Intralesional cytokines in chronic oxazolone-induced contact sensitivity suggest roles for tumor necrosis factor alpha and interleukin-4.

An analysis was conducted of the cytokine profile and inflammatory response in oxazolone sensitized mouse skin. Following exposure to oxazolone, the intralesional production of inflammatory cytokines was demonstrable at the levels of both mRNA and protein. An initial challenge led to a transient increase in tumor necrosis factor-alpha production followed predominately by the T helper (Th)1 cytokine, interferon-gamma. There was a minimal production of interleukin-4, a Th2 cytokine. Continued exposure to oxazolone led to a downregulation of interferon-gamma and an upregulation of interleukin-4 production. A strong relationship was found between interleukin-4 and the inflammatory response, as measured by ear thickness. Similar experiments conducted in mast cell-deficient mice revealed reduced neutrophil influx but only minor changes in cytokine profile. An irritant response induced by chronic exposure of mouse skin to phorbol ester did not reveal any significant interferon-gamma or interleukin-4 response but was characterized by a tumor necrosis factor-alpha response that correlated with the inflammatory response. These observations suggest that the major source of interferon-gamma and interleukin-4 in the oxazolone response may be the infiltrating lymphocytes; whereas the tumor necrosis factor-alpha may result from the local irritation seen with both oxazolone and phorbol ester. At the end of 4 wk of chronic exposure to oxazolone, it was found that serum IgE levels had significantly increased. Histologic analysis of the skin lesion revealed that a mixed infiltrate including eosinophils developed upon repeat exposure to oxazolone. These findings are consistent with an early predominate Th1 response that is reduced and largely replaced with a Th2 response upon chronic T cell activation.

Bicyclic N-hydroxyurea inhibitors of 5-lipoxygenase: pharmacodynamic, pharmacokinetic, and in vitro metabolic studies characterizing N-hydroxy-N-(2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl)urea.

A series of N-hydroxyurea derivatives have been prepared and examined as inhibitors of 5-lipoxygenase. Oral activity was established by examining the inhibition of LTB4 biosynthesis in an ex vivo assay in the mouse. The pharmacodynamic performance in the mouse of selected compounds was assessed using an ex vivo LTB4 assay and an adoptive peritoneal anaphylaxis assay at extended pretreat times. Compounds with an extended duration of action were re-examined as the individual enantiomers in the ex vivo assay, and the (S) enantiomer of N-hydroxy-N-[2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl]urea, (+)-1a (SB 202235), was selected as the compound with the best overall profile. Higher plasma concentrations and longer plasma half-lives were found for (+)-1a relative to its enantiomer in the mouse, monkey, and dog. In vitro metabolic studies in mouse liver microsomes established enantiospecific glucuronidation as a likely mechanism for the observed differences between the enantiomers of 1a. Enantioselective glucuronidation favoring (-)-1a was also found in human liver microsomes.

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.

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.

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.

In vitro and in vivo pharmacological characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with anti-inflammatory activity.

Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.6 nM) and to membranes of RBL 2H3 cells expressing the LTB4 receptor (RBL 2H3-LTB4R; IC50 = 154 nM). This compound demonstrated competitive antagonism of LTB4- and 12-(R)-HETE-induced Ca2+ mobilization responses in human neutrophils (IC50s of 131 nM and 105 nM, respectively) and inhibited LTB4-induced Ca2+ mobilization in human cultured keratinocytes (IC50 = 61 nM), RBL 2H3-LTB4R cells (IC50 = 255 nM) and mouse neutrophils (IC50 = 410 nM). SB 201993 showed weak LTD4-receptor binding affinity (Ki = 1.9 microM) and inhibited 5-lipoxygenase (IC50 of 3.6 microM), both in vitro and ex vivo. In vivo, SB 201993 inhibited LTB4-induced neutrophil infiltration in mouse skin and produced dose-related, long lasting topical anti-inflammatory activity against the fluid and cellular phases of arachidonic acid-induced mouse ear inflammation (ED50 of 580 microg/ear and 390 microg/ear, respectively). Similarly, anti-inflammatory activity was also observed in the murine phorbol ester-induced cutaneous inflammation model (ED50 of 770 and 730 microg/ear, respectively, against the fluid and cellular phases). These results indicate that SB 201993 blocks the actions of LTB4 and 12-(R)-HETE and inhibits a variety of inflammatory responses; and thus may be a useful compound to evaluate the role of these mediators in disease models.

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.

Phlogistic activity of leukotriene D4 in the mouse.

