Jinzi Zheng

Multimodal Contrast Agent for Combined Computed Tomography and Magnetic Resonance Imaging Applications

Objective:The objective of this study was to examine the feasibility of a multimodal system to effectively induce and maintain contrast enhancement in both computed tomography (CT) and magnetic resonance (MR) for radiation therapy applications. Materials and Methods:The physicochemical characteristics of a liposome-encapsulated iohexol and gadoteridol formulation were assessed in terms of agent loading efficiencies, size and morphology, in vitro stability, and release kinetics. The imaging properties of the liposome formulation were assessed based on T1 and T2 relaxivity measurements and in vitro CT and MR imaging in a phantom. A preliminary imaging-based evaluation of the in vivo stability of this multimodal contrast agent was also performed in a lupine model. Results:The average agent loading levels achieved were 26.5 ± 3.8 mg/mL for iodine and 6.6 ± 1.5 mg/mL for gadolinium. These concentrations correspond to approximately 10% of that found in the commercially available preparations of each of these agents. However, this liposome-based formulation is expected to have a smaller volume of distribution and prolonged circulation lifetime in vivo. This multimodal system was found to have high agent retention in vitro, which translated into maintained contrast enhancement (up to 3 days) and stability in vivo. Conclusions:This study demonstrated the feasibility of engineering a multimodal contrast agent with prolonged contrast enhancement in vivo for use in CT and MR. This contrast agent may serve as a valuable tool for cardiovascular imaging as well as image registration and guidance applications in radiation therapy.

APN/CD13-targeting as a strategy to alter the tumor accumulation of liposomes

Targeting angiogenic vasculature has been validated as a viable approach for cancer imaging and therapy. The tumor vasculature-specific ligand asparagine-glycine-arginine (NGR) peptide targets the isoform of aminopeptidase N (APN, also referred to as CD13) that is expressed on the endothelial cells in angiogenic vessels such as the neovasculature in tumors. APN/CD13 has become widely recognized as a rational target for therapeutic development and several NGR-conjugated agents are now in pre-clinical and clinical development. In the current study, a CT image-based approach is used to evaluate the in vivo performance of several NGR conjugated liposome formulations that vary in terms of NGR density and PEG spacer arm length. Indeed, for the first time it is demonstrated that CT imaging can be used for quantitative and longitudinal assessment of the pharmacokinetics and biodistribution of an actively targeted liposome formulation containing an iodinated agent. In comparison to conventional methods, the CT image guided drug delivery approach enables visualization of the intratumoral distribution of liposomes and quantification of the fraction of tumor occupied by the vesicles over time. This study is the first to use CT for molecular imaging.

The translocator protein ligand [18F]DPA-714 images glioma and activated microglia in vivo

PurposeIn recent years there has been an increase in the development of radioligands targeting the 18-kDa translocator protein (TSPO). TSPO expression is well documented in activated microglia and serves as a biomarker for imaging neuroinflammation. In addition, TSPO has also been reported to be overexpressed in a number of cancer cell lines and human tumours including glioma. Here we investigated the use of [18F]DPA-714, a new TSPO positron emission tomography (PET) radioligand to image glioma in vivo.MethodsWe studied the uptake of [18F]DPA-714 in three different rat strains implanted with 9L rat glioma cells: Fischer (F), Wistar (W) and Sprague Dawley (SD) rats. Dynamic [18F]DPA-714 PET imaging, kinetic modelling of PET data and in vivo displacement studies using unlabelled DPA-714 and PK11195 were performed. Validation of TSPO expression in 9L glioma cell lines and intracranial 9L gliomas were investigated using Western blotting and immunohistochemistry of brain tissue sections.ResultsAll rats showed significant [18F]DPA-714 PET accumulation at the site of 9L tumour implantation compared to the contralateral brain hemisphere with a difference in uptake among the three strains (F > W > SD). The radiotracer showed high specificity for TSPO as demonstrated by the significant reduction of [18F]DPA-714 binding in the tumour after administration of unlabelled DPA-714 or PK11195. TSPO expression was confirmed by Western blotting in 9L cells in vitro and by immunohistochemistry ex vivo.ConclusionThe TSPO radioligand [18F]DPA-714 can be used for PET imaging of intracranial 9L glioma in different rat strains. This preclinical study demonstrates the feasibility of employing [18F]DPA-714 as an alternative radiotracer to image human glioma.

