JP2002047211A - Combination of fat-soluble substance with physiologically active high polymer and method for introduction into cell nulceus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for introducing a high polymer capable of entering a cell nucleus and exhibiting a physiological activity in the cell nucleus without passing through a cytoplasm sol. SOLUTION: This method comprises chemically binding the high polymer capable of exhibiting the physiological activity in the cell nucleus to a fat- soluble substance and introducing the high polymer into the nucleus of the cell to be a target. The combination is used therefore. The carrier comprises the fat-soluble substance and is capable of making the high polymer capable of exhibiting the physiological activity in the cell nucleus migrate into the nucleus of the target cell. Furthermore, the pharmaceutical composition comprises the combination prepared by chemically binding the high polymer capable of exhibiting the physiological activity in the cell nucleus to the fat- soluble substance and used for introducing the high polymer into the nucleus of the target cell and is useful for therapy, prophylaxis or treatment of various diseases by an abnormality in the nucleus of the cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for introducing a macromolecular substance exhibiting a physiological activity in a cell nucleus into a nucleus of a target cell. More specifically, the present invention is a method of chemically binding a polymer substance exhibiting a biological activity in a cell nucleus and a fat-soluble substance, and introducing the polymer substance into a nucleus of a target cell, And a conjugate therefor. Also,
TECHNICAL FIELD The present invention relates to a carrier for transferring a polymer substance, which is composed of a fat-soluble substance and exhibits a physiological activity in a cell nucleus, into a nucleus of a target cell. Furthermore, the present invention includes a conjugate for introducing the macromolecular substance into the nucleus of the target cell, in which a macromolecular substance exhibiting a biological activity in the cell nucleus is chemically bonded to a fat-soluble substance. The present invention relates to a pharmaceutical composition for treating, preventing or treating various diseases caused by abnormalities in the nucleus of cells.

[0002]

2. Description of the Related Art Cells are the smallest functional units that make up living organisms. Cells are made up of the outermost cell membrane, the cytoplasm within the cell membrane, and the nucleus. There are a wide variety of "organelles" in the cytoplasm, which make up about 50% of the cytoplasm. The portion of the cytoplasm other than the organelles is called the "cytosol", of which about 70-90% is water.
The nucleus is a relatively large structure present inside the cell and can be observed with an optical microscope. The nucleus is surrounded by two membranes resembling the cell membrane, the nuclear envelope. The nuclear envelope has many pores called "nuclear envelope pores" from which substances necessary for the nucleus are taken in, and substances such as mRNA are released. The nucleus contains genes that play a central role in the genetic information, and the substances that can enter the nucleus in order to protect them are severely restricted. Nuclear pore is a physical pore having a diameter of about 100 nm, and a nuclear pore complex consisting of 50 to 100 kinds of proteins is present in the inner periphery thereof. The size is believed to be up to 26-28 nm.

[0003] Since the nuclear pore is a physical pore, the inside of the nucleus and the cytosol form a continuous phase, and the molecular size is about 9%.
Sub-nm materials can pass through relatively freely, whereas larger materials up to 26-28 nm have nuclei.
Transport is performed by the cytoplasmic transport mechanism (Nigg, EA
(1997) Nature, 386: 779-787). The protein transported into the nucleus has a transport signal “nuclear localization signal (NLS)” from the cytoplasm to the nucleus, and the protein transported from the nucleus to the cytoplasm has a “nuclear export signal (NES)”. have.

[0004] The protein transported into the nucleus forms a stable complex in the cytoplasm with essential factors called importin α and importin β centering on the nuclear localization signal (NLS) and passes through the nuclear pore. . That is, first, importin α recognizes and binds to NLS of a protein to form a nuclear import protein-importin α complex, and importin β binds to this complex to form a heterotrimer containing a nuclear import protein. It is thought to be formed and pass through the nuclear pore by interaction with the nuclear pore complex of importin β (Yoneda, Biological Science, 50 (6), 531-538).
(1999)).

