JP5651426B2 - Chlorine derivative - Google Patents

Description

  The present invention relates to a novel chlorin derivative or a pharmacologically acceptable salt thereof, a skin disease therapeutic agent for use in photophysical chemical diagnosis / treatment (PDT: Photodynamic Therapy) containing them as an active ingredient, and antibacterial It relates to the agent.

  As a new treatment method for cancer, a photophysicochemical diagnosis / treatment method (hereinafter referred to as “photodynamic therapy”: PDT: Photodynamic Therapy) is performed. In this method, a certain porphyrin derivative is administered by a method such as intravenous injection and selectively accumulated in cancer (tumor) tissue, and then only the cancer tissue is selectively destroyed by irradiation with laser light. Is a treatment method that uses two characteristics of selective accumulation in a cancer tissue and a photosensitizing action of a porphyrin derivative.

The present inventors have been diligently researching a porphyrin derivative that can be used for this PDT, and have provided an iminochlorin aspartic acid derivative referred to as ATX-S10.
Among the iminochlorin aspartic acid derivatives or pharmacologically acceptable salts thereof, particularly a compound that is a sodium salt, that is, a compound that is named ATX-S10 · Na, has an ability to accumulate in cancer tissue, as well as neovascularization. It is a compound that has a remarkably high selective accumulation property. Therefore, it has been confirmed that it is extremely effective as a therapeutic agent for PDT for the treatment of tumors using its excellent characteristics (Patent Document 1).

  On the other hand, inflammatory keratosis, which is caused by inflammation caused by new blood vessels, is caused by the spread of blood vessels in the dermis part of the skin and the invasion of white blood cells such as lymphocytes into the skin and the skin epidermis. Is a skin symptom in which "keratosis" that causes thickening of the stratum corneum and thickening of the stratum corneum occurs simultaneously. Anti-inflammatory agents such as steroids, epidermal growth inhibitors such as retinoids, and UV therapy (PUVA therapy) are implemented. However, it is not a definitive treatment leading to complete cure. In recent years, it has been found that treatment with PDT using 5-aminolevulinic acid hydrochloride (5-ALA) is effective. However, 5-ALA is a biosynthetic precursor of protoporphyrin IX, and a sufficient therapeutic effect can be obtained because of the compound characteristics of protoporphyrin IX, that is, low neovascularization and the longest wavelength absorption edge is 630 nm. I can't.

  One of the keratosis is actinic keratosis. This actinic keratosis is also referred to as senile keratosis, and is said to be a “cancer precursor”, one step before cancer because it may progress to skin cancer in the future. It is a keratosis caused by long-term exposure to sunlight that damages the nuclear DNA DNA of skin cells and makes repair difficult with aging. Symptoms are crisp, slightly raised erythema, or gray or brown pigment spots that appear on the skin surface and seem to resemble eczema or warts. It is preferable to treat and excise. Treatment with surgery, excision with a carbon dioxide laser, cryotherapy, anticancer agent-containing ointment, etc. are carried out, but treatment with PDT is also effective.

  By the way, as a result of the study by the present inventors, ATX-S10 · Na proposed by the present inventors is excellent in accumulation in inflammatory cells caused by new blood vessels other than cancer and ophthalmology, and applied PDT. As a skin disease therapeutic agent, it was proved to be effective as a therapeutic agent for inflammatory keratosis (dermatitis such as psoriasis), and as a skin disease therapeutic agent applying PTX as ATX-S10 · Na A patent application has already been filed for this point (Patent Document 2).

However, the ointment containing ATX-S10 · Na has low skin permeability, and the stratum corneum must be removed by tape stripping in order to reach the effective therapeutic amount in the affected area.
Therefore, the present inventors have studied to develop a skin disease treatment agent for PDT that does not require tape stripping and reduces the burden on the patient. That is, what is effective as a skin disease treatment agent used in PDT therapy requires that the drug itself has high skin permeability and that it is fat-soluble so that it can be uniformly dissolved and dispersed in the base as an ointment. Is done. Based on this point of view, we synthesized several anionic chlorin derivatives and examined their effects. As a result, we found that these new anionic chlorin derivatives were extremely effective. An application has been filed (Patent Document 3).

International Publication WO 98/14453 Japanese Patent Laid-Open No. 2004-026717 JP 2009-263317 A

  This time, as a result of examining the pharmacological effect of the previously proposed anionic chlorin derivative, it is effective not only for skin diseases but also for infectious diseases, and is extremely effective as an antibacterial agent for PDT. It was confirmed that the cationic chlorin derivative is effective not only as an ointment but also as an external preparation for PDT, such as a lotion, for the treatment of infectious diseases, and the present invention has been completed.

