N-desmethyl Imatinib Research Articles

Therapeutic drug monitoring of imatinib: is there a rationale for also quantifying its active metabolite ?

Therapeutic drug monitoring of imatinib is widely performed to individualize imatinib dosage. While N-desmethyl imatinib is an active metabolite of imatinib, its concentrations are not routinely determined. Imatinib and N-desmethyl imatinib trough plasma concentrations at steady-state were obtained from 295 patients with either chronic myeloid leukemia or gastrointestinal stromal tumor to see whether N-desmethyl imatinib provided additional information. Pharmacokinetic data were analyzed using a nonlinear mixed effect approach. Correlations between several pharmacokinetic metrics of drug exposure were evaluated. The mean value of the N-desmethyl imatinib/imatinib ratio of trough concentrations was 0.31 with half of the values between 0.23-0.37. N-desmethyl imatinib and total (i.e., N-desmethyl imatinib plus imatinib) trough plasma concentrations or area-under-the curve values were closely correlated with imatinib values. The distribution of imatinib or total concentrations between patients requiring imatinib dosage adjustment, or not, were similar. These results do not clearly support routine N-desmethyl monitoring since it does not provide additional information to imatinib data.

Effect of recombinant CYP3A4 variants and interaction on imatinib metabolism in vitro

The aim of this study was to investigate the catalytic activity of 26 Cytochrome P450 3A4 (CYP3A4) variants and drug interactions on imatinib metabolism in recombinant insect microsomes. This study was designed with an appropriate incubation system and carried out in the constant temperature water. By using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to measure the quantities of its metabolite N-desmethyl imatinib, to elucidate the impacts of the CYP3A4 genetic polymorphism and drug interactions on the metabolism of imatinib. Consequently, as compared to CYP3A4.1, the intrinsic clearance (CLint) values of the variations were dramatically changed, rising from 2.34 % to 120.57 %. CYP3A4.14 showed an increase in CLint in comparison to CYP3A4.1, and the remaining 24 variants demonstrated decreases in catalytic activity for the metabolism of imatinib. In addition, the metabolism of imatinib was decreased to varied degrees by ketoconazole, itraconazole, and fluconazole in CYP3A4.1 and CYP3A4.18. Moreover, most of CYP3A4 variants showed similar trend of enzyme activity under different substrates of imatinib and cabozantinib, except 6 variants (CYP3A4.3,.4,.10,.15,.29 and.31). The first study of the effects of 26 CYP3A4 variants on imatinib metabolism will contribute to the clinical evaluation of imatinib and help personalize therapy in clinical settings.

Layer-by-layer fabrication of covalent organic frameworks on stainless steel needles as solid-phase microextraction probe coupled with electrospray ionization mass spectrometry for enrichment and determination of tyrosine kinase inhibitors in biosamples

Sunitinib, N-desmethyl imatinib, dasatinib, imatinib, and bosutinib are tyrosine kinase inhibitors (TKIs) that are commonly employed in the treatment of a multitude of cancers. However, the inappropriate concentrations of TKIs can result in ineffective treatment or the emergence of multiple adverse effects. Consequently, the development of a rapid and sensitive analytical method for TKIs is of paramount importance for the safe administration of drugs. In this work, solid-phase microextraction (SPME) probe combined with an electrospray ionization mass spectrometry (ESI-MS) coupling platform was constructed for rapid and sensitive determination of TKIs. The covalent organic frameworks (COFs) coated SPME probe was made of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (TAPT) and 2,5-dibutoxyterephthalaldehyde (DBTA) by in-situ layer-by-layer chemical bonding synthesis strategy. The TAPT-DBTA-SPME probe exhibited several advantageous properties which rendered it suitable for the enrichment of TKIs. Under the optimal conditions, the developed analytical method demonstrated a broad linear range (0.05–500.00 µg/L), a low limit of detection (0.02 µg/L) and a high enrichment factor (51–203) for TKIs. The developed analytical method was successfully applied to a pharmacokinetic study of TKIs in mouse plasma and tissue matrix, demonstrating that the proposed analytical method has promise for clinical applications and metabolic monitoring.

Physiologically based pharmacokinetic modeling of imatinib and N-desmethyl imatinib for drug-drug interaction predictions.

