Development of a rapid screen for detecting and differentiating immunomodulatory vs. anti-inflammatory compounds in rats.

Polyarthritis can be induced in rats using a synthetic adjuvant, N,N-dioctyldecyl-N', N-bis(2-hydroxyethyl) propanediamine (LA) suspended in oil. The disease is morphologically indistinguishable from the classic adjuvant arthritis induced by Freund's complete adjuvant (FCA). LA injection (7.5 mg/animal) consistently induced paw swelling, splenomegaly and fibrinogen level increases at certain time points. Studies evaluating various protocols and parameters determined that a 15 day assay where agents administered from days 9 through 13, would differentiate immunomodulatory and anti-inflammatory compounds. Parameters utilized were body weight, paw volumes, spleen weights, and fibrinogen levels. Immunomodulatory agents reduce paw swelling, splenomegaly and in some cases fibrinogen levels. NSAIDS reduce paw swelling, increase splenomegaly and have no effect on fibrinogen levels. These results indicate that compounds active in the traditional FCA assay can be detected and differentiated with respect to anti-inflammatory vs. immunomodulatory activity in a rapid screen.

Radiographic studies of the effect of benoxaprofen on bone damage in the adjuvant arthritic rat.

The ability of benoxaprofen to suppress bone damage associated with adjuvant-induced arthritis in rats was studied radiographically over a 49-day period. Benoxaprofen was administered orally at a daily dose of 30 mg/kg. Treated and control groups of rats were examined at seven-day intervals from 14 through 49 days. Benoxaprofen showed significant suppression of bone damage at all time intervals. Benoxaprofen was compared in a radiographic bone study with other nonsteroidal anti-inflammatory agents. Benoxaprofen at doses of 10-40 mg/kg/day orally, administered from days 15 to 39 of the disease, was more effective in suppressing bone damage exhibiting toxic effects than other agents. This modification of the experimental disease process by benoxaprofen may be due in part to its reported ability to suppress monocyte migration into inflammatory sites.

Comparative effects of benoxaprofen and other anti-inflammatory drugs on bone damage in the adjuvant arthritic rat.

Benoxaprofen, a new nonsteroidal anti-inflammatory agent recently marketed as an an antirheumatic drug, has been reported to suppress leucocyte migration into inflammatory sites, possibly by its reported inhibition of leukotriene synthesis. Benoxaprofen is also a weak to moderate inhibitor of prostaglandin synthesis. The effect of benoxaprofen was examined on adjuvant-induced arthritis in rats by radiographic assessment of bone damage. The effect of benoxaprofen was compared to other nonsteroidal anti-inflammatory agents, considered to act primarily by the inhibition of synthesis and/or release of prostaglandins. Drugs were administered from the 15th to the 30th day after induction of the adjuvant disease ('established adjuvant'). Radiographs of adjuvant rats showed extensive bone damage that was markedly suppressed by 30-40 mg/kg of benoxaprofen. Benoxaprofen exerted a dose-related inhibition of bone damage. There was less suppression of bone damage by comparable doses of phenylbutazone, oxyphenbutazone, ibuprofen, fenbufen, naproxen, tolectin and sulindac. Indomethacin, piroxicam and flurbiprofen were nearly as effective but only in doses that produced adverse effects or death in rats. Benoxaprofen may modify the progression of the experimental arthritic disease through a suppression of leucocyte migration.

Anti-inflammatory activity of pergolide, a dopamine receptor agonist.