  Therefore, the present invention relates to chlorin derivatives that can be used for PDT (Photodynamic Therapy: photophysical chemical diagnosis / treatment method), and further external preparations for PDT containing these chlorin derivatives as active ingredients, in particular, skin disease therapeutic agents, and antibacterial agents. It is an object to provide an agent.

  As a basic aspect of the present invention for solving such a problem, the following formula (I):

[Where:
X represents O,
Y is, - (CH ₂₎ represent _n,
R is —OH, —O (CH ₂ ) _m CH ₃ , —O (CH ₂ ) _m —OH, or —NHCH ₂ —Z.
(Where Z is

Represents
n and m represent an integer of 0 to 10]
Or a pharmacologically acceptable salt thereof.
[However, in the A-type derivative, R is —OH, —O (CH ₂ ) _m CH ₃ and —O (CH ₂ ) _m —OH]]

  As one more specific aspect, the present invention provides the compound represented by the formula (I):

[Where:
X represents O,
Y is, - (CH ₂₎ represent _n,
R is —NHCH ₂ —Z
(Z is

Represents
n represents an integer of 0 to 10]
Or a pharmacologically acceptable salt thereof.

  As another specific embodiment, the present invention provides that the formula (I) is represented by the following formula (I) -B type:

(Wherein X, Y and R are the same as defined above)
Or a pharmacologically acceptable salt thereof.

  Furthermore, this invention is a skin disease therapeutic agent for photodynamic therapy (PDT: Photodynamic Therapy) which contains said chlorin derivative or its pharmacologically acceptable salt as an active ingredient as another aspect.

  Further, the present invention provides, as yet another aspect, the following formula (I):

[Where:
X represents O,
Y is, - (CH ₂₎ represent _n,
R is —OH, —O (CH ₂ ) _m CH ₃ , —O (CH ₂ ) _m —OH, or —NHCH ₂ —Z.
(Where Z is

Represents
n and m represent an integer of 0 to 10]
The antibacterial agent for photodynamic therapy (PDT: Photodynamic Therapy) which contains the chlorin derivative shown by these, or its pharmacologically acceptable salt as an active ingredient.

  More specifically, in the present invention, the formula (I) is represented by the following formula (I) -A type and formula (I) -B type:

(In each formula, X, Y and R are the same as defined above)
It is an antibacterial agent for PDT.

  More specifically, the present invention is a skin disease treatment agent for PDT described above or an antibacterial agent in the form of an ointment or lotion.

  That is, the basic aspect of the present invention is that the chlorin derivative represented by the above formula (I), particularly the (I) -A type and (I) -B type chlorin derivatives, or pharmacologically acceptable thereof. This is characterized by the use of PDT to treat skin diseases and infections with PDT.

The chlorin derivative provided by the present invention or a pharmacologically acceptable salt thereof is an anionic or cationic chlorin derivative, and an external preparation such as an ointment or a lotion containing these chlorin derivatives is tapes. It does not require tripping and has good permeability to the affected area of the skin, and therefore does not burden the patient in PDT therapy in the treatment of skin diseases.
Further, since tape stripping is not required, the treatment itself has an advantage that it can be easily performed, and a new treatment system can be provided.

Furthermore, it is effective as an antibacterial agent for PDT for the treatment of infectious diseases. In particular, since the cationic chlorin derivative has high water solubility, it can be used not only as a non-aqueous external preparation but also as an aqueous external preparation. is there.
In addition, antibacterial agents, antibiotics, and the like that are used in the treatment of infectious diseases today are easily resistant to drugs. However, in the PDT treatment system of the present invention, such problems of drug resistance do not occur. In that respect, the advantages are very specific.

10 is a graph showing results in Test Example 4. 10 is a photograph showing symptoms before irradiation in Test Example 5. 10 is a photograph showing symptoms after irradiation in Test Example 5.

10 is a photograph before laser irradiation (150 J / cm ² ) with a TONS 501B type-containing ointment in Test Example 7. FIG. It is the photograph in the test example 7 48 hours after laser irradiation (150 J / cm < ² >) by the TONS 501B type | mold containing ointment. 10 is a photograph before laser irradiation (100 J / cm ² ) with a TONS 501B-type ointment in Test Example 7. It is the photograph in the test example 7 24 hours after laser irradiation (100 J / cm < ² >) by the TONS 501B type | mold containing ointment.

