JP3360152B2 - Cell damage inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a preparation containing a pterin derivative (including a neopterin derivative) as an active ingredient, which is effective for preventing and treating extrinsic and endogenous cell damage and for protecting cells.

[0002]

2. Description of the Related Art As the population ages, senile diseases such as dementia have become social problems. All diseases are due to the impairment of various organs as a result of the cells becoming damaged or degenerated or lost in some way and their original function is reduced or lost. Therefore, it is consensus that the suppression of cell function deterioration and cell death against various disorders is effective for treatment. Damage stress to cells includes familial genetic dysfunction syndrome, cell destruction due to physical stress, trophic factor depletion and cell damage due to free radicals seen in ischemia and hyperoxia disorder, and autoimmune diseases. Cell damage caused by killer T cells and the like, drug-induced disorders caused by pesticides, poisons, and deleterious substances, and viral disorders found in acquired immunodeficiency syndrome (AIDS) and the like.

Conventionally, various drugs have been used for treatment of these disorders. For example, treatment with a preparation such as superoxide dismutase or catalase has been considered effective for the treatment of disorders caused by free radicals, but the effective time of these enzymes in vivo is short and a large amount of administration is required. Further, since the enzyme is a protein, it has antigenicity, and when long-term administration is required, there are concerns about side effects such as anaphylactic shock caused as a result of an antigen-antibody reaction, and there is a problem in safety.

[0004] Interferon-β having antitumor activity and antiviral activity cannot be used for those with a history of hypersensitivity to biological products such as vaccines. On the other hand, regarding pterin derivatives,
JP-A-63-297326 discloses a pteridine relating to a method for producing a pharmaceutical composition having anti-amnesic activity, and JP-A-1-86887 discloses a pteridine derivative and a preparation containing these having antiarrhythmic and diuretic effects. . Also,
Recently, tetrahydrobiopterin (sapropterin hydrochloride) has been approved as a drug for the treatment of hereditary atypical hyperphenylalaninemia.

Furthermore, it has been recognized that pterin derivative preparations have excellent effects on the treatment of diabetes (type I), ischemia-reperfusion injury, liver injury and the like (Japanese Patent Application No. 04-17591).
No. 7). In addition, it has been confirmed that NPH4 is also effective for suppressing cancer metastasis and treating side effects of anticancer drugs (Japanese Patent Application No. 04-304525). However, it has not been found in academic journals or patent publications that pterin derivatives show a preventive and inhibitory effect on cell damage.

[0006] In the life sciences in general, various cells are cultured and used for analyzing biological phenomena and producing substances. However, culturing fragile cells has many difficulties with current technology. One of the problems is the operation of changing the cell culture medium to supply the cells with nutrients. As cell damage due to medium exchange, it is considered that damage is caused by generation of active oxygen due to entrainment of oxygen, physical shock by water flow, depletion of autocrine trophic factors, and the like. When culturing for a long period of time, it is necessary to constantly change the medium, and the medium replacement, which should be performed for cell protection by nutrient supplementation and waste removal, would rather damage cells. In particular,
Primarily cultured nerve cells, cells of the nervous system such as oligodendrocytes, and cells of the immune system are generally a group of cells that are highly susceptible to damage caused by medium exchange. Also, hybridomas producing monoclonal antibodies sometimes cause a decrease or denaturation of antibody production ability due to disturbance of medium exchange. These cells are very expensive cells due to their usefulness and fragility. Therefore, it is necessary to reduce the obstacles associated with the exchange of medium for precious cells. However, drugs that suppress such disability have not been studied so far.

[0007]

For example, cells such as nerve cells having a low self-renewal ability are very vulnerable to trophic factor depletion, damage to cell membranes by free radicals, physical shock, toxic substances, and the like. Easily leads to cell death and, consequently, tissue and organ dysfunction.
Therefore, an object of the present invention is to search for a substance having high safety and acting stably even in a living body and to formulate a formulation in order to contribute to effective treatment caused by these cell disorders.

[0008]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, the pterin derivative preparations were originally fragile cells and cells with reduced functions.
In addition to showing cytoprotective effects, it has cytotoxicity-inhibiting activity against various disorders, and furthermore, pterin derivatives and their salts can be easily added to cell culture media due to their high water solubility, and are formulated as injections. The present invention has been completed by clarifying what can be done. That is, the first of the present invention is:
Next formula

[0009]

Embedded image

(Wherein R ₁ , R ₂ and R ₅ , R ₆ represent H or together represent a double bond, and R ₃ , R ₄ represent H, alkyl, hydroxyalkyl, respectively). The present invention relates to a cytotoxic inhibitor containing a pterin derivative as a main component. The second aspect of the present invention relates to a cytoprotective agent containing a pterin derivative represented by the above formula as a main component, and is preferably added for cell protection in cell culture.

