Efficacy of treatment after measurable diabeticlike retinopathy in galactose-fed rats.

PURPOSE: To determine whether the diabeticlike retinal microangiopathies of the galactose-fed rat model could be ameliorated if intervention by withdrawal of the galactose diet or treatment with the aldose reductase inhibitor AL-3152 was initiated after quantifiable microangiopathies had occurred. METHODS: Weanling male Sprague-Dawley rats were randomized into five groups and fed for up to 24 months Purina laboratory chow (#5001) plus 50% starch (control [CON]), 50% D-galactose (galactose [GAL]), 50% D-galactose with AL-3152 (approximately 14 mg/kg per day) (prevention [PRV]), 50% D-galactose for 6 months followed by intervention with the inhibitor (intervention [INT]), or 50% D-galactose for 6 months followed by replacement with the 50% starch diet (withdrawal [GWD]). In rats on experimental diets and killed after 6, 18, and 24 months, one retina was prepared for transmission electron microscopy; the other was used for vessel wholemounts using elastase digestion. Capillary images were analyzed by computer morphometry. RESULTS: At 6 months, the GAL rats exhibited statistically significant (P < 0.05) increases over CON rats in mean capillary basement membrane thickness, capillary density, and dilated channels. These parameters tended to increase with time in most groups, and the differences between GAL and age-matched CON rats were maintained at the 18- and 24-month endpoints. Although the microangiopathies were ameliorated by AL-3152 treatment from the onset (PRV), intervention after 6 months of galactosemia with either galactose withdrawal (GWD) or addition of inhibitor (INT) showed amelioration in only some parameters at 18 months and no statistically significant benefit at the 24-month endpoint. CONCLUSIONS: Amelioration of galactose-induced retinal microangiopathies with AL-3152 in the prevention group suggests an efficacious application of aldose reductase inhibitors in treating diabetic retinopathy, provided treatment can begin soon after the onset of diabetes. Intervention after some of the earliest microscopic lesions neither halted progression of the angiopathy nor provided appreciable benefit at the 24-month follow-up.

Effects of two new aldose reductase inhibitors, AL-1567 and AL-1576, in diabetic rats.

Two new potent aldose reductase inhibitors, AL-1567 (DL-spiro(2-fluoro-9H-fluoren-9,4'-imidazolidine)-2',5'-dione) and AL-1576 (spiro-(2,7-difluoro-9H-fluoren-9,4'-imidazolidine)2',5'-dione), have been characterized with respect to in vitro activity toward rat lens and human placental aldose reductase and in vivo activity in uncontrolled, severely diabetic rats dosed acutely with the compounds. The IC50 values for inhibition of rat lens aldose reductase are 2.7 X 10(-8) mol/L for AL-1567 and 8.5 X 10(-9) mol/L for AL-1576; very similar IC50 values were measured for each compound with the human placental enzyme. When the compounds were administered orally once per day to 3-week diabetic rats for a period of eight days, the ED50 values for normalization of lens sorbitol levels were 0.60 mg/kg for AL-1567 and 0.05 mg/kg for AL-1576, and for normalization of sciatic nerve sorbitol levels; 0.22 mg/kg for AL-1567 and 0.04 mg/kg for AL-1576. Compared with published data on other aldose reductase inhibitors evaluated in very similar diabetic rat models, both compounds have unusually high activity in lens, and AL-1576 appears to be the most active such compound in both lens and sciatic nerve reported thus far. The evidence linking increased sorbitol pathway activity to diabetic complications, such as cataract and neuropathy in animal models, suggests that aldose reductase inhibitors will be useful therapeutic agents in human diabetics.

High-performance liquid chromatographic assay of the aldose reductase inhibitor spiro-(2-fluoro-9H-fluorene-9,4'-imidazolidine)-2',5'-dione (AL01567) in plasma and urine and its pharmacokinetics in humans.

