RESUMO
Atopic dermatitis (AD) treatment has largely relied on non-specific broad immunosuppressants despite their long-term toxicities until the approval of dupilumab, which blocks IL-4 signaling to target Th2 cell responses. Here, we report the discovery of compound 4aa, a novel compound derived from the structure of chlorophyll a, and the efficacy of chlorophyll a to alleviate AD symptoms by oral administration in human AD patients. 4aa downregulated GATA3 and IL-4 in differentiating Th2 cells by potently blocking IL-4 receptor dimerization. In the murine model, oral administration of 4aa reduced the clinical severity of symptoms and scratching behavior by 76% and 72%, respectively. Notably, the elevated serum levels of Th2 cytokines reduced to levels similar to those in the normal group after oral administration of 4aa. Additionally, the toxicological studies showed favorable safety profiles and good tolerance. In conclusion, 4aa may be applied for novel therapeutic developments for patients with AD.
Assuntos
Dermatite Atópica , Humanos , Camundongos , Animais , Dermatite Atópica/tratamento farmacológico , Células Th2 , Clorofila A , Interleucina-4 , Citocinas , Diferenciação CelularRESUMO
This study aimed to develop and validate a sensitive liquid chromatography-coupled tandem mass spectrometry method for the quantification of LDD-2614, an indirubin derivative and novel FLT3 inhibitor, in rat plasma. In addition, the developed analytical method was applied to observe the pharmacokinetic properties of LDD-2614. Chromatographic separation was achieved on a Luna omega C18 column using a mixture of water and acetonitrile, both containing 0.1% formic acid. Quantitation was performed using positive electrospray ionization in a multiple reaction monitoring (MRM) mode. The MRM transitions were optimized as m/z 426.2â113.1 for LDD-2614 and m/z 390.2â113.1 for LDD-2633 (internal standard), and the lower limit of quantification (LLOQ) for LDD-2614 was determined as 0.1 ng/mL. Including the LLOQ, the nine-point calibration curve was linear with a correlation coefficient greater than 0.9991. Inter- and intraday accuracies (RE) ranged from -3.19% to 8.72%, and the precision was within 9.02%. All validation results (accuracy, precision, matrix effect, recovery, stability, and dilution integrity) met the acceptance criteria of the U.S. Food and Drug Administration and the Korea Ministry of Food and Drug Safety guidelines. The proposed method was validated and demonstrated to be suitable for the quantification of LDD-2614 for pharmacokinetics studies.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Indóis/farmacocinética , Oximas/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Espectrometria de Massas em Tandem/métodos , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Administração Intravenosa , Administração Oral , Animais , Calibragem , Cromatografia Líquida de Alta Pressão/instrumentação , Indóis/administração & dosagem , Indóis/química , Indóis/farmacologia , Masculino , Oximas/administração & dosagem , Oximas/química , Oximas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/instrumentaçãoRESUMO
Benzo[a]pyrene (BaP) is a well-known carcinogen formed during the cooking process. Although BaP exposure has been implicated as one of the risk factors for lung cancer in animals and humans, there are only limited data on BaP-induced gastrointestinal cancer. Therefore, this study investigated the protective effects of curcumin on BaP-induced DNA damage in rat stomach tissues. BaP (20 mg/kg/day) and curcumin (50, 100, or 200 mg/kg) were administered daily to Sprague-Dawley rats by oral gavage over 30 days. Curcumin was pre-administered before BaP exposure. All rats were euthanized, and liver, kidney, and stomach tissues were removed at 24 h after the last treatment. We observed that aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose levels were significantly reduced in rats treated with high dose co-administration of curcumin (200 mg/kg) compared to BaP alone. The expression levels of cytochrome P450 (CYP) 1A1 and CYP1B1 were significantly increased in the liver of rats treated with BaP. However, co-administration of curcumin (200 mg/kg) with BaP markedly reduced CYP1A1 expression in a dose-dependent manner. Furthermore, plasma levels of BaP-diolepoxide (BPDE) and BaP metabolites were significantly reduced by co-administration of curcumin (200 mg/kg). Additionally, co-administration of curcumin (200 mg/kg) with BaP significantly reduced the formation of BPDE-I-DNA and 8-hydroxydeoxy guanosine (8-OHdG) adducts in the liver, kidney, and stomach tissues. The inhibition of these adduct formations were more prominent in the stomach tissues than in the liver. Overall, our observations suggest that curcumin might inhibit BaP-induced gastrointestinal tumorigenesis and shows promise as a chemopreventive agent.
