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The 16th GCC Closed Forum was held in Orlando, FL, USA, on 23 June 2023. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: IS response, flow cytometry, changes to the bioanalytical industry, NGS assays, biomarker assay for tissues, dPCR validation, immunogenicity harmonization and ICH M10 implementation. Conclusions and consensus from discussions of these topics are included in this article.
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Biomarcadores , Citometría de Flujo , Citometría de Flujo/normas , Citometría de Flujo/métodos , Biomarcadores/análisis , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
We have previously reported that hexamethylene bis-acetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound estrogen receptor α (ERα) and the androgen receptor (AR) by disrupting the interaction between these receptors and positive transcriptional elongation factor b (P-TEFb) and attenuating RNA polymerase II (RNAPII) phosphorylation at serine 2. Functional consequences of the inhibition of transcriptional activity of ERα and AR by HEXIM1 include the inhibition of ERα- and AR-dependent gene expression, respectively, and the resulting attenuation of breast cancer (BCa) and prostate cancer (PCa) cell proliferation and growth. In our present study, we determined that HEXIM1 inhibited AKR1C3 expression in BCa and PCa cells. AKR1C3, also known as 17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 5, is a key enzyme involved in the synthesis of 17ß-estradiol (E2) and 5-dihydrotestosterone (DHT). Downregulation of AKR1C3 by HEXIM1 influenced E2 and DHT production, estrogen- and androgen-dependent gene expression, and cell proliferation. Our studies indicate that HEXIM1 has the unique ability to inhibit both the transcriptional activity of the ER and AR and the synthesis of the endogenous ligands of these receptors.
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Dihidrotestosterona/metabolismo , Regulación hacia Abajo , Estradiol/metabolismo , Receptor alfa de Estrógeno/metabolismo , Regulación de la Expresión Génica , Proteínas de Unión al ARN/biosíntesis , Receptores Androgénicos/metabolismo , Factores de Transcripción/biosíntesis , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Estrógenos/metabolismo , Femenino , Humanos , Ligandos , Células MCF-7 , Masculino , Neoplasias de la Próstata/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Aim: To report the development and validation of an LC-MS/MS method for the simultaneous determination of unconjugated payload DM4 and its metabolite S-methyl-DM4 in human plasma. Methodology: A workflow of protein precipitation followed by reduction and solid phase extraction was employed to remove antibody-maytansinoid conjugates from plasma matrix, release DM4 from endogenous conjugates, and generate a clean sample extract for analysis, respectively. Sodium adduct species of both analytes were selected for multiple reaction monitoring to meet the assay sensitivity requirement in liquid chromatography with tandem mass spectrometry. Conclusion: The method was fully validated for a dynamic range of 0.100-50.0 ng/ml for both analytes along with desired stability and acceptable incurred sample reanalysis.
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Inmunoconjugados/sangre , Maitansina/sangre , Cromatografía Liquida , Humanos , Inmunoconjugados/química , Inmunoconjugados/metabolismo , Maitansina/análogos & derivados , Maitansina/metabolismo , Espectrometría de Masas en TándemRESUMEN
Mucoadhesive polymers have an essential role in drug localization and target-specific actions in oral delivery systems. The current work aims to develop and characterize a new mucoadhesive polysaccharide polymer (thiolated xanthan gum-TXG and S-Protected thiolated xanthan gum-STX) that was further utilized for the preparation of repaglinide mucoadhesive tablets. The thiolation of xanthan gum was carried out by ester formation through the reaction of the hydroxyl group of xanthan gum and the carboxyl group of thioglycolic acid. Synthesis of TXG was optimized using central composite design, and TXG prepared using 5.303 moles/L of TGA and 6.075 g/L of xanthan gum can accomplish the prerequisites of the optimized formulation. Consequently, TXG was further combined with aromatic 2-mercapto-nicotinic acid to synthesize STX. TXG and STX were further studied for Fourier-transform infrared spectroscopy, rheological investigations, and Ellman's assay (to quantify the number of thiol/disulfide groups). A substantial rise in the viscosity of STX might be due to increased interactions of macromolecules liable for improving the mucosal adhesion strength of thiolated gum. STX was proven safe with the support of cytotoxic study data. Mucoadhesive formulations of repaglinide-containing STX showed the highest ex vivo mucoadhesion strength (12.78 g-RSX-1 and 17.57 g- RSX-2) and residence time (>16 h). The improved cross-linkage and cohesive nature of the matrix in the thiolated and S-protected thiolated formulations was responsible for the controlled release of repaglinide over 16 h. The pharmacokinetic study revealed the greater AUC (area under the curve) and long half-life with the RSX-2 formulation, confirming that formulations based on S-protected thiomers can be favorable drug systems for enhancing the bioavailability of low-solubility drugs.
