KITS FOR INDENTIFYING AND TREATING SUBJECTS RESISTANT TO ACETYL SALICYLIC ACID

Abstract

In alternative embodiments, the invention provides compositions and methods for overcoming or diminishing or preventing Growth Factor Inhibitor resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor inhibitor, comprising for example, administration of a combination of a TBK1 inhibitor and an RTK inhibitor. In alternative embodiments, the cell is a tumor cell, a cancer cell or a dysfunctional cell. In alternative embodiments, the invention provides compositions and methods for determining: whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or, which individuals or patients would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor and at least one compound, composition or formulation used to practice a method of the invention, such as an NFKB inhibitor, such as a lenalidomide or a REVIAMID, or IKK inhibitor; or an inhibitor of Galectin-3.

Claims

1. A kit for detecting in vitro ASA resistance in a subject by detection of a subject whose platelets show high levels of Multidrug Resistance Protein-4 (MRP4) expression as compared to levels of MRP4 expression found in normal aspirin-responding individuals, comprising reagents for measuring or quantifying levels of expressed MRP4 channel protein.

2. The kit of claim 1, wherein the reagents comprise primer oligonucleotides for detecting the presence of and quantifying mRNA for MRP4 channel protein, and optionally further comprising reagents for processing a sample comprising the subject's platelets for detecting the presence of and quantifying mRNA.

3. The kit of claim 1, wherein the reagents comprise a reagent or reagents for a polymerase chain reaction (PCR) or a Real-Time PCR (RT-PCR).

4. The kit of claim 1, wherein the reagents comprise reagents for carrying out a Western Blot assay, and optionally further comprising reagents for processing a sample comprising the subject's platelets for a Western Blot assay.

5. The kit of claim 4, wherein the reagents for carrying out a Western Blot assay comprise monoclonal or polyclonal antibodies that specifically bind to MRP4 channel protein.

6. The kit of claim 1, further comprising a standard preparation of MRP4 channel protein at known concentration.

7. The kit of claim 1, further comprising primer oligonucleotides for detecting mRNA for the protein GADPH.

8. The kit of claim 1, further comprising reagents and materials for carrying out platelet separation from other corpuscolated bodies present in a biological fluid and for quantifying non-platelet contamination in a separated platelets preparation.

9. The kit of claim 1, further comprising instructions for use, wherein the instructions comprise detecting the presence of and quantifying levels of MRP4 channel protein in a sample comprising the subject's platelets.

10. The kit of claim 9, wherein the instruction comprise: (i) taking a sample of biological fluid or platelet from the subject, (ii) measuring the amount of Multidrug Resistance Protein-4 (MRP4) protein or messenger RNA (mRNA) in a platelet-comprising biological fluid or a platelet sample, (iii) comparing the amount of MRP4 protein or MRP4 mRNA in the sample to the amount of MRP4 protein or MRP4 mRNA present in one or a plurality of normal subject controls, and (iv) selecting the subject as an acetylsalicalic acid (ASA)-resistant subject if the amount of MRP4 protein or MRP4 mRNA present in the subject is greater than the amount of MRP4 protein or MRP4 mRNA present in the one or a plurality of normal subject controls.

11. The kit of claim 4, wherein the instruction comprise measuring the amount of MRP4 protein by means of a Western Blot by making a comparison with a control standard comprising an MRP4 protein.

12. The kit of claim 10, wherein the subject is selected as an acetylsalicalic acid (ASA)-resistant subject if the amount of MRP4 protein present in the subject is greater than 10% the amount of MRP4 protein in the one or a plurality of normal subject controls.

