KR20190001096A - Biomarkers for the determination of CYP3A enzyme activity - Google Patents

Abstract

The present invention relates to hydroxy acylcarnitine, a novel biomarker for measuring an activity of CYP3A enzymes. A method of measuring the activity of CYP3A enzymes of the present invention can estimate changes in hepatic CYP3A metabolism in non-invasive and effective ways by detecting hydroxy acylcarnitine in urine. Also, it is possible to provide information important for drug development and drug interaction to be useful for patient-specific drug treatment.

Description

[0002] Biomarkers for the determination of CYP3A enzyme activity [

The present invention relates to a novel biomarker for measuring the activity of a CYP3A enzyme.

Human cytochrome P450 (CYP) 3A, a drug metabolizing enzyme, accounts for about 40% of the total CYP in human liver microsomes. CYP3A is an important drug metabolizing enzyme that has a wide substrate specificity and is involved in many drug metabolisms of about 50% or more. Looking at changes in CYP3A metabolism by drugs during the development phase of new drugs provides important information for drug metabolism and enables predictions of drug interactions. Due to the increased use of multiple drug therapies, the assessment of CYP3A mediated drug-drug interactions (DDI) is crucial to drug development and clinical trials.

The metabolism of CYP3A enzymes is highly variable in humans, so customized drug therapy according to individual CYP3A metabolism ability can optimize treatment. Conventional CYP3A metabolic assay was measured by the degree of exposure or clearance of the probe drug by administering a probe drug selectively metabolized by CYP3A to humans. However, this method is disadvantageous in that it is an invasive method in which unnecessary drugs are administered into the human body and blood concentration is required to be obtained through blood collection.

Specifically, CYP3A metabolic activity is traditionally assessed by the exposure or clearance rate of the probe drug measured in plasma or urine by administering a probe drug that is selectively metabolized by CYP3A, The relative ratio was assessed as a metabolic ratio. We also used a noninvasive method to measure ¹³ CO ₂ in the respiration after the erythromycin medication, the probe drug, or to measure the concentration or metabolite ratio of endogenous metabolites without drug administration. However, the use of probe drugs to measure drug concentrations in the bloodstream is invasive and not suitable for clinical use. As a result, a noninvasive method using an endogenous substance specifically metabolized by CYP3A has been proposed. Representative endogenous metabolites include 6beta-hydroxycortisol and 4beta-hydroxycholesterol. In particular, studies have examined whether the concentration of 6 beta-hydroxycortisol or 6 beta-hydroxycortisol / cortisol metabolite ratio in the urine reflects the individual's CYP3A metabolic ability, but the diurnal variation, And these endogenous substances were not suitable for use as CYP3A metabolic predictive markers. In addition, the half-life of 4-beta-hydroxycholesterol is very long, ranging from 60 to 17 days, and it is difficult to reflect the individualized CYP3A metabolic activity which is dynamically changed as in the case of enzyme inhibition. Therefore, there is a need to develop a biomarker capable of accurately and easily evaluating the metabolism of CYP3A enzyme non-invasively.

A problem to be solved by the present invention is to provide a biomarker capable of non-invasively measuring the metabolic ability of CYP3A enzyme and a method for non-invasively measuring the metabolic ability of CYP3A enzyme using the biomarker.

The inventors of the present invention have developed a global metabolite analysis method using liquid chromatogram time of flight mass spectrometry (LC-TOF / MS) in order to overcome the problems of the prior art and develop new markers in addition to existing steroid series markers To develop a new CYP3A endogenous metabolite marker, thereby completing the present invention.

Specifically, the inventors of the present invention have developed markers that can predict the degree of CYP3A induction and inhibition from healthy male and female urine using midazolam (probe drug), ketoconazole (for inhibition) and rifampicin (for induction) . Global metabolite analysis revealed eight urinary metabolites related to CYP3A activity, among which five acylcarnitines containing a hydroxy-unsaturated heavy chain fatty acid were found as new markers for CYP3A4. Further, the present inventors derived an equation for predicting the midazolam clearance (CL) based on the marker and compared the result with the experimental results, it was confirmed that the hydroxy-unsaturated heavy chain fatty acid can predict the activity of the CYP3A4 enzyme.

The present invention provides a method for measuring the activity of a CYP3A enzyme comprising the step of detecting hydroxyacylcarnitine from a biological sample. The hydroxyacylcarnitine is a biomarker or a diagnostic marker for measuring the activity of a CYP3A enzyme and means that a change in the activity of the CYP3A enzyme can be determined when the drug is metabolized in the body.

