Bioanalysis of erlotinib, its O-demethylated metabolites OSI-413 and OSI-420, and other metabolites by liquid chromatography-tandem mass spectrometry with additional ion mobility identification.

Erlotinib is a first-generation epithelial growth factor receptor inhibitor used in the treatment of non-small cellular lung cancers. Our previously published method on a Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer for the quantitation of erlotinib, OSI-420, and OSI-413 and some other kinase inhibitors was transferred to a more sensitive Sciex QTRAP5500 system. Both methods showed comparable performance in the previous range (5-5000 and 1-1000 ng/mL for erlotinib and OSI-420) with comparable accuracies and precisions (98.9-106.2 vs 98.7.0-104.0, and 3.7-13.4 vs 4.6-13.2), and a high level of agreement between the methods (R2 = 0.9984 and 0.9951) for the quality control samples. The new system however was also capable of quantifying lower concentrations of both erlotinib and OSI-420 (0.5 and 0.1 ng/mL) with sufficient accuracy and precision. Along with the increased sensitivity we included the semi-quantitative determination of additional erlotinib metabolites M2, M3, M5, M6, M7, M8, M9, M10, M11, M12, M16 (hydroxy-erlotinib), M17, M18, M19, M20, M21 in a 0.1-1000 ng/mL range to the method. With a simple crash, dilute, and shoot sample preparation with acetonitrile and a 4.5 min analytical run time the method outperformed most other published methods in speed and simplicity and was suitable for TDM. Further, enhancement of the understanding of the pharmacokinetics of erlotinib and its metabolites was demonstrated.

Extrahepatic metabolism of ibrutinib.

Ibrutinib is a first-in-class Bruton's kinase inhibitor used in the treatment of multiple lymphomas. In addition to CYP3A4-mediated metabolism, glutathione conjugation can be observed. Subsequently, metabolism of the conjugates and finally their excretion in feces and urine occurs. These metabolites, however, can reach substantial concentrations in human subjects, especially when CYP3A4 is inhibited. Ibrutinib has unexplained nephrotoxicity and high metabolite concentrations are also found in kidneys of Cyp3a knockout mice. Here, a mechanism is proposed where the intermediate cysteine metabolite is bioactivated. The metabolism of ibrutinib through this glutathione cycle was confirmed in cultured human renal proximal tubule cells. Ibrutinib-mediated toxicity was enhanced in-vitro by inhibitors of breast cancer resistance protein (BCRP), P-glycoprotein (P-gp) and multidrug resistance protein (MRP). This was a result of accumulating cysteine metabolite levels due to efflux inhibition. Finally, through inhibition of downstream metabolism, it was shown now that direct conjugation was responsible for cysteine metabolite toxicity.

Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT) with Enhanced Activity.

Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide to form N-methylnicotinamide. Overexpression of NNMT is associated with a variety of diseases, including a number of cancers and metabolic disorders, suggesting a role for NNMT as a potential therapeutic target. By structural modification of a lead NNMT inhibitor previously developed in our group, we prepared a diverse library of inhibitors to probe the different regions of the enzyme's active site. This investigation revealed that incorporation of a naphthalene moiety, intended to bind the hydrophobic nicotinamide binding pocket via π-π stacking interactions, significantly increases the activity of bisubstrate-like NNMT inhibitors (half-maximal inhibitory concentration 1.41 µM). These findings are further supported by isothermal titration calorimetry binding assays as well as modeling studies. The most active NNMT inhibitor identified in the present study demonstrated a dose-dependent inhibitory effect on the cell proliferation of the HSC-2 human oral cancer cell line.

Brain accumulation of osimertinib and its active metabolite AZ5104 is restricted by ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein).

