RESUMO
Cytotoxic chemotherapy or radiotherapy of cancer is limited by serious, sometimes life-threatening, side effects that arise from toxicities to sensitive normal cells because the therapies are not selective for malignant cells. So how can selectivity be improved? One strategy is to couple the therapeutics to antibodies or other ligands that recognize tumour-associated antigens. This increases the exposure of the malignant cells, and reduces the exposure of normal cells, to the ligand-targeted therapeutics.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Humanos , Imunoglobulina G/metabolismo , Imunotoxinas/uso terapêutico , Ligantes , Modelos Biológicos , Polímeros/química , Ligação Proteica , RadioimunoterapiaRESUMO
A family of 3-methoxypoly(ethylene glycol)-vinyl ether-1,2-dioleylglycerol (mPEG-VE-DOG) lipopolymer conjugates, designed on the basis of DFT calculations to possess a wide range of proton affinities, was synthesized and tested for their hydrolysis kinetics in neutral and acidic buffers. Extruded â¼100 nm liposomes containing these constructs in ≥90 mol % 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) produced dispersions that retained their calcein cargo for more than 2 days at pH 7.5, but released the encapsulated contents over a wide range of time scales as a function of the electronic properties of the vinyl ether linkage, the solution pH, and the mPEG-VE-DOG composition in the membrane. The in vivo performance of two different 90:10 DOPE:mPEG-VE-DOG compositions was also evaluated for blood circulation time and biodistribution in mice, using (125)I-tyraminylinulin as a label. The pharmacokinetic profiles gave a t(1/2) of 7 and 3 h for 90:10 DOPE:ST302 and 90:10 DOPE:ST502, respectively, with the liposomes being cleared predominantly by liver and spleen uptake. The behavior of these DOPE:mPEG-VE-DOG formulations is consistent with their relative rates of vinyl ether hydrolysis, i.e., the more acid-sensitive mPEG-VE-DOG derivatives produced faster leakage rates from DOPE:mPEG-VE-DOG liposomes, but decreased the blood circulation times in mice. These findings suggest that the vinyl ether-based PEG-lipid derivatives are promising agents for stabilizing acid-sensitive DOPE liposomes to produce formulations with a priori control over their pH responsiveness in vitro. Our data also suggest, however, that the same factors that contribute to enhanced acid sensitivity of the DOPE:mPEG-VE-DOG dispersions are also likely responsible for their reduced pharmacokinetic profiles.
Assuntos
Diglicerídeos/farmacocinética , Lipídeos/síntese química , Lipossomos , Fosfatidiletanolaminas/farmacocinética , Polietilenoglicóis/farmacocinética , Compostos de Vinila/farmacocinética , Ácidos/metabolismo , Animais , Tempo de Circulação Sanguínea , Feminino , Concentração de Íons de Hidrogênio , Hidrólise , Lipídeos/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Distribuição TecidualRESUMO
RNA interference molecules have some advantages as cancer therapeutics, including a proved efficacy on both wild-type (WT) and mutated transcripts and an extremely high sequence-specificity. The most significant hurdle to be overcome if exogenous small interfering RNAs (siRNA) is to be used therapeutically is the specific, effective, nontoxic delivery of siRNA to its intracellular site of action. At present, human applications are confined almost exclusively to targets within the liver, where the delivery systems naturally accumulate, and extra-hepatic targets remain a challenge. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that has recently been shown to contribute to the cell growth and progression of human neuroblastoma (NB). We investigated its potential as a therapeutic target in NB by generating anti-GD2-targeted nanoparticles that carry ALK-directed siRNA, which are specifically and efficiently delivered to GD2-expressing NB cells. Relative to free ALK-siRNA, anti-GD2-targeted liposomal formulations of ALK-siRNA had low plasma clearance, increased siRNA stability, and improved binding, uptake, silencing and induction of cell death, and specificity for NB cells. In NB xenografts, intravenous (i.v.) injection of the targeted ALK-siRNA liposomes showed gene-specific antitumor activity with no side effects. ALK-selective siRNA entrapped in anti-GD2-targeted nanoparticles is a promising new modality for NB treatment.
