KR100658184B1 - A cocktail kit for high-throughput evaluation of human cytochrome P450 enzyme - Google Patents

Abstract

The present invention relates to a cocktail kit for rapid evaluation of cytochrome P450 enzyme activity. More specifically, the present invention includes a probe drug consisting of caffeine, losartan, omeprazole, dextrometolpan, and midazolam, and characterized in that the drug metabolism activity of five cytochrome P450 isoenzymes can be simultaneously measured at high speed. The present invention relates to a cocktail kit for rapid evaluation of cytochrome P450 enzyme activity.

Cytochrome P450 Enzyme, Drug Metabolism Activity, Probe Drug

Description

Cocktail kit for high-throughput evaluation of human cytochrome P450 enzyme

1 is a graph comparing metabolic activity index (oral cleaning rate) of CYP3A enzyme when midazolam alone and five drug cocktails are administered. Each horizontal line and bar represents the mean ± standard deviation.

FIG. 2 is a graph showing the correlation between midazolam concentration and the area under the 12 hour midazolam curve (AUC _0-12h ) in blood samples collected 4 hours after oral administration of midazolam. Dotted line represents 95% confidence interval.

Figure 3 is a graph comparing the concentration of midazolam in blood samples collected at 4 hours when midazolam alone and five drug cocktails. Each horizontal line and bar represents the mean ± standard deviation.

Figure 4 is a graph comparing the CYP2C9 metabolic activity index (Rosatan / E-3174) when the losartan alone and five drug cocktails. Each horizontal line and bar represents the mean ± standard deviation.

Figure 5 shows the metabolic activity index of CYP2C9 enzyme (Rosatan / E-3174) in urine samples collected between 0-8 hours after oral administration of losartan and the metabolic activity index of CYP2C9 enzyme in blood samples (Losatan / E-3174) is a graph showing the correlation between the _{area under the} curve (AUC _0-12h ). Dotted line represents 95% confidence interval.

Figure 6 shows the metabolic activity index of CYP2C9 enzyme (Rosatan / E-3147) in urine samples collected between 0-12 hours after oral administration of losartan and the metabolic activity index of CYP2C9 enzyme in blood samples (Losatan / E-3174) is a graph showing the correlation between the _{area under the} curve (AUC _0-12h ). Dotted line represents 95% confidence interval.

Figure 7 is a graph comparing the metabolic activity index (paraxanthin / caffeine) of the CYP1A2 enzyme when administered three drug cocktails and administered five drug cocktails. Each horizontal line and bar represents the mean ± standard deviation.

FIG. 8 shows the area under the curve of the metabolic activity index (paraxanthin / caffeine) of CYP1A2 enzyme and the metabolic activity index (paraxanthin / caffeine) of CYP1A2 enzyme in blood samples collected 4 hours after oral administration of caffeine AUC _0-12h ) is a graph showing the correlation between the ratio. Dotted line represents 95% confidence interval.

9 is a comparison of metabolic activity indicators (omeprazole / 5-OH omeprazole) of CYP2C19 enzyme measured using blood samples collected at three hours when three drug cocktails and five drug cocktails were administered. It is a graph. Each horizontal line and bar represents the mean ± standard deviation.

FIG. 10 shows metabolic activity index of CYP2C19 enzyme (omeprazole / 5-OH omeprazole) in blood samples collected 3 hours after oral administration of omeprazole and metabolic activity index of CYP2C19 enzyme in blood samples (omeprazole / 5-OH omeprazole) It is a graph showing the correlation between the _{area under the} curve (AUC _0-12h ). Dotted line represents 95% confidence interval.

FIG. 11 shows metabolic activity index of CYP2C19 enzyme (omeprazole / 5-OH omeprazole) in blood samples collected 4 hours after oral administration of omeprazole and metabolic activity index of CYP2C19 enzyme in blood samples (omeprazole / 5-OH omeprazole) It is a graph showing the correlation between the _{area under the} curve (AUC _0-12h ). Dotted line represents 95% confidence interval.

12 is a comparison of metabolic activity index (omeprazole / 5-OH omeprazole) of the CYP2C19 enzyme measured by blood samples collected at three hours when three drug cocktails and five drug cocktails were administered. It is a graph. Each horizontal line and bar represents the mean ± standard deviation.

FIG. 13 is a graph comparing CYP2D6 enzyme metabolic activity index (Log [dextrometol / dextropan]) in urine samples when three drug cocktails were administered and five drug cocktails were administered. Each horizontal line and bar represents the mean ± standard deviation.

The present invention relates to an agent cocktail kit for rapid evaluation of cytochrome P450 enzyme activity. More specifically, the present invention includes a probe drug consisting of caffeine, losartan, omeprazole, dextrometolpan, and midazolam, and characterized in that the drug metabolism activity of five cytochrome P450 isoenzymes can be simultaneously measured at high speed. It relates to a cocktail kit for the rapid evaluation of cytochrome P450 enzyme activity.

Cytochrome P450 (hereinafter referred to as CYP) enzyme is involved in the metabolism of various clinically useful drugs, and exists in a complex form composed 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, evaluation of activity of CYP isozymes is very important and can be applied not only in academic fields such as drug interaction studies and genotyping phenotyping but also in industrial applications such as new drug development.

