WO2014057699A1

WO2014057699A1 - Pharmacokinetic evaluation method and kit for pharmacokinetic evaluation

Info

Publication number: WO2014057699A1
Application number: PCT/JP2013/060904
Authority: WO
Inventors: 山下　伸二; 祥江政岡; 一天富樫
Original assignee: 株式会社住化分析センター
Priority date: 2012-10-09
Filing date: 2013-04-11
Publication date: 2014-04-17
Also published as: JPWO2014057699A1

Abstract

The problem of the present invention is to provide a pharmacokinetic evaluation method and kit for pharmacokinetic evaluation with which accurate pharmacokinetic evaluation can be performed in one measurement without using a radioactive substance. The problem is solved by a pharmacokinetic evaluation method and kit for pharmacokinetic evaluation with which pharmacokinetic data is obtained from changes over time in the blood concentration in a blood sample collected from a subject following extravascular administration of a compound followed by intravenous administration of the stable isotope substitute of the compound.

Description

Pharmacokinetic evaluation method and pharmacokinetic evaluation kit

The present invention relates to a pharmacokinetic evaluation method and a pharmacokinetic evaluation kit.

In drug development, hundreds of thousands of candidate drug compounds are narrowed down to about several at the stage of non-clinical trials and transferred to clinical trials. However, even those compounds that have entered clinical trials may be abandoned due to problems associated with pharmacokinetics, ie drug absorption, distribution, metabolism, and excretion. If development is abandoned in clinical trials due to pharmacokinetic problems after gathering a lot of necessary data, the development time and development costs up to that time are wasted.

Therefore, it is extremely important to perform pharmacokinetic evaluation at the stage of non-clinical trials or at the initial stage of clinical trials. Bioavailability (bioavailability) is known as a basic pharmacokinetic parameter serving as a pharmacokinetic index. The bioavailability is a constant that represents the rate at which an extravascularly administered drug reaches the systemic circulation, and is calculated with the bioavailability when the drug is administered intravenously as 100%. .

Conventionally, as a method for calculating bioavailability, the blood concentration of the drug when the drug is administered extravascularly by a method such as oral administration and the blood of the drug when the same drug is separately administered intravenously A method of calculating from the medium concentration is known.

On the other hand, the microdose test, in which a very small amount of drug candidates that do not require consideration of acute or chronic toxicity, is administered to a subject in an extremely small amount intravenously, is attracting attention as a method for evaluating pharmacokinetics safely and in a short period of time. Collecting.

As a method for obtaining a more accurate bioavailability by applying the above microdose test, a small amount of [ ¹⁴ C] radiolabel is injected at the same time as the oral administration of the drug, and a biological test is performed in one test. A method for obtaining a scientific availability has been proposed (Non-patent Document 1). This method is used overseas in human clinical trials for microdose clinical trials in which a small amount of [ ¹⁴ C] radiolabel is intravenously administered simultaneously with oral administration of a clinical dose of a test drug.

However, the method described in Non-Patent Document 1 has a great resistance to radioactive substances in Japan, and it takes a lot of time and money to manufacture an intravenous preparation labeled with a radioactive substance and to ensure its safety. The use is not progressing because of this. As for microdose clinical trials, “Ministry of Health, Labor and Welfare” provided “Guidance on Implementation of Microdose Clinical Trials” in June 2008, but it has not been conducted yet.

In order to measure a drug labeled with a radioactive substance, an accelerator mass spectrometer (AMS: Accelerator-Mass-Spectrometry, hereinafter referred to as AMS in this specification) or a positron emission tomography apparatus (PET) is used. : Positron Emission Tomography (hereinafter sometimes referred to as PET in this specification)), but these require large-scale facilities and special equipment for using radioactive materials, and can be measured. There is also a problem that facilities are limited.

The present invention has been made to solve the above-described problems, and its purpose is to perform an accurate pharmacokinetic evaluation in one measurement by one test without using a radioactive substance. The object is to provide a pharmacokinetic evaluation method and a pharmacokinetic evaluation kit.

In order to solve the above problems, the pharmacokinetic evaluation method according to the present invention is a test in which a stable isotope substitute in which the same compound as the above compound is labeled with a stable isotope is administered intravenously after the compound is administered extravascularly. From the step of measuring the concentration of the compound and the stable isotope substitute in a blood sample collected over time from the body, and the time course of the blood concentration measured in the step of measuring, the drug of the compound It is characterized by including a step of acquiring dynamic data.

In order to solve the above problems, a kit for evaluating pharmacokinetics according to the present invention includes (a) a compound to be evaluated, and (b) a stable isotope substituent obtained by labeling the same compound as the above compound with a stable isotope. (C) a first step in which the compound is administered extravascularly, a second step in which a stable isotope substitute in which the same compound as the compound is labeled with a stable isotope is administered intravenously after the compound is extravascularly administered, A third step of measuring the time course of blood concentration of the compound and the stable isotope substitute, and a fourth step of obtaining pharmacokinetic data of the compound from the time course of blood concentration measured in the third step And an instruction sheet for performing pharmacokinetic evaluation according to the process.