Assuntos
Curcumina/farmacologia , Dano ao DNA , Estômago/patologia , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Animais , Benzo(a)pireno , Peso Corporal/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Adutos de DNA/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Metaboloma , Tamanho do Órgão/efeitos dos fármacos , Ratos Sprague-Dawley , Estômago/efeitos dos fármacosRESUMO
A liquid chromatography-triple quadrupole mass spectrometric (LC-MS/MS) method was developed and validated for the determination of 5-nitro-5'-hydroxy-indirubin-3'-oxime (AGM-130) in human plasma to support a microdose clinical trial. The method consisted of a liquid-liquid extraction for sample preparation and LC-MS/MS analysis in the positive ion mode using TurboIonSpray(TM) for analysis. d3 -AGM-130 was used as the internal standard. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 10-2000 pg/mL for AGM-130. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 96.6% with a precision (coefficient of variation, CV) of 4.4%. For quality control samples at 30, 160 and 1600 pg/mL, the between run CV was ≤5.0 %. Between-run accuracy ranged from 98.1 to 101.0%. AGM-130 was stable in 50% acetonitrile for 168 h at 4°C and 6 h at room temperature. AGM-130 was also stable in human plasma at room temperature for 6 h and through three freeze-thaw cycles. The variability of selected samples for the incurred sample reanalysis was ≤12.7% when compared with the original sample concentrations. This validated LC-MS/MS method for determination of AGM-130 was used to support a phase 0 microdose clinical trial.
Assuntos
Indóis/administração & dosagem , Indóis/sangue , Oximas/administração & dosagem , Oximas/sangue , Cromatografia Líquida/métodos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Feminino , Humanos , Indóis/farmacocinética , Modelos Lineares , Extração Líquido-Líquido , Masculino , Oximas/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodosRESUMO
Red ginseng (RG) is the top-selling functional food in Korea, but is not recommended for use in hypertensive patients. This study was performed to determine the pharmacokinetic (PK) interaction between RG and amlodipine, an antihypertensive drug. RG (0, 0.5, 1, or 2 g/kg/d) was administered orally for 2 wk, and then amlodipine (10 mg/kg) was given orally, to Sprague-Dawley (SD) rats. Blood was collected at 0.08, 0.25, 1, 1.5, 2, 3, 6, 12, and 24 h after amlodipine administration. In intravenous (iv) study, RG (0, 1, or 2 g/kg/d) was administered orally to SD rats for 2 wk, followed by amlodipine (2 mg/kg) intravenously (iv). Plasma concentrations of amlodipine were analyzed using a high-pressure liquid chromatography-tandem mass system (LC-MS/MS). Oral administration of amlodipine produced an increase of time to maximum plasma concentration (tmax: 2.6, 4.1, 8.3, and 8.9 h at 0, 0.5, 1, and 2 g/kg/d, respectively), and a decrease of maximum plasma concentration (Cmax: 278.5, 212.4, 232.1, and 238.7 ng/ml at 0, 0.5, 1, and 2 g/kg/d, respectively.). However, the area under the concentration-time curve from time 0 to 24 h measurable concentration (AUC0-24 h was 3487.4, 2895.4, 3158.2, and 3495 ng/h/ml at 0, 0.5, 1, and 2 g/kg/d respectively) was not significantly changed among the different dose groups. Administration of amlodipine iv produced no significant changes in the apparent terminal half-life, volume of distribution, and AUC0-24 hr among the different dose groups. These results suggest that RG induced negligible influence on amlodipine pharmacokinetically in rats.
