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.

BMS-229724 is a tight-binding inhibitor of cytosolic phospholipase A2 that acts at the lipid/water interface and possesses anti-inflammatory activity in skin inflammation models.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. BMS-229724 (4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethyl-sulfonyl]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone) was found to be a selective inhibitor of cPLA2 (IC50 = 2.8 microM) in that it did not inhibit secreted phospholipase A2 in vitro, nor phospholipase C and phospholipase D in cells. The compound was active in inhibiting arachidonate and eicosanoid production in U937 cells, neutrophils, platelets, monocytes, and mast cells. With a synthetic covesicle substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (K(I)*(app)) was determined to be 1. 10(-5) mol% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.35 mol%. The unit of concentration in the interface is mole fraction (or mol%), which is related to the surface concentration of substrate, rather than bulk concentration that has units of molarity. Thus, BMS-229724 represents a novel inhibitor of cPLA2, which partitions into the phospholipid bilayer and competes with phospholipid substrate for the active site. This potent inhibition of the enzyme translated into anti-inflammatory activity when applied topically (5%, w/v) to a phorbol ester-induced chronic inflammation model in mouse ears, inhibiting edema and neutrophil infiltration, as well as prostaglandin and leukotriene levels in the skin. In hairless guinea pigs, BMS-229724 was active orally (10 mg/kg) in a UVB-induced skin erythema model in hairless guinea pigs.

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.

Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-dimethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 microM). It was over 70 times more selective for the cPLA2 as compared with the human nonpancreatic secreted phospholipase A2, and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn -glycero-3-phosphomethanol containing 6-10 mol % of 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.097 +/- 0.032 mol % versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.3 +/- 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller KI*app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [3H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [3H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.

A beta-lactam inhibitor of cytosolic phospholipase A2 which acts in a competitive, reversible manner at the lipid/water interface.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 3,3-Dimethyl-6-(3-lauroylureido)-7-oxo-4-thia-1-azabicyclo[3,2,0] heptane-2-carboxylic acid (1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 72 microM). This beta-lactam did not inhibit other phospholipases, including the human nonpancreatic secreted phospholipase A2. The inhibition of cPLA2 was found not to be time-dependent. This, along with the observation that the degradation of the inhibitor was not catalyzed by the enzyme, demonstrates that the inhibition does not result from the formation of an acyl-enzyme intermediate with the active site serine residue. Moreover, the ring-opened form of 1 is also able to inhibit cPLA2 with near-equal potency. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol containing 6-10 mole percent of 1-palmitoyl-2-[1-14C]-arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition was defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.5 +/- 0.1 mole% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.4 +/- 0.1 mole%. Thus, 1 represents a novel structural class of inhibitors of cPLA2 which partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site.

Leukotriene B4 stimulates the release of arachidonate in human neutrophils via the action of cytosolic phospholipase A2.

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation and is involved in the receptor-mediated activation of a number of leukocyte responses including degranulation, superoxide formation, and chemotaxis. In the present research, stimulation of unprimed polymorphonuclear leukocytes (neutrophils) with LTB4 results in the transient release of arachidonate as measured by mass. This release of arachidonate was maximal at an LTB4 concentration of 50-75 nM and peaked at 45 s after stimulation with LTB4. The transient nature of this release can be attributed, in part, to a fast (< 60 s) metabolism of the added LTB4. Moreover, the inhibition of the reacylation of the released arachidonate with thimerosal results in greater than 4-times as much arachidonate released. Thus, a rapid reacylation of the released arachidonate also contributes to the transient nature of its measured release. Multiple additions of LTB4, which would be expected to more closely resemble the situation in vivo where the cell may come into contact with an environment where LTB4 is in near constant supply, yielded a more sustained release of arachidonate. No release of [3H]arachidonate was observed when using [3H]arachidonate-labeled cells. This indicates that the release of arachidonate as measured by mass is most probably the result of hydrolysis of arachidonate-containing phosphatidylethanolamine within the cell since the radiolabeled arachidonate is almost exclusively incorporated into phosphatidylcholine and phosphatidylinositol pools under the non-equilibrium radiolabeling conditions used. Consistent with the role of cytosolic phospholipase A2 (cPLA2) in the release of arachidonate, potent inhibition of the LTB4-stimulated release was observed with methylarachidonylfluorophosphonate, an inhibitor of cPLA2 (IC50 of 1 microM). The bromoenol lactone of the calcium-independent phosphospholipase A2. failed to affect LTB4-stimulated release of arachidonate in these cells.

