Discovery of a potent, metabolically stabilized resorcylic lactone as an anti-inflammatory lead.

With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.

Production and pharmacologic modulation of the granulocyte-associated allergic responses to ovalbumin in murine skin models induced by injecting ovalbumin-specific Th1 or Th2 cells.

Because interferon-gamma, interleukin-4, and interleukin-5 have been identified at the mRNA and protein levels in the lesional skin of patients with atopic dermatitis, we investigated the roles played by granulocytes as effector cells in allergic inflammation by using two unique murine skin models. In vitro generated Th1 and Th2 cells from naïve splenocytes of antiovalbumin T cell receptor transgenic BALB/C mice were adoptively transferred with ovalbumin into the ear pinnae or air-pouches produced in the back skin of naïve, nontransgenic BALB/C mice. The injection of Th1 cells with ovalbumin induced delayed type ear swelling that peaked at 48 h, whereas that of Th2 resulted in ear swelling that peaked at a much earlier time, 24 h. Histologic study of the swollen ear skin and granulocytes recruited into the air-pouch demonstrated that, although the Th1-induced inflammation caused a neutrophil-predominant infiltrate with few eosinophils, larger numbers of eosinophils accumulated in the Th2-induced inflammation. Using these murine models, we further evaluated the effects of drugs used for the treatment of atopic diseases. The results showed that FK506 administration could effectively reduce skin inflammation induced by either Th cells. Interestingly, the neutrophil elastase inhibitor ONO-6818 efficiently inhibited Th1-induced inflammation. In contrast, a leukotriene receptor antagonist, ONO-1078, specifically suppressed Th2-induced inflammation. We also found that each ONO drug exerted direct influence on specified granulocytes, as neither affected in vitro production of relevant Th cytokines. Thus, we succeeded in developing animal skin inflammation models in which we can evaluate the contribution of protein antigen-specific Th1 or Th2 cells through the action of granulocytic effector cells.

Synthesis and antiinflammatory activities of 3-(3,5-di-tert-butyl-4-hydroxybenzylidene)pyrrolidin-2-ones.

A series of 3-(3,5-di-tert-butyl-4-hydroxybenzylidene)pyrrolidin-2-ones was synthesized and evaluated as candidate antiinflammatory/analgesic agents as well as dual inhibitors of prostaglandin and leukotriene synthesis. Some compounds that showed dual inhibitory activity were found to possess equipotent antiinflammatory activities to indomethacin, with reduced ulcerogenic effects. One of the compounds, N-methoxy-3-(3,5-di-tert-butyl-4-hydroxybenzylidene)pyrrolidin-2-o ne, was found to have a wider safety margin than indomethacin or piroxicam, and was selected for detailed evaluation as a candidate drug for clinical application.

A novel orally active inhibitor of IL-1 generation: synthesis and structure-activity relationships of 3-(4-hydroxy-1-naphthalenyl)-2-propenoic acid derivatives.

A new series of 3-(4-hydroxy-1-naphthalenyl)-2-propenoic acids was prepared and the inhibitory activities of its members on IL-1 generation were evaluated both by in vitro systems using human monocytes and/or rat exudated macrophages stimulated with LPS, and by an in vivo system using the rat CMC-LPS air-pouch model. Many compounds in this series were found to be potent inhibitors of IL-1 generation both in vitro and in vivo. Structure-activity relationships indicated that in the rat CMC-LPS air-pouch model by oral administration the (Z)-2-substituted propenoic acids with 3-alkoxy, 5-alkyl, and 4-hydroxy substituents on the naphthalene ring exhibit optimal inhibition. Among the compounds evaluated, (Z)-3-(5-ethyl-4-hydroxy-3-methoxy-1-naphthalenyl)-2-methyl-2-propeno ic acid (20a), which inhibited IL-1 generation from human monocytes with an IC50 value of 3.0 microM and had an IC50 value of 1.4 microM for rat exudated macrophages, showed the most potent inhibitory activity in the rat CMC-LPS model by oral administration. Compound 20a also showed antiinflammatory effects in animal models of inflammation.

