Oxidized Hyaluronic Acid Hydrogels as a Carrier for Constant-Release Clenbuterol Against High-Fat Diet-Induced Obesity in Mice.

The global obesity population is increasing year-by-year, and the related cost is sharply increasing annually. There are several methods available to combat obesity; however, there is a lack of a single tool that is both safe and efficacious. The use of Clenbuterol in bodybuilding and by professional athletes is controversial owing to its side effects, including hepatotoxicity. This study administered Clenbuterol at a much lower dose than the established safety level, and rather than through oral administration, the treatments were delivered through controlled-release intra-adipose injection. The different dosing and mode of administration will lower the risk of side effects, increase the safety profile, and could facilitate use in the anti-obesity market. A thermo-sensitive hydrogel was used as the carrier uploaded with Clenbuterol to achieve controlled-release. In the in vitro study, the developed new formulae were not cytotoxic to 3T3-L1 cells and could inhibit lipogenesis effectively. In the animal study, the mice were fed a high-fat diet and treated with Clenbuterol by oral administration, or injected with Clenbuterol-modified hyaluronate hydrogel (HAC) regularly. Both groups showed reduction in whole-body, visceral, and gonadal fat contents and body weight. The abdominal fat was analyzed using MRI imaging in adipose mode and water mode. The abdominal fat ratio in the mice treated with normal diet and those given intra-adipose injections with HAC had the lowest value among the test groups. The mice treated with high-fat diet (HFD) showed the highest value of 53.78%. The chronic toxicity in-vivo test proved that controlled-release injections of 2-10 µg Clenbuterol daily were safe, as demonstrated in the blood elements and serological analyses. This study developed a new and promising method for anti-obesity treatment, using a monthly intra-adipose controlled-release injection of HAC. The developed new formulae of Clenbuterol not only effectively decreased body weight and body fat content but also inhibited lipogenesis on the harvested visceral tissue and reduced adipose tissue around the gonadal fat area. The side effects induced by traditional oral administration of Clenbuterol were not observed in this research; this has excellent potential to be a useful tool for future obesity treatment without safety concerns.

Beta-2 Adrenergic and Glucocorticoid Receptor Agonists Modulate Ozone-Induced Pulmonary Protein Leakage and Inflammation in Healthy and Adrenalectomized Rats.

We have shown that acute ozone inhalation activates sympathetic-adrenal-medullary and hypothalamus-pituitary-adrenal stress axes, and adrenalectomy (AD) inhibits ozone-induced lung injury and inflammation. Therefore, we hypothesized that stress hormone receptor agonists (ß2 adrenergic-ß2AR and glucocorticoid-GR) will restore the ozone injury phenotype in AD, while exacerbating effects in sham-surgery (SH) rats. Male Wistar Kyoto rats that underwent SH or AD were treated with vehicles (saline + corn oil) or ß2AR agonist clenbuterol (CLEN, 0.2 mg/kg, i.p.) + GR agonist dexamethasone (DEX, 2 mg/kg, s.c.) for 1 day and immediately prior to each day of exposure to filtered air or ozone (0.8 ppm, 4 h/day for 1 or 2 days). Ozone-induced increases in PenH and peak-expiratory flow were exacerbated in CLEN+DEX-treated SH and AD rats. CLEN+DEX affected breath waveform in all rats. Ozone exposure in vehicle-treated SH rats increased bronchoalveolar lavage fluid (BALF) protein, N-acetyl glucosaminidase activity (macrophage activation), neutrophils, and lung cytokine expression while reducing circulating lymphocyte subpopulations. AD reduced these ozone effects in vehicle-treated rats. At the doses used herein, CLEN+DEX treatment reversed the protection offered by AD and exacerbated most ozone-induced lung effects while diminishing circulating lymphocytes. CLEN+DEX in air-exposed SH rats also induced marked protein leakage and reduced circulating lymphocytes but did not increase BALF neutrophils. In conclusion, circulating stress hormones and their receptors mediate ozone-induced vascular leakage and inflammatory cell trafficking to the lung. Those receiving ß2AR and GR agonists for chronic pulmonary diseases, or with increased circulating stress hormones due to psychosocial stresses, might have altered sensitivity to air pollution.

Clenbuterol Hydrochloride.

