3-Mercapto-5H-1,2,4-Triazino[5,6-b]Indole-5-Acetic Acid (Cemtirestat) Alleviates Symptoms of Peripheral Diabetic Neuropathy in Zucker Diabetic Fatty (ZDF) Rats: A Role of Aldose Reductase

Peripheral neuropathy is the most prevalent chronic complication of diabetes mellitus. Good glycemic control can delay the appearance of neuropathic symptoms in diabetic patients but it is not sufficient to prevent or cure the disease. Therefore therapeutic approaches should focus on attenuation of pathogenetic mechanisms responsible for the nerve injury. Considering the role of polyol pathway in the etiology of diabetic neuropathy, we evaluated the effect of a novel efficient and selective aldose reductase inhibitor, 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (cemtirestat), on symptoms of diabetic peripheral neuropathy in Zucker Diabetic Fatty (ZDF) rats. Since the age of 5 months, male ZDF rats were orally administered cemtirestat, 2.5 and 7.5 mg/kg/day, for two following months. Thermal hypoalgesia was evaluated by tail flick and hot plate tests. Tactile allodynia was determined by a von Frey flexible filament test. Two-month treatment of ZDF rats with cemtirestat (i) did not affect physical and glycemic status of the animals; (ii) partially inhibited sorbitol accumulation in red blood cells and the sciatic nerve; (iii) markedly decreased plasma levels of thiobarbituric acid reactive substances; (iv) normalized symptoms of peripheral neuropathy with high significance. The presented findings indicate that inhibition of aldose reductase by cemtirestat is not solely responsible for the recorded improvement of the behavioral responses. In future studies, potential effects of cemtirestat on consequences of diabetes that are not exclusively dependent on glucose metabolism via polyol pathway should be taken into consideration.

     

2,4-dinitrophenylhydrazine carbonyl assay in metal-catalysed protein glycoxidation

Using an experimental in vitro glycation model, long-term incubations of bovine serum albumin with glucose (fructose) resulted in a significant increase in protein content of 2,4-dinitrophenylhydrazine (DNPH)-reactive carbonyl groups, which could be strongly inhibited by anaerobiosis and metal chelation. The pattern of yields of the protein-bound DNPH was not in accordance with that of the sugar-derived carbonyls determined as the ketoamine Amadori product. In spite of the fact that the contribution of the final advanced glycation end-products to the total DNPH-reactivity of glycation-altered protein remains unclear, the present results stress the need of oxidative steps in formation of most of the DNPH-reactive carbonyl compounds generated by glycation. The results provide evidence that, in protein glycoxidation, the DNPH assay is selective enough to discriminate between protein-bound carbonyls produced by metal-catalysed oxidations and those formed in the early glycation steps.     

Oxidative modification of serum albumin in an experimental glycation model of diabetes mellitus in vitro: effect of the pyridoindole antioxidant stobadine

Under conditions of an experimental in vitro glycation model, the pyridoindole antioxidant stobadine significantly inhibited glycation-related fluorescence changes of bovine serum albumin as well as the yield of 2,4-dinitrophenylhydrazine-reactive carbonyls with an efficacy comparable to that of the reference antioxidants Trolox C and 2-keto-4-methiolbutyric acid, and more efficiently than did aminoguanidine. Since stobadine did not affect the covalent binding of glucose, the protective effect may be explained by the ability of the drug to eliminate free radical intermediates of glyco-oxidation reactions, operative after the preceding glycation steps.     

Cardiac effects of endothelin-1 (ET-1) and related C terminal peptide fragment: increased inotropy or contribution to heart failure?

The contrasting pattern of cardiac inotropy induced by human peptide endothelin-1 (ET-1) has not been satisfactorily explained. It is not clear whether ET-1 is primarily responsible for increased myocardial ET-1 expression and release with resultant inotropic effects, or for the induction of myocardial hypertrophy and heart failure. There are at least two subtypes of endothelin receptors (ET(A) and ET(B)) and the inotropic effects of ET-1 differ depending on the receptor involved. Along with some other groups, we reported significant subtype-ET(B) endothelin receptor down-regulation in human cardiac cells preincubated with endothelin agonists (Drímal et al. 1999, 2000). The present study was therefore designed to clarify the subtype-selective mechanisms underlying the inotropic response to ET-1 and to its ET(B)-selective fragment (8-21)ET-1 in the isolated rat heart. The hearts were subjected to (1-21)ET-1 and to (8-21)ET-1, or to 30 min of stop-flow ischemia followed by 40 min of reperfusion, both before and after selective blockade of endothelin receptors.The present study revealed that both peptides, ET-1 and its (8-21)ET-1 fragment, significantly reduced coronary blood flow in nmolar and higher concentrations. The concomitant negative inotropy and chronotropy were marked after ET-1, while the infusion of the ET-1(8-21) fragment produced a slight but significant positive inotropic effect. Among the four endothelin antagonists tested in continuous infusion only the non-selective PD145065 and ET(B1/B2) selective BQ788 (in molar concentrations) slightly reduced the early contractile dysfunction of the heart induced by ischemia, whereas ET(A)-selective PD155080 partially protected the rat heart on reperfusion.     

