64 results on '"Kirpotin DB"'
Search Results
2. Characterization of highly stable liposomal and immunoliposomal formulations of vincristine and vinblastine.
- Author
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Noble CO, Guo Z, Hayes ME, Marks JD, Park JW, Benz CC, Kirpotin DB, Drummond DC, Noble, Charles O, Guo, Zexiong, Hayes, Mark E, Marks, James D, Park, John W, Benz, Christopher C, Kirpotin, Dmitri B, and Drummond, Daryl C
- Abstract
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. [ABSTRACT FROM AUTHOR]- Published
- 2009
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3. Drug Stability and Minimized Acid-/Drug-Catalyzed Phospholipid Degradation in Liposomal Irinotecan.
- Author
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Kirpotin DB, Hayes ME, Noble CO, Huang ZR, Wani K, Moore D, Kesper K, Brien DO, and Drummond DC
- Subjects
- Irinotecan, Drug Stability, Tissue Distribution, Camptothecin, Catalysis, Liposomes, Phospholipids
- Abstract
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., Competing Interests: Declaration of Competing Interest D.B.K., M.E.H., C.O.N., Z.R.H., K.W., D.M., K.K., D.O., and D.C.D. were all employees and stock option holders in Merrimack Pharmaceuticals at the time the work was completed. D.B.K., K.W., K.K., and D.C.D. currently own stock and stock options in Merrimack Pharmaceuticals. D.C.D. is currently a consultant to Merrimack Pharmaceuticals and Ipsen, and K.W. is currently an employee of and owns stock in Ipsen., (Published by Elsevier Inc.)
- Published
- 2023
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4. Development of disulfide-stabilized Fabs for targeting of antibody-directed nanotherapeutics.
- Author
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Geddie ML, Kirpotin DB, Kohli N, Kornaga T, Boll B, Razlog M, Drummond DC, and Lugovskoy AA
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- Disulfides chemistry, Immunoglobulin Fab Fragments chemistry, Immunoglobulin Fab Fragments genetics, Nanoparticles chemistry, Single-Chain Antibodies
- Abstract
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.
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- 2022
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5. Targeting EphA2 in Bladder Cancer Using a Novel Antibody-Directed Nanotherapeutic.
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Kamoun W, Swindell E, Pien C, Luus L, Cain J, Pham M, Kandela I, Huang ZR, Tipparaju SK, Koshkaryev A, Askoxylakis V, Kirpotin DB, Bloom T, Mino-Kenudson M, Marks JD, Zalutskaya A, Bshara W, Morrison C, and Drummond DC
- Abstract
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.
- Published
- 2020
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6. Formulation optimization of an ephrin A2 targeted immunoliposome encapsulating reversibly modified taxane prodrugs.
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Huang ZR, Tipparaju SK, Kirpotin DB, Pien C, Kornaga T, Noble CO, Koshkaryev A, Tran J, Kamoun WS, and Drummond DC
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- A549 Cells, Animals, Antineoplastic Agents chemistry, Antineoplastic Agents pharmacokinetics, Antineoplastic Agents pharmacology, Bridged-Ring Compounds chemistry, Bridged-Ring Compounds pharmacokinetics, Bridged-Ring Compounds pharmacology, Cell Line, Tumor, Drug Compounding, Drug Liberation, Female, Humans, Liposomes, Mice, Nude, Particle Size, Prodrugs chemistry, Prodrugs pharmacokinetics, Prodrugs pharmacology, Protein Binding, Taxoids chemistry, Taxoids pharmacokinetics, Taxoids pharmacology, Tumor Cells, Cultured, Xenograft Model Antitumor Assays, Antineoplastic Agents administration & dosage, Bridged-Ring Compounds administration & dosage, Drug Carriers chemistry, Ephrin-A2 metabolism, Nanoparticles chemistry, Prodrugs administration & dosage, Taxoids administration & dosage
- Abstract
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 114 mol% taxane per mole phospholipid) and subsequent stability (>3 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 (0.69 nM) apparent equilibrium dissociation constant toward the extracellular domain of the ephrin A2 receptor, long circulation half-life (8-12 h) in mouse plasma, a release rate dependent on intraliposomal drug concentration and stable long-term storage. At an equitoxic dose of 50 mg taxane/kg, ephrin A2-targeted liposomal prodrug showed greater antitumor activity than 10 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)., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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7. Antitumour activity and tolerability of an EphA2-targeted nanotherapeutic in multiple mouse models.
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Kamoun WS, Kirpotin DB, Huang ZR, Tipparaju SK, Noble CO, Hayes ME, Luus L, Koshkaryev A, Kim J, Olivier K, Kornaga T, Oyama S, Askoxylakis V, Pien C, Kuesters G, Dumont N, Lugovskoy AA, Schihl SA, Wilton JH, Geddie ML, Suchy J, Grabow S, Kohli N, Reynolds CP, Blaydes R, Zhou Y, Sawyer AJ, Marks JD, and Drummond DC
- Subjects
- Animals, Antineoplastic Agents pharmacology, Bridged-Ring Compounds pharmacology, Bridged-Ring Compounds therapeutic use, Cell Line, Tumor, Cell Proliferation drug effects, Disease Models, Animal, Docetaxel blood, Docetaxel chemistry, Docetaxel pharmacokinetics, Docetaxel therapeutic use, Humans, Liposomes, Mice, Inbred NOD, Mice, SCID, Taxoids pharmacology, Taxoids therapeutic use, Tissue Distribution drug effects, Tumor Burden drug effects, Xenograft Model Antitumor Assays, Antineoplastic Agents therapeutic use, Nanoparticles therapeutic use, Receptor, EphA2 metabolism
- Abstract
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.
- Published
- 2019
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8. 64 Cu-MM-302 Positron Emission Tomography Quantifies Variability of Enhanced Permeability and Retention of Nanoparticles in Relation to Treatment Response in Patients with Metastatic Breast Cancer.
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Lee H, Shields AF, Siegel BA, Miller KD, Krop I, Ma CX, LoRusso PM, Munster PN, Campbell K, Gaddy DF, Leonard SC, Geretti E, Blocker SJ, Kirpotin DB, Moyo V, Wickham TJ, and Hendriks BS
- Subjects
- Adolescent, Adult, Aged, Bone Neoplasms diagnostic imaging, Bone Neoplasms pathology, Bone Neoplasms secondary, Brain Neoplasms diagnostic imaging, Brain Neoplasms pathology, Brain Neoplasms secondary, Breast Neoplasms blood, Breast Neoplasms diagnostic imaging, Breast Neoplasms pathology, Cell Membrane Permeability drug effects, Cell Membrane Permeability radiation effects, Copper Radioisotopes administration & dosage, Copper Radioisotopes chemistry, Cyclophosphamide administration & dosage, Doxorubicin administration & dosage, Doxorubicin chemistry, Female, Humans, Liver diagnostic imaging, Liver drug effects, Middle Aged, Nanoparticles chemistry, Neoplasm Metastasis, Polyethylene Glycols administration & dosage, Polyethylene Glycols chemistry, Positron Emission Tomography Computed Tomography, Receptor, ErbB-2 blood, Spleen diagnostic imaging, Spleen pathology, Trastuzumab administration & dosage, Bone Neoplasms drug therapy, Brain Neoplasms drug therapy, Breast Neoplasms drug therapy, Doxorubicin analogs & derivatives, Nanoparticles administration & dosage
- Abstract
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
64 Cu-labeled nanoparticle,64 Cu-MM-302 (64 Cu-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 of64 Cu-MM-302 as part of a clinical trial of MM-302 plus trastuzumab with and without cyclophosphamide (NCT01304797). Results: Significant background uptake of64 Cu-MM-302 was observed in liver and spleen. Tumor accumulation of64 Cu-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 of64 Cu-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, high64 Cu-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 ., (©2017 American Association for Cancer Research.)- Published
- 2017
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9. Improving the developability of an anti-EphA2 single-chain variable fragment for nanoparticle targeting.
