CA2275544A1 - Lebetin peptides as platelet aggregation inhibitors - Google Patents
Lebetin peptides as platelet aggregation inhibitors Download PDFInfo
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- CA2275544A1 CA2275544A1 CA002275544A CA2275544A CA2275544A1 CA 2275544 A1 CA2275544 A1 CA 2275544A1 CA 002275544 A CA002275544 A CA 002275544A CA 2275544 A CA2275544 A CA 2275544A CA 2275544 A1 CA2275544 A1 CA 2275544A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
Lebetin peptides having the amino acid sequence: (X)mNKPPKKGPPNG(CFGHKIDRIGSHSGLGCNKVDDNKG)n(X)p where m=0 to 6, n=0 or 1, p=0 to 4 and each X is an amino acid residue are effective agents for the inhibition of platelet aggregation, and may be useful for the treatment of thrombosis or thromboembolism.
Description
TITLE
A Lebetin Peptide which inhibits Platelet Aggregation DESCRIPTION
The invention relates to a peptide derived from lebetin, and to its use in methods for the treatment of thrombosis or thromboembolism.
Several components isolated from snake venom, e.g. platelet aggregation inhibitors, phospholipases and ADPases, thrombin-like enzymes and fibrinolytic enzymes, affect hemostasis by interfering with the coagulation and platelet aggregation processes.
Platelet aggregation involves a complex network of cell surface adhesion proteins, one of which is GPIIb/IIIa. GPIIb/IIIa binds fibrinogen and this binding is inhibited by proteins ( "disintegrins") isolated from snake venom and containing an RGD
sequence.
Fibrinogen-GPIIb/IIIa interaction is the final step of a complex cascade of biochemical reactions and cell morphological changes, including activation of platelets which become competent to bind fibrinogen, changes in shape, secretion of the granular content and aggregation. These events are induced by platelet aggregation agonists and each of these may be a target for anti-aggregation agents.
Barbouche et al (FEBS Letters 392 (1996) 6-10] have recently isolated a new inhibitor of platelet aggregation from Vipera lebetina venom. This isolate, lebetin, lacks the RGD sequence of the disintegrins. Lebetin is composed of two groups of related peptides, lebetin 1 and lebetin 2. Lebetin 1 is a mixture of two proline and lysine rich peptides, one (sLla) of 13 amino acid residues and the other (sL113) of 12 amino acid residues, the same sequence but lacking the N-terminal glycine of sLla. sLla and sL113 have the following amino acid sequences:
sLl« GDNKPPKKGPPNG (SEQ ID NO 1), and sL113 DNKPPKKGPPNG (SEQ ID NO 2).
Lebetin 2 also consists of two peptides, one of 38 amino acid residues (sL2a) and the other of 37 amino acid residues (sL213) . sL2a has sL 1 a at its N-terminal and a 25 amino acid residue peptide with one disulphide bridge at its C-terminal. sL213 has the same sequence but lacking the N-terminal glycine of sL2a.
The invention provides a peptide (hereinafter called sLl ;~) having the amino acid AMENDED SHEET
_7_ . .
sequence NKPPKKGPPNG (SEQ ID NO 3).
sLly has proven more effective as an inhibitor of platelet aggregation than native lebetin 1 or its components sLla and sL113.
The invention also includes within its scope a modified sLly peptide in which one or more of the amino acid residues is a D-amino acid residue. D amino acids last longer in vivo because they are harder for peptidase to cut, but the L amino acids have better activity.
Moreover, peptide analogues, synthetic constructs using the carbon skeleton of peptides but omitting the -CONH- peptide bonds, can be employed in place of peptides.
Thus, it should be understood that the invention also includes within its scope a modified sLl°~
peptide in which at least one of the amino acid residues is replaced by its decarboxamido analogue. It is believed that such peptide analogues will be more resistant to peptidase and last longer in vivo.
Synthetic methods 1. Manual Synthesis of the lebetin 1 peptides using Boc-chemistry The synthetic lebetin 1 peptides sLl a (Glyl-Gly 13), sL113 (Asp2-Gly 13) and sLly (Asn3-Gly 13) were assembled manually by the solid phase technique (R. B.
