Additional studies on triamcinolone acetonide use and misuse in sports: Elimination profile after intranasal and high-dose intramuscular administrations.

Triamcinolone acetonide (TA) is a glucocorticoid (GC) widely used in sports medicine. GCs are prohibited in sports competitions by oral, intramuscular (IM), intravenous and rectal administrations, and they are allowed by other routes considered of local action such as intranasal administration (INT). We examined the urinary profiles of TA and its metabolites after INT and high-dose IM administrations. We also measured concentrations of TA and cortisol (CORT) in plasma following IM administration. TA was administered to healthy volunteers using INT route (220 µg/day for 3 days, n = 4 males and 4 females) or IM route (single dose of 40 mg, n = 4 males and 4 females and single dose 80 mg, n = 4 males). Urine and plasma samples were collected before and after administration at different time periods, and were analysed by liquid chromatography-tandem mass spectrometry. TA concentrations in urine were constant during 23 days after IM injection (range 1.4-129.0 ng/mL), and were very low after INT administration (range 0.0-3.5 ng/mL). For 6ß-hydroxy-triamcinolone, the main TA metabolite, higher concentrations were detected (0.0-93.7 ng/mL and 15.7-973.9 ng/mL after INT and IM administrations, respectively). On the other hand, TA was detected in all plasma samples collected during 23 days after IM administration (range 0.2-5.7 ng/mL). CORT levels were largely suppressed after IM injection, and were recovered in a dose-dependent manner. In view of the results obtained, we propose a reporting level of 5 ng/mL for TA to distinguish forbidden from allowed TA administrations in sports. We also suggest that other GCs with faster urinary elimination from the body should be considered for IM therapies in out-of-competition rather than TA, in order to reduce the possibility of reporting false adverse analytical findings.

Elimination profile of triamcinolone hexacetonide and its metabolites in human urine and plasma after a single intra-articular administration.

Triamcinolone hexacetonide (THA) is a synthetic glucocorticoid (GC) used by intra-articular (IA) administration. GCs are prohibited in sports competitions by systemic routes, and they are allowed by other routes considered of local action (IA administration, among others). The aim of the present work was to study the metabolic profile of THA in urine and plasma following IA administration. Eight patients (4 males and 4 females) with knee osteoarthritis received an IA dose of THA (40 mg) in the knee joint. Spot urine and plasma samples were collected before injection and at different time periods up to day 23 and 10 post-administration, respectively. The samples were analysed by liquid chromatography-tandem mass spectrometry. Neither THA nor specific THA metabolites were detected in urine. Triamcinolone acetonide (TA) and 6ß-hydroxy-triamcinolone acetonide were the main urinary metabolites. Maximum concentrations wereobtained between 24 and 48 h after administration. Using the reporting level of 30 ng/mL to distinguish allowed from forbidden administrations of GCs, a large number of false adverse analytical findings would be reported up to day 4. On the other hand, TA was detected in all plasma samples collected up to day 10 after administration. THA was also detected in plasma but at lower concentrations. The detection of plasma THA would be an unequivocal proof to demonstrate IA use of THA. A reversible decrease was observed in plasma concentrations of cortisol in some of the patients, indicating a systemic effect of the drug.

Evaluation of the reporting level to detect triamcinolone acetonide misuse in sports.

Triamcinolone acetonide (TA) is prohibited in sport competitions using systemic administrations (e.g., intramuscular, IM), and it is allowed by other routes (e.g., intranasal, IN, or topical, TOP). A reporting level of 30 ng/mL is used to discriminate between forbidden and allowed administrations. We examined urinary profiles of TA metabolites after TOP, IN and IM administrations to evaluate the suitability of the current reporting level and to define the best criteria to discriminate between these administrations. TA was administered to healthy volunteers by different routes: a single IM dose (n=2), IN doses for three days (n=6), and TOP doses for five days followed by a single IM dose (n=8). Urine samples were collected at different time intervals and they were analyzed by liquid chromatography-tandem mass spectrometry to measure TA and eight metabolites. After TOP and IN administrations, concentrations of the metabolites were significantly lower (p<0.05) than after IM administrations. Concentrations of TA after IM administration were lower than 30 ng/mL for all volunteers (range 0.7-29.7 ng/mL), and they were lower than 5 ng/mL after multiple IN or TOP doses (0.1-3.6 ng/mL and 0-1.7 ng/mL, respectively). For 6ß-hydroxy-TA, the main TA metabolite, greater concentrations were obtained: 10.7-469.1 ng/mL, 2.2-90.6 ng/mL and 0-57.2 ng/mL after IM, IN and TOP administrations, respectively. These results suggest that the current reporting level is not suitable to detect forbidden IM administration of TA. A lower concentration of the parent drug or the use of specific metabolites could discriminate IM from TOP or IN administrations.

