WO2016121738A1 - Method for drug evaluation of cilostazol - Google Patents

Abstract

　Provided is a novel and practical method for drug evaluation of cilostazol.　A method for drug evaluation of cilostazol is used, which uses a thrombin receptor activating reagent and a cAMP-increasing reagent. A platelet function measuring method that includes a step in which a thrombin receptor activating reagent and a cAMP-increasing reagent are brought into contact with a sample originating from blood of patients administered with cilostazol may also be used.

Description

Method for evaluating the efficacy of cilostazol

The present invention relates to a method for evaluating the efficacy of cilostazol.

Antiplatelet drugs such as aspirin, clopidogrel, cilostazol are used for secondary prevention of cerebral infarction or myocardial infarction. In stent treatment, treatment using these two or three agents in combination is performed. Evaluation of the efficacy of these antiplatelet drugs is carried out based on the respective action mechanisms (Non-patent Document 1). Specifically, a platelet activating reagent corresponding to the mechanism of action of the antiplatelet drug is allowed to act on platelets in the patient's blood after administration of the antiplatelet drug, and the platelet aggregation rate is measured.

For example, the efficacy of aspirin (cyclooxygenase 1 inhibitor) is evaluated by measuring the platelet aggregation rate by causing the platelet activation reagent arachidonic acid (a substance metabolized to prostanoids by cyclooxygenase 1) to act on the patient's platelets. Can be implemented. At this time, if the platelet aggregation rate is low, it can be determined that the drug is effective.

As platelet activation reagents used for evaluating the efficacy of antiplatelet drugs, arachidonic acid is used for aspirin, and ADP or ADP + PGE1 is used for clopidogrel.

In Non-Patent Document 2, sodium arachidonic acid is used as a platelet activating reagent used for evaluating the efficacy of cilostazol.

"Practical monitoring of drug efficacy using platelet agglutination test in antiplatelet therapy." Katsuo Sato., Acupuncture and Thrombus Journal 17: 424-429, 2006. "Platelet aggregometry in the presence of PGE (1) provides a reliable method for cilostazol monitoring." Satoh et al., Thromb Res. 2012 Oc; 130 (4): 616-21.

However, in Non-Patent Document 2 above, since sodium arachidonic acid is used as a platelet activating reagent, inhibition of platelet aggregation is observed not only with cilostazol but also with aspirin. Therefore, the efficacy of cilostazol could not be monitored during drug combination therapy with aspirin.

In addition, as described in Examples described later, the inventors of the present application tried to apply epinephrine and CRP, which are platelet activating reagents, to a cilostazol drug efficacy evaluation system. However, epinephrine has great individual differences and is not practical. CRP did not change with or without cilostazol.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel and practical method for evaluating the efficacy of cilostazol.

As described in Examples below, the inventors of the present application caused thrombin receptor activating reagent and cAMP increasing reagent to act on the blood of patients taking cilostazol, and the platelet aggregation was significantly suppressed. I found out. That is, the efficacy of cilostazol could be evaluated with the thrombin receptor activating reagent and cAMP increasing reagent. And based on this knowledge, this invention was completed.

That is, according to one aspect of the present invention, there is provided a method for evaluating the efficacy of cilostazol using a thrombin receptor activating reagent and a cAMP increasing reagent. If this method is used, the efficacy evaluation of cilostazol can be performed. Moreover, the thrombin receptor activating reagent which is a platelet activating reagent is substantially not affected by aspirin. Therefore, even when the patient is using cilostazol and aspirin in combination, the efficacy of cilostazol can be evaluated.

Also, according to one aspect of the present invention, there is provided a method for measuring platelet function, comprising a step of contacting a thrombin receptor activating reagent and a cAMP increasing reagent with a blood-derived sample of a patient taking cilostazol. If this method is used, the platelet function of a patient taking cilostazol can be measured.

Also, according to one aspect of the present invention, there is provided a kit for evaluating the efficacy of cilostazol, comprising a thrombin receptor activating reagent and a cAMP increasing reagent. If this kit is used, the efficacy of cilostazol can be evaluated.

