Enhanced Unique Pattern of Hematopoietic Cell Mobilization Induced by the CXCR4 Antagonist 4F-Benzoyl-TN14003

Albumin, the most abundant plasma protein, not only controls osmotic blood pressure, but also serves as a carrier for various small molecules, including pharmaceuticals. Its impact on pharmacological properties of many drugs has been extensively studied over decades. Here, we focus on its interaction with the following mobilizing agents: Granulocyte-colony stimulating factor (G-CSF) and AMD3100, where such analyses are lacking. These compounds are widely used for hematopoietic stem cell mobilization of healthy donors or patients. Using albumin-deficient (Alb−/−) mice, we studied the contribution of albumin to mobilization outcomes. Mobilization with the bicyclam CXCR4 antagonist AMD3100 was attenuated in Alb−/− mice compared to wild-type littermates. By contrast, mobilization with recombinant human G-CSF (rhG-CSF), administered twice daily over a five-day course, was significantly increased in Alb−/− mice. In terms of a mechanism, we show that rhG-CSF bioavailability in the bone marrow is significantly improved in Alb−/− mice, compared to wild-type (WT) littermates, where rhG-CSF levels dramatically drop within a few hours of the injection. These observations likely explain the favorable mobilization outcomes with split-dose versus single-dose administration of rhG-CSF to healthy donors.

Download Full-text

Sphingosine-1-phosphate facilitates trafficking of hematopoietic stem cells and their mobilization by CXCR4 antagonists in mice

Blood ◽

10.1182/blood-2011-04-348904 ◽

2012 ◽

Vol 119 (3) ◽

pp. 707-716 ◽

Cited By ~ 88

Author(s):

Julius G. Juarez ◽

Nadia Harun ◽

Marilyn Thien ◽

Robert Welschinger ◽

Rana Baraz ◽

...

Keyword(s):

Stem Cells ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Progenitor Cell ◽

Hematopoietic Progenitors ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Sphingosine 1 Phosphate ◽

Cxcr4 Antagonists ◽

Cell Mobilization

Abstract CXCL12 and VCAM1 retain hematopoietic stem cells (HSCs) in the BM, but the factors mediating HSC egress from the BM to the blood are not known. The sphingosine-1-phosphate receptor 1 (S1P1) is expressed on HSCs, and S1P facilitates the egress of committed hematopoietic progenitors from the BM into the blood. In the present study, we show that both the S1P gradient between the BM and the blood and the expression of S1P1 are essential for optimal HSC mobilization by CXCR4 antagonists, including AMD3100, and for the trafficking of HSCs during steady-state hematopoiesis. We also demonstrate that the S1P1 agonist SEW2871 increases AMD3100-induced HSC and progenitor cell mobilization. These results suggest that the combination of a CXCR4 antagonist and a S1P1 agonist may prove to be sufficient for mobilizing HSCs in normal donors for transplantation purposes, potentially providing a single mobilization procedure and eliminating the need to expose normal donors to G-CSF with its associated side effects.

Download Full-text

Modulating Cholesterol Levels on Bone Marrow Cells Affects Endothelial and Hematopoietic Cell Mobilization and Differentiation.

Blood ◽

10.1182/blood.v110.11.1413.1413 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1413-1413

Author(s):

