KR20140009783A - Minidose cocktail kit for high-throughput evaluation of human cytochrome p450 enzyme - Google Patents

Abstract

The present invention relates to a minidose cocktail kit for high-throughput evaluation of human cytochrome P450 enzyme activities and, more specifically, to a minidose cocktail kit for high-throughput evaluation of human cytochrome P450 enzyme activities which includes probe drugs including caffein, losartan, omeprazole, dextromethorphan, and midazolam and concurrently measures drug metabolizing activities of five kinds of cytochrome P450 isoenzymes at high speed. The minidose cocktail kit according to the present invention does not show interaction between probe drugs and safely and concurrently measures metabolizing activities of five kinds of cytochrome P450 at high speed without side effects caused by dose reduction. Especially, the minidose cocktail kit according to the present invention simultaneously administrates, has short time for collecting biological materials and concurrently analyzes all kinds of drugs, thereby enhancing convenience and practicality. In addition, the minidose cocktail kit introduces a novel concept to a method used as a bioactive marker for CYP enzyme in order to minimize number of blood drawing and increase accuracy.

Description

Minimalose cocktail kit for high-throughput evaluation of human cytochrome P450 enzyme}

The present invention relates to a minimum dose cocktail kit for high speed evaluation of cytochrome P450 enzyme activity and a high speed evaluation method of cytochrome P450 enzyme activity using the same.

Cytochrome P450 (Cytorcrome P450, CYP) enzymes are involved in the metabolism of various clinically useful drugs, and exist in the complex form of several isoenzymes in the human body instead of a single form. In particular, humans have isoenzymes such as CYP1A2, 2C9, 2C19, 2D6, 2E1 and 3A.

On the other hand, the activity of the CYP isozymes is showing a variety of differences between individuals. Differences in enzyme activity among individuals are largely due to a variety of genetic polymorphisms, including Single Nucleotide Polymorphisms (SNPs), and several acquired factors. Acquired factors include food, cytokines, hormones, and various drugs, which can increase or inhibit the metabolic enzyme activity. The difference in activity of CYP isozymes is a major factor that causes individual differences in blood drug concentration, and depending on the drug may cause serious side effects. Therefore, the evaluation of activity of CYP isozymes is very important and can be applied not only in academic fields such as drug interaction research and genotype phenotyping but also in industrial applications such as new drug development.

In vivo to measure the activity of current drug metabolism enzymes ( in vivo) and in vitro (in Several techniques in vitro have been developed. In vivo techniques include the use of probes or marker drugs, which are selectively metabolized to specific CYP enzymes, to measure them individually for each CYP enzyme, and to measure the activity of drug metabolic enzymes by simultaneously administering multiple probes. There is a 'cocktail' method. Both methods require the determination of unchanged parent compounds and metabolites in plasma or urine, and a metabolic enzyme index that adequately reflects metabolic activity for each CYP enzyme. Should be used.

However, the in vivo use of these drugs can be accompanied by many problems in several aspects such as drug side effects, analytical procedures, and costs. Therefore, the selection of appropriate probe drug is very important in measuring metabolic activity of CYP enzyme.

Recently, the cocktail method of simultaneously administering a variety of probe drugs has been in the spotlight because of the advantages of real-time evaluation of enzyme activity in terms of efficiency and reliability. The greatest advantage of the cocktail method is that one clinical trial can simultaneously evaluate the activity of isoenzymes in multiple pathways. This allows the rapid evaluation of enzyme activity in drug interaction studies or phenotypic tests. However, the pharmacokinetic and pharmacodynamic interactions between the probes must be taken into account when using the cocktail method, as opposed to measuring them individually for each enzyme. For example, caffeine, a CYP1A2 substrate drug, has been reported to significantly reduce blood flow in the liver, altering the clearance rate of potentially high extraction rates of substances, caffeine, chlorzoxazone ), Dextromethorphan and chloroguanide have been reported to interact with each other. Therefore, before using the cocktail method, it is necessary to confirm that there is no interaction in the combination of the probe drugs. In addition, it is important to verify that the probe drug is redundantly substrated for other CYP enzymes. Therefore, the choice of drug and proper selection of enzyme activity indicators by metabolic enzyme are very important factors in designing cocktails. It is also important to establish appropriate analytical methods. Due to the nature of the cocktail method, a single test evaluates metabolic enzyme activity. Therefore, a multi-component simultaneous assay is required to measure several drugs in a single biological sample.

On the other hand, many cocktail methods have been developed since the "Pittsburgh cocktail" was announced in 1997 as a cocktail method for measuring the drug metabolic activity of CYP enzymes (Frye RF et al., Clin Pharmacol). Ther , 62: 365-376, 1997). Currently known representative cocktail methods are the “Cooperstown cocktail” and the “Karolinska cocktail”, both of which have advantages and disadvantages (Streetman DS. Et al., Clin) . Pharmacol Ther , 70: 455-461, 2001; Christensen M. et al., Clin Pharmacol Ther , 73: 517-528, 2003). In the Cooperstown cocktail method, caffeine (substrate of CYP1A2), omeprazole (substrate of CYP2C19), dextrometolpan (substrate of CYP2D6) and intravenous midazolam (substrate of CYP3A) were used as probes and the drugs interacted. Appeared to be missing. However, when using intravenous midazolam, as in the Cooperstown cocktail, the metabolic activity of CYP3A, including enzymes expressed in the gastrointestinal tract, cannot be determined and the metabolic activity of CYP2C9, an important CYP enzyme, cannot be evaluated. To complement this, the Cooperstown 5 + 1 cocktail method was added to warfarin to the existing Cooperstown cocktail to evaluate the metabolic activity of CYP2C9 (Chainuvati S. et al., Clin Pharmacol Ther , 74: 437-447, 2003). However, when warfarin is used, there are risk factors such as bleeding.