According to the present invention, a practical medicinal evaluation of cilostazol can be performed.

FIG. 1 is a diagram showing the measurement results of platelet aggregation ability using VerifyNow in an in vitro experiment. FIG. 2 is a diagram showing the measurement results of platelet aggregation ability using VerifyNow in an ex-vivo experiment. FIG. 3 is a diagram showing the measurement results of platelet aggregation ability using VerifyNow in an ex-vivo experiment. FIG. 4 is a diagram showing measurement results of platelet aggregation ability using AG-10 in an ex-vivo experiment. FIG. 5 is a diagram showing the measurement results of platelet aggregation ability using AG-10 in an ex-vivo experiment. FIG. 6 is a diagram showing the results of examining individual differences in platelet aggregation reaction. FIG. 7 is a diagram showing the measurement results of platelet aggregation ability when CRP is used.

Hereinafter, embodiments of the present invention will be described in detail. In addition, in order to avoid the repetition complexity about the same content, description is abbreviate | omitted suitably.

One embodiment of the present invention is a method for evaluating the efficacy of cilostazol using a thrombin receptor activating reagent and a cAMP increasing reagent. If this method is used, the efficacy evaluation of cilostazol can be performed. Moreover, the thrombin receptor activating reagent which is a platelet activating reagent is substantially not affected by aspirin. Therefore, even when the patient is using cilostazol and aspirin in combination, the efficacy of cilostazol can be evaluated.

The platelet activation level may be measured by, for example, an absorbance method, an electric resistance method, a thrombus formation method, a screen filter method, a plastic bead column method, a flow cytometry method, a Western blot method, or an ELISA method. Absorbance method, for example, put blood sample (for example, whole blood or platelet rich plasma (PRP)) in cuvette and add platelet activating substance to add platelet aggregation, change light transmittance (absorbance) This can be done by monitoring with At this time, the permeability of platelet poor plasma (PPP) may be set to 100%. In the screen filter method, the whole blood can be measured without separating the PRP.

For example, a multiplate analyzer (Dynabyte medical, Munich, Germany) can be used for the electrical resistance method. The multiplate analyzer is a whole blood platelet aggregation ability measuring device based on the electric resistance method (Can et al., Thromb Res. 2010 Apr; 125 (4): e132-7.). The outline is as follows, for example. Blood is added to a measurement cell equipped with a pair of electrodes, and the electrical resistance between the electrodes is measured under stirring conditions. Due to platelet activation, platelets adhere and aggregate on the electrode, which changes the electrical resistance. This change in electrical resistance is converted into an arbitrary unit set by the manufacturer to give an indicator of platelet aggregation ability. This indicator is plotted on the vertical axis and time is plotted on the horizontal axis, and the area under the aggregation curve; AUC _a ) evaluates platelet aggregation ability. Although specific operation is not specifically limited, For example, you may perform according to the following procedure according to an instruction manual. That is, 300 μl of hirudin-treated blood is placed in a measurement cuvette, followed by adding an equal amount of physiological saline and heating at 37 ° C. for 3 minutes. Next, add 3 μM cilostazol and warm for 2 minutes, then add 9.4 nM PGE1 (ADPtestHS) and warm for another 2 minutes. Platelet aggregation is induced with TRAP as a platelet activator, and the platelet aggregation ability is measured for 6 minutes.