Pergolide, a dopamine agonist effective in the treatment of Parkinson's disease, has been shown to have anti-inflammatory activity in the carrageenan paw edema assay in rats at p.o. doses greater than or equal to 0.3 mg/kg. Studies were done to investigate the mechanism of action and to determine the pharmacologic significance of this finding. Because pergolide elevates circulating glucocorticoids, the effect of pergolide on carrageenan-induced paw swelling was assessed in adrenalectomized rats. Pergolide retained its anti-inflammatory activity in adrenalectomized carrageenan-injected rats, thus eliminating corticosterone induction as a possible mechanism of action. Pergolide treatment also did not decrease thromboxane B2, prostaglandin E2 or leukotriene B4 production, ruling out direct effects on arachnoid acid inflammatory mediators. Interactions with the autonomic nervous system were suggested, in that an alpha adrenergic agonist (clonidine) mimicked the activity of pergolide in the carrageenan assay, and an alpha adrenergic antagonist (phenoxybenzamine) blocked the anti-inflammatory activity of pergolide in this assay. Dopamine receptor antagonists (haloperidol or sulpiride) partially inhibited the effect of pergolide in the carrageenan model. However, the peripherally restricted dopamine antagonist, domperidone, was ineffective, suggesting that a central dopamine receptor was involved in the effect. Experiments in chronic inflammation models such as lipoidal-amine induced arthritis in rats and picryl chloride-induced delayed type hypersensitivity in mice also revealed an anti-inflammatory effect of pergolide. Activity in the carrageenan system and the lipoidalamine model demonstrated that the anti-inflammatory effects of pergolide were separable from potential immunosuppressive effects. Multiple dose studies indicated that tolerance might develop to the anti-inflammatory effect of pergolide.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-inflammatory effects of LY221068 and LY269415.

LY221068, 5-[[3,5-bis(1,1-dimethylethyl)-4-hydroxy phenyl]methylene]-3-(dimethylamino)-4-thiazolidinone, and the monomethylamino analog, LY269415, are anti-oxidants and potent inhibitors of iron dependent lipid peroxidation and 5-lipoxygenase enzyme. Since oxygen radical species, lipid peroxides and products of the arachidonic acid cascade have been implicated as important mediators in a variety of inflammatory diseases including arthritis, LY221068 and LY269415 were studied in the Freund's Complete Adjuvant Induced Arthritis (FCA) model in rats. The compounds were administered orally and inhibition of bone damage and paw swelling of both the injected and uninjected paws was assessed. At 50 mg/kg p.o., LY221068 inhibited soft tissue swelling in the uninjected paw by 72% while LY269415 at 25 mg/kg p.o. exhibited 74% inhibition. Bone damage was also significantly inhibited by both compounds. In a dose response study, the minimum effective dose for LY221068 was 10 mg/kg p.o. and for LY269415 was 5 mg/kg p.o. In the established FCA model in rats, LY221068 at 50 mg/kg p.o. inhibited the uninjected paw swelling by 71% while LY269415 at 25 mg/kg p.o. inhibited 70%. These results suggest that LY221068 and LY269415 may be useful in the treatment of arthritis.

Effect of weight manipulation on bone loss due to ovariectomy and the protective effects of estrogen in the rat.

While characterizing the effects of estrogen on an ovariectomized (OVX) rat model of bone loss, we examined several weight-matching regimens [e.g., ad libitum (feed bins continually full), weight matched (rate of weight gain for OVX and Sham-OVX groups was equalized), and weight restricted (weight gain rates for all groups were equalized to that of estrogen-treated OVX rats)] for possible effects. Bone loss following ovariectomy is primarily the result of an increase in bone resorption and is extremely sensitive to the effects of estrogens. Thus, in all of our analyses, treatment with 17 beta-estradiol served as a positive control for the prevention of bone loss. Each weight-matching study had three groups: control (Sham-OVX), OVX, and OVX + 17 beta-estradiol (0.1 mg/kg/day), and lasted for either 2, 4, or 6 weeks. Throughout the study, each Sprague Dawley rat was weighed every other day, and following sacrifice, a femur was removed for bone mineral density (BMD) analysis at the distal metaphysis by single photon absorptiometry. Following 2 weeks of dietary modifications, no significant differences were detected in BMD among the ad lib or weight matched groups. However, an estradiol-preventable reduction in BMD in restricted OVX rats was detected at 2 weeks postovariectomy. Additionally, OVX rats in all three dietary regimens displayed an estrogen-preventable reduction in proximal femur BMD at 4 and 6 weeks postovariectomy. These results indicate that a 4-week rat ovariectomized model of bone loss, under conditions of ad libitum feeding, shows great potential for pharmacologic manipulation.

Anti-inflammatory activity of BF389, a Di-T-butylphenol, in animal models of arthritis.

