TW201317260A - Use of PEDF-derived polypeptides for treating alopecia and/or hair depigmentation - Google Patents

Abstract

Disclosed herein is a synthetic peptide, which has an amino acid sequence that has 20-44 amino acid residues. The synthetic peptide has at least 80% amino acid sequence identity to SEQ ID NO: 10, and includes at least 20 consecutive residues that has at least 90% amino acid sequence identity to residues 16-35 of SEQ ID NO: 10. Also disclosed herein are pharmaceutical compositions containing the synthetic peptide; and uses thereof. According to various embodiments of the present disclosure, the synthetic peptide is useful in treating alopecia and/or hair depigmentation in a subject.

Description

Use of pigment epithelium-derived factor-derived multi-peptide to treat baldness and/or hair bleaching

The present invention is a treatment for alopecia and/or hair discoloration, and in particular for the treatment of baldness and/or hair discoloration using a synthetic peptide derived from a pigment epithelium-derived factor (PEDF).

Alopecia refers to the phenomenon that hair is abnormally detached from the head or body. Baldness can be roughly classified into cicatricial and non-cicatricial alopecia; the former usually involves permanent, irreversible damage to the hair follicle and replaces the hair follicle with scar tissue, while the latter does not. Although baldness does not affect the patient's life, it may affect the appearance of the individual and may therefore affect the individual's social interaction, self-esteem and mental health.

Non-sexual baldness includes different types such as anagen effluvium, telogen effluvium, androgenic alopecia (also known as male alopecia), and alopecia areata. The causes of different types of baldness are different, and their effects on hair follicle tissue are also different.

Growth during hair loss means that when the hair follicle is still in the anagen, the mitosis of the matrix cell abruptly terminates, thereby making it impossible for the hair follicle to make hair or to produce hair that is prone to breakage. This condition is common in patients undergoing chemotherapy or radiation therapy; in general, the hair follicles gradually return to their original growth cycle after stopping treatment. The hair will start to grow again, but the color and texture of the hair may change.

The resting period refers to the situation in which the hair follicles in the growing period enter the rest period early, thus causing the hair to fall off. There are a variety of stress-related factors that can contribute to this phenomenon, including diet imbalances, drugs (such as beta blockers, calcium channel blockers, antidepressants (antidepressants). ), non-steroidal anti-inflammatories, etc., production, high fever, major surgery, thyroid dysfunction, and serious infections, chronic diseases and psychological stress. It usually occurs 3 to 4 months after these factors occur, and abnormal hair loss begins to occur; and within about 6 to 12 months, the hair gradually recovers.

Male baldness is the most common form of baldness in humans and occurs in both men and women, although female patients usually have a lower incidence of hair loss than male patients. The true mechanism of male baldness is still not fully understood. It is generally believed that hair follicles are too sensitive to the androgen derivative Dihydrotestosterone (DHT), which causes hair follicle atrophy and hair follicle cell growth to shorten, leading to easy formation. Club hair.

There are currently two drugs approved by the US Food & Drug Administration (FDA) for the treatment of male baldness, namely Minoxidil (trade name: ROGAINE ^® ) and Fiesta (finasteride; Named Rope PROPECIA ^® ). Minoxide is a potassium channel antagonist that can affect hair follicles by prolonging the growth phase of hair follicles, promoting hair follicle growth from rest period, and promoting hair follicle enlargement; in addition to male baldness, it can also be used to treat rounds. Baldness and baldness caused by chemotherapy. Fistain is a competitive inhibitor of type 2 5α-reductase, which blocks the conversion of testosterone to dihydrotestosterone, thus achieving the effect of treating male baldness. Although both drugs can treat some of the baldness symptoms, they must be used continuously to maintain the therapeutic effect. In addition, long-term use of these two drugs can cause side effects to patients; such as Minoxidide can cause skin irritation (such as redness, itching, scaling, etc.), decreased libido, increased heart rate, difficulty breathing, weight gain and Hypertrichosis; Fistin may cause redness, enlarged breasts, pain in the testicles, decreased libido, and sexual dysfunction.

The hair needs to undergo a process of pigmentation in order to present an appropriate hair color. When the supply of melanin from melanocytes is reduced, the newly grown hair cannot retain the normal color, but presents a lighter color or even white; this phenomenon is called hair depigmentation. ). Factors such as aging, stress, and disease (such as piebaldism) may cause the hair to discolor and appear grayish white. In addition, patients who receive chemotherapy or radiation therapy often develop discoloration after re-existing hair after treatment. There are currently no effective methods or drugs to improve the problem of hair discoloration, so most patients can only accept this phenomenon, or by dyeing hair or using a wig to cover the gray hair.

In view of the above, there is a need in the related art to provide a drug and method capable of effectively treating the severity of symptoms such as alopecia and/or hair discoloration.

SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an

The present invention is based, at least in part, on the discovery that PEDF-derived synthetic peptides are capable of protecting hair follicles and are therefore effective in preventing and/or alleviating the occurrence of hair loss and/or hair discoloration in a body. Specifically, the results of the experimental examples presented in the present specification show that a variety of PEDF-derived synthetic peptides can inhibit hair follicle dystrophy and hair follicle cell apoptosis, and can prevent melanocyte stem cells in hair follicles. , referred to as MSC), early differentiation. Thus, the PEDF-derived synthetic peptide proposed herein can be used as an active compound for preventing and/or alleviating hair loss and/or hair discoloration.

In view of this, one aspect of the present invention relates to the use of a PEDF-derived synthetic peptide which can be used to prepare a medicament for treating baldness and/or hair discoloration in a body.

According to various embodiments of the present specification, the synthetic peptide is 20-44 amino acid residues in length and has a sequence of at least 80% and sequence number: 10 the same. Furthermore, the above amino acid sequence comprises at least 20 contiguous amino acid residues, the sequence of which is at least 90% identical to the amino acid residues 16-35 of SEQ ID NO: 10, such that the synthetic peptide can be used to treat a body. The symptoms of baldness and/or hair discoloration.

In an optional embodiment, the above synthetic peptide has at least four consecutive amino acids having the same sequence as the amino acid residues 16-19 of SEQ ID NO: 10 (i.e., the SLGA sequence). Non-limiting examples of such synthetic peptides include amino acid sequences such as: SEQ ID NO: 10 (44-mer), SEQ ID NO: 1 (39-mer), SEQ ID NO: 2 (34-mer), SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24-mer), SEQ ID NO: 6 (20-mer), SEQ ID NO: 8 (MO 29-mer) and SEQ ID NO: 9 (MO 20-mer) By. According to some embodiments of the invention, the amino acid sequence of the above synthetic peptide is SEQ ID NO: 10 (44-mer), SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24-mer) or SEQ ID NO: :6 (20-mer) is shown as either.

According to various embodiments of the present invention, the baldness symptoms are alopecia areata in the growing season (eg, baldness caused by chemotherapy or radiation therapy), and alopecia areata (eg, by stress, diet, Baldness caused by drugs or infections or baldness caused by male alopecia.

In one embodiment, the hair bleaching symptom refers to hair discoloration caused by chemotherapy or radiation therapy. In another embodiment, the hair bleaching symptom refers to hair discoloration caused by stress or aging.

According to various embodiments of the invention, the individual may be any mammal, including a human.

In another aspect of the invention, a method for treating a body appears A topical pharmaceutical composition (hereinafter referred to as a pharmaceutical composition) for alopecia and/or hair discoloration. The individual can be any mammal, including a human.

According to an embodiment of the present invention, the pharmaceutical composition comprises any of the synthetic peptides according to the above aspect/embodiment of the present invention, and the synthetic peptide is present in an amount sufficient to treat the baldness and/or hair discoloration symptoms of the individual. The pharmaceutical composition also includes a pharmaceutically acceptable excipient that can be used to carry the synthetic peptide.

According to various embodiments of the invention, the pharmaceutical composition may be in any of the following dosage forms: solution, spray, aerosol, foam, cream, lotion, cream, gel or dressing.