Clenbuterol (Broncodil and trade) is a direct-acting sympathomimetic agent with mainly beta-adrenergic activity and a selective action on ß2 receptors (a ß2 agonist). It has properties similar to those of salbutamol. It is used as a bronchodilator in the management of reversible airways obstruction, as in asthma and in certain patients with chronic obstructive pulmonary disease. The uses, applications, and the synthetic pathways of this drug are outlined. Physical characteristics including: ionization constant, solubility, X-ray powder diffraction pattern, thermal methods of analysis, UV spectrum, IR spectrum, mass spectrum are all produced. This profile also includes the monograph of British Pharmacopoeia, together with several reported analytical methods including spectrophotometric, electrochemical, chromatographic, immunochemical methods, and capillary electrophoretic methods. The stability, the pharmacokinetic behavior, and the pharmacology of the drug are also provided.

Skeletal Muscle-specific G Protein-coupled Receptor Kinase 2 Ablation Alters Isolated Skeletal Muscle Mechanics and Enhances Clenbuterol-stimulated Hypertrophy.

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a ß2-adrenergic receptor (ß2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as ß2AR-induced hypertrophy.

Quantification of trantinterol, its two metabolites and their primary conjugated metabolites in human plasma by ultra-high-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.

A highly rapid, selective and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to simultaneously determine trantinterol, its major phase-I metabolites and their primary conjugated metabolites in human plasma. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation. The separation was carried out on an ACQUITY UPLC™ BEH C18 column with methanol/0.2% formic acid (30:70, v/v) as the mobile phase at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in selective reaction monitoring (SRM) mode with the use of an electrospray ionization (ESI) source. The linear calibration curves for trantinterol, tert-butyl hydroxylated trantinterol (tert-OH-trantinterol) and 1-carbonyl trantinterol (trantinterol-COOH) were obtained in the concentration ranges of 0.200-250, 0.108-4.00 and 0.0840-5.02 ng/mL, respectively (r(2)≥0.99). The intra- and inter-day precision (relative standard deviation, RSD) values were less than 13%, and the accuracy (relative error, RE) was within ±9.9%, as determined from quality control (QC) samples for the analytes. The concentrations of conjugated forms of trantinterol and tert-OH- trantinterol in plasma were determined using selective enzyme hydrolysis. The method described herein was fully validated and successfully applied for the pharmacokinetic study of trantinterol in healthy volunteers after oral administration.

An LC-MS/MS method for simultaneous determination of trantinterol and its major metabolite in rat plasma and its application to a comparative pharmacokinetic study.

Trantinterol is a novel ß2-adrenoceptor agonist, currently undergoing clinical trials for the treatment of asthma. We developed and validated an liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of trantinterol and its major metabolite, 1-carbonyl trantinterol (SPFFCOOH), in rat plasma. Aliquots (100µL) of heparinized plasma samples were processed by protein precipitation with acetonitrile. Chromatographic separation used an Acquity UPLC BEH C18 column (2.1mm×50mm, 1.7µm) and acetonitrile-0.1% formic acid (20:80, v/v) as mobile phase, at a flow rate of 0.25mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple-reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The precursor-to-product ion transitions m/z 310.9→m/z 237.9 for trantinterol, m/z 324.9→m/z 251.9 for SPFFCOOH and m/z 368.0→m/z 294.0 for bambuterol (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentration of 0.25-100ng/mL for both trantinterol and SPFFCOOH. The intra- and inter-day precision (relative standard deviations, RSD) values were below 15% and accuracy (relative error, RE) was from -4.3% to 6.6% at all quality control (QC) levels. The method was successfully applied to compare the pharmacokinetics of trantinterol and SPFFCOOH in male and female Wistar rats after a single oral administration of trantinterol.

Pharmacokinetics of trantinterol and its metabolite in healthy elderly and young subjects.

