JP2010265239A - Method for purifying and concentrating antimalarial medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To newly invent an antibody having bonding abilities to a target peroxide derivative-type antimalarial agent and to provide a method for selectively concentrating and purifying the target peroxide derivative-type antimalarial agent by applying an antigen-antibody reaction utilizing the invented antibody. <P>SOLUTION: An antibody exhibiting cross-reactivity to the peroxide derivative-type antimalarial agent is selected from among antibodies against 3-(12-(2-carboxyethyl)-9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro-[5.8]tetradec-9-yl)-propionic acid having an analogous structure to the peroxide derivative as an antibody exhibiting bonding abilities to the target peroxide derivative-type antimalarial agent, for example, 3-(3-methyl-1,2,4,5-tetraoxa-spiro[5.5]undec-3-yl)-propionic acid ethyl ester, and then the target peroxide derivative is caused to selectively bond to the selected antibody via an antigen-antibody reaction thereby to concentrate and purify the target antimalarial agent. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for selectively concentrating and purifying a peroxide derivative using an antibody having an ability to bind to the peroxide derivative, which is used as an active ingredient of an antimalarial drug.

  Malaria is a protozoal infection in which Plasmodium spp. Is the pathogen. When infected with malaria, it presents symptoms such as high fever, headache, and nausea. If malignant, it may cause consciousness disorder or kidney failure, resulting in death. Since the infection of the pathogen Plasmodium spp. Is mediated by Anopheles spp., The infected area is widely distributed from the tropics to the subtropics where Anopheles lives.

  Human pathogens include P. falciparum, P. vivax, P. malariae, and Oval malaria (P. ovale). There are four types. In particular, malaria caused by Plasmodium falciparum has severe symptoms. With the expansion of the subtropical zone caused by global warming, the habitat of Anopheles larvae has expanded. As a result, malaria infected areas due to Plasmodium falciparum have expanded.

  In recent years, Chinhao drugs derived from Kampo medicines are said to have few side effects and drug resistance, and are widely used as first-line drugs for the treatment of malaria. The Chinhaos drug is a peroxide derivative represented by artemisinin, its derivatives and analogs, and having a dioxy bond (—O—O—) in the molecule. In addition, artemisinin has the following structure.

  The artemisinin-based antimalarial agent has a drawback in that the duration of drug efficacy is short. Therefore, in the treatment of falciparum malaria, it is used in combination with an artemisinin-based antimalarial agent and lumefantrin. Lumefunthrin has a half-life of 3 to 6 days. A treatment method for malaria that uses an artemisinin-based antimalarial agent in combination with another antimalarial agent is called ACT (artemisinin-based combination therapy).

  In addition, in the case of conventional peroxide-type antimalarial drugs, the dose is selected in a range where the blood concentration is a maximum of 20 μM (Non-patent Document 3).

Chemistry Letters, Vol.35, No.10, p.1126-1127 (2006) Organic & Biomolecular Chemistry Vol.4, p.4431-4436 (2006) Malaria Journal 2008, 7: 9

  On the other hand, new peroxide derivative type antimalarial agents are currently being developed for the purpose of prolonging the duration of drug efficacy. A new peroxide derivative-type antimalarial agent, which is being developed, is a chemically synthesized compound, and is required to be purified to a high purity when used as a pharmaceutical product. In particular, in order to reduce production costs, it is desired to develop a purification method capable of achieving high-purity purification of the target peroxide derivative-type antimalarial agent with fewer steps.

  Various chromatographic methods are used as means for purifying compounds used as pharmaceuticals. In particular, a technique of selectively concentrating and purifying a target compound by applying an affinity chromatography method showing high selectivity to the target compound is suitably used.

  For example, as a means for isolating and purifying various peptide compounds, when a specific antibody against the target peptide compound is available, the target peptide compound is bound to the specific antibody by an antigen-antibody reaction. Separation and purification methods are widely used. The complex formed between the antigenic compound and the specific antibody by the antigen-antibody reaction is in a dissociation equilibrium state. The binding between the antigenic compound and its specific antibody is an intermolecular bond, and the antigenic compound can be dissociated and recovered from the formed complex by acting a binding dissociator.

  Peroxide-derivative antimalarial agents are low molecular weight compounds and do not themselves exhibit immunogenicity, so antibodies specific for the subject peroxide-derivative antimalarial agents are generally not available, Affinity chromatography methods using antibodies have not been applied. If an antibody having a selective binding ability to the peroxide derivative-type antimalarial agent is available, the affinity chromatography method using the antibody having the selective binding ability is used for the peroxidation method. This is a technique for selectively concentrating and purifying a product derivative type antimalarial agent.

  In fact, the anti-malarial drug itself is a prerequisite that it must be a drug that can be administered repeatedly for a long period of time until the pathogenic malaria parasite in the infected body is completely removed. It is essential not to show primordiality. Therefore, production of a specific antibody against a peroxide derivative-type antimalarial agent that does not itself exhibit immunogenicity has not been made so far.

  The present invention solves the aforementioned problems. That is, an object of the present invention is to newly create an antibody having a binding ability to a target peroxide derivative-type antimalarial agent, and apply the antigen-antibody reaction using the created antibody. It is an object of the present invention to provide an affinity chromatography purification method capable of selectively concentrating and purifying the peroxide derivative-type antimalarial agent.

  The object of the present invention is, for example, to newly create an antibody capable of binding to a peroxide represented by the following formula (I) as a target peroxide derivative type antimalarial agent, The present invention provides an affinity chromatography purification method capable of selectively concentrating and purifying a peroxide derivative-type antimalarial agent represented by the formula (I) by utilizing an antigen-antibody reaction by utilizing the antigen-antibody reaction. There is.

  3- (3-Methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propionic acid ethyl ester (3- (3-methyl-1,2,4,5-tetraoxa- spiro [5.5] undec-3-yl) -propanoic acid ethyl ester):

  In order to solve the above-mentioned problems, the present inventors first studied a method for newly creating an antibody having a binding ability to a target peroxide derivative-type antimalarial agent.

  It has already been found that the subject peroxide-derivative antimalarial agents themselves are not immunogenic. On the other hand, even a low molecular weight compound having no immunogenicity may function as an immunogen when the low molecular weight compound is bound onto a carrier protein to form a modified protein modified with the low molecular weight compound. (Non-patent document 1: Chemistry Letters, Vol. 35, No. 10, p. 1126-1127 (2006)). There are many cases in which antibodies that show specific reactivity to low molecular weight compounds that do not have immunogenicity using this technique have been produced, but they are not effective against all low molecular weight compounds. That is, in the modified protein, whether or not the site to which the low molecular weight compound is bound actually shows immunogenicity depends on the three-dimensional structure of the site, and is effective for all low molecular weight compounds. It's not that.

  In fact, the present inventors also tried to immunize mice using, for example, a modified protein obtained by binding a carboxylic acid moiety constituting an ester compound of formula (I) onto a carrier protein. However, the present inventors have not succeeded in creating an antibody having reactivity with the carboxylic acid moiety.

  Furthermore, the present inventors have noted that in an antigen-antibody reaction, an antibody may exhibit a phenomenon that exhibits reactivity with a substance having a structure similar to the original antigen, so-called cross-reactivity. did. That is, in place of the target low molecular weight compound, when many kinds of specific antibodies against an antigen having a structure similar to the low molecular weight compound are created, Thus, it was conceived that there may be an antibody showing cross-reactivity.

  The inventors of the present invention actually have a characteristic structure in the ester compound of the formula (I), which is a carboxylic acid moiety, and among the many kinds of low molecular weight compounds having structural similarity with the carboxylic acid moiety. The inventors searched for an immunogenicity of the resulting modified protein when bound on a carrier protein. Next, among the many types of antibodies created by immunizing mice using the modified protein as an immunogen, there is an antibody that exhibits cross-reactivity with the ester compound of formula (I), A search was made for no. When the above two-step search process was carried out, fortunately, when the compound represented by the following formula (II) was bound on the carrier protein, the resulting modified protein showed immunogenicity, It has been found that antibodies exhibiting cross-reactivity exist among many types of antibodies created by immunizing mice using the modified protein as an immunogen.

  3- [12- (2-Carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl] -propionic acid (3- [ 12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl] -propanoic acid)

  Specifically, an antibody against a compound represented by the formula (II) created by immunizing a mouse using a modified protein obtained by binding a compound represented by the formula (II) on a carrier protein as an immunogen. Producing a group of hybridoma cell lines to produce, and selecting from this group of hybridoma cell lines several hybridoma cell lines that produce antibodies that exhibit cross-reactivity with the ester compound of formula (I) did it.

  The monoclonal antibodies produced by several of the selected hybridoma cell lines do not exhibit cross-reactivity with at least the endogenous substance of the mouse used as the immunized animal, but the compound represented by the formula (II) and the formula (I It was confirmed that the compound had reactivity to the ester compound. In addition, the inventors have found that the monoclonal antibody produced by several selected hybridoma cell lines forms a complex with at least the ester compound of formula (I), for example, a binding dissociator such as acetonitryl It was also confirmed that the ester compound of formula (I) can be dissociated from the complex by acting.

  In addition, when a mouse is immunized using the modified protein as an immunogen, multiple types of specific antibodies against the compound represented by formula (II) are present in the blood of the mouse. Plasma prepared from blood collected from this immunized mouse contains a specific polyclonal antibody against the compound represented by formula (II). It was also confirmed that a specific polyclonal antibody against the compound represented by the formula (II) also showed cross-reactivity with the ester compound of the formula (I).

  In addition to the series of findings described above, the present inventors include a raw material compound used in the process of synthesizing the ester compound of formula (I) in a specific polyclonal antibody against the compound represented by formula (II). In addition, it was also confirmed that no antibody showing binding ability to by-products was contained. In conclusion, in addition to the monoclonal antibodies produced by several selected hybridoma cell lines, a specific polyclonal antibody against the compound represented by the above formula (II) also selectively binds the ester compound of the formula (I), It was confirmed that it can be used for the purpose of concentration and isolation.

  Specifically, a monoclonal antibody produced by several selected hybridoma cell lines or a specific polyclonal antibody against a compound represented by the formula (II) collected from an immunized mouse is immobilized on a carrier, and an affinity chromatograph is obtained. -It was verified that an ester compound of formula (I) can be selectively concentrated and isolated by preparing a column agent and applying an affinity chromatograph column agent using the antibody.

  The present inventors have completed the present invention based on the above-described series of findings and verification results.

That is, a method for concentrating and purifying a peroxide derivative-type antimalarial drug according to the present invention is as follows.
Ester compound having the structure represented by the following formula (I) contained in the solution: ethyl 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propionate A method for concentrating and purifying an ester (3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propanoic acid ethyl ester),

A solution containing an ester compound having the structure represented by the formula (I) in an antibody-immobilized carrier obtained by immobilizing an antibody having a binding ability to the ester compound having the structure represented by the formula (I) on the surface of the carrier. Contacting the ester compound having the structure represented by the formula (I) with an antibody immobilized on the surface of the antibody-immobilized carrier by antigen-antibody reaction,
Separating the solution from the antibody-immobilized carrier;
After the separation step, the antibody-immobilized carrier is brought into contact with an eluate containing a binding dissociation agent, and is bound to the antibody immobilized on the surface of the antibody-immobilized carrier. Dissociating an ester compound having the structure shown by the action of the bond dissociator;
After the dissociation step, recovering the eluate containing an ester compound having a structure represented by the formula (I), which is transferred into the eluate containing the binding dissociator;
Separating and concentrating the ester compound having the structure represented by the formula (I) contained in the recovered eluate from the eluate, and concentrating and purifying the method. .

in particular,
An antibody having a binding ability to an ester compound having the structure represented by the formula (I) is:
An antibody against a low molecular weight compound having a structure similar to the characteristic structure in the ester compound having the structure represented by the formula (I), and cross-linked to the ester compound having the structure represented by the formula (I) It is preferable to have reactivity.

that time,
The low molecular weight compound having a structure similar to the characteristic structure in the ester compound having the structure represented by the formula (I) is a dicarboxylic acid compound having a structure represented by the following formula (II): 3- [12 -(2-Carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl] -propionic acid (3- [12- (2 -carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl] -propanoic acid).

In the above configuration,
In the first aspect of the present invention,
An antibody having a binding ability to an ester compound having the structure represented by the formula (I) is:
A modified protein obtained by binding a low molecular weight compound having a structure similar to the characteristic structure in the ester compound having the structure represented by the above formula (I) onto a carrier protein is used as an immunogen, and other than humans. A polyclonal antibody to the low molecular weight compound created by immunizing a mammal, and an antibody having cross-reactivity to the ester compound having the structure represented by the formula (I).

In the second embodiment of the present invention,
An antibody having a binding ability to an ester compound having the structure represented by the formula (I) is:
A modified protein obtained by binding a low molecular weight compound having a structure similar to the characteristic structure in the ester compound having the structure represented by the above formula (I) onto a carrier protein is used as an immunogen, and other than humans. A monoclonal antibody to the low molecular weight compound created by immunizing a mammal, and an antibody having cross-reactivity to the ester compound having the structure represented by the formula (I).

In the first form of the present invention, and in the second form,
The non-human mammal is preferably a mouse.

  Further, in a modified protein obtained by binding the low molecular compound on a carrier protein, it is preferable to select Keyhole Limpet Hemocyanin as the carrier protein.

In the first form of the present invention, and in the second form,
A compound having a carboxyl group (—COOH) in the molecule is selected as a low molecular weight compound having a structure similar to the characteristic structure of the ester compound having the structure represented by the formula (I),
A modified protein obtained by binding a compound having a carboxyl group (—COOH) in the molecule onto a carrier protein comprises the carboxyl group (—COOH) and an amino group (—NH ₂ ) on the carrier protein. It is preferable that a compound having a carboxyl group (—COOH) in the molecule is bonded via an amide bond (—CO—NH—).

At that time, an amide bond (—CO—NH—) is formed between the carboxyl group (—COOH) and the amino group (—NH ₂ ) on the carrier protein using a carbodiimide method. It is preferable.

In the concentration and purification method according to the present invention,
As an eluent containing the bond dissociator,
It is desirable to use an aqueous solution containing a hydrophilic organic solvent capable of dissolving the ester compound having the structure represented by the formula (I) as the bond dissociator.