Ana Gomes ◽

Rita Fragoso ◽

Catia Igreja ◽

Sergio Dias

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Progenitor Cells ◽

B Lymphocytes ◽

Peripheral Blood ◽

Progenitor Cell ◽

Myeloid Cells ◽

Hematopoietic Cell ◽

Hematopoietic Differentiation ◽

Cell Mobilization

Abstract A decrease in cholesterol (CH) levels associated with hematological malignancies, such as acute leukemia, correlates with Hematopoietic suppression. Moreover, patients with mevalonate kinase deficiency (which results in hypocholesterolemia) have besides neurological defects, hepatosplenomegaly, thrombocytopenia, anemia and eventually progress into Myelodisplasic Syndromes/Leukemia. For this study, we hypothesized that CH levels might affect hematopoietic differentiation (acting directly on hematopoietic stem cells, HSC) or hematopoietic cell mobilization. For this purpose we started by studying the bone marrow (BM) expression pattern of CD36 and ApoE, 2 proteins involved in CH cellular transport, in BM samples of normal adult mice. Immunofluorescent staining showed that CD36 and ApoE are both expressed within the BM microenvironment, being particularly evident in BM sinusoids and also small subsets of hematopoietic cells. Next, we took advantage of CD36 and ApoEKO models to study their hematological parameters and BM cellular contents. To do so, CD36/ApoEKO mice and their counterparts were euthanized and BM and peripheral blood cells were collected, stained for CD11b (myeloid cells), B220 (B lymphocytes), Sca1 (progenitor cells), Flk1 (endothelial cells) and analyzed by FACS. FACS analysis of BM cells revealed that KO and WT mice had similar progenitor cells and B lymphocytes percentage, but substantially less myeloid cells. Peripheral blood analysis revealed a substantial increase in circulating B lymphocytes, and a decrease in circulating progenitor cells and myeloid cells. Regarding circulating endothelial cells, no differences were detected between KO and WT mice. However, ApoEKO mice had increased levels of BM endothelial cells and progenitor cells. As B lymphocytes are increased in the periphery and diminished in the BM, CD36 deficiency (which results in diminished CH uptake) seems to promote B lymphocyte exit from the BM. An opposing effect seems to occur in the progenitor cell populations, since less percentage in the peripheral blood might imply a failure in their exit from the BM. Alternatively, altered CH levels may affect selectively progenitor cell (subsets) differentiation. In order to investigate the role of CH modulation in hematopoietic differentiation/commitment, progenitor cells (Lin-Sca+) from CD36 WT mice were cultured in a methylcellulose hematopoietic differentiation assay and also cultured in endothelial differentiation conditions in the presence/absence of a CH lowering agent (Pravastatin). At day 9 of differentiation, cells were collected and expression of Sca1, CD11b and Flk1 was analyzed by flow cytometry. Reduced CH availability, as a result of Pravastatin treatment, resulted in substantial lower levels of differentiated endothelial cells, suggesting a blockade in this differentiation process, and in a reduced number of progenitor cells, suggesting progenitor cell death. Notably, the percentage of myeloid cells was not affected by Pravastatin treatment. Taken together, we suggest that CH influences not only trafficking between BM and peripheral blood, influences endothelial cell differentiation, and might also influence BM recovery to injury by diminishing progenitor cell number available for subsequent hematopoiesis. Current studies aim at comparing the BM hematopoietic recovery of CD36, ApoEKO vs control in response to sublethal irradiation, and the contribution of the BM endothelial compartment in this process.

Download Full-text

ALL Cells Mobilized by AMD3100 Are More Proliferative, and Remain In the Circulation Longer Than Normal HSC, Enhancing the Action of Vincristine

Blood ◽

10.1182/blood.v116.21.2137.2137 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2137-2137 ◽

Cited By ~ 1

Author(s):

Linda J. Bendall ◽

Robert Welschinger ◽

Florian Liedtke ◽

Carole Ford ◽

Aileen Dela Pena ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Bone Marrow Microenvironment ◽

Hematopoietic Progenitors ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Cell Mobilization ◽

