Pharmacokinetics and pharmacodynamics of acepromazine in horses

P. J. Marroum, PhD; A. I. Webb, BVSc, PhD; Gina Aeschbacher, Dr Med Vet; S. H. Curry, PhD, DSc (Med)

Summary A specific, sensitive, reverse-phase high-per­

formance liquid chromatographic assay for aceprom­azine, with analytic sensitivity as low as 5 ng/ml of plasma, and electrochemical detection with an oxi­dation potential of 0. 7 V, was used to study the phar­macokinetics of acepromazine given at a dosage of 0.15 mg/kg of body weight in horses. The relation between effect and pharmacokinetics of the drug was examined. The effects studied included those on blood pressure, pulse, PCV, measures of respiration function, and sedation. Intravenously administered doses led to a biphasic concentration decay pattern with an a-phase distribution half-life of< 3 minutes. The J3-phase half-life was in the range of 50 to 150 minutes. The CNS effects peaked at 20 minutes after administration, and the hemodynamic effects peaked at 100 minutes. In all horses, the most sensitive var­iable was the PCV, which decreased by up to 20% (P < 0.0001). Systolic, diastolic, and mean blood pres­sures decreased (P < 0.0001); heart rate was unchan­ged (P > 0.05). Neither blood gas tensions nor blood pH changed noticeably (P > 0.05). In all horses stud­ied, acepromazine had a significant (P < 0.0001) sed­ative effect, as observed by posture and alertness. None of the observed pharmacodynamic effects cor­related well with plasma acepromazine concentra­tion. These effects persisted beyond the time of detectable acepromazine concentration, indicating that they might be caused by active metabolites, or that their timing could result from complex phar­macokinetic compartment influences.

Acepromazine is a sedative/tranquilizer com­monly used in horses, cats, and dogs. Its pharmaco-

Received for publication Mar 12, 1993. From the Department of Pharmaceutics (College of Phar­

macy), and the Department of Medical Science (College of Veter­inary Medicine), Health Science Center, University of Florida, Gainesville, FL 31610.

Dr. Marroum's present address is Division of Biopharmaceu­tics, US Food and Drug Administration, 5600 Fishers Lane, Rock­ville, MD 20857. Dr. Curry's present address is Fison's Pharma­ceuticals, Divisional Research and Development, Jefferson Road, PO Box 1710, Rochester, NY 14603-1710.

Published as Florida Agricultural Experimental Station Journal . Series No. R-034251.

Address reprint requests to Dr. Curry.

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logic effects are similar to those of other pheno­thiazines, but it is considered more potent than the prototype drug, chlorpromazine.1 Despite its wide­spread use, little is known of the metabolism and pharmacokinetics of acepromazine. In fact, it appears that only 3 relevant reports have been published.2-4

In 1 study, 2 acepromazine was detected in horses for 8 hours after IV administration of 0 .3 mg/kg of body weight.2 The plasma decay was biexponential, with mean a-phase half-life of 4.2 minutes and mean J3-phase half-life of 184.8 minutes. The apparent vol­ume of distribution was 6.6 L/kg. Protein binding exceeded 99%. Drug distribution was approximately even between plasma and RBC. Dewey et al3 reported that the major urinary metabolite of acepromazine in mares is unconjugated 2-(1-hydroxyethyl) promazine sulfoxide. Conjugated 7-hydroxy-acepromazine and conjugated 2-(1-hydroxyethyl)-7-hydroxy-promazine also were isolated. Miller et al4 studied oral admin­istration of a paste form of acepromazine and its tim­ing in relation to feeding.

The objectives of the study reported here were to use a high-peformance liquid chromatography (HPLC) assay for acepromazine in biological fluids, and to describe the pharmacokinetic profile after IV administration of 0.15 mg/kg in horses. Parallel stud­ies of the pharmacokinetic properties of the drug and various pharmacodynamic measurements, such as blood pressure, heart rate, degree of sedation, PCV, and arterial blood gas tensions, were conducted.

