Diagnosis of thromboembolism

Abstract

A kit comprising a predetermined amount of tissue factor and phospholipids in a container; a predetermined amount of the tissue factor, phospholipids and activated protein C in a second container; and a container comprising a test solution; uses of the kit; and methods for predicting the risk of pulmonary thromboembolism.

Description

[0001] DIAGNOSIS OF THROMBOEMBOLIA

[0002] technical field

[0003] The present invention relates to a diagnostic method for predicting the risk of vascular thromboembolism in a patient, in particular a female patient taking estrogens.

[0004] Previous art

[0005] The measurement of endogenous thrombin potential (ETP) is known and, performed under specific conditions, can reflect resistance to activated protein C (APCr).

[0006] However, measurement methods are not standardized between laboratories, and inventors have observed significant variations in results depending on the reagents used, the test procedure, and even the batches. Thus, despite efforts (see, for example, Douxfils et al., Clin Chem Lab Med, 2020) to reduce these numerous variations (development of the nAPCsr ratio), the measurement of ETP and / or APCr is not used as a diagnostic tool, for example, for assessing the risk of vascular thromboembolism.

[0007] Thus, this diagnosis is currently managed by taking into account genetic factors, such as the Factor V mutation, or family history, or is not even performed, forcing the doctor to prescribe estrogen-based treatment blindly, even if it means adapting it after an unfortunate episode of vascular thromboembolism in his patient.

[0008] Given the potentially devastating impact of vascular thromboembolism, the development of a reliable diagnostic tool is a necessity.

[0009] Brief summary of the invention

[0010] A first aspect of the present invention relates to a kit comprising a predetermined quantity of tissue factor and phospholipids in one container, a predetermined quantity of tissue factor, phospholipids and activated protein C in a second container, said predetermined quantity of activated protein C being the quantity which, after contact of said activated protein C with said quantity of tissue factor and phospholipids for one hour and then with a standardization plasma, allows inhibition of a predetermined value of thrombin production by said standardization plasma.

[0011] Preferably, the predetermined value of inhibition is between 85 and 95%, preferably between 88 and 92%, preferably 90%.

[0012] Advantageously, the kit also includes a container containing a reference plasma, preferably from a different origin than the standardization plasma, for the standardization of measurements.

[0013] An associated aspect of the present invention relates to the use of this kit for predicting the risk of vascular thromboembolism in a patient or for determining the risk inherent in a new treatment impacting estrogen receptors.

[0014] An associated aspect of the present invention relates to a method for predicting the risk of vascular thromboembolism in a patient, comprising measuring the thrombin production of a plasma sample of said patient in the presence of a predetermined amount of tissue factor and phospholipids, measuring the thrombin production of a plasma sample of said patient in the presence of a predetermined amount of tissue factor, phospholipids and a predetermined amount of activated protein C, measuring the thrombin production of a standard plasma sample in the presence of a predetermined amount of tissue factor and phospholipids, measuring the thrombin production of a sample of said standard plasma in the presence of a predetermined amount of tissue factor, phospholipids and a predetermined amount of activated protein C,said activated protein C being brought into contact with said tissue factor and phospholipids for a predetermined time and / or at least 45 minutes before being brought into contact with said plasma sample.

[0015] Preferably, in this method, the patient is a woman, advantageously a woman likely to receive hormonal treatment, preferably treatment based on estrogens, estrogen receptor agonists and estrogen receptor modulators, or a woman who is pregnant or likely to become pregnant.

[0016] The method is advantageous when the woman is under treatment for vasomotor problems related to menopause, osteoporosis, vaginal atrophy, endometriosis, polycystic ovary syndrome, uterine fibroids, dysfunctional hormonal cycle regulation, and therapeutic treatment of androgenic acne.

[0017] The method is also advantageous as a risk prediction for a woman who regularly flies.

[0018] Thus, a complementary aspect of the present invention relates to an antithrombotic treatment administered to patients considered at risk by the above method, said antithrombotic treatment preferably being an antiplatelet treatment or an anticogaulant, preferably heparin or a fragment of heparin.

[0019] Thus, another complementary aspect of the present invention relates to estradiol E2 or estetrol (E4) for use in a patient likely to take treatment based on artificial agonists and / or estrogen receptor modulators and predicted as at risk by the above method, or an aromatase inhibitor for use against ER+ breast cancer in a patient predicted as at risk by the above method, as well as epidermal estrogens for use in the treatment of side effects associated with menopause in a patient likely to take artificial estrogens or estrogen receptor modulators orally, said patient being predicted as at risk by the above method.

