the 3.0- to 3.75-mmol/l group occurred at six hours, with the experimental intraocular pressure decreasing 2.9 mm Hg compared to a mean intraocular pressure increase of 0.1 mm Hg in the control group (n = 19). Concentrations of ethacrynic acid less than 3.0 mmol/l did not provide reliable reduction of intraocular pressure, whereas concentrations greater than 3.75 mmol/l caused a greater incidence and severity of corneal edema. We believe that the intracameral injection of ethacrynic acid can reliably and safely reduce intraocular pressure in living monkey eyes, and that this drug deserves further investigation as a potential antiglaucomatous agent.

[Deafness, induced by sodium ethacrynate in guinea pigs, alleviated by microwave treatment]. / Amélioration de la surdité induite par l'étacrynate de sodium chez le cobaye, à l'aide d'un traitement par micro-onde.

Microwave is used to treat temporal hearing loss caused by intravenous injection of the ethacrynic acid in guinea pigs. The recovery of hearing is much faster in the treated groups than in the control group. The article proposes possible mechanism of the effects against the ethacrynic acid induced deafness and assume that the result of this research can provide an experimental basis for treatment of some perceptive deafness due to ischemia of stria vascularis of the cochlea.

Physiologic and rheologic effects of the antisickling agent ethacrynic acid and its N-butylated derivative on normal and sickle erythrocytes.

Ethacrynic acid, a loop diuretic, has been shown to inhibit hemoglobin S polymerization. Until now, however, most studies were performed using purified solutions of hemoglobin S. The experiments reported here were designed to examine the effects of ethacrynic acid and its n-butryic acid derivative on the rheological and physiological properties of intact red blood cells. Using net and unidirectional flux measurements, both agents were shown to cause ion and water loss from normal and sickle erythrocytes. Since cell shrinkage adversely influences red cell rheology, it is unlikely that this class of compounds, despite its ability to inhibit hemoglobin S polymerization, will prove useful in the treatment of sickle cell disease.

Phase I study of thiotepa in combination with the glutathione transferase inhibitor ethacrynic acid.

The glutathione transferases comprise a family of isoenzymes, one or more of which are involved in the conjugation of alkylating agents to glutathione (GSH). Increased GSH transferase activity has been shown to underlie acquired resistance to several alkylating agents. Ethacrynic acid inhibits the isoenzymes of GSH transferase with 50% inhibitory concentration values ranging from 0.3 to 6.0 microM and has been shown to restore sensitivity to alkylating agents in drug-resistant animal tumor models. We entered 27 previously treated patients with advanced cancer on a study of ethacrynic acid (25 to 75 mg/m2 p.o. every 6 h for 3 doses) and thiotepa (30 to 55 mg/m2 i.v. 1 h after the second dose of ethacrynic acid). The major toxicity of ethacrynic acid was diuresis, which was observed at every dose level; in addition, severe metabolic abnormalities occurred at 75 mg/m2. At 50 mg/m2, the diuretic effects were manageable. Myelosuppression was the most important effect of the combination. Two of seven courses of ethacrynic acid, 50 mg/m2, and thiotepa, 55 mg/m2, were associated with grade 3 or 4 neutropenia and/or thrombocytopenia. Nausea/vomiting greater than or equal to grade 2 was observed in 16% of courses. GSH transferase activity was assayed spectrophotometrically in the peripheral mononuclear cells of all patients. At each dose level, activity decreased following ethacrynic acid administration, with recovery by 6 h. Administration of ethacrynic acid, 50 mg/m2, resulted in a mean nadir of transferase activity of 37% of control. The pharmacokinetics of thiotepa and its principal metabolite TEPA were studied in 23 patients. The plasma disappearance of thiotepa fit a two-compartment open model with a terminal half-life of approximately 2 h. Plasma TEPA levels peaked at a mean of 2.16 h following thiotepa administration. The harmonic mean terminal half-life of TEPA was 10.4 h, and the TEPA area under the curve (AUC) did not increase with increasing thiotepa dose. The AUC of thiotepa was approximately twice, and the clearance about one-half, of the values obtained in a previous study of single agent thiotepa. The AUC of TEPA was lower than that previously observed. The data suggest that ethacrynic acid inhibits enzymes involved in the metabolic disposition of thiotepa, including its oxidative desulfuration to TEPA. The severity of the platelet toxicity was correlated with the AUC of thiotepa, but not with that of TEPA. This combination of thiotepa and ethacrynic acid will be tested further in Phase II trials.

Protective effect of electrical stimulation in the deafened guinea pig cochlea.

The effect of chronic intrascalar electrical stimulation on the spiral ganglion cell survival of the ototoxically deafened guinea pig was investigated. Immediately after ototoxic drug administration, unilateral sinusoidal (1 kHz) charge-balanced electrical stimulation on a 50% duty cycle was administered for 2 hours per day, 5 days per week, at intensities from 0 (control) to 400 microAmp via an implanted scala tympani electrode. The relationship of electrically evoked middle latency response (EMLR) to stimulation protocol and cell survival was studied. At 9 weeks post-drug treatment, the animals were killed and temporal bones were prepared for morphometric analysis of spiral ganglion cell density. The subjects showed essentially complete elimination of outer hair sensory cells, with minimal remaining inner hair cells confined to apical turns. Variable loss of spiral ganglion cell populations was observed, which related to electrical stimulation. In animals that received daily unilaterally electrical stimulation, statistically significant increases in survival of spiral ganglion cells were observed in the stimulated ear, compared to the nonstimulated ear-particularly in basal cochlear regions near the electrode. Spiral ganglion cell density was a function of stimulation current intensity level. Moreover, the slope of the amplitude input/output (I/O) function of the EMLR was found to be dependent on stimulating current level. The effect of stimulation on induced survival may be dependent on a number of mechanisms, including metabolic effects of direct activation of "deafferented" spiral ganglion cells. These data support the suggestion that implantation may provide optimal benefits when performed shortly after deafness.

[Synergism between noise and ototoxic drugs: new experimental data]. / Synergies entre bruit et médicaments ototoxiques: nouvelles données expérimentales.

Results of experiments investigating, in the guinea pig, the effects of combinations of gentamicin (GM), ethacrynic acid (EA) and acoustic stimulations are reported. They indicate that functional and morphological alterations develop only after GM has penetrated specifically into the sensory cells, that this penetration is indirectly facilitated by EA, since it is known to increase GM concentration in endolymph, and directly by functional depolarization of the cell, as it occurs during normal acoustic stimulation.