Benzodiazepines are commonly used during the perioperative period as preanesthetics, adjunct anesthesia induction agents, or both.1–4 Some anesthetists favor the use of midazolam over other sedatives because of its minimal effects on the cardiopulmonary system and its muscle relaxant and antiseizure properties, and because it allows for dose reduction of other induction and anesthetic agents.4–7 Midazolam increases the GABAA receptor channel opening frequency by enhancing the activity of GABA on the GABAA receptor complex. This activity increases chloride conductance through the GABAA receptor, which hyperpolarizes cell membranes and reduces neuronal excitability.5,8 Although benzodiazepines are sedatives, a paradoxical excitatory effect has been reported when those drugs are administered alone to humans,9,10 cats,11 horses,12 and dogs.13,14
In small ruminants, benzodiazepines provide reliable sedation with minimal cardiovascular and gastrointestinal tract effects.15,16 In sheep, midazolam in particular provides mediation of antinociception at the level of the spinal cord.17,18 Therefore, in small ruminants, benzodiazepines are commonly chosen for use as premedications before surgical procedures or sedatives for diagnostic procedures.
The pharmacokinetics of midazolam following IV administration has been described in humans,19,20 dogs,13,21 alpacas,22 horses,12 rabbits,23 and pigs24 and after IM administration in humans,25 dogs,21 alpacas,22 and guinea pigs.26 To our knowledge, the pharmacokinetics of midazolam has not been described in sheep. The objective of the study reported here was to determine the pharmacokinetics and pharmacodynamics of midazolam following IV and IM administration of a single dose to sheep. We hypothesized that, in healthy sheep, the absorption of midazolam following IM administration would be rapid and complete and that IV administration of midazolam would result in more profound ataxia and sedation of shorter duration, compared with IM administration of the drug.
Materials and Methods
Animals
All study protocols were reviewed and approved by the Ross University School of Veterinary Medicine Institutional Animal Care and Use Committee. The study was conducted during the period of December 2014 through February 2015 on the island of St Kitts. Eight healthy adult mixed-breed rams with a mean ± SD body weight of 37.4 ± 2.3 kg were used for the study. Each sheep was determined to be healthy on the basis of clinical history and the results of a physical examination, CBC, and serum biochemical analysis. Prior to study initiation, sheep were dewormed, vaccinated, and housed as a group in a covered outside pen with ad libitum access to hay and water for an acclimation period of 3 weeks.
Study design
The study had a block-assigned, crossover design. Sheep were administered midazolam (0.5 mg/kg) by the IV route (MIV treatment) initially and 7 days later were administered the same dose of the drug by the IM route (MIM treatment). The dose of midazolam administered was chosen on the basis of the investigators’ clinical experience with use of the drug for sedating and anesthetizing sheep. For each sheep following midazolam administration, blood samples were collected for determination of plasma midazolam and 1-hydroxymidazolam concentrations and heart rate, respiratory rate, rectal temperature, and levels of sedation and ataxia were assessed at predetermined times for 1,440 minutes (24 hours).
Study protocol
The morning of midazolam administration, each sheep was individually weighed on an electronic standing scale. They were then moved to a sampling pen (4 × 4 m), which was adjacent to the pen where they were housed. Each sheep was loosely restrained with a halter and lead rope to facilitate sample collection and assessment before and for 90 minutes after midazolam administration, after which they were allowed to roam unrestrained in the pen and provided free access to hay and water. At least 3 sheep were restrained side by side to reduce stress associated with separation from flock mates. The restraint did not hinder assessment of sedation or ataxia because the sheep had sufficient room to lie down and sway.
For each sheep prior to the MIV treatment, the hair over each jugular vein was clipped, and an 18-gauge, 3-inch cathetera was aseptically placed in each vein. When the MIM treatment was administered, a catheter was aseptically placed only in the left jugular vein. All catheters were secured with tissue adhesive and sutured to the skin. The catheters placed in the left jugular vein were used exclusively for collection of blood samples. The catheters placed in the right jugular vein were used to administer midazolam during the MIV treatment and were removed 30 minutes after drug administration.
For the MIV treatment, midazolam hydrochlorideb (0.5 mg/kg) was administered via the catheter in the right jugular vein over 15 seconds, after which the catheter was flushed with 5 mL of heparinized saline (0.9% NaCl) solution to ensure that none of the drug was left in the catheter. For the MIM treatment, midazolam (0.5 mg/kg) was injected into the cervical muscles on the right side of the neck.
For each sheep, a blood sample (8 mL) was obtained immediately before (baseline; 0 minutes) and at 3, 5, 10, 15, 30, 45, 60, 90, 120, 240, 480, 720, and 1,440 minutes after midazolam administration. All samples except those collected at 720 and 1,440 minutes after midazolam administration were collected from the catheter in the left jugular vein. To avoid sample dilution, 3 mL of blood (presample blood) was aspirated into a 3-mL syringe that had been flushed with heparinized saline solution (heparinized syringe). The blood sample for analysis was aspirated into a sterile unheparinized 10-mL syringe. Then, the presample blood was administered back through the catheter, and the catheter was flushed with 3 mL of heparinized saline solution. The blood samples collected at 720 and 1,440 minutes after midazolam administration were obtained by venipuncture of the left jugular vein cranial to the catheter site with a 20-gauge, 1-inch needle attached to a sterile unheparinized 10-mL syringe. All blood samples were immediately transferred to blood collection tubesc that contained lithium heparin as an anticoagulant and stored on crushed ice prior to centrifugation. Samples were centrifuged at 1,000 × g for 10 minutes within 1 hour after collection. Plasma was harvested from each sample and stored at −80°C until analyzed for midazolam and 1-hyrdoxymidazolam concentrations.
For each sheep, heart and respiratory rates were determined at baseline and 5, 10, 15, 30, 45, 60, 90, 120, 240, 480, 720, and 1,440 minutes after midazolam administration. Heart rate was determined by cardiac auscultation, and respiratory rate was determined by visual observation of thoracic excursion; each was counted for 30 seconds at each designated time. Rectal temperature was measured with a digital thermometer at baseline and 15, 45, 90, 240, 480, 720, and 1,440 minutes after midazolam administration.
The levels of sedation and ataxia were recorded for each sheep at baseline and 3, 5, 10, 15, 30, 45, 60, 90, 120, 240, 480, 720, and 1,440 minutes after midazolam administration. Sedation (Appendix 1) and ataxia (Appendix 2) were semiquantitatively assessed by use of 5-point scoring systems adapted from those used to assess sedation and ataxia in horses following midazolam administration in another study.12 All scores were assigned by the same investigator (BTS) prior to measurement of heart rate, respiratory rate, and rectal temperature and blood sample collection.