OBJECTIVE: The aim of this study was to investigate and compare the pharmacokinetics of trantinterol and its active amphoteric carboxylic acid metabolite (1-carbonyl trantinterol) between the healthy elderly and young subjects. METHODS: This was a single-center, open-label, parallel-group study completed by 22 healthy subjects (≥65 years (the elderly group); 18-45 years (the young group); 9 males and 2 females per age group) receiving single oral dose of 50 µg trantinterol tablets. Blood samples were taken at intervals up to 48 hours post-dose. RESULTS: In both groups, maximum plasma concentration of trantinterol was researched at 0.9 hours, while the tmax of 1-carbonyl trantinterol differed slightly. Trantinterol Cmax and AUClast were higher in the elderly group than the young group, by 27% (90% CI, 0.95-1.69) and 77% (90% CI, 1.25-2.51), respectively. For 1-carbonyl trantinterol, Cmax, and AUClast were also higher, by 36% (90% CI, 1.04-1.78) and 71% (90% CI, 1.27-2.30), respectively, in the elderly group. The CL/F and V/F of trantinterol and 1-carbonyl trantinterol were significantly lower in the elderly group, while t1/2 of both did not show significant differences. CL/F of trantinterol and 1-carbonyl trantinterol were found to significantly correlate inversely with age, and positively with the baseline creatinine clearance. CONCLUSIONS: A single dose of 50 µg trantinterol was well tolerated. Significant changes in Cmax and AUC of trantinterol and 1-carbony trantinterol were seen in the elderly and may be clinically important.

Determination of trantinterol enantiomers in human plasma by high-performance liquid chromatography - tandem mass spectrometry using vancomycin chiral stationary phase and solid phase extraction and stereoselective pharmacokinetic application.

A sensitive and enantioselective vancomycin chiral stationary phase high-performance liquid chromatography-tandem mass spectrometry method was developed for the determination of trantinterol enantiomers in human plasma. Baseline resolution was achieved using the vancomycin chiral stationary phase known as Chirobiotic V with polar ionic mobile phase consisting of acetonitrile-methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation of trantinterol samples from plasma. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. The calibration curve was linear in a concentration range from 0.0606 to 30.3 ng/mL in plasma, with the lower limit of quantification of 0.0606 ng/mL. The intra- and interday precision (relative standard deviation) values were within 9.7% and the accuracy (relative error) was from -6.6 to 7.2% at all quality control levels. The method was successfully applied to a study of stereoselective pharmacokinetics in human.

Simultaneous Determination of Trantinterol and One of Its Major Metabolites, 1-Carbonyl Trantinterol, in Human Plasma by LC-MS-MS.

A highly selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of trantinterol and one of its major metabolites, 1-carbonyl trantinterol, in human plasma. An Oasis MCX 96-well solid-phase extraction cartridge and a SeQuantTM ZIC(®)-HILIC LC column were used for sample preparation and chromatographic separation, respectively. The analytes were monitored by a QTrap 5500 mass spectrometer with positive electrospray ionization. Multiple reaction monitoring was used for quantification using the precursor to product ion pairs of m/z 311.1 → 237.9 (trantinterol), m/z 325.1 → 251.9 (1-carbonyl trantinterol) and m/z 368.4 → 294.0 (bambuterol as internal standard). The assay had a calibration range from 0.2 to 50 pg/mL and a lower limit of quantification of 0.2 pg/mL for both trantinterol and 1-carbonyl trantinterol. The inter-day and intra-day precisions were <12.0% and the accuracies were within the range of 87.1-111%. The mean recovery ranged from 82.0 to 97.7% and internal standard normalized matrix effect from 0.813 to 0.899. The analytes were stable under all tested conditions. This validated method was successfully applied to a pilot pharmacokinetic study in healthy subjects administered a single 50 µg oral dose.

Comparison of accumulation of clenbuterol and salbutamol residues in animal internal tissues, non-pigmented eyes and hair.

The aim of the study was to compare the accumulation of ß-adrenergic agonist residues clenbuterol (CLB) and salbutamol (SAL) in internal tissues, non-pigmented eyes and hair of laboratory animals repeatedly administered with CLB and SAL during 7 days. Experimental albino guinea pigs (n = 20) were treated with CLB (n = 10) and SAL (n = 10) in anabolic doses of 0.25 and 2.5 mg/kg, whereas the control animal group (n = 10) was left untreated. Methodology validation showed that the ELISA assay to be suitable for ß-agonists' semiquantitative determination. The results revealed a significantly higher (P < 0.05) accumulation potential of CLB in comparison with SAL in all investigated tissues. Despite of their lack of pigmentation and the applied dose, the highest residual CLB concentrations were determined in the eyes of the studied animals, followed by their hair, liver, lungs, kidney, heart and adipose and muscle tissue, whereas residual SAL concentrations found in the eyes and hair of the administered animals did not significantly differ (P > 0.05) from those obtained in their internal tissues.

Enantioselective disposition of clenbuterol in rats.