Assuntos
Neuroblastoma/enzimologia , Neuroblastoma/terapia , RNA Interferente Pequeno/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Animais , Western Blotting , Linhagem Celular , Inativação Gênica/fisiologia , Humanos , Camundongos , Camundongos Nus , Nanopartículas/química , Neuroblastoma/genética , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is involved in the pathogenesis of different types of human cancers, including neuroblastoma (NB). In NB, ALK overexpression, or point mutations, are associated with poor prognosis and advanced stage disease. Inhibition of ALK kinase activity by small-molecule inhibitors in lung cancers carrying ALK translocations has shown therapeutic potential. However, secondary mutations may occur that, generate tumor resistance to ALK inhibitors. To overcome resistance to ALK inhibitors in NB, we adopted an alternative RNA interference (RNAi)-based therapeutic strategy that is able to knockdown ALK, regardless of its genetic status [mutated, amplified, wild-type (WT)]. NB cell lines, transduced by lentiviral short hairpin RNA (shRNA), showed reduced proliferation and increased apoptosis when ALK was knocked down. In mice, a nanodelivery system for ALK-specific small interfering RNA (siRNA), based on the conjugation of antibodies directed against the NB-selective marker GD(2) to liposomes, showed strong ALK knockdown in vivo in NB cells, which resulted in cell growth arrest, apoptosis, and prolonged survival. ALK knockdown was associated with marked reductions in vascular endothelial growth factor (VEGF) secretion, blood vessel density, and matrix metalloproteinases (MMPs) expression in vivo, suggesting a role for ALK in NB-induced neoangiogenesis and tumor invasion, confirming this gene as a fundamental oncogene in NB.
Assuntos
Apoptose , Mutação/genética , Neovascularização Patológica/prevenção & controle , Neuroblastoma/irrigação sanguínea , Neuroblastoma/terapia , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Quinase do Linfoma Anaplásico , Animais , Western Blotting , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Gangliosídeos/imunologia , Gangliosídeos/metabolismo , Células HeLa , Humanos , Técnicas Imunoenzimáticas , Lipossomos , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Nus , Camundongos SCID , Neuroblastoma/mortalidade , Fosforilação , Interferência de RNA , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Taxa de SobrevidaRESUMO
Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are major phospholipids in mammalian membranes. In liver, PC is synthesized via the choline pathway or by methylation of PE via phosphatidylethanolamine N-methyltransferase (PEMT). Pemt(-/-) mice fed a choline-deficient (CD) diet develop rapid steatohepatitis leading to liver failure. Steatosis is observed in CD mice that lack both PEMT and multiple drug-resistant protein 2 (MDR2), required for PC secretion into bile. We demonstrate that liver failure in CD-Pemt(-/-) mice is due to loss of membrane integrity caused by a decreased PC/PE ratio. The CD-Mdr2(-/-)/Pemt(-/-) mice escape liver failure by maintaining a normal PC/PE ratio. Manipulation of PC/PE levels suggests that this ratio is a key regulator of cell membrane integrity and plays a role in the progression of steatosis into steatohepatitis. The results have clinical implications as patients with nonalcoholic steatohepatitis have a decreased ratio of PC to PE compared to control livers.
Assuntos
Membrana Celular/metabolismo , Fígado Gorduroso/metabolismo , Falência Hepática Aguda/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Membrana Celular/patologia , Deficiência de Colina/complicações , Deficiência de Colina/metabolismo , Deficiência de Colina/patologia , Progressão da Doença , Fígado Gorduroso/etiologia , Fígado Gorduroso/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Falência Hepática Aguda/etiologia , Falência Hepática Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
The safe and effective delivery of anticancer agents to diseased tissues is one of the significant challenges in cancer therapy. Conventional anticancer agents are generally cytotoxins with poor pharmacokinetics and bioavailability. Nanocarriers are nanosized particles designed for the selectivity of anticancer drugs and gene transport to tumors. They are small enough to extravasate into solid tumors, where they slowly release their therapeutic load by passive leakage or biodegradation. Using smart nanocarriers, the rate of release of the entrapped therapeutic(s) can be increased, and greater exposure of the tumor cells to the therapeutics can be achieved when the nanocarriers are exposed to certain internally (enzymes, pH, and temperature) or externally (light, magnetic field, and ultrasound) applied stimuli that trigger the release of their load in a safe and controlled manner, spatially and temporally. This review gives a comprehensive overview of recent research findings on the different types of stimuli-responsive nanocarriers and their application in cancer treatment with a particular focus on ultrasound.
