Identification and characterization of the enzymes responsible for the metabolism of the non-steroidal anti-inflammatory drugs, flunixin meglumine and phenylbutazone, in horses.

The in vivo metabolism and pharmacokinetics of flunixin meglumine and phenylbutazone have been extensively characterized; however, there are no published reports describing the in vitro metabolism, specifically the enzymes responsible for the biotransformation of these compounds in horses. Due to their widespread use and, therefore, increased potential for drug-drug interactions and widespread differences in drug disposition, this study aims to build on the limited current knowledge regarding P450-mediated metabolism in horses. Drugs were incubated with equine liver microsomes and a panel of recombinant equine P450s. Incubation of phenylbutazone in microsomes generated oxyphenbutazone and gamma-hydroxy phenylbutazone. Microsomal incubations with flunixin meglumine generated 5-OH flunixin, with a kinetic profile suggestive of substrate inhibition. In recombinant P450 assays, equine CYP3A97 was the only enzyme capable of generating oxyphenbutazone while several members of the equine CYP3A family and CYP1A1 were capable of catalyzing the biotransformation of flunixin to 5-OH flunixin. Flunixin meglumine metabolism by CYP1A1 and CYP3A93 showed a profile characteristic of biphasic kinetics, suggesting two substrate binding sites. The current study identifies specific enzymes responsible for the metabolism of two NSAIDs in horses and provides the basis for future study of drug-drug interactions and identification of reasons for varying pharmacokinetics between horses.

Human Serum Albumin Aggregation/Fibrillation and its Abilities to Drugs Binding.

Human serum albumin (HSA) is a protein that transports neutral and acid ligands in the organism. Depending on the environment's pH conditions, HSA can take one of the five isomeric forms that change its conformation. HSA can form aggregates resembling those in vitro formed from amyloid at physiological pH (neutral and acidic). Not surprisingly, the main goal of the research was aggregation/fibrillation of HSA, the study of the physicochemical properties of formed amyloid fibrils using thioflavin T (ThT) and the analysis of ligand binding to aggregated/fibrillated albumin in the presence of dansyl-l-glutamine (dGlu), dansyl-l-proline (dPro), phenylbutazone (Phb) and ketoprofen (Ket). Solutions of human serum albumin, both non-modified and modified, were examined with the use of fluorescence, absorption and circular dichroism (CD) spectroscopy. The experiments conducted allowed observation of changes in the structure of incubated HSA (HSAINC) in relation to nonmodified HSA (HSAFR). The formed aggregates/fibrillation differed in structure from HSA monomers and dimers. Based on CD spectroscopy, previously absent ßstructural constructs have been registered. Whereas, using fluorescence spectroscopy, the association constants differing for fresh and incubated HSA solutions in the presence of dansyl-amino acids and markers for binding sites were calculated and allowed observation of the conformational changes in HSA molecule.

Oxidative Alteration of Trp-214 and Lys-199 in Human Serum Albumin Increases Binding Affinity with Phenylbutazone: A Combined Experimental and Computational Investigation.

Human serum albumin (HSA) is a target for reactive oxygen species (ROS), and alterations of its physiological functions caused by oxidation is a current issue. In this work, the amino-acid residues Trp-214 and Lys-199, which are located at site I of HSA, were experimentally and computationally oxidized, and the effect on the binding constant with phenylbutazone was measured. HSA was submitted to two mild oxidizing reagents, taurine monochloramine (Tau-NHCl) and taurine dibromamine (Tau-NBr2). The oxidation of Trp-214 provoked spectroscopic alterations in the protein which were consistent with the formation of N'-formylkynurenine. It was found that the oxidation of HSA by Tau-NBr2, but not by Tau-NHCl, provoked a significant increase in the association constant with phenylbutazone. The alterations of Trp-214 and Lys-199 were modeled and simulated by changing these residues using the putative oxidation products. Based on the Amber score function, the interaction energy was measured, and it showed that, while native HSA presented an interaction energy of -21.3 kJ/mol, HSA with Trp-214 altered to N'-formylkynurenine resulted in an energy of -28.4 kJ/mol, and HSA with Lys-199 altered to its carbonylated form resulted in an energy of -33.9 kJ/mol. In summary, these experimental and theoretical findings show that oxidative alterations of amino-acid residues at site I of HSA affect its binding efficacy.

