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We developed and validated an assay utilizing a liquid chromatography–tandem mass spectrometry technique to quantify the KRAS inhibitor adagrasib in mouse plasma and seven tissue‐related matrices. The straightforward protein precipitation technique was selected to extract adagrasib and the internal standard salinomycin from the matrices. Gradient elution of acetonitrile and water modified with 0.5% (v/v) ammonium hydroxide and 0.02% (v/v) acetic acid on a C18 column at a flow rate of 0.6 ml/min was applied to separate the analytes. Both adagrasib and salinomycin were detected with a triple quadrupole mass spectrometer with positive electrospray ionization in a selected reaction monitoring mode. A linear calibration range of 2–2,000 ng/ml of adagrasib was demonstrated during the validation. In addition, the reported precision values (intra‐ and inter‐day) were between 3.5 and 14.9%, while the accuracy values were 85.5–111.0% for all tested levels in all investigated matrices. Adagrasib in mouse plasma was reported to have good stability at room temperature, while adagrasib in tissue‐related matrices was stable on ice for up to 4 h (matrix dependent). Finally, this method was successfully applied to determine the pharmacokinetic profile and tissue distribution of adagrasib in wild‐type mice. [ABSTRACT FROM AUTHOR]

EAI045 is a tyrosine kinase inhibitor (TKI) that targets the mutant epidermal growth factor receptor (EGFR). It was developed to control resistance to available EGFR TKIs. In this study, a major metabolite of EAI045, (5‐fluoro‐2‐hydroxyphenyl)(1‐oxo‐1,3‐dihydro‐2H‐isoindol‐2‐yl)acetic acid (PIA), was discovered as a hydrolysis product of the parent drug. A validated assay for both analytes in mouse plasma and tissue homogenates from brain, kidney, liver, lung, spleen, and small intestine with content was set up using LC–MS/MS. Samples were prepared by protein precipitation with acetonitrile and with PLX4720 as internal standard. Separation was performed on a bridged ethylene hybrid C18 column by gradient elution with 0.1% v/v formic acid and methanol. Using positive electrospray, detection was performed in selected reaction monitoring mode. A linear calibration range of 2–2,000 ng/ml was used and validated for both analytes. Precision values ranged between 2.0 and 7.5% for EAI045 and between 2.2 and 12.1% for the metabolite, and accuracy values were between 91.1 and 107.6% for EAI045 and between 87.6 and 100.6% for the metabolite. Both analytes were sufficiently stable under the relevant analytical conditions. Finally, the assay was applied to analyze mouse plasma and tissue levels in a pharmacokinetic study in FVB/NRj wild‐type female mice treated with oral EAI045. [ABSTRACT FROM AUTHOR]

Proximal tubular damage is an important prognostic determinant in various chronic kidney diseases (CKDs). Currently available diagnostic methods do not allow for early disease detection and are neither efficient. Indoxyl sulfate (IS) is an endogenous metabolite and protein‐bound uremic toxin that is eliminated via renal secretion, but accumulates in plasma during tubular dysfunction. Therefore, it may be suitable as a tubular function marker. To evaluate this, a fast bioanalytical method was developed and validated for IS in various species and a kidney cell line using LC–MS/MS. An isotope‐labeled IS potassium salt as an internal standard and acetonitrile (ACN) as a protein precipitant were used for sample pretreatment. The analyte was separated on a Polaris 3 C18‐A column by gradient elution using 0.1% formic acid in water and ACN, and detected by negative electrospray ionization in selected reaction monitoring mode. The within‐day (≤ 4.0%) and between‐day (≤ 4.3%) precisions and accuracies (97.7 to 107.3%) were within the acceptable range. The analyte showed sufficient stability at all conditions investigated. Finally, applying this assay, significantly higher plasma and lower urine concentrations of IS were observed in mice with diabetic nephropathy with tubular damage, which encourages validation toward its use as a biomarker. [ABSTRACT FROM AUTHOR]

