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1.
AAPS J ; 22(2): 53, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32124093

RESUMO

Static in vitro cell culture studies cannot capture the dynamic concentration profiles of drugs, nutrients, and other factors that cells experience in physiological systems. This limits the confidence in the translational relevance of in vitro experiments and increases the reliance on empirical testing of exposure-response relationships and dose optimization in animal models during preclinical drug development, introducing additional challenges owing to species-specific differences in drug pharmacokinetics (PK) and pharmacodynamics (PD). Here, we describe the development of a microfluidic cell culture device that enables perfusion of cells under 2D or 3D culture conditions with temporally programmable concentration profiles. Proof-of-concept studies using doxorubicin and gemcitabine demonstrated the ability of the microfluidic PK-PD device to examine dose- and time-dependent effects of doxorubicin as well as schedule-dependent effects of doxorubicin and gemcitabine combination therapy on cell viability using both step-wise drug concentration profiles and species-specific (i.e., mouse, human) drug PK profiles. The results demonstrate the importance of including physiologically relevant dynamic drug exposure profiles during in vitro drug testing to more accurately mimic in vivo drug effects, thereby improving translatability across nonclinical studies and reducing the reliance on animal models during drug development.

2.
PLoS One ; 14(5): e0217276, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31112568

RESUMO

Cancer cells harness immune checkpoints such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase 1 (IDO1) to evade immune control. Checkpoint inhibitors have demonstrated durable anti-tumor efficacy in human and preclinical models. Liver toxicity is one of the common immune-related adverse events associated with checkpoint inhibitors (CPIs) and its frequency and severity often increase significantly during CPI combination therapies. We aim to develop a mouse model to elucidate the immune mechanisms of CPI-associated liver toxicity. Co-administration of CTLA-4 blocking antibody, 9D9, and/or an IDO1 inhibitor, epacadostat in wild-type and PD-1-/- mice (to simulate the effect of PD1 blockade) synergistically induced liver injury and immune cell infiltration. Infiltrated cells were primarily composed of CD8+ T cells and positively associated with hepatocyte necrosis. Strikingly, sites of hepatocyte necrosis were frequently surrounded by clusters of mononuclear immune cells. CPI treatments resulted in increased expression of genes associated with hepatocyte cell death, leukocyte migration and T cell activation in the liver. In conclusion, blockade of immune checkpoints PD-1, CTLA-4, and IDO1 act synergistically to enhance T cell infiltration and activity in the liver, leading to hepatocyte death.


Assuntos
Antígeno CTLA-4/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Fígado/imunologia , Fígado/lesões , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Modelos Animais de Doenças , Feminino , Hepatócitos/patologia , Humanos , Ipilimumab/administração & dosagem , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nivolumabe/administração & dosagem , Oximas/administração & dosagem , Receptor de Morte Celular Programada 1/deficiência , Receptor de Morte Celular Programada 1/genética , Sulfonamidas/administração & dosagem
3.
J Pharm Sci ; 108(9): 3124-3129, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31136767

RESUMO

Microdialysis is a technique that utilizes a semipermeable membrane to sample analytes present within tissue interstitial fluid. Analyte-specific calibration is required for quantitative microdialysis, but these calibration methods are tedious, require significant technical skill, and often cannot be performed jointly with the experimental measurements. Here, we describe a method using retrodialysis with stable-isotope-labeled analytes that enables simultaneous calibration and quantification for in vivo tumor microdialysis. Isotope-labeled amino acids relevant to immuno-metabolism in the tumor microenvironment (tryptophan, kynurenine, glutamine, and glutamate) were added to the microdialysis perfusate, and microdialysis probes were inserted in subcutaneous CT26 and MC38 tumors in mice. The levels of both the endogenous and isotope-labeled amino acids in the perfusate outlet were quantified using LC-MS/MS. Plasma and tumor tissue samples were also collected from the same mice and amino acid levels quantified using LC-MS/MS. Amino acids which showed statistically significant differences between the CT26-bearing and MC38-bearing mice in tumor lysate (tryptophan, kynurenine, and glutamine) and plasma (glutamate) were not the same as those identified as significantly different in tumor interstitial fluid (kynurenine and glutamate), underscoring how microdialysis can provide unique and complementary insights into tumor and immune metabolism within the tumor microenvironment.

