ABSTRACT
Interferon-inducible human oligoadenylate synthetase-like (OASL) and its mouse ortholog, Oasl2, enhance RNA-sensor RIG-I-mediated type I interferon (IFN) induction and inhibit RNA virus replication. Here, we show that OASL and Oasl2 have the opposite effect in the context of DNA virus infection. In Oasl2-/- mice and OASL-deficient human cells, DNA viruses such as vaccinia, herpes simplex, and adenovirus induced increased IFN production, which resulted in reduced virus replication and pathology. Correspondingly, ectopic expression of OASL in human cells inhibited IFN induction through the cGAS-STING DNA-sensing pathway. cGAS was necessary for the reduced DNA virus replication observed in OASL-deficient cells. OASL directly and specifically bound to cGAS independently of double-stranded DNA, resulting in a non-competitive inhibition of the second messenger cyclic GMP-AMP production. Our findings define distinct mechanisms by which OASL differentially regulates host IFN responses during RNA and DNA virus infection and identify OASL as a negative-feedback regulator of cGAS.
Subject(s)
2',5'-Oligoadenylate Synthetase/metabolism , DNA Virus Infections/immunology , DNA Viruses/physiology , RNA Virus Infections/immunology , RNA Viruses/immunology , 2',5'-Oligoadenylate Synthetase/genetics , Animals , Cyclic AMP/metabolism , Humans , Interferon Type I/genetics , Interferon Type I/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nucleotidyltransferases/metabolism , RNA, Small Interfering/genetics , Signal Transduction , THP-1 Cells , Virus ReplicationABSTRACT
To support a phase 1 trial in patients with lymphomas, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for tazemetostat quantitation in 20 µL of human plasma. After protein precipitation, chromatographic separation employed a Kinetex C18 column and a gradient of 0.1% formic acid in both water and acetonitrile, during a 3-min run time. Detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. Validation was based on the latest Food and Drug Administration guidance. With a stable isotopic internal standard, the assay was linear within the range of 10-5000 ng/mL and proved to be accurate (91.9%-103.7%) and precise (<4.4% imprecision). Recovery varied between 93.3% and 121.1%, and matrix effect ranged from -25.5% to -4.9%. Hemolysis, lipemia, and dilution did not impact quantitation. Plasma stability was confirmed after three freeze-thaw cycles, 24 h at room temperature, and 4 months at -80°C. Incurred sample reanalysis yielded 94.4% samples within 20% difference (n = 36). External validation showed a mean bias of -11.1%. Pharmacokinetic (PK) data obtained from three patients suggested variable concentration time profiles, warranting collection of further data. The assay proved to be suitable for tazemetostat quantitation in human plasma and will support clinical studies by defining tazemetostat PKs.
Subject(s)
Tandem Mass Spectrometry , Humans , Tandem Mass Spectrometry/methods , Reproducibility of Results , Chromatography, Liquid/methods , Drug Stability , Linear Models , Sensitivity and SpecificityABSTRACT
Fluoropyridine-based chemotherapy remains the most widely used treatment for colorectal cancer (CRC). In this study, we investigated the mechanism by which the natural product Scutellaria baicalensis (Huang Qin; HQ) and one of its main components baicalin enhanced 5-fluorouracil (5-FU) antitumor activity against CRC. Cell proliferation assays, cell cycle analysis, reverse-phase protein array (RPPA) analysis, immunoblot analysis, and qRT-PCR were performed to investigate the mechanism(s) of action of HQ and its active components on growth of CRC cells. HQ exhibited in vitro antiproliferative activity against drug resistant human CRC cells, against human and mouse CRC cells with different genetic backgrounds and normal human colon epithelial cells. In vivo animal models were used to document the antitumor activity of HQ and baicalin. The mechanism of growth inhibitory activity of HQ is due to inhibition of proliferative signaling pathways including the CDK-RB pathway. In addition, HQ enhanced the antitumor effects of 5-FU and capecitabine in vivo. Furthermore, we identified baicalin as an active component of HQ. The combination of baicalin and 5-FU demonstrated synergistic activity against 5-FU-resistant RKO-R10 cells. The combination significantly inhibited in vivo tumor growth greater than each treatment alone. RPPA results showed that the signaling pathway alterations in CRC cells were similar following HQ and baicalin treatment. Together, these results indicate that HQ and its component baicalin enhance the effect of 5-fluorouracil-based chemotherapy via inhibition of CDK-RB pathway. These findings may provide the rational basis for developing agents that can overcome the development of cellular drug resistance. Video Abstract.
