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.

Nonsteroidal anti-inflammatory drug sensitizes Mycobacterium tuberculosis to endogenous and exogenous antimicrobials.

Existing drugs are slow to eradicate Mycobacterium tuberculosis (Mtb) in patients and have failed to control tuberculosis globally. One reason may be that host conditions impair Mtb's replication, reducing its sensitivity to most antiinfectives. We devised a high-throughput screen for compounds that kill Mtb when its replication has been halted by reactive nitrogen intermediates (RNIs), acid, hypoxia, and a fatty acid carbon source. At concentrations routinely achieved in human blood, oxyphenbutazone (OPB), an inexpensive anti-inflammatory drug, was selectively mycobactericidal to nonreplicating (NR) Mtb. Its cidal activity depended on mild acid and was augmented by RNIs and fatty acid. Acid and RNIs fostered OPB's 4-hydroxylation. The resultant 4-butyl-4-hydroxy-1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (4-OH-OPB) killed both replicating and NR Mtb, including Mtb resistant to standard drugs. 4-OH-OPB depleted flavins and formed covalent adducts with N-acetyl-cysteine and mycothiol. 4-OH-OPB killed Mtb synergistically with oxidants and several antituberculosis drugs. Thus, conditions that block Mtb's replication modify OPB and enhance its cidal action. Modified OPB kills both replicating and NR Mtb and sensitizes both to host-derived and medicinal antimycobacterial agents.

Structural and ligand-binding properties of serum albumin species interacting with a biomembrane interface.

In the process of drug development, preclinical testing using experimental animals is an important aspect, for verification of the efficacy and safety of a drug. Serum albumin is a major binding protein for endogenous and exogenous ligands and regulates their distribution in various tissues. In this study, the structural and drug-binding properties of albumins on a biomembrane surface were investigated using reverse micelles as a model membrane. In reverse micelles, the secondary structures of all albumins were found, to varying degrees, to be intermediate between the native and denatured states. The tertiary structures of human and bovine albumin were similar to those of the native and intermediate states, respectively, whereas those of the dog, rabbit, and rat were in a denatured state. Thus, bovine albumin is an appropriate model for studying structural changes in human albumin in a membrane-water phase. Binding studies also showed the presence of species difference in the change in binding capacity of albumins during their interaction with reverse micelles. Among the albumins, rat albumin appears to be a good model for the protein-mediated drug uptake of human albumin in a biomembrane environment. These findings are significant in terms of the appropriate extrapolation of pharmacokinetics and pharmacodynamics data in various animals to humans.

Phospholipase A2 as a target protein for nonsteroidal anti-inflammatory drugs (NSAIDS): crystal structure of the complex formed between phospholipase A2 and oxyphenbutazone at 1.6 A resolution.

Phospholipase A(2) (PLA(2); EC 3.1.1.4) is a key enzyme involved in the production of proinflammatory mediators known as eicosanoids. The binding of the substrate to PLA(2) occurs through a well-formed hydrophobic channel. To determine the viability of PLA(2) as a target molecule for the structure-based drug design against inflammation, arthritis, and rheumatism, the crystal structure of the complex of PLA(2) with a known anti-inflammatory compound oxyphenbutazone (OPB), which has been determined at 1.6 A resolution. The structure has been refined to an R factor of 0.209. The structure contains 1 molecule each of PLA(2) and OPB with 2 sulfate ions and 111 water molecules. The binding studies using surface plasmon resonance show that OPB binds to PLA(2) with a dissociation constant of 6.4 x 10(-8) M. The structure determination has revealed the presence of an OPB molecule at the binding site of PLA(2). It fits well in the binding region, thus displaying a high level of complementarity. The structure also indicates that OPB works as a competitive inhibitor. A large number of hydrophobic interactions between the enzyme and the OPB molecule have been observed. The hydrophobic interactions involving residues Tyr(52) and Lys(69) with OPB are particularly noteworthy. Other residues of the hydrophobic channel such as Leu(3), Phe(5), Met(8), Ile(9), and Ala(18) are also interacting extensively with the inhibitor. The crystal structure clearly reveals that the binding of OPB to PLA(2) is specific in nature and possibly suggests that the basis of its anti-inflammatory effects may be due to its binding to PLA(2) as well.

Pharmacokinetic studies of flunixin meglumine and phenylbutazone in plasma, exudate and transudate in sheep.

