Bioavailability, multiple-dose pharmacokinetics, and biotransformation of the aldose reductase inhibitor zopolrestat in dogs

Zopolrestat (Alond) is a new drug that is being evaluated as an aldose reductase inhibitor for the treatment of diabetic complications. The bioavailability in dogs of a 2 mg/kg oral dose of zopolrestat was 97.2%. In a 1-year, multiple-dose, pharmacokinetic study, systemic exposure increased with increasing dose (50, 100, and 200 mg/kg/day), and there were no consistent changes in exposure with multiple dosing. Renal clearance at 1 year appeared to be higher in males. The magnitude of the potential gender difference in exposure was relatively small and was unlikely to have had a meaningful impact on the pharmacokinetics of zopolrestat in dogs. In studies with bile duct-cannulated dogs, radioactivity from [14C]zopolrestat was primarily eliminated as unchanged drug and acyl glucuronide in the bile and feces (77.3% of the dose) and in urine (18.3% of the dose). The concentrations of acyl glucuronide in urine and feces were approximately 50% of the zopolrestat concentrations. Minor metabolites (each accounting for <1% of the dose) included those resulting from hydroxylation of the phthalazinone ring and glutathione conjugation of the benzothiazole ring.     

An aldose reductase inhibitor and aminoguanidine prevent vascular endothelial growth factor expression in rats with long-term galactosemia

OBJECTIVE: To study the effects of an aldose reductase inhibitor (ARI-509, Wyeth-Ayerst, Princeton, NJ) and aminoguanidine (AMG), agents that have been reported to prevent or delay diabetic retinopathy, on retinal vascular abnormalities and the immunocytochemical expression in the retina of vascular endothelial growth factor (VEGF) in rats maintained for up to 2 years on a 50% galactose diet. METHODS: Albino rats were placed on a control diet, a diet containing 50% galactose, or the 50% galactose diet containing either ARI-509 or AMG. Treatment with ARI-509 or AMG was initiated at the beginning of the experiment or after 12 months of galactose feeding. After 22 to 24 months, the rats were killed and the retinal vasculature from half of one eye was isolated by trypsin-elastase digestion for semiquantitative evaluation of retinal vascular lesions. The other half of the retina was prepared for immunocytochemistry and stained for the presence of VEGF, factor VIII, vimentin, and glial fibrillary acidic protein. Red blood cells, sciatic nerves, and a portion of the retina from the second eye were assayed for glucose, galactose, fructose, sorbitol, galactitol, and myo-inositol. Red blood cells were also assayed for galactosylated hemoglobin. RESULTS: Galactose-fed animals developed a vascular retinopathy characterized by severe cellular loss in the retinal capillaries and intensification of periodic acid-Schiff staining of the vascular basement membranes. Some animals also displayed dilation and hypercellularity of vessels in the posterior retina. These changes were substantially reduced in animals receiving ARI-509 from the beginning of the galactose diet, but were unaffected in all of the other treatment groups. None of the rats receiving ARI-509 or AMG treatment, whether initiated from the onset or after 12 months of galactosemia, demonstrated VEGF immunoreactivity. With the exception of the animals receiving ARI-509 from the beginning of the experiment, all of the galactose-fed animals developed dense cataracts within 6 weeks of the beginning of the galactose diet. Galactitol levels in animals receiving ARI-509 were 86% to 93% lower in red blood cells, retina, and sciatic nerve than those in the other galactose-fed groups. CONCLUSIONS: Although ARI-509 and AMG have different abilities to delay or prevent the diabetic-like retinopathy in galactosemic rats, even when substantial retinal microvascular acellularity occurs, both drugs prevent the immunocytochemical expression of VEGF. These results suggest that factors other than hypoxia may be responsible for VEGF expression in the retina, and that aldose reductase inhibitors and AMG have potential roles in preventing such expression and, thus, perhaps preventing retinal neovascularization.     