[0020] Another aspect of the present invention relates to a method for predicting the risk of vascular thromboembolism of a drug candidate comprising: measuring thrombin production from a plasma sample in several patients to whom said drug candidate has been administered, in the presence of a predetermined amount of tissue factor and phospholipids; measuring thrombin production from a plasma sample for each of said patients in the presence of a predetermined amount of tissue factor, phospholipids and a predetermined amount of activated protein C; measuring thrombin production from a standard plasma sample in the presence of a predetermined amount of tissue factor and phospholipids; measuring thrombin production from a sample of said standard plasma in the presence of a predetermined amount of tissue factor and a predetermined amount of activated protein C.said activated protein C being brought into contact with said tissue factor and phospholipids for a predetermined time and / or at least 45 minutes before being brought into contact with said plasma sample.

[0021] Another aspect of the present invention relates to a method for predicting the risk of thromboembolism in a patient treated with artificial estrogen receptor modulators, comprising measuring (i) the thrombin production of a plasma sample from said patient in the presence of a predetermined amount of tissue factor and phospholipids, measuring (ii) the thrombin production of a plasma sample from said patient in the presence of a predetermined amount of tissue factor, phospholipids, and a predetermined amount of activated protein C, measuring (iii) the thrombin production of a standard plasma sample in the presence of a predetermined amount of tissue factor and phospholipids, and measuring (iii) the thrombin production of a sample of said standard plasma in the presence of a predetermined amount of tissue factor, phospholipids, and a predetermined amount of activated protein C.said activated protein C being brought into contact with said tissue factor and phospholipids for a predetermined time and / or at least 45 minutes before said measurement, to calculate the combined ratio, being the ratio between (I) and (II) and the ratio between (III) and (III), and to divide the first ratio by the second; to calculate the risk for the patient based on the comparison of the result of said calculation with a predetermined value, preferably being the value associated with the treatment administered to said patient or the risk threshold value.

[0022] Advantageously, if the predetermined value is exceeded, this method is repeated after a period of at least 10 days during which the patient has not received artificial estrogen receptor modulators, which allows the calculation of a second combined ratio, being the ratio between (I) and (II) and the ratio between (III) and (IIII), and dividing the first ratio with the second, the measurements being taken after said period of at least 10 days.

[0023] An associated aspect of the present method is the use of estrogen E2 or estetrol E4 in patients for whom the first combined ratio is greater than the predetermined value, and the second combined ratio is less than the predetermined value.

[0024] Conversely, a final aspect of the present invention relates to an estrogen-free treatment for use in patients for whom the first combined ratio is greater than the predetermined value, and the second combined ratio is greater than the predetermined value.

[0025] Brief description of the drawings

[0026] Other features, details and advantages of the invention will become apparent from the description given below, by way of non-limitation and with reference to the drawings and examples.

[0027] Figure 1 represents the relationship between activated protein C resistance and the risk of venous thromboembolism following estrogen-progestin combination therapy.

[0028] Figure 2 represents the loss of activated protein C activity over time, with or without a one-hour pre-incubation.

[0029] Figure 3 represents the decision tree based on the measured risk

[0030] Detailed description of an embodiment of the invention

[0031] The inventors concluded that the risk of vascular thromboembolism is serious, often misdiagnosed, and too frequent. Indeed, they evaluated the available specific tests and algorithms and concluded that these only accounted for a portion of the risks, resulting in a kind of empirical approach whereby vascular thromboembolism in a patient is considered an inherent risk that must be accepted.

[0032] Given that this practice can have very serious consequences for the patient and / or will mobilize significant clinical resources, this risk, if it can be reduced through a finally accurate diagnosis, must be.

[0033] To this end, the inventors had an intuition of a global type of risk prediction diagnosis and noticed that the integration of this diagnosis made it possible, in some cases, to highlight an exponential risk, when the different risk factors, known or not necessarily, combine.

[0034] Thanks to this, a patient identified as "at risk" will no longer take estrogen receptor-modulating compounds associated with greater risks, even if these are more convenient (e.g., tamoxifen versus aromatase inhibitors for treating ER+ breast cancer, or estradiol (E2) instead of estetrol (E4) for managing vasomotor symptoms of menopause) or less expensive (e.g., synthetic estrogen receptor agonists (ethinylestradiol, EE) versus estetrol (E4) or estradiol (E2)). Furthermore, in some cases, non-pharmacological or pharmacological measures such as anticoagulation therapy may be recommended for an at-risk patient.