Materials and Methods The following analytic grade materials were

used: acepromazine maleate8; trimeprazine tartrateb;

acetonitrile, hexane, ammonium acetate, and sodium acetatec; hexamethyldisilazined; sodium heparin e; and sodium chloride.f

Apparatus-The HPLC system consisted of a sol­vent delivery system, an automatic injector, and a data module,g a recorder, h and an electrochemical detector.i The HPLC column was nitrile-bonded (13 cm X 5 µmi).

a Fort Dodge Laboratories, Fort Dodge, Iowa. b Smith Klfue & French Laboratories, Philadelphia, Pa. c Fisher Scientific, Pittsburgh, Pa. d SCM Specialty Chemicals, Gainesville, Fla. e Lyphomed Inc, Rosemont, ill. f Kendall McGaw Laboratories, Inc, Irvine, Calif. g Model 6000, WISP 710~ and model M730, Waters Asso-

ciates, Millford, Mass. h Recordall 5000, Fisher Scientific, Pittsbur~ Pa. l Model 5100A Colochem, ESA Inc, Bedford, Mass. 1 Zorbax CN, Mac-Mod Analytical Inc, Chadds Ford, Pa.

Am J Vet Res, Vol SS, No. 10, October 1994

Analysis of acepromazine in biological fluids-Sam­ples (0.5 to 2 ml, depending on availability, and ac­curately measured) of biological fluids, such as plasma, were alkalinized by addition of 0.1 ml of lN NaOH. An appropriate amount of the internal stan­dard trimeprazine (to a concentration of 150 ng/ml) was added. This alkaline sample was then extracted with 5 ml of hexane for 1 hour, using a mechanical shaker that caused inversion of the tube with a fre­quency of at least 30 cycles/min. After centrifugation, an aliquot of the hexane phase was removed and evaporated to dryness at 25 C under a constant stream of nitrogen. If any emulsion persisted after centrifugation, the contents of the tube were gently stirred with a glass rod, followed by further centri­fugation. The residue was redissolved in an appro­priate volume of mobile phase, usually 250 µl, and an aliquot of this reconstituted residue was injected into the chromatographic system. The mobile phase consisted of 75:25 acetonitrile-0.lM acetate buffer, pH 4.75. The flow rate was 1.2 ml/min. The oxidation potential was set at 0.7 V for the analytic cell and 0.75 V for the guard cell.

Phannacokinetics of acepromazine after N admin­istration of 0.15 mg/kg-The kinetic studies were con­ducted, using 4 female and 2 male adult horses that had been donated to the College of Veterinary Med­icine at the University of Florida. Five horses were used in the pharmacokinetic and the pharmacody­namic studies. One horse (No. 6) was only used in pharmacokinetic studies. Ages ranged from 2 to 20 years, with that that for horse 6 unknown. Four were Thoroughbred, and 2 were Arabian. Body weight ranged from 389 to 543 kg. All were thought to be healthy at the time of the investigation, but during the course of the investigation, horse 1 was found to have a vegetative lesion on the aortic valve, on the basis of echocardiography indicating regurgitation, and later postmortem examination revealing marked pulmonary congestion. Prior to the study, horses were acclimatized to their surroundings. Aceproma­zine maleate solution (concentration, 0.5 mg/ml) was injected into the right jugular vein, and blood samples from the left jugular vein were withdrawn through a 14-gauge, 5-inch Teflon over-the-needle catheter into heparinized evacuated tubes. Catheters were flushed with heparinized saline solution. Sample col­lection times were 0 (predos~) and 1.5, 3, 4.5, 6, 7.5, 9, 12, 15, 18, 24, 30, 45, 60, 75, 90, 120, 150, 180, 210, 240, 300, and 360 minutes after injection or a selec­tion of times thereof. Each venous blood sample, consisting of 5 ml, was used to measure PCV, then was centrifuged immediately afterward at 800 X g to separate plasma and RBC. Samples from 4 of the horses (2 male and 2 female) also were collected from the arterial line installed for blood gas analysis.

Blood pressure measurements-Systemic systolic, diastolic, and mean blood pressures were obtained by transducing the pressures obtained by use of a per­cutaneous catheter placed in the transfacial artery. The transducer was powered by a multichannel oscillo-· scope with digital pressure and heart rate displays.

Am J Vet Res, Vol 55, No. 10, October 1994

Heart rate-The heart rate was measured by the rate counter on the multichannel oscilloscope that determined the number of arterial pulse waves per 10-second period and gave a beats-per-minute count output.