Effects of an aldose reductase inhibitor, SNK-860, on the histopathological changes of retinal tissues in a streptozotocin-induced diabetic rat model

The efficacy of two protocols for the prevention of relapse using Mitomycin-C and alpha-Interferon in 56 patients with surface bladder carcinoma (stages Ta-T1) treated with TUR was compared in a prospective study. Relapse percentages and rates, related to the tumours' presentation characteristics (single, multiple, primary, recurrent), as well as their systemic and local toxicity, were evaluated. The study of statistical significance is made using the squared-chi test, and it is completed with a Fisher's test for groups containing few elements for accuracy. The results show no statistical differences (log rank p = 0.313) between the two groups of endovesical therapy, both confirming to be effective as adjuvant therapy to TUR in surface bladder tumours.     

The absence of synergism between the effects of an aldose reductase inhibitor, epalrestat, and a vasodilator, cilostazol, on the nerve conduction slowing and the myelinated fiber atrophy in streptozotocin-induced diabetic rats

The preventive effects of combined or separate treatment for 10 weeks with an aldose reductase inhibitor, epalrestat (50 mg/kg/day), and a vasodilator, cilostazol (30 mg/kg/day), on nerve conduction deficits and morphometric alterations were examined in streptozotocin-induced diabetic rats. The average motor nerve conduction velocities (MNCV) in the tail nerve of the untreated diabetic (DM) group, the group treated with epalrestat (ES), the group treated with cilostazol (CZ), the group with both agents together (ES&CZ), and the normal control group were 34.7, 37.7, 39.3, 39.0 and 42.1 m/s, respectively. All treatments partially but significantly prevented a reduction in MNCV. The MNCV in the ES&CZ group was almost the same as in the CZ group. In a morphometric study of the sural nerve, the DM group showed a reduction in the average diameter of myelinated fiber and in occupancy (percentage of the fascicular area occupied by myelinated fibers), and a shift in the diameter-frequency histogram to smaller diameters. Only the CZ group showed evidence of a partial but significant preventive effect on the decrease in occupancy. In the CS and ES&CZ groups, there was a significant tendency away from the shift of histograms to smaller diameters. The ES&CZ group did not show any fewer morphometric changes than the CZ group. Thus, there was no synergism between the effects of epalrestat and cilostazol on the development of experimental diabetic neuropathy. This finding may provide a useful clue to the mechanisms of action of ES and CZ in diabetic neuropathy.     

Vasoreactivity and prostacyclin release in streptozotocin-diabetic rats: effects of insulin or aldose reductase inhibition

1. Alterations in vasoreactivity and endothelial cell function could underlie some of the vascular abnormalities in diabetes. To examine aspects of these phenomena we studied the effects of 4-6 weeks streptozotocin-induced diabetes in the rat on basal and angiotensin II (AII)-stimulated prostacyclin release from isolated lung, perfused at constant flow. In addition, pressure was monitored throughout the lung perfusion as an index of vasomotor tone. 2. The experiment also included lungs from groups of diabetic rats treated with either insulin or an aldose reductase inhibitor (imirestat), to determine whether these treatments influenced the development of any defects seen in untreated diabetes. 3. Despite some indication of a trend towards reduced prostacyclin release in lungs from diabetic rats, neither the basal nor AII-stimulated release was significantly different from that seen in tissues from control animals. There were no significant differences between groups in the average basal perfusion pressure and in either the absolute pressure response to AII or the time of this peak. 4. The area under the perfusion pressure curve during AII infusion was greater in lungs from diabetic animals than in controls indicating a prolonged vasoconstrictor response. This increased pressor response may indicate increased sensitivity of diabetic tissue to AII or a reduced production of vasodilators in response to the vasoconstriction. 5. Whichever mechanism was responsible, this alteration was prevented by insulin treatment but not by aldose reductase inhibition, implicating mechanisms probably unrelated to exaggerated polyol pathway flux.     

