RESUMO
Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Células-Tronco Hematopoéticas , Humanos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Regulação da Expressão Gênica , Hematopoese/genética , Cromatina/metabolismoRESUMO
Principal components analysis (PCA), linear discriminant analysis (LDA), and quadratic discriminant analysis (QDA) were used to develop a multistep classification procedure for determining the presence of ignitable liquid residue in fire debris and assigning any ignitable liquid residue present into the classes defined under the American Society for Testing and Materials (ASTM) E 1618-10 standard method. A multistep classification procedure was tested by cross-validation based on model data sets comprised of the time-averaged mass spectra (also referred to as total ion spectra) of commercial ignitable liquids and pyrolysis products from common building materials and household furnishings (referred to simply as substrates). Fire debris samples from laboratory-scale and field test burns were also used to test the model. The optimal model's true-positive rate was 81.3% for cross-validation samples and 70.9% for fire debris samples. The false-positive rate was 9.9% for cross-validation samples and 8.9% for fire debris samples.
RESUMO
Graft-versus-host disease (GVHD) is a major contributor to transplant-related mortality and morbidity after allogeneic stem cell transplantation. Despite advancements in tissue-typing techniques, conditioning regimens, and therapeutic intervention, the incidence rate of GVHD remains high. GVHD is caused by alloreactive donor T cells that infiltrate and destroy host tissues (e.g., skin, liver, and gut). Therefore, GVHD is prevented and treated with therapeutics that suppress proinflammatory cytokines and T-cell function (e.g., cyclosporine, glucocorticoids). Here we report that the small molecule inhibitor of glycogen synthase kinase 3, 6-bromoindirubin 3'-oxime (BIO), prevents lethal GVHD in a humanized xenograft model in mice. BIO treatment did not affect donor T-cell engraftment, but suppressed their activation and attenuated bone marrow and liver destruction mediated by activated donor T cells. Glycogen synthase kinase 3 inhibition modulated the Th1/Th2 cytokine profile in vitro and suppressed activation of signal transducers and activators of transcription 1 and 3 signaling pathways both in vitro and in vivo. Importantly, human T cells derived from BIO-treated mice were able to mediate anti-tumor effects in vitro, and BIO did not affect stem cell engraftment and multilineage reconstitution in a mouse model of transplantation. These data demonstrate that inhibition of glycogen synthase kinase 3 can potentially abrogate GVHD without compromising the efficacy of transplantation.
Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Doença Enxerto-Hospedeiro/prevenção & controle , Indóis/farmacologia , Oximas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Transplante de Células-Tronco Hematopoéticas , Humanos , Indóis/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Oximas/uso terapêutico , Fosforilação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos T/imunologiaRESUMO
Glycogen synthase kinase-3ß (GSK-3ß) has been identified as an important regulator of stem cell function acting through activation of the wingless (Wnt) pathway. Here, we report that treatment with an inhibitor of GSK-3ß, 6-bromoindirubin 3'-oxime (BIO) delayed cell cycle progression by increasing cell cycle time. BIO treatment resulted in the accumulation of late dividing cells enriched with primitive progenitor cells retaining the ability for sustained proliferation. In vivo analysis using a Non-obese diabetic/severe combined immunodeficient (NOD/SCID) transplantation model has demonstrated that pretreatment with BIO promotes engraftment of ex vivo-expanded hematopoietic stem cells. BIO enhanced the engraftment of myeloid, lymphoid and primitive stem cell compartments. Limiting dilution analysis of SCID repopulating cells (SRC) revealed that BIO treatment increased human chimerism without increasing SRC frequency. Clonogenic analysis of human cells derived from the bone marrow of transplant recipient mice demonstrated that a higher level of human chimerism and cellularity was related to increased regeneration per SRC unit. Gene expression analysis showed that treatment with BIO did not modulate the expression of canonical Wnt target genes upregulated during cytokine-induced cell proliferation. BIO increased the expression of several genes regulating Notch and Tie2 signaling downregulated during ex vivo expansion, suggesting a role in improving stem cell engraftment. In addition, treatment with BIO upregulated CDK inhibitor p57 and downregulated cyclin D1, providing a possible mechanism for the delay seen in cell cycle progression. We conclude that transient, pharmacologic inhibition of GSK-3ß provides a novel approach to improve engraftment of expanded HSC after stem cell transplantation.
