CTT Technology

Prostate Specific Membrane Antigen (PSMA) , a zinc metallopeptidase also known as folate hydrolase or glutamate carboxypeptidase, has cleavage specificity for the C-terminal glutamic acid residues of folate. PSMA is a transmembrane protein with an extracellular region containing the catalytic domain of the enzyme and an internal filamin binding domain (inside of the cell). PSMA is up-regulated and strongly expressed on the surface of prostate cancer cells. PSMA is also expressed on the newly formed blood vessels in a variety of non-prostatic solid malignancies but is minimally expressed on normal tissue. Significant up regulation of PSMA expression was noted in patients with metastatic disease as compared with those with localized prostate cancer and in localized disease compared with benign prostate tissue. The correlation of PSMA expression with advanced disease and its expression on new blood vessels of many different cancers have suggested a role for PSMA in promoting cancer invasion and metastases.

CTT considers PSMA an ideal target to image the extent and location of metastatic disease and to help define and monitor treatment strategies. To this end, we have identified innovative small molecules that readily bind and image prostate cancer expressing PSMA, in animal models. These imaging agents have unique properties and are rapidly undergoing preclinical and clinical development. The market for a prostate-specific imaging agent that recognizes early as well advanced stage disease is significant and the use of a PSMA-directed imaging agent could be expanded to vascularized solid tumors making the market potential substantial.

Various chemical scaffolds have been developed as inhibitors of this enzyme. However, the binding of high-affinity small-molecule inhibitors to PSMA has not yet been exploited for prostate cancer detection or treatment strategies. Current imaging techniques lack sensitivity and specificity and PSMA has remained largely untapped as a target for novel detection strategies.

CTT has developed a flexible methodology for the synthesis of various PSMA phosphoramidate peptidomimetic inhibitors and identified a lead compound based on inhibitor potency--CTT-54. This inhibitor possesses a free N-terminal amine for functionalization with amine-reactive payloads. Functionalization of this inhibitors occurs under mild conditions that are typical for protein and peptide chemistry. CTT-54 (also known as LW-54) exhibits tight and irreversible binding and exhibited time-dependent inhibition that is essentially complete by 6 minutes. In subsequent experiments, we observed an apparent IC50 for CTT-54 of 0.46 nM. CTT has prepared analogs of increasing complexity. Relevant payloads that have been coupled through the N-terminal amine of this inhibitor are: amine-reactive fluorescent dyes; F-18 labeled prosthetic groups and the radiolabel 99Tc for in vitro and in vivo imaging. Subsequent in vitro and in vivo results have demonstrated the efficacy of the first generation phosphoramidates inhibitors as viable PSMA targeting groups.

It is known that PSMA undergoes internalization, especially when bound with antibodies. We confirmed by fluorescence microscopy that our fluorescent PSMA inhibitors were internalized at body temperature as early as 30 min into PSMA expressing LNCAP prostate tumor cells. Maximum internalization is obtained after an incubation period of about 2 hours. After incubation periods of up to 16 hours, the cells look similar to those after 150 minute, indicating tumor labeling agents based on this framework have the potential to be retained in their target cells. Neither surface labeling nor internalization was observed for PC-3 cell under the same conditions. CTT believes that the property of irreversible or slowly reversible binding for our targeting agents is key to what appears to be a nearly quantitative internalization of these agents into tumor cells. This is unlike the Trofex compound which appears to be rapidly lost from the tumor. The IC50 values for fluorescent and biotinylated PSMA inhibitors that demonstrated LNCaP cell labeling and internalization were approximately 0.3 nM.

The 18F labeled phosphoramidate prototype probe was evaluated in vivo using tumors grown in mice. Prostate cancer cell lines, the PSMA expressing LNCaP and the PSMA negative PC-3 tumors were injected into the right shoulder of the animals and palpable tumors were imaged. The synthesized 18F-labeled phosphoramidate prototype probe at 50-100 µCi dose range was administered through tail vein injection. The animals were imaged by a microPET/CT imaging system (Inveon, Seimens, Germany) at 0, 60 and 120 min. Our imaging data show that there is a significant tumor uptake of the agent by the LNCaP PSMA+ model with imaging at 120 min post-injection. In contrast, there was no detectable tumor signal in the PC-3 PSMA- model. Time activity curves of the kidney, muscle and tumor demonstrate that uptake in the tumor is stable over the course of the study while the kidney (excretion) and muscle (non-specific uptake) uptake wash-out. In addition, the specificity of the 18F-labeled-CTT-54 to PSMA was evident and demonstrated by the competition study with PSMA.

Dramatic improvement of technology for diagnostic imaging of prostate cancer is critically important for patient management during disease progression. Desirable properties for such technology involve targeting a cell surface marker highly expressed on prostate cancer cells with low expression levels elsewhere. PSMA is an ideal prostate cancer cellular target that can be targeted by a variety of agents having suitable PSMA binding specificity. The results describe a series of phosphoramidate derivatives having high specific binding affinity to PSMA at levels equal to or superior to that observed with PSMA-specific monoclonal antibodies. This class of PSMA-specific phosphoramidate derivatives is particularly well suited for in vivo diagnostic and therapeutic applications. In particular, these low molecular weight targeting agents (100- or more times smaller than an antibody) that bind to the extracellular catalytic domain of the PSMA protein will have increased tumor penetrability. They are non-biological and can be conveniently chemically synthesized. High affinity, irreversibly binding inhibitors have been identified and were shown to bind to PSMA expressed on cultured cells and in prostate cancer tumors grown in nude mice. Irreversible binding is a particularly desirable property for targeting prostate tumor cells. In sharp contrast to reversible inhibitors, irreversible inhibitors remain bound to prostate cancer cells for extended periods and are internalized into the cell leading to an increasing intracellular concentration that is ideal for delivery of a diagnostic or therapeutic payload.