Nonsteroidal selective glucocorticoid modulators: the effect of C-5 alkyl substitution on the transcriptional activation/repression profile of 2,5-dihydro-10-methoxy-2,2,4-trimethyl-1H-[1]benzopyrano[3,4-f]quinolines.

The preparation and characterization of a series of selective glucocorticoid receptor modulators are described. The preliminary structure-activity relationship of nonaromatic C-5 substitution on the tetracyclic quinoline core showed a preference for small lipophilic side chains. Proper substitution at this position maintained the transcriptional repression of proinflammatory transcription factors while diminishing the transcriptional activation activity of the ligand/glucocorticoid receptor complex. The optimal compounds described in this study were the allyl analogue 18 and cyclopentyl analogue 32. These candidates showed slightly less potent, highly efficacious E-selectin repression with significantly reduced levels of glucocorticoid response element activation in reporter gene assays vs prednisolone. Allyl analogue 18 was evaluated in vivo. An oral dose of 18 showed an ED(50) = 1.7 mg/kg as compared to 1.2 mg/kg for prednisolone in the Sephadex-induced pulmonary eosinophilia model and an ED(50) = 15 mg/kg vs 4 mg/kg for prednisolone in the carrageenan-induced paw edema model.

Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia.

BACKGROUND: The excitatory amino acid domoic acid, a glutamate and kainic acid analog, is the causative agent of amnesic shellfish poisoning in humans. No studies to our knowledge have investigated the potential contribution to short-term neurotoxicity of the brain microglia, a cell type that constitutes circa 10% of the total glial population in the brain. We tested the hypothesis that a short-term in vitro exposure to domoic acid, might lead to the activation of rat neonatal microglia and the concomitant release of the putative neurotoxic mediators tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinases-2 and-9 (MMP-2 and -9) and superoxide anion (O2-). RESULTS: In vitro, domoic acid [10 microM-1 mM] was significantly neurotoxic to primary cerebellar granule neurons. Although neonatal rat microglia expressed ionotropic glutamate GluR4 receptors, exposure during 6 hours to domoic acid [10 microM-1 mM] had no significant effect on viability. By four hours, LPS (10 ng/mL) stimulated an increase in TNF-alpha mRNA and a 2,233 % increase in TNF-alpha protein In contrast, domoic acid (1 mM) induced a slight rise in TNF-alpha expression and a 53 % increase (p < 0.01) of immunoreactive TNF-alpha protein. Furthermore, though less potent than LPS, a 4-hour treatment with domoic acid (1 mM) yielded a 757% (p < 0.01) increase in MMP-9 release, but had no effect on MMP-2. Finally, while PMA (phorbol 12-myristate 13-acetate) stimulated O2- generation was elevated in 6 hour LPS-primed microglia, a similar pretreatment with domoic acid (1 mM) did not prime O2- release. CONCLUSIONS: To our knowledge this is the first experimental evidence that domoic acid, at in vitro concentrations that are toxic to neuronal cells, can trigger a release of statistically significant amounts of TNF-alpha and MMP-9 by brain microglia. These observations are of considerable pathophysiological significance because domoic acid activates rat microglia several days after in vivo administration.

A new spin on an old model: in vivo evaluation of disease progression by magnetic resonance imaging with respect to standard inflammatory parameters and histopathology in the adjuvant arthritic rat.

OBJECTIVE: To noninvasively examine the pathogenesis of rat adjuvant-induced arthritis (AIA) by magnetic resonance imaging (MRI), and to correlate MRI indices of disease progression with classic inflammatory parameters and histologic evaluation. METHODS: AIA was established in male Lewis rats following subcutaneous injection in the right hindpaw with 0.5 mg of heat-killed Mycobacterium butyricum suspended in light mineral oil. In vivo MRI evaluations of soft tissue and bony changes in AIA rats with matched histopathology were correlated with changes in left hindpaw volumes, circulating leukocytes, acute-phase reactants, and urinary collagen crosslinks throughout the disease process. RESULTS: MRI of arthritic tibiotarsal joints of the uninjected left hindpaws from AIA rats demonstrated 2 distinct phases of disease activity. The first phase, apparent between days 10 and 18, was characterized by periarticular inflammation with marked synovitis, synovial fibroplasia, and distension of the joint capsule into the surrounding tissue. The secondary phase, occurring between days 18 and 30, was marked by continued soft tissue inflammation, periostitis with osteolysis, and periosteal new bone formation progressing to a state of near complete ankylosis by day 30. These 2 phases of disease activity observed by MRI paralleled biochemical, cellular, and histologic markers of disease progression. CONCLUSION: MRI can be used to noninvasively detect, monitor, and quantify the chronic synovitis and progressive destruction of soft tissue and bone in live AIA rats, thereby improving the ability to evaluate disease progression in this preclinical animal model of rheumatoid arthritis.

