Design, synthesis, and biological activity of diiminoisoindolines as complement component 3a antagonists.

The failure to fully regulate the inflammation response has been linked to diseases such as rheumatoid arthritis, septic shock syndrome, and asthma. The human complement system initiates and regulates the inflammation response through a cascade of regulatory factors. Complement Component 3a (C3a) is an essential regulatory factor and inhibiting its binding to a C3a receptor will diminish the inflammation response by disrupting the cascade. We report the design, synthesis, in vitro and in vivo activity of diiminoisoindolines as C3a antagonists.

Increased expression of serine palmitoyltransferase (SPT) in balloon-injured rat carotid artery.

The response to vascular injury is a complex wound healing response involving cell proliferation, migration, remodeling and inflammation. In the present studies we employed a rat balloon angioplasty model of vascular injury to investigate the potential role of sphingolipid signaling in the response to vascular injury. The enzyme serine palmitoyltransferase (SPT) catalyzes the first committed step in de novo sphingolipid biosynthesis. We observed marked upregulation of expression of both SPT subunits in actively proliferating cells in injured vessels. This enhanced SPT expression occurs in de-differentiated fibroblasts and proliferating vascular smooth muscle cells. The upregulation is particularly apparent in the proliferating luminal edge of the neointima and the adventitial de-differentiated fibroblasts and may serve as a hallmark of this process. The possible functional consequences of this enzyme upregulation and its role in the response to vascular injury are suggested but remain to be determined.

1,3-Diarylcycloalkanopyrazoles and diphenyl hydrazides as selective inhibitors of cyclooxygenase-2.

Novel 1,3-diarylcycloalkanopyrazoles 1, and diphenyl hydrazides 2 were identified as selective inhibitors of cyclooxygenase-2. The 1,3-diaryl substitution pattern of the pyrazole ring in 1 differentiates these compounds from most of the known selective COX-2 inhibitors that contain two aryl rings at the adjacent positions on a heterocyclic or a phenyl ring. Similarly, the two phenyl rings in 2 are also separated by three atoms. SAR of both phenyl rings in 1 and 2, and the aliphatic ring in 1 will be discussed.

N-hydroxyurea and hydroxamic acid inhibitors of cyclooxygenase and 5-lipoxygenase.

Two series of compounds (1 and 2) having structural features of the dual COX/5-LO inhibitor tepoxalin and the 5-LO inhibitor ABT-761 were prepared. Many of these hybrid compounds are potent COX and 5-LO inhibitors; two compounds (1a and 2t) inhibit eicosanoid biosynthesis in an ex vivo assay.

Monitoring Inositol-Specific Phospholipase C Activity Using a Phospholipid FlashPlate(R).

Inositol-specific phospholipase Cs(PLCs) are a group of enzymes involved in the signal transduction pathway of many plasma membrane receptor mediated events. We developed a modified solid surface to capture [(3)H] PIP(2) onto the Basic FlashPlate(R) in order to monitor PLC activity. Our results clearly demonstrate the utility of [(3)H] PIP(2)-Coated Phospholipid FlashPlate(R) microtiter plates for assessing PLC activity for HTS of receptor-coupled functional assays. The results show that PLC activity can be measured easily from a variety of sources including purified recombinant enzyme preparations, crude HL60 cell lysates and permeabilized A431 human carcinoma cells. Moreover, this format provides a surface comparable to that used for classical solution based radiolabeled mixed phospholipid micelle studies and illustrates the feasibility of this assay for measuring PLC activation in a variety of different drug screening assays.

Evaluation of the antiinflammatory activity of a dual cyclooxygenase-2 selective/5-lipoxygenase inhibitor, RWJ 63556, in a canine model of inflammation.

