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1.
Eur J Pharmacol ; 417(3): 223-30, 2001 Apr 13.
Article in English | MEDLINE | ID: mdl-11334854

ABSTRACT

Increased polyol pathway flux has been linked to nerve complications in diabetic rats, which are attenuated by aldose reductase inhibitors, defective nitric oxide-mediated vasodilation being a particular target. Diabetes also elevates the endothelial angiotensin system, increasing vasa nervorum vasoconstriction. The aim was to assess whether promotion of vasodilation by treatment with the aldose reductase inhibitor, ZD5522 (3',5'-dimethyl-4'-nitromethylsulphonyl-2-(2-tolyl)acetanilide), coupled with reduced vasoconstriction using the angiotensin-converting enzyme inhibitor, lisinopril, interacted positively to improve neurovascular function. After 8 weeks of streptozotocin-induced diabetes, sciatic nerve blood flow and motor conduction velocity were 51% and 21% reduced, respectively. Two weeks of lisinopril treatment dose-dependently corrected the conduction deficit (ED(50) approximately 0.9 mg kg(-1)). Low-dose lisinopril (0.3 mg kg(-1)) or ZD5522 (0.25 mg kg(-1)) had modest corrective (10-20%) effects on nerve conduction and perfusion. However, when combined, blood flow and conduction velocity reached the nondiabetic range. The ZD5522 dose used gave a approximately 45% nerve sorbitol reduction but had no significant effect on fructose content; lisinopril co-treatment did not alter ZD5522 action on polyols. Thus, there was a marked neurovascular synergistic interaction between angiotensin-converting enzyme and aldose reductase inhibition in diabetic rats. This points to a potential therapeutic benefit, which requires evaluation in clinical trials.


Subject(s)
Acetanilides/pharmacology , Aldehyde Reductase/antagonists & inhibitors , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Diabetes Mellitus, Experimental/enzymology , Lisinopril/pharmacology , Sciatic Nerve/drug effects , Sulfones/pharmacology , Acetanilides/therapeutic use , Aldehyde Reductase/metabolism , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Blood Glucose/analysis , Body Weight , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/physiopathology , Dose-Response Relationship, Drug , Drug Synergism , Lisinopril/therapeutic use , Neural Conduction/drug effects , Rats , Regional Blood Flow/drug effects , Sciatic Nerve/blood supply , Sciatic Nerve/physiology , Sulfones/therapeutic use , Vasodilation/drug effects
2.
J Med Chem ; 43(26): 4964-72, 2000 Dec 28.
Article in English | MEDLINE | ID: mdl-11150166

ABSTRACT

A novel series of 4-piperidinopyridines and 4-piperidinopyrimidines showed potent and selective inhibition of rat 2,3-oxidosqualene cyclase-lanosterol synthase (OSC) (e.g. 26 IC(50) rat = 398 +/- 25 nM, human = 112 +/- 25 nM) and gave selective oral inhibition of rat cholesterol biosynthesis (26 ED(80) = 1.2 +/- 0.3 mg/kg, n = 5; HMGCoA reductase inhibitor simvastatin ED(80) = 1.2 +/- 0.3 mg/kg, n = 5). The piperidinopyrimidine OSC inhibitors have a significantly lower pK(a) than the corresponding pyridine or the previously reported quinuclidine OSC inhibitor series. This indicates that other novel OSC inhibitors may be found in analogues of this series across a broader pK(a) range (6.0-9.0). These series may yield novel hypocholesterolemic agents for the treatment of cardiovascular disease.


Subject(s)
Anticholesteremic Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Intramolecular Transferases/antagonists & inhibitors , Piperazines/chemical synthesis , Piperidines/chemical synthesis , Pyridines/chemical synthesis , Pyrimidines/chemical synthesis , Administration, Oral , Animals , Anticholesteremic Agents/chemistry , Anticholesteremic Agents/pharmacology , Cholesterol/biosynthesis , Chromatography, High Pressure Liquid , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Female , In Vitro Techniques , Microsomes/drug effects , Microsomes/enzymology , Piperazines/chemistry , Piperazines/pharmacology , Piperidines/chemistry , Piperidines/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Rats , Structure-Activity Relationship
3.
J Med Chem ; 42(7): 1306-11, 1999 Apr 08.
Article in English | MEDLINE | ID: mdl-10197973

ABSTRACT

Novel 3-substituted quinuclidine inhibitors of cholesterol biosynthesis are reported. Compounds were optimized against oxidosqualene cyclase-lanosterol synthase (OSC) inhibition in vivo, rather than by the conventional optimization of structure-activity relationship information based on in vitro OSC inhibition. Thus, examination of HPLC lipid profiles from orally dosed rats showed cholesterol biosynthetic intermediates and whether cholesterol levels were reduced. A new substituted quinuclidine pharmacophore 18a-c was rapidly found for the inhibition of OSC, and the most promising inhibitors were validated by the confirmation of potent OSC inhibition. Compound 16 gave an IC50 value of 83 +/- 11 nM for human and an IC50 value of 124 +/- 14 nM, for rat, coupled with oral and selective inhibition of cholesterol biosynthesis derived from OSC inhibition (rat, ED50 = 1.3 +/- 0.7 mg/kg, n = 5; marmoset, 15 mg/kg dose, n = 3, caused complete inhibition). These 3-substituted quinuclidines, which were derived from a quinuclidine series previously known to inhibit cholesterol biosynthesis at the squalene synthase step, may afford a novel series of hypocholesterolemic agents acting by the inhibition of OSC.