Quantitative CT Imaging of the Spatial and Temporal Distribution of Liposomes in a Rabbit Tumor Model

Successful employment of noninvasive imaging techniques to quantitatively assess the in vivo pharmacokinetics and biodistribution of nanoparticle drug delivery systems will facilitate the rational design of novel targeted drug carriers. This study reports on the bulk organ/tissue (liver, kidneys, spleen, tumor and blood) and intratumoral distribution of liposomes containing iohexol and gadoteridol over a 14-day period in VX2 sarcoma-bearing New Zealand White rabbits using computed tomography (CT). The vascular half-life of the liposomes was found to be 63.6 +/- 5.8 h and the maximum tumor-to-muscle iodine concentration ratio of 11.9 +/- 6.0 was measured 7 days postinjection with 1.13 +/- 0.29% ID of liposomes accumulating at the tumor site. The liposomes achieved their highest intratumoral distribution volume ratio at 48 h postadministration, occupying 72 +/- 5% of the total tumor volume. This investigation demonstrated the feasibility of using CT to perform quantitative, volumetric and longitudinal assessment of the pharmacokinetics and biodistribution of iodinated liposomes with sensitivities in the range of microg/cm3 while maintaining the ability to identify boundaries of anatomical structures at submillimeter resolution and with imaging time of less than one minute per scan. If successfully approved for clinical adoption, the use of CT imaging to monitor nanoparticulate drug delivery will provide an opportunity for online adjustment of therapeutic regimens and implementation of personalized medicine.

Targeting Focal Adhesion Kinase with Dominant-Negative FRNK or Hsp90 Inhibitor 17-DMAG Suppresses Tumor Growth and Metastasis of SiHa Cervical Xenografts

Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase and key modulator of integrin signaling, is widely expressed in different tissues and cell types. Recent evidence indicates a central function of FAK in neoplasia where the kinase contributes to cell proliferation, resistance to apoptosis and anoikis, invasiveness, and metastasis. FAK, like other signaling kinases, is dependent on the chaperone heat shock protein 90 (Hsp90) for its stability and proper function. Thus, inhibition of Hsp90 might be a way of disrupting FAK signaling and, consequently, tumor progression. FAK is expressed in high-grade squamous intraepithelial lesions and metastatic cervical carcinomas but not in nonneoplastic cervical mucosa. In SiHa, a cervical cancer cell line with characteristics of epithelial-to-mesenchymal transition, the stable expression of dominant-negative FAK-related nonkinase decreases anchorage independence and delays xenograft growth. FAK-related nonkinase as well as the Hsp90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin both negatively interfere with FAK signaling and focal adhesion turnover. Short-term 17-dimethylaminoethylamino-17-demethoxygeldanamycin treatment prolongs survival in a SiHa lung metastasis model and chronic administration suppresses tumor growth as well as metastatic spread in orthotopic xenografts. Taken together, our data suggest that FAK is of importance for tumor progression in cervical cancer and that disruption of FAK signaling by Hsp90 inhibition might be an avenue to restrain tumor growth as well as metastatic spread.

