ABSTRACT
Plerixafor is a CXCR4 antagonist approved in 2008 by the FDA for hematopoietic stem cell collection. Subsequently, plerixafor has shown promise as a potential pathogen-agnostic immunomodulator in a variety of preclinical animal models. Additionally, investigator-led studies demonstrated plerixafor prevents viral and bacterial infections in patients with WHIM syndrome, a rare immunodeficiency with aberrant CXCR4 signaling. Here, we investigated whether plerixafor could be repurposed to treat sepsis or severe wound infections, either alone or as an adjunct therapy. In a Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced zebrafish sepsis model, plerixafor reduced sepsis mortality and morbidity assessed by tail edema. There was a U-shaped response curve with the greatest effect seen at 0.1 µM concentration. We used Acinetobacter baumannii infection in a neutropenic murine thigh infection model. Plerixafor did not show reduced bacterial growth at 24 h in the mouse thigh model, nor did it amplify the effects of a rifampin antibiotic therapy, in varying regimens. While plerixafor did not mitigate or treat bacterial wound infections in mice, it did reduce sepsis mortality in zebra fish. The observed mortality reduction in our LPS model of zebrafish was consistent with prior research demonstrating a mortality benefit in a murine model of sepsis. However, based on our results, plerixafor is unlikely to be successful as an adjunct therapy for wound infections. Further research is needed to better define the scope of plerixafor as a pathogen-agnostic therapy. Future directions may include the use of longer acting CXCR4 antagonists, biased CXCR4 signaling, and optimization of animal models.
Subject(s)
Benzylamines , Cyclams , Disease Models, Animal , Heterocyclic Compounds , Receptors, CXCR4 , Sepsis , Zebrafish , Animals , Cyclams/pharmacology , Cyclams/administration & dosage , Benzylamines/pharmacology , Sepsis/drug therapy , Sepsis/microbiology , Heterocyclic Compounds/pharmacology , Heterocyclic Compounds/administration & dosage , Mice , Receptors, CXCR4/antagonists & inhibitors , Receptors, CXCR4/metabolism , Thigh/microbiology , Acinetobacter Infections/drug therapy , Acinetobacter Infections/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/isolation & purification , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Female , Lipopolysaccharides , Wound Infection/microbiology , Wound Infection/drug therapy , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic useABSTRACT
The interest in mercury radioisotopes, 197mHg (t1/2 = 23.8 h) and 197gHg (t1/2 = 64.14 h), has recently been reignited by the dual diagnostic and therapeutic nature of their nuclear decays. These isotopes emit γ-rays suitable for single photon emission computed tomography imaging and Auger electrons which can be exploited for treating small and metastatic tumors. However, the clinical utilization of 197m/gHg radionuclides is obstructed by the lack of chelators capable of securely binding them to tumor-seeking vectors. This work aims to address this challenge by investigating a series of chemically tailored macrocyclic platforms with sulfur-containing side arms, namely, 1,4,7,10-tetrakis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO4S), 1,4,7-tris[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO3S), and 1,7-bis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,10-diacetic acid (DO2A2S). 1,4,7,10-Tetrazacyclododecane-1,4,7,10-tetracetic acid (DOTA), the widest explored chelator in nuclear medicine, and the nonfunctionalized backbone 1,4,7,10-tetrazacyclododecane (cyclen) were considered as well to shed light on the role of the sulfanyl arms in the metal coordination. To this purpose, a comprehensive experimental and theoretical study encompassing aqueous coordination chemistry investigations through potentiometry, nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and density functional theory (DFT) calculations, as well as concentration- and temperature-dependent [197m/gHg]Hg2+ radiolabeling and in vitro stability assays in human serum was conducted. The obtained results reveal that the investigated chelators rapidly complex Hg2+ in aqueous media, forming extremely thermodynamically stable 1:1 metal-to-ligand complexes with superior stabilities compared to those of DOTA or cyclen. These complexes exhibited 6- to 8-fold coordination environments, with donors statically bound to the metal center, as evidenced by the presence of 1H-199Hg spin-spin coupling via NMR. A similar octacoordinated environment was also found for DOTA in both solution and solid state, but in this case, multiple slowly exchanging conformers were detected at ambient temperature. The sulfur-rich ligands quantitatively incorporate cyclotron-produced [197m/gHg]Hg2+ under relatively mild reaction conditions (pH = 7 and T = 50 °C), with the resulting radioactive complexes exhibiting decent stability in human serum (up to 75% after 24 h). By developing viable chelators and understanding the impact of structural modifications, our research addresses the scarcity of suitable chelating agents for 197m/gHg, offering promise for its future in vivo application as a theranostic Auger-emitter radiometal.
