WO2012110843A1 - Methods and pharmaceutical compositions for promoting fibrinolysis and thrombolysis - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for promoting fibrinolysis and thrombolysis in a subject in need thereof. More particularly the present invention relates to an inhibitor of protease nexin-1 (PN-1) for use in a method for promoting fibrinolysis in a subject in need thereof.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR PROMOTING

FIBRINOLYSIS AND THROMBOLYSIS

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for promoting fibrinolysis and thrombolysis in a subject in need thereof.

BACKGROUND OF THE INVENTION:

Vascular injury and subsequent thrombus formation are key events in the pathogenesis of athero thrombosis and venous thromboembolism. The serine proteases, urokinase- and tissue-type plasminogen activators (uPA and tPA respectively), generate plasmin which drives fibrinolysis. The thrombolytic actions of these proteases are critical for clot dissolution. Their properties have numerous therapeutic applications, including fibrinolysis for ST elevation myocardial infarction (STEMI). Direct recanalization of an occluded vessel by primary angioplasty became the preferred reperfusion strategy in STEMI subjects. Thrombolysis remains however, an option of reperfusion therapy in early STEMI presenters. Despite early administration of recombinant tPA in STEMI presenters, fibrinolysis fails to achieve myocardial reperfusion in one out of two subjects and is associated with poor clinical outcome¹. This phenomenon is of considerable clinical importance in the setting of acute myocardial infarction, because early restoration of normal blood flow is strongly associated with improved survival. A few factors have been identified to be involved in this inter- individual heterogeneity, such as age, delay between symptom onset and fibrinolytic therapy, smoking habit, infarct size and site².

Plasminogen activator inhibitor type-1 (PAI-1) is a serine protease inhibitor which is present in plasma and in platelet a-granules. An increased plasma concentration of PAI-1 has been associated with recurrent myocardial infarction³' ⁴. In humans, platelet PAI-1 is assumed to be a major contributor to the stabilization of the thrombus, by inhibiting endogenous fibrinolysis⁵' ⁶. However, platelets have also been shown to inhibit fibrinolysis by PAI-1 - independent mechanisms⁷, and the individual role of other serpins, in the thrombolytic process has not yet been defined. Protease nexin-1 (PN-1), also known as SERPINE2, deserves special attention since it has been shown in vitro to inhibit significantly uPA, tPA and plasmin. PN-1 is barely detectable in plasma⁸ but is produced by various cell types⁹, and interestingly, stored in the a-granules of platelets¹⁰. However the role of PN-1 in fibrinolysis and thrombolysis has not yet been investigated.

SUMMARY OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for promoting fibrinolysis and thrombolysis in a subject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION:

Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin and thrombin. PN-1 is barely detectable in plasma, but we have recently shown that PN-1 is present within the alpha-granules of platelets. The role of platelet PN-1 in fibrinolysis was investigated by the inventors using human platelets incubated with a blocking antibody and platelets from PN-1 -deficient mice. The inventors showed by using fibrin-agar zymography and fibrin matrix that platelet PN-1 inhibited both the generation of plasmin by fibrin-bound tPA, and the activity of fibrin-bound plasmin itself. Rotational thromboelastometry (ROTEM^®) and laser scanning confocal microscopy were used to demonstrate that PN-1 blockade or deficiency resulted in increased clot lysis and in an acceleration of the lysis front. PN-1 is thus a major determinant of the lysis-resistance of platelet-rich clots (PRCs). Moreover, in an original murine model in which thrombolysis induced by tPA can be measured directly in situ, we observed that vascular recanalization was significantly increased in PN-1 -deficient mice. Surprisingly, general physical health, after tP A- induced thrombolysis, was much better in PN-1 -deficient than in wild-type mice. The results reveal that platelet PN- 1 can be considered as a new important regulator of thrombolysis in vivo. Inhibition of PN-1 is thus predicted to promote endogenous and exogenous t-PA-mediated fibrinolysis, and may enhance the therapeutic efficacy of thrombolytic agents.

Accordingly the present invention relates to an inhibitor of protease nexin-1 (PN-1) for use in a method for promoting fibrinolysis in a subject in need thereof. In a particular embodiment the fibrinolysis is a t-PA-mediated fibrinolysis.

Accordingly the present invention relates to an inhibitor of protease nexin-1 (PN-1) for use in a method for promoting thrombolysis in a subject in need thereof. As used herein the term "subject" refers to any subject (preferably human) afflicted with an ischemic condition.

The term "ischemic conditions" refers to any conditions that result from a restriction in blood supply in at least one organ or tissue due to a clot. Theses conditions typically results from the obstruction of a blood vessel by a clot. For example ischemic conditions include but are not limited to renal ischemia, retinal ischemia, brain ischemia and myocardial ischemia. More particularly, the term includes but it is not limited to coronary artery bypass graft surgery, global cerebral ischemia due to cardiac arrest, focal cerebral infarction, cerebral hemorrhage, hemorrhage infarction, hypertensive hemorrhage, hemorrhage due to rupture of intracranial vascular abnormalities, subarachnoid hemorrhage due to rupture of intracranial arterial aneurysms, hypertensive encephalopathy, carotid stenosis or occlusion leading to cerebral ischemia, cardiogenic thromboembolism, spinal stroke and spinal cord injury, diseases of cerebral blood vessels: e.g., atherosclerosis, vasculitis, macular degeneration, myocardial infarction, cardiac ischemia and superaventicular tachyarrhytmia.