[0035] Thus, a first object of the present invention relates to a kit comprising: a predetermined quantity of tissue factor and phospholipids in a first container; a predetermined quantity of tissue factor, phospholipids and activated protein C in a second container (preferably in dry form); preferably a container comprising a reference plasma, said predetermined quantity of activated protein C being the quantity which, after contact of said activated protein C with said quantity of tissue factor and phospholipids for one hour and then with a standardization plasma, allows an inhibition of a predetermined value of thrombin production by said first standard plasma.In the context of the present invention (kit and methods), contacting activated protein C with tissue factor (and phospholipids) means an active step at the end of which the protein C is formulated in solution with the tissue factor (and phospholipids), allowing these substances to act on (or against) plasma coagulation. This can be done either (i) by mixing a solution containing activated protein C but not tissue factor with a solution containing tissue factor (and phospholipids) but not activated protein C, or (ii) by dissolving these active substances, which were previously present together in a vial but in dry form; the second option is, in fact, simpler in practice.

[0036] Advantageously, in this kit, the predetermined quantity of activated protein C has been validated by contact with (first) healthy plasma, and this validation is performed for each batch. Indeed, the inventors observed batch-to-batch variation at this level. Thus, even if different sources of activated protein C are used, exactly the same activity will be incorporated into each kit, so that the combined ratio (nAPCsr), which can be measured due to the implementation of the kit, will provide an absolute value. This will allow for a precise assessment of a patient's risk profile, either before hormonal treatment, during treatment, or for a patient in other conditions (e.g.pregnancy, air travel), but this will also allow the evaluation of the risk profile of new hormonal combinations, which will strengthen the veracity of clinical trials or even make them obsolete in some cases, for example if the risk of vascular thromboembolism is too high.

[0037] This standardization plasma is preferably carefully validated and will be advantageously reused over time. For example, it is a plasma pool from at least 20, 30, 40, 50, or 60 healthy patients (men and / or women, preferably both men and women).

[0038] Preferably, this standardized plasma is a pool of plasmas from individuals not taking estrogen. Furthermore, these patients do not have Protein S deficiency, Factor V mutations, or G20210a mutations. Similarly, the plasmas were obtained from volunteers who were not experiencing bleeding, were not taking antiplatelet or anticoagulant medication, or any substances with a similar effect, and, for women, were neither pregnant nor menopausal. Indeed, the inventors observed that incorporating even a single "resistant" plasma into the pool, even a pool of 30 different plasmas, and thus even with a marked dilution of the unintended effect, skews the results.

[0039] This standardization plasma can be frozen (e.g., at -70°C) in aliquot form, which will enhance standardization, including over time. Advantageously, another pool of reference plasma can be compared and will generate the same thrombin production values ​​or, at a minimum, identical values, subject to a correction factor that can be easily calculated, for example, via non-linear regression tracking inhibition as a function of activated protein C concentration.

[0040] The involvement, at least at some point, of a plasma pool in which characteristics such as ETP (endogenous thrombin potential, in other words the amount of thrombin produced by the sample after the addition of tissue factor and phospholipids) and the percentage of inhibition by activated protein C are controlled and validated, rather than relying exclusively on commercial plasma that is not sufficiently characterized, or whose full set of characteristics is not provided, is advantageous since the characteristics of commercial plasma could change over time and not reflect pre-analytical conditions similar to those obtained in patients.

[0041] Similarly, the predetermined quantity of tissue factor and phospholipids is carefully fixed and standardized. Otherwise, there is a risk of creating a wide diversity of activities between batches, which would prevent the determination of the combined ratio (nAPCsr) and would not allow comparisons with the standard values ​​generated by the inventors. In the context of the present invention, the tissue factor is always formulated in the presence of phospholipids. In the context of the present invention, the tissue factor can be the complete human protein or that of another mammal, but also an active fragment thereof, that is, a fragment capable of triggering thrombin generation via a mechanism that can be inhibited by activated protein C.

[0042] It should be noted that, in the present invention, the plasma samples (reference, standardization, or those to be analyzed) are preferably "platelet-poor" samples, and / or have fewer than 10,000 platelets / platelet count. Thus, even if the extracellular, soluble fraction of tissue factor is used, potentially eliminating the need for phospholipids, the use of platelet-poor plasma necessitates the addition of phospholipids to enable coagulation under optimal conditions. Preferably, the kit is used in conjunction with a thrombin production detection system, advantageously using a predetermined quantity of a fluorogenic substrate specifically cleaved by thrombin (preferably to chromogenic substrates or mechanical methods for measuring coagulation over time).

