Repair of UV-Induced DNA Damage Independent of Nucleotide Excision Repair Is Masked by MUTYH.

DNA lesions caused by UV damage are thought to be repaired solely by the nucleotide excision repair (NER) pathway in human cells. Patients carrying mutations within genes functioning in this pathway display a range of pathologies, including an increased susceptibility to cancer, premature aging, and neurological defects. There are currently no curative therapies available. Here we performed a high-throughput chemical screen for agents that could alleviate the cellular sensitivity of NER-deficient cells to UV-induced DNA damage. This led to the identification of the clinically approved anti-diabetic drug acetohexamide, which promoted clearance of UV-induced DNA damage without the accumulation of chromosomal aberrations, hence promoting cellular survival. Acetohexamide exerted this protective function by antagonizing expression of the DNA glycosylase, MUTYH. Together, our data reveal the existence of an NER-independent mechanism to remove UV-induced DNA damage and prevent cell death.

Quantitative Evaluation of the Contribution of Each Aldo-Keto Reductase and Short-Chain Dehydrogenase/Reductase Isoform to Reduction Reactions of Compounds Containing a Ketone Group in the Human Liver.

Enzymes of the aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase superfamilies are involved in the reduction of compounds containing a ketone group. In most cases, multiple isoforms appear to be involved in the reduction of a compound, and the enzyme(s) that are responsible for the reaction in the human liver have not been elucidated. The purpose of this study was to quantitatively evaluate the contribution of each isoform to reduction reactions in the human liver. Recombinant cytosolic isoforms were constructed, i.e., AKR1C1, AKR1C2, AKR1C3, AKR1C4, and carbonyl reductase 1 (CBR1), and a microsomal isoform, 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1), and their contributions to the reduction of 10 compounds were examined by extrapolating the relative expression of each reductase protein in human liver preparations to recombinant systems quantified by liquid chromatography-mass spectrometry. The reductase activities for acetohexamide, doxorubicin, haloperidol, loxoprofen, naloxone, oxcarbazepine, and pentoxifylline were predominantly catalyzed by cytosolic isoforms, and the sum of the contributions of individual cytosolic reductases was almost 100%. Interestingly, AKR1C3 showed the highest contribution to acetohexamide and loxoprofen reduction, although previous studies have revealed that CBR1 mainly metabolizes them. The reductase activities of bupropion, ketoprofen, and tolperisone were catalyzed by microsomal isoform(s), and the contributions of HSD11B1 were calculated to be 41%, 32%, and 104%, respectively. To our knowledge, this is the first study to quantitatively evaluate the contribution of each reductase to the reduction of drugs in the human liver. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to determine the contribution of aldo-keto reductase (AKR)-1C1, AKR1C2, AKR1C3, AKR1C4, carbonyl reductase 1, and 11ß-hydroxysteroid dehydrogenase type 1 to drug reductions in the human liver by utilizing the relative expression factor approach. This study found that AKR1C3 contributes to the reduction of compounds at higher-than-expected rates.

In Vitro Investigations of Acetohexamide Binding to Glycated Serum Albumin in the Presence of Fatty Acid.

The interaction of drugs with human serum albumin (HSA) is an important element of therapy. Albumin affects the distribution of the drug substance in the body, as well as its pharmacokinetic and pharmacodynamic properties. On the one hand, inflammation and protein glycation, directly associated with many pathological conditions and old age, can cause structural and functional modification of HSA, causing binding disorders. On the other hand, the widespread availability of various dietary supplements that affect the content of fatty acids in the body means that knowledge of the binding activity of transporting proteins, especially in people with chronic diseases, e.g., diabetes, will achieve satisfactory results of the selected therapy. Therefore, the aim of the present study was to evaluate the effect of a mixture of fatty acids (FA) with different saturated and unsaturated acids on the affinity of acetohexamide (AH), a drug with hypoglycaemic activity for glycated albumin, simulating the state of diabetes in the body. Based on fluorescence studies, we can conclude that the presence of both saturated and unsaturated FA disturbs the binding of AH to glycated albumin. Acetohexamide binds more strongly to defatted albumin than to albumin in the presence of fatty acids. The competitive binding of AH and FA to albumin may influence the concentration of free drug fraction and thus its therapeutic effect.