Subject(s)
Colorectal Neoplasms , Fluorouracil , Humans , Animals , Mice , Fluorouracil/pharmacology , Fluorouracil/therapeutic use , Scutellaria baicalensis , Signal Transduction , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Cell Proliferation , Cell Line, TumorABSTRACT
Ataxia-telangiectasia-mutated and Rad3-related (ATR) is master regulator of the DNA-damage response that, through multiple mechanisms, can promote cancer cell survival in response to replication stress from sources, including chemotherapy and radiation. Elimusertib (BAY-1895344) is an orally available small-molecule ATR inhibitor currently in preclinical and clinical development for cancer treatment. To support these studies and define elimusertib pharmacokinetics, we developed a HPLC-MS method for its quantitation. A 50-µL volume of plasma was subjected to acetonitrile protein precipitation and then chromatographic separation using a Phenomenex Polar-RP column (2 × 50 mm, 4 µm) and a gradient mobile phase consisting of 0.1% formic acid in acetonitrile and water during a 7-min run time. Mass spectrometric detection was achieved using a SCIEX 4000 triple-stage mass spectrometer with electrospray positive-mode ionization. With a stable isotopic internal standard, the assay was linear from 30 to 5000 ng/mL and proved to be both accurate (93.5-108.2%) and precise (<6.3% coefficient of variation) fulfilling criteria from the Food and Drug Administration guidance on bioanalytical method validation. This LC-MS/MS assay will support several ongoing clinical studies by defining elimusertib pharmacokinetics.
Subject(s)
Ataxia Telangiectasia , Tandem Mass Spectrometry , Acetonitriles , Chromatography, Liquid/methods , DNA , Humans , Protein Kinase Inhibitors , Reproducibility of Results , Tandem Mass Spectrometry/methods , WaterABSTRACT
BACKGROUND: 5-Fluorouracil (5-FU) remains the most widely used agent to treat colorectal cancer (CRC). However, its clinical efficacy is currently limited by the development of drug resistance. Traditional Chinese Herbal Medicine (TCM) has been shown to enhance the efficacy of standard anticancer agents. However, there are only a limited number of well-controlled preclinical and clinical studies documenting the potential benefit of TCM. Herein, we screened a series of TCM formulas in in vitro and in vivo animal models to identify biologically active formulas that were effective against CRC. METHODS: Cell proliferation and clonogenic assays, cell cycle analysis, immunoblot analysis and qRT-PCR were performed to investigate the mechanism(s) of action of the most active formula Huang-Qin-Ge-Gen-Tang (HQGGT) on growth of human CRC cells. In vivo animal models were used to document the antitumor activity of HQGGT alone and HQGGT in combination with 5-FU. RESULTS: We identified HQGGT, which suppressed the in vivo growth of human colon cancer HT-29 xenografts without associated toxicities. HQGGT displayed anti-proliferative activity against a wide range of CRC cell lines. This growth suppression correlated with induction of apoptosis. HQGGT enhanced the cytotoxicity of 5-FU against human 5-FU-resistant cells (H630R1) and mouse colon cancer cells (MC38). Our studies showed that the mechanism of action of this synergism was the result of suppression of thymidylate synthase (TS) expression by HQGGT. We analyzed different batches of HQGGT and observed consistent chemical fingerprints and biological activity. Finally, we show that orally administered HQGGT significantly enhanced the antitumor effect of 5-FU in mice bearing MC38 xenografts. CONCLUSIONS: These findings provide support for the potential role of HQGGT as a novel modulator of fluoropyrimidine chemotherapy in the treatment of CRC.