Escherichia coli lipopolysaccharide potentiation and inhibition of rat neonatal microglia superoxide anion generation: correlation with prior lactic dehydrogenase, nitric oxide, tumor necrosis factor-alpha, thromboxane B2, and metalloprotease release.

The effects of lipopolysaccharide (LPS) on the central nervous system, one of the first organs to be affected by sepsis, are still incompletely understood. Rat microglia (BMphi) constitute the main leukocyte-dependent source of reactive oxygen species in the central nervous system. The in vitro effect of LPS on agonist-stimulated superoxide (O2-) generation from BMphi appears controversial. Our purpose was to determine the time- and concentration-dependent effect of Escherichia coil LPS on phorbol-12 myristate 13-acetate-stimulated O2- generation from BMphi. Our results demonstrate that BMphi O2- generation in vitro peaked 17 h after stimulation of with .3 ng/mL LPS. Furthermore, stimulation of BMphi with LPS for 17 h resulted in the following concentration-dependent responses: .1-1 ng/mL LPS induced no prior mediator generation but potently enhanced subsequent phorbol-12 myristate 13-acetate-stimulated O2- generation; 3-10 ng/mL LPS caused nitric oxide, tumor necrosis factor-alpha (TNF-alpha), thromboxane B2 and matrix metalloproteinase-9 release although partially inhibiting ensuing phorbol-12 myristate 13-acetate-stimulated O2- generation; 30-100 ng/mL LPS, maximized nitric oxide, TNF-alpha, thromboxane B2, matrix metalloproteinase-9 generation with concomitant lactic dehydrogenase release although strongly deactivating successive phorbol-12 myristate 13-acetate-stimulated O2 production. Our in vitro studies suggest that enhanced release of these four mediators (nitric oxide, TNF-alpha, thromboxane B2, and matrix metalloproteinase-9) during stimulation of BMphi with LPS might play a critical role in the subsequent ability of BMphi to generate O2- in vivo. Potential clinical implications of our findings are suggested by the fact that LPS levels similar to the ones used in this study have been observed in cerebrospinal fluid both in Gram-negative meningitis and sepsis.

Pharmacological characterization of the pseudopterosins: novel anti-inflammatory natural products isolated from the Caribbean soft coral, Pseudopterogorgia elisabethae.

Pseudopterosin E (PSE), a C-10 linked fucose glycoside and pseudopterosin A (PSA), a C-9 xylose glycoside isolated from the marine gorgonian Pseudopterogorgia elisabethae were both effective in reducing PMA-induced mouse ear edema when administered topically (ED50 (microg/ear) PSE(38), PSA(8)) or systemically (ED50 (mg/kg, i.p.) PSE (14), PSA (32)). Both compounds exhibited in vivo analgesic activity in phenyl-p-benzoquinone-induced writhing (ED50 (mg/kg, i.p.) PSE(14), PSA(4). PSE inhibited zymosan-induced writhing (ED50 = 6 mg/kg, i.p.), with a concomitant dose-dependent inhibition of peritoneal exudate 6-keto-prostaglandin F1alpha (ED50 = 24 mg/kg) and leukotriene C4 (ED50 = 24 mg/kg). In vitro, the pseudopterosins were inactive as inhibitors of phospholipase A2, cyclooxygenase, cytokine release, or as regulators of adhesion molecule expression. PSA inhibited prostaglandin E2 and leukotriene C4 production in zymosan-stimulated murine peritoneal macrophages (IC50 = 4 microM and 1 microM, respectively); however, PSE was much less effective. These data suggest that the pseudopterosins may mediate their anti-inflammatory effects by inhibiting eicosanoid release from inflammatory cells in a concentration and dose-dependent manner.

