The structure of human aldose reductase bound to the inhibitor IDD384.

The crystallographic structure of the complex between human aldose reductase (AR2) and one of its inhibitors, IDD384, has been solved at 1.7 A resolution from crystals obtained at pH 5.0. This structure shows that the binding of the inhibitor's hydrophilic head to the catalytic residues Tyr48 and His110 differs from that found previously with porcine AR2. The difference is attributed to a change in the protonation state of the inhibitor (pK(a) = 4.52) when soaked with crystals of human (at pH 5.0) or pig lens AR2 (at pH 6.2). This work demonstrates how strongly the detailed binding of the inhibitor's polar head depends on its protonation state.

Tissue selective inhibition of prostaglandin biosynthesis by etodolac.

Inflammation is a complex process involving numerous mediators. Because prostaglandins (PG) have been implicated as mediators in all stages of inflammation, inhibition of their synthesis provides the basis for the therapeutic effects of nonsteroidal antiinflammatory drugs (NSAID). Treatment with NSAID is usually accompanied by gastric side effects, attributed to interference with the formation of cytoprotective PG in gastric mucosa. An ideal NSAID should inhibit PG synthesis at the site(s) of inflammation but not in gastric mucosa. Experimental and clinical data support the view that this criterion has been met by etodolac, a structurally distinct NSAID. Thus, in rats and humans with rheumatoid arthritis, longterm daily administration of etodolac at effective antiinflammatory dosages (3 mg/kg in rats; 600 mg in humans) had no effect on PGF2 and prostacyclin levels in gastric mucosa. In contrast, significant decreases in gastric PG levels occurred with antiinflammatory doses of aspirin, naproxen, and piroxicam. Cyclooxygenase (COX), the pivotal enzyme in PG biosynthesis, exists in 2 isoforms: constitutive COX-1, which produces the PG required for maintenance of normal cell activity (e.g., gastric cytoprotection), and COX-2, which is induced in restricted tissue-specific fashion (e.g., by inflammatory stimuli). The antiinflammatory action of NSAID may result from inhibition of COX-2, whereas their gastric side effects may result in large part from inhibition of COX-1; thus, a preferred NSAID should inhibit COX-2 but not COX-1. Results show that etodolac has 10-fold selectivity for COX-2 and indicate that etodolac's pharmacotherapeutic efficacy can be explained by its demonstrably selective inhibition of COX-2, amplified by its favorable tissular pharmacokinetics. The sparing of COX-1 activity in gastric mucosa gives rise to etodolac's noted gastric tolerance.

Aminoguanidine does not inhibit aldose reductase activity in galactose-fed rats.

Aminoguanidine, nucleophilic hydrazine derivative, has been shown to inhibit diamine oxidase, the formation of advanced glycation endproducts, nitric oxide synthase, and catalase. Prompted by the reports that aminoguanidine also inhibits aldose reductase (AR), we have investigated the effect of aminoguanidine, 1,3-diaminoguanidine, and methylguanidine on AR activity in vitro, and in vivo. In vitro, we have measured the inhibition of AR isolated from bovine lenses; in vivo, we have examined the effect on the galactitol levels in the red blood cells, sciatic nerve, retina, and lens of rats administered the test compounds for 11 days in the drinking water and, for the last 4 days, given access to a 20% galactose diet. Two known, structurally distinct AR inhibitors, tolrestat and compound WAY-121,509, were used as reference. In vitro, at concentrations up to 1.0 mmol/L, none of the tested guanidine derivatives had any effect on AR. As a corollary, in vivo, at doses ranging from 201 to 349 mg/kg/day, none of the guanidine derivatives affected tissular galactitol levels. We conclude that, in short-term galactose-fed rats, at the doses tested, aminoguanidine, 1,3-diaminoguanidine, and methylguanidine do not inhibit AR.

Tolrestat pharmacokinetics in rat peripheral nerve.