- Author
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Geddie ML, Kohli N, Kirpotin DB, Razlog M, Jiao Y, Kornaga T, Rennard R, Xu L, Schoerberl B, Marks JD, Drummond DC, and Lugovskoy AA
- Subjects
- Animals, Humans, Immunoglobulin Variable Region immunology, Protein Engineering methods, Protein Stability, Receptor, EphA2, Single-Chain Antibodies biosynthesis, Drug Delivery Systems methods, Ephrin-A2 immunology, Immunoconjugates immunology, Nanoparticles, Single-Chain Antibodies immunology
- Abstract
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.
- Published
- 2017
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10. Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle.
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Zhang K, Geddie ML, Kohli N, Kornaga T, Kirpotin DB, Jiao Y, Rennard R, Drummond DC, Nielsen UB, Xu L, and Lugovskoy AA
- Subjects
- Amino Acid Substitution, Animals, Antibodies, Neoplasm chemistry, Antibodies, Neoplasm genetics, Antibodies, Neoplasm immunology, Antibodies, Neoplasm pharmacology, Cell Line, Tumor, Humans, Mice, Neoplasms immunology, Recombinant Proteins, Cytotoxins chemistry, Cytotoxins pharmacology, Drug Delivery Systems methods, Nanoparticles chemistry, Neoplasms drug therapy, Single-Chain Antibodies chemistry, Single-Chain Antibodies genetics, Single-Chain Antibodies immunology, Single-Chain Antibodies pharmacology
- Abstract
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.
- Published
- 2015
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11. Pharmacokinetics, tumor accumulation and antitumor activity of nanoliposomal irinotecan following systemic treatment of intracranial tumors.
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Noble CO, Krauze MT, Drummond DC, Forsayeth J, Hayes ME, Beyer J, Hadaczek P, Berger MS, Kirpotin DB, Bankiewicz KS, and Park JW
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- Animals, Antineoplastic Agents, Phytogenic administration & dosage, Antineoplastic Agents, Phytogenic pharmacokinetics, Brain Neoplasms metabolism, Camptothecin administration & dosage, Camptothecin pharmacokinetics, Camptothecin therapeutic use, Irinotecan, Rats, Antineoplastic Agents, Phytogenic therapeutic use, Brain Neoplasms drug therapy, Camptothecin analogs & derivatives, Liposomes, Nanostructures
- Abstract
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.
- Published
- 2014
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12. Convection-enhanced delivery of targeted quantum dot-immunoliposome hybrid nanoparticles to intracranial brain tumor models.
- Author
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Weng KC, Hashizume R, Noble CO, Serwer LP, Drummond DC, Kirpotin DB, Kuwabara AM, Chao LX, Chen FF, James CD, and Park JW
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- Animals, Brain metabolism, Brain Neoplasms pathology, Cell Line, Tumor, Convection, Female, Glioblastoma pathology, Humans, Liposomes analysis, Mice, Mice, Inbred BALB C, Mice, Nude, Quantum Dots analysis, Brain pathology, Brain Neoplasms metabolism, Drug Delivery Systems, ErbB Receptors metabolism, Glioblastoma metabolism, Liposomes metabolism, Quantum Dots metabolism
- Abstract
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.
- Published
- 2013
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13. Comparing routes of delivery for nanoliposomal irinotecan shows superior anti-tumor activity of local administration in treating intracranial glioblastoma xenografts.
- Author
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Chen PY, Ozawa T, Drummond DC, Kalra A, Fitzgerald JB, Kirpotin DB, Wei KC, Butowski N, Prados MD, Berger MS, Forsayeth JR, Bankiewicz K, and James CD
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- Animals, Brain Neoplasms mortality, Brain Neoplasms pathology, Camptothecin administration & dosage, Convection, Drug Administration Routes, Female, Glioblastoma mortality, Glioblastoma pathology, History, Ancient, Humans, Immunoenzyme Techniques, Injections, Intraperitoneal, Irinotecan, Mice, Mice, Nude, Survival Rate, Tumor Cells, Cultured, Xenograft Model Antitumor Assays, Antineoplastic Agents, Phytogenic administration & dosage, Brain Neoplasms drug therapy, Camptothecin analogs & derivatives, Drug Delivery Systems, Glioblastoma drug therapy, Liposomes, Nanoparticles
- Abstract
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.
- Published
- 2013
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14. Building and characterizing antibody-targeted lipidic nanotherapeutics.
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Kirpotin DB, Noble CO, Hayes ME, Huang Z, Kornaga T, Zhou Y, Nielsen UB, Marks JD, and Drummond DC
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- Animals, Antibodies, Monoclonal metabolism, Antibodies, Monoclonal pharmacokinetics, Antibody Affinity, Antineoplastic Agents pharmacokinetics, Drug Compounding methods, Drug Stability, Humans, Immunoconjugates chemistry, Immunoconjugates pharmacokinetics, Immunoglobulin Fragments chemistry, Kinetics, Lipids chemistry, Liposomes immunology, Mice, Neoplasms immunology, Neoplasms metabolism, Neoplasms pathology, Particle Size, Polyethylene Glycols chemistry, Protein Engineering methods, Rats, Sulfhydryl Reagents chemistry, Antineoplastic Agents metabolism, Drug Delivery Systems methods, Immunoconjugates metabolism, Immunoglobulin Fragments metabolism, Liposomes metabolism, Nanomedicine methods, Neoplasms drug therapy
- Abstract
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., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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15. Investigation of intravenous delivery of nanoliposomal topotecan for activity against orthotopic glioblastoma xenografts.
- Author
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Serwer LP, Noble CO, Michaud K, Drummond DC, Kirpotin DB, Ozawa T, Prados MD, Park JW, and James CD
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- Animals, Brain Neoplasms mortality, Brain Neoplasms pathology, Female, Glioblastoma mortality, Glioblastoma pathology, Humans, Immunoenzyme Techniques, Luminescent Measurements, Mice, Mice, Nude, Survival Rate, Tissue Distribution, Topoisomerase I Inhibitors pharmacokinetics, Topotecan pharmacokinetics, Tumor Cells, Cultured, Xenograft Model Antitumor Assays, Brain Neoplasms drug therapy, Drug Delivery Systems, Glioblastoma drug therapy, Liposomes, Nanotechnology, Topoisomerase I Inhibitors therapeutic use, Topotecan therapeutic use
- Abstract
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.
- Published
- 2011
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16. Canine spontaneous glioma: a translational model system for convection-enhanced delivery.
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Dickinson PJ, LeCouteur RA, Higgins RJ, Bringas JR, Larson RF, Yamashita Y, Krauze MT, Forsayeth J, Noble CO, Drummond DC, Kirpotin DB, Park JW, Berger MS, and Bankiewicz KS
- Subjects
- Animals, Brain Neoplasms pathology, Brain Neoplasms veterinary, Camptothecin administration & dosage, Convection, Disease Models, Animal, Dogs, Glioma pathology, Glioma veterinary, Irinotecan, Liposomes, Magnetic Resonance Imaging, Antineoplastic Agents, Phytogenic administration & dosage, Brain Neoplasms drug therapy, Camptothecin analogs & derivatives, Drug Delivery Systems methods, Glioma drug therapy, Nanoparticles
- Abstract
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.
- Published
- 2010
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17. Development of a highly stable and targetable nanoliposomal formulation of topotecan.
- Author
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Drummond DC, Noble CO, Guo Z, Hayes ME, Connolly-Ingram C, Gabriel BS, Hann B, Liu B, Park JW, Hong K, Benz CC, Marks JD, and Kirpotin DB
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- Animals, Antineoplastic Agents administration & dosage, Antineoplastic Agents pharmacokinetics, Antineoplastic Agents therapeutic use, Cell Line, Tumor, Chemistry, Pharmaceutical, Drug Stability, ErbB Receptors antagonists & inhibitors, Female, Humans, Liposomes, Mice, Mice, Nude, Molecular Structure, Neoplasms, Experimental drug therapy, Receptor, ErbB-2 antagonists & inhibitors, Time Factors, Topotecan administration & dosage, Topotecan pharmacokinetics, Topotecan therapeutic use, Xenograft Model Antitumor Assays, Antineoplastic Agents chemistry, Nanostructures chemistry, Topotecan chemistry
- Abstract
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.