Blood gas analysis-Systemic arterial blood gas tensions and pH were measured from samples col­lected anaerobically through the arterial catheter into a heparinized plastic syringe. These samples were an­alyzedk within 2 hours of collection.

Sedative effects-Such effects were evaluated, us­ing a scale devised for the purpose of this work (Ap­pendix). Degree of sedation, general behavior, posture, and general alertness of each horse were evaluated. All horses were rated by the same person to avoid any observer-dependent subjective differ­ences. Rating was on an open basis, without blinding.

Evaluation and fitting of phannacokinetic data­Plasma concentrations of acepromazine were evalu­ated, using commercial software packages.Lm After IV administered doses, the equation for the linear sum of exponentials was:

Cpt = Ae-a.t + Be-~t

where Cp is the plasma concentration, A, B, a, and ~are constants, and tis time in minutes. Goodness­of-fit parameters were the sum of squared residuals, sum of weighted squared residuals, and correlation.

Statistical review of phannacodynamic data-Raw data were transformed to percentages of the control preacepromazine values, which were taken as 100%. The data for each variable were then compared, using a commercial statistical packagen in a one-way re­peated-measures ANOVA, with subsequent break­down of the source of significant differences, using Dunnett's multiple-range test.

Results and Discussion Results of the study reported here complement

and extend the study that Ballard et al2 · conducted some 10 years ago. Those investigators used a gas chromatographic assay and calculated group mean pharmacokinetic constants without error estimates for venous plasma from a population of male horses, whereas by use of a HPLC method, we were able to calculate pharmacokinetic constants for individual horses, and for venous and arterial plasma data. Bal­lard et al focused their attention on penile protrusion and measurement of one particular behavioral phe­nomenon after training. We studied 5 spontaneous behavioral responses, using a rating scale, in addition to studying eating. Ballard et al studied protein bind­ing and RBC partitioning of acepromazine; we focused on cardiovascular observations, and on blood gas analysis to assess any effects on respiration.

k IL1304 Blood Gas Analyzer, Instrumentation Laboratories, Lexin~on, Mass.

1 RSTRIP, Micromath Scientific Software, Salt Lake Oty, Utah.

mpCNQNLIN, Statistical Consultants Inc, Lexington, Ky. n STATP~ Northwest Analytic Inc, Portland, Ore.

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300

250

e Cl 200 .5. c 0

! 150

c GI

100 u c 0 0

50

0 0 50 100 150 200

Time (min)

Figure I -Venous plasma concentrations (ng/ml) vs time (min) in 6 horses after iv administration of acepromazine maleate (0.15 mg/kg of body weight).

High-perfonnance liquid chromatography assay­The coefficient of variation for repeated assays of plasma containing known quantities of acepromazine varied from 16.5 to 3.89%. The coefficients of variation for interday and intraday variation were in similar range; calibration was conducted daily. A typical cali­bration curve is described by the following equation:

y = -0.01044 ± 0.0279 + (0.595 ± 0.0258) x

where y is the peak height ratio and x is the concen­tration of acepromazine in the plasma. The correla­tion coefficient was 0.993. The high coefficient of

Table I -Individual armacokinetic variables for ace

2 Variable Venous Arterial Venous

Dose (mg) 65 65 82

a (llmin) 0.53 0.59 0.15

~ (1/min) 0.008 0.007 0.013

A (ng/ml) 327 365 139

B (ng/ml) 38.8 35.3 21.9

ti12a (min) 1.30 1.17 4.43

ti12~ (min) 81.6 90.2 51.7

CO (ng/ml) 366 401 161

AUC (ng/min/ml) 5,187 5,228 2,529

AUMC (ng/min2/ml) 540,100 601,450 128,050

MRT (min) 104 115 50

Vd.. (L) 1,303 1,430 1,621

Vd..fkg(L) 3.03 3.33 3.0

Vdp.(L) 1,566 1,776 2,463

Vdp.lkg(L) 3.65 4.14 4.53

Vd.:c(L) 177 162 503

Vcl,;dkg(L) 0.41 0.37 0.92

Otot{L/min) 12.5 12.4 32.0

Otot/k~(ml/min) 29.2 28.9 59.0

variation was attributable to the fact that phenothi­azines, being lipophilic, have a large degree of glass binding and this renders their assay difficult.5 The method assayed total (free plus bound) concentration in plasma, with linearity from 5 ng/ml to at least 200 ng/ml.