Subject(s)
Drugs, Chinese Herbal/pharmacology , E2F1 Transcription Factor/metabolism , Fluorouracil/pharmacology , Signal Transduction/drug effects , Thymidylate Synthase/metabolism , Animals , Apoptosis/drug effects , Cell Line , Cell Proliferation/drug effects , Chromatography, High Pressure Liquid , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Drug Synergism , Drugs, Chinese Herbal/analysis , Drugs, Chinese Herbal/therapeutic use , Female , Fluorouracil/therapeutic use , HT29 Cells , Humans , Mice , Mice, Nude , Transplantation, HeterologousABSTRACT
AIM: Inducers and inhibitors of CYP3A, such as ritonavir and efavirenz, may be used as part of the highly active antiretroviral therapy (HAART) to treat HIV patients. HIV patients with chronic myeloid leukemia or gastrointestinal stromal tumour may need imatinib, a CYP3A4 substrate with known exposure response-relationships. Administration of imatinib to patients on ritonavir or efavirenz may result in altered imatinib exposure leading to increased toxicity or failure of therapy, respectively. We used primary human hepatocyte cultures to evaluate the magnitude of interaction between imatinib and ritonavir/efavirenz. METHODS: Hepatocytes were pre-treated with vehicle, ritonavir, ketoconazole, efavirenz or rifampicin, and the metabolism of imatinib was characterized over time. Concentrations of imatinib and metabolite were quantitated in combined lysate and medium, using LC-MS. RESULTS: The predicted changes in imatinib CLoral (95% CI) with ketoconazole, ritonavir, rifampicin and efavirenz were 4.0-fold (0, 9.2) lower, 2.8-fold (0.04, 5.5) lower, 2.9-fold (2.2, 3.5) higher and 2.0-fold (0.42, 3.5) higher, respectively. These predictions were in good agreement with clinical single dose drug-drug interaction studies, but not with reports of imatinib interactions at steady-state. Alterations in metabolism were similar after acute or chronic imatinib exposure. CONCLUSIONS: In vitro human hepatocytes predicted increased clearance of imatinib with inducers and decreased clearance with inhibitors of CYP enzymes. The impact of HAART on imatinib may depend on whether it is being initiated or has already been dosed chronically in patients. Therapeutic drug monitoring may have a role in optimizing imatinib therapy in this patient population.
Subject(s)
Benzoxazines/pharmacology , Hepatocytes/drug effects , Imatinib Mesylate/metabolism , Imatinib Mesylate/pharmacokinetics , Ketoconazole/pharmacology , Rifampin/pharmacology , Ritonavir/pharmacology , Adolescent , Adult , Aged , Alkynes , Anti-HIV Agents/pharmacology , Cyclopropanes , Cytochrome P-450 CYP3A Inducers/pharmacology , Cytochrome P-450 CYP3A Inhibitors/pharmacology , Drug Interactions , Female , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Male , Middle Aged , Primary Cell Culture , Young AdultABSTRACT
BACKGROUND: Imatinib pharmacokinetic variability and the relationship of trough concentrations with clinical outcomes have been extensively reported. Although physical methods to quantitate imatinib exist, they are not widely available for routine use. An automated homogenous immunoassay for imatinib has been developed, facilitating routine imatinib testing. METHODS: Imatinib-selective monoclonal antibodies, without substantial cross-reactivity to the N-desmethyl metabolite or N-desmethyl conjugates, were produced. The antibodies were conjugated to 200 nm particles to develop immunoassay reagents on the Beckman Coulter AU480 analyzer. These reagents were analytically validated using Clinical Laboratory Standards Institute protocols. Method comparison to liquid chromatography tandem mass spectrometry (LC-MS/MS) was conducted using 77 plasma samples collected from subjects receiving imatinib. RESULTS: The assay requires 4 µL of sample without pretreatment. The nonlinear calibration curve ranges from 0 to 3000 ng/mL. With automated sample dilution, concentrations of up to 9000 ng/mL can be quantitated. The AU480 produces the first result in 10 minutes and up to 400 tests per hour. Repeatability ranged from 2.0% to 6.0% coefficient of variation, and within-laboratory reproducibility ranged from 2.9% to 7.4% coefficient of variation. Standard curve stability was 2 weeks and on-board reagent stability was 6 weeks. For clinical samples with imatinib concentrations from 438 to 2691 ng/mL, method comparison with LC-MS/MS gave a slope of 0.995 with a y-intercept of 24.3 and a correlation coefficient of 0.978. CONCLUSIONS: The immunoassay is suitable for quantitating imatinib in human plasma, demonstrating good correlation with a physical method. Testing for optimal imatinib exposure can now be performed on routine clinical analyzers.
