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WO1995012615A1 - Agents anti-hemostatiques tires du necator americanus - Google Patents

Agents anti-hemostatiques tires du necator americanus Download PDF

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Publication number
WO1995012615A1
WO1995012615A1 PCT/GB1994/002406 GB9402406W WO9512615A1 WO 1995012615 A1 WO1995012615 A1 WO 1995012615A1 GB 9402406 W GB9402406 W GB 9402406W WO 9512615 A1 WO9512615 A1 WO 9512615A1
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WO
WIPO (PCT)
Prior art keywords
excretory
necator americanus
products
secretory
human
Prior art date
Application number
PCT/GB1994/002406
Other languages
English (en)
Inventor
David Idris Pritchard
Original Assignee
The University Of Nottingham
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by The University Of Nottingham filed Critical The University Of Nottingham
Publication of WO1995012615A1 publication Critical patent/WO1995012615A1/fr

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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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
    • 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 relates to antihaemostatic agents for the treatment of human thrombotic disorders.
  • heparin is a common anticoagulant
  • fibrin-rich blood clots may be dissolved using tissue plasminogen activator, streptokinase and urokinase.
  • tissue plasminogen activator streptokinase
  • urokinase urokinase
  • the present invention seeks to provide novel antihaemostatic agents.
  • an excretory-secretory product of the human hookworm Necator americanus for use as an active pharmaceutical substance .
  • the invention further provides for the use of excretory-secretory products of the human hookworm Necator americanus for the manufacture of an antihaemostatic composition.
  • the invention further provides an antihaemostatic composition
  • an antihaemostatic composition comprising a pharmaceutically acceptable diluent or carrier and an excretory-secretory product of the human hookworm Necator americanus.
  • the invention further provides an antihaemostatic composition
  • an antihaemostatic composition comprising a pharmaceutically acceptable diluent or carrier and an active ingredient obtained from an excretory-secretory product of the human hookworm Necator americanus.
  • the active ingredient may be obtained directly, or by chemical synthesis, or by a genetic engineering technique .
  • the invention further provides excretory-secretory products or derivatives thereof of the human hookworm Necator americanus for use as an inhibitor of platelet aggregation .
  • the invention further provides excretory-secretory products or derivatives thereof of the human hookworm Necator americanus for use as an inhibitor of Factor Xa activity.
  • the invention further provides derivatives of excretory-secretory products of the human hookworm Necator americanus for use as an inhibitor of platelet dense granule release.
  • Fig. 1 shows the effect of Necator americanus excretory-secretrory products on human plasma clotting times
  • Fig. 2 shows the effects of Necator americanus excretory-secretory products on human plasma stypven clotting time
  • Fig. 3 shows the effects of Necator americanus on human Factor Xa activity
  • Fig. 4 shows the effects of Necator americanus excretory-secretory products on platelet aggregation in human plasma mediated respectively by collagen, adenosine diphosphate (ADP), thrombin and platelet activating factor (PAF);
  • ADP adenosine diphosphate
  • PAF platelet activating factor
  • Fig. 5 shows the effects of Necator americanus excretory-secretory products on platelet granule release .
  • Fig. 6 shows the degradation of human fibrinogen by Necator americanus excretory-secretory products. PREPARATION OF NECATOR AMERICANUS EXCRETORY-SECRETORY (ES) PRODUCTS
  • Necator americanus is passaged in DSN hamsters. Faecal cultured from infected animals provide infective (L3) larvae, which are then used to infect neonates percutaneously .
  • Adult worms are routinely harvested from the small intestines of infected hamsters 5 weeks post-infection. The ileum of the infected hamster is removed, opened longitudinally, and placed in Hanks' saline at 37°C. As worms release their hold on the mucosa, they are carefully removed, thoroughly washed, and cleansed in Hanks' saline containing 100 iu/ml penicillin and 100 ug/ml streptomycin. Cleansed worms are examined under a dissecting microscope, and undamaged worms retained.
  • worms are added to RPMI 1640, containing penicillin and streptomycin. The worms are then cultured for 16 hrs, and supernates removed for analysis of thro bolytic and anticoagulant activities.
  • Culture supernatants are filtered through 0.2 urn Minisort NML filters (Sartorius) to remove eggs that may have been deposited during the culture period.
  • Concentration of supernatants is carried out using centrifugal separation methods.
  • the bulk of the culture media is removed using Macrosep Centrifugal Concentrators (Flowgen) with a cut off point of 10K.
  • Final concentration and separation into 2 fractions according to MW is carried out using Centricon micro concentrators (Amincon) with cut off points of 30K and 10K.
  • Imidazole saline buffer (Sigma) is used to dilute the supernatant at this stage to aid in separation.