4.
Gene Regul Syst Bio ; 11: 1177625017710941, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28804243

RESUMO

Reduction in low-density lipoprotein cholesterol (LDL-C) is associated with decreased risk for cardiovascular disease. Alirocumab, an antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9), significantly reduces LDL-C. Here, we report development of a quantitative systems pharmacology (QSP) model integrating peripheral and liver cholesterol metabolism, as well as PCSK9 function, to examine the mechanisms of action of alirocumab and other lipid-lowering therapies, including statins. The model predicts changes in LDL-C and other lipids that are consistent with effects observed in clinical trials of single or combined treatments of alirocumab and other treatments. An exploratory model to examine the effects of lipid levels on plaque dynamics was also developed. The QSP platform, on further development and qualification, may support dose optimization and clinical trial design for PCSK9 inhibitors and lipid-modulating drugs. It may also improve our understanding of factors affecting therapeutic responses in different phenotypes of dyslipidemia and cardiovascular disease.

5.
Mol Pharm ; 11(11): 3965-73, 2014 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-24779727

RESUMO

Engineered antibody fragments offer faster delivery with retained tumor specificity and rapid clearance from nontumor tissues. Here, we demonstrate that positron emission tomography (PET) based detection of prostate specific membrane antigen (PSMA) in prostatic tumor models using engineered bivalent antibodies built on single chain fragments (scFv) derived from the intact antibody, huJ591, offers similar tumor delineating properties but with the advantage of rapid targeting and imaging. (89)Zr-radiolabeled huJ591 scFv (dimeric scFv-CH3; (89)Zr-Mb) and cysteine diabodies (dimeric scFv; (89)Zr-Cys-Db) demonstrated internalization and similar Kds (∼2 nM) compared to (89)Zr-huJ591 in PSMA(+) cells. Tissue distribution assays established the specificities of both (89)Zr-Mb and (89)Zr-Cys-Db for PSMA(+) xenografts (6.2 ± 2.5% ID/g and 10.2 ± 3.4% ID/g at 12 h p.i. respectively), while minimal accumulation in PSMA(-) tumors was observed. From the PET images, (89)Zr-Mb and (89)Zr-Cys-Db exhibited faster blood clearance than the parent huJ591 while tumor-to-muscle ratios for all probes show comparable values across all time points. Ex vivo autoradiography and histology assessed the distribution of the probes within the tumor. Imaging PSMA-expressing prostate tumors with smaller antibody fragments offers rapid tumor accumulation and accelerated clearance; hence, shortened wait periods between tracer administration and high-contrast tumor imaging and lower dose-related toxicity are potentially realized.


Assuntos
Anticorpos Monoclonais , Antígenos de Superfície/imunologia , Glutamato Carboxipeptidase II/imunologia , Imagem Molecular/métodos , Neoplasias da Próstata/diagnóstico por imagem , Compostos Radiofarmacêuticos , Anticorpos de Cadeia Única , Zircônio , Animais , Anticorpos Monoclonais/farmacocinética , Humanos , Fragmentos de Imunoglobulinas , Radioisótopos do Iodo/farmacocinética , Masculino , Camundongos , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Compostos Radiofarmacêuticos/farmacocinética , Anticorpos de Cadeia Única/farmacocinética , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Zircônio/farmacocinética
6.
J Biomed Opt ; 18(10): 101304, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23752742

RESUMO

Antibody fragments including diabodies have more desirable pharmacokinetic characteristics than whole antibodies. An activatable optical imaging probe based on a cys-diabody targeting prostate-specific membrane antigen conjugated with the near-infrared fluorophore, indocyanine green (ICG), was designed such that it can only be activated when bound to the tumor, leading to high signal-to-background ratios. We employed short polyethylene glycol (PEG) linkers between the ICG and the reactive functional group (Sulfo-OSu group), resulting in covalent conjugation of ICG to the cys-diabody, which led to lower dissociation of ICG from cys-diabody early after injection, reducing hepatic uptake. However, unexpectedly, high and long-term fluorescence was observed in the kidneys, liver, and blood pool more than 1 h after injection of the cys-diabody PEG-ICG conjugate. A biodistribution study using I125-labeled cys-diabody-ICG showed immediate uptake in the kidneys followed by a rapid decrease, while gastric activity increased due to released radioiodine during rapid cys-diabody-ICG catabolism in the kidneys. To avoid this catabolic pathway, it would be preferable to use antibody fragments large enough not to be filtered through glomerulus or to conjugate the fragments with fluorescent dyes that are readily excreted into urine when cleaved from the cys-diabody to achieve high tumor-specific detection.


