KR102423955B1 - A Peptide having impotence treatment activity and the Composition Comprising the Same - Google Patents

Abstract

The present invention relates to a composition for preventing and treating erectile dysfunction, and more particularly, to a composition for preventing and treating erectile dysfunction, including a peptide derived from telomerase, effective for treating and preventing erectile dysfunction symptoms. The peptide according to the present invention exhibits an effect of treating erectile dysfunction symptoms by inhibiting the activity of the PDE-5 enzyme, thereby providing a method for preventing and treating erectile dysfunction.

Description

A peptide having impotence treatment activity and a composition comprising the same {A Peptide having impotence treatment activity and the Composition Comprising the Same}

The present invention relates to a peptide having an effect of preventing and treating erectile dysfunction and a composition comprising the same, and more particularly, to a peptide derived from telomerase and inhibiting the activity of the PDE-5 enzyme, which is an enzyme related to erectile dysfunction. It relates to a composition for preventing and treating erectile dysfunction effective for treating impotence.

Erectile Dysfunction (Erectile Dysfunction) is a condition in which the erection required for a satisfactory sexual life is not sufficient or obtained but not maintained. If these symptoms persist for more than 3 months, it is diagnosed as erectile dysfunction.

Erectile dysfunction is largely divided into psychogenic (mental fault) and temperamental (temperamental fault) according to the cause. Temperament is also divided into neurological abnormalities, endocrine (hormonal) abnormalities, arterial abnormalities, and cavernous abnormalities, and others are subdivided by drugs or accompanying diseases. Recently, organic erectile dysfunction is the most common, and can be largely treated with oral and injection therapy and surgical therapy.

The erection mechanism is as follows. The sexually stimulated cerebrum sends commands to the spinal cord, pelvis, and genitals via nerves to dilate arterial vessels for an erection. Nitric oxide is secreted from the vascular endothelial cells according to the command, and CTP is converted to c-GMP. When blood rushes in and the gap between the cavernous bodies narrows, the passage of blood flowing in through the arteries into the veins is blocked, resulting in an erection.

Among erectile dysfunction drugs, those that act on the PDE-5 (Phosphodiesterase-5) enzyme have the function of maintaining an erection by inhibiting PDE, an enzyme that breaks down c-GMP, which relaxes smooth muscles and enables blood flow inflow.

However, erectile dysfunction drugs (sildenafil, vardenafil, tadenafil, etc.) that have been released to date have the ability to inhibit PDE-5 enzymes, and not only act on PDE-5 but also on other PDE enzymes, resulting in various side effects. For example, PDE1 is mainly distributed in blood vessels, heart, and brain, PDE6 is in the retina, and PDE11 is mainly distributed in skeletal muscle and testes. Inhibition of PDE1 may cause headache, and inhibition of PDE6 may result in color blindness (cyanosis). Inhibition of PDE11 may cause back pain, myalgia, and hypospermia.

Therefore, if only PDE-5 is inhibited, or if other side effects are hardly observed while having a PDE-5 inhibitory effect, treatment for erectile dysfunction can be performed without fatal side effects.

WO 2011055917 A2

Under this background, the present inventors have completed the present invention as a result of earnest efforts to develop a peptide composition for the treatment of erectile dysfunction having an excellent PDE-5 inhibitory effect while having few side effects.

An object of the present invention is to provide a composition for treating erectile dysfunction that is effective and has no side effects.

According to one aspect of the present invention, erectile dysfunction prevention comprising at least one selected from the group consisting of a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having at least 80% sequence homology with the amino acid sequence, or a peptide that is a fragment thereof and therapeutic compositions are provided.

In the composition according to the present invention, the fragment of the peptide may be a fragment consisting of three or more amino acids.

In the composition according to the present invention, the erectile dysfunction may be due to PDE-5 enzyme activity.

In the composition according to the present invention, the composition may be a pharmaceutical composition.

According to another aspect of the present invention, there is provided a method for preventing erectile dysfunction comprising administering a pharmaceutically effective amount of the composition to an individual suffering from erectile dysfunction.