Subject(s)
Cyclams , Macrocyclic Compounds , Humans , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/chemical synthesis , Mercury/chemistry , Sulfur/chemistry , Radioisotopes/chemistry , Molecular Structure , Electrons , Coordination Complexes/chemistry , Coordination Complexes/chemical synthesis , Heterocyclic Compounds/chemistry , Theranostic NanomedicineABSTRACT
ABSTRACT: We have reported the direct repair of the sickle cell mutation in vivo in a disease model using vectorized prime editors after hematopoietic stem cell (HSC) mobilization with granulocyte colony-stimulating factor (G-CSF)/AMD3100. The use of G-CSF for HSC mobilization is a hurdle for the clinical translation of this approach. Here, we tested a G-CSF-free mobilization regimen using WU-106, an inhibitor of integrin α4ß1, plus AMD3100 for in vivo HSC prime editing in sickle cell disease (SCD) mice. Mobilization with WU-106 + AMD3100 in SCD mice was rapid and efficient. In contrast to the G-CSF/AMD3100 approach, mobilization of activated granulocytes and elevation of the key proinflammatory cytokine interleukin-6 in the serum were minimal. The combination of WU-106 + AMD3100 mobilization and IV injection of the prime editing vector together with in vivo selection resulted in â¼23% correction of the SCD mutation in the bone marrow and peripheral blood cells of SCD mice. The treated mice demonstrated phenotypic correction, as reflected by normalized blood parameters and spleen size. Editing frequencies were significantly increased (29%) in secondary recipients, indicating the preferential mobilization/transduction of long-term repopulating HSCs. Using this approach, we found <1% undesired insertions/deletions and no detectable off-target editing at the top-scored potential sites. Our study shows that in vivo transduction to treat SCD can now be done within 2 hours involving only simple IV injections with a good safety profile. The same-day mobilization regimen makes in vivo HSC gene therapy more attractive for resource-poor settings, where SCD does the most damage.
Subject(s)
Anemia, Sickle Cell , Genetic Therapy , Granulocyte Colony-Stimulating Factor , Hematopoietic Stem Cell Mobilization , Animals , Humans , Mice , Anemia, Sickle Cell/therapy , Anemia, Sickle Cell/genetics , Benzylamines , Cyclams/pharmacology , Cyclams/therapeutic use , Disease Models, Animal , Gene Editing , Genetic Therapy/methods , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cell Mobilization/methods , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/metabolism , Heterocyclic Compounds/pharmacology , Heterocyclic Compounds/therapeutic useSubject(s)
Benzylamines , Bone Marrow , Cyclams , Heterocyclic Compounds , Multiple Myeloma , Receptors, CXCR4 , Cyclams/pharmacology , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Receptors, CXCR4/antagonists & inhibitors , Receptors, CXCR4/metabolism , Benzylamines/pharmacology , Heterocyclic Compounds/pharmacology , Heterocyclic Compounds/therapeutic use , Bone Marrow/pathology , Bone Marrow/drug effects , Bone Marrow/metabolism , Tumor Microenvironment/drug effectsABSTRACT
A ligature-induced periodontitis model was established in wild-type and CD146CreERT2; RosatdTomato mice to explore the function of pericytes in alveolar bone formation. We found that during periodontitis progression and periodontal wound healing, CD146+/NG2+ pericytes were enriched in the periodontal tissue areas, which could migrate to the alveolar bone surface and colocalize with ALP+/OCN+ osteoblasts. Chemokine C-X-C motif receptor 4 (CXCR4) inhibition using AMD3100 blocked CD146-Cre+ pericyte migration and osteogenesis, as well as further exacerbated periodontitis-associated bone loss. Next, primary pericytes were sorted out by magnetic-activated cell sorting and demonstrated that C-X-C motif chemokine ligand 12 (CXCL12) promotes pericyte migration and osteogenesis via CXCL12-CXCR4-Rac1 signaling. Finally, the local administration of an adeno-associated virus for Rac1 overexpression in NG2+ pericytes promotes osteoblast differentiation of pericytes and increases alveolar bone volume in periodontitis. Thus, our results provided the evidence that pericytes may migrate and osteogenesis via the CXCL12-CXCR4-Rac1 axis during the pathological process of periodontitis.
Subject(s)
Cell Movement , Chemokine CXCL12 , Osteogenesis , Pericytes , Periodontitis , Receptors, CXCR4 , Animals , Osteogenesis/physiology , Cell Movement/physiology , Mice , Chemokine CXCL12/metabolism , Receptors, CXCR4/metabolism , Alveolar Bone Loss , Signal Transduction/physiology , rac1 GTP-Binding Protein/metabolism , Disease Models, Animal , CD146 Antigen , Osteoblasts , Cell Differentiation , Cyclams , BenzylaminesABSTRACT
OBJECTIVE: Multiple myeloma (MM) is the leading indication of autologous hematopoietic stem cell transplantation. The aim of this study was to determine the incidence of mobilization failure and characterize the risk factors associated with poor mobilization (PM) of MM patients in novel therapies era. METHODS: We conducted a retrospective study of 211 MM patients who received their first peripheral blood stem cells (PBSC) mobilization at our single center. The following data were collected: age, gender, clinical stage, disease status, complete blood cell count, induction regimen, CD34+ cell count in peripheral blood (PB), and PBSC collections. RESULTS: In addition to conventional drugs, 22 (10.4%) patients received daratumumab containing induction, and 33 (15.6%) patients used plerixafor for poor mobilization (pre-apheresis PB CD34+ cells <20/µL). Failure of collection occurred in 24 (11.4%) patients and was correlated with low white blood cell (WBC), ≥3 cycles of lenalidomide treatment before mobilization, steady-state mobilization and nouse of plerixafor are associated with mobilization failure. Daratumumab-based induction treatment ≥2 courses, albumin >41 g/L before mobilization, and steady-state mobilization were risk factors for PM in subgroups of patients treated with lenalidomide for <3 courses. In addition, Hepatitis B virus infection at baseline, thalassemia and measurable residual disease positivity were recognized as predictive factors for PM in subset of chemo-mobilization patients. CONCLUSION: In addition to some well-recognized risk factors, baseline WBC count and daratumumab exposure ≥2 courses before mobilization were revealed as the predictive factors of mobilization failure, providing consultation for preemptive use of plerixafor.