Osimertinib is an irreversible EGFR inhibitor registered for advanced NSCLC patients whose tumors harbor recurrent somatic activating mutations in EGFR (EGFRm+) or the frequently occurring EGFR-T790M resistance mutation. Using in vitro transport assays and appropriate knockout and transgenic mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 transport osimertinib and whether they influence the oral availability and brain accumulation of osimertinib and its most active metabolite, AZ5104. In vitro, human ABCB1 and mouse Abcg2 modestly transported osimertinib. In mice, Abcb1a/1b, with a minor contribution of Abcg2, markedly limited the brain accumulation of osimertinib and AZ5104. However, no effect of the ABC transporters was seen on osimertinib oral availability. In spite of up to 6-fold higher brain accumulation, we observed no acute toxicity signs of oral osimertinib in Abcb1a/1b;Abcg2 knockout mice. Interestingly, even in wild-type mice the intrinsic brain penetration of osimertinib was already relatively high, which may help to explain the documented partial efficacy of this drug against brain metastases. No substantial effects of mouse Cyp3a knockout or transgenic human CYP3A4 overexpression on oral osimertinib pharmacokinetics were observed, presumably due to a dominant role of mouse Cyp2d enzymes in osimertinib metabolism. Our results suggest that pharmacological inhibition of ABCB1 and ABCG2 during osimertinib therapy might potentially be considered to further benefit patients with brain (micro-)metastases positioned behind an intact blood-brain barrier, or with substantial expression of these transporters in the tumor cells, without invoking a high toxicity risk.

P-Glycoprotein (MDR1/ABCB1) Restricts Brain Penetration of the Bruton's Tyrosine Kinase Inhibitor Ibrutinib, While Cytochrome P450-3A (CYP3A) Limits Its Oral Bioavailability.

Ibrutinib (Imbruvica), an oral tyrosine kinase inhibitor (TKI) approved for treatment of B-cell malignancies, irreversibly inhibits the Bruton's tyrosine kinase (BTK). Its abundant metabolite, dihydrodiol-ibrutinib (ibrutinib-DiOH), which is primarily formed by CYP3A, has a 10-fold reduced BTK inhibitory activity. Using in vitro transport assays and genetically modified mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 and the multidrug-metabolizing CYP3A enzyme family can affect the oral bioavailability and tissue disposition of ibrutinib and ibrutinib-DiOH. In vitro, ibrutinib was transported moderately by human ABCB1 and mouse Abcg2 but not detectably by human ABCG2. In mice, Abcb1 markedly restricted the brain penetration of ibrutinib and ibrutinib-DiOH, either alone or in combination with Abcg2, resulting in 4.5- and 5.9-fold increases in ibrutinib brain-to-plasma ratios in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice relative to wild-type mice. Abcb1 and/or Abcg2 did not obviously restrict ibrutinib oral bioavailability, but Cyp3a deficiency increased the ibrutinib plasma AUC by 9.7-fold compared to wild-type mice. This increase was mostly reversed (5.1-fold reduction) by transgenic human CYP3A4 overexpression, with roughly equal contributions of intestinal and hepatic CYP3A4 metabolism. Our results suggest that pharmacological inhibition of ABCB1 during ibrutinib therapy might benefit patients with malignancies or (micro)metastases positioned behind an intact blood-brain barrier, or with substantial expression of this transporter in the malignant cells. Moreover, given the strong in vivo impact of CYP3A, inhibitors or inducers of this enzyme family will likely strongly affect ibrutinib oral bioavailability and, thus, its therapeutic efficacy, as well as its toxicity risks.

Quantification of cystine in human renal proximal tubule cells using liquid chromatography-tandem mass spectrometry.

Nephropathic cystinosis is characterized by abnormal intralysosomal accumulation of cystine throughout the body, causing irreversible damage to various organs, particularly the kidneys. Cysteamine, the currently available treatment, can reduce lysosomal cystine and postpone disease progression. However, cysteamine poses serious side effects and does not address all of the symptoms of cystinosis. To screen for new treatment options, a rapid and reliable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to quantify cystine in conditionally immortalized human proximal tubular epithelial cells (ciPTEC). The ciPTEC were treated with N-ethylmaleimide, lysed and deproteinized with 15% (w/v) sulfosalicylic acid. Subsequently, cystine was measured using deuterium-labeled cystine-D4, as the internal standard. The assay developed demonstrated linearity to at least 20 µmol/L with a good precision. Accuracies were between 97.3 and 102.9% for both cell extracts and whole cell samples. Cystine was sufficiently stable under all relevant analytical conditions. The assay was successfully applied to determine cystine levels in both healthy and cystinotic ciPTEC. Control cells showed clearly distinguishable cystine levels compared with cystinotic cells treated with or without cysteamine. The method developed provides a fast and reliable quantification of cystine, and is applicable to screen for potential drugs that could reverse cystinotic symptoms in human kidney cells.

