CA2068312A1 - Agent for neutralising the procoagulant activity associated with tumor cells - Google Patents
Agent for neutralising the procoagulant activity associated with tumor cellsInfo
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- CA2068312A1 CA2068312A1 CA002068312A CA2068312A CA2068312A1 CA 2068312 A1 CA2068312 A1 CA 2068312A1 CA 002068312 A CA002068312 A CA 002068312A CA 2068312 A CA2068312 A CA 2068312A CA 2068312 A1 CA2068312 A1 CA 2068312A1
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- leu
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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Abstract
(57) Abstract The object of the present invention is the use of an anticoagulant protein of the annexine family as an agent for preventing the metastasis of tumors.
Description
S012821J.31 ~gent for neutralising the tumour cell-associated procoaqulant activity The present invention relates to the use of an anticoagulant protein from the family of annexines as an agent for preventing the metastasis of tumours.
The formation of metastases has proved to be a particular problem in tumour diseases. The mechanism of this phenomenon, referred to as metastasizing, would appear to proceed as follows-tumour cells swamp the circulatory system of the organism (intravasculation) and are carried in the bloodstream. These malignant cells interact with the platelets and the plasma coagulation factors and activate the haemostatic system. The surface-dependent procoagulant activity known of tum;our cells results in the formation of thrombin and finally the fibrin polymer. Moreover, these same cells also interact with other host cells. Then the tumour cells surrounded by clumps of platelets and threads of fibrin become lodged in capillary vessels. As a result of the formation of the fibrin web around the surface of the tumour cells specific tumour antigens are concealed, which would otherwise cause an immunological reaction resulting in the destruction of the tumour cells. It is there~ore precisely this ability of the tumour cell to surround itself with a fibrin network which is responsible for the metastatic activity of these cells. Tumour enzymes then attack the vascular wall proteolytically, enabling the tumour cells to leave the circulatory system (extravasculation); secondary tumours are produced.
Clearly, the crucial factor of this mechanism is the ability of the circulating tumour cells to cause formation of a fibrin net which serves to lodge the cell in the capillary vessel and/or escape from the immune defence of the organism. Falanga et al. (Biochemistry 24, 5558-5567 (1985) isolated and characterised a special cancer procoagulant which was capable of triggeriny coagulation by activating factor X. Other cancer procoa~ulants which directly activate factor X
have also been isolated. A summary of the research in the field of activating the coagulation system by tumour cells is found in Cancer ~etastasis Reviews 3, 99-116 (1984).
The aim of the present invention was therefore to provide an active substance directed against the procoagulant activity of tumour cells.
The formation of fibrin is the last step in a cascade of enzymatic reactions in which thrombin is formed, the thrombin finally converting fibrinogen into ~ibrin. Various procoagulant reactions such as, for example, the activation of prothrombin by factors Xa and Va are catalysed by phospholipid surfaces to~which~the clotting factors bind. Not every kind of phospholipid is capable of stimulating coagulation. The charge on the phospholipid surface appears to determine the degree of influence. Negatively charged phospholipids such as phosphatidylserine have a high procoagulatory effect.
Among the proteins which bind to phospholipids and interfere with processes dependent on phospholipid surfaces, there is a family which is Ca2+-dependent in its bonding to phospholipids.
This family, which is also known as the annexines, includes in addition to lipocortin I, calpactin I, protein II, lipocortin III, p67-calelectrin, the vascular anticoagulant protein (VAC) and IBC, PAP~
PAP I, PP4, endonexine II and lipocortin V. Derivatives of these proteins also have these properties.
The structural features common to the annexines are probably the reason for their similar Ca2+ and phospholipid binding properties. Although this general quality applies to all annexines, there is a clear ~ ?~
individuality in terms of ~heir affinity ~or Ca2+ and the various kinds of phospholipids.
The physiological ~UnCtiQnS of the annexines are concerned with membrane-associated processes. The basic mechanism of the coagulation-inhibitiny effect of VAC
has been recognised as the inhibition of the catalytic capacity of phospholipids as a result of the binding of VAC to their surface, thereby preventiny the formation of the clot-promoting complex on their surface.
Other annexines are capable of inhibiting blood clottiny, but VAC appears to be the most effective inhibitors.
Surprisingly, it has been found that annexines, especially VAC, inhibit the procoagulant activity of tumour cells.f The neutralising effect of annexines on the procoagulant activity has been demonstrated on various colon-tumou~ cells. The procoagulant activity of a certain amount of colon cells was inhi~ited by VAC~ in dosage-dependent manner (Fig. 2). From the correlation between the number of cells and the clotting time (Fig. 1) it has been calculated that 210 nM VAC~
neutralise 99% of the procoagulant activity of 6X106 cells/ml. When colon cells neutralised by VAC~ were centrifuged in the presence of Ca2+ and then washed with T~SA, they showed no procoagulant activity in the coagulation assay. If, however, the liquid contained EDTA instead of Ca2+, the cells showed normal procoagulant activity.
These results indicate that VAC~ neutralises the procoagulant activity associated with tumour cells by binding, Ca2+-dependently, to surface sites which play a crucial role in this procoagulant activity. Very probably, phospholipids also participate in the procoayulant activity associated with tumour cells since, up till now, no other surface receptors have been found for VAC~.
Thus, VAC~ inhibits the procoa~ulant activity of tumour cells by being bouncl to the sites on the surfaces of tumour cells which participate in the initiation and/or propagation of the reactions which lead to thrombin and finally to the formation of fibrin. Since not only V~C~ but also the other annexines bind to phospholipids in the Ca2`~~dependent manner, the results obtained with VAC~ should also be applicable to these other annexines.