13. The kit of claim 12, wherein the subject is selected as an acetylsalicalic acid (ASA)-resistant subject if the amount of MRP4 protein present in the subject is greater than 20% the amount of MRP4 protein present in the one or a plurality of normal subject controls.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 and FIG. 2 graphically present the results obtained which are representative of the elimination of ASA from platelets by means of the MRP4s, achieved by measuring the concentrations of ASA and its metabolite salicylic acid in the cytosol (FIG. 1) and in the platelet granules (FIG. 2) which had been incubated with aspirin after different treatments, such as inhibitors of the transport mediated by the MRP4s (dipyridamole and MK-571), or systems that reduced the active transport of such channels (incubation at 4 C. or with ATP scavengers) and recording the percentage obtained as compared to platelets or granules treated with ASA alone.

[0037] FIG. 2 graphically present the results obtained which are representative of the elimination of ASA from platelets by means of the MRP4s, achieved by measuring the concentrations of ASA and its metabolite salicylic acid in platelet granules.

[0038] FIG. 3 illustrates the release of .sup.14C-labelled ASA after activation of platelets radiolabelled with thrombin.

[0039] FIG. 4 illustrates the MRP4 expression in a platelet protein lysate of healthy volunteers as compared to patients undergoing bypass surgery at 5 days after the operation (upper panels). In lower panels there are reported control patterns obtained using an anti.actin monoclonal antibody.

[0040] FIG. 5A illustrates electron microscopy images from experiments in healthy patients conducted using the immunogold technique, which reveals the location of the channel by means of electron microscopy.

[0041] FIG. 5B illustrates electron microscopy images from experiments in resistant patients conducted using the immunogold technique, which reveals the location of the channel by means of electron microscopy.

[0042] Like reference symbols in the various drawings indicate like elements.

[0043] Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.

DETAILED DESCRIPTION

[0044] Provided are combinations of acetylsalicylic acid, its derivatives and physiologically acceptable salts, and MRP4 channel inhibitors, formulable in co-administration, is used for the treatment of ASA-resistant patients, as defined above.

[0045] The medicament according to the present invention can be obtained by mixing the active ingredients or their corresponding derivatives and pharmaceutically acceptable salts with excipients suitable for compositions for oral or parenteral administration, or for intramuscular, venous, per os or nasal administration. All the excipients, diluents, vehicles and/or adjuvants necessary for the preparation are ones with which the expert in the field will be familiar.

[0046] More specifically, what is meant by the term composition is a combination of active ingredients that is capable of producing a pharmacological effect as indicated above. The composition includes-in addition to the active ingredients-excipients, diluents, vehicles and/or adjuvants which alone are not capable of producing a pharmacological effect on the subject receiving the composition. These excipients, diluents, vehicles and adjuvants are well known to the experts in the field and can be selected from among the group of substances such as water, saline solution, glycerol, ethanol, emulsifying agents, buffer substances, preservatives and the like.

[0047] Examples of pharmaceutical compositions which may comprise the combination of active ingredients according to the invention are those in solid, semisolid or liquid form, in the form of lozenges, capsules, pills, tablets, pastilles, granulates, syrups, ampoules, drops, solutions, suspensions, emulsions, in unitary dosage forms or in separate doses, ready-to-use syrups or ex tempore units, creams and gels. Other examples of formulations are those for parenteral use, injectable forms for intramuscular, subcutaneous, transdermal or intravenous administration.

[0048] The compositions can be administered per os or parenterally, rectally or cutaneously.

[0049] Co-administration means a pack containing unitary or separate administration forms of the active ingredients, accompanied by the instructions for simultaneous coordinated administration or administration in succession of the active ingredients according to the dosage prescribed by the physician.

[0050] Administration doses, posologies and protocols are decided by the physician on the basis of his or her own experience, the severity of the disease and the patient's general condition and age.

[0051] Patients manifesting ASA resistance according to the present invention present a higher platelet expression of MRP4 compared to healthy controls (FIG. 4).