In the present invention, the hydroxyacylcarnitine is preferably selected from the group consisting of Hydroxyoctenoyl carnitine, Hydroxydecadienoyl carnitine, Hydroxyundecadienoyl carnitine and Hydroxydenedecanoyl carnitine, (Hydroxyundecenoyl carnitine). Preferably, the hydroxyacylcarnitine may be hydroxyacylcarnitine hydrolyzed at the omega or omega-1 position. The hydroxyacylcarnitine biomarkers may be used singly or in combination of two or more, and may be used together with existing detection methods, if necessary. Those skilled in the art (hereinafter referred to as a person skilled in the art) will be able to select a combination of markers satisfying a desired sensitivity and specificity through analysis using a biological sample from a male or female subject, You can do it.

In the present invention, the biological sample may be a body fluid such as urine, sweat, saliva and tears, whole blood, plasma, serum, cells or tissue, and preferably the biological sample may be urine. The biomarker used in the method of measuring CYP3A enzyme activity of the present invention is an endogenous metabolite reflecting the metabolic activity of CYP3A, and it is possible to obtain a sample by non-invasive method through urine and there is no need to administer unnecessary drug.

In the present invention, detection includes quantitative and / or qualitative analysis, including detection of presence or absence and concentration measurement, which methods are well known in the art and will be readily apparent to those skilled in the art, You will be able to choose.

The present invention also provides a marker composition for evaluating CYP3A enzyme activity comprising hydroxyacylcarnitine. In the present invention, the hydroxyacylcarnitine is hydroxyoctenoyl carnitine, hydroxydecadienoyl carnitine, hydroxydecadienoyl carnitine, hydroxydecadienoyl carnitine, hydroxydecadienoyl carnitine, carnitine, and hydroxydecenoyl carnitine. < Desc / Clms Page number 7 > The hydroxyacylcarnitine in the marker composition for evaluating CYP3A enzyme activity can be detected by, for example, liquid chromatography-mass spectrometry (LC / MS) or high performance liquid chromatography (HPLC).

In addition, the present invention provides a kit for evaluating CYP3A enzyme activity, comprising the marker composition for evaluating CYP3A enzyme activity. The kit can measure the activity of CYP3A enzyme through quantitative and / or qualitative analysis of hydroxyacylcarnitine from a biological sample to be tested.

In addition, the present invention provides a method for evaluating bio-metabolism of a drug, comprising the step of detecting and quantitating hydroxyacylcarnitine from urine to evaluate CYP3A enzyme activity so as to provide information necessary for evaluation of drug metabolism. The urine is urine separated from the patient, especially the first urine in the morning, which is concentrated urine, and the concentration of the metabolites is high, so that it is easy to detect. The present invention enables the qualitative and quantitative analysis of hydroxyacylcarnitine through the above detection methods, and by comparing the activity of CYP3A enzyme with that of the control group, bioactivity of the drug can be evaluated.

The method for measuring CYP3A enzyme activity of the present invention can non-invasively and effectively evaluate changes in hepatic CYP3A metabolism by detecting hydroxyacylcarnitine in the urine.

In addition, the biomarker for CYP3A enzyme activity assay according to the present invention can easily identify changes in CYP3A metabolic activity, thereby providing important information on drug development and drug interaction, .