Assuntos
Anlodipino/farmacocinética , Anti-Hipertensivos/farmacocinética , Interações Ervas-Drogas , Panax/química , Administração Oral , Anlodipino/administração & dosagem , Animais , Anti-Hipertensivos/administração & dosagem , Cromatografia Líquida de Alta Pressão , Meia-Vida , Hipertensão/tratamento farmacológico , Masculino , Ratos , Ratos Sprague-Dawley , República da Coreia , Espectrometria de Massas em TandemRESUMO
PURPOSE: The oncoprotein KAI1 C-terminal interacting tetraspanin (KITENIN; vang-like 1) promotes cell metastasis, invasion, and angiogenesis, resulting in shorter survival times in cancer patients. Here, we aimed to determine the effects of KITENIN on the energy metabolism of human colorectal cancer cells. EXPERIMENTAL DESIGN: The effects of KITENIN on energy metabolism were evaluated using in vitro assays. The GEPIA web tool was used to extrapolate the clinical relevance of KITENIN in cancer cell metabolism. The bioavailability and effect of the disintegrator of KITENIN complex compounds were evaluated by LC-MS, in vivo animal assay. RESULTS: KITENIN markedly upregulated the glycolytic proton efflux rate and aerobic glycolysis by increasing the expression of GLUT1, HK2, PKM2, and LDHA. ß-catenin, CD44, CyclinD1 and HIF-1A, including c-Myc, were upregulated by KITENIN expression. In addition, KITENIN promoted nuclear PKM2 and PKM2-induced transactivation, which in turn, increased the expression of downstream mediators. This was found to be mediated through an effect of c-Myc on the transcription of hnRNP isoforms and a switch to the M2 isoform of pyruvate kinase, which increased aerobic glycolysis. The disintegration of KITENIN complex by silencing the KITENIN or MYO1D downregulated aerobic glycolysis. The disintegrator of KITENIN complex compound DKC1125 and its optimized form, DKC-C14S, exhibited the inhibition activity of KITENIN-mediated aerobic glycolysis in vitro and in vivo. CONCLUSIONS: The oncoprotein KITENIN induces PKM2-mediated aerobic glycolysis by upregulating the c-Myc/hnRNPs axis.
RESUMO
BACKGROUND: Immunological contexture differs across malignancies, and understanding it in the tumor microenvironment (TME) is essential for development of new anticancer agents in order to achieve synergistic effects with anti-programmed cell death protein-1 (PD-1) therapy. TYRO3, AXL, and MERTK receptors are bi-expressed in both cancer and immune cells, and thus emerge as promising targets for therapeutic intervention. Whereas AXL and MERTK have been extensively studied, the role of TYRO3, in the TME, is still undetermined. METHODS: Here, we screened the TYRO3-focused chemical library consisting of 208 compounds and presented a potent and highly selective TYRO3 inhibitor, KRCT87. We explored the role of TYRO3 using mouse engrafting MC38 or 4T1 tumors. We validated the results using flow cytometry, RNA sequencing analysis, gene knockdown or overexpression, ex vivo immune cells isolation from mouse models, immunoblotting and quantitative PCR. Flow cytometry was used for the quantification of cell populations and immunophenotyping of macrophages and T cells. Co-cultures of macrophages and T cells were performed to verify the role of CCN1 in the tumors. RESULTS: TYRO3 blockade boosts antitumor immune responses in both the tumor-draining lymph nodes and tumors in MC38-syngeneic mice models. Moreover, the combination of KRCT87 and anti-PD-1 therapy exerts significant synergistic antitumor effects in anti-PD-1-non-responsive 4T1-syngeneic model. Mechanistically, we demonstrated that inhibition of TYRO3-driven CCN1 secretion fosters macrophages into M1-skewing phenotypes, thereby triggering antitumor T-cell responses. CCN1 overexpression in MC38 tumors diminishes responsiveness to anti-PD-1 therapy. CONCLUSIONS: The activated TYRO3-CCN1 axis in cancer could dampen anti-PD-1 therapy responses. These findings highlight the potential of TYRO3 blockade to improve the clinical outcomes of anti-PD-1 therapy.
