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T C Hohman

Publications and source records attributed to T C Hohman.

At least 37 records · Page 2Linked to original sources
PubMed1996

Diabetic-like retinopathy ameliorated with the aldose reductase inhibitor WAY-121,509.

PURPOSE: To evaluate the efficacy of WAY-121,509, a potent new aldose reductase inhibitor (ARI), in preventing the retinopathy that develops in the galactose-fed rat model of diabetic ocular complications. METHODS: Sprague-Dawley rats were randomized into treatment and duration groups and fed diets with either 50% starch of 50% galactose with or without WAY-121,509 (25 mg/kg body weight per day). Progression of cataracts was monitored by slit-lamp biomicroscopy. After duration of 4, 8, 16, and 24 months, levels of plasma glucose and glycated hemoglobin, as well as erythrocyte and retinal galactose and galactitol, were measured in rats in each group. Retinal vasculatures of the 24-month rats were isolated by elastase digestion and analyzed by computer-assisted morphometry. RESULTS: Mature, diabetic-like cataracts developed within 5 weeks in all the galactose-fed, untreated rats, but only nonprogressive anterior cortical opacities were present in lenses of 85% of the ARI-treated galactosemic animals after 3 months. Plasma glucose remained the same in all groups. Erythrocyte and retinal galactose and glycated (galactosylated) hemoglobin were elevated with galactosemia and were unaffected by ARI treatment. Erythrocyte and retinal galactitol levels were decreased by 91% and 95%, respectively, with inhibitor treatment. At 24 months, capillary length, width, density, the number of microaneurysms, and the percent of capillary length involved in intraretinal microvascular abnormalities, expressed as hypercellular channels with diameters > 20 microns, were significantly increased by galactosemia and were attenuated in the galactose-fed, ARI-treated group. CONCLUSIONS: A dose of WAY-121,509 sufficient to reduce retinal polyol levels by 95% ameliorated the development of galactose-induced cataracts and diabetic-like retinopathy but was insufficient to prevent early lens opacifications or all the diabetic-like retinal microangiopathies.

Aldehyde Reductase↗
PubMed1995

Residues affecting the catalysis and inhibition of rat lens aldose reductase.

Aldose reductase (AR), the first enzyme of the polyol pathway, has been implicated in diabetic complications. Results of recent clinical studies have shown that compounds that inhibit aldose reductase (ARIs) and block the flux of glucose through the polyol pathway have provided benefit to diabetic neuropathic patients. Since many ARIs show broad substrate specificity, emphasis on the structure-function properties of the AR enzyme will help in the refinement and design of future inhibitors. To this end, catalysis and inhibition of rat lens aldose reductase was examined following site-directed mutagenesis. Replacement of tyrosine 48 with phenylalanine (Y48F) resulted in an enzyme form with less than 0.25% activity with DL-glyceraldehyde and no detectable activity with p-nitrobenzaldehyde or xylose, although circular dichroism spectra and NADPH binding affinity were similar to wild-type AR. Mutation of histidine 110 to glutamine (H110Q) also resulted in a less active protein with an approximate 3-fold decrease in kcat for the reduction of DL-glyceraldehyde; slight or no activity was measured with other substrates and an increase of 195-fold over wild type was observed in the Km for glyceraldehyde. H110Q was less sensitive to inhibition by aldose reductase inhibitors. The most dramatic change was seen with imeristat, which showed an 1800-fold increase in IC50. Mutation of cysteine 298 to serine (C298S) affected enzyme function by increasing kcat 2- to 4-fold and increasing Km 15- to 48-fold, with DL-glyceraldehyde, p-nitrobenzaldehyde or xylose as substrates. As a result kcat/Km, catalytic efficiency, dropped to approx. 10% of control. Inhibition of C298S was not noticeably different from wild type. Substitution of histidine 187 or 200 with glutamine (H187Q, H200Q) had little effect on AR catalysis or inhibition. Based on structural and mutagenesis studies of human AR and the conservation of amino acids between human and rat, these data would indicate that Y48, H110, and C298 are important residues in the active site of rat AR and that Y48 is most likely the proton donor during substrate reduction by rat lens aldose reductase. In addition, these studies indicate that mutagenesis of H110 also affects aldose reductase inhibition.