Subject(s)
Anticholesteremic Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Intramolecular Transferases/antagonists & inhibitors , Lipid Metabolism , Quinuclidines/chemical synthesis , Administration, Oral , Animals , Anticholesteremic Agents/chemistry , Anticholesteremic Agents/pharmacology , Callithrix , Chromatography, High Pressure Liquid , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Lipids/analysis , Liver/chemistry , Liver/drug effects , Male , Microsomes/drug effects , Microsomes/metabolism , Quinuclidines/chemistry , Quinuclidines/pharmacology , Rats , Structure-Activity Relationship
4.
Biochem Pharmacol ; 51(11): 1477-87, 1996 Jun 14.
Article in English | MEDLINE | ID: mdl-8630089

ABSTRACT

Squalene synthase (SQS) is a key enzyme in the biosynthetic pathway for cholesterol and is a target for improved agents to lower plasma levels of low-density lipoprotein (LDL). A series of novel 3' substituted quinuclidines have been discovered as inhibitors of the rat liver microsomal enzyme. In this study, we demonstrate the inhibitory effects in vitro and in vivo, of two examples of the series. When microsomes were preincubated with compounds, before addition of substrate, both 3-(biphenyl-4-yl)quinuclidine (BPQ) and 3-(biphenyl-4-yl)-3-hydroxyquinuclidine (BPQ-OH) were found to cause biphasic inhibition of the enzyme with apparent inhibition constants (K'i) for the sensitive phases of 12 nM and 15 nM, respectively. The K'i values for the insensitive phases were 1.8 microM and 2.9 microM, respectively. The two examples inhibited equally both steps of the SQS-catalysed reaction, as shown by parallel inhibition of 3H+ release and labelled squalene formation from [1-3H]farnesyl pyrophosphate (FPP). BPQ and BPQ-OH were shown to be inhibitors of hepatic sterol synthesis from mevalonate with ED50 values of 10.6 and 7.1 mg/kg, respectively, after acute oral administration to the rat. BPQ-OH was chosen for further study and, to determine its selectivity of effect on the mevalonate pathway in vivo, the effect of a dose of 70 mg/kg on the pattern of labelled mevalonate incorporation into the various lipid fractions of the rat liver was examined. As expected, the incorporation into squalene and sterol products was inhibited by about 70%. An appearance of label in fractions corresponding to farnesyl and geranylgeranylpyrophosphates, as well as the corresponding alcohols, was observed in treated but not control animals. In addition, the administration of compound resulted in the appearance of peaks of mevalonate-derived radioactivity in an acidic fraction believed to represent metabolites of farnesol. Such results are consistent with inhibition of the mevalonate pathway at, and not before, SQS. In contrast, there was a significant increase in the incorporation of labelled mevalonate into ubiquinone 10, and the synthesis of dolichols was apparently unchanged. The results suggest a specific effect of BPQ-OH on rat liver SQS. The compound is, therefore, an interesting lead for further investigation of this class of compounds.


Subject(s)
Enzyme Inhibitors/pharmacology , Farnesyl-Diphosphate Farnesyltransferase/antagonists & inhibitors , Liver/enzymology , Quinuclidines/pharmacology , Animals , Female , Kinetics , Liver/drug effects , Mevalonic Acid/analogs & derivatives , Mevalonic Acid/metabolism , Rats , Rats, Inbred Strains , Sensitivity and Specificity , Tritium
5.
NMR Biomed ; 8(3): 133-8, 1995 May.
Article in English | MEDLINE | ID: mdl-8580001

ABSTRACT

We report 13C NMR measurements of the flux through aldose reductase in isolated rat sciatic nerve, and its inhibition by an aldose reductase inhibitor of the sulphonylnitromethane class. [1-13C] galactose was used as substrate, and the rate of production of [1-13C] dulcitol was measured. Quantitation required the use both of internal extracellular, and external, standards. The mean net forward flux (+/- SD) was 20 +/- 11 nmol/(mL nerve water)/min (n = 10). In the presence of the inhibitor, flux was reduced significantly (p < 0.001) to 13% of control. Since dulcitol is symmetrical, an estimate of the backward flux, to [6-13C] galactose, is also possible; under our conditions, this was negligible.


