Chemicals and reagents

Acalabrutinib, ACP-5862, tirabrutinib (internal standard, IS), and 21 cardiovascular drugs including finerenone, were obtained from Shanghai Canspec Scientific Instruments Co., Ltd. (Shanghai, China), with purity levels exceeding 98%. The essential reagents nicotinamide adenine dinucleotide phosphate (NADPH) and human liver microsomes (HLM) were sourced from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) and iPhase Pharmaceutical Services Co., Ltd. (Beijing, China), respectively. Recombinant human CYP3A4 (rCYP3A4) was given by Beijing Hospital. The chromatographic reagents in the experiment, including HPLC-grade acetonitrile and methanol, together with formic acid, were procured from Merck Company (Darmstadt, Germany) and Anaqua Chemicals Supply (ACS, American), respectively. The purity of the other solvents utilized in the experiment exceeded 99.9%.

Experimental equipment and its parameters

All analytes were quantified using an UPLC-MS/MS system. Chromatographic separation was carried out on a Waters Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm). The column temperature was set at 40 °C, the flow rate was 0.4 mL/min, and the total gradient run time was 2 min. The mobile phases used in the assay were 0.1% aqueous formic acid (A) and acetonitrile (B). A Waters Xevo TQ-S triple quadrupole mass spectrometer (Milford, MA, USA) was employed for detection and operated in positive ion mode. Quantification was achieved by multiple reaction monitoring (MRM), with transitions of m/z 466.04 → 372.11 for acalabrutinib, m/z 481.89 → 388.14 for ACP-5862 and m/z 455.32 → 320.21 for IS (Tirabrutinib), respectively, as summarized in Table 1. The mass spectra and corresponding chemical structures of acalabrutinib and ACP-5862 were presented in Fig. 1.

Table 1 The quantitative ion pairs and related parameters of acalabrutinib, its metabolite ACP-5862 and ISFig. 1

Mass spectra and chemical structures of acalabrutinib (A) and ACP-5862 (B) in this study

Preparation of rat liver microsomes (RLM)

Livers were collected from six healthy rats, and initial rinsed with phosphate-buffered saline (PBS) solution after undergoing perfusion. Subsequently, 0.25 M sucrose buffer solution was added to the livers and kept at 4 °C, and the liver tissues were mechanically homogenized prior to further processing. Then, they were centrifuged at a rotational speed of 11,000 rpm for 15 min. After this operation was repeated twice, the supernatant was centrifuged at 75,600 × g for 2 h, and the precipitate was resuspended in PBS solution and stored at −80 °C. The determination of protein concentration was executed through the utilization of the Bradford protein Assay kit (Thermo Scientific, Waltham, MA, USA).

Assessment of Michaelis–Menten constant (Km) in liver microsomes and rCYP3A4

An enzyme incubation mixture (200 μL) was prepared containing rCYP3A4 or RLM/HLM (0.3 mg/mL), acalabrutinib, 1 × PBS buffer, and 1 mM NADPH. Concentration gradients of acalabrutinib were 1, 2, 5, 10, 20, 80, 100 μM in RLM, 1, 2, 5, 10, 20, 100, 150 μM in HLM and 1, 2, 5, 10, 20, 40, 60 μM in rCYP3A4, respectively. After 5 min of pre-incubation at 37 °C, 10 μL of NADPH was added, followed by further incubation for 30 min and termination by storage at −80 °C. After 2 h of freezing, samples were processed by protein precipitation using acetonitrile (400 μL), with 20 μL of IS working solution (200 ng/mL) incorporated during sample preparation. Then, the samples were subjected to vortex mixing for 2 min after complete thawing, centrifuged at a rotational speed of 13,000 rpm for 10 min at 4 °C, an aliquot (100 μL) of supernatant was transferred for subsequent UPLC-MS/MS determination.

Assessment of acalabrutinib inhibition and its mechanism by cardiovascular analogues in different in vitro metabolic systems

After determining the Km value, an enzymatic reaction was performed in 200 μL system using rCYP3A4 or RLM/HLM (0.3 mg/mL), acalabrutinib, 1 × PBS buffer, 1 mM NADPH, and 100 μM of potential inhibition drugs. Information on the 21 cardiovascular drugs was provided in Supplementary Table S1. Inhibitory effects were compared based on relative enzyme activity. Relative activity is defined as the ratio of metabolite production in the presence and absence of the inhibitor. Inhibitors exhibiting more than 20% relative activity were not subjected to the next step of the experiment.

