RESUMO
Folate receptor (FR)-ß has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid-derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity. Using FR-expressing macrophages, the effect of EC0565 on mTOR signaling and cellular proliferation was studied. The pharmacokinetics, metabolism and bioavailability of EC0565 were studied in normal rats. The in vivo activity of EC0565 was assessed in rats with adjuvant arthritis, a "macrophage-rich" model with close resemblance to rheumatoid arthritis. EC0565 forms micellar aggregates in physiological buffers and demonstrates good water solubility as well as strong multivalent FR-binding capacity. EC0565 inhibited mTOR signaling in rat macrophages at nanomolar concentrations and induced G0/G1 cell cycle arrest in serum-starved RAW264.7 cells. Subcutaneously administered EC0565 in rats displayed good bioavailability and a relatively long half-life (~12 h). When given at 250 nmol/kg, EC0565 selectively inhibited proliferating cell nuclear antigen expression in thioglycollate-stimulated rat peritoneal cells. With limited dosing regimens, the antiarthritic activity of EC0565 was found superior to that of etanercept, everolimus and a nontargeted everolimus analog. The in vivo activity of EC0565 was also comparable to that of a folate-targeted aminopterin. Folate-targeted mTOR inhibition may be an effective way of suppressing activated macrophages in sites of inflammation, especially in nutrient-deprived conditions, such as in the arthritic joints. Further investigation and improvement upon the physical and biochemical properties of EC0565 are warranted.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Everolimo/análogos & derivados , Everolimo/administração & dosagem , Ácido Fólico/análogos & derivados , Ácido Fólico/administração & dosagem , Inflamação/tratamento farmacológico , Serina-Treonina Quinases TOR/genética , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Artrite Experimental/tratamento farmacológico , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Proliferação de Células/efeitos dos fármacos , Everolimo/química , Receptor 2 de Folato/genética , Receptor 2 de Folato/metabolismo , Ácido Fólico/química , Humanos , Inflamação/genética , Inflamação/patologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
During a phase I trial of EC145 (a folate-targeted vinca alkaloid conjugate), constipation was identified as the dose-limiting toxicity, probably from a nonfolate receptor-related liver clearance process capable of releasing unconjugated vinca alkaloid from EC145 and shuttling it to the bile. Here, we report on the selective placement of novel carbohydrate segments (1-amino-1-deoxy-glucitolyl-γ-glutamate) spaced in-between the folate and vinca alkaloid moieties of EC145, which yielded a new agent (EC0489) that is equipotent but less toxic than EC145. Whereas both compounds could cure tumor-bearing mice reproducibly, EC0489 differed from EC145 with i) a shorter elimination half-life, ii) approximately 70% decrease in bile clearance, iii) a 4-fold increase in urinary excretion, and iv) improved tolerability in rodents. This combination of improvements justified the clinical evaluation of EC0489 where currently administered dose levels have exceeded the maximal tolerated dose of EC145 by approximately 70%, thereby reflecting the translational benefits to this new approach.
Assuntos
Antineoplásicos/farmacocinética , Ácido Fólico/análogos & derivados , Fígado/metabolismo , Alcaloides de Vinca/farmacocinética , Animais , Antineoplásicos/toxicidade , Relação Dose-Resposta a Droga , Descoberta de Drogas , Feminino , Receptores de Folato com Âncoras de GPI/fisiologia , Ácido Fólico/farmacocinética , Ácido Fólico/toxicidade , Rim/metabolismo , Masculino , Taxa de Depuração Metabólica , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Alcaloides de Vinca/toxicidadeRESUMO
BACKGROUND: Alcohol is a significant risk factor for the development of hepatocellular carcinoma (HCC). To date, no rodent model has demonstrated the formation of hepatic neoplasia in the setting of chronic alcohol consumption alone. METHODS: We investigated whether rats selectively bred for high alcohol preference (P rats), allowed free access to water, or water and 10% (v/v) alcohol, for 6, 12, or 18 months, develop hepatic neoplasia. RESULTS: At necropsy, liver tumor incidence and multiplicity were significantly increased in 18-month alcohol-consuming versus water-consuming P rats. These data were confirmed histologically by glutathione-S-transferase pi-class (GSTp) staining. Phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK) staining was also increased in the sinusoidal lining cells within livers of alcohol-consuming versus water only P rats. In addition, cytochrome p450IIE1 (CYP2E1) mRNA, protein expression/activity, and intrahepatic oxidative stress were significantly increased in alcohol-consuming P rat livers versus water only. In contrast, acetaldehyde dehydrogenase expression decreased in alcohol-consuming versus water only P rats. No significant difference in alcohol dehydrogenase expression was detected. CONCLUSIONS: These data demonstrate that chronic alcohol consumption is associated with hepatic neoplasia, MAPK/ERK activation, increased CYP2E1 activity, and intrahepatic oxidative stress in P rats. As these rats are well characterized as a model of alcoholism, these findings identify a novel rodent model of alcohol or "alcoholism"-induced liver neoplasia.
