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PubMed2004

New ethacrynic acid derivatives as potent cytoskeletal modulators in trabecular meshwork cells.

A series of ethacrynic acid (ECA) derivatives were synthesized and examined for ocular hypotensive activity. Efficacy was evaluated in a cell-shape assay, using human trabecular meshwork cells, and cytotoxicity in a (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, using cultured bovine trabecular meshwork cells. Many of the derivatives demonstrated efficacy equal to or greater than that of ECA. SA9000 was selected as the most promising candidate for a novel ocular hypotensive drug with few side effects.

Animals↗
PubMed1995

Effect of daily topical ethacrynic acid on aqueous humor dynamics in monkeys.

We determined the effect of 1.5% ethacrynic acid (ECA) in ointment on intraocular pressure (IOP) and outflow facility following 5 days of topical treatment in ocular normotensive cynomolgus monkeys. Twelve monkeys received a 1 cm strip of ointment containing ECA in one eye but without ECA in the other once daily for 5 days. On Day 1 and Day 5 IOP was measured immediately before and 1 and 3 h after treatment. Outflow facility (perfusion) was determined 3.5 h after treatment on Day 5. The ECA-vehicle IOP differences averaged -2.8 +/- 0.8 (S.E.M) (p < 0.01), -1.7 +/- 0.6 (p < 0.02) and -3.7 +/- 0.7 mm Hg (p < 0.001), equivalent to IOP reductions of 21 +/- 6% (p < 0.01), 13 +/- 5% (p < 0.02) and 26 +/- 5% (p < 0.002) respectively, at 0, 1 and 3 h respectively after treatment on Day 5. Facility averaged 40 +/- 15% higher (p < 0.03) in the ECA-treated compared to the vehicle-treated eyes 4 h after treatment on Day 5.

Administration, Topical↗
PubMed1991

The initial-rate kinetics of mouse glutathione S-transferase YfYf. Evidence for an allosteric site for ethacrynic acid.

Mouse glutathione S-transferase GST YfYf (an orthologue of GST P or 7-7 in the rat and of GST pi in the human) was found to have a subunit Mr of 24,500 and cross-reacted with anti-(rat GST YfYf). N-Terminal analysis showed a close similarity to the rat, human and bovine orthologues. On isoelectric focusing the native enzyme had a pI of 8.3 and a pI of 7.3 in the presence of urea. Initial-rate studies with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates and inhibition studies with the product of the enzyme-catalysed conjugation of CDNB and GSH, S-(2,4-dinitrophenyl)glutathione, indicated a rapid-equilibrium random mechanism for the enzyme. The diuretic drug ethacrynic acid was found to be simultaneously a competitive inhibitor and an uncompetitive activator of the enzyme (with CDNB as the substrate whose concentration was varied). By using a computer simulation program (EKPLOT) a model was developed that would explain the experimental data. It is proposed that ethacrynic acid can compete with CDNB at the active site but simultaneously bind to an allosteric site on the enzyme, causing an elevation in the Vmax. for the conjugation of CDNB and GSH. The implications of such an activation mechanism for an enzyme potentially conjugating a range of xenobiotic compounds are discussed.

Allosteric Site↗
PubMed1995

Structural analysis of human alpha-class glutathione transferase A1-1 in the apo-form and in complexes with ethacrynic acid and its glutathione conjugate.

BACKGROUND: Glutathione transferases (GSTs) constitute a family of isoenzymes that catalyze the conjugation of the tripeptide glutathione with a wide variety of hydrophobic compounds bearing an electrophilic functional group. Recently, a number of X-ray structures have been reported which have defined both the glutathione- and the substrate-binding sites in these enzymes. The structure of the glutathione-free enzyme from a mammalian source has not, however, been reported previously. RESULTS: We have solved structures of a human alpha-class GST, isoenzyme A1-1, both in the unliganded form and in complexes with the inhibitor ethacrynic acid and its glutathione conjugate. These structures have been refined to resolutions of 2.5 A, 2.7 A and 2.0 A respectively. Both forms of the inhibitor are clearly present in the associated electron density. CONCLUSIONS: The major differences among the three structures reported here involve the C-terminal alpha-helix, which is a characteristic of the alpha-class enzyme. This helix forms a lid over the active site when the hydrophobic substrate binding site (H-site) is occupied but it is otherwise disordered. Ethacrynic acid appears to bind in a non-productive mode in the absence of the coenzyme glutathione.