It is the photograph before 48 hours after laser irradiation (100 J / cm < ² >) by the TONS 501B type | mold ointment in Test Example 7. FIG. It is the photograph in 96 hours after laser irradiation (100 J / cm < ² >) by the TONS 501B type | mold ointment in Test Example 7. FIG.

It is the photograph in the test example 7 before the laser irradiation by the TONS 504A type containing ointment. It is the photograph in the test example 7 24 hours after laser irradiation (100 J / cm < ² >) by the TONS 504A type | mold ointment. It is the photograph in 48 hours after laser irradiation (100J / cm < ² >) by the TONS 504A type | mold ointment in Test Example 7. FIG. It is the photograph in 96 hours after laser irradiation (100 J / cm < ² >) with the TONS 504A type | mold ointment in Test Example 7. FIG.

A chlorin derivative represented by formula (I) or a pharmacologically acceptable salt thereof, specifically a chlorin derivative of formula (I) -A or formula (I) -B (provided by the present invention) Hereinafter, these may be referred to simply as “chlorine derivatives”).
This chlorin derivative is different from ATX-S10, which is an iminochlorin aspartic acid derivative that has been provided by the present inventors, so that it has a high skin permeability and is fat-soluble or water-soluble. Dissolves and disperses uniformly in the base, and the stability of the ointment itself is extremely good. Also, it dissolves and disperses uniformly in the preparation as a water-soluble lotion, and the stability of the preparation itself is also very good. Is.

  Among such chlorin derivatives represented by the formula (I), in particular, the A type and the B type represented by the following formula (Ia):

Furthermore, the A type and the B type represented by the following formula (Ib):

Or a pharmacologically acceptable salt thereof, specifically, a compound represented by the following abbreviation.

TONS 501B type: A chlorin derivative in which X is O and Y is —CH ₂ — in the formula (Ia) B type.

TONS 502A type: Formula (Ia) A chlorin derivative in which A is A and X is O and Y is —CH ₂ —.

TONS 503B type: A chlorin derivative in which X is O and Y is —CH ₂ — in the formula (Ib) B type.

TONS 504A type: Chlorine derivative in which X is O and Y is —CH ₂ — in formula (Ib) A type.

These chlorin derivatives can be obtained, for example, as follows.
That is, it can be prepared by dissolving the corresponding photoprotoporphyrin derivative in a suitable organic solvent and reacting it with a diol compound such as 1,3-propanediol or ethylene glycol.

  The organic solvent used for the reaction is not particularly limited and can be arbitrarily selected as long as it does not directly affect the reaction. Specific examples include aromatic hydrocarbons such as benzene, toluene and xylene, and ether solvents such as ether, tetrahydrofuran and dioxane. Of these, tetrahydrofuran is preferably used.

The reaction temperature and reaction time are also not particularly limited, and the stirring treatment is preferably performed at 0 to 60 ° C., preferably at room temperature for about 0.5 to 10 hours.
The reaction requires the presence of a mild acid catalyst, and examples of such an acid catalyst include methanesulfonic acid, toluenesulfonic acid, p-toluenesulfonic acid and the like, and among them, p-toluene. By using sulfonic acid, the target chlorin derivative can be obtained in high yield.

  Specific examples of some of the preparation methods are shown below.

Production Example 1: Preparation of an anionic chlorin derivative wherein X is O and Y is —CH ₂ — in the formula (Ia) type B (preparation of TONS 501B type)
1.0 g of photoprotoporphyrin dimethyl ester (P-Me B type) is dissolved in 50 mL of tetrahydrofuran, and 5.0 mL of 1,3-propanediol and 300 mg of p-toluenesulfonic acid monohydrate are added to this solution. Stir for hours. After completion of the stirring, 1% aqueous sodium hydrogen carbonate solution was added and the deposited precipitate was collected by filtration and recrystallized from ethyl acetate to obtain 0.9 g of a novel anionic chlorin derivative (TONS 501B type).
When the obtained derivative was subjected to UV-VIS spectrum analysis, it showed a longest wavelength absorption edge of 662.5 nm and was subjected to mass spectrometry by FAB-MS. As a result, m / z: 681 was shown, and the intended anionic chlorin It was confirmed to be a derivative.
This structure was supported by NMR spectral analysis.