- Published
- 2010
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18. Improved pharmacokinetics and efficacy of a highly stable nanoliposomal vinorelbine.
- Author
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Drummond DC, Noble CO, Guo Z, Hayes ME, Park JW, Ou CJ, Tseng YL, Hong K, and Kirpotin DB
- Subjects
- Drug Carriers, Drug Stability, Humans, Liposomes, Nanoparticles, Phospholipids blood, Tritium, Vinblastine chemistry, Vinblastine pharmacokinetics, Vinorelbine, Vinblastine analogs & derivatives
- Abstract
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.
- Published
- 2009
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19. Pharmacokinetics and in vivo drug release rates in liposomal nanocarrier development.
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Drummond DC, Noble CO, Hayes ME, Park JW, and Kirpotin DB
- Subjects
- Animals, Area Under Curve, Half-Life, Tissue Distribution, Drug Carriers, Liposomes, Nanoparticles, Pharmacokinetics
- Abstract
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.
- Published
- 2008
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20. Targeted tumor cell internalization and imaging of multifunctional quantum dot-conjugated immunoliposomes in vitro and in vivo.
- Author
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Weng KC, Noble CO, Papahadjopoulos-Sternberg B, Chen FF, Drummond DC, Kirpotin DB, Wang D, Hom YK, Hann B, and Park JW
- Subjects
- Animals, Cell Line, Tumor, Freeze Fracturing, Humans, Mice, Microscopy, Electron, Liposomes, Quantum Dots
- Abstract
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.
- Published
- 2008
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21. Canine model of convection-enhanced delivery of liposomes containing CPT-11 monitored with real-time magnetic resonance imaging: laboratory investigation.
- Author
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Dickinson PJ, LeCouteur RA, Higgins RJ, Bringas JR, Roberts B, Larson RF, Yamashita Y, Krauze M, Noble CO, Drummond D, Kirpotin DB, Park JW, Berger MS, and Bankiewicz KS
- Subjects
- Animals, Brain metabolism, Camptothecin administration & dosage, Camptothecin pharmacokinetics, Camptothecin toxicity, Dogs, Environmental Monitoring, Female, Fluorescence, Gadolinium, Irinotecan, Liposomes, Nanoparticles, Camptothecin analogs & derivatives, Magnetic Resonance Imaging
- Abstract
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.
- Published
- 2008
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22. Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies.
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An F, Drummond DC, Wilson S, Kirpotin DB, Nishimura SL, Broaddus VC, and Liu B
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- Antineoplastic Agents administration & dosage, Cell Line, Tumor, Humans, Mesothelioma immunology, Antibodies immunology, Antineoplastic Agents therapeutic use, Immunoglobulin Fragments immunology, Mesothelioma drug therapy
- Abstract
Mesothelioma is a malignancy of the mesothelium and current treatments are generally ineffective. One promising area of anticancer drug development is to explore tumor susceptibility to targeted therapy. To achieve efficient, targeted intracellular delivery of therapeutic agents to mesothelioma cells, we selected a naive human single-chain (scFv) phage antibody display library directly on the surface of live mesothelioma cells to identify internalizing antibodies that target mesothelioma-associated cell surface antigens. We have identified a panel of internalizing scFvs that bind to mesothelioma cell lines derived from both epithelioid (M28) and sarcomatous (VAMT-1) types of this disease. Most importantly, these antibodies stain mesothelioma cells in situ and therefore define a panel of clinically represented tumor antigens. We have further exploited the internalizing function of these scFvs to achieve targeted intracellular drug delivery to mesothelioma cells. We showed that scFv-targeted immunoliposomes were efficiently and specifically taken up by both epithelioid and sarcomatous mesothelioma cells, but not control cells, and immunoliposomes encapsulating the small-molecule drug topotecan caused targeted killing of both types of mesothelioma cells in vitro.
- Published
- 2008
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23. Anti-CD166 single chain antibody-mediated intracellular delivery of liposomal drugs to prostate cancer cells.
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Roth A, Drummond DC, Conrad F, Hayes ME, Kirpotin DB, Benz CC, Marks JD, and Liu B
- Subjects
- Antibiotics, Antineoplastic administration & dosage, Antineoplastic Agents administration & dosage, Antineoplastic Agents, Phytogenic administration & dosage, Cell Communication immunology, Cell Line, Tumor drug effects, Cell Survival drug effects, Doxorubicin administration & dosage, Flow Cytometry, Humans, Immunoglobulin Fragments immunology, Ligands, Liposomes, Male, Prostatic Neoplasms pathology, Radiation-Sensitizing Agents administration & dosage, Topotecan administration & dosage, Vinblastine administration & dosage, Vinblastine analogs & derivatives, Vinorelbine, Activated-Leukocyte Cell Adhesion Molecule immunology, Antibodies, Monoclonal administration & dosage, Drug Delivery Systems, Prostatic Neoplasms drug therapy
- Abstract
Targeted delivery of small-molecule drugs has the potential to enhance selective killing of tumor cells. We have identified previously an internalizing single chain [single chain variable fragment (scFv)] antibody that targets prostate cancer cells and identified the target antigen as CD166. We report here the development of immunoliposomes using this anti-CD166 scFv (H3). We studied the effects of a panel of intracellularly delivered, anti-CD166 immunoliposomal small-molecule drugs on prostate cancer cells. Immunoliposomal formulations of topotecan, vinorelbine, and doxorubicin each showed efficient and targeted uptake by three prostate cancer cell lines (Du-145, PC3, and LNCaP). H3-immunoliposomal topotecan was the most effective in cytotoxicity assays on all three tumor cell lines, showing improved cytotoxic activity compared with nontargeted liposomal topotecan. Other drugs such as liposomal doxorubicin were highly effective against LNCaP but not PC3 or Du-145 cells, despite efficient intracellular delivery. Post-internalization events thus modulate the overall efficacy of intracellularly delivered liposomal drugs, contributing in some cases to the lower than expected activity in a cell line-dependent manner. Further studies on intracellular tracking of endocytosed liposomal drugs will help identify and overcome the barriers limiting the potency of liposomal drugs.
- Published
- 2007
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24. Convection-enhanced delivery of nanoliposomal CPT-11 (irinotecan) and PEGylated liposomal doxorubicin (Doxil) in rodent intracranial brain tumor xenografts.
- Author
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Krauze MT, Noble CO, Kawaguchi T, Drummond D, Kirpotin DB, Yamashita Y, Kullberg E, Forsayeth J, Park JW, and Bankiewicz KS
- Subjects
- Animals, Antineoplastic Combined Chemotherapy Protocols adverse effects, Antineoplastic Combined Chemotherapy Protocols pharmacokinetics, Camptothecin administration & dosage, Camptothecin adverse effects, Camptothecin pharmacokinetics, Cell Line, Tumor, Convection, Doxorubicin adverse effects, Doxorubicin pharmacokinetics, Drug Synergism, Half-Life, Humans, Irinotecan, Liposomes, Male, Nanoparticles, Neoplasm Transplantation, Rats, Rats, Sprague-Dawley, Tissue Distribution, Xenograft Model Antitumor Assays, Antineoplastic Combined Chemotherapy Protocols administration & dosage, Brain Neoplasms drug therapy, Camptothecin analogs & derivatives, Doxorubicin administration & dosage
- Abstract
We have previously shown that convection-enhanced delivery (CED) of highly stable nanoparticle/liposome agents encapsulating chemotherapeutic drugs is effective against intracranial rodent brain tumor xenografts. In this study, we have evaluated the combination of a newly developed nanoparticle/liposome containing the topoisomerase I inhibitor CPT-11 (nanoliposomal CPT-11 [nLs-CPT-11]), and PEGylated liposomal doxorubicin (Doxil) containing the topoisomerase II inhibitor doxorubicin. Both drugs were detectable in the CNS for more than 36 days after a single CED application. Tissue half-life was 16.7 days for nLs-CPT-11 and 10.9 days for Doxil. The combination of the two agents produced synergistic cytotoxicity in vitro. In vivo in U251MG and U87MG intracranial rodent xenograft models, CED of the combination was also more efficacious than either agent used singly. Analysis of the parameters involved in this approach indicated that tissue pharmacokinetics, tumor microanatomy, and biochemical interactions of the drugs all contributed to the therapeutic efficacy observed. These findings have implications for further clinical applications of CED-based treatment of brain tumors.