As described above, among substances present in the cytoplasm, substances having a relatively small size (9 nm or less) can freely enter the nucleus, but high-molecular substances such as large proteins usually enter the nucleus. And is transported by the nuclear-cytoplasmic transport mechanism described above, but with a size of 28
Substances of nm or more cannot pass through the nuclear pore. However, Maul et al. (1978) reported that simian virus 40 (SV40)
It suggested that the uptake (about 45 nm) vacuole and the outer nuclear membrane were fused. We used SV40 markers for cell membrane markers.
(1986) demonstrated that the uptake vacuole and the nuclear envelope fused together were fused, and that the uptake vacuole was a single bilayer diaphragm found in a part of the nuclear envelope consisting of the nuclear double membrane. Suggests that substances that are normally taken up by lysosomes and do not enter the nucleoplasm enter the nucleus (T. Nishimura, et al., Cel.
l Structure and Function, 11, 135-141 (1991)).

In this pathway, the macromolecular substance does not pass through the cytosol, and it has been shown that there is another nucleus-cytoplasmic pathway that does not pass through the nuclear pore. In order to prove this, studies using genetically modified baculovirus have been made. This wild-type strain acquires a viral envelope in the nucleus and is incorporated into polyhedrin (protein).
When the insects of the infected host die, the cells can break apart and go outside, infecting the next host. On the other hand, the strain in which the polyhedrin gene is modified is not surrounded by polyhedrin in the nucleus, so it is necessary to go out of the cell and infect the next host. Therefore, instead of acquiring the envelope in the nucleus, it is necessary to acquire it at the cell membrane,
For that, it must move from the nucleus to the cytoplasm. However, the size that can pass through the nuclear pore is up to 26-28 nm, and a virus particle (nucleoca) having a diameter of 45 nm is used.
psid) cannot pass through the nuclear pore and has no "nuclear export signal (NES)". Therefore, it is necessary to form a small pore different from the nuclear pore and to pass through it to the cytoplasm, and it is considered that there is a pathway between the nucleus and the cytoplasm that does not pass through the nuclear pore.

[0007] Transferring such a large substance into the nucleus of a cell has been extremely difficult due to the nuclear defense system. On the other hand, in autoimmune diseases such as systemic lupus erythematosus, antinuclear antibodies are observed.
The present inventors used IgG ghost and HVJ to obtain IgG.
Was injected into the cytoplasm, but IgG did not translocate to the nucleus.
In this method, the injected substance is cytosol
(Furusawa et al., Method in Cell
Biology, Vol. 14, 1976, p. 75). Synthetic I in which a nuclear localization signal is linked to IgG
While gG translocates from the cytoplasm to the nucleus, IgG cannot cross the cell membrane. As a method for introducing a large-sized physiologically active substance such as IgG into the nucleus, a method of injecting various substances into cultured cells by microinjection has been adopted, but this method is not applicable to living organisms. It is difficult and does not provide a realistic treatment.

Further, the present inventors have observed that colloidal gold to which silver sensitizer has been applied, and that intravenous 2 nm colloidal gold-labeled testosterone-bovine serum albumin (B
SA) The conjugate or the hydrocortisone-BSA conjugate was observed to enter the hormone target cell nucleus of the rat (Nishimura and Ichihara, Cell Struct. Funct., 22: 4).
33-442. (1997); Nishimura and Nakano, Cell Struct.
Funct., 22: 621-629. (1997) ,; Nishimura and Nakano,
Cell Struct. Funct., 24: 227-235. (1999)). It has been suggested that the transfer of the steroid hormone-BSA conjugate to the target cell nucleus is determined at the level of the cell membrane, and it has been reported that the BSA transferred to the cell nucleus maintains antigenicity (Analysis Society of March 1999; 1999 August Cell Biology Society).

As described above, the present inventors have shown that albumin bound to a steroid hormone translocates into the nucleus while maintaining its antigenicity. However, albumin is a substance having a physiological activity in the nucleus. Not a matter in the nucleus. How to transfer a large-sized substance (polymeric substance) having physiological activity in the nucleus such as IgG into the nucleus has not been solved yet.