Subject(s)
Benzylamines , Cyclams , Hematopoietic Stem Cell Mobilization , Multiple Myeloma , Humans , Multiple Myeloma/therapy , Hematopoietic Stem Cell Mobilization/methods , Female , Male , Middle Aged , Retrospective Studies , Aged , Adult , Cyclams/therapeutic use , Cyclams/pharmacology , Benzylamines/therapeutic use , Peripheral Blood Stem Cells/metabolism , Risk Factors , Antibodies, Monoclonal/therapeutic use , Lenalidomide/therapeutic use , Lenalidomide/administration & dosage , Heterocyclic Compounds/administration & dosage , Heterocyclic Compounds/therapeutic use , Peripheral Blood Stem Cell Transplantation/methods , Transplantation, AutologousABSTRACT
BACKGROUND: Stem cell therapy is a promising alternative for inflammatory diseases and tissue injury treatment. Exogenous delivery of mesenchymal stem cells is associated with instant blood-mediated inflammatory reactions, mechanical stress during administration, and replicative senescence or change in phenotype during long-term culture in vitro. In this study, we aimed to mobilize endogenous hematopoietic stem cells (HSCs) using AMD-3100 and provide local immune suppression using FK506, an immunosuppressive drug, for the treatment of inflammatory bowel diseases. METHODS: Reactive oxygen species (ROS)-responsive FK506-loaded thioketal microspheres were prepared by emulsification solvent-evaporation method. Thioketal vehicle based FK506 microspheres and AMD3100 were co-administered into male C57BL6/J mice with dextran sulfate sodium (DSS) induced colitis. The effect of FK506-loaded thioketal microspheres in colitis mice were evaluated using disease severity index, myeloperoxidase activity, histology, flow cytometry, and gene expression by qRT-PCR. RESULTS: The delivery of AMD-3100 enhanced mobilization of HSCs from the bone marrow into the inflamed colon of mice. Furthermore, targeted oral delivery of FK506 in an inflamed colon inhibited the immune activation in the colon. In the DSS-induced colitis mouse model, the combination of AMD-3100 and FK506-loaded thioketal microspheres ameliorated the disease, decreased immune cell infiltration and activation, and improved body weight, colon length, and epithelial healing process. CONCLUSION: This study shows that the significant increase in the percentage of mobilized hematopoietic stem cells in the combination therapy of AMD and oral FK506 microspheres may contribute to a synergistic therapeutic effect. Thus, low-dose local delivery of FK506 combined with AMD3100 could be a promising alternative treatment for inflammatory bowel diseases.
Subject(s)
Benzylamines , Colitis , Cyclams , Dextran Sulfate , Mice, Inbred C57BL , Tacrolimus , Animals , Colitis/chemically induced , Colitis/therapy , Colitis/drug therapy , Colitis/pathology , Mice , Male , Cyclams/pharmacology , Cyclams/therapeutic use , Tacrolimus/pharmacology , Tacrolimus/therapeutic use , Hematopoietic Stem Cell Mobilization/methods , Heterocyclic Compounds/pharmacology , Heterocyclic Compounds/therapeutic use , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Disease Models, Animal , Immunosuppression Therapy , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/therapeutic use , Microspheres , Reactive Oxygen Species/metabolismSubject(s)
Drug Approval , Primary Immunodeficiency Diseases , Receptors, CXCR4 , United States Food and Drug Administration , Humans , Receptors, CXCR4/antagonists & inhibitors , United States , Primary Immunodeficiency Diseases/drug therapy , Immune System Diseases/drug therapy , Cyclams/therapeutic use , Cyclams/pharmacology , Warts/drug therapy , Heterocyclic Compounds, 3-Ring/therapeutic use , Benzylamines/therapeutic use , Benzylamines/pharmacologyABSTRACT
PURPOSE: This study aimed to evaluate the potential of astragalus polysaccharide (APS) pretreatment in enhancing the homing and anti-peritoneal fibrosis capabilities of bone marrow mesenchymal stromal cells (BMSCs) and to elucidate the underlying mechanisms. METHODS: Forty male Sprague-Dawley rats were allocated into four groups: control, peritoneal dialysis fluid (PDF), PDF + BMSCs, and PDF + APSBMSCs (APS-pre-treated BMSCs). A peritoneal fibrosis model was induced using PDF. Dil-labeled BMSCs were administered intravenously. Post-transplantation, BMSC homing to the peritoneum and pathological alterations were assessed. Stromal cell-derived factor-1 (SDF-1) levels were quantified via enzyme-linked immunosorbent assay (ELISA), while CXCR4 expression in BMSCs was determined using PCR and immunofluorescence. Additionally, a co-culture system involving BMSCs and peritoneal mesothelial cells (PMCs) was established using a Transwell setup to examine the in vitro effects of APS on BMSC migration and therapeutic efficacy, with the CXCR4 inhibitor AMD3100 deployed to dissect the role of the SDF-1/CXCR4 