KR102535976B1 - Analysis method of drug biodistribution using fluorescence image and mass spectrometry - Google Patents

Abstract

The present invention relates to a method for complementarily confirming the biodistribution of drugs using fluorescence images and mass spectrometry, and the method for complementarily confirming the biodistribution of drugs using fluorescence images and mass spectrometry according to the present invention , By comparing and reviewing fluorescence images and mass spectrometry results, errors can be detected and reliable analysis results can be obtained, so there is an effect of significantly improving the accuracy when confirming the biodistribution of drugs.

Description

Method for mutually confirming the biodistribution of drugs using fluorescence image and mass spectrometry {Analysis method of drug biodistribution using fluorescence image and mass spectrometry}

The present invention relates to a method for mutually confirming the biodistribution of drugs using fluorescence images and mass spectrometry.

Curcumin is a natural hydrophobic polyphenolic component isolated from herbs such as the rhizome of turmeric. Although curcumin exhibits various pharmacological effects and stability, its application is limited due to poor solubility and rapid degradation in vivo. Additionally, curcumin is rapidly metabolized to curcumin derivatives including ferulic acid, dihydroferulic acid and curcumin glucuronide which reduce its pharmacological activity.

Since the bioavailability and biodistribution of curcumin in vivo are very important for predicting pharmacological availability in target organs, after tissue extraction, high-performance liquid chromatography (HPLC) and mass spectrometry (mass spectrometry) It is currently being studied using various analysis methods such as spectrometry (MS).

In particular, the use of mass spectrometry is characterized by low background signals, no false-positive signals, multiplexing capability, and intrinsic label-free detection compared to other analytical methods. detections and high sensitivity.

However, mass spectrometry is often unreliable because it requires an extraction process that requires an extraction method optimized for each tissue and organ. Furthermore, considering curcumin's low stability at pH in vivo, it is important to analyze it without an extraction process. Previous studies have reported that more than 60% of curcumin was degraded after incubation at pH 7.2 for 5 minutes.

On the other hand, since curcumin naturally shows fluorescence, it can be directly observed by optical fluorescence imaging without an extraction process. The optical fluorescence imaging method has numerous advantages in the in vivo detection of biomolecules, such as ease of use, rapid detection, and real-time observation of biomolecules without time consuming extraction procedures.

However, optical fluorescence imaging methods have some challenges, such as false-positives due to autofluorescence, high signal-to-noise ratios and low sensitivity.

Therefore, the present inventors confirmed that the accuracy of confirming the biodistribution of pharmaceutical components such as curcumin can be improved by taking the advantages of each analysis method and overcoming the disadvantages by complementing fluorescence image analysis and mass spectrometry. The invention was completed.

US Patent Publication 2011-0117025

An object of the present invention is to provide a complementary method for confirming the biodistribution of drugs using fluorescence imaging and mass spectrometry.

Another object of the present invention is to provide a biodistribution diagram of a drug obtained by the above method.

Another object of the present invention is to provide a method of modifying a drug to increase the distribution to a target organ by using the biodistribution of the drug.

Another object of the present invention is to provide a method for binding a drug delivery system to a drug in order to increase the distribution to a target organ using the biodistribution of the drug.

In order to achieve the above purpose,

The present invention administers drugs to non-human animals, then euthanizes them, removes each organ to prepare an experimental group, and euthanizes non-human animals without administering drugs to prepare a control group by removing each organ. step (step 1);

Obtaining a primary analysis result of quantifying the autofluorescence of each organ in the control group in step 1 by fluorescence image analysis and quantifying the drug distributed in each organ in the experimental group in step 1 by fluorescence image analysis ( step 2);

Obtaining a secondary analysis result by quantifying the drug distributed in each organ by mass spectrometry of the extract obtained from each organ of the experimental group for which the primary analysis result was obtained in step 2 (step 3); and

Improving the accuracy of biodistribution results of drugs by comparing the ratio of the results of each organ in the experimental group in the first analysis result with the ratio of the results in each organ in the experimental group in the second analysis result (step 4);

It provides a method for confirming the biodistribution of pharmaceuticals using fluorescence images and mass spectrometry, including a complementary method.

In addition, the present invention provides a biodistribution diagram of the drug obtained by the above method.

Furthermore, the present invention provides a method of modifying a medicine to increase the distribution to a target organ by using the biodistribution of the medicine.

In addition, the present invention provides a method of binding a drug delivery system to a drug in order to increase the distribution to a target organ using the biodistribution of the drug.