[0010]

DISCLOSURE OF THE INVENTION The present invention provides a simple and efficient method for introducing a macromolecular substance exhibiting a biological activity into a cell nucleus without passing through a cytosol. . The present invention also provides a novel conjugate for introducing a macromolecular substance exhibiting a biological activity in a cell nucleus into a cell nucleus. Also, the present invention
An object of the present invention is to provide a carrier for transferring a high molecular substance exhibiting a physiological activity in a cell nucleus into a nucleus of a target cell. Further, the present invention provides a method for treating various diseases in which the cause of the disease exists in the nucleus of a cell, and a therapeutic composition or therapeutic agent therefor.

[0011]

SUMMARY OF THE INVENTION The present invention relates to a method for binding a polymer substance having a physiological activity in a cell nucleus to a fat-soluble substance and introducing the polymer substance into a nucleus of a target cell. In addition, the present invention relates to a conjugate in which a polymer substance exhibiting a physiological activity in a cell nucleus is chemically bound to a liposoluble substance for introducing the polymer substance into the nucleus of a target cell. In addition, the present invention relates to a carrier for transferring a polymer substance, which is composed of a fat-soluble substance and exhibits a physiological activity in a cell nucleus, to the nucleus of a target cell. Furthermore, the present invention relates to a pharmaceutical composition comprising the above-mentioned conjugate of the present invention and a pharmaceutically acceptable carrier.

The present invention is based on the finding shown by the present inventors that a polymer substance can be transferred from the outside of a cell to the nucleus of a cell using a fat-soluble substance. Such transfer of the macromolecular substance into the nucleus was a finding that could not be explained by the transfer mechanism between the nucleus and the cytoplasm by the conventional transport signal. The present invention is based on the findings of the present inventors, and attempted to introduce a macromolecular substance exhibiting a physiological activity in the nucleus of a cell from outside the cell into the nucleus of the cell.

The present inventors have developed immunoglobulin G (IgG) as a macromolecule that exerts physiological activity in the nucleus of cells.
And steroid hormones, more specifically hydrocortisone, were selected as fat-soluble substances, and steroid hormone-IgG conjugates composed of these were prepared. Various methods can be adopted as a method for producing the conjugate. For example, a condensing agent (for example, a hydrocortisone-21-hemisuccinate ester) is added to a pyridine-containing aqueous solution.
Carbodiimides are also preferred. ) And add I
An aqueous solution of gG was added to obtain a conjugate in which both were bound by an amide bond or the like.

A physiological saline solution of the obtained hydrocortisone-IgG conjugate (HC-IgG) was injected into the tail vein of a rat. Two and three hours later, the rats were perfused and fixed, the liver was removed and fixed in a fixative. Thereafter, this was rapidly frozen with liquid nitrogen to prepare a frozen section, and the frozen section was fixed with methanol for 20 minutes and washed with a phosphate buffer (PBS). This was reacted with normal rabbit serum, and then reacted with an antibody produced from a supernatant obtained by mixing and reacting a FITC-labeled anti-bovine IgG antibody (immune animal rabbit, IgG fraction) with normal rat liver powder. Fig. 1 shows the result of observation with a confocal laser microscope.
This is shown in the pictures replacing the drawings Nos. Figures 1-3 show 2 after administration
The rats were perfused and fixed after time, and FIGS. 4 to 8 show the rats after perfusion and fixed 3 hours after administration.

FIGS. 1 and 2 show the results 2 hours after the administration. In hepatocytes, the hepatocyte surface (FIG. 2), vesicles approaching the nucleus (FIGS. 1 and 2),
Further, it can be seen that the fluorescence of the FITC-labeled antibody is observed in the liver cell nucleus (FIG. 2). Vesicles are involved in vesicle transport of substances inside cells, and transport particles inside the cells by wrapping them in membranes. In FIG. 1, the fluorescence of the FITC-labeled antibody was observed in the vesicles approaching the nucleus, indicating that the administered HC-IgG was taken up by the cells, enveloped in the membrane, and transported by the vesicles. is there. Endocytosis causes endocytic vesicles (endoc)
The substances taken up by ytotic vesiclea) are usually decomposed by lysosomes in the cells.
Surprisingly, the conjugate of the present invention translocated into the nucleus (see FIG. 2). FIG. 3 shows the results 2 hours after the administration.
This indicates that fluorescence was observed in the nucleus of one hepatocyte.

