In vitro effects of bisphosphonates on chemotaxis, phagocytosis, and oxidative burst of neutrophil granulocytes.

OBJECTIVES: Bisphosphonate-associated osteonecrosis of the jaws is a serious side effect that mainly occurs in patients receiving highly potent, nitrogen-containing bisphosphonates. Usually the diagnosis is made due to exposed bone and a nonhealing wound. Neutrophil granulocytes are essential for sufficient wound healing; therefore, the influence of different bisphosphonates on neutrophil granulocytes was the focus of this study. MATERIAL AND METHODS: The effect of nitrogen-containing bisphosphonates (ibandronate, pamidronate, and zoledronate) and one non-nitrogen-containing bisphosphonate (clodronate) on chemotaxis, phagocytosis, and oxidative burst of neutrophil granulocytes in human whole blood was analyzed using standard cytometric flow assays. RESULTS: Chemotaxis of neutrophils was reduced by almost 50 % when cells were treated with ibandronate and zoledronate. All tested nitrogen-containing bisphosphonates moderately increased the percentage of phagocytizing neutrophils, whereas the percentage of oxidizing cells was extremely affected. Zoledronate increased the oxidative burst activity even at low concentrations. Treatment with ibandronate and pamidronate reached the same level, but only in at least 10 times the higher concentrations. The maximal burst activity of a single cell reached nearly 150 % compared to control. In this case, zoledronate also caused maximal effects even at low concentrations. Clodronate did not show any effects. CONCLUSION: The results show a proinflammatory effect of the nitrogen-containing effect on neutrophil granulocytes which might contribute to the development of osteonecrosis. CLINICAL RELEVANCE: The altered neutrophil defense might play a key role in the pathogenesis of bisphosphonate-associated osteonecrosis of the jaws, although the underlying causation between inflammatory reaction and the development of necrosis is yet unknown.

Diaryl-dithiolanes and -isothiazoles: COX-1/COX-2 and 5-LOX-inhibitory, *OH scavenging and anti-adhesive activities.

Three series of non-steroidal anti-inflammatory drugs (NSAIDs) inhibiting the cyclooxygenase/5-lipoxygenase (COX/5-LOX) pathways as such as formation of hydroxyl radicals and adhesion were prepared: 4,5-diaryl isothiazoles, 4,5-diaryl 3H-1,2-dithiole-3-thiones and 4,5-diaryl 3H-1,2-dithiole-3-ones. The aim of the present study was to develop substances which can intervene into the inflammatory processes via different mechanisms of action as multiple target non-steroidal anti-inflammatory drugs (MTNSAIDs) with increased anti-inflammatory potential. The current lead 11a was evaluated in COX-1/2, 5-LOX and (*)OH scavenging in vitro assays and in a static adhesion assay where it proved to inhibit adhesion. Moreover, 11a treatment attenuated expression of macrophage adhesion molecule-1 (Mac-1) on extravasated polymorphonuclear leukocytes (PMNs) which indicates that the activation was reduced. The assays used are predictive for the in vivo efficacy of test compounds as shown for 11a in a peritonitis model of acute inflammation in mice. Thus, the novel 5-LOX/COX and (*)OH inhibitor 11a possesses anti-inflammatory activity that, in addition to COX/5-LOX inhibition, implicates effects on leukocyte-endothelial interactions.

Investigations concerning the correlation of COX-1 inhibitory and hydroxyl radical scavenging activity.

The aim was to study the COX-1 inhibiting efficacy in context with hydroxyl radical scavenging properties of compounds bearing a carboxylic acid and ester function, respectively. In general, the acids are more potent radical scavengers than the corresponding esters but there is no clear correlation with their COX-1 inhibiting potencies. A feasible scavenging mechanism of carboxylic acids is discussed.

Effects of Cyclooxygenase Inhibitors on Apoptotic Neuroretinal Cells.