Phannacokinetics-The plasma concentration pro­files for all horses after administration of 0.15 mg of acepromazine/kg conferred on the body the characteristics of a two-compartment model (Fig 1; Ta­ble 1). The distribution half-life ranged from 0.12 to 4.43 minutes, indicating existence of fast distribution into a shallow compartment after the drug entered the blood. In venous plasma, the terminal half-life ranged from 51.7 to 148.5 minutes, indicating larger variation and slow elimination from the body. The apparent vol­ume of the central compartment ranged from 137 to 503 L. These values were larger than the volume of blood in horses (70 ml/kg of body weight), indicating that acepromazine is widely distributed through the tissues. Mean total body clearance of acepromazine was found to be 21.1 L/min, which was not signifi­cantly different from cardiac output in horses (approx 18 L/min), probably indicating that this drug does not undergo nonflow-dependent metabolism, although it could also indicate acepromazine metabolism in the blood.

There are differences and similarities in the pharmacokinetic data obtained by Ballard et al, 2 and

romazine in 6 horses after rv administration of the dru

Horse No.

3 4 5 6 Venous Venous Venous Arterial Venous

58 69 58 58 76

0.37 0.32 0.49 0.17 0.72

0.004 0.017 0.022 0.011 0.010

236 199 205 61 470

32.5 27.2 29.1 6.0 76.1

1.88 2.15 1.40 3.97 0.96

148.5 39.4 31.5 59.4 67.6

268 226 234 67 546

7,608 2,169 1,744 872 8,077

1,495,200 90,044 61,317 46,759 725,540

196 41 35 53 89

1,497 1,320 1,169 2,559 834

3.85 2.87 3.0 6.57 1.64

1,906 1,871 1,512 6,041 928

4.9 4.06 3.88 15.52 1.82

216 304 248 865 137

0.55 0.66 0.63 2.22 0.27

7.6 31.8 33.2 66.4 9.2

19.6 69.1 85.4 170.8 18.2

a = Rate constant of plasma concentration decay in initial rapid phase. ~ = Rate constant of plasma concentration decay in later slower phase. A = Intercept of a-phase decay at y a.xis of graph of concentration vs. time. B = Intercept of ~-phase decay at y a.xis of graph of concentration vs time. h12a = Half-life of the initial phase of relatively rapid plasma level decay, 0.693 divided by a. t1/2J = Half-life of the late phase of relatively slow plasma level decay, 0.693 divided by p. CO = (A + B). AUC = Area under curve of graph of concentration vs time. AUMC = Area under curve of first moment plot. MRT = Mean resistence time. Vd.. = Apparent volume of distribution at steady state. Vd.,lkg = Vd. divided by weight of horse. Vdp., = Apparent volume of pheripheral compartment at steady state. Vdp,,lkg = Vdp,, divided by weight of horse. Vdcc = Apparent volume of central compartment. Vdcc/kg = Vdcc divided by weight of horse. Otot = Total body clearance. Ototfkg = Otot per kg of body weight of horse.

1430 Am J Vet Res, Vol 55, No. 10, October 1994

0 40 80 120

Time(mln)

160 200 240

Figure 2-Mean ( ± SEM) PO/ vs time after acepromazine administration.

150

Ci :::z:: 0 E 100 .§. e ::I Ill Ill e ~ 50 0 0 iii

0 40 80 120

Time (min)

160 200 240

Figure 3-Mean ( ± SEMJ blood pressure vs time after ace­promazine administration.

0 40 80 120

Time (min)

160 200 240

Figure 4-Mean ( ± SEMJ heart rate (HR} VS time after ace­promazine administration.

by us. For example, we recorded a higher value for the pharmacokinetic A-intercept, possibly reflecting earlier collection of blood samples. The values of a and ~ are somewhat different, as might be expected with different doses used in the 2 investigations, or sex or history of the animals. All other calculated var-

Am J Vet Re~, Vol SS, No. 10, October 1994

3

! 0 u

Cl)

c .2 iii 2 "1:::1 GI

Cl)

c I'll GI :ii

0 60 120

Time (min)

180 240

Figure 5-Mean sedation score vs time after acepromazine administration.

