Louis J. Datko

Comparison of inflammatory changes in established type II collagen- and adjuvant-induced arthritis using outbred wistar rats

Type II collagen- and adjuvant-induced arthritis in outbred Wistar rats were compared using parameters that measured the inflammatory response, cellular and humoral immunity, blood protein changes, drug metabolism and histopathological and bony changes of the inflamed paws. There was a lesser incidence (40-70%) and severity of collagen disease than the adjuvant model (incidence approximately 100%). The use of MDP increased the incidence and severity of collagen arthritis. The acute phase protein response (plasma fibrinogen) was similar in both models during the peak of inflammatory response. Drug metabolism was inhibited in both type II collagen boosted with MDP or M. butyricum sensitized rats with arthritis; however, arthritic rats sensitized with collagen alone produced no inhibition. Only collagen arthritic rats produced type II collagen antibody and exhibited delayed hypersensitivity to type II collagen. Bony changes as assessed by radiographic evaluation were more severe in adjuvant arthritic rats than in the collagen arthritic model; histopathological findings from these animals confirmed this observation. The primary lesions in both models were periosteal reaction of the bone and ankylosis. Several classes of antiarthritic drugs were compared in both models using paw edema measurements and bony changes by radiographic evaluation. Drugs with inhibitory activity in both models were indomethacin, methylprednisolone, D-penicillamine and gold sodium thiomalate. Levamisole, chloroquine and auranofin were inactive in both models.

Effect of a 5-lipoxygenase (5-LO)/cyclooxygenase (CO) inhibitor, WY-47, 288, on cutaneous models of inflammation.

WY-47,288 (2-[(1-naphthalenyloxy)methyl]quinoline) demonstrated topical antiinflammatory activity in several animal models of skin inflammation. Application of WY-47,288 to mouse ear surfaces inhibited arachidonic acid (ED50=0.3 mg/ear) and tetradecanoylphorbol acetate (TPA)-induced inflammation (40% at 1 mg/ear). Administration of WY-47,288 (1 mg/ear) at 30 min and 5 h after TPA reduced ear edema and epidermal proliferation by 50%. WY-47,288 also inhibited oxazolone-induced contact hypersensitivity in mouse ears (ED50=0.4 mg/ear) and UVB-induced guinea pig skin erythema (ED50≈0.25 mg/spot). These antiinflammatory effects may be due to inhibition of 5-lipoxygenase (5-LO) and cyclooxygenase (CO) since the synthesis of 5-LO and CO products by rat neutrophils and mouse macrophages was dose-dependently reduced by WY-47,288. By contrast, WY-47,288 demonstrated no appreciable inhibition of 12-LO (rabbit platelet), 15-LO (soybean) or phospholipase A2 (human platelet). Furthermore, no systemic adverse effects were observed after topical, parenteral or oral administration of WY-47,288, suggesting that WY-47,288 is a safe topical 5-LO/CO inhibitor for treating skin inflammation.

Interaction studies of tilomisole, aspirin, and naproxen in acute and chronic inflammation with assessment of gastrointestinal irritancy in the rat

The effect of combination NSAID therapy of tilomisole with aspirin or naproxen was studied in rats with carrageenan-induced paw edema and established adjuvant arthritis. Inflamed paws were measured using mercury plethysmography and the arthritic paws were X-rayed to determine any bony/soft tissue changes. The gastrointestinal tract was also examined for bleeding and ulceration. Tilomisole had a less potent acute anti-inflammatory effect than aspirin or naproxen, but produced no significant gastrointestinal damage. A significant reduction in anti-inflammatory activity was observed with the tilomisole/aspirin combination in acute inflammation. Only additive interactions were observed with the naproxen inhibition. In the established arthritis assay, a significant synergistic anti-inflammatory response, i.e. both inhibition of paw edema and bone erosion, was also observed with the 80 and 93% tilomisole/naproxen combinations. The gastric ulcerogenic effect of the combination paralleled its increased activity. The synergism between tilomisole and naproxen in thischronic arthritic model may be due to enhanced cyclooxygenase inhibitory activity. These drug interaction studies suggest possible interactions in human clinical trials of rheumatoid arthritis.

