JPH092955A - Preventing and therapeutic medicinal composition for psoriasis - Google Patents

Abstract

PURPOSE: To obtain a suppressant, comprising an ecdysteroid analog, having both differentiation promoting and division suppressing actions on keratinocytes sufficient to ameliorate psoriasis and high safety and effective in preventing and treating psoriasis. CONSTITUTION: This medicinal composition comprises an ecdysteroid analog, preferably the one selected from polyporusterone A of formula I, polyporusterone B of formula II, 20-hydroxyecdysone of formula III, ponasterone A of formula IV and ponasteroside A of formula V. Furthermore, the analog in the case of, e.g. the polyporusterone A or polyporusterone B is obtained by extracting a natural substance such as Polyporus umbellatus FRIES with a polar solvent, then treating the resultant extract solution by column chromatography, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a keratinocyte differentiation-promoting / mitotic-suppressing agent comprising an ecdysteroid analog and a pharmaceutical composition for preventing or treating psoriasis, which contains the agent as an active ingredient.

[0002]

2. Description of the Related Art Psoriasis is one of the illnesses whose causes are difficult to treat. At present, steroids are often used for the treatment of psoriasis, but the use of steroids can lead to serious side effects and therefore their use requires extreme caution. In addition, vitamin A derivatives such as retinoids have come to be used for the treatment of psoriasis,
Due to teratogenic problems, the use of retinoids may be limited and has not been an effective means of treating psoriasis.

By the way, as a drug for treating psoriasis,
It is known that a substance having a vitamin D-like action, that is, a substance having both actions of promoting differentiation of keratinocytes and inhibiting keratinocyte division is substantially effective. ing. However, a substance having such an action is not well known, has a stronger action than that of vitamin D with respect to the above action, and does not induce serious side effects such as hypercalcemia occurring when vitamin D is administered. Despite the long-awaited appearance of substances, no substance having the action of vitamin D or higher has been found.

As described above, conventional drugs for treating psoriasis have a certain degree of therapeutic effect, but their use is limited due to the side effects, and even if there is no problem in safety, they can be treated. If the effect is not sufficient, there is a problem in practicality such as usability and effectiveness. Therefore, development of a safe therapeutic drug for psoriasis with excellent effect of preventing or treating psoriasis and no side effect is desired. It was

Here, the pharmaceutical composition for preventing or treating psoriasis used in the present specification is not a disease in which psoriasis spreads to many parts at one time, but it initially develops in a very limited site and is present in the body. Due to its nature of spreading to psoriasis, it may be administered in the early stages of onset to prevent the exacerbation of psoriasis symptoms, or it may have spread to partial psoriasis symptoms or already in many parts at the onset of psoriasis. A pharmaceutical composition having a function of curing the symptoms of psoriasis by administration thereof.

On the other hand, although it is known that ecdysteroid analogs have a hair growth promoting action and a skin-beautifying action, this ecdysteroid analog promotes differentiation of keratinocytes and keratinocyte It has not been known at all that it also has an action of suppressing division, and there are no reports of using these for the prevention or treatment of psoriasis.

[0007]

The present invention has been made from the above viewpoint, has a keratinocyte differentiation promoting action and a keratinocyte division inhibitory action sufficient to ameliorate psoriasis, and is highly safe. It is an object of the present invention to provide a drug having the above-mentioned property and a pharmaceutical composition for preventing or treating psoriasis, which is effective for the prevention and treatment of psoriasis and can be used safely for a long period of time.

[0008]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have sought a substance having both of the above-mentioned actions with the keratinocyte differentiation promoting action and the keratinocyte division suppressing action as indicators. As a result of intensive studies, ecdysteroid analogs, particularly polyporsterone A, polyporsterone B, 20-hydroxyecdysone, ponasterone A, ponasteroside A and the like, promote the differentiation of keratinocytes and keratinocytes. The present invention has been completed by discovering that it is excellent in the action of suppressing the division of

That is, the present invention is a keratinocyte differentiation-promoting / mitotic-suppressing agent comprising an ecdysteroid analog and a pharmaceutical composition for preventing or treating psoriasis, which contains the agent as an active ingredient.