Clenbuterol is a long-acting ß2-adrenoceptor agonist and bronchodilator that is used for the treatment of asthma, but the desired activities reside almost exclusively in the (-)-R-enantiomer. This study examined enantioselectivity in the disposition of clenbuterol following administration of clenbuterol racemate to rats. Concentrations of clenbuterol enantiomers in plasma, urine and bile were determined by LC-MS/MS assay with a Chirobiotic T column. This method was confirmed to show high sensitivity, specificity and precision, and clenbuterol enantiomers in 0.1 ml volumes of plasma were precisely quantified at concentrations as low as 0.25 ng/ml. The pharmacokinetic profiles of clenbuterol enantiomers following intravenous and intraduodenal administration of clenbuterol racemate (2 mg/kg) in rats were significantly different. The distribution volume of (-)-R-clenbuterol (9.17 l/kg) was significantly higher than that of (+)-S-clenbuterol (4.14 l/kg). The total body clearance of (-)-R-clenbuterol (13.5 ml/min/kg) was significantly higher than that of the (+)-S-enantiomer (11.5 ml/min/kg). An in situ absorption study in jejunal loops showed no difference in the residual amount between the (-)-R- and (+)-S-enantiomers. Urinary clearance was the same for the two enantiomers, but biliary excretion of (-)-R-clenbuterol was higher than that of the (+)-S-enantiomer. The fractions of free (non-protein-bound) (-)-R- and (+)-S-clenbuterol in rat plasma were 48.8% and 33.1%, respectively. These results indicated that there are differences in the distribution and excretion of the clenbuterol enantiomers, and these may be predominantly due to enantioselective protein binding.

Detection, pharmacokinetics and cardiac effects following administration of clenbuterol to exercised horses.

REASONS FOR PERFORMING STUDY: The use of clenbuterol in performance horses necessitates the establishment of appropriate withdrawal times. OBJECTIVES: To describe plasma and urine concentrations of clenbuterol following administration of 2 commonly used dosing regimens to racing fit Thoroughbreds. STUDY DESIGN: Experimental. METHODS: Twenty-two horses received an oral dose of 0.8 µg/kg bwt of clenbuterol twice daily for 30 days. A second group of 6 horses received clenbuterol according to the escalating dose protocol on the manufacturer's label. Blood and urine samples were collected prior to, throughout and at various times up to 35 days post administration of the final dose. Drug concentrations were measured using liquid chromatography-mass spectrometry, and plasma data were analysed using noncompartmental analysis. Behavioural and physiological effects were monitored and heart rate was recorded throughout the course of the study. RESULTS: Clenbuterol plasma concentrations were below the limit of quantification (10 pg/ml) of the assay by Day 4 in all horses receiving the chronic low-dose regimen and by Day 7 in 5 of 6 horses receiving the escalating dosing protocol. Urine clenbuterol concentrations fell below the limit of quantification of the assay between Days 21 and 28 in all 22 horses in the low-dose group and in 5 of 6 of the horses in the escalating dose group. Muscle fasciculations, sweating and transient increases in heart rate were noted in a small number of horses following clenbuterol administration, but tolerance to these effects occurred rapidly. CONCLUSIONS AND POTENTIAL RELEVANCE: Establishment of appropriate withdrawal times for specific racing jurisdictions depends upon the threshold adopted by that specific jurisdiction. This study extends previous studies describing the pharmacokinetics of clenbuterol and describes plasma and urine concentrations following administration of 2 commonly used dosing regimens to racing fit Thoroughbreds, which will allow jurisdictions to establish withdrawal times in order to prevent inadvertent positive regulatory findings.

Bidirectional chiral inversion of trantinterol enantiomers after separate doses to rats.