Assuntos
Consumo de Bebidas Alcoólicas/patologia , Carcinoma Hepatocelular/induzido quimicamente , Etanol/toxicidade , Neoplasias Hepáticas/induzido quimicamente , Animais , Carcinoma Hepatocelular/patologia , Citocromo P-450 CYP2E1/metabolismo , Etanol/administração & dosagem , Neoplasias Hepáticas/patologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Animais , Estresse Oxidativo/efeitos dos fármacos , RatosRESUMO
Provoked by sterile/nonsterile insults, prolonged monocyte mobilization and uncontrolled monocyte/macrophage activation can pose imminent or impending harm to the affected organs. Curiously, folate receptor beta (FRß), with subnanomolar affinity for the vitamin folic acid (FA), is upregulated during immune activation in hematopoietic cells of the myeloid lineage. This phenomenon has inspired a strong interest in exploring FRß-directed diagnostics/therapeutics. Previously, we have reported that FA-targeted aminopterin (AMT) therapy can modulate macrophage function and effectively treat animal models of inflammation. Our current investigation of a lead compound (EC2319) leads to discovery of a highly FR-specific mechanism of action independent of the root causes against inflammatory monocytes. We further show that EC2319 suppresses interleukin-6/interleukin-1ß release by FRß+ monocytes in a triple co-culture leukemic model of cytokine release syndrome with anti-CD19 chimeric antigen receptor T cells. Because of its chemical stability and metabolically activated linker, EC2319 demonstrates favorable pharmacokinetic characteristics and cross-species translatability to support future pre-clinical and clinical development.
Assuntos
Aminopterina/farmacologia , Síndrome da Liberação de Citocina/prevenção & controle , Receptor 2 de Folato/genética , Antagonistas do Ácido Fólico/farmacologia , Ácido Fólico/metabolismo , Macrófagos/efeitos dos fármacos , Animais , Antígenos CD19/genética , Antígenos CD19/imunologia , Células CHO , Cricetulus , Síndrome da Liberação de Citocina/genética , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Feminino , Receptor 1 de Folato/antagonistas & inibidores , Receptor 1 de Folato/genética , Receptor 1 de Folato/imunologia , Receptor 2 de Folato/antagonistas & inibidores , Receptor 2 de Folato/imunologia , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Modelos Biológicos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/patologia , Células RAW 264.7 , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The extracellular signal-regulated (ERK), mitogen-activated protein kinase (p42/p44 MAPK) pathway is up-regulated in hepatocellular carcinoma (HCC). Molecular targeting of this critical mitogenic pathway may have therapeutic potential for the treatment of HCC; however, chemoresistance to long-term therapy may develop. In the present study, we employed small-molecule MAPK kinase (MEK) inhibitors, including U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] and PD184161 (Neoplasia 8:1-8, 2006), in HepG2 and Hep3B human HCC cell lines to identify potential mechanism(s) of resistance. U0126 dose-dependently suppressed ERK phosphorylation at both 1- and 24-h time points in HepG2 cells, previously shown to be sensitive to growth inhibition by U0126. In contrast, ERK phosphorylation was only decreased at the 1-h time point but not at 24 h in the more resistant Hep3B cells. It is interesting that the lack of prolonged phospho-ERK suppression was associated with MEK hyperphosphorylation in Hep3B cells. Several MEK/ERK pathway intermediates were up-regulated in Hep3B cells; furthermore, transfection of Raf-1 small interfering RNA to suppress MEK/ERK pathway activation sensitized Hep3B cells to U0126. MEK inhibitor resistance was independent of p53 or hepatitis Bx protein status. Finally, we showed that combining two chemically distinct MEK inhibitors enhanced growth inhibition and apoptosis compared with the single agents. Taken together, these results suggest that up-regulated expression or activity of the MEK/ERK pathway contributes to MEK inhibitor resistance in HCC cells. Our findings also provide preclinical evidence suggesting that the status of the MEK/ERK pathway in patients may predict response to MEK/ERK-targeted therapeutics.