According to another aspect of the present invention, there is provided a method for treating erectile dysfunction comprising administering a pharmaceutically effective amount of the composition to a subject suffering from erectile dysfunction.

According to another aspect of the present invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having 80% or more sequence homology with the amino acid sequence, or a peptide, which is a fragment thereof, is prepared for the prevention and treatment of erectile dysfunction pharmaceutical composition The intended use is provided.

The peptide having the sequence of SEQ ID NO: according to the present invention, or a peptide or a fragment having a sequence having 80% homology with the sequence, has a PDE-5 inhibitory effect, thereby treating erectile dysfunction by PDE-5 activity or It is expected to provide preventive measures.

The peptide having the sequence of SEQ ID NO: according to the present invention, or a peptide or a fragment having a sequence having 80% homology with the sequence, has a PDE-5 inhibitory effect, thereby treating erectile dysfunction by PDE-5 activity or It is expected to provide preventive measures.

The present invention can apply various transformations and can have various embodiments. Hereinafter, the present invention will be described in more detail. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Telomere (telomere) is a genetic material repeatedly present at the ends of chromosomes, it is known to prevent damage to the chromosome or the binding to other chromosomes. Each time a cell divides, the length of the telomere decreases little by little. If there is more than a certain number of cell divisions, the telomere becomes very short, and the cell stops dividing and dies. On the other hand, it is known that lengthening telomeres prolongs the lifespan of cells. For example, in cancer cells, an enzyme called telomerase is secreted to prevent shortening of telomeres, so it is known that cancer cells can continue to proliferate without dying. The present inventors have completed the present invention by confirming that the peptide derived from telomerase is effective for preventing and treating erectile dysfunction.

In one aspect of the present invention, the peptide of SEQ ID NO: 1, a peptide fragment of SEQ ID NO: 1, or a peptide having at least 80% sequence homology with the peptide sequence is telomerase, specifically human ( Homo sapiens ) telomerase. derived peptides.

The peptides disclosed herein may include peptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence homology. In addition, the peptides disclosed herein include a peptide comprising SEQ ID NO: 1 or fragments thereof and one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids or a peptide in which 7 or more amino acids have been changed.

In one aspect of the present invention, the amino acid change belongs to a property that causes the physicochemical properties of the peptide to be altered. For example, amino acid changes can be performed to improve the thermostability of the peptide, alter substrate specificity, change the optimum pH, and the like.

As used herein, "amino acid" includes the 22 standard amino acids that are naturally incorporated into peptides, as well as D-isomeric and modified amino acids. Accordingly, in one aspect of the present invention, the peptide may be a peptide comprising a D-amino acid. Meanwhile, in another aspect of the present invention, the peptide may include post-translational modified non-standard amino acids and the like. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, e.g., acetylation, myristoylation, and palmitoylation), alkylation ), carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, change in chemical properties (e.g., beta-elimination deimidation) , deamidation) and structural changes (eg, formation of disulfide bridges). Also includes changes in amino acids caused by chemical reactions that occur in the course of binding with crosslinkers to form peptide conjugates, such as changes in amino acids, such as changes in amino groups, carboxyl groups or side chains.

The peptides disclosed herein may be wild-type peptides identified and isolated from a pristine source. On the other hand, the peptide disclosed herein may be an artificial variant, comprising an amino acid sequence in which one or more amino acids are substituted, deleted and/or inserted as compared to the peptide fragments of SEQ ID NO: 1. Amino acid changes in wild-type polypeptides as well as in artificial variants include conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein. Examples of conservative substitutions are basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions that generally do not alter specific activity are known in the art. The most common exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly, and vice versa. Other examples of conservative substitutions are given in the table below.