Phosphorylation and calcium influx are not sufficient for the activation of cytosolic phospholipase A2 in U937 cells: requirement for a Gi alpha-type G-protein.

Differentiation with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line toward a monocyte/granulocyte-like cell results in the cell acquiring an ability to release arachidonate upon stimulation. In contrast, the calcium ionophore ionomycin was able to stimulate phospholipase C, as measured by inositol 1,4,5-trisphosphate formation, to equal extents in both undifferentiated and dBcAMP-differentiated U937 cells. The role and regulation of cytosolic phospholipase A2 (cPLA2) in the production of arachidonate in these cells when either the chemotactic peptide fMLP or ionomycin are used as stimulus were investigated. The ionomycin- and fMLP-stimulated release of arachidonate were sensitive to the cPLA2 inhibitor arachidonyl trifluoromethylketone (IC50 values of 32 and 18 microM, respectively), but were not inhibited by E-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2 H-pyran-2-one, a bromoenol lactone inhibitor of the calcium-independent phospholipase A2. These results, coupled with the inhibition of ionomycin-induced arachidonate production by electroporation of differentiated cells to introduce an anti-cPLA2, demonstrate that the cPLA2 is the enzyme responsible for arachidonate release in differentiated cells. SDS-PAGE and immunoblot analysis of differentiated cells showed the cells to contain both phosphorylated and unphosphorylated forms of cPLA2 (ratio of about 2: 3). Surprisingly, undifferentiated cells contain 30% more enzyme than differentiated cells and contain a higher percentage (approximately 75%) of the phosphorylated in the absence of stimulation. The inability of undifferentiated cells to produce arachidonate is not due to insufficient intracellular calcium concentrations since ionomycin induces large (820-940 nM) influxes of intracellular calcium in both differentiated and undifferentiated cells. This demonstrates that phosphorylation of cPLA2 andan influx of intracellular calcium are not sufficient to activate the enzyme to produce arachidonate. Instead, activation of a pertussis toxin-sensitive Gi alpha-type G-protein is required as evidenced by the production of arachidonate in undifferentiated cells stimulated with mastoparan, an activator of Gi alpha subunits, in combination with ionomycin. This activation of a Gi alpha-type G-protein is independent of modulations of adenylyl cyclase activity since cellular cAMP levels were not modulated upon treatment with mastoparan and ionomycin.

Sulfated galactocerebrosides as potential antiinflammatory agents.

Native sulfatides, as well as many sulfated glycolipids, have been shown to avidly bind to the selectin receptors. In vivo, native sulfatides significantly block activity in selectin-dependent inflammatory responses. The fact that nonsulfated galactocerebrosides did not inhibit selectin-mediated adhesion identified a critical role for the anionic sulfate residue. We therefore initiated a program to evaluate the activity of position isomers. This study showed a binding selectivity for the positions 2 and 3 of the sulfate group on the carbohydrate ring as well as enhanced activity for the disulfated analogs. Furthermore, it was discovered that the attachment of lipophilic substituents on the carbohydrate ring was tolerated, consistent with the presence of a lipophilic pocket in the binding activity. This resulted in compounds with a 6-fold increased potency.

BMS-190394, a selectin inhibitor, prevents rat cutaneous inflammatory reactions.