Figure 1 shows a scheme of a non-target metabolite assay method for determining urine biomarkers exhibiting CYP3A activity.
Figure 2 shows the PCA score plane derived from the UPLC-TOF MS non-target analysis of male and female control, inhibition and induction groups.
FIG. 3 is a graph showing the CYP3A inhibition, the control group, and the induction phase of car C8: 1-OH I / cr, (b) car C8: ) car C11: 2-OH / cr, (e) car C11: 1-OH / cr, (f) 6β-OH cortisol, (g) 16 α -OH TS / cr, and (h) 16 α -OH AS / cr indicates the concentration of urine CYP3A marker.
(B) car C8: 1-OH II / cr, (c) car C10: 2-OH / cr, (d) car C11 : 2-OH / cr, ( e) car C11: 1-OH / cr, (f) 6β-OH cortisol, (g) 16 α -OH TS / cr, and ^{(h) 16 α -OH AS /} cr between .
Figure 5a shows the measurement of ω- and ω-1 hydroxylation of C8 fatty acids by CYP3A4, and Figure 5b shows the measurement of ω- or ω-1 hydroxylation of C10 fatty acids of CYP3A4.
Figure 6 shows (a) omega- and (b) omega-1 hydroxylation by inhibition of 2-octenoic acid (2.5 mM) CYP3A4 (), CYP4A11 (), or CYP4F2 () superosomal ketoconazole. The percentage of 2-octenoic acid (a) ω- and (b) ω-1 hydroxide remaining in each super-cotton compared to the control is determined by the presence of ketoconazole.
Figure 7 shows the dynamic characterization of 2-octenoic acid (a) omega- and (b) omega-1 hydroxide by CYP3A4 (), CYP4A11 (), or CYP4F2 () SuperSom. Each point represents the mean of the double measurements. The line represents a function that is consistent with the Michaelis-Menten model for (a) ω- and (b) ω-1 2-octenoic hydroxide.
FIG. 8 is a graph comparing Ln (CL) = 1.4443-0.5559 * sex + 0.1637 * ln (Car C8: 1_2 / Cr + 1) + 0.09661 * ln (Car C10: 2 / Cr +1) + 0.1261 * ln (Car C11: 1 / Cr + 1) + 0.1191 * ln (6b-OH cortisol / Cr + 1).
Figure 9 shows the identification and characterization of carnitine C8: 1-OH.
Figure 10 shows the identification and characterization of carnitine C10: 2-OH.
Figure 11 shows the identification and characterization of carnitine C11: 2-OH and carnitine C11: 1-OH.
Figure 12 shows the identification and characterization of 6β-OH cortisol.
Figure 13 shows the identification and characterization of the 16 [alpha] -OH TS.
Figure 14 shows the identification and characterization of 16 [alpha] -OH.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

One. Experimental Example

Experimental Example  1: subjects

A total of 26 healthy men participated in the experiment, but then withdrew with consent from two others. Sixteen healthy women participated in the experiment, but then withdrew with consent of four. Thus, 24 men and 12 women performed this study. Twenty - four males were aged between 20 and 38 years (mean ± SD, 26.5 ± 4.6 years) and weighed 56.8 to 87.3 kg (mean ± SD, 69.2 ± 7.7 kg). Twelve women were between 20 and 30 years of age (mean ± SD, 23.9 ± 4.1 years) and weighing between 46.0 and 59.6 kg (mean ± SD, 51.3 ± 4.4 kg). Pharmacokinetics (PK, pharmacokinetics) and metabolite analysis were performed on those who completed this study. No serious or unexpected side effects were observed.

Experimental Example  2: Pharmacokinetics of midazolam (PK)

The PK parameters of midazolam were used as a standard to predict CYP3A activity. Stepwise descriptive statistics of the PK parameters of midazolam are shown in Table 1 below.

After coadministration with either midazolam alone intravenous administration or steady state ketoconazole or rifampicin, pharmacokinetic parameters parameter Midazolam alone Midazolam + ketoconazole Midazolam + rifampicin male† female‡ male† female‡ male† female‡ AUClast / dose (ng * h / mL / mg) 36.21 + - 11.47 42.7 ± 8.76 102.54 + - 22.48 204.2 ± 43.1 16.15 ± 3.08 20.9 ± 3.58 GMR (90% CI) - - 2.89 (2.57-3.25) 1.45 (1.38-1.53) 0.46 (0.41-0.51) 0.71 (0.68-0.76) Clearance (l / h) 30.08 + - 9.04 23.1 ± 4.26 10.26 ± 2.59 5.01 ± 1.19 64.16 + - 12.44 47.8 ± 9.05 t1 / 2 (h) 3.92 ± 1.40 3.98 ± 0.72 9.61 + - 2.98 7.88 ± 1.52 2.69 0.70 2.93 ± 1.01

After 4 days of ketoconazole treatment, the mean CL of midazolam was reduced to ~ 30% and ~ 20%, respectively, compared to the mean CL after treatment with midazolam alone in both males and females. On the other hand, mean midazolam CL of male and female increased more than two times after 10 days of treatment with rifampicin compared with the control. Pharmacokinetic parameters did not differ between the CYP3A5 or NR1I2 genotypes during the three study periods (change analysis, P> 0.05).

Experimental Example  3: Global metabolism analysis

Urine samples from inhibition, control, and induction steps were analyzed via UPLC / QTOF running in both cation and anion mode. Figure 1 shows a scheme for screening markers exhibiting CYP3A activity. A total of 7,535 ions were produced in the cation and anion modes using MarkerLynx. To remove the metabolites of ketoconazole or rifampicin and to select appropriate endogenous markers with good strength, more than 20% of all subjects at all stages removed ions when the intensity was zero. For this step, 1,479 ions were removed and 6,056 ions were analyzed by multivariate analysis. After data standardization, PCA was performed to classify the metabolic phenotype. Unaffected principal component analysis (PCA) showed a clear difference between the inhibition, control and induction phases in both male and female subjects (FIG. 2). PCA's QC sample clustering proved the stability and reproducibility of the analytical platform. To determine the endogenous metabolite markers associated with the inhibition and induction of CYP3A, a relative change and metabolism function of less than 0.5 times the inhibition and more than 2 times the induction phase were selected as compared to the control phase (FIG. 1). The number of potential markers selected was 27. Among these, 8 endogenous markers were identified and quantified.