RESUMO
PURPOSE: In vivo evaluation of good manufacturing practice-grade targeted liposomal doxorubicin (TVT-DOX), bound to a CD13 isoform expressed on the vasculature of solid tumors, in human tumor xenografts of neuroblastoma, ovarian cancer, and lung cancer. EXPERIMENTAL DESIGN: Mice were implanted with lung, ovarian, or neuroblastoma tumor cells via the pulmonary, peritoneal, or orthotopic (adrenal gland) routes, respectively, and treated, at different days post inoculation, with multiple doses of doxorubicin, administered either free or encapsulated in untargeted liposomes (Caelyx) or in TVT-DOX. The effect of TVT-DOX treatment on tumor cell proliferation, viability, apoptosis, and angiogenesis was studied by immunohistochemical analyses of neoplastic tissues and using the chick embryo chorioallantoic membrane assay. RESULTS: Compared with the three control groups (no doxorubicin, free doxorubicin, or Caelyx), statistically significant improvements in survival was seen in all three animal models following treatment with 5 mg/kg (maximum tolerated dose) of TVT-DOX, with long-term survivors occurring in the neuroblastoma group; increased survival was also seen at a dose of 1.7 mg/kg in mice bearing neuroblastoma or ovarian cancer. Minimal residual disease after surgical removal of neuroblastoma primary mass, and the enhanced response to TVT-DOX, was visualized and quantified by bioluminescence imaging and with magnetic resonance imaging. When treated with TVT-DOX, compared with Caelyx, all three tumor models, as assayed by immunohistochemistry and chorioallantoic membrane, showed statistically significant reductions in cell proliferation, blood vessel density, and microvessel area, showing increased cell apoptosis. CONCLUSION: TVT-DOX should be evaluated as a novel angiostatic strategy for adjuvant therapy of solid tumors.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Animais , Antígenos CD13/metabolismo , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Imuno-Histoquímica , Lipossomos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma, the most common solid tumor of infancy derived from the sympathetic nervous system, continues to present a formidable clinical challenge. Sterically stabilized immunoliposomes (SIL) have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. We showed that SIL loaded with doxorubicin (DXR) and targeted to the disialoganglioside receptor GD(2) [aGD(2)-SIL(DXR)] led to a selective inhibition of the metastatic growth of experimental models of human neuroblastoma. By coupling NGR peptides that target the angiogenic endothelial cell marker aminopeptidase N to the surface of DXR-loaded liposomes [NGR-SL(DXR)], we obtained tumor regression, pronounced destruction of the tumor vasculature, and prolonged survival of orthotopic neuroblastoma xenografts. Here, we showed good liposome stability, long circulation times, and enhanced time-dependent tumor accumulation of both the carrier and the drug. Antivascular effects against animal models of lung and ovarian cancer were shown for formulations of NGR-SL(DXR). In the chick embryo chorioallantoic assay, NGR-SL(DXR) substantially reduced the angiogenic potential of various neuroblastoma xenografts, with synergistic inhibition observed for the combination of NGR-SL(DXR) with aGD(2)-SIL(DXR). A significant improvement in antitumor effects was seen in neuroblastoma-bearing animal models when treated with the combined formulations compared with control mice or mice treated with either tumor- or vascular-targeted liposomal formulations, administered separately. The combined treatment resulted in a dramatic inhibition of tumor endothelial cell density. Long-term survivors were obtained only in animals treated with the combined tumor- and vascular-targeted formulations, confirming the pivotal role of combination therapies in treating aggressive metastatic neuroblastoma.
Assuntos
Doxorrubicina/administração & dosagem , Proteínas da Mielina/metabolismo , Neuroblastoma/irrigação sanguínea , Neuroblastoma/tratamento farmacológico , Receptores de Superfície Celular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/farmacocinética , Feminino , Proteínas Ligadas por GPI , Gangliosídeos/administração & dosagem , Gangliosídeos/metabolismo , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Camundongos SCID , Proteínas da Mielina/administração & dosagem , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neuroblastoma/metabolismo , Receptor Nogo 1 , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacocinética , Receptores de Superfície Celular/administração & dosagem , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Site-specific delivery of anticancer agents to tumors represents a promising therapeutic strategy because it increases efficacy and reduces toxicity to normal tissues compared with untargeted drugs. Sterically stabilized immunoliposomes (SIL), guided by antibodies that specifically bind to well internalizing antigens on the tumor cell surface, are effective nanoscale delivery systems capable of accumulating large quantities of anticancer agents at the tumor site. The epithelial cell adhesion molecule (EpCAM) holds major promise as a target for antibody-based cancer therapy due to its abundant expression in many solid tumors and its limited distribution in normal tissues. We generated EpCAM-directed immunoliposomes by covalently coupling the humanized single-chain Fv antibody fragment 4D5MOCB to the surface of sterically stabilized liposomes loaded with the anticancer agent doxorubicin. In vitro, the doxorubicin-loaded immunoliposomes (SIL-Dox) showed efficient cell binding and internalization and were significantly more cytotoxic against EpCAM-positive tumor cells than nontargeted liposomes (SL-Dox). In athymic mice bearing established human tumor xenografts, pharmacokinetic and biodistribution analysis of SIL-Dox revealed long circulation times in the blood with a half-life of 11 h and effective time-dependent tumor localization, resulting in up to 15% injected dose per gram tissue. These favorable pharmacokinetic properties translated into potent antitumor activity, which resulted in significant growth inhibition (compared with control mice), and was more pronounced than that of doxorubicin alone and nontargeted SL-Dox at low, nontoxic doses. Our data show the promise of EpCAM-directed nanovesicular drug delivery for targeted therapy of solid tumors.