Selectin binding is the first step in extravasation of leukocytes through the endothelium. Infiltration of leukocytes is a hallmark of an inflammatory response. Blockade of selectin-dependent adhesion, therefore, represents a specific mechanism-based anti-inflammatory strategy. We have used the natural product sulfatide, one of the selectin ligands, as a template to design a novel selectin antagonist. BMS-190394, a structural analog of sulfatide, is an inhibitor of cell binding to P-, E- and L-selectin-Ig fusion proteins. BMS-190394 also inhibits binding mediated by native P-selectin expressed on the surface of activated platelets. Pharmacokinetic analysis of BMS-190394 showed that the compound remained in circulation with a T1/2 of 7 hr, long enough to inhibit the development of an acute inflammatory response. The in vitro activity and pharmacokinetic profile of this selectin-blocking compound led to the determination of its in vivo anti-inflammatory activity. BMS-190394 was a potent inhibitor of the dermal immune complex-induced reverse passive Arthus reaction in rats when delivered by the i.v. or i.p. route. The ED50 of the compound in the reverse passive Arthus reaction compares favorably to that for dexamethasone. BMS-190394 was also an effective inhibitor of the delayed-type hypersensitivity reaction in the rat. Compared with previous reports of the use of antibodies and complex oligosaccharides to inhibit the activity of the selectins, this low-molecular-weight inhibitor of the selectins presents a novel class of anti-inflammatory agents.

Cooperativity and binding in the mechanism of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to be responsible for the receptor-regulated release of arachidonic acid from phospholipid pools. The enzyme was assayed using vesicles containing arachidonate-containing phospholipid substrate, such as 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) or 1-stearoyl-2-arachidonoylphosphatidylinositol (SAPI), dispersed within vesicles of 1,2-dimyristoylphosphatidylmethanol (DMPM). We report here that the enzyme shows an apparent cooperative effect with respect to the mole fraction of arachidonate-containing phospholipids within these covesicles. The data can be fit to a modified Hill equation yielding Hill coefficients, n, of 2-3. This effect is unusual in that it is dependent on the nature of the sn-2 ester as opposed to the phosphoglycerol head group. This cooperativity is independent of both the concentration of glycerol, which greatly increases enzyme activity and stability, and the concentration of calcium, which facilitates the fusion of the covesicles. Surprisingly, 1-palmitoyl-2-arachidonoylphosphatidylethanolamine (PAPE) does not show the same cooperative effect, although the rate at which it is hydrolyzed is much greater when PAPC is present. Moreover, PAPE has a dissociation constant from the active site (KD* = 0.7 mol %) which is comparable to that of PAPC and SAPI (KD* values of 0.3 and 0.3 mol %, respectively). These results are consistent with the presence of an allosteric site that, when occupied, induces a change in the enzyme which facilitates enzymatic hydrolysis. If so, PAPC and SAPI, but not PAPE, must be able to bind to this allosteric site. Alternatively, this effect may result from changes in the physical nature of the bilayer which result upon increasing the bilayer concentration of arachidonate-containing phospholipids. This previously unobserved effect may represent another mechanism by which cells can regulate the activity of cPLA2.

Phorbol ester-induced upregulation of polymorphonuclear leukocyte P-selectin ligand expression.

Proinflammatory stimuli cause the vascular endothelium to express P-selectin that tethers leukocytes by binding surface glycoprotein carbohydrate. While the activation of polymorphonuclear leukocytes (PMN) is associated with upregulation of the beta 2-integrins, there is little known about the regulated expression of the ligand for the endothelial P-selectin. We have used a soluble chimeric P-selectin protein as a probe for the expression of ligand on the surface of the PMN. Treatment with phorbol ester for more than 20 min stimulated P-selectin ligand expression. The upregulation of beta 2-integrin expression was affected in a similar manner. The mechanism of selectin ligand upregulation did not involve de novo protein synthesis, and may involve translocation of membranes containing performed intracellular ligand. C5a, which is generated in response to complement activation in vivo, also stimulated selectin ligand upregulation. Degranulation induced by nigericin increased ligand expression, and TNF-alpha treatment resulted in a modest upregulation.

In-vivo activity of retinoid esters in skin is related to in-vitro hydrolysis rate.