Apoenzymes↗
PubMed1992

Modulation of mitomycin C resistance by glutathione transferase inhibitor ethacrynic acid.

This study was undertaken to elucidate the mechanism(s) of cross-resistance (4.9-fold) to mitomycin C (MMC) in a multi-drug-resistant cell line, P388/R-84. Intracellular accumulation of MMC by sensitive (P388/S) and P388/R-84 cells was comparable. Despite a 32% reduction in NADPH cytochrome P-450 reductase activity (responsible for MMC activation) in P388/R-84 cells, the rate of MMC bio-reduction by sensitive and resistant cells was similar. These results suggested that MMC resistance in P388/R-84 cell line must depend on factors other than impaired drug accumulation or bio-activation. Recent studies suggest that glutathione transferase (GST) dependent drug detoxification also contributes to cellular resistance of a variety of alkylating agents. Even though overexpression of GST has been noted in some MMC resistant tumor cells, it is not known if its level affects sensitivity to MMC. We have, therefore, determined the effect of ethacrynic acid (an inhibitor of GST activity) treatment on MMC cytotoxicity in P388/R-84 cells, which have about 2-fold higher GST activity than P388/S cells. The IC50 value for the inhibition of GST activity in vitro by ethacrynic acid (EA) was 16.5 microM (5 micrograms/ml). A depletion in intracellular GSH was also observed by treating P388/R-84 cells with EA alone or in combination with MMC. A non-toxic concentration of EA (1 microgram/ml; 3.3 microM) increased MMC cytotoxicity by 36% in P388/R-84 cells. MMC cytotoxicity was increased 2-fold by EA treatment in glutathione (GSH)-depleted P388/R-84 cells. These results suggest that GST mediated drug inactivation may represent another important mechanism of MMC resistance.

Animals↗
PubMed1978

Stability of pediatric liquid dosage forms of ethacrynic acid, indomethacin, methyldopate hydrochloride, prednisone and spironolactone.

The stability of liquid dosage forms of ethacrynic acid (1 mg/ml), indomethacin (2 mg/ml), methyldopate hydrochloride (25 mg/ml), prednisone (0.5 mg/ml) and spironolactone (2 mg/ml), which often are compounded extemporaneously, was studied. One or two liquid dosage forms of each of the five drugs was prepared with the pure drug or the powder from a commercial dosage form using aqueous sorbitol or simple syrup alone or with a 10% (v/v) solution of alcohol in water. The dosage forms were stored at 24 C in amber-colored bottles for 21-224 days and assayed by various methods. All solutions studied were stable for at least 84 days. A solution was considered stable if it retained 90% of its drug concentration. Except for the prednisone solution, all solutions were stable for at least 164 days; however, the solution of methyldopate hydrochloride prepared from the pure drug became discolored after 98 days. The liquid dosage forms studied have limited stability but can be used by the pharmacist when extemporaneous oral solutions of these drugs are needed.

Child↗
PubMed1988

Glutathione depletion in the guinea pig and its effect on the acute cochlear toxicity of ethacrynic acid.

