RESUMO
Impaired blood flow in the tumor vascular bed caused by structurally and functionally abnormal blood vessels not only hinders the delivery of chemotherapeutic agents but also aggravates tumor hypoxia, making the tumor cells further resistant to antineoplastic drugs. Therefore, normalization of tumor blood vessels may be an important approach to increase therapeutic efficacy in the treatment of cancer patients. As blood vessels are supplied by sympathetic nerves containing dopamine (DA), and DA regulates functions of normal blood vessels through its receptors present in these vessels, we investigated the effect of DA on tumor vasculature. Here we report loss of sympathetic innervation and endogenous DA in abnormal and immature tumor blood vessels in malignant colon and prostate tumor tissues. In contrast, exogenous administration of DA normalizes the morphology and improves the functions of these vessels by acting on pericytes and endothelial cells, the two major cellular components of blood vessels. DA acts through its D(2) receptors present in these cells to up-regulate directly the expression of angiopoietin 1 (Ang1) in pericytes and the expression of the zinc finger transcriptional factor, Krüppel-like factor-2 (KLF2) in tumor endothelial cells. Importantly, this vessel stabilization by DA also significantly increases the concentration of anticancer drug in tumor tissues. These results show a relationship between vascular stabilization and a neurotransmitter and indicate that DA or its D(2) receptor-specific agonists can be an option for the treatment of cancer and disorders in which normalization of blood vessels may have therapeutic benefits.
Assuntos
Angiopoietina-1/biossíntese , Vasos Sanguíneos/metabolismo , Dopamina/metabolismo , Células Endoteliais/citologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/biossíntese , Pericitos/metabolismo , Regulação para Cima , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Hipóxia/metabolismo , Camundongos , Microscopia Confocal/métodos , Neovascularização PatológicaRESUMO
Nevirapine is one of the most extensively prescribed antiretrovirals worldwide. The present analyses used data and specimens from two prior studies to characterize and compare plasma nevirapine phase I metabolite profiles following a single 200-mg oral dose of nevirapine in 10 HIV-negative African Americans and a steady-state 200-mg twice-daily dose in 10 HIV-infected Cambodians. Nevirapine was assayed by high-performance liquid chromatography (HPLC). The 2-, 3-, 8- and 12-hydroxy and 4-carboxy metabolites of nevirapine were assayed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Pharmacokinetic parameters were calculated by noncompartmental analysis. The metabolic index for each metabolite was defined as the ratio of the metabolite area under the concentration-time curve (AUC) to the nevirapine AUC. Every metabolite concentration was much less than the corresponding nevirapine concentration. The predominant metabolite after single dose and at steady state was 12-hydroxynevirapine. From single dose to steady state, the metabolic index increased for 3-hydroxynevirapine (P < 0.01) but decreased for 2-hydroxynevirapine (P < 0.001). The 3-hydroxynevirapine metabolic index was correlated with nevirapine apparent clearance (P < 0.001). These findings are consistent with induction of CYP2B6 (3-hydroxy metabolite) and a possible inhibition of CYP3A (2-hydroxy metabolite), although these are preliminary data. There were no such changes in metabolic indexes for 12-hydroxynevirapine or 4-carboxynevirapine. Two subjects with the CYP2B6 *6*6 genetic polymorphism had metabolic indexes in the same range as other subjects. These results suggest that nevirapine metabolite profiles change over time under the influence of enzyme induction, enzyme inhibition, and host genetics. Further work is warranted to elucidate nevirapine biotransformation pathways and implications for drug efficacy and toxicity.