- Published
- 2007
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25. Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells.
- Author
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Zhou Y, Drummond DC, Zou H, Hayes ME, Adams GP, Kirpotin DB, and Marks JD
- Subjects
- Amino Acid Sequence, Antibodies chemistry, Antibodies genetics, Antibodies metabolism, Cell Line, Tumor, Cell Survival drug effects, Epidermal Growth Factor metabolism, ErbB Receptors genetics, Gene Expression Regulation, Humans, Liposomes immunology, Molecular Sequence Data, Mutation genetics, Neoplasms genetics, Neoplasms pathology, Sequence Alignment, Solubility, Topotecan toxicity, Antibodies immunology, Antibody Affinity immunology, ErbB Receptors immunology, ErbB Receptors metabolism, Nanoparticles, Neoplasms immunology, Neoplasms metabolism
- Abstract
To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.
- Published
- 2007
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26. Identification and characterization of tumor antigens by using antibody phage display and intrabody strategies.
- Author
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Goenaga AL, Zhou Y, Legay C, Bougherara H, Huang L, Liu B, Drummond DC, Kirpotin DB, Auclair C, Marks JD, and Poul MA
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Neoplasm genetics, Antibody Specificity immunology, Antigens, Neoplasm chemistry, CHO Cells, Cell Adhesion, Cell Line, Tumor, Cell Proliferation, Chromatography, Liquid, Cricetinae, Cricetulus, Down-Regulation, Endocytosis, Epitopes immunology, Humans, Immunoglobulin Variable Region immunology, Mass Spectrometry, Molecular Sequence Data, Neoplasm Proteins metabolism, Phenotype, Protein Binding, Antibodies, Neoplasm immunology, Antigens, Neoplasm immunology, Antigens, Neoplasm isolation & purification, Peptide Library
- Abstract
To generate a panel of antibodies binding human breast cancers, a human single chain Fv phage display library was selected for rapid internalization into the SK-BR-3 breast cancer cell line. Thirteen unique antibodies were identified within the 55 cell binding antibodies studied, all of them showing specific staining of tumor cells compare to normal epithelial cells. Two of the antibodies bound the ErbB2 oncogene while 6 bound the tumor marker transferrin receptor (TfR). By developing a scFv immunoprecipitation method, we were able to use LC-MS/MS to identify the antigen bound by one of the antibodies (3GA5) as FPRP (prostaglandin F2alpha receptor-regulatory protein)/EWI-F/CD9P-1 (CD9 partner 1) an Ig superfamily member that has been described to interact directly with CD9 and CD81 tetraspanins and to be overexpressed in adherent cancer cell lines. Although the 3GA5 scFv had no direct anti-proliferative effect, intracellular expression of the scFv was able to knockdown CD9P-1 expression and could be used to further define the role of the tetraspanin system in proliferation and metastasis. Moreover, the 3GA5 scFv was rapidly internalized into breast tumor cells and could have potential for the targeted delivery of cytotoxic agents to breast cancers. This study is the proof of principle that the direct selection of phage antibody libraries on tumor cells can effectively lead to the identification and functional characterization of relevant tumor markers.
- Published
- 2007
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27. Increased target specificity of anti-HER2 genospheres by modification of surface charge and degree of PEGylation.
- Author
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Hayes ME, Drummond DC, Hong K, Zheng WW, Khorosheva VA, Cohen JA, C O N 4th, Park JW, Marks JD, Benz CC, and Kirpotin DB
- Subjects
- Antibodies chemistry, Biomedical Engineering, Humans, Models, Biological, Sensitivity and Specificity, Surface Properties, Transfection, Tumor Cells, Cultured, Drug Delivery Systems methods, Nanoparticles chemistry, Polyethylene Glycols chemistry, Receptor, ErbB-2 immunology
- Abstract
Genospheres are cationic lipid-nucleic acid nanoparticles prepared by the assembly of the lipids and nucleic acids from an aqueous/organic liquid monophase that independently dissolves the components, where the resultant particles are homogeneously sized (70-110 nm), with efficiently incorporated and protected DNA. In the present study, we demonstrate pH-dependent modulation of the Genosphere surface charge using pH-titratable lipids. By incorporation of the lipids with titratable anionic or imidazole headgroups, Genospheres with neutral or anionic surface charge at neutral pH were produced and compared for cellular uptake and transfection of a reporter gene (luciferase) in culture of breast cancer cells. The extent of particle-cell association was also studied by fluorescent microscopy and quantified by cytofluorometery. The effects of Genosphere surface modification with poly(ethylene glycol) (molecular weight 2000) at low (0.5 mol %) and high (5 mol %) grafting densities, as well as the effects of HER2-receptor-directed targeting by an internalizable anti-HER2 scFv F5, linked via PEG spacer, were also studied. Inclusion in the Genosphere formulation of pH-titratable lipids CHEMS (cholesteryl hemisuccinate), CHIM (1-(3-(cholesteryloxycarbonylamino)propyl)imidazole), or DSGG (1,2-distearoyl-sn-glycero-3-hemiglutarate) rendered the particles surface-charge neutral or slightly anionic at neutral pH, and cationic at mildly acidic pH, as shown by zeta-potential measurements. In HER2-targeted systems, transfection activity and target specificity with HER2-overexpressing SKBR-3 breast cancer cells were dependent on Genosphere surface charge and PEGylation. The highest target specificity correlated with low cationic charge at neutral pH, while incorporation of 5 mol % PEG-lipid had only minor effects on Genosphere-cell association, internalization, and transfection activity. The implications of this work for potential in vivo applications are discussed.
- Published
- 2006
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28. A novel assay for monitoring internalization of nanocarrier coupled antibodies.
- Author
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Nielsen UB, Kirpotin DB, Pickering EM, Drummond DC, and Marks JD
- Subjects
- Antineoplastic Agents metabolism, Cell Death, Cell Line, Tumor, Doxorubicin chemistry, Histidine metabolism, Humans, Immunoglobulin Variable Region immunology, Ligands, Lipid Bilayers chemistry, Lipid Metabolism, Methotrexate chemistry, Nickel metabolism, Oligopeptides metabolism, Receptor, ErbB-2 immunology, Sensitivity and Specificity, Antibodies, Neoplasm administration & dosage, Antibodies, Neoplasm analysis, Immunologic Techniques, Liposomes administration & dosage, Liposomes metabolism
- Abstract
Background: Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA., Results: The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay., Conclusion: The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.
- Published
- 2006
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29. Convection-enhanced delivery of Ls-TPT enables an effective, continuous, low-dose chemotherapy against malignant glioma xenograft model.
- Author
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Saito R, Krauze MT, Noble CO, Drummond DC, Kirpotin DB, Berger MS, Park JW, and Bankiewicz KS
- Subjects
- Animals, Brain Neoplasms pathology, Cell Line, Tumor, Dose-Response Relationship, Drug, Glioma pathology, Humans, Liposomes, Male, Rats, Rats, Nude, Brain Neoplasms drug therapy, Convection, Drug Delivery Systems methods, Glioma drug therapy, Topotecan administration & dosage, Xenograft Model Antitumor Assays methods
- Abstract
Treatment of malignant gliomas represents one of the most formidable challenges in oncology. The combination of surgery, radiation, and chemotherapy yields median survivals of less than one year. Here we demonstrate the use of a minimally invasive surgical technique, convection-enhanced delivery (CED), for local administration of a novel nanoparticle liposome containing topotecan. CED of this liposomal topotecan (Ls-TPT) resulted in extended brain tissue retention (t1/2 = 1.5 days), whereas free topotecan was rapidly cleared (t1/2 = 0.1 days) after CED. The favorable pharmacokinetic profile of extended topotecan release for about seven days, along with biodistribution featuring perivascular accumulation of the nanoparticles, provided, in addition to the known topoisomerase I inhibition, an effective antiangiogenic therapy. In the rat intracranial U87MG tumor model, vascular targeting of Ls-TPT with CED was associated with reductions in laminin expression and vascular density compared to free topotecan or control treatments. A single CED treatment on day 7 showed that free topotecan conferred no survival benefit versus control. However, Ls-TPT produced a significant (P = 0.0002) survival benefit, with six of seven complete cures. Larger U87MG tumors, where CED of Ls-TPT on day 12 resulted in one of six cures, indicated the necessity to cover the entire tumor with the infused therapeutic agent. CED of Ls-TPT was also efficacious in the intracranial U251MG tumor model (P = 0.0005 versus control). We conclude that the combination of a novel nanoparticle Ls-TPT and CED administration was very effective in treating experimental brain tumors.