Assuntos
Carcinoma Hepatocelular/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Regulação para Cima/fisiologia , Compostos de Anilina/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Butadienos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas ras/metabolismoRESUMO
Chimeric antigen receptor (CAR)-T cell therapy has transformed pediatric oncology by producing high remission rates and potent effects in CD19+ B-cell malignancies. This scenario is ideal as CD19 expression is homogeneous and human blood provides a favorable environment for CAR-T cells to thrive and destroy cancer cells (along with normal B cells). Yet, CAR-T cell therapies for solid tumors remain challenged by fewer tumor targets and poor CAR-T cell performances in a hostile tumor microenvironment. For acute myeloid leukemia and childhood solid tumors such as osteosarcoma, the primary treatment is systemic chemotherapy that often falls short of expectation especially for relapsed and refractory conditions. We aim to develop a CAR-T adaptor molecule (CAM)-based therapy that uses a bispecific small-molecule ligand EC17, fluorescein isothiocyanate (FITC) conjugated with folic acid, to redirect FITC-specific CAR-T cells against folate receptor (FR)-positive tumors. As previously confirmed in rodents as well as in human clinical studies, EC17 penetrates solid tumors within minutes and is retained due to high affinity for the FR, whereas unbound EC17 rapidly clears from the blood and from receptor-negative tissues. When combined with a rationally designed CAR construct, EC17 CAM was shown to trigger CAR-modified T cell activation and cytolytic activity with a low FR threshold against tumor targets. However, maximal cytolytic potential correlated with (i) functional FR levels (in a semi-log fashion), (ii) the amount of effector cells present, and (iii) tumors' natural sensitivity to T cell mediated killing. In tumor-bearing mice, administration of EC17 CAM was the key to drive CAR-T cell activation, proliferation, and persistence against FR+ pediatric hematologic and solid tumors. In our modeling systems, cytokine release syndrome (CRS) was induced under specific conditions, but the risk of severe CRS could be easily mitigated or prevented by applying intermittent dosing and/or dose-titration strategies for the EC17 CAM. Our approach offers the flexibility of antigen control, prevents T cell exhaustion, and provides additional safety mechanisms including rapid reversal of severe CRS with intravenous sodium fluorescein. In this paper, we summarize the translational aspects of our technology in support of clinical development.
RESUMO
Hepatocellular carcinoma (HCC) causes 600,000 mortalities per year worldwide. Previous studies from our lab provide evidence for altered mitogen-activated protein kinase and extracellular signal-regulated kinase kinase (MEK) signaling in HCC pathogenesis. We hypothesized that pharmacologic targeting of MEK may prevent HCC. Transforming growth factor-alpha-transgenic mice (CD1-MT42) exposed to diethylnitrosamine were randomized to 20 (trial I) or 35 (trial II) weeks of MEK inhibitor PD0325901 (1, 10 mg/kg) or control via orogastric gavage. Ten HCC (44%) formed in trial I controls versus 0 in treatment arms (p<0.05). Fourteen HCC (50%) formed in trial II controls versus 1 (9%) in treatment arms (p<0.05). Mean HCC volume was 578 mm3 in control versus 46 mm3 in the single tumor formed in trial II. In trial I, foci of altered hepatocytes (FAH) formed in 78% of control versus 40% and 0% (1 and 10 mg/kg PD0325901) in treatment arms (p<0.05). In trial II, incidence of FAH was 80% in control versus 20% and 50% (1 and 10 mg/kg PD0325901) in treatment arms (p<0.05). Hepatocyte expression of phosphorylated extracellular signal-regulated kinase dose-dependently decreased in trial I but remained the same in trial II. Control and treated HCC demonstrated similar proliferation rates, but apoptosis appeared increased with treatment. MEK targeting is effective HCC chemoprevention, perhaps by lowering the apoptotic threshold.
Assuntos
Benzamidas/uso terapêutico , Carcinoma Hepatocelular/prevenção & controle , Quimioprevenção/métodos , Difenilamina/análogos & derivados , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Neoplasias Hepáticas Experimentais/prevenção & controle , Fator de Crescimento Transformador alfa/metabolismo , Alquilantes/toxicidade , Animais , Apoptose/efeitos dos fármacos , Benzamidas/administração & dosagem , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Dietilnitrosamina/toxicidade , Difenilamina/administração & dosagem , Difenilamina/uso terapêutico , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Seguimentos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos TransgênicosRESUMO
PURPOSE: The purpose was to determine the safety and antitumor activity of a folate-tubulysin conjugate (EC0531) in a relevant preclinical animal model, dogs with naturally-occurring invasive urothelial carcinoma (iUC). Canine iUC is an aggressive cancer with high folate receptor (FR) expression similar to that in certain forms of human cancer. EXPERIMENTAL DESIGN: A 3+3 dose escalation study of EC0531 (starting dose 0.2 mg/kg given intravenously at two-week intervals) was performed in dogs with iUC expressing high levels of FRs (>50% positive tumor cells). Pharmacokinetic (PK) analysis was performed, and the maximum tolerated dose (MTD) was determined. The dose cohort at the MTD was expanded to determine antitumor activity. RESULTS: The MTD of EC0531 was 0.26 mg/kg every two weeks, with grade 3-4 neutropenia and gastrointestinal toxicity observed at higher doses. Treatment at the MTD was well tolerated. Clinical benefit was found in 20 of 28 dogs (71%), including three dogs with partial remission and 17 dogs with stable disease. Plasma EC0531 concentrations in the dogs far exceeded those required to inhibit proliferation of FR-expressing cell in vitro. Unlike human neutrophils, canine neutrophils were found to express FRs, which contributes to the neutropenia at higher doses of EC0531 in dogs. CONCLUSION: EC0531 was well tolerated and had good antitumor activity in dogs with iUC. It is likely that humans will tolerate higher, potentially more effective doses of folate-tubulysin without myelotoxicity because of the absence of FRs on human neutrophils. The results clearly justify the evaluation of folate-tubulysin in human clinical trials.