In a particular embodiment, the subject is diagnosed with a coronary disorder, more particularly the subject has been diagnosed as presenting one of the following coronary disorders:

· chronic ischemic disorders without myocardial necrosis, such as stable or effort angina pectoris;

acute ischemic disorders without myocardial necrosis, such as unstable angina pectoris;

ischemic disorders with myocardial necrosis, such as ST segment elevation myocardial infarction or non-ST segment elevation myocardial infarction.

The present invention also relates to an inhibitor of protease nexin-1 (PN-1) for use in a method for enhancing therapeutic efficacy of a thrombolytic agent. Currently available thrombolyic agents include reteplase (r-PA or Retavase), alteplase (t-PA or Activase), urokinase (Abbokinase), prourokinase, anisoylated purified streptokinase activator complex (APSAC), and streptokinase.

The present invention also relates to a combination of an inhibitor of PN-1 and a thrombolytic agent for use in a method for treating an ischemic condition in a subject in need thereof. In a particular embodiment the combination may further comprise an inhibitor of plasminogen activator inhibitor type-1.

A used herein the term "inhibitor of protease nexin-1 (PN-1)" refers to any compound able to inhibit PN-1 activity. More particularly the inhibitor of PN-1 refers to a compound that inhibits the activity of PN-1 consisting in the inhibition of the generation of plasmin by fibrin- bound tPA, and in the inhibition of the activity of fibrin-bound plasmin itself. The inhibition of the generation of plasmin by fibrin-bound tPA, and in the inhibition of the activity of fibrin-bound plasmin itself may be evaluated according to any well known method in the art. Typically said inhibitions may be evaluated according to the methods described in the EXAMPLE that may be performed in the presence of the compound to be tested. For example, plasminogen activation by tPA, may be measured on a fibrin surface, in the presence of recombinant PN-1 and the compound to be tested. First, tPA is incubated for 1 hour on fibrin-coated plates, the excess of unbound tPA being eliminated. PN-1 is subsequently added to the fibrin-coated plates and the excess discarded. Finally the compound to be tested in added. Plasmin generation induced by the residual fibrin-bound tPA was is then determined after addition of plasminogen with the chromogenic substrate CBS0065. The rate of plasmin generation by tPA is finally compared to the rate measured in the absence to the compound to be tested wherein a higher rate is indicative that said compound is an inhibitor of PN-1.

In one embodiment, the inhibitor of PN-1 is a low molecular weight antagonist, e. g. a small organic molecule. The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

Alternatively, the inhibitor of PN-1 may consist in an antibody (the term including "antibody portion").

In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of PN-1. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immuno stimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes. Briefly, the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in PN-1. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice : Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody.

This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al, I. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immuniz ation o f the s e mi c e (e . g . , Xeno Mous e (Ab g enix) , HuMAb mi c e (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non- human sequences. The present invention also includes so-called single chain antibodies. The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.

In another embodiment, the antibody according to the invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody®". According to the invention, sdAb can particularly be llama sdAb.

In another embodiment the inhibitor of PN-1 is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996). Then after raising aptamers directed against PN-ls as above described, the skilled man in the art can easily select those inhibiting PN-1.

In another embodiment the inhibitor of PN-1 may consist in a polypeptide. In specific embodiments, it is contemplated that polypeptides according to the invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify bio distribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify bio distribution.

A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify bio distribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

Polyethylene glycol (PEG), has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydro xyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri- functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e- amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold- limiting glomular filtration (e.g., less than 45 kDa).

In addition, to the polymer backbone being important in maintaining circulatory half- life, and bio distribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of bio distribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes (see e.g., technologies of established by VectraMed, Plainsboro, N.J.). Such linkers may be used in modifying the polypeptides described herein for therapeutic delivery.

According to the invention, the polypeptides may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art.

The polypeptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. The polypeptides of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433A from Applied Biosystems Inc. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art.

As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides.

A further object of the invention relates to pharmaceutical compositions comprising an inhibitor of PN-1 for any use according to the invention.

Typically, the inhibitor of PN-1 may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The inhibitor of PN-1 can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium mono stearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In addition to the compounds of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

The pharmaceutical compositions according to the invention may also comprises a thrombolytic agent and even further an inhibitor of plasminogen activator inhibitor type-1.

The present invention also relates to the use of an inhibitor of PN-1 for the preparation of biomaterials or medical delivery devices selected among endovascular prostheses, such as stents, bypass grafts, internal patches around the vascular tube, external patches around the vascular tube, vascular cuff, and angioplasty catheter.

In this respect, the invention relates more particularly to biomaterials or medical delivery devices as mentioned above, coated with such inhibitor of PN-1 as defined above, said biomaterials or medical devices being selected among endovascular prostheses, such as stents, bypass grafts, internal patches around the vascular tube, external patches around the vascular tube, vascular cuff, and angioplasty catheter. Such a local biomaterial or medical delivery device can be used to reduce stenosis as an adjunct to revascularizing, bypass or grafting procedures performed in any vascular location including coronary arteries, carotid arteries, renal arteries, peripheral arteries, cerebral arteries or any other arterial or venous location, to reduce anastomic stenosis such as in the case of arterial-venous dialysis access with or without polytetrafluoro- ethylene grafting and with or without stenting, or in conjunction with any other heart or transplantation procedures, or congenital vascular interventions.

