JP2000198748A - New medicine for genetic treatment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a genetic treatment medicine especially useful as an antitumor agent having high safety and giving excellent treating effects, while both of ganciclovir and acyclovir having been known as antitumor agents for genetic treatments have strong toxicity and are insufficient in medicinal effects. SOLUTION: This medicine for genetic treatments contains (-)-9-[1'S,2'R-bis(hydroxymethyl)cyclopropan-1'-yl]methylguanine as an active ingredient. This medicine is greatly excellent in usefulness when considering medicinal effects and safety in comparison with ganciclovir and acyclovir having been known as antitumor agents, and can be expected on practical uses as a genetic treatment medicine for treating, improving, preventing, or the like, other diseases as well as the tumor. The guanine derivative can be used as the medicine for the genetic treatments, and can further be expected to be an antitumor medicine, a medicine for inhibiting restenosis after PTCA or a medicine for controlling host diseases against transplants.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel gene therapy drug for tumors, restenosis after PTCA, and graft-versus-host disease, and more particularly to (-)-9- [1'S, 2'R-bis (hydroxymethyl) ) Cyclopropan-1′-yl] methylguanine or a drug for gene therapy containing a derivative that can be converted into the guanine derivative in the body of an animal, and more particularly, in a gene therapy, the guanine derivative is preferably used as a gene for phosphoric acid The present invention relates to an agent for gene therapy containing the guanine derivative, which is used together with a gene of an enzyme to be converted, more preferably a thymidine kinase gene, particularly a thymidine kinase gene of a virus (such as herpes).

[0002]

2. Description of the Related Art Clinical techniques for gene therapy for cancer include (1) direct targeting of cancer-related genes such as introduction of a p53 gene which is a tumor suppressor gene or an antisense gene of an oncogene into cancer cells. , (2) Introducing various cytokine genes and allogeneic tissue antigen genes into tumor-infiltrating lymphocytes (TIL) and cancer cells to enhance immunity against cancer, (3) Herpes virus thymidine kinase into cancer cells Introducing a gene (HSV-TK gene) to kill cancer cells by administering ganciclovir (GCV), a therapeutic agent for herpes virus, and introducing a suicide gene to cancer cells to make cancer cells drug-sensitive And (4) a method in which a multidrug resistance gene is introduced into hematopoietic cells to enhance the resistance to anticancer drugs, and then a large amount of chemotherapy is applied to cancer (Eiki Matsushita et al. (4): 433-4
38, 1997. ).

[0003] Using such a technique, a clinical protocol has been approved for various cancers such as melanoma, brain tumor, breast cancer, colon cancer, lung cancer, ovarian cancer, and renal cancer. The use of the above method (3) for hepatocellular carcinoma has been reported early (Huber BE, Richards CA, Krenisky TA: Re:
troviral mediated gene therapy for the treatment of
hepatocellular carcinoma: an innovative approach
for cancer therapy.Proc Natl Acad Sci USA 88: 80
39-8043, 1991. ), And clinical reports on brain tumors have recently been found (Ram Z. et al., Nat. Med. 3 (1
2), p1354-1361, 1997. ).

[0004]

As described above, the gene therapy is a therapy in which a gene is introduced into a human body, and various methods have been proposed, but none of them is a perfect method. Therefore, various methods have been studied, particularly the drug, the gene and the vector carrying the same, but at present, the research on the gene therapy drug used in combination with the method has not been advanced much. For example, ganciclovir has been reported (ST Clair MH, LambeCU, Furman PA
: Inhibition by ganciclovir of cell growth and DN
A synthesis of cells biochemically tranformed with
herpesvirus genetic information. Antimicro Agents
Chemother 31: 844-849, 1987. ) However, ganciclovir has myelosuppression and reproductive toxicity, which is a dose limiting factor. Acyclovir (ACV) is known as the same guanine antiviral agent as ganciclovir, but acyclovir has a very weak therapeutic effect. Under such circumstances, there is a need for the development of a gene therapy drug that is highly safe and has an excellent therapeutic effect, particularly as an antitumor agent.

[0005] An object of the present invention is to develop an excellent drug for gene therapy, and particularly to a drug having high utility for effectively exhibiting its effect in the human body, particularly from the aspect of both efficacy and safety. is there.