An investigation of the phlogistic activity of LTD4 in the mouse was accomplished by examination of its ability to cause increased capillary permeability and edema formation following subcutaneous administration. It was observed that nanogram quantities of LTD4 caused edema and increased capillary permeability in a dose-related manner. The increase in capillary permeability was not inhibited by pretreatment with indomethacin and thus was unrelated to the production of cyclo-oxygenase products. These data suggest that LTD4 can mediate the edematous phase of the inflammatory response in the mouse and illustrate the sensitivity of this species to LTD4.

Effect of selective phosphodiesterase type IV inhibitor, rolipram, on fluid and cellular phases of inflammatory response.

The antiinflammatory activity of rolipram, a selective inhibitor of the cyclic AMP-specific phosphodiesterase (PDE IV), was studied. Rolipram did not inhibit 5-lipoxygenase activity but did inhibit human monocyte production of leukotriene B4 (LTB4, IC50 3.5 microM). Likewise, murine mast cell release of leukotriene C4 and histamine was inhibited. In vivo, rolipram inhibited arachidonic acid-induced inflammation in the mouse, while the low Km-cyclic-GMP PDE inhibitor, zaprinast, did not inhibit. Rolipram had a modest effect on LTB4 production in the mouse, but markedly reduced LTB4-induced PMN infiltration. Beta-adrenergic receptor activation of adenylate cyclase was important for rolipram antiinflammatory activity since beta blockade abrogated arachidonic acid-induced inflammation. Thus, the antiinflammatory profile of rolipram is novel and may result from inhibition of PMN function and perhaps vasoactive amine release and leukotriene biosynthesis. These actions may be dependent upon endogenous beta-adrenergic activity and are likely mediated through inhibition of PDE IV.

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.

Microprocessor-assisted plethysmograph for the measurement of mouse paw volume.

A sensitive and reproducible method of measuring mouse paw volume was developed by interfacing a Mettler DeltaRange top-loading balance with a microcomputer. This methodology combined ease of operation and precision with the advantages of computer-controlled data processing and archivable storage of data.

Analgetic activity of SK&F 105809, a dual inhibitor of arachidonic acid metabolism.

The analgetic activity of inhibitors of 5-lipoxygenase (5-LO) and cyclooxygenase (CO) was investigated using rat Randall-Selitto (RS) hyperalgesia and mouse phenylbenzoquinone (PBQ)-induced abdominal constriction assays. Using the RS assay, the CO inhibitors indomethacin, naproxen, and ibuprofen all effectively reduced hyperalgesia; whereas, the inhibitors of leukotriene production, MK886 and phenidone were inactive. SK&F 105809, a dual inhibitor of 5-LO/CO, significantly reduced hyperalgesia. In the PBQ assay, CO inhibitors were active, SK&F 105809 was nearly as potent as naproxen, and MK886 and phenidone were found to be active. Thus, improved analgetic activity appeared to result from inhibition of 5-LO and CO; whereas, in the RS assay, only CO inhibitors and SK&F 105809 were clearly effective. These results suggest that dual inhibitors, and in particular, SK&F 105809 may be more efficient analgesic agents than selective CO inhibitors in clinical situations in which 5-LO products play a significant role.

Inhibition of contact sensitivity in human CD4+ transgenic mice by human CD4-specific monoclonal antibodies: CD4+ T-cell depletion is not required.

Clenoliximab and keliximab are monkey/human chimeric monoclonal antibodies (mAbs) of the immunoglobulin G4 (IgG4) and IgG1 isotypes, respectively, that recognize the same epitope on human CD4. The two mAbs possess identical idiotypes and exhibit equal affinities for CD4. Upon administration of these mAbs to mice that express a human CD4 transgene, but not mouse CD4 (HuCD4/Tg mice), clenoliximab and keliximab exhibited similar kinetics of binding to CD4, and induced the same degree of CD4 modulation from the cell surface, although only keliximab mediated CD4+ T-cell depletion. Epicutaneous sensitization and challenge of HuCD4/Tg mice with the hapten oxazolone resulted in a contact sensitivity response characterized by tissue swelling, and the presence of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the local tissue. Administration of a single 2-mg dose of either clenoliximab or keliximab to HuCD4/Tg mice prior to sensitization significantly reduced post-challenge tissue swelling, and levels of IFN-gamma and IL-4, indicating that CD4+ T-cell depletion is not required for anti-CD4 mAb-mediated inhibition of contact sensitivity. Administration of either mAb prior to challenge failed to inhibit the contact sensitivity response, indicating differential sensitivity of the afferent and efferent phases of the response to inhibition by CD4-specific mAbs. Collectively, these data indicate that CD4 functions as a positive regulatory molecule in the contact sensitivity response.

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 were 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 cycloxygenase (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 profile as candidates for therapy of chronic inflammatory diseases, specifically those mediated by cytokines (e.g. IL-1, TNF) and eicosanoids.