As described above, the method for evaluating pharmacokinetics according to the present invention is a method in which a stable isotope substitution product obtained by intravenously administering a stable isotope substitute obtained by labeling the same compound as the above compound with a stable isotope after the compound has been administered extravascularly. Pharmacokinetic data of the compound from the step of measuring the concentration of the compound and the stable isotope substitute in the blood sample collected and the time course of the blood concentration measured in the step of measuring Since the acquisition step is included, the pharmacokinetic evaluation can be performed without using a radioactive substance. Then, blood collected over time from a subject to whom a stable isotope substitute labeled with a stable isotope is labeled with a stable isotope while the compound administered extravascularly is present in the blood. When measuring the concentration of the compound and the stable isotope substituent in the sample, there is an effect that accurate pharmacokinetic evaluation can be performed by one measurement by one test. .

The pharmacokinetic evaluation kit according to the present invention includes, as described above, (a) a compound to be evaluated, and (b) a stable isotope substitute obtained by labeling the same compound as the above compound with a stable isotope, (C) a first step in which the compound is administered extravascularly, a second step in which a stable isotope substituent in which the same compound as the compound is labeled with a stable isotope is intravenously administered after the compound is administered extravascularly, The third step of measuring the time course of the blood concentration of the compound and the stable isotope substitute, and the fourth step of acquiring pharmacokinetic data of the compound from the time course of the blood concentration measured in the third step Thus, there is an instruction that the pharmacokinetic evaluation is performed, so that the pharmacokinetic evaluation can be performed without using a radioactive substance. Then, blood collected over time from a subject to whom a stable isotope substitute labeled with a stable isotope is labeled with a stable isotope while the compound administered extravascularly is present in the blood. When measuring the concentration of the compound and the stable isotope substituent in the sample, there is an effect that accurate pharmacokinetic evaluation can be performed by one measurement by one test. .

In Reference Example 1, it is a figure which shows the result of having conducted the in vitro metabolic experiment of chlorpromazine and its deuterium substitution product. In Reference Example 2, it is a figure which shows the result of having performed the in vivo disappearance clearance experiment of chlorpromazine and its deuterium substitution. In Example 1, it is a figure which shows the blood concentration-time curve when performing the microdose test by [oral dosage: intravenous dosage] = [100: 1]. In Example 2, it is a figure which shows the blood concentration-time curve when the test by [oral dosage: intravenous dosage] = [10: 1] was done. In Comparative Example 1, it is a diagram showing a blood concentration-time curve of chlorpromazine administered orally. In Comparative Examples 1 and 2, it is a figure which shows the blood concentration-time curve of the chlorpromazine administered intravenously, or its deuterium substitution.

Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to this, and can be implemented in a mode in which various modifications are made within the described range. Unless otherwise specified in this specification, “A to B” representing a numerical range means “A or more (including A and greater than A)” and “B or less (including B and less than B)”.

(I) Pharmacokinetic evaluation method according to the present invention The pharmacokinetic evaluation method according to the present invention is the intravenous administration of a stable isotope substitute obtained by labeling the same compound as the above compound with a stable isotope after the compound has been administered extravascularly. From the step of measuring the concentration of the compound and the stable isotope substitute in the blood sample collected over time from the obtained subject, and the change over time in the blood concentration measured in the step of measuring, Obtaining pharmacokinetic data of the compound. Hereinafter, the step in which the compound is administered extravascularly is the first step, the step in which the stable isotope substitute labeled with the stable isotope is compounded intravenously is the second step, and the compound is administered extravascularly. Of the compound and the stable isotope substitute in a blood sample collected over time from a subject to which a stable isotope substitute labeled with the same compound as the above compound was intravenously administered. The step of measuring the concentration will be described as the third step, and the step of obtaining the pharmacokinetic data of the compound from the change over time in the blood concentration measured in the step of measuring will be described as the fourth step.

(I-1) First Step In the first step, a compound that is a target for pharmacokinetic evaluation is administered to a subject extravascularly. Here, the compound which is the target of the pharmacokinetic evaluation method according to the present invention is usually a drug candidate compound or a compound used as a drug. Such a compound is not particularly limited as long as it is a compound containing an atom having a stable isotope. More specifically, the compound preferably contains at least one of a hydrogen atom, a carbon atom, a nitrogen atom, and an oxygen atom.

The above compound has such a number that the molecular weight changes by 3 or more, more preferably the molecular weight increases by 3 or more when an atom having a stable isotope in the molecule is substituted with the stable isotope of the atom. It is more preferable that it contains. Thereby, the said compound and the compound substituted with the stable isotope can be distinguished and analyzed.

The above compound is more preferably a low molecular compound. More specifically, the molecular weight of the above compound is more preferably 100 to 2000, and further preferably 100 to 1000. The molecular weight of a compound used as a drug is often 100 or more. Moreover, since the molecular weight is 2000 or less, such a compound is preferable because it is easily absorbed by extravascular administration.

The above compound is not particularly limited, and examples thereof include chlorpromazine, phenytoin, nifedipine, diltiazem, nicardipine, erythromycin, simvastatin, atorvastatin and the like.

In this step, the compound is administered extravascularly by the intended administration method for pharmacokinetic evaluation. For drugs that are expected to have systemic effects, the amount that reaches the systemic circulation is involved in drug treatment. The above-mentioned compounds administered extravascularly are incompletely absorbed or are metabolized and excreted before reaching the systemic circulating blood, so the amount reaching the systemic circulating blood decreases, and all the systemic circulating blood It is not always possible to reach In the pharmacokinetic evaluation method according to the present invention, for example, the drug of a compound is used with the bioavailability (bioavailability), which is a pharmacokinetic parameter indicating the rate at which extra-vascularly administered drug reaches the systemic circulation blood as an index. Perform dynamic evaluation.

