JPH0446121A - Agent for inhibiting multiplication of herpes virus - Google Patents

Abstract

PURPOSE:To provide the subject multiplication inhibitor useful for preventing and treating diseases caused by human herpes virus-6 (HHV-6) and immune- deficient state diseases by compounding a tetrapyrrole derivative or a salt thereof. CONSTITUTION:The objective inhibitor contains as an active ingredient a tetrapyrrole derivative which has a basic structure of the formula (R1, R2 are OH, substituted OH) and may have substituents (preferably lower alkyl, alkenyl, carboxyalkyl, etc.,) at the 2, 3, 7, 8, 12, 13, 17 and 18 positions thereof or a pharmacologicallly acceptable salt thereof, preferably biliverdin, The compound specifically inhibits the multiplication of a virus causing cataplectic anthema and the multiplication of HHV-6 causing diseases in immune-deficient states.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a herpesvirus growth inhibitor, and more particularly to a human herpesvirus-6 growth inhibitor containing a tetravirol derivative as an active ingredient.

[Prior Art and Problems to be Solved by the Invention] Herpesviruses are characterized by a lifelong latent infection and frequent reactivation when the virus becomes immunocompromised. Given these characteristics, it is predicted that herpesviruses will become more of a problem in the future due to immunosuppressive therapy used in organ transplants, cell transplants, etc., and with the increase in the number of AIDS (acquired immunodeficiency syndrome) patients. It's a virus. Conventionally, human herpesviruses include herpes simplex virus, varicella/herpes zoster virus, cytomegalovirus, and E.B.
(Epstein-Barr) virus was known. Recently, a new herpesvirus was isolated from the peripheral blood of patients with lymphatic disease, and subsequently, viruses with the same characteristics were successively isolated from AIDS patients. This herpesvirus infects lymphocytes (particularly T lymphocytes), and is different from conventional herpesviruses in terms of antigenicity, DNA structure, and specific cell tropism, so it is classified as a human herpesvirus. 6 (human herpe)
svlrus-6, hereinafter referred to as 1HHV-6). Subsequent research revealed that WHV-6 is the virus responsible for the sudden rash (X, YaI
Iranishl et at.

Lancet j: 1065-1067, 1988)
It has also been suggested that HHV-6 infection is involved in rejection reactions in patients who have received kidney transplants (T,
0kuno etat, Transplantat
ion, 49: 519-522.1990), and what has attracted further attention is that, as mentioned above, HHV-6
As shown in isolation from IDS patients,
This is the relationship between HHV-6 and the progress of AIDS. That is, H
HV-6 primarily infects CD4/1926 cells,
Takahashi et al, J.

Vlology, 63: 3181-3163.1
989), and the AIDS virus (HIV-1)
Iuman Immunodeficiency Vir
It is known that P, Lu5so e
tal, Nature, 337: 370-37
3.1989). Furthermore, HHV-6 infection is associated with HIV-1
It has also been reported that Ensoli plays an important role in the progression from HIV-1 infection to AlDS by promoting gene expression (B, Ensoli et at.
The EMBO journal, 8: 3019-
3027.1989). Thus, HHV-6
It has been suggested that it is closely related to the expression and progression of IDS and acts as a cofactor.

As described above, HHV-6 acts as a virus that causes sudden rashes and as a trigger for diseases that develop in immunodeficiency states, and it is important to suppress the proliferation of WHV-6 and HHV-6-infected cells in clinical practice. has important significance.

The present invention was created in view of the above-mentioned problems, and was completed after the inventors of the present invention repeatedly conducted various studies and discovered that a specific tetravirol derivative can suppress the proliferation of HHV-6. The object of the present invention is to provide an HHV-6 growth inhibitor.

[Means for Solving the Problems] WHV- of the present invention made to solve the above problems.
The growth inhibitor of No. 6 has a basic structure shown by the following structural formula (1), and has the following structural formula (2nd, 3rd, 7th, 8th, and
The active ingredient is a tetravirol derivative which may have substituents at the 12th, 13th, 17th and 18th positions, or a pharmacologically acceptable salt thereof.

