3.8. In Vivo Pharmacokinetics Studies in Rats

Healthy female and male Sprague Dawley rats of 7 weeks age weighing 140–240 g (Charles River, Denmark), were used for the experiments. The rats were housed with free access to water and food in groups of two rats per cage under controlled environmental conditions (constant temperature and humidity with a 12 h day-night cycle). All animal experimental work was carried out under the protocol approved by the Danish Animal Experiments Inspectorate (approval no. 2014-15-0201-00031), and all the procedures used were refined to provide maximal comfort and minimal stress to the animals.

All animals were fasted for approximately 12 h prior to administration of the drug formulations and food was provided to rats approximately 6 h after drug administration, although free access to water was allowed during fasting. The nine groups studied, each consisting of 6–8 rats, were assigned randomly and included two groups receiving an intravenous bolus injection of either DTX or BIC corresponding to 1 mg/rat (4–7 mg/kg) dissolved in saline: DMSO (50:50 v/v) with 1% Tween 80, administered in the tail vein. The remaining seven groups were orally administered with DTX or BIC or a combination of these at a fixed dose of 30 mg/kg for DTX and 16 mg/kg for BIC. For DTX, four groups of animals were studied, one group administered with crystalline DTX (crystalline), one group administered with a physical mixture of crystalline DTX and BIC (physical mix), one group administered with amorphous DTX (amorphous) and one group administered with the co-amorphous mixture of DTX and BIC (co-amorphous). For BIC three groups of animals were studied, one group administered with crystalline BIC (crystalline), one groups administered with a physical mixture of crystalline DTX and BIC (physical mix) and one group administered with the co-amorphous mixture of DTX and BIC (co-amorphous). Drug powders were suspended in 0.5 mL PBS and the suspension was immediately administered to the rats using a flexible cannula. Blood samples (150–200 µL) were collected in EDTA coated tubes from puncturing of the tail vein at 0.5, 1, 2, 4, 6 and 24 h after drug administration. The rats were euthanized by cervical dislocation after collection of the final blood sample. Plasma samples were obtained by centrifugation of blood samples at 3600 g (10 min, 4 °C) and transferred into microtubes. The plasma samples were stored at −80 °C until used for further analysis.