Thrombin receptors and their antagonists: an update on the patent literature

transcript

1. Introduction

2. PAR-1 antagonists

3. PAR-3 and -4 antagonists

4. Combination of PAR

antagonists

5. Expert opinion

Review

Thrombin receptors and theirantagonists: an update on thepatent literatureGiuseppe Cirino† & Beatrice Severino†Universita di Napoli Federico II, Departmrnt of Experimental Pharmacology, Napoli 80131, Italy

Importance of the field: Thrombin plays a central role in cardiovascular

inflammation. Most of the cellular responses to thrombin are mediated by

cell surface protease-activated receptors (PARs). Several preclinical studies

indicate that PARs are potential targets for treating cardiovascular diseases

such as thrombosis, atherosclerosis and restenosis. Among PARs, PAR-1 has

emerged as an important therapeutic target.

Areas covered in this review: This review covers recent advances in the devel-

opment of thrombin receptors antagonists. It is focused on the search for

PAR-1 antagonists as this is at the moment the most promising and attractive

target. However, some early promising studies on PAR-3 and -4 antagonists

are also reported.

What the reader will gain: The review has been written in order to give to the

reader hints and references that cover, in our opinion, the most interesting

and/or promising approaches in this research field.

Take home message: Research on PAR-1 antagonists has finally led to

good clinical candidates such as SCH-530348 (Schering-Plough) and E-5555

(Eisai Co.). Clinical trials clearly demonstrate that development of PAR1 antag-

onists is not only possible but most likely will lead to development of anti-

platelet drugs as well as of drugs useful for the treatment of inflammatory,

proliferative and neurodegenerative diseases.

Keywords: cardiovascular diseases, PAR, platelets, thrombin receptors antagonists

Expert Opin. Ther. Patents [Early Online]

1. Introduction

Proteases such as thrombin, trypsin and tissue kallikreins regulate cell signaling bycleaving and activating a family of G-protein-coupled protease-activated receptors(PARs 1 -- 4) via exposure of a tethered receptor-triggering ligand. Thrombincleaves the N-terminal domain of PAR-1, -3 and -4 being 10 -- 100 times morepotent at PAR-1 than at PAR-4 [1]. PAR-1 is a high affinity receptor for platelet acti-vation at low thrombin concentrations whereas PAR-4 appears to play a supportive‘backup’ function for thrombin-induced platelet activation mediating signaling onlyat high thrombin concentrations. Thus, PAR-1 is the major thrombin-activatedreceptor in human and non-human primate’s platelets and its activation plays amajor role in mediating platelet aggregation, cell proliferation, inflammatoryresponses and neurodegeneration [2-7]. This suggests that PAR-1 represents anattractive drug discovery target for the possible treatment of various disorderssuch as thrombosis, restenosis, atherosclerosis, inflammation, cancer metastasisand stroke [8-13]. However, it should be clear to the reader that PAR-1 activationis not always detrimental. It is known, in fact, that the anti-inflammatory and cyto-protective effects of activated protein C (APC) are elicited by APC-mediatedPAR-1 activation [14,15]. Most of the patents reported so far are related to thediscovery of different classes of molecules that show PAR-1 antagonist properties

while there are only few inventions related to PAR-3 and -4antagonists. Most likely, the research on selective antagoniststo these receptors has been impaired by the lack of appropriatetools that only at the present have started to emerge.

2. PAR-1 antagonists

Although several experimental evidences support the crucialinvolvement of PAR-1 receptor in different diseases such asinflammatory diseases and cancer, to date, the majority ofpublished studies on PAR-1 antagonists concern theiremployment as antithrombotic agents. It is important topoint out to the reader that additional challenges associatedwith the search of PAR-1 antagonists arose from the differentdistribution of thrombin receptors or even the absence ofPAR-1 in the platelets of several test animals. For example,PAR-1 is absent in platelets of rats and dogs and the presenceof the additional thrombin receptors, PAR-3 and -4, differsamong species [16].PAR-1 antagonists can be classified into two major

chemical groups, peptide/peptidomimetic derivatives andnon-peptide based derivatives. Design and preparation ofpeptide-mimetics or small organic molecules with PAR-1antagonist activity has been very challenging. Indeed, the teth-ered ligand binding mechanism is energetically preferred; inaddition, very little information about conformation of thereceptor is currently available.

2.1 Peptide/peptidomimetic PAR-1 antagonistsExtensive structure--activity relationships studies have beenperformed using as a model the sequence of the small peptidesthat selectively activate the thrombin receptor namely,SFLLRN or SFLLR. These sequences are commonly refereedas PAR-1 activating peptide (PAR-1-AP) and have beenwidely used in the current literature. The structure--activity

relationships studies have provided a basic understanding ofthe peptide ligand side chain structural requirements and ontheir relative tolerance with regard to their interactions withthe human thrombin receptor. A ‘three-point model’, consti-tuted by the ammonium group, the center of the benzene ring(phenylalanine residue) and the central carbon of the guani-dine group (arginine residue), has been used in conjunctionwith different molecular templates such as azole [17],indole [18], indazole [19] and benzimidazolone [20] to designcandidate peptide-mimetic structures. In particular, a break-through came from an indole-based series, RWJ-53052 (1)being a prototype [21,22]. This compound inhibited humanplatelet aggregation induced by both PAR-1-AP (IC50 =0.49 µM) and thrombin (IC50 = 2.0 µM) and was selectivetoward collagen as trigger of platelet aggregation. However,it had modest affinity for PAR-1 in a binding assay. Theapplication of an iterative approach for optimization led toseveral peptide-mimetic embodying 6-aminoindole and6-aminoindazole nuclei that met the spatial requirementsfor displaying the three key substituents in the correctthree-dimensional configuration. Introduction of thesescaffolds led to some active compounds exemplified byRWJ-56110 (2) and RWJ-58259 (3) [21-23]. These com-pounds showed potent and selective PAR-1 antagonist prop-erties. They did not display any PAR-1 agonist activity anddid not interact directly with thrombin. RWJ-56110 andRWJ-58259 bind to PAR-1 and doing this interferes withcalcium mobilization and cellular functions associatedwith PAR-1 only. Indeed, RWJ-56110 did not interact withPAR-2, -3 or -4.

