Changes In Biodistribution Research Articles

In vivo selection of synthetic nucleocapsids for tissue targeting.

Controlling the biodistribution of protein- and nanoparticle-based therapeutic formulations remains challenging. In vivo library selection is an effective method for identifying constructs that exhibit desired distribution behavior; library variants can be selected based on their ability to localize to the tissue or compartment of interest despite complex physiological challenges. Here, we describe further development of an in vivo library selection platform based on self-assembling protein nanoparticles encapsulating their own mRNA genomes (synthetic nucleocapsids or synNCs). We tested two distinct libraries: a low-diversity library composed of synNC surface mutations (45 variants) and a high-diversity library composed of synNCs displaying miniproteins with binder-like properties (6.2 million variants). While we did not identify any variants from the low-diversity surface library that yielded therapeutically relevant changes in biodistribution, the high-diversity miniprotein display library yielded variants that shifted accumulation toward lungs or muscles in just two rounds of in vivo selection. Our approach should contribute to achieving specific tissue homing patterns and identifying targeting ligands for diseases of interest.

Exploration of surface chemistry effects on the biodistribution and pharmacokinetics of dual-ligand luminescent gold nanoparticles

Understanding how surface chemistry influences gold nanoparticle (AuNP) biodistribution and pharmacokinetics is critical for the design of Au-based nanoprobes for in vivo sensing and imaging. AuNPs prepared using a single surface ligand have been widely studied; however, limited work has been reported for luminescent AuNPs decorated with multiple surface ligands. Herein, we describe the synthesis and characterization of four dual-ligand luminescent AuNPs prepared using a combination of four thiol-containing ligands (GSH, MUA, tDK, tDY). The impact of surface chemistry on in vivo biodistribution and pharmacokinetics was then investigated for each dual-ligand AuNP type using a mouse model. Our results indicate that variation of one of the two surface ligands can exert a significant influence on the pharmacokinetics and biodistribution of the AuNPs. Interestingly, we observed changes in biodistribution in the lung and digestive organs, as well as blood circulation time. These results are expected to inform the future de novo design of dual-ligand luminescent AuNPs for in vivo applications.

Robustness of Biology-Guided Radiotherapy Delivery to PET Biodistribution Changes within Target

<h3>Purpose/Objective(s)</h3> Biology-guided radiotherapy (BgRT) is a novel tracked dose delivery modality that uses positron emission tomography (PET) emissions to deliver hypofractionated planned fluence to the target. This study evaluated PET biodistribution changes within target during BgRT delivery. <h3>Materials/Methods</h3> Two phantom experiments were designed to evaluate the FDG signal variations and dosimetric effects under stationary target and moving target on a non-clinical investigational radiotherapy system. For the stationary case, a custom built cylindrical 4-D detector phantom insert with a spherical target of 22 mm in diameter containing 4 quadrants that can be filled independently was used to mimic homogenous and non-homogenous FDG uptake conditions. A C-shape insert was used as a PET avid OAR. For simulating PET signal loss within tumor: BgRT plan was created for the spherical target with all quadrants filled and the plan was delivered when ¾ volume of the target was PET-avid. For the PET signal gain within tumor scenario: BgRT plan was created for the same spherical CTV structure where ¾ volume of the target was filled with FDG, mimicking non-homogeneous uptake, and delivered to a full volume PET-avid target. The dosimetric accuracy of BgRT delivery was evaluated using 3%/3 mm Gamma criterion using 4-D detector measurements. Target biodistribution changes under motion was evaluated by creating a BgRT plan with an FDG filled 26 mm diameter spherical target as a CTV and C-shape OAR moving independently (target:3D respiratory, OAR: sinusoidal motions) within a large water filled phantom. During BgRT delivery the PET avid target insert was switched to a smaller 22 mm spherical insert, simulating 40% uniform PET distribution reduction within 26mm CTV. For the moving target experiment, BgRT delivered dose was evaluated using the radiographic film under the condition of CTV coverage ≥97% and maximum dose ≤130%. For all the experiments, the target and the C-shaped OAR were filled with FDG at a ratio of 8:1 with respect to the water background mimicking normal tissue uptake, and a 5 mm CTV to PTV expansion to be targeted with a prescription of 50Gy in 5 fractions. <h3>Results</h3> For the stationary scenarios, BgRT delivery at remissive and progressive conditions achieved gamma pass-rate of 93.2 and 97.7%, respectively. The CTV coverage goals for the motion experiment were met where the minimum CTV dose was 103.3% and the maximum dose was 123.8%. <h3>Conclusion</h3> This study evaluated the biodistribution changes within CTV from planning to delivery suggesting the BgRT delivery is robust with respect to daily variations of PET signal within the PTV under these experimental conditions. Clinical feasibility of BgRT needs further evaluation.

