Paul L. Stanley

Mouse skin inflammation induced by multiple topical applications of 12-O-Tetradecanoylphorbol-13-Acetate

It is well known that applications of a single dose of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse ears induces an acute inflammatory reaction consisting of erythema, edema and polymorphonuclear leukocyte (PMN) infiltration. We report here that multiple topical applications of TPA to mouse ears produce a prolonged inflammatory reaction characterized by increases in ear weight, inflammatory cell infiltration and epidermal hyperplasia. TPA was applied 5 times over 10 days to mouse ears. Epidermal thickness and PMN infiltration (myeloperoxidase content) increased 3- and 160-fold, respectively, by day 3 and remained elevated over control values throughout the test period. Ear weight was elevated from day 1 and remained high. Hydrocortisone 17-valerate and betamethasone dipropionate significantly reduced all three parameters of inflammation. Indomethacin and two other cyclo-oxygenase inhibitors, and an antihistamine had little or no effect on any of the parameters. This chronic skin inflammation model may be more relevant for evaluating anti-inflammatory compounds than the acute TPA model because the test compounds are applied after the inflammatory lesion is established, which mirrors the use of clinical anti-inflammatory drugs. Also this model may be more selective than the acute TPA model for compounds which affect leukotriene production since other pharmacological agents which are active in the acute model are not active in the multiple-application model.

Temporal infiltration of leukocyte subsets into mouse skin inflamed with phorbol ester

We have previously shown that multiple topical applications, over 11 days, of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a persistent inflammatory reaction characterized by edema, cell infiltration and epidermal hyperplasia. In order to characterize the cell infiltrate during the establishment of this inflammatory reaction, immunohistochemistry was performed using two monoclonal antibodies: MOMA-2, a macrophage antibody and Thy-1, a pan T-cell antibody. The level of polymorphonuclear leukocytes (PMNs) peaked by day 3 at 160-fold over nontreated controls and then subsided to a 30-fold elevation on days 7–10. By day 4, the number of macrophages increased 2.9-fold over the nontreated control and by day 10 were elevated 6.0-fold over the nontreated control. In comparison, the number of T-cells present by day 7 was significantly elevated 9.5-fold over the nontreated group and peaked at day 8 with a 19-fold elevation relative to nontreated controls. Topical treatment of animals with hydrocortisone valerate resulted in a dramatic (>60%) reduction in the number of T-cells present in the tissue. In contrast, there was no effect of the steroid on the number of macrophages present in the tissue. The identification of specific cell types and their time course of infiltration is consistent with the development of a chronic inflammatory lesion.

Novel mimics of sialyl Lewis X: design, synthesis and biological activity of a series of 2- and 3-malonate substituted galactoconjugates.

A series of potent inhibitors of P-selectin as potential anti-inflammatory agents is reported. These compounds are derivatives of galactocerebrosides bearing a malonate side chain in positions 2 and 3 of the galactose moiety. Based on the binding mode of sialyl Lewis X, the two acidic groups of the malonate are designed to form ionic interactions with two important lysines in the active site of P-selectin, Lys113 and Lys111. On the other hand, the 4- and 6-hydroxy groups on the galactose ring are arranged to chelate the calcium ion in the P-selectin active site. The synthesis and the biological activity of this series of compounds are described. Lead compounds having a greater potency than sialyl Lewis X are identified.

Biaryl diacid inhibitors of human s-PLA2 with anti-inflammatory activity.

Twenty-four hydrophobic dicarboxylic acids are described which were evaluated as inhibitors of 14 kDa human platelet phospholipase A2 (HP-PLA2). In general, biarylacetic acid derivatives were found to be more active than biaryl acids or biarylpropanoic acids. More potent inhibitors were obtained when hydrophobic groups were attached to the biaryl acid nucleus using an olefin linkage as compared to an ether linkage. Compounds with larger hydrophobic groups were usually more potent inhibitors of HP-PLA2. Five of the compounds disclosed in this report (2, 4, 28, 36b and 36i) were found to possess significant anti-inflammatory activity in a phorbol ester induced mouse ear edema model of chronic inflammation.

Biochemical and pharmacological properties of a new topical anti-inflammatory compound, 9-phenylnonanohydroxamic acid (BMY 30094)

Drugs which block the biosynthesis of leukotrienes and prostaglandins may have potential in the treatment of psoriasis and other skin diseases. The biochemical and anti-inflammatory activity of 9-phenylnonanohydroxamic acid (BMY 30094) is described. BMY 30094 inhibited human neutrophil 5-lipoxygenase with an IC50 of 5.7 μM. BMY 30094 also blocked human platelet cyclooxygenase and lipoxygenase with IC50 values of 15.2 and 15.0 μM, respectively. Topical application of this compound blocked arachidonic acid and 12-O-tetradecanoylphorbol ester-induced mouse skin inflammation with activity comparable to that observed for lonapalene. The topical ED50 for BMY 30094 in the arachidonic acid-induced inflammation model is 2.2 μmoles/ear. In the sub-cutaneous carrageenan sponge assay in rats, BMY 30094 blocked LTB4 and PGE2 production and inhibited neutrophil migration. This compound would be a useful tool to determine the role of arachidonic acid metabolites in the etiology of inflammatory dermatoses.

Dicarboxylic acid inhibitors of phospholipase A2

Abstract Ten diacids were synthesized via a simple regio- and stereoselective aldol reaction. All of these compounds were good to excellent inhibitors of 14 kDa human platelet PLA2, and many of these derivatives displayed activity in a phorbol-ester induced mouse ear edema assay. One of the new compounds reported here was selected for further development as a potential antipsoriasis agent.