Gastrointestinal stromal tumor (GIST), commonly occurring in the gastrointestinal tract, is a mesenchymal cell tumor usually driven by activating mutations in the receptor tyrosine kinase proto-oncogene, KIT, or the platelet-derived growth factor receptor α (PDGFRα) (Mehren and Joensuu, 2018; Di Vito et al., 2023; Zalcberg, 2021). Tyrosine kinase inhibitors (TKIs) such as imatinib, sunitinib and regorafenib serve as the first, second, and third line treatment, respectively, by targeting KIT and PDGFRA. Additionally, avapritinib is specifically utilized for GIST cases harboring PDGFRA exon 18 mutations. These therapies have demonstrated promise in extending patient survival and improving outcomes. They do not completely prevent secondary mutations that cause tumor progression over time (Mehren and Joensuu, 2018; Di Vito et al., 2023; Janku et al., 2020; Naito et al., 2023).

By inhibiting kinase signaling of primary and secondary-resistant KIT and PDGFRA mutations by a dual action mechanism, ripretinib is a broad-spectrum switch-controlled kinase inhibitor that offers a highly favorable safety and efficacy profile in patients with advanced GISTs in clinical studies (Smith et al., 2019; Dhillon, 2020). In the phase III INTRIGUE trial, ripretinib demonstrated comparable efficacy to sunitinib in patients with disease progression or intolerance to imatinib (median progression-free survival (PFS) in KIT exon 11 intent-to-treat (ITT) populations, 8.3 versus 7.0 months, respectively, P = 0.36), with less adverse events and improved tolerance compared to sunitinib (Bauer et al., 2022). In a phase 2, multicenter, randomized, open-label bridging study of the INTRIGUE study conducted in China, PFS was longer for ripretinib compared to sunitinib in the KIT exon 11 ITT population (median PFS not reached for ripretinib versus 4.9 months for sunitinib, P = 0.03) (Li et al., 2023). Given its proven efficacy and safety demonstrated in clinical trials, ripretinib has been approved by the National Medical Products Administration of China as well as the U.S. Food and Drug Administration for treating advanced GISTs in adult patients who have undergone three or more kinase inhibitors, including imatinib. Furthermore, ripretinib is also recommended as an alternative second-line therapy in the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology for Gastrointestinal Mesenchymal Tumors Version 1.2024 and the 2023 edition of Guidelines of Chinese Society of Clinical Oncology (CSCO) for the Diagnosis and Treatment of Gastrointestinal Mesenchymal Tumors (Janku et al., 2020; Smith et al., 2019; Klug et al., 2022; Blay et al., 2020; Bauer et al., 2022; National Comprehensive Cancer Network, 2024; China Clinical Oncology Society Guidelines Working Committee, 2023). In addition, ripretinib demonstrated varying activity in inhibiting different exon locations of KIT mutations in vitro, which was also observed in the mutational subgroup assessment from the INTRGUE study (Smith et al., 2019; Heinrich et al., 2024). Improved PFS was observed with sunitinib compared to ripretinib in patients with only KIT exon 11 + 13/14 mutations (median, 15.0 versus 4.0 months). Conversely, ripretinib showed better PFS versus sunitinib in patients with only KIT exon 11 + 17/18 mutations (median, 14.2 versus 1.5 months) (Heinrich et al., 2024). Ongoing research has been conducted continuously in different mutation types to maximize the efficacy and safety of ripretinib for patients with GIST.

Ripreintib is primarily metabolized by N-demethylation, producing the active metabolite DP-5439. The anti-tumor activity of DP-5439 is similar to ripretinib, and both undergo hepatic metabolism primarily via the CYP3A4 enzyme (Li et al., 2022b; Pan et al., 2023). Clinical pharmacokinetic (PK) studies revealed considerable variability in PK parameters among patients, with maximum observed plasma concentration (C_max) and area under the concentration-time curve from time 0–24 h (AUC_0-24h) of both ripretinib and DP-5439 increasing proportionally to the dose within a certain dose range, whose variability (coefficient of variation, CV%) amounted to 35 ∼ 60% (Janku et al., 2020). Preclinical studies indicated that the inhibitory effect of ripretinib on KIT was both concentration-dependent and time-dependent, suggesting that higher in vivo concentrations correlated with increased effectiveness in inhibiting KIT phosphorylation. Additionally, dose escalation had demonstrated improved tumor regression and enhanced survival rates (Smith et al., 2019; Dhillon, 2020). This was confirmed in its phase I clinical study and the INVICTUS phase III trial, where patients initially receiving 150 mg QD orally experienced disease progression (PD) showed renewed benefit after dose escalation to 150 mg BID (Blay et al., 2020; Janku et al., 2020). This suggested that increased exposure of ripretinib correlated with improved efficacy (George et al., 2021; Zalcberg et al., 2021). Furthermore, considering the differences in ripretinib effects across various mutations observed in in vitro and in vivo studies, it can be speculated that the exposure to ripretinib may vary among these mutational subgroups, warranting further investigation.