Subject(s)
Aldehyde Reductase/metabolism , Enzyme Inhibitors/pharmacology , Magnetic Resonance Spectroscopy , Nitroparaffins/pharmacology , Sciatic Nerve/enzymology , Sulfones/pharmacology , Aldehyde Reductase/antagonists & inhibitors , Animals , Carbon Isotopes , Galactitol/analysis , Galactitol/metabolism , Galactose/metabolism , In Vitro Techniques , Male , Rats , Rats, Wistar
6.
Biochem Pharmacol ; 49(8): 1043-9, 1995 Apr 18.
Article in English | MEDLINE | ID: mdl-7748183

ABSTRACT

Aldose reductase (aldehyde reductase 2) catalyses the conversion of glucose to sorbitol, and methylglyoxal to acetol. Treatment with aldose reductase inhibitors (ARIs) is a potential approach to decrease the development of diabetic complications. The sulphonylnitromethanes are a recently discovered class of aldose reductase inhibitors, first exemplified by ICI215918. We now describe enzyme kinetic characterization of a second sulphonylnitromethane, 3',5'-dimethyl-4'-nitromethylsulphonyl-2-(2-tolyl)acetanilide (ZD5522), which is at least 10-fold more potent against bovine lens aldose reductase in vitro and which also has a greater efficacy for reduction of rat nerve sorbitol levels in vivo (ED95 = 2.8 mg kg-1 for ZD5522 and 20 mg kg-1 for ICI 215918). ZD5522 follows pure noncompetitive kinetics against bovine lens aldose reductase when either glucose or methylglyoxal is varied (K(is) = K(ii) = 7.2 and 4.3 nM, respectively). This contrasts with ICI 215918 which is an uncompetitive inhibitor (K(ii) = 100 nM) of bovine lens aldose reductase when glucose is varied. Against human recombinant aldose reductase, ZD5522 displays mixed noncompetitive kinetics with respect to both substrates (K(is) = 41 nM, K(ii) = 8 nM with glucose and K(is) = 52 nM, K(ii) = 3.8 nM with methylglyoxal). This is the first report of the effects of a sulphonylnitromethane on either human aldose reductase or utilization of methylglyoxal. These results are discussed with reference to a Di Iso Ordered Bi Bi mechanism for aldose reductase, where the inhibitors compete with binding of both the aldehyde substrate and alcohol product. This model may explain why aldose reductase inhibitors follow noncompetitive or uncompetitive kinetics with respect to aldehyde substrates, and X-ray crystallography paradoxically locates an ARI within the substrate binding site. Aldehyde reductase (aldehyde reductase 1) is closely related to aldose reductase. Inhibition of bovine kidney aldehyde reductase by ZD5522 follows uncompetitive kinetics with respect to glucuronate (K(ii) = 39 nM), indicating a selectivity greater than 5-fold for bovine aldose reductase relative to aldehyde reductase.


Subject(s)
Acetanilides/pharmacology , Aldehyde Reductase/antagonists & inhibitors , Lens, Crystalline/enzymology , Sulfones/pharmacology , Animals , Cattle , Dose-Response Relationship, Drug , Glucose/metabolism , Humans , Kidney/enzymology , Kinetics , NADP , Pyruvaldehyde/metabolism , Recombinant Proteins/antagonists & inhibitors
7.
Diabetologia ; 37(7): 651-63, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7958535

ABSTRACT

We examined the effects of aldose reductase inhibition on nerve biochemistry and function, blood flow and endoneurial oxygenation in experimental diabetes mellitus. After 1 month untreated diabetes in rats, treatment with the novel sulphonylnitromethane aldose reductase inhibitor, ZENECA ZD5522, prevented a progressive increase in sciatic nerve resistance to hypoxic conduction failure (p < 0.05). Motor conduction velocity deficits after 4 months untreated diabetes were rapidly returned to normal within 12 days (p < 0.0001) by ZD5522 treatment. Following 2-months untreated diabetes, examination of 1 month ZD5522 treatment dose-response relationships for correction of nerve sorbitol and fructose accumulations and reduction in myo-inositol concentration, sciatic motor and saphenous sensory conduction velocity and sciatic blood flow by laser-Doppler flowmetry revealed poor agreement between nerve function and biochemical indices. In addition, polyol accumulation differed between sciatic and saphenous nerves, the latter showing ten-fold lower sorbitol concentrations. Laser-Doppler blood flow was 60% decreased by untreated diabetes (p < 0.001) and there was a strong correlation between ZD5522-mediated increases in blood flow and conduction velocity (p < 0.0001). Measurement of nutritive endoneurial blood flow by microelectrode polarography and hydrogen clearance showed 44% and 45% deficits for 1 and 2 months untreated diabetes (p < 0.001) that were prevented by ponalrestat and ZD5522 treatments, respectively. In contrast, 2 months myo-inositol treatment from diabetes induction did not prevent reduction in blood flow or sciatic motor conduction velocity. A 37% reduction in endoneurial oxygen tension after 2 months diabetes (p < 0.001) was completely prevented by ZD5522 treatment (p < 0.001). The data show that a very high degree of polyol pathway blockade is necessary to correct nerve functional deficits and that aldose reductase inhibitors have a neurovascular action that does not depend on restoration of nerve myo-inositol.