Novel therapeutic approaches to xeroderma pigmentosum.

BACKGROUND: The study of xeroderma pigmentosum has yielded unforeseen advances regarding how defects in the nucleotide excision repair pathway result in this devastating disease, but development of therapeutic strategies has trailed behind the mechanistic discoveries. OBJECTIVES: This review aims to cover clinical presentation, molecular mechanisms and current management, and highlights more recent insights into targeting the deficiencies secondary to the DNA repair defects to prevent skin cancer and/or neurological degeneration. METHODS: This review article discusses novel therapeutic approaches to xeroderma pigmentosum that focus on metabolic defects downstream of nucleotide excision repair. RESULTS: Current research demonstrates that specific sulfonylureas promote clearance of DNA damage and increase resistance to ultraviolet radiation in a cellular model of xeroderma pigmentosum. Moreover, nicotinamide attenuates the effects of ultraviolet radiation in cells, and caloric restriction decreases DNA damage burden in animal models of xeroderma pigmentosum. CONCLUSIONS: Clinical management of patients with xeroderma pigmentosum still focuses on preventative avoidance of sun exposure as opposed to therapies that would improve the patients' condition; thus, novel approaches to this disease are warranted.

Modification of Drug Crystallization by Cyclodextrins in Pre-formulation Study.

Controlling drug crystallization is one of the important issues in pre-formulation study. In recent years, advanced approaches including the use of tailor-made additives have gathered considerable attention to control crystallization behavior of drugs. This review focuses on the use of hydrophilic cyclodextrins (CDs) as additives for controlling drug crystallization. CDs affect the crystallization of drugs in solution and in solid state based on a host-guest interaction. For example, 2,6-di-O-methyl-ß-CD and 2-hydroxybutyl-ß-CD suppressed solution-mediated transition of drugs during crystallization by the host-guest interaction; as a result, metastable forms selectively precipitated in solution. The use of CDs in crystal engineering provided an opportunity for the detection of a new polymorph by changing the crystallization pathway. It was also possible to modify crystal morphology (i.e., crystal habit) by selective suppression of crystal growth on a certain direction based on the host-gust interaction. For solid formulation, stable amorphous drug/CDs complex under humid conditions was prepared using two different CDs. An overview of some recent progress in the use of CDs in crystal engineering and in amorphous formulation is described in this review.

In vitro inhibition of breast cancer spheroid-induced lymphendothelial defects resembling intravasation into the lymphatic vasculature by acetohexamide, isoxsuprine, nifedipin and proadifen.

BACKGROUND: As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration. METHODS: A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called 'circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis. RESULTS: Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation. CONCLUSION: The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.

Rabbit dehydrogenase/reductase SDR family member 11 (DHRS11): Its identity with acetohexamide reductase with broad substrate specificity and inhibitor sensitivity, different from human DHRS11.