Determination of plasma midazolam and 1-hydroxymidazolam concentrations
Plasma concentrations of midazolam and its metabolite, 1-hydroxymidazolam, were determined by use of a reverse-phase HPLC system that consisted of an integrated solvent and sample management moduled and UV detector.e Separation was attained with a 5-μm C18 columnf (3.9 × 150 mm) protected by a 5-μm C18 guard column.f The mobile phase was an isocratic mixture of 0.01M sodium acetate (pH, 4.7) with concentrated glacial acetic acid and acetonitrile (66:34). The isocratic mixture was prepared daily with double-distilled deionized water that was passed through a 0.22-μm filter and degassed before use. The flow rate was 1.3 mL/min, and UV absorbance was measured at 220 nm.
Frozen plasma samples were thawed and vortexed. For each sample, 1 mL of plasma was mixed with 250 μL of 7.5M sodium hydroxide and 6 mL of methylene chloride in a 15-mL screw-cap tube. The tube was capped and rocked for 30 minutes and then centrifuged for 20 minutes at 1,000 × g. The organic layer was removed and placed into a clean glass tube and evaporated with nitrogen gas. The residue was reconstituted with 250 μL of the mobile phase mixture and placed in HPLC vials; 100 μL of the resulting mixture was injected into the HPLC system.
Standard curves were prepared for both midazolam and 1-hydroxymidazolam by spiking blank plasma samples with either the drug or metabolite to achieve a linear concentration range of 10 to 2,500 ng/mL. The mean recovery was 94% for both midazolam and 1-hydroxymidazolam. Intra-assay variability ranged from 0.6% to 5.0% for midazolam and 0.8% to 5.7% for 1-hydroxymidazolam, and interassay variability ranged from 1.8% to 8.6% for midazolam and 3.9% to 7.6% for 1-hydroxymidazolam. The lower limit of quantification was 10 ng/mL for midazolam and 5 ng/mL for 1-hydroxymidazolam.
Pharmacokinetic analysis
Plasma midazolam and 1-hydroxymidazolam concentrations were analyzed for each study subject by compartmental and noncompartmental methods with a commercially available pharmacokinetics software program.g The goodness of fit of the proposed models was assessed by visual examination of line fits and residual plots and the Akaike information criterion as described.27 Data were weighted by use of the 1/yhat2 method (where yhat is the variance of the plasma concentration of the drug or metabolite of interest) to improve the line fit and residual plots. For the MIM treatment, plasma midazolam and 1-hydroxymidazolam data were analyzed with noncompartmental analysis. For the MIV treatment, plasma midazolam and 1-hydroxymidazolam data were analyzed with noncompartmental and compartmental analyses. These analyses yielded values for the elimination rate constant, plasma t1/2, Cmax, tmax, apparent volume of distribution based on the AUC, apparent volume of distribution at steady state, total body clearance, and AUC0-∞. The AUCs and area under the first moment curves were calculated by use of the log-linear trapezoidal rule. The MRT was calculated as the area under the first moment curve from time 0 to infinity divided by the AUC0-∞. The absolute systemic bioavailability of midazolam was calculated as the AUC0-∞ for the MIM treatment divided by the AUC0-∞ for the MIV treatment.
Statistical analysis
The data distributions for the variables and parameters assessed were evaluated for normality by use of the Shapiro-Wilk test. Normally distributed variables and parameters were reported as the mean ± SD with the exception of t1/2, which was reported as the harmonic mean ± pseudo-SD. Ordinal variables were reported as the median (range) and proportion of population. Two-way repeated-measures ANOVA models followed by Bonferroni tests for multiple comparisons were used to compare heart rate, respiratory rate, and rectal temperature over time after midazolam administration (time) within each treatment (MIV and MIM) and to compare sedation and ataxia scores between the 2 treatments and over time. The interaction between treatment and time was evaluated in all models. The trapezoidal method was used to calculate the AUC for time 0 to 15 minutes (AUC0–15), AUC for time 15 to 60 minutes (AUC15–60), and AUC for time 60 to 1,440 minutes (AUC60–1,440) for both sedation and ataxia, and those parameters were compared between treatments by use of the Friedman test followed by the Dunn test. Times to peak sedation and ataxia were compared between treatments by use of Mantel-Cox survival analysis. Sigmoidal concentration-response curves were constructed for sedation and ataxia scores. The EC50s, Hill slopes, and associated 95% CIs for sedation and ataxia were computed and compared between treatments by use of the extra sum-of-squares F test. Coefficients of determination (R2) were also determined for each treatment. All analyses were performed with commercially available statistical software,h and values of P ≤ 0.05 were considered significant.
Results
Pharmacokinetics
The mean plasma midazolam and 1-hydroxymidazolam concentrations over time for the MIV and MIM treatments were plotted (Figure 1). For both the MIV and MIM treatments, midazolam was detected in the plasma of all 8 sheep 3 minutes after drug administration. 1-Hydroxymidazolam was first detected in the plasma of 7 and 1 sheep at 3 and 5 minutes, respectively, after IV administration of midazolam and in the plasma of 5, 2, and 1 sheep at 10, 15, and 30 minutes, respectively, after IM administration of midazolam. For the MIV treatment, midazolam and 1-hydroxymidazolam were last detected in the plasma of 7 sheep at 120 and 240 minutes, respectively, after drug administration. After MIV treatment, 1 sheep had midazolam and 1-hydroxymidazolam last detected in the plasma at 480 minutes. For the MIM treatment, midazolam and 1-hydroxymidazolam were detected in the plasma of all 8 sheep at 240 minutes after drug administration; however, at 480 minutes after drug administration, only 2 and 4 sheep had detectable plasma concentrations of midazolam and 1-hydroxymidazolam, respectively.