FIGS. 4, 5 and 6 show the results 3 hours after the administration. In FIG. 4, fluorescence was observed along the vesicle membrane of the hepatocytes inside, and further, the hepatocyte nuclei were observed. Some fluorescence was observed. In FIG. 5, fluorescence was observed in vesicles approaching the hepatocyte nucleus, and further, fluorescence was observed in a part of the hepatocyte nucleus. In FIG. 6, fluorescence was observed in vesicles approaching the hepatocyte nucleus, and fluorescence was observed in almost the entire area of the nucleus. 7 and 8 show the results three hours after the administration. In FIG. 7, fluorescence was observed in the nucleus of the hepatic stellate cell, and in FIG. Fluorescence was observed in possible parts. Fluorescence was also observed in the nuclei of lymphocytes.

From the above, the conjugate of the present invention is taken up by vesicles into cells of a living body and transported by vesicles.
It was observed that at least a part thereof was not decomposed by the lysosome and translocated into the nucleus. This means that a polymer substance that exerts a biological activity in the cell nucleus can be directly introduced into the nucleus by binding a polymer substance that exerts a physiological activity in the cell nucleus to a fat-soluble substance to form a conjugate. In addition to the fact that fluorescence was observed in the nucleus due to the antigen-antibody reaction, the conjugate of the polymer substance that exerts physiological activity in the cell nucleus of the present invention and a fat-soluble substance was It demonstrates that it can be introduced into the nucleus of target cells without losing sex.
Moreover, the method of the present invention does not use a conventional transport signal, but rather a new transport pathway into the nucleus by formation of vesicles different from the transport pathway between the nucleus and cytoplasm in a conventional protein. Conceivable.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors previously studied the transfer of simian virus 40 (SV40) from the extracellular to the nucleus, and the uptake vacuole induced by SV40 was expressed in the outer nuclear membrane. It also reported that it also fused with the septum found in a part of the nuclear bilayer membrane. When the vacuole fused with the outer nuclear membrane, virus particles were transported to the nuclear envelope, and when fused with the septum, virus particles could enter the nucleoplasm. Steroid hormone-bovine albumin conjugate is also S
It has been suggested to enter the nucleus in a manner similar to V40. The pathway by which the conjugate of the present invention is translocated into the nucleus of the target cell is also considered to be almost the same as those pathways suggested by the present inventors.

Therefore, the conjugate of the present invention is an agent for introducing a macromolecular substance exhibiting a physiological activity in the cell nucleus from the outside of the cell into the nucleus of the cell. The fat-soluble substance used in the present invention is a carrier for introducing a high-molecular substance having a physiological activity into the nucleus of a target cell from outside the cell.

The present invention has found that a lipid-soluble substance can be used as a carrier (transport carrier) for introducing a polymer substance having a physiological activity in a cell nucleus into a nucleus of a target cell. The lipophilic substance of the present invention is a substance that can serve as a carrier (transport carrier) for introducing a polymer substance having a physiological activity in a cell nucleus into the nucleus of a target cell, Any compound having a functional group capable of forming a chemical bond directly with or via a linker moiety may be used. As a preferable fat-soluble substance of the present invention, a fat-soluble substance which itself has the ability to transfer to the nucleus is preferable. As long as it has such properties, it is preferable that the substance does not have a physiological activity on the target cell.However, a substance that does not significantly affect the biological activity of the polymer substance that exerts the biological activity in the cell nucleus is preferable. It may have a physiological activity. The molecular weight of the fat-soluble substance of the present invention is not particularly limited, but since the substance itself to be transferred into the nucleus has a high molecular weight, the fat-soluble substance of the present invention preferably has a molecular weight that is not so large. In addition, since a target cell may be selected depending on the fat-soluble substance, it is important to select a suitable fat-soluble substance in relation to the target cell.

