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Journal of Chromatography. A Apr 2011This report examined the use of silica monoliths in affinity microcolumns containing human serum albumin (HSA) to measure the dissociation rates for various drugs from...
This report examined the use of silica monoliths in affinity microcolumns containing human serum albumin (HSA) to measure the dissociation rates for various drugs from this protein. Immobilized HSA and control monolith columns with dimensions of 1 mm × 4.6 mm i.d. were prepared for this work and used with a noncompetitive peak decay method. Several drugs known to bind HSA were examined, such as warfarin, diazepam, imipramine, acetohexamide, and tolbutamide. Items that were studied and optimized in this method included the sample volume, sample concentration, and elution flow rate. It was found that flow rates up to 10 mL/min could be used in this approach. Work with HSA silica monoliths at these high flow rates made it possible to provide dissociation rate constants for drugs such as warfarin in less than 40s. The dissociation rate constants that were measured gave good agreement with values reported in the literature or that had been obtained with other solutes that had similar binding affinities for HSA. This approach is a general one that should be useful in examining the dissociation of other drugs from HSA and in providing a high-throughput method for screening drug-protein interactions.
Topics: Chromatography, Affinity; High-Throughput Screening Assays; Humans; Immobilized Proteins; Kinetics; Pharmaceutical Preparations; Protein Binding; Serum Albumin; Silicon Dioxide
PubMed: 20956006
DOI: 10.1016/j.chroma.2010.09.070 -
Journal of Chromatography. B,... Oct 2010Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the...
Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used high-performance affinity chromatography to examine the changes in acetohexamide-HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (K(a)) for the high affinity interactions was in the range of 1.2-2.0×10(5)M(-1) and increased in moving from normal HSA to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The K(a) for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the K(a) for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA.
Topics: Acetohexamide; Chromatography, Affinity; Chromatography, High Pressure Liquid; Diabetes Mellitus; Glycation End Products, Advanced; Humans; Hypoglycemic Agents; Protein Binding; Regression Analysis; Serum Albumin; Tryptophan; Warfarin; Glycated Serum Albumin
PubMed: 20829128
DOI: 10.1016/j.jchromb.2010.08.021 -
Journal of Chromatography. B,... Jun 2010Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed...
Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high-affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (+/-0.2) x 10(5) and 3.5 (+/-3.0) x 10(2) M(-1) for acetohexamide and values of 8.7 (+/-0.6) x 10(4) and 8.1 (+/-1.7) x 10(3) M(-1) for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high-affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug-binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (+/-0.1) x 10(5) and 4.3 (+/-0.3) x 10(4) M(-1), respectively, at 37 degrees C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (+/-0.2) x 10(4) and 5.3 (+/-0.2) x 10(4) M(-1), respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions.
Topics: Acetohexamide; Chromatography, Affinity; Humans; Hypoglycemic Agents; Immobilized Proteins; Kinetics; Models, Chemical; Nonlinear Dynamics; Protein Binding; Serum Albumin; Tolbutamide
PubMed: 20435530
DOI: 10.1016/j.jchromb.2010.04.019 -
Biological & Pharmaceutical Bulletin Jan 2005Acetohexamide (AH) is reduced to its alcohol metabolite by carbonyl reductase. We have previously shown that carbonyl reductase present in the liver microsomes of rats... (Comparative Study)
Comparative Study
Acetohexamide (AH) is reduced to its alcohol metabolite by carbonyl reductase. We have previously shown that carbonyl reductase present in the liver microsomes of rats is a male-specific and androgen-dependent enzyme. In the present study, the role of microsomal carbonyl reductase in the pharmacokinetics of AH was examined in male Wistar-Imamichi (WI) and Sprague-Dawley (SD) rats after its intravenous administration. AH was eliminated more slowly from plasma in the WI strain, which lacks most of the microsomal carbonyl reductase, than in the SD strain. Furthermore, several pharmacokinetic parameters were derived from the data for the plasma concentrations of AH. The plasma clearance (CL(p)) of AH (72.8+/-11.2 ml/h/kg) in male WI rats was significantly smaller than that (105.5+/-11.1 ml/h/kg) in male SD rats. Testectomy caused a marked decrease, from 105.5+/-11.1 to 44.3+/-11.8 ml/h/kg, in the CL(p) of AH in male SD rats. These results indicate that microsomal carbonyl reductase plays a critical role in the differential pharmacokinetics of AH in male WI and SD rats.
Topics: Acetohexamide; Alcohol Oxidoreductases; Aldehyde Reductase; Aldo-Keto Reductases; Animals; Female; Male; Microsomes; Rats; Rats, Sprague-Dawley; Rats, Wistar; Species Specificity
PubMed: 15635190
DOI: 10.1248/bpb.28.185 -
Yakugaku Zasshi : Journal of the... Jan 2001We examined physiological and genetic factors affecting acetohexamide reductase (AHR) and 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) activities in liver...
[Strain-, sex- and species-related differences of acetohexamide reductase and 20 beta-hydroxysteroid dehydrogenase activities in liver microsomes of experimental animals].
We examined physiological and genetic factors affecting acetohexamide reductase (AHR) and 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) activities in liver microsomes of experimental animals. Pronounced strain-related differences were found in both activities of AHR and 20 beta-HSD present in liver microsomes of male rats. Among rat strains tested in this study, even though a Wistar-Imamichi (WIM) rat strain was taken to lack AHR activity, it exhibited a significant 20 beta-HSD activity. These findings appeared to be in conflict with our conclusion reported so far, which AHR and 20 beta-HSD present in liver microsomes of male rats are identical enzymes. Thus the reason for this discrepancy was discussed. Furthermore, AHR and 20 beta-HSD activities were little or not observed in liver microsomes of female rats or male experimental animals other than the rat, indicating the existence of sex- and species-related differences in these two enzyme activities.
Topics: 20-Hydroxysteroid Dehydrogenases; Alcohol Oxidoreductases; Animals; Female; Male; Mice; Microsomes, Liver; Rabbits; Rats; Rats, Inbred Strains; Sex Characteristics; Species Specificity
PubMed: 11201165
DOI: 10.1248/yakushi.121.85 -
Journal of Biochemistry Apr 1997The structural requirements of acetohexamide reductases purified from rabbit liver, kidney, and heart for substrates and inhibitors were examined. Acetohexamide, an oral... (Comparative Study)
Comparative Study
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.
Topics: Acetohexamide; Alcohol Oxidoreductases; Animals; Binding Sites; Carbutamide; Chlorpropamide; Enzyme Inhibitors; Hypoglycemic Agents; Kidney; Kinetics; Liver; Myocardium; Oxidation-Reduction; Rabbits; Structure-Activity Relationship; Substrate Specificity; Tolbutamide; Triazines
PubMed: 9163521
DOI: 10.1093/oxfordjournals.jbchem.a021643 -
Journal of Biochemistry Apr 1996An enzyme catalyzing the metabolic reduction of acetohexamide [4-acetyl-N-(cyclohexyl-carbamoyl)benzenesulfonamide], an oral antidiabetic drug, was purified to...
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.
Topics: Acetohexamide; Alcohol Oxidoreductases; Animals; Catalysis; Enzyme Inhibitors; Male; Molecular Weight; Myocardium; Phenobarbital; Quercetin; Rabbits; Substrate Specificity
PubMed: 8743564
DOI: 10.1093/oxfordjournals.jbchem.a021291 -
National Cancer Institute... 1978A bioassay of acetohexamide for possible carcinogenicity was conducted by administering the test chemical in feed to Fischer 344 rats and B6C3F1 mice. Groups of 35 rats...
A bioassay of acetohexamide for possible carcinogenicity was conducted by administering the test chemical in feed to Fischer 344 rats and B6C3F1 mice. Groups of 35 rats of each sex were administered acetohexamide in the diet at one of two doses, either 10,000 or 20,000 ppm, for 103 weeks and then observed for 2 to 4 additional weeks. Matched controls consisted of 15 untreated rats of each sex. All surviving rats were killed at 105 to 107 weeks. Groups of 35 mice of each sex were administered acetohexamide at one of two doses for 103 weeks and then observed for 4 or 5 additional weeks. Time-weighted average doses were 6,359 or 12,718 ppm. Matched controls consisted of 15 untreated mice of each sex. All surviving mice were killed at 107 or 108 weeks. Mean body weights of the dosed rats and mice of both sexes were lower than those of the corresponding matched controls throughout the study, and the depressions in weight were dose related. Except for the female mice, sufficient numbers of animals survived long enough to be at risk for development of late-appearing tumors. In the rats, the only tumor occurring with greater incidence in dosed than in matched-control animals was leukemia (males: matched controls 0/15, low-dose 10/35, high-dose 4/35; females: matched controls 0/14, low-dose 7/35, high-dose 4/34). Only the incidence in the low-dose males was statistically significant (P=0.018). All of these animals had undifferentiated (mononuclear cell) leukemia, which commonly occurs spontaneously in Fischer 344 rats, except for two with lymphocytic leukemia. The incidence of combined leukemia and lymphoma in historical-control male rats at this laboratory in the bioassay program to date is 24/235 (10.2%), which is higher than that in the matched controls. Thus, the incidence in the low-dose males cannot be clearly associated with administration of the test chemical. In the mice, the only neoplasms that occurred at a higher incidence in dosed groups than in matched controls were lymphomas in the males, but the incidences were not statistically significant (matched controls 1/15, low-dose 9/35, high-dose 3/34). These types of lesions are found commonly in untreated B6C3F1 mice. The incidence of lymphomas in the historical-control male mice is 28/536 (5.2%). It is concluded that under the conditions of this bioassay, acetohexamide was not carcinogenic for either Fischer 344 rats or B6C3F1 mice.
PubMed: 12830247
DOI: No ID Found -
Brain Research Feb 1977In further studies on axonally transported protein in the goldfish visual system, the turnover of rapidly transported [3H]proline-labeled protein was examined. It was...
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.
Topics: Acetohexamide; Animals; Asparagine; Axonal Transport; Axons; Cycloheximide; Goldfish; Hydroxyproline; Leucine; Nerve Tissue Proteins; Optic Nerve; Proline; Superior Colliculi
PubMed: 65202
DOI: 10.1016/0006-8993(77)90292-x -
The Tohoku Journal of Experimental... Aug 1974
Topics: Acetohexamide; Blood Glucose; Cholesterol; Clofibrate; Diabetes Mellitus; Fatty Acids, Nonesterified; Female; Humans; Lipoprotein Lipase; Male; Middle Aged; Phospholipids; Triglycerides
PubMed: 4446025
DOI: 10.1620/tjem.113.343