Several techniques have been developed in vivo and in vitro to measure the activity of drug metabolism enzymes. 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' way. 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, including 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 recently because of the advantage of enabling the evaluation of enzyme activity in real time in terms of efficiency and reliability. The biggest advantage of the cocktail method is that one clinical trial can evaluate the activity of isoenzymes in multiple pathways simultaneously. This allows the rapid evaluation of enzyme activity in drug interaction studies or phenotypic tests. However, pharmacokinetic and pharmacodynamic interactions between probes must be considered in the use of cocktails, as opposed to the method of individual determination 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 is used to evaluate metabolic enzyme activity. Therefore, a multi-component simultaneous assay is required to measure several drugs in one 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 "Cooperstown cocktail" and "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 compensate for this, the Cooperstown 5 + 1 cocktail method added 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 have been studying for many years to develop a new cocktail clinical trial technology that can complement the problems of the existing cocktail method while simultaneously evaluating various types of drug metabolizing enzyme activity, and caffeine, which has been reported to have no drug interaction in the past, By developing oral midazolam and losartan, three drug cocktails, omeprazole and dextrometolphan, were developed.The drug cocktails were used to rapidly increase the drug metabolism activity of five cytochrome P450 isozymes without side effects. This invention was completed by confirming that it can evaluate.

Accordingly, it is an object of the present invention to provide an agent cocktail kit for rapid evaluation of cytochrome P450 enzyme activity comprising a probe drug consisting of caffeine, losartan, omeprazole, dextrometolpan and midazolam.

In order to achieve the above object, the present invention provides a kit for measuring the drug metabolic activity of the cytochrome P450 enzyme comprising a probe drug consisting of caffeine, losartan, omeprazole, dextrometolpan and midazolam.

Hereinafter, the present invention will be described in detail.

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

The 'probe drug' is a drug that is a selective substrate of each cytochrome P450 isoenzyme, and means that the drug metabolism activity of the cytochrome P450 enzyme can be measured 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, a large amount of blood was inevitable to analyze the pharmacokinetic properties of warfarin because there is no established metabolic enzyme activity index.

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 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 in the present invention used a method of oral administration of an injection. Therefore, the probes in the present invention is preferably administered orally.

In addition, the probe drug according to the present invention may be administered simultaneously or sequentially. That is, the probe drug may be administered simultaneously with one ingredient before, after and / or with another ingredient.

More preferably, the probe drug according to the present invention may be administered by mixing caffeine, losartan, omeprazole and midazolam at the same time, and then dextrometolpan after a predetermined time more preferably 4 hours. Separation of dextrometol plate dosing time in consideration of the gastrointestinal motility reduction effect of the drug, it is considered that there is no big problem even when the probes according to the present invention is administered simultaneously with reference to the conventional literature. (Streetman DS et al., Clin Pharmacol Ther , 68: 375-383, 2000; Christensen M. et al., Clin Pharmacol Ther , 73: 517-528, 2003).

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 an effective dose that can evaluate the activity of the cytochrome P450 enzyme and a low dose in a range that does not cause side effects in vivo. Should be Preferably, the adult may include 50-200 mg of caffeine, 25-100 mg of losartan, 10-40 mg of omeprazole, 10-50 mg of dextrometolphan, and 1-5 mg of midazolam. Most preferably, it may include 93 mg of caffeine, 50 mg of losartan, 20 mg of omeprazole, 30 mg of dextrometolphan and 2 mg of midazolam.

In an embodiment of the present invention, 2 mg of midazolam (15 mg / 3 ml) is diluted orally administered to 100% apple juice, and at this time, one tablet of caffeine (93 mg), one capsule of omeprazole (20 mg), and one losartan tablet (50 mg) ) Was administered together. Four hours after dosing, two tablets of dextrometol (15 mg per tablet) were administered with water (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 carried out 4 hours after administration, urine collection is preferably performed between 0 to 8 hours after administration.

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.

CYP1A2 (probe drug: caffeine) = Paraxanthin / caffeine concentration in plasma taken 4 hours after dosing

CYP3A (probe drug: midazolam) = midazolam concentration in plasma taken 4 hours after dosing

CYP2C9 (probe drug: Rosatan) = concentration of Rosatan / E-3174 in urine collected up to 8 hours after dosing

CYP2C19 (probe drug: omeprazole) = omeprazole concentration / 5-OH omeprazole concentration taken 4 hours after administration

CYP2D6 (probe drug: dextrometol) = log in urine collected up to 8 hours after dosing

As described above, in one embodiment of the present invention, blood collected 4 hours after administration was used as a sample for measuring the activity of the CYP1A2 enzyme and the CYP2C19 enzyme. This is correlated well with the _area under the 12 hour curve (AUC _0-12h ) in the blood sample obtained from the blood sample taken at 4 hours, as verified through an embodiment of the present invention. Since it is shown (see Fig. 8 and 11), the blood sample collected at the 4 hours was found to be a suitable sample for measuring the drug metabolic activity of each enzyme.

