HK1228253B - Composition for treating and preventing benign prostatic hyperplasia - Google Patents

Description

Composition for treating and preventing benign prostatic hyperplasia

Technical Field

The present invention relates to a composition for treating and preventing benign prostatic hyperplasia. More particularly, the present invention relates to a composition comprising a peptide derived from telomerase and the composition is used for treating and preventing benign prostatic hyperplasia.

Background Art

Benign prostatic hyperplasia (BPH) is the most common aging disease in men, often accompanied by lower urinary tract symptoms. Symptoms begin in men in their 40s, but most clinical symptoms develop in their late 50s. BPH can cause sexual dysfunction, leading to decreased quality of life. Treatment and surgery for BPH can also affect sexual function.

BPH, which causes hyperplasia, is dependent on male hormones. In particular, male hormones are essential for the inhibition of normal cell proliferation and apoptosis in the prostate. The most well-known internal cause is aging. The prostate enlarges due to aging and normal testicular function. As a prostate-dependent male hormone, testosterone plays an important role in the growth and differentiation of the prostate and is metabolized by 5α-reductase to produce dihydrotestosterone (DHT), which plays an important role in prostate growth and the expression of prostate genes.

External factors that cause prostate growth include androgens, estrogens, glucocorticoids, and substances related to endocrine enzymes, which are induced by diet and the environment. The physiological effects of these external factors occur through various growth factor peptides.

BPH occurs in the early 20s to late 40s and is caused by histological changes in which androgens and estrogens act synergistically to cause BPH. With aging, the estrogen/DHT ratio increases, and then BPH increases.

At the same time, it is well known that the prostate grows until the early 20s and maintains its size until the age of 50, which depends on a very complex interaction that allows the prostate to maintain its balance, such as endogenous growth factors, signaling pathways, cell cycle regulation, cell division and apoptosis. If cell cycle regulators are altered, BPH may be induced.

Genetic factors are a major factor influencing BPH. Patients with a family history of BPH are reported to have an increased risk of BPH of over 60%. It has also been reported that treatment with 5α-reductase inhibitors is less effective in patients with a family history of BPH. This is because, in this case, BPH relies on an androgen-independent pathway.

Surgery and medication are available for the treatment of BPH. For medication, drug administration is adjusted based on the patient's age and clinical course. Recently, the number of BPH patients has increased significantly in Korea and globally, and the prevalence of younger patients is also increasing. Numerous medications are used for treatment, but their use is limited by side effects.

Sulpiride is a type 2 dopamine receptor antagonist commonly used as a drug for the treatment of depression. Dopamine is an intermediate produced during the synthesis of epinephrine and norepinephrine and is an inhibitory neurotransmitter. Sulpiride inhibits the binding of dopamine to its receptors, which inhibits the secretion of prolactin, which acts as a dopaminergic factor, and increases the concentration of prolactin in the blood. The increased prolactin level caused by continuous administration of sulpiride can cause hyperprolactinemia.

Prolactin is reported to be associated with the development and regulation of prostate hyperplasia, prostate cancer, and BPH. It is also known that prolactin synergizes with androgens to increase prostate proliferation. As another mechanism, prolactin is also known to act as a stress hormone to increase the expression of 5α-reductase and induce prostate hyperplasia. Prolactin is a non-steroidal factor that is associated with prostate hyperplasia and the induction of BPH. With age, prolactin increases and testosterone levels decrease. Prolactin is reported to induce BPH in the elderly. In rats and humans, prolactin is reported to be associated with prostate hyperplasia and differentiation. According to this report, prolactin is believed to be induced by receptors through signal transduction pathways.

Prior art documents

Patent documents

KR 2011-0062943 A

KR 2011-0057049 A

EP 1020190 A3

Non-patent documents

MCCONNELL, John D., et al. "The effect of finasteride on the risk of acute urinary retention and the need for surgical treatment among men with benignprostatic hyperplasia", New England Journal of Medicine, 1998, Volume 338, Issue 9, Pages 557-563.

Detailed Description of the Invention

Technical issues

Then, the present inventors attempted to develop a composition for treating and preventing BPH, which has minimal side effects and excellent therapeutic effects, and completed the present invention.

The inventors of the present invention have found that a peptide derived from telomerase can have excellent effects for treating and preventing BPH and have completed the present invention.

An object of the present invention is to provide a composition effective in treating and preventing BPH.

Technical Solution

In order to solve the above technical problems, according to the present invention, a composition comprising a peptide or a peptide fragment for treating and preventing BPH is provided, wherein the peptide or peptide fragment comprises the sequence of SEQ ID NO: 1 (hereinafter referred to as "PEP1", "GV1001" or "GV") or a sequence having 80% or more homology with SEQ ID NO: 1.