Merrifield, 1986) on Boc-aminoacyl-Pam resin (0.5 mmol, substitution 0.67-0.82 mequiv of amino group per gram). The following side-chain protecting groups for trifunctional Boc-amino acids were used: cyclohexyl (CHex) for Asp, 2-chlorobenzyloxy (C1Z) for Lys and 2-bromocarbobenzoxy (BrZ) for Tyr. The synthesis cycle used for incorporation of each Boc-amino acids was: ( 1 ) dichloromethane (DCM) wash, (2 x 0.5 min); (2) 65 % trifluoroacetic acid (TFA) in DCM for deprotection step, 2 min and 13 min; (3) DCM wash, 0.6 min; isopropanol wash, 0.5 min; (4) DCM wash, (2 x 0.5 min); (5) N-methylpyrrolidone (NMP) wash, (2 x 0.5 min); (6) Boc amino acid (4 equiv) and PyBOP (4 equiv) in NMP and (7) diisopropylethylamine (DIEA) (8 equiv) for Boc-as coupling, 3 min; and (8) two NMP washes, 1 min. Each coupling step was monitored by using the qualitative ninhydrin test and recoupling was performed as necessary. Unreacted amino groups detected by ninhydrin after two consecutive couplings were blocked by acetylation withy 50 % acetic anhydride in DCM for 10 min.
At the end of the assembly and after the last deprotection step, the peptidyl resin was dried under vacuum. The high hydrogen fluoride (HF) procedure was achieved for AMENDED SHEET
deprotection and cleavage from the resin using 10 % p-cresol per volume as a scavenger. After removal of HF in vacuo, the resin was washed with cold diethylether and the peptide extracted with water. The crude peptides were purified by reversed-phase preparative medium-pressure liquid chromatography (MPLC) (Labomatic, C 18 HD-SIL 15-22 ~cm, 26 x 313 mm) using a 90-min linear Gradient of acetonitrile in 0.1 % (by vol . ) TFA/H~ O from 0 to 30 % , at a flow rate of 10 mllmin with UV detection at 206 nm. The homogeneity of the fractions was assessed by analytical HPLC (Merck, C 18 Lichrospher, 4 x 125 mm). Fractions containing homogeneous peptides ( > 99 % ) were pooled and lyophilized.
Experimental Results Platelets were prepared from 0.2M EDTA treated blood samples. Human platelets were prepared as follows: blood was collected in vials containing a sodium citrate/
dextrose ( 1: 5 v/v j mixture from a donor who had not taken any drug for at least 1 week.
Platelets were resuspended in Tyrod's buffer pH 7.4 at a final concentration of 3 x 108 cells/ml prior to the assay which was performed at 37°C with stirring in an aggregometer. For anti-aggregation activity assays, washed platelets ( 1.2 x cells/400 ~1) were incubated at 37 ° C for 2 min with peptides, and then stimulated with the agonist. The aggregation was monitored by recording the change in light transmission. The concentration of peptide giving 50 %o inhibition of platelet aggregation (ICSo) was determined from the dose responsive curve.
1. In vitro anti platelet a~Rre~ation by lebetins.
Rabbit platelets (3 x 108 cells/ml) were incubated with lebetin 1 for 2 minutes at 37 ° C .
Agonists were then added. Figure 1 shows the inhibition by native lebetin 1 of rabbit platelet aggregation induced by 0.04 IU/ml thrombin (plotted as ~, 10-7 M
PAF-acether (plotted as D) and 5 ~,g/ml collagen (plotted as ~). Rabbit platelet aggregation induced by thrombin, PAF-acether and collagen was inhibited by lebetin 1 with ICSOS of 125, 48 and 27 nM respectively.
Human platelets (3 x 108 cells/ml) were incubated with native lebetin 1 (plotted as ~ in Figure 2) for 2 minutes at 37°C. Thrombin was then added. Native lebetin 1 inhibited thrombin induced aggregation of human platelets with an ICSO of 590 nM.
Rabbit platelets (3 x 108 cells/ml) were incubated with sLla (plotted as 1 in Figure 3), sL113 (plotted as 1 in Figure 3) and sLly (plotted as ~ in Figure 3) for 2 minutes at AMENDED SHEET
_4- " .
37°C. 0.04 IU/ml Thrombin was then added. Rabbit platelet aggregation induced by thrombin was inhibited by sLla, sL113 and sLly with ICSOS of 23, 10 and 7 nVI
respectively.
Rabbit platelets (3 x 10g cells/ml) were incubated with sLla (plotted as 1 in Figure 4A), sL113 (plotted as ~ in Figure 4B) or sLly (plotted as ~ in Figure 4A) for minutes at 37°C. 5 ~,g/ml of collagen was then added.