Assuntos
Fármacos Anti-HIV/farmacocinética , Infecções por HIV/tratamento farmacológico , Nevirapina/farmacocinética , Adulto , Negro ou Afro-Americano , Fármacos Anti-HIV/sangue , Área Sob a Curva , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Povo Asiático , Biotransformação , Estudos de Casos e Controles , Cromatografia Líquida , Citocromo P-450 CYP2B6 , Esquema de Medicação , Feminino , Infecções por HIV/etnologia , Infecções por HIV/microbiologia , HIV-1/efeitos dos fármacos , Humanos , Masculino , Nevirapina/sangue , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Polimorfismo Genético , Espectrometria de Massas em TandemRESUMO
PURPOSE: Prolonged exposure of cancer cells to triapine, an inhibitor of ribonucleotide reductase, followed by gemcitabine enhances gemcitabine activity in vitro. Fixed-dose-rate gemcitabine (FDR-G) has improved efficacy compared to standard-dose. We conducted a phase I trial to determine the maximum tolerated dose (MTD), safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of prolonged triapine infusion followed by FDR-G. EXPERIMENTAL DESIGN: Triapine was given as a 24-hour infusion, immediately followed by FDR-G (1000 mg/m(2) over 100-minute). Initially, this combination was administered days 1 and 8 of a 21-day cycle (Arm A, triapine starting dose 120 mg); but because of myelosuppression, it was changed to days 1 and 15 of a 28-day cycle (Arm B, starting dose of triapine 75 mg). Triapine steady-state concentrations (Css) and circulating ribonucleotide reductase M2-subunit (RRM2) were measured. RESULTS: Thirty-six patients were enrolled. The MTD was determined to be triapine 90 mg (24-hour infusion) immediately followed by gemcitabine 1000 mg/m(2) (100-minute infusion), every 2 weeks of a 4-week cycle. DLTs included grade 4 thrombocytopenia, leukopenia and neutropenia. The treatment was well tolerated with fatigue, nausea/vomiting, fever, transaminitis, and cytopenias being the most common toxicities. Among 30 evaluable patients, 1 had a partial response and 15 had stable disease. Triapine PK was similar, although more variable, compared to previous studies using doses normalized to body-surface-area. Steady decline in circulating levels of RRM2 may correlate with outcome. CONCLUSIONS: This combination was well tolerated and showed evidence of preliminary activity in this heavily pretreated patient population, including prior gemcitabine failure.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia/induzido quimicamente , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/análogos & derivados , Feminino , Humanos , Leucopenia/induzido quimicamente , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/sangue , Piridinas/administração & dosagem , Piridinas/efeitos adversos , Piridinas/farmacocinética , Ribonucleosídeo Difosfato Redutase/sangue , Tiossemicarbazonas/administração & dosagem , Tiossemicarbazonas/efeitos adversos , Tiossemicarbazonas/farmacocinética , Trombocitopenia/induzido quimicamente , GencitabinaRESUMO
Systemic delivery of fenretinide in oral cancer chemoprevention trials has been largely unsuccessful due to dose-limiting toxicities and subtherapeutic intraoral drug levels. Local drug delivery, however, provides site-specific therapeutically relevant levels while minimizing systemic exposure. These studies evaluated the pharmacokinetic and growth-modulatory parameters of fenretinide mucoadhesive patch application on rabbit buccal mucosa. Fenretinide and blank-control patches were placed on right/left buccal mucosa, respectively, in eight rabbits (30 min, q.d., 10 days). No clinical or histological deleterious effects occurred. LC-MS/MS analyses of post-treatment samples revealed a delivery gradient with highest fenretinide levels achieved at the patch-mucosal interface (no metabolites), pharmacologically active levels in fenretinide-treated oral mucosa (mean: 5.65 µM; trace amounts of 4-oxo-4-HPR) and undetectable sera levels. Epithelial markers for cell proliferation (Ki-67), terminal differentiation (transglutaminase 1-TGase1) and glucuronidation (UDP-glucuronosyltransferase1A1-UGT1A1) exhibited fenretinide concentration-specific relationships (elevated TGase1 and UGT1A1 levels <5 µM, reduced Ki-67 indices >5 µM) relative to blank-treated epithelium. All fenretinide-treated tissues showed significantly increased intraepithelial apoptosis (TUNEL) positivity, implying activation of intersecting apoptotic and differentiation pathways. Human oral mucosal correlative studies showed substantial interdonor variations in levels of the enzyme (cytochrome P450 3A4-CYP3A4) responsible for conversion of fenretinide to its highly active metabolite, 4-oxo-4-HPR. Complementary in vitro assays in human oral keratinocytes revealed fenretinide and 4-oxo-4-HPR's preferential suppression of DNA synthesis in dysplastic as opposed to normal oral keratinocytes. Collectively, these data showed that mucoadhesive patch-mediated fenretinide delivery is a viable strategy to reintroduce a compound known to induce keratinocyte differentiation to human oral cancer chemoprevention trials.