In the case of Karolinska cocktail, the metabolic activity of CYP 1A2, 2C9, 2C19, 2D6 and 3A was measured using caffeine, losartan, omeprazole, debrisoquin and quinine as probes, respectively. The Karolinska cocktail has the advantage of being able to see the activity of five enzymes at the same time, but debrisoquine and quinine are not licensed in Korea as a drug that is not practically limited in the country as well as its use worldwide. Not universal In addition, the simultaneous use of Rosatan and debrisoquine, which have a blood pressure-lowering effect, can cause unexpected drug side effects.

Therefore, the present inventors used caffeine, losartan, omeprazole, dextrometolpan, and midazolam as the components that are easy to apply and have no drug interaction, and used the drug metabolism of five cytochrome P450 isoenzymes. The present invention has been completed by devising a method for evaluating the activity at the same time without any side effects at high speed.

Republic of Korea Patent Publication 10-2008-0005556

Therefore, an object of the present invention is a high-speed evaluation of cytochrome P450 enzyme activity comprising a probe drug consisting of 5 to 15 mg of caffeine, 1 to 5 mg of losartan, 10 to 500 μg of omeprazole, 1 to 5 mg of dextrometolpan, and 10 to 500 μg of midazolam. To provide a minimum dose cocktail kit for.

Another object of the present invention is from a subject except a human to which a probe drug consisting of 5 to 15 mg of caffeine, 1 to 5 mg of losartan, 10 to 500 μg of omeprazole, 1 to 5 mg of dextrometolpan and 10 to 500 μg of midazolam, is administered.

(a) collecting blood or urine;

(b) to provide a rapid evaluation method of cytochrome P450 enzyme activity comprising analyzing the concentration of drugs and metabolites in the blood or urine collected by LC-MS / MS system.

In order to achieve the above object, the present invention comprises a probe drug consisting of 5 to 15 mg of caffeine, 1 to 5 mg of losartan, 10 to 500 μg of omeprazole, 1 to 5 mg of dextrometolpan and 10 to 500 μg of midazolam, cytochrome P450 Minimum dose cocktail kits are provided for rapid assessment of enzyme activity.

In one embodiment of the present invention, the cytochrome P450 enzyme may be selected from the group consisting of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A enzyme.

In one embodiment of the present invention, the probe drug may be a capsule formulation that can be administered orally.

In one embodiment of the present invention, the probe drug may be administered simultaneously (simultaneous).

In the present invention,

From subjects, except for humans, who received probes consisting of 5-15 mg caffeine, 1-5 mg losartan, 10-500 μg omeprazole, 1-5 mg dextrometolpan, and 10-500 μg midazolam,

(a) collecting blood or urine;

(b) caffeine, lotan, omeprazole, dextrometolpan and midazolam contained in the collected blood or urine; Or analyzing their metabolites with an LC-MS / MS system and calculating a ratio to provide a high-speed evaluation method of cytochrome P450 enzyme activity.

In one embodiment of the invention, the blood may be collected three times at 2, 3 and 4 hours after the administration of the probe drug.

In one embodiment of the present invention, urine may be collected once every 4 hours after administration of the probe drug.

In one embodiment of the present invention, the metabolite of caffeine is paraxanthin, the metabolite of losartan is EXP3174, the metabolite of omeprazole is 5-OH omeprazole, and the metabolite of dextrometol is dextrose. The metabolite of midazolam may be 1-'OH midazolam.

In one embodiment of the present invention, the step of calculating the ratio in step (b) is carried out by measuring the metabolites and metabolites that are not metabolized after administration of the probe drug, blood or urine when the mother drug is caffeine Calculate the excretion ratio of paraxanthin / caffeine excretion contained in, and, if the parent drug is Rosatan, the area under the curve of EXP3174 contained in blood or urine (AUC _{2 -4h} ) / curve of _Rosatan ever (AUC _{2 -4h)} calculate the ratio, and the base when the drug is omeprazole, when the curve of the 5-hydroxy-omeprazole (5-hydroxyomeprazole) contained in the blood or urine enemy (AUC _2-4h) / omeprazole curve of Calculate the area of _lower surface area (AUC _{2 -4h} ), and if the parent drug is dextrometol, the area under the curve of the dextrorphan contained in blood or urine (AUC _{2 -4h} ) / dextrome If the curve of the enemy tolpan dextromethorphan (AUC _{2 -4h)} calculating a ratio, wherein the parent drug If the midazolam, the curve of the 1'-OH midazolam contained in blood or urine enemy (AUC _{2 -4h)} / the curve of midazolam can be to calculate the enemy (AUC _{2 -4h)} ratio.

The minimum dose cocktail kit for rapid assessment of cytochrome P450 enzyme activity comprising five drug cocktails according to the present invention is administered at a dose of about 10-100 times higher than the commonly used therapeutic doses without showing interactions between the probes. By reducing the dose, there is an advantage that can measure the metabolic activity of five cytochrome P450 enzymes at high speed safely without side effects. In particular, in the case of the existing cocktail cocktail, the number of doses and the biological sample collection time is inconvenient in concentration analysis, etc., but the minimum dose cocktail of the present invention is concurrently administered, the biological sample collection time is short, and all drugs are simultaneously analyzed. It is possible in terms of convenience and practicality. In addition, a new concept can be introduced into the method used as an indicator of the activity of the CYP enzyme, thereby increasing the accuracy of the analysis while minimizing the number of blood samples.