Human dehydrogenase/reductase SDR family member 11 (DHRS11) has been recently reported to be an NADP+-dependent 3(17)ß-hydroxysteroid dehydrogenase, and its orthologs are predicted in genomic analyses of various animals. Among them, the amino acid sequence of predicted rabbit DHRS11 shares 92% identity with that of human DHRS11 and matches peptide sequences (composed of total 87 amino acids) of rabbit heart acetohexamide reductase (RHAR) previously reported. However, the physiological role of RHAR remains unknown, because its known substrates are only acetohexamide and 1,4-naphthoquinone. To elucidate whether the two rabbit enzymes are identical, we have isolated the cDNA for rabbit DHRS11, which was abundantly detected in the brain, heart, kidney and intestine by RT-PCR. The recombinant rabbit DHRS11 reduced acetohexamide and 1,4-naphthoquinone, and was inhibited by tolbutamide and phenobarbital (RHAR-specific inhibitors), demonstrating its identity with RHAR. Rabbit DHRS11 also reduced α-dicarbonyl compounds, aldehydes and aromatic ketones (acetylbenzenes and acetylpyridines), and exhibited 3(17)ß-hydroxysteroid dehydrogenase activity. It was competitively inhibited not only by tolbutamide and phenobarbital, but also more potently by several non-steroidal anti-inflammatory drugs such as diclofenac and sulindac. The broad substrate specificity and inhibitor sensitivity were different from those of human DHRS11, which did not reduce aliphatic aldehydes and aromatic ketones despite its higher 3(17)ß-hydroxysteroid dehydrogenase activity, and was insensitive to tolbutamide, phenobarbital and diclofenac. The site-directed mutagenesis of Thr163 and Val200 in human DHRS11 to the corresponding residues (Gly and Leu, respectively) in rabbit DHRS11 suggested that these residues are pertinent to the differences in properties of rabbit and human DHRS11s.

The effect of sulfonylurea drugs on rabbit myocardial contractility, canine purkinje fiber automaticity, and adenyl cyclase activity from rabbit and human hearts.

Long-term clinical studies have associated tolbutamide therapy with an increased incidence of cardiovascular deaths. The effects of this and other sulfonylurea drugs on contractility and rate of isolated rabbit atria, automaticity of isolated dog Purkinje fibers, and adenyl cyclase activity in particulate preparations of rabbit and human hearts were studied. At concentrations that are attained clinically, tolbutamide (10 mg/100 ml) increased contractility of driven rabbit atria to 124+/-5% of control, acetohexamide (3.9 mg/100 ml) to 140+/-5%, chlorpropamide (8.3 mg/100 ml) to 139+/-6%, and tolazamide (3.1 mg/100 ml) to 119+/-6%. These effects were accentuated in the presence of 2.5 x 10(-4) M theophylline and were not blocked by 1 x 10(-5) M propranolol. Adenyl cyclase was activated by each of these drugs at concentrations below those which increase contractility. The drugs also increased the rate and slope of phase 4 depolarization in spontaneously beating Purkinje fibers, but did not alter the spontaneous rate of isolated rabbit atria. Since inotropic and chronotropic stimulation can be deleterious in some clinical settings, these findings may be of significance in interpretation of cardiovascular mortality data.

Kinetic studies on the reduction of acetohexamide catalyzed by carbonyl reductase from rabbit kidney.

The kinetic mechanism for the reduction of acetohexamide catalyzed by carbonyl reductase from rabbit kidney was investigated. The initial velocity and product inhibition studies indicated that the enzymatic reaction follows an ordered Bi Bi mechanism, in which NADPH binds to the enzyme first and NADP leaves last. This kinetic mechanism was confirmed on the basis of the dead-end inhibition by Cibacron Blue and the binding of NADPH and NADP to the free enzyme. However, whether or not coenzyme-induced isomerization is involved in the enzymatic reaction remains to be clarified. In kinetic studies of inhibition of the enzyme by therapeutically active drugs, indomethacin and befunolol were found to be noncompetitive and competitive inhibitors, respectively, with respect to acetohexamide.

Effects of sulfonylureas on membrane-bound low Km cyclic AMP phosphodiesterase in rat fat cells.

