CA2021953A1 - Fibrinogen receptor antagonists - Google Patents

Abstract

7879P/ 7/28/89: Fl 17981 TITLE OF THE INVENTION FIBRINOGEN RECEPTOR ANTAGONISTS A fibrinogen receptor antagonist of the formula: wherein A, R, R1, D, E and X-Y are preferably defined as follows A is acylamido; R is H or methyl; R1 is H or methyl; X-Y is S-S, CH2-S-S or CH2CH2; E is COOH; and D is L-arginine.

Description

7/28/89: Fl 1 - . 17981 TITLE OF T~E INVENTLg~
FIBRINOGEN RECEPTOR ANTAGONI STS

BA(;E~;R~:)UND OF T~;. INVEN~
The invention relates generally to modulating cell adhesion a~d to inhibiting the binding of ~ibrinogen and other protein6 to blood platelets, and inhibiting the aggregation of blood platelet specifically to the IIb/IIIa fibr~nQgen receptor ~ite. Fibrinogen î6 a glycoprotein, pre~ent in blood plasma, which participate~ in platelet aggregation an~ in ~ibrin formation. Platelet~ are cell-like anucleated ~ragment~, found in the blood of all mammal~, which participate in b~o~d coagulation.
Inter~ction of fibrinogen with the lIb/IIIa receptor site i6 known ~o be e~ential for normal p~atelet function.

-.:

7t28/89: Fl When a blood veB~el i6 damaged, platelet~
adhere to the di~rupted subendothelial ~ur~ace. The adherent platelet~ ~ubsequently release biologically active con~tituent~ and aggregate. Aggregation i~
initiated by the binding of agonists, such a6 S thrombin, epinephrine, or ADP to specific platelet membrane receptors. Stimulation by agoni~t6 results in exposure of latent ~ibrinogen receptor~ on the platelet surface, and binding of ~ibrinogen to the glycoprotein IIb/IIIa complex.
Attempts have been made to use natural products and Rynthetic peptide~ to ~tudy the ~echani~m of platelet aggregation and adhesion.
Rouælah~i and Pierschbacher, S~ience. 1987,

2~, pp. 491-497, describe adhe~ive proteins ~uch as fibronectin, vitronectin, osteopontin, collagen~, thrombospondin, fibrinogen, an~ von Willebrand factor present in extracellular matrices and in the blood.
The proteinR contain the tripeptide arginine-glycine-a~partic acid a6 their cell recognition site. The tripeptide~ are recognized by at least one member of a family of ~tructurally related receptor~, integrinæ, which are heterodimeric proteins with two membrane-~panning ~ubunit~. The authors ~tate that the conformation of the tr~pcptide sequence in the individual proteing may be critical to recognition specifieity.
Cheresh, Pr~c. ~at'l, ~r.a~. Sci.~, 1987, 84, pp. 6471-6475, de~cribes an Arg-~ly-A~p directed adhe~ion r~ceptor expressed by human endothelial cell~ that i8 ~tructurally sim~lar to the IIb/IlIa complex on platelet~ but antigenically and 7/28/89: Fl functionally di~tinct. The receptor i6 directly involved in endothelial cell attachment to fibrinogen, von Willebrand ~actor, and Yitronectin.
Pierschbacher and Rouslahti, J. of Biol.
Ch~mL, 1987, 2~2. 36, pp. 17294-17298 de6cribe stereochemical influence of the sequence Arg-Gly-Asp-Xaa, where gaa is one o~ tbe 20 natural L-amino acids other than ~et, Cy~, ~i8, Trp or Gly on bindin~
~pecificity of peptides containing the tripeptide sequence Arg-Gly-Asp. The authors showed that lo cyclization of the sequence Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala (where Pen i~ penicillamine), by forming a disulfide bridge betl~een Pen and Cys, rendered the peptide ineffective at inhibiting attachment to fibronecti~. In PrQ~ s~L Acad.
Sci. USA, 1984, ~1. pp. 5985-5988, the ~ame authors de~cribe variants of the cell recognition 6ite of fibronectin that retain attachment-promoting activity. The tetrapeptide Arg-Gly-Asp-Ser i~
de~cribed as the minimal ~tructure reco~nized by cells in the large, adhesive ~lycopr~tein fibronectin.
Peptides having portion~ -Ar~-&ly-Asp-Ser- are described in U.S. Patent No~. 4,589,881 and 4,614,517. Peptide6 having portion~ -Arg-Gly-A~p-R
wherein R is selected from Thr or Cys or other amino acid having the same cell-attachment activity aR
fibronectin, are described i~ ~.S. Patent No.
4,~78,079.
Ruggeri et al., Proc. Nat'l. ~cad.-~i. U~Q, l9B6, ~, pp. 5708-5712, de~cribe~ a serie~ of synthetic peptides, de~lgned i~ lengths to 16 residue~, that contain the ~eguence Arg-Gly-A~p-Val~