Experimental Example  4: CYP3A Biomarker  discrimination

To identify the structure of the 27 potential markers, tandem MS cleavage patterns and elemental composition were determined. Of these, five markers were identified as acylcarnitines with a m / z 85 fission pattern in cationic mode and a formula consistent with the highest scoring formula of the elemental composition generated from MassLynx (Fig. 9-11). The exact positions of the double bonds and hydroxyl groups have not been completely identified as compared to the correct compounds due to the absence of reference standards for hydroxyl-unsaturated acylcarnitines. However, the potential position of the double bond of Car (carnitine) 8: 1-OH and Car 10: 2-OH is related to the reference standard of saturated acyl carnitine estimated by the MS / MS pattern of urinary acyl carnitine And 10). MS / MS spectra of the identified urinary unsaturated acyl carnitine and related reference standards of saturated acyl carnitine are shown in Figures 9-11. Urinary 6? -OH cortisol was identified by comparing its exact compound with the MS / MS spectrum (FIG. 12). Identification of urine 16α-OH TS and 16α-OH AS is as follows (FIGS. 13 and 14): (1) MS / MS cleavage pattern of sulfate binding ( m / z 96 in negative ion mode) Present; (2) the peak area of the sulfated metabolite decreases and the peak of the desulfurized metabolite appears in de-binding urine that is not present in the empty urine; And (3) urine decoupling metabolites and the correct compound (16α-OH T or 16α-OH A) have the same MS / MS spectral pattern.

Experimental Example  5: CYP3A4  of mine Octenic  Position selective hydroxide

All acylcarnitine markers include hydroxylated unsaturated heavy chain fatty acids (MCFA, C8-C12). Octanoic acid, 2-octenoic acid, 3-octenoic acid and 7-octenoic acid were cultured in CYP3A4 supersomes (Fig. 5A) to identify the location selectivity of MCFA hydroxylation by CYP3A4. The chromatogram of the hydroxoated octanoic acid or octenoic acid indicates that octanoic acid and 7-octenoic acid were not hydroxylated. However, 2- and 3-octenoic acid is hydroxylated by CYP3A4, indicating that the saturated fatty acids are not hydroxylated, but unsaturated fatty acids are ω- and ω-1 hydroxylated by CYP3A4 (AD in FIG. Four hydroxylated octenoic acids (peaks 1-4 in B and C in Fig. 5a) were not compared to the correct hydroxyoctenoic acid due to the absence of commercially available compounds. However, the terminal ω-hydroxylated fatty acid tended to retain a smaller amount than the front position-hydroxylated fatty acid in the reversed phase column. Thus, peaks 1 and 3 were identified as omega -hydroxylated octenoic acid, and peaks 2 and 4 were presumed as omega-1 hydroxylated octenoic acid. The MS / MS spectra of the hydroxylated 2- and 3-octenoic acids were similar to the correct 8-hydroxyoctanoic acid (EI in Figure 5a).

Experimental Example  6: CYP3A  Inhibitory Effect of ω- and ω-1 Hydroxylation by Suppressor, Ketoconazole

The CYP4 family catalyzes the ω- and ω-1 hydroxylation of saturated, branched chains and unsaturated fatty acids. In this study, ketokonazole, a CYP3A-specific inhibitor, was incubated with CYP3A4, CYP4A11 or CYP4F2 recombinant enzyme with 2-octenoic acid (2.5 mM) to determine whether the acylcarnitine marker was altered by CYP3A activity. Ketoconazole inhibits the formation of ω- and ω-1 hydroxy-2-octenoic acid in CYP3A4 with a half maximum inhibitory concentration (IC ₅₀ ) value of 213.9 μM and 41.6 μM, respectively, and a maximum inhibition of 27-40% 6). However, ketoconazole has reduced the ω- and ω-1 hydroxylation of 2-octenoic acid by 5-14% in CYP4A11 and CYP4F2, indicating that ketoconazole has a weak or no inhibitory effect on CYP4 enzymes.