For illustration purpose, such endovascular prostheses and methods for coating an inhibitor of PN-1 thereto are more particularly described in WO2005094916, or are those currently used in the art. The compounds used for the coating of the prostheses should preferentially permit a controlled release of said inhibitor. Said compounds could be polymers (such as sutures, polycarbonate, Hydron, and Elvax), biopolymers/biomatrices (such as alginate,fucans, collagen-based matrices, heparan sulfate) or synthetic compounds such as synthetic heparan sulfate-like molecules or combinations thereof. Other xamples of polymeric materials may include biocompatible degradable materials, e. g. lactone-based polyesters orcopolyesters, e. g. polylactide ; polylactide-glycolide ;polycaprolactone- glycolide ; polyortho esters ; polyanhydrides ; polyamino acids ; polysaccharides ;polyphospha- zenes; poly (ether-ester) copolymers, e. g. PEO-PLLA, or mixtures thereof; and biocompatible non- degrading materials, e. g. polydimethylsiloxane ; poly (ethylene-vinylacetate) ; acrylate based polymers or coplymers, e. g. polybutylmethacrylate, poly (hydroxyethyl methyl- methacrylate) ; polyvinyl pyrrolidinone ;fluorinated polymers such as polytetrafluo ethylene ; cellulose esters. When a polymeric matrix is used, it may comprise 2 layers, e. g. a base layer in which said inhibitor is incorporated, such as ethylene-co-vinylacetate and polybutylmethacrylate, and a top coat, such as polybutylmethacrylate, which acts as a diffusion-control of said inhibitor. Alternatively, said inhibitor may be comprised in the base layer and the adjunct may be incorporated in the outlayer, or vice versa.

Such biomaterial or medical delivery device may be biodegradable or may be made of metal or alloy, e. g. Ni and Ti, or another stable substance when intented for permanent use. The inhibitor of the invention may also be entrapped into the metal of the stent or graft body which has been modified to contain micropores or channels. Also internal patches around the vascular tube, external patches around the vascular tube, or vascular cuff made of polymer or other biocompatible materials as disclosed above that contain the inhibitor of the invention may also be used for local delivery.

Said biomaterial or medical delivery device allow the inhibitor releasing from said biomaterial or medical delivery device over time and entering the surrounding tissue. Said releasing may occur during 1 month to 1 year. The local delivery according to the present invention allows for high concentration of the inhibitor of the invention at the disease site with low concentration of circulating compound. The amount of said inhibitor used for such local delivery applications will vary depending on the compounds used, the condition to be treated and the desired effect. For purposes of the invention, a therapeutically effective amount will be administered.

The local administration of said biomaterial or medical delivery device preferably takes place at or near the vascular lesions sites. The administration may be by one or more of the following routes: via catheter or other intravascular delivery system, intranasally, intrabronchially, interperitoneally or eosophagal. Stents are commonly used as a tubular structure left inside the lumen of a duct to relieve an obstruction. They may be inserted into the duct lumen in a non-expanded form and are then expanded autonomously (self-expanding stents) or with the aid of a second device in situ, e. g. a catheter-mounted angioplasty balloon which is inflated within the stenosed vessel or body passageway in order to shear and disrupt the obstructions associated with the wall components of the vessel and to obtain an enlarged lumen. A further aspect of the invention relates to a method of determining whether the patient will respond to thrombolytic agents which comprises the step of analyzing a biological sample from said patient for:

(i) detecting the presence of a mutation in the gene encoding for PN-1 and/or its associated promoter, and/or

(ii) determining the level of expression of the gene encoding for PN-1.

As used herein, the term "biological sample" refers to any sample from a patient such as blood or serum.

Typical techniques for detecting a mutation in the gene encoding for PN-1 may include restriction fragment length polymorphism, hybridisation techniques, DNA sequencing, exonuclease resistance, microsequencing, solid phase extension using ddNTPs, extension in solution using ddNTPs, oligonucleotide assays, methods for detecting single nucleotide polymorphism such as dynamic allele-specific hybridisation, ligation chain reaction, mini-sequencing, DNA "chips", allele-specific oligonucleotide hybridisation with single or dual-labelled probes merged with PCR or with molecular beacons, and others.

Analyzing the expression of the gene encoding for PN-1 may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed nucleic acid or translated protein.

In a preferred embodiment, the expression of the gene encoding for PN-1 is assessed by analyzing the expression of mRNA transcript or mRNA precursors, such as nascent RNA, of said gene. Said analysis can be assessed by preparing mRNA/cDNA from cells in a biological sample from a patient, and hybridizing the mRNA/cDNA with a reference polynucleotide. The prepared mRNA/cDNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TaqMan), and probes arrays such as GeneChip(TM) DNA Arrays (AFF YMETRIX).

Advantageously, the analysis of the expression level of mRNA transcribed from the gene encoding for PN-1 involves the process of nucleic acid amplification, e. g., by RT-PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (BARANY, Proc. Natl. Acad. Sci. USA, vol.88, p: 189-193, 1991), self sustained sequence replication (GUATELLI et al, Proc. Natl. Acad. Sci. USA, vol.57, p: 1874-1878, 1990), transcriptional amplification system (KWOH et al, 1989, Proc. Natl. Acad. Sci. USA, vol.86, p: 1173-1177, 1989), Q-Beta Replicase (LIZARDI et al, Biol. Technology, vol.6, p: 1197, 1988), rolling circle replication (U. S. Patent No. 5,854, 033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5 Or 3 'regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

In another preferred embodiment, the expression of the gene encoding for PN-1 is assessed by analyzing the expression of the protein translated from said gene. Said analysis can be assessed using an antibody (e.g., a radio-labeled, chromophore- labeled, fluorophore- labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin- streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for PN- 1.