Subject(s)
Acetanilides/pharmacology , Aldehyde Reductase/antagonists & inhibitors , Diabetes Mellitus, Experimental/physiopathology , Oxygen/metabolism , Sciatic Nerve/physiology , Sulfones/pharmacology , Animals , Blood Flow Velocity/drug effects , Blood Glucose/metabolism , Dose-Response Relationship, Drug , Inositol/metabolism , Male , Motor Neurons/drug effects , Motor Neurons/physiology , Neural Conduction/drug effects , Neural Conduction/physiology , Perfusion , Peripheral Nerves/blood supply , Peripheral Nerves/physiology , Rats , Rats, Sprague-Dawley , Sciatic Nerve/blood supply
8.
Br J Pharmacol ; 111(1): 42-8, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8012723

ABSTRACT

1. Noradrenaline sensitivity and relaxation to acetylcholine were investigated in the isolated perfused mesentery and in aortic rings of control and streptozotocin (STZ)-induced (50 mg kg-1) diabetic Charles River rats. 2. In addition, noradrenaline sensitivity and acetylcholine relaxation were similarly assessed in streptozotocin-induced diabetic rats treated from the time of onset of diabetes with the aldose reductase inhibitor, ponalrestat (100 mg kg-1 day-1). 3. The untreated diabetic rats (2-10 weeks after injection of STZ) demonstrated enhanced vascular sensitivity to noradrenaline in the perfused mesenteric arterial tree, compared with age matched controls (pEC50 [-log concentration (M)]: diabetic 5.62 +/- 0.09, n = 18, versus control 5.23 +/- 0.07, n = 16, P < 0.01). 4. Acetylcholine-induced relaxation was significantly impaired in the perfused mesentery of the diabetic animals compared to controls (pED50 [-log dose (mol)]: diabetic 9.87 +/- 0.10, n = 20, versus controls, 10.29 +/- 0.09, n = 20, P < 0.05). 5. In contrast, the aortic ring preparations demonstrated no significant functional differences between the diabetic and control groups in response to either noradrenaline (pEC50: diabetic 7.66 +/- 0.08, n = 15, versus controls 7.55 +/- 0.06, n = 15, NS), or acetylcholine (pEC50: diabetics 7.30 +/- 0.06, n = 15, versus controls 7.40 +/- 0.09, n = 15, NS). 6. Treatment with the aldose reductase inhibitor, ponalrestat, did not affect the increased vascular reactivity to noradrenaline, or impaired relaxation to acetylcholine in the perfused mesentery.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Diabetes Mellitus, Experimental/physiopathology , Endothelium, Vascular/drug effects , Muscle, Smooth, Vascular/drug effects , Phthalazines/pharmacology , Acetylcholine/pharmacology , Animals , Aorta/drug effects , Aorta/physiopathology , Diabetes Mellitus, Experimental/drug therapy , Endothelium, Vascular/physiopathology , Female , In Vitro Techniques , Mesenteric Arteries/drug effects , Mesenteric Arteries/physiopathology , Muscle Contraction/drug effects , Muscle Relaxation/drug effects , Muscle, Smooth, Vascular/physiopathology , Norepinephrine/pharmacology , Phthalazines/therapeutic use , Rats , Vascular Resistance/drug effects
9.
Adv Exp Med Biol ; 328: 301-11, 1993.
Article in English | MEDLINE | ID: mdl-8493907

ABSTRACT

Aldose reductase (aldehyde reductase 2, ALR2) is often isolated as a mixture of two forms which are sensitive (ALR2S), or insensitive (ALR2I), to inhibitors. We show that ICI 215918 ((2-6-dimethylphenylsulphonyl)-nitromethane) follows either noncompetitive, or uncompetitive kinetics with respect to aldehyde for ALR2S, or the closely related enzyme, aldehyde reductase (aldehyde reductase 1, ALR1). Similar behaviour is exhibited by two other structural types of aldose reductase inhibitor (ARI), spirohydantoins and acetic acids, when either aldehyde, or NADPH is varied. For ALR2S, we have demonstrated kinetic competition between a sulphonylnitromethane, an acetic acid and a spirohydantoin. Thus, different ARIs probably have overlapping binding sites. Published studies imply that ALR2 follows an ordered mechanism where coenzyme binds first and induces a reversible conformation change (E.NADPH-->E*.NADPH). Reduction of aldehyde appears rate-limited by the step E*.NADP+-->E.NADP+. Spontaneous activation converts ALR2S into ALR2I and increases kcat. This must be associated with acceleration of the rate-determining step. We now propose the following hypothesis to explain characteristics of ARIs. (1) Inhibitors preferentially bind to the E* conformation. (2) The ARI binding site contains residues in common with that for aldehyde substrates. When aldehyde is varied, uncompetitive inhibition arises from association at the site for alcohol product in the E*.NADP+ complex which has little affinity for the substrate. Any competitive inhibition arises from use of the aldehyde site in the E*.NADPH complex. (3) Acceleration of the E*.NADP+-->E.NADP+ step upon activation of ALR2 reduces steady state levels of E* and so decreases sensitivity to ARIs.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Nitroparaffins/pharmacology , Sulfones/pharmacology , Acetates/pharmacology , Aldehyde Reductase/chemistry , Aldehyde Reductase/metabolism , Binding Sites , Binding, Competitive , Humans , Hydantoins/pharmacology , In Vitro Techniques , Kinetics , Models, Chemical
10.
Biochem Pharmacol ; 42(11): 2115-23, 1991 Nov 06.
Article in English | MEDLINE | ID: mdl-1958230

