Nabil Hanna

Inhibition of monocyte IL-1 production by the anti-inflammatory compound, SK&F 86002

The effects of several anti-inflammatory/anti-arthritic drugs on the in vitro production of interleukin-1 (IL-1) by human monocytes were examined. SK&F 86002, a novel dihydroimidazo thiazoline which inhibits both 5-lipoxygenase- and cyclooxygenase-mediated arachidonate metabolism was shown to be a potent inhibitor of IL-1 production by LPS-stimulated human monocytes. The inhibition was dose-dependent (IC50 = 1.30 +/- 1 microM), reversible and was independent of the concentration or type of stimulus used. The compound also inhibited cell-associated IL-1 activity. The compound did not exert general inhibitory effects on such parameters as adherence, cytotoxic function and protein synthesis of the monocytes. Other cyclooxygenase and/or 5-lipoxygenase inhibitors of arachidonic acid metabolism tested, with the exception of nordihydroguaiaretic acid, were inactive in inhibiting monocyte IL-1 production suggesting that the inhibition of IL-1 production by 86002 may be dissociated from its inhibition of the fatty acid oxygenases. The inhibition of IL-1 production by SK&F 86002 adds another facet of drug action contributing to its spectrum of anti-inflammatory activities.

SK&F 86002: A structurally novel anti-inflammatory agent that inhibits lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid

The effects of SK&F 86002 [5-(4-pyridyl)-6 (4-fluorophenyl)-2,3-dihydroimidazo (2,1-b) thiazole] on the generation of eicosanoids in vitro and on inflammatory responses in vivo are described and compared to other non-steroidal anti-inflammatory drugs. SK&F 86002 inhibited prostaglandin H2 (PGH2) synthase activity (IC50 120 microM) as well as prostanoid production by rat basophilic leukemia (RBL-1) cells (IC50 70 microM) and its sonicate (IC50 100 microM) and human monocytes (IC50 1 microM). In addition, SK&F 86002 inhibited the generation of dihydroxyeicosatetraenoic acid (diHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE) by a high speed supernatant fraction of RBL-1 cells (IC50 10 microM). Cellular production of 5-lipoxygenase products was inhibited by SK&F 86002 as measured by leukotriene B4 (LTB4) generation from human neutrophils (IC50 20 microM), leukotriene C4 (LTC4) generation by human monocytes (IC50 20 microM), and 5-HETE production by RBL-1 cells (IC50 40 microM). The in vivo profile of anti-inflammatory activity of SK&F 86002 supports the dual inhibition of arachidonate metabolism as indicated by its activity in inflammation models that are insensitive to selective cyclooxygenase inhibitors. The responses of arachidonic-acid-induced edema in the mouse ear and rat paw, as well as the cell infiltration induced by carrageenan in the mouse peritoneum and by arachidonic acid in the rat air pouch, were inhibited by SK&F 86002 and phenidone but not by the selective cyclooxygenase inhibitors naproxen and indomethacin.

Pharmacologic characterization of the antiinflammatory properties of a new dual inhibitor of lipoxygenase and cyclooxygenase

SK&F 86002 [6-(4-fluorophenyl)2,3-dihydro-5-(4-pyridinyl)imidazo(2,1-b)thiazole], a dual inhibitor of arachidonic acid metabolism, administered orally to rats prevented the development of carrageenan-induced edema, immune- and nonimmune-mediated inflammation of adjuvant-induced arthritis (AA) and reduced established inflammation in AA and collagen type II-induced arthritis. A similar profile of activity was observed following treatment with the cyclooxygenase inhibitor, indomethacin. However, unlike other nonsteroidal antiinflammatory drugs, SK&F 86002 exhibited antiinflammatory activity in inflammation models that are insensitive to cyclooxygenase inhibitors such as the established inflammation in carrageenan-induced edema and the edema induced by arachidonic acid and platelet activating factor. Moreover, SK&F 86002, but not indomethacin, inhibited the immune-mediated inflammatory responses evoked in sensitized animals by challenge with purified protein derivative. In addition, SK&F 86002 produced dose-related analgesia in mice, which was not reversed by the narcotic antagonist, naltexone. SK&F 86002 thus represents an orally active antiarthritic and analgesic compound with novel antiinflammatory properties.

Arachidonic acid-induced inflammation: inhibition by dual inhibitor of arachidonic acid metabolism, SK&F 86002.

