RESUMO
Therapeutics at or close to the nanoscale, such as liposomal irinotecan, offer significant promise for the treatment of solid tumors. Their potential advantage over the unencapsulated or free form of the drug is due in part to their altered biodistribution. For slow and sustained release, significant optimization of formulation is needed to achieve the required level of stability and allow long-term storage of the drug product. Gradient-based liposomal formulation of camptothecins such as irinotecan poses unique challenges owing to the camptothecin- and acid-catalyzed hydrolysis of phospholipid esters in the inner monolayer of the liposomal membrane. We demonstrated that a narrow set of conditions related to the external pH, temperature, intraliposomal concentration, identity of the drug-trapping agent, physical form of the drug inside the liposomes, and final drug load have a marked impact on the stability of the liposome phospholipid membrane. The physical form of the drug inside the liposome was shown to be an insoluble gel with an irinotecan-to-sulfate ratio approximating 1:1, reducing the potential for irinotecan-catalyzed phospholipid hydrolysis in the internal phospholipid monolayer. As a result of this work, a stable and active liposome formulation has been developed that maintains phospholipid chemical stability following long-term storage at 2-8°C.
Assuntos
Lipossomos , Fosfolipídeos , Irinotecano , Estabilidade de Medicamentos , Distribuição Tecidual , Camptotecina , CatáliseRESUMO
Antibody-directed nanotherapeutics (ADNs) represent a promising delivery platform for selective delivery of an encapsulated drug payload to the site of disease that improves the therapeutic index. Although both single-chain Fv (scFv) and Fab antibody fragments have been used for targeting, no platform approach applicable to any target has emerged. scFv can suffer from intrinsic instability, and the Fabs are challenging to use due to native disulfide over-reduction and resulting impurities at the end of the conjugation process. This occurs because of the close proximity of the disulfide bond connecting the heavy and light chain to the free cysteine at the C-terminus, which is commonly used as the conjugation site. Here we show that by engineering an alternative heavy chain-light chain disulfide within the Fab, we can maintain efficient conjugation while eliminating the process impurities and retaining stability. We have demonstrated the utility of this technology for efficient ADN delivery and internalization for a series of targets, including EphA2, EGFR, and ErbB2. We expect that this technology will be broadly applicable for targeting of nanoparticle encapsulated payloads, including DNA, mRNA, and small molecules.
Assuntos
Nanopartículas , Anticorpos de Cadeia Única , Dissulfetos/química , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Nanopartículas/químicaRESUMO
Ephrin receptor A2 (EphA2) is a member of the Ephrin/Eph receptor cell-to-cell signaling family of molecules, and it plays a key role in cell proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, and its expression is in many cases associated with poor prognosis. We recently developed a novel EphA2-targeting antibody-directed nanotherapeutic encapsulating a labile prodrug of docetaxel (EphA2-ILs-DTXp) for the treatment of EphA2-expressing malignancies. Here, we characterized the expression of EphA2 in bladder cancer using immunohistochemistry in 177 human bladder cancer samples and determined the preclinical efficacy of EphA2-ILs-DTXp in four EphA2-positive patient-derived xenograft (PDX) models of the disease, either as a monotherapy, or in combination with gemcitabine. EphA2 expression was detected in 80-100% of bladder cancer samples and correlated with shorter patient survival. EphA2 was found to be expressed in tumor cells and/or tumor-associated blood vessels in both primary and metastatic lesions with a concordance rate of approximately 90%. The EphA2-targeted antibody-directed nanotherapeutic EphA2-ILs-DTXp controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing in all four EphA2-positive bladder cancer PDX models. Combination of EphA2-ILs-DTXp and gemcitabine in one PDX model led to improved tumor growth control compared to monotherapies or the combination of free docetaxel and gemcitabine. These data demonstrating the prevalence of EphA2 in bladder cancers and efficacy of EphA2-ILs-DTXp in PDX models support the clinical exploration of EphA2 targeting in bladder cancer.
