J. vet. Pharmacol. Therap. 38, 301--304. doi: 10.1111/jvp.12192.

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Pharmacokinetics and physiologic effects of alprazolam after a single oral dose in healthy mares D. M. WONG* J. L. DAVIS



C. J. ALCOTT*

Wong, D. M., Davis, J. L., Alcott, C. J., Hepworth-Warren, K. L., Galow-Kersh, N. L., Rice, S., Coetzee, J. F. Pharmacokinetics and physiologic effects of alprazolam after a single oral dose in healthy mares. J. vet. Pharmacol. Therap. 38, 301–304.

K. L. HEPWORTH-WARREN* N. L. GALOW-KERSH* S. RICE ‡ & J. F. COETZEE ‡ *Department of Veterinary Clinical Sciences, Lloyd Veterinary Medical Center, College of Veterinary Medicine, Iowa State University, Ames, IA, USA; †Department of Veterinary Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; ‡Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA

The objective of this study was to evaluate the pharmacokinetic properties and physiologic effects of a single oral dose of alprazolam in horses. Seven adult female horses received an oral administration of alprazolam at a dosage of 0.04 mg/kg body weight. Blood samples were collected at various time points and assayed for alprazolam and its metabolite, a-hydroxyalprazolam, using liquid chromatography/mass spectrometry. Pharmacokinetic disposition of alprazolam was analyzed by a one-compartmental approach. Mean plasma pharmacokinetic parameters (SD) following single-dose administration of alprazolam were as follows: Cmax 14.76  3.72 ng/mL and area under the curve (AUC0–∞) 358.77  76.26 ngh/mL. Median (range) Tmax was 3 h (1–12 h). Alpha-hydroxyalprazolam concentrations were detected in each horse, although concentrations were low (Cmax 1.36  0.28 ng/mL). Repeat physical examinations and assessment of the degree of sedation and ataxia were performed every 12 h to evaluate for adverse effects. Oral alprazolam tablets were absorbed in adult horses and no clinically relevant adverse events were observed. Further evaluation of repeated dosing and safety of administration of alprazolam to horses is warranted. (Paper received 2 September 2014; accepted for publication 7 November 2014) Jennifer L. Davis, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC 27607, USA. E-mail: [email protected]

Alprazolam (Xanaxâ, Pfizer, New York, NY, USA; 8-chloro-1methyl-6-phenyl-4H-s-triazolo benzodiazepine) is a benzodiazepine derivative used for anxiety disorders, panic attacks, and depression in people, with optimum results achieved at serum concentrations between 20 and 40 ng/mL (Greenblatt et al., 1993; Verster & Volkerts, 2004). The exact mechanism of action of benzodiazepines is unknown, but alprazolam readily crosses the blood brain barrier, enters the central nervous system, and nonselectively binds to the gamma-amino butyric acidA (GABAA)–benzodiazepine receptor complex (Verster & Volkerts, 2004). At the receptor, alprazolam increases the influx of chloride ions resulting in hyperpolarization of neuronal membranes; the inhibition exerted by GABA results in inhibition of neuronal excitability and slowing of brain activity, thus producing sedation (Costa, 1998; Verster & Volkerts, 2004). GABA also interacts with other neurotransmitters (e.g., serotonergic and noradrenergic pathways) resulting in its clinical efficacy as an anxiolytic and antidepressant (Verster & Volkerts, 2004). Pharmacokinetic properties of other benzodi© 2014 John Wiley & Sons Ltd

azepine drugs (diazepam, midazolam) have been investigated in the horse, particularly for use in anesthetic protocols, but no information is available for alprazolam (Muir et al., 1982; Hubbell et al., 2013). The investigators of this study have used oral alprazolam as an adjunctive method to facilitate maternal acceptance of neonatal foals when the mare demonstrates aggression towards her own progeny and to facilitate mare–foal bonding and fostering of orphan foals to nurse mares (Wong, D.M., Alcott, C.J., Davis, J.L., Hepworth, K.L., Wulf, L. & Coet-zee, J.H., under review). Alprazolam may potentially also be used as a sedative/anxiolytic in horses with behavioral abnormalities. The objective of this study is to provide preliminary pharmacokinetic information of a single dose of alprazolam administered orally to healthy mares. Seven healthy adult mares (Quarter Horses [n = 5], Paint Horse [1], Arabian Horse [1]) with a mean age of 11 years (range 3–24 years) and mean body weight of 509 kg (range 444–584 kg) were used in this study. Health status was based on acceptable physical examination parameters, complete blood 301