  For example, acetonitrile can be selected as a hydrophilic organic solvent that can dissolve the ester compound having the structure represented by the formula (I).

On the other hand, an antibody-immobilized carrier obtained by immobilizing an antibody having a binding ability to an ester compound having the structure represented by the formula (I) on the surface of a carrier,
The base material of the carrier is silica gel, and the surface of the silica gel base material is modified with protein A or protein G.
The antibody can be immobilized on the carrier surface in a form that is achieved by immobilizing the antibody molecule with protein A or protein G that is modifying the surface of the silica gel substrate.

In the second embodiment of the present invention,
As an antibody having a binding ability to an ester compound having the structure represented by the formula (I),
It is preferable to use a monoclonal antibody against a dicarboxylic acid compound having a structure represented by the above formula (II) produced by a hybridoma cell line: NECP-C57Z 15B-1E (FERM ABP-11126).

  In the method for concentrating and purifying a peroxide derivative type antimalarial drug according to the present invention, a monoclonal antibody against a low molecular weight compound having a structure having a characteristic structure and similarity to the peroxide derivative type antimalarial drug, Alternatively, using a polyclonal antibody, the monoclonal antibody or the cross-reactivity of the polyclonal antibody to the peroxide derivative type antimalarial drug, the antigen derivative reaction of the target peroxide derivative type Antimalarial drugs can be selectively combined with antibodies, concentrated and purified. For example, if the subject peroxide derivative type antimalarial drug is 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propionic acid ethyl ester (3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propanoic acid ethyl ester), it has a structure with similarity to the ester compound. Molecular compound: 3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl] -propionic acid ( 3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl] -propanoic acid) monoclonal antibody, Alternatively, using a polyclonal antibody, the monoclonal antibody or the cross-reactivity of the polyclonal antibody to the peroxide derivative type antimalarial drug, the antigen derivative reaction of the target peroxide derivative type Antimalarial The drug can be selectively bound to the antibody, concentrated and purified. In particular, by applying the method for concentrating and purifying a peroxide derivative type antimalarial drug according to the present invention, high purity purification of the target peroxide derivative type antimalarial drug produced by chemical synthesis is performed. It becomes possible to perform with good reproducibility.

3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa which can be used in the concentration and purification method according to the first embodiment of the present invention -Spiro- [5.8] tetradec-9-yl] -propionic acid (3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [ 5.8] tetradec-9-yl] -propanoic acid), a peroxide derivative antimalarial drug of interest, 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] Cross-reactivity to undec-3-yl) -propionic acid ethyl ester (3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propanoic acid ethyl ester) It is a graph which shows the result evaluated by ELISA method. 3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa which can be used in the concentration and purification method according to the second embodiment of the present invention -Spiro- [5.8] tetradec-9-yl] -propionic acid (3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [ 5.8] tetradec-9-yl] -propanoic acid), a target peroxide derivative antimalarial drug, 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] Cross-reactivity to undec-3-yl) -propionic acid ethyl ester (3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propanoic acid ethyl ester) It is a graph which shows the result evaluated by ELISA method.

  Hereinafter, a method for concentrating and purifying a peroxide derivative type antimalarial drug according to the present invention will be described in detail.

  First, the antibody having the ability to bind to a peroxide derivative antimalarial drug used in the method for concentrating and purifying a peroxide derivative antimalarial drug according to the present invention, and a production method thereof will be described in more detail. To do.

  As a means of creating antibodies against low molecular weight organic compounds, if the target low molecular weight organic compound itself does not show immunogenicity, the modified carrier obtained by binding the target low molecular weight organic compound on the carrier protein There is a technique of using a protein as an immunogen (Non-patent Document 1: Chemistry Letters, Vol. 35, No. 10, p. 1126-1127 (2006)).

Specifically, a low molecular weight organic compound has a reactive functional group such as an amino group (—NH ₂ ), a hydroxyl group (—OH), a sulfanyl group (—SH), or a carboxyl group (—COOH). In such a case, the reactive functional group can be used to covalently link organic compounds having other reactive functional groups. The carrier protein has a three-dimensional structure composed of a peptide chain in which a plurality of amino acid residues are linked, but on its surface, an amino acid residue having a reactive functional group on the side chain There are several. Therefore, it has a low molecular weight that has a reactive functional group by utilizing a reactive functional group on the side chain of an amino acid residue that has a three-dimensional structure and exists on the surface of the carrier protein. It is possible to bind organic compounds. The modified carrier protein, which is modified on the surface by a low molecular weight organic compound, is a non-natural protein molecule and is recognized as a foreign substance that is different from the endogenous protein molecule of the mammal itself. Is frequent. In particular, a site that has been modified due to a low molecular weight organic compound is highly immunogenic. When the modified carrier protein surface modified with a low molecular weight organic compound exhibits immunogenicity, when the mammal is immunized with the modified carrier protein, the modified carrier Specific antibodies against proteins are created.

  There may be a plurality of sites (antigenic determinants) that exert immunogenicity on the surface of the modified carrier protein. In that case, multiple types of antibodies specific to each of the plurality of immunogenic sites (antigenic determinants) are created. Among the multiple types of antibodies that are specific to modified carriers and proteins that have been created, there are antibodies that use the low molecular weight organic compounds themselves used for their modification as sites that exhibit immunogenicity (antigenic determinants) The frequency of doing is high. By screening based on the binding ability to the low molecular weight organic compound itself used for modification, an antibody that uses the low molecular weight organic compound itself used for modification as an immunogenic site (antigenic determinant) It is possible to sort.

  However, if the mammal to be immunized already has an antibody that exhibits high cross-reactivity with the antigenic determinant present on the surface of the modified carrier protein, the antigenic determination that exhibits this cross-reactivity is determined. Creation of new antibodies against the group does not occur. That is, when an immune reaction to the modified carrier protein is possible using the high cross-reactivity exhibited by the antibody already held by the mammal to be immunized, the antigen determinant exhibiting this cross-reactivity, Creation of new antibodies does not occur.

  Furthermore, even when the modified site functions as a site that exhibits immunogenicity (antigenic determinant), the antibody specific for the antigenic determinant has a low molecular weight used for the modification. There are many cases where the binding ability to the organic compound itself is not high.

  In other words, using the technique of binding the target low molecular weight organic compound on the carrier protein and using the resulting modified carrier protein as an immunogen, it is specific to the low molecular weight organic compound itself used for modification. Whether or not a specific antibody can be created depends on the following factors. Specifically, it depends on the three-dimensional structure of the target low molecular weight organic compound itself, the combination with the carrier protein to be used, the binding form on the carrier protein, the selection of the modification site, and the above four factors. is doing.

  In fact, since the three-dimensional structure of the target low molecular weight organic compound itself has already been determined, whether or not an appropriate combination can be selected for the remaining three factors depends largely on chance. . In other words, the binding form on the carrier protein and its modification site depend on the type of carrier protein used, and are limited by the type of reactive functional group possessed by the target low molecular weight organic compound. The Depending on the three-dimensional structure of the target low molecular weight organic compound itself, an appropriate combination may not be selected for the remaining three factors.

  On the other hand, when the peroxide derivative-type antimalarial drug itself does not have a reactive functional group in the molecule, the target low molecular weight organic compound is bound to the carrier protein. Therefore, it is not possible to apply a method of using the obtained modified carrier protein as an immunogen. Of course, the peroxide-derivative antimalarial drug is a low molecular weight organic compound and does not itself exhibit immunogenicity.

  Therefore, in the present invention, a large number of antibodies against low molecular weight compounds having a structure similar to the characteristic structure and similarity of the target peroxide derivative type antimalarial drug are created, and the structure has the similarities. Among many antibodies against low molecular weight compounds, a method of selecting an antibody having cross-reactivity with a target peroxide derivative type antimalarial drug is employed.

  The following is an ester compound having the structure represented by the following formula (I): 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] as a target peroxide derivative type antimalarial drug. The method for producing the antibody used in the present invention will be described more specifically by taking, as an example, undec-3-yl) -propionic acid ethyl ester.

  The structural feature of the ester compound represented by formula (I) is its spiro ring structure itself. Among the low-molecular-weight peroxide compounds having the spiro ring structure and having a reactive functional group in the molecule, the toxicity to mammals is not high, and when bound on a carrier protein, We searched for those that could maintain the spiro ring structure.

  As a result of the search, a dicarboxylic acid compound having a structure represented by the following formula (II) among a group of low molecular weight peroxide type compounds that are being investigated for use as antimalarial drugs having low toxicity: 3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl] -propionic acid (3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl] -propanoic acid) as a candidate satisfying the above requirements ,chosen.

In the present invention, a reactive functional group present on the surface of a carrier protein is utilized by utilizing a carboxyl group (—COOH), which is a reactive functional group present in the molecule of a dicarboxylic acid compound having the structure represented by formula (II). It is preferable to select a form to be bonded to the surface of the carrier protein by forming an amide bond (—CO—NH—) with a group, particularly an amino group (—NH ₂ ).

  At that time, the carrier protein is a method of binding the target low molecular weight organic compound on the carrier protein and using the resulting modified carrier protein as an immunogen (Non-patent Document 1: Chemistry Letters, Vol. 35, No. 10, p. 1126-1127 (2006)), various carrier proteins already used can be used. As the carrier protein, bovine serum albumin, bovine thyroglobulin, Keyhole Limpet Hemocyanin and the like can be suitably used.

  When a target low molecular weight organic compound is bound on a carrier protein and the resulting modified carrier protein is used as an immunogen, a specific antibody against the modified carrier protein is used in an immunized mammal. Is created. In this case, since the carrier protein itself to be used generally has immunogenicity, in addition to the antibody specific to the modified site in the modified carrier protein, it is specific to the antigenic determinant of the carrier protein itself. New antibodies are also created.

  Considering this point, it is preferable that the mixing ratio of unmodified carrier / protein is low with respect to the modified carrier / protein used for immunization. Of course, it is more preferable to use a modified carrier protein that does not contain unmodified carrier protein.

  On the other hand, on the carrier protein to be used, there are generally a plurality of sites (modifiable sites) where the target low molecular weight organic compound can be bound and modified. Each of these modifiable sites has a reactive functional group, but generally there is a difference in reactivity. Therefore, the binding of the low molecular weight organic compound of interest proceeds preferentially from the highly reactive modifiable site, and the low molecular weight organic compound of interest is consumed. , The efficiency with which the low molecular weight organic compound of interest is bound tends to be further reduced. In order to achieve binding of the low molecular weight organic compound of interest to all of the plurality of modifiable sites present on the carrier protein, the amount of the low molecular weight organic compound of interest used in the reaction must be multiple. It is desirable to select a considerably excessive amount relative to the total number of modifiable sites. For example, when there are N modifiable sites on the carrier protein, the amount of the low molecular weight organic compound to be used for the reaction requires a minimum of N molecules per carrier protein molecule. However, it is desirable to select at least 50 molecules or more, preferably 60 molecules or more.

  The immunization can be performed by, for example, administering a solution containing an effective amount of a modified carrier protein combined with a low molecular weight organic compound of the subject to a mammal to be immunized by injection. desirable. As the administration form by injection, subcutaneous injection, intradermal injection, intravenous injection, or intraperitoneal administration can be used. The solution is usually administered by subcutaneous injection. At this time, it is preferable to add various adjuvants to a solution containing an effective amount of the modified carrier protein. Usually, an adjuvant that has been conventionally used for immunization can be used as the adjuvant. Available adjuvants include Freund's complete adjuvant, water-in-oil-in-water emulsion, oil-in-water emulsion, liposome, aluminum hydroxide gel, and silica adjuvant. Freund's complete adjuvant is widely used and can be suitably used in the present invention. For example, during the first immunization (sensitization), it is preferable to use Freund's complete adjuvant as the adjuvant.

  In the immunization, booster immunization is performed when a predetermined period has elapsed after the first immunization (sensitization). In this additional immunization, it is preferable to add various adjuvants to a solution containing an effective amount of the modified carrier protein. For example, it is preferable to use Freund's complete adjuvant as the adjuvant at the time of booster immunization, but a considerable effect can be obtained by using Freund's incomplete adjuvant.

  After the first immunization operation (sensitization), the booster immunization to be performed is preferably performed a plurality of times. It is desirable to perform booster immunization when the antibody concentration in the blood shows a maximum due to the immune response to the previous immunization operation (sensitization) and the antibody concentration is decreasing. The number of days until the antibody concentration in the blood reaches the maximum after the last immunization (sensitization) usually depends on the metabolic rate in the body of the immunogen used. Thus, the booster interval depends on the type of immunogen used, the type of immunized animal of interest, and its health condition. When a small animal such as a mouse is used as an immunized animal, a form in which booster immunization is performed after the first immunization (sensitization), for example, 2 weeks, 4 weeks, 6 weeks, or 8 weeks can be selected.

  Scientifically, the type of mammal to be immunized is not limited, but is selected from mammals other than humans from an ethical viewpoint. When creating hybridoma cells that produce monoclonal antibodies, mice, rats, goats, and the like can be selected as mammals other than humans that can be used for mammals to be immunized.

  As a mammal to be immunized, a mammal that already holds an antibody showing cross-reactivity with the modified carrier protein is not preferable. That is, it is generally preferable that the mammal to be immunized is an individual who has no acquired immunity. In view of the above requirements, it is preferable to use mammals grown after birth in an environment that is essentially free from exposure to various immunogenic substances. Alternatively, it is preferable to use a mammal having a short period until it grows to the extent that it can be subjected to immunization after giving birth. In consideration of these conditions, it is more preferable to use small pedigree mammals used for medical research. Specifically, mice, rats, rabbits and the like that are used for the creation of various novel antibodies are preferable, and in particular, mice or rats, and more preferably mice are used.

  Prior to immunization, the non-human mammal used as an immunized animal is used to produce the modified carrier protein, the immunogenicity of the carrier protein itself, and a specific antibody against the carrier protein It is desirable to investigate in advance the immunization conditions for which is created. A method of binding a target low molecular weight organic compound on the above carrier protein and using the resulting modified carrier protein as an immunogen (Non-patent Document 1: Chemistry Letters, Vol. 35, No. 10, p. 1126-1127 (2006)), regarding the various carrier proteins already used, the above matters have already been described for mice, rats, rabbits, etc., which are used for the creation of various novel antibodies. It has been investigated and reports are available.