RESUMO
Ephrin A2 targeted immunoliposomes incorporating pH-sensitive taxane prodrugs were developed for sustained delivery of active drug to solid tumors. Here we describe the systematic formulation development and characterization of these immunoliposomes. We synthesized both paclitaxel and docetaxel prodrugs to formulate as ephrin A2-targeted liposomes stabilized in the aqueous core with sucroseoctasulfate (SOS). The optimized lipid formulation was comprised of egg-sphingomyelin, cholesterol, and polyethylene glycol distearoyl glycerol (PEG-DSG). The formulations examined had a high efficiency of prodrug encapsulation (as high as 114â¯mol% taxane per mole phospholipid) and subsequent stability (>3â¯years at 2-8⯰C). The taxane prodrug was stabilized with extraliposomal citric acid and subsequently loaded into liposomes containing a gradient of SOS, resulting in highly stable SOS-drug complexes being formed inside the liposome. The internal prodrug and SOS concentrations were optimized for their impact on in vivo drug release and drug degradation. Cryo-electron microscope images revealed dense prodrug-SOS complex in the aqueous core of the immunoliposomes. Ephrin A2-targeted taxane liposomes exhibited sub-nanomolar (0.69â¯nM) apparent equilibrium dissociation constant toward the extracellular domain of the ephrin A2 receptor, long circulation half-life (8-12â¯h) in mouse plasma, a release rate dependent on intraliposomal drug concentration and stable long-term storage. At an equitoxic dose of 50â¯mg taxane/kg, ephrin A2-targeted liposomal prodrug showed greater antitumor activity than 10â¯mg/kg of docetaxel in A549 non-small cell lung, as well as MDA-MB-436 and SUM149 triple negative breast cancer xenograft models. The lead molecule entered a Phase I clinical trial in patients with solid tumors (NCT03076372).
Assuntos
Antineoplásicos/administração & dosagem , Hidrocarbonetos Aromáticos com Pontes/administração & dosagem , Portadores de Fármacos/química , Efrina-A2/metabolismo , Nanopartículas/química , Pró-Fármacos/administração & dosagem , Taxoides/administração & dosagem , Células A549 , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/farmacocinética , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular Tumoral , Composição de Medicamentos , Liberação Controlada de Fármacos , Feminino , Humanos , Lipossomos , Camundongos Nus , Tamanho da Partícula , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Ligação Proteica , Taxoides/química , Taxoides/farmacocinética , Taxoides/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Nanopartículas/uso terapêutico , Receptor EphA2/metabolismo , Animais , Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Docetaxel/sangue , Docetaxel/química , Docetaxel/farmacocinética , Docetaxel/uso terapêutico , Humanos , Lipossomos , Camundongos Endogâmicos NOD , Camundongos SCID , Taxoides/farmacologia , Taxoides/uso terapêutico , Distribuição Tecidual/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Purpose: Therapeutic nanoparticles are designed to deliver their drug payloads through enhanced permeability and retention (EPR) in solid tumors. The extent of EPR and its variability in human tumors is highly debated and has been proposed as an explanation for variable responses to therapeutic nanoparticles in clinical studies.Experimental Design: We assessed the EPR effect in patients using a 64Cu-labeled nanoparticle, 64Cu-MM-302 (64Cu-labeled HER2-targeted PEGylated liposomal doxorubicin), and imaging by PET/CT. Nineteen patients with HER2-positive metastatic breast cancer underwent 2 to 3 PET/CT scans postadministration of 64Cu-MM-302 as part of a clinical trial of MM-302 plus trastuzumab with and without cyclophosphamide (NCT01304797).Results: Significant background uptake of 64Cu-MM-302 was observed in liver and spleen. Tumor accumulation of 64Cu-MM-302 at 24 to 48 hours varied 35-fold (0.52-18.5 %ID/kg), including deposition in bone and brain lesions, and was independent of systemic plasma exposure. Computational analysis quantified rates of deposition and washout, indicating peak liposome deposition at 24 to 48 hours. Patients were classified on the basis of 64Cu-MM-302 lesion deposition using a cut-off point that is comparable with a response threshold in preclinical studies. In a retrospective exploratory analysis of patient outcomes relating to drug levels in tumor lesions, high 64Cu-MM-302 deposition was associated with more favorable treatment outcomes (HR = 0.42).Conclusions: These findings provide important evidence and quantification of the EPR effect in human metastatic tumors and support imaging nanoparticle deposition in tumors as a potential means to identify patients well suited for treatment with therapeutic nanoparticles. Clin Cancer Res; 23(15); 4190-202. ©2017 AACR.