Assuntos
Corantes Fluorescentes/química , Verde de Indocianina/química , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Animais , Anticorpos/química , Anticorpos/metabolismo , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Feminino , Corantes Fluorescentes/farmacocinética , Glutamato Carboxipeptidase II/imunologia , Glutamato Carboxipeptidase II/metabolismo , Verde de Indocianina/farmacocinética , Cinética , Camundongos , Camundongos Nus , Polietilenoglicóis , Distribuição Tecidual
7.
Bioconjug Chem ; 19(9): 1927-37, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18720981

RESUMO

Increasing interest in the use of radiolabeled antibodies for cancer imaging and therapy drives the need for more efficient production of the antibody conjugates. Here, we illustrate a method for rapid and efficient production of radiolabeled antibody conjugates using vacuum diafiltration guided by mathematical modeling. We apply this technique to the production of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated antibodies at the milligram and gram production scale and achieve radiolabeling efficiencies >95% using In-111. Using vacuum diafiltration, antibody-chelate conjugation and purification can be accomplished within the same vessel, and the entire process can be completed in <24 h. Vacuum diafiltration also offers safer and gentler processing conditions by eliminating the need to keep the retentate vessel under positive pressure through applied gas pressure or shear-inducing restriction points in the retentate flow path. Experimental data and mathematical model calculations suggest there exists a weak binding affinity (approximately 10(4)M(-1)) between the charged chelate molecules (e.g., DOTA) and the antibodies that slows the removal of excess chelate during purification. By analyzing the radiolabeling efficiency as a function of the number of diavolumes, we demonstrate the importance of balancing the removal of free chelate with the introduction of metal contaminants from the diafiltration buffer and also illustrate how to optimize radiolabeling of antibody conjugates under a variety of operating conditions. This methodology is applicable to the production of antibody conjugates in general.


Assuntos
Anticorpos Monoclonais , Filtração/métodos , Compostos Heterocíclicos com 1 Anel/química , Imunoconjugados/química , Marcação por Isótopo/métodos , Radioimunoterapia/métodos , Compostos Radiofarmacêuticos/química , Animais , Quelantes/química , Humanos , Modelos Teóricos , Vácuo
8.
Biotechnol Bioeng ; 99(4): 975-85, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17929316

RESUMO

This study addresses issues of relevance for siRNA nanoparticle delivery by investigating the functional impact of tumor-specific targeting and dosing schedule. The investigations are performed using an experimental system involving a syngeneic mouse cancer model and a theoretical system based on our previously described mathematical model of siRNA delivery and function. A/J mice bearing subcutaneous Neuro2A tumors approximately 100 mm(3) in size were treated by intravenous injection with siRNA-containing nanoparticles formed with cyclodextrin-containing polycations (CDP). Three consecutive daily doses of transferrin (Tf)-targeted nanoparticles carrying 2.5 mg/kg of two different siRNA sequences targeting ribonucleotide reductase subunit M2 (RRM2) slowed tumor growth, whereas non-targeted nanoparticles were significantly less effective when given at the same dose. Furthermore, administration of the three doses on consecutive days or every 3 days did not lead to statistically significant differences in tumor growth delay. Mathematical model calculations of siRNA-mediated target protein knockdown and tumor growth inhibition are used to elucidate possible mechanisms to explain the observed effects and to provide guidelines for designing more effective siRNA-based treatment regimens regardless of delivery methodology and tumor type.


Assuntos
Marcação de Genes/métodos , Terapia Genética/métodos , Nanopartículas/administração & dosagem , Neuroblastoma/genética , Neuroblastoma/terapia , RNA Interferente Pequeno/administração & dosagem , Transferrina/genética , Animais , Proliferação de Células , Relação Dose-Resposta a Droga , Feminino , Injeções Intravenosas , Camundongos , Neuroblastoma/patologia
9.
Proc Natl Acad Sci U S A ; 104(39): 15549-54, 2007 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-17875985

RESUMO

Targeted delivery represents a promising approach for the development of safer and more effective therapeutics for oncology applications. Although macromolecules accumulate nonspecifically in tumors through the enhanced permeability and retention (EPR) effect, previous studies using nanoparticles to deliver chemotherapeutics or siRNA demonstrated that attachment of cell-specific targeting ligands to the surface of nanoparticles leads to enhanced potency relative to nontargeted formulations. Here, we use positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the 5' end of the siRNA molecules allows labeling with (64)Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2A s.c. tumors before and after PET imaging enables correlation of functional efficacy with biodistribution data. Although both nontargeted and transferrin-targeted siRNA nanoparticles exhibit similar biodistribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduce tumor luciferase activity by approximately 50% relative to nontargeted siRNA nanoparticles 1 d after injection. Compartmental modeling is used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Optimization of internalization may therefore be key for the development of effective nanoparticle-based targeted therapeutics.


