A quantitative approach to analysing cortisol response in the horse.

The cortisol response to glucocorticoid intervention has, in spite of several studies in horses, not been fully characterized with regard to the determinants of onset, intensity and duration of response. Therefore, dexamethasone and cortisol response data were collected in a study applying a constant rate infusion regimen of dexamethasone (0.17, 1.7 and 17 µg/kg) to six Standardbreds. Plasma was analysed for dexamethasone and cortisol concentrations using UHPLC-MS/MS. Dexamethasone displayed linear kinetics within the concentration range studied. A turnover model of oscillatory behaviour accurately mimicked cortisol data. The mean baseline concentration range was 34-57 µg/L, the fractional turnover rate 0.47-1.5 1/h, the amplitude parameter 6.8-24 µg/L, the maximum inhibitory capacity 0.77-0.97, the drug potency 6-65 ng/L and the sigmoidicity factor 0.7-30. This analysis provided a better understanding of the time course of the cortisol response in horses. This includes baseline variability within and between horses and determinants of the equilibrium concentration-response relationship. The analysis also challenged a protocol for a dexamethasone suppression test design and indicated future improvement to increase the predictability of the test.

Plasma concentration-dependent suppression of endogenous hydrocortisone in the horse after intramuscular administration of dexamethasone-21-isonicotinate.

Detection times and screening limits (SL) are methods used to ensure that the performance of horses in equestrian sports is not altered by drugs. Drug concentration-response relationship and knowledge of concentration-time profiles in both plasma and urine are required. In this study, dexamethasone plasma and urine concentration-time profiles were investigated. Endogenous hydrocortisone plasma concentrations and their relationship to dexamethasone plasma concentrations were also explored. A single dose of dexamethasone-21-isonicotinate suspension (0.03 mg/kg) was administered intramuscularly to six horses. Plasma was analysed for dexamethasone and hydrocortisone and urine for dexamethasone, using UPLC-MS/MS. Dexamethasone was quantifiable in plasma for 8.3 ± 2.9 days (LLOQ: 0.025 µg/L) and in urine for 9.8 ± 3.1 days (LLOQ: 0.15 µg/L). Maximum observed dexamethasone concentration in plasma was 0.61 ± 0.12 µg/L and in urine 4.2 ± 0.9 µg/L. Terminal plasma half-life was 38.7 ± 19 h. Hydrocortisone was significantly suppressed for 140 h. The plasma half-life of hydrocortisone was 2.7 ± 1.3 h. Dexamethasone potency, efficacy and sigmoidicity factor for hydrocortisone suppression were 0.06 ± 0.04 µg/L, 0.95 ± 0.04 and 6.2 ± 4.6, respectively. Hydrocortisone suppression relates to the plasma concentration of dexamethasone. Thus, determination of irrelevant plasma concentrations and SL is possible. Future research will determine whether hydrocortisone suppression can be used as a biomarker of the clinical effect of dexamethasone.

Methadone in healthy goats - pharmacokinetics, behaviour and blood pressure.

The pharmacokinetics and effects of the opioid methadone on behaviour, arterial blood pressure, heart rate and haematocrit were studied in goats. Two goats received methadone (0.2mg/kg) intravenously and the terminal half-life was 88 and 91 min, the volume of distribution 8.4 and 6.1L/kg, and clearance 86 and 123 mL/min/kg. In a crossover study eight goats received methadone (0.6 mg/kg) or 0.15M NaCl subcutaneously (SC). After SC administration bioavailability was complete and the terminal half-life was 215 ± 84 min (mean ± SD), Tmax 31 ± 15 min and Cmax 45 ±11 ng/mL. Blood pressure and haematocrit increased while heart rate did not change. The goats did not ruminate and they climbed, scratched, gnawed and showed tail-flicking after SC methadone in contrast to NaCl administration. The use of methadone in goats may be restricted due to the inhibition of rumination and the rather short half-life.

Pharmacokinetics of meloxicam in adult goats and its analgesic effect in disbudded kids.

