JP6469767B2 - Use of PEDF-derived polypeptides for preventing and / or improving skin aging - Google Patents

Description

(Background of the Invention)
(1. Field of Invention)

The present disclosure relates to the prevention and / or improvement of skin aging. In particular, the disclosed invention
It relates to the use of PEDF-derived polypeptides for preventing and / or improving skin aging.

(2. Explanation of related technology)

Human skin ages with time like other living tissues. Skin aging leads to the formation of wrinkles and wrinkles, thinning of the skin, skin discoloration or hyperpigmentation, and loss of firmness and elasticity. Depending on the cause, skin aging can be divided into intrinsic aging and extrinsic aging. Endogenous aging, or real age aging, affects almost all internal organs and is primarily regulated by the genetic structure of the subject. Exogenous aging is the result of exposure to various environmental factors, primarily ultraviolet (UV) radiation, and is therefore often referred to as photoaging. In areas exposed to the sun, such as the face, neck, and back of the hand, the combination of endogenous aging and photoaging damage can produce more significant changes.

Collagen is a fibrous extracellular insoluble protein that constitutes the main component of connective tissue. Among the various types of collagen, type I and type III are mainly related. Type I collagen is the major structural protein in human skin, making up more than 90% of the dry weight of the skin, whereas type III collagen is also widely distributed throughout the body and is dominant in fetal tissue is there. As the skin ages, the total collagen content and the ratio of type I collagen to type III collagen gradually decreases and the extracellular matrix (ECM) begins to be disorganized. For example, both endogenous aging and ultraviolet B wave (UVB) exposure attenuate the regeneration of collagen fibers (especially type I collagen). UVB exposure also upregulates the production of several collagenolytic enzymes known as matrix metalloproteases (MMPs). MM in humans
P, specifically collagenase and gelatinase, are induced within hours of UVB exposure. All such events will likely result in a change in the extracellular matrix composition of the dermis.

There are many treatments available for aging skin. For example, the importance of collagen in the aging process has led to the development of many collagen-containing topical products. Other ingredients, such as retinoic acid, vitamin C and hyaluronic acid, are used in cosmetics by claiming that they can stimulate collagen synthesis. Some cosmetic techniques (eg, laser skin resurfacing) are also effective in reducing facial wrinkles and skin irregularities.

Despite the numerous solutions proposed to reduce signs of aging or stimulate skin regeneration, they all evoke an intrinsic repair mechanism of the skin (i.e., collagen synthesis) It is impossible to do. In view of the foregoing, there remains a need in the art for a means of effectively treating skin aging, particularly skin photoaging.

(Overview)
The following provides a brief overview of the present disclosure to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key / critical elements of the invention or delineate the scope of the invention. Its sole purpose is to provide certain concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

The present disclosure shows that synthetic peptides derived from pigment epithelium-derived factor (PEDF) stimulate collagen synthesis, promote dermal fibroblast proliferation, and down-regulate UVB-induced MMP-1 expression, resulting in these Is based at least on the discovery that it is effective in preventing and / or improving skin aging in a subject. Therefore, the PEDF-derived synthetic peptide of the present invention is useful as a drug or pharmaceutical for skin aging.

Accordingly, in one aspect, the present disclosure relates to a synthetic peptide for preventing and / or ameliorating skin aging in a subject.

According to an embodiment of the present disclosure, the synthetic peptide is 20-39 amino acid residues long,
And having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. The amino acid sequence also comprises at least 20 consecutive residues that are at least 90% identical to residues 11-30 of SEQ ID NO: 1, so that the synthetic peptide prevents skin aging in a subject And / or useful to improve.

According to any embodiment of the present disclosure, at least 4 consecutive residues of the synthetic peptide are identical to residues 11-14 of SEQ ID NO: 1. Non-limiting examples of such synthetic peptides include 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-me)
Those having the amino acid sequence of r). In some embodiments of the disclosure, the amino acid sequence of the synthetic peptide is SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24-mer), or SEQ ID NO: 6 (2
0-mer).

  According to various embodiments of the present disclosure, skin aging is caused by UV irradiation.

According to various embodiments of the present disclosure, the subject can be any animal classified as a mammal, including a human.

In another aspect, the present disclosure relates to a pharmaceutical composition for preventing and / or ameliorating skin aging in a subject. The subject can be any animal classified as a mammal, including a human.

According to one embodiment of the present disclosure, the pharmaceutical composition comprises a synthetic peptide according to any of the foregoing aspects / embodiments, and the synthetic peptide is effective enough to treat skin aging in a subject. Present in quantity. The pharmaceutical composition also includes a pharmaceutically acceptable excipient for the synthetic peptide.

In certain optional embodiments, the pharmaceutical composition further comprises at least one penetration enhancer that facilitates the flow of the synthetic peptide or pharmaceutical composition.

In various embodiments of the present disclosure, the pharmaceutical composition can be formulated into solutions, sprays, aerosols, foams, creams, lotions, ointments, gels, or patches.

In yet another aspect, the present invention relates to a method for preventing and / or ameliorating skin aging in a subject. The subject can be any animal classified as a mammal, including a human.

In one embodiment, the method comprises administering to the subject an effective amount of a synthetic peptide according to any of the foregoing aspects / embodiments for transdermal delivery of the synthetic peptide to the subject's dermis.

According to any embodiment, the synthetic peptide is formulated into a pharmaceutical composition according to the previous aspect / embodiment of the present disclosure. In one example, the pharmaceutical composition may be administered topically to the subject's skin. In any embodiment, the method comprises external stimulation (e.g., to the subject's skin) prior to, concurrently with, or after topical administration of the pharmaceutical composition to facilitate transdermal delivery of the synthetic peptide or pharmaceutical composition. Mechanical, electrical, thermal, ultrasonic, or high frequency stimulation). Further optionally, the pharmaceutical composition can further comprise at least one on-permeation enhancer to facilitate the flow of the synthetic peptide or pharmaceutical composition.

  According to certain embodiments of the present disclosure, skin aging is caused by UV irradiation.

Many of the attendant features and advantages of the present disclosure will become better understood with reference to the following detailed description considered in conjunction with the accompanying drawings.

(Brief description of the drawings)
The file of this patent and application contains at least one drawing made in color. Copies of this patent or patent application publication with color drawings will be accepted after request and payment of the necessary fee:
It will be provided by the secretariat.

The description of the present invention will be better understood from the following detailed description read in light of the accompanying drawings:

FIG. 1 provides images of H & E stained sections (left panel) and fluorescence images (middle and right panels) of skin specimens according to one test example of the present disclosure;

FIG. 2 provides a representative image of a skin tissue section stained with Masson Trichrome to highlight collagen fibers, according to another test example of the present disclosure;

FIG. 3 provides representative immunostained images of primary dermal fibroblasts (vimentin: green; and BrdU: red) and an image integrated with nuclear stained Hoechst 33258 (blue);

FIG. 4 provides representative photographs illustrating mouse wing formation in each experimental condition;

FIG. 5 provides a representative image of a skin tissue section stained with Masson Trichrome to highlight collagen fibers according to yet another test example of the present disclosure.

(Description)
The detailed description provided below in connection with the accompanying drawings is intended as a description of embodiments of the invention and is not intended to represent the only forms in which embodiments of the invention may be constructed or utilized. This description describes the function of the example and the sequence of steps for configuring and operating the example. But,
Different examples may implement the same or equivalent functions and sequences.

For convenience, certain terms used throughout the application (including the description, examples, and appended claims) are collected here. Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings that are commonly understood and used by those skilled in the art. Unless otherwise required by context, singular terms shall include the same plural form and plural terms shall include the singular form. Specifically, in the present specification and claims, the singular forms “a” and “an” include plural referents unless the context clearly indicates otherwise.