axis and its downstream impacts. RESULTS: In vivo and in vitro experiments confirmed that APS pre-treatment notably facilitated the targeted homing of BMSCs to the peritoneal tissue of PDF-treated rats, thereby amplifying their therapeutic impact. PDF exposure markedly increased SDF-1 levels in peritoneal and serum samples, which encouraged the migration of CXCR4-positive BMSCs. Inhibition of the SDF-1/CXCR4 axis through AMD3100 application diminished BMSC migration, consequently attenuating their therapeutic response to peritoneal mesenchyme-to-mesothelial transition (MMT). Furthermore, APS upregulated CXCR4 expression in BMSCs, intensified the activation of the SDF-1/CXCR4 axis's downstream pathways, and partially reversed the AMD3100-induced effects. CONCLUSION: APS augments the SDF-1/CXCR4 axis's downstream pathway activation by increasing CXCR4 expression in BMSCs. This action bolsters the targeted homing of BMSCs to the peritoneal tissue and amplifies their suppressive influence on MMT, thereby improving peritoneal fibrosis.
Subject(s)
Astragalus Plant , Chemokine CXCL12 , Mesenchymal Stem Cells , Peritoneal Fibrosis , Polysaccharides , Rats, Sprague-Dawley , Receptors, CXCR4 , Animals , Receptors, CXCR4/metabolism , Chemokine CXCL12/metabolism , Rats , Male , Peritoneal Fibrosis/drug therapy , Peritoneal Fibrosis/metabolism , Polysaccharides/pharmacology , Mesenchymal Stem Cells/drug effects , Disease Models, Animal , Cyclams/pharmacologyABSTRACT
Most transplant-eligible multiple myeloma (MM) patients undergo autologous peripheral blood stem cell collection (PBSC) using G-CSF with on-demand plerixafor (G ± P). Chemomobilization (CM) can be used as a salvage regimen after G ± P failure or for debulking residual tumor burden ahead of autologous peripheral blood stem cell transplantation (ASCT). Prior studies utilizing cyclophosphamide-based CM have not shown long-term benefits. At our center, intensive CM (ICM) using a PACE- or HyperCVAD-based regimen has been used to mitigate "excessive" residual disease based on plasma cell (PC) burden or MM-related biomarkers. Given the lack of efficacy of non-ICM, we sought to determine the impact of ICM on event-free survival (EFS), defined as death, progressive disease, or unplanned treatment escalation. We performed a retrospective study of newly diagnosed MM patients who collected autologous PBSCs with the intent to proceed immediately to ASCT at our center between 7/2020 and 2/2023. Patients were excluded if they underwent a tandem autologous or sequential autologous-allogeneic transplant, had primary PC leukemia, received non-ICM treatment (i.e., cyclophosphamide and/or etoposide), or had previously failed G ± P mobilization. To appropriately evaluate the impact of ICM among those who potentially could have received it, we utilized a propensity score matching (PSM) approach whereby ICM patients were compared to a cohort of non-CM patients matched on pre-ASCT factors most strongly associated with the receipt of ICM. Of 451 patients identified, 61 (13.5%) received ICM (PACE-based, n = 45; hyper-CVAD-based, n = 16). Post-ICM/pre-ASCT, 11 patients (18%) required admission for neutropenic fever and/or infection. Among 51 evaluable patients, the overall response rate was 31%; however, 46 of 55 evaluable patients (84%) saw a reduction in M-spike and/or involved free light chains. Among those evaluated with longitudinal peripheral blood flow cytometry (n = 8), 5 patients (63%) cleared circulating blood PCs post-ICM. Compared to patients mobilized with non-CM, ICM patients collected a slightly greater median number of CD34+ cells (10.8 versus 10.2 × 106/kg, P = .018). The median follow-up was 30.6 months post-ASCT. In a PSM multivariable analysis, ICM was associated with significantly improved EFS (hazard ratio [HR] 0.30, 95% CI 0.14 to 0.67, P = .003), but not improved OS (HR 0.38, 95% CI 0.10 to 1.44, P = .2). ICM was associated with longer post-ASCT inpatient duration (+4.1 days, 95% CI, 2.4 to 5.8, P < .001), more febrile days (+0.96 days, 95% CI 0.50 to 1.4, P < .001), impaired platelet engraftment (HR 0.23, 95% CI 0.06 to 0.87, P = .031), more bacteremia (OR 3.41, 95% CI 1.20 to 9.31, P = .018), and increased antibiotic usage (cefepime: +2.3 doses, 95% CI 0.39 to 4.1, P = .018; vancomycin: +1.0 doses, 95% CI 0.23 to 1.8, P = .012). ICM was independently associated with improved EFS in a matched analysis involving MM patients with excessive disease burden at pre-ASCT workup. This benefit came at the cost of longer inpatient duration, more febrile days, greater incidence of bacteremia, and increased antibiotic usage in the immediate post-ASCT setting. Our findings suggest that ICM could be considered for a subset of MM patients, but its use must be weighed carefully against additional toxicity.