[0043] Preferably, in this kit, the inhibition of thrombin production (the ratio of thrombin production without versus with activated protein C) is between 85 and 95%, preferably between 88 and 92%, and preferably around 90%. Indeed, the inventors observed that the combined ratio (nAPCsr) exhibits the highest resolution for values ​​around 90%.

[0044] In this kit, the amount of activated protein C to be incorporated is calculated based on its inhibitory activity measured one hour after dissolution (in the presence of tissue factor and phospholipids). The inventors were surprised to find that protein C activity decreases rapidly upon dissolution, stabilizing after approximately one hour. Therefore, they chose to standardize the product based on this observation, which provides robust and reliable measurements and reduces operational constraints compared to an alternative where the dissolution time must be perfectly controlled and standardized, which is difficult in practice.

[0045] Advantageously, this kit, and the method described below, are validated, used or practiced at a constant and fixed temperature, for example 22°C, or 25°C, or even 37°C. The inventors have indeed noticed that the measurements and, above all, the ratios obtained were strongly influenced by the temperature, preferably at 37°C.

[0046] Preferably, this kit includes a container of reference plasma, here called "reference plasma" (the purpose of the "standardization plasma," which is not included in the kit, is to standardize the kit upstream; thus, preferably, the standardization plasma and the reference plasma are from different sources, the reference plasma being lyophilized), which may be from a different source than the first standard plasma. This allows for the normalization of measurements via the combined ratio (nAPCsr). The reference plasma is advantageously a commercial plasma, in practice another pool of healthy plasmas, such as Cryocheck™ Pooled Normal Plasma from Cryopep or reagent 2 from the STG®-ThromboScreen kit from Stago Diagnostica.

[0047] Advantageously, the reference plasma is calibrated, for example, when a new batch is used, using the standard plasma. This kit is advantageous for predicting the risk of vascular thromboembolism in a patient or for determining the risk inherent in a new treatment that impacts estrogen receptors. A related aspect of this is therefore the use of this kit for these purposes.

[0048] In the context of the present invention, preferably, "treatment having an impact on estrogen receptors" means any molecule (natural or artificial) capable of binding to this receptor, whether to the membrane surface fraction, the nuclear fraction or both fractions, and modifying its signaling, whether by an antagonistic effect, an agonist effect (preferred), or both, antagonistic at one dose and agonist at another dose, or even by an allosteric modulator, or even by an inverse agonist molecule (binds and directs the expression of genes having a metabolic effect opposite to that caused by the endogenous ligand).

[0049] A related aspect concerns a method for predicting the risk of vascular thromboembolism in a patient, comprising measuring thrombin production from a plasma sample of said patient in the presence of a predetermined amount of tissue factor (associated with phospholipids), measuring thrombin production from a plasma sample of said patient in the presence of a predetermined amount of tissue factor (associated with phospholipids) and a predetermined amount of activated protein C, measuring thrombin production from a plasma sample (reference) in the presence of a predetermined amount of tissue factor (associated with phospholipids), measuring thrombin production from a plasma sample (reference) in the presence of a predetermined amount of tissue factor (associated with phospholipids) and a predetermined amount of activated protein C,said activated protein C being brought into contact with said tissue factor for: a predetermined time and / or at least 45 minutes before said measurement (i.e., contact with plasma and measurement of thrombin production), preferably at least one hour before said measurement.

[0050] As described above for the kit, the inventors observed that the activity of protein C decreases immediately after reconstitution, but stabilizes over time. Therefore, the waiting time for the method advantageously reflects the amount of activated protein C incorporated into the kit, which is a function of the residual activity one hour after reconstitution.

[0051] Thus, the method can be used if there is precise control of the time after dissolution or after a sufficient waiting period. The inventors noted that 45 minutes of stabilization of activated Protein C mixed with Tissue Factor and phospholipids already allows for initial stabilization, although waiting for one hour ensures optimal stabilization.

[0052] In practice, it is simpler to wait 45 minutes to an hour than to carry out the tests under perfectly controlled time conditions.

[0053] The implementation of this process advantageously allows the calculation of the combined nAPCsr ratio (Douxfils, 2020), but, this time, under perfectly standardized conditions (variation between batches, management of the loss of activity of the activated protein C).