The method for mutually confirming the biodistribution of drugs using fluorescence images and mass spectrometry according to the present invention can compare and review the fluorescence images and mass spectrometry results to detect errors and obtain highly reliable analysis results. There is an effect that can significantly improve the accuracy when confirming the biodistribution chart.

1 is a schematic diagram of a method for mutually confirming the biodistribution of curcumin after administering it to a living body using an optical fluorescence image analyzer and a mass spectrometer.
2 is an optical fluorescence image of each organ excised after intravenous administration of curcumin (25 mg/kg) (before administration (control), 1 hour after administration (Curcumin)).
3 is a graph quantifying the fluorescence intensity of each organ excised after intravenous administration of curcumin (25 mg/kg) (▲: curcumin, ●: internal standard (acetonitrile)).
4 is a mass spectrometry spectrum of each organ excised after intravenous administration of curcumin (25 mg/kg), and is a graph showing total flux. For reference, the peaks of curcumin and internal standard (acetonitrile) appear clearly at m/z 369.13 and 397.16 [M + H] ⁺ respectively in the curcumin-untreated organs.
5 is a graph showing the quantification of curcumin content from mass spectrometry results of each organ excised after intravenous administration of curcumin (25 mg/kg).

Hereinafter, the present invention will be described in detail.

How to check the biodistribution of pharmaceuticals

The present invention administers drugs to non-human animals, then euthanizes them, removes each organ to prepare an experimental group, and euthanizes non-human animals without administering drugs to prepare a control group by removing each organ. step (step 1);

Obtaining a primary analysis result of quantifying the autofluorescence of each organ in the control group in step 1 by fluorescence image analysis and quantifying the drug distributed in each organ in the experimental group in step 1 by fluorescence image analysis ( step 2);

Obtaining a secondary analysis result by quantifying the drug distributed in each organ by mass spectrometry of the extract obtained from each organ of the experimental group for which the primary analysis result was obtained in step 2 (step 3); and

Improving the accuracy of biodistribution results of drugs by comparing the ratio of the results of each organ in the experimental group in the first analysis result with the ratio of the results in each organ in the experimental group in the second analysis result (step 4);

It provides a method for confirming the biodistribution of pharmaceuticals using fluorescence images and mass spectrometry, including a complementary method.

In the method according to the present invention, step 1 is performed by administering a drug to non-human animals, then euthanasia, removing each organ to prepare an experimental group, and euthanizing non-human animals without administering drugs to them. This is the step of preparing a control group by removing each organ.

The drug in step 1 may use either a compound that exhibits fluorescence by itself or a compound that does not show fluorescence by itself. It can be used to indicate. All known methods can be used without any limitation as a method for modifying to show fluorescence, and it is preferable to modify the drug in a form that hardly affects the natural pharmacological effect of the drug. If a method of labeling a drug with a fluorescent substance is used, there is a problem in that the original biodistribution of the drug cannot be obtained due to the labeled fluorescent substance.

As an example of the drug, a plant-derived ingredient, preferably a small molecule drug, can be used, and in the present invention, curcumin is used as an example of a plant-derived ingredient showing fluorescence. In general, since small molecule drugs are metabolized very rapidly in vivo, a rapid analysis method is required to confirm their biodistribution.

In the method according to the present invention, in step 2, the autofluorescence of each organ is quantified by fluorescence image analysis of the control group of step 1, and the fluorescence image analysis of the experimental group of step 1 is performed. This is the step of obtaining the primary analysis result of quantifying the drug.

As the fluorescence image analysis method, in vivo optical fluorescence imaging analysis may be used.

Here, since each organ exhibits its own autofluorescence, errors may occur during fluorescence imaging, so it is necessary to complement the results of mass spectrometry in the next step 3.

In the method according to the present invention, step 3 is a step of obtaining a secondary analysis result of quantifying the drug distributed in each organ by mass spectrometry of an extract obtained from each organ of the experimental group for which the primary analysis result was obtained in step 2. am.

As the mass spectrometry method, MALDI-TOF (Matrix Associated Laser Desorption Ionization - Time Of Flight) mass spectrometry may be used.

The extract can be obtained by homogenizing each organ, treating with an enzyme, and extracting from the supernatant after centrifugation. In addition, using two types of immiscible solvents, a desired component may be separated into one layer and extracted.

In the method according to the present invention, the step 4 compares the ratio of the results of each organ in the experimental group in the first analysis result and the ratio of the results in each organ in the experimental group in the second analysis result to each other to determine the biodistribution of the drug. This step improves the accuracy of the results.

