Development of UPLC-MS/MS method for studying the pharmacokinetic interactions of fuzuloparib with curcumin in rats.

Fuzuloparib is a novel orally bioactive poly-ADP-ribose polymerase inhibitor (PARPi), which was approved by the Chinese Regulatory Agency (CRA) in 2020 for the treatment of platinum-sensitive recurrent ovarian, fallopian tube, and primary peritoneal cancers. This study firstly presents a rapid and accurate ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for analyzing the levels of fuzuloparib and its major metabolite (SHR165202), and to investigate drug-drug interaction between fuzuloparib and curcumin in vitro and in vivo studies. After protein precipitation with acetonitrile, mobile phase consisted of acetonitrile and 0.1 % formic acid with a gradient elution was used to successfully separate fuzuloparib, SHR165202 and talazoparib (internal standard, IS). The results indicated that fuzuloparib and SHR165202 had good linearity over the calibration range of 2-50 ng/mL and 1-20 ng/mL, respectively. The precision, accuracy, stability, matrix effect, and extraction recovery required for methodological validation all complied with the requirements of the Bioanalytical Method Validation Guidelines. In vitro microsome incubation experiments, curcumin exhibited inhibitory effect on fuzuloparib in both rat liver microsomes (RLM) and human liver microsomes (HLM) with half-maximal inhibitory concentration (IC50) value of 10.54 µM and 47.64 µM, respectively, and the corresponding mechanism was non-competitive. Furthermore, the inhibitory mechanism of curcumin on fuzuloparib was validated through molecular docking. In pharmacokinetic experiments in rats, curcumin significantly altered the plasma exposure of fuzuloparib, resulting in significant increases in AUC(0-t) and Cmax of fuzuloparib and a significant decrease in CLz/F. Moreover, the metabolite SHR165202 showed significant increases in AUC(0-t), AUC(0-∞), Tmax and Cmax and a significant decrease in CLz/F. This further supports the notion that curcumin could inhibit the metabolism of fuzuloparib. Therefore, when co-administering fuzuloparib and curcumin in clinic, it is recommended to monitor plasma levels of fuzuloparib and pay close attention to adverse effects. If necessary, the dose of fuzuloparib needs to be reduced.

Evaluation of the inhibitory effect of quercetin on the pharmacokinetics of tucatinib in rats by a novel UPLC-MS/MS assay.

CONTEXT: Tucatinib (CYP2C8 substrate) and quercetin (CYP2C8 inhibitor) are two common drugs for the treatment of cancer. However, the effect of quercetin on the metabolism of tucatinib remains unknown. OBJECTIVE: We validated a sensitive method to quantify tucatinib levels in rat plasma based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), which was successfully employed to explore the effect of quercetin on tucatinib pharmacokinetics in rats. MATERIALS AND METHODS: An Acquity UPLC BEH C18 column was applied to achieve the separation of tucatinib and internal standard (IS) talazoparib after protein precipitation with acetonitrile. Then, we used this assay to investigate the effect of different doses of quercetin (25, 50 and 100 mg/kg) on the exposure of orally administered tucatinib (30 mg/kg) in 24 Sprague-Dawley (SD) rats, which were randomly divided into three quercetin pre-treated groups and one control group (n = 6). RESULTS: Our developed assay was verified in all aspects of bioanalytical method validation, involving lower limit of quantification (LLOQ), selectivity, accuracy and precision, calibration curve, extraction recovery, matrix effect and stability. After pre-treatment with 100 mg/kg quercetin, AUC0→t, AUC0→∞ and Cmax of tucatinib were remarkably increased by 75.4%, 75.8% and 59.1% (p < 0.05), respectively, while CLz/F was decreased significantly by 47.3% (p < 0.05) when compared with oral administration of 30 mg/kg tucatinib alone. This change is dose-dependent. CONCLUSIONS: This study will help better understand the pharmacokinetic properties of tucatinib with concurrent use with quercetin, and more clinical verifications were inspired to confirm whether this interaction has clinical significance in humans.