Production Example 2: Preparation of an anionic chlorin derivative wherein X is O and Y is —CH ₂ — in the formula (Ia) type A (preparation of TONS 502A type)
1.0 g of photoprotoporphyrin dimethyl ester (P-Me A type) is dissolved in 50 mL of tetrahydrofuran, and 4.8 mL of 1,3-propanediol and 150 mg of p-toluenesulfonic acid monohydrate are added to this solution. Stir for hours. After the completion of stirring, 1% aqueous sodium hydrogen carbonate solution was added and the deposited precipitate was collected by filtration and purified by silica gel column chromatography to obtain 0.9 g of a novel anionic chlorin derivative (TONS 502A type).
When the obtained derivative was subjected to UV-VIS spectrum analysis, it showed a longest wavelength absorption edge of 662 nm, and mass spectrometry was performed by FAB-MS. As a result, m / z: 681 was shown, and the target anionic chlorin derivative was obtained. It was confirmed that there was.
This structure was supported by NMR spectral analysis.

Production Example 3: Preparation of a free form (ester free form) of an anionic chlorin derivative in which X is O and Y is —CH ₂ — in formula (Ia) type B (preparation of TONS-H 501B type) )
1.0 g of the novel anionic chlorin derivative (TONS 501B type) obtained in Production Example 1 was dissolved in 20 mL of dimethylformamide, and 10 mL of a 5 mol / L aqueous sodium hydroxide solution was added while cooling, followed by stirring for 1 hour. After completion of the reaction, 100 mL of ethanol and then 30 mL of ethyl acetate were added for precipitation, and after filtration, a precipitate was obtained.
The resulting precipitate was purified by reprecipitation with 1 mL of water and 50 mL of acetonitrile, and the precipitate was collected by filtration and dried to give a free form (ester free form) sodium salt (TONS-Na 501B type). 0.9 g was obtained.
1 mL of water is added to the obtained sodium salt to dissolve it, and 200 mL of 10% aqueous citric acid solution is added for reprecipitation. After filtration, the precipitate is washed with water, dried, dissolved in 5 mL of pyridine, and 30 mL of ethyl acetate and 50 mL of n-hexane. The precipitate was collected by filtration and dried to obtain 0.8 g of the desired free chlorin derivative (TONS-H 501B type).

Production Example 4: Preparation of a free form (ester free form) of an anionic chlorin derivative in which X is O and Y is —CH ₂ — in formula (Ia) type A (preparation of TONS-H 502A type )
In accordance with the method of Production Example 3, 1.0 g of the novel anionic chlorin derivative (TONS 502A type) obtained in Production Example 2 was dissolved in 50 mL of pyridine, and 10 mL of 1 mol / L sodium hydroxide aqueous solution was added while cooling to room temperature. And stirred for 1 hour. After completion of the reaction, 200 mL of 10% aqueous citric acid solution was added to precipitate, and after filtration, washed with water, dried, dissolved in 5 mL of pyridine, reprecipitated with 30 mL of ethyl acetate and 50 mL of n-hexane, and these were collected by filtration. After drying, 0.8 g of the desired free chlorin derivative (TONS-H 502A type) was obtained.

Production Example 5: Formula In (I-b) B-type, X is O, Y -CH ₂ - (Preparation of TONS 503B type) in which the preparation of cationic chlorin derivative
Dissolve 1.0 g of the free chlorin derivative (TONS-H 501B type) obtained in Production Example 3 in 50 mL of dimethylacetone, add 0.7 mL of 3- (aminomethyl) pyridine, and further add WSC · HCl (N, N-dimethyl). 2.0 g of aminopropylcarbodiimide hydrochloride) was added, and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the precipitate is collected by filtration with 200 mL of cold water, and the resulting precipitate is washed with water, dried, and purified by silica gel column chromatography (elution solvent: dichloromethane / methanol) to give a pyridine-supported amidochlorin derivative ( 0.4 g ( TONS-Py 501B type) was obtained (FAB-MS, m / z: 834) .
1.0 g of the resulting pyridine-supported amidochlorine derivative (TONS-Py 501B type) was dissolved in 10 mL of dichloromethane, 0.2 g of methyl iodide was added, and the mixture was stirred overnight at room temperature. The obtained reaction solution was heated and concentrated at 40 ° C. or lower to obtain 0.4 g of a cationic chlorin derivative (TONS 503B type).
When UV-VIS spectrum analysis was performed on this derivative, the longest wavelength absorption edge was 666 nm, and mass spectrometry was performed by FAB-MS. As a result, m / z: 835 (m / z M-CH ₃ I × 2) was obtained. And was confirmed to be the desired cationic chlorin derivative.