For the thrombus formation measurement method, for example, Total Thrombus-formation Analysis System (T-TAS; Fujimori Industrial Co., Ltd., Tokyo) can be used. T-TAS is a device that analyzes platelet function in the bloodstream, and is a system that evaluates the thrombus formation process in the microchip based on the pressure increase of the blood pump (Hosokawa et al., Microvasc Res. 2012 Mar; 83 (2): 154-61.). Although specific operation is not specifically limited, For example, you may perform according to the following procedure according to an instruction manual. Dispense 320 μl of hirudin-treated blood into the reservoir, cover it with a cap, and connect a liquid pump with a pressure sensor attached. The liquid flow is measured by setting, for example, 12 μl / min and 24 μl / min. The shear stresses correspond to, for example, 1000 s ⁻¹ and 2000 s ⁻¹ , respectively. Twenty-five capillaries having a width of 40 μm and a depth of 40 μm are engraved in the microchip, and collagen, which is a component of the subendothelial tissue, is applied to the bottom surface. When blood passes through this region, platelets are activated by contact with collagen to form a thrombus. Since the blood flow path becomes smaller with the formation of a thrombus, the pressure of the liquid feeding pump increases. The pressure is measured with the baseline 30 seconds after the start of liquid delivery, the time when the pressure rises 10 kPa from this baseline pressure is the clogging start time (T ₁₀ ), the time when the pressure rises 80 kPa from the baseline pressure is the clogging time (T ₈₀ ), The pressure change is defined as the area under the pressure curve (AUC _p ) drawn with the vertical axis representing time and the horizontal axis representing time, and AUC _p is evaluated as an index of thrombus formation.

The platelet activation level may be measured using a measurement method using whole blood as a measurement sample. At this time, as a measurement principle, for example, aggregation, shear stress-induced aggregation, adhesion / aggregation, protein phosphorylation, and the like can be used. As the detection principle, for example, turbidity, electrical resistance, pressure, occlusion time, platelet count reduction, surface occupancy, flow cytometry, filter pressure, ELISA, or the like can be used. As a device that can utilize these principles, for example, a whole blood aggregometer can be used. Whole blood aggregometers are, for example, VerifyNow (Accumetrics), Platelet Function Analyzer (Siemens Healthcare Diagnostics Inc.), multiplate (Dynabyte), Plateletworks (Helena Laboratories), Impact cone and plate (let) analyzer (Dia) ISS Co., Ltd.). In order to know the phosphorylation level of VASP, flow cytometry method and Western blot method can be mentioned. The blood volume to be used may be, for example, 0.01, 0.1, 1, 10, 100, 1000, or 10000 μL / measurement, and may be in the range of any two values thereof.

In one embodiment of the present invention, the “drug efficacy evaluation method” comprises (i) a step of administering cilostazol to a patient, (ii) a step of obtaining blood from the patient, (iii) a blood-derived sample of the patient, and thrombin reception A step of bringing a body activation reagent and a cAMP increasing reagent into contact with each other to prepare a mixed solution, or (iv) a step of measuring the platelet activation level of the mixed solution may be included. In step (iii) above, the blood-derived sample and the thrombin receptor activating reagent may be contacted, and then the blood-derived sample and the cAMP increasing reagent may be contacted, and the contact treatment is performed in the reverse order. Alternatively, the contact treatment may be performed at the same time. In addition, the process employ | adopted by the medicinal effect evaluation method in the above or below is employable also for a platelet function measuring method.

In addition, the method for evaluating drug efficacy includes (v) a step of obtaining blood from a patient not administered cilostazol, (vi) a step of obtaining blood from the patient, (vii) a blood-derived sample of the patient, and thrombin receptor activation. A step of bringing a reagent and a cAMP increasing reagent into contact with each other to prepare a mixed solution, or (viii) a step of measuring a platelet activation level of the mixed solution. Non-administration includes before administration.

The drug efficacy evaluation method may include (ix) a step of comparing the platelet activation levels of the mixed solution (vii) and the mixed solution (iii). In addition, the drug efficacy evaluation method (x) evaluates that cilostazol is effective for a patient when the platelet activation level of the mixed solution of (iii) is smaller than the platelet activation level of the mixed solution of (vii) Process. At this time, the platelet activation level of the mixed solution of (iii) is, for example, 0.9, 0.7, 0.5, 0.3, 0.1 times or less, or 0 times the platelet activation level of the mixed solution of (vii). At this time, cilostazol may be evaluated as effective for the patient.

In addition, the above-mentioned drug efficacy evaluation method comprises (xi) a step of obtaining blood from a patient after cilostazol administration, or (xii) contacting the blood-derived sample of the patient with a thrombin receptor activating reagent to prepare a mixed solution Process.

