Pyridinyl imidazoles inhibit IL-1 and TNF production at the protein level

The mechanism by which SK&F 86002 and other pyridinyl imidazoles inhibit the production of IL-1 and TNF from LPS-stimulated human monocytes was examined. Inhibition of IL-1 and TNF production was found to depend on the time of addition of SK&F 86002, with diminishing effect when added more than 2 h after LPS stimulation. Analysis of Western blots confirmed that both intracellular IL-1 and extracellular TNF were significantly reduced in response to SK&F 86002, but these reductions were not paralleled by changes in IL-1 and TNF mRNA.³⁵S methionine pulse and pulse-chase studies on IL-1 biosynthesis suggest that significant inhibition by SK&F 86002 and related compounds occurs at the translational level.     

Inhibition of CD44, CD45 and LFA-3 mediated cytokine release from human monocytes by SK&F 86002 and pentoxifylline

Compounds from two distinct pharmacological classes namely, SK&F 86002 and pentoxifylline, were examined for their effects on TNFα and IL-1β release by human monocytes stimulated with LPS or monoclonal antibodies to three cell surface glycoproteins, CD44, CD45 and LFA-3 (LFA-3 is also known as CD58). SK&F 86002, an inhibitor of 5-LO and CO in arachidonic acid metabolism, inhibited LPS-induced release of TNFα and IL-1β with an IC₅₀ of 1 μM. At this dose, it also inhibited by >50%, release of both cytokines induced by the three monoclonal antibodies. Pentoxifylline, a methylxanthine derivative with phosphodiesterase inhibitory activity, selectivity inhibited LPS-induced TNFα release with an IC₅₀ of 100 μM. TNFα and IL-1β release mediated by the monoclonal antibodies were inhibited by less than 30% in the presence of 100 μM pentoxifylline. These results suggest that (a) LPS induced cytokine release shares a common step with the physiologically relevant stimuli (involving cross-linking of cell surface receptors), and that this pathway is sensitive to inhibition by SK&F 86002 and, (b) SK&F 86002 is more potent than pentoxifylline in inhibiting TNFα and IL-1β release induced by both stimuli.     

Effect of SK&F 86002 on cytokine production by human monocytes

Human monocytes respond to a variety of stimuliin vitro by producing a number of physiologically important macromolecules including the cytokines. SK&F 86002, a dual inhibitor of the arachidonate metabolism, has been shown to inhibit LPS induced IL-1 production in human monocytes. We examined its effect on the production of other cytokines which are coordinately expressed as a result of LPS stimulation such as tumor necrosis factor alpha (TNF), alpha interferon (IFN-A), interferon beta-2 (IL-6) and granulocyte colony stimulating factor (g-CSF). The IC₅₀ of SK&F 86002 for the TNF production was 5–8 M, and >20M for the other three cytokines. These IC₅₀s were significantly higher than that previously reported for IL-1 production (1–2M). Taken together these data indicate that the inhibitory effect of SK&F 86002 on IL-1 production is selective and the production of cytokines in drug treated monocytes can be differentially affected.     

Inhibition of inflammatory cell infiltration by bicyclic imidazoles,SK&F 86002 and SK&F 104493

The mode of action of the dual inhibitors of eicosanoid metabolism, SK&F 86002 and SK&F 104493 was evaluated on inflammatory cell infiltration induced in mice by carrageenan, monosodium urate crystals, and arachidonic acid. The results were compared to those seen with standard antiinflammatory compounds. Inflammatory cell infiltration was inhibited by SK&F 86002, SK&F 104493, colchicine, and phenidone but not naproxen. In vivo, PMN infiltration induced by LTB₄ was inhibited by colchicine but not by SK&F 86002, SK&F 104493, or phenidone treatment. Similarly, in vitro chemotaxis to LTB₄ was not inhibited by SK&F 86002. The 5-lipoxygenase inhibitors, SK&F 86002, SK&F 104493, and phenidone inhibited LTB₄ production in vivo as well as inflammatory cell infiltration induced by arachidonic acid. The data are consistent with the suggestion that the bicyclic imidazoles inhibit PMN infiltration by virtue of inhibition of LTB₄ production.     

Differential effects of the bicyclic imidazoles on cytokine biosynthesis in human monocytes and endothelial cells

The effects of bicyclic imidazoles on human monocyte and endothelial cell cytokine production were examined. These compounds constitute the CSAID^TM class of anti-inflammatories and are inhibitors of cytokine biosynthesis. The bicyclic imidazoles differ from glucocorticoids and phosphodiesterase inhibitors in their chemical structure as well as pharmacological profile. At optimal concentrations of LPS (50 ng/ml), SK&F 86002, a prototypic compound, inhibited IL-1 and TNF but not g-CSF or IRAP production in human monocytes. At suboptimal concentrations of LPS (50 pg/ml), IL-6 and IL-8 production were also inhibited. Inhibition of cytokine biosynthesis was stimulus independent. For example, induction of IL-1 or TNF expression by phosphatase 1 and 2A inhibitors (Okadaic acid or Calyculin A) and Vitamin D3-dependent induction of IL-1 or TNF was also inhibited. In addition, IL-8 production, but not ICAM/E-Selectin expression in IL-1-stimulated HUVEC, was inhibited at similar IC₅₀s. Taken together, the bicyclic imidazoles inhibit cytokine production selectively in a stimulus and cell type independent manner.     

