Antiplatelet and antithrombotic activity of RWJ-53308, a novel orally active glycoprotein IIb/IIIa antagonist.

RWJ-53308 is a novel nonpeptide glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist that inhibits fibrinogen binding to GPIIb/IIIa with an IC(50) of 0.4+/-0.3 nM. RWJ-53308 inhibits thrombin-induced platelet aggregation in human gel-filtered platelets (IC(50)=60+/-12 nM) and platelet aggregation in human platelet-rich plasma (PRP) in response to collagen, arachidonic acid, ADP, and SFLLRN-NH(2) (IC(50)=60+/-10, 150+/-30, 70+/-4, and 160+/-80 nM, respectively). The potency of RWJ-53308 in dog and guinea pig PRP is similar to human PRP. RWJ-53308 inhibits ex vivo collagen- and ADP-induced platelet aggregation in conscious dogs for up to 4 h following 0.3 mg/kg iv, and through 4 and 6 h following 1 and 3 mg/kg po. Oral bioavailability is 16+/-7%. RWJ-53308 reduces thrombus weight in a canine arteriovenous (AV) shunt model following intravenous (0.01-0.1 mg/kg) and oral (3 mg/kg) administration. In a guinea pig carotid artery pinch-injury model, RWJ-53308 completely suppresses thrombus-induced cyclic flow reductions (CFR) at 0.7 mg/kg iv. RWJ-53308 also blocks thrombus formation in photoactivation- and ferric chloride-induced models of thrombosis in guinea pigs at 0.3 and 1 mg/kg iv, respectively. In summary, RWJ-53308 is a potent orally active GPIIb/IIIa antagonist that may be useful for both acute and chronic treatment of arterial thrombotic disorders.

1,2,4-triazolo[3,4-a]pyridine as a novel, constrained template for fibrinogen receptor (GPIIb/IIIa) antagonists.

Conformationally constrained analogues of the GPIIb/IIIa antagonist elarofiban (RWJ-53308) have been synthesized and biologically evaluated. The 1,2,4-triazolo[3,4-a]pyridine scaffold provided potent antagonists with favorable pharmacodynamic and pharmacokinetic attributes in dogs. Compounds 12a and 13a exhibited enhancements in oral bioavailability, t(1/2), and ex vivo duration of action (inhibition of ADP-induced platelet aggregation) relative to elarofiban.

Antithrombotic properties of RWJ-50353, a potent and novel thrombin inhibitor.

The antithrombotic, anticoagulant, and kinetic properties of RWJ-50353, a novel, reversible, active-site-directed thrombin inhibitor, were evaluated. RWJ-50353 inhibited the catalytic activity of human alpha-thrombin with a K(i) of 0.19+/-0.02 nM. It showed a 16-fold selectivity relative to inhibition of trypsin and at least 330-fold selectivity relative to inhibition of other biologically important serine proteases. In a gel-filtered platelet preparation, RWJ-50353 inhibited alpha-thrombin-induced aggregation with an IC(50) of 32+/-6 nM. In a canine arteriovenous shunt antithrombotic model, RWJ-50353 demonstrated a significant dose-related (0.1-1.0 mg/kg, i.v.) reduction in thrombus formation with 50% inhibition (ID(50)) obtained at 0.46+/-0.1 mg/kg. In a rabbit deep vein thrombosis model, RWJ-50353 dose-dependently (0.1-1. 0 mg/kg, i.v.) reduced thrombus formation with an ID(50) of 0.25+/-0. 03 mg/kg. The antithrombotic activity in both of these models was associated with only mild prolongations in bleeding time and coagulation parameters. These results demonstrate that RWJ-50353 is a potent, selective thrombin inhibitor that is an effective antithrombotic agent after intravenous administration in models of arterial and venous thrombosis and may be useful in the management of various thrombotic disorders.

Characterization of a monoclonal antibody specific for HMW subunits of glutenin and its use to investigate glutenin polymers.