Sterically Shielded Hydrophilic Analogs of Indocyanine Green.

A modular synthetic process enables two or four shielding arms to be appended strategically over the fluorochromes of near-infrared cyanine heptamethine dyes to create hydrophilic analogs of clinically approved indocyanine green. A key synthetic step is the facile substitution of a heptamethine 4'-Cl atom by a phenol bearing two triethylene glycol chains. The lead compound is a heptamethine dye with four shielding arms, and a series of comparative spectroscopy studies showed that the shielding arms (a) increased dye photostability and chemical stability and (b) inhibited dye self-aggregation and association with albumin protein. In mice, the dye cleared from the blood primarily through the renal pathway rather than the biliary pathway for ICG. This change in biodistribution reflects the much smaller hydrodynamic diameter of the shielded hydrophilic ICG analog compared to the 67 kDa size of the ICG/albumin complex. An attractive feature of versatile synthetic chemistry is the capability to systematically alter the dye's hydrodynamic diameter. The sterically shielded hydrophilic ICG dye platform is well-suited for immediate incorporation into dynamic contrast-enhanced (DCE) spectroscopy or imaging protocols using the same cameras and detectors that have been optimized for ICG.

Genetically modified macrophages accomplish targeted gene delivery to the inflamed brain in transgenic Parkin Q311X(A) mice: importance of administration routes

Cell-based drug delivery systems have generated an increasing interest in recent years. We previously demonstrated that systemically administered macrophages deliver therapeutics to CNS, including glial cell line-derived neurotrophic factor (GDNF), and produce potent effects in Parkinson’s disease (PD) mouse models. Herein, we report fundamental changes in biodistribution and brain bioavailability of macrophage-based formulations upon different routes of administration: intravenous, intraperitoneal, or intrathecal injections. The brain accumulation of adoptively transferred macrophages was evaluated by various imaging methods in transgenic Parkin Q311(X)A mice and compared with those in healthy wild type littermates. Neuroinflammation manifested in PD mice warranted targeting macrophages to the brain for each route of administration. The maximum amount of cell-carriers in the brain, up to 8.1% ID/g, was recorded followed a single intrathecal injection. GDNF-transfected macrophages administered through intrathecal route provided significant increases of GDNF levels in different brain sub-regions, including midbrain, cerebellum, frontal cortex, and pons. No significant offsite toxicity of the cell-based formulations in mouse brain and peripheral organs was observed. Overall, intrathecal injection appeared to be the optimal administration route for genetically modified macrophages, which accomplished targeted gene delivery, and significant expression of reporter and therapeutic genes in the brain.

Changes in biodistribution on 68Ga-DOTA-Octreotate PET/CT after long acting somatostatin analogue therapy in neuroendocrine tumour patients may result in pseudoprogression

BackgroundTo evaluate the effects of long-acting somatostatin analogue (SSA) therapy on 68Ga-DOTA-octreotate (GaTate) uptake at physiological and metastatic sites in neuroendocrine tumour (NET) patients.MethodsTwenty-one patients who underwent GaTate PET/CT before and after commencement of SSA therapy were reviewed. Maximum standardized uptake values (SUVmax) were measured in normal organs. Changes in uptake of 49 metastatic lesions in 12 patients with stable disease were also compared. Serum chromogranin-A (CgA) levels were available for correlation between scans in 17/21 patients.ResultsMean thyroid, spleen and liver SUVmax decreased significantly following SSA therapy from a baseline of 5.9 to 3.5, 30.3 to 23.1 and 10.3 to 8.0, respectively (p = < 0.0001 for all). Pituitary SUVmax increased from 10.2 to 11.0 (p = 0.004) whereas adrenal and salivary gland SUVmax did not change. Tumour SUVmax increased in 7 of 12 patients with stable disease; CgA was stable or decreasing in 5 of these patients. 30/49 (61%) metastatic lesions had an increase in SUVmax and lesion-to-liver uptake ratio increased in 40/49 (82%) following SSA therapy.ConclusionLong-acting SSA therapy decreases GaTate uptake in the thyroid, spleen and liver but in most cases increases intensity of uptake within metastases. This has significant implications for interpretation of GaTate PET/CT following commencement of therapy as increased intensity alone may not represent true progression. Our findings also suggest pre-dosing with SSA prior to PRRT may enable higher doses to be delivered to tumour whilst decreasing dose to normal tissues.