Said analysis can be assessed by a variety of techniques well known from one of skill in the art including, but not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (RIA). The method of the invention may comprise comparing the level of expression of the gene encoding for PN-1 in a biological sample from a patient with a reference expression level of said gene. A difference in the level measured in said patient and said reference level is indicative whether the patient will respond or not to thrombolytic agents. The reference level of expression of the gene encoding for PN-1 may be the level of expression of said gene determined in a biological sample of a patient who responds to said thrombolytic agents. The reference level of expression of the gene encoding for PN-1 may also be the level of expression of said gene determined in a biological sample of a patient who does not respond to said thrombolytic agents. The invention will be further illustrated by the following examples. However, these examples should not be interpreted in any way as limiting the scope of the present invention.

EXAMPLE:

Materials and methods:

Animals

PN-1 -deficient mice (PN-1-/-) come from Pr D. Monard's laboratory (FMI, Basel, Switzerland) and were back-crossed for 12 generations into the C57BL/6 line¹¹. Experimental animals were 8-16 weeks of age. Heterozygous mating generated PN-1-/- and wild-type mice (WT). Mice were bred and maintained in our own laboratory (Paris, France). All animals were genotyped by PCR. All experiments were performed in accordance with European legislation on the protection of animals.

Methods

Preparation of washed platelets

Human platelets

Human blood from healthy adult volunteers was collected into 1/10 vol. ACD-A (38 mM citric acid, 60 mM sodium citrate, 136 mM glucose). Washed platelets were isolated as previously described¹².

Mouse platelets

Blood was collected from anesthetized mice by cardiac puncture into syringes containing 1/10 vol. ACD-C (130 mM citric acid, 124 mM sodium citrate, 110 mM glucose). Washed platelets were isolated as previously described¹⁰.

Binding of tPA and plasmin to fibrin matrices, and measurement of plasmin generation or activity

Fibrin matrices in 96-well plates were prepared as previously described¹³. The functionality of this fibrin surface was determined by measuring the activation of plasminogen by fibrin-bound t-PA, or the activity of fibrin-bound plasmin itself.

SDS-polyacrylamide gel electrophoresis and zymography Platelets (5^χ 10 /mL in reaction buffer) were activated by PAR1-AP (PARI -activating peptide, SFLLRN, NeoMPS) (50 μΜ) for human platelets or by PAR4-AP (PAR4-activating peptide, AYPGKF, NeoMPS) (250 μΜ) for mouse platelets, for 30 minutes at 37°C. Control samples were obtained by incubating platelets for the same time with buffer. At the end of the incubation, samples were centrifuged and the supernatants (secreted fraction) were removed for analysis. The secreted fractions were incubated with recombinant tPA (10 IU/ml) or plasmin (0.25μΜ) for 30 minutes at 37°C in the presence or absence of the blocking anti-PN- 1 (generous gift from Dr D.Hantai, Inserm U582, Paris) or anti-PAI-1 IgGs (MA-33B8-307; Molecular Innovations). Proteins were first separated on a 10% SDS-polyacrylamide gel. After incubation with 2% Triton X-I00, the gel was then overlaid on a fibrin-plasminogen (200 nM)-agar gel, for tPA activity measurement, or on a fibrin-agar gel for plasmin activity, as previously described¹⁴. Zymograms were allowed to develop at 37°C during 24 hours and photographed at regular intervals using dark-ground illumination. Zymograms were stained with blue-coomassie¹⁵.

Clot formation and fibrinolysis ex vivo

Human PRP was obtained from citrated blood by centrifugation at 120g during 15 minutes. PRP was adjusted at 10⁸ platelets/ml in platelet free plasma and supplemented with 75μg/ml FITC-fibrinogen. For mouse PRCs, citrated human platelet-free plasma was mixed with murine washed platelets to a concentration of 8 x 10⁸/ml. Samples were incubated with irrelevant-IgG or the blocking anti-PN-1 IgG or/and anti-PAI-1 IgG both at 100 μg/ml and recalcified with 10 mM CaCl₂ in glass tubes. After retraction, clots were removed, blotted and weighed. To assess fibrinolysis, clots were incubated in Hanks buffer (Sigma) for 24 hours at 37°C. The supernatant was removed, and the fluorescence released from the clot was measured in a spectrofluorometer¹⁶. The remaining clots were blotted and reweighed to calculate the loss of clot weight, and then were totally dissolved to calculate the fluorescence remained in the clot.

Fibrinolysis experiments: microscopic lysis velocity by laser scanning confocal microscopy

Citrated human or mice PRP was adjusted at 10⁸ platelets/ml and supplemented with Alexa 488-fibrinogen (Invitrogen). Human PRP was incubated with control IgG (Jackson immunoresearch) or blocking anti-PN-1 IgG (100 μg/ml) and PRCs were obtained by adding tissue factor (TF, Innovin 1/5 (v/v)) (Diagnostica Stago) and 10 mM CaCl₂ in microchambers as previously described¹⁷. PRP from WT or PN-1 -/- mice was clotted in the same conditions. Clots were scanned with a LEICA confocal laser scanning microscope linked to a Leica inverted microscope equipped with a x63 water immersion objective. Scans were collected in a format of 5 12 x5 12 pixels with 1024 gradations of intensity. Recombinant tissue plasminogen activator (rtPA, 26nM) (Alteplase, Boerhinger) was loaded at the edge of the labelled PRC. The edge of the clot was visualized with the confocal microscope set up in the reflection mode. Scanning was performed at a magnification 125 ^x 125 μιη every 15 seconds for 30 minutes. The velocity of the lysis front was determined from confocal microscope images and analysed with image J software.