Assuntos
Moléculas de Adesão Celular/antagonistas & inibidores , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Células Epiteliais/metabolismo , Nanotecnologia/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antígenos de Neoplasias/metabolismo , Disponibilidade Biológica , Moléculas de Adesão Celular/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colesterol/análogos & derivados , Colesterol/metabolismo , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Endocitose/efeitos dos fármacos , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Lipossomos , CamundongosRESUMO
The central problem in cancer chemotherapy is the severe toxic side effects of anticancer drugs on healthy tissues. The use of liposomes as drug delivery vehicles for antitumour therapeutics has great potential to revolutionise the future of cancer therapy. As tumour architecture causes liposomes to preferentially accumulate at the tumour site, their use as drug carriers results in the localization of a greater amount of the loaded drug at the tumour site, thus improving cancer therapy and reducing the harmful non-specific side effects of chemotherapeutics. In addition, targeting of liposomal anticancer drugs to antigens expressed or over-expressed on tumour cells provides a very efficient system for increasing the therapeutic indices of the drugs. Animal models allow detailed examination of molecular and physiological basis of diseases and offer a frontline testing system for studying the involvement of specific genes and the efficacy of novel therapeutic approaches. Until recently, the most resorted experimental model of paediatric Neuroblastoma (NB) tumour is the subcutaneous xenograft in nude mice. However, the main disadvantage of this animal model is that it does not reflect the metastatic potential of NB cells, ultimately responsible for poor patient survival. A more realistic view of the clinical potential of targeted therapies could be obtained if a tumour model were available that better reflects the growth of advanced NB in children (i.e. large adrenal gland tumours and multiple small metastatic lesions). All current data support this concept and recommend that orthotopic implantation of tumour cells in recipient animals is mandatory for studies of tumour progression, angiogenesis, invasion, and metastasis. This review will focus on the description of the most clinically relevant animal models established to test the efficacy of targeted liposomal anti-tumour formulations for the treatment of Neuroblastoma.
Assuntos
Antineoplásicos/administração & dosagem , Neuroblastoma/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Humanos , Ligantes , Lipossomos , Transplante de Neoplasias , Neuroblastoma/imunologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/uso terapêutico , Transplante HeterólogoRESUMO
Nanoscale drug delivery systems, such as sterically stabilized immunoliposomes binding to internalizing tumor-associated antigens, can increase therapeutic efficacy and reduce toxicity to normal tissues compared with nontargeted liposomes. The epithelial cell adhesion molecule (EpCAM) is of interest as a ligand for targeted drug delivery because it is abundantly expressed in solid tumors but shows limited distribution in normal tissues. To generate EpCAM-specific immunoliposomes for targeted cancer therapy, the humanized single-chain Fv antibody fragment 4D5MOCB was covalently linked to the exterior of coated cationic liposomes. As anticancer agent, we encapsulated the previously described antisense oligonucleotide 4625 specific for both bcl-2 and bcl-xL. The EpCAM-targeted immunoliposomes (SIL25) showed specific binding to EpCAM-overexpressing tumor cells, with a 10- to 20-fold increase in binding compared with nontargeted control liposomes. No enhanced binding was observed on EpCAM-negative control cells. On cell binding, SIL25 was efficiently internalized by receptor-mediated endocytosis, ultimately leading to down-regulation of both bcl-2 and bcl-xL expression on both the mRNA and protein level, which resulted in enhanced tumor cell apoptosis. In combination experiments, the use of SIL25 led to a 2- to 5-fold sensitization of EpCAM-positive tumor cells of diverse origin to death induction by doxorubicin. Our data show the promise of EpCAM-specific drug delivery systems, such as antisense-loaded immunoliposomes, for targeted cancer therapy.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Doxorrubicina/farmacologia , Neoplasias/terapia , Oligonucleotídeos Antissenso/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína bcl-X/genética , Antígenos de Neoplasias/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo , Sistemas de Liberação de Medicamentos , Endocitose/fisiologia , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Lipossomos/administração & dosagem , Lipossomos/metabolismo , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/terapia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Oligonucleotídeos Antissenso/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/terapia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/biossínteseRESUMO
