RESUMO
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
PURPOSE: The improvement of postoperative pain control plays an important role in recovery outcomes and patient satisfaction. Multilamellar vesicles ropivacaine, MVR, is being developed to sustain the release of ropivacaine in situ while maintaining the local concentration of ropivacaine within the therapeutic window. METHODS: These studies summarized the processes of MVR formulation development and the evaluation of its releasing profile in vitro and the pharmacokinetics and anesthetic effect in vivo. RESULTS: The MVR demonstrates a sustained-release profile in an in vitro serum environment model after 24 hrs of incubation which translates in the in vivo rat pharmacokinetic profile of ropivacaine as a prolonged half-life that is 10-fold longer in duration than plain ropivacaine solution. The anesthetic effect of single-dose MVR is apparent by providing a prolonged analgesia effect compared to plain ropivacaine solution in an in vivo guinea pig pin-prick wheal model after a single intracutaneous injection. From a safety evaluation, MVR is well tolerated after a subcutaneously injection at a dose level of 20 mg/kg in rats, with no observable changes in clinical observation, body weight, organ weight, hematology and serum chemistry analysis. CONCLUSION: These results suggest that single administration of MVR is a promising candidate in postoperative pain management.
Assuntos
Portadores de Fármacos/química , Composição de Medicamentos , Manejo da Dor , Ropivacaina/uso terapêutico , Anestésicos Locais/administração & dosagem , Anestésicos Locais/farmacocinética , Anestésicos Locais/uso terapêutico , Animais , Liberação Controlada de Fármacos , Feminino , Cobaias , Humanos , Concentração de Íons de Hidrogênio , Masculino , Tamanho do Órgão , Dor Pós-Operatória/tratamento farmacológico , Ratos Sprague-Dawley , Ropivacaina/administração & dosagem , Ropivacaina/efeitos adversos , Ropivacaina/farmacocinética , Fatores de TempoRESUMO
Liposome formulations of camptothecins have been actively pursued because of the potential for significant pharmacologic advantages from successful drug delivery of this important class of anticancer drugs. We describe nanoliposomal CPT-11, a novel nanoparticle/liposome construct containing CPT-11 (irinotecan) with unprecedented drug loading efficiency and in vivo drug retention. Using a modified gradient loading method featuring a sterically hindered amine with highly charged, multivalent anionic trapping agents, either polymeric (polyphosphate) or nonpolymeric (sucrose octasulfate), liposomes were capable of entrapping CPT-11 at extremely high drug-to-lipid ratios (>800 g CPT-11/mol phospholipid) and retaining encapsulated drug in vivo with a half-life of drug release in the circulation of 56.8 hours. CPT-11 was also protected from hydrolysis to the inactive carboxylate form and from metabolic conversion to SN-38 while circulating. The maximum tolerated dose in normal mice was determined to be 80 mg/kg for free CPT-11 and >320 mg/kg for nanoliposomal CPT-11. Nanoliposomal CPT-11 showed markedly superior efficacy when compared with free CPT-11 in human breast (BT474) and colon (HT29) cancer xenograft models. This study shows that intraliposomal stabilization of CPT-11 using a polymeric or highly charged, nonpolymeric polyanionic trapping agent results in a markedly active antitumor agent with low toxicity.
Assuntos
Camptotecina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/química , Nanoestruturas/química , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Camptotecina/administração & dosagem , Camptotecina/química , Camptotecina/farmacocinética , Camptotecina/toxicidade , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Estabilidade de Medicamentos , Feminino , Células HT29 , Humanos , Irinotecano , Lipossomos/administração & dosagem , Lipossomos/farmacocinética , Lipossomos/toxicidade , Camundongos , Camundongos Nus , Nanoestruturas/toxicidade , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We describe evidence for a novel mechanism of monoclonal antibody (MAb)-directed nanoparticle (immunoliposome) targeting to solid tumors in vivo. Long-circulating immunoliposomes targeted to HER2 (ErbB2, Neu) were prepared by the conjugation of anti-HER2 MAb fragments (Fab' or single chain Fv) to liposome-grafted polyethylene glycol chains. MAb fragment conjugation did not affect the biodistribution or long-circulating properties of i.v.-administered liposomes. However, antibody-directed targeting also did not increase the tumor localization of immunoliposomes, as both targeted and nontargeted liposomes achieved similarly high levels (7-8% injected dose/g tumor tissue) of tumor tissue accumulation in HER2-overexpressing breast cancer xenografts (BT-474). Studies using colloidal gold-labeled liposomes showed the accumulation of anti-HER2 immunoliposomes within cancer cells, whereas matched nontargeted liposomes were located predominantly in extracellular stroma or within macrophages. A similar pattern of stromal accumulation without cancer cell internalization was observed for anti-HER2 immunoliposomes in non-HER2-overexpressing breast cancer xenografts (MCF-7). Flow cytometry of disaggregated tumors posttreatment with either liposomes or immunoliposomes showed up to 6-fold greater intracellular uptake in cancer cells due to targeting. Thus, in contrast to nontargeted liposomes, anti-HER2 immunoliposomes achieved intracellular drug delivery via MAb-mediated endocytosis, and this, rather than increased uptake in tumor tissue, was correlated with superior antitumor activity. Immunoliposomes capable of selective internalization in cancer cells in vivo may provide new opportunities for drug delivery.