Subject(s)
Imatinib Mesylate/blood , Imatinib Mesylate/immunology , Immunoassay/methods , Antibodies, Monoclonal/immunology , Automation , Calibration , Chromatography, High Pressure Liquid , Humans , Limit of Detection , Tandem Mass SpectrometryABSTRACT
Large display screens are common in supervisory tasks, meaning that alerts are often perceived in peripheral vision. Five air traffic control notification designs were evaluated in their ability to capture attention during an ongoing supervisory task, as well as their impact on the primary task. A range of performance measures, eye-tracking and subjective reports showed that colour, even animated, was less effective than movement, and notifications sometimes went unnoticed. Designs that drew attention to the notified aircraft by a pulsating box, concentric circles or the opacity of the background resulted in faster perception and no missed notifications. However, the latter two designs were intrusive and impaired primary task performance, while the simpler animated box captured attention without an overhead cognitive cost. These results highlight the need for a holistic approach to evaluation, achieving a balance between the benefits for one aspect of performance against the potential costs for another. Practitioner summary: We performed a holistic examination of air traffic control notification designs regarding their ability to capture attention during an ongoing supervisory task. The combination of performance, eye-tracking and subjective measurements demonstrated that the best design achieved a balance between attentional power and the overhead cognitive cost to primary task performance.
Subject(s)
Attention , Aviation , Aviation/instrumentation , Aviation/methods , Data Display , Equipment Design , Eye Movement Measurements , Eye Movements , Humans , Task Performance and AnalysisABSTRACT
This study aims to determine whether indirect touch device can be used to interact with graphical objects displayed on another screen in an air traffic control (ATC) context. The introduction of such a device likely requires an adaptation of the sensory-motor system. The operator has to simultaneously perform movements on the horizontal plane while assessing them on the vertical plane. Thirty-six right-handed participants performed movement training with either constant or variable practice and with or without visual feedback of the displacement of their actions. Participants then performed a test phase without visual feedback. Performance improved in both practice conditions, but accuracy was higher with visual feedback. During the test phase, movement time was longer for those who had practiced with feedback, suggesting an element of dependency. However, this 'cost' of feedback did not extend to movement accuracy. Finally, participants who had received variable training performed better in the test phase, but accuracy was still unsatisfactory. We conclude that continuous visual feedback on the stylus position is necessary if tablets are to be introduced in ATC.
Subject(s)
Aviation/instrumentation , Touch , User-Computer Interface , Adult , Aviation/education , Aviation/methods , Feedback , Humans , Psychomotor PerformanceABSTRACT
BACKGROUND: The Ataxia Telangiectasia and Rad3-related (ATR) protein complex is an apical initiator of DNA damage response pathways. Several ATR inhibitors (ATRi) are in clinical development including berzosertib (formerly M6620, VX-970). Although clinical studies have examined plasma pharmacokinetics (PK) in humans, little is known regarding dose/exposure relationships and tissue distribution. To understand these concepts, we extensively characterized the PK of berzosertib in mouse plasma and tissues. METHODS: A highly sensitive LC-MS/MS method was utilized to quantitate berzosertib in plasma and tissues. Dose proportionality was assessed in female BALB/c mice following single IV doses (2, 6, 20 or 60 mg/kg). A more extensive PK study was conducted in tumor-bearing mice following a single IV dose of 20 mg/kg to evaluate distribution to tissues. PK parameters were calculated by non-compartmental analysis (NCA). A compartmental model was developed to describe the PK behavior of berzosertib. Plasma protein binding was determined in vitro. RESULTS: Increased doses of berzosertib were associated with less than proportional increases in early plasma concentrations and greater than proportional increase in tissue exposure, attributable to saturation of plasma protein binding. Berzosertib extensively distributed into bone marrow, tumor, thymus, and lymph nodes, however; brain and spinal cord exposure was less than plasma. CONCLUSION: The nonlinear PK of berzosertib displayed here can be attributed to saturation of plasma protein binding and occurred at concentrations close to those observed in clinical trials. Our results will help to understand preclinical pharmacodynamic and toxicity data and to inform optimal dosing and deployment of berzosertib.