Modification of aldose reductase by S-nitrosoglutathione

Kinetic and structural changes in recombinant human aldose reductase (AR) due to modification by S-nitrosoglutathione (GSNO) were investigated. Incubation of the enzyme with 10-50 microM GSNO led to a time- and concentration-dependent inactivation of the enzyme, with a second-order rate constant of 0.087 +/- 0.009 M-1 min-1. However, upon exhaustive modification, 30-40% of the enzyme activity was retained. The non-inactivated enzyme displayed a 2-3-fold change in Km for NADPH and Km fordl-glyceraldehyde, whereas the Km for the lipid peroxidation product, 4-hydroxy-2-trans nonenal (HNE), was comparable to that of the untreated enzyme. The residual activity of the enzyme after GSNO treatment was less sensitive to inhibition by the active site inhibitor sorbinil or to activation by sulfate. Significantly higher catalytic activity was retained when the enzyme was modified in the presence of NADPH, suggesting relatively low reactivity of the E-NADPH complex with GSNO. The modification site was identified using site-directed mutants in which each of the solvent-exposed cysteines of the enzyme was replaced individually by serine. The mutant C298S was insensitive to GSNO, whereas the sensitivity of the mutants C303S and C80S was comparable to that of the wild-type enzyme. Electrospray ionization mass spectroscopy of the GSNO-modified enzyme revealed a major modified species (70% of the protein) with a molecular mass that was 306 Da higher than that of the untreated enzyme, which is consistent with the addition of a single glutathione molecule to the enzyme. The remaining 30% of the protein displayed a molecular mass that was not significantly different from that of the native enzyme. No nitrosated forms of the enzyme were observed. These results suggest that inactivation of AR by GSNO is due to the selective formation of a single mixed disulfide between glutathione and Cys-298 located at the NADP(H)-binding site of the enzyme.     

Molecular modeling studies of the binding modes of aldose reductase inhibitors at the active site of human aldose reductase

Molecular modeling studies using the CHARMM method have been conducted to study the binding modes of aldose reductase inhibitors at the active site of aldose reductase. The energy minimized structures of aldose reductase with six structurally diverse inhibitors (spirofluorene-9,5'-imidazolidine-2',4'-dione (1), 9-fluoreneacetic acid (2), AL1576 (3), 2,7-difluoro-9-fluoreneacetic acid (4), FK366 (5), and Epalrestat (9)) indicate that the side chains of Tyr48, His110, and Trp111 can form numerous hydrogen bonds with either the carboxylate or the hydantoin group of the inhibitors while the side chains of Trp20, Trp111, and Phe122 are positioned to form aromatic-aromatic interactions. Of the three residues (Tyr 48, His 110, and Trp 111) that can form hydrogen bonds with the ionized portion of aldose reductase inhibitors, protonated His110 appears to play an important role in directing charged inhibitors to bind at the active site through charge interaction. Based on the binding mode of the inhibitors and their observed inhibitory activities, pharmacophore requirements for aldose reductase inhibitors are discussed.     

Tissue distribution, metabolism, and clearance of the convulsant trimethylolpropane phosphate in rats

The distribution, metabolism, and clearance of trimethylolpropane phosphate (TMPP), a potent, bicyclophosphate, gamma-aminobutyric acid-ergic convulsant, were studied in male Fischer-344 rats. Intraperitoneal administration of TMPP was compared with oral gavage with respect to rates of absorption, distribution, and clearance. Distribution of TMPP to major body tissues was evaluated for the first 24 hr after administration or, in the case of regional brain distribution, immediately after the first TMPP-induced clinical seizure. Samples purified from the urine, feces, and bile of rats exposed to TMPP, as well as from rat liver microsomes incubated with TMPP in vitro, were analyzed for possible phase I and phase II metabolism, using HPLC. The disposition and clearance of TMPP in the blood and major body tissues were measured. TMPP was found to be well distributed to highly vascularized tissue compartments, with little retention >24 hr after administration. TMPP was eliminated through the urine and feces as the parent compound, with no evidence of phase I or phase II metabolism. TMPP was rapidly cleared from the blood during the first 30 min after exposure, with slower clearance of >87% of the drug during the following 8-hr period and >99.5% clearance by 100 hr after injection. Repeated daily exposure to TMPP for up to 5 successive days resulted in no measurable accumulation in the brain or other major tissue compartments. Possible mechanisms for TMPP-induced, short- and long-term, neurobehavioral modulation are discussed.     