In addition, in one embodiment of the present invention, urine collected between 0-8 hours after dosing was used as a sample for measuring the activity of the CYP2D6 enzyme and the CYP2C9 enzyme. This was correlated well with the _area under the 12 hour curve (AUC _0-12h ) in the blood sample in the urine sample collected between 0-8 hours, as verified through one embodiment of the present invention. Since it is shown (see Fig. 5), the urine samples collected up to 8 hours was found to be a suitable sample for measuring the drug metabolic activity of each enzyme.

On the other hand, one of the limitations of the cocktail method for measuring enzyme activity is that several blood collections are required to obtain the oral cleaning rate after administration of the probe drug, midazolam, for the measurement of enzyme activity of CYP3A. This is a major limitation in that the cocktail method aims at high speed measurement of CYP enzyme activity. In one embodiment of the present invention, blood collected 4 hours after administration was used as a sample for measuring the activity of the CYP3A enzyme. As shown through one embodiment of the present invention, this is a blood sample collected four hours after dosing with one subject each administered 12 cases of midazolam alone and one cocktail administered to 12 subjects Since it shows a good correlation between the concentration of zolam and the area under the curve (AUC) (see Fig. 2), it can be seen that the blood sample taken at 4 hours is a suitable sample for measuring the drug metabolic activity of the CYP3A enzyme. there was.

On the other hand, the present inventors have been found that the simultaneous use of caffeine, omeprazole and dextromephanpan in a kit comprising five drugs according to the present invention do not interact with each other in the Cooperstown cocktail. On the other hand, midazolam has been administered through the intravenous administration method, which has a disadvantage in that it does not reflect the activity of the CYP3A enzyme expressed in the gastrointestinal tract as described above. Therefore, the present inventors adopted a method of oral administration of midazolam, and unlike the intravenous administration of midazolam, it is necessary to evaluate this part because there is a possibility of interaction with other drugs at the drug absorption site during oral administration. Rosatan included in the kit composition of the present invention also needs to be evaluated for the interaction when co-administered with other cocktail drugs.

Therefore, the present inventors compared the metabolic activity index of each enzyme when the five drug cocktails according to the present invention were administered with midazolam alone, losartan alone, and three drug cocktails (caffeine, omeprazole, and dextrometol). (See Example 5).

As a result, the drug metabolism activity of the enzyme was not statistically different between the cocktail administration and the single drug administration and the three drug cocktail administration according to the present invention, the cocktail administration of the five drugs of the present invention It did not cause any side effects.

Therefore, the kit for analyzing the drug metabolism activity of the cytochrome P450 enzyme according to the present invention, characterized in that the use of caffeine, losartan, omeprazole, dextromemethane and midazolam at the same time as a probe drug does not show interaction between the probe drugs Without the side effect, there is an advantage that can simultaneously measure the metabolic activity of the five cytochrome P450 enzymes.

In addition, in the case of using the kit according to the present invention, there is an advantage that the metabolic activity of cytochrome P450 enzyme can be easily measured at a high speed by collecting blood once every 4 hours after the drug administration and collecting urine for up to 8 hours.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Example 1>

Selection of Subjects

Among the subjects recruited through the clinical trial announcement, the attending physician should participate in the clinical trial based on the subject's background, physical examination, vital symptoms (temperature, blood pressure, pulse rate), electrocardiogram test, and laboratory test results. The subjects were healthy Korean male volunteers between 20 and 45 years of age who deemed appropriate and had written informed consent to participate in clinical trials. As a result, a total of 12 subjects, except the dropout subjects, participated in the clinical trial. The mean age of the patients was 21.3 ± 0.8 years old, average body weight was 66.8 ± 5.4 kg, and height was 172.8 ± 5.5 cm. The clinical trial was conducted after approval of the Institutional Review Board of Busan Paik Hospital.

<Example 2>

Establishment of test drug and administration method

To assess CYPIA2, CYP2C9, CYP2C19, CYP2D6 and CYP3A enzyme activities, caffeine, losartan, omeparazol, dextrometolphan and midazolam were used as the respective probes. The composition, formulation, name and content of each test drug are as follows (Table 1).

Ingredients, formulations, names and contents of test drugs Ingredient Name product name Pharmaceutical companies Formulation content Midazolam Bovine midazolam injection Bukwang Chemical Injection 15mg / 3ml Caffeine Cafe soft handjob Eisai Co., LTD refine 93 mg / tablet Omeprazole Losec capsule Yuhan Corporation Capsule 20mg / capsule Dextrometol Rummy Handjob Roche Korea refine 15 mg / tablet Rosatan Koza Jung MS D Korea refine 50 mg / tablet

As a test drug administration method, the subjects selected in Example <1-1> were performed according to the open, randomized, balanced, and lattice-based four-dimensional cross test method. That is, four groups of three subjects each were assigned to a total of 12 subjects according to a pre-filled randomized schedule, and A (midazolam 2mg alone), B (rosatan 50mg alone), C (caffeine 93mg, omeprazole 20mg and dex) Clinical trials were divided into four drug regimens: 30 mg of trometholpan and 1 mg of midazol, caffeine 93 mg, omeprazole 20 mg, losartan 50 mg, and 15 mg of dextrometol. (Table 2). There was a one week break between each phase.