ABSTRACT
Diffuse gliomas are epigenetically dysregulated, immunologically cold, and fatal tumors characterized by mutations in isocitrate dehydrogenase (IDH). Although IDH mutations yield a uniquely immunosuppressive tumor microenvironment, the regulatory mechanisms that drive the immune landscape of IDH mutant (IDHm) gliomas remain unknown. Here, we reveal that transcriptional repression of retinoic acid (RA) pathway signaling impairs both innate and adaptive immune surveillance in IDHm glioma through epigenetic silencing of retinol binding protein 1 (RBP1) and induces a profound anti-inflammatory landscape marked by loss of inflammatory cell states and infiltration of suppressive myeloid phenotypes. Restorative retinoic acid therapy in murine glioma models promotes clonal CD4 + T cell expansion and induces tumor regression in IDHm, but not IDH wildtype (IDHwt), gliomas. Our findings provide a mechanistic rationale for RA immunotherapy in IDHm glioma and is the basis for an ongoing investigator-initiated, single-center clinical trial investigating all-trans retinoic acid (ATRA) in recurrent IDHm human subjects.
ABSTRACT
Erlotinib is approved for the treatment of non-small cell lung and pancreatic cancers, and is metabolized by CYP3A4. Inducers and inhibitors of CYP3A enzymes such as ritonavir and efavirenz, respectively, may be used as part of the highly active antiretroviral therapy drugs to treat patients with human immunodeficiency virus (HIV). When HIV patients with a malignancy need treatment with erlotinib, there is a potential of as-yet-undefined drug-drug interaction. We evaluated these interactions using human hepatocytes benchmarked against the interaction of erlotinib with ketoconazole and rifampin, the archetype cytochrome P450 inhibitor and inducer, respectively. Hepatocytes were treated with vehicle [0.1% dimethylsulfoxide, ritonavir (10 µM)], ketoconazole (10 µM), efavirenz (10 µM), or rifampin (10 µM) for 4 days. On day 5, erlotinib (5 µM) was incubated with the above agents for another 24-48 hours. Concentrations of erlotinib and O-desmethyl erlotinib were quantitated in collected samples (combined lysate and medium) using liquid chromatography and tandem mass spectrometry. The half-life (t(½)) of erlotinib increased from 10.6 ± 2.6 to 153 ± 80 and 23.9 ± 4.8 hours, respectively, upon treatment with ritonavir and ketoconazole. The apparent intrinsic clearance (C(Lint, app)) of erlotinib was lowered 16-fold by ritonavir and 1.9-fold by ketoconazole. Efavirenz and rifampin decreased t1/2 of erlotinib from 10.3 ± 1.1 to 5.0 ± 1.5 and 3.4 ± 0.2 hours, respectively. Efavirenz and rifampin increased the C(Lint, app) of erlotinib by 2.2- and 2-fold, respectively. Our results suggest that to achieve desired drug exposure, the clinically used dose (150 mg daily) of erlotinib may have to be significantly reduced (25 mg every other day) or increased (300 mg daily), respectively, when ritonavir or efavirenz is coadministered.
Subject(s)
Benzoxazines/therapeutic use , Drug Interactions/physiology , HIV Infections/drug therapy , Hepatocytes/metabolism , Neoplasms/drug therapy , Quinazolines/therapeutic use , Ritonavir/therapeutic use , 14-alpha Demethylase Inhibitors/therapeutic use , Adult , Aged , Alkynes , Anti-HIV Agents/metabolism , Anti-HIV Agents/therapeutic use , Cyclopropanes , Erlotinib Hydrochloride , Female , HIV Infections/metabolism , HIV Infections/virology , HIV Protease Inhibitors/metabolism , HIV Protease Inhibitors/therapeutic use , Half-Life , Hepatocytes/drug effects , Humans , Ketoconazole/therapeutic use , Male , Middle Aged , Neoplasms/metabolism , Neoplasms/virology , Nucleic Acid Synthesis Inhibitors/therapeutic use , Quinazolines/metabolism , Rifampin/therapeutic useABSTRACT
The purpose of this study was to investigate the possibility to integrate a free head motion eye-tracking system as input device in air traffic control (ATC) activity. Sixteen participants used an eye tracker to select targets displayed on a screen as quickly and accurately as possible. We assessed the impact of the presence of visual feedback about gaze position and the method of target selection on selection performance under different difficulty levels induced by variations in target size and target-to-target separation. We tend to consider that the combined use of gaze dwell-time selection and continuous eye-gaze feedback was the best condition as it suits naturally with gaze displacement over the ATC display and free the hands of the controller, despite a small cost in terms of selection speed. In addition, target size had a greater impact on accuracy and selection time than target distance. These findings provide guidelines on possible further implementation of eye tracking in ATC everyday activity. PRACTITIONER SUMMARY: We investigated the possibility to integrate a free head motion eye-tracking system as input device in air traffic control (ATC). We found that the combined use of gaze dwell-time selection and continuous eye-gaze feedback allowed the best performance and that target size had a greater impact on performance than target distance.