Heat-activated thermosensitive liposomal cisplatin (HTLC) results in effective growth delay of cervical carcinoma in mice

Cisplatin (CDDP) has been identified as the primary chemotherapeutic agent for the treatment of cervical cancer, but dose limiting toxicity is a key issue associated with its clinical application. A suite of liposome formulations of CDDP has been developed in efforts to reduce systemic toxicity, but their therapeutic advantage over the free drug has been modest due to insufficient drug release at the tumor site. This report describes the development of a novel heat-activated thermosensitive liposome formulation containing CDDP (HTLC) designed to release approximately 90% of the loaded drug in less than 5min under mild heating conditions (42°C). Physico-chemical characteristics of HTLC were assessed in terms of gel to liquid crystalline phase transition temperature (Tm), drug loading efficiency, particle size, and stability. The pharmacokinetic profile and biodistribution of HTLC in non-tumor-bearing mice were evaluated over a 24h period. A sophisticated spatio-temporal elucidation of HTLC release in tumor-bearing mice was achieved by way of real-time monitoring using a magnetic resonance (MR) imaging protocol, wherein a custom-built laser-based conformal heat source was applied at the tumor volume to trigger the release of HTLC co-encapsulated with the MR contrast agent gadoteridol (Gd-HP-DO3A). MR thermometry (MRT) demonstrated that a relatively uniform temperature distribution was achieved in the tumor volume using the external laser-based heating setup. In mice bearing subcutaneously-implanted ME-180 cervical tumors, the combination of HTLC and heat resulted in a 2-fold increase in tumor drug levels at 1h post-administration compared to HTLC without heating. Furthermore, the overall tumor accumulation levels for the HTLC groups (with and without heat) at 1h post-injection were significantly higher than the corresponding free CDDP group. This translated into a significant improvement in therapeutic efficacy evaluated as tumor growth delay (p<0.05) for the heated HTLC treatment group compared to the unheated HTLC, heated or unheated free CDDP, and saline groups. Overall, findings from this study demonstrate that a heat-activated, triggered release formulation of CDDP results in a significant enhancement in the therapeutic index of this drug.

Cyclophosphamide-Mediated Tumor Priming for Enhanced Delivery and Antitumor Activity of HER2-Targeted Liposomal Doxorubicin (MM-302)

Given the bulky nature of nanotherapeutics relative to small molecules, it is hypothesized that effective tumor delivery and penetration are critical barriers to their clinical activity. HER2-targeted PEGylated liposomal doxorubicin (MM-302, HER2-tPLD) is an antibody–liposomal drug conjugate designed to deliver doxorubicin to HER2-overexpressing cancer cells while limiting uptake into nontarget cells. In this work, we demonstrate that the administration and appropriate dose sequencing of cyclophosphamide can improve subsequent MM-302 delivery and enhance antitumor activity in preclinical models without negatively affecting nontarget tissues, such as the heart and skin. We demonstrate that this effect is critically dependent on the timing of cyclophosphamide administration. Furthermore, the effect was found to be unique to cyclophosphamide and related analogues, and not shared by other agents, such as taxanes or eribulin, under the conditions examined. Analysis of the cyclophosphamide-treated tumors suggests that the mechanism for improved MM-302 delivery involves the induction of tumor cell apoptosis, reduction of overall tumor cell density, substantial lowering of interstitial fluid pressure, and increasing vascular perfusion. The novel dosing strategy for cyclophosphamide described herein is readily translatable to standard clinical regimens, represents a potentially significant advance in addressing the drug delivery challenge, and may have broad applicability for nanomedicines. This work formed the basis for clinical evaluation of cyclophosphamide for improving liposome deposition as part of an ongoing phase I clinical trial of MM-302 in HER2-positive metastatic breast cancer. Mol Cancer Ther; 14(9); 2060–71. ©2015 AACR.

Differential expression of the 18 kDa translocator protein (TSPO) by neoplastic and inflammatory cells in mouse tumors of breast cancer.

Tumor-associated inflammation has been linked to angiogenesis, metastasis and poor prognosis. The 18 kDa translocator protein (TSPO), also known as the peripheral benzodiazepine receptor (PBR), is expressed in activated immune cells such as macrophages, but also in a number of cancer cell lines such as those of breast cancer. There is an increasing clinical interest in TSPO expression as it has been proposed as a poor prognostic factor for survival in lymph-node negative breast cancer patients. This study aims to assess of the presence of neoplastic cell-associated TSPO and tumor macrophage-associated TSPO in mouse xenografts generated from the MDA-MB-231 and the MCF-7 breast cancer cell lines, as well as 25 different breast tumors originally derived from patient-tissue but propagated in mice using two antibodies, each specific to either the human or the murine form of TSPO. Autoradiography with the TSPO ligand [¹⁸F]DPA-714 and immunohistochemistry were also performed on the excised tumor tissues from the MDA-MB-231, MCF-7 and one of the patient-derived xenografts (HBCx-12B). High TSPO expression (either cancer or stromal cell-associated, or both) was measured in 20/25 (80%) of the patient-derived breast cancer xenografts. [¹⁸F]DPA-714 showed displaceable binding to both the human and murine TSPO on tumor tissue sections. Immunohistochemistry demonstrated that a significant portion of the tumor stromal TSPO expression colocalized with F4/80 positive macrophages cells. This study constitutes a first report of the tumor TSPO expression by mixed cell populations, and it may have important implications for cancer biology as well as for the development of imaging and therapeutic ligands targeted to TSPO.

A Mathematical Model of the Enhanced Permeability and Retention Effect for Liposome Transport in Solid Tumors

The discovery of the enhanced permeability and retention (EPR) effect has resulted in the development of nanomedicines, including liposome-based formulations of drugs, as cancer therapies. The use of liposomes has resulted in substantial increases in accumulation of drugs in solid tumors; yet, significant improvements in therapeutic efficacy have yet to be achieved. Imaging of the tumor accumulation of liposomes has revealed that this poor or variable performance is in part due to heterogeneous inter-subject and intra-tumoral liposome accumulation, which occurs as a result of an abnormal transport microenvironment. A mathematical model that relates liposome accumulation to the underlying transport properties in solid tumors could provide insight into inter and intra-tumoral variations in the EPR effect. In this paper, we present a theoretical framework to describe liposome transport in solid tumors. The mathematical model is based on biophysical transport equations that describe pressure driven fluid flow across blood vessels and through the tumor interstitium. The model was validated by direct comparison with computed tomography measurements of tumor accumulation of liposomes in three preclinical tumor models. The mathematical model was fit to liposome accumulation curves producing predictions of transport parameters that reflect the tumor microenvironment. Notably, all fits had a high coefficient of determination and predictions of interstitial fluid pressure agreed with previously published independent measurements made in the same tumor type. Furthermore, it was demonstrated that the model attributed inter-subject heterogeneity in liposome accumulation to variations in peak interstitial fluid pressure. These findings highlight the relationship between transvascular and interstitial flow dynamics and variations in the EPR effect. In conclusion, we have presented a theoretical framework that predicts inter-subject and intra-tumoral variations in the EPR effect based on fundamental properties of the tumor microenvironment and forms the basis for transport modeling of liposome drug delivery.

A gradient-loadable 64Cu-chelator for quantifying tumor deposition kinetics of nanoliposomal therapeutics by positron emission tomography

Effective drug delivery to tumors is a barrier to treatment with nanomedicines. Non-invasively tracking liposome biodistribution and tumor deposition in patients may provide insight into identifying patients that are well-suited for liposomal therapies. We describe a novel gradient-loadable chelator, 4-DEAP-ATSC, for incorporating (64)Cu into liposomal therapeutics for positron emission tomographic (PET). (64)Cu chelated to 4-DEAP-ATSC (>94%) was loaded into PEGylated liposomal doxorubicin (PLD) and HER2-targeted PLD (MM-302) with efficiencies >90%. (64)Cu-MM-302 was stable in human plasma for at least 48h. PET/CT imaging of xenografts injected with (64)Cu-MM-302 revealed biodistribution profiles that were quantitatively consistent with tissue-based analysis, and tumor (64)Cu positively correlated with liposomal drug deposition. This loading technique transforms liposomal therapeutics into theranostics and is currently being applied in a clinical trial (NCT01304797) to non-invasively quantify MM-302 tumor deposition, and evaluate its potential as a prognostic tool for predicting treatment outcome of nanomedicines.