Cycle Dependent

Abstract Abstract 2137 The chemokine CXCL12, and its receptor CXCR4, play an essential role in homing and engraftment of normal hematopoietic cells in the bone marrow, with the CXCR4 antagonist AMD3100 inducing the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. We have previously demonstrated that AMD3100 similarly induces the mobilization of acute lymphoblastic leukemia (ALL) cells into the peripheral blood. The bone marrow microenvironment is thought to provide a protective niche for ALL cells, contributing to chemo-resistance. As a result, compounds that disrupt leukemic cell interactions with the bone marrow microenvironment are of interest as chemo-sensitizing agents. However, the mobilization of normal hematopoietic stem and progenitor cells may also increase bone marrow toxicity. To better evaluate how such mobilizing agents affect normal hematopoietic progenitors and ALL cells, the temporal response of ALL cells to the CXCR4 antagonist AMD3100 was compared to that of normal hematopoietic progenitor cells using a NOD/SCID xenograft model of ALL and BALB/c mice respectively. ALL cells from all 7 pre-B ALL xenografts were mobilized into the peripheral blood by AMD3100. Mobilization was apparent 1 hour and maximal 3 hours after drug administration, similar to that observed for normal hematopoietic progenitors. However, ALL cells remained in the circulation for longer than normal hematopoietic progenitors. The number of ALL cells in the circulation remained significantly elevated in 6 of 7 xenografts examined, 6 hours post AMD3100 administration, a time point by which circulating normal hematopoietic progenitor levels had returned to baseline. No correlation between the expression of the chemokine receptor CXCR4 or the adhesion molecules VLA-4, VLA-5 or CD44, and the extent or duration of ALL cell mobilization was detected. In contrast, the overall motility of the ALL cells in chemotaxis assays was predictive of the extent of ALL cell mobilization. This was not due to CXCL12-specific chemotaxis because the association was lost when correction for background motility was undertaken. In addition, AMD3100 increased the proportion of actively cells ALL cells in the peripheral blood. This did not appear to be due to selective mobilization of cycling cells but reflected the more proliferative nature of bone marrow as compared to peripheral blood ALL cells. This is in contrast to the selective mobilization of quiescent normal hematopoietic stem and progenitor cells by AMD3100. Consistent with these findings, the addition of AMD3100 to the cell cycle dependent drug vincristine, increased the efficacy of this agent in NOD/SCID mice engrafted with ALL. Overall, this suggests that ALL cells will be more sensitive to effects of agents that disrupt interactions with the bone marrow microenvironment than normal progenitors, and that combining agents that disrupt ALL retention in the bone marrow may increase the therapeutic effect of cell cycle dependent chemotherapeutic agents. Disclosures: Bendall: Genzyme: Honoraria.

Download Full-text

First Results of a Phase-II Study with the New CXCR4 Antagonist POL6326 to Mobilize Hematopoietic Stem Cells (HSC) In Multiple Myeloma (MM)

Blood ◽

10.1182/blood.v116.21.824.824 ◽

2010 ◽

Vol 116 (21) ◽

pp. 824-824 ◽

Cited By ~ 9

Author(s):

Stefan Schmitt ◽

Niels Weinhold ◽

Klaus Dembowsky ◽

Kai Neben ◽

Mathias Witzens-Harig ◽

...

Keyword(s):

Stem Cells ◽

Dose Escalation ◽

Peripheral Blood ◽

Tumour Cells ◽

Tolerability Profile ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Minimum Number ◽