Mean ± SD plasma midazolam (triangles) and 1-hydroxymidzolam (circles) concentrations for 8 healthy adult mixed-breed rams at various times after administration of midazolam (0.5 mg/kg) by the IV route (MIV treatment; A) and then by the IM route (MIM treatment; B) 7 days later in a crossover study. Midazolam administration was designated 0 minutes. Midazolam and 1-hydroxymidazolam could not be quantified in all samples at all times. In panel A, the mean ± SD plasma midazolam concentration represented values for all 8 sheep at 3, 5, 10, 15, 30, 45, 60, and 90 minutes; 7 sheep at 120 minutes; and 4 sheep at 240 minutes; and the mean ± SD 1-hydroxymidazolam concentration represented values for 7 sheep at 3 and 120 minutes; 8 sheep at 5, 10, 15, 30, 45, 60, and 90 minutes; and 3 sheep at 240 minutes. In panel B, the mean ± SD plasma midazolam concentration represented values for all 8 sheep at all times up to 240 minutes and 2 sheep at 480 minutes; and the mean ± SD 1-hydroxymidazolam concentration represented values for 5 sheep at 10 minutes, 7 sheep at 15 minutes, 4 sheep at 480 minutes, and all 8 sheep all other times.
Citation: American Journal of Veterinary Research 78, 5; 10.2460/ajvr.78.5.539
Mean ± SD plasma midazolam (triangles) and 1-hydroxymidzolam (circles) concentrations for 8 healthy adult mixed-breed rams at various times after administration of midazolam (0.5 mg/kg) by the IV route (MIV treatment; A) and then by the IM route (MIM treatment; B) 7 days later in a crossover study. Midazolam administration was designated 0 minutes. Midazolam and 1-hydroxymidazolam could not be quantified in all samples at all times. In panel A, the mean ± SD plasma midazolam concentration represented values for all 8 sheep at 3, 5, 10, 15, 30, 45, 60, and 90 minutes; 7 sheep at 120 minutes; and 4 sheep at 240 minutes; and the mean ± SD 1-hydroxymidazolam concentration represented values for 7 sheep at 3 and 120 minutes; 8 sheep at 5, 10, 15, 30, 45, 60, and 90 minutes; and 3 sheep at 240 minutes. In panel B, the mean ± SD plasma midazolam concentration represented values for all 8 sheep at all times up to 240 minutes and 2 sheep at 480 minutes; and the mean ± SD 1-hydroxymidazolam concentration represented values for 5 sheep at 10 minutes, 7 sheep at 15 minutes, 4 sheep at 480 minutes, and all 8 sheep all other times.
Citation: American Journal of Veterinary Research 78, 5; 10.2460/ajvr.78.5.539
Mean ± SD plasma midazolam (triangles) and 1-hydroxymidzolam (circles) concentrations for 8 healthy adult mixed-breed rams at various times after administration of midazolam (0.5 mg/kg) by the IV route (MIV treatment; A) and then by the IM route (MIM treatment; B) 7 days later in a crossover study. Midazolam administration was designated 0 minutes. Midazolam and 1-hydroxymidazolam could not be quantified in all samples at all times. In panel A, the mean ± SD plasma midazolam concentration represented values for all 8 sheep at 3, 5, 10, 15, 30, 45, 60, and 90 minutes; 7 sheep at 120 minutes; and 4 sheep at 240 minutes; and the mean ± SD 1-hydroxymidazolam concentration represented values for 7 sheep at 3 and 120 minutes; 8 sheep at 5, 10, 15, 30, 45, 60, and 90 minutes; and 3 sheep at 240 minutes. In panel B, the mean ± SD plasma midazolam concentration represented values for all 8 sheep at all times up to 240 minutes and 2 sheep at 480 minutes; and the mean ± SD 1-hydroxymidazolam concentration represented values for 5 sheep at 10 minutes, 7 sheep at 15 minutes, 4 sheep at 480 minutes, and all 8 sheep all other times.
Citation: American Journal of Veterinary Research 78, 5; 10.2460/ajvr.78.5.539
The pharmacokinetic parameters for midazolam were summarized (Table 1). The harmonic mean ± pseudo-SD t1/2 for midazolam (0.47 ± 0.19 hours) and 1-hydroxymidazolam (0.68 ± 0.29 hours) for the MIV treatment was approximately half the harmonic mean ± pseudo-SD t1/2 of midazolam (0.94 ± 0.23 hours) and 1-hydroxymidazolam (1.19 ± 0.43 hours) for the MIM treatment, which suggested that plasma concentrations of midazolam and 1-hydroxymidazolam declined twice as fast after IV administration of midazolam, compared with IM administration of the drug. The mean ± SD AUC0-∞ for midazolam (1,396 ± 463 h·ng/mL) and 1-hydroxymidazolam (1,995 ± 993 h·ng/mL) for the MIM treatment was approximately 3 times that for midazolam (423 ± 143 h·ng/mL) and 1-hyrdoxymidazolam (477 ± 216 h·ng/mL) for the MIV treatment. Thus, the mean ± SD systemic bioavailability of midazolam was very high (352 ± 148%). The mean ± SD MRT for midazolam (1.59 ± 0.35 hours) and 1-hydroxymidazolam (2.39 ± 0.68 hours) for the MIM treatment was approximately twice that for midazolam (0.66 ± 0.19 hours) and 1-hydroxymidazolam (1.21 ± 0.42 hours) for the MIV treatment.
Pharmacokinetic parameters for midazolam and 1-hydroxymidazolam in the plasma of 8 healthy adult mixed-breed rams following administration of midazolam (0.5 mg/kg) by the IV route (MIV) and then by the IM route (MIM) 7 days later in a crossover study.
| MIV | MIM | |||
|---|---|---|---|---|
| Parameter | Midazolam | 1-Hydroxymidazolam | Midazolam | 1-Hydroxymidazolam |
| t1/2 (h) | 0.47 ± 0.19* | 0.68 ± 0.29* | 0.94 ± 0.23* | 1.19 ± 0.43* |
| λz (1/h) | 1.46 ± 0.56 | 1.02 ± 0.44 | 0.74 ± 0.18 | 0.58 ± 0.21 |
| C0 (ng/mL) | 1,390 ± 574 | — | — | — |
| tmax (h) | — | 0.28 ± 0.23 | 0.46 ± 0.26 | 1.19 ± 0.44 |
| Cmax (ng/mL) | — | 422 ± 184 | 820 ± 268 | 785 ± 418 |
| CL (mL/h/kg) | 1,272 ± 310 | — | — | — |
| Vd(ss) (mL/kg) | 838 ± 330 | — | — | — |
| Vd(area) (mL/kg) | 1,015 ± 560 | — | — | — |
| AUC0-∞ (h·ng/mL) | 423 ± 143 | 477 ± 216 | 1,396 ± 463 | 1,995 ± 993 |
| MRT0-∞ (h) | 0.66 ± 0.19 | 1.21 ± 0.42 | 1.59 ± 0.35 | 2.39 ± 0.68 |
| A (ng/mL) | 1,116 ± 722 | — | — | — |
| B (ng/mL) | 360 ± 223 | — | — | — |
| α (1/h) | 12.42 ± 8.44 | — | — | — |
| β (1/h) | 1.11 ± 0.52 | — | — | — |
| t1/2α (h) | 0.09 ± 0.07* | — | — | — |
| t1/2β (h) | 0.79 ± 0.44* | — | — | — |
| F (%) | — | — | 352 ± 148 | — |
Values represent the mean ± SD unless otherwise indicated.