The clinical efficacy of an aldose reductase (AR) inhibitor in diabetic polyneuropathy depends on its bioavailability at the site(s) of AR in peripheral nerves. Accordingly, the link between the concentration of the AR inhibitor, tolrestat, and the extent of its inhibition of the AR-catalyzed polyol production was investigated in sciatic nerves of galactosemic rats. Tolrestat was administered by gavage (1 x 150 mg/kg, or 5, and 15 mg/kg/day for 15 days to attain steady state as estimated from the 53-h half-life of tolrestat determined in rat nerve); subsequently, at six time intervals, ranging from 4 to 59 days, rats were given access for 4 days to a 20% galactose diet, and killed. At every time point, the composite tolrestat concentration in the nerve correlated with the percentage decrease in nerve galactitol (r = 0.857, p = 0.0015). Because the latter should reflect the extent of nerve AR inhibition by tolrestat, the concentration of "free" tolrestat available at the site(s) of AR in the nerve was estimated from the tolrestat concentration/percent AR inhibition plot obtained in vitro. The estimated amount of tolrestat present at the site(s) of nerve AR represented 0.4% of the composite tolrestat concentration measured in the nerve. The results support the view that the effectiveness of an AR inhibitor in peripheral nerve depends on its pharmacokinetics in the nerve, i.e., on its uptake, nonspecific binding to cellular constituents, and elimination.

Aldose reductase inhibitors as pathobiochemical probes.

In tissues susceptible to damage from chronic diabetes, excess glucose is metabolized by aldose reductase (AR) to sorbitol. Originally, AR-catalyzed sorbitol formation (and accumulation) was found in the diabetic lens; the cataractogenicity of this process was proven by preventing cataract formation with an AR inhibitor (ARI). These findings were extended to the hypothesis that, in diabetic tissues, excessive intracellular sorbitol formation initiates a cascade of metabolic abnormalities which gradually progress to loss of functional and structural integrity. The pivotal role of AR as a trigger for such abnormalities was established by preventing their occurrence in diabetic animals treated with an ARI. By inference, this led to the concept that inhibition of AR should prevent, arrest, and, possibly, reverse the development of late diabetic sequelae. In addition to motivating drug-oriented research, the ARI concept provided a rationale for the use of ARIs as experimental tools to probe the pathogenesis of diabetic complications. By helping to elucidate the metabolic, functional, and structural ramifications of the AR-catalyzed disposal of excess glucose in diabetic schemes, and in addition, by helping to define the applicability of animal models for the study of early functional pathogenic alterations occurring in diabetic subjects, ARIs may enable the discrimination in diabetic tissue of arrestible and reversible from the irreversible abnormalities.

Theoretical mechanism for the gastrointestinal safety of etodolac: selective sparing of cytoprotective prostaglandins.

The high concentrations of pro-inflammatory prostaglandins (PGs) produced in the joint during the initial stage of inflammation can be decreased by inhibiting their biosynthesis with nonsteroidal anti-inflammatory drugs (NSAIDs). The commonly encountered gastrointestinal intolerance in human subjects treated with NSAIDs is generally attributed to inhibition of PG synthesis in gastric mucosa, where the natural role of locally biosynthesized PGs is to protect the mucosa from necrosis upon exposure to noxious agents. The action of an ideal NSAID should therefore be tissue specific, i.e., it should inhibit PG formation at the sites of inflammation but not in gastric mucosa, where PGs are needed for cytoprotection. We believe that etodolac, a new, structurally distinct NSAID, meets this criterion, inhibiting PG synthesis in a tissue-specific fashion. Experimental data supporting this interpretation were obtained in rats by demonstrating that daily administration of orally effective anti-inflammatory doses of etodolac had no significant effect on gastric mucosal PGE2 or prostacyclin levels (measured as the stable metabolite, 6-keto-PGF1 alpha). In contrast, naproxen, piroxicam, and aspirin caused a statistically significant PG depletion. The results obtained in rats thus support the view that the favorable gastrointestinal safety profile of etodolac in human patients may be attributable to selective sparing of the cytoprotective PGs in gastric mucosa.

Prevention of urinary albumin excretion in 6 month streptozocin-diabetic rats with the aldose reductase inhibitor tolrestat.

Recent clinical data strongly suggest that elevated urinary albumin excretion (UAE) identifies diabetic subjects at risk of developing nephropathy. Elevated UAE is attributed to increased transglomerular pressure, which is associated with poor metabolic control in rats. Because excess glucose in diabetes is metabolized via the polyol pathway, we were interested in whether the diabetes-induced elevation in UAE in rats could be prevented by inhibiting aldose reductase (AR), the first enzyme in the polyol pathway, with the AR inhibitor tolrestat. In fact, in rats made diabetic with streptozocin (35 mg/kg IV), treatment for 6 months with tolrestat (25 mg/kg/day in the diet) prevented both sorbitol accumulation in the kidney and the increase in UAE. Sorbitol accumulation and the increased UAE were not associated with statistically significant mesangial expansion, and the thickening of glomerular basement membranes was not affected by tolrestat treatment. The authors conclude that the 4.7-fold elevation in UAE in chronically diabetic rats is linked to the increased flux of glucose through the polyol pathway since it was prevented by inhibiting aldose reductase with tolrestat.