(In the formula, R1 and R2 each represent a hydroxyl group or a substituted hydroxyl group) In the above structural formula (I), for convenience, symbols A to D are attached in order from the left ring. In addition, in the A ring and D ring at both ends of structural formula (1), when the group R1 and/or R2 is a hydroxyl group, the ring has an enol-keto tautomerism represented by the following partial structural formula (J and mountain). It is widely known to those skilled in the art that

(a) (b) In this specification, such tautomers will be represented by the partial structural formula (al) for convenience.

The active ingredient of the HHV-6 proliferation inhibitor of the present invention has a basic structure represented by the above structural formula (1), and the 2nd position of the formula,
It is a tetravirol derivative or a pharmacologically acceptable salt thereof which may have substituents at the 3rd, 7th, 8th, 12th, 13th, 17th and 18th positions. Pharmacologically acceptable salts include inorganic metal salts such as alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (magnesium salts, calcium salts, etc.), ammonium salts, and organic bases. Salts (e.g. trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, etc.)
, organic acid salts (e.g., formates, acetates, maleates,
(tartrate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.), inorganic acid salts (eg, hydrochloride, hydrobromide, sulfate, phosphate, etc.), and the like.

Also, the 2nd, 3rd, 7th, 8th, 12th, and 1st positions of structural formula H
Examples of groups that can be substituted at the 3rd, 17th, and 18th positions include various substituents, such as lower alkyl groups (e.g., methyl, ethyl, propyl, butyl, isobutyl, tertiary butyl). , pentyl, hexyl, etc.),
Alkenyl groups (e.g. vinyl, allyl, 1-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl,
phytyl, farnesyl, etc.), carboxyalkyl groups and esterified carboxyalkyl groups (e.g.
carboxymethyl, carboxyethyl, methoxycarbonylmethyl, ethoxycarbonylethyl, glucuronide carbonylethyl, etc.).

Examples of substituted hydroxyl groups for R1 and R2 include acyloxy groups (e.g., acetoxy, propionyloxy, benzoyloxy, etc.), alkoxy groups (e.g.,
methoxy, ethoxy, propoxy, impropoxy, butoxy, tertiary butoxy, pentyloxy, hexyloxy, etc.).

The tetravirol derivative, which is the active ingredient of the antiproliferative agent of the present invention, can be collected from natural ingredients such as bile pigments, or may be artificially synthesized. A particularly suitable compound is biliverdin (hereinafter referred to as BV), which has the advantage of being excellent in action, being a normal metabolite, and having few side effects.

In the antiproliferative agent of the present invention, the dosage of the active ingredient is appropriately selected and determined depending on the patient's age, weight, symptoms, degree of disease, administration route, administration schedule, formulation form, etc. For example, oral administration Generally 1 to 30 per day
It is considered to be about 0 mg/kg body weight.

The growth inhibitor of the present invention can be administered either orally or parenterally. For administration, the active ingredient can be mixed with a solid or liquid non-toxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration, injection, etc., and administered in the form of a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders, and capsules, liquid preparations such as solutions, suspensions, and emulsions, and lyophilized preparations. It can be prepared by conventional means. Examples of the above pharmaceutical non-toxic carriers include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acids, gelatin, and albumin. , water, physiological saline, and the like. Further, if necessary, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, and isotonic agents can be appropriately added. More specifically, when making an oral preparation, for example, the active ingredient is made into a suspension using a dispersant such as a nonionic surfactant, and sugar, sugar alcohol, silicic anhydride, and a nonionic surfactant are used. After adding a disintegrant such as an active agent, the powder is lyophilized and prepared into any dosage form (e.g., capsule, powder, coarse granule, granule, etc.) according to a conventional method.
Tablets, liquid preparations, etc.) are obtained by formulating them into JP-A-60-208910 and JP-A-61-2796.
By using the liposome formation method described in Publication No. 5, etc., a liposome preparation can be obtained.

In addition, when preparing an intravenous formulation, the active ingredient may be, for example,
It can be obtained by formulating it using conventional means such as emulsification with a surfactant, inclusion with cyclodextrin, liposome formation, and fat emulsion formation.

[Actions and Effects of the Invention] The tetravirol derivative, which is an active ingredient of the proliferation inhibitor of the present invention, has the effect of specifically suppressing the proliferation of WHV-6. Therefore, the antiproliferative agent of the present invention containing the tetrapyrrole derivative as an active ingredient is extremely useful for the prevention and treatment of diseases caused by HHV-6 and diseases caused by immunodeficiency.

