JP2002080494A - New anti-depilation peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a functional peptide effective for Gepilation, especially as an adverse effect of an anticancer agent. SOLUTION: fMLP and MLW and fMLW which are its derivative peptide are found to have an inhibitory effect on depilation by an anticancer agent. Consequently a new anti-depilation agent is obtained by using these peptides.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an anti-alopecia agent containing a peptide effective for hair loss by an anti-cancer agent or the like.

[0002]

BACKGROUND OF THE INVENTION For the treatment of cancer, anticancer drugs are widely used alone or in conjunction with surgical removal and radiation therapy. However, while the use of appropriate anticancer agents is effective in treating cancer or prolonging life, it is presently unavoidable to cause some side effects. Alopecia also occurs quite frequently as one of its side effects. Although alopecia is not directly life-threatening and does not cause physical distress, it has a very large effect on the patient's mental state, and its reduction is desired in clinical settings.

[0003] Met-Ile-Thr-Le derived from soybean
u (MITL) and some peptides containing the MITL sequence in the molecule have an immune system activating action (promoting phagocytosis, promoting active oxygen, promoting secretion of tumor lethal factor). (Kaihei 7-224093 and Tokuhei 8-73374). The present inventors have determined that the above peptide is an anticancer agent, etoposide.
It has been reported in the past that the hair loss caused by (etoposide) was suppressed (JP-A-9-249535).

[0004] As a food intended for hair growth, there is known a food containing an extract of seaweed, kakinoha and kelp. Also, an oral alopecia agent comprising an oolong tea extract as an active ingredient capable of suppressing, improving or preventing alopecia as a side effect of an anticancer agent is known (Japanese Patent Application Laid-Open No. 9-3).
09840). In addition, as an oral hair growth drug, Typ
Finasteride, an eII5α reductase inhibitor, is known (J. of).
American Academy of Derma
strategy (1998) 39, 578-589). However, since this drug is a drug that acts on androgen metabolism, there are concerns about side effects, and there are many precautions and restrictions on use.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel anti-hair loss agent which is effective for hair loss, especially for hair loss as a side effect of an anticancer agent, and has high safety and efficacy. It is to be. Since the most effective drug may differ depending on individual differences, it is expected that providing a new anti-hair loss agent will enable a wider range of responses.

[0006]

As described above, the present inventors have reported that MITL, an immunostimulatory peptide obtained from soy protein, prevents hair loss by an anticancer drug.
In the course of elucidating the mechanism of action, the immune-promoting action of MITL was confirmed to be formyl-Met-Leu-Phe (fML
P) It has been found for the first time that the receptor is mediated and that fMLP itself has an anti-hair loss effect. Although fMLP is a known substance known as a chemotactic peptide, its anti-hair loss activity was not known before the present invention. Also, formyl-Met-Leu-Tr, which is a peptide in which Phe of fMLP is substituted by Trp,
p (fMLW) and Met-Leu-Trp (MLW)
Also had an anti-hair loss effect. Met mentioned above is methionine, Leu is leucine, Phe is phenylalanine, and Trp is tryptophan. All such amino acids are in the L-form.

[0007]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a formyl-Met.
Novel peptides which are derivatives of -Leu-Phe (fMLP), Met-Leu-Trp (MLW) and form
yl-Met-Leu-Trp (fMLW). f
MLP and these novel peptides are particularly effective in preventing hair loss, which is a side effect of anticancer drugs. For the above-mentioned hair loss preventing effect, an anti-hair loss agent containing fMLP as an active ingredient can be prepared. MLW and fMLW
Since they also have a hair loss preventing effect, anti-alopecia agents containing these peptides as active ingredients can also be prepared. Since it has not been known that fMLP has an anti-hair loss effect, it is possible to provide a hair removal agent by an unprecedented mechanism according to the present invention.

[0008] As an anticancer agent to prevent hair loss as a side effect by co-administering the functional peptide of the present invention, a plant alkaloid podophyllin compound,
Etoposide can be mentioned. In addition, other plant alkaloid anticancer drugs such as vincristine, vinblastine, vindesine, methotrexate, 5-fluorouracil, 5-fluorodeoxyuridine, tegafur, carmofur, cytosine abavinoside, cyclocytidine, 6
-Mercaptopurine, 6-mercaptopurine riboside,
Antimetabolic anticancer agents such as 6-thioguanine, nitrogen mustard, cyclophosphamide, nimustine, ranimustine, alkylating anticancer agents such as carbone, adriamycin, daunorubicin, daunomycin, achracinomycin A, actinomycin D, mitomycin C, chromomycin a _3, bleomycin, peplomycin, neocarzinostatin, antibiotic-based anti-cancer agents such as O Romo clarithromycin, other, L- asparaginase, cisplatin, estramustine, picibanil, krestin, lentinan, schizophyllan, levamisole, bestatin, Horufeni Anticancer agents such as sinol can also be mentioned as anticancer agents that prevent hair loss by co-administering the functional peptide of the present invention.

