EP0412115A4 - Hybrid peptides and methods of their use - Google Patents
Hybrid peptides and methods of their useInfo
- Publication number
- EP0412115A4 EP0412115A4 EP19890905845 EP89905845A EP0412115A4 EP 0412115 A4 EP0412115 A4 EP 0412115A4 EP 19890905845 EP19890905845 EP 19890905845 EP 89905845 A EP89905845 A EP 89905845A EP 0412115 A4 EP0412115 A4 EP 0412115A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- hybrid
- platelets
- analogue
- interaction
- fibrinogen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention was made, in part, in the course of work under Research Grant HL-33014 from the National Institutes of Health, United States Public Health Service.
- the present invention relates to synthetic peptide analogues of active sites on proteins and their uses. More particularly, the invention relates to hybrid linear peptides which have sites that mimic at least two interaction sites of proteins with receptor sites, e.g., receptor sites for fibrinogen on platelets.
- the interaction between platelets and plasma proteins such as fibrinogen has been extensively studied. Separate platelet receptor recognition domains or interaction sites have been located on the gamma chain and alpha chain of fibrinogen. See Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D. D., and Doolittle, R. F., Proc. Natl. Aead. Sci. U.S.A. 79. p. 2068 (1982).
- the platelet receptor recognition domain on the gamma chain is located between residues 400-411 ( ⁇ 400-411), and the alpha chain platelet receptor recognition domain is thought to be related to two loci ( ⁇ 95-97 and ⁇ 572-575).
- the sequences on the gamma chain (HHLGGAKQAGDV) are not homologous to the common areas flanking the sequence RGD on the alpha chain.
- the native human fibrinogen alpha chain fragment encompassing residues ⁇ 518-584 and the gamma chain fragment encompassing residues ⁇ 385-411 can each independently inhibit the ADP- or thrombin-induced interaction between the glycoprotein fibrinogen receptor on platelets and fibrinogen.
- synthetic peptide analogues of the alpha chain can inhibit gamma chain binding and visa-versa.
- the use of synthetic peptide analogues in platelet-binding systems, as well as their medical applications, is further discussed in United States Patents Nos. 4,661,471, 4,666,884, and 4,703,039, all based on applications of some of the present inventors.
- Fibrinogen, fibrin, and von Willebrand factor are important in the formation of hemostatic platelet plugs and the initiation of thrombotic lesions. Blockage caused by these plugs and the damage caused by thrombotic lesions are major factors in heart disease and stroke.
- Much research has been directed towards developing drugs that will dissolve already formed blood clots but many of these drugs, e.g., see recent reports on the use of streptokinase and tissue plasminogen activator, have their drawbacks. While enzymatic methods of dissolving clots may help minimize the after-effects of heart attacks, a pharmaceutical preparation which will inhibit platelet adhesion and clumping may prevent the initial blockage of blood vessels responsible for cardiac or cerebral infarction.
- the manufacture and use of molecules which promote platelet aggregation, or adhesion to blood vessels may have a variety of uses.
- a number of patients e.g., some bleeders, may be missing fibrinogen or von Willebrand factor due to genetic deficiencies or due excess consumption in circulation.
- a synthetic platelet aggregation or adhesion promoting molecule can assist in platelet plug formation and attachment of plugs to blood vessels to arrest bleeding and help these patients to lead normal lives.
- an object of the invention is to provide hybrid linear peptide analogues which mimic at least two distinct protein domains which interact with receptor sites on platelets.
- Another object of the invention is to provide a method in inhibiting the interaction between fibrinogen, von Willebrand factor, and other adhesive proteins with platelets by the use of hybrid linear peptide analogues of the interaction domains of the adhesive proteins.
- a further object of the invention is to provide synthetic aggregation or adhesion promoting molecules formed of multimers of hybrid linear peptide analogues of at least two interaction domains on fibrinogen or von Willebrand factor.
- the present invention features hybrid linear peptides which have interaction sites that are analogous to interaction sites on a native protein. These interaction sites on the native protein will react with a receptor recognition site, e.g., a platelet receptor recognition site for the protein. The interaction sites are physically distinct in the native protein.
- the invention features a hybrid linear peptide of the form X 1 -X 2 where X 1 is an analogue of a first interaction site on a protein for a first receptor domain of that protein and X 2 is an analogue for a second interaction site on the protein for a second receptor domain.