Harmonic mean ± pseudo-SD.
α = Distribution constant. β = Elimination constant. λz = Elimination rate constant. A = Distribution rate intercept. B = Elimination rate intercept. C0 = Plasma drug concentration extrapolated to time 0. Ci = Clearance. F = Absolute systemic bioavailability. t1/2α = Distribution half-life. t1/2β = Elimination half-life. Vd(area) = Apparent volume of distribution based on AUC. Vd(ss) = Volume of distribution at steady state. — = Not calculated.
Physiologic variables
Descriptive data for heart rate, respiratory rate, and rectal temperature immediately before and at various times after midazolam administration were summarized (Table 2). Results of the 2-way repeated-measures ANOVA indicated that sheep, treatment, and the interaction between treatment and time accounted for 11%, 44%, and 8%, respectively, of the total variation for heart rate, all of which were significant (P < 0.001). The mean heart rate did not differ significantly from baseline at any time after midazolam administration for either treatment.
Summary statistics for heart rate, respiratory rate, rectal temperature, sedation, and ataxia in 8 healthy adult mixed-breed rams immediately before (baseline; 0 minutes) and at various times following administration of midazolam (0.5 mg/kg) by the IV route (MIV) and then by the IM route (MIM) 7 days later in a crossover study.
| Time after midazolam administration (min) | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Variable | Treatment | 0 | 3 | 5 | 10 | 15 | 30 | 45 | 60 | 90 | 120 | 240 | 480 | 720 | 1,440 |
| Heart rate | MIV | 93 ± 9 | — | 109 ± 13 | 109 ± 26 | 110 ± 19 | 113 ± 24 | 104 ± 14 | 101 ± 13 | 86 ± 13 | 91 ± 11 | 96 ± 12 | 106 ± 15 | 96 ± 13 | 99 ± 24 |
| (beats/min) | MIM | 64 ± 13 | — | 66 ± 13 | 67 ± 10 | 67 ± 18 | 75 ± 14 | 71 ± 12 | 70 ± 11 | 84 ± 18 | 68 ± 8 | 81 ± 17 | 76 ± 11 | 82 ± 19 | 69 ± 14 |
| Respiratory | MIV | 66 ± 20 | — | 35 ± 12* | 37 ± 12* | 34 ± 13* | 53 ± 15 | 48 ± 26 | 44 ± 14* | 57 ± 23 | 63 ± 20 | 70 ± 21 | 54 ± 18 | 48 ± 11 | 55 ± 15 |
| rate (breaths/min) | MIM | 32 ± 13 | — | 30 ± 8 | 36 ± 10 | 32 ± 16 | 33 ± 11 | 32 ± 5 | 33 ± 10 | 34 ± 6 | 36 ± 16 | 46 ± 27 | 40 ± 14 | 35 ± 13 | 32 ± 7 |
| Rectal | MIV | 39.8 ± 0.2 | — | — | — | 39.6 ± 0.3 | — | 39.5 ± 0.3 | — | 39.7 ± 0.3 | — | 39.8 ± 0.3 | 39.6 ± 0.4 | 39.5 ± 0.5 | 39.2 ± 0.2* |
| temperature (°C) | MIM | 38.8 ± 0.2 | — | — | — | 38.9 ± 0.4 | — | 38.9 ± 0.6 | — | 39.1 ± 0.5 | — | 39.4 ± 0.2* | 39.3 ± 0.3* | 39.6 ± 0.6* | 38.9 ± 0.5 |
| Sedation score | MIV | 0 (0–0) | 3.5 (1–4)*† | 4 (2–4)*† | 3.5 (2–4)*† | 3.5 (2–4*† | 0.5 (0–2) | 0.5 (0–2) | 0 (0–1) | 0 (0–1) | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0 (0–0) |
| MIM | 0 (0–0) | 0 (0–1) | 0 (0–1) | 0 (0–1) | 0 (0–1) | 1 (0–2)* | 0 (0–2) | 0 (0–1) | 0 (0–1) | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0 (0–0) | |
| Ataxia score | MIV | 0 (0–0) | 4 (3–4)*† | 4 (3–4)*† | 4 (4–4)*† | 4 (1–4)*† | 1.5 (1–2)*1 | (0–1)* | 0.5 (0–1) | 0 (0–1) | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0 (0–0) |
| MIM | 0 (0–0) | 0 (0–0) | 0 (0–1) | 1 (0–2)* | 1 (0–3)* | 1 (0–2)* | 1 (0–2)* | 1 (0–1)* | 0 (0–1) | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0 (0–0) | |
Values represent the mean ± SD or median (range). At each data acquisition time, each sheep was assigned semiquantitative sedation and ataxia scores on a scale of 0 to 4 (where 0 = no sedation or ataxia and 4 = profound sedation or ataxia) by the same investigator. The sedation and ataxia scores were assigned prior to measurement of heart rate, respiratory rate, and rectal temperature and blood sample collection.
Within a row, value differs significantly (P ≤ 0.05) from the baseline value.
Within a time within a variable, value for the MIV treatment differs significantly (P ≤ 0.05) from the corresponding value for the MIM treatment.
— = Not measured.
Sheep, treatment, time, and the interaction between treatment and time accounted for 20%, 19%, 13%, and 7%, respectively, of the total variation for respiratory rate, all of which were significant (P ≤ 0.009). For the MIV treatment, the mean respiratory rate at 5, 10, 15, and 60 minutes after drug administration was significantly decreased from that at baseline (P < 0.001 for all comparisons) and 240 minutes after drug administration (P ≤ 0.018 for all comparisons); similarly, the mean respiratory rate at 5, 10, and 15 minutes after drug administration was significantly (P ≤ 0.018 for all comparisons) decreased from that at 120 minutes after drug administration. The mean respiratory rate did not differ significantly among any of the data acquisition times for the MIM treatment.