The first-generation tyrosine kinase inhibitor imatinib has revolutionized the development of targeted cancer therapy and remains among the frontline treatments, for example, against chronic myeloid leukemia. As a substrate of cytochrome P450 (CYP) 2C8, CYP3A4, and various transporters, imatinib is highly susceptible to drug-drug interactions (DDIs) when co-administered with corresponding perpetrator drugs. Additionally, imatinib and its main metabolite N-desmethyl imatinib (NDMI) act as inhibitors of CYP2C8, CYP2D6, and CYP3A4 affecting their own metabolism as well as the exposure of co-medications. This work presents the development of a parent-metabolite whole-body physiologically based pharmacokinetic (PBPK) model for imatinib and NDMI used for the investigation and prediction of different DDI scenarios centered around imatinib as both a victim and perpetrator drug. Model development was performed in PK-Sim® using a total of 60 plasma concentration-time profiles of imatinib and NDMI in healthy subjects and cancer patients. Metabolism of both compounds was integrated via CYP2C8 and CYP3A4, with imatinib additionally transported via P-glycoprotein. The subsequently developed DDI network demonstrated good predictive performance. DDIs involving imatinib and NDMI were simulated with perpetrator drugs rifampicin, ketoconazole, and gemfibrozil as well as victim drugs simvastatin and metoprolol. Overall, 12/12 predicted DDI area under the curve determined between first and last plasma concentration measurements (AUClast) ratios and 12/12 predicted DDI maximum plasma concentration (Cmax) ratios were within twofold of the respective observed ratios. Potential applications of the final model include model-informed drug development or the support of model-informed precision dosing.

Development of a simultaneous quantification method of imatinib and sunitinib and their main metabolites and its application in patients with gastrointestinal stromal tumor.

Correlations between plasma concentrations of imatinib and sunitinib with efficacy and toxicity have been established. It is crucial to develop a sensitive and precise method for determining the plasma concentrations of imatinib and sunitinib, along with their active metabolites, to facilitate therapeutic drug monitoring and individualized therapy. Plasma samples were separated on an Agilent ZORBAX SB-C18 chromatographic column using gradient elution. Quantification was performed using a mass spectrometer equipped with electrospray ionization in multiple reaction monitoring. The analysis time was 18 min per run, with all analytes and internal standards eluting within 8 min. The calibration range was 25-4000 ng/mL for imatinib, 5-800 ng/mL for N-desmethyl imatinib (CGP74588), and 2.5-400 ng/mL for sunitinib and N-desethyl sunitinib (SU12662). Intra- and inter-assay precision were both below 15%, and accuracy ranged between 90.0% and 101.9%. The method was successfully applied to determine blood samples from 120 patients with gastrointestinal stromal tumors who received imatinib (n = 115) and sunitinib (n = 5). It has been validated as linear, accurate, precise, and robust, making it suitable for therapeutic drug monitoring of imatinib and sunitinib in routine clinical practice.

Pharmacokinetic Interaction Between Imatinib and Metformin in Rats.

Imatinib is primarily transported into the liver by organic cation transporter 1 (OCT1), organic anion transporting polypeptide 1B3 (OATP1B3), and novel organic cation transporter 2 (OCTN2), which is the first step in the metabolic and elimination of imatinib. Patients taking imatinib may concurrently take metformin, a substrate for OCT1. Drug-drug interactions (DDI) may occur between imatinib and metformin, affecting the clinical efficacy of imatinib. This experiment aimed to investigate the pharmacokinetic effects of metformin on imatinib and its active metabolism of N-desmethyl imatinib in rats. Twenty healthy Sprague-Dawley rats were selected and randomly divided into control and experimental groups (10 rats per group). The control group was orally administered imatinib (30 mg/kg) for 14 days, and the experimental group was orally co-administered imatinib (30 mg/kg) and metformin (200 mg/kg) for 14 days. The plasma concentrations of imatinib and N-desmethyl imatinib in rats were determined by ultra-performance liquid chromatography-mass spectrometry. Pharmacokinetic parameters were calculated by DAS2.0 software. After single-dose co-administration of imatinib and metformin on day 1, the AUC0-24 (area under the plasma concentration-time curve) and Cmax (maximum concentration) of imatinib and the MRT (mean residence time) and Cmax of N-desmethyl imatinib in the experimental group were significantly decreased compared with the control group (P < 0.05). After multiple-dose co-administration of imatinib and metformin for 14 days, the AUC0-24 and Cmax of both imatinib and N-desmethyl imatinib were significantly decreased in the experimental group (P < 0.05). With both single and multiple co-administration doses, metformin significantly changed the pharmacokinetic parameters of imatinib and N-desmethyl imatinib. The results suggest that care should be taken when metformin and imatinib are co-administered.