Owing to the fact that the annexines inhibit the procoagulant activity inherent in the tumour cells, they prevent the encapsulation of the tumour cells in a fibrin net; an absolute necessity for the metastatic activity of the tumour cells. Because of the neutralising effect the annexines are therefore capable of preventing the metastasizing of tumour cells and are consequently potent active substances with an anti-metastatic activity. VAC is a~particularly suitable active substance of this kind.
The objective of the invention is therefore achieved by the use of an anticoagulant protein selected from the family of annexines.
The active substances which can be used according to the invention, particularly V~C, may occur not only in the free form but also in the form of their salts, particularly the pharmaceutically acceptable salts.
Since they contain a number of amino acid groups with free amino groups, the compounds according to the invention may,~for example, occur in the form of acid addition salts. The acid addition salts may be, in particular, physiologic211y acceptable salts with conventional, therapeutically acceptable acids; examples of inorganic acids include the hydrohalic acids such as hydrochloric acid, but also sulphuric and phosphoric or pyrophosphoric acid; examples of organic acids include, in particular, sulphonic acids such as benzene- or p-toluene-sulphonic acid or lower alkanesulphonic acids such as methanesulphonic, as well as carboxyllc acids such as acetic, lactic, palmitic and stearic acid, malic, tartaric, ascorbic and citric acid. Since the compounds also contain amino acid groups having free carboxyl yroups, they may also occur as metal salts, especially alkali metal or alkaline earth metal salts, e.g. sodium, calcium or magnesium salts, or as ammonium salts, derived from ammonia or a physiologically acceptable, organic nitrogenous base~ However, since they also contain free carboxyl groups and free amino groups, they may also occur as an internal salt.
The agents accordinq to the invention may be administered, for example, by parenteral route, e.g.
intravenously, intracutaneously, subcutaneously or intramuscularly, or topically.
The size of the individual doses and the dosage plan can best be determined by individual assessment of the particular case: the methods required to determine the relevant factors are well known to those skilled in the art. In normal circumstances, for injection, the therapeutically effective amount of the drug according to the invention will be within the range from about 0.005 to about 0.5 mg/kg of body weight. The range from about 0.01 mg/kg to about 0.1 mg/kg, more especially from about 0.01 to about 0.05 mg/kg of body weight, is particularly preferred. The drugs are administered by intravenous, intramuscular or subcutaneous injection.
Accordingly, pharmaceutical preparations for parenteral administration in the form of single doses contain about 0.4 to about 7.5 mg of active substance per dose, depending on the route of administration. The pharmaceutical compositions according to the invention may also contain in addition to the active substances a buffer, e.g. a phosphate buffer, intended to maintain the pH at between about 3.5 and ~, as well as sodium chloride, mannitol or sorbitol to achieve an isotonic solution. They may occur in freeze-dried or dissolved form, whilst solutions may contain an antibacterially active preservative, e.g. 0.2 to 0.3% methyl or ethyl 4-hydroxybenzoate. A preparation for topical use may take the form of an aqueous solution, lotion or gel, an oily solution or suspension, or a greasy or, more particularly, emulsified ointment. A preparation in the form of an aqueous solution may be obtained, for example, by dissolving the active substances according to the invention in an aqueous buffer solution at pH 4 to 6.5 and, if necessary, adding another active substance, e.g. an antiinflammatory agent, and/or a polymeric adhesive agent, e.g. polyvinylpyrrolidone, and/or a preservative. The concentration of active substance is about 0.1 to about 1.5 mg, preferably 0.25 to 1.0 mg, in 10 ml of a solution or 10 g of a gel.
An oily preparation for topical use is obtained, for example, by suspending the active substances according to the invention in an oil, optionally with the addition of swelling agents such as aluminium stearate and/or surface-active agents (surfactants), the HLB (hydrophilic-lipophilic balance) of which is less than 10, such as fatty acid monoesters of polyhydric alcohols, e.g. glycerol monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate. A greasy ointment is obtained, for example, by suspendiny the active substances according to the invention in a spreadable grease base, optionally with the addition of a surfactant having an HLB of less than 10. An emulsified ointment is obtained by triturating an aqueous solution of the active substances according to the invention in a soft, spreadable grease base with the addition of a surfactant having an HLB of less than 10. The topical preparations may also contain preservatives. The concentration of the active substance is 0.1 to 1.5 mg, preferably 0.25 to 1.0 mg, in about 10 g of base.
The invention relates to an agent selected from the 2 ~ d ,~
group of annexine~, and the natural.ly occur-~ing derivatives or analoyues thereof or those prepared synthetically or by genetlc enyineering, optionally with excipients and/or carriers and/or stabilisers for neutralising the tumour cell-associated procoayulant activity;
agent for neutralising the tumour cell-associated procoagulant activity, characterised in that the annexine corresponds to the formula Met Ala Gln Val Leu Arg Gly Thr v21 Thr ASp Phe Pro Gly Plle 3v Asp Glu Arg Ala Asp Ala XX Thr Leu Arg Lys Ala Met Lys Gly ~ 40 . 45 Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu Ile Val Ala Leu Met Lys Pro Ser Arg Leu Tyr Asp Ala Tyr Glu Leu Lys His Ala Leu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu Ile Ile Ala Ser Arg Thr Pro Glu ~ s~,~ r~
Glu Leu Arg Ala l:le Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp ?ro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala Gln Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr~Asp Glu Glu Lys Phe Ile 200 ~ 205 210 Thr Ile Phe Gly Thr Arg Ser Val Ser H~is Leu Arg Lys Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val Val Lys Ser Ile Arg Ser Ile Pro Al.a Tyr Leu Ala Glu Thr Leu Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val Met Val Ser Arg Ser Glu Ile Asp Le~ Phe Asn Ile Arg Lys Glu Phe Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile ~JL~ et Lys Gly Asp Thr Ser Gly Asp Tyr L~s Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp *
wherein XX represents Glu or Asp and optionally at position 1 the methionine is cleaved and the alanine at position 2 is optionally blocked and/or there may optionally be aggregations caused by intermolecular disulphide bridges, for example, between the cysteines at position 316, or the biologically active variants or derivatives thereof, optionally with excipients and/or carriers and/or stabilisers.
The present invention also rel.ates to:
an anticoagulant~;selected from the group of the annexines and thè naturally occurring derivatives or analogues thereof or those prepared synthetically or by genetic engineering, optionally with excipients and/or carriers and/or stabilisers for pr-eventing the metastasizing of tumours;
pharmaceutical compositions for preventing the metastasizing of tumours, characterised in that the anticoagulant corresponds to the formula Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp Glu Arg Ala Asp Ala XX Thr Leu Arg Lys Ala Met Lys Gl.y Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly ~5 go Lys Phe G].u Lys I,eu Ile Val Ala Leu Met Lys Pro Ser Arg Leu 100 lOS
Tyr Asp Ala Tyr Glu I.eu Lys His Ala Leu Lys Gly Ala Gly Thr 110 llS 120 Asn Glu Lys Val Leu Thr Glu Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala Gln Ala Leu Phe Gln Ala Gly Glu I,eu Lys Trp Gly Thr Asp Glu Glu Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile 3~-?J ~ ~
230 ~35 240 Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr I,eu Ile 275 2~0 2~5 Arg Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg Lys Glu Phe Arg Lys Asn Phe Ala Thr-Ser Leu Tyr Ser Met Ile Lys Gly Asp Thr Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp x wherein XX represents Glu or Asp and optionally at position 1 the methionine is cleaved and the alanine at position ~ is optlonally blocked and/or there may optionally be aggregations caused by intermolecular disulphide bridges, for example, between the cysteines at position 316, or the biologically active variants or derivatives thereof, optionally with excipients and/or carriers and/or stabilisers.
I'he present invention also relates to:
the use of an anticoagulant selected from the group of the annexines and the naturally occurring derivatives or analogues thereof or those prepared synthetically or by genetic engineering, optionally with excipients and/or carriers and/or stabilisers for preventing the metastasizing of tumours;
Use of an anticoagulant selected from the group of annexines and the naturally occurring derivatives or analogues thereof or tho~e prepared synthetically or by genetic englneering, optiona]ly with excipients and/or carriers and/or stabilisers for preventing metastasizing, wherein the antlcoagulant corresponds to the formula 1 5 10 ].5 Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp Glu Arg Ala Asp Ala XX Thr Leu Arg Lys Ala l~et Lys Gly ~5 Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu Ile Val Ala Leu Met Lys Pro Ser Arg Leu -100 . 105 Tyr Asp Ala Tyr Glu Leu Lys His Ala I.eu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp Pro Asp Ala 170 175 1~0 Gly Ile Asp Glu Ala Gln Va]. Glu Gln Asp Ala G].n Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile Asp Arg Glu Thr Ser Gly~Asn Leu Glu Gln Leu Leu Leu Ala Val Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg 290 2gs 300 Lys Glu Phe Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met I le Lys Gly Asp Thr Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp ?
wherein XX represents G]u or Asp and optionally at position l the methionine is cleaved and the alanine at position 2 is optionally blocked and/or there may optional]y ~e aggregations caused by intermolecular disulphide bridges, for example, between the cysteines at posi-tion 316, or the biologically active variants or derivatives thereof;
Use of an anticoagulant selected from the group of the annexines and the naturally occurring derivatives or analogues thereof or those prepared by synthetically or by genetic engineering, for the preparation of a pharmaceutical composition for preventing the metastasizing of tumours;
Use of an anticoagulant selected from the group of the annexines and the naturally occurring derivatives or analogues thereof or those prepare~ synthetically or by genetic engineering, for the preparation:of a pharmaceutical composition for preventing metastasizing, wherein the anticoagulant corresponds to the formula l 5 lO 15 Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp Glu Arg Ala Asp Ala XX Thr Leu Ar~ L~s Ala Met Lys Gly ~5 Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly 2~
-go Lys Phe Glu Lys Leu Ile Val Ala Leu Met Lys Pro Ser Arg l,eu Tyr Asp Ala Tyr Glu Leu Lys His Ala Leu Lys Gly Ala Gly Thr ~Srl ~lU l-ys val Leu lnL ~lu 1l~ lle Ala ~er Ary lnL ~'IO ~lU
Glu Leu Arg Ala Ile Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser 1~0 145 150 Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln 155 160 1~5 Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp Pro Asp Ala 170 : 175 180 Gly Ile Asp Glu ~la Gln Val Glu Gln Asp Ala Gln Ala Leu Phe 1~5 190 195 Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp G]u Glu Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu 260 2~5 270 Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile 7.75 280 235 Arg Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg ~yS ~lU i~ne ~rg ~JyS Asn ~ne Ala lhl ~eL LeU lyl .~eL l~le~
Lys Gly Asp Thr Ser Gly Asp Tyr-Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp *
wherein XX represents Glu or Asp and optionally at position 1 the methionine is cleaved and the alanine at position 2 is optionally blocked and/or there may optionally be aggregations caused by intermolecular disulphide bridges, for example, between the cysteines at position 316, or the biologically active variants or derivatives thereof.
The Example which follows is intended to illustrate the invention without restricting it.
Materials and Methods VAC~ was prepared analogously to either EPA
0 181 465 or EPA 0 293 567. The following tests were carried out with VACQ. However, the results should also apply to the other annexines, especially VAC~.
Colon tumour cells were cultivated in a tissue flask in RPMI 16~0 supplemented with 10% foetal calves' serum. The cells were harvested by wiping the bottom of the flask with a rubber wiper. The collected cells were washed with a TBSA buffer (50 mM Tris/HCl, 100 mM NaCl, 4.5 mg glucose/ml, 0.5 mg bovine serum albumin/ml, 2 mM
CaCl2, pH 7.9) and finally suspended in this buffer for the tests. The viability of these cells was tested with tryptan-blue exclusion. More than 95% of the cells were viable after this procedure.
Coagulation assay: The procoagulant activity of the colon cells was measured by adding a certain number of washed cells to 50 ~1 of citrated platelet-free plasma (PFP). This mixture was stirred for two minutes at 37'C. Coagulation was initiated by the addition of CaCl2 (the final concentration was 10 mM). The final volume of the mixture was 500 ~1. Coagulation was observed turbidimetrica]ly using a "Payton dual wave aggregometer".
Results Procoaqulant activitv associated with colon cells:
When washed colon cells were added to citrated PFP
and the plasma was recalcified, a coagulation time was observed which was dependent on the number of cells present (Fig. 1). In the absence of the cells the coagulation time was more than 15 minutes, whereas 8X106 cells/ml reduced this coagulation time to about 2 minutes. This procoagulant activity was caused by the 2 1~ J
intact cells; centrifuging the cells resulted in coprecipi ation of the procoagulant activity.
Neutrall~ation of the procoaqulant activltY associated _ith colon cells by VAC~:
The procoagulant activity of a certain number of colon cells was inhibited by VAC~ in dosage-dependent manner (Fig. 2). F'rom the correlation between the number of cells and the coagulation time (Fig. l) it was calculated that 210 nM of VAC~ neutralised 99% of the procoagulant activity of 6X106 cells/ml. When colon cells neutralised by VAC~ were centrifuged in the presence of Ca2+ and then washed with TBSA, they showed no procoagulant activity in the coagulation assay. If, however, the liquid contained EDTA instead of Ca2r, the cells exhibited normal procoagulant activity.
d.r Leqe d accoml~anYinq the dr w~
Fig. 1 Procoagulant activity associated with colon cells.
Citrated platelet-free plasma (PFP) was diluted with TBSA containing the specified number of colon eells.
After 3 minutes incubation at 37~C, CaCl2 was added (final concentration 10 mM) and the coagulation time was recorded.
Fig. 2 Effeet of VAC~ on the proeoagulant activity of eolon cells. Citrated platelet-free plasma (PFP) was diluted with TBSA eontaininy the specified amount of VAC~ and a eertain number of colon eells (up to a final concentration of 6X106 eells/ml). After 3 minutes incubation at 37C, eoagulation was started by the addition of CaCl2 (final eoneentration 10 mM~. The eoagulation time measured was eompared with Fig. 1 and extrapolated to the number of procoagulant-aetive eolon cells.
The formation of metastases has proved to be a particular problem in tumour diseases. The mechanism of this phenomenon, referred to as metastasizing, would appear to proceed as follows-tumour cells swamp the circulatory system of the organism (intravasculation) and are carried in the bloodstream. These malignant cells interact with the platelets and the plasma coagulation factors and activate the haemostatic system. The surface-dependent procoagulant activity known of tum;our cells results in the formation of thrombin and finally the fibrin polymer. Moreover, these same cells also interact with other host cells. Then the tumour cells surrounded by clumps of platelets and threads of fibrin become lodged in capillary vessels. As a result of the formation of the fibrin web around the surface of the tumour cells specific tumour antigens are concealed, which would otherwise cause an immunological reaction resulting in the destruction of the tumour cells. It is there~ore precisely this ability of the tumour cell to surround itself with a fibrin network which is responsible for the metastatic activity of these cells. Tumour enzymes then attack the vascular wall proteolytically, enabling the tumour cells to leave the circulatory system (extravasculation); secondary tumours are produced.
Clearly, the crucial factor of this mechanism is the ability of the circulating tumour cells to cause formation of a fibrin net which serves to lodge the cell in the capillary vessel and/or escape from the immune defence of the organism. Falanga et al. (Biochemistry 24, 5558-5567 (1985) isolated and characterised a special cancer procoagulant which was capable of triggeriny coagulation by activating factor X. Other cancer procoa~ulants which directly activate factor X
have also been isolated. A summary of the research in the field of activating the coagulation system by tumour cells is found in Cancer ~etastasis Reviews 3, 99-116 (1984).
The aim of the present invention was therefore to provide an active substance directed against the procoagulant activity of tumour cells.
The formation of fibrin is the last step in a cascade of enzymatic reactions in which thrombin is formed, the thrombin finally converting fibrinogen into ~ibrin. Various procoagulant reactions such as, for example, the activation of prothrombin by factors Xa and Va are catalysed by phospholipid surfaces to~which~the clotting factors bind. Not every kind of phospholipid is capable of stimulating coagulation. The charge on the phospholipid surface appears to determine the degree of influence. Negatively charged phospholipids such as phosphatidylserine have a high procoagulatory effect.
Among the proteins which bind to phospholipids and interfere with processes dependent on phospholipid surfaces, there is a family which is Ca2+-dependent in its bonding to phospholipids.
This family, which is also known as the annexines, includes in addition to lipocortin I, calpactin I, protein II, lipocortin III, p67-calelectrin, the vascular anticoagulant protein (VAC) and IBC, PAP~
PAP I, PP4, endonexine II and lipocortin V. Derivatives of these proteins also have these properties.
The structural features common to the annexines are probably the reason for their similar Ca2+ and phospholipid binding properties. Although this general quality applies to all annexines, there is a clear ~ ?~
individuality in terms of ~heir affinity ~or Ca2+ and the various kinds of phospholipids.
The physiological ~UnCtiQnS of the annexines are concerned with membrane-associated processes. The basic mechanism of the coagulation-inhibitiny effect of VAC
has been recognised as the inhibition of the catalytic capacity of phospholipids as a result of the binding of VAC to their surface, thereby preventiny the formation of the clot-promoting complex on their surface.
Other annexines are capable of inhibiting blood clottiny, but VAC appears to be the most effective inhibitors.