The absence of antibodies to type II collagen in established adjuvant arthritis in rats

Utilizing an adjuvant arthritis model in rats, we examined humoral immunity to collagen and inflammation in animals with active disease and during drug therapy. Humoral immunity to types I or II collagen was not detected in the sera of rats with advanced adjuvant arthritis; this was in marked contrast to rats with type II collagen-induced arthritis which possessed serum antibodies to native and denatured type II collagen. Hind paw edema and bone pathology were monitored as parameters of inflammation. A new investigational drug, Wy-41,770, was most effective in reducing all of these aspects of inflammatory disease while indomethacin, methylprednisolone, andd-penicillamine caused a less significant diminution of only some of these parameters of inflammation. Antibodies to collagen were not detected in the sera of rats treated with the drugs under study. These data demonstrate that adjuvant arthritis can occur in rats in the absence of antibodies to types I or II collagen.

The antiinflammatory profile of (5H-dibenzo[a,d]-cyclohepten-5-ylidene)acetic acid (WY-41,770), an agent possessing weak prostaglandin synthetase inhibitory activity that is devoid of gastric side effects.

Wy-41,770 [(5H-dibenzo[a,d]cyclohepten-5-ylidene)acetic acid], a novel acrylic acid, was compared to indomethacin and aspirin in standard antiinflammatory, analgesic and antipyretic animal models. The acute antiinflammatory, analgesic and antipyretic activity of Wy-41,770 (oral ED50s 50–170 mg/kg) was similar to aspirin; however, it was considerably more potent orally in adjuvant arthritis in the rat (ED50, 16 mg/kg) and urate-induced synovitis in the dog (ED50, 4.5 mg/kg). Wy-41,770 was a weak inhibitor of prostaglandin biosynthesis and did not inhibit either 5- or 15-lipoxygenase. Furthermore, the cellular migration characteristic of carrageenan pleurisy was not affected by Wy-41,770. Unlike a majority of NSAIDs, it produced no gastric irritation in rats after either acute or chronic oral administration over the range 400–800 mg/kg. The major mechanism of action of Wy-41,770 has yet to be identified but does not seem to involve interference of arachidonic acid metabolism.

Immunomodulation of delayed hypersensitivity to methylated bovine serum albumin (MBSA) in mice using subliminal and normal sensitization procedures.

Delayed hypersensitivity (DH) was induced in the footpads of mice sensitized to methylated bovine serum albumin (MBSA). The magnitude of this DH response increased with increasing sensitizing concentration of MBSA. Levamisole administered 1 hr prior to MBSA challenge stimulated the DH response and this was optimal using subliminal sensitizing concentrations of antigen. A number of antirheumatic agents, immunomodulators mediator antagonists and antiallergics were subsequently examined using the subliminal sensitizing concentration of MBSA. The same drugs were also evaluated using a normal sensitizing procedure. These studies indicate that the sensitizing concentration of antigen is critical in establishing whether a drug will stimulate or suppress a DH response.

Humoral and Cellular Immunologic Responses in Collagen-Induced Arthritis in Rats: Their Correlation with Severity of Arthritis

Collagen arthritis in rats has a well defined humoral and cellular immunologic response to type II collagen, the inciting antigen. Like other chronic models of inflammation, considerable variation exists in terms of severity and incidence. We have attempted to correlate the inflammatory response as measured by paw volume, with serum type II collagen antibody and skin delayed hypersensitivity (DH) to type II collagen. Surprisingly, the incidence and severity of collagen arthritis, induced in the presence of MDP to increase incidence of the disease, are neither correlated with type II collagen antibody nor DH to type II collagen. However, tarsometatarsal bone erosion is significantly correlated with paw edema. Further studies will be necessary to elucidate the role of both humoral and cellular immune responses in the development of type II collagen arthritis in the rat.

