Effects of the CYP3A inhibitors, voriconazole, itraconazole, and fluconazole on the pharmacokinetics of osimertinib in rats

Materials

OSIM (≥98.0%) was purchased from the National Institute for Food and Drug Control (Beijing, China). VCZ (≥98.0%), ICZ (≥98.0%), and FCZ (≥98.0%) were provided by Zhejiang Provincial People’s Hospital (Hangzhou, China). Nilotinib (≥99.0%), carboxymethyl cellulose sodium, isopropanol, and formic acid of LC-MS grade were purchased from Aladdin Industrial Corporation (Shanghai, China). Acetonitrile and methanol were purchased from Merck (HPLC grade; Darmstadt, Germany). Ultra-pure water was produced with a Millipore Synergy system (Merck, Darmstadt, Germany). The rat tail vein fixator (Yuyan, China) was provided by the Laboratory Animal Center of Zhejiang Provincial People’s Hospital.

Calibration curve, quality control, and internal standard samples

Two separate stock solutions of OSIM, 0.5 mg/mL, were produced in methanol and stored at −40 °C. The first stock solution was used to prepare working solutions for the calibration curve by serial dilution in methanol at concentrations ranging from 20 ng/mL to 5000 ng/mL. Calibration curve samples consisting of eight non-zero concentrations were obtained by spiking aliquots of 90 µL blank plasma with 10 µL working solutions of calibration solutions (90:10, v/v). Quality control (LLOQ; low, medium, and high QC) samples were prepared in the same manner at concentrations of 20,50, 2000, and 4000 ng/mL. Internal standard (IS) nilotinib was prepared in acetonitrile at a final concentration of 10 ng/mL.

Chromatography and mass spectrometry

A liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) system coupled to a Jasper™ HPLC and Triple Quad™ 4500MD and equipped with an electrospray ionization (ESI) interface source (AB Sciex, Redwood City, CA, USA) was used for the analysis. The separation was achieved on a Waters XBridge C₁₈ C18 column (3.5 µm, 2.1 × 100 mm; Waters Corp., Milford, MA, USA) maintained at 45 °C. Mobile phase A was 0.2% formic acid in water, and mobile phase B was acetonitrile. The gradient elution procedure was optimized as follows: 20% B (0–0.5 min), 20–40% (0.5–1.0 min B), 40–90% B (1.0–2.0 min), 90% B (2.0–3.0 min), and 90–20% B (3.1–3.2 min) at a flow rate of 0.50 mL/min for 4.0 min. Carry-over was minimized with methanol-acetonitrile-isopropanol-water solution (1:1:1:1, v/v/v/v) before and after the injection procedure.

The source parameters were set as follows: ion spray voltage: 5.5 kV, source temperature: 550 °C, GS1: 50psi, GS2: 55 psi, curtain gas: 30 psi, collision gas: 9 psi, entrance potential: 10 V. Multiple reaction monitoring (MRM) was performed in the positive ion mod, and monitoring was carried out for two ion pairs: m/z 500.0 → m/z 72.1 for OSIM (DP = 153 V, CE = 35 V), and m/z 530.0 →m/z 289.1 for IS (DP = 151 V, CE = 41 V). The MS/MS spectra of the ion fragments are illustrated in Fig. 1. The manually tuned MS parameters are provided in Table S1. Data acquisition and quantification were carried out with Analyst™ MD 1.6.3 and MultiQuant™ MD 3.0.2 software (AB Sciex, Redwood City, CA, USA), respectively.

Sample preparation

The 100 µL of plasma samples (calibration curve, quality control, and rat plasma samples) were precipitated with 100 µL IS (10 ng/mL) and 200 µL acetonitrile. The blank plasma samples were prepared by mixing 100 µL blank plasma and 300 µL acetonitrile. Subsequently, all samples were vortex-mixed for 30 s and were centrifuged at 14,000 × g for 10 min. Finally, each sample was reconstituted with an aliquot of mobile phase A. The sample was transferred to a 0.8 mL 96-well plate, of which 5 µL was injected into HPLC–MS/MS system for analysis.

Pharmacokinetic study

Twenty-four adult male Sprague-Dawley rats (220 ± 20 g) with specific pathogen free were supplied by the Laboratory Animal Center of Zhejiang Provincial People’s Hospital. All rats were healthy and did not involve genetic modification status, genotype, and any previous procedures. The rats were housed at an animal laboratory (temperature 24 ± 2 °C, relative humidity 50 ± 10%, 12-hour light-dark cycle) for one week as an adaptation period. The study was performed in accordance with the Animal Care and Use Committee of Zhejiang Provincial People’s Hospital (number: A20220030).

OSIM, VCZ, ICZ, and FCZ were prepared in 0.3% carboxymethyl cellulose sodium (CMC-Na). After 12 h of fasting and free water consumption, the rats were randomly separated into four groups, and the sample size was based on a similar study using as few rats as possible (n = 6): control (equivalent volume of 0.3% CMC-Na), VCZ (20 mg/kg), ICZ (20 mg/kg), and FCZ (20 mg/kg) groups. Only those responsible for treatment management researchers know this group well. Each group was housed in a single cage. The rats in the four groups were orally dosed once daily with 0.3% CMC-Na, VCZ, ICZ, or FCZ, respectively; they reached steady-state exposure after five continuous days of dosing. Thirty minutes after the last administration, each rat received a single oral dose of 10 mg/kg of OSIM. All rats received an oral gavage dose of approximately equivalent volume treatment regimens (one mL). Rats were restrained on the rat fixator on the laboratory bench. Blood samples (300 µL) from the tail veins were collected in heparin tubes at 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 24, and 48 h after OSIM administration. Plasma samples were then separated by centrifugation (3,500 × g) for 10 min, and the resultant plasma was frozen at −30 °C prior to analysis.

