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The pharmacokinetic profile of selected NSAIDs in southern black rhinoceros ( Diceros bicornis minor ) were studied. Phenylbutazone (PBZ), meloxicam (MEL), and firocoxib (FIR) were administered orally to five captive, black rhinoceros, and blood was collected at predetermined time points for NSAID quantification and noncompartmental pharmacokinetic (PK) analysis. Phenylbutazone 4.0 mg/kg PO q12h for three doses, MEL 0.3 mg/kg PO q24h administered twice, and a single oral dose of FIR 0.1 mg/kg, were tested with a minimum washout time of 2 wk. PBZ reached a median (range) peak concentration (C max ) of 9.42 (2.74-11.5) g/ml at a mean (range) time (T max ) of 6.00 (4.00 to >12.00) h, and the median (range) elimination half-life (T 1/2 ) was 6.07 (3.95-6.49) h. Phenylbutazone pharmacokinetic parameters for black rhinoceros in this study were similar to domestic horses. Meloxicam reached a median (range) C max of 0.576 (0.357-0.655) µg/ml at a median (range) time (T max ) of 6.00 (4.00-12.00) h; the median (range) T 1/2 of MEL was 14.0 (12.4-17.9) h. These results demonstrate that once-daily administration of MEL at 0.3 mg/kg resulted in a serum concentration of greater than 0.200 µg/ml from 2 to 24 h in four animals, which is within the analgesic range (0.200-0.400 µg/ml) for this drug in other species postulated by other studies. A single dose of firocoxib (0.1 mg/kg) reached a median (range) peak concentration (C max ) of 15.7 (9.65-17.3) ng/ml at a median (range) T max of 4.00 (4.00-6.00) h. The median (range) elimination T 1/2 of FIR was 4.96 (4.47-6.51) h, which is faster than in the horse. The data suggest that extrapolation from equine FIR dosage recommendations is inappropriate for black rhinoceros.

Objective: To evaluate the effects of aging on phenylbutazone (PBZ) disposition in older horses (≥ 25 years old) compared to young adults (4 to 10 years old) by characterizing the pharmacokinetic profile of PBZ and its active metabolite, oxyphenbutazone (OPBZ), following a 2.2-mg/kg dose, IV. We hypothesized that the disposition of PBZ will be affected by age., Animals: 16 healthy horses (8 young adults aged 4 to 10 years and 8 geriatric horses ≥ 25 years old)., Methods: Horses were administered a single 2.2-mg/kg PBZ dose, IV. Plasma samples were collected at designated time points and frozen at -80 °C until assayed using liquid chromatography-tandem mass spectrometry. Pharmacokinetic analyses were performed using Phoenix WinNonlin, version 8.0 (Certara). Both clinical and pharmacokinetic data were compared between age groups using independent samples t tests, with P < .05 considered significant., Results: Baseline characteristics did not differ between groups, with the exception of age, weight, and plasma total solids. Plasma concentrations of PBZ were best described by a two-compartment model. The maximum plasma concentration of OPBZ was reached at 5 hours for both age groups, and the metabolite-to-parent-drug area-under-the-curve ratios were approximately 20% for both groups. None of the pharmacokinetic parameters of PBZ or its metabolite, OPBZ, differed significantly between age groups., Clinical Relevance: The hypothesis was rejected as there was no significant difference in PBZ disposition in young-adult horses compared to geriatric horses. Our data do not support the need for dose adjustments of PBZ in clinically healthy geriatric horses.

Phenylbutazone (PBZ) is a potent mon-steroidal anti-inflammatory drug used commonly in performance horses. The objectives of the current study were to describe blood and urine concentrations and the pharmacokinetics of PBZ and its metabolites following intravenous (IV) and oral administration and to describe the duration of pharmacodynamic effect. To that end, 17 horses received an IV administration and 18 horses an oral administration of 2 g of PBZ. Blood and urine samples were collected prior to and for up to 96 hours post drug administration. Whole blood samples were collected at various time points and challenged with lipopolysaccharide or calcium ionophore to induce ex vivo synthesis of eicosanoids. Concentrations of PBZ and eicosanoids were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-compartmental pharmacokinetic analysis performed on concentration data from IV and oral administration. Serum concentrations of PBZ and its metabolites were below the limit of quantitation at 96 hours post administration. The volume of distribution at steady state, systemic clearance, and terminal half-life was 0.194 ± 0.019 L/kg, 23.9 ± 4.48 mL/h/kg, and 10.9 ± 5.32 hours, respectively. The terminal half-life following oral administration was 13.4 ± 3.01 (paste) and 15.1 ± 3.96 hours (tablets). Stimulation of PBZ treated whole blood with lipopolysaccharide and calcium ionophore resulted in an inhibition of TXB 2 , PGE 2 , LTB 4 and 15-HETE production for a prolonged period of time post drug administration. The results of this study suggest that PBZ has a prolonged anti-inflammatory following IV or oral administration of 2 g to horses., (© 2018 John Wiley & Sons, Ltd.)

Phenylbutazone is a non-steroidal anti-inflammatory drug licensed for use in horses to treat musculoskeletal disorders. It is not permitted in the European Union for use in animals destined for the food chain. Official statistics provided by the European Food Safety Authority (EFSA) show that 0.18% of bovines tested in the European Union between 2008 and 2014 for non-steroidal anti-inflammatory drugs were non-compliant, with phenylbutazone representing over 28% of these. Anecdotal evidence suggests animals that have not been treated with the drug may have produced non-compliant samples, possibly through some form of contamination. In this study, ultra-high-performance liquid chromatography coupled with mass-spectrometric detection was applied to bovine plasma samples to determine if detectable residues (CCα = 0.28 ng ml -1 ) may occur in untreated animals as a result of environmental contamination through normal farming practice. The study demonstrates that waste from animals treated with phenylbutazone, and spread on an area of pasture, can contaminate untreated bovines grazing the pasture many weeks later. It was determined that this contamination, which can persist over a significant period, may be due to the ingestion of as little as 30 μg phenylbutazone by a 500 kg bullock.

Methocarbamol (MCBL) is commonly used in performance horses for the treatment of skeletal muscle disorders. Current regulatory recommendations for show horses and racehorses are based on a single oral dose of 5 g, although doses in excess of this are often administered. The goal of the current study was to characterize the disposition of MCBL following higher dose administration and administration in combination with another commonly used drug in performance horses, phenylbutazone (PBZ). Exercised Thoroughbred horses were administered various doses of MCBL as a sole agent and MCBL in combination with PBZ. Blood samples were collected at various times, concentrations of MCBL and PBZ measured using LC-MS/MS and pharmacokinetic parameters calculated using compartmental analysis. Following administration of 15 g of MCBL, either as part of a single- or multiple-dose regimen, a number of horses exceeded the Association of Racing Commissioners International and the United States Equestrian Federation's recommended regulatory threshold at the recommended withdrawal time. There was not a significant difference between horses that received only MCBL and those that received MCBL and PBZ. Results of the current study support an extended withdrawal guideline when doses in excess of 5 g are administered., (© 2016 John Wiley & Sons Ltd.)