There is controversy as to whether or not the acute cochlear toxicity of ethacrynic acid (EA) is dependent upon its metabolic conversion to EA-cysteine via conjugation with glutathione. In order to investigate this we examined the acute effects of EA on cochlear potentials in guinea pigs in which glutathione levels were decreased by prior administration of (+/-)-buthionine sulphoximine (BSO), an inhibitor of glutamylcysteine synthetase. First, we determined the effects of BSO on hepatic and renal glutathione levels in the guinea pig. Guinea pigs (pigmented animals of both sexes or male albino animals) were killed at intervals up to 72 hr after i.p. administration of 1.6 g kg-1 BSO. Livers, and also kidneys in the case of pigmented guinea pigs, were removed and total glutathione (GSH + GSSG) measured. Glutathione levels reached a nadir in the liver at 24-48 hr (11% of control) and in the kidneys at 24 hr (14% of control) after administration of BSO. Hepatic but not renal levels approached control values by 72 hr. There were no sex or strain differences. Pigmented guinea pigs were anaesthetised and their endocochlear potential and a.c. cochlear potential in response to a 4 kHz tone were measured using an intracochlear microelectrode. The depression of these potentials by i.v. administration of 60 mg kg-1 EA was not affected by administration of 1.6 g kg-1 BSO 24 hr earlier, despite profound depletion of glutathione. Also prior p.o. administration of N-acetyl-L-cysteine did not affect hepatic glutathione levels nor modify the toxicity of EA. These results suggest that the acute cochlear toxicity of EA is not altered by glutathione depletion, a finding which argues against a role for the metabolic activation of EA in its ototoxicity.

Animals↗
PubMed1996

Transport and metabolism of glutathione conjugates of menadione and ethacrynic acid in confluent monolayers of rat renal proximal tubular cells.

Confluent monolayers of primary rat renal proximal tubular (RPT) cells were used to compare transepithelial transport and concomitant metabolism of two different glutathione (GSH) S-conjugates. For the GSH-conjugated quinone compound, [35S]GSH-conjugated menadione (MGNQ), no specific transepithelial transport was observed. Most likely, [35S]MGNQ passed the monolayer via paracellular leakage as the result of a reduction in monolayer integrity due to toxicity via extensive redox cycling of the quinone under the culture conditions. RPT cell monolayers metabolise MGNQ into a cysteinylglycine conjugate, which after intramolecular cyclization yields 2H-(3-glycinyl)-9-hydroxy-10-methyl-1,4-naphthothiazine. Acivicin, an inhibitor of gamma-glutamyltranspeptidase, inhibited the formation of this 1,4-napthothiazine adduct. The second product formed is 1,4-napthothiazine formed by loss of glycine via the action of dipeptidases. Similarly, no basolateral (B) to apical (A) transport of a GSH-conjugated alpha, beta unsaturated ketone, [14C]ethacrynic acid (EASG), occurred. However, net transport of [14C] radioactivity could be observed from A=>B direction. After 8 h, 23% of total [14C] radioactivity was transported from the apical to the basolateral chamber. In both the apical and basolateral chambers, free, unconjugated ethacrynic acid (EA) was observed. gamma GT-mediated metabolism of EASG to the much more unstable cysteinylglycine conjugate leads to relatively large amounts of free EA. Thus, the GSH conjugate is not transported but rather the cysteine adduct and/or free, unconjugated EA. In agreement with this, acivicin reduced A=>B transport of EASG and inhibited the formation of free EA. In conclusion, the confluent monolayers of RPT cells do not or no longer possess active basolateral transport systems for GSH conjugates. However, they are still quite useful for studying biotransformation reactions of thioether conjugates.

Animals↗
PubMed1997

Characterization of chicken-liver glutathione S-transferase (GST) A1-1 and A2-2 isoenzymes and their site-directed mutants heterologously expressed in Escherichia coli: identification of Lys-15 and Ser-208 on cGSTA1-1 as residues interacting with ethacrynic acid.