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/análogos & derivados , Nanopartículas/administração & dosagem , Adolescente , Adulto , Idoso , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos da radiação , Radioisótopos de Cobre/administração & dosagem , Radioisótopos de Cobre/química , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Feminino , Humanos , Fígado/diagnóstico por imagem , Fígado/efeitos dos fármacos , Pessoa de Meia-Idade , Nanopartículas/química , Metástase Neoplásica , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Receptor ErbB-2/sangue , Baço/diagnóstico por imagem , Baço/patologia , Trastuzumab/administração & dosagemRESUMO
Antibody-targeted nanoparticles have great promise as anti-cancer drugs; however, substantial developmental challenges of antibody modules prevent many candidates from reaching the clinic. Here, we describe a robust strategy for developing an EphA2-targeting antibody fragment for immunoliposomal drug delivery. A highly bioactive single-chain variable fragment (scFv) was engineered to overcome developmental liabilities, including low thermostability and weak binding to affinity purification resins. Improved thermostability was achieved by modifying the framework of the scFv, and complementarity-determining region (CDR)-H2 was modified to increase binding to protein A resins. The results of our engineering campaigns demonstrate that it is possible, using focused design strategies, to rapidly improve the stability and manufacturing characteristics of an antibody fragment for use as a component of a novel therapeutic construct.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Efrina-A2/imunologia , Imunoconjugados/imunologia , Nanopartículas , Anticorpos de Cadeia Única/imunologia , Animais , Humanos , Região Variável de Imunoglobulina/imunologia , Engenharia de Proteínas/métodos , Estabilidade Proteica , Receptor EphA2 , Anticorpos de Cadeia Única/biossínteseRESUMO
Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.
Assuntos
Citotoxinas , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Neoplasias/tratamento farmacológico , Anticorpos de Cadeia Única , Substituição de Aminoácidos , Animais , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Citotoxinas/química , Citotoxinas/farmacologia , Humanos , Camundongos , Neoplasias/imunologia , Proteínas Recombinantes , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
AIM: We sought to evaluate nanoliposomal irinotecan as an intravenous treatment in an orthotopic brain tumor model. MATERIALS & METHODS: Nanoliposomal irinotecan was administered intravenously in the intracranial U87MG brain tumor model in mice, and irinotecan and SN-38 levels were analyzed in malignant and normal tissues. Therapy studies were performed in comparison to free irinotecan and control treatments. RESULTS: Tissue analysis demonstrated favorable properties for nanoliposomal irinotecan, including a 10.9-fold increase in tumor AUC for drug compared with free irinotecan and 35-fold selectivity for tumor versus normal tissue exposure. As a therapy for orthotopic brain tumors, nanoliposomal irinotecan showed a mean survival time of 54.2 versus 29.5 days for free irinotecan. A total of 33% of the animals receiving nanoliposomal irinotecan showed no residual tumor by study end compared with no survivors in the other groups. CONCLUSION: Nanoliposomal irinotecan administered systemically provides significant pharmacologic advantages and may be an efficacious therapy for brain tumors.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Camptotecina/análogos & derivados , Lipossomos , Nanoestruturas , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacocinética , Neoplasias Encefálicas/metabolismo , Camptotecina/administração & dosagem , Camptotecina/farmacocinética , Camptotecina/uso terapêutico , Irinotecano , RatosRESUMO