Human platelets (3 x 10g cells/ml) were incubated with sLla (plotted as 1 in Figure SA), sL113 (plotted as ~ in Figure 5A) or sLly (plotted as ~ in Figure SB) for minutes at 37°C. 0.04 IU/ml Thrombin was then added. Human platelet aggregation induced by thrombin was inhibited by sLla, sL113 and sLly with ICSOS of 140, 32 and 3 nl~i respectively.
Results are shown in the Figures as mentioned above, and also in the Table below.
TABLE
Inhibition of Platelet Aggregation induced by Thrombin Rabbit Human Platelets Platelets Optimal Optimal ICSO ICSO
Inhibition Inhibition Concentration Concentration ( % (~) ) (~) Lebetin 125 - - 590 - -sLla 23 50 106 140 55 170 sL113 10 87 106 32 49 170 sLly 7 93 33 3 73 8 sLly appeared to be about 20 (ICSO=7nM) and 200 (ICSO=3nM) times more active than native lebetin 1 for the inhibition of either rabbit (ICSo=125nM) or human (ICSO=590nM) platelet aggregation (induced by thrombin) respectively. sLly was also notably more active than sLla or sL113 on both rabbit and human platelets.
2. Inhibition by lebetins of collagen-induced thrombocytopenia in rats sLla (plotted as D in Figure 6), sL113 (plotted as O in Figure 6) or sLl~~
(plotted as O
in Figure 6) were injected into the left jugular vein of anesthetised rats.
After 2 AMENDED SHEET
minutes, 1 mg/kg body weight of collagen was injected into the jugular, and 1 minute later blood was sampled from the right carotid and platelet rich plasma was prepared.
The percentage of inhibition is the ratio of platelet counts in rats which received lebetins against those which received saline solution. The EDSOS were 10.7, 3.2 and 3.1 nmol/kg for sLla, sL113 and sLll respectively.
3. Study of toxicity in vivo sLly (100 ~cg) was devoid of toxicity in Swiss mice (20 ~ 2 g) after injection whether intracerebroventricularly, intraperitoneally or subcutaneously.
These results suggest that sLly will be useful for the treatment of thrombosis or thromboembolism in veins and arteries, and accordingly the invention further provides a method for the treatment of thrombosis or thromboembolism in the veins or arteries of a patient, the method comprising administering to the patient an effective amount of the peptide sLly. In particular, it is envisaged that the method of treatment will be suitable for anti-thrombotic therapy in animals and humans for venous thrombosis, coronary ischaemic event, pulmonary embolism, and in the pre-operative, operative and post-operative periods of endovascular examination and of cardiovascular surgery.
It is also envisaged that the peptide sLly will be useful for prophylactic anticoagulant therapy including the prevention of restenosis after transluminal angioplasty, for the development of coagulation tests and platelet functional exploration, and for vascular imaging by the injection of tracers.
AMENDED SHEET
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAI1~IE: Armel S.A.
(B) STREET: 50 rue Basse (C) CITY: Steinsel (E) COUNTRY: Luxembourg (F) POSTAL CODE (ZIP): L-7307 (ii) TITLE OF INVENTION: A Lebetin Peptides which inhibits Platelet Aggregation (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM:
(.A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9627116.8 (B) FILING DATE: 31-DEC-1996 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Gly Asp Asn Lys Sro Pro Lys Lys Gly PrOo Pro Asn Gly AMENDED SHEET
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Asp Asn Lys Pro Pro Lys Lys Gly Pro Pro Asn Gly (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Asn Lys Pro Pro Lys Lys Giy Pro Pro Asn Gly AMENDED SHEET
A Lebetin Peptide which inhibits Platelet Aggregation DESCRIPTION
The invention relates to a peptide derived from lebetin, and to its use in methods for the treatment of thrombosis or thromboembolism.
Several components isolated from snake venom, e.g. platelet aggregation inhibitors, phospholipases and ADPases, thrombin-like enzymes and fibrinolytic enzymes, affect hemostasis by interfering with the coagulation and platelet aggregation processes.
Platelet aggregation involves a complex network of cell surface adhesion proteins, one of which is GPIIb/IIIa. GPIIb/IIIa binds fibrinogen and this binding is inhibited by proteins ( "disintegrins") isolated from snake venom and containing an RGD
sequence.