Glaucoma is characterized by a loss of retinal ganglion cells (RGC) which is associated with a decrease of visual function. Neuroprotective agents as a new therapeutic strategy could prevent the remaining neurons from apoptotic cell death. Previous studies have shown the involvement of the Cyclooxygenase (COX)-2 signalling in the apoptotic death of neurons. Herein we investigated the neuroprotective effect of COX-1/COX-2- and selective COX-2- inhibitors on apoptotic. R28, a neuroretinal cell line and determined the PGE(2) levels by ELISA. Furthermore we investigated differences in protein expression in the cells after exposure to elevated pressure compared to untreated cells by ProteinChip analysis.In addition, a protein profiling study of the cells after exposure to elevated pressure was performed. The protein expression profiles were measured by SELDI-TOF (Surface Enhanced Laser Desorption/Ionization-time of flight) Protein Chips. The protein identification was performed by mass spectrometry (MS).It could be shown that COX-2 inhibition significantly prevented the cells from apoptosis and reduced the PGE(2) concentrations. Selective COX-2 inhibitors were significant more potent than non-selective inhibitors or COX-1 inhibitors. We found differently expressed protein patterns in neuroretinal cells cultured at atmospheric pressure compared to those cells exposed to elevated pressure with or without celecoxib respectively. We identified three biomarkers, ubiquitin, HSP10 and NDKB, which were differently expressed in the groups. However, our data indicates a distinct neuroprotective effect of COX-2 inhibition. The local treatment with selective COX-2 inhibitors might provide an innovative strategy of therapeutic intervention for glaucoma.

Investigations concerning the COX/5-LOX inhibiting and hydroxyl radical scavenging potencies of novel 4,5-diaryl isoselenazoles.

The aim of this study was to investigate 4,5-diaryl isoselenazoles as multiple target non-steroidal anti-inflammatory drugs (MTNSAIDs) which can intervene into the inflammatory processes via different mechanisms of action creating a new class of compounds. Here we describe the synthesis of COX/LOX inhibitors which additionally reduce the level of reactive oxygen species, such as hydroxyl radicals which are well known for supporting inflammation processes in Parkinson's disease, Alzheimer's disease and rheumatoid arthritis.

A novel class of potent nonglycosidic and nonpeptidic pan-selectin inhibitors.

An early step of the inflammatory response, the rolling of leukocytes on activated endothelial cells, is mediated by selectin/carbohydrate interactions. The tetrasaccharide sialy Lewisx is a ligand for E-, P-, and L-selectin and therefore serves as a lead structure for the development of analogues. A combination of synthesis and structure-based design allowed rapid optimization. The current lead 2a was evaluated in our E-selectin cell flow chamber assay where it proved to inhibit rolling and adhesion with an IC50 of 28+/-7 microM. The assays used are predictive for the in vivo efficacy of test compounds as shown for 2a in a proteose peptone induced peritonitis model of acute inflammation in mice.

ACE inhibition lowers angiotensin-II-induced monocyte adhesion to HUVEC by reduction of p65 translocation and AT 1 expression.

Angiotensin-converting enzyme (ACE) inhibitors interfere with several key events of vascular inflammation resulting in impressive reductions in coronary vascular events. However, in human arteries ACE inhibitors block the production of angiotensin II (AngII) incompletely because of the involvement of alternative pathways in local AngII formation. Therefore, our study concentrated on the presumed modulation by ACE inhibition of local AngII-mediated inflammatory actions by a mechanism independent of blockage of AngII formation. We analyzed the effect of the ACE inhibitor ramiprilat on AngII-dependent cell adhesion molecule (CAM) expression and adhesion of monocytic THP-1 cells to endothelial cells. AngII induced upregulation of P-selectin, VCAM-1 and ICAM-1 on endothelial cells via activation of AT 1, which was correlated with enhanced THP-1 adhesion in flow chamber assays. Both enhanced adhesion and adhesion molecule expression were significantly reduced by pretreatment with ramiprilat. Ramiprilat reduced AT 1 expression on endothelial cells and decreased the AngII-induced p65 translocation into the nucleus. Diminished AT 1 expression and adhesion molecule expression in response to ramiprilat treatment were partially reversed after incubation with a bradykinin 2 receptor antagonist, suggesting that elevated bradykinin levels under ACE inhibition may be involved in the beneficial effect of ACE inhibitors. Thus, modulation of the local AngII system by ramiprilat may at least in part contribute to the benefits of ACE inhibition in the treatment of atherosclerotic diseases.

Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions.

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca(2+) that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.

Characterization of a computerized assay for rapid and easy determination of leukocyte adhesion to endothelial cells.

