Hemopexin as an Inhibitor of Hemolysis-Induced Complement Activation.

Hemopexin is the main plasmatic scavenger of cell-free heme, released in the context of intravascular hemolysis or major cell injury. Heme is indispensable for the oxygen transport by hemoglobin but when released outside of the erythrocytes it becomes a danger-associated molecular pattern, contributing to tissue injury. One of the mechanisms of pro-inflammatory action of heme is to activate the innate immune complement cascade. Therefore, we hypothesized that injection of hemopexin will prevent hemolysis-induced complement activation. Human plasma-derived hemopexin is compatible with the heme clearance machinery of the mice. 100 or 500 mg/kg of hemopexin was injected in C57Bl/6 mice before treatment with phenylhydrazine (inducer of erythrocytes lysis) or with PBS as a control. Blood was taken at different timepoints to determine the pharmacokinetic of injected hemopexin in presence and absence of hemolysis. Complement activation was determined in plasma, by the C3 cleavage (western blot) and in the kidneys (immunofluorescence). Kidney injury was evaluated by urea and creatinine in plasma and renal NGAL and HO-1 gene expression were measured. The pharmacokinetic properties of hemopexin (mass spectrometry) in the hemolytic mice were affected by the target-mediated drug disposition phenomenon due to the high affinity of binding of hemopexin to heme. Hemolysis induced complement overactivation and signs of mild renal dysfunction at 6 h, which were prevented by hemopexin, except for the NGAL upregulation. The heme-degrading capacity of the kidney, measured by the HO-1 expression, was not affected by the treatment. These results encourage further studies of hemopexin as a therapeutic agent in models of diseases with heme overload.

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.

POL5551, a novel and potent CXCR4 antagonist, enhances sensitivity to chemotherapy in pediatric ALL.

The importance of the cell surface receptor CXCR4 and the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) is well-established in normal and malignant hematopoiesis. The Protein Epitope Mimetic POL5551 is a novel and potent antagonist of CXCR4. POL5551 efficiently mobilizes hematopoietic stem and progenitor cells, but its effects in acute lymphoblastic leukemia (ALL) have not been reported. Here, we demonstrate that POL5551 is a potent antagonist of CXCR4 in pre-B and T cell ALL cell lines and pediatric ALL primary samples. POL5551 has activity at nanomolar concentrations in decreasing CXCR4 antibody binding, blocking SDF-1α-mediated phosphorylation of ERK1/2, inhibiting SDF-1α-induced chemotaxis, and reversing stromal-mediated protection from chemotherapy. POL5551 is significantly more effective at inhibiting CXCR4 antibody binding than the FDA-approved CXCR4 inhibitor plerixafor in ALL cell lines and primary samples. We also show that treatment with POL5551 in vitro and cytarabine +/- POL5551 in vivo modulates surface expression of adhesion molecules, findings that may guide the optimal clinical use of POL5551. Finally, we demonstrate that POL5551 increases sensitivity to cytarabine in a xenograft model of a high-risk pediatric ALL, infant MLL-rearranged (MLL-R) ALL. Therefore, disruption of the CXCR4/SDF-1 axis with POL5551 may improve outcomes in children with high-risk ALL.

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.

Pharmacokinetics of paracetamol in Göttingen minipigs: in vivo studies and modeling to elucidate physiological determinants of absorption.

PURPOSE: Onset and rate of gastric emptying are important determinants of drug absorption after oral dosing. Therefore, robust estimates of these parameters are needed in physiologically based absorption models to predict reliably plasma concentration time profiles. For human and some other laboratory animals, reasonable parameterization of gastric emptying has been established. However gastric emptying is less well characterized in minipigs, a large animal model rapidly gaining importance in pharmaceutical research. METHODS: A pharmacokinetic crossover study using different dosage forms of paracetamol (intravenous and oral solution, capsule and tablet) was conducted in four male and four female Göttingen minipigs after an overnight fast. Deconvolution analysis was performed to determine the absorption kinetics. Estimated lag times and first order gastric emptying parameters were incorporated in a previously published PBPK model of the minipig and simulations verified. Postmortem assessments of minipig stomachs were made after different fasting protocols. RESULTS: Fraction of dose absorbed vs. time profiles showed high interindividual variability, comparable to human fed state absorption. Mean gastric transit times were determined to be 0.63 h, 1.36 h, and 0.73 h for solution, capsules, and tablets, respectively. Postmortem assessment confirmed that minipig stomachs were not empty after an overnight fast. CONCLUSIONS: Gastric transit times in overnight fasted minipigs are longer than those observed in humans. This is most likely caused by delayed and incomplete food emptying and further work is needed to develop feasible and effective fasting protocols for minipigs.

