Materials and Methods

Animals—Twelve healthy fit actively competing adult Thoroughbred racehorses (7 geldings and 5 mares) with a mean ± SD body weight of 444.6 ± 49.6 kg and that ranged from 3 to 6 years of age were provided for use in in the study.^a Horses were assessed as healthy and free of cardiovascular disease on the basis of results of physical examination and auscultation. Horses did not receive any sedative or analgesic agents for at least 72 hours prior to commencement of the study. Horses continued to be exercised throughout the sample collection period, except for the day of drug administration, during which no exercise was performed. Food was withheld for 12 hours before and for approximately 6 hours after drug administration. Water was available ad libitum throughout the study. The study was approved by the Institutional Animal Care and Use Committee of the University of California-Davis.

Drug administration—The targeted dose of detomidine^b was 0.04 mg/kg, which was the recommended dose. Each horse was weighed immediately before drug administration, and the dose was determined in accordance with the dosing table provided in the package insert. The gel was administered sublingually by inserting the syringe tip into the side of each horse's mouth and positioning the tip beneath the tongue. Horses were observed for any loss of drug from the oral cavity as a result of swallowing or expulsion from the mouth.

Sample collection—Blood samples were collected at time 0 (before drug administration) and at 15, 30, and 45 minutes and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 18, 24, 48, and 72 hours after drug administration. Samples were collected via direct venipuncture of a jugular vein; each sample (10 mL) was placed into a heparin-containing blood tube. Samples were centrifuged at 3,000 × g for 10 minutes. Plasma was immediately transferred into storage cryovials and stored at −20°C until analysis. Urine samples were collected via midstream catch on days 1, 2, 3, 4, and 5 after detomidine administration. Urine samples were stored at −20°C for approximately 1 week until analysis.

Additional blood samples (10 mL) were collected via direct venipuncture of a jugular vein; samples were placed into heparin-containing blood tubes and used for analysis of plasma glucose concentrations, PCV, and plasma protein concentrations. Samples for plasma glucose analysis were collected at 0, 15, 30, and 45 minutes and 1, 1.5, 2, 3, 4, and 6 hours after drug administration. Samples were immediately placed on ice until centrifugation at 3,000 × g for 10 minutes at 4°C. Hematologic analyses were performed at the Clinical Pathology Laboratory of the William R. Pritchard Veterinary Medical Teaching Hospital of the University of California-Davis via their standard protocol for glucose analysis.

Samples for PCV and plasma protein determination were collected at 0, 15, 30, and 45 minutes and 1, 1.5, 2, 2.5, 3, and 4 hours after drug administration into heparinized syringes. The PCV was measured via a microhematocrit method, and plasma protein concentration was measured via a refractometer.

Physiologic responses—Each horse was equipped with a Holter monitor^c for continuous long-term recording of heart rate and rhythm. Heart rate and rhythm were recorded for a minimum of 30 minutes before and 4 hours after drug administration. The percentage of atrial signals blocked by the AV node before and after detomidine administration was calculated by use of the following equation: ([No. of atrial beats/min − No. of ventricular beats/min]/No. of atrial beats/min) × 100. In addition, chin-to-ground distance was monitored for 6 hours after detomidine administration by measuring the distance from the horse's muzzle to the ground. Any adverse behavior or effects were recorded at each sample collection time. Frequency of urination and defecation as well as fecal consistency was recorded throughout the sample collection period.

Statistical analysis—Data were summarized as mean ± SD. Statistical analyses were performed by use of commercially available software^d to assess significant differences in physiologic variables, PCV, and plasma protein and glucose concentrations before and after detomidine administration for individual horses. Raw data for all physiologic variables as well as glucose concentration, PCV, and plasma protein concentration were evaluated for normality by use of the Shapiro-Wilk test and then logarithmically transformed as necessary to bring the residual distribution in close agreement with a normal distribution. Data for all variables were subsequently analyzed by use of a mixed-model ANOVA with repeated measures. Values of P < 0.05 were considered significant.

Detomidine analysis—Quantitative analyses were performed on a triple quadrupole mass spectrometer^e coupled with a turbulent flow chromatography system^f (used in laminar flow mode). Chromatography was performed by use of a C₁₈ column^g (10 cm × 2.1 mm; particle size, 3 μm) and a linear gradient of acetonitrile in water with a constant flow rate of 0.35 mL of 0.2% formic acid/min. The acetonitrile concentration was held at 1.0% for 0.33 minutes, increased to 15% for 3.34 minutes, and finally increased to 95% for 5.17 minutes. Before analysis, plasma samples, standards, and quality-control samples were allowed to thaw at 27°C. Plasma proteins were extracted by precipitation via the addition of 0.5 mL of acetonitrile containing medetomidine and carboxylic acid–detomidine-d4 (20 ng/mL) as internal standards. All samples were vortex mixed for 30 seconds, followed by centrifugation (1,800 × g for 5 minutes). Injection volumes were 40.0 μL.

