4586 A specific peptide inhibitor CTTHWGFTLC (CTT1) of gelatinases (MMP-2 and MMP-9) has been isolated from phage display peptide libraries (Koivunen et al . 1999, Nature Biotechnology 17, 768-774). This cyclic decapeptide was shown to prevent tumor growth and to improve survival of nude mice bearing human tumors. We have designed a more hydrophilic derivative of CTT1-peptide. Here, we have studied the pharmacokinetics of this novel CTT2-peptide and shown that CTT2 exhibits lower liver accumulation than the original phage display derived peptide. CTT2 accumulation was then screened in 12 different gelatinase expressing human xenograft tumors. High accumulation of CTT2 was seen in ovarian xenografts, and the highest accumulation was observed in serous type ovarian carcinoma where tumor/muscle ratio was 35 at 180 min after peptide injection. The peptide did not accumulate significantly in any other tissues studied. To generate tumor targeted liposomes (CTT2-SL), the CTT2-peptide was coupled covalently on the surface of doxorubicin loaded liposomes (SL). After single i.v. dose the CTT2-SL showed pharmacokinetics that were indistinguishable from that of SL in xenograft mice. Furthermore, CTT2-SL retains non-immunogenicity of SL. A biodistribution study showed that tumor AUC of doxorubicin encapsulated inside CTT2-SL is 40% greater than that of doxorubicin encapsulated inside SL. Finally, therapeutic efficacy of CTT2-SL was compared to that of SL. Mice were inoculated with ovarian carcinoma cells that were shown to be sensitive to doxorubicin in in vitro assay. Afterwards, the mice were treated systemically three times in three day intervals with CTT2-SL or SL (9mg/kg doxorubicin equivalents). The life span of mice treated with CTT2-SL increased 35% when compared to SL treated mice. We conclude that CTT-liposomes represent a promising technology to treat gelatinase expressing solid tumors. Based on these results, the development of CTT-SL peptidoliposomes toward clinical trials is underway.
The development of molecularly targeted probes that exhibit high biostability, biocompatibility, and efficient clearance profiles is key to optimizing biodistribution and transport across biological barriers. Further, coupling probes designed to meet these criteria with high-sensitivity, quantitative imaging strategies is mandatory for ensuring early in vivo tumor detection and timely treatment response. These challenges have often only been examined individually, impeding the clinical translation of fluorescent probes. By simultaneously optimizing these design criteria, we created a new generation of near-infrared fluorescent core-shell silica-based nanoparticles (C dots) tuned to hydrodynamic diameters of 3.3 and 6.0 nm with improved photophysical characteristics over the parent dye. A neutral organic coating prevented adsorption of serum proteins and facilitated efficient urinary excretion. Detailed particle biodistribution studies were performed using more quantitative ex vivo fluorescence detection protocols and combined optical-PET imaging. The results suggest that this new generation of C dots constitutes a promising clinically translatable materials platform which may be adapted for tumor targeting and treatment.
Nanoparticle-based materials, such as drug delivery vehicles and diagnostic probes, currently under evaluation in oncology clinical trials are largely not tumor selective. To be clinically successful, the next generation of nanoparticle agents should be tumor selective, nontoxic, and exhibit favorable targeting and clearance profiles. Developing probes meeting these criteria is challenging, requiring comprehensive in vivo evaluations. Here, we describe our full characterization of an approximately 7-nm diameter multimodal silica nanoparticle, exhibiting what we believe to be a unique combination of structural, optical, and biological properties. This ultrasmall cancer-selective silica particle was recently approved for a first-in-human clinical trial. Optimized for efficient renal clearance, it concurrently achieved specific tumor targeting. Dye-encapsulating particles, surface functionalized with cyclic arginine-glycine-aspartic acid peptide ligands and radioiodine, exhibited high-affinity/avidity binding, favorable tumor-to-blood residence time ratios, and enhanced tumor-selective accumulation in αvβ3 integrin-expressing melanoma xenografts in mice. Further, the sensitive, real-time detection and imaging of lymphatic drainage patterns, particle clearance rates, nodal metastases, and differential tumor burden in a large-animal model of melanoma highlighted the distinct potential advantage of this multimodal platform for staging metastatic disease in the clinical setting.