Prevention of neural myoinositol depletion in diabetic rats by aldose reductase inhibition with tolrestat.

The effect of the aldose reductase inhibitor tolrestat on the sugar and polyol contents in the sciatic nerve was investigated in male Wistar and Sprague-Dawley rats rendered diabetic with streptozocin. At a daily oral dose of 5 mg/kg, given for 10 days before and for 14 days after streptozocin injection, tolrestat completely prevented the accumulation of sorbitol and the depletion of myoinositol.

Relationship between plasma propranolol concentration and dose in young, healthy volunteers.

The relationship between dose and bioavailability of propranolol was determined in 12 healthy male subjects. The doses employed were 10, 40, 80 or 160 mg of propranolol HCl given three times daily for 4 days. Daily minimum plasma levels showed that there was no accumulation of the drug. In all subjects, non-linear relationships were observed between peak plasma level, or area under the plasma concentration-time curve, and dose. The between subject variation of these parameters was not dose related.

The metabolic disposition of acifran, a new antihyperlipidemic agent, in rats and dogs.

The metabolic disposition of the antihyperlipidemic agent acifran (AY-25, 712) was determined in rats and dogs. The synthesis of 14C-labelled acifran is described. Serum levels of 14C and acifran were measured in rats and dogs after p.o. and i.v. administration of 14C-acifran at a dose of 10 mg/kg. Over 80% of the 14C in serum was due to acifran. The drug was rapidly absorbed and the pharmacokinetics, unaffected by increasing the dose or by daily multiple doses, were characterized by a two-compartment open model. Food reduced the bioavailability of acifran by 27% in the dog. About 65% of the dose was absorbed in rats, and at least 88% in dogs. The elimination t 1/2 of acifran from serum was 1.5 h in the rat and 3 h in the dog. Acifran was partially bound to serum proteins, man greater than rat greater than dog; the drug was found to displace protein-bound warfarin in rat and dog, but not in human serum. Radioactivity did not tend to accumulate in tissues, except for the kidney, where the 14C concentration was five times higher than in the serum; elimination of 14C from all the tissues was similar to that from serum. Most of the absorbed dose was excreted in the urine. Acifran did not undergo enterohepatic circulation in the rat. Virtually all the urinary 14C in both species was due to the unchanged compound. In conclusion, the disposition of acifran was similar in rats and dogs. The drug was rapidly absorbed and eliminated, and underwent no detectable biotransformation. There was no tissue retention and excretion was mainly in the urine.

Prevention of basement membrane thickening in retinal capillaries by a novel inhibitor of aldose reductase, tolrestat.

A diabetic-like thickening of retinal capillary basement membranes induced in rats fed for 207 consecutive days a diet containing 50% galactose was prevented by the addition to the diet of tolrestat, a potent, structurally novel inhibitor of aldose reductase. Analysis of electron micrographs (X 25,000) of capillaries from the outer plexiform layer of the retina by computer planimetry showed that the basement membranes were approximately twofold thicker in rats fed galactose than in those fed either a standard diet or a diet containing galactose and tolrestat in doses of 43 or 57 mg/kg/day. The thickening of basement membranes in galactose-fed rats was accompanied by other ultrastructural alterations mimicking changes typical of diabetic microangiopathy, such as multilamination and the formation of vacuoles and dense inclusions. Therefore, the galactosemic rat represents a useful model for studying basement membrane-related complications of diabetes and their possible prevention by aldose reductase inhibitors.

Disposition and biotransformation of 14C-etodolac in man.

Four human subjects were given a capsule containing 200 mg of 14C-etodolac. At the peak (two hours after dosing), most of the radioactivity in serum was due to etodolac; subsequently, metabolites gradually appeared. The elimination half-life of etodolac from serum averaged six hours. Etodolac was greater than 99% bound to human serum proteins. An average of 73% of the dose was excreted in the urine and 14% in faeces within seven days, with 61% appearing in the urine during the first 24 h. Microbial transformation of etodolac was employed to biosynthesize sufficient amounts of two urinary metabolites to facilitate structure elucidation. Five metabolites, representing 65% of the radioactivity in urine collected 0-24 h after dosing (61% of the dose was excreted in urine within 24 h), were isolated and characterized by t.l.c., g.c., h.p.l.c., n.m.r (1H and 13C) and m.s. Most of the identified urinary components were conjugates of etodolac and three hydroxylated metabolites (6-hydroxyetodolac, 7-hydroxyetodolac and 8-(1-hydroxyethyl)etodolac). Two metabolites were identified as glucuronyl ester conjugates of etodolac and 7-hydroxyetodolac; the former represented about 20% of the urinary radioactivity. False positive tests for bilirubin in urine of patients treated with etodolac were found to be due to the two phenolic metabolites.