ABSTRACT

Many of the complications of diabetes seem to be due to aldose reductase (aldehyde reductase 2, ALR2) catalysing the increased conversion of glucose to sorbitol. Therapy with aldose reductase inhibitors (ARIs) could, therefore, decrease the development of diabetic complications. (2,6-Dimethylphenylsulphonyl)nitromethane (ICI 215918) is an example from a newly discovered class of ARIs, and we here describe its kinetic properties. Preparations of bovine lens ALR2 exhibit biphasic kinetics with respect to glucose and various inhibitors including ICI 215918. The inhibitor sensitive form (ALR2S) has a higher affinity for glucose than does the inhibitor insensitive form (ALR2I). Only ALR2S was characterized in detail because ALR2I activity is very low at physiological levels of glucose and is difficult to measure with accuracy. Aldehyde reductase (ALR1) is the most closely related enzyme to ALR2. Inhibition of ALR1 was, therefore, investigated in order to assess the specificity of ICI 215918. The values of Ki and Kies (dissociation constants for inhibitor from enzyme-inhibitor and enzyme-inhibitor-substrate complexes, respectively) for ICI 215918 with bovine kidney ALR1 and bovine lens ALR2S have been determined. When glucose is varied, the compound is an uncompetitive inhibitor of ALR2S (Kies = 0.10 microM and Ki is much greater than Kies), indicating that ICI 215918 associates with an allosteric site on the enzyme. These kinetic characteristics would cause a decrease in the concentration required to give 50% inhibition when glucose levels rise during hyperglycaemia. ICI 215918 is a mixed noncompetitive inhibitor of ALR1 (Ki = 10 microM and Kies = 1.8 microM) when glucuronate is varied. Thus, the compound has up to 100-fold specificity in favour of ALR2S relative to ALR1. Therapeutic interest has now centred upon at least three distinct structural types of ARIs: spirohydantoins, acetic acids and sulphonylnitromethanes. Using one representative of each type, we have demonstrated kinetic competition for inhibition of ALR2S. This observation strongly suggests that the different inhibitors use overlapping binding sites.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Nitroparaffins/pharmacology , Sulfones/pharmacology , Animals , Binding Sites , Cattle , Kidney/enzymology , Kinetics
11.
Metabolism ; 40(10): 1084-7, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1943734

ABSTRACT

Glucose, polyol (sorbitol, fructose), and myo-inositol levels were estimated in peripheral nerve tissue of the diabetic mutant mouse [C57/BL/Ks (db/db)]. At 26 and 40 weeks of age, there was significant accumulation of glucose, sorbitol, and fructose. Tissue myo-inositol levels were lower than those in age-matched control animals at 40 weeks, but not at 26 weeks. Polyol changes in mouse nerve were less marked than those in rat nerve. In dorsal root ganglia, there was also an increase in glucose, sorbitol, and fructose, with a decrease in myo-inositol concentration. These findings show that there is increased aldose reductase activity in peripheral nerve tissue of the diabetic mutant mouse.


Subject(s)
Aldehyde Reductase/metabolism , Diabetes Mellitus, Experimental/metabolism , Ganglia, Spinal/metabolism , Inositol/metabolism , Sciatic Nerve/metabolism , Animals , Blood Glucose/analysis , Body Weight , Diabetes Mellitus, Experimental/genetics , Glucose/metabolism , Male , Mice , Mice, Mutant Strains , Polymers/metabolism , Rats
12.
Diabetes ; 39(6): 690-6, 1990 Jun.
Article in English | MEDLINE | ID: mdl-2140802