Assuntos
Fenretinida/administração & dosagem , Neoplasias Bucais/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimioprevenção/métodos , Citocromo P-450 CYP3A/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Fenretinida/análogos & derivados , Fenretinida/metabolismo , Fenretinida/farmacocinética , Glucuronosiltransferase/metabolismo , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/metabolismo , Neoplasias Bucais/metabolismo , CoelhosRESUMO
MicroRNAs (miRs) are deregulated in cancer and leukemia. Restoring aberrantly downregulated tumor suppressor miRs or antagonizing overexpressed oncogenic miRs in malignant cells by synthetic RNA oligonucleotides represents a potentially novel therapeutic approach in cancer and leukemia. However, given the complex networking and concurrent deregulation of miRs in malignant cells, an effective approach may require concurrent targeting of multiple miRs. Cassette dosing involves simultaneous administration of a mixture of oligonucleotides from the same or different structural classes. However, information on cassette dosing pharmacokinetics, tissue distribution and bioactivity of synthetic miRs is lacking. In this study, three synthetic 2'-methoxyphosphorothioate-miRs (2'-MeOPSmiR16-1, 2'-MeOPSmiR29b and 2'-MeOPSantagomiR155) were administered iv to C57BL/6 mice as a mixture, each at 7.5 mg/kg. Analysis of concentrations of individual miR in plasma and major organ tissues (bone marrow, spleen, liver, brain, heart, kidney and lung) was performed. The mRNA and protein levels of miR's biotargets were monitored sequentially after dosing up to 24 h. Our results demonstrated that these synthetic miRs retain their different individual pharmacokinetic properties and all display three-compartmental pharmacokinetics. 2'-MeOPSmiR16-1 has the longest plasma gamma half-life of 2508 min and lowest total body clearance of 0.0054 L/min·kg, whereas 2'-MeOPSmiR29b has the shortest gamma half-life of 510.6 min and highest total body clearance of 0.042 L/min·kg. The tissue concentrations of all three 2'-MeOPS-modified miR(s)/antagomiR were measurable from 5 min to at least 24 h after dosing, indicating that these concurrently delivered oligonucleotides can reach organ tissues. Importantly, there were biological activities of the concurrently administered miRs which persisted, as shown by the downregulation of specific targets in tested tissues, albeit with variations. Brain was one of the most sensitive tissues with respect to downregulation of mRNA and protein levels of four measured biotargets (e.g., Bcl-2, Mcl-1, DNMT3a and DNMT3b) despite its relatively low miR/antagomiRs levels. We conclude that cassette dosing is applicable to 2'-MeOPS-modified synthetic miRs that are tissue-deliverable and biofunctional without any additional formulation requirement. This study supports future exploration of miR-involved combination therapies.
Assuntos
MicroRNAs/síntese química , MicroRNAs/farmacocinética , Animais , Western Blotting , Encéfalo/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Ensaio de Imunoadsorção Enzimática , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/química , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DNA Metiltransferase 3BRESUMO
Reactivation of tumor suppressor genes (TSGs) involved in carcinogenesis by nontoxic bioactive food component represents a promising strategy for cancer chemoprevention. Recently, curcumin has been demonstrated to inhibit a bacterial DNA methyltransferase (M. Sss I) activity, induce global DNA hypomethylation in leukemia cells, and reactivate several hypermethylation silenced genes in lung and prostate cancer cells. Herein, we demonstrated that curcumin can enhance the mRNA and protein levels of ras-association domain family protein 1A (RASSF1A), 1 hypermethylation-silenced TSG, and decrease its promoter methylation in breast cancer cells. Mechanistic study demonstrated that curcumin can decrease DNA methylation activity of nuclear extract and downregulate the mRNA and protein levels of DNMT1 in MCF-7 cells, which may be associated with curcumin-induced disruption of NF-κB/Sp1 complex bound to the promoter region of DNMT1. Altogether, this study reveals a novel molecular mechanism of curcumin as a chemo-preventive agent for breast cancer through hypomethylation reactivation of RASSF1A.
Assuntos
Neoplasias da Mama/prevenção & controle , Curcumina/farmacologia , Ativação Transcricional/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Animais , Neoplasias da Mama/química , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , Proteínas Repressoras/análise , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/análise , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aberrant methylation of tumor suppressor genes can lead to their silencing in many cancers. TSC-22 is a gene silenced in several solid tumors, but its function and the mechanism(s) responsible for its silencing are largely unknown. Here we demonstrate that the TSC-22 promoter is methylated in primary mouse T or natural killer (NK) large granular lymphocyte (LGL) leukemia and this is associated with down-regulation or silencing of TSC-22 expression. The TSC-22 deregulation was reversed in vivo by a 5-aza-2'-deoxycytidine therapy of T or NK LGL leukemia, which significantly increased survival of the mice bearing this disease. Ectopic expression of TSC-22 in mouse leukemia or lymphoma cell lines resulted in delayed in vivo tumor formation. Targeted disruption of TSC-22 in wild-type mice enhanced proliferation and in vivo repopulation efficiency of hematopoietic precursor cells (HPCs). Collectively, our data suggest that TSC-22 normally contributes to the regulation of HPC function and is a putative tumor suppressor gene that is hypermethylated and silenced in T or NK LGL leukemia.
Assuntos
Movimento Celular/genética , Proliferação de Células , Células-Tronco Hematopoéticas/fisiologia , Leucemia Linfocítica Granular Grande/genética , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Animais , Células Cultivadas , Metilação de DNA , Epigênese Genética/fisiologia , Regulação Leucêmica da Expressão Gênica , Inativação Gênica/fisiologia , Genes Supressores de Tumor , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras GenéticasRESUMO
Aberrant DNA hypermethylation contributes to myeloid leukemogenesis by silencing structurally normal genes involved in hematopoiesis. MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression by targeting protein-coding mRNAs. Recently, miRNAs have been shown to play a role as both targets and effectors in gene hypermethylation and silencing in malignant cells. In the current study, we showed that enforced expression of miR-29b in acute myeloid leukemia cells resulted in marked reduction of the expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B at both RNA and protein levels. This in turn led to decrease in global DNA methylation and reexpression of p15(INK4b) and ESR1 via promoter DNA hypomethylation. Although down-regulation of DNMT3A and DNMT3B was the result of a direct interaction of miR-29b with the 3' untranslated regions of these genes, no predicted miR-29b interaction sites were found in the DNMT1 3' untranslated regions. Further experiments revealed that miR-29b down-regulates DNMT1 indirectly by targeting Sp1, a transactivator of the DNMT1 gene. Altogether, these data provide novel functional links between miRNAs and aberrant DNA hypermethylation in acute myeloid leukemia and suggest a potentially therapeutic use of synthetic miR-29b oligonucleotides as effective hypomethylating compounds.
Assuntos
DNA (Citosina-5-)-Metiltransferases/biossíntese , Metilação de DNA/genética , Regulação Leucêmica da Expressão Gênica , Genes Supressores de Tumor , Leucemia Mieloide/genética , MicroRNAs/genética , RNA Neoplásico/genética , Regiões 3' não Traduzidas/genética , Doença Aguda , Diferenciação Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p15/biossíntese , Inibidor de Quinase Dependente de Ciclina p15/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Regulação para Baixo/genética , Indução Enzimática/genética , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/genética , Vetores Genéticos/genética , Humanos , Vírus da Imunodeficiência Felina/genética , Leucemia Mieloide/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , RNA Neoplásico/biossíntese , Fator de Transcrição Sp1/antagonistas & inibidores , DNA Metiltransferase 3BRESUMO
Targeting aberrant DNA hypermethylation in chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma (NHL) with decitabine may reverse epigenetic silencing in B-cell malignancies. Twenty patients were enrolled in two phase I trials to determine the minimum effective pharmacological dose of decitabine in patients with relapsed/refractory CLL (n = 16) and NHL (n = 4). Patients received 1-3 cycles of decitabine. Dose-limiting toxicity (DLT) was observed in 2 of 4 CLL and 2 of 2 NHL patients receiving decitabine at 15 mg/m(2) per d days 1-10, consisting of grade 3-4 thrombocytopenia and hyperbilirubinaemia. Six patients with CLL received decitabine at 10 mg/m(2) per d days 1-10 without DLT; however, re-expression of methylated genes or changes in global DNA methylation were not observed. Therefore, a 5-day decitabine schedule was examined. With 15 mg/m(2) per d decitabine days 1-5, DLT occurred in 2 of 6 CLL and 2 of 2 NHL patients, consisting of grade 3-4 neutropenia, thrombocytopenia, and febrile neutropenia. Eight patients had stable disease. In 17 patients, there were no significant changes in genome-wide methylation or in target gene re-expression. In conclusion, dose-limiting myelosuppression and infectious complications prevented dose escalation of decitabine to levels associated with changes in global methylation or gene re-expression in CLL and NHL.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Azacitidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/genética , Linfoma não Hodgkin/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/sangue , Antimetabólitos Antineoplásicos/uso terapêutico , Antimetabólitos Antineoplásicos/toxicidade , Azacitidina/administração & dosagem , Azacitidina/sangue , Azacitidina/uso terapêutico , Azacitidina/toxicidade , DNA de Neoplasias/efeitos dos fármacos , Decitabina , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Linfoma não Hodgkin/sangue , Linfoma não Hodgkin/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Neutropenia/induzido quimicamente , Trombocitopenia/induzido quimicamente , Resultado do TratamentoRESUMO
A highly sensitive and specific LC-MS/MS assay was developed and validated to quantify nevirapine (NVP) and its five metabolites [2-, 3-, 8-, 12-hydroxyl NVP (OHNVP) and 4-carboxyl NVP (CANVP)] simultaneously in baboon serum and the assay was used to characterize their pharmacokinetic studies of an oral-dose escalation study in baboon. The lower limit of quantification (LLOQ) for NVP and its four hydroxyl nevirapine metabolites was 1.0 ng/mL and for 4-CANVP was 5.0 ng/mL. The between-run and within-run precisions and accuracies at four quality control concentrations (1, 5, 50 and 500 ng/mL) were evaluated in baboon serum with less than 14% variation and 93-114% accuracies (n = 6), except for the LLOQ for 2-OHNVP, which had an accuracy of 115.8% for between-run validation. The pharmacokinetics of NVP and its five metabolites in non-pregnant baboons by a single-dose escalation study were also profiled. The major metabolites detected were 4-CANVP and 12-OHNVP. 3-OHNVP and 2-OHNVP were the minor metabolites with only a trace amount of 2-OHNVP detected in some pharmacokinetic samples. No 8-OHNVP was observed in all of the pharmacokinetic samples. In addition, the fragmentation for the four hydroxyl metabolite isomers is also discussed.
Assuntos
Fármacos Anti-HIV/sangue , Fármacos Anti-HIV/farmacocinética , Cromatografia Líquida/métodos , Nevirapina/sangue , Nevirapina/farmacocinética , Espectrometria de Massas em Tandem/métodos , Animais , Fármacos Anti-HIV/metabolismo , Feminino , Nevirapina/metabolismo , Papio , Sensibilidade e EspecificidadeRESUMO
Hypermethylation of 5'-cytosine-guanosine islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys(38) of p65 to inhibit nuclear factor-kappaB activation and exhibit anti-tumor activity in human malignancies. In this article, we report that parthenolide 1) inhibits DNA methyltransferase 1 (DNMT1) with an IC(50) of 3.5 microM, possibly through alkylation of the proximal thiolate of Cys(1226) of the catalytic domain by its gamma-methylene lactone, and 2) down-regulates DNMT1 expression possibly associated with its SubG(1) cell-cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in vitro and in vivo global DNA hypomethylation. Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.