Positive doping results caused by the single-dose local injection of triamcinolone acetonide.

Triamcinolone acetonide (TA) is classified as an S9 glucocorticoid in the 2014 Prohibited List published by the World Anti-Doping Agency, which caused it to be prohibited in-competition when administered orally, intravenously, intramuscularly or rectally. The Minimum Required Performance Level (MRPL) for the detection and identification of glucocorticoids is 30 ng/mL. Other common local injection routes, such as intraarticular, intratendinous, or intrabursal injection, are not prohibited. The purpose of this study was to analyze the TA and triamcinolone (T) concentrations in urine after a single injection of TA in patients to determine if it would produce a positive result. This study was performed on 40 patients with sports injuries or joint pains. TA was administered locally (doses varied from 12 to 80 mg). Samples were extracted using a solid-phase extraction column, followed by hydrolysis and liquid extraction using diethyl ether. The elution solvents were collected and dried. The dried residue was reconstituted and assayed by performing liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive ionization mode using electrospray ionization and multiple-reaction monitoring as the acquisition mode. The results demonstrated that the concentrations of both TA and T in urine exceeded the MRPL (30 ng/mL) after a single local injection. We obtained positive results for TA in 25 patients, and a positive result for T in one patient. Furthermore, the metabolic situation of TA, a long-acting glucocorticoid, was not an exact linear model. The highest concentrations of TA and T appeared 1-4h after injection. This information could be useful for limiting the misuse of TA by athletes. We suggest that athletes be aware when using TA injections during a competition period and obtain approval for therapeutic use exemption prior to using TA.

Detection and characterization of triamcinolone acetonide metabolites in human urine by liquid chromatography/tandem mass spectrometry after intramuscular administration.

RATIONALE: Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. intramuscular, IM), whereas they are allowed using other ways of administration. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of triamcinolone acetonide (TA), one of the most used glucocorticosteroids, was studied using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). METHODS: Urine samples obtained after IM administration of TA were analyzed using two sample treatments: (a) hydrolysis with ß-glucuronidase enzymes and liquid-liquid extraction under alkaline conditions, and (b) liquid-liquid extraction under acidic conditions. The extracts were analyzed by LC/MS/MS. RESULTS: TA, commercially available metabolites (6ß-hydroxytriamcinolone acetonide, 6ß-OH-TA, and triamcinolone), and their C20-reduced derivatives showed characteristic fragmentation behavior. Besides common product ions and neutral losses for corticosteroids containing fluorine, additional characteristic neutral losses (58 Da, loss of acetone; 44 Da, loss of acetaldehyde) were observed in positive electrospray ionization. Based on that behavior, two complementary approaches were applied to detect TA metabolites: (a) open detection by precursor ion and neutral loss scan methods and (b) targeted detection by selected reaction monitoring methods (SRM) containing theoretical ion transitions of the potential metabolites. Two main compounds, TA and 6ß-OH-TA, and nine minor potential metabolites, were detected by open screening methods. Using SRM, two additional metabolites were detected. Some of the metabolites were characterized using reference standards and, for the rest of metabolites, feasible structures were proposed based on mass spectrometric data. CONCLUSIONS: Metabolites resulting from hydroxylation in C-6, oxidation of the 11-hydroxyl group, reduction of the Δ(4) double bond and oxidation of the side chain were detected. Some of them have not been previously described. Excretion profiles of the detected metabolites after IM administration are presented.

Cushing's syndrome after intra-articular and intradermal administration of triamcinolone acetonide in three pediatric patients.