Background and Purpose: Larotrectinib is a FDA-approved oral small-molecule inhibitor for treatment of neurotrophic tropomyosin receptor kinase fusion-positive cancer. We here investigated the functions of the multidrug efflux transporters ABCB1 and ABCG2, the SLCO1A/1B (OATP1A/1B) uptake transporters, and the multispecific drug-metabolizing enzyme CYP3A in larotrectinib pharmacokinetic behaviour.Experimental Approach: In vitro, transepithelial drug transport and uptake assays were performed. In vivo, larotrectinib (10 mg·kg-1 ) was administered orally to relevant genetically modified mouse models. Cell medium, plasma samples, and organ homogenates were measured by a sensitive and specific LC-MS/MS larotrectinib assay.Key Results: In vitro, larotrectinib was avidly transported by human (h) ABCB1 and mouse (m) Abcg2 efficiently by hABCG2 and modestly by hOATP1A2. In vivo, both mAbcb1a/1b and mAbcg2 markedly limited larotrectinib oral availability and brain and testis accumulation (by 2.1-fold, 10.4-fold, and 2.7-fold, respectively), with mAbcb1a/1b playing a more prominent role. mOatp1a/1b also restricted larotrectinib oral availability (by 3.8-fold) and overall tissue exposure, apparently by mediating substantial uptake into the liver, thus likely facilitating hepatobiliary excretion. Additionally, larotrectinib is an excellent substrate of CYP3A, which restricts the oral availability of larotrectinib and hence its tissue exposure.Conclusions and Implications: ABCG2 and especially ABCB1 limit the oral availability and brain and testis penetration of larotrectinib, while OATP1A/1B transporters restrict its systemic exposure by mediating hepatic uptake, thus allowing hepatobiliary excretion. CYP3A-mediated metabolism can strongly limit larotrectinib oral availability and hence its tissue concentrations. These insights may be useful in the further clinical development of larotrectinib. [ABSTRACT FROM AUTHOR]

Lorlatinib (PF‐06463922) is a promising oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor currently in Phase III clinical trials for treatment of non‐small‐cell lung cancer (NSCLC) containing an ALK rearrangement. With therapy‐resistant brain metastases a major concern in NSCLC, lorlatinib was designed to have high membrane and blood–brain barrier permeability. We investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, and the multispecific drug‐metabolizing enzyme CYP3A in plasma pharmacokinetics and tissue distribution of lorlatinib using genetically modified mouse strains. In vitro, human ABCB1 and mouse Abcg2 modestly transported lorlatinib. Following oral lorlatinib administration (at 10 mg/kg), brain accumulation of lorlatinib, while relatively high in wild‐type mice, was still fourfold increased in Abcb1a/1b−/− and Abcb1a/1b;Abcg2−/− mice, but not in single Abcg2−/− mice. Lorlatinib plasma levels were not altered. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar increased the brain accumulation of lorlatinib in wild‐type mice fourfold, that is, to the same level as in Abcb1a/1b;Abcg2−/− mice, without altering plasma exposure. Similar results were obtained for lorlatinib testis accumulation. In Cyp3a−/− mice, the plasma exposure of lorlatinib was increased 1.3‐fold, but was then twofold reduced upon transgenic overexpression of human CYP3A4 in liver and intestine, whereas relative tissue distribution of lorlatinib remained unaltered. Our data indicate that lorlatinib brain accumulation is substantially limited by P‐glycoprotein/ABCB1 in the blood–brain barrier, but this can be effectively reversed by elacridar coadministration. Moreover, oral availability of lorlatinib is markedly restricted by CYP3A4 activity. These insights may be used in optimizing the therapeutic application of lorlatinib. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