Comparison of Tl-201 chloride and Ga-67 citrate scintigraphy in the diagnosis of thyroid tumor: concise communication.

Twenty-five patients with thyroid tumors were scintigraphed with both Tl-201 chloride and Ga-67 citrate. All cases showed a focal area of decreased activity with I-131 or pertechnetate (Tc-99m), and each had a histological diagnosis after surgery or excisional biopsy. From the data we conclude the following: (1) Tumors giving a positive scan with Tl-201 chloride but negative results using Ga-67 citrate prove to be differentiated carcinoma or poorly differentiated adenoma. (2) All tumors that are positive with Ga-67 are highly malignant types, and if these tumors are negative by Tl-201, undifferentiated carcinoma is suggested. (3) Ga-67 citrate scintigraphy is a useful procedure in locating distant metastases, in determining the area to be irradiated, and in judging the effect of therapy on undifferentiated carcinoma.

[Behavior of drug resistant fecal coliforms and R plasmids in a wastewater treatment plant].

Fecal coliforms were isolated from the inlet, the primary sedimentation tank, the activated sludge digestion tank, the final settling tank, the outlet and the return activated sludge drain at the municipal wastewater plant in Ube City, and examined for drug resistance and presence of R plasmids. Drug concentrations employed to distinguish resistant isolates from sensitive isolates were 25 micrograms/ml for tetracycline, kanamycin, chloramphenicol and streptomycin, 50 micrograms/ml for ampicillin, nalidixic acid and rifampicin, and 200 micrograms/ml for sulfisoxazole, respectively. Of a total of 900 isolates, 45.7% were drug resistant and 51.1% of them carried R plasmids. The further along that wastewater had progressed through the treatment process the greater the tendency was for appearance of the multiresistant isolates. These isolates also were shown to simultaneously carry transferable R plasmids. Observed resistant patterns of R plasmids were mainly multiple and encoded to resistance to tetracycline, chloramphenicol, streptomycin and sulfisoxazole. It became clear that multiplication of R plasmids took place in the activated sludge digestion tank. This study show that drug resistance transfer mediated by these R plasmids may occur in actual wastewater treatment plants.

A novel antagonist of the prostaglandin E(2) EP(4) receptor inhibits Th1 differentiation and Th17 expansion and is orally active in arthritis models.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is an autoimmune disorder involving subsets of activated T cells, in particular T helper (Th) 1 and Th17 cells, which infiltrate and damage tissues and induce inflammation. Prostaglandin E(2) (PGE(2)) enhances the Th17 response, exacerbates collagen-induced arthritis (CIA) and promotes inflammatory pain. The current study investigated whether selective antagonism of the PGE(2) EP(4) receptor would suppress Th1/Th17 cell development and inflammatory arthritis in animal models of RA. EXPERIMENTAL APPROACH: Effects of PGE(2) and a novel EP(4) receptor antagonist ER-819762 on Th1 differentiation, interleukin-23 (IL-23) production by dendritic cells (DCs), and Th17 development were assessed in vitro. The effect of ER-819762 was evaluated in CIA and glucose-6-phosphate isomerase (GPI)-induced arthritis models. In addition, the effects of ER-819762 on pain were evaluated in a model of chronic inflammatory pain induced by complete Freund's adjuvant (CFA) in the rat. KEY RESULTS: Stimulation of the EP(4) receptor enhanced Th1 differentiation via phosphatidylinositol 3 kinase signalling, selectively promoted Th17 cell expansion, and induced IL-23 secretion by activated DCs, effects suppressed by ER-819762 or anti-PGE(2) antibody. Oral administration of ER-19762 suppressed Th1 and Th17 cytokine production, suppressed disease in collagen- and GPI-induced arthritis in mice, and suppressed CFA-induced inflammatory pain in rats. CONCLUSION AND IMPLICATIONS: PGE(2) stimulates EP(4) receptors to promote Th1 differentiation and Th17 expansion and is critically involved in development of arthritis in two animal models. Selective suppression of EP(4) receptor signalling may have therapeutic value in RA both by modifying inflammatory arthritis and by relieving pain.