Subject(s)
Multiple Myeloma , Transplantation, Autologous , Humans , Multiple Myeloma/therapy , Male , Middle Aged , Female , Retrospective Studies , Aged , Peripheral Blood Stem Cell Transplantation/methods , Cyclams/therapeutic use , Cyclams/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Benzylamines , Granulocyte Colony-Stimulating Factor/administration & dosage , Granulocyte Colony-Stimulating Factor/therapeutic use , Adult , Hematopoietic Stem Cell Transplantation/methodsABSTRACT
BACKGROUND: Increasing indications for cellular therapy collections have stressed our healthcare system, with autologous collections having a longer than desired wait time until apheresis collection. This quality improvement initiative was undertaken to accommodate more patients within existing resources. STUDY DESIGN AND METHODS: Patients with multiple myeloma who underwent autologous peripheral blood stem cell collection from October 2022 to April 2023 were included. Demographic, mobilization, laboratory, and apheresis data were retrospectively collected from the medical record. RESULTS: This cohort included 120 patients (49.2% male), with a median age of 60 years. All received G-CSF and 95% received pre-emptive Plerixafor approximately 18 hours pre-collection. Most (79%) had collection goals of at least 8 × 106/kg CD34 cells, with 63% over 70 years old having this high collection goal (despite 20 years of institutional data showing <1% over 70 years old have a second transplant). With collection efficiencies of 55.9%, 44% of patients achieved their collection goal in a single day apheresis collection. A platelet count <150 × 103/µL on the day of collection was a predictor for poor mobilization; among 27 patients with a low baseline platelet count, 17 did not achieve the collection goal and 2 failed to collect a transplantable dose. CONCLUSIONS: With minor collection goal adjustments, 15% of all collection appointments could have been avoided over this 6-month period. Other strategies to accommodate more patients include mobilization modifications (Plerixafor timing or substituting a longer acting drug), utilizing platelet counts to predict mobilization, and modifying apheresis collection volumes or schedule templates.
Subject(s)
Benzylamines , Cyclams , Granulocyte Colony-Stimulating Factor , Hematopoietic Stem Cell Mobilization , Multiple Myeloma , Transplantation, Autologous , Humans , Multiple Myeloma/therapy , Cyclams/pharmacology , Cyclams/therapeutic use , Middle Aged , Male , Female , Granulocyte Colony-Stimulating Factor/administration & dosage , Hematopoietic Stem Cell Mobilization/methods , Aged , Retrospective Studies , Blood Component Removal/methods , Heterocyclic Compounds/administration & dosage , Heterocyclic Compounds/therapeutic use , Adult , Peripheral Blood Stem Cell Transplantation/methods , Platelet CountABSTRACT
Regenerating gene family member 4 (Reg4) has been implicated in acute pancreatitis, but its precise functions and involved mechanisms have remained unclear. Herein, we sought to investigate the contribution of Reg4 to the pathogenesis of pancreatitis and evaluate its therapeutic effects in experimental pancreatitis. In acute pancreatitis, Reg4 deletion increases inflammatory infiltrates and mitochondrial cell death and decreases autophagy recovery, which are rescued by the administration of recombinant Reg4 (rReg4) protein. In chronic pancreatitis, Reg4 deficiency aggravates inflammation and fibrosis and inhibits compensatory cell proliferation. Moreover, C-X-C motif ligand 12 (CXCL12)/C-X-C motif receptor 4 (CXCR4) axis is sustained and activated in Reg4-deficient pancreas. The detrimental effects of Reg4 deletion are attenuated by the administration of the approved CXCR4 antagonist plerixafor (AMD3100). Mechanistically, Reg4 mediates its function in pancreatitis potentially via binding its receptor exostosin-like glycosyltransferase 3 (Extl3). In conclusion, our findings suggest that Reg4 exerts a therapeutic effect during pancreatitis by limiting inflammation and fibrosis and improving cellular regeneration.