Therapeutic Drug Monitoring of Imatinib and N-Desmethyl Imatinib in Chronic Myeloid Leukemia Patients Using LC-MS/MS in a Cohort Study.

Imatinib is an oral tyrosine kinase inhibitor (TKI) and first-line therapy for patients with chronic myeloid leukemia (CML). There is a positive correlation between serum imatinib concentrations and treatment response. However, the specific relationship between the blood concentration of imatinib and its influencing factors remains unclear. This study collected basic information from 102 patients using imatinib as first-line treatment for CML. Further, we analyzed the individual differences in imatinib concentration and explored its influencing factors. Through intra-day and inter-day precision studies, we found that the precision for the imatinib assay methodology was within ±13% and that the recovery rate was above 85%. There is notable individual variation in the blood concentration of imatinib; the recommended treatment concentration is 860-1500ng/mL, with only 41.40% of patients achieving this concentration. Also, there was a negative correlation between age and imatinib trough concentration (Ctrough ), as is observed between age and N-desmethyl imatinib. Moreover, compared with the adolescent group, the serum imatinib Ctrough for groups aged 17-47 and 48-68years was significantly reduced. Further analysis shows that imatinib Ctrough values reaching therapeutic concentrations (59%) increased dramatically for patients with CML aged 17-47years. Moreover, groups dosed with 400mg/day resulted in therapeutic imatinib concentrations for 68% of patients with CML, which was the best performance. The established method was validated, with acceptable accuracy, precision, linearity, and stability, as required, and then successfully applied to the therapeutic drug monitoring of imatinib. Age, dose, and metabolites can influence the imatinib concentration and its therapeutic effect in patients with CML.

A Study to Explore the Role of IDH1 (R132) Mutation on Imatinib Toxicity and Effect of ABCG2/OCT1 Expression on N-Desmethyl Imatinib Plasma Level in Egyptian Chronic Myeloid Leukemia Patients.

Imatinib mesylate (IM) is the gold standard for treatment of Chronic Myeloid Leukemia (CML). This study aimed to gain more knowledge of the altered PK, pharmacogenetic factors, and gene expression leading to variable IM levels. Fifty patients with chronic phase-CML were enrolled in this study and divided as 25 responders and 25 non-responders (patients are directly recruited after response assessment). HPLC/MS/MS was used to determine trough and peak concentration of imatinib and N-desmethyl imatinib in the blood. PCR-RFLP technique was used to detect IDH1 gene mutation (R132). The median value of IM trough level was significantly higher, the P/T ratio was significantly lower and the α-1-acid glycoprotein (AGP) was significantly higher among responders compared to non-responders (P=0.007, 0.009 and 0.048, respectively). Higher N-desmethyl imatinib peak plasma concentration was observed with low mRNA expression of ABCG2 and OCT1 (P=0.01 and 0.037, respectively). IDH1 R132 gene mutation was associated with a significant increase in toxicities (P=0.028). In conclusion, IM trough level, P/T ratio and AGP was significantly higher in responders. In addition, ABCG2 and OCT1 gene expression may affect the interindividual PK variation. Although a prospective study with a larger patient population is necessary to validate these findings. IDH1 mutation is a predictor of increased toxicity with IM treatment.

Development of a Sensitive and High-Throughput Assay for Simultaneous Quantification of 5 Tyrosine Kinase Inhibitors and 2 Active Metabolites in Human Plasma Using Ultra-high Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry.

Breakpoint cluster region-Abelson (BCR-ABL) tyrosine kinase inhibitors (TKIs) demonstrate improved therapeutic efficacy in chronic myeloid leukemia (CML). However, drug-drug interactions, nonadherence, and host-related factors may influence plasma concentrations. Therefore, therapeutic drug monitoring may be necessary for patients presenting inadequate treatment responses or adverse events. Herein, the authors aimed to develop a more sensitive and high-throughput method than those previously reported to simultaneously quantify 5 TKIs (imatinib, nilotinib, dasatinib, bosutinib, and ponatinib) and 2 active metabolites (N-desmethyl imatinib and N-desmethyl ponatinib) using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Plasma samples were prepared according to a solid-phase extraction protocol using an Oasis MCX µElution plate. The assay fulfilled the requirements of the US Food and Drug Administration for assay validation, with a lower limit of quantification of 0.2 ng/mL for dasatinib, 0.3 ng/mL for N-desmethyl ponatinib, 0.5 ng/mL for N-desmethyl imatinib, bosutinib, and ponatinib, and 2.5 ng/mL for imatinib and nilotinib. Within-batch and batch-to-batch precision at the lower limit of quantification and quality control levels were within 14.3% and 10.9%, respectively. Within-batch and batch-to-batch accuracies ranged from 15.5% to 13.0% and 5.70% to 7.03%, respectively. A positive electrospray ionization mode was used with a run time of 6.0 minutes. The assay applicability was verified by the successful measurement of 78 clinical samples from patients undergoing CML therapy. The method allows assessment of trough concentrations of TKIs and active metabolites in patients with CML, and hence can be used to assess blood samples in routine clinical settings.