Some formulations of liposomal doxorubicin with intermediate rates of drug release have shown increased levels of toxicity in mice. Because antibody-mediated targeting of liposomal drugs influences the pharmacokinetics, mechanism of uptake, and selectivity of the associated drugs, we hypothesized that anti-CD19-mediated targeting of liposomal doxorubicin might moderate the toxicity of the problem formulations. Phosphatidylcholine/cholesterol liposomal formulations of doxorubicin having faster, intermediate, and slower drug release rates were prepared by altering the fatty acyl chain length or degree of saturation of the phosphatidylcholine component. Pharmacokinetic and biodistribution studies and in vivo drug release rates were determined in mice using liposomes dual labeled with [3H]cholesteryl hexadecylether and [14C]doxorubicin. Therapeutic studies were done in xenograft models of human B lymphoma (Namalwa cells). The rate of clearance of the liposomal lipid was similar for all formulations (average t1/2, 18 hours), but the rate of clearance of doxorubicin was dependent on the release rate of the formulation (t1/2, 2-315 hours). Liposomes with the slowest drug release rates showed no toxicity and exhibited therapeutic activity that was superior to the other formulations when targeted with anti-CD19; liposomes with the most rapid drug release rates also showed no toxicity but showed little therapeutic effect even when targeted. Liposomes with intermediate drug release rates exhibited varying degrees of toxicity. The toxicities could be reduced and even overcome by targeting with anti-CD19 antibodies. For these formulations, therapeutic effects were intermediate between those found for liposomes with the fastest and slowest drug release rates.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antígenos CD19/imunologia , Doxorrubicina/uso terapêutico , Linfoma de Células B/tratamento farmacológico , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacocinética , Anticorpos Monoclonais/química , Área Sob a Curva , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Lipossomos/química , Linfoma de Células B/patologia , Camundongos , Camundongos SCID , Fosfolipídeos/química , Análise de Sobrevida , Distribuição Tecidual , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Pharmacokinetic studies on liposomal drugs have previously measured total drug levels in tumors, which include non-bioavailable drug. However, drugs must be released from liposomes to have activity. We have developed a method for measuring levels of bioavailable (released) doxorubicin in vivo in tumors that will allow therapeutic activity to be correlated with bioavailable drug levels. EXPERIMENTAL DESIGN: Mice orthotopically implanted with mammary carcinoma (4T1) were injected i.v. 10 days after implantation with free doxorubicin or formulations of liposomal doxorubicin with different drug release rates. Tumors were excised at various times after injection, and total tumor doxorubicin levels were determined by acidified isopropanol extraction of whole tumor homogenates. Bioavailable doxorubicin levels were determined by extraction of doxorubicin from isolated tumor nuclei. RESULTS: Free doxorubicin had high levels of bioavailability in tumor tissue; 95% of the total doxorubicin in tumors was bound to nuclear DNA by 24 hours after injection. Administration of Doxil, a slow release liposomal formulation of doxorubicin, gave an area under the time-versus-concentration curve (AUC) for total doxorubicin 7 days after injection that was 87-fold higher than that obtained for free doxorubicin, and 49% of the liposomal doxorubicin was bioavailable. For liposomes with a more rapid doxorubicin release rate, by 7 days after injection, the AUC(0-7 days) for total doxorubicin was only 14-fold higher than that for free doxorubicin and only 27% of liposomal doxorubicin was bioavailable. CONCLUSIONS: This technique allows correlations to be made between drug bioavailability and therapeutic activity and will help in the rational design of drug carriers.