In the composition for treating and preventing BPH according to the present invention, the fragment may contain more than 3 amino acids.

In the composition for treating and preventing BPH according to the present invention, the peptide may be included at a concentration of 0.01 mg to 1 mg, preferably 0.56 mg (4 nmol peptide/kg body weight).

In the composition for treating and preventing BPH according to the present invention, the composition may be a pharmaceutical composition.

In the composition for treating and preventing BPH according to the present invention, the composition may be a food composition.

According to another embodiment of the present invention, there is provided a method for treating and preventing BPH by administering a composition for treating and preventing BPH to a subject in need thereof.

In the method for treating and preventing BPH according to the present invention, administration of the composition may be performed three times a week.

Effects of the Invention

The composition according to the present invention, comprising a peptide having a sequence of SEQ ID NO: 1 or a peptide having a sequence having 80% or more homology to SEQ ID NO: 1, has excellent effects and fewer side effects for treating and preventing BPH.

Description of the drawings

FIG1 shows photographs of the process of removing a target organ to measure its weight.

FIG2 shows electrophoresis photographs in an experiment to verify the therapeutic effect of PEP1 on BPH, which shows the results of the effect on 5α-reductase expression in the ventral prostate of each experimental group by using RT-PCR.

FIG. 3 shows a graph showing the results of the experiment to verify the therapeutic effect of PEP1 on BPH, showing the weight of the seminal vesicles measured in each experimental group.

FIG. 4 shows a graph showing the results of an experiment to verify the therapeutic effect of PEP1 on BPH, showing the prostate weight measured in each experimental group.

FIG. 5 shows graphs showing the amount of cell proliferation after treatment with PEP1 in a stromal cell line (WPMY-1) of a BPH-induced animal model.

FIG. 6 shows graphs showing the amount of cell proliferation after treatment with PEP1 in an epithelial cell line (RWPE-1) of a BPH-induced animal model.

FIG. 7 shows a graph showing that the binding ability of PEP1 to the androgen receptor was measured by using a PEP1-FITC (fluorescein isothiocyanate) conjugate in a stromal cell line (WPMY-1) of a BPH-induced animal model.

FIG. 8 shows a graph showing that the binding ability of PEP1 to the androgen receptor was measured by using a PEP1-FITC (fluorescein isothiocyanate) conjugate in an epidermal cell line (RWPE-1) of a BPH-induced animal model.

FIG. 9 shows electrophoresis photographs showing the effect of PEP1 on PCNA (proliferating cell nuclear antigen) expression, which is increased in a BPH-induced model.

FIG. 10 shows a photograph of immunostaining showing the effect of PEP1 on Ki67 (MK67) expression, which increases in a BPH-induced model when BPH is induced.

FIG. 11 shows photographs showing the results of an experiment on a BPH animal model, showing the effect of PEP1 on cells related to BPH tissues by H&E staining.

FIG. 12 shows photographs showing the results of a BPH animal model experiment showing the effect of PEP1 on BPH tissue-related cells by Masson's trichrome staining.

FIG. 13 shows a graph showing changes in body weight of animals in an experiment to determine the effects of PEP1 in an animal model of BPH.

FIG. 14 shows graphs showing changes in prostate weight of a BPH animal model in an experiment to determine the effects of PEP1 in the animal model.

FIG. 15 shows a graph showing changes in the weight of the seminal vesicles of the animals in an experiment to determine the effects of PEP1 in an animal model of BPH.

Best Mode for Carrying Out the Invention

Because the present invention is applicable to a wide variety of applications and variations, the present invention will be described in greater detail below. However, this is not intended to limit the scope of practical applications; it should be understood that the subject matter is intended to encompass all variations, equivalents, and alternatives of the concepts and techniques. In describing the present invention, any detailed description of prior art will be omitted if it is deemed to undermine the underlying principles of the present invention.

Telomeres are known to be repetitive sequences of genetic material found at the ends of chromosomes that prevent chromosome damage or merging into other chromosomes. Telomere length shortens with each cell division, and after a certain number of cell divisions, telomeres become so shortened that the cell stops dividing and dies. On the other hand, it is known that extending telomeres can extend cell lifespan. For example, cancer cells produce an enzyme called telomerase, which prevents telomere shortening, thereby promoting the proliferation of cancer cells. The present inventors have identified a peptide derived from telomerase that is effective in treating and preventing BPH, leading to the completion of the present invention.

