Pharmacokinetics of buprenorphine in pregnant sheep after intravenous injection

Abstract Buprenorphine is a semi‐synthetic opioid, widely used in the maintenance treatment for opioid‐dependent pregnant women. Limited data exist on the pharmacokinetics of buprenorphine in pregnancy. We conducted a pharmacokinetic study to determine the pharmacokinetics of intravenous buprenorphine in pregnant sheep. Fourteen pregnant sheep in late gestation received 10 µg/kg of buprenorphine as an intravenous bolus injection. Plasma samples were collected up to 48 h after administration. Buprenorphine and its metabolite, norbuprenorphine, were quantified from plasma using a LC/MS/MS method, with lower limits of quantification of 0.01 µg/L and 0.04 µg/L for buprenorphine and norbuprenorphine, respectively. The pharmacokinetic parameters were calculated using noncompartmental analysis. The pharmacokinetic parameters, median (minimum−maximum), were Cmax 4.31 µg/L (1.93–15.5), AUC inf 2.89 h*µg/L (1.72–40.2), CL 3.39 L/h/kg (0.25–6.02), terminal t½ 1.75 h (1.07–31.0), Vss 8.04 L/kg (1.05–49.3). Norbuprenorphine was undetected in all plasma samples. The median clearance in pregnant sheep was higher than previously reported for nonpregnant sheep and human (male) subjects. Our sensitive analytical method was able to detect long terminal half‐lives for six subjects, and a wide between‐subject variability in the study population. Significance statement: Buprenorphine is widely used for the treatment of opioid use disorder in pregnancy. However, limited data exist on the pharmacokinetics of buprenorphine during pregnancy. As this type of study cannot be done in humans due to ethical reasons, we conducted a study in pregnant sheep. This study provides pharmacokinetic data on buprenorphine in pregnant sheep and helps us to understand the pharmacokinetics of the drug in humans.


| INTRODUC TI ON
Buprenorphine (BUP) is a partial agonist at µ-opioid receptor and has antagonist effects at δ-and ĸ-opioid receptors. 1,2 Agonistic interactions with the opioid receptor-like 1 receptor could also contribute to the antinociceptive effect. 1,3 Due to high affinity and low dissociation rate from the opioid receptors, BUP is classified as a long-acting opioid. 1,4 BUP is a small, highly lipophilic compound that is highly bound to plasma proteins (96%). 5 BUP is metabolized in liver via CYP3A4 by N-dealkylation into its main active metabolite, norbuprenorphine (NBUP). BUP and NBUP are further glucuronidated by uridine 5'-diphospho-glucuronosyltransferase (UGT) UGT1A1, UGT1A3, and UGT2B7 into BUP-3-glucuronide and NBUP-3glucuronide. BUP and its metabolites are mainly eliminated through feces, and 10-30% of the administered dose is excreted into urine as water-soluble metabolites. 6,7 Buprenorphine has been used for the treatment of moderate-tosevere pain and opioid use disorder since 1996. During pregnancy, BUP is not recommended for pain management but is commonly used in opioid substitution treatment for opioid-addicted pregnant women. Despite the wide use, the pharmacokinetics of BUP during pregnancy is poorly understood. 7 Several physiological and body composition changes during pregnancy could affect the pharmacokinetic of BUP. These include increased cardiac output, plasma volume, total body water and glomerular filtration rate, changes in the expression and activity of metabolizing enzymes (CYP3A4 and UGTs), and decreased protein binding. 8 After sublingual administration, BUP exposure (area under the plasma concentration curve, AUC) is lower during pregnancy than postpartum in women receiving BUP for substitution treatment of opioid dependence. 9,10 In humans, BUP metabolic ratios (AUC of NBUP and NBUP-glucuronide to the AUC of BUP) are higher during pregnancy compared with postpartum period. 11 These findings may indicate an increased systemic clearance of BUP in pregnancy.
However, the pharmacokinetic data on BUP during pregnancy are sparse. In this study, we have determined the basic pharmacokinetic parameters of BUP in pregnant sheep after intravenous (IV) administration, for future reference in consecutive studies on BUP central nervous system permeation in the sheep model, and to eventually help us understand BUP pharmacokinetics in humans during pregnancy. We used a pregnant sheep model, as this type of study could not be conducted in humans, due to ethical reasons. In this study, 14 sheep were in the late stages of their pregnancies. Sheep was chosen as the animal model for the study, since the resemblance in size and weight is close to that of humans. Also, even though the gestational period is half of that of humans, the sheep fetus is of similar size and weight in late gestation as the human fetus. The size of the animal makes cannulation easy and enables sufficient blood samples to be collected from the animal. Another advantage with sheep is that they adapt to the laboratory environment and to handling fast and show little to no signs of stress during acclimation and experimentation. These features offer significant advantages in obstetric studies compared to rodent species. However, no animal model is exactly similar to humans. Even though the basic function of the placenta (e.g., hormone secretion, and transfer of nutrients and drugs) and hormonal profiles (e.g., estrogens and progesterone) are similar to all mammals during pregnancy, the placental interface differs between species and can affect the pharmacokinetics of a drug. In sheep, the placenta has one layer of maternal uterine endothelium and one layer of trophoblasts that separate the maternal and fetal circulation (epitheliochorial placenta), whereas, in humans, the maternal blood is separated from the fetal circulation with only one layer of trophoblasts (hemomonochorial placenta). 12