Surprisingly, it has been found that annexines, especially VAC, inhibit the procoagulant activity of tumour cells.f The neutralising effect of annexines on the procoagulant activity has been demonstrated on various colon-tumou~ cells. The procoagulant activity of a certain amount of colon cells was inhi~ited by VAC~ in dosage-dependent manner (Fig. 2). From the correlation between the number of cells and the clotting time (Fig. 1) it has been calculated that 210 nM VAC~
neutralise 99% of the procoagulant activity of 6X106 cells/ml. When colon cells neutralised by VAC~ were centrifuged in the presence of Ca2+ and then washed with T~SA, they showed no procoagulant activity in the coagulation assay. If, however, the liquid contained EDTA instead of Ca2+, the cells showed normal procoagulant activity.
These results indicate that VAC~ neutralises the procoagulant activity associated with tumour cells by binding, Ca2+-dependently, to surface sites which play a crucial role in this procoagulant activity. Very probably, phospholipids also participate in the procoayulant activity associated with tumour cells since, up till now, no other surface receptors have been found for VAC~.
Thus, VAC~ inhibits the procoa~ulant activity of tumour cells by being bouncl to the sites on the surfaces of tumour cells which participate in the initiation and/or propagation of the reactions which lead to thrombin and finally to the formation of fibrin. Since not only V~C~ but also the other annexines bind to phospholipids in the Ca2`~~dependent manner, the results obtained with VAC~ should also be applicable to these other annexines.
Owing to the fact that the annexines inhibit the procoagulant activity inherent in the tumour cells, they prevent the encapsulation of the tumour cells in a fibrin net; an absolute necessity for the metastatic activity of the tumour cells. Because of the neutralising effect the annexines are therefore capable of preventing the metastasizing of tumour cells and are consequently potent active substances with an anti-metastatic activity. VAC is a~particularly suitable active substance of this kind.
The objective of the invention is therefore achieved by the use of an anticoagulant protein selected from the family of annexines.
The active substances which can be used according to the invention, particularly V~C, may occur not only in the free form but also in the form of their salts, particularly the pharmaceutically acceptable salts.
Since they contain a number of amino acid groups with free amino groups, the compounds according to the invention may,~for example, occur in the form of acid addition salts. The acid addition salts may be, in particular, physiologic211y acceptable salts with conventional, therapeutically acceptable acids; examples of inorganic acids include the hydrohalic acids such as hydrochloric acid, but also sulphuric and phosphoric or pyrophosphoric acid; examples of organic acids include, in particular, sulphonic acids such as benzene- or p-toluene-sulphonic acid or lower alkanesulphonic acids such as methanesulphonic, as well as carboxyllc acids such as acetic, lactic, palmitic and stearic acid, malic, tartaric, ascorbic and citric acid. Since the compounds also contain amino acid groups having free carboxyl yroups, they may also occur as metal salts, especially alkali metal or alkaline earth metal salts, e.g. sodium, calcium or magnesium salts, or as ammonium salts, derived from ammonia or a physiologically acceptable, organic nitrogenous base~ However, since they also contain free carboxyl groups and free amino groups, they may also occur as an internal salt.
The agents accordinq to the invention may be administered, for example, by parenteral route, e.g.
intravenously, intracutaneously, subcutaneously or intramuscularly, or topically.
The size of the individual doses and the dosage plan can best be determined by individual assessment of the particular case: the methods required to determine the relevant factors are well known to those skilled in the art. In normal circumstances, for injection, the therapeutically effective amount of the drug according to the invention will be within the range from about 0.005 to about 0.5 mg/kg of body weight. The range from about 0.01 mg/kg to about 0.1 mg/kg, more especially from about 0.01 to about 0.05 mg/kg of body weight, is particularly preferred. The drugs are administered by intravenous, intramuscular or subcutaneous injection.
Accordingly, pharmaceutical preparations for parenteral administration in the form of single doses contain about 0.4 to about 7.5 mg of active substance per dose, depending on the route of administration. The pharmaceutical compositions according to the invention may also contain in addition to the active substances a buffer, e.g. a phosphate buffer, intended to maintain the pH at between about 3.5 and ~, as well as sodium chloride, mannitol or sorbitol to achieve an isotonic solution. They may occur in freeze-dried or dissolved form, whilst solutions may contain an antibacterially active preservative, e.g. 0.2 to 0.3% methyl or ethyl 4-hydroxybenzoate. A preparation for topical use may take the form of an aqueous solution, lotion or gel, an oily solution or suspension, or a greasy or, more particularly, emulsified ointment. A preparation in the form of an aqueous solution may be obtained, for example, by dissolving the active substances according to the invention in an aqueous buffer solution at pH 4 to 6.5 and, if necessary, adding another active substance, e.g. an antiinflammatory agent, and/or a polymeric adhesive agent, e.g. polyvinylpyrrolidone, and/or a preservative. The concentration of active substance is about 0.1 to about 1.5 mg, preferably 0.25 to 1.0 mg, in 10 ml of a solution or 10 g of a gel.
An oily preparation for topical use is obtained, for example, by suspending the active substances according to the invention in an oil, optionally with the addition of swelling agents such as aluminium stearate and/or surface-active agents (surfactants), the HLB (hydrophilic-lipophilic balance) of which is less than 10, such as fatty acid monoesters of polyhydric alcohols, e.g. glycerol monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate. A greasy ointment is obtained, for example, by suspendiny the active substances according to the invention in a spreadable grease base, optionally with the addition of a surfactant having an HLB of less than 10. An emulsified ointment is obtained by triturating an aqueous solution of the active substances according to the invention in a soft, spreadable grease base with the addition of a surfactant having an HLB of less than 10. The topical preparations may also contain preservatives. The concentration of the active substance is 0.1 to 1.5 mg, preferably 0.25 to 1.0 mg, in about 10 g of base.