The pharmacokinetics and analgesic effect of the nonsteroidal anti-inflammatory drug meloxicam (0.5 mg/kg) in goats were investigated. In a randomized, cross-over design the pharmacokinetic parameters were investigated in adult goats (n = 8) after single intravenous and oral administration. The analgesic effect was evaluated in kids using a randomized, placebo controlled and blinded protocol. Kids received meloxicam (n = 6) once daily and their siblings (n = 5) got isotonic NaCl intramuscularly while still anaesthetized after cautery disbudding and injections were repeated on three consecutive days. In the adult goats after intravenous administration the terminal half-life was 10.9 ± 1.7 h, steady-state volume of distribution was 0.245 ± 0.06 L/kg, and total body clearance was 17.9 ± 4.3 mL/h/kg. After oral administration bioavailability was 79 ± 19%, C(max) was 736 ± 184 ng/mL, T(max) was 15 ±5 h, although the terminal half-life was similar to the intravenous value, 11.8 ± 1.7 h. Signs of pain using a visual analogue scale were smaller in kids treated with meloxicam compared with kids treated with placebo on the first day after disbudding, but subsequently no difference in pain was noticeable. Plasma cortisol and glucose concentrations did not differ between the two groups.

Clinical signs and etiology of adverse reactions to procaine benzylpenicillin and sodium/potassium benzylpenicillin in horses.

Case reports of 59 horses reacting adversely to procaine benzylpenicillin or to sodium or potassium benzylpenicillin in Sweden in 2003-2005 were obtained through contacts with horse-owners. For the assessment of the reports, various parameters were evaluated, such as the times to the reactions, information on previous penicillin treatment, the clinical signs and the actions taken in the reacting horses. Among the reports, two horses had received sodium or potassium benzylpenicillin intravenously, whereas the remaining 57 horses had been treated with procaine benzylpenicillin intramuscularly. Allergy may underlie the adverse reactions in the horses given sodium and potassium benzylpenicillin, and in a few of the horses given procaine benzylpenicillin. However, in most horses in the latter group, the clinical signs may be due to the toxic effects of procaine. In these horses, the dominating clinical signs were locomotor and behavioral changes. Some risk factors may enhance the probability that horses react to procaine. One is repeated injections, which increase the likelihood of intravascular administration and also may increase the sensitivity to procaine due to neuronal sensitization (kindling). Procaine is rapidly hydrolyzed by plasma esterases to nontoxic metabolites. When high amounts of procaine enter the circulation, the hydrolyzing capacity may be exceeded and toxicity occurs. Analyses of plasma esterases from reacting horses showed lower activity than in nonreacting control horses. Low esterase activity may increase the possibility of procaine toxicity and constitute another risk factor.

Cetirizine in horses: pharmacokinetics and effect of ivermectin pretreatment.

The pharmacokinetics of the histamine H(1)-antagonist cetirizine and the effects of pretreatment with the antiparasitic macrocyclic lactone ivermectin on the pharmacokinetics of cetirizine were studied in horses. After oral administration of cetirizine at 0.2 mg/kg bw, the mean terminal half-life was 3.4 h (range 2.9-3.7 h) and the maximal plasma concentration 132 ng/mL (101-196 ng/mL). The time to reach maximal plasma concentration was 0.7 h (0.5-0.8 h). Ivermectin (0.2 mg/kg bw) given orally 1.5 h before cetirizine did not affect its pharmacokinetics. However, ivermectin pretreatment 12 h before cetirizine increased the area under the plasma concentration-time curve by 60%. The maximal plasma concentration, terminal half-life and mean residence time also increased significantly following the 12 h pretreatment. Ivermectin is an inhibitor of P-glycoprotein, which is a major drug efflux transporter in cellular membranes at various sites. The elevated plasma levels of cetirizine following the pretreatment with ivermectin may mainly be due to decreased renal secretion, related to inhibition of the P-glycoprotein in the proximal tubular cells of the kidney. The pharmacokinetic properties of cetirizine have characteristics which are suitable for an antihistamine, and this substance may be a useful drug in horses.

Clinical pharmacology of buprenorphine in healthy, lactating goats.

