Frank Carey

The local anti-inflammatory action of dexamethasone in the rat carrageenin oedema model is reversed by an antiserum to lipocortin 1

1 A local pre‐injection of 1 μg dexamethasone sodium phosphate strongly inhibited (> 60% inhibition at 3 h; P < 0.001 at all time points) the development of carrageenin‐induced paw oedema in the rat induced by a subplantar injection of 0.1 ml, 2% carrageenin. 2 Coinjection of a polyclonal rabbit antiserum raised against human 1–188 recombinant lipocortin 1, which also recognised the rat protein, reversed the inhibitory action of dexamethasone (P < 0.05 at 4 h and 5 h). At the highest volume used (40 μl) control antisera were without any effect. 3 These data further support the concept that lipocortin 1 is involved in the anti‐inflammatory mechanism of action of the glucocorticoids.

The role of lipocortin‐1 in dexamethasone‐induced suppression of PGE2 and TNFα release from human peripheral blood mononuclear cells

1 Lipocortin‐1 and its N‐terminal derivatives exert potent inhibitory actions in various models of acute inflammation. The present study examined the ability of lipocortin (LC)‐1 to suppress the release of the acute pro‐inflammatory mediators, tumour necrosis factor (TNFα) and prostaglandin E2 (PGE2) from human peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or recombinant human interleukin‐1β (rhIL‐1β). 2 LPS (10 μg ml−1)‐stimulated release of TNFα and PGE2 from PBMC was significantly inhibited by (4 h) co‐incubation of the cells with 10−6 m dexamethasone (Dex), but not with 10−9 m to 10−7 m of a N‐terminal fragment (amino acids 1–188) of recombinant human LC‐1 (LC‐1 fragment). However, Dex suppression of LPS‐stimulated TNFα and PGE2 secretion from PBMC was reversed when polyclonal antibody to LC‐1 fragment (1:10,000 dilution) was included in the medium. rhIL‐1β (5times10−8 m)‐stimulated release of TNFα and PGE2 from PBMC (after 18 h) was abolished by co‐incubation of the cells with 10−7 m LC‐1 fragment. 3 After incubation with Dex (4 h), cellular proteins from PBMC were immunoblotted using anti‐LC‐1 fragment antibody (which showed no cross‐reactivity with human annexins 2 to 6). Dex caused no increase in immunoreactive (ir)LC‐1 content of PBMC, although there was a three fold increase in the amount of a lower mass species with LC‐1‐like immunoreactivity. This was accompanied by the appearance of irLC‐1 in the extracellular medium. 4 The results of the present study implicate endogenous LC‐1 in glucocorticoid suppression of TNFα and PGE2 release from human PBMC and suggest an extracellular site of action for LC‐1. LC‐1 may also inhibit rhIL‐1β‐stimulated TNFα and PGE2 secretion from PBMC.

Evidence for the presence and location of annexins in human platelets

In this study the identity of annexins in human platelets has been determined together with their ability to be released by agents which induce platelet degranulation. The presence of proteins cross-reacting to antibodies against annexins I and V was detected in human platelets. However, the study provided evidence that these annexins are not located on the surface of the plasma membrane in a Ca++ dependent manner. Moreover, activation of platelets with several agents which induced platelet degranulation did not cause release of annexins I or V as determined by both immunoblotting and ELISA.

Simple procedure for measuring the pharmacodynamics and analgesic potential of lipoxygenase inhibitors

A model is described for determining the pharmacodynamics of inhibitors of arachidonate metabolism in mice. Bioavailability and selectivity were assessed by ex vivo RIA of TXB2, LTB4, and 12-HETE from ionophore-challenged blood. Inhibition of LTB4 and 12-HETE was measured using a single LTB4 RIA, following extraction and separation of these eicosanoids from plasma. Separation on cyanopropyl mini-columns yielded hexane/ether and methanol fractions, which contained 12-HETE and LTB4, respectively. Analgesic efficacy was measured by inhibition of phenylbenzoquinone-induced abdominal constriction. The NSAIDs, indomethacin ibuprofen, flurbiprofen, and benoxaprofen, were analgesic and selective cyclo-oxygenase inhibitors. BW775C was also analgesic, but inhibited cyclo-oxygenase, 5-lipoxygenase and 12-HETE formation. Other in vitro 5-lipoxygenase inhibitors, NDGA, quercetin, and nafazatrom, were inactive in vivo, although NDGA reduced abdominal constrictions. The results indicate that this model has utility in determining the mechanism/selectivity of action and analgesic potential of 5-lipoxygenase inhibitors.

