No abstract

The 'late complications' of diabetes mellitus, i.e., nephropathy, neuropathy and retinopathy are firmly rooted in inadequate control of blood glucose: hyperglycaemia. Hyperglycaemia causes elevated cytosolic glucose and/or rates of glucose metabolism, i.e., 'hyperglysolia,' within cells of vulnerable tissues. Although the molecular basis for the pathogenic effects of hyperglysolia remains to be proven, substantial evidence points to a key role for increased glucose metabolism through a cytosolic enzyme, aldose reductase (AR). Recent human genetic and biochemical data link polymorphisms of the AR gene (technically called the AR2 gene) and elevated tissue levels of AR with strongly altered risks for diabetic complications. Despite several genetic reports failing to confirm such an association, there are now ten concordant reports from five continents that certain polymorphisms of the AR gene are associated with an ~ 3- to 20-fold higher risk for diabetic complications. Moreover, in US and European diabetic study populations the principle allele of the AR gene associated with elevated disease risk, the Z-2 allele, correlates with an ~ 2- to 3-fold increase in AR expression. These results, together with recent clinical, experimental and pharmacological data, provide powerful new support for the rationale for research and development of aldose reductase inhibitors (ARIs) targeted at slowing the progression of diabetic complications. Although past clinical trials of ARIs have been disappointing, this has stemmed from overly optimistic expectations, inadequate trial designs and lack of pharmacological robustness and/or acceptable systemic toleration of the agents tested. However, a more realistic and encouraging perspective for therapeutic expectations for ARIs has arisen from recent data revealing that the seemingly modest short-term effects of intensified glycaemic control and of pancreatic transplantation are followed by substantial long-term benefits on diabetic complications. In addition, robust inhibition of AR in human nerve has recently yielded dose-dependent efficacy on nerve structure and function. Thus, the quest for well-tolerated, potent ARIs continues to be a worthy and more urgent objective than ever before.

A new working hypothesis that there is a hitherto unrecognized binding site on the aldose reductase (AR) enzyme with strong affinity for benzothiazoles was pursued for the design of novel, potent aldose reductase inhibitors (ARIs). The first application of this hypothesis led to a novel series of 3,4-dihydro-4-oxo-3-(benzothiazolylmethyl)-1-phthalazineacetic+ + + acids. The parent of this series (207) was a potent inhibitor of AR from human placenta (IC50 = 1.9 x 10(-8) M) and was orally active in preventing sorbitol accumulation in rat sciatic nerve, in an acute test of diabetic complications (ED50 = 18.5 mg/kg). Optimization of this lead through medicinal chemical rationale, including analogy from other drug series, led to more potent congeners of 207 and culminated in the design of 3,4-dihydro-4-oxo-3-[[5-(trifluoromethyl)-2-benzothiazolyl] methyl]-1-phthalazineacetic acid (216, CP-73,850, zopolrestat). Zopolrestat was found to be more potent than 207, both in vitro and in vivo. Its IC50 against AR and ED50 in the acute test were 3.1 x 10(-9)M and 3.6 mg/kg, respectively. Its ED50s in reversing already elevated sorbitol accumulation in rat sciatic nerve, retina, and lens in a chronic test were 1.9, 17.6, and 18.4 mg/kg, respectively. It was well absorbed in diabetic patients, resulting in high blood level, showed a highly favorable plasma half-life (27.5 h), and is undergoing further clinical evaluation. An assortment of synthetic methods used for the construction of benzothiazoles, including an efficient synthesis of zopolrestat, is described. Structure-activity relationships in the new series are discussed.

Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.

These experiments were undertaken to assess the importance of cytoplasmic (c) sorbitol oxidation versus mitochondrial (m) pyruvate oxidation in mediating neural and vascular dysfunction attributable to hyperglycemia in diabetic rats. Increased oxidation of sorbitol is coupled to enzymatic reduction of free oxidized NAD(+)c to reduced NADHc, manifested by an increased ratio of NADH to NAD(+)c. Likewise, increased oxidation of pyruvate is coupled to reduction of NAD(+)m to NADHm, which increases the NADH/NAD(+)m ratio. Specific inhibitors of sorbitol production or sorbitol oxidation normalized: increased diabetic nerve NADH/NAD(+)c, impaired nerve-conduction velocity, and vascular dysfunction in sciatic nerve, retina, and aorta; however, they had little or no impact on increased NADH/NAD(+)m. These observations provide, for the first time, strong in vivo evidence for the primacy of sorbitol oxidation versus. pyruvate oxidation in mediating the metabolic imbalances, impaired nerve conduction, and vascular dysfunction evoked by diabetes. These findings are consistent with (a) the fact that oxidation of sorbitol produces "prooxidant" NADHc uncoupled from subsequent production of "antioxidant" pyruvate required for reoxidation of NADHc to NAD(+)c by lactate dehydrogenase, and (b) the hypothesis that neural and vascular dysfunction in early diabetes are caused primarily by increased NADHc, which fuels superoxide production by NADH-driven oxidases.