The effects of sulfonylureas and a biguanide on membrane-bound low Km cyclic AMP phosphodiesterase and lipolysis were examined in rat fat cells. Pharmacologically active sulfonylureas, such as tolbutamide (10 mM), acetohexamide (10 mM) and glibenclamide (200 microM) activated the phosphodiesterase when incubated with fat cells and suppressed lipolysis induced by isoproterenol. However, neither of these actions was observed in the presence of a pharmacologically inactive sulfonylurea, carboxytolbutamide (10 mM) and a biguanide, buformin (500 microM). Tolbutamide (0.5-10 mM) activated the enzyme, concentration dependently, and this manner of activation appears to coincide with that of the suppressive effect on the lipolysis. The time course of the enzyme activation was similar to that seen with insulin. Km, optimal pH and sensitivity to temperature of the enzyme from tolbutamide-treated cells were the same as those of the enzyme from control and insulin-treated cells. Direct incubation of the enzyme from control cells with tolbutamide did not affect the activity, while as little as 10 microM 3-isobutyl-1-methylxanthine markedly inhibited the enzyme. Tolbutamide continued to activate the enzyme in cells in which insulin receptor had been destroyed by trypsin-pretreatment. These results are compatible with the idea that the enzyme activated by sulfonylurea and that activated by insulin may be the same species of phosphodiesterase and that the antilipolytic action of sulfonylurea may be mediated by the activation of the enzyme which does not occur through the insulin receptor.

The effect of chronic oral antidiabetic therapy on insulin and glucagon responses to a meal.

Nineteen maturity-onset diabetic patients receiving oral hypoglycemic therapy in a university diabetes clinic completed a study to assess the efficacy of the oral agents and to determine their effects on pancreatic islet hormone secretion. All patients were receiving sulfonylureas, and seven were also receiving phenformin. The subjects were studied as outpatients in the clinic setting on four different occasions with collections of a baseline blood sample before a standard breakfast and a second sampling two hours postprandially, twice while on their prescribed medication and twice after having been withdrawn from the medication. The values obtained during the two studies on the two studies off medications were reproducible for each subject. Analysis of the results by paired differences revealed that mean 24-hour urine glucose values deteriorated significantly (p less than 0.005) after oral antidiabetic therapy was withdrawn; similarly, mean plasma glucose values, both at baseline and two hours postprandially, rose significantly (p less than 0.001) when subjects were off medication. Baseline serum insulin values were not changed, but postprandial levels were significantly higher on oral agents (p less than 0.005). Plasma immunoreactive glucagon was significantly lower both at baseline (p less than 0.02) and postprandially (p less than 0.005) when the subjects were on their antidiabetic medications. During the trial off medication, 16 patients became symptomatic, with three of these developing symptoms severe enough to require hospitalization. It is apparent from this study that oral hypoglycemic medications can play a role in controlling symptoms in maturity-onset diabetic patients and that the beneficial effect of these agents on hyperglycemia may, in part, be explained by their stimulation of endogenous insulin secretion and partial suppression of endogenous glucagon.

The effects of long-term therapy with oral hypoglycemic agents on the oral glucose tolerance test dynamics in male chemical diabetics.

The effect of fixed doses of oral hypoglycemic agents and placebo (diet alone) on the blood glucose, serum insulin, triglyceride, and cholesterol responses during oral glucose tolerance tests done annually for up to four years' follow-up was studied, in a double-blind manner, in five groups of mild male chemical diabetics. The drugs used were chlorpropamide (100 mg. O.D.), tolbutamide (500 mg. b.i.d.), phenformin (50 mg. O.D.), acetohexamide (250 mg. O.D.), and placebo. Each subject was given an individualized diet aimed at attaining and maintaining ideal weight. Comparison by chi-square analysis between the placebo group and each of the drug groups showed (a) no significant differences with regard to the number of subjects with normal glucose tolerance in each of the tests and (b) no change in the insulin secretion dynamics. Comparison between the initial test and each of the subsequent tests within each group showed (a) a greater number of subjects with normal glucose tolerance in the first follow-up test in the chlorpropamide group only, (b) no change in the insulin secretion dynamics except in the chlorpropamide group, where there was an increased insulin/glucose ratio in the first follow-up test, and (c) no change in the fasting serum triglyceride and cholesterol levels.

Fasting plus prandial insulin supplements improve insulin secretory ability in NIDDM subjects.