(2-Benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-8-yl)-acetic acid: an aldose reductase inhibitor and antioxidant of zwitterionic nature

Novel carboxymethylated pyridoindoles, characterized by antioxidant activity combined with the ability to inhibit aldose reductase, represent an example of a multitarget approach to the treatment of diabetic complications - severe diabetes-related health disorders of multifunctional nature. One of the novel carboxymethylated pyridoindoles, (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-8-yl)-acetic acid (compound 1), was found to inhibit aldose reductase with the IC(50) value 18.2 ± 1.2 μM. Owing to aldose reductase pharmacophore requirements for an acidic proton, most aldose reductase inhibitors contain an acetic acid moiety, ionized at physiological pH, resulting in poor bioavailability of the drugs. The presence of a basicity center at the tertiary nitrogen of the carboxymethylated pyridoindoles, in addition to the acidic carboxylic function, predisposes these compounds to form double-charged zwitterionic species. The zwitterionic nature of compound 1 may remarkably affect its pH-lipophilicity profile allowing for increased membrane penetration in the pH region around its isoelectric point, which lies close to the physiological pH 7.4. In the first part of this study, the presence of zwitterionic species was experimentally proved by the concentration-dependent effect of sodium 1-hexanesulphonate on the distribution profile of compound 1. Then a series of experiments was performed in the cellular system of isolated erythrocytes in vitro. Isolated rat erythrocytes exposed to peroxyl radicals, generated in the solution by decomposition of the hydrophilic azoinitiator AAPH or intracellularly by decay of lipophilic t-BuOOH, underwent progressive hemolysis. The onset of the hemolysis was shifted from the starting zero point by the time interval assigned as a lag period. In the presence of compound 1 the lag period was significantly prolonged. Finally, under conditions of a short-term experiment in STZ-diabetic rats in vivo, increase in sorbitol levels in erythrocytes was recorded. Compound 1 administered in the dose 50mg/kg/day (i.g.) significantly decreased the sorbitol level in the erythrocytes. To conclude, the physico-chemical proof of the zwitterionic nature of compound 1 was established and the results obtained in isolated red blood cells indicated good cellular availability of the compound. In addition, in diabetic rats, sorbitol accumulation in red blood cells was significantly inhibited by compound 1 administered intra-gastrically, suggesting its ready uptake into the central compartment. The zwitterionic principle thus may have significant consequences for increased bioavailability of drugs bearing an acidic function.     

Effect of the pyridoindole antioxidant stobadine on the cardiac Na(+),K(+)-ATPase in rats with streptozotocin-induced diabetes

In the present study we examined the effect of dietary supplementation with the pyridoindole antioxidant stobadine on functional properties of the cardiac Na(+),K(+)-ATPase in diabetic rats. Diabetes lasting sixteen weeks which was induced by a single i.v. dose of streptozotocin (55 mg x kg(-1)) was followed by decrease in the enzyme activity. Evaluation of kinetic parameters revealed a statistically significant decrease in the maximum velocity (Vmax) (32% for ATP-activation, 33% for Na(+)-activation), indicating a diabetes-induced diminution of the number of active enzyme molecules in cardiac sarcolemma. The ATP-binding properties of the enzyme were not affected by diabetes as suggested by statistically insignificant changes in the value of Michaelis-Menten constant, K(M (ATP)). On the other hand, the affinity to sodium decreased as suggested by 54% increase in the K(M (Na+)) value. This impairment in the affinity of the Na(+)-binding site together with decreased number of active Na(+),K(+)-ATPase molecules are probably responsible for the deteriorated enzyme function in hearts of diabetic animals. Administration of stobadine to diabetic rats dramatically improved the function of cardiac Na(+),K(+)-ATPase with regard to Na(+)-handling, as documented by statistically significant elevation of Vmax by 66 and 47% decrease in K(M (Na+)). Our data suggest that stobadine may prevent the diabetes-induced deterioration of cardiac Na(+),K(+)-ATPase, thus enabling to preserve its normal function in regulation of intracellular homeostasis of Na(+) and K(+) ions.     