Data analysis

Experimental data were given as mean ± standard deviation (SD). The mean concentration levels of OSIM versus time profile were drawn using GraphPad Prism 9.4 (GraphPad Software, Inc., San Diego, CA, USA). Non-compartmental pharmacokinetic parameters of concentration–time data were processed by PKSolver 2.0 (China Pharmaceutical University, Nanjing, China), and the NCA Extravascular module was employed to derive pharmacokinetic parameters for each treatment separately, including C_max, AUC_0–t, AUC_0–inf, T_max, t_1/2, MRT_0–inf, V_z/F, and Cl/F. SPSS 26.0 was used for statistical analysis (IBM, Armonk, USA). Significant differences among the groups were identified using one-way ANOVA and Tukey test. P < 0.05 was considered significant.

Method validation

The chromatographic conditions were carefully optimized to provide the best peak shape and highest response. The retention times were 1.82 and 2.08 min for OSIM and IS, respectively. Representative chromatograms are presented in Fig. 2. The calibration curve was linear from 2 to 500 ng/mL, with correlation coefficients (r²) of over 0.99.

Intra- and inter-day precision and accuracy were measured in sextuplicate at the LLOQ, LQC, MQC, and HQC levels, on three consecutive days. The precision (coefficient of variation, %CV) and accuracy (relative error, %RE) for all QC samples should be within ± 15% (± 20% for LLOQ). For all QC samples, the average %RE ranged from 88.45 to 110.41%, and the intra- and inter-day %CV was less than 7.73% (Table S2).

Extraction recovery and matrix effect were determined using three concentrations of QC samples in five replicates. The peak area ratio of QC obtained in blank plasma pre-extraction was compared to that prepared in samples spiked post-extraction for extraction recovery. The matrix factor was estimated by comparing the peak area ratio of QC generated in samples spiked post-extraction to that of pure solutions at the same nominal concentration. The mean extraction recovery of OSIM ranged from 96.25 to 100.67%, with the %CV ≤ 2.50% (Table S3). The IS-normalized matrix effect values for OSIM ranged from 97.26 to 105.48% and the %CV was not higher than 4.69% (Table S3).

The stability of the method was evaluated by determining the concentrations of LQC, MQC, and HQC; the evaluation was performed in quadruplicate. Bench-top stability was tested by placing the pretreatment samples at room temperature (−25 °C) for 6 h, autosampler stability was assessed by placing the prepared sample in the injector for 24 h, freeze-thaw stability was determined to be adequate over at least three cycles (from −30 °C to 25 °C), and long-term stability was assessed to be at least one month before sample preparation (at −30 °C for 30 d). For all stability results, the average %RE of OSIM ranged from −11.04% to 7.48% across the three QC concentrations, with the %CV less than 6.42% (Table S4).

Carry-over was evaluated by injecting four blank plasma samples over the upper limit of quantitation (ULOQ, 500 ng/mL). The peak area of the blank plasma sample should not exceed 20% of that recorded at LLOQ for OSIM and should be less than 5% of IS. The calculated carry-overs for OSIM and IS were 4.44% and 0.11%, respectively (Table S5).

The above results demonstrated that the assay met the required standards and can be applied to the pharmacokinetic study of OSIM.

Effect of VCZ, ICZ, and FCZ on the pharmacokinetic profile of OSIM

The mean concentration–time profiles of OSIM alone and OSIM combined with VCZ, ICZ, and FCZ are presented in Fig. 3. The main pharmacokinetic parameters for OSIM are listed in Table 1. The results of the Tukey test are represented in Fig. S1.

These results revealed significant differences in pharmacokinetic parameters between the control and the VCZ or FCZ groups, especially for the values of C_max, AUC_0–t, AUC_0–inf, V_z/F, and Cl/F (P < 0.05). Concomitant administration of OSIM with VCZ demonstrated an increase in C_max and AUC_0–t of OSIM by 58.04% and 62.56%, respectively compared with those of administration of OSIM alone. Conversely, the Cl/F of OSIM decreased by 37.74% in VCZ group. The T_max value of OSIM in the VCZ group increased by 17.67%, but the t_1/2, MRT_0–inf, and V_z/F values were decreased by 42.56%,10.25%, and 64.03%, respectively compared with the control. These results implied that VCZ contributed to enhanced systemic exposure and decreased clearance of OSIM. When OSIM was co-administered with FCZ, the Cl/F value of OSIM in the FCZ group decreased by 0.49-fold, but the values of C_max and AUC_0–t were enhanced by approximately 0.53- and 1.01-fold, respectively. In contrast, co-administration with OSIM and FCZ did not significantly alter the t_1/2 and T_max of orally administered OSIM. Following the administration of OSIM with ICZ, the t_1/2 and V_z/F of OSIM decreased by 25.41% and 38.08%, respectively (P < 0.05), but other parameters (C_max, T_max, AUC, MRT, and Cl/F) were not significantly different compared with the control group.