HK1168363B - Novel peptide and use thereof - Google Patents

Description

Novel peptides and uses thereof

Technical Field

The present invention relates to novel peptides which are effective in the treatment and/or prevention of degenerative disc diseases, the treatment of body organ fibrosis, the treatment of cancer, the treatment of glomerulosclerosis or similar diseases.

Background

Degenerative Disc Disease (DDD) is the cause of chronic lower back pain, a pathological condition that accompanies lower back pain that is caused by intervertebral disc rupture and cracking due to degenerative or shortened discs that age and dehydrate, particularly in the nucleus pulposus. Degeneration of the intervertebral disc is characterized by abnormal nerve growth and vascular proliferation, as well as changes in cell number and function (cell clustering, necrosis, apoptosis, etc.). One of the molecular characteristics of a degenerated disc is a reduction in aggrecan. Aggrecan plays an important role in the weight bearing of the disc, and loss of aggrecan results in a decrease in disc osmotic pressure and thus the loss of water, thus exacerbating the degeneration of the existing disc, which comprises the presence of the annulus fibrosus and has a major impact on other spinal structures and functions, such as degeneration and hypertrophy of the facet joints and ligamentum flavum.

As current therapies for treating pathological chronic lower back pain including degenerative disc disease, there are medical procedures including analgesia, rehabilitation training therapy and the like. Unfortunately, these treatments suffer from frequent recurrence of the disease, require long and significant effort to treat the associated disease, and risk of possible complications due to prolonged drug treatment.

If there is no good effect even after treatment of the disease using such long-term, conservative treatment, the patient will inevitably receive surgical treatment. Typical surgical treatments include conventional lumbar fusion procedures involving the thorough removal of affected disc tissue and the insertion of bone grafts to the target injury site, as well as recently designed artificial disc insertions. However, these surgical approaches have various drawbacks, such as being relatively expensive and also having the potential for surgical-induced early and late surgical complications. For example, lumbar fusion procedures often require periodic redo due to degeneration of adjacent discs. Artificial discs developed to reduce this disadvantage have not provided satisfactory results in long-term follow-up studies. Therefore, no artificial disc surgery is currently performed. As described above, it is very difficult to treat chronic lower back pain caused by degenerative disc disease. To address this situation, various experimental therapies have been attempted as alternatives to conservative and surgical therapies to achieve disc regeneration while minimizing degeneration of the disc itself.

In recent years, several biological therapies for the treatment of disc degeneration have been tried, for example, methods of up-regulating the production of important matrix proteins such as aggrecan, methods of down-regulating catabolism induced by proinflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-alpha) (Ahn, SH et al, Spine 27: 911-, 2005).

These biological treatment methods have been mainly performed abroad. A popular approach of great interest is the direct injection of bone growth factors (bone morphogenetic proteins, BMPs) into the disc or the transplantation of disc cells injected with therapeutic genes (Masuda K et al, Spine 31: 742-. However, this method is only a method for achieving physical change of the structure of the intervertebral disc through regeneration of the human body, and does not alleviate or relieve pain of a patient, and may cause deterioration of a neurological state due to pressing nerves if the intervertebral disc is overgrown.

Meanwhile, TGF-beta 1 signaling pathway is known to be associated with fibrosis, apoptosis, angiogenesis, invasion and metastasis of tumor cells, and thus inhibition of TGF-beta 1 signaling pathway may be a viable measure for the treatment of body organ fibrosis, cancer and/or glomerulosclerosis (Prud' homme GJ, Lab Invest 87: 1077-.

For this reason, there is a need to develop a new biomaterial which is effective for degenerative disc diseases by promoting disc regeneration while minimizing degeneration of the disc itself, and is capable of treating body organ fibrosis, cancer, glomerulosclerosis, or the like by inhibiting TGF- β 1 signaling pathway.

Disclosure of Invention

Technical problem

The present invention intends to provide a novel peptide capable of promoting disc regeneration while minimizing degeneration of the disc itself.

Further, the present invention intends to provide an effective composition for treating fibrosis of body organs, cancer, or glomerulosclerosis.

Technical scheme

The present invention provides a polypeptide comprising SEQ ID NO:1 (LQVVYLH) or a variant or a pharmaceutically acceptable salt thereof.

In SEQ ID NO:1, L, Q, V, Y and H represent leucine (Leu), glutamic acid (Gln), valine (Val), tyrosine (Tyr) and histidine (His), respectively.

The respective amino acid components of the peptide may be in L-form, D-form, and/or DL-form, which are included in the amino acid component of the peptide of the present invention.

A variant is a form in which the structure of the peptide of the present invention is partially altered due to spontaneous or artificial changes without causing any alteration in the main activity of the peptide. For example, it may be a form in which SEQ ID NO:1 by substitution of one or more amino acids of glutamine, tyrosine and histidine with other amino acids. A preferred form is the form set forth in SEQ ID NO:1 with asparagine, phenylalanine or tryptophan, and/or lysine or arginine for histidine. Glutamine and asparagine belong to the group of amino acids that contain a terminal amide group. Tyrosine, phenylalanine and tryptophan belong to the group of aromatic amino acids containing aromatic side chains. Histidine, lysine and arginine belong to the group of basic amino acids containing strongly polar side chains which make them highly hydrophilic. The same group of amino acids is considered to have the same or similar biochemical characteristics (size, shape, charge, ability to form hydrogen bonds, or chemical reactivity).