Here, extravascular administration is not particularly limited as long as it is an administration method other than intravascular administration, for example, oral administration, intraperitoneal administration, intramuscular administration, transdermal administration, respiratory administration, ophthalmic administration, It is intended to include nasal administration or subcutaneous administration.

Further, the amount of the compound to be administered extravascularly is not particularly limited, but it is preferably not less than the amount of drug effect from the viewpoint of performing pharmacokinetic evaluation. In a clinical trial, when the pharmacokinetic evaluation according to the present invention is simultaneously performed, the amount of the compound administered extravascularly may be a clinical dose.

(I-2) Second Step In the second step, after the compound is administered extravascularly, a stable isotope substitute obtained by labeling the same compound as the above compound with a stable isotope is intravenously administered. In this step, “administered intravenously” means that the stable isotope substitute is intravenously administered to a subject to which the compound has been administered extravascularly in the first step.

Here, “after the compound is administered extravascularly” may be after the extravascular administration, but the compound administered extravascularly is present in the blood, ie, administered extravascularly. More preferably, it is carried out before the compound has completely disappeared from the blood, and it is even more preferable that the blood concentration of the compound reaches the maximum blood concentration. The above-mentioned compound administered extravascularly increases in blood concentration depending on the degree of absorption in the subject. On the other hand, the administered compound disappears by metabolism or degradation, and its blood concentration decreases. When the absorption rate and disappearance rate of the above-mentioned compound are equal, the blood concentration becomes the maximum value. The concentration of the compound at this time is called the maximum blood concentration.

The time at which the blood concentration of the above compound reaches the maximum blood concentration, that is, the time to reach the maximum blood concentration can be determined, for example, by the following method. That is, at a plurality of time points after the compound is administered extravascularly, the concentration of the compound is measured for blood collected from the subject, and the horizontal axis represents time and the vertical axis represents the compound blood concentration. The time when the blood concentration of the above compound reaches the maximum value is defined as the time for reaching the maximum blood concentration.

The vicinity of the blood concentration of the above compound reaching the maximum blood concentration is more preferably 10 minutes before the maximum blood concentration arrival time to 60 minutes after the maximum blood concentration arrival time, and the maximum blood concentration reached. Particularly preferred is within 60 minutes after the time.

“After the compound is administered extravascularly” usually means 30 minutes to 360 minutes after the time when the compound is administered extravascularly, more preferably 60 minutes to 240 minutes later.

Thus, the compound in a blood sample collected over time from a subject to which the stable isotope substitute was administered intravenously while the compound administered extravascularly is present in the blood, and By measuring the concentration of the stable isotope substitute, the change in the blood concentration of the compound and the stable isotope substitute with time can be measured in a single measurement.

In addition, while the compound administered extravascularly is present in the blood, a stable isotope substitution product obtained by labeling the same compound as the above compound with a stable isotope is administered intravenously, so that Pharmacokinetic evaluation is performed under the same conditions as in the case of extravascular administration (the blood concentration of the compound, metabolism, respiration, and excretion). be able to. Therefore, a more accurate pharmacokinetic assessment can be performed in a single measurement by a single test.

The present invention aims to provide a pharmacokinetic evaluation method capable of performing accurate pharmacokinetic evaluation in one measurement by one test without using a radioactive substance. The compound in a blood sample collected over time from a subject to whom a stable isotope substitute labeled with a stable isotope is labeled with a stable isotope after the compound is administered extravascularly; and The problem is solved by measuring the concentration of the stable isotope substitution product. That is, according to the above configuration, since the compound and its stable isotope substitute can be analyzed separately, the concentration of the compound and the stable isotope substitute in a blood sample simultaneously containing these compounds can be analyzed. Can be measured simultaneously. In addition to the configuration of the present invention in which a stable isotope substituent obtained by labeling the same compound as the above compound with a stable isotope is administered intravenously after the compound is administered extravascularly, the compound administered extravascularly is treated with blood. After completely disappearing from the inside, the present invention can also be used in an embodiment in which a stable isotope substituent obtained by labeling the same compound as the above compound with a stable isotope is intravenously administered. In such an embodiment, pharmacokinetic evaluation cannot be performed by a single measurement, but pharmacokinetic evaluation can be performed without using a radioactive substance. Therefore, this embodiment is also included in the present invention.

In this step, a stable isotope substitute obtained by labeling the same compound as the above-mentioned compound administered extravascularly in the first step with a stable isotope is intravenously administered. Here, in the present invention, the stable isotope is not particularly limited as long as it is a non-radioactive isotope. More preferably, ² H (D), ¹³ C, ¹⁵ N, or ¹⁸ O can be suitably used as such a stable isotope. These elements are suitable for labeling because they are usually included in drugs or their candidate compounds. The “stable isotope substituted with the same compound as the above compound labeled with a stable isotope” refers to a compound in which an atom contained in the same compound as the above compound is substituted with a stable isotope of the atom. More specifically, a substituted product in which at least one hydrogen atom of the same compound as the above compound containing a hydrogen atom is substituted with ² H (D), at least one carbon atom of the same compound as the above compound containing a carbon atom A substituent in which part is substituted with ¹³ C, a substituent in which at least one nitrogen atom of the same compound as the above compound containing a nitrogen atom is substituted with ¹⁵ N, an oxygen atom of the same compound as in the above compound containing an oxygen atom A substituted product in which at least one part is substituted with ¹⁸ O. The substituted atom may be one type or a combination of two or more types.

