C-X-C Motif Chemokine Receptor 4 Blockade Promotes Tissue Repair After Myocardial Infarction by Enhancing Regulatory T Cell Mobilization and Immune-Regulatory Function.

BACKGROUND: Acute myocardial infarction (MI) elicits an inflammatory response that drives tissue repair and adverse cardiac remodeling. Inflammatory cell trafficking after MI is controlled by C-X-C motif chemokine ligand 12 (CXCL12) and its receptor, C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 antagonists mobilize inflammatory cells and promote infarct repair, but the cellular mechanisms are unclear. METHODS: We investigated the therapeutic potential and mode of action of the peptidic macrocycle CXCR4 antagonist POL5551 in mice with reperfused MI. We applied cell depletion and adoptive transfer strategies using lymphocyte-deficient Rag1 knockout mice; DEREG mice, which express a diphtheria toxin receptor-enhanced green fluorescent protein fusion protein under the control of the promoter/enhancer region of the regulatory T (Treg) cell-restricted Foxp3 transcription factor; and dendritic cell-depleted CD11c-Cre iDTR mice. Translational potential was explored in a porcine model of reperfused MI using serial contrast-enhanced magnetic resonance imaging. RESULTS: Intraperitoneal POL5551 injections in wild-type mice (8 mg/kg at 2, 4, 6, and 8 days) enhanced angiogenesis in the infarct border zone, reduced scar size, and attenuated left ventricular remodeling and contractile dysfunction at 28 days. Treatment effects were absent in splenectomized wild-type mice, Rag1 knockout mice, and Treg cell-depleted DEREG mice. Conversely, treatment effects could be transferred into infarcted splenectomized wild-type mice by transplanting splenic Treg cells from POL5551-treated infarcted DEREG mice. Instructive cues provided by infarct-primed dendritic cells were required for POL5551 treatment effects. POL5551 injections mobilized Treg cells into the peripheral blood, followed by enhanced Treg cell accumulation in the infarcted region. Neutrophils, monocytes, and lymphocytes displayed similar mobilization kinetics, but their cardiac recruitment was not affected. POL5551, however, attenuated inflammatory gene expression in monocytes and macrophages in the infarcted region via Treg cells. Intravenous infusion of the clinical-stage POL5551 analogue POL6326 (3 mg/kg at 4, 6, 8, and 10 days) decreased infarct volume and improved left ventricular ejection fraction in pigs. CONCLUSIONS: These data confirm CXCR4 blockade as a promising treatment strategy after MI. We identify dendritic cell-primed splenic Treg cells as the central arbiters of these therapeutic effects and thereby delineate a pharmacological strategy to promote infarct repair by augmenting Treg cell function in vivo.

Pharmacokinetics and Safety of Intravenous Murepavadin Infusion in Healthy Adult Subjects Administered Single and Multiple Ascending Doses.

Murepavadin is the first in class of the outer membrane protein-targeting antibiotics (OMPTA) and a pathogen-specific peptidomimetic antibacterial with a novel, nonlytic mechanism of action targeting Pseudomonas aeruginosa Murepavadin is being developed for the treatment of hospital-acquired bacterial pneumonia (HABP) and ventilator-associated bacterial pneumonia (VABP). The pharmacokinetics (PK) and safety of single and multiple doses of murepavadin were investigated in healthy male subjects. Part A of the study was a double-blind, randomized, placebo-controlled, single-ascending-dose investigation in 10 sequential cohorts where each cohort comprised 6 healthy male subjects; 4 subjects were randomized to murepavadin, and 2 subjects were randomized to placebo. Part B was a double-blind, randomized, placebo-controlled, multiple-ascending-dose investigation in 3 sequential cohorts. After a single dose of murepavadin, the geometric mean half-life (2.52 to 5.30 h), the total clearance (80.1 to 114 ml/h/kg), and the volume of distribution (415 to 724 ml/kg) were consistent across dose levels. The pharmacokinetics of the dosing regimens evaluated were dose proportional and linear. Murepavadin was well tolerated, adverse events were transient and generally mild, and no dose-limiting toxicity was identified.

Multifaceted C-X-C Chemokine Receptor 4 (CXCR4) Inhibition Interferes with Anti-Vascular Endothelial Growth Factor Therapy-Induced Glioma Dissemination.