A Validated 2D-LC-UV Method for Simultaneous Determination of Imatinib and N-desmethylimatinib in Plasma and its Clinical Application for Therapeutic Drug Monitoring with GIST Patients

Background: The trough concentration (Cmin) of Imatinib (IM) is closely related to the treatment outcomes and adverse reactions of patients with gastrointestinal stromal tumors (GIST). However, the drug plasma level has great inter- and intra-individual variability, and therapeutic drug monitoring (TDM) is highly recommended. Objective: To develop a novel, simple, and economical two-dimensional liquid chromatography method with the ultraviolet detector (2D-LC-UV) for simultaneous determination of IM and its major active metabolite, N-desmethyl imatinib (NDIM) in human plasma, and then apply the method for TDM of the drug. Methods: The sample was processed by simple protein precipitation. Two target analytes were separated on the one-dimension column, captured on the middle column, and then transferred to the two-dimension column for further analysis. The detection was performed at 264 nm. The column temperature was maintained at 40˚C and the injection volume was 500 μL. Totally 32 plasma samples were obtained from patients with GIST who were receiving IM. Results: IM and NDIM were separated well from other impurities and the entire analytical time for each run was 12.0 min. The calibration curves had good linearity in the range of 33.5-2678.4 ng/mL for IM, and 20.0-1600.0 ng/mL for NDIM, respectively. The extraction efficiency was more than 95%. The acceptable accuracy, precision, recovery and stability were also obtained. The Cmin of the drug in patients was measured with the validated method. Conclusion: The novel 2D-LC-UV method is simple, stable, highly automated and independent of specialized technicians, which greatly increases the real-time capability of routine TDM for IM in hospital.

Method development and validation for simultaneous determination of six tyrosine kinase inhibitors and two active metabolites in human plasma/serum using UPLC–MS/MS for therapeutic drug monitoring

Over the past decades, therapeutic drug monitoring (TDM) of tyrosine kinase inhibitors (TKIs) and their main active metabolites has shown benefits in improving treatment efficacy and safety. Therefore, a sensitive, simple and economical ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of six oral tyrosine kinase inhibitors (TKIs) and two active metabolites, including imatinib (IMA), N-desmethyl imatinib (NDIMA), sunitinib (SUNI), N-desethyl sunitinib (NDSUNI), regorafenib (REGO), nilotinib (NILO), dasatinib (DASA) and osimertinib (OSI) in human plasma/serum for therapeutic drug monitoring. The plasma/serum samples were deproteinated with acetonitrile after spiking with two deuterated internal standards (ISs, [2H8]-imatinib and [2H10]-sunitinib) and separated on a 40 °C ACQUITY UPLC® BEH C18 column (1.7 µm, 2.1 mm × 50 mm). The mobile phase was composed of acetonitrile (solution A) and water-formic acid-ammonium acetate (1 M) (994:1:5, v/v/v, solution B). Gradient elution was applied at a flow rate of 0.4 mL/min. Detection was carried out using a Triple Quad 5500 tandem mass spectrometer coupled with an electrospray ionization (ESI) source in positive mode. The method was validated over the calibration curve (CV) range of 2–400 ng/mL for NDSUNI and DASA, 2.5–500 ng/mL for SUNI, 10–2000 ng/mL NDIMA and OSI, 20–4000 ng/mL for NILO, 30–6000 ng/mL for REGO and 50–10000 ng/mL for IMA using linear regression and 1/x2 weighting. No difference was observed in the matrix effect (ME) among blank human plasma, hemolytic plasma, lipemic plasma, plasma spiked with ten commonly used drugs by cancer patients, or serum samples in general. The standard curve fitted by two standard curves of serum and plasma showed good linearity, and the precision and accuracy results of QC samples in both plasma and serum were acceptable and the concentration was comparable. To provide a clinical reference for the operation, the stability of the whole process from sample collection to drug detection was verified. SUNI and NDSUNI showed obvious photoisomerization under light exposure. Therefore, strict light protection was applied for all sample collection and handling steps of SUNI and NDSUNI. Compared with heparin anticoagulant tubes, the stability of the eight compounds in both whole blood and plasma was better in K3-EDTA and sodium citrate anticoagulant tubes. Given that all the analytes were stable in plasma at 4 °C for 48 h and in whole blood at room temperature for 48 h but OSI and REGO were unstable in whole blood and plasma at room temperature, samples should be centrifuged as soon as possible to be preserved as plasma at 4 °C when OSI or REGO is detected. In conclusion, this validated method can provide support for clinical practice, such as therapeutic drug monitoring (TDM) and pharmacokinetic studies of these six TKIs and two active metabolites.