Preferred fat-soluble substances of the present invention include fat-soluble hormones such as thyroid hormone, fat-soluble vitamins such as vitamin A and vitamin D, and steroid hormones. Preferred steroid hormones include sex hormones such as male and female hormones,
Corticosteroids and the like. More specifically,
Testosterone, progesterone, estrogen, aldosterone, cortisone, hydrocortisone and the like. The steroid hormones of the present invention include not only the above-mentioned natural steroid hormones, but also synthetic steroid hormones, natural or synthetic steroids having steroid hormone-like physiological activity, or substances having other chemical structures. Examples of substances having a chemical structure other than synthetic steroids having steroid hormone-like physiological activity include endocrine disrupting substances (environmental hormones).

The polymer substance of the present invention which exerts a physiological activity in the cell nucleus may be a protein or a non-protein substance as long as it exerts a physiological activity in the nucleus. , Proteins are preferred. The protein may be any protein, such as an enzyme, an antibody, an antigen, or a base sequence recognizing substance such as DNA, or a modified form thereof, as long as it can exert a required function in the nucleus. Preferred proteins include IgG and I.
antibodies such as gM.

In the conjugate of the present invention, which is a conjugate for introducing a physiologically active substance into the nucleus of a target cell, in which a polymer substance exhibiting a physiological activity in the cell nucleus is chemically bound to a fat-soluble substance. When a functional group capable of reacting with both can be used as the group for bonding both, direct bonding can be used, or a group such as a linker can be introduced to bond the two. Such a linker group has, at both ends, a functional group capable of binding to a “polymer substance exhibiting a biological activity in a cell nucleus” and a functional group capable of binding to a “lipid-soluble substance”. 1-50 carbon atoms between groups, 1-2
Those having 0 or about 1 to 10 chain or cyclic groups are exemplified. One or more carbon atoms in the carbon chain may be replaced by another heteroatom such as an oxygen atom, a nitrogen atom or a sulfur atom, and one or two carbon atoms in the carbon chain may be substituted. Other groups may be substituted at the above positions. The functional groups at both ends of the linker group may be those capable of causing a chemical reaction without impairing the physiological activity, and include, for example, a carboxyl group, an amino group, and a hydroxyl group.

The method for forming a chemical bond can be performed by a method generally used for peptide synthesis or the like. For example, an amino group and a carboxyl group can be condensed using a carbodiimide-based condensing agent such as DCC. If necessary, it can be protected by using a protecting group used in the synthesis of another functional group peptide in the substance.

The target cells of the present invention include all cells that are targets of fat-soluble substances, for example, liver cells such as hepatocytes, germ cells such as testis cells, and immune system cells such as lymphocytes. No.

The conjugate of the present invention for introducing a physiologically active substance into a nucleus of a target cell, in which a polymer substance exhibiting a biological activity in a cell nucleus is chemically bound to a fat-soluble substance,
The compositions can be formulated with conventional pharmaceutically acceptable carriers. The pharmaceutical composition of the present invention comprising the conjugate and a pharmaceutically acceptable carrier can be administered orally or parenterally, but parenteral administration is generally preferred. Since the active ingredient of the pharmaceutical composition of the present invention contains a protein, when it is formulated as an oral preparation to prevent the degradation of the protein in the gastrointestinal tract, it may be used in the stomach and small intestine as in a large intestine preparation. It is preferable to apply a coating for preventing the decomposition of the active ingredient.

As described above, the conjugate of the present invention can be introduced into the target cell nucleus by binding a high-molecular substance having a physiological activity, which does not normally enter the cell nucleus, to a fat-soluble substance, thereby allowing the action of the high-molecular substance having a physiological activity. And can be applied in a wide range of fields. An application example thereof will be illustrated more specifically. For example, in the experimental example described above, hydrocortisone intravenously injected into rats-
The bovine IgG conjugate selectively entered the hepatocyte nucleus and hepatic stellate cell nucleus. By replacing the bovine IgG with the antinuclear antibody, the antinuclear antibody controls the function of the cells. Therefore, by administering this intravenously to a liver cancer patient, it can be used as a therapeutic agent for liver cancer. .