[Examples] Hereinafter, the present invention will be explained in more detail based on Examples and Test Examples, but the present invention is not limited to these Examples.

Example I BV (20 g) was suspended in 5 W/V% polyoxyethylene polyoxypropylene glycol aqueous solution 50 yij and wet-pulverized using glass beads.
Sucrose fatty acid ester 3 was added to the obtained pulverized liquid 50'11.
0 g was added, frozen in a dry ice/methanol bath, and dried to prepare a lyophilized preparation.

Example 2 BV (1.8 mg) in 12.5% (V/V) dimethyl β
The mixture was heated and dispersed in Wittebusol W-35 halving base (45 mg) containing type cyclodextrin, and then halved portions were prepared by a conventional method.

Test Example The effect of the HHV-6 proliferation inhibitor of the present invention was tested by infecting MT-4 cells, a T lymphocyte cell line, with HHV-6 and testing B.
The test was conducted using a method to examine inhibition of HHV-6 growth by applying V to the cells. The methods and test results of the BV cytotoxicity test for MT4 cells and the HHV-6 growth inhibition test are shown below.

Test Example Cytotoxicity test of BV on MT-4 cells The cytotoxicity test of BV on MT-4 cells was conducted using a 0.1% trivan blue dye exclusion test.

That is, MT-4 cells were treated with various concentrations of BV (BV concentration:
0.10.20 and 40μg/111) and Fe
2 (fetal bovine serum, FC3 concentration 2 2%, 10% and 20
%) in RPMI-1640 medium for 2 days at 37°C. Then, immediately check the number of viable cells using a microscope.
The cell survival rate (%) was determined by calculating the ratio to the initial cell number. The results are shown in Table 1.

In addition, after BV was dissolved in methanol, it was added to the culture medium, and the final methanol concentration was adjusted to 0.25% (
The same applies to the following tests). In addition, MT-4 cells prepared by the method described in Test Example 2 (1)-■ below were used.

As shown in Table 1, cell viability was determined by FC3 in the medium.
Although it depends on the concentration, in a medium containing 10% or more Fe2, the cell survival rate did not substantially decrease even at a BV concentration of 40 μg/11, indicating that the cytotoxicity of BV to MT-4 cells was low. It became clear.

Test Example 2 Test of BV's growth inhibitory effect on WHV-6 In order to investigate the growth inhibitory effect of BV on WHV-6, a test was conducted using MT-4 cells infected with WHV-6. The materials used in the test, the test method, and the test results are as follows.

(1) Materials ■ Preparation of virus (WHV-6) Tantric blood mononuclear cells infected with HHV-6/H5T species isolated from patients with sudden rash were precipitated by centrifugation.
PBS containing 0.2% gelatin and 5% sucrose (
(phosphate buffered saline) (5 X 106 cells/yl). The suspension was sonicated for 30 seconds, then centrifuged at 2,000 x g for 20 minutes, and the resulting supernatant was stored at -70°C until use.

■Preparation of MT-4 cells MT-4 cells were prepared using the method previously used by Yoshi et al.
hietal, GANN Monogr, C2n
Cer Res, 28: 219-228゜1982
) using cells prepared according to 10% heat-inactivated F
C5SL-RPMI with glutamine and kanamycin
-1640 medium at 37°C.

■Preparation of a cell monolayer (ionolayer) A monolayer of MT-4 cells was prepared using the method of Asada et al. (H).

Asada et al., J.
cal Mlcrobiology, 27:22
04-2207.1989). Briefly, monolayers of cells were prepared on spot glass slides coated with poly-L-lysine dissolved in PBS. Poly-L-lysine solution (25 μD) was placed on each spot on the slide and incubated for 120 minutes at room temperature, followed by three washes with PBS.

On the other hand, MT-4 cells were washed three times and then suspended in Fe2-free RPMI-1640 medium (2.0×10
6 cells/11). The above cell suspension (25 μg) was applied to each spot coated with poly-L-lysine, and
Incubated at ℃ for 40 minutes. Unbound cells were then removed by washing three times with Fe2-free RPMI-1640 medium.

