Application of arachidonic acid (AA) (0.1-4 mg) to the ears of mice produces immediate vasodilatation and erythema (5 min) followed by the abrupt development of edema which is maximal at 40-60 min. The onset of edema coincides with extravasation of protein and leukocytes. After 1 h, the edema begins to wane rapidly and the inflammatory cells leave the tissue so that by 6 h the ears have returned to near normal except for residual erythema. During the period 6-48 h, AA-treated ears show a greatly diminished response with respect to edema and cell infiltrate when AA is applied a second time. Inhibitor studies show that the inflammatory response is due to formation of AA metabolites via both the cyclooxygenase and lipoxygenase pathways. Under appropriate conditions, AA-induced ear edema can be used as a model to screen for compounds showing in vivo lipoxygenase inhibitory activity. Although relatively large doses of AA were applied topically, there was only a modest stimulation of epidermal DNA synthesis and mitotic index with no consequent hyperplasia. Although arachidonic acid is capable of eliciting most aspects of an inflammatory response, the reaction is abrupt in onset and of short duration. Additional factors appear to be required to produce a prolonged inflammatory response with associated tissue destruction, or inflammatory cell activation and immobilization in situ.

12-0-Tetradecanoylphorbol acetate (TPA) applied to mouse ears rapidly induces an edema which is maximal by 6 hr but has substantially waned by 24 hr. (This is in contrast to many inflammatory agents that cause a prolonged edema lasting many days.) Reapplication of TPA at 16-24 hr will not provoke a second edematous response although increased erythema is evident. Arachidonic acid (AA) applied to mouse ears (4 mg) provokes an even more rapid edema which is maximal at 1 hr and has substantially waned by 6 hr. Reapplication of AA at 3-24 hr also will not provoke a second edematous response although, again, increased erythema does result. Pretreatment of ears with AA results in inhibition of the edema response to subsequent application of TPA, and TPA pretreatment moderately inhibits a subsequent response to AA. TPA-induced edema can be delayed by agents such as naproxen, an inhibitor of AA cyclooxygenase. In contrast, AA-induced edema is inhibited only by agents, such as phenidone, that inhibit both cyclooxygenase and lipoxygenase. The data suggest that the edemas result from interaction of the products of the cyclooxygenase and lipoxygenase pathways of AA metabolism. The lack of secondary edema response appears to be related to the inability of TPA or AA to reinduce vascular permeability. The effect is specific to AA and TPA; responses to xylene or anthralin are unaffected by TPA or AA pretreatment. It is postulated that the tachyphylactic effects observed involve lipoxygenase metabolites of AA.

Peptidyl (acyloxy)methyl ketones, previously established as potent irreversible inhibitors of the cysteine proteinase cathepsin B in vitro, were investigated and optimized for their inhibitory activity in vivo. Incorporation of polar or charged functional groups in the inhibitor structure afforded effective cathepsin B inhibition, following dosing to rats. The most effective inhibitor, Z-Phe-Lys-CH2OCO-(2,4,6-Me3)Ph (8), was found to give ED50 values of 18 mg/kg po (orally) and 5.0 mg/kg ip (intraperitoneally) at 4-5 h postdose, and 2.4 mg/kg sc (subcutaneously) at 24 h postdose, for liver cathepsin B inhibition (measured ex vivo). The subcutaneous route of administration of (acyloxy)methyl ketone 8 also provided potent cathepsin B inhibition in certain peripheral tissues (e.g., ED50 1.0 mg/kg for skeletal muscle, 0.1 mg/kg for heart). These investigations demonstrate that peptidyl (acyloxy)methyl ketones such as 8 have promise as tools for the characterization of in vivo biochemical processes and as therapeutic agents.

Corticosteroid-induced dermal atrophy has been studied in the rat using daily application of ethanolic solutions to small areas of flank skin. After 12 days of treatment, the degree of atrophy was determined by comparing the weights of skin plugs (16 mm diameter) taken from the treated areas with contralaterally paired control areas. Doses can be adjusted so that systemic effects are minimized and only local effects are observed. Hydrocortisone, hydrocortisone butyrate, dexamethasone, betamethasone, desonide and triamcinolone acetonide all produce atrophy in the rat, and the degree of thinning is dose dependent. Potencies in the dermal atrophy assay compare directly with topical anti-inflammatory potencies in the rat, and the presence of fluorine in the steroid molecule is not a determining factor in the production of atrophy.

Epidermal strips, free of sebaceous gland and hair follicle contamination, were prepared from mouse tail skin. Epidermal homogenates synthesized prostaglandins and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenously added [1-14C]arachidonic acid. The effects of pH, assay time, substrate concentration, and several selective inhibitors upon the lipoxygenase and cyclooxygenase pathways were determined. Ultracentrifugation of the crude homogenate at 105,000 g sedimented both activities, and pellet 12-HETE synthesis increased 2-fold relative to the crude homogenate. Recombination of the 105,000 g pellet and supernatant gave yields of prostaglandins and 12-HETE essentially equivalent to that of crude homogenate. When tested in homogenate with 4.5 microM arachidonic acid, anthralin specifically inhibited 12-HETE production with IC50 of 50.0 microM; no significant effect against cyclooxygenase was observed over the dose range of 2-200 microM. 1,8-Dihydroxy-9,10-anthraquinone (DHAQ) also specifically inhibited 12-HETE synthesis, but the dose response curve was flatter and maximum inhibition was only 55% at 200 microM. 6-Chloro-2,3-dihydroxy-1,4-naphthoquinone (CDNQ), an agent with topical antipsoriatic activity, also inhibited 12-HETE synthesis with an IC50 of 25 microM, but simultaneously stimulated prostaglandin production, up to 2.5-fold at 200 microM. When tested with washed human platelets, anthralin again specifically inhibited 12-HETE production with an IC50 of 10 microM, while DHAQ inhibited lipoxygenase activity by only 40% at 25 microM. When tested in platelets, CDNQ gave 33% inhibition of 12-HETE production at 200 microM, although prostaglandin synthesis was stimulated over the range of 25-200 microM. It is proposed that certain antipsoriatic agents may exert their action through modulation of arachidonic acid metabolism.