Recent developments in the chromatographic bioanalysis of approved kinase inhibitor drugs in oncology.

In recent years (2010-present) there has been an increase in the number of publications reporting the development, validation and use of bioanalytical methods in the rapidly expanding drug class of small molecule protein kinase inhibitors. Most reports describe the technological set-up of the methods that have allowed for drug concentration measurements from various sample types. This includes plasma, dried blood-spot, and tissue-analysis. Also method development, exploration of various techniques, as well as measurement and identification of metabolites were addressed. For the bioanalysis, a variety of sample-pretreatment methods like protein-precipitation, liquid-liquid extraction, and solid-phase extraction have been employed, all varying in complexity, cleanliness and time-consumption. Chromatographic separation, nowadays, is more focused on separating components from ion-suppressive effects, since for MS/MS detection, various components do not have to be baseline separated. For detection multiple types of detectors were used, ranging from state-of-the-art high resolution, and tandem mass spectrometry with low picogram per milliliter detection limits to the classical UV-detector with several nanograms per milliliter limits. As new bioanalytical methods have arisen that do rely on chromatographic separation, for example for high-throughput analysis, these are addressed in this review as well.

Liquid chromatography-tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma.

A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was performed in a range from 1 to 1000ng/ml, with the lowest level corresponding to the lower limit of quantitation. Gradient elution was performed on a 1.8µm particle trifunctional bonded C18 column by 1% (v/v) formic acid in water, and acetonitrile as mobile phase. The analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer after positive ionization with the heated electrospray interface. Within-day precisions ranged from 3.4 to 10.3%, and between-day precisions from 3.8 to 10.4%, accuracies were 95.5-102.8%. Plasma (either lithium heparin or sodium EDTA) pretreatment was performed by salting-out assisted liquid-liquid extraction using acetonitrile and magnesium sulfate. This method was used to analyze the osimertinib blood plasma levels of five adult patients with metastatic T790M mutated non-small cellular lung carcinoma for therapeutic drug monitoring purposes.

Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction.

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out assisted liquid-liquid extraction (SALLE) with acetonitrile, magnesium chloride and a stable isotopically labeled internal standard. After dilution, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was completely validated for plasma in a 0.5-500ng/ml calibration range with r(2)=0.995±0.002 (n=6) using linear regression with the inverse square of the concentration as the weighting factor for the calibration. Within-run precisions (n=18) were 2.7-11.7% and between-run (3 runs; n=18) precisions 3.0-14.5%. Accuracies were between 96-109% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of afatinib to female FVB mice.

Liquid chromatography-tandem mass spectrometric assay for the simultaneous determination of the irreversible BTK inhibitor ibrutinib and its dihydrodiol-metabolite in plasma and its application in mouse pharmacokinetic studies.

A validated simple, fast and sensitive bio-analytical assay for ibrutinib and its dihydrodiol metabolite in human and mouse plasma was set up. Sample preparation was performed by protein precipitation, and addition of the respective deuterated internal standards, followed by LC-MS/MS analysis. Separation was performed on a 3.5 µm particle-size, bridged ethylene hybrid column with gradient elution by 0.1% v/v formic acid and acetonitrile. The full eluate was transferred to an electrospray interface in positive ionization mode, and subsequently analyzed by a triple quadrupole mass spectrometer by selected reaction monitoring. The assay was validated in a 5-5000 ng/ml calibration range. Both ibrutinib and dihydrodiol-ibrutinib were deemed stable under refrigerated or frozen storage conditions. At room temperature, ibrutinib showed a not earlier described instability, and revealed rapid degradation at 37 °C. Finally, the assay was used for a pharmacokinetic study of plasma levels in treated FVB mice.