Inhibitory effect of carvedilol on bedaquiline metabolism in vitro and in vivo

Chemicals and reagents

Bedaquiline, M2, and fluconazole (internal standard, IS) were bought from Yingxin Biotechnology Co., Ltd. (Shanghai, China). Various drugs (andrographolide, amiodarone hydrochloride, apigenin, apixaban, artemether, astragalin, baicalin, berberine, bergenin, betaine, bosentan, carvedilol, cangrelor, cariprazine, chrysin, cimetidine, dabigatran, daidzein, daphnoretin, genistein, glimepiride, hesperetin, isorhamnetin, kaempferol, limonin, imipramine hydrochloride, losartan, lovastatin, lycopene, mexiletine hydrochloride, naringenin, naringin, nifedipine, quercetin, quinidine, PF-04971729, piperine, propafenone, resveratrol, rivaroxaban, rutin, selexipag, silibinin, sitagliptin, sophocarpine, sophoridine, verapamil hydrochloride, vericiguat, warfarin, 2-(4-hydroxyphenyl) acetic acid) were provided by Shanghai Chuangsai Technology Co., Ltd. (Shanghai, China). RLM and HLM were prepared by our team (Wang et al., 2015). Recombinant human CYP3A4 was obtained from the Beijing Hospital (Beijing, China). Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was obtained from Roche Pharmaceutical, Ltd. (Basel, Switzerland). The remaining chemicals and reagents were of analytical grade.

Equipment and operation conditions

The concentrations of bedaquiline and its metabolite (M2) were detected using UPLC-MS/MS, which was equipped with a Waters Acquity UPLC system (Milford, MA, USA) and a Waters Xevo TQS triple quadrupole tandem mass spectrometer (Milford, MA, USA). Chromatographic separations of the analytes were performed on the Bridged Ethylene Hybrid (BEH) C18 column (2.1 × 50 mm, 1.7 µm; Milford, MA, USA), and it was operated at a column temperature of 40 °C. The mobile phases were acetonitrile (ACN, a) and 0.1% formic acid (b) at 0.30 mL/min gradient elution for 2.0 min. The gradient conditions were as follows: 90% B (0–0.5 min), 90%–10% B (0.5–1.0 min), 10% B (1.0–1.4 min), 10%–90% B (1.4–1.5 min), and the last 90% B (1.5–2.0 min) was maintained for balance. The temperature of the auto-sample injector was set at 4 °C, and the sample volume was 1.0 µL for each run. Mass spectral information of the analytes was obtained by selective response monitoring in the positive ion mode. To improve the sensitivity and specificity of the analysis, the transitions of bedaquiline, M2, and IS were m/z 555.00 > 58.08, m/z 540.93 > 479.92, and m/z 307.14 > 238.14, respectively.

In vitro experiments

200 µL enzymatic reaction system contained 1.0 M phosphate buffered saline (PBS), 1.0 mM NADPH, 0.3 mg/mL RLM or 0.3 mg/mL HLM or 0.5 pmol CYP3A4, 0–100 µM bedaquiline to determine the K_m (Michaelis–Menten constant) values of bedaquiline. The mixture without NADPH was pre-incubated at 37 °C for 5 min, and then 20 mM NADPH was added to activate the reaction. After incubation for 30 min, the reaction was stopped by rapid cooling to −80 °C. Next, 500 µL ACN and 20 µL IS (200 ng/mL) were added to precipitate protein before the samples melted. The mixture was vortexed for 2 min and centrifuged at 13,000 rpm for 10 min. Finally, 100 µL supernatant was collected for UPLC-MS/MS analysis (Lin et al., 2019).

To detect the possible DDIs with bedaquiline, the 200 µL system was kept the same, and the K_m value was used as the concentration of bedaquiline in the RLM system to determine the inhibitory effect of 50 kinds of drugs (the concentration of each drug as the inhibitor was 100 µM) on bedaquiline metabolism.

To explore the half-maximal inhibitory concentration (IC₅₀), the concentrations of carvedilol were set at 0, 0.01, 0.1, 1, 10, 25, 50, and 100 µM, while bedaquiline was set at 10 µM in RLM, 80 µM in HLM, and 0.8 µM in CYP3A4, according to their K_m values, respectively. The incubation system consisted of 0.3 mg/mL RLM or 0.3 mg/mL HLM or 0.5 pmol CYP3A4, 1.0 mM NADPH, bedaquiline, carvedilol, and 1.0 M phosphate-buffered saline buffer to the final volume of 200 µL.

To investigate the potential inhibitory mechanism, the concentrations of carvedilol and bedaquiline were determined based on IC₅₀ and K_m values, respectively. Carvedilol’s concentrations were 0, 7.5, 15, and 30 µM in RLM, 0, 1.9, 3.8, and 7.6 µM in HLM, and 0, 0.4, 0.8, and 1.6 µM in CYP3A4, while the concentrations of bedaquiline were 2.5, 5, 10, and 20 µM or 20.8, 41.5, 83, and 166 µM or 2.5, 5, 10, and 20 µM in RLM or HLM or CYP3A4, respectively. The sample was prepared following the experiment mentioned above.

To study the time-dependent inhibition (TDI), the incubation system was the same as that described for detecting possible drug interactions with bedaquiline. The mixture with or without NADPH was incubated at 37 °C for 30 min, where the concentration of carvedilol was set as 0, 0.01, 0.1, 1, 10, 25, 50, and 100 µM. Thereafter, bedaquiline (the corresponding K_m values: 10 µM in RLM, 80 µM in HLM, and 0.8 µM in CYP3A4) was added and incubated for another 30 min. The following procedures were the same as the experiment above.