Effects of Firocoxib, Flunixin Meglumine, and Phenylbutazone on Platelet Function and Thromboxane Synthesis in Healthy Horses.

OBJECTIVE: Determine the effects of nonsteroidal anti-inflammatory drugs (NSAID) on platelet function and thromboxane synthesis immediately after drug administration and following 5 days of NSAID administration in healthy horses. STUDY DESIGN: Randomized cross-over study. ANIMALS: Healthy adult horses (n=9; 6 geldings and 3 mares). METHODS: Horses received either flunixin meglumine (1.1 mg/kg IV every 12 hours), phenylbutazone (2.2 mg/kg IV every 12 hours), or firocoxib (loading dose of 0.27 mg/kg IV on day 1, then 0.09 mg/kg IV every 24 hours for 4 days) for a total of 5 days. Blood samples were collected prior to drug administration (day 0), 1 hour after initial NSAID administration (day 1), and then 1 hour post-NSAID administration on day 5. Platelet function was assessed using turbidimetric aggregometry and a platelet function analyzer. Serum thromboxane B2 concentrations were determined by commercial ELISA kit. A minimum 14 day washout period occurred between trials. RESULTS: At 1 hour and 5 days postadministration of firocoxib, flunixin meglumine, or phenylbutazone, there was no significant effect on platelet aggregation or function using turbidimetric aggregometry or a platelet function analyzer. There was, however, a significant decrease in thromboxane synthesis at 1 hour and 5 days postadministration of flunixin meglumine and phenylbutazone that was not seen with firocoxib. CONCLUSION: Preoperative administration of flunixin meglumine, phenylbutazone, or firocoxib should not inhibit platelet function based on our model. The clinical implications of decreased thromboxane B2 synthesis following flunixin meglumine and phenylbutazone administration are undetermined.

Phenylbutazone Oxidation via Cu,Zn-SOD Peroxidase Activity: An EPR Study.

We investigated the effect of Cu,Zn-superoxide dismutase (Cu,Zn-SOD)-peroxidase activity on the oxidation of the nonsteroidal anti-inflammatory drug phenylbutazone (PBZ). We utilized electron paramagnetic resonance (EPR) spectroscopy to detect free radical intermediates of PBZ, UV-vis spectrophotometry to monitor PBZ oxidation, oxygen analysis to determine the involvement of C-centered radicals, and LC/MS to determine the resulting metabolites. Using EPR spectroscopy and spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we found that the spin adduct of CO3(•-) (DMPO/(•)OH) was attenuated with increasing PBZ concentrations. The resulting PBZ radical, which was assigned as a carbon-centered radical based on computer simulation of hyperfine splitting constants, was trapped by both DMPO and MNP spin traps. Similar to Cu,Zn-SOD-peroxidase activity, an identical PBZ carbon-centered radical was also detected with the presence of both myeloperoxidase (MPO/H2O2) and horseradish peroxidase (HRP/H2O2). Oxygen analysis revealed depletion in oxygen levels when PBZ was oxidized by SOD peroxidase-activity, further supporting carbon radical formation. In addition, UV-vis spectra showed that the λmax for PBZ (λ = 260 nm) declined in intensity and shifted to a new peak that was similar to the spectrum for 4-hydroxy-PBZ when oxidized by Cu,Zn-SOD-peroxidase activity. LC/MS evidence supported the formation of 4-hydroxy-PBZ when compared to that of a standard, and 4-hydroperoxy-PBZ was also detected in significant yield. These findings together indicate that the carbonate radical, a product of SOD peroxidase activity, appears to play a role in PBZ metabolism. Interestingly, these results are similar to findings from heme peroxidase enzymes, and the context of this metabolic pathway is discussed in terms of a mechanism for PBZ-induced toxicity.