Fibrinogen-GPIIb/IIIa interaction is the final step of a complex cascade of biochemical reactions and cell morphological changes, including activation of platelets which become competent to bind fibrinogen, changes in shape, secretion of the granular content and aggregation. These events are induced by platelet aggregation agonists and each of these may be a target for anti-aggregation agents.
Barbouche et al (FEBS Letters 392 (1996) 6-10] have recently isolated a new inhibitor of platelet aggregation from Vipera lebetina venom. This isolate, lebetin, lacks the RGD sequence of the disintegrins. Lebetin is composed of two groups of related peptides, lebetin 1 and lebetin 2. Lebetin 1 is a mixture of two proline and lysine rich peptides, one (sLla) of 13 amino acid residues and the other (sL113) of 12 amino acid residues, the same sequence but lacking the N-terminal glycine of sLla. sLla and sL113 have the following amino acid sequences:
sLl« GDNKPPKKGPPNG (SEQ ID NO 1), and sL113 DNKPPKKGPPNG (SEQ ID NO 2).
Lebetin 2 also consists of two peptides, one of 38 amino acid residues (sL2a) and the other of 37 amino acid residues (sL213) . sL2a has sL 1 a at its N-terminal and a 25 amino acid residue peptide with one disulphide bridge at its C-terminal. sL213 has the same sequence but lacking the N-terminal glycine of sL2a.
The invention provides a peptide (hereinafter called sLl ;~) having the amino acid AMENDED SHEET
_7_ . .
sequence NKPPKKGPPNG (SEQ ID NO 3).
sLly has proven more effective as an inhibitor of platelet aggregation than native lebetin 1 or its components sLla and sL113.
The invention also includes within its scope a modified sLly peptide in which one or more of the amino acid residues is a D-amino acid residue. D amino acids last longer in vivo because they are harder for peptidase to cut, but the L amino acids have better activity.
Moreover, peptide analogues, synthetic constructs using the carbon skeleton of peptides but omitting the -CONH- peptide bonds, can be employed in place of peptides.
Thus, it should be understood that the invention also includes within its scope a modified sLl°~
peptide in which at least one of the amino acid residues is replaced by its decarboxamido analogue. It is believed that such peptide analogues will be more resistant to peptidase and last longer in vivo.
Synthetic methods 1. Manual Synthesis of the lebetin 1 peptides using Boc-chemistry The synthetic lebetin 1 peptides sLl a (Glyl-Gly 13), sL113 (Asp2-Gly 13) and sLly (Asn3-Gly 13) were assembled manually by the solid phase technique (R. B.
Merrifield, 1986) on Boc-aminoacyl-Pam resin (0.5 mmol, substitution 0.67-0.82 mequiv of amino group per gram). The following side-chain protecting groups for trifunctional Boc-amino acids were used: cyclohexyl (CHex) for Asp, 2-chlorobenzyloxy (C1Z) for Lys and 2-bromocarbobenzoxy (BrZ) for Tyr. The synthesis cycle used for incorporation of each Boc-amino acids was: ( 1 ) dichloromethane (DCM) wash, (2 x 0.5 min); (2) 65 % trifluoroacetic acid (TFA) in DCM for deprotection step, 2 min and 13 min; (3) DCM wash, 0.6 min; isopropanol wash, 0.5 min; (4) DCM wash, (2 x 0.5 min); (5) N-methylpyrrolidone (NMP) wash, (2 x 0.5 min); (6) Boc amino acid (4 equiv) and PyBOP (4 equiv) in NMP and (7) diisopropylethylamine (DIEA) (8 equiv) for Boc-as coupling, 3 min; and (8) two NMP washes, 1 min. Each coupling step was monitored by using the qualitative ninhydrin test and recoupling was performed as necessary. Unreacted amino groups detected by ninhydrin after two consecutive couplings were blocked by acetylation withy 50 % acetic anhydride in DCM for 10 min.
At the end of the assembly and after the last deprotection step, the peptidyl resin was dried under vacuum. The high hydrogen fluoride (HF) procedure was achieved for AMENDED SHEET
deprotection and cleavage from the resin using 10 % p-cresol per volume as a scavenger. After removal of HF in vacuo, the resin was washed with cold diethylether and the peptide extracted with water. The crude peptides were purified by reversed-phase preparative medium-pressure liquid chromatography (MPLC) (Labomatic, C 18 HD-SIL 15-22 ~cm, 26 x 313 mm) using a 90-min linear Gradient of acetonitrile in 0.1 % (by vol . ) TFA/H~ O from 0 to 30 % , at a flow rate of 10 mllmin with UV detection at 206 nm. The homogeneity of the fractions was assessed by analytical HPLC (Merck, C 18 Lichrospher, 4 x 125 mm). Fractions containing homogeneous peptides ( > 99 % ) were pooled and lyophilized.