, ~

7/28/89: Fl which inhibit fibrinogen binding to platelet~.
While it i~ ~nown that the tripeptide sequence Arg-Gly-A~p i~ present in certain polypeptide~ which can duplicate or inhibit the cell attachment-promotlng effects o~ fibronectin and vitronectin, the tripeptide Arg-Gly-Asp has low activity. There is little understanding o~ the in~luence on binding specificity of other amino acids in the polypeptide. Applicants have prepared small cyclic pentapeptideR which contain the $ripeptide 6equence Arg-Gly-Asp which are active platelet aggregation inhibitor~.

SUMMARX OF T~E INvENTIQ~
The present invention iæ a ~ibrinogen receptor antagoni~t of the following structure:

R R~
R ~ X-Y ~ R
A ~ D-Gly-A6p-N~ E

wherein:
A i~ ~, acylamido, aminoacylamido or N-methylaminoacyl~mido;
R and Rl are independe~t7y ~, methyl, ethyl or a lower al~yl group haYin~ 3 to 5 carbons;
~-~ i8 S-S, UI2-S, S-CH2, CH2CH2, 1:~2CH2CH2, CE12-S~S, CH2-S-S-ClI
S-S-C~2;

- ~

', . ! `

`, 5 ~ ~

7879P/ - 5 - . 17981 7/28/89: Fl D i~ an L-i~omer of arginine, homo-arginine, guanido ~minobutyric acid or guanido aminopropionic acid;
E is ~, COO~, CON~2, COM~R2, CoNR3~4~
wherein R i8 an alkyl group having 1 to 4 carbon atoms, R3R4 i 8 an alkyl group having 1 to 4 carbon atoms or NR3R4 i8 a secondary amino acid, or ~N- NH
N
lo N ~

More preferred compounds are those where:
A is acylamide;
R i~ ~ or methyl;
Rl is ~ or methyl;
X-Y is S-S, C~2-S-S or C~2C~ ;
E i~ COO~; and D is L arginine.
More pre~erred compounds are:
2D i ) Ac-Cy~-Arg-Gly-Asp-Cy~-N~2;
ii) Ac-(D-Cys)-Arg-Gly-Asp-Cy~-M~2;
iii) Ac-~en-Arg-Gly-A~p-Cy~-N~2;
iv) Ac-(homoCy~)-Arg-Gly-A~p-C~6-N~2;
v) Ac-Cys-Arg-Çly-Asp-Pen-N~2;
2s vi~ Ac-C ~ ~ y8-O~;
vi~ Ac-Pen-Arg-Gly-A~p-Cys-O~;
viii) Ac-Cys-Arg-Gly-Asp-~y~-Pro-N~2;
iX ~ _ _ ~ Arg-Gly-As~-N~

x) Cy~-Arg-Gly-Asp-~y~-OH

7/28/89: Fl xi) Ac-(N-methyl-Cy6)-Arg-Gly-Asp-Cys-O~
~ii) Ac-Cys-Arg-Gly-Asp-(D-Cys)-NH2 ~:iii) Ac-Cys-Arg-Giy-A8p-Pen-O~I
~iv) Ac-Pen-Arg-Gly-Asp-Pen-NH2 ~v) Ac-(D-Pen)-Arg-Gly-Asp-Cys-O~
S ~vi) Ac-Cy~-Arg-Gly-Asp-(bomo-Cy~)-N~2 (TGA) xvii) Ac-Cys-(D-Arg)-Gly-Asp-Cys-NH2 All amino acids are L-isomers unle~
otherwise indicated.
The mo~t preferred cyclic peptide~ are those in the acid form, ~pecifically compound~ vi, vii, x, xi, xiii, and ~v.
Pen is penicillamine, which iB repre~ented by: :
c~3 1~ C~3tSII
~0~ .