The presence of horse meat in food products destined for human consumption and labelled as beef has raised several concerns of public interest. This review deals solely with one aspect of these concerns; samples of equine tissue from horses destined for the human food chain have tested positive for the non-steroidal anti-inflammatory drug, phenylbutazone. The safety of some or all such foods for human consumers is a major concern, because it was shown many years ago that phenylbutazone therapy in humans can be associated with life threatening blood dyscrasias. As an initial basis for assessing the potential toxicity of foods containing phenylbutazone and its metabolites, this article reviews (1) the pharmacokinetic, pharmacodynamic, metabolic and toxicological profiles of phenylbutazone, with particular reference to horses and humans; (2) toxicity data in laboratory animals; (3) phenylbutazone residues in food producing species, and (4) as a preliminary assessment, the potential hazard associated with the consumption of horse meat containing phenylbutazone and its metabolites. Since phenylbutazone cannot be classified as a carcinogenic substance in humans, and noting that blood dyscrasias in humans are likely to be dose and treatment duration-dependent, the illegal and erratic presence of trace amount residues of phenylbutazone in horse meat is not a public health issue., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Understanding the dendrimer-drug interaction is of great importance to design and optimize the dendrimer-based drug delivery system. Using atomistic molecular dynamics (MD) simulations, we have analyzed the release pattern of four ligands (two soluble drugs, namely, salicylic acid (Sal), L-alanine (Ala), and two insoluble drugs, namely, phenylbutazone (Pbz) and primidone (Prim)), which were initially encapsulated inside the ethylenediamine (EDA) cored polyamidoamine (PAMAM) dendrimer using the docking method. We have computed the potential of mean force (PMF) variation with generation 5 (G5)-PAMAM dendrimer complexed with drug molecules using umbrella sampling. From our calculated PMF values, we observe that soluble drugs (Sal and Ala) have lower energy barriers than insoluble drugs (Pbz and Prim). The order of ease of release pattern for these drugs from G5 protonated PAMAM dendrimer was found to be Ala > Sal > Prim > Pbz. In the case of insoluble drugs (Prim and Pbz), because of larger size, we observe much nonpolar contribution, and thus, their larger energy barriers can be reasoned to van der Waals contribution. From the hydrogen bonding analysis of the four PAMAM-drug complexes under study, we found intermolecular hydrogen bonding to show less significant contribution to the free energy barrier. Another interesting feature appears while calculating the PMF profile of G5NP (nonprotonated)-PAMAM-Pbz and G5NP (nonprotonated)-PAMAM-Sal complex. The PMF was found to be less when the drug is bound to nonprotonated dendrimer compared to the protonated dendrimer. Our results suggest that encapsulation of the drug molecule into the host PAMAM dendrimer should be carried out at higher pH values (near pH 10). When such complex enters the human body, the pH is around 7.4 and at that physiological pH, the dendrimer holds the drug tightly. Hence the release of drug can occur at a controlled rate into the bloodstream. Thus, our findings provide a microscopic picture of the encapsulation and controlled release of drugs in the case of dendrimer-based host-guest systems.

Reason for Performing Study: Using an adjustable heart bar shoe model of foot pain, the objective of this study was to test the hypothesis that the combined use of phenylbutazone (PBZ) and flunixin meglumine (FM) would prove more efficacious in alleviating lameness than either drug alone., Materials and Methods: One hour after induction of lameness at weekly intervals, 8 healthy adult Thoroughbred horses randomly underwent one of 4 i.v. treatments: saline (SAL) placebo (1 ml/45 kg bwt), PBZ (4.4 mg/kg bwt), FM (1.1 mg/kg bwt) or PBZ+FM (at the same dosages as given individually). Heart rate (HR) and lameness score (LS) responses were assessed in a blinded manner every 20 min for 5 h after lameness induction and then hourly for 12 h after treatment. Jugular venous blood samples were obtained at -1, 0, 0.05, 1, 2, 4, 6, 8, 10 and 12 h and subsequently analysed for drug concentrations. Repeated measures ANOVA and post hoc Tukey's test were used to identify analgesic effects at a significance level of P<0.05., Results: Heart rate was lower in all nonsteroidal anti-inflammatory drug (NSAID)-treated trials from 2 h to 10 h post treatment (P<0.05). Analgesic effects of FM and PBZ+FM, as evidenced by decreases in HR, lasted for 12 h post treatment (P<0.05). Lameness score decreased earlier in PBZ and PBZ+FM trials than in FM trials (P<0.05) and the analgesic effect on LS lasted for 12 h post treatment for all NSAID trials (P<0.05). Peak PBZ plasma concentration was 73.7 ± 6.0 and 77.9 ± 5.5 µg/ml. Peak FM concentration was 12.0 ± 0.8 and 13.7 ± 1.0 µg/ml., Conclusions: It was concluded that the combination of PBZ+FM was not more effective than either PBZ or FM alone. These data do not support the hypothesis that the combination is more efficacious at these dosages than either drug alone in this model of acute foot pain., (© 2011 EVJ Ltd.)

Reasons for Performing Study: Equine antidoping rules were established to prevent a horse's performance being altered after the administration of prohibited substances, including approved drugs used for legitimate treatment. Veterinarians have to advise owners or trainers on appropriate withholding times to guarantee that their horses may safely compete after drug administration. In order to propose tailored withdrawal times, several horse organisations released detection time (DT) values, for the main veterinary drugs used in horses. One of the possible limits to the information provided by published DTs in horses is the fact that they are determined from classic pharmacokinetic studies performed at rest under laboratory conditions. In field conditions, training and exercise programmes may have an influence on drug elimination., Methods: Dexamethasone (DMX) and phenylbutazone (PBZ) have been quantified in plasma and urine after solid phase extraction. The kinetic disposition of DXM (8 microg/kg) and PBZ (8 mg/kg) administered by i.v. route in 8 horses, was investigated in rest conditions and during a standardised 3 h test exercise according to a cross-over design., Objectives: The aim of the present study was to compare the kinetic disposition of 2 test drugs, DMX and PBZ in rest vs. exercising conditions., Results: It was shown in 8 horses that a sustained 3 h of mild exercise slightly decreased the plasma clearance of both drugs (about 25% for DXM and 37% for PBZ) and this is mainly explained by the significant decrease of the corresponding hepatic clearance. In addition, as the volume of distribution was correlatively decreased, the plasma terminal half-life, which is a hybrid parameter of plasma clearance and volume of distribution, remains unchanged overall., Conclusion and Potential Relevance: Establishing DTs or withdrawal times (WTs) are relevant as plasma and urine half-lives, but not clearance, are the main determinants of DT length. Veterinarians may realistically decide upon a WT for a legitimate drug based on the corresponding DT obtained under resting conditions providing this drug has a low hepatic extraction ratio and a safety margin is added to allow for all possible sources of variability.