Development and validation of an UPLC-MS/MS method for simultaneous determination of fifteen targeted anti-cancer drugs in human plasma and its application in therapeutic drug monitoring

In this study, an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously detect 15 targeted anti-cancer drugs: aletinib, afatinib, apatinib, icotinib, dasatinib, erlotinib, gefitinib, crizotinib, lapatinib, regorafenib, ceritinib, sorafenib, vemurafenib, imatinib, and N-desmethyl imatinib. Plasma samples were processed using a new magnetic solid phase extraction technique to extract each drug. The 15 analytes and four isotope internal standards were separated using an Agilent Eclipse XDB-C18 column (50.0 × 2.1 mm, 1.7 µm) with water containing 0.1% formic acid and acetonitrile as the mobile phase. The method verification included specificity, calibration curves, carryover, accuracy, crosstalk, precision, stability, recovery, dilution integrity, and matrix effects. The results showed that the developed UPLC-MS/MS method met the requirements of the U.S. Food and Drug Administration guidelines for methodological validation and could be used to monitor plasma concentrations. The response function was established for concentration range of 2.5–2500.0 ng/mL for aletinib, afatinib, apatinib, icotinib, dasatinib, crizotinib, regorafenib, vemurafenib, and N-desmethyl imatinib and 10.0–10,000.0 ng/mL for erlotinib, ceritinib, imatinib, sorafenib, gefitinib, and lapatinib, with a coeffificient of correlation of > 0.9977 for all the compounds. The precision and accuracy of all the analytes were < 6.88% and 5.29%, respectively. The percentage recovery and matrix effect of all the analytes were 91.3–103% and 93.8–102% for three QC concentrations levels. The recovery and matrix effect for all the ISs ranged from 93.7% to 98.8% and 94.6–101%. Meanwhile, we also found that the plasma concentrations of these targeted anti-cancer drugs showed large individual differences, which is not conducive to the treatment of tumors. Therefore, therapeutic drug monitoring (TDM) of these 15 targeted anti-cancer drugs is necessary, and this method could be used for TDM and exploration of pharmacokinetics of the aforementioned 15 targeted anti-cancer drugs.

High-resolution AP-SMALDI MSI as a tool for drug imaging in Schistosoma mansoni

Schistosoma mansoni is a parasitic flatworm causing schistosomiasis, an infectious disease affecting several hundred million people worldwide. Schistosomes live dioeciously, and upon pairing with the male, the female starts massive egg production, which causes pathology. Praziquantel (PZQ) is the only drug used, but it has an inherent risk of resistance development. Therefore, alternatives are needed. In the context of drug repurposing, the cancer drug imatinib was tested, showing high efficacy against S. mansoni in vitro. Besides the gonads, imatinib mainly affected the integrity of the intestine in males and females. In this study, we investigated the potential uptake and distribution of imatinib in adult schistosomes including its distribution kinetics. To this end, we applied for the first time atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) for drug imaging in paired S. mansoni. Our results indicate that imatinib was present in the esophagus and intestine of the male as early as 20 min after in vitro exposure, suggesting an oral uptake route. After one hour, the drug was also found inside the paired female. The detection of the main metabolite, N-desmethyl imatinib, indicated metabolization of the drug. Additionally, a marker signal for the female ovary was successfully applied to facilitate further conclusions regarding organ tropism of imatinib. Our results demonstrate that AP-SMALDI MSI is a useful method to study the uptake, tissue distribution, and metabolization of imatinib in S. mansoni. The results suggest using AP-SMALDI MSI also for investigating other antiparasitic compounds and their metabolites in schistosomes and other parasites.Graphical abstract

Method development for determination of imatinib and its major metabolite, N‐desmethyl imatinib, in biological and environmental samples by SA–SHS–LPME and HPLC