The peptide of the present invention can be obtained by a peptide synthesis method. That is, in a liquid phase method or a solid phase method, which is a method usually used for peptide synthesis, a raw material having a reactive carboxyl group corresponding to one of two types of fragments bisected at an arbitrary position of a peptide bond, A raw material having a reactive amino group corresponding to the fragment, and 2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetramethyl
It can be condensed using a method using an active ester such as uronium hexafluorophosphate (HBTU), a method using carbodiimide, or the like. When the resulting condensate has a protecting group, it can be produced also by removing the protecting group.

In this reaction step, functional groups which should not participate in the reaction are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl (B
z), t-butyloxycarbonyl (Boc), p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl group-protecting agent include groups capable of forming an alkyl ester, a benzyl ester, and the like. In the case of the solid phase method, the C-terminal carboxyl group is a chlorotrityl resin, a chloromethyl resin, an oxymethyl resin, It is bound to a carrier such as an alkoxybenzyl alcohol resin. The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.

After completion of the condensation reaction, the protecting group is removed,
In the case of the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. Further, the peptide of the present invention is purified according to a usual method. For example, ion exchange chromatography,
Examples include reversed-phase liquid chromatography and affinity chromatography. The synthesis of the synthesized peptide is analyzed by a protein sequencer that reads the amino acid sequence from the C-terminal by the Edman degradation method, GC-MS, or the like.

Next, the case of using as a medicine will be described. The administration route of MLW may be any of oral administration, parenteral administration, and rectal administration, and can be administered orally or parenterally. fMLP and fML
W is not suitable for oral administration because it is not absorbed from the intestinal tract. The dose of the peptide varies depending on the type of the compound, the administration method, the condition of the patient, the age, etc., but is usually 0.1 mg / kg to 1000 mg per day.
mg / kg, preferably 1 mg / kg to 100 mg / k
g. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing with a preparation carrier. As the pharmaceutical carrier, a substance which is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.

Specifically, examples of such substances include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch,
Carboxymethylcellulose calcium, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan Fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water, etc. No.

[0014] Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, and injections.
These preparations are prepared according to a conventional method. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another suitable solvent at the time of use. Tablets and granules may be coated by a known method. In the case of an injection, the peptide of the present invention is prepared by dissolving the peptide in water, but may be dissolved in a physiological saline solution or a glucose solution if necessary, or may be added with a buffer or a preservative. Good.

These preparations contain the peptide of the present invention in a concentration of 0.1%.
It can be contained at a rate of 01% to 100% by weight, preferably 1 to 90% by weight. These formulations may also contain other components of therapeutic value.

To prepare a solid preparation for oral administration, the active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, and silicic acid anhydride are mixed together to form a powder, or if necessary, Then, a binder such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone and the like, a disintegrant such as carboxymethylcellulose and calcium carboxymethylcellulose are added, and granulated by wet or dry granulation. In order to produce tablets, these powders and granules may be compressed as they are or by adding a lubricant such as magnesium stearate or talc. These granules or tablets should be coated with an enteric base such as hydroxypropylmethylcellulose phthalate, methacrylic acid-methyl methacrylate polymer and enteric coated, or coated with ethylcellulose, carnauba wax, hydrogenated oil, etc. to form a sustained release preparation. Can also. To produce capsules, powders or granules are filled into hard capsules, or the active ingredient is dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, or the like, and then coated with a gelatin film to form soft capsules. Can be.

To prepare a liquid preparation for oral administration, the active ingredient and a sweetening agent such as sucrose, sorbitol, and glycerin are dissolved in water, and a clear syrup, an essential oil, ethanol and the like are added, and an elixir is added. Alternatively, gum arabic, tragacanth, polysorbate 80, sodium carboxymethylcellulose and the like may be added to form an emulsion or suspension. These liquid preparations may optionally contain a flavoring agent,
You may add a coloring agent, a preservative, etc.

In order to prepare an injection, the active ingredient may be added, if necessary, to a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, Dissolve it in distilled water for injection together with an isotonic agent such as glucose, filter aseptically and fill in ampoules, or add mannitol, dextrin,
Cyclodextrin, gelatin, etc. may be added and freeze-dried in vacuum to obtain a working-solution type injection. Also, reticin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like can be added to the active ingredient and emulsified in water to prepare an emulsion for injection.

To prepare a rectal preparation, the active ingredient is dissolved in a suppository base such as cocoa butter, fatty acid tri-, di- and monoglycerides, polyethylene glycol and the like, dissolved and poured into a mold, cooled or cooled. The components may be dissolved in polyethylene glycol, soybean oil, or the like, and then coated with a gelatin film.

To prepare an external preparation for the skin, the active ingredient is added to white petrolatum, beeswax, liquid paraffin, polyethylene glycol, etc. and, if necessary, humidified and kneaded to form an ointment, or rosin, alkyl acrylate After kneading with an adhesive such as an ester polymer, it is spread on a non-woven fabric such as a polyalkyl to form a tape.