Aldehyde Reductase↗
PubMed1995

Na,K-ATPase response to osmotic stress in primary dog lens epithelial cells.

PURPOSE: Na,K-ATPase activity increases in lens cells exposed to hypertonic stress. To test whether the increase in activity involves stimulation of Na,K-ATPase expression, dog lens epithelial cells were subjected to hypertonic stress, and the time course of Na,K-ATPase protein and mRNA response was measured. METHODS: Primary cultures of dog lens epithelial cells were maintained in isotonic or hypertonic media over the course of several days. Rubidium-86 uptake measurements, immunoreactive protein, and northern blot analysis were performed. RESULTS: Dog lens epithelial cells exposed to hypertonic stress from culture medium supplemented with 150 mM NaCl or 250 mM cellobiose showed a twofold increase in Na,K-ATPase activity. The increase in activity was blocked by cycloheximide and was reversible when the cells were returned to isotonic medium. This activity was unaffected by the aldose reductase inhibitor, tolrestat. Na,K-ATPase protein and mRNA levels increased in cells exposed to medium containing 150 mM NaCl. Northern blot analysis showed that the alpha-1 and beta-1 mRNA levels increased as early as 6 hours and maximally increased 1.5-fold to twofold by 12 to 24 hours. CONCLUSIONS: Elevation of Na,K-ATPase activity in dog lens epithelial cells exposed to hypertonic stress was associated with increased expression of Na,K-ATPase subunit mRNAs and was dependent on protein synthesis. These results suggest that upregulation of the enzyme activity is the result of an induction of Na,K-ATPase.

Animals↗
PubMed1994

Novel spirosuccinimide aldose reductase inhibitors derived from isoquinoline-1,3-diones: 2-[(4-bromo-2-fluorophenyl)methyl]-6- fluorospiro[isoquinoline-4(1H),3'-pyrrolidine]-1,2',3,5'(2H)-tetrone and congeners. 1.

The high concentrations of plasma glucose formed during diabetic hyperglycemia rapidly translate into high levels of glucose in tissues where glucose uptake is independent of insulin. In these tissues that include the lens, retina, nerve, and kidney, this excess glucose enters the sorbitol (polyol) pathway. The first enzyme in this pathway, aldose reductase, reduces glucose to sorbitol. The diabetes-induced increased flux of glucose through the polyol pathway is believed to play an important role in the development of certain chronic complications of diabetes mellitus. Compounds that inhibit aldose reductase activity and block the flux of glucose through the polyol pathway prevent the development of neuropathy and nephropathy in diabetic animals and interrupt the progression of neuropathy in diabetic patients. Here we describe the preparation and characterization of novel aldose reductase inhibitors. These spiro[isoquinoline-4(1H),3'-pyrrolidine]-1,2',3,5'-(2H)-tetrones, based on the isoquinoline-1,3-dione framework, were evaluated in vitro for their ability to inhibit glyceraldehyde reduction, using a partially purified bovine lens aldose reductase preparation, and in vivo for their ability to inhibit galactitol accumulation in the lens and sciatic nerve of galactose-fed rats. Substitution at the N-2 position of the isoquinoline-1,3-dione framework with diverse structural substituents (i.e., aralkyl, benzothiazolylmethyl, methyl) produced several excellent series of ARIs. Optimization of these new series of spirosuccinimides through structure-activity relationship (SAR) studies, including analogy from other drug series (ponalrestat, zopolrestat), led to the design of the clinical candidate 2-[(4-bromo-2-fluorophenyl)methyl]-6-fluorospiro[isoquinoline-4(1H ),3'- pyrrolidine]-1,2',3,5'(2H)-tetrone (41). Compound 41 exhibited exceptional oral potency in two animal models of diabetic complications, the 14-day galactose-fed and streptozocin-induced diabetic rats, with ED50 values for the sciatic nerve of 0.1 and 0.09 mg/kg/day, respectively. Both enantiomeric forms of 41 exhibited similar inhibitory activity in both in vitro and in vivo assays possibly due to their rapid interconversion. In an ex vivo experiment, the pharmacodynamic effect of 41 in the plasma of rats and dogs, after a single dose, appeared to be comparable to that of tolrestat.

Aldehyde Reductase↗
PubMed1994

N-substituted spirosuccinimide, spiropyridazine, spiroazetidine, and acetic acid aldose reductase inhibitors derived from isoquinoline-1,3-diones. 2.

The isoquinoline-1,3-dione framework featured in our clinical candidate (1) and its congener was used as the template in the design of several new series of aldose reductase inhibitors (ARIs). These series included N'-substituted spirosuccinimide, spiropyridazine, spiroazetidine, and acetic acid analogues. Compounds within these series were evaluated in vitro for their ability to inhibit glyceraldehyde reduction by bovine lens aldose reductase and in vivo by their ability to inhibit galactitol accumulation in the lens and sciatic nerve of galactose-fed rats. The N'-amino- and N'-alkyl-substituted spiro[isoquinoline-4(1H),3'-pyrrolidine]-1,2',3,5'(2H)- tetrones 6 exhibited high oral potency, even though they were devoid of any intrinsic activity for the aldose reductase enzyme. Similar results were observed for the closely related spiropyridazines 8. Both of these groups are also considered to be prodrugs since they exhibited good oral potency, even though they were devoid of any intrinsic activity for the aldose reductase enzyme. In contrast, the isoquinoline-1,3-dione acetic acids 9 exhibited very high intrinsic activity for the aldose reductase enzyme, although minimal or no in vivo activity. The absence of in vivo activity for some of these compounds may be due to poor tissue penetration. In support of this suggestion, the more lipophilic acetyl alkyl carbamate derivatives of these isoquinoline-1,3-dione acetic acids, exhibited enhanced oral potency. The spiroazetidines 7 exhibited good activity for the aldoe reductase enzyme in both the in vitro and in vivo assays. The findings of this study demonstrate the utility of the isoquinoline-1,3-dione framework, as a versatile template for the design of divese series of potent ARIs.

Acetates↗
PubMed1994

Galactosemia produces ARI-preventable nodal changes similar to those of diabetic neuropathy.

The present study was designed to examine the development of structural changes, characteristic of diabetic neuropathy, in chronic galactosemia and their responsiveness to inhibition of the polyol-pathway. Sprague-Dawley rats weighing 70-90 g were given a 50% galactose diet continued for 4 or 8 months. Half of these animals were simultaneously given the aldose reductase inhibitor (ARI) WAY 121-509. ARI-treatment normalized galactitol and myoinositol levels in the sciatic nerve. At 4 months, sciatic nerve conduction velocity (NCV) in galactosemic rats was reduced by 30% which was prevented in ARI-treated rats. At 8 months galactosemia reduced NCV to 58% of control values, while ARI-treatment for 8 months improved NCV to 71% of control values. ARI-treatment prevented in galactosemic rats nodal structural changes characteristic of diabetic neuropathy, whereas axonal atrophy was not affected by ARI-treatment, which may in part account for the only partial prevention of the NCV slowing at 8 months. Nerve fiber regeneration was increased 4-fold in ARI-treated rats compared with untreated galactosemic rats. These data suggest that chronic galactosemia produces a neuropathy structurally similar to diabetic neuropathy. The lack of an ARI-treatment effect on axonal atrophy suggests that this defect is not polyol related in galactosemia.

Aldehyde Reductase↗
PubMed1994

Impairment of afferent arteriolar myogenic responsiveness in the galactose-fed rat.