A salting-out-assisted switchable hydrophilicity solvent-based liquid phase microextraction (SA-SHS-LPME) was developed for the separation and determination of trace amounts of imatinib and N-desmethyl imatinib in biological and environmental samples by HPLC-UV. Triethylamine as a hydrophobic compound and protonated triethylamine carbonate as a hydrophilic one were switched by the addition or elimination of CO2 . The use of NaOH resulted in the elimination of CO2 from the sample solution, which led to the conversion of P-TEA-C into triethylamine (TEA) and as a result, the analytes was extracted and entered the TEA phase. The salting out was performed to speed up the formation of the TEA in the shape of fine droplets in the specimen solution. Furthermore, the impact of several momentous factors that influence the recovery of the extraction was investigated. Under the optimum conditions, the limit of detection and limit of quantification were obtained in ranges of 0.03-0.05 and 0.1-0.15 μg L-1 for imatinib and 0.04-0.06 and 0.13-0.20 μg L-1 for N-desmethyl imatinib, respectively. The preconcentration factor was 250. Inter- and intraday precision (RSD, n = 5) was <5%. In the case of imatinib and N-desmethyl imatinib in biological and environmental specimens, a range of 97.0-102% was obtained as the recovery.

Development and clinical validation of a simple and fast UPLC-ESI-MS/MS method for simultaneous quantification of nine kinase inhibitors and two antiandrogen drugs in human plasma: Interest for their therapeutic drug monitoring

Kinase inhibitors (KIs) and antiandrogen drugs (AAs) are oral anticancer drugs with narrow therapeutic index that exhibit high inter- and intra-individual variability. We describe here a UPLC-MS/MS method for the simultaneous quantification of nine KIs: cobimetinib, dasatinib, ibrutinib, imatinib, nilotinib, palbociclib, ruxolitinib, sorafenib and vemurafenib; two active metabolites of them: N-desmethyl imatinib, N-oxide sorafenib; and two AAs: abiraterone and enzalutamide; with short pre-treatment and run time in order to be easily used in clinical practice for their therapeutic drug monitoring (TDM) and facilitating pharmacokinetics and pharmacokinetics/pharmacodynamics studies. Plasma samples were prepared by a single-step protein precipitation. Analytes were separated on a Waters Acquity UPLC® T3 HSS C18 column by non-linear gradient elution; with subsequent detection by Xevo® TQD triple quadrupole tandem mass spectrometer in a positive ionization mode. Analysis time was 2.8 min per run, and all analytes eluted within 1.46–1.97 minutes. The analytical performance of the method in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, dilution integrity and stability of analytes under different conditions met all criteria for a bioanalytical method for the quantification of drugs. The calibration curves were linear over the range of 1−500 ng/mL for abiraterone, dasatinib and ibrutinib; 5−500 ng/mL for cobimetinib and palbociclib; 10−5,000 ng/mL for imatinib, N-desmethyl imatinib, nilotinib, sorafenib, N-oxide sorafenib and ruxolitinib; 100−50,000 ng/mL for enzalutamide and 100−100,000 ng/mL for vemurafenib with coefficient of correlation above 0.995 for all analytes. This novel method was successfully applied to TDM in clinical practice.

Method Development and Validation for Simultaneous Determination of Six Tyrosine Kinase Inhibitors and Two Active Metabolites in Human Plasma Using UPLC–MS/MS for Therapeutic Drug Monitoring