Detection and quantification were performed by use of highly selective reaction monitoring of the initial precursor ion for detomidine (m/z, 187). The response for the major product ions for detomidine (m/z, 52, 54, and 81) was plotted and peaks at the proper retention time integrated by use of software.^h The software was used to generate calibration curves and quantitate these analytes in all samples. Detection and quantification were performed by use of transitions of the initial precursor ion for hydroxydetomidine (m/z, 203) and carboxydetomidine (m/z, 217). The response for the product ions for hydroxydetomidine (m/z, 54, 81, and 185) and carboxydetomidine (m/z, 81, 144, and 199) were plotted and peaks at the proper retention time integrated by use of software.^h The software was used to generate calibration curves and quantitate these analytes in all samples.

Concentrations of detomidine and both principle metabolites for each sample (eg, calibrators, quality-control samples, and unknowns) were determined via an internal standard method by use of the peak area ratio and linear regression analysis. The response for detomidine was linear and yielded values of R² ≥ 0.99. The technique was optimized to provide an LOQ in plasma of 0.05, 0.1, and 0.5 ng/mL for detomidine, hydroxydetomidine, and carboxydetomidine, respectively. The LOQ in urine was 0.5 ng/mL for all compounds. For detomidine, the intraday accuracy (percentage of nominal concentration) was 100%, 104%, 106%, and 99% for 0.25, 1.5, 7.5, and 12.5 ng/mL, respectively. Interday accuracy was 95.4% and 97.8% for 0.75 and 7.5 ng/mL, respectively. Intraday precision (percentage of relative SD) was 9%, 2%, 5%, and 4% for 0.25, 1.5, 7.5, and 12.5 ng/mL, respectively. Precision and accuracy were < 10% for hydroxydetomidine and carboxydetomidine.

Pharmacokinetic calculations—Nonlinear least squares regression was performed on plasma detomidine and carboxydetomidine concentrations by use of commercially available software.ⁱ Data were analyzed by use of a noncompartmental approach. The AUC was extrapolated to infinity by use of the last measured plasma concentration divided by the terminal slope. The Cmax was obtained directly from the data. Pharmacokinetic parameters for all 12 horses were reported as mean ± SD.

Results

Pharmacokinetics—The LOQ for liquid chromatography–mass spectrometry analysis of plasma samples was 0.02, 0.1, and 0.5 ng/mL for detomidine, carboxydetomidine, and hydroxydetomidine, respectively. Detomidine plasma concentrations (mean and range) were summarized (Table 1). Both carboxydetomidine and hydroxydetomidine were detected in the plasma samples; however, the hydroxydetomidine concentration was below the LOQ at all time points. Concentrations of detomidine and its metabolites were below the LOD by 24 hours and 3 days after drug administration for plasma and urine, respectively. Results of noncompartmental analysis for a number of pharmacokinetic parameters for detomidine and carboxydetomidine were summarized (Table 2). Carboxydetomidine and hydroxydetomine were detected in urine samples (Table 3).

Physiologic responses—Physiologic responses after administration of detomidine gel were variable among horses. All horses developed signs of sedation after detomidine administration, as evidenced by a decrease in head height (chin-to-ground distance) relative to preadministration values (Figure 1) and results of behavioral observations. However, the degree of sedation was variable among individual horses. The mean percentage decrease in chin-to-ground distance was plotted against detomidine concentration for the 12 horses. There was a slight lag between maximum detomidine concentrations and the maximal decrease in chin to ground distance. Six horses had moderate amounts of sweat, especially around the head and neck, and 7 horses had frequent periods of voluminous urination. One horse tended to snore throughout the period of sedation. Signs of sedation persisted for approximately 2 hours after drug administration in all horses.

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Mean ± SD percentage change in chin-to-ground distance (expressed as the change from the value for time 0) with respect to time (A) and mean percentage decrease in chin-to-ground distance (expressed relative to the value for time 0) with respect to detomidine plasma concentration (B) after sublingual administration of 0.04 mg of detomidine/kg to 12 horses. Time 0 was immediately before drug administration. For panel B, the time at which the sample was collected (number of hours after drug administration) is indicated next to each data point. *Value differs significantly (P < 0.05) from the value for time 0.

Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1378

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The mean change in heart rate at all time points, expressed as the change from the value before detomidine administration (ie, time 0), was calculated and plotted (Figure 2). Heart rate decreased significantly after detomidine administration, relative to the value for time 0, for all horses. The mean ± SD maximal decrease in heart rate (10 ± 5 beats/min) was at approximately 45 minutes and correlated with Cmax. The percentage of atrial signals blocked by the AV node following sublingual administration of detomidine was plotted (Figure 3). Atrioventricular conduction disturbances were recorded for 2 horses prior to drug administration. For these horses, AV blocks were again recorded after administration (10 minutes for 1 horse and 15 minutes for the second horse) and persisted throughout the duration of the recording period for both horses. Atrioventricular conduction disturbances were also recorded for 5 other horses following administration of detomidine (20 minutes for 1 horse, 30 minutes for 2 horses, and 45 minutes for 2 horses). The AV blockade appeared to be resolved by the end of the recording period for these 5 horses. The maximal percentage of blocked AV signals correlated with Cmax.

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Mean ± SD change in heart rate (expressed as the change from time 0) with respect to time (A) and mean decrease in heart rate (expressed relative to the value for time 0) with respect to detomidine plasma concentration (B) after sublingual administration of 0.04 mg of detomidine/kg to 12 horses. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1378

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Mean ± SD percentage of AV blocks (expressed as the change from time 0) with respect to time (A) and mean percentage of AV blocks (expressed relative to the value for time 0) with respect to detomidine plasma concentration (B) after sublingual administration of 0.04 mg of detomidine/kg to 12 horses. The percentage of AV blocks was calculated by use of the following equation: ([No. of atrial beats/min − No. of ventricular beats/min]/No. of atrial beats/min) × 100. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1378

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Changes in plasma glucose concentrations (Figure 4), PCV (Figure 5), and plasma protein concentrations (Figure 6), compared with the respective values at time 0, were plotted. The PCV was significantly different from the value at time 0 beginning at 45 minutes and continuing until 3 hours after drug administration. Significant changes in plasma protein concentrations were not detected after detomidine administration.

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Mean ± SD change in plasma glucose concentration (from value at time 0) with respect to time after sublingual administration of 0.04 mg of detomidine/kg to 12 horses. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1378

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Mean ± SD change in PCV (from value at time 0) with respect to time after sublingual administration of 0.04 mg of detomidine/kg to 12 horses. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1378

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Mean ± SD change in plasma total protein concentration (from value at time 0) with respect to time after sublingual administration of 0.04 mg of detomidine/kg to 12 horses. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 72, 10; 10.2460/ajvr.72.10.1378

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Discussion

The primary objective of the study reported here was to characterize the pharmacokinetics of a novel detomidine gel product after sublingual administration to horses, with the ultimate goal of providing appropriate withdrawal guidelines prior to a performance activity. A similar study⁶ on the pharmacokinetics of detomidine following sublingual administration of detomidine gel in horses has been conducted. Although it appears that the 2 studies were essentially conducted concurrently, there are a few important differences between the 2 studies. In the other study,⁶ sample collection was terminated at 8 hours after drug administration, when detomidine concentrations could still be detected, which makes it less than ideal for establishing withdrawal guidelines for horses in competitions. Additionally, because elimination of drugs has been reported to differ between sedentary and fit horses, in the present study, we used a representative population of young actively competing racehorses, as opposed to older sedentary research horses.

In the study reported here, the highest mean ± SD plasma detomidine concentration was 168 ± 83.7 ng/mL, which indicated that detomidine was absorbed well from the sublingual mucosa into the systemic circulation. This concentration is much higher than values reported for IV administration of 0.04 mg/kg (68.8 ng/mL)¹ and 0.03 mg/kg (74.4 ng/mL)³ and IM administration of 0.03 mg/kg (6.9 ng/mL).³ In previous studies as well as in the study reported here, a jugular vein was used for sample collection. Although the sample collection site was the same for all studies, in the case of sublingual administration, it is important to remember that drainage from the submucosal region is via the jugular vein; therefore, collection of samples via a jugular vein at the initial time points was conducted immediately following absorption. Thus, sample collection was performed prior to drug distribution and before any systemic metabolism took place, which offers a possible explanation for the higher plasma concentrations after sublingual administration in the present study, compared with results for other routes of administration. However, this would only affect the initial concentrations during the drug absorption phase and not the concentrations at later time points. Additionally, although the Cmax after IV administration has been reported as 68.8 ng/mL for 0.04 mg/kg¹ and 74.4 ng/mL for 0.03 mg/kg,³ both of which were detected at 5 minutes after administration, the true Cmax would be the administered dose in the case of IV administration, whereby the drug is administered directly into the systemic circulation. Because detomidine is rapidly distributed following IV administration, plasma concentrations measured at 5 minutes after administration may underestimate the true peak plasma concentration. The Cmax in the present study also differed substantially from that reported in another study⁶ (4 ng/mL). This is also likely a result of the site used for collection of samples because the lateral thoracic vein was used for sample collection in that other study.⁶