[0021]

Now, the present invention will be described more specifically below with reference to working examples. (Synthesis of peptide) Commercially available Fmoc-Trp (Boc)
-Wang resin (substitution rate 0.50 meq / g) 0.60
g to a PS3 peptide synthesizer (Protein Tec)
(made by Hnologies, Inc.), and the novel peptide was synthesized as follows. First, the above resin was placed in a reaction vessel, and 1 mmol of Fmoc-Le
u and 1 mmol of HBTU as an activator for 10 m
1 of dimethylformamide containing 0.4 M of N-methylmorpholine was added to the reaction vessel, and the mixture was stirred and reacted at room temperature for 20 minutes.

The Fmoc group was removed from the obtained resin in 20 ml of dimethylformamide containing 20% by weight of piperidine, and Fmoc-Met was coupled in the same manner as in the above-described method of coupling Fmoc-Leu. Met-Leu-Trp (Boc) -resin was obtained.
In case of further formylation, this resin and 1 mmol
Was reacted with 1 mmol of HBTU in the same manner as described above. The resin was added at room temperature in 10 ml of deprotection solution (82% trifluoroacetic acid, 5% thioanisole, 3% ethanediol, 2% ethyl methyl sulfide, 3% phenol, 5% water). Stir for 1 hour to release the peptide from the resin.

To this was added 40 ml of cold ether to precipitate the peptide, which was further washed three times with cold ether to obtain a crude peptide. This is supplied to an ODS column (Cosmosi
15C ₁₈ -AR, 20 × 250 mm), developed and purified with a linear concentration gradient of acetonitrile containing 0.1% by weight of trifluoroacetic acid by reverse phase chromatography.
Met-Leu-Trp (MLW) was obtained. The product was analyzed using a protein sequencer (Model 477A, manufactured by Applied Biosystems), and as a result, the product was found to have the above composition. formyl-Met-Leu-Tr
p (fMLW) was similarly obtained. As for fMLP, a commercial product of Sigma was used.

(Evaluation of anti-hair loss activity) As a specimen, a 6-day-old SD rat was used. This sample was prepared using the following (A) to
(D) was divided into four groups. Each group had one mother and was allowed to feed freely. The mother was allowed to inoculate the solid food CE2 made by CLEA Japan freely, and was allowed to drink freely. Drug administration is 1
I went once a day between 10 am and 11 am. The peptide is administered once a day according to the protocol of intraperitoneal administration (ip) for 10 days from birth for 4 days, and oral administration (po) for 6 days from birth for 8 days. Was. Etoposide, 20 mg / ml injection 0.9%
Dilute to 2 mg / ml with physiological saline and add 1.5 mg / k
It was administered intraperitoneally at a dose of g body weight for three consecutive days starting on day 11 of birth. (A) Etoposide (Nippon Kayaku's lastet: anticancer agent)
Single administration group (B) Etoposide and fMLP combination administration group (C) Etoposide and MLW combination administration group (D) Etoposide and fMLW combination administration group

At the age of 20 days, the degree of hair loss was examined by visual observation under the naked eye for the administration samples of the above four groups. The determination of the degree of hair removal was performed according to the following criteria. Table 1 shows the test results. Note that the results shown in Table 1 are average values obtained by performing a test at each concentration at n = 4. Hair removal judgment criteria 0: 0 to 10% hair removal 1:11 to 30% hair removal 2:31 to 50% hair removal 3:51 to 70% hair removal 4:71 to 90% hair removal 5:91 to 100% hair removal

[0026]

[Table 1]

From the results shown in Table 1, the known substance fMLP
Completely suppressed hair loss by the anticancer agent etoposide by intraperitoneal administration at 10 mg / kg for 4 days. MLW, a newly synthesized substance, showed almost the same effect by intraperitoneal administration at 30 mg / kg × 4 days or oral administration at 100 mg / kg. FMLW is 10 mg / kg ×
Intraperitoneal administration for 4 days also completely suppressed hair loss by etoposide. The anti-hair loss effect of fMLP was determined by IL
(D) PT which is a -1 (interleukin 1) inhibitor
Was intraperitoneally administered. This finding, f
This suggests that the anti-hair loss effect of MLP is mediated by IL-1.

[0028]

Industrial Applicability It has been shown that fMLP and its derivative peptides, MLW and fMLW, have an effect of suppressing hair loss by an anticancer drug. Therefore, according to the present invention, a novel anti-hair loss agent can be provided by using these peptides.

Claims (5)

[Claims] 1. A novel peptide represented by Met-Leu-Trp. 2. formyl-Met-Leu-Trp
A novel peptide represented by: 3. formyl-Met-Leu-Phe
An anti-alopecia agent comprising, as an active ingredient, a peptide represented by the formula: 4. An anti-alopecia agent comprising a peptide represented by Met-Leu-Trp as an active ingredient. 5. A formyl-Met-Leu-Trp
An anti-alopecia agent comprising, as an active ingredient, a peptide represented by the formula: