Double-Targeting Using a TrkC Ligand Conjugated to Dipyrrometheneboron Difluoride (BODIPY) Based Photodynamic Therapy (PDT) Agent

A molecule <b>1</b> (IY-IY-PDT) was designed to contain a fragment (IY-IY) that targets the TrkC receptor and a photosensitizer that acts as an agent for photodynamic therapy (PDT). Molecule <b>1</b> had submicromolar photocytotoxicities to cells that were engineered to stably express TrkC (NIH3T3-TrkC) or that naturally express high levels of TrkC (SY5Y neuroblastoma lines). Control experiments showed that <b>1</b> is not cytotoxic in the dark and has significantly less photocytotoxicity toward cells that do not express TrkC (NIH3T3-WT). Other controls featuring a similar agent <b>2</b> (YI-YI-PDT), which is identical and isomeric with <b>1</b> except that the targeting region is scrambled (a YI-YI motif, see text), showed that <b>1</b> is considerably more photocytotoxic than <b>2</b> on TrkC⁺ cells. Imaging live TrkC⁺ cells after treatment with a fluorescent agent <b>1</b> (IY-IY-PDT) proved that <b>1</b> permeates into TrkC⁺ cells and is localized in the lysosomes. This observation indirectly indicates that agent <b>1</b> enters the cells via the TrkC receptor. Consistent with this, the dose-dependent PDT effects of <b>1</b> can be competitively reduced by the natural TrkC ligand, neurotrophin NT3

Small Molecule Ligands for Active Targeting of TrkC-Expressing Tumor Cells

A small molecule motif was used in “active targeting” to deliver cytotoxic substances into tumor cells that express the TrkC receptor. Underlying this study was the hypothesis that internalization of targeted conjugates into cells would be facile if mediated by receptor binding and receptor–ligand internalization. Initial experiments using 6-mercaptopurine gave encouraging data but demonstrated the importance of maintaining solubility and high cytotoxicity. Conjugates of the targeting agent with a cytotoxic rosamine (similar to a rhodamine) were more successful. Targeting of TrkC was observed, validated in a series of competition experiments featuring other TrkC ligands, and accumulation into lysosomes was observed, as expected for receptor-mediated internalization

Novel Small Molecule Probes for Metastatic Melanoma

Actively targeting probe <b>1b</b>, an unsymmetrical bivalent dipeptide mimic, selectively bound melanoma over healthy skin tissue in histological samples from patients and Sinclair swine. Modifications to <b>1b</b> gave agents <b>2</b>–<b>4</b> that contain a near-IR aza-BODIPY fluor. Contrary to our expectations, symmetrical probe <b>3</b> gave the highest melanoma-to-healthy skin selectivity in histochemistry and experiments with live cells; this was surprising because <b>2</b>, not <b>3</b>, is unsymmetrical like the original lead <b>1</b>. Optical imaging of <b>3</b> in a mouse melanoma model failed to show tumor accumulation <i>in vivo</i>, but the probe did selectively accumulate in the tumor (some in lung and less in the liver) as proven by analysis of the organs post mortem

Targeted PDT Agent Eradicates TrkC Expressing Tumors via Photodynamic Therapy (PDT)

This contribution features a small molecule that binds TrkC (tropomyosin receptor kinase C) receptor that tends to be overexpressed in metastatic breast cancer cells but not in other breast cancer cells. A sensitizer for ¹O₂ production conjugated to this structure gives <b>1</b>-<b>PDT</b> for photodynamic therapy. Isomeric <b>2</b>-<b>PDT</b> does not bind TrkC and was used as a control throughout; similarly, TrkC– cancer cells were used to calibrate enhanced killing of TrkC+ cells. Ex vivo, <b>1</b>- and <b>2</b>-<b>PDT</b> where only cytotoxic when illuminated, and <b>1</b>-<b>PDT</b>, gave higher cell death for TrkC+ breast cancer cells. A 1 h administration-to-illumination delay gave optimal TrkC+/TrkC–-photocytotoxicity, and distribution studies showed the same delay was appropriate in vivo. In Balb/c mice, a maximum tolerated dose of 20 mg/kg was determined for <b>1</b>-<b>PDT</b>. <b>1</b>- and <b>2</b>-<b>PDT</b> (single, 2 or 10 mg/kg doses and one illumination, throughout) had similar effects on implanted TrkC– tumors, and like those of <b>2</b>-<b>PDT</b> on TrkC+ tumors. In contrast, <b>1</b>-<b>PDT</b> caused dramatic TrkC+ tumor volume reduction (96% from initial) relative to the TrkC– tumors or <b>2</b>-<b>PDT</b> in TrkC+ models. Moreover, 71% of the mice treated with 10 mg/kg 1-PDT (<i>n</i> = 7) showed full tumor remission and survived until 90 days with no metastasis to key organs