  In addition, a method of binding a target low molecular weight organic compound on the carrier protein and using the resulting modified carrier protein as an immunogen (Non-patent Document 1: Chemistry Letters, Vol. 35, No. 10, p.1126-1127 (2006)), referring to the successful examples already reported, the immunization of modified carrier proteins for mice, rats, rabbits, etc., which are used for the creation of various new antibodies It is also possible to estimate the effective amount with considerable accuracy.

  It is desirable to select the procedure of immunization for mice, rats, rabbits, etc., which are used to create various novel antibodies, in accordance with the procedures used in the successful examples already reported.

  When a mouse or rat is selected as the mammal to be immunized, the age at which the first immunization operation (sensitization) is performed is selected in consideration of the number of subsequent immunizations and the interval thereof. Specifically, it is necessary to verify that an antibody specific for the immunogen is present in the blood of the immunized animal after completion of multiple boosters. Therefore, it is preferable to select an age at which the first immunization operation (sensitization) is performed so that the immunized animal does not reach an age at which the ability to produce antibodies decreases at the time of completing multiple boosters. . When the period until the end of multiple boosts after the first immunization (sensitization) is selected to be about 8 weeks, in mice or rats, the age at which the first immunization (sensitization) is performed is: It is preferable to select the range of 10 to 15 weeks of age, and it is usually more preferable to select the range of about 12 weeks of age. It is known that mice or rats have the ability to produce sufficient antibodies when they reach about 12 weeks of age, and the ability to create new antibodies is the highest.

  The dicarboxylic acid compound having the structure represented by the formula (II) is a known compound, and its synthesis method has already been reported in the literature (Non-patent Document 2: Organic & Biomolecular Chemistry Vol. 4, p.4431- 4436 (2006)). Since the dicarboxylic acid compound having the structure represented by the formula (II) is solid at room temperature, the reaction for binding the compound on the carrier protein is performed using a reaction solvent capable of dissolving the compound. There is a need. On the other hand, the carrier protein used may undergo denaturation depending on the type of solvent. Therefore, it is necessary to select a reaction solvent capable of dissolving the compound without causing denaturation of the carrier protein used.

Conventionally, dicarboxylic acids having a structure represented by the above formula (II) from various reaction solvents used in preparing modified carrier protein by binding a target low molecular weight organic compound on carrier protein. It is preferable to select a reaction solvent capable of dissolving the acid compound. In fact, when the selection of the reaction solvent was advanced, dimethyl sulfone (DMSO: (CH ₃ ) ₂ SO) and a borate buffer were selected as particularly preferable reaction solvents.

Dimethylsulfone (DMSO: (CH ₃ ) ₂ SO) is a non-aqueous solvent, but can be dissolved without denaturing the carrier protein, and is equivalent to a dicarboxylic acid compound having the structure shown in formula (II) It is a solvent that can be dissolved at a high concentration.

  Further, the boric acid buffer solution has a pH range in which the buffering action can be exerted in the range of 6.8 to 9.2, and is a solvent capable of dissolving the dicarboxylic acid compound having the structure represented by the above formula (II). As the system, it is usually preferable to select a composition in which the pH is selected in the range of 8.2 to 8.7, particularly a composition in which the pH can be adjusted to around 8.5.

Reactive functional groups present on the surface of the carrier protein using a carboxyl group (—COOH), which is a reactive functional group present in the molecule of the dicarboxylic acid compound having the structure represented by the formula (II), In the case of forming an amide bond (—CO—NH—) with an amino group (—NH ₂ ), it is preferable to use an amide bond forming method using carbodiimide. In amide bond formation using carbodiimide, N, N′-dicyclohexylcarbodiimide (DCC), N, N′-diisopropylcarbodiimide (DIC), N- [3- (dimethylamino) propyl] -N ′ is used as a binder carbodiimide. -Ethylcarbodiimide (EDC), N- [3- (dimethylamino) propyl] -N'-ethylcarbodiimide hydrochloride (EDAC), etc. can be utilized. The amount of the binder carbodiimide is preferably selected in the range of 5 to 20 molecules per molecule of the dicarboxylic acid compound having the structure represented by the formula (II).

On the other hand, the amount of the dicarboxylic acid compound having the structure represented by the formula (II) is determined based on the total number of amino groups (—NH ₂ ) exposed on the surface of the carrier protein. At that time, when the total number of amino groups (—NH ₂ ) exposed on the surface of one molecule of the carrier protein is N, the amount of the dicarboxylic acid compound having the structure represented by the formula (II) is used. It is preferable to select in the range of N × 3 molecules to N × 10 molecules per protein molecule. As a result, a modified carrier protein is prepared in which a dicarboxylic acid compound having a structure represented by the formula (II) is bound in a range of 1/2 × N molecule to N molecule per molecule of the carrier protein. It is desirable to do.

  In the present invention, a modified carrier protein obtained by binding a target low molecular weight organic compound on a carrier protein is used as an immunogen to be used for the above-described immunization. Among the multiple types of antibodies that are newly created by immunization and specific for the modified carrier protein, it is also high against the low molecular weight organic compound itself that is not bound to the carrier protein used. First, it is verified that there is actually an antibody showing reactivity.

  After completion of the above-mentioned last round of booster immunization, when a significant increase in the blood concentration of an antibody specific for the modified carrier protein used as an immunogen is found, blood is collected from the immunized animal and collected. Antiserum is prepared from the collected blood. Among the multiple antibodies specific to the modified carrier protein contained in the antiserum, high reactivity with the low molecular weight organic compound itself that is not bound to the carrier protein used It is verified that an antibody showing is actually present.

  That is, the presence or absence of a polyclonal antibody having the low molecular weight organic compound itself as an antigenic determinant is verified. An enzyme immunoassay (ELISA method) is preferably used as a means for verifying the presence of an antibody that specifically binds to a specific antigenic determinant in antisera containing multiple types of antibodies. The The enzyme immunoassay (ELISA method) uses a specific reactivity of an antibody to a specific antigenic determinant, and therefore has high selectivity, particularly for a specific antigenic determinant contained in the antiserum. When the antibody concentration is unknown, the antibody titer can be easily evaluated.

  In the present invention, in order to detect an antibody that is not bound to the carrier protein used and shows high reactivity with the low molecular weight organic compound itself, it is used in an enzyme immunoassay (ELISA method). As an antigen, another kind of modified carrier protein in which the low molecular weight organic compound is bound to the surface of another kind of carrier protein is used. Of course, it is necessary that the different type of carrier protein itself does not react with a plurality of types of antibodies specific to the modified carrier protein.

  The carrier protein used for the preparation of the immunogen and the other kind of carrier protein used for the preparation of the antigen used in the enzyme immunoassay (ELISA method) are preferably used for the preparation of the immunogen. It is preferable to select a combination of two different carrier proteins from the group of carrier proteins.

  In the carrier protein combination, antigenic determinants of the carrier protein itself are usually different, and multiple types of antibodies specific to the modified carrier protein react with the different carrier protein itself. The possibility of showing sex can be eliminated. Further, in the combination of the two different carrier proteins described above, it is very unlikely that the local structure (partial amino acid sequence) of the site capable of binding the low molecular weight organic compound substantially matches. Therefore, in the above-mentioned combination, another type of modified carrier protein in which the low molecular weight organic compound is bound to the surface of another type of carrier protein among a plurality of types of antibodies specific to the modified carrier protein of the immunogen. An antibody that exhibits binding ability to can be regarded as an antibody that binds to the low molecular weight organic compound itself.

  In particular, bovine serum albumin, which is widely used as a blocking protein, is selected as another type of carrier protein used for the preparation of the antigen used in the enzyme immunoassay (ELISA method), while the production of the immunogen It is more preferable to select a general-purpose carrier protein other than bovine serum albumin, such as Keyhole Limpet Hemocyanin, as the carrier protein used in the above. When bovine serum albumin is selected as another type of carrier protein used for the production of a modified carrier / protein type antigen used in the enzyme immunoassay (ELISA method), the modified carrier / protein type antigen The phenomenon of non-selective binding of antibody molecules to the bovine serum albumin moiety is also eliminated. Further, the modified carrier protein type antigen using bovine serum albumin as a carrier protein can be immobilized on an ELISA plate at a high density.

  After verifying that the obtained antiserum contains an antibody having reactivity to the low molecular weight organic compound itself, which is used to produce an immunogen, the reactivity to the low molecular weight organic compound itself It is verified whether a polyclonal antibody having a cross-reactivity with a target peroxide derivative-type antimalarial drug, for example, an ester compound (TATP2) having a structure represented by the formula (I) itself To do.

  In the present invention, the verification of the cross-reactivity of the antibody preferably uses a competitive reaction of the two antigens with the antibody.

  A target peroxide derivative type antimalarial drug, for example, an ester compound (TATP2) itself having a structure represented by the formula (I) is a low molecular weight organic compound, and when performing an antigen-antibody reaction with an antibody, That binding is believed to be achieved by one of the complementarity determining sites of the antibody molecule. In addition, when a low molecular weight organic compound having a similar structure used for the production of a modified carrier / protein used for the above-described immunization is also subjected to an antigen-antibody reaction with an antibody, its binding is caused by an antibody molecule. It is thought that this is achieved by one of the complementarity determining sites.

  Therefore, when an antibody has cross-reactivity, the complementarity determining site of the antibody molecule involved in binding to a low molecular weight organic compound having a similar structure, which is used to produce a modified carrier protein of an immunogen And the target peroxide derivative type antimalarial drug, for example, the ester compound (TATP2) represented by the formula (I) itself, may be coincident with the complementarity determining site of the antibody molecule. Extremely high. In that case, when a target peroxide derivative-type antimalarial drug, for example, an ester compound (TATP2) represented by the formula (I) is bound to the complementarity determining site of the antibody molecule, the modified carrier protein of the immunogen It inhibits the binding of low molecular weight organic compounds having a similar structure that are used in the production of. By utilizing this phenomenon of competitive inhibition, it is possible to verify whether or not a specific complementarity determining site of the antibody molecule exhibits cross-reactivity.

  Specifically, the low molecular weight compound having the similar structure used for the verification of the reactivity to the low molecular weight organic compound itself having a similar structure, which is used for the production of the modified carrier / protein of the above immunogen. Another type of modified carrier protein having an organic compound bound to the surface of another type of carrier protein is immobilized on an ELISA plate. On the other hand, the target peroxide derivative-type antimalarial drug, for example, the ester compound (TATP2) represented by the formula (I) contains a polyclonal antibody in the reaction solution for carrying out the antigen-antibody reaction in the ELISA method. Dissolve with antiserum.

  When the above competitive reaction proceeds, the cross-reactive antibody present in the reaction solution is a target peroxide derivative-type antimalarial drug, for example, an ester compound (TATP2) represented by the formula (I) As a result of the antigen-antibody reaction with the antibody, the amount of antibody molecules immobilized is reduced through the antigen-antibody reaction with the modified carrier / protein type antigen immobilized on the plate. Due to this competitive reaction, the decrease in the amount of the immobilized antibody through the antigen-antibody reaction with the modified carrier protein type antigen immobilized on the plate is determined by enzyme immunoassay (ELISA method). Detect by applying.

  By using this technique, the polyclonal antibody against the low molecular weight organic compound itself having a similar structure used in the production of the modified carrier protein of the above-mentioned immunogen contained in the antiserum, It is possible to verify that an antimalarial agent of a peroxide derivative type, for example, an antibody showing cross-reactivity with the ester compound (TATP2) represented by the formula (I) is included.

  In other words, the antiserum verified as described above comprises a target peroxide derivative-type antimalarial drug, for example, a polyclonal antibody having binding ability to an ester compound (TATP2) represented by the formula (I).

  Antibodies of immunized animals that have been verified to produce cross-reactive antibodies with the target peroxide derivative type antimalarial drug, for example, the ester compound (TATP2) represented by the formula (I) A production cell group is collected, and this antibody-producing cell group is fused with a cell of a cell line derived from myeloma to produce a group of hybridoma cells.

  Usually, a spleen cell group is prepared by removing the spleen from the immunized animal that has been verified as described above. This spleen cell group and myeloma-derived cell line cells are fused to produce a group of hybridoma cells.

  The myeloma-derived cell line used for the above-mentioned cell fusion needs to be compatible with the spleen cell derived from the immunized animal to be fused. The proliferation ability of hybridoma cells created by cell fusion depends on the myeloma-derived cell line used for cell fusion, and it is preferable to use a myeloma-derived cell line with excellent proliferation ability. .

  For example, when a mouse is selected as the immunized animal, the myeloma-derived cell line used for cell fusion is a mouse myeloma-derived cell line such as P3X63 Ag8.653, P3X63Ag8U, Sp2 / O Ag14, FO.1, S194 / 5. XX0 BU. 1 or the like is preferably used. In particular, the use of the cell line P3X63Ag8U has a high proliferation ability of the hybridoma cells to be created, and the antibody molecules produced by the hybridoma cells are all antibodies that have been properly assembled, and antibodies that have not been assembled yet More preferred because it does not contain molecular fragments.

  For example, when a rat is selected as the immunized animal, the myeloma-derived cell line used for cell fusion is the rat myeloma-derived cell line 210, RCY3. Ag1.2.3, YB2 / 0, etc. are mentioned.

  Examples of the cell fusion technique for creating the hybridoma cell include a polyethylene glycol method, a method using Sendai virus, and a method using an electric current. The polyethylene glycol method is more suitable for the present invention because it has low cytotoxicity, is easy to fuse, and particularly has high reproducibility. That is, in the present invention, among a group of hybridoma cells that produce a specific monoclonal antibody against a modified carrier protein of an immunogen, a target peroxide derivative-type antimalarial drug, for example, formula (I) It is necessary to select hybridoma cells that produce a monoclonal antibody having cross-reactivity with the ester compound (TATP2) shown. Since the frequency of inclusion of hybridoma cells producing the above-mentioned monoclonal antibody having cross-reactivity among a group of hybridoma cells to be created is never high, the number of cell lines (number of populations) of a group of hybridoma cells to be screened Need to be larger. Therefore, it is preferable to select a cell fusion method with higher reproducibility, and the polyethylene glycol method meets the above-mentioned requirements.

