Cutaneous responses to 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE).

The responses to 12-HETE in normal human skin have been investigated by means of intradermal and topical administration in 15 subjects. Intradermal infusion of 12-HETE produced a neutrophil polymorphonuclear and mononuclear infiltrate in the dermis. Topical administration resulted in a dose-related erythematous response to 200 ng-50 micrograms. This was accompanied by a neutrophil and mononuclear dermal infiltrate at 6 and 24 h after application. In addition, collections of neutrophils were present in the epidermis in 4 of 10 subjects biopsied at 6 h and in all patients biopsied 24 h after topical application. Intradermal and topical application of 9-hydroxyoctadecadienoic acid (9-HODD), a chemically similar but chemokinetically inactive substance, did not produce neutrophil infiltration of the epidermis, nor did the chemical irritant nonanoic acid. The results suggest that the cellular infiltrates produced in vivo in humans by 12-HETE are due to its chemoattractant properties and are not the result of a nonspecific inflammatory response.

Computer-aided design and synthesis of 5-substituted tryptamines and their pharmacology at the 5-HT1D receptor: discovery of compounds with potential anti-migraine properties.

The design and synthesis of a series of novel 5-substituted tryptamines with pharmacological activity at 5-HT1D and other monoamine receptors is described. Structural modifications of N- and C-linked (principally hydantoin) analogues at the 5-position were synthesized and their pharmacological activities were utilized to deduce significant steric and electrostatic requirements of the 5-HT1D and 5-HT2A receptor subtypes. Conformations of the active molecules were computed which, when overlaid, suggested a pharmacophore hypothesis which was consistent with the affinity and selectivity measured at 5-HT1D and 5-HT2A receptors. This pharmacophore is composed of a protonated amine site, an aromatic site, a hydrophobic pocket, and two hydrogen-bonding sites. A "selectivity site" was also identified which, if occupied, induced sensitivity for 5-HT1D over 5-HT2A in this series of molecules. The development and use of the pharmacophore models in compound design is described. In addition, the physicochemical constraints of molecular size and hydrophobicity required for efficient oral absorption are discussed. Utilizing the pharmacophore model in conjunction with the physicochemical constraints of molecular size and log DpH7.4 led to the discovery of 311C90 (6), a new selective 5-HT1D agonist with good oral absorption and potential use in the treatment of migraine.

Contrasting in vitro lymphocyte chemotactic activity of the hydroxyl enantiomers of 12-hydroxy-5,8,10,14-eicosatetraenoic acid.

1. The chemotactic activity of 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(R)-HETE), 12(S)-HETE and leukotriene B4 (LTB4) for human mixed peripheral blood lymphocytes has been assessed in a 48-well microchemotaxis assay. Responses to the standard lymphocyte chemoattractants, zymosan-activated plasma, casein and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were also measured. 2. 12(R)-HETE was shown to be chemotactic for lymphocytes over the range 5 x 10(-7) to 5 x 10(-5) M. In contrast, negligible chemotactic responses to 12(S)-HETE were obtained. 3. LTB4 was 200 times more potent than 12(R)-HETE as a lymphocyte chemoattractant, although maximal responses to the two agonists were similar. 4. 12(R)-HETE and LTB4, which are present in extracts of samples from the skin lesions of psoriasis, may be, at least in part, responsible for the lymphocyte infiltrate which is a characteristic feature of this disease.

Psoriatic skin lesions contain a novel lipid neutrophil chemokinetic compound which is distinct from known chemoattractant eicosanoids.

1. Lipid extracts of scale from the lesions of the skin disease psoriasis were purified by high performance liquid chromatography (h.p.l.c.). Assay of fractions by an agarose microdroplet method showed the presence of a novel neutrophil chemokinetic compound which possessed the chromatographic properties of a monohydroxy fatty acid, yet was distinct from the chemoattractant eicosanoid, 12-hydroxyeicosatetraenoic acid, previously isolated in psoriasis. 2. The novel, material, termed compound X, was also detected in fractions collected on h.p.l.c. of extracts of chamber fluid samples obtained from abraded psoriatic lesions, but was not detectable in samples from clinically normal skin. 3. Comparison of the straight and reversed phase h.p.l.c. retention times of compound X with those of a range of standard monohydroxy fatty acids, together with further analysis by gas chromatography-mass spectrometry and assay of selected standards for neutrophil chemokinetic activity, failed to reveal the structural identity of compound X. 4. The finding of a further compound in psoriatic lesions, which stimulates neutrophil movement, highlights the complexity of inflammatory mediator production in this disease.