A monoclonal antibody, IFRN 1602, has been developed to a synthetic peptide based on the sequence (94)GSVTCPQQV(101) of HMW subunit 1Dx5. The antibody bound strongly to the synthetic peptide based on the cognate sequence of HMW subunit 1Dx2 which contains a serine instead of a cysteine residue. However, it recognized the immunizing peptide by enzyme-linked immunosorbent assay (ELISA) only poorly, probably because the peptide exists as a disulfide-bonded dimer under the assay conditions. From immunoblotting studies against a wide range of wheat varieties, IFRN 1602 was shown to primarily recognize x-type HMW subunits of glutenin encoded on chromosomes 1A and 1D, cross-reacting weakly with the 1A and 1D y-type subunits. It did not bind to any of the 1B-encoded subunits. The Mab also recognized a small number of polypeptides of greater mobility than HMW subunits which were not visible on the stained gels and occurred only in the presence of specific 1A and 1D x-type HMW subunits. Such polypeptides were not present in a preparation of recombinant subunit 2, suggesting that they are modified forms of the subunits which arise in the seed perhaps by processing of the associated subunits. When used to probe partially reduced glutenin, IFRN 1602 bound to 1Dx5-1Dy10 dimers. As the Mab reacted primarily with Cys(97) of 1Dx5 in a reduced form, these data suggest that this residue is not involved in either intra- or intermolecular disulfide bond in the HMW subunit dimers. Thus, Cys(97) of 1Dx5 may be present in gluten in a reduced form, involved in intramolecular disulfide bonds, or linking of the HMW subunit dimers into larger polymers.

Potent, orally active GPIIb/IIIa antagonists containing a nipecotic acid subunit. Structure-activity studies leading to the discovery of RWJ-53308.

Although intravenously administered antiplatelet fibrinogen receptor (GPIIb/IIIa) antagonists have become established in the acute-care clinical setting for the prevention of thrombosis, orally administered drugs for chronic use are still under development. Herein, we present details from our exploration of structure-activity surrounding the prototype fibrinogen receptor antagonist RWJ-50042 (racemate of 1), which was derived from a unique approach involving the gamma-chain of fibrinogen (Hoekstra et al. J. Med. Chem. 1995, 38, 1582). Our analogue studies culminated in the discovery of RWJ-53308 (2), a potent, orally active GPIIb/IIIa antagonist. To progress from RWJ-50042 to a suitable candidate for clinical development, we conducted a series of optimization cycles that employed solid-phase parallel synthesis for the rapid, efficient preparation of nearly 250 analogues, which were assayed for fibrinogen receptor affinity and inhibition of platelet aggregation induced by four different activators. This strategy produced several promising analogues for advanced study, including 3-(3,4-methylenedioxybenzene)-beta-amino acid analogue 3 (significant improved in vivo potency) and 3-(3-pyridyl)-beta-amino acid 2 (significantly improved potency, oral absorption, and duration of action). In dogs, 2 displayed significant ex vivo antiplatelet activity on oral administration at 1.0 mg/kg, 16% systemic oral bioavailability, minimal metabolic transformation, and an excellent safety profile. Additionally, 2 was found to be efficacious in three in vivo thrombosis models: canine arteriovenous (AV) shunt (0.01-0.1 mg/kg, iv), guinea pig photoactivation-induced injury (0.3-3 mg/kg, iv), and guinea pig ferric chloride-induced injury (0.3-1 mg/kg, iv). On the basis of its noteworthy preclinical data, RWJ-53308 (2) was selected for clinical evaluation.

Monoclonal antibody probe for assessing beer foam stabilizing proteins.

A monoclonal antibody (Mab; IFRN 1625) has been produced, which is specific for the most hydrophobic polypeptides responsible for foam stabilization. The binding characteristics of the Mab suggest that it is the conformation of certain hydrophobic polypeptides which is important for foam stabilization. An enzyme-linked immunosorbent assay (ELISA) for assessing the foam-positive form of the foam-stabilizing polypeptides in beer was developed using IFRN 1625. A good correlation was obtained between ELISA determination of foam-stabilizing polypeptides and an empirical means of determining foaming, that is, the Rudin head retention values, for a collection of beers of various foam qualities. Application of the ELISA to different stages of the brewing process showed that the amounts of foam-positive polypeptides increased during barley germination. During the brewing process the proportion of foam-positive polypeptides present after fermentation increased slightly, although a large amount was lost along with other beer proteins during subsequent steps, such as filtering. The present study demonstrates that the amounts of beer polypeptide present in a foam-positive form have a direct relationship with the foaming potential of beer, that their levels are altered by processing, and that there is potential for greater quality control.

Thrombin receptor (PAR-1) antagonists. Heterocycle-based peptidomimetics of the SFLLR agonist motif.

The thrombin receptor (PAR-1) is activated by alpha-thrombin to stimulate various cell types, including platelets, through the tethered-ligand sequence SFLLRN. A series of azole-based carboxamides, designed after SFLLR, were synthesized and evaluated in vitro. The compounds inhibited platelet aggregation induced by SFLLRN-NH2 or alpha-thrombin, and blocked the binding of [3H]-S-(p-F-Phe)-Har-L-Har-KY-NH2 to a CHRF membrane preparation of PAR-1. Oxazole 30 bound to PAR-1 with an IC50 of 1.6 microM, and gave IC50 values of 25 microM and 6.6 microM against alpha-thrombin- and SFLLRN-NH2-induced platelet aggregation, respectively.

Cyclotheonamide derivatives: synthesis and thrombin inhibition. Exploration of specific structure-function issues.

Macrocyclic pentapeptide analogues (5-9) of the sponge natural product cyclotheonamide A (CtA, 3) were prepared by means of our convergent [3 + 2] synthetic protocol, in which a late-stage primary amine group is available for substitution (Maryanoff et al. Proc. Natl Acad. Sci. U.S.A. 1993, 90, 8048). These analogues, as well as CtA and cyclotheonamide B (CtB, 4), were examined for their ability to inhibit the serine protease alpha-thrombin, in comparison with suitable reference standards. We characterized Michaelis-Menten and slow-binding kinetics for the cyclotheonamide derivatives. An attempt was made to utilize the unoccupied hydrophobic S3 subsite of thrombin via analogues 5 and 6. Also, removal of the hydroxyphenyl group, which is thought to be involved in an aromatic stacking interaction with Trp60D of thrombin, was explored via analogue 9. The importance of the alpha-keto and olefin groups was examined via 7 and 8, respectively. The relationship of structure and function with the analogues proved to be less predictable than anticipated.

Molecular basis for the inhibition of human alpha-thrombin by the macrocyclic peptide cyclotheonamide A.

The macrocyclic peptide cyclotheonamide A (CtA), isolated from the marine sponge Theonella sp., represents an unusual class of serine protease inhibitor. A complex of this inhibitor with human alpha-thrombin, a protease central to the bioregulation of thrombosis and hemostasis, was studied by x-ray crystallography. This work (2.3-A resolution) confirms the structure of CtA and reveals intimate details about its molecular recognition within the enzyme active site. Interactions due to the "Pro-Arg motif" (Arg occupancy of the S1 specificity pocket; formation of a hydrogen-bonded two-strand antiparallel beta-sheet with Ser214-Gly216) and the alpha-keto amide group of CtA are primarily responsible for binding to thrombin, with the alpha-keto amide serving as a transition-state analogue. A special interaction with the "insertion loop" of thrombin (Tyr60A-Thr60I) is manifested through engagement of the hydroxyphenyl group of CtA with Trp60D as part of an "aromatic stacking chain." Biochemical inhibition data (Ki values at 37 degrees C) were obtained for CtA with thrombin and a diverse collection of serine proteases. Thus, CtA is just a moderate inhibitor of human alpha-thrombin (Ki = 0.18 microM) but a potent inhibitor of trypsin (Ki = 0.023 microM) and streptokinase (Ki = 0.035 microM). The relative lack of potency of CtA as a thrombin inhibitor is discussed with respect to certain structural features of the enzyme complex. We also report the total synthesis of CtA, by a convergent [2 + 3] fragment-condensation approach, to serve the preparation of cyclotheonamide analogues for structure-function studies.