Streptococcal cell wall-induced arthritis. Requirements for neutrophils, P-selectin, intercellular adhesion molecule-1, and macrophage-inflammatory protein-2.

Immune arthritis in rat ankle joints was induced by intra-articular injection of streptococcal cell was extract (SCW), followed 21 days later by i.v. injection of SCW. This results in a monoarticular arthritis characterized by an influx of neutrophils and mononuclear cells, a 35-fold increase in urinary excretion of 8-hydroxy-deoxyguanosine (8-OH-dGUA; an index of free radical production), ankle edema, and joint damage/destruction. Neutrophil depletion substantially reduced the intensity of ankle edema. Ab-induced blockade of P-selectin or ICAM-1 also reduced the intensity of ankle edema and the influx of neutrophils. Blockade of TNF-alpha or IL-1 resulted in nearly complete and persistent reduction in ankle edema and profound reductions in the accumulation of neutrophils and mononuclear cells in affected joints. Finally, blocking of macrophage-inflammatory protein-2 reduced ankle edema and neutrophil accumulation during the first 2 days after i.v. challenge with SCW. These data indicate that SCW-induced arthritis is neutrophil dependent and that the recruitment of neutrophils and subsequent joint edema requires ICAM-1, P-selectin, and macrophage-inflammatory protein-2, as well as TNF-alpha and IL-1.

The effects of the phospholipase A2 inhibitor, manoalide, on cartilage degradation, stromelysin expression, and synovial fluid cell count induced by intraarticular injection of human recombinant interleukin-1 alpha in the rabbit.

OBJECTIVE: To evaluate the effects of the phospholipase A2 (PLA2) inhibitor manoalide on cartilage degradation, stromelysin expression, and inflammatory cell accumulation in rabbits treated intraarticularly with recombinant human interleukin-1 alpha (rHuIL-1 alpha). METHODS: Rabbits were given an intraarticular injection of rHuIL-1 alpha. At various time points over a 24-hour period, the rabbits were euthanized and the articular space was lavaged with sterile PBS. The proteoglycan content of the lavage fluid was measured using a dimethylmethylene blue assay. PLA2 activity and differential cell counts were also measured. The femur was removed and cartilage proteoglycan content determined. In some experiments, levels of synovial stromelysin messenger RNA (mRNA) were assessed. Manoalide or vehicle was administered 30 minutes before the rHuIL-1 alpha injection. RESULTS: The rHuIL-1 alpha-induced arthritic response is characterized by significant accumulation of inflammatory cells, loss of proteoglycan from the condylar cartilage, and induction of mRNA for stromelysin. PLA2 activity was also elevated in synovial fluids from rHuIL-1 alpha-injected joints. Pretreatment with manoalide (0.3 mg/joint) significantly inhibited PLA2 activity in the synovial fluid, prevented the loss of proteoglycan from the condylar cartilage, and reduced proteoglycan levels in lavage fluids. However, manoalide either had no effect on, or stimulated, cell accumulation. To assess the relationship between the induction of PLA2 and stromelysin, levels of stromelysin mRNA were measured in synovial tissue from manoalide- and vehicle-treated joints. Stromelysin message levels were significantly suppressed in a dose-dependent manner. CONCLUSION: These studies demonstrate that manoalide is a potent inhibitor of inflammation and cartilage catabolism, and suggest that PLA2 is involved in the pathophysiology of rHuIL-1 alpha-induced arthritis in rabbits.

Anti-inflammatory properties of Gö 6850: a selective inhibitor of protein kinase C.