In step 4, when the ratio of the results of each organ in the experimental group in the first analysis result and the ratio of the results in each organ in the experimental group in the second analysis result are similar, both the fluorescence image analysis and mass spectrometry results are considered to be reliable. can judge

In addition, in the case of an organ in which the autofluorescence intensity of the control group is low in the fluorescence image analysis result in step 2, the mass spectrometry result in step 4 may be discarded and the fluorescence image analysis result may be judged to be reliable.

Furthermore, in the case of an organ in which the autofluorescence intensity of the control group is high in the fluorescence image analysis result in step 2, the fluorescence image analysis result in step 4 is discarded and the mass spectrometry result can be judged to be reliable.

Therefore, the method for mutually confirming the biodistribution of drugs using fluorescence images and mass spectrometry according to the present invention can detect errors and obtain highly reliable analysis results by comparing and reviewing fluorescence images and mass spectrometry results, There is an effect of significantly improving the accuracy when confirming the biodistribution of drugs (see Experimental Example 1, Table 1).

biodistribution

The present invention provides a biodistribution diagram of the drug obtained by the above method.

Examples of the medicines are as described above, and when various medicines are administered to a living body, a biodistribution map of each organ is provided, which can provide important information for research and development of medicines.

medicines to reform method

The present invention provides a method of modifying a drug to increase the distribution to a target organ using the biodistribution of the drug.

The term "modification" does not mean binding other compounds, but means modifying the structure of an active ingredient compound of a drug.

The drug to be developed can be modified in a form in which the degree of distribution to the organ in which the pharmacological effect of the drug is to be exhibited increases, and all known methods can be used for the modification method.

How to bind a drug delivery system to a drug

The present invention provides a method of binding a drug delivery system to a drug in order to increase the distribution to a target organ using the biodistribution of the drug.

In order to increase the distribution of the drug to be developed to the organ where the pharmacological effect is to be exhibited, a drug delivery system suitable for this can be chemically and/or physically bonded to the drug, and all known methods for combining the drug delivery system can be used. available.

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

experimental material

Curcumin, dimethyl sulfoxide (DMSO), ethyl acetate (EA), α-cyano-4-hydroxycinnamic acid (CHCA), acetonitrile (ACN), sodium dodecyl sulfate (SDS), and kolliphor was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used. A phosphate-buffered saline (PBS) solution was purchased from Gibco BRL (Grand Island, NY, USA) and used. ICR mice (six-week-old, female) were purchased from Orient Bio Inc. (Seongnam, Korea).

< manufacturing example 1> dimethylation curcumin Preparation of (internal standard)

Dimethylated curcumin was prepared as an internal standard using a method disclosed in a conventional literature (Bioorganic & Medicinal Chemistry, 2011, 19(12), pp. 3793-3800).

< Experimental example 1> of curcumin Biodistribution evaluation ( Optical fluorescence image Complementary analysis and mass spectrometry results)

After intravenous administration of curcumin to the mouse tail, each organ of the euthanized mouse was removed and optical fluorescence image analysis was performed to quantitatively first analyze the curcumin distributed in each organ, and immediately mass spectrometry to each organ. After quantitative secondary analysis of the distributed curcumin, the biodistribution of curcumin was quantitatively evaluated by complementing the optical fluorescence image analysis result and the mass spectrometry result obtained above.

1 is a schematic diagram of a method for mutually confirming the biodistribution of curcumin after administering it to a living body using an optical fluorescence image analyzer and a mass spectrometer.

of curcumin Optical fluorescence image analyze

Curcumin dissolved in DMSO was diluted in PBS containing 15% each of Kolliphor and DMSO, and then injected into the tail vein of ICR mice (25 mg/kg). In order to observe the biodistribution of curcumin 1 hour after curcumin administration, the organs of the euthanized mice were removed and washed with PBS. Each organ was observed at Exitation 430nm and Emission 509nm using an In Vivo Imaging System (IVIS, Caliper Life Sciences Lumina II, Massachusetts, USA). The total flux of each organ was analyzed using IVIS, and the total flux of curcumin alone was obtained by subtracting the total flux of the general organ from the total flux of the organ to which curcumin was administered in order to exclude errors due to autofluorescence. Organs were stored at -70°C until the next mass spectrometry analysis.

2 is an optical fluorescence image of each organ excised after intravenous administration of curcumin (25 mg/kg) (before administration (control), 1 hour after administration (Curcumin)).