Injuries sustained by horses during racing have been considered as an unavoidable part of horse racing. Many factors may be associated with the musculoskeletal injuries of Thoroughbred race horses. This study surveyed the amounts of nonsteroidal anti-inflammatory agents (NSAIDs) in injured horse's biological system (plasma) at Kentucky racetracks from January 1, 1995 through December 31, 1996. During that period, there were 84 catastrophic cases (euthanized horses) and 126 noncatastrophic cases. Plasma concentrations of NSAIDs were determined by High Performance Liquid Chromatography in injured and control horses. The possible role of anti-inflammatory agents in musculoskeletal injuries of Thoroughbred race horses was investigated by comparing the apparent concentrations of NSAIDs in injured horses to concentrations in control horses. The plasma concentrations of phenylbutazone and flunixin were higher in injured horses than in control horses. Most injured and control horses did not have a detectable level of naproxen in their plasma samples. Further studies must be carried out to determine whether horses with higher plasma concentrations of NSAIDs have an altered risk of musculoskeletal injuries compared with other horses.

A sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated for screening, quantification, and confirmation of phenylbutazone and oxyphenbutazone in equine plasma. Analytes were recovered from plasma by liquid-liquid extraction followed by separation in a reversed-phase column and identification by mass spectrometry with selected reaction monitoring in negative electrospray ionization mode. Extraction recovery for both analytes was >80%. Limits of detection, quantification, and confirmation for both analytes were 0.01 microg/mL (S/N>or= 3), 0.05 microg/mL, and 0.05 microg/mL, respectively. The assay with d9-labeled phenylbutazone as internal standard (IS) was linear over a range of 0.05-20 microg/mL (r2>0.995). Intra- and interday precision in terms of coefficient of variation was less than 15%. Intra- and interday accuracy (bias%) was within 80-120%. Hemolysis of red blood cells decreased analyte signal intensity but did not affect quantification results because an isotope-labeled IS was used. Analytes were stable in plasma for 24 h at room temperature, 9 days at 4 degrees C, and 45 days at -20 degrees C and -70 degrees C. The method was successfully used in screening, quantification, and confirmation of phenylbutazone in post-competition plasma samples obtained from racehorses. The method is simple, rapid, and reliably reproducible.

Direct-injection LC-LC hybrid tandem MS methods have been developed for undertaking broad-based screening for acidic drugs in protein-precipitated plasma and neutral doping agents in equine urine. In both analyses, analytes present in the matrix were trapped using a HLB extraction column before being refocused and separated on a Chromolith RP-18e monolithic analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Each method has been optimised by the adoption of a mobile phase and gradient that was tailored to enhance ionisation in the MS source while maintaining good chromatographic behaviour for the majority of the target drugs. The analytical column eluent was fed into the heated nebulizer (HN) part of the Duospray interface attached to a 4000 QTRAP mass spectrometer. Information dependent acquisition (IDA) with dynamic background subtraction (DBS) was configured to trigger a sensitive enhanced product ion (EPI) scan when a multiple reaction monitoring (MRM) survey scan signal exceeded the defined criteria. Ninety-one percent of acidic drugs in protein-precipitated plasma and 80% of the neutral compounds in equine urine were detected when spiked at 10 ng/ml.

The effect of feeding Sporobolus and Rhodes hay on phenylbutazone (4 g) relative absorption was examined in six camels using a two-period, two-sequence, two-treatment crossover design. Serum concentration of the drug was measured by high performance liquid chromatography. The measured values (means+/-SD) for Rhodes and Sporobolus hay, respectively, were Cmax 35.59+/-22.36 and 36.55+/-18.99 microg/mL, Tmax 26+/-2.53 and 26.3+/-1.97 h and AUC0-72 h 1552+/-872.6 and 1621+/-903.6 microg h/mL. Broad plateau concentrations of phenylbutazone in serum were observed between 12 and 36 h. There was no significant difference in any parameter between the two feeding regimens. Multiple peaks in serum concentration-time curve were observed, regardless of the type of grass available to and the animals prior to drug administration. It was concluded that the phasic absorption of phenylbutazone was a particular feature of hay feeding in camels, and the Sporobolus hay can be fed to camels without any effect on the rate and extent of phenylbutazone absorption compared to Rhodes grass hay.

Ultracentrifugation methods have been widely used for the determination of the free fraction of compounds in plasma, especially for lipophilic compounds. To estimate the effect of contaminated proteins in the "protein-free phase" fraction, 200 microL of human plasma was separated into three layers by ultracentrifugation at 436,000g for 140 min with a table-top ultracentrifuge. Twenty microliters of the middle layer was taken as the protein-free fraction. Major contaminated proteins were analyzed by liquid chromatography/electrospray ionization/tandem mass spectrometry (LC/ESI/MS/MS) and identified as albumin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, apolipoprotein E, and apolipoprotein A-1. alpha-1-acid glycoprotein was not detected. Contamination of albumin was 0.13% of that in plasma. Simulation analysis demonstrated that at an actual free fraction of 1% (protein binding ratio of 99%), the extent of overestimation of free fraction was just 13% and the apparent free fraction was 1.13%. Human plasma protein binding ratios of 10 drugs estimated by this method correlated well with reported values determined by other methods, such as ultrafiltration and equilibrium dialysis, with a correlation factor of 0.98 and a slope of 0.99. Collectively, our results indicate the reliability of this micro-scale ultracentrifugation technique for the evaluation of the protein binding of drugs despite a little contamination of albumin., (Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association.)

The pregnant Holstein cow and her newborn calf were evaluated as an animal model to study in utero and for lactational drug transfer and offspring exposure. A nonsteroidal antiinflammatory drug, phenylbutazone, and an antiparasitic drug, ivermectin, were tested in the model. Prior to parturition, pregnant cows were dosed orally to steady state with phenylbutazone at 4 g/day or given a single subcutaneous injection of 200 microg ivermectin/kg body wt. The level of drug transferred to calves exposed in utero, in utero combined with lactational exposure, and via lactational exposure only, was measured from days 1 through 7 postpartum. At birth the plasma level in phenylbutazone-exposed calves was approximately one-half the dam's steady-state level. For ivermectin-exposed calves, plasma levels were at or below the limit of quantitation (0.5 ng/ml) at birth, suggesting that placental transfer of ivermectin is limited in the cow. For both drugs, rapid accumulation of the drug in calf plasma occurred with lactational exposure to a mean daily dose of 2 microg ivermectin/kg body wt or 0.1 mg phenylbutazone/kg body wt/day for the first 7 days of life. The accumulation observed in the newborn calf is attributed to the lipid solubility and long elimination half-lives of these drugs. These results demonstrate that drug transfer and offspring exposure can be studied using the cow-calf model. The data also highlight the importance of considering not only the dose but also physicochemical characteristics and pharmacokinetics of the drug in the offspring when evaluating the safety of a newborn's exposure to a drug in breast milk.