AIM: The aim of this work is to evaluate combining targeting strategy and convection-enhanced delivery in brain tumor models by imaging quantum dot-immunoliposome hybrid nanoparticles. MATERIALS & METHODS: An EGF receptor-targeted, quantum dot-immunoliposome hybrid nanoparticle (QD-IL) was synthesized. In vitro uptake was measured by flow cytometry and intracellular localization was imaged by confocal microscopy. In the in vivo study, QD-ILs were delivered to intracranial xenografts via convection-enhanced delivery and fluorescence was monitored noninvasively in real-time. RESULTS: QD-ILs exhibited specific and efficient uptake in vitro and exhibited approximately 1.3- to 5.0-fold higher total fluorescence compared with nontargeted counterpart in intracranial brain tumor xenografts in vivo. CONCLUSION: QD-ILs serve as an effective imaging agent in vitro and in vivo, and the data suggest that ligand-directed liposomal nanoparticles in conjunction with convection-enhanced delivery may offer therapeutic benefits for glioblastoma treatment as a result of specific and efficient uptake by malignant cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Encéfalo/patologia , Sistemas de Liberação de Medicamentos , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Lipossomos/metabolismo , Pontos Quânticos/metabolismo , Animais , Encéfalo/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Convecção , Feminino , Glioblastoma/patologia , Humanos , Lipossomos/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pontos Quânticos/análiseRESUMO
BACKGROUND: Liposomal drug packaging is well established as an effective means for increasing drug half-life, sustaining drug activity, and increasing drug efficacy, whether administered locally or distally to the site of disease. However, information regarding the relative effectiveness of peripheral (distal) versus local administration of liposomal therapeutics is limited. This issue is of importance with respect to the treatment of central nervous system cancer, for which the blood-brain barrier presents a significant challenge in achieving sufficient drug concentration in tumors to provide treatment benefit for patients. METHODS: We compared the anti-tumor activity and efficacy of a nanoliposomal formulation of irinotecan when delivered peripherally by vascular route with intratumoral administration by convection-enhanced delivery (CED) for treating intracranial glioblastoma xenografts in athymic mice. RESULTS: Our results show significantly greater anti-tumor activity and survival benefit from CED of nanoliposomal irinotecan. In 2 of 3 efficacy experiments, there were animal subjects that experienced apparent cure of tumor from local administration of therapy, as indicated by a lack of detectable intracranial tumor through bioluminescence imaging and histopathologic analysis. Results from investigating the effectiveness of combination therapy with nanoliposomal irinotecan plus radiation revealed that CED administration of irinotecan plus radiation conferred greater survival benefit than did irinotecan or radiation monotherapy and also when compared with radiation plus vascularly administered irinotecan. CONCLUSIONS: Our results indicate that liposomal formulation plus direct intratumoral administration of therapeutic are important for maximizing the anti-tumor effects of irinotecan and support clinical trial evaluation of this therapeutic plus route of administration combination.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Camptotecina/análogos & derivados , Sistemas de Liberação de Medicamentos , Glioblastoma/tratamento farmacológico , Lipossomos , Nanopartículas , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Camptotecina/administração & dosagem , Convecção , Vias de Administração de Medicamentos , Feminino , Glioblastoma/mortalidade , Glioblastoma/patologia , História Antiga , Humanos , Técnicas Imunoenzimáticas , Injeções Intraperitoneais , Irinotecano , Camundongos , Camundongos Nus , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immunoliposomes provide a complementary, and in many instances advantageous, drug delivery strategy to antibody-drug conjugates. Their high carrying capacity of 20,000-150,000 drug molecules/liposome, allows for the use of a significantly broader range of moderate-to-high potency small molecule drugs when compared to the comparably few subnanomolar potency maytansinoid- and auristatin-based immunoconjugates. The multivalent display of 5-100 antibody fragments/liposome results in an avidity effect that can make use of even moderate affinity antibodies, as well as a cross-linking of cell surface receptors to induce the internalization required for intracellular drug release and subsequent activity. The underlying liposomal drug must be effectively engineered for long circulating pharmacokinetics and stable in vivo drug retention in order to allow for the drug to be efficiently delivered to the target tissue and take advantage of the site-specific bioavailability provided for by the targeting arm. In this chapter, we describe the rationale for engineering stable immunoliposome-based therapeutics, methods required for preparation of immunoliposomes, as well as for their physicochemical and in vivo characterization.