As shown in FIG. 2 , it was found that the fluorescence signal significantly increased after curcumin administration compared to before curcumin administration in the brain, lung, gall bladder, and pancreas among various organs. On the other hand, negligible fluorescence was observed in the spleen and kidney. On the other hand, in the curcumin untreated group (control), it was found that the liver, gall bladder, and pancreas showed clear fluorescence values due to organ autofluorescence.

3 is a graph quantifying the fluorescence intensity of each organ excised after intravenous administration of curcumin (25 mg/kg) (▲: curcumin, ●: internal standard (acetonitrile)).

As shown in Figure 3, the gall bladder and pancreas showed high fluorescence intensity after administration of curcumin. However, the fluorescence intensity did not significantly increase in the liver.

of curcumin mass spectrometry

Mass spectrometry was performed immediately upon completion of the fluorescence image analysis.

In order to quantify the amount of curcumin distributed in the brain, lung, liver, gallbladder, spleen, pancreas, and kidney, PBS (pH 7.4, organs: PBS = 1:3, w/v) was added and organs were homogenized. 200 μL of 10% SDS and 500 μL of Ethyl Acetate were mixed with 500 μL of the homogenate and sonicated for 10 minutes. The supernatant obtained by centrifuging the mixture at 6000 rpm for 2 minutes was extracted three more times and then dried at room temperature.

Add 20 μL of an internal standard solution (5 μM in Acetonitrile) to the dried organ extract. After mixing this sample and α-cyano-4-hydroxycinnamic acid at the same volume ratio, 1.5 μL of this mixture was analyzed at 19 kV, 50 Hz using an Autoflex Ⅲ MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). . All spectra were obtained as an average of 500 shots using α-cyano-4-hydroxycinnamic acid (5 mg/ml in Acetonitrile) as a matrix. In this experiment, curcumin at various concentrations (200 nM to 200 μM, 10 μL in Acetonitrile) was mixed with 10 μM dimethylated curcumin and used as a calibration curve.

4 is a mass spectrometry spectrum of each organ excised after intravenous administration of curcumin (25 mg/kg), and is a graph showing total flux. For reference, the peaks of curcumin and internal standard (acetonitrile) appear clearly at m/z 369.13 and 397.16 [M + H] ⁺ respectively in the curcumin-untreated organs.

5 is a graph showing the quantification of curcumin content from mass spectrometry results of each organ excised after intravenous administration of curcumin (25 mg/kg).

As shown in Figs. 4 and 5, the lung, liver, gall bladder and pancreas showed clear curcumin content, while other organs showed little curcumin content.

Optical fluorescence image Complementary analysis and mass spectrometry results

In the case of optical fluorescence image analysis, errors may occur due to autofluorescence in each organ, and in the case of mass spectrometry, errors may occur due to curcumin being lost during the extraction process from each organ or curcumin being decomposed during the preparation for extraction. can

Therefore, in the optical fluorescence image analysis and mass spectrometry results obtained above, the distribution amount of curcumin in each organ was compared and reviewed, and if the ratio of the two analysis results is significantly different from that of other organs, an error in one of the two analysis results detected, and compared with other organs to complement each other.

Table 1 below shows the optical fluorescence image analysis and mass spectrometry results together.

long time Optical fluorescence image total flux
IVIS (×10 ⁸ , Photon/Sec) mass spectrometry
(μg/g) Brain 3.998±0.058 0.037±0.002 Heart 1.370±0.999 not determined Lung 4.889±0.213 0.771±0.035 Liver 8.695±8.096 1.693±0.035 Gall bladder 33.073±4.397 > 10 Jira (Spleen) 0.927±0.879 0.037±0.012 Interest (Pancreas) 18.741±11.092 3.075±0.225 Kidney 2.790±1.869 0.041±0.005

*IVIS (in vivo imaging system)

As shown in Table 1 above, in both analysis results, Gall bladder and Pancreas showed high curcumin content. In the brain and lung, as a result of optical fluorescence image analysis, the total flux of curcumin was similar, whereas in the mass spectrometry, the brain showed a slight curcumin content. As a result of mass spectrometry, curcumin content was found to be about 20 times higher in the lung than in the brain. These results are thought to be due to the insufficient extraction of curcumin from the brain using the current extraction process.