In one embodiment of the present invention, a peptide of the amino acid sequence of SEQ ID NO: 1, a peptide fragment of the above peptide, or a peptide having more than 80% sequence homology to the amino acid sequence of the above peptide comprises telomerase, in particular telomerase from humans. The peptides disclosed herein may include peptides or fragments thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology to the peptide of SEQ ID NO: 1. In addition, the peptides disclosed herein may include peptides that differ from SEQ ID NO: 1 or a fragment thereof in at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 converted amino acids, at least 6 converted amino acids, or at least 7 amino acids.

In one embodiment of the present invention, amino acid changes include modifications of the physical and chemical properties of the peptide. For example, amino acid modifications can be made to increase the thermal stability of the peptide, change substrate specificity, and change the optimal pH.

The term "amino acid" herein includes not only the 22 standard amino acids that naturally constitute peptides, but also D-isomers and modified amino acids. Therefore, in a specific embodiment of the present invention, the peptides herein include peptides having D-type amino acids. On the other hand, the peptides may include non-standard amino acids, such as those that are post-translationally modified. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including acetylation, myristoylation, palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitination, modification of chemical properties (e.g., β-elimination deamidation, deamidation) and structural modifications (e.g., formation of disulfide bonds). At the same time, changes in amino acids include changes in amino acids that occur due to chemical reactions during the binding process using a cross-linking agent for forming a peptide conjugate, such as changes in amino groups, carboxyl groups and side chains.

The peptides disclosed herein can be wild-type peptides identified and isolated from natural sources. On the other hand, when compared with SEQ ID NO: 1 or its fragments, the peptides disclosed herein can be artificial variants comprising one or more amino acid substitutions, deletions and/or insertions. Amino acid changes in wild-type polypeptides (not only artificial variants) include conservative substitutions of the folding and/or amino acids that do not significantly affect the activity of the protein. Examples of conservative substitutions can be within the following groups: 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 change a specific activity are known in the art. The most common transformations 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 their reverse transformations. Other examples of conservative substitutions are shown in Table 1 below:

[Table 1]

Original amino acids Examples of 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

Significant changes in the biological properties of peptides can be achieved by selecting substitutions that differ significantly in their efficacy: (a) in maintaining the structure of the polypeptide backbone (e.g., sheet-like or helical three-dimensional structure) in the region of substitution, (b) in maintaining the charge or hydrophobicity of the molecule in the target region, or (c) in maintaining the bulk of the side chain. Natural residues are divided into the following groups based on their general side chain properties:

(1) Hydrophobicity: 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) Aromatic: trp, tyr, phe.

Non-conservative substitutions can be made by exchanging a member of the above-mentioned group for a member of a different group. Any cysteine residue that is not relevant to maintaining the proper three-dimensional structure of the peptide can generally be substituted with a serine to increase the oxidative stability of the molecule and avoid undesirable cross-linking. Conversely, increased stability can be achieved by adding cysteine bonds to the peptide.

Another type of amino acid variant of a peptide is one with an altered glycosylation pattern. The term "alteration" herein means deleting at least one sugar residue present in the peptide and/or adding at least one glycosylation residue not present in the peptide.

Glycosylation of peptides is usually N-linked or O-linked. The term "N-linked" herein refers to the connection of a sugar residue to the side chain of an asparagine residue. As tripeptide sequences, asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid except proline) are recognition sequences for enzymatically connecting a sugar residue to the asparagine side chain. Therefore, when one of these tripeptide sequences is present in a polypeptide, a potential glycosylation site is created. "O-linked glycosylation" means that one of the sugars, N-acetylgalactosamine, galactose, or xylose, is connected to a hydroxyamino acid. The hydroxyamino acid is most typically serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

Glycosylation sites are conveniently added to the polypeptide by altering the amino acid sequence to contain the above-described tripeptide sequence (for N-linked glycosylation sites). The alteration can be made by adding at least one residue from serine or threonine to the first antibody sequence or by substituting these residues (for O-linked glycosylation sites).

At the same time, according to the present invention, peptides comprising the amino acid sequence of SEQ ID NO: 1, peptides comprising an amino acid sequence having greater than 80% homology to the aforementioned sequence, or fragments thereof have the advantages of low toxicity and high stability in living matter. SEQ ID NO: 1, as used herein, is a 16-amino acid peptide derived from telomerase.

SEQ ID NO: 1EARPALLTSRLRFIPK

In one embodiment of the present invention, a composition for treating and preventing BPH is provided, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide comprising an amino acid sequence having greater than 80% homology with the above sequence, or a fragment of the above peptide.

In one embodiment of the present invention, the composition may have application to all animals including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs and monkeys.

In one embodiment of the present invention, a pharmaceutical composition for treating and preventing BPH is provided, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide comprising an amino acid sequence having greater than 80% homology to the aforementioned sequence, or a fragment of the aforementioned peptide. The pharmaceutical composition according to one embodiment of the present invention can be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramedullary, epidurally, or subcutaneously.