According to an optional embodiment of the invention, the synthetic peptide is present in the pharmaceutical composition in an amount of from about 0.1 to 100 μM; preferably from about 1 to 25 μM.

Another aspect of the invention is directed to a method of treating alopecia and/or hair discoloration in a body. The individual/patient can be any mammal, including a human.

In one embodiment, the method comprises administering to the individual an effective amount of a synthetic peptide according to the above aspects/embodiments of the invention. According to various embodiments of the invention, the synthetic peptide described above may be subjected to topical delivery or systemic delivery; preferably topical topical administration.

According to an optional embodiment of the present invention, the above synthetic peptide can be formulated into a topical surface-administered pharmaceutical composition according to the above aspect/embodiment of the present invention. In one embodiment, the pharmaceutical composition can be applied to the one On the scalp of the body.

The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, will be readily apparent to those skilled in the art of the invention.

The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in this specification covers the plural of the noun in the case of no conflict with the context; the plural noun of the noun is also included in the plural noun used.

Although numerical values and parameter boundaries are used to define a broad range of values for the present invention, the relevant values of the specific embodiments have been presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Or, the word "about" represents the acceptable standard error of the actual value falling on the mean. Within the skill of the art to which the invention pertains, it will be considered by those of ordinary skill in the art. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method.

The term "peptide" as used herein refers to a polymer composed of amino acid residues. The word "synthetic peptide" means that the peptide does not contain intact protein molecules found in nature. The reason why such a peptide is "synthetic" is that it is obtained by humans using technical means such as chemical synthesis, recombinant genetic techniques or segmentation of the entire antigen. In the present specification, any amino acid residue based on the position in a peptide is counted from the N-terminus of the peptide.

The term "stem cell" as used herein means that the cell retains the ability to proliferate without substantial differentiation under certain conditions and is capable of differentiating into more specific or highly differentiated cells under certain conditions.

The various parts of the word "proliferation" or "proliferation" herein refer to the situation in which the number of cells in a population increases through cell division.

"Percent % amino acid sequence identity" as used herein with respect to the synthetic peptide sequence means the amino acid residue of the candidate synthetic peptide and the amino acid of a reference polypeptide The percentage of residues that are identical. When performing the above alignment, the candidate synthetic peptide can be side by side with the reference polypeptide, and a gap is introduced as necessary to form the highest sequence similarity of the two sequences, and when calculating the similarity, the Amino acid residues of conservative substitutions are taken into account. A variety of methods are available for the above-described side-by-side, such as publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art to which the present invention pertains may select appropriate parameters and calculation methods when performing side-by-side to obtain an optimal arrangement. In the present specification, sequence comparison between diamine acid sequences is carried out using the protein-protein BLAST analysis software Blastp provided by the National Center for Biotechnology Information (NCBI). Amino acid sequence similarity of a candidate amino acid sequence A compared to a reference amino acid sequence B (also referred to herein as sequence A and sequence B have a specific percentage (%) of amino acid sequence similarity Degree) is calculated as follows: Where X is the identity of the same amino acid residue obtained by aligning the sequences A and B with Blastp software, and Y is the total number of amino acid residues of the shorter of the A and B sequences.

In this specification, the various parts of speech and tense (such as treat, treating or treatment) refer to the prophylactic (eg, prophylactic), healing or palliative treatment to achieve the desired pharmacy. And / or physiological effects. In the preferred case, the above effect means partial or complete It can cure or prevent the occurrence of baldness or hair discoloration in individuals. Furthermore, the term "therapeutic" may be used to refer to the administration or administration of a PEDF-derived synthetic peptide or pharmaceutical composition presented herein to a body which may have a medical condition, symptom, disease or abnormality or ease associated with the condition. Suffering from a condition that partially or completely alleviates, improves, or alleviates, or delays, arrests, delays, and/or reduces the incidence of one or more symptoms or characteristics of a particular abnormality and/or condition . Individuals who are not indicative of signs of disease, abnormality, and/or condition and/or individuals presenting early signs may also be treated with a view to reducing the risk of developing pathological changes associated with the disease, disorder, and/or condition. Here, the above symptoms, diseases, abnormalities or conditions refer to alopecia or hair discoloration. In the present specification, when one or more symptoms or clinical indicators are reduced, the treatment is generally considered to be "effective."

As used herein, the term "effective amount" means an amount of a component sufficient to induce a desired response or effect. The term "therapeutically effective amount" means that the therapeutic agent is present in an amount sufficient to produce the desired "effective treatment" as defined above. The particular therapeutically effective amount depends on a variety of factors, such as the particular condition being treated, the physiological condition of the individual (eg, the individual's weight, age, or sex), the type of mammal or animal being treated, the duration of treatment, current therapy (if The nature of any) and the specific formulation and structure of the compound or derivative thereof used. A therapeutically effective amount also means that the toxic or negative effect of the compound or composition is less than the positive effect of the compound or composition.

"injection", "dosing" or "administration" (application or Administration, as used herein, refers to the administration of one or more effective amounts of the PEDF-derived synthetic peptide or pharmaceutical composition to an individual to treat baldness and/or hair discoloration. According to an embodiment of the present invention, a preferred mode of administration is topical topical administration; that is, the PEDF-derived synthetic peptide or pharmaceutical composition is topically applied to the biological surface of the individual (eg, skin, Scalp or hair) and allow it to reach the target area (eg, hair follicles) to prevent or alleviate baldness and/or hair discoloration.

The term "excipient" as used herein refers to any inert substance (including powder or solution) which can be used as a vehicle/carrier for the PEDF-derived synthetic peptide. Excipients are generally safe, non-toxic materials, and broadly include any material that can be used in the pharmaceutical industry to prepare pharmaceutical compositions, such as fillers, diluents, coagulants, binders, lubricants, glidants, stabilizers. , colorants, sizing agents, etc.

In the present specification, "pharmaceutically acceptable" means that it is suitable for use in humans and/or animals and does not cause undue side effects (such as toxicity, irritation, and toxicity) at a reasonable benefit/risk ratio. Allergic reaction). In addition, each excipient must be compatible with the other ingredients in the pharmaceutical formulation to be an "acceptable" ingredient.

The term "subject" as used herein refers to a mammal (including a human) that can receive the synthetic peptide, pharmaceutical composition and/or method of the present invention for treatment. Unless otherwise indicated, "individual" generally includes males and females.

Animal hair growth follows a fixed growth cycle. In the anagen, cells located in the hair bulb divide rapidly and differentiate into hair shafts; on average, human scalp hair grows. The long-term growth period of mouse hair is about 3-5 years, and the average growth period of mouse hair is about 11 days. After the growth phase, the hair enters the catagen and stops growing. About 70% of the hair follicles in this stage undergo apoptosis and resorption, thus producing club hair; the degeneration period is A transitional phase with a relatively short duration, and the degenerative phase of human scalp hair averages about 1-2 weeks. Then the hair will enter the telogen, during which the hair follicles are completely at rest, and the hair will fall off after the hair is fully formed; the human scalp hair rests for an average of about 3 months; compared to the hair in the growing period The hair that is in the rest period (ie, burst) can be easily pulled up and easily peeled off.

The outer bulge bulge (or bulge) contains hair follicle stem cells (FSCs) and melanocyte stem cells (MSCs), which are responsible for hair regeneration and hair staining, respectively. Factors such as human aging, environmental oxidants, hair follicle disease, chemotherapy, and even the genetic makeup of an individual may affect the stem cell characteristics of hair follicle stem cells and/or melanocyte stem cells, thereby affecting the normal growth cycle of their descendants (progeny). Causes baldness or hair to discolor (eg, gray or white hair).

Pigment epithelium-derived factor (PEDF) is a multifunctional secreted protein with anti-angiogenic, anti-tumorigenic and neurotrophic functions. . The human PEDF protein (SEQ ID NO: 11) is a secreted protein of approximately 50 kDa with 418 amino acids. The 34-mer fragment of PEDF (corresponding to residues 44-77 of PEDF) and the 44-mer fragment are known. Residues Nos. 78-121 of PEDF; SEQ ID NO: 10) have anti-angiogenic and neurotrophic properties, respectively.