RWJ-56110 was determined to be a direct inhibitor ofPAR-1 activation and internalization, without affectingPAR-1 N-terminal cleavage. Therefore, both RWJ-56110and RWJ-58259 clearly interrupt the binding of a tetheredligand to its receptor. RWJ-58259 demonstrated antireste-notic activity in vivo in a rat balloon angioplasty model andantithrombotic activity in a cynomolgus monkey arterialinjury model [24].

A more recent approach to receptor inhibition has arisenfrom the observation that N-palmitoylated peptides, termedpepducins, based on the third intracellular loop of certainGPCRs can cause activation and/or inhibition of G-proteinsignaling only in the presence of the parent GPCR [25].Attachment of a palmitate lipid to peptides based on theN-terminal portion of the i3 loop of PAR-1 yieldedP1pal-12 pepducin, whose sequence is pal-RCLSSSA-VANRS-NH2. This peptide lacked agonist activity, but wasa full antagonist of PAR-1-dependent inositol phosphateproduction and Ca2+ signaling in platelets and recombinantsystems [26,27]. More recently, it has been demonstrated thatMMP-1 directly activates PAR-1 [28], which was found to behighly upregulated in invasive ovarian carcinomas in vitro.Starting from these observations, pepducins P1pal-12 andP1pal-7, whose sequence is pal-KKSRALF-NH2, were testedin xenograft models of advanced peritoneal ovarian

Article highlights.

. Protease-activated receptors (PARs) are a family ofGPCRs. Of the four members so far identified, PAR-1,-3 and -4 are activated by thrombin.

. PAR-1 has been shown to be involved in mediatingplatelet aggregation, cell proliferation, inflammatoryresponses and neurodegeneration.

. Early attempts to develop PAR-1 antagonists failedbecause of problems of poor efficacy, possibly due tothe tethered ligand intramolecular binding that followsenzymatic cleavage of the extracellular loop of PAR-1.

. PAR-1 antagonists, so far developed, can be chemicallyclassified into two major groups, peptide/peptidomimetic antagonists and non-peptideantagonists.

. Two PAR-1 antagonists, SCH-530348 from Schering-Plough and E-5555 from Eisai Co., are in clinical trialsfor the prevention of arterial thrombosis.

This box summarizes key points contained in the article.

Thrombin receptors and their antagonists: an update on the patent literature

2 Expert Opin. Ther. Patents (2010) 20(7)

cancer. P1pal-7 derives from the carboxylterminal thirdintracellular loop and acts as a full antagonist of PAR-1.Administration for 6-weeks intraperitoneal of the PAR-1pepducin reduced production of ascites and angiogenesis.Furthermore, when administered in combination with doce-taxel it inhibited metastatic progression of peritoneal ovariancancer [29].

2.2 Non-peptide based PAR-1 antagonistsSeveral authors have patented low molecular mass thrombinreceptor antagonists resulting from the examination ofstructure--activity relationships performed on peptide-mimeticantagonists or from high-throughput screening. These studieshave determined that it was possible to obtain simpler struc-tures of lower molecular mass which retained the antago-nist activity. These derivatives were initially screened in aradioligand binding assay using tritiated alanine-p-fluorophe-nylalanine-arginine-cyclo-hexylalanine-homoarginine-[3H]phenylalanine amide, [3H]ha-PAR-AP. Promising compounds

were further characterized by functional assays such as plateletaggregation assay, cytosolic Ca+2 measurement assay and cellproliferation assay. Among the non-peptide thrombin receptorantagonists, the pyrroloquinazoline analogues were firstreported with good PAR-1 affinity and promising activity infunctional assays. In particular, SCH-79797 (4), first reportedby Ahn et al. in 1999 [30], inhibited PAR-1-AP induced plateletaggregation with an IC50 of 56 nM but only transiently inhib-ited platelet aggregation induced by thrombin. In addition, ithas been described a cardioprotective effect, related to its abilityin inhibiting PAR-1. Indeed, SCH-79797 attenuates myocar-dial injury and dysfunction related to myocardial ischemia/reperfusion injury in an in vivo experimental protocol. Interest-ingly, the compound was active both when given before(preventive protocol) or during ischemia (therapeutic proto-col) [31]. Recently, SCH-79797 has been tested in an in vivoangiogenesis model providing direct evidence that PAR-1 isinvolved in the initiation of the angiogenic cascade [32].SCH-79797 has also shown a remarkable antiproliferative

1 (RWJ-53052)

H2N NH

X = CH 2 (RWJ-56110)X = N 3 (RWJ-58259)

