Karen J. Wong

A New Approach in the Preparation of Dendrimer-Based Bifunctional Diethylenetriaminepentaacetic Acid MR Contrast Agent Derivatives

In this paper, we report a new method to prepare and characterize a contrast agent based on a fourth-generation (G4) polyamidoamine (PAMAM) dendrimer conjugated to the gadolinium complex of the bifunctional diethylenetriamine pentaacetic acid derivative (1B4M-DTPA). The method involves preforming the metal-ligand chelate in alcohol prior to conjugation to the dendrimer. The dendrimer-based agent was purified by a Sephadex G-25 column and characterized by elemental analysis. The analysis and SE-HPLC data gave a chelate to dendrimer ratio of 30:1 suggesting conjugation at approximately every other amine terminal on the dendrimer. Molar relaxivity of the agent measured at pH 7.4 displayed a higher value than that of the analogous G4 dendrimer based agent prepared by the postmetal incorporation method (r(1) = 26.9 vs 13.9 mM(-1) s(-1) at 3 T and 22 degrees C). This is hypothesized to be due to the higher hydrophobicity of this conjugate and the lack of available charged carboxylate groups from noncomplexed free ligands that might coordinate to the metal and thus also reduce water exchange sites. Additionally, the distribution populations of compounds that result from the postmetal incorporation route are eliminated from the current product simplifying characterization as quality control issues pertaining to the production of such agents for clinical use as MR contrast agents. In vivo imaging in mice showed a reasonably fast clearance (t(1/2) = 24 min) suggesting a viable agent for use in clinical application.

Multimodality Therapy: Potentiation of High Linear Energy Transfer Radiation with Paclitaxel for the Treatment of Disseminated Peritoneal Disease

Purpose: Studies herein explore paclitaxel enhancement of the therapeutic efficacy of α-particle-targeted radiation therapy. Experimental Design: Athymic mice bearing 3 day i.p. LS-174T xenografts were treated with 300 or 600 μg paclitaxel at 24 h before, concurrently, or 24 h after [213Bi] or [212Pb]trastuzumab. Results: Paclitaxel (300 or 600 μg) followed 24 h later with [213Bi]trastuzumab (500 μCi) provided no therapeutic enhancement. Paclitaxel (300 μg) administered concurrently with [213Bi]trastuzumab or [213Bi]HuIgG resulted in median survival of 93 and 37 days, respectively; no difference was observed with 600 μg paclitaxel. Mice receiving just [213Bi]trastuzumab or [213Bi]HuIgG or left untreated had a median survival of 31, 21, and 15 days, respectively, 23 days for just either paclitaxel dose alone. Paclitaxel (300 or 600 μg) given 24 h after [213Bi]trastuzumab increased median survival to 100 and 135 days, respectively. The greatest improvement in median survival (198 days) was obtained with two weekly doses of paclitaxel (600 μg) followed by [213Bi]trastuzumab. Studies were also conducted investigating paclitaxel administered 24 h before, concurrently, or 24 h after [212Pb]trastuzumab (10 μCi). The 300 μg paclitaxel 24 h before radioimmunotherapy (RIT) failed to provide benefit, whereas 600 μg extended the median survival from 44 to 171 days. Conclusions: These results suggest that regimens combining chemotherapeutics and high linear energy transfer (LET) RIT may have tremendous potential in the management and treatment of cancer patients. Dose dependency and administration order appear to be critical factors requiring careful investigation.

Preparation and long-term biodistribution studies of a PAMAM dendrimer G5–Gd-BnDOTA conjugate for lymphatic imaging

AIMS To demonstrate the use of gadolinium (Gd)-labeled dendrimers as lymphatic imaging agents and establish the long-term biodistribution (90-day) of this type of agent in mice. MATERIALS & METHODS A G5-Gd-BnDOTA dendrimer was prepared and injected into mice and monkeys for MR lymphangiography, and long-term biodistribution of the conjugate was studied. RESULTS Administration of G5-Gd-BnDOTA in mice demonstrated a rapid uptake in the deep lymphatic system while injection in monkeys showed enhanced internal iliac nodes, indicating its general utility for lymphatic tracking. Biodistribution studies to 90 days showed that gadolinium conjugate is slowly being eliminated from the liver and other organs. CONCLUSION The use of G5-Gd-BnDOTA holds great promise for lymphatic imaging, but its slow clearance from the body might hamper its eventual clinical translation.