This specification is based, at least in part, on the discovery that synthetic peptides from 44-mer PEDF are capable of protecting hair follicles through a variety of mechanisms. For example, the synthetic peptide can inhibit hair follicle atrophy and hair follicle cell apoptosis, prevent melanocyte stem cells in hair follicles from early differentiation into melanocytes, and prevent melanin clumping. Without being bound by a particular theory, it is believed that these protective mechanisms help maintain the stem cell characteristics of stem cells in hair follicles and/or allow the stem cells of these stem cells to be in a normal growth cycle, thereby enabling the hair follicle to continue to be healthy. Normal hair, including hair that is less likely to fall abnormally and/or retain the original hair color. Another innovative feature of the present invention is that the synthetic peptide proposed herein has only 20 to 44 amino acid residues, which is much shorter than the full length PEDF described in the prior art; therefore, the present invention overcomes the traditional protein in clinical practice. Difficulties often encountered in use, such as high manufacturing costs, low bioavailability, and poor pharmacokinetics. Thus, these synthetic peptides can be used to treat baldness and/or hair discoloration.

In view of this, an aspect of the present invention relates to the use of a PEDF-derived synthetic peptide which can be used to prepare a medicament for treating alopecia and/or hair discoloration in a body. Another aspect of the present invention relates to the use of the above PEDF-derived synthetic peptide for treating alopecia and/or hair discoloration in a body; and in yet another aspect of the present invention, the above PEDF-derived synthetic peptide is in the treatment of The individual has the use of baldness and/or hair bleaching. In addition to the above uses, an aspect of the present invention also provides for the treatment of an object A method of bleaching baldness and/or hair. Various embodiments suitable for the above aspects are described below.

According to various embodiments of the present specification, the synthetic peptide has 20-44 amino acid residues, and its amino acid sequence has at least 80% amino acid sequence similarity to SEQ ID NO: 10 (VLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGT). For example, the synthetic peptide can be similar to the amino acid sequence of SEQ ID NO: 10 by about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93. , 94, 95, 96, 97, 98, 99 or 100%. In addition, the synthetic peptide comprises at least 20 contiguous amino acid residues having at least 90% amino acid sequence similarity to SEQ ID NO: 10 16-35 residues. Specifically, the 20 consecutive amino acid residues may have a similarity to the amino acid sequence of SEQ ID NO: 10 16-35 residues of about 90, 91, 92, 93, 94, 95, 96, 97. , 98, 99 or 100%.

In one embodiment of the invention, the synthetic peptide is the 44-mer described above. The inventors of the present invention have previously conducted experiments (e.g., U.S. Patent Application Serial No. 13/428,996, filed on Jun. The experimental examples provided in this specification show that there are several other short PEDF synthetic peptides from this 44-mer that can be used to treat alopecia and/or hair discoloration. In particular, Taiwan Patent Application No. 100138168 discloses that the above PEDF 44-mer is effective in preventing and/or alleviating alopecia and hair discoloration caused by chemotherapy or stress, and preventing and/or easing male baldness.

For example, from the experimental examples of the following and the prior application, the 39-mer synthetic peptide represented by SEQ ID NO: 1 (LSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGT) is effective for treating alopecia and/or hair loss. This 39-mer synthetic peptide corresponds to amino acid residues 83-121 of human PEDF; and is a short peptide fragment derived from the above 44-mer. According to the calculation method of the amino acid sequence similarity described above, the amino acid sequence similarity of the 39-mer to the 44-mer is 100%, and the amino acid residues 11-30 of the 39-mer are The amino acid sequence similarity to the amino acid residue of No. 16-35 of the 44-mer is also 100%.

Further, from the experimental examples of the following and the prior applications, the 34-mer synthetic peptide as shown in SEQ ID NO: 2 (ALSALSLGAEQRTESIIHRALYYDLISSPDIHGT) is also effective for treating alopecia and/or hair loss. The 34-mer synthetic peptide corresponds to amino acid residues 88-121 of human PEDF; its similarity to the amino acid sequence of the 44-mer is 100%, and the amine of the 6--25 of the 34-mer The amino acid sequence similarity to the amino acid sequence of the 16-35 amino acid residue of the 44-mer is also 100%.

Furthermore, the following experimental examples demonstrate that the 29-mer synthetic peptide as shown by SEQ ID NO: 3 (SLGAEQRTESIIHRALYYDLISSPDIHGT) is effective in preventing and/or alleviating the symptoms of male baldness in an individual. In addition, it can be inferred from the experimental examples of the following and the prior application that 29-mer can also be used to treat baldness and/or hair discoloration caused by chemotherapy or stress. This 29-mer synthetic peptide corresponds to amino acid residues 93-121 of human PEDF; its similarity to the amino acid sequence of 44-mer is 100%, and the amine of No. 1-20 of 29-mer Amino acid with a base acid residue and a 44-mer amino acid residue of No. 16-35 The sequence similarity is also 100%.

As demonstrated in some of the experiments below, a 24-mer synthetic peptide is also effective in preventing and/or alleviating the symptoms of male baldness in an individual. In addition, it can be inferred from the experimental examples of the following and the prior application that 24-mer can also be used to treat baldness and/or hair discoloration caused by chemotherapy or stress. The sequence of this 24-mer is shown in SEQ ID NO: 5 (SLGAEQRTESIIHRALYYDLISSP) and corresponds to amino acid residues of human PEDF No. 93-116. In addition, the 24-mer sequence is identical to the amino acid residues 16-35 of the 44-mer (amino acid sequence similarity: 100%) and is similar to the amino acid sequence similarity of the 44-mer. It is also 100%.

Some experiments in this specification demonstrate that the 20-mer as shown in SEQ ID NO: 6 (SLGAEQRTESIIHRALYYDL) is also effective in preventing and/or alleviating the symptoms of male baldness in an individual. In addition, it can be inferred from the experimental examples of the following application that the 20-mer can also be used for the treatment of chemotherapy or stress-induced alopecia and/or hair discoloration. The sequence of this 20-mer corresponds to the amino acid residue of human PEDF No. 93-112, which is identical to the amino acid residue of No. 16-35 of the 44-mer (amino acid sequence similarity: 100%) And its similarity to the amino acid sequence of the 44-mer is also 100%.

The experimental examples presented in this case and the prior application also show that two other short peptides derived from mouse PEDF are also effective in treating male alopecia or baldness caused by chemotherapy or stress and/or Hair color falls off. The first synthetic peptide derived from mouse is referred to in this specification as Mo 29-mer, the sequence of which is shown in SEQ ID NO: 8 (SLGAEHRTESVIHRALYYDLITNPDIHST), this sequence The degree of similarity to the amino acid sequence of the 44-mer was 83%, and the similarity of the first 20 amino acid residues to the amino acid sequence of the 16-35 residues of the 44-mer was 90%. Another synthetic peptide derived from mouse PEDF is called Mo 20-mer and its sequence is shown as SEQ ID NO: 9 (SLGAEHRTESVIHRALYYDL). The similarity of the amino acid sequence of the Mo 20-mer to the 44-mer or 44-mer amino acid residues of the 44-mer was 90%.

In an optional embodiment, the PEDF-derived synthetic peptide has at least four consecutive amino acids having the same sequence as the amino acid residues 16-19 of SEQ ID NO: 10. The experimental results conducted by the inventors of the present invention show that the amino acid residues No. 16-19 of the sequence of SEQ ID NO: 10 (i.e., SLGA) play an important role in maintaining the physiological function of the short PEDF synthetic peptide. For example, the various experimental examples presented below show that the 18-mer synthetic peptide (EQRTESIIHRALYYDLIS; SEQ ID NO: 7) without this SLGA fragment does not provide protection to the individual's hair follicles. In addition, it can be found from the experimental examples contained in the present application and the prior application that the 25-mer synthetic peptide (EQRTESIIHRALYYDLISSPDIHGT; SEQ ID NO: 4) containing no SLGA sequence is also ineffective in treating alopecia and/or hair discoloration.