Podocyte EphB4 signaling helps recovery from glomerular injury.

Eph receptor tyrosine kinases and their ligands (ephrins) have a pivotal role in the homeostasis of many adult organs and are widely expressed in the kidney. Glomerular diseases beginning with mesangiolysis can recover, with podocytes having a critical role in this healing process. We studied here the role of Eph signaling in glomerular disease recovery following mesangiolytic Thy1.1 nephritis in rats. EphB4 and ephrinBs were expressed in healthy glomerular podocytes and were upregulated during Thy1.1 nephritis, with EphB4 strongly phosphorylated around day 9. Treatment with NPV-BHG712, an inhibitor of EphB4 phosphorylation, did not cause glomerular changes in control animals. Nephritic animals treated with vehicle did not have morphological evidence of podocyte injury or loss; however, application of this inhibitor to nephritic rats induced glomerular microaneurysms, podocyte damage, and loss. Prolonged NPV-BHG712 treatment resulted in increased albuminuria and dysregulated mesangial recovery. Additionally, NPV-BHG712 inhibited capillary repair by intussusceptive angiogenesis (an alternative to sprouting angiogenesis), indicating a previously unrecognized role of podocytes in regulating intussusceptive vessel splitting. Thus, our results identify EphB4 signaling as a pathway allowing podocytes to survive transient capillary collapse during glomerular disease.

The effects of PTK787/ZK222584, an inhibitor of VEGFR and PDGFRß pathways, on intussusceptive angiogenesis and glomerular recovery from Thy1.1 nephritis.

The aim of our study was to investigate the phenomenon of intussusceptive angiogenesis with a focus on its molecular regulation by vascular endothelial growth factor receptor (VEGFR)/platelet-derived growth factor receptor ß (PDGFRß) pathways and biological significance for glomerular recovery after acute injury. Glomerular healing by intussusception was examined in a particular setting of Thy1.1 nephritis, where the lysis of mesangial cells results in an initial collapse and successive rebuilding of glomerular capillary structure. Restoration of capillary structure after induction of Thy1.1 nephritis occurred by intussusceptive angiogenesis resulting in i) rapid expansion of the capillary plexus with reinstatement of the glomerular filtration surface and ii) restoration of the archetypical glomerular vascular pattern. Glomerular capillaries of nephritic rats after combined VEGFR2 and PDGFRß inhibition by PTK787/ZK222584 (PTK/ZK) were tortuous and irregular. However, the onset of intussusceptive angiogenesis was influenced only after long-term PTK/ZK treatment, providing an important insight into differential molecular regulation between sprouting and intussusceptive angiogenesis. PTK/ZK treatment abolished α-smooth muscle actin and tensin expression by injured mesangial cells, impaired glomerular filtration of microspheres, and led to the reduction of glomerular volume and the presence of multiple hemorrhages detectable in the tubular system. Collectively, treatment of nephritic patients with PTK/ZK compound is not recommended.

Lipopolysaccharide induces intestinal glucocorticoid synthesis in a TNFalpha-dependent manner.

Stringent control of immune responses in the intestinal mucosa is critical for the maintenance of immune homeostasis and prevention of tissue damage, such as observed during inflammatory bowel disease. Intestinal epithelial cells, primarily thought to form a simple physical barrier, critically regulate intestinal immune cell functions by producing immunoregulatory glucocorticoids on T-cell activation. In this study we investigated whether stimulation of cells of the innate immune system results in the induction of intestinal glucocorticoids synthesis and what role TNF-alpha plays in this process. Stimulation of the innate immune system with lipopolysaccharide (LPS) led to an up-regulation of colonic steroidogenic enzymes and the induction of intestinal glucocorticoid synthesis. The observed induction was dependent on macrophage effector functions, as depletion of macrophages using clodronate-containing liposomes, but not absence of T and B cells, inhibited intestinal glucocorticoid synthesis. LPS-induced glucocorticoid synthesis was critically dependent on TNF-alpha as it was significantly decreased in TNF-alpha-deficient animals. Both TNF receptor-1 and -2 were found to be equally involved in LPS- and T-cell-induced intestinal GC synthesis. These results describe a novel and critical role of TNF-alpha in immune cell-induced intestinal glucocorticoid synthesis.

The human brain endothelial cell line hCMEC/D3 as a human blood-brain barrier model for drug transport studies.