Bioaccumulation and Biodistribution of Selenium in Metamorphosing Tadpoles.

Selenium is an important macronutrient with a very narrow margin between essentiality and toxicity. Amphibians are hypothesized to be particularly sensitive due to the potential for metamorphosis-driven mobilization, which could transfer or concentrate contaminant burdens within specific organs. We explored the potential role of tissue degeneration and remodeling during anuran metamorphosis as a mechanism for altering tissue-specific Se burdens. Limnodynastes peronii tadpoles were exposed to dissolved 75Se (as selenite) for 7 days and depurated until completion of metamorphosis. Bioaccumulation and retention kinetics were assessed in whole tadpoles and excised tissues using gamma spectroscopy, and temporal changes in biodistribution were assessed using autoradiography. Tadpoles retained Se throughout metamorphosis, and partitioned the element predominantly within digestive and excretory tissues, including livers > mesonephros > guts > gallbladder. Importantly, our results demonstrate that Se biodistribution varies significantly throughout development. This is indicative of tissue transference, and particularly in tissues developing de novo after depuration. To the best of our knowledge, this is the first study demonstrating Se transference during metamorphic tissue remodelling. Further research is warranted to explore the fate and metabolism of Se (and other metal and metalloids) during anuran development and the implications of transference for influencing toxicity.

PET Imaging of Tumor Uptake of a Biocompatible C60 Fullerene Drug Delivery Vector

Traditional chemotherapy in the form of cytotoxic drugs is one of the standards in cancer treatment, but it is hindered by various barriers to drug delivery throughout the cancer microenvironment. Recently, nanoparticles have been of great interest as drug delivery vectors because they can be tailored for specific functions to increase the amount of administered drug in solid tumors. In particular, biocompatible C60 fullerene has potential as a drug delivery scaffold since it has been shown to penetrate very restrictive physiological membranes (e.g. blood-brain-barrier and nuclear membrane)1. In addition, radiofrequency (RF) irradiation can also be combined with C60 fullerene-based drug delivery to increase the permeability of solid tumors to improve material uptake in cancerous tissue2. Our long-term goal is to determine the biodistribution of a biocompatible C60 material, as well as other pharmacokinetic data in both small and large animal models in order to evaluate its potential as a clinical drug vector for anti-cancer drugs. In this work, we have synthesized a C60 derivative with a metal chelating agent covalently bound to the C60 cage (Figure 1). The chelating agent of this material can be easily radiolabeled with 64Cu2+ (β+ emitter; t1/2 = 12 h) and tracked in vivo using positron emission tomography (PET) imaging. Because of its extreme sensitivity and routine clinical use, PET is an ideal imaging technique for studying pharmacokinetic properties and tumor accumulation of a C60 fullerene delivery vector, for both preclinical and possible future clinical studies. The material has been synthesized following an optimized six-step synthesis and the stability of the 64Cu2+ ion in the chelate has been evaluated by challenge with biological media. Animal biodistribution data will be determined using PET imaging in tumor- and non-tumor-bearing murine models and possibly porcine models as well. Our primary focus is on pancreatic cancer, since clinical treatment of this disease is presently ineffective3. Future studies include examining changes in biodistribution and exploring the potential to achieve increased selectivity for cancerous tissue with RF irradiation, as well as expanding to other tumor models. Acknowledgements: We gratefully acknowledge The Welch Foundation (C-0627) and the Kanzius Cancer Research Foundation for support of this work, as well as the Department of Chemistry and the Smalley-Curl Institute at Rice University and the Department of Surgery at Baylor College of Medicine. Additionally, the work is supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1450681 (N.G.Z.).

Targeted PRINT Hydrogels: The Role of Nanoparticle Size and Ligand Density on Cell Association, Biodistribution, and Tumor Accumulation.

In this Letter, we varied targeting ligand density of an EGFR binding affibody on the surface of two different hydrogel PRINT nanoparticles (80 nm × 320 and 55 nm × 60 nm) and monitored effects on target-cell association, off-target phagocytic uptake, biodistribution, and tumor accumulation. Interestingly, variations in ligand density only significantly altered in vitro internalization rates for the 80 nm × 320 nm particle. However, in vivo, both particle sizes experienced significant changes in biodistribution and pharmacokinetics as a function of ligand density. Overall, nanoparticle size and passive accumulation were the dominant factors eliciting tumor sequestration.

Mass spectrometry imaging reveals new biological roles for choline esters and Tyrian purple precursors in muricid molluscs.