Assuntos
Antineoplásicos/farmacologia , Metilação de DNA/efeitos dos fármacos , Lactonas/farmacologia , Sesquiterpenos/farmacologia , Alquilação , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Domínio Catalítico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Cisteína/metabolismo , Citocinas/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Humanos , Immunoblotting , Lactonas/química , Lactonas/uso terapêutico , Camundongos , Camundongos Nus , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Sesquiterpenos/química , Sesquiterpenos/uso terapêutico , Fator de Transcrição Sp1/metabolismo , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Promoter hypermethylation-associated tumor suppressor gene (TSG) silencing has been explored as a therapeutic target for hypomethylating agents. Promoter methylation change may serve as a pharmacodynamic endpoint for evaluation of the efficacy of these agents and predict the patient's clinical response. Here a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed for quantitative regional DNA methylation analysis using the molar ratio of 5-methyl-2'-deoxycytidine (5mdC) to 2'-deoxycytidine (2dC) in the enzymatic hydrolysate of fully methylated bisulfite-converted polymerase chain reaction (PCR) amplicons as the methylation indicator. The assay can differentiate 5% of promoter methylation level with an intraday precision ranging from 3 to 16% using two TSGs: HIN-1 and RASSF1A. This method was applied to characterize decitabine-induced promoter DNA methylation changes of these two TSGs in a breast cancer MCF-7 cell line. Promoter methylation of these TSGs was found to decrease in a dose-dependent manner. Correspondingly, the expression of these TSGs was enhanced. The sensitivity and reproducibility of the method make it a valuable tool for specific gene methylation analysis that could aid characterization of hypomethylating activity on specific genes by hypomethylating agents in a clinical setting.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Metilação de DNA , Espectrometria de Massas em Tandem/métodos , Azacitidina/análogos & derivados , Azacitidina/química , Sequência de Bases , Linhagem Celular Tumoral , DNA/química , Decitabina , Desoxicitidina/análogos & derivados , Desoxicitidina/análise , Desoxicitidina/química , Humanos , Regiões Promotoras Genéticas , Reprodutibilidade dos TestesRESUMO
PURPOSE: To simultaneously quantify intracellular nucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP) pools and to assess their changes produced by interfering with ribonucleotide reductase (RNR) expression in leukemia cells. METHODS: A HPLC-MS/MS system was used to quantify intracellular NTP and dNTP pools. RESULTS: The assay was linear between 50 nM, the lower limit of quantification (LLOQ), and 10 muM in cell lysate. The within-day coefficients of variation (CVs, n = 5) were found to be 12.0-18.0% at the LLOQ and 3.0-9.0% between 500 and 5,000 nM for dNTPs and 8.0-15.0% and 2.0-6.0% for NTPs. The between-day CVs (n = 5) were 9.0-13.0% and 3.0-11.0% for dNTPs and 9.0-13.0% and 3.0-6.0% for NTPs. The within-day accuracy values were 93.0-119.0% for both NTPs and dNTPs. ATP overlapped with dGTP and they were analyzed as a composite. This method was applied to measure basal intracellular dNTPs/NTPs in five leukemia cell lines exposed to the RNR antisense GTI-2040. Following drug treatment, dCTP and dATP levels were found to decrease significantly in MV4-11 and K562 cells. Additionally, perturbation of dNTP/NTP levels in bone marrow sample of a patient treated with GTI-2040 was detected. CONCLUSIONS: This method provides a practical tool to measure intracellular dNTP/NTP levels in cells and clinical samples.
Assuntos
Cromatografia Líquida/métodos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Leucemia/metabolismo , Leucemia/terapia , Nucleotídeos/análise , Espectrometria de Massas em Tandem/métodos , Antimetabólitos Antineoplásicos/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Citarabina/uso terapêutico , Humanos , Modelos Lineares , Nucleotídeos/isolamento & purificação , Oligodesoxirribonucleotídeos/uso terapêutico , Sensibilidade e EspecificidadeRESUMO
Molecular docking of the interaction of curcumin and DNMT1 suggested that curcumin covalently blocks the catalytic thiolate of C1226 of DNMT1 to exert its inhibitory effect. This was validated by showing that curcumin inhibits the activity of M. SssI with an IC(50) of 30 nM, but no inhibitory activity of hexahydrocurcumin up to 100 microM. In addition, curcumin can induce global DNA hypomethylation in a leukemia cell line.