Experimental Results Platelets were prepared from 0.2M EDTA treated blood samples. Human platelets were prepared as follows: blood was collected in vials containing a sodium citrate/
dextrose ( 1: 5 v/v j mixture from a donor who had not taken any drug for at least 1 week.
Platelets were resuspended in Tyrod's buffer pH 7.4 at a final concentration of 3 x 108 cells/ml prior to the assay which was performed at 37°C with stirring in an aggregometer. For anti-aggregation activity assays, washed platelets ( 1.2 x cells/400 ~1) were incubated at 37 ° C for 2 min with peptides, and then stimulated with the agonist. The aggregation was monitored by recording the change in light transmission. The concentration of peptide giving 50 %o inhibition of platelet aggregation (ICSo) was determined from the dose responsive curve.
1. In vitro anti platelet a~Rre~ation by lebetins.
Rabbit platelets (3 x 108 cells/ml) were incubated with lebetin 1 for 2 minutes at 37 ° C .
Agonists were then added. Figure 1 shows the inhibition by native lebetin 1 of rabbit platelet aggregation induced by 0.04 IU/ml thrombin (plotted as ~, 10-7 M
PAF-acether (plotted as D) and 5 ~,g/ml collagen (plotted as ~). Rabbit platelet aggregation induced by thrombin, PAF-acether and collagen was inhibited by lebetin 1 with ICSOS of 125, 48 and 27 nM respectively.
Human platelets (3 x 108 cells/ml) were incubated with native lebetin 1 (plotted as ~ in Figure 2) for 2 minutes at 37°C. Thrombin was then added. Native lebetin 1 inhibited thrombin induced aggregation of human platelets with an ICSO of 590 nM.
Rabbit platelets (3 x 108 cells/ml) were incubated with sLla (plotted as 1 in Figure 3), sL113 (plotted as 1 in Figure 3) and sLly (plotted as ~ in Figure 3) for 2 minutes at AMENDED SHEET
_4- " .
37°C. 0.04 IU/ml Thrombin was then added. Rabbit platelet aggregation induced by thrombin was inhibited by sLla, sL113 and sLly with ICSOS of 23, 10 and 7 nVI
respectively.
Rabbit platelets (3 x 10g cells/ml) were incubated with sLla (plotted as 1 in Figure 4A), sL113 (plotted as ~ in Figure 4B) or sLly (plotted as ~ in Figure 4A) for minutes at 37°C. 5 ~,g/ml of collagen was then added.
Human platelets (3 x 10g cells/ml) were incubated with sLla (plotted as 1 in Figure SA), sL113 (plotted as ~ in Figure 5A) or sLly (plotted as ~ in Figure SB) for minutes at 37°C. 0.04 IU/ml Thrombin was then added. Human platelet aggregation induced by thrombin was inhibited by sLla, sL113 and sLly with ICSOS of 140, 32 and 3 nl~i respectively.
Results are shown in the Figures as mentioned above, and also in the Table below.
TABLE
Inhibition of Platelet Aggregation induced by Thrombin Rabbit Human Platelets Platelets Optimal Optimal ICSO ICSO
Inhibition Inhibition Concentration Concentration ( % (~) ) (~) Lebetin 125 - - 590 - -sLla 23 50 106 140 55 170 sL113 10 87 106 32 49 170 sLly 7 93 33 3 73 8 sLly appeared to be about 20 (ICSO=7nM) and 200 (ICSO=3nM) times more active than native lebetin 1 for the inhibition of either rabbit (ICSo=125nM) or human (ICSO=590nM) platelet aggregation (induced by thrombin) respectively. sLly was also notably more active than sLla or sL113 on both rabbit and human platelets.
2. Inhibition by lebetins of collagen-induced thrombocytopenia in rats sLla (plotted as D in Figure 6), sL113 (plotted as O in Figure 6) or sLl~~
(plotted as O
in Figure 6) were injected into the left jugular vein of anesthetised rats.