RESUMO
Chemotherapy to date has not been effective in the treatment of human hepatocellular carcinoma. More effective treatment strategies may involve combinations of agents with activity against hepatocellular carcinoma. Parthenolide, a nuclear factor-kappaB (NF-kappaB) inhibitor, and NS398, a cyclooxygenase (COX)-2 inhibitor, have been shown to individually suppress the growth of hepatocellular carcinoma cells in vitro. To investigate their effects in combination, three human hepatocellular carcinoma lines (Hep3B, HepG2, and PLC) were treated with parthenolide and/or NS398. Parthenolide (0.1-10 micromol/L) and NS398 (1-100 micromol/L) each caused concentration-dependent growth inhibition in all cell lines. The addition of parthenolide to NS398 reduced the concentration of NS398 required to inhibit hepatocellular carcinoma growth. Because parthenolide and COX-2 inhibitors have been reported to influence NF-kappaB activity, the effects on this pathway were investigated. The combination of parthenolide/NS398 inhibited phosphorylation of the NF-kappaB-inhibitory protein IkappaBalpha and increased total IkappaBalpha levels. NF-kappaB DNA-binding and transcriptional activities were inhibited more by the combination than the single agents in Hep3B and HepG2 cells but not in PLC cells. The response of PLC cells to NS398 was augmented by p65 small interfering RNA to inhibit NF-kappaB p65 protein expression. The combination of parthenolide/NS398 increased apoptosis only in PLC cells, suggesting that the combination may decrease the apoptotic threshold in these cells. In Hep3B and HepG2 cells, combination treatment with NS398/parthenolide altered the cell cycle distribution resulting in more G0-G1 accumulation. Cyclin D1 levels were further decreased by combination treatment in all cell lines, correlating with the cell cycle alterations. Our results suggest that parthenolide may be effective in combination with COX-2 inhibitors for the treatment of hepatocellular carcinoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Interfase/efeitos dos fármacos , Nitrobenzenos/farmacologia , Sesquiterpenos/farmacologia , Sulfonamidas/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Proteínas de Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Proteínas de Ligação a DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Proteínas I-kappa B/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Nitrobenzenos/uso terapêutico , Sesquiterpenos/uso terapêutico , Sulfonamidas/uso terapêutico , Transcrição Gênica/efeitos dos fármacosRESUMO
PURPOSE: EC0305 represents a folate-tubulysin B construct capable of specifically eradicating folate receptor (FR)-positive subcutaneous tumors from mice (Leamon et al., Cancer Res 68:9839-9844, 8). Herein we report on the use of multiple polar carbohydrate segments (e.g. 1-amino-1-deoxy-glucitolyl-γ-glutamate) placed in-between the folate and tubulysin B moieties of EC0305 creating a new conjugate, herein referred to as EC0531, with more desirable biological properties. METHODS: The synthesis of EC0531 and its tritium-labeled counterpart are described. EC0531's affinity for FR binding and specific cytotoxic activity was assessed using standard in vitro assays. Human tumor xenografts were used to directly compare EC0305 and EC0531's antitumor activity. Finally, bile duct cannulated, female Sprague-Dawley rats were used to compare hepatobiliary clearance of these two targeted chemotherapeutic agents. RESULTS: EC0531 tightly binds to the FR with an affinity about half that of folic acid. It was found to specifically inhibit the growth of FR+ cells (IC50 of ~2 nM) in a dose-dependent manner. Using 3H-labeled compounds, more than a 12-fold higher amount of tubulysin was measured in a FR + human tumor xenograft compared to the unconjugated drug, a finding that explains, in part, why EC0531 displays curative activity, whereas the unconjugated tubulysin agent is essentially inactive. EC0531 was found to produce greater FR-specific anti-tumor activity at lower dose levels than EC0305; furthermore, EC0531's maximum tolerated dose level was significantly higher than that of EC0305, likely because EC0531's saccharopeptidic-based spacer allows for ~sixfold reduction in hepatic clearance. CONCLUSIONS: These data provide additional evidence that the therapeutic range of targeted small-molecule drug conjugates can be favorably increased using molecular spacers constructed with 1-amino-1-deoxy-glucitolyl-γ-glutamate residues.