Cirino & Severino

Expert Opin. Ther. Patents (2010) 20(7) 3

effect in several cell lines suggesting another potential clinicaluse. However, the observation that it was able to slow the pro-liferation rate of mouse PAR-1 null cells implies that this phar-macological effect most likely is not mediated byPAR-1 inhibition [33].Maryanoff et al. have patented a structurally novel class of

aminomethyl--pyrroloquinazoline compounds in that an ami-nomethyl group has been positioned on the pyrrolo portion ofthe pyrroloquinazoline scaffold [34]. Several series of aryl-isoxazolyl-amines were described [35]. Some of the resultantmolecules were potent PAR-1 antagonists that were effectiveagainst both PAR-1-AP- and thrombin-stimulated receptoractivations. These compounds do not inhibit the proteolyticeffects of thrombin but rather interfere with the intramolecu-lar binding of the tethered ligand (SFLLRN) to the trans-membrane portion of the thrombin receptor. The sameauthors have patented other compounds as thrombin receptorantagonists, for example, pyrrolidinecarboxamides, piperidi-necarboxamides andN-aryl-N¢-arylethylurea derivatives [36,37].These compounds displayed submicromolar IC50 values in aPAR-1 radioligand binding assay and in PAR-1-AP induced

5-HT secretion functional assays. PAR-1 antagonists basedon cyclic guanidine and amidine templates have been alsopatented [38-40]. The monocyclic guanidine derivatives seemto have only modest potency against the PAR-1 receptor,while the bicyclic amidine derivatives are generally morepotent. Indeed, a PAR-1 antagonist, based on the bicyclicamidine motif, E-5555 (5), has been reported to inhibit plate-let aggregation with no change in bleeding time in Phase Istudies and it is currently under development as a potentialtreatment for critical care acute coronary syndrome. Fivedrug--drug interaction studies, a food effect study and aPhase II proof-of-concept trial in patients with coronaryartery disease have been completed [41]. Moreover, thein vitro effects of E-5555 on platelet function beyondPAR-1 blockade have been investigated in healthy volunteersand in patients with coronary artery disease treated withaspirin with or without clopidogrel. E-5555 in vitro moder-ately but significantly inhibits platelet activity beyondPAR-1 blockade. The obtained data demonstrated that theantiplatelet potency of aspirin alone and the combinationof aspirin and clopidogrel may be enhanced by E-5555

6 (SCH-530348)

4 (SCH-79797) 5 (E-5555)

OCH3t-Bu

H3CH2CO

providing rationale for a synergistic use. Therefore, the selec-tive blockade of platelet receptors suggests unique antiplateletproperties of E-5555 as a potential addition to current antith-rombotic regimens [42].

2.3 Non-peptide based PAR-1 antagonists: himbacine

derivativesSeveral patents related to PAR-1 antagonists based on the corestructure of the natural product himbacine have beenreported [43-45]. Initial structure--activity relationships studieswere directed at optimizing the substitution pattern on thepyridine ring. The newly synthesized compounds were testedby in vitro binding assays, performed by using purified humanplatelet membranes as PAR-1 source and tritiated high-affinity thrombin receptor activating peptide as ligand. Explo-ration of the C-7 region of the tricyclic motif led to a furtheroptimization of himbacine-derived PAR-1 antagonists allow-ing the discovery of SCH-530348 (6), which showedKi = 8.1 ± 1.1 nM in the in vitro binding assays [46].

SCH-530348 is characterized by an excellent oral bioavail-ability in multiple species (33% in rats and 86% in monkeys).SCH-530348 has high potency in a series of in vitro func-tional assays; it inhibits thrombin-induced platelet aggrega-tion with an IC50 = 47 nM, PAR1-AP-induced plateletaggregation with an IC50 = 25 nM and has a potent oral activ-ity in an ex vivo cynomologus monkey model of plateletaggregation. In this model, a complete inhibition ofplatelet aggregation is achieved for 24 h post dosing withpartial recovery occurring at 48 h at a dose of 0.1 mg/kg.Finally, SCH-530348 has been also evaluated in patientsundergoing non-urgent percutaneous coronary intervention.SCH-530348 was generally well tolerated and did not causeincreased thrombolysis in myocardial infarction (TIMI)bleeding, even when administered in association with aspirinand clopidogrel [47]. These results have allowed the design ofa large Phase III program, TRA.CER, a multi-center, ran-domized, double-blind, placebo-controlled study [48]. A partof this study, named TRAP degrees P-TIMI 50 trial, hasbeen designed in order to evaluate the efficacy and safety ofSCH-530348 during long-term treatment of patients withestablished atherosclerotic disease receiving standard therapy(n = 27,000). The outcome of this study will give informationon whether the PAR-1 receptor is a valuable target for

reducing major cardiovascular events with a favorable safetyprofile in patients with established atherosclerosis [49]. Insummary, the TRA.CER study will define efficacy andsafety of SCH-530348 in the treatment of high-risk patientswith non-ST-segment elevation, acute coronary syndromeand high-risk features. Recently Hezi-Yamit and Wong [50]

have described methods and medical devices used to deliverSCH-530348 locally to vasculature in treating and/orpreventing cardiovascular conditions including, but notlimited to, restenosis, in-stent restenosis, thrombosis andin-stent thrombosis [50].

Further modifications performed on himbacine corehave led to the recently patented series of monocyclic,bicyclic, tricyclic, spiroheterocyclic-decalin and oxazoloiso-quinolinic himbacine derivatives as thrombin receptorantagonists [51-55].