BACKGROUND: Intra-articular and intradermal steroids are often used for their antiinflammatory effect. There is limited experience with intra-articular and intralesional administration of corticosteroids in the pediatric age group. DESIGN/METHODS: We performed a retrospective chart review of 3 pediatric patients who developed Cushing's syndrome after local administration of triamcinolone acetonide (TCA). RESULTS: Two females 9 and 17 years old, received intra-articular injections of TCA. One patient received multiple injections of TCA into the interphalangeal joints (cumulative dose: 120 mg), whereas the other received a single injection of 40 mg, a dose that is considered to be in the therapeutic range, into the hip joint. The third patient, a 7-year-old female, received multiple intralesional injections of TCA. These patients developed signs and symptoms of hypercortisolism that appeared 4 to 6 weeks after local administration of TCA and lasted for 4 to 6 months after the last dose of TCA. TCA was detectable in the plasma and urine by the liquid chromatography/tandem mass spectrometry method 4 to 5 months after the last dose of the steroid. CONCLUSIONS: We noted evidence for Cushing's syndrome in 3 pediatric patients after intra-articular or intradermal administration of TCA. One of them had received a therapeutic dose of TCA. The possibility of hypothalamic-pituitary-adrenal axis suppression should be considered in patients who have received intra-articular or intradermal steroid injections, particularly in those who have had multiple or relatively high doses.

Analysis of corticosteroids in equine urine by liquid chromatography-mass spectrometry.

A liquid chromatography-mass spectrometry (LC-MS) method for the analysis of corticosteroids in equine urine was developed. Corticosteroid conjugates were hydrolysed with beta-glucuronidase; free and enzyme-released corticosteroids were then extracted from the samples with ethyl acetate followed by a base wash. The isolated corticosteroids were detected by LC-MS and confirmed by LC-MS-MS in the positive atmospheric pressure chemical ionisation mode. Twenty-three corticosteroids (comprising hydrocortisone, deoxycorticosterone and 21 synthetic corticosteroids), each at 5 ng/ml in urine, could easily be analysed in 10 min.

A mass balance study to evaluate the biotransformation and excretion of [14C]-triamcinolone acetonide following oral administration.

The principle objective of this study was to characterize the absorption, metabolism, and disposition of orally administered [14C]-triamcinolone acetonide. Six healthy male subjects each received a single 100 microCi (approximately 800 micrograms) oral dose of [14C]-triamcinolone acetonide. Plasma, urine, and fecal samples were collected at selected times and analyzed for triamcinolone acetonide and [14C]-derived radioactivity. Plasma protein binding of triamcinolone acetonide was also determined. Metabolite profiling and identification were carried out in plasma and excreta. Principle metabolites were assessed for activity with in vitro anti-inflammatory models. [14C]-triamcinolone acetonide was found to be systemically absorbed following oral administration. The presystemic metabolism and clearance of triamcinolone acetonide were extensive, with only a small fraction of the total plasma radioactivity being made up of triamcinolone acetonide. Little to no parent compound was detected in the plasma 24 hours after administration. Most of the urinary and fecally [14C]-derived radioactivity was also excreted within 24 and 72 hours postdose, respectively. Mean plasma protein binding of triamcinolone acetonide was constant, predictable, and a relatively low 68% over a 24-fold range of plasma concentrations. Three principle metabolites of triamcinolone acetonide were profiled in plasma, urine, and feces. These metabolites were identified as 6 beta-hydroxy triamcinolone, 21-carboxylic acid triamcinolone acetonide, and 6 beta-hydroxy-21-oic triamcinolone acetonide. All three metabolites failed to show any concentration-dependent effects in anti-inflammatory models evaluating IL-5-sustained eosinophil viability and IgE-induced basophil histamine release.

Circadian changes in the pharmacokinetics of triamcinolone acetonide in rabbits.

Circadian changes in the pharmacokinetics of triamcinolone acetonide were investigated in rabbits after a single intravenous administration of the drug, 1.0 mg/kg, at 08:00 and 20:00. At 20:00 hrs, the area under the plasma concentration-time curve from time zero to time infinity was significantly greater (46% increase), and this could be due to significantly slower total body clearance (31% decrease) than that at 08:00. The terminal half-life was significantly longer (82%) at 20:00. However, the apparent volume of distribution at steady state and total amount of unchanged drug excreted in 12 hr urine were not significantly different between two groups of rabbits.

Determination of triamcinolone acetonide in equine serum and urine by liquid chromatography-atmospheric pressure ionization mass spectrometry.