Production Example 6: Preparation of a cationic chlorin derivative in formula (Ib) A type, wherein X is O and Y is -CH _2- (preparation of TONS 504A type)
1.0 g of the free chlorin derivative (TONS-H 502A type) obtained in Production Example 4 was suspended and dissolved in 50 mL of acetonitrile, and 2.0 g of WSC · HCl (N, N-dimethylaminopropylcarbodiimide hydrochloride) was added. The reaction was allowed to stir at room temperature for 3 hours. At the end of the reaction, 0.7 mL of 3- (aminomethyl) pyridine was added to obtain a reaction product (confirmed by TLC). 200 mL of water was added to the resulting product, and the deposited precipitate was collected by filtration, washed successively with 60 mL of water, then with 20 mL of 1% aqueous citric acid solution and 100 mL of water, dried, and dried, and amide-supported amidochlorin derivative (TONS-Py 502A 0.9 g was obtained.
The obtained pyridine-supported amidochlorin derivative (TONS-Py 502A type) was dissolved in a mixture of chloroform (20 mL) and acetonitrile (10 mL), and then 0.7 g of methyl iodide was added and stirred overnight at room temperature. After the reaction (confirmed by TLC), it was evaporated to dryness at 30 ° C. on a rotary evaporator. This was naturally dried to obtain 1.0 g of a cationic chlorin derivative (TONS 504A type).
When UV-VIS spectrum analysis was performed on this derivative, the longest wavelength absorption edge was 666 nm, and mass spectrometry was performed by FAB-MS. As a result, m / z: 835 (m / z M-CH ₃ I × 2) was obtained. And was confirmed to be the desired cationic chlorin derivative.
This structure was supported by NMR spectral analysis.

In the present invention, these chlorin derivatives are prescribed as a skin disease therapeutic agent for PDT or an external preparation such as an antibacterial agent.
An external preparation as a skin disease therapeutic agent or antibacterial agent is prescribed in a dosage form such as a non-aqueous ointment, an aqueous ointment, or a lotion. Various bases can be mentioned as the ointment base, and the formulation of an ointment preparation that can ensure the stability of the preparation over time was examined, and it was found that a non-aqueous ointment base could be a good ointment. .
Specific examples of such non-aqueous ointment bases include FAPG (H) ointment base, FAPG (K) ointment base, PEG ointment base (macrogol ointment base), and PEG-PG ointment base. , Petrolatum ointment base, SR petrolatum ointment base, SR petrolatum-IPM ointment base, plastibase ointment base.
In addition, since the chlorin derivative of this invention includes both anionic and cationic chlorin derivatives, it cannot be overemphasized that not only a non-aqueous ointment base but an aqueous ointment may be sufficient.

  Specifically, the FAPG (H) ointment base is an ointment base made of stearyl alcohol, stearic acid and propylene glycol. The FAPG (K) ointment base is an ointment base made of stearyl alcohol, stearic acid and polyethylene glycol 400. The PEG ointment base (macrogol ointment base) is an ointment base composed of polyethylene glycol 400 and polyethylene glycol 4000, and the PEG-PG ointment base is an ointment base composed of propylene glycol and polyethylene glycol 4000.

  The petrolatum ointment base is an ointment base composed of petrolatum and liquid paraffin, and the SR petrolatum ointment base is an ointment base composed of SR petrolatum (5% salicylic acid-petroleum) and SR petrolatum- The IPM ointment base is an ointment base composed of SR petrolatum and isopropyl myristate. Furthermore, the plastibase ointment base is an ointment base made of plastibase (95% liquid paraffin and 5% polyethylene resin).

It should be noted that the ointment base used in the present invention is not limited to those described above, and it is needless to say that an ointment base generally used in pharmacology can be used. It is also acceptable to add other stabilizers pharmacologically or pharmacologically and transdermal absorption enhancers such as l-menthol.
According to the study by the present inventors, it has been found that the macrogol ointment base is particularly preferable among the above-described ointment bases.

  Moreover, as a lotion agent, it can be prescribed | regulated as a general lotion agent, and it turned out that a macrogol containing lotion agent is especially preferable especially.

  In the present invention, the amount of the chlorin derivative represented by the formula (I) to be contained in these preparations is such that the chlorin derivative as the compounded active ingredient is absorbed percutaneously and accumulated at the diseased site, and is targeted by irradiation with a laser beam. An amount sufficient to kill cells or bacteria may be added. According to the study by the present inventors, it was found that a sufficient effect can be obtained if the blending amount is 0.1 to 20% by weight based on the weight of the preparation.