In addition, the number of substitutions with a stable isotope in the “stable isotope substituted with the same compound as the above compound with a stable isotope” is the number of substitutions such that the molecular weight of the compound changes by 3 or more, more preferably the molecular weight of the compound The number of substitutions is preferably 3 or more. That is, the stable isotope substitute administered in the second step has a molecular weight more preferably 3 or more compared to the compound administered in the first step, more preferably the molecular weight changes compared to the compound. It is preferably substituted with a stable isotope so as to be 3 or more. In the natural world, there can be compounds with molecular weights of 2 or less, so if the molecular weight has changed by 3 or more, or the molecular weight has increased by 3 or more, the concentration is analyzed by distinguishing it from compounds existing in nature. This is because it can be done.

The method for producing such a stable isotope substituent is not particularly limited, and can be suitably produced using a conventionally known method. Alternatively, a commercially available stable isotope substitute may be used as the stable isotope substitute.

Further, the dose of the stable isotope substitute administered intravenously in this step is greater than 0 and less than or equal to 1/10 by weight with respect to the dose of the compound administered in the first step. It is more preferable that the toxicity to the subject can be suppressed, so that it is preferably 1 / 100,000 to 1/100, more preferably 1/10000 to 1/100, and more preferably 1/1000 to 1/100 is particularly preferable. When the dose of the stable isotope substitute administered intravenously is 1/10 or less by weight with respect to the dose of the compound administered in the first step, stable isotope substitution is achieved. Pharmacokinetic evaluation can be performed from the blood concentration of the body. In addition, since the dose of the stable isotope substitute administered intravenously is a value greater than 0 in weight ratio to the dose of the compound administered in the first step, The blood concentration of the administered stable isotope substitute can be determined, and pharmacokinetic evaluation can be performed.

In addition, the dose of the above-mentioned stable isotope substitute administered intravenously in this step is more preferably 1 / 10,000 to 1/100 of the amount of drug effect in terms of weight ratio. Thereby, pharmacokinetic evaluation can be performed from the blood concentration of a stable isotope substitute without expressing toxicity to a subject.

In this way, pharmacokinetic evaluation can be performed safely and in a short period of time by using a microdose test in which a trace amount of compound that does not require consideration of acute or chronic toxicity is intravenously administered to a subject. It becomes.

In addition, a clinical dose of a compound was administered extravascularly, and a stable isotope substitute labeled with the same compound as the above compound was administered intravenously while the extravasated compound was present in the blood. By measuring the concentration of the compound and the stable isotope substitute in a blood sample collected over time from a subject, intravenous administration under conditions at the time of extravascular administration, that is, under clinical dose loading conditions Administration allows pharmacokinetic evaluation under the same conditions as clinical dose loading conditions (compound blood concentration, metabolism, respiration and excretion) in the subject. Moreover, it can be performed by one test and one measurement. Conventionally, as a problem in a microdose test in which a very small amount of a compound is administered alone, there is no correlation with a clinical dose. This is because the results obtained from the microdose test cannot be used because the condition in the subject is different between the dose used for the microdose test and the clinical dose. For example, since the proportional relationship between the dose of the compound and the blood concentration does not hold between the blood concentration obtained in the microdose test and the blood concentration obtained at the clinical dose (no linearity), the microdose test There was a problem that the data of the above could not be used for clinical dose reference. In this step, the above-mentioned problems in the microdose test are avoided by intravenously administering the stable isotope substitute to the subject to which a clinical dose is administered extravascularly while the compound is present in the blood. be able to.

The second step in which the stable isotope substitute is administered intravenously is preferably performed after the compound is administered extravascularly. However, the present invention is not limited to this. For example, the compound is administered extravascularly on different days. And the step of intravenously administering the stable isotope substitute may be performed.

(I-3) Third Step In the third step, the time course of the blood concentration of the compound and the stable isotope-substituted product is measured. Specifically, in the third step, after the compound was administered extravascularly, a stable isotope substitute obtained by labeling the same compound as the above compound with a stable isotope was collected over time from a subject who was intravenously administered. The concentration of the compound and the stable isotope substitute in the blood sample is measured. That is, from the subject, blood of the compound and the stable isotope substitute in blood collected at a plurality of time points after extravascular administration of the compound and at a plurality of time points after intravenous administration of the stable isotope substitute. Measure the concentration.

At this time, when a stable isotope substitute obtained by labeling the same compound as the above compound with a stable isotope is intravenously administered while the compound administered extravascularly is present in the blood, the compound is removed from the blood vessel. Since blood samples collected over time after administration and blood samples collected over time after intravenous administration of stable isotope substitutes can be performed simultaneously, pharmacokinetic evaluation is performed once per test. It can be done by measuring.

Here, in the present specification, the blood concentration may be a plasma concentration or a serum concentration. Pharmacokinetic evaluation can be performed from any concentration.

In this step, the method for measuring the time-dependent change in the blood concentration of the compound and the stable isotope substitute is particularly limited as long as it is a method capable of separating and quantifying the compound and the stable isotope substitute. is not.