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Receptor 1 de Folato/efeitos dos fármacos , Ácido Fólico/análogos & derivados , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ácidos Pipecólicos/química , Animais , Antineoplásicos/farmacocinética , Bile/metabolismo , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Feminino , Ácido Fólico/metabolismo , Ácido Fólico/farmacologia , Humanos , Marcação por Isótopo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aflatoxin B1 (AFB1) is a potent dietary hepatocarcinogen in animals and probably in humans. Mutations (and altered expression) of the tumor suppresser gene p53 have been observed in liver tumors from patients exposed to high dietary AFB1. Inhalation of AFB1-laden grain dusts has been associated with an increased incidence of lung cancer in humans as well. We examined the effects of low concentrations of AFB1 on the expression of p53 and MDM2 in human bronchial epithelial cells (BEAS-2B) transfected with cDNA for either cytochrome P450 (CYP) 1A2 (B-CMV1A2) or CYP 3A4 (B3A4), two isozymes that are responsible for AFB1 activation in human liver and possibly the lung. Untreated B-CMV1A2 and B3A4 cells constitutively expressed p53. Exposure to a range (0.015-15 microM for 30 min) of AFB1 concentrations caused a concentration-dependent decline in p53 expression in B-CMV1A2 cells, and to a lesser extent, in B3A4 cells. The AFB1-mediated decrease in p53 continued for at least 12 h after 30-min exposures to 1.5 muM AFB(1). Mirroring the decrease in p53 expression was a concentration-dependent increase in the expression of the 76-kDa MDM2 isoform in B-CMV1A2 and B-3A4 cells. Interestingly, AFB1 did not induce DNA laddering, an indicator of apoptotic cell death, but proteolytic activation of caspase-3 was detected in AFB1-treated B-CVM1A2 cells. In total, these data show that low, environmentally-relevant concentrations of AFB1 alter the expression of p53 and MDM2 in these human lung cells, and that cells that stably express CYP 1A2 were more susceptible to this effect than nontransfected, or 3A4-expressing cells.
Assuntos
Aflatoxina B1/toxicidade , Apoptose/efeitos dos fármacos , Carcinógenos Ambientais/toxicidade , Sistema Enzimático do Citocromo P-450/biossíntese , Pulmão/efeitos dos fármacos , Proteína Supressora de Tumor p53/biossíntese , Western Blotting , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Pulmão/citologia , Pulmão/metabolismo , Fatores de TempoRESUMO
EC1456 is a novel folate-targeted small molecule drug conjugate of tubulysin B hydrazide being developed as an anticancer agent for patients with advanced solid tumors expressing the folate receptor. To try and correlate circulating systemic levels of EC1456 and its metabolites to tumor concentrations and potentially develop a PK/PD model, a sensitive bioanalytical method was developed and validated for the quantitation of the analytes in KB tumor homogenates. The method involved homogenizing tumors with buffer containing N-maleoyl-ß-alanine, mannitol and acetic acid, precipitation of the homogenate with acetone followed by heating at 55°C for 1h to convert tubulysin B hydrazide to its corresponding hydrazone. The extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method demonstrated good inter-day (3 runs, n=18) accuracy (-2.3% to 7.3%) and precision (1.7% to 10.3%) for all three analytes. Stability was established for three freeze-thaw cycles, 4h on the bench-top on ice, 20h in the autosampler at 8°C and for at least 46days frozen at -70°C. This method was successfully used to determine concentration of EC1456 and its metabolites tubulysin B hydrazide and tubulysin B in tumor homogenates in preliminary experiments with KB tumor bearing mice dosed intravenously with EC1456.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácido Fólico/metabolismo , Neoplasias/metabolismo , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Espectrometria de Massas em Tandem/métodos , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oligopeptídeos/metabolismo , Ácidos Pipecólicos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
1,3-Dichloropropene (1,3-D) is a soil fumigant used primarily for preplanting control of parasitic nematodes. In a previous chronic dietary exposure study, 1,3-D induced an increased incidence of hepatocellular adenomas in male rats at a dose of 25 mg/kg/day. Although the mechanism for tumor induction in the rat liver by 1,3-D has not been specifically elucidated, available data suggested that the observed liver tumorigenesis was through a nongenotoxic mode of action at the tumor promotion stage. Fischer 344 rats containing preneoplastic lesions were treated (via gavage) with 25 mg/kg/day 1,3-D or 80 mg/kg/day phenobarbital (PB) for 30 days and 60 days, or for 30 days followed by a 30-day recovery period (no compound exposure). Following treatment, placental form glutathione S-transferase (GSTP) positive and GSTP-negative liver focal lesions were quantitated as to size and number. 1,3-D treatment had no effect on GSTP-positive foci number or relative size but significantly increased the number, labeling index, and relative size of GSTP-negative focal lesions (identified by H and E staining) after 30 and 60 days of treatment. Following the 30-day recovery period, the number, labeling index, and relative size of the GSTP-negative lesions in 1,3-D-treated animals returned to control levels. As expected, PB treatment produced an increase in number and relative size of the GSTP-positive lesions. The results of this study are consistent with 1,3-D inducing liver carcinogenesis through a nongenotoxic mode of action by functioning as a tumor promoter specifically through induction of a non-GSTP staining focal hepatocyte population.
