Janet E. Merritt

OCTIMIBATE, A POTENT NON-PROSTANOID INHIBITOR OF PLATELET AGGREGATION, ACTS VIA THE PROSTACYCLIN RECEPTOR

1 Octimibate, 8‐[(1,4,5‐triphenyl‐1H‐imidazol‐2‐yl)oxy]octanoic acid, is reported to have antithrombotic properties. This is in addition to its antihyperlipidaemic effects which are due to inhibition of acyl‐CoA: cholesterol acyltransferase (ACAT). The aim of this study was to investigate the mechanism of the antithrombotic effect of octimibate, and to determine whether the effects of octimibate are mediated through prostacyclin receptors. 2 In suspensions of washed (plasma‐free) human platelets, octimibate is a potent inhibitor of aggregation; its IC50 is approx. 10 nm for inhibition of aggregation stimulated by several different agonists, including U46619 and ADP. The inhibitory effects of octimibate on aggregation are not competitive with the stimulatory agonist; the maximal response is suppressed but there is no obvious shift in potency of the agonist. In platelet‐rich plasma, octimibate inhibits agonist‐stimulated aggregation with an IC50 of approx. 200 nm. 3 Octimibate also inhibits agonist‐stimulated rises in the cytosolic free calcium concentration, [Ca2+]i, in platelets. Both Ca2+ influx and release from intracellular stores are inhibited. The effects of octimibate on aggregation and [Ca2+]i are typical of agents that act via elevation of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP). Similar effects are seen with forskolin, prostacyclin (PG12) and iloprost (a stable PG12 mimetic). 4 Octimibate increases cyclic AMP concentrations in platelets and increases the cyclic AMP‐dependent protein kinase activity ratio. Octimibate stimulates adenylyl cyclase activity in human platelet membranes, with an EC50 of 200 nm. The maximal achievable activation of adenylyl cyclase by octimibate is 60% of that obtainable with iloprost. Octimibate has no effect on the cyclic GMP‐inhibited phosphodiesterase (phosphodiesterase‐III), which is the major cyclic AMP‐degrading enzyme in human platelets. 5 Octimibate inhibits, apparently competitively, the binding of [3H]‐iloprost (a stable PG12 mimetic) to platelet membranes; the estimated Ki is 150 nm. 6 The platelets of different species show considerable differences in the apparent potency of their inhibition of aggregation by octimibate; platelets from cynomolgus monkeys are 3 fold more sensitive than those from humans, while rat, cat and cow platelets are 50, 100, and 250 fold less sensitive than human platelets. The sensitivity of these different species to iloprost, however, varies over a range of only 10 fold with no obvious difference between primates and non‐primates. 7 Octimibate appears to be a potent agonist (aggregation), or partial agonist (adenylyl cyclase), at prostacyclin receptors and is the first non‐prostanoid agent of this type to be identified. The species differences in relative potency of octimibate and iloprost may reflect the existence of receptor subtypes.

The bisindolylmaleimide protein kinase C inhibitor, Ro 32-2241, reverses multidrug resistance in KB tumour cells

Abstract Ro 32-2241 is a bisindolylmaleimide that selectively inhibits protein kinase C (PKC) as compared with other protein kinases. Experiments were carried out to examine its potential as a multidrug resistance-reversing agent. Ro 32-2241 inhibited efflux, and increased accumulation, of [3H]-daunomycin in multidrug-resistant (MDR) KB-8-5 and KB-8-5-11 cells and had no effect on drug-sensitive KB-3-1 cells. Ro 32-2241 completely reversed the doxorubicin resistance of KB-8-5 and KB-8-5-11 cells, showing no effect on the sensitivity of drug-sensitive KB-3-1 cells. The potency of Ro 32-2241 was comparable with that of cyclosporin A and better than that of verapamil, known modulators of multidrug resistance. Ro 32-2241 also completely reversed the taxol resistance of KB-8-5 cells and partially reversed the resistance of KB-8-5-11 cells. Vinblastine resistance was also partially reversed. Mechanistic experiments were carried out to determine whether Ro 32-2241 interacted with P-glycoprotein (Pgp) directly. Increased efflux of [14C]-Ro 32-2241 was seen with the more resistant KB-8-5-11 cells (although the percentage effluxed was very low as compared with [3H]-daunomycin), suggesting that Ro 32-2241 can act as a substrate for Pgp. Direct interaction of Ro 32-2241 with Pgp was confirmed by demonstration that it inhibited binding of [3H]-azidopine to Pgp in KB-8-5-11 membranes. In conclusion, Ro 32-2241, acting directly on Pgp (rather than, or in addition to, an effect on PKC), is effective in reducing or reversing resistance to doxorubicin, taxol and vinblastine in human tumour cells with a clinically relevant degree of MDR. However, results of in vivo experiments conducted to investigate the effects of Ro 32-2241 on resistance to doxorubicin suggest that it may not be possible to achieve sufficiently high levels of Ro 32-2241 in vivo to modulate MDR.