In the phase I dose-escalation trial for ripretinib, no maximum tolerated dose (MTD) was identified. However, dose-limiting toxicities (DLTs) such as elevated lipase and creatine kinase were observed in the 100 mg BID, 200 mg BID, and 150 mg QD dose groups. Adverse events including myalgia, muscle spasms, palmar-plantar erythrodysesthesia (PPES), and hypertension exhibited a dose-dependent increase, indicating potential dose-escalation-related toxicity with ripretinib (Janku et al., 2020). This highlights the importance of exploring ripretinib exposure to mitigate adverse events. Consequently, therapeutic drug monitoring (TDM) of ripretinib could be clinically significant for patients. Moreover, further investigation into the pharmacokinetic and pharmacodynamic properties of ripretinib is essential to enhance patient outcomes and facilitate its clinical utility.

Several quantification methods for ripretinib in beagle dogs and rats plasmas have been developed. An ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) was established by Wang et al. (2021) to measure ripretinib levels in beagle dogs, and the impact of voriconazole and itraconazole on the PK of ripretinib was investigated. Similarly, Mudavath and Ashok (2023) developed a LC-MS/MS method to quantify ripretinib in rat plasma. However, few methods remain for monitoring blood concentrations of ripretnib and DP-5439 in human plasma. Li et al. (2022b) reported the effect of CYP3A initiative, CYP3A inspiration, and gastric acid reduction on the pharmacokinetics of ripretinib without detailed quantification methods. Qian et al. (2024) established an LC-MS/MS method for determining blood concentration in human plasma ripretnib and DP-5439. Therefore, the purpose of this study was to develop and validate an LC-MS/MS method for quantifying ripretinib and DP-5439 in human plasma. Subsequently, this method was then applied to clinical samples to assess its practical applicability in GIST patients.

This study established and validated an LC-MS/MS method for the determination of ripretinib and its active metabolite DP-5439 in human plasma. The established method was initially implemented for quantifying ripretinib levels in plasma samples from 33 patients who received ripretinib at our hospital and for the pharmacokinetics studies in 4 patients.

To acquire mass spectral data, MRM mode with ESI+ was chosen, consistent with previously published methods for detection in beagles and rats. In the UPLC-MS/MS method developed by Wang et al. (2021) for quantifying ripretinib in beagle dog plasma, the ion transitions were monitored at 509.93→416.85 for ripretinib. While the LC-MS/MS method established by Mudavath and Ashok (2023) in rat plasma employed 510.09→94.06. In the LC-MS/MS method established by Qian et al. (2024) in human plasma, the ion transitions were monitored at 510.1→417.1 for ripretinib and 496.0→403.1 for DP-5439. In this study, ripretinib was monitored with the ion transitions of 510.1→417, and DP-5439 was 496.11→ 402.9, consistent with the results of Qian et al.

In this study, D8-ripretinib, a deuterium substituent analogue of ripretinib, was utilized as the internal standard. This isotopic internal standard closely mirrored the structure of both ripretinib and DP-5439, resulting in a retention time similar to that of ripretinib. This congruence significantly reduced errors arising from matrix interference and disparities in ionization characteristics among analytes. Simultaneously, during LC-MS/MS detection, structurally similar substances can induce ion suppression, potentially compromising detection accuracy. Therefore, we assessed the performance of IS solutions at concentrations of 10.0 μg/mL, 5.0 μg/mL, and 2.0 μg/mL. The results indicated that using an IS concentration of 2.0 μg/L minimized ion suppression, leading to notable improvements in the linearity of the standard curve and significant enhancements in both the accuracy and precision of quality control measures. Furthermore, our method provided a wider linear range (10–5,000 μg/L both for ripretinib and DP-5439) compared to the existing method (Qian et al., 7.5–3,000 μg/L for ripretinib and 10–4,000 ng/mL for DP-5439), thus accommodating the varied demands of clinical sample analysis. The mobile phases used in this method were 0.1% formic acid in water (phase A) and acetonitrile (phase B), similar to that of Wang et al.; however, Qian et al. added 5 mM ammonium formate to mobile phase A to enhance the peak shapes of the analytes. Meanwhile, various chromatographic conditions were explored to improve the specificity and selectivity of ripretinib and DP-5439, including elution methods and gradient elution times. Both gradient elution and isocratic elution (10% A phase: 90% B phase) were assessed. The results indicated that isocratic elution caused peak tailing, while gradient elution facilitated superior separation of distinct analytes. Following evaluations at elution times of 5, 6, and 7 min, it was determined that a 6-min elution time yielded enhanced substance separation and higher response values in a shorter period. Compared to the method of Qian et al. (2024), the analysis time of this method is slightly longer (6 min versus 4.7 min). However, based on the simple preparation of the mobile phase, convenient operation, and lesser internal standard usage, the method of this study made improvements in economic practicability and clinical applications, which may reduce detection costs.