original amino acids Exemplary residue substitutions Preferred residue substitutions Ala (A) val; leu; ile Val Arg (R) lys; gln; asn Lys Asn (N) gln; his; asp, lys; arg Gln Asp (D) glu; asn Glu Cys (C) ser; ala Ser Gln (Q) asn; glu Asn Glu (E) asp; gln Asp Gly (G) Ala Ala His (H) asn; gln; lys; arg Arg Ile (I) leu; val; met; ala; phe; norleucine Leu Leu (L) norleucine; ile ; val; met; ala; phe Ile Lys (K) arg; gln; asn Arg Met (M) leu; phe; ile Leu Phe (F) leu; val; ile; ala; tyr Tyr Pro (P) Ala Ala Ser (S) thr Thr Thr (T) Ser Ser Trp (W) tyr; phe Tyr Tyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu; met; phe; ala; norleucine Leu

Substantial modifications in the biological properties of the peptides may include (a) their effect on maintaining the structure of the polypeptide backbone within the region of substitution, e.g., a sheet or helical conformation, (b) the charge of the molecule at the target site. or by selecting substituents that differ significantly in their effect in maintaining hydrophobicity, or (c) in maintaining the bulk of the side chains. Natural residues are divided into the following groups based on common side chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilicity: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that affect chain orientation: gly, pro; and

(6) Aromatics: trp, tyr, phe.

Non-conservative substitutions will be made by exchanging a member of one of these classes for another class. Any cysteine residue not involved in maintaining the proper conformational structure of the peptide may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the peptide to improve its stability.

Another type of amino acid variant of a peptide is one in which the glycosylation pattern of the antibody is altered. A change refers to the deletion of one or more carbohydrate moieties found in the peptide and/or the addition of one or more glycosylation sites not present in the peptide.

Glycosylation of peptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine moiety. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrate moieties to the asparagine side chain. Thus, the presence of one of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine can also be used.

Addition of glycosylation sites to the peptide is conveniently accomplished by changing the amino acid sequence to contain one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). Such changes may also be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

In addition, the peptide having the sequence of SEQ ID NO: 1 according to one aspect of the present invention, a peptide fragment of the sequence of SEQ ID NO: 1, or a peptide having at least 80% sequence homology with the peptide sequence has low intracellular toxicity and in vivo stability This has the advantage of being high. SEQ ID NO: 1 in the present invention is a telomerase-derived peptide consisting of 16 amino acids as follows.

The peptides set forth in SEQ ID NO: 1 are shown in Table 2 below. "Names" in Table 2 below are named to distinguish peptides. In one aspect of the present invention, the peptide set forth in SEQ ID NO: 1 represents the entire peptide of human telomerase. In another aspect of the present invention, a peptide having the sequence of SEQ ID NO: 1, a peptide fragment of the sequence of SEQ ID NO: 1, or a peptide having at least 80% sequence homology with the peptide sequence is selected from among the peptides included in telomerase. It includes "synthetic peptides" synthesized by selecting the peptide of the position. SEQ ID NO: 2 shows the amino acid sequence of the entire telomerase.

SEQ ID NO: name telomerase phase location order length One pep1 [611-626] EARPALLTSRLRFIPK 16 aa 2 [1-1132] MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLQRRVGDDVLVHLLARCALFVLVAPRSCAYQRPGRRSVGRRSVGLARPGRRSVGLPSCAYQW
GAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGT TLTALEAAANPALPSDFKTILD 1132 aa

In one aspect of the present invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having 80% or more sequence homology with the amino acid sequence, or a fragment thereof, a peptide having efficacy in preventing and treating erectile dysfunction, as an active ingredient It provides a pharmaceutical composition comprising

In one aspect, the pharmaceutical composition for preventing and treating erectile dysfunction according to an aspect of the present invention is a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having at least 80% sequence homology with the amino acid sequence, or a fragment thereof. The peptide may be included in an amount of 0.01 mg/mL to 0.1 mg/mL, 1 mg/mL, 10 mg/mL, and 100 mg/mL, but if there is a difference in the effect depending on the dose, it may be appropriately adjusted. When included in the above range or less, it may be appropriate to exhibit the intended effect of the present invention, as well as satisfy both stability and safety of the composition, and it may be appropriate to include in the above range in terms of cost-effectiveness. .