[0006]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and as a result, a compound represented by the following structural formula (1): (-)-9- [1'S, 2 'R-bis (hydroxymethyl) cyclopropan-1'-yl] methylguanine or a derivative that can be converted to the compound in an animal body (hereinafter sometimes abbreviated as “guanine derivative”) is used for gene therapy. Used as a drug component, the gene to be used is preferably a gene for an enzyme that phosphorylates the guanine derivative, in particular, the gene is preferably a thymidine kinase gene of a virus such as herpes, particularly HSV-1,
By using the guanine derivative together with the thymidine kinase gene of HSV-2 or VZV, the guanine derivative is phosphorylated in an animal such as a human, particularly in a cell at a diseased site such as a tumor cell, in particular, triphosphorylated, thereby inhibiting the growth of a DNA chain to be inhibited. It exhibits cytotoxicity by stopping, and also exerts an inhibitory effect on adjacent target cells in the same manner. Therefore, this guanine derivative is a gene for tumor, post-PTCA restenosis, and graft-versus-host disease. That it can be used as a therapeutic drug,
Based on this finding, the present invention has been completed.

[0007]

Embedded image

That is, the present invention relates to (-)-9- [1'S, 2'R-bis (hydroxymethyl) cyclopropan-1'-yl] methylguanine or a derivative which can be converted to the compound in an animal body ( Guanine derivative) and can be used for the treatment of tumor, restenosis after PTCA or graft-versus-host disease. The drug for gene therapy of the present invention includes the following inventions.

1. The above drug, wherein the gene therapy is a therapy by suicide gene therapy. 2. A therapeutic agent for humans, preferably for the treatment of tumor, restenosis after PTCA, and graft-versus-host disease ("treatment" used in the present invention includes treatment, improvement, prevention of deterioration, prevention, etc.) The use according to claim 1, wherein the use of the guanine derivative is included.). 3. 0.001 to 10,000 m per person per day
g / kg for parenteral administration, especially for intravenous administration, or 0.00
The above agent, which is for oral administration of 5 to 50,000 mg / kg.

[0010] 4. The above agent, wherein the gene used for gene therapy is a gene for an enzyme that phosphorylates the guanine derivative, preferably a thymidine kinase gene, more preferably a viral thymidine kinase gene. 5. The above agent, wherein the gene of the enzyme that phosphorylates the guanine derivative is a herpes (including HSV-1, HSV-2 and VZV) thymidine kinase gene. 6. The above agent, wherein the thymidine kinase gene is a gene for introducing into a target cell with a viral vector.

[0011] As the gene of the enzyme, a viral thymidine kinase gene such as the above herpes is preferably employed. The gene can be transferred to a cell required by using a vector (such as a viral vector) appropriate for the target cell. It can be transported or introduced directly into the required cells in the body, for example, by liposomes. 7. The above agent, wherein the guanine derivative is a derivative of the compound of the above formula (1), wherein at least one of the hydroxyl groups of the hydroxymethyl group is esterified with an amino acid.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the drug for gene therapy of the present invention will be described. The drug for gene therapy of the present invention is a drug used for gene therapy, and is used for treatment, that is, for treatment, improvement, and prevention of various diseases using a conventionally known method or a gene therapy method to be developed in the future. Drug The drug of the present invention is a guanine derivative (-)-9- [1'S,
2′R-bis (hydroxymethyl) cyclopropan-1′-yl] methylguanine or a derivative convertible to the compound in the animal body, preferably a gene for an enzyme that phosphorylates the compound, Preferably, it is a gene therapy drug used together with the thymidine kinase gene, particularly preferably the thymidine kinase gene of a virus (herpes etc. described above).

The agent for gene therapy of the present invention can be used in various gene therapy methods, preferably for introducing a suicide gene into target cells such as tumor cells and suppressing the target cells. At this time, the guanine derivative used in the present invention is used as a prodrug, and the guanine derivative used in the present invention is preferably converted into triphosphoric acid in a target cell by the action of an enzyme gene to form a drug. It is. In this case, it is necessary to select a suicide gene and a prodrug. Preferably, herpes simplex thymidine kinase can be used as the suicide gene. The use of ganciclovir was reported in this method (Ram
Z. et al., See above report. ), The drug for gene therapy of the present invention can be prepared and used according to this method.