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Cardiovascular diseases are a leading cause of mortality across the globe, and transplant surgeries are not always successful since it is not always possible to replace most of the damaged heart tissues, for example in myocardial infarction. Chitosan, a natural polysaccharide, is an important biomaterial for many biomedical and pharmaceutical industries. Based on the origin, degree of deacetylation, structure, and biological functions, chitosan has emerged for vital tissue engineering applications. Recent studies reported that chitosan coupled with innovative technologies helped to load or deliver drugs or stem cells to repair the damaged heart tissue not just in a myocardial infarction but even in other cardiac therapies. Herein, we outlined the latest advances in cardiac tissue engineering mediated by chitosan overcoming the barriers in cardiac diseases. We reviewed in vitro and in vivo data reported dealing with drug delivery systems, scaffolds, or carriers fabricated using chitosan for stem cell therapy essential in cardiac tissue engineering. This comprehensive review also summarizes the properties of chitosan as a biomaterial substrate having sufficient mechanical stability that can stimulate the native collagen fibril structure for differentiating pluripotent stem cells and mesenchymal stem cells into cardiomyocytes for cardiac tissue engineering.
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PIK3CA encodes the p110α catalytic subunit of PI3K and is frequently mutated in human cancers, including â¼30% of colorectal cancer. Oncogenic mutations in PIK3CA render colorectal cancers more dependent on glutamine. Here we report that the glutaminase inhibitor CB-839 preferentially inhibits xenograft growth of PIK3CA-mutant, but not wild-type (WT), colorectal cancers. Moreover, the combination of CB-839 and 5-fluorouracil (5-FU) induces PIK3CA-mutant tumor regression in xenograft models. CB-839 treatment increased reactive oxygen species and caused nuclear translocation of Nrf2, which in turn upregulated mRNA expression of uridine phosphorylase 1 (UPP1). UPP1 facilitated the conversion of 5-FU to its active compound, thereby enhancing the inhibition of thymidylate synthase. Consistently, knockout of UPP1 abrogated the tumor inhibitory effect of combined CB-839 and 5-FU administration. A phase I clinical trial showed that the combination of CB-839 and capecitabine, a prodrug of 5-FU, was well tolerated at biologically-active doses. Although not designed to test efficacy, an exploratory analysis of the phase I data showed a trend that PIK3CA-mutant patients with colorectal cancer might derive greater benefit from this treatment strategy as compared with PIK3CA WT patients with colorectal cancer. These results effectively demonstrate that targeting glutamine metabolism may be an effective approach for treating patients with PIK3CA-mutant colorectal cancers and warrants further clinical evaluation. SIGNIFICANCE: Preclinical and clinical trial data suggest that the combination of CB-839 with capecitabine could serve as an effective treatment for PIK3CA-mutant colorectal cancers.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bencenoacetamidas/administración & dosificación , Fosfatidilinositol 3-Quinasa Clase I/genética , Neoplasias Colorrectales/tratamiento farmacológico , Fluorouracilo/administración & dosificación , Tiadiazoles/administración & dosificación , Adulto , Animales , Bencenoacetamidas/efectos adversos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Relación Dosis-Respuesta a Droga , Femenino , Fluorouracilo/efectos adversos , Humanos , Masculino , Dosis Máxima Tolerada , Ratones , Persona de Mediana Edad , Tiadiazoles/efectos adversos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Androgens play a vital role in prostate cancer development, and their elimination and blockade are essential in the disease management. DHT is the key ligand for androgen receptor (AR) in the prostate. It is locally synthesized from testosterone. In the prostate, DHT is predominantly metabolized to α-diol and ß-diol. Recent studies indicate that impaired DHT catabolism is associated with prostate cancer, signifying the necessity of a sensitive quantitative method for the determination of DHT and its metabolites. In this work, an LC-MS/MS method for the simultaneous quantification of DHT and its metabolites was developed and validated. Steroid-free sera were prepared and used for the preparation of sera calibrators and quality controls (QCs). DHT and its metabolites along with their respective stable heavy isotope labeled analytes representing internal standards were first extracted with methyl tertiary-butyl ether (MTBE) and derivatized with picolinic acid (PA). The derivatized analytes were then extracted again with MTBE, dried under nitrogen and reconstituted in the mobile phase (80% methanol and 0.2% formic acid in water). Baseline chromatographic separation of the derivatized analytes was achieved isocratically on XTerra C18 column (2.1â¯×â¯100â¯mm) using the mobile phase at a flow rate of 0.25â¯mL/min. Quantitation was performed using multiple-reaction-monitoring mode with positive electrospray ionization. The method has calibration ranges from 0.0500â¯ng/mL to 50.0â¯ng/mL for DHT and its two metabolites with acceptable assay precision, accuracy, recovery, and matrix factor. It was applied to the determination of DHT and its metabolites in an animal study.
