WO1998029436A2 - Lebetin peptides as platelet aggregation inhibitors - Google Patents

Lebetin peptides as platelet aggregation inhibitors Download PDF

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Publication number
WO1998029436A2
WO1998029436A2 PCT/EP1997/007335 EP9707335W WO9829436A2 WO 1998029436 A2 WO1998029436 A2 WO 1998029436A2 EP 9707335 W EP9707335 W EP 9707335W WO 9829436 A2 WO9829436 A2 WO 9829436A2
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Prior art keywords
peptide
lebetin
peptides
amino acid
platelet aggregation
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PCT/EP1997/007335
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French (fr)
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WO1998029436A3 (en
Inventor
Hervé Rochat
Kamel Mabrouk
Mohammed El-Ayeb
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Armel S.A.
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Priority to CA002275544A priority Critical patent/CA2275544A1/en
Priority to AU58612/98A priority patent/AU5861298A/en
Publication of WO1998029436A2 publication Critical patent/WO1998029436A2/en
Publication of WO1998029436A3 publication Critical patent/WO1998029436A3/en

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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
    • 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
    • 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
    • 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 invention relates to peptides derived from lebetin, and to their use in methods for the treatment of thrombosis or thromboembolism.
  • 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.
  • Lebetin A new inhibitor of platelet aggregation has recently been isolated 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 of 13 amino acid residues (lebetin l ⁇ ) and the other of 12 amino acid residues (lebetin IB), the same sequence but lacking the N-terminal glycine of Lebetin l ⁇ .
  • Lebetin 2 also consists of two peptides, one of 38 amino acid residues (lebetin 2 ⁇ ) and the other of 37 amino acid residues (lebetin 2 ⁇ ).
  • Lebetin 2 ⁇ has lebetin l ⁇ at its N-terminal and a 25 amino acid residue peptide with one disulphide bridge at its C-terminal.
  • Lebetin 2 ⁇ has the same sequence but lacking the N-terminal glycine of lebetin 2 ⁇ .
  • the invention provides peptides having the amino acid sequence
  • the synthetic peptides according to the invention have proven more effective as ... inhibitors of platelet aggregation than natural lebetin or its components lebetin 1 and lebetin 2.
  • Preferred peptides according to the invention include: sLl ⁇ GDNKPPKKGPPNG (SEQ ID NO 1), sLl ⁇ DNKPPKKGPPNG (SEQ ID NO 2), sLl ⁇ NKPPKKGPPNG (SEQ ID NO 3), sLl ⁇ l3 YNKPPKKGPPNG (SEQ ID NO 4),
  • Peptides can include D or L-amino acid residues.
  • D amino acids last longer in vivo because they are harder for peptidase to cut, but the L amino acids have better activity.
  • peptide analogues synthetic constructs using the carbon skeleton of peptides but omitting the -CONH- peptide bonds, can be employed in place of peptides.
  • references to peptides herein may also be taken to include peptide analogues. It is believed that peptide analogues will be more resistant to peptidase and last longer in vivo.
  • the synthetic lebetin 1 peptides sLl ⁇ (Glyl-Glyl3), sLl ⁇ (Asp2-Glyl3), sLl ⁇ (Asn3-Glyl3) and sLl ⁇ l3 (Tyr2-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).
  • Boc-amino acids 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, 0
  • the high hydrogen fluoride (HF) procedure was achieved for 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, C18 HD-SIL 15-22 ⁇ m, 26 x 313 mm) using a 90-min linear gradient of acetonitrile in 0.1 % (by vol.) TFA/H 2 O from 0 to 30%, at a flow rate of 10 ml/min with UV detection at 206 nm. The homogeneity of the fractions was assessed by analytical HPLC (Merck, C18 Lichrospher, 4 x 125 mm). Fractions containing homogeneous peptides ( > 99%) were pooled and lyophilized.
  • MPLC reversed
  • Stepwise elongation of the synthetic lebetin 2 peptides sL2 ⁇ (Glyl-Gly38) and sL2 ⁇ (Asp2-Gly38) was carried out on 0.35 mmol of HMP resin (0.96 mmol of hydroxyl sites) (Wang S.S. , 1973) using an automated peptide synthesizer (Model 433A, Applied Biosystems Inc.).
  • lebetin 2 analogue sL2 ⁇ (Asp2-Gly38) aliquots of peptide-resin were removed at the corresponding cycle.
  • Trifunctional amino acids were chain protected as follows: trityl (Trt) for Cys, His and Asn; t-butyl (t-Bu) for Ser and Asp; Boc for Lys and pentamethylchroman (Pmc) for Arg.
  • Trt trityl
  • t-Bu t-butyl
  • Boc Boc
  • Lys pentamethylchroman
  • Arg Arg
  • Each coupling cycle comprised (i) Deblocking of the ⁇ -amino group by piperidine (18 and 20% in NMP for 3 and 8 min); following deprotection the resin was washed with NMKP (5 x 1 min); (ii) double coupling in NMP of the Fmoc-amino acids (1 mmOl) as their hydroxybenzotriazole (HOBt) active esters preformed in the cartridge using
  • the peptidyl resins 0.91 g for sL2 ⁇ (Glyl-Gly38) and 0.69 g for sL2 ⁇ (Asp2-Gly38) were cleaved and deprotected by 2-h treatment at 25 °C with TFA containing 5 % thioanisole 5 % ethanedithiol in a final volume of 10 ml/g of peptidyl resin.
  • the peptide mixtures were then filtered to remove resins and the filtrates were precipitated and washed twice by adding cold diethylether.
  • the resulting crude peptides were pelleted by centrifugation (2,500 g; 10 min) and the final pellets were dissolved in H 2 O.
  • the reduced peptides [0.47 g for sL2 ⁇ (Glyl-Gly38) and 0.36 g for sL2 ⁇ (Asp2-Gly38)] were dissolved at 10 mg/ml in 0.2 M Tris-HCl buffer, pH 8, and stirred under air to follow folding (48 h, 25°C).
  • the oxidized peptides (50-mg batches) were purified by preparative reversed-phase HPLC (Perkin- Elmer, ODS 20 ⁇ m, 100 x 10 mm) by elution with a 2-h linear gradient of 0.1 % TFA/H 2 O (A) and 70% acetonitrile in water containing 0.1 % TFA (B) from 0 to 50%, at a flow rate of 6 ml/min with UV detection at 230 nm. Fractions were collected and analysed by analytical HPLC. Fractions containing purified peptides ( >99%) were pooled and lyophilized.
  • preparative reversed-phase HPLC Perkin- Elmer, ODS 20 ⁇ m, 100 x 10 mm
  • 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) 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 10 8 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 10 8 cells/400 ⁇ l) 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% inhibition of platelet aggregation (IC 50 ) was determined from the dose responsive curve. 1. In vitro anti platelet aggregation by lebetins.
  • FIGS. 1A and IB show the inhibition by native lebetins 1 and 2 respectively 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 •).
  • Human platelets (3 x 10 8 cells/ml) were incubated with native lebetin 1 (plotted as ⁇ in Figure 2) or native lebetin 2 (plotted as O in Figure 2) for 2 minutes at 37 °C. Thrombin was then added. Native lebetin 1 and native lebetin 2 inhibited thrombin induced aggregation of human platelets with IC 50 S of 590 and 100 nM respectively.
  • Rabbit platelets (3 x 10 8 cells/ml) were incubated with sLl ⁇ (plotted as ⁇ in Figure 3), sLl ⁇ (plotted as A in Figure 3), sLl ⁇ (plotted as • in Figure 3) and sLl ⁇ l3 for 2 minutes at 37 °C. 0.04 IU/ml Thrombin was then added.
  • Rabbit platelet aggregation induced by thrombin was inhibited by sLl ⁇ , sLl ⁇ and sLl ⁇ with IC 50 S of 23, 10 and 7 nM respectively.
  • sLl ⁇ l3 gave a maximum inhibition of 49%.
  • Rabbit platelets (3 x 10 8 cells/ml) were incubated with sLl ⁇ (plotted as ⁇ in Figure 4A), sLl ⁇ (plotted as ⁇ in Figure 4B) or sLl ⁇ (plotted as • in Figure 4A) for 2 minutes at 37 °C. 5 ⁇ g/ml of collagen was then added.
  • Human platelets (3 x 10 8 cells/ml) were incubated with sLl ⁇ (plotted as ⁇ in Figure 5 A), sLl ⁇ (plotted as ⁇ in Figure 5 A) or sLl ⁇ (plotted as • in Figure 5B) for 2 minutes at 37 °C. 0.04 IU/ml Thrombin was then added. Human platelet aggregation induced by thrombin was inhibited by sLl ⁇ , sLl ⁇ and sLl ⁇ with IC 50 S of 140, 32 and 3 nM respectively.
  • Rabbit platelets (3 x 10 8 cells/ml) were incubated with sL2 ⁇ (plotted as D in Figure 6) or sL2 ⁇ (plotted as ⁇ in Figure 6) for 2 minutes at 37 °C. 0.04 IU/ml Thrombin was then added. Rabbit platelet aggregation induced by thrombin was inhibited by sL2 ⁇ and sL2 ⁇ with IC 50 S of 0.2 and 0.9 nM respectively.
  • sLl ⁇ (plotted as D in Figure 7), sLl ⁇ (plotted as ⁇ in Figure 7) or sLl ⁇ (plotted as O in Figure 7) were injected into the left jugular vein of anesthetised rats. After 2 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 ED 50 S were 10.7, 3.2 and 3.1 nmol/kg for sLl ⁇ , sLl ⁇ and sLl ⁇ respectively.
  • the lebetin peptides (100 ⁇ g) were devoid of toxicity in Swiss mice (20 + 2 g) after injection whether intracerebroventricularly, intraperitoneally or subcutaneously.
  • 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 a peptide according to the invention.
  • 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 endo vascular examination and of cardiovascular surgery.
  • the peptides 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.
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO : 2:
  • Lys lie Asp Arg lie Gly Ser His Ser Gly Leu Gly Cys Asn Lys Val 20 25 30

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

Lebetin Peptides as Platelet Aggregation Inhibitors
DESCRIPTION
The invention relates to peptides derived from lebetin, and to their 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.
A new inhibitor of platelet aggregation has recently been isolated 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 of 13 amino acid residues (lebetin lα) and the other of 12 amino acid residues (lebetin IB), the same sequence but lacking the N-terminal glycine of Lebetin lα. Lebetin 2 also consists of two peptides, one of 38 amino acid residues (lebetin 2ά) and the other of 37 amino acid residues (lebetin 2β). Lebetin 2α has lebetin lα at its N-terminal and a 25 amino acid residue peptide with one disulphide bridge at its C-terminal. Lebetin 2β has the same sequence but lacking the N-terminal glycine of lebetin 2α.
The invention provides peptides having the amino acid sequence
(X)mNKPPKKGPPNG(CFGHKIDRIGSHSGLGCNKVDDNKG)n(X)p wherein m is from 0 to 6, n is 0 or 1, p is from 0 to 4, and each X independently of each other X represents an amino acid residue.
The synthetic peptides according to the invention have proven more effective as ... inhibitors of platelet aggregation than natural lebetin or its components lebetin 1 and lebetin 2.
Preferred peptides according to the invention include: sLlα GDNKPPKKGPPNG (SEQ ID NO 1), sLlβ DNKPPKKGPPNG (SEQ ID NO 2), sLlγ NKPPKKGPPNG (SEQ ID NO 3), sLlγl3 YNKPPKKGPPNG (SEQ ID NO 4),
sL2α GDNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG
I I
(SEQ ID NO 5), and sL2B DNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG
I 1
(SEQ ID NO 6)
Peptides can include D or L-amino acid residues. 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 references to peptides herein may also be taken to include peptide analogues. It is believed that 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 sLlα (Glyl-Glyl3), sLlβ (Asp2-Glyl3), sLlγ (Asn3-Glyl3) and sLlγl3 (Tyr2-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, 0
(2 x 0.5 min); (6) Boc amino acid (4 equiv) and PyBOP (4 equiv) in NMP and (7) dusopropylethylamine (DIEA) (8 equiv) for Boc-aa 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 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, C18 HD-SIL 15-22 μm, 26 x 313 mm) using a 90-min linear gradient of acetonitrile in 0.1 % (by vol.) TFA/H2O from 0 to 30%, at a flow rate of 10 ml/min with UV detection at 206 nm. The homogeneity of the fractions was assessed by analytical HPLC (Merck, C18 Lichrospher, 4 x 125 mm). Fractions containing homogeneous peptides ( > 99%) were pooled and lyophilized.
2. Automated synthesis of the Lebetin 2 peptides using Fmoc-chemistry
Stepwise elongation of the synthetic lebetin 2 peptides sL2α (Glyl-Gly38) and sL2β (Asp2-Gly38) was carried out on 0.35 mmol of HMP resin (0.96 mmol of hydroxyl sites) (Wang S.S. , 1973) using an automated peptide synthesizer (Model 433A, Applied Biosystems Inc.). To obtain lebetin 2 analogue sL2β (Asp2-Gly38) aliquots of peptide-resin were removed at the corresponding cycle. Trifunctional amino acids were chain protected as follows: trityl (Trt) for Cys, His and Asn; t-butyl (t-Bu) for Ser and Asp; Boc for Lys and pentamethylchroman (Pmc) for Arg. To load the first amino acid into HMP resin the Fmoc aa (lmmOl) dissolved in NMP/DCM was mixed with 1.0 M DCC/NMP (1 ml) and 0.1 equiv of DMAP for 30 min. After washing with 50% MeOH/DCM (2 x) and DCM (5 x) the resin was capped with benzoic anhydride (3 rnmOl) and washed with NMP (3 x). Each coupling cycle comprised (i) Deblocking of the α-amino group by piperidine (18 and 20% in NMP for 3 and 8 min); following deprotection the resin was washed with NMKP (5 x 1 min); (ii) double coupling in NMP of the Fmoc-amino acids (1 mmOl) as their hydroxybenzotriazole (HOBt) active esters preformed in the cartridge using
[2-( 1 H-benzotrizol- 1 -yl)-l , 1 , 2-tetramethyluronium hexafluorophosphate] (HBTU) (0.45 M HBTU/HOBt in DMF and 2 M DIEA) (2 x 20 min). The deprotection and coupling steps were repeated until the peptide synthesis was completed. After manual deprotection of the last N-terminal Fmoc group, the peptidyl resins was washed with NMP, followed by DCM, and dried under vacuum. The peptidyl resins 0.91 g for sL2α (Glyl-Gly38) and 0.69 g for sL2β (Asp2-Gly38) were cleaved and deprotected by 2-h treatment at 25 °C with TFA containing 5 % thioanisole 5 % ethanedithiol in a final volume of 10 ml/g of peptidyl resin. The peptide mixtures were then filtered to remove resins and the filtrates were precipitated and washed twice by adding cold diethylether. The resulting crude peptides were pelleted by centrifugation (2,500 g; 10 min) and the final pellets were dissolved in H2O. After lyophilisation, the reduced peptides [0.47 g for sL2α (Glyl-Gly38) and 0.36 g for sL2β (Asp2-Gly38)] were dissolved at 10 mg/ml in 0.2 M Tris-HCl buffer, pH 8, and stirred under air to follow folding (48 h, 25°C). The oxidation process was monitored every 60 min by analytical reversed-phase HPLC on a Merck C18 column (Lichrospher 5 μ, 4 x 250 mm) using a 50-min linear gradient of acetronitrile in 0.1 % (by vol.) TFA/H2O from 10 to 35% at a flow rate of 1 ml/min (λ = 230 nm). After filtration, the oxidized peptides (50-mg batches) were purified by preparative reversed-phase HPLC (Perkin- Elmer, ODS 20 μm, 100 x 10 mm) by elution with a 2-h linear gradient of 0.1 % TFA/H2O (A) and 70% acetonitrile in water containing 0.1 % TFA (B) from 0 to 50%, at a flow rate of 6 ml/min with UV detection at 230 nm. Fractions were collected and analysed by analytical HPLC. Fractions containing purified peptides ( >99%) were pooled and lyophilized. The chemical identity of synthetic lebetin 2 peptides sL2α (Glyl-Gly38) and sL2β (As[2-Gly38) was confirmed by (i) coelution with natural lebetin 2 after simultaneous injection of both products in analytical reversed-phase HPLC; (ii) amino acid analysis after acid hydrolysis (6 N HC1/1 % (mass/vol.) phenol, 20 h, 120°C); and (iii) electrospray mass spectrometry.
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) 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 108 cells/400 μl) 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% inhibition of platelet aggregation (IC50) was determined from the dose responsive curve. 1. In vitro anti platelet aggregation by lebetins.
Rabbit platelets (3 x 108 cells/ml) were incubated with lebetin 1 or lebetin 2 for 2 minutes at 37°C. Agonists were then added. Figures 1A and IB show the inhibition by native lebetins 1 and 2 respectively of rabbit platelet aggregation induced by 0.04 IU/ml thrombin (plotted as ■), 10"7 M PAF-acether (plotted as D) and 5 μg/ml collagen (plotted as •). Rabbit platelet aggregation induced by thrombin, PAF-acether and collagen was inhibited by lebetin 1 with IC50S of 125, 48 and 27 nM respectively and by lebetin 2 with IC50S of 8, 48 and 5 nM respectively.
Human platelets (3 x 108 cells/ml) were incubated with native lebetin 1 (plotted as ♦ in Figure 2) or native lebetin 2 (plotted as O in Figure 2) for 2 minutes at 37 °C. Thrombin was then added. Native lebetin 1 and native lebetin 2 inhibited thrombin induced aggregation of human platelets with IC50S of 590 and 100 nM respectively.
Rabbit platelets (3 x 108 cells/ml) were incubated with sLlα (plotted as ■ in Figure 3), sLlβ (plotted as A in Figure 3), sLlγ (plotted as • in Figure 3) and sLlγl3 for 2 minutes at 37 °C. 0.04 IU/ml Thrombin was then added. Rabbit platelet aggregation induced by thrombin was inhibited by sLlα, sLlβ and sLlγ with IC50S of 23, 10 and 7 nM respectively. sLlγl3 gave a maximum inhibition of 49%.