  The created group of hybridoma cells are dispersed, dispensed into a microplate, and grown under known culture conditions appropriately selected according to the utilized myeloma cell line. For a group of hybridoma cell lines established by the culture described above, for the monoclonal antibodies produced by each hybridoma cell line, the desired peroxide derivative type antimalarial drug, for example, formula (I) It is verified whether the antibody shows cross-reactivity with the ester compound (TATP2) shown.

  For a group of hybridoma cell lines established by culture, the culture supernatant of each hybridoma cell line is collected. The culture supernatant of each hybridoma cell line contains a monoclonal antibody produced by the cell line.

  Monoclonal antibodies contained in the culture supernatant of each hybridoma cell line have reactivity to low molecular weight organic compounds themselves with similar structures used for the production of modified carrier proteins used for immunization. First, it is verified whether it has or not.

  The verification is based on an enzyme immunoassay (ELISA method) in which a low molecular weight organic compound having a similar structure is bound to the surface of another type of carrier protein and another type of modified carrier protein is used as an antigen. Verification methods can be used. The specific measurement method is in principle the same as the verification regarding the reactivity of the polyclonal antibody contained in the antiserum.

  By this verification, a monoclonal antibody having reactivity to a low molecular weight organic compound itself having a similar structure, used for production of a modified carrier protein used for immunization, from a group of hybridoma cell lines. A cell line of hybridoma cells that produces is selected. With respect to the group of cell lines of hybridoma cells selected in this primary screening, the monoclonal antibody produced by the cell line of hybridoma cells is the target peroxide derivative type antimalarial drug, that is, the formula (I) It is verified whether or not the antibody shows cross-reactivity to the ester compound (TATP2) shown.

  The verification regarding the cross-reactivity can be performed by applying the same method in principle as the verification regarding the cross-reactivity of the polyclonal antibody contained in the antiserum.

  By verifying the cross-reactivity with the target peroxide derivative type antimalarial drug, that is, the ester compound (TATP2) represented by the formula (I) (secondary screening), the target peroxide derivative type antimalarial drug A cell line of a hybridoma cell that produces a monoclonal antibody exhibiting cross-reactivity with a malaria drug, ie, an ester compound (TATP2) represented by the formula (I) is selected. The type of monoclonal antibody selected depends on the antibody type specificity exhibited by the anti-Ig antibody used in the enzyme immunoassay (ELISA method).

  By using the cell line of the hybridoma cell selected by this secondary screening, it has a binding ability to a target peroxide derivative type antimalarial drug, for example, an ester compound (TATP2) represented by the formula (I) Monoclonal antibodies can be produced.

  The selected hybridoma cell line can be cultured in vitro, the culture supernatant can be collected, and the contained monoclonal antibody can be purified. Further, when the selected hybridoma cell line is inoculated into the abdominal cavity of a mammal other than a human used for immunization, it grows in the abdominal cavity and accumulates the monoclonal antibody produced in the ascites. Thereafter, the ascites can be collected and the contained monoclonal antibody can be purified.

  For purification of monoclonal antibodies contained in ascites or culture supernatant, for example, DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method, etc. Purification to a purity of Desirable purity is 95% or more, more preferably 98% or more. For example, a target peroxide derivative-type antimalarial drug, for example, a monoclonal antibody having a binding ability to the ester compound (TATP2) represented by the formula (I) is concentrated in the peroxide derivative-type antimalarial drug, When used for purification, if an antibody having reactivity to contaminants is mixed in, it becomes a factor for reducing the concentration efficiency and purification purity. Considering this point, it is desirable to carry out purification to the above purity.

  In addition, an antibody having binding ability to the peroxide derivative type antimalarial drug according to the present invention, for example, an antibody having binding ability to the ester compound (TATP2) represented by the above formula (I), particularly the formula (II) In the antibody having the cross-reactivity to the ester compound (TATP2) represented by the formula (I) selected from the antibodies to the dicarboxylic acid compound (TATP3) represented by the following, the cross-reactivity to the peroxide of the following formula (III) That do not substantially exhibit the

  That is, an antibody that exhibits cross-reactivity with the peroxide of the formula (III) is characterized by the ester compound (TATP2) represented by the formula (I) and the dicarboxylic acid compound (TATP3) represented by the formula (II). May not have specific reactivity to a spiro ring structure. When the antibody having the binding ability to the ester compound (TATP2) represented by the formula (I) is used for the purpose of selectively detecting the ester compound (TATP2) represented by the formula (I), it is specific to the spiro ring structure. It is necessary to have high reactivity. From this point of view, an antibody that exhibits cross-reactivity with the peroxide of formula (III) is not necessarily an ester compound (TATP2) represented by formula (I) and a dicarboxylic acid compound (TATP3) represented by formula (II). ), Which cannot be judged as having a specific reactivity to the spiro ring structure.

  The fact that the cross-reactivity to the peroxide of the formula (III) is not substantially shown means that the cross-reactivity to the ester compound (TATP2) represented by the formula (I) is used as a reference to the peroxide of the formula (III). It means that the cross-reactivity is 1/20 or less. More preferably, the cross-reactivity to the peroxide of formula (III) is 1/100 or less based on the cross-reactivity to the ester compound (TATP2) represented by formula (I).

(Trust number)
In addition,
As a hybridoma cell producing a monoclonal antibody having a binding ability to the ester compound (TATP2) represented by the formula (I) used in the present invention,
Hybridoma cell line: NECP-C57Z 15B-1E is based on the Budapest Treaty. National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center In addition, an international deposit (May 12, 2009) has been made.

  The hybridoma cell line is a hybridoma cell line created and selected by the procedure disclosed in the second embodiment to be described later. The hybridoma cell line: NECP-C57Z 15B-1E is a hybridoma cell line that produces the monoclonal antibody mAb-m004 described below.

  In the present invention, it is preferable to use an affinity chromatography concentration and purification method having the following constitution by utilizing a monoclonal antibody having binding ability to the ester compound (TATP2) represented by the formula (I).

The method for concentrating and purifying a peroxide derivative type antimalarial drug according to the present invention is a method for concentrating and purifying a target peroxide derivative type antimalarial drug contained in a solution using an antigen-antibody reaction. It is. The concentration and purification method using affinity chromatography is usually
An antibody-immobilized carrier obtained by immobilizing an antibody capable of binding to the subject peroxide derivative-type antimalarial drug on a carrier surface, and a solution containing the subject peroxide-derivative type antimalarial drug A step of bringing the subject peroxide derivative-type antimalarial drug into contact with an antibody immobilized on the surface of the antibody-immobilized carrier by antigen-antibody reaction; and
Separating the solution from the antibody-immobilized carrier;
After the separation step, the subject peroxide, which is bound to the antibody immobilized on the surface of the antibody-immobilized carrier, is brought into contact with the antibody-immobilized carrier with an eluate containing a binding dissociator. Dissociating a derivative antimalarial agent by the action of the binding dissociator;
After the dissociation step, the step of recovering the eluate containing the subject peroxide derivative-type antimalarial agent, which has migrated into the eluate containing the binding dissociator;
Separating the target peroxide derivative-type antimalarial drug contained in the recovered eluate from the eluate and concentrating the antimalarial drug.

  The antibody-immobilized carrier obtained by immobilizing an antibody having a binding ability to the target peroxide derivative-type antimalarial drug on the surface of the carrier is preferably prepared using the following method.

  Since the target peroxide derivative type antimalarial drug is the ester compound (TATP2) represented by the formula (I), the binding to the ester compound (TATP2) represented by the formula (I) prepared according to the above method An antibody having the ability is used.

  At that time, in the first form of the present invention, the structure similar to the characteristic structure of the ester compound (TATP2) represented by the above formula (I) prepared by immunizing a mammal other than human according to the above-mentioned procedure is used. This is a polyclonal antibody against a low molecular compound having a structure having a property, and an antibody having cross-reactivity with the ester compound (TATP2) represented by the formula (I) is used.

  The second aspect of the present invention comprises the characteristic structure and similarity of the ester compound (TATP2) represented by the above formula (I) prepared by immunizing a mammal other than human according to the above procedure. A monoclonal antibody against a low molecular weight compound having the above structure, and an antibody having cross-reactivity with the ester compound (TATP2) represented by the formula (I) is used.

  In general, a polyclonal antibody is composed of a plurality of types of antibodies having differences in antigen-antibody reactivity with a target antigen, and the composition ratio often varies. On the other hand, a monoclonal antibody is essentially a single type of antibody, and antigen-antibody reactivity with a target antigen is uniform. In view of this point, it is generally desirable to use monoclonal antibodies.

  When an antibody is immobilized on a carrier surface to produce an antibody-immobilized carrier, the antibody (immunoglobulin) molecule is usually immobilized on the carrier surface using the constant region of the antibody (immunoglobulin) molecule, that is, the Fc site of the antibody. The immobilization on the surface of the carrier is performed through a linker that binds to the constant region of the antibody (immunoglobulin) molecule, that is, the Fc site of the antibody. The linker is modified in advance on the surface of the carrier, and this linker is bound to the Fc site of the antibody.

  Therefore, it is preferable that the immobilization carrier itself is obtained by modifying the linker in advance on the surface of a carrier mainly composed of silica gel or nitrocellulose, which is widely used for producing conventional antibody immobilization carriers. As the linker, protein G and protein A, which are widely used for immobilizing antibody molecules, can be suitably used.

  By the antigen-antibody reaction with the antibody on the antibody-immobilized carrier, the bound antigen molecule is dissociated from the antibody by contacting with an eluate containing a binding dissociator. In general, strong acids, strong alkalis, ureas, chelating agents, surfactants, organic solvents, and the like are used as bond dissociating agents used for dissociating the bond between an antibody and an antigen molecule. On the other hand, the use of a bond dissociator having a function of dissociating the bond between an antibody and the linker is generally undesirable. Of the above bond dissociators, those that can be used in combination can be used in combination of two or more.

  As the bond dissociating agent for the ester compound (TATP2) represented by the formula (I), those having no reactivity to the ester compound (TATP2) itself represented by the formula (I) are used. That is, since the ester compound (TATP2) itself represented by the formula (I) is a peroxide derivative, it is preferable to use an organic solvent as a bond dissociator.

  In particular, it is preferable to use an organic solvent capable of dissociating the ester compound (TATP2) represented by the formula (I) that is bound to the antibody without dissociating the bond between the antibody and the linker. . When used as an eluent, the organic solvent used as a bond dissociator is in the form of an aqueous solution. Therefore, an organic solvent that forms a uniform solution with water is preferable. Organic solvents that can be suitably used as a bond dissociator for the ester compound (TATP2) represented by the formula (I) include acetonitrile, dimethyl sulfone, ethanol, formamide, and the like. In particular, acetonitrile is less likely to cause denaturation of antibody (immunoglobulin) molecules, is a good solvent for the ester compound (TATP2) represented by the formula (I), and is more preferable as a bond dissociator.

  After the elution step, the target peroxide derivative-type antimalarial drug contained in the recovered eluate is separated from the eluate and concentrated.

  Specifically, in the recovered eluate, contaminants that do not bind to the antibody are removed. As a result, the ratio of the ester compound (TATP2) represented by the formula (I) in the solute substance is relatively high. Yes. In the eluate, in addition to the ester compound (TATP2) represented by the formula (I), a solvent component constituting an aqueous solution is contained. By removing the solvent component contained in the eluate, the ester compound (TATP2) represented by the formula (I) is concentrated. The organic solvent that can be suitably used as the above bond dissociator can be removed by transpiration, and is also a preferable bond dissociator in that respect.

  Note that a technique of using antigen-antibody reaction to bind and separate antigen molecules contained in a sample solution from antibody molecules is also used, for example, in a surface plasmon measurement apparatus. In the surface plasmon measurement device, the binding of antigen molecules contained in the sample solution using the immobilized antibody is also a special form of the method for concentrating and purifying a peroxide derivative type antimalarial drug according to the present invention. It corresponds. At that time, as a surface plasmon measuring device, for example, a BIACORE series device using a surface plasmon manufactured by Biacore Co., Ltd. can be suitably used in terms of operability and reliability.

  The peroxide derivative type antimalarial drug concentration and purification method according to the present invention will be described in more detail below with reference to typical embodiments.

  Specific examples of the embodiments illustrated below are examples of the best mode of the present invention, but the technical scope of the present invention is not limited to the modes illustrated in the specific examples.

(First embodiment)
In the method for concentrating and purifying a peroxide derivative-type antimalarial drug according to the first embodiment of the present invention described below, as a peroxide derivative-type antimalarial drug to be concentrated and purified, the following formula is used. Ester compound having the structure shown in (I): 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propionic acid ethyl ester (3- (3-methyl -1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propanoic acid ethyl ester).

(I) Synthesis of ester compound having structure shown in formula (I) Structure shown in formula (I) which is a peroxide derivative type antimalarial drug to be concentrated and purified in the first embodiment of the present invention. Ester compound having 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] andec-3-yl) -propionic acid ethyl ester (3- (3-methyl-1,2,4 , 5-tetraoxa-spiro [5.5] undec-3-yl) -propanoic acid ethyl ester) will be described. Hereinafter, the ester compound having the structure represented by the formula (I) is abbreviated as TATP2.

  A method for synthesizing an ester compound (TATP2) having a structure represented by the formula (I) has already been reported in the literature (Non-Patent Document 2: Organic & Biomolecular Chemistry Vol. 4, p.4431-4436 (2006)). A method for purifying an ester compound (TATP2) having a structure represented by the formula (I) to be synthesized and a method for evaluating its purity are also disclosed in this document.

  On the other hand, 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] andeck-, which corresponds to the carboxylic acid moiety constituting the ester compound (TATP2) having the structure represented by the formula (I) Synthesis of 3-yl) -propionic acid (3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propanoic acid) is also performed in accordance with the disclosure of the above literature. be able to.

  The ester compound (TATP2) having the structure represented by the formula (I) can be administered to humans as a peroxide derivative-type antimalarial agent that can be used for the treatment of P. falciparum malaria. In that case, since the blood concentration of the conventional peroxide derivative type antimalarial drug is selected in the range of 20 μM at the maximum (Non-patent Document 3), the structure shown in the formula (I) in human blood The concentration of the ester compound having TA (TATP2) is also preferably selected within the above range.

(Ii) Synthesis of dicarboxylic acid compound having structure represented by formula (II) Formula (II) having a structure having characteristic structure and similarity to ester compound (TATP2) having the structure represented by formula (I). ) Dicarboxylic acid compound having the structure shown in: 3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9 -Il] -propionic acid (3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl]- The synthesis method of propanoic acid will be described. Hereinafter, the dicarboxylic acid compound having the structure represented by the formula (II) is abbreviated as TATP3.