Any of the conventional techniques described herein can be utilized to synthesize the synthetic peptides described herein, such as t-BOC or EMOC, to protect alpha-amino groups. Both methods employ a stepwise synthesis starting with the C-terminus of the peptide, each time adding an amino acid. It can also be synthesized by other known solid phase peptide synthesis (SPPS) methods. Synthetic peptides as described herein.

Also included within the scope of the invention are other synthetic peptides that have conservative variations compared to 39-mer. Here, the term "conservative substitution" refers to the replacement of a residue with another biologically similar residue. Illustrative of conservative substitutions include substitutions of hydrophilic residues (such as isoleucine, valine, leucine, and methionine) with each other, or similar polar residues (such as arginine and lysine; Or glutamic acid and aspartate) replacement with each other, and other similar modes of substitution. "Conservative substitution" as used herein also refers to the use of a substituted amino acid to replace an unsubstituted amino acid, as long as the antibody reactive with the substituted amino acid can also react with the original amine. The base acid can be used for an immune reaction.

According to various embodiments of the present invention, the baldness symptoms are alopecia areata in the growing season (eg, baldness caused by chemotherapy or radiation therapy), and alopecia areata (eg, by stress, diet, Baldness caused by drugs or infections or baldness caused by male alopecia.

In certain embodiments, the hair bleaching symptom refers to hair discoloration caused by chemotherapy or radiation therapy. In other embodiments, the hair bleaching symptom refers to hair discoloration caused by stress or aging.

The PEDF multipeptide fragments described herein are useful for treating baldness and/or hair discoloration in mammals. The mammals include, but are not limited to, humans, primates other than humans, murines, and the like. In a preferred embodiment, the object is a human.

The various synthetic peptides described in the above embodiments may also be formulated into a topical surface-administered pharmaceutical composition for treating alopecia and/or hair bleaching of an individual. Phenomenon; this topical surface-administered pharmaceutical composition is within the scope of another aspect of the invention.

According to an embodiment of the present invention, the above topical surface-administered pharmaceutical composition comprises any of the synthetic peptides according to the above aspect/embodiment of the present invention, and the synthetic peptide is present in an amount sufficient to treat the baldness and/or hair of the individual. Decolorization symptoms. The topical surface-administered pharmaceutical composition also includes a pharmaceutically acceptable excipient that can be used to carry the synthetic peptide.

The above pharmaceutical compositions can be prepared according to established pharmaceutical procedures, such as those described in Remington's Pharmaceutical Sciences (17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa (1985).).

In selecting a pharmaceutically acceptable excipient suitable for delivery of a synthetic peptide, it is primarily necessary to consider the mode of administration of the topical surface-administered pharmaceutical composition. According to an optional embodiment of the invention, the topical surface application pharmaceutical composition can be contacted with the biological surface of the subject (eg, skin, scalp or hair) by spraying, smearing, massaging or coating or other means. The synthetic peptide contained therein can be actively absorbed through the hair follicle (ie, through a transappendageal route) or through a membrane (eg, a transcellular or intercellular pathway). A target site (eg, a hair follicle) that reaches the body of the subject.

In the preparation of the above topical surface-administered pharmaceutical composition, various dermatologically acceptable inert excipients can be used; for example, water, ethyl alcohol, polyvinyl pyrrolidone, propylene glycol, minerals Mineral oil, stearyl alcohol Alcohol), ointment base and other materials that form a gel.

Optionally, the pharmaceutical compositions of the present invention may also comprise one or more pharmaceutically (especially dermatologically) acceptable additives well known to those of ordinary skill in the art to improve drug delivery efficiency and improvement. User experience. The above optional ingredients include, but are not limited to, one or more of the following ingredients: drying agents, anti-itch agents, anti-foaming agents, buffers, Neutralizing agents, pH adjusting agents, coloring agents, discoloring agents, emollients, emulsifying agents, emulsion stabilizers ), viscosity builders, humectants, odorants, preservatives, antioxidants, chemical stabilizers, thickening agents, Stiffening agents, suspending agents, and the like.

According to various embodiments of the present invention, the pharmaceutical composition can be formulated into any of the following dosage forms: a solution, a spray, an aerosol, a foam, a cream, Lotion, ointment, gel or patch.

According to an optional embodiment of the invention, the synthetic peptide is present in the pharmaceutical composition in an amount of from about 0.1 to 100 μM; preferably from about 1 to 25 μM. For example, the content of the synthetic peptide can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μM. Specifically, the dose concentration for the approximately 20 gram mouse was used in the experimental examples below to be about 25 μM. The person skilled in the art can calculate the human equivalent dose (HEQ) based on the dose of the mouse administered in the present specification; for example, according to the guidelines proposed by the US Food and Health Administration (title) "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers").

In an optional embodiment, the synthetic peptide can also be formulated in a sustained release dosage form to ensure that the pharmaceutical composition provides a longer-lasting therapeutic effect. For example, the synthetic peptide can be first packaged in a carrier and then formulated into the desired dosage form. Such carriers are, for example, liposomes, microspheres or nanosomes. In certain embodiments, these vesicles-forming carriers (e.g., liposomes) can also facilitate delivery efficiency of the synthetic peptides or pharmaceutical compositions set forth herein.

In other optional embodiments, the synthetic peptide can be first coated, then coated, impregnated or otherwise left on the surface of a substrate and/or in internal pores. To get a dressing. The substrate described herein may be made from one or more natural materials and/or one or more synthetic materials such as collagen, hyaluronic acid, alginate or chitin. Glycan (chitosan) or a derivative thereof; a synthetic material such as polyurethane, polyglycolic acid or polyvinyl pyrrolidone or a derivative thereof. The coating material and/or the substrate may have suitable bioadhesive properties to facilitate attachment of the dressing to the biological surface (eg, scalp) of the site to be treated, and not to the hair of the portion when the dressing is removed. Growth has a negative impact.

In yet another aspect, the invention provides a method for treating alopecia and/or hair discoloration in a body. The individual can be any mammal, including a human.

In one embodiment, the above method comprises administering to the individual a therapeutically effective amount of a synthetic peptide according to the above aspect/embodiment of the invention. According to various embodiments of the invention, the above synthetic peptides can be delivered using local or systemic methods of administration; in the preferred case, topical administration is utilized. In a more preferred embodiment, the synthetic peptide can be topically administered to the biological surface of the patient (such as the skin, scalp or hair) such that the synthetic peptide can reach the target site (eg, hair follicle) Promotes hair growth and/or hair coloration.

In an optional embodiment, the synthetic peptide can be formulated into a topical surface-administered pharmaceutical composition as described above in the Aspects/Examples of the present invention. In one embodiment, the pharmaceutical compositions presented herein can be applied to the biological surface of the individual, such as the skin, scalp or hair.

In addition, other methods of use (such as specific humidity or temperature) can be used in implementing the methods presented herein. For example, in an optional embodiment, it can be administered under relatively warm and humid conditions to promote absorption of the drug.

According to various embodiments of the invention, the individual being treated may have alopecia areata in the growing season (eg, baldness caused by chemotherapy or radiation therapy), and alopecia (eg, by stress, diet, Baldness caused by drugs or infections or baldness caused by male alopecia. In certain embodiments, the subject being treated may develop hair discoloration symptoms as a result of receiving chemotherapy or radiation therapy. In other embodiments, the subject being treated may have discoloration of the hair due to oxidative stress or aging. In addition, the individual may also have alopecia and/or hair discoloration due to his or her individual genetic makeup.

A number of experimental examples are set forth below to illustrate certain aspects of the present invention, and the present invention may be practiced by those of ordinary skill in the art. These examples should not be construed as limiting the scope of the invention. It is believed that the skilled artisan, after reading the description set forth herein, may fully utilize and practice the invention without undue interpretation. All publications cited herein are hereby incorporated by reference in their entirety.

