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, 11β-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 11β-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.

Lupus nephritis (LN) is the most common and significant complication of systemic lupus erythematosus (SLE) due to its poor prognosis and mortality rates in SLE patients. There is a critical need for new drugs as the pathogenesis of LN remains to be elucidated and immunosuppressive therapy comes with many deficiencies. In this study, 23 hub genes (IFI6, PLSCR1, XAF1, IFI16, IFI44, MX1, IFI44L, IFIT3, IFIT2, IFI27, DDX58, EIF2AK2, IFITM1, RTP4, IFITM3, TRIM22, PARP12, IFIH1, OAS1, HERC6, RSAD2, DDX60, and MX2) were identified through bioinformatics and network analysis and are closely related to interferon production and function. Interestingly, immune cell infiltration analysis and correlation analysis demonstrate a positive correlation between the expression of 23 hub genes and monocyte infiltration in glomeruli and M2 macrophage infiltration in the tubulointerstitium of LN patients. Additionally, the CTD database, DsigDB database, and DREIMT database were used to explore the bridging role of genes in chemicals and LN as well as the potential influence of these chemicals on immune cells. After comparison and discussion, six small molecules (Acetohexamide, Suloctidil, Terfenadine, Prochlorperazine, Mefloquine, and Triprolidine) were selected for their potential ability in treating lupus nephritis.

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disease caused by a defect in the DNA repair system, exhibiting skin cancer on sun exposure. As it is an incurable disease, therapeutic strategies of this disease are critical. This review article takes an attempt to explore the current therapeutic advancements in XP. Different approaches including sun avoidance; surgical removal of cancerous lesions; laser and photodynamic therapy; use of retinoid, 5-fluorouracil, imiquimod, photolyase, and antioxidant; interferon therapy and gene therapy are chosen by doctors and patients to lessen the adverse effects of this disease. Among these options, sun avoidance, use of 5-fluorouracil and imiquimod, and interferon therapy are effective. However, some approaches including laser and photodynamic therapy, and the use of retinoids are effective against skin cancer having severe side effects. Furthermore, surgical removal of cancerous lesions and use of antioxidants are considered to be effective against this disease; however, efficacies of these are not experimentally determined. In addition, some approaches including oral vismodegib, immunotherapy, nicotinamide, acetohexamide, glimepiride-restricted diet are found to be effective to minimize the complications secondary to defects in the nucleotide excision repair (NER) system and also enhance the NER, which are under experimental level yet. Besides these, gene therapy, including the introduction of missing genes and genome edition, may be a promising approach to combat this disease, which is also not well established now. In the near future, these approaches may be effective tools to manage XP.

We aim to identify the common genes, biological pathways, and treatment targets for primary Sjögren's syndrome patients with varying degrees of fatigue features. We select datasets about transcriptomic analyses of primary Sjögren's syndrome (pSS) patients with different degrees of fatigue features and normal controls in peripheral blood. We identify common differentially expressed genes (DEGs) to find shared pathways and treatment targets for pSS patients with fatigue and design a protein-protein interaction (PPI) network by some practical bioinformatic tools. And hub genes are detected based on the PPI network. We perform biological pathway analysis of common genes by Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Lastly, potential treatment targets for pSS patients with fatigue are found by the Enrichr platform. We discovered that 27 DEGs are identified in pSS patients with fatigue features and the severe fatigued pSS-specific gene is RTP4. DEGs are mainly localized in the mitochondria, endosomes, endoplasmic reticulum, and cytoplasm and are involved in the biological process by which interferon acts on cells and cells defend themselves against viruses. Molecular functions mainly involve the process of RNA synthesis. The DEGs of pSS are involved in the signaling pathways of viruses such as hepatitis C, influenza A, measles, and EBV. Acetohexamide PC3 UP, suloctidil HL60 UP, prenylamine HL60 UP, and chlorophyllin CTD 00000324 are the four most polygenic drug molecules. PSS patients with fatigue features have specific gene regulation, and chlorophyllin may alleviate fatigue symptoms in pSS patients.

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.