Assuntos
Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Indóis/farmacologia , Oximas/farmacologia , Proteínas Wnt/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimerismo/efeitos dos fármacos , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Sangue Fetal/citologia , Citometria de Fluxo , Glicogênio Sintase Quinase 3 beta , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transdução de Sinais/genéticaRESUMO
OBJECTIVE: The objective of this study was to investigate the effect of small molecule inhibitors of glycogen synthase kinase-3ß (GSK-3ß) on leukemia cell growth and survival. MATERIALS AND METHODS: Analysis of cytotoxicity and cell proliferation was conducted using the MTS assay, cell-cycle analysis, and division tracking. Apoptosis was investigated by Annexin-V/7-aminoactinomycin D and caspase-3 expression. The effect of GSK-3ß inhibitors was also tested in vivo in an animal model of leukemia. Gene expression analysis was performed to identify the genes modulated by GSK-3ß inhibition in leukemia cells. RESULTS: GSK-3ß inhibitors suppress cell growth and induce apoptosis in seven leukemia cell lines of diverse origin, four acute myeloid leukemia, one myelodysplastic syndrome, and one acute lymphoblastic leukemia samples. GSK-3ß inhibitors are cytotoxic for rapidly dividing clonogenic leukemia blasts, and higher doses of the inhibitors are needed to eliminate primitive leukemia progenitor/stem cells. Slow cell-division rate, low drug uptake, and interaction with bone marrow stroma make leukemia cells more resistant to apoptosis induced by GSK-3ß inhibitors. Global gene expression analysis combined with functional approaches identified multiple genes and specific signaling pathways modulated by GSK-3ß inhibition. An important role for Bcl2 in the regulation of apoptosis induced by GSK-3ß inhibitors was defined by expression analysis and confirmed by using pharmacological inhibitors of the protein. In vivo administration of GSK-3ß inhibitors delayed tumor formation in a mouse leukemia model. GSK-3ß inhibitors did not affect hematopoietic recovery following irradiation. CONCLUSIONS: Our data support further evaluation of GSK-3ß inhibitors as promising novel agents for therapeutic intervention in leukemia and warrant clinical investigation in leukemia patients.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Leucemia/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HL-60 , Humanos , Indóis/farmacologia , Células Jurkat , Células K562 , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Oximas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted alpha therapy utilizes alpha-emitting radionuclides conjugated to monoclonal antibodies to allow specific irradiation of cancer cells whilst sparing normal, healthy tissues. The mutagenic potential of (213)Bi conjugated to a human melanoma antigen-specific antibody (9.2.27) was examined using an in vivo transgenic mouse model containing multiple copies of a lacZ target gene in every cell, allowing the quantification and comparison of mutagenesis in different organs. Mice received an ip injection of 16.65 MBq of (213)Bi-cDTPA-9.2.27, and were sacrificed at 24 h, 1 w and 4 w post-injection. Pharmacokinetic studies gave the absorbed and effective doses for each organ. The mutant frequency and mutant spectra were analysed for the brain, spleen and kidneys. The brain and spleen did not show significant increases in induced mutation frequencies compared to spontaneous background levels or changes in mutant spectra, these results being independent of p53 status. However, elevated mutation frequencies and persistent size change mutations were observed in the kidneys, but are not significant at the p = 0.05 level. The effect of p53 status was also evident, as p53 heterozygotes displayed higher mutation frequencies than their wild-type counterparts, suggesting a reduction in the p53 gene may lead to an increased susceptibility to mutagenesis. These effects were time dependent and levels returned to those of the controls at 4 w post-irradiation, albeit with a predominant residue of size mutations. These effects were observed at activities very much higher than those expected for the therapy of human patients. As such, the induction of secondary cancer with the (213)Bi-cDTPA-9.2.27 alpha immunoconjugate is not expected to be a significant problem in the clinic.