[0052] A diagnostic method for the in-vitro detection of ASA-resistant subjects is claimed in claim 1. A way to carry out the method according to the present invention comprises the following steps:

[0053] a. Take a sample of biological fluid from the patient, generally purified venous peripheral blood, from which a platelet protein lysate is obtained which, after purification, i.e. through electrophoresis, is put in contact with anti-MRP4 mono- or polyclonal antibodies (available on the market) in conditions and for a time period sufficient for the formation of a complex between antigen (MRP4) and antibody;

[0054] b. Quantify the presence of the characteristic band of the MRP4s, the identification can be done by means of Western Blot by making a comparison with a control standard made up of the MRP4 protein.

[0055] The Western Blot technique is well known to the expert in the field, who, on the basis of the present description and his or her knowledge, can easily identify the optimal conditions for implementing the method.

[0056] Experts in the field are familiar with the definitions of polyclonal antibody and monoclonal antibody.

[0057] Another mode of identification is based on the study of platelet messenger RNA responsible for the synthesis of this protein (MRP4), which is greater in resistant subjects than in healthy subjects. In this case the determination can be done using the Southern Blot technique or Real-Time PCR (RT-PCR), both of which are well known to the expert in the field, who will easily identify the necessary restriction enzymes and the subsequent conditions for electrophoresis.

[0058] A detection kit according to the present invention may advantageously contain, in addition to the material for performing the assay (assay test tubes, titration plate wells, reagents, etc.) and the explanations relating to the method of use, anti-MRP4 antibodies for an evaluation assay by means of Western Blot; or specific restriction enzymes for the MRP4s (particularly profitable the TaqMan Gene Expression Assay kit) to be quantitatively analyzed to determine platelet mRNA by Southern Blot or Real-Time PCR.

[0059] The kit can suitably further comprise: [0060] a system for the purification of platelets from the other granular blood components (Platelet GelSep of BioCytex for purifications carried out with centrifugation methods on gel); and [0061] Reagents capable of quantifying the interferences due to non platelet components in the cellular preparation to be used in diagnosis, such as monoclonal antibodies or anti CD45 restriction enzymes which reveal the presence of leucocytes.

[0062] As for the evaluation of the results that can be obtained with the method of the restriction enzymes, we can hypothesize that values higher than 20 UA (Arbitrare Units), preferably higher than 25 UA, more preferably higher than 30 UA, and even more preferably higher than 35 UA of threshold cycles of MRP4/GAPDH are values that identify resistant patients. Considering the pre-analytic and analytic variabilities of the lab data obtained with the kit for mRNA, it is recommended to create your own normal values, that can be obtained after analyzing at least 15 healthy volunteers. As for the kit with western blot, we suggest the acquisition of data from resistant patients together with those of healthy volunteers. So, it is better add to the kit a standard preparation of MRP4 with a known value and the patient will be considered positive with a proteic densitometry higher than 10%, preferably 20%, better 35% and much better 50% compared to the standard.

[0063] Through the method of analysis of the invention and the corresponding kit, it is possible to identify and monitor the subjects whose platelets show high levels of MRP4, this is of fundamental importance in reducing the level of therapeutic failures linked to aspirin chronic treatment. The kit of the invention advantageously allows to identify both patients ASA resistant and subjects who show such resistance because of genetic causes.

[0064] On the basis of the hypotheses advanced, the inventors have experimentally demonstrated that ASA can be conveyed via MRP4 channels and in patients with increased platelet expression of this protein, a reduced capability of ASA in reducing the platelet functionality is present and as a consequence inhibition of MRP4-mediated transport increases the therapeutic capacity induced by ASA.