Assuntos
Adenoma de Células Hepáticas/induzido quimicamente , Compostos Alílicos/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Neoplasias Hepáticas/induzido quimicamente , Fígado/efeitos dos fármacos , Lesões Pré-Cancerosas/induzido quimicamente , Adenoma de Células Hepáticas/metabolismo , Adenoma de Células Hepáticas/patologia , Animais , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Replicação do DNA/efeitos dos fármacos , Glutationa/metabolismo , Glutationa S-Transferase pi/metabolismo , Hidrocarbonetos Clorados , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fenobarbital/toxicidade , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Ratos Endogâmicos F344 , Medição de Risco , Fatores de TempoRESUMO
The mycotoxin aflatoxin B(1) (AFB(1)) is a hepatocarcinogen in many animal models and probably a human carcinogen. Besides being a dietary carcinogen, AFB(1) has been detected in dusts generated in the processing and transportation of AFB(1)-contaminated products. Inhalation of grain dusts contaminated with AFB(1) may be a risk factor in human lung cancer. Aflatoxin B(1) requires cytochrome P-450 (CYP)-mediated activation to form cytotoxic and DNA-reactive intermediates, and this activation in human liver is mediated by the CYP 1A2 and 3A4 isoforms. Which isoforms are important in AFB(1) activation in human lung is not well understood. To investigate whether these CYPs can activate AFB(1) at low, environmentally relevant concentrations in human lung cells, SV40 immortalized human bronchial epithelial cells (BEAS-2B) that were transfected with cDNA for CYPs 3A4 (B3A4) or 1A2 (B-CMV1A2) were used. B-CMV1A2 cultured in 15 nM AFB(1) produced the AFB(1)-glutathione conjugate (AFB(1)-GSH) and aflatoxin M(1) (AFM(1)), while B3A4 cells produced only aflatoxin Q(1) (AFQ(1)) at 0.15 microM AFB(1). Nontransfected BEAS-2B cells produced no metabolites, even at 1.5 mM AFB(1). Microsomes prepared from B-CMV1A2 and B3A4 cells activated AFB(1) to AFB(1) 8,9-epoxide (AFBO), while those from BEAS-2B cells did not produce AFBO. Cytosol from all three cell types was ineffective at glutathione S-transferase (GST)-mediated trapping of enzymatically generated AFB(1) 8,9-epoxide. B-CMV1A2 cells were 100-fold more sensitive to AFB(1) compared to B3A4 cells, and were 6000-fold more sensitive than control BEAS-2B cells. Western immunoblots confirmed that only B-CMV1A2 cells expressed CYP 1A2 protein, while CYP 3A4 was only in B3A4 cells. B-CMV1A2 cells were the most sensitive to AFB(1), followed by B3A4 cells. CYP 3A4, which has been predicted to activate AFB(1) primarily at higher AFB(1) concentrations, was also responsible for significant AFB(1) toxicity at low concentrations. These data indicate that human lung cells expressing these CYP isoforms are capable of activating AFB(1), even at environmentally relevant concentrations.
Assuntos
Aflatoxina B1/metabolismo , Aflatoxina B1/toxicidade , Citocromo P-450 CYP1A2/farmacologia , Sistema Enzimático do Citocromo P-450/farmacologia , Pulmão/citologia , Oxigenases de Função Mista/farmacologia , Western Blotting , Técnicas de Cultura de Células , Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar , Células Epiteliais , Humanos , Isoenzimas , Pulmão/efeitos dos fármacos , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , TransfecçãoRESUMO
Folate receptors (FR) may be of use for targeted delivery of cytotoxic drugs in invasive urothelial carcinoma (iUC), for which improved therapy is needed. FR expression and function in iUC were explored and the antitumor activity and toxicity of a folate-targeted vinblastine conjugate were evaluated in dogs with naturally occurring iUC, an excellent model for human iUC. FR immunohistochemistry was carried out on iUC and normal human and dog bladder tissues together with nuclear scintigraphy in dogs to monitor iUC folate uptake. Dose escalation of a folate-targeted vinblastine compound, EC0905, was conducted in dogs with biopsy-confirmed, FR-positive iUC. FRs were detected by immunohistochemistry (PU17) in most primary iUC and many nodal and lung metastases from dogs, and scintigraphy confirmed folate uptake in both primary and metastatic lesions. The maximum tolerated dose of EC0905 in dogs was 0.25 mg/kg IV weekly, with neutropenia at higher doses. Tumor responses included partial remission (≥ 50% reduction in tumor volume) in five dogs and stable disease (<50% change in tumor volume) in four dogs. Immunoreactivity to PU17 was similar in humans (78% of primary iUC, 80% of nodal metastases). Less immunoreactivity to mab343 (22% of cases) occurred. FR-ß was noted in 21% of human iUC cases. Our findings suggest folate-targeted therapy holds considerable promise for treating iUC, where FR-ß may be important in addition to FR-α.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células de Transição/tratamento farmacológico , Receptores de Folato com Âncoras de GPI/antagonistas & inibidores , Neoplasias da Bexiga Urinária/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Cães , Feminino , Ácido Fólico/análogos & derivados , Ácido Fólico/uso terapêutico , Ácido Fólico/toxicidade , Humanos , Masculino , Dose Máxima Tolerável , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Projetos Piloto , Vimblastina/análogos & derivados , Vimblastina/uso terapêutico , Vimblastina/toxicidadeRESUMO