Integrated analysis of gut metabolome, microbiome, and exfoliome data in an equine model of intestinal injury.

BACKGROUND: The equine gastrointestinal (GI) microbiome has been described in the context of various diseases. The observed changes, however, have not been linked to host function and therefore it remains unclear how specific changes in the microbiome alter cellular and molecular pathways within the GI tract. Further, non-invasive techniques to examine the host gene expression profile of the GI mucosa have been described in horses but not evaluated in response to interventions. Therefore, the objectives of our study were to (1) profile gene expression and metabolomic changes in an equine model of non-steroidal anti-inflammatory drug (NSAID)-induced intestinal inflammation and (2) apply computational data integration methods to examine host-microbiota interactions. METHODS: Twenty horses were randomly assigned to 1 of 2 groups (n = 10): control (placebo paste) or NSAID (phenylbutazone 4.4 mg/kg orally once daily for 9 days). Fecal samples were collected on days 0 and 10 and analyzed with respect to microbiota (16S rDNA gene sequencing), metabolomic (untargeted metabolites), and host exfoliated cell transcriptomic (exfoliome) changes. Data were analyzed and integrated using a variety of computational techniques, and underlying regulatory mechanisms were inferred from features that were commonly identified by all computational approaches. RESULTS: Phenylbutazone induced alterations in the microbiota, metabolome, and host transcriptome. Data integration identified correlation of specific bacterial genera with expression of several genes and metabolites that were linked to oxidative stress. Concomitant microbiota and metabolite changes resulted in the initiation of endoplasmic reticulum stress and unfolded protein response within the intestinal mucosa. CONCLUSIONS: Results of integrative analysis identified an important role for oxidative stress, and subsequent cell signaling responses, in a large animal model of GI inflammation. The computational approaches for combining non-invasive platforms for unbiased assessment of host GI responses (e.g., exfoliomics) with metabolomic and microbiota changes have broad application for the field of gastroenterology. Video Abstract.

Non-antibiotic pharmaceutical phenylbutazone binding to MexR reduces the antibiotic susceptibility of Pseudomonas aeruginosa.

Antimicrobial resistance has been an increasingly serious threat to global public health. The contribution of non-antibiotic pharmaceuticals to the development of antibiotic resistance has been overlooked. Our study found that the anti-inflammatory drug phenylbutazone could protect P. aeruginosa against antibiotic mediated killing by binding to the efflux pump regulator MexR. In this study, antibiotic activity against P. aeruginosa alone or in combination with phenylbutazone was evaluated in vitro and in vivo. Resazurin accumulation assay, transcriptomic sequencing, and PISA assay were conducted to explore the underlying mechanism for the reduced antibiotic susceptibility caused by phenylbutazone. Then EMSA, ITC, molecular dynamic simulations, and amino acid substitutions were used to investigate the interactions between phenylbutazone and MexR. We found that phenylbutazone could reduce the susceptibility of P. aeruginosa to multiple antibiotics, including parts of ß-lactams, fluoroquinolones, tetracyclines, and macrolides. Phenylbutazone could directly bind to MexR, then promote MexR dissociating from the mexA-mexR intergenic region and de-repress the expression of MexAB-OprM efflux pump. The overexpressed MexAB-OprM pump resulted in the reduced antibiotic susceptibility. And the His41 and Arg21 residues of MexR were involved in the phenylbutazone-MexR interaction. We hope this study would imply the potential risk of antibiotic resistance caused by non-antibiotic pharmaceuticals.

Genetic control of drug levels in man: phenylbutazone.