Sterile perforated polyethylene spheres (wiffle golf balls) were implanted s.c. in beagle dogs. A local inflammatory reaction was elicited within the spheres by injecting carrageenan. Changes in leukocyte count, prostaglandin E2, thromboxane B2 and leukotriene B4 levels were monitored in fluid samples collected over a 24-hr period. Blood samples were also collected at various time points and analyzed for prostaglandin E2 and leukotriene B4 production after ex vivo calcium ionophore treatment. Effects of standard antiinflammatory agents (aspirin, indomethacin, dexamethasone, tenidap and zileuton) and newer cyclooxygenase-2 (COX-2) selective agents (nimesulide, nabumetone and SC-58125) were determined after oral administration. Ex vivo inhibition of cyclooxygenase product synthesis (prostaglandin E2, thromboxane B2) in whole blood was used as an indicator of activity for the constitutive COX-1 isoform, although inhibition of the synthesis of these mediators in the chamber exudate during an inflammatory process is believed to represent COX-2 inhibition. Treatment effects on leukotriene B4 production were also determined both ex vivo in whole blood and in the fluid. All of the compounds tested, except aspirin, inhibited leukocyte infiltration into the fluid exudate. Inhibitors that exert their effects on both isozymes of cyclooxygenase attenuate production of cyclooxygenase metabolites in both the inflammatory exudate and in peripheral blood ex vivo, although COX-2 selective inhibitors only demonstrated activity in the exudate. A 5-lipoxygenase inhibitor (zileuton), a corticosteroid (dexamethasone) and a dual COX-2 selective/5-lipoxygenase inhibitor (RWJ 63556) had similar profiles in that they all inhibited cell infiltration and eicosanoid production in the fluid and also attenuated leukotriene B4 production in both the fluid and blood.

Effects of tepoxalin, a dual inhibitor of cyclooxygenase/5-lipoxygenase, on events associated with NSAID-induced gastrointestinal inflammation.

Prostaglandins and thromboxanes are products of arachidonic acid metabolism via the cyclooxygenase (CO) enzyme and are responsible for the pain and swelling common to sites of inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the production of these substances and are used in the treatment of inflammatory diseases such as arthritis. However, one of the major side-effects of NSAID therapy is gastric ulceration. It is possible that inhibition of prostaglandin production and a related increase in the formation of leukotrienes via the 5-lipoxygenase (5-LO) enzymatic pathway are responsible for attracting inflammatory cells, causing local sites of inflammation and producing ulceration. To determine the effects of 5-LO inhibition on this hypothesis, studies were performed in rats to evaluate the effects of tepoxalin, a dual CO/LO inhibitor on leukotriene B4 levels in gastric mucosa and neutrophil adhesion in mesenteric venules. In rats, chronic oral administration of an NSAID, indomethacin (2 mg/kg daily over 4 days), resulted in 40% mortality, accompanied by intestinal adhesions and perforations when evaluated 24 h after the fourth dose of drug. Additionally, neutrophil adhesion was increased in the mesenteric venules and cell infiltration was evident in the mesenteric interstitium. These gastrointestinal side-effects were inhibited in a separate group of rats administered tepoxalin (20 mg/kg, p.o) 30 min prior to each daily indomethacin treatment. Further studies were performed to determine tepoxalin's effects on early events associated with NSAID-induced gastrointestinal inflammation, including neutrophil adhesion, lipid peroxide generation and LTB4 production. Indomethacin (100 mg/kg, p.o.) produced elevated levels of LTB4 in rat gastric mucosa 90 min after administration. Additionally, neutrophil adhesion in mesenteric venules was increased at this dose and with the administration of another NSAID, naproxen. No generation of lipid peroxides was evident in the gastric mucosa at this timepoint. Tepoxalin (up to 400 mg/kg, p.o.) did not have an effects on gastric mucosal LTB4 generation and lipid peroxide levels. A decrease in neutrophil adhesion was observed at the highest dose. In another study, pretreatment with tepoxalin (ED50=7.5 mg/kg, p.o.) or the selective 5-LO inhibitor zileuton (100 mg/kg, p.o.) prevented the increases in gastric mucosal LTB4 levels and neutrophil adhesion induced by indomethacin (100 mg/kg, p.o.). These data suggest that LO inhibition may play a vital role in the prevention of NSAID-induced gastric inflammation, providing insight into the lack of ulcerogenicity with tepoxalin and new approaches to anti-inflammatory therapy which may prevent gastric side effects.