1 is a graph showing the blood concentration profile of caffeine, a metabolite, and paraxanthin, a CYP1A2 probe drug, after administration of a cocktail drug.
2 is a graph showing the blood concentration profile of Rosatan and its metabolite EXP3174, which is a CYP2C9 probe drug, after administration of the cocktail drug.
3 is a graph showing the blood concentration profile of omeprazole and its metabolite 5-hydroxyomeprazole, which is a probe drug of CYP2C19, after administration of a cocktail drug.
Figure 4 is a diagram showing the blood concentration profile of dextromephan and its metabolite dextrophan (Dextrorphan) of the CYP2D6 probe drug over time after the administration of the cocktail drug.
FIG. 5 is a graph showing the blood concentration profile of midazolam, which is a CYP3A probe drug, and its metabolite 1'-hydroxymidazolam (1'-Hydroxymidazolam) with time after the administration of the cocktail drug.

In order to achieve the above object, the present invention comprises a probe drug consisting of caffeine, losartan, omeprazole, dextrometolpan and midazolam and simultaneously measure the drug metabolic activity of five cytochrome P450 isoenzymes Provided is a minimum dose cocktail kit for rapid assessment of cytochrome P450 enzyme activity characterized in that it is capable of.

Recently, microdosing, which is administered at a dose that satisfies a maximum of 100 ug or less and less than a hundredth of the therapeutic dose showing a pharmacological effect, has been progressed. The potential risks to the subjects, such as sedation effects, are extremely low, so the study can be conducted with less clinical safety data and subjects, which is a cost-effective study that saves time and resources (Ajay Madan, 2009, Lionel D. Lewis, 2009). . However, when a small amount of drug is administered, there may be a problem that the drug concentration is too low to be analyzed, and thus, in the case of the minidosing method of administering a dose lower than the normal dose, even if the current standard of microdosing is not met. Attempts are being made. The application of the minimum dose test method can minimize the incidence of adverse drug reactions that may occur when administering existing cocktail doses, and further reduce the likelihood of mutual interfering effects of related metabolic enzymes that may occur during normal dose administration. It is expected that the activity of the enzyme can be evaluated. To date, however, cocktails with minimal doses of probes have not been tested. Based on the above, the present invention provides an improved cocktail using 1/10 to 1/100 doses of the normal doses of caffeine, dextrometol, losartan, omeprazole, and midazolam, which are indicator drugs of existing cocktails. The method was developed and encapsulated in each active pharmaceutical ingredient (API) with the help of Yuhan Co., Ltd. for minimum dose administration. In this case, the metabolic enzyme activity is more sensitively reflected and the side effects are less sensitive. It happened. At the same time, drug concentration measurements were developed to provide a detailed assessment of the pharmacokinetics of the drug at the minimum dose. By developing an analytical method that maximizes analytical sensitivity and analysis time, the LC-MS / MS instrument was able to quantify low concentrations in pg / mL, thus enabling minidose studies.

The kit according to the present invention has five drugs, more specifically caffeine as a probe of CYP1A2 enzyme, losartan as a probe of CYP2C9 enzyme, omeprazole as a probe of CYP2C19 enzyme, dextromemethane as a probe of CYP2D6 and CYP3A It contains all of the midazolam as a probe drug of the enzyme, characterized in that the drug metabolism activity of the CYP1A2, CYP3A, CYP2C9, CYP2C19 and CYP2D6 enzyme can be measured at high speed simultaneously.

The 'probe drug' refers to a drug that is a selective substrate of each cytochrome P450 isoenzyme and can measure the drug metabolic activity of the cytochrome P450 enzyme without causing side effects in vivo.

Caffeine, one of the probes, has been widely used as a substrate drug for measuring the activity of the CYP1A2 enzyme in the cytochrome P450 enzyme. The caffeine is metabolized to paraxanthine by the CYP1A2 enzyme.

The losartan has been reported as a probe with high sensitivity and specificity in measuring activity of CYP2C9 enzyme in cytochrome P450 enzyme. E-3174, the carboxylic acid metabolite of losartan, is the only active metabolite specifically produced by CYP2C9. On the other hand, as a probe drug for measuring the activity of the CYP2C9 enzyme, tolbutamide (tolbutamide), phenytoin (phenytoin), warfarin (warfarin) and diclofenac (diclofenac) has been used. In Cooperstown 5 + 1 cocktail, warfarin was used as a probe to measure CYP2C9 enzyme activity, but it was very dangerous because of side effects such as bleeding. In addition, due to the long half-life, the practicality of application is inferior, and since there is no established metabolic enzyme activity index, a large amount of blood collection is inevitable to analyze the pharmacokinetic properties of warfarin.

The omeprazole is a drug widely used in the treatment of gastric ulcer and is known to have almost no side effects. In the present invention, it was used for the purpose of measuring the activity of the CYP2C19 enzyme. Omeprazole is metabolized to 5-OH omeprazole by the CYP2C19 enzyme. Mephinitoin, omeprazole, and chloroguanide are used as the probe drug for measuring the enzyme activity of CYP2C19, and the S / R ratio of mepinitoin is mainly used. However, in the case of mepinitoin, there is a strong sedation, and in the case of a person with enhanced metabolic ability, the concentration of S-mepinitoin in urine is very low and the stability of urine to the S-mepinitoin polymer is questioned.