To examine how insulin secretory ability is modified by strict glycemic control in non-insulin-dependent diabetes mellitus (NIDDM) subjects, basal and/or prandial insulin was supplemented for 4 wk in 24 diabetic subjects who were secondary failures to sulfonylurea treatment. One intermediate-acting insulin injection a day (n = 7) failed to suppress the rise in plasma C-peptide after meals and did not improve plasma C-peptide responses during a posttreatment oral glucose challenge. Continuous subcutaneous insulin infusion with a premeal bolus (n = 8) suppressed both fasting and meal-related rises in C-peptide and improved C-peptide response during the posttreatment oral glucose challenge. Daily insulin requirements during the 4 wk of treatment were reduced significantly by 52%. A short-acting insulin injection before each meal (n = 9) without basal supplementation suppressed the prandial rise in C-peptide and was associated with a significant reduction in daily insulin requirements during 4 wk of treatment by 28%. Diabetic subjects whose fasting and prandial hyperglycemia were less than 140 and less than 200 mg/dl, respectively, showed a significantly higher C-peptide response during oral glucose challenge after treatment than those whose insulin treatment only normalized (less than 200 mg/dl) prandial but not basal hyperglycemia (greater than 140 mg/dl). These results suggest that a short-term period of meal-related insulin treatment (which normalized prandial glycemia) increases residual beta-cell function in NIDDM subjects who failed long-term sulfonylurea administration. A basal insulin supplement alone was not effective. The effectiveness of a prandial insulin supplement may have been further improved by a combined basal and meal-related treatment program.

[Studies for analyzing the prohibited ingredients such as acetohexamide in cosmetics].

Acetohexamide (AH) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method for AH was investigated by HPLC. The lotion or milky lotion of 0.5g was put into a 10-ml volumetric flask. After adding 1.0ml of AH solution at 50 microg/ml into the volumetric flask, the mixture was made up to 10ml with methanol as the testing solution. Creams were procedured as follows; 0.5 g of cream was put into a 10-ml volumetric flask. After adding 1.0ml of tetrahydrofuran into the volumetric flask, the mixture was stirred for several minutes and the ingredients of the creams were dissolved. After adding 1.0ml of AH solution at 50 microg/ml into the volumetric flask, the mixture was made up to 10ml with methanol. One milliliter of the mixture including AH at 5 microg/ml was exactly put into a test tube with a cap and then 1 ml of water and 1 ml of hexane were added. After shaking vigorously, stand for several minutes. After centrifuging, the hexane layer was eliminated and the residual mixture was used as the test solution. The testing solution of 20 microl was analyzed by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250mm), the mixture of acetonitrile and 50 mmol/l phosphate buffer(pH 5.3)(3:1) and the detection wavelength of 247 nm. The working curve from 0.5 to 6.0 microg/ml showed a linear line between the concentrations of AH and the peak areas. There was no interference of peak of AH with the ingredients such as methylparaben, ethylparaben in the lotions, milky lotion and creams.

The pharmacology of sulfonylureas.

In this report we review the pharmacology of the hypoglycemic sulfonylurea drugs. The early work with sulfonylureas is briefly described. The pharmacokinetics of first-generation sulfonylureas, such as tolbutamide, chlorpropamide, acetohexamide and tolazamide, are described. The first-generation sulfonylureas are compared with second-generation sulfonylureas such as glyburide, glipizide and glibornuride. These latter drugs have a more nonpolar or lipophilic side chain, which results in a marked increase in their hypoglycemic potency. Because of the low serum concentration required for effective therapy, it is necessary to measure the serum concentration of second-generation sulfonylureas by gas-liquid chromatography or radioimmunoassay. The second-generation sulfonylureas do not produce facial flushing after ethanol ingestion (Antabuse effect) and are not uricosuric. Glyburide (but not glipizide or glibornuride) has been evaluated for its effect on water excretion. Glyburide not only does not increase water retention but in fact also increases free water clearance. The second-generation sulfonylureas bind to human serum albumin by nonionic forces in contrast with tolbutamide and chlorpropamide which bind by ionic forces. Thus, anionic drugs such as phenylbutazone, warfarin and salicylate do not displace glyburide from albumin as they displace tolbutamide and chlorpropamide. Therefore, it may be safer to administer the second-generation sulfonylureas than the more polar sulfonylureas when concurrent administration of other pharmacologic agents is likely. The sulfonylurea drugs lower plasma glucose concentrations in diabetic patients by stimulating insulin secretion and by potentiating the biologic effect of the insulin on such tissues as skeletal muscle, fat and liver. The mechanism of the latter so-called extra-pancreatic effect may be activated by increasing the deficient numbers of insulin receptors on muscle, fat or liver cells.

Pharmacologically active drug metabolites: therapeutic and toxic activities, plasma and urine data in man, accumulation in renal failure.

Drugs that are administered to man may be biotransformed to yield metabolites that are pharmacologically active. The therapeutic and toxic activities of drug metabolites and the species in which this activity was demonstrated are compiled for the metabolites of 58 drugs. The metabolite to parent drug ratio in the plasma of non-uraemic man and the percentage urinary excretion of the metabolite in non-uraemic man are also tabulated. Those active metabolites with significant pharmacological activity and high plasma levels, both relative to that of the parent drug, will probably contribute substantially to the pharmacological effect ascribed to the parent drug. Active metabolites may accumulate in patients with end stage renal disease if renal excretion is a major elimination pathway for the metabolite. This is true even if the active metabolite is a minor metabolite of the parent drug, as long as the minor metabolite is not further biotransformed and is mainly excreted in the urine. Minor metabolite accumulation may also occur if it is further biotransformed by a pathway inhibited in uraemia. Some clinical examples of the accumulation of active drug metabolites in patients with renal failure are: (a) The abolition of premature ventricular contractions and prevention of paroxysmal atrial tachycardia in some cardiac patients with poor renal function treated with procainamide are associated with high levels of N-acetylprocainamide. (b) The severe irritability and twitching seen in a uraemic patient treated with pethidine (meperidine) are associated with high levels of norpethidine. (c) The severe muscle weakness and tenderness seen in patients with renal failure receiving clofibrate are associated with excessive accumulation of the free acid metabolite of clofibrate. (d) Patients with severe renal insufficiency taking allopurinol appear to experience a higher incidence of side reactions, possibly due to the accumulation of oxipurinol. (e) Accumulation of free and acetylated sulphonamides in patients with renal failure is associated with an increase in toxic side-effects (severe nausea and vomiting, evanescent macular rash). (f) Peripheral neuritis seen after nitrofurantoin therapy in patients with impaired renal function is thought to be due to accumulation of a toxic metabolite. The high incidence of adverse drug reactions seen in patients with renal failure may for some drugs be explained in part, as the above examples illustrate, by the accumulation of active drug metabolites. Monitoring plasma levels of drugs can be an important guide to therapy. However, if a drug has an active metabolite, determination of parent drug alone may cause misleading interpretations of blood level measurements. The plasma level of the active metabolite should also be determined and its time-action characteristics taken into account in any clinical decisions based on drug level monitoring.

Purification and catalytic properties of a novel acetohexamide-reducing enzyme from rabbit heart.

An enzyme catalyzing the metabolic reduction of acetohexamide [4-acetyl-N-(cyclohexyl-carbamoyl)benzenesulfonamide], an oral antidiabetic drug, was purified to homogeneity from the cytosolic fraction of rabbit heart. The molecular mass of the purified enzyme was estimated to be 110 kDa by gel filtration and nondenaturing PAGE and 28 kDa by SDS-PAGE, suggesting that the enzyme is composed of four identical-size subunits. 4-Benzoyl-pyridine and p-nitroacetophenone, typical substrates of carbonyl reductase [EC 1.1.1.184], were not reduced by the enzyme. Of drugs with a ketone group tested, only acetohexamide was a good substrate of the enzyme. the enzyme effectively reduced analogs substituted with various alkyl groups instead of the cyclohexyl group in acetohexamide, although it had little or no ability to reduce analogs substituted with various alkyl groups instead of the methyl group in acetohexamide. The enzyme was inhibited not only by quercetin, a well-known inhibitor of carbonyl reductase, but also by phenobarbital, a potent inhibitor of aldehyde reductase [EC 1.1.1.2]. These results indicate that the enzyme purified from rabbit heart is a novel enzyme responsible for the reduction of acetohexamide and its analogs.

Characterization of acetohexamide reductases purified from rabbit liver, kidney, and heart: structural requirements for substrates and inhibitors.

The structural requirements of acetohexamide reductases purified from rabbit liver, kidney, and heart for substrates and inhibitors were examined. Acetohexamide, an oral antidiabetic drug with a ketone group, and analogs of it with various alkyl groups instead of the cyclohexyl group were used as substrates for these three enzymes. The results obtained as to substrate specificity suggested that the nature of the substrate-binding region of the heart enzyme is markedly different from those of the substrate-binding regions of the liver and kidney enzymes. Tolbutamide, which has no ketone group within its chemical structure, strongly inhibited the heart enzyme, whereas it had little ability to inhibit the liver or kidney enzyme. The inhibition of the heart enzyme by tolbutamide was competitive with respect to acetohexamide and uncompetitive with respect to NADPH. Furthermore, tolbutamide analogs with n-pentyl and n-hexyl groups instead of the n-butyl group exhibited very pronounced inhibition of only the heart enzyme. Therefore, it is reasonable to postulate that the heart enzyme, unlike the liver and kidney ones, has a cleft of a strongly hydrophobic nature near its substrate-binding region, and that this hydrophobic cleft plays a critical role in the interaction of the heart enzyme with the cyclohexyl group of acetohexamide.

Reutilization of precursor following axonal transport of [3H]proline-labeled protein.

In further studies on axonally transported protein in the goldfish visual system, the turnover of rapidly transported [3H]proline-labeled protein was examined. It was found that: (1) a fraction of the rapidly transported protein has a relatively short half-life; (2) [3H]proline released following proteolysis of transported protein is efficiently reutilized for tectal protein synthesis, as inferred from an increased labeling of nuclear protein in the contralateral tectum (COT) relative to that in the ipsilateral tectum (IOT); (3) a small amount of [3H]proline arrives in the COT by axonal flow of the free amino acid; and (4) [3H]leucine and [3H]asparagine are less efficiently reutilized than [3H]proline. These findings may relate to the phenomenon of transneuronal transfer of radioactivity which has been observed with [3H]proline as precursor. The extensive reutilization of [3H]proline may account for part or all of the labeling at secondary synaptic sites. The results suggest that asparagine may be highly suitable for radioautographic identification of primary neuronal fields.

(R)-ACX is a novel sufonylurea compound with potent, quick and short-lasting hypoglycemic activity.

We investigated the mechanism of the hypoglycemic effect of (R)-4-(1-acetoxyethyl)-N-(cyclohexylcarbamoyl)benzene-sulfonamide [(R)-acetoxyhexamide; (R)-ACX], a new sulfonylurea compound. (R)-ACX potently stimulated the release of insulin from cultured pancreatic beta-cells (HIT T15 cells), established from hamster islet cells SV40-transformed. When (R)-ACX was orally administered to fasted rats, it decreased the plasma glucose level in a dose-dependent manner. The hypoglycemic effect of (R)-ACX was quick and short lasting, as compared to that of acetohexamide and glibenclamide. The quick and short-lasting hypoglycemic effect of (R)-ACX was thought likely to result from rapid absorption of (R)-ACX and rapid elimination of (R)-ACX and its metabolite, (R)-hydroxyhexamide. Furthermore, (R)-ACX was found to suppress the increase of blood glucose level due to starch loading in fasted mice. (R)-ACX may be useful in the control of postprandial hyperglycemia to patients with non-insulin-dependent diabetic mellitus.