Inhibitory effects of voriconazole, itraconazole and fluconazole on the pharmacokinetic profiles of ivosidenib in rats by UHPLC-MS/MS.

Ivosidenib, as an oral mutant isocitrate dehydrogenase 1 (mIDH1) inhibitor, was awarded approval in the USA for the targeted therapy of relapsed or refractory acute myeloid leukemia (AML) in adult patients, who also had a susceptible enzyme to mIDH1. The aim of our present study was to develop and validate an accurate and fast assay based on the ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technique for the quantification of ivosidenib in plasma and to investigate the possible effects of different CYP3A4 inhibitors (voriconazole, itraconazole and fluconazole) on ivosidenib metabolism in rats. After the fast protein crash with acetonitrile, chromatographic separation of ivosidenib and erlotinib (used as the internal standard in this experiment, IS) was accomplished using an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column, and detection of the analyte was also performed using a Xevo TQ-S triple quadrupole tandem mass spectrometer in the positive ion electrospray ionization (ESI) interface. The assay showed enough linearity over a 0.5-6000 ng/mL calibration range. The application of the validated bioanalytical method based on the UHPLC-MS/MS technique was further successfully exhibited in an animal study of the drug-drug interaction between ivosidenib (50 mg/kg) and voriconazole (20 mg/kg)/itraconazole (20 mg/kg)/fluconazole (20 mg/kg) in rats. Voriconazole, itraconazole and fluconazole increased the exposure of ivosidenib in plasma by different degrees and also had a potential inhibitory effect on the metabolism of ivosidenib. Thus, a dose reduction or interruption of ivosidenib may be important to guide the practice of clinical medicine.

Development of a novel and quick UPLC-MS/MS method for the pharmacokinetic analysis of duvelisib in beagle dogs.

Duvelisib, a new oral phosphoinositide-3-kinase (PI3K)-δ and PI3K-γ inhibitor, was recently approved in the USA as the therapeutic drug for patients with the diseases of relapsed or refractory chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). In the present study of our research, a quick and simple bioanalytical method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique was fully explored and established for the quantification of plasma duvelisib concentrations from beagle dog in which gilteritinib was used as the internal standard (IS). After a simple and quick protein precipitation treated with acetonitrile, the chromatographic separation of the analyte was carried out on an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 µm) conducted in a gradient elution procedure where acetonitrile (solvent A) and 0.1 % formic acid in water (solvent B) consisted as the mobile phase. The measurements of the analyte and IS were explored using a XEVO TQS triple quadrupole tandem mass spectrometer, which was comprised with electrospray ionization (ESI) source in positive ion mode. Selected reaction monitoring (SRM) mode was employed to detect the parent-to-daughter ion transitions as follows: m/z 416.88 → 281.88 for duvelisib, and m/z 553.09 → 436.01 for IS, respectively. The assay was successfully established in the calibration range from 0.5 to 3000 ng/mL for duvelisib, where the lower limit of quantification (LLOQ) was set at 0.5 ng/mL. The precisions of intra-day and inter-day for duvelisib were all below 12.6 %, and the accuracies were from -2.5% to 14.1%. Both matrix effect and mean recovery of the analyte and IS were all acceptable, and the analyte was stable during the assay and storage in dog plasma samples. The novel established bioanalytical method based on UPLC-MS/MS technique was effectively employed to the investigation of the pharmacokinetic profile of duvelisib in beagle dogs following a 1.34 mg/kg single dose of oral administration.

Drug-drug interaction study of imatinib and voriconazole in vitro and in vivo.