No effective targeted therapy is currently available for NRAS mutant melanoma. Experimental MEK inhibition is rather toxic and has only limited efficacy in clinical trials. At least in part, this is caused by the emergence of drug resistance, which is commonly seen for single agent treatment and shortens clinical responses. Therefore, there is a dire need to identify effective companion drug targets for NRAS mutant melanoma. Here, we show that at concentrations where single drugs had little effect, ROCK inhibitors GSK269962A or Fasudil, in combination with either MEK inhibitor GSK1120212 (Trametinib) or ERK inhibitor SCH772984 cooperatively caused proliferation inhibition and cell death in vitro. Simultaneous inhibition of MEK and ROCK caused induction of Bim EL, PARP, and Puma, and hence apoptosis. In vivo , MEK and ROCK inhibition suppressed growth of established tumors. Our findings warrant clinical investigation of the effectiveness of combinatorial targeting of MAPK/ ERK and ROCK in NRAS mutant melanoma. [ABSTRACT FROM AUTHOR]

ABSTRACT Regorafenib has recently been approved for the treatment of colorectal cancer. A bioanalytical liquid chromatography-tandem mass spectrometric assay for this multikinase inhibitor was developed and validated in plasma. The concentration range of the assay was 25-25,000 ng/mL. Protein precipitation with acetonitrile was used as sample pre-treatment with sorafenib as internal standard. The extract was diluted with methanol (25%, v/v) and then injected onto the sub-2 µm particle, bridged ethylsilicia hybrid trifunctional bonded C18 column. Isocratic elution using 0.02% (v/v) formic acid in a methanol-water mixture was used. Compounds were monitored by a triple quadrupole mass spectrometer in the selected reaction monitoring mode after positive electrospray ionization. Double logarithmic calibration was used; within-day precisions, between-day precisions, and accuracies were 3.2-9.2, 4.1-12.3 and 94.8-103.0%, respectively. High drug stability was observed under all relevant storage conditions. The assay was used to measure drug concentrations in a pharmacokinetic study in wild-type FVB mice. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Crizotinib is an oral tyrosine kinase inhibitor approved for treating patients with non-small cell lung cancer (NSCLC) containing an anaplastic lymphoma kinase (ALK) rearrangement. We used knockout mice to study the roles of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in plasma pharmacokinetics and brain accumulation of oral crizotinib, and the feasibility of improving crizotinib kinetics using coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. In vitro, crizotinib was a good transport substrate of human ABCB1, but not of human ABCG2 or murine Abcg2. With low-dose oral crizotinib (5 mg/kg), Abcb1a/1b−/− and Abcb1a/1b;Abcg2−/− mice had an approximately twofold higher plasma AUC than wild-type mice, and a markedly (∼40-fold) higher brain accumulation at 24 hr. Also at 4 hr, crizotinib brain concentrations were ∼25-fold, and brain-to-plasma ratios ∼14-fold higher in Abcb1a/1b−/− and Abcb1a/1b;Abcg2−/− mice than in wild-type mice. High-dose oral crizotinib (50 mg/kg) resulted in comparable plasma pharmacokinetics between wild-type and Abcb1a/1b−/− mice, suggesting saturation of intestinal Abcb1. Nonetheless, brain accumulation at 24 hr was still ∼70-fold higher in Abcb1a/1b−/− than in wild-type mice. Importantly, oral elacridar coadministration increased the plasma and brain concentrations and brain-to-plasma ratios of crizotinib in wild-type mice, equaling the levels in Abcb1a/1b;Abcg2−/− mice. Our results indicate that crizotinib oral availability and brain accumulation were primarily restricted by Abcb1 at a non-saturating dose, and that coadministration of elacridar with crizotinib could substantially increase crizotinib oral availability and delivery to the brain. This principle might be used to enhance therapeutic efficacy of crizotinib against brain metastases in NSCLC patients. [ABSTRACT FROM AUTHOR]