Assuntos
Partículas alfa/uso terapêutico , Anticorpos Monoclonais/farmacologia , Imunoconjugados/farmacologia , Actínio/química , Animais , Anticorpos Monoclonais/farmacocinética , Encéfalo/metabolismo , Encéfalo/efeitos da radiação , Modelos Animais de Doenças , Humanos , Imunoconjugados/farmacocinética , Rim/metabolismo , Rim/efeitos da radiação , Óperon Lac , Camundongos , Camundongos Transgênicos , Mutagênese/efeitos da radiação , Baço/metabolismo , Baço/efeitos da radiação , Proteína Supressora de Tumor p53/genéticaRESUMO
PURPOSE: To investigate the therapeutic potential of 213Bilabeled multiple targeted alpha-radioimmunoconjugates for treating prostate cancer (CaP) micrometastases in mouse models. EXPERIMENTAL DESIGN: PC-3 CaP cells were implanted s.c., in the prostate, and intratibially in NODSCID mice. The expression of multiple tumor-associated antigens on tumor xenografts and micrometastases was detected by immunohistochemistry. Targeting vectors were two monoclonal antibodies, and a plasminogen activator inhibitor type 2 that binds to cell surface urokinase plasminogen activator, labeled with 213Bi using standard methodology. In vivo efficacy of multiple alpha conjugates (MTAT) at different activities was evaluated in these mouse models. Tumor growth was monitored during observations and local regional lymph node metastases were assessed at the end of experiments. RESULTS: The take rate of PC-3 cells was 100% for each route of injection. The tumor-associated antigens (MUC1, urokinase plasminogen activator, and BLCA-38) were heterogeneously expressed on primary tumors and metastatic cancer clusters at transit. A single i.p. injection of MTAT (test) at high and low doses caused regression of the growth of primary tumors and prevented local lymph node metastases in a concentration-dependent fashion; it also caused cancer cells to undergo necrosis and apoptosis. CONCLUSIONS: Our results suggest that MTAT can impede primary PC-3 CaP growth at three different sites in vivo through induction of apoptosis, and can prevent the spread of cancer cells and target lymph node micrometastases in a concentration-dependent manner. MTAT, by targeting multiple antigens, can overcome heterogeneous antigen expression to kill small CaP cell clusters, thus providing a potent therapy for micrometastases.
Assuntos
Bismuto , Imunoconjugados/uso terapêutico , Neoplasias da Próstata/terapia , Animais , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Metástase Neoplásica/prevenção & controle , Transplante de Neoplasias , Neoplasias da Próstata/patologia , Radioisótopos/uso terapêutico , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
Bevacizumab, a humanized anti-VEGF monoclonal antibody has shown promise in various clinical trials. We report the development and testing of Bi-213 (an alpha-emitting radionuclide) labeled bevacizumab for in vitro and in vivo studies using two different chelators viz cDTPA and CHX-A''. The developed labeling method showed high labeling yields of 93.6% and 89.7% for cDTPA and CHX-A'' respectively and the results were reproducible. The in vitro and in vivo stability tests were carried out using Bi-213 and long half-life Bismuth isotope (Bi-205/Bi-206) for pharmacokinetics. The in vitro results showed remarkable stability of the radiolabeled bevacizumab regardless of the chelator. The in vivo pharmacokinetics studies however, showed that the uptake and retention of cDTPA-bevacizumab was significantly higher in kidneys (p-value 0.02) and lower in liver and spleen (p-value <0.0001 and 0.0009 respectively). The values for the two conjugates compared well in blood but the longer term data for CHX-A'' conjugate showed slow clearance resulting in a significantly longer blood half-life of the product (211 hours compared to 4 hours for cDTPA-bevacizumab). Preliminary in vivo results showed increased efficacy of the combination therapy compared to bevacizumab only. The tumor area (mm(2)) decreased from 24.8 +/- 3.6 and 12.8 +/- 1.7 for 1 and 3 mg/kg cold bevacizumab only to 6.5 +/- 0.7 and 7.5 +/- 4.8 when single dose of 333 MBq/kg of (213)Bi-bevacizumab was administered as combination therapy. In conclusion it can be said that stable radiolabeled bevacizumab conjugates can be prepared with Bi-213 with either chelators used. The shorter blood half-life with cDTPA-bevacizumab may not be a major concern with Bi-213 as its own half-life is 46 minutes only. The combination therapy proved superior to bevacizumab alone therapy, a phenomenon that can be particularly useful in cancers where bevacizumab