Sheep, treatment, time, and the interaction between treatment and time accounted for 24%, 21%, 10 %, and 12%, respectively, of the total variation for rectal temperature, all of which were significant (P < 0.004). For the MIV treatment, the mean rectal temperature at 1,440 minutes after drug administration was significantly (P = 0.002) decreased from that at baseline. For the MIM treatment, the mean rectal temperature at 240, 480, and 720 minutes after drug administration was significantly (P ≤ 0.032 for all comparisons) increased, compared with that at baseline and 15, 45, and 1,440 minutes after drug administration; similarly, the mean rectal temperature at 720 minutes after drug administration was significantly (P = 0.032) increased from that at 90 minutes after drug administration.
Behavioral variables
Descriptive data (Table 2) and the frequency distribution of the semiquantitative scores for sedation (Table 3) and ataxia (Table 4) immediately before and at various times after midazolam administration were summarized. The respective relationships between plasma midazolam concentration and sedation and ataxia scores were plotted (Figure 2). Intravenous administration of midazolam resulted in profound sedation (lateral or sternal recumbency; sedation score, 4) within 5 minutes for 5 of the 8 sheep, and all sheep regained the ability to stand within 30 minutes after drug administration. Conversely, none of the sheep became profoundly sedate following IM administration of midazolam. Treatment, time, and the interaction between treatment and time accounted for 13%, 40%, and 31%, respectively, of the total variation for sedation score, all of which were significant (P ≤ 0.001). For the MIV treatment, the median sedation score was significantly (P < 0.001 for all comparisons) greater than that at baseline at 3, 5, 10, and 15 minutes after drug administration. For the MIM treatment, the median sedation score was significantly (P = 0.013) greater than that at baseline only at 30 minutes after drug administration. The median sedation score for the MIV treatment was significantly (P < 0.001 for all comparisons) greater than that for the MIM treatment at 3, 5, 10, and 15 minutes after drug administration.
Concentration-response plots that depict individual data points and sigmoidal curves (solid lines) and associated 95% CIs (dotted lines) for sedation (A) and ataxia (B) following IV (asterisks) and IM (circles) administration of midazolam (0.5 mg/kg) to the 8 healthy adult mixed-breed rams of Figure 1. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 78, 5; 10.2460/ajvr.78.5.539
Concentration-response plots that depict individual data points and sigmoidal curves (solid lines) and associated 95% CIs (dotted lines) for sedation (A) and ataxia (B) following IV (asterisks) and IM (circles) administration of midazolam (0.5 mg/kg) to the 8 healthy adult mixed-breed rams of Figure 1. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 78, 5; 10.2460/ajvr.78.5.539
Concentration-response plots that depict individual data points and sigmoidal curves (solid lines) and associated 95% CIs (dotted lines) for sedation (A) and ataxia (B) following IV (asterisks) and IM (circles) administration of midazolam (0.5 mg/kg) to the 8 healthy adult mixed-breed rams of Figure 1. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 78, 5; 10.2460/ajvr.78.5.539
Number of rams from Table 2 assigned each sedation score immediately before (baseline; 0 minutes) and at various times following midazolam administration.
| Time after midazolam administration (min) | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Score | Treatment | 0 | 3 | 5 | 10 | 15 | 30 | 45 | 60 | 90 | 120 | 240 | 480 | 720 | 1,440 |
| 0 | MIV | 8 | 0*† | 0*† | 0*† | 0*† | 4 | 4 | 5 | 7 | 8 | 8 | 8 | 8 | 8 |
| MIM | 8 | 7 | 5 | 6 | 5 | 2 | 5 | 5 | 7 | 8 | 8 | 8 | 8 | 8 | |
| 1 | MIV | 0 | 1 | 0 | 0 | 0 | 2 | 3 | 3 | 1 | 0 | 0 | 0 | 0 | 0 |
| MIM | 0 | 1 | 3 | 2 | 3 | 5* | 2 | 3 | 1 | 0 | 0 | 0 | 0 | 0 | |
| 2 | MIV | 0 | 1 | 1 | 2 | 3 | 2 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| MIM | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 3 | MIV | 0 | 2 | 2 | 2 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| MIM | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 4 | MIV | 0 | 4 | 5 | 4 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| MIM | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Within a timepoint within a score, value for the MIV treatment differs significantly (P ≤ 0.05) from the corresponding value for the MIM treatment.
See Table 2 for remainder of key.
Number of rams from Table 2 assigned each ataxia score immediately before (baseline; 0 minutes) and at various times following midazolam administration.
| Time after midazolam administration (min) | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Score | Treatment | 0 | 3 | 5 | 10 | 15 | 30 | 45 | 60 | 90 | 120 | 240 | 480 | 720 | 1,440 |
| 0 | MIV | 8 | 0*† | 0*† | 0*† | 0*† | 0* | 1* | 4 | 7 | 8 | 8 | 8 | 8 | 8 |
| MIM | 8 | 8 | 5 | 2* | 1* | 2* | 2* | 2* | 5 | 8 | 8 | 8 | 8 | 8 | |
| 1 | MIV | 0 | 0 | 0 | 0 | 1 | 4 | 7* | 4 | 1 | 0 | 0 | 0 | 0 | 0 |
| MIM | 0 | 0 | 3 | 4 | 4 | 3 | 3 | 6* | 3 | 0 | 0 | 0 | 0 | 0 | |
| 2 | MIV | 0 | 0 | 0 | 0 | 1 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| MIM | 0 | 0 | 0 | 2 | 2 | 3 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 3 | MIV | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| MIM | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 4 | MIV | 0 | 7*† | 7*† | 8*† | 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| MIM | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
See Tables 2 and 3 for key.
The mean ranks for the sedation AUC differed significantly (P < 0.001) between the MIV and MIM treatments. The mean rank for the sedation AUC0–15 for the MIV treatment (43) was significantly (P = 0.002) greater than that for the MIM treatment (17). However, the mean ranks for the sedation AUC15–60 (40.5) and AUC60–1,440 (18.5) for the MIV treatment did not differ significantly from the mean ranks for the sedation AUC15–60 (29.5) and AUC60–1,440 (19.5) for the MIM treatment. The estimated time to peak sedation for the MIV treatment (4 minutes) was significantly (P = 0.015) less than that for the MIM treatment (22.5 minutes). The sedation EC50 (274 ng/mL; 95% CI, 220 to 340 ng/mL) and Hill slope (3.66; 95°% CI, 1.00 to 6.33) for the MIV treatment did not differ significantly from the sedation EC50 (235 ng/mL; 95% CI, 160 to 343 ng/mL; P = 0.853) and Hill slope (12.43; 95°% CI, −36.34 to 61.19; P = 0.389) for the MIM treatment. The R2 was 0.6787 and 0.1312 for the MIV and MIM treatments, respectively.