Assuntos
Antineoplásicos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Imunoconjugados/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Lipossomos/metabolismo , Nanomedicina/métodos , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos , Antineoplásicos/farmacocinética , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Fragmentos de Imunoglobulinas/química , Cinética , Lipídeos/química , Lipossomos/imunologia , Camundongos , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Tamanho da Partícula , Polietilenoglicóis/química , Engenharia de Proteínas/métodos , Ratos , Reagentes de Sulfidrila/químicaRESUMO
Achieving effective treatment outcomes for patients with glioblastoma (GBM) has been impeded by many obstacles, including the pharmacokinetic limitations of antitumor agents, such as topotecan (TPT). Here, we demonstrate that intravenous administration of a novel nanoliposomal formulation of TPT (nLS-TPT) extends the survival of mice with intracranial GBM xenografts, relative to administration of free TPT, because of improved biodistribution and pharmacokinetics of the liposome-formulated drug. In 3 distinct orthotopic GBM models, 3 weeks of biweekly intravenous therapy with nLS-TPT was sufficient to delay tumor growth and significantly extend animal survival, compared with treatment with free TPT (P ≤ .03 for each tumor tested). Analysis of intracranial tumors showed increased activation of cleaved caspase-3 and increased DNA fragmentation, both indicators of apoptotic response to treatment with nLS-TPT. These results demonstrate that intravenous delivery of nLS-TPT is a promising strategy in the treatment of GBM and support clinical investigation of this therapeutic approach.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Glioblastoma/tratamento farmacológico , Lipossomos , Nanotecnologia , Inibidores da Topoisomerase I/uso terapêutico , Topotecan/uso terapêutico , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Feminino , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Técnicas Imunoenzimáticas , Medições Luminescentes , Camundongos , Camundongos Nus , Taxa de Sobrevida , Distribuição Tecidual , Inibidores da Topoisomerase I/farmacocinética , Topotecan/farmacocinética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Canine spontaneous intracranial tumors bear striking similarities to their human tumor counterparts and have the potential to provide a large animal model system for more realistic validation of novel therapies typically developed in small rodent models. We used spontaneously occurring canine gliomas to investigate the use of convection-enhanced delivery (CED) of liposomal nanoparticles, containing topoisomerase inhibitor CPT-11. To facilitate visualization of intratumoral infusions by real-time magnetic resonance imaging (MRI), we included identically formulated liposomes loaded with Gadoteridol. Real-time MRI defined distribution of infusate within both tumor and normal brain tissues. The most important limiting factor for volume of distribution within tumor tissue was the leakage of infusate into ventricular or subarachnoid spaces. Decreased tumor volume, tumor necrosis, and modulation of tumor phenotype correlated with volume of distribution of infusate (Vd), infusion location, and leakage as determined by real-time MRI and histopathology. This study demonstrates the potential for canine spontaneous gliomas as a model system for the validation and development of novel therapeutic strategies for human brain tumors. Data obtained from infusions monitored in real time in a large, spontaneous tumor may provide information, allowing more accurate prediction and optimization of infusion parameters. Variability in Vd between tumors strongly suggests that real-time imaging should be an essential component of CED therapeutic trials to allow minimization of inappropriate infusions and accurate assessment of clinical outcomes.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Camptotecina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Glioma/tratamento farmacológico , Nanopartículas , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/veterinária , Camptotecina/administração & dosagem , Convecção , Modelos Animais de Doenças , Cães , Glioma/patologia , Glioma/veterinária , Irinotecano , Lipossomos , Imageamento por Ressonância MagnéticaRESUMO
Topotecan (TPT), a highly active anticancer camptothecin drug, would benefit from nanocarrier-mediated site-specific and intracellular delivery because of a labile lactone ring whose hydrolysis inactivates the drug, poor cellular uptake resulting from both lactone hydrolysis and a titratable phenol hydroxyl, and the schedule-dependency of its efficacy due to its mechanism of action. We have encapsulated topotecan in liposomes using transmembrane gradients of triethylammonium salts of polyphosphate (Pn) or sucroseoctasulfate (SOS). Circulation lifetimes were prolonged, and the rate of drug release in vivo depended on the drug load (T(1/2)=5.4 h vs. 11.2 h for 124 and 260 g TPT/mol PL, respectively) and the nature of intraliposomal drug complexing agent used to stabilize the nanoliposome formulation (T(1/2)=11.2 h vs. 27.3 h for Pn and SOS, respectively). Anti-EGFR and anti-HER2-immunoliposomal formulations dramatically increased uptake of topotecan compared to nontargeted nanoliposomal topotecan and poorly permeable free topotecan in receptor-overexpressing cancer cell lines, with a corresponding increase in cytotoxicity in multiple breast cancer cell lines and improved antitumor activity against HER2-overexpressing human breast cancer (BT474) xenografts. We conclude that stabilization of topotecan in nanoliposomes significantly improves the targetability and pharmacokinetic profile of topotecan, allowing for highly active formulations against solid tumors and immunotargeting to cancer-overexpressing cell surface receptors.