Escherichia coli-expressed chicken-liver glutathione S-transferase, cGSTA1-1, displays high ethacrynic acid (EA)-conjugating activity. Molecular modelling of cGSTA1-1 with EA in the substrate binding site reveals that the side chain of Phe-111 protrudes into the substrate binding site and possibly interacts with EA. Replacement of Phe-111 with alanine resulted in an enzyme (F111A mutant) with a 4.5-fold increase in EA-conjugating activity (9.2 mmol/min per mg), and an incremental Gibbs free energy (DeltaDeltaG) of 4.0 kJ/mol lower than that of the wild-type cGSTA1-1. Two other amino acid residues that possibly interact with EA are Ser-208 and Lys-15. Substitution of Ser-208 with methionine generated a cGSTA1-1(F111AS208M) double mutant that has low EA-conjugating activity (2.0 mmol/min per mg) and an incremental Gibbs free energy of +3.9 kJ/mol greater than the cGSTA1-1(F111A) single mutant. The cGSTA1-1(F111A) mutant, with an additional Lys-15-to-leucine substitution, lost 90% of the EA-conjugating activity (0.55 mmol/min per mg). The Km values of the cGSTA1-1(F111A) and cGSTA1-1(F111AK15L) mutants for EA are nearly identical. The wild-type cGSTA2-2 isoenzyme has a low EA-conjugating activity (0.56 mmol/min per mg). The kcat of this reaction can be increased 2. 5-fold by substituting Arg-15 and Glu-104 with lysine and glycine respectively. The KmEA of the cGSTA2-2(R15KE104G) double mutant is nearly identical with that of the wild-type enzyme. Another double mutant, cGSTA2-2(E104GL208S), has a KmEA that is 3.3-fold lower and a kcat that is 1.8-fold higher than that of the wild-type enzyme. These results, taken together, illustrate the interactions of Lys-15 and Ser-208 on cGSTA1-1 with EA.

Amino Acid Sequence↗
PubMedDate not supplied

Evidence for intracochlear impedance changes following ethacrynic acid administration.

The effects of intra-arterial 30-, 40-, and 50-mg/kg doses of ethacrynic acid upon cochlear function in guinea pigs were studied for periods of three to five hours. Cochlear potentials recorded in the first turn included the endocochlear potential, whole nerve response, cochlear microphonics, and summating potentials in scala media, scala tympani, and scala vestibuli. Evidence of organ of Corti damage at 50 mg/kg was found in addition to electrical impedance changes in the cochlear membranes at all dose levels.

Animals↗
PubMed1991

High-performance liquid chromatographic determination of ethacrynic acid in human plasma.

A high-performance liquid chromatographic method for the determination of ethacrynic acid (EA) in human plasma is described. Plasma was prepared for analysis by addition of 4-(2,4-dichlorophenoxy)-butyric acid as an internal standard followed by acidification with hydrochloric acid and extraction with ethyl acetate. Separation was by isocratic reversed-phase chromatography, the column effluent was monitored at 280 nm and quantitation was performed using peak-area ratios. The linear range for EA determination was from 0.5 to 25 micrograms/ml with a lower limit of detection of 0.1 microgram/ml. The reported method is convenient, sensitive and reproducible, illustrating its usefulness for pharmacokinetic studies.

Acetates↗
PubMed1970

[Ethacrynic acid].

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Ethacrynic Acid↗
PubMedDate not supplied

The delayed effects of ethacrynic acid on the stria vascularis of the guinea pig.

Guinea pigs were administered 40 mg ethacrynic acid per kg b.wt. and sacrificed at 30-48 minutes, 3-4 hours, 2 or 7 days post-drug. Cochlear potentials (EP and CP) were monitored before sacrifice. At 30-48 minutes, the potentials had decreased considerably, and a marked edema plus cytological changes were visible in the stria vascularis. The potentials had recovered to about 75% of their original value at 3-4 hours; some cell recovery was visible, but the edema was still present. Potentials recorded from the basal turn were normal at 2 and 7 days, although some strial cells showed deterioration.

