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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.
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Proteínas de la Ataxia Telangiectasia Mutada , Relación Dosis-Respuesta a Droga , Ratones Endogámicos BALB C , Animales , Femenino , Ratones , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Distribución Tisular , Pirazinas/farmacocinética , Pirazinas/administración & dosificación , Espectrometría de Masas en Tándem , Humanos , Pirazoles/farmacocinética , Pirazoles/administración & dosificación , Dinámicas no Lineales , Administración Intravenosa , IsoxazolesRESUMEN
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.
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Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los Resultados , Cromatografía Liquida/métodos , Estabilidad de Medicamentos , Modelos Lineales , Sensibilidad y EspecificidadRESUMEN
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.
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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.
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Neoplasias Colorrectales , Fluorouracilo , Humanos , Animales , Ratones , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Scutellaria baicalensis , Transducción de Señal , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Proliferación Celular , Línea Celular TumoralRESUMEN
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.
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Antineoplásicos , Ratones , Ratas , Masculino , Femenino , Animales , Ratones Desnudos , Antineoplásicos/toxicidad , Bencimidazoles/farmacología , Androstadienos/farmacologíaRESUMEN
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.
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Antineoplásicos , Neoplasias de la Próstata , Masculino , Animales , Humanos , Ratones , Línea Celular Tumoral , Glutamato Carboxipeptidasa II/metabolismo , Antígenos de Superficie/metabolismo , Neoplasias de la Próstata/patología , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacocinética , Albúminas/uso terapéuticoRESUMEN
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.
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Ataxia Telangiectasia , Espectrometría de Masas en Tándem , Acetonitrilos , Cromatografía Liquida/métodos , ADN , Humanos , Inhibidores de Proteínas Quinasas , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodos , AguaRESUMEN
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.
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Inhibidores de Proteínas Quinasas , Espectrometría de Masas en Tándem , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Disponibilidad Biológica , Cromatografía Liquida , Humanos , Ratones , Inhibidores de Proteínas Quinasas/farmacocinética , Distribución TisularRESUMEN
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.
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Indoles/farmacocinética , Modelos Biológicos , Morfolinas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/farmacocinética , Sulfonamidas/farmacocinética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Disponibilidad Biológica , Cromatografía Liquida , Relación Dosis-Respuesta a Droga , Femenino , Indoles/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Morfolinas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirimidinas/administración & dosificación , Sulfonamidas/administración & dosificación , Espectrometría de Masas en Tándem , Distribución TisularRESUMEN
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.
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Anilidas , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Humanos , Piridinas , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
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.
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Preparaciones Farmacéuticas , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Técnicas de Dilución del Indicador , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: To address multidrug resistance, we developed engineered Cationic Antimicrobial Peptides (eCAPs). Lead eCAP WLBU2 displays potent activity against drug-resistant bacteria and effectively treats lethal bacterial infections in mice, reducing bacterial loads to undetectable levels in diverse organs. OBJECTIVE: To support the development of WLBU2, we conducted a mass balance study. METHODS: CD1 mice were administered 10, 15, 20 and 30 mg/kg of QDx5 WLBU2 or a single dose of [14C]-WLBU2 at 15 mg/kg IV. Tolerability, tissue distribution and excretion were evaluated with liquid scintillation and HPLC-radiochromatography. RESULTS: The maximum tolerated dose of WLBU2 is 20 mg/kg IV. We could account for greater than >96% of the radioactivity distributed within mouse tissues at 5 and 15 min. By 24h, only ~40-50% of radioactivity remained in the mice. The greatest % of the dose was present in liver, accounting for ~35% of radioactivity at 5 and 15 min, and ~ 8% of radioactivity remained at 24h. High radioactivity was also present in kidneys, plasma, red blood cells and lungs, while less than 0.2% of radioactivity was present in brain, fat, or skeletal muscle. Urinary and fecal excretion accounted for 12.5 and 2.2% of radioactivity at 24h. CONCLUSION: WLBU2 distributes widely to mouse tissues and is rapidly cleared with a terminal radioactivity half-life of 22 h, a clearance of 27.4 mL/h/kg, and a distribution volume of 0.94 L/kg. At 2-100 µg-eq/g, the concentrations of 14C-WLBU2 appear high enough in the tissues to account for the inhibition of microbial growth.
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Péptidos Catiónicos Antimicrobianos , Infecciones Bacterianas , Animales , Péptidos Antimicrobianos , Radioisótopos de Carbono , RatonesRESUMEN
PURPOSE: Carboplatin dose is calculated based on kidney function, commonly estimated with imperfect creatinine-based formulae. Iohexol is used to measure glomerular filtration rate (GFR) and allows calculation of a more appropriate carboplatin dose. To address potential concerns that iohexol administered during a course of chemotherapy impacts that therapy, we performed in vitro and in vivo pharmacokinetic drug-drug interaction evaluations of iohexol. METHODS: Carboplatin was administered IV to female mice at 60 mg/kg with or without iohexol at 300 mg/kg. Plasma ultrafiltrate, kidney and bone marrow platinum was quantitated by atomic absorption spectrophotometry. Paclitaxel microsomal and gemcitabine cytosolic metabolism as well as metabolism of CYP and UGT probes was assessed with and without iohexol at 300 µg/mL by LC-MS/MS. RESULTS: In vivo carboplatin exposure was not significantly affected by iohexol co-administration (platinum AUC combination vs alone: plasma ultrafiltrate 1,791 vs 1920 µg/mL min; kidney 8367 vs 9757 µg/g min; bone marrow 12.7 vs 12.7 µg/mg-protein min). Paclitaxel microsomal metabolism was not impacted (combination vs alone: 6-α-OH-paclitaxel 38.3 versus 39.4 ng/mL/60 min; 3-p-OH-paclitaxel 26.2 versus 27.7 ng/mL/60 min). Gemcitabine human cytosolic elimination was not impacted (AUC combination vs gemcitabine alone: dFdU 24.1 versus 23.7 µg/mL/30 min). Iohexol displayed no relevant inhibition of the CYP and UGT enzymes in human liver microsomes. CONCLUSIONS: Iohexol is unlikely to affect the clinical pharmacokinetics of carboplatin, paclitaxel, gemcitabine, or other agents used in combination with carboplatin treatment. Measuring GFR with iohexol to better dose carboplatin is unlikely to alter the safety or efficacy of chemotherapy through pharmacokinetic drug-drug interactions.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Carboplatino/farmacocinética , Medios de Contraste/farmacocinética , Yohexol/farmacocinética , Administración Intravenosa , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Área Bajo la Curva , Médula Ósea/química , Carboplatino/administración & dosificación , Medios de Contraste/administración & dosificación , Creatinina , Sistema Enzimático del Citocromo P-450/metabolismo , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacocinética , Cálculo de Dosificación de Drogas , Interacciones Farmacológicas , Femenino , Tasa de Filtración Glomerular , Glucuronosiltransferasa/metabolismo , Humanos , Yohexol/administración & dosificación , Riñón/química , Riñón/metabolismo , Tasa de Depuración Metabólica , Ratones , Microsomas Hepáticos , Modelos Animales , Paclitaxel/administración & dosificación , Paclitaxel/farmacocinética , Organismos Libres de Patógenos Específicos , Espectrometría de Masas en Tándem , Distribución Tisular , GemcitabinaRESUMEN
We developed a high-performance liquid chromatography mass spectrometry method for quantitating iohexol in 50 µL human plasma. After acetonitrile protein precipitation, chromatographic separation was achieved with a Shodex Asahipak NH2P-50 2D (5 µm, 2 × 150 mm) column and a gradient of 0.1 % formic acid in acetonitrile and 0.1 % formic acid in water over a 10 min run time. Mass spectrometric detection was performed on a Micromass Quatromicro triple-stage bench-top mass spectrometer with electrospray, positive-mode ionization. The assay was linear from 1 to 500 µg/mL for iohexol, proved to be accurate (101.3-102.1 %) and precise (<3.4 %CV), and fulfilled Food and Drug Administration (FDA) criteria for bioanalytical method validation. Recovery from plasma was 53.1-64.2 % and matrix effect was trivial (-3.4 to -1.3 %). Plasma freeze thaw stability (97.4-99.4 %), stability for 5 months at -80 °C (95.5-103.3 %), and stability for 4 h at room temperature (100.6-103.3 %) were all acceptable. This validated assay using a deuterated internal standard will be an important tool in measuring iohexol clearance and determining glomerular filtration rate (GFR) in patients.
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Yohexol , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Humanos , Yohexol/análisis , Reproducibilidad de los ResultadosRESUMEN
Poor tumor penetration and highly immunosuppressive tumor microenvironment are two major factors that limit the therapeutic efficacy for the treatment of pancreatic ductal adenocarcinoma (PDA). In this work, a redox-responsive gemcitabine (GEM)-conjugated polymer, PGEM, was employed as a tumor penetrating nanocarrier to co-load an immunomodulating agent (NLG919, an inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1) and a chemotherapeutic drug (paclitaxel (PTX)) for immunochemo combination therapy. The NLG919/PTX co-loaded micelles showed very small size of ~15 nm. In vivo tumor imaging study indicated that PGEM was much more effective than the relatively large-sized POEG-co-PVD nanoparticles (~160 nm) in deep tumor penetration and could reach the core of the pancreatic tumor. PTX formulated in the PGEM carrier showed improved tumor inhibition effect compared with PGEM alone. Incorporation of NLG919 in the formulation led to a more immunoactive tumor microenvironment with significantly decreased percentage of Treg cells, and increased percentages of CD4+ IFNγ+ T and CD8+ IFNγ+ T cells. PGEM micelles co-loaded with PTX and NLG919 showed the best anti-tumor activity in pancreatic (PANC02) as well as two other tumor models compared to PGEM micelles loaded with PTX or NLG919 alone, suggesting that codelivery of NLG919 and PTX via PGEM may represent an effective strategy for immunochemotherapy of PDA as well as other types of cancers. STATEMENT OF SIGNIFICANCE: In order to effectively accumulate and penetrate the PDA that is poorly vascularized and enriched with dense fibrotic stroma, the size of nanomedicine has to be well controlled. Here, we reported an immunochemotherapy regimen based on co-delivery of GEM, PTX and IDO1 inhibitor NLG919 through an ultra-small sized GEM-based nanocarrier (PGEM). We demonstrated that the PGEM carrier was effective in accumulating and penetrating into PDA tumors. Besides, PGEM co-loaded with PTX and NLG9 induced an improved anti-tumor immune response and was highly efficacious in inhibiting tumor growth as well as in prolonging the survival rate in PANC02 xenograft model. Our work represents a potential strategy for enhancing PDA tumor penetration and immunochemotherapy.
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Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Portadores de Fármacos/química , Imidazoles/uso terapéutico , Isoindoles/uso terapéutico , Paclitaxel/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Antineoplásicos/química , Línea Celular Tumoral , Desoxicitidina/química , Desoxicitidina/uso terapéutico , Liberación de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Femenino , Imidazoles/química , Inmunidad/efectos de los fármacos , Inmunoterapia/métodos , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Isoindoles/química , Ratones Endogámicos BALB C , Micelas , Paclitaxel/química , Polietilenglicoles/química , Profármacos/química , Profármacos/uso terapéutico , GemcitabinaRESUMEN
We have developed a high performance liquid chromatography mass spectrometry method for quantitating paclitaxel and its 6-alpha-OH and 3-para-OH metabolites in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex synergy polar reverse phase (4 µm, 2 mm × 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 ABI SCIEX 4000Q with electrospray, positive-mode ionization. The assay was linear from 10-10,000 ng/mL for paclitaxel and 1-1000 ng/mL for both metabolites and proved to be accurate (94.3-110.4%) and precise (<11.3%CV). Recovery from plasma was 59.3-91.3% and matrix effect was negligible (-3.5 to 6.2%). Plasma freeze thaw stability (90.2-107.0%), stability for 37 months at -80 °C (89.4-112.6%), and stability for 4 h at room temperature (87.7-100.0%) were all acceptable. This assay will be an essential tool to further define the metabolism and pharmacology of paclitaxel and metabolites in the clinical setting. The assay may be utilized for therapeutic drug monitoring of paclitaxel and may also reveal the CYP2C8 and CYP3A4 activity phenotype of patients.
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Antineoplásicos Fitogénicos/sangre , Recolección de Muestras de Sangre/métodos , Monitoreo de Drogas/métodos , Paclitaxel/sangre , Antineoplásicos Fitogénicos/metabolismo , Antineoplásicos Fitogénicos/farmacocinética , Cromatografía Líquida de Alta Presión/métodos , Ensayos Clínicos Fase I como Asunto , Citocromo P-450 CYP2C8/metabolismo , Citocromo P-450 CYP3A/metabolismo , Estabilidad de Medicamentos , Humanos , Paclitaxel/metabolismo , Paclitaxel/farmacocinética , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
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.
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2',5'-Oligoadenilato Sintetasa/metabolismo , Infecciones por Virus ADN/inmunología , Virus ADN/fisiología , Infecciones por Virus ARN/inmunología , Virus ARN/inmunología , 2',5'-Oligoadenilato Sintetasa/genética , Animales , AMP Cíclico/metabolismo , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleotidiltransferasas/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Células THP-1 , Replicación ViralRESUMEN
The gastrointestinal (GI) epithelium is the fastest renewing adult tissue and is maintained by tissue-specific stem cells. Treatment-induced GI side effects are a major dose-limiting factor for chemotherapy and abdominal radiotherapy and can decrease the quality of life in cancer patients and survivors. p53 is a key regulator of the DNA damage response, and its activation results in stimulus- and cell type-specific outcomes via distinct effectors. We demonstrate that p53-dependent PUMA induction mediates chemotherapy-induced intestinal injury in mice. Genetic ablation of Puma, but not of p53, protects against chemotherapy-induced lethal GI injury. Blocking chemotherapy-induced loss of LGR5+ stem cells by Puma KO or a small-molecule PUMA inhibitor (PUMAi) prevents perturbation of the stem cell niche, rapid activation of WNT and NOTCH signaling, and stem cell exhaustion during repeated exposures. PUMAi also protects human and mouse colonic organoids against chemotherapy-induced apoptosis and damage but does not protect cancer cells in vitro or in vivo. Therefore, targeting PUMA is a promising strategy for normal intestinal chemoprotection because it selectively blocks p53-dependent stem cell loss but leaves p53-dependent protective effects intact.
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Células Madre/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Muerte Celular/genética , Muerte Celular/fisiología , Intestinos/citología , Irinotecán/efectos adversos , Ratones , Ratones Noqueados , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
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.
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Medicamentos Herbarios Chinos/farmacología , Factor de Transcripción E2F1/metabolismo , Fluorouracilo/farmacología , Transducción de Señal/efectos de los fármacos , Timidilato Sintasa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Sinergismo Farmacológico , Medicamentos Herbarios Chinos/análisis , Medicamentos Herbarios Chinos/uso terapéutico , Femenino , Fluorouracilo/uso terapéutico , Células HT29 , Humanos , Ratones , Ratones Desnudos , Trasplante HeterólogoRESUMEN
JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC-MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50µL plasma was followed by isocratic reverse phase chromatography for a 6min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1 to 1000ng/mL with appropriate accuracy (97.1-107.6%) and precision (3.7-12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2-136.1% with moderate ionization enhancement (10.9-39.5%). Plasma freeze-thaw stability (98.8-104.2%), stability for 13.5 months at -80°C (93.1-105.6%), and stability for 4h at room temperature (94.2-97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20mg/kg JP4-039 and will be extended to other studies of this agent.