A novel inhibitor of IL-1 generation, E5090: in vivo inhibitory effect on the generation of IL-1-like factor and on granuloma formation in an air-pouch model.

The in vivo production of IL-1-like activity was investigated in the exudate of a rat air-pouch inflammatory model. An inflammatory reaction was induced by LPS injection into the air-pouch. IL-1 activity in the exudate reached the maximum level at 4 h and then rapidly decreased until 8 h after the injection of LPS. Orally administered E5090 and prednisolone dose-dependently inhibited the generation of IL-1 activity. Both compounds also suppressed chronic granuloma formation in parallel with the IL-1 inhibition. On the other hand, indomethacin had no effect on either IL-1 generation or granuloma formation in spite of the complete inhibition of PGE2 generation. These results suggest that E5090 inhibits the production of IL-1-like activity in the exudate and exhibits steroid-like antiinflammatory effects.

Application of adjuvant-induced local hyperthermia for evaluation of anti-inflammatory drugs.

A recently established method for determining the surface temperature of adjuvant-inflamed rat's paw was used to examine the anti-inflammatory potencies of various nonsteroidal and steroidal anti-inflammatory drugs, which are currently in clinical use. The results were compared with those obtained by the carrageenin paw edema method and with the inhibitory potencies on prostaglandin E2 generation. The clinically effective single doses of the drugs were better correlated with the effective doses on local hyperthermia than with those on carrageenin paw edema, or with the inhibitory activities of the drugs on prostaglandin E2 generation from cultured rat synovial cells. Although care should be taken to avoid false-positive anti-inflammatory effects arising from vasoconstrictor activity or toxicity, determination of lowering effects on local hyperthermia in this inflammatory model may be useful for the evaluation of anti-inflammatory compounds.

Ultrafast dynamics in aqueous polyacrylamide solutions.

We have investigated the ultrafast dynamics of aqueous polyacrylamide ([-CH(2)CH(CONH(2))-](n), or PAAm) solutions using femtosecond optical heterodyne-detected Raman-induced Kerr effect spectroscopy (OHD-RIKES). The observed aqueous PAAm dynamics are nearly identical for both M(w) = 1500 and 10 000. Aqueous propionamide (CH(3)CH(2)CONH(2), or PrAm) solutions were also studied, because PrAm is an exact model for the PAAm constitutional repeat unit (CRU). The longest time scale dynamics observed for both aqueous PAAm and PrAm solutions occur in the 4-10 ps range. Over the range of concentrations from 0 to 40 wt %, the picosecond reorientation time constants for the aqueous PAAm and PrAm solutions scale linearly with the solution concentration, despite the fact that the solution shear viscosities vary exponentially from 1 to 264 cP. For a given value of solution concentration in weight percent, constant ratios of measured reorientation time constants for PAAm to PrAm are obtained. This ratio of PAAm to PrAm reorientation time constants is equal to the ratio of the volume for the PAAm constitutional repeat unit (-CH(2)CHCONH(2)-) to the molecular volume of PrAm. For these reasons, we assign the polymer reorientation dynamics to motions of the entire constitutional repeat unit, not only side group motions. Simple molecular dynamics simulations of H[-CH(2)CH(CONH(2))-](7)H in a periodic box with 180 water molecules support this assignment. Amide-amide and amide-water hydrogen-bonding interactions lead to strongly oscillatory femtosecond dynamics in the Kerr transients, peaking at 80, 410, and 750 fs.

Quantification of 35S-labeled proteoglycans complexed to alcian blue by rapid filtration in multiwell plates.