The form of oral administration may be, but is not limited to, tablets, pills, soft or hard capsules, granules, powders, solutions or emulsions. The form of parenteral administration may be, but is not limited to, injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays.

In one embodiment of the present invention, the pharmaceutical composition may include additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavorings or sweeteners, if necessary. In one embodiment of the present invention, the pharmaceutical composition may be manufactured by conventional industrial methods in the art.

In one embodiment of the invention, the dosage of the active ingredient of the pharmaceutical composition can be changed according to the patient's age, sex, weight, pathology and state, route of administration or the judgment of the prescriber. The dosage according to the factors can be determined within the level of those skilled in the art, and the daily dose can be, for example, but not limited to: 0.01 μg/kg/day to 10g/kg/day, particularly 0.1 μg/kg/day to 1mg/kg/day, more particularly 1 μg/kg/day to 0.1g/kg/day, more particularly 1 μg/kg/day to 10mg/kg/day, preferably 1 μg/kg/day to 1mg/kg/day, preferably 0.005mg/kg/day to 0.05mg/kg/day, most preferably 0.01mg/kg/day, but if different according to the effect of the applied dosage, it can be adjusted. For adults, the preferred applied dosage is 0.1mg to 1mg, preferably 0.4mg to 0.6mg, and the most preferred dosage is 0.56mg.

In one embodiment of the present invention, the pharmaceutical composition may be administered, but is not limited to, 1 to 3 times a day.

In one embodiment of the present invention, the composition may contain 0.01 g/L to 1 kg/L, particularly 0.1 g/L to 100 g/L, and more particularly 1 g/L to 10 g/L of a peptide comprising at least one of the amino acid sequences of SEQ ID NO: 1, a peptide comprising an amino acid sequence having at least 80% homology to the aforementioned sequence, or a fragment thereof. When the content of the peptide is within the aforementioned range, both safety and stability of the composition can be met, and the aforementioned range is suitable in terms of cost-effectiveness.

In one embodiment of the present invention, a food composition for treating and preventing BPH is provided, which comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide comprising an amino acid sequence having greater than 80% homology with the above sequence, or a fragment of the above peptide.

In one embodiment of the present invention, food composition is not limited to a particular form, but can be, for example, tablet, granule, powder, liquid and solid form. In addition to the active ingredient, each form can be formed by those skilled in the art selecting suitable industrial common ingredients, and can be combined with other ingredients to produce a synergistic effect.

The terms used herein are intended to describe embodiments, but not to limit the present invention. The absence of a number in front of a term does not limit the quantity, but rather indicates that more than one of the term may be used. The terms "comprising," "having," "including," and "containing" should be understood as open-ended (i.e., "including but not limited to").

The reason why values are mentioned as ranges is simply because it is more convenient to describe them in ranges than as individual numbers. Unless otherwise specified, each individual value is understood to be described separately and incorporated into the specification. The thresholds of all ranges are inclusive and independently combinable.

Unless otherwise provided or clearly contradictory in the context, all methods proposed herein are performed in the appropriate order. Unless otherwise provided or clearly contradictory in the context, all methods proposed herein are performed in the appropriate order. Unless otherwise provided, the use of "any one embodiment" and "all embodiments" or exemplary language (e.g., "such as" and "as") is used to more clearly describe the present invention, rather than to limit the scope of the present invention. Any language herein other than the claims should not be interpreted as a prerequisite for the present invention. Unless otherwise defined, the scientific and technological terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

The preferred embodiments of the present invention include the best embodiments of the present invention known to the inventors. Variations of the preferred embodiments may become apparent to those skilled in the art after reading the above description. The inventors of the present invention hope that those skilled in the art can make full use of the variations and that the present invention may be implemented in other ways than those listed herein. Therefore, the present invention includes equivalents, modifications and variations of the key points of the invention set out in the claims, as permitted by patent law. In addition, all possible variations within any combination of the above components are included in the present invention, unless otherwise expressly stated or contrary to the context. Although the present invention has been described and illustrated by exemplary embodiments, it will be clear to those skilled in the art that various changes in form and detail may be made without departing from the spirit of the present invention and the scope defined by the claims hereinafter.

For establishing the embodiments of the present invention

Hereinafter, the present disclosure will be described in detail through embodiments and test examples. However, the following embodiments and test examples are for illustrative purposes only, and it is obvious to those skilled in the art that the scope of the present disclosure is not limited by the embodiments and test examples.