The pharmacokinetics and the effects of the opioid buprenorphine on behavior, cardiovascular parameters, plasma concentrations of cortisol and vasopressin were studied in the goat. After intravenous injection at a dosage of 0.02 mg/kg bw, the terminal half-life was 73.8+/-19.9 min (mean+/-SD), the apparent volume of distribution 5.22+/-1.01 L/kg, and total body clearance 79.1+/-18.5 mL/min/kg. After intramuscular administration of buprenorphine at the same dosage, bioavailability was complete and clearance was 54.7+/-16.6 mL/min/kg. Heart rate, blood pressure and concentrations of cortisol and vasopressin in plasma increased after drug administration. The goats became agitated and stopped ruminating. The effects were more pronounced the first time the animals received the drug, especially the influence on the hormone levels. The concentrations of cortisol and vasopressin in plasma remained unaffected after the second dose despite a wash-out period of 3-6 weeks. Buprenorphine may be an unsuitable drug in goats because of the profound inhibition of rumination and the agitation it causes. The short half-life of buprenorphine may limit its use if long-term analgesia is required but be advantageous if a short acting drug is desirable.

Fexofenadine in horses: pharmacokinetics, pharmacodynamics and effect of ivermectin pretreatment.

The pharmacokinetics and the effects on inhibition of histamine-induced cutaneous wheal formation of the histamine H1-antagonist fexofenadine were studied in horse. The effect of ivermectin pretreatment on the pharmacokinetics of fexofenadine was also examined. After intravenous infusion of fexofenadine at 0.7 mg/kg bw the mean terminal half-life was 2.4 h (range: 2.0-2.7 h), the apparent volume of distribution 0.8 L/kg (0.5-0.9 L/kg), and the total body clearance 0.8 L/h/kg (0.6-1.2 L/h/kg). After oral administration of fexofenadine at 10 mg/kg bw bioavailability was 2.6% (1.9-2.9%). Ivermectin pretreatment (0.2 mg/kg, p.o.) 12 h before oral fexofenadine decreased the bioavailability to 1.5% (1.4-2.1%). In addition, the area under the plasma concentration-time curve decreased 27%. Ivermectin did not affect the pharmacokinetics of i.v. administered fexofenadine. Ivermectin may influence fexofenadine absorption by interfering in intestinal efflux and influx pumps, such as P-glycoprotein and the organic anion transport polypeptide family. Oral and i.v. fexofenadine significantly decreased histamine-induced wheal formation, with a maximal duration of 6 h. A pharmacokinetic/pharmacodynamic link model indicated that fexofenadine in horse has antihistaminic effects at low plasma concentrations (EC50 = 16 ng/mL). However, oral treatments of horses with fexofenadine may not be suitable due to the low bioavailability.

Pharmacokinetics and pharmacodynamics of clemastine in healthy horses.

Clemastine is an H1 antagonist used in certain allergic disorders in humans and tentatively also in horses, although the pharmacology of the drug in this species has not yet been investigated. In the present study we determined basic pharmacokinetic parameters and compared the effect of the drug measured as inhibition of histamine-induced cutaneous wheal formation in six horses. The most prominent feature of drug disposition after intravenous dose of 50 microg/kg bw was a very rapid initial decline in plasma concentration, followed by a terminal phase with a half-life of 5.4 h. The volume of distribution was large, Vss = 3.8 L/kg, and the total body clearance 0.79 L/h kg. Notably, oral bioavailability was only 3.4%. There was a strong relationship between plasma concentrations and effect. The effect maximum (measured as reduction in histamine-induced cutaneous wheal formation) was 65% (compared with controls where saline was injected) and the effect duration after i.v. dose was approximately 5 h. The effect after oral dose of 200 microg/kg was minor. The results indicate that clemastine is not appropriate for oral administration to horses because of low bioavailability. When using repeated i.v. administration, the drug has to be administered at least three to four times daily to maintain therapeutic plasma concentrations because of the short half-life. However, if sufficient plasma concentrations are maintained the drug is efficacious in reducing histamine-induced wheal formations.

Muscarinic receptors in equine airways.

The distribution of muscarinic receptors in equine airways was investigated using autoradiography. Frozen sections of tissue from six different levels in the bronchial tree, from the trachea to the distal bronchioles, were incubated in vitro with 1.5 nmol/L of the muscarinic receptor antagonist 1-[N-methyl-3H]scopolamine methyl chloride (3H-NMS). In addition, the subtype pattern of muscarinic receptors was investigated in equine tracheal smooth muscle using radioligand binding with methoctramine, tripinamidc, 4-DAMP-methiodide and pirenzipine as competitors against the binding of 1.3 nmol/L 3H-NMS. The autoradiograms showed specific labelling indicating a high density of muscarinic receptors in smooth-muscle tissue in all levels of the airway tree investigated. Besides muscle tissue, subepithelial glands were the only structures specifically labelled. The dominating subtypes in tracheal smooth muscle investigated with radioligand binding studies were found to be M2 and M4, as both methoctramine (pKd = 8.5) and tripinamide (pKd = 8.6 and 6.7 for two different sites) showed high affinity. The density of the M3-muscarinic receptor subtype was low, but this subtype could be detected with statistical significance when methoctramine was used as the competitor against 3H-NMS binding.

