Frank Osterkamp

Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

Background: Tissue factor pathway inhibitor (TFPI) inhibits coagulation factors Xa and VIIa. Results: A de novo synthesized 20-mer peptide that binds to TFPI was structurally and functionally characterized. Conclusion: The peptide binds to the Kunitz domain 1 of TFPI and blocks inhibition of factor Xa and factor VIIa by TFPI. Significance: The peptide can potentially prevent bleeding in hemophilia patients. Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nm). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nm) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nm). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.

Comparative Evaluation of the Biodistribution Profiles of a Series of Nonpeptidic Neurotensin Receptor-1 Antagonists Reveals a Promising Candidate for Theranostic Applications

Neurotensin receptor-1 (NTR1) is a promising target for diagnostic imaging and targeted radionuclide therapy. The aim of this study was to evaluate the biodistribution profiles of a series of newly developed diarylpyrazole-based NTR1 antagonists regarding their suitability as diagnostic and potentially radiotherapeutic agents. Methods: 3BP-227, 3BP-228, and 3BP-483 were labeled with 111In and injected intravenously into NTR1-positive HT29 xenograft–bearing nude mice. At 3, 6, 12, and 24 h after administration, SPECT/CT images were acquired or mice were sacrificed for ex vivo determination of tissue-associated radioactivity. Results: High-contrast tumor visualization in SPECT/CT images was achieved using the 3 compounds of this study. Ex vivo biodistribution studies confirmed a high and persistent tumor uptake, peaking at 6 h after injection for 111In-3BP-227 (8.4 ± 3.1 percentage injected dose per gram [%ID/g]) and at 3 h after injection for 111In-3BP-228 (10.2 ± 5.3 %ID/g) and 111In-3BP-483 (1.9 ± 0.8 %ID/g). Tumor–to–normal-tissue ratios obtained with 111In-3BP-227 and 111In-3BP-228 were consistently greater than 1. Conclusion: On the basis of the superior biodistribution profile compared with previously reported radiolabeled NTR1 ligands, 111In-3BP-227 is an ideal candidate for further development as a theranostic tracer.

SAR of N-phenyl piperidine based oral integrin α5β1 antagonists

Recently, a new class of selective integrin alpha5beta1inhibitors consisting of a heterocyclic based scaffold was published. Herein the SAR and pharmacokinetic profiles of N-phenyl piperidine derivatives are described.

Discovery of orally available integrin α5β1 antagonists

Previous research within our laboratories identified the 3-hydroxypyrrolidine scaffold 1 as a new and selective integrin alpha5beta1 inhibitor class which was designed for local administration. Herein the discovery of new orally available integrin alpha5beta1 inhibitor scaffolds for potential systemic treatment is described.

Proof of Therapeutic Efficacy of a Novel 177Lu-Labeled Neurotensin Receptor 1-Antagonist in a Colon Carcinoma Xenograft Model

Increased expression of neurotensin receptor 1 (NTR1) has been shown in a large number of tumor entities such as pancreatic or colon carcinoma. Hence, this receptor is a promising target for diagnostic imaging and radioligand therapy. Using the favorable biodistribution data of the NTR1-targeting agent 111In-3BP-227, we investigated the therapeutic effect of its 177Lu-labeled analog on the tumor growth of NTR1-positive HT29 colon carcinoma xenografts. Methods: 3BP-227 was labeled with 177Lu. To assess its biodistribution properties, SPECT and CT scans of HT29-xenografted nude mice injected with 177Lu-3BP-227 were acquired, and ex vivo tissue activity was determined. To evaluate therapeutic efficacy, 2 groups of mice received the radiopharmaceutical in a median dose of either 165 MBq (129–232 MBq, n = 10) or 110 MBq (82–116 MBq, n = 10), whereas control mice were injected with vehicle (n = 10). Tumor sizes and body weights were monitored for up to 49 d. Renal function and histologic morphology were evaluated. Results: Whole-body SPECT/CT images allowed clear tumor visualization with low background activity and high tumor-to-kidney and -liver ratios. Ex vivo biodistribution data confirmed high and persistent uptake of 177Lu-3BP-227 in HT29 tumors (19.0 ± 3.6 vs. 2.7 ± 1.6 percentage injected dose per gram at 3 and 69 h after injection, respectively). The application of 177Lu-3BP-227 resulted in a distinct delay of tumor growth. Median tumor doubling time for controls was 5.5 d (interquartile range [IQR], 2.8–7.0), compared with 17.5 d (IQR, 5.5–22.5 d) for the 110-MBq and 41.0 d (IQR, 27.5–55.0) for the 165-MBg group. Compared with controls, median relative tumor volume at day 23 after injection was reduced by 55% (P = 0.034) in the 110-MBq and by 88% (P < 0.01) in the 165-MBq group. Renal histology and clinical chemistry results did not differ between radiotherapy groups and controls, suggesting absence of therapy-induced acute renal damage. Conclusion: These data demonstrate that the novel NTR1-targeting theranostic agent 3BP-227 is an effective and promising candidate for radioligand therapy, with a favorable preliminary safety profile and high potential for clinical translation.

177Lu-3BP-227 for Neurotensin Receptor 1–Targeted Therapy of Metastatic Pancreatic Adenocarcinoma: First Clinical Results

Neurotensin receptor 1 (NTR1) is overexpressed in ductal pancreatic adenocarcinoma, which is still one of the deadliest cancers, with a very poor prognosis. Eligible patients were offered salvage radiopharmaceutical therapy with the novel NTR1 antagonist 177Lu-3BP-227. Methods: Six patients with confirmed ductal pancreatic adenocarcinoma who had exhausted all other treatment options received 177Lu-3BP-227 for evaluation of NTR1 expression in vivo. Three patients received treatment activities of 5.1–7.5 GBq. Results: Administration of 177Lu-3BP-227 was well tolerated by all patients. The kidneys were identified as the dose-limiting organ. The most severe adverse event was reversible grade 2 anemia. One patient achieved a partial response and experienced significant improvement of symptoms and quality of life. This patient survived 13 mo from diagnosis and 11 mo from the start of 177Lu-3BP-227 therapy. Conclusion: This initial report provides clinical evidence of the feasibility of treatment of ductal pancreatic adenocarcinoma using 177Lu-3BP-227.