The drug efficacy evaluation method may include (xiii) a step of comparing the platelet activation level of the mixed solution (xii) and the mixed solution (iii). In addition, the above-described method for evaluating the efficacy of the anti-platelet drug is effective for a patient when the platelet activation level of the mixed solution of (xiv) (iii) is smaller than the platelet activation level of the mixed solution of (xii). A step of evaluating may be included. At this time, the platelet activation level of the mixed solution (iii) is, for example, 0.9, 0.7, 0.5, 0.3, 0.1 times or less, or 0 times the platelet activation level of the mixed solution (xii). At that time, the antiplatelet drug may be evaluated as effective for the patient.

In addition, the drug efficacy evaluation method may include a step of evaluating that the antiplatelet drug is effective for the patient when the platelet activation level of the mixed solution (iii) is lower than a predetermined threshold. The platelet activation level may be measured using, for example, a platelet aggregation measuring device.

In addition, the above-described method for evaluating drug efficacy may include a step of leaving a mixed solution of a blood-derived sample, a thrombin receptor activating reagent, and a cAMP increasing reagent. The standing time may be, for example, 0, 0.1, 0.5, 1, 2, 3, 5, or 10 minutes or more, and may be within the range of any two values thereof.

Further, the above-described method for evaluating the efficacy may include a step of mixing the patient's blood and a blood anticoagulant. The blood anticoagulant may be, for example, sodium citrate. Moreover, the said medicinal effect evaluation method may include the process of keeping a patient's blood in the state which is not coagulated. Moreover, the above-mentioned drug efficacy evaluation method may measure immediately after collecting blood without mixing the patient's blood and the blood anticoagulant.

In addition, the method for evaluating drug efficacy includes (xv) a step of administering a second antiplatelet agent different from cilostazol to the patient, (xvi) a step of obtaining blood from the patient, (xvii) a blood-derived sample of the patient, A step of contacting a thrombin receptor activating reagent and a cAMP increasing reagent to prepare a mixed solution, or (xviii) the platelet activation level of the mixed solution of (iii) above is the platelet activity of the mixed solution of (xvii) above A step of assessing that cilostazol is effective for the patient when the level is less than the activation level. At this time, the platelet activation level of the mixed solution (iii) is, for example, 0.9, 0.7, 0.5, 0.3, 0.1 times or less, or 0 times the platelet activation level of the mixed solution (xvii). At this time, cilostazol may be evaluated as effective for the patient. The antiplatelet drug of (i) and the second antiplatelet drug may be antiplatelet drugs having different pharmacological actions. The patients (i), (v), and (xi) may be the same or different. The “acquisition” includes, for example, collection by injection. The “contact” includes, for example, mixing.

Moreover, the above-mentioned medicinal effect evaluation method may include a step of preparing a blood-derived sample from a patient's blood. In one embodiment of the present invention, the “blood-derived sample” or “blood sample” includes, for example, whole blood or PRP. The blood-derived sample may be, for example, artificially created blood. The blood-derived sample may be in a state of being accommodated in a test container, a tube, or a cuvette, for example.

In addition, the above-mentioned method for evaluating the efficacy is a step of preparing PRP from the blood of a patient, a step of contacting PRP with a thrombin receptor activating reagent and a cAMP increasing reagent, and preparing a mixed solution, or a platelet activation level of the mixed solution The step of measuring. PRP can be obtained, for example, by centrifuging blood. The centrifugal force of the centrifuge may be, for example, 50, 100, 500, 1000, 5000, or 10000 g, and may be in the range of any two values thereof. The time of centrifugation may be 0.01, 0.1, 1, 10, or 15 minutes, and may be in the range of any two of them.

Moreover, the method for evaluating the efficacy of the drug may include a step of preparing PPP from the blood of the patient. PPP can be obtained, for example, by centrifuging blood. The centrifugal force of the centrifugal separation may be, for example, 500, 1000, 1500, 2000, 3000, 4000, 10000, or 30000 g, and may be in the range of any two values thereof. The time of centrifugation may be 0.01, 0.1, 1, 5, 10, 12, 15, or 20 minutes, and may be within the range of any two of them. In the above-described method for evaluating drug efficacy, the platelet activation level may be measured, for example, in vitro or ex vivo.