BMS-181163 (4-acetamidophenyl retinoate, previously reported as BMY-30123), the acetamidophenyl ester of all-trans-retinoic acid (tRA), is topically active in various retinoid-sensitive animal models, but was recently shown to be ineffective for the treatment of acne in patients. To determine whether BMS-181163 functions as a prodrug of tRA in mice but not in man, the relative rates of ester hydrolysis in mouse and human skin homogenates were determined. In-vitro hydrolysis assays showed that BMS-181163 was substantially hydrolysed in mouse skin homogenates and minimally in human skin preparations. In addition, a series of phenyl esters of tRA and several known active synthetic retinoids (Ch-80: (E)-4-[3-oxo-3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1 - propenyl] benzoic acid; CD-271: 6-[3-(1-adamantyl)-4-methyoxyphenyl]-2-naphthoic acid; and TTNPB: (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl] benzoic acid) was prepared and hydrolysis rates and in-vivo (rhino mouse utriculi reduction) activities were compared. The hydrolysis rates of the six test retinoid phenyl esters, ranging from 0.06 to 2.0 h-1 were found to correlate with the in-vivo activity. Those esters (BMS-181163 and acetamidophenyl esters of Ch-80 and TTNPB) with a higher hydrolysis rate exhibited in-vivo activity only slightly lower than their parent free acid retinoids. In contrast, the three phenyl esters with a hydrolysis rate less than 0.3 h-1 were inactive in-vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of inhibition of human nonpancreatic secreted phospholipase A2 by the anti-inflammatory agent BMS-181162.

Many important mediators of inflammation result from the liberation of free arachidonic acid from phospholipid pools which is thought to result from the action of phospholipase A2 (PLA2). It is believed, therefore, that the inhibition of PLA2 would be an important treatment in many inflammatory disease states. The anti-inflammatory agent BMS-181162 (4-(3'-carboxyphenyl)-3,7-dimethyl-9-(2",6",6"-trimethyl-1"-cyclohexenyl )-2Z,4E , 6E,8E-nonatetraenoic acid) selectively inhibits PLA2 and has been shown to block arachidonic acid release in whole cells. The mechanism of inhibition of human non-pancreatic-secreted PLA2 by BMS-181162 is investigated in this paper. A scooting mode assay in which the enzyme is irreversibly bound to vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol containing 5 mol % of 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine, was used to characterize the inhibition. With this assay system, BMS-181162 inhibited the enzyme in a dose-dependent manner. Compounds which inhibit in the scooting mode have been shown to be competitive inhibitors in the interface (Gelb, M. H., Berg, O., and Jain, M. K. (1991) Curr. Op. Struct. Biol. 1, 836-843). This was verified by demonstrating that the inhibition was not due to the desorption of the enzyme from the lipid-water interface. Additionally, the compound did not measurably affect the rate of association onto the vesicles. Therefore, the inhibition was not the result of a modulation of the bilayer morphology nor an interaction with the interfacial binding site on the enzyme. The degree of inhibition was dependent on the reaction volume which indicates that the inhibitor is only partially partitioned into the bilayer. After compensating for this partitioning, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the interface. The equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*) was determined to be 0.013 mol fraction, thus demonstrating that BMS-181162 represents a novel structural class of tight-binding competitive inhibitors of human nonpancreatic secreted PLA2. Using Escherichia coli membranes as substrate, to which the enzyme binds to the interface reversibly, the inhibition showed a nonclassical kinetic pattern which is also consistent with a partial partitioning of the inhibitor into the bilayer. This was verified by a direct measurement of the amount of inhibitor remaining in solution. The implications for in vivo efficacy which result from this mechanism are discussed.

Gene expression of retinoic acid receptors and cellular retinoic acid-binding proteins in rhino and hairless mouse skin.