The invention relates to an agent selected from the 2 ~ d ,~
group of annexine~, and the natural.ly occur-~ing derivatives or analoyues thereof or those prepared synthetically or by genetlc enyineering, optionally with excipients and/or carriers and/or stabilisers for neutralising the tumour cell-associated procoayulant activity;
agent for neutralising the tumour cell-associated procoagulant activity, characterised in that the annexine corresponds to the formula Met Ala Gln Val Leu Arg Gly Thr v21 Thr ASp Phe Pro Gly Plle 3v Asp Glu Arg Ala Asp Ala XX Thr Leu Arg Lys Ala Met Lys Gly ~ 40 . 45 Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu Ile Val Ala Leu Met Lys Pro Ser Arg Leu Tyr Asp Ala Tyr Glu Leu Lys His Ala Leu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu Ile Ile Ala Ser Arg Thr Pro Glu ~ s~,~ r~
Glu Leu Arg Ala l:le Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp ?ro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala Gln Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr~Asp Glu Glu Lys Phe Ile 200 ~ 205 210 Thr Ile Phe Gly Thr Arg Ser Val Ser H~is Leu Arg Lys Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val Val Lys Ser Ile Arg Ser Ile Pro Al.a Tyr Leu Ala Glu Thr Leu Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val Met Val Ser Arg Ser Glu Ile Asp Le~ Phe Asn Ile Arg Lys Glu Phe Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile ~JL~ et Lys Gly Asp Thr Ser Gly Asp Tyr L~s Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp *
wherein XX represents Glu or Asp and optionally at position 1 the methionine is cleaved and the alanine at position 2 is optionally blocked and/or there may optionally be aggregations caused by intermolecular disulphide bridges, for example, between the cysteines at position 316, or the biologically active variants or derivatives thereof, optionally with excipients and/or carriers and/or stabilisers.
The present invention also rel.ates to:
an anticoagulant~;selected from the group of the annexines and thè naturally occurring derivatives or analogues thereof or those prepared synthetically or by genetic engineering, optionally with excipients and/or carriers and/or stabilisers for pr-eventing the metastasizing of tumours;
pharmaceutical compositions for preventing the metastasizing of tumours, characterised in that the anticoagulant corresponds to the formula Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp Glu Arg Ala Asp Ala XX Thr Leu Arg Lys Ala Met Lys Gl.y Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly ~5 go Lys Phe G].u Lys I,eu Ile Val Ala Leu Met Lys Pro Ser Arg Leu 100 lOS
Tyr Asp Ala Tyr Glu I.eu Lys His Ala Leu Lys Gly Ala Gly Thr 110 llS 120 Asn Glu Lys Val Leu Thr Glu Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala Gln Ala Leu Phe Gln Ala Gly Glu I,eu Lys Trp Gly Thr Asp Glu Glu Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile 3~-?J ~ ~
230 ~35 240 Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr I,eu Ile 275 2~0 2~5 Arg Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg Lys Glu Phe Arg Lys Asn Phe Ala Thr-Ser Leu Tyr Ser Met Ile Lys Gly Asp Thr Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp x wherein XX represents Glu or Asp and optionally at position 1 the methionine is cleaved and the alanine at position ~ is optlonally blocked and/or there may optionally be aggregations caused by intermolecular disulphide bridges, for example, between the cysteines at position 316, or the biologically active variants or derivatives thereof, optionally with excipients and/or carriers and/or stabilisers.
I'he present invention also relates to:
the use of an anticoagulant selected from the group of the annexines and the naturally occurring derivatives or analogues thereof or those prepared synthetically or by genetic engineering, optionally with excipients and/or carriers and/or stabilisers for preventing the metastasizing of tumours;
Use of an anticoagulant selected from the group of annexines and the naturally occurring derivatives or analogues thereof or tho~e prepared synthetically or by genetic englneering, optiona]ly with excipients and/or carriers and/or stabilisers for preventing metastasizing, wherein the antlcoagulant corresponds to the formula 1 5 10 ].5 Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp Glu Arg Ala Asp Ala XX Thr Leu Arg Lys Ala l~et Lys Gly ~5 Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu Ile Val Ala Leu Met Lys Pro Ser Arg Leu -100 . 105 Tyr Asp Ala Tyr Glu Leu Lys His Ala I.eu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp Pro Asp Ala 170 175 1~0 Gly Ile Asp Glu Ala Gln Va]. Glu Gln Asp Ala G].n Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile Asp Arg Glu Thr Ser Gly~Asn Leu Glu Gln Leu Leu Leu Ala Val Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg 290 2gs 300 Lys Glu Phe Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met I le Lys Gly Asp Thr Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp ?
wherein XX represents G]u or Asp and optionally at position l the methionine is cleaved and the alanine at position 2 is optionally blocked and/or there may optional]y ~e aggregations caused by intermolecular disulphide bridges, for example, between the cysteines at posi-tion 316, or the biologically active variants or derivatives thereof;
Use of an anticoagulant selected from the group of the annexines and the naturally occurring derivatives or analogues thereof or those prepared by synthetically or by genetic engineering, for the preparation of a pharmaceutical composition for preventing the metastasizing of tumours;
Use of an anticoagulant selected from the group of the annexines and the naturally occurring derivatives or analogues thereof or those prepare~ synthetically or by genetic engineering, for the preparation:of a pharmaceutical composition for preventing metastasizing, wherein the anticoagulant corresponds to the formula l 5 lO 15 Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp Glu Arg Ala Asp Ala XX Thr Leu Ar~ L~s Ala Met Lys Gly ~5 Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly 2~
-go Lys Phe Glu Lys Leu Ile Val Ala Leu Met Lys Pro Ser Arg l,eu Tyr Asp Ala Tyr Glu Leu Lys His Ala Leu Lys Gly Ala Gly Thr ~Srl ~lU l-ys val Leu lnL ~lu 1l~ lle Ala ~er Ary lnL ~'IO ~lU
Glu Leu Arg Ala Ile Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser 1~0 145 150 Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln 155 160 1~5 Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp Pro Asp Ala 170 : 175 180 Gly Ile Asp Glu ~la Gln Val Glu Gln Asp Ala Gln Ala Leu Phe 1~5 190 195 Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp G]u Glu Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu 260 2~5 270 Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile 7.75 280 235 Arg Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg ~yS ~lU i~ne ~rg ~JyS Asn ~ne Ala lhl ~eL LeU lyl .~eL l~le~
Lys Gly Asp Thr Ser Gly Asp Tyr-Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp *
wherein XX represents Glu or Asp and optionally at position 1 the methionine is cleaved and the alanine at position 2 is optionally blocked and/or there may optionally be aggregations caused by intermolecular disulphide bridges, for example, between the cysteines at position 316, or the biologically active variants or derivatives thereof.