[0011] Examples of the pterin derivative used in these preparations include the following compounds. Pterin (R ₁ , R ₂ and R ₅ and R ₆ together represent a double bond, R ₃ and R ₄ represent H), 5,6,7,8-tetrahydropterin (R _{1 to} R ₆ are H), pterincarboxylic acid (R ₁ , R ₂ and R ₅ , R ₆ together represent a double bond, R ₃ represents COOH, R ₄ represents H), biopterin (R ₁ , R ₂ and R ₅ and R ₆ together form a double bond,
R ₃ represents CH (OH) CH (OH) CH ₃ , and R ₄ represents H
), 7,8-dihydrobiopterin (R ₁ and R ₂ together represent a double bond, and R ₃ is CH (OH) CH
(OH) CH ₃ , and R ₄ , R ₅ and R ₆ represent H),
5,6,7,8-tetrahydrobiopterin (R _1,
R ₂ , R ₄ , R ₅ and R ₆ represent H, and R ₃ represents CH (OH) C
H (OH) CH ₃ ), sepiapterin (R ₁ , R ₂
Are together a double bond, and R ₃ is COCH (OH) CH ₃
And R ₄ , R ₅ and R ₆ represent H), 1 ′, 2′-diacetyl-5,6,7,8-tetrahydrobiopterin (R ₁ , R ₂ , R ₄ , R ₅ , R ₆ Represents H, and R ₃ represents CH (O
Ac) CH (OAc) CH is _3), 6-methyl -
5,6,7,8-tetrahydropterin (R ₁ , R ₂ , R
₄ , R ₅ and R ₆ represent H, and R ₃ represents CH ₃ . ). In the formula, R ₃ represents D-erythro, L-threo, D
-Threo or L-erythro type may be used.

Among the pterin derivatives, neopterin derivatives include, for example, neopterin (R ₁ , R ₂ and R ₅ , R ₆ together represent a double bond, R ₄ represents H), 7, 8, -dihydroneopterin (R ₁ and R ₂ together represent a double bond and R ₄ , R ₅ and R ₆ represent H), 5,6,7,8-tetrahydroneopterin (R
₁ , R ₂ , R ₄ , R ₅ , and R ₆ represent H). In the formula, the 6-hydroxypropyl moiety is D-erythro, L-
Any of threo, D-threo and L-erythro type may be used.

The pterin derivative can be used as it is, but is produced as an inorganic acid salt such as hydrochloride or sulfate or an organic acid salt such as camphorsulfonic acid, tartaric acid, malic acid or oxalic acid. It can be the main agent of an oral preparation. Pterin derivatives can be prepared by known methods (eg,
Bernhard Schiriks, Jost H. Bieri und Max Viscontin
i, Helvetica Chimica Acta -Vol. 60, Fasc. 1 (1977)
-Nr. 24, -Vol. 61, Fasc. 7 (1978)-Nr. 257 and the production method introduced in JP-A-63-297326). Further, the neopterin derivative can be prepared by a known method (for example, M. Viscontini, R. Provenzale, S. et al.
Ohlgart und J. Mallevialle, Helvetica Chimica Acta
-Vol. 53, Fasc. 5 (1970)-Nr. 139, Bernhard Schiri
ks, Jost H. Bieri und Max Viscontini, Helvetica Ch
imica Acta -Vol. 59, Fasc. 1 (1976)-Nr. 27, Rembo
ld, Buschman, (1963), Ber., 96, 1406).

Pharmaceutical dosage forms include:
It can be used as powders, fine granules, granules, pills, tablets, capsules and suspensions, and as injections, it is manufactured by encapsulating in liquid solutions or liposomes or microcapsules. The adjuvants mixed with tablets, fine granules, capsules and the like are as follows. Gum arabic, tragacanth, gelatin, sucrose, methylcellulose, binders such as polyvinylpyrrolidone, lactose, sucrose, starch, dextrin,
Excipients such as microcrystalline cellulose, talc, titanium oxide, inorganic salts, disintegrants such as carboxymethylcellulose, sodium alginate, gelatinized starch, corn starch, lubricants such as talc, magnesium stearate, wax, Sugars, sweeteners like saccharin,
Citric acid, glutamic acid, peppermint, reddish oil,
In the case of flavoring agents such as cherry and capsules, in addition to the above materials, liquid carriers such as vegetable oils and fats can be contained. If necessary, dyes permitted for medical use are used.

Further, in order to change the physical form of the preparation, for example, as an enteric coating agent, known celluloses such as cellulose acetate phthalate, or acrylic materials such as methacrylic acid and ethyl acrylate copolymer. Is used. Tablets can be coated with shellac and sugar. Solutions such as syrups and elixirs can contain, in addition to the active substance, sucrose as a sweetening agent and methyl or propylparabens as preservatives, a dye and cherry or flavoring agents such as orange flavor. If necessary, surfactants, emulsifying or suspending agents such as methylcellulose, gum arabic and tragacanth can be used.