Resistance to antiangiogenic therapy in glioblastoma (GBM) patients may involve hypoxia-induced expression of C-X-C motif chemokine receptor 4 (CXCR4) on invading tumor cells, macrophage/microglial cells (MGCs), and glioma stem cells (GSCs). We determined whether antagonizing CXCR4 with POL5551 disrupts anti-vascular endothelial growth factor (VEGF) therapy-induced glioma growth and dissemination. Mice bearing orthotopic CT-2A or GL261 gliomas received POL5551 and/or anti-VEGF antibody B20-4.1.1. Brain tissue was analyzed for tumor volume, invasiveness, hypoxia, vascular density, proliferation, apoptosis, GSCs, and MGCs. Glioma cells were evaluated for CXCR4 expression and polymorphism and POL5551's effects on CXCR4 ligand binding, cell viability, and migration. No CXCR4 mutations were identified. POL5551 inhibited CXCR4 binding to its ligand, stromal cell-derived factor-1α, and reduced hypoxia- and stromal cell-derived factor-1α-mediated migration dose-dependently but minimally affected cell viability. In vivo, B20-4.1.1 increased hypoxic foci and invasiveness, as seen in GBM patients receiving anti-VEGF therapy. Combination of POL5551 and B20-4.1.1 reduced both glioma invasiveness by 16% to 39% and vascular density compared to B20-4.1.1 alone in both glioma models. Reduced populations of GSCs and MGCs were also seen in CT-2A tumors. POL5551 concentrations, evaluated by mass spectrometry, were higher in tumors than in neighboring brain tissues, likely accounting for the results. Inhibition of CXCR4-regulated tumoral, stem cell, and immune mechanisms by adjunctive CXCR4 antagonists may help overcome antiangiogenic therapy resistance, benefiting GBM patients.

The Role of Neutrophil Elastase Inhibitors in Lung Diseases.

In many respiratory diseases characterized by an intense inflammatory response, the balance between proteolytic enzymes (proteases, including elastases) and their inhibitors (proteinases inhibitors) is not neutral. Excess activity of neutrophil elastase (NE) and similar proteases has been reported to cause tissue damage and to alter the remodeling process in many clinical conditions such as pneumonia, respiratory distress, and acute lung injury (ALI). Several experimental NE inhibitors have been tested in preclinical and clinical studies of different conditions of inflammatory lung injury such as ALI and pneumonia, with contrasting results. This study reviews the literature regarding NE inhibitors in the field of respiratory diseases and reflects on possible future developments. In particular, we highlight potential gaps in the scientific evidence and discuss potential strategies for focusing investigation on antielastases in clinical practice through the selection of targeted populations and proper outcomes.

Continuous blockade of CXCR4 results in dramatic mobilization and expansion of hematopoietic stem and progenitor cells.

Interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 plays a critical role in the retention and migration of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of the CXCR4/CXCL12 axis on the HSPC compartment were investigated by using 3 structurally unrelated small molecule CXCR4 antagonists. A >10-fold increase in mobilization efficiency was achieved by administering the antagonists as a subcutaneous continuous infusion for 2 weeks compared to a single bolus injection. A concurrent increase in self-renewing proliferation leading to a twofold to fourfold expansion of the HSPC pool in the BM was observed. The expanded BM showed a distinct repopulating advantage when tested in serial competitive transplantation experiments. Furthermore, major changes within the HSPC niche associated with previously described HSPC expansion strategies were not detected in bones treated with a CXCR4 antagonist infusion. Our data suggest that prolonged but reversible pharmacologic blockade of the CXCR4/CXCL12 axis represents an approach that releases HSPC with efficiency superior to any other known mobilization strategy and may also serve as an effective method to expand the BM HSPC pool.

CXCR4 Protein Epitope Mimetic Antagonist POL5551 Disrupts Metastasis and Enhances Chemotherapy Effect in Triple-Negative Breast Cancer.

The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow-disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a "chemotherapy framing" dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases.

Combined VEGF and CXCR4 antagonism targets the GBM stem cell population and synergistically improves survival in an intracranial mouse model of glioblastoma.

Glioblastoma recurrence involves the persistence of a subpopulation of cells with enhanced tumor-initiating capacity (TIC) that reside within the perivascular space, or niche (PVN). Anti-angiogenic therapies may prevent the formation of new PVN but have not prevented recurrence in clinical trials, suggesting they cannot abrogate TIC activity. We hypothesized that combining anti-angiogenic therapy with blockade of PVN function would have superior anti-tumor activity. We tested this hypothesis in an established intracranial xenograft model of GBM using a monoclonal antibody specific for murine and human VEGF (mcr84) and a Protein Epitope Mimetic (PEM) CXCR4 antagonist, POL5551. When doses of POL5551 were increased to overcome an mcr84-induced improvement in vascular barrier function, combinatorial therapy significantly inhibited intracranial tumor growth and improved survival. Anti-tumor activity was associated with significant changes in tumor cell proliferation and apoptosis, and a reduction in the numbers of perivascular cells expressing the TIC marker nestin. A direct effect on TICs was demonstrated for POL5551, but not mcr84, in three primary patient-derived GBM isolates. These findings indicate that targeting the structure and function of the PVN has superior anti-tumor effect and provide a strong rationale for clinical evaluation of POL5551 and Avastin in patients with GBM.