The composition according to one aspect of the present invention can be applied to all animals including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs or monkeys.

In one aspect of the present invention, the composition comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having at least 80% sequence homology with the amino acid sequence, or a fragment thereof, a peptide having efficacy in preventing and treating erectile dysfunction. It provides a pharmaceutical composition comprising. The pharmaceutical composition according to one aspect of the present invention may be administered orally, rectal, transdermally, intravenously, intramuscularly, intraperitoneally, intramedullary, intrathecally or subcutaneously.

Formulations for oral administration may be tablets, pills, soft or hard capsules, granules, powders, solutions, or emulsions, but are not limited thereto. Formulations for parenteral administration may be injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches, or sprays, but is not limited thereto.

The pharmaceutical composition according to one aspect of the present invention may contain additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavoring agents or sweeteners, if necessary. The pharmaceutical composition according to one aspect of the present invention may be prepared by a conventional method in the art.

The active ingredient of the pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber. Determination of the dosage based on these factors is within the level of one skilled in the art, and the daily dose thereof is for example 0.1 μg/kg/day to 100 g/kg/day, specifically 10 μg/kg/day to 10 g/kg / day, more specifically, 100 μg/kg/day to 1 g/kg/day, and more specifically 500 μg/kg/day to 100 mg/kg/day, but the difference in effect depending on the dose If it is visible, it can be appropriately adjusted. The pharmaceutical composition according to one aspect of the present invention may be administered 1 to 3 times a day, but is not limited thereto.

In one aspect of the present invention, the composition comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having 80% or more sequence homology with the amino acid sequence, or a peptide fragment thereof as an active ingredient. Prevention of erectile dysfunction and A therapeutic composition is provided.

The formulation of the composition according to one aspect of the present invention is not particularly limited, but may be formulated as, for example, tablets, granules, powders, liquids, solid formulations, and the like. Each formulation can be formulated by appropriately selecting components commonly used in the field other than the active ingredient without difficulty depending on the formulation or purpose of use, and when applied simultaneously with other raw materials, a synergistic effect may occur.

The terminology used herein is intended for the purpose of describing particular embodiments only and is not intended to limit the present invention. A term with a number omitted before a noun is not intended to limit the quantity, but rather indicates the presence of one or more of the mentioned noun item. The terms “comprising,” “having,” and “containing” are to be construed as open terms (ie, meaning “including but not limited to”).

Recitation of a range of values is merely an easy alternative to recitation of each separate number falling within that range, and unless it is explicitly stated that it is not, each discrete number is individually recited in the specification. It is incorporated herein as is. All ranges end values are included within that range and are independently combinable.

All methods mentioned herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any one and all embodiments or exemplary language (eg, “such as”) is merely to better describe the invention and not to limit the scope of the invention, unless included in the claims. It is not meant to be limiting. No language in the specification should be construed as constituting any non-claimed element as essential to the practice of the invention. Unless otherwise defined, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

Preferred embodiments of the invention include the most optimal mode known to the inventor for carrying out the invention. Variations of the preferred embodiments will become apparent to those skilled in the art upon reading the preceding description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventor expects the invention to be practiced otherwise than as described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by patent law. Moreover, within all possible variations any combination of the above-mentioned elements is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. While the present invention has been particularly shown and described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the following claims .

Hereinafter, the configuration and effect of the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are provided only for the purpose of illustration to help the understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

Example 1: Synthesis of peptides

1. Synthesis of peptides

The peptide of SEQ ID NO: 1 (hereinafter referred to as "PEP1") was prepared according to a conventionally known solid-phase peptide synthesis method. Specifically, peptides were synthesized by coupling amino acids one by one from the C-terminus through Fmoc solid phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, Korea). As follows, the first amino acid at the C-terminus of the peptides attached to the resin was used. For example:

NH ₂ -Lys(Boc)-2-chloro-Trityl Resin

NH ₂ -Ala-2-chloro-Trityl Resin

NH ₂ -Arg(Pbf)-2-chloro-Trityl Resin

Trt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6, Pbf (2,2,4,6, 7-pentamethyl dihydro-benzofuran-5-sulfonyl) and the like were used. For example:

Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc- Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Met-OH, Fmoc -Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ahx-OH, Trt-Mercaptoacetic acid.

