The potential of polymeric nanoparticles to increase the immunogenicity of the hapten clenbuterol

Micro-and nanoparticles prepared front the biodegradable and biocompatible polymers poly(lactide-co-glycolide) (PLGA) and polymetylmethacrylate (PMMA) have been successfully used as immunopotentiating antigen delivery systems. In our study, this approach was used to improve polyclonal antibody production to clenbuterol (CBL), a model hapten. PLGA and PMMA nanoparticles were loaded with either CBL alone or with a clenbuterol-transferrin conjugate (CBL-Tfn) and administered subcutaneously to mice. PLGA nano-particles were administered with or without the saponin adjuvant Quil A. The anti-CBL titres present in experimental sera were determined by an enzyme immunoassay (ELISA). CBL-Tfn-loaded PLGA nanoparticles co-administered with Quil A had obvious advantages immmunologically over the currently used method of raising antibodies to CBL (the positive control). The combined adjuvanticity of Quil A and PLGA nanoparticles resulted in a positive response in all four of the mice tested and in higher antibody titles than were seen in the positive control group. Furthermore, the sustained release of immunogen from the nanoparticles permitted a reduction in immunizing frequency over the 15-week study period.

Active immunisation of lambs with a monoclonal antibody against clenbuterol

An experiment was undertaken with 50 Texel x Suffolk-Cheviot lambs (54+/-8.8 days of age) to investigate the effects of active immunisation with a murine monoclonal antibody against clenburerol on growth and carcass characteristics. Animals on treatments 1 and 2 each received 0.1 mg of clenbuterol antibody while animals on treatments 3 and 4 received 0.1 mg of antibody encapsulated within a synthetic polymer. Diethylaminoethyl (DEAE)-dextran was used as the adjuvant in treatments 1 and 3 and saponin in treatments 2 and 4. Control animals were immunised with saponin only. Four immunisations were given at 4-week intervals. Animals were slaughtered 3 weeks after the final immunisation. Each vaccine evoked a similar level of antibody response while the control group showed no titres. Lamb growth rate did not vary significantly between the vaccinated and control groups. Dressing proportion was higher (P

RESIDUES OF CLENBUTEROL IN CATTLE RECEIVING THERAPEUTIC DOSES - IMPLICATIONS FOR DIFFERENTIATING BETWEEN LEGAL AND ILLEGAL USE

Clenbuterol (CBL) can be used legally in the treatment of respiratory diseases and illegally as a growth promoter in animals, Liver and eye have previously been shown to be effective matrices for the detection of residual concentrations of the drug.

PATHOLOGY AND RESIDUES IN VEAL CALVES TREATED EXPERIMENTALLY WITH CLENBUTEROL

Six veal calves were medicated with clenbuterol at 20 mu g kg bodyweightl day(-1) for 42 days before they were slaughtered, to evaluate the lesions and residues in target organs. Compared with six unmedicated calves the most noticeable changes were tracheal dilatation, decreased uterine weight, slight mucous hypersecretion in the uterus and vagina and depletion of liver glycogen. The highest concentrations of clenbuterol (62 to 128 ng/g(-1)) were recorded in the choroid/retina, and the aqueous humour had the lowest concentration (0.5 to 2.4 ng ml(-1)). The residue concentrations were higher than the maximum residue level set for clenbuterol (0.5 ng g(-1))

OBSERVATIONS ON THE EFFECTS OF LONG-TERM WITHDRAWAL ON CARCASS COMPOSITION AND RESIDUE CONCENTRATIONS IN CLENBUTEROL-MEDICATED CATTLE

The detection of the illegal use of clenbuterol (CBL) as a growth promoter has relied on detecting residual concentrations of the drug in body fluids or tissues. Analysis of retinal extracts has recently been shown to considerably extend the detection period following withdrawal. The withdrawal periods required to eliminate residues from the liver and retina were investigated by medicating 20 cattle with CBL for 30 days; 6 control animals remained unmedicated. Residual concentrations were monitored throughout this period and for the subsequent 140 days. Concurrent changes in muscle areas and backfat thicknesses were recorded by ultrasound.

EFFECTIVE LABORATORY MONITORING FOR THE ABUSE OF THE BETA-AGONIST CLENBUTEROL IN CATTLE

The use of the beta-agonist clenbuterol (CBL) as a growth promoter has been outlawed in European meat production. The detection of its illegal use is dependent on CBL residues persisting in animal tissues for longer than the withdrawal times given by abusers. A comparison of urine, bile and liver matrices indicated that analysis of the liver offered the best possibility for CBL detection. However, an experimental study showed that CBL detection following withdrawal could be further extended (up to 56 d) if the retina was used as the target tissue. Analysis of 703 retina and liver samples from cattle suspected of CBL medication revealed that 96 cattle had CBL residues present in their retinas, only 46 of these were liver positive. There were no instances of liver CBL residues being detected without the associated retina also being positive.