[0065] The fact that ASA can be transported into the dense granules or extruded from the cell via the MRP4s is demonstrated by the following experimental results:

[0066] 1) inhibition of MRP4-mediated transport increases the ability of platelets to retain ASA in the cytosol; in fact, if the platelets are pretreated with dipyridamole or MK-571 10 minutes before incubation of the platelets with ASA 50 microM, the cytosolic concentrations of acetylsalicylic acid (ASA) or of its metabolite salicylic acid are 285% and 293% greater, respectively, in the platelets pretreated with dipyridamole 100 microM, whereas they are 208% and 202%, respectively, in the platelets treated with MK-571 as compared to platelets treated with the solvent alone both of dipyridamole and of MK-571 (DMSO, dimethylsulphoxide) (see FIG. 1). Dipyridamole, in addition to inhibiting the transport mediated by these channels, is also an inhibitor of platelet activity, in that it increases the platelet concentrations of AMPc, inhibiting phosphodiesterase isoform V (PDE typeV). The inventors have demonstrated that the action of dipyridamole in inhibiting MRP4-mediated transport does not occur via inhibition of PDE type V, in that another inhibitor of this enzyme, 1-(3-chlorophenylamino)-4-phenylphthalazine (MY-5445), molecular formula C.sub.2OH.sub.14ClN.sub.3, does not increase the cytosolic concentrations of ASA or salicylate (110% and 120%, respectively) (see FIG. 1);

[0067] 2) treatment of the platelets with ASA at a temperature below body temperature (4 C.) increases the cytosolic concentrations of ASA and salicylate in the platelets by 240% and 160%, respectively: this demonstrates that the MRP4-mediated transport of ASA is efficient at body temperature (approximately 37 C.) (see FIG. 1);

[0068] 3) ASA is internalized within the platelet granules via the MRP4s; this effect was demonstrated by assaying ASA in the granules after these organelles had been treated with ASA; that the transport can also occur via the MRP4s is demonstrated by the fact that it is necessary that the transport should occur at 37 C. and in the presence of ATP in the external medium, in that MRP4-mediated transport is an active transport. In fact, the maximum concentration reached was obtained if ATP was added to the external medium and the incubation with ASA was done at 37 C., whereas it was substantially reduced if the incubation of ASA was done at 4 C. (53.6%), in the presence of apyrase (56.1%), an enzyme which degrades ATP, or if the platelets were pretreated with dipyridamole (41.7%) 10 minutes prior to the addition of ASA (see FIG. 2);

[0069] 4) The platelets release ASA after activation with thrombin, and this release is absent if pretreatment with dipyridamole is performed. On incubating the platelets with radiolabelled ASA we were able to verify that if this platelet preparation was activated with thrombin, it was possible to assay the radiolabelled substance in the external medium; this effect is due to the fact that, after activation, the platelets release the contents of the granules to the outside of the cells, such as ADP, ATP, Ca2+, etc. The demonstration that ASA enters the granules is provided by the fact that, in the case of pretreatment of the platelets with dipyridamole, the release of radiolabelled ASA was absent, whereas the release of the other granular components was entirely normal (1.80.3 nmoles of ATP released by the platelets pretreated with dipyridamole as against 1.90.1 nmoles of ATP released by the control platelets) (see FIG. 3);

[0070] 5) in platelets from megakarocytes in culture in which expression of the channel was suppressed by blocking RNA transcription by means of specific siRNA for MRP4, the platelets treated with ASA presented intracellular ASA and salicylate values which were 159% and 144% higher, respectively, than those of a platelet population that was similar but treated with the vehicle alone (lipofectamine) and incubated with the same concentration and for the same time with ASA.

[0071] Another important conclusion which the inventors arrived at through the experiments conducted is that by reducing MRP4-mediated transport it proves possible to enhance the action of ASA in inhibiting COX-1. In fact, when dipyridamole or MK-571 is added prior to treatment with ASA, the production of thromboxane, a prostaglandin produced by platelets as a result of activation of COX-1, induced by thrombin, is less than that of platelets which are aspirinated but not pretreated with MRP4-mediated transport inhibitors. The thrombin-induced production of thromboxane of the aspirinated platelets was 343.7 ng/10.sup.8 cells, whereas, if the MRP4-mediated transport was inhibited by means of dipyridamole or MK-571, thromboxane production was down to 142.7 ng/10.sup.8 cells, a 58% reduction, and to 140.22 ng/10.sup.8 cells, a 59.2% reduction, respectively.