The CXCR4 antagonist POL5551 is equally effective as sirolimus in reducing neointima formation without impairing re-endothelialisation.

Impaired endothelial recovery after the implantation of drug-eluting stents is a major concern because of the increased risk for late stent thrombosis. The disruption of the chemokine axis CXCL12/CXCR4 inhibits neointima formation by blocking the recruitment of smooth muscle progenitor cells. To directly compare a CXCR4-targeting treatment strategy with drugs that are currently used for stent coating, we studied the effects of the CXCR4 antagonist POL5551 and the drug sirolimus on neointima formation. Apolipoprotein E-deficient mice were treated with POL5551 or sirolimus continuously for 28 days after a carotid wire injury. POL5551 inhibited neointima formation by 63% (for a dosage of 2 mg/kg/day) and by 70% (for a dosage of 20 mg/kg/day). In comparison, sirolimus reduced the neointimal area by 69%. In contrast to treatment with POL5551 during the first three days after injury, injection of POL5551 (20 mg/kg) once per day for 28 days diminished neointimal hyperplasia by 53%. An analysis of the cellular composition of the neointima showed a reduction in the relative smooth muscle cell (SMC) and macrophage content in mice that had been treated with a high dose of POL5551. In contrast, the diminished SMC content after sirolimus treatment was associated with a neointimal enrichment of macrophages. Furthermore, endothelial recovery was impaired by sirolimus, but not by POL5551. Therefore, the inhibition of CXCR4 by POL5551 is equally effective in preventing neointima formation as sirolimus, but POL5551 might be more beneficial because treatment with it results in a more stable lesion phenotype and because it does not impair re-endothelialisation.

Cardiomyocyte cell cycle activation ameliorates fibrosis in the atrium.

MHC-TGFcys33ser transgenic mice have elevated levels of active transforming growth factor (TGF)-beta1 in the myocardium. Previous studies have shown that these animals develop atrial, but not ventricular, fibrosis. Here we show that atrial fibrosis was accompanied with cardiomyocyte apoptosis. Although similar levels of cardiomyocyte apoptosis were present in the right and left atria of MHC-TGFcys33ser hearts, the extent of fibrosis was more pronounced in the right atrium. Thus, additional factors influence the degree of atrial fibrosis in this model. Tritiated thymidine incorporation studies revealed cardiomyocyte cell cycle activity in left atrial cardiomyocytes, but not in right atrial cardiomyocytes. These observations suggested that cardiomyocyte cell cycle activation ameliorated the severity of atrial fibrosis. To directly test this hypothesis, MHC-TGFcys33ser mice were crossed with MHC-cycD2 mice (which have constitutive cardiomyocyte cell cycle activity in the right atrium). Mice inheriting both transgenes exhibited right atrial cardiomyocyte cell cycle activity and a concomitant reduction in the severity of right atrial fibrosis, despite the presence of a similar level of cardiomyocyte apoptosis as was observed in mice inheriting the MHC-TGFcys33ser transgene alone. These data support the notion that cardiomyocyte cell cycle induction can antagonize fibrosis in the myocardium.

Expression, purification, biochemical and pharmacological characterization of a recombinant aprotinin variant.

Aprotinin (GAS 9087-70-1) is known as a potent inhibitor of serine proteases such as trypsin, plasmin, tissue and plasma kallikrein. In this study, an aprotinin variant was designed by means of rationale mutagenesis that differs from aprotinin by two amino acids in the active site and by seven amino acids in the backbone. The recombinant protein is expressed in a secretory yeast system enabling large scale production. A purification procedure was developed to yield high amounts of pure and correctly processed aprotinin variant. The changes in the active site of the aprotinin variant increase the potency towards inhibition of plasma kallikrein whereas the inhibition of plasmin is only marginally reduced. The net charge of the molecule is reduced from the basic (IP 10.5) to the neutral range (IP 5.6). The recombinant aprotinin variant shows a decrease of immunogenicity in several models. No cross-reactivity with human and rabbit antibodies directed against aprotinin was observed both in in vivo and in ex vivo studies. In addition, the variant is more potent in a rat brain edema model of acute subdural hematoma compared to aprotinin.