As a coupling reagent, HBTU[2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetamethylaminium hexafluorophosphate] / HOBt [N-Hydroxxybenzotriazole] /NMM [4-Methylmorpholine] is used did. For Fmoc removal, piperidine in DMF was used in 20% DMF. Cleavage Cocktail [TFA (trifluoroacetic acid) /TIS (triisopropylsilane) / EDT (ethaneedithiol) / H ₂ O=92.5/2.5/2.5/2.5] is used to separate the synthesized peptide from the resin and remove the protecting group of the residue did.

Each peptide was synthesized by repeating the process of reacting the respective amino acids with the starting amino acid to which the amino acid protecting group is bound to the solid support, washing with a solvent, and then deprotecting. After the synthesized peptide was cut off from the resin, it was purified by HPLC, and whether it was synthesized was confirmed by MS and freeze-dried.

As a result of high performance liquid chromatography on the peptides used in this example, the purity of all peptides was 95% or higher.

A detailed process for preparing the peptide PEP 1 will be described as follows.

1) Coupling

NH ₂ -Lys(Boc)-2-chloro-Trityl Resin The protected amino acid (8 equivalents) and coupling reagent HBTU (8 equivalents)/HOBt (8 equivalents)/NMM (16 equivalents) were dissolved in DMF and added , and reacted for 2 hours at room temperature, followed by washing with DMF, MeOH, and DMF.

2) Fmoc deprotection

Piperidine in DMF of 20% was added, and the reaction was performed twice at room temperature for 5 minutes, followed by washing with DMF, MeOH, and DMF.

3) Reaction of 1 and 2 was repeated to obtain peptide backbone NH ₂ -E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(Boc) -2-chloro-Trityl Resin) was made.

4) Cleavage: A cleavage cocktail was added to the synthesized peptide resin to separate the peptide from the resin.

5) After adding cooling diethyl ether to the obtained mixture, centrifuge to precipitate the obtained peptide.

6) After purification by Prep-HPLC, molecular weight was confirmed by LC/MS and frozen to prepare powder.

Example 2: Confirmation of PDE-5 enzyme inhibitory effect of PEP1

In order to verify the efficacy of preventing and treating erectile dysfunction of PEP1, the inhibitory effect of the PDE-5 enzyme known to cause erectile dysfunction was confirmed. PDE-5 enzyme activity inhibitory effect was measured through the PDE-5 inhibition assay (inhibition assay) through the ELISA method. Statistical processing was performed to derive the IC ₅₀ (the half maximal inhibitory concentration) value so that the measured inhibitory effect could be compared with existing inhibitors of PDE-5 enzymes.

Preparation of experimental reagents and materials

The inhibition rate of the PDE-5 enzyme was measured using an ELISA Kit (BML-AK800-0001, Cyclic Nucleotide Phosphdiesterase Assay Kit, Enzo life Science).

The reagents used are as follows. PDE Enzyme (BMLKI4560020), 5'Nucleotidase (BMLKI3075000), 3',5'cAMP SUBSTRATE (BMLKI1800002), 3',5'cGMP SUBSTRATE (BMLKI1900002), BIOMOL GREENTM REAGENT (BMLAK1119090), 5'AMP STANDARD (BMLAK1119090), 5'AMP STANDARD 5'GMP STANDARD (BMLKI1820001), INHIBITOR (IBMX) (BMLPD1409090), PDE ASSAY BUFFER (BMLKI1810040).