Assuntos
Antibióticos Antineoplásicos/farmacocinética , Núcleo Celular/metabolismo , Doxorrubicina/farmacocinética , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/metabolismo , Animais , Antibióticos Antineoplásicos/química , Área Sob a Curva , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Doxorrubicina/química , Feminino , Lipossomos/química , Camundongos , Camundongos Endogâmicos BALB C , Fatores de TempoRESUMO
Direct experimental proof has been sought for the hypothesis that liposomal drugs targeted against internalizing epitopes (e.g., CD19) will have higher therapeutic efficacies than those targeted against noninternalizing epitopes (e.g., CD20). Anti-CD19-targeted liposomes were rapidly internalized into human B-lymphoma (Namalwa) cells, whereas those targeted with anti-CD20 were not internalized. Similar in vitro binding and cytotoxicity were observed for anti-CD19-targeted and anti-CD20-targeted liposomal formulations of doxorubicin (DXR). Therapeutic experiments were performed in severe combined immunodeficient mice inoculated i.v. with Namalwa cells. Administration of single i.v. doses of DXR-loaded anti-CD19-targeted liposomes resulted in significantly greater survival times than either DXR-loaded anti-CD20-targeted liposomes or nontargeted liposomes. The therapeutic advantage of targeting internalizing versus noninternalizing epitopes has been directly demonstrated.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antígenos CD19/imunologia , Antígenos CD20/imunologia , Epitopos/metabolismo , Imunotoxinas/farmacocinética , Lipossomos/farmacocinética , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Doxorrubicina/imunologia , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Epitopos/imunologia , Feminino , Humanos , Imunofenotipagem , Imunotoxinas/imunologia , Imunotoxinas/farmacologia , Lipossomos/imunologia , Lipossomos/farmacologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Camundongos , Camundongos SCID , Microscopia Confocal , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma (NB) is the most common extracranial solid tumor in children. Intensive therapeutic intervention does not prolong the overall disease-free survival rate for this tumor. NB tumor, but not normal tissues, overexpress the disialoganglioside (GD(2)) at the cell surface. Anti-GD(2) whole antibodies (aGD(2)) or their corresponding Fab' fragments were covalently coupled to Stealth immunoliposomes (aGD(2)-SIL or Fab'-SIL), and their binding to GD(2)-positive NB cells was measured. Cytotoxic effects of immunoliposomes loaded with doxorubicin (DXR) were determined. Radiolabelled immunoliposomes were used to evaluate pharmacokinetics (PK). The effectiveness of different liposomal formulations of DXR was tested against a metastatic model of human NB in nude mice. aGD(2)-SIL and Fab'-SIL showed concentration-dependent specific binding and uptake by GD(2)-positive NB cells. DXR entrapped in aGD(2)-SIL or Fab'-SIL (aGD(2)-SIL[DXR], Fab'-SIL[DXR]) showed higher cytotoxicities than nontargeted liposomes (SL[DXR]). DXR-loaded Fab'-SIL (Fab'-SIL[DXR]) also showed specific binding, uptake, and cytotoxic effects on several GD(2)-positive NB cells in vitro. PK studies showed that Fab'-SIL had long-circulating profiles in blood compared with aGD(2)-SIL, with the PK profile for Fab'-SIL being almost identical to that obtained with nontargeted Stealth liposomes. In vivo, long-term survivors were obtained in mice treated with Fab'-SIL[DXR] but not in untreated animals, or those treated with free aGD(2) Fab', Fab'-SIL (no drug), free-DXR, or nontargeted Stealth liposomes[DXR] (no antibody; P < 0.0001). Immunoliposomes containing DXR prevented the establishment and growth of the tumor in all of the organs examined. In conclusion, Fab'-SIL[DXR] formulations led to the total inhibition of metastatic growth of human NB in a nude mouse metastatic model. This formulation should receive clinical evaluation as adjuvant therapy of NB.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Gangliosídeos/imunologia , Fragmentos Fab das Imunoglobulinas , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Animais , Divisão Celular/efeitos dos fármacos , Doxorrubicina/uso terapêutico , Portadores de Fármacos , Humanos , Lipossomos , Camundongos , Camundongos Nus , Células Tumorais CultivadasRESUMO
The poor selective toxicity of chemotherapeutic anticancer drugs leads to dose-limiting side effects that compromise clinical outcome. Solid tumors recruit new blood vessels to support tumor growth, and unique epitopes expressed on tumor endothelial cells can function as targets for the anti-angiogenic therapy of cancer. An NGR peptide that targets aminopeptidase N, a marker of angiogenic endothelial cells, was coupled to the surface of liposomal doxorubicin (NGR-SL[DXR]) and was used to treat orthotopic neuroblastoma (NB) xenografts in SCID mice. Pharmacokinetic studies indicated that liposomes coupled to NGR peptide had long-circulating profiles in blood. Their uptake into NB tumor was time dependent, being at least 10 times higher than that of nontargeted liposomes (SL[DXR]) after 24 h, with DXR spreading outside the blood vessels and into the tumors. No uptake was observed into tumors of mice treated with the mismatched peptide ARA-targeted SL[DXR]. Tumor-specific DXR uptake was completely blocked when mice were coinjected with a 50-fold molar excess of the soluble NGR peptide. Adrenal tumor-bearing mice treated with 2 mg/kg/week/x3 of NGR-SL[DXR] partly outlived the control mice (P < 0.001), whereas