Such a measurement is performed, for example, by liquid chromatography mass spectrometry (hereinafter sometimes referred to as “LC / MS” in the present specification), liquid chromatography tandem mass spectrometry (hereinafter referred to as “LC / MS”). / MS / MS ”) and the like. According to these analysis methods, a stable isotope substitute and the above-mentioned compound in blood collected from a subject can be separated and quantified. Therefore, pharmacokinetic evaluation can be performed using these blood concentrations. The measurement in this step also has the advantage that pharmacokinetic evaluation can be performed relatively easily because it does not require special equipment such as AMS and PET unlike the conventional microdose clinical trial using radioactive materials. Have.

In addition, the method for preparing the collected blood sample, the method for measuring the blood concentration, the interval and the number of times of blood collection for measuring changes over time are not particularly limited, and a conventionally known method can be appropriately selected and used. That's fine.

(I-4) Fourth Step In the fourth step, pharmacokinetic data of the compound is obtained from the change over time in the blood concentration measured in the third step. In the present invention, the method for obtaining pharmacokinetic data is not particularly limited as long as it can be performed using the time-dependent change in blood concentration measured in the third step. Examples of the method for obtaining pharmacokinetic data include a method for calculating bioavailability (bioavailability) which is a pharmacokinetic parameter.

“Bioavailability” is a constant representing the rate at which an administered drug reaches the systemic circulation, and is calculated assuming that the bioavailability when the drug is administered intravenously is 100%. Is done. On the other hand, when the drug is administered by a route other than intravenous administration, the bioavailability decreases because it is affected by absorption, metabolism, excretion and the like before reaching the systemic circulating blood. The bioavailability is calculated using the area surrounded by the blood concentration and the horizontal axis (time axis) (the area under the blood concentration-time curve: AUC), and is calculated by the following equation.
F = ([AUC] po / [AUC] iv) / (DOSEpo / DOSEiv) (1)
Where F is the absolute bioavailability, [AUC] po is the area under the blood concentration-time curve of the compound administered extravascularly, and [AUC] iv is the blood of the stable isotope substitute administered intravenously. The area under the medium concentration-time curve, DOSEpo, represents the dose of compound administered extravascularly. DOSEiv represents the dose of a stable isotope substitute administered intravenously.

Moreover, the pharmacokinetic evaluation method according to the present invention can be suitably used in a microdose clinical trial. Thereby, the information regarding the pharmacokinetics of the drug candidate compound can be obtained at the initial stage of clinical development of the drug. In addition, two doses of clinical and microdose can be performed in one test, and pharmacokinetic data can be acquired.

(II) Pharmacokinetic evaluation kit according to the present invention The present invention also includes a pharmacokinetic evaluation kit,
(A) a compound to be evaluated;
(B) a stable isotope substituent obtained by labeling the same compound as the above compound with a stable isotope,
(C) a first step in which the compound is administered extravascularly, a second step in which a stable isotope substituent in which the same compound as the compound is labeled with a stable isotope is intravenously administered after the compound is administered extravascularly, A third step of measuring the time course of the blood concentration of the compound and the stable isotope substitute, and a fourth step of acquiring pharmacokinetic data of the compound from the time course of the blood concentration measured in the third step A kit including an instruction to perform pharmacokinetic evaluation according to the process is also included.

The compound to be evaluated and the stable isotope substituent obtained by labeling the same compound as the above compound with a stable isotope are as described in (I) above.

The kit according to the present invention is the same as the above compound after the compound to be evaluated and the stable isotope substituted with the same compound as the above compound labeled with a stable isotope, and further after the compound is administered extravascularly. A second step of intravenously administering a stable isotope substituent labeled with a stable isotope, a third step of measuring the time course of blood concentration of the compound and the stable isotope substitute, and a third step. It is only necessary to include an instruction to perform pharmacokinetic evaluation in the fourth step of acquiring pharmacokinetic data of the compound from the change in blood concentration measured in the step.

The first to fourth steps in the above instruction are also as described in the above (I), so the description is omitted here.

(III) Summary In order to solve the above problems, the pharmacokinetic evaluation method according to the present invention is characterized in that a stable isotope substituent obtained by labeling the same compound as the above compound with a stable isotope after intravenous administration of the compound is intravenously administered. From the step of measuring the concentration of the compound and the stable isotope substitute in the blood sample collected over time from the administered subject, and the time course of the blood concentration measured in the step of measuring, It includes a step of obtaining pharmacokinetic data of a compound.

According to the above configuration, there is an effect that pharmacokinetic evaluation can be performed without using a radioactive substance. Then, blood collected over time from a subject to whom a stable isotope substitute labeled with a stable isotope is labeled with a stable isotope while the compound administered extravascularly is present in the blood. When measuring the concentration of the compound and the stable isotope substitute in the sample, there is an effect that accurate pharmacokinetic evaluation can be performed by one measurement by one test.

In the pharmacokinetic evaluation method according to the present invention, the dose of the stable isotope substituent administered intravenously is greater than 0 by weight ratio with respect to the dose of the compound administered extravascularly. It is preferable that it is 10 or less.

In the pharmacokinetic evaluation method according to the present invention, the stable isotope is preferably ² H (D), ¹³ C, ¹⁵ N or ¹⁸ O.