Experimental example Materials and Methods <material>

DMEM medium (Dulbecco's modified Eagle's medium), fetal bovine serum (FBS) and 0.25% trypsin were purchased from Invitrogen (Carlsbad, CA). Dihydrochitinone, ethanol, dimethyl sulfoxide (DMSO), insulin, hydrocortisone, bovine blood Bovine serum albumin (BSA), 5-bromo-2'-deoxyuridine (BrdU), Hoechst 33258 dye and Hoechst 33342 dye were purchased from Sigma-Aldrich ( St.Louis, MO). Anti-BrdU antibodies were purchased from GeneTex (Taipei, Taiwan). Anti-c-kit antibody (No. 3074) was purchased from Cell Signaling Technology, Inc. (Beverley, MA). A tyrosinase antibody (No. sc-7833) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Various fluorescent dye-binding secondary antibodies were purchased from BioLegend (San Diego, CA). Hematoxylin and eosin (H&E) dyes were purchased from Merck (Rayway, NJ, USA).

FITC-bound 29-mer and other short synthetic peptides (including 44-mer (SEQ ID NO: 10), 29-mer (SEQ ID NO: 3), 24-mer (SEQ ID NO: 5), 20-mer (SEQ ID NO: :6), 18-mer (SEQ ID NO: 7), and Mo 20-mer (SEQ ID NO: 9)) were purchased from GenScript (Piscataway, NJ), and after synthesis, their N-terminal was acetylated and The C-terminus was amidated to enhance stability and was characterized by mass spectrometry (purity >95%).

<animal>

All animals used in the examples contained in this specification were housed in a cage with temperature and humidity control at a temperature of about 24 ° C to 25 ° C and a light dark cycle of 12: 12 hours. Drinking water and standard rodent feed are provided for the meal during the test. The experimental plans were approved by the Ethics Committee of the Ma Rong Memorial Hospital (New Taipei City, Taiwan) and followed the national animal protection regulations.

<cream or gel formula>

Each PEDF-derived synthetic peptide (hereinafter referred to as PEDF peptide, including FITC-conjugated 29-mer, 44-mer, 29-mer, 24--) was prepared using dimethyl sulfoxide (DMSO) as a solvent. Mer, 20-mer, 18-mer and Mo 20-mer) were made into a 5 mM stock solution and stored at -20 °C for subsequent use.

Prior to use, the stock solution containing 44-mer was diluted into working dilution and mixed with antibiotic ointment to obtain an antibiotic ointment containing 25 μM of 44-mer. The above antibiotic ointment contains 5 mg of neomycin sulfate and 12.5 mg of bacitracin per gram. As for the other PEDF peptides, 5 μl of the above 5 mM PEDF peptide storage solution was gradually diluted with ethanol (30% w/w in distilled water), and then slowly added 1% (w/w) 1- Octadecanoic acid monoglyceride (a skin penetration enhancer) with 1% (w/w) light mineral oil; followed by 5% (w/w) hydroxypropyl methylcellulose for final The volume was 1 ml. After the addition of hydroxypropylmethylcellulose, the solution began to undergo a gelling reaction. Upon completion of the reaction, a gel containing 25 μM of PEDF peptide was obtained. In addition, the PEDF peptide was replaced with DMSO to prepare an ointment or gel for the control group. The 29-mer (hereinafter referred to as FITC-29-mer) combined with FITC was also prepared in the same manner to observe the distribution of FITC-29-mer in the hair follicle. The ointment or gel prepared above is used immediately after preparation.

For topical topical administration, 350 mg contains 25 μM of PEDF The peptide or gel of the peptide is gently applied to the back skin of each mouse.

<Histology, Immunofluorescence and Quantitative Analysis>

The back skin of the mouse parallel to the vertebral line was collected to obtain a sample containing the longitudinal extent of the hair follicle. After the back skin was fixed with 4% formaldehyde, it was embedded in solid paraffin blocks, and the tissue was cut into sheets of about 5-μm to obtain longitudinal sections. The paraffin was removed in xylene before use and the sample was returned to the series with a concentration gradient of ethanol. General histological observations were performed using H&E staining. Digital micrographs (fixed magnification: 100 x) were taken from representative regions of the sample using a Zeiss fluorescence microscope (Jena, Germany).

To analyze the morphogenesis of the hair follicles, the animals were euthanized on the 15th day after depilation. According to the classification of murine hair follicle tissue accepted in the art (see: Müller-Röver et al., Journal of Investigative Dermatology (2001) 117, 3-15), hair follicles at different stages during tissue formation are evaluated. percentage. At least 24 longitudinal hair follicle sections were taken from 6-10 DHT treated mice. To determine the catagen retardation associated with PEDF peptides in DHT-treated animals, select 5 to 7 consecutive microscopic fields from a single representative skin slice, and then use computer-assisted These microscopic fields were combined by an image analyzer (Adobe Photoshop CS3 10.0).

Prior to immunostaining analysis, de-waxed tissue sections were treated with 10% goat serum for about 1 hour for blocking. Anti-c-kit primary antibody (Dilution ratio 1:100) and anti-tyrosinase antibody (dilution ratio 1:100) were incubated at 37 ° C for 2 hours, and then combined with appropriate fluorescent dye goat IgG antibody (dilution ratio 1: 500) Incubate for 1 hour at room temperature. The sample was then contacted with Hoechst 33258 dye for about 7 minutes for counter staining of the nuclei, and the stained nuclei appeared blue.

Frozen sections of mouse vibrissae follicles were washed three times with PBS. The paraffin was removed from the paraffin-embedded sample by 4% formaldehyde and the sample was returned to the sample with a series of concentration gradients of ethanol. Next, a TdT-mediated dUTP nick-end labeling (TUNEL) kit (available from Roche Molecular Biochemicals, Indianapolis, IN) was used and used according to the manufacturer. Description for dyeing. Contrast staining (blue) of the nuclei was performed with Hoechst 33258 stain to observe the number of cells. The nucleus was calculated by randomly selecting 10 fields from three different sample chambers using a Zeiss fluorescence microscope (fixed magnification: 400×; 10 fields per sample); and images were captured using the Zeiss software.

After fixation with 4% paraformaldehyde, the cells were placed in cold methanol for about 2 minutes, followed by treatment with 1 N hydrogen chloride at room temperature for about 1 hour, followed by immunofluorescence staining. BrdU-labeled DNA was detected using an anti-BrdU multi-strain antibody and rose-cross-conjugated anti-rabbit IgG antibody.

In the animal test, BrdU was dissolved in DMSO to obtain a stock solution of BrdU at a concentration of 80 mM. 10 μl of BrdU stock solution and 90 μl of PBS solution were mixed and intraperitoneally injected into mice. The mice were euthanized after 16 hours. The paraffin was removed from the paraffin-embedded sample by 4% formaldehyde and the sample was returned to the sample with a series of concentration gradients of ethanol. The tentacle hair follicles were washed three times with PBS, and then treated with 1 N hydrogen chloride at room temperature for about 1 hour, followed by immunofluorescence analysis.

<cell culture>

The mouse hair follicle hair follicles were cultured as follows. A whisker pad was cut from the face of the mouse and placed in a petri dish containing a mixture of PBS and 1% antibiotic. Each of the tentacles hair follicles separated from the whisker pad was placed in a 24-well tray (3 whisker hair follicles per well) and cultured in DMEM medium; insulin (10 μg/ml) and hydrogen cortex were added to the medium. Ketone (10 ng/ml), 1% penicillin/streptomycin antibiotic mixture and L-glutamine (2 mM); culture conditions were 37 ° C and 5% carbon dioxide. 10-15 hair follicles were observed under each experimental condition and repeated three times. DHT and PEDF peptide were dissolved in DMSO and added to the above hair follicle medium respectively, and insulin and hydrocorticosterone were added to the culture solution, and the final concentration of DMSO was 0.05% or less.