In one embodiment of the present invention, the “reagent” is not limited in its use or form, and may be, for example, a substance or drug having pharmacological activity. The form of the reagent may be, for example, a dried product (for example, powder), a solution, a solid phase in a container (for example, a plastic container), or the like. The reagent may be prepared by further mixing an arbitrary aqueous solution (for example, a buffer solution), an additive, or a carrier with the thrombin receptor activating reagent or the cAMP increasing reagent.

In one embodiment of the present invention, the “platelet activating reagent” includes a reagent having an action of activating platelet function or a reagent having an action of promoting platelet aggregation.

In one embodiment of the present invention, “thrombin receptor activating reagent” refers to, for example, TRAP (Thrombin Receptor Activating Peptide), thrombin, or a derivative thereof that activates thrombin receptor, or a salt or solvate thereof. Including. The efficacy of cilostazol depends on the mechanism of action of the reagents used to evaluate the efficacy, but they all have the same mechanism of activating the thrombin receptor and should be used in combination with cAMP increasing reagents. Thus, evaluation of the efficacy of cilostazol can be realized. TRAP derivatives include iso-TRAP. iso-TRAP can be purchased from, for example, Multiple® Peptide® Systems. Derivatives include modifications. From the viewpoint of the sensitivity of cilostazol drug efficacy evaluation, TRAP or iso-TRAP is particularly preferable. The amino acid sequence of TRAP is, for example, SFLLRN (SEQ ID NO: 1).

In one embodiment of the present invention, the “cAMP increasing reagent” includes a reagent having an action of increasing the amount of cAMP in platelets. The cAMP increasing reagent includes, for example, PGE1, PGI2, forskolin, Dibutyryl-cAMP, beraprost, limaprost alphadex, or derivatives thereof that increase the amount of cAMP, or salts or solvates thereof. The efficacy of cilostazol depends on the mechanism of action of the reagents used to evaluate the efficacy, but they all have the same mechanism of increasing cAMP, so they are used in combination with a thrombin receptor activation reagent. Thus, evaluation of the efficacy of cilostazol can be realized. In addition, PGE1 is particularly preferable from the viewpoint of sensitivity for evaluating the efficacy of cilostazol.

When the platelet function is measured, the concentration per thrombin receptor activating reagent or cAMP increasing reagent in the mixture (iv) may be a sufficient amount to activate the platelet function. This concentration may be, for example, 0.001, 0.01, 0.1, 1, 5, 10, 30, 50, 100, 300, 500, or 1000 μM or less, or 0.01, 0.1, 1, or 10 U / ml or less. , Any two of them may be within the range of values.

In one embodiment of the present invention, the “antiplatelet drug” includes a drug that suppresses the function of platelets. Antiplatelet agents include ischemic disease therapeutic agents, thrombus therapeutic agents, embolic therapeutic agents, or blood flow disorder therapeutic agents.

The dose of the antiplatelet drug may be, for example, 0.00001, 0.0001, 0.001, 1, 10, or 50 mg / kg body weight, and may be in the range of any two of them. Alternatively, it may be 0.001, 0.01, 1, 10, 100, 200, or 400 mg / time, and may be within the range of any two of them. The number of administrations may be 1, 2, 3, 4 times / day (or week, month), and may be within the range of any two of them. The antiplatelet drug may be administered in combination of two or more drugs. The administration method may be, for example, oral administration or intravenous administration.

One embodiment of the present invention is a kit for evaluating the efficacy of cilostazol, comprising a thrombin receptor activating reagent and a cAMP increasing reagent. By using this kit, the medicinal efficacy of cilostazol can be evaluated by measuring the platelet aggregation level of the blood sample of the patient. Moreover, the thrombin receptor activating reagent which is a platelet activating reagent is substantially not affected by aspirin. Therefore, even when the patient is using cilostazol and aspirin in combination, the efficacy of cilostazol can be evaluated. In the embodiment of the present invention, the form of the “kit” is not particularly limited, and includes, for example, a cocktail reagent.