Cell Mobilization

Abstract Abstract 824 In multiple myeloma (MM), the second most common hematological malignancy, high-dose therapy followed by autologous CD34+ stem cell transplantation (ABSCT) is therapy of choice for younger patients. Standard treatment to mobilize hematopoietic stem cells (HSC) is either G-CSF alone or combined with chemotherapy. In the last years the antagonism of the CXCR4 receptor has been identified as a potent mechanism of HSC release from the bone marrow compartment. This mobilization by CXCR4 antagonists is more direct and more rapid than by G-CSF given over 4 to 6 days, and the combination of G-CSF plus a CXCR4 antagonist is superior to G-CSF alone. Furthermore, HSC mobilized using G-CSF and a CXCR4 antagonist have been shown to result in a rapid and sustained engraftment post-transplantation. POL6326 is a novel, potent and selective CXCR4 antagonist based on the PEM (Protein Epitope Mimetics) technology for intravenous application. In a previous Phase I study in 52 healthy volunteers POL6326 has been demonstrated to effectively mobilize CD34+ stem cells and was very well tolerated. The goal of the Phase II study in newly diagnosed myeloma patients reported here was to test to what extent POL6326 can be used as monotherapy, infused over 1 or 2h, to mobilize sufficient HSC for subsequent autologous transplantation. In our study the minimum number of HSC required for ABSCT was 2 × 106 CD34+ cells/kg body weight (BW). All patients also received CAD/G-CSF (cyclophosphamide, doxorubicin and dexamethasone) about 10 days after POL6326 and leukapheresis. The second goal was to determine tumour cell mobilization by polymerase chain-reaction after POL6326 or CAD/G-CSF respectively. Here we report the first data of this ongoing study. In all doses tested, up to 1200 μg/kg BW over 2h, POL6326 was safe and very well tolerated. During a total number of 38 infusions the only minor adverse event possibly related to study drug was a discrete pruritus of CTC grade 1 at the infusion site during POL6326 administration on two consecutive days. It resolved spontaneously and did not require any medication. This excellent safety and tolerability profile warrants further dose escalation in the ongoing trial. After first diagnosis, patients received 3 cycles of induction chemotherapy with bortezomib (or thalidomide respectively in one single subject) plus doxorubicin and dexamethasone. 3 weeks after the last injection of induction treatment HSC were mobilized with POL6326 given as a 1 or 2h infusion on up to 4 consecutive days according to an intra-individual dose escalation scheme (from 600 to 1200 μg/kg) with consecutive leukapheresis 30 min after end of infusion. In all patients (n=16) we observed an elevation of leukocytes and HSC compared to baseline. In 66% of patients sufficient stem cells were mobilized with POL6326 for ABSCT (with a mean of 2.5 × 106 CD34+ cells/kg BW). In 75 % of these subjects 2 leukapheresis cycles were sufficient to reach the minimum number of HSC. In all patients undergoing ABSCT with HSC mobilized by POL6326, the engraftment was successful. The time to reconstitution ranged from 10 to 19 days (mean 14 days) and was comparable to ABSCT with CAD/G-CSF-mobilized HSC (historical mean of 14 days and a range from 6 to 31 days). Five patients were evaluated for the presence of tumour cells in peripheral blood before mobilization and in leukapheresis products after treatment with POL6326 and CAD/G-CSF. In 2 of these patients we could not detect any tumour cells, neither in all leukapheresis products nor in all peripheral blood samples. In 2 patients a minor contamination with less than 0.0001% of tumour cells was detected both in the peripheral blood before POL6326 and in the leukapheresis products. Finally, 1 patient showed a similarly minor (<0.0001%) percentage of tumour cells only in the apheresis product after POL6326 treatment. However, a significant mobilization of tumour cells (0.001%) was detected after CAD plus G-CSF in this patient. Due to the excellent safety and tolerability profile observed for POL6326 in this study the next steps include further dose escalation. These initial data with low doses of POL6326 indicate that this novel CXCR4 antagonist holds the promise to be eventually used as a stand alone therapy not requiring the application of G-CSF. The preliminary findings of the absence or very low counts of tumour cells in the leukapheresis product generated with POL6326 warrant further investigation. Disclosures: Off Label Use: POL6326 as new CXCR4 antagonist for experimental CD34+ hematopoietic stem cell mobilization. Dembowsky:Polyphor Ltd: Employment. Braun:Cytonet Heidelberg GmbH: Employment. Ludin:Polyphor Ltd: Employment.

Download Full-text

Potent Stem Cell Mobilization with the Novel CXCR4 Antagonist POL6326 - Results of a Phase IIa Dose Escalation Study in Comparison to G-CSF

Blood ◽

10.1182/blood.v126.23.511.511 ◽

2015 ◽

Vol 126 (23) ◽

pp. 511-511 ◽

Cited By ~ 1

Author(s):

Darja Karpova ◽

Susanne Brauninger ◽

Eliza Wiercinska ◽

Ariane Kraemer ◽

Belinda Stock ◽

...