The Example which follows is intended to illustrate the invention without restricting it.
Materials and Methods VAC~ was prepared analogously to either EPA
0 181 465 or EPA 0 293 567. The following tests were carried out with VACQ. However, the results should also apply to the other annexines, especially VAC~.
Colon tumour cells were cultivated in a tissue flask in RPMI 16~0 supplemented with 10% foetal calves' serum. The cells were harvested by wiping the bottom of the flask with a rubber wiper. The collected cells were washed with a TBSA buffer (50 mM Tris/HCl, 100 mM NaCl, 4.5 mg glucose/ml, 0.5 mg bovine serum albumin/ml, 2 mM
CaCl2, pH 7.9) and finally suspended in this buffer for the tests. The viability of these cells was tested with tryptan-blue exclusion. More than 95% of the cells were viable after this procedure.
Coagulation assay: The procoagulant activity of the colon cells was measured by adding a certain number of washed cells to 50 ~1 of citrated platelet-free plasma (PFP). This mixture was stirred for two minutes at 37'C. Coagulation was initiated by the addition of CaCl2 (the final concentration was 10 mM). The final volume of the mixture was 500 ~1. Coagulation was observed turbidimetrica]ly using a "Payton dual wave aggregometer".
Results Procoaqulant activitv associated with colon cells:
When washed colon cells were added to citrated PFP
and the plasma was recalcified, a coagulation time was observed which was dependent on the number of cells present (Fig. 1). In the absence of the cells the coagulation time was more than 15 minutes, whereas 8X106 cells/ml reduced this coagulation time to about 2 minutes. This procoagulant activity was caused by the 2 1~ J
intact cells; centrifuging the cells resulted in coprecipi ation of the procoagulant activity.
Neutrall~ation of the procoaqulant activltY associated _ith colon cells by VAC~:
The procoagulant activity of a certain number of colon cells was inhibited by VAC~ in dosage-dependent manner (Fig. 2). F'rom the correlation between the number of cells and the coagulation time (Fig. l) it was calculated that 210 nM of VAC~ neutralised 99% of the procoagulant activity of 6X106 cells/ml. When colon cells neutralised by VAC~ were centrifuged in the presence of Ca2+ and then washed with TBSA, they showed no procoagulant activity in the coagulation assay. If, however, the liquid contained EDTA instead of Ca2r, the cells exhibited normal procoagulant activity.
d.r Leqe d accoml~anYinq the dr w~
Fig. 1 Procoagulant activity associated with colon cells.
Citrated platelet-free plasma (PFP) was diluted with TBSA containing the specified number of colon eells.
After 3 minutes incubation at 37~C, CaCl2 was added (final concentration 10 mM) and the coagulation time was recorded.
Fig. 2 Effeet of VAC~ on the proeoagulant activity of eolon cells. Citrated platelet-free plasma (PFP) was diluted with TBSA eontaininy the specified amount of VAC~ and a eertain number of colon eells (up to a final concentration of 6X106 eells/ml). After 3 minutes incubation at 37C, eoagulation was started by the addition of CaCl2 (final eoneentration 10 mM~. The eoagulation time measured was eompared with Fig. 1 and extrapolated to the number of procoagulant-aetive eolon cells.
Claims (4)
1. Use of an anticoagulant selected from the group of the annexines and the derivatives or analogues thereof, naturally occurring or prepared synthetically or by genetic engineering, optionally with excipients and/or carriers and/or stabilisers for preventing the metastasizing of tumours.
2. Use of an anticoagulant according to claim 3, wherein the anticoagulant corresponds to the formula wherein XX represents Glu or Asp and optionally at position 1 the methionine is cleaved and the alanine at position 2 is optionally blocked and/or there may optionally be aggregations caused by intermolecular disulphide bridges, for example, between the cysteines at position 316, or the biologically active variants or derivatives thereof.
3. Use of an anticoagulant selected from the group of.
the annexines and the derivatives and analogues thereof, naturally occurring or produced synthetically or by genetic engineering, for the preparation of a pharmaceutical composition for preventing the metastasizing of tumours.
the annexines and the derivatives and analogues thereof, naturally occurring or produced synthetically or by genetic engineering, for the preparation of a pharmaceutical composition for preventing the metastasizing of tumours.