The numerical ranges and parameters describing the broad scope of the invention are approximate values,
The numerical values described in the examples are reported as accurately as possible. But any number
It inherently contains certain errors that necessarily arise from the standard deviation found in each test measurement. Also, as used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about” means within an acceptable standard error of the mean, as judged by those skilled in the art. Except for specific examples / test examples, or unless otherwise specified, such as those disclosed herein for material amounts, times, temperatures, operating conditions, proportions of amounts, and the like. , Numerical range, quantity,
All values and percentages are to be understood as being modified in all cases by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and appended claims are approximate values that may vary if desired. At a minimum, each numeric parameter should be interpreted at least by considering the number of significant digits reported and applying normal rounding techniques.

As used herein, the term “peptide” refers to a polymer of amino acid residues. The term “synthetic peptide” as used herein means a peptide that does not contain a naturally occurring full-length protein molecule. Peptides are “synthetic” in that they can be produced by human intervention using techniques such as chemical synthesis, recombinant gene technology, or full-length protein fragmentation. Throughout this disclosure, the position of any particular amino acid residue within a peptide is numbered starting from the N-terminus of the peptide.

As used herein, “proliferating” and “proliferation” refer to an increase in the number of cells in a population due to cell division.

式中、Xは、配列アラインメントプログラムBLASTのAとBのアラインメントにおいて、この
プログラムによって同一のマッチとしてスコア化されるアミノ酸残基の数であり、Yは、A
又はB内のいずれか短い方のアミノ酸残基の総数である。 “Percentage of amino acid sequence identity (%) with respect to the synthetic polypeptide sequences identified herein.
Is a specific alignment that does not consider any conservative substitutions as part of sequence identity after aligning the sequences and introducing gaps if necessary to obtain the greatest percentage of sequence identity. Defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a polypeptide sequence. Alignments for the purpose of determining percent sequence identity are available in various ways within the skill of the art and are publicly available, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software Can be implemented using computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, sequence comparison between two amino acid sequences was performed by the computer program Blastp (protein-protein BLAST) provided online by the National Center for Biotechnology Information (NCBI). The percentage of amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (or a given amino acid sequence in which the amino acid sequence identity to a given amino acid sequence B is some percent (Which can be expressed as A) is calculated by the following formula:

Where X is the number of amino acid residues scored as identical matches by this program in the alignment of A and B of the sequence alignment program BLAST, and Y is A
Or the total number of amino acid residues in B, whichever is shorter.

The term `` treating '' as used herein partially or completely alleviates, ameliorates, reduces, delays the onset of one or more signs or features associated with skin aging,
Synthetic peptides or pharmaceutical compositions of the present disclosure to subjects with signs of skin aging or predisposition to skin aging for the purpose of preventing progression, reducing severity and / or reducing incidence Application or administration. Treatment generally includes 1 as the term is defined herein.
“Effective” when the above symptoms or clinical markers are reduced.

The term “effective amount” as used herein refers to the amount of an ingredient sufficient to effect the desired reaction. The specific effective amount is the particular condition being treated, the physical condition of the patient (e.g., the patient's weight, age, or gender), the type of mammal or animal being treated, the duration of the treatment, if any It will vary depending on factors such as the characteristics of the combination therapy and the specific formulation used. An effective amount is also an amount by which the therapeutically beneficial effect of the compound or composition exceeds any toxic or deleterious effects.

The terms “application” or “administration” are used interchangeably herein and refer to a means of providing a subject with a synthetic peptide or pharmaceutical composition of the present invention to prevent and / or ameliorate skin aging. According to various embodiments of the present disclosure, transdermal delivery is a preferred delivery route. For example, the synthetic peptide or pharmaceutical composition of the present invention is topically applied to the subject's skin, so that the synthetic peptide or pharmaceutical composition can be used to prevent and / or ameliorate skin aging.
For example, the dermis is reached.

The term “excipient” as used herein means any inert material (such as a powder or liquid) that forms a vehicle / carrier for the synthetic PEDF peptides of the present disclosure. Excipients are generally safe, non-toxic and, in a broad sense, fillers, diluents, flocculants, binders, lubricants, glidants, stabilizers, colorants, wetting agents, disintegrants. Any known substance in the pharmaceutical industry useful for preparing pharmaceutical compositions can be included.

As used herein, a “pharmaceutically acceptable” ingredient is a human and / or without excessive adverse side effects (such as toxicity, irritation, and allergic reactions) that are balanced with a reasonable benefit / risk ratio. Or an ingredient suitable for use in animals. Each excipient must also be “acceptable” in the sense of being compatible with the other ingredients of the pharmaceutical formulation.
The carrier can be in the form of a solid, semi-solid, or liquid diluent, cream, or capsule.

The term “subject” refers to a mammal, including a human species, that is treatable with the synthetic peptides, compositions, and / or methods of the present invention. The term “subject” is intended to refer to both men and women unless one gender is specifically indicated.

As used herein, the term `` transdermal '' refers to an active substance (e.g.
This synthetic peptide or pharmaceutical composition).

Pigment epithelium-derived factor (PEDF) is a multifunctional secreted protein having an anti-angiogenic function, an anti-tumor forming function, and a neurotrophic function. Human PEDF protein (SEQ ID NO: 11) is a secreted protein having a size of approximately 50 kDa and a length of 418 amino acids. 34-mer fragment of PEDF (residues 44-7
The 7) and 44-mer fragments (residues 78-121: SEQ ID NO: 10) have been determined to have anti-angiogenic and neurotrophic properties, respectively.

The present disclosure is based at least on the discovery that synthetic peptides derived from 44-mer PEDF can protect the skin from damage of skin aging through various mechanisms. Examples of this disclosure demonstrate that synthetic peptides can increase collagen levels in the dermis, induce proliferation of dermal fibroblasts, and reduce the expression of MMP-1 (UVB-induced collagenase) I have to. Another inventive feature of the present invention is that this synthetic peptide is much shorter than full-length PEDF (up to 39 amino acid residues), which results in high manufacturing costs, low bioavailability, and poor pharmacokinetics. It is to overcome the limitations associated with the clinical use of conventional protein drugs including. Therefore,
This synthetic peptide is useful for preventing and / or improving skin aging.

Accordingly, in one aspect, the present disclosure relates to a synthetic peptide that prevents and / or ameliorates skin aging in a subject.

のアミノ酸配列とのアミノ酸配列同一性が、少なくとも80％である。例えば、合成ペプチ
ドは、配列番号:1とのアミノ酸配列同一性が、約80、81、82、83、84、85、86、87、88、
89、90、91、92、93、94、95、96、97、98、99、又は100％であり得る。また、合成ペプ
チドは、配列番号:1の残基11〜30と少なくとも90％同一である少なくとも20個の連続的な
残基を含む。具体的には、この20個の連続的なアミノ酸残基は、配列番号:1の残基11〜30
とのアミノ酸配列同一性が、約90、91、92、93、94、95、96、97、98、99、又は100％で
あり得る。 According to an embodiment of the present disclosure, the synthetic peptide is 20-39 amino acid residues in length,
and

Amino acid sequence identity with the amino acid sequence of at least 80%. For example, the synthetic peptide has an amino acid sequence identity with SEQ ID NO: 1 of about 80, 81, 82, 83, 84, 85, 86, 87, 88,
It can be 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%. The synthetic peptide also comprises at least 20 consecutive residues that are at least 90% identical to residues 11-30 of SEQ ID NO: 1. Specifically, the 20 consecutive amino acid residues are residues 11-30 of SEQ ID NO: 1.
The amino acid sequence identity to can be about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

In one embodiment, the synthetic peptide has the sequence of SEQ ID NO: 1 which is 39 amino acids in length. This synthetic peptide is referred to as 39-mer in the following description. This 39-mer peptide corresponds to residues 83-121 of human PEDF, and thus this is known to be the known PEDF 44-mer (residues 78-121 of PEDF).
Is a short variant derived from

Previous experiments performed by the inventors, such as those disclosed in co-pending application US 13 / 428,996 (incorporated herein by reference in its entirety) and the experiments provided below, Several short synthetic PEDF peptides derived from this 39-mer have been shown to be able to prevent and / or ameliorate skin aging in subjects.