One embodiment of the present invention is a cocktail reagent for evaluating the efficacy of cilostazol, comprising a thrombin receptor activating reagent and a cAMP increasing reagent. This cocktail reagent can be prepared by mixing a thrombin receptor activating reagent and a cAMP increasing reagent. This cocktail reagent can be used to evaluate the efficacy of cilostazol by measuring the platelet activation level of patient samples. Moreover, the thrombin receptor activating reagent which is a platelet activating reagent is substantially not affected by aspirin. Therefore, even when the patient is using cilostazol and aspirin in combination, the efficacy of cilostazol can be evaluated. The thrombin receptor activating reagent or cAMP increasing reagent content in the kit or cocktail reagent may be a sufficient amount to activate the platelet function.

One embodiment of the present invention is a method for measuring platelet function, comprising a step of contacting a thrombin receptor activating reagent and a cAMP increasing reagent with a blood-derived sample of a patient taking cilostazol. By using this measurement method, the platelet function of a patient taking cilostazol can be measured by measuring the platelet activation level of a patient sample. Moreover, the thrombin receptor activating reagent which is a platelet activating reagent is substantially not affected by aspirin. Therefore, even when the patient is using cilostazol and aspirin in combination, the efficacy of cilostazol can be evaluated.

One embodiment of the present invention is a method for treating a disease including a step of administering cilostazol to a patient who has undergone a drug efficacy evaluation by the drug efficacy evaluation method according to any one of the above embodiments. The patients include, for example, patients for whom cilostazol has been evaluated as effective. Such diseases include, for example, diseases treatable with cilostazol. The disease includes, for example, a disease based on increased platelet function. The disease includes, for example, infarctions, ischemic symptoms, or diseases based on them. The diseases based on them include, for example, ulcers. The treatment method includes a recurrence suppression method or a prevention method. The dose in the above-mentioned treatment method may be a dose adjusted based on the drug efficacy evaluation result. One embodiment of the present invention is a method for treating a disease, comprising the step of administering an antiplatelet drug other than cilostazol to a patient for whom cilostazol was not evaluated as effective by the drug efficacy evaluation method according to any of the above embodiments. is there.

It should be noted that various detailed embodiments relating to the above-described drug efficacy evaluation method can also be applied to the above-described kit, cocktail reagent, platelet function measurement method, and treatment method.

In one embodiment of the present invention, the “patient” may be, for example, a patient who has developed a symptom associated with increased platelet function, a patient who has a possibility of developing it, or a patient who has been diagnosed as having a possibility of developing a disease. Good. Symptoms associated with increased platelet function include, for example, ischemic diseases, thrombi, emboli, or blood flow disorders. The patient may be a human or a mammal other than a human (eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee). Included).

Also, the patient may be a patient who has been or will be administered an antiplatelet drug. The patient may be a patient who has been or will be administered cilostazol. The patient may be a patient who has been or will be administered cilostazol and an antiplatelet drug other than cilostazol. In addition, the patient may be a patient receiving combination therapy with cilostazol and an antiplatelet drug other than cilostazol. Combination therapy includes medical methods in which two or more drugs are administered to a patient. The interval at which two or more drugs are administered to the patient may be the interval employed in general combination therapy. This interval may be, for example, simultaneously or within a certain period. The fixed period may be, for example, 1 to 24 hours, 1 to 28 days, 1 to 6 months, 1 to 30 years, or may be within the range of any two of them.

The antiplatelet drugs other than cilostazol include, for example, a platelet activation signal inhibitor or a platelet inhibitory signal enhancer. Platelet activation signal inhibitors include, for example, arachidonic acid metabolism inhibitors, ADP receptor inhibitors, GPIIb / IIIa inhibitors, serotonin receptor inhibitors, GPIb / IX / V inhibitors, or GPVI inhibitors. Arachidonic acid metabolism inhibitors include, for example, acetylsalicylic acid (aspirin), ozagrel sodium (ozagrel), eicosapentaenoic acid, etc. (in parentheses are examples of trade names; the same applies hereinafter). ADP receptor inhibitors include, for example, thienopyridines. Thienopyridine drugs include, for example, ticlopidine hydrochloride (panarzine), clopidogrel sulfate (Plavix), and prasugrel hydrochloride (effient). Serotonin receptor inhibitors include, for example, sarpogrelate hydrochloride (Amprag).