Two selective high-performance liquid chromatographic (HPLC) methods have been developed for the quantitative determination of spiro-(2-fluoro-9H-fluorene-9,4'-imidazolidine)-2',5'-dione (AL01567; 1) in plasma and urine, with an assay sensitivity of 0.25 micrograms/mL for plasma and 0.13 micrograms/mL for urine. The plasma assay procedure involved precipitation of proteins with acetonitrile followed by dilution with water. The diluted supernatant was analyzed on an ODS column eluting with acetonitrile:0.5% phosphoric acid (30:70) adjusted to pH 7.2 with concentrated ammonium hydroxide. The urine assay procedure involved extraction of 1 with 10% n-butanol in hexane, followed by back extraction with 0.05 M sodium hydroxide. The basic extract was neutralized and analyzed on a phenyl column eluting with acetonitrile:10 mM potassium phosphate (30:70; monobasic, pH 5.6). The pharmacokinetics of 1 was investigated in humans following single and multiple oral doses. The elimination half-life from 12 normal subjects following single 100-400-mg oral doses was independent of dose, and the overall mean half-life was 66 +/- 9 h. The overall mean oral clearance (assuming a bioavailability of 100%) was 11 +/- 3 mL/min, and the mean apparent volume of distribution was 59 +/- 13 L. The mean urinary recovery of intact drug during the first 24 h after dosing was 1.2 +/- 0.4% of the administered dose. During once daily 100-mg oral dosing of 1 to five subjects for 21 d, plasma concentrations of 1 reached apparent steady-state by 7 d.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione depletion in the lens of galactosemic and diabetic rats.

Depletion of lens glutathione (GSH) occurs quickly and drastically following induction of diabetes or galactosemia in rats as well as in lens culture. The explanation for this dramatic loss of GSH has been investigated by many laboratories but the solution has been elusive. There are several possible causes for the change in the reducing power of the lens under hyperglycemia. (a) The enzyme glutathione reductase which reduces oxidized glutathione to GSH is inhibited. (b) The cofactor NADPH which both the aldose reductase of polyol pathway and glutathione reductase require becomes depleted under hyperglycemia to the point that there is an insufficient amount for glutathione reduction. (c) Membrane permeability is increased, due to osmotic-induced lens hydration. We explored all the above possibilities in the mechanism of GSH depletion and studied the effect of aldose reductase inhibitor (ARI) on osmotic change. We found that under hyperglycemic condition, there was no change in the enzyme glutathione reductase activity. There was an initial drop in NADPH level but there was sufficient remaining for glutathione reductase use. Both NADPH and glutathione depletion could be prevented completely by ARI. In addition, ARI could also prevent any hyperglycemic-induced abnormal transport and leakage of amino acids. We have therefore concluded that only the decreased membrane transport of amino acids which are needed for glutathione biosynthesis and the simultaneous loss of GSH through leaky membrane as initiated by the polyol pathway can be responsible for the drastic GSH depletion.

Metabolism of the aldose reductase inhibitor ALO1567 in man.

1. The metabolism of the aldose reductase inhibitor, ALO1567, was studied in man. The major biotransformation pathway was aromatic hydroxylation followed by glucuronide conjugation. 2. Hydroxylation occurred at several positions on the fluorene ring. The major metabolite was identified as the 7-hydroxy analogue of ALO1567 and three minor metabolites were characterized as positional isomers of the 7-hydroxy metabolite. 3. Oxidative defluorination and metabolism on the hydantoin ring were also indicated as minor pathways. 4. The capacity of normal subjects to oxidize ALO1567 was indicated by the urinary ratio of the parent drug to the 7-hydroxy metabolite after daily oral administration of 100 mg and 200 mg of ALO1567. Most subjects having higher ALO1567 plasma concentrations showed higher ratios.

The prevention of biochemical changes in lens, retina, and nerve of galactosemic dogs by the aldose reductase inhibitor AL01576.