Action Potentials↗
PubMed1974

The effect of ethacrynic acid on the guinea-pig and rat isolated vas deferens.

1 The effect of ethacrynic acid (EA) was studied on guinea-pig and rat vas deferens in vitro.2 EA contracted the guinea-pig but not the rat vas deferens in a dose-dependent manner (50-800 mug/ml). Tyramine caused contraction in 10 out of 18 guinea-pig vas deferens; EA caused contraction in 17 of the preparations which did not respond to tyramine. Repeated doses of EA produced tachyphylaxis, but there was no cross tachyphylaxis to tyramine.3 The contractions produced by EA were prevented by phentolamine or reserpine pretreatment and potentiated by cocaine. A low concentration of desipramine (3 ng/ml) potentiated and higher concentrations (0.6 and 3.0 mug/ml) inhibited the response of vas deferens to EA.4 Hexamethonium (100 mug/ml) or atropine (0.1 mug/ml) did not inhibit the effect of EA, excluding the nicotinic and muscarinic receptors as the sites of action.5 The effect of noradrenaline (NA) on the guinea-pig and rat vas deferens was enhanced by EA pretreatment, which may be due to inhibition of NA uptake.6 It is concluded that EA releases NA from guinea-pig vas deferens. The mechanism of release seems to be different from that of tyramine.

Animals↗
PubMed1975

Ethacrynic acid. Effects on the cochlear potentials in normal and high blood oxygen.

The effect of ethacrynic acid (EA) at different blood O(2) saturations on cochlear potentials of guinea pigs was investigated. All 18 young healthy guinea pigs received 50 mg/kg/h of EA intravenously and were divided into three groups: first group, normal (90.00+/-6.30-86.17+/-4.83 mm Hg); second group, lower Po(2) (78.00+/-4.74-70.00+/-4.42 mm Hg); and third group, high Po(2) (174.40+/-13.41-179.00+/-26.15 mm Hg). The partial pressure of oxygen (Po(2)), the partial pressure of carbon dioxide (Pco(2)), and the pH of the blood were measured before EA administration and at the end of the experiment (3 h later) by drawing blood samples from the contralateral carotid artery. Cochlear potentials-endocochlear potential (EP), cochlear microphonics (CM), and action potentials (AP)-were recorded by standard methods from the first turn of the cochlea. Experimental data seem to indicate that elevation of the Po(2) to 174-179 mm Hg during relatively high doses of EA treatment prevents the declines in cochlear potentials which were observed in the first and second groups (normal and lower Po(2)), and preserves active ion transport which is responsible for the generation of cochlear potentials. These data suggest a means by which to reduce the ototoxic effect of EA and possibly indicates a method of treatment for hearing loss which developed after the administration of EA.

Action Potentials↗
PubMed1978

The effects of ethacrynic acid upon the potassium concentration in guinea pig cochlear fluids.

After i.v. injection of 50 mg/kg ethacrynic acid (EA), potassium concentration in the endolymph (Ke+) measured with K+-specific microelectrodes decreases by 10 mM at the most and endocochlear potential falls to negative values. Potassium concentration in the perilymph (Kp+) generally does not change, but sometimes a transient decrease in Kp+ level of about 0.5 mM was observed, presumably due to the electrogenic effect of the time-related decrease of the endocochlear potential. When anoxia is induced approximately 120 min after EA administration Ke+ slowly decreases. The decrease in Ke+ 50 min after the arrest of ventilation is smaller when compared with the Ke+ anoxic decrease without preceding EA administration. The endocochlear potential, which falls to negative values during anoxia after EA administration, does not return to the zero level as in the case when only anoxia is applied. Similarly, during anoxia, which follows EA administration, the perilymphatic Ke+ concentration increases more slowly than in the case when only anoxia is introduced. It is assumed from the results that EA abolishes activity of the positive electrogenic K+ pump and reduces the passive permeability of the walls of the cochlear duct to the potassium ions.

Animals↗