Over the past decades, therapeutic drug monitoring (TDM) of tyrosine kinase inhibitors (TKIs) and their main active metabolites has shown benefits in improving treatment efficacy and safety. Therefore, a sensitive, simple and economical ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of six oral tyrosine kinase inhibitors (TKIs) and two active metabolites, including imatinib (IMA), N-desmethyl imatinib (NDIMA), sunitinib (SUNI), N-desethyl sunitinib (NDSUNI), regorafenib (REGO), nilotinib (NILO), dasatinib (DASA) and osimertinib (OSI) in human plasma for therapeutic drug monitoring. The plasma samples were deproteinated with acetonitrile after spiking with two deuterated internal standards (ISs, [ 2 H 8 ]-imatinib and [ 2 H 10 ]-sunitinib) and separated on a 40 °C ACQUITY UPLC® BEH C 18 column (1.7 μm, 2.1 mm×50 mm). The mobile phase was composed of acetonitrile (solution A) and water-formic acid-ammonium acetate (1M)(994:1:5, v/v/v, solution B). Gradient elution was applied at a flow rate of 0.4 mL/min. Detection was carried out using a Triple Quad 5500 tandem mass spectrometer coupled with an electrospray ionization (ESI) source in positive mode. The method was validated over the calibration curve (CV) range of 2-400 ng/mL for NDSUNI and DASA, 2.5-500 ng/mL for SUNI, 10-2000 ng/mL NDIMA and OSI, 20-4000 ng/mL for NILO, 30-6000 ng/mL for REGO and 50-10000 ng/mL for IMA using linear regression and 1/x 2 weighting. No difference was observed in the matrix effect (ME) among blank human plasma, hemolytic plasma and lipemic plasma samples in general. And the intra- and inter-batch precision and accuracy of all eight components were acceptable. To provide a clinical reference for the operation, the stability of the whole process from sample collection to drug detection was verified. SUNI and NDSUNI showed obvious photoisomerization under light exposure. Therefore, strict light protection was applied for all sample collection and handling steps of SUNI and NDSUNI. Compared with heparin anticoagulant tubes, the stability of the eight compounds in both whole blood and plasma was better in K3-EDTA and sodium citrate anticoagulant tubes. Given that all the analytes were stable in plasma at 4 °C for 48 h and in whole blood at room temperature for 48 h but OSI and REGO were unstable in whole blood and plasma at room temperature, samples should be centrifuged as soon as possible to be preserved as plasma at 4 °C when OSI or REGO is detected. In conclusion, this validated method can provide support for clinical practice, such as therapeutic drug monitoring (TDM) and pharmacokinetic studies of these six TKIs and two active metabolites.

Development and Validation of an UPLC-MS/MS Method for Simultaneous Determination of Fifteen Targeted Anti-Cancer Drugs in Human Plasma and Its Application in Therapeutic Drug Monitoring

In this study, an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously detect 15 targeted anti-cancer drugs: aletinib, afatinib, apatinib, icotinib, dasatinib, erlotinib, gefitinib, crizotinib, lapatinib, regorafenib, ceritinib, sorafenib, vemurafenib, imatinib, and N-desmethyl imatinib. Plasma samples were processed using a new magnetic solid phase extraction technique to extract each drug. The 15 analytes and four isotope internal standards were separated using an Agilent Eclipse XDB-C18 column (50.0 × 2.1 mm, 1.7 μm) with water containing 0.1% formic acid and acetonitrile as the mobile phase. The method verification included specificity, calibration curves, carryover, accuracy, crosstalk, precision, stability, recovery, dilution integrity, and matrix effects. The results showed that the developed UPLC-MS/MS method met the requirements of the U.S. Food and Drug Administration guidelines for methodological validation and could be used to monitor plasma concentrations. Meanwhile, we also found that the plasma concentrations of these targeted anti-cancer drugs showed large individual differences, which is not conducive to the treatment of tumors. Therefore, therapeutic drug monitoring of these 15 targeted anti-cancer drugs is necessary to improve the efficacy and reduce the side effects of cancer treatment.

Targeting Kinases in Fasciola hepatica: Anthelminthic Effects and Tissue Distribution of Selected Kinase Inhibitors.

Protein kinases have been discussed as promising druggable targets in various parasitic helminths. New drugs are also needed for control of fascioliasis, a food-borne trematode infection and worldwide spread zoonosis, caused by the liver fluke Fasciola hepatica and related species. In this study, we intended to move protein kinases more into the spotlight of Fasciola drug research and characterized the fasciolicidal activity of two small-molecule inhibitors from human cancer research: the Abelson tyrosine kinase (ABL-TK) inhibitor imatinib and the polo-like 1 (PLK1) inhibitor BI2536. BI2536 reduced viability of 4-week-old immature flukes in vitro, while adult worms showed a blockade of egg production. Together with a significantly higher transcriptional expression of PLK1 in adult compared to immature worms, this argues for a role of PLK1 in fluke reproduction. Both fluke stages expressed ABL1-TK transcripts at similar high levels and were affected by imatinib. To study the uptake kinetic and tissue distribution of imatinib in F. hepatica, we applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) for the first time in this parasite. Drug imaging revealed the accumulation of imatinib in different fluke tissues from 20 min to 12 h of exposure. Furthermore, we show that imatinib is metabolized to N-desmethyl imatinib by F. hepatica, a bioactive metabolite also found in humans. Besides the vitellarium, gastrodermal tissue showed strong signal intensities. In situ hybridization demonstrated the gastrodermal presence of abl1 transcripts. Finally, we assessed transcriptional changes of physiologically important genes in imatinib-treated flukes. Moderately increased transcript levels of a gene encoding a multidrug resistance protein were detected, which may reflect an attempt to defend against imatinib. Increased expression levels of the cell cycle dependently expressed histone h2b and of two genes encoding superoxide dismutases (SODs) were also observed. In summary, our pilot study demonstrated cross-stage activity of imatinib but not BI2536 against immature and adult F. hepatica in vitro; a fast incorporation of imatinib within minutes, probably via the oral route; and imatinib-induced expression changes of physiologically relevant genes. We conclude that kinases are worth analyzing in more detail to evaluate the potential as therapeutic targets in F. hepatica.