Myocardial distribution and biotransformation in vitro and in vivo of nicorandil in rats, with special reference to mitochondria

This study reports subcellular localization of nicorandil in the myocardium and metabolism in mitochondria after oral dosing of 3 mg/kg nicorandil to rats. In the in vitro experiments, nicorandil, which was incubated with tissue homogenates (liver, kidney, heart, and small intestine), was metabolized to its denitrated compound, SG-86, and unknown substances. In the absence of a NADPH-generating system in the heart, the metabolic activity existed only in the mitochondrial fraction, but not in cytosolic and microsomal fractions. In the presence of the system, the activity in the mitochondrial fraction became much higher. To examine subcellular distribution of nicorandil in the myocardium, [14C]nicorandil was orally given to rats. Fifteen minutes after oral dosing of 3 mg/kg [14C]nicorandil, of which myocardial concentration reached a peak, nicorandil and SG-86 were found in mitochondrial fractions as well as in cytosolic and microsomal ones of the heart. Electron-microscopic autoradiograms, 15 min after oral dosing of 3 mg/kg [3H]nicorandil to rats, also showed the existence of the silver grains (showing radioactivity) in mitochondria of the heart. We conclude that nicorandil given orally is distributed in mitochondria of the heart, being partly transformed into SG-86, and that the myocardial mitochondria may be a potential site of action of nicorandil, an opener of KATP channels, which have been demonstrated to be present in this subcellular particle.     

Characterization of the human aldose reductase gene promoter

The promoter region of the human aldose reductase gene has been identified upstream of the translation start ATG codon. The promoter contains a TATA box, a CCAAT promoter element, and three Sp1 protein binding consensus sequences upstream of the capsite. A 640-base pair insert spanning +31 to -609 directs expression of the reporter gene chloramphenicol acetyltransferase in an orientation-specific manner in transfected Hep G2 cells. The promoter activity remained constant with deletions from base pairs -609 to -186. The TATA and the CCAAT consensus sequences show significant promoter activity, whereas the three Sp1 binding consensus sequences, individually, have no significant promoter activity. A GA-rich region (-186 to -146) contains two CGGAAA/G motifs, which show promoter activity and interaction with Hep G2 nuclear extract and GA-binding proteins (GABP alpha and GABP beta 1) as shown by mobility shift assays and DNase I footprinting. Similar cis-elements in herpes simplex virus type 1 interact with rat liver GABP and the viral VP16 protein to mediate the induction of immediate early viral genes. A GC-rich region (-87 to -31) is identified by mobility shift assay, and a consensus sequence of an androgen response element is present at -396 to -382. The human aldose reductase promoter, thus, has regulatory response elements that may be important during early development and puberty. These regulatory elements may play a significant role in the development of certain diabetic complications.     

Synthesis and aldose reductase inhibitory activity of benzoyl-amino acid derivatives

Discoid lupus erythematosus (DLE) is an uncommon disease in childhood. We present two patients initially diagnosed as impetigo and photosensitive eczema with impetigo, respectively, who failed to respond to topical and systemic antistaphylococcal agents and in whom a diagnosis of discoid lupus erythematosus subsequently became apparent.     