The chemokinetic response of psoriatic and normal polymorphonuclear leukocytes to arachidonic acid lipoxygenase products.

Polymorphonuclear leukocytes (PMN) from ten patients with chronic stable plaque psoriasis, five of whom had more than 40% skin involvement and five with less than 20% involvement, responded in a dose-related fashion to stimulation with the arachidonic acid lipoxygenase products 5- and 12-hydroxyeicosatetraenoic acid (5- and 12-HETE) and leukotriene B4 (LTB4) in an in vitro chemokinesis assay. There was no significant difference in either the random migration or the chemokinetic response of psoriatic PMN to LTB4 when compared to the responses of PMN from a group of age- and sex-matched healthy controls. Psoriatic PMN migrated further in response to low doses of 5- and 12-HETE although the distance moved after maximal stimulation was no different to that observed in controls. No significant difference was observed in the responses of PMN obtained from patients with less than 20% skin involvement when compared to those with more extensive psoriasis. The small differences measured between the chemokinetic responses of psoriatic and control PMN to the lipoxygenase products tested are unlikely to be of pathogenetic significance.

Stereochemical difference between 12-hydroxy-5,8,10,14-eicosatetraenoic acid in platelets and psoriatic lesions.

Stereochemical analysis of 12-hydroxy-5,8,10,14-eicosatetraenoic acid derived from the lesional scale of patients with psoriasis is reported. Resolution of the C-12 hydroxyl enantiomers was carried out by high pressure liquid chromatography of their diastereomeric methyl ester dehydroabietyl urethane derivatives. The 'psoriasis derived" compound was shown to be stereochemically distinct from the platelet 12(S)-enantiomer as its derivative co-chromatographed with the 12(R)-diastereomer.

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid is a chemoattractant for human polymorphonuclear leucocytes in vitro.

Increased amounts of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) are found in the lesional skin of patients with the skin disease psoriasis when compared to clinically normal skin. Stereochemical analysis has recently shown that the 12-HETE present in lesional psoriatic scale is the (R), and not the (S) hydroxyl enantiomer, produced by platelets. Since the chemoattractant activity of 12(R)-HETE has not previously been described, the (R) and (S) hydroxyl enantiomers of 12-HETE have now been synthesised and their chemokinetic activity compared in vitro. 12(R)-HETE, was more potent than 12(S)-HETE as a chemokinetic agent for human polymorphonuclear leucocytes but 2000 times less potent than leukotriene B4. In contrast to results obtained with the 12-HETE enantiomers, the chemoattractant compound 5(S)-HETE was found to be more potent than the 5(R) hydroxyl enantiomer. Thus, the configuration of the hydroxyl group appears to be of importance to the chemokinetic activity of the HETEs, and the increased potency of the 12(R) enantiomer may enhance its significance as a mediator of inflammation in psoriasis.

Lipoxygenase products. A novel gas chromatographic-mass spectrometric assay for monohydroxy fatty acids.

We present a novel mass spectrometric method for the profiling of monohydroxy fatty acids and illustrate its use for the analysis of lesional skin of patients with psoriasis. By combination of vapour phase hydrogenation and gas chromatography-mass spectrometry with selected ion monitoring the position of the substituted hydroxyl group of each fatty acid was determined in their methyl ester trimethylsilyl ether derivatives. Reduction was instantaneous and quantitative allowing detection of less than 100 picogram of each compound. Biological extracts, derivatised as methyl ester tert.-butyldimethylsilyl ethers, were purified by reversed-phase high-performance liquid chromatography. Separation was dependent on carbon chain length and degree of unsaturation, but not on the position of the silyl ether group. Subsequent conversion of the tert.-butyldimethylsilyl ethers to trimethylsilyl ethers facilitated detection of each of the positional hydroxyl isomers as described. Distinction between double bond isomers was possible when they were separated on gas chromatography prior to reduction.

Use of high-performance liquid chromatography-thermospray mass spectrometry and gas chromatography-electron-impact mass spectrometry in the identification of the metabolites of alpha-methylacetohydroxamic acids, potential anti-asthmatic agents.