doses > 3 mg/kg/week/x3 were toxic. Histopathological analysis of cryosections taken from treated mice revealed pronounced destruction of the tumor vasculature with a marked decreased in vessel density. Double staining of tumors with terminal deoxynucleotidyl transferase-mediated nick end labeling and antifactor VIII antibody or antihuman NB demonstrated endothelial cell apoptosis in the vasculature, as well as increased tumor cell apoptosis. Moreover, mice injected with 3 mg/kg/week/x3 of NGR-SL[DXR] displayed rapid tumor regression, as well as inhibition of metastases growth (P = 0.0002). One day after the third treatment, four of six mice showed no evidence of tumors, and the two others showed a >80% reduction in tumor mass and a >90% suppression of blood vessel density (P < 0.01). In contrast, mice treated with ARA-SL[DXR] formed large well-vascularized tumors. Finally, a metronomic administration of NGR-SL[DXR] (1 mg/kg/every other 2 days x 9) induced complete tumor eradication in all animals (P < 0.0001). Our strategy markedly enhanced the therapeutic index of DXR and enabled metronomic administration of therapeutic doses. A dual mechanism of action is proposed: indirect tumor cell kill via the destruction of tumor endothelium by NGR-targeted liposomes and direct tumor cell kill via localization of liposomal DXR to the tumor interstitial space. This combined strategy has the potential to overcome some major limitations of conventional chemotherapy.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Doxorrubicina/administração & dosagem , Neovascularização Patológica/tratamento farmacológico , Neuroblastoma/irrigação sanguínea , Neuroblastoma/tratamento farmacológico , Sequência de Aminoácidos , Inibidores da Angiogênese/farmacocinética , Animais , Antígenos CD13/metabolismo , Doxorrubicina/farmacocinética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Feminino , Humanos , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The pharmacokinetics (PK), biodistribution (BD), and therapeutic activity of pegylated liposomal doxorubicin formulations with different drug release rates were studied in an orthotopic 4T1 murine mammary carcinoma model. The focus of these experiments was to study the effects of different release rates on the accumulation of liposomal lipid and doxorubicin (DXR) into the tumor and cutaneous tissues of mice (skin and paws). These tissues were chosen because the clinical formulation of pegylated liposomal doxorubicin (Caelyx)/Doxi) causes mucocutaneous reactions such as palmar-plantar erythrodysesthesia (PPE). Liposomes with different doxorubicin (DXR) leakage rates were prepared by altering liposome fluidity through changing the fatty acyl chain length and/or degree of saturation of the phosphatidylcholine component of the liposome. Liposomes with fast, intermediate, and slow rates of drug release were studied. The plasma PK of the liposomal lipid was similar for all formulations, while the plasma PK of the DXR component was dependent on the liposome formulation. Liposomal lipid accumulated to similar levels in tumor and cutaneous tissues for all three formulations tested, while the liposomes with the slowest rates of DXR release produced the highest DXR concentrations in both cutaneous tissues and in tumor. Liposomes with the fastest drug release rates resulted in low DXR concentrations in cutaneous tissues and tumor. The formulation with intermediate release rates produced unexpected toxicity that was not related to the lipid content of the formulation. The liposomes with the slowest rate of drug leakage had the best therapeutic activity of the formulations tested.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/farmacologia , Doxorrubicina/farmacocinética , Lipossomos/química , Polietilenoglicóis/química , Animais , Antibióticos Antineoplásicos/sangue , Antibióticos Antineoplásicos/química , Área Sob a Curva , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Doxorrubicina/sangue , Doxorrubicina/química , Feminino , Meia-Vida , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Fosfatidilcolinas/química , Distribuição Tecidual , Temperatura de TransiçãoRESUMO
The influence of diameter on the pharmacokinetic and biodistribution of STEALTH liposomes into the tumor (4T1 murine mammary carcinoma) and cutaneous tissues (skin and paws) of mice was studied to ascertain the time course of liposome accumulation and to determine if a preferential accumulation of liposomes into tumor over skin or paws could be achieved by altering liposome size. These tissues were chosen as the dose-limiting toxicity for Caelyx/Doxil in humans is palmar-plantar erythrodysesthesia, a cutaneous toxicity. We examined liposomes of four diameters: 82, 101, 154, or 241 nm. Liposomes with the three smallest diameters showed similar accumulation profiles that were significantly higher than the largest liposomes in all three tissues of interest. We were unable to achieve a preferential accumulation of liposomes into tumor over skin or paws based on size alone, as evidenced by the tumor to skin and tumor to paw ratios. However, there were differences in the time courses of liposome accumulation in these three tissues. Liposome levels plateaued in tumors and paws within 24 h, whereas skin levels plateaued between 24 and 48 h. The therapeutic activity of liposomal doxorubicin of three diameters (100, 157, and 255 nm) was tested in the same model. All formulations delayed tumor growth, with liposomes of 100 or 157 nm being equally efficacious and superior to liposomes of 255 nm.