Effect of tolrestat on red blood cell sorbitol levels in patients with diabetes.

The effect of the aldose reductase inhibitor, tolrestat, on red blood cell (RBC) sorbitol levels was studied in 23 patients with diabetes after oral dosing with tolrestat, 25 or 100 mg b.i.d. The mean (+/- SE) RBC sorbitol levels (measured 12 hours after the preceding dose) after 3, 7, and 13 days of dosing decreased after both dose levels. After 25 mg tolrestat the RBC sorbitol levels fell from 25.1 +/- 4.0 to 20.0 +/- 5.7 nmol/gm hemoglobin (21%) and after 100 mg tolrestat the level fell from 26.7 +/- 3.7 to 11.4 +/- 1.7 nmol/gm hemoglobin (57%; P less than 0.001). This latter RBC sorbitol concentration is similar to levels in individuals without diabetes. At both dosage levels the maximum decrease in RBC sorbitol levels occurred after only 3 days of dosing. Tolrestat had no effect on plasma glucose or hemoglobin A1 concentrations. The overall mean plasma unbound drug concentration measured 12 hours after 100 mg tolrestat (11.7 +/- 3.0 ng/ml; 3.3 X 10(-8) mol/L) was similar to the median inhibitory level (3 X 10(-8) mol/L) of tolrestat for sorbitol accumulation in human RBCs incubated in a high-glucose medium. Our results demonstrate the systemic bioavailability of tolrestat and its aldose reductase inhibitory activity in erythrocytes of patients with diabetes.

The effects of a new aldose reductase inhibitor (tolrestat) in galactosemic and diabetic rats.

Tolrestat(N-[[5-(trifluoromethyl)-6-methoxy-1-naphthalenyl] thioxomethyl]-N-methylglycine; AY-27,773; Alredase) is a potent, structurally novel inhibitor of aldose reductase (AR). In vitro, tolrestat inhibited in dose-dependent fashion the AR from bovine lenses (IC50, 3.5 X 10(-8) mol/L) and the formation of sorbitol in human RBC incubated with glucose (IC50, 3 X 10(-8) mol/L). Upon administration with the diet to rats made galactosemic or diabetic, tolrestat decreased, in a dose-related fashion, the accumulation of galactitol or sorbitol in the sciatic nerve and lens. The effectiveness of tolrestat depended upon the experimental conditions and tended to be higher in less severe galactosemia and after suitable pretreatment, particularly in galactosemic rats, resulting in ID50 of 5 mg/kg/d in the sciatic nerve and 12-15 mg/kg/d in the lens. Tolrestat also decreased, in dose-related manner, the RBC sorbitol levels in normal and in streptozotocin diabetic rats; in the latter, at less than 2 mg/kg/d, the RBC sorbitol was reduced to control levels.

Metabolic disposition and pharmacokinetics of the aldose reductase inhibitor tolrestat in rats, dogs, and monkeys.

The metabolic disposition and pharmacokinetics of the aldose reductase inhibitor tolrestat were studied in rats, dogs, and assamese and capuchin monkeys. In addition, the ocular penetration of tolrestat was examined in rabbits. The bioavailability of tolrestat was 81% in rats and 68% in dogs. In contrast to rats, a major proportion of the serum 14C in dogs and monkeys was due to unchanged drug. The terminal elimination half-life of tolrestat in serum was 3.5 hr in rats, 11 hr in dogs, and 9 hr in monkeys; in both dogs and monkeys, the total body clearance was 200 ml/kg X hr, and the volume of distribution was 3 liters/kg. In rats and dogs, serum tolrestat concentrations were similar after single and multiple po doses, and were linearly dose-related up to 25 mg/kg, but increased disproportionately at higher doses. Tolrestat was at least 98% bound to rat and dog serum proteins. Except for organs associated with absorption and elimination, tissue 14C levels were lower than in serum of rats and capuchin monkeys, and there was no tissue 14C accumulation. The 14C from topically applied 14C-tolrestat readily penetrated into the eyes of rabbits. Liver microsomal cytochrome P-450 was virtually unaltered in tolrestat-treated rats. Tolrestat (and/or its metabolites) underwent enterohepatic circulation in rats. Most of the 14C from 14C-tolrestat administered po and iv to rats and dogs was excreted in the feces. Based on 14C excretion, the absorption of tolrestat was 84% in rats and 82% in dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance of the diabetic rat nerve to ischemic inactivation.