Phenylbutazone was administered to seven pairs of identical (monozygotic) twins and to seven pairs of fraternal (dizygotic) twins. Individual half-lives ranged from 1.2 to 7.3 days. Subjects with identical genotypes (monozygotic twins) exhibited very similar phenylbutazone half-lives; significantly greater differences in half-life occurred in dizygotic twins. The previously established large variations among individuals in phenylbutazone metabolism appear to be genetically, rather than environmentally, determined.

Enhancement of transdermal delivery of phenylbutazone from liposomal gel formulations through deer skin.

Phenylbutazone (PBZ) is a nonsteroidal anti-inflammatory drug used in the treatment of chronic pain and arthritis. Topical and transdermal administration of PBZ would be beneficial in large animals in terms of minimizing gastro-intestinal ulcerations and other side effects, easy administration to legs and joints and minimizing the dose to reduce systemic toxicity of the drug. A topical liposomal preparation with different concentrations of a mono-substituted alkyl amide (MSA) and PBZ was formulated. The formulations were evaluated by in vitro skin-permeation kinetics through deer skin using Franz diffusion cells. By increasing drug loading from 1% to 5% w/w, the steady-state flux (microg/cm(2)/h) of PBZ was increased twofold (P < 0.001). Similarly, by increasing the MSA concentration from 0% to 4%, the steady-state flux (microg/cm(2)/h) of PBZ was increased twofold (P < 0.001). Overall, by increasing the drug load and the use of an appropriate amount of the penetration enhancer, the steady-state flux of PBZ through skin was increased fourfold (P < 0.001). MSA at both 2% and 4% w/w concentrations significantly increased the skin levels of PBZ as compared with control (P < 0.05). In conclusion, MSA served as an effective skin-penetration enhancer in the liposomal gel of PBZ for deer.

A critical view on the analysis of fluorescence quenching data for determining ligand-protein binding affinity.

The past decade has seen an increase in the number of research papers on ligand binding to proteins based on fluorescence spectroscopy. In most cases, determination of the binding affinity is made by analyzing the quenching of protein fluorescence induced by the ligand. However, many such articles, even those published in reputed journals, suffer from several mistakes with regard to analysis of fluorescence quenching data. Using the binding of phenylbutazone to human serum albumin as a model, we consider some of these mistakes and show how they affect the values of the association constant. In particular, the failure to correct for the inner filter effect and the use of unsuitable equations are discussed. Ligand binding data presented in these articles should be treated with caution, especially in the absence of data from complementary techniques.

Role of Herborn (K240E) and Milano Slow (D375H) human serum albumin variants towards binding of phenylbutazone and ibuprofen.

Human serum albumin (HSA) is the binding cargo in blood plasma. The binding of drugs to HSA determines the pharmacokinetics and pharmacodynamics of the drugs. There are 67 natural genetic variants of HSA were reported in literature. Studying the effect of albumin modifications on drug binding helps to treat the patients with proper medication. In the present study, we have aimed to understand the effect of two natural variants of HSA, such as Herborn (K240E) and Milano Slow (D375H) on the binding of phenylbutazone and ibuprofen. For this, we have generated K240E and D375H mutants and also double mutant (K240E/D375H) of HSA using site directed mutagenesis. The recombinant HSA and its variants were expressed in Pichia pastoris. The interaction of HSA and its variants to phenylbutazone and ibuprofen was studied using fluorescence spectroscopy. Our results showed that there is no significant effect of K240E and D375H mutations on phenylbutazone and ibuprofen binding. But the effect is significant when both the mutations were there in a single protein (K240E/D375H). Further, the CD spectroscopy data showed that there is no effect of phenylbutazone and ibuprofen binding on the conformation of protein, except in case of D375H, where there is a conformational change in the binding pocket with the ibuprofen binding.

Influence of Antipsychotic Drug Risperidone on Human Serum Albumin Affinity to Organic Anions.