Phenylbutazone was administered intravenously to a group of 11 beef steers at a dosage of 6 mg/kg of body weight. Whole plasma and protein-free plasma were analyzed for phenylbutazone residues. Pharmacokinetic parameters of total and free phenylbutazone in plasma were calculated using a noncompartmental method. In regards to whole plasma data, the mean volume of distribution at steady state (Vss), was 140 mL/kg body weight, with a mean (+/-SEM) terminal elimination half-life (t1/2) of 34 +/- 9 h. The mean clearance was 3.2 mL/h/kg body weight. The Vss, as determined from the protein-free plasma fraction, was 54093 mL/kg body weight. This larger Vss of free phenylbutazone compared with total plasma phenylbutazone was attributed to a high degree of plasma protein binding, as well as the greater penetration of free phenylbutazone into tissues. The mean t1/2 of free phenylbutazone was 35 +/- 12 h. This similarity to the t1/2 estimated from total plasma phenylbutazone data is attributed to an equilibrium between free and plasma phenylbutazone during the terminal elimination phase. The pharmacokinetic parameters of free and total plasma phenylbutazone in beef steers are statistically similar to those previously reported for lactating dairy cows.

The current performance of analytical techniques used for drug control in horses lead the Regulatory Authorities to decide whether trace levels of drugs legitimately used for therapeutic medication should or should not be reported. Here, we propose a well-ordered and nonexperimental pharmacokinetic/pharmacodynamic approach for the determination of irrelevant drug plasma (IPC) and urine concentrations (IUC). The published plasma clearance is used to transform an effective (marketed) dose into an effective concentration (EPC). EPC is transformed into an IPC by applying a safety factor (SF). This method is based on several assumptions (eg, drug effects reversibly driven by plasma concentration, linearity of drug disposition). The suitability of the computed IPC and IUC can be checked by calculating the residual amount of drug at IPC and computing a minimal drug withdrawal time. It is concluded that controlling the drug effect (using drug or any analyte concentration as a marker) rather than the drug exposure will be more demanding and also makes urine a less than ideal matrix.

The purpose of this study was to investigate the possible presence of phenylbutazone in plasma samples from fighting bulls killed in 2nd and 3rd category bullrings in the province of Salamanca (Spain) in 1998, 1999 and 2000. For quantitative and qualitative determination, a high-performance liquid chromatograph was used, equipped with a photodiode-array detector and setting wavelengths at 240, 254 and 284 nm. The mobile phase optimized for the simultaneous detection of dexamethasone, betamethasone, flunixin and phenylbutazone, was 0.01 M acetic acid pH 3 in methanol (35:65 v/v) at a flow rate of 1 ml/min. Plasma samples were deproteinized with 400 microl of acetonitrile and 20 microl of the supernatant were injected directly into the chromatographic system equipped with a Lichrospher 60 RP select B column and guard column. For the quantitative analysis, standard calibration curves were made in a concentration range between 0.25 and 30 microg/ml, using betamethasone as internal standard. The retention time of phenylbutazone was 8.7 +/- 0.2 min and recovery was 83%. The detection and quantification limits were 0.016 and 0.029, respectively for A=240 nm. The study results show that 17 of the 74 samples analyzed in 1998, 18 of those from 1999 and 10 of those from 2000 were positive for phenylbutazone.

Objective: To describe the pharmacokinetics of phenylbutazone and oxyphenbutazone after IV administration in miniature donkeys., Animals: 6 clinically normal miniature donkeys., Procedure: Blood samples were collected before and 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 300, 360, and 480 minutes after IV administration of phenylbutazone (4.4 mg/kg of body weight). Serum was analyzed in triplicate by use of high-performance liquid chromatography for determination of phenylbutazone and oxyphenbutazone concentrations. The serum concentration-time curve for each donkey was analyzed separately to estimate model-independent pharmacokinetic variables., Results: Serum concentrations decreased rapidly after IV administration of phenylbutazone, and they reached undetectable concentrations within 4 hours. Values for mean residence time ranged from 0.5 to 3.0 hours (median, 1.1 hour), whereas total body clearance ranged from 4.2 to 7.5 ml/kg/min (mean, 5.8 ml/kg/min). Oxyphenbutazone appeared rapidly in the serum; time to peak concentration ranged from 13 to 41 minutes (mean, 26.4 minutes), and peak concentration in serum ranged from 2.8 to 4.0 mg/ml (mean, 3.5 microg/ml)., Conclusion and Clinical Relevance: Clearance of phenylbutazone in miniature donkeys after injection of a single dose (4.4 mg/kg, IV) is rapid. Compared with horses, miniature donkeys may require more frequent administration of phenylbutazone to achieve therapeutic efficacy.

Objective: To determine the effects of phenylbutazone (PBZ) on bone activity and bone formation in horses., Animals: 12 healthy 1- to 2-year-old horses., Procedures: Biopsy was performed to obtain unicortical bone specimens from 1 tibia on day 0 and from the contralateral tibia on day 14. Fluorochromic markers were administered IV 2 days prior to and on days 0, 10, 15, and 25 after biopsy was performed. Six horses received PBZ (4.4 mg/kg of body weight, PO, q 12 h) and 6 horses were used as controls. All horses were euthanatized on day 30 and tissues from biopsy sites, with adjacent cortical bone, were collected. Osteonal density and activity, mineral apposition rate (MAR), and percentage of mineralized tissue filling the biopsy-induced defects in cortical bone were assessed. Serum samples from all horses were analyzed for bone-specific alkaline phosphatase activity and concentration of PBZ., Results: MAR was significantly decreased in horses treated with PBZ. Regional acceleratory phenomenon was observed in cortical bone in both groups but was significantly decreased in horses treated with PBZ. Osteonal activity was similar at all time points in all horses. In control horses, percentage of mineralized tissue filling the cortical defects was significantly greater in defects present for 30 days, compared with defects present for 14 days. Differences in percentage of mineralized tissue were not detected in horses treated with PBZ., Conclusions and Clinical Relevance: PBZ decreased MAR in cortical bone and appeared to decrease healing rate of cortical defects in horses.

The binding of the anti-inflammatory agent kebuzone (CAS 853-34-9) to human serum albumin (HSA) was examined by means of high performance liquid affinity chromatography. Phenylbutazone and diazepam, the binding properties of which were an object of our previous work, were used as markers for the major supposed binding sites on HSA. It was established that kebuzone binds to the high-affinity phenylbutazone binding sites as well as to the low-affinity diazepam binding sites. The estimated values of the equilibrium affinity constants were 5.9 x 10(4) l/mol and 1.7 x 10(3) l/mol, respectively, suggesting a low binding affinity for kebuzone. A stepwise mechanism of the binding process could be proposed: primary at phenylbutazone sites, and secondary at the diazepam sites. The binding behavior of kebuzone was very similar to that of the structurally related compound phenylbutazone. The observed lower affinity is well explained in view of the lower lipophilicity of kebuzone.

Ethyl acetate extracts of equine serum, containing 0-5 microg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile-water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 microg/ml for HYD, DEX and IND, 0.5 microg/ml for PB and 1 microg/ml for OPB, despite the occurrence of significant degradation of OPB and PB during the analysis) and precision (intra-assay and inter-assay C.V.'s of about 3-6 and 9-15%, respectively) of the method appeared appropriate for anti-doping control of racehorses.