Table 2-Pharmacodynamic effects after administration of ace romazine maleate 0.15 m /k , rv

Period over which

variable Signifi- was

Variable Effect cance• affected Pulse No effect NS Systolic blood

pressure Lowers p < 0.0001 50 to 150 min Diastolic blood

pressure Lowers p < 0.0001 12 to 210 min Mean blood

pressure Lowers p < 0.0001 10 to 360 min Arterial pH No effect NS

Paoi No effect NS Pa~ No effect NS ... PCT Lowers p < 0.0001 10 to 360 min Head carriage Lowers head p < 0.0001 6 to 75 min Random

movement Decreases movement p < 0.0001 12 to 20 min

Reaction to pin pricks Reduced

reactivity p < 0.0001 62 to 120 min Eating Ceases p < 0.0001 4.5 to 90 min Eyelid position Eyelid droops p < 0.0001 6 to 75 min Reaction to noise No effect NS

• Significance was tested, using ANOVA for identifying principal effects and Dunnett's multiple-range test for characterizing the period that each variable was affected.

NS = not significant.

iables were similarly affected because they were based on the same 2 sets of raw data. Unfortunately, Ballard et al did not define their pharmacokirtetic terms with sufficient precision to facilitate definitive comparisons.

Packed cell volume-The percentage change in PCV vs time was plotted (Fig 2). At this dose, ace­promazine had a profound and significant effect on the PCV, because, in all horses, it decreased by 20%. This decrease was not immediate, but gradual, and reached the lowest value after 6 hours. It is possible that it might have been further reduced, but such was not observed owing to the fact that the study was stopped after 6 hours. The mechanism of action of this decrease is thought to be attributable to the a­adrenolytic activity of acepromazine together with depression of the vasomotor center, causing splenic relaxation with consequent erythrocyte'sequestration leading to a decrease in hematocrit. 6-9 ·

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Cardiovascu.lar and hemodynamic effects-Change in mean blood pressure against time (Fig 3), as well as change in heart rate (Fig 4) were graphed. In all horses, there was a marked gradual and significant decrease in systolic blood pressure, which peaked be­tween 60 and 90 minutes after. acepromazine admin­istration. The same trend was observed for diastolic, systolic, and mean blood pressures.

The decrease in blood pressure might have been attributable to a direct effect on the heart and blood vessels and to indirect effects through actions on CNS and autonomic reflexes. Reflex tachycardia would be expected to result from such induction of hypoten­sion. However, it can be seen (Fig 4) that heart rate was actually decreased, although not to an important extent. Any decrease in heart rate would have been attributable to either a direct adrenergic blocking ef­fect in the heart or to some sort of centrally mediated action, which would have to have been stronger than the reflex tachycardia. Even though blood pressure and heart rate changed, there was no effect on either blood gas tensions or physiologic pH. This is probably attributable to the fact that acepromazine had no ef­fect on respiratory rate. Lack of significant changes in arterial pH and arterial carbon dioxide and oxygen tensions may reflect a compensatory increase in tidal volume to maintain an appropriate minute alveolar ventilation.

In this study, the effect of acepromazine on blood pressure was confounded by inclusion of horse 1 that had previously unsuspected cardiac failure. Also, the between-horse variation, which was appar­ently greater than the variation for the effects of ace­promazine, made precise conclusions difficult. For example, slowness of the acepromazine effect on sys­tolic pressure to become obvious was attributable to maintenance of blood pressure at or above control values for nearly an hour in 2 of the 5 horses. One of these 2 horses (No. 1) had similar resistance to the overall trend in maintaining control PCV value for nearly 20 minutes. A possible explanation for these effects might be high sympathetic tone in the face of borderline cardiac failure, increased blood volume, or both. Data on these possibilities were not collected.

Effects on the CNS-Acepromazine induced con­siderable sedation, as judged by the subjective scoring of head carriage, movement, drooping of eyelids, ces­sation of feeding, and response to pin pricks (raw data not presented). Reactivity to noise was not af­fected. Maximal effect was at 20 minutes after dosing (Fig 5).