After 2 AMENDED SHEET
minutes, 1 mg/kg body weight of collagen was injected into the jugular, and 1 minute later blood was sampled from the right carotid and platelet rich plasma was prepared.
The percentage of inhibition is the ratio of platelet counts in rats which received lebetins against those which received saline solution. The EDSOS were 10.7, 3.2 and 3.1 nmol/kg for sLla, sL113 and sLll respectively.
3. Study of toxicity in vivo sLly (100 ~cg) was devoid of toxicity in Swiss mice (20 ~ 2 g) after injection whether intracerebroventricularly, intraperitoneally or subcutaneously.
These results suggest that sLly will be useful for the treatment of thrombosis or thromboembolism in veins and arteries, and accordingly the invention further provides a method for the treatment of thrombosis or thromboembolism in the veins or arteries of a patient, the method comprising administering to the patient an effective amount of the peptide sLly. In particular, it is envisaged that the method of treatment will be suitable for anti-thrombotic therapy in animals and humans for venous thrombosis, coronary ischaemic event, pulmonary embolism, and in the pre-operative, operative and post-operative periods of endovascular examination and of cardiovascular surgery.
It is also envisaged that the peptide sLly will be useful for prophylactic anticoagulant therapy including the prevention of restenosis after transluminal angioplasty, for the development of coagulation tests and platelet functional exploration, and for vascular imaging by the injection of tracers.
AMENDED SHEET
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAI1~IE: Armel S.A.
(B) STREET: 50 rue Basse (C) CITY: Steinsel (E) COUNTRY: Luxembourg (F) POSTAL CODE (ZIP): L-7307 (ii) TITLE OF INVENTION: A Lebetin Peptides which inhibits Platelet Aggregation (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM:
(.A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9627116.8 (B) FILING DATE: 31-DEC-1996 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Gly Asp Asn Lys Sro Pro Lys Lys Gly PrOo Pro Asn Gly AMENDED SHEET
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Asp Asn Lys Pro Pro Lys Lys Gly Pro Pro Asn Gly (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Asn Lys Pro Pro Lys Lys Giy Pro Pro Asn Gly AMENDED SHEET
Claims (6)
1. The peptide NKPPKKGPPNG (SEQ ID NO 3), also known as sL1.gamma..
2. A peptide according to claim 1 in which at least one of the amino acid residues is a D-amino acid residue.
3. A peptide according to any claim 1 or claim 2 in which at least one of the amino acid residues is replaced by its decarboxamido analogue.
4. A medicament comprising a peptide according to any preceding claim in admixture with a pharmaceutically acceptable diluent or carrier.
5. A method for the treatment of thrombosis or thromboembolism in the veins or arteries of a patient, the method comprising administering to the patient an effective amount of a peptide according to any of claims 1 to 3.
6. A method for the treatment of thrombosis or thromboembolism in the veins or arteries of a patient, the method comprising administering to the patient an effective amount of a medicament according to claim 4.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9627116.8A GB9627116D0 (en) | 1996-12-31 | 1996-12-31 | Lebetin peptides as platelet aggregation inhibitors |
| GB9627116.8 | 1996-12-31 | ||
| PCT/EP1997/007335 WO1998029436A2 (en) | 1996-12-31 | 1997-12-30 | Lebetin peptides as platelet aggregation inhibitors |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2275544A1 true CA2275544A1 (en) | 1998-07-09 |
Family
ID=10805141
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002275544A Withdrawn CA2275544A1 (en) | 1996-12-31 | 1997-12-30 | Lebetin peptides as platelet aggregation inhibitors |
Country Status (4)
| Country | Link |
|---|---|
| AU (1) | AU5861298A (en) |
| CA (1) | CA2275544A1 (en) |
| GB (1) | GB9627116D0 (en) |
| WO (1) | WO1998029436A2 (en) |
-
1996
- 1996-12-31 GB GBGB9627116.8A patent/GB9627116D0/en active Pending
-
1997
- 1997-12-30 CA CA002275544A patent/CA2275544A1/en not_active Withdrawn
- 1997-12-30 WO PCT/EP1997/007335 patent/WO1998029436A2/en active Application Filing
- 1997-12-30 AU AU58612/98A patent/AU5861298A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU5861298A (en) | 1998-07-31 |
| WO1998029436A3 (en) | 1998-09-03 |
| WO1998029436A2 (en) | 1998-07-09 |
| GB9627116D0 (en) | 1997-02-19 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AZWI | Withdrawn application |