Changes in the function and ultrastructure of vessels in the rat model of multiple low dose streptozotocin-induced diabetes

In this study we investigated functional changes in the femoral artery and ultrastructural alterations in mesenteric vessels and capillaries in the rat model of multiple low dose streptozotocin (STZ)-induced diabetes. Participation of oxidative stress in this model of diabetes was established by studying the effect of the pyridoindole antioxidant stobadine (STB) on diabetes-induced impairment. Experimental diabetes was induced by i.v. bolus of STZ (20 mg/kg) given for three consecutive days to male rats. At the 12(th) week following STZ administration, the animals revealed typical signs of diabetes, such as polyphagia, polydypsia and polyuria. There was no weight gain in the diabetic groups throughout the experiment. No exitus occurred in any group. Diabetes was characterised with high levels of plasma glucose, no significant changes in lipid metabolism, decreased serum levels of glutathione, increased serum levels of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (NAGA), injured endothelial relaxant capacity of the femoral artery and alterations in ultrastructure of mesenteric arteries and capillaries. Antioxidant STB in the dose of 25 mg/kg body weight i.p. (5 times per week) did not influence glucose levels, however, it mitigated biochemical, functional and ultrastructural changes induced by diabetes, suggesting a role of reactive oxygen species in diabetes-induced tissue damage.     

Reactive oxygen species induced smooth muscle responses in the intestine, vessels and airways and the effect of antioxidants

Numerous experimental data confirm the importance of reactive oxygen species (ROS) in physiological activities of smooth muscles and in the pathogenesis of various diseases with altered function of smooth muscles. The present study shows that smooth muscles of the intestine, airways and vessels, as well as their epithelium, endothelium and innervations, might be important targets of the ROS action. We demonstrated differences among the actions of various ROS (endogenous, exogenous, produced enzymatically, non-enzymatically) as well as among their actions in different smooth muscle tissues. Our results indicate that ROS are involved in changes in muscle tone, membrane conductance, calcium homeostasis, calcium-dependent processes, as well as in eicosanoid and nitric oxide metabolism. The effects of antioxidative enzymes (superoxide dismutase, catalase), of several drugs of natural origin (e.g. Kampo Medicines) and synthetic agents (e.g. stobadine, nitrosopine, ACE inhibitors) suggest that smooth muscle tissues are useful models to study ROS action and drug intervention in ROS induced injuries.     

Structural aspects of antioxidant activity of substituted pyridoindoles

Stobadine and its two structural analogues, dehydrostobadine and N-acetylated stobadine were used to examine how structural alteration in the close proximity of the indolic nitrogen would influence the antioxidant activity of the substituted pyridoindoles. The compounds were tested for their efficiency to scavenge stable free radicals of alpha,alpha'-diphenyl-beta-picrylhydrazyl as well as for their ability to prevent 2,2'-azobis-(2-amidinopropane)hydrochloride induced peroxidation of dioleoyl phosphatidylcholine liposomes. The results proved that the substituted pyridoindoles can act as potent scavengers of peroxyl radicals both in aqueous and lipid phases, the antioxidant activity being comparable with that of Trolox. Structural changes in the proximity of the indolic nitrogen were found crucial for the radical scavenging efficiency: aromatisation of the pyridoindole skeleton in dehydrostobadine lowered the antioxidant activity, while acetylation of the indolic nitrogen completely abolished the ability to scavenge peroxyl radicals. The results are in agreement with the notion that the antioxidant activity of stobadine and of the related pyridoindoles may be mediated via the indolic nitrogen centre. When stobadine and Trolox were present simultaneously in liposomal incubations, Trolox spared stobadine in a dose-dependent manner; a direct interaction of Trolox with stobadinyl radical appears to be a plausible explanation with possible consequences for the antioxidant capacity of stobadine under in vivo conditions, where re-cycling of stobadine by vitamin E might occur.     

Effect of the pyridoindole antioxidant stobadine on ATP-utilisation by renal Na,K-ATPase in rats with streptozotocin-induced diabetes

The effect of the pyridoindole antioxidant stobadine on diabetes-induced changes of Na,K-ATPase, especially those concerning the utilisation of its substrate ATP, was investigated. Sixteen weeks of streptozotocin-induced diabetes (single i.v. dose of streptozotocin; 55 mg/kg) was followed by decrease in the enzyme activity. This effect was emphasised in the presence of higher concentrations of substrate and in the presence of 8 mmol x l(-1) ATP it represented 20%. It might be a consequence of altered functional properties of Na,K-ATPase as suggested by 20% decrease in the V(max) value along with decrease in the K(m) value by 20%. Administration of 0.05% (w/w) stobadine in the diet to diabetic rats improved the function of renal Na,K-ATPase with respect to utilisation of ATP as suggested by significant increase in the enzyme activity in the whole concentration range of ATP investigated as a consequence of V(max) elevation to the level comparable to absolute controls. In conclusion, stobadine may play a positive role in restoring the functional properties of renal Na,K-ATPase, especially concerning the utilisation of energy derived from hydrolysis of ATP, improving thus the maintenance of ionic homeostasis during diabetes.