20HomoCysteine i~ represented by:
S~

I OH
N~2/
O

, The invention also include~ compo~itions, compri~ing fibr;nogen receptor antagoniæt peptide~ o~
the present invention and one or more pharmacologically acceptable carrierÆ, e.g. ~aline, at a pharmacologically acceptable pH, e.g. 7.4, which .
.. . .
:' . . ' ' 7/2~/89: ~l are suitable for continuous intravenou~ or oral o~
intravenous bolus administration for promoting i~hibition of platelet aggregation.
The invention al80 includes method~ for inhibiting platelet aggregation which comprise administering to a patient, eit~er by continuous intravenous or oral or intravenou6 bolus method, an effective amount of a composition of the pre~ent invention~

~ETAILED D~RIRTIQ~ ~F T~E INVENTION
Compounds of the invention are fibrinogen receptor antagonist6 which inhibit fibrinogen induced platelet aggregation~ These compounds are prepared by solid phase synthesi~ which is well known in the art, or by liquid method well known in the art ~Neurath, Hill & Boeder, Eds~ "The Proteins" 3rd Edition, Vol~ II, Academic Pre~, 1976)~
The compound~ have a relatively ~hort duration of activity which makes them de6irable for use in therapeutic treatments where prevention Q~
platele~ aggreation over a ~hort period o~ time i~
de6irable~ The compound~ are al~o particularly advantageous because they do not ~i~nificantly deplete the platelet coun~.
Compound6 of the invention may be prepared u~in~ 601id phaæe peptide ~ynthe~is, such as that described by Merrifield, J. Am. Chem. Soct~ 85, 2149 (1964~, although other e~uivalent chemical syntheses known in the ~rt can al~o be used, such as the ~ynthe6es of ~oughten, PrQc. Natl. Ac~l. S~iLt 82, 5132 (1985). Solid-pha~e synthe~iR is commenced from f .~ ~J ~,' i..3 7/28/89: Fl the C-terminus of the peptide ~y coupling a protected amino acid to a ~uitable resin, as generally ~et forth in U.S. Patent No. 4,244,946, issued Jan. 21, 1982 to Rivier et al., the disclo6ure of which i6 hereby incorporated by reference. Liquid method can be u~ed a~ de6cribed by Neu~oth et al. Chapter 2, pp.
106-253 .Example~ o~ ~y~thesi~ of thi~ general type are ~et ~orth in U.S. ~atent Nos. 4,305,872 and 4,316,891.
In 6yntheEizing these polypeptides, the carboxyl terminal amino acid, having it~ alpha-amino group suitably protected, i8 coupled to a chloromethylated poly~tyrene resin or the like.
After removal of the alpha-amino protecting group, as by using trifluoroacet;c acid in methylene chloride, the next ~tep in the syntheæi~ i~ ready to proceed.
Other standard cleaving reagents and conditions for the removal of ~pecific amino protecting groups may be u~ed, as de~cribed in the open literature.
The remainin~ alpha-amino- and Eide-chain-p~otected amino acids are the~ coupled by condensation Rtepwiæe in the de6ired order to obtain an intermediate compound connected to the re~in.
The conteneation between two amino acid~, or an amino acid and a peptide, or a peptide and a peptide can bç carried out according to the usual condensation method~ ~uch as azide method, mi~ed acid anhydride method, DCC ~dicyclohexyl-carbodiimide) method, BOP (benzotriazole-l-yloxytris (dim~t~ylamino) phosphonium hexafluorophosphate method, active estex method (p-nitrophenyl e~ter method, N-hydroxy~ucciniC acid lmido ester method, 7/2B/89: Fl cyanomethyl e~ter method, etc.), Woodward reagent K
method, carbonyldiimidazol method, oxidatin-reduction method. In the c~e of elongating the peptide chain in the solid phase method, ~he peptide is attached to an in~oluble carrier at the C-terminal amino acid.
For insoluble carriers, ~hose which react with the carboxy group of the C-termina~ amino acid to form a bond which i~ readily cleaved later, for example, halomethyl resin 6uch as chloromethyl re~in and bromomethyl resin, hydroxymethyl resin, aminomethyl reæin, benzhydrylamine re~in, and t-alkyloxy-carbonylhydrazide resin can be used.
Common to chemical æyntheses o~ peptides is the protection of the reactive ~ide-chain groups of the variou~ amino acid moieties with ~uitable protecting groups at that site until the group i5 ultimately removed after the chain has been completely assembled. Al~o commo~ i~ the protection of the alpha-ami~o ~roup on an amino acid or a fragment while that entity react6 at the carboxyl group followed by the selective removal of the alpha-amino-protecting group to allow subæequent reaction to take place at that location.
Accordingly, it i~ common that, a~ a 6tep i~ the synthe~is, an intermediate compound i8 produced which include~ eaeh of the amino acid re~idue~ located in the deæiret sequence in the peptide chai~ with various oP these re idues having side-cha~n protecting group~. The~e protecting groupæ are then commonly removed sub6tantially at the ~ame time 80 a8 39 to produce the desired resultant product following purification.

