JP2010145113A - Method for determining internal kinetics, and method for preparing library of compound favorable in internal kinetics using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method which can efficiently determine a possibility of an inspection target as a drug from an aspect of internal kinetics in a mammal without requiring much labor in the searching study stage of the development of medicines or experiments with animals using the mammal, a method for preparing a compound library favorable in internal kinetics constituted of a compound afv in internal kinetics by the method of the determination, and the compound library. <P>SOLUTION: In the method for determining internal kinetics, the inspection target is administered to the larva of a holometabolous insect to evaluate the internal kinetics in the larva of the holometabolous insect of the compound in the inspection target or a substance formed by the internal chemical change of the compound in the inspection target of the larva and the quality of the internal kinetics of the compound in the inspection target or the substance formed by the internal chemical change of the compound in the inspection target in the mammal is determined from the evaluation result. In the method for preparing the compound library, the compound in the inspection target is selected using the method for determining internal kinetics to prepare the compound library using the compound in the inspection target as a constituent element. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for determining the pharmacokinetics in mammals of an efficient compound or substance for the development of a new pharmaceutical, and a library comprising a compound or substance having a good pharmacokinetics found by the determination method as a constituent element. The present invention relates to a production method and a library produced by the production method.

  In drug development, the exploratory research stage of drug candidate compounds (lead compounds), which is an upstream process, tends to be a bottleneck in overall drug development. When inappropriate lead compounds are selected in the exploratory research stage, or when the selection is insufficient, the pharmacokinetics of drugs performed in mammals such as mice in the downstream process, such as absorption A · This is because the loss associated with confirmation of the distribution D, metabolism M, excretion E (hereinafter abbreviated as “ADME”), etc. becomes enormous, and the development itself is often neglected before results are achieved.

  However, selecting a lead compound by examining the pharmacokinetics and the like using mammals at the exploratory research stage is a heavy burden and generates a bottleneck. Therefore, at the exploratory research stage, it is possible to efficiently examine the problems that must be cleared as pharmaceuticals such as pharmacokinetics, and to establish a method that enables selection of more appropriate lead compounds. is important. If this is possible, losses in downstream processes can be greatly reduced in the future, such as shortening the development period, reducing development costs, and reducing labor-intensive work, leading to overcoming bottlenecks in drug development. Because.

  However, there is hardly any technique that focuses on selecting the one with good pharmacokinetics, and the concept of a compound library with good pharmacokinetics itself was not clear.

  However, in the exploratory research stage, a method for efficiently examining not only the drug efficacy but also the pharmacokinetics etc. has been proposed. For example, Patent Document 1 discloses that at the exploratory research stage, three types of tests are performed: a bioactivity test, a lead compound selection test, and an pharmacokinetic evaluation test using an enzyme reaction test using a human-derived cytochrome P450 enzyme. A screening method for lead compounds that can reduce the burden on downstream processes has been proposed.

  On the other hand, as a method for improving the efficiency of the exploratory research stage itself, screening using a commercially available compound library (for example, Patent Document 2) and screening using a compound library by combinatorial chemistry (Combichem) are often performed. . However, since the structural diversity of the compounds constituting the underlying library is low, and the compounds themselves have little additional information such as drug efficacy information, the expected results may not be obtained.

  Thus, in order to efficiently select lead compounds with higher accuracy in accordance with the purpose, various methods for preparing various compound libraries having a complex structure and various compound libraries prepared by the methods have been proposed. For example, Patent Document 3 describes the disadvantages of chemical synthesis methods that are unsuitable for the synthesis of complex compounds, and bioconversion by enzymes that are easy to obtain compounds having complex structures but have high substrate specificity and difficult to obtain various reaction products. In order to eliminate the disadvantages of the method, a method for preparing a compound library using a biocombchem characterized by using a plurality of enzyme species has been reported. This method has been proposed as a method that can be applied to the production of natural product libraries containing a wide variety of complex compounds.

  In particular, many compounds derived from natural products have a variety of biologically significant structures acquired in the course of evolution, and are considered highly likely to have beneficial biological activities that lead to pharmacological activity. In fact, traditional Chinese medicines and traditional medicines that are effectively used today are made from natural products. Therefore, a library of compounds derived from natural products can be a library more suitable for drug discovery than a synthetic compound library synthesized at random. Nevertheless, it is said that a compound derived from a natural product is difficult to make into a library because of complicated sample preparation, requiring a device for confirmation of activity, and difficult to obtain a derivative. For this reason, natural product libraries have become important targets for commerce as they are of high value.

  Therefore, Patent Document 4 describes a method for efficiently forming a library of not only compounds derived from natural products but also derivatives thereof. The method involves adding various reaction reagents to a culture solution of a microorganism that produces an organic compound and reacting them, and adding a culture solution containing the organic compound and the reaction reagent produced by the microorganism to a culture solution that does not contain the reaction reagent. Comparative analysis is performed to identify and recover compounds that are not contained in the culture medium without the addition of reaction reagents, and make them a component compound of the compound library. It has been made easier.

  However, there are few useful libraries of compounds derived from natural products, and there has been almost no technology for producing libraries of compounds derived from natural products, particularly focusing on pharmacokinetics.

  On the other hand, the present inventor conducted experiments to replace mammals in various situations, such as using a silkworm larva (a silkworm), a fully transformed insect larva, in a method for screening a compound having antibacterial activity against pathogenic microorganisms. It has been reported that it can be used as an animal (for example, Patent Document 5). However, regarding the pharmacokinetics, including the report of the present inventor, there was nothing that was linked to the creation of a library by looking at the correlation between larvae of fully transformed insects and mammals.

JP 2004-313101 A Special table 2001-518053 gazette JP 2006-129836 A JP 2006-087392 A JP 2007-327964 A

  The pharmacokinetic evaluation method of Patent Document 1 can give certain information on the pharmacokinetics of the evaluation target, but the information is obtained only by a limited pharmacokinetic evaluation model system using human-derived cytochrome P450 enzyme. This information is insufficient compared to the information on pharmacokinetics when evaluated in animal experiments with mammals. Moreover, although the method of patent document 3 and 4 has the advantage that the compound which is hard to be made into a library by the conventional method can be added to a library, it evaluates a pharmacokinetics and evaluates as a compound and a substance with good pharmacokinetics. Has not been.