ABSTRACT

There is controversy over the efficacy of aldose reductase inhibitors in preventing the development of peripheral nerve lesions in experimental diabetes. This study was designed to show whether long-term (28-wk) inhibition of aldose reductase by ponalrestat influences structural changes in peripheral sensory nerve in rats with chronic streptozocin-induced diabetes. Sciatic nerve levels of sorbitol and fructose were significantly reduced but not completely normalized by ponalrestat treatment. myo-Inositol levels, which tended to decrease in diabetic rats, were significantly increased by ponalrestat treatment and exceeded the level in nondiabetic control rats (P less than 0.01). Ponalrestat treatment significantly increased nerve conduction velocity over the 28 wk of treatment (P less than 0.05), but levels remained well below those of control rats. Structural analysis of sural nerve of diabetic rats disclosed significant preventive effects of ponalrestat on the reduction in myelinated nerve fiber size and fiber occupancy. Axon-fiber size ratio was also preserved in the ponalrestat-treated group. However, diffuse deposition of glycogen and increased glycogenosomes within axons were not influenced by ponalrestat treatment. In contrast to the effect on myelinated nerve fibers, morphometry of unmyelinated nerve fibers did not reveal a significant effect of ponalrestat treatment. These results suggest that chronic treatment with an aldose reductase inhibitor has beneficial effects on the peripheral sensory nerve of experimentally diabetic rats. The effects were primarily on myelinated rather than unmyelinated nerve fibers.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Diabetic Neuropathies/etiology , Sugar Alcohol Dehydrogenases/antagonists & inhibitors , Animals , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Experimental/physiopathology , Inositol/metabolism , Male , Nerve Fibers/metabolism , Nerve Fibers/pathology , Nerve Fibers, Myelinated/metabolism , Nerve Fibers, Myelinated/pathology , Neural Conduction , Rats , Rats, Inbred Strains , Sorbitol/metabolism , Streptozocin , Sural Nerve/metabolism , Sural Nerve/pathology , Sural Nerve/physiopathology , Time Factors
13.
Biochem Pharmacol ; 39(2): 337-46, 1990 Jan 15.
Article in English | MEDLINE | ID: mdl-2105733

ABSTRACT

Many of the complications of diabetes appear to be closely linked to increased conversion of tissue glucose to sorbitol which is catalysed by aldose reductase (aldehyde reductase 2, ALR2). Inhibition of ALR2 could, therefore, lead to a reduction in the development of diabetic complications. Ponalrestat ["Statil" (a trademark, the property of Imperical Chemical Industries PLC), "Prodiax" (a trademark, the property of Merck, Sharp and Dohme), ICI 128436, MK538] inhibits ALR2 from a number of sources. Until now, the mechanism of this inhibition has not been fully elucidated. In this paper, we present a detailed mechanism for inhibition of bovine lens ALR2 by ponalrestat. Treatment of humans with some ALR2 inhibitors leads to side-effects, some of which may result from interactions with other enzymes. Aldehyde reductase (ALR1) is probably the most closely related enzyme to ALR2. Inhibition of ALR1 from bovine kidney was, therefore, investigated in order to assess the specificity of ponalrestat. The values of Ki and Kies (apparent dissociation constants for inhibitor from enzyme-inhibitor and enzyme-inhibitor-substrate complexes, respectively) for the interactions of ponalrestat with ALR1 and ALR2 has been calculated by non-linear fitting of kinetic data. These values indicate that ponalrestat does not compete with binding of glucose of NADPH to ALR2, nor with binding of glucuronate or NADPH to ALR1. Lack of competition and the structural dissimilarity of substrates and inhibitor make it unlikely that ponalrestat will utilize substrate binding sites on other enzymes, and so produce undesirable side-effects via such a mechanism. Ponalrestat is a potent inhibitor (Ki = Kies = 7.7 nM) of ALR2 and follows a pure noncompetitive mechanism with respect to glucose. Efficacy, therefore, will not be decreased by development of hyperglycaemia. The compound is a mixed noncompetitive inhibitor of ALR1 when glucuronate is varied. The values of Ki and Kies are 60 microM and 3 microM, respectively, so that inhibition tends towards uncompetitive. The selectivity of ponalrestat in favour of ALR2, therefore, lies in the range 390 to 7,800-fold, being higher at lower concentrations of glucuronate. The high selectivity of ponalrestat in favour of ALR2 rather than ALR1 suggests that the compound is unlikely to inhibit other enzymes which have less homology with ALR2.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Kidney/enzymology , Lens, Crystalline/enzymology , Phthalazines/pharmacology , Pyridazines/pharmacology , Sugar Alcohol Dehydrogenases/antagonists & inhibitors , Animals , Cattle , Dose-Response Relationship, Drug , Glucose/pharmacology , Glucuronates/pharmacology , Glucuronic Acid , Hyperglycemia/enzymology , Isoenzymes/antagonists & inhibitors , Kinetics , Phthalazines/metabolism , Protein Binding , Software , Substrate Specificity
14.
Histochemistry ; 94(6): 659-66, 1990.
Article in English | MEDLINE | ID: mdl-1704001