[0072] The finding that platelets from megakaryocytes in culture, in which expression of the channel had been suppressed by treatment with siRNA, if aspirinated, presented a 51% reduced production of thromboxane as compared to platelets from megakaryocytes in culture treated with the siRNA vehicle alone clearly demonstrates that, if the platelets do not present this channel, aspirin inhibits COX-1 more effectively.

[0073] The correlation between ASA resistance and MRP4 is also evident from the fact that patients who appear to be more resistant to the action of ASA, such as, for example, those undergoing bypass surgery, diabetics and patients on chronic treatment with ASA, have a different platelet MRP4 expression as compared to healthy volunteers (FIG. 4). In fact, in all the tested patients a band related to MRP4 is observed (Jedlitschky et al. Blood 2004, 104, 3603-3610) which is densitometrically higher when compared with healthy controls.

[0074] We can hypothesize that in the platelets of resistant patients there is a greater expression of the channel. This hypothesis is confirmed by the experiments conducted using the immunogold technique (FIGS. 5A and 5B), where we saw that resistant patients present an increased platelet concentration of the channel with respect to healthy volunteers. As additional confirmation of the fact that the patients that are less sensitive to the action of aspirin in reducing the platelet response, we have verified for them that the platelet MRP4 genic expression, calculated by quantifying the specific mRNA, is increased by 214% in platelets from patients subjected to bypass as compared with platelets from healthy volunteers.

[0075] Finally we have demonstrated that by reducing the MRP4 mediated transport in platelets obtained from highly ASA resistant patients, an increase in the therapeutic efficacy of aspirin is obtained.

[0076] n fact, in platelets obtained from patients subjected to aortho coronaric bypass and pretreated in-vitro with dipirydamole before aspirin, the thromboxane production induced by thrombin was 35% with respect to the platelets treated with aspirin alone (499.8247.7 ng/10.sup.8 cells versus 1437.0549.1 ng/10.sup.8 cells).

[0077] To evaluate the MRP4-mRNA platelet values capable of indicating the patients resistant to aspirin, dosages in healthy volunteers and patients were performed at 5 days from bypass operation and we verified that the healthy volunteers showed values of 21.112.8 EM 3.2 mean 23.2 min 4.1 Mx 37.5 UA of threshold cycles of MRP4/GAPDH. It can be hypothesize that values higher than 20 UA, preferably higher than 25 UA, more preferably higher than 30 UA, much more preferably higher than 35 UA are values that identify resistant patients. Considering the pre-analytic and analytic variabilities of the lab data obtained with the kit for mRNA, it is recommended to create your own normal values, that can be obtained after analyzing at least 15 healthy volunteers. As for the kit with western blot, our data demonstrated that resistant patients showed a protein expression higher than 20% (densitometry carried out with the computer program ImageJ, NIH, Bethesda, USA) in comparison with the controls of the healthy volunteers and patients studied at the same time.

[0078] e can hypothesize that the higher expression of these channels reduces the pharmacological effects of aspirin in platelets in that it reduces its intacellular entrapment.

[0079] It is for this reason that the identification of patients whose platelets show high levels of MRP4 is of fundamental importance in reducing the level of therapeutic failures linked to aspirin chronic treatment.