Assuntos
Microambiente Tumoral , Camundongos , Animais , c-Mer Tirosina Quinase , Linhagem Celular Tumoral , Modelos Animais de DoençasRESUMO
Acute myeloid leukemia (AML) is a prevalent form of leukemia in adults. As its survival rate is low, there is an urgent need for new therapeutic options. In AML, FMS-like tyrosine kinase 3 (FLT3) mutations are common and have negative outcomes. However, current FLT3-targeting agents, Midostaurin and Gilteritinib, face two significant issues, specifically the emergence of acquired resistance and drug-related adverse events leading to treatment failure. Rearranged during transfection (RET), meanwhile, is a proto-oncogene linked to various types of cancer, but its role in AML has been limited. A previous study showed that activation of RET kinase enhances FLT3 protein stability, leading to the promotion of AML cell proliferation. However, no drugs are currently available that target both FLT3 and RET. This study introduces PLM-101, a new therapeutic option derived from the traditional Chinese medicine indigo naturalis with potent in vitro and in vivo anti-leukemic activities. PLM-101 potently inhibits FLT3 kinase and induces its autophagic degradation via RET inhibition, providing a superior mechanism to that of FLT3 single-targeting agents. Single- and repeated-dose toxicity tests conducted in the present study showed no significant drug-related adverse effects. This study is the first to present a new FLT3/RET dual-targeting inhibitor, PLM-101, that shows potent anti-leukemic activity and fewer adverse effects. PLM-101, therefore, should be considered for use as a potential therapeutic agent for AML.
Assuntos
Leucemia Mieloide Aguda , Tirosina Quinase 3 Semelhante a fms , Adulto , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Leucemia Mieloide Aguda/metabolismo , Inibidores de Proteínas Quinases/efeitos adversos , Mutação , Proteínas Proto-Oncogênicas c-ret/genéticaRESUMO
The objective of this study is to report the effects of cysteine on the pharmacokinetics of intravenous and oral docetaxel in rats with protein-calorie malnutrition (PCM). The in vivo pharmacokinetics and in vitro hepatic/intestinal metabolism of docetaxel were assessed using control, CC (control with cysteine), PCM and PCMC (PCM with cysteine) rats. The effects of cysteine on the intestinal absorption of docetaxel were further investigated through in vitro transport studies using rat intestine and Caco-2 cell monolayers. The AUCs (the areas under the plasma concentration-time curve from time zero to time infinity) of intravenous docetaxel in PCM rats were significantly greater than in the control rats because of the significant decrease in the hepatic CYP3A. In PCMC rats, the elevated AUCs in PCM rats returned to control levels. The AUC(0-6 h)s of oral docetaxel in PCM rats were significantly smaller than that in the control rats, mainly due to the decrease in gastrointestinal absorption. In CC and PCMC rats, oral cysteine supplement enhanced the gastrointestinal absorption of docetaxel probably via intestinal P-gp inhibition. If the present rat data could be expressed to humans, the alterations in docetaxel absorption and metabolism should be considered in designing a dosage regimen for cancer patients with PCM state after cysteine supplement.
Assuntos
Cisteína/uso terapêutico , Desnutrição Proteico-Calórica/tratamento farmacológico , Taxoides/farmacocinética , Administração Oral , Animais , Transporte Biológico/efeitos dos fármacos , Análise Química do Sangue , Proteínas Sanguíneas/metabolismo , Células CACO-2 , Docetaxel , Relação Dose-Resposta a Droga , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Ingestão de Energia/efeitos dos fármacos , Humanos , Injeções Intravenosas , Rim/efeitos dos fármacos , Rim/patologia , Cinética , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Desnutrição Proteico-Calórica/sangue , Desnutrição Proteico-Calórica/urina , Ratos , Ratos Sprague-Dawley , Rodamina 123/metabolismo , Taxoides/administração & dosagem , Taxoides/farmacologia , Fatores de Tempo , Aumento de Peso/efeitos dos fármacosRESUMO
The transition from a chemotherapy-responsive cancer to a chemotherapy-resistant one is accompanied by increased expression of multidrug resistance 1 (MDR1, p-glycoprotein), which plays an important role in the efflux from the target cell of many anticancer agents. We recently showed that a Forkhead box-containing protein of the O subfamily 1 (FoxO1) is a key regulator of MDR1 gene transcription. Because nuclear localization of FoxO1 is regulated by silent information regulator two ortholog 1 (SIRT1) deacetylase, we wondered whether SIRT1 dominates MDR1 gene expression in breast cancer cells. Overexpression of SIRT1 enhanced both FoxO reporter activity and nuclear levels of FoxO1. Protein expression of MDR1 and gene transcriptional activity were also up-regulated by SIRT1 overexpression. In addition, SIRT1 inhibition reduced both nuclear FoxO1 levels and MDR1 expression in doxorubicin-resistant breast cancer cells (MCF-7/ADR) cells. A potent SIRT1 inhibitor, amurensin G (from Vitis amurensis), was identified by screening plant extracts and bioassay-guided fractionation. The compound suppressed FoxO1 activity and MDR1 expression in MCF-7/ADR cells. Moreover, pretreatment of MCF-7/ADR cells with 1 µg/ml amurensin G for 24 h increased cellular uptake of doxorubicin and restored the responsiveness of MCF-7/ADR cells to doxorubicin. In xenograft studies, injection of 10 mg/kg i.p. amurensin G substantially restored the ability of doxorubicin to inhibit MCF-7/ADR-induced tumor growth. These results suggest that SIRT1 is a potential therapeutic target of MDR1-mediated chemoresistance and that it may be possible to develop amurensin G as a useful agent for chemoresistance reversal.