ROTEM^® Modified Rotation Thrombelastogram Analyzer

Citrated PRP was obtained and adjusted at 10⁸ platelets/ml as described above. ROTEM^® analysis was performed in pre-warmed ROTEM^® cup containing 300 μΐ of PRP in presence of control IgG or the blocking anti-PN-1 or/and anti-PAI-1 IgG both at 100 μg/ml. Clotting was initiated by the addition of TF (Innovin 1/5 (v/v)), CaCl₂ (10 mM). Fibrinolysis was initiated by the addition of human r-tPA (0.5 nM) (Alteplase, Boerhinger) or mouse r-tPA (30 nM) (Molecular Innovation). The fibrinolytic response by rtPA was assessed using ROTEM^® software, thereby providing the lysis rate at 60 minutes in each condition. Dorsal skinfold chamber

Dorsal skinfold chambers were implanted in 10- to 12-week-old mice (25 to 30 g body weight) anesthetized by intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine in saline solution as previously described¹⁸. Briefly, a patch of dorsal hair was removed, and two titanium frames were positioned so as to sandwich the extended double layer of skin. One layer of betadine-cleaned skin was completely removed in a circular area of 13 mm in diameter, and the remaining layer, consisting of epidermis, subcutaneous tissue, and striated skin muscle, was covered with a 12-mm glass coverslip incorporated in the frame. Following surgery, mice were injected subcutaneously with buprenorphine (0.05 mg/kg) and then again 8-12 h later. The animals tolerated the chambers well and showed no sign of discomfort. After a 48 h-period of recovery from surgery, preparations fulfilling the criteria of intact microcirculation and showing no signs of inflammation were utilized for thrombosis and thrombolysis experiments.

Real-time intravital imaging of thrombus formation and thrombolysis Mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine and vascular injury was induced by placing a Whatman filter paper strip (1 x 0.5 mm) saturated with 15 % FeCl3 (Sigma) over venules (ranging from 130 to 160 μιη diameter) in dorsal skinfold chambers for 3 minutes. Thrombus formation following vessel injury was examined in real- time by monitoring the accumulation of rhodamine 6G (Sigma) (3 mg/kg mouse)-labelled platelets using an inverted fluorescence microscope (Axio Observer, Carl Zeiss Microimaging GmbH, Germany) with a 5x objective connected to a Hamamatsu Orca-R2 charge-coupled device video camera. Platelet deposition and thrombus growth in injured venules were monitored until vessel occlusion defined as a complete arrest of blood flow for at least 5 minutes. Immediately after vessel occlusion, 20 μΐ of saline containing rtPA (80 μΜ) and hirudin (10 μΜ) (Serbio) were applied topically in the chamber to enhance thrombolysis, and prevent rethrombosis. Thrombolysis was analyzed by measuring the occurrence of recanalization of occluded venules, the time to recanalization, and the decrease in thrombus area at 30 minutes and 1 hour after rtPA treatment. A total of 13 venules in 7 PN1 -/- mice and 13 venules in 7 WT mice were studied. Data acquisition and analysis were done using the Axio vision software (Carl Zeiss Microimaging GmbH, Germany).

Statistical analysis

Results are shown as means ± SEM. Students t test was used for in vitro experiments with recombinant PN-1, in vitro experiments of wild-type and PN-1 -deficient mice, and for lysis front velocity experiments. The one-way ANOVA followed by Dunnett's test was used when comparisons of anti-PN-1 IgG or anti-PAI IgG groups versus Control IgG were performed. A linear mixed-effects model (LME) was used for the analysis of in vivo thrombolysis. A P value less than or equal to 0.05 was considered significant.

Results

PN-1 inhibits plasminogen activation by fibrin-bound tPA

Plasminogen activation by tPA, was measured on a fibrin surface, in the presence or absence of recombinant PN-1. First, tPA was incubated for 1 hour on fibrin-coated plates, the excess of unbound tPA being eliminated. PN-1 was subsequently added to the fibrin-coated plates and the excess discarded. Plasmin generation induced by the residual fibrin-bound tPA was then determined after addition of plasminogen with the chromogenic substrate CBS0065. The initial rate of plasmin generation by tPA decreased by ~ 2fold in the presence of PN-1 : 2.7 ± 0.3 nM and 1.3 ± 0.1 nM plasmin were generated, respectively in the absence and presence of PN-1.

tPA-induced fibrin degradation was measured by fibrin-plasminogen-agar zymography with platelet releasates. Recombinant tPA induces a lysis area reflecting fibrinolytic activity relative to the amount of plasmin converted from plasminogen by tPA. As expected, the fibrin zymography lysis band corresponding to tPA was reduced by recombinant PN-1. No reduction in tPA induced- lysis area was observed after tPA incubation with the supernatant of resting human platelets. In contrast, when tPA was incubated with the secretion products of activated human platelets, the fibrin zymography lysis t-PA-band was barely detected, indicating the secretion of tPA inhibitor(s) by activated platelets. To determine whether PN-1 contributed to fibrinolysis inhibition, zymography experiments were performed in the presence of a PN-1 -blocking antibody. tPA activity was restored in the presence of the anti-PN-1 IgG but not in the presence of an irrelevant IgG. To confirm these findings, the same experiments were performed with platelets from PN-1 -deficient mice and their littermate controls. Incubation of tPA with the secretion products of activated platelets from WT mice resulted in an almost complete inhibition of lysis. On the contrary, the products secreted by platelets from PN-1-/- did not decrease the tP A- induced lysis area. Together, these data demonstrate that PN-1 has the remarkable capacity to inhibit the generation of plasmin induced by tPA bound to fibrin.