- the receptor can be a glycoprotein or any other type of receptor which reacts with a linear protein fragment.
- the domains or interaction sites are distinct on the native form of the protein.
- the preferred receptor is the glycoprotein receptor site on human platelets for fibrinogen, fibrin, von Willebrand factor, or other adhesive proteins.
- Fibrinogen is the preferred protein whose sites form the basis of the analogues and X 1 and X 2 are preferably analogues of the interaction sites for platelets on the gamma and alpha chains, respectively.
- the X 1 -X 2 Preferred hybrid peptide has, therefore, at least a portion of the platelet receptor recognition domain from the gamma chain near its amino terminal end and a portion of the platelet recognition receptor domain from the alpha chain near the carboxyl terminal end. Alternatively, the arrangements of these domains can be reversed.
- the most preferred X 2 or carboxyl terminal portion for fibrinogen-platelet analogue systems is a tetrapeptide selected from a group consisting of RGDV, RGDN, RGDF, RGDY, RGDS, RGDM, and RGDC.
- the most preferred X 1 portion is a linear peptide fragment of 2-13 residues comprising an active portion of the sequence HHLGGAKQAGDV or substitute analogues thereof.
- the symbols designating the amino acids are those standard in peptide chemistry and are set forth in Table I.
- the invention also features methods of inhibiting the interaction of platelets with native adhesive proteins, e.g., fibrinogen, primarily as an antiaggregation mechanism.
- This method has the steps of forming the hybrid linear peptide of the invention, reacting the hybrid peptide with the platelets, and exposing the platelets to the adhesive protein. Pre-incubation or pre-reaction of the peptides with the platelets and exposure of the peptides to the adhesive protein allows the hybrid peptides to interact with the platelets, thereby filling the receptor site, and prohibiting the adhesive protein platelet interaction.
- This type of procedure can be used to inhibit the interaction of fibrinogen, fibrin, von Willebrand factor, and other adhesive protein with platelets, thereby inhibiting hemostatic plug formation and adhesive of platelets to vessel walls.
- the invention further features a method of promoting aggregation and/or adhesion of platelets as part of a hemostatic plug formation process.
- This method is based on the use of multimers of the hybrid peptide of the invention to form synthetic aggregation or adhesion-promoting molecules. These multimers are either cross-linked plural copies of the hybrid or plural hybrids may be linked to a protein or other analogous backbone. In either case, the multimers have the ability to react with receptors on separate mole Icules, thereby cross-linking the molecules.
- the method of the invention starts by manufacturing these synthetic aggregation or adhesion promoting molecules and then incubating the platelets with these molecules under conditions which promote the interaction between the receptors and the synthetic molecules. Conditions which help promote this type of adhesive interaction include activation of platelets with ADP, thrombin epinephrine, or other agonists.
- Figure 1 illustrates that both the fragment corresponding to the platelet receptor recognition site on the alpha chain (RGDS) and the fragment corresponding to the platelet receptor recognition site on the gamma chain ( ⁇ 400-411) will inhibit the aggregation of platelets by gamma chain or alpha chain multimers;
- FIG. 1 shows the inhibition of binding of
- Figure 3 illustrates the inhibition of binding of 125 I-Fibrinogen by seven different hybrid peptides within the scope of the invention.
- the present invention is based, in part, on the finding that platelet receptor recognition domains located on the gamma and alpha chains of human fibrinogen are functionally cross-inhibited by synthetic peptides derived from these chains despite the lack of homologous amino acid sequences.
- Figure 1 illustrates this phenomenon of cross-inhibition by synthetic peptide analogues of sequences present in the gamma and alpha chains.
- Figure 1A shows the inhibition of the interaction between platelets and gamma chain multimers
- Figure IB shows the inhibition of the interaction of platelets and alpha chain multimers.
- Figure 1A illustrates an experiment whereby platelets were pre-mixed with a buffer, a fragment corresponding to the alpha chain platelet receptor recognition domain of fibrinogen (RGDS), or the gamma chain platelet receptor recognition domain of fibrinogen ( ⁇ 400-411).