Beneficial effects of SK&F 105685 in rat adjuvant arthritis: prophylactic and therapeutic effects on disease parameter progression.

Prophylactic administration of SK&F 105685 (N, N-dimethyl-8,8,-dipropyl-2-azaspiro[4.5]decane-2-propanamine dihydrochloride) at 30 mg/kg/day inhibited hindpaw lesions in adjuvant arthritic (AA) rats following 16 (84% inhibition) and 24 (70% inhibition) days of treatment. In a therapeutic protocol, where dosing was initiated on day 10 after disease induction, SK&F 105685 (20 mg/kg/day) effectively halted disease progression, and the inflammatory lesion was suppressed by 70% following treatment for 20 days. Histological evaluation of the joint periarticular soft tissue, bone and articulation (joint space and joint surface) from therapeutically treated rats showed a clear beneficial effect. Most rats presented moderate lesions rather than the severe lesions seen in the AA animals. AA control rats and AA rats treated prophylactically or therapeutically with SK&F 105685 had significant and similar increases in their total white blood cell (WBC), neutrophil, monocyte and platelet counts. Although increases were seen in the absolute number of neutrophils and platelets, there were no differences in the levels of leukotriene B4 (LTB4) and hydroxyheptadecatrienoic acid (HHT), per cell when these cells were stimulated with the calcium ionophore A23187. These results, describing the beneficial effects of SK&F 105685 administered therapeutically to the AA rat, indicate that this compound possesses properties desirable of an anti-arthritic agent and may potentially modify the disease outcome.

Effects of scalaradial, a type II phospholipase A2 inhibitor, on human neutrophil arachidonic acid mobilization and lipid mediator formation.

The role of scalaradial (SLD) (or its 12-epi analog), a marine natural product purified from sponge (Cacospongia mollior) in human neutrophil (polymorphonuclear leukocyte, PMN) arachidonic acid metabolism was studied. SLD potently inhibited human recombinant (rh) type II-14 kDa-phospholipase A2 (PLA2) (IC50 = 0.07 microM) but displayed weak inhibition of U937 cell, 85 kDa-PLA2 (IC50 = 20 microM). Sn-2 acylhydrolytic activity expressed in human PMN acid extract, that was completely neutralized by anti-rh type II-14 kDa-PLA2 monoclonal antibody, was inhibited by SLD in a concentration-dependent manner (IC50 = 35 microM). Exposure of human PMN to SLD resulted in a concentration-dependent inhibition of calcium ionophore (A23187)-induced leukotriene B4 release (IC50 = 0.1-0.6 microM). In contrast to the action of selective 5-lipoxygenase inhibitors (WY 50295 or zileuton), SLD decreased A23187-induced PMN liberation of arachidonic acid mass and platelet-activating factor biosynthesis (IC50 = 1-2 microM). PMN acetyltransferase activity was not significantly affected by SLD suggesting that platelet-activating factor inhibition was due, predominantly, to inactivation of PLA2 activity. In vivo, topical application of SLD on mouse ear treated with phorbol ester, not only inhibited edema formation but also the increase in myeloperoxidase activity (an index of cellular infiltration). SLD had little or no effect on arachidonic acid-induced ear edema or myeloperoxidase which is consistent with an action on PLA2. Take together these data suggest that the predominant mechanism of SLD is via inhibition of 14 kDa-like-PLA2 and suggests that this enzyme may participate in PMN arachidonic acid liberation and lipid mediator formation.

SB 207499 (Ariflo), a second generation phosphodiesterase 4 inhibitor, reduces tumor necrosis factor alpha and interleukin-4 production in vivo.

The ability of the second generation phosphodiesterase 4 inhibitor SB 207499 (Ariflo), [c-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)-r-l-cyclohexane carboxylic acid], to inhibit inflammatory cytokine production in vivo was evaluated and compared to that of rolipram, a first generation phosphodiesterase 4 inhibitor. To examine human tumor necrosis factor alpha (TNFalpha) production, human monocytes were adoptively transferred into Balb/c mice and challenged with lipopolysaccharide (LPS). In this model, SB 207499 inhibited human TNFalpha production with oral ED50 of 4.9 mg/kg. Similarly, R-rolipram inhibited human TNFalpha production with an ED50 of 5.1 mg/kg, p.o. In contrast to their equipotent activity against TNFalpha production, SB 207499 (ED50 = 2.3 mg/kg, p.o.) was 10-fold less potent than R-rolipram (ED50 = 0.23 mg/kg, p.o.) in reversing reserpine-induced hypothermia, a model of antidepressant activity. In time course studies, SB 207499 (30 mg/kg, p.o.) inhibited TNFalpha production for at least 10 hr; substantial plasma concentrations of SB 207499 were detected over the same interval. The ability of SB 207499 to modulate interleukin-4 production in vivo was assessed in a chronic oxazolone-induced contact sensitivity model in Balb/c mice. In this model, topical administration of SB 207499 (1000 microgram) inhibited intralesional concentrations of interleukin-4 (55%; P <.01). The results demonstrate that SB 207499 is a potent inhibitor of inflammatory cytokine production in a variety of settings in vivo. Moreover, although it is as potent as R-rolipram in inhibiting TNFalpha production, it has substantially less central nervous system activity. Thus SB 207499 represents an excellent candidate with which to evaluate the antiinflammatory potential of PDE4 inhibitors.