PN-1 inhibits fibrin-bound plasmin

Degradation of fibrin by the serine protease, plasmin, is a step in the fibrinolysis process where PN-1 can also play an important role. To test this hypothesis, plasmin activity was measured on a fibrin surface, in the presence or absence of recombinant PN-1. The initial rate of substrate hydrolysis induced by fibrin-bound plasmin decreased by ~10-fold in the presence of PN-1. Fibrin-bound plasmin activity was thus drastically inhibited by PN-1.

Plasmin-induced fibrin degradation was measured by using fibrin-agar zymography. Similarly to the results obtained with tPA, we observed that the secretion products of activated platelets inhibited plasmin-induced lysis. This inhibition was completely prevented by the blocking anti-PN-1 antibody. Fibrin-agar zymography was also performed with platelets from PN-1 -deficient mice and their littermate controls. Incubation of plasmin with the secretion products of activated platelets from WT mice resulted in an almost complete inhibition of lysis. On the contrary, the products secreted by platelets from PN-1-/- mice did not reduce plasmin- induced lysis area. Our results thus demonstrate that PN-1 secreted by activated platelets is able to inhibit the fibrinolysis induced by fibrin-bound plasmin

Platelet PN-1 limits PRC lysis

To test the functional effect of PN-1 on endogenous clot lysis, human platelet-rich plasma (PRP) containing FITC-fibrinogen, was preincubated with a control IgG or the blocking anti-PN-1 IgG before clotting. Fibrinolysis was then assessed by clot weight loss and fluorescence release from the clot after 24 hours at 37°C. In the presence of a control IgG, clot weight loss was 7 ± 1 mg, whereas preincubation with the anti-PN-1 IgG resulted in a large increase in clot weight loss, reaching 27 ± 2 mg. A blocking anti-PAI-1 IgG also enhanced clot weight loss by 17 ± 5 mg, although this increase was not statistically significant. The combination of both blocking antibodies resulted in a large increase in clot weight loss, reaching 46 ± 10 mg. The percentage of FITC released from the clots was also significantly higher in the presence of the anti-PN-1 IgG (37 ± 2 %) than in the presence of an irrelevant IgG (26 ± 1 %). The same experiments were performed with PRP from WT and PN-1 -deficient mice. Clot weight loss was greater for fibrinolysis with PN-1-/- clots (55 ± 6 mg) than with WT clots (31 ± 2 mg), and the percentage of released fluorescence was higher for PN-1 -/- (89 ± 3 %) than for WT clots (64 ± 3 %). Together, these results show that, in the absence of PN-1, endogenous tPA-induced clot lysis is enhanced within 24 hours, indicating that platelet PN-1 is a regulator of endogenous clot lysis.

The effect of PN-1 inhibition or PN-1 -deficiency on clot lysis was further investigated using a ROTEM^® analyser. An exogenous supplement of a subthreshold lytic concentration of tPA (0.5 nM) was used to induce clot lysis. The percentage of tPA-induced clot lysis was minimal in the presence of a control IgG, reaching 16 ± 2%, while it was greatly increased in presence of the anti-PN-1 IgG, reaching 42 ± 5%. A blocking anti-PAI-1 IgG also has an increased tendency for clot lysis by 28 ± 5 %, although it was statistically insignificant. The combination of both blocking antibodies resulted in an almost complete clot lysis at 60 minutes (91 ± 1 % of lysis). To substantiate these results, experiments were also performed using mouse platelets. The ROTEM^® tracing showed that a subthreshold concentration of tPA induced lysis of WT clots by 56 ± 7 % whereas lysis of PN-1 -deficient clots was almost complete (84 ± 8 %) under our experimental conditions. These results indicate that both PN-1 and PAI-1 released by activated platelets contribute to inhibit tPA-induced clot lysis.

Platelet PN-1 reduces the velocity of clot lysis We visualized the lysis front of PRC by using laser scanning confocal microscopy. Addition of tPA at the edge of the microchambers of PRC initiated lysis with a straight and sharp front moving across the entire fibrin surface. A significant increase in the lysis front velocity was observed in the presence of the blocking anti-PN-1 IgG with an average rate of 22.5 ± 2.8 μηι/ηιήηιΐε compared to the control IgG 1 1.8 ± 1.6 μηι/ηιήηιΐε , (P< 0.01 n=5 ). To confirm these findings, the same experiments were performed with clots from PN-1 -deficient mice and their littermate controls. As observed with human clots, addition of tPA resulted in an acceleration of the lysis front in PN-1 -deficient clots with a rate of 16.0 ± 1.5 μηι/ηώηιΐε versus 10.3 ± 0.9 μΓη/ηώηιΐε with the WT clots (P< 0.05 n=5 ). tPA-induced thrombolysis is enhanced in PN 1-/- mice