Urine and serum samples collected from four standard-bred mares after 30-mg intraarticular administrations of triamcinolone acetonide were analyzed using combined high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry. Maximum triamcinolone acetonide concentrations of 32.3, 14.8, 24.3, and 29.4 ng/mL in the urine and 2.7, 1.9, 2.3, and 2.5 ng/mL in the serum samples were observed. The peak concentrations of the drug were detected approximately 22 h (urine) and 12 h (serum) after administration. The drug elimination profiles for both urine and serum are presented and discussed.

Further evaluation of a new penetration enhancer, HPE-101.

The penetration enhancer, 1-[2-(decylthio)ethyl]azacyclopentan-2-one (HPE-101), significantly enhanced the excretion of topically applied [14C]indomethacin when dissolved in dipropylene glycol, triethylene glycol, diethylene glycol, 1,3-butylene glycol, trimethylene glycol, glycerin, water, silicone or triethanolamine, but not when dissolved in ethanol, isopropyl alcohol, oleyl alcohol, olive oil, peppermint oil, isopropyl myristate or hexylene glycol. HPE-101 significantly enhanced the excrection of [14C]indomethacin, [14C]nicotinic acid, [14C]5-fluorouracil, [3H]oestradiol and [3H]triamcinolone acetonide, but not that of [3H]testosterone. HPE-101 also significantly enhanced the excretion of [14C]indomethacin applied to intact skin of rabbit, guinea-pig and rat, and to tape-stripped skin of guinea-pig, but did not enhance the excretion of [14C]indomethacin applied to tape-stripped skin of rat or rabbit.

Studies of pharmacokinetics and therapeutic effects of glucocorticoids entrapped in liposomes after intraarticular application in healthy rabbits and in rabbits with antigen-induced arthritis.

Dexamethasone palmitate (DMP) entrapped in liposomes of defined sizes was administered intraarticularly in healthy rabbits and in rabbits with antigen-induced arthritis. The pharmacokinetics and therapeutic effect of liposomal DMP were measured and compared with corresponding experiments using microcrystalline triamcinolone acetonide (TAC). The small DMP liposomes (diameter 160 nm) showed a greater decrease in joint circumference than the 3-times-higher dose of microcrystalline TAC. Moreover, about 98% of administered TAC had already disappeared from the joint 6 h after injection, whereas about 36% of liposomal DMP was still measured in synovial fluid and synovium at the same time. Increasing the vesicle diameter from 160 to 750 nm (large liposomes) improved the retention of DMP by a factor of 2.6 within 48 h after injection in healthy rabbits. In addition, none of the liposomal glucocorticoid preparations ever suppressed the endogenous plasma cortisol level, which is in contrast to the suppression measured after administration of the microcrystalline preparation.

The metabolic fate of triamcinolone acetonide in laboratory animals.

The metabolic fate of 9-fluoro-11beta,16alpha,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione cyclic 16,17-acetal with 2(-14)C-acetone, triamcinolone acetonide (TA) was studied in rabbits, dogs, monkeys and rats and found to be qualitatively similar in all species. In the dog, rat and monkey the major excretory route was the feces irrespective of the mode of administration. In the rabbit the excreted radioactivity was equally distributed between urine and feces. The metabolites were isolated by preparative thin layer chromatography, located by autoradiography, eluted and analyzed by MS, IR, UV and NMR. The major metabolites of triamcinolone acetonide (TA) were identified as the C-21 carboxylic acids of TA and of the 6beta hydroxy-TA, (6BETA-OH-TA) and the previously identified (1,2) 6beta-OH-TA. In addition MS and UV data indicate the presence of 9-fluoro-11beta,16alpha, 17-trihydroxy-3,20-dioxo-1,4,6-pregnatrien-21-oic acid cyclic 16,17 acetal with 2(-14)C-acetone.

Penetration, permeation, and absorption of triamcinolone acetonide in normal and psoriatic skin.

Penetration studies of radiolabelled Triamcinolone acetonide from ointment or cream preparations revealed that in cases of normal as well as psoriatic skin 70-90% of the applied substance remains on the surface. Normal horny layer stores up to 30% of the steroid. Nevertheless, a rapid penetration into the living layers of the skin is observed, whereby the epidermal concentrations reach levels between 5-10(-6) and 3-10(-5) M (mol per liter of tissue). The excretion in the urine took more than 72 h after removal of the excess of substance from the skin. In psoriatic skin, the epidermal and dermal concentrations were 3-10 times higher than in normal skin. This increase lies in the same range as the one resulting from removal of the horny layer by stripping prior to the application, as reported earlier.