Flunixin meglumine (FM, 1.1 mg/kg) and phenylbutazone (PBZ, 4.4 mg/kg) were administered intravenously (i.v.) as a single dose to eight sheep prepared with subcutaneous (s.c.) tissue-cages in which an acute inflammatory reaction was stimulated with carrageenan. Pharmacokinetics of FM, PBZ and its active metabolite oxyphenbutazone (OPBZ) in plasma, exudate and transudate were investigated. Plasma kinetics showed that FM had an elimination half-life (t1/2beta) of 2.48 +/- 0.12 h and an area under the concentration - time curve (AUC) of 30.61 +/- 3.41 Lg/mL x h. Elimination of PBZ from plasma was slow (t1/2beta = 17.92 +/- 1.74 h, AUC = 968.04 +/- microg/mL x h). Both FM and PBZ distributed well into exudate and transudate although penetration was slow, indicated by maximal drug concentration (Cmax) for FM of 1.82 +/- 0.22 microg/mL at 5.50 +/- 0.73 h (exudate) and 1.58 +/- 0.30 microg/mL at 8.00 h (transudate), and Cmax for PBZ of 22.32 +/- 1.29 microg/mL at 9.50 +/- 0.73 h (exudate) and 22.07 +/- 1.57 microg/mL at 11.50 +/- 1.92 h (transudate), and a high mean tissue-cage fluids:plasma AUClast ratio obtained in the FM and PBZ groups (80-98%). These values are higher than previous reports in horses and calves using the same or higher dose rates. Elimination of FM and PBZ from exudate and transudate was slower than from plasma. Consequently the drug concentrations in plasma were initially higher and subsequently lower than in exudate and transudate.

Determination of binding conformations of drugs to human serum albumin by transferred nuclear overhauser effect measurements and conformational analyses using high-temperature molecular dynamics calculations.

The binding conformations of oxyphenbutazone (OXY), Nepsilon-dansyl-L-lysine (DNS-LYS), and furosemide (FU) to human serum albumin (HSA) have been investigated by molecular dynamics (MD) calculations and transferred nuclear Overhauser effect (TRNOE) measurements. We have combined distance information obtained from the Conformational Analyzer with Molecular Dynamics And Sampling (CAMDAS) calculation and experimental NOE spectroscopy measurements to determine a "binding conformation" for each drug which binds to site I of HSA. For OXY, only one conformer (conf9) among the conformer set generated by MD calculation satisfied the distance restraint conditions obtained from TRNOE measurements. For DNS-LYS and FU, 17 and 5 conformers satisfied distance restraint conditions, respectively. The structure of conf9 of OXY was taken as a "template" to choose binding conformers for DNS-LYS and FU. By fitting the "template" to the 17 conformers of DNS-LYS and 5 conformers of FU, we could efficiently obtain one binding conformer for DNS-LYS (conf144) and FU (conf26). It is suggested from the feature of the binding conformation that the three-dimensional location of a hydrophobic aromatic ring, alkyl chain, and electronegative functional group is important for binding to site I of HSA. This method, which combines MD calculations and NOE information, is thought to be effective for determining the binding conformation of drugs to HSA.

Pharmacokinetics of phenylbutazone in camels.

OBJECTIVE: To document disposition variables of phenylbutazone and its metabolite, oxyphenbutazone, in camels (Camelus dromedarius) after single i.v. bolus administration of phenylbutazone, with a view to making recommendation on avoiding violative residues in racing camels. ANIMALS: 6 healthy camels (4 males, 2 females), 5 to 7 years old, and weighing from 350 to 450 kg. PROCEDURE: Blood samples were collected to 0, 5, 10, 15, 45, and 60 minutes and at 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 26, 28, 30, 40, 48, 50, 53, and 60 hours after i.v. administration of 4.5 mg of phenylbutazone per kg of body weight. Urine was obtained in fractions during the entire blood sample collection period. Serum and urine phenylbutazone concentrations were measured by high-performance liquid chromatography; assay sensitivity was 100 ng/ml. Serum oxyphenbutazone concentration was measured by gas chromatography/mass spectrometry; assay sensitivity was 10 ng/ml. RESULTS: Disposition of phenylbutazone was best described by a two-compartment open model. Mean +/- SEM elimination half-life was 13.44 +/- 0.44 hours. Total body clearance was 12.63 +/- 1.64 mg/kg/h. Renal clearance was between 0.3 and 0.4% of total body clearance. The elimination half-life of oxyphenbutazone was 23.9 +/- 2.09 hours. CONCLUSIONS: The elimination half-life and total body clearance of phenylbutazone in camels are intermediate between reported values in horses and cattle. Extrapolation of a dosage regimen from either species to camels is, therefore, not appropriate. Elimination of phenylbutazone in camels is mainly via metabolism. Owing to the long half-life of phenylbutazone and of oxyphenbutazone, and to the zero drug concentration regulation adopted by the racing commissioner in the United Arab Emirates, practicing veterinarians would be advised not to use phenylbutazone in camels for at least 7 days prior to racing.