Despite significant advances in chemical ecology, the biodistribution, temporal changes and ecological function of most marine secondary metabolites remain unknown. One such example is the association between choline esters and Tyrian purple precursors in muricid molluscs. Mass spectrometry imaging (MSI) on nano-structured surfaces has emerged as a sophisticated platform for spatial analysis of low molecular mass metabolites in heterogeneous tissues, ideal for low abundant secondary metabolites. Here we applied desorption-ionisation on porous silicon (DIOS) to examine in situ changes in biodistribution over the reproductive cycle. DIOS-MSI showed muscle-relaxing choline ester murexine to co-localise with tyrindoxyl sulfate in the biosynthetic hypobranchial glands. But during egg-laying, murexine was transferred to the capsule gland, and then to the egg capsules, where chemical ripening resulted in Tyrian purple formation. Murexine was found to tranquilise the larvae and may relax the reproductive tract. This study shows that DIOS-MSI is a powerful tool that can provide new insights into marine chemo-ecology.

Injection site radioactivity of (99m)Tc-labeled mannosylated dextran for sentinel lymph node mapping.

The high and persistent radioactivity at the injection site hinders the accuracy and expansion of sentinel lymph node (SLN) mapping. We investigated the mechanism underlying the undesirable radioactivity after subcutaneous injection of (99m)Tc-labeled mannosylated dextran ((99m)Tc(CO)3-DCM20), a SLN mapping agent targeting mannose receptors on macrophages and dendritic cells, in a mouse model. Biodistribution studies were performed 1 h after subcutaneous injection of (99m)Tc(CO)3-DCM20 from the rear footpad of mice in the presence of varying molar amounts of DCM20 or DC15, a modified dextran without mannose. Biodistribution studies were also conducted after subcutaneous injection of [(125)I]radioiodinated mannosyl-neoglycoalbumin ((125)I-NMA) from the rear footpad. The distribution of fluorescence-labeled DCM20 and DC15 at the injection site was also compared 1 h after subcutaneous injection by immunofluorescent histochemistry. The radioactivity levels of (99m)Tc(CO)3-DCM20 at the injection site and popliteal lymph node, a SLN in this model, decreased with an increase in the molar amounts of DCM20, whereas no significant changes in biodistribution were observed after injection of (99m)Tc(CO)3-DCM20 with varying molar amounts of DC15. (125)I-NMA exhibited rapid elimination of radioactivity from both the popliteal lymph node and the injection site. The fluorescence-labeled DCM20 colocalized well with CD68-positive cells such as macrophages and dendritic cells at the injection site. While partial colocalization was observed between DC15 and CD68-positive cells, the signal intensity was very weak. These findings suggest that specific binding of (99m)Tc(CO)3-DCM20 to the mannose receptor on macrophages and dendritic cells would be responsible for the sustained radioactivity levels at the injection site. These results also imply that discriminated blockage of (99m)Tc(CO)3-DCM20 binding to mannose receptors at the injection sites would reduce the radioactivity at the injection site.

Effects of nanoparticle size and gestational age on maternal biodistribution and toxicity of gold nanoparticles in pregnant mice

Gold nanoparticles (GNPs) have considerable applications in biomedicine, such as in bio-sensing, bio-imaging, drug delivery and photothermal therapeutics. However, currently there are limited information regarding the impact of pregnancy on their biodistribution, elimination and toxicity. In this study, we investigated the biodistribution and potential toxic effects of different-sized GNPs (1.5, 4.5, 13, 30 and 70nm in diameter) in non-pregnant and pregnant mice at different gestational ages (E5.5, 7.5, 9.5, 11.5 and 13.5). 5h after intravenous injection, GNPs exhibited size-dependent biodistribution profiles; however, regardless of size, no significant biodistribution changes were observed between non-pregnant and pregnant mice. Kinetic studies showed that 4.5nm GNPs were primarily excreted through urine within 5h, whereas 30nm GNPs had a more prolonged blood circulation time. No apparent toxic effects (e.g., increased mortality, altered behavior, reduced animal weight, abnormal organ morphology or reduced pregnancy duration) were observed with different-sized GNPs in pregnant mice. However, treatment with 30nm GNPs induced mild emphysema-like changes in lungs of pregnant mice. These results indicated that the maternal biodistribution patterns of GNPs in pregnant mice depended on particle size, but not gestational age; organ-specific adverse effects may arise with treatment with some GNPs according to their size.