Thromboxane synthase inhibition: Implications for prostaglandin endoperoxide metabolism: I Characterisation of an acute intravenous challenge model to measure prostaglandin endoperoxide metabolism

It has been proposed that thromboxane synthase inhibition (TXSI) may be a useful form of anti-thrombotic therapy and that this is due, in part, to redirection of PGH2 metabolism in favour of PGI2, a potent vasodilator and anti-platelet agent. While redirection has been observed ex vivo there are conflicting reports of its occurrence in vivo. We now describe the characterisation of an acute intravenous challenge model using thrombin, collagen, arachidonic acid (AA) and PGH2 for the study of PGH2 metabolism. Following challenge, plasma concentrations of TXB2, 6-oxo-PGF1 alpha, alleged metabolites of PGI2 (PGI2m) and PGE2 were measured by radioimmunoassay (RIA). Thrombin and collagen challenge resulted in a dose-related increase in plasma TXB2 while AA and PGH2, in addition, elevated 6-oxo-PGF1 alpha and PGI2m. Injection of PGH2 elevated 6-oxo-PGF1 alpha, PGI2m, TXB2 and PGE2 levels. Experimental conditions were defined such that challenge with thrombin (40 NIH units kg-1), collagen (100 micrograms kg-1), AA (1 mg kg-1) and PGH2 (5 micrograms kg-1) and measurement of eicosanoids 0.5 min following challenge were optimal for detection of redirection of PGH2 metabolism in vivo. The identity of immunoreactive TXB2 and 6-oxo-PGF1 alpha was further supported by experiments in which the extracted immunoreactive eicosanoids co-eluted with authentic [3H]standards when subject to reverse phase high performance liquid chromatography (RPHPLC). Evidence is also presented that the levels of plasma eicosanoids measured in this model reflect in vivo biosynthesis.

Thromboxane synthase inhibition: Implications for prostagland endoperoxide metabolism II testing the ‘redirection hypothesis’ in an acute intravenous challenge model

In the preceding paper we described the characterisation of an acute intravenous challenge model for the evaluation of the effects of thromboxane synthase inhibition (TXSI) on eicosanoid metabolism. Herein we describe the biochemical pharmacology of two TXSI and aspirin in this model. Both TXSI caused significant inhibition of plasma TXB2 in vivo without elevation of 6-oxo-PGF1 alpha levels. Similar results were obtained when combined levels of 6-oxo-PGF1 alpha,13,14 dihydro 6-oxo-PGF1 alpha,13,14 dihydro 6,15-dioxo-PGF1 alpha and 6-oxo-PGE1 were measured as an index of PGI2 biosynthesis (PGI2m). Thus no evidence of in vivo redirection of PGH2 to PGI2 was found. Ex vivo experiments performed in serum gave an apparent stimulation of immunoreactive 6-oxo-PGF1 alpha following TXSI but RPHPLC analysis of extracted serum showed that this stimulation was accounted for by increase in a product co-eluting with [3H]PGF2 alpha. The implications of these findings in relation to TXSI and receptor antagonists are discussed.

Radioimmunoassay of Eicosanoids: Its Application in the Development of Anti-Inflammatory and Analgesic Drugs

Membrane-bound avachidonic acid is a precursor for PG and LT biosynthesis*. Members of this family of autocoids have been implicated in pathophysiology including regulation of microvascular blood flow and permeability, leukocyte chemotaxis, hyperalgesia and anaphylaxis. Consequently, in recent years considerable effort has been expended in the search for agents that block the synthesis and antagonize the actions of these autocoids. Most of the biologically stable arachidonic metabolites can now be measured by RIA, and this technique finds widespread use in the search for AI and analgesic drugs. Within a mechanistic approach to new drug development, RIA of eicosanoids can be used at the pre-clinical and clinical stages of drug development and now plays an integral part in hypothesis testing and in the progression of pharmacological curiosities to effective medicines.