N-(5-substituted) thiophene-2-alkylsulfonamides as potent inhibitors of 5-lipoxygenase.

Compound 4k N-[5-(4-fluoro)phenoxythien-2-yl]methanesulfonamide is representative of a new class of potent inhibitors of 5-lipoxygenase (5-LO). These versatile compounds exhibit dose-dependent inhibition of 5-LO with IC50s ranging from 20-100 nM in the rat basophilic leukemia (RBL-1) cell homogenate assay and submicromolar IC50s in both the RBL-1 and human peripheral blood leukocyte (PBL) whole cell assays. Compound 4k also showed significant anti-inflammatory activity in the adjuvant arthritic rat at an oral dose of 3 mg/kg.

Pharmacokinetics and pharmacodynamics of tepoxalin after single oral dose administration to healthy volunteers.

This study was conducted to examine the pharmacokinetics and pharmacodynamics of tepoxalin in healthy volunteers, an antiinflammatory compound that inhibits cyclooxygenase and lipoxygenase. Tepoxalin was absorbed after oral administration of single doses from 35 to 300 mg, after which it was rapidly converted to an acidic metabolite, RWJ 20142, which inhibits cyclooxygenase but not lipoxygenase. The areas under the concentration-time curve (AUC) of tepoxalin and RWJ 20142 in plasma increased in a dose-dependent fashion. Administration of the lowest dose of tepoxalin completely inhibited whole blood cyclooxygenase for the entire period of observation. This inhibition correlated closely with that of secretion and aggregation induced by collagen of platelets obtained from these subjects. Similarly, administration of tepoxalin was associated with significant inhibition of lipoxygenase in whole blood. Lipoxygenase was inhibited a maximum of 60% in a time-dependent fashion, and the duration of inhibition was dose-dependent. These studies demonstrate that tepoxalin inhibits whole blood cyclooxygenase, lipoxygenase, and platelet function after oral administration in humans.

Cytokine-modulating activity of tepoxalin, a new potential antirheumatic.

Tepoxalin is a new dual cyclooxygenase/5-lipoxygenase anti-inflammatory compound currently under clinical investigation. It has been shown to possess anti-inflammatory activity in a variety of animal models and more recently to inhibit IL-2 induced signal transduction. The current study was conducted to evaluate the cytokine modulating activity of tepoxalin and the role of iron in these effects. In human peripheral blood mononuclear cells (PBMC) stimulated with OKT3/PMA, tepoxalin inhibited lymphocyte proliferation with an IC50 of 6 microM. Additionally, it inhibited the production of LTB4 (IC50 = 0.5 microM) and the cytokines IL-2, IL-6 and TNF alpha (IC50 = 10-12 microM). Cytotoxicity was not demonstrated at these concentrations. Add-back experiments with either cytokines (IL-2 or IL-6), LTB4 or conditioned media failed to restore the proliferative response in the presence of tepoxalin. However, the concurrent addition of iron (in the form of ferrous or ferric chloride and other iron salts) reversed the inhibition of proliferation caused by tepoxalin. Tepoxalin also inhibits the activation of NF kappa B, a transcription factor which acts on several cytokine genes. Tepoxalin's effect on NF kappa B is also reversed by the addition of iron salts. These data suggest that the action of tepoxalin to inhibit proliferation in PBMC may be at least in part due to its ability to reduce the amount of available iron resulting in decreased activation of NF kappa B and subsequent inhibition of cytokine production.

A primary immunodeficiency syndrome in Shar-Pei dogs.