Effects of pyridinyl imidazole compounds on murine TNF-α production

The effects of SK&F 86002 and other pyridinyl imidazole compounds on murine cytokine production were investigated.In vitro, SK&F 86002 inhibited LPS stimulated TNF-α production by the RAW 264.7 cell line and by oil elicited peritoneal macrophages with an IC₅₀ of 5μM. In general, the activity was reflective of previous results obtained with human monocytes as SK&F 86002 and its analogs demonstrated identical rank order potency for TNF-α inhibition in both species. These compounds also inhibited TNF-αin vivo in a murine model of endotoxin shock. Following oral administration, SK&F 86002 and its analogs reduced serum TNF-α levels by >80% and afforded 100% protection from lethality. In contrast, tenidap, a novel anti-inflammatory drug, had minimal to no effect on murine TNF-α production in the same assays. These data further extend the pharmacological profile of the pyridinyl imidazoles by demonstrating that these compounds potently inhibit murine TNF-α production bothin vitro andin vivo.     

Pyridinyl imidazoles inhibit the inflammatory phase of delayed type hypersensitivity reactions without affecting T-dependent immune responses

The effects of pyridinyl imidazoles, specifically SK&F 105809 and its metabolite, on normal T-cell and B-cell mediated immune responses examined and compared to the fungal macrolide immunosuppressives, cyclosporin A, FK506 and rapamycin and to the corticosteroid, dexamethasone. The orally active prodrug SK&F 105809 {2-(4-methylsulfinylphenyl)-3(-4-pyridyl-6,7-dihydro-[5H]-pyrrolo [1,2-a] imidazole and its metabolite, SK&F 105561 {2-(4-methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole are dual 5-lipoxygenase (5-LO) and cyclxygenase (CO) inhibitors with potent anti-inflammatory and cytokine (IL-1/TNF) suppressive activities. The anti-inflammatory activity of SK&F 105809 and its metabolite were evaluated in an antigen-specific murine model of delayed type hypersensitivity (DTH) response, where they were found to affect only the inflammatory and not the induction phase of this response. In contrast, these compounds and other pyridinyl imidazoles (SK&F 86002 and its analog, SK&F 104351) exhibited no immunosuppressive activity under conditions where the macrolide rapamycin and the corticosteroid dexamethasone abrogated both the cellular and humoral immune responses. Thus, the ability of pyridinyl imidazoles to attenuate independently the inflammatory components of the disease without causing generalized immunosuppression enhances their profiles as candiadates for theraphy of chronic inflammatory diseases, specifically those mediated by cytokines (e.g. IL-1, TNF) and eicosanoids.     

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.     

The anti-inflammatory effect of the SOCC blocker SK&F 96365 on mouse lymphocytes after stimulation by Con A or PMA/ionomycin

SK&F 96365, 51-(beta-[3-(p-methoxyphenyl)-propyloxy]-p-methoxyphenethyl)-1H-imidazole hydrochloride, has emerged as a useful pharmacological tool in the study of store-operated Ca²⁺ entry (SOCE). But the precise molecular mechanism and effect of SK&F 96365 on mouse lymphocytes are still not well determined. This study investigated the pharmacological profile of SK&F 96365 on mouse lymphocytes stimulated by mitogen concanavalin A (Con A) or by a combination of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) and a calcium ionophore, ionomycin in vitro. Our results showed that SK&F 96365 pre-treatment diminished the cytosolic calcium rise on lymphocytes induced by ionomycin, PMA/ionomycin, and thapsigargin (TG), respectively. CFDA-SE staining results showed that SK&F 96365 (5-20 μM) inhibited both Con A- and PMA/ionomycin-induced lymphocytes proliferation in a time- and dose-dependent manner. Upon the same stimulation, SK&F 96365 inhibited the expression of CD69 and CD25 on CD3⁺ T lymphocytes in a dose-dependent manner. The cell cycle analyzing results showed that SK&F 96365 caused a G0/G1 phase cell cycle arrest on both Con A- and PMA/ionomycin-activated lymphocytes in a dose-dependent manner. In addition, SK&F 96365 induced a decrease in mitochondrial membrane potential (ΔΨm) and promoted mitochondrial permeability transition (MPT) in both Con A- and PMA/ionomycin-activated lymphocytes. Furthermore, SK&F 96365 significantly inhibited the production of proinflammatory cytokines (interferon (IFN)-γ and tumor necrosis factor (TNF)), and the anti-inflammatory cytokine (IL-10) on both Con A- and PMA/ionomycin-activated lymphocytes. SK&F 96365 did not induce a statistically significant increase in levels of proinflammatory IL-6 and monocyte chemoattractant protein-1 (MCP-1) but of IL-12p70 upon the stimulation of Con A, whereas these three cytokines were markedly inhibited by it upon the stimulation of PMA/ionomycin. This finding revealed that SK&F 96365 exhibited an anti-inflammatory effect on mouse lymphocytes both upon the stimulation of Con A and PMA/ionomycin, and the precise mechanism of SK&F 96365 inhibiting Con A-activated lymphocytes proliferation is different from PMA/ionomycin.     