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle.

1. Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (alpha/ss) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. 2. Through a high-throughput screen we identified BAY 58-2667, an amino dicarboxylic acid which potently activates sGC in an NO-independent manner. In contrast to NO, YC-1 and BAY 41-2272, the sGC stimulators described recently, BAY 58-2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem deficient. 3. Binding studies with radiolabelled BAY 58-2667 show a high affinity site on the enzyme. 4. Using photoaffinity labelling studies we identified the amino acids 371 (alpha-subunit) and 231 - 310 (ss-subunit) as target regions for BAY 58-2667. 5. sGC activation by BAY 58-2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not influenced by nitrate tolerance. 6. BAY 58-2667 shows a potent antihypertensive effect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic effects of BAY 58-2667 and GTN are very similar on the arterial and venous system. 7. This novel type of sGC activator is a valuable research tool and may offer a new approach for treating cardiovascular diseases.

Pharmacological actions of a novel NO-independent guanylyl cyclase stimulator, BAY 41-8543: in vitro studies.

BAY 41-8543 is a novel, highly specific and so far the most potent NO-independent stimulator of sGC. Here we report the effects of BAY 41-8543 on the isolated enzyme, endothelial cells, platelets, isolated vessels and Langendorff heart preparation. BAY 41-8543 stimulates the recombinant sGC concentration-dependently from 0.0001 microM to 100 microM up to 92-fold. In combination, BAY 41-8543 and NO have synergistic effects over a wide range of concentrations. Similar results are shown in implying that BAY 41-8543 stimulates the sGC directly and furthermore makes the enzyme more sensitive to its endogenous activator NO. In vitro, BAY 41-8543 is a potent relaxing agent of aortas, saphenous arteries, coronary arteries and veins with IC(50)-values in the nM range. In the rat heart Langendorff preparation, BAY 41-8543 potently reduces coronary perfusion pressure from 10(-9) to 10(-6) g ml(-1) without any effect on left ventricular pressure and heart rate. BAY 41-8543 is effective even under nitrate tolerance conditions proved by the same vasorelaxing effect on aortic rings taken either from normal or nitrate-tolerant rats. BAY 41-8543 is a potent inhibitor of collagen-mediated aggregation in washed human platelets (IC(50)=0.09 microM). In plasma, BAY 41-8543 inhibits collagen-mediated aggregation better than ADP-induced aggregation, but has no effect on the thrombin pathway. BAY 41-8543 is also a potent direct stimulator of the cyclic GMP/PKG/VASP pathway in platelets and synergizes with NO over a wide range of concentrations. These results suggest that BAY 41-8543 is on the one hand an invaluable tool for studying sGC signaling in vitro and on the other hand its unique profile may offer a novel approach for treating cardiovascular diseases.

Cardiovascular actions of a novel NO-independent guanylyl cyclase stimulator, BAY 41-8543: in vivo studies.

BAY 41-8543 is a novel non-NO-based stimulator of sGC. This study investigates the acute effects of BAY 41-8543 on haemodynamics in anaesthetized rats and dogs, its long-term effects in conscious hypertension rat models and its antiplatelet effects. In anaesthetized dogs, intravenous injections of BAY 41-8543 (3 - 100 microg kg(-1)) caused a dose-dependent decrease in blood pressure and cardiac oxygen consumption as well as an increase in coronary blood flow and heart rate. In anaesthetized normotensive rats, BAY 41-8543 produced a dose-dependent and long-lasting blood pressure lowering effect after intravenous (3 - 300 microg kg(-1)) and oral (0.1 - 1 mg kg(-1)) administration. A dose-dependent and long-lasting decrease in blood pressure was also observed in conscious spontaneously hypertensive rats with a threshold dose of 0.1 mg kg(-1) p.o. After 3 mg kg(-1) the antihypertensive effect lasted for nearly 24 h. After multiple dosages, BAY 41-8543 did not develop tachyphylaxis in SHR. BAY 41-8543 prolonged the rat tail bleeding time and reduced thrombosis in the FeCl(3) thrombosis model after oral administration. In a low NO, high renin rat model of hypertension, BAY 41-8543 prevented the increase in blood pressure evoked by L-NAME and reveals a kidney protective effect. In this model, the overall beneficial effects of BAY 41-8543 manifested as both antiplatelet effect and vasodilatation were reflected in a significant reduction in mortality. The pharmacological profile of BAY 41-8543 suggests therefore that this compound has the potential to be an important research tool for in vivo investigations in the sGC/cGMP field and it also has the potential of being a unique clinical utility for treatment of cardiovascular diseases.