SB 239063, a p38 MAPK inhibitor, reduces neutrophilia, inflammatory cytokines, MMP-9, and fibrosis in lung.

The effects of a second generation p38 mitogen-activated protein kinase (MAPK) inhibitor, SB 239063 [trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridim idi n-4-yl)imidazole; IC(50) = 44 nM vs. p38 alpha], were assessed in models that represent different pathological aspects of chronic obstructive pulmonary disease (COPD) [airway neutrophilia, enhanced cytokine formation and increased matrix metalloproteinase (MMP)-9 activity] and in a model of lung fibrosis. Airway neutrophil infiltration and interleukin (IL)-6 levels, assessed by bronchoalveolar lavage 48 h after lipopolysaccharide (LPS) inhalation, were inhibited dose dependently by 3-30 mg/kg of SB 239063 given orally twice a day. In addition, SB 239063 (30 mg/kg orally) attenuated IL-6 bronchoalveolar lavage fluid concentrations (>90% inhibition) and MMP-9 activity (64% inhibition) assessed 6 h after LPS exposure. In guinea pig cultured alveolar macrophages, SB 239063 inhibited LPS-induced IL-6 production (IC(50) of 362 nM). In a bleomycin-induced pulmonary fibrosis model in rats, treatment with SB 239063 (2.4 or 4.8 mg/day via osmotic pump) significantly inhibited bleomycin-induced right ventricular hypertrophy (indicative of secondary pulmonary hypertension) and increases in lung hydroxyproline synthesis (indicative of collagen synthesis and fibrosis). Therefore, SB 239063 demonstrates activity against a range of sequelae commonly associated with COPD and fibrosis, supporting the therapeutic potential of p38 MAPK inhibitors such as SB 239063 in chronic airway disease.

Nonpeptide tachykinin receptor antagonists: I. Pharmacological and pharmacokinetic characterization of SB 223412, a novel, potent and selective neurokinin-3 receptor antagonist.

The in vitro and in vivo pharmacological profile of SB 223412 [(S)-(-)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carbo xamide], a novel human NK-3 (hNK-3) receptor antagonist, is described. SB 223412 demonstrated enantioselective affinity for inhibition of [125I][MePhe7]neurokinin B (NKB) binding to membranes of CHO cells expressing the hNK-3 receptor (CHO hNK-3). SB 223412, the (S)-isomer, (Ki = 1.0 nM), has similar affinity as the natural ligand, NKB (Ki = 0.8 nM) and another nonpeptide NK-3 receptor antagonist, SR 142801 (Ki = 1.2 nM). SB 223412 was selective for hNK-3 receptors compared with hNK-1 (>10,000-fold selective) and hNK-2 receptors (>140-fold selective), and selectivity was further demonstrated by its lack of effect, in concentrations up to 1 or 10 microM, in >60 receptor, enzyme and ion channel assays. SB 223412 enantioselectively inhibited the NKB-induced Ca++ mobilization in HEK 293 cells stably expressing the hNK-3 receptor. SB 223412 (10-1,000 nM) produced concentration-dependent rightward shifts in NKB-induced Ca++ mobilization concentration-response curves with a Kb value of 3 nM. In addition, SB 223412 antagonized senktide-induced contraction in the isolated rabbit iris sphincter muscle (Kb = 1.6 nM). In mice, oral administration of SB 223412 produced dose-dependent inhibition of behavioral responses induced by the NK-3 receptor-selective agonist, senktide (ED50 = 12.2 mg/kg). Pharmacokinetic evaluation of SB 223412 in rat and dog indicated low plasma clearance, oral bioavailability and high and sustained plasma concentrations after 4 to 8 mg/kg oral dosages. The preclinical profile of SB 223412 (high affinity, selectivity, reversibility and oral activity) suggests that it will be a useful tool compound to define the physiological and pathophysiological roles of NK-3 receptors.