Peak detomidine plasma concentrations were rapidly achieved after sublingual administration, with mean ± SD Tmax at 36 ± 10 minutes after drug administration. This is in stark contrast to the Tmax of 1.83 hours reported in that other study,⁶ which again is likely a result of differences in the site of sample collection. As reported in another study,³ Tmax after IV and IM administration was 2.1 and 77.0 minutes, respectively. The mean ± SD half-life of elimination of detomidine following sublingual administration was 1.5 ± 1 hours, which is in close agreement with that reported in 1 study⁶ but longer than that after IV (26.4 minutes) and IM (53.4 minutes) administration in another study.³ Concentrations of detomidine as well as its metabolites in urine samples were below the LOD by 3 days after administration. This is similar to results in another study⁷ in which investigators used an ELISA; in that study,⁷ the detomidine concentration was below the LOD by 72 hours after drug administration.

Although detomidine appeared to be absorbed well following sublingual administration, there was a great degree of variability in Cmax and Tmax among horses. In the present study, the wide individual variability can be attributed to a number of factors. We tried to ensure that the entire dose was delivered and remained under the tongue, and no loss of drug was observed; however, it is possible that some of the dose was lost via expulsion from the mouth or swallowing. Additionally, it is possible that small amounts may have been swallowed following administration, which would have subjected the detomidine to metabolism by enzymes in the gastrointestinal tract wall, first-pass effects, or both. In that case, the swallowed drug (parent compound) would not reach the systemic circulation, thus decreasing the plasma concentration of detomidine in those horses. Differences in the pH of the oral cavity are another potential explanation for the variability in absorption and subsequent plasma concentrations among horses. Detomidine is a lipophilic weak base with an acid dissociation constant of 7.2; thus, absorption is favored in an alkaline environment. Although the pH of the oral cavity in horses tends to be alkaline, it was not measured in the present study, which makes it possible that slight differences among horses could have contributed to differences in absorption and the subsequent wide differences in plasma detomidine concentrations. At the later time points (after absorption), differences in the rate of metabolism and elimination among horses may also have been a factor.

Sedative effects of detomidine, as assessed via the chin-to-ground distance, have been described following sublingual^6,7 and parenteral² administration. Decreases of 70%² and 51%⁸ have been reported after IV administration of detomidine at 0.03 and 0.02 mg/kg, respectively. A 45% maximal decrease in head height, relative to baseline values, was observed following IM administration of 0.03 mg of detomidine/kg.² Similar to results of the present study, sublingual administration of the injectable or gel detomidine formulation at 0.04 mg of detomidine/kg caused a significant decrease in the chin-to-ground distance.^5,6 In all 3 studies, the maximal decrease was observed at 60 minutes after detomidine administration; however, the magnitude of the decrease varied slightly among the studies. Investigators in 1 study⁵ reported a maximal decrease in muzzle-to-floor distance of nearly 50%, compared with a maximal decrease in head height of 40% in another study⁶ and a maximal decrease of 28.4% in the present study.