Phenylbutazone in racing greyhounds: plasma and urinary residues 24 and 48 hours after a single intravenous administration.

The concentrations of phenylbutazone (PBZ), oxyphenbutazone (OPBZ) and gammahydroxyphenylbutazone (OHPBZ) in plasma and urine from 50 Greyhounds 24 and 48 h after the intravenous administration of a single dose of PBZ (30 mg/kg) were measured. The 24 h plasma concentrations of OPBZ and OHPBZ, the 48 h plasma concentration of OHPBZ and the 24 h urinary concentration of PBZ were normally distributed, while log transformations were required before the 24 h plasma concentration of PBZ and the 24 and 48 h urinary concentrations of OPBZ and OHPBZ became normally distributed. The 95%, 99%, 99.9% and 99.99% upper predicted confidence intervals for both 24 h and 48 h plasma and urinary concentrations demonstrated wide potential variation in the concentration of the analytes should PBZ be administered to Greyhounds. The 24 h plasma and urinary concentrations of PBZ were weakly correlated, but no similar relationship existed for OPBZ or OHPBZ. The urinary concentrations of each analyte were not affected by the trainer or sex of the Greyhound or the urinary pH. We conclude that it would be impossible to predict the timing of the PBZ administration or the plasma concentration of PBZ from the measurement of the concentration of PBZ in a single sample of urine.

A drug release-facilitating mechanism for human serum albumin in reverse micelles: requirement of a structural switch.

We measured the binding of a drug oxyphenylbutazone to the N-terminal peptic fragment of human serum albumin in 0.1 M Tris buffer, pH 8.0 (Kass = 2.4 10(5) M-1) and in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate and buffer in isooctane (Kass. = 2.7 10(5) M-1). In the absence of any measured change in conformation of the fragment in reverse micelles, the peptide affinity for the drug is not decreased, in contrast to what is observed in intact albumin (HSA) under similar conditions. The interaction and the subsequent unfolding of HSA at the membrane-mimetic interface, constitutes thus a drug release-facilitating mechanism.

Ligand binding at membrane mimetic interfaces. Human serum albumin in reverse micelles.

The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenylbutazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2-ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane-water interface. Upon micellar incorporation, the dansylsarcosine-albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin-albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 x 10(4) M-1 cm-1. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged (Ka approximately 1.0 x 10(5) M-1), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant. These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344 nm in buffer to 327 nm in sodium bis(2-ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N-bromosuccinimide, indicate perturbations of the sole tryptophan-214 microenvironment. However, the distance between the indole residue and tyrosine-411 does not seem substantially modified by the 15% decrease affecting the alpha helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane-like interface which generates two protein subpopulations differing in their membrane-surface and ligand affinities. Overall and local conformational changes, originating from this surface-induced effect, may thus constitute a ligand-release facilitating mechanism acting at cellular membrane levels.

The fatty-acid-induced conformational states of human serum albumin investigated by means of multiple co-binding of protons and oleic acid.

The binding of oleic acid to human serum albumin causes progressive changes in (a) the pK of some amino acid residues, as detected by pH-stat titration and (b) the induced molar ellipticities of albumin-bound drugs (diazepam and oxyphenbutazone), as measured by c.d. It is concluded that albumin undergoes several conformational transitions as the amount of oleic acid bound increases from 0 to about 9 molecules/molecule of protein. At least three different conformations of the protein seem to be involved. These conformations can be linked with the three classes of oleic acid-binding sites on albumin.

Pharmacokinetics, metabolism and excretion of phenylbutazone in cattle following intravenous, intramuscular and oral administration.

The absorption, metabolism and urinary excretion of phenylbutazone were investigated in six adult cattle in a cross-over study involving administration intravenously, intramuscularly and orally at a dose rate of 4.4 mg kg-1. Following intravenous injection plasma disposition was described by a three compartment open model with mean elimination half-life (t1/2 beta) and clearance (ClB) values of 35.9 hours and 2.77 ml kg-1 h-1, respectively. Somewhat longer t1/2 beta values were obtained after oral and intramuscular dosing and these may have resulted from sequestration within and slow absorption from the gastrointestinal tract and continual uptake from intramuscular sites following precipitation as a depot. Absorption was more complete after intramuscular than after oral dosing; area under curve values were almost twice as high for the intramuscular route. Double peaks in the plasma concentration time curves after oral dosing were recorded in some cows. These may have resulted from drug adsorption on to and subsequent desorption from hay or as a consequence of enterohepatic shunting. There was no evidence for opening of the oesophageal groove and direct passage of the drug into the abomasum. Two hydroxylated metabolites of phenylbutazone, oxyphenbutazone and gamma-hydroxyphenylbutazone were detected in trace amounts in plasma for 72 hours and in much higher concentrations in urine for 168 hours. Approximate urine:plasma (U/P) concentration ratios for the metabolites approached and occasionally exceeded the U/P ratio for endogenous creatinine, indicating poor reabsorption and, possibly, tubular secretion. Cumulative urinary excretion data indicated that the hydroxylated derivatives of phenylbutazone are probably formed more slowly in cattle than in horses.