A multiple immunodeficiency, involving antibody- and cell-mediated responses in 10 Chinese Shar-Pei (CSP) dogs is described. Abnormal levels of serum IgM and IgA in most cases, and IgG in fewer cases characterized the immunoglobulin deficiencies. Decreased in vitro proliferative responses of pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC) were found in nine cases. Clinical presentation involved several organ systems and was associated with recurrent infections and malignancy. Sera from affected dogs suppressed PWM-stimulated cell proliferation of affected and normal dogs, but not cultures stimulated with PWM followed by recombinant IL-2 (rIL-2). In vitro supplementation of PBMC cultures with immunomodulatory guanosine analogs (GA) resulted in increased de novo IgG and/or interleukin-6 (IL-6) synthesis. Cells from five immunodeficient dogs showed in vitro evidence of GA- or rIL-2-dependent enhanced immunological responses. Since rIL-2-mediated activation of the IL-2 receptor and GA-mediated immunomodulation are reported to act through protein kinase C (PKC)-independent pathways, it is concluded that the IL-2 receptor is functional in these dogs and that cell activation through alternative pathways may restore immune responses in affected CSP dogs.

Tepoxalin, a novel immunosuppressive agent with a different mechanism of action from cyclosporin A.

Tepoxalin, a compound previously identified as a dual cyclooxygenase/lipoxygenase (CO/LO) inhibitor, is a potent inhibitor of T cell proliferation. Comparing the suppressive effects of tepoxalin and cyclosporin A (CsA) on OKT3-, PMA-, IL-2-, and PMA+ionomycin-induced T cell proliferations revealed marked differences in the mechanism of action between the two compounds. Whereas CsA was most effective in suppressing OKT3-stimulated proliferation, tepoxalin was more potent in inhibiting PMA-, PMA+ionomycin-, and IL-2-induced proliferation. Quantitative PCR (QPCR) assays used to detect cytokine messages showed that tepoxalin blocked IL-2 mRNA transcription in PMA- and PMA+ionomycin-, but not OKT3-stimulated T cells whereas CsA was most potent in inhibiting OKT3-induced IL-2 mRNA induction in these cells. Both tepoxalin and CsA did not inhibit the expression of IL-2R; however, only tepoxalin, but not CsA, inhibited the proliferation of IL-2-dependent blasts and the transcription of IFN-gamma, an IL-2-dependent target gene. Moreover, addition of exogenous IL-2 restored OKT3-induced proliferation to CsA- but not tepoxalin-treated cells. These data suggest that tepoxalin, but not CsA, suppressed T cell proliferation by inhibiting IL-2-induced signal transduction. Consistent with these findings, tepoxalin, unlike CsA, which was most potent when added at the initiation of OKT3 stimulation, was equally active, regardless of whether it was added at the beginning or 48 h after culture initiation. The difference in mechanism of action between tepoxalin and CsA was confirmed further by the synergistic suppressive effects on T cell proliferation upon co-administration of the two compounds.

Tepoxalin: a dual cyclooxygenase/5-lipoxygenase inhibitor of arachidonic acid metabolism with potent anti-inflammatory activity and a favorable gastrointestinal profile.

