Generation and Characterization of Novel VLA-4 Inhibitors for Stem Cell Mobilization in Combination with a CXCR2 Agonist

Introduction: The integrin very-late antigen-4 (VLA-4) is comprised of an alpha4 chain (CD49d) that is non-covalently associated with the beta1 integrin chain (CD29). VLA-4 plays an important role in the homing and retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. We previously reported (Karpova et al. Blood 2016;128:659) a novel strategy and pathway for rapid and potent mobilization of HSPCs in mice using a combination of a VLA-4 inhibitor and the truncated isoform of the CXCR2 agonist Gro-beta (tGro-β, SB-251353). These studies were completed with a novel VLA-4 inhibitor, CWHM-823, that exhibited increased potency and/or solubility relative to the previously described comparators BIO5192 (α4β1-specific) and firategrast (α4β1 and α4β7 dual-specific). Here we extended these findings by generating and characterizing additional novel VLA-4 inhibitors.

Methods: We designed and synthesized 21 novel VLA-4 inhibitors with the intent of further improving potency toward VLA-4 and achieving a more prolonged mobilization effect in vivo. All novel VLA-4 antagonists were assayed for potency by measuring their inhibition of binding of a soluble VCAM/Fc chimeric protein (sVCAM-1) to VLA-4 expressing human G2 acute lymphoblastic leukemia (ALL) or murine 5TGM1 multiple myeloma cells by flow cytometry. To evaluate the mobilization ofHSPCs, DBA/2 mice were left untreated (nonmobilized) or treated witha VLA-4 inhibitor alone (5 mg/kg, SC) or in combination with tGro-β (2.5 mg/kg, SC). The number of circulating white blood cells (WBCs), colony forming units (CFU), lineage-Sca-1+c-kit+ (LSK) cells and myeloid and lymphoid cell subsets in the peripheral blood were determined at baseline, 0.5h and 2h after treatment.

Results: Given the encouraging results with CWHM-823, we designed, synthesized and tested the potency of 21 additional VLA-4 inhibitors to provide further structure-activity relationships. We observed that CWHM-823 (IC₅₀ = 4 nM) was nearly 50-fold more potent than firategrast (IC₅₀ = 198 nM), 7.5-fold more potent than zaurategrast (IC₅₀ = 30.7 nM) and 6-fold less potent than BIO5192 (IC₅₀ = 0.7 nM) at inhibiting the binding of sVCAM-1 to G2 ALL cells. Nine of our 21 novel inhibitors proved to be more potent than CWHM-823 at inhibiting the binding of sVCAM-1 to G2 ALL cells. Encouragingly, 4 of these inhibitors were equipotent to BIO5192 at inhibiting sVCAM-1 binding to G2 ALL or 5TGM1 myeloma cells (IC₅₀ < 1 nM). Initial mobilization studies in mice indicate that single 5 mg/kg injections of these compounds effectively mobilize murine CFUs and LSK cells to levels comparable to that attained with BIO5192. The combination of tGro-β with the best inhibitors resulted in a dramatic increase in circulating CFU and LSK numbers when compared to treatment with single agents (3-5 fold). Further, combination therapy induced an approximately 2-fold increase in total circulating WBCs with pan mobilization of B cells (3.5-fold), monocytes (3.2-fold), plasmacytoid DCs (3-fold), neutrophils (2.5-fold) and T cells (1.4-fold). Similar to BIO5192 and CWHM-823, mobilization with tGro-β and the new compounds was rapid, peaking at 15-30 minutes after injection. However, in contrast to all of the VLA-4 inhibitors that we have tested in vivo to date, one inhibitor (compound 1611) maintained peak levels of circulating CFUs and LSK cells at 2h after injection. This prolonged CFU and LSK cell mobilization exhibited by these new analogues is quite encouraging, and additional analogues based upon these findings are currently being synthesized.

Summary: Currently available small molecule VLA-4 antagonists (BIO5192, firategrast, and zaurategrast) have suffered from poor bioavailability and a short half-life. These preliminary data demonstrate that our novel analogs are exceptionally potent ligands for VLA-4, soluble and efficacious in mobilization of stem cells (CFU and LSK cells) in vivo, and thus are suitable leads for optimization to maximize their pharmacodynamic effect in vivo.