302 D. M. Wong et al.

counts, and serum biochemistry profiles. Horses were housed in stalls and allowed access to free-choice grass hay and water during the study, which was approved by Iowa State University’s Institutional Animal Care and Use Committee. After a 24-h acclimatization period, base-line (Time 0) physical examination, including assessment of the heart rate, respiratory rate, rectal temperature, and intestinal borborygmi was performed along with a sedation score and ataxia score. These were also reported at 12, 24, 36, and 48 h after drug administration. The sedation and ataxia scores were performed by a single observer based on previously described criteria. Briefly, the sedation score criteria included 0 = no sedation (normal frequency and velocity of movement, ear and neck carriage, eye alertness, stance); 1 = mild sedation (slightly decreased frequency and velocity of movement, lower ear and neck carriage, slight base-wide stance); 2 = moderate sedation (moderately decreased frequency and velocity of movement, obvious ear tip separation, increased base-wide stance, appearance of crossed legs, buckled knees and/or fetlocks); and 3 = deep sedation (markedly decreased frequency and velocity of movement, pronounced ear tip separation, markedly lower neck carriage, markedly increased base-wide stance, increased occurrence and severity of crossed legs, buckled knees, and/or fetlocks) whereas the ataxia score was based on the following criteria evaluated in the box stall and during ambulation: 0 – none; 1 – stable, swaying slightly; 2 – swaying, leaning; and 3 – swaying, leaning, crossing limbs, buckling (Solano et al., 2009; Wojtasiak-Wypart et al., 2012). Each horse was administered 0.04 mg/kg of alprazolam. Tablets were pulverized, mixed in 60 mL of water, and administered via nasogastric tube. The tube was flushed with 1.5 L of water to ensure all medication was delivered. Blood (12 mL) was collected from a jugular catheter just prior to drug administration (Time 0) and at 15, 30, and 45 min and 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h after dosing for determination of alprazolam concentrations. Blood was placed in clot tubes, allowed to clot, centrifuged, and then serum was harvested and stored at 80 °C in plastic vials until further analysis. Concentrations of alprazolam and its active metabolite, a-hydroxyalprazolam, were determined via liquid chromatography–mass spectrometry (LC-MS) in positive ion mode using fragment patterns of parent pseudomolecular ions 309 and 325, respectively, for quantification. Hydroxyalprazolam-D5 was used as an internal standard. Frozen samples or standards were thawed at room temperature and aliquoted into glass tubes. One milliliter of plasma sample containing 1 ng of internal standard was adjusted to pH 9.0. Five milliliters of methyl tert-butyl ether (MTBE) was added to each tube and then mixed and centrifuged at 671 g for 20 min. The organic layer was transferred to a clean glass tube while the remaining aqueous layer was re-extracted with another 5 mL of MTBE, mixed, centrifuged, and the organic layer transferred to a glass collection tube. The samples were evaporated to dryness at 48 °C under a stream of nitrogen, reconstituted with 100 lL 25% (v/v) acetonitrile in water and 50 lL ultrapure water and transferred into an injection vial for LC-MS/MS analysis.

Separation was achieved on a phenyl–hexyl column and guard column with mobile phases consisting of acetonitrile and water, each containing 0.1% formic acid. Using this method, standard curves were linear from 0.2 to 50 ng/mL for both compounds, with a coefficient of determination (R2) of >0.99 and measured values were within 15% of the actual values. Data were subjected to compartmental modeling (Phoenix WinNonlin 6.1; Pharsight Corporation, Cary, NC, USA) using an extravascular one-compartment model. This model was chosen based on visual inspection of a log-linear plot of the concentration vs. time curves to determine the best-fit model, as well as analysis of the Akaike’s inclusion criteria. Parameters reported include area under the serum concentration vs. time curve extrapolated to infinity (AUC0–∞), absorption and elimination rate constants, and half-life of absorption and elimination (k01 t1/2 and k10 t1/2, respectively). The true bioavailability (F) of alprazolam could not be calculated due to the lack of an accompanying i.v. dose. Therefore, results for volume of distribution and clearance are reported as Vd/F and Cl/ F, respectively. Values for maximum plasma concentration (Cmax) and time to maximum concentration (Tmax) are reported directly from the data. Using parameters derived from initial pharmacokinetic models, computerized dosing simulations were run to determine a dosage regimen that would result in serum concentrations within the therapeutic range determined for humans (20–40 ng/mL). Repeated measures ANOVA followed by post hoc pairwise comparison with Tukey adjustment was used to evaluate for differences in temperature, respiration rate and heart rate across time. Friedman’s test and post hoc Wilcoxon signed rank sum test were used to evaluate sedation and ataxia scores. Alprazolam was rapidly detected in serum, with concentrations above the LOQ at 15 min in four of seven horses and 30 min in all horses. The metabolite was detected in all horses, starting between 30 min and 2 h after parent drug administration. Alprazolam and a-hydroxyalprazolam serum concentration vs. time curves are depicted in Fig. 1. Maximum serum concentrations of alprazolam (14.76  3.72 ng/mL) occurred