Structural and kinetic determinants of aldehyde reduction by aldose reductase

Aldose reductase (AR) is a member of the aldo-keto reductase superfamily. Due to its ability to catalyze the formation of sorbitol from glucose during hyperglycemic and hypertonic stress, the aldose-reducing property of AR has been accepted as its main physiological and pathological function. Nonetheless, AR is a poor catalyst for glucose reduction and displays active-site properties unexpected of a carbohydrate-binding protein. We, therefore, examined the catalytic properties of AR with a series of naturally occurring aldehydes, compatible in their hydrophobicity to the large apolar active site of the enzyme. Our results show that recombinant human AR is an efficient catalyst for the reduction of medium- to long-chain unbranched saturated and unsaturated aldehydes. The enzyme displayed selective preference for saturated aldehydes, such as hexanal, and unsaturated aldehydes, such as trans-2-octenal and nonenal as well as their 4-hydroxy derivatives. Short-chain aldehydes such as propanal and acrolein were reduced less efficiently. Branched derivatives of acrolein or its glutathione conjugate (GS-propanal) were, however, reduced with high efficiency. In the absence of NADPH, the alpha, beta unsaturated aldehydes caused covalent modification of the enzyme. On the basis of electrospray mass spectrometric analysis of the wild-type and site-directed mutants of AR (in which the solvent exposed cysteines were individually replaced with serine), the site of modification was identified to be the active-site residue, Cys 298. The unsaturated aldehydes, however, did not modify the enzyme bound to NADPH and did not inactivate the enzyme during catalysis. Modeling studies indicate that the large hydrophobic active site of AR can accommodate a large number of aldehydes without changes in the structure of the binding site or movement of side chains. High hydrophobicity due to long alkyl chains or apolar substituents appears to stabilize the interaction of the aldehyde substrates with the enzyme. Apparently, such hydrophobic interactions provide substrate selectivity and catalytic efficiency of the order achievable by hydrogen bonding. Since several of the aldehydes reduced by AR are either environmental and pharmacological pollutants or products of lipid peroxidation, the present studies provide the basis of future investigations on the role of AR in regulating aldehyde metabolism particularly under pathological states associated with oxidative stress and/or aldehyde toxicity.     

Pharmacokinetics of methyl palmoxirate, an inhibitor of beta-oxidation, in rats and humans

Recent studies from our laboratory have shown that methyl palmoxirate (MEP), an inhibitor of mitochondrial beta-oxidation of long chain fatty acids, can be used to increase incorporation of radiolabeled palmitic acid into brain lipids and reduce beta-oxidation of the fatty acid. Thus, MEP allows the use of carbon labeled palmitate for studying brain lipid metabolism in animals and humans by quantitative autoradiography or positron emission tomography (PET). As it is essential to pretreat human subjects with an acute dose of MEP prior to intravenous injection of [1-11C]palmitate for PET scanning, this study was undertaken to determine the plasma elimination half-life of MEP in rats and human subjects and to provide insight about the drug's absorption and metabolism. A gas chromatographic method was developed to measure MEP in body fluids. Following oral administration of MEP to rats (2.5 and 10 mg/kg) and to humans, the unmetabolized drug could not be detected in plasma or urine (sensitivity of detection was 1 ng). However, when MEP was injected intravenously (10 mg/kg) in rats, a peak initial concentration could be measured in plasma (7.7 microg/mL), the clearance of the drug from plasma was rapid (t1/2 = 0.6 min), which indicates that MEP readily enters tissue lipid pools or is metabolized like long-chain fatty acids. As no adverse experience occured in the 11 human subjects studied, oral administration of a single dose of MEP was safe under the conditions of this study and may be used to increase the incorporation of positron labeled palmitic acid for studying brain lipid metabolism in vivo by PET.     

Noncovalent enzyme-substrate interactions in the catalytic mechanism of yeast aldose reductase

Cases of Burkitt's lymphoma (BL) from north-western Iran were investigated for the usage and somatic mutational pattern of their immunoglobulin variable region genes. Potentially functional V(H) genes were amplified from 6/12 of the tumour masses and all of these were derived from the V(H)3 family, with 4/6 being derived from the most commonly used V(H)3 family member, V3-23. All of the tumour sequences were mutated from their germline counterparts, to varying degrees, with a mean level of 5.8%, indicating that the cell of origin had encountered the germinal centre. Intraclonal sequence heterogeneity was also evident in 4/6 of the lymphomas, showing that the tumour cells had undergone further somatic mutation following neoplastic transformation. Analysis of the five potentially functional mutated V(H) sequences showed a significant clustering of replacement mutations in the complementarity-determining region 2, consistent with a role for antigen in selection of tumour cell sequences. The pattern of extensive somatic mutation, and intraclonal variation, in these mainly EBV+ve tumours, was similar to that previously reported in V(H) sequences of EBV+ve endemic BL (eBL) and EBV-ve sporadic BL (sBL), with the mean level of somatic mutation lying between those reported for eBL (7.7%) and sBL (4.0%). However, VH gene bias and the distribution of mutations in the Iranian cases showed features which differed from those reported for endemic or sporadic BL.     