Previous studies from our laboratory have demonstrated impaired afferent arteriolar responsiveness to pressure in rats 4-6 weeks after the induction of diabetes mellitus. Although the responsible mechanisms mediating this renal autoregulatory defect have not been fully defined, increased polyol metabolism has been implicated as a possible factor involved in the pathogenesis of diabetic complications. We therefore investigated the possible role of this metabolic disturbance in renal autoregulation using the galactose-fed rat, a model characterized by increased polyol pathway activity independent of hyperglycemia or insulin deficiency. Hydronephrosis was induced to permit direct visualization of renal microvessels. Pressure-induced vasoconstriction of afferent arterioles was assessed by quantitating vessel diameter following stepwise increments of renal perfusion pressure (RAP; from 80 to 180 mm Hg) in the hydronephrotic kidneys from control rats and rats fed a 50% galactose diet for 2 or 4 weeks. Vessel diameters were measured from video images by computer-assisted image processing. Control rats exhibited progressive afferent arteriolar vasoconstriction when RAP was increased from 80 to 180 mm Hg (-17.3% +/- 1.0%; P < 0.001). In contrast, myogenic responses to increases in pressure were absent in the afferent arterioles of rats fed a 50% galactose diet for either 2 (-4.1% +/- 1.9%; not significant) or 4 weeks (-2.9 +/- 3.4%; not significant). Our demonstration that the impairment of afferent arteriolar responsiveness to increasing RAP in the normoglycemic galactose-fed rat was identical to that observed in the STZ-diabetic rat suggests that increased polyol accumulation may contribute to the impairment of renal autoregulation in the diabetic rat.

Animals↗
PubMed1992

Novel spirosuccinimides with incorporated isoindolone and benzisothiazole 1,1-dioxide moieties as aldose reductase inhibitors and antihyperglycemic agents.

Compounds from two novel series of spirosuccinimides were prepared. Analogs of series 2 possessed a spiro-fused isoindolone moiety while those of series 3 contained a spiro-fused benzisothiazole S,S-dioxide group. These compounds were evaluated as aldose reductase inhibitors (ARI) in vitro by their ability to inhibit glyceraldehyde reduction using a partially purified bovine lens aldose reductase preparation and in vivo as inhibitors of galactitol accumulation in the lens, sciatic nerve, and diaphragm of galactose-fed rats. Many members from the isoindolone series 2, particularly those containing an isoindolone N-methyl moiety, showed good in vitro and in vivo potency. The most potent member, the 6-chloro analog 32, was resolved, and aldose reductase activity was found to reside almost exclusively in the (+)-enantiomer. Compound 32 was approximately equipotent in the sciatic nerve of the galactose-fed rat to other cyclic imide ARI's of similar in vitro activity, namely sorbinil and ADN-138 and also to tolrestat, an acetic acid-based ARI (ED50's 4-8 mg/kg). Compounds from both series, 2 and 3, were also found to lower plasma glucose levels of genetically obese db/db and ob/ob mice with potency similar to that of ciglitazone. However, members from these series failed to lower insulin levels of the ob/ob mouse at the doses tested.

Aldehyde Reductase↗
PubMed1992

Hypertonic stress induces alpha B-crystallin expression.

Alpha B-crystallin, a major lens protein, was induced in primary cultures of dog lens epithelial cells and glomerular endothelial cells when they were grown under conditions of hypertonic stress. With Western blot analysis using a specific alpha B-crystallin antibody, we observed a significant increase in the concentration of alpha B-crystallin protein in cells grown for 4-6 days in media supplemented with 150 mM NaCl or 250 mM cellobiose. These supplements increased the osmolarity of the medium from 300 to 550-600 mosmol kg-1. Alpha B-crystallin mRNA was also increased reaching a maximum four-fold increase in lens and 16-fold increase in kidney cells within 1-2 days. These studies demonstrate a type of regulation of alpha B-crystallin expression in cells from lenticular and non-lenticular tissues.

Animals↗
PubMed1991

Intervention with the aldose reductase inhibitor, tolrestat, in renal and retinal lesions of streptozotocin-diabetic rats.

The progressive increase in urinary albumin excretion, which precedes the development of diabetic nephropathy, can be prevented in diabetic rats if the aldose reductase inhibitor, tolrestat, is administered at the initiation and throughout the duration of hyperglycaemia. We therefore determined the ability of tolrestat to intervene in the further progression of already established urinary albumin excretion of streptozotocin-diabetic female Wistar rats. Two months after streptozotocin injection, diabetic rats were grouped as low-urinary albumin excretion (0.2-1.0 mg albumin/day) or high-urinary albumin excretion (1.9-5.9 mg albumin/day), at which time tolrestat intervention (25 mg/kg per day) was begun for half of the diabetic rats in each urinary albumin excretion group. After six months of treatment tolrestat caused a significant reduction in the urinary albumin excretion rate of the low-urinary albumin excretion group only. The diabetes-induced rise of total urinary protein in both groups was significantly reduced by tolrestat. Furthermore, the diabetes-induced increase (49%) in the thickness of the basement membranes of retinal capillaries from the outer plexiform layer was significantly diminished by tolrestat administration. In conclusion, intervention therapy with the aldose reductase inhibitor, tolrestat, can reduce the progression of urinary albumin excretion and retinal basement membrane thickening in long-term diabetic rats.

