John Ambler

Role of Thromboxane A2

When platelets are stimulated to aggregate and secrete by a sub-maximal concentration of an agonist, the platelets on the outside of an aggregate appear more degranulated than those on the inside (1) (fig. 1). In the example shown, aggregation of rabbit platelet-rich plasma (PRP) was induced by a sub-maximal concentration of collagen.

CGS 12970: a novel, long acting thromboxane synthetase inhibitor.

1 CGS 12970 is a potent selective inhibitor of human platelet thromboxane synthestase in vitro (IC50 = 12 nM). It is four orders of magnitude less potent as an inhibitor of sheep seminal vesicle cyclo‐oxygenase, bovine aorta prostacyclin synthetase and human leucocyte 15‐lipoxygenase. 2 The compound inhibited collagen‐induced thromboxane B2 production by human platelets in vitro without an effect on the accompanying platelet aggregation induced by collagen, ADP, platelet activating factor, thrombin, arachidonic acid or the prostaglandin mimetic, U 46619. 3 Administration of CGS 12970 to rats inhibited collagen‐induced thromboxane B2 production but had no effect on platelet aggregation ex vivo. It also had no effect on platelet aggregation induced by thrombin or on plasma clotting times. 4 A single oral dose of 1 or 3 mg kg−1 to rabbits inhibited thromboxane B2 production in clotting blood ex vivo for 12 or 24 h respectively. 5 CGS 12970 inhibited thromboxane B2 production in vivo induced by intravenous administration of collagen to rats or calcium ionophore to guinea‐pigs. In both cases there was a concomitant elevation of immunoreactive 6‐keto‐prostaglandin F1α but no effect on the induced thrombocytopenia. 6 As with other thromboxane synthetase inhibitors, CGS 12970 prolonged tail bleeding time in the rat. However, CGS 12970 was not as potent as other thromboxane synthetase inhibitors in this test. 7 In addition to these usual effects of thromboxane synthetase inhibitors, CGS 12970 inhibited the thrombocytopenia induced by the Forssman reaction or ADP administration. In these tests the effect of the compound was of short duration. 8 CGS 12970 had no effect on the thrombocytopenia associated with the Arthus reaction which distinguishes it from cyclo‐oxygenase inhibitors. It also had no effect on thrombus formation on a cotton thread in an arteriovenous shunt in the rat.

The effects of recombinant desulphatohirudin on arterial thrombosis in rats.

The role of thrombin in the formation of arterial thrombi is poorly understood. With the new availability of the specific thrombin inhibitor, recombinant desulphatohirudin, in large quantities this is now under investigation. A new model of arterial thrombosis in rats is described where a thrombus is formed on a mechanically injured vessel in vivo. Both platelet and fibrin deposition is inhibited by a recombinant hirudin (CGP 39393) at doses which prolong the activated partial thromboplastin time (APTT) to no more than 3-4 times the control level. In contrast, both unfractionated heparin and Fragmin only inhibit thrombosis when the APTT is excessively prolonged (i.e. to greater than 15 times the control value). It is concluded that CGP 39393 is an effective antithrombotic in arterial thrombosis at lower levels of anticoagulation than either heparin or Fragmin.

The discovery of orally available thrombin inhibitors: studies towards the optimisation of CGH1668.

The chemical optimisation of CGH1668 1 is described employing an in vivo model of absorption to determine the influence on bioavailability of single point modifications to five key molecular templates. The discovery of an orally bioavailable and selective thrombin inhibitor, 24, highlights the utility of this approach.

A comparison between platelet aggregation and atp secretion induced by collagen and by paf acether and their inhibition by phenylbutazone, sulphinpyrazone and its metabolites

Abstract 1. 1. Guinea pig platelet aggregation and ATP secretion induced by collagen and platelet-activating factor (PAF-acether) has been compared using a lumiaggregometer. 2. 2. Dose response curves for collagen-induced platelet aggregation occurred at slightly lower collagen concentrations than those for ATP secretion. In contrast, when PAF acether was the agonist, ATP secretion only occurred at agonist concentrations which induced nearly maximal platelet aggregation rate. 3. 3. Phenylbutazone, sulphinpyrazone and its metabolites inhibited both the aggregation and secretion simultaneously when this was induced by collagen with the rank potency order G 25671> G 31442 > phenylbutazone > sulphinpyrazone. As expected this parallels their potency as inhibitors of cyclooxygenase. 4. 4. The compounds only inhibited PAF acether-induced aggregation and ATP secretion at much higher concentrations. All of the sulphinpyrazone metabolites were equipotent in this case but phenylbutazone was ineffective. 5. 5. It is concluded that the phenyl group on the side chain of the sulphinpyrazone-like compounds could be important for inhibition of PAF-acether-induced platelet activation. 6. 6. The plasma concentrations of sulphinpyrazone and its metabolites required to inhibit PAF-acether-induced platelet activation are very high compared to those achieved in man. Thus inhibition of PAF acether-induced platelet aggregation is unlikely to be an important mechanism of action in man.

the design and synthesis of thrombin inhibitors: the introduction of in vivo efficacy and oral bioavailability into benzthiazolylalanine inhibitors

The further optimisation of the novel lead compound CGH752 (Fig. 1) is described. By introducing various substituents into the 6-position of the 3,3-dimethyltetrahydroquinoline (DMTHQS) ring we have been able to favourably affect the in vitro and in vivo activity, and the pharmacokinetics of such compounds. One of the inhibitors synthesised (CGH1484) is bioavailable and shows efficacy in animal models of thrombosis.

The design and synthesis of thrombin inhibitors: analogues of MD805 containing non-polar surrogates for arginine at the P1 position

A series of monocyclic and bicyclic amino acids have been synthesised and incorporated into thrombin inhibitors based on CGH728, an analogue of the Mitsubishi compound MD805. Benzthiazolylalanine (Bta) was found to be a good non-polar substitute for arginine at the P1 position, yielding compounds with low nanomolar potency and good selectivity for thrombin.

Studies towards the identification of potent, selective and bioavailable thrombin inhibitors.

The application of selection criteria, based on potency and physicochemical parameters, to a candidate library of thrombin inhibitors is described. The utility of the approach is exemplified by the discovery of a potent, selective and bioavailable thrombin inhibitor 62.

The discovery of orally available thrombin inhibitors: optimisation of the P1 pharmacophore.

Thrombin inhibitors have been designed with the replacement of the strongly basic guanidine P1 pharmocophore with a group that exploits the lipophilicity of the S1 pocket. The approach has lead to the discovery of potent thrombin inhibitors demonstrating good intra-duodenal absorption.

Optimisation of the P2 pharmacophore in a series of thrombin inhibitors: Ion-dipole interactions with lysine 60G

The optimisation of the P2 pharmacophore in a series of thrombin inhibitors is described. The interaction of a number of piperidine P2 functionalities with lysine 60G of thrombin is explored with reference to the crystal structure of inhibitor enzyme complexes. A primary ion-dipole interaction between the terminal P2 side chain group and lysine 60G is evoked to explain the SAR in this series.