Subject(s)
Aviation/instrumentation , Eye Movements , Head Movements , Pattern Recognition, Visual , Adult , Attention , Discrimination, Psychological , Feedback , Female , Fixation, Ocular , Humans , Male , Orientation , Reaction Time , Size Perception , User-Computer InterfaceABSTRACT
VNPP433-3ß (compound 2, (3ß-(1H-imidazole-1-yl)-17-(1H-benzimidazole-1-yl)-androsta-5,16-diene), a multitarget anticancer agent has emerged as our lead next generation galeterone analogs (NGGA). Compound 2 is currently in development as potential new therapeutic for prostate and pancreatic cancers. The preliminary toxicity study reveals that the compound 2 was better tolerated by the normal male CD-1 mice than the male Nude mice. The maximum tolerated dose (MTD) in the Nude mice was estimated to be between 25 < 50 mg/kg. After oral dosing of compound 2 to male and female rats, the plasma concentration versus time curves were very consistent between animals and the AUClast increased with dose. Many plasmas concentration versus time curves profiles were nearly flat over 24 hr., suggesting extended absorption from the GI tract. Consequently, reliable values for half-life and AUCinf were not determined. Calculated oral bioavailability (using oral AUClast and excluding the outlier IV animal) ranged from 32 to 47 %. This should be considered a minimum value since the contribution to true AUC beyond 24 hr. is clearly not zero. Clearly, these toxicology and pharmacokinetics parameters pave the way for understanding the anticancer pharmacological actions and provide a meaningful basis for further preclinical development and eventual clinical development.
Subject(s)
Antineoplastic Agents , Mice , Rats , Male , Female , Animals , Mice, Nude , Antineoplastic Agents/toxicity , Benzimidazoles/pharmacology , Androstadienes/pharmacologyABSTRACT
PURPOSE: Ataxia telangiectasia and Rad3-related (ATR) initiates and regulates cellular responses to DNA damage, such as those caused by cancer treatments. Several ATR inhibitors (ATRi) are in clinical development including AZD6738. Therapeutic indices among ATRi may differ as a result of varying potencies and concentrations at both tumor and off-target sites. Additionally, AZD6738 contributes to anti-tumor immune responses necessitating evaluation of exposure at immunological sites. METHODS: Using mouse models and a highly sensitive LC-MS/MS assay, the pharmacokinetics of AZD6738 were studied, including dose linearity, bioavailability, metabolism, and tissue distribution in tumor-bearing mice. RESULTS: Initial studies identified dose-dependent bioavailability, with greater than proportional increases in exposure as dose increased resulting in a ~ twofold increase in bioavailability between the lowest and highest investigated doses. These behaviors were successfully captured with a compartmental PK model. Analysis of metabolite PK revealed decreasing metabolic ratios with increasing dose, indicative of saturable first-pass metabolism. Further analysis revealed that intestinal and gut metabolism contribute to metabolism and these saturable mechanisms. Studies of tumor and tissue distribution found rapid and extensive drug distribution to most tissues except brain and spinal cord. CONCLUSION: The complex non-linear behavior of AZD6738 PK in mice was due to pre-systemic saturation and which appears to be recapitulated clinically at low doses. PK reported here will allow future correlation of tissue related toxicities with drug exposure as well as exposure with immunological responses. These results can also be compared with those from similar studies of other ATRi to contrast drug exposure with responses.