- RGDS alpha chain platelet receptor recognition domain of fibrinogen
- ⁇ 400-411 gamma chain platelet receptor recognition domain of fibrinogen
- 5 ⁇ M of gamma chain multimers were added to the treated platelets and the transmission percentage was measured.
- the buffer had no effect on the percent transmission while the ⁇ 400-411 fragment clearly inhibited the interaction between the gamma chain multimers and the platelets. This is expected since the ⁇ 400-411 fragment is the interaction site on the gamma chain.
- the alpha chain fragment (RGDS) was equally effective in inhibiting the interaction with the gamma chain. multimers.
- Concentrations of a dodecapeptide ( ⁇ 400-411) ranging from 5-60 ⁇ M and concentrations of a tetrapeptide (RGDS) ranging from 5-15 ⁇ M were mixed as shown to test for inhibition of radiolabelled fibrinogen binding to platelets.
- Purified fibrinogen, iodinated with 125 I using standard iodine monochloride methods and having a specific radioactivity of 3 ⁇ 10 7 cpm/mg was used.
- the labelled fibrinogen (33 ⁇ g/0.5 mL) was added, followed by 5 ⁇ M ADP.
- the binding experiments were carried out a room temperature, without stirring, in a final volume of 0.5 mL which contained 1 ⁇ 10 8 platelets.
- the results of Table II are shown as percent binding of a control value determined by a test without any peptide fragment added.
- the peptides were synthesized by solid phase methods either manually, see Kloczewiak et al., Biochemistry. 23., p. 1767 (1984), or with a Biosearch BSOO. Peptide Synthesizer. The peptides were cleaved from the resin with HF and concentrations identified using standard procedures (see Kloczewiak et al., Ibid.). The inhibition of aggregation of platelet-rich plasma was measured photometrically in a Payton dual channel aggregometer (Payton Associates, Buffalo, New York).
- Aggregation was measured after the addition of ADP (5 ⁇ M) using a percentage of maximum transmission (Tmax) and rate (slope value) which represented a change in one minute along a tangent line to the steepest increase in light transmission. Timmons et al.. Trans. Assoc. Am. Phys., 99, p. 226 (1986).
- Binding of 125 I-Fibrinogen was carried out as previously described, with a five minute incubation of the peptide and the plasma-free platelets in HEPES buffer followed by treatment with ADP and fibrinogen.
- hybrid peptide or “synthetic peptide” means and includes hybrid or synthetic peptides made using any procedure including classical peptide synthesis and recombinant techniques.
- IC 50 values are the concentration of peptide at which there is 50% inhibition of fibrinogen binding.
- Table III also illustrates the IC 50 for aggregation of ADP-treated platelet-rich plasma by the same peptides. These values are 300 ⁇ M and 50 ⁇ M, respectively.
- a variety of hybrids having at least a portion of the platelet receptor recognition domain ⁇ f the gamma chain or analogue thereof and the RGD portion of the platelet receptor recognition domain from the alpha chain were prepared using standard solid phase methods.
- the terminal phenylalanine (F) or serine (S) of the alpha chain domain was replaced with a valine (V) because of the known effectiveness of valine at the terminal end of the ⁇ 400-411 domain.
- Figures 3A-G showed testing with a variety of the hybrid peptides of the invention.
- the peptide shown has the analogue of the domain from the alpha chain at the amino rather than carboxyl terminal end. In comparison to the other results, based particularly on inhibition of
- Hybrid peptides having a large portion of the gamma chain component e.g., HHLGGAKQAGDSRGDV or HHLGGAKQAGDVGRGDV, yield improvement (decrease) in the ratio of the amount necessary to inhibit
- the aggregation-preventing amount is as low, or lower, then just the alpha tetrapeptide itself, a surprising finding. This is even more important since a problem with using just an alpha fragment is that the tetrapeptide fragment exerts a significant effect on the integrity of the vascular endothelium in regard to its interaction with the extra cellular matrix. Chen et al., J. Cell Biol.. 105. p. 1885 (1987). In vivo, this effect may result in endothelial desquamation and/or impaired endothelialization of the denuded vascular surfaces.
- the resulting hybrid may lose its reactivity towards vascular endothelium so it may be possible to produce hybrids having enhanced inhibitory potency while retaining the significant features of the gamma chain domain, e.g., fibrinogen and platelet specificity and protection of vascular endothelium.