  There is a library that consists of compounds that are evaluated as to how the drug moves in the body before the drug exerts its effects, and whose pharmacokinetics are poor, so that it is not repelled in the downstream process. If the library can be used to screen lead compounds, the loss generated in the downstream process can be greatly reduced. In particular, if the evaluation of pharmacokinetics is carried out in a library comprising a natural product-derived compound that is difficult to be a library, its utility value is considered to be very high.

  However, as described above, animal experiments with mammals that are necessary for the evaluation of pharmacokinetics are too expensive to carry out, and the loss when the results are not good is too great. A library composed of a compound or substance evaluated as a good compound or substance as a constituent element has never been provided, regardless of whether it is derived from a chemically synthesized product or a natural product.

  The present invention has been made in view of the above-mentioned background art, and its problem is that it is a subject represented by ADME without requiring much labor and animal experiments by mammals in the exploratory research stage, which is an upstream process of drug development. Provided is a method capable of determining the possibility of a test subject, particularly a natural product-derived test subject, as a medicine from the viewpoint of pharmacokinetics in mammals, and also comprises a compound or substance having good pharmacokinetics by the method It is an object of the present invention to provide a method and library for preparing a compound or substance library with good pharmacokinetics.

  As a result of intensive studies for solving the above-mentioned problems, the present inventors have found that 7-hydroxy-4-methylcoumarin, which is a pharmacokinetic model drug for mammals, has undergone deethoxylation by cytochrome P450 enzyme in the silkworm body. Then, after glucose conjugation reaction, it was found that it was excreted by the same metabolic pathway and metabolic mechanism as mammals. They also found that the therapeutic effects of drugs and the quality of pharmacokinetics are well correlated.

  Therefore, when an extract from a natural product was administered to a silkworm and the pharmacokinetics of the extract in a silkworm body fluid was examined after a lapse of a certain time, a stable compound in the silkworm body fluid was analyzed and the analysis result of the extract itself. By detecting / identifying by comparing, etc., “compound in the test object” (hereinafter sometimes simply referred to as “compound”) or “compound in the test object is chemically changed in the silkworm body. It was found that the quality of the pharmacokinetics of the “stabilized substance” (hereinafter sometimes simply referred to as “substance”) can be determined.

  In addition, it was confirmed that the above compounds and substances can be recovered from silkworm body fluids, purified and identified relatively easily, and used to create new libraries of compounds and substances with stable pharmacokinetics. I found out that I can do it. From these findings and the knowledge that the pharmacokinetics of the drug in the previous mammal and the pharmacokinetics of the drug in the silkworm are in good agreement, the “method of determining the pharmacokinetics in the mammal of the test subject” of the present invention, The inventors have completed “a method for preparing a compound or substance library with good pharmacokinetics” and “a compound or substance library with good pharmacokinetics”.

That is, the present invention
1) Completely transformed form of a substance produced by administering a test object to a larva of a completely transformed insect and subjecting the compound in the test object or a compound in the test object to a chemical change in the body The pharmacokinetics in insect larvae are evaluated, and from the evaluation results, the pharmacokinetics in mammals of the compound in the test object or the substance produced by the chemical change in the test object in the body is determined. A method for determining pharmacokinetics, characterized by:

2) The following steps:
(A) a step of administering a test subject to a fully transformed insect larva,
(B) elapse of time;
(C) collecting a body fluid of the larvae of the fully transformed insect;
(D) identifying the substance in the body fluid collected in (c),
The method for determining pharmacokinetics according to 1).

3) The following steps,
(X) identifying a compound in the test object;
(A) a step of administering a test subject to a fully transformed insect larva,
(B) elapse of time;
(C) collecting a body fluid of the larvae of the fully transformed insect;
(D) identifying the substance in the body fluid collected in (c),
(E) comparing the compound in the test object specified in (x) with the substance in the body fluid specified in (d);
The method for determining pharmacokinetics according to 1) or 2).

4) The pharmacokinetic determination method according to any one of 1) to 3), wherein the completely transformed insect larva is a silkworm.

5) The pharmacokinetic determination method according to any one of 1) to 4), wherein the test subject is a natural product.

6) A method for preparing a library of compounds having favorable pharmacokinetics in mammals, wherein the pharmacokinetic determination method according to any one of 1) to 5) is used to produce a fully transformed insect larva in the body. A method for producing a library, comprising selecting a compound in the test subject that is stable or stable upon receiving a chemical change in the body of a larva of a completely transformed insect.

7) A method for preparing a library of compounds with favorable pharmacokinetics in mammals, comprising the following steps:
(X) identifying a compound in the test object;
(A) a step of administering a test subject to a fully transformed insect larva,
(B) elapse of time;
(C) collecting a body fluid of the larvae of the fully transformed insect;
(D) identifying the substance in the body fluid collected in (c),
(E) comparing the compound in the test object specified in (x) with the substance in the body fluid specified in (d);
(F) Among the compounds in the test object identified in (x), the compound is stable in the body of a fully transformed insect larvae or stable in response to a chemical change in the body of a completely transformed insect larva The method for producing a library according to 6), wherein a compound to be selected is selected as a constituent element.

8) A method for preparing a library of substances having good pharmacokinetics in mammals, and using the pharmacokinetic determination method according to any one of 1) to 5), thereby producing a fully metamorphic insect larva in the body. A method for preparing a library, comprising selecting a substance in the body fluid that exists stably as a constituent element.

9) A library of compounds having favorable pharmacokinetics in mammals produced using the method for producing a library according to 6) or 7).

10) A library of substances having favorable pharmacokinetics in mammals produced using the method for producing a library according to 8).
Etc.

  According to the present invention, the pharmacokinetic aspect represented by ADME in mammals is not required without requiring much labor in the exploratory research stage, which is an upstream process of drug development, and animal experiments by mammals having ethical problems. From the above, it is possible to provide a method capable of determining the possibility of a test subject, particularly a test subject derived from a natural product, as a medicine.

  Further, it is possible to provide a method for preparing a library of compounds or substances composed of “a compound or substance having good pharmacokinetics in mammals” determined to be good by the above method for determining the quality of pharmacokinetics, By using a compound or a library of the substance, it is possible to develop a pharmaceutical that is more efficient and less lossy than before. The library of compounds or substances with good pharmacokinetics of the present invention makes it possible to screen lead compounds and substances with good pharmacokinetics efficiently in the exploratory research stage of the upstream process in drug development. Many losses that have occurred can be reduced.

  Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the technical scope of the present invention.

  The completely transformed insect used in the present invention refers to an insect that undergoes the growth process of eggs, larvae, pupae and adults. Examples of the completely metamorphic insect include insects belonging to the order Lepidoptera (butterflies, moths, etc.), Diptera (flies, etc.), Hymenoptera (bees, ants, etc.), Coleoptera (beetles, etc.) and the like. In the present invention, these completely transformed insect larvae are used as test animals.

  The type of larvae is not particularly limited and can be appropriately selected according to the purpose of the test. However, it is in the form of a worm, facilitating accurate drug administration by injection, etc., organ removal, and fecal analysis. What has a magnitude | size is preferable. Larvae such as moths, butterflies and beetles are preferred in that many have a size suitable for such a demand.

Further, as such larvae, silkworm larvae (hereinafter abbreviated as “silkworm”) are particularly preferred from the following points.
(1) It is easy to obtain.
(2) A breeding method has already been established, and there is convenience in breeding.
(3) The properties similar to the internal organs and organs of mammals such as humans have been known to some extent in previous studies.
(4) Genetic lines have been established and genetic homogeneity has been maintained.
(5) Since it is relatively large, slowly moving, and substantially hairless, it is easy to administer drugs, such as being quantitatively injectable, and blood and the like are also easily collected.
(6) Having a relatively large organ, the organ can be taken out and the contained substance can be quantified.
(7) It is cheaper than mice, rats, etc., and can keep a large number of individuals in a small space, and there are few ethical problems.
(8) Evaluation can be performed even when there is only a small amount of the test target substance.
(9) It is easy to align individuals in the same state, such as aligning the age and mass of individuals.

  Among the above, after (5), it is a characteristic that can be said to be general larvae of completely transformed insects, and it is clear that larvae of completely transformed insects are excellent as experimental animals, not limited to silkworms.

  The fully metamorphic insect has a simple form specialized in feeding, represented by caterpillars, and has an age with a clear separation by several moltings. Therefore, by aligning the age period, it is possible to accurately align the growth state of the individuals used in the test, and it is possible to select the most appropriate age period according to the purpose. In addition, it has many excellent characteristics as a test animal that it is easy to grow because of its slow movement and is easy to inject drugs and the like. In contrast, imperfectly transformed insects, such as mantis and grasshoppers, have the same form as adults that are not suitable for injection since the time of larvae. There are many disadvantages such as active and quantitative administration by injection is difficult, and there is little blood to be used for analysis, so it cannot be used in the present invention.

  In the present invention, the size and age of the larvae may be selected from the types of larvae, larvae forms, administration methods, instruments used, operational viewpoints, etc., and are not particularly limited. It is preferable to have a size that facilitates administration of blood, organs, feces and the like necessary for studying pharmacokinetics. However, the preferred size varies depending on the administration method of the test subject, the purpose of the test, and the like. For example, in the case of mixing with food, administration is possible even with the size of the 1st instar larva if it is a silkworm, so that the 1st instar larva can be started immediately when it is raised from an egg. Is preferred. However, it is preferable to use larvae 3 years of age or older in consideration of administration of an object to be examined using a syringe or the like, removal of an organ, collection of blood, analysis of feces, and the like. In this case, 4th to 5th instar larvae are more preferable, and 5th instar larvae which are the final ages are particularly preferable.

  The size of the fully transformed insect larva used as a test animal is not particularly limited, but the length is 1 cm from the viewpoint of ease of administration of the test substance, organ removal, blood collection, feces analysis, etc. The above larvae are preferable, 1.5 cm or more and 15 cm or less are more preferable, and 2 cm or more and 10 cm or less are particularly preferable.

  The sample preparation method, administration method, administration route, etc. of the test subject may be selected in the same manner as in mammals, but the fully transformed insect larvae are open vasculature and there is no distinction between body fluid and blood Therefore, when the pharmacokinetics is examined by administration into a mammal, such as intravenously, subcutaneously or intraperitoneally, it is preferable to administer it in blood unless there are special circumstances in the case of a fully transformed insect larva. Administration into blood can be performed, for example, by injection into the first abdominal leg or the like.

  In addition, when the pharmacokinetics by oral administration is studied in mammals, administration into the intestinal tract may be performed for larvae of completely transformed insects. A drug that exerts a pharmacological action by oral administration is highly useful because the burden on the patient is small. Therefore, it is one of the advantages of the present invention that the pharmacokinetics of the test subject absorbed from the digestive tract by intestinal administration can be easily examined. Moreover, depending on the case, it is possible not only to enter the intestine but also to administer the subject to be tested orally by mixing it with the feed.

  In both cases of intra-blood administration and intestinal administration, as a medium for dissolving or dispersing the test subject, those usually used for administration to mammals can be used.For example, when the subject is water-soluble, It is preferable to use pure water, physiological saline, etc., and in the case of oil solubility, vegetable oils such as olive oil are used.

  In the present invention, no matter what kind of investigation is performed, it is possible to study at a low cost without taking up space, and there are few ethical problems. Therefore, an experiment on the pharmacokinetics of a subject to be examined can be performed on a number of subjects to be examined and under a number of conditions that cannot be practically performed in mammals.

  In addition, by using a model compound whose pharmacokinetics has already been determined in larvae of fully transformed insects as a control, by determining the pharmacokinetics of the test compound at the same time as determining the pharmacokinetics of the model compound, The reliability of the pharmacokinetic determination method can be confirmed. Performing such a confirmation test using a mammal as an experimental animal has not only great resource problems such as complicated operations and cost problems, but also ethical problems. However, according to the present invention, since there are few such problems, it is possible to carry out a “reliability confirmation test” or the like that could not be substantially performed until now.

  In the following, the method for determining the pharmacokinetics of mammals using larvae of fully transformed insects based on silkworms will be described, but there is no difference in the basic embodiment when using larvae of other completely transformed insects. Each item can be implemented by performing the same or similar method with reference to the case of silkworm.

  The variety of silkworms used in the method for determining pharmacokinetics of the present invention is not particularly limited, and for example, hybrids such as Hu, Yo x Tukuba, and Ne can be used. In addition, since there are many varieties, it is possible to select an appropriate variety according to the content of the test. Silkworms may be used by growing from fertilized eggs depending on the content of the test, or the test may be carried out by obtaining larvae of the required age.