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Antibióticos Antineoplásicos/farmacologia , Dibenzocicloeptenos/farmacologia , Doxorrubicina/farmacologia , Resorcinóis/farmacologia , Sirtuína 1/antagonistas & inibidores , Estilbenos/farmacologia , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/fisiologia , Humanos , Metanol , Camundongos , Camundongos Nus , Transplante de Neoplasias , Extratos Vegetais/farmacologia , Relação Estrutura-Atividade , Transplante Heterólogo , VitisRESUMO
FMS-like receptor tyrosine kinase-3 (FLT3) is expressed on acute leukemia cells and is implicated in the survival, proliferation and differentiation of hematopoietic cells in most acute myeloid leukemia (AML) patients. Despite recent achievements in the development of FLT3-targeted small-molecule drugs, there are still unmet medical needs related to kinase selectivity and the progression of some mutant forms of FLT3. Herein, we describe the discovery of novel orally available type 1 FLT3 inhibitors from structure-activity relationship (SAR) studies for the optimization of indirubin derivatives with biological and pharmacokinetic profiles as potential therapeutic agents for AML. The SAR exploration provided important structural insights into the key substituents for potent inhibitory activities of FLT3 and in MV4-11 cells. The profile of the most optimized inhibitor (36) showed IC50 values of 0.87 and 0.32 nM against FLT3 and FLT3/D835Y, respectively, along with potent inhibition against MV4-11 and FLT3/D835Y expressed MOLM14 cells with a GI50 value of 1.0 and 1.87 nM, respectively. With the high oral bioavailability of 42.6%, compound 36 displayed significant in vivo antitumor activity by oral administration of 20 mg/kg once daily dosing schedule for 21 days in a mouse xenograft model. The molecular docking study of 36 in the homology model of the DFG-in conformation of FLT3 resulted in a reasonable binding mode in type 1 kinases similar to the reported type 1 FLT3 inhibitors Crenolanib and Gilteritinib.
Assuntos
Desenho de Fármacos , Indóis/química , Indóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Oximas/química , Oximas/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Indóis/administração & dosagem , Indóis/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Simulação de Acoplamento Molecular , Oximas/administração & dosagem , Oximas/metabolismo , Fosforilação/efeitos dos fármacos , Conformação Proteica , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
BACKGROUND: Acitretin and etretinate are potentially teratogenic. Many people taking acitretin for psoriasis have donated blood during the deferral period in Korea. Therefore, many of the blood products from these donors treated with acitretin have been circulated in Korea. STUDY DESIGN AND METHODS: A high-performance liquid chromatography system (HP 1050, Agilent Technologies) was used to measure the drug concentrations in five blood products and in patients. Sixty patients taking acitretin were enrolled to determine their plasma drug levels. Forty-one female patients were recruited to investigate the residual plasma levels of acitretin and etretinate in relation to their teratogenicity. We calculated the elimination rate of acitretin and etretinate during the manufacturing process. RESULTS: Sixty individuals taking acitretin expressed variable acitretin (<2.0-206.8 ng/mL) and etretinate levels (<2.0-9.1 ng/mL). All patients that had a transfusion had concentrations of acitretin and etretinate lower than the lower limit of quantification (LLOQ; 2 ng/mL). The concentrations of acitretin and etretinate in five blood products were less than the LLOQ. Approximately 98.84 percent (log value, 1.94) of the acitretin and 99.93 percent (log value, 3.14) of the etretinate was eliminated during the manufacturing process of albumin. More than 99.99 percent (log values, 5.95-15.76) of acitretin and etretinate was eliminated during the manufacturing processing of immunoglobulin and blood coagulation factors. CONCLUSIONS: We confirmed the effective manufacturing processing of various blood products. We also demonstrated that individuals receiving transfusions with blood products originating from donors treated with acitretin were not at risk for significant exposure to the acitretin and etretinate.