4D cross test design group Step 1 Step 2 Step 3 Step 4 Group 1 A D B C Group 2 B A C D Group 3 C B D A Group 4 D C A B

More specifically, the drug regimen A was administered orally by diluting 2 mg of midazolam (15 mg / 3 ml of fluoride midazolam injection) in 50 ml of 100% apple juice, and the containers used were rinsed twice using 90 ml of water. . Drug regimen B was orally administered with Rosatan (cosa tablet 50 mg) with 240 ml of water. Medication C was administered 1 capsule of caffeine (caffeine soft tablets 93mg) and 1 capsule of omeprazole (loseccapsule 20mg) with 240ml of water, and 240ml of 2 tablets of dextrometol (15mg of rumira tablets) 4 hours after administration. Dosing with water. Drug D was administered orally by diluting 2 mg of midazolam (15 mg / 3 ml of buoyant midazolam injection) in 50 ml of 100% apple juice, and the containers used were rinsed twice with 90 ml of water. At this time, 1 tablet of caffeine (cafe soft tablets 93 mg), 1 capsule of omeprazole (losec capsule 20 mg), and 1 tablet of losartan (cosa tablet 50 mg) were taken together. Was dosed with 240 ml of water.

As a result, all subjects completed the clinical trial successfully, and no side effects were observed during drug administration alone or during cocktail administration.

< Example 3>

Concentration analysis of drugs and their metabolites in urine and plasma of subjects

After administering the drug according to the method of Example 2 to the subject of Example 1, the concentration of dextrometolpan in urine and the concentration of losartan in the urine and plasma samples were measured by high pressure liquid chromatography (HPLC). In addition, the concentrations of midazolam, caffeine, and omeprazole in plasma samples were analyzed using an LC / MS / MS system. Among them, caffeine and omeprazole were extracted at one time and analyzed simultaneously.

<3-1> Collection of Urine and Blood Samples

Blood sampling was performed from the intravenous catheter (Angiocath PlusTM, 20GA, 1.16IN, 1.1mm 30mm, Boin Medical, Korea) mounted on the upper arm, taking 10ml of control blood immediately before administration. At 15, 30, 60, 90, 120, 180, 240, 300, 360, 480, 600 and 720 minutes after dosing, 10 ml of blood were collected in heparinized tubes (Vacutainer, Becton & Dickinson, USA). . Blood samples were collected and centrifuged at 4 ° C. and 3000 rpm. Plasma was collected in microcentrifuge tubes (Stockwell Scientific, USA) and stored at −80 ° C. until analysis.

Urine was collected 20 ml of control urine immediately prior to dosing and collected between 0 and 4 hours, between 4 and 8 hours, and between 8 and 12 hours after dosing. The urine collected over time was measured and recorded using a volumetric cylinder, and 20 ml of urine was collected in conical centrifuge tubes (BDFalcon, Becton & Dickinson, USA) and stored at -80 ° C until analysis.

<3-2> Analysis of dextrometol and dextrose in urine samples

The concentration analysis of dextrometol and its metabolite, dextrose, in urine samples was measured by HPLC using a modified method (Schadel M. et al., J Clin Psychopharmacol , 15 (4): 263-269). , 1995; Kristensen HT.J Pharm Biomed Anal , 6: 211-220, 1988). The HPLC system consisted of Gilson's 307 pump, 234 autoinjector, Waters' 2475 fluorescence detector and Linchrosorb RP-8 (10 μm, 200 mm 4 mm, Merk, USA) column. As a mobile phase, 10 mM KH ₂ PO ₄ buffer: methanol: acetonitrile (570: 200: 200, v / v) was used, and the flow rate was 0.8 ml / min. Dextromethol and dextrose were separated from the column using a fluorescence detector (Ex 200 nm, Em 304 nm).

To determine the concentrations of dextrometol and dextrose in urine samples, first, 1000 μl of urine sample collected from the subject and 500 μl of 0.1 M acetic acid buffer were mixed, and 50 μl of 10,000 unit β-glucuronidase was added thereto. The reaction was carried out in a 37 ℃ water bath for 18 hours. Then, 50 µl of an internal standard (levallorphan 5 µg / ml) was added to the reaction solution, and the mixture was gently mixed. Then, 500 µl of saturated carbonate solution and 5 ml of hexane: butanol (98: 5, v / v) solution were added subsequently. And shake extraction for 10 minutes using Multi Reax (Heidolph, Germany). Thereafter, the organic layer was transferred to a new test tube by centrifugation at 3000 rpm for 10 minutes, dried using Speed-Vac (Savant, USA), redissolved in 200 μl of methanol, and 25 μl was injected into an HPLC system. The quantitative limit concentrations of dextrometol and dextropan measured by the method were 5 and 100 ng / ml, respectively, and a standard calibration curve was obtained from the ratio of the peak areas of dextrometol and dextrose and the internal standard. It was. The standard calibration curves showed a straight line at 5 to 250 ng / ml and 100 to 5000 ng / ml for dextrometol and dextrose. Using the standard calibration curve, the concentrations of dextrometol and dextrose in urine samples were determined.