  When determining the pharmacokinetics of a test subject using a silkworm, it is preferable to use a 5th instar larva having a large body as described above. However, it is also possible to use 3rd or 4th instar larvae when it is desired to secure larvae to be used for the test earlier, such as when raised from eggs.

  In the pharmacokinetic determination method of the present invention, the subject to be administered to the silkworm includes a single compound whose structure has already been confirmed, a single compound whose structure has not been confirmed, and a mixture thereof. Such a mixture is not particularly limited, and examples thereof include a mixture of synthetic compounds and a mixture derived from a natural product.

  More specifically, such natural products are described as follows: extracts from plants such as flowers, leaves, roots, stems, seeds, buds, pollen, dried products thereof; microorganisms and fungus extracts; animal blood, urine And extracts from organs, etc .; extracts from culture supernatants of plants, microorganisms and various cells; extracts from minerals, petroleum, soil and the like. Moreover, the mixture in the middle of refinement | purification, such as a fraction at the time of isolate | separating those extracts by chromatography etc., is also contained.

  In addition, the test subject used in the pharmacokinetic determination method of the present invention is obtained by subjecting the above extract to further artificial processing such as esterification, hydrolysis, hydrogenation, alkylation, fermentation treatment, and enzymatic degradation treatment. Also included.

  The determination target of the pharmacokinetic determination method of the present invention includes not only the compound in the administered test subject but also the substance after undergoing a chemical change in the silkworm body. Here, “compound” means what is originally contained in the test subject to be administered, and “substance” means that the compound in the test subject is stable due to chemical changes in silkworms. This means something that has become a The compound and substance need to be specified, but the term “specified” means a compound or substance that has been confirmed to be reproducible under certain conditions. However, it does not mean only a single compound with a clear chemical structure. For example, the compound in the fraction which shows the fixed retention time of the chromatography under a fixed condition, and high molecular substances, such as the protein which shows a fixed fraction pattern, are also contained.

  In addition, in the method for determining pharmacokinetics in mammals such as the test subject shown in 2) of the present invention, the pharmacokinetics is good in the silkworm by administering the test subject to silkworms and allowing time to pass after administration. Or not, and the result is evaluated as a pharmacokinetic determination result in mammals. Therefore, it is important to allow time to confirm the change in the body after the subject is administered to the silkworm. After that, the body fluid is collected, and the substance present in the body fluid is analyzed and specified, so that "the pharmacokinetics of the compound in the test subject in mammals" or "the compound in the test subject is silkworm. The quality of the pharmacokinetics in mammals of substances produced by chemical changes in the body can be determined.

  In order to confirm whether or not the pharmacokinetics is stable after administering the test subject to the silkworm in the step (a), the specific mode of each step is (b) as a step (in a fasted state if necessary, ) Allow a certain time to elapse. The elapsed time after administration is not particularly limited, but both in blood administration and intestinal administration are preferably in the range of 4 to 24 hours, more preferably in the range of 5 to 14 hours, and in the range of 6 to 10 hours. Particularly preferred. If the time lapse is too short, the ADME of the test object in the silkworm body may be incomplete, and non-specific compounds and substances may be identified. On the other hand, if the time lapse is too long, There are cases where all the test objects cannot be collected due to excretion, etc., and there is a case where there is no particular necessity just because the need to pay attention to the maintenance management of silkworms and the grasp of the state is increased.

  In studies using antibiotics, drugs that do not show therapeutic effects in mammals do not show therapeutic effects in silkworms, and drugs that show therapeutic effects in silkworms have good pharmacokinetics and a blood half-life of 6 hours or more. (See Test Examples 1 and 2, Table 1, FIG. 9 and FIG. 10). These results indicate that good pharmacokinetics is a very important factor in showing pharmacological action.

  After a certain period of time, silkworm body fluids are collected in step (c). The collected body fluid is subjected to a known pretreatment according to the analysis object and the analysis method, such as deproteinization, as necessary, and is subjected to component analysis for specifying the substance in the body fluid in the step (d).

  (D) In the step “step for identifying a substance in the body fluid”, the means is not particularly limited, and a known method is used. Specific examples include high performance liquid chromatography (HPLC), thin layer chromatography (TLC), gel filtration chromatography, and the like. For example, if the development condition, time, position, etc. are determined by chromatography or the like, the substance can be identified, and if there is reproducibility, the substance can be identified even if the chemical structural formula of the substance is unknown. The method for determining the chemical structure is also not particularly limited. After the compound or substance is fractionated and purified using the above-mentioned means, various mass spectra; UV, visible, infrared absorption spectra; instrumental analysis methods such as nuclear magnetic resonance spectra Can be used.

  In addition, the method for determining the pharmacokinetics of a mammal such as a test subject shown in 3) of the present invention is performed by subjecting the test subject to various analysis / identification means such as chromatography as the step (x) in advance. In this aspect, the test subject is administered to the silkworm after the compound in the test subject is specified and information about the compound in the test subject is arranged. Also in this case, steps (a), (b), (c), and (d) are performed, a body fluid is collected after elapse of time after administration, and substances in the body fluid are specified by various analysis / identification means. . This is a method for determining the pharmacokinetics by comparing and analyzing information such as chromatographic data on the substance in the body fluid and information on the compound in the previous test object as step (e).

  Specifically, an example will be described below. For example, when it is specified by the step (x) that compounds A, B, C and D are contained in the test object, the substance found in the body fluid of silkworm and the previous test object By comparing various analysis / identification results with the compounds (A, B, C and D) in the product, B ′ (a substance that can be confirmed that B has undergone a chemical change in the body) from the silkworm body fluid, and If D is identified as a stable substance and A and C are not found in the body fluid of silkworm or the abundance is clearly reduced, B and D are determined to be compounds with good pharmacokinetics, and A And C are determined to be compounds with poor pharmacokinetics. B 'is determined to be a substance with good pharmacokinetics.

  A more specific method for determining a substance with good pharmacokinetics in a body fluid after administration of a test subject is, for example, (a) “Negative control such as physiological saline is administered to a silkworm, and after a certain time has elapsed. Fractionation of the extract from the collected silkworm body fluid ”and (b)“ Fractionation of the extract from the silkworm body fluid collected after the test subject is administered for a certain period of time ”respectively. This can be done by comparing the results of (b) and (c) fractionation patterns obtained by "fractionation operation of the test subject itself".