In the pharmacokinetic evaluation method according to the present invention, the stable isotope substitute administered intravenously is more preferably administered extravascularly such that the molecular weight changes by 3 or more compared to the compound administered extravascularly. It is preferably substituted with a stable isotope so that the molecular weight is 3 or more larger than that of the above compound.

In the pharmacokinetic evaluation method according to the present invention, in a blood sample collected over time from a subject to which the stable isotope substitute was administered intravenously while the compound administered extravascularly is present in the blood. It is preferable to measure the change in blood concentration of the compound and the stable isotope substitute with time by measuring the concentration of the compound and the stable isotope substitute in a single measurement.

In the pharmacokinetic evaluation method according to the present invention, the amount of the compound administered extravascularly is preferably a clinical dose.

In the pharmacokinetic evaluation method according to the present invention, it is preferable to calculate the bioavailability of the compound.

In the method for selecting drug candidate compounds according to the present invention, it is preferable to use the results obtained by the pharmacokinetic evaluation method.

In order to solve the above problems, a kit for evaluating pharmacokinetics according to the present invention includes (a) a compound to be evaluated, and (b) a stable isotope substituent obtained by labeling the same compound as the above compound with a stable isotope. (C) a first step in which the compound is administered extravascularly, and a second step in which a stable isotope substituent in which the same compound as the compound is labeled with a stable isotope is intravenously administered after the compound is extravascularly administered. A third step of measuring the time course of blood concentration of the compound and the stable isotope substitute, and a fourth step of obtaining pharmacokinetic data of the compound from the time course of blood concentration measured in the third step And an instruction sheet for performing pharmacokinetic evaluation according to the process.

In the pharmacokinetic evaluation kit according to the present invention, it is preferable that the stable isotope substitute is greater than 0 and 1/10 or less by weight with respect to the compound to be evaluated.

In the pharmacokinetic evaluation kit according to the present invention, the stable isotope is preferably ² H (D), ¹³ C, ¹⁵ N or ¹⁸ O.

In the pharmacokinetic evaluation kit according to the present invention, the stable isotope substitute preferably has a molecular weight of 3 compared to the compound to be evaluated so that the molecular weight changes by 3 or more compared to the compound to be evaluated. It is preferable to be substituted with a stable isotope so as to be larger.

In the pharmacokinetic evaluation kit according to the present invention, in the second step, a stable isotope substitute obtained by labeling the same compound as the above compound with a stable isotope while the compound administered extravascularly is present in the blood. It is preferably administered intravenously, and in the third step, measurement of the time course of blood concentration of the compound and the stable isotope substitute is preferably performed in a single measurement.

In the pharmacokinetic evaluation kit according to the present invention, in the first step, the amount of the compound to be administered is preferably a clinical dose.

The present invention will be described more specifically based on examples and FIGS. 1 to 6, but the present invention is not limited to this.

First, the reagents, materials used, and measurement conditions used in this example will be described.

(Reagent used)
As a model drug, chlorpromazine represented by the following formula (1) (hereinafter, sometimes referred to as “CPZ” in the present specification, molecular weight: 319), and heavy hydrogen in which 6 hydrogen atoms are substituted by deuterium. Chlorpromazine-d ₆ (hereinafter, sometimes referred to as “CPZ-d ₆ ” in the present specification) (molecular weight: 325) represented by the following formula (2), which is a hydrogen substitution product, was used.

CPZ was purchased from SIGMA-ALDRICH, and CPZ-d ₆ was purchased from LKT Laboratories.

For other reagents used in this example, commercially available special grade products were used.

(Materials used)
SD male rats were purchased from Shimizu Experimental Materials Co., Ltd. SD rat liver microsomes were purchased from XenoTech.

(Measurement condition)
The measurement of the blood concentration of the target compound in Reference Example 1 and Reference Example 2 and Comparative Examples 1 and 2 was performed using LC / MS / MS (LC: Acquity UPLC (Waters), MS: Acquity TQD (Waters)). The measurement conditions shown in Table 1 below were performed.

The measurement of the blood concentration of the target compound in Examples 1 and 2 is shown in Table 2 below using LC / MS / MS (LC: LC-20A system (Shimadzu Corporation), MS: AP15000 (AB Sciex)). The measurement was performed under the conditions shown.

[Reference Example 1: In vitro metabolism experiment of CPZ and its deuterium substitutes]
In order to examine whether the substitution of the target compound with deuterium affects liver metabolism, in vitro metabolism experiments of CPZ and its deuterium substitute were performed. Specifically, CPZ and its deuterium substitute were exposed to SD rat liver microsome in an SD rat liver microsome incubation solution, and the disappearance of CPZ and its deuterium substitute due to liver metabolism was measured by LC / MS / MS. And measured.

A final concentration of CPZ of 10 ng / mL, a final concentration of SD rat liver microsomes of 0.2 mg / mL, a final concentration of phosphate buffer (pH 7.4) of 0.1 M, and MgCl ₂ These were mixed so that the final concentration was 3.3 mM to prepare a reaction solution for measuring the metabolic activity of CPZ. A NADPH solution was prepared by mixing so that the final concentration of NADHP was 1.3 mM and the final concentration of MgCl ₂ was 3.3 mM. The prepared metabolic activity measurement reaction solution was preincubated at 37 ° C. for 5 minutes, and then the NADPH solution was added to the preincubated metabolic activity measurement reaction solution to start the reaction. After incubating at 37 ° C. for a fixed time, a certain amount (0.05 mL) of the incubated reaction solution was taken out at a plurality of time points and added to ice-cooled acetonitrile (0.45 mL) to stop the reaction. The reaction solution after stopping the reaction was centrifuged, and the concentration of CPZ in the supernatant was measured using LC / MS / MS.