alone has shown limited success like prostate cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Bismuto/uso terapêutico , Imunoconjugados/uso terapêutico , Radioisótopos/uso terapêutico , Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Bevacizumab , Linhagem Celular Tumoral , Quelantes/farmacologia , Ensaios Clínicos como Assunto , Humanos , Ácido Pentético/farmacologia , Controle de Qualidade , Fatores de Tempo , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Ex vivo expansion of cord blood cells generally results in reduced stem cell activity in vivo. Glycogen synthase kinase-3beta (GSK-3beta) regulates the degradation of beta-catenin, a critical regulator of hematopoietic stem cells (HSCs). Here we show that GSK-3beta inhibition activates beta-catenin in cord blood CD34(+) cells and upregulates beta-catenin transcriptional targets c-myc and HoxB4, both known to regulate HSC self-renewal. GSK-3beta inhibition resulted in delayed ex vivo expansion of CD34(+) cells, yet enhanced the preservation of stem cell activity as tested in long-term culture with bone marrow stroma. Delayed cell cycling, reduced apoptosis, and increased adherence of hematopoietic progenitor cells to bone marrow stroma were observed in these long-term cultures treated with GSK-3beta inhibitor. This improved adherence to stroma was mediated via upregulation of CXCR4. In addition, GSK-3beta inhibition preserved severe combined immunodeficiency (SCID) repopulating cells as tested in the nonobese diabetic/SCID mouse model. Our data suggest the involvement of GSK-3beta inhibition in the preservation of HSC and their interaction with the bone marrow environment. Methods for the inhibition of GSK-3beta may be developed for clinical ex vivo expansion of HSC for transplantation. In addition, GSK-3beta inhibition suppressed leukemic cell growth via the induction of apoptosis mediated by the downregulation of survivin. Modulators of GSK-3beta may increase the range of novel drugs that specifically kill leukemic cells while sparing normal stem cells.
Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Células-Tronco Hematopoéticas/enzimologia , Leucemia/enzimologia , Leucemia/patologia , Animais , Antígenos CD34/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 beta , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Indóis/farmacologia , Camundongos , Camundongos SCID , Oximas/farmacologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Fatores de Tempo , Cordão Umbilical/citologia , Cordão Umbilical/efeitos dos fármacos , Cordão Umbilical/enzimologia , beta Catenina/metabolismoRESUMO
PURPOSE: Control of ovarian cancer (OC) ascites remains a major objective in post-surgical treatment. The aim of this study was to investigate the effect of targeted alpha therapy (TAT) for the control of ascites in an OC ascites mouse model; the biodistribution of (213)Bi-C595 and its long term toxicity. METHODS: The expression of tumor-associated antigen mucin-1 (MUC-1) in OVCAR3 ascites cells in mice and OC cancer tissues in patients was detected by indirect immmunostaining. The monoclonal antibody (MAb) C595 was labeled with (213)Bi using the chelator cDTPA to form the alpha-immunoconjugate (AIC). Mice were injected with different concentrations of AIC by i.p administration. Changes in tumor progression were assessed by measurement of the circumference of the abdomen. RESULTS: MUC-1 is strongly expressed in 73% of OC tissues. At 9 days post-cell inoculation in mice, a single injection of 355 MBq/kg of (213)Bi-C595 can prolong survival by 25 days. A high tumor: blood ratio (5.8) was found in biodistribution study. The maximum tolerance dose (MTD) was more than 1180 MBq/kg up to 21 weeks. CONCLUSIONS: C595 is a specific targeting vector for ovarian cancer cells, which show a high percentage of expression of MUC1. (213)Bi-C595 can effectively target and kill ovarian cancer cells in vitro and in vivo. (213)Bi-C595 is the recommended alpha conjugate for a Phase I clinical trial for ovarian cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Bismuto/uso terapêutico , Mucina-1/imunologia , Neoplasias Ovarianas/radioterapia , Radioimunoterapia , Partículas alfa , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Rim/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mucina-1/análise , Neoplasias Ovarianas/mortalidade , Radioimunoterapia/efeitos adversos , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ionizing radiation causes structural chromosomal aberrations, a proportion of which give rise to chromosome fragments without spindle