Normal eight month old beagle dogs were fed a diet of 30% galactose for a period of two weeks. One group of dogs was untreated while three other groups were orally dosed with 0.25, 1.0, and 5.0 mg/kg of the aldose reductase inhibitor (ARI), AL01576. No visible changes were observed in the lens but glutathione (GSH) and inositol were depleted while dulcitol was elevated. These biochemical changes closely parallel those found in the (two week) streptozotocin induced diabetic rat. In contrast with the diabetic rat model, retina and nerve inositol was not found to differ from normal in spite of significant dulcitol accumulation. Plasma AL01576 was found to be inversely correlated with lens dulcitol concentration. When plasma AL0P1576 concentration was greater than 1 microgram/mL (5 mg/kg), there was a 95% reduction in dulcitol concentration (relative to untreated), while concentrations of 0.2 to 0.2 mg/mL (1 mg/kg) of AL01576 resulted in a dulcitol reduction of at least 70%. Retina and nerve dulcitol concentrations of galactosemic dogs were similarly diminished by AL01576 treatment. The dog model exhibits a biochemical profile of change and responsiveness to ARI therapy similar to that observed in hyperglycemic rats. Changes in retina morphology in diabetic and galactosemic dogs has been shown to closely resemble those occurring in human diabetics; these early biochemical events may also parallel.

The effect of an aldose reductase inhibitor on lens phosphorylcholine under hyperglycemic conditions: biochemical and NMR studies.

Phosphorylcholine (P-choline) is a precursor of the phospholipids in the lens membrane. A human lens normally contains approx. 1 mM P-choline but this is significantly lowered in some cataractous lenses. A normal rat lens contains a very high concentration (11 mM). We found that rat lens P-choline was depleted drastically when the lenses were exposed to hyperglycemic conditions either in culture, with galactose or xylose, or in vivo by streptozotocin-induced diabetes. The lens P-choline level was measured by fractionating the organic phosphates in the lens homogenate using an ion exchange column, or by quantitating the P-choline 31P NMR intensity in intact lenses. The results of both the chemical method and the noninvasive method agreed remarkably well. Besides the change in P-choline, the choline influx was also drastically reduced both in lenses from diabetic rats and in lenses incubated with 30 mM xylose. In addition, the ATP concentration was greatly diminished under similar conditions. The changes in P-choline, choline, and ATP could all be prevented in the presence of an aldose reductase inhibitor (ARI). It is thus concluded that these changes in phospholipid precursors may result from lenticular membrane defects caused by hyperglycemic stress. The effect of the lowered precursors on lipid biosynthesis was observed, and surprisingly showed a more rapid phospholipid-biosynthesis in the 2-week diabetic rat lens than in the 3-day diabetic rat lens.

Comparison of the pharmacokinetics and pharmacodynamics of the aldose reductase inhibitors, AL03152 (RS), AL03802 (R), and AL03803 (S).

The pharmacokinetics of AL03152 (RS) and its enantiomers, AL03802 (R) and AL03803 (S), were studied in the Sprague-Dawley rat following intravenous bolus administration. The enantiomers had differing pharmacokinetic profiles, while the racemic compound exhibited pharmacokinetic parameters approximating the mean values of the individual enantiomers. The total clearance (CLT) values of the two enantiomers were similar, but the intrinsic clearance (Cl(int)) was much greater for the S-enantiomer than for the R-enantiomer. The volume of distribution (Vss) for AL03802 (R) was threefold greater than that for AL03803 (S). The stereoselectivity in Vss could not be totally accounted for by the slight difference in serum protein binding of the isomers and resulted in a difference in the half-lives of the enantiomers. Only the R-isomer exhibited a persistent terminal elimination phase, consistent with more extensive tissue binding than the S-isomer. AL03152 enantiomers were equivalent in potency assessed from in vitro IC50 values toward rat lens aldose reductase and rat kidney L-hexonate dehydrogenase and lens EC50 values in diabetic rats.