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Cromatografía Liquida/métodos , Dihidrotestosterona/sangre , Espectrometría de Masas en Tándem/métodos , Animales , Dihidrotestosterona/metabolismo , Modelos Lineales , Masculino , Ratones , Ácidos Picolínicos/química , Neoplasias de la Próstata , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Purpose: (i) To investigate the expression of the E3 ligase, RNF126, in human invasive breast cancer and its links with breast cancer outcomes; and (ii) to test the hypothesis that RNF126 determines the efficacy of inhibitors targeting the cell-cycle checkpoint kinase, CHEK1.Experimental Design: A retrospective analysis by immunohistochemistry (IHC) compared RNF126 staining in 110 invasive breast cancer and 78 paired adjacent normal tissues with clinicopathologic data. Whether RNF126 controls CHEK1 expression was determined by chromatin immunoprecipitation and a CHEK1 promoter driven luciferase reporter. Staining for these two proteins by IHC using tissue microarrays was also conducted. Cell killing/replication stress induced by CHEK1 inhibition was evaluated in cells, with or without RNF126 knockdown, by MTT/colony formation, replication stress biomarker immunostaining and DNA fiber assays.Results: RNF126 protein expression was elevated in breast cancer tissue samples. RNF126 was associated with a poor clinical outcome after multivariate analysis and was an independent predictor. RNF126 promotes CHEK1 transcript expression. Critically, a strong correlation between RNF126 and CHEK1 proteins was identified in breast cancer tissue and cell lines. The inhibition of CHEK1 induced a greater cell killing and a higher level of replication stress in breast cancer cells expressing RNF126 compared to RNF126 depleted cells.Conclusions: RNF126 protein is highly expressed in invasive breast cancer tissue. The high expression of RNF126 is an independent predictor of a poor prognosis in invasive breast cancer and is considered a potential biomarker of a cancer's responsiveness to CHEK1 inhibitors. CHEK1 inhibition targets breast cancer cells expressing higher levels of RNF126 by enhancing replication stress. Clin Cancer Res; 24(7); 1629-43. ©2018 AACR.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Ubiquitina-Proteína Ligasas/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Estudios de Cohortes , Replicación del ADN/genética , Femenino , Humanos , Inmunohistoquímica/métodos , Células MCF-7 , Pronóstico , Regiones Promotoras Genéticas/genética , Estudios RetrospectivosRESUMEN
Radiotherapy (RT) remains a standard therapeutic modality for breast cancer patients. However, intrinsic or acquired resistance limits the efficacy of RT. Here, we demonstrate that CHK1 inhibitor AZD7762 alone significantly inhibited the growth of radioresistant breast cancer cells (RBCC). Given the critical role of ATR/CHK1 signaling in suppressing oncogene-induced replication stress (RS), we hypothesize that CHK1 inhibition leads to the specific killing for RBCC due to its abrogation in the suppression of RS induced by oncogenes. In agreement, the expression of oncogenes c-Myc/CDC25A/c-Src/H-ras/E2F1 and DNA damage response (DDR) proteins ATR/CHK1/BRCA1/CtIP were elevated in RBCC. AZD7762 exposure led to significantly higher levels of RS in RBCC, compared to the parental cells. The mechanisms by which CHK1 inhibition led to specific increase of RS in RBCC were related to the interruptions in the replication fork dynamics and the homologous recombination (HR). In summary, RBCC activate oncogenic pathways and thus depend upon mechanisms controlled by CHK1 signaling to maintain RS under control for survival. Our study provided the first example where upregulating RS by CHK1 inhibitor contributes to the specific killing of RBCC, and highlight the importance of the CHK1 as a potential target for treatment of radioresistant cancer cells.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/radioterapia , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Reparación del ADN/genética , Recombinación Homóloga/genética , Humanos , Células MCF-7 , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Tiofenos/farmacología , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
5-fluorodeoxyuridine (5-FdU, floxuridine) is active against multiple cancers through the inhibition of thymidylate synthase, which consequently introduces uracil and 5-FU incorporation into the genome. Uracil DNA glycosylase (UDG) is one of the main enzymes responsible for the removal of uracil and 5-FU. However, how exactly UDG mediates cellular sensitivity to 5-FdU, and if so whether it is through its ability to remove uracil and 5-FU have not been well characterized. In this study, we report that UDG depletion led to incorporation of uracil and 5-FU in DNA following 5-FdU treatment and significantly enhanced 5-FdU's cytotoxicity in cancer cell lines. Co-treatment, but not post-treatment with thymidine prevented cell death of UDG depleted cells by 5-FdU, indicating that the enhanced cytotoxicity is due to the retention of uracil and 5-FU in genomic DNA in the absence of UDG. Furthermore, UDG depleted cells were arrested at late G1 and early S phase by 5-FdU, followed by accumulation of sub-G1 population indicating cell death. Mechanistically, 5-FdU dramatically reduced DNA replication speed in UDG depleted cells. UDG depletion also greatly enhanced DNA damage as shown by γH2AX foci formation. Notably, the increased γH2AX foci formation was not suppressed by caspase inhibitor treatment, suggesting that DNA damage precedes cell death induced by 5-FdU. Together, these data provide novel mechanistic insights into the roles of UDG in DNA replication, damage repair, and cell death in response to 5-FdU and suggest that UDG is a target for improving the anticancer effect of this agent.