The present invention will be described in detail below. (1) Keratinocyte differentiation promoting / mitotic inhibitor of the present invention The keratinocyte differentiation promoting / mitotic inhibitor of the present invention comprises an ecdysteroid analog. The ecdysteroid analog is a generic term for steroids having a structure similar to that of ecdysteroids, which are metamorphic hormones such as ecdysone involved in molting of insects, and the like. Examples of ecdysteroid analogs that can be:
Examples include paristerone, ponasterone, inocosterone, maxisterone, epiecdysone, pterosterone, tachysterone, piperidone, hydroxyecdysone, podoecdysone, sidasterone, and their glycosides, and their acetylated and substituted benzoyl acylated products. To be Further, those obtained by modifying the free hydroxyl group possessed by paristerone or the like by a usual means, for example, compounds obtained by acylation, alkylation, phenylation or phenylalkyl etherification, etc. may be mentioned as the ecdysteroid analogs. it can.

As mentioned above, a large number of compounds can be mentioned as ecdysteroid analogs, but as the keratinocyte differentiation promoting / mitotic inhibitor of the present invention, one of these compounds can be used alone. It is also possible to use two or more of these as a mixture. Among such ecdysteroid analogs, in the present invention, polyporsterone A represented by the following formula (1), polyporsterone B represented by the following formula (2), and the following formula (3 20-hydroxyecdysone represented by the formula (4), ponasterone A represented by the formula (4) below, and ponasteroside A represented by the formula (5) below.

[0012]

[Chemical 6]

[0013]

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[0014]

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[0015]

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[0016]

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Here, all of the above ecdysteroid analogs are known compounds and they are easily available. For example, since many ecdysteroid analogs are present in natural animals and plants, they can be obtained by extracting ecdysteroid analogs from these animals and plants. For example, polyporsterone A and polyporsterone B are ponasterone A from Chorei (Polyporus umbellatus FRIES).
Is obtained by extracting from Podocarpus nakaii and Ponasteroside A by extracting from bracken (Pteridium aquilinum). As a method for obtaining an ecdysteroid analog from a natural product such as the above-mentioned animals and plants, the starting natural product material is extracted with a polar solvent, and the resulting extract is subjected to silica gel, ODS, alumina, ion exchange resin, etc. A general method of applying column chromatography using the as a carrier may be used.

Further, ecdysteroid analogs thus obtained from animals and plants are chemically induced by a conventional method,
For example, a derivative of the above compound can be obtained by acylation, alkylation, phenylation or phenylalkyl etherification.

The ecdysteroid analogs used in the present invention may be produced in this manner, but since many of them are already on the market, they promote differentiation of keratinocytes of the present invention. It is also possible to use these commercially available products as mitotic inhibitors. As described below, each of these ecdysteroid analogs has both an action of promoting keratinocyte differentiation and an action of suppressing keratinocyte division as in the case of vitamin D. Both effects are superior to vitamin D as a whole, and are highly safe. (2) Pharmaceutical composition of the present invention The pharmaceutical composition of the present invention is one or two of the keratinocyte differentiation-promoting / mitotic-suppressing agents consisting of the above ecdysteroid analogs.
A pharmaceutical composition for preventing or treating psoriasis, which comprises one or more species as active ingredients.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a dosage form used as a usual pharmaceutical composition,
For example, oral administration agents such as powders, tablets, granules, capsules, drenches, sublingual agents, rectal administration agents, ointments, creams,
It can be used in any dosage form such as an external preparation for skin such as a liquid preparation, an injectable preparation such as a sterile solution and a suspension. Further, among these dosage forms, a more preferable dosage form in the present invention is a skin external preparation that can be directly administered to the affected area.