Assuntos
Acitretina/sangue , Produtos Biológicos/química , Doadores de Sangue , Transfusão de Sangue , Etretinato/sangue , Acitretina/administração & dosagem , Acitretina/farmacocinética , Acitretina/uso terapêutico , Adolescente , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Contaminação de Medicamentos , Etretinato/administração & dosagem , Etretinato/farmacocinética , Etretinato/uso terapêutico , Feminino , Meia-Vida , Humanos , Masculino , Pessoa de Meia-Idade , Psoríase/sangue , Psoríase/tratamento farmacológico , Teratogênicos , Reação TransfusionalRESUMO
BACKGROUND: Ligand-dependent activation of the G-protein coupled receptor 119 (GPR119) lowers blood glucose via glucose-dependent insulin secretion and intestinal glucagon-like peptide-1 production. However, the function of GPR119 in cancer cells has not been studied. METHODS: GPR119 expression was assessed by real-time qPCR and immunohistochemistry in human breast cancer cell lines and breast cancer tissues. Cell proliferation and cell cycle analyses were performed by Incucyte® live cell analysis system and flow cutometry, respectively. Autophagy activity was estimeated by western blottings and LC3-GFP transfection. RESULTS: mRNA or protein expression of GPR119 was detected in 9 cancer cell lines and 19 tissue samples. Cotreatment with GPR119 agonist (MBX-2982 or GSK1292263) significantly potentiated gefitinib-induced cell growth inhibition in gefitinib-insensitive MCF-7 and MDA-MB-231 breast cancer cells. We observed that caspase-3/7 activity was enhanced with the downregulation of Bcl-2 in MCF-7 cells exposed to MBX-2982. Gefitinib-induced autophagy is related with cancer cell survival and chemoresistance. GPR119 agonists inhibit gefitinib-induced autophagosome formation in MCF-7 and MDA-MB-231 cells. MBX-2982 also caused a metabolic shift to enhanced glycolysis accompanied by reduced mitochondrial oxidative phosphorylation. MBX-2982 increased intracellular (~ 2.5 mM) and extracellular lactate (~ 20 mM) content. Gefitinib-mediated autophagy was suppressed by 20 mM lactate in MCF-7 cells. CONCLUSIONS: GPR119 agonists reduced mitochondrial OXPHOS and stimulated glycolysis in breast cancer cells, with consequent overproduction of lactate that inhibited autophagosome formation. Because autophagy is crucial for the survival of cancer cells exposed to TKIs, GPR119 agonists potentiated the anticancer effects of TKIs.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Gefitinibe/farmacologia , Ácido Láctico/metabolismo , Mesilatos/farmacologia , Oxidiazóis/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Tetrazóis/farmacologia , Tiazóis/farmacologia , Animais , Autofagia/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptores Acoplados a Proteínas G/metabolismo , TransfecçãoRESUMO
Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the management of neuropathic pain because P2X3 receptors are predominantly localized on small to medium diameter C- and Aδ-fiber primary afferent neurons, which are related to the pain-sensing system. In this study, 5-hydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro biological activities by two-electrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3 receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin (calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the corresponding methyl ester analogue, 28, which could be converted to compound 29 in vivo, was investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic effects, with ED50 values of 2.62 and 2.93 mg/kg in spinal nerve ligation and chemotherapy-induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.