As a result of analysis using HPLC in the previous literature (Journal of Colloid and Interface Science, 2011, 354(1), pp. 116-123), the content of curcumin in the lung was clearly higher than that in the brain. Furthermore, curcumin in the conventional literature (Journal of Colloid and Interface Science, 2011, 354 (1), pp. 116-123) and other conventional literature (International journal of pharmaceutics, 2011, 416 (1), pp. 331-338) The biodistribution of curcumin in the kidney 1 hour after intravenous administration was different. The difference between these biodistribution results and those of the prior literature is that curcumin is easily decomposed during the extraction process, and furthermore, the extraction efficiency from the lung and brain is different even when using the same extraction protocol. Additionally, it is clear that the curcumin content of the liver was confirmed by mass spectrometry, since autofluorescence is highly detected in the case of the liver in FIG. 2 .

Summary of experimental results

The biodistribution of curcumin in the heart, spleen and kidney was similar in both optical fluorescence image analysis and mass spectrometry. It was found that the biodistribution of curcumin was reliable in both analysis methods.

In the case of the brain, the biodistribution of curcumin was significantly observed only in the result of optical fluorescence image analysis, and was insignificant in the result of mass spectrometry. It is believed that this is because it is difficult to extract curcumin from the brain and curcumin is decomposed during the extraction process. Therefore, it was found that the biodistribution of curcumin in the brain was reliable in the optical fluorescence image analysis result.

In the case of liver, gall bladder and pancreas where autofluorescence is high, it was found that mass spectrometry results of curcumin's biodistribution are reliable.

Therefore, complementary analysis of the results of optical fluorescence imaging and mass spectrometry has the effect of improving the accuracy when confirming the biodistribution of fluorescent polyphenols such as curcumin and the like (in particular, small molecule drugs). If the active ingredient whose biodistribution is to be confirmed does not show fluorescence, it can be modified and used to show fluorescence, so the scope of application of the method of the present invention is not limited to pharmaceutical active ingredients showing fluorescence.

Claims (14)

Administering drugs to non-human animals, then euthanizing and removing each organ to prepare an experimental group, and preparing a control group by euthanizing non-human animals without administering drugs and removing each organ (step One);
The step of obtaining a primary analysis result of quantifying the autofluorescence of each organ in the control group in step 1 by fluorescence image analysis and quantifying the drug distributed in each organ in the experimental group in step 1 by fluorescence image analysis ( step 2);
Obtaining a secondary analysis result by quantifying the drug distributed in each organ by mass spectrometry of the extract obtained from each organ of the experimental group for which the primary analysis result was obtained in step 2 (step 3); and
Improving the accuracy of biodistribution results of drugs by comparing the ratio of the results of each organ in the experimental group in the first analysis result with the ratio of the results in each organ in the experimental group in the second analysis result (step 4); including,
In the case of an organ in which the autofluorescence intensity of the control group is low in the fluorescence image analysis result in step 2, the mass spectrometry result in step 4 is discarded and the fluorescence image analysis result is judged to be reliable.
A method for mutually confirming the biodistribution of drugs using fluorescence image and mass spectrometry.
According to claim 1,
In step 4, when the ratio of the results of each organ in the experimental group in the first analysis result and the ratio of the results in each organ in the experimental group in the second analysis result are similar, both the fluorescence image analysis and mass spectrometry results are considered to be reliable. A method characterized by judging.
delete According to claim 1,
In the case of an organ in which the autofluorescence intensity of the control group is high in the fluorescence image analysis result in step 2, the fluorescence image analysis result in step 4 is discarded and the mass spectrometry result is judged to be reliable. .
According to claim 1,
The method according to claim 1, wherein the drug in step 1 is a compound that exhibits fluorescence by itself or a compound that does not exhibit fluorescence by itself.
According to claim 5,
The method characterized in that the compound that does not exhibit fluorescence by itself is not labeled with a fluorescent substance, but modified to exhibit fluorescence by itself.
According to claim 5,
The method according to claim 1, wherein the medicinal product is a plant-derived ingredient.
According to claim 5,
The method, characterized in that the drug is a small molecule drug (small molecule drug).
According to claim 1,
The method, characterized in that the fluorescence image analysis is an in vivo optical fluorescence imaging analysis.
According to claim 1,
Wherein the mass spectrometry is MALDI-TOF (Matrix Associated Laser Desorption Ionization - Time Of Flight) mass spectrometry.
According to claim 1,
The method characterized in that the extract of step 3 is obtained by extracting from the supernatant obtained by homogenizing and centrifuging each organ.
Biodistribution diagram of the drug obtained by the method of claim 1.
A method of modifying a drug to increase the distribution to a target organ using the biodistribution map of claim 12.
A method of binding a drug delivery system to a drug to increase the distribution to a target organ using the biodistribution map of the drug according to claim 12.

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