Profound ataxia was observed in all 8 sheep following IV administration of midazolam, which resolved within 30 minutes after drug administration. Profound ataxia was not observed in any sheep following IM administration of midazolam. Treatment, time, and the interaction between treatment and time accounted for 10%, 50%, and 30%, respectively, of the total variation for ataxia, all of which were significant (P < 0.001). The median ataxia score was significantly (P ≤ 0.013 for all comparisons) increased from baseline at 3, 5, 10, 15, 30, and 45 minutes after drug administration for the MIV treatment and at 10, 15, 30, 45, and 60 minutes after drug administration for the MIM treatment. The median ataxia score for the MIV treatment was significantly (P < 0.001 for all comparisons) greater than that for the MIM treatment at 3, 5, 10, and 15 minutes after drug administration.
The mean ranks for the ataxia AUC differed significantly (P < 0.001) between the MIV and MIM treatments. The mean rank for the ataxia AUC0–15 for the MIV treatment (36) was significantly (P = 0.019) greater than that for the MIM treatment (15.5). However, the mean ranks for the ataxia AUC15–60 (46) and AUC60–1,440 (14.5) for the MIV treatment did not differ significantly from the mean ranks for the ataxia AUC15–60 (34) and AUC60–1,440 (22) for the MIM treatment. The estimated time to peak ataxia for the MIV treatment (3 minutes) was significantly (P < 0.001) less than that for the MIM treatment (15 minutes). The ataxia EC50 (235 ng/mL; 95% CI, 197 to 280 ng/mL) and Hill slope (3.32; 95% CI, 1.60 to 5.05) for the MIV treatment did not differ significantly from the ataxia EC50 (235 ng/mL; 95% CI, 160 to 343 ng/mL; P = 0.884) and Hill slope (12.43; 95% CI, −36.34 to 61.19; P = 0.277) for the MIM treatment. The R2 was 0.8000 and 0.1312 for the MIV and MIM treatments, respectively.
Of the 8 study sheep, hypersalivation was observed in 6 and 4 after IV and IM administration of midazolam, respectively. The onset of hypersalivation ranged from 5 to 60 minutes after drug administration for the MIV treatment and from 18 to 45 minutes after drug administration for the MIM treatment. Of the 6 sheep that developed hypersalivation following the MIV treatment, only 1 developed the condition > 15 minutes after drug administration. The duration of hypersalivation ranged from 5 to 15 minutes for both treatments. Agitation (thrashing, rolling, vocalizing, and restlessness) was observed in 2 sheep after IM administration of midazolam. One sheep developed agitation 15 minutes after midazolam administration, and the condition lasted for 28 minutes. The other sheep developed agitation 10 minutes after midazolam administration, and the condition lasted for 5 minutes.
Discussion
In the present study, midazolam and its metabolite, 1-hydroxymidazolam, were detectable in the plasma following IV and IM administration of a single dose (0.5 mg/kg) of the drug to healthy adult sheep. Midazolam was rapidly absorbed and distributed and appeared to undergo monophasic elimination. Following IV administration, the apparent volume of distribution for the intact drug exceeded the blood volume, which was indicative of substantial tissue distribution. The midazolam mean volume of distribution at steady state (838 mL/kg) following IV administration for the sheep of this study was similar to that reported for humans (760 to 1,140 mL/kg),19,20 dogs (680 mL/kg),21 alpacas (525 mL/kg),22 and rabbits (831 mL/kg).23 The elimination half-life of midazolam (0.79 hours) was fairly short for the sheep of this study and was comparable to the elimination half-life for dogs (1.05 to 1.2 hours)13,21 and rabbits (0.5 hours)23 but less than that for alpacas (1.6 hours),22 horses (6.8 hours),12 and humans (2.29 hours).19,20 The clearance of midazolam from the sheep of this study (1,272 mL/h/kg) was rapid and similar to that for dogs (1,620 mL/h/kg)13 and rabbits (951 mL/h/kg)23 but substantially quicker than that reported for alpacas (678 mL/h/kg),22 humans (294 to 383 mL/h/kg),19,20,28 and the dogs of another study (606 mL/h/kg).21 The difference in the clearance of midazolam reported for the dogs of the 2 aforementioned studies13,21 was likely attributable to differences in the dose of midazolam administered (0.5 mg/kg13 vs 0.2 mg/kg21).
The midazolam clearance rate for the sheep of the present study was within the estimated portal venous blood flow for that species.29 That finding suggested that, in sheep, midazolam is metabolized primarily by the liver, which is consistent with the metabolism of benzodiazepines in humans and other domestic animal species.13 Midazolam undergoes hydroxylation by cytochrome P450, and the main biotransformation product is 1-hydroxymidazolam.5,30 For the sheep of the present study, 1-hydroxymidazolam was detected in the plasma sooner after IV midazolam administration than after IM midazolam administration, and the mean tmax for the MIM treatment was approximately 4 times that for the MIV treatment (Table 1). However, the mean Cmax for 1-hydroxymidazolam for the MIM treatment (785 ng/mL) was almost twice that for the MIV treatment (422 ng/mL). Nevertheless, the contribution of 1-hydroxymidazolam to the clinical effects of midazolam is considered minimal.20,21
Midazolam was rapidly absorbed following IM administration and was detected in the plasma of all sheep within 3 minutes after injection. However, the mean tmax for midazolam for the MIM treatment (0.46 hours [approx 28 minutes]) was longer than the mean tmax reported for humans (17.5 minutes),25 dogs (7.8 and < 15 minutes),13,21 and guinea pigs (1.66 and 2.91 minutes)26; and the mean Cmax (820 ng/mL) was approximately 1.5 to 8 times that reported for dogs (200 and 549 ng/mL),13,21 guinea pigs (436.6 and 535.1 ng/mL),26 alpacas (411 ng/mL),22 and humans (100.5 ng/mL).25
Intramuscular administration of midazolam to the sheep of the present study was associated with a high mean systemic bioavailability (352%). The mean systemic bioavailability of midazolam reported for dogs (> 90%),13 humans (106%),25 and alpacas (92%)22 suggests that the systemic bioavailability of midazolam is almost complete (ie, 100%) after IM administration in many species; however, in 1 study,21 IM administration of midazolam to dogs was associated with incomplete absorption and a fairly low systemic bioavailability (50%). Many factors, including study design, can affect the systemic bioavailability of a drug. Regardless of the species, IV administration of a drug during the first phase of a study followed by extravascular administration of the same drug during the second phase of that study can result in a period effect owing to interoccasion variation in the clearance of the drug that leads to overestimation of the drug availability.31 Overestimation of the systemic bioavailability of a drug can also result from an insufficient washout period. We do not believe that the high systemic bioavailability for midazolam calculated for the sheep of this study was an artifact of an insufficient washout period between IV and IM administration of the drug because the 7-day washout period was > 10 times the mean t1/2 for the MIV treatment. It is possible that experimental error occurred during sample collection, preparation, or conservation. An absolute systemic bioavailability > 100% for a drug following IM administration can result from overestimation of the AUC owing to use of a linear trapezoidal method for its calculation or excessive extrapolation of the AUC. In the present study, a log-linear trapezoidal method was used to calculate the AUC and extrapolation was < 5% of the total AUC, which should have minimized overestimation.31 Therefore, we are uncertain which factors contributed to the high systemic bioavailability of midazolam following IM administration to the sheep of this study.