  A method for synthesizing a dicarboxylic acid compound (TATP3) having a structure represented by the formula (II) has already been reported in the literature (Non-patent Document 2: Organic & Biomolecular Chemistry Vol. 4, p.4431-4436 (2006)). . A method for purifying a dicarboxylic acid compound (TATP3) having a structure represented by the formula (II) to be synthesized and a method for evaluating the purity thereof are also disclosed in this document.

  In the dicarboxylic acid compound (TATP3) having the structure represented by the formula (II), the spiro ring structure: carbon at the 9-position on the ring of 7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradecane Both the atom and the carbon atom at the 12-position are asymmetric centers (chiral centers). Regarding the configuration of the 9th carbon atom and the 12th carbon atom, the meso-type dicarboxylic acid compound (TATP3) that can be expressed as (9R, 12S) has a three-dimensional structure having two-fold rotational symmetry. Yes. Regarding the configuration of the carbon atom at the 9th position and the carbon atom at the 12th position, the cis-form dicarboxylic acid compound (TATP3) that can be expressed as (9R, 12R) has a three-dimensional structure having a symmetry plane.

  In the first embodiment, a mixture of the two stereoisomers is used. In addition, the dicarboxylic acid compound (TATP3) itself having a structure represented by the formula (II) does not exhibit immunogenicity.

  A dicarboxylic acid compound (TATP3) having a structure represented by the formula (II) is also a kind of peroxide derivative type antimalarial agent that can be used for the treatment of P. falciparum malaria. In addition, since the blood concentration of the conventional peroxide derivative type antimalarial drug is selected in the range of 20 μM at the maximum (Non-patent Document 3), the dicarboxylic acid compound (TATP3) having the structure shown in the formula (II) ), The blood concentration is preferably selected within the above range in order to exert its therapeutic effect.

(Iii) Immobilization of the dicarboxylic acid compound (TATP3) having the structure shown in Formula (II) on the carrier protein Keyhole Limpet Hemocyanin is selected as the carrier protein.

  A modified carrier protein was prepared by binding a dicarboxylic acid compound (TATP3) represented by the formula (II) on the carrier protein by a carbodiimide method. In the first embodiment, the following two modified carrier proteins were prepared and used as immunogens for immunization.

(iii-a) Preparation of Modified Carrier Protein (TATP3-KLH-DMSO Immunogen) Using DMSO ((CH ₃ ) ₂ SO: Wako Pure Chemical Industries, Ltd.) as a reaction solvent, keyhole limpet hemocyanin ( A dicarboxylic acid compound (TATP3) represented by the formula (II) is bound on Keyhole Limpet Hemocyanin by the carbodiimide method to prepare a modified carrier protein (TATP3-KLH-DMSO immunogen). In the bond formation by the carbodiimide method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC · HCl) (manufactured by Wako Pure Chemical Industries, Ltd.) is used as the binder carbodiimide.

  TATP3 dissolved in DMSO, 10 mg / 0.3 ml, and keyhole limpet hemocyanin (manufactured by Sigma Aldrich Japan) dissolved in DMSO, 10 mg / 1.7 ml are mixed. After mixing, the binder carbodiimide, 50 mg / 0.5 ml, dissolved in DMSO is added.

  A reaction solution using DMSO as a reaction solvent was allowed to stand at room temperature for 2 hours, and then allowed to stand at 4 ° C. for 12 hours to carry out the reaction. 0.1 ml of 1M glycine buffer (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 8 was added to stop the reaction. The modified carrier protein (TATP3-KLH-DMSO immunogen) and the unreacted carrier protein contained in the solution were purified by dialysis with PBS (manufactured by Wako Pure Chemical Industries, Ltd.). A protein solution containing the prepared modified carrier protein (TATP3-KLH-DMSO immunogen) and unreacted carrier protein was used as a TATP3-DMSO (TATP3-KLH-DMSO immunogen) solution.

Under the reaction conditions using the above-described binder carbodiimide, the carboxyl group (—COOH) of the dicarboxylic acid compound (TATP3) represented by the formula (II) with respect to the amino group (—NH ₂ ) exposed on the surface of the carrier protein. Is used to form an amide bond (—CO—NH—), thereby binding the dicarboxylic acid compound (TATP3) represented by the formula (II).

  In addition, on the modified carrier protein (TATP3-KLH-DMSO immunogen) prepared under the above reaction conditions, the dicarboxylic acid compound (TATP3) represented by the formula (II) binds on average at 150 to 200 sites. is doing. That is, this corresponds to a state in which TATP3 is bound to the amino group of the lysine residue side chain on the surface of the carrier protein KLH.

(iii-b) Preparation of a modified carrier protein (TATP3-KLH-borate buffer immunogen) Using a borate buffer at pH 8.5 as a reaction solvent, a formula on Keyhole Limpet Hemocyanin A dicarboxylic acid compound (TATP3) shown in (II) is bound by a carbodiimide method to prepare a modified carrier protein (TATP3-KLH-borate buffer immunogen). In the bond formation by the carbodiimide method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC · HCl) (manufactured by Wako Pure Chemical Industries, Ltd.) is used as the binder carbodiimide. The pH 8.5 borate buffer is 0.992 g boric acid (Wako Pure Chemical Industries), 1.906 g borax (Wako Pure Chemical Industries), and 2.628 g NaCl (Wako Pure Chemical Industries). Is a buffer solution in which pH is adjusted to 8.5 by dissolving NaOH in 180 ml of pure water and adding NaOH.

  TATP3, 10 mg / (0.3 ml DMOS + 0.2 ml borate buffer) dissolved in a mixed solution of borate buffer and DMSO and keyhole limpet hemocyanin, 10 mg / 1.5 ml dissolved in borate buffer are mixed. After mixing, the binder carbodiimide, 50 mg / 0.5 ml, dissolved in borate buffer is added.

  A reaction solution using the borate buffer as a reaction solvent was allowed to stand at room temperature for 2 hours, and then allowed to stand at 4 ° C. for 12 hours to carry out the reaction. 0.1 ml of 1M glycine buffer (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 8 was added to stop the reaction. The modified carrier protein (TATP3-KLH-DMSO immunogen) and the unreacted carrier protein contained in the solution were purified by dialysis with PBS (manufactured by Wako Pure Chemical Industries, Ltd.). A protein solution containing the prepared modified carrier protein (TATP3-KLH-borate buffer immunogen) and an unreacted carrier protein is converted into a TATP3-borate buffer (TATP3-KLH-borate buffer immunogen) solution. It was.

Under the reaction conditions using the above-described binder carbodiimide, the carboxyl group (—COOH) of the dicarboxylic acid compound (TATP3) represented by the formula (II) with respect to the amino group (—NH ₂ ) exposed on the surface of the carrier protein. Is used to form an amide bond (—CO—NH—), thereby binding the dicarboxylic acid compound (TATP3) represented by the formula (II).

  On the modified carrier protein (TATP3-KLH-borate buffer immunogen) prepared under the above reaction conditions, the dicarboxylic acid compound (TATP3) represented by the formula (II) averages 150 to 200. The place is connected.

(Iv) Preparation of Modified Carrier Protein for Antigen Modified with Dicarboxylic Acid Compound (TATP3) Represented by Formula (II) Bovine serum albumin (BSA) is selected as the carrier protein.

  A modified carrier protein was prepared by binding a dicarboxylic acid compound (TATP3) represented by the formula (II) on the carrier protein by a carbodiimide method. In the first embodiment, the following modified carrier protein was prepared and used as an antigen to be used for confirmation of antibody reactivity.

Preparation of Modified Carrier Protein for Antigen (TATP3-BSA Antigen) Using a borate buffer at pH 8.5 as a reaction solvent, a dicarboxylic acid compound (TATP3) represented by the formula (II) on bovine serum albumin (BSA) The modified carrier protein (TATP3-BSA antigen) is prepared by binding by the carbodiimide method. In the bond formation by the carbodiimide method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC · HCl) (manufactured by Wako Pure Chemical Industries, Ltd.) is used as the binder carbodiimide. The pH 8.5 borate buffer is 0.992 g boric acid (Wako Pure Chemical Industries), 1.906 g borax (Wako Pure Chemical Industries), and 2.628 g NaCl (Wako Pure Chemical Industries). Is a buffer solution in which pH is adjusted to 8.5 by dissolving NaOH in 180 ml of pure water and adding NaOH.

  TATP3 dissolved in DMSO, 10 mg / 0.1 ml, bovine serum albumin (BSA) dissolved in pure water, 30 mg / 1.5 ml, and borate buffer 0.9 ml are mixed. After mixing, the binder carbodiimide, 50 mg / 0.25 ml, dissolved in borate buffer is added.

  A reaction solution using the borate buffer as a reaction solvent was allowed to stand at room temperature for 5 hours for reaction. 0.3 ml of 1M glycine buffer (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 8 was added to stop the reaction. The modified carrier protein (TATP3-BSA antigen) and the unreacted carrier protein contained in the solution were purified by dialysis with PBS (manufactured by Wako Pure Chemical Industries, Ltd.). A protein solution containing the prepared modified carrier protein (TATP3-BSA antigen) and unreacted carrier protein was used as a TATP3-BSA (TATP3-BSA antigen) solution.

(V) Immunization using a modified carrier protein for an immunogen Sensitization is performed on the target non-human mammal using the two modified carrier proteins for immunogen. In the first embodiment, a mouse (SLC: C57BL / 6) is selected as a mammal other than a human to be immunized.

  For immunization, a solution prepared by mixing the above-mentioned two modified immunogen carriers and proteins and adding Freund's complete adjuvant (Funakoshi Co., Ltd.) is used. The composition of the solution was 0.01 ml TATP3-DMSO (TATP3-KLH-DMSO immunogen) solution, 0.01 ml TATP3-borate buffer (TATP3-KLH-borate buffer immunogen) solution, 0.07 ml PBS and 0.01 ml of Freund's complete adjuvant (Funakoshi) are mixed uniformly.

  Sensitization (immune manipulation) was performed by subcutaneously injecting 1.0 ml of the solution into a 12-week-old mouse (SLC: C57BL / 6). On the 22nd, 35th, and 49th days after the first sensitization (first day), 1.0 ml of the solution was subcutaneously injected, and booster immunization was performed.

  On the 66th day after the first sensitization (first day), blood was collected from the immunized mice. Antiserum was prepared from the collected blood.

(Vi) Verification of cross-reactivity of polyclonal antibody contained in the obtained antiserum The polyclonal antibody contained in the obtained antiserum exhibits cross-reactivity with the ester compound (TATP2) represented by the formula (I) This is verified using a competitive ELISA method.

  The polyclonal antibody contained in the obtained antiserum is presumed to be a mixture of two or more types of antibodies specific to the above-mentioned two modified immunogen carriers and proteins used for immunization. First, it is verified that an antibody exhibiting a specific reactivity with the dicarboxylic acid compound (TATP3) itself represented by the formula (II) is present in the polyclonal antibody.

  Specifically, as a carrier protein, the modified carrier protein for antigen (TATP3-BSA antigen) prepared using bovine serum albumin (BSA) instead of Keyhole Limpet Hemocyanin (Keyhole Limpet Hemocyanin) The presence of a reactive antibody is verified using an ELISA method.

  Using a TATP3-BSA (TATP3-BSA antigen) solution diluted 1000-fold, the TATP3-BSA (TATP3-BSA antigen) is immobilized on a plate for ELISA measurement by a natural adsorption method. After washing away and removing unadsorbed protein, 50 μl of PBS is added to the plate for ELISA measurement. Next, 50 μl of a solution obtained by diluting the obtained antiserum 100 times with PBS is added. The antibody is reacted with the TATP3-BSA (TATP3-BSA antigen) after standing at room temperature for 2 hours.

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 3 times with 100 μl of each PBS. After the washing, 50 μl of an anti-mouse IgG-POD labeled antibody solution (Funakoshi) diluted 2000 times is added to the plate. The antibody that has been allowed to stand at room temperature for 1 hour and reacted with the antigen-modified carrier protein (TATP3-BSA antigen) on the plate is reacted with an anti-mouse IgG-POD-labeled antibody.

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 4 times with 100 μl of each PBS. After the washing, 50 μl of ELISA peroxidase substrate (TMBZ, Funakoshi) solution is added to the plate. After carrying out an enzyme reaction for 60 minutes with the labeling enzyme peroxidase of the anti-mouse IgG-POD labeled antibody, 50 μl of 1N sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added to stop the reaction. The concentration of the reaction product by the enzyme reaction is determined by measuring the absorbance at 450 nm. In FIG. 1, the measurement result denoted as “buffer” corresponds to the amount of antibody reacted with TATP3-BSA (TATP3-BSA antigen).

  The above-mentioned antibody having reactivity with TATP3-BSA (TATP3-BSA antigen) reacts with the dicarboxylic acid compound (TATP3) itself represented by the formula (II) bound on bovine serum albumin (BSA). It is an antibody. Therefore, it was verified that the polyclonal antibody contained in the obtained antiserum contains an antibody having a specific reactivity with the dicarboxylic acid compound (TATP3) itself represented by the formula (II).

  Next, among the plurality of types of antibodies that are reactive with the dicarboxylic acid compound (TATP3) itself represented by the formula (II) contained in the obtained antiserum polyclonal antibody, an ester compound represented by the formula (I) ( The presence of an antibody showing cross-reactivity to TATP2) is verified using a competitive ELISA method.

  Using a TATP3-BSA (TATP3-BSA antigen) solution diluted 1000-fold, the TATP3-BSA (TATP3-BSA antigen) is immobilized on a plate for ELISA measurement by a natural adsorption method. After washing and removing unadsorbed protein, 50 μl of a PBS solution of TATP2 is added to the plate for ELISA measurement so that the final concentration becomes 100 ppm. Next, 50 μl of a solution obtained by diluting the obtained antiserum 100 times with PBS is added. The antibody is reacted with the TATP3-BSA (TATP3-BSA antigen) after standing at room temperature for 2 hours. The final concentration of 100 ppm of TATP2 contained in the reaction solution corresponds to 0.41 mM.