The dextrometol plate was used as a probe drug of the CYP2D6 enzyme in the present invention, and as the probe drug of the CYP2D6 enzyme, codeine, metoprolol, desipramine, and debrisoquin, in addition to dextrometholpan, were used. (debrisoquin) is widely used. However, debrisoquine is a drug that lowers blood pressure, which is not currently available in Korea, and debrisoquine synergizes with Rosatan, an antihypertensive agent used as a probe drug of CYP2C9 in the present invention, causing serious side effects. There is one danger.

The midazolam was used as a probe drug of the CYP3A enzyme in the present invention. The CYP3A enzyme is widely distributed not only in the liver but also in the gastrointestinal tract. However, the majority of studies for measuring enzyme activity of CYP3A use intravenous midazolam. As described above, when intravenous midazolam is injected into the blood without passing through the gastrointestinal tract, metabolism occurs in the liver without undergoing the metabolic process by the CYP3A enzyme expressed in the gastrointestinal tract. Therefore, the activity of the CYP3A enzyme expressed in the gastrointestinal tract does not reflect at all. In consideration of this, the midazolam oral administration method was used in the present invention.

In addition, the probe drug according to the present invention can be administered simultaneously (simultaneous). Although the administration time of dextrometolphan was separated in consideration of the gastrointestinal motility effect of the drug in the existing reported cocktails, it would not be a big problem even if the probes according to the present invention were administered simultaneously when referring to the conventional literatures. (Streetman DS et al., Clin Pharmacol Ther , 68: 375-383, 2000; Christensen M. et al., Clin Pharmacol Ther , 73: 517-528, 2003), since the present invention used a tenth dose of the normal dose, the simultaneous administration method was used to maximize practicality and convenience.

The dose of the probe drug included in the kit for measuring the drug metabolism activity of the cytochrome P450 enzyme according to the present invention is a dose capable of analyzing the concentration of the drug and its metabolites so that the activity of the cytochrome P450 enzyme can be evaluated, and the side effects in vivo. It should be a low dose in the range that does not cause. Preferably, the range was set to 200 mg, caffeine 100 mg, octaprazole 40 mg, dextrometolpan 50 mg and midazolam 5 mg or less, and more than the minimum dose that can be analyzed based on the adult. Based on this, 10 mg of caffeine, 2 mg of losartan, 200 μg of omeprazole, 2 mg of dextrometolphan, and 100 μg of midazolam were used, which is about 1/10 the caffeine dose and 25 minutes of losartan compared to the previously reported cocktail kit. It is a dose of 1, omeprazole 1/100, dextromemethane 1/15, and midazolam 1/20.

Therefore, in the present invention, the use of low-dose drugs reduces the possibility of interacting with five drugs, enables precise phenotyping of the CYP450 enzyme, increases the drug convenience of test subjects when administered at reduced doses, and the frequency of occurrence of side effects. Can be a safe way.

In one embodiment of the present invention, one capsule of caffeine (10 mg), one capsule of losartan (2 mg), one capsule of omeprazole (200 μg), one capsule of dextrometol plate (2 mg), and one capsule of midazolam (100 μg) are used together. Oral administration (see Example 2).

By using the kit according to the present invention, the drug metabolic activity of the five cytochrome P450 isozymes can be simultaneously measured at high speed according to the following method.

(a) administering to a mammal a probe drug consisting of caffeine, losartan, omeprazole, dextrometolphan and midazolam,

(b) taking a biological sample from the mammal of step (a),

(c) measuring the concentration of the probe drug and its metabolite of step (a) in the biological sample of step (b) and calculating the enzyme activity from the metabolic activity indicator.

In step (b), the biological sample may be blood or urine of the subject. In addition, the blood collection of the subject in step (b) is preferably performed at 2, 3, 4 hours after administration, and urine collection is preferably performed between 0-4 hours, preferably 4 hours after administration. Do.

Shorter dosing times and collection times can increase the compliance of the test subject and improve the accuracy of the analytical results.

The metabolic activity indicator in step (c) can be calculated from the ratio and the metabolites and metabolites that are not metabolized in biological samples, ie blood or urine of the subject. In the present invention, the following metabolic activity index was used to evaluate the activity of each cytochrome P450 enzyme.

In this way, one analysis method can be used to simultaneously analyze five kinds, which reduces the cost and time of analysis, thereby increasing convenience and analyzing five drugs with high sensitivity using the minimum dose.

In addition, we introduced a new concept in the method of using CYP enzymes as a phenotyping index, and the AUC of the parent drug and metabolite at a limited time (between 2 and 4 hours) with the parent drug and metabolite AUCt ratio. Using the limited AUC ratio, the concept of increasing the accuracy of typing while minimizing the number of blood samples was introduced.

CYP1A2 (probe drug: caffeine) = excretion of paraxanthine in urine collected up to 4 hours after administration / excretion of caffeine

CYP2C9 (probe drugs: losartan) = The curve of the EXP3174 during one of plasma collected in the second 2,3,4 hours after dosing ever when ever curves (AUC _{2 -4h)} of (AUC _{2 -4h)} / losartan

CYP2C19 (probe drug: omeprazole) = area under the curve of 5-hydroxyomeprazole (AUC _{2 -4h} ) / area under the curve of omeprazole (AUC 2,3,4 hours after dosing) _{2 -4h} )

CYP2D6 (probe medications: dextromethorphan tolpan) = the curve of the plasma collected in the second one of dextrorphan 2,3,4 hours after dosing enemy of the curve (AUC _{2 -4h)} / dextromethorphan ever (AUC _{2 -4h)}

CYP3A (probe drug: midazolam) = excretion of 1'-OH midazolam in urine collected up to 4 hours after dosing / excretion of midazolam