Fig. 1. Alprazolam and a-hydroxyalprazolam concentration–time profiles after oral administration. Values are means  SD (n = 7). © 2014 John Wiley & Sons Ltd

Alprazolam in horses 303 Table 1. One-compartmental pharmacokinetic parameters for alprazolam (0.04 mg/kg) and its metabolite a-hydroxyalprazolam given orally to healthy adult horses (n = 7) a-hydroxyalprazolam

Alprazolam

Pharmacokinetic variable

Mean

Tmax (h)* Cmax (ng/mL) k01 (h 1) k10 (h 1) k01 t1/2 (h) k10 t1/2 (h) AUC0–∞ (hng/mL) Vd/F (L/kg) Cl/F (mL/kg/min)

3† 14.76† 1.42 0.04 0.55 16.35 358.77 2.66 1.93

SD

Mean

SD

1–12 3.72 0.58 0.01 0.19 3.63 76.26 0.46 0.43

12.29† 1.36† 0.24 0.04 5.89 28.38 71.11 – –

5.71 0.28 0.26 0.02 4.04 30.42 65.33 – –

Tmax, time to maximum concentration; Cmax, maximum concentration; k01, first-order absorption rate constant; k10, first-order elimination rate constant; k01 t1/2, half-life of absorption; k10 t1/2, half-life of elimination; AUC0–∞, area under the concentration–time curve extrapolated to infinity; Vd/F, apparent volume of distribution dependent on bioavailability; Cl/F, clearance dependent on bioavailability. *Tmax reported as median and range. †Derived from the plasma concentration curves.

Fig. 2. Nonparametric superposition modeling of repeated dosing of alprazolam to maintain concentrations between 20 and 40 ng/mL. In this scenario, a loading dose of 0.1 mg/kg was used, followed by 0.04 mg/kg q12 h.

at a median time of 3 h (range 1–12 h). The elimination half-life of alprazolam in horses is long at 16.35  3.63 h. Maximum a-hydroxyalprazolam concentrations reached 1.36  0.28 ng/mL at a median time of 12 h (range 6–24 h) and metabolite concentrations accounted for only 16.5% of the total AUC. Other pertinent pharmacokinetic parameters are summarized in Table 1. Based on computer simulations, a loading dose of 0.1 mg/kg, followed by 0.04 mg/kg q12 h orally would result in serum concentrations of alprazolam between 20 and 40 ng/mL (Fig. 2); however, it must be clearly recognized that this therapeutic range is established for people and the effects of this drug concentration range has not be investigated in horses. Mean values for physical examination parameters are reported in Table 2. No adverse events were noted in any horse although two horses developed fevers (102.2–104.2°F) between the 24 and 48 h time points; collectively, however, no significant difference in rectal temperature was documented and these two horses appeared clinically healthy. A statistically significant increase in heart rate was noted between baseline and 12 h (P = 0.0016). No statistical differences in respiratory rate were detected across time. Sedation and ataxia scores were 0 for all time points except for the 12 h time. The median sedation and ataxia scores at 12 h were both 0 with a range of 0–1, indicating slight changes in behavior in some horses. No statistically significant differences were detected with the sedation score, but a significant difference was detected for the ataxia score across time (P = 0.017). However, post hoc Wilcoxon signed rank sum test comparing time 0 to each time point (12, 24, 36, 48 h) revealed no significant differences between time 0 and specific time points. Results of this study indicate that alprazolam is absorbed following oral administration in adult horses, with minimal induction of sedation and ataxia at a dose of 0.04 mg/kg. In people, alprazolam has a fast onset of action and excellent oral bioavailability with 90% of alprazolam absorbed after oral administration; a single 1 mg dose of alprazolam resulted in a Cmax between 12 and 22 ng/mL within 1–2 h after administration (Verster & Volkerts, 2004). The study reported here confirms that a single dose of oral alprazolam results in similar serum concentrations in horses but a median time of 3 h is necessary to reach this concentration. Although a therapeutic dose has not been established in horses, alprazolam has been