The mean t1/2 and MRT for both midazolam and 1-hydroxymidazolam for the MIM treatment were approximately twice those for the MIV treatment. This indicated that plasma concentrations of midazolam and 1-hydroxymidazolam declined twice as fast after IV administration as after IM administration. That finding could have been the result of faster distribution, metabolism, or elimination of midazolam following IV administration relative to IM administration or some rate-limiting effect that affected midazolam absorption following IM administration.
Benzodiazepines cause minimal cardiac depression and mild, dose-dependent respiratory depression.32,33 Midazolam administration was not associated with significant alterations in heart rate during the present study, a finding that was consistent with the results of another study,12 in which the heart rate of horses did not vary significantly after IV administration of midazolam. Conversely, the heart rate of sheep7,34 increased by 7% and 47% and that of dogs32 increased by 15% after IV administration of midazolam, and the heart rate of goats35 increased by 25% and 40% after IM administration of midazolam. In pigs, the heart rate decreased by 20% after IM midazolam administration.36
For the sheep of the present study, the mean respiratory rate was decreased approximately 50% from baseline following IV administration of midazolam, and that decrease lasted for approximately 15 minutes; however, the mean respiratory rate did not decrease significantly following IM administration of midazolam (Table 2). That finding suggested that, in sheep, IV administration of midazolam causes respiratory depression, which correlated well with the dose-dependent respiratory depression associated with IV administration of midazolam in sheep of another study.7 Similar decreases in the mean respiratory rate as those observed during the MIV treatment of this study were reported in pigs36 and goats35 after IM administration of midazolam but were not reported for horses12 after IV administration of midazolam. In short, midazolam administration to sheep was not associated with any significant alterations in heart rate or respiratory rate, other than a transient decrease in respiratory rate following IV administration of the drug.
Although the mean heart and respiratory rates varied throughout the present study, those observed during the MIM treatment were within the respective reference ranges for those variables in healthy adult sheep at rest.37 The mean heart rate for the MIV treatment was 35% to 65% greater than the corresponding heart rate for the MIM treatment at most of the data acquisition times. The mean baseline respiratory rate for the MIV treatment was twice that for the MIM treatment, and the mean respiratory rate for the MIV treatment after drug administration was frequently 50% to 75% greater than that for the MIM treatment at the corresponding data acquisition time.
The mean rectal temperatures for the sheep of the present study were within the reference range for healthy adult sheep, and the changes observed were considered normal diurnal variations37 despite the fact that some were significant. In another study7 involving sheep, a significant decrease (magnitude, 0.42°C) in mean rectal temperature was observed after midazolam administration, which the investigators attributed to heat loss associated with midazolam-induced vasodilation. In the present study, the significant alterations in mean rectal temperature did not appear to be associated with midazolam administration and were not considered clinically important. Rather, we attributed the variations in rectal temperature to weather conditions or stress.
In the present study, the mean heart rate, respiratory rate, and rectal temperature at baseline for the MIV treatment were numerically higher than those for the MIM treatment. That finding was most likely a reflection of stress associated with the environmental conditions and restraint owing to the lack of randomization in treatment order. The sheep were exposed to the experimental conditions for the first time during the MIV treatment and may have been more excited or stressed than during the subsequent MIM treatment, which could have caused an increase in sympathetic tone or catecholamine release. Random variability might also have contributed to the differences in the physiologic measurements between the 2 treatments. Therefore, heart rate, respiratory rate, and rectal temperature were not compared between the 2 treatments. It is possible that, had the sheep been acclimated to the experimental conditions or the environmental conditions for a longer duration or had the treatment order been randomized, the mean values for the physiologic variables would have been similar for both treatments, at least at baseline.
In small ruminants, benzodiazepines provide reliable sedation that is often accompanied by ataxia.7,16,17,35 Similar to dogs,13 cats,11 goats,15 pigs,36 and alpacas,22 the extent of midazolam-induced sedation and ataxia in the sheep of the present study was more profound and developed more quickly following IV administration of the drug than following IM administration of the drug. This was expected because the initial plasma midazolam concentrations after IV administration were higher than those after IM administration, which contributed to a greater and more rapid uptake of the drug by the CSF. Within 15 minutes after IV midazolam administration, many of the sheep of the present study developed profound sedation and ataxia, were unable to stand unaided, and became recumbent. Intravenous administration of midazolam to sheep in other studies7,17 was associated with similar results. In contrast, IV administration of midazolam was associated with sedation and loss of consciousness in humans28 and ataxia without sedation in horses.12 In the present study, IM administration of midazolam was associated with only mild sedation and mild to moderate ataxia, and those effects were transient and most evident within 30 minutes after drug administration. In goats, IM administration of midazolam rapidly induces sedation followed by recumbency.35 In general, the sheep of the present study had resumed clinically normal behavior or had only minimal sedation and ataxia within 60 minutes after IM administration of midazolam. Although the sedation and ataxia scores were assigned by the same investigator throughout the study, that person was aware of (ie, was not blinded to) the route and time of midazolam administration, which might have biased score assignment.
The R2s for the plasma midazolam concentration-response curves for sedation and ataxia were relatively high following IV administration, compared with those following IM administration, which suggested that IV administration of midazolam had a concentration-dependent effect on sedation and ataxia. However, the same did not hold true for IM administration of midazolam. Even though the mean plasma midazolam concentrations achieved for the MIM treatment were generally greater than those achieved for the MIV treatment (Figure 1), the median sedation and ataxia scores for the MIM treatment were generally less than those for the MIV treatment at similar plasma midazolam concentrations. Although the investigator who assigned the sedation and ataxia scores was not blinded to the treatments, 5 of the 8 sheep became recumbent during the MIV treatment, whereas none of the sheep became recumbent during the MIM treatment, which definitely influenced the sedation and ataxia scores. The effect of route of midazolam administration on sedation and ataxia in sheep requires further investigation.