BACKGROUND: Risperidone is an antipsychotic drug. In blood, this drug binds mainly to human serum albumin (HSA) and is also transported by HSA. METHOD: To study certain details of the interaction between risperidone and HSA, a fluorescent dye CAPIDAN was used as a reporter. This dye specifically fluoresces from HSA in serum and is highly sensitive to structural changes in HSA including pathology-induced changes. Interaction of CAPIDAN with HSA has been studied using time-resolved fluorescence techniques. RESULT: The addition of phenylbutazone, a marker for the HSA drug-binding site I, leads to displacement of CAPIDAN from this site due to direct competition between phenylbutazone and the dye. The addition of risperidone induces a response of CAPIDAN fluorescence that is highly similar to its response to phenylbutazone. This response depends strongly on ionic strength and is very similar in both cases, phenylbutazone and risperidone. This similarity suggests that risperidone binds to HSA in the region of site I. In this site, the risperidone molecule probably covers the positive charge of Arginine 218 or Arginine 222 preventing their interaction with the CAPIDAN negatively charged carboxyl group. This effect was observed both in isolated HSA and in serum, suggesting similarity of the interaction. CONCLUSION: Thus, risperidone is able to prevent binding of organic anions (i.e. CAPIDAN as a drug-like molecule) to HSA.

Competitive binding of bilirubin and drugs to human serum albumin studied by enzymatic oxidation.

The mechanism of drug-induced displacement of bilirubin from the blood into tissues was studied. A model of simple, competitive binding of bilirubin and drug to one site on serum albumin was established. Variations of the free bilirubin concentration after addition of drugs were studied in vitro by measuring velocities of oxidation with hydrogen peroxide and horseradish peroxidase. In all cases, the results were in agreement with the model. The competitive effects of 20 drugs were measured and expressed quantitatively as binding constants to the bilirubin site on human serum albumin. Several drugs caused changes of the bilirubin-albumin light absorption spectrum, indicating simultaneous binding of both ligands, without an effect on the free bilirubin concentration. Noncompetitive site-to-site effects on bilirubin binding could not be demonstrated. An equation is proposed for calculation of the maximal displacing effect of a drug from knowledge of its plasma concentration, the above-determined binding constant, and the degree of protein binding of the drug. Comparison of these results with previous observations of bilirubin displacement in newborn humans and in experimental animals indicates a general agreement with a simple competitive mechanism of binding of bilirubin and drug to one site on the albumin molecule. Binding of drugs to other, noncompetitive sites is common.

Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug-competition analysis.

Human serum albumin (HSA) is an abundant plasma protein that transports a wide variety of drugs and endogenous compounds. The complex binding capacity of HSA has made it a challenging system to study in detail but in order to develop our understanding of the interactions between ligands for HSA, the locations and relative affinities of different ligand binding sites must be determined. Albumin possesses multiple binding sites for its primary physiological ligand, non-esterified fatty acids (FA). Previously, titration of BSA with (13)C-labeled FA revealed multiple chemical shifts and allowed identification of a subset of three chemical shifts that were associated with high affinity FA binding. Recent crystallographic studies of HSA have mapped at least seven FA binding sites for long-chain FA and delineated the overlap with binding sites for drugs and other endogenous compounds. We aim to correlate NMR and structural data for FA to provide a more complete description of the binding capacity of HSA. Our recent mutagenesis studies allowed us to identify two high affinity binding sites in domain III of HSA. Here, we use NMR to study the binding of (13)C-carboxyl labeled palmitate to HSA in the presence and absence of competitor ligands to complete the correlation of NMR chemical shifts with specific structural binding sites. We carefully selected ligands with specific binding sites identified by crystallography and used them, either singly or in combination, to compete with [(13)C]palmitate for binding to HSA. We show that FA sites 2, 4 and 5 bind FA with high affinity, while sites 1, 3, 6 and 7 exhibit low affinity for FA, thus providing the first complete determination of relative affinities of all seven long-chain FA sites on HSA. Our results also yield direct insights into the interactions between FA and other ligands.