The rhino mouse comedolytic model and the hairless mouse photoaging model are established animal models for screening the in vivo activity of retinoids. However, the expression of the retinoic acid receptors (RARs) and cellular retinoic acid-binding proteins (CRABPs), known to regulate retinoid activity, is not completely understood in these mouse mutants. For this purpose, mRNA was isolated from rhino and hairless mouse skin and the gene expression of the RARs and CRABPs was measured by Northern blot hybridization. Results showed that RAR gamma was the predominantly expressed RAR in both mouse strains. Two isoforms of RAR gamma, RAR gamma 1 and RAR gamma 2, were detected with RAR gamma 1 being the more strongly expressed. RAR alpha was also detected, but to a lesser degree than RAR gamma. RAR beta expression was not detectable by our methodology. Additionally, topical treatment of these mice with 0.1% all-trans-retinoic acid (tRA) cream resulted in no significant alteration in the expression of the RAR genes. By contrast, CRABP-II was induced 2-4 fold by topical tRA treatment. CRABP-I, expressed to a lesser degree than CRABP-II, was not inducible. The relative expression of the RARs, CRABPs, and inducibility of CRABP-II by tRA in both rhino and hairless mouse skin paralleled that reported for human and mouse skin. These observations suggest that the altered phenotype observed in the rhino mouse most likely does not result from an altered expression level of these genes. The results also support these two animals as models for evaluating the therapeutic potential of retinoids.

Inhibitor of phospholipase A2 blocks eicosanoid and platelet activating factor biosynthesis and has topical anti-inflammatory activity.

The effect of a phospholipase A2 (PLA2) inhibitor on leukotriene, prostaglandin and platelet activating factor (PAF) biosynthesis in isolated cells and in vivo was determined. BMS-181162, [4(3'-carboxyphenyl)-3,7-dimethyl-9(2",6",6"-trimethyl-1"- cyclohexenyl)2Z,4E,6E,8E-nonatetraenoic acid], reversibly inhibited the 14-kdalton PLA2 purified from human synovial fluid with an IC50 of 8 microns. In A23187-stimulated human polymorphonuclear leukocytes (PMNs), BMS-181162 blocked arachidonic acid release with an IC50 of 10 microns. Leukotriene B4 and PAF biosynthesis in these cells was also inhibited. In a phorbol ester-induced chronic mouse skin inflammation model, topically applied BMS-181162 markedly lowered the tissue levels of leukotriene B4 and prostaglandin E2 and dose-dependently inhibited leukocyte infiltration (ED50 = 180 micrograms per ear). BMS-181162 is an inhibitor of PLA2 and may prove to be a useful tool in the delineation of the role of PLA2 in the inflammatory process.

Inhibition of Immune Complex-Induced Inflammation by A small Molecular Weight Selectin Antagonist.

The anti-inflammatory effect of a small molecular weight antagonist of P- and E-selectin-dependent cell adhesion was examined. The glycolipid sulphatide was shown to block the adherence of thrombin-activated rat platelets to HL-60 cells. This interaction is known to be dependent on P-selectin. The rat dermal reverse passive Arthus reaction was used to assess the effect of sulphatide on a neutrophil dependent inflammatory response. Sulphatide dosedependently blocked both the vascular permeability increase and cell infiltration after intraperitoneal administration. These results show that a small molecular weight compound which blocks P- and E-selectin dependent adhesion in vitro can effectively block the inflammation due to immune complex deposition. A compound with this type of profile may have therapeutic potential in the treatment of immune complex mediated diseases.

Combination of a dual 5-lipoxygenase/cyclooxygenase inhibitor with a glucocorticoid results in synergistic topical antiinflammatory activity without inducing skin atrophy.

Synthetic glucocorticosteroids are widely used in clinical dermatology in the treatment of a number of inflammatory skin disorders. However, cutaneous side effects such as induction of skin atrophy are among the factors that limit the chronic use of this class of agents. We have found that a combination of a dual 5-lipoxygenase/cyclooxygenase inhibitor (9-phenylnonanohydroxamic acid, BMY 30094) and a glucocorticosteroid (hydrocortisone valerate) has synergistic antiinflammatory activity. Topical application of a 0.2% solution of hydrocortisone valerate alone inhibited phorbol ester-induced mouse skin inflammation by 92%. A 1% solution of BMY 30094 alone or a low dose of hydrocortisone valerate (0.005%) had no significant effect on the inflammatory reaction. A combination of 1% BMY 30094 and 0.005% hydrocortisone valerate inhibited the inflammation by 76%. This latter combination produced no signs of skin atrophy in rats after 28 days of application. In contrast, a 0.1% dose of hydrocortisone valerate produced significant skin atrophy. These findings suggest that it is possible to reduce the skin atrophy potential of glucocorticoids while maintaining the antiinflammatory activity using a novel drug combination.