Dasatinib, a new-generation Src and platelet-derived growth factor receptor (PDGFR) inhibitor, is currently under evaluation in high-grade glioma clinical trials. To achieve optimum physicochemical and/or biologic properties, alternative drug delivery vehicles may be needed. We used a novel fluorinated dasatinib derivative (F-SKI249380), in combination with nanocarrier vehicles and metabolic imaging tools (microPET) to evaluate drug delivery and uptake in a platelet-derived growth factor B (PDGFB)-driven genetically engineered mouse model (GEMM) of high-grade glioma. We assessed dasatinib survival benefit on the basis of measured tumor volumes. Using brain tumor cells derived from PDGFB-driven gliomas, dose-dependent uptake and time-dependent inhibitory effects of F-SKI249380 on biologic activity were investigated and compared with the parent drug. PDGFR receptor status and tumor-specific targeting were non-invasively evaluated in vivo using 18F-SKI249380 and 18F-SKI249380-containing micellar and liposomal nanoformulations. A statistically significant survival benefit was found using dasatinib (95 mg/kg) versus saline vehicle (P < .001) in tumor volume-matched GEMM pairs. Competitive binding and treatment assays revealed comparable biologic properties for F-SKI249380 and the parent drug. In vivo, Significantly higher tumor uptake was observed for 18F-SKI249380-containing micelle formulations [4.9 percentage of the injected dose per gram tissue (%ID/g); P = .002] compared to control values (1.6%ID/g). Saturation studies using excess cold dasatinib showed marked reduction of tumor uptake values to levels in normal brain (1.5%ID/g), consistent with in vivo binding specificity. Using 18F-SKI249380-containing micelles as radiotracers to estimate therapeutic dosing requirements, we calculated intratumoral drug concentrations (24–60 nM) that were comparable to in vitro 50% inhibitory concentration values. 18F-SKI249380 is a PDGFR-selective tracer, which demonstrates improved delivery to PDGFB-driven high-grade gliomas and facilitates treatment planning when coupled with nanoformulations and quantitative PET imaging approaches.
Tumors express MMP-2 and MMP-9 gelatinases, which are involved in the formation of tumor vasculature. This suggests that a tumor and its surrounding neovasculature can be visualized by a sensitive gelatinase recognition method. We have studied tumor radioimaging using a gelatinase inhibitory peptide CTTHWGFTLC (CTT), which in a mouse model targets the tumor site following an intravenous injection. We determined a solution NMR structure of CTT and its retro-inversion peptide, and prepared 125I and 99mTc-labelled CTT peptide derivatives. Radiolabelled CTT inhibited gelatinases in vitro, and homed to a tumor xenograft in mice. In normal mice, CTT was instead rapidly cleared from the circulation mainly through the kidney and, after 24 h, no significant radioactivity was accumulated in healthy tissues. Gamma camera imaging of a primary tumor in live mice was obtained with double-labelled liposomes, which were coated with 99mTc-CTT and encapsulated with 125I albumin. CTT also targeted liposomes to the lungs of tumor-bearing mice, which may indicate the existence of non-visible lung micrometastases. Our studies suggest that selective gelatinase-targeting compounds could be useful in the early detection and imaging of primary tumors and metastases.
