Pharmacokinetics of intravenous flumetasone and effects on plasma hydrocortisone concentrations and inflammatory mediators in the horse.

BACKGROUND: Flumetasone is a potent corticosteroid reportedly used in horses to decrease inflammation associated with strenuous exercise. There are currently no reports describing the use of this drug in horses. OBJECTIVES: To describe the pharmacokinetics and effects on cortisol and eicosanoid concentrations, following administration of flumetasone to exercised horses. STUDY DESIGN: Parallel design. METHODS: Twelve exercised horses received a single i.v. administration of 5 mg of flumetasone. Blood and urine samples were collected before and for 72 h post-drug administration for determination of flumetasone and cortisol concentrations. Whole blood samples were collected at various time and challenged with lipopolysaccharide, calcium ionophore or methanol to induce ex vivo synthesis of eicosanoids. Concentrations of flumetasone, cortisol and eicosanoids were measured using LC-MS/MS and pharmacokinetic/pharmacodynamic analysis performed. RESULTS: Flumetasone was detected for 23.5 ± 1.73 h in blood. The volume of distribution at steady state, systemic clearance and elimination half-life was 5.90 ± 0.200 L/kg, 30.7 ± 0.166 mL/min/kg and 4.84 ± 0.83 h respectively. Cortisol concentrations were still suppressed at last time point collected (72 h). For cortisol, Kin , Kout and the t1/2out were 30.3 ± 1.56 ng/mL × h, 0.331 ± 0.02 1/h and 2.1 h respectively. Stimulation with lipopolysaccharide resulted in a decrease in TXB2 , PGF2 , LTB4 , 15-HETE and 5-HETE for up to 72 h and PGE2 for 24 h post-flumetasone administration. Stimulation of whole blood with calcium ionophore resulted in a decrease in LTB4 for up to 6 h and 15-HETE at 8 h. MAIN LIMITATIONS: Lack of sample collection for determination of biomarker concentrations beyond 72 h and the use of a single sample for determination of baseline cortisol concentrations. CONCLUSIONS: Flumetasone is rapidly cleared from blood following administration to horses. It is a potent anti-inflammatory with prolonged effects on production of cortisol and other inflammatory mediators.

High throughput HPLC-ESI-MS method for the quantitation of dexamethasone in blood plasma.

Dexamethasone is a synthetic glucocorticoid with potent anti-inflammatory properties. However, its administration causes significant side effects, specially in long-term therapy. A new approach for limiting adverse effects consists in the slow and constant deliver of this drug, using dexamethasone-21-phosphate-loaded erythrocytes (RBC) as circulating bioreactors converting the non-diffusible dexamethasone-21-phosphate into the diffusible dexamethasone. In order to evaluate the real possibility to use this new method of administration, a simple, cheap and rapid assay was set to manage a large number of samples originating from clinical studies. Due to the sample complexity and analite polarity, electrospray mass spectrometry (MS) is the most powerful technique to achieve qualitative and quantitative data. In order to overcome the complex, time-consuming and expensive LC-MS/MS methods reported in the literature in the present work a standard fluxes HPLC-ESI-MS method was set up for quantitative evaluation of dexamethasone. Thanks to the extraction ion chromatogram (XIC) feature of the software, it was possible to obtain sharp profiles for dexamethasone (DXM) and for the employed internal standard (IS) flumethasone (FM), in spite of the extremely complicated chromatogram obtained after HPLC separation of acetonitrile extracted plasma sample, thus avoiding the use of the expensive deuterated internal standard. This enabled us to obtain a linear response curve, allowing the quantification of DXM from blood samples at the picomoles level.

Determination of flumethasone in calf urine and serum by liquid chromatography-tandem mass spectrometry.

Corticosteroids can be illegally administered to cattle as growth promoting agents to improve meat production. We developed a liquid chromatography-atmospheric pressure ionization mass spectrometry-mass spectrometry (LC-MS-MS) method able to identify and quantify flumethasone, one of the most potent fluorinated synthetic corticosteroid, in serum and urine from treated calves. The analyte was purified from urine (conjugated and free, following enzymatic hydrolysis) and from serum by C18 solid-phase and liquid-liquid extractions, then analyzed by LC-MS-MS monitoring the product ions of an abundant precursor (SRM in negative ionization mode). Results on flumethasone residues in biological fluids in three calves treated at different levels are presented. This method allowed the detection of flumethasone in bovine urine and serum at the 30-pg/ml level.

Use of enzyme immunoassay and reverse-phase high-performance liquid chromatography to detect and confirm identity of dexamethasone in equine blood.