Keyword(s):

Stem Cell ◽

Healthy Volunteer ◽

Dose Level ◽

Dose Escalation ◽

The Novel ◽

Cxcr4 Antagonist ◽

Cd34 Cells ◽

Cell Mobilization ◽

Over Time

Abstract Background: Stem cell mobilization (SCM) with G-CSF is efficient but - although overall safe - inconvenient because of the five-day injection regime and certain contraindications. Side effects, sometimes severe, are frequent. These disadvantages fuel the quest for alternative mobilizing agents. Mobilization with the CXCR4-inhibitor plerixafor is rapid, albeit insufficiently efficacious on its own. POL6326, a potent 2nd generation macrocycle CXCR4 antagonist, has demonstrated rapid mobilization kinetics and efficacy in mice. We herein report the results of a Phase IIa dose escalation trial where SCM in response to POL6326 was compared with G-CSF in healthy volunteer stem cell donors. Methods: In this Phase IIa open label trial, healthy volunteer stem cell donors with average mobilization (121±7 CD34+ cells/μL, MW±SEM)after a five-day course of G-CSF, and a wash-out period of at least 6 weeks, received POL6326 at 500-2500 µg/kg as a single 2-hour i.v. infusion. Safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) were assessed in 3-10 subjects/dose group. Subgroups received two doses of POL6326, 1000 and 2500 µg/kg or 1500 and 2500 µg/kg, at least 2 weeks apart (paired intra-individual analysis). For PK and PD blood samples were collected before (0) and at 2, 3, 4, 6, 8 and 24 hrs after infusion start. Complete blood count, CD34+, CFU-C count and PK were assessed at all time points. At 0, 4, 8 and 24 hrs extensive phenotyping of mobilized mature and immature leukocyte subsets was performed. Eight to 14 days after treatment volunteers underwent extensive clinical and laboratory follow-up. Results: POL6326 was very well tolerated. Several volunteers experienced a mild urticarial or itchy macular rash which responded well to H1/H2 blockade. Rating of tolerability/adverse events by volunteers (questionnaire) compared favourably with G-CSF administration. Exposure (Cmax, AUC) was dose-linear. At all doses tested POL6326 mobilized CD34+ progenitor cells and colony-forming cells (CFU-C, Figure 1) exceeding reported peak mobilization with plerixafor in donors at all except the lowest dose levels. In this dataset mobilization after doses of 2000 or 2500 µg/kg did not appear meaningfully stronger than after 1500 µg/kg. The SCM response for CD34+ cells to doses ≥1500 µg/kg was 36.9±2.4/µL (mean±SEM), or 1/3 that of G-CSF (y=0.324x). Good SCM with G-CSF was predictive of good SCM with POL6326 (r=0.63). One/5.7 POL6326-mobilized CD34+ cells was clonogenic (G-CSF: 1 CFU-C/3.4 CD34+ cells) possibly indicating a more immature phenotype of CD34+ cells mobilized by POL6326. POL6326 caused mixed leucocytosis with peak values in the mid-20K/µL. B-lymphocytosis was more and neutrophilia and monocytosis were less pronounced after POL6326 than G-CSF. Compared to G-CSF the subset of plasmocytoid dendritic cell progenitors (pDC) was enriched to a distinct population within the CD34+ cells following SCM with POL6326 as previously described for plerixafor. At the 24 h time point, blood values were well on their way towards normal, and at follow-up all laboratory values had normalized. Summary/Conclusions: The novel CXCR4-antagonist POL6326 is safe, well tolerated, and provides efficient mobilization of HSPCs. Based on the number of mobilized CD34+ cells at higher doses in this study, we conclude that a standard dose of 4x10E6 CD34+ cells/kg can be extracted with a single apheresis for most recipients unless their body weight significantly exceeds the donor weight. However, exploration of alternative dosing regimens may provide even higher mobilization responses. POL6326 can be an effective mobilizing agent for allogeneic donors, including subjects with contra-indications to G-CSF. Figure 1. Mobilization of CD34+ cells (left) and CFU-C (right) over time is shown (mean±SEM for each dose level of POL6326). Figure 1. Mobilization of CD34+ cells (left) and CFU-C (right) over time is shown (mean±SEM for each dose level of POL6326). Figure 2. Figure 2. Disclosures Escot: Polyphor Ltd.: Employment. Douglas:Polyphor Ltd.: Employment. Romagnoli:Polyphor Ltd.: Employment. Chevalier:Polyphor Ltd.: Employment. Dembowsky:Polyphor Ltd.: Consultancy. Hooftman:Polyphor Ltd.: Employment. Bonig:Polyphor Ltd.: Research Funding.

