Ethical approval for this study was obtained from the Research Ethics Committee: Animal Care and Use at the University of Stellenbosch, South Africa. Protocol ethical approval number SU-ACUD16-00025.
3.2.1 Experimental location
This study was conducted on Ngongoni farm (25°31’25.2” S, 31°06’50.8” E), outside Nelspruit in Mpumalanga, South Africa. The region is characterized by an average temperature of 19.8 °C (minimum 14.6 °C and maximum 23.6 °C), and annual rainfall of 796 mm (summer rainfall region; range: 11 mm in June to 130 mm in January) (Climate: Nelspruit).
3.2.2 Experimental animals and husbandry
This study was carried out using six healthy female indigenous goats (Capra hircus), with an average weight of 20.8 ± 1.88 kg (range: 17.7 to 23 kg). Only six animals were used due to the high cost of the blood analysis. The number of animals were also based on that used in other pharmacokinetic studies, where six subjects were deemed sufficient to yield the desired pharmacokinetic results (Kreuder et al., 2012; Kuusela et al., 2000; Carroll et al., 2001; Cole et al., 2006; Shukla et al., 2007; Wasfi et al., 2012; Aarnes et al., 2013; Hubbell et al., 2013;
Schwartz et al., 2013). The animals were detained in a large pen surrounded by wire fencing (Figure 3.1A), which contained a small enclosure for the conduction of veterinary procedures (Figure 3.1B). The animals were provided with fresh water and lucerne hay ad libitum prior to the start of the trial.. Feed was withheld for 12 hrs prior to the start of the trial and during blood sampling to standardize conditions, while water was provided ad libitum. On the day of the trial, the goats were herded into the small enclosure within the pen. After blood sampling was completed, the animals received fresh lucerne hay ad libitum. The animals were checked for diseases before being brought onto the farm and were continuously monitored for their health via the veterinarian throughout their time in captivity.
Figure 3.1 (A) The pen used for housing indigenous goats used during the pharmacokinetic study of midazolam, and (B) the handling facility where veterinary procedures were performed.
3.2.3 Administration of pharmaceutical substances
Midazolam was administered by the IM route at a dose of 0.8 mg midazolam/kg body weight (BWt) and a concentration of 20 mg/mL (Wildlife Pharmaceuticals SA (Pty) Ltd, White River, South Africa) into the right quadriceps femoris muscle of five of the trial animals. The dosage was selected based on previous dosages of midazolam used in goats (Stegmann, 1998, 1999; Stegmann & Bester, 2001).
The remaining goat received the drug intravenously via the auricular vein in order to determine the absolute bioavailability. The volume (in mL) midazolam to be administered per goat was calculated based on their respective body weights as per guidelines of the manufacturer. All goats were weighed and ear-tagged prior to the study.
Goats were selected at random and physically restrained for blood sampling.
3.2.4 Blood sampling and processing
Blood samples were collected from the jugular vein from each goat at time 0 (immediately prior to midazolam administration), 30 min, 1 hr, 2 hrs, 4 hrs, 6 hrs, 12 hrs and 24 hrs following midazolam administration. The blood samples were collected using an 18-gauge sterile needle and 20 mL syringe, with the blood being transferred to sterile 10 mL serum vacutainer tubes (BD Vacutainer® Plus plastic serum tubes, 16 mm x 100 mm, 10 mL, Clot Activator, Ref nr 367896). A total volume of 30 mL was collected from each goat per sampling session. Two extra 10 mL blood samples were collected per goat with sample 0 for calibration of the gas spectrometry machine at Labserve (Pty) Ltd (Nelspruit, Mpumalanga, South Africa). The time at which each sample was collected per goat was noted on a data sheet (Appendix A).
The blood samples were centrifuged (Heraeus Biofuge A 1217) at 5000 rpm for 10 min, as soon as possible after collection. The serum in each centrifuged blood sample was then drawn up using an 18-gauge sterile needle and a 3 mL syringe. The serum was then stored in a marked 5 mL cryotube at ~4ºC until collection. A minimum of 5 mL serum was collected per goat for each sampling interval. The serum samples were sent to Labserve (Pty) Ltd (Nelspruit, Mpumalanga, South Africa) for analyses with gas chromatography mass spectrometry (GC-MS).
A B
3.2.5 Gas chromatography mass spectrometry assay
The goat serum samples were analysed via GC-MS by Labserve (Pty) Ltd. Due to the confidential nature of the protocol developed for the assay of midazolam, only a brief explanation is provided of the assay process. The analysis entailed the extraction of each serum sample using an organic solvent. Salt was used to remove moisture and proteins. Diazepam was used as an internal standard to compensate for any losses during extraction or on the instrument. GC-MS was performed with liquid injections. Each serum sample was extracted with acetonitrile in the presence of magnesium sulphate. An aliquot of the organic phase was analysed on the GC-MS in Single Ion Monitoring (SIM) mode. A split/splitless injector was used with helium serving as the mobile phase.