DIFFERENT SENSITIVITIES OF NEUTROPHIL RESPONSES TO A SELECTIVE PROTEIN KINASE C INHIBITOR, RO 31-8425 ; REDUNDANCY IN SIGNAL TRANSDUCTION

Previous studies implicating a role for protein kinase C (PKC) in mediating stimulation of cellular responses by physiological agonists have relied on use of non-specific inhibitors or direct stimulation of PKC by phorbol esters. However, much of this evidence is questionable. Here, we have investigated the effects of a potent and selective PKC inhibitor, Ro 31-8425, on three different responses of human neutrophils stimulated by either a physiological agonist, C5a, or a phorbol ester, PMA. The responses studied were superoxide generation, collagenase secretion and adhesion to endothelial cells. In each case, the PMA-stimulated response was more sensitive to inhibition than the C5a-stimulated response. Even the PMA-stimulated responses differed in their sensitivity to inhibition, with superoxide production being the most sensitive and adhesion at least sensitive. The different sensitivities of the PMA stimulated responses suggest that, although activation of PKC stimulates the responses, either different degrees of activation or different isozymes are required for the different responses. The lower sensitivity of the C5a-stimulated responses in each case suggests that PKC activation, if needed at all, is not rate limiting in these signal transduction pathways. These results emphasize the redundancy in intracellular signal transduction.

Primate vascular responses to octimibate, a non-prostanoid agonist at the prostacyclin receptor.

1 Octimibate is a potent inhibitor of human platelet aggregation, and appears to act (at least in part) through the prostacyclin receptor, as described in the preceding paper. Here, the vascular effects, both in vitro and in vivo, of octimibate have been compared to those of the stable prostacyclin (PGI2) mimetic, iloprost. Since octimibate shows extensive species variation and is potent at inhibiting platelet aggregation in primates, all of the experiments reported here have been carried out with primate tissue or in vivo in cynomolgus monkeys. 2 Activation of adenylyl cyclase in human lung membranes appears to involve stimulation of the vascular PGI2 receptor. Octimibate, as well as iloprost, stimulates adenylyl cyclase in this preparation. The EC50 values for iloprost and octimibate are 50 nm and 340 nm respectively. These values are similar to those seen with human platelet membranes. As with platelets, the maximal activation achievable with octimibate is 60% of that seen with iloprost. This result suggests that octimibate is a partial agonist for stimulation of adenylyl cyclase. 3 Iloprost (10–100 nm) relaxes human coronary and mesenteric artery precontracted with KC1, and also relaxes cynomolgus monkey aorta precontracted with phenylephrine. Octimibate appears to be a partial agonist for relaxation of human coronary artery precontracted with KC1; the intrinsic activity of octimibate (10 μm) is 0.15 compared to iloprost, and octimibate surmountably antagonizes the relaxant effects of iloprost with a Kp of 200 nm. Octimibate (up to 10 μm) evokes only weak relaxation of human mesenteric artery (precontracted with KC1) and cynomolgus monkey aorta (precontracted with phenylephrine). 4 The effects of iloprost and octimibate were compared in vivo in cynomolgus monkeys. In addition to inhibiting ex vivo platelet aggregation, both compounds cause hypotension with little effect on heart rate. The dose‐response curves for inhibition of ex vivo platelet aggregation and a fall in mean arterial blood pressure were compared. The dose‐separation (i.e., the relative differences in effective concentrations) for the two responses is similar with both iloprost and octimibate. 5 Since the periberal resistance vessels are intimately involved in regulation of systemic arterial blood pressure, the effects of both agents were tested on human peripheral resistance vessels (150–400 μm diameter) in vitro. These vessels are relaxed by both iloprost and octimibate following precontraction with KC1. The IC50 value for iloprost is 44 nm, and 1.7 μm octimibate evokes 50% of the maximal relaxation obtained with iloprost. Thus, the relative potencies of the two compounds in relaxing human subcutaneous resistance vessels are similar to their relative potencies in inhibiting platelet responses. This result correlates with the lack of platelet versus vascular selectivity seen with the in vivo monkey studies. 6 These results suggest that octimibate, a partial agonist at the prostacyclin receptor, is unable to discriminate between platelet and vascular prostacyclin receptors in primates.

The involvement of calcium and protein kinase C in modulating agonist-stimulated chemotaxis of human neutrophils

Chemotaxis of human neutrophils in response to a gradient of the chemotactic peptide, fmet-leu-phe (FMLP), was measured by the under-agarose technique. The dose-response curve for FMLP was biphasic; low concentrations were stimulatory, and the response was reduced at higher concentrations. The response to FMLP was partially inhibited (about 50%) in the absence of extracellular Ca2 (EGTA added). NiCl2 dose-dependently inhibited FMLP-stimulated chemotaxis in the presence of extracellular Ca2+; the maximum inhibition obtainable with NiCl2 was similar to that with the absence of extracellular Ca2+. These results suggest that FMLP-stimulated chemotaxis is, at least partially, dependent on stimulation of Ca2+ influx. The phorbol ester, PMA, dose-dependently inhibited chemotaxis; the response was almost completely inhibited by 10 nM PMA. This result indicates that activation of protein kinase C inhibits chemotaxis. These results are discussed in relation to the physiological responses of neutrophils.