Suxibuzone (SBZ), a nonsteroidal anti-inflammatory drug, was administered to 6 horses at a dose rate of 7.5 mg/kg bwt by intravenous (i.v.) route. Plasma and synovial fluid concentrations of suxibuzone and its main active metabolites, phenylbutazone (PBZ) and oxyphenbutazone (OPBZ), were measured simultaneously by a sensitive and specific high-performance liquid chromatographic method. The pharmacokinetic parameters were determined by noncompartmental analysis. Plasma SBZ concentrations rapidly decreased and were not detectable beyond 20 min after treatment. The parent drug was not detected in any synovial fluid samples. Average maximum plasma concentrations of PBZ (16.43 microg/ml) and OPBZ (2.37 microg/ml) were attained at 0.76 and 7.17 h, respectively. The mean residence time (MRT) of PBZ was 6.96 h in plasma. Oxyphenbutazone plasma concentrations were below those reached by phenylbutazone during the first 12 h after suxibuzone administration, even though its values were detectable for at least 24 h (MRT = 10.65 h). Plasma concentrations of PBZ and OPBZ exceeding EC50 and IC50 of TXB2 and PGE2 were reached by at least 12 h. Synovial fluid concentrations of PBZ and OPBZ were 2.87+/-0.37 microg/ml and 0.97+/-0.08 microg/ml at 9 h after suxibuzone administration and exceeded IC50 of PGE2 for at least this time. In the present study, suxibuzone was well tolerated following i.v. injection.

A disposition and bioequivalence study with a suxibuzone granulated and a suxibuzone paste oral formulation was performed in horses. Suxibuzone (SBZ) is a nonsteroidal anti-inflammatory drug, which was administered to horses (n = 6) at a dosage of 19 mg/kg bwt by the oral route (p.o.) in a two period cross-over design. Suxibuzone is very rapidly transformed into its main active metabolites, phenylbutazone (PBZ) and oxyphenbutazone (OPBZ). Therefore plasma and synovial fluid concentrations of SBZ, PBZ and OPBZ were simultaneously measured by a sensitive and specific high-performance liquid chromatographic method. The pharmacokinetic parameters were determined by noncompartmental analysis. Suxibuzone could not be detected in any plasma and synovial fluid samples (< 0.04 microgram/mL). Plasma PBZ and OPBZ concentrations were detected between 30 min and 72 h after granulate and paste administration. Mean plasma concentration of PBZ peaked at 5 h (34.5 +/- 6.7 micrograms/mL) and at 7 h (38.8 +/- 8.4 micrograms/mL), and mean area under the concentration-time curve (AUC0-->LOQ) was 608.0 +/- 162.2 micrograms.h/mL and 656.6 +/- 149.7 micrograms.h/mL after granulate and paste administration, respectively. Mean plasma concentration of OPBZ increased to 5-6.7 micrograms/mL, with the maximum concentration (Cmax) appearing between 9 and 12 h after administration of both formulations. The AUCs0-->LOQ for OPBZ were also similar (141.8 +/- 48.3 micrograms.h/mL granulate vs. 171.4 +/- 45.0 micrograms.h/mL paste). It was concluded that the suxibuzone products were bioequivalent with respect to PBZ. For OPBZ, the 95% confidence intervals of the pharmacokinetic parameters were within the acceptable range of 80-125%. The paste formulation provided greater bioavailability of PBZ and OPBZ.

The quantitative parameters characterizing the binding of phenylbutazone (CAS 50-33-9) to human serum albumin are determined using the circular dichroism titration method and a combined mathematical approach. The values of affinity constants obtained are in good agreement with those found in the literature. The characterization and identification of binding sites is also performed. A conclusion is derived that phenylbutazone can be used as a model compound for investigation of ligand-macromolecule interactions.

A liquid chromatographic procedure using UV detection was coupled with ultrafiltration for the quantitation of free phenylbutazone in bovine plasma, in the range of 20 ng/ml to 2.0 microg/ml. Whole plasma samples (0.5 to 1 ml) were placed in a 2-ml centrifugal concentrator with a molecular-mass cut-off membrane of 10000 and centrifuged at 4500 g for 2 h at 4 degrees C using a fixed angle rotor. The ultrafiltrate was transferred to an LC vial with a 200-microl insert and 100 microl was injected into an LC system. The chromatographic system used a C18 reversed-phase column connected to a UV detector set at 264 nm. The mobile phase was 0.2 M sodium phosphate buffer (pH 7)-methanol (1:1). Recoveries of phenylbutazone from protein-free plasma water fortified at levels of 20 ng/ml to 2 microg/ml ranged from 91 to 93%, with relative standard deviations (R.S.D.s) ranging from 1 to 4%. The concentration of incurred non-protein bound phenylbutazone obtained from a cow intravenously dosed twice with 2 g phenylbutazone, 8 h apart, was 111, 26 and 11 ng/ml for 2, 72 and 104 h post first phenylbutazone dose, respectively.

The reversible binding of ethacrynic acid was characterized by a difference circular dichroism method. A 2/1 stoichiometry was determined for the [drug]/[HSA] (human serum albumin) complex. The reversible binding of ethacrynic acid to HSA determines direct competition with ligands that selectivity bind to site II and to the fatty acid site. Furthermore, indirect competition was shown for ligands for site I (anti-cooperative) and to site III (cooperative).

Phenylbutazone was administered intravenously (i.v.) to a group of four lactating cows at a dosage of 6 mg/kg body weight. Whole plasma, protein-free plasma and milk were analysed for phenylbutazone residues. Pharmacokinetic parameters of total and free phenylbutazone in plasma were calculated using a non compartmental method. In regards to whole plasma data, the mean volume of distribution at steady state (Vss), was 147 mL/kg body weight, with a mean (+/-SEM) terminal elimination half-life (t1/2) of 40+/-6 h. The mean clearance (Cl) was 3 mL/h/kg body weight. The Vss as determined from the protein-free plasma fraction was 50021 mL/kg body weight. This larger Vss of free phenylbutazone compared to total plasma phenylbutazone was attributed to a high degree of plasma protein binding, as well as the greater penetration of free phenylbutazone into tissues. The mean t1/2 of free phenylbutazone was 39+/-5 h. This similarity to the t1/2 estimated from total plasma phenylbutazone data is attributed to an equilibrium between free and plasma phenylbutazone during the terminal elimination phase. Mean t1/2 as determined from milk, applying a urinary excretion rate model, was 47+/-4 h. Milk clearance of phenylbutazone was 0.009 mL/h/kg body weight, or about 0.34% of total body clearance. Furthermore, evidence suggests that phenylbutazone either binds to milk proteins, or is actively transported into milk, as its concentration in milk was greater than that predicted due to a simple partitioning from plasma into milk.

