sorbinil and imirestat

The effects of aldose reductase inhibitors on lens protein modifications induced by naphthalene-1,2-dihydrodiol were investigated in vitro to confirm the role of aldose reductase on naphthalene cataract formation. HPLC analysis of naphthalene-1, 2-dihydrodiol incubated with aldose reductase and NAD+indicated the formation of a metabolite peak corresponding to 1,2-naphthoquinone. Soluble proteins from rat lenses prepared by gel filtration of crude lens extracts through Sephadex PD-10, incubated with naphthalene-1, 2-dihydrodiol in the presence of NAD+displayed an absorbance ca 450 nm and their spectra were essentially identical to those of 1, 2-naphthoquinone-protein adducts. Similar spectra were also obtained from proteins isolated from the intact rat lens after in vitro incubation in medium containing naphthalene-1,2-dihydrodiol. The spectra obtained from lens proteins incubated with 1, 2-dihydroxynaphthalene were distinct from those of either naphthalene-1,2-dihydrodiol or 1,2-naphthoquinone. Aldose reductase inhibitors possessing either hydantoin or carboxylic acid groups prevented protein modification induced by naphthalene-1, 2-dihydrodiol but not protein modification induced by 1, 2-dihydroxynaphthalene or 1,2-naphthoquinone. Therefore, the metabolite formed from naphthalene-1,2-dihydrodiol by aldose reductase is 1,2-naphthoquinone. Lens proteins modified by naphthalene-1,2-dihydrodiol appear essentially identical to protein adducts formed with 1,2-naphthoquinone and their formation can be prevented by both hydantoin and carboxylic acid containing aldose reductase inhibitors.

Topics: Aldehyde Reductase; Animals; Cataract; Chromatography, High Pressure Liquid; Crystallins; Enzyme Inhibitors; Fluorenes; Hydantoins; Imidazoles; Imidazolidines; Lens, Crystalline; Naphthalenes; Naphthols; Naphthoquinones; Phthalazines; Rats; Spectrophotometry

Topics: Aldehyde Reductase; Animals; Enzyme Inhibitors; Eye Proteins; Fluorenes; Hydantoins; Imidazoles; Imidazolidines; Lens, Crystalline; Naphthalenes; Naphthoquinones; Oxidoreductases; Phthalazines; Rats

Naphthalene-induced cataract in rat lenses can be completely prevented by AL01576, an aldose reductase inhibitor (ARI). In an attempt to understand the mechanism of this inhibition, several ARIs were examined to compare their efficacies in preventing naphthalene cataract, using both in vitro and in vivo models. Two classes of ARIs were tested: One group including AL01576, AL04114 (a AL01576 analog) and Sorbinil contained the spirohydantoin group, while Tolrestat contained a carboxylic acid group. Furthermore, to clarify if aldose reductase played a role in naphthalene-induced cataractogenesis in addition to its role in sugar cataract formation, a new dual cataract model was established for ARI evaluations. This was achieved by feeding rats simultaneously with high galactose and naphthalene or incubating rat lenses in culture media containing high galactose and naphthalene dihydrodiol. Under these conditions, both cortical cataract and perinuclear cataract developed in the same lens. It was found that at the same dosage of 10 mg/kg/day, both AL01576 and AL04114 completely prevented all morphological and biochemical changes in the lenses of naphthalene-fed rats. Sorbinil was less efficacious, while Tolrestat was inactive. AL01576 showed a dose-response effect in preventing naphthalene cataract and at 10 mg/kg/day, it was also effective as an intervention agent after cataractogenesis had begun. With the dual cataract model, Tolrestat prevented the high galactose-induced cortical cataract but showed no protection against the naphthalene-induced perinuclear cataract. AL01576, on the other hand, prevented both cataract formations. Results for dulcitol and glutathione levels were in good agreement with the morphological findings. AL04114, and ARI as potent as AL01576 but without its property for cytochrome P-450 inhibition, displayed similar efficacy in preventing naphthalene cataract. Based on these results, it was concluded that the prevention of the naphthalene cataract probably results from inhibition of the conversion of naphthalene dihydrodiol to 1,2-dihydroxynaphthalene and that the effect of the ARIs cannot be explained by their inhibition of the dihydrodiol dehydrogenase activity of aldose reductase.