Inactivation of alpha1-antiproteinase induced by phenylbutazone: participation of peroxyl radicals and hydroperoxide.

To clarify the action of a side-effect of phenylbutazone, we investigated the inactivation of alpha(1)-antiproteinase induced by phenylbutazone in the presence of horseradish peroxidase (HRP) and H(2)O(2) (HRP-H(2)O(2)). The activity of alpha(1)-antiproteinase was rapidly lost during the interaction of phenylbutazone with HRP-H(2)O(2) under aerobic conditions. Phenylbutazone showed a marked spectral change under aerobic conditions but not under anaerobic conditions. Spin trap agents were very effective in inhibiting alpha(1)-antiproteinase inactivation induced by phenylbutazone. Oxidation of phenylbutazone was stopped by catalase, but the inactivation reaction of alpha(1)-antiproteinase proceeded even after removal of H(2)O(2) in the reaction mixture. Formation of the peroxidative product from phenylbutazone was detected by iodometric assay. These results indicate that both peroxyl radicals and the peroxidative product of phenylbutazone participated in the inactivation of alpha(1)-antiproteinase. Other anti-inflammatory drugs did not inactivate alpha(1)-antiproteinase during interaction with HRP-H(2)O(2). Inactivation of alpha(1)-antiproteinase may contribute to serious side effects of phenylbutazone.

Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis.

Human serum albumin (HSA), transporting protein, is exposed during its life to numerous factors that cause its functions become impaired. One of the basic factors --glycation of HSA--occurs in diabetes and may affect HSA-drug binding. Accumulation of advanced glycation end-products (AGEs) leads to diseases e.g. diabetic and non-diabetic cardiovascular diseases, Alzheimer disease, renal disfunction and in normal aging. The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSA(GLC)) with HSA glycated by fructose (gHSA(FRC)). We focused on presenting the differences between gHSA(FRC) and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively. At an excitation and an emission wavelength of λex 335nm and λem 420nm, respectively the increase of fluorescence intensity and the blue-shift of maximum fluorescence was observed. It indicates that the glycation products decreases the polarity microenvironment around the fluorophores. Analysis of red-edge excitation shift method showed that the red-shift for gHSA(FRC) is higher than for HSA. Non-enzymatic glycation also caused, that the Trp residue of gHSA(FRC) becomes less accessible for the negatively charged quencher (I(-)), KSV value is smaller for gHSA(FRC) than for HSA. TNS fluorescent measurement demonstrated the decrease of hydrophobicity in the glycated albumin. KSV constants for gHSA-PHB systems are higher than for the unmodified serum albumin, while KSV values for gHSA-KP systems are only slightly lower than that obtained for HSA-KP. The affinity of PHB to the glycated HSA is stronger than to the non-glycated in the first class binding sites within subdomain IIA, in the vicinity of Trp-214. Ketoprofen bound to unmodified human serum albumin stronger than for glycated albumin and one class of binding sites is observed (Scatchard linear plots).

Analysis of phenylbutazone residues in horse tissues with and without enzyme-hydrolysis by LC-MS/MS.

Phenylbutazone (PBZ) is permitted to be used for the treatment of musculoskeletal pain and inflammation in race horses but it is not approved for use in horses destined for human consumption. In a recent study initiated in our laboratory to study the disposition of PBZ and its oxyphenbutazone (OXPBZ) metabolite in equine tissues, we compared the effect of an additional enzymatic hydrolysis step with ß-glucuronidase on the results of the analysis for PBZ without enzymatic hydrolysis. Incurred tissue samples obtained from a female horse dosed with PBZ at 8.8 mg/kg for 3 days and sacrificed 6 days following the last administration were used for this study. Liver, kidney, and muscle tissues were collected, extracted, cleaned up on a silica-based solid-phase extraction (SPE) preceded by a weak-anion exchange SPE and analyzed with our in-house validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for PBZ and OXPBZ. Addition of the hydrolysis step resulted in a significant increase in recovery of both PBZ and OXPBZ residues. © 2016 Her Majesty the Queen in Right of Canada. Drug Testing and Analysis © 2016 John Wiley & Sons, Ltd.