The term AUC used in the present invention is an abbreviation of area under the curve, and means the area under the curve in the time-concentration graph, and thus, the unit becomes the time * concentration and is the most representative indicator of pharmacokinetic parameters. to be. In the existing cocktail cocktail, the AUC ratio was shown to have a good correlation with the concentration ratio at 4 hours, and then the AUC ratio was replaced with the concentration ratio at 4 hours, but the concentration of 2,3,4 hours was the minimum dose cocktail according to the present invention. The limited AUC ratio was used as a more reliable and stable result than the one-point concentration ratio.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

Selection of Subjects

The subjects of the clinical trial were the health examinations (checkup, blood pressure, 12-lead ECG, physical examination, blood, urine test, serum test, etc.) set and performed by the doctor in charge among the test subjects recruited through the clinical trial announcement. Based on the results of the screening item), it is appropriate to participate in the clinical trial, and the written consent to participate in the clinical trial by the free doctor is 20 to 50 years of age and is within 20% of the ideal weight using the Broca formula. The subjects were healthy Korean male volunteers. As a result, a total of 26 subjects except dropout subjects participated in the clinical trial. The clinical trial was conducted after approval of the Institutional Review Board of Busan Paik Hospital.

However, apart from the clinical trial agreement, the authors received the written consent of the genetic test agreement, and blood was collected for genotyping at the time of medical examination, and the subjects who had the genotype as shown in Table 1 through the drug metabolic genotyping were excluded.

CYP450 causes a difference in metabolic activity among individuals according to its genetic polymorphism. In particular, the differences in in vivo kinetics of substrate drugs related to various gene mutations of CYP450, which are mainly involved in drug metabolism, are well known, and there are also reports of specific gene mutations in which activity of CYP450 isozyme is observed to be reduced or almost absent. . When evaluating major CYP450 metabolic activity in normal subjects as in this study, consideration should be given to the diversity of metabolic activity by genotype, especially when evaluating metabolic activity indicators reflecting CYP2D6, CYP2C9, and CYP2C19 isozymes. Care must be taken (Huang et al, USFDA draft guidance). Therefore, the metabolic activity of CYP450 is not properly reflected in the subjects with the genes of the metabolic lowering group showing the relative decrease in metabolic activity. Therefore, the present study has previously reported (shon et al, 2002, Yoon et al. In 2001, Roh et al, 1996, Kim et al, 2002, Lee et al, 2006), genetic variants belonging to the CYP450 metabolic lowering group found in Koreans were selected and subjects who had them were excluded.

< Example  2>

Test agent  And establishment of a method of administration

To assess CYPIA2, CYP2C9, CYP2C19, CYP2D6 and CYP3A enzyme activities, caffeine, losartan, omeparazol, dextrometolphan and midazolam were used as the respective probes. The cocktail to be applied in this test was prepared with the ingredients and contents shown in Table 2 below, and the dose was the minimum dose estimated in the range capable of concentration analysis in blood after taking each drug according to analytical conditions previously established by LC-MS / MS. It was set as. In other words, the doses of four drugs, midazolam, losartan, dextromethorphan, and omeprazole, were determined to be sufficient to be analyzed at the last pharmacokinetic blood collection. In the case of caffeine, the dose was increased based on the remaining concentration of caffeine since it is already present in the human body even without taking the drug. As a result, the dose of each drug used in the experiments of the present invention used a much smaller dose than that used in the past, thus reducing the possibility of the five drugs interacting due to the use of this minimum dose, Increasing phenotyping accuracy, reducing the dose of the drug increases the convenience of the subject, and reduces the frequency of side effects.

 Dosages and formulations of the test drug Ingredients Volume Formulation Pharmaceutical companies Caffeine 10 mg Capsule Yuhan Corporation Losartan 2 mg Capsule Yuhan Corporation Omeprazole 200ug Capsule Yuhan Corporation Dextromethorphan 2 mg Capsule Yuhan Corporation Midazolam 100ug Capsule Yuhan Corporation

The trial was conducted in two cohorts, open, randomized, and in a single sequence. Cohort A was administered in the following order: Omeprazole (OMP), Rosatan (LST), and Dextrometolphan (DXT). Dog probes were taken as cocktails (CKTs), and cohort B was given midazolam (MDZ) and caffeine (CAF) individually, followed by cocktails (CKTs). When assigning probe drugs to cohorts, omeprazole is a weak inhibitor of CYP3A (Ko et al., 1997), and because Rosatan and dextrometolpan metabolize CYP3A in addition to metabolism by CYP2C9 and CYP2D6 (Guo Wang, 2009, C Funck-Brentano, 2005) consisting of midazolam and other cohorts. Since the known half-life of probes is up to 5 hours, a 48-hour drug period is set in consideration of the period during which the drug can be lost after administration of the drug and the safety of the subject, and the dose of the subject assigned to each cohort. All clinical trials were planned to be hospitalized for safety and safety management. The test was designed as shown in Table 3 and Table 4 below.