- Published
- 2006
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30. Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models.
- Author
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Kirpotin DB, Drummond DC, Shao Y, Shalaby MR, Hong K, Nielsen UB, Marks JD, Benz CC, and Park JW
- Subjects
- Animals, Antibodies, Monoclonal administration & dosage, Antibodies, Monoclonal immunology, Antibodies, Monoclonal pharmacokinetics, Breast Neoplasms drug therapy, Breast Neoplasms metabolism, Female, Humans, Liposomes chemistry, Mice, Receptor, ErbB-2 biosynthesis, Transplantation, Heterologous, Breast Neoplasms immunology, Liposomes immunology, Liposomes pharmacokinetics, Nanostructures, Receptor, ErbB-2 immunology
- Abstract
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.
- Published
- 2006
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- View/download PDF
31. Tissue affinity of the infusate affects the distribution volume during convection-enhanced delivery into rodent brains: implications for local drug delivery.
- Author
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Saito R, Krauze MT, Noble CO, Tamas M, Drummond DC, Kirpotin DB, Berger MS, Park JW, and Bankiewicz KS
- Subjects
- Animals, Antineoplastic Agents administration & dosage, Antineoplastic Agents pharmacokinetics, Antineoplastic Agents therapeutic use, Brain Neoplasms drug therapy, Chemistry, Pharmaceutical, Convection, Drug Compounding, Electrochemistry, Excipients, Liposomes, Male, Nanostructures, Particle Size, Pharmaceutical Vehicles, Polyethylene Glycols chemistry, Rats, Rats, Sprague-Dawley, Stereoisomerism, Brain physiology, Drug Delivery Systems instrumentation, Pharmaceutical Preparations administration & dosage
- Abstract
Convection-enhanced delivery (CED) is a recently developed technique for local delivery of agents to a large volume of tissue in the central nervous system (CNS). We have previously reported that this technique can be applied to CNS delivery of nanoparticles including viruses and liposomes. In this paper, we describe the impact of key physical and chemical properties of infused molecules on the extent of CED-mediated delivery. For simple infusates, CED distribution was significantly increased if the infusate was more hydrophilic or had less tissue affinity. Encapsulation of tissue-affinitive molecules by neutral liposomes significantly increased their tissue distribution. The poorer brain distribution observed with cationic liposomes, due to their greater tissue affinity, was completely overcome by PEGylation, which provides steric stabilization and reduced surface charge. Finally, liposomal encapsulation of doxorubicin reduced its tissue affinity and substantially increased its distribution within brain tumor tissue. Taken together, the physical and chemical properties of drugs, small molecules and macromolecular carriers determine the tissue affinity of the infusate and strongly affect the distribution of locally applied agents. Thus, an increased and more predictable tissue distribution can be achieved by reducing the tissue affinity of the infusate using appropriately engineered liposomes or other nanoparticles.
- Published
- 2006
- Full Text
- View/download PDF
32. Genospheres: self-assembling nucleic acid-lipid nanoparticles suitable for targeted gene delivery.
- Author
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Hayes ME, Drummond DC, Kirpotin DB, Zheng WW, Noble CO, Park JW, Marks JD, Benz CC, and Hong K
- Subjects
- Drug Carriers, Gene Targeting, Genetic Therapy instrumentation, Humans, Liposomes, Microscopy, Electron, Nanostructures, Phosphatidylethanolamines, Polyethylene Glycols, DNA administration & dosage, Genetic Therapy methods, Immunoglobulin Variable Region genetics, Nanotechnology methods, Receptor, ErbB-2 immunology
- Abstract
We describe the assembly of a cationic lipid-nucleic acid nanoparticle from a liquid monophase containing water and a water miscible organic solvent where both lipid and DNA components are separately soluble prior to their combination. Upon removal of the organic solvent, stable and homogenously sized (70-100 nm) lipid-nucleic acid nanoparticles (Genospheres) were formed. The low accessibility (<15%) of the nanoparticle-encapsulated DNA to a DNA intercalating dye indicated well-protected nucleic acids and high DNA incorporation efficiencies. It was demonstrated that Genospheres could be stably stored under a variety of conditions including a lyophilized state where no appreciable increase in particle size or DNA accessibility was observed following reconstitution. Finally, Genospheres were made target-specific by insertion of an antibody-lipopolymer (anti-HER2 scFv (F5)-PEG-DSPE) conjugate into the particle. The target specificity (>100-fold) in HER2 overexpressing SK-BR-3 breast cancer cells was dependent on the degree of PEGylation, where the incorporation of high amounts of PEG-lipid on the particle surface (up to 5 mol%) had only a minor effect on the transfection activity of the targeted Genospheres. In summary, this work describes a novel, readily scalable method for preparing highly stable immunotargeted nucleic acid delivery vehicles capable of achieving a high degree of specific transfection activity.
- Published
- 2006
- Full Text
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33. Assembly of nucleic acid-lipid nanoparticles from aqueous-organic monophases.
- Author
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Hayes ME, Drummond DC, Hong K, Park JW, Marks JD, and Kirpotin DB
- Subjects
- Base Sequence, DNA analysis, Indicators and Reagents, Oligodeoxyribonucleotides chemistry, Organic Chemicals, Phosphatidylcholines genetics, Plasmids, Receptor, ErbB-2 genetics, Solvents, Water, Lipids analysis, Nanostructures analysis, Phosphatidylcholines analysis
- Abstract
Effective, reproducible, and scalable methods for DNA-lipid assembly are important for the success of non-viral vectors in in vivo gene therapy. We hypothesized DNA-lipid assembly would be optimal if started from a liquid monophase where both DNA and lipids separately form molecular or micellar solutions prior to mixing, without preexisting condensed lipid phases, thus allowing DNA-lipid assembly under conditions close to equilibrium. Previously, we found that mixing plasmid DNA, 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), cholesterol and a cationic lipid, 1, 2-dioleoyl-3-(trimethylammonio) propane (DOTAP) in 50% (v/v) aqueous ethanol spontaneously produced an optically transparent solution. Upon ethanol removal, DNA-lipid nanoparticles (Genospheres) were formed. For comparison with well-known technologies, different DNA-lipid particles were prepared by interaction of plasmid DNA and stable or ethanol-destabilized lipid vesicles by combining the components in water or 30% (v/v) aqueous ethanol, respectively. Among the three studied DNA-lipid assembly methods, only Genospheres combined the properties of small size (less than or around 100 nm), high incorporation of both lipid and DNA, high degree of DNA protection (dye accessibility 5-12%), a narrow distribution of particle density and when immuno-targeted, the highest transfection efficiency in HER2-overexpressing cells in vitro. We conclude that the Genosphere assembly methodology offers advantages for the development of effective, scalable and targetable non-viral gene delivery vectors.