The sterile composition for an injection is prepared by dissolving or suspending the water-soluble active ingredient in water for injection as it is, and dissolving or suspending the active ingredient which is hardly soluble in water by using the emulsifier or suspending agent. , Palm oil, peanut oil, natural vegetable oils such as cottonseed oil, dissolved or suspended in propylene glycol or ethyl oleate, etc., if necessary, phosphates, acetates, buffers such as borates, salt, Isotonic agents such as glucose, soothing agents such as benzyl alcohol, procaine hydrochloride, preservatives, antioxidants such as ascorbic acid, dyes and the like can be added. Natural lecithin,
The drug may be encapsulated in a synthetic lecithin such as dipalmitoyl or distearoylresin, and liposome-ized to prepare an injection. In addition, microcapsules coated with ethylcellulose, polyisobutylene or the like can be used as oral or injectable preparations.

As an external preparation, vaseline,
Using paraffin, fats and oils, lanolin, macrogol, wax, etc., if necessary, in addition to the above-mentioned antioxidants, preservatives, suspending agents, fragrances, glycerin, D-sorbitol, propylene glycol Surfactants such as humectants, polysorbate 80, and sorbitan sesquioleate can also be included.

[0018]

The present invention will be described below in detail with reference to examples. Production Example 1 Production of Tablet of Pterin Derivative Main Formulation Tablet (1) Neopterin (2-amino-4-oxo-6- (D-erythro-1 ′, 2 ′, 3′-trihydroxypropyl) -pteridine) 10 g (2) Lactose 90 g (3) Corn starch 29 g ( 4) Magnesium stearate 1g 1000 tablets 130g Ingredients (1), (2) and 17g (3) are mixed and 7g of ingredients
Granulate with the paste made from (3), add 5 g of component (3) and component (4) to the granules and compress the mixture with a compression tablet machine to a diameter of 7 containing 10 mg of component (1) per tablet.
1000 tablets of mm were produced.

[0019] Mix all the ingredients and fill into 1,000 gelatin capsules No. 3 (12th edition of the Japanese Pharmacopoeia), 10m of ingredient (1) per capsule
A capsule containing g was prepared.

[0020] 3. Injection (1) 5,6,7,8-tetrahydroneopterin dihydrochloride 12 g (2) Sodium chloride 9 g Components (1) and (2) are dissolved in distilled water (1000 ml), and 1 ml per 1000 brown ampoules. The mixture was dispensed, replaced with nitrogen gas, and sealed. All steps were performed under aseptic conditions.

[0021] 4. External preparation (1) 5,6,7,8-tetrahydroneopterin 3 g (2) Polyethylene glycol 40 g (3) Stearate 3.1 g (4) Glyceryl stearate 7.7 g (5) Biphenyl alcohol 8.5 g (6 ) Squalene 12.3 g (7) Glycerin trioctanoate 12.3 g (8) Propyl paraben 0.1 g (9) Methyl paraben 0.1 g (10) Disodium EDTA 0.3 g (11) Dipropylene glycol 7.7 g (12) Citric acid 0.2 g (13) 1.4 g of sodium citrate The component (1) was dissolved or suspended in water, and 100 g of an ointment was produced together with other components according to a conventional method.

Example 1 Among the pterin derivative preparations produced in the same manner as in Production Example 1, 5,6,7,8-tetrahydro-D-neopterin (NPH4), 5,6,7,8-tetrahydro-6- Various effects were tested on formulations using biopterin (BPH4), 5,6,7,8-tetrahydropterin (PTH4), and 5,6,7,8-tetrahydro-6,7-dimethylpterin (DPH4).

1) Protective effect against cell damage caused by medium exchange (Method) Nerve cells prepared from rat fetal cerebral cortex
In a minimum essential medium (MEM) containing 10% fetal bovine serum on a well culture dish,
After culturing for 4 days, serum-free MEM containing insulin, transferrin, sodium selenate, and putrescine (ITS
Pterin derivatives (NPH4, BPH4,
PTH4, DPH4) dissolved sample solution (0.25ml
/ Well) and incubated at 37 ° C., 5% CO ₂ . One in which the medium was replaced with only ITSP-MEM was used as a test control.

By replacing the medium containing fetal calf serum with a serum-free medium, cell damage due to depletion of trophic factors, generation of free radicals due to physical shock at the time of addition of the medium and entrapment of oxygen, and the like can be considered. The concentration of each drug in the test was 100 μM or 100 units / ml. The effect of the pterin derivative was determined by ITSP-ME containing 100 μM pterin derivative.
All medium exchanges were performed at M. After completing the medium exchange, 37 ° C,
After 24 hours of incubation at 5% CO _{2, the} degree of neuronal damage was measured. The degree of damage was evaluated by measuring the lactate dehydrogenase activity in the culture supernatant after incubation for 24 hours, obtaining a relative value with the control being 100%. Furthermore, after removing the supernatant, 500 μl / well ITSP-ME
M was added, an MTT assay was performed, the mitochondrial enzyme activity of the cells was measured, and the relative value to the control was evaluated.