の配列を有する34-mer合成ペプチドは、対象における皮膚老化を予防及び/又は改善する
のに有効である。この34-merペプチドは、ヒトPEDFの残基88〜121に相当する。上で提供
された2つの所与の配列間の配列同一性の割合を推定するためのプロセスによれば、この3
4-merは、39-merとのアミノ酸配列同一性が100％であり、34-merの第6〜第25アミノ酸残
基は、39-merのアミノ酸残基11〜30とのアミノ酸配列同一性が100％である。 For example, based on experiments disclosed in both the prior application and the present application,

A 34-mer synthetic peptide having the sequence of is effective in preventing and / or ameliorating skin aging in a subject. This 34-mer peptide corresponds to residues 88-121 of human PEDF. According to the process for estimating the percent sequence identity between the two given sequences provided above, this 3
4-mer has 100% amino acid sequence identity with 39-mer, and the 6-th to 25th amino acid residues of 34-mer have amino acid sequence identity with amino acid residues 11-30 of 39-mer Is 100%.

の配列を有する29-mer合成ペプチドは、対象における皮膚老化を予防及び/又は改善する
のに有効であることが確認されている。この29-merペプチドは、39-merに対する100％ア
ミノ酸配列同一性を有するヒトPEDFの残基93〜121に相当する。また、29-merの第1〜第20
アミノ酸残基は、39-merのアミノ酸残基11〜30とのアミノ酸配列同一性が100％である。 Furthermore, according to the various examples below,

A 29-mer synthetic peptide having the sequence of has been confirmed to be effective in preventing and / or ameliorating skin aging in a subject. This 29-mer peptide corresponds to residues 93 to 121 of human PEDF with 100% amino acid sequence identity to the 39-mer. Also, 29-mer 1st to 20th
The amino acid residues have 100% amino acid sequence identity with the 39-mer amino acid residues 11-30.

の配列を有する。この24-merペプチドは、39-merとのアミノ酸配列同一性が100％であり
、その最初の20個のアミノ酸残基は、39-merのアミノ酸残基11〜30とのアミノ酸配列同一
性が100％である。 In some examples, a 24-mer has been found to be effective in preventing and / or ameliorating skin aging in a subject. This 24-mer corresponds to residues 93 to 116 of human PEDF,

It has the arrangement | sequence. This 24-mer peptide has 100% amino acid sequence identity with 39-mer, and the first 20 amino acid residues have amino acid sequence identity with 39-mer amino acid residues 11-30. 100%.

の配列を有する。この20-merペプチドは、39-merのアミノ酸残基11〜30と完全に同一(100
％アミノ酸配列同一性)であり、且つ39-merとのアミノ酸配列同一性が100％である。 In other examples, it has been established that a 20-mer can prevent and / or ameliorate skin aging in a subject. This 20-mer corresponds to residues 93 to 112 of human PEDF,

It has the arrangement | sequence. This 20-mer peptide is completely identical to amino acid residues 11-30 of 39-mer (100
% Amino acid sequence identity) and 100% amino acid sequence identity with the 39-mer.

の配列を有し、これは、39-merとのアミノ酸配列同一性が83％であり、且つその最初の20
アミノ酸残基の、39-merの11〜30アミノ酸残基とのアミノ酸配列同一性が90％である。別
のマウス由来のペプチド、Mo 20-merは、

の配列を有する。Mo 20-merは、39-mer又は39-merの11〜30アミノ酸残基のいずれかとの
アミノ酸配列同一性が90％である。 Based on mouse PEDF, based on experiments disclosed in both prior application and this application
Two synthetic peptides can also prevent and / or ameliorate skin aging in a subject. The first mouse-derived peptide is referred to in this disclosure as “Mo 29-mer”. Mo 29-mer

Which has 83% amino acid sequence identity with the 39-mer and its first 20
Amino acid sequence identity with the 39-mer 11-30 amino acid residues is 90%. Another mouse-derived peptide, Mo 20-mer,

It has the arrangement | sequence. Mo 20-mer has 90% amino acid sequence identity with either 39-mer or 11-30 amino acid residues of 39-mer.

は、対象における皮膚老化に対するいかなる保護も引き起こすことができない。また、先
願と本出願との両方において開示されている実験に基づくと、SLGA残基を有しない25-mer
ペプチド

は、対象における皮膚老化を予防及び/又は改善しないであろうことが示唆される。 Optionally, the synthetic peptide comprises 4 consecutive residues that are identical to residues 11-14 of SEQ ID NO: 1. Residues 11-14 of SEQ ID NO: 1 (ie SLGA) are believed to play an important role in maintaining the biological function of the short PEDF peptide. For example, according to the various examples provided below, an 18-mer peptide without SLGA residues

Cannot cause any protection against skin aging in the subject. Also, based on experiments disclosed in both the prior application and the present application, a 25-mer having no SLGA residue
peptide

Suggests that it will not prevent and / or ameliorate skin aging in the subject.

The synthetic peptides of the present invention can be synthesized by commonly used methods such as t-BOC or FMOC protection of the α-amino group. Both methods involve a step-wise synthesis where a single amino acid is added at each step, starting from the C-terminus of the peptide. The peptide of the present invention can also be synthesized by a well-known solid phase peptide synthesis method.

Other synthetic peptides with conservative variations on the 39-mer are also contemplated. The term "conservative deformation"
As used herein indicates the replacement of an amino acid residue by another biologically similar residue. Examples of conservative variations include substitution from one hydrophobic residue to another hydrophobic residue, such as isoleucine, valine, leucine, or methionine, or another polar residue to one polar residue. Substituent substitution, for example, lysine to arginine, aspartic acid to glutamic acid, asparagine to glutamine, and the like. The term “conservative variation” also refers to a substituted amino acid in place of the parent amino acid that is not substituted, provided that the antibody produced against the substituted polypeptide also immunoreacts with the unsubstituted polypeptide. Including the use of

According to various embodiments of the present disclosure, skin aging can be caused by UV (particularly UVB) irradiation.

According to various embodiments of the present disclosure, the subject may be any animal classified as a mammal, including humans.

Synthetic peptides according to the foregoing embodiments can be formulated into a pharmaceutical composition for preventing and / or ameliorating skin aging in a subject, which is included in other aspects of the present disclosure.

According to one embodiment of the present disclosure, the pharmaceutical composition comprises a synthetic peptide according to any of the foregoing aspects / embodiments, and the synthetic peptide is used to prevent and / or improve skin aging in a subject. Present in a sufficiently effective amount. The pharmaceutical composition is also for synthetic peptides,
Also included are pharmaceutically acceptable excipients.

Pharmaceutical compositions are described in “Remington's Pharmaceutical Sciences”, 17th.
Edition, edited by Alfonoso R. Gennaro, Mack Publishing, Pennsylvania, Easton (1985)
Prepared according to acceptable pharmaceutical practice, as described in

The selection of a pharmaceutically acceptable excipient to be used with a synthetic peptide is basically determined by the manner in which the pharmaceutical composition will be administered. According to one optional embodiment of the present disclosure, the pharmaceutical composition or the synthetic peptide contained therein is delivered transdermally to the subject's dermis and consequently excipients suitable for the means are used. A wide variety of dermatological acceptable inert excipients well known in the art can be utilized. Typical inert excipients can be, for example, water, ethyl alcohol, polyvinyl pyrrolidone, propylene glycol, mineral oil, stearyl alcohol, and gel-generating materials.