Experimental Example  7: CYP3A4 , CYP4A11  And On CYP4F2  Measurement of ω- and ω-1 Hydroxylation Activity

The kinetics of ω- and ω-1 hydroxylation of 2-octenoic acid by CYP3A4, CYP4A11 and CYP4F2 were further investigated using recombinantly expressed enzymes. The kinetic parameters were measured by nonlinear regression analysis using the Michaelis-Menten model. CYP3A4, CYP4A11 and CYP4F2 all showed the ability to catalyze the formation of omega- and omega-1 hydroxy-2-octenoic acid (Fig. 7). The most effective enzymes for 2-octenoic acid ω- and ω-1 hydroxylation are CYP3A4, which produces a 1.43 mM value and a lowest Km value of 1.20 mM and a highest Vmax / Km ratio of 0.84 and 0.82, respectively (Table 2).

Dynamics of ω- and ω-1 hydroxylation of 2-octenoic acid by CYP3A4, CYP4A11 and CYP4F2 enzyme Km (mM) Vmax
(pmol / min / pmol P450) Vmax / Km ω omega-1 ω omega-1 ω omega-1 CYP3A4 1.43 2.99 1.20 2.46 0.84 0.82 CYP4A11 64.22 7.34 27.96 4.36 0.44 0.59 CYP4F2 4.89 8.52 2.53 4.28 0.52 0.50

The most effective enzymes for omega-hydroxylation are CYP4A2 followed by CYP4F2 (Vmax / Km of 0.52) and CYP4A11 (Vmax / km of 0.44). The calculated Km and Vmax of ω-1 hydroxyl on CYP4A11 (Km 7.34 mM and Vmax 7.63 pmol / min / pmol P450) and CYP4F2 (Km 20.59 mM and Vmax 6.63 pmol / min / pmol P450) did. In addition, the calculated Vmax / Km for ω-1 hydroxide was also similar for CYP4A11 (Vmax / Km 0.38) and CYP4F2 (Vmax / Km 0.32) and about 0.5 times lower for CYP3A4 than CYP4A11.

Experimental Example  8: CYP3A  Metabolism according to active state Marker  change

Eight intrinsic urine metabolites with a significant association with CYP3A activity were (semi -) quantified during each treatment period. Figure 3 shows changes in urine concentrations of eight metabolites that show a significant increase with increased CYP3A activity. The changes in magnification compared to the control group are shown in Table 3.

Changes in the mean concentration of metabolites in the CYP3A inhibition and induction phase compared to the identified urine CYP3A biomarker and control, and correlation with midazolam CL Metabolite RT
(min) Mass ( m / z ) Change in magnification in men Change in magnification in women Correlation coefficient with MIDACOLAM CL Inhibitory phase Induction phase Inhibitory phase Induction phase r P Car C8: 1-OH I 4.93 302.1962 0.81 ▲ 13.16 0.63 ▲ 7.91 0.608 <0.001 Car C8: 1-OH II 5.14 302.1968 $ 0.19 ▲ 6.11 ▼ 0.07 ▲ 6.21 0.788 <0.001 Car C10: 2-OH 7.67 328.2125 0.83 ▲ 6.41 0.29 ▲ 4.76 0.797 <0.001 Car C11: 2-OH 7.45 342.2277 $ 0.33 ▲ 2.33 $ 0.18 ▲ 2.68 0.801 <0.001 Car C11: 1-OH 7.75 344.2437 ▼ 0.37 ▲ 2.79 ▼ 0.20 ▲ 3.44 0.801 <0.001 6β-OH Cortisol 7.00 379.2121 $ 0.10 ▲ 5.18 $ 0.14 ▲ 5.82 0.772 <0.001 16α-OH T sul 8.78 383.1525 ▼ 0.45 ▲ 2.77 $ 0.16 ▲ 2.48 0.855 <0.001 16α-OHAN sul 8.39 385.1679 $ 0.17 - 3.74 $ 0.18 ▲ 2.21 0.493 <0.001

Said data indicating a change in magnification as compared to a control; ( P <0.001, P <0.05), and statistical significance ( P <0.001, P <0.05)

All metabolites were significantly reduced and increased in the CYP3A inhibition and CYP3A induction phases, respectively, except for the inhibition step of carnitine C8: 1-OH I and Car C10: 2-OH, respectively. The largest increases were observed in Car C8: 1-OH I (13.16 times in males and 7.91 times in females) and in Car C8: 1-OH II (6.11 times in males and 6.21 times in females). 6β-OH cortisol and Car C8: 1-OH II were shown to be the greatest decrease (less than 0.2-fold change in both males and females) in the CYP3A-inhibited phase.