When the agent of the present invention is used for gene therapy, treatment of human and other animal diseases (the treatment, improvement,
Including use for prevention and prevention of deterioration. The present invention can be widely used for gene therapy for the purpose of (1), that is, for treatment, improvement, prevention of deterioration, prevention and the like of diseases. As a disease to which this method is applied, any disease can be used as long as it is a gene-utilizing disease. However, it can be expected to be preferably used for various tumors, restenosis after PTCA, graft-versus-host disease, and the like. The tumor includes benign and malignant tumors and cancers occurring at various sites. Regarding graft-versus-host disease, in organ transplantation, the types of organs and cells are not particularly limited.

The guanine derivative used in the present invention as an active ingredient of a drug can be used in the free form represented by the structural formula (1), but may be a pharmaceutically acceptable salt, ester or other pharmaceutically acceptable salt. In the form of a related derivative or other derivative that can be converted into the compound represented by the structural formula (1) in the body of an animal such as a patient, for example, in the compound represented by the structural formula (1), both or one of the hydroxyl groups of the hydroxymethyl group is Derivatives esterified with amino acids or isomers in which the carbonyl group present on the guanine ring resonates with the hydroxyl group and the hydroxyl group is substituted with a hydrogen atom (having a purine ring) (JP-A-5-78357, 6-22798)
2 and JP-A-7-188231. ) Can be used, and all of these forms are also included in the guanine derivative used as the active ingredient of the drug of the present invention.

When administered to humans, both oral and parenteral administration are possible. In the case of manufacturing such a preparation, a required oral or parenteral preparation can be manufactured using various known preparation techniques.

The amount to be used is appropriately selected depending on the condition, severity, health condition, etc. of the patient. In the case of parenteral administration, especially intravenous administration, preferably about 0.001 to 10,000 mg / kg per day. , More preferably about 0.01 to 1000 mg / kg, and even more preferably about 0.1 to 20 mg / kg. Similarly, when administered orally, it is preferably 0.00
About 5 to 50,000 mg / kg, more preferably about 0.0
About 5 to 5000 mg / kg, more preferably 0.1 to
About 2000 mg / kg can be used. 1 per day
The administration can be divided into several times to several times.
It may be administered 1 to 3 times per day. It can be administered intermittently or intensively depending on the symptoms and the like.

When the agent of the present invention is administered, it is necessary to introduce the virus thymidine kinase gene. Particularly, when a virus vector-producing cell is locally transplanted, it is necessary to sufficiently spread the virus vector to the affected part. Therefore, it is preferable to administer the above drug after transplantation of vector-producing cells.

To introduce the thymidine kinase gene,
Conventionally known introduction methods and introduction methods such as vectors to be developed in the future can be adopted. Tumors (see International Patent Application No. 9-504518), restenosis after PTCA (see International Application No. 9-504558), and HSV-1
For the introduction of thymidine kinase, in particular, a method of infecting a virus vector systemically or locally can be considered, such as a method of directly injecting a virus vector solution to be used, a method of directly transplanting a virus vector producing cell into a tumor, There is also a method of improving local infectivity of a virus vector by wrapping and retaining the virus vector in a hydrogel. Furthermore, for the indication in the case of graft-versus-host disease, there is also a method of ex vivo gene introduction into a graft taken from a donor, followed by transplantation into a patient (see WO 97/45142).

In addition to viral vectors, various known methods such as direct gene injection into cells, particularly into cell nuclei, erythrocyte ghosts, liposomes, polycations, and immunogenes (antibodies in which the target gene is conjugated to an antibody) are also known. A method of locally delivering the HSV-1 thymidine kinase gene by the physical preparation technique described above can also be adopted.

Although various reports have been found on the above, recently published patent information (International Application Publication No. WO93 / 10218,
WO96 / 05321, WO97 / 45142 and WO97 /
See 440065. ) Or gene therapy using ganciclovir (Ram Z. et a
l., Therapy of malignant brain tumors by intratumo
ral implantation of retroviral vector-producing ce
lls, Nature Medicine, vol. 3, No. 12, 1354-1361, 199
See 7th magnitude. ) Can also be referred to. In this case, it is helpful to use the drug component (guanine derivative) used in the present invention instead of ganciclovir as the drug component.