Background: Nanoparticle imaging and tracking the release of the loaded material from the nanoparticle system have attracted significant attention in recent years. If the release of the loaded molecules could be monitored reliably in vivo, it would speed up the development of drug delivery systems remarkably. Methods: Here, we test a system that uses indocyanine green (ICG) as a fluorescent agent for studying release kinetics in vitro and in vivo from the lipid iron nanoparticle delivery system. The ICG spectral properties like its concentration dependence, sensitivity and the fluctuation of the absorption and emission wavelengths can be utilized for gathering information about the change of the ICG surrounding. Results: We have found that the absorption, fluorescence, and photoacoustic spectra of ICG in lipid iron nanoparticles differ from the spectra of ICG in pure water and plasma. We followed the ICG containing liposomal nanoparticle uptake into squamous carcinoma cells (SCC) by fluorescence microscopy and the in vivo uptake into SCC tumors in an orthotopic xenograft nude mouse model under a surgical microscope. Conclusion: Absorption and emission properties of ICG in the different solvent environment, like in plasma and human serum albumin, differ from those in aqueous solution. Photoacoustic spectral imaging confirmed a peak shift towards longer wavelengths and an intensity increase of ICG when bound to the lipids. The SCC cells showed that the ICG containing liposomes bind to the cell surface but are not internalized in the SCC-9 cells after 60 minutes of incubation. We also showed here that ICG containing liposomal nanoparticles can be traced under a surgical camera in vivo in orthotopic SCC xenografts in mice.
The poor prognosis associated with malignant melanoma has not changed substantially over the past 30 years. Targeted molecular therapies, such as immunotherapy, have shown promise but suffer from resistance and off-target toxicities, underscoring the need for alternative therapeutic strategies that can be used in combination with existing protocols. Moreover, peptides targeting melanoma-specific markers, like the melanocortin-1 receptor (MC1-R), for imaging and therapy exhibit high renal uptake that limits clinical translation. In the current study, the application of ultrasmall fluorescent (Cy5) silica nanoparticles (C′ dots), conjugated with MC1-R targeting alpha melanocyte stimulating hormone (αMSH) peptides on the polyethylene glycol (PEG) coated surface, is examined for melanoma-selective imaging. αMSH peptide sequences, evaluated for conjugation to the PEG-Cy5-C′ dot nanoparticles, bound to MC1-R with high affinity and targeted melanoma in syngenetic and xenografted melanoma mouse models. Results demonstrated a 10-fold improvement in MC1-R affinity over the native peptide alone following surface attachment of the optimal αMSH peptide. Systematic in vivo studies further demonstrated favorable in vivo renal clearance kinetics as well as receptor-mediated tumor cell internalization of as-developed radiolabeled particle tracers in B16F10 melanoma bearing mice. These findings highlight the ability of αMSH-PEG-Cy5-C′ dots to overcome previous hurdles that prevented clinical translation of peptide and antibody-based melanoma probes and reveal the potential of αMSH-PEG-Cy5-C′ dots for melanoma-selective imaging, image-guided surgery, and therapeutic applications.
Matrix metalloproteinases (MMP) are strongly associated with cancer progession. Broad-spectrum MMP inhibition is rarely beneficial clinically due to adverse effects. Of all MMPs, the gelatinases are associated with the spread of several types of cancer, including oral carcinoma. We have developed gelatinase-specific peptides, as well as their fusion with green fluorescent protein (GFP), capable of effectively targeting carcinomas.Effects on tumor growth and lymphatic micrometastatic spread in vivo was studied by use of HSC-3-cell xenografted athymic nude mice. Antigelatinolytic mono- vs. polytherapies, as well as biological activity of peptide-GFP fusion, were also analyzed in vivo.Antigelatinolytic therapy effectively inhibited growth of xenografted tumors in mice but the proportion of enlarged lymph nodes remained the same; antigelatinolytic polytherapy seemed not to potentiate the antitumor effects. The peptide-GFP chimera sustained its activity in vivo and effectively homed to the primary tumors.Peptide gelatinase inhibitors are effective in inhibiting primary tumor growth but alone do not prevent the spread of carcinoma cells; however, their bioactive GFP fusion is a candidate for tumor characterization and imaging.