Topics: Aldehyde Reductase; Animals; Cataract; Culture Techniques; Disease Models, Animal; Enzyme Inhibitors; Fluorenes; Galactose; Hydantoins; Imidazoles; Imidazolidines; Male; Naphthalenes; Rats; Spiro Compounds

Sorbitol formation in rat lenses incubated with high levels of glucose was related to activation of aldose reductase (AR). The hyperglycaemia-activated aldose reductase was inhibited by alpha-lipoic (thioctic) acid, O-phenanthroline and aldose reductase inhibitors (ARIs) including Zeopolastat (ZPLS), Sorbinil (SBN) and AL-1576. This study also examined ARIs for the ability to chelate metal ions. We found that ARIs suppress copper-dependent ascorbate oxidation, lipid peroxidation and hydrogen peroxide production in erythrocytes. ARIs also increased partition of copper ions into noctanol, which indicates formation of lipophilic complexes. Our data support the hypothesis that transition metals may be involved in activation of the polyol (aldose reductase) pathway. Also, ARIs function as metal-chelating antioxidants that may contribute to their therapeutic role for diabetic complications.

Topics: 1-Octanol; Aldehyde Reductase; Animals; Ascorbic Acid; Chelating Agents; Copper; Enzyme Activation; Enzyme Inhibitors; Erythrocytes; Fluorenes; Glucose; Humans; Hydantoins; Hydrogen Peroxide; Hyperglycemia; Imidazoles; Imidazolidines; In Vitro Techniques; Ions; Lens, Crystalline; Lipid Peroxidation; NADP; Octanols; Oxidation-Reduction; Phenanthrolines; Rats; Thioctic Acid

It has been demonstrated that activation of aldose reductase (AR; EC 1.1.1.21) in diabetic tissues plays an important role in the pathogenesis of diabetic complications. In the present study, the effects of non-enzymatic glycation of recombinant human AR (rhAR) on enzyme activity and affinity for its substrate (glyceraldehyde), co-factor (NADPH) and inhibitors (ARI; Sorbinil, Tolrestat, AL-1576 and Statil) were examined. Although rhAR was successfully non-enzymatically glycated with HPLC-purified [3H]D-glucose, the Michaelis constant (Km) and catalytic efficiency (Kcat/Km) for glyceraldehyde, the Km for NADPH and the inhibitor constant (Ki) for ARI did not change. These results suggest that the mechanism of AR activation and its insensitivity to inhibition observed in diabetic tissues cannot be attributed to its non-enzymatic glycation.

Topics: Aldehyde Reductase; Enzyme Inhibitors; Fluorenes; Glucose; Glycosylation; Humans; Hydantoins; Imidazoles; Imidazolidines; Kinetics; Naphthalenes; Phthalazines; Recombinant Proteins; Substrate Specificity; Transfection

The role of His42, His188, and Lys263 residues in the catalytic action of human aldose reductase was investigated in association with various inhibitors of this enzyme by site-directed mutagenesis. While mutations at His42- greater than Gln, His42- greater than Tyr, His188- greater than Gln, and His188- greater than Tyr brought small change in the kinetic parameters, Lys263- greater than Glu mutation markedly increased the Km value for the substrate DL-glyceraldehyde by a factor of 60. Lys263- greater than Met substitution resulted in approximately 14 fold elevation of Km for the substrate. By contrast, mutation of Lys263- greater than Arg significantly decreased the Km for the substrate with concomitant reduction in kcat. Moderate increase in Km values for the cofactor NADPH was demonstrated for mutated enzymes. These results are indicative of the possible role of Lys263 in the substrate binding through electrostatic interaction. The inhibitor constants (Ki) for structurally diverse aldose reductase inhibitors against mutated enzymes demonstrated different degree of alteration, indicating binding sites of aldose reductase inhibitors on the enzyme molecule vary from one another, and some of the sites are more closely correlated with the physicochemical property of Lys263.

Topics: Aldehyde Reductase; Amino Acid Sequence; Base Sequence; Binding Sites; Cloning, Molecular; Fluorenes; Humans; Hydantoins; Imidazoles; Imidazolidines; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; NADP; Naphthalenes; Structure-Activity Relationship

Human lens epithelial (HLE) cells in tissue culture accumulated significant levels of galactitol when they were cultured for 72 hr in medium containing 30 mM D-galactose. Polyol accumulation was accompanied by the appearance of vacuoles as seen by transmission electron microscopy. The number and size of intracellular vacuoles increased when the culture period was extended to 7 days. In addition, polyol accumulation was accompanied by loss of myoinositol. None of these changes occurred in cells exposed to 30 M L-galactose which is not a substrate for aldose reductase. The accumulation of galactitol, intracellular vacuole formation and loss of myoinositol observed in D-galactose-exposed cells were prevented by the inclusion of the aldose reductase inhibitor, sorbinil, in the culture medium. Comparison of the relative efficacies of two aldose reductase inhibitors indicate that AL 1576 is nearly 20 times more potent than sorbinil in inhibiting the human lens enzyme. It is concluded that vacuole formation in HLE cells is due to the osmotic effect of polyol formation brought about by the action of aldose reductase and that the etiology of human diabetic cataract may also involve the polyol pathway.