In addition, for those suffering from breast cancer that is sensitive to female hormones, IgG (antinuclear antibody) that binds to breast cancer cell nuclei and suppresses the proliferation of the cells is bound to female hormones and injected into the patient intravenously. By administering
gG enters the nucleus, binds to the antigen, reduces the function of breast cancer cells, and can be used as a therapeutic agent for breast cancer. Since the lipophilic substance-protein conjugate of the present invention can pass through the basement membrane, it can be used as a macromolecular substance that exerts bioactivity in the cell nucleus regardless of where cancer cells sensitive to the lipophilic bioactive substance are transferred. The function of cancer cells can be affected only by intravenously injecting a conjugate of a fat-soluble physiologically active substance, and it can be used as an agent for preventing cancer metastasis.

Further, since the polymer substance cannot freely pass through the cell membrane, it does not normally enter the cell nucleus. At present, it is necessary to use a micromanipulator to introduce a polymer substance into cultured cells. Furthermore, it was not considered that a macromolecular substance having a physiological activity could be introduced into the target cell nucleus at the individual level. As a suggestion that a macromolecular substance such as a protein is transferred into a cell nucleus at an individual level, there is a report on a lipid-soluble bioactive substance binding protein such as a steroid hormone. Fat-soluble physiologically active substances such as steroid hormones bind to binding proteins and circulate in the bloodstream. It has been thought that free lipophilic physiologically active substances cross the cell membrane and exert their effects by binding to nuclear receptors (two-step the
ory). There have been reports suggesting that such a fat-soluble physiologically active substance-binding protein such as a steroid hormone is present in a target cell nucleus.

However, since these studies have confirmed using isotopes, it is unclear whether the protein translocated to the nucleus without losing its properties. In addition, since macromolecular substances cannot freely pass through cell membranes, the pathway of translocation to the nucleus is unknown. Isotopically labeled atoms, such as [
It merely reports that [ ¹²⁵ I] and [ ³ H] were present there, but does not clearly elucidate the mechanism of translocation of cells into the nucleus. In addition, proteins in "lipid-soluble bioactive substance-binding proteins such as steroid hormones" target lipid-soluble bioactive substances that are hardly soluble in water via the bloodstream, rather than macromolecules that exert bioactivity in the nucleus. It is a protein that acts as a carrier to carry to cells, and is different from the technical idea of the present invention.

[0031] The inventors have also reported that Simian virus 40
The extracellular to nuclear translocation of (simian virus 40 (SV40)) was studied and it was reported that the uptake vacuoles induced by SV40 also fused with the septum found in the outer nuclear membrane and part of the nuclear bilayer. When the vacuole fused with the outer nuclear membrane, virus particles were transported to the nuclear envelope, and when fused with the septum, virus particles could enter the nucleoplasm.
Although it has been suggested that the steroid hormone-bovine albumin conjugate also enters the nucleus in the same manner as SV40, it has been suggested that a polymer substance capable of exhibiting a biological activity in the nucleus can be transferred into the nucleus while maintaining its physiological activity. Illustrated according to the invention. Therefore, by using the conjugate of the present invention, the mechanism of transport of substances in the cell, such as the mechanism of translocation into the nucleus, the mechanism of uptake of fat-soluble substances into cells, and the mechanism of transport in cells. It can be used to elucidate various mechanisms.

Furthermore, at present, it is necessary to use a micromanipulator to introduce a macromolecular substance into cultured cells, and erythrocyte is used as a method for introducing a macromolecular substance into a large number of cultured cell nuclei, particularly into the nuclei of the cells. Although a method using a membrane-based carrier such as ghost or liposome has been reported, this method could not pass through a basement membrane because a membrane-based substance was used as a carrier. The present invention discloses for the first time that a fat-soluble substance, preferably a fat-soluble bioactive substance, can be used to easily introduce a bioactive polymer substance into the target cell nucleus from outside the cell, and By using the fat-soluble substance of the present invention as a carrier, it is possible to pass through the basement membrane. Furthermore, the method of the present invention makes it possible to transfer a macromolecular substance into the cell nucleus of a cell at the individual level. This indicates that a liposoluble substance, preferably a lipophilic physiologically active substance, can be easily used as a carrier for introducing a macromolecular substance having a physiological activity from outside the cell into a target cell, preferably into the nucleus of the cell. ing.
Accordingly, the present invention provides a fat-soluble substance, preferably a fat-soluble physiologically active substance, from the outside of a cell to a target cell, preferably into the nucleus of a cell, for introducing a macromolecular substance capable of exerting a physiological activity in the nucleus. It also provides a carrier (introducing agent or carrier for transfer).