Assuntos
Curcumina/farmacologia , Metilação de DNA , Catálise , Domínio Catalítico , Linhagem Celular Tumoral , Curcumina/análogos & derivados , Curcumina/química , Citosina/química , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Leucemia/tratamento farmacológico , Modelos Químicos , Conformação MolecularRESUMO
PURPOSE: Inhibition of ribonucleotide reductase reduces the availability of the endogenous pool of deoxycytidine and may increase cytarabine (AraC) cytotoxicity. We performed a phase I dose escalation trial of AraC combined with GTI-2040, a 20-mer antisense oligonucleotide shown in preclinical studies to decrease levels of the R2 subunit of ribonucleotide reductase, to determine the maximum tolerated dose in adults with relapsed/refractory acute myeloid leukemia. EXPERIMENTAL DESIGN: Twenty-three adults (ages 18-59 years) were enrolled in this dose escalation phase I trial, receiving high-dose AraC twice daily combined with infusional GTI-2040. An ELISA-based assay measured plasma and intracellular concentrations of GTI-2040. R2 protein changes were evaluated by immunoblotting in pretreatment and post-treatment bone marrow samples. RESULTS: The maximum tolerated dose was 5 mg/kg/d GTI-2040 (days 1-6) and 3 g/m2/dose AraC every 12 hours for 8 doses. Neurotoxicity was dose limiting. Eight patients (35%) achieved complete remission. Mean bone marrow intracellular concentration of GTI-2040 were higher at 120 hours than at 24 hours from the start of GTI-2040 (P = 0.002), suggesting intracellular drug accumulation over time. Reductions in bone marrow levels of R2 protein (>50%) were observed at 24 and 120 hours. Higher baseline R2 protein expression (P = 0.03) and reductions after 24 hours of GTI-2040 (P = 0.04) were associated with complete remission. CONCLUSIONS: GTI-2040 and high-dose AraC were coadministered safely with successful reduction of the intended R2 target and encouraging clinical results. The clinical efficacy of this combination will be tested in an upcoming phase II study.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Citarabina/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Oligodesoxirribonucleotídeos/administração & dosagem , Ribonucleotídeo Redutases/antagonistas & inibidores , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Citarabina/efeitos adversos , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/efeitos adversos , Oligodesoxirribonucleotídeos/farmacocinética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/efeitos adversos , Resultado do TratamentoRESUMO
DNA hypermethylation is a common finding in malignant cells and has been explored as a therapeutic target for hypomethylating agents (e.g., decitabine). Detection of changes in DNA methylation might serve as a pharmacodynamic endpoint to establish the biological activity of these agents and predict clinical response. We developed and validated a rapid, sensitive and specific LC-MS/MS method for determination of global DNA methylation (GDM) in vitro and in vivo. Ratios of 5-methyl-2'-deoxycytidine (5mdC) to the internal standard 2-deoxyguanosine (2dG) in mass signal were used to quantify GDM levels. The assay was validated in a linear range from 40 fmol to 200 pmol 5mdC. The intra-day precision values ranged from 2.8 to 9.9% and the inter-day values from 1.1 to 15.0%. The accuracy of the assay varied between 96.7 and 109.5%. This method was initially applied for characterization of decitabine-induced GDM changes in in-vitro-treated leukemia cells. Following exposure to 2.5 microM decitabine, GDM decreased to approximately 50% of the baseline value. The clinical applicability of this method was then demonstrated in bone marrow samples from patients with acute myeloid leukemia treated with decitabine. Our data support the use of our LC-MS/MS method for clinical pharmacodynamic determination of changes in GDM in vivo.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Cromatografia Líquida/métodos , Metilação de DNA/efeitos dos fármacos , Leucemia Mieloide/tratamento farmacológico , Espectrometria de Massas em Tandem/métodos , Doença Aguda , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , Ensaios Clínicos Fase I como Assunto , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Decitabina , Desoxicitidina/análogos & derivados , Desoxicitidina/análise , Desoxiguanosina/análise , Humanos , Leucemia Mieloide/enzimologia , Leucemia Mieloide/genéticaRESUMO
Enzyme kinetics of GTI-2040 (5'-GGC TAA ATC GCT CCA CCA AG-3'), a phosphorothioate ribonucleotide reductase antisense, were investigated for the first time in 3' exonuclease solution and human liver microsomes (HLMs), using the ion-pair high-performance liquid chromatogram method for quantification of the parent drug and two major 3'N-1 and 3'N-2 metabolites. Enzyme kinetics of GTI-2040 in 3'-exonuclease solution were found to be well characterized by the Michaelis-Menten model, using the sum of formation rates of 3'N-1 and 3'N-2 (approximately total metabolism) because of sequential metabolism. In HLMs, a biphasic binding was observed for GTI-2040 with high- and low-affinity constants (K(d)s) of 0.03 and 3.8 microM, respectively. Enzyme kinetics of GTI-2040 in HLMs were found to deviate from Michaelis-Menten kinetics when the total GTI-2040 substrate was used. However, after correction for the unbound fractions, the formation