[0054] Standardization offers the advantage of comparing the values ​​measured for a person (a patient) to known values, such as baseline values, risk thresholds, normal values ​​associated with a given hormonal treatment, etc.

[0055] This method is particularly useful when the patient is a woman, especially for a woman likely to receive hormonal treatment, preferably treatment based on estrogens, estrogen receptor agonists or estrogen receptor modulators (see above for a more complete description of these concepts).

[0056] In this case, the method will allow the evaluation of the risk profile for the patient and represents a basis for the decision to possibly adapt the treatment in favor of less risky treatments: the test makes it possible to avoid a patient at risk taking hormonal cocktails that the inventors have determined to be the most risky, and thus to avoid an exponential risk (i) for a patient and (ii) at the level of a population.

[0057] For example, this method is used when a woman is undergoing hormonal treatment (estrogens) to manage vasomotor symptoms related to menopause, in vitro fertilization following unsuccessful natural conception, osteoporosis, vaginal atrophy, endometriosis, polycystic ovary syndrome and uterine fibroids, regulation of dysfunctional hormonal cycles, and the therapeutic management of androgenic acne in women. This method is also useful when a woman is not taking estrogens and is pregnant or may become pregnant.Indeed, a significant risk for a patient, if measured, can be managed either through an adapted diet, or through targeted physical activity or by taking anticoagulants, for example at the end of pregnancy, or even by wearing compression stockings; measuring the risk of pulmonary thromboembolism allows for balancing with the risk inherent in taking anticoagulants, this balance being made to the benefit of the patient, but only possible if the risk of venous thromboembolism is precisely defined.

[0058] Alternatively, this method is also useful for women who fly regularly. Indeed, air travel is also a risk factor for pulmonary thromboembolism, and the increased risk can be reduced by flying less, wearing compression stockings, or, occasionally, taking antithrombotic medication.

[0059] A preferred antithrombotic (anticoagulant) is heparin or a heparin fragment. Other antithrombotics are possible, including oral anticoagulants or antiplatelet agents.

[0060] An associated aspect of the present invention is estradiol E2 or estetrol (E4) for use in a patient likely to be taking treatment based on artificial agonists and / or estrogen receptor modulators and predicted to be at risk by the above method.

[0061] As described above, when a risk is determined to be high, the simplest option is to reduce the risk at the estrogen level and, in the case of administering hormonal treatments including estrogens, to favour E2 or E4, which are shown by the inventors to be less problematic.

[0062] Another related aspect of the present invention is an aromatase inhibitor (rather than tamoxifen) for use against ER+ breast cancer in a patient predicted to be at risk by the above method.

[0063] Indeed, tamoxifen-based treatment for this type of cancer is usually the most widespread because it is better tolerated or more convenient.

[0064] However, if the risk profile for venous thromboembolism is too high, the risk-benefit balance now favors the choice of drugs currently used as second-line therapy, since, again, the exponential risk will be avoided. Another aspect of the present invention is the application of estrogens to the epidermis for use in a patient likely to be taking artificial estrogens or oral estrogen receptor modulators, said patient being predicted to be at risk by the method described above. This is particularly relevant for the treatment of side effects associated with menopause.

[0065] Skin application is preferably via a patch, or via a gel or spray. It involves application to the patient's epidermis and not to other tissues of endodermal origin, even those sometimes considered external.

[0066] Another related aspect of the present invention is a method for predicting the risk of venous thromboembolism of a drug candidate in a patient comprising either: measuring the thrombin production of a plasma sample of said patient in the presence of a predetermined amount of tissue factor, measuring the thrombin production of a plasma sample of said patient in the presence of a predetermined amount of tissue factor and a predetermined amount of activated protein C, measuring the thrombin production of a standard plasma sample in the presence of a predetermined amount of tissue factor, or measuring the thrombin production of a sample of said standard plasma in the presence of a predetermined amount of tissue factor and a predetermined amount of activated protein C.said activated protein C being brought into contact with said tissue factor for a predetermined time and / or at least 45 minutes before said measurement (i.e., contact with plasma and measurement of thrombin production); or, where the drug candidate is a novel combination of molecules whose risk has already been determined individually, calculating the risk of the combination by multiplying the known risk associated with one molecule by the risk associated with the other molecule, or combinations of molecules.

[0067] The first alternative of this method, repeated on a few dozen patients, will allow a quick, simplified estimation of the risk associated with the developed hormonal combination.