The chiral inversion and pharmacokinetics of two enantiomers of trantinterol, a new ß2 agonist, were studied in rats dosed (+)- or (-)-trantinterol separately. Plasma concentrations of (+)- and (-)-trantinterol were measured by chiral stationary phase liquid chromatography tandem mass spectroscopy (LC-MS/MS). The apparent inversion ratio was calculated as the ratio of AUC0-t of (-)-trantinterol or (+)-trantinterol inverted from their antipodes to the sum of the AUC0-t of (-)- and (+)-trantinterol. Following single intravenous administration, both given enantiomers declined in similar plasma concentrations, suggesting that the two enantiomers have approximately the same disposition kinetics by the route of intravenous administration. However, after single oral administration, plasma concentrations of uninverted (-)-trantinterol at many timepoints were significantly higher than those of uninverted (+)-trantinterol, suggesting that the two enantiomers undergo apparently different absorption or metabolism after oral administration. Significant bidirectional chiral inversion occurred after intravenous and oral administration of (+)- or (-)-trantinterol. After dosing with optically pure enantiomer, the concentration of the administered enantiomer predominated in vivo. The AUC0-36 of (+)-trantinterol after intravenous and oral dosing of (-)-trantinterol were 16.6 ± 5.2 and 33.3 ± 16%, respectively of those of total [(+) + (-)] trantinterol. The AUC0-36 of (-)-trantinterol after intravenous and oral dosing of (+)-trantinterol were 19.6 ± 8.8 and 37.9 ± 4.5%, respectively, of those of total [(-) + (+)] trantinterol. After intravenous administration of (+)- and (-)-trantinterol the chiral inversion ratios of the two enantiomers were not significantly different and similar results were found for oral administration. The extent of chiral inversion after intravenous administration was apparently lower, indicating that the bidirectional chiral inversion was not only systemic but also presystemic.

An electrochemical biosensor for clenbuterol detection and pharmacokinetics investigation.

Clenbuterol is a member of ß2 adrenergic agonists, which is widely used not only as a food additive for livestocks, but also a kind of stimulant for athletes; however, the abuse of clenbuterol may pose a significant negative impact on human health. Since it is highly required to develop fast, sensitive and cost-effective method to determine clenbuterol level in the suspected urine or blood, we herein have fabricated an electrochemical biosensor for the determination of clenbuterol. Measurement of the species with the proposed biosensor can also have the advantages of simplicity, high sensitivity and selectivity. Moreover, the sensor can be directly used for clenbuterol determination in rat urine. We have further studied the pharmacokinetics of clenbuterol by using this proposed electrochemical biosensor, so a new tool to investigate pharmacokinetic is developed in this work.

Accumulation of ß-agonists clenbuterol and salbutamol in black and white mouse hair.

Hair has been shown to be an excellent site for the accumulation of different drugs including ß-agonists, and therefore, it would be an appropriate matrix for surveillance for the presence of drug residues. The aim of this study was to determine concentrations and to compare accumulation of two different ß-agonists in black and white mice hair by use of ELISA as a screening quantitative method. The study included 200 8-week-old white and black mice. One group of black mice and one group of white mice were treated with clenbuterol in a dose of 2.5 mg/kg body mass per os for 28 days. Other animals were treated in the same way with salbutamol. The highest (±SD) clenbuterol concentration of 631.4 ± 23.5 ng/g in black hair and 228.5 ± 156.2 ng/g in white hair was determined on day 1 of treatment withdrawal. Study results revealed the black-to-white hair ratio of clenbuterol accumulation to be 1:2-1:4 and of salbutamol accumulation 1:1.4. The mean (±SD) salbutamol concentrations determined on day 1 of treatment withdrawal was 23.9 ± 0.9 ng/g and 16.4 ± 1.1 ng/g in black and white hair samples, respectively. The study demonstrated that residues could be determined in hair samples even after a 30-day withdrawal period.

Determination of clenbuterol residues in retinal tissue of food-producing pigs.

The aim of the present study was to assess the persistence of clenbuterol residues in retinal tissue of pigs after repeated administration in a growth-promoting dose, using enzyme-linked immunosorbent assay (ELISA) as a screening method for quantitative determination. A growth-promoting dose of clenbuterol (20 µg/kg body mass per day) was administered orally to the experimental group (n = 6) for 21 days, whereas control animals (n = 3) were left untreated. Clenbuterol-treated pigs were randomly sacrificed (n = 3) on days 0 and 45 of treatment discontinuation, and clenbuterol residues were determined in retinal tissue dissected from the eye. ELISA was found to be acceptable for quantitative determination of clenbuterol in retinal samples because previous method validation yielded mean recovery values of 84.3-96.5% with variation coefficients < 14%. The mean (± SD) retinal clenbuterol concentration was 1874 ± 114 ng/g immediately upon clenbuterol withdrawal (day 0) and 73 ± 4 ng/g on the last day post-withdrawal (day 45). Study results pointed to a very high potential of clenbuterol accumulation in retinal tissue and marked persistence of clenbuterol residues upon anabolic dose administration, suggesting retinal tissue to be a very useful matrix for effective control of residual clenbuterol in food-producing pigs.