Metabolism, excretion, pharmacokinetics and tissue residues of phenylbutazone in the horse.

The pharmacokinetics, metabolism, excretion and tissue residues of phenylbutazone (PBZ) in the horse were studied following both intravenous and oral administration of the drug at a dose rate of 4.4 mg/kg. A 72-hour blood sampling schedule failed to demonstrate a third exponential phase; the plasma disposition following intravenous injection being described by a two compartment open model, with the following elimination phase parameters: beta = 0.13h-1, t1/2 beta = 5.46h, Vdarea = 0.141 1/kg and C1B = 17.9 ml/kg/h. The hydroxylated metabolites oxyphenbutazone (OPBZ) and gamma-hydroxyphenylbutazone (OHPBZ) were present in detectable concentrations in plasma for 72 and 24 h, respectively. After 36 h OPBZ concentrations exceeded plasma PBZ concentrations. In urine the principal metabolites were OPBZ and OHPBZ but smaller concentrations of another compound, probably gamma-hydroxyoxyphenbutazone (OHOPBZ), were also detected. The percentages of the administered dose recovered from urine were 30.7, 39.0 and 40.3 after 24, 48 and 72 h from the time of injection. Recovery of PBZ and its metabolites from urine was significantly reduced in the first 24 h after oral dosing when the horses had free access to hay, probably as a result of markedly delayed absorption, but this did not occur in animals deprived of food for a few hours before and after dosing. Determination of approximate values of urine/plasma (U/P) concentration ratios for PBZ and its metabolites relative to endogenous creatinine U/P concentration ratio suggested that PBZ was filtered in small amounts only because of the high degree of plasma protein binding and then excreted by diffusion trapping in the alkaline urine. Much higher U/P ratios were obtained for the hydroxylated derivatives, and one at least (OHPBZ) was secreted into urine.

Absorption of phenylbutazone from a paste formulation administered orally to the horse.

The absorption pattern of phenylbutazone was studied in five horses during administration of the drug in a paste formulation on days 1, 5, 8 and 12 of a 12-day dosing schedule. Since two or more plasma concentration peaks were usually obtained following each oral dose, it was concluded that phasic absorption was a particular feature of the oil:water formulation of the product. Possible causes of this unusual absorption pattern are discussed and the therapeutic implications of both phasic absorption and the recorded values of Cmax, tmax and AUC024 for phenylbutazone and its active metabolite oxyphenbutazone are considered.

Phenylbutazone and oxyphenbutazone distribution into tissue fluids in the horse.

The clinically recommended dose rate of phenylbutazone (4.4 mg/kg) was administered intravenously as a single dose to five Welsh Mountain ponies. Distribution of phenylbutazone and its active metabolite oxyphenbutazone into body fluids was studied by measuring concentrations in plasma, tissue-cage fluid, peritoneal fluid and acute inflammatory exudate harvested from a polyester sponge model of inflammation. The ready penetration of phenylbutazone into inflammatory exudate was demonstrated by the relatively high mean value for Cmax of 12.4 micrograms/ml occurring at a time of 4.6 h and a mean AUC0-24 of 128 microgram X h/ml. A high mean exudate:plasma AUC0-24 ratio of 0.83 was recorded. Plasma:exudate concentration ratios for phenylbutazone were initially greater than and subsequently less than one; the slower clearance from exudate was indicated by approximate t1/2 beta) values of 4.8 and 24 h for plasma and exudate, respectively. These findings may help to explain the relatively long duration of action of phenylbutazone, in spite of a plasma elimination half-life of less than 5 h. Lower values of Cmax and AUC0-24 for phenylbutazone passage into peritoneal fluid (6.3 micrograms/ml and 45 micrograms X h/ml) were recorded, and a limited number of sampling times indicated a similar degree of penetration as into tissue cage fluid. Mean concentrations of oxyphenbutazone in all fluids were lower than phenylbutazone concentrations at all times, but ready penetration of the metabolite into body fluids, especially into inflammatory exudate, occurred suggesting that oxyphenbutazone may contribute to the anti-inflammatory effect. The hyperaemia of acute inflammation and the high protein levels in inflammatory exudate may both assist passage of phenylbutazone and oxyphenbutazone into exudate.