  In this case, the pharmacokinetics of the compound in the test object can be determined, and the pharmacokinetics of the compound in which the compound in the test object is stabilized by some chemical change in the silkworm body can be determined. . For example, in the above example, Compound B is not found in the body fluid of silkworm, but can be found in the body fluid of silkworm as B ′ (a substance that can be confirmed that B has been chemically changed in the body). Compound B is determined to be a compound with good pharmacokinetics.

  On the other hand, it exists in the fraction pattern of the test subject itself, and it is not observed in the fraction pattern of the extract from the silkworm body fluid collected after the test subject is administered for a certain time. It can be confirmed that the weakened peak is that of an unstable compound in the body fluid. It can be determined that the compound in such a test subject does not have good pharmacokinetics.

  According to the pharmacokinetic determination method in mammals such as the test subject of the present invention described above, the compound in the test subject determined to have good pharmacokinetics as being stable in the silkworm body, or in the test subject The compounds that are stable and have good pharmacokinetics due to chemical changes in the body fluids of silkworms are all the compounds of the compound library with good pharmacokinetics in mammals according to the invention described in 6) above. It can be a component of a library produced by the production method. As a result, it is possible to provide a “method for preparing a compound library with favorable pharmacokinetics in mammals” that enables selection of effective lead compounds that have never existed in the early stage of drug development, which is an exploratory research stage. it can.

  In addition, the method shown in 7) performs the steps (x) to (e) in the same manner as in the invention 3), and the analysis / identification result of the compound in the test object and the substance in the body fluid. The analysis and identification results are compared with each other to determine the pharmacokinetics of the compound and substance specified in each. (F) As a step, among the compounds in the test object specified in (x), in the silkworm body Select compounds that are stable or stable after undergoing chemical changes in the silkworm, and each of which has been determined to have good pharmacokinetics, and constitute a component of a “compound library with good pharmacokinetics in mammals” This method is also a method for producing a “compound library with good pharmacokinetics in mammals”.

  In the above method, the compound in the test object is separated and identified, and the previous compound is collected from the test object and used as a component of the compound library of the present invention. On the other hand, the method shown in 8) above is characterized by separating and identifying a substance having good pharmacokinetics from silkworm body fluid, and using the collected substance as a component of a library of substances having good pharmacokinetics in mammals. This is a method for preparing a substance library.

  An important feature of the method for preparing a compound or substance library with good pharmacokinetics as described above is that the test subject is administered into the silkworm body and time is allowed to elapse so that the compound in the test subject etc. Is based on the new discovery that ADMA undergoes metabolism, absorption, etc., similar to the pharmacokinetics in the mammalian body in the silkworm.

  In general, compound libraries used for drug development include primary libraries and secondary libraries. Primary libraries are compound mixtures and natural products created by combichem, etc. that have not been screened for exploratory research. The secondary library is composed of compounds that have been screened and selected from various properties suitable for drug discovery from the primary library as indicators. is there. Therefore, the secondary library is created by spending a great deal of labor, cost, and time due to mammalian experiments and the like. Especially for natural products, it is difficult to produce a primary library due to the fact that many extraction and purification operations are more complicated than synthetic products to evaluate them. Few secondary libraries are made available.

  However, according to the method for preparing a compound or substance library having good pharmacokinetics in the mammal of the present invention, the determination of the pharmacokinetics of a compound or substance is greatly saved in labor, cost, and time compared to conventional methods. Even if the target is a natural product, a library having the same value as the secondary library can be easily prepared. In addition, as described above, in the present invention, the subject to be examined can be determined for pharmacokinetics without any special pretreatment, so the system using cultured cells may affect the results. Extracts from natural products that are likely to contain high contaminants can also be used as test objects without problems.

  Since libraries used for normal drug development, particularly natural product libraries, are mostly primary libraries, they contain many compounds and the like that have poor pharmacokinetics. The confirmation of pharmacokinetics is usually performed in animal experiments using mammals, and is often performed at a relatively downstream exploratory research stage to prevent waste and loss. However, since it was found that the pharmacokinetics in the downstream process were not good, being excluded from candidates for drug development resulted in many losses, such as wasted labor, and the cause of the bottleneck of overall drug development. It also becomes. A library prepared using the present invention has an effect of suppressing the occurrence of the above loss. Therefore, as the test object of the present invention, an extract from a natural product can be said to be a particularly preferable test object.

  The compound in the test subject whose pharmacokinetics was confirmed to be stable by the method of determining pharmacokinetics of the present invention is the substance stabilized from the test subject and chemical change in the body fluid. It can be recovered, purified, and identified by a known method from the body fluid of silkworm after a certain time has elapsed after administration of the test subject. Specifically, for example, the previous test object or body fluid is extracted with an extraction solvent such as water, acetone, ethyl acetate, methanol, hexane, ether, dichloromethane, supercritical fluid, and the extraction solvent is removed. A compound or substance with good pharmacokinetics can be obtained by combining fractionation chromatography such as preparative HPLC, and other various methods for purification and purification, and can be used for various analyzes and applications.

  The compound or substance thus obtained can be stored, and can be used as a “compound or substance constituting a compound or substance library having good pharmacokinetics in mammals” of the present invention. In addition, even if the actual compound or substance does not exist as a sample and the chemical structure is unknown, if the compound or substance is specified in the above-mentioned meaning, the person who obtained the library Since the compound or substance can be obtained and used by the above method, such a case is also included in the library of the present invention.

  The above-described pharmacokinetic determination method for determining the quality of pharmacokinetics in mammals, a method for preparing a library of compounds or substances having good pharmacokinetics in mammals, and a mammal prepared using the method for preparing the library The library of compounds or substances with good pharmacokinetics can be efficiently screened for lead compounds and substances with good pharmacokinetics in the exploratory research stage of the upstream process in drug development. Many losses can be reduced. In addition, since the present invention can be evaluated by oral administration and can be administered to the subject by dietary administration such as mixed with food, it is not limited to drug development, and functional food ingredients with good pharmacokinetics. It can also be used for development.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated more concretely, this invention is not limited to these Examples, unless the summary is exceeded.