The human keratinocyte strain HaCaT was cultured in 10% FBS-DMEM at 37 ° C, and the cells were seeded on 5% FBS-DMEM, and then the cells were treated with PEDF peptide or recombinant human TGFβ1 (R&D Systems).

<Statistical Analysis>

The results are expressed as mean ± standard error of the mean (SEM). Statistical comparisons were made using one-way ANOVA analysis. Statistically significant differences were obtained at P < 0.05 unless otherwise stated.

Experimental example 1 PEDF peptide can prevent/alleviate hair loss and hair bleaching caused by chemotherapy

An animal model of baldness and hair decolorization induced by cyclophosphamide (CYP) in C57BL/6 mice has been widely used to study and simulate baldness and hair discoloration (white hair) caused by human chemotherapy. This test used 6-week-old female C57BL/6 mice with normal black hair (ie, the terminal hair shaft contained a large amount of melanin). A total of 80 mice were used in four independent experiments. In this test, only mice in which the hair growth cycle was in the resting phase were used. Since the production of pigment in mice (also known as melanogenesis) occurs only in the hair follicles in the growth phase, the hair follicles of the C57BL/6 mice in the resting period do not have melanin production, so the skin on the back is uniformly pink. The hair removal of the back of the mouse allows the hair follicles in the rest period to enter the growth phase. In order to obtain mice with identical hair growth cycles, this experiment carefully cuts the hair on the back of resting mice (pink skin) using an electric scissors. On the 9th day after depilation, the hair entered the growth phase, and it was observed that the skin color of the mouse changed from pink (resting skin) to gray (pre-growth VI).

On the 9th day after depilation, the mice were intraperitoneally injected with a mixture of zoletil (6 mg per kg body weight) and xylazine (3 mg per kg body weight) to anesthetize the experimental mice. Randomly divide mice Assigned to different experimental groups and given the following treatments. Mice in the CYP/carrier group received an intraperitoneal injection of the anticancer drug CYP (150 mg per kg body weight, CYP dissolved in water before the experiment) and a 350 mg antibiotic ointment on the back skin. Mice in the CYP/44-mer group received an intraperitoneal injection of an anticancer drug (150 mg/kg body weight) and a 350 mg 44-mer antibiotic ointment on the back. Carrier and 44-mer mice I did not receive CYP intraperitoneal injection, only 350 mg of antibiotic ointment and 350 mg of 44-mer antibiotic ointment. After that, apply 350 mg of appropriate antibiotic ointment (with or without 44-) to the back of each group for 7 consecutive days. Mer) In the blank control group, the mice did not receive CYP injections and did not apply any antibiotic ointment.

A representative photograph of Figure 1 presents the mice on each group on day 9 after depilation (ie, day 0 after CYP injection), on day 16 after depilation (ie, on day 7 after CYP injection) and on the 45th after depilation Appearance of days (ie, day 36 after CYP injection). Comparing the CYP/vector group with the CYP/44-mer group, it was found that the CYP/44-mer mice were superior to the CYP/vehicle group on day 7 and day 36 after CYP injection.

It is known that administration of 150 mg of CYP per kilogram of body weight in mice can induce apoptosis of hair follicle keratinocytes and atrophy of hair follicles in mice. Therefore, apoptotic cells were stained by TUNEL staining to evaluate the protective effect of PEDF peptide on hair follicle keratinocytes. The representative photographs in Fig. 2 show the results of TUNEL fluorescent staining (green); the results of the quantitative analysis are summarized in Table 1. The number of TUNEL-positive cells is represented by a labeling index, which is the number of labeled cells divided by the total number of cells, and expressed as a percentage (%) said.

The above results showed that TUNNEL-positive cells were significantly reduced in mice treated with the 44-mer ointment compared to mice treated only with cream. It can be seen that administration of the PEDF peptide protects the hair follicle keratinocytes from the damage caused by chemotherapeutic drugs (e.g., CYP), and thus can be used to prevent and/or alleviate hair loss caused by chemotherapy.

Melanocyte stem cells (MSCs) are responsible for supplying differentiated descendant cells (melanocytes and melanocytes) to the hair roots and regulating the differentiation cycle of their descendants. Recent studies have pointed out that when MSCs in stem cell niches are abnormally differentiated into mature melanocytes, the number of MSCs is reduced, and individuals will irreversibly produce gray hair. This phenomenon is called atopic. Ectopically pigmented melanocytes (EPM).

Only melanin located in the hair bulb can dye the hair shaft. Specifically, melanin granules produced by melanocytes in the hair bulb are delivered to keratinocytes to form a stained hair shaft. In addition, fully differentiated hair follicle melanocytes undergo apoptosis during the degenerative phase, while self-wilting Removed from the shrunken hair follicle. When the hair follicles have dystrophic anagen, the melanin content in the hair bulb during the growth period will be abnormally reduced and unevenly distributed, and melanin clump will occur. Dystrophic catagen refers to the phenomenon of melanin aggregation in the hair follicle where the melanin should not appear (ie, the part other than the hair bulb).

Referring again to Figure 1, mice treated with CYP/vessel had more and more pronounced gray-white hair on day 36 after CYP injection; whereas mice treated with CYP/44-mer had darker hair color. . These results imply that MSCs may be damaged by CYP in the CYP and CYP/carrier groups and thus unable to supply sufficient melanocytes to produce melanin.

MSCs can express the melanocyte lineage marker-c-kit, and mature melanocytes can express tyrosinase and c-kit. Thus, the activity of stem cells in hair follicles was observed here by staining with an anti-c-kit antibody and an anti-tyrosinase antibody. The representative immunostained photographs in Fig. 3 show the results of staining analysis. As can be seen from the figure, in the CYP/carrier group, the expression of c-kit and tyrosinase can hardly be detected, indicating The activity of MSCs in the hairs is relatively low. In contrast, in the CYP/44-mer group of mice, the performance of c-kit and tyrosinase was observed. Taken together, these data show that the PEDF peptide proposed here can effectively protect MSC from CYP.

In summary, the results in Experimental Example 1 show that the 44-mer PEDF peptide can effectively reduce hair follicle atrophy, melanin aggregation, hair follicle and keratinocyte apoptosis caused by CYP. Thus, 44-mer PEDF peptide It is possible to prevent and/or alleviate hair loss and/or hair discoloration caused by chemotherapy. Furthermore, from the results of previous studies by the inventors of the present invention and the results of Experimental Example 3 below, it was found that the 39-mer derived from this 44-mer and various short peptide fragments derived from 39-mer are similar to 44-mer. Physiological activity. It can be inferred that these short PEDF synthetic peptides (including SEQ ID NO: 1 (39-mer), SEQ ID NO: 2 (34-mer), SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24 -mer), SEQ ID NO: 6 (20-mer), SEQ ID NO: 8 (MO 29-mer) and SEQ ID NO: 9 (MO 20-mer) are also effective in treating baldness and/or chemotherapy-induced alopecia and/or Hair is discolored.

Experimental example 2 PEDF peptide can prevent/alleviate pressure-induced hair loss and hair bleaching

A variety of environmental and endogenous factors are known to cause Oxidative stress and thus accelerate the aging process; such factors as ultraviolet light, radiation, inflammation, or psychoemotional stress. In addition, the genetic makeup of an individual can also affect an individual's tolerance to these factors. Studies have shown that hydrogen peroxide is present in milligrams in old hair follicles. In addition, it has been demonstrated in animals that such extraneous oxidative stress can increase the proportion of melanocyte apoptosis in hair follicles, which ultimately leads to hair whitening.

This experimental example explores the relationship between stress caused by external factors (such as oxides) and graying of hair loss feathers. Mice were selected and depilated according to the method described in Experimental Example 1. On the 9th day after depilation, mice were subcutaneously injected with 10 ml of 1% hydrogen peroxide (H ₂ O ₂ ) per kg of body weight. In addition, 350 mg antibiotic ointment (H ₂ O ₂ /carrier group) or 44-mer antibiotic ointment (H ₂ O ₂ /44-mer group) was applied to the back skin of the mouse at the same time as the injection.