Relationships between biophasic disposition and pharmacokinetic behavior in nonsteroid antiinflammatory drugs.

The time course of biophasic amounts of two nonsteroid antiinflammatory drugs (naproxen and oxyphenbutazone) after an intravenous dose of both drugs in rats (5 and 20 mg/kg, respectively) and their relationships with the drug amounts in central and peripheral compartments as defined through blood level data obtained after the same intravenous dose of the drug, are described. To assess the first point, the carrageenin-induced rat paw edema method is employed. Through previously established dose-response curves obtained after administration of different i.v. doses, the conversion of responses to biophasic amounts is achieved by means of the corresponding model equations. To calculate kinetic parameters through the fitting of the concentration-time data, a least-squares method based on the Marquardt algorithm is used on a computer. It can be concluded that the biophasic compartment cannot be identified as the "central" or the "peripheral" kinetic ones, being, however, the output biophasic rate constant not significantly different from the terminal disposition rate constant, beta or lambda 2.

A study on the allosteric interaction between the major binding sites of human serum albumin using microcalorimetry.

The binding of warfarin and oxyphenbutazone to albumin has been studied at pH 6.8 and pH 9.2 by measuring the heat of binding of these ligands to their high-affinity binding sites on albumin (delta Ho'1). The -delta Ho'1 values for the binding of warfarin at pH 6.8 and 9.2 and oxyphenbutazone at pH 6.8 and 9.2 were found to be 16.9(+/- 0.6), 28.8(+/- 0.6), 10.5(+/- 0.4) and 17.4(+/- 0.6) kJmol-1, respectively. The Gibbs energies (delta Go'1) corresponding to these delta Ho'1 values cover a much smaller range. The pH dependences of delta Go'1 and delta Ho'1 are explained in terms of pK shifts in the albumin upon binding warfarin or oxyphenbutazone. Diazepam, which binds to a site on albumin which is different from the warfarin-oxyphenbutazone binding site, increases - delta Ho'1 for the binding of warfarin and oxyphenbutazone to albumin at pH 6.8, but it does not influence the -delta Ho'1 at pH 9.2. This phenomenon may be attributed to an allosteric interaction between the diazepam binding site and the warfarin binding site. This allosteric interaction must have its origin in a phenomenon other than the N-B transition.

[Influence of tobacco on the serum levels and pharmacokinetics of phenylbutazone and oxyphenbutazone]. / Influence du tabac sur les taux sériques et la pharmacocinétique de la phénylbutazone et de l'oxyphenbutazone.

The serum levels of a number of drugs are reduced by smoking. The authors wanted to see whether this phenomenon occurred with phenylbutazone. With this objective, they compared the changes in serum levels of phenylbutazone and oxyphenbutazone, the hydroxylated metabolite of phenylbutazone, during ten days of administration, in 9 smokers and in 6 non-smokers. Blood samples were taken every day at 8:00 a.m. and the PB and OPB were assayed by high performance liquid chromatography. The comparison of the mean serum levels of PB in smokers and non-smokers revealed a statistically significant reduction (p less than or equal to 0.05) of this level in the smoker, between D5 and D10. Comparison of the mean serum levels of OPB in smokers and non-smokers revealed a statistically significant reduction of this level in the smoker, but only on days 9 and 10. When we compare these results with those reported in the literature for other drugs, it would appear that tobacco smoke accelerates the metabolism of phenylbutazone and oxyphenbutazone by a mechanism of enzyme induction. It is proposed that the therapeutic activity of phenylbutazone is reduced in the smoker, but this has not been confirmed by a reliable clinical evaluation in this initial study.

Diffusion of oxyphenbutazone into synovial fluid, synovial tissue, joint cartilage and cerebrospinal fluid.

The diffusion of oxyphenbutazone into synovial and cerebrospinal fluids and synovium and joint cartilage was investigated in 25 patients receiving short-term treatment. In the synovial fluid, the mean oxyphenbutazone concentration, was 57.1 +/- 13.4% of the plasma level, due to its excellent diffusion into the joint cavity. In synovial tissue, the oxyphenbutazone level was higher in patients with severe inflammation than in those with no or little inflammation. Penetration into joint cartilage is less than into synovial tissue. In cerebrospinal fluid the concentration was close to the level of free plasma oxyphenbutazone. The findings show increased diffusion of oxyphenbutazone towards its site of action in inflammation.