Topics: Aldehyde Reductase; Cells, Cultured; Epithelial Cells; Epithelium; Fluorenes; Galactitol; Galactose; Humans; Hydantoins; Imidazoles; Imidazolidines; Inositol; Lens, Crystalline; Microscopy, Electron; Microscopy, Phase-Contrast; Polymers; Reference Values; Stereoisomerism; Vacuoles

Transition metal-catalysed oxidations have been implicated in the complications of diabetes. We report here that some experimental inhibitors of the enzyme aldose reductase (implicated in diabetes mellitus via its ability to catalyse glucose reduction to sorbitol) are also potent inhibitors of transition metal-catalysed ascorbate oxidation. The inhibition appears to be dependent upon the presence of a spirohydantoin group. It is conceivable that the copper- and iron-binding capacity of these compounds may contribute to some of their observed biological effects and may provide a starting point for a new generation of experimental drugs for the treatment of diabetes mellitus.

Topics: Aldehyde Reductase; Antioxidants; Ascorbic Acid; Copper; Diabetes Mellitus; Dose-Response Relationship, Drug; Fluorenes; Humans; Hydantoins; Imidazoles; Imidazolidines; Iron; Oxidation-Reduction; Structure-Activity Relationship

This study investigated hemodynamic changes in diabetic rats and their relationship to changes in vascular albumin permeation and increased metabolism of glucose to sorbitol. The effects of 6 wk of streptozocin-induced diabetes and three structurally different inhibitors of aldose reductase were examined on 1) regional blood flow (assessed with 15-microns 85Sr-labeled microspheres) and vascular permeation by 125I-labeled bovine serum albumin (BSA) and 2) glomerular filtration rate (assessed by plasma clearance of 57Co-labeled EDTA) and urinary albumin excretion (determined by radial immunodiffusion assay). In diabetic rats, blood flow was significantly increased in ocular tissues (anterior uvea, posterior uvea, retina, and optic nerve), sciatic nerve, kidney, new granulation tissue, cecum, and brain. 125I-BSA permeation was increased in all of these tissues except brain. Glomerular filtration rate and 24-h urinary albumin excretion were increased 2- and 29-fold, respectively, in diabetic rats. All three aldose reductase inhibitors completely prevented or markedly reduced these hemodynamic and vascular filtration changes and increases in tissue sorbitol levels in the anterior uvea, posterior uvea, retina, sciatic nerve, and granulation tissue. These observations indicate that early diabetes-induced hemodynamic changes and increased vascular albumin permeation and urinary albumin excretion are aldose reductase-linked phenomena. Discordant effects of aldose reductase inhibitors on blood flow and vascular albumin permeation in some tissues suggest that increased vascular albumin permeation is not entirely attributable to hemodynamic changes. We hypothesize that 1) increases in blood flow may reflect impaired contractile function of smooth muscle cells in resistance arterioles and 2) increases in vascular 125I-BSA permeation and urinary albumin excretion reflect impaired vascular barrier functional integrity in addition to increased hydraulic conductance secondary to microvascular hypertension associated with decreased vascular resistance.

Topics: Albuminuria; Aldehyde Reductase; Animals; Cardiac Output; Diabetes Mellitus, Experimental; Fluorenes; Glomerular Filtration Rate; Hemodynamics; Hydantoins; Imidazoles; Imidazolidines; Iodine Radioisotopes; Male; Naphthalenes; Rats; Rats, Inbred Strains; Reference Values; Regional Blood Flow; Serum Albumin, Bovine; Sorbitol; Strontium; Sugar Alcohol Dehydrogenases; Vascular Resistance

In the presence of 10(-8) M concentrations of the aldose reductase inhibitor AL 1576, there is a 20-30% increase in the rate of hydrolysis of near-saturating concentrations of ATP by bovine renal Na+-K+-ATPase. When bovine renal Na+-K+-ATPase is reacted with glucose 6-phosphate in the presence of 10(-8) M concentrations of AL 1576 or 10(-6) M concentrations of a second aldose reductase inhibitor, sorbinil, glucosylation occurs. Whereas sorbinil has no effect on ATP hydrolysis by the glucosylated Na+-K+-ATPase, 10(-8) M AL 1576 causes a shift in the kinetics of hydrolysis of ATP from substrate inhibition to normal substrate activation. The aldose reductase inhibitors interact with the enzyme at the low-affinity ATP-binding site.

Topics: Aldehyde Reductase; Animals; Cattle; Dose-Response Relationship, Drug; Fluorenes; Fluorescein-5-isothiocyanate; Fluoresceins; Glycosylation; Hydantoins; Imidazoles; Imidazolidines; Kidney Medulla; Sodium-Potassium-Exchanging ATPase; Sugar Alcohol Dehydrogenases; Thiocyanates