In addition to the keratinocyte differentiation-promoting / mitotic-suppressing agent of the present invention, which is an active ingredient, the pharmaceutical composition of the present invention may contain optional components for formulation which are usually used in pharmaceutical compositions. Is possible. A method for producing the pharmaceutical composition for psoriasis of the present invention, which comprises the keratinocyte differentiation promoting / mitotic inhibitor of the present invention and optional components as described above, according to a conventional method for producing a pharmaceutical composition. It should be manufactured.

The optional components used in the pharmaceutical composition of the present invention include one or more generally acceptable pharmaceutically acceptable vehicles, vehicles, carriers, excipients, binders, disintegrants, lubricants, Examples thereof include flavoring agents, coloring agents, coating agents, emulsifying / solubilizing / dispersing agents, bulking agents, sugar coating agents, pH adjusting agents, isotonic agents, stabilizers and the like. For example, the above-mentioned keratinocyte differentiation promoting / mitotic inhibitor and corn starch, a binder such as gelatin, an excipient such as microcrystalline cellulose, a potato starch, a disintegrating agent such as sodium carboxymethyl cellulose, a flavoring agent such as lactose and sucrose. The agent and the like can be dispensed to prepare powder, tablets, granules, and capsules. In addition, in the case of an injection, distilled water for injection, polyethylene glycol or the like is used as a solvent, or a dispersant, a buffer, a preservative, an antioxidant or the like may be added thereto as required. . When used as an external preparation, in addition to the above-mentioned keratinocyte differentiation promoter / mitotic inhibitor, lower alcohols such as ethanol and isopropanol, thickeners, oils and fats, higher alcohols, higher fatty acids, and surfactants , An antiseptic, an antioxidant, an ultraviolet absorber, a coloring agent, a fragrance, a water-soluble polymer, powders, and an adhesive can be added.

The injection may be prepared by solubilizing or dispersing it in an aqueous carrier or other inert diluent in advance, or may be prepared by adding the aqueous carrier or the like at the time of use. Further, the injection route may be any of intravenous injection, arterial injection, portal vein injection, subcutaneous injection, intramuscular injection, and intraperitoneal injection. As a method for administering such an injection, bolus administration or drip administration may be used.

The preferred dose of the pharmaceutical composition of the present invention varies depending on the physical condition, body type, symptom, etc., but is generally 10 to 1000 mg for oral administration and 1 to 500 mg for injection. In the case of transdermal administration, an appropriate amount of 0.1 to 20% by weight blended to spread evenly on the affected area can be administered once or several times a day.
Within this range, the pharmaceutical composition of the present invention can be safely used.

Such a pharmaceutical composition for the prevention or treatment of psoriasis of the present invention can be administered at an early stage of the onset of psoriasis such that the symptoms of psoriasis are still mild or have not spread to the whole body. , The symptoms of psoriasis spread to many parts,
It works to prevent the symptoms from becoming more severe, and it also works to cure the symptoms in a mild manner. In addition, even for patients who have already spread to many parts of the body or whose symptoms have become severe, the administration of the pharmaceutical composition of the present invention can reduce the symptoms of psoriasis or partially or totally. It is possible to treat the disappearance of various symptoms.

[0026]

Embodiments of the present invention will be described below. First, examples of the keratinocyte differentiation promoting / mitotic inhibitor of the present invention will be described.

[0027]

Example 1 Polyporsterone A, Polyporsterone B To 100 kg of Chorei, a 10-fold amount of 50% aqueous ethanol solution was added, and heated under reflux for 2 hours with stirring to extract soluble components. The obtained extract was filtered to remove insoluble matter, and the filtrate was concentrated to obtain an extract concentrate.

After this was dispersed in 20 L of water, an equal amount of diethyl ether was further added to separate the layers. Only the diethyl ether layer was taken out from this, and after concentration under reduced pressure, a silica gel column (elution solvent; chloroform: methanol =
1: 0 → 0: 1) followed by preparative liquid chromatography equipped with an ODS column.
8 mg of polyporsterone A and 276 mg of polyporsterone B were obtained.