The platelet inhibitory signal enhancer includes, for example, a PGl2 derivative, a PGE1 derivative, or an adenosine reabsorption inhibitor. PGl2 derivatives include, for example, beraprost sodium (Dorner). PGE1 derivatives include, for example, Limaprost® alphadex (Opalmon). Adenosine reabsorption inhibitors include, for example, dipyridamole (Persantin). The antiplatelet drug other than cilostazol may be one or more antiplatelet drugs. In this specification, “two or more agents” may be 2, 3, 4, or 5 agents or more, and may be in the range of any two of them.

All publications and publications (patents or patent applications) cited in this specification are incorporated by reference in their entirety.

In this specification, “or” is used when “at least one or more” of the items listed in the text can be adopted. The same applies to “or”. In this specification, when “in the range of two values” is specified, the range includes the two values themselves. In the present specification, “A to B” means A or more and B or less.

As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above can also be employ | adopted. Moreover, it is also possible to adopt a combination of the configurations described in the above embodiments.

Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited thereto.

<Example 1>
1) Reagents and Materials Prostaglandin E1 (PGE1), a cAMP increasing reagent, was purchased from Cayman Chemical Company (MI, USA). Cilostazol was provided by Otsuka Pharmaceutical Co., Ltd. The VerifyNow IIb / IIIa cassette was purchased from Accumetrics (CA, USA). A vacuum blood collection tube containing sodium citrate (blood anticoagulant) having a calcium chelating action was purchased from Nipro Corporation (Osaka).

2) Blood collection and measurement samples In in vitro experiments, the subjects were healthy adults who did not take drugs within 2 weeks, or patients with cerebral infarction who did not take antiplatelet drugs within 2 weeks. In ex vivo experiments, patients with cerebral infarction taking cilostazol, aspirin, and clopidogrel as antiplatelet drugs (depending on experimental system).

The blood of the subject was collected in a vacuum blood collection tube containing sodium citrate. At this time, it was performed so that 3.2% sodium citrate and blood had a ratio of 1: 9. Whole blood was used for measurement by VerifyNow. Platelet rich plasma (PRP) was used for measurement by AG-10. This PRP was prepared by centrifuging whole blood at 900 rpm for 10 minutes. Further, platelet poor plasma (PPP) was prepared by further centrifuging at 3000 rpm for 10 minutes.

3) Measurement of platelet aggregation ability with VerifyNow VerifyNow (Accumetrics, CA, USA) is a whole blood platelet aggregation ability measurement device based on the absorbance method. For the platelet aggregation capacity measurement system, a dedicated cassette for VerifyNow GPIIb / IIIa assay in which iso-TRAP (iso-thrombin receptor activation peptide), which is a thrombin receptor activation reagent, was encapsulated was used. The measurement was performed according to the instruction manual.

3-1) In vitro experiment After setting the above-mentioned dedicated cassette in VerifyNow, 3 μM cilostazol was added to a blood collection tube containing blood and allowed to stand at room temperature for 2 minutes, and PGE1 was added and allowed to stand for another 2 minutes. When this blood collection tube is inserted into a dedicated cassette, blood is automatically sucked and measurement is started, and the result is output after a predetermined time has elapsed. The measurement results were displayed in PAU (Platelet Aggregation Units), which is an arbitrary unit set by the manufacturer, and this value was used to evaluate platelet aggregation (see Fig. 1). As a result, when TARP and PGE1 were used, platelet aggregation was significantly suppressed when cilostazol was present compared to when it was absent.

3-2) Ex vivo experiment (single administration)
The subjects were patients who received a single dose of cilostazol 100 mg. PGE1 was added to the blood collection tube containing the subject's blood, allowed to stand for 2 minutes, and inserted into the dedicated cassette for measurement (see FIG. 2). As a result, when TARP and PGE1 were used, platelet aggregation was significantly suppressed after taking cilostazol compared to before taking.