Cimetidine was administered intravenously and by the intragastric route to six mares at a dose of 4.0 mg/kg of body weight (bw). Specific and sensitive high performance liquid chromatographic methods for the determination of cimetidine in horse plasma and urine and cimetidine sulfoxide in urine are described. Plasma cimetidine concentration vs. time data were analysed by non-linear least squares regression analysis to determine pharmacokinetic parameter estimates. The median (range) plasma clearance (Cl) was 8.20 (4.96-10.2) mL/min.kg of body weight, that of the steady-state volume of distribution (Vdss) was 0.771 (0.521-1.15) L/kg bw, and that of the terminal elimination half-life (t1/2 beta) was 92.4 (70.6-125) minutes. The median (range) renal clearance of cimetidine was 4.08 (2.19-6.23) mL/min.kg bw or 55.4 (36.3-81.8)% of the corresponding plasma clearance. Cimetidine sulfoxide was excreted in urine and its urinary excretion through 8 h accounted for 12.0 (9.8-16.6)% of the plasma clearance of cimetidine. The median (range) extent of intragastric bioavailability was 14.4 (6.82-21.8)% and the maximum plasma concentration after intragastric administration was 0.31 (0.24-0.50) microgram/mL. Intravenous cimetidine had no effect on the disposition of intravenous phenylbutazone or its metabolites except that the maximum plasma concentration of gamma-hydroxyphenylbutazone was less after cimetidine treatment.

A direct injection HPLC method has been developed for the determination of phenylbutazone and its active metabolite oxyphenylbutazone in serum using a semipermeable surface (SPS) column. The method is easy to perform and requires 20 microliters of a filtered serum sample. The chromatographic time is less than 13 min using a mobile phase of 15:85 v/v acetonitrile-0.05M phosphate buffer pH 7.5. The method was linear in the range 0.5-20 micrograms ml-1 (r > 0.99, n = 6) with R.S.D. less than 6%. Interday and intraday variability were found to be less than 8.3%. The limit of quantitation and detection were 0.5 and 0.25 microgram ml-1 (s/n > 3), respectively, for both drug and metabolite.

Phenylbutazone was administered intravenously and intramuscularly at a dosage rate of 4.4 mg/kg to a group of 6 female camels in a two-period crossover study. After intravenous (i.v.) administration, disposition was characterised by a two-compartment open model, with a low volume of distribution (0.174 l.kg-1), and distribution and elimination half-lives of 0.43 and 12.51 h, respectively. After intramuscular (i.m.) dosing absorption was relatively rapid with absorption half-time and time of maximal concentration values of 1.14 and 3.95 h, respectively. Plateau concentrations of phenylbutazone in plasma were obtained between 2 and 12 h and mean bioavailability was 97%, although this was subject to wide inter-animal differences. Plasma concentrations of the phenylbutazone metabolite, oxyphenbutazone, were low after iv dosing and generally undetectable after im administration, indicating that it is unlikely to contribute significantly to the pharmacological effects produced by phenylbutazone administration. An indication was obtained that phenylbutazone inhibited the ex vivo synthesis of serum thromboxane B2 (TxB2) for 24h after i.v. dosing, but this finding requires confirmation.

Phenylbutazone was administered intravenously and orally to six goats as a single dose of 4.4 mg/kg and its disposition and bioavailability and the disposition of its active metabolite, oxyphenbutazone, in plasma were investigated. The effect of the administration of the drug of oxyphenbutazone on ex vivo serum thromboxane (TX)B2 generation in platelets was also studied. Phenylbutazone was eliminated slowly with mean (se) elimination half-lives (t1/2 beta) of 15.3 (1.15) hours and 22.0 (3.32) hours after intravenous and oral administration, respectively. The bioavailability of phenylbutazone paste administered orally was 61 (7) per cent (corrected by the t1/2 beta) and relatively slow absorption was observed, as indicated by a time of maximum drug concentration (tmax) of 3.47 (0.39) hours and a mean absorption time (MAT) of 10.4 (8.61) hours. The concentration of oxyphenbutazone in plasma was low and the ratio of the areas under the curve (AUC) of oxyphenbutazone to phenylbutazone was approximately 0.02:1 after both intravenous and oral administration. Thromboxane B2 generation in the platelets was significantly inhibited (P < 0.05) from one to 12 hours after intravenous administration and from two to 12 hours after oral administration. The results suggest that phenylbutazone is a potentially useful non-steroidal anti-inflammatory drug for use in goats by either route of administration.

Objective: To compare plasma disposition of phenylbutazone and its metabolite oxyphenbutazone after i.v. administration of phenylbutazone in horses and donkeys., Animals: 4 clinically normal horses and 6 clinically normal donkeys., Procedure: Blood samples were collected from each animal at time 0 (before) and 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 300, 360, and 480 minutes after i.v. administration of a bolus dose of phenylbutazone. Serum was analyzed in triplicate by use of high-performance liquid chromatography for determination of phenylbutazone and oxyphenbutazone concentrations. The serum concentration-time curve for each horse and donkey was analyzed separately to estimate model-independent pharmacokinetic variables., Results: Significant differences were found in several pharmacokinetic variables of phenylbutazone and oxyphenbutazone in horses, compared with donkeys. Mean total body clearance of phenylbutazone in horses was fivefold less than that in donkeys (29.3 and 170.3 ml/kg/h, respectively). Mean values for area under the curve and mean residence time in horses (118.3 micrograms/h/ml and 3.6 hours, respectively) were significantly greater than values in donkeys (28.3 micrograms/h/ml and 1.7 hours, respectively). Mean values for apparent volume of distribution at steady state were not significantly different between horses and donkeys. For oxyphenbutazone, mean time to peak concentration in donkeys was significantly less than that in horses (1.6 and 6.4 hours, respectively)., Conclusion: Phenylbutazone clearance in donkeys was higher than that in horses, and appearance of the metabolite oxyphenbutazone in serum was more rapid in donkeys than in horses, indicating that hepatic metabolism of phenylbutazone is more rapid in donkeys than in horses., Clinical Relevance: Because serum concentration of phenylbutazone after single i.v. bolus administration (4.4 mg/kg of body weight) decreases more rapidly in donkeys, compared with horses, phenylbutazone may require more frequent administration in donkeys to achieve therapeutic efficacy.

Applicability of high-performance frontal analysis (HPFA) to the stereoselective study of drug-drug interaction upon plasma protein binding has been investigated. Racemic warfarin and phenylbutazone were used as model drugs. An on-line HPFA/HPLC system consisting of a HPFA column (diol-silica column), an extraction column, and a chiral separation column was developed, and human serum albumin solution containing racemic warfarin and/or phenylbutazone was injected directly to the HPFA column. When the injection volume was large enough, the binding equilibrium in the sample solution was reproduced in the column, and consequently a plateau region appeared on the chromatogram. This plateau region contains unbound drug(s). A given volume of eluent in the plateau part was transferred into the extraction column by column-switching. The concentrated drug(s) was then transferred to the chiral separation column to determine the unbound concentrations of the enantiomers and/or the competitor. The results agreed with those obtained by a conventional ultrafiltration-HPLC method. The influence of phenylbutazone upon the protein binding of warfarin is enantioselective. In warfarin and human serum albumin mixed solution, the unbound concentration of (R)-warfarin was 1.22 times higher than that of the S-isomer. By addition of phenylbutazone, the unbound concentration of (S)-warfarin increased more than that of (R)-warfarin, resulting in the reversed enantioselectivity, i.e., the unbound concentration of (S)-warfarin became 1.19 times larger than that of (R)-warfarin. The present method was also applicable to human plasma samples.

