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-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.

Evaluation of retinoid lactones as topical therapeutic agents in dermatology.

PURPOSE: Optimization of the therapeutic ratio of analogs of the topically active 11-cis, 13-cis-12-hydroxymethylretinoic acid, delta-lactone (1) relative to antihyperproliferation and antihyperkeratinization vs. toxicity. METHODS: Nine analogs of 1, in which variations were made in the lipophilic cyclohexenyl moiety or in the lactone ring, were evaluated for topical activity against hyperkeratinization, inhibition of TPA-induced DNA synthesis and for skin irritation. RESULTS: Although more potent lactones than the parent lactone 1 were identified, none possessed the favorable therapeutic ratio associated with 1. CONCLUSIONS: The delta-lactone 1 possesses unique molecular features responsible for its desirable therapeutic ratio as an antihyperproliferative and antihyperkeratotic agent. In view of its very low systemic retinoid toxicity and the absence of any systemic toxicity, this lactone may be a good candidate for use in the topical treatment of acne.

Evaluation of retinoids as therapeutic agents in dermatology.

Evaluation of 13-cis-12-substituted analogues of retinoic acid in a series of dermatologic screens has revealed that structural modifications can lead to selectivity and specificity. An analogue, 11-cis,13-cis-12-hydroxymethylretinoic acid, delta-lactone, has been found to have good activity and to be devoid of topical and systemic toxicity.

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 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.

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.

The pharmacology of a novel topical retinoid, BMY 30123: comparison with tretinoin.

Preclinical studies pertaining to the pharmacology and toxicology of BMY 30123 (4-acetamidophenyl retinoate) are reported. BMY 30123 is a novel compound which has topical retinoid activity. This compound exhibits lower toxicity, both local and systemic, than other clinically used topical retinoids such as tretinoin (all-trans retinoic acid) in animal models. BMY 30123 is effective in a number of retinoid sensitive skin models including the rhino mouse utriculi reduction assay, the mouse epidermal hyperplasia model and in the suppression of DNA synthesis in mouse skin stimulated with phorbol ester. BMY 30123 was equipotent with tretinoin in these topical models. In the rhino mouse model the ED30 values for BMY 30123 and tretinoin were 0.037 and 0.015 mM, respectively. In addition, BMY 30123 was active in the UVB-induced photodamaged mouse model, another retinoid sensitive model. One of the problems associated with topically applied tretinoin is local irritation. Therefore, for topical therapy to be optimal, it is important to reduce or minimize local irritation. Repeated applications of BMY 30123 to rabbit skin resulted in low skin irritation. The first perceptible signs of skin irritation produced by BMY 30123 occurred at a dose 10 times higher than that observed for tretinoin. BMY 30123 also exhibits low retinoid activity after oral or i.p. administration in mice and produced no signs of hypervitaminosis A-related toxicity at twenty times the no effect dose of tretinoin. Because retinoids are effective modulators of epidermal growth and differentiation, this compound should be useful for the treatment of cutaneous disorders that exhibit altered epidermal differentiation such as acne, psoriasis, ichthyosis and epithelial tumours.(ABSTRACT TRUNCATED AT 250 WORDS)

The Yucatan miniature swine as an in vivo model for screening skin depigmentation.

The usefulness of the Yucatan miniature pig as a screen for skin dipigmenting activity by topical application was evaluated with standard compounds. This is a naturally occurring breed of swine with light brown to dark brown skin that is relatively hairless. The skin morphology, including the pattern of pigment distribution, in this breed of swine closely resembles the human skin. Test compounds examined in this study included the three standard compounds with known clinical depigmenting activity, hydroquinone (HQ), 4-hydroxyanisole (4HA) and tert-butyl catechol (TBC), each at a 5% concentration. Test materials in 25 microliters of propylene glycol/ethanol (50:50) were applied topically twice daily, 7 days a week for 90 days to test sites on each side of the dorsal mid-line. Test sites were graded weekly for variation in pigmentation and local irritation. After 90 days of test material application, skin biopsies of the test sites were taken for histological evaluation. Topical application of HQ, 4HA and TBC promoted marked skin depigmentation which was substantiated by reductions of pigment and melanocytes observed on microscopic examination. While both HQ and TBC produced marked local irritation, 4HA was only mildly irritating. These results suggest that the Yucatan pig, could be a potentially useful model for screening compounds with skin depigmenting activity.

BMY 30047: a novel topically active retinoid with low local and systemic toxicity.