Experimental Example  9: with Midazolam CL CYP3A Marker  correlation

To assess the usefulness of the metabolites as endogenous markers of CYP3A, each midazolam CL was compared. The log-transformed urine concentration correlated significantly ( P <0.001) with the log-transformed midazolam CL (Figure 4): Car C8: 1-OH I / cr, r = 0.608; Car C8: 1-OH II / cr, r = 0.788; Car (carnitine) C10: 2-OH / cr, r = 0.797; Car C11: 2-OH / cr, r = 0.801; Car C11: 1-OH / cr, r = 0.801; 6 [beta] -OH cortisol / cr, r = 0.772; 16 [alpha] -OH TS / cr, r = 0.855; 16 [alpha] -OH AS / cr, r = 0.493.

Experimental Example  10: Selection of models for prediction of midazolam CL

Urinary metabolites with significant correlation with midazolam CL were considered as covariates in the midazolam CL prediction equation. Reverse removal was used to select the variables. The detailed model selection procedure is shown in Table 4 below.

Model selection process for Midazolam clearance satisfying the lowest Akaike information criterion (AIC) value Model Blank variables Removed Blank variables AIC One OH-TS, 16a-OH-AS, CYP, NR, SEX, Car C8: 1_1, Car C8: 1_2, Car C10: 2, Car C11: 16a-OH-AS 84.8 2 Carboxamide, CYP, NR, SEX, Car C8: 1_1, Car C8: 1_2, Car C10: 2, Car C11: Car C8: 1_1 80.0 3 Carboxamide, CYP, NR, SEX, Car C8: 1_2, Car C10: 2, Car C11: 2, Car C11: 1,6b- CYP 76.2 4 NR, SEX, Car C8: 1_2, Car C10: 2, Car C11: 2, Car C11: 1,6b-OH-cortisol, 16a-OH-TS 16a-OH-TS 72.6 5 NR, SEX, Car C8: 1_2, Car C10: 2, Car C11: 2, Car C11: 1,6b-OH-cortisol Car C11: 1 69.5 6 NR, SEX, Car C8: 1_2, Car C10: 2, Car C11: 2,6b-OH-cortisol NR 66.6 Last SEX, Car C8: 1_2, Car C11: 1,6b-OH-cortisol, 16a-OH-TS 16a-OH-TS

All markers were corrected for creatinine concentration. CYP, CYP3A5, NR, NRl2; Car, acyl carnitine

Sex, acylcarnitine C8: 1_2, acylcarnitine C11: 1, 6β-OH-cortisol and 16a-OH-testosterone were selected as the final covariate. The final model developed is as follows:

Ln (Car C11: 1 / Cr + 1) + 0.09661 * ln (CL) = 1.4443-0.5559 * gender + 0.1637 * ln (Car C8: 1) + 0.1191 * ln (6? -OH cortisol / Cr + 1)

Gender: Male = 0, Female = 1

The final model matched the data with a high correlation coefficient square (r ² = 0891) as shown in FIG.

2. Experimental Method

(1) Research design

This study was conducted at the clinical trial center of Seoul National University College of Medicine. The experimental protocol was approved by the Review Committee of the Seoul National University Hospital. All procedures were carried out in accordance with the principles of the Helsinki Declaration (59 ^th World Medical Association General Assembly, Seoul, October 2008) and the GCP.

Each subject received midazolam intravenously during three study phases: Phase I, control phase, evaluation of the PK of midazolam (i.v., 1 mg); Phase II, CYP3A-induction phase, changes in midazolam (i.v., 1 mg) PK induced by pretreatment with 400 mg of ketoconazole 400 mg daily for 4 days; And Phase III, CYP3A-inhibited phase, and midazolam (i.v., 2.5 mg) PK were administered daily for 10 days with a 600 mg oral dose of rifampicin. To minimize the effects of the menstrual cycle on steroid levels, the three stages of female volunteers were separated by their menstrual cycle; Midazolam was administered at the same stage of the menstrual cycle of each subject. For metabolite analysis, urine samples were collected 24 hours prior to administration of midazolam at 12 hour intervals.

(2) Subjects

Healthy men and women from 20 to 50 years old participated in this study. Their body mass index was 17.0-28.0 kg / m2, and the male weight was over 50kg and the female was between 45-85kg. All subjects agreed to the contraceptive use during the study and agreed in writing prior to selection. Subjects underwent general clinical trials (clinical chemistry, blood tests and urinalysis), history, physical examination, vital sign, 12 lead ECG, serum test (Hepatitis B surface antigen, anti-hepatitis virus, anti-HIV). Subjects who took the drug immediately before the study were excluded. From 3 days before the first drug administration until the last day of the study, ingestion of grapefruit, grapefruit products or caffeinated beverages was not allowed.