The resistance of the action potential to ischemic inactivation observed in diabetic patients has been reproduced in vivo in rat rendered diabetic with streptozotocin and, acutely, in normal rats given p.o. a load of glucose. The resistance phenomenon was not detected in galactosemic rats. The preservation of the action potential was reversed by the administration of insulin, but not by treatment with an aldose reductase (AR) inhibitor. The ischemic resistance is attributed to the metabolic availability of excess glucose to the nerve. AR does not appear to be involved in the phenomenon.

Prevention of cataract development in severely galactosemic rats by the aldose reductase inhibitor, tolrestat.

With a fixed time period of galactose feeding, the rate of appearance of lenticular opacities depended on the severity of galactosemia, while with a fixed amount of galactose fed, the rate was time dependent. The capacity of tolrestat, a structurally novel inhibitor of aldose reductase (AR), to control cataract development was assessed in rats fed 30-50% galactose with the diet for 7 to 277 days. In rats fed 30% galactose for 31 days, the controlling effect of tolrestat was dose dependent, and no cataracts were detected at a dose of 35 mg/kg/day. In rats given tolrestat with the diet for 14 days, then rendered severely galactosemic with a diet containing 50% galactose, and subjected to continued treatment with tolrestat at a dose of 43 mg/kg/day, no changes were detected by slit-lamp microscopy after 207 days. The preventive effect was also dose dependent. In view of the established similarity in the pathogenesis of galactosemic and diabetic cataracts, the results obtained with tolrestat support its potential for controlling cataract development in diabetics.

Etodolac: effect on prostaglandin concentrations in gastric mucosa of rats.

Etodolac is a structurally novel compound exhibiting potent analgesic and anti-inflammatory activity in laboratory animals and man, with excellent G. I. tolerance. Like other nonsteroidal anti-inflammatory drugs (NSAIDs) etodolac inhibits prostaglandin (PG) biosynthesis. In view of the cytoprotective role of PGE2, we have investigated in normal rats the effect of etodolac on the gastric mucosal concentration of PGE2 as well as of 6-keto-PGF1 alpha, the stable metabolite of prostacyclin; naproxen and piroxicam served as reference NSAIDs. The orally effective anti-inflammatory doses in the chronic arthritic rat model (3 mg/kg for etodolac and naproxen; 0.5 mg/kg for piroxicam), and their arbitrarily selected multiples of 10 were used. Rats were killed at 1, 2, 6 and 24 hr after single doses and the PG concentrations were measured by RIA. With the low dose, 2 and 6 hr after dosing, etodolac diminished the PGE2 concentration by 20-25% (vs control) while naproxen and piroxicam caused a fall of 53-65%; the difference between etodolac and the untreated control group is not statistically significant but the difference between etodolac and both piroxicam and naproxen is significant (p less than 0.001). At the high doses, the lowering in PGE2 was similar after all three drugs, i.e. about 70% at 1 and 2 hr; 50% at 6 hr, and 20-50% at 24 hr after dosing. Except for the consistently smaller reduction of concentrations after etodolac, the effects on 6-keto-PGF1 alpha concentration followed a similar pattern but the differences are not significant. The lack of the G.I. irritation of etodolac in rats and man at therapeutically effective doses may be attributed to the benefits of the relatively short-lived and slight decrease in gastric mucosal PGE2 concentrations found in this study.

Tolrestat kinetics.

The kinetics of tolrestat, a potent inhibitor of aldose reductase, were examined. Serum concentrations of tolrestat and of total 14C were measured after dosing normal subjects and subjects with diabetes with 14C-labeled tolrestat. In normal subjects, tolrestat was rapidly absorbed and disappearance from serum was biphasic. Distribution and elimination t 1/2s were approximately 2 and 10 to 12 hr, respectively, after single and multiple doses. Unchanged tolrestat accounted for the major portion of 14C in serum. Radioactivity was rapidly and completely excreted in urine and feces in an approximate ratio of 2:1. Findings were much the same in subjects with diabetes. In normal subjects, the kinetics of oral tolrestat were independent of dose in the 10 to 800 mg range. Repetitive dosing did not result in unexpected cumulation. Tolrestat was more than 99% bound to serum protein; it did not compete with warfarin for binding sites but was displaced to some extent by high concentrations of tolbutamide or salicylate.