Characterization of the aflatoxin B1-binding site of rat albumin.

A fluorescence-enhancement method was used to investigate the non-covalent interaction between aflatoxin B1 and rat albumin. Solvent-induced shifts in the emission spectrum of aflatoxin B1 provided evidence that the aflatoxin B1-binding site of rat albumin is a highly nonpolar environment. A dissociation constant of 20 microM was determined at 20 degrees C. The possibility that aflatoxin B1 binds one of the three major drug sites of albumin was investigated by ligand-displacement experiments. Mechanisms whereby marker ligands displace aflatoxin B1 were further investigated by comparing the experimental binding parameters with those derived theoretically, assuming competitive binding. The results indicate that: aflatoxin B1 and phenylbutazone compete for a common high-affinity site on rat albumin; high-affinity binding of aflatoxin B1 and site-II marker ligands takes place independently; aflatoxin B1 does not compete with either cholate or warfarin for the same high-affinity site, but the simultaneous binding of warfarin or cholate negatively modulates the binding of aflatoxin B1 to albumin. Fluorescence energy-transfer studies show that the lone tryptophan residue, Trp-214, is not associated with the aflatoxin B1-binding site.

Characterization of site I on human serum albumin: concept about the structure of a drug binding site.

Human serum albumin (HSA) possesses at least three sites or areas for high-affinity binding of drugs. Of these sites, site I was investigated by series of ultrafiltration and equilibrium dialysis experiments. Three ligands, acenocoumarol, dansyl-L-asparagine (DNSA) and n-butyl p-aminobenzoate (n-butyl p-ABE) were employed as marker ligands. Each ligand binds to a single high-affinity site on HSA, and binding studies with different pairs of the ligands revealed independent high-affinity binding. Preliminary displacement studies performed with the typical site I binding drugs warfarin, phenylbutazone and iodipamide showed different displacement patterns of the three marker ligands. These studies were followed by stringent competition experiments involving all possible combinations of the three test ligands themselves and of these and the three marker ligands. On the basis of the results obtained it seems that the acenocoumarol and DNSA binding regions correspond to the warfarin and azapropazone binding regions, respectively, of site I reported by others (Fehske, Schläfer, Wollert and Müller (1982) Mol. Pharmacol. 21, 387-393). The new binding region, represented by n-butyl p-ABE, is probably located adjacent to the acenocoumarol binding region but apart from that of DNSA. We have elaborated a model for binding site I in which we propose novel nomenclatures, region Ia, Ib, and Ic for the acenocoumarol, DNSA and n-butyl p-ABE binding regions, respectively. Furthermore, the relation between these regions and the high-affinity binding sites for other drugs have been discussed.

Drug metabolism in normal children, lead-poisoned children, and normal adults.

Drug-metabolizing capacities were determined in 10 normal adults and 10 children in the age range from 1 to 8 years. Among the latter, 2 were normal and 8 had biochemical evidence of lead poisoning but no clinical expression of plumbism. There were no differences between the 2 normal children and the 8 lead-poisoned children in their capacities to metabolize two test drugs, antipyrine and phenylbutazone. The mean antipyrine half-life in the whole group of 10 children, 6.63 hr, was significantly lower than the mean half-life of 13.58 obtained in adults. The mean phenylbutazone half-lives in the children and adults, 1.68 and 3.16 days, respectively, also differed significantly. Thus children in the age range studied appear to metabolize drugs at almost twice the rate of adults which differs from findings in animals in which drug-metabolizing capacities increase with maturation. In two other children who showed clinical as well as biochemical manifestations of acute plumbism, antipyrine half-lives were signficantly longer than normal and therapy with EDTA led to restitution toward normal.