<Type of silkworm, breeding conditions>
Silkworm fertilized eggs (a hybrid, Hu • Yo x Tukuba • Ne) were purchased from Ehime Seed Co., Ltd. The hatched larvae were given artificial feed silk mate 2S (manufactured by Nippon Nosan Kogyo Co., Ltd.) at room temperature to grow to 5th instar larvae. As the breeding container, a square type No. 2 petri dish (manufactured by Eiken Equipment Co., Ltd.) was used from the egg to the second instar larva, and the disposable plastic food pack (Food Pack FD Daifuka, Chuo Chemical Co., Ltd.) was used thereafter. The breeding temperature was 27 ° C.

Example 1
<Isolation and purification of compounds with good pharmacokinetics derived from summer tangerine leaf extract>
(1) Preparation of sample for administration and sample for purification of test object After pulverizing 100 g of summer tangerine leaves, it was extracted with acetone, and the extract was vacuum concentrated and extracted with water. The aqueous extract was filtered through a filter having a pore size of 0.45 μm to obtain a sample for administration to silkworm, a sample for HPLC analysis, and a sample for preparative HPLC.

(2) Administration of summer tangerine leaf extract to silkworm After fasting a 5-year-old silkworm for 1 day, the above-mentioned sample for administration is diluted to 50 mg / mL with physiological saline, and 0.05 mL of the sample is added to silkworm body fluid. It was administered by intra-medium administration. After administration, silkworm body fluids were collected after 8 hours in the fasted state. Two silkworms were used in one condition.

(3) Preparation of sample for HPLC analysis from silkworm body fluid after administration of test subject To 0.1 mL of body fluid collected in (2) above, an equal amount of acetone is added to perform acetone extraction, and after centrifugation, The extracted supernatant was dried and 0.05 mL of physiological saline was added to prepare a sample for HPLC analysis. In addition, a sample for HPLC analysis was prepared in the same manner from a silkworm body fluid administered with physiological saline and similarly aged.

(4) HPLC analysis HPLC analysis uses a PEGASIL ODS 4.6 mmφX250 mm column, and the analysis conditions are 10% acetonitrile isocratic 15 minutes 10% -100% acetonitrile linear gradient 20 minutes 100% acetonitrile wash 20 minutes flow rate 1 mL / Min Sample 1 mg / mL Injection volume 5 μL The measurement was performed at a measurement wavelength of 215 nm, 254 nm, and 360 nm.

  As a result, fraction 2 compound UV: 283.4 nm, 329.9 nm, and fraction 4 compound UV: 252.6 nm, 347.9 nm were confirmed as compounds having good pharmacokinetics (stable). See FIGS.

(5) Implementation of preparative HPLC <Preparation of crudely purified fraction>
115 mL of the filtered sample of (1) was roughly purified by preparative HPLC under the following conditions to obtain 275.7 mg of a crudely purified fraction (fraction 3 in FIG. 2).
PEGASIL ODS 2 mmφX250 mm column Purification conditions: 10% acetonitrile isocratic 15 minutes 10% -100% acetonitrile linear gradient 20 minutes flow rate 9 mL / min 100% acetonitrile wash 20 minutes.

<Repurification of the roughly purified fraction (fraction 3 in FIG. 2) by preparative HPLC>
PEGASIL ODS 2φX25 cm column Purification conditions; 15% acetonitrile isocratic 185 minutes Flow rate 9 mL / min 100% acetonitrile wash 20 minutes.
Fractions 1 to 7 were fractionated, and fractions 2 and 4 were determined to have good pharmacokinetics (comparative) by comparative examination of fraction patterns, and were subjected to subsequent purification steps by preparative TLC. See FIG.

(6) Preparative TLC <Purification of fraction 2 and fraction 4>
Fraction 2 (12.02 mg) and fraction 4 (6.38 mg) were purified by preparative TLC (silica gel 60F ₂₅₄ Merck), developing solvent chloroform / methanol = 2/1, fraction 2 compound 6.38 mg, fraction 4 3.98 mg of compound was obtained. See FIG.

Example 2
<Investigation using model crude drugs for determination of pharmacokinetics and library development by intestinal administration>
Based on the results of the screening, it was suggested that a compound with stable pharmacokinetics was present by intestinal administration, and the herbal medicine blue skin was used as a model. We verified the method for determining pharmacokinetics and the method for creating libraries of compounds with stable pharmacokinetics. Citrus reticulata Branco is a crude drug based on the blue immature pericarp of Ponkan and Satsuma mandarin, and is known to have a healthy stomach effect.

(1) Preparation of sample for administration / purification sample 100 g of green skin is extracted with 2 L of 50% acetone, and a portion of the fraction obtained by removing acetone is used as an administration sample, and the remaining 1 L is an equal amount. Solvent extraction was performed with n-hexane to prepare a sample for purification.

(2) Administration of green skin extract to silkworm 500 μL of the above green skin acetone extract was administered by injection into the intestine of the silkworm. After 6 hours from the administration, body fluid was collected, and a sample for HPLC analysis was prepared in the same manner as in Example 1, (3). In addition, a sample for HPLC analysis was similarly prepared from a body fluid of a silkworm administered with physiological saline.

(3) HPLC analysis A PEGASIL ODS 4.5φX250 mm column was used, and the analysis conditions were 10% acetonitrile isocratic 15 minutes 10% -100% acetonitrile linear gradient 20 minutes 100% acetonitrile washing 15 minutes flow rate 1 mL / min sample 1 mg / ML Injection amount 5 μL The measurement was performed at a measurement wavelength of 215 nm, 254 nm, and 360 nm. See FIG.

(4) Acquisition of crude purified fraction by preparative HPLC Preparative HPLC was carried out under the following conditions to obtain 11.6 mg of crude purified fraction A, 21.1 mg of B, and 23.5 mg of C. Preparative conditions: PEGASIL ODS 20φX250 mm column 40% acetonitrile isocratic flow rate 9 mL / min.

(5) Purification by preparative TLC of each crudely purified fraction _Obtained in (3) using preparative TLC (25HPTLC plate RP-18F _254S Merck), developing solvent n-hexane / ethyl acetate = _3/1 Each fraction was purified to obtain purified fraction A 8.5 mg, B16.3 mg, and C19.1 mg. Each of these fractions was further purified by preparative TLC (silica gel 60F ₂₅₄ Merck) and developing solvent chloroform / methanol = 10/1. As a final purified product, 5.8 mg of compound A which was absorbed from the intestinal tract and stable in pharmacokinetics was obtained. Compound B (10.2 mg) and Compound C (17.2 mg) were obtained. FIG. 7 shows the operation flow of the second embodiment.