Example 1. Synthesis of peptides

The peptide of SEQ ID NO: 1 was synthesized according to conventional methods of solid-phase peptide synthesis. More specifically, the peptide was synthesized by Fmoc solid-phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon ROK) by coupling each amino acid from the C-terminus. Peptides whose first amino acid was attached to the resin at the C-terminus were used as follows:

NH2-Lys(Boc)-2-chlorotrityl resin

NH2-Ala-2-chlorotrityl resin

NH2-Arg(Pbf)-2-chlorotrityl resin

All amino acids used to synthesize peptides were protected at the N-terminus by Fmoc, and the amino acid residues were protected by Trt, Boc, t-Bu (t-butyl ester), Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) that are soluble in acid. Examples include the following:

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(tB u)-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.

HBTU [2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate]/HOBt [N-hydroxybenzotriazole]/NMM [4-methylmorpholine] were used as coupling reagents. A 20% piperidine solution in DMF was used to remove Fmoc. To remove protecting groups from residues or to cleave the synthesized peptide from the resin, a cleavage mixture [TFA (trifluoroacetic acid)/TIS (triisopropylsilane)/H ₂ O = 92.5/2.5/2.5] was used.

Peptide synthesis was performed using a solid-phase support and repeating the following process: starting with amino acid protection, separate reaction of each amino acid, washing with a solvent, and deprotection. Each peptide was synthesized by using a solid-phase support to bind to a starting amino acid having an amino acid protecting group, reacting the corresponding amino acid, washing with a solvent, and deprotecting, and repeating this process. After release from the resin, the synthesized peptide was purified by HPLC, verified by mass spectrometry, lyophilized, and the synthesis was verified by MS, and then lyophilized.

The purity of the prepared peptide was found to be 95% or higher by high performance liquid chromatography.

The specific synthesis process of PEP 1 can be as follows:

1) Coupling

The amino acid (8 eq) protected with NH2 _- Lys(Boc)-2-chlorotrityl resin was mixed with coupling reagents HBTU (8 eq)/HOBt (8 eq)/NMM (16 eq) dissolved in DMF and incubated at room temperature (RT) for 2 hours. After incubation, the reaction mixture was washed with DMF, MeOH, and DMF in sequence.

2) Fmoc deprotection

A 20% piperidine solution in DMF was added and incubated at RT for 5 min twice, followed by sequential washing with DMF, MeOH, and DMF.

3) The basic framework of the peptide, NH ₂ -E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(Boc)-2-chlorotrityl resin, was prepared by repeating the above reactions 1) and 2).

4) Cleavage: Add cleavage mixture to the completed peptide to cleave the peptide from the resin.

5) Pre-cooled diethyl ether was added to the obtained mixture, followed by centrifugation to precipitate the collected peptide.

6) After purification by Prep-HPLC, the molecular weight was confirmed by LC/MS, and lyophilized to produce a powder form.

Example 2: Validation of the effect of PEP1 on BPH by using a BPH-induced animal model

Preparation of BPH-induced animal model

1) As an androgen, testosterone is the most commonly used hormone in the body. However, the most effective hormone among the androgens related to prostate development is 5α-dihydrotestosterone (DHT), which is produced by combining testosterone and 5α-reductase. In rats, when sulpiride was administered at a concentration of 40 mg/kg for 30 days, it inhibited type 2 dopamine receptors in the body to increase the concentration of prolactin and induced hyperprolactinemia to activate 5α-reductase, and it showed a synergistic effect by reacting with testosterone. It is reported that DHT produced by hyperprolactinemia produces more weight gain in the lateral lobes of the prostate than in the dorsal or ventral lobes. Based on this fact, experiments using only PEP1 according to Example 1 or co-administered with other test materials to induce BPH animal models were conducted as follows. Mature Sprague-Dawley male rats (6 weeks old) were purchased from the Jae-il Laboratory Animal Center and raised for one week (7 weeks old, 49 days) for purification and then used in experiments. To induce BPH, sulpiride (40 mg/kg) was orally administered once a day for 30 days. Each experiment followed the results of the previous experiment (Van Coppenolle et al., 2001). Each animal was administered the test material daily, starting at 10:00 AM. Following administration of the test material, each animal was observed daily for general condition and specific symptoms. Prior to administration of the test material, each animal's body weight was measured and recorded.