To determine whether the antifibrino lytic effect of PN-1 is of in vivo relevance, we have developed in mice, a method in which thrombolysis can be measured by intravital microscopy using the dorsal skinfold chamber model. We compared the efficiency of tPA- induced thrombolysis in WT and PN-1 -/- mice. Topical application of FeCl3 over venules ranging from 130 to 160 μιη in diameter was used to induce vascular injury leading to occlusive thrombosis. While there was no significant difference in the occlusive thrombus size/area between WT and PN-1 -/- mice (34163μιη² ± 5459 μιη² vs 31656 ± 4709μιη², n = 13 vessels from 7 mice per group), the time to reach complete occlusion was significantly reduced in PN-1 -/- mice compared to WT mice, in agreement with data obtained in the mesenteric vessel thrombosis model ¹⁰. During the 24 hours following complete arrest of blood flow, spontaneous recanalization was observed in only 2 of 13 vessels out of 7 PN 1-/- mice and in none of the 13 occluded vessels from WT mice. This indicates that spontaneous thrombolysis following FeCl3 injury is a slow process in both WT and PN1-/- mice. In order to accelerate thrombolysis, tPA was directly added to the chamber 5 minutes following complete vessel occlusion. Hirudin was simultaneously added to prevent rethrombosis. In WT mice, the mean time to recanalization following tPA treatment was superior to 1 hour while it was of 13 ± 2 minutes in PN-1 -/- mice (n= 7 mice). Furthermore, 1 hour after tPA treatment, the incidence of recanalization was 15 % (2 of 13 vessels) in WT mice and reached 92 % (12 of 13 vessels) in PN1 -/- mice. Thirty minutes after tPA treatment, thrombus size remained unchanged in WT mice (101.6 ± 7.2 % of initial size) whereas it was significantly reduced in PN-1 -/- mice (56.1 ± 8.5 % of initial size). At 1 hour post-tPA treatment, the thrombus size was reduced in WT but this reduction was less important than in PN-1 -/- mice (76.7 ± 6.3 % vs 42.8 ± 9.5 % of initial size). Altogether, these results confirm that PN1 is a potent inhibitor of tPA-induced thrombolysis in vivo.

After the thrombolysis experiments, mice were kept under observation for 24 hours and euthanized. Four hours after tPA treatment, all vessels occluded by FeCl₃ injury were recanalized in both WT and PN1 -/- mice. Interestingly, all PN-1 -deficient mice (7 out of 7) remained healthy the day following thrombolytic treatment, whereas 71% (5 out of 7) of WT mice were apathetic and showed signs of respiratory distress.

Discussion: In humans, platelet PAI-1 released locally after platelet activation is assumed to be a major contributor to the stabilization of the thrombus by inhibiting endogenous fibrinolysis ^{5 6}. However, PAI-1 -independent mechanisms have also been proposed to contribute to platelet-dependent inhibition of fibrinolysis ¹. The existence of other non-PAI-1 proteinases inhibitors able to reduce plasminogen activation and/or plasmin activity has previously been suggested ¹⁹. Our study also suggests a less important role for PAI- and reveals that an additional serpin plays an important role in inhibiting plasminogen activators and plasmin. Indeed, we show here for the first time that PN-1, which can accumulate at the sites of vascular injury due to its presence in platelets ¹⁰, is an important player in the control of fibrinolysis. The fact that PN-1 can down-regulate both plasmin generation and plasmin activity on the fibrin matrix highlights the potential influence of PN-1 on fibrinolysis. Indeed, the fibrin matrix is largely recognized as an essential actor in the fibrinolysis process. It is well known that tPA-mediated plasminogen activation is dependent on fibrin, which restricts fibrinolysis to the site of thrombus ²⁰. Importantly, when bound to fibrin, tPA is protected from inhibition by PAI-1 ²¹ ' ²². The inhibition of tPA by PAI-1 is decreased by 80-90 percent in the presence of fibrin, because PAI-1 has no access to the catalytic domain of fibrin-bound tPA ²³. Moreover, the rate of inactivation of plasmin by a2-antiplasmin slows down very significantly when plasmin is bound to fibrin ²⁴. Thus, whereas serine proteases of the fibrin- bound plasminergic system are "protected" from their principal inhibitors, platelet PN-1 appears to be one inhibitor capable of blocking them in situ. The blocking PAI-1 antibody alone led to a non significant increase in clot lysis, in agreement with previous data demonstrating that PAI-1 -deficiency induced only mild hyperfibrino lysis ¹⁹. This suggests that PAI-1 alone is not sufficient in regulating the lysis of platelet-rich clots. The higher fibrinolytic capacity observed in the presence of both PN-1 and PAI-1 blocking antibodies supports a synergic involvement of both proteins in the regulation of clot lysis. Moreover, platelet PN-1 can influence the lysis of fibrin clots generated spontaneously from PRP, without any exogenous tPA, but also after addition of recombinant tPA, indicating that PN-1 is inhibitory not only on endogenous but also on exogenous tPA-mediated lysis. These points are of clinical relevance: first, because endogenous fibrinolysis is known to play a pivotal role in the evolution of thrombotic cardiovascular diseases and second, because this may relate to the failure of optimal reperfusion in approximately one half of STEMI patients who are treated with fibrinolytic agents. A polymorphism in PN-1 could possibly explain the heterogeneity in the therapeutic efficacy of thrombolytic agents. Moreover, the fact that the lysis front moves faster when the PRC is devoid of PN-1, may imply that PRCs are refractory to tPA-induced lysis in a PN-1 -dependent manner and that platelet PN-1 may have a critical impact at the level of fibers in the fibrin clot. Further experiments are needed to clarify this potential implication of platelet PN-1 on clot structure.