Due to the limited solubility of ripretinib, DP-5439 and D8-ripretinib in both water and methanol, DMSO was used to enhance solubility (Huang et al., 2022). Subsequently, the agents were diluted with methanol-water to prepare the working solution and samples. However, pure methanol solution exhibited high viscosity at room temperature, potentially leading to analyte adsorption onto container walls and resulting in greater inaccuracies. Therefore, to prevent the precipitation of analytes and ensure the stability of samples, it was crucial to prepare the working solution with the highest feasible concentration of methanol. After experimenting with various ratios of methanol-water, it was determined that an 85% methanol-water mixture reduced wall adhesion while maintaining a sufficient concentration of methanol, thereby minimizing solvent effects. For pretreatment, acetonitrile was selected as the protein precipitant in this study for its convenience and cost-effectiveness in therapeutic drug monitoring.

In method validation, we did not assess matrix effects in hyperlipidemic and hemolytic matrices, unlike the study by Qian et al. However, Qian et al. (2024) only reported data from 15 patients receiving 150 mg of ripretinib. In contrast, our study evaluated the method’s applicability on a larger sample size, presenting concentration results for the 150 mg and 300 mg dosage groups. In this study, thorough blood sampling for pharmacokinetic curve analysis was performed on four patients, providing a preliminary description of the pharmacokinetics of ripretinib.

The PK of ripretinib had been preliminarily explored in its phase I clinical study, revealing significant inter-patient variability following ripretinib administration. The geometric mean C_min (CV%) reached 284 (62.5%) μg/L for ripretinib and 546 (78.2%) μg/L for DP-5439 after patients attained steady-state levels on a regimen of ripretinib 150 mg QD for more than 15 days. For those who received ripretinib 150 mg BID continuously, the geometric mean C_min (CV%) of ripretinib and DP-5439 were 968 (113.8%) μg/L and 1,590 (93.8%) μg/L, respectively (Janku et al., 2020). In this study, the total median C_min (range) of patients treated with 150 mg QD for more than 15 consecutive days was 1129.46 (140.95 ∼ 2981.39) μg/L, of which the median C_min (range) of ripretinib was 398.50 (66.98 ∼ 1458.91) μg/L, and 654.74 (30.71 ∼ 1522.48) μg/L for DP-5439, slightly surpassing the patient levels in phase I trial (Janku et al., 2020). Xu et al. (2023) conducted a study monitoring the plasma concentration of ripretinib in Chinese patients. Among 42 patients, the C_min of ripretinib was 406.55 ± 272.52 (mean ± SD) μg/L, comprising 378.08 ± 226.3 μg/L for 150 mg QD and 863.67 ± 511.7 μg/L for 150 mg BID. Compared to Hao Xu et al.’s study, the C_min of ripretinib in this study was marginally elevated. This discrepancy might stem from potential bias attributed to ripretinib high PK parameter variability and the limited patient cohort size in this study.

In the phase I clinical study, the geometric mean C_max (CV%) reached 761 (31.8%) μg/L for ripretinib and 804 (45.5%) μg/L for DP-5439 among those receiving 15 consecutive days on a regimen of 150 mg QD (Janku et al., 2020). However, In the Chinese patient cohort, the C_max (range) of ripretinib and DP-5439 was observed at 833 (308-1700) μg/L and 1,250 (276-1930) μg/L, respectively (Li et al., 2022a). In this study, C_max for all three patients taking 150 mg QD ripretinib was within the range of data from the phase I clinical study and the Chinese patient cohort. Besides, the median AUC_0-12h of ripretinib and DP-5439 for 150 mg QD dosage in this study were also consistent with that of the Chinese patient cohort (median AUC_0-12h(range): ripretinib: 6,610 (2,760–15,900) μg/L·h; DP-5439: 11,400 (2,450–18,600) μg/L·h). For patient 4 with 150 mg BID, C_max (ripretinib: 812.98 μg/L, DP-5439: 1,249.39 μg/L) and AUC_0-12h (ripretinib: 7,055.65 μg/L·h, DP-5439: 12,601.33 μg/L·h) were slightly below the geometric mean C_max (CV%) (ripretinib: 1,290 (79.1%) μg/L, DP-5439: 1,800 (85.9%) μg/L) and AUC_0-12h (CV%) (ripretinib: 7,929 (97.7%) μg/L·h, DP-5439: 15,646 (110.3%) μg/L·h) of the Phase I clinical study (Janku et al., 2020).

However, some limitations remain in this study. Due to the small sample size of this study and the short follow-up time, it is unsatisfactory to analyze the correlation based on concentration results and clinical outcomes. In addition, the same high inter-individual variability of PK parameters as in the clinical trial could also be observed in the four patients with intensive blood collection. This also provides a reference for the subsequent pharmacokinetic study of ripretinib and demonstrates new challenges. Further studies are being conducted to expose the correlation between plasma concentrations of ripretinib and clinical benefit in patients with GISTs.

In conclusion, an LC-MS/MS method capable of concurrently quantifying ripretinib and its metabolite DP-5439 in human plasma was established. Following thorough validation, the method was effectively utilized in PK investigations and TDM of ripretinib among 33 Chinese patients with advanced GISTs. This method provided a swift, straightforward, and precise assay for ripretinib TDM and PK evaluations within clinical settings.