attachment organelles. When a cell divides, some of these fragments are excluded from the main daughter nuclei and form small nuclei within the cytoplasm. The cytokinesis-block micronucleus assay allows these micronuclei (MN) to be counted, providing an in situ biological dosimeter. In this study, we evaluated the micronucleus frequency in peripheral blood lymphocytes after in vitro incubation with the alpha conjugates (213)BiI(3) and (213)Bi-9.2.27 (AIC). Lymphocytes were inoculated in vitro AIC for 3 h. Further, we report the first MN measurements in melanoma patients after targeted alpha therapy (TAT) with (213)Bi-9.2.27. Patients were injected with 260-360 MBq of AIC, and blood samples taken at 3 h, 2 weeks and 4 weeks post-treatment. Absorbed dose (MIRD) and effective total body dose (PED) were calculated. The MN frequency in lymphocytes was similar for equal in vitro incubation activities of (213)BiI(3) and (213)Bi-9.2.27 (P=0.5), indicating that there is no selective targeting of lymphocytes by the alpha conjugates. After inoculation with 10-1200 kBq mL-1 of AIC, there was a substantial activity-related increase in MN. The number of MN in the blood of treated patients peaked at 3 h post-TAT, slowly returning to baseline levels by 4 weeks. The mean photon equivalent dose (PED) is 0.43 Gy (SD 0.15) and the mean MIRD calculated absorbed dose is 0.11 Gy (SD 0.03), giving an RBE=4+/-0.4 for this study.
Assuntos
Bioensaio/métodos , Citocinese/efeitos da radiação , Linfócitos/citologia , Linfócitos/efeitos da radiação , Testes para Micronúcleos/métodos , Radiometria/métodos , Radioterapia/métodos , Idoso , Idoso de 80 Anos ou mais , Partículas alfa/uso terapêutico , Feminino , Humanos , Masculino , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Pessoa de Meia-Idade , Doses de Radiação , Eficiência Biológica RelativaRESUMO
Targeted alpha therapy (TAT) is an emerging therapeutic modality, thought to be best suited to cancer micrometastases, leukaemia and lymphoma. TAT has not been indicated for solid tumours. However, several melanoma patients in a phase 1 clinical trial of systemic targeted alpha therapy for melanoma experienced marked regression of subcutaneous and internal tumours. This response cannot be ascribed to killing of all cancer cells in the tumours by targeted alpha therapy. These new observations support the original hypothesis that tumours can be regressed by a mechanism called tumour anti-vascular alpha therapy. This effect depends on the expression of targeted receptors by capillary pericytes and contiguous cancer cells, and on the short-range and high-energy transfer of alpha radiation.
Assuntos
Partículas alfa , Melanoma/radioterapia , Metástase Neoplásica/radioterapia , Radioterapia/métodos , Neoplasias Cutâneas/radioterapia , Neoplasias Cutâneas/terapia , Bismuto/uso terapêutico , Ensaios Clínicos como Assunto , Relação Dose-Resposta à Radiação , Humanos , Cinética , Radioisótopos/uso terapêutico , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVES: The plasminogen activator inhibitor type 2 (PAI2) when labelled with (213)Bi forms the (213)Bi-PAI2 alpha conjugate (AC). This AC has been shown to be efficacious in preclinical studies with breast, ovarian, prostate and pancreatic cancers. The objectives of this study were to investigate the pharmacokinetics and in vivo stability of (213)Bi-PAI2 in mice, its toxicity in mice and rabbits; and to determine whether a prior injection of a metal chelator (Ca-DTPA) or lysine can reduce toxicity by decreasing renal uptake. METHODS: Two chelators (CHX-A"-DTPA and cDTPA) were used for preparation of the (213)Bi-PAI2 conjugate, for intraperitoneal administration in mice and ear vein injection in rabbits. The mice were sacrificed at different time points for pharmacokinetic studies. Blood and organs were collected for toxicity studies for all groups. RESULTS: Both chelators were found to have similar %ID/g in the kidneys over four hours. Mice and rabbits did not show any short term toxicity over 13 weeks at 1420 MBq/kg and 120 MBq/kg (213)Bi-PAI2 respectively. Kidney uptake was decreased three fold by lysine. Radiation nephropathy was observed at 20-30 weeks in mice, leading to severe weight loss, whereas severe and widespread renal tubular necrosis was observed at 13 weeks in rabbits. CONCLUSIONS: Radiation nephropathy is the dose limiting toxicity observed in mice and rabbits. Lysine can reduce kidney uptake by three fold. Based on long-term monitoring, the maximum tolerance doses (MTD) are 350 and 120 MBq/kg for mice and rabbits respectively.