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-β-CD and 2-hydroxybutyl-β-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 this study, we attempted to improve the non-aqueous titration method using N,N-dimethylformamide (DMF) in the Japanese Pharmacopoeia seventeenth edition (JP XVII) for advancement in experimental safety. As an alternative solvent for DMF, we demonstrate titrations using dimethyl sulfoxide (DMSO), which has similar properties as and much higher safety than DMF. Five drugs (i.e., acetohexamide, glibenclamide, sulfamethoxazole, tranilast, and furosemide) listed in JP XVII use DMF as a solvent for titrations with sodium hydroxide standard solution. For these drugs, we examined whether DMF can be replaced with DMSO in quantitative analyses. As a result, a quantification similar to that of the Pharmacopoeia protocol is possible by simply replacing DMF with DMSO or using a mixed solvent of DMSO and water.

Type 2 diabetes mellitus (T2DM) is associated with a higher risk of cancer and cancer-related mortality. Increased blood glucose and insulin levels in T2DM patients may be, at least in part, responsible for this effect. Indeed, lowering glucose and/or insulin levels pharmacologically appears to reduce cancer risk and progression, as has been demonstrated for the biguanide metformin in observational studies. Studies investigating the influence of sulfonylurea derivatives (SUs) on cancer risk have provided conflicting results, partly due to comparisons with metformin. Furthermore, little attention has been paid to within-class differences in systemic and off-target effects of the SUs. The aim of this systematic review is to discuss the available preclinical and clinical evidence on how the different SUs influence cancer development and risk. Databases including PubMed, Cochrane, Database of Abstracts on Reviews and Effectiveness, and trial registries were systematically searched for available clinical and preclinical evidence on within-class differences of SUs and cancer risk. The overall preclinical and clinical evidence suggest that the influence of SUs on cancer risk in T2DM patients differs between the various SUs. Potential mechanisms include differing affinities for the sulfonylurea receptors and thus differential systemic insulin exposure and off-target anti-cancer effects mediated for example through potassium transporters and drug export pumps. Preclinical evidence supports potential anti-cancer effects of SUs, which are of interest for further studies and potentially repurposing of SUs. At this time, the evidence on differences in cancer risk between SUs is not strong enough to guide clinical decision making.

Human dehydrogenase/reductase SDR family member 11 (DHRS11) has been recently reported to be an NADP+-dependent 3(17)β-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)β-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)β-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.

Because no information is available on the use of acetohexamide during breastfeeding, an alternate drug may be preferred, especially while nursing a newborn or preterm infant. Monitor breastfed infants for signs of hypoglycemia such as jitteriness, excessive sleepiness, poor feeding, seizures cyanosis, apnea, or hypothermia. If there is concern, monitoring of the breastfed infant's blood glucose is advisable during maternal therapy with hypoglycemic agents.[1][2]

The first generation sulfonylureas include acetohexamide, chlorpropamide, tolazamide and tolbutamide, oral hypoglycemic agents that are used in therapy of type 2 diabetes. They are well known, but rare causes of clinically apparent liver injury.

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.

Diabetes is one of the most widespread diseases characterized by a deficiency in the production of insulin or its ineffectiveness. As a result, the increased concentrations of glucose in the blood lead not only to damage to many of the body's systems but also cause the nonenzymatic glycation of plasma proteins affecting their drug binding. Since the binding ability influences its pharmacokinetics and pharmacodynamics, this is a very important issue in the development of new drugs and personalized medicine. In this study, capillary electrophoresis-frontal analysis was used to evaluate the affinities between human serum albumin or its glycated form and the first generation of sulfonylurea antidiabetics, since their inadequate concentration may induce hypoglycaemia or on the contrary hyperglycaemia. The binding constants decrease in the sequence acetohexamide > tolbutamide > chlorpropamide > carbutamide both for normal and glycated human serum albumins, with glycated giving lower values. These results provide a more quantitative picture of how these drugs bind with normal and modified human serum albumin and indicate capillary electrophoresis-frontal analysis to be another tool for examining the changes arising from modifications of albumin, or any other protein, with all its benefits like short analysis time, small sample requirement, and automation.

Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research.

