Targeting Tissue Factor to Tumor Vasculature to Induce Tumor Infarction.

Besides its central functional role in coagulation, TF has been described as being operational in the development of malignancies and is currently being studied as a possible therapeutic tool against cancer. One of the avenues being explored is retargeting TF or its truncated extracellular part (tTF) to the tumor vasculature to induce tumor vessel occlusion and tumor infarction. To this end, multiple structures on tumor vascular wall cells have been studied at which tTF has been aimed via antibodies, derivatives, or as bifunctional fusion protein through targeting peptides. Among these targets were vascular adhesion molecules, oncofetal variants of fibronectin, prostate-specific membrane antigens, vascular endothelial growth factor receptors and co-receptors, integrins, fibroblast activation proteins, NG2 proteoglycan, microthrombus-associated fibrin-fibronectin, and aminopeptidase N. Targeting was also attempted toward cellular membranes within an acidic milieu or toward necrotic tumor areas. tTF-NGR, targeting tTF primarily at aminopeptidase N on angiogenic endothelial cells, was the first drug candidate from this emerging class of coaguligands translated to clinical studies in cancer patients. Upon completion of a phase I study, tTF-NGR entered randomized studies in oncology to test the therapeutic impact of this novel therapeutic modality.

Targeting Kca3.1 Channels in Cancer.

The Kca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca2+ concentration. These K+ channels are widely expressed in many organs and involved in many pathologies. In particular, Kca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of Kca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of Kca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using Kca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to Kca3.1 channels in order to provide a deeper understanding of Kca3.1 channel pharmacology. Alltogether, Kca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.

Synthesis and biological evaluation of PET tracers designed for imaging of calcium activated potassium channel 3.1 (KCa3.1) channels in vivo.

Expression of the Ca2+ activated potassium channel 3.1 (KCa3.1) channel (also known as the Gàrdos channel) is dysregulated in many tumor entities and has predictive power with respect to patient survival. Therefore, a positron emission tomography (PET) tracer targeting this ion channel could serve as a potential diagnostic tool by imaging the KCa3.1 channel in vivo. It was envisaged to synthesize [18F]senicapoc ([18F]1) since senicapoc (1) shows high affinity and excellent selectivity towards the KCa3.1 channels. Because problems occurred during 18F-fluorination, the [18F]fluoroethoxy senicapoc derivative [18F]28 was synthesized to generate an alternative PET tracer targeting the KCa3.1 channel. Inhibition of the KCa3.1 channel by 28 was confirmed by patch clamp experiments. In vitro stability in mouse and human serum was shown for 28. Furthermore, biodistribution experiments in wild type mice were performed. Since [18F]fluoride was detected in vivo after application of [18F]28, an in vitro metabolism study was conducted. A potential degradation route of fluoroethoxy derivatives in vivo was found which in general should be taken into account when designing new PET tracers for different targets with a [18F]fluoroethoxy moiety as well as when using the popular prosthetic group [18F]fluoroethyl tosylate for the alkylation of phenols.

Animal Safety, Toxicology, and Pharmacokinetic Studies According to the ICH S9 Guideline for a Novel Fusion Protein tTF-NGR Targeting Procoagulatory Activity into Tumor Vasculature: Are Results Predictive for Humans?

BACKGROUND: CD-13 targeted tissue factor tTF-NGR is a fusion protein selectively inducing occlusion of tumor vasculature with resulting tumor infarction. Mechanistic and pharmacodynamic studies have shown broad anti-tumor therapeutic effects in xenograft models. METHODS: After successful Good Manufacturing Practice (GMP) production and before translation into clinical phase I, ICH S9 (S6) guideline-conforming animal safety, toxicology, and pharmacokinetic (PK) studies were requested by the federal drug authority in accordance with European and US regulations. RESULTS: These studies were performed in mice, rats, guinea pigs, and beagle dogs. Results of the recently completed clinical phase I trial in end-stage cancer patients showed only limited predictive value of these non-clinical studies for patient tolerability and safety in phase I. CONCLUSIONS: Although this experience cannot be generalized, alternative pathways with seamless clinical phase 0 microdosing-phase I dose escalation studies are endorsed for anticancer drug development and translation into the clinic.

Co-staining of KCa 3.1 Channels in NSCLC Cells with a Small-Molecule Fluorescent Probe and Antibody-Based Indirect Immunofluorescence.

The Ca2+ activated potassium channel 3.1 (KCa 3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of KCa 3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes 1 and 2 were synthesized by connecting a dimethylpyrrole-based BODIPY dye with a derivative of the KCa 3.1 channel inhibitor senicapoc via linkers of different length. Patch-clamp experiments revealed the inhibition of KCa 3.1 channels by the probes confirming interaction with the channel. Both probes 1 and 2 were able to stain KCa 3.1 channels in non-small-cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre-incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes 1 and 2. Staining of the channel with the fluorescently labeled senicapoc derivatives 1 or 2 or with antibody-based indirect immunofluorescence yielded identical or very similar densities of stained KCa 3.1 channels. However, co-staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes 1 and 2 label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes 1 and 2 bind within the open channel.

Synthesis of Small-Molecule Fluorescent Probes for the In†Vitro Imaging of Calcium-Activated Potassium Channel KCa 3.1.

Small-molecule probes for the in vitro imaging of KCa 3.1 channel-expressing cells were developed. Senicapoc, showing high affinity and selectivity for the KCa 3.1 channels, was chosen as the targeting component. BODIPY dyes 15-20 were synthesized and connected by a CuI -catalyzed azide-alkyne [3+2]cycloaddition with propargyl ether senicapoc derivative 8, yielding fluorescently labeled ligands 21-26. The dimethylpyrrole-based imaging probes 25 and 26 allow staining of KCa 3.1 channels in NSCLC cells. The specificity was shown by removing the punctate staining pattern by pre-incubation with senicapoc. The density of KCa 3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells. Molecular modeling showed binding of the senicapoc-targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.