Then, except that the CPZ and _{CPZ-d 6} in the same manner, was carried out in vitro metabolism experiments of _{CPZ-d 6.}

Here, the above-mentioned “final concentration” refers to the concentration in the reaction solution after the NADPH solution is added to the pre-incubated metabolic activity measurement reaction solution.

The results are shown in FIG.

In FIG. 1, the vertical axis indicates the concentration of CPZ or CPZ-d _{6 in} the reaction solution, and the horizontal axis indicates the time from the start of the reaction. In Table 3, the metabolic rate is a value obtained by dC / dt = k * C (0). Where dC / dt is the metabolic rate, k is the disappearance rate constant (−ln (drug unchanged concentration in the reaction solution)) and the slope of the straight line obtained from the time, and C (0) is the initial drug concentration in the reaction solution. To express. In addition, n = 4 indicates that the same experiment was performed four times and an average was obtained. As shown in Table 3, since no significant difference was observed in the metabolic rate and metabolic rate of CPZ-d ₆ of CPZ, due to the fact that the subject compounds have been substituted by deuterium, effects on liver metabolism It turned out that it was not admitted.

[Reference Example 2: In vivo disappearance clearance experiment of CPZ and its deuterium substitutes]
In order to examine whether the substitution of the target compound with deuterium affects the elimination clearance, an in vivo elimination clearance experiment of CPZ and its deuterium substitution was performed.

Specifically, 0.02 mg of CPZ was intravenously administered to SD male rats. Thereafter, blood was collected at multiple time points, and the plasma concentration of CPZ was measured using LC / MS / MS. Then, except that the CPZ and _{CPZ-d 6} in the same manner, was carried out in vivo loss clearance experiment of _{CPZ-d 6.} The results are shown in FIG.

FIG. 2 is a graph showing a blood concentration-time curve, in which the vertical axis represents plasma concentration and the horizontal axis represents time since intravenous administration. In Table 4, t _1/2 represents a half-life, k _el represents a drug elimination rate constant, CL _tot represents systemic clearance, and AUC _0-∞ represents the area under the blood concentration-time curve. N represents the number of samples. Systemic clearance refers to the amount of drug metabolized and excreted in a certain time converted to volume. As shown in Table 4, since there was no significant difference between CPZ and CPZ-d ₆ , the disappearance clearance of CPZ-d ₆ substituted by deuterium is equivalent to CPZ. It was confirmed.

From the results of Reference Examples 1 and 2, it was confirmed that no isotope effect was observed in the pharmacokinetics.

[Example 1: Evaluation of bioavailability by microdose test with [oral dose: intravenous dose] = [100: 1]]
CPZ 0.2 mg was orally administered to SD male rats. The amount of CPZ administered orally was determined by converting the dose administered to humans from the body weight of SD rats. 1.5 hours after oral administration, were administered _{CPZ-d 6} of 0.002mg is one-hundredth of the oral dose intravenously to the same SD rats. Blood was collected at multiple time points and the blood levels of CPZ and CPZ-d ₆ were measured. Specifically, extracted CPZ, the CPZ-d ₆ and the internal standard from the plasma separated from blood were bled with diethyl ether under alkaline conditions by the addition of ammonium bicarbonate buffer. After evaporation to dryness, redissolve in methanol / 1% formic acid solution (1: 1, v / v), measure the amount of CPZ and CPZ-d ₆ using LC / MS / MS, and calculate blood concentration did. The obtained results are shown in FIG. Further, Table 5 shows the bioavailability calculated by the above formula (1) from the relationship between the area under the blood concentration-time curve (AUC _0-∞ ) obtained from FIG. 3 and the dose.

In FIG. 3, the vertical axis represents plasma concentration (unit: ng / mL), and the horizontal axis represents time after oral administration of CPZ. In Table 5, AUC _0-∞ represents the area under the blood concentration-time curve, BA represents bioavailability, and n represents the number of samples collected at one time point. PO represents oral administration, and IV represents intravenous administration.

[Example 2: Evaluation of bioavailability by test with [oral dose: intravenous dose] = [10: 1]]
CPZ 0.2 mg was orally administered to SD male rats. The amount of CPZ administered orally was determined by converting the dose administered to humans from the body weight of SD rats. 1.5 hours after oral administration, were administered _{CPZ-d 6} of 0.02mg is one tenth of an oral dosage intravenously. Blood was collected at multiple time points, and the blood concentrations of CPZ and CPZ-d ₆ were measured in the same manner as in Example 1. The obtained results are shown in FIG. In FIG. 4, the vertical axis represents the plasma concentration of CPZ (unit: ng / mL), and the horizontal axis represents the time after oral administration of CPZ. However, the plasma concentration of _{CPZ-d 6,} the vertical axis represents the plasma concentration / 10 of _{CPZ-d 6.} Further, Table 5 shows the bioavailability calculated by the above equation (1) from the relationship between the area under the blood concentration-time curve (AUC _0-∞ ) obtained from FIG. 4 and the dose.