When the blending amount is less than 0.1% by weight, the intended therapeutic effect cannot be increased, and even when blended in an amount of 20.0% by weight or more, no further effect can be obtained.
The blending amount cannot be specified in general depending on the type of the active ingredient to be contained, and the stability of the active ingredient to be contained is not greatly affected by the concentration of the active ingredient and the base to be used. Within the range, various changes can be made according to the application.

In order to produce an external preparation such as an ointment preparation of the present invention, a general method for preparing a general external preparation is used, and a chlorin derivative as an active ingredient may be uniformly kneaded in a preparation base.
Moreover, what is necessary is just to knead | mix many components uniformly as needed.

When the preparation of the present invention obtained as described above is used for PDT, it can be effectively applied directly to a skin disease site such as actinic keratosis, inflammatory keratosis, epidermis cancer, infection, etc. Then, the chlorin derivative is accumulated, and then the site can be effectively treated by light irradiation with a laser beam or the like.
In application of external preparations such as ointments and lotions, the chlorin derivative provided by the present invention does not require tape stripping and has good permeability to the affected skin area. However, there is an advantage that the treatment itself can be easily performed without burdening the patient.
In applying an external preparation such as an ointment or lotion, it is more effective to keep the application site sealed in order to obtain an ODT effect (occlusive dressing technique).

  Moreover, it turned out that the chlorin derivative of this invention is effective with respect to various infectious diseases as an antibacterial agent for PDT. Such infections include infections not only in humans but also in various animals. Malassezia otitis externa in dogs, cats, etc. due to yeast-like fungi, nail infections, infections caused by fungi such as athlete's foot, MRSA, Used for diagnosis and treatment of various infections caused by Pseudomonas aeruginosa, Escherichia coli, etc., dental infections such as periodontal disease due to periodontal disease bacteria, other tuberculosis or virus infections, and PDT for sputum can do.

Various laser beams and lamps can be used for light irradiation at a diseased site after applying a preparation containing a chlorin derivative provided by the present invention. For example, a titanium sapphire laser, a semiconductor laser, an OPO-YAG laser, a xenon lamp, a halogen lamp, a metal halide lamp, a light emitting diode, or the like can be used. Among them, it is more effective to use a semiconductor laser or an LED light source for a chlorin derivative having an absorption wavelength near 660 to 670 nm.

  Thus, after applying the ointment preparation, lotion preparation and the like of the present invention to the diseased site, and percutaneously absorbing the active ingredient, the diseased site is irradiated with light to effectively treat the skin disease or infectious disease. can do.

  Hereinafter, the present invention will be described in detail with specific formulation examples, test examples, and the like, but the present invention is not limited to these examples.

Test Example 1: Thermal stability test of anionic chlorin derivatives (TONS 501B type and TONS 502A type) < Stability to high-pressure steam (autoclave) treatment>
The chlorin derivative of the present invention is autoclaved for sterilization when it is formulated.
Therefore, the stability to high-pressure steam (autoclave) treatment was examined.
20 mg each of anionic chlorin derivatives (TONS 501B type and TONS 502A type) were subjected to high-pressure steam (autoclave) treatment at 121 ° C./20 minutes.
When the TLC analysis and the HPLC analysis were performed on the sample after the high-pressure steam treatment, no decomposition product was confirmed, and none of the samples was decomposed under the high-pressure steam (autoclave) treatment, and it was confirmed that the sample was stable.

  Specific formulation examples of ointments and lotions containing the chlorin derivative of formula (I) provided by the present invention are described below.

Formulation Example 1: PEG ointment base (macrogol ointment)
Polyethylene glycol 4000 (PEG 4000, average molecular weight of about 3,000; manufactured by Wako Pure Chemical Industries, Ltd.) 3.0 g, polyethylene glycol 400 (PEG 400, average molecular weight of about 360-440; manufactured by Wako Pure Chemical Industries, Ltd.) 7.0 g and l- Menthol (0.1 g) was heated and dissolved and mixed, and 1.0 g of each of the anionic chlorin derivatives (TONS 501B type and TONS 502A type) obtained in the above production example was added and mixed. The mixture was allowed to cool with stirring to obtain each macrogol ointment.
Similarly, macrogol ointment was obtained for cationic chlorin derivatives (TONS 503B type and TONS 504A type).