Gold nanorod-mediated hyperthermia enhances the efficacy of HPMA copolymer-90Y conjugates in treatment of prostate tumors

The treatment of prostate cancer using a radiotherapeutic (90)Y labeled N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer can be enhanced with localized tumor hyperthermia. An (111)In labeled HPMA copolymer system for single photon emission computerized tomography (SPECT) was developed to observe the biodistribution changes associated with hyperthermia. Efficacy studies were conducted in prostate tumor bearing mice using the (90)Y HPMA copolymer with hyperthermia. HPMA copolymers containing 1, 4, 7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were synthesized by reversible addition-fragmentation transfer (RAFT) copolymerization and subsequently labeled with either (111)In for imaging or (90)Y for efficacy studies. Radiolabel stability was characterized in vitro with mouse serum. Imaging and efficacy studies were conducted in DU145 prostate tumor bearing mice. Imaging was performed using single photon emission computerized tomography (SPECT). Localized mild tumor hyperthermia was achieved by plasmonic photothermal therapy using gold nanorods. HPMA copolymer-DOTA conjugates demonstrated efficient labeling and stability for both radionuclides. Imaging analysis showed a marked increase of radiolabeled copolymer within the hyperthermia treated prostate tumors, with no significant accumulation in non-targeted tissues. The greatest reduction in tumor growth was observed in the hyperthermia treated tumors with (90)Y HPMA copolymer conjugates. Histological analysis confirmed treatment efficacy and safety. HPMA copolymer-DOTA conjugates radiolabeled with both the imaging and treatment radioisotopes, when combined with hyperthermia can serve as an image guided approach for efficacious treatment of prostate tumors.

Metabolism of Epidermal Growth Factor Receptor Targeting Probe [11C]PD153035: Impact on Biodistribution and Tumor Uptake in Rats

Several tracers have been evaluated as probes for noninvasive epidermal growth factor receptor (EGFR) quantification with PET. One of the most promising candidates is the (11)C-labeled analog of the EGFR tyrosine kinase inhibitor PD153035. However, previous in vitro studies indicated extensive metabolism of the tracer, which could be disadvantageous for the assessment of receptor density in vivo. The aim of this study was to investigate the in vivo metabolism of [(11)C]PD153035 to determine whether alterations in metabolite formation are accompanied by changes in biodistribution and tumor uptake. EGFR-overexpressing human epidermoid carcinoma xenografts in rats were used in all examinations of tumor uptake. Cytochrome P450 enzymes of subfamilies CYP2D and CYP3A were inhibited before intravenous injection of [(11)C]PD153035 into healthy and tumor-bearing male rats. Samples were taken from arterial blood and urine, and the occurrence of radioactive metabolites was assessed with radio-high-performance liquid chromatography. Dynamic PET examinations of healthy and tumor-bearing animals were performed. In 1 rat, the effect of local intraarterial administration was examined. [(11)C]PD153035 labeled at position 6 was metabolized extensively in vivo in male rats, resulting in very low levels of the intact tracer in plasma only minutes after injection. The major identified radiolabeled metabolites found were the N-oxide and metabolites arising from O demethylation at position 7. They were reduced by inhibition of CYP2D and CYP3A enzymes. PET revealed enzyme activity-dependent changes in the radioactivity distribution in the liver and tumors. Local administration of [(11)C]PD153035 greatly increased radioactivity levels in the adjacent tumor compared with levels typically found after intravenous administration. The highest tumor-to-muscle ratio at 60 min after intravenous injection was found in the untreated animals, whereas the overall highest ratio was found in the tumor near the intraarterial administration site. We suggest that the metabolism of [(11)C]PD153035 should be taken into consideration when this tracer is used to quantify EGFR expression, as our results indicated that the distribution of radioactivity to EGFR-overexpressing tumors was affected by the rate of metabolism and the route of administration.

A phase I imaging and pharmacodynamic trial of CS-1008 in patients (pts) with metastatic colorectal cancer (mCRC).