[Comparative Example 1]
CPZ 0.2 mg was orally administered to SD male rats. In addition, 0.02 mg of CPZ was intravenously administered to separate SD male rats. Blood was collected at multiple time points, and the blood concentration of CPZ was measured. Specifically, extracted CPZ, the CPZ-d ₆ and the internal standard from the plasma separated from blood were bled with diethyl ether under alkaline conditions by the addition of ammonium bicarbonate buffer. After evaporation to dryness, the sample was redissolved in a methanol / 1% formic acid solution (1: 1, v / v), the amount of CPZ was measured using LC / MS / MS, and the blood concentration was calculated. The obtained results are shown in FIGS. Further, Table 6 shows bioavailability calculated from the relationship between the area under the blood concentration-time curve (AUC _0-∞ ) obtained from FIG. 6 and the dose of CPZ administered orally.

5 and 6, the vertical axis indicates plasma concentration, and the horizontal axis indicates time after drug administration. In Table 6, AUC _0-∞ represents the area under the blood concentration curve, BA represents bioavailability, and n represents the number of samples collected at one time point. PO represents oral administration, and IV represents intravenous administration.

[Comparative Example 2]
In Comparative Example 1, 0.02 mg of CPZ-d ₆ was intravenously administered to separate SD male rats. Blood was collected at each elapsed time, and the blood concentration of CPZ-d ₆ was measured in the same manner as in Comparative Example 1. The obtained result is shown in FIG. Further, Table 6 shows bioavailability calculated from the relationship between the area under the blood concentration-time curve (AUC _0-∞ ) obtained from FIG. 6 and the dose of CPZ administered orally.

According to the present invention, there is an effect that an accurate pharmacokinetic evaluation can be performed by one measurement by one test without using a radioactive substance. Therefore, it becomes easy to obtain information on the pharmacokinetics of the drug candidate compound before the clinical trial of the pharmaceutical or at the initial stage of the clinical trial, and improvement in the development efficiency of the pharmaceutical can be expected.

Claims

In a blood sample collected over time from a subject to whom a stable isotope substitute, which is labeled with a stable isotope after the compound has been administered extravascularly, is intravenously administered,
A step of measuring the concentration of the compound and the stable isotope substitute, and a step of obtaining pharmacokinetic data of the compound from a change over time in blood concentration measured in the step of measuring. Pharmacokinetic evaluation method.
The dose of the stable isotope substitute administered intravenously is greater than 0 and less than or equal to 1/10 by weight with respect to the dose of the compound administered extravascularly. Item 4. The pharmacokinetic evaluation method according to Item 1.
The stable isotope, 2 H (D), 13 C, 15 N or 18 Pharmacokinetic evaluation method according to claim 1 or 2, characterized in that it is O.
The stable isotope substitute administered intravenously is substituted with a stable isotope so as to have a molecular weight of 3 or more as compared with the compound administered extravascularly. The pharmacokinetic evaluation method according to any one of the above.
In a blood sample collected over time from a subject to which the stable isotope substitute was administered intravenously while the compound administered extravascularly is present in the blood,
The measurement of the time-dependent change in the blood concentration of the compound and the stable isotope substitute is performed in a single measurement by measuring the concentration of the compound and the stable isotope substitute. Item 5. The method for evaluating pharmacokinetics according to any one of Items 1 to 4.
The pharmacokinetic evaluation method according to any one of claims 1 to 5, wherein the amount of the compound administered extravascularly is a clinical dose.
The method for evaluating pharmacokinetics according to any one of claims 1 to 6, wherein the bioavailability of the compound is calculated.
A method for selecting drug candidate compounds, comprising using the result obtained by the pharmacokinetic evaluation method according to any one of claims 1 to 7.
A pharmacokinetic evaluation kit comprising:
(A) a compound to be evaluated;
(B) a stable isotope substituent obtained by labeling the same compound as the above compound with a stable isotope,
(C) a first step in which the compound is administered extravascularly;
A second step of intravenously administering a stable isotope substitute obtained by labeling the same compound as the above compound with a stable isotope after the compound is administered extravascularly;
The third step of measuring the time course of blood concentration of the compound and the stable isotope substitute, and the fourth step of acquiring pharmacokinetic data of the compound from the time course of blood concentration measured in the third step Instructions to perform pharmacokinetic evaluation according to the process,
A kit comprising:
The kit according to claim 9, wherein the stable isotope substitution product is greater than 0 and 1/10 or less by weight with respect to the compound to be evaluated.
The stable isotope, 2 H (D), 13 C, 15 N or 18 kit according to claim 9 or 10, characterized in that it is O.
The stable isotope substitution product according to any one of claims 9 to 11, wherein the stable isotope substitution product is substituted with a stable isotope so as to have a molecular weight of 3 or more as compared with a compound to be evaluated. Kit.
The second step involves intravenous administration of a stable isotope substitute obtained by labeling the same compound as the above compound with a stable isotope while the compound administered extravascularly is present in the blood,
The third step according to any one of claims 9 to 12, wherein the measurement of the time course of the blood concentration of the compound and the stable isotope-substituted product is performed in a single measurement. kit.
The kit according to any one of claims 9 to 13, wherein the compound to be administered in the first step is a clinical dose.