The representative photographs of Fig. 4 show the appearance of each group of mice on the 9th day after depilation (the day of H ₂ O ₂ injection) and the 31st day after depilation (the 22nd day after H ₂ O ₂ injection). It can be seen from Fig. 4 that the hair loss and hair whitening of the mice treated with H ₂ O ₂ /carrier were more severe than those of the H ₂ O ₂ /44-mer treated mice. From this, it can be seen that the 44-mer PEDF polypeptide fragment described herein is effective for preventing and/or alleviating hair loss and hair whitening caused by pressure (e.g., oxidative stress) or aging.

Further, from the results of previous studies by the inventors of the present invention and the results of Experimental Example 3 below, it was found that various short peptide fragments derived from this 44-mer and 44-mer have similar physiological activities. It can be inferred that these short PEDF synthetic peptides (including SEQ ID NO: 1 (39-mer), SEQ ID NO: 2 (34-mer), SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24 -mer), SEQ ID NO: 6 (20-mer), SEQ ID NO: 8 (MO 29-mer) and SEQ ID NO: 9 (MO 20-mer)) can also effectively treat baldness caused by stress and/or Hair is discolored.

Experimental example 3 PEDF peptide can prevent / alleviate male baldness

A number of in vitro and in vivo analyses were performed in this experimental example to evaluate the effect of the PEDF peptide proposed herein on male baldness.

Experimental example 3.1 PEDF peptide can antagonize DHT-induced hair follicle growth arrest and keratinocyte apoptosis in vitro

PEDF peptide (29-mer, 24-mer, 20-mer or Mo 20-mer) was added to the tissue culture of C57BL/6 male mouse tentative hair follicles for 16 hours, respectively, and then 100 nM DHT was added for further culture. 48 hours. Tissue analysis at high magnification revealed that DHT caused irregular hair matrix cells and melanin aggregation compared to untreated hair follicles (Fig. 5). In contrast, in the 20-mer PEDF-treated culture, in most areas of the hair matrix, the arrangement of keratinocytes was regular and there were fewer abnormal melanin particles (Fig. 5).

Immunological analysis of frozen section samples was performed to detect TUNEL- and BrdU-positive cells to assess apoptosis and cell proliferation of HF epithelial cells. The results of the quantitative analysis are shown in Table 2, wherein the labeling index (%) of TUNEL- and BrdU-positive cells is the number of labeled cells divided by the total number of cells, and expressed as a percentage (%).

^# P <0.05 compared to the untreated group; ^## P <0.002 compared to the DHT/carrier group.

As can be seen from Table 2, in vitro, the proportion of spontaneous apoptosis of keratinocytes in the hair matrix is very low (1.7 ± 0.76%; untreated group); but after 100 nM DHT treatment, around the skin (dermal) The keratinocytes near papilla (DP) have a large number of deaths (28.5 ± 4.90%; DHT/vector group). In addition, the hair follicle hair follicles treated with the PEDF peptides (eg, 29-mer, 24-mer, 20-mer, or Mo 20-mer) presented herein have fewer TUNEL-positive keratinocytes than carriers or 18- Mer PEDF peptide handler. These results show that the PEDF treatment proposed in the present specification can effectively reduce DHT-induced keratinocyte apoptosis.

From the results of Table 2, it was also found that the cell division of keratinocytes in the hair matrix was also inhibited by DHT compared to the untreated group of hair follicles (untreated group: 15.8 ± 2.08%; DHT/carrier group: 9.0 ± 1.92%). ). On the other hand, cells treated with the PEDF peptide described herein have a higher BrdU labeling index, meaning that this treatment improves the proliferative activity of hair follicle keratinocytes.

DHT is known to induce the expression of transforming growth factor-β1 (TGF-β1) in dermal papilla cells (DPCs), which is a degenerative phase inducer that inhibits males. The hair growth of bald patients and the growth of hair follicle keratinocytes. To further confirm whether the PEDF-derived peptide proposed here can protect keratinocytes from DHT-mediated growth inhibition, the cell division of human epithelial keratinocytes (HaCaT) is analyzed by introducing BrdU to TGF-β1 and/or PEDF. The reaction of the peptide. The results of the quantitative analysis are summarized in Table 3, in which the labeling index (%) of BrdU-positive cells is labeled. The number of cells divided by the total number of cells is expressed as a percentage (%).

As shown in Table 3, the labeling index of BrdU-positive cells in HaCaT cells without any treatment group was 23.6 ± 2.84%; in comparison, HaCaT treated with TGF-β1 (4 ng/ml, 24 hours) The proportion of BrdU-positive cells in the cells was greatly reduced, and only 9.9 ± 2.23%; that is, TGF-β1 treatment did inhibit the proliferation of HaCaT. In the TGF-β1/20-mer group, cells were treated with 20-mer (50 nM) for 16 hours, followed by TGF-β1 (4 ng/ml, 24 hours); as can be seen from the results in Table 3, At this time, TGF-β1 could not inhibit the proliferative activity of HaCaT cells.

In summary, PEDF-derived peptides can counteract the negative effects of DHT (and TGFβ1) on inhibiting keratinocyte proliferation.

Experimental Example 3.2 PEDF peptide can block DHT-induced degenerative development in vivo

In vivo assays were performed using a 5α-dihydrosterolone (DHT) animal model. In this test, 6-week-old male C57BL/6 mice (body weight 15-20 g) with a hair growth cycle in a resting period were used, and 6 to 10 mice were used in each experimental group. After anesthetizing the mice, use a melted wax/sabi mixture to make the small The hair is removed from the hair to induce long-term. DHT was dissolved in DMSO to give a 50 mg/ml stock solution. On day 11 after depilation, DHT solution (1.5 mg of DHT was mixed with 200 μl of olive oil; about 30 mg per kilogram of body weight) was subcutaneously injected into the mouse from the depilated back area. After DHT injection, a 350 mg carrier gel or 350 mg of a gel containing 25 μM of PEDF peptide (29-mer, 24-mer, 20-mer or 18-mer) was applied to the skin of the back of the mouse. And administered once a day for three consecutive days.

Tissue analysis was performed on day 15 after depilation (ie, day 4 after DHT injection), and representative photographs in panel 6 show that in untreated mice, the hair follicles were mainly in the growth phase and the degeneration period was observed. Spontaneous progression; in contrast, in DHT/vectors and DHT/18-mer, mouse hair follicles have entered a degenerative phase under the induction of DHT. On the other hand, in the mice treated with 29-mer and 20-mer, the hair follicles were mainly in the growth phase, and this result showed that the accelerated development of the degenerative phase due to DHT was blocked. For the different experimental groups, the hair follicles in each growth cycle were quantitatively analyzed, and the results are summarized in Table 4.

The data in Table 4 indicate that in the DHT/29-mer, DHT/24-mer and DHT/20-mer groups, the mouse hair follicles have a significantly higher proportion in the growth phase and early in the degenerative phase (degraded phase I to Phase III). On the other hand, in the DHT/vector and DHT/18-mer groups, the hair follicles of mice are mostly in the middle to late stages of degeneration (ie, stage IV to VIII of degenerative phase).

In order to confirm the results of the above-mentioned tissue type analysis, BrdU and TUNEL staining analysis was also carried out, and the results of the quantitative analysis are summarized in Table 5, in which the hair matrix (HM) and the outer root sheath (outer root sheath, In the ORS), the number of TUNNEL-positive cells in each hair follicle, and the marker index (%) of BrdU-positive cells in the hair bulb.