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.     

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.Portions of this work were presented at the 70th Annual Meeting of the Federation of American Societies for Experimental Biology, St. Louis, MO, April 14, 1986, and the Second World Conference on Inflammation, Monte Carlo, March 19–22, 1986.     

Effects of pyridinyl imidazole compounds on murine TNF-α production

The effects of SK&F 86002 and other pyridinyl imidazole compounds on murine cytokine production were investigated.In vitro, SK&F 86002 inhibited LPS stimulated TNF- production by the RAW 264.7 cell line and by oil elicited peritoneal macrophages with an IC₅₀ of 5M. In general, the activity was reflective of previous results obtained with human monocytes as SK&F 86002 and its analogs demonstrated identical rank order potency for TNF- inhibition in both species. These compounds also inhibited TNF-in vivo in a murine model of endotoxin shock. Following oral administration, SK&F 86002 and its analogs reduced serum TNF- levels by >80% and afforded 100% protection from lethality. In contrast, tenidap, a novel anti-inflammatory drug, had minimal to no effect on murine TNF- production in the same assays. These data further extend the pharmacological profile of the pyridinyl imidazoles by demonstrating that these compounds potently inhibit murine TNF- production bothin vitro andin vivo.     

Induction of plasma exudation and inflammatory cell infiltration by leukotriene C4 and leukotriene B4 in mouse peritonitis

Leukotriene induction of the fluid and cellular phases of the inflammatory response in the mouse was evaluated. Intraperitoneal injection of leukotriene C₄ (LTC₄ 250 ng) led to dye extravasation but not polymorphonuclear leukocyte (PMN) infiltration, whereas injection of leukotriene B₄ (LTB₄ 250 ng), led to PMN infiltration but not dye extravasation. The injection of both leukotrienes did not result in synergy. LTC₄ did not appear to induce significant release or formation of chemotactic mediators, but the dye extravasation induced by LTC₄ was inhibited by the vasoactive amine antagonist cyproheptadine and not by the eicosanoid inhibitors phenidone or naproxen. The response was markedly inhibited by the cytokine and eicosanoid inhibitors SK&F 86002 and SK&F 104493. PMN infiltration induced by LTB₄ was not inhibited by SK&F 86002 or phenidone but was abrogated by colchicine treatment. LTB₄ in this model did not appear to cause release or formation of vasoactive mediators. These leukotrienes appeared to be independent, complementary, and sufficient to mount a complete inflammatory response in the mouse.     

Comparative biochemical and morphometric changes associated with induction of the hepatic mixed function oxidase system in the rat.

This study characterized the induction of the rat hepatic cytochrome P-450-dependent mixed function oxidase system by SK&F 86002 [6-(4'-fluorophenyl)-5-(4'-pyridyl)-2,3-dihydroimidazo-(2,1-b)thia zole], an inhibitor of both the cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism. The induction characteristics of SK&F 86002 were compared to those of the classical inducer, phenobarbital, and morphological features of both SK&F 86002 and phenobarbital induced hepatocellular hypertrophy were quantitated. Rats were administered either SK&F 86002 (6, 18, or 60 mg/kg/day, po) or phenobarbital (8, 24, 80 mg/kg/day, ip) for 3 or 14 consecutive days. Liver to body weight ratio, total hepatic microsomal protein and cytochrome P-450 content, ethoxycoumarin-O-deethylase (ECOD) and leukotriene B4(LTB4) omega- and omega-1 hydroxylase were measured. Ultrastructural morphometry of the liver from control, and high dose SK&F 86002 (60 mg/kg/day) and phenobarbital (80 mg/kg/day) treated rats was completed. On day 3, phenobarbital increased liver to body weight ratio but only at the 80 mg/kg/day dosage; microsomal protein content was unchanged. ECOD activity increased in a dose-dependent fashion. LTB4 omega- and omega-1 hydroxylase activities were unaffected. Administration of SK&F 86002 for 3 days increased the liver to body weight ratio at both the 18 and 60 mg/kg/day dosage; microsomal protein content was unchanged. ECOD activity was significantly increased by the 60 mg/kg/day dosages of SK&F 86002. On day 14, phenobarbital increased the liver to body weight ratio and microsomal protein content but again only at the 80 mg/kg/day dosage. Cytochrome P-450 content was increased by all dosages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of SK&F 105685, a novel anti-arthritic agent,on immune function in the dog