2583 Background: Death receptor 5 (DR5) is a member of the TNFR superfamily that initiates the extrinsic apoptotic pathway. CS-1008 is a humanised, monoclonal IgG1 agonistic antibody (Ab) to human DR5 created by CDR grafting of the murine Ab TRA-8. The aim of this study was to investigate the impact of CS-1008 dose on biodistribution, quantitative tumor uptake and anti-tumor response in pts with mCRC. Methods: Pts with mCRC were treated with weekly IV CS-1008 in 5 non-sequential cohorts (Co). Different loading doses were used on days 1 and 8 (0.2 to 6 mg/kg), followed by a weekly dose of 2 mg/kg. D1 and D36 doses were trace-labeled with 111In, followed by whole-body planar and regional SPECT imaging over 10 days. Primary endpoints: initial biodistribution, pharmacokinetic (PK) and tumor uptake following single infusion; changes in biodistribution, PK and tumor uptake following sequential doses. Secondary endpoints: tumor response; changes in tumor metabolism by FDG-PET; serum apoptosis biomarkers and tumor response markers. Results: 19 pts (median age 64 yrs; M:F 11:8; 2-6 prior chemotherapy regimens) were enrolled as follows: Co 1, n=2; Co 2, n=4; Co 3, n=5; Co 4, n=3; and Co 5, n=5. Tumor uptake was variable: 7 pts had no uptake, 11 pts had uptake in all sites of disease but liver, 1 pt showed liver uptake. Tumor uptake and PK were not affected by dose or repeated drug administration. No anti-CS-1008 Ab responses were detected. DR5 expression in archived samples did not correlate with uptake or response. 111In‐CS‐1008 biodistribution showed gradual blood pool clearance and no abnormal uptake in normal tissue. Mean % change in SUVmax from baseline in lesions with uptake was higher than in those with no uptake. There were 8 SD, 1 PR and 10 PD. Duration of PR was 3.7 months (mo). Mean duration of SD was 4 mo. Disease control rate (SD + PR) in pts with uptake was 58% vs 28% of pts with no uptake. Lesions with no uptake were more likely to progress, with a PD risk of 3.4 times higher than those lesions with uptake. Conclusions: Tumor DR5 expression, assessed by 111In-CS-1008 imaging, revealed real-time heterogeneous DR5 expression and appeared to be a promising predictive imaging biomarker of clinical benefit in pts with mCRC receiving CS-1008. Clinical trial information: NCT01220999.

Abstract 1174: A phase I imaging and pharmacodynamic trial of CS-1008 in patients (pts) with metastatic colorectal cancer (mCRC).

Abstract Background: Death receptor 5 (DR5) is a member of the TNFR superfamily that initiates the extrinsic apoptotic pathway by activating caspases. CS-1008 is a humanised, monoclonal IgG1 agonistic antibody to human DR5 created by CDR grafting of the murine antibody TRA-8. The aim of this study was to investigate the impact of CS-1008 dose on biodistribution, quantitative tumor uptake and anti-tumor response in patients with mCRC. Methods: Pts with mCRC who had received at least 1 course of chemotherapy (CT) were treated with weekly IV CS-1008 infusions in 5 non-sequential cohorts (Co). Different loading doses were used on days 1 and 8 (Co 1: 0.2 and 6 mg/kg; Co 2: 1 and 6 mg/kg; Co 3: 2 and 6 mg/kg; Co 4: 4 and 4mg/kg; Co 5: 6 and 2 mg/kg), followed by a weekly CS-1008 dose of 2 mg/kg. Cycle 1 encompassed 7 wks of therapy (D1 and D36 doses trace-labeled with 111In); additional weekly CS-1008 was scheduled as 4-wk cycles. Primary endpoints were to determine (1) the influence of CS-1008 dose on initial biodistribution, pharmacokinetic (PK) and tumor uptake of 111In-CS-1008 following single infusion; (2) changes in biodistribution, PK and tumor uptake following sequential doses. Secondary endpoints were to determine (1) anti-tumor response; (2) changes in tumor metabolism; (3) serum apoptosis biomarkers and serum tumor response markers. Results: Nineteen pts with a median age of 64 years and 2-6 prior CT lines were enrolled as follows: Co 1, 2 pts; Co 2, 4 pts; Co 3, 5 pts; Co 4, 3 pts; and Co 5, 5 pts. Twelve pts showed tumor uptake of 111In-CS-1008, 3 at each of the 1, 2, 4 and 6 mg/kg D1 dose levels. 111In-CS-1008 uptake in tumor was variable: some pts showed no uptake, in others uptake was observed in all measurable lesions. Liver metastases showed poor uptake of CS-1008. No significant differences were observed in tumor uptake between D1 and D36, and no effect of dose on tumor uptake was seen. DR5 expression in archived samples did not correlate with 111In-CS-1008 uptake, nor with clinical outcome. 111In-CS-1008 biodistribution showed gradual blood pool clearance and no discernible abnormal uptake in normal tissue. CS-1008 PK was not affected by dose or repeated drug administration. At restaging, there were 8 SD, 1 PR and 10 PD. The duration of PR was 3.7 months (mos). The mean duration of SD was 4 mos (range, 2.6-6.7 mos). Among the group of pts who showed CS-1008 uptake in tumor, 58% had clinical benefit (SD or PR), compared with 28% of pts in the group with no tumor uptake. The lesions that showed CS-1008 uptake were less likely to progress even in pts with overall PD at restaging. Conclusions: 111In-CS-1008 uptake in tumor predicts SD or PR. Tumor DR5 expression, assessed by 111In-CS-1008 imaging, reveals real-time heterogeneous DR5 expression, both on a per pt and on a lesion by lesion basis, and appears to be a promising predictive imaging biomarker of clinical benefit in pts with mCRC receiving CS-1008. Citation Format: Marika Ciprotti, Niall C. Tebbutt, Fook T. Lee, Sze T. Lee, Dave C. McKee, Graeme J. O'Keefe, Sylvia J. Gong, Geoffrey Chong, Hui K. Gan, Wendie Hopkins, Bridget Chappell, Nancy Y. Guo, Fiona E. Smyth, Archie N. Tse, Mendel Jansen, Manabu Matsumura, Rira Watanabe, Robert A. Beckman, Jon Greenberg, Andrew M. Scott. A phase I imaging and pharmacodynamic trial of CS-1008 in patients (pts) with metastatic colorectal cancer (mCRC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1174. doi:10.1158/1538-7445.AM2013-1174

Biodistribution and Pharmacokinetic Studies of a Porphyrin Dimer Photosensitizer (Oxdime) by Fluorescence Imaging and Spectroscopy in Mice Bearing Xenograft Tumors

Herein, we present a study of the pharmacokinetics and biodistribution of a butadiyne-linked conjugated porphyrin dimer (Oxdime) designed to have high near-infrared (NIR) 2-photon absorption cross-section for photodynamic therapy (PDT). Changes in biodistribution over time were monitored in mice carrying B16-F10 melanoma xenografts, following intravenous injection. Using fluorescence imaging of live animals and analyzing isolated organs ex vivo at different time points between 30 min and 24 h after injection, accumulation of Oxdime was measured in several organs (heart, kidney and liver) and in tumor. The concentration in the plasma was about 5-10 times higher than in other tissues. The fluorescence signal peaked at 3-12 h after injection in most tissues, including the tumor and the plasma. The change in the fluorescence emission spectrum of the sensitizer over time was also monitored and a shift in the maximum from 800 to 740 nm was observed over 24 h, showing that the Oxdime is metabolized. Significant quantities accumulated in the tumor, indicating that this PDT sensitizer may be promising for cancer treatment.

Molecular Imaging and Pharmacokinetic Analysis of Carbon-11 Labeled Antisense Oligonucleotide LY2181308 in Cancer Patients

Antisense oligonucleotides (ASOs) have potential as anti-cancer agents by specifically modulating genes involved in tumorigenesis. However, little is known about ASO biodistribution and tissue pharmacokinetics (PKs) in humans, including whether sufficient delivery to target tumor tissue may be achieved. In this preliminary study in human subjects, we used combined positron emission and computed tomography (PET-CT) imaging and subsequent modeling analysis of acquired dynamic data, to examine the in vivo biodistribution and PK properties of LY2181308 - a second generation ASO which targets the apoptosis inhibitor protein survivin. Following radiolabeling of LY2181308 with methylated carbon-11 ([(11)C]methylated-LY2181308), micro-doses (<1mg) were administered to three patients with solid tumors enrolled in a phase I trial. Moderate uptake of [(11)C]methylated-LY2181308 was observed in tumors (mean=32.5ng*h /mL, per mg administered intravenously). Highest uptake was seen in kidney and liver and lowest uptake was seen in lung and muscle. One patient underwent repeat analysis on day 15 of multiple dose therapy, during administration of LY2181308 (750mg), when altered tissue PKs and a favorable change in biodistribution was seen. [(11)C]methylated-LY2181308 exposure increased in tumor, lung and muscle, whereas renal and hepatic exposure decreased. This suggests that biological barriers to ASO tumor uptake seen at micro-doses were overcome by therapeutic dosing. In addition, (18)F-labeled fluorodeoxyglucose (FDG) scans carried out in the same patient before and after treatment showed up to 40% decreased tumor metabolism. For the development of anti-cancer ASOs, the results provide evidence of LY2181308 tumor tissue delivery and add valuable in vivo pharmacological information. For the development of novel therapeutic agents in general, the study exemplifies the merits of applying PET imaging methodology early in clinical investigations.

Biodisposition and metabolism of [18F]fluorocholine in 9L glioma cells and 9L glioma-bearing fisher rats

[(18)F]Fluorocholine ([(18)F]FCH) was developed as an analog of [(11)C]choline for tumor imaging; however, its metabolic handling remains ill defined. In this study, the metabolism of [(18)F]FCH is evaluated in cultured 9L glioma cells and Fisher 344 rats bearing 9L glioma tumors. 9L glioma cells were incubated with [(18)F]FCH and [(14)C]choline under normoxic and hypoxic (1% O(2)) conditions and analyzed for metabolic fate. [(18)F]FCH and [(14)C]choline kinetics and metabolism were studied in Fisher 344 rats bearing subcutaneous 9L tumors. [(18)F]FCH and [(14)C]choline were similarly metabolized in 9L cells in both normoxic and hypoxic conditions over a 2-h incubation period. In normoxia, radioactivity was predominantly in phosphorylated form for both tracers after 5-min incubation. In hypoxia, the tracers remained mainly in nonmetabolized form at early timepoints (<20 min). Slow dephosphorylation of intracellular [(18)F]phosphofluorocholine (0.043-0.060 min(-1)) and [(14)C]phosphocholine (0.072-0.088 min(-1)) was evidenced via efflux measurements. In rat, both [(18)F]FCH and [(14)C]choline showed high renal and hepatic uptake. Blood clearance of both tracers was rapid with oxidative metabolites, [(18)F]fluorobetaine and [(14)C]betaine, representing the majority of radiolabel in plasma after 5 min postinjection. Oxidation (in liver) and lipid incorporation (in lung) were somewhat slower for [(18)F]FCH relative to [(14)C]choline. The majority of radiolabel in hypoxic subcutaneous tumor, as in hypoxic cultured 9L cells, was found as nonmetabolized [(18)F]FCH and [(14)C]choline. [(18)F]FCH mimics choline uptake and metabolism by 9L glioma cells and tumors. However, subtle changes in biodistribution, oxidative metabolism, dephosphorylation, lipid incorporation, and renal excretion show moderate effects of the presence of the radiofluorine atom in [(18)F]FCH. The decrease in phosphorylation of exogenous choline by cancer cells should be considered in interpretation of positron emission tomography images in characteristically hypoxic tumors.

23. T Cell Carriers for Gene Delivery to Tumors

Recurrent colorectal adenocarcinoma can be treated by surgery and chemotherapy, but effects on length of survival time are generally modest. Therefore, identification of new therapeutic modalities is of great importance to improve the survival of these patients. Adenoviral gene delivery systems have shown promise as cancer therapeutics, but insufficient or non-selective transduction of tumor cells remains an issue. Targeting adenoviral vectors (Ads) to tumor-associated antigens, such as carcinoembryonic antigen (CEA, which is overexpressed in colon carcinomas), may overcome this problem. Ads can be targeted using %targeting adapter molecules', such as sCAR-scFv fusion proteins. One part of these fusion proteins consists of the ectodomain of the Coxsackie and Adenovirus Receptor (sCAR, the primary receptor for Ad), which binds to the adenoviral particle. The other part consists of a CEA recognizing modality, such as a single chain antibody (scFv). In vitro, these fusion proteins have already shown great promise for targeting Ads to cancer cells. However, availability of animal models in which CEA is expressed is limited. This has hampered the subsequent in vivo characterization of these sCAR-scFv fusion proteins. We hypothesized that we could overcome these problems by using a transgenic hCAR mouse (a kind gift of Dr. S. Petterson, Sweden), in combination with Ads encoding CEA under transcriptional control of the endothelial specific flt-1 promoter to induce CEA in the pulmonary vasculature of these mice. In this manner we could define key Ad targeting issues, independent of specific tumor biology factors. First, Ad biodistribution in hCAR mice was determined by injecting a GFP labeled Ad (AdIXEGFP) in the inferior vena cava. Compared to wild type mice, hCAR mice showed an increased uptake of AdIXEGFP particles in the lungs. Second, transgene biodistribution was determined by administering a luciferase-encoding Ad (AdCMVLuc) in the tail vein. As expected from the change in biodistribution, hCAR mice showed an increase in luciferase activity in the lungs. However, luciferase expression in the liver, the main organ of ectopic transgene expression after systemic Ad administration, was high as well. To increase selectivity of transgene expression in the lungs, we employed the endothelial specific promoter flt-1. This resulted in significantly reduced liver luciferase expression, thereby increasing the lung/liver ratio by several orders of magnitude. Finally, AdfltCEA was administered to study cellular distribution of CEA using immunohistochemical techniques, showing that CEA transgene expression in the lungs was largely confined to pulmonary endothelial cells. Thus, selective expression of CEA in the pulmonary vasculature by use of the here-described in vivo system provides a valuable tool for transductional targeting studies. The targeting adapter molecule sCAR-scFv, recognizing CEA, can now be tested for its targeting potential in this %transient transgenic' mouse model. This is of vital importance before translation to a clinical setting for the treatment of colon carcinoma can be endeavored.