<3-3> Analysis of Rosatan and E-3147 Concentrations in Urine and Plasma Samples

Analysis of the concentration of losartan and its metabolites, E3147, in urine samples was determined using HPLC by modifying known methods (Gonzalez L. et al., J Chromatogr A , 949: 49-60, 2002; Polinko M. et al. , J Pharm Biomed Anal , 33: 73-84, 2003). The HPLC system consisted of Gilson's 307 pump, 234 autoinjector, Waters' 2475 fluorescence detector and Zorbax C ₈ (5 μm, 150 μs 4.6 mm, Agilent Technologies, USA) column. As a mobile phase, acetonitrile: 0.2% formic acid (37: 63, v / v) was used, and the flow rate was 1.0 ml / min. Rosatan and E-3147 separated from the column were detected using a fluorescence detector (Ex. 250 nm, Em 370 nm).

In order to measure the concentration of losartan and E-3174 in urine samples, first, 500 ml 0.5 ml of formic acid and 25 µl of internal standard (napthoxyacetic acid 1000ng / ml) were added to a 10 ml volume test tube and mixed lightly. Then 5 ml of a CH ₂ Cl ₂ : ether (40:60, v / v) solution was then added and shaken for 10 minutes using Multi Reax (Heidolph, Germany). After centrifugation at 3000 rpm for 10 minutes, the organic layer was transferred to a new test tube, dried using Speed-Vac (Savant, USA), redissolved in 200 µl of 20% acetonitrile, and 25 µl was injected into the HPLC system. When using this method, the measurable limit concentration of Rosatin and E-3174 was 5ng / ml, and a standard calibration curve was obtained from the peak area ratio of Rosatin and E-3174 and the internal standard. The standard calibration curve showed linearity at 5 to 1000 ng / ml. From this standard calibration curve, the concentrations of Rosatin and E-3174 in urine were determined.

The concentrations of losartan and E-3174 in plasma samples were measured using the same conditions and extraction methods as in urine sample analysis. When using this method, the measurable limit concentration of Rosatan and E-3174 was 5ng / ml, and the standard calibration curve was obtained from the peak area ratio of Rosatan and E-3174 and the internal standard. The standard calibration curve showed linearity at 5 to 1000 ng / ml. The standard calibration curve was used to determine the concentrations of losartan and E-3174 in plasma.

<3-4> Midazolam, Caffeine, Omeprazole and Their Metabolites in Plasma Samples analysis

Analysis of midazolam, caffeine, omeprazole and its metabolites in plasma samples was measured using LC / MS / MS with a modification of known methods (Kashuba et al., Clin Pharmacol Ther , 64 (3): 269-277, 1998; Streetman et al., Pharmacogenetics , 10: 187-216, 2000; Christensen M et al., Clin Pharmacol Ther , 73: 517-528, 2003). The system used was an Agilent 1100 series HPLC system (Agilent Technologies, USA) and a PE SCIEX API 3000 LC / MS / MS system (Applied Bio Systems, Canada). The HPLC column was Luna C ₁₈ (3 μm, 2 μs 50 mm, Phenomenex Inc. USA), and the mobile phase was acetonitrile: 0.1% formic acid (30: 70, v / v), and the flow rate was 0.2 ml. It was / min. 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 3.

Analytical conditions of midazolam, caffeine, omeprazole and each metabolite enzyme Analytes Precursor ions (m / z) Product Ion (m / z) Collision energy (ev) CYP3A Midazolam 326 291 35 CYP1A2 Caffeine 194 138 28 Paraxanthin 180 110 35 CYP2C19 Omeprazole 346 198 35 5-OH omeprazole 362 214 35 IS Phenacetin 180 110 35

For the analysis of midazolam, first add 10 µl of internal standard (1 μM of phenacetin) to 0.5 ml of plasma and mix gently. Then, 600 µl of 2M NaOH solution and diethyl ether: methylene chloride (6: 4, v / v) 5 ml of solution was then added and shake extracted for 10 minutes using Multi Reax (Heidolph, Germany). After centrifugation at 3000 rpm for 10 minutes, the organic layer was transferred to a new test tube, dried using Speed-Vac (Savant, USA), redissolved in 200 µl of 30% acetonitrile and injected into 10 µl LC / MS / MS system. It was. When the above method was used, the limit of quantitation of midazolam was 1 ng / ml, and a standard calibration curve was obtained from the peak area ratio of the midazolam internal standard in plasma samples. The standard calibration curve showed linearity at 1-50 ng / ml. The concentration of midazolam in blood was determined using the standard calibration curve.

Extraction method for the concentration analysis of caffeine, omeprazole and its metabolites is almost the same as the midazolam analysis method, and both drugs were extracted at once. That is, 10 µl of internal standard (0.5 µM of phenacetin) is added to 0.5 ml of plasma and gently mixed. 1000 µl of 0.5 M NaH ₂ PO ₄ solution and diethyl ether: methylene chloride (6: 4, v / v) 5 ml of solution was then added and shaken for 10 minutes using Multi Reax (Heidolph, Germany). After centrifugation at 3000 rpm for 10 minutes, the organic layer was transferred to a new test tube, dried using Speed-Vac (Savant, USA), redissolved in 200 µl of 30% acetonitrile and injected into 10 µl LC / MS / MS system. It was.