This paper describes a rapid filtration assay for the quantification of 35S-labeled proteoglycans and/or 35S-labeled glycosaminoglycans in a large number of samples. Separation of 35S-labeled proteoglycans and 35S-labeled glycosaminoglycans from unincorporated [35S]sulfate is effected by forming insoluble complexes between alcian blue and the glycosaminoglycan moieties of the proteoglycans and then filtering the solutions through "Durapore membrane" discs (0.45 microns pore size) fitted in a 96-well plate. Following brief rinsing steps, the discs are punched out and 35S-labeled macromolecules retained on the membrane are then quantified by scintillation counting. In this rapid filtration assay, the relationship between the amount of [35S]-aggrecan applied and radioactivity measured was linear over a broad range of concentrations (2-800 micrograms aggrecan/ml). The amount of 35S-labeled proteoglycans measured in media and 4 M guanidine HCl extracts of articular cartilage and three different chondrocyte culture systems (monolayer, agarose gel, and alginate bead) ranged between 90 and 101% of the value obtained by sieve chromatography on Sephadex G-25. The presence in samples of unlabeled proteoglycans (up to 1 mg/ml), bovine serum albumin (up to 4 mg/ml), DNA (up to 20 micrograms/ml), serum (up to 30%), or guanidine hydrochloride at 4 M did not affect recovery of 35S-labeled proteoglycans measurably. CPM values obtained for 35S-labeled proteoglycans or 35S-labeled glycosaminoglycans quantified by chromatography on Sephadex G-25 and the filtration assay showed a strong linear relationship (r > 0.99) irrespective of the type of culture medium, extract, or digest used.

A novel inhibitor of IL-1 generation, E5090: in vitro inhibitory effects on the generation of IL-1 by human monocytes.

E5090 is an orally active inhibitor of IL-1 generation, being converted in vivo into the pharmacologically active deacetylated form (DA-E5090). In vitro effects of DA-E5090 on the generation of IL-1 by human monocytes stimulated with LPS were examined. DA-E5090 inhibited both IL-1 alpha and IL-1 beta generation by human monocytes stimulated with 1 microgram/ml of LPS in a dose dependent-manner (1-10 microM), as determined by LAF assay and ELISA. Northern blotting analysis indicated that DA-E5090 inhibits transcription of IL-1 alpha and IL-1 beta m-RNAs.

Determination of inflamed paw surface temperature in rats.

The surface temperature of a rat's paw is greatly affected by heat transfer because of its contacts with the body, as well as several environmental conditions. To establish a methodology for determining the surface temperature of the inflamed rat's paw, we devised an individual cage with a pair of rolling rods. The rods were set to protect contacts of paws with the body and were devised to roll easily when rats put their feet on the rods. When a rat was transferred to the individual cage with rolling rods from a group cage, the surface temperature of a normal or inflamed paw fell gradually and reached equilibrium within 10 minutes. The application of the rolling rods markedly narrowed the range of fluctuation of paw surface temperatures at the equilibrium, and increased the temperature difference between inflamed and noninflamed paws. We compared local hyperthermia and swelling of inflamed paws, and tested the effects of indomethacin. The results suggest that the local hyperthermia is a useful index of inflammation and a sensitive parameter for the pharmacological evaluation of antiinflammatory drugs.

Interleukin-1-like activity in the exudate of air-pouch inflammation in rats, and its participation in granuloma formation.

The in vivo production of lymphocyte activating factor (LAF) activity was investigated in the exudate of the rat air-pouch inflammation model. An inflammatory reaction was induced by lipopolysaccharide (LPS) injection into the air-pouch, and the time course of LAF activity in the exudate was investigated. LAF activity in the exudate reached a peak by 6 h, and rapidly decreased at 10 to 48 h after the LPS injection. Dexamethasone revealed strong inhibitory action on LAF activity and granuloma formation. On the other hand, indomethacin could not inhibit either of the phenomena. In conclusion, LAF (IL-1) is rapidly produced after the onset of inflammation and may participate in the subsequent granuloma formation.