SK&F 105685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4,5]decane-2-propanamine dihydrochloride) is a novel azaspirane with beneficial activity in animal models of autoimmune disease such as adjuvant-induced arthritis and experimental encephalomyelitis in the Lewis rat and lupus-like disease in the MRL mouse. The activity of SK&F 105685 in these models is associated with the induction of non-specific suppressor cell (SC) activity as defined by the ability of cells from drug-treated animals to inhibit the proliferative response of lymphocytes from control animals to concanavalin A. To evaluate the immunotoxicologic potential of SK&F 105685, the effect on immune function of one month of dosing with 1 mg/kg/day of SK&F 105685 was examined in the dog. Differential blood cell counts and ex vivo immune function assays were performed using blood collected before dosing on days 1 (baseline), 15 and 29, of the study. Immune function assays were performed on spleen cells on day 30. Under the conditions of the study, SK&F 105685 displayed pharmacological activity as demonstrated by the induction of splenic SC activity. The drug did not affect the total number or relative percentages of the various white blood cell types present in peripheral blood and did not cause generalized immunosuppression. The ability of peripheral blood lymphocytes or spleen cells to produce IL-2 or proliferate in response to mitogenic stimulation was not affected by drug treatment. SK&F 105685 also failed to affect the candidacidal activity of polymorphonuclear leucocytes and spleen cells indicating that it is unlikely to compromise nonspecific resistance to infection. SK&F 105685 however, was able to inhibit the generation of a specific in vitro antibody response to sheep red blood cells (SRBC) by splenocytes from treated animals. Inhibition of the anti-SRBC antibody response was also observed upon addition of the drug to normal spleen cells. Addition of the drug at different time points during the culture period indicated that SK&F 105685 was interfering with an event(s) occurring during the first 72 h of culture. Taken together, these results suggest that, in a therapeutic setting, SK&F 105685 in unlikely to compromise the immune status of the host as it can down-regulate a specific immune response without causing generalized immunosuppression.     

Effects of SK&F 93479 on experimentally induced ventricular arrhythmias in dogs,rats and mice

The effects of SK&F 93479, a potent histamine H₂-receptor antagonist, on ventricular arrhythmias induced by coronary artery ligation in dogs and rats, and by aconitine infusion in mice were investigated. It was found that SK&F 93479 in large doses, significantly prevented the occurrence of spontaneous ventricular fibrillation and the changes in ventricular fibrillation threshold following coronary artery ligation in dogs. In rats subjected to ligation of the main left coronary artery, it significantly reduced the incidence of ventricular fibrillation, and significantly prolonged the time of onset of ventricular tachycardia and ventricular fibrillation. On the contrary, SK&F 93479 did not significantly alter the incidence or the time of onset of cardiac dysrhythmias caused by aconitine infusion in mice. These findings suggest that SK&F 93479 lacks non-specific antiarrhythmic activity and that its protective effects against coronary artery ligation may be mediated by its histamine H₂-receptor antagonizing action. They also support the hypothesis that histamine may contribute to the genesis of ventricular arrhythmias resulting from acute myocardial ischaemia.     

The Eft of Dual Inhibitor, Sk&F 86002, On Helper T Cell Functions

SK&F 86002, a mixed cyclooxygenase-lipoxygenase inhibitor, was examined for its effects on helper T cell functions. The drug was found to inhibit concanavalin A-induced mitogenesis of splenic T cells (IC₅₀ = 13 mμm), the mixed lymphocyte response (IC₅₀ = 16 mμM), and proliferation of antigen specific T cells (cloned line, IC₅₀ = 11 mμM; uncloned line, IC₅₀ = 13 mμM). In contrast, another mixed cycloxygenase-lipoxygenase inhibitor, BW775c, did not have such effects at non-cytotoxic levels. These T cell functions are believed to be dependent on the effects of elaborated IL-1. SK&F 86002 has been shown to inhibit the production of mature IL-1 (IC₅₀ = 1 mμM), possibly accounting for the anti-inflammatory effects of the drug in rheumatoid arthritis models. In an in vivo model of contact sensitivity, SK&F 86002 was able to inhibir mouse footpad swelling, demonstrating additional anti-inflammatory activity. As an inhibitor of IL-1 synthesis or release, SK&F 86002 may be useful for the treatment of T cell-dependent inflammation.