Assuntos
Ciclodextrinas/administração & dosagem , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Neoplasias/terapia , RNA Interferente Pequeno/metabolismo , Animais , Química Farmacêutica/métodos , Ciclodextrinas/química , Portadores de Fármacos , Compostos Heterocíclicos com 1 Anel/química , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Tomografia por Emissão de Pósitrons/métodos , Tecnologia Farmacêutica/métodos
10.
Clin Cancer Res ; 13(7): 2207-15, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17404105

RESUMO

PURPOSE: Ribonucleotide reductase (RR) is a therapeutic target for DNA replication-dependent diseases such as cancer. Here, a potent small interfering RNA (siRNA) duplex against the M2 subunit of RR (RRM2) is developed and shown to reduce the growth potential of cancer cells both in vitro and in vivo. EXPERIMENTAL DESIGN: Three anti-RRM2 siRNAs were identified via computational methods, and the potency of these and additional "tiling" duplexes was analyzed in cultured cells via cotransfections using a RRM2-luciferase fusion construct. Knockdown of RRM2 by the best duplex candidates was confirmed directly by Western blotting. The effect of potent duplexes on cell growth was investigated by a real-time cell electronic sensing assay. Finally, duplex performance was tested in vivo in luciferase-expressing cells via whole animal bioluminescence imaging. RESULTS: Moderate anti-RRM2 effects are observed from the three duplexes identified by computational methods. However, the tiling experiments yielded an extremely potent duplex (siR2B+5). This duplex achieves significant knockdown of RRM2 protein in cultured cells and has pronounced antiproliferative activity. S.c. tumors of cells that had been transfected with siR2B+5 preinjection grew slower than those of control cells. CONCLUSIONS: An anti-RRM2 siRNA duplex is identified that exhibits significant antiproliferative activity in cancer cells of varying human type and species (mouse, rat, monkey); these findings suggest that this duplex is a promising candidate for therapeutic development.


Assuntos
Terapia Genética/métodos , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/genética , Ribonucleosídeo Difosfato Redutase/antagonistas & inibidores , Ribonucleosídeo Difosfato Redutase/genética , Animais , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Transfecção
11.
Bioconjug Chem ; 18(2): 456-68, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17326672

RESUMO

Nucleic acid-based therapeutics have the potential to provide potent and highly specific treatments for a variety of human ailments. However, systemic delivery continues to be a significant hurdle to success. Multifunctional nanoparticles are being investigated as systemic, nonviral delivery systems, and here, we describe the physicochemical and biological characterization of cyclodextrin-containing polycations (CDP) and their nanoparticles formed with nucleic acids including plasmid DNA (pDNA) and small interfering RNA (siRNA). These polycation/nucleic acid complexes can be tuned by formulation conditions to yield particles with sizes ranging from 60 to 150 nm, zeta potentials from 10 to 30 mV, and molecular weights from approximately 7 x 107 to 1 x 109 g mol-1 as determined by light scattering techniques. Inclusion complexes formed between adamantane (AD)-containing molecules and the beta-cyclodextrin molecules enable the modular attachment of poly(ethylene glycol) (AD-PEG) conjugates for steric stabilization and targeting ligands (AD-PEG-transferrin) for cell-specific targeting. A 70 nm particle can contain approximately 10 000 CDP polymer chains, approximately 2000 siRNA molecules, approximately 4000 AD-PEG5000 molecules, and approximately 100 AD-PEG5000-Tf molecules; this represents a significant payload of siRNA and a large ratio of siRNA to targeting ligand (20:1). The particles protect the nucleic acid payload from nuclease degradation, do not aggregate at physiological salt concentrations, and cause minimal erythrocyte aggregation and complement fixation at the concentrations typically used for in vivo application. Uptake of the nucleic acid-containing particles by HeLa cells is measured by flow cytometry and visualized by confocal microscopy. Competitive uptake experiments show that the transferrin-targeted particles display enhanced affinity for the transferrin receptor through avidity effects (multiligand binding). Functional efficacy of the delivered pDNA and siRNA is demonstrated through luciferase reporter protein expression and knockdown, respectively. The analysis of the CDP delivery vehicle provides insights that can be applied to the design of targeted nucleic acid delivery vehicles in general.