Dual-Modality Positron Emission Tomography/Optical Image-Guided Photodynamic Cancer Therapy with Chlorin e6-Containing Nanomicelles

Multifunctional nanoparticles with combined diagnostic and therapeutic functions show great promise in nanomedicine. Herein, we develop an organic photodynamic therapy (PDT) system based on polyethylene glycol (PEG)-coated nanomicelles conjugated with ∼20% chlorin e6 (PEG-Ce 6 nanomicelles), which functions as an optical imaging agent, as well as a PDT agent. The formed PEG-Ce 6 nanomicelles with the size of ∼20 nm were highly stable in various physiological solutions for a long time. Moreover, Ce 6 can also be a ⁶⁴Cu chelating agent for <i>in vivo</i> positron emission tomography (PET). By simply mixing, more than 90% of ⁶⁴Cu was chelator-free labeled on PEG-Ce 6 nanomicelles, and they also showed high stability in serum conditions. Both fluorescence imaging and PET imaging revealed that PEG-Ce 6 nanomicelles displayed high tumor uptake (13.7 ± 2.2%ID/g) after intravenous injection into tumor-bearing mice at the 48 h time point. In addition, PEG-Ce 6 nanomicelles exhibited excellent PDT properties upon laser irradiation, confirming the theranostic properties of PEG-Ce 6 nanomicelles for imaging and treatment of cancer. In addition, PDT was not shown to render any appreciable toxicity. This work presents a theranostic platform based on polymer nanomicelles with great potential in multimodality imaging-guided photodynamic cancer therapy

ImmunoPET Imaging of Insulin-Like Growth Factor 1 Receptor in a Subcutaneous Mouse Model of Pancreatic Cancer

The role of insulin-like growth factor-1 receptor (IGF-1R) in cancer tumorigenesis was established decades ago, yet there are limited studies evaluating the imaging and therapeutic properties of anti-IGF-1R antibodies. Noninvasive imaging of IGF-1R may allow for optimized patient stratification and monitoring of therapeutic response in patients. Herein, this study reports the development of a Zirconium-89 (⁸⁹Zr)-labeled anti-IGF-1R antibody (⁸⁹Zr-Df-1A2G11) for PET imaging of pancreatic cancer. Successful chelation and radiolabeling of the antibody resulted in a highly stable construct that could be used for imaging IGF-1R expressing tumors in vivo. Western blot and flow cytometry studies showed that MIA PaCa-2, BxPC-3, and AsPC-1 pancreatic cancer cell lines expressed high, moderate, and low levels of IGF-1R, respectively. These three pancreatic cancer cell lines were subcutaneously implanted into mice. By employing the PET imaging technique, the tumor accumulation of ⁸⁹Zr-Df-1A2G11 was found to be dependent on the level of IGF-1R expression. Tumor accumulation of ⁸⁹Zr-Df-1A2G11 was 8.24 ± 0.51, 5.80 ± 0.54, and 4.30 ± 0.42 percentage of the injected dose (%ID/g) in MIA PaCa-2, BxPC-3, and AsPC-1-derived tumor models at 120 h postinjection, respectively (<i>n</i> = 4). Biodistribution studies and ex vivo immunohistochemistry confirmed these findings. In addition, ⁸⁹Zr-labeled nonspecific human IgG (⁸⁹Zr-Df-IgG) displayed minimal uptake in IGF-1R positive MIA PaCa-2 tumor xenografts (3.63 ± 0.95%ID/g at 120 h postinjection; <i>n</i> = 4), demonstrating that ⁸⁹Zr-Df-1A2G11 accumulation was highly specific. This study provides initial evidence that our ⁸⁹Zr-labeled IGF-1R-targeted antibody may be employed for imaging a wide range of malignancies. Antibodies may be tracked in vivo for several days to weeks with ⁸⁹Zr, which may enhance image contrast due to decreased background signal. In addition, the principles outlined in this study can be employed for identifying patients that may benefit from anti-IGF-1R therapy