Assuntos
Neoplasias da Mama/imunologia , Lipossomos/imunologia , Lipossomos/farmacocinética , Nanoestruturas , Receptor ErbB-2/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Feminino , Humanos , Lipossomos/química , Camundongos , Receptor ErbB-2/biossíntese , Transplante HeterólogoRESUMO
We previously reported the development of epidermal growth factor receptor (EGFR)-targeted immunoliposomes that bind and internalize in tumor cells which overexpress EGFR and/or mutant EGFR variant III (EGFRvIII), enabling intracellular delivery of potent anticancer agents in vitro. We now describe in vivo proof-of-concept for this approach for the delivery of multiple anticancer drugs in EGFR-overexpressing tumor models. Anti-EGFR immunoliposomes were constructed modularly with Fab' fragments of cetuximab (IMC-C225), covalently linked to liposomes containing probes and/or anticancer drugs. Pharmacokinetic and biodistribution studies confirmed long circulation times (t(1/2) = 21 hours) and efficient accumulation in tumors (up to 15% ID/g) irrespective of the presence of the targeting ligand. Although total accumulations of anti-EGFR immunoliposomes and nontargeted liposomes in EGFR-overexpressing tumors were comparable, only immunoliposomes internalized extensively within tumor cells (92% of analyzed cells versus <5% for nontargeted liposomes), indicating different mechanisms of delivery at the cellular level. In vivo therapy studies in a series of xenograft models featuring overexpression of EGFR and/or EGFRvIII showed the superiority of immunoliposomal delivery of encapsulated drugs, which included doxorubicin, epirubicin, and vinorelbine. For each of these drugs, anti-EGFR immunoliposome delivery showed significant antitumor effects and was significantly superior to all other treatments, including the corresponding free or liposomal drug (P < 0.001-0.003). We conclude that anti-EGFR immunoliposomes provide efficient and targeted drug delivery of anticancer compounds and may represent a useful new treatment approach for tumors that overexpress the EGFR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptores ErbB/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Imunoconjugados/uso terapêutico , Lipossomos/administração & dosagem , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Cetuximab , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Epirubicina/administração & dosagem , Receptores ErbB/genética , Receptores ErbB/imunologia , Feminino , Glioblastoma/imunologia , Glioblastoma/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Transfecção , Transplante Heterólogo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Vimblastina/administração & dosagem , Vimblastina/análogos & derivados , VinorelbinaRESUMO
We hypothesized that immunoliposomes (ILs) that target epidermal growth factor receptor (EGFR) and/or its truncated variant EGFRvIII can be constructed to provide efficient intracellular drug delivery in tumor cells overexpressing these receptors. Monoclonal antibody fragments included Fab' fragments derived from C225, which binds both EGFR and EGFRvIII, or novel anti-EGFR scFv C10, which binds EGFR only. Monoclonal antibody fragments were covalently linked to liposomes containing various reporters or drugs. ILs were evaluated for specific binding, internalization, and cytotoxicity in EGFR/EGFRvIII-overexpressing cell lines in vitro. Flow cytometry and fluorescence microscopy showed that EGFR-targeted ILs, but not nontargeted liposomes or irrelevant ILs, were efficiently bound and internalized by EGFR-overexpressing cells, including glioma cells (U-87), carcinoma cells (A-431 and MDA-MB-468), and EGFRvIII stable transfectants (NR-6M). Furthermore, EGFR-targeted ILs did not bind to non-EGFR-overexpressing cells (MCF-7 and parental NR-6). ILs showed 3 orders of magnitude greater accumulation in NR-6-EGFRvIII stable transfectants versus parental NR-6 cells. Quantitative internalization studies indicated binding of EGFR-targeted ILs to target cells within 5 min, followed by intracellular accumulation beginning at 15 min; total uptake reached approximately 13,000 ILs/cell. ILs were used to deliver cytotoxic drugs doxorubicin, vinorelbine, or methotrexate to EGFR/EGFRvIII-overexpressing target cells in vitro. In each case, the IL agent was significantly more cytotoxic than the corresponding nontargeted liposomal drug in target cells, whereas it was equivalent in cells lacking EGFR/EGFRvIII overexpression. We conclude that EGFR-targeted ILs provide efficient and targeted delivery of anticancer drugs in cells overexpressing EGFR or EGFRvIII.