Assuntos
Antineoplásicos/química , Nanoestruturas/química , Topotecan/química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Química Farmacêutica , Estabilidade de Medicamentos , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Lipossomos , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Fatores de Tempo , Topotecan/administração & dosagem , Topotecan/farmacocinética , Topotecan/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Liposome and immunoliposome formulations of two vinca alkaloids, vincristine and vinblastine, were prepared using intraliposomal triethylammonium sucroseoctasulfate and examined for their ability to stabilize the drug for targeted drug delivery in vivo. METHODS: The pharmacokinetics of both the encapsulated drug (vincristine or vinblastine) and liposomal carrier were examined in Sprague Dawley rats, and the in vivo drug release rates determined. Anti-HER2 immunoliposomal vincristine was prepared from a human anti-HER2/neu scFv and studied for targeted cytotoxic activity in cell culture, and antitumor efficacy in vivo. RESULTS: Nanoliposome formulations of vincristine and vinblastine demonstrated similar pharmacokinetic profiles for the liposomal carrier, but increased clearance for liposome encapsulated vinblastine (t (1/2) = 9.7 h) relative to vincristine (t (1/2) = 18.5 h). Immunoliposome formulations of vincristine targeted to HER2 using an anti-HER2 scFv antibody fragment displayed a marked enhancement in cytotoxicity when compared to non-targeted liposomal vincristine control; 63- or 253-fold for BT474 and SKBR3 breast cancer cells, respectively. Target-specific activity was also demonstrated in HER2-overexpressing human tumor xenografts, where the HER2-targeted formulation was significantly more efficacious than either free vincristine or non-targeted liposomal vincristine. CONCLUSIONS: These results demonstrate that active targeting of solid tumors with liposomal formulations of vincristine is possible when the resulting immunoliposomes are sufficiently stabilized.
Assuntos
Antineoplásicos/farmacologia , Lipossomos , Vimblastina/farmacologia , Vincristina/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Camundongos , Ratos , Ratos Sprague-Dawley , Transplante Heterólogo , Vimblastina/administração & dosagem , Vimblastina/farmacocinética , Vincristina/administração & dosagem , Vincristina/farmacocinéticaRESUMO
Effective liposomal formulations of vinorelbine (5' nor-anhydro-vinblastine; VRL) have been elusive due to vinorelbine's hydrophobic structure and resulting difficulty in stabilizing the drug inside the nanocarrier. Triethylammonium salts of several polyanionic trapping agents were used initially to prepare minimally pegylated nanoliposomal vinorelbine formulations with a wide range of drug release rates. Sulfate, poly(phosphate), and sucrose octasulfate were used to stabilize vinorelbine intraliposomally while in circulation, with varying degrees of effectiveness. The release rate of vinorelbine from the liposomal carrier was affected by both the chemical nature of the trapping agent and the resulting drug-to-lipid ratio, with liposomes prepared using sucrose octasulfate displaying the longest half-life in circulation (9.4 h) and in vivo retention in the nanoparticle (t(1/2) = 27.2 h). Efficacy was considerably improved in both a human colon carcinoma (HT-29) and a murine (C-26) colon carcinoma model when vinorelbine was stably encapsulated in liposomes using triethylammonium sucrose octasulfate. Early difficulties in preparing highly pegylated formulations were later overcome by substituting a neutral distearoylglycerol anchor for the more commonly used anionic distearoylphosphatidylethanolamine anchor. The new pegylated nanoliposomal vinorelbine displayed high encapsulation efficiency and in vivo drug retention, and it was highly active against human breast and lung tumor xenografts. Acute toxicity of the drug in immunocompetent mice slightly decreased upon encapsulation in liposomes, with a maximum tolerated dose of 17.5 mg VRL/kg for free vinorelbine and 23.8 mg VRL/kg for nanoliposomal vinorelbine. Our results demonstrate that a highly active, stable, and long-circulating liposomal vinorelbine can be prepared and warrants further study in the treatment of cancer.