[0068] Furthermore, according to the second branch of the alternative, the risk associated with new combinations of known molecules can advantageously be determined directly through a simple mathematical operation, starting from the risk of each molecule taken individually. This represents a saving of time and energy and even avoids clinical trials, thus eliminating the inherent risk to volunteers or patients. This also allows for faster adoption of therapeutic innovations, such as new molecules with a lower risk of venous thromboembolism as determined by the aforementioned test.

[0069] Another aspect of the invention is a method for predicting the (intrinsic) risk of thromboembolism in a patient before starting treatment with estrogen receptor-acting derivatives, comprising measuring (i) thrombin production from a plasma sample of said patient in the presence of a predetermined amount of tissue factor, measuring (ii) thrombin production from a plasma sample of said patient in the presence of a predetermined amount of tissue factor and a predetermined amount of activated protein C, measuring (iii) thrombin production from a reference plasma sample in the presence of a predetermined amount of tissue factor, measuring (iii) thrombin production from a sample of said reference plasma in the presence of a predetermined amount of tissue factor and a predetermined amount of activated protein C,said activated protein C being brought into contact with said tissue factor for a predetermined time and / or at least 45 minutes before said measurement (i.e., contact with plasma and measurement of thrombin production), to calculate the combined ratio (nAPCsr), being the ratio between (I) and (II) and the ratio between (III) and (IIII), and to divide the first ratio by the second; to calculate the risk for the patient based on the comparison of the result of said calculation with a predetermined value, preferably the average value of a population, for example a value of 3.

[0070] This method allows for the rapid identification of whether the patient has a prothrombotic profile and avoids administering synthetic estrogen products.

[0071] Thus, if the predetermined value for a given molecule is exceeded, the method is repeated after a period of at least 10 days during which the patient has not received artificial estrogen receptor modulators. This will allow the calculation of a second combined ratio (nAPCsr), being the ratio between (I) and (II) and the ratio between (III) and (III) above, and dividing the first ratio with the second, the measurements being taken after said period of at least 10 days.

[0072] This will allow for the definition of the type of risk involved and, if necessary, the administration of estrogen E2 or estetrol E4 to patients for whom the first ratio is higher than the predetermined value, and the second ratio is lower than the predetermined value. Indeed, these two molecules have a lower risk profile, which avoids the exponential nature of the risk while allowing for minor adjustments to the treatment.

[0073] However, patients for whom the second combined ratio (nAPCsr) remains too high should preferably stop taking estrogens.

[0074] Another aspect of the present invention is a method for predicting the general risk of thromboembolism in a patient receiving treatment comprising an estrogen receptor modulator, comprising measuring (i) thrombin production from a plasma sample of said patient in the presence of a predetermined amount of tissue factor, measuring (ii) thrombin production from a plasma sample of said patient in the presence of a predetermined amount of tissue factor and a predetermined amount of activated protein C, measuring (iii) thrombin production from a standard plasma sample in the presence of a predetermined amount of tissue factor, measuring (iii) thrombin production from a sample of said standard plasma in the presence of a predetermined amount of tissue factor and a predetermined amount of activated protein C,said activated protein C being brought into contact with said tissue factor for a predetermined time and / or at least 45 minutes before said measurement (i.e., contact with plasma and measurement of thrombin production), the ratio between (I) and (II) and the ratio between (III) and (III) are calculated, and the first ratio is divided by the second (obtaining the nAPCsr ratio). The risk for the patient is then calculated based on the result of said calculation, using a known or predetermined value for said treatment comprising an estrogen receptor modulator, preferably the values ​​listed in Tables 1 and 2 below. Indeed, the inventors have developed comparisons of risk profiles for numerous combinations available on the market. A patient taking a given hormonal combination may have a risk lower than or equal to the average, but also, unfortunately, a higher risk. Thus, the method makes it possible to determine,Even for patients taking hormonal treatments, if their risk factor, independent of hormonal treatment, is significant, then a less risky treatment or a switch to estrogen-free treatment should be prioritized.

[0075] Other features and advantages of the present invention will be derived from the following non-limiting description, and with reference to the drawings and examples.

[0076] Examples.

[0077] It is understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made to it without departing from the scope of the attached claims.

[0078] Example 1:

[0079] Kinetics of the loss of activity of activated protein C.

[0080] The inventors prepared 15 µl aliquots of activated protein C at a final concentration of 0.2 mg / ml in Tris buffer, pH 7.4 (Sigma), diluted 100% in distilled water (PureLab). These aliquots are stored at a temperature below -72°C.