[0080] The final conclusion is that ASA can be eliminated from platelets by means of MRP4-mediated transport: greater expression of these channels would therefore be responsible for the reduced cellular accumulation of the drug with a consequent reduction of the pharmacological effect. An illustrative but by no means exhaustive list of MRP4s, includes, in addition to dipyridamole [2,6-bis-(diethanolamino)-4,8-dipiperidino-(5,4-d)-pyrimidine] and mopidamole [2,6-bis-(diethanolamino)-8-piperidino-(5,4-d)-pyrimidine]: MK-571; pyrimido-pyrimidine derivatives such as those described in U.S. Pat. No. 4,694,024; purine analogues suc as the 6-oxo-purines and pyrazol[3,4-d] pirimidines (described in J. Comb. Chem 2007, 9, 210-218); quinoline derivatives, such as mefloquine, amodiaquine, chloroquine, primaquine, quinidine and quinine, cilostazol and quinolinone derivatives, polyphenols, particularly those of a natural origin and those of a synthetic one, such as those described in FEBS J. September 2005, 272 (18): 4725-4740; diclofenac, celecoxib, d1-buthionine-(S,R)-sulphoximine (BSO), probenecide, S-(2,4-dinitro-phenyl)-glutathione (DNP-SG), N-acetyl-(2,4-dinitrophenyl) cysteine (NAc-DNP-Cys), -naphthyl--D-gluco-ronide, p-nitrophenyl--D-glucoronide, flurbiprofen, ibuprofen, indomethacin, indoprofen, ketoprofen, diclofenac, celecoxib, rofecoxib, disulphiram; corresponding derivatives and physiologically acceptable salts of said compounds, and mixtures thereof.

[0081] They all exert the action of reducing MRP4-mediated transport, thus enhancing the inhibitory effect of ASA on platelet COX-1.

[0082] The concentration of dipyridamole necessary in in-vitro studies is high, but could be lower in vivo and range from 75 to 500 mg, preferably 75 to 200 mg.

[0083] This new mechanism of action of dipyridamole or other inhibitors of MRP4-mediated transport may offer various advantages, such as reducing the effect referred to as ASA resistance, thus enhancing the therapeutic efficacy of this drug in the diseases described above; it may also offer the major advantage of being able to reduce the pharmacologically active doses in all diseases requiring chronic treatment, thus reducing the complications of ASA treatment, and above all its gastrolesivity.

[0084] The new-generation molecules which will be capable of reducing MRP4-mediated transport, in addition to reducing the phenomenon of resistance to chemotherapy agents and antiviral agents in oncological diseases, will also offer the advantage, as demonstrated by the inventors, of improving chronic aspirin treatment in all diseases requiring such treatment.

[0085] The advantages deriving from the combined use of treatment with ASA plus inhibitors of MRP4-mediated transport are the following:

[0086] 1) reduction of complications in cardio- and cerebrovascular diseases, obliterating arteriopathies of the peripheral vessels, retinopathy, atrial fibrillation, deep vein thrombosis, cardiac rhythm diseases, thrombocytosis, gestosis of pregnancy, recurrent miscarriages, and ASA treatment of patients with a family history of various types of tumours, such as colon cancer, to reduce the incidence of these diseases;

[0087] 2) Reduction of cardio- and cerebrovascular accidents (therapeutic failure) in patients on treatment with ASA alone in primary prevention (patients at high risk of atherothrombotic diseases); in secondary prevention (patients previously suffering a thrombotic event);

[0088] 3) reduction of the daily dose of ASA with a reduction of side effects;

[0089] 4) possibility of administering ASA to patients who cannot take large amounts of it, due to gastric sensitivity to the drug, but who need treatment for lengthy periods, such as patients affected by chronic inflammatory diseases;

[0090] 5) possibility of administering ASA at a reduced dose for short periods and thus of reducing the risk of NSAID-induced gastric damage.

[0091] The following examples are given to illustrate the invention and must not be considered as limitative of its scope.

Selection of the Patients

[0092] The following patients were enrolled for the study:

[0093] healthy volunteers who had declared they had not taken any drug in the 15 days before the blood tests;

[0094] patients under chronic aspirin therapy (100-160 mg/die) for at least two months.

[0095] patients who suffered from diabetes mellitus type II, that had been diagnosed according to the International Society of Diabetes guidelines (Diagnosis and classification of diabetes mellitus. Diabetes Care 2006; 29 Suppl 1:S43-8);

[0096] patients at 5 days from an aortic-coronary-bypass.