Subject(s)
Fibrosis , Mitochondria , Pancreatitis-Associated Proteins , Pancreatitis , Receptors, CXCR4 , Animals , Pancreatitis-Associated Proteins/metabolism , Pancreatitis-Associated Proteins/genetics , Mitochondria/metabolism , Mitochondria/pathology , Pancreatitis/pathology , Pancreatitis/metabolism , Mice , Receptors, CXCR4/metabolism , Receptors, CXCR4/genetics , Humans , Mice, Inbred C57BL , Cyclams/pharmacology , Male , Mice, Knockout , Benzylamines/pharmacology , Chemokine CXCL12/metabolism , Cell Proliferation , Signal Transduction , Autophagy , Pancreas/pathology , Pancreas/metabolism , Cell DeathABSTRACT
In this work, we experimentally investigate the potency of high pressure to drive a protein toward an excited state where an inhibitor targeted for this state can bind. Ras proteins are small GTPases cycling between active GTP-bound and inactive GDP-bound states. Various states of GTP-bound Ras in active conformation coexist in solution, amongst them, state 2 which binds to effectors, and state 1, weakly populated at ambient conditions, which has a low affinity for effectors. Zn2+-cyclen is an allosteric inhibitor of Ras protein, designed to bind specifically to the state 1. In H-Ras(wt).Mg2+.GppNHp crystals soaked with Zn2+-cyclen, no binding could be observed, as expected in the state 2 conformation which is the dominant state at ambient pressure. Interestingly, Zn2+-cyclen binding is observed at 500â MPa pressure, close to the nucleotide, in Ras protein that is driven by pressure to a state 1 conformer. The unknown binding mode of Zn2+-cyclen to H-Ras can thus be fully characterized in atomic details. As a more general conjunction from our study, high pressure x-ray crystallography turns out to be a powerful method to induce transitions allowing drug binding in proteins that are in low-populated conformations at ambient conditions, enabling the design of specific inhibitors.
Subject(s)
Cyclams , Zinc , Zinc/chemistry , Zinc/metabolism , Crystallography, X-Ray , Cyclams/chemistry , Cyclams/pharmacology , Allosteric Regulation , Pressure , Protein Binding , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Humans , Binding SitesABSTRACT
PURPOSE: Radiopharmaceutical therapies targeting fibroblast activation protein (FAP) have shown promising efficacy against many tumor types. But radiopharmaceuticals alone in most cases are insufficient to completely eradicate tumor cells, which can partially be attributed to the protective interplay between tumor cells and cancer-associated fibroblasts (CAFs). The C-X-C chemokine receptor type 4/C-X-C motif chemokine 12 (CXCR4/CXCL12) interaction plays an important role in orchestrating tumor cells and CAFs. We hereby investigated the feasibility and efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2, a FAP-targeting radiopharmaceutical, in combination with AMD3100, a CXCR4 antagonist, in a preclinical murine model of triple-negative breast cancer (TNBC). METHODS: Public database was first interrogated to reveal the correlation between CAFs' scores and the prognosis of TNBC patients, as well as the expression levels of FAP and CXCR4 in normal tissues and tumors. In vitro therapeutic efficacy regarding cell proliferation, migration, and colony formation was assessed in BALB/3T3 fibroblasts and 4T1 murine breast cancer cells. In vivo therapeutic efficacy was longitudinally monitored using serial 18F-FDG, [18F]AlF-NOTA-FAPI-04, and [68Ga]Ga-DOTA-Pentixafor PET/CT scans and validated using tumor sections through immunohistochemical staining of Ki-67, α-SMA, CXCR4, and CXCL12. Intratumoral abundance of myeloid-derived suppressive cells (MDSCs) was analyzed using flow cytometry in accordance with the PET/CT schedules. Treatment toxicity was evaluated by examining major organs including heart, lung, liver, kidney, and spleen. RESULTS: CAFs' scores negatively correlated with the survival of TNBC patients (p < 0.05). The expression of CXCR4 and FAP was both significantly higher in tumors than in normal tissues. The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 significantly suppressed cell proliferation, migration, and colony formation in cell culture, and exhibited synergistic effects in 4T1 tumor models along with a decreased number of MDSCs. PET/CT imaging revealed lowest tumor accumulation of 18F-FDG and [18F]AlF-NOTA-FAPI-04 on day 13 and day 14 after treatment started, both of which gradually increased at later time points. A similar trend was observed in the IHC staining of Ki-67, α-SMA, and CXCL12. CONCLUSION: The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 is a feasible treatment against TNBC with minimal toxicity in main organs.