[0029]

Example 2 Ponasterone A 10 L of methanol was added to 1 kg of a dried product of Togaribamaki, and the mixture was heated under reflux for 2 hours with stirring to extract soluble components. After the obtained extract was filtered to remove insoluble matter, the filtrate was subjected to Diaion HP-20 column chromatography (Mitsubishi Kasei, elution solvent; water: methanol 80: 2).
0), silica gel column chromatography (elution solvent; chloroform: methanol = 100: 0 → 0: 10).
0), followed by preparative high performance liquid chromatography equipped with an ODS column (elution solvent; acetonitrile: water = 1)
(0: 90 → 90: 10) to obtain 2 g of ponasterone A.

[0030]

Example 3 Ponasteroside A Using 1 kg of the dried product of bracken, the same extraction and purification treatments as in Example 2 were carried out to obtain 10 mg of ponasteroside A. <Evaluation of Keratinocyte Differentiation Promoting / Division Inhibitor of the Present Invention> As the keratinocyte differentiation promoting / mitotic inhibitor of the present invention, polyporsterone A, polyporsterone B obtained in each of the above Examples, Using ponasterone A, ponasteroside A and commercially available 20-hydroxyecdysone (manufactured by Sigma), the effect of promoting keratinocyte differentiation, the effect of suppressing keratinocyte division and safety were evaluated. (1) Effect on differentiation of keratinocytes Using a normal human epidermal keratinocyte culture kit (Morinaga Biochemical Research Laboratories), promotion and division of keratinocytes of the present invention against cell differentiation of normal human keratinocytes The action of the inhibitor was examined in two experiments, a method of adding calcium to the medium and a method of not adding calcium.

First, calcium is added to the medium. The normal human epidermal keratinocytes are seeded in a 24-well multiplate at a density of 2 × 10 ⁴ cells / well, and MCDB153 medium attached to the kit. After culturing for about 3 days at 50 ° C and confirming that it was semi-confluent, the medium was added to MCDB15 containing Ca at a concentration of 2 mM and polyporsterone A in DMSO at 0.1%.
The medium was changed to 3 and the cells were further cultured for 6 days. The concentration of polyporsterone A in the culture medium in this culture was various concentrations shown in Table 1. On day 6 after changing the medium, the cultures were trypsinized into single cells and the total number of cells in the wells was counted. After that, add 1% SDS,
After treatment with 1% β-mercaptoethanol (BME) for 20 minutes at room temperature, the number of remaining cells resistant to SDS and BME treatment, that is, the number of cells that formed a fully differentiated conicified envelope was measured. I counted each. This number was divided by the total number of cells in each well, and the obtained value was defined as the cornified envelope formation rate.

Further, as a control, all tests were carried out in the same manner except that a medium obtained by removing the polyporsterone A from the above-mentioned medium was used as the medium after the change in the above-mentioned test, and the control The fido envelope formation rate was calculated. Polyporsterone A
The cornified envelope formation rate of 24 wells of the administration group was divided by the average cornified envelope formation rate of 24 wells of the control group, and the obtained value was used as the keratinocyte differentiation promoting value.

In addition, a similar test was performed for polyporsterone A.
Instead, polyporsterone B, 20-hydroxyec
Using Dison, Ponasterone A, Ponasteroside A
Each was performed and for comparison, polyporsterone A
Instead of active vitamin D _ThreeIs 1α, 25-jihi
Droxy vitamin D_ThreePerform the same test using
In the same manner as in
The differentiation promoting value of the activated cells was determined. Table 1 shows the results. What
Note that M in the table is a differentiation promoting / mitotic inhibitor of each keratinocyte.
It represents the molar concentration relative to the medium.

[0034]

[Table 1] Next, according to the method in which calcium was not added to the medium, the normal human epidermal keratinocytes were added to a 6-well multi-plate in an amount of 2
After inoculating at a density of × 10 ⁴ cells / well and culturing for 40 hours in the MCDB153 medium attached to the kit, the medium was
The DMSO solution of polyporsterone A was changed to MCDB153 medium supplemented with 0.1%, and the cells were further cultured for 48 hours.
The concentrations of polyporsterone A in the culture medium in this culture were various concentrations shown in Table 2.