(Results) FIG. 1 shows the cell damage caused by medium exchange and the protective effect of drug addition. NPH4, B
In the medium containing PH4, PTH4, and DPH4, the damage caused by medium exchange was clearly reduced as compared with the medium containing nothing. Among them, NPH4, BPH4, and DPH4 exhibited strong neuroprotective effects. Since this action had no protective effect even when a large amount of SOD and catalase were added simultaneously, it was considered to be a protective action different from the active oxygen scavenging action. Thus, as one of the indicators of mitochondrial activity, N-
The effect of PH4 was measured (FIG. 2). NPH4 showed higher MTT reducing ability than the control. Simultaneous addition of SOD and catalase did not have such an effect. NP
H4 was considered to have a mitochondrial activating effect.

2) Inhibitory effect on nerve cell death from hydroxyl radical damage caused by hydrogen peroxide and iron sulfate (Method) Cortical neurons were prepared and cultured in the same manner as in 1) of Example 1. Cells cultured for 2 to 4 days were subjected to ITSP-
Replace with MEM (0.25 ml / well), 37 ° C, 5% CO ₂
For 30 minutes. Thereafter, the medium was removed, and the ITSP-MEM containing the pterin derivative, hydrogen peroxide and iron sulfate were mixed immediately before, and the mixture was immediately added to the cultured cells at 0.25 ml / well. After incubation at 37 ° C. and 5% CO _{2 for} 24 hours, the degree of neuronal damage was measured. The degree of damage was evaluated by measuring the lactate dehydrogenase activity in the culture supernatant after incubation for 24 hours, obtaining a relative value with the control being 100%.

(Results) The results are shown in FIG. Addition of hydrogen peroxide and iron sulfate to neurons caused cytotoxicity in a concentration-dependent manner. When 100 μM of NPH4 and BPH4 were simultaneously added, the death of nerve cells was suppressed. Also,
After the injury was first given for 30 or 60 minutes with ITSP-MEM containing 100 μM hydrogen peroxide and iron sulfate, the medium was removed and N
Even when PH4 was acted on, the cell death inhibitory effect was exhibited (FIG. 4). Furthermore, even before the neuronal cell was loaded with 100 μM NPH4 for 30 minutes before the loading of the injury with hydrogen peroxide and iron sulfate, the cell death inhibitory effect was exhibited. Therefore, it is clear that pterins reduce severe damage that causes nerve cell death in this way, and are effective for prevention and treatment.

[0028]

Industrial Applicability According to the present invention, a pterin derivative is used as an active ingredient, a cytotoxicity inhibitor effective for the prevention and treatment of exogenous and endogenous cytotoxicity, and a method for protecting cells by adding to a medium during cell culture. It was possible to provide a cytoprotective agent having an effect.

[Brief description of the drawings]

FIG. 1 is a graph showing a neuroprotective effect of a pterin derivative on cell damage caused by medium exchange.

FIG. 2 is a graph showing the effect of a pterin derivative on neuronal mitochondrial activity for a medium exchange disorder.

FIG. 3 is a graph showing the damage caused by hydrogen peroxide + iron sulfate and the cell death inhibitory effect of a pterin derivative.

FIG. 4 is a graph showing the administration of NPH4 before and after impairment of hydrogen peroxide + iron sulfate and the effect of inhibiting neuronal cell death. In the figure, all the loads of injury were performed by adding 100 μM hydrogen peroxide and 1 μM iron sulfate. NPH4 was dissolved in the medium and added. **; p <0.01, *; p <0.03

Continuation of the front page (56) Reference European Patent Application Publication No. 481791 (EP, A1) Biochemical Pharmology, May 25, 1993, Vol. 45, no. 10, p. 1953-1958 FEBS LETTERS, Vol. 2, 3, p. 163-166, 1992 (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 475/04 A61K 31/519 C12N 5/06 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] (1) A general formula: (Wherein, R ₁ , R ₂ and R ₅ , R ₆ are H or together represent a double bond, and R ₃ , R ₄ are each H, alkyl,
Shows hydroxyalkyl. ) A nerve cell damage inhibitor comprising a pterin derivative as a main component. 2. A compound of the general formula: (Wherein, R ₁ , R ₂ and R ₅ , R ₆ are H or together represent a double bond, and R ₃ , R ₄ are each H, alkyl,
Shows hydroxyalkyl. ) A neuroprotective agent for cell culture in a cell culture comprising a pterin derivative as the main component.