Dehydrogenase/reductase (SDR family) member 3 (DHRS3), also known as retinal short-chain dehydrogenase/reductase (retSDR1) is a member of SDR16C family. This family is thought to be NADP(H) dependent and to have multiple substrates; however, to date, only all-trans-retinal has been identified as a DHRS3 substrate. The reductive reaction catalysed by DHRS3 seems to be physiological, and recent studies proved the importance of DHRS3 for maintaining suitable retinoic acid levels during embryonic development in vivo. Although it seems that DHRS3 is an important protein, knowledge of the protein and its properties is quite limited, with the majority of information being more than 15 years old. This study aimed to generate a more comprehensive characterisation of the DHRS3 protein. Recombinant enzyme was prepared and demonstrated to be a microsomal, integral-membrane protein with the C-terminus oriented towards the cytosol, consistent with its preference of NADPH as a cofactor. It was determined that DHRS3 also participates in the metabolism of other endogenous compounds, such as androstenedione, estrone, and DL-glyceraldehyde, and in the biotransformation of xenobiotics (e.g., NNK and acetohexamide) in addition to all-trans-retinal. Purified and reconstituted enzyme was prepared for the first time and will be used for further studies. Expression of DHRS3 was shown at the level of both mRNA and protein in the human liver, testis and small intestine. This new information could open other areas of DHRS3 protein research.

Diabetes mellitus is a life threatening disease and scientists are doing their best to find a cost effective and permanent treatment of this malady. The recent trend is to control the disease by target base inhibiting of enzymes or proteins. Secreted frizzled-related protein 4 (SFRP4) is found to cause five times more risk of diabetes when expressed above average levels. This study was therefore designed to analyze the SFRP4 and to find its potential inhibitors. SFRP4 was analyzed by bio-informatics tools of sequence tool and structure tool. A total of three potential inhibitors of SFRP4 were found, namely cyclothiazide, clopamide and perindopril. These inhibitors showed significant interactions with SFRP4 as compared to other inhibitors as well as control (acetohexamide). The findings suggest the possible treatment of diabetes mellitus type 2 by inhibiting the SFRP4 using the inhibitors cyclothiazide, clopamide and perindopril.

The metabolic reduction of nabumetone was examined by inhibition and correlation studies using human liver microsomes and cytosol. This reduction was observed in both fractions, with the V(max) values for reduction activity being approximately fourfold higher, and the V(max)/K(m) values approximately three-fold higher, in the microsomes than in the cytosol. The reduction of nabumetone was inhibited by 18β-glycyrrhetinic acid, an 11β-hydroxysteroid dehydrogenase (11β-HSD) inhibitor, in the microsomal fraction. The reduction activity was also inhibited by quercetin and menadione [carbonyl reductase (CBR) inhibitors], and by phenolphthalein and medroxyprogesterone acetate [potent inhibitors of aldo-keto reductase (AKR) 1C1, 1C2 and 1C4] in the cytosol. A good correlation (r² = 0.93) was observed between the reduction of nabumetone and of cortisone, as a marker of 11β-HSD activity, in the microsomal fractions. There was also an excellent relationship between reduction of nabumetone and of the AKR1C substrates, acetohexamide, and ethacrynic acid (r 2 = 0.92 and 0.93, respectively), in the cytosol fractions. However, a poor correlation was observed between the formation of 4-(6-methoxy-2-naphthyl)-butan-2-ol (MNBO) from nabumetone and CBR activity (with 4-benzoyl pyridine reduction as a CBR substrate) in the cytosol fractions (r² = 0.24). These findings indicate that nabumetone may be metabolized by 11β-HSD in human liver microsomes, and primarily by AKR1C4 in human liver cytosol, although multiple enzymes in the AKR1C subfamily may be involved. It cannot be completely denied that CBR is involved to some extent in the formation of MNBO from nabumetone in the cytosol fraction.

The presence of elevated glucose concentrations in diabetes is a metabolic change that leads to an increase in the amount of non-enzymatic glycation that occurs for serum proteins. One protein that is affected by this process is the main serum protein, human serum albumin (HSA), which is also an important carrier agent for many drugs and fatty acids in the circulatory system. Sulfonylureas drugs, used to treat type 2 diabetes, are known to have significant binding to HSA. This study employed ultrafiltration and high-performance affinity chromatography to examine the effects of HSA glycation on the interactions of several sulfonylurea drugs (i.e., acetohexamide, tolbutamide and gliclazide) with fatty acids, whose concentrations in serum are also affected by diabetes. Similar overall changes in binding were noted for these drugs with normal HSA or glycated HSA and in the presence of the fatty acids. For most of the tested drugs, the addition of physiological levels of the fatty acids to normal HSA and glycated HSA produced weaker binding. At low fatty acid concentrations, many of these systems followed a direct competition model while others involved a mixed-mode interaction. In some cases, there was a change in the interaction mechanism between normal HSA and glycated HSA, as seen with linoleic acid. Systems with only direct competition also gave notable changes in the affinities of fatty acids at their sites of drug competition when comparing normal HSA and glycated HSA. This research demonstrated the importance of considering how changes in the concentrations and types of metabolites (e.g., in this case, glucose and fatty acids) can alter the function of a protein such as HSA and its ability to interact with drugs or other agents.