Subject(s)
Chemokine CXCL12 , Receptors, CXCR4 , Triple Negative Breast Neoplasms , Receptors, CXCR4/metabolism , Receptors, CXCR4/antagonists & inhibitors , Triple Negative Breast Neoplasms/diagnostic imaging , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/radiotherapy , Animals , Mice , Chemokine CXCL12/metabolism , Humans , Cell Line, Tumor , Female , Cyclams/pharmacology , Cyclams/therapeutic use , Lutetium , Benzylamines/pharmacology , Heterocyclic Compounds/pharmacology , Heterocyclic Compounds/chemistry , Radiopharmaceuticals/therapeutic use , Radiopharmaceuticals/pharmacology , Endopeptidases , Cell Proliferation/drug effects , Gelatinases/metabolism , Membrane Proteins/metabolism , Serine Endopeptidases/metabolismABSTRACT
The laminar flow profiles in microfluidic systems coupled to rapid diffusion at flow streamlines have been widely utilized to create well-controlled chemical gradients in cell cultures for spatially directing cell migration. However, within hydrogel-based closed microfluidic systems of limited depth (≤0.1 mm), the biomechanical cues for the cell culture are dominated by cell interactions with channel surfaces rather than with the hydrogel microenvironment. Also, leaching of poly(dimethylsiloxane) (PDMS) constituents in closed systems and the adsorption of small molecules to PDMS alter chemotactic profiles. To address these limitations, we present the patterning and integration of a PDMS-free open fluidic system, wherein the cell-laden hydrogel directly adjoins longitudinal channels that are designed to create chemotactic gradients across the 3D culture width, while maintaining uniformity across its â¼1 mm depth to enhance cell-biomaterial interactions. This hydrogel-based open fluidic system is assessed for its ability to direct migration of U87 glioma cells using a hybrid hydrogel that includes hyaluronic acid (HA) to mimic the brain tumor microenvironment and gelatin methacrylate (GelMA) to offer the adhesion motifs for promoting cell migration. Chemotactic gradients to induce cell migration across the hydrogel width are assessed using the chemokine CXCL12, and its inhibition by AMD3100 is validated. This open-top hydrogel-based fluidic system to deliver chemoattractant cues over square-centimeter-scale areas and millimeter-scale depths can potentially serve as a robust screening platform to assess emerging glioma models and chemotherapeutic agents to eradicate them.
Subject(s)
Cell Movement , Chemotaxis , Glioma , Hydrogels , Humans , Glioma/pathology , Glioma/metabolism , Cell Movement/drug effects , Hydrogels/chemistry , Hydrogels/pharmacology , Chemotaxis/drug effects , Cell Line, Tumor , Cell Culture Techniques, Three Dimensional/methods , Tumor Microenvironment/drug effects , Chemokine CXCL12/pharmacology , Chemokine CXCL12/metabolism , Cyclams/pharmacology , Cyclams/chemistry , Cell Culture Techniques/methods , Hyaluronic Acid/chemistry , Hyaluronic Acid/pharmacology , Gelatin/chemistry , Benzylamines/pharmacology , Benzylamines/chemistry , Brain Neoplasms/pathology , Brain Neoplasms/metabolismABSTRACT
Autologous hematopoietic progenitor cell transplantation (ASCT) has been used for more than five decades to treat malignant and non-malignant diseases. Successful engraftment after high-dose chemotherapy relies on the ability to collect sufficient CD34 + hematopoietic progenitor cells (HPCs), typically from peripheral blood after mobilization. Commonly, either granulocyte colony-stimulating factor (G-CSF) alone as a single agent (i.e. steady-state mobilization) or G-CSF after chemotherapy is administered to collect adequate numbers of HPCs (minimum ≥2 × 106 CD34 + cells/kg for one ASCT; optimal up to 5 × 106 CD34 + cells/kg). However, a significant proportion of patients fail successful HPC mobilization, which is commonly defined as a CD34+ cell count below 10-15/µL after at least 4 days of 10 µg/kg b.w. G-CSF alone, or after chemo-mobilization in combination with 5-10 µg/kg b.w. G-CSF. In these situations plerixafor, a chemokine receptor inhibitor (CXCR4) can be used to enhance HPC collection in patients with multiple myeloma and malignant lymphoma whose cells mobilize poorly. Risk factors for poor mobilization have been evaluated and several strategies (e.g. plerixafor to rescue the mobilization approach or pre-emptive use) have been suggested to optimize mobilization, especially in patients at risk. This manuscript discusses the risk factors of poor CD34+ mobilization and summarizes the current strategies to optimize mobilization and HPC collection.
Subject(s)
Hematopoietic Stem Cell Mobilization , Humans , Hematopoietic Stem Cell Mobilization/methods , Hematopoietic Stem Cell Transplantation/methods , Granulocyte Colony-Stimulating Factor/therapeutic use , Cyclams/pharmacology , Cyclams/therapeutic use , BenzylaminesABSTRACT
BACKGROUND: Despite recent advances in the treatment of multiple myeloma, high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT) remains an essential therapeutic keystone. As for the stem cell mobilization procedure, different regimens have been established, usually consisting of a cycle of chemotherapy followed by application of granulocyte-colony stimulating factor (G-CSF), although febrile neutropenia is a common complication. Following national guidelines, our institution decided to primarily use G-CSF only mobilization during the COVID-19 pandemic to minimize the patients' risk of infection and to reduce the burden on the health system. STUDY DESIGN AND METHODS: In this retrospective single-center analysis, the efficacy and safety of G-CSF only mobilization was evaluated and compared to a historic control cohort undergoing chemotherapy-based mobilization by cyclophosphamide and etoposide (CE) plus G-CSF. RESULTS: Although G-CSF only was associated with a higher need for plerixafor administration (p < .0001) and a higher number of apheresis sessions per patient (p = .0002), we were able to collect the target dose of hematopoietic stem cells in the majority of our patients. CE mobilization achieved higher hematopoietic stem cell yields (p = .0015) and shorter apheresis sessions (p < .0001) yet was accompanied by an increased risk of febrile neutropenia (p < .0001). There was no difference in engraftment after ASCT. DISCUSSION: G-CSF only mobilization is a useful option in selected patients with comorbidities and an increased risk of serious infections, especially in the wintertime or in future pandemics.