ABSTRACT

We have previously shown depletion of nerves and neuropeptides in skin biopsies of diabetic patients, even in the absence of clinical signs and symptoms of sensory and autonomic neuropathy, but were unable to examine the changes occurring at an early stage of the disease. Therefore, the distribution and relative density of peptide-containing nerves was studied in streptozotocin-treated rats in order to assess the progression of neural changes in the initial stages of diabetes. Skin samples dissected from the lip and footpad of diabetic rats, 2, 4, 8 and 12 weeks after streptozotocin injection and age matched controls were sectioned and were immunostained with antisera to the neuropeptides substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), and to a general neural marker, protein gene product 9.5 (PGP 9.5). No change was apparent in the distribution or relative density of immunoreactive cutaneous nerve fibres 2, 4 and 8 weeks after streptozotocin treatment. By 12 weeks there was a marked increase in the number of CGRP-immunoreactive fibres present in epidermis and dermis, and of VIP-immunoreactive fibres around sweat glands and blood vessels. A parallel increase was seen in nerves displaying PGP 9.5 immunoreactivity. No differences were detected in nerves immunoreactive for either substance P in the epidermis and dermis, and NPY around blood vessels. The alterations in the peptide immunoreactivities may be similar in the initial stages of human diabetes.


Subject(s)
Calcitonin Gene-Related Peptide/analysis , Diabetes Mellitus, Experimental/metabolism , Diabetic Neuropathies/physiopathology , Nerve Fibers/chemistry , Skin/innervation , Vasoactive Intestinal Peptide/analysis , Animals , Fluorescent Antibody Technique , Male , Neuropeptide Y/analysis , Neuropeptides/analysis , Rats , Rats, Inbred Strains , Substance P/analysis , Ubiquitin Thiolesterase
16.
Biochem J ; 240(1): 233-7, 1986 Nov 15.
Article in English | MEDLINE | ID: mdl-3030278

ABSTRACT

The production of polyols in vitro by highly purified aldose reductase (EC 1.1.1.21) was monitored by g.l.c. In the presence of NADPH aldose reductase reduced glucose, galactose and xylose to the respective polyols sorbitol, galactitol and xylitol. The rates of formation of these polyols closely mirrored the Km values for the substrates obtained from kinetic measurements that monitored the rate of disappearance of NADPH. No polyol production occurred in the absence of purified aldose of purified aldose reductase, and analysis by g.l.c. revealed only the presence of unchanged monosaccharides. Addition of the aldose reductase inhibitor sorbinil to purified rat lens aldose reductase incubated with xylose in the presence of NADPH resulted in decreased xylitol production. However, aldose reductase inhibitors produced no effect in altering the rate of Nitro Blue Tetrazolium formation from either glucose or xylose, indicating that the observed inhibition in vitro does not result from a free-radical-scavenger effect.


Subject(s)
Aldehyde Reductase/isolation & purification , Lens, Crystalline/enzymology , Sugar Alcohol Dehydrogenases/isolation & purification , Aldehyde Reductase/antagonists & inhibitors , Animals , Chromatography, Gas , Electrophoresis, Polyacrylamide Gel , Kinetics , Polymers/metabolism , Rats , Superoxides/metabolism
17.
J Neurol Sci ; 74(1): 55-67, 1986 Jun.
Article in English | MEDLINE | ID: mdl-3522807

ABSTRACT

Diabetes mellitus was induced in rats by the administration of streptozotocin and observations have been made over a period of 2 months in 3 groups of animals: controls, untreated diabetics and diabetics treated with continuous subcutaneous insulin infusion (CSII) therapy, using a 14-day Alzet osmotic minipump. Optimal control of day-to-day and 24-h blood glucose levels was achieved in diabetic animals treated with CSII. Body weight and skeletal growth, assessed by measurements of tibial length, were decreased in untreated diabetic rats and were normalized by insulin treatment. The concentrations of glucose, sorbitol and fructose in the nerves of diabetic animals were significantly increased and that of myoinositol significantly decreased; CSII therapy normalized these levels to those of age-matched controls. External myelinated fibre diameter in the tibial nerve was significantly less in untreated diabetic rats as compared with age-matched controls. In the insulin-treated group, fibre diameter significantly increased as compared with untreated diabetics and there was no significant difference between insulin-treated and control animals. Teased fibre preparations from the tibial nerve revealed very few abnormal fibres in all the three groups and no significant difference was detected between any of the groups. Continuous subcutaneous insulin infusion therapy, therefore, corrected biochemical abnormalities and also normalized myelinated fibre diameter in the peripheral nerves of experimental diabetic animals. The paradoxical excess of axonal degeneration that has been reported with conventional insulin treatment was not observed.


Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetic Neuropathies/prevention & control , Insulin/therapeutic use , Peripheral Nerves/pathology , Animals , Blood Glucose/analysis , Body Height , Body Weight , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Insulin/administration & dosage , Male , Peripheral Nerves/analysis , Rats , Rats, Inbred Strains
18.
Metabolism ; 34(4): 336-44, 1985 Apr.
Article in English | MEDLINE | ID: mdl-3920474

ABSTRACT

ICI 128,436 (3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-ylacetic acid) is a chemically novel, potent inhibitor of aldose reductase. It inhibits partially purified aldose reductase isolated from a number of sources including human tissue (human lens - IC50 2.0 X 10(-8) mol/L). Dulcitol accumulation in erythrocytes and sciatic nerves of galactose loaded rats was inhibited by five days of treatment with ICI 128,436 (oral ED50's 2.21 mg/kg and 8.56 mg/kg, respectively). On oral administration for five days to streptozotocin diabetic rats, ICI 128,436, reduced sorbitol levels in sciatic nerve, lens, retina, and renal cortex. The ED50 for inhibition of nerve sorbitol accumulation was 5 mg/kg. The effect of a single dose of ICI 128,436 in diabetic rats was prolonged, with little increase in nerve sorbitol for 48 hours. No tolerance to the ability of ICI 128,436 to reduce nerve sorbitol was found on treatment for 74 days. ICI 128,436 was effective in rodent models of the neural and lenticular complications of diabetes. At doses as low as 25 mg/kg/d it completely prevented the development of cataracts in diabetic rats. The deterioration in motor nerve conduction velocity velocity found in diabetic rats was ameliorated by treatment with ICI 128,436 (3.125 mg/kg/d). Thus, ICI 128,436 constitutes a chemically novel aldose reductase inhibitor that is now being assessed for therapeutic value in the diabetic patient.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Cataract/prevention & control , Diabetes Mellitus, Experimental/physiopathology , Neural Conduction/drug effects , Phthalazines/pharmacology , Pyridazines/pharmacology , Sugar Alcohol Dehydrogenases/antagonists & inhibitors , Animals , Cataract/etiology , Cattle , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/metabolism , Dogs , Erythrocytes/metabolism , Fructose/metabolism , Galactitol/blood , Galactitol/metabolism , Humans , In Vitro Techniques , Kidney Cortex/drug effects , Kidney Cortex/metabolism , Lens, Crystalline/drug effects , Lens, Crystalline/enzymology , Male , Motor Neurons/drug effects , Rats , Retina/drug effects , Retina/metabolism , Sciatic Nerve/drug effects , Sciatic Nerve/metabolism , Sorbitol/metabolism
19.
Exp Eye Res ; 36(5): 751-60, 1983 May.
Article in English | MEDLINE | ID: mdl-6406254

ABSTRACT

UNLABELLED: To investigate the aetiology of complications secondary to experimental diabetes in the rat, the concentrations of glucose, sorbitol, fructose, fructose-1-phosphate, lactate and inositol were measured in the retina and sciatic nerve of streptozotocin-diabetic and normal rats treated for 22 days with ICI 105552 (1-(3,4-dichlorobenzyl)-3-methyl-1,2-dihydro-2-oxoquinol-4-ylacetic acid, sodium salt; 50 mg/kg body wt daily), an inhibitor of aldose reductase (E.C. 1.1.1.21) the first enzyme of the sorbitol pathway. In the diabetic nerve, where accumulation of sorbitol may be pathogenic, treatment with ICI 105552 reduced the accumulations of sorbitol (70%), fructose (47%) and lactate (34%) without affecting glucose, fructose-1-phosphate or inositol. In the nerves of controls, the inhibitor reduced both sorbitol (23%) and fructose (20%) levels without other effects. In the diabetic retina where accumulation of sorbitol or lactate might be pathogenic, treatment with ICI 105552 had no statistically significant effect upon the concentrations of glucose, sorbitol, fructose, fructose-1-phosphate or inositol. However, the inhibitor reduced the concentration of lactate to below the non-diabetic level. In the retinas of controls, dosage with ICI 105552 reduced the concentration of sorbitol by 36% without other effects. THE RESULTS: (1) demonstrated that ICI 105552 was a potent inhibitor of aldose reductase in sciatic nerve and suggested that a proportion of nerve lactate in diabetes could result from sorbitol pathway activity; (2) implied that flux through the sorbitol pathway, eventually to form lactate, increases in diabetic retina; and (3) indicated, by the drug's reduction of retinal lactate concentration, that inhibitors of aldose reductase might be of potential use in diabetic retinopathy.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Diabetes Mellitus, Experimental/metabolism , Quinolines/pharmacology , Quinolones , Retina/metabolism , Sugar Alcohol Dehydrogenases/antagonists & inhibitors , Animals , Fructosephosphates/metabolism , In Vitro Techniques , Lactates/metabolism , Male , Rats , Rats, Inbred Strains , Sciatic Nerve/metabolism , Sorbitol/metabolism , Sucrose/metabolism
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