A new polymorph of acetohexamide (Form VI) was prepared via the formation of a complex with 2-hydoxybutyl-β-cyclodextrin (HB-β-CD) in aqueous solution. An alkaline solution of acetohexamide and HB-β-CD was adjusted to pH 4.0 by titration with hydrochloric acid. The resulting opaque solution was filtered through paper and allowed to stand at 4°C for 24h. The resulting precipitate was isolated on a filter and analyzed for polymorph content by powder X-ray diffractometry and thermal analysis. The diffraction pattern and thermal behavior of the precipitate was different from those of previously reported acetohexamide polymorphs (Forms I, III, IV and V), indicating that a new polymorph of the drug, i.e. Form VI was produced. This new polymorph was fairly stable against conversion to a stable form even at accelerated storage conditions. Crystalline Form VI was highly soluble in water and dissolved more rapidly than the other known polymorphs. This property was reflected in the blood concentrations of the drug after oral administration to rats.

Sulfonylureas are widely used oral drugs in the treatment of diabetes mellitus. They function by the inhibition of ATP-sensitive K+ channels in pancreatic β-cells, which are thus considered the 'classical' sulfonylurea receptor. Next to the ATP-sensitive K+ channels, additional sulfonylurea-interacting proteins were identified, which might contribute to the physiological effects of this drug family. Most recently, Epac2 (exchange protein directly activated by cAMP 2) was added to the list of sulfonylurea receptors. However, this finding caused controversy in the literature. The critical discussion of the present paper comes to the conclusion that sulfonylureas are not able to activate Epac2 directly and are unlikely to bind to Epac2. Increased blood glucose levels after food intake result in the secretion of insulin from pancreatic β-cells. Glucose levels are detected 'indirectly' by β-cells: owing to increased glycolysis rates, the ratio of cellular ATP/ADP increases and causes the closure of ATP-sensitive K+ channels. In consequence, cells depolarize and voltage-dependent Ca2+ channels open to cause an increase in the cellular Ca2+ concentration. Finally, Ca2+ induces the fusion of insulin-containing granules with the plasma membrane. Sulfonylureas, such as tolbutamide, glibenclamide or acetohexamide, form a class of orally applicable drugs used in the treatment of non-insulin-dependent diabetes mellitus.

This study examined the use of frontal analysis and high-performance affinity chromatography for detecting heterogeneous binding in biomolecular interactions, using the binding of acetohexamide with human serum albumin (HSA) as a model. It was found through the use of this model system and chromatographic theory that double-reciprocal plots could be used more easily than traditional isotherms for the initial detection of binding site heterogeneity. The deviations from linearity that were seen in double-reciprocal plots as a result of heterogeneity were a function of the analyte concentration, the relative affinities of the binding sites in the system and the amount of each type of site that was present. The size of these deviations was determined and compared under various conditions. Plots were also generated to show what experimental conditions would be needed to observe these deviations for general heterogeneous systems or for cases in which some preliminary information was available on the extent of binding heterogeneity. The methods developed in this work for the detection of binding heterogeneity are not limited to drug interactions with HSA but could be applied to other types of drug-protein binding or to additional biological systems with heterogeneous binding.

This report examines the use of high-performance affinity chromatography as a screening tool for studying the change in binding by sulfonylurea drugs to the protein human serum albumin (HSA) during diabetes. The effects of both the non-enzymatic glycation of HSA and the presence of fatty acids on these interactions were considered using a zonal elution format. It was found that there was a significant increase (i.e., 2.7- to 3.6-fold) in the relative retention of several sulfonylurea drugs (i.e., acetohexamide, tolbutamide, glybenclamide and gliclazide) on columns containing normal versus glycated HSA. The addition of various long chain fatty acids to the mobile phase gave the same trend in retention for the tested drugs on both the HSA and glycated HSA columns, generally leading to lower binding. Most of the fatty acids examined produced similar or moderately different relative shifts in retention; however, palmitic acid was found to produce a much larger change in retention on columns containing glycated HSA versus normal HSA under the conditions used in this study.

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.

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.

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.

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.

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.