An enzyme immunoassay (EIA) was developed for detection of dexamethasone in equine blood. Dexamethasone 21-hemisuccinate-bovine serum albumin was used for immunization of rabbits, and prednisolone 21-hemisuccinate-horseradish peroxidase was used as enzyme conjugate. The assay had sensitivity in the low-picogram range (detection limit, 0.3 pg/well, 50% inhibition of binding at 4.5 +/- 0.7 pg/well). Apart from cortisol, which was recognized by the antiserum at concentration > 8.5 ng/ml, the dexamethasone antiserum failed to interfere with endogenous steroids, but cross-reacted with triamcinolone, flumethasone, and betamethasone. Thus, the antiserum was used to perform simultaneous screening for these synthetic glucocorticoids and to confirm their identity by combining reverse-phase high-performance liquid chromatography (RP-HPLC) and EIA. The immunoreactivity obtained by direct serum measurements was characterized by means of 2 independent RP-HPLC systems. Serum extracts were submitted to RP-HPLC systems I and II, and the fractions were tested by EIA. Immunoreactive peaks were identified by comparing their retention time with that of the standard glucocorticoids used for calibration. Coinjection of an internal standard (methylprednisolone) in RP-HPLC system II yielded reproducible relative retention times. The effectiveness of the test system was evaluated, using blood from a horse treated with commonly used veterinary preparations of dexamethasone. Administration of the free alcohol of dexamethasone and of dexamethasone 21-trioxaundecanoate, both given IV, was detected, and the identity of each was confirmed for up to 48 hours. Intramuscular administration of dexamethasone 21-isonicotinate was continued for at least 14 days after injection of a therapeutic dose.(ABSTRACT TRUNCATED AT 250 WORDS)

[Detection of dexamethasone in horses]. / Nachweis von Dexamethason im Pferd.

Due to their marked antiinflammatory effect, synthetic corticosteroids are used to mask illness, especially lameness in horses. The detection of these drugs in equine body fluids requires accurate methods, particularly where misuse of corticosteroids is suspected. Gas chromatography/mass spectrometry (GC/MS) is well established as a reliable technique for the identification of drugs in biological fluids. Using GC/MS, we determined dexamethasone levels in horse urine and serum after intravenous application of a therapeutic dose. Dexamethasone was detectable, in serum for up to six hours, and in urine for up to 32 hours, after its administration. These findings indicate that serum measurements are unreliable for the detection of corticosteroid abuse, and demonstrate urine to be a more suitable body fluid for investigation. Nevertheless, it should be emphasized that, regardless of the technique employed, the clinical effects of dexamethasone last longer than 32 hours; thus, failure to detect dexamethasone does not disprove corticosteroid abuse.

The administration of flumethasone, by three different routes, its measurement in the plasma and some effects on wool growth in Merino wethers.

Flumethasone was given to Merino wethers weighing 30-50 kg at rates of 0.62-1.35 mg/kg(0.75) by intravenous (experiments 1 and 2), intraruminal (experiment 4) and subcutaneous (experiment 5) routes over 8 days. In experiment 3, 1.2 mg flumethasone/kg(0.75) was given intravenously over 4, 5 or 6 days. The plasma concentration profiles showed concentrations in the order: intravenous greater than subcutaneous greater than intraruminal. Plasma concentration patterns usually were highest during the first 48 h of infusion followed by relatively stable values. This last feature was not evident in experiments when the rate of hormone infusion was increased. Estimates of the metabolic clearance rates for flumethasone in experiments 1, 2 and 5 were 200-700 ml/min during the equilibrium concentration periods. The effects of flumethasone on some aspects of wool growth revealed interactions between the routes of administration, the period of dosage and the rate of wool growth in the recipients. In experiments 1 and 2 intravenous infusion of 1.20-1.33 mg flumethasone/kg(0.75) caused the shedding of all wool fibres about 30 days after treatment. Some effects of dosing sheep with flumethasone at a time when wool growth was decreasing were also observed in experiment 2. Flumethasone given at a rate of 1.2 mg/kg(0.75) over 4, 5 or 6 days caused the shedding of only some wool fibres which were firmly retained on the sheep by the continuous fibres. Intraruminal and subcutaneous infusions of 0.62-1.35 mg flumethasone/kg(0.75) had similar results to the last in the majority of animals although in a few cases no discontinuity of wool fibres was observed. Recovery in wool growth was observed after treatment. Animals regained their pretreatment wool growth in experiments 1, 4 and 5 by 60 days after treatment and probably equalled at that time wool growth in controls. Recovery was retarded in some individuals in experiment 2 and in some groups in experiment 3. In experiment 1, 21 days wool growth was estimated to have been lost. Some aspects of complete versus partial shedding of wool fibres are discussed particularly with reference to wool harvesting. Some similarities in the appearance of fleeces of steroid-treated sheep and naturally shedding animals are also discussed. In some experiments, particularly when the infusion rate of flumethasone was increased (experiment 3), the sheep showed temporary but significant feed refusals during, but more commonly after, treatment. Speculative discussion as to the metabolic causes of this response is included.