Based on the results of the previous step of experiments, the drugs with higher inhibitory effects, finerenone and ticagrelor, were selected for further evaluation of their inhibitory potency in RLM using the half-maximal inhibitory concentration (IC50) assay. A 200 μL incubation system consistent with the previous step was used, in which inhibitor concentrations were set at 0, 0.01, 0.1, 1, 10, 25, 50, and 100 μM to obtain IC50 values. We also determined the IC50 value of finerenone in HLM and rCYP3A4 with the same concentration gradient as in RLM.

In addition, an investigation was conducted to ascertain the type of inhibitory mechanism of finerenone on acalabrutinib, where the concentration gradient of finerenone and acalabrutinib was established in the RLM/HLM and rCYP3A4 system based on the corresponding IC50 of finerenone and the corresponding Km of acalabrutinib, respectively. The gradients were shown below, in RLM, the concentrations of acalabrutinib were 8.87, 17.74, 35.48, and 44.35 μM, and the concentrations of finerenone were 0, 3.50, 7.00, and 17.50 μM. In HLM, the concentrations of acalabrutinib were 2.12, 4.23, 8.46, and 16.92 μM, and the concentrations of finerenone were 0, 10.65, 21.29, and 42.58 μM. In rCYP3A4, the concentrations of acalabrutinib were 1.52, 3.03, 4.55, and 7.06 μM, and the concentrations of finerenone were 0, 1.76, 3.52, and 5.28 μM. The incubation conditions and sample post-treatment procedures of the inhibitor screening experiments were consistent with those of the Km experiments, and the same experimental conditions were used for the IC50 assay and the mechanism of inhibition studies.

Animal experiments

Ten male Sprague–Dawley rats weighing 180–200 g were utilized for the experiments, with animals sourced from the Animal Experimentation Center of the First Affiliated Hospital of Wenzhou Medical University (Zhejiang, China). Animal procedures were performed in accordance with established animal welfare guidelines and were designed to follow the principles of reduction, replacement, and refinement.

Healthy rats were randomly divided into Group A (control group, 5.0 mg/kg acalabrutinib) (Xu et al. 2022) and Group B (experiment group, 5.0 mg/kg acalabrutinib + 2.0 mg/kg finerenone), with 5 rats in each group. And, the solvent for all drugs dissolved was corn oil. Fasting was required for 12 h per to the start of the experiment, but unrestricted water intake was allowed. During the experiment, rats in Group B were orally administered 2.0 mg/kg of finerenone (Bui et al. 2024), and 30 min later, Groups A and B were orally administered 5.0 mg/kg of acalabrutinib. Blood was collected via the tail vein at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 6, 8, and 24 h following the administration of the drug. The fresh blood samples were anticoagulated with sodium heparin at the appropriate ratio and subsequently centrifuged for 10 min at 8,000 rpm and 4 °C. Then 100 μL of supernatant was aspirated, and protein precipitation was carried out by adding 300 μL of acetonitrile together with 10 μL of IS working solution, followed by complete vortexing. The mixture was then centrifuged at 13,000 rpm for 10 min. The supernatant was extracted and used for the determination of the concentration of each analyte by UPLC-MS/MS method.

Molecular docking

Chemical structures of acalabrutinib and finerenone were retrieved from PubChem as SDF files (https://pubchem.ncbi.nlm.nih.gov/). In addition, the crystal structure of the CYP3A4 protein was retrieved from the Protein Data Bank (PDB ID: 5ET8) (https://www.rcsb.org/). The target protein was then processed using AutoDock Tools 1.5.7, and PyMOL was used to remove crystalline water molecules and original ligands, followed by polar hydrogen assignment. The number of docking operations was 200. During the docking process, the CYP3A4 active site was centered, where a 40 Å × 40 Å × 40 Å grid box was set up and AutoDock was used for semi-flexible docking, allowing the ligand conformation to change while maintaining receptor rigidity.

Data analysis

Enzyme kinetic parameters, including Km, IC₅₀, Ki, and αKi, were calculated using GraphPad Prism (version 9.5.1). Pharmacokinetic parameters of acalabrutinib and ACP-5862 in rat plasma were derived using Drugs and Statistics (DAS, version 3.0). Statistical analyses were conducted in SPSS (version 27.0), and the Student's t-test was employed for parametric testing, with p < 0.05 considered indicative of statistical significance.