Assuntos
Analgésicos não Narcóticos/farmacologia , Neuralgia/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/farmacologia , Piridinas/farmacologia , Analgésicos não Narcóticos/síntese química , Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/farmacocinética , Animais , Antineoplásicos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Células HEK293 , Humanos , Ligadura , Masculino , Camundongos , Estrutura Molecular , Neuralgia/metabolismo , Oócitos , Técnicas de Patch-Clamp , Permeabilidade , Antagonistas do Receptor Purinérgico P2X/síntese química , Antagonistas do Receptor Purinérgico P2X/química , Antagonistas do Receptor Purinérgico P2X/farmacocinética , Piridinas/síntese química , Piridinas/química , Piridinas/farmacocinética , Ratos , Receptores Purinérgicos P2X3/metabolismo , Nervos Espinhais , Relação Estrutura-Atividade , XenopusRESUMO
We developed and validated a simple, rapid, and accurate HPLC-MS/MS method with simple protein precipitation for the determination of orphenadrine. Injection-to-injection running time was 3 min with a retention time of orphenadrine of 1.1 min. The linear assay range was 1-200 ng/mL (r2 > 0.99). The intra- and inter-assay imprecisions were CV 0.6-4.2% and CV 1.6-6.1%, respectively. The accuracy, extraction recovery, specificity and stability were satisfactory. Using the measured plasma concentrations of orphenadrine in 24 healthy subjects, pharmacokinetic profiles of orphenadrine were evaluated (AUC(0-72,) 1565+/-731 ng h/mL, Cmax 82.8+/-26.2 ng/mL, Tmax 3.0+/-0.9 h, elimination half-life 25.8+/-10.3 h).
Assuntos
Orfenadrina/sangue , Orfenadrina/farmacocinética , Administração Oral , Precipitação Química , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Meia-Vida , Humanos , Espectrometria de Massas , Orfenadrina/química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A bioassay-guided fractionation of the ethylacetate soluble fraction from the flower buds of Tussilago farfara L. (Compositae) yielded two flavonoids, quercetin 3-O-beta-L-arabinopyranoside and quercetin 3-O-beta-D-glucopyranoside. These two sugar conjugates of quercetin exhibited higher antioxidative activity than their aglycone, quercetin by NBT superoxide scavenging assay. Moreover, treatment with quercetin 3-O-beta-L-arabinopyranoside significantly increased the total glutathione (GSH) contents and the protein level of gamma-glutamylcysteine ligase (gamma-GCL), a key enzyme required for glutathione (GSH) synthesis in a rat hepatocyte cell line. Subcellular fractionation and reporter gene analysis using antioxidant response element (ARE) construct revealed that quercetin 3-O-beta-L-arabinopyranoside increased the level of nuclear Nrf2 and reporter activity, and that these were associated with the induction of the gamma-GCL gene. After 24 h incubation of cells with quercetin 3-O-beta-L-arabinopyranoside, 23% of the glycoside was converted to its aglycone, quercetin, but gamma-GCL was not induced by 7 microM (23%) quercetin. These results suggest that the two quercetin-glycosides isolated from T. farfara L. have direct antioxidative properties, and that quercetin 3-O-beta-L-arabinopyranoside increases the cellular GSH level by inducing the gamma-GCL gene. These novel effects of quercetin-glycosides are suggestive to underlie the potential putative chemopreventive effects of T. farfara L.