- Published
- 2006
- Full Text
- View/download PDF
34. Development of a highly active nanoliposomal irinotecan using a novel intraliposomal stabilization strategy.
- Author
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Drummond DC, Noble CO, Guo Z, Hong K, Park JW, and Kirpotin DB
- Subjects
- Animals, Breast Neoplasms drug therapy, Breast Neoplasms metabolism, Camptothecin administration & dosage, Camptothecin chemistry, Camptothecin pharmacokinetics, Camptothecin toxicity, Colonic Neoplasms drug therapy, Colonic Neoplasms metabolism, Drug Stability, Female, HT29 Cells, Humans, Irinotecan, Liposomes administration & dosage, Liposomes pharmacokinetics, Liposomes toxicity, Mice, Mice, Nude, Nanostructures toxicity, Rats, Rats, Sprague-Dawley, Xenograft Model Antitumor Assays, Camptothecin analogs & derivatives, Drug Delivery Systems methods, Liposomes chemistry, Nanostructures chemistry
- Abstract
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.
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- 2006
- Full Text
- View/download PDF
35. Novel nanoliposomal CPT-11 infused by convection-enhanced delivery in intracranial tumors: pharmacology and efficacy.
- Author
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Noble CO, Krauze MT, Drummond DC, Yamashita Y, Saito R, Berger MS, Kirpotin DB, Bankiewicz KS, and Park JW
- Subjects
- Animals, Brain Neoplasms metabolism, Camptothecin administration & dosage, Camptothecin chemistry, Camptothecin pharmacokinetics, Camptothecin toxicity, Cell Line, Tumor, Convection, Humans, Irinotecan, Liposomes administration & dosage, Liposomes chemistry, Liposomes pharmacokinetics, Liposomes toxicity, Male, Nanostructures chemistry, Nanostructures toxicity, Phospholipids administration & dosage, Phospholipids chemistry, Phospholipids pharmacokinetics, Phospholipids toxicity, Rats, Rats, Sprague-Dawley, Xenograft Model Antitumor Assays, Brain Neoplasms drug therapy, Camptothecin analogs & derivatives, Drug Delivery Systems methods
- Abstract
We hypothesized that combining convection-enhanced delivery (CED) with a novel, highly stable nanoparticle/liposome containing CPT-11 (nanoliposomal CPT-11) would provide a dual drug delivery strategy for brain tumor treatment. Following CED in rat brains, tissue retention of nanoliposomal CPT-11 was greatly prolonged, with >20% injected dose remaining at 12 days for all doses. Tissue residence was dose dependent, with doses of 60 microg (3 mg/mL), 0.8 mg (40 mg/mL), and 1.6 mg (80 mg/mL) resulting in tissue half-life (t(1/2)) of 6.7, 10.7, and 19.7 days, respectively. In contrast, CED of free CPT-11 resulted in rapid drug clearance (tissue t(1/2) = 0.3 day). At equivalent CED doses, nanoliposomal CPT-11 increased area under the time-concentration curve by 25-fold and tissue t(1/2) by 22-fold over free CPT-11; CED in intracranial U87 glioma xenografts showed even longer tumor retention (tissue t(1/2) = 43 days). Plasma levels were undetectable following CED of nanoliposomal CPT-11. Importantly, prolonged exposure to nanoliposomal CPT-11 resulted in no measurable central nervous system (CNS) toxicity at any dose tested (0.06-1.6 mg/rat), whereas CED of free CPT-11 induced severe CNS toxicity at 0.4 mg/rat. In the intracranial U87 glioma xenograft model, a single CED infusion of nanoliposomal CPT-11 at 1.6 mg resulted in significantly improved median survival (>100 days) compared with CED of control liposomes (19.5 days; P = 4.9 x 10(-5)) or free drug (28.5 days; P = 0.011). We conclude that CED of nanoliposomal CPT-11 greatly prolonged tissue residence while also substantially reducing toxicity, resulting in a highly effective treatment strategy in preclinical brain tumor models.
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- 2006
- Full Text
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36. Epidermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo.
- Author
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Mamot C, Drummond DC, Noble CO, Kallab V, Guo Z, Hong K, Kirpotin DB, and Park JW
- Subjects
- Animals, Antibodies, Monoclonal administration & dosage, Antibodies, Monoclonal, Humanized, Antineoplastic Combined Chemotherapy Protocols pharmacokinetics, Brain Neoplasms drug therapy, Brain Neoplasms immunology, Brain Neoplasms metabolism, Breast Neoplasms immunology, Breast Neoplasms metabolism, Cetuximab, Doxorubicin administration & dosage, Drug Delivery Systems, Epirubicin administration & dosage, ErbB Receptors genetics, ErbB Receptors immunology, Female, Glioblastoma immunology, Glioblastoma metabolism, Humans, Immunoglobulin Fab Fragments immunology, Mice, Mice, Nude, Rats, Rats, Sprague-Dawley, Transfection, Transplantation, Heterologous, Tumor Cells, Cultured drug effects, Tumor Cells, Cultured metabolism, Vinblastine administration & dosage, Vinblastine analogs & derivatives, Vinorelbine, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Breast Neoplasms drug therapy, ErbB Receptors drug effects, Glioblastoma drug therapy, Immunoconjugates therapeutic use, Liposomes administration & dosage
- Abstract
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.
- Published
- 2005
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37. Gadolinium-loaded liposomes allow for real-time magnetic resonance imaging of convection-enhanced delivery in the primate brain.
- Author
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Saito R, Krauze MT, Bringas JR, Noble C, McKnight TR, Jackson P, Wendland MF, Mamot C, Drummond DC, Kirpotin DB, Hong K, Berger MS, Park JW, and Bankiewicz KS
- Subjects
- Animals, Image Processing, Computer-Assisted methods, Macaca fascicularis, Male, Statistics as Topic, Time Factors, Tissue Distribution, Brain metabolism, Convection, Drug Delivery Systems, Gadolinium administration & dosage, Liposomes metabolism, Magnetic Resonance Imaging
- Abstract
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.
- Published
- 2005
- Full Text
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38. Enhanced pharmacodynamic and antitumor properties of a histone deacetylase inhibitor encapsulated in liposomes or ErbB2-targeted immunoliposomes.
- Author
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Drummond DC, Marx C, Guo Z, Scott G, Noble C, Wang D, Pallavicini M, Kirpotin DB, and Benz CC
- Subjects
- Animals, Area Under Curve, Blotting, Northern, Blotting, Western, Capsules, Cell Line, Tumor, Cell Survival drug effects, Dose-Response Relationship, Drug, Female, Histone Deacetylase Inhibitors, Histone Deacetylases metabolism, Humans, Hydroxamic Acids pharmacokinetics, Hydroxamic Acids therapeutic use, Liposomes immunology, Mice, Mice, Nude, Neoplasms, Experimental genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Rats, Sprague-Dawley, Receptor, ErbB-2 genetics, Receptor, ErbB-2 metabolism, Xenograft Model Antitumor Assays, Hydroxamic Acids pharmacology, Neoplasms, Experimental prevention & control, Receptor, ErbB-2 immunology
- Abstract
ErbB2-overexpressing human cancers represent potentially sensitive targets for therapy by candidate histone deacetylase (HDAC) inhibitors as we have shown that HDAC inhibitors can selectively reduce ErbB2 expression by repressing the ErbB2 promoter and accelerating the decay of cytoplasmic ErbB2 transcripts. To extend these in vitro findings and enhance the in vivo pharmacodynamic properties of HDAC inhibitors, we stably encapsulated a potent hydroxamate-based HDAC inhibitor (LAQ824) within long-circulating liposomes (Ls-LAQ824) and immunoliposomes (ILs-LAQ824) bearing >10,000 LAQ824 molecules per nanovesicle. Liposomal LAQ824 exhibits prolonged in vivo stability and, unlike free LAQ824, circulates with a half-life of 10.8 hours following a single i.v. injection. Three weekly i.v. injections of 20 to 25 mg/kg Ls-LAQ824 in nude mice with ErbB2 overexpressing BT-474 breast tumor xenografts significantly impairs tumor growth, and administration of ErbB2-targeted ILs-LAQ824 may further improve this antitumor activity. Studies of tumor-bearing mice 24 hours after single treatment indicate that: (a) >10% of injected liposomal LAQ824 is still circulating (whereas free LAQ824 is undetectable in the blood after 15 minutes); and (b) tumor uptake of Ls-LAQ824 and ILs-LAQ824 is >3% injected drug per gram of tumor, producing levels of acetylated tumor histones that are 5- to 10-fold increased over those following free LAQ824 or saline treatments and resulting in concordantly reduced levels of tumor ErbB2 mRNA. These preclinical results support the clinical evaluation of HDAC inhibitors against ErbB2-overexpressing malignancies, and further indicate that encapsulation into targeted and nontargeted liposomes substantially improves the in vivo pharmacokinetics, tumor uptake, and antitumor properties of hydroxamate-based HDAC inhibitors.