Combination of 4-hydroxyanisole and all-trans retinoic acid produces synergistic skin depigmentation in swine.

A combination of 4-hydroxyanisole (4HA) and all-trans retinoic acid (TRA) was found to synergistically cause moderate to complete depigmentation of Yucatan swine skin. Two hyperpigmentation models were used: Natural dark-skinned swine, a potential model for melasma-like disorders, and ultraviolet light-stimulated hyperpigmentation, a model of solar lentigines. Test materials were applied twice daily, 5 d/week, to dorsal flank skin. Application sites were graded at weekly intervals for skin color using a 0 to 4 grading scale. After 8 weeks of treatment of naturally dark swine skin, a combination of 2% 4HA and 0.01% TRA produced grade 2 hypopigmentation (definite but moderate hypopigmentation). In contrast, 2% 4HA alone or 0.01% TRA alone did not produce significant hypopigmentation. After cessation of treatment, the 4HA/TRA-treated sites reverted to normal color within 7-12 weeks. The 4HA/TRA combination completely reversed the hyperpigmentation induced by ultraviolet light after 8 weeks of treatment. In vitro skin-penetration studies using hairless mouse and human skin show that skin penetration of 4HA was not significantly affected by adding 0.01% TRA. These data suggest that the observed synergy is not due to enhanced bioavailability of 4HA. We have demonstrated that combining low concentrations of 4HA and TRA results in effective skin lightening without causing irreversible depigmentation and with minimal local skin irritation.

Effects of all-trans retinoic acid on glycosaminoglycan synthesis in photodamaged hairless mouse skin.

All-trans retinoic acid (tRA) was previously shown to be active in wrinkle reduction in the hairless mouse photoaging model. To address the questions of whether tRA also alters glycosaminoglycan (GAG) synthesis and whether observed wrinkle effacement can be attributed to changes in total GAG production, the effects of tRA on de novo GAG synthesis were examined in this model. Sulfated glycosaminoglycans (S-GAG) and the non-sulfated hyaluronic acid (HA) labeled with [3H]-glucosamine or [35S]-sulfate were found to diffuse differentially into the medium during the labeling period in the control animals (9% and 35% of total incorporated label for HA and S-GAG, respectively). Furthermore, the diffusion of HA into media was significantly changed after tRA treatment (from 9% to 24%), but no alteration was observed in the diffusion of S-GAG. Separation of epidermis and dermis indicated that the additional HA in medium after retinoid treatment primarily originated from the dermis. When incorporated label from the medium and skin fractions was combined, both labeling protocols revealed that 10 weeks of tRA treatment did not increase the total (medium plus skin) de novo synthesis of either HA or S-GAG. Wrinkle effacement as induced by retinoids in the photodamaged mouse skin therefore can not be related to an increased total GAG synthesis.

Swine as a model of skin inflammation. Phospholipase A2-induced inflammation.

A predictive animal model of skin inflammation is needed for the development of potential therapeutic agents. The existing models of inflammation rely on animals whose skin physiology or biochemistry differs significantly from human. The objective of this investigation was to evaluate the swine as a potential model of inflammation, because its skin has been recognized to exhibit morphologic and functional similarities to human skin. In the swine, an inflammatory response was produced following intradermal injection of snake venom phospholipase A2 (PLA2). This response was characterized by transient erythema (2-3 h) and microscopic changes of cell infiltration, epidermal hyperplasia, and dermal damage, which were apparent two days after PLA2 and peaked by day 7. In general, these microscopic changes persisted up to 21 days. Treatment with the antiinflammatory steroid, betamethasone dipropionate (Diprolene), gave a significant reduction of the inflammatory responses. Heat-inactivated PLA2, ovalbumin, or saline did not provoke this reaction, although PLA2 inactivated by bromophenacyl bromide alkylation did produce an inflammatory response. The alkylated PLA2 was also able to provoke an inflammatory response in the mouse paw edema assay. These results demonstrate that PLA2 can stimulate an inflammatory response in the swine skin, but that phospholipid hydrolytic activity is not required.