Assuntos
Vimblastina/análogos & derivados , Portadores de Fármacos , Estabilidade de Medicamentos , Humanos , Lipossomos , Nanopartículas , Fosfolipídeos/sangue , Trítio , Vimblastina/química , Vimblastina/farmacocinética , VinorelbinaRESUMO
Targeted drug delivery systems that combine imaging and therapeutic modalities in a single macromolecular construct may offer advantages in the development and application of nanomedicines. To incorporate the unique optical properties of luminescent quantum dots (QDs) into immunoliposomes for cancer diagnosis and treatment, we describe the synthesis, biophysical characterization, tumor cell-selective internalization, and anticancer drug delivery of QD-conjugated immunoliposome-based nanoparticles (QD-ILs). Pharmacokinetic properties and in vivo imaging capability of QD-ILs were also investigated. Freeze-fracture electron microscopy was used to visualize naked QDs, liposome controls, nontargeted QD-conjugated liposomes (QD-Ls), and QD-ILs. QD-ILs prepared by insertion of anti-HER2 scFv exhibited efficient receptor-mediated endocytosis in HER2-overexpressing SK-BR-3 and MCF-7/HER2 cells but not in control MCF-7 cells as analyzed by flow cytometry and confocal microscopy. In contrast, nontargeted QD-Ls showed minimal binding and uptake in these cells. Doxorubicin-loaded QD-ILs showed efficient anticancer activity, while no cytotoxicity was observed for QD-ILs without chemotherapeutic payload. In athymic mice, QD-ILs significantly prolonged circulation of QDs, exhibiting a plasma terminal half-life ( t 1/2) of approximately 2.9 h as compared to free QDs with t 1/2 < 10 min. In MCF-7/HER2 xenograft models, localization of QD-ILs at tumor sites was confirmed by in vivo fluorescence imaging.
Assuntos
Lipossomos , Pontos Quânticos , Animais , Linhagem Celular Tumoral , Técnica de Fratura por Congelamento , Humanos , Camundongos , Microscopia EletrônicaRESUMO
OBJECT: Many factors relating to the safety and efficacy of convection-enhanced delivery (CED) into intracranial tumors are poorly understood. To investigate these factors further and establish a more clinically relevant large animal model, with the potential to investigate CED in large, spontaneous tumors, the authors developed a magnetic resonance (MR) imaging-compatible system for CED of liposomal nanoparticles into the canine brain, incorporating real-time MR imaging. Additionally any possible toxicity of liposomes containing Gd and the chemotherapeutic agent irinotecan (CPT-11) was assessed following direct intraparenchymal delivery. METHODS: Four healthy laboratory dogs were infused with liposomes containing Gd, rhodamine, or CPT-11. Convection-enhanced delivery was monitored in real time by sequential MR imaging, and the volumes of distribution were calculated from MR images and histological sections. Assessment of any toxicity was based on clinical and histopathological evaluation. Convection-enhanced delivery resulted in robust volumes of distribution in both gray and white matter, and real-time MR imaging allowed accurate calculation of volumes and pathways of distribution. RESULTS: Infusion variability was greatest in the gray matter, and was associated with leakage into ventricular or subarachnoid spaces. Complications were minimal and included mild transient proprioceptive deficits, focal hemorrhage in 1 dog, and focal, mild perivascular, nonsuppurative encephalitis in 1 dog. CONCLUSIONS: Convection-enhanced delivery of liposomal Gd/CPT-11 is associated with minimal adverse effects in a large animal model, and further assessment for use in clinical patients is warranted. Future studies investigating real-time monitored CED in spontaneous gliomas in canines are feasible and will provide a unique, clinically relevant large animal translational model for testing this and other therapeutic strategies.
Assuntos
Camptotecina/análogos & derivados , Imageamento por Ressonância Magnética , Animais , Encéfalo/metabolismo , Camptotecina/administração & dosagem , Camptotecina/farmacocinética , Camptotecina/toxicidade , Cães , Monitoramento Ambiental , Feminino , Fluorescência , Gadolínio , Irinotecano , Lipossomos , NanopartículasRESUMO
Liposomes represent a widely varied and malleable class of drug carriers generally characterized by the presence of one or more amphiphile bilayers enclosing an interior aqueous space. Thus, the pharmacological profile of a particular liposomal drug formulation is a function not only of the properties of the encapsulated drug, but to a significant extent of the pharmacokinetics, biodistribution, and drug release rates of the individual carrier. Various physicochemical properties of the liposomal carriers, the drug encapsulation and retention strategies utilized, and the properties of the drugs chosen for encapsulation, all play an important role in determining the effectiveness of a particular liposomal drug. These properties should be carefully tailored to the specific drug, and to the application for which the therapeutic is being designed. Liposomal carriers are also amenable to additional modifications, including the conjugation of targeting ligands or environment-sensitive triggers for increasing the bioavailability of the drug specifically at the site of disease. This review describes the rationale for selecting optimal strategies of liposomal drug formulations with respect to drug encapsulation, retention, and release, and how these strategies can be applied to maximize therapeutic benefit in vivo.