■Test method and results During and/or after adsorption (infection) of HHV-6 to MT-4 cells, MT-4 cells were treated with BV to determine the growth inhibitory effect against HHV-6 using immunofluorescence. Antibody (I
FA) method is used to form a focus (rocus-for
ming) number.

More specifically, BV treatment of MT-4 cells involves injecting a monolayer of cells into RPMI during and/or after adsorption of HHV-6.
BV (
Manufactured by Sigma, BV concentration: 0.10.20 and 40μg
/xi). Also, MT
For adsorption (infection) of HHV-6 to -4 cells, a monolayer on each spot slide was coated with HHV-6 prepared in (1)-■ above.
-6 to approximately 100 focus forming units (2 per spot)
The following (al
~(treated as in C1, then incubated.

(al) Infected in the above BV-containing medium (hereinafter referred to as drug medium) at 4°C for 2 hours, washed, and then 10%
Using RPMI-1640 medium containing FC3 (hereinafter referred to as control medium), in a Co2 incubator,
The system was incubated at 37°C for 2 days (hereinafter, this system was
(referred to as adsorption drug processing system).

Mountain) After infecting in drug medium at 4℃ for 2 hours, washing,
Next, a drug culture medium was used to incubate under the same conditions as above (al) (hereinafter, this system will be referred to as the drug treatment system during adsorption and after adsorption).

(After infecting in Cl control medium at 4°C for 2 hours, washing, and then incubating with drug medium under the same conditions as above (hereinafter, this system is referred to as post-adsorption drug treatment system)) .

After treatment and incubation as described above (al-(C)), cells were fixed and stained with indirect immunofluorescent antibody (IFA) method. The IFA method was performed according to the method of Asada et al. - A mouse monoclonal antibody was used as the secondary antibody, and a fluorescein-isothiocyanate-labeled goat antibody was used as the secondary antibody.

Measure the number of foci that emit fluorescent light, calculate the ratio to the number of foci in a system that does not contain BV (control),
It was expressed as focus formation rate (%).

Therefore, the lower the focus formation rate, the higher the effect of suppressing virus proliferation. The results are shown in Table 2.

Table 2 As shown in Table 2, “drug treatment system during adsorption” and “
In the drug treatment system during and after adsorption, the focus formation rate decreased significantly, indicating that the proliferation of HHV6 was suppressed by BV.
Depends on concentration.

However, in the "adsorption plate drug treatment system", the effect of suppressing virus proliferation was reduced compared to the above two systems.

MT-4 cells were pretreated with BV in 12 wells (2' diameter
12++m) multiwell plate (manufactured by Corning Glass). RPMI containing 10% FC3-
MT-4 cells <2.0×1.06 cells/l) in 1640 medium were treated with BV (IClug/11) at 4°C and 37°C for 0.5.1 and 2 hours. After treatment, cells were washed three times and treated with Fe2-free RPMI-16.
40 medium (2.0x106 cells/11).

Next, a cell monolayer was prepared in the same manner as in (1)-■ above.

Each monolayer on the spot slide contains RPM containing approximately 200 focus-forming units (20 μg/spot) of HHV6.
I-1640 medium (containing 2% FCS) was inoculated. After inoculation, spot slides were placed in a CO2 incubator for 3
After incubation at 7°C for 2 days, cells were fixed and stained as described above.

The number of fluorescent focuses was measured, and the ratio to the number of focuses in a system not pretreated with BV (control) was determined, which was defined as the focus formation rate (%). Therefore, the lower the focus formation rate, the higher the effect of suppressing virus proliferation. The results are shown in Table 3.

It was found that the proliferation of HHV-6 was significantly suppressed by pretreatment with .

The results in Tables 2 and 3 suggest that the growth-inhibiting effect of BV against HHV6 is based on suppressing the early stages of virus infection, such as the binding and invasion of virus particles into cells.

Claims (1)

[Claims] 1. It has a basic structure represented by the following structural formula, and substituents are present at the 2nd, 3rd, 7th, 8th, 12th, 13th, 17th, and 18th positions of the structural formula. An agent for inhibiting the growth of human herpesvirus-6, the active ingredient being a tetrapyrrole derivative or a pharmacologically acceptable salt thereof, which may have the following. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R_1 and R_2 each represent a hydroxyl group or a substituted hydroxyl group)