2) Test materials and doses administered

Sulpiride, a test material, was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and used in the experiment. The present inventors administered sulpiride (40 mg/kg) by continuous intraperitoneal injection once daily for 60 days to induce BPH through hyperprolactinemia. Before each administration of the test material, sulpiride was first dissolved in 0.1N HCl solution and then neutralized to pH 7.0 using 0.1N NaOH solution. For the combined administration group, PEP1 and finasteride according to Example 1 were administered by intraperitoneal injection after the administration of sulpiride. PEP1 (0.01 mg/kg, 0.1 mg/kg, 1 mg/kg and 10 mg/kg) was freshly prepared before use and administered by subcutaneous injection. Finasteride was prepared daily using 15% ethanol/corn oil (v/v) as a carrier. The administered dose was calculated based on a concentration of 0.5 ml/kg, reflecting the body weight measured daily. The administration of the 7 groups was completed in accordance with Table 2 below to verify the effect of PEP1 on BPH.

[Table 2]

(i.p = intraperitoneal, s.c = subcutaneous)

1) For the BPH-induced model, after the experiment of administering PEP1 and test materials, the animal organs were collected, preserved and their weights were measured

24 hours after administration of the test material on day 60, all animals were anesthetized with ether, and then blood was collected from the abdominal aorta to separate the serum. The separated serum was stored at -80°C for hormone analysis.

For all animals, after the exit of the test prepuce separation (PPS), the accessory gonads such as glans penis (Gp), seminal vesicle and coagulation glands (SV), ventral prostate (VP), bulbourethral glands (CpG), levator ani plus bulbocavernosus muscle (LABC) were sequentially isolated from the animal body. The detailed isolation process followed the OECD protocol.

As mentioned in Figure 1, for isolating Gp, by clamping the Gp portion using forceps and cutting the preputial separation line. As mentioned in Figure 1, for lungs, after separating the bladder from the abdominal muscle layer, the left and right lung lobules covered with lipid layer were exposed, the bladder was exposed to SV, lipid was separated from the left and right lung lobules by using forceps, and the left lung lobule was cut from the urethra by using micro forceps, and the right lung lobule was cut after being exposed from the urethra by using surgical forceps. As mentioned in Figure 1, for SV containing coagulated glands, a paper towel was prepared under the SV to classify the muscle, lipid layer and glands. The bottom of the SV containing the seminiferous tubules connected to the urethra was fixed by using a clamp to prevent leakage during the removal of the seminal vesicle. After removing the lipid, the associated accessory organs were cleaned, the clamp was removed and the seminal vesicle was placed on a plate to measure its weight.

2) Effect of PEP1 administration on 5α-reductase expression in a BPH-induced animal model

After 60 days of administration of sulpiride and the test materials and collection of the ventral prostate, the effects of sulpiride on 5α-reductase expression were determined by RT-PCR. Specifically, total RNA was isolated from the ventral prostate (25 mg) and resuspended in DEPC-treated water. RNA was then quantified using a spectrophotometer. First-strand cDNA was synthesized using the method of Torres and Ortega (2004). The PCR protocol was: 94°C denaturation (30 sec), 55°C annealing (30 sec), and 72°C extension (30 sec) for 30 to 35 cycles. GAPDH was used as a control and quantified by electrophoresis; its expression level was not altered by the use of other drugs. As a result, sulpiride administration inhibited the increase in 5α-reductase levels in the PEP1-administered group in a dose-dependent manner, and the inhibitory effect was greater in the high-dose PEP1-administered group (GV10, 10 mg PEP1-administered group) than in the finasteride-administered group (see Figure 2). Therefore, PEP1 can provide a dose-dependent treatment and enhance its effect on BPH by inhibiting 5α-reductase.

3) Effects of PEP1 administration on organs in BPH-induced test animal models

In the following Table 3, the effect of peptide PEP1 on the weight of the seminal vesicles, the weight of the prostate and the prostate index in each group of experiments is reported. The prostate index described in Table 3 was calculated by using the formula "body weight/final prostate weight".

[Table 3]

The results described in Table 3 were converted into a graph. That is, in a BPH-induced animal model, after administering PEP1 and finasteride (5 mg/kg) and then sulpiride, the seminal vesicle weight was observed. The graph shows that when a high dose of PEP1 (10 mg/kg) was administered, the seminal vesicle weight was significantly reduced (see Figure 3). At the same time, in a BPH-induced animal model, when sulpiride and PEP1 were co-administered, a significant reduction in prostate weight was shown (see Figure 4). A P value of less than 0.05 was considered significant.

Therefore, the results of Example 2 indicate that administration of PEP1 to a sulpiride-induced BPH animal model can effectively reduce 5α-reductase expression, spermatovesicle weight, and prostate weight in a dose-dependent manner. Therefore, administration of PEP1 can effectively treat and improve BPH-related disease symptoms caused by 5α-reductase expression and reproductive organ weight.