The effect of inflammation on the disposition of phenylbutazone (PBZ) was investigated in Thoroughbred horses. An initial study (n = 5) in which PBZ (8.8 mg/kg) was injected intravenously twice, 5 weeks apart, suggested that the administration of PBZ would not affect the plasma kinetics of a subsequent dose. Two other groups of horses were given PBZ at either 8.8 mg/kg (n = 5) or 4.4 mg/kg (n = 4). Soft tissue inflammation was then induced by the injection of Freud's adjuvant and the administration of PBZ was repeated at a dose level equivalent to, but five weeks later than, the initial dose. Inflammation did not appear to affect the plasma kinetics or the urinary excretion of PBZ and its metabolites, oxyphenbutazone (OPBZ) or hydroxyphenylbutazone (OHPBZ) when PBZ was administered at 8.8 mg/kg. However, small but significant increases (P < 0.05) in total body clearance (CLB; 29.2 +/- 3.9 vs. 43.8 +/- 8.1 mL/ h.kg) and the volume of distribution, calculated by area (Vd(area); 0.18+/- 0.05 vs. 0.25 +/- 0.03 L/kg) or at steady-state (Vd(SS); 0.17 +/- 0.04 vs. 0.25 +/- 0.03 L/ kg), were obtained in horses after adjuvant injection, compared to controls, when PBZ was administered at 4.4 mg/kg which corresponded to relatively higher tissues concentrations and lower plasma concentrations (calculated) at the time of maximum peripheral PBZ concentration. Soft tissue inflammation also induced a significantly (P < 0.05) higher amount of OPBZ in the urine 18 h after PBZ administration but the total urinary excretion of analytes over 48 h was unchanged. These results have possible implications regarding the administration of PBZ to the horse close to race-day.

This study examined the pharmacokinetics of steady-state phenylbutazone and single bolus intravenous gentamicin when administered together in the horse. The trial design was completed as a cross-over with seven thoroughbred horses. In the first phase each horse received 2.2 mg/kg gentamicin intravenously. After a 2-week washout, each horse received 4.4 mg/kg phenylbutazone intravenously every 24 h for 5 days. On the fourth day each horse received gentamicin as before. Plasma was harvested for gentamicin concentration determination by fluorescence polarization immunoassay and for phenylbutazone concentration determination by high-performance liquid chromatography. All gentamicin data were best approximated by a two-compartment open model using sequential, weighted non-linear regression. Pharmacokinetic parameters were calculated using model-dependent formulae. Phenylbutazone data were analysed by non-compartmental methods. Phenylbutazone induced a 49% increase in the rate of gentamicin return to the central compartment from peripheral tissues (k21) (P < 0.05) and there was a trend to a 24% increase in k12 (P = 0.052). The gentamicin elimination half-life was decreased 23% and the Vd(urea) was reduced by 26%. No induction by gentamicin of changes in phenylbutazone pharmacokinetics were detected. In summary, phenylbutazone induced changes to the rate and extent of distribution and elimination of gentamicin. Therefore, care should be exercised in the use of aminoglycosides in equine patients concurrently maintained on phenylbutazone.

Objective: To develop a sensitive, rugged high-performance liquid chromatography (HPLC) method for the measurement of phenylbutazone (PBZ) in equine sera, using a rapid, nonevaporative extraction technique., Sample Population: Sera from 5 nonexercising adult horses., Procedure: After addition of sodium chloride and acetonitrile to serum samples, reverse-phase HPLC analysis for PBZ and oxyphenbutazone (OXY) was performed directly on extracts, using diode array UV spectrophotometric detection. Probenecid was used as an internal standard. Data were evaluated by standard means of statistical analysis., Results: Recoveries of PBZ, OXY, and probenecid from spiked samples were acceptable (ie, > or = 80%) and within run retention times were reproducible. Chromatograms were free of interfering substances, and linearity of calibration curves was observed throughout operational ranges. Coefficients of variation at each fortified PBZ concentration were in the 5 to 10% range. The method was applicable to analysis of PBZ and OXY in serum extracts from horses dosed with PBZ (4.4 mg/kg of body weight, IV) in a controlled environment. Track samples analyzed by use of this method and a conventional liquid/liquid extraction method gave comparable results (mean deviation, 1.6%) for PBZ concentrations., Conclusion: The HPLC protocol described is suitable for measuring PBZ and OXY in equine sera to regulate PBZ administration in horses involved in pari-mutuel racing.

A HPLC method for quantification of kebuzone and its metabolites in whole blood was developed. The compounds and the internal standard were isolated from blood by solid-phase extraction on a C-18 cartridge. A blood sample was to be hemolyzed before extraction. HPLC was performed on a C-18 column with the mobile phase composed of methanol/water acidified to pH 2.7 and UV absorbance detection at 247 nm. This method has been successfully applied to a pharmacokinetic study of kebuzone and its metabolites in rabbits.

In a study in the horse, the disposition, the pharmacokinetic parameters and the absorption rates of 3 formulations of phenylbutazone (injection solution, powder and paste suspension) have been determined. After i.v. injection, the half-life time of phenylbutazone has been determined to be 6.6-6.7 h. After oral administration, the absorption of phenylbutazone was found to be faster after administration via stomach tube than after direct application into the mouth. The absorption rat constant of the paste suspension was found to be higher than that of the powder (1.797-2.304 h-1 vs. 0.656-1.197 h-1). The maximal plasma concentrations were attained within 0.5-3 hours with all formulations and were dose-dependent. The elimination of phenylbutazone was not dependent on the dose and formulation applied. The bioavailability of the studied powder and paste formulation was calculated to be 55.1-96.7%. Generally, the bioavailability was higher following administration of the drug via stomach tube. In some horses, the disposition pattern was atypical and delayed absorption was found. This may be due to adsorption of phenylbutazone on the food with subsequent release in the large intestine. The minimal therapeutic level of 5 micrograms/ml was attained 0.2-0.8 hours after administration of the drug and lasted 8.2-22 hours depending on the dose applied. The paste suspension of phenylbutazone may provide a good therapeutic tool for horses, which normally refuse to take powder supplemented food. At the end the authors report on their good clinical results in using Butasan-Paste with 30 ill horses.