The human brain endothelial capillary cell line hCMEC/D3 has been developed recently as a model for the human blood-brain barrier. In this study a further characterization of this model was performed with special emphasis on permeability properties and active drug transport. Para- or transcellular permeabilities (P(e)) of inulin (0.74 x 10(-3) cm/min), sucrose (1.60 x 10(-3) cm/min), lucifer yellow (1.33 x 10(-3) cm/min), morphine (5.36 x 10(-3) cm/min), propranolol (4.49 x 10(-3) cm/min) and midazolam (5.13 x 10(-3) cm/min) were measured. By addition of human serum the passive permeability of sucrose could be reduced significantly by up to 39%. Furthermore, the expression of a variety of drug transporters (ABCB1, ABCG2, ABCC1-5) as well as the human transferrin receptor was demonstrated on the mRNA level. ABCB1, ABCG2 and transferrin receptor proteins were detected and functional activity of ABCB1, ABCG2 and the ABCC family was quantified in efflux experiments. Furthermore, ABCB1-mediated bidirectional transport of rhodamine 123 was studied. The transport rate from the apical to the basolateral compartment was significantly lower than that in the inverse direction, indicating directed p-glycoprotein transport. The results of this study demonstrate the usefulness of the hCMEC/D3 cell line as an in vitro model to study drug transport at the level of the human blood-brain barrier.

Drug targeting using OX7-immunoliposomes: correlation between Thy1.1 antigen expression and tissue distribution in the rat.

OX7 monoclonal antibody F((ab')2) fragments directed against Thy1.1 antigen can be used for drug targeting by coupling to the surface of drug-loaded liposomes. Such OX7-conjugated immunoliposomes (OX7-IL) were used recently for drug delivery to rat glomerular mesangial cells, which are characterized by a high level of Thy1.1 antigen expression. In the present study, the relationship between OX7-IL tissue distribution and target Thy1.1 antigen localization in different organs in rat was investigated. Western blot and immunohistofluorescence analysis revealed a very high Thy1.1 expression in brain cortex and striatum, thymus and renal glomeruli. Moderate Thy1.1 levels were observed in the collecting ducts of kidney, lung tissue and spleen. Thy1.1 was not detected in liver and heart. There was a poor correlation between Thy1.1 expression levels and organ distribution of fluorescence- or (14)C-labeled OX7-IL. The highest overall organ density of OX7-IL was observed in the spleen, followed by lung, liver and kidney. Heart and brain remained negative. With respect to intra-organ distribution, a localized and distinct signal was observed in renal glomerular mesangial cells only. As a consequence, acute pharmacological (i.e. toxic) effects of doxorubicin-loaded OX7-IL were limited to renal glomeruli. The competition with unbound OX7 monoclonal antibody F((ab')2) fragments demonstrated that the observed tissue distribution and acute pharmacological effects of OX7-IL were mediated specifically by the conjugated OX7 antibody. It is concluded that both the high target antigen density and the absence of endothelial barriers are needed to allow for tissue-specific accumulation and pharmacological effects of OX7-IL. The liposomal drug delivery strategy used is therefore specific toward renal glomeruli and can be expected to reduce the risk of unwanted side effects in other tissues.

A new drug-release method using the Staudinger ligation.

Many drugs induce severe side-effects caused by their lack of selectivity. One way to overcome this problem is to design a specific system which releases a free drug in a controlled manner. Herein we describe a new way to liberate a drug from a prodrug using the Staudinger ligation as the trigger.

Immunoliposome targeting to mesangial cells: a promising strategy for specific drug delivery to the kidney.

Mesangial cell-mediated nephropathies are a frequent cause of ESRD. Specific drug delivery to mesangial cells might be more effective and better tolerated than existing systemic treatments. Rat mesangial cells are characterized by Thy1.1 antigen expression. Therefore, OX7-coupled immunoliposomes (OX7-IL) were prepared by coupling liposomes with F(ab') fragments of OX7 mAb directed against Thy1.1 antigen. As the glomerular endothelium is fenestrated and no basement membrane separates glomerular capillaries from the mesangium, mesangial cells represent a particularly suitable target for drug delivery by OX7-IL. Therefore, the targeting efficacy of OX7-IL to mesangial cells was investigated. Specific targeting in vitro was obtained, and intravenous injection of OX-7-IL to rats showed a specific targeting of all mesangial cells in both kidneys. OX7-IL showed marked accumulation in the cytoplasm of rat mesangial cells, both in vitro and in vivo. This renal targeting was blocked when free OX7 F((ab')2) fragments were co-administered with OX7-IL. Rats that were given a single intravenous injection of low-dose doxorubicin encapsulated in OX7-IL showed extensive glomerular damage, whereas other parts of the kidney and other organs were spared. Free doxorubicin and the liposomal formulation of this agent had no effect. Thus, immunoliposomes are a very promising delivery system for the treatment of kidney diseases.