Albuminuria↗
PubMed1990

Induction of aldose reductase expression in rat kidney mesangial cells and Chinese hamster ovary cells under hypertonic conditions.

Rat kidney cortex mesangial cells (MES) and Chinese hamster ovary cells (CHO) responded to hypertonicity (600 mosmol/kg) in culture by accumulating sorbitol. The accumulation of sorbitol was due to increased aldose reductase (AR) activity, apparently brought about by increased levels of AR mRNA and protein. The levels of AR mRNA increased approximately 60-fold in MES cells and 30-fold in CHO cells by 24 h in culture media (300 mosmol/kg supplemented with 150 mM NaCl, 600 mosmol/kg total). AR activity also markedly increased (14- to 16-fold above control), but MES took 4 days and CHO 6 days to reach this maximum. Other osmolytes, raffinose and sorbitol (at concentrations of 250 to 300 mM) elicited the same response as that of 150 mM NaCl. These data show that AR expression is induced in MES and CHO cells under hypertonic conditions. Of special interest is the induction of large amounts of AR in rat kidney cortex mesangial cells, a target tissue of diabetes and a site where excessive accumulation of sorbitol is suspected to be a critical factor in diabetic nephropathy.

Aldehyde Reductase↗
PubMed1989

Polyol and vacuole formation in cultured canine lens epithelial cells.

Polyol accumulation and myo-inositol depletion were accompanied by extensive vacuole formation in cultured canine lens epithelial cells that were incubated for up to 96 hr in growth medium supplemented with 30 mM D-galactose or 30 mM D-glucose. These changes did not occur in cells incubated in a hypergalactosemic or hyperglycemic medium which also contained an aldose reductase inhibitor (20 microM sorbinil). In addition, these changes were not observed in lens cells incubated in growth medium supplemented with either 30 mM mannitol, which is known to enter cells only slowly, or in 30 mM L-galactose, which is not a substrate for aldose reductase. The vacuoles were visible at the ultrastructural level after 6 hr of incubation in 30 mM D-galactose and increased in both number and size with time. These vacuoles had a unique fine structure. They did not result from swelling of mitochondria or other cell organelles. As demonstrated cytochemically, they did not represent either lysosomes or Golgi saccules. The proliferation pattern of cells incubated with 30 mM D-galactose was clearly different from that of control cells, but approached normal when an aldose reductase inhibitor was added to the incubation medium. Together these findings suggest that vacuole formation and altered cell proliferation were caused by polyol accumulation and/or myo-inositol loss, both of which result from aldose reductase activity.

Aldehyde Reductase↗
PubMed1989

Aldose reductase and polyol in cultured pericytes of human retinal capillaries.

Aldose reductase has been found in several tissues involved in the various complications of long-term diabetes. Since the loss of intramural pericytes is typical of early microangiopathy of the diabetic retina, the presence of aldose reductase and its activity were investigated in pericytes cultured from capillaries of human retinas. Verification that contaminating cells had been removed was made on the basis of the pericytes' distinctive appearance in culture, inability to internalize acetylated-low-density lipoprotein, and immunoreactivity for muscle actin. Using pure cultures of human pericytes, the presence of aldose reductase was demonstrated immunohistochemically with antibodies directed against human placental aldose reductase, and aldose reductase activity was shown biochemically by monitoring the accumulating of xylitol in cells incubated with xylose. Xylitol accumulation was inhibited by the inclusion of an aldose reductase inhibitor in the xylose-containing medium.

Adult↗
PubMed1989

Diabetic-like retinopathy in rats prevented with an aldose reductase inhibitor.