  When the antigen-antibody reaction progresses between TATP2 and the antibody that are added to the solution on the plate during the reaction, TATP3-BSA immobilized on the plate (TATP3-BSA antigen) and Competition with the antigen-antibody reaction. As a result, the amount of antibody immobilized on the plate is reduced through an antigen-antibody reaction with TATP3-BSA (TATP3-BSA antigen) immobilized on the plate.

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 4 times with 100 μl of each PBS. After the washing, 50 μl of ELISA peroxidase substrate (TMBZ, Funakoshi) solution is added to the plate. After carrying out an enzyme reaction for 60 minutes with the labeling enzyme peroxidase of the anti-mouse IgG-POD labeled antibody, 50 μl of 1N sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added to stop the reaction. The concentration of the reaction product by the enzyme reaction is determined by measuring the absorbance at 450 nm.

  Moreover, it replaces with the PBS solution of TATP2, PBS is added, and it reacts similarly.

  Using a TATP3-BSA (TATP3-BSA antigen) solution diluted 1000-fold, the TATP3-BSA (TATP3-BSA antigen) is immobilized on a plate for ELISA measurement by a natural adsorption method. After washing away and removing unadsorbed protein, 50 μl of PBS is added to the plate for ELISA measurement. Next, 50 μl of a solution obtained by diluting the obtained antiserum 100 times with PBS is added. The antibody is reacted with the TATP3-BSA (TATP3-BSA antigen) after standing at room temperature for 2 hours.

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 4 times with 100 μl of each PBS. After the washing, 50 μl of ELISA peroxidase substrate (TMBZ, Funakoshi) solution is added to the plate. After carrying out an enzyme reaction for 60 minutes with the labeling enzyme peroxidase of the anti-mouse IgG-POD labeled antibody, 50 μl of 1N sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added to stop the reaction. The concentration of the reaction product by the enzyme reaction is determined by measuring the absorbance at 450 nm. In FIG. 1, the measurement result expressed as “TATP2” corresponds to the amount of antibody reacted with TATP3-BSA (TATP3-BSA antigen) in the situation where the above-mentioned TATP2 coexists.

  The result shown in FIG. 1 shows that the antigen-antibody reaction progresses between TATP2 and the antibody that are added to the solution on the plate during the above reaction, and is immobilized on the plate. It clearly shows that an antigen-antibody reaction with BSA (TATP3-BSA antigen) and competition have occurred. That is, among a plurality of types of antibodies having specific reactivity to the dicarboxylic acid compound (TATP3) itself represented by the formula (II) contained in the polyclonal antibody, the antibody against the ester compound (TATP2) represented by the formula (I) It was verified that antibodies showing cross reactivity were present.

  Therefore, it was verified that the obtained antiserum polyclonal antibody contained an antibody exhibiting cross-reactivity with the ester compound (TATP2) represented by the formula (I).

  The anti-mouse IgG-POD-labeled antibody has specificity to the mouse IgG1-type antibody, and the type of antibody showing cross-reactivity with the ester compound (TATP2) represented by the formula (I) is IgG1-type. It was confirmed that.

  FIG. 1 also shows the results of verification of four types of antisera, that is, antiserum prepared from blood collected from four different mice immunized on the same schedule, Plate 1 to Plate 4. .

  An antibody-immobilized carrier is prepared using an antiserum that has been verified to contain an antibody exhibiting cross-reactivity with the ester compound (TATP2) represented by the formula (I). A polyclonal antibody contained in the antiserum is immobilized on the surface of the prepared antibody immobilization carrier. The antibody contained in the antiserum was immobilized on a silica gel modified with protein A (IgG Purification kit-A manufactured by Dojindo Laboratories). The immobilization method followed the supplier's instruction manual attached to the silica gel modified with protein A. The immobilized antibody is about 0.2 mg.

  As an unpurified solution containing the ester compound (TATP2) represented by the formula (I), it has already been reported in the literature (Non-patent Document 2: Organic & Biomolecular Chemistry Vol. 4, p.4431-4436 (2006)). During the synthesis process, after completion of the synthesis reaction, a liquid that has not been concentrated is used. The yield of the synthesis reaction was 18%. The next reaction product is contained.

  The prepared antibody-immobilized carrier is put into about 1 ml of this unpurified solution and left at room temperature for 30 minutes. Filter and collect the antibody-immobilized carrier (silica gel) in the liquid with filter paper. The recovered antibody-immobilized carrier (silica gel) is put into a 20% (v / v) acetonitrile solution and allowed to stand at room temperature for 30 minutes. It is then filtered to recover a 20% (v / v) acetonitrile solution.

  The collected acetonitrile solution is dried, and the solute component dissolved in the solution is collected as a solid. About the compound which comprises the fractionated solid substance, the mass spectrometry spectrum was measured with the electrospray ionization method. In addition, a crystal of a compound constituting the solid was prepared, and X-ray crystal analysis was performed. As a result, the mass spectrometric spectrum of the compound constituting the fractionated solid was obtained as the target 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl)- It was consistent with the mass spectrometry spectrum of ethyl propionate. From the results of X-ray crystallographic analysis, it was found to be the target 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propionic acid ethyl ester. Verified.

  Therefore, by using the antibody-immobilized carrier prepared using the polyclonal antibody contained in the antiserum, the ester compound (TATP2) represented by the formula (I) contained in the unpurified solution can be obtained. It was confirmed that concentration and purification can be performed.

(Second embodiment)
In the second embodiment of the present invention described below, the ester compound (TATP2) represented by the above formula (I) is selected as the peroxide derivative type antimalarial drug to be concentrated and purified.

  In the second embodiment, in the first embodiment described above, the obtained antiserum polyclonal antibody contains an antibody exhibiting cross-reactivity to the ester compound (TATP2) represented by the formula (I). Using a group of mouse antibody-producing cells that have been verified, hybridoma cells producing a monoclonal antibody that exhibits cross-reactivity with the ester compound (TATP2) represented by the formula (I) are prepared.

  According to the procedure described in the first embodiment, blood is collected from the immunized mouse on the 66th day after the first sensitization (first day), antiserum is prepared from the collected blood, and the antiserum It is verified that an antibody showing cross-reactivity with the ester compound (TATP2) represented by the formula (I) is present in the polyclonal antibody. After the first sensitization (first day) after this verification, the spleen is extracted from the mouse on the 66th day to prepare spleen cells.

The prepared mouse spleen cells and P3-X63-Ag8-U mouse myeloma cells in a ratio of 5: 1 cells in RPMI 1640 medium (manufactured by Invitrogen), 50% polyethylene glycol (sum) In the presence of Kogyo Pharmaceutical Co., Ltd.), the mixture is mixed at 37 ° C. for 2 minutes to cause cell fusion. After the cell fusion treatment, the resulting hybridoma cells are suspended in a HAT medium (20% fetal bovine serum) and then dispensed into a microplate. The hybridoma cells dispensed on the microplate are cultured in a carbon dioxide incubator at 37 ° C. and 5% CO ₂ . During the culture, half of the medium is replaced with fresh HT medium (10% fetal calf serum) once every four days.

  The HAT medium is obtained by adding an appropriate amount of HAT supplement (manufactured by Invitrogen) to RPMI 1640 medium. In the second embodiment, 20 μl of HAT supplement is added per 1 ml of RPMI 1640 medium.

  The HT medium is obtained by adding an appropriate amount of HT supplement (manufactured by Invitrogen) to RPMI1640 medium. In the second embodiment, 20 μl of HT supplement is added per 1 ml of RPMI 1640 medium.

  Under the above culture conditions, the hybridoma cells dispensed on the microplate were cultured for 2 weeks to establish hybridoma cell lines, respectively.

  Next, whether or not the monoclonal antibody produced in the culture supernatant of each hybridoma cell line is a monoclonal antibody having a specific reactivity with the dicarboxylic acid compound (TATP3) itself represented by the formula (II) Was confirmed. Furthermore, among those that have been verified to be monoclonal antibodies having specific reactivity with the dicarboxylic acid compound (TATP3) itself represented by the formula (II), a cross-reaction with the ester compound (TATP2) represented by the formula (I) The antibody which shows sex was selected.

(a) Verification of whether or not the dicarboxylic acid compound (TATP3) itself represented by formula (II) is a monoclonal antibody having specific reactivity Specifically, it is contained in the culture supernatant of each hybridoma cell line An ELISA method is used to determine whether the monoclonal antibody is a monoclonal antibody produced using bovine serum albumin (BSA) and having reactivity with the modified carrier protein for antigen (TATP3-BSA antigen). To verify.

  Using a TATP3-BSA (TATP3-BSA antigen) solution diluted 1000-fold, the TATP3-BSA (TATP3-BSA antigen) is immobilized on a plate for ELISA measurement by a natural adsorption method. After washing away and removing unadsorbed protein, 50 μl of PBS is added to the plate for ELISA measurement. Next, 50 μl of a solution obtained by diluting the culture supernatant of each hybridoma cell line 100 times with PBS is added. The mixture is allowed to stand at room temperature for 2 hours, and the monoclonal antibody is reacted with the TATP3-BSA (TATP3-BSA antigen).

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 3 times with 100 μl of each PBS. After the washing, 50 μl of an anti-mouse IgG-POD labeled antibody solution (Funakoshi) diluted 2000 times is added to the plate. The antibody that has been allowed to stand at room temperature for 1 hour and reacted with the antigen-modified carrier protein (TATP3-BSA antigen) on the plate is reacted with an anti-mouse IgG-POD-labeled antibody.

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 4 times with 100 μl of each PBS. After the washing, 50 μl of ELISA peroxidase substrate (TMBZ, Funakoshi) solution is added to the plate. After carrying out an enzyme reaction for 60 minutes with the labeling enzyme peroxidase of the anti-mouse IgG-POD labeled antibody, 50 μl of 1N sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added to stop the reaction. The concentration of the reaction product by the enzyme reaction is determined by measuring the absorbance at 450 nm.

  Using a TATP3-BSA (TATP3-BSA antigen) solution diluted 1000-fold, the TATP3-BSA (TATP3-BSA antigen) is immobilized on a plate for ELISA measurement by a natural adsorption method. After washing away and removing unadsorbed protein, 50 μl of PBS is added to the plate for ELISA measurement. Next, 50 μl of a solution obtained by diluting the culture supernatant of each hybridoma cell line 100 times with PBS is added. The mixture is allowed to stand at room temperature for 2 hours, and the monoclonal antibody is reacted with the TATP3-BSA (TATP3-BSA antigen).

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 3 times with 100 μl of each PBS. After the washing, 50 μl of an anti-mouse IgG-POD labeled antibody solution (Funakoshi) diluted 2000 times is added to the plate. The antibody that has been allowed to stand at room temperature for 1 hour and reacted with the antigen-modified carrier protein (TATP3-BSA antigen) on the plate is reacted with an anti-mouse IgG-POD-labeled antibody.

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 4 times with 100 μl of each PBS. After the washing, 50 μl of ELISA peroxidase substrate (TMBZ, Funakoshi) solution is added to the plate. After carrying out an enzyme reaction for 60 minutes with the labeling enzyme peroxidase of the anti-mouse IgG-POD labeled antibody, 50 μl of 1N sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added to stop the reaction. The concentration of the reaction product by the enzyme reaction is determined by measuring the absorbance at 450 nm.

  The above monoclonal antibody having reactivity with TATP3-BSA (TATP3-BSA antigen) reacts with the dicarboxylic acid compound (TATP3) itself represented by the formula (II) bound on bovine serum albumin (BSA). It is a monoclonal antibody.

  According to this screening procedure, it was verified whether or not a monoclonal antibody having specific reactivity with the dicarboxylic acid compound (TATP3) itself represented by the formula (II) exists in the culture supernatant of each hybridoma cell line. . As a result, it was confirmed that, out of a total of 13 clones of the hybridoma cell line, 13 clones produced monoclonal antibodies that reacted with the dicarboxylic acid compound (TATP3) itself represented by the formula (II).

(b) Selection of monoclonal antibody showing cross-reactivity to ester compound (TATP2) shown in formula (I) The dicarboxylic acid compound (TATP3) shown in formula (II) itself selected in the primary screening (a) Monoclonal antibodies exhibiting cross-reactivity to the ester compound (TATP2) represented by formula (I) are selected from a plurality of monoclonal antibodies exhibiting reactivity using a competitive ELISA method.

  Using a TATP3-BSA (TATP3-BSA antigen) solution diluted 1000-fold, the TATP3-BSA (TATP3-BSA antigen) is immobilized on a plate for ELISA measurement by a natural adsorption method. After washing and removing unadsorbed protein, 50 μl of a PBS solution of TATP2 is added to the plate for ELISA measurement so that the final concentration becomes 100 ppm. Subsequently, 50 μl of a solution obtained by diluting the culture supernatant of each selected hybridoma cell line 100 times with PBS is added. The antibody is reacted with the TATP3-BSA (TATP3-BSA antigen) after standing at room temperature for 2 hours. The final concentration of 100 ppm of TATP2 (molecular weight 274.3) contained in the reaction solution corresponds to 0.41 mM.

  When the antigen-antibody reaction progresses between TATP2 and the antibody that are added to the solution on the plate during the reaction, TATP3-BSA immobilized on the plate (TATP3-BSA antigen) and Competition with the antigen-antibody reaction. As a result, the amount of antibody immobilized on the plate is reduced through an antigen-antibody reaction with TATP3-BSA (TATP3-BSA antigen) immobilized on the plate.

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 4 times with 100 μl of each PBS. After the washing, 50 μl of ELISA peroxidase substrate (TMBZ, Funakoshi) solution is added to the plate. After carrying out an enzyme reaction for 60 minutes with the labeling enzyme peroxidase of the anti-mouse IgG-POD labeled antibody, 50 μl of 1N sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added to stop the reaction. The concentration of the reaction product by the enzyme reaction is determined by measuring the absorbance at 450 nm.

  Compared with the case where TATP2 is not added to the solution on the plate during the antigen-antibody reaction, when TATP2 is added, a result showing a decrease in the concentration of the reaction product by the enzyme reaction is obtained. When added, it is judged that competition has occurred. That is, when such competition occurs, the monoclonal antibody contained in the culture supernatant of the hybridoma cell line can be determined as a monoclonal antibody exhibiting cross-reactivity with TATP2.