In vivo experiments

Sprague-Dawley (SD) male rats (200 ± 10 g) were provided by the Animal Experimental Center of The First Affiliated Hospital of Wenzhou Medical University (Zhejiang, China) and were housed in an animal experimental center with a temperature range of 20–26 °C, relative humidity of 55% ± 15%, and a 12 h/day light/dark cycle. Before the experiment, the rats were fasted for 12 h and allowed free access to water. All animal experiments followed the National Research Council’s Guide for the Care and Use of Laboratory Animals. Furthermore, the study protocol followed the ARRIVE guidelines and was approved by the Laboratory Animal Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University (Ethics approval number: WYYY-IACUC-AEC-2023-046).

Eight SD rats were randomly divided into two groups (n = 4): a single group (group A) and a combined group (group B). Bedaquiline and carvedilol were dissolved with 0.5% carboxymethylcellulose sodium salt (CMC-Na) solution to the desired concentrations. Group B was orally administered carvedilol (5 mg/kg), while group A administered an equivalent volume of 0.5% CMC-Na solution by gavage. Thirty minutes later, both groups administered oral doses of bedaquiline (10 mg/kg) (Kotwal et al., 2020). The time points of the collected tail vein blood samples were 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 h after bedaquiline administration. Finally, conform to the AVMA guidelines for the euthanasia of animals, eight SD rats were given 2% isoflurane inhalation anesthesia followed by 5% isoflurane concentration. Plasma (100 µL) was mixed with 300 µL ACN and 20 µL IS (200 ng/mL), vortexed, and centrifuged thoroughly, and the supernatant (100 µL) was obtained for UPLC-MS/MS detection.

Statistical analysis

The Michaelis–Menten, IC₅₀, Lineweaver–Burk plots and mean plasma concentration–time curves were plotted by GraphPad Prism software (version 9.5; GraphPad Software Inc., CA, USA). The pharmacokinetic parameters were obtained by non-compartmental analysis using Drug and Statistics software (version 3.0; Shanghai University of Traditional Chinese Medicine, Shanghai, China). All data were expressed as mean ± standard deviation (SD) and analyzed statistically using the Statistical Package for the Social Sciences software (version 24.0; SPSS Inc., Chicago, IL, United States). P <  0.05 was considered statistically significant.

UPLC-MS/MS analysis

The UPLC-MS/MS chromatograms of bedaquiline, M2, and IS (fluconazole) under three different conditions are presented in Fig. 1. The retention times of bedaquiline, M2, and IS were 1.36, 1.35, and 1.17 min, respectively. No endogenous interferences were observed to affect the determination of bedaquiline, M2, and IS. Moreover, the precision and accuracy of this method were both less than 15%. The concentration range of the calibration standard curve of bedaquiline was 1–5,000 ng/mL, and M2 was 1–500 ng/mL, with correlation coefficients over 0.999. The lower limit of quantification (LLOQ) of bedaquiline and M2 was 1 ng/mL. In addition, the stability, matrix effect (ME), and extraction recovery of samples in this method were all accorded with the requirements.

Carvedilol inhibited the metabolism of bedaquiline in vitro

According to Fig. 2, bedaquiline was a self-inhibiting substrate, and carvedilol exhibited the most powerful restraining power of 50 drugs, with 81.88% inhibition of bedaquiline. Then, combined with the result of IC₅₀, carvedilol strongly inhibited the metabolism of bedaquiline, with IC₅₀ values of 15.35 ± 0.43 µM in RLM, 7.55 ± 0.74 µM in HLM, and 0.79 ± 0.05 µM in CYP3A4, respectively (Fig. 3). Therefore, we next conducted to explore the underlying mechanisms. The IC₅₀ shift was used to assess the existence of TDI. Commonly, if the decrease in enzyme activity in the presence of NADPH (+NADPH) was greater than that in the absence of NADPH (–NADPH), TDI was suspected, and an IC₅₀ shift fold >10 was a time-dependent inhibition (Jin et al., 2015). As exhibited in Fig. 4, IC_50(−NADPH)/IC_50(+NADPH) was 1.18, 1.92, and 5.00 for RLM, HLM, and CYP3A4, respectively. Moreover, the Lineweaver–Burk plot intersected in the second quadrant in RLM, exhibited a set of parallel lines in HLM, and intersected in the third quadrant of CYP3A4 (Fig. 5). Accordingly, these data demonstrated that carvedilol inhibited the metabolism of bedaquiline was all not in a time-dependent inhibition, and the inhibition type was mixed of non-competitive + competitive, un-competitive, and mixed of non-competitive + un-competitive, with the αK_i values of 61.32, 8.44, and 0.13 in RLM, HLM, and CYP3A4, respectively (Table 1).

Carvedilol changed the main pharmacokinetic parameters of bedaquiline in vivo

The mean concentration–time curves of bedaquiline and M2 were demonstrated in Fig. 6. Based on Tables 2 and 3, we found that carvedilol could raise the AUC_(0−t), AUC_(0−∞), and C_max values of bedaquiline by 2.15–2.28 fold and declined the CL_z/F of bedaquiline by about 4.33 fold. Furthermore, carvedilol prolonged the T_max of bedaquiline while shortening its t_1/2z. However, there was a non-significant difference in M2 when bedaquiline was used in combination with carvedilol compared to the single-used.