| Animals
The animal transport, husbandry, and experimental procedures were carried out according to the Finnish national legislation and the EU directive 2010/63/EU. 15,16 The study protocol was approved Animals were monitored throughout the study by veterinarians, animal technicians and the research team for signs of distress, pain, injuries, or diseases. Actions were taken immediately to improve the well-being of the animals when needed.

| Buprenorphine administration and sample collection
Prior to the drug administration, sheep's both external jugular veins were cannulated. The area was shaved, cleaned with soap, and disinfected with ethanol solution before cannulation. The right-side jugular vein cannula was used for blood sampling and the left side for BUP administration.
Buprenorphine (Vetergesic vet 0.3 mg/ml; Ceva Santé Animale) dose of 10 µg/kg BUP-free base was diluted in 10 ml 0.9% saline (sodium chloride) and given IV as a 1-min injection through the left side cannula. The dose was based on a clinically administered dose for sheep. 17 The BUP dose was well tolerated in all animals.
Blood samples, 4 ml, were collected prior to the BUP administration and then, at 5, 15, 30, 45, and 60 min, and at 2, 4, 7, 10, 24, 30, and 48 h after the IV administration. After blood sampling, the jugular vein cannula was flushed with 20 ml 0.9% NaCl and then with 2 ml 50 IU/ml heparin solution. The blood samples were collected in heparinized plasma tubes and centrifuged at 2000g for 10 min.
Plasma was divided into two cryotubes and stored first at −35°C and then moved to −85°C until analysis.

| Buprenorphine quantification
The plasma concentrations are expressed as BUP-free base. Plasma

| Data analysis
The pharmacokinetic parameters for noncompartmental analy-

| RE SULTS
Individual BUP plasma concentration curves are shown in Figure 1, and pharmacokinetic parameters in Table 1, respectively.
Wide between-subject variability was observed in C max (range

| DISCUSS ION
The novelty of our study was that, to our knowledge, this is the first pharmacokinetic study of IV BUP in pregnant sheep, and the first sheep study, where blood samples were collected up to 48 h after IV BUP administration. A long sampling period and a highly sensitive quantification method allowed us to obtain more precise pharmacokinetic data during the elimination phase. The use of pregnant sheep was justified as it provided necessary nonclinical data for assessing potential risks to pregnant women and fetuses. Due to ethical reasons, this type of study could not be carried out in humans without prior nonclinical data. High between-subject variability in   There are limitations in our study. Due to study site logistics, we were unable to perform the study at different stages of the pregnancy, prior to the pregnancy, or postpartum. This would have provided us with a deeper understanding of the effect of pregnancy on the BUP pharmacokinetics in our study population.
Additionally, we did not have access to the fetus at the time of the IV study, and thus were unable to determine the fetal exposure of BUP after the injection. The results of this pilot study should be considered preliminary and should be used with caution in a clinical setting. The strength of our study was a highly sensitive analytical method with relatively low LLOQ and a long sampling period that allow us to measure plasma concentration for a longer time period to gain a more precise understanding of the pharmacokinetics of BUP in pregnant sheep.
In conclusion, we uncovered the basic pharmacokinetics of

ACK N OWLED G EM ENTS
We appreciate Biocentre Finland and Biocentre Kuopio for supporting LC-MS laboratory facility. We thank the personnel of Oulu Laboratory Animal Centre, University of Oulu for the help to conduct animal research. We appreciate the help of Ms. Erica Armstrong in laboratory assistance and revision of grammar. This is a part of the KuBiCo study for HK.