Questionable role of leukotriene B4 in monosodium urate (MSU)-induced synovitis in the dog

Monosodium urate (MSU)-induced synovitis in the dogs stifle (knee joint) is similar to an acute gouty attack in man in which a loss of function of the joint correlates with massive influx of neutrophils and the release of an assortment of inflammatory mediators (e.g. histamine, bradykinin, lysosomal enzymes, complement and eicosanoids) into the synovial space. We found in the urate-induced inflammatory exudates 3 hr post MSU the following: 88 million leukocytes/ml (approximately 95% neutrophils) and eicosanoid concentrations of LTB4, LTC4, and PGE2 of less than 0.1, 1.4 and 20 ng/ml, respectively. Isotonic saline injected knee joints at 3 hr contained 5 million leukocytes/ml (approximately 95% neutrophils) and concentrations of LTB4, LTC4, and PGE2 of less than 0.1, 0.7 and 0.2 ng/ml, respectively. Intrasynovial injections of 1 microgram LTB4, 10 micrograms PGE2 or the combination of LTB4 and PGE2 produced no reduction of paw pressure for up to 3 hr. Leukocyte concentrations measured at 3 hr in joints injected with these arachidonic acids metabolites were similar to saline controls. These results question the role of LTB4 as a chemotactic and inflammatory mediator in urate-induced synovitis in the dog but confirm the importance of PGE2 and possibly LTC4 in this model.

An automated microcomputer-based system for determining canine paw pressure quantitatively in the dog synovitis model

The automated system for measuring canine paw pressure is a very useful tool for the evaluation of nonsteroid antiinflammatory drugs such as indomethacin and ibuprofen in the dog synovitis model. The apparatus has been designed to give the operator control over the dogs posture while measuring hind-paw pressure. The stability of the measuring platform permits reproducible measurement of paw pressures. The software package performs direct data recording and data reduction and eliminates tedious manual calculations. Subsequently, the software produces a printout summarizing the experimental results in tabular and graphic form.

Immunomodulating activity of Wy-41,770 (5H-dibenzo[A,D]cyclohepten-5-ylidene) acetic acid.

The immunomodulatory effects of Wy-41,770 (5H-dibenzo[a,d]cyclohepten-5-ylidene) acetic acid, were compared to levamisole and indomethacin in several in vivo models. In the Jerne plaque assay, Wy-41,770 (1 and 100 mg/kg, p.o.) administered on day 1 after sensitization suppressed IgM plaque forming cells (PFC) while levamisole was active when given on days 1 and 2 after sensitization. In contrast, indomethacin administered on days 2 and 3 after sensitization increased PFC. In the rat experimental allergic encephalomyelitis (EAE) model, Wy-41,770 reduced limb paralysis at 10 and 100 mg/kg, p.o. when dosed before sensitization. Indomethacin was active too when predosed in the rat EAE model. In the methylated bovine serum albumin model (MBSA) delayed hypersensitivity (DH) model in mouse, Wy-41,770 (10 mg/kg, p.o.) given on day 1 prior to sensitization and day 2 after sensitization in subliminally sensitized animals augmented the DH response while inhibiting the subliminal DH response when administered at 6 hr after challenge. Levamisole showed similar activity in this subliminal model while indomethacin given 6 hr post challenge was inhibitory. All three drugs were inactive in mice normally sensitized to MBSA at the same drug regimens. In guinea pigs, subliminally sensitized to tuberculin, Wy-41,770 (10 and 100 mg/kg, p.o.) and levamisole augmented the DH response. No changes in DH response were observed for both drugs in normally sensitized guinea pigs. In the rat adjuvant arthritic model, Wy-41,770 (5 and 15 mg/kg, p.o.) inhibited day 16 uninjected paw edema and restored significantly the depressed proliferative responses to mitogen by spleen cells taken from the same arthritic rats at day 16. The moderate immunomodulatory activity of Wy-41,770 may contribute along with its antiinflammatory activity, towards the treatment of arthritic diseases.