The gene therapy drug of the present invention preferably exerts an effect on the treatment of tumors regardless of various types of tumors, malignant and non-malignant, but may be used in combination with the above-mentioned thymidine kinase and various cytokines such as interleukins. It can also be carried on vectors. The method is described in JP-T-Hei 9-5.
The description in the specification of JP 04518 is referred to. Tokiwa Table 9
In the specification of 504518, the method of using a vector is particularly useful, but when a cytokine is not used in the present invention, it is possible to refer to a part other than the part relating to the combined use of the cytokine. In particular, the moieties that use ganciclovir as an interacting agent can be helpful for the use of the guanine derivatives used in the present invention. Therefore, the description contents that are useful for reference of the present invention regarding the use of a vector by thymidine kinase and the like are incorporated in the present specification and constitute a part of the present specification.

When the drug for gene therapy of the present invention is used, it can be used alone, but at the same time, it inhibits the growth of the target tumor cell by using the drug or therapy / treatment on other tumor cells in combination. Can interfere with or destroy it.

In addition to the drug for gene therapy, a guanine derivative (-)-9- [1'S, 2'R-bis (hydroxymethyl) cyclopropan-1'-yl] methylguanine or the compound in an animal body An antitumor containing said convertible derivative,
An agent for suppressing restenosis after PTCA or controlling graft-versus-host disease is also included in the present invention. In this case, the drug for gene therapy is described as an example of the present invention: an antitumor, antiarteriosclerotic or antiallergic drug containing the guanine derivative.

As a detailed description of the drug other than gene therapy, that is, the invention of the drug for controlling antitumor, restenosis after PTCA or controlling graft versus host disease, guanine derivatives used in the drug component, Description of its dosage form, formulation, daily dose, antitumor as indication, indications such as suppression of restenosis after PTCA or control of graft versus host disease,
As described in the description of the drug for gene therapy. That is, in the description of the drug for gene therapy, all descriptions other than the description on the gene to be used and the method of using the gene (including the use of the vector gene) are incorporated in the description of the present invention unless particularly unnecessary or inconsistent. Is done.

[0026]

The present invention will be specifically described below with reference to examples and comparative examples.

(Example 1) Cytotoxicity against HSV-1 thymidine kinase gene transfected cells
The cytotoxicity test of the guanine derivative (free form of the compound represented by the structural formula (1)), ganciclovir and acyclovir (Comparative Examples 1 and 1 ′) used in the present invention was performed on HSV-1 thymidine kinase gene-transfected cells. went.

The cells were derived from human small cell lung tumor derived RERF-LC-LC-
RERF-LC-MA / LTRNL cells prepared by infecting MA cells with a viral vector having a thymidine kinase gene incorporated into a vector derived from Moloney mouse leukemiavirus were used. RERF-LC-MA / LTRNL cells were used as control cells into which the HSV-1 thymidine kinase gene was not introduced. The method for producing these cells and the measurement of cytotoxicity were determined by the method reported by Hasegawa et al. (Hasegawa Y. et a
l., Am. J. Respir. Cell. Mol. Biol. 8 (6) (1993)
See p655-661. A). Based on this cytotoxicity measurement method, MTT (3- (4,5-dimethylthiazol-2-yl)-
Cytotoxicity was confirmed by a colorimetric method using 2,5-diphenyl-tetrazolium bromide).

[0029] RERF-LC-MA / LTRNL cells were cultured in a culture medium (E-ME
M, 10% FBS) and seeded 2 × 10 ³ cells per well in a 96-well plate. Next, the drug components (guanine derivative), ganciclovir and acyclovir used in the present invention were added to the wells at various concentrations. Next, the cells were cultured in a humid environment at 37 ° C. and 5% CO ₂ for 4 days.
On the fourth day, cytotoxicity was confirmed by a colorimetric assay using MTT.