48 hours after changing the medium, the cultures were trypsinized into single cells and the total number of cells in the wells was counted. After that, 1% SDS, 1% β
20 with mercaptoethanol (BME) at room temperature
After the treatment for a minute, the number of remaining cells resistant to the SDS and BME treatment, that is, the cells that formed a fully differentiated conified envelope, was counted for each well. This number was divided by the total number of cells in each well, and the obtained value was defined as the cornified envelope formation rate.

Further, as a control, all tests were carried out in the same manner except that a medium obtained by removing polyporsterone A from the above-mentioned medium was used as the medium after the change in the above-mentioned test, and the control cone was prepared in the same manner as above. The fido envelope formation rate was calculated. Polyporsterone A
The cornified envelope formation rate of 24 wells of the administration group was divided by the average cornified envelope formation rate of 24 wells of the control group, and the obtained value was used as the keratinocyte differentiation promoting value.

Further, a similar test was conducted using polyporsterone B, 20-hydroxyecdysone, ponasterone A and ponasteroside A instead of polyporsterone A, and differentiation of each keratinocyte was carried out in the same manner as above. The keratinocyte differentiation promoting value of the promoter / mitotic inhibitor was determined. Table 2 shows the results. In the table, M represents the molar concentration of each keratinocyte differentiation promoting / mitotic inhibitor with respect to the medium.

[0038]

[Table 2] As is clear from these results, the ecdysteroid analog, which is a keratinocyte differentiation promoting / mitotic inhibitor of the present invention,
It promotes the differentiation of keratinocytes regardless of the presence or absence of calcium, and its differentiation promoting action is vitamin D.
Turns out to be better. (2) Effect on keratinocyte cell division Using a normal human epidermal keratinocyte culture kit (manufactured by Morinaga Biochemical Laboratories), promoting differentiation of the keratinocytes of the present invention against cell division of normal human keratinocytes The action of mitotic inhibitors was investigated.

The normal human epidermal keratinocytes were seeded in a 24-well multiplate at a density of 2 × 10 ⁴ cells / well and cultured in MCDB153 medium attached to the kit for 24 hours. Then, the medium was added with polyporsterone A in DMSO.
The solution was changed to MCDB153 medium containing 0.1%,
After further culturing for 2 days, the amount of DNA in each well was measured as follows. The concentrations of polyporsterone A in the culture medium in this culture were various concentrations shown in Table 3.

Cultured cells in the wells were treated with 50 μg / mL protein kinase K, 0.25% sodium N-lauroyl sarcosinate, and 25 mg / mL EDTA.
Was dissolved in 100 μL of an aqueous solution containing and heated at 60 ° C. for 1 hour. After completion of heating, 3 mL of an aqueous solution (pH 7.0) containing 10 mM Tris-hydrochloric acid, 10 mM EDTA, and 100 mM NaCl was added and mixed, and further 3 μg / mL HOECHST, 10 mM Tris-hydrochloric acid, 10 mM EDTA and 100 mM NaCl
(PH 7.0) was added and mixed, and then the fluorescence intensity was measured to quantify the DNA amount. This work is instructed by the manual of the kit, and all the above reagents are also attached to the measurement kit.

Further, as a control, all tests were carried out in the same manner except that a medium obtained by removing the polyporsterone A from the above-mentioned medium was used as the medium after the change in the above-mentioned test, and the amount of the control DNA Was measured. Mean D of 24 wells of the polyporsterone A administration group
The NA amount was divided by the average DNA amount of 24 wells in the control group, and the obtained value was used as the keratinocyte division inhibition value.

Further, similar tests were carried out using polyporsterone B and 20-hydroxyecdysone instead of polyporsterone A, respectively, and for the comparison, active vitamin D ₃ was used instead of polyporsterone A. Is 1
A similar test was conducted using α, 25-dihydroxyvitamin D ₃ , and in the same manner as above, promotion of differentiation of each keratinocyte
The mitotic inhibitory value of keratinocytes of the mitotic inhibitor was determined. The results are shown in Table 3. In addition, M in the table indicates differentiation promotion of each keratinocyte
It represents the molar concentration of the mitotic inhibitor with respect to the medium.