- Published
- 2005
- Full Text
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39. Preclinical manufacture of anti-HER2 liposome-inserting, scFv-PEG-lipid conjugate. 2. Conjugate micelle identity, purity, stability, and potency analysis.
- Author
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Nellis DF, Giardina SL, Janini GM, Shenoy SR, Marks JD, Tsai R, Drummond DC, Hong K, Park JW, Ouellette TF, Perkins SC, and Kirpotin DB
- Subjects
- Calorimetry, Differential Scanning, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange methods, Electrophoresis, Polyacrylamide Gel, Lysophospholipase metabolism, Micelles, Models, Biological, Molecular Weight, Particle Size, Receptor, ErbB-2 antagonists & inhibitors, Receptor, ErbB-2 immunology, Sequence Analysis, Protein, Temperature, Time Factors, Immunoglobulin Fragments chemistry, Liposomes chemistry
- Abstract
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.
- Published
- 2005
- Full Text
- View/download PDF
40. Clinical development of histone deacetylase inhibitors as anticancer agents.
- Author
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Drummond DC, Noble CO, Kirpotin DB, Guo Z, Scott GK, and Benz CC
- Subjects
- Animals, Antineoplastic Agents chemistry, Antineoplastic Agents pharmacology, Histone Deacetylases metabolism, Humans, Protease Inhibitors chemistry, Protease Inhibitors pharmacology, Antineoplastic Agents therapeutic use, Histone Deacetylase Inhibitors, Protease Inhibitors therapeutic use
- Abstract
Acetylation is a key posttranslational modification of many proteins responsible for regulating critical intracellular pathways. Although histones are the most thoroughly studied of acetylated protein substrates, histone acetyltransferases (HATs) and deacetylases (HDACs) are also responsible for modifying the activity of diverse types of nonhistone proteins, including transcription factors and signal transduction mediators. HDACs have emerged as uncredentialed molecular targets for the development of enzymatic inhibitors to treat human cancer, and six structurally distinct drug classes have been identified with in vivo bioavailability and intracellular capability to inhibit many of the known mammalian members representing the two general types of NAD+-independent yeast HDACs, Rpd3 (HDACs 1, 2, 3, 8) and Hda1 (HDACs 4, 5, 6, 7, 9a, 9b, 10). Initial clinical trials indicate that HDAC inhibitors from several different structural classes are very well tolerated and exhibit clinical activity against a variety of human malignancies; however, the molecular basis for their anticancer selectivity remains largely unknown. HDAC inhibitors have also shown preclinical promise when combined with other therapeutic agents, and innovative drug delivery strategies, including liposome encapsulation, may further enhance their clinical development and anticancer potential. An improved understanding of the mechanistic role of specific HDACs in human tumorigenesis, as well as the identification of more specific HDAC inhibitors, will likely accelerate the clinical development and broaden the future scope and utility of HDAC inhibitors for cancer treatment.
- Published
- 2005
- Full Text
- View/download PDF
41. Preclinical manufacture of an anti-HER2 scFv-PEG-DSPE, liposome-inserting conjugate. 1. Gram-scale production and purification.
- Author
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Nellis DF, Ekstrom DL, Kirpotin DB, Zhu J, Andersson R, Broadt TL, Ouellette TF, Perkins SC, Roach JM, Drummond DC, Hong K, Marks JD, Park JW, and Giardina SL
- Subjects
- Amino Acid Sequence, Cell Division physiology, Cells, Cultured, Electrophoresis, Polyacrylamide Gel, Escherichia coli chemistry, Escherichia coli cytology, Micelles, Molecular Conformation, Molecular Sequence Data, Phosphates metabolism, Receptor, ErbB-2 antagonists & inhibitors, Receptor, ErbB-2 immunology, Time Factors, Immunoglobulin Fragments biosynthesis, Immunoglobulin Fragments isolation & purification, Liposomes isolation & purification, Liposomes metabolism
- Abstract
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.
- Published
- 2005
- Full Text
- View/download PDF
42. Development of ligand-targeted liposomes for cancer therapy.
- Author
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Noble CO, Kirpotin DB, Hayes ME, Mamot C, Hong K, Park JW, Benz CC, Marks JD, and Drummond DC
- Subjects
- Animals, Humans, Ligands, Liposomes, Neoplasms genetics, Neoplasms metabolism, Antineoplastic Agents administration & dosage, Antineoplastic Agents metabolism, Drug Delivery Systems methods, Drug Delivery Systems trends, Neoplasms drug therapy
- Abstract
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.
- Published
- 2004
- Full Text
- View/download PDF
43. Extensive distribution of liposomes in rodent brains and brain tumors following convection-enhanced delivery.
- Author
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Mamot C, Nguyen JB, Pourdehnad M, Hadaczek P, Saito R, Bringas JR, Drummond DC, Hong K, Kirpotin DB, McKnight T, Berger MS, Park JW, and Bankiewicz KS
- Subjects
- Animals, Convection, Gadolinium administration & dosage, Gadolinium pharmacokinetics, Glioma metabolism, Liposomes administration & dosage, Magnetic Resonance Imaging, Mannitol administration & dosage, Mice, Rats, Sensitivity and Specificity, Transplantation, Heterologous, Brain metabolism, Brain Neoplasms metabolism, Drug Delivery Systems, Liposomes pharmacokinetics
- Abstract
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.
- Published
- 2004
- Full Text
- View/download PDF
44. Distribution of liposomes into brain and rat brain tumor models by convection-enhanced delivery monitored with magnetic resonance imaging.
- Author
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Saito R, Bringas JR, McKnight TR, Wendland MF, Mamot C, Drummond DC, Kirpotin DB, Park JW, Berger MS, and Bankiewicz KS
- Subjects
- Animals, Antibiotics, Antineoplastic administration & dosage, Antibiotics, Antineoplastic pharmacokinetics, Carbocyanines administration & dosage, Carbocyanines pharmacology, Convection, Doxorubicin administration & dosage, Doxorubicin pharmacokinetics, Fluorescent Dyes administration & dosage, Fluorescent Dyes pharmacokinetics, Gadolinium administration & dosage, Gadolinium pharmacokinetics, Glioma metabolism, Gliosarcoma metabolism, Liposomes administration & dosage, Liposomes toxicity, Magnetic Resonance Imaging, Male, Rats, Rats, Sprague-Dawley, Tissue and Organ Procurement, Brain metabolism, Brain Neoplasms metabolism, Liposomes pharmacokinetics
- Abstract
Although liposomes have been used as a vehicle for delivery of therapeutic agents in oncology, their efficacy in targeting brain tumors has been limited due to poor penetration through the blood-brain barrier. Because convection-enhanced delivery (CED) of liposomes may improve the therapeutic index for targeting brain tumors, we conducted a three-stage study: stage 1 established the feasibility of using in vivo magnetic resonance imaging (MRI) to confirm adequate liposomal distribution within targeted regions in normal rat brain. Liposomes colabeled with gadolinium (Gd) and a fluorescent indicator, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'-disulfonic acid [DiI-DS; formally DiIC(18)(3)-DS], were administered by CED into striatal regions. The minimum concentration of Gd needed for monitoring, correlation of infused volume with distribution volume, clearance of infused liposome containing Gd and DiI-DS (Lip/Gd/DiI-DS), and potential local toxicity were evaluated. After determination of adequate conditions for MRI detection in normal brain, stage 2 evaluated the feasibility of in vivo MRI monitoring of liposomal distribution in C6 and 9L-2 rat glioma models. In both models, the distribution of Lip/Gd/DiI-DS covering the tumor mass was well defined and monitored with MRI. Stage 3 was designed to develop a clinically relevant treatment strategy in the 9L-2 model by infusing liposome containing Gd (Lip/Gd), prepared in the same size as Lip/Gd/DiI-DS, with Doxil, a liposomal drug of similar size used to treat several cancers. MRI detection of Lip/Gd coadministered with Doxil provided optimum CED parameters for complete coverage of 9L-2 tumors. By permitting in vivo monitoring of therapeutic distribution in brain tumors, this technique optimizes local drug delivery and may provide a basis for clinical applications in the treatment of malignant glioma.