The quantitative limit concentration of caffeine and paraxanthin measurable using this method was 5 ng / ml. A standard calibration curve was obtained from the peak area ratios of caffeine, paraxanthin and internal standards. The standard calibration curve showed linearity at 5 to 1000 ng / ml. The standard calibration curve was used to determine the concentrations of caffeine and paraxanthin in the blood.

The quantitative limit concentrations of omeprazole and 5-OH omeprazole were 5 ng / ml and 1.25 ng / ml, respectively, and standard calibration curves were obtained from the peak area ratios of omeprazole and 5-OH omeprazole and internal standards. The standard calibration curves showed linearity at 5-1600 ng / ml and 1.25-600 ng / ml, respectively. The standard calibration curve was used to determine the concentrations of omeprazole and 5-OH omeprazole in the blood.

<Example 4>

Metabolic Activity Evaluation of CYP Enzymes

<4-1> Calculation of Metabolic Activity Indicators of CYP 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 the CYP1A2 enzyme was calculated from the ratio of caffeine to the concentration of paraxanthin in plasma taken 4 hours after the administration of caffeine (CYP1A2 = 4-h plasma paraxanthin / caffeine concentration). It was calculated from the ratio of the concentration of E-3174 to the concentration of Rosatin in urine collected between 0 and 8 hours after losartan dosing (CYP2C9 = 0-8 hours urinary Rosatin concentration / E-3174 concentration).

In addition, the metabolic activity of CYP2C19 enzyme was calculated from the ratio of 5-OH omeprazole concentration to omeprazole concentration in plasma taken 4 hours after omeprazole administration (CYP2C19 = 3-h plasma omeprazole / 5-OH omeprazole) and CYP2D6 enzyme. The metabolic activity of was calculated from the log value of the molar concentration of dextropan to the molar concentration of dextrometol in urine collected from 0 to 8 hours after dextrometol plate administration (CYP2D6 = 0-8 hours urine). Log [dextrometol / dextropan] molar concentration ratio).

The metabolic activity of CYP3A enzyme was calculated from the concentration of midazolam in plasma taken 4 hours after the administration of midazolam (CYP3A = 4-h plasma midazolam concentration) or as oral cleaning rate through pharmacokinetic analysis of midazolam ( CYP3A = oral clearance). In other words, the pharmacokinetic properties of midazolam were obtained from non-compartment analysis using WinNonlin ^® (Pharsight Co. ver. 4.0.1), a pharmacokinetic analysis program. The loss rate constant (K _el ) of midazolam was obtained from the linear regression analysis by the least-squares method, and the area under the curve (AUC _0-last ) until the last blood collection time was calculated from the trapezoidal formula. The AUC _inf under the curve from 0 hours to infinity time was obtained from the formula of AUC _0-last + C _last / K _el and the oral cleaning rate (Cl / F) was calculated from the formula of Dose / AUC _inf .

<4-2> Statistical Analysis

Indicative of metabolic enzyme activity and single administration (in the case of drug use A and B) or three drug cocktails during simultaneous administration of the five drug cocktails calculated in accordance with the method described in <4-1>. A nonparametric Wilcoxon signed rank test was performed to test for differences from the metabolic activity indicators in the city (in case of drug use C). 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 recognized as statistically significant when p <0.05 using SAS (Ver. 8.01) and SPSS (Ver. 10.07).

As a result, the average and 95% confidence intervals of the individual metabolic enzyme activity indicators obtained by the single drug alone or three drug cocktails and five drug cocktails are shown in Tables 4 and 5.

Mean and 95% confidence intervals (n = 12) of individual metabolic activity indicators for individual drug alone and five drug cocktails enzyme Phenotype Index Individual medication alone 5 drug cocktails Average 95% CI Average 95% CI P value CYP3A Oral cleaning rate 129 100-157 123 96-149 .480 CYP2C9 Rosatin / E-3174 0.83 0.63 to 1.03 0.78 0.50 to 1.05 .583

Mean and 95% confidence intervals (n = 12) of individual metabolic enzyme activity indicators for three drug cocktails and five drug cocktails enzyme Phenotype Index 3 drug cocktails 5 drug cocktails Average 95% CI Average 95% CI P value CYP1A2 Paraxanthin / Caffeine 0.38 0.32 to 0.42 0.37 0.28-0.46 .583 CYP2C19 Omeprazole / 5-OH omeprazole (n = 11) 2.49 1.62-3.36 2.59 1.80-3.37 .248 CYP2D6 Log [DMP / DP] -2.24 -2.6 to-1.88 -2.39 -2.81 to -1.97 .136

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.