[0043]

[Table 3] From this result, it is understood that the ecdysteroid analog, which is the keratinocyte differentiation promoting / mitotic inhibitor of the present invention, has a keratinocyte mitotic inhibitory action, and this action is superior to vitamin D.

When combined with the results of the keratinocyte differentiation promoting experiment described above, the ecdysteroid analog, which is the keratinocyte differentiation promoting / mitotic inhibitor of the present invention, has the same action as that of vitamin D (horn). It has both the action of promoting differentiation of keratinocytes and the action of suppressing keratinocyte division), but it can be said that the action is superior to that of vitamin D. (3) Safety test 6 groups of 6 Hartley white guinea pigs (female, body weight 300-350 g) were shaved on the backs and stripped with gum tape 3 times, and then 1 group was treated with petrolatum as control 1 group contains Vaseline containing polyporsterone A at a concentration of 0.5%, and the remaining 1 group contains 1α, 25-dihydroxyvitamin D ₃ at a concentration of 0.5% for comparison Vaseline is 0.
Closed patch of 05g each. After 48 hours, the patch was removed, the state of the patch test portion on the back was observed, and evaluation was performed using the following Draize criterion.

++: Erythema accompanied by edema is observed +: Clear erythema is observed ±: Slight erythema is observed −: No reaction result is shown in Table 4 as the number of guinea pigs for each judgment.

[0046]

[Table 4] From this result, it is clear that the ecdysteroid analog which is a keratinocyte differentiation promoting / mitotic inhibitor of the present invention has high safety.

Next, promotion of differentiation of the keratinocytes of the present invention described above
Examples of the pharmaceutical composition of the present invention containing a mitotic inhibitor will be described. The blending amount represents parts by weight.

[0048]

Examples 4 and 5 Granules The ingredients shown in Table 5 were placed in a Grad granulator, and 50% ethanol was gradually added to the granules while rotating at a low speed until granulation was performed. This was blow-dried at 40 ° C. for 48 hours, and then sieved,
The particles were sized to obtain granules.

[0049]

[Table 5] The two types of granules obtained in the above examples were treated 10 times 3 times a day.
When 0 mg each was administered to two psoriasis patients,
Both of them improved their symptoms within 1 week after the start of administration.

[0050]

[Examples 6 and 7] External preparation for skin An external preparation for skin was prepared by stirring and precipitating the ingredients shown in Table 6 at room temperature.

[0051]

[Table 6] When two types of external preparations for skin obtained in the above-mentioned examples were applied to two affected psoriasis patients twice a day on the affected area, respectively, the psoriasis was cured 5 days after the start of application.

[0052]

INDUSTRIAL APPLICABILITY The ecdysteroid analog, which is a keratinocyte differentiation promoting / mitotic inhibitor of the present invention, has a keratinocyte differentiation promoting effect and a keratinocyte mitotic suppressing effect sufficient to improve psoriasis. And has high safety. In addition, the pharmaceutical composition of the present invention containing it is effective for the prevention and treatment of psoriasis and can be used safely for a long period of time.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Kunio Tsuji 1375-11 Ikeda, Shizuoka City, Shizuoka Prefecture

Claims (3)

[Claims] 1. A keratinocyte differentiation-promoting / mitotic inhibitor consisting of an ecdysteroid analog. 2. An ecdysteroid analog has polyporsterone A represented by the following formula (1), polyporsterone B represented by the following formula (2), and 20 represented by the following formula (3). The keratinocyte differentiation promoting / mitotic inhibitor according to claim 1, which is selected from hydroxyecdysone, ponasterone A represented by the following formula (4), and ponasteroside A represented by the following formula (5). Embedded image Embedded image Embedded image Embedded image Embedded image 3. A pharmaceutical composition for preventing or treating psoriasis, which comprises, as an active ingredient, the keratinocyte differentiation-promoting / mitotic-suppressing agent of claim 1 or 2.