(6) A, B, C, structural analysis of each compound <Molecular weight>
Each purified compound was subjected to FAB-MS spectral mode to determine the molecular weight. As a result, Compound A was determined to have a molecular weight of 402, Compound B to a molecular weight of 432, and Compound C to a molecular weight of 372.

<Structural analysis>
Structural analysis was performed using ¹ H and ¹³ C-NMR spectra, and spectra showing the presence of characteristic hydrogen and carbon derived from a methoxy group and the presence of hydrogen and carbon derived from an aromatic ring were observed for each compound.

From the above results, compounds A, B, and C are known compounds, compound A is nobiletin with molecular formula C ₂₁ H ₂₂ O ₈ , and compound B is heptamethoxyflavone with molecular formula C ₂₂ H ₂₄ O ₉ (Heptamethyflavone). compound C was identified respectively tangeretin molecular formula _{C 20 H 20 O 7 (tangeretin} ). The chemical structural formula of the identified compound is shown in FIG.

<Consideration of results of Example 1 and Example 2>
From the results of Example 1 and Example 2, the biological extract, which is the test object of this time, is administered into silkworm blood (in body fluid) or in the intestinal tract, and the substance in the silkworm body fluid is biologically extracted after a certain period of time. It was confirmed that the quality of the pharmacokinetics of the compound in the test object can be judged by comparative analysis with the object itself. It was also confirmed that the compound could be identified by purifying the relevant compound from the fraction that was confirmed to have good and stable pharmacokinetics and structural analysis. Therefore, it has been confirmed that these compounds are constituent compounds of a compound library having good (stable) pharmacokinetics according to the present invention.

  Here, the three compounds obtained in Example 2 are all polymethoxyflavones having a flavonoid skeleton (see FIG. 8), and these group of compounds are anti-inflammatory, antioxidant, and antitumor effects. Pharmacological activity has been reported. Furthermore, due to its diverse physiological activities, its pharmacokinetics have attracted attention. For nobiletin and tangeretin, several studies have been reported to evaluate the pharmacokinetics in mammalian rats (for example, Murakami). A., Kawahara S., Takahashi Y. et al., Biosci Biotechnol Biochem. 2001, 65, 194-7). And this document suggests that nobiletin has a blood half-life of 24 hours or more.

  The result of the substance confirmed in the body fluid after 6 hours after administration to the silkworm obtained in Example 2 indicates that the pharmacokinetics of nobiletin and tangeretin reported in the rat are good (stable). Consistent with the report, this is evidence that the method of using the fully transformed insect larvae of the present invention can be a method for determining pharmacokinetics in mammals.

  The above results are based on studies on the pharmacokinetics of biological extracts or natural products. It is difficult to make a biological extract into a library, and it has been studied from the point that it is easy to clarify the advantages of the present invention. The pharmacokinetics of a synthetic compound obtained by combichem or the like can be determined in the same manner in the present invention. Needless to say. A library of compounds having good pharmacokinetics based on the determination result of pharmacokinetics of the present invention can also be applied to any test object.

  In order to show that the results of pharmacokinetics obtained using silkworms, one of the fully transformed insect larvae, can replace the results of pharmacokinetics obtained in mammals, The results of a study comparing the pharmacokinetics of various antibiotics and examining the relationship between therapeutic effects and pharmacokinetics are described.

Test example 1
<Examination of pharmacokinetics of antibiotics that do not show therapeutic effects on silkworms (excluding topical use)>
[the purpose]
It was confirmed that three antibiotics that show therapeutic effects when used externally in mammals but do not show therapeutic effects when administered in blood do not show therapeutic effects on silkworms (see Table 1 below). .

  Therefore, it was examined whether antibiotics that do not show therapeutic effects when administered to the body of mammals are likely to disappear from the silkworm body fluid (blood).

[Method]
Drug solutions 50 μL and 100 μL (in the case of Na fusidate) were administered to silkworms (body weight 1.8 g to 2.0 g) on the second day of age 5 in the blood, and body fluids were collected over time. The drug was extracted by adding an equal volume of acetone or methanol to the body fluid. The extract was dried and dissolved in 50% methanol equivalent to the collected body fluid, and then HPLC analysis was performed to determine the drug concentration in the body fluid. The changes in body fluid concentration were graphed from the elapsed time after administration and the drug concentration, and the pharmacokinetic parameters of each drug were calculated.

<Dose: Study drug>
100 μg / silkworm: sodium fusidate

<HPLC analysis conditions>
Column: PEGASIL ODS R1251
Moving bed: acetonitrile / methanol / 10 mM phosphoric acid = 5/2/3
Detection: 235 nm absorption

[result]
Sodium fusidate had a short half-life in silkworm bodies. A graph in which the blood half-life of sodium fusidate was calculated to be 15 minutes is shown in FIG.

Test example 2
<Examination of pharmacokinetics of antibiotics with therapeutic effects on silkworm>
[the purpose]
Contrary to Test Example 1, it has been confirmed that antibiotics having a therapeutic effect in mammals also have a therapeutic effect on silkworms (vancomycin, amphotericin B in Table 1). Therefore, it was examined whether therapeutically effective antibiotics exist stably in the silkworm body fluid (blood).

[Method]
Administration of the drug, changes in concentration in the body fluid, and calculation of pharmacokinetic parameters were performed in the same manner as in Test Example 1.

<Dose: Study drug>
100 μg / silk: vancomycin, tetracycline, chloramphenicol 50 μg / silk: fluconazole

<HPLC analysis conditions>
Column: PEGASIL ODS R1251
Moving layer: acetonitrile / 50 mM sodium phosphate = 8/92 vancomycin methanol / 5 mM EDTA = 3/7 tetracycline methanol / 20 mM potassium phosphate = 35/65 chloramphenicol acetonitrile / 25 mM acetate buffer (pH 5) = 1/4 fluconazole

[result]
The results are shown in Table 2. The distribution volume (Vdss) values were in good agreement between silkworms and mammals. In addition, the half-life in blood was 6 hours or longer for both silkworms and mammals. FIG. 10 is a graph plotting changes in blood concentrations of vancomycin and chloramphenicol in silkworms among the examined drugs.