Protein kinase C (PKC) regulates a variety of signal transduction events implicated in the pathogenesis of inflammation, including the biosynthesis of inflammatory cytokines and superoxide and the activation of phospholipase A2. Because of the significant role of PKC in these inflammatory processes, we evaluated a specific and potent inhibitor of C kinase for efficacy in several in vitro and in vivo murine models of inflammation. Unlike the relatively nonspecific kinase inhibitor staurosporine, the bisindolylmaleimide 3-[1-[-3-(dimethylaminopropyl]-1H-indol-3-yl]- 4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (Gö 6850) demonstrated increased selectivity for C kinase in purified enzyme assays (respective IC50 values (microM) for Gö 6850 and staurosporine: protein kinase C (0.032, 0.009); myosin light-chain kinase (0.6, 0.01); protein kinase G (4.6, 0.018); protein kinase A (33, 0.04); tyrosine kinase1 (94, 0.4); tyrosine kinase2 (> 100, > 1)). Topically applied Gö 6850 inhibited phorbol myristate acetate-induced edema, neutrophil influx and vascular permeability in murine epidermis in a dose- and time-dependent manner at levels comparable to indomethacin. In a murine model of delayed type hypersensitivity, Gö 6850 inhibited dinitrofluorobenzene-induced contact dermatitis with and ID50 value of 150 micrograms/ear. Cellular studies in mouse peritoneal macrophages demonstrated that Gö 6850 was a potent inhibitor of phorbol myristate acetate-induced prostaglandin E2 production. Superoxide production in phorbol myristate acetate-stimulated murine neutrophils was also inhibited by Gö 6850 (IC50 = 88 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of CD11b/CD18 expression in human neutrophils by phospholipase A2.

Recent evidence suggests that phospholipase A2 (PLA2)-derived lipid mediators may regulate a number of neutrophil responses including degranulation and adhesion. In view of the potential role of PLA2 in stimulus-secretion coupling, we examined the relationship between PLA2 activation and the surface expression of CD11b/CD18 (MAC-1) in human polymorphonuclear leukocytes (hPMNL), including the functional consequences of PLA2 inactivation on MAC-1-dependent adhesion. The selective inhibition of PLA2 by the marine natural products manoalide (MLD) and scalaradial (SLD) blocks [3H]arachidonic acid (AA) release in calcium ionophore A23187-stimulated neutrophils, and also inhibits secretion of specific and azurophilic granule constituents. Additional studies demonstrate that MLD, SLD, and other less potent PLA2 inhibitors such as 4-bromophenacylbromide and nordihydroguiaretic acid inhibit the surface expression of MAC-1 (IC50: MLD, 0.33 microM; SLD, 0.23 microM; 4-bromophenacylbromide, 2.8 microM; NDGA, 3.5 microM) at concentrations similar to those at which they inhibit [3H]AA release. Inhibitors of cyclooxygenase, 5-lipoxygenase, protein kinase C, or calcium channel antagonists have no effect on MAC-1 expression. PLA2 inactivation also prevents MAC-1 up-regulation in hPMNL stimulated with FMLP, IL-8, TNF-alpha, PMA, or platelet activating factor. In FMLP-stimulated hPMNL, under conditions in which no secondary granule constituents are secreted, MAC-1 and alkaline phosphatase up-regulation from intracellular granules is inhibited by MLD and SLD. Functional assays also demonstrate that MLD and SLD block MAC-1-dependent adhesion of activated neutrophils to keyhole limpet hemocyanin at concentrations that block the surface expression of MAC-1. [3H]AA release and MAC-1 expression in MLD and SLD-treated hPMNL could be recovered in the presence of 1 mM hydroxylamine in a time-dependent fashion, consistent with reported data that MLD and SLD inactivate PLA2 through Schiff base formation. In summary, these data emphasize the role of PLA2 as a key regulator of MAC-1 expression in models of neutrophil adhesion.

Fuscoside: an anti-inflammatory marine natural product which selectively inhibits 5-lipoxygenase. Part I: Physiological and biochemical studies in murine inflammatory models.