The effect of grape seed and green tea extracts on the pharmacokinetics of imatinib and its main metabolite, N-desmethyl imatinib, in rats

BackgroundImatinib is mainly metabolized by CYP3A4 and to a lesser extent by other isoenzymes, with N-desmethyl imatinib being its major equipotent metabolite. Being a CYP3A4 substrate, imatinib co-administration with CYP3A4 modulators would change its pharmacokinetic profile. The cancer chemoprevention potential and anticancer efficacy of many herbal products such as grape seed (GS) and green tea (GT) extracts had led to an increase in their concomitant use with anticancer agents. GS and GT extracts were demonstrated to be potent inhibitors of CYP3A4. The aim of this study is to investigate the effect of standardized GS and/or GT extracts at two different doses on the pharmacokinetics of imatinib and its metabolite, N-desmethyl imatinib, in SD-rats.MethodsStandardized GS and/or GT extracts were administered orally once daily for 21 days, at low (l) and high (h) doses, 50 and 100 mg/kg, respectively, before the administration of a single intragastric dose of imatinib. Plasma samples were collected and analyzed for imatinib and N-desmethyl imatinib concentrations using LC-MS/MS method, then their non-compartmental pharmacokinetic parameters were determined.Resultsh-GS dose significantly decreased imatinib’s Cmax and the {mathrm{AUC}}_0^{infty } by 61.1 and 72.2%, respectively. Similar effects on N-desmethyl imatinib’s exposure were observed as well, in addition to a significant increase in its clearance by 3.7-fold. l-GT caused a significant decrease in imatinib’s Cmax and {mathrm{AUC}}_0^{infty } by 53.6 and 63.5%, respectively, with more significant effects on N-desmethyl imatinib’s exposure, which exhibited a significant decrease by 79.2 and 81.1%, respectively. h-GT showed similar effects as those of l-GT on the kinetics of imatinib and its metabolite. However, when these extracts were co-administered at low doses, no significant effects were shown on the pharmacokinetics of imatinib and its metabolite. Nevertheless, increasing the dose caused a significant decrease in Cmax of N-desmethyl imatinib by 71.5%.ConclusionsThese results demonstrated that the pharmacokinetics of imatinib and N-desmethyl imatinib had been significantly affected by GS and/or GT extracts, which could be partially explained by the inhibition of CYP3A-mediated metabolism. However, the involvement of other kinetic pathways such as other isoenzymes, efflux and uptake transporters could be involved and should be characterized.Graphical abstract

Quantification of imatinib and related compounds using capillary electrophoresis-tandem mass spectrometry with field-amplified sample stacking.

A quantification method for imatinib (IM), its major metabolite N-desmethyl imatinib (NDI), and a degradation by-product was developed using CE-MS combined with an online concentration technique. The use of multiple reaction monitoring (MRM)-MS/MS further improved the sensitivity of this technology. Liquid-liquid extraction (LLE) using tertiary butyl methyl ether yielded high recovery and reproducibility for the pretreatment of serum samples. The recovery rate exceeded 83% for all three analytes, and was 90% for IM. To improve quantification results, a conductivity-induced online analyte concentration technique, field-amplified sample stacking (FASS), was used. The S/N ratios were improved at least 10-fold when compared with conventional capillary zone electrophoresis. The detection limits were 0.2ng/mL for IM, 0.4ng/mL for NDI, and 4ng/mL for the degradation by-product. These results are superior to those previously obtained by other reported methods. The new method was validated in terms of its selectivity, intra- and interday repeatability and accuracy, and sample storage stability, following the guidelines issued by the European Medicines Agency. Considering the convenient pretreatment procedure (LLE), superior sensitivity, and fast analysis speed (<15min), this method can be useful in the determination of imatinib levels in blood.