Formulation Example 2: Lotion (containing macrogol)
10.0 g of polyethylene glycol 400 (PEG 400, average molecular weight of about 360 to 440; manufactured by Wako Pure Chemical Industries, Ltd.) and 0.1 g of l-menthol were heated and dissolved and mixed, and the anionic chlorin derivative obtained in the above production example was then mixed there. 1.0 g of each (TONS 501B type and TONS 502A type) was added and mixed. The mixture was allowed to cool with stirring to obtain a lotion containing each macrogol.
Similarly, lotions containing macrogol were obtained for cationic chlorin derivatives (TONS 503B type and TONS 504A type).

Test Example 2: Stability test The macrogol ointment or lotion containing the chlorin derivative of the present invention obtained above is stored in a polypropylene container in a refrigerator for 3 months to determine the purity of the active ingredient. The stability was measured by HPLC. Purity before the start of storage and purity after storage for 3 months were determined by area percentage by HPLC.
As a result, it was found that the purity of the contained active ingredient (anionic chlorin derivative and cationic chlorin derivative) was not lowered and was stable.

  Next, the actual therapeutic effect of PDT will be described for a preparation containing the novel chlorin derivative provided by the present invention.

Test Example 3: In vitro test using Staphylococcus aureus (gram-negative bacteria) <Method>
After enrichment of Staphylococcus aureus, the number of bacteria was adjusted to 1 × 10 ⁶ cfu / mL with sterile distilled water, and 500 μL was dispensed into a standard agar medium dish. The test solution (TONS 504A type) was prepared to be 0, 0.1, 1, and 10 μL (dissolved in sterile distilled water).
Next, this petri dish was irradiated with light (Super Lyzer-PDT-D1; wavelength: 600-700 nm, 2.3 W, manufactured by Tokyo Medical Research Institute: total irradiation dose: 5, 10 and 20 J / cm ² ). After irradiation, a 10-fold diluted bacterial solution was applied to an agar medium, cultured for 24 hours at 37 ° C., and the number of colonies was counted.
<Result>
When the drug concentration was 0.1 μ / mL or more, a significant decrease in the number of colonies was observed at all of 5, 10 and 20 J / cm ² , indicating an antibacterial effect.
The results are shown in Table 1 below.

Test Example 4: In Vivo Drug Efficacy Test Using Malassezia External Otitis (Dog) <Symptoms>
Beagle dog (10 years old, weight 9.4 kg). Yeast-like fungi (at earwax smear) were confirmed in both ears, and itching in both ears was observed.
<Method>
As a control (no drug application), Super Lyzer-PDT-D1 (wavelength: 600-700 nm, 2.3 W, manufactured by Tokyo Medical Research) was used for the left ear, and the output was 20%, 10 seconds at intervals of 1 second. Irradiated for 1 minute.
On the other hand, for the right ear, 1% TONS 501B type lotion obtained in prescription 2 was applied to the entire external auditory canal, and immediately after using Super Lyzer-PDT-D1, the output was 20%, and 1 second for 2 seconds. Irradiated for 10 minutes at intervals.
After the treatment, no treatment such as washing the inside of the ear canal was performed. For both ears, the earwax smear was taken before treatment, 2 days after treatment, and 7 days after treatment, and the number of malassezia per visual field was measured for 10 visual fields, and the effect was confirmed by the bacteria reduction rate.

<Result>
A 1% TONS 501B type lotion was found to be effective against malachitic otitis externa.
Similarly, when tested using a 1% lotion preparation of TONS 504A using dogs suffering from other malachitic otitis externa, it was found to be equally effective.
These results are shown in FIG.

Test Example 5: In vivo drug efficacy test using malassessy otitis externa (dog) (16 cases)
<Symptoms>
The test was conducted after confirming the dog and fungus species.
<Method>
In the same manner as in Test Example 4 above, 1% TONS 501A type lotion obtained in prescription 2 was applied to the ears of affected dogs, and immediately after using Super Lyzer-PDT-D1, 1.8 W, Irradiation was carried out for 7 minutes at intervals of 1 second every 2 seconds.
Subsequent follow-up observations showed good results for each dog.
The results are summarized in Table 2 below.
Moreover, the concrete symptom healing condition about the 1 example was shown in FIG.2 and FIG.3. FIG. 2 shows symptoms before irradiation, and FIG. 3 is a photograph of a dog's ear showing symptoms after irradiation.

Test Example 6: Preparation of papilloma (papilloma) model mouse 0.2 μM 7,12-dimethylbenzanthracene (DMBA) was applied once to the back of a 12-week-old female hairless mouse (HOS: Hr Shizuoka Laboratory Center). After 2 weeks, 0.02 μM 12-O-tetradecanoyl phorbol-13-acetate (TPA) was applied twice a week.
As a result, many papillomas having a diameter of 1 to 2 mm appeared after 2 months.