rate of total metabolites could be described by Michaelis-Menten kinetics. Using the free substrate fraction, the K(m) and V(max) of GTI-2040 were determined to be 6.33 +/- 3.2 microM and 16.5 +/- 8.4 nmol/mg/h, respectively. Using these values, in vitro hepatic intrinsic clearance (CL(int)) in HLM was estimated to be 2.61 +/- 0.56 ml/h. The CL(int) was then used to predict GTI-2040's in vivo intrinsic clearance in humans by a microsomal protein scaling factor, which gave a mean value of 182.7 l/h, representing 24.1% of the observed in vivo mean scaled hepatic intrinsic clearance of 758.7 l/h in patients with acute myeloid leukemia. We concluded that the saturable nonspecific binding of GTI-2040 in HLMs complicated the interpretation of its enzyme kinetics, and scaled intrinsic clearance from HLMs only partially predicted the in vivo intrinsic clearance.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Oligodesoxirribonucleotídeos/metabolismo , Oligonucleotídeos Fosforotioatos/metabolismo , Ribonucleotídeo Redutases/metabolismo , Animais , Crotalus , Feminino , Humanos , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/metabolismo , Cinética , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/farmacologia , Oligonucleotídeos Fosforotioatos/administração & dosagem , Oligonucleotídeos Fosforotioatos/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidoresRESUMO
In our study, we have established a novel liquid-driven co-flow focusing (LDCF) process to fabricate curcumin (CUR)-loaded poly (lactic-co-glycolic acid) (PLGA) microparticles (CPMs). LDCF-CPMs of size 20.26 ± 2.37 µm have high encapsulation efficiency (>70%) and were intended for application in ovarian cancer by intraperitoneal (IP) administration. LDCF-CPMs have smooth surface with narrow size distribution and a core-shell structured verified by confocal microscopy which can be precisely controlled by changing the flow rates of focusing, outer and inner phases. The LDCF-CPMs reveal the physiochemical stability with sustained release profile corresponding to 95% CUR release over a period of 14 days in an in vitro release medium. Moreover, LDCF-CPMs were testified for cytotoxicity against SKOV-3 ovarian cancer cell lines and peritoneal delivery advantages by animal experiments. The pharmacokinetics of LDCF-CPMs in rats following IP injection shows slow systemic absorption with mean residence time (MRT) of 13.54 h in comparison with 9.82 and 6.74 h for SE-CPMs and free CUR, respectively. In addition, IP delivery of CUR can expose the ovarian tumour to higher concentration for a longer duration by programming the thickness of the shell. The study provides compelling evidence for LDCF-CPMs having high therapeutic opportunity in the treatment of peritoneal cancers, such as ovarian, that reside in the peritoneal cavity.
Assuntos
Antineoplásicos Fitogênicos , Curcumina , Nanopartículas , Neoplasias Ovarianas , Ácido Poliglicólico , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/farmacologia , Curcumina/química , Curcumina/farmacocinética , Curcumina/farmacologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Feminino , Humanos , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Tamanho da Partícula , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacocinética , Ácido Poliglicólico/farmacologia , Ratos , Ratos Wistar , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A specific and sensitive HPLC-MS/MS method for the quantitative determination of valproic acid (VPA) and its metabolite, 2-propyl-4-pentenoic acid in human plasma has been developed, using VPA-d15 as the internal standard. The method was based on pre-column derivatization using 4-dimethylaminobenzylamine dihydrochloride. The derivatives were separated with a gradient elution and quantified by positive electrospray ionization with multiple reaction monitoring. The assay provides routine quantification limits of 200 ng/mL for VPA and 20 ng/mL for 4-ene VPA with within- and between-day coefficients of variation of <10%. This method has been applied to the analysis of plasma samples obtained from patients treated with this drug.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácidos Graxos Monoinsaturados/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Ácido Valproico/sangue , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Ácido Valproico/farmacocinéticaRESUMO
Histone deacetylase inhibitors (HDACi) have proven activity in hematologic malignancies, and their FDA approval in multiple myeloma (MM) and T-cell lymphoma highlights the need for further development of this drug class. We investigated AR-42, an oral pan-HDACi, in a first-in-man phase 1 dose escalation clinical trial. Overall, treatment was well tolerated, no DLTs were evident, and the MTD was defined as 40 mg dosed three times weekly for three weeks of a 28-day cycle. One patient each with MM and mantle cell lymphoma demonstrated disease control for 19 and 27 months (ongoing), respectively. Treatment was associated with reduction of serum CD44, a transmembrane glycoprotein associated with steroid and immunomodulatory drug resistance in MM. Our findings indicate that AR-42 is safe and that further investigation of AR-42 in combination regimens for the treatment of patients with lymphoma and MM is warranted. TRIAL REGISTRATION: http://clinicaltrials.gov/ct2/show/NCT01129193.