Quantification was performed using the Internal Standard method (Immelman, personal communication, 4 August, 2016).
3.2.6 Statistical analyses
The serum concentrations of midazolam in the pharmacokinetic study were analysed with the PK solver add-on in Microsoft Excel (2016). A non-linear one-compartmental analysis was used to fit the mean serum time-concentration data to standard pharmacokinetic models and to generate pharmacokinetic data. Predicted versus observed concentrations as a function of time and estimates of pharmacokinetic parameters were generated from the analysis of the concentration-time data. The following pharmacokinetic parameters were generated:
Elimination rate constant (ke), ratio of clearance to bioavailability, the ratio of volume of distribution to bioavailability (CL/Vd), peak plasma concentration (Cmax), time to peak plasma concentration (Tmax), the area under the plasma concentration curve (AUCt), the area under the plasma concentration curve from zero to infinity (AUC0-∞), area under the first moment curve (AUMC) and the mean residence time (MRT).
The AUC following IV administration of midazolam to one of the goats was also obtained with the PK Solver add-on in Microsoft Excel (2016) via a add-one-compartmental analysis, for the purpose of calculating the absolute bioavailability of midazolam when administered by the IM route. The absolute bioavailability of the IM administered midazolam was calculated from the AUC ratio determined following IM and IV administration of midazolam using Equation 3.1:
Eq. 3.1: 𝐹 =𝐴𝑈𝐶𝐴𝑈𝐶𝐼.𝑀.
𝐼.𝑉.
The following pharmacokinetic parameters were calculated:
Peak plasma concentration (Cmax): The maximum concentration reached by a drug in the plasma of an animal following administration (Urso et al., 2002).
Time to peak plasma concentration (Tmax): The amount of time it takes for the maximum plasma concertation of a drug to be reached in an animal (Urso et al., 2002).
Elimination rate constant (ke): The drug fraction that is eliminated per unit of time in an animal (Clarke &
Trim, 2013).
Elimination half-life (t1/2β): The time necessary for a drug’s plasma concentration to reach half its original value (Clarke & Trim, 2013).
Ratio of volume of distribution to bioavailability (V/F): The apparent volume of distribution after non-IV administration of a drug. The apparent volume of distribution refers to the theoretical volume that would be required to contain the total amount of the drug at the same concentration observed in the plasma of an animal after administration (Gepts,1998). The volume of distribution and bioavailability (F) of drugs administered via non-IV routes cannot be determined on their own, only their ratio.
Ratio of clearance to bioavailability (CL/F): The apparent total clearance of a drug from plasma after oral administration. The total clearance of a drug is the measurement of the volume of plasma from which a drug is completely eliminated per time unit. The total clearance and bioavailability (F) of drugs administered via non-IV routes cannot be determined on their own, only their ratio (Gepts, 1998)
The area under the plasma concentration curve (AUC0-t): The calculated area under the plasma drug-concentration profile of a drug, which is a reflection of the total drug exposure of an animal’s body after administration (Urso et al., 2002).
The area under the plasma concentration curve from zero to infinity (AUC0-∞): The area under the plasma drug-concentration profile of a drug extrapolated to infinity (Urso et al., 2002).
The area under the first moment curve (AUMC): The area under the product of the concentration and time against the time curve (Gabrielsson & Weiner, 2012).
The mean residence time (MRT): The mean amount of time a drug remains in an animal’s body (Yamaoka et al., 1978).
Absolute bioavailability (F): A drug’s bioavailability when administered via non-IV route in comparison with the bioavailability of the drug when administered via the IV route. Bioavailability refers to the fraction of the original drug that enters circulation after administration (Urso et al., 2002).
The AUC following IV administration of midazolam was calculated using the PK Solver add-on in Microsoft Excel (2016) following a one-compartmental analysis approach for the purpose of calculating the absolute bioavailability of midazolam when administered by the IM route. The fit weight of the IV administered midazolam data was calculated from the observed plasma concentrations using the following equation:
𝑊 = 1
𝐶𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑
The absolute bioavailability of the IM administered midazolam was calculated from the AUC ratio determined following IM and IV administration of midazolam using the following equation:
𝐹 =𝐴𝑈𝐶𝐼.𝑀.
𝐴𝑈𝐶𝐼.𝑉.