We report on a facile and rapid computerized in-vitro assay for the quantification of leukocyte adhesion to endothelial cells under static conditions using bovine polymorphonuclear neutrophils (PMN) or human leukaemic Mono Mac 6 cells (MM6) and bovine aorta endothelial cells (BAEC). Images of leukocytes adherent to BAEC monolayers grown in microtiter plates were obtained by a digital camera attached to a conventional microscope and transferred to the public domain NIH ImageJ program for analysis. Using individually adapted program routines adherent leukocytes are easily discriminated and reproducibly quantified. The results obtained with our assay correspond to previous findings and demonstrate the suitability of the described procedure, which can easily be adapted to further standards as proven by the use of two different leukocyte species. This assay lends itself to the screening of pharmacological substances with different mechanism of action that might act on either leukocytes or endothelial cells.

Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease.

Inflammation is a fundamental response to tissue injury and invasion of pathogens, but it is detrimental in clinically important inflammatory disorders. Leukocytes are key players in the inflammatory response because of their antimicrobial, secretory and phagocytic activities. They are recruited to the inflamed tissue by sequential adhesive interactions between leukocytes and the endothelium that are mediated by cell-adhesion molecules (CAMs) on the surface of the interacting cells. The effects of many anti-inflammatory drugs can be ascribed, in part, to inhibition of the expression of CAMs. However, in the search for more selective and potent drugs for clinically important diseases such as multiple sclerosis, asthma, rheumatoid arthritis, inflammatory bowel disease, allergies and atherosclerosis, direct inhibition of the function of CAMs has attracted increasing interest. In recent years, the development of synthetic antagonists has provided better opportunities for drug targeting. Future advances in this field hold new prospects for therapeutic intervention in human inflammatory disorders.

Convergent synthesis and preliminary biological evaluations of the stilbenolignan (+/-)-aiphanol and various congeners.

Treatment of an equimolar mixture of stilbene 7 and cinnamyl alcohol 8 with silver carbonate in acetone-benzene afforded a ca. 2:1:2:1 mixture of the stilbenolignan (+/-)-aiphanol (1) and congeners 2-4 each of which show significant anti-angiogenic and COX-2 inhibitory properties.

Inhibitory activity of tryptanthrin on prostaglandin and leukotriene synthesis.

The indolo[2,1- b]quinazoline alkaloid tryptanthrin has previously been identified as the cyclooxygenase-2 (COX-2) inhibitory principle in the extract ZE550 prepared from the medicinal plant Isatis tinctoria (Brassicaceae). We here investigated the potential inhibitory activity of tryptanthrin and ZE550 on COX-2, COX-1 in cellular and cell-free systems. A certain degree of selectivity towards COX-2 was observed when COX-1-dependent formation of thromboxane B(2) (TxB(2)) in HEL cells and COX-2-dependent formation of 6-ketoprostaglandin F(1alpha) (6-keto-PGF(1alpha)) in Mono Mac 6 and RAW 264.7 cells were compared. Preferential inhibition of COX-2 by two orders of magnitude was found in phorbol myristate acetate (PMA) activated bovine aortic coronary endothelial cells (BAECs). Assays with purified COX isoenzymes from sheep confirmed the high selectivity towards COX-2. The leukotriene B(4) (LTB(4)) release from calcium ionophore-stimulated human granulocytes (neutrophils) was used as a model to determine 5-lipoxygenase (5-LOX) activity. Tryptanthrin and the extract ZE550 inhibited LTB(4) release in a dose dependent manner and with a potency comparable to that of the clinically used 5-LOX inhibitor zileuton.

Cyclooxygenase-1/2 (COX-1/COX-2) and 5-lipoxygenase (5-LOX) inhibitors of the 6,7-diaryl-2,3-1H-dihydropyrrolizine type.

A series of 6,7-diaryl-2,3-1H-dihydropyrrolizines was prepared as COX-1/COX-2 and 5-LOX inhibitors. The inhibition of COX-1 was evaluated using intact bovine platelets as the enzyme source, whereas LPS-stimulated human monocytes served as the enzyme source for inducible COX-2. The determination of arachidonic metabolites was performed by HPLC for COX-1 and RIA for COX-2. The balance between COX-1/COX-2 and 5-LOX inhibition can be shifted by modifying the substitution pattern of the phenyl moiety at the 6- and 7-position of the pyrrolizine nucleus. Structure-activity relationships are discussed.