7/28/89: ~1 The applicable protective groups for protecting the alpha-and omega-side chain amino group~ are exemplified 6uch as benzylo~y-carbonylthereinafter abbreviatet a~ Z), isonicotinyloxy- carbonyl (iNOC), o-chlorobenzyl-S oxycarbonyl ~Z(2-Cl)], p-nitrobenzylo~ycarbonyl ~Z(N02)], p-methoxybenzyl- oxyc,arbonyl ~Z(OMe)~, t-butoxycarbonyl (Boc), t-amylo~ycarbonyl (Aoc), isobor~ylo~ycarbonyl, ada~antyloxycarbonyl, 2-(4-biphenyl)-2- propyloxy- carbonyl (RPOC), 9-fluorenylfethoxycarbonyl (Fmoc), methyl~ulfonyl-ethoxycarbonyl (MBC), trifluor~acetyl, phthalyl, formyl, 2-nitrophenylsulphenyl (NPS), diphenyl-phosphinothioyl (Ppt), dimethylphosph- inothioyl (Mpt) and the like.
As protectiYe group~ for carbo~y group there can be exemplified, for example, benzyl ester (OBzl), cyclohexyl ester (Chx) 4-nitro~enzyl ester (ONb), t-butyl ester (OBut),4-pyrityl~ethyl ester (OPic), and the like. It is de~irable that ~pecific amino acids such as arginine, cysteine, and ~erine possessing a functional group other than amino and carboxyl groups are protected by a ~uitable protective grQup a~ occa~ion demand~. For example, the guanidi~o grvup in ar~inine may be protected with 2s ni~r~, p-toluene-sulfonyl, ~enzylo~ycarbonyl, adamantylo~ycarbonyl, p-methoxybenzene~ul~onyl, 4-methoxy-2, 6-dimethylbenzene~ul~onyl ~Mds), 1,3,5-trimethylphenyl~ulfonyl (Mts), and the like. The thiol group in cy~teine may be protected 30 with benzyl, p-methoxybenzyl, tr~phenylmethyl, acetylaminomethyl, ethylcarbamoyl, 4-methylbenzyl, J J,'. ~

7879P/ - ll - 17981 7/28/89: Fl 2,4,6-trimethylbenzyl (Tmb) etc., and the hydroxyl group in serine can be protected with benzyl, t butyl, acetyl, tetrahydropyranyl etc.
Stewart and Young, "Solid Phase Peptide Synthe~is", Pierce Chemical Company, Roc~ford, IL
(1984) provide~ detailed information regarding procedures for preparing peptide~. Protection of a-amino groups iB de~cribed on pages 14~18, ~nd ~ide-chain blockage is described on pages 18-28. A
table of protecting groups for amine, hydroxyl and ~ulfhydryl functions i8 provide~ on pages 14g~151.
These deRcriptions are hereby incorp~rated by reference.
After the desired amino-acid sequence ha6 been completed, the intermediate peptide iB removed from the resin support by treatment with a reage~t, æuch as liquid ~F, which not only cleaves the peptide from the re~in, but also cleaves all the remaining protecting groups ~rom the side chain which do not interfere in the cyclizatio~ reaction. Potentially reactive 6ide chains functionalities are protected with blocking groups which are ~table to ~F. The peptides are cyclized by any one of ~everal known procedure6 ~6ee Schroder and Lubke, "The Peptides:
Method~ of Peptide Synthe~ie" Vol. I, Academic Pre~, New ~or~ (1965>, pp. 271-286, the conte~ts of which are hereby incorporated by ref~rence), e.g. by forming a di~ulfide bridge between the cystei~e residue~ uæing iodine in AcO~, or air oxidation at p~
8 in dilute N~4 OAc buffer. The polypeptide can then be purified by gel permeation followed by preparative ~PLC, as de~cribed in Rivier et al., 7/28/89: Fl Peptides: Structure and Biological Function (1979) pp. 125-128.

$ynthe6i~_~f~ Ae.etyl-Cys-A~-Gly-A~ ys-NH2 S B~c (~ butylcarbonyl) and PMB
(p-metho~ybenzyl) protected cy~teine ~c combined with N~2-NBH ~ (4-methylbenzhydryla~ine-cr~s~linked poly~tyrene resin~ to form PMB
lo Boc-Cy~ ~ N~-MBH
The alpha-amino Boc protecting group i~ remo~ed (while Cy~ remains protected by PMB) u~ing t~ifluoroacetic acid and methylene chloride. The deprotected cy~teine i5 neutralized with dii~opropylethylamine in dimethylformamide. Boc-pro-tectet Asp ~benzyl) (Asp ~Bzl)) i~ then coupled to sy~teine ~ediated by dicyclohexylcarbodiimide, and then deprotected with trifluoroacetic acid and methylene chloride. Asp i8 then neutralized wigh dii~opropylethylamine. Following th;~ stepwi~e procedure of coupling with dicyclohexylcarbodiimide, deprotection with trifluoroacetic icid and methylene ehloride, and neutralization with diisopropylethylamine, Boe-protected Gly~ Arg and Cy~
re~idues are coupled in succession. Ar~ i~
additionally protected with 4-toluenesulfonyl (Arg (TQ8~), and the ~inal Cy8 residue is again additionally protected ~y p-methoxybenzyl. The ~inal Cy~ i8 then acetylated with acetic anhydride.

'~

, 5; ~3 7/28/89: ~1 After acetylation, the following peptide re~in i~
formed:
PMB Tos Bzl PMB
Acetyl-Cy6-Arg-Gly-A~p-Cys-N~-MB ~.
Cleavage of the peptide from the re~in i8 achieved uein~ ~F/ani~ole (9:1 (v/v)) to ~orm H H
Acetyl-Cy~-Arg-Gly-A~p-Cy~-NH2 A cyclic 6tructure i6 formed by formation of a di6ulfide bridge between the cysteine residues. The pep~ide i~ dissolved in 50-80~/o HOAc:~20 at room temperature, and the solution ~tirred during rapid addition of a solution of 12-15 equivalent~ iodi~e in AcOH to a final concentration of 2.25 m~/ml of iodine. After 1-2 hour~ reaction time, excess I2 and AcOH are removed by rotary evaporation under vacuum and the aqueous solution containing the cyclized peptide is purified u~ing preparati~e ~PLC
in O~lZo TFA H20-C~3CN gradient. The final TFA ~alt product i~ eonverted to ~OAc salt by passing through ion exchange column BioRad AG3-~4A (acetate cycle>.
The fini~hed peptide is:

Acetyl-Cy~-Arg-Gly-Asp-Cy6-M~2 ~ ~OAc salt As an alternative to d~o~ing the peptide in 50-89% HOAc-~20 at ~oom temperature, the free S~
peptide i8 di6~01ved in 1-5% ~OAc at a concentration of approximately 2 ~g./ml. and made to approximately pH 7-8.5 with concentration NH~OH. Cyclization i~
- accompli~hed under bri8~ stirring (preferably with a 7~79P/ -- 14 -- 179 7/28/8Y: Fl ~mall bit of copper wire added to accelerate the reaction) during a period of 1-4 hour~ at ~5. The reactiion mixture i8 then concentrated a~ before Sy~h~si~ o~ c~lQ-(Aha-Al~L~ y~
Starting with Boc-Gly-0-Pam ~, the alpha-amino Boc protectin~ group i8 removed usin~ trifluoroacetic acid and methylene chloride. The deprotected glycine is neutralized with diisopropylethylamine and dimethylformamide. Boc-protected Arg (Tos) ~ then coupled to Gly mediated by l-hydro~y-~enzotriazole, and then deprotect~d with trifluoro-acetic acid and methylene chloride. Arg is then neutralized with diisopropylethylamine and dimethylformamide. Boc-protected Aha and Asp (N02Bz) are the 6ucces~ively coupled, Aha to Arg and Asp to Aha, following the step~ise procedure of coupling with dicyclohexylcar~odiimide, deprote~tion with trifluoroacetic acid and methylene chloride, and neutralization with tii~opropylethylamine, to form:

N2BZ To~
Boc-Asp-Aha-Arg-Gly-O-Pam ~) Clsavage of the peptide from the resin i~
achieved u~ing ~F/anisole (9:1 (v/v)~ to form:
~0 Bz EF salt ~ o A6p-Aha-Arg-Gly-0~
A cycl~c structure i8 then formed aB ~ollows:
The llnear peptide ~8 treated with N-ethyl-N'-(3-dimethylaminopropyl)carbod$imide, l-hydroxybe~zotriazole, dimethylformamide and , . , . , ~

' ~ r~ 3 7/28/89: Fl N-methylmorpholine to form NBz cyclo (Asp-Aha-Arg-Gly3 and finally deprotected with Zn/HOAc or ~2/Pd on ~harcoal to yield _ Arg-~ly-Asp-NH

The cyclized peptide i6 purified u~ing gel permeation with 50% aqueous ~OAc and HPLC in 0.1% TFA
H20-C~3CN gradient. The fin~l TFA salt product is converted to ~OAc ~alt by pa~sing through ion exchange column BioRad AG3-X4A (acetate cyele).

lS .The~eutic Treatm~nt Peptides of the invention may be used for inhibiting integrin protein-complex function relating to cell-attachment activity. They may be administered to patient~ where inhibiti~n of human or ~O mammalian platelet aggregation or adhe~ion iE de~ired.
Polypeptide~ of the inve~tion are eliminated from circulation rapidly and are particularly uæeful in inhibiting platelet aggregation in ~ituations where a strong antithrombotic of ~hort duration of effectivenes~ i~ needed. Thu~, they may find utili~y in surgery on peIipheral arteries (arterial graftæ~
carotid endarterectomy) and in card iovaRcul ar 8urgery where manipulatio~ o~ ar~erieæ and organs, and/or the interaction of platelet~ with arti~ieial ~ur~aces, lead~ to platelet aggregation and con~umption. The aggregated platelet~ may form thrombi and 7879P/ - 16 - 179~1 7/28/89: ~l th~omboemboli. Polypeptides of the invention ~ay be adminirtered to the6e ~urgical patients to prevent the formation o~ thrombi and thro~boemboli.
Extracorporeal circulation iR routinely used for cardiovascular surgery in order to o~ygenate blood. PlAtelets adhere to surfaces of the extracorporeal circui~. Adhesion i~ dependent ~n the interaction between GPlIb/IIIa on the platelet ~embrane~ and fibrinogen ad60rbed to the ~ur~ace of the circuit. (Gluszko et al., ~mer~ J. Ph~iQl~
lo 1987, 2$2:~, pp 615-621). Platelet~ relea~ed from artificial ~urfaceR show impaired hemo~tatic function. Polypeptides of the invention may be adminis~ered to prevent adhesion.
Other applications of these polypeptides include prevention of platelet thrombosi~, thromboembolism and reocclusion during and a~ter thrombolytic therapy and prevention o~ platelet thrombosi~, thromboemboli~m and reocclu~ion after angioplasty o~ coronary and other arterie~ and after coronary art~ry bypas~ procedureE. Polypeptides of the invention may also be u~ed to prevent myocardial infarction.
The~e polypeptide~ may be administered by any convenient ~ean~ which will re~ult i~ it~ delivery into the blood ~tream in ~ubstantial amount including continuou~ intravenous or bolu~ i~jection or oral methods. Compogitions of ~he invention include peptide~ of the invention and pharmacologically acceptable carrier~, e.g. ~aline, at a p~ level e.g.

3~ 7 4, ~uitable for achieving inhibition of platelet aggregation. They may be combined with thrombolytic ''';J

7~79P/ - 17 - 17981 7/28/89: Fl agents such as pla~minogen activators or streptokinase in order to inhibit platelet aggregation. They may al60 be combined with anticoagulants such as heparin, aspirin or warfarin.
Intravenou~ admini~tration is presently contemplated as the pre~erred admini~tration route. They are soluble in water, and ~ay therefore be effectively ad~inistered i~ æolution.
In one e~emplary application, a suitable amount o~ peptide is intravenously administered to a lo heart attack victim undergoin~ angiopla~ty.
Administration occur~ during or ~everal minutes prior to angioplasty, and i~ in an amount ~ufficient to inhibit platelet aggregation, e.g. a~ amount which achieve~ a steady ~tate plasma concentration of between about 0.05-30 ~M per kilo, preferably between about 0.3-3 ~M per kilo. When thi~ amount i6 achieved, an infu~ion of between about 1-100 ~M
per kilo per min., preferably between about 10-30 ~M per kilo per min. i~ maintained to inhibit platelet aggregation. Should the patient need to undergo bypas~ ~urgery, administration may be ~topped immediately and ~ill not cau~e complication~ during ~ur~ery that would be cauged by other materials ~uch a~ aspirin or monoclonal antibodie~, the effects of which last hours a~ter ce6satio~ o~ administratio~.
The pre~ent inYention al ~o includes a pharmaceutical composit~on eompri~ing peptldes of the pre~ent inventio~ and tis~ue-type ~la~minogen ac~ivator or strepto~inase. The in~ention also includes a method for promoting t~ro~bolysi~ and preventing reocclu~ion in a patient which compri~es ,; ", ~ :: . .. .

r~ ' 7~

7/28/89: Fl admini~tering to the patient an effectiYe amount of compositions of the invention.
The present invention may be embodied in other specific forms without departing ~rom the ~pirit or essential attributes thereof. Thus, the specific examples described above ~hould not be interpreted as limiting the scope of the present invention.

,

Claims (13)

7/28/89: Fl WHAT IS CLAIMED IS:

1. A fibrinogen receptor antagonist compound of the following structure:

wherein:
A is H, acylamido, aminoacylamido or N-methylaminoacylamido;
R and R1 are independently H, methyl, ethyl or a lower alkyl group having 3 to 4 carbons;
X-Y is S-S, CH2-S, S-CH2, CH2CH2, CH2CH2CH2, CH2-S-S, CH2-S-S-CH2, S-S-CH2;
D is an L isomer of arginine, homo-arginine, guanido aminobutyric acid or guanido aminopropionic acid;
E is H, COOH, CONH2, CONHR2, CONR3R4, wherein R2 is an alkyl group having 1 to 4 carbon atomE, R3R4 is an alkyl group having 1 to 4 carbon atoms or NR3R is a secondary amino acid, or 7/28/89: Fl

2. A compound of Claim 1 wherein:
A is acylamide;
R is H or methyl;
R1 is H or methyl;
X-Y is S-S, CH2-S-S or CH2CH2;
E is H, COOH;
D is L-arginine.

3. A compound of Claim 1 which is:

4. A compound of Claim 1 which is:

5. A compound of Claim 1 which is:

6. A compound of Claim 1 which is:

7. A compound of Claim 1 which is:

8. A compound of Claim 1 which is:

9. A compound of Claim 1 which is:

10. A compound of Claim 1 which is:
7/28/89: Fl

11. A compound of Claim 1 which is:

12. A compound of Claim 1 which is:

13. A composition for inhibiting fibrinogen-induced platelet aggregation in a mammal comprising a peptide of claim 1 and a pharmaceutically acceptable carrier.