A liquid chromatography-tandem mass spectrometric assay for the determination of the antiretroviral nucleoside emtricitabine in human plasma was developed and validated using a simple sample pre-treatment procedure. After addition of 5′-deoxy-5-fluorocytidine as the internal standard and protein precipitation with acetonitrile, the supernatant was directly injected in the isocratic chromatographic system using a polar embedded reversed-phase column and formic acid in water-methanol as the eluent. The eluate was completely led into an electrospray interface with positive ionization and the analytes were quantified using triple quadrupole mass spectrometry. The assay was validated in the range 5-5000 ng/mL. Intra-day precisions were ≤7% and inter-day precisions were ≤10%. Accuracies between 92 and 99% were found. The analytes were chemically stable under all relevant conditions and the assay was applied in the analysis of plasma samples of HIV-infected patients treated with the drug. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Atazanavir is the most recently introduced protease inhibitor for the suppression of the anti-human immunodefficiency virus. A sensitive and selective reversed-phase liquid chromatographic assay for this drug in human plasma has been developed and validated. Atazanavir was isolated from a 500 µL plasma sample using liquid-liquid extraction with dichloromethane. After evaporation and reconstitution of the extract the sample was analysed using liquid chromatography and ultraviolet detection at 280 nm. In the evaluated concentration range (44-4395 ng[sol ]mL atazanavir), intra-day precisions were ≤7% and inter-day precisions were ≤14%. Accuracies between 96 and 106% were found. The lower limit of quantification was 44 ng[sol ]mL with an intra-day precision of 7%, an inter-day precision of 14% and an accuracy of 87%. There was no interference from 32 tested potentially co-administrated drugs and metabolites. The usefulness of the assay was demonstrated for samples obtained from an HIV-infected patient treated with atazanavir. Copyright © 2005 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The stimulant effect of caffeine, as an additive in diacetylmorphine preparations for study purposes, may interfere with the pharmacodynamic effects of diacetylmorphine. In order to obtain insight into the pharmacology of caffeine after inhalation in heroin users, the pharmacokinetics of caffeine and its dimethylxanthine metabolites were studied. The objectives were to establish the population pharmacokinetics under these exceptional circumstances and to compare the results to published data regarding intravenous and oral administration in healthy volunteers. Diacetylmorphine preparations containing 100 mg of caffeine were used by 10 persons by inhalation. Plasma concentrations of caffeine, theobromine, paraxanthine and theophylline were measured by high performance liquid chromatography. Non-linear mixed effects modelling was used to estimate population pharmacokinetic parameters. The model was evaluated by the jack-knife procedure. Caffeine was rapidly and effectively absorbed after inhalation. Population pharmacokinetics of caffeine and its dimethylxanthine metabolites could adequately and simultaneously be described by a linear multi-compartment model. The volume of distribution for the central compartment was estimated to be 45.7 l and the apparent elimination rate constant of caffeine at 8 hr after inhalation was 0.150 hr−1 for a typical individual. The bioavailability was approximately 60%. The presented model adequately describes the population pharmacokinetics of caffeine and its dimethylxanthine metabolites after inhalation of the caffeine sublimate of a 100 mg tablet. Validation proved the stability of the model. Pharmacokinetics of caffeine after inhalation and intravenous administration are to a large extent similar. The bioavailability of inhaled caffeine is approximately 60% in experienced smokers. [ABSTRACT FROM AUTHOR]

Tipranavir is the most recently introduced protease inhibitor for the suppression of the human immunodeficiency virus (HIV). A selective reversed-phase liquid chromatographic assay, previously developed for atazanavir, has been extended and validated for tipranavir in plasma. Compounds were isolated from a 500 µL plasma sample using liquid-liquid extraction with dichloromethane. After evaporation and reconstitution of the extract the sample was analysed using reversed-phase liquid chromatography and ultra violet detection at 280 nm. In the evaluated concentration range (0.2-50 µg/mL tipranavir), intra-day precisions were ≤8% and inter-day precisions were ≤10%. Accuracies between 95 and 108% were found. The clinical applicability of the assay was demonstrated in an HIV-infected patient who ingested 500 mg tipranavir bid in combination with 100 mg ritonavir. Copyright © 2006 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]