The antiinflammatory activity of the structurally novel dual inhibitor of arachidonic acid metabolism, SK&F 86002 was evaluated using arachidonic acid-induced edema and inflammatory cell infiltration. Histological examination demonstrated extensive subcutaneous edema and neutrophil (PMN) accumulation in perivascular and interstitial locations one hour after application of arachidonic acid to the ear. SK&F 86002 and, to a lesser extent, phenidone demonstrated potent inhibition of this inflammatory response following oral and topical administration. Nordihydroguaiaretic acid (NDGA) displayed only topical activity. The selective cyclooxygenase inhibitors ibuprofen and naproxen were either inactive or stimulated ear swelling, Histological evaluation of the lesion in drug-treated animals revealed that SK&F 86002 impaired edema formation and caused a significant reduction in numbers of infiltrating neutrophils. Using arachidonic acid-induced peritoneal exudation, a reduction in the cellular infiltrate was observed after oral treatment with SK&F 86002 or phenidone, but not with naproxen. Taken together, these data illustrate the potent antiinflammatory effects of SK&F 86002 and support the suggestion that 5-lipoxygenase products play a significant role in both the edematous and cellular phases of arachidonic acid-induced inflammation.

Pharmacology of the pyrroloimidazole, SK&F 105809—I: Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid

SK&F 105809 [2-(4- methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2- a] imidazole] was determined to be a prodrug for the sulfide metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole] which inhibited interleukin-1 (IL-1) production in vitro and both 5-lipoxygenase (5-LO) and prostaglandin H (PGH) synthase activities in vitro and ex vivo. SK&F 105561 inhibited partially purified 5-LO with a half-maximal concentration (IC50) of 3 microM. This inhibition was reversible, independent of preincubation time, and dependent on the concentration of the substrate arachidonic acid. SK&F 105561 also inhibited purified PGH synthase with the potency dependent on the level of peroxidase activity. The IC50 was 100 microM in the absence of peroxidase activity, whereas an IC50 of 3 microM was observed in the presence of peroxidase activity. Using human monocytes, SK&F 105561 inhibited A23187-stimulated prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production with IC50 values of 0.1 and 2 microM, respectively. In addition, IL-1 production by lipopolysaccharide-stimulated human monocytes was also inhibited (IC50 2 microM). Oral administration of SK&F 105809 to rats resulted in a dose-related generation of SK&F 105561 and in the inhibition of thromboxane B2 and LTB4 production ex vivo with a half-maximal dose (ED50) of 15 and 60 mg/kg, respectively. SK&F 105561 showed weak inhibitory activity on 12-lipoxygenase with an IC50 of greater than 200 microM. Neither SK&F 105561 nor SK&F 105809 inhibited the stimulated-turnover of arachidonic acid-containing phospholipids in human monocytes or the activity of cell-free phospholipases A2 and C. Moreover, neither SK&F 105561 nor SK&F 105809 antagonized the binding of LTB4 or leukotriene D4 to membrane receptors. From these results, SK&F 105561, the active principle of SK&F 105809, acts as an inhibitor of both inflammatory cytokine and eicosanoid production.

Pharmacology of the pyrroloimidazole, SK&F 105809—II: Antiinflammatory activity and inhibition of mediator production in vivo

SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)- 6,7-dihydro-[5H]-pyrrolo[1,2,a] imidazole] demonstrated unique antiinflammatory activities in murine models that are resistant to selective cyclooxygenase (CO) inhibitors. Both edema and inflammatory cell infiltration induced by the topical application of arachidonic acid to the mouse ear were decreased by SK&F 105809 (ED50 values of 44 mg/kg, p.o.). Polymorphonuclear leukocyte (PMN) infiltration following the intraperitoneal injection of either monosodium urate crystal or carrageenan was inhibited with ED50 values of 64 and 72 mg/kg, p.o., respectively. These inflammatory responses were unaffected by the selective cyclooxygenase inhibitor naproxen. SK&F 105809 also inhibited leukotriene B4 (LTB4) and prostaglandin E2 production in vivo in arachidonic acid-induced inflammatory exudates (ED50 values of 41 and 15 mg/kg, p.o., respectively). The inhibition of LTB4 production preceded the inhibition of PMN infiltration. The impact of inhibition of both 5-lipoxygenase (5-LO) and CO was seen with platelet-activating factor-induced vascular permeability which was inhibited markedly by SK&F 105809. However, the 5-LO inhibitor, phenidone, only strongly inhibited when coadministered with the selective CO inhibitor, indomethacin. In spite of a short half-life (14-18 min) for both SK&F 105809 and the active metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole], the pharmacological activity lasted at least 1.5 hr. The biochemical evidence of inhibition of interleukin-1 (IL-1) production and 5-LO and CO activity, in vitro, by the metabolite (SK&F 105561) seen in the companion paper (Marshall PJ, Griswold DE, Breton J. Webb EF, Hillegass LM, Sarau HM, Newton J Jr, Lee JC, Bender PE and Hanna N, Pharmacology of the pyrroloimidazole, SK&F 105809--I. Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid. Biochem Pharmacol 42: 813-824, 1991) and inhibition of the fluid and cellular phases of the inflammatory response, in vivo, by SK&F 105809 suggest that this compound possesses a unique profile of activity.