Exclusion Criteria

[0097] The following patients were excluded from the research:

[0098] 1) those who had taken, in the last 15 days, drugs that interfered with the platelet function (steroidal and non-steroidal anti-inflammatory drugs, thienopiridines, anticoagulant agents);

[0099] 2) those who suffered from pathologies that could alter the platelet functionality;

[0100] 3) those who showed a number of leucocytes in the PRP higher than 0.210.sup.3 cells/microl.

Preparation of the Biological Samples; Platelet Rich Plasma (PRP), Platelet Poor Plasma (PPP)

[0101] Venous blood of patients was taken in a test tube with 3,8% sodium citrate (dilution 1:10).

[0102] The blood sample with citrate sodium was centrifugated at 200 g for 15 minutes at room temperature, thus obtaining as supernatant platelet rich plasma (PRP). The PRP was then centrifugated at 200 g for 5 minutes in order to improve the purification from whole blood in the supernatant.

[0103] The PRP was then subjected to platelet count through blood cell count Celltac Auto Nihon Kohden (MEK 8118K). The platelets were then moved away from plasma through centrifugation of PRP, acidified at pH 6.5 with ACD (citric acid, sodium citrate and glucose 1/10 v/v), at 800g for 10 min. and resuspended in Tyrode's buffer (CaCl.sub.2 0.20 g/L, KCl 0.20 g/L, MgCl.sub.2 g/L, MgCl.sub.2 g/L, NaCl 8.00 g/L, NaH.sub.2PO.sub.4*H.sub.2O), at pH 7.35 and charged with glucose and albumin. The preparation of a dense rich platelet granule suspension was made according to the method reported by (Fukami M H et al. J. Cell. Biol. 1978, 77, 389-99) and the degree of purity was verified through mepacrine Fluorimetric probe and the purity percentage was checked through the cytofluorimeter. If the concentration of the dense granules compared to the other organelles was lower than 80%, the suspension of platelet granules was not used for the study.

Treatment of Platelets with Aspirin

[0104] The resuspended platelets in the Tyrode's buffer at the concentration of 2.510.sup.8 cells/ml were treated with aspirin (50 microM) at 10 C. for 15 min and then kept at 37 C. or at 4 C. for 15 min.

Reduction of the MRP4 Mediated Transport

[0105] MRP4 inhibitors were used such as Dipyridamole and MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxo-propyl]-thio]-methyl]thio]-propanoic acid), both at 100 microM and incubated 30 min at RT before aspirin addition.

[0106] In the rich platelet dense granule suspension, ATP and ASA were added to the preparation, then it was kept at 37 C. for 15 min so a to reduce the transpost mediated by MRP4; the suspension was pretreated either with apyrase, an ATP scavenger substance or with Dipyridamole 30 min before adding aspirin, or the treatment with aspirin was made at 4 C.

Measurement of the Cytosolic Concentrations of ASA and of its Metabolite, Salicylic Acid (SA)

[0107] The entrapment of aspirin inside platelets or in the dense granules was measured through the method of Gaspari and Locatelli (Ther Drug Monit. 1987, 9, 243-7).

Platelet Secretion of ASA Marked with (.SUP.14.C), in Thrombin Activated Platelets

[0108] Aspirin labelled with .sub.14C (American Radiolabeled Chemicals, Inc. Saint Louis, USA) was added to the platelet suspension for 15 min at 37 C. After removing the excess of the substance marked by double centrifugation, the platelets were activated with Thrombin (0.2 U/ml) and then centrifugated. The reduction of the transport mediated by MRP4 was made through the preincubation of the platelet preparation with Dipyridamole 30 min before aspirin addition. The results are reported by evaluating the percentage of the radioactive present in the supernatant compared to the total radioactivity of the platelet preparation. ATP secretion was analyzed as a control through the luminometric method with commercial kit (Chronolog, Hevertown, Pa., USA).