Table 2. Physical examination parameters along with sedation and ataxia scores in seven adult mares orally administered 0.04 mg/kg of alprazolam Parameter Temperature (°F) Heart rate (beats/min) Respiratory rate (resp/min) Sedation score Ataxia score

Baseline

12 h

24 h

36 h

48 h

99.4  0.4 41  6 25  12 0 0

100.6  1.1 47  8* 17  3 0 (0–1) 0 (0–1)

100.6  1.2 43  7 19  6 0 0

100.7  2 41  9 18  5 0 0

100.6  1.7 42  9 16  3 0 0

Results for temperature, heart rate, and respiratory rate are reported as mean  standard deviation; results for sedation and ataxia score reported as median and range. *Significant difference between baseline and 24 h. © 2014 John Wiley & Sons Ltd

304 D. M. Wong et al.

used to successfully modulate aggressive behavior in a mare towards her foal at a dose of 0.035 mg/kg p.o. q8–12 h which resulted in serum concentrations ranging between 16 and 35 ng/mL (Wong, D.M., Alcott, C.J., Davis, J.L., Hepworth, K.L., Wulf, L. & Coet-zee, J.H., under review). Given the long half-life of alprazolam noted here, steady-state concentrations would not be reached until approximately 81 h (3.5 days). For animals with more aggressive behavior or anxiety, a loading dose may help reach therapeutic concentrations more rapidly, allowing for a faster effect. Based on results of this report, a suggested loading dose is 0.1 mg/kg p.o., followed by maintenance therapy of 0.04 mg/kg p.o. q12 h. This higher dose has not been investigated and might potentially induce sedation, thus it is likely the dosage regimen may need to be tapered for the individual horse and the proposed purpose. In people, the potency of a-hydroxyalprazolam is approximately half that of alprazolam (Package Insert). This fact, combined with the low serum concentrations of the metabolite measured in horses make it unlikely that the metabolite significantly contributes to the overall effectiveness of alprazolam in horses. In this study, the single oral dose of alprazolam was well tolerated in adult horses with no changes in physical examination parameters considered clinically or biologically important. Two mares developed transient fever, but were otherwise alert and healthy. The exact cause of the fever is unknown, but may be related to acquisition of a respiratory virus from another horse, as the study horses were exposed to a number of other horses in the hospital. Fever is not a reported side effect of any of the benzodiazepines, including alprazolam, when administrated to people; thus, fever in these two mares as a result of alprazolam administration is unlikely (Jonas & Cohon, 1993; Charney et al., 2001; Verster & Volkerts, 2004). Side effects of alprazolam administration in people include drowsiness, fatigue, weakness, ataxia and sedation (Jonas & Cohon, 1993; Verster & Volkerts, 2004). Based on the study reported here, mild sedation and ataxia may occur with a single administration of alprazolam at the dose used in this study. Sedation was reported after multiple doses of alprazolam administration in a case report involving an aggressive mare (Wong, D.M., Alcott, C.J., Davis, J.L., Hepworth, K.L., Wulf, L. & Coet-zee, J.H., under review). Therefore, it is possible that

multiple oral doses of alprazolam may result in sedation and ataxia and warrants further study. Nonetheless, alprazolam has a wide safety margin evidenced by the very high median lethal dose of alprazolam that will kill 50% of rats (LD50 331– 2171 mg/kg; Package Insert). In conclusion, a single oral dose of alprazolam was bioavailable and tolerated in horses. Additional investigations evaluating absorption, pharmacokinetics, efficacy and safety with repeated dosing are warranted.

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© 2014 John Wiley & Sons Ltd

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Pharmacokinetics and physiologic effects of alprazolam after a single oral dose in healthy mares.

The objective of this study was to evaluate the pharmacokinetic properties and physiologic effects of a single oral dose of alprazolam in horses. Seve...
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