(3) Chemicals and reagents

Formic acid, sulfatase (type H-1 of Helix pomatia ) was purchased from Sigma Aldrich (St. Louis, Mo.). HPLC-grade solvents (methanol, acetonitrile and water) were purchased from JT Baker (Center Valley, PA). Semi-quantified acylcarnitines (octanoyl carnitine and decanoyl carnitine) and fatty acids (octanoic acid, 2-octenoic acid, 3-octenoic acid, 7-octenoic acid and 8-hydroxyoctanoic acid) Sigma Aldrich. The internal standard used, purchased from the CDN isotope (Pointe-Claire, Quebec, Canada) is as follows; Octanoic acid noik -2,2-d _2, octanoyl carnitine -L- -d3, decanoyl -L- carnitine -d3, -d4 cortisol, testosterone sulfate -d3, -d4 and creatinine. For solid phase extraction (SPE), an Oasis HLB cartridge (3 mL, 60 mg; Waters, Milford, MA) was used.

(4) Global Metabolite  Global metabolomics

The urine sample was thawed on ice and centrifuged at 15,000 x g for 20 minutes at 4 ° C to remove particles. The supernatant was diluted in 2 volumes of water. Using a Waters High Performance Liquid Chromatography (UPLC) system, a 3 μL aliquot of the sample was injected into a reversed phase 2.1 × 100 mm ACQUITY 1.8 μm HSS T3. The gradient mobile phase contained methanol (B) containing 0.1% formic acid (A) and 0.1 formic acid. Each sample was analyzed for 20 minutes at a flow rate of 0.4 mL / min. The gradient consisted of 5% B for 1 minute, 5-30% B for 1-8 minutes, 30-70% B for 8-13 minutes, and 95% B for 14 minutes while maintaining for 2 minutes. The sample was then equilibrated to 95% A for 3.5 minutes before the next injection. The Waters Xevo G2 Flight Time Mass Spectrometer (TOF-MS) was operated in cation and anion electrospray ionization (ESI + and ESI-) modes. To obtain constantly different variables, a mixed sample of urine was prepared by mixing partial samples of individual samples. A duplicate (QC) of a mixed urine sample was obtained by a series of injections and random injection of data. Metabolomic data were imported into EZinfo 2.0 software (Umetrics, Umea, Sweden) for multivariate analysis (Pareto-scale, pareto-scaled). An unmonitored multivariate statistical approach, Principal Component Analysis (PCA), was conducted to investigate intrinsic changes in the group and assess clustering behavior among groups. The clustering of QC samples in PCA was evaluated to demonstrate platform stability and reproducibility.

(5) CYP3A Biomarker  discrimination

We further investigated the elemental composition of ions through MassLynx, MS fragmentation, database search, detoxification of urea metabolites, and validation using true standards. In addition, the human Metabolome database (HMDB, http://www.hmdb.ca/) and the Metlin database (https://metlin.scripps.edu/index.php) can be used to identify the mass basis Respectively. Metabolites with a fragmentation pattern of sulfate binding were digested with enzymes and the free form of each metabolite was confirmed by MS fragmentation. The fragmentation pattern of the sulfated compound has a fragmented pattern peak at m / z 96.96 in anion mode. The decoupling system for urine metabolites included: 1 mL of urine, 50 μL of β-glucuronidase / aryl sulfatase, and 500 μL of phosphate buffer (pH 7.4 ). A blank containing buffer instead of enzyme was used as a control. The mixture was incubated at 40 ° C for 20 hours. After centrifugation at 14,000 rpm for 5 minutes, the supernatant was extracted with an Oasis HLB solid phase extraction cartridge connected to a peristaltic pump. After the sample was loaded into the cartridge, the cartridge was washed with 3 ml of water and eluted twice with 2 ml of methanol. The mixed methanol eluate was evaporated at room temperature under nitrogen, and the dried residue was reconstituted with 100 μL of methanol. A 4 microliter aliquot was injected into the UPLC / QTOF system.