Repeated administration of trimethoprim/sulfadiazine in the horse--pharmacokinetics, plasma protein binding and influence on the intestinal microflora.

Six healthy adult horses were given repeated administrations of trimethoprim/ sulfadiazine (TMP/SDZ) intravenously (i.v.) (2.5 mg/kg TMP and 12.5 mg/kg SDZ) and orally (p.o.) as a paste (5 mg/kg TMP and 25 mg/kg SDZ). Both formulations were given twice daily for 5 days, with a 3-week interval between i.v. and oral administration. The influence of the drug combination on the intestinal microflora was examined and the plasma concentrations, pharmacokinetic parameters and plasma protein binding were determined. There were no major changes in the bacterial intestinal flora and no clinical evidence of gastrointestinal disturbances following the i.v. and oral TMP/SDZ administration. An initial reduction in the number of coliform bacteria during the treatment was notable, though with no evident difference between i.v. and oral treatment. The minimum concentration during a dose interval at steady state (Cminss), the elimination half-life (t1/2beta) and the mean residence time (MRT) were significantly greater after oral administration compared to i.v. for both TMP and SDZ. The plasma protein binding was measured to be 20% for SDZ and 35% for TMP. Oral administration of TMP/SDZ in a dose of 30 mg/kg given twice daily in the form of paste appeared as a satisfactory method for obtaining plasma levels above MIC (minimum inhibitory concentration in vitro) values during the interdosing interval.

A comparison between clenbuterol, salbutamol and terbutaline in relation to receptor binding and in vitro relaxation of equine tracheal muscle.

Beta2-adrenoceptor agonists are used as bronchodilators in both humans and horses. Of these drugs, clenbuterol is the one most frequently used when treating chronic obstructive pulmonary disease in the horse, while salbutamol and terbutaline are used in the treatment of human asthma. Little is known of the properties of the latter two drugs in equine medicine. We have compared salbutamol and terbutaline with clenbuterol in relation to their ability to relax muscle strips from equine tracheal muscle, precontracted with 40 nM carbachol, in tissue chambers. The affinities of these drugs to the beta2-adrenoceptors in homogenates of the same muscle tissue were also examined. These experiments were performed with radioligand binding studies using the very potent beta-adrenoceptor antagonist 125I-cyanopindolol. The three drugs were almost equipotent in relaxing the muscle strips. The EC50-values for salbutamol, terbutaline and clenbuterol were 5.6, 13.8 and 2.1 nM, respectively, and all three drugs relaxed the preparations completely. In the competitive binding study, however, the Kd-value of clenbuterol was much lower (24 nM) than that of salbutamol and terbutaline (1100 nM and 3900 nM, respectively). The amount of receptors bound at the EC50-value of clenbuterol was 8% compared to less than 1% for salbutamol and terbutaline. This indicates a lower intrinsic efficacy of clenbuterol than of the other two drugs. The beta-adrenoceptor density was 45 +/- 14.3 fmol/mg protein (mean +/- SD) and the Kd-value of 125I-cyanopindolol was 11.4 +/- 3.3 pM.

Distribution studies of theophylline: microdialysis in rat and horse and whole body autoradiography in rat.

After intravenous administration of theophylline, microdialysis has been used for studying the non protein bound theophylline concentration in blood and in lung tissue in the rat as well as in two horses. The distribution pattern of 14C-theophylline in the rat was also investigated. When the distribution of theophylline was completed the time course of free drug in the interstitial fluid in lung tissue was in good agreement with the total concentration-time profile in plasma in both species. In the rat the free concentration of theophylline in the lung was slightly lower than the free concentration in the blood from 40 to 300 min. The in vivo protein binding in blood was 48.8 +/- 6.2% in the rats (n = 9) and 8-25% in the horses (n = 2). The whole body autoradiography study in rat showed that the concentration of radioactivity in the lung followed the blood concentration very closely up to 24 h after injection. The effect of theophylline in the lung can be assumed to be related to the plasma concentration of theophylline, since the concentration-time profile in plasma reflects the time course in the lung.