Subject(s)
Indoles/pharmacokinetics , Models, Biological , Morpholines/pharmacokinetics , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidines/pharmacokinetics , Sulfonamides/pharmacokinetics , Animals , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Biological Availability , Chromatography, Liquid , Dose-Response Relationship, Drug , Female , Indoles/administration & dosage , Mice , Mice, Inbred BALB C , Morpholines/administration & dosage , Protein Kinase Inhibitors/administration & dosage , Pyrimidines/administration & dosage , Sulfonamides/administration & dosage , Tandem Mass Spectrometry , Tissue DistributionABSTRACT
PURPOSE: Ataxia Telangiectasia and Rad3-related (ATR) is a pivotal component of the DNA damage response and repair pathways that is activated in responses to cytotoxic cancer treatments. Several ATR inhibitors (ATRi) are in development that block the ATR mediated DNA repair and enhance the damage associated with cytotoxic therapy. BAY-1895344 (elimusertib) is an orally available ATRi with preclinical efficacy that is in clinical development. Little is known about the pharmacokinetics (PK) which is of interest, because tissue exposure and ATR inhibition may relate to toxicities or responses. METHODS: To evaluate BAY-1895344 PK, a sensitive LC-MS/MS method was utilized for quantitation in mouse plasma and tissues. PK studies in mice were first conducted to determine dose linearity. In vivo metabolites were identified and analyzed semi-quantitatively. A compartmental PK model was developed to describe PK behavior. An extensive PK study was then conducted in tumor-bearing mice to quantitate tissue distribution for relevant tissues. RESULTS: Dose linearity was observed from 1 to 10 mg/kg PO, while at 40 mg/kg PO bioavailability increased approximately fourfold due to saturation of first-pass metabolism, as suggested by metabolite analyses and a developed compartmental model. Longer half-lives in PO treated mice compared to IV treated mice indicated absorption-rate limited elimination. Tissue distribution varied but showed extensive distribution to bone marrow, brain, and spinal cord. CONCLUSIONS: Complex PK behavior was limited to absorption processes which may not be recapitulated clinically. Tissue partition coefficients may be used to contrast ATR inhibitors with respect to their efficacy and toxicity.
Subject(s)
Protein Kinase Inhibitors , Tandem Mass Spectrometry , Animals , Ataxia Telangiectasia Mutated Proteins , Biological Availability , Chromatography, Liquid , Humans , Mice , Protein Kinase Inhibitors/pharmacokinetics , Tissue DistributionABSTRACT
To support a phase III randomized trial of the multi-targeted tyrosine kinase inhibitor cabozantinib in neuroendocrine tumors, we developed a high-performance liquid chromatography mass spectrometry method to quantitate cabozantinib in 50 µL of human plasma. After acetonitrile protein precipitation, chromatographic separation was achieved with a Phenomenex synergy polar reverse phase (4 µm, 2 × 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and 0.1% formic acid in water over a 5-min run time. Detection was performed on a Quattromicro quadrupole mass spectrometer with electrospray, positive-mode ionization. The assay was linear over the concentration range 50-5000 ng/mL and proved to be accurate (103.4-105.4%) and precise (<5.0%CV). Hemolysis (10% RBC) and use of heparin as anticoagulant did not impact quantitation. Recovery from plasma varied between 103.0-107.7% and matrix effect was -47.5 to -41.3%. Plasma freeze-thaw stability (97.7-104.9%), stability for 3 months at -80°C (103.4-111.4%), and stability for 4 h at room temperature (100.1-104.9%) were all acceptable. Incurred sample reanalysis of (N = 64) passed: 100% samples within 20% difference, -0.7% median difference and 1.1% median absolute difference. External validation showed a bias of less than 1.1%. This assay will help further define the clinical pharmacokinetics of cabozantinib.
Subject(s)
Anilides , Tandem Mass Spectrometry , Chromatography, Liquid/methods , Humans , Pyridines , Reproducibility of Results , Tandem Mass Spectrometry/methodsABSTRACT
New targeted chemotherapeutics are urgently needed to minimize off-target toxicity and reduce the high-mortality rate associated with metastatic prostate cancer. Herein, we report on the modular synthesis, pharmacokinetics, and efficacy of two small-molecule-drug conjugates (SMDC) targeted to prostate-specific membrane antigen (PSMA) incorporating either: (i) a cathepsin-B-cleavable valine-citrulline (Val-Cit), or (ii) an acid-cleavable phosphoramidate linker. Crucial components used in the design of the conjugates include: (i) CTT1298, a nanomolar affinity ligand that binds irreversibly to PSMA and has proven in past studies to rapidly internalize and shuttle payloads into PSMA-expressing prostate cancer cells, (ii) MMAE, a known potent cytotoxic payload, and (iii) an albumin-binder, proven to improve residence time of drug conjugates. At dose of 0.8 mg/kg (â¼250 nmol/kg), the two SMDCs showed significant efficacy in a PSMA(+) PC3-PIP mouse model of human prostate cancer compared with controls, without inducing systemic toxicity. Though localization of the SMDCs was observed in tissues apart from the tumor, release of MMAE was observed predominantly in tumor tissue, at levels that were 2-3 orders of magnitude higher than non-target tissues. Furthermore, SMDC2, which incorporated a novel pH-responsive phosporamidate linker, demonstrated significantly improved efficacy over SMDC1 that has a Val-Cit linker, with a 100% survival over 90 days and 4 out of 8 mice showing complete tumor growth inhibition after 6 weekly doses of 0.8 mg/kg (244 nmol/kg). Our findings demonstrate the potential of irreversible PSMA inhibitors combined with pH-responsive linkers as a way to specifically deliver chemotherapeutic drugs to prostate cancer tumors with minimal toxicity.