Potassium depletion modulates aldose reductase mRNA in rat renal inner medulla

The organic osmolytes present in renal inner medullary cells balance the extracellular hyperosmolality and protect the cell against the effects of high salts and urea. We previously demonstrated that a renal concentrating defect due to potassium depletion was associated with a decrease in organic osmolytes including sorbitol. However, we could not determine whether a reduction in medullary organic osmolyte would be cause or effect of urine concentration defect associated with potassium depletion. We focused on the synthesis of sorbitol catalyzed by the enzyme, aldose reductase. To clarify whether the treatment of potassium depletion would affect aldose reductase when extracellular tonicity, and medullary sodium or potassium was maintained at the level of control rats, we administered a hypertonic solution of NaCl or KCl to potassium-depleted rats and evaluated aldose reductase enzymatic activity and mRNA abundance as well as the medullary contents of organic osmolytes. Either infusion significantly reduced tissue sodium content in potassium-depleted rats. With KCl infusion protocol but not that of NaCl, sorbitol as well as aldose reductase mRNA abundance increased to the control level. Medullary contents of other organic osmolytes exhibited a pattern similar to sorbitol. Data suggested that aldose reductase mRNA abundance was reduced in potassium depletion irrespective of medullary sodium content. A decrease in sorbitol level may precede a urinary concentrating defect. Our finding constitutes the first demonstration of the relationship between a potassium deficiency and the abundance of aldose reductase mRNA, an osmoregulatory protein in the kidney.     

Effects of one year of treatment with pravastatin, an HMG-CoA reductase inhibitor, on lipoprotein a

Lipoprotein a [Lp(a)] has emerged as a critical factor in the assessment of cardiovascular risk. In the study reported here, Lp(a) concentrations were monitored in patients taking pravastatin, a new hydrophilic, HMG-CoA reductase inhibitor. A cohort of patients with frozen plasma aliquots at baseline, week 12 of the double-blind therapy, and week 48 of open-label therapy (1 years' treatment) was selected from 306 participants in a phase 2 dose-ranging study of pravastatin. The 125 men and women in the cohort had mean low-density lipoprotein cholesterol (LDL-C) concentrations of at least 150 mg/dL (3.88 mmol/L), and mean plasma triglyceride concentrations less than 250 mg/dL (2.82 mmol/L) during the baseline diet phase. During the double-blind phase, 46 patients received placebo, and 79 received pravastatin 10, 20, or 40 mg daily. Only the 79 pravastatin-treated patients in the cohort continued in the 48-week open-label study of pravastatin. During the double-blind phase, Lp(a) decreased 4.6% in patients taking placebo, and 0.4% in patients taking pravastatin. Net change was not significant. At week 48, in the patients taking pravastatin, Lp(a) had increased 2.4%, a difference that again was not statistically significant. Low-density lipoprotein cholesterol (-33.6%), total cholesterol (-25.6%), triglycerides (-19.9%), high-density lipoprotein cholesterol (HDL-C) (+7.0%), apolipoprotein A-I (+13.3%), and apolipoprotein B (-33.0%) changed significantly (P < .01). Among 19 patients with baseline Lp(a) levels greater than 30 mg/dL, Lp(a) decreased insignificantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

HMG CoA reductase inhibitor accelerates aging effect on diaphragm mitochondrial respiratory function in rats

We examined effects of pravastatin on age-related changes in mitochondrial function in rats. Decline in the activity of complex I of the mitochondrial electron transport chain was observed in diaphragm and psoai major in rats aged 35 and 55 weeks, and that of complex IV in rats aged 55 weeks. Pravastatin accelerated significantly age-related decline in the activity of complex I of diaphragm mitochondria, though pravastatin did not show significant effect on normally observed age-associated decline in the activities of complex IV of psoai major and diaphragm mitochondria. Aging effect on mitochondrial respiratory function was not observed on heart muscle and liver in rats up to 55 weeks old, and pravastatin did not effect significantly heart and liver mitochondrial respiratory function. From these results, careful clinical examination on respiratory muscle function should be necessary in patients treated with pravastatin particularly in elderly patients.