  According to this screening procedure, an ester compound (TATP2) represented by the formula (I) was selected from a plurality of monoclonal antibodies reactive with the dicarboxylic acid compound (TATP3) itself represented by the formula (II) selected in the primary screening (a). Were selected for monoclonal antibodies showing cross-reactivity.

  As a result, a plurality of types of monoclonal antibodies showing cross-reactivity with the ester compound (TATP2) represented by the formula (I) were selected. FIG. 2 shows, as an example, the measurement results for four monoclonal antibodies: mAb-m001 to mAb-m004 among a plurality of monoclonal antibodies selected by this secondary screening.

  In FIG. 2, the measurement result expressed as “buffer” is the measurement result by the ELISA method in the primary screening described above (a), that is, TATP3-BSA (TATP3-BSA antigen) in the situation where TATP2 does not exist. On the other hand, it corresponds to the amount of the reacted monoclonal antibody. In FIG. 2, the measurement result expressed as “100 ppm TATP2” corresponds to the amount of monoclonal antibody reacted with TATP3-BSA (TATP3-BSA antigen) in the situation where the above-mentioned TATP2 coexists. Yes.

  The results shown in FIG. 2 show that TATP3 immobilized on the plate because the antigen-antibody reaction proceeds between TATP2 and the monoclonal antibody added to the solution on the plate during the above reaction. -It clearly shows that an antigen-antibody reaction with BSA (TATP3-BSA antigen) and competition have occurred. That is, the four types of monoclonal antibodies are monoclonal antibodies that are reactive with the dicarboxylic acid compound (TATP3) itself represented by the formula (II), and are further cross-reactive with the ester compound (TATP2) represented by the formula (I). It is a result which verifies that it is also a monoclonal antibody which shows this.

  Therefore, the monoclonal antibody selected by the primary screening (a) and the secondary screening (b) is a monoclonal antibody exhibiting cross-reactivity with the ester compound (TATP2) represented by the formula (I). Was confirmed.

In addition, the result shown in FIG. 2 shows that the ester compound (TATP2) represented by the formula (I) is added to a final concentration of 0.41 mM in the reaction solution for performing the antigen-antibody reaction.
In mAb-m001 to mAbm004, the result suggests that at least about 90% is bound to TATP2.

  An antibody-immobilized carrier is prepared using the mAb-m002 antibody, which is selected by the above screening, and exhibits a cross-reactivity with the ester compound (TATP2) represented by the formula (I). The mAb-m002 antibody was immobilized on a silica gel modified with protein A (IgG Purification kit-A manufactured by Dojindo Laboratories). The immobilization method followed the supplier's instruction manual attached to the silica gel modified with protein A. The immobilized antibody is about 0.2 mg.

  As an unpurified solution containing the ester compound (TATP2) represented by the formula (I), it has already been reported in the literature (Non-patent Document 2: Organic & Biomolecular Chemistry Vol. 4, p.4431-4436 (2006)). During the synthesis process, after completion of the synthesis reaction, a liquid that has not been concentrated is used. The yield of the synthesis reaction was 18%. The next reaction product is contained.

  The prepared antibody-immobilized carrier is put into about 1 ml of this unpurified solution and left at room temperature for 30 minutes. Filter and collect the antibody-immobilized carrier (silica gel) in the liquid with filter paper. The recovered antibody-immobilized carrier (silica gel) is put into a 20% (v / v) acetonitrile solution and allowed to stand at room temperature for 30 minutes. It is then filtered to recover a 20% (v / v) acetonitrile solution.

  The collected acetonitrile solution is dried, and the solute component dissolved in the solution is collected as a solid. About the compound which comprises the fractionated solid substance, the mass spectrometry spectrum was measured with the electrospray ionization method. In addition, a crystal of a compound constituting the solid was prepared, and X-ray crystal analysis was performed. As a result, the mass spectrometric spectrum of the compound constituting the fractionated solid was obtained as the target 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl)- It was consistent with the mass spectrometry spectrum of ethyl propionate. From the results of X-ray crystallographic analysis, it was found to be the target 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propionic acid ethyl ester. Verified.

  Therefore, concentration of the ester compound (TATP2) represented by the formula (I) contained in the unpurified solution by using the antibody-immobilized carrier prepared using the monoclonal antibody, mAb-m002 antibody, It was confirmed that purification could be performed.

(Reference Example 1)
In Reference Example 1, antibody immobilization was performed using the mAb-m003 antibody, which was selected by the screening of the second embodiment, and exhibited a cross-reactivity to the ester compound (TATP2) represented by the formula (I). A carrier is prepared. The mAb-m003 antibody was immobilized on a silica gel modified with protein A (IgG Purification kit-A manufactured by Dojindo Laboratories). The immobilization method followed the supplier's instruction manual attached to the silica gel modified with protein A. The immobilized antibody is about 0.2 mg.

An unpurified solution containing the dicarboxylic acid compound (TATP3) represented by the formula (II) has already been reported in the literature (Non-patent Document 2: Organic & Biomolecular Chemistry Vol. 4, p.4431-4436 (2006)). During the synthesis process, after completion of the synthesis reaction, a liquid that has not been concentrated is used. The yield of the synthesis reaction is 88%, and the unpurified solution is the remaining raw material compound other than the dicarboxylic acid compound (TATP3) represented by the formula (II) in methanol (CH ₃ OH) as a reaction solvent. , Side reaction products are contained.

  The prepared antibody-immobilized carrier is put into about 1 ml of this unpurified solution and left at room temperature for 30 minutes. Filter and collect the antibody-immobilized carrier (silica gel) in the liquid with filter paper. The recovered antibody-immobilized carrier (silica gel) is put into a 20% (v / v) acetonitrile solution and allowed to stand at room temperature for 30 minutes. It is then filtered to recover a 20% (v / v) acetonitrile solution.

  The collected acetonitrile solution is dried, and the solute component dissolved in the solution is collected as a solid. About the compound which comprises the fractionated solid substance, the mass spectrometry spectrum was measured with the electrospray ionization method. In addition, a crystal of a compound constituting the solid was prepared, and X-ray crystal analysis was performed. As a result, the mass spectrometric spectrum of the compound constituting the fractionated solid was 3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa. It was consistent with the mass spectrometric spectrum of -spiro- [5.8] tetradec-9-yl] -propionic acid (TATP3). From the results of X-ray crystallography, the target 3- [12- (2-carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8 ] Tetradec-9-yl] -propionic acid (TATP3).

  Therefore, concentration of the dicarboxylic acid compound (TATP3) represented by the formula (II) contained in the unpurified solution by using the antibody-immobilized carrier prepared using the monoclonal antibody, mAb-m003 antibody. It was confirmed that purification was possible.

(Reference Example 2)
In this Reference Example 2, the carboxylic acid compound constituting the ester compound (TATP2) represented by the formula (I): 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl An attempt was made to create an antibody capable of binding to the ester compound (TATP2) represented by the formula (I) using a modified carrier protein obtained by binding propionic acid to the carrier protein.

(D-1) Preparation of carboxylic acid compound (TATP2 ′): 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propionic acid TATP2 ′) is also a known compound, and its synthesis method has already been reported in the literature (Non-Patent Document 2: Organic & Biomolecular Chemistry Vol. 4, p.4431-4436 (2006)).

  Specifically, it is prepared from the ester compound (TATP2) represented by the formula (I) by converting the ester bond (—CO—OEt) to a carboxyl group (—COOH) using KOH. Then, purification was performed and the carboxylic acid compound (TATP2 ') was recovered.

(D-2) Preparation of Modified Carrier Protein for Immunogen Modified with Carboxylic Acid Compound (TATP2 ′) Keyhole Limpet Hemocyanin is selected as the carrier protein.

  A modified carrier protein was prepared by binding the carboxylic acid compound (TATP2 ') onto the carrier protein by a carbodiimide method. Also in this reference example, the following two modified carrier proteins were prepared and used as immunogens for immunization.

(d-2-a) Preparation of Modified Carrier Protein (TATP2′-KLH-DMSO Immunogen) Using DMSO ((CH ₃ ) ₂ SO: Wako Pure Chemical Industries, Ltd.) as a reaction solvent, keyhole phosphorus The carboxylic acid compound (TATP2 ′) is bound on Keyhole Limpet Hemocyanin by the carbodiimide method to prepare a modified carrier protein (TATP2′-KLH-DMSO immunogen). In the bond formation by the carbodiimide method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC · HCl) (manufactured by Wako Pure Chemical Industries, Ltd.) is used as the binder carbodiimide.

  The carboxylic acid compound (TATP2 ') dissolved in DMSO, 10 mg / 0.3 ml, and keyhole limpet hemocyanin (manufactured by Sigma Aldrich Japan), 10 mg / 1.7 ml dissolved in DMSO are mixed. After mixing, the binder carbodiimide, 50 mg / 0.5 ml, dissolved in DMSO is added.

  A reaction solution using DMSO as a reaction solvent was allowed to stand at room temperature for 2 hours, and then allowed to stand at 4 ° C. for 12 hours to carry out the reaction. 0.1 ml of 1M glycine buffer (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 8 was added to stop the reaction. The modified carrier protein (TATP2'-KLH-DMSO immunogen) and the unreacted carrier protein contained in the solution were purified by dialysis with PBS (manufactured by Wako Pure Chemical Industries, Ltd.). A protein solution containing the prepared modified carrier protein (TATP2'-KLH-DMSO immunogen) and unreacted carrier protein was used as a TATP2'-DMSO (TATP2'-KLH-DMSO immunogen) solution.

In the reaction conditions using the binder carbodiimide, the carboxyl group (—COOH) of the carboxylic acid compound (TATP2 ′) is used for the amino group (—NH ₂ ) exposed on the surface of the carrier protein. By forming an amide bond (—CO—NH—), the carboxylic acid compound (TATP2 ′) is bound.

  In addition, on the modified carrier protein (TATP2′-KLH-DMSO immunogen) prepared under the above reaction conditions, the target carboxylic acid compound (TATP2 ′) binds on average 150 to 200 sites. is doing.

(d-2-b) Preparation of Modified Carrier Protein (TATP2′-KLH-Borate Buffer Immunogen) Using a borate buffer at pH 8.5 as a reaction solvent, Keyhole Limpet Hemocyanin The carboxylic acid compound (TATP2 ′) is bound thereto by a carbodiimide method to prepare a modified carrier protein (TATP2′-KLH-borate buffer immunogen). In the bond formation by the carbodiimide method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC · HCl) (manufactured by Wako Pure Chemical Industries, Ltd.) is used as the binder carbodiimide. The pH 8.5 borate buffer is 0.992 g boric acid (Wako Pure Chemical Industries), 1.906 g borax (Wako Pure Chemical Industries), and 2.628 g NaCl (Wako Pure Chemical Industries). Is a buffer solution in which pH is adjusted to 8.5 by dissolving NaOH in 180 ml of pure water and adding NaOH.

  The carboxylic acid compound (TATP2 ′), 10 mg / (0.3 ml DMOS + 0.2 ml borate buffer) dissolved in a mixed solution of borate buffer and DMSO, and keyhole limpet hemocyanin, 10 mg / 1. Mix with 5 ml. After mixing, the binder carbodiimide, 50 mg / 0.5 ml, dissolved in borate buffer is added.

  A reaction solution using the borate buffer as a reaction solvent was allowed to stand at room temperature for 2 hours, and then allowed to stand at 4 ° C. for 12 hours to carry out the reaction. 0.1 ml of 1M glycine buffer (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 8 was added to stop the reaction. The modified carrier protein (TATP2'-KLH-DMSO immunogen) and the unreacted carrier protein contained in the solution were purified by dialysis with PBS (manufactured by Wako Pure Chemical Industries, Ltd.). A protein solution containing the prepared modified carrier protein (TATP2′-KLH-borate buffer immunogen) and unreacted carrier protein is treated with TATP2′-borate buffer (TATP2′-KLH-borate buffer immunogen). Raw) solution.

In the reaction conditions using the binder carbodiimide, the carboxyl group (—COOH) of the carboxylic acid compound (TATP2 ′) is used for the amino group (—NH ₂ ) exposed on the surface of the carrier protein. By forming an amide bond (—CO—NH—), the carboxylic acid compound (TATP2 ′) is bound.

  In addition, on the modified carrier protein (TATP2′-KLH-borate buffer immunogen) prepared under the above reaction conditions, the target carboxylic acid compound (TATP2 ′) averages 150 to 200 sites. Are connected.

  In addition, the modified carrier protein (TATP2′-KLH-DMSO immunogen) and the modified carrier protein (TATP2′-KLH-borate buffer immunogen) prepared under the above two reaction conditions are It was confirmed that the antigen-antibody reaction was carried out with the monoclonal antibodies mAb-m001 to mAbm004 created in the above embodiment.

(D-3) Preparation of Modified Carrier Protein for Antigen Modified with the Carboxylic Acid Compound (TATP2 ′) Bovine serum albumin (BSA) is selected as the carrier protein.

  A modified carrier protein was prepared by binding the carboxylic acid compound (TATP2 ') onto the carrier protein by a carbodiimide method. In this reference example, the following modified carrier protein was prepared and used as an antigen to be used for confirmation of antibody reactivity.

Preparation of Modified Carrier Protein for Antigen (TATP2′-BSA Antigen) Dicarboxylic acid compound (TATP3) represented by formula (II) on bovine serum albumin (BSA) using borate buffer at pH 8.5 as a reaction solvent Are bound by a carbodiimide method to prepare a modified carrier protein (TATP3-BSA antigen). In the bond formation by the carbodiimide method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC · HCl) (manufactured by Wako Pure Chemical Industries, Ltd.) is used as the binder carbodiimide. The pH 8.5 borate buffer is 0.992 g boric acid (Wako Pure Chemical Industries), 1.906 g borax (Wako Pure Chemical Industries), and 2.628 g NaCl (Wako Pure Chemical Industries). Is a buffer solution in which pH is adjusted to 8.5 by dissolving NaOH in 180 ml of pure water and adding NaOH.

  The carboxylic acid compound (TATP2 ') dissolved in DMSO, 10 mg / 0.1 ml, bovine serum albumin (BSA) dissolved in pure water, 30 mg / 1.5 ml, and borate buffer 0.9 ml are mixed. After mixing, the binder carbodiimide, 50 mg / 0.25 ml, dissolved in borate buffer is added.