Test Example 7: Treatment effect of ointment using papilloma model mouse by PDT therapy <Method>
In the papilloma part of the papilloma model mouse prepared in Test Example 6 above, a macroin which contains the chlorin derivative of the present invention (TONS 501B type, TONS 502A type, TONS 503B type, and TONS 504A type) prepared in Formulation Example 1 as an active ingredient Goal ointment was applied to the extent of red beans, the application site was kept sealed for 3 hours, and then the ointment was wiped off.
The papilloma part from which the ointment was removed was irradiated with a laser using a semiconductor laser (150 mW / cm ² , 670 nm, LD670-05; manufactured by Hamamatsu Photonics).
After irradiation, the naked eye was appropriately observed.

<Result>
As for anionic chlorin derivatives (TONS 501B type and TONS 502A type), papilloma dropped off and disappeared on the 4th day in both light irradiation 100 J / cm ^{2 and} 150 J / cm ² .
In addition, reduction of papilloma was observed even with light irradiation of 50 J / cm ² .
On the other hand, in the case of cationic chlorin derivatives (TONS 503B type and TONS 504A type) and as a control only with light irradiation (200 J / cm ² ), no effect was shown.
The results are shown in FIGS.

Each figure is a photograph of a papilloma (papilloma) portion of a papilloma model mouse.
4 and 6 are photographs showing the results of control in which laser irradiation (100 J / cm ² and 150 J / cm ² ) was performed without applying the ointment of the present invention.
FIG. 5 is a photograph 48 hours after laser irradiation (150 J / cm ² ), and FIGS. 7 to 9 are photographs 24, 48 and 96 hours after laser irradiation (100 J / cm ² ). By applying an ointment for treating skin diseases (anionic chlorin derivative: containing TONS 501B type) and performing PDT therapy, papilloma shrinkage was observed even after 48 hours of irradiation, and papilloma disappeared completely after 96 hours of irradiation. Was.

On the other hand, FIGS. 10 to 13 show the results of using the cationic chlorin derivative (TONS 504A type) -containing ointment of the present invention before laser irradiation (100 J / cm ² ) (FIG. 10) and 24 hours after irradiation. The disappearance of papilloma was not confirmed even after 48 hours (FIG. 11), 48 hours (FIG. 12) and 96 hours (FIG. 13).

As described above, the present invention provides an anionic chlorin derivative and a cationic chlorin derivative used as photodynamic therapy (PDT), and the chlorin derivative provided by the present invention comprises a skin-applied ointment preparation, By doing so, the skin permeability (percutaneous absorbability) is very good. Therefore, by applying to skin diseases such as actinic keratosis, inflammatory keratosis, epidermis cancer, infectious diseases, such as papilloma, chlorin derivatives are effectively accumulated at the diseased site, and laser irradiation Can effectively treat skin diseases.
In addition, as an antibacterial agent for PDT, it exhibits antibacterial activity against various bacteria. Antibiotics and the like that are used for the treatment of infectious diseases today tend to easily cause drug resistance. In the PDT diagnosis / treatment system, the advantage is extremely specific in that such a drug resistance problem does not occur, and its medical value is great.

Claims (4)

Formula (I):

[Where:
X represents O,
Y is, - (CH ₂₎ represent _n,
R is —OH, —O (CH ₂ ) _m CH ₃ , —O (CH ₂ ) _m —OH, or —NHCH ₂ —Z.
(Where Z is

Represents
n and m represent an integer of 0 to 10]
Or a pharmacologically acceptable salt thereof.
[However, in the A-type derivative, R is —OH, —O (CH ₂ ) _m CH ₃ and —O (CH ₂ ) _m —OH]] Formula (I) is represented by the following formula (I) -A type:

[Where:
X represents O,
Y is, - (CH ₂₎ represent _n,
R is —NHCH ₂ —Z
(Z is

Represents
n represents an integer of 0 to 10]
The chlorin derivative according to claim 1 or a pharmacologically acceptable salt thereof. Formula (I) is represented by the following formula (I) -B type:

(Wherein X, Y and R are the same as defined in claim 1)
The chlorin derivative according to claim 1 or a pharmacologically acceptable salt thereof.   A skin disease treatment agent for photodynamic therapy (PDT) comprising the chlorin derivative according to any one of claims 1 to 3 or a pharmacologically acceptable salt thereof as an active ingredient.

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