Assuntos
Lipossomos , Neoplasias Experimentais/metabolismo , Farmacocinética , Pele/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Distribuição TecidualRESUMO
PURPOSE: We have reported previously that successful immunoliposomal drug therapy with liposomal doxorubicin (DXR) against xenograft B-lymphoma models required targeting against an internalizing B-cell antigen, CD19 (P. Sapra and T. M. Allen. Cancer Res 2002;62:7190-4.). Here we compare targeting of immunoliposomal formulations of DXR with vincristine (VCR) targeted against CD19 versus a noninternalizing (CD20) epitope. We also examine the effect of targeting immunoliposomes with antibody combinations in an attempt to increase the total number of binding sites (apparent antigen density) at the target cell surface. EXPERIMENTAL DESIGN: Cell association of immunoliposomes (CD19-targeted, CD20-targeted, or combinations of the two) with human B-cell lymphoma (Namalwa) cells were studied using radiolabeled liposomes. Therapeutic efficacy of the same formulations was determined in a severe combined immunodeficient murine model. RESULTS: Therapeutic results in severe combined immunodeficient mice bearing Namalwa cells administered anti-CD20-targeted liposomal DXR were barely improved over those found for nontargeted liposomal DXR or free DXR but, surprisingly, administration of anti-CD20-targeted liposomal VCR resulted in a significantly improved therapeutic outcome compared with nontargeted liposomal VCR, free VCR, or anti-CD20-targeted liposomal DXR. Treatment of murine B lymphoma with single injections of combinations of anti-CD19- and anti-CD20-targeted liposomal VCR led to cures in 70% of mice. However, mice injected with similar combinations of liposomal DXR did not have improved survival rates over anti-CD19-targeted liposomal DXR by itself. CONCLUSIONS: The success of immunoliposomal therapy in combination regimens varies with the type of encapsulated drug and the nature of the target epitopes.
Assuntos
Antígenos CD19/química , Antígenos CD20/química , Antineoplásicos/uso terapêutico , Terapia Combinada , Imunoterapia/métodos , Lipossomos/química , Linfoma de Células B/terapia , Animais , Antibióticos Antineoplásicos/uso terapêutico , Anticorpos Monoclonais/química , Sobrevivência Celular , Relação Dose-Resposta a Droga , Doxorrubicina/uso terapêutico , Portadores de Fármacos , Epitopos , Feminino , Ligantes , Camundongos , Camundongos SCID , Transplante de Neoplasias , Resultado do Tratamento , Vincristina/uso terapêuticoRESUMO
PURPOSE: Monoclonal antibody-mediated targeting of liposomal anticancer drugs to surface antigens expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. In a murine model of human B-cell lymphoma, we have made in vitro and in vivo comparisons of long-circulating sterically stabilized (Stealth) immunoliposome (SIL) formulations of two anticancer drugs, vincristine (VCR) and doxorubicin (DXR), with different mechanisms of action and drug release rates. EXPERIMENTAL DESIGN: SIL formulations of VCR or DXR were conjugated to the monoclonal antibody anti-CD19 (SIL[alphaCD19]) or its Fab' fragments (SIL[Fab']). Specific binding of SILs to Namalwa cells was studied using radiolabeled liposomes, and cytotoxicities of DXR- or VCR-loaded SILs were quantitated by a tetrazolium assay. Pharmacokinetic and drug leakage experiments were performed in mice using dual-labeled liposomes, and the therapeutic responses of SILs were evaluated in a Namalwa (human B lymphoma) cell xenograft model. RESULTS: SIL[alphaCD19] or SIL[Fab'] had higher association with and cytotoxicity against Namalwa cells than nontargeted liposomes. SIL[Fab'] had longer circulation times than SIL[alphaCD19], and VCR had faster release rates from the liposomes than DXR. SIL formulations of either VCR or DXR had significantly better therapeutic outcomes than nontargeted liposomes or free drugs. SILs loaded with VCR were superior to those loaded with DXR. SIL[Fab'] had better therapeutic outcomes than SIL[alphaCD19] for the drug DXR but were equally efficacious for the drug VCR. CONCLUSIONS: Treatment of a B lymphoma model with single injections of anti-CD19-targeted liposomal formulations of VCR resulted in high levels of response and long-term survivors. Responses to anti-CD19-targeted liposomal DXR were more modest, although the longer circulation times of SIL[Fab'] versus SIL[alphaCD19] led to superior therapeutics for DXR-loaded immunoliposomes.