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.     

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.     

Effect of ethacrynic acid on the course of cytotoxic glomerulonephritis]

Ethacrynic acid (10 mg/kg subcutaneously 15 days) significantly facilitated the experimental glomerulonephritis in rats. The efficacy of diuretic was confirmed by the improvement of the functional characteristics (the degree of proteinuria, the plasma contents of protein and urea, the creatinine excretion), and by the results of the histological investigation, especially at the second phase of the pathological process.     

Stimulation of renal tubular transport by ethacrynic acid in rats of different ages.

The transport system for organic acids in the kidney is not fully developed in the neonatal period. The effect of repeated administrations of ethacrynic acid on the renal excretion of p-aminohippurate (PAH) was studied in rats of different ages. Pretreatment with ethacrynic acid was followed by an increase in the renal excretion of PAH in 33-, 55-, 105- and 240-day-old rats but not in newborn rats. In 55-day-old rats the increase in renal excretion of PAH after pretreatment with ethacrynic acid was not associated with any consistent change of the glomerular filtration rate. It is concluded from these results that the stimulation of transport processes in the kidney by ethacrynic acid and some other drugs is linked with their affinity to tissue proteins.     

Immediate short-time effects of ethacrynic acid on frog gastric mucosa in chloride-free solutions.

The presence of ethacrynic acid in the nutrient sulphate solution produces, generally in less than 3 min, a decrease in resistance accompanied by an increase in transmucosal PD. These results strongly suggest that the dominant effect of ethacrynic acid is not on the Na+ pathway of the nutrient membrane, but without further knowledge of the resistance of other ionic pathways, it cannot be inferred whether the effect predominates on the K+ pathway as in Cl- media.     

Culture duration alters the glutathione content and sensitivity to ethacrynic acid of rat hepatocyte monolayer cultures

Many of the differentiated functions of hepatocytes are lost in culture, yet addition of certain medium supplements can aid in the retention of differentiated character. Therefore, the effect of time in monolayer culture on rat hepatocyte glutathione (GSH) synthesis and sensitivity to the GSH detoxicated xenobiotic ethacrynic acid was examined in cultures with and without medium supplementation by transferrin and sodium selenite. GSH content was found to be about 12 nmol/µg DNA at 4 hr in culture and to approximately triple by 24 hr. Intracellular GSH levels continued to increase in transferrin/sodium selenite-supplemented cultures, from 32 to 41.6 nmol/µg DNA, while GSH levels in unsupplemented cultures declined to 18 nmol/µg DNA. However, the rate of GSH synthesis after diethylmaleate depletion was found to decrease from 4.2 to 2.8 nmol/hr/µg DNA at 4 and 24 hr after inoculation, respectively. GSH repletion rate increased to 3.9 nmol/hr/µg DNA at 48 hr. The GSH accumulation rate after depletion in supplemented cultures did not vary significantly over the initial 48 hr. Incubation for 3 hr with 100 µM ethacrynic acid (EA) did not elicit an increase in LDH leakage in hepatocyte monolayers after 4 or 48 hr in culture or in cultures with supplemented medium at any time point tested. Cultures 24 hr in medium without transferrin/sodium selenite supplementation exhibited significant LDH leakage after 3 hr of EA treatment. Over the 3 hr EA treatment, intracellular GSH content was decreased in all cultures. Only in the 24 hr unsupplemented cultures did GSH depletion exceed the 90% level previously associated with depletion of the mitochondrial pool of GSH and EA toxicity in hepatocytes. The experiments show that during the redifferentiation of hepatocytes in culture, a transient period occurs when apparent GSH synthesis is depressed and enhanced sensitivity to GSH-detoxicated compounds is observed. This period of increased sensitivity is prevented or at least delayed by inclusion of supplemental transferrin and sodium selenite, suggesting that redifferentiation can be regulated by extracellular influences.Abbreviations CYSSG cysteine-glutathione mixed disulfide - DEM diethyl maleate - EA ethacrynic acid - GSH reduced glutathione - GSSG oxidized glutathione - HBS HEPES buffered saline - HWME hepatocyte Williams' Medium E (WME with insulin, corticosterone and 0.5 mM methionine) - LDH lactate dehydrogenase - TS-HWME transferrin/sodium selenite-supplemented HWME - WME Williams' Medium E     

Reversible binding of ethacrynic acid to human serum albumin: Difference circular dichroism study

The reversible binding of ethacrynic acid was characterized by a difference circular dichroism method. A 2/1 stoichiometry was determined for the [drug]/[HSA] (human serum albumin) complex. The reversible binding of ethacrynic acid to HSA determines direct competition with ligands that selectivity bind to site II and to the fatty acid site. Furthermore, indirect competition was shown for ligands for site I (anticooperative) and to site III (cooperative). Chirality 11:33–38, 1999. © 1999 Wiley‐Liss, Inc.