Abstract
Background
Pharmacologic disruption of the CXCL12/CXCR4 axis induces hematopoietic stem and progenitor cell (HSPC) mobilization. Unfortunately, there is still a significant portion of patients who fail to mobilize an adequate number of HSPCs to proceed to transplantation. For example, one-third of healthy donors mobilized with the CXCR4 antagonist plerixafor (Mozobil, AMD3100) alone require more than one apheresis to obtain the minimal number of CD34+ HSPCs necessary for transplantation (Devine et al. Blood 2008). Therefore, efforts to discover and develop more potent inhibitors of the CXCR4/CXCL12 axis continue. Methods: We tested four nonpeptide CXCR4 antagonists (ALT1188, 1187, 1128, and 1228) with low molecular weights and a novel and simple scaffold: an azacarbazole, linked via a short chain alkyldiamine to a tetrahydroquinoline. This template incorporates the critical features necessary for CXCR4 inhibition, while eliminating the toxic metal-chelating properties of AMD3100. Specificity was tested using CXCR4 monoclonal antibody (mAb) clones 12G5 (binds to first and second extracellular domains) or 1D9 (binds to the N-terminus) and CXCL12-mediated transmigration assays. To evaluate the mobilization of HSPC progenitors, wild-type or splenectomized mice were left untreated (unmobilized) or treated with ALT1188 (0.6 to 20 mg/kg), AMD3100 (5 mg/kg), G-CSF (250 mg/kg/d x 5 d) or combinations. Total WBC, colony forming units (CFU), and peripheral blood cell subsets were determined at different time points. For competitive repopulation cell assays, PBMCs from 1.5, 1 or 0.5 mL of blood from CD45.2+ unmobilized or mobilized C57BL/6 mice were mixed with 2x105 CD45.1+/CD45.2+ C57BL/6 BM cells and injected into lethally irradiated CD45.1+ C57BL/6 recipients. Results: Initial mobilization studies with the four test compounds showed that ALT1188 induced the greatest and most prolonged mobilization of mouse CFUs. Therefore ALT1188 was selected as the lead compound for further testing. Similar to AMD3100, ALT1188 inhibited the binding of CXCR4 mAb clone 12G5 (IC50 = 0.93 nM) but not clone 1D9 and completely blocked CXCL12-induced transwell migration of human CD34+ HSPCs and G2 acute lymphoblastic leukemia cells. Mobilization of mouse CFUs by AMD3100 was maximal within 1 to 3 hours, and circulating HSPCs returned to baseline levels by 6 hours. In contrast, a single injection of an equivalent dose of ALT1188 mobilized 3-fold more CFUs with peak numbers of circulating cells being maintained at 6 hours post-injection. We have completed ALT1188 dose response studies (peak mobilization with 2.5 to 5 mg/kg; maximum tolerated dose is 20 mg/kg) and demonstrated that the prolonged and enhanced mobilization of murine HSPCs by ALT1188 compared to AMD3100 is maintained in three different mouse strains (BALB/C, C57BL/6 and DBA). When combined with G-CSF, ALT1188 mobilized 1.7-fold (P = 0.04) and 4-fold (P = 0.008) more CFU than the combination of G-CSF and AMD3100 at 1 and 3 hours, respectively. Analysis of mobilized peripheral blood subsets showed that, like AMD3100, ALT1188 induced pan mobilization of B and T cells, neutrophils, monocytes and plasmacytoid DCs. Furthermore, analysis of Sca-1+c-kit+lineage- (SKL) cells showed that ALT1188 mobilized 5-fold more SKL cells than AMD3100 at 3 hours after inhibitor administration. Ongoing competitive transplant studies show that ALT1188-mobilized cells produce significantly higher chimerism than cells mobilized by AMD3100 at day 35 after transplant (P = 0.01). In separate studies, we found that the maximum number of circulating CFU is reached after 3 to 4 days when AMD3100 or ALT1188 are administered by continuous infusion via osmotic minipumps in nonsplenectomized mice. Overall, continuous infusion of ALT1188 mobilized 5-fold higher CFU than a single bolus injection of the drug and 2.7-fold greater CFU than continuous AMD3100 administration in normal mice. Surprisingly, AMD3100 and ALT1188 showed remarkably different mobilization kinetics in splenectomized animals. While AMD3100 still required 4 days to reach peak circulating CFU levels, ALT1188 induced maximum CFU mobilization within a single day of pump infusion. Summary: ALT1188 induces increased and prolonged mobilization of murine HSPC compared to AMD3100. The potential of ALT1188 to mobilize human CD34+ HSPCs remains to be determined.
Disclosures:
Tahirovic: Altiris Therapeutics: Employment. Bridges:Altiris Therapeutics, Inc: Employment. Gooding:Altiris Therapeutics: Employment, Equity Ownership. Dipersio:Altiris Therapeutics: Research Funding.
Comparative Study of Circadian Variation in Numbers of Peripheral Blood Cells among Mouse Strains: Unique Feature of C3H/HeN Mice