Synthesis and SAR of 6-substituted purine derivatives as novel selective positive inotropes.

A series of purine derivatives was prepared and examined for selective inotropic activity in vitro and in vivo. Thioether-linked derivatives were superior to their oxygen and nitrogen isosteres. Substitution of electron-withdrawing groups on the benzhydryl moiety of these agents increased potency. The best compound of the study, 17 (carsatrin), was examined further and demonstrated selective oral activity as a positive inotrope. These compounds are presumed to act by affecting the kinetics of the cardiac sodium channel by analogy to the prototypic agent DPI 201106 (1). Their high selectivity for increasing contractile force and dP/dt without affecting blood pressure or heart rate is consistent with this mechanism. Carsatrin (17) was selected as a potential development candidate.

Comparison of the action of types A and F botulinum toxin at the rat neuromuscular junction.

Blockade of neuromuscular transmission was produced in the lower hind limb of the rat by local injection of either type A or type F botulinum toxin (BoTx). At 1, 3, 7, and 10 days after injection, the extensor digitorum longus (edl) nerve-muscle preparation was excised and analyzed for alterations in muscle mechanical properties or spontaneous and nerve stimulus-evoked quantal transmitter release. Muscles receiving type A toxin were paralyzed up to and including 7 days after injection. Muscles treated with type F toxin, although completely paralyzed at 1 and 3 days after injection, twitched in response to nerve stimulation by 7 days. Both toxins induced a marked decrease in the frequency of miniature end-plate potentials, but type A did so to a greater extent. Between 1 and 3 days after toxin injection nerve impulse-evoked transmitter release was reduced in both type A- and type F-treated muscles. Evoked release was temperature sensitive in type A-treated muscles but not in those treated with type F. 3,4-Diaminopyridine (3,4-DAP), a compound which increases nerve-evoked transmitter release by increasing Ca2+ influx, was more effective in reversing the paralysis in type A than in type F-treated muscles. 3,4-DAP induced asynchronous end-plate potentials in response to nerve stimulation in type F-paralyzed muscles, but not in muscles treated with type A. Amidination of the amino groups (presumably lysine) on the toxin by treatment with ethylacetimidate increased the potency and efficacy of only type F BoTx. The results show that type F BoTx differs from type A, mainly by its lower potency, efficacy, shorter duration of action, and by being less effectively antagonized by 3,4-DAP.

Comparison of the effects of botulinum neurotoxin types A and E at the rat neuromuscular junction.

Local blockade of transmitter release was produced by s.c. injection of purified botulinum neurotoxin (NT) types A or E above the tibialis anterior muscle of adult male rats. Extensor digitorum longus nerve-muscle preparation was examined for toxin-induced alterations in single twitch and tetanic tension (in situ) or transmitter release (in vitro). For both single twitch and tetanic tension, muscles treated with type E NT recovered from an initial partial paralysis (induced with 56 mouse LD50) or full paralysis (induced with 565 mouse LD50) by 7 days after NT injection, while those treated with only 5 mouse LD50 of type A remained either fully or partially paralysed through 10 days. Also, miniature end-plate potential frequency and mean quantal content were reduced for a longer period of time and/or to a greater extent for muscles treated with type A NT than for those treated with type E. The present results are consistent with the observed higher specific toxicity (i.p. injections in mice) for type A NT than for type E, although these differences may be exaggerated after s.c. injections. The differences in the paralytic effect between types A and E may be determined by differences in amino acid sequence, which causes type E to dissociate more easily from its site of action and/or be detoxified more rapidly. The clinical implications of these findings are discussed.