  • platelet rich plasma contains all the factors necessary to activate prothrombin by the intrinsic pathway.
  • the rate of clot ⁇ ting is a measure of the overall coagulant activity dev ⁇ eloped and this will be decreased if there is inhibition of any intrinsic pathway factor or factor-complex.
  • PRP platelet poor plasma
  • platelet substitute phospholipid emulsion
  • the test is carried out in a water bath at 37°C.
  • the 50 ul PPP in a previously unused glass test tube, 25 ul Celite (485 w/v in 0.85? ⁇ NaCl) and 25 ul phospholipid substitute (Rabbit brain cephalin (RVC): 1 vial in 5 ml 0.85? ⁇ NaCl) is added.
  • RVC phospholipid substitute
  • the tube is agitated to disperse the Celite.
  • 50 ul warmed CaClpit (0.025M) is then added and the timing started. At 2-3 second intervals, the tube is tilted and observed for the formation of a clot.
  • the PPP is preincubated for 10 minutes with varying concentrations of ES products (volumes being made up with saline). As delayed clotting time is often followed by the formation of a poor clot, the aggregation of Celite particles (caused by fibrin formation) is taken to indicate clot formation. If no clot is formed within 3 minutes, the timing is terminated.
  • the ES products of N. americanus have a dose-dependent effect on APTT (see figure 1).
  • Tissue thromboplastin tissue factor
  • factor VII a phospholipoprotein
  • the test is carried out in a water bath at 37°C.
  • SUBSTITUTE SHEET (RULE 26 ⁇ Equal volumes of thromboplastin solution and 0.025M CaCl_ are mixed and stored in the water bath.
  • the thromboplastin obtained from Sigma Diagnostics - 1 vial reconstituted with 2 ml dionized water). 50 ul citrated PPP is warmed in a clean unused test tube, 100 ul thromboplastin/CaCl- mixture added, and the timing started. (The tube is tilted every 2-3 seconds and the clotting time noted).
  • the PPP is first incubated for 10 minutes with varying concentrations of ES products (volumes being made up with saline). A prolonged clotting time or no clot formation is taken as an indication of inhibition in the extrinsic pathway by hookworm ES products.
  • the ES products of N. americanus have a dose-dependent effect on PT (see figure 1).
  • Stypven clotting time is the accelerated clotting time of recalcified plasma when mixed with Russell's Viper Venom (RVV). In the presence of brain phospholipid, RVV activates factor X directly.
  • the test is carried out in a water bath at 37°C. 50 ul human PPP is warmed in a clean unused test tube, and 25 ul RVV (in cephalin solution) added.
  • RVV in cephalin solution
  • the RVV in cephalin was obtained from Sigma Diagnostics - 1 vial was dissolved in 3 ml 0.85? ⁇ NaCl). After 3 minutes incubation, 50 ul 0.025M CaClweb is added, and the time s tar ted .
  • hookworm ES products The effect of hookworm ES products on Stypven Time is assessed by adding varying concentrations of ES products to the mixture after incubation with RVV and allowing a further incubation period of 10 minutes. 50 ul of CaCl_ is then added and the timing started. Prolongation of clotting time is taken as an indication of inhibition in the common pathway of coagulation by hookworm ES products.
  • Necator ES products prolong human plasma Stypven clotting time (see figure 2).
  • a synthetic oligopeptide substrate possessing a fluorescent group commercially used to measure activated factor X, was used to confirm factor Xa activity.
  • Factor Xa splits the AMC.HC1 from the carboxyl terminal of this substrate molecule causing it to fluoresce. The enzyme activity can thus be assessed using a fluorimeter .
  • Necator ES products The effect of Necator ES products on fibrinogenolysis was investigated by incubating human fibrinogen and Necator ES products over timed intervals then examining the degradation products using SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), 40ug aliquots of fibrinogen were incubated with 10ug Necator ES products at 37°C for 1,3,5 and 24 hour periods. At the end of each incubation period, the samples were run under reducing conditions on a 7-12. ⁇ gradient gel. This was run at 30volts overnight, at 200volts until completion, and then stained with Coomassie brilliant blue R-250.
  • SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis
  • Fibrinogenolysis occured during incubation of human fibrinogen with Necator americanus ES products (Fig. 6). After one hour incubation, degradation of fibrinogen was clearly seen. Prolonged incubation times caused increading amounts of degradation, and after 24 hours most of the 40ug protein sample was degraded.
  • excretory-secretory products of the human hookworm prevent haemostasis by various strategies, including inhibition of coagulation factor Xa, a pivotal component of the clotting cascade. Platelet activation is also affected.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne l'utilisation de produits excréteurs-secréteurs (ES) de l'ankylostome de l'homme, le Necator americanus, comme agents anti-hémostatiques. En effet, ces produits inhibent l'activité du facteur de coagulation Xa, ainsi que l'agrégation plaquettaire.
PCT/GB1994/002406 1993-11-02 1994-11-02 Agents anti-hemostatiques tires du necator americanus WO1995012615A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9322576.1 1993-11-02
GB939322576A GB9322576D0 (en) 1993-11-02 1993-11-02 Antihaemostatic agents

Publications (1)

Publication Number Publication Date
WO1995012615A1 true WO1995012615A1 (fr) 1995-05-11

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PCT/GB1994/002406 WO1995012615A1 (fr) 1993-11-02 1994-11-02 Agents anti-hemostatiques tires du necator americanus

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GB (1) GB9322576D0 (fr)
WO (1) WO1995012615A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996032479A1 (fr) * 1995-04-10 1996-10-17 Yale University Vaccin contre l'ankylostome
US5863894A (en) * 1995-06-05 1999-01-26 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5864009A (en) * 1994-10-18 1999-01-26 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5866542A (en) * 1994-10-18 1999-02-02 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5866543A (en) * 1995-06-05 1999-02-02 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5872098A (en) * 1995-06-05 1999-02-16 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5955294A (en) * 1994-10-18 1999-09-21 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6040441A (en) * 1994-10-18 2000-03-21 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
WO2000041706A1 (fr) * 1999-01-15 2000-07-20 University Of Nottingham Agents favorisant l'apoptose
WO2000058341A1 (fr) * 1999-03-31 2000-10-05 Yale University Inhibiteur des plaquettes d'ankylostome

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BEHNKE ET AL: "HUMAN PARASITIC DISEASES.VOLUME 4.HOOKWORM INFECTIONS (GILLES AND BALL,EDS.).9.AN OVERVIEW.PAGES 217-237", 1991, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM *
CARR ET AL: "ANTIGEN EXPRESSION DURING DEVELOPMENT OF THE HUMAN HOOKWORM,NECATOR AMERICANUS (NEMATODA)", PARASITE IMMUNOLOGY, vol. 9, no. 2, March 1987 (1987-03-01), OXFORD,GB, pages 219 - 234 *
CARR ET AL: "IDENTIFICATION OF HOOKWORM (NECATOR AMERICANUS) ANTIGENS AND THEIR TRANSLATION IN VITRO", MOLECULAR AND BIOCHEMICAL PARASITOLOGY, vol. 19, no. 3, 1986, AMSTERDAM, pages 251 - 258 *
KUMAR ET AL: "THE PARTIAL CHARACTERIZATION OF PROTEASES PRESENT IN THE EXCRETORY/SECRETORY PRODUCTS AND EXSHEATHING FLUID OF THE INFECTIVE (L3) LARVA OF NECATOR AMERICANUS", INTERNATIONAL JOURNAL FOR PARASITOLOGY, vol. 22, no. 5, 1992, OXFORD,GB, pages 563 - 572 *
PRITCHARD ET AL: "EPIDEMIOLOGY AND IMMUNOLOGY OF NECATOR AMERICANUS INFECTION IN A COMMUNITY IN PAPUA NEW GUINEA:HUMORAL RESPONSES TO EXCRETORY-SECRETORY AND CUTICULAR COLLAGEN ANTIGENS", PARASITOLOGY, vol. 100, no. PT.2, April 1990 (1990-04-01), CAMBRIDGE,GB, pages 317 - 326 *
PRITCHARD ET AL: "NECATOR AMERICANUS SECRETORY ACETYLCHOLINESTERASE AND ITS PURIFICATION FROM EXCRETORY-SECRETORY PRODUCTS BY AFFINITY CHROMATOGRAPHY", PARASITE IMMUNOLOGY, vol. 13, no. 2, March 1991 (1991-03-01), OXFORD,GB, pages 187 - 199 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5945275A (en) * 1994-10-18 1999-08-31 Corvas International, Inc. Nematode-extracted anticoagulant protein
US6872808B1 (en) 1994-10-18 2005-03-29 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US5864009A (en) * 1994-10-18 1999-01-26 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5866542A (en) * 1994-10-18 1999-02-02 Corvas International, Inc. Nematode-extracted anticoagulant protein
US6534629B1 (en) 1994-10-18 2003-03-18 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6096877A (en) * 1994-10-18 2000-08-01 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US5955294A (en) * 1994-10-18 1999-09-21 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6040441A (en) * 1994-10-18 2000-03-21 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6046318A (en) * 1994-10-18 2000-04-04 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6087487A (en) * 1994-10-18 2000-07-11 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6090916A (en) * 1994-10-18 2000-07-18 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
US6121435A (en) * 1994-10-18 2000-09-19 Corvas International, Inc. Nematode-extracted serine protease inhibitors and anticoagulant proteins
WO1996032479A1 (fr) * 1995-04-10 1996-10-17 Yale University Vaccin contre l'ankylostome
US5866543A (en) * 1995-06-05 1999-02-02 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5872098A (en) * 1995-06-05 1999-02-16 Corvas International, Inc. Nematode-extracted anticoagulant protein
US5863894A (en) * 1995-06-05 1999-01-26 Corvas International, Inc. Nematode-extracted anticoagulant protein
WO2000041706A1 (fr) * 1999-01-15 2000-07-20 University Of Nottingham Agents favorisant l'apoptose
WO2000058341A1 (fr) * 1999-03-31 2000-10-05 Yale University Inhibiteur des plaquettes d'ankylostome

Also Published As

Publication number Publication date
GB9322576D0 (en) 1993-12-22

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