[0081] For each test, an aliquot will be thawed and diluted in the same diluted Tris buffer.

[0082] On the other hand, for each test, two vials of tissue factor (STG- Thromboscreen) are taken, then reconstituted in 2x 980 pl of aqua injectabila.

[0083] In one of the vials, 20 pl of the reconstituted activated protein C solution will be added immediately; in the other, 20 pl of the diluted Tris.

[0084] As shown in Figure 1, nAPCsr was measured 1 hour after the first reconstitution (reconstitution of TF+PL), and APC was added 30 minutes before the start of the analyses (another 30 minutes elapsed between the addition and the methodological steps). Therefore, there is almost no stabilization time since the reagent (TF+PL+APC) is used directly to fill the multiwell plate. In contrast, in the second experiment, APC was added directly during the reconstitution of TF+PL, and the TF+PL+APC reconstitution took place over 1 hour, which ensured more stable results. It is primarily the intermediate resistance conditions that are most affected, even though these are the conditions for which the added value of the test is greatest.

[0085] On the other hand, a pool of lyophilized reference plasma is reconstituted in 1 ml of aqua injectabila and left to stabilize for a minimum of 30 minutes.

[0086] The plasmas are then mixed with tissue factor solutions (with or without APC) and thrombin production is measured.

[0087] The inventors noted that activated protein C lost up to 10% of its activity (which, given the use of ratios and correction factors, no longer allows standardization) in the most delicate cases; however, this loss of activity is obtained during the first hour, and is stable thereafter, so that it is possible to ensure a constant and reliable activity of the inhibitory power of activated protein C, by means of activation (making contact) with the tissue factor and a sufficiently long stabilization time.

[0088] Conversely, if activated protein C is directly brought into contact with the composition including plasma and tissue factor, the results fluctuate greatly.

[0089] Example 2:

[0090] Variation in the effect of activated protein C depending on the batches of commercial plasma.

[0091] Tested batch Correction factor

[0092] A 3,156

[0093] B 3,001

[0094] C 1.78

[0095] D 2.36

[0096] E 2.34

[0097] F 2.78

[0098] The inventors therefore observed that commercially available "healthy" plasmas exhibit very high variations, necessitating calibration. Otherwise, the values ​​obtained could only be used in a very limited context and would not allow for comparisons between patients, or in relation to treatments received or to be administered. Example 3:

[0099] Risk factor of various contraceptives including synthetic estrogen receptor modulators or estradiol E2 or estetrol E4.

[0100] The inventors collected plasma samples from patients not taking oral contraceptives along with those taking them. The estrogen receptor modulator was ethinyl estradiol (EE) in each case, in combination with various progestins, as shown in Table 1 below.

[0101] Table 1. Relative risk of venous thromboembolism (VTE) associated with the use of oral contraceptives (COCs):

[0102] From this, the inventors conclude that the dose of EE increases the relative risk of thromboembolism, while progestins with a more pronounced androgenic effect increase this risk less.

[0103] Substituting EE with E2 reduces this risk by approximately 50%.

[0104] The inventors then reproduced these experiments, including a test with estetrol (E4), and the results are presented in Table 2 below or in Figure 2. Table 2:

[0105] Example 4:

[0106] Decision tree for administering an oral contraceptive to a patient (see Figure 3).

Claims

DEMANDS 1. A kit comprising a predetermined quantity of tissue factor and phospholipids in one container, a predetermined quantity of tissue factor, phospholipids and activated protein C in a second container, said predetermined quantity of activated protein C being the quantity which, after contact of said activated protein C with said quantity of tissue factor and phospholipids for one hour and then with standardization plasma, enables inhibition of a predetermined value of thrombin production by said standardization plasma.

2. The kit according to claim 1, wherein the inhibition is between 85 and 95%, preferably between 88 and 92%, preferably 90%.

3. The kit according to claim 1 or 2 further comprising a container including a reference plasma, preferably from a different origin than the standardization plasma, for the standardization of measurements.

4. A method for predicting the risk of vascular thromboembolism in a patient comprising measuring the thrombin production of a plasma sample of said patient in the presence of a predetermined amount of tissue factor and phospholipids, measuring the thrombin production of a plasma sample of said patient in the presence of a predetermined amount of tissue factor, phospholipids and a predetermined amount of activated protein C, measuring the thrombin production of a standard plasma sample in the presence of a predetermined amount of tissue factor and phospholipids, measuring the thrombin production of a sample of said standard plasma in the presence of a predetermined amount of tissue factor, phospholipids and a predetermined amount of activated protein C,said activated protein C being brought into contact with said tissue factor and phospholipids for a predetermined time and / or at least 45 minutes before being brought into contact with said plasma sample.