Background: In clinical practice, common problem polypharmacy could result in the increased risks of drug-drug interactions (DDIs). Co-administered imatinib (IMA) and voriconazole (VOR) as one treatment protocol in cancer patients with fungal infections are common.Purpose: The aim of the present study was to assess the potential DDIs associated with the concurrent use of IMA and VOR in rat liver microsomes (RLMs) and in rats.Methods and results: The concentration levels of IMA, VOR, and their metabolites N-desmethyl IMA (CGP74588) and N-oxide voriconazole (N-oxide VOR) were determined by ultra performance liquid chromatography-tandem mass spectrometry. In vitro study of RLMs, VOR inhibited the IMA metabolism with the half-maximal inhibitory concentration (IC50) of 105.20 µM, while IC50 for IMA against VOR was 61.30 µM. After co-administered IMA and VOR in rats, the C max of IMA was increased significantly, while the AUC0→t, AUC0→∞, and C max of CGP74588 were decreased significantly. In addition, similar results were also found that the main pharmacokinetic parameters (AUC0→t, AUC0→∞, MRT0→∞, T max, and C max) of VOR were increased significantly, while the AUC0→t, AUC0→∞, and C max of N-oxide VOR were decreased significantly. Incorporation of all the results indicated that both drugs had a inhibitory effect on each other's metabolism in vitro and in vivo.Conclusion: Thus, it is of great value to monitor the concomitant use of IMA and VOR in the clinic to reduce the risks of unexpected clinical outcomes.

Functional Characterization of 22 CYP3A4 Protein Variants to Metabolize Ibrutinib In Vitro.

Cytochrome P450 3A4 (CYP3A4) is quantitatively the most important P450 enzyme in adults. It is suggested that CYP3A4 genetic polymorphisms may influence the rate of the metabolism and elimination of CYP3A4 substrates in human beings. Ibrutinib is an anticancer drug and primarily metabolized by CYP3A4. The aim of this study was to systematically investigate the effects of 22 CYP3A4 protein variants on the metabolism of ibrutinib in vitro. When compared with wild-type CYP3A4.1, two variants (CYP3A4.17 and CYP3A4.24) had no detectable enzyme activity; five variants (CYP3A4.10, .11, .18, .23 and .33) exhibited no significant differences; another five variants (CYP3A4.3, .4, .9, .19 and .34) showed increased intrinsic clearance values, while the remaining nine variants (CYP3A4.2, .5, .14, .15, .16, .28, .29, .31 and .32) displayed decreased enzymatic activities in different degrees. As the first study of 22 CYP3A4 protein variants in ibrutinib metabolism, these comprehensive data may help in the clinical assessment of the metabolism and elimination of ibrutinib and also offer a reference to the personalized treatment of ibrutinib in clinic.

Intestinal and peripheral fibrinogen-like protein 2 expression in inflammatory bowel disease.

BACKGROUND: Fibrinogen-like protein 2 (FGL2), a new member of the fibrinogen-like family, has recently been identified as a novel immunosuppressive molecule. AIM: The purpose of this work was to investigate intestinal and peripheral expression of FGL2 in patients with inflammatory bowel disease (IBD), mainly ulcerative colitis (UC) and Crohn's disease (CD). METHODS: FGL2 expression in mucosal biopsies from three groups (UC group (n = 61), CD group (n = 54), and controls group (n = 35)) was detected by immunohistochemistry. Concentrations of FGL2 in plasma from 50 UC patients, 45 CD patients, and 30 controls were analyzed by enzyme-linked immunosorbent assay. Western blot of FGL2 protein and real-time fluorescent quantitative PCR of FGL2 mRNA expression by peripheral mononuclear cells was performed. Correlations of FGL2 expression with disease type, activity, and location, and with measured laboratory data, including C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), were examined. RESULTS: Intestinal and peripheral FGL2 protein data showed that FGL2 expression was significantly up-regulated in both UC and CD patients compared with controls (P < 0.001). Expression of FGL2 was higher in UC and CD patients with active disease than in those with inactive disease (P < 0.001). Moreover, FGL2 mRNA expression was significantly higher in patients with active disease than in those with inactive disease (P < 0.050). Expression of FGL2 protein was correlated with disease activity indices, CRP levels, and ESR levels. CONCLUSION: Expression of FGL2 was up-regulated in IBD patients with active disease. Measurement of FGL2 may be used as a helpful biomarker for understanding immunopathogenesis and for assessment of IBD.