The peptides and variants thereof may have the general formula (I):

L-X₁-VV-X₂-L-X₃ (I)

wherein, X₁Is Q or N; x₂Is Y, F or W; x₃Is H, K or R; l is leucine, Q is glutamine, N is asparagine, V is valine, Y is tyrosine, F is phenylalanine, W is tryptophan, H is histidine, K is lysine and R is arginine.

Examples of pharmaceutically acceptable salts may include hydrochloride, sulfate, phosphate, lactate, maleate, fumarate, oxalate, mesylate, p-toluenesulfonate and the like.

Further, the present invention provides a pharmaceutical use of the peptide of the present invention or a variant or a pharmaceutically acceptable salt thereof. The medical applications include therapeutic and/or prophylactic applications for degenerative disc disease, therapeutic applications for body organ fibrosis, therapeutic applications for cancer, and therapeutic applications for glomerulosclerosis. Treating fibrosis of a body organ, cancer, or glomerulosclerosis by inhibiting the signaling pathway of transforming growth factor beta 1 (TGF-beta 1).

TGF- β is known to be a highly pleiotropic cytokine that plays important roles in apoptosis regulation, angiogenesis, wound healing, immunomodulation and tumor biology. There are three subtypes of TGF- β: TGF-. beta.1, TGF-. beta.2, and TGF-. beta.3. The same receptor was used for all three subtypes. The TGF- β receptor has three components: type I (RI or ALK5), type II (RII) and type III (RIII or β glycans). TGF-. beta.s (all subtypes) bind to and recruit RIII, which is then phosphorylated to form a heterotetrameric serine/threonine protein kinase complex. RI in turn phosphorylates Smad2 and Smad3 (receptor-associated Smads (R-Smads)), and then forms a heteromeric complex with Smad4, which translocates into the nucleus to bind to DNA and regulate transcription (Prud' homme GJ, Lab Invest 87: 1077-1091, 2007).

The present invention uses the term "inhibiting TGF- β 1 signaling pathway" to mean that TGF- β 1 fails to bind to the receptor, then Smad2 and Smad3 fail to undergo phosphorylation and therefore fail to form a complex with Smad4, such that the complex fails to translocate into the nucleus and regulate transcription.

The present invention provides a composition for treating and/or preventing degenerative disc disease, comprising the peptide of the present invention or a variant or pharmaceutically acceptable salt thereof.

Further, the present invention provides a composition for treating fibrosis in a body organ, the composition comprising the peptide of the present invention or a variant or a pharmaceutically acceptable salt thereof.

Further, the present invention provides a composition for treating cancer, the composition comprising the peptide of the present invention or a variant or a pharmaceutically acceptable salt thereof.

Further, the present invention provides a composition for treating glomerulosclerosis, the composition comprising a peptide of the present invention or a variant or a pharmaceutically acceptable salt thereof.

The peptide of the present invention can be prepared by a method commonly used for peptide synthesis. For example, The peptide can be prepared by those methods described by Schroder and lubk (The Peptides, vol.1, Academic Press, New York, 1965) and The like, and can be prepared by liquid phase synthesis or solid phase synthesis.

Examples of the method of forming a peptide bond include an azide method, an acid chloride method, a symmetrical acid anhydride method, a mixed acid anhydride method, a carbodiimide-additive method, an activated ester method, a carbonyldiimidazole method, an oxidation-reduction method, and a method using a wood wad reagent K. In the synthesis of peptides, a cystine group can be formed by using a cystine derivative, or after a peptide chain is formed by a conventional method, a cysteine group in the peptide chain is converted into a cystine group.

Before the synthesis reaction is carried out, a carboxyl group, an amino group, a guanidino group, a hydroxyl group, etc. which do not participate in the reaction can be protected, and the carboxyl group and the amino group which participate in the synthesis reaction can be activated by a method known in the art.

Examples of the protecting group for the carboxyl group may include ester-forming groups such as methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl and cyclohexyl.

Examples of the protecting group for an amino group may include a benzyloxycarbonyl group, a tert-butoxycarbonyl group, an isobornyloxycarbonyl group and/or a 9-fluorenylmethoxycarbonyl group.

Examples of the protective group for a guanidino group may include a nitro group, a benzyloxycarbonyl group, a tosyl group, a p-methoxybenzenesulfonyl group and/or a 4-methoxy-2, 3, 6-trimethylbenzenesulfonyl group.

Examples of the protective group for the hydroxyl group may include a tert-butyl group, a benzyl group, a tetrahydropyranyl group and/or an acetyl group.

Examples of carboxy activated forms may include symmetrical anhydrides, azides, and activated esters (esters formed with alcohols such as pentachlorophenol, 2, 4-dinitrophenol, hydroxyacetonitrile, p-nitrophenol, N-hydroxy-5-norbornene-2, 3-dicarboximide, N-hydroxysuccinimide, N-hydroxyphthalimide, and 1-hydroxybenzotriazole).

An example of an activated amino group is an amine phosphate.

The reaction is carried out in a solvent such as chloroform, dichloromethane, ethyl acetate, N-dimethylformamide, dimethyl sulfoxide, pyrimidine, dioxane, tetrahydrofuran, water, methanol or a mixture thereof.

The temperature of the reaction may be in the range generally employed for the reaction of about-30 to 50 ℃.

The reaction for removing the protecting group of the peptide of the present invention may vary depending on the type of the protecting group, but should be