Two of the 8 sheep of the present study became agitated soon after IM administration of midazolam. Agitation is not uncommon after benzodiazepine administration in domestic animal species, particularly healthy young individuals.38 The condition was transient and did not compromise the welfare of the affected sheep, and both animals safely completed the study. Agitation has been described after sole administration of midazolam to humans,9,10 cats,11 horses,12 and dogs.6,13,14 Hypersalivation was observed in 6 and 4 sheep following IV and IM midazolam administration, respectively. Hypersalivation associated with midazolam administration has been described in dogs13 and sheep of other studies.17,34 The factors that contribute to the development of agitation and hypersalivation following midazolam administration have yet to be defined.
The present study had some limitations. It had a block-assigned design. The 8 sheep all received midazolam by the IV route and then by the IM route 7 days later; however, within each treatment (MIV and MIM), the order in which the sheep received midazolam was randomized. Ideally, the order in which the MIV and MIV treatments were administered should have been randomized so each animal had an equal opportunity to receive either the MIV or MIM treatment initially. The block-assigned design may have caused an aberrant increase in the heart rate and respiratory rate during the MIV (initial) treatment owing to stress associated with novel exposure to the experimental conditions. Therefore, the heart and respiratory rates recorded for this study should be interpreted with caution, and additional studies are necessary to elucidate the cardiopulmonary effects of midazolam following IV and IM administration in sheep. Differences in the baseline physiologic variables between the MIV and MIM treatments might have altered the pharmacokinetics of midazolam. Changes in cardiac output alter hepatic blood flow, which can significantly affect the pharmacokinetics and pharmacodynamics of a drug.39 Because cardiac output is the product of heart rate and stroke volume, the marked discrepancy in the baseline heart rate between the MIV and MIM treatments may have affected the systemic bioavailability of midazolam calculated in the present study. The sheep of this study underwent a 3-week acclimation period to ensure that they were familiar with the experimental conditions. This acclimation included restraint for simulation of catheter placement, physical examination, and blood collection on a daily basis. We thought that 3 weeks was sufficient to acclimatize the sheep to the experimental conditions; however, a longer acclimation period may have been necessary. Although the same investigator assigned sedation and ataxia scores to all sheep throughout the duration of the study, that person was not blinded to the route and time after midazolam administration, which could have biased the subjective assessment of sedation and ataxia. Nevertheless, on the basis of our clinical experience, the extent of sedation and ataxia observed in the sheep of this study was similar to that observed in sheep following midazolam administration in clinical practice. The study population consisted of rams only. Sex affects the pharmacokinetic profiles of some drugs in sheep.40 In humans, gender has been significantly associated with the pharmacokinetic profile for midazolam after IV41 but not IM42 administration. Therefore, the pharmacokinetic profile for midazolam in rams may differ from that for ewes.
Results of the present study indicated that midazolam and its metabolite, 1-hydroxymidazolam, became detectable in the plasma soon after IV and IM administration of a single dose (0.5 mg/kg) of the drug to healthy adult sheep. The physiologic and behavioral effects induced by midazolam were typical of those described for benzodiazepines in ruminants. Additional studies are necessary to further investigate the effects of midazolam on the cardiopulmonary system of sheep. Findings suggested midazolam will be a suitable sedative for sheep when short periods of sedation are required, and IM administration may be a viable alternative when IV administration is not possible.
Acknowledgments
Supported by the Ross University School of Veterinary Medicine. The authors declare that there were no conflicts of interest. The authors thank Alexandra Jordan, Michaela Witcher, Kathy Alston, and Crace Lewis for technical assistance.
ABBREVIATIONS
| AUC | Area under the concentration-time curve |
| AUC0-∞ | Area under the concentration-time curve from time 0 to infinity |
| CI | Confidence interval |
| Cmax | Maximum plasma concentration |
| EC50 | Plasma midazolam concentration required to achieve 50% of the maximal effect |
| GABA | γ-Aminobutyric acid |
| HPLC | High-performance liquid chromatography |
| MRT | Mean residence time |
| t1/2 | Terminal half-life |
| Tmax | Time to maximum plasma concentration |
Footnotes
Terumo Medical Corp, Somerset, NJ.
Midazolam HCl injection USP (5 mg/mL), Hospira, Lake Forest, Ill.
BD, Franklin Lakes, NJ.
2695 Separations Module, Waters, Milford, Mass.
2487 Dual Wavelength Absorbance Detector, Waters, Milford, Mass.
Symmetry Shield, Waters, Milford, Mass.
Phoenix WinNolin, version 6.4, Centera LP, Princeton, NJ.
GraphPad Software Inc, La Jolla, Calif.
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Appendix 1
Description of the 5-point scale used to assess sedation in 8 healthy adult mixed-breed rams that were administered midazolam (0.5 mg/kg) by the IV route and then by the IM route 7 days later in a crossover study.
| Score | Degree of sedation | Definition |
|---|---|---|
| 0 | None | Appropriate (clinically normal) response to auditory stimulus (clapping), alert, and aware. |
| 1 | Mild | Slight decrease in movement and response to auditory stimulus; head maintained in neutral position or muzzle lowered to the point of the shoulder. |
| 2 | Moderate | Moderate decrease in movement, moderately wide-based stance, muzzle lowered to the level of the carpus, and slight drooping of eyelids and lips. |
| 3 | Marked | Marked decrease in movement and response to auditory stimulus, marked wide-based stance, head dropped to the ground, marked drooping of eyelids and lips, and markedly drowsy. |
| 4 | Profound | No response to auditory stimulus; recumbent. |
Appendix 2
Description of the 5-point scale used to assess ataxia in 8 healthy adult mixed-breed rams that were administered midazolam (0.5 mg/kg) by the IV route and then by the IM route 7 days later in a crossover study.
| Score | Degree of sedation | Definition |
|---|---|---|
| 0 | None | No difficulties walking or turning; able to stand without swaying. |
| 1 | Mild | Slight staggering when taking multiple steps or turning; able to stand in natural position with a sway. |
| 2 | Moderate | Visibly abnormal gate with considerable staggering; able to stand with feet in an unnatural position. |
| 3 | Marked | Severe staggering; unable to stand without intermittent support of wall. |
| 4 | Profound | Unable to walk or stand without support. |