<Consideration of Test Example 1 and Test Example 2>
Bifonazole, clotrimazole, and Na fusidate are effective as topical drugs in mammals, so it is thought that they disappear rapidly from body fluids (blood) in mammals. It was also observed that disappearance was rapid in silkworms. On the other hand, drugs such as vancomycin, which have a therapeutic effect other than topical drugs, exist stably in the body in mammals, but also exist stably in body fluids (blood) in silkworms.

  From the above test examples, it was found that the pharmacokinetics of antibiotics using silkworms are in good agreement with those of mammals. Moreover, it was confirmed that the therapeutic effect which is not as an external medicine reflects well the state of pharmacokinetics in both silkworms and mammals.

  Moreover, the pharmacokinetics in the silkworm of the compound having a pharmacological action in the green skin extract obtained from Example 2 was consistent with the pharmacokinetics of mammals. Therefore, the method for determining the pharmacokinetics of a drug using a silkworm, which is one of the larvae of a completely transformed insect, can replace the pharmacokinetics in a mammal and can be used as a method for determining the pharmacokinetics in a mammal.

  The method for determining the pharmacokinetics of a test object using a fully transformed insect larva of the present invention can be used for determining the pharmacokinetics of a test object in mammals, is simple, cost-effective, and ethical. Since there are few problems, it can be used as a determination test of the pharmacokinetics of the test subject in mammals, which was not performed in the exploratory research stage of conventional drug development. In addition, the determination method of the pharmacokinetics of the present invention can be used as a method for preparing a library of compounds or substances having good pharmacokinetics in mammals. It is widely used as a library.

It is the figure which compared each HPLC pattern of the humor extract of a summer tangerine leaf extract, the body fluid extract of the silkworm which administered physiological saline, and the body fluid extract of the silkworm which administered summer mandarin orange leaf extract. It is a HPLC pattern comparison figure of a summer tangerine leaf extract and a summer tangerine leaf extract administration silkworm body fluid extract, It is a figure which shows the peak which the compound considered to be stable in a silkworm body fluid, and the compound considered to be unstable. It is the figure which examined the fraction of the stable substance and the unstable substance in a silkworm body fluid by comparing the HPLC pattern of a summer tangerine leaf extract and a silkworm body fluid extract administered with a summer tangerine leaf extract. It is the figure which compared seven fractions fractionated by preparative HPLC of a crudely purified fraction between a summer tangerine leaf extract and a summer tangerine leaf extract administration silkworm body fluid extract. It is a figure which shows the chromatogram which the two substances derived from the summer tangerine leaf and judged to be stable in a silkworm body fluid show. In Example 2, it is the figure which compared the HPLC pattern which the silkworm body fluid extract which administered the herbal extract of the herbal medicine showed, and the HPLC pattern which shows the silkworm body fluid extract which the physiological saline solution administration. FIG. 10 is a diagram illustrating a flow of operations according to the second embodiment. It is a figure which shows the chemical structural formula of three compounds determined by structural analysis that it was determined that the pharmacokinetics in Example 2 was favorable. It is a figure which shows the data at the time of determining the half life in the silkworm body fluid (blood) of the sodium fusidate which examined the pharmacokinetics in Test Example 1. FIG. It is a figure which shows the data at the time of determining the half life in the silkworm body fluid (blood) of vancomycin and chloramphenicol which show a therapeutic effect with respect to a mammal.

Claims (10)

  A test object is administered to a larvae of a completely transformed insect, and the compound in the test object or a substance produced by a chemical change in the body of the compound in the tested object Evaluate the pharmacokinetics in larvae and determine the quality of the pharmacokinetics in mammals of the compound in the test object or the substance produced by the chemical change in the test object A pharmacokinetic determination method characterized by: The following steps,
(A) a step of administering a test subject to a fully transformed insect larva,
(B) elapse of time;
(C) collecting a body fluid of the larvae of the fully transformed insect;
(D) identifying the substance in the body fluid collected in (c),
The method for determining pharmacokinetics according to claim 1. The following steps,
(X) identifying a compound in the test object;
(A) a step of administering a test subject to a fully transformed insect larva,
(B) elapse of time;
(C) collecting a body fluid of the larvae of the fully transformed insect;
(D) identifying the substance in the body fluid collected in (c),
(E) comparing the compound in the test object specified in (x) with the substance in the body fluid specified in (d);
The pharmacokinetics determination method of Claim 1 or Claim 2 containing this.   The pharmacokinetic determination method according to any one of claims 1 to 3, wherein the larva of the completely transformed insect is a silkworm.   The pharmacokinetic determination method according to claim 1, wherein the test object is a natural product.   A method for preparing a library of compounds with favorable pharmacokinetics in mammals, wherein the larvae of fully transformed insects are obtained by using the pharmacokinetic determination method according to any one of claims 1 to 5. Preparation of a library characterized by selecting a compound in the above test object that is stable in the body of the animal or stable in response to a chemical change in the body of a fully transformed insect larva Method. A method for preparing a library of compounds with favorable pharmacokinetics in mammals, comprising the following steps:
(X) identifying a compound in the test object;
(A) a step of administering a test subject to a fully transformed insect larva,
(B) elapse of time;
(C) collecting a body fluid of the larvae of the fully transformed insect;
(D) identifying the substance in the body fluid collected in (c),
(E) comparing the compound in the test object specified in (x) with the substance in the body fluid specified in (d);
(F) Among the compounds in the test object specified in (x), it is stable in the body of the fully transformed insect larvae or is stable by receiving a chemical change in the body of the completely transformed insect larva The method for producing a library according to claim 6, wherein a compound to be selected is selected as a component.   A method for preparing a library of substances having favorable pharmacokinetics in mammals, wherein a larva of a fully transformed insect is obtained by using the pharmacokinetic determination method according to any one of claims 1 to 5. A method for producing a library, comprising selecting a substance in the body fluid that is stably present in the body of the body as a component thereof.   A library of compounds having favorable pharmacokinetics in a mammal produced using the method for producing a library according to claim 6 or 7.   A library of substances having favorable pharmacokinetics in a mammal produced using the method for producing a library according to claim 8.

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