Mean and 95% Confidence Intervals for Metabolic Enzyme Activity Index of Individual Drug Administration for Five Drug Cocktail Administration enzyme Phenotype Index 5 drugs / individual drugs 95% CI CYP3A Oral cleaning rate 0.98 0.83 to 1.13 CYP2C9 Rosatan / E-3174 0.97 0.70 to 1.24

 Mean and 95% Confidence Intervals for Metabolic Enzyme Indices at Three Drug Doses for Five Drug Cocktails enzyme Phenotype Index 5 drugs / 3 drugs 95% CI CYP1A2 Paraxanthin / Caffeine 0.98 0.81 to 1.14 CYP2C19 Omeprazole / 5-OH omeprazole (n = 11) 1.08 0.98-1.17 CYP2D6 Log [DMP / DP] 1.07 0.98-1.15

<4-3> Comparison of Metabolic Activity Indicators of CYP3A Enzyme During Drug Administration and Cocktail Administration

The midazolam oral cleaning rate averaged 129L / h (95% confidence interval, 100-157L / h) when administered midazolam alone, and averaged 123L / h (95% confidence interval, 96-149L) when administered five drug cocktails. / h) (Table 4). Therefore, when the midazolam alone administration was compared with the administration of the five drug cocktails, there was no significant difference between the two doses (p = 0.480). In addition, the administration of the five drug cocktails did not appear to increase or decrease the metabolic activity of the CYP3A enzyme compared to the single administration (Fig. 1). Furthermore, the 95% confidence interval for the ratio of the oral cleaning rate to the administration of five drugs for cocktail administration was 0.83 to 1.13, indicating that there was no difference in enzyme activity between the two doses (Table 6).

In addition, to check whether the ratio of blood samples was adequate for 4 hours after administration as an indicator of metabolic activity, 4 subjects were administered with 12 individual subjects alone and 5 drug cocktails. The correlation between the concentration of midazolam and the area under the 12-hour midazolam curve (AUC _0-12h ) in the blood samples collected was examined (n = 24). As a result, r ² = 0.863 and p <0.001 showed good correlation (FIG. 2).

The blood concentrations of midazolam collected at 4 hours were 1.39 (95% confidence interval, 1.04 to 1.74) and 1.37 (95% confidence interval, 1.03 to 1.71) between the two doses, respectively, when administered alone and with cocktails of five drugs. There was no significant difference (P = 0.875) (FIG. 3).

<4-4> Comparison of CYP2C9 Metabolic Enzyme Activity Indicators During Drug Administration and Cocktail Administration

The CYP2C9 metabolic enzyme activity index (Rosatan / E-3147) also averaged 0.83 (95% confidence interval, 0.63 to 1.08) and 0.78 (95% confidence interval, 0.50 to 1.05, respectively) when administered alone or with cocktails of five drugs. ) Showed no statistically significant difference between the two doses (P = 0.583) (Table 4). On the other hand, the variation of the metabolic enzyme activity index slightly increased when the cocktails were administered to five drugs, but the overall metabolism did not show a tendency to increase or decrease (FIG. 4). In addition, the 95% confidence interval for metabolic enzyme activity index ratio was 0.70 to 1.24, showing no difference between the two doses and a bias toward one side (Table 6).

Furthermore, to determine whether the ratio of using urine specimens from 0 to 8 hours as an indicator of metabolic activity was appropriate, 12 subjects were treated with a single drug and five drug cocktails as one individual. The correlation between the losartan / E-3174 ratio in urine samples taken between 8 h and the 12 h losartan / E3174 area under the curve (AUC _{0-12 h} ) in blood samples was examined (n = 24). As a result, r ² = 0.726 and p <0.001 showed good correlation (FIG. 5). There was also a good correlation between the Lostan / E-3147 ratio and the 12-hour Lostan / E-3174 area under the curve (AUC) ratio in urine collected between 8 and 12 hours (r ² = 0.693). , p <0.001) (FIG. 6). Therefore, it is considered that both urine samples collected between 0 and 8 hours and urine samples collected between 0 and 12 hours can be used for the calculation of metabolic activity indicators. On the other hand, considering the simplicity of the test procedure, it is more appropriate to use urine samples collected between 0 and 8 hours.

<4-5> Comparison of CYP1A2 Metabolizing Enzyme Indices with Three or Five Drug Cocktails

In the case of CYP1A2 metabolic activity index (paraxanthin / caffeine), the average of 0.38 (95% confidence interval, 0.32-0.42) and 0.37 (95% confidence interval, 0.28-0) for the cocktail administration of three drugs and the cocktail administration of five drugs, respectively 0.46), there was no statistically significant difference between the two doses (P = 0.583) (Table 5). In addition, as in the case of CYP2C9, the variation of metabolic enzyme activity index was slightly increased when cocktails of five drugs were compared with that of cocktails of three drugs, but the overall metabolism tends to increase or decrease. (FIG. 7). Furthermore, the 95% confidence interval for the ratio of metabolic activity indicators did not show a difference between the two doses (0.81 to 1.14) (Table 7).

To determine whether it is appropriate to use the blood sample collected 4 hours after the administration as a metabolic activity indicator, 3 subjects and 5 drug cocktails were administered to 12 subjects. The correlation between the _{concentration of paraxanthin} / caffeine in blood collected over time and the area under the 12 hour _paraxanthin / caffeine curve (AUC _0-12h ) in blood samples was examined (n = 24). As a result, r ² = 0.949, p <0.001 showed a good correlation (Fig. 8). Therefore, it was found that the ratio of paraxanthin / caffeine in blood collected at 4 hours was a good indicator reflecting the enzyme metabolic activity.