4. Use of an anticoagulant according to claim 5, wherein the anticoagulant corresponds to the formula wherein XX represents Glu or Asp and optionally at position 1 the methionine is cleaved and the alanine at position 2 is optionally blocked and/or there may optionally be aggregations caused by intermolecular disulphide bridges, for example, between the cysteines at position 316, or the biologically active variants or derivatives thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3937607A DE3937607A1 (en) | 1989-11-11 | 1989-11-11 | USE OF AN ANTI-COAGULAR TREATMENT FOR TUMOR DISEASES |
| DEP3937607.9 | 1989-11-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2068312A1 true CA2068312A1 (en) | 1991-05-12 |
Family
ID=6393365
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002068312A Abandoned CA2068312A1 (en) | 1989-11-11 | 1990-11-08 | Agent for neutralising the procoagulant activity associated with tumor cells |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0500602B1 (en) |
| JP (1) | JPH05501876A (en) |
| AT (1) | ATE120371T1 (en) |
| CA (1) | CA2068312A1 (en) |
| DE (2) | DE3937607A1 (en) |
| DK (1) | DK0500602T3 (en) |
| ES (1) | ES2071833T3 (en) |
| HU (1) | HU208636B (en) |
| WO (1) | WO1991007187A1 (en) |
Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3664727B2 (en) * | 1994-01-24 | 2005-06-29 | ネオルクス コーポレイション | Radiolabeled annexin |
| US5968477A (en) * | 1994-01-24 | 1999-10-19 | Neorx Corporation | Radiolabeled annexin conjugates with hexose and a chelator |
| US20030220233A1 (en) | 1994-01-24 | 2003-11-27 | Neorx Corporation | Radiolabeled annexins |
| EP0765166B1 (en) * | 1994-06-16 | 2003-05-21 | Neorx Corporation | Radiolabeled annexin-galactose conjugates |
| EP0799050B1 (en) * | 1994-12-07 | 2004-08-11 | Neorx Corporation | Radiolabeled annexin-galactose cluster conjugates |
| EP1486509A3 (en) * | 1994-12-07 | 2005-03-23 | Neorx Corporation | Radiolabeled annexin-galactose cluster conjugates |
| US5935860A (en) | 1995-03-07 | 1999-08-10 | The George Washington University | Use of uteroglobin expression as a molecular marker for prostatic intraepithelial neoplasia |
| US6509316B2 (en) | 1995-03-07 | 2003-01-21 | George Washington University | Pharmaceutical compositions, methods, and kits for treatment and diagnosis of lung cancer |
| US5696092A (en) * | 1995-03-07 | 1997-12-09 | George Washington University | Methods and compositions for inhibiting metastasis of epithelial cell-derived cancers |
| US6288039B1 (en) | 1995-03-07 | 2001-09-11 | George Washington University | Pharmaceutical compositions methods and kits for treatment and diagnosis of breast cancer |
| EP1520588B1 (en) | 1998-07-13 | 2014-12-24 | Board Of Regents, The University Of Texas System | Uses of antibodies to aminophospholipids for cancer treatment |
| US6818213B1 (en) | 1998-07-13 | 2004-11-16 | Board Of Regents, The University Of Texas System | Cancer treatment compositions comprising therapeutic conjugates that bind to aminophospholipids |
| AU771224B2 (en) | 1998-07-13 | 2004-03-18 | Board Of Regents, The University Of Texas System | Cancer treatment methods using antibodies to aminophospholipids |
| US6406693B1 (en) | 1998-07-13 | 2002-06-18 | Board Of Regents, The University Of Texas System | Cancer treatment methods using antibodies to aminophospholipids |
| IL140700A0 (en) | 1998-07-13 | 2002-02-10 | Univ Texas | Cancer treatment methods using therapeutic conjugates that bind to aminophospholipids |
| US7635676B2 (en) | 2001-02-21 | 2009-12-22 | Alavita Pharmaccuticals, Inc. | Modified annexin proteins and methods for their use in organ transplantation |
| EP1839670A3 (en) | 2001-02-21 | 2009-11-11 | Alavita Pharmaceuticals, Inc. | Modified Annexin Proteins and their use against thrombosis |
| US7635680B2 (en) | 2001-02-21 | 2009-12-22 | Alavita Pharmaceuticals, Inc. | Attenuation of reperfusion injury |
| US7645739B2 (en) | 2001-02-21 | 2010-01-12 | Alavita Pharmaceuticals, Inc. | Modified annexin compositions and methods of using same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PT87083B (en) * | 1987-03-28 | 1992-07-31 | Boehringer Ingelheim Int | A PROCESS FOR THE PREPARATION OF A VASCULAR ANTICOAGULANT PROTEIN, OF DNA THAT CODIFIES FOR THIS PROTEIN AND OF PHARMACEUTICAL COMPOSITIONS CONTAINING IT |
-
1989
- 1989-11-11 DE DE3937607A patent/DE3937607A1/en not_active Withdrawn
-
1990
- 1990-11-08 AT AT90916090T patent/ATE120371T1/en active
- 1990-11-08 CA CA002068312A patent/CA2068312A1/en not_active Abandoned
- 1990-11-08 WO PCT/EP1990/001864 patent/WO1991007187A1/en active IP Right Grant
- 1990-11-08 DE DE59008813T patent/DE59008813D1/en not_active Expired - Fee Related
- 1990-11-08 JP JP2514759A patent/JPH05501876A/en active Pending
- 1990-11-08 HU HU9201552A patent/HU208636B/en not_active IP Right Cessation
- 1990-11-08 ES ES90916090T patent/ES2071833T3/en not_active Expired - Lifetime
- 1990-11-08 DK DK90916090.5T patent/DK0500602T3/en active
- 1990-11-08 EP EP90916090A patent/EP0500602B1/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| DK0500602T3 (en) | 1995-07-10 |
| HU208636B (en) | 1993-12-28 |
| JPH05501876A (en) | 1993-04-08 |
| ATE120371T1 (en) | 1995-04-15 |
| EP0500602A1 (en) | 1992-09-02 |
| DE59008813D1 (en) | 1995-05-04 |
| ES2071833T3 (en) | 1995-07-01 |
| EP0500602B1 (en) | 1995-03-29 |
| HUT61202A (en) | 1992-12-28 |
| WO1991007187A1 (en) | 1991-05-30 |
| HU9201552D0 (en) | 1992-08-28 |
| DE3937607A1 (en) | 1991-05-16 |
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