(6) CYP3A Biomarker  Quantification

OH, C8: 1-OH I, C8: 1-OH II, C10: 2-OH, C11: 2-OH and C11: OH AS was quantified using TargetLynx (Waters Corp.). In the absence of reference standards for unsaturated or hydroxyacylcarnitines, the associated saturated acylcarnitines were used for the slope of the standard curve, i.e. carnitine C8: 0 for C8: 1-OH1 and carnitine for C8: And carnitine C10: 0 for carnitine of C10: 2-OH, C11: 2-OH and 11: 1-OH. Creatinine was also quantified for use as a general standard for the actual concentration of each urine biomarker. 50 microliters of urine supernatant was diluted with 150 μL of the 5 international standard mixture of 500 ng / mL carnitine C8: 0- d _3, carnitine C10: 0- d _3, testosterone sulfate - d _3, 600 ng / mL Of cortisol- d ₄ , and 1 μg / mL of creatinine- d ₃ . The concentration of each biomarker in urine was determined from the calibration curve using linear regression analysis. All determined correlation coefficients were> 0.98 for each biomarker and the resulting concentration was expressed as ng / mg creatinine (normalized).

(7) Microsomes  Microsomal incubations

All samples were double processed. Octenoic acid dissolved in acetonitrile was added to a reaction mixture (final volume 100 μl) containing 1.4 mM NADPH in phosphate buffer (pH 7.4) and 20 pmol CYP3A4, CYP4A11, or CYP4F2 supersomes. The reaction was initiated by addition of NADPH (final concentration of 1.5 mmol / l). After 1 hour incubation at 37 ° C, 300 μl of 500 ng / ml octanoic acid- d ₂ in methanol was added to stop the reaction. The dynamic parameters (Km and Vmax) for the supercohomatic hydroxy 2-octenoic acid were determined using 2-octenoic acid concentrations of 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 10.0, 20.0, 40.0, and 60.0 mmol / Respectively. IC50 values for super-cotton ketoconazole were determined using 2.5-octenoic acid from 2.5 mM hydroxy-2-octenoic acid. The concentrations of ketoconazole in the cultures were 0 (ACN vehicle control), 10, 50, 125, 250, 500, 1000, and 2000 μmol / l. For semi-quantitative analysis of ω- and ω-1 hydroxy-2-octenoic acid, calibration curves of 8-hydroxyoctanoic acid were constructed between 6 ng / mL and 6500 ng / mL.

(8) Data and statistical analysis

Pharmacokinetic (PK) parameters and demographic characteristics were analyzed using descriptive statistics. The PK parameters were evaluated for the effect of the two genotypes (CYP3A5 and NR1I2) using the ANOVA. All metabolites correlated with midazolam CL were entered into a multivariate linear regression analysis model to meet the lowest Akaike's information criterion (AIC) values. Endogenous metabolites were analyzed using a mixed effect model with individuals as a fixed effect and random effects on the relative, log-transformed concentrations of the metabolites. Taking into account the metabolites quantified below LLOQ, the value of ln (metabolite + 1) was used. All statistical analyzes were performed using SAS version 9.3 (SAS Korea, Seoul, Korea). Km, Vmax, and Clint for hydroxylation of 2-octenoic acid in CYP3A4, CYP4A11, and CYP4F2 superosomes were obtained by applying the Michaelis-Menten equation to the data using GraphPad Prism. IC50 values were obtained using a sigmoid dose-response (inhibition) model. Data are presented as means ± SD. Statistical analysis was performed using Excel (Microsoft Office 2013, Redmond, WA).

Claims (8)

A method for measuring the activity of a CYP3A enzyme comprising the step of detecting hydroxyacylcarnitine from a biological sample The pharmaceutical composition according to claim 1, wherein the hydroxyacylcarnitine is hydroxyoctenoyl carnitine, hydroxydecadienoyl carnitine, hydroxydecadienoyl carnitine (hydroxydecadienoyl carnitine) hydrolyzed at the? Or? Wherein the activity of the CYP3A enzyme is at least one selected from the group consisting of Hydroxyundecadienoyl carnitine and Hydroxyundecenoyl carnitine. The method according to claim 1, wherein the biological sample is a urine CYP3A enzyme. A marker composition for evaluating CYP3A enzyme activity comprising hydroxyacylcarnitine. 5. The pharmaceutical composition according to claim 4, wherein the hydroxyacylcarnitine is hydroxyoctenoyl carnitine, hydroxydecadienoyl carnitine, hydroxydecadienoyl carnitine, hydroxydecadienoyl carnitine, Wherein the marker composition is one or more selected from the group consisting of Hydroxyundecadienoyl carnitine and Hydroxyundecenoyl carnitine. 5. The marker composition according to claim 4, wherein the hydroxyacylcarnitine is detected by liquid chromatography mass spectrometry. A kit for evaluating the activity of CYP3A enzyme comprising the marker composition for evaluating CYP3A enzyme activity according to claim 4. And assessing CYP3A enzyme activity by detecting hydroxyacyl carnitine from urine.

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