In another approach, the synthetic peptide can be initially encapsulated by a vesicle-forming vehicle (such as a liposome, microsphere, or nanosome) and then formulated into the desired dosage form. These vesicle-forming vehicles can facilitate the transdermal delivery rate of the synthetic peptides. In addition, they can provide sustained release dosage forms to ensure a more prolonged therapeutic effect of the treatment.

Alternatively or in addition, the pharmaceutical composition can further contain any penetration enhancer that can enhance the transdermal delivery rate of the synthetic peptide or pharmaceutical composition. For example, penetration enhancers can disrupt the highly ordered structure of stratum corneum lipids, interact with intercellular proteins, or have drugs, co-enhancers or vehicles in the stratum corneum. Can act to improve distribution. Examples of penetration enhancers include sulfoxides (e.g. dimethyl sulfoxide (DMSO) and dimethylformamide (DMF)), azone (e.g. 1-dodecylazacycloheptan-2-one), pyrrolidone (e.g. N-methyl-2- Pyrrolidone), oils (e.g. terpenes and L-menthol), oxazolidinones (e.g. 4-decyloxazolidine-2-one),
Fatty acids (e.g. lauric acid, myristic acid and capric acid), glycols (e.g. diethylene glycol and tetraethylene glycol), surfactants (e.g. polyoxyethylene-2-oleyl ether and polyoxyethylene-2-stearyl) Ethers), pore-forming peptides (eg, magainin), and cell-permeable peptides (eg, transportan and penetratin), but are not limited to these.

Further optionally, the pharmaceutical composition of the present invention may also be prepared by various pharmaceutical-
It may also contain acceptable additives, especially for dermatology-). Examples of the additive include a drying agent, an anti-pruritic agent, an antifoaming agent, a buffering agent, a neutralizing agent, a pH adjusting agent, a coloring agent, a decoloring agent, a relaxation agent, an emulsifying agent, an emulsion stabilizer, a viscosity increasing agent, Examples include, but are not limited to, flavoring agents, preservatives, antioxidants, chemical stabilizers, thickeners, curing agents, or suspending agents.

In various embodiments of the present disclosure, the pharmaceutical composition comprises various agents suitable for transdermal delivery, such as solutions, sprays, aerosols, foams, creams, lotions, ointments, gels, or patches. It can be formulated into a form.

According to any embodiment of the present disclosure, the synthetic peptide is about 0.1-100 μm in a pharmaceutical composition.
M, and preferably present in an amount of about 1-25 μM. For example, the concentration of the synthetic peptide is 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, 2
It can be 5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μM. Specifically, the concentration used for mice (body weight about 20 g) in the following test examples is about 25 μM. One skilled in the art can calculate the human equivalent (HEQ) for the synthetic peptide or pharmaceutical composition based on the animal dosage provided herein. For example, the U.S. Food and Drug Administration (FDA) stated, `` Estimating the Maximum Safe Starting Dose in Initial Clinical Trial for the Treatment of Healthy Adult Volunteers.
"Clinical Trials for Therapeutics in Adult Healthy Volunteers").

In yet another aspect, the present invention relates to a method for preventing and / or ameliorating skin aging in a subject. The subject may be any animal classified as a mammal, including humans. In accordance with the principles and spirit of the present disclosure, skin aging is caused by UV irradiation.

In one embodiment, the method comprises administering to the subject an effective amount of the synthetic peptide according to any of the foregoing aspects / embodiments, such that the synthetic peptide is delivered transdermally to the dermis of the subject. Including.

According to any embodiment, the synthetic peptide is formulated into a pharmaceutical composition according to the foregoing aspect / embodiment of the present disclosure.

In one example, the pharmaceutical composition may be administered topically to the subject's skin, and the synthetic peptide or pharmaceutical composition transdermally into the subject's dermis with or without the aid of an external stimulus. Delivered.

In the event that no external stimulus is applied, the pharmaceutical composition is applied topically, smeared, massaged, sprayed or otherwise applied onto the subject's skin. Can. In this case, the pharmaceutical composition preferably contains at least one optional penetration enhancer as discussed above. Alternatively or in addition, the synthetic peptide may be encapsulated and formulated as taught above to improve the transdermal delivery rate of the synthetic peptide.

Many external stimuli have been found to increase skin permeability to compounds (eg, the present synthetic peptides). Such external stimuli include, but are not limited to, mechanical, electrical, thermal, ultrasonic, or high frequency stimuli. In practice, external stimuli may be applied to the skin before, simultaneously with, or after application of the pharmaceutical application. Similarly, pharmaceutical compositions or synthetic peptides delivered with the aid of such external stimuli can optionally contain the aforementioned penetration enhancers and / or vesicle-forming vehicles.

In one embodiment, a microstructured applicator (to provide mechanical stimulation to the skin)
A microneedle system or a microchannel system) is utilized. The microneedle system can comprise a plurality of solid or hollow microneedles that are approximately 100-1000 μM in length. For solid microneedle systems, the synthetic peptide or pharmaceutical composition is coated on the tip of each microneedle, and upon application, the microneedle penetrates the stratum corneum and remains in the skin, thereby causing the synthetic peptide Alternatively, the pharmaceutical composition is delivered to the dermis. For hollow microneedle systems, a pharmaceutical composition formulated in liquid form is loaded into the internal cavity of each microneedle, and upon application, the microneedle penetrates the skin and from this device to the dermis of the skin Allows liquid flow. Alternatively, the pharmaceutical composition is applied topically to the skin, after which a microchannel system having a large number of microneedles is pressed against the skin so that the microneedles penetrate less than 100 microns into the skin. Thus, a number of microchannels are formed on the skin that allow transdermal delivery of the pharmaceutical composition. Additionally or alternatively, the pharmaceutical composition is applied topically after the microchannel is formed.

In addition, a synthetic peptide or topical pharmaceutical composition according to any embodiment of the present disclosure can be administered under certain conditions (eg, certain humidity or temperature). For example, administration in a moist and warm environment can facilitate the rate of transdermal delivery of synthetic peptides.

In order to clarify certain aspects of the present invention and to assist one of ordinary skill in practicing the present invention, the following examples are provided. These examples should in no way be construed as limiting the scope of the invention in any way. Without further details, one skilled in the art will be able to make the most of the invention based on the description herein. All publications cited herein are hereby incorporated by reference in their entirety.

(Example)
(Materials and methods)

(material)

Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), 0.25% Trypsin, Invit
Purchased from rogen (Carlsbad, CA). Light mineral oil, glycerol, dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), 5-bromo-2'-deoxyuridine (BrdU
), Hoechst 33258 dye, Hoechst 33342 dye, anti-β-actin antibody and other chemicals were all obtained from Sigma-Aldrich (St. Louis, MO). Dispase II and type I collagenase were obtained from Roche (Indianapolis, IN). Anti-BrdU antibody (GTX42641)
Anti-MMP-1 antibody and anti-vimentin antibody (GTX100619) were purchased from GeneTex (Taipei, Taiwan). Anti-collagen 1A1 antibody is available from Santa Cruz Biotechnology (Sant, CA).
a Cruz). All fluorescent dye-conjugated secondary antibodies were purchased from BioLegend (San Diego, Calif.). Hematoxylin and eosin (H & E) dyes were purchased from Merck (Rayway, NJ, USA).