Evaluation of Platinum Chemotherapy in Combination with HER2-Targeted α-Particle Radiation

The studies described herein assess the potential of combining platinum-based chemotherapy with high-linear energy transfer (LET) α-particle-targeted radiation therapy using trastuzumab as the delivery vehicle. An initial study explored the combination of cisplatin with (213)Bi-trastuzumab in the LS-174T i.p. xenograft model. This initial study determined the administration sequence of cisplatin and (213)Bi-trastuzumab. Cisplatin coinjected with (213)Bi-trastuzumab increased the median survival (MS) to 90 days versus 65 days for (213)Bi-trastuzumab alone. Toxicity was observed with a weight loss of 17.6% in some of the combined treatment groups. Carboplatin proved to be better tolerated. Maximal therapeutic benefit, that is, a 5.1-fold increase in MS, was obtained in the group injected with (213)Bi-trastuzumab, followed by carboplatin 24 hours later. This was further improved by administration of multiple weekly doses of carboplatin. The MS achieved with administration of 3 doses of carboplatin was 180 days versus 60 days with (213)Bi-trastuzumab alone. The combination of carboplatin with (212)Pb radioimmunotherapy was also evaluated. The therapeutic efficacy of (212)Pb-trastuzumab (58-day MS) increased when the mice were pretreated with carboplatin 24 hours prior (157-day MS). These results again demonstrate the necessity of empirically determining the administration sequence when combining therapeutic modalities.

Two-Step Synthesis of Galactosylated Human Serum Albumin as a Targeted Optical Imaging Agent for Peritoneal Carcinomatosis

An optical probe, RG-(gal)(28)GSA, was synthesized to improve the detection of peritoneal implants by targeting the beta-d-galactose receptors highly expressed on the cell surface of a wide variety of cancers arising from the ovary, pancreas, colon, and stomach. Evaluation of RG-(gal)(28)GSA, RG-(gal)(20)GSA, glucose-analogue RG-(glu)(28)GSA, and control RG-HSA demonstrates specificity for the galactose, binding to several human adenocarcinoma cell lines, and cellular internalization. Studies using peritoneally disseminated SHIN3 xenografts in mice also confirmed a preference for galactose with the ability to detect submillimeter size lesions. Preliminary toxicity study for RG-(gal)(28)GSA using Balb/c mice reveal no toxic effects up to 100x of the standard imaging dose of 1 mg/kg administered either intraperitoneally or intravenously. These data indicate that RG-(gal)(28)GSA can selectively target a variety of human adenocarcinomas, can improve intraoperative or endoscopic tumor detection and resection, and may have little or no toxic in vivo effects; hence, it may be clinically translatable.

Synthesis of dendrimer-based biotin radiopharmaceuticals to enhance whole-body clearance

To synthesize a biotin radiopharmaceutical that clears rapidly, dendrimer was used as a carrier and conjugated with succinimidyl 3-[(125)I]iodobenzoate and tetrafluorophenyl norbiotinamidosuccinate. Then, succinic anhydride was used to reduce its pI. In mice, the non-succinylated product showed high liver (67% ID/g) and kidney (44% ID/g) uptakes and whole-body retention (94% ID) at 20 min that persisted for 12 hr. The corresponding organ uptakes (22% and 11% ID/g) and the whole-body retention (47% ID) were drastically reduced by succinylation (p<0.0001). Lysine co-injection further lowered renal uptake.