For the standard solution used in the experiment, the standard solution dilution buffer was left at room temperature for 10 minutes, a stock solution of 20 ng/mL was prepared, and then 500 μL of distilled water was sequentially placed in 8 tubes in the same manner as in FIG. (Distilled Water, D.W.) was added and diluted to prepare a standard solution having eight concentrations (0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, 20) and nine tubes with distilled water.

To prepare a calibration curve, a standard solution of sildenafil to be used as a standard solution is dissolved in phosphate buffered saline (PBS) at a concentration of 0.046 to 100 μg/L, similar to the method for preparing the standard solution in step 8. Nine tubes with concentration and PBS only were prepared. At this time, the initial stock solution of sildenafil was prepared by using methanol as a solvent at a concentration of 10 mg/L.

Experimental method

The ELISA assay for measuring the inhibition rate was as follows.

A standard, a blank, and an experimental sample were determined and displayed in a 96-well plate. Seven wells were set for the test substances (sildenafil and PEP1), and 1 well was set for the blank. 20 μL of cAMP or cGMP (0.5 mM) was added to each well. Assay buffer was added to each well of the control (control, 15 μL), test substance (5 μL) and inhibitor (IBMX, 5 μL) wells, respectively. After adding 10 μL of 5-nucleotidase, add 10 μL of the test substance. 5 μL of PDE-5 enzyme (4 mU/μL) was added to each well and reacted at 37° C. for 90 minutes. To end the reaction, 100 μL of BIOMOL GREEN reagent was added and the reaction was allowed to develop for 20-30 minutes. After completion of the reaction, absorbance was measured using a microplate reader with a wavelength of 620 nm.

Statistical processing

All data were expressed as mean ± standard error (mean ± SEM), and statistical processing was performed by ANOVA test. SigmaStat statistical program was used and statistical analysis was performed using Kruskal-Wallis test and Mann-Whitney U-test. In addition, the comparison between each group was determined to be statistically significant when the p-value was less than 0.05 by performing a post-hoc test (Tukey test).

Experimental results and analysis

The results measured through the above experimental method were divided into the following two methods.

PDE-5 enzyme inhibition ratio according to the concentration of PEP1 was measured and expressed as a percentage (%) (see FIG. 2).

The PDE-5 enzyme activity according to the treatment concentration of PEP1 was set to 100% as a control, indicating a relative activity ratio (see FIG. 3 ).

As a result of the experiment, as the concentration of PEP1 increased, the inhibition rate of PDE-5 enzyme increased, and the activity ratio of PDE-5 enzyme decreased. The IC ₅₀ value of PEP1 for inhibition and activity reduction for PDE-5 was 9.5 mg/mL.

Through the above experimental examples, it can be seen that the composition comprising PEP1 and PEP1, which are peptides according to the present invention, has an effect of inhibiting the PDE-5 enzyme, and using it to treat erectile dysfunction due to the activity of the PDE-5 enzyme It is judged that it is possible to develop a therapeutic agent or a treatment for erectile dysfunction.

<110> KIM, sangjae Gemvax & KAEL Co.,LTD. <120> A Peptide having impotence treatment activity and the Composition Comprising the Same <130> DP-2015-0004 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 16 <212> PRT <213> homo sapiens <400> 1 Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys 1 5 10 15 <210> 2 <211> 1132 <212> PRT <213> homo sapiens <400> 2 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gly Gly Ser Trp 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990 Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln 1010 1015 1020 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala 1025 1030 1035 1040 Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu 1045 1050 1055 Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp 1060 1065 1070 Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr 1075 1080 1085 Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser 1090 1095 1100 Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn 1105 1110 1115 1120 Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130

Claims (8)

A composition for preventing and treating erectile dysfunction comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.
delete The composition for preventing and treating erectile dysfunction according to claim 1, wherein the erectile dysfunction is caused by PDE-5 enzyme activity.
The composition for preventing and treating erectile dysfunction according to claim 1, wherein the composition inhibits PDE-5 enzyme activity.
The composition for preventing and treating erectile dysfunction according to claim 3 or 4, wherein the composition is a pharmaceutical composition.
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