Induction of non-specific suppressor cells in normal Lewis rats by a novel azaspirane SK&F 105685.

SK&F 105685 (N,N-Dimethyl-8,8-dipropyl-2-azaspiro[4,5]decane-2- propanamine dihydrochloride) is a novel azaspirane which has therapeutic activity in rat models of autoimmune disease. In this study, we have demonstrated that SK&F 105685 is a potent inducer of non-specific suppressor cells (SC). Oral administration of 15-30 mg/kg/day results in the generation of SC in the spleen, lymph nodes and bone marrow, but not the thymus of Lewis rats. Splenic SC suppress Con-A-induced proliferation in co-culture assays at effector-responder ratios of 1:1 to 1:64. SC are radiation resistant (2000 R), non-T, non-B cells, partially adherent to plastic surfaces and are enriched in a 1.07 g/ml fraction of a Percoll density gradient. Their activity is increased, rather than ablated, by indomethacin. No definitive changes in Ig+, OX-19+, OX-8+, W3/25+ or asialo GM1+ cells could be detected in the spleens of treated rats compared to control untreated animals. Elevated levels of both radiation-sensitive and radiation-resistant suppressor cells were found in the bone marrow of treated rats in addition to the radiosensitive SC normally present in this tissue.

Effects of prostaglandins and cAMP levels on monocyte IL-1 production.

The effects of prostaglandin E2 (PGE2), as well as other cAMP-elevating agents, on thein vitro production of interleukin-1 (IL-1) by human monocytes (HM) were examined. Exposure toE. coli lipopolysacharide (LPS) resulted in a dose- and time-dependent increase of IL-1 activity in monocytes culture supernatants. Maximal levels of secreted IL-1 in response to 10 ng LPS/ml were obtained at 18 h. PGE1, PGE2, cholera toxin (CT) and the phosphodiesterase inhibitor, isobutylmethylxanthin (IBMX), when added with LPS, resulted in a dose-dependent increase in cellular cAMP and in secreted IL-1. Maximal levels of secreted IL-1 were 2.5–5.0-fold over LPS alone. When CT or PGE2 was added with IBMX a further increase was observed. These agents exhibited marginal effect on cell-associated IL-1. Maximum cAMP levels was acheived at 10 min in response to either PGE1 or PGE2 and returned to near basal levels after 18 h. While PGE1 elevated cAMP to a larger extent than PGE2 (58- vs. 30-fold) the latter resulted in a higher levels of secreted IL-1. Elevated cAMP persisted throughout the entire culture period in response to CT (4–6-fold) or IBMX (7-fold). Thus, we conclude that in adherent HM, IL-1 production is potentiated and not inhibited by prostaglandins or agents that elevate cellular cAMP.

Macrophage activation in rat models of inflammation and arthritis: Determination of markers of stages of activation

Disease-associated alterations in macrophage functions were assessed by investigating the stages of activation of peritoneal macrophages obtained from adjuvant-induced arthritic rats. The stages of activation were established by defining several functional parameters in macrophages obtained from normal, sterile-irritant injected and Propionibacterium acnes injected animals. Peritoneal macrophages taken from arthritic rats 17 days post adjuvant injection displayed parameters characteristic of activated, but not elicited or resident macrophages. Specifically, an increased number of macrophages was recovered from arthritic rats which spread readily in culture, exhibited enhanced Fc receptor-mediated phagocytosis, increased leucine aminopeptidase ectoenzyme activity, enhanced secretion of prostaglandin E2 and interleukin 1, and ability to lyse tumor cells spontaneously. In addition, these macrophages were impaired in their ability to secrete superoxide anion. These data demonstrate distinct differences in parameters of peritoneal macrophage activation in rats compared to mice and that macrophage activation is associated with disease progression in adjuvant-induced arthritic rats.

Inhibition of animal models of autoimmune disease and the induction of non-specific suppressor cells by SK&F 105685 and related azaspiranes

SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine+ ++ dihydrochloride), administered orally to adjuvant arthritic (AA) rats inhibited immune-mediated hindpaw inflammation with an ED50 of 20 mg/kg/day. Both prophylactic and therapeutic administration were effective in this model. In addition, SK&F 105685 inhibited skin wheal responses to purified protein derivative (PPD) of tuberculin in AA rats and the development of hindleg paralysis associated with experimental allergic encephalomyelitis (EAE). Spleens of normal rats treated with SK&F 105685 were found to contain a population(s) of suppressor cells (SC) which inhibited the response of normal cells to Concanavalin A (Con A) in co-culture assays. The association between SC induction and anti-arthritic activity was determined by evaluating a series of chemically related azaspiranes in the AA rat model and for SC induction in normal rats. A statistically significant correlation was demonstrated (r = 0.79, P less than 0.001), indicating that SC induction may be responsible for the therapeutic activity of these compounds.