Comparing the untreated group with the DHT/carrier group, it can be seen that the TUNEL-positive apoptotic cells in the hair matrix and the outer root sheath are significantly increased, and the proliferative activity of the hair follicles near the growth period is decreased. These results show that DHT injection promotes hair follicles. Degraded development. After DHT injection and 20-mer, 24-mer In the 29-mer treated mice, there were almost no TUNEL-positive apoptotic cells (less than 1 in each hair follicle), and a large number of BrdU-positive keratinocytes were observed, confirming the proposed PEDF peptide can antagonize the effect of DHT on hair follicle degeneration.

In summary, the inhibition of cell proliferation and apoptosis of hair follicle keratinocytes can prove that DHT can cause degenerative development, while the surface surface is administered with the PEDF-derived peptides proposed by the present invention (such as 29-mer, 24-mer and 20-). Mer) can hinder the development of the above-mentioned degradation period. It can be seen that the PEDF-derived peptide proposed herein can block DHT-induced premature anagen to catagen transition.

Experimental Example 3.3 PEDF peptide can maintain hair follicle development in vivo

In order to evaluate the efficacy of PEDF peptides in restarting hair growth, C57BL/6 mice were selected according to the method described in Experimental Example 3.2 and DHT animal models were established, and DHT was administered twice a week to the mice during the test. Solution. After the first DHT injection, apply 350 mg of the carrier gel or 350 mg of the gel to the surface of the gel containing 25 μM of PEDF peptide (29-mer, 24-mer, 20-mer or 18-mer). The back skin of the rats was administered once a day for 14 consecutive days. The change in hair was recorded using a digital camera and quantitative analysis was performed on the 18th day after depilation (see Figure 7 for representative photos). First, the percentage of the black area (ie, the portion where the hair grows again) is calculated in the hair removal area, and the score of hair regrowth after DHT injection is evaluated according to the following criteria: 0% (no hair growth) 0 Points -; <20% of 1 point; 20-39% of 2 points; 40-59% of 3 points; 60-79% of 4 points Points; 80-100% of 5 points. The number of hair bulbs per unit length (1 mm) in the deep subcutis was also calculated according to the method described in Materials and Methods. The above quantitative analysis results are summarized in Table 6.

The data shown in Figures 7 and 6 indicate that back hair removal can induce hair follicles to enter the growth phase (untreated group; hair growth fraction 5 ± 0). Conversely, on day 18 after depilation, hair growth was significantly inhibited in DHT/vehicle-treated and DHT/18-mer-treated mice (hair growth fractions were all less than 2); this phenomenon is widely accepted in male alopecia animals. In the model, it is the expected result. On the other hand, DHT-injected mice showed significant improvement in hair growth inhibition after daily topical application of 29-mer, 24-mer or 20-mer, and significant hair growth was observed. . The results of statistical analysis showed that mice treated with DHT and receiving 29-mer, 24-mer and 20-mer had better hair growth score after depilation compared to the control group of DHT/vector, and this one The difference is statistically significant.

Continuing with reference to Table 6, mice treated with 29-mer, 24-mer or 20-mer were in deep subcutaneous tissue compared to the DHT/vector and DHT/18-mer groups. The number of hair balls inside is also significantly higher (1.6 times). These results show that the PEDF peptide proposed here can protect the hair follicle, so that most hair follicles can escape the influence of DHT and enter the growth phase.

In summary, various data in Experimental Example 3 (including Experimental Examples 3.1 to 3.3) confirmed that the PEDF peptide proposed herein is effective for treating DHT-induced hair loss. Therefore, administration of the PEDF peptide in the form of topical topical administration can prevent or alleviate hair loss caused by male alopecia.

The present disclosure demonstrates for the first time that administration of a short PEDF synthetic peptide in the form of topical topical administration has a therapeutic effect on hair follicles. The topical surface application method proposed herein is not only safer but also relatively inexpensive compared to the traditional delivery of a full-length PEDF peptide by intravenous or intramuscular injection.

Although the embodiments of the present invention are disclosed in the above embodiments, the present invention is not intended to limit the invention, and the present invention may be practiced without departing from the spirit and scope of the invention. Various changes and modifications may be made thereto, and the scope of the invention is defined by the scope of the appended claims.

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood. The description of the drawing is as follows. The representative photograph of FIG. 1 shows the mice of each group in the experimental example 1 of the present invention in CYP. Appearance on days 0, 7, and 36 after injection, and high magnification micrographs corresponding to the box marks in the figure; representative immunostained photographs on Fig. 2 show apoptosis in TUNNEL positive in experimental example 1 of the present invention Hair follicle keratinocytes (green), nuclear staining by Hoechst 33258 (blue), original magnification: ×1000; representative immunostained photograph of Figure 3 shows hairy tyrosinase (green) in experimental example 1 of the present invention And the expression and distribution of c-kit (red), the cell nucleus was stained by Hoechst 33258 (blue), the original magnification: ×1000; the representative photograph of Fig. 4 shows the mice of each group in Experimental Example 2 of the present invention in H ₂ Appearance on days 0 and 22 after O ₂ injection; Figure 5 is a representative photograph of H&E-stained male mouse tentative hair follicle samples according to experimental example 3.1 of the present invention (ORS: outer root sheath; IRS: inner root sheath; HM: hair matrix; DP: dermal papilla), photo above Magnification: ×100, the original photograph original magnification: ×400; Figure 6 is a representative photograph of H&E staining of the back skin sample after 4 days of subcutaneous injection of DHT solution in male mice of Experimental Example 3.2 according to the present invention, original magnification Representative photographs of: ×100; and Fig. 7 show the appearance of the mice of each group in Example 3.3 of the present invention on days 0 and 18 after depilation and representative photographs of H&E-stained skin, original magnification: ×100.

<110> Taiwan's Presbyterian Church, Ma Rong Memorial Social Enterprise Foundation, Ma Rong Memorial Hospital

<120> Use of pigment epithelium-derived factor-derived multi-peptide to treat baldness and/or hair bleaching

<130> P2693-1-TW

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<170> PatentIn version 3.5

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Claims (10)

A use of a synthetic peptide for the preparation of a medicament for treating alopecia and/or hair discoloration in a body, the synthetic peptide being an amino group having a length of 20-44 amino acid residues An acid sequence consisting of the amino acid sequence having a nucleotide sequence similarity to SEQ ID NO: 10 of at least 80%, and the amino acid sequence comprising at least 20 contiguous amino acid residues, which are SEQ ID NO: The 16-35 amino acid residue of 10 has at least 90% amino acid sequence similarity. The use of claim 1, wherein the synthetic peptide has at least four consecutive amino acid residues identical to the 16-19 amino acid residues of SEQ ID NO: 10. The use according to claim 2, wherein the amino acid sequence of the synthetic peptide is SEQ ID NO: 10 (44-mer), SEQ ID NO: 1 (39-mer), SEQ ID NO: 2 (34-mer) , SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24-mer), SEQ ID NO: 6 (20-mer), SEQ ID NO: 8 (MO 29-mer) or SEQ ID NO: 9 (MO 20- The amino acid sequence shown by mer). The use according to claim 1, wherein the baldness is a baldness caused by alopecia or a baldness caused by male alopecia. The use of claim 1, wherein the hair bleaching refers to hair bleaching caused by chemotherapy, radiation therapy, stress, heredity or aging. A topical surface-administered pharmaceutical composition for treating baldness and/or hair discoloration in a body, comprising: an effective amount of the synthetic peptide according to any one of claims 1 to 3; and a pharmaceutically Acceptable excipients. The topical surface-administered pharmaceutical composition according to claim 6, wherein the topical surface-administered pharmaceutical composition is in the form of a solution, a spray, an aerosol, a foam, a cream, an emulsion, a cream, a gel or a dressing. The topical surface-administered pharmaceutical composition according to claim 7, wherein the synthetic peptide is contained in an amount of from 0.1 to 100 μM. The topical surface-administered pharmaceutical composition according to claim 6, wherein the baldness is a baldness caused by alopecia or alopecia caused by male alopecia. The topical surface-administered pharmaceutical composition according to claim 6, wherein the hair bleaching refers to hair bleaching caused by chemotherapy, radiation therapy, stress, heredity or aging.