FITC-conjugated 29-mer, and 29-mer (SEQ ID NO: 3), 24-mer (SEQ ID NO: 5), 20-mer (SEQ ID NO: 6), and 18-mer (SEQ ID NO: 7). Other short synthetic PEDF peptides, including, were synthesized and modified by acetylation at the NH ₂ terminus and amidation at the COOH terminus. The modified peptide was subsequently characterized by mass spectrometry (> 95% purity) (GenScript, Piscataway, NJ). Each PEDF-derived short synthetic peptide (29-mer, 24-mer, or 20-mer; hereinafter PEDF peptide) is reconstituted in DMSO as a stock solution (5 mM),
And stored at -20 ° C until further use.

The skin ointment was prepared by mixing white petrolatum (10% w / w), glycerol (10% w / w), and light mineral oil (1% w / w) in distilled water. For the preparation of an ointment containing PEDF peptide, 5 μl of PEDF peptide stock solution was dissolved in 1 g of ointment. For an ointment for use as a vehicle control, 5 μl of DMSO was dissolved in 1 g of ointment.

(animal)

All animals used in the embodiments of the present disclosure are temperature controlled (24-25 ° C.) and
12:12 Breeding in an animal room under a light-dark cycle. Standard laboratory feed and tap water were freely available. The experimental procedure is Mackay Memorial Hospital Review Board (Taiwan (ROC),
Approved by New Taipei City) and implemented according to domestic animal welfare regulations.

(Isolation and culture of mouse dermis-derived dermal fibroblasts)

Full thickness skin tissue was obtained from the back of C57BL / 6 mice. The subcutaneous tissue was carefully excised from the skin and washed three times in sterile phosphate-buffered saline (PBS). Next, the tissue
Cut to 1-2 mm ³ ). After digestion with 0.1% dispase overnight at 4 ° C., the epidermal layer was removed, and the remaining dermis was further digested with 0.1% type I collagenase at 37 ° C. for an additional 4 hours. The digested cells are then collected by centrifugation (400 g for 5 minutes), 10% FBS, 2 mM L-glutamine,
And resuspended in high-glucose DMEM supplemented with 1% penicillin-streptomycin antibiotic mixture. Cells were seeded at 1 × 10 ³ cells / cm ² on tissue culture plates (Falcon Labware, NJ, USA) and maintained at 37 ° C. with 5% CO ₂ . After 24 hours, the medium was discarded, the remaining non-adherent cells were removed, and the cell culture was filled with fresh medium.
For passage, nearly confluent cells were collected using 0.25% trypsin / EDTA.

(WS-1 cell culture)

Human skin fibroblast WS-1 cell line was purchased from Food Industry Research and Development Organization
Development Institute) (Hsin-Chu, Taiwan). WS-1 cells were maintained as a monolayer in culture flasks at 37 ° C. in a humidified incubator with 5% CO ₂ . These are 2mM
10 supplemented with L-glutamine, 0.1 mM non-essential amino acid, 1% penicillin / streptomycin
Grown in% FBS-DMEM. For passage, nearly confluent cells were subcultured with 0.25% trypsin / EDTA.

(Tissue diagnosis)

Skin samples were fixed with 4% paraformaldehyde, dehydrated with a graded series of ethanol and paraffinized. The tissue was sliced into 5 μm sections. Prior to use, the fixed samples were deparaffinized in xylene and rehydrated in a graded series of ethanol.

General histology was performed using hematoxylin and eosin (H & E) dye. Deparaffinized skin tissue sections were stained using Masson Trichrome (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions; the sections were examined and Nikon Ec
Photographed with a lipse 80i optical microscope (original magnification, x400). For semi-quantitative analysis of the collagen area, 10 fields from each slide were randomly selected under a light microscope, and the blue-stained area (mm ² / mm ² ) per total area of the cross section was image- Pro Plus 4.5.1 system (Media Cybernetic
s).

(Immunofluorescence measurement and BrdU staining)

After fixation with 4% paraformaldehyde, the cells were exposed to cold methanol for 2 minutes and then 1N
Treatment with HCl for 1 hour at room temperature followed by immunofluorescence staining.

For animal experiments, BrdU was reconstituted as a stock solution (80 mM) in DMSO. PBS 90μl
Mice were injected with 10 μl of BrdU stock solution intraperitoneally and euthanized 16 hours later. Formalin-fixed, paraffin-embedded skin specimens were deparaffinized in xylene and rehydrated in a graded series of ethanol. Thereafter, the specimens were exposed to 1N HCl for 1 hour at room temperature, and subsequently subjected to an immunofluorescence measurement test.

BrdU-labeled DNA was detected with polyclonal anti-BrdU antibody and rhodamine-conjugated donkey anti-rabbit IgG. Nuclei were counterstained with Hoechst 33258 (blue).

(Immunoblot analysis)

Skin tissue was treated with 20 mM HEPES (pH 7.4), 150 mM NaCl, 1% SDS, 1 mM ethylenediaminetetraacetic acid (E
DTA), 5 mM phenylmethanesulfonyl fluoride (PMSF), and 1 mM dithiothreitol (DTT)
), Freshly added protease inhibitor cocktail (Roche, Indianapolis, Ind.) In ice-cold lysis buffer containing 1% Triton X-100 and homogenized. The homogenate was then centrifuged at 12,000g for 30 minutes at 4 ° C, after which the supernatant was collected and stored at -70 ° C. The protein content in the lysate was determined using the Bradford assay. A whole cell lysate was prepared using the lysis buffer described above. An aliquot of the lysate was then resolved on SDS-PAGE, electrotransferred onto a poly (vinylidene difluoride) (PVDF) membrane (Millipore, Bedford, Mass., USA) and proceeded to immunoblot analysis.

The antibodies used in the immunoblot test were anti-type I collagen 1A1 antibody (1: 1000 dilution), anti-MMP-
1 antibody (1: 500 dilution) and anti-β-actin antibody (1: 10000 dilution) were included. Proteins of interest include suitable IgG-HRP secondary antibodies and ECL reagents (Amersham, Illinois, USA, Arlington H
eights). X-ray film is modeled on model GS-700 Image Densitometer (Bio-Ra
d Laboratories, Inc., Hercules, Calif.) and analyzed using Labworks 4.0 software. For quantification, blots from at least 3 independent experiments were used.

(RNA extraction and reverse transcription-polymerase chain reaction)

；アンチセンス、

；PCR産物：201bp；及び、マウスグリセルアルデヒド-3-リン酸デヒドロゲナーゼ(GAPDH
；寄託番号；M32599)のセンス、

；及び、アンチセンス、

；PCR産物：223bpであった。これらのPCR産物を、臭化エチジウムを含有する2％アガロー
スゲル中で電気泳動させ、UV照明によって視覚化した。FUJI LAS-3000システム及びMulti
Gauge Ver. 1.01ソフトウェア(富士フイルム社、日本、東京)を使用して、PCR産物の強
度を濃度測定的に定量化した。 Total RNA was extracted from cells using TRIzol (Invitrogen) and RNase-free DNase I (Qia
Gen, Santa Clarita, CA) to remove genomic DNA and then RNA
Purification was performed using a purification kit (Dynabeads; Invitrogen). 1μ recovered from primary dermal fibroblasts
g total RNA was added at 42 ° C. with 200 units of expanded reverse transcriptase (Roche, Mannheim, Germany) in 20 μl reaction buffer containing 0.25 μg random primer and 0.8 mM dNTP.
Time was reverse transcribed into cDNA. 2 μl of cDNA was used as a template in the PCR reaction. PCR was performed using 15 μl EconoTaq® PLUS GREEN 2 × Master Mix (Lucigen®),
Performed in a 30 μl reaction volume containing 1 μM of each primer and 2 μl of template DNA. 18 ~
22 cycles of amplification reaction (denaturation, 20 seconds, 94 ° C; annealing, 30 seconds, 57 ° C; and polymerization, 40 seconds, 7 ° C
CDNA was synthesized at 2 ° C. The number of cycles for the primer set was chosen to be within the linear range of amplification. The specific PCR primer sequence is mouse MMP-13 (deposit number: NM_00860).
7) sense,

Antisense,

PCR product: 201 bp; and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH
Sense of deposit number; M32599)

And antisense,

PCR product: 223 bp. These PCR products were electrophoresed in a 2% agarose gel containing ethidium bromide and visualized by UV illumination. FUJI LAS-3000 system and Multi
The intensity of PCR products was quantified densitometrically using Gauge Ver. 1.01 software (Fujifilm, Tokyo, Japan).

