Don E. Griswold

Inflammatory mediators of experimental colitis in rats.

Colonic inflammation was induced in rats by intracolonic administration of 0.25 ml of 50% ethanol containing 30 mg of trinitrobenzene sulfonic acid (TNB). Control rats were treated with 0.25 ml of 50% ethanol or with 30 mg of TNB in 0.25 ml of saline. After 24 h, mucosal ulceration and hemorrhage were observed in TNB/ethanol-, 50% ethanol-, and to a lesser extent, in TNB/saline-treated rats. After 1 wk, mucosal damage was completely resolved in the 50% ethanol and TNB/saline-treated rats but the lesions in the TNB/ethanol-treated rats persisted and progressed to a chronic active inflammatory process after 3 wk. Myeloperoxidase activity was significantly elevated in mucosal scrapings from all treatment groups at all time intervals when macroscopic and microscopic mucosal injury was evident. Interleukin-1 was found to be the most sensitive indicator of mucosal inflammation, and its mucosal values correlated with myeloperoxidase activity. Leukotriene B4 was increased in control rats at 1 wk and in TNB/ethanol-treated rats at all time intervals. The maximal increase in leukotriene B4 was observed at 1 wk. Thromboxane B2 generation was reduced while platelet activating factor generation was not increased in TNB/ethanol-treated rats. These results indicate that in this TNB/ethanol model of gut inflammation, myeloperoxidase activity and interleukin-1 are reliable and sensitive indicators of colonic inflammation, and that thromboxane B2 is not involved in the acute lesions, whereas leukotriene B4 appears in the chronic active inflammatory response.

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.

Biologic actions and pharmacokinetic studies of auranofin

The preclinical profiles of auranofin (Ridaura), an oral chrysotherapeutic agent, parenteral gold sodium thiomalate, gold thioglucose, and their respective ligands were compared. Auranofin was more effective than gold sodium thiomalate in suppressing inflammation and stimulating cell-mediated immunity. In contrast to gold sodium thiomalate and gold thioglucose, auranofin inhibited cellular release of lysosomal enzymes, antibody-dependent cellular cytotoxicity, production of antibodies in adjuvant arthritic rats, and antibodies involved in cytotoxicity reactions. The respective ligands were without significant biologic activity. In rats, a higher fraction of gold was associated with blood cells after auranofin administration than after gold sodium thiomalate. The absorption, distribution, metabolism, and excretion of auranofin are uniquely different from other gold compounds.

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.

Method for quantification of myocardial infarction and inflammatory cell infiltration in rat cardiac tissue

A method to quantitate both creatine phosphokinase (CPK) and myeloperoxidase (MPO) activity from the same cardiac tissue homogenate preparation is described. Depletion of CPK specific activity is used to quantitate myocardial infarct size, while MPO activity is utilized as a marker for polymorphonuclear leukocyte and monocyte infiltration into inflammatory sites. However, the standard assay systems are not compatible, necessitating the use of different groups of animals for these two parameters. This leads to an increase in cost, effort, and variability. The described method utilized a standard CPK methodology. It was found that interference in the MPO assay was likely caused by 2-mercaptoethanol present in the homogenate buffer (IC50 = 90 microM). Washing of the 30 K X g pellet followed by rehomogenization restored the MPO activity. Negligible MPO activity was found in the original supernatant or washes. Through the use of this technique, MPO activity was measured in the hearts of myocardial infarcted animals. The results indicated that MPO activity generated from CPK homogenate pellets compared favorably to the activity seen using standard methodology homogenates. The procedure described thus allowed the simultaneous determination of myocardial CPK specific activity and MPO activity, resulting in decreased animal usage and potentially less variability.

Immunopharmacology of gold sodium thiomalate and auranofin (SK&F D-39162): Effects on cell-mediated immunity

The effects of gold sodium thiomalate (GST) and auranofin (SK&F D-39162) on cell-mediated immunity were investigated using oxazolone-induced contact sensitivity and delayed hypersensitivity to sheep red blood cells. C57Bl mice were sensitized to oxazolone on day 0 and challenged either 45 or 72 h later. The resulting paw edema was read plethysmographically 24 h after challenge. GST and auranofin both were capable of stimulating oxazollone-induced contact sensitivity which was compromised by using a shortened sensitization period (45 h). Auranofin but not GST stimulated the response to oxazolone in immunosuppressed mice, but neither agent significantly altered the uncompromised response in normal mice. The stimulatory effect of auranofin and GST on cell-mediated immunity was corroborated using SRBC to induce delayed hypersensitivity. Comparison of blood Au levels revealed that gold in the form of auranofin was approximately 4 × more effective in stimulating cell-mediated immunity than was gold in the form of GST. These results were suggested to be due to the possible stimulation by gold of T effector as well as T suppressor lymphocytes, thus explaining the condition dependency of the immunoregulation.

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.

Evaluation of the effect of Evan's blue and triphenyltetrazolium chloride dyes on myeloperoxidase activity in canine cardiac tissue

Myeloperoxidase (MPO) activity in postinfarction, dual-stained canine tissue in the presence of Evans Blue (EB) and Triphenyltetrazolium chloride (TTC) was evaluated. Perfusion of EB and TTC allows quantification of the area of necrosis, area-at-risk of infarction and noninvolved, normal tissue postinfarction. EB in cardiac tissue has been reported to interfere with MPO activity used to measure polymorphonuclear leukocyte (PMN) infiltration, thus requiring that infarct size and MPO activity be measured in separate groups of animals. Admixtures of EB- or TTC-stained canine cardiac tissue extracts with MPO homogenates were found to have similar MPO activity. Addition of a constant amount of EB- or TTC-stained tissue to a standard curve of MPO activity failed to influence the concentration-activity relationship. Furthermore, EB in the presence of inflammatory cell infiltration in vivo in the mouse did not alter MPO activity. Thus, neither EB nor TTC significantly interfered with the measurement of MPO activity so that EB and/or TTC-stained tissue can be utilized to examine the role of PMNs in myocardial infarction.