Two potential anti-asthmatic alpha-methylacetohydroxamic acids, compound 1 and compound II were metabolised to two major products (metabolite 1 and metabolite 2) after oral dosing to rabbits. Metabolite 1, extracted under acid conditions from the plasma and urine of dosed animals, was identified as a glucuronide by incubation with beta-glucuronidase and subsequent high-performance liquid chromatographic-mass spectrometric (HPLC-MS) analysis of the aglycone. HPLC-MS analysis of metabolite 2 suggested that it was the acetamide, however, unequivocal identification was obtained by further analysis using gas chromatography-mass spectrometry (GC-MS) of its trimethylsilyl derivative and by comparison with the mass spectra of the authentic acetamides. This study shows the advantages of combining HPLC-MS with other techniques such as GC-MS for the identification of metabolites.

Proinflammatory properties of unsaturated fatty acids and their monohydroxy metabolites.

The proinflammatory effects of unsaturated fatty acids and, where appropriate, their monohydroxy derivatives, have been investigated both by application to human skin and with respect to human polymorphonuclear leukocyte (PMN) migration. Of the fatty acids applied to the skin only eicosapentaenoic and arachidonic acids (EPA; AA) produced consistent, measurable erythema. The monohydroxy derivatives of the two fatty acids also caused erythema, the 12-hydroxy isomers being the most potent. Chemokinetic activity towards PMNs was observed in the presence of AA, EPA and alpha-linolenic acid using an agarose microdroplet chemokinesis assay. In contrast to their in vivo properties, the 5-hydroxy isomers of AA and EPA were the most potent, being approximately 10 times more chemokinetically active than the other isomers. Quantification of the hydroxyeicosatetraenoic and hydroxyeicosapentaenoic acids (HETEs; HEPEs) in the lesional skin of psoriatic patients demonstrated that, of the metabolites measured, 12-HETE was present in the greatest amounts. Twenty five times more 12-HETE than 12- or 15-HEPE was detected, these being the most abundant of the HEPEs formed. The monohydroxy derivatives of AA and EPA may contribute to the inflammatory changes observed in psoriasis. The HETEs appear to be of greater importance than the HEPEs in view of the relative amounts present.

Structure-activity relationship investigations of a potent and selective benzodiazepine oxytocin antagonist.

We have investigated the structure-activity relationships of the 1- and 3-substituents and replacements of the 5-phenyl group of GW405212X 1, a potent selective oxytocin antagonist. The effect of these modifications on oxytocin binding antagonism and on pharmacokinetic parameters is reported.

The identification of hydroxy fatty acids in psoriatic skin.

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B4 and monohydroxy-eicosatetraenoic acid (HETE)-like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified: 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10(-4)M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.

Topical steroid treatment reduces arachidonic acid and leukotriene B4 in lesional skin of psoriasis.

Topical clobetasol propionate or vehicle ointment was applied daily for 3 days to psoriatic plaques on eight patients. Skin chamber exudates from untreated, steroid and vehicle treated lesions were assayed for arachidonic acid (AA), leukotriene B4 (LTB4), prostaglandin E2 (PGE2) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) before, and at 24 h and 72 h after treatment. Significant reductions in AA and LTB4 were observed at 72 h in steroid treated lesions. The reduction in 12-HETE levels observed after steroid treatment was not statistically significant. PGE2 levels in lesional psoriatic skin were unaltered. The reduction of AA, and LTB4 was associated with clinical improvement of psoriasis.

Neutrophil aggregating and chemokinetic properties of a 5,12,20-trihydroxy-6,8,10,14-eicosatetraenoic acid isolated from human leukocytes.

Suspensions of human peripheral blood leukocytes were incubated with arachidonic acid and calcium ionophore A23187. Acidic lipid extracts were purified by silicic acid chromatography and reversed phase high performance liquid chromatography. A polar metabolite with an ultraviolet absorption spectrum identical to that of leukotriene B4 was identified by gas chromatography and mass spectrometry as a 5,12,20-trihydroxy-6,8,10,14-eicosatetraenoic acid. The data indicated that this compound was the 20-hydroxy metabolite of leukotriene B4. The compound showed chemokinetic activity towards human polymorphonuclear leukocytes and aggregating activity towards rat peritoneal polymorphonuclear leukocytes. Its activity was greater than that of 5-hydroxy-6,8,11,14-eicosatetraenoic acid but less than that of leukotriene B4. It is concluded that the 15,12,20-trihydroxyeicosatetraenoic acid may play a role as a mediator of inflammatory reactions.