Assuntos
Ciclodextrinas/química , Portadores de Fármacos/química , Nanopartículas , Ácidos Nucleicos/química , Poliaminas/química , Transferrina/química , Adamantano/metabolismo , Animais , Bovinos , Ciclodextrinas/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Ensaio de Desvio de Mobilidade Eletroforética , Agregação Eritrocítica , Técnicas de Transferência de Genes , Células HeLa , Humanos , Ácidos Nucleicos/metabolismo , Plasmídeos , Poliaminas/metabolismo , Polietilenoglicóis/química , RNA Interferente Pequeno , Receptores da Transferrina/metabolismo , Transferrina/metabolismo
12.
Biotechnol Bioeng ; 97(4): 909-21, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17154307

RESUMO

Small interfering RNA (siRNA) molecules achieve sequence-specific gene silencing through the RNA interference (RNAi) mechanism. Here, live-cell and live-animal bioluminescent imaging (BLI) is used to directly compare luciferase knockdown by unmodified and nuclease-stabilized siRNAs in rapidly (HeLa) and slowly (CCD-1074Sk) dividing cells to reveal the impact of cell division and siRNA nuclease stability on the kinetics of siRNA-mediated gene silencing. Luciferase knockdown using unmodified siRNAs lasts approximately 1 week in HeLa cells and up to 1 month in CCD-1074Sk cells. There is a slight increase in the duration of luciferase knockdown by nuclease-stabilized siRNAs relative to unmodified siRNAs after cationic lipid transfection, but this difference is not observed after electroporation. In BALB/cJ mice, a fourfold increase in maximum luciferase knockdown is observed after hydrodynamic injection (HDI) of nuclease-stabilized siRNAs relative to unmodified siRNAs, yet the overall kinetics of the recovery after knockdown are nearly identical. By using a mathematical model of siRNA-mediated gene silencing, the trends observed in the experimental data can be duplicated by changing model parameters that affect the stability of the siRNAs before they reach the cytosolic compartment. Based on these findings, we hypothesize that the stabilization advantages of nuclease-stabilized siRNAs originate primarily from effects prior to and during internalization before the siRNAs can interact with the intracellular RNAi machinery.


Assuntos
Endonucleases/metabolismo , Inativação Gênica , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Eletroporação , Estabilidade Enzimática , Células HeLa , Humanos , Técnicas In Vitro , Cinética , Luciferases/genética , Substâncias Luminescentes , Medições Luminescentes , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transfecção
13.
Clin Cancer Res ; 12(5): 1606-14, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16533788

RESUMO

Preclinical efficacy of i.v. IT-101, a nanoparticulate conjugate of 20(S)-camptothecin and a cyclodextrin-based polymer, was investigated in several mouse xenografts. The effects of different multiple dosing schedules on tumor growth of LS174T colon carcinoma xenografts are elucidated. All multiple dosing schedules administered over 15 to 19 days resulted in enhanced efficacy compared with untreated or single-dose groups. Further improvements in antitumor efficacy were not observed when the dosing frequency was increased from three weekly doses to five doses at 4-day intervals or 5 days of daily dosing followed by 2 days without dosing repeated in three cycles using similar cumulative doses. This observation was attributed to the extended release characteristics of camptothecin from the polymer. Antitumor efficacy was further evaluated in mice bearing six different s.c. xenografts (LS174T and HT29 colorectal cancer, H1299 non-small-cell lung cancer, H69 small-cell lung cancer, Panc-1 pancreatic cancer, and MDA-MB-231 breast cancer) and one disseminated xenograft (TC71-luc Ewing's sarcoma). In all cases, a single treatment cycle of three weekly doses of IT-101 resulted in a significant antitumor effect. Complete tumor regression was observed in all animals bearing H1299 tumors and in the majority of animals with disseminated Ewing's sarcoma tumors. Importantly, IT-101 is effective in a number of tumors that are resistant to treatment with irinotecan (MDA-MB-231, Panc-1, and HT29), consistent with the hypothesis that polymeric drug conjugates may be able to overcome certain kinds of multidrug resistance. Taken together, these results indicate that IT-101 has good tolerability and antitumor activity against a wide range of tumors.