Clenbuterol plasma concentrations after repeated oral administration and its effects on cardio-respiratory and blood lactate responses to exercise in healthy Standardbred horses.

To evaluate the effects of clenbuterol on cardio-respiratory parameters and blood lactate relation to exercise tolerance, experimental horses performed standardized exercise tests on a high-speed treadmill before and after administration of the drug. Clenbuterol was administered in feed to six healthy Standardbreds at a dose rate of 0.8 micrograms/kg b.wt twice daily for 5.5 days. Each horse was tested twice, without and with a respiratory mask, during two consecutive days. One week elapsed between the baseline tests without drug and the tests with clenbuterol treatment (each horse served as its own control). The results show an unchanged heart rate response to exercise 2 h after the last clenbuterol administration. The blood lactate response and the arterial oxygen tension during exercise did not differ before and after drug treatment. The oxygen uptake as well as pulmonary ventilation relative to the work load performed was essentially unaffected. The arterial pH during exercise was significantly increased (P less than 0.05) following clenbuterol treatment. Plasma levels of clenbuterol were maximal 2 h post-administration with values between 0.45 and 0.75 ng/ml. The plasma half-life of elimination was 10.4 h (+/- 2.25 SD). In conclusion, clenbuterol did not cause any major effects on the cardio-respiratory and blood lactate parameters studied in healthy horses performing submaximal exercise tolerance tests.

Relaxant effects of theophylline and clenbuterol on tracheal smooth muscle from horse and rat in vitro.

A comparison between the relaxant effects of clenbuterol and theophylline on horse tracheal smooth muscle has been made in vitro. Rat tracheal smooth muscle was also investigated as a reference. The tracheal preparations were initially contracted with carbachol since the smooth muscle did not spontaneously develop tone. The response of the carbachol-contracted preparations to theophylline was the same in the two species. The response to clenbuterol varied. In only five out of eleven horses were the tracheal smooth muscles sensitive to clenbuterol (mean pD2 = 7.92 M). In the remaining six horses the tracheal smooth muscles were insensitive to clenbuterol (mean pD2 = 3.59 M), yet the preparations responded well to theophylline with complete relaxation. All rat tracheal preparations were insensitive to clenbuterol.

Pharmacokinetics and cardio-respiratory effects of oral theophylline in exercised horses.

The pharmacokinetics of theophylline at rest and the effects on cardio-respiratory and blood lactate responses to exercise were investigated after repeated oral administrations in six healthy Standardbred horses. A dose of 5 mg/kg body weight was administered every 12 h. The binding of theophylline to plasma protein was also determined. There was good agreement between predicted and observed plasma concentrations of theophylline at steady state. The mean half-life of elimination was shown to be 17.0 +/- 2.5 h, the mean half life of absorption was 1.6 +/- 1.8 h, the apparent volume of distribution was 852 +/- 99.0 ml/kg and total plasma clearance 0.61 +/- 0.08 ml/kg/min. Theophylline showed very low plasma protein binding (12%). The heart rate and blood lactate levels, during and after exercise, were significantly increased during theophylline-treatment. There was an increase of the arterial oxygen tension after exercise and the arterial carbon dioxide values before and after exercise were significantly lower than the premedication values. No severe adverse effects of the drug were noted. The recommended oral dose is therefore 5 mg/kg every 12 h but due to inter-individual variation, an adjustment of the dose may be necessary. The changes in the studied exercise parameters indicate that the performance capacity may be impaired by theophylline in the healthy horse.

Pharmacokinetic studies of theophylline in horses.

The pharmacokinetics of theophylline were determined in Standardbred trotters after single intravenous and oral administration. A bi-exponential equation was fitted to the intravenous data and a tri-exponential equation to the oral data. The biological half-life of theophylline was found to be 14.8 h, the volume of distribution 1.02 l/kg and the total plasma clearance 0.86 ml/kg/min. The oral absorption of the drug was complete (bioavailability 108%) and rapid (absorption half-life 0.4 h).