Assuntos
Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Receptores ErbB/efeitos dos fármacos , Imunoconjugados/administração & dosagem , Metotrexato/administração & dosagem , Proteínas de Neoplasias/efeitos dos fármacos , Vimblastina/análogos & derivados , Vimblastina/administração & dosagem , Adenocarcinoma/patologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/administração & dosagem , Antineoplásicos/sangue , Neoplasias Encefálicas/patologia , Neoplasias da Mama/patologia , Carcinoma de Células Escamosas/patologia , Doxorrubicina/sangue , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Receptores ErbB/genética , Receptores ErbB/imunologia , Feminino , Glioblastoma/patologia , Humanos , Imunoconjugados/sangue , Fragmentos Fab das Imunoglobulinas/imunologia , Lipossomos/administração & dosagem , Lipossomos/sangue , Proteínas de Neoplasias/imunologia , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Vinorelbina , Neoplasias Vulvares/patologiaRESUMO
Many targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell. To generate single chain Fv (scFv) antibodies capable of triggering receptor-mediated endocytosis, we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell. Using this methodology, we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library. For this work, an immunotherapeutic was generated from one of these scFv (F5), which bound to ErbB2 (HER2/neu). The F5 scFv was reengineered with a C-terminal cysteine, expressed at high levels in Escherichia coli, and coupled to sterically stabilized liposomes. F5 anti-ErbB2 immunoliposomes were immunoreactive as determined by surface plasmon resonance (SPR) and were avidly internalized by ErbB2-expressing tumor cell lines in proportion to the levels of ErbB2 expression. F5-scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin. This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Lipossomos , Receptor ErbB-2/imunologia , Animais , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/uso terapêutico , Anticorpos , Modelos Animais de Doenças , Doxorrubicina/farmacocinética , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Endocitose , Endossomos , Feminino , Cinética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Receptor ErbB-2/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein-lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 degrees C. Traditional methods for characterizing monodisperse protein species were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2) and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) after addition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation with phospholipase B selectively removed DSPE lipid groups and dispersed the conjugate prior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4 vol %) and n-butanol (4.5 vol %) to buffers for salt-gradient cation exchange chromatography provided gentler, nonenzymatic dispersion, resulting in well-resolved peaks. This method was used to assess stability, identify contaminants, establish lot-to-lot comparability, and determine the average chromatographic purity (93%) for conjugate lots, described previously. The F5cys amino acid content was confirmed after conjugation. The expected conjugate avidity for immobilized HER-2/neu was measured by bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were made by conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody-directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Together these developed assays established that the manufacturing method as described in the first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE having sufficient purity, stability, and functionality for use in preclinical toxicology investigations.
Assuntos
Fragmentos de Imunoglobulinas/química , Lipossomos/química , Varredura Diferencial de Calorimetria , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel de Poliacrilamida , Lisofosfolipase/metabolismo , Micelas , Modelos Biológicos , Peso Molecular , Tamanho da Partícula , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Análise de Sequência de Proteína , Temperatura , Fatores de TempoRESUMO
A GMP-compliant process is described for producing F5cys-PEG-lipid conjugate. This material fuses with preformed, drug-loaded liposomes, to form "immunoliposomes" that bind to HER2/neu overexpressing carcinomas, stimulates drug internalization, and ideally improves the encapsulated drug's therapeutic index. The soluble, single-chain, variable region antibody fragment, designated F5cys, was produced in E. coli strain RV308 using high-density cultures. Affinity adsorption onto horizontally tumbled Streamline rProtein-A resin robustly recovered F5cys from high-pressure-disrupted, whole-cell homogenates. Two product-related impurity classes were identified: F5cys with mid-sequence discontinuities and F5cys with remnants of a pelB leader peptide. Low-pressure cation exchange chromatography, conducted at elevated pH under reducing conditions, enriched target F5cys relative to these impurities and prepared a C-terminal cysteine for conjugation. Site-directed conjugation, conducted at pH 5.9 +/- 0.1 with reaction monitoring and cysteine quenching, yielded F5cys-MP-PEG(2000)-DSPE. Low-pressure size exclusion chromatography separated spontaneously formed, high-molecular-weight conjugate micelles from low-molecular-weight impurities. When formulated at 1-2 mg/mL in 10 mM trisodium citrate, 10% sucrose (w/v), at pH 6.4 (HCl), the conjugate was stable when stored below -70 degrees C. Six scale-up lots were compared. The largest 40-L culture produced enough F5cys to manufacture 2,085 mg of conjugate, enough to support planned preclinical and future clinical trials. The conjugate was 93% pure, as measured by polyacrylamide gel electrophoresis. Impurities were primarily identified as product-related. Residual endotoxin, rProtein A, and genomic DNA, were at acceptable levels. This study successfully addressed a necessary step in the scale-up of immunoliposome-encapsulated therapeutics.
Assuntos
Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/isolamento & purificação , Lipossomos/isolamento & purificação , Lipossomos/metabolismo , Sequência de Aminoácidos , Divisão Celular/fisiologia , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/citologia , Micelas , Conformação Molecular , Dados de Sequência Molecular , Fosfatos/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Fatores de TempoRESUMO
PURPOSE: Anti-HER2 immunoliposomes combine the tumor-targeting of certain anti-HER2 monoclonal antibodies (MAbs) with the pharmacokinetic and drug delivery capabilities of sterically stabilized liposomes. We previously showed that anti-HER2 immunoliposomes bind efficiently to and internalize in HER2-overexpressing cells in vitro, resulting in intracellular drug delivery. EXPERIMENTAL DESIGN: Here we describe the pharmacokinetics and therapeutic efficacy of anti-HER2 immunoliposomes containing doxorubicin (dox) in a series of animal models. RESULTS: Immunoliposomes displayed long circulation that was identical to that of sterically stabilized liposomes in single- and multiple-dose studies in normal rats. Anti-HER2 immunoliposome-dox produced marked therapeutic results in four different HER2-overexpressing tumor xenograft models, including growth inhibition, regression, and cures. These results demonstrated that encapsulation of dox in anti-HER2 immunoliposomes greatly increased its therapeutic index, both by increasing antitumor efficacy and by reducing systemic toxicity. Immunoliposome-dox was significantly superior to all other treatment conditions tested, including free dox, liposomal dox, and anti-HER2 MAb (trastuzumab). When compared with liposomal dox in eight separate therapy studies in HER2-overexpressing models, immunoliposome delivery produced significantly superior antitumor efficacy in each study (P < 0.0001 to 0.04). Anti-HER2 immunoliposome-dox containing either recombinant human MAb HER2-Fab' or scFv C6.5 yielded comparable therapeutic efficacy. Cure rates for immunoliposome-dox reached 50% (11 of 21) with optimized immunoliposomes and Matrigel-free tumors and overall was 16% (18 of 115) versus no cures (0 of 124) with free dox or liposomal dox. Finally, anti-HER2 immunoliposome-dox was also superior to combinations consisting of free MAb plus free dox or free MAb plus liposomal dox. CONCLUSIONS: Anti-HER2 immunoliposomes produced enhanced antitumor efficacy via targeted delivery.
Assuntos
Anticorpos Monoclonais/imunologia , Antineoplásicos/uso terapêutico , Doxorrubicina/uso terapêutico , Receptor ErbB-2/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Área Sob a Curva , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Sinergismo Farmacológico , Humanos , Fragmentos de Imunoglobulinas/administração & dosagem , Fragmentos de Imunoglobulinas/imunologia , Lipossomos/farmacocinética , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
Phagocytes of the reticuloendothelial system are important in clearing systemic infection; however, the role of the reticuloendothelial system in the response to localized infection is not well-documented. The major goals of this study were to investigate the roles of phagocytes in the reticuloendothelial system in terms of bacterial clearance and inflammatory modulation in sepsis caused by Pseudomonas pneumonia. Macrophages in liver and spleen were depleted by administering liposome encapsulated dichloromethylene diphosphonate (clodronate) intravenously 36 h before the instillation of Pseudomonas aeruginosa into the lungs of anesthetized rabbits. Blood samples were analyzed for bacteria and cytokine concentrations. Lung injury was assessed by the bidirectional flux of albumin and by wet-to-dry weight ratios. Blood pressure and cardiac outputs decreased more rapidly and bacteremia occurred earlier in the clodronate-treated rabbits compared with the nondepleted rabbits. Plasma TNF-alpha (1.08 +/- 0.54 vs. 0.08 +/- 0.02 ng/ml) and IL-8 (6.8 +/- 1.5 vs. 0.0 +/- 0.0 ng/ml) were higher in the depleted rabbits. The concentration of IL-10 in liver of the macrophage-depleted rabbits was significantly lower than in normal rabbits at 5 h. Treatment of macrophage-depleted rabbits with intravenous IL-10 reduced plasma proinflammatory cytokine concentrations and reduced the decline in blood pressure and cardiac output. These results show that macrophages in the reticuloendothelial system have critical roles in controlling systemic bacteremia and reducing systemic inflammation, thereby limiting the systemic effects of a severe pulmonary bacterial infection.
Assuntos
Sistema Fagocitário Mononuclear/metabolismo , Fagócitos/fisiologia , Pneumonia Bacteriana/etiologia , Pneumonia Bacteriana/mortalidade , Infecções por Pseudomonas/etiologia , Infecções por Pseudomonas/mortalidade , Animais , Bacteriemia/etiologia , Bacteriemia/mortalidade , Pressão Sanguínea , Ácido Clodrônico/administração & dosagem , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Lipossomos , Fígado/citologia , Fígado/imunologia , Fígado/microbiologia , Lesão Pulmonar/etiologia , Lesão Pulmonar/patologia , Macrófagos/fisiologia , Masculino , Pseudomonas aeruginosa/patogenicidade , Coelhos , Baço/citologia , Baço/imunologia , Baço/microbiologia , Taxa de SobrevidaRESUMO
Drug delivery to brain tumors has long posed a major challenge. Convection-enhanced delivery (CED) has been developed as a drug delivery strategy to overcome this difficulty. Ideally, direct visualization of the tissue distribution of drugs infused by CED would assure successful delivery of therapeutic agents to the brain tumor while minimizing exposure of the normal brain. We previously developed a magnetic resonance imaging (MRI)-based method to visualize the distribution of liposomal agents after CED in rodent brains. In the present study, CED of liposomes was further examined in the non-human primate brain (n = 6). Liposomes containing Gadoteridol, DiI-DS, and rhodamine were infused in corona radiata, putamen nucleus, and brain stem. Volume of distribution was analyzed for all delivery locations by histology and MR imaging. Real-time MRI monitoring of liposomes containing gadolinium allowed direct visualization of a robust distribution. MRI of liposomal gadolinium was highly accurate at determining tissue distribution, as confirmed by comparison with histological results from concomitant administration of fluorescent liposomes. Linear correlation for liposomal infusions between infusion volume and distribution volume was established in all targeted locations. We conclude that an integrated strategy combining liposome/nanoparticle technology, CED, and MRI may provide new opportunities for the treatment of brain tumors. Our ability to directly monitor and to control local delivery of liposomal drugs will most likely result in greater clinical efficacy when using CED in management of patients.
Assuntos
Encéfalo/metabolismo , Convecção , Sistemas de Liberação de Medicamentos , Gadolínio/administração & dosagem , Lipossomos/metabolismo , Imageamento por Ressonância Magnética , Animais , Processamento de Imagem Assistida por Computador/métodos , Macaca fascicularis , Masculino , Estatística como Assunto , Fatores de Tempo , Distribuição TecidualRESUMO
Drug resistance remains an important obstacle towards better outcomes in the treatment of cancer. One general approach to overcome this problem has been to inhibit specific resistance mechanisms, such as P-glycoprotein (PGP)-mediated drug efflux, using small molecule agents or other therapeutic strategies. Alternatively, drug delivery approaches using liposomes or other carriers can in principle target drugs to tumor tissue, tumor cells, or even compartments within tumor cells. By increasing bioavailability of drugs at sites of action, these approaches may provide therapeutic advantages, including enhanced efficacy against resistant tumors. Current liposomal anthracyclines have achieved clinical use in cancer treatment by providing efficient encapsulation of drug in stable and non-reactive carriers, and there is evidence indicating potential benefit in some clinical settings involving resistant tumors. Other liposome-based strategies include constructs designed to be taken up by tumor cells, as well as further modifications to allow triggered drug release. These approaches seek to overcome drug resistance by more efficient delivery to tumor cells, and in some cases by concomitant avoidance or inhibition of drug efflux mechanisms. Newer agents employ molecular targeting, such as immunoliposomes using antibody-directed binding and internalization. These agents selectively deliver drug to tumor cells, can efficiently internalize for intracellular drug release, and can potentially enhance both efficacy and safety.
Assuntos
Antineoplásicos/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lipossomos/administração & dosagem , Antineoplásicos/farmacocinética , Sistemas de Liberação de Medicamentos , HumanosRESUMO
Topotecan (TPT), a topoisomerase I inhibitor, is presently undergoing clinical evaluation worldwide. Previous studies have shown that entrapping TPT within multi-lamellar vesicle liposome can stabilize the lactone moiety, which is structurally important for biological activity. However, low drug:lipid ratios due to the amphipathic character and small entrapment volume in the unilamellar vesicle limits the development of pharmaceutically acceptable liposomal formulation. With an aim to improve on this drawback, we herein describe a method that utilizes the ammonium sulfate gradient to entrap TPT into liposomes. By this method, the encapsulation efficiency was over 90% and a drug:lipid molar ratio as high as 1:5.4 was reached. In comparison with free drug, liposome-encapsulated TPT is more stable in physiological conditions and shows higher in vitro cytotoxicity. Because of increased blood circulation time, the initial plasma concentration and area under the plasma concentration of liposomal drugs were 14 and 40 times, respectively, of those of free drug. Furthermore, liposome encapsulation enhanced the antitumor activity of TPT in syngeneic murine C-26 and human HTB-9 xenograft models in vivo. At a dose of 5 mg/kg, the tumor growth delay of liposomal formulation was significantly than that of free TPT. Based on these results, we believe that this liposomal TPT formulation is worthy of further clinical study.
Assuntos
Antineoplásicos/administração & dosagem , Composição de Medicamentos/métodos , Topotecan/administração & dosagem , Algoritmos , Sulfato de Amônio/química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Portadores de Fármacos , Estabilidade de Medicamentos , Humanos , Indicadores e Reagentes , Lactonas/química , Lipossomos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Topotecan/farmacocinética , Topotecan/uso terapêutico , Células Tumorais CultivadasRESUMO
The continued evolution of targeted liposomal therapeutics has resulted in new agents with remarkable antitumour efficacy and relatively mild toxicity profiles. A careful selection of the ligand is necessary to reduce immunogenicity, retain extended circulation lifetimes, target tumour-specific cell surface epitopes, and induce internalisation and subsequent release of the therapeutic substance from the liposome. Methods for assembling targeted liposomes, including a novel micellar insertion technology, for incorporation of targeting molecules that efficiently transforms a non-targeted liposomal therapeutic to a targeted one, greatly assist the translation of targeted liposome technology into the clinic. Targeting strategies with liposomes directed at solid tumours and vascular targets are discussed. The authors believe the development of ligand-targeted liposomes is now in the advanced stage and offers unique and important advantages among other targeted therapies. Anti-HER2 immunoliposomal doxorubicin is awaiting Phase I clinical trials, the results of which should provide new insights into the promise of ligand-targeted liposomal therapies.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/tendências , Neoplasias/tratamento farmacológico , Animais , Humanos , Ligantes , Lipossomos , Neoplasias/genética , Neoplasias/metabolismoRESUMO
Liposomes labeled with various markers were subjected to local-regional administration with either direct injection or convection-enhanced delivery (CED) into rodent brains and brain tumor models. Direct injection of liposomes containing attached or encapsulated fluorochromes and/or encapsulated gold particles indicated that tissue localization of liposomes could be sensitively and specifically detected in the central nervous system (CNS). When CED was applied, liposomes achieved extensive and efficient distribution within normal mouse brains. Co-infusion of mannitol further increased tissue penetration of liposomes. Liposomes were also loaded with gadodiamide to monitor their CNS distribution in rats by magnetic resonance imaging (MRI). CED-infused liposomes were readily seen on MRI scans as large regions of intense signal at 2 h, and more diffuse regions at 24 h. Finally, labeled liposomes were infused via CED into tumor tissue in glioma xenograft models in rodent hosts. In intracranial U-87 glioma xenografts, CED-infused liposomes had distributed throughout tumor tissue, including extension into surrounding normal tissue. Greater penetration was observed using 40 versus 90 nm liposomes, as well as with mannitol co-infusion. To our knowledge, this is the first report of CED infusion of liposomes into the CNS. We conclude that CED of liposomes in the CNS is a feasible approach, and offers a promising strategy for targeting therapeutic agents to brain tumors.