The effects of α₂-adrenergic receptor agonists, specifically detomidine, on heart rate and rhythm have been described.^2,8,9 In 1 study,⁹ investigators reported a mean decrease in heart rate of 10 beats/min following IV administration of 0.02 mg of detomidine/kg. A mean decrease of 18 beats/min was reported following IV administration of 0.03 mg of detomidine/kg in another study²; however, in that same study, no significant effect on heart rate was observed following IM administration of the same dose. Because higher plasma concentrations are achieved after IV administration than after IM administration, this suggests a dose-dependent effect. In the present study, a minimal but significant decrease in heart rate of 10 beats/min was detected, which is in close agreement with the mean ± SD value reported following sublingual administration in another study⁵ (9 ± 2.6 beats/min). In addition to its effects on heart rate, detomidine has effects on AV conduction when administered to horses. An increased incidence of AV blocks has been reported following epidural administration⁸ of detomidine and after IV infusion.¹⁰ Furthermore, in the experience of our laboratory group, there is an increased incidence as well as exacerbation of preexisting AV blocks following IV administration of 0.03 mg of detomidine/kg. It has been postulated⁸ that the bradycardia and conduction disturbances observed following detomidine administration may be attributable to a centrally mediated decrease in peripheral sympathetic tone, presynaptic inhibition of norepinephrine release from fibers innervating the heart, or enhancement of vagal reflexes.

Although slight, there was an obvious hysteresis between plasma detomidine concentrations and effects on the chin-to-ground distance in the present study. Effects on chin-to-ground distance were observed within minutes after detomidine administration, with maximal effects achieved at 1 hour after administration. Because the chin-to-ground distance is a centrally mediated effect, the lag phase between Cmax (45 minutes) and sedation effects (ie, head height) was most likely a reflection of the time required for distribution into the CNS. Although the only way to directly correlate drug concentrations to effects on sedation would be to measure concentrations at the site of action (ie, brain), it is the opinion of the authors' that by 45 to 60 minutes after administration, absorption should be nearly complete. The lag in time to peak plasma concentrations (1.83 hours) and maximal sedation as determined by use of the chin-to-ground distance (60 minutes) was slightly greater in another study.⁶ The discrepancy between peak plasma concentrations and time to peak sedation in the 2 studies was likely a reflection of the differences in the site of blood collection. A lag effect similar to that detected in the present study has been described following IV and IM administration. Investigators in 1 study² reported peak detomidine plasma concentrations at 1.5 minutes, with maximal clinical effects 4 to 20 minutes after administration. Another possible explanation for the hysteresis or lag in physiologic effect with respect to plasma detomidine concentrations would be the presence of an active metabolite. However, we believe this to be unlikely because detomidine metabolites are believed to be inactive.

Conversely, effects on the heart appeared to directly correlate with detomidine plasma concentrations, with maximal effects observed as Cmax was reached. The difference in time of maximal changes in cardiovascular variables versus the chin-to-ground distance was most likely a result of the route of administration. Following sublingual administration, a drug is apt to reach the heart prior to distribution to the brain because the mucosal capillaries drain directly into the jugular veins, which run directly to the heart. However, blood must travel throughout the body before reaching the brain. Furthermore, this suggests that although head height is centrally mediated, effects on the cardiovascular system may be mediated by peripheral α₂-adrenergic receptors.

To the authors' knowledge, there are no reports in which the effect of detomidine on plasma glucose concentrations has been described. However, other α₂-adrenergic receptor agonists, such as xylazine, can have a marked effect on plasma glucose concentrations in horses. Hyperglycemia has been detected in a number of animal species, including dogs,¹¹ cats,¹² sheep,¹³ cattle,^14,15 and horses,^16,17 and has been attributed to inhibition of insulin release from the pancreas. In the present study, there was no apparent pattern for glucose concentrations over the 6-hour sample collection period. Additionally, there was large variability among horses. There was a significant decrease in plasma glucose concentrations at 15 and 30 minutes after detomidine administration. However, because this was contrary to what would be expected on the basis of the effects of other α₂-adrenergic receptor agonists, it is possible that it was not a drug-related phenomenon and simply a result of food being withheld from the horses before and throughout the glucose-monitoring period. Because this was believed to be the first study in which investigators assessed the effects of detomidine on plasma glucose concentrations, additional studies will be necessary to fully characterize these effects in horses.

On the basis of the high plasma concentrations, detomidine appeared to have been absorbed well when administered via the sublingual route. The half-life of elimination following sublingual administration was prolonged, compared with elimination after IV and IM administration, with detectable concentrations of detomidine or its metabolites in the plasma for up to 24 hours after administration. Additionally, concentrations of the parent compound and metabolites were below the LOD in urine by 3 days after drug administration. On the basis of the pharmacokinetics described here, a 48-hour and 3-day withdrawal period for detection in plasma and urine samples, respectively, should be adopted for sublingual administration of detomidine gel. Additionally, although no specific procedures were undertaken in the present study, detomidine gel appeared to induce a moderate degree of sedation, as indicated by the chin-to-ground distance, subjective observations of behavior, and effects on heart rate and rhythm.