The earliest histopathologic signs of diabetic retinopathy include selective loss of intramural pericytes and thickening of capillary basement membranes. Previous evidence from animal models indicated that aldose reductase inhibitors could prevent these capillary wall lesions, but only recently have aldose reductase inhibitors been tested for prevention of the subsequent retinal complications of diabetes, such as microaneurysms. In the present study, Sprague-Dawley rats were fed diets containing 50% galactose with or without an aldose reductase inhibitor (tolrestat). After 28 months of galactose feeding, the retinal capillaries in whole mounts exhibited a marked increase in periodic acid-Schiff (PAS) staining, extensive pericyte loss, endothelial cell proliferation, acellularity, diffuse dilation, occluded lumens, microaneurysms, and complex microvascular abnormalities including gross dilation and formation of multiple shunt networks. The PAS hyperchromaticity of basement membrane material and pericyte loss occurred throughout the retinal vasculature, while while the microaneurysms and complex lesions were limited to the capillaries of the central and paracentral retina. The changes were associated with both the arterial and venous portions of the capillary plexus. Treatment with orally administered tolrestat prevented essentially all of the vessel abnormalities. Thus, long-term galactose feeding of rats induced microvascular lesions simulating those occurring in background diabetic retinopathy in humans, and these lesions were prevented by treatment with an aldose reductase inhibitor.

Aldehyde Reductase↗
PubMed1988

Characterization of mRNA and genes for aldose reductase in rat.

Aldose reductase (AR; E.C. 1. 1. 1. 21) has been implicated in a variety of diabetic complications. To investigate the expression of this enzyme in target tissues susceptible to such complications, mRNA encoding AR was characterized by Northern blot hybridization in various tissues and cultured cell preparations. The size of mRNA for AR (approximately 1500 bases) was in good agreement with the size determined by sequence analysis. A cDNA probe for AR from rat lens hybridized to the same size species of RNA isolated from cultured dog lens epithelial cells, cultured human retinal capillary pericytes (mural cells), and Y 79 human retinoblastoma cells. In rat tissues, a substantial amount of mRNA was expressed not only in lens, but also in retina, sciatic nerve and kidney medulla. AR mRNA seemed to be less abundant in rat skeletal muscle and brain, and was scarcely present in liver. Furthermore, Southern blot analysis of rat genomic DNA indicated that there are multiple sequences related to that for AR, probably indicating the existence of a multi-gene family.

Aldehyde Reductase↗
PubMed1988

Purification of plasma membrane from Acanthamoeba castellanii.

A simple method for isolation of plasma membrane from Acanthamoeba using self-generating gradients of Percoll is described. To obtain a membrane marker, intact amoebae were radioiodinated and the distribution of the radiolabel was followed through the plasma membrane isolation procedure. The purity of isolated plasma membrane was assessed by enrichment of radiolabel, by electron microscopy, and by enzymatic assays for contaminating membranes. As judged from enrichment of radiolabel, a 37-fold purification of plasma membrane was obtained. We estimate that 80% of the total protein was from plasma membrane and 10% from membrane-associated actin.

Acanthamoeba↗
PubMed1987

Relationship between proteins encoded by three human gamma-crystallin genes and distinct polypeptides in the eye lens.

Although individual gamma-crystallins from the human eye lens have not been successfully purified and sequenced, most of the genes coding for these lens-specific structural proteins have been cloned and characterized. To investigate the relationship between these genes and the gamma-crystallins of the human lens, we made use of mouse cell lines which contain stably integrated copies of the coding sequences for three of the human gamma-crystallin genes coupled to the human metallothionein IIA promoter. The proteins produced by these hybrid genes in cell culture were detected immunologically and compared by physical characteristics with the gamma-crystallins from the human lens. The protein encoded by the G3 gene showed properties identical to those of the 21,000-molecular-weight gamma-crystallin from 11-month-old lens. The protein isolated from the cells expressing the G4 gene was similar to a 19,000-molecular-weight lens gamma-crystallin, while gene G5 encodes a highly basic gamma-crystallin which may be synthesized in only limited amounts in the human lens. These correlations provide a basis for future investigations on the relationship between putative mutations in human gamma-crystallin genes and altered proteins in hereditary lens cataracts.

Animals↗