RERF-L expressing HSV-1 thymidine kinase
The IC50 values of the guanine derivative, ganciclovir, and acyclovir used in the present invention for C-MA / LTRNL cells were 0.66 μg / ml, 3.2 μg / ml, and 125 μg / ml or more, respectively (see FIG. 1). On the other hand, RERF-L of control
The IC50 values of the guanine derivatives, ganciclovir and acyclovir used in the present invention for C-MA / LTRNL cells are as follows:
They were 135 μg / ml, 201 μg / ml and 250 μg / ml or more, respectively. From these results, it was found that the guanine derivative used in the present invention was superior in cytotoxic effect as compared with the conventional product.

Example 2 Generation of Mouse Granulocyte Macrophage Progenitor Cells (CFU-GM)
Long inhibitory effect The guanine derivative (free form of the compound represented by the structural formula (1)), ganciclovir and acyclovir (Comparative Examples 2 and 2 ′) used in the present invention were treated with mouse granulocyte macrophage precursor cells (CFU-GM). ) Were tested for growth inhibitory effects.

Colony formation of mouse granulocyte macrophage precursor cells (CFU-GM) was carried out by the method of Okano et al.
See, e.g., Transplantation 48 (1989) p495-498. ).

The growth inhibitory effect of CFU-GM was measured as follows. Mouse C57BL / 6N Crj Bone marrow cells were prepared from 10-week-old female femur bone marrow, and guanine derivatives at various concentrations were prepared.
Drug-containing semi-solid medium containing ganciclovir or acyclovir (Iscove's modified Dulbecco's medium, 0.
8% carboxymethylcellulose, 20% FBS, 200unit / ml
Mouse interleukin-3) was seeded at a concentration of 4 × 10 ⁴ cells / ml. 37 ° C, 5% CO ₂ in a humid environment on a 35 mm plate
For 7 days, and the number of appearing CFU-GM colonies was counted under a phase-contrast microscope.

A guanine derivative for colony formation of mouse granulocyte macrophage progenitor cells (CFU-GM),
The IC50 values of ganciclovir and acyclovir are respectively
They were 66 μg / ml or more, 18 μg / ml and 26 μg / ml (see FIG. 2). From the above results, it is expected that the guanine derivative used in the present invention has lower bone marrow toxicity when administered than ganciclovir or acyclovir, and this indicates that the safety is extremely high.

As is clear from the results of the above Examples and Comparative Examples, particularly the results of FIGS. 1 and 2, the drug of the present invention, that is, the drug for gene therapy containing a guanine derivative, considers both therapeutic effect and safety. Then, it turns out that it is extremely excellent in utility compared with conventionally known ganciclovir and acyclovir.

[0036]

According to the present invention, (-)-9- [1'S, 2'R-bis (hydroxymethyl) cyclopropan-1'-yl] methylguanine (including salts and derivatives which can be converted into the compound in the body) .)
The drug for gene therapy containing is superior in terms of usefulness in consideration of both drug efficacy and safety as an antitumor agent, in comparison with the case where conventionally known ganciclovir or acyclovir is used. In addition to various types of tumors at various sites as well as other treatments, it can be expected to be used widely for drugs such as treatment, improvement, prevention of deterioration, prevention and prevention, especially for humans, such as restenosis after PTCA and graft-versus-host disease.

Further, the guanine derivative can be used as the above-mentioned drug for gene therapy, and is widely used for treating, improving, preventing deterioration, such as tumor, restenosis after PTCA, and graft-versus-host disease.
It can also be used as an active ingredient of drugs for prevention and the like.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of a cytotoxicity test on HSV-1 thymidine kinase gene-transfected cells performed in Example 1. In the figures,-○-: the present invention;-◇-: acyclovir; and-▲-: ganciclovir.

FIG. 2 shows the results of a test on the growth inhibitory effect of mouse granulocyte macrophage progenitor cells (CFU-GM) performed in Example 2. In the figure,-●-: the present invention;-□
-: Acyclovir; and-△-: ganciclovir.

Claims (4)

[Claims] (1) It is characterized by containing (-)-9- [1'S, 2'R-bis (hydroxymethyl) cyclopropan-1'-yl] methylguanine or a derivative which can be converted into the compound in an animal body. For gene therapy. 2. The drug according to claim 1, wherein said gene therapy is treatment by suicide gene therapy. 3. The medicament according to claim 1, which is used for treating any one of a tumor, restenosis after PTCA, and graft-versus-host disease. 4. The drug according to claim 1, wherein the gene used for the gene therapy is a thymidine kinase gene.