More specifically, subjects belonging to Cohort A took one capsule of 200 ug of omeprazole together with 240 ml of water at about 8 am on the day of the test on an empty stomach after 10 pm on the day before the first dose of the test drug. After 48 hours of absence, two capsules of 2 mg of Rosatan were taken with 240 ml of water around 8 am on the third day after 10 pm on the second day. On the 4th day after the same drug holiday was maintained on an empty stomach after 10 pm, and on day 5 at 8 am one capsule of 2 mg of dextrometolphan was taken with 240 ml of water. On the 6th day, the dogs were kept on an empty stomach from 10 pm and on the 7th morning, 5 capsules (caffeine 10 mg, losartan 2 mg, omeprazole 200 ug, dextrometolphan 2 mg, midazolam 100 ug) were simultaneously taken with 240 ml of water. Subjects who belonged to Cohort B took one capsule of 100 ug of midazolam with 240 ml of water at about 8 am on the day of the test on an empty stomach after 10 pm on the day before the first dose of the test drug. After 48 hours of absence, two capsules of caffeine 10mg were taken with 240ml of water around 8 am on the third day after 10 pm on the second day. On the 4th day after the same drug period, fasting was maintained after 10 pm, and 5 capsules (caffeine 10 mg, losartan 2 mg, omeprazole 200 ug, dextrometolphan 2 mg, midazolam 100 ug) simultaneously at 8 am on day 5 240 ml of water. Taken with.

As a result, all subjects completed the clinical trial successfully, and no side effects were observed during drug administration alone or during cocktail administration. Therefore, by reducing the number of dosing and sampling time can increase the compliance of the test subjects, it is possible to increase the accuracy of the analysis results.

< Example 3>

Drugs in urine and plasma of subjects and their Metabolite  Concentration analysis

After administering the drug to the subject of Example 1 according to the method of Example 2, the concentration of drugs in urine and plasma samples was measured using a liquid chromatography-mass spectrometer (LC-MS / MS). In the previously reported cocktails, the five drug concentrations were quantified in four analyzes, whereas in this study, all drugs were quantified in one analysis, which makes it easier and faster to perform drug analysis than conventional methods. Could know.

<3-1> Collection of Urine and Blood Samples

Plasma and urine samples for concentration measurement and pharmacokinetic profile of probes are collected until 12 hours after dosing based on blood collection and urine collection time established in the existing Inje cockail (Korean Patent 658,184). In order to measure the concentration change according to the minimum dose in detail, the number of blood collections was designed to 14 times, and urine collection was performed at all drug administrations, in addition to rosatan and dextrometol, which are known metabolic activity indicators using drug concentrations in urine. This was done to measure the change in concentration in urine.

-Blood collection point: 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12 hours, 14 times

-Collection point: 0, 0-4, 4-8, 8-12 hours, total 4 times

When blood samples were taken continuously, the catheter was inserted into the antecubital or forearm vein, and the catheter is kept open with saline, which is removed earlier after the final blood sample or, if requested by the subject. . The first 0.5-1.0 mL of blood was collected and discarded before each sample so that saline did not artificially dilute the sample. When collecting blood, take 7cc of whole blood, put it in heparinized vacutainer tube, immediately centrifuge at 3000rpm for 15 minutes in refrigerated (2 ~ 4 ℃) centrifuge, take plasma and transfer 1mL each to 1.5mL volume eppendorf vial until measuring the concentration. Frozen storage at 80 ℃. At the time of urine collection, urine up to 12 hours after administration is collected in storage containers hourly (0 ~ 4, 4 ~ 8, 8 ~ 12hr), and then shaken well to measure the volume of urine. 10 mL each was added to (15 mL) and stored at -80 ° C until the concentration was measured with standard urine (0 hr).

<3-2> plasma sample Probe Drugs  Concentration Analysis Method

Simultaneous analysis of caffeine, losartan, omeprazole, dextromemethane, midazolam and their metabolites and internal standards (IS) in plasma samples was determined using the following method using LC-MS / MS. . In contrast to the existing drug cocktails, five drug concentrations were quantified in four analyzes. In this study, all drugs were quantified in one analysis. The system used an Agilent 1100 series HPLC system (Agilent Technologies, USA) and an AB QTrap 5500 triple quadrupole mass spectrometer (Applied Bio Systems, Canada) LC-MS / MS system. The HPLC column was Xbridge MS C ₁₈ (3 μm, 2.1 × 100 mm, Waters Corp. USA), and the mobile phase was (A) distilled water (0.1% formic acid) and (B) acetonitrile (0.1% formic acid) was used to elute under the gradient conditions shown in Table 5. All drugs were ionized in positive ion mode and measured in multiple reaction monitoring (MRM) mode. MRM assay conditions for drugs and metabolites are shown in Table 6.

Gradient condition of mobile phase Total time (min) Flow rate (μl / min) A (%) B (%) 0 0 200 70 30 One One 200 70 30 2 4 200 30 70 3 8 200 30 70 4 9 200 70 30 5 18 200 70 30

Analytical Conditions for Caffeine, Rosatan, Omeprazole, Dextrometolphan, Midazolam and Each Metabolite enzyme Analyte Precursor ions (m / z) Product Ion (m / z) Collision energy (ev) CYP1A2 Caffeine 195.0 138.3 30 Paraxanthine 181.0 124.4 30 CYP2C9 Losartan 423.2 207.1 34 EXP3174 437.2 235.1 24 CYP2C19 Omeprazole 346.2 198.1 17 5-Hydroxyomeprazole 362.2 214.1 17 CYP2D6 Dextromethorphan 272.2 171.2 50 Dextrorphan 258.2 157.2 50 CYP3A Midazolam 326.2 291.2 40 1’-Hydroxymidazolam 342.2 203.0 40 IS Propranolol 260 116.5 24

For the analysis of drugs, first add 50 µl of internal standard (propranolol 200ng / ml) to 0.5 ml of plasma, mix gently, add 3 ml of ethyl acetate and use Multi Reax (Heidolph, Germany). The extract was shaken for 10 minutes. After centrifugation at 3000 rpm for 10 minutes, the organic layer was transferred to a new test tube, dried for 1 hour using Speed-Vac (Savant, USA), and redissolved in 100 μl of methanol (A). Meanwhile, 100 μl of 1 mole of acetic acid was added to the remaining aqueous layer, which resembled the organic layer, and 3 ml of ethyl acetate was added thereto, followed by shaking extraction for 10 minutes using Multi Reax. Thereafter, the organic layer obtained by centrifugation at 3000 rpm for 10 minutes was transferred to a new test tube, dried for 1 hour using Speed-Vac, and redissolved in 100 μl of methanol (B). Thereafter, 80 μl of each of (A) and (B) was removed, mixed into 1.5 mL tubes (Axygen, USA), and 5 μl of the same was injected into the LC-MS / MS system.