Rabbit platelets (3 x 108 cells/ml) were incubated with sLlα (plotted as ■ in Figure 4A), sLlβ (plotted as ▲ in Figure 4B) or sLlγ (plotted as • in Figure 4A) for 2 minutes at 37 °C. 5 μg/ml of collagen was then added.
Human platelets (3 x 108 cells/ml) were incubated with sLlα (plotted as ■ in Figure 5 A), sLlβ (plotted as ▲ in Figure 5 A) or sLlγ (plotted as • in Figure 5B) for 2 minutes at 37 °C. 0.04 IU/ml Thrombin was then added. Human platelet aggregation induced by thrombin was inhibited by sLlα, sLlβ and sLlγ with IC50S of 140, 32 and 3 nM respectively.
Rabbit platelets (3 x 108 cells/ml) were incubated with sL2α (plotted as D in Figure 6) or sL2β (plotted as ■ in Figure 6) for 2 minutes at 37 °C. 0.04 IU/ml Thrombin was then added. Rabbit platelet aggregation induced by thrombin was inhibited by sL2α and sL2β with IC50S of 0.2 and 0.9 nM respectively.
Results are shown in the Figures as mentioned above, and also in the Table below. TABLE
Inhibition of Platelet Aggregation induced by Thrombin
Figure imgf000008_0001
sLlγ was more active than sLlα or sLlβ on both rabbit and human platelets, and also appeared to be about 20 (ICso=7nM) and 200 (ICso=3nM) times more active than native lebetin 1 for the inhibition of either rabbit (IC5o= 125nM) or human (IC5o=590nM) platelet aggregation (induced by thrombin) respectively. Although less active than sLlγ, sLlα and sLlβ were also more active than native lebetin 1 in this test.
Similarly, sL2α and sL2β were about 40 (IC50=0.2nM) and 9 (IC50=0.9nM) times more active than native lebetin 2 (ICso=8nM) for the inhibition of rabbit platelet aggregation induced by thrombin.
However, it was found that shortening sLlγ by even one amino acid residue, whether from the N-terminal or the C-terminal, resulted only in weak inhibition of platelet aggregation.
2. Inhibition by lebetins of collagen-induced thrombocytopenia in rats
sLlα (plotted as D in Figure 7), sLlβ (plotted as Δ in Figure 7) or sLlγ (plotted as O in Figure 7) were injected into the left jugular vein of anesthetised rats. After 2 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 ED50S were 10.7, 3.2 and 3.1 nmol/kg for sLlα, sLlβ and sLlγ respectively.
3. Study of toxicity in vivo
The lebetin peptides (100 μg) were devoid of toxicity in Swiss mice (20 + 2 g) after injection whether intracerebroventricularly, intraperitoneally or subcutaneously.
These results suggest that the peptides according to the invention 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 a peptide according to the invention. 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 endo vascular examination and of cardiovascular surgery. It is also envisaged that the peptides 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.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Armel S.A.
(B) STREET: 50 rue Basse
(C) CITY: Steinsel
(E) COUNTRY: Luxembourg
(F) POSTAL CODE (ZIP) : L-7307
(ii) TITLE OF INVENTION: Lebetin Peptides as Platelet Aggregation Inhibitors
(iii) NUMBER OF SEQUENCES: 6
(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 Pro Pro Lys Lys Gly Pro Pro Asn Gly 1 5 10
(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 1 5 10
(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 Gly Pro Pro Asn Gly 1 5 10 2 ) INFORMATION FOR SEQ ID NO : 4:
(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: 4:
Tyr Asn Lys Pro Pro Lys Lys Gly Pro Pro Asn Gly 1 5 10
(2) INFORMATION FOR SEQ ID NO : 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 Gly Asp Asn Lys Pro Pro Lys Lys Gly Pro Pro Asn Gly Cys Phe Gly 1 5 10 15
His Lys lie Asp Arg lie Gly Ser His Ser Gly Leu Gly Cys Asn Lys 20 25 30
Val Asp Asp Asn Lys Gly 35
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6:
Asp Asn Lys Pro Pro Lys Lys Gly Pro Pro Asn Gly Cys Phe Gly His 1 5 10 15
Lys lie Asp Arg lie Gly Ser His Ser Gly Leu Gly Cys Asn Lys Val 20 25 30
Asp Asp Asn Lys Gly 35

Claims

1. A peptide having the amino acid sequence
(X)mNKPPKKGPPNG(CFGHKIDRIGSHSGLGCNKVDDNKG)n(X)p wherein m is from 0 to 6, n is 0 or 1, p is from 0 to 4, and each X independently of each other X represents an amino acid residue.
2. The peptide GDNKPPKKGPPNG, also known as sLl╬▒.
3. The peptide DNKPPKKGPPNG, also known as sLl╬▓.
4. The peptide NKPPKKGPPNG, also known as sLl╬│.
5. The peptide YNKPPKKGPPNG, also known as sLl╬│l3.
6. The peptide GDNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG, also known as sL2╬▒.
7. The peptide DNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG, also known as sL2╬▓.
8. A peptide according to any preceding claim in which at least one of the amino acid residues is a D-amino acid residue.
9. A peptide according to any of claims 1 to 7 in which at least one of the amino acid residues is replaced by its decarboxamido analogue.
10. A medicament comprising a peptide according to any preceding claim in admixture with a pharmaceutically acceptable diluent or carrier.
11. Use of a peptide according to any of claims 1 to 9 for the preparation of a medicament for the treatment of thrombosis or thromboembolism in veins or arteries.
12. 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 9.
3. 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 10.
PCT/EP1997/007335 1996-12-31 1997-12-30 Lebetin peptides as platelet aggregation inhibitors WO1998029436A2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CA002275544A CA2275544A1 (en) 1996-12-31 1997-12-30 Lebetin peptides as platelet aggregation inhibitors
AU58612/98A AU5861298A (en) 1996-12-31 1997-12-30 Lebetin peptides as platelet aggregation inhibitors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9627116.8 1996-12-31
GBGB9627116.8A GB9627116D0 (en) 1996-12-31 1996-12-31 Lebetin peptides as platelet aggregation inhibitors

Publications (2)

Publication Number Publication Date
WO1998029436A2 true WO1998029436A2 (en) 1998-07-09
WO1998029436A3 WO1998029436A3 (en) 1998-09-03

Family

ID=10805141

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1997/007335 WO1998029436A2 (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)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
R BARBOUCHE ET AL.: "Novel anti-platelet aggregation polypeptides from Vipera lebetina venom; isolation and characterization" FEBS LETTERS., vol. 392, no. 1, 19 August 1996, AMSTERDAM NL, pages 6-10, XP002066590 *

Also Published As

Publication number Publication date
CA2275544A1 (en) 1998-07-09
AU5861298A (en) 1998-07-31
WO1998029436A3 (en) 1998-09-03
GB9627116D0 (en) 1997-02-19

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