Subject(s)
Antineoplastic Agents , Prostatic Neoplasms , Male , Animals , Humans , Mice , Cell Line, Tumor , Glutamate Carboxypeptidase II/metabolism , Antigens, Surface/metabolism , Prostatic Neoplasms/pathology , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacokinetics , Albumins/therapeutic useABSTRACT
Liposomes, such as pegylated-liposomal CKD-602 (S-CKD602), undergo catabolism by macrophages and dendritic cells (DCs) of the reticuloendothelial system (RES). The relationship between plasma and tumor disposition of S-CKD602 and RES was evaluated in mice bearing A375 melanoma or SKOV-3 ovarian xenografts. Area under the concentration-time curves (AUCs) of liposomal encapsulated, released, and sum total (encapsulated + released) CKD-602 in plasma, tumor, and tumor extracellular fluid (ECF) were estimated. A375 and SKOV-3 tumors were stained with cd11b and cd11c antibodies as measures of macrophages and DC. The plasma disposition of S-CKD602 was similar in both xenograft models. The ratio of tumor sum total AUC to plasma sum total AUC was 1.7-fold higher in mice bearing human SKOV-3 xenografts, compared with A375. The ratio of tumor ECF AUC to tumor sum total AUC was 2-fold higher in mice bearing human SKOV-3 xenografts, compared with A375. The staining of cd11c was 4.5-fold higher in SKOV-3, compared with A375 (P < 0.0001). The increased tumor delivery and release of CKD-602 from S-CKD602 in the ovarian xenografts, compared with the melanoma xenografts, was consistent with increased cd11c staining, suggesting that variability in the RES may affect the tumor disposition of liposomal agents.
Subject(s)
Camptothecin/analogs & derivatives , Mononuclear Phagocyte System/drug effects , Topoisomerase I Inhibitors/pharmacokinetics , Animals , Area Under Curve , Camptothecin/pharmacokinetics , Camptothecin/pharmacology , Chromatography, Liquid , Disease Models, Animal , Drug Evaluation, Preclinical , Humans , Mass Spectrometry , Mice , Topoisomerase I Inhibitors/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
AIM: We developed a generic high-performance liquid chromatography mass spectrometry approach for quantitation of small molecule compounds without availability of isotopically labelled standard. METHODS: The assay utilized 50 µL of plasma and offers 8 potential internal standards (IS): acetaminophen, veliparib, busulfan, neratinib, erlotinib, abiraterone, bicalutamide, and paclitaxel. Preparation consisted of acetonitrile protein precipitation and aqueous dilution in a 96 well-plate format. Chromatographic separation was achieved with a Kinetex C18 reverse phase (2.6 µm, 2 mm x 50 mm) column and a gradient of 0.1 % formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an AB SCIEX4000QTRAP with electrospray, positive-mode ionization. Performance of the generic approach was evaluated with seven drugs (LMP744, olaparib, cabozantinib, triapine, ixabepilone, berzosertib, eribulin) for which validated assays were available. RESULTS: The 8 IS covered a range of polarity, size, and ionization; eluted over the range of chromatographic retention times; were quantitatively extracted; and suffered limited matrix effects. The generic approach proved to be linear for test drugs evaluated over at least 3 orders of magnitude starting at 1-10 ng/mL, with extension of assay ranges with analyte isotopologue MRM channels. At a bias of less than 16 % and precision within 15 %, the assay performance was acceptable. CONCLUSION: The generic approach has become a useful tool to further define the pharmacology of drugs studied in our laboratory and may be utilized as described, or as starting point to develop drug-specific assays with more extensive performance characterization.