RESIDUES OF THE BETA-AGONIST CLENBUTEROL IN TISSUES OF MEDICATED FARM-ANIMALS

Reports of the illegal use of clenbuterol as a growth promotant prompted the development of a competitive enzyme immunoassay for this drug. This procedure was utilized to study the elimination of clenbuterol from tissues in sheep medicated with both therapeutic and growth-promoting doses of the drug. The results indicated that prior to removal of medication clenbuterol was widely distributed throughout the animal tissues. However as the withdrawal periods increased fluid targets such as urine and bile became less effective at detecting clenbuterol usage. At both therapeutic and growth-enhancing concentrations of clenbuterol liver samples remained positive up to the maximum withdrawal time given in this experiment (15 days). Concentrations of clenbuterol likely to cause food poisoning (> 100 ng/g) were only detected in liver samples taken prior to the removal of medication. The highest recorded concentration of clenbuterol in muscle was 22.5 ng/g.

DETECTION OF CLENBUTEROL RESIDUES IN BOVINE LIVER, MUSCLE, RETINA AND URINE USING GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

A gas chromatographic/mass spectrometric method is described for the detection of clenbuterol residues in liver, muscle, urine and retina. Tissue samples are first digested using protease and any clenbuterol present is extracted using a simple liquid/liquid extraction procedure. The dried extracts are then derivatized using methylboronic acid and the derivatives are subjected to gas chromatography/mass spectrometry on a magnetic sector instrument. The detection limit of the assay is 0.05 ng g-1 clenbuterol in liver, muscle or urine using a 10 g sample size, and 4 ng g-1 in retina using a 0.5 g sample size. The assay is made very specific by using selected ion monitoring of three ions at a resolution of 3500 and by ion ratio measurements. The precision and reproducibility of the assay are enhanced by the use of a deuterated internal standard, with a typical coefficient of variation of 3%.

Monitoring for clenbuterol abuse in N Ireland 1989-1994

Laboratory testing in N. Ireland for the illegal growth promoting agent, clenbuterol (CBL), is centralized at the Veterinary Sciences Division, Belfast. During the past 6 years a variety of testing schemes have evolved to determine the level of abuse of this drug in the local meat industry.

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Effect of acute administration of Clenbuterol on athletic performance in horses

The aim of the study was to determine the effect of clenbuterol on the anaerobic-threshold of horses on a tread-mill with increasing physical stress, measuring heart rate (HR) and blood levels of lactate, glucose, and insulin. Twelve Arabian horses. were submitted to two physical tests separated by a 10-day interval. Clenbuterol (CL) at 0.8 mu g/kg or saline (control-C) was administered intravenously 30 minutes, before the test. The treadmill exercise test consisted of an initial warmup followed by a gradually increasing effort. There was no statistical difference in either V-2 or V-4 (velocity at which plasma lactate concentration reached 4 and 2 mmol/L, respectively) between the two-experimental groups. For the CL group, V-200, V-180, V-160, and V-140 (velocity at which the rate heart is 140, 160, 180, and 200 beats/minute, respectively) decreased significantly. At rest as well as times 4, 6, and 10 minutes, insulin levels were higher in the group that recieved clenbuterol (P < .05). Contrary to what was expected, apparently, there was no improvement in aerobic metabolism in animals when given a therapeutic dose of the bronchodilator. The elevated heart rate observed could have been attributable to the stimulation of cardiac beta(1) adrenoceptors and the increased insulin levels to the stimulation of pancreatic beta(2) receptors.

Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt

Goncalves DA, Silveira WA, Lira EC, Gra a FA, Paula-Gomes S, Zanon NM, Kettelhut IC, Navegantes LC. Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt. Am J Physiol Endocrinol Metab 302: E123-E133, 2012. First published September 27, 2011; doi:10.1152/ajpendo.00188.2011.-Although it is well known that administration of the selective beta(2)-adrenergic agonist clenbuterol (CB) protects muscle following denervation (DEN), the underlying molecular mechanism remains unclear. We report that in vivo treatment with CB (3 mg/kg sc) for 3 days induces antiproteolytic effects in normal and denervated rat soleus muscle via distinct mechanisms. In normal soleus muscle, CB treatment stimulates protein synthesis, inhibits Ca(2+)-dependent proteolysis, and increases the levels of calpastatin protein. On the other hand, the administration of CB to DEN rats ameliorates the loss of muscle mass, enhances the rate of protein synthesis, attenuates hyperactivation of proteasomal and lysosomal proteolysis, and suppresses the transcription of the lysosomal protease cathepsin L and of atrogin-1/MAFbx and MuRF1, two ubiquitin (Ub) ligases involved in muscle atrophy. These effects were not associated with alterations in either IGF-I content or Akt phosphorylation levels. In isolated muscles, CB (10(-6) M) treatment significantly attenuated DEN-induced overall proteolysis and upregulation in the mRNA levels of the Ub ligases. Similar responses were observed in denervated muscles exposed to 6-BNZ-cAMP (500 mu M), a PKA activator. The in vitro addition of triciribine (10 mu M), a selective Akt inhibitor, did not block the inhibitory effects of CB on proteolysis and Ub ligase mRNA levels. These data indicate that short-term treatment with CB mitigates DEN-induced atrophy of the soleus muscle through the stimulation of protein synthesis, downregulation of cathepsin L and Ub ligases, and consequent inhibition of lysosomal and proteasomal activities and that these effects are independent of Akt and possibly mediated by the cAMP/PKA signaling pathway.

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Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease.

Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation-independent mannose-6-phosphate receptor (CI-MPR)-mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA-knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA-KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI-MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β2-agonist treatment enhanced CI-MPR-mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.