The onset of action for the sedative effects was faster than the onset of hemodynamic effects, and recovery from sedation was quick. These sedative and CNS effects are thought to be centrally mediated and are thought to be attributable to the antagonism of dopamine-mediated synaptic transmission.

All sedation scores, except reactivity to noise, were affected by acepromazine, as would be expected from use of an acknowledged tranquilizer (Table 2). Failure of . reactivity to noise may reflect the coarse method chosen; 2 of the 5 horses did not have be-

1432

havioral effects when a hay manger was tipped over causing considerable noise. Horse 4, in particular, was outwardly calm, but its pulse nearly trebled over 2 minutes. Only 1 of our horses was male, and we did not observe any effects on the penis that have been observed by others who studied all male sub­jects. This effect reported by other investigators usu­ally is observed about 10 minutes after administration of acepromazine and has persisted throughout study periods.

Pharmacodynamic-pharmacokinetic correlation­N one of the observed pharmacokinetic variables cor­related well with plasma concentration of aceprom­azine. The peak effects were observed at a later time, while the concentration of acepromazine was de­creasing. This might have been attributable to the fact that these pharmacologic effects are mediated by an active metabolite of acepromazine, and thus, there is a lag time for the formation of sufficient amounts of metabolites to exert a noticeable pharmacologic ef­fect, or it might have been attributable to the fact that the receptors are located in a deep pharmacokinetic compartment, so that a finite time would be neces­sary for the drug to reach sufficient concentration in the relevant biophase. These possibilities remain to be investigated by means of appropriate modeling techniques.

Appendix-Rating scale for evaluating sedative ef­fects

Five functions were observed. For movement/ proprioception, scale criteria and scores were: none (collapsed or ready to collapse), 4; very lethargic, 3; slightly affected, 2; normal for environment, 1; more than normal activity for environment, 0. For reaction to noise, scale criteria! and scores were: no reaction, 4; marked effect of drug, 3; slight effect of drug, 2; normal reaction, 1; excess reaction, 0. For reaction to pin prick, scale criteria and scores were: no reaction 4; marked effect of drug, 3; slight effect of drug, 2; normal reaction, 1; excess reaction, 0. For head car­riage scale, criteria and scores were: head down, 4; neck horizontal, 3; head up, but less than normal, 2; normal, 1; excess movement/elevation, 0. For eyelid droop, scale criteria and scores were: closed, 4; half­closed, 3; slight closure, 2; normal, 1; excessively wide-eyed, 0. At each time point, each horse was rated on the five 5-point scales and an average score for that time point was calculated. For Figure 5, the average scores for all of the horses at the respective times were calculated, together with data for the co­efficient of variation at each time point.

References 1. Veterinary phannaceuticals and biologicals, 6th ed, Lenexa,

Kan: Veterinary Medical Publishing Co, 1989;858.

2. Ballard S, Shults T, Kownaski AA, et al. The pharmaco­kinetics, pharmacological responses and behavioral effects of ace­promazine in the horse. J Vet Phannacol Ther 1982;5:21-31.

3. Dewey EA, Maylin G~ Ebel JG, et al. The metabolism of promazine and acetylpromazine in the horse. Drug Metab Dispos 1981;9:30'-36.

Am J Vet Res, Vol 55, No. 10, October 1994

4. Miller PJ, Martin ICA, Kohnke ~ et al. Responses of horses to acepromazine maleate administered orally in a paste. Res Vet Sci 1987;42:318-325.

5. Marroum PJ, Curry SH. Red blood cell partioning, protein binding and lipophilicity of six phenothiazines. f Phann Philrmac.ol 1993;45:39-42.

6. Muir WW, Skarda RT, Sheehan W. Hemodynamic and respiratory effects of a xylazine-acepromazine drug combination in horses. Am] Vet Res 1979;40:1518-1522.

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7. Parry BW, Anderson GA, Gay CC. Hypotension in the horse induced by acepromazine maleate. Aust Vet] 1979;59:148-151.

8. Parry BW, Anderson GA Influence of acepromazine mal­eate on the equine haematocrit.] Vet Pharmac.ol Ther 1983;6:121-126.

9. Mackenzie G, Snow DJ. An evaluation of chemical re­straining agents in the horse. Vet Rec 1977;101:30-33.

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