According to the present invention, a macromolecule capable of exerting a bioactivity in a target cell nucleus from the outside of a cell can be easily introduced by using a fat-soluble substance such as a steroid hormone, preferably a fat-soluble physiologically active substance. The present invention discloses for the first time what can be done, and the present invention covers everything utilizing such technical ideas. And, by the obvious knowledge of those skilled in the art, the technical idea disclosed by the present invention,
It is obvious to those skilled in the art that the present invention can be widely applied to expression of functions in normal cells and abnormal cells, inhibition of expression of functions, and the like in a wide range of fields such as medicine, agriculture, and biology.

[0034]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (Hydrocortisone)
tisone) -Production of IgG conjugate) 16 mg of hydrocortisone-21-hemisuccinate (HC) was dissolved in 600 µl of pyridine: water (1: 1) at an ice temperature.
This was treated with 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDAC (1-ethyl-3 (3-
dimethylaminopropyl) -carbodiimido hydrochlorid
e)) It was added to a container containing 103 mg under ice cooling. After the addition is completed, the mixture is stirred, and the bovine immunoglobulin G is added thereto.
A solution of 20 mg of (Bovine immunoglobulin G (IgG)) dissolved in 500 μl of water at an ice temperature was added, and the mixture was further stirred for 3 minutes. After allowing this to stand overnight at room temperature, the contents were placed in a dialysis tube, and dialyzed against a 30% aqueous solution of pyridine for 3 days. During the dialysis, the external solution was changed 10 times or more. Obtained hydrocortisone (Hydrocortisone)-
The IgG conjugate was lyophilized and stored at 4 ° C.

Example 2 (Hydrocortisone)
tisone) -Preparation of administration solution of IgG conjugate) Hydrocortisone (Hydrocortiso) obtained in Example 1
ne) -IgG conjugate (HC-IgG) (10 mg) was placed in a glass homogenizer, and 1000 μl of 10% glycerin in physiological saline was added to eliminate clumps. Further, 1000 μl of physiological saline was added to the homogenizer, and HC-
The concentration of IgG was 5 m in 5% glycerin saline.
g / ml.

Example 3 (Cell distribution test) The solution prepared in Example 2 was administered via the tail vein of an adrenalectomized rat (10 weeks old, male, 5 days after excision). After administration,
After 2 and 3 hours, the rats were perfused and fixed, the liver was removed, placed in fixative, and cut into approximately 5 mm squares. 15% of this
The mixture was allowed to stand in a sucrose solution at 4 ° C. overnight, and snap-frozen in liquid nitrogen. After cryosectioning, the cryosections were washed with methanol for 20 minutes.
After fixing with phosphate buffer (PBS), the cells were reacted with normal rabbit serum. Then, FITC-labeled anti-bovine I
Immunostaining was performed with a gG antibody (immune animal rabbit, IgG fraction). The FITC-labeled anti-bovine IgG antibody used here was mixed with normal rat liver powder, reacted at 4 ° C. for 1 hour, centrifuged, and the supernatant was used as a FITC-labeled anti-bovine IgG antibody. Immunostained sections in PBS
After washing with, these were observed with a confocal laser microscope. The results are shown in FIGS. 1 to 8 as photographs replacing the drawings.
FIGS. 1 to 3 show the results of perfusion-fixation of rats 2 hours after administration, and FIGS. 4 to 8 show the results of perfusion-fixation of rats 3 hours after administration. From these results, it was found that the hydrocortisone-IgG conjugate of the present invention was introduced into the nucleus of hepatocytes and hepatic stellate cells without impairing its physiological activity. .

[0038]

In the past, even if a macromolecular substance was added to the extracellular fluid, the macromolecular substance could not be introduced into the nucleus of the target cell. The present invention makes it possible to easily introduce a macromolecular substance into the nucleus of a target cell by binding a bioactive macromolecular substance to a fat-soluble substance. It is possible to do. The nucleus of a cell plays a central role in the cell, such as controlling the expression of genetic information, and the method of the present invention makes it possible to directly introduce a physiologically active substance into the nucleus of a cell without impairing its physiological activity. It is possible to directly control the central functions of cells such as controlling the expression of genetic information. In addition, according to the method of the present invention, a macromolecular substance capable of exerting a physiological activity in the nucleus of a cell can be easily introduced. Method can be used.

[Brief description of the drawings]

FIG. 1 shows the results 2 hours after the administration of the conjugate of the present invention. In the hepatocytes, fluorescence of FITC-labeled IgG was observed in vesicles approaching the nucleus. Is a photograph replacing a drawing.

FIG. 2 shows two hours after administration of the conjugate of the present invention. In hepatocytes, FITC was added to the hepatocyte surface, vesicles approaching the nucleus, and hepatocyte nucleus.
It is a photograph instead of a drawing which shows that the fluorescence of labeled IgG was recognized.

FIG. 3 is a photograph instead of a drawing, showing that two hours after administration of the conjugate of the present invention, fluorescence was observed in nuclei in two hepatocytes.

FIG. 4 shows 3 hours after administration of the conjugate of the present invention, in which fluorescence is observed along the inside of the vesicle membrane of hepatocytes, and further, a part of the hepatocyte nucleus. 3 is a photograph instead of a drawing, showing that fluorescence was observed.

FIG. 5 shows the results 3 hours after administration of the conjugate of the present invention, in which fluorescence was observed in vesicles approaching the hepatic nucleus, and further, fluorescence was observed in a part of the hepatic nucleus. Is a photograph replacing a drawing.

FIG. 6 shows the results obtained 3 hours after administration of the conjugate of the present invention, in which fluorescence was observed in vesicles approaching the hepatocyte nucleus, and fluorescence was observed in almost the entire area of the nucleus. , Is a photograph replacing the drawing.

FIG. 7 is a photograph instead of a drawing, showing that fluorescence was observed in nuclei of hepatic stellate cells three hours after administration of the conjugate of the present invention.

FIG. 8 is a photograph instead of a drawing, showing that fluorescence was observed in a portion considered to be a nuclear membrane along the outer periphery of a hepatocyte nucleus 3 hours after administration of the conjugate of the present invention. It is.

Claims (18)

[Claims] 1. A method in which a polymer substance exhibiting a physiological activity in a cell nucleus is bound to a fat-soluble substance, and the polymer substance is introduced into a nucleus of a target cell. 2. The method according to claim 1, wherein the fat-soluble substance is a steroid hormone. 3. The method according to claim 1, wherein the polymer substance exhibiting a physiological activity in a cell nucleus is an antibody. 4. The method according to claim 3, wherein the antibody is IgG. 5. The method according to claim 3, wherein the antibody is IgM. 6. The target cell is a hepatocyte.
The method according to any of the above. 7. The target cell is a germ cell.
5. The method according to any one of 5. 8. A conjugate for introducing a polymer substance into a nucleus of a target cell, wherein the polymer substance having a physiological activity in a cell nucleus is chemically bonded to a fat-soluble substance. 9. The conjugate according to claim 8, wherein the polymer substance having a physiological activity in the cell nucleus is directly bound to a fat-soluble substance. 10. The conjugate according to claim 8, wherein a polymer substance exhibiting a physiological activity in a cell nucleus and a liposoluble substance are chemically bonded via a linker portion. 11. The conjugate according to claim 8, wherein the fat-soluble substance is a steroid hormone. 12. The conjugate according to any one of claims 8 to 11, wherein the polymer substance exhibiting a biological activity in a cell nucleus is an antibody. 13. The conjugate according to claim 12, wherein the antibody is an IgG. 14. The conjugate according to claim 12, wherein the antibody is IgM. 15. A carrier comprising a fat-soluble substance for transferring a macromolecular substance exhibiting a physiological activity in a cell nucleus into the nucleus of a target cell. 16. The carrier according to claim 15, wherein the fat-soluble substance is a steroid hormone. 17. The carrier according to claim 15, wherein the polymer substance exhibiting a physiological activity in a cell nucleus is an antibody. 18. A pharmaceutical composition comprising the conjugate according to any one of claims 8 to 14 and a pharmaceutically acceptable carrier.