Assuntos
Camptotecina/uso terapêutico , Ciclodextrinas/química , Modelos Animais de Doenças , Polímeros/química , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Neoplasias da Mama/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Nus , Nanotecnologia , Neoplasias Pancreáticas/tratamento farmacológico , Sarcoma de Ewing/tratamento farmacológico , Células Tumorais Cultivadas
14.
Nucleic Acids Res ; 34(1): 322-33, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16410612

RESUMO

Small interfering RNA (siRNA) molecules are potent effectors of post-transcriptional gene silencing. Using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function, the effects of target-specific and treatment-specific parameters on siRNA-mediated gene silencing are monitored in cells stably expressing the firefly luciferase protein. In vitro, luciferase protein levels recover to pre-treatment values within <1 week in rapidly dividing cell lines, but take longer than 3 weeks to return to steady-state levels in nondividing fibroblasts. Similar results are observed in vivo, with knockdown lasting approximately 10 days in subcutaneous tumors in A/J mice and 3-4 weeks in the nondividing hepatocytes of BALB/c mice. These data indicate that dilution due to cell division, and not intracellular siRNA half-life, governs the duration of gene silencing under these conditions. To demonstrate the practical use of the model in treatment design, model calculations are used to predict the dosing schedule required to maintain persistent silencing of target proteins with different half-lives in rapidly dividing or nondividing cells. The approach of bioluminescent imaging combined with mathematical modeling provides useful insights into siRNA function and may help expedite the translation of siRNA into clinically relevant therapeutics for disease treatment and management.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Linhagem Celular , Proliferação de Células , Hepatócitos/metabolismo , Cinética , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/genética , Substâncias Luminescentes , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , RNA Interferente Pequeno/química
15.
Cancer Res ; 65(19): 8984-92, 2005 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16204072

RESUMO

The development of effective, systemic therapies for metastatic cancer is highly desired. We show here that the systemic delivery of sequence-specific small interfering RNA (siRNA) against the EWS-FLI1 gene product by a targeted, nonviral delivery system dramatically inhibits tumor growth in a murine model of metastatic Ewing's sarcoma. The nonviral delivery system uses a cyclodextrin-containing polycation to bind and protect siRNA and transferrin as a targeting ligand for delivery to transferrin receptor-expressing tumor cells. Removal of the targeting ligand or the use of a control siRNA sequence eliminates the antitumor effects. Additionally, no abnormalities in interleukin-12 and IFN-alpha, liver and kidney function tests, complete blood counts, or pathology of major organs are observed from long-term, low-pressure, low-volume tail-vein administrations. These data provide strong evidence for the safety and efficacy of this targeted, nonviral siRNA delivery system.


Assuntos
Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Sarcoma de Ewing/terapia , Animais , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Inativação Gênica , Luciferases/biossíntese , Luciferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/genética , RNA Interferente Pequeno/toxicidade , Proteína EWS de Ligação a RNA , Receptores da Transferrina/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Transdução Genética
16.
J Am Soc Nephrol ; 15(7): 1927-35, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15213283

RESUMO

Clinical hemodialysis systems achieve high single pass extraction of small solutes that are not bound to plasma proteins. But they clear protein-bound solutes much less effectively. This study examines the extent to which clearance of a protein-bound test solute is improved by increasing the dialyzer mass transfer area coefficient (KoA) and the dialysate flow rate (Qd). A reservoir containing test solutes and artificial plasma with albumin concentration approximately 4 g/dl was dialyzed with a standard clinical dialysate delivery system. The clearance of phenol red (ClPR) was compared with the clearances of urea and creatinine at a plasma flow rate (Qp) of 200 ml/min with varying values of KoA and Qd. ClPR increased from 11 +/- 2 ml/min to 23 +/- 2 ml/min when KoA for phenol red, KoAPR, was increased from 238 to 640 ml/min and Qd was increased from 286 +/- 6 ml/min to 734 +/- 9 ml/min. Increasing either KoAPR or Qd alone had lesser effects. Clearance values for phenol red were much lower than clearance values for the unbound solutes urea and creatinine, which ranged from 150 to 200 ml/min and were less affected by varying KoA and Qd. A mathematical model was developed to predict ClPR from values of Qp, Qd, the fraction of phenol red bound to albumin (94% +/- 1%) and KoAPR. The model accurately predicts the pattern of measured results and shows further that ClPR can be made to approach Qp only by very large increases in both KoAPR and Qd.


Assuntos
Albuminas/metabolismo , Falência Renal Crônica/terapia , Diálise Renal/métodos , Soluções para Diálise , Hemofiltração , Humanos , Membranas Artificiais , Modelos Estatísticos , Modelos Teóricos , Fenolsulfonaftaleína/química , Fenolsulfonaftaleína/farmacologia , Ligação Proteica , Proteínas/química , Fatores de Tempo , Ureia/metabolismo
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