Determination of pharmacokinetic data from plasma often is not sufficient in order to predict or to explain the clinical efficacy of drugs, e.g., in non steroidal antiinflammatory drugs (NSAID) with short elimination half lives in plasma a discrepancy appears between elimination half life and clinical effect. We have studied the tissue kinetics of a drug having a long elimination half life (naproxen) in comparison with phenylbutazone and flunixin, having a short elimination half life in dogs after oral application. Tissue fluid was obtained by using so called "tissue cages" implanted laterally in the neck. An inflammatory response was produced by injecting 1-2 ml of 2% carrageenan into the chamber. The drug concentrations were determined throughout the experiments both in plasma and tissue fluid. Naproxen concentrations (elimination half life of about 72 h in dogs) achieved equilibrium between tissue fluid and plasma concentration. In the case of phenylbutazone, concentration in tissue fluid declined with a definitely longer half life than in plasma and exceeded plasma concentrations after some hours. Flunixin concentrations in plasma and exudate attained equilibrium after 2-5 h. Of clinical importance is the finding that after this time flunixin accumulated in inflammatory exudate and the concentration in exudate exceeded that in plasma two to ten times, then declined with an almost identical half-life. The number of leukocytes in exudate as a measure of inflammation was reduced during 2 days after phenylbutazone, 3-4 days after flunixin and 5-7 days after naproxen. This allows therapy with a lower dosage than calculated on the basis of plasma concentrations in the case of phenylbutazone and flunixin, possibly also with other NSAIDs having a short plasma half life.

A method is described for the qualitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 microliters were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 microgram/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 microgram/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.

Five days after the induction of acute systemic inflammation in greyhounds by intramuscular and subcutaneous injections of Freund's adjuvant, the hepatic concentrations of cytochromes P-450 and b5, the activities of the hepatic microsomal enzymes aniline p-hydroxylase and aminopyrine n-demethylase and the disposition and urinary excretion of phenylbutazone were determined. The mean plasma concentrations of phenylbutazone after intravenous administration were described by the bi-exponential equations: Cp = 144.2e-34.6t + 171.5e-0.104t for five normal greyhounds and Cp = 113.6e-16.13t + 163.1e-0.108t for five febrile greyhounds. The elimination half-lives, total body clearances and apparent volumes of distribution were 6.7 hours, 18.4 ml kg-1 hour-1 and 0.18 litre kg-1, for the normal greyhounds, and 6.4 hours, 19.5 ml kg-1 hour-1 and 0.18 litre kg-1, for the febrile greyhounds. There were no significant differences between the pharmacokinetic parameters describing the distribution and elimination of phenylbutazone, or between the quantities of phenylbutazone, oxyphenbutazone and hydroxyphenylbutazone excreted in the urine. In the febrile greyhounds, there were significant decreases in the hepatic microsomal concentrations of cytochromes P-450 and b5 and in the activities of aniline p-hydroxylase and aminopyrine n-demethylase.

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.

Intramuscular drug administration can lead to more or less extensive muscle damage. The aim of the present study was to show the possibility of quantitating, in vivo and non-invasively, the equivalence of muscle destroyed by the administration of a test drug (phenylbutazone) known for its injurious properties. Creatine kinase (CK) kinetic parameters (clearance, volume of distribution) were measured in 6 sheep after an iv administration of muscle CK homogenate. In the same 6 sheep, CK release after iv and im 8 mg phenylbutazone/kg was measured. The calculated total CK released, based on the CK plasma clearance (0.28 mL/kg/min) and area under the curve of CK activity after im phenylbutazone administration was 191 +/- 140 U/kg. By relating this quantity to that of CK gluteal muscle (5114 +/- 891 U/g), it was calculated that im phenylbutazone administration was able to destroy an equivalence of 2.4 +/- 2.1 g of muscle. For the 2 main sites of im administration (neck and gluteal muscle), general equations are proposed to calculate the equivalence of muscle destroyed in sheep when only plasma CK activity following a test drug administration is available.

The separation of phenylbutazone (PB) and its major metabolite oxyphenbutazone (OPB) using supercritical fluid chromatography (SFC) has been investigated. The separations were studied on octadecylsilane, silica and cyano packed columns with 5% methanol in carbon dioxide as mobile phase and detection at 240 nm. The octadecylsilane column showed the most favourable chromatographic parameters for the analysis of the analytes. Recoveries of PB and OPB from spiked human serum were in the 82-83% range using solid phase extraction on an ODS cartridge. Limits of detection of the SFC assay were 0.1 microgram ml-1 for PB and 1.0 microgram ml-1 for OPB. Accuracy and precision of the method were in the 0.24-4.94% range for PB and OPB. The SFC method was directly comparable to an HPLC assay of the same analytes. The SFC method was also applied to a commercial 100 mg dosage form of PB with good recovery of PB.

Phenylbutazone given during the perisurgical period has been reported to increase the intensity and duration of thiamylal anaesthesia in horses. A possible mechanism of competitive plasma protein binding has been suggested. The purpose of the present study was to experimentally reproduce the phenomenon of increased intensity and/or duration of thiamylal anaesthesia and to determine if there is competitive displacement of plasma protein bound thiamylal by phenylbutazone. Six ponies each received one of three treatments, 11 mg/kg intravenous (i.v.) thiamylal; 8.8 mg/kg i.v. phenylbutazone; and 11 mg/kg i.v. thiamylal with 8.8 mg/kg i.v. phenylbutazone given 9 min later. Thirteen blood samples were collected from 0 time through 600 min following drug administration and plasma drug concentrations quantified by high performance liquid chromatography. The pharmacokinetics of thiamylal and phenylbutazone were best described by three- and two-compartment models, respectively. There were no significant differences in pharmacokinetic parameters for thiamylal in the presence of phenylbutazone. However, there were differences in phenylbutazone pharmacokinetics when preceded by thiamylal administration. Unbound phenylbutazone concentrations were increased at 171, 231 and 351 min when given with thiamylal, accompanied by decreases in per cent bound phenylbutazone (P < 0.05). There were also significant (P < 0.05) changes in per cent plasma protein binding of thiamylal and phenylbutazone between 120 and 360 min, when in combination. No changes in intensity or duration of anaesthesia were observed.

The effect of lead exposure on phenylbutazone kinetics was studied in rats. The biological half-life (t1/2) of phenylbutazone was determined from the plasma level versus time curve in 3 groups of rats given (i) 10 mg/kg lead orally for 8 weeks (ii) 100 mg/kg single oral dose and (iii) no lead, after oral administration of 100 mg/kg phenylbutazone to all rats. The t1/2 of the drug was found to be 33% lower on chronic lead exposure and 46% higher on acute exposure than in unexposed control rats. This variation in the t1/2 values of the two different groups of rats indicates that probably phenylbutazone metabolism varies with the period of lead exposure.

Single doses (2.2 mg/kg of body weight) of phenylbutazone (PBZ) were administered IV to 6 neonatal horses (5 to 17 hours old at time of dosing). Plasma concentrations of PBZ and its metabolite oxyphenbutazone were monitored serially for 120 hours after drug administration. Pharmacokinetic variables were calculated, using 1- and 2-compartment open models. Descriptive equations from the best model for each foal were then used to derive model-independent variables describing PBZ disposition. Median volume of distribution at steady-state was 0.274 L/kg (range, 0.190 to 0.401 L/kg). Median terminal half-life was 7.4 (6.4 to 22.1) hours, and median total plasma clearance of PBZ for foals in this study was 0.018 L/kg/h (range, 0.013 to 0.038 L/kg/h). Volume of distribution was larger, half-life was longer, and total clearance was lower, compared with similar values reported for administration of PBZ to adult horses.