The biological and biochemical pharmacology of fuscoside, a novel anti-inflammatory marine natural product isolated from the Caribbean gorgonian Eunicea fusca, has recently been characterized using murine (part I) and human (part II) models of inflammation. Topically applied fuscoside (FSD) effectively inhibits phorbol myristate acetate (PMA)-induced edema in mouse ears at levels comparable with indomethacin over a 3.3-hr exposure period, and is significantly more efficacious than indomethacin over 24 hr in the PMA model. Histological preparations and quantification of the neutrophil-specific marker, myeloperoxidase, demonstrate that FSD inhibits neutrophil infiltration into PMA-induced regions of edema and inflammation. In systemic studies, where FSD is injected i.p. before the topical application of PMA, negligible effects on ear inflammation are observed. FSD does not inhibit bee venom or human synovial fluid phospholipase A2 up to concentrations of 500 microM. In calcium ionophore-activated cultures of mouse peritoneal macrophages, FSD selectively and irreversibly inhibits leukotriene C4 biosynthesis (IC50 = 8 microM), yet has negligible effects on prostaglandin E2 production. FSD is also without effect on the conversion of arachidonic acid to prostaglandin E2 by ram seminal vesicle cyclooxygenase. Chromatographic and spectroscopic studies suggest that FSD is not metabolized, and that drug uptake/binding by macrophages is time dependent, saturable and independent of active transport mechanisms. These studies represent the first report of an anti-inflammatory marine natural product that selectively inhibits leukotriene biosynthesis.

Fuscoside: an anti-inflammatory marine natural product which selectively inhibits 5-lipoxygenase. Part II: Biochemical studies in the human neutrophil.

Fuscoside (FSD) is a potent and long-lasting anti-inflammatory drug that selectively inhibits leukotriene production in murine models of inflammation. In the present study, the effects of FSD on the lipoxygenase pathways in human polymorphonuclear leukocytes are explored in order to better understand the mechanism of action of this novel drug. In adherent and suspended polymorphonuclear leukocytes, FSD irreversibly inhibits leukotriene B4 (LTB4) synthesis (IC50 = 10 microM) and the release of 14C-labeled LTB4 from neutrophils prelabeled with [14C]arachidonic acid. Unlike the reversible 5-lipoxygenase inhibitor L-651,896, FSD has no observable effect on LTB4 biosynthesis in whole blood, but does express activity as blood is successively diluted. In 10,000 x g supernatants of human platelets and polymorphonuclear leukocytes, FSD does not inhibit platelet 12-lipoxygenase, but is extremely effective in inhibiting the metabolism of arachidonic acid and 5-hydroperoxyeicosatetraenoic acid to LTB4 via neutrophil 5-lipoxygenase. FSD has no effect on the conversion of leukotriene A4 to LTB4 in this system. Interestingly, concurrent with FSD inhibition of leukotriene synthesis is a concentration-dependent increase in 5-hydroxyeicosatetraenoic acid, suggesting that FSD may selectively inhibit the leukotriene A4 synthase activity associated with human 5-lipoxygenase. FSD is therefore representative of a new class of nonantioxidant 5-lipoxygenase inhibitors that may be effective local therapeutic agents in the management of diseases such as psoriasis, arthritis and inflammatory bowel and lung diseases.

Inactivation of human synovial fluid phospholipase A2 by the marine natural product, manoalide.

The marine natural product, manoalide (MLD), was investigated to determine if this drug inhibited purified human synovial fluid phospholipase A2 (HSF-PLA2). Utilizing classical Michaelis-Menten kinetics, apparent Km and Vmax values for HSF-PLA2 of 1.34 mM and 0.47 mumol [3H]palmitic acid released/min/mg protein were obtained using dipalmitoylphosphatidylcholine (DPPC) as the substrate, and 38.0 microM and 18.8 mumol [3H]arachidonic acid released/min/mg protein with Escherichia coli as a natural substrate. These kinetic parameters were utilized subsequently to evaluate the inhibitory effects of manoalide on HSF-PLA2. Inhibition of HSF-PLA2 by MLD was concentration and time dependent with IC50 values of 0.2 and 0.02 microM for DPPC and E. coli respectively. Dialysis studies and examination of DPPC or E. coli hydrolysis versus enzyme concentration indicate that MLD is an irreversible inhibitor of HSF-PLA2. Substrate specificity was also examined in the absence and presence of MLD using dipalmitoylphosphatidylethanolamine (DPPE) as a substrate. MLD inhibited the hydrolysis of DPPE (greater than 90% inhibition at 2 microM), and preliminary results indicate that DPPC was more readily hydrolyzed than DPPE under the substrate conditions of the assay. While the cellular source of secreted HSF-PLA2 is unknown, these studies indicate that MLD can inactivate secreted phospholipase A2 isolated from patients with inflammatory joint disease.