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The interaction between the chemokine receptor CXCR4 and its ligand, CXCL12, has been shown in pre-clinical models to facilitate tumour metastasis and to be involved in tissue repair and in recruitment of hematopoietic precursors. AMD3100, a potent CXCR4 antagonist, has been approved by FDA for stem cell mobilization in patients with multiple myeloma and non-Hodgkin lymphoma. Several other CXCR4 inhibitors are in clinical development (BTK140, CTCE-9908, MDX-1338 and POL6326), with the aim of inhibiting the development and progression of metastasis. In the rational design of CXCR4 inhibitors, a new class of cyclic peptide CXCL12-based CXCR4 inhibitors was discovered (IT Patent M12010A 000096; PCT WO2011/092575 A1) and validated for efficacy in in vitro and in vivo models. Among the tested peptides RFF-1 showed potent and specific inhibitory effects on CXCR4-mediated functions in multiple in vitro assays (P-ERK induction, migration and calcium mobilization CXCL12-induced and binding to CXCR4). C57/BL mice were injected with B16 melanoma cells transfected with human CXCR4. A 4,5 fold reduction in lung metastases was observed after 10 days treatment with 2 mg/kg intraperitoneally (i.p.) RFF-1 compared to the CXCR4 inhibitor AMD3100 (1.25mg/kg i.p), the powerful CXCR4 inhibitor not suitable for prolonged administration, that reduced lung metastases of about 4 fold. In another model of mouse osteosarcoma, K7M2 murine cells, were injected in Balb/C mice. A 3 fold reduction in lung metastases was observed after 15 days treatment with 10 mg/kg i.p. RFF-1 whereas AMD3100 treatment (2.5mg/kg i.p.) lead to 1.8 fold increase in lung metastases. Additionally, a 30% reduction in primary cell growth was achieved in a subcutaneous model of human renal cancer SN12C cells when i.p. treated with 2 mg/kg RFF-1. With the aim to evaluate RFF-1 efficacy in hematopoietic stem cell (HSC) mobilization DBA/2 healthy mice were injected with a single s.c. RFF-1 dose and peripheral blood HSC mobilization was evaluated by colony forming unit assay. Treatment with 30 mg/kg RFF-1 results in rapid, potent and durable mobilization of HSC reaching faster the peak compared to 20 mg/kg AMD3100. These results have prompted us to pursue the clinical development of peptide RFF-1. A first in human Phase I trial is planned to determine the maximal tolerated dose and recommended Phase 2 dose, toxicity profile, pharmacokinetics and antitumor activity of RFF-1 in patients with refractory solid tumors. The Phase I-II study of a novel CXCR4 antagonist provides the opportunity to develop settings in diseases where there is increasing evidence of a role for CXCR4 in the development and progression of metastases such as relapsed glioblastoma, neoadjuvant colorectal and breast cancer, and advanced prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4645. doi:1538-7445.AM2012-4645

Introduction: The CXCR4 inhibitor AMD3100 is FDA-approved to provide stem cell mobilization for autologous transplantation in NHL and Multiple Myeloma patients. Newly designed CXCR4 inhibitory molecules, named Hys-7 inv, Phe-7 inv and Hys-OH, were recently discovered and validated in in vitro and in vivo studies (patent MI2010A-000093). Aim of the study was to evaluate the role of new CXCR4 inhibitors in hematopoietic stem cell and neutrophil mobilization. Experimental procedures: 60 DBA/2 female were injected subcutaneously with 5 or 30 mg/kg of CXCR4 antagonist in comparison with AMD3100 (3 or 20 mg/kg). Blood was collected at 1, 2, 6 and 24 hours from the treatment. Neutrophilis were evaluated trough flow cytometry (Ly-6G+ cells) and colony forming unit assay (CFU) of the mobilized stem cell were performed. Bone marrow was also evaluated through flow cytometry and cytological analysis. Results: Hys-7 inv, Phe-7 inv and Hys-OH, cyclic peptides CXCR4 antagonists, induced potent stem cell and neutrophils mobilization compared with AMD3100. Treatment resulted in a rapid and dose related increment in neutrophils count as well as hematopoietic stem and progenitor cells increase in peripheral blood (2 to 5 fold increase). Although Hys-7inv and Hys-OH induced neutrophils increment similar to AMD3100 the kinetic was different. AMD3100 (20 mg/kg) mobilized Ly-6G cells within 1 hour but transiently (no increase at 24 hours); instead Hys-7inv and Hys-OH mobilized Ly-6G within 2 hours lasting up to 24 hours. Interestingly, Phe-7 inv increased neutrophils mobilization within 1 hour lasting up to 24 hours. AMD3100 treated bone marrow showed an increase in progenitor cells myelo-committed (from 1.5 to 3 fold increase) and polimorphonuclear cells (PMN) (1.5 to 2.5 fold increase) and a reduction in mature-lymphocyte (0.4 to 0.28 fold increase); CXCR4 inhibitor peptides increased the immature myelo-committed progenitor cells (from 2 to 3 fold increase VS control) compared with AMD3100 (1.5 fold increase VS control) reducing PMN (1.5 to 1.65 fold increase) compared to AMD3100 (2.5 fold increase). CFU showed increased colonies in peripheral blood from mice treated with cyclic-peptides (from 2.5 to 4 fold increase) compared to AMD3100 (3.5 fold increase); the highest number of CFU was reached earlier in mice treated with Hys-7inv, Phe-7 inv and Hys-OH rapidly (within 1h) versus AMD3100 (6 hours). Conclusions: In vivo studies demonstrated that three newly synthesized CXCR4 inhibitors increased neutrophil and hematopoietic stem cells release from the bone marrow with different kinetic and duration compared to the FDA approved stem cell mobilizer AMD3100. Ongoing experiments are evaluating advantages in mobilizing precursors cells with CXCR4 cyclic-peptides antagonists compared to AMD3100. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 394. doi:10.1158/1538-7445.AM2011-394