2.4. In Vivo Pharmacokinetic Study in Rats

Sprague Dawley male rats were supplied by Charles River (Sulzfeld, Germany). At the start of the pharmacokinetic study, the rats weighed 350 to 450 g and were 9 to 11 weeks old. Polysulfone cages with corn cob bedding material were used for group housing in airconditioned (20–24 °C) rooms with a 12 h light cycle. To enrich the environment, rats had access to Aspen wood block (Datesand, UK) and Rodent retreat (Bio-Serv, Flemington, NJ, USA). An acclimatization period of at least 4 days was applied before starting the pharmacokinetic study. Food and water were available ad libitum throughout the study.

The guidelines of the Janssen Pharmaceutica (Beerse, Belgium) Animal Ethics Committee, the local Belgium laws controlling the use of experimental animals, and the EC Directive 2010/63/EU were followed.

Based on body weight, 15 rats were divided into 5 groups (3 animals per group) (Table 2). Rats of groups 1 to 4 received a dorsal SC injection of 0.4 mL/kg or a dose of 96.8 mg/kg bedaquiline fumarate salt (equivalent to 80 mg/kg bedaquiline free base, and further referred to as 80 mg eq./kg bedaquiline fumarate salt) of ISG formulations 1 to 4, respectively. In group 5, rats received a dorsal SC injection of 0.8 mL/kg of formulation 5, corresponding to a dose of 4.84 mg/kg bedaquiline fumarate salt (equivalent to 4 mg/kg bedaquiline free base, and further referred to as 4 mg eq./kg bedaquiline fumarate salt).

Pharmacokinetic study design in rats.

ISG: in situ forming gel; SC: subcutaneous injection.

At time points ranging from 0.5 h to 168 days after dosing, 32 µL of blood was collected via the tail vein into Vitrex micro hematocrit tubes “soda lime glass” containing potassium ethylenediaminetetraacetic acid (K2.EDTA). Samples were placed on melting ice until centrifugation for approximately 10 min at 1500× g and 5 °C. Plasma samples of 10 µL were collected with Vitrex end to end pipettes in FluidX tubes and were stored at −20 °C until bioanalysis (see Section 2.4.3). Pharmacokinetic parameters were calculated by non-compartmental analysis using Phoenix™ WinNonlin^® (Certara, Princeton, NJ, USA). A two-tailed homoscedastic t-test was applied for statistical comparisons between groups.

The bioanalytical method was developed internally at Janssen. Plasma samples collected during the in vivo study in rats (see Section 2.4.2) were processed by washing out the Vitrex end to end pipettes using 20 µL methanol and 400 µL internal standard solution (5 ng/mL of 6-deuterium labelled bedaquiline in acetonitrile/water 80/20% (v/v)) into the FluidX tubes. Bedaquiline calibration standards of 0.4 to 1000 ng/mL were prepared in rat plasma and processed in the same way as the study samples. The FluidX tubes were shaken for 10 min using an orbital shaker and were centrifuged for 3 min at 2500× g. The supernatant (150 µL) was transferred to a 96-deepwell plate to determine the levels of bedaquiline after injection in a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. A Shimadzu LC30AD HPLC instrument with an SIL-HTC autosampler (Shimadzu Scientific Instruments, Columbia, MD, USA) and a Waters BEH C18 50 × 2.1 mm, 1.7 µm column maintained at 50 °C was coupled to an API4000™ or 5500™ triple quadrupole mass spectrometer (AB Sciex, Toronto, ON, Canada) equipped with Turbo Ionspray source operated at 400 °C. Mobile phases (A) 0.01 M ammonium formate pH 4.0 and (B) methanol were eluted via the following gradient, mixing (A) and (B), at a flow rate of 0.6 mL/min: the percentage of (B) was increased from 55% to 80% during the first 3 min, was further increased to 98% in 0.01 min, and kept stable at 98% for 0.99 min. Thereafter, the percentage of (B) was again reduced to 55% in 0.01 min and kept stable at 55% for 0.99 min, resulting in a total run time of 5 min. The MS was operated in the positive ion mode using the TurboIonSpray™-interface (electrospray ionization) and was optimized for the quantification of bedaquiline. Multiple reaction monitoring (MRM) was applied with transitions of m/z 555.2 → 58 for bedaquiline and m/z 561.2 → 64 for the 6-deuterium labelled internal standard and a collision energy of 71 eV. The LC-MS/MS results of the calibration standards were used to generate a calibration curve: peak area ratios of bedaquiline to its internal standard were plotted versus corresponding bedaquiline concentrations, and a linear regression model with 1/x² weighting was fitted to these data. Bedaquiline concentrations of the study samples were calculated by interpolation from the calibration curve.

Illness, abnormal behaviour or unusual appearance, untoward clinical signs, toxic or pharmacological response, and moribund state or mortality were checked daily for each rat in groups 1 to 5.