Imaging the Met Receptor Tyrosine Kinase (Met) and Assessing Tumor Responses to a Met Tyrosine Kinase Inhibitor in Human Xenograft Mouse Models with a [99mTc] (AH-113018) or CY 5** (AH-112543) Labeled Peptide

Developing an imaging agent targeting the hepatocyte growth factor receptor protein (Met) status of cancerous lesions would aid in the diagnosis and monitoring of Met-targeted tyrosine kinase inhibitors (TKIS). A peptide targeting Met labeled with [99mTc] had high affinity in vitro (Kd = 3.3 nM) and detected relative changes in Met in human cancer cell lines. In vivo [99mTc]-Met peptide (AH-113018) was retained in Met-expressing tumors, and high-expressing Met tumors (MKN-45) were easily visualized and quantitated using singlephoton emission computed tomography or optical imaging. In further studies, MKN-45 mouse xenografts treated with PHA 665752 (Met TKI) or vehicle were monitored weekly for tumor responses by [99mTc]-Met peptide imaging and measurement of tumor volumes. Tumor uptake of [“mTc]-Met peptide was significantly decreased as early as 1 week after PHA 665752 treatment, corresponding to decreases in tumor volumes. These results were comparable to Cy5**-Met peptide (AH-112543) fluorescence imaging using the same treatment model. [99mTc] or Cy5**-Met peptide tumor uptake was further validated by histologic (necrosis, apoptosis) and immunoassay (total Met, p Met, and plasma shed Met) assessments in imaged and nonimaged cohorts. These data suggest that [99mTc] or Cy5**-Met peptide imaging may have clinical diagnostic, prognostic, and therapeutic monitoring applications.

Cerenkov radiation-induced photoimmunotherapy with 18F-FDG

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with toxicity induced by photoabsorbers after irradiation with NIR light. A limitation of NIR-PIT is the inability to deliver NIR light to a tumor located deep inside the body. Cerenkov radiation (CR) is the ultraviolet and blue light that is produced by a charged particle traveling through a dielectric medium faster than the speed of light in that medium and is commonly produced during radioactive decay. Here, we demonstrate the feasibility of using CR generated by 18F-FDG accumulated in tumors to induce photoimmunotherapy. Methods: Using A431-luc cells, we evaluated the therapeutic effects of CR-PIT in vitro and in vivo using bioluminescence imaging. Results: CR-PIT showed significant suppression of tumor size, but the decrease of bioluminescence after CR-PIT was not observed consistently over the entire time course. Conclusion: Although CR-PIT can induce tumor killing deep within body, it is less effective than NIR-PIT, possibly related to the relatively lower efficiency of short wavelength light than NIR.

Comparison of planar, PET and well-counter measurements of total tumor radioactivity in a mouse xenograft model

INTRODUCTION Quantitative small animal radionuclide imaging studies are often carried out with the intention of estimating the total radioactivity content of various tissues such as the radioactivity content of mouse xenograft tumors exposed to putative diagnostic or therapeutic agents. We show that for at least one specific application, positron projection imaging (PPI) and PET yield comparable estimates of absolute total tumor activity and that both of these estimates are highly correlated with direct well-counting of these same tumors. These findings further suggest that in this particular application, PPI is a far more efficient data acquisition and processing methodology than PET. METHODS Forty-one athymic mice were implanted with PC3 human prostate cancer cells transfected with prostate-specific membrane antigen (PSMA (+)) and one additional animal (for a total of 42) with a control blank vector (PSMA (-)). All animals were injected with [18F] DCFPyl, a ligand for PSMA, and imaged for total tumor radioactivity with PET and PPI. The tumors were then removed, assayed by well counting for total radioactivity and the values between these methods intercompared. RESULTS PET, PPI and well-counter estimates of total tumor radioactivity were highly correlated (R2>0.98) with regression line slopes near unity (0.95<slope≤1.02) and intercepts near zero (-0.001MBq≤intercept ≤0.004MBq). CONCLUSION Total mouse xenograft tumor radioactivity can be measured with PET or PPI with an accuracy comparable to well counting if certain experimental and pharmacokinetic conditions are met. In this particular application, PPI is significantly more efficient than PET in making these measurements.