<4-6> Comparison of CYP2C19 Metabolic Enzyme Activity Indicators During Cocktail Administration of Three or Five Drugs

CYP2C19 metabolic enzyme activity index (omeprazole / 5-OH omeprazole) was averaged at 2.49 (95% confidence interval, 1.62 to 3.36) and 2.59 (95% confidence interval, 1.80 to 3. 3.37), there was no statistically significant difference between the two doses (P = 0.248) (Table 5).

In addition, the metabolic activity of the CYP2C19 enzyme was not increased or decreased when the five drug cocktails were administered compared with the three drug cocktails (FIG. 9). Furthermore, in the 95% confidence interval estimate for the ratio of metabolic enzyme activity indicators, there was no difference between the two doses (0.81 to 1.14) (Table 7).

To determine whether it is appropriate to use the blood sample collected three hours after the administration as a metabolic activity indicator, three subjects and five drug cocktails were administered to 12 subjects. The correlation between the ratio of omeprazole / 5-OH omeprazole concentration in blood taken over time and the area under the curve of 12 hour omeprazole / 5-OH omeprazole in blood (AUC _0-12h ) was examined (n = 24). As a result, r ² = 0.865 and p <0.001 showed good correlation (FIG. 10). On the other hand, omeprazole was not detected in one subject from blood collected at 2 hours. Therefore, the blood collected at the second hour was judged to be an inappropriate sample. In addition, the correlation between the ratio of omeprazole / 5-OH omeprazole concentration in blood collected at 4 hours and the area under the curve (AUC _0-12h ) at 12 hours omeprazole / 5-OH omeprazole in blood samples was compared. 0.908, p <0.001 showed good correlation (FIG. 11). On the other hand, an appropriate sample for measuring CYP1A2 enzyme activity using a cocktail as a probe drug was selected as blood 4 hours after dosing.As a test sample, blood collected 4 hours after dosing was used as an appropriate sample for measuring CYP2C19 enzyme activity. Was judged to be reasonable.

The omeprazole / 5-OH omeprazole concentration ratio in the blood collected at the 4 hour period was 2.09 (95% confidence interval, 1.18 to 3.01) and 2.21 (95% confidence interval, respectively) for three drug cocktails and five drug cocktails, respectively. 1.35 to 3.08), there was no significant difference between the two doses (P = 0.328) (FIG. 12). In addition, since the 95% confidence interval of the ratio of five drug cocktails for the three drug cocktails included 1 from 0.89 to 1.36, there was no difference in the metabolic activity index between the two doses.

<4-7> Comparison of CYP2D6 Metabolic Enzyme Activity Indicators in Three or Five Drug Administrations

The CYP2D6 metabolic activity index (log [dextrometolpan / dextrose]) averaged -2.24 (95% confidence interval, -26 to -1.88) and -2.39 for three drug cocktails and five drug cocktails, respectively. There was no statistically significant difference between the two doses (95% confidence interval, -2.81 to -1.97) (P = 0.136) (Table 5). In addition, the metabolic activity of the CYP2D6 enzyme was not increased or decreased when the five drug cocktails were administered compared with the three drug cocktails (FIG. 13). The 95% confidence interval estimate for the ratio of metabolic enzyme activity indicators showed no difference between the two doses (0.98-1.15) (Table 7).

From the above experimental results, the metabolic enzyme activity index at the time of cocktail administration of the five drugs did not show a statistically significant difference compared with the metabolic enzyme activity index at the cocktail administration of the three drugs alone. In addition, when the 95% confidence interval for the ratio of metabolic enzyme activity index between each dose was included all 1, it was found that there is no difference between the metabolic enzyme activity index between each dose.

Inje cocktail kit for high-speed evaluation of cytochrome P450 enzyme activity comprising five drug cocktails according to the present invention simultaneously measures the metabolic activity of the five cytochrome P450 enzymes without any side effects without showing interactions between the probes There is an advantage to this. Particularly, in the case of the conventional cocktail, side effects due to the use of warfarin may occur, but the Inje cocktail may be used without the occurrence of such side effects. Inje cocktails require minimal invasive sampling (one time for 4 hours and once for 8 hours of urine) and have the advantage of reducing analysis time through simultaneous analysis using LC / MS / MS.

Claims (6)

Cytochrome P450 efficacies comprising a probe drug consisting of caffeine 50-200 mg, losartan 25-100 mg, omeprazole 10-40 mg, dextrometolphan 10-50 mg, and midazolam 1-5 mg Cocktail kit for fast evaluation of bovine activity. The cocktail kit for rapid evaluation of cytochrome P450 enzyme activity according to claim 1, wherein the cytochrome P450 enzyme is selected from the group consisting of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A enzymes. The cytosol according to claim 1, wherein the probe drug is orally administered. Cocktail kit for fast evaluation of chromium P450 enzyme activity. The cocktail kit for rapid evaluation of cytochrome P450 enzyme activity according to claim 1, wherein the probe drug is administered simultaneously or sequentially. 5. The cocktail kit for rapid evaluation of cytochrome P450 enzyme activity according to claim 4, characterized in that dextrometolpan is administered 4 hours after simultaneously administering caffeine, losartan, omeprazole and midazolam. delete

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