  A reaction solution using the borate buffer as a reaction solvent was allowed to stand at room temperature for 5 hours for reaction. 0.3 ml of 1M glycine buffer (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 8 was added to stop the reaction. The modified carrier protein (TATP2'-BSA antigen) and unreacted carrier protein contained in the solution were purified by dialysis with PBS (manufactured by Wako Pure Chemical Industries, Ltd.). A protein solution containing the prepared modified carrier protein (TATP2'-BSA antigen) and unreacted carrier protein was used as a TATP2'-BSA (TATP2'-BSA antigen) solution.

  In addition, it was confirmed that the modified carrier protein (TATP2′-BSA antigen) produced under the above reaction conditions undergoes an antigen-antibody reaction with the monoclonal antibodies mAb-m001 to mAbm004 created in the second embodiment. did.

(D-4) Immunization using a modified carrier protein for immunogen The modified carrier protein (TATP2′-KLH-DMSO immunogen) and the modified carrier protein (TATP2) are applied to the target non-human mammal. Sensitization is performed using '-KLH-borate buffer immunogen). Also in this reference example, a mouse (SLC: C57BL / 6) is selected as a mammal other than a human to be immunized.

  For immunization, the modified carrier protein (TATP2′-KLH-DMSO immunogen) and the modified carrier protein (TATP2′-KLH-borate buffer immunogen) are mixed, and Freund's complete adjuvant (Funakoshi) ) Is used. The composition of the solution was 0.01 ml of TATP2′-DMSO (TATP2′-KLH-DMSO immunogen) solution, 0.01 ml of TATP2′-borate buffer (TATP2′-KLH-borate buffer immunogen) solution, 0.07 ml PBS and 0.01 ml Freund's complete adjuvant (manufactured by Funakoshi) were mixed uniformly.

  Sensitization (immune manipulation) was performed by subcutaneously injecting 1.0 ml of the solution into a 12-week-old mouse (SLC: C57BL / 6). On the 22nd, 35th, and 49th days after the first sensitization (first day), 1.0 ml of the solution was subcutaneously injected, and booster immunization was performed.

  On the 66th day after the first sensitization (first day), blood was collected from the immunized mice. Serum was prepared from the collected blood.

(D-5) Verification of the reactivity of the antibody contained in the obtained serum The ELISA method indicates that the antibody contained in the obtained serum exhibits reactivity with the target carboxylic acid compound (TATP2 ′). Verify using.

  Antibodies in the obtained serum are specific to the above modified carrier protein (TATP2′-KLH-DMSO immunogen) and modified carrier protein (TATP2′-KLH-borate buffer immunogen) used for immunization. It is estimated that multiple types of typical antibodies are mixed. It is verified that an antibody exhibiting a specific reactivity with the target carboxylic acid compound (TATP2 ') itself is present in the polyclonal antibody.

  Specifically, the modified carrier protein for antigen (TATP2′-BSA antigen) produced using bovine serum albumin (BSA) instead of Keyhole Limpet Hemocyanin as a carrier protein. It is verified by using the ELISA method that an antibody having reactivity with is present.

  Using a TATP2'-BSA (TATP2'-BSA antigen) solution diluted 1000 times, the TATP2'-BSA (TATP2'-BSA antigen) is immobilized on a plate for ELISA measurement by a natural adsorption method. After washing away and removing unadsorbed protein, 50 μl of PBS is added to the plate for ELISA measurement. Next, 50 μl of a solution obtained by diluting the obtained antiserum 100 times with PBS is added. The antibody is reacted with the TATP2'-BSA (TATP2'-BSA antigen) after standing at room temperature for 2 hours.

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 3 times with 100 μl of each PBS. After the washing, 50 μl of an anti-mouse IgG-POD labeled antibody solution (Funakoshi) diluted 2000 times is added to the plate. The antibody that has been allowed to stand at room temperature for 1 hour and reacted with the antigen-modified carrier protein (TATP2'-BSA antigen) on the plate is reacted with an anti-mouse IgG-POD-labeled antibody.

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 4 times with 100 μl of each PBS. After the washing, 50 μl of ELISA peroxidase substrate (TMBZ, Funakoshi) solution is added to the plate. After carrying out an enzyme reaction for 60 minutes with the labeling enzyme peroxidase of the anti-mouse IgG-POD labeled antibody, 50 μl of 1N sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added to stop the reaction. The concentration of the reaction product by the enzyme reaction is determined by measuring the absorbance at 450 nm.

  When the reactivity was verified by the above-mentioned ELISA method, the presence of the reaction product by the enzyme reaction was not confirmed. That is, a result indicating the presence of an IgG antibody showing reactivity to a modified carrier protein for antigen (TATP2'-BSA antigen) was not obtained.

  Therefore, depending on the immunization operation using the modified carrier protein (TATP2′-KLH-DMSO immunogen) and the modified carrier protein (TATP2′-KLH-borate buffer immunogen), the target carboxylic acid compound ( It was judged that no IgG antibody showing specific reactivity to TATP2 ′) itself was created.

  That is, depending on the immunization operation using the modified carrier protein (TATP2′-KLH-DMSO immunogen) and the modified carrier protein (TATP2′-KLH-borate buffer immunogen), the ester represented by the formula (I) It was judged that no IgG antibody showing the binding ability to the compound (TATP2) was created.

(Reference Example 3)
In this Reference Example 3, with respect to the monoclonal antibody mAb-m004 described in the second embodiment, the peroxide (TATP) of the formula (III) is based on the cross-reactivity to the ester compound (TATP2) represented by the formula (I). ) Cross-reactivity is verified to be 1/20 or less.

  First, the peroxide of formula (III) (TATP) is dissolved in acetonitrile as a solvent to prepare a TATP solution having a concentration of 100 ppm. The concentration of 100 ppm corresponds to about 0.45 mM.

  Also in this reference example 3, the reaction conditions described in the second embodiment are used.

  Using a TATP3-BSA (TATP3-BSA antigen) solution diluted 1000-fold, the TATP3-BSA (TATP3-BSA antigen) is immobilized on a plate for ELISA measurement by a natural adsorption method. After washing and removing unadsorbed protein, 50 μl of the TATP solution is added to the ELISA measurement plate. Subsequently, 50 μl of mAb-m004 solution (antibody concentration: 3.8 mg / ml) is added. The mixture is allowed to stand at 36 ° C. for 30 minutes, and in the presence of TATP, the monoclonal antibody mAb-m004 is reacted with TATP3-BSA immobilized on the plate (TATP3-BSA antigen).

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 3 times with 100 μl of each PBS. After the washing, 50 μl of an anti-mouse IgG-POD labeled antibody solution (Funakoshi) diluted 2000 times is added to the plate. The anti-mouse IgG-POD labeled antibody is allowed to react with the antibody mAb-m004 that has been allowed to stand at room temperature for 1 hour and reacted with the modified carrier protein for antigen (TATP3-BSA antigen) on the plate.

  After completion of the reaction, the solution on the plate for ELISA measurement is removed, and the plate is washed 4 times with 100 μl of each PBS. After the washing, 50 μl of ELISA peroxidase substrate (TMBZ, Funakoshi) solution is added to the plate. After carrying out an enzyme reaction for 60 minutes with the labeling enzyme peroxidase of the anti-mouse IgG-POD labeled antibody, 50 μl of 1N sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added to stop the reaction. The concentration of the reaction product by the enzyme reaction is determined by measuring the absorbance at 450 nm.

  When the TATP acetonitrile solution was used, the absorbance at 450 nm resulting from the reaction product of the enzyme reaction was 2.996.

  As described in Reference Example 2, when a control borate buffer was used, the absorbance at 450 nm resulting from the reaction product of the enzyme reaction was 3.031.

  Therefore, the amount of decrease in absorbance at 450 nm due to antigen-antibody reaction between TATP added to the reaction solution and antibody mAb-m004 is estimated to be only 0.035.

  Referring to the results described in Reference Example 2, when the serum sample estimated to contain about 0.41 mM of the ester compound (TATP2) represented by formula (I) was used, the reaction by the enzyme reaction The absorbance at 450 nm attributable to the product was 2.510. Therefore, the amount of decrease in absorbance at 450 nm due to the antigen-antibody reaction between TATP2 contained in the serum sample and the antibody mAb-m004 was 0.521.

Considering the TATP concentration in the TATP acetonitrile solution: about 0.45 mM and the TATP2 concentration contained in the serum sample: about 0.41 mM,
The cross-reactivity exhibited by the monoclonal antibody mAb-m004 is
Based on the cross-reactivity to TATP2, the cross-reactivity to TATP is determined to be at least 1/20 or less.

  Therefore, it is verified that the monoclonal antibody mAb-m004 has cross-reactivity with the ester compound (TATP2) represented by the formula (I) but does not substantially exhibit cross-reactivity with the peroxide of the formula (III). It was done.

  The method for concentrating and purifying a peroxide derivative type antimalarial drug according to the present invention is a target peroxide derivative type antimalarial drug, for example, 3- (3-methyl-1,2,4,5- Tetraoxa-spiro [5.5] undec-3-yl) -propionic acid ethyl ester (3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propanoic acid ethyl ester ) Can be suitably used for the purpose of purifying to the high purity required for pharmaceuticals.

(Trust number)
As a hybridoma cell producing a monoclonal antibody having a binding ability to the ester compound (TATP2) represented by the formula (I) used in the present invention,
Hybridoma cell line: NECP-C57Z 15B-1E is based on the Budapest Treaty. National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center In addition, an international deposit (May 12, 2009) has been made.

Claims (13)

Ester compound having the structure represented by the following formula (I) contained in the solution: ethyl 3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propionate A method for concentrating and purifying an ester (3- (3-methyl-1,2,4,5-tetraoxa-spiro [5.5] undec-3-yl) -propanoic acid ethyl ester),

A solution containing an ester compound having the structure represented by the formula (I) in an antibody-immobilized carrier obtained by immobilizing an antibody having a binding ability to the ester compound having the structure represented by the formula (I) on the surface of the carrier. Contacting the ester compound having the structure represented by the formula (I) with an antibody immobilized on the surface of the antibody-immobilized carrier by antigen-antibody reaction,
Separating the solution from the antibody-immobilized carrier;
After the separation step, the antibody-immobilized carrier is brought into contact with an eluate containing a binding dissociation agent, and is bound to the antibody immobilized on the surface of the antibody-immobilized carrier. Dissociating an ester compound having the structure shown by the action of the bond dissociator;
After the dissociation step, recovering the eluate containing an ester compound having a structure represented by the formula (I), which is transferred into the eluate containing the binding dissociator;
Separating and concentrating the ester compound having the structure represented by the formula (I) contained in the recovered eluate from the eluate, and concentrating and purifying the method. An antibody having a binding ability to an ester compound having the structure represented by the formula (I) is:
An antibody against a low molecular weight compound having a structure similar to the characteristic structure in the ester compound having the structure represented by the formula (I), and cross-linked to the ester compound having the structure represented by the formula (I) The concentration and purification method according to claim 1, which has reactivity. The low molecular weight compound having a structure similar to the characteristic structure in the ester compound having the structure represented by the formula (I) is a dicarboxylic acid compound having a structure represented by the following formula (II): 3- [12 -(2-Carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl] -propionic acid (3- [12- (2 -carboxyethyl) -9,12-dimethyl-7,8,10,11,13,14-hexaoxa-spiro- [5.8] tetradec-9-yl] -propanoic acid)

The concentration and purification method according to claim 2. An antibody having a binding ability to an ester compound having the structure represented by the formula (I) is:
A modified protein obtained by binding a low molecular weight compound having a structure similar to the characteristic structure in the ester compound having the structure represented by the above formula (I) onto a carrier protein is used as an immunogen, and other than humans. A polyclonal antibody to the low molecular weight compound created by immunizing a mammal, and an antibody having cross-reactivity to the ester compound having the structure represented by the formula (I) Item 4. The concentration and purification method according to Item 2 or 3. An antibody having a binding ability to an ester compound having the structure represented by the formula (I) is:
A modified protein obtained by binding a low molecular weight compound having a structure similar to the characteristic structure in the ester compound having the structure represented by the above formula (I) onto a carrier protein is used as an immunogen, and other than humans. A monoclonal antibody against the low molecular weight compound created by immunizing a mammal, wherein the antibody has cross-reactivity with an ester compound having the structure represented by the formula (I). Item 4. The concentration and purification method according to Item 2 or 3. The concentration and purification method according to any one of claims 2 to 5, wherein the non-human mammal is a mouse. 7. The modified protein obtained by binding the low molecular weight compound on a carrier protein, wherein the carrier protein is selected from keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin). Concentration and purification method. A compound having a carboxyl group (—COOH) in the molecule is selected as a low molecular weight compound having a structure similar to the characteristic structure of the ester compound having the structure represented by the formula (I),
A modified protein obtained by binding a compound having a carboxyl group (—COOH) in the molecule onto a carrier protein comprises the carboxyl group (—COOH) and an amino group (—NH ₂ ) on the carrier protein. 8. The compound having a carboxyl group (—COOH) in the molecule is bonded through an amide bond (—CO—NH—) between the compounds. The concentration and purification method according to 1. Formation of an amide bond (—CO—NH—) between the carboxyl group (—COOH) and an amino group (—NH ₂ ) on the carrier protein is performed using a carbodiimide method. The concentration and purification method according to claim 8. As an eluent containing the bond dissociator,
The aqueous solution containing a hydrophilic organic solvent capable of dissolving the ester compound having the structure represented by the formula (I) as the bond dissociator is used. The concentration and purification method according to 1. The concentration and purification method according to claim 10, wherein acetonitrile is selected as a hydrophilic organic solvent capable of dissolving the ester compound having the structure represented by the formula (I). An antibody-immobilized carrier obtained by immobilizing an antibody having a binding ability to an ester compound having the structure represented by the formula (I) on the surface of a carrier,
The base material of the carrier is silica gel, and the surface of the silica gel base material is modified with protein A or protein G.
The immobilization of the antibody on the surface of the carrier is achieved by immobilization of the antibody molecule with protein A or protein G modifying the surface of the silica gel substrate. The concentration and purification method according to any one of the above. As an antibody having a binding ability to an ester compound having the structure represented by the formula (I),
6. The enrichment according to claim 5, wherein a monoclonal antibody against a dicarboxylic acid compound having a structure represented by the formula (II) produced by a hybridoma cell line: NECP-C57Z 15B-1E (FERM ABP-11126) is used. , Purification method.

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