Assuntos
Bismuto/farmacocinética , Bismuto/toxicidade , Inibidor 2 de Ativador de Plasminogênio/farmacocinética , Inibidor 2 de Ativador de Plasminogênio/toxicidade , Radioimunoterapia/métodos , Radioisótopos/toxicidade , Partículas alfa , Animais , Quelantes/farmacologia , Feminino , Rim/patologia , Oncologia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Coelhos , Radioisótopos/química , Fatores de TempoRESUMO
PURPOSE: The aim is to assess toxicity and response of systemic alpha therapy for metastatic melanoma. EXPERIMENTAL DESIGN: This is an open-labelled Phase 1 dose escalation study to establish the effective dose of the alpha-immunoconjugate (213)Bi-cDTPA-9.2.27 mAb (AIC). Tools used to investigate the effects were physical examination; imaging of tumors; pathology; GFR; CT and changes in tumor marker. Responses were assessed using RECIST criteria. RESULTS AND DISCUSSION: Twenty-two patients with stage IV melanoma/in-transit metastasis were treated with activities of 55-947 MBq. Using RECIST criteria 50% showed stable disease and 14% showed partial response. One patient (6%) showed near complete response and was retreated because of an excellent response to the initial treatment. Another patient showed response in his tumor on mandible and reduction in lung lesions. Overall 30% showed progressive disease. The tumor marker melanoma inhibitory activity protein (MIA) showed reductions over eight weeks in most of the patients. The disparity of dose with responders is discussed. No toxicity was observed over the range of administered activities. CONCLUSION: Observation of responses without any toxicity indicates that targeted alpha therapy has the potential to be a safe and effective therapeutic approach for metastatic melanoma.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/uso terapêutico , Melanoma/tratamento farmacológico , Ácido Pentético/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/toxicidade , Biomarcadores Tumorais , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Imunoterapia/métodos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
OBJECTIVES: The key objective of the study was to determine the single and multiple dose toxicity and efficacy of Bismuth-213 labeled PAI2 based targeted alpha therapy by selectively targeting uPA and uPAR in regressing prostate cancer in a nude mouse model. Targeted alpha therapy (TAT) is an experimental therapeutic modality for cancer. Another objective was to compare the in vivo pharmacokinetics using two different chelators to form the radioisotope-protein construct. METHODS: The single and multiple intra-peritoneal (IP) dose toxicity and efficacy of 213BiPAI2 was investigated in nude mice and its toxicity in rabbits. CD 31 staining for vasculature and uPA expression were measured at different stages of tumor growth. The pharmacokinetics of the chelators cDTPA and CHX-A'' were measured. RESULTS: All TAT regimes were well tolerated in mice and rabbits on biochemical and haematological examination. Capillaries became evident at six days post-cell inoculation. uPA expression was positive at all stages of tumour growth. No significant differences were observed between cDTPA and CHX-A. '' Inhibition of tumour growth was observed at 947 and 1421 MBq/kg single dose injection at three days post-PC3 cell inoculation. The three day post-inoculation multiple dose regime gave complete tumour growth inhibition at a total dose of 947 MBq/kg given on five successive days. Mice treated at 6, 12 and 18 days post-inoculation showed significantly slower tumour growth compared to controls. CONCLUSIONS: The efficacy of single and multiple dose TAT in mice was demonstrated within tolerance limits, the multiple dose regime being no more toxic than the single dose. Either of the two chelators could be used for 213Bi studies.
