The effect of inflammation on the disposition of phenylbutazone in thoroughbred horses.

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.

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry.

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.

Disposition and urinary excretion of phenylbutazone in normal and febrile greyhounds.

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.

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.

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.

Effects of phenylbutazone and oxyphenbutazone on basic drug detection in high performance thin layer chromatographic systems.

Interference or 'masking' in thin layer chromatography occurs when the presence of one drug on a thin layer plate physically obscures or interferes with the detection of another drug. We investigated the ability of phenylbutazone and oxyphenbutazone to mask or interfere with the detection by high performance thin layer chromatography (HPTLC) of basic drugs used illegally in horse racing. Of fifty-five basic drugs called 'positive' since 1981 by laboratories affiliated with the Association of Official Racing Chemists (AORC), forty did not comigrate with phenylbutazone or oxyphenbutazone and could not, therefore, be masked. When 75 micrograms/ml of oxyphenbutazone was spiked into urine samples, subjected to an extraction procedure for basic drugs, and then run in our routine HPTLC systems, no 'spots' due to oxyphenbutazone appeared. 'Masking' by oxyphenbutazone, therefore, did not and could not occur in our test systems. When phenylbutazone at a concentration of 30 micrograms/ml was spiked into urine samples and run in the routine HPTLC system, phenylbutazone spots were visible under ultraviolet light and after certain specific oversprays were used to visualize basic drugs. These spots, however, did not interfere with routine thin layer testing for basic drugs. It was concluded that phenylbutazone and oxyphenbutazone had no significant ability to interfere with detection of the parent forms of these basic drugs under the conditions described in these experiments.

Plasma and serum concentrations of phenylbutazone and oxyphenbutazone in racing Thoroughbreds 24 hours after treatment with various dosage regimens.

The plasma and serum concentrations of phenylbutazone (PBZ) and oxyphenbutazone were measured in 158 Thoroughbred horses after various doses of PBZ wer given. All horses were competing or training at racetracks in various parts of the country. All horses used in the study had not been given PBZ 24 hours before they were placed on a specific dosage schedule. Samples were collected 24 hours after the last PBZ administration. Four grams of PBZ were given daily by stomach tube, paste, or tablet for 3 days. On day 4, 24 hours before sample collection, an IV dose of 2 g of PBZ was given, regardless of the dose and method of administration. The 24-hour PBZ plasma concentrations were 3.51, 6.13, and 6.40 micrograms/ml, respectively. After 2 g of PBZ was administered IV daily for 4 days, the plasma PBZ concentration was 4.16 g/ml; after a single 2-g IV administration, the serum concentration was 0.87 g/ml. Concentrations of oxyphenbutazone were 3.35 (stomach tube), 4.29 (paste), 3.60 (tablet), 3.65 (4-day IV), and 1.11 g/ml (single IV). A significant relationship was not found between the serum and the urinary concentrations at this 24-hour measurement. Split samples sent to various laboratories confirmed the stability of high-performance liquid chromatography as a method of analysis.

Pharmacokinetics of phenylbutazone in two age groups of ponies: a preliminary study.

A clinical dose rate (4.4 mg/kg bodyweight) of phenylbutazone was administered intravenously and orally to six Welsh mountain ponies to provide data on the pharmacokinetics and bioavailability of the drug. In three, three-year-old ponies, clearance of the drug from plasma after intravenous administration was almost twice as rapid as in three ponies aged eight to 10 years. After oral administration, plasma phenylbutazone levels were greater in the older ponies, the area under the plasma concentration time curve being almost twice as high. This did not result from more efficient absorption but from slower plasma clearance. The fractional absorption of phenylbutazone was similar in young and older ponies, 0.78 and 0.75, respectively. The 24 hour urinary excretion of phenylbutazone and its hydroxylated metabolites, oxyphenbutazone and gamma-hydroxyphenylbutazone, accounted for approximately 25 per cent of the administered intravenous dose in both young and older ponies. The possible fate(s) of the remaining 75 per cent were considered.

Phenylbutazone kinetics and metabolite concentrations in the horse after five days of administration.

Phenylbutazone (PBZ) was administered (8.8 mg/kg of body weight) every 24 hours for 5 consecutive days, orally for the first 4 days and IV on day 5. The half-life (t 1/2) after this daily administration was 6.2 hours and the volume of distribution was 0.152 +/- 0.014 L/kg; the bioavailability after oral administration was 91.8 +/- 2.5%. The plasma concentration of PBZ at experimental hour (EH) 24 (24 hours after the 1st oral dose) was 1.7 +/- 0.39 micrograms/ml and increased to 4.2 +/- 0.29 micrograms/ml at EH 48 (24 hours after the 2nd oral dose). Values at EH 72, 96, and 120 (24 hours after administration of oral doses 3 and 4, and IV dose 5, respectively) were 4.8 +/- 0.62 micrograms/ml, 5.3 +/- 0.84 micrograms/ml, and 4.3 +/- 1.1 micrograms/ml, respectively. Significant increases (P less than 0.05) were measured between EH 24 and 48 with no changes during the subsequent 3 days. Changes in the urinary concentrations of PBZ were not seen over the 5-day dosing period. There were increases in the metabolites oxyphenbutazone and the alpha-alcohol during the 5-day dosing period. There was a delay in the appearance of alpha-alcohol in urine after oral and IV administration.

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and gamma-hydroxy-phenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene--cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a muBondapak C18 column using a mobile phase of methanol--0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5% min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultraviolet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for gamma-hydroxyphenylbutazone was 0.05 microgram/ml. A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.