- 4,661,471 and 4,666,884 leads to the possibility that the same procedure could be used for making molecules which inhibit von Willebrand factor, fibrin, or other adhesive protein platelet interaction, or, in fact, allow for the formation of synthetic adhesive proteins. In fact, it may be possible to mix domains, e.g., place domains from different peptides on the same hybrid to achieve new and distinct effects.
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18803988A | 1988-04-29 | 1988-04-29 | |
US188039 | 1988-04-29 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0412115A1 EP0412115A1 (en) | 1991-02-13 |
EP0412115A4 true EP0412115A4 (en) | 1991-11-13 |
Family
ID=22691550
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19890905845 Withdrawn EP0412115A4 (en) | 1988-04-29 | 1989-04-25 | Hybrid peptides and methods of their use |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0412115A4 (en) |
JP (1) | JPH03505087A (en) |
AU (1) | AU3569189A (en) |
WO (1) | WO1989010135A1 (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5610148A (en) * | 1991-01-18 | 1997-03-11 | University College London | Macroscopically oriented cell adhesion protein for wound treatment |
US5629287A (en) * | 1991-01-18 | 1997-05-13 | University College London | Depot formulations |
US5645815A (en) | 1991-02-08 | 1997-07-08 | Diatide, Inc. | Radiolabled compounds for thrombus imaging |
JP2774378B2 (en) * | 1991-02-08 | 1998-07-09 | ダイアテク,インコーポレイテッド | Technetium-99m labeled polypeptide for imaging |
AU677208B2 (en) * | 1992-05-21 | 1997-04-17 | Cis Bio International | Technetium-99m labeled peptides for thrombus imaging |
ES2182833T3 (en) * | 1992-10-02 | 2003-03-16 | Diatide Inc | MULTIMERENT POLYVALENT ANTITROMBOTIC AGENTS. |
US5750088A (en) | 1993-03-30 | 1998-05-12 | The Dupont Merck Pharmaceutical Company | Stable hydrazones linked to a peptide moiety as reagents for the preparation of radiopharmaceuticals |
US6808698B1 (en) | 1999-03-26 | 2004-10-26 | Bristol-Myers Squibb Pharma Company | Method for localization of blood clots |
US6685914B1 (en) | 1999-09-13 | 2004-02-03 | Bristol-Myers Squibb Pharma Company | Macrocyclic chelants for metallopharmaceuticals |
US7317104B2 (en) | 2003-06-13 | 2008-01-08 | Bristol-Myers Squibb Pharma Company | Chelants and macrocyclic metal complex radiopharmaceuticals thereof |
US7319149B2 (en) | 2003-06-13 | 2008-01-15 | Bristol-Myers Squibb Pharma Company | Chelants and macrocyclic metal complex radiopharmaceuticals thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4578079A (en) * | 1982-08-04 | 1986-03-25 | La Jolla Cancer Research Foundation | Tetrapeptide |
US4683291A (en) * | 1985-10-28 | 1987-07-28 | Scripps Clinic And Research Foundation | Platelet binding inhibitors |
-
1989
- 1989-04-25 WO PCT/US1989/001742 patent/WO1989010135A1/en not_active Application Discontinuation
- 1989-04-25 AU AU35691/89A patent/AU3569189A/en not_active Abandoned
- 1989-04-25 EP EP19890905845 patent/EP0412115A4/en not_active Withdrawn
- 1989-04-25 JP JP1505894A patent/JPH03505087A/en active Pending
Non-Patent Citations (3)
Title |
---|
CELL, vol. 48, 13th March 1987, pages 867-873; S.A. SANTORO et al.: "Competition for related but nonidentical binding sites on the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins" * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, USA, vol. 83, 1986, pages 5708-5712, Washington, US; Z.M. RUGGERI et al.: "Inhibition of platelet function with synthetic peptides designed to be high-affinity antagonists of fibrinogen binding to platelets" * |
See also references of WO8910135A1 * |
Also Published As
Publication number | Publication date |
---|---|
JPH03505087A (en) | 1991-11-07 |
AU3569189A (en) | 1989-11-24 |
EP0412115A1 (en) | 1991-02-13 |
WO1989010135A1 (en) | 1989-11-02 |
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