(statistics)

The results were expressed as mean ± standard error of the mean (SEM). One-way ANO for statistical comparison
VA was used. P <0.05 was considered significant unless specified otherwise.

(Example 1)

(Transdermal delivery of PEDF peptide)

Fluorescein (FITC) -conjugated 29-mer peptide was used to study transdermal delivery of the PEDF peptide in vivo.

Initially, dorsal hairs of 8 week old male C57BL / 6 mice were removed using a melted wax / rosin mixture (1: 1) under general anesthesia. The next day, an ointment containing FITC-conjugated 29-mer peptide (25 μM) was applied to the dorsal skin of the mice, followed by a microneedle patch (3M ™ microchannel skin system; provided by 3M Company (Singapore)) Was gently pressed against the dorsal skin, and a microneedle was allowed to penetrate the skin less than 100 microns (FITC-29mer / MN group). Control group (FITC-29mer / CT
), The mice received ointment and did not receive subsequent microneedle treatment. After 24 hours, skin was collected, fixed, frozen, and examined with a fluorescence microscope. A representative image of three independent experiments (original magnification, x400) is provided in FIG.

The H & E stained section in the left panel of FIG. 1 highlights the epidermis and dermis of the specimen. The fluorescence images in the center panel and right panel of FIG. 1 illustrate the distribution of FITC-conjugated 29-mer across the skin. In particular, the highly recognizable green signal from fluorescein beyond the epidermis and dermis in the FITC-29mer / MN group indicates that the FITC-conjugated 29-mer peptide was successfully delivered to the dermis. In contrast, in the FITC-29mer / CT group, a green signal was barely detectable in the dermis, but most of the fluorescein signal remained in the epidermis.

(Example 2)

(PEDF peptide increases the collagen content of the dermis)

* ビヒクル/MN群に対し、P＜0.05。** ビヒクル/CT群に対し、P＜0.002。 Collagen is a major matrix component of the dermis and forms a tough fibrous structure that provides the elastic properties of the skin. This test example examined whether the PEDF peptide increased the collagen content of the dermis. Mice that were depilated as described in Example 1 were randomly assigned to several experimental groups and treated as described below. For each mouse, one side of the dorsal midline (approximately 2 cm ² ) is coated with 50 μl ointment (29-mer, 20-mer, or DMSO-containing ointment), followed by microneedle treatment; On the other side of the line (about 2 cm ² ), 50 μl of the same ointment was applied and no microneedle treatment was performed. This treatment was given twice a week for 2 weeks and the skin was left uncovered and collected 14 days after the first treatment. Skin specimens were stained for dermal collagen with Masson Trichrome staining (blue); representative images are provided in FIG. The stained collagen area of the skin is quantified as described above in the section “Materials and Methods” and the results are
The vehicle / MN area was normalized. The level of type I collagen (COL1A1) was examined by immunoblot analysis as described above, and the results were normalized to the level of the vehicle / CT group.
Quantitative results are summarized in Table 1.

* P <0.05 vs vehicle / MN group. ** P <0.002 for vehicle / CT group.

The image in FIG. 2 shows the 29-mer combined with the microneedle treatment as evidenced by the more prominent blue staining in the 29-mer / MN and 20-mer / MN sections when compared to the vehicle / MN sections. Alternatively, 20-mer treatments indicate that collagen levels in the dermis are increased. In addition, the vehicle
By comparing the / CT section and the vehicle / MN section, we can
It has been found that it has no effect on collagen levels. Vehicle / CT, 20-mer / CT
, And there is no significant difference in collagen levels between sections of 29-mer / CT, indicating that collagen levels do not vary significantly between subjects.

Type I collagen (COL1A1) is a major component of dermal collagen, and its immunoblot analysis shows that 29-mer / MN and 20-mer / MN treatments have COL1A1 levels compared to that of control treatments (vehicle / CT). It points out that it produces a significant increase.

(Example 3)

(PEDF peptide promotes dermal fibroblast proliferation)

Fibroblasts are the primary collagen-producing cells in human skin, and thus in vitro and in vivo analyzes were performed to evaluate the effect of the PEDF peptide on dermal fibroblast proliferation.

(Example 3.1)

(PEDF peptide promotes dermal fibroblast proliferation in vitro)

* 未処置細胞に対し、P＜0.002。 Primary mouse dermal fibroblasts were isolated and cultured as described in the “Materials and Methods” section. Dermal fibroblasts passaged twice are seeded onto gelatin-coated slides in 6-well plates at a density of 2 × 10 ⁵ cells per well and grown in growth medium (DMEM + 10% FBS) for 24 hours. Cultured, then 1% FBS alone (control group) or 1% FBS plus an additional 50 nM PEDF-derived peptide (ie 29-mer, 24-mer, 20-mer, 18-mer, Mo 29-mer, Alternatively, the basal growth medium containing Mo 20-mer) was replaced and cultured for 24 hours. For the BrdU labeling assay, BrdU (final concentration 10 μM) was added to the culture for 2 hours. Fibroblasts were also stained with the fibroblast marker vimentin; a representative image was provided in FIG. The levels of BrdU- and vimentin-double positive cells were expressed as the ratio of BrdU- and vimentin-double positive cells per total vimentin-positive cells (BrdU / vimentin labeling index) and the results are summarized in Table 2.

* P <0.002 for untreated cells.

In summary, PEDF-derived peptides according to embodiments of the present disclosure (e.g., 29-mer, 24-mer,
Dermal fibroblasts cultured in medium containing 20-mer and two mouse homolog peptides) proliferated more (about 5 times) than those cultured in control medium (Table 2). In contrast, treatment of 18-mer without the “SLGA” residue failed to induce such effects.

(Example 3.2)

(PEDF peptide promotes dermal fibroblast proliferation in vivo)

* ビヒクル/MN群に対し、P＜0.002。 The specimen from the previous Example 2 is also stained for the fibroblast marker vimentin,
Vimentin-positive cells were counted under a microscope.

* P <0.002 for vehicle / MN group.

The data in Table 3 shows that samples treated with 29-mer / MN and 20-mer / MN are significantly more in the dermis region than those treated with vehicle / MN and vehicle alone (ie, vehicle / CT). It is pointed out that it has a number of fibroblasts. This observation suggests that transdermal delivery of the PEDF peptide is positively related to fibroblast density in the dermis.

* ビヒクル/MN群に対し、P＜0.05。 To investigate whether transdermal delivery of this PEDF peptide promotes dermal fibroblast proliferation in vivo, mice were injected intraperitoneally with BrdU and treated with PEDF peptide / MN-treated or vehicle / MN-treated 16 Euthanized after hours. Skin sections were treated with vimentin (green) and BrdU (red
). The level of BrdU- and vimentin-double positive cells was expressed as the ratio of BrdU- and vimentin-double positive cells per total vimentin-positive cells (BrdU / vimentin labeling index) and the results are summarized in Table 4.

* P <0.05 vs vehicle / MN group.

As can be seen in Table 4, the fibroblasts in the skin sections from the 29-mer / MN group and the 20-mer / MN group had more fibroblasts than the sections treated with DMSO-containing ointment (vehicle / MN). Proliferating. This observation points out that transdermal delivery of the synthetic peptide is effective in promoting the proliferation activity of dermal fibroblasts. This increased proliferation of dermal fibroblasts is consistent with the finding that collagen content is improved by transdermal delivery of the PEDF peptide (Table 1, above).