Example 3: Verifying the effect of PEP1 on BPH by observing the changes of DHT on prostate stromal cells and epithelial cells

1) Test cell preparation and experimental process

Testosterone is converted into DHT by 5α-reductase after being injected into the body, which induces prostate cell proliferation and causes BPH. Based on this, an experiment was conducted to observe its effect on the proliferation of prostate cell lines by using the administration of PEP1 according to Example 1. As cell lines, WPMY-1 (prostate stromal cell line) and RWPE-1 (prostate epithelial cell line) from animal models were used. For the experiment, WPMY-1 (2.5×10 ³ cells) and RWPE-1 (1×10 ⁴ cells) were seeded into 96-well plates with different experimental groups as shown in Table 4 to observe proliferation changes. After aspirating the culture medium, the proliferation changes were measured by injecting 10 μL of CCK-8 solution into each well of the culture medium and then measuring the optical density at a wavelength of 450 nm for 1 to 4 hours.

2) Confirm observations and impacts

In the non-DHT-treated groups (Groups 1-3), there were no significant differences between the WPMY-1 and RWPE-1 groups without PEP1 (Group 1) and those with PEP1 (Groups 2 and 3). In the DHT-treated groups (Groups 4-6), there were significant differences between the non-PEP1 group (Group 4) and those with PEP1 (Groups 5 and 6), with the PEP1-treated groups showing significant inhibition of prostate cell proliferation (see Table 4 and Figures 5 and 6). Therefore, PEP1 can effectively inhibit prostate cell proliferation, which affects DHT-induced BPH.

[Table 4]

Treatment conditions for each cell line

Example 4: Verification of PEP1's binding ability to androgen receptor and its mechanism of inhibiting BPH

1) Preparation of test cells and experimental procedures

DHT produced by 5α-reductase promotes the proliferation of prostate cells by binding to androgen receptors and causes BPH. Based on this, an experiment was conducted using the administration of PEP1 according to Example 1 to observe its effect on the proliferation of prostate cell lines. As cell lines, WPMY-1 and RWPE-1 from animal models were used. WPMY-1 and RWPE-1 were divided into an anti-androgen receptor group and an isotype control group, and a competition test was performed by placing PEP1-FITC (fluorescein isothiocyanate) therein, incubating with each antibody, and measuring the results of the fluorescence value. The fluorescence value was measured by using a flow cytometry method.

2) Confirm observations and impacts

For each WPMY-1 and RWPE-1, the fluorescence values were measured in the following situations: one situation in which PEP1 was first reacted with an anti-androgen receptor isotype control antibody (competition with the antibody, rightmost peak), another situation in which PEP1 was reacted with an anti-androgen receptor antibody (competition with the antibody, middle peak), and another situation in which PEP1 was not reacted with either antibody that was not conjugated to FITC (leftmost peak) (see Figures 7 and 8). In the case of competition with the anti-androgen receptor isotype control antibody, PEP1 binds to the anti-androgen receptor, so the fluorescence value of the PEP1-FITC conjugate increases (the peak shifts to the right side of the histogram in the figure). In the case of competition with the anti-androgen receptor, PEP1 binds to the anti-androgen receptor more weakly, so the fluorescence value decreases (the peak shifts to the left side of the histogram in the figure). Therefore, considering that PEP1 inhibits BPH induced by DHT binding to the anti-androgen receptor, PEP1 can affect BPH by directly binding to the anti-androgen receptor.

Example 5: Verification of the effectiveness of PEP1 against BPH in vivo using a BPH-induced animal model

1) Prepare test animals

The experiment used 6 to 8 week old male C57BL/6 (n=10/group) mice, which were raised in an SPF (specific pathogen-free) area of the laboratory animal laboratory of Seoul National University College of Medicine. 50 mg of testosterone enanthate (TE, purchased from EVERPharma Hena GmbH, Germany) and 0.5 mg of estradiol valerate (purchased from EVER Pharma Hena GmbH, Germany) for injection were mixed into a 70 μl volume micro-osmotic pump (Alzet pump, purchased from DURECT Corporation, USA), which was implanted in the back of the mouse under anesthesia. The pump was designed to release hormones to the mouse at a concentration of 0.11 μl per hour for 28 days (2 weeks) by utilizing the osmotic phenomenon.

2) Test materials and dosage

As test materials, testosterone and finasteride were used. An animal model was prepared, and each subject (25 g mouse model) was subcutaneously (injected) daily with 250 μg of PEP1 described in Example 1 and 2500 μg of finasteride (in DMSO or cyclodextrin, purchased from Sigma Aldrich, USA). Two weeks after the injection of the test materials (four weeks after the pump was implanted into the animal model), blood was collected from the supraorbital vein and centrifuged at 14,000 rpm at 4°C for 30 minutes to separate the serum, and the prostate was extracted and frozen in -70°C liquid nitrogen or fixed in a fixative. The experimental test groups are described in Table 5 below.