Assuntos
Bismuto/farmacologia , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Neoplasias da Próstata/terapia , Radioisótopos/farmacologia , Animais , Quelantes/farmacologia , Cicloeximida/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Ácido Pentético/farmacologia , Inibidor 2 de Ativador de Plasminogênio/química , Coelhos , Inibidores de Serina Proteinase/farmacologiaRESUMO
PURPOSE: The urokinase plasminogen activator (uPA) and its receptor (uPAR) are expressed by pancreatic cancer cells and can be targeted by the plasminogen activator inhibitor type 2 (PAI2). We have labeled PAI2 with (213)Bi to form the alpha conjugate (AC), and have studied its in vitro cytotoxicity and in vivo efficacy. METHODS AND MATERIALS: The expression of uPA/uPAR on pancreatic cell lines, human pancreatic cancer tissues, lymph node metastases, and mouse xenografts were detected by immunohistochemistry, confocal microscopy, and flow cytometry. Cytotoxicity was assessed by the MTS and TUNEL assay. At 2 days post-cancer cell subcutaneous inoculation, mice were injected with AC by local or systemic injection. RESULTS: uPA/uPAR is strongly expressed on pancreatic cancer cell lines and cancer tissues. The AC can target and kill cancer cells in vitro in a concentration-dependent fashion. Some 90% of TUNEL positive cells were found after incubation with 1.2 MBq/ml of AC. A single local injection of approximately 222 MBq/kg 2 days post-cell inoculation can completely inhibit tumor growth over 12 weeks, and an intraperitoneal injection of 111 MBq/kg causes significant tumor growth delay. CONCLUSIONS: (213)Bi-PAI2 can specifically target pancreatic cancer cells in vitro and inhibit tumor growth in vivo. (213)Bi-PAI2 may be a useful agent for the treatment of post-surgical pancreatic cancer patients with minimum residual disease.
Assuntos
Bismuto/uso terapêutico , Metástase Neoplásica/radioterapia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/radioterapia , Inibidor 2 de Ativador de Plasminogênio/uso terapêutico , Radioisótopos/uso terapêutico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Microscopia Confocal , Compostos Radiofarmacêuticos/uso terapêutico , Transplante Heterólogo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Targeted alpha therapy (TAT) offers the potential to inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The practicality and efficacy of TAT is tested by in vitro and in vivo studies in melanoma, leukaemia, colorectal, breast and prostate cancers, and by a phase 1 trial of intralesional TAT for melanoma. The alpha-emitting radioisotope used is Bi-213, which is eluted from the Ac-225 generator and chelated to a cancer specific monoclonal antibody (mab) or protein (e.g. plasminogen activator inhibitor-2 PAI2) to form the alpha-conjugate (AC). Stable alpha-ACs have been produced which have been tested for specificity and cytotoxicity in vitro against melanoma (9.2.27 mab), leukaemia (WM60), colorectal (C30.6), breast (PAI2, herceptin), ovarian (PAI2, herceptin, C595), prostate (PAI2, J591) and pancreatic (PAI2, C595) cancers. Subcutaneous inoculation of 1-1.5 million human cancer cells into the flanks of nude mice causes tumours to grow in all mice. Tumour growth is compared for untreated controls, nonspecific AC and specific AC, for local (subcutaneous) and systemic (tail vein or intraperitoneal) injection models. The 213Bi-9.2.27 AC is injected into secondary skin melanomas in stage 4 patients in a dose escalation study to determine the effective tolerance dose, and to measure kinematics to obtain the equivalent dose to organs. In vitro studies show that TAT is one to two orders of magnitude more cytotoxic to targeted cells than non-specific ACs, specific beta emitting conjugates or free isotopes. In vivo local TAT at 2 days post-inoculation completely prevents tumour formation for all cancers tested so far. Intra-lesional TAT can completely regress advanced sc melanoma but is less successful for breast and prostate cancers. Systemic TAT inhibits the growth of sc melanoma xenografts and gives almost complete control of breast and prostate cancer tumour growth. Intralesional doses up to 450 microCi in human patients are effective in regressing melanomas, with no concomitant complications. These results point to the application of local and systemic TAT in the management of secondary cancer. Results of the phase 1 clinical trial of TAT of subcutaneous, secondary melanoma indicate proof of the principle that TAT can make tumours in patients regress.