Download Full-text

Unexpected Evidence That Inducible Nitric Oxide Synthase (iNOS) Is a Negative Regulator of Hematopoietic Stem/Progenitor Cell Mobilization

Blood ◽

10.1182/blood.v128.22.3372.3372 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3372-3372

Author(s):

Mateusz Adamiak ◽

Ahmed Abdelbaset-Ismail ◽

Marcin Wysoczynski ◽

Ahmed Abdel-Latif ◽

Joseph B Moore ◽

...

Keyword(s):

Nitric Oxide ◽

Cell Migration ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Hematopoietic Cell ◽

Negative Regulator ◽

Heme Oxygenase 1 ◽

Cell Trafficking ◽

Cell Mobilization

Abstract Background . Nitric oxide (NO) is gaseous free radical molecule involved in several biological processes related to inflammation, tissue damage, and infections. NO is synthesized by three isoforms of nitric oxide synthetase: two constitutively expressed isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS), and one isoform (iNOS) that is induced in response to inflammation. Interestingly, iNOS activity is enhanced during complement cascade (ComC) activation, which is a crucial element of innate immunity, and its cleavage fragments C3 and C5 (C3a and C5a, respectively) orchestrate the egress of HSPCs from BM into peripheral blood (PB). Mobilization studies in eNOS-/- mice revealed that the lack of constitutively expressed eNOS in endothelium reduces vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increases murine mortality after myelosuppression (Nature Med. 2003, 9:1370-6). However, no mobilization studies have been performed so far to assess the role of inducible hematopoietic cell-expressed iNOS in the mobilization of HSPCs. Because of this research void, we became interested in the potential role of iNOS in mobilization of HSPCs. This interest had two motivations. First, it was demonstrated in a recent paper that NO inhibits migration of granulocytes and monocytes, and it is known that these cells are crucial in facilitating egress of HSPCs across the bone marrow (BM)-peripheral blood (PB) barrier. Second, NO, which promotes inflammation, may have anti-inflammatory activity, and inflammation itself is one of the driving forces in HSPC mobilization. Aim of the study. To fill in this knowledge gap, we focused on the role of iNOS in the trafficking of hematopoietic cells as well as mobilization and homing of HSPCs. Materials and Methods. iNOS was upregulated or downregulated in established hematopoietic cell lines to study migration and adhesion of these cells. HSPC mobilization studies were performed in iNOS-/- mice, and, in parallel, homing and engraftment of iNOS-/- BM cells was evaluated in wild type animals. Since activation of the ComC is negatively regulated by heme oxygenase 1 (HO-1), and the ComC may downregulate HO-1, we tested the effect of HO-1 on iNOS-mediated cell trafficking. Results. Our results indicate that iNOS is a negative regulator of hematopoietic cell migration and prevents egress of HSPCs into PB during the mobilization process. At the molecular level, downregulation of iNOS resulted in downregulation of HO-1, and, vice versa, upregulation of iNOS enhanced HO-1 activity. Since, as we observed recently, HO-1 is a negative regulator of cell migration, the inhibitory effects of iNOS on the mobilization of HSPCs can be at least partially explained by its enhancing the HO-1 level in BM cells. Moreover, our studies, in which we generated hematopoietic chimeras, revealed that the iNOS effect depends on its expression in hematopoietic cells. Conclusions. We identified iNOS as a negative regulator of cell trafficking and mobilization of HSPCs. Further studies are needed to establish the mutual relationship between activation of iNOS and activation of HO-1 in hematopoietic cells, as both inducible enzymes are potent negative regulators of HSPC trafficking and together and regulate stem cell mobilization and homing. Therefore, inhibition of iNOS or HO-1 in HSPCs by employing small molecular inhibitors could find practical clinical application. Disclosures No relevant conflicts of interest to declare.

Download Full-text