Effect of clenbuterol on apoptosis, adipogenesis, and lipolysis in adipocytes

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Clenbuterol, a beta2-adrenergic receptor (Beta2-AR) selective agonist, has been shown to decrease body fat in animals and can induce apoptosis in adipose tissue in mice. We hypothesized that direct actions of a Beta -adrenergic receptor agonist on adipocytes could trigger the observed apoptotic effect. The hypothesis was inspected by investigating the direct effect of clenbuterol on apoptosis,adipogenesis, and lipolysis in vitro using the 3T3-L1 cell line and rat primary adipocytes. Cells were treated with (…) (AU)

Detection of urine and blood clenbuterol following short-term oral administration in the horse.

BACKGROUND AND AIM: The pharmacokinetics of clenbuterol in equine urine and blood was investigated. MATERIAL AND METHODS: Urine and blood samples were collected following 3-day multiple oral administrations. The samples were examined using enzyme-linked immunosorbent assay and further confirmed by solid phase extraction and capillary electrophoresis. RESULTS: Urinary clenbuterol was detectable until day 14 after the last dose. The urinary excretion of clenbuterol was characterized by a biphasic pattern. The half-lives of the bi-exponential elimination (t(1/2alpha) and t(1/2beta)) for urinary clenbuterol were about 12.1 and 48 hours. After a single oral administration (4 microg/kg) of clenbuterol, the half-life of serum clenbuterol was approximately 11.4 hours.

Accumulation of the beta(2)-adrenergic agonist clenbuterol in mouse dark hair.

The aim of the present study was to evaluate the suitability of dark hair as a matrix for determination of the beta(2)-adrenergic agonist clenbuterol residues using previously validated enzyme-linked immunosorbent assay (ELISA) as a screening method for its quantitative determination. The experimental group of mice (n = 60) were treated with two different anabolic dosages of clenbuterol for 15 days, whereas the control group of animals (n = 30) was left completely untreated. Hair samples were collected on days 0, 5, 10, and 15 of treatment. Validation of the ELISA analytical procedure showed good recovery (mean recovery 74%) with an acceptable intra-assay variation in individual measurements for all hair samples to which 5, 10, and 50 ng/g clenbuterol were added (CV < 10%). Low blank levels of clenbuterol (2.4 +/- 0.6 ng/g) were measured in hair of untreated mice, whereas significantly higher clenbuterol concentrations rising proportionally with the time of treatment were recorded in hair of mice treated with lower (6.5 mg/kg body mass) and higher (12.5 mg/kg body mass) dose of clenbuterol. The peak hair concentration of clenbuterol measured on the last day of treatment (day 15) was 1553.9 +/- 140.1 ng/g and 6248.3 +/- 589.4 ng/g in the lower and higher dose group, respectively. Study results clearly indicated dark hair as a pigmented tissue to have a high accumulation potential for clenbuterol residues, thus being the target matrix of choice for detection of clenbuterol abuse as an anabolic in meat production.

Synthesis, structure characterization, and enzyme screening of clenbuterol glucuronides.

Two clenbuterol O-glucuronide diastereomers were synthesized by the Koenigs-Knorr reaction. Structures and glucuronidation sites of the glucuronides were characterized by tandem mass spectrometry and nuclear magnetic resonance spectroscopy. The two diastereomers were used as standard compounds in studies of stereoselective glucuronidation of clenbuterol with liver microsomes from different species and with 15 human recombinant UDP-glucuronosyltransferases. In this study, chemical and enzymatic reactions produced only O-glucuronides of clenbuterol, although on the basis of the chemical structure of the aglycone, both O- and N-glucuronides of clenbuterol could be formed. Differences in the production of diastereomers of clenbuterol glucuronides were observed among liver microsomes from the various animals. Dog and bovine liver microsomes were significantly active, and also stereoselective, each producing only one but a different diastereomer. Liver microsomes from rabbit and rat were also rather actively glucuronidating clenbuterol, but human, pig, and moose liver microsomes produced only minor amounts of glucuronides. Human liver microsomes produced only one clenbuterol glucuronide diastereomer, and the same was true of the human UDP-glucuronosyltransferases that were active (formation of glucuronide: 1A9 > 1A10 >> 1A7). The marked differences in the stereoselective glucuronidation of clenbuterol show that UDP-glucuronosyltransferases in the livers of different animals do not have the same functions, activities, or distribution. This needs to be taken into account, particularly in toxicology testing.