2.4 Non-peptide based PAR-1 antagonists: piperazine

and pyridazine derivativesPerez et al. have recently patented two series of derivativessuch as phenylpentadienoylpiperazines [56] and cinnamoylpi-perazines [57]. Many of the reported molecules inhibitedSFLLR-induced human platelet aggregation and are activeby both intravenous (i.v.) and oral administration routes ina rat thrombosis model without any significant impact onbleeding time. However, these results are difficult to interpretas rat platelets do not express PAR-1. Two of the describedcompounds, for example, F16618 (7) and F16357 (8), arethe most promising in terms of antithrombotic activity andADME profile. Indeed, these compounds inhibit SFLLR-induced human platelet aggregation and display antithrom-botic activity in an arteriovenous shunt model in the ratafter i.v. or oral administration [58]. Because rat platelets donot express PAR-1, the antithrombotic effect displayed inthis thrombosis model implies that these compoundscan either act on the other PAR receptors or interfere withother signaling mechanism(s). Further pharmacological char-acterization has suggested a possible use of these compoundsas possible core structure to develop drugs to preventand/or treat atrial fibrillation. In this context, it has beenshown that F16357, administered at a dose of 40 mg/kg/daily for 2 months, induced a significant reduction in thevolume of the left auricle compared to untreated animals [59].

7 (F16618) 8 (F16357)

Cirino & Severino

More recently, Heinelt et al. have patented some triazolo-pyridazines. In the patent are reported 15-examples of com-pounds that have IC50 values ranging from 0.098 to 24 µMin PAR-1 inhibition assays [60].

3. PAR-3 and -4 antagonists

As previously mentioned, the patent literature is less rich ofinventions regarding the other two thrombin receptors,namely PAR-3 and -4. This deficiency is mainly due to thelack of specific tools and to the limited availability of antago-nists that specifically bind these receptors. In addition, the factthat PAR-3 and -4 have been so far considered as ‘accessory’receptors has in some way impaired the research and at thepresent there are few papers that have attempted to investigateand possibly define a role for these two receptors.

3.1 PAR-3 antagonistsPAR-3 is usually considered as a cofactor for PAR-4 activationin human platelets that might serve to enhance the specificityof thrombin’s actions. Recently, AstraZeneca has patented aninvention based, in part, on the surprising finding that, in thepresence of pro-inflammatory cytokines, such as TNF-a orIL-lb, thrombin interacts with PAR-3. Indeed, in this experi-mental condition, resembling a pathological condition sus-tained by an inflammatory state, PAR-3 is highly expressedin lung fibroblasts. This enhanced expression results in theinduction of GM-CSF to a level that is found in asthma.This observation has made the basis for a possible use ofPAR-3 antagonists in the treatment of asthma [61].

3.2 Small-molecule compounds as PAR-4 antagonistsIn order to identify heterocyclic structures as PAR-4 anta-gonists, a screening of chemical compounds directed towardsthe evaluation of their ability to inhibit platelet aggregationallowed the identification of the indazole derivativeYD-3 (9) [62]. Several studies have demonstrated that YD-3is able to inhibit PAR-4-dependent platelet activation andthromboxane formation aggregation [63,64]. YD-3 inhibitsthe thrombin-induced signal via Ras/extracellular-signal-regu-lated kinase, which critically influences cell proliferation invascular smooth muscle cells in vitro, attenuating, also, therestenosis after balloon angioplasty in vivo [65]. More recently,several series of derivatives have been prepared usingYD-3 as lead compound in order to establish completestructure--activity relationships and to explore their activityas anti-angiogenic agents [66,67]. The structure--activityrelationship studies have demonstrated that both the4¢¢-ethoxycarbonyl and 1-benzyl groups on YD-3 contributeto the selective anti-PAR-4 activity. Any modification of the4¢¢-ethoxycarbonyl group decreased the activity supportingthe evidence that this moiety is the major contributing func-tional group for the antiplatelet activity. The introduction ofchloro, fluorine or methoxy group onto the 1-benzyl group

of YD-3 maintained its selective and potent anti-PAR-4activity, but led to considerable fluctuation in potency.

YD-3 and a series of related compounds were tested fortheir effects on VEGF-induced cell proliferation, and on neo-vascular formation in vivo. This study allowed the identifica-tion of some derivatives showing superior potency to thepositive control YD-3. Analysis of the anti-angiogenic activityof the compounds led to the important finding that the intro-duction of Cl, CH3 or OCH3 group into the para-positionof the N2-benzyl group of the tested indazole derivativessignificantly increases their anti-angiogenic activity.

3.3 Pepducins as PAR-4 antagonistsThe application of the pepducin approach in the search ofPAR-4 antagonists has led to the identification of a selectivePAR-4 antagonist, named P4pal10, whose sequence is pal-SGRRYGHALR-NH2 [25-27]. Mouse platelets, treated withP4pal10, participate to a significantly lesser extent inthrombus initiation and growth when compared with non-treated platelets. This finding supports the notion that oneof the most important mechanisms of PAR-pepducins’action is the prevention of platelet--platelet (thrombus) inter-actions [68]. P4pal10 and a structurally unrelated PAR-4antagonist, trans-cinnamoyl-YPGKF-NH2, were used toassess the potential cardioprotective role of PAR-4 antago-nists [69]. P4pal10 treatment decreased infarct size whengiven before, during and after ischemia in an in vivo model.This cardioprotective effect was due to the unmasking ofadenosine receptor signaling, supporting the hypothesis ofa coupling between thrombin receptors and adenosine recep-tors. PAR-4 has been also shown to be involved in inflam-mation. However, the involvement of PAR-4 receptors ininflammation processes has been recently revisited testingthe effects of PAR-4 inhibition in a model of systemicinflammation and disseminated intravascular coagulation(Shwartzman reaction) [70]. This study has provided circum-stantial evidence that the primary cellular target of theP4pal10 may be neutrophils, rather than platelets or endo-thelial cells. Thus, a current hypothesis is that the beneficialeffect of PAR-4 inhibition could be based on a reduced neu-trophil infiltration into the site of the inflammation. Thislatter hypothesis suggests a possible development for theseantagonists in the treatment of systemic inflammation.