[Table 5]

3) Measure the reduction of factors that induce BPH

Inducing BPH increases PCNA (proliferating cell nuclear antigen, an essential protein for replication) and Ki67 (MK167, an essential protein for cell proliferation) in prostate tissue. Based on this, the effectiveness of PEP1 in inhibiting PCNA and Ki67 expression was tested in a BPH-induced mouse model. PCNA was measured using 2D-gel electrophoresis using proteins extracted from prostate tissue cells, and the expression level in the tissue was detected by immunostaining to measure Ki67. As a result, the expression of PCNA and Ki67 increased in the prostate tissue of BPH-induced animals and decreased in the group treated with PEP1 (see Figures 9 and 10). Therefore, PEP1 inhibits BPH-inducing factors and is effective for treating and improving BPH.

4) Measure tissue changes associated with induced BPH

It is known that BPH is induced by the abnormal proliferation of stromal cells and epithelial cells that make up the prostate gland. Based on this, in order to detect the changes caused by PEP1 in the prostate tissue of the BPH-induced animal model, the BPH-induced animal model was subjected to histological analysis. The changes in general tissue were detected using the H&E staining method, and the inflammatory reaction level was determined using the Masson trichrome staining method and the shape of the cell nucleus was more clearly detected. As a result, it was shown that the epithelial layer of the BPH-induced group was thicker than that of the control group, but in the PEP1-treated group, it was shown that the epithelial layer was arranged in a regular order similar to that of the control group, and the thickness of the epithelium was thinner than that of the BPH-induced group (see Figures 11 and 12). Therefore, PEP1 can effectively restore the changes in BPH-induced tissue and convert the tissue into normal tissue that does not show BPH.

5) Measure changes in BPH-related organs

BPH can be detected by changes in the weight of the prostate and seminal vesicle. Based on this, in order to detect the effect of PEP1 on the weight of the prostate and seminal vesicle (which directly show the symptoms related to BPH), body weight, prostate weight and seminal vesicle weight were measured in a BPH-induced animal model. The measurement results are shown in Table 5 (see Figures 13, 14 and 15) as a chart by group. No changes in overall body weight were shown, but compared with the hormone-treated group, the prostate weight of the PEP1-treated group was significantly reduced, and the reduction in the PEP1-treated group was comparable to the results of the group administered finasteride (known as a therapeutic drug for BPH), so this confirms that the reduction in the PEP1-treated group is significant. For the seminal vesicle, the weight of the seminal vesicle in the PEP1-treated group was less than that in the hormone-treated group. Therefore, PEP1 can effectively and significantly reduce the weight of organs with BPH symptoms.

In all of the above examples, in vitro and in vivo experiments in BPH-induced animal models demonstrated that PEP1 has a significant therapeutic effect on BPH-related inducing factors, hormone receptors, and parenchymal reproductive organs. Therefore, PEP1 is believed to be effective for treating, ameliorating, and preventing BPH, and there is great potential for developing PEP1 into a composition and method for treating BPH.

Sequence List Free Text

The 1132 amino acids of the complete telomerase sequence

MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSTRSYLPNTVTDALRGSGAWGLLL RRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVEEAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRCFCVVSPARPAEEAT SLEGALSGTRHSHPSVGRGHHAGPPSTSRPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAG VCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTET TFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLREIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELY FVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDM ENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQ VNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

Claims (9)

1. A composition for treating and preventing benign prostatic hyperplasia, wherein the composition comprises a peptide having the amino acid sequence of SEQ ID NO:1. 2. The composition of claim 1, wherein the composition comprises one or more additives selected from the group consisting of: diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, fragrances, or sweeteners. 3. The composition of claim 1, wherein the composition comprises 0.01 mg to 1 mg of the peptide. 4. The composition of claim 1, wherein the composition comprises 0.56 mg of the peptide. 5. The composition of claim 1, wherein the composition is a pharmaceutical composition. 6. The composition of claim 1, wherein the composition is a food composition. 7. Use of the composition of any one of claims 1 to 6 in the preparation of a medicament for treating and preventing benign prostatic hyperplasia, wherein the treatment and prevention comprises the step of administering the composition to a subject in need of treatment. 8. The application as described in claim 7, wherein the composition is administered at a dose of 0.005 mg/kg to 0.05 mg/kg per administration. 9. The application as claimed in claim 7, wherein the composition is administered every 2 weeks during a total treatment duration of 12 weeks, wherein the administration is completed on a day during weeks 0, 2, 4, 6, 8, 10 and 12, and each administration of the composition is 0.56 mg, which is equivalent to 4 nmol peptide/kg body weight.