Tepoxalin [5-(4-chlorophenyl)-N-hydroxy-(4-methoxyphenyl)-N-methyl-1H- pyrazole-3-propanamide] is a potent inhibitor of sheep seminal vesicle cyclooxygenase (CO) (IC50 = 4.6 microM), rat basophilic leukemia cell (RBL-1) lysate CO (IC50 = 2.85 microM) and CO from intact RBL-1 cells (IC50 = 4.2 microM). The compound inhibits the production of thromboxane B2 (TxB2) in Ca++ ionophore A-23187-stimulated human peripheral blood leukocytes (HPBL; IC50 = 0.01 microM) and human whole blood (IC50 = 0.08 microM) and is a potent inhibitor of epinephrine-induced human platelet aggregation (IC50 = 0.045 microM). Tepoxalin inhibits lipoxygenase (LO) in RBL-1 lysates (IC50 = 0.15 microM) and intact RBL-1 cells (IC50 = 1.7 microM) and inhibits the generation of leukotriene B4 (LTB4) in calcium ionophore A-23187-stimulated HPBL (IC50 = 0.07 microM) and human whole blood (IC50 = 1.57 microM). Human platelet 12-LO (IC50 = 3.0 microM) is inhibited, but 15-LO is only weakly so (IC50 = 157 microM). In vivo, tepoxalin, administered orally, demonstrated potent anti-inflammatory activity in the established adjuvant arthritic rat (ED50 = 3.5 mg/kg) and potent analgesic activity in the acetic acid abdominal construction assay in mice (ED50 = 0.45 mg/kg). In an ex vivo whole blood eicosanoid production assay, tepoxalin produces a dose-related inhibition of prostaglandin (PG) and LT production in dogs (PGF2 alpha - ED50 = 0.015 mg/kg; LTB4 - ED50 = 2.37 mg/kg) and adjuvant arthritic rats following oral administration. In adjuvant arthritic rats, tepoxalin is devoid of ulcerogenic activity within its anti-inflammatory therapeutic range (1-33 mg/kg p.o.) and does not exhibit ulcerogenic activity in normal rats at doses lower than 100 mg/kg (UD50 = 173 mg/kg p.o.). Tepoxalin represents a new class of anti-inflammatory drugs which may exhibit less gastrointestinal toxicity and may be efficacious in immunoinflammatory disease states where excessive PG and LT production has been implicated and may offer a significant alternative to nonsteroidal and corticosteroidal anti-inflammatory therapy.

Small-molecule immunostimulants. Synthesis and activity of 7,8-disubstituted guanosines and structurally related compounds.

A series of 7,8-disubstituted guanosine derivatives was designed and prepared as potential B-cell-selective activators of the humoral immune response. These compounds were evaluated for their ability to act as B-cell mitogens and to augment the antibody response of B cells to sheep red blood cell (SRBC) challenge (adjuvanticity). In addition, they were tested for their ability to stimulate the natural killer (NK) cell response in murine in vitro cell assays. Certain of the compounds demonstrated in vivo activity when administered either intravenously, subcutaneously, or orally. Analogues with a medium-length alkyl chain (2-4 carbons, 5-7) on the 7-position of 7-alkyl-8-oxoguanosines were found to be particularly potent. Compounds bearing hydroxyalkyl, aminoalkyl, or substituted aminoalkyl substituents on this 7-position were weakly active. However, benzyl groups, including those substituted with heteroatoms (e.g., p-nitrobenzyl, 14), were active. Oxo, thioxo, and seleno groups on C-8 of the guanosine ring all imparted strong activity, whereas other larger substituents did not (e.g., N = CN). Stereochemical inversion of the 2'-hydroxyl on the ribose ring in this series, giving arabinose analogue 70, lessened activity. However, removal of the 2'-hydroxyl, either with (64) or without (73) removal of the 3'-hydroxyl, resulted in excellent activity and improved solubility; 64 also displayed good oral in vivo activity as well. A series of ketals involving the 2',3'-hydroxyls were prepared; certain of the nonpolar ketals (e.g., 48) were remarkably active, pointing to an ancillary hydrophobic binding region that can augment activity. 5'-Phosphate derivative 57 was fairly active, and acyclovir analogue 90 displayed good NK-selective activity: other N-9 sugar mimetics were also active (97-104), although this activity did not carry over into the human B-cell assay. A total of 80 compounds were prepared and evaluated for their immunostimulating activity. Within this group, compounds could be divided into those that were active in all three assays, those that displayed some measure of selectivity for the adjuvanticity assay, and those that preferentially activated NK responses. Because of its overall biological profile and ease of synthesis, 7-allyl-8-oxoguanosine (6; loxoribine, RWJ-21757) was chosen for further development. It is among the most potent compounds evaluated in the three biological assays.

Synthesis and 5-lipoxygenase inhibitory activities of some novel 2-substituted 5-benzofuran hydroxamic acids.