The retention times, standard calibration curve ranges, linearity of standard calibration curves, and quantitative limit concentrations of the materials using the above method are shown in Table 7, and the standard calibration curves were calculated from the peak area ratios of drug and internal standard in plasma samples. Obtained. The standard calibration curve was used to determine the concentrations of drugs and their metabolites in the blood. The concentrations of drugs and metabolites in the blood with time during the cocktail administration are shown in FIGS. 1 to 5.

Retention time, standard calibration curve range, linearity and quantitative limit concentration of analyte Analyte Retention time (min) Standard calibration curve range (ng / ml) Correlation Coefficient ( R ² ) Quantitative limit concentration (ng / mL) Caffeine 2.2 5-500 0.9992 ± 0.0006 5 Paraxanthine 2.2 5-500 0.9994 ± 0.0004 5 Losartan 8.5 0.1-40 0.9972 ± 0.0013 0.1 EXP3174 9.4 0.1-40 0.9976 ± 0.0010 0.1 Omeprazole 2.9 0.05-20 0.9986 ± 0.0014 0.05 5-Hydroxyomeprazole 2.5 0.05-20 0.9990 ± 0.0004 0.05 Dextromethorphan 6.3 0.008-0.8 0.9997 ± 0.0003 0.008 Dextrorphan 2.7 0.008-0.8 0.9993 ± 0.0007 0.008 Midazolam 6.2 0.01-1 0.9997 ± 0.0002 0.01 1′- Hydroxymidazolam 5.3 0.04-4 0.9997 ± 0.0002 0.04

<3-3> caffeine and urine samples Paraxanthin ( paraxanthine ) Concentration Analysis Method

In order to measure the concentration of caffeine and paraxanthin in urine samples, 200 μl of urine sample and 190 μl of 0.1M acetic acid buffer were first mixed from the subject, and 10 μl of 10,000unit β-glucuronidase was added thereto for 90 minutes in a 55 ° C. water bath. Reacted. Thereafter, 20 µl of an internal standard (propranolol 10 µg / ml) was added to the reaction mixture, and the mixture was gently mixed, followed by 800 µl of methanol, followed by shaking extraction for 10 minutes using Multi Reax. After centrifugation for 5 minutes at 3000rpm 2μL was injected into the LC-MS / MS system after protein precipitation. Mobile phase conditions and MRM conditions are analyzed by the conditions in Tables 5 and 6. The quantitative limit concentrations of caffeine and paraxanthin measurable by this method were both 0.5 μg / ml, and a standard calibration curve was obtained from the ratio of the peak areas of caffeine and paraxanthin to internal standards. The standard calibration curve showed linearity at 0.5-50 μg / ml for both caffeine and paraxanthine. The concentration of caffeine and paraxanthin in urine samples was determined using the standard calibration curve.

Thus, since one analysis method of the present invention can be analyzed at the same time, the analysis cost and time can be analyzed at the same time, not only increased convenience, but also five drugs can be analyzed with high sensitivity using the minimum dose.

< Example  4>

CYP  Evaluation of Metabolic Activity of Enzymes

<4-1> CYP  Calculation of Metabolic Activity Index of Enzymes

To evaluate the metabolic activity of CYP enzymes, metabolic enzyme markers were calculated using ratios of pharmacokinetics and parent drug in urine or plasma according to the target enzyme.

The metabolic activity of CYP1A2 enzyme was calculated as the ratio of paraxanthine excretion to caffeine excretion from 0 to 4 hours after caffeine administration.

The metabolic activity of the CYP2C9 enzyme was the ratio of the area under the curve (AUC _{2 -4h} ) of _EXP3174 to the area under the curve (AUC _{2 -4h} ) of the losartan in plasma collected at 2, 3 and 4 hours after dosing. Calculated as

The metabolic activity of the enzyme CYP2C19 is omeprazole dosage after 2, 3, when the curve of omeprazole of the plasma collected in the fourth time - when the curve of the 5-hydroxy-omeprazole for the (AUC _{2 -4h)} enemy (AUC _{2 -4h)} Calculated as a ratio value.

If the metabolic activity of CYP2D6 enzyme dextromethorphan tolpan when the curve of the plasma dextromethorphan tolpan of collected in 2, 3 and 4 hours after administration of Dex curve trawl board for ever (AUC _{2 -4h)} enemy (AUC _{2 -4h} ) was calculated as the ratio value.

The metabolic activity of CYP3A enzyme was calculated as the ratio of the excretion of 1'-OH midazolam to the excretion of midazolam in urine collected from 0 to 4 hours after midazolam administration.

Using these indicators, the present invention maintains a minimum amount of blood collection by using the AUC (limited AUC) ratio of the parent drug and the metabolite at a limited time (between 2-4 hours) in addition to the parent drug and metabolite AUCt ratio. At the same time, we introduced the concept of increasing the accuracy of typing.