- Published
- 2004
- Full Text
- View/download PDF
45. Mapping tumor epitope space by direct selection of single-chain Fv antibody libraries on prostate cancer cells.
- Author
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Liu B, Conrad F, Cooperberg MR, Kirpotin DB, and Marks JD
- Subjects
- Antibodies, Neoplasm, Cell Division immunology, Cell Line, Tumor, Endocytosis, Humans, Immunoglobulin Fragments, Male, Prostatic Neoplasms pathology, Epitopes analysis, Peptide Library, Prostatic Neoplasms immunology
- Abstract
The identification of tumor-specific cell surface antigens is a critical step toward the development of targeted therapeutics for cancer. The epitope space at the tumor cell surface is highly complex, composed of proteins, carbohydrates, and other membrane-associated determinants including post-translational modification products, which are difficult to probe by approaches based on gene expression. This epitope space can be efficiently mapped by complementary monoclonal antibodies. By selecting human antibody gene diversity libraries directly on the surface of prostate cancer cells, we have taken a functional approach to identifying fully human, tumor-specific monoclonal antibodies without prior knowledge of their target antigens. Selection conditions have been optimized to favor tumor-specific antibody binding and internalization. To date, we have discovered >90 monoclonal antibodies that specifically bind and enter prostate cancer cells, with little or no binding to control cells. These antibodies are able to efficiently deliver intracellular payloads when attached to nanoparticles such as liposomes. In addition, a subset of the antibodies displayed intrinsic antiproliferative activity. These tumor-specific internalizing antibodies are likely to be useful for targeted therapeutics either alone or in combination with effector molecules. The antigens they bind constitute a tumor-specific internalizing epitope space that is likely to play a significant role in cancer cell homeostasis. Targeting components of this epitope space may facilitate development of immunotherapeutic and small molecule-based strategies as well as the use of other therapeutic agents that rely upon delivery to the interior of the tumor cell.
- Published
- 2004
- Full Text
- View/download PDF
46. Liposome-based approaches to overcome anticancer drug resistance.
- Author
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Mamot C, Drummond DC, Hong K, Kirpotin DB, and Park JW
- Subjects
- Antineoplastic Agents pharmacokinetics, Drug Delivery Systems, Humans, Antineoplastic Agents administration & dosage, Drug Resistance, Neoplasm drug effects, Liposomes administration & dosage
- Abstract
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.
- Published
- 2003
- Full Text
- View/download PDF
47. Epidermal growth factor receptor (EGFR)-targeted immunoliposomes mediate specific and efficient drug delivery to EGFR- and EGFRvIII-overexpressing tumor cells.
- Author
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Mamot C, Drummond DC, Greiser U, Hong K, Kirpotin DB, Marks JD, and Park JW
- Subjects
- Adenocarcinoma pathology, Antibodies, Monoclonal administration & dosage, Antibodies, Monoclonal therapeutic use, Antibodies, Neoplasm administration & dosage, Antineoplastic Agents blood, Brain Neoplasms pathology, Breast Neoplasms pathology, Carcinoma, Squamous Cell pathology, Doxorubicin blood, Drug Delivery Systems, Drug Design, ErbB Receptors genetics, ErbB Receptors immunology, Female, Glioblastoma pathology, Humans, Immunoconjugates blood, Immunoglobulin Fab Fragments immunology, Liposomes administration & dosage, Liposomes blood, Neoplasm Proteins immunology, Transfection, Tumor Cells, Cultured drug effects, Tumor Cells, Cultured metabolism, Vinorelbine, Vulvar Neoplasms pathology, Antineoplastic Agents administration & dosage, Doxorubicin administration & dosage, ErbB Receptors drug effects, Immunoconjugates administration & dosage, Methotrexate administration & dosage, Neoplasm Proteins drug effects, Vinblastine administration & dosage, Vinblastine analogs & derivatives
- Abstract
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.
- Published
- 2003
48. Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis.
- Author
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Nielsen UB, Kirpotin DB, Pickering EM, Hong K, Park JW, Refaat Shalaby M, Shao Y, Benz CC, and Marks JD
- Subjects
- Animals, Antibiotics, Antineoplastic pharmacokinetics, Antibiotics, Antineoplastic therapeutic use, Antibodies, Disease Models, Animal, Doxorubicin pharmacokinetics, Doxorubicin therapeutic use, Drug Delivery Systems, Endocytosis, Endosomes, Female, Kinetics, Mice, Mice, Nude, Neoplasm Transplantation, Neoplasms, Experimental drug therapy, Neoplasms, Experimental metabolism, Receptor, ErbB-2 genetics, Xenograft Model Antitumor Assays, Antibiotics, Antineoplastic administration & dosage, Doxorubicin administration & dosage, Liposomes, Receptor, ErbB-2 immunology
- Abstract
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.
- Published
- 2002
- Full Text
- View/download PDF
49. Anti-HER2 immunoliposomes: enhanced efficacy attributable to targeted delivery.
- Author
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Park JW, Hong K, Kirpotin DB, Colbern G, Shalaby R, Baselga J, Shao Y, Nielsen UB, Marks JD, Moore D, Papahadjopoulos D, and Benz CC
- Subjects
- Animals, Antibodies, Monoclonal administration & dosage, Antineoplastic Agents administration & dosage, Antineoplastic Agents pharmacokinetics, Area Under Curve, Breast Neoplasms drug therapy, Breast Neoplasms pathology, Dose-Response Relationship, Drug, Doxorubicin administration & dosage, Doxorubicin pharmacokinetics, Drug Delivery Systems methods, Drug Synergism, Humans, Immunoglobulin Fragments administration & dosage, Immunoglobulin Fragments immunology, Liposomes pharmacokinetics, Mammary Neoplasms, Experimental drug therapy, Mammary Neoplasms, Experimental pathology, Mice, Mice, Nude, Neoplasm Transplantation, Rats, Rats, Sprague-Dawley, Treatment Outcome, Tumor Cells, Cultured, Xenograft Model Antitumor Assays, Antibodies, Monoclonal immunology, Antineoplastic Agents therapeutic use, Doxorubicin therapeutic use, Receptor, ErbB-2 immunology
- Abstract
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.
- Published
- 2002
50. Tumor targeting using anti-her2 immunoliposomes.
- Author
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Park JW, Kirpotin DB, Hong K, Shalaby R, Shao Y, Nielsen UB, Marks JD, Papahadjopoulos D, and Benz CC
- Subjects
- Animals, Antibiotics, Antineoplastic administration & dosage, Antibiotics, Antineoplastic pharmacokinetics, Antibodies, Monoclonal administration & dosage, Antibodies, Neoplasm administration & dosage, Doxorubicin administration & dosage, Doxorubicin pharmacokinetics, Drug Stability, Genetic Therapy methods, Humans, Immunoglobulin Fragments immunology, Liposomes, Rats, Antibodies, Monoclonal immunology, Antibodies, Neoplasm immunology, Genes, erbB-2 immunology, Neoplasms immunology
- Abstract
We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes (P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.0-8.5% of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidal-gold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while non-targeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2-overexpression, no difference in tumor cell uptake was seen, with both ILs and non-targeted liposomes accumulating within stroma. Thus, anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, anti-HER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizing anti-HER2 phage antibodies; modifications of the scFv expression construct to support large scale production and clinical use; and development of methods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed and drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct consists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox suggest that this strategy may provide optimal delivery of anthracycline-based chemotherapy to HER2-overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumab+anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents.
- Published
- 2001
- Full Text
- View/download PDF
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