5. Method according to claim 4 wherein the patient is a woman.

6. Method according to claim 5 wherein the woman is likely to receive hormonal treatment, preferably treatment based on estrogens, estrogen receptor agonists and estrogen receptor modulators.

7. Method according to claim 5 wherein the woman is pregnant or likely to become pregnant.

8. Method according to claim 5 or 6 wherein the woman is under treatment for the treatment of vasomotor problems related to menopause, osteoporosis, vaginal atrophy, endometriosis, polycystic ovary syndrome, uterine fibroids, regulation of dysfunctional hormonal cycle, and therapeutic treatment of androgenic acne in a patient.

9. Method according to claim 5 or 6 in which the woman takes the plane.

10. An antithrombotic treatment administered to patients considered at risk by the method according to any of the preceding claims 4 to 9, said antithrombotic treatment preferably being an antiaggregant treatment or an anticogaulant, preferably heparin or a fragment of heparin. 1 1. Estradiol E2 or estetrol (E4) for use in a patient likely to be taking treatment based on artificial agonists and / or estrogen receptor modulators and predicted to be at risk by the method according to any of the preceding claims 4 to 6 and 8-9.

12. An aromatase inhibitor for use against ER+ breast cancer in a patient predicted to be at risk by the method according to any of the preceding claims 4 to 9.

13. Estrogens administered epidermally for use in the treatment of side effects associated with menopause in a patient likely to be taking artificial estrogens or oral estrogen receptor modulators, said patient being predicted to be at risk by the method according to any one of the preceding claims 4 to 6 and 8-9.

14. A method for predicting the risk of vascular thromboembolism of a drug candidate comprising: measuring thrombin production from a plasma sample in several patients to whom said drug candidate has been administered, in in the presence of a predetermined amount of tissue factor and phospholipids, measure the thrombin production of a plasma sample for each of said patients in the presence of a predetermined amount of tissue factor, phospholipids and a predetermined amount of activated protein C, measure the thrombin production of a standard plasma sample in the presence of a predetermined amount of tissue factor and phospholipids, measure the thrombin production of a sample of said standard plasma in the presence of a predetermined amount of tissue factor and a predetermined amount of activated protein C, said activated protein C being brought into contact with said tissue factor and phospholipids for a predetermined time and / or at least 45 minutes before being brought into contact with said plasma sample.

15. Use of the kit according to any of the preceding claims 1 to 3 for predicting the risk of vascular thromboembolism in a patient or for determining the risk inherent in a new treatment having an impact on estrogen receptors.

16. A method for predicting the risk of thromboembolism in a patient treated with artificial estrogen receptor modulators comprising measuring (i) thrombin production from a plasma sample of said patient in the presence of a predetermined amount of tissue factor and phospholipids, measuring (ii) thrombin production from a plasma sample of said patient in the presence of a predetermined amount of tissue factor, phospholipids and a predetermined amount of activated protein C, measuring (iii) thrombin production from a standard plasma sample in the presence of a predetermined amount of tissue factor and phospholipids, measuring (iii) thrombin production from a sample of said standard plasma in the presence of a predetermined amount of tissue factor, phospholipids and a predetermined amount of activated protein C, said activated protein C being brought into contact with said tissue factor and phospholipids for a predetermined time and / or at least 45 minutes before said measurement, to calculate the combined ratio, being the ratio between (I) and (II) and the ratio between (III) and (III), and to divide the first ratio with the second; to calculate the risk for the patient based on the comparison of the result of said calculation with a predetermined value being preferably the value associated with the treatment administered to said patient or the risk threshold value.

17. The method according to claim 16 which, in the event of exceeding the predetermined value, is repeated after a period of at least 10 days during which the patient has not received artificial estrogen receptor modulators, and allowing the calculation of a second combined ratio, being the ratio between (I) and (II) and the ratio between (III) and (III), and dividing the first ratio with the second, the measurements being taken after said period of at least 10 days.

18. Estrogen E2 or Estetrol E4 for use in patients according to claim 17 for which the first combined ratio is greater than the predetermined value, and the second combined ratio is less than the predetermined value.

19. An estrogen-free treatment for use in patients according to claim 17 in which the first combined ratio is greater than the predetermined value, and the second combined ratio is greater than the predetermined value.

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