Biofor 389 (BF389), dihydro-4-[[3,5-bis(1,1-dimethyl)-4-hydroxyphenyl] methylene]-2-methyl-2H-1,2-oxazin-3(4H)-one, was tested for anti-inflammatory activity in various animal models of arthritis. Initial evaluation in the lipoidalamine (LA) arthritis model in rats (5-day dosing protocol) resulted in an oral ED50 of 4.9 mg/kg for inhibition of paw swelling. No effects on splenomegaly were observed, suggesting that the compound was efficacious as a result of anti-inflammatory rather than immunomodulatory effects. BF389 was efficacious in interleukin 1 (IL-1)-enhanced type II collagen arthritis in rats (oral ED50 less than 1.0 mg/kg) as assessed by paw volume measurement and histologic evaluation of joints. Mice with IL-1-enhanced type II collagen arthritis given 30 mg/kg of BF 389 had significantly lower histological scores for joint damage than did untreated controls. Normal rats given single oral doses of BF389 had significant suppression of arachidonate-stimulated whole blood prostaglandin E2 and thromboxane B2 production 2 hr postdosing (ED50 = 0.1 mg/kg). Leukotriene B4 production in these animals was not decreased. After it became apparent that the compound was a potent inhibitor of prostaglandin production in vivo, a study was done to compare the efficacy and toxicity of BF389 with several currently marketed nonsteroidal anti-inflammatory drugs, piroxicam, naproxen and diclofenac. Lipoidalamine-injected rats were given daily oral doses of BF389 or the comparators for 21 days. Quantitation of effects on arthritis on day 21 resulted in ED50 values of 0.9 mg/kg (BF389), 3.9 mg/kg (naproxen), 4.9 mg/kg (diclofenac) and 0.6 mg/kg (piroxicam).(ABSTRACT TRUNCATED AT 250 WORDS)

The anti-inflammatory effects of LY178002 and LY256548.

LY178002 (5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene-4-thiazolidinon e) and its N-methyl analog, LY256548, inhibit the enzymatic activity of phospholipase A2, 5-lipoxygenase and fatty acid cycloxygenase. They also inhibit leukotriene B4 production from human polymorphonuclear leukocytes stimulated with the calcium ionophore A23187. Since products of the arachidonic acid cascade have been implicated as important mediators in a variety of inflammatory diseases including arthritis, LY178002 and LY256548 were studied in the Freund's Complete Adjuvant-Induced Arthritis (FCA) model in rats. The compounds were administered orally and inhibition of bone damage and paw swelling was assessed of both the injected and uninjected paws. At 50 mg/kg LY178002 inhibited soft tissue swelling in the uninjected paw by 81% while LY256548 exhibited 57% inhibition. Bone damage was also significantly inhibited by both compounds. A dose response was conducted. The minimum effective dose for LY178002 was 10 mg/kg p.o. In the established FCA model LY178002 at 50 mg/kg p.o. inhibited the uninjected paw swelling by 75% while LY256548 did not show this level of activity. These results suggest that LY178002 and LY256548 may be useful in the treatment of arthritis.

Correlation of anti-inflammatory activity with peak tissue rather than peak plasma levels of BF389.

BF389 (Biofor 389) is a potent anti-inflammatory agent in various animal models including adjuvant and type II collagen arthritis in rats. In vitro assays indicate that this compound is a mixed inhibitor of arachidonic acid metabolism. The compound was evaluated for effects on the acute inflammatory response in the carrageenan paw edema assay in rats using a standard protocol in which the animals were given single or multiple (daily for 5 days) p.o. doses of the compound. Carrageenan was injected into the footpad of each animal 1 hr after dosing and volumes of both hind paws were determined 3 hr later. Basal serum prostaglandin (PG) E2 levels and PGE2 levels in arachidonate-stimulated blood from these same animals also were measured. No anti-inflammatory activity was observed in BF389-treated rats despite the occurrence of profound suppression of basal and stimulated PGE2 production. Animals treated with conventional nonsteroidal anti-inflammatory drugs in this assay had both suppressed PGE2 production and significant anti-inflammatory activity. In another study, groups of rats were given p.o. doses of 1, 10, 100 or 250 mg/kg of BF389 for 5 days in order to determine peak concentrations, T1/2s and total areas under the plasma (tissue) drug concentration curves in plasma and paws. Peak plasma concentrations occurred 2 to 4 hr postdosing and the half-life was 8 to 15 hr over the 1- to 250-mg/kg/day dose range. Peak paw concentrations occurred 6 to 12 hr postdosing and the levels were 9- to 14-fold greater in the paw than in the plasma.(ABSTRACT TRUNCATED AT 250 WORDS)