In the treatment of various dermatological disorders, topically applied retinoids have potential therapeutic use with the advantage of improved localized activity and lower toxicity over systemically administered retinoids. However, most retinoids cause a significant degree of local irritation. In the present study, the ability to produce local activity with low local irritation potential was evaluated with a novel retinoic acid derivative. BMY 30047 (11-cis, 13-cis-12-hydroxymethylretinoic acid delta-lactone) is one of a series of retinoic acid derivatives in which the carboxyl function of the polar end was modified with the aim of achieving reduced local irritation and systemic toxicity while retaining the local therapeutic effect. BMY 30047 was evaluated and compared with all-trans retinoic acid for topical retinoid activity in several preclinical assay systems, including the utricle reduction assay in rhino mice, 12-o-tetradecanoylphorbol 13-acetate ester-stimulated ornithine decarboxylase induction in hairless mice and the UV light-induced photodamaged skin model in hairless mice. BMY 30047 was assessed for retinoid-type side effects by evaluating the skin irritation potential in rabbits after repeated topical application, and hypervitaminosis A-inducing potential in mice after i.p. injection. BMY 30047 demonstrated significant topical retinoid activity in several in vivo models with less skin irritation potential relative to the most used clinical concentrations of all-trans retinoic acid. BMY 30047 also showed very little systemic activity and did not produce any evidence of hypervitaminosis A syndrome at systemic doses 20 times greater than the no-effect dose of all-trans retinoic acid.

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 microM. BMY 30094 also blocked human platelet cyclooxygenase and lipoxygenase with IC50 values of 15.2 and 15.0 microM, 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 mumoles/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.

Effects of retinoids on phorbol ester-stimulated epidermal DNA synthesis and hyperplasia in hairless mice.

We investigated the effects of the retinoids, all-trans retinoic acid (t-RA), 13-cis retinoic acid, etretinate, and arotinoid ethyl ester, on 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced DNA synthesis, and epidermal hyperplasia in hairless mouse skin. Topical application of these retinoids produced dose-dependent inhibition of the TPA-induced epidermal DNA synthesis as measured by [3H]thymidine incorporation at 15 h after TPA application. However, this inhibition was only transient and did not affect the corresponding increase in epidermal cell layers measured at 40 or 70 h after TPA application. Fluocinonide also inhibited the epidermal DNA synthesis and failed to block TPA-induced epidermal hyperplasia. However, fluocinonide did effectively suppress the inflammation caused by TPA. In this paper we have shown that the suppression of TPA-stimulated DNA synthesis is a general property of topically applied retinoids. The biologic significance of a temporary suppression of TPA-stimulated epidermal DNA synthesis by the retinoids and fluocinonide is not understood at this time.

UVB-induced pigmentation in hairless mice as an in vivo assay for topical skin-depigmenting activity.

Several reports have demonstrated that exposure to ultraviolet light elicits increased pigmentation in the skin of the Skh:HR2 mouse. We have reexamined this model to assess its potential as a screen for compounds with skin-depigmenting activity. The application of the previously reported ultraviolet light-B (UVB) doses led to marked necrotic damage to the skin which greatly diminished the usefulness of the model for drug testing. We have modified this model by exposing the mice to a progressively increasing dose of UVB that promotes pigmentation with a marked reduction of skin irritation. Furthermore, for compound evaluation, we preselected only those mice which developed signs of increased pigmentation after the first week of UVB exposure. This was critical for any meaningful compound evaluation, since only about 50% of the mice eventually showed signs of increased pigmentation with UVB. Our modifications make it possible to use this model for evaluating new compounds with skin-depigmenting activity. The validity of this method has been examined with a number of compounds including hydroquinone, 4-hydroxyanisole, kojic acid and all-trans retinoic acid, all with known depigmenting activity.

Effects of ruthenium red and KCL on responses of guinea pig umbilical veins.

5-Hydroxytryptamine (10(-5) M), norepinephrine (10(-5) M) and acetylcholine (10(-5) M) induced maximal contractions in isolated guinea pig umbilical vein strips. Pretreatment with ruthenium red (2.7--90 mug/ml) for ten minutes reduced 5-hydroxytryptamine and norepinephrine responses in a dose-related manner but did not affect acetylcholine-induced responses. Ruthenium red alone did not produce contractions. In the presence of KC1 (125 MM) the responses to norepinephrine and acetylcholine were enhanced significantly. The sensitivity of umbilical veins to ruthenium red may be useful in determining to what extent various vasoactive agents utilize extracellular calcium to induce contractions.

L- -methyl- -hydrazino- -(3,4-dihydroxyphenyl)propionic acid: relative lack of antidecarboxylase activity in adrenals.

In rats previously treated with a monoamine oxidase inhibitor, the administration of 5-hydroxytryptophan results in increases in concentrations of 5-hydroxytryptamine in kidney, brain, and adrenal glands. When the peripheral L-aromatic amino acid decarboxylase inhibitor, L-alpha-methyl-alpha-hydrazino-beta-(3,4-dihydroxyphenyl)propionic acid (HMD) is administered prior to 5-hydroxytryptophan, the concentration of 5-hydroxytryptamine in kidneys does not rise, that of the brain increases slightly, and that of the adrenal rises markedly. This indicates that although the adrenal gland is a peripheral organ, it does not respond in the typical manner to the antidecarboxylase action of HMD. These results suggest that HMD does not gain free access into the adrenal medulla and that a possible "blood-adrenal barrier" may exist to this compound.