A series of 2-substituted benzofuran hydroxyamic acids were synthesized as rigid analogs of simple (benzyloxy)phenyl hydroxamates, evaluated for their in vitro and in vivo 5-lipoxygenase activity and found to be potent inhibitors of the enzyme. Substituents which enhanced lipophilicity near the 2-position of the benzofuran nucleus increased inhibitor potency but reduced oral activity. Incorporation of small polar substituents such as methoxymethylene, hydroxymethylene, and amino (urea) on the acyl group led to more consistent oral activity. The most potent inhibitors of this series in vitro were N-hydroxy-N-[1-(2-phenyl-5-benzofuranyl)-ethyl]furancarboxamide (12) and methyl 5-[N-hydroxy-N-[1-(2-(3,4,5-trimethoxyphenyl)-5-benzofuranyl]ethyl]-5- oxopentanoate (17), both with IC50 values of 40 nM, and in vivo the most potent compound was N-hydroxy-N-[1-(2-phenyl-5-benzofuranyl)ethyl]urea, 20, with an ED50 = 10.3 mg/kg.

Tissue-selective inhibition of prostaglandin synthesis in rat by tepoxalin: anti-inflammatory without gastropathy?

BACKGROUND: Inhibition of prostaglandin synthesis is likely a primary mechanism for both the anti-inflammatory and ulcerogenic effects of nonsteroidal anti-inflammatory drugs (NSAIDs). The present study examined the mechanism underlying the ability of a novel anti-inflammatory drug, tepoxalin, to suppress prostaglandin synthesis without inducing gastric mucosal injury. METHODS: The effects on prostaglandin synthesis by various tissues of tepoxalin, diclofenac, and indomethacin were examined in vivo and in vitro. These compounds were also studied in two inflammation models. The capacity of indomethacin and tepoxalin to induced antral ulceration in the rabbit was compared. RESULTS: In most tissues, tepoxalin was a weaker inhibitor of prostaglandin synthesis than the two NSAIDs. However, at a site of peripheral inflammation, tepoxalin was comparable with the NSAIDs in suppressing prostaglandin synthesis and in exerting anti-inflammatory effects. Indomethacin induced penetrating antral ulcers in rabbits whereas tepoxalin produced no detectable mucosal injury. CONCLUSIONS: The ability of tepoxalin to suppress inflammation without causing gastric mucosal injury appears to be related to its differential suppression of prostaglandin synthesis in various tissues. Compounds that selectively inhibit prostaglandin synthesis at sites of inflammation may represent a class of anti-inflammatory drugs without detrimental effects on the stomach.

Comparison of the ulcerogenic properties of tepoxalin with those of non-steroidal anti-inflammatory drugs (NSAIDs).

The ability of tepoxalin to render the gastric mucosa susceptible to injury by a topically applied irritant was compared to that of indomethacin, naproxen and diclofenac. While the three NSAIDs significantly increased the extent of mucosal damage at doses in the 1-30 mg/kg range, tepoxalin failed to significantly augment damage at doses of up to 300 mg/kg. Daily treatment with tepoxalin (10-100 mg/kg) for 4 days also did not significantly affect the susceptibility of the gastric mucosa to damage. The absence of ulcerogenic properties of tepoxalin at doses previously shown to be anti-inflammatory may be related to its relative lack of activity as an inhibitor of gastric prostaglandin synthesis, or to its 5-lipoxygenase inhibitory activity.

Rapid detection of antigen-specific mouse IgE by enzyme-linked immunosorbent assay (ELISA).

A rapid, sensitive, antigen-specific mouse IgE capture ELISA is described. A monoclonal rat anti-mouse IgE was used as the capture antibody, and a DNP-coupled BSA-biotinylated conjugate along with a peroxidase-avidin-biotin complex was utilized as the detection system. The lower detection limit of this assay is 8.5 ng/ml of antigen-specific IgE. With some modifications, this assay can be employed to screen for antigen specific antibodies of other isotypes and subtypes.