<4-2> Statistical Analysis

Wilcoxon rank sum test (Nonparametric test) to test the difference between the metabolic activity index at the same time of five drug cocktails and the metabolase activity index at the time of single administration calculated according to the method described in <4-1> ( Wilcoxon signed rank test) was performed. Normality test was performed using Shapiro-Wilk's value, and paired t test was performed on data with normality. In addition, the significance of the difference in metabolic activity between two doses was tested by interval estimation for the ratio of metabolic activity index between doses. All data are presented as mean and 95% confidence intervals. The above analysis was considered to be statistically significant when p <0.05 using SAS (Ver. 4.3), a statistical analysis program. Also, linear regression analysis was performed to evaluate the linear relationship between metabolic activity index and AUC (AUC ratio) of each probe.

As a result, the average ratio of individual metabolic enzyme activity indicators and 95% confidence intervals for five drug cocktails versus individual drug administrations is shown in Table 8.

Average ratio and 95% confidence intervals of individual metabolic enzyme activity indicators for five drug cocktails versus individual drug alone (n = 13) enzyme Phenotype Index Average ratio 95% confidence interval (%) P value CYP1A2 Excretion of paraxanthine / excretion of caffeine up to 4 hours in urine 0.89 69-114 0.153 CYP2C9 If the curve of EXP3174 in blood plasma ever the curve of (AUC _{2 -4h)} / losartan enemy (AUC _{2 -4h)} 1.01 82-126 0.845 CYP2C19 Area under the curve of 5-hydroxyomeprazole in plasma (AUC _{2 -4h} ) / _{Area under} the curve of omeprazole (AUC _{2 -4h} ) 1.07 74-155 0.359 CYP2D6 Area under the curve of dextrorphan in plasma (AUC _2-4h ) / _{Area under} the curve of dextromethorphan (AUC _{2 -4h} ) 1.19 73-195 0.455 CYP3A Excretion of 1'-hydroxymidazolam up to 4 hours in urine / excretion of midazolam 0.91 83-177 0.269

In addition, the mean and 95% confidence intervals of the ratios of metabolic enzyme activity indicators for individual drug alone or three drug cocktails for the five drug cocktails are shown in Tables 6 and 7.

Therefore, using the minimum dose cocktail kit for high-speed evaluation of CYP450 enzyme activity, the activity of CYP450 enzyme can be analyzed with high sensitivity, and the use of the minimum dose reduces side effects, increases convenience, and safety, compared to the previously reported cocktail kit. It can be seen that it is an excellent analysis method.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (9)

Minimum dose cocktail for rapid evaluation of cytochrome P450 enzyme activity, comprising a probe drug consisting of 5-15 mg caffeine, 1-5 mg losartan, 10-500 μg omeprazole, 1-5 mg dextromemethane, and 10-500 μg midazolam Kit. The method of claim 1,
Minimal dose cocktail kit for the rapid evaluation of cytochrome P450 enzyme activity, characterized in that the cytochrome P450 enzyme is selected from the group consisting of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A enzyme. The method of claim 1,
The probe drug is a minimum dose cocktail kit for the rapid evaluation of cytochrome P450 enzyme activity, characterized in that oral administration of the capsule formulation. The method of claim 1,
Minimal dose cocktail kit for the rapid evaluation of cytochrome P450 enzyme activity, characterized in that the probe drug is administered simultaneously (simultaneous). From subjects, except for humans, who received probes consisting of 5-15 mg caffeine, 1-5 mg losartan, 10-500 μg omeprazole, 1-5 mg dextrometolpan, and 10-500 μg midazolam,
(a) collecting blood or urine;
(b) caffeine, lotan, omeprazole, dextrometolpan and midazolam contained in the collected blood or urine; Or analyzing their metabolites in an LC-MS / MS system and calculating the ratios of the cytochrome P450 enzyme activity. The method of claim 5,
Blood is collected three times at 2, 3, and 4 hours after the administration of the probe drug, high-speed evaluation method of cytochrome P450 enzyme activity. The method of claim 5,
Urine is a high-speed method for evaluating cytochrome P450 enzyme activity, characterized in that it was collected once 4 hours after administration of the probe drug. The method of claim 5,
The metabolite of caffeine is paraxanthin, the metabolite of losartan is EXP3174, the metabolite of omeprazole is 5-OH omeprazole, the metabolite of dextrometol is dextrose, the metabolite of midazolam is 1- A high-speed evaluation method of cytochrome P450 enzyme activity, characterized in that 'OH midazolam. The method of claim 5,
The step of calculating the ratio in step (b) is performed by measuring the metabolites and metabolites that are not metabolized after administration of the probe drug,
If the mother drug is caffeine, calculate the excretion ratio of paraxanthin / caffeine excreted in blood or urine,
And wherein the parent compound is calculated to losartan the case, when the curve of EXP3174 in the blood or urine containing enemy (AUC _{2 -4h)} / the curve of losartan enemy (AUC _{2 -4h)} ratio,
Wherein the parent drug is the case of omeprazole, the curve of the 5-hydroxy-omeprazole (5-hydroxyomeprazole) contained in the blood or urine enemy (AUC _{2 -4h)} / the curve of omeprazole and calculating a ratio enemy (AUC _2-4h) ,
Wherein the parent drug is dextromethorphan tolpan a case, if a curve of the text contained in the blood or urine plate (dextrorphan) enemy (AUC _{2 -4h)} / dextromethorphan the curve of methoxy tolpan dextromethorphan enemy (AUC _2-4h) ratio And calculate
When the mother drug is midazolam, the excretion ratio of 1'-OH midazolam / midazolam excretion ratio in the blood or urine, characterized in that the rapid evaluation method of cytochrome P450 enzyme activity.

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