Evaluation of the Biologic Efficacy of Aspirin

[0109] The evaluation of the biologic efficacy of aspirin in platelets was made by studying Thromboxane A2 production, a specific marker of the cyclooxygenase pathway in platelets. Aspirin and/or Dipyridamole treated platelets (see above) were activated with thrombin 1 U/ml for 1 h at 37 C., and then centrifugated 1 min at 8.000 g. Thromboxane B2, a stable metabolite of TXA.sub.2, was measured in the supernatant through a commercial ELISA kit (Cayman, chemicals Co).

Evaluation of the Platelet Expression of MRP4 Through Western Blot

[0110] The platelet expression of the MRP4 was studied through an electrophoretic technique and the subsequent transfer of protein on membrane through Western Blotting, from a volume of platelet rich plasma (PRP) containing 510.sup.8 platelets.

[0111] After carefully removing the supernatant, two platelet washings were made through suspension in Tyrode's buffer (pH 6.35) and centrifugation at 8000 g for 2 min.

[0112] The platelet pellet was resuspended in 200 l lysis buffer (RIPA buffer) and kept in ice for one hour.

[0113] Then, the Laemly buffer solution 5 and beta-mercaptoethanol 5% were added and the sample was kept at 100 C. for 5 min. After 15 min in ice, the sample was centrifugated at 12000 g for 4 min and the supernatant was then used for the electrophoresis on a polyacrilamide gel at 4-12% containing SDS 1% (SDS-PAGE). A marker containing a mixture of coloured proteins at known molecolar weight were used in order to identify the proteins dimensions.

[0114] Then the proteins were transferred through Western Blot on a cellulose membrane.

[0115] The identification of the protein was made through primary polyclonal antibodies heading against the MRP4 (Santa-Criz and Alexis), and secondary antibodies conjugated with the peroxidase and then treated with ECL (a solution containing a chemoluminescent substrate), for the production of light that can be detected with photographic film.

Evaluation of the MRP4 Platelet Expression Through Immunogold Micrography

[0116] Platelets were fixed with paraformaldehyde (2%) and embedded in 10% gelatin and solidified on ice. After the preparation was frozen in liquid nitrogen and cryosectioned using an Ultracut EM FC (Leica). Ultrathin cryosections were thus treated with polyclonal-antibody anti MRP4 (Alexis) followed by the addition of 10 nm diameter proteinA-colloidal gold conjugates of. After labeling all ultrathin cryosections, the preparations were fixed in 1% glutharaldheyd and stained with a solution of methyl cellulose (2%) and uranyl acetate (0.4%) to observe it under electron-microscopy.

Platelet Preparation from Megakariocytes in Culture Treated with si-mRNA

[0117] Platelets from megakariocytes in culture were prepared according to the method by Guerriero et al (Journal of Cell Science 119, 744-752, 2006).The transfection with siRNA was made according to the method by Dharmacon Research using the SMARTpool product of four siRNAs for MRP4. 3 days after transfection the produced platelets from megakariocytes were isolated through centrifugation, then aspirinated, and then aspirin entrapment and the pharmacological efficacy were measured (see above). Platelets from megakariocytes treated with only vehicle used for the transfection of siRNA were utilized as a control.

Measurement of the Platelet MRP4-mRNA. Concentration

[0118] Platelet DNA prepared with the reversal-PCR method according to the method by Paul el al. (Methods Mol Biol. 2004; 273:435-54) was amplified by using primers anti MRP4 e GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), both obtained from Applied Biosystems, at standard cyclic thermic conditions. In parallel standard cDNA curves have been made for the quantization of the results. The results are reported as expression of the levels of MRP4-mRNA standardized with GAPDH-mRNA. Other mRNA constant platelet standards can also be used.

[0119] A number of embodiments as provided herein have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.