(Example 3.3)

(PEDF peptide promotes dermal fibroblast proliferation in photodamaged skin in vivo)

UVB irradiation causes fragmentation of the dermal collagen matrix, which is known to induce cell cycle arrest of fibroblasts. In this test example, we examined whether transdermal delivery of the PEDF peptide was effective in restoring the proliferative activity of fibroblasts damaged by UVB irradiation.

* 正常群に対し、P＜0.01；** UVB/ビヒクル群に対し、P＜0.05。 Ultraviolet B wave (UVB) on the back of a 6 week old female nude mouse (BALB / cAnN.Cg-Foxn1nu / CrlNarl)
A bank of 5 UVB lamps (model XL-1000B, Spectrolinker ™ instrument; emission peak: 312 nm) placed approximately 20 cm above the animal's back 5 times a week for 8 weeks. Used and irradiated. During UVB exposure, the mice moved freely within the cage. Minimum erythema formation dose (MED)
The irradiation intensity expressed as is set to 1 MED for the first two weeks (60 mJ / cm ² ), and from the third week to the fourth of the experiment
Subjected week until 2MED (120mJ / cm ^2), from week 5 until 3MED (180mJ / cm ²⁾ over the first 6 weeks, from week 7 toward Week 8 to 4MED (240mJ / cm ^2), was gradually increased. After 8 weeks of UVB exposure, the animals exhibited noticeable wrinkles and were subsequently randomly divided into the following 4 groups with 3 mice in each group. In the UVB / vehicle, UVB / 29-mer, UVB / 20-mer, and UVB / 18-mer groups, ointment (DMSO, 29-
350 μl) (containing mer, 20-mer or 18-mer PEDF) was applied to the dorsal skin, after which transdermal delivery was achieved by pressing the ointment-treated skin with a microneedle patch. Similarly, mice that were not subjected to UVB irradiation were used as controls (normal). Ointment / microneedle treatment was given twice a week for 2 weeks. Mice received BrdU intraperitoneally and were euthanized 16 hours after the last treatment. Skin sections were stained for vimentin and BrdU. The level of BrdU- and vimentin-double positive cells was expressed as the ratio of BrdU- and vimentin-double positive cells per total vimentin-positive cells (BrdU / vimentin labeling index). The quantitative results are summarized in Table 5.

* P <0.01 for normal group; ** P <0.05 for UVB / vehicle group.

As can be seen in Table 5, UVB exposure reduced dermal fibroblast proliferative activity by approximately 50% compared to skin not exposed to UVB irradiation (UVB / vehicle: 2.1 ± 1.1% vs. Normal: 4.1 ± 0.8%). On the other hand, transdermal delivery of 29-mer and 20-mer after UVB exposure enhanced the proliferation activity of dermal fibroblasts by about 2.5 times compared to that of UVB / vehicle group. It is noted that the proliferative activity in 29-mer or 20-mer treated animals is even higher than that of animals not irradiated with UVB, but the difference is less significant. In contrast, an 18-mer lacking an important “SLGA residue” failed to promote dermal fibroblast proliferation.

In conclusion, the data presented in Example 3 (including Examples 3.1 to 3.3) show that the PEDF peptide is not only damaged by normal skin tissue (i.e., tissue that has not been treated with UVB irradiation), but also photodamaged. Have been shown to be effective in promoting the proliferation of dermal fibroblasts in fresh skin tissue (ie, tissues with wrinkles that have been treated with UVB irradiation and noted as a result of exposure). Since fibroblasts are involved in collagen synthesis, improved fibroblast proliferation activity and the resulting higher fibroblast density can then promote collagen synthesis. Thus, the present PEDF peptide is capable of inducing an endogenous repair mechanism of the skin for collagen synthesis.

(Example 4)

(PEDF peptide reduces UVB-induced MMP-1 expression)

MMP-1 (interstitial collagenase) is the only enzyme capable of catalyzing the cleavage of collagen triple helices of type I and type III collagen. Furthermore, MMP-1 is a major collagenolytic enzyme present in human skin and is upregulated in response to UV irradiation.
Rodents lack the MMP-1 gene, and MMP-1 is functionally replaced by MMP-13 in these animals. Therefore, this test example aims to elucidate whether this synthetic peptide reduces the expression of MMP-1 and MMP-13 induced by UVB exposure.

According to our previous studies, cell viability was greater than 95% relative to control viability after exposure to 15 mJ / cm ^{2 of} UVB irradiation (data not shown). Human dermal fibroblasts (WS-1 cells) and primary mouse dermal fibroblasts pretreated with 100 nM 29-mer for 24 hours did not cause significant changes in cell viability (data not shown) and trans It did not cause a significant change in cell migration assayed by the well assay (data not shown). The present inventors have found that UV irradiation at this dose leads to an increase in MMP expression level. Therefore,
The amount of exposure was set to 15 mJ / cm ² in the following experiment.

* ビヒクル/UVB群に対し、P＜0.02。 WS-1 was seeded in 6-well plates at a density of 1 × 10 ⁵ cells per well and 10% FBS-D
Growth was performed in MEM growth medium for 24 hours, followed by an additional 24 hours incubation with 1% FBS-DMEM supplemented or not supplemented with 100 nM PEDF peptide. Cells were washed twice with phosphate buffered saline (PBS), coated with PBS and exposed to UVB irradiation. UVB treatment is performed in a cell culture hood using a UVB lamp (Model XL-1000B, Spectrolinker (trademark)), with a final irradiation dose of 15 mJ
Performed at / cm ² for about 100 seconds. Cell viability was assayed using the trypan blue exclusion assay (> 95%). After UVB irradiation, PBS was replaced with 1% FBS-DMEM with or without PEDF peptide and incubated for a further 48 hours, then protein extracts were assayed by Western blot for expression of MMP-1 protein. Cells that were not subjected to UVB irradiation were used as controls (normal). The expression level of MMP-1 is the expression level in the vehicle / UVB group (set to 100%)
Table 6 summarizes the quantitative results.

* P <0.02 for vehicle / UVB group.

By comparing the vehicle / UVB group with the normal group, we noted that UVB irradiation resulted in a significant increase in MMP-1 protein expression (100% vs. about 5 in the normal group). %). However, preincubation of fibroblasts with 29-mer, 24-mer or 20-mer significantly reduced MMP-1 protein levels to about half of the vehicle / UVB group.

* ビヒクル/UVB群に対し、P＜0.0001。 Primary mouse dermal fibroblasts passaged twice in monolayer cultures are incubated for 24 hours with 1% FBS-DMEM supplemented or not supplemented with 100 nM PEDF peptide, followed by UVB irradiation as described above. Provided. After UVB irradiation, PBS was replaced with 1% FBS-DMEM with or without PEDF peptide and incubated for an additional 24 hours, after which total cellular RNA was extracted for subsequent RT-PCR analysis. The expression level of MMP-13 mRNA was compared with the expression level in the vehicle / UVB group (100
Table 7 summarizes the quantitative results.

* P <0.0001 for vehicle / UVB group.

Similar to previous findings in Table 6, MMP-13 expression levels were also up-regulated by UVB exposure (vehicle / UVB group vs. normal group). Such up-regulation is also
In cells pretreated with PEDF peptide, it was substantially reduced (29-mer / UVB, 24-
mer / UVB, and 20-mer / UVB vs. vehicle / UVB). In contrast, 18-m without the “SLGA residue”
er was unable to reduce UVB-induced upregulation.