4. Combination of PAR antagonists

Recent inventions have been patented related to the combina-tion of PAR-4 antagonists with PAR-2 or -1 antagonists.The first association represents a method for preventingand/or treating itch by administration of the PAR-4 antago-nist that can be combined with a PAR-2 antagonist. Theinventors discovered that PAR-4 is a key effector of the itchsensation caused by a cysteine protease that was isolated andidentified from the plant Mucuna pruriens, also known ascowhage, referred to as mucunain. It has also been discovered

that mucunain cleaves the PAR-4 and -2, which then induceinflux of calcium into the affected cells triggering acascade of events that leads to the sensation of itch [71].

More recently has been patented an invention related tothe use of an effective amount of at least one PAR-1 antago-nist, at least one PAR-4 antagonist and, optionally, an effec-tive amount of at least one cardiovascular agent for thetreatment of diseases associated with thrombosis, inflamma-tory disorders, fibrotic disorders, cardiovascular disordersand cancer [72].

5. Expert opinion

Thrombin has a central role in platelet aggregation and coag-ulation. The discovery of thrombin receptors has now madeclear that thrombin plays a central role in the link betweencoagulation and inflammation. In particular, the findingthat the thrombin receptors are widely expressed in vascularcells has led in past 10 years to determine that thrombin,through its receptors, is also involved in cardiovascular com-plications such as atherosclerosis, restenosis and neointimalformation. Platelets are the main players in atherothrombo-sis and they represent a key target for pharmacotherapy. Atthe present, the most widely used drug is aspirin that signif-icantly reduces the risk of major cardiovascular events in ath-erosclerotic patients. Another drug that is widely used isclopidogrel, an inhibitor of the P2Y12 adenosine diphos-phate receptor. This inhibitor is now widely used in clinicalpractice in association with aspirin in acute coronarysyndromes because it has been shown that the association issignificantly more efficacious than aspirin alone in reducingthe risk of cardiovascular events. However, the risk ofbleeding linked to this therapy represents an unmet needfor pharmacotherapy.

Pharmaceutical research in identifying a PAR-1 antagonisthas been somewhat limited for several reasons. First, studyingPAR functions with proteinase activation has been challengingespecially in systems where more than one receptor is expressed.Noteworthy, standard species used in preclinical research, suchas mice, rats, rabbits and dogs, do not express PAR-1 on theirplatelets and are thus not suitable for evaluation of PAR-1

antagonists. This problem has been overcome using nonhumanprimates that express PAR-1 on their platelets such as the cyn-omolgus monkey. Indeed, it has been characterized an ex vivomodel of platelet aggregation in conscious cynomolgus mon-keys [73] that has allowed verifying the pharmacokinetic profile,and in particular the oral bioavailability, of the compoundsshowing interesting results in the in vitro assays. Other limita-tions to the development of PARs antagonists derived from:i) the limited information about the conformation of the recep-tors and ii) the tethered ligand binding mechanism that is ener-getically preferred. This original feature determines that a highaffinity antagonist may not be sufficient in order to effectivelycompete with the intramolecular binding mechanism; it couldbe necessary to identify a compound with a slow dissociationrate from the receptor.

There are two main issues that make the development ofPAR1 antagonist an attractive therapeutic target: i) acute cor-onary syndromes are characterized by thrombin formationthat persists despite the above described therapy and ii) aPAR-1 antagonist does not compromise the proteolyticactions of thrombin and does not interfere with platelet acti-vation mediated by other agonists and so thrombin’s coagu-lant and anticoagulant properties are preserved. Thus, theantiplatelet action of a PAR1 antagonist has the potential togive an efficacious platelet inhibition accompanied by areduced suppression of the normal hemostasis. In otherwords, this approach has the potential for developing drugswith a more favorable clinical profile, where the efficacy iskept and the risk of bleeding is reduced. In addition, severalexperimental data suggest a pivotal role for PAR-1 in orches-trating the interplay between coagulation and inflammationin different experimental models [74,75]. These findings raisethe possibility that other potential beneficial effect(s) couldbe obtained in cardiovascular diseases by this new classof drugs.

A proof of concept of what is stated above is given bySCH-530348 developed by Schering-Plough [76]. This himba-cine derivative potently antagonizes PAR-1 but leavesuntouched the procoagulant/anticoagulant function ofthrombin. The compound has high bioavailability and in aPhase II clinical trial on patients undergoing percutaneouscoronary intervention, where it has been added to standardtherapy with aspirin and clopidogrel, did not increase majoror minor TIMI bleeding. Phase III trials are ongoing andwill give more information that most likely will boost furtherpreclinical and clinical research [49].

In conclusion, PAR-1 antagonism appears to be a promis-ing therapeutic target that could lead to drugs with cardiovas-cular protective effects lacking the undesired increasedbleeding associated with other antiplatelet therapies.

Declaration of interest

The author states no conflict of interest and has received nopayment in preparation of this manuscript.