PURPOSE: EC145 (vintafolide), a conjugate of folic acid and the vinca alkaloid desacetylvinblastine hydrazide (DAVLBH), is a ligand for the folate receptor (FR), with activity against FR-positive tumor xenografts in vivo. This phase I study determined the maximum-tolerated dose (MTD) of EC145 administered as a bolus intravenous injection or 1-hour infusion in patients with refractory solid tumors. PATIENTS AND METHODS: EC145 was administered as a bolus injection or 1-hour infusion on days 1, 3, and 5 and days 15, 17, and 19 of each 28-day cycle with dose escalation in cohorts of three to six patients until the MTD was identified. Plasma pharmacokinetics were determined on days 1 and 3 of the first cycle. RESULTS: The MTD of EC145 was 2.5 mg when administered as either a bolus injection or 1-hour infusion. Constipation was the dose-limiting toxicity with both routes. Constipation, nausea, fatigue, and vomiting were the most commonly reported adverse events. One partial response to therapy was observed in a patient with metastatic ovarian cancer. CONCLUSION: EC145 administered by bolus injection or as a 1-hour infusion at a dose of 2.5 mg on days 1, 3, and 5 and days 15, 17, and 19 of a 28-day cycle has an acceptable safety profile in patients with advanced cancer. On the basis of these findings, phase II studies of EC145 have been initiated in patients with advanced epithelial ovarian cancer and non-small-cell lung cancer.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Ácido Fólico/análogos & derivados , Dose Máxima Tolerável , Neoplasias/tratamento farmacológico , Alcaloides de Vinca/administração & dosagem , Alcaloides de Vinca/efeitos adversos , Adulto , Idoso , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Constipação Intestinal/induzido quimicamente , Esquema de Medicação , Resistencia a Medicamentos Antineoplásicos , Fadiga/induzido quimicamente , Feminino , Ácido Fólico/administração & dosagem , Ácido Fólico/efeitos adversos , Ácido Fólico/sangue , Ácido Fólico/farmacocinética , Humanos , Infusões Intravenosas , Injeções Intravenosas , Masculino , Pessoa de Meia-Idade , Náusea/induzido quimicamente , Neoplasias/sangue , Índice de Gravidade de Doença , Alcaloides de Vinca/sangue , Alcaloides de Vinca/farmacocinética , Vômito/induzido quimicamenteRESUMO
INTRODUCTION: Folate receptor (FR)-expressing macrophages have been shown to accumulate at sites of inflammation, where they promote development of inflammatory symptoms. To target such a macrophage population, we designed and evaluated the biologic activity of EC0746, a novel folic acid conjugate of the highly potent antifolate, aminopterin. METHODS: Using a FR-positive subclone of murine macrophage-derived RAW264.7 cells and rat thioglycollate-elicited macrophages, we studied the effect of EC0746 on dihydrofolate reductase activity, cell proliferation, and cellular response towards bacterial lipopolysaccharide as well as IFNγ activation. The EC0746 anti-inflammatory activity, pharmacokinetics, and toxicity were also evaluated in normal rats or in rats with adjuvant-induced arthritis; that is, a FR-positive macrophage model that closely resembles rheumatoid arthritis in humans. RESULTS: EC0746 suppresses the proliferation of RAW264.7 cells and prevents the ability of nonproliferating rat macrophages to respond to inflammatory stimuli. In the macrophage-rich rat arthritis model, brief treatment with subcutaneously administered EC0746 is shown to mediate an FR-specific anti-inflammatory response that is more potent than either orally administered methotrexate or subcutaneously delivered etanercept. More importantly, EC0746 therapy is also shown to be ~40-fold less toxic than unmodified aminopterin, with fewer bone marrow and gastrointestinal problems. CONCLUSIONS: EC0746 is the first high FR-binding dihydrofolate reductase inhibitor that demonstrates FR-specific anti-inflammatory activities both in vitro and in vivo. Our data reveal that a relatively toxic anti-inflammatory drug, such as aminopterin, can be targeted with folic acid to inflammatory macrophages and thereby relieve inflammatory symptoms with greatly reduced toxicity.