PN-1 appears to be a particularly important actor both in the development and in the dissolution of a thrombus. Indeed, PN-1 is involved in thrombus generation and extension by its capacity to inhibit thrombin-mediated fibrin formation and platelet activation ¹⁰, and we demonstrate here that PN-1 is also involved in thrombolysis by its capacity to inhibit the local generation and activity of plasmin. Because of these opposing effects, it was of great interest to analyze the effect of PN-1 -deficiency in the process of thrombus dissolution, in vivo. For this purpose, we have developed an original murine model of in vivo thrombolysis associating ferric chloride injury and the dorsal skinfold chamber model. This approach is a reproducible method to quantify thrombus formation and lysis induced by a topical application of tPA. This device has the great advantage of allowing direct visualization, via intravital video- microscopy, of thrombus formation but also, which is the originality of our model, of thrombus lysis in living animals. We observed that tPA-triggered PRCs are more readily lysed in PN-1 -deficient mice than in WT mice, with both the rate and the extent of recanalization being increased in PN-1-/- mice. Our data thus demonstrate the important role of PN-1 in mediating the resistance of PRC to lysis. We also observed that WT mice poorly survived thrombolysis and exhibited a global organ failure syndrome, in contrast to PN-1 -deficient mice which supported well the procedure without exhibiting any clinical manifestations.

The fact that platelet PN-1 is so important to protect the developing thrombus from premature lysis may explain the reason why the role of PAI-1 in thrombolysis resistance is a subject of controversy. Indeed, none of the previous investigations studying PAI-1 role in thrombolysis failure took into account the contribution of PN-1. We suggest here that endogenous PN-1 can play an important role in the failure of thrombolytic therapy to restore arterial blood flow. Clearly, our findings should be considered in the design of new therapeutic strategies, which should include the inhibition of PN-1 by antibodies or synthetic compounds to improve the therapeutic efficacy of thrombolytic agents.

Mechanical desobstruction of the occluded coronary artery by primary angioplasty has become the gold standard method for myocardial reperfusion in patients with ST-elevation myocardial infarction (STEMI). Indeed, early coronary reocclusion or failure to restore coronary patency after thrombolysis is a major limitation leading to frequent percutaneous coronary intervention. As a consequence, thrombolysis has become the first line reperfusion therapy in early STEMI presenters (<3 hours) who cannot be fast transferred to the catheterization laboratory (<2hours). Plasminogen Activator Inhibitor of type-1 (PAI-1) is assumed to play a role in thrombolysis failure. The data reported in the present study demonstrate for the first time that another protein present in platelet, protease nexin-1 (PN-1), which is known to inhibit significantly uPA, tPA and plasmin, possesses efficient antifibrino lytic properties. Platelet PN-1 is shown to inhibit both the generation of plasmin by fibrin-bound tPA but also the activity of fibrin-bound plasmin itself. The remarkable protective effects of PNl toward premature lysis of the developing thrombus may thus represent an unknown and underestimated mechanism of thrombolysis regulation in vivo. PN- 1 provides a further dimension in our understanding of the regulation of the plasminergic system and opens an interesting perspective for future clinical investigations looking at pharmacological thrombolysis. PN-1 thus represents a target to improve the therapeutic applications of thrombolytic agents. REFERENCES:

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Claims

CLAIMS:

1. An inhibitor of protease nexin-1 (PN-1) for use in a method for promoting fibrinolysis in a subject in need thereof.

2. The inhibitor of PN-1 for use according to claim 1 wherein said fibrinolysis is a t-PA- mediated fibrinolysis.

3. An inhibitor of protease nexin-1 (PN-1 ) for use in a method for promoting thrombolysis in a subject in need thereof.

4. The inhibitor of PN-1 for use according to any of claims 1 to 3 wherein said subject is afflicted with an ischemic condition selected from the group consisting of renal ischemia, retinal ischemia, brain ischemia and myocardial ischemia.

5. The inhibitor of PN-1 for use according to claim 4 wherein said subject has been diagnosed with a coronary disorder.

6. The inhibitor of PN-1 for use according to claim 5 wherein said subject has been diagnosed a ST segment elevation myocardial infarction.

7. The inhibitor of PN-1 for use according to any of claims 1 to 6 wherein said inhibitor is selected from the group consisting of small organic molecules, antibodies, aptamers or polypeptides.

8. An inhibitor of protease nexin-1 (PN-1) for use in a method for enhancing therapeutic efficacy of a thrombolytic agent.

9. The inhibitor of PN-1 for use according to claim 8 wherein said thrombolyic agents is selected from the group consisting of reteplase (r-PA or Retavase), alteplase (t-PA or Activase), urokinase (Abbokinase), prourokinase, anisoylated purified streptokinase activator complex (APSAC), and streptokinase.

10. A combination of an inhibitor of PN-1 and a thrombolytic agent for use in a method for treating an ischemic condition in a subject in need thereof.

11. The combination according to claim 10 which further comprises an inhibitor of plasminogen activator inhibitor type-1.