These results suggest that the PEDF peptide can protect human dermal fibroblasts against UVB-induced MMP-1 induction. The destruction of type I collagen by MMP-1
It is an important contributor to photoaging. Therefore, the present PEDF peptide can be used as a photoaging inhibitor.

(Example 5)

(PEDF peptide reduces UV-induced wrinkles)

The formation of wrinkles is closely related to repeated exposure to UVB radiation. Specifically, UVB irradiation induces collagen destruction and inhibits procollagen biosynthesis in the dermis; this results in collagen loss and results in wrinkle formation. In this test example, we examined the anti-wrinkle effect of the PEDF peptide in nude (ie hairless) mice.皺
Induced by 8 weeks of UVB irradiation following the procedure shown in Example 3.3 above. Mouse appearance, UVB
Before irradiation (normal), after UVB irradiation (UVB / UT), and after treatment with each ointment for 2 weeks (UVB / vehicle, UVB
/ 29-mer, UVB / 20-mer and UVB / 18-mer); representative photographs are provided in FIG.

As illustrated in FIG. 4, mice exhibited a noticeable amount of deep and long wrinkles after UVB irradiation.
(Normal group, vs. UVB / UT group). Mice treated with vehicle- or 18-mer-containing ointments also have deep and long wrinkles, indicating that 18-mer cannot reduce wrinkles formed as a result of UVB irradiation It suggests. In contrast, the PEDF peptides (e.g. 29-mer and 20-me
Mice treated with r) have fewer wrinkle lines and are thinner than those observed in the UVB / UT, UVB / vehicle, and UVB / 18-mer groups.

* 未処置の正常群に対し、P＜0.0001。
** UVB/ビヒクル群に対し、P＜0.001。 Skin specimens were subjected to Masson trichrome staining to approximate their collagen content and a representative photograph was provided in FIG. 5 (original magnification, × 200). The level of type I collagen (COL1A1)
Examined by immunoblot analysis, the results were normalized to the level of the untreated normal group (set to 100%). The quantitative results are summarized in Table 8.

* P <0.0001 vs untreated normal group.
** P <0.001 for UVB / vehicle group.

With reference to both FIG. 5 and Table 8, it can be seen that repeated UVB irradiation (UVB / UT) significantly reduced collagen signal and COL1A1 levels in the dermis as compared to that of normal mice in the untreated normal group. In contrast, transdermal delivery of the PEDF peptide (e.g. UVB / 29-mer group and UVB / 20-mer
Group) significantly enhanced both collagen and COL1AL levels in the dermis compared to that of the UVB / vehicle group. These findings indicate that transdermal delivery of the PEDF peptide can induce collagen synthesis in UVB-damaged dermis, which is justified by reduced wing formation in mice treated with the PEDF peptide. Suggest that it can be.

The present disclosure first reveals that transdermal delivery of short synthetic PEDF peptides has a protective effect against skin aging. Compared to intravenous or intramuscular delivery of vectors expressing full-length PEDF peptides, transdermal delivery of short synthetic PEDF peptides is a safe and inexpensive strategy.

It will be appreciated that the above description of the embodiments is given for illustrative purposes only and that various modifications can be made by those skilled in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. While various embodiments of the invention have been described above with some specificity or with respect to one or more individual embodiments, those skilled in the art will depart from the spirit and scope of the invention. Rather, many modifications may be made to the disclosed embodiments.
The present application provides an invention having the following configuration.
(Configuration 1)
Amino acid sequence that is 20-39 amino acid residues in length and has at least 80% amino acid sequence identity with SEQ ID NO: 1, and amino acid sequence identity with residues 11-30 of SEQ ID NO: 1 Use of a synthetic peptide for preventing and / or ameliorating skin aging in a subject consisting of said amino acid sequence comprising at least 20 consecutive residues, wherein is at least 90%.
(Configuration 2)
The use according to configuration 1, wherein at least 4 consecutive residues of the synthetic peptide are identical to residues 11-14 of SEQ ID NO: 1.
(Configuration 3)
The use according to configuration 1, wherein the skin aging is photoaging.
(Configuration 4)
The use according to configuration 1, wherein the subject is a human.
(Configuration 5)
An effective amount of the synthetic peptide of composition 1; and
Pharmaceutically acceptable excipient
A pharmaceutical composition for preventing and / or improving skin aging in a subject.
(Configuration 6)
The pharmaceutical composition according to configuration 5, wherein at least 4 consecutive residues of the synthetic peptide are the same as residues 11 to 14 of SEQ ID NO: 1.
(Configuration 7)
6. The pharmaceutical composition according to constitution 5, wherein the pharmaceutical composition is in the form of a liquid, a spray, an aerosol, a foam, a cream, a lotion, an ointment, a gel, or a patch.
(Configuration 8)
6. The pharmaceutical composition according to constitution 5, further comprising a penetration enhancer.
(Configuration 9)
6. The pharmaceutical composition according to constitution 5, wherein the skin aging is photoaging.
(Configuration 10)
6. The pharmaceutical composition according to constitution 5, wherein the subject is a human.
(Configuration 11)
A method for preventing and / or improving skin aging in a subject comprising:
The method comprising administering to the subject an effective amount of the synthetic peptide by transdermal delivery of the synthetic peptide of configuration 1 to the dermis of the subject.
(Configuration 12)
12. The method of configuration 11, wherein at least 4 consecutive residues of the synthetic peptide are identical to residues 11-14 of SEQ ID NO: 1.
(Configuration 13)
12. The method of configuration 11, wherein the synthetic peptide is formulated into a pharmaceutical composition comprising the synthetic peptide and a pharmaceutically acceptable excipient.
(Configuration 14)
14. The method according to configuration 13, wherein the pharmaceutical composition is in the form of a liquid, a spray, an aerosol, a foam, a cream, a lotion, an ointment, a gel, or a patch.
(Configuration 15)
14. The method of configuration 13, wherein the pharmaceutical composition is administered topically to the subject's skin.
(Configuration 16)
16. The method of configuration 15, further comprising applying an external stimulus to the skin of the subject prior to, simultaneously with, or after topical administration of the pharmaceutical composition, thereby facilitating transdermal delivery of the synthetic peptide. .
(Configuration 17)
The method of configuration 16, wherein the external stimulus is a mechanical, electrical, thermal, ultrasonic, or high frequency stimulus.
(Configuration 18)
The method of configuration 16, wherein the pharmaceutical composition further comprises a penetration enhancer.
(Configuration 19)
12. The method according to configuration 11, wherein the skin aging is photoaging.
(Configuration 20)
12. The method of configuration 11, wherein the subject is a human.

Claims (9)

A pharmaceutical composition for preventing and / or improving skin aging in a subject comprising a synthetic peptide consisting of an amino acid sequence having a length of 20 to 29 amino acid residues,
The pharmaceutical composition, wherein the amino acid sequence comprises at least an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9.   The pharmaceutical composition according to claim 1, wherein the amino acid sequence of the synthetic peptide comprises SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6. The skin aging is a photoaging claim 1 or a pharmaceutical composition according to any one claim of 2. The pharmaceutical composition according to any one of claims 1 to 3 , wherein the subject is a human. The pharmaceutical composition according to any one of claims 1 to 4 , comprising an effective amount of the synthetic peptide and a pharmaceutically acceptable excipient. The pharmaceutical composition according to any one of claims 1 to 5 , which is in the form of a liquid, a spray, an aerosol, a foam, a cream, a lotion, an ointment, a gel, or a patch. The pharmaceutical composition according to any one of claims 1 to 6 , further comprising a penetration enhancer. By transdermal delivery the synthetic peptide effective amount into the dermis of the subject, an effective amount of the synthetic peptide is configured to administering to said subject, in any one of claims 1-7 Pharmaceutical composition. 9. A pharmaceutical composition according to claim 8 , configured to be administered topically to the skin of the subject.

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