Assuntos
Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Tussilago/química , Animais , Linhagem Celular , Indução Enzimática/fisiologia , Flavonoides/química , Flores/química , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/biossíntese , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Glutationa Transferase/biossíntese , Glutationa Transferase/metabolismo , Glicosídeos/química , Hepatócitos , Conformação Molecular , Fator 2 Relacionado a NF-E2/metabolismo , Nitroazul de Tetrazólio/química , Ressonância Magnética Nuclear Biomolecular , Ratos , Superóxidos/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
The discovery of new biomarkers for early detection of drug-induced acute kidney injury (AKI) is clinically important. In this study, sensitive metabolomic biomarkers identified in the urine of rats were used to detect cisplatin-induced AKI. Cisplatin (10 mg kg(-1), i.p.) was administered to Sprague-Dawley rats, which were subsequently euthanized after 1, 3 or 5 days. In cisplatin-treated rats, mild histopathological alterations were noted at day 1, and these changes were severe at days 3 and 5. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels were significantly increased at days 3 and 5. The levels of new urinary protein-based biomarkers, including kidney injury molecule-1 (KIM-1), glutathione S-transferase-α (GST-α), tissue inhibitor of metalloproteinase-1 (TIMP-1), vascular endothelial growth factor (VEGF), calbindin, clusterin, neutrophil, neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin, were significantly elevated at days 3 and 5. Among urinary metabolites, trigonelline and 3-indoxylsulfate (3-IS) levels were significantly decreased in urine collected from cisplatin-treated rats prior to histological kidney damage. However, carbon tetrachloride (CCl4), a hepatotoxicant, did not affect these urinary biomarkers. Trigonelline is closely associated with GSH depletion and results in insufficient antioxidant capacity against cisplatin-induced AKI. The predominant cisplatin-induced AKI marker appeared to be reduced in urinary 3-IS levels. Because 3-IS is predominantly excreted via active secretion in proximal tubules, a decrease is indicative of tubular damage. Further, urinary excretion of 3-IS levels was markedly reduced in patients with AKI compared to normal subjects. The area under the curve receiver operating characteristics (AUC-ROC) for 3-IS was higher than for SCr, BUN, lactate dehydrogenase (LDH), total protein, and glucose. Therefore, low urinary or high serum 3-IS levels may be more useful for early detection of AKI than conventional biomarkers.
Assuntos
Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/urina , Metaboloma , Metabolômica , Injúria Renal Aguda/etiologia , Animais , Biomarcadores , Biópsia , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Metabolômica/métodos , Espectroscopia de Prótons por Ressonância Magnética , Curva ROC , RatosRESUMO
Liver cirrhosis (LC) is a chronic disease with high mortality rate. In the United States and Western world as well as Asian countries, LC is the major leading cause of death by disease. Yet, no effective therapeutic agent is available for LC treatment. Laboratory cirrhotic rats produced by dimethylnitrosamine administrations simulate the clinical features of human LC such as mortality, ascites, hepatic parenchymal cell destruction, and formation of connective tissue and nodular regeneration, providing a preclinical model to evaluate therapeutic efficacy of drugs and the underlying mechanisms. Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione] has been used clinically and is of little toxicity. Comprehensive mechanistic and phase IIa clinical studies supported the notion that oltipraz exerts chemopreventive effects against chemical carcinogenesis. We report here that oltipraz within the clinical dose range regenerates cirrhotic liver in the established LC rats as a result of reduction of the intensities of cirrhotic nodules, elimination of accumulated extracellular matrix, and inactivation of stellate cells, thereby improving survival rate. We also reveal that activation of CCAAT/enhancer binding protein by oltipraz inhibits transforming growth factor b1 gene expression in stellate cells, which provides a molecular target for pharmacological treatment of LC. Oltipraz is the first therapeutic agent that regenerates cirrhotic liver.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Cirrose Hepática Experimental/tratamento farmacológico , Regeneração Hepática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Pirazinas/farmacologia , Animais , Matriz Extracelular/efeitos dos fármacos , Fígado/citologia , Fígado/fisiopatologia , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Modelos Biológicos , Pirazinas/uso terapêutico , RNA Mensageiro/biossíntese , Ratos , Análise de Sobrevida , Tionas , Tiofenos , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1RESUMO
We have isolated four different phenylethanoid glycosides (purpureaside A, desrhamnosyl acteoside, calceolarioside B and plantainoside D) from the leaves of Digitalis purpurea (foxglove). The effects of these glycosides on activator protein-1 (AP-1)-mediated inducible nitric oxide synthase (iNOS) gene expression in the Raw264.7 macrophage cell line have been studied. Of these four glycosides, purpureaside A potently inhibited iNOS induction by lipopolysaccharide (LPS). Increase in iNOS mRNA by LPS was completely suppressed by purpureaside A. Purpureaside A did not significantly affect LPS-inducible nuclear factor-kappaB (NF-kappaB) activation or the nuclear translocation of p65. Moreover, a reporter gene assay using AP-1 specific luciferase reporter revealed that the enhanced activity of AP-1 by LPS was completely abolished in cells treated with purpureaside A. These results demonstrated that purpureaside A inhibited LPS-inducible iNOS expression in macrophages through the suppression of AP-1, but not of NF-kappaB.