Subject(s)
Cyclophosphamide , Etoposide , Granulocyte Colony-Stimulating Factor , Hematopoietic Stem Cell Mobilization , Multiple Myeloma , Transplantation, Autologous , Adult , Female , Humans , Male , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Benzylamines , COVID-19 , Cyclams/therapeutic use , Cyclams/pharmacology , Cyclophosphamide/therapeutic use , Cyclophosphamide/administration & dosage , Etoposide/therapeutic use , Etoposide/administration & dosage , Granulocyte Colony-Stimulating Factor/administration & dosage , Granulocyte Colony-Stimulating Factor/therapeutic use , Hematopoietic Stem Cell Mobilization/methods , Hematopoietic Stem Cell Transplantation/methods , Multiple Myeloma/therapy , Retrospective Studies , SARS-CoV-2ABSTRACT
OBJECTIVES: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.
Subject(s)
Benzylamines , Cyclams , Lipopolysaccharides , Periodontal Ligament , Humans , Periodontal Ligament/metabolism , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , Core Binding Factor Alpha 1 Subunit/metabolism , Receptors, CXCR4/metabolism , Tumor Necrosis Factor-alpha/metabolism , Interleukin-6/metabolism , Interleukin-6/pharmacology , Osteogenesis , Signal Transduction , Inflammation/metabolism , Stem Cells , RNA, Messenger/metabolism , Apoptosis , Cell Proliferation , Stromal Cells/metabolism , Cell Differentiation , Cells, CulturedABSTRACT
The chemokine receptor CXCR4 is implicated in multiple diseases including inflammatory disorders, cancer growth and metastasis, and HIV/AIDS. CXCR4 targeting has been evaluated in treating cancer metastasis and therapy resistance. Cyclam derivatives, most notably AMD3100 (Plerixafor™), are a common motif in small molecule CXCR4 antagonists. However, AMD3100 has not been shown to be effective in cancer treatment as an individual agent. Configurational restriction and transition metal complex formation increases receptor binding affinity and residence time. In the present study, we have synthesized novel trans-IV locked cyclam-based CXCR4 inhibitors, a previously unexploited configuration, and demonstrated their higher affinity for CXCR4 binding and CXCL12-mediated signaling inhibition compared to AMD3100. These results pave the way for even more potent CXCR4 inhibitors that may provide significant efficacy in cancer therapy.
Subject(s)
Coordination Complexes , Cyclams , Heterocyclic Compounds , Benzylamines , Coordination Complexes/pharmacology , Heterocyclic Compounds/pharmacology , Heterocyclic Compounds/chemistry , Receptors, CXCR4/antagonists & inhibitorsABSTRACT
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is characterized by an immune-suppressive microenvironment, which contributes to tumor progression, metastasis, and immunotherapy resistance. Identification of HCC-intrinsic factors regulating the immunosuppressive microenvironment is urgently needed. Here, we aimed to elucidate the role of SYR-Related High-Mobility Group Box 18 (SOX18) in inducing immunosuppression and to validate novel combination strategies for SOX18-mediated HCC progression and metastasis. METHODS: The role of SOX18 in HCC was investigated in orthotopic allografts and diethylinitrosamine/carbon tetrachloride-induced spontaneous models by using murine cell lines, adeno-associated virus 8, and hepatocyte-specific knockin and knockout mice. The immune cellular composition in the HCC microenvironment was evaluated by flow cytometry and immunofluorescence. RESULTS: SOX18 overexpression promoted the infiltration of tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) while diminishing cytotoxic T cells to facilitate HCC progression and metastasis in cell-derived allografts and chemically induced HCC models. Mechanistically, transforming growth factor-beta 1 (TGF-ß1) upregulated SOX18 expression by activating the Smad2/3 complex. SOX18 transactivated chemokine (C-X-C motif) ligand 12 (CXCL12) and programmed death ligand 1 (PD-L1) to induce the immunosuppressive microenvironment. CXCL12 knockdown significantly attenuated SOX18-induced TAMs and Tregs accumulation and HCC dissemination. Antagonism of chemokine receptor 4 (CXCR4), the cognate receptor of CXCL12, or selective knockout of CXCR4 in TAMs or Tregs likewise abolished SOX18-mediated effects. TGFßR1 inhibitor Vactosertib or CXCR4 inhibitor AMD3100 in combination with anti-PD-L1 dramatically inhibited SOX18-mediated HCC progression and metastasis. CONCLUSIONS: SOX18 promoted the accumulation of immunosuppressive TAMs and Tregs in the microenvironment by transactivating CXCL12 and PD-L1. CXCR4 inhibitor or TGFßR1 inhibitor in synergy with anti-PD-L1 represented a promising combination strategy to suppress HCC progression and metastasis.