CO2C2H5

9 (YD-3)

Cirino & Severino

Bibliography

1. Holinstat M, Voss B, Bilodeau ML, et al.

Protease-activated receptors differentially

regulate human platelet activation through

a phosphatidic acid-dependent pathway.

Mol Pharmacol 2007;71:686-94

2. McNamara CA, Sarmbock IJ,

Gimple LW, et al. Thrombin stimulates

proliferation of cultured rat aortic

smooth muscle cells by a proteolytically

activated receptor. J Clin Invest

1993;9:94-8

3. Coughlin SR. Protease-activated receptors

in hemostasis, thrombosis and vascular

biology. J Thromb Haemost

2005;3:1800-14

4. Cenac N, Cellars L, Steinhoff M, et al.

Proteinase-activated receptor-1 is an

anti-inflammatory signal for colitis

mediated by a type 2 immune response.

Inflamm Bowel Dis 2005;11:792-8

5. Ramachandran R and Hollenberg MD.

Proteinases and signalling:

pathophysiological and therapeutic

implications via PARs and more.

Br J Pharmacol 2008;53(S1):S263-82

6. Kaufmann R, Rahn S, Pollrich K, et al.

Thrombin-mediated hepatocellular

carcinoma cell migration: cooperative

action via proteinase-activated receptors

1 and 4. J Cell Physiol

2007;211(3):699-707

7. Choi MS, Kim YE, Lee WJ, et al.

Activation of protease-activated

receptor1 mediates induction of matrix

metalloproteinase-9 by thrombin in rat

primary astrocytes. Brain Res Bull

2008;76(4):368-75

8. Vergnolle N, Wallace JL, Bunnett NW,

et al. Protease-activated receptors in

inflammation, neuronal signaling and

pain. Trends Pharmacol Sci

2001;22:146-52

9. Asokananthan N, Graham PT,

Stewart DJ, et al. Activation of

protease-activated receptor (PAR)-1,

PAR-2, and PAR-4 stimulates IL-6, IL-8,

and prostaglandin E2 release from

human respiratory epithelial cells.

J Immunol 2002;168:3577-85

10. Derian CK, Damiano BP, Addo MF,

et al. Blockade of the thrombin receptor

protease-activated receptor-1 with a

small-molecule antagonist prevents

thrombus formation and vascular

occlusion in nonhuman primates.

J Pharmacol Exp Ther 2003;304:855-61

11. Darmoul D, Gratio V, Devaud H, et al.

Aberrant expression and activation of the

thrombin receptor protease-activated

receptor-1 induces cell proliferation and

motility in human colon cancer cells.

Am J Pathol 2003;162:1503-13

12. Vergnolle N, Cellars L, Mencarelli A,

et al. A role for proteinase-activated

receptor--1 in inflammatory bowel

diseases. J Clin Invest 2004;114:1444-56

13. Hamilton JR. Protease-activated receptors

as targets for antiplatelet therapy.

Blood Rev 2009;23:61-5

14. Riewald M, Petrovan RJ, Donner A,

Ruf W. Activated protein C signals

through the thrombin receptor PAR-1 in

endothelial cells. J Endotoxin Res

2003;9(5):317-21

15. Feistritzer C, Riewald M. Endothelial

barrier protection by activated protein C

through PAR-1-dependent sphingosine

1-phosphate receptor-1 crossactivation.

Blood 2005;105(8):3178-84

16. Maryanoff BE. Adventures in drug

discovery: potent agents based on ligands

for cell-surface receptors. Acc Chem Res

2006;39:831-40

17. Hoekstra WJ, Hulshizer BL. Preparation

of azole peptidomimetics as thrombin

receptor antagonists. US6017890; 2000

18. Hoekstra WJ, Maryanoff BE,

McComsey DF, Zhang H. Preparation

of novel indole peptidomimetics as

thrombin receptor antagonists.

WO2001000657; 2001

19. Zhang H, Maryanoff BE, Pandey A,

Scarborough RM. Preparation of

indazole peptidomimetics as thrombin

receptor antagonists. WO2001000656;

20. Zhang H, Maryanoff BE, Mccomsey DF,

White KB. Preparation of

benzimidazolone peptidomimetics as

WO2001000659; 2001

21. Andrade-Gordon P, Maryanoff BE,

Derian CK, et al. Design, synthesis, and

biological characterization of a

peptide-mimetic antagonist for a

tethered-ligand receptor. Proc Natl Acad

Sci USA 1999;96:12257-62

22. Zhang HC, McComsey DF, White KB,

et al. Thrombin receptor (PAR-1)

antagonists. Solid-phase synthesis of

indole-based peptide mimetics by

anchoring to a secondary amide.

Bioorg Med Chem Lett

2001;11(16):2105-09

23. Zhang HC, Derian CK,

Andrade-Gordon P, et al. Discovery and

optimization of a novel series of

thrombin receptor (PAR-1) antagonists:

potent, selective peptide mimetics based

on indole and indazole templates.

J Med Chem 2001;44:1021-4

24. Maryanoff BE, Zhang H,

Andrade-Gordon P, Derian CK.

Discovery of potent peptide-mimetic

antagonists for the human thrombin

receptor, protease-activated receptor-1

(PAR-1). Curr Med Chem 2003;1:13-36

25. Kuliopulos A, Covic L. G protein

coupled receptor (GPCR) agonists and

antagonists and methods of activating

and inhibiting GPCR using the same.

US6864229; 2005

26. Covic L, Gresser AL, Talavera J, et al.

Activation and inhibition of G

protein-coupled receptors by

cell-penetrating membrane tethered

peptides. PNAS 2002;99:643-8

27. Covic L, Misra M, Badar J, et al.

Pepducin-based intervention of

thrombin-receptor signaling and systemic

platelet activation. Nat Med

2002;8(10);1161-5

28. Boire A, Covic L, Agarwal A, et al.

PAR-1 is a matrix

metalloprotease-1 receptor that promotes

invasion and tumorigenesis of breast

cancer cells. Cell 2005;120:303-13

29. Agarwal A, Covic L, Sevigny LM, et al.

Targeting a metalloprotease-PAR-1

signaling system with cell-penetrating

pepducins inhibits angiogenesis, ascites,

and progression of ovarian cancer.

Mol Cancer Ther 2008;7(9):2746-57

30. Ahn H, Arik L, Boykow G, et al.

Structure-activity relationships of

pyrroloquinazolines as thrombin receptor

antagonists. Bioorg Med Chem Lett

1999;9:2073-8

31. Strande JL, Hsu A, Su J, et al. SCH

79797, a selective PAR-1 antagonist,

limits myocardial ischemia/reperfusion

injury in rat hearts. Basic Res Cardiol

2007;102:350-8

32. Zania P, Kritikou S, Flordellis CS, et al.

Blockade of angiogenesis by small

molecule antagonists to protease-activated

receptor-1: association with endothelial

cell growth suppression and induction of

apoptosis. J Pharmacol Exp Ther

2006;318:246-54

33. Di Serio C, Pellerito S, Duarte M, et al.

Protease-activated receptor 1-selective

antagonist SCH79797 inhibits cell

proliferation and induces apoptosis by a

protease-activated receptor 1-independent

mechanism. Basic Clin

Pharmacol Toxicol 2007;101:63-9

34. Maryanoff BE, Zhang H,

McComsey DF.

Aminomethyl-pyrroloquinazoline

compounds as thrombin receptor

antagonists. WO2002068425; 2002

35. Barrow JC, Connolly T, Freidinger RM,

et al. Preparation of

aryl-isoxazolyl-amines and use as

GB2356198; 2001

36. Nantermet PG, Barrow JC, Selnick HG.

Preparation of pyrrolidinecarboxamides,

piperidinecarboxamides, and related

compounds as thrombin receptor

antagonists. US2001044454; 2001

37. Barrow JC, Nantermet PG, Selnick HG,

et al. Preparation of N-aryl-N¢-arylethylurea derivatives as thrombin

receptor antagonists. US2002007045;

38. Suzuki S, Kotake M, Miyamoto M, et al.

Preparation of cyclic amidine derivatives

as thrombin receptor antagonists.

WO2002085850; 2002

Preparation of 2-iminopyrrolidine

derivatives as thrombin receptor

Preparation of 2-iminoimidazole

derivatives as thrombin receptor

41. Husted S. New developments in oral

antiplatelet therapy. Eur Heart J Suppl

2007;9:D20-27

42. Serebruany VL, Kogushi M,

Dastros-Pitei D, et al. The in-vitro

effects of E5555, a protease-activated

receptor (PAR)-1 antagonist, on platelet

biomarkers in healthy volunteers and

patients with coronary artery disease.

Thromb Haemost 2009;102(1):111-9

43. Chackalamannil S, Asberom T, Xia Y,

et al. Preparation of himbacine analogs as

US6063847; 2000

44. Chackalamannil S, Chelliah MV,

Clasby MC, Xia Y. Preparation of

himbacine analogues as thrombin

receptor antagonists. WO2003033501;

45. Chelliah MV, Chackalamannil S, Xia Y,

et al. Preparation of constrained

himbacine analogs as thrombin receptor

46. Chackalamannil S, Wang Y,

Greenlee WJ, et al. Discovery of a novel,

orally active himbacine-based thrombin

receptor antagonist (SCH 530348) with

potent antiplatelet activity. J Med Chem

2008;51:3061-4

47. Becker RC, Moliterno DJ, Jennings LK,

et al. Safety and tolerability of SCH

530348 in patients undergoing

non-urgent percutaneous coronary

intervention: a randomised, double-blind,

placebo-controlled phase II study. Lancet

2009;14(373):919-28

48. The TRA*CER Executive and Steering

Committees. The Thrombin Receptor

Antagonist for Clinical Event Reduction

in Acute Coronary Syndrome

(TRA*CER) trial: study design and

rationale. Am Heart J

2009;158(3):327-34

49. Morrow D, Scirica B, Fox K, et al.

Evaluation of a novel antiplatelet agent

for secondary prevention in patients with

a history of atherosclerotic disease: design

and rationale for the thrombin-receptor

antagonist in secondary prevention of

atherothrombotic ischemic events

(TRA 2 degrees P)-TIMI 50 trial.

Am Heart J 2009;158(3):335-41

50. Hezi-Yamit A, Wong J. Local delivery of

PAR-1 antagonists to treat vascular

complications. US2009297576; 2009

51. Chackalamannil S, Wang Y.

Oxazoloisoquinoline derivatives as

thrombin receptor antagonists and their

preparation, pharmaceutical compositions

and use in the treatment of diseases.

US2007149518; 2007

52. Chackalamannil S, Chelliah MV, Xia Y.

Spiroheterocyclic-decalin compounds as

thrombin receptor antagonists and their

preparation and pharmaceutical

compostions. WO2006105217; 2006

53. Chelliah MV, Chackalamannil S, Xia Y.

Monocyclic and bicyclic himbacine

derivatives useful as thrombin receptor

54. Xia Y, Chelliah MV, Chackalamannil S.

Fused ring thrombin receptor

55. Chackalamannil S, Chelliah MV,

Wang Y, Xia Y. Preparation of bicyclic

and tricyclic himbacine derivatives as

WO2008042422; 2008

56. Perez M, Lamothe M, Le Grand B,

Letienne R. Preparation of

phenylpentadienoyl derivatives, especially

phenylpentadienoylpiperazines, as

proteinase-activated receptor

1 antagonists. FR2902427; 2007

57. Perez M, Lamothe M, Le Grand B,

Letienne R. Preparation of

cinnamoylpiperazines as

proteinase-activated receptor

1 antagonists. FR2902426; 2007

58. Perez M, Lamothe M, Maraval C, et al.

Discovery of novel protease activated

receptors 1 antagonists with potent

antithrombotic activity in vivo.

J Med Chem 2009;52(19):5826-36

59. Perez M, Le Grand B, Letienne R.

Protease activated receptor-1 antagonists

for the treatment of atrial fibrillation.

WO2008155335; 2008

60. Heinelt U, Wehner V, Herrmann M,

et al. Preparation of triazolopyridazines as

proteinase activated receptor (PAR-1)

61. Beri R. The use and identification of

antagonists of protease-activated receptor

3 (PAR-3) for the treatment of asthma.

WO2006054931; 2006

62. Wu CC, Huang SW, Hwang TL, et al.

YD-3, a novel inhibitor of

protease-induced platelet activation.

Br J Pharmacol 2000;130:1289-96

63. Wu CC, Hwang TL, Liao CH, et al.

Selective inhibition of protease-activated

receptor 4-dependent platelet activation

by YD-3. Thromb Haemost

2002;87:1026-33

64. Wu CC, Hwang TL, Liao CH, et al.

The role of PAR-4 in thrombin-induced

thromboxane production in human

platelets. Thromb Haemost

2003;90:299-308

Cirino & Severino

65. Peng CY, Pan SL, Guh JH, et al. The

indazole derivative YD-3 inhibits

thrombin-induced vascular smooth

muscle cell proliferation and attenuates

intimal thickening after balloon injury.

Thromb Haemost 2004;92:1232-9

66. Huang LJ, Shih ML, Chen HS, et al.

Synthesis of N2-(substituted benzyl)-3-

(4-methylphenyl)indazoles as novel

anti-angiogenic agents.

Bioorg Med Chem 2006;14(2):528-36

67. Chen HS, Kuo SC, Teng CM, et al.

Synthesis and antiplatelet activity of ethyl

4-(1-benzyl-1H-indazol-3-yl)benzoate

(YD-3) derivatives. Bioorg Med Chem

2008;16(3):1262-78

68. Wielders SJH, Bennaghmouch A,

Reutelingsperger CPM, et al.

Anticoagulant and antithrombotic

properties of intracellular

protease-activated receptor antagonists.

J Thromb Haemost 2007;5:571-6

69. Strande JL, Hsu A, Su J, et al. Inhibiting

protease-activated receptor 4 limits

myocardial ischemia/reperfusion injury in

rat hearts by unmasking adenosine

signalling. J Pharmacol Exp Ther

2008;324(3):1045-54

70. Slofstra SH, Bijlsma MF, Groot AP,

et al. Protease-activated

receptor-4 inhibition protects from

multiorgan failure in a murine model of

systemic inflammation. Blood

2007;110(9):3176-82

71. Lerner E, Reddy VB. Method for

preventing and treating itch by

administering a G protein-coupled

protease activated receptor 4 antagonist.

WO2008086069; 2008

72. Xia Y, Chackalamannil S. Combination

therapies comprising PAR-1 antagonists

with PAR-4 antagonists for treatment of

diseases assocd. with thrombosis.

WO200912410; 2009

73. Chackalamannil S, Xia Y, Greenlee WJ,

et al. Discovery of potent orally active

thrombin receptor (protease activated

receptor 1) antagonists as novel

antithrombotic agents. J Med Chem

2005;48:5884-87

74. Howell DC, Goldsack NR, Marshall RP,

et al. Direct thrombin inhibition reduces

lung collagen, accumulation, and

connective tissue growth factor

mRNA levels in bleomycin-induced

pulmonary fibrosis. Am J Pathol

2001;159:1383-95

75. Fiorucci S, Antonelli E, Distrutti E, et al.

PAR-1 Antagonism protects against

experimental liver fibrosis. Role of

proteinase receptors in stellate cell

activation. Hepatology 2004;39:365-75

76. Oestreich J. SCH-530348, a thrombin

receptor (PAR-1) antagonist for the

prevention and treatment of

atherothrombosis. Curr Opin

Investig Drugs 2009;10:988-96

AffiliationGiuseppe Cirino†1 PhD FBPharmacolS &

Beatrice Severino2 PhD†Author for correspondence1Professor,

University of Naples Federico II,

Department of Experimental Pharmacology,

Via Domenico Montesano 49,

Napoli 80131, Italy

Tel: +39 81678442; Fax: +39 81678442;

E-mail: cirino@unina.it2University of Naples Federico II,

Department of Medicinal Chemistry,

Via Domenico Montesano 49,

Napoli 80131, Italy

Thrombin receptors and their antagonists: an update on the patent literature

Documents