3-2) Ex vivo experiment (continuous administration)
The subject was a cerebral infarction patient who was transported urgently, and collected blood for measuring platelet aggregation before taking cilostazol before starting treatment. Thereafter, treatment for cerebral infarction was performed, and patients who continued to take cilostazol alone (50 to 150 mg / day) for 2 weeks or longer, collected blood after taking cilostazol (1 hour 30 minutes to 3 hours 15 minutes) Platelet aggregation ability was measured using a cassette (see FIG. 3). As a result, platelet aggregation before taking cilostazol was 241 ± 55 PAU for 3nM PGE1 and 192 ± 55 PAU for 10nM PGE1, compared to the platelet aggregation in the absence of PGE1 (221 ± 37 PAU). I couldn't. On the other hand, compared with the platelet aggregation ability in the absence of PGE1 after taking cilostazol (238 ± 56 PAU), 3nM PGE1 was 153 ± 52 PAU and 10nM PGE1 was 60 ± 29 PAU, showing significant suppression.

4) Measurement of platelet aggregation ability with AG-10 Platelet aggregation ability measurement device AG-10 (Kowa Co., Ltd.) was used. The blood was used before taking the antiplatelet drug or 2 hours after taking the antiplatelet drug. Measurement was performed by adding 300 μL of PPP to the cuvette, and the transmittance of PPP was set to 100%. After adding 300 μL of PRP to the cuvette and heating at 37 ° C. for 2 minutes, the cuvette was set on AG-10 and the transmittance was set to 0%. The measurement was started by pressing the start button. After 30 seconds, a platelet activating reagent was added and the platelet aggregation ability was measured for 5 minutes. This measurement was performed in an ex vivo experiment. Before setting the cuvette, PGE1 was added to PRP and allowed to stand for 2 minutes before starting the measurement.

As the platelet activating reagent, TRAP (thrombin receptor activating peptide), CRP (collagen related peptide), and epinephrine were used, and the measurement data were evaluated by the maximum aggregation rate (%). For TRAP and CRP, individual differences in platelet aggregation were examined using blood from healthy individuals. The above results are shown in FIGS. 4 and 5, the TRAP concentrations were 50 μM and 30 μM, respectively. In FIG. 7, “without cilostazol” means a patient group receiving one or two of aspirin and clopidogrel. “With cilostazol” means a group of patients receiving one or two of aspirin and clopidogrel in addition to cilostazol.

As can be seen from FIGS. 4 and 5, when TARP and PGE1 were used, platelet aggregation was significantly suppressed after taking cilostazol compared to before taking. Further, as can be seen from FIG. 6, when TRAP was used, individual differences were significantly smaller than epinephrine, and stable platelet reactivity was exhibited. As can be seen from FIG. 7, platelet aggregation was not significantly suppressed when CRP was used.

From the above results, it was revealed that the efficacy of cilostazol can be evaluated by using a thrombin receptor activating reagent and a cAMP increasing reagent. In addition, it was revealed that this evaluation system has remarkably small individual differences. In addition, because TRAP is not affected by aspirin or the like due to its mechanism, it is possible to evaluate the efficacy of cilostazol even when administered in combination with aspirin or the like.

In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Claims (6)

A method for evaluating the efficacy of cilostazol using a thrombin receptor activating reagent and a cAMP increasing reagent. 2. The method according to claim 1, comprising a step of bringing a thrombin receptor activating reagent and a cAMP increasing reagent into contact with a blood-derived sample. 3. The method according to claim 2, wherein the blood-derived sample is a blood-derived sample of a patient taking cilostazol. 4. The method according to claim 2, wherein the blood-derived sample is a blood-derived sample of a patient who has been administered cilostazol and an antiplatelet drug other than cilostazol. A method for measuring platelet function, comprising a step of bringing a thrombin receptor activating reagent and a cAMP increasing reagent into contact with a blood-derived sample of a patient taking cilostazol. A kit for evaluating the efficacy of cilostazol containing a thrombin receptor activating reagent and a cAMP increasing reagent.

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