Assuntos
Aminopterina/análogos & derivados , Aminopterina/farmacologia , Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Antagonistas do Ácido Fólico/farmacologia , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacologia , Aminopterina/síntese química , Aminopterina/farmacocinética , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacocinética , Feminino , Receptores de Folato com Âncoras de GPI/efeitos dos fármacos , Ácido Fólico/síntese química , Ácido Fólico/farmacocinética , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacocinética , Macrófagos/efeitos dos fármacos , Camundongos , Ratos , Ratos Endogâmicos LewRESUMO
The clinical pharmacokinetics and exposure-toxicity relationship were determined for EC145, a conjugate of folic acid and the Vinca alkaloid desacetylvinblastine hydrazide (DAVLBH), in cancer patients. EC145 plasma concentration and toxicity data were obtained from a first-in-man phase I study and analyzed by nonlinear mixed effect modeling with NONMEM. EC145 concentration-time profile after intravenous administration was well described by a 2-compartment model with a first-order elimination process from the central compartment. BSA was identified as a significant covariate on EC145 clearance, accounting for 14.6% of interindividual variation on EC145 clearance. Population estimates for the clearance, steady-state volume of distribution, distribution, and elimination half-lives were 56.1 L/h, 26.1 L, 6 minutes, and 26 minutes, respectively. Constipation and peripheral neuropathy were the most common and clinically relevant toxicities. The clearance and area under the concentration-time curve (AUC) were significant predictors for the incidence of EC145-induced constipation but not peripheral neuropathy. In conclusion, EC145 is rapidly distributed and eliminated in cancer patients. BSA is a statistically significant covariate on EC145 clearance, but its clinical relevance remains to be defined. EC145-induced constipation occurs at a higher frequency in the patients with lower EC145 clearance, where the drug exposure tends to be higher.
Assuntos
Ácido Fólico/análogos & derivados , Neoplasias/metabolismo , Alcaloides de Vinca/efeitos adversos , Alcaloides de Vinca/farmacocinética , Adulto , Idoso , Superfície Corporal , Proteínas de Transporte/metabolismo , Constipação Intestinal/induzido quimicamente , Feminino , Receptores de Folato com Âncoras de GPI , Ácido Fólico/administração & dosagem , Ácido Fólico/efeitos adversos , Ácido Fólico/sangue , Ácido Fólico/farmacocinética , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Dinâmica não Linear , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Receptores de Superfície Celular/metabolismo , Vimblastina/análogos & derivados , Vimblastina/sangue , Alcaloides de Vinca/administração & dosagem , Alcaloides de Vinca/sangueRESUMO
Cancer vaccine/immunotherapy rarely involves systemic administration of an immunogenic compound to an actively immunized host. We have developed such a strategy that utilizes folate to deliver antigenic haptens [e.g., fluorescein (FITC) and dinitrophenyl] to folate receptor-positive tumors in a hapten-pre-vaccinated host. Here, we investigated the safety of this novel approach and developed strategies to prevent drug-related hypersensitivity. Using FITC as the model hapten, we identified a potential source of allergic species in folate-FITC preparations by LC-MS/MS. In mice and guinea pigs, we tested the significance of this impurity by passive cutaneous anaphylaxis and active systemic anaphylaxis assays. We studied the effect of immunogen (e.g., KLH-FITC) dose and derived a desensitization regimen that was further evaluated in a murine tumor model. Administration of folate-FITC with low multi-haptenated contaminants (e.g. bis-FITC) resulted in hypersensitivity in underimmunized animals. However, this drug-related hypersensitivity may be independently prevented by (1) increasing the immunogen dose and/or (2) desensitizing animals with folate-FITC during vaccination. In addition, such manipulation in vivo did not appear to negatively alter the effectiveness of immunotherapy. This study provided confidence on the safety of folate-hapten-targeted cancer immunotherapy in an actively immunized host.
Assuntos
Hipersensibilidade a Drogas/prevenção & controle , Antagonistas do Ácido Fólico/uso terapêutico , Haptenos/uso terapêutico , Imunoterapia , Neoplasias/terapia , Animais , Linhagem Celular Tumoral , Feminino , Antagonistas do Ácido Fólico/efeitos adversos , Cobaias , Haptenos/efeitos adversos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológicoRESUMO
Herein we report on the potencies of 4 related folate-conjugated tubulysins constructed with either tubulysin B hydrazide (EC0305), tubulysin A hydrazide (EC0510), the N,O-acetal derivative of natural tubulysins (EC0317) or a tubulysin B ester (EC0302). Our results confirmed that EC0305 is the most favorable conjugate of the group due to its potent antitumor activity [100% cures at 1 micromol/kg, three times a week (TIW) for 2 weeks] and its favorably low toxicity profile. In contrast, the natural tubulysin B drug proved to be inactive against a human nasopharyngeal tumor model when administered at doses near to or greater than the maximum tolerated dose (MTD). When tested against more chemoresistant folate receptor expressing M109 and 4T1-cl2 tumors, EC0305 displayed superior antitumor activity over a previously disclosed folate conjugate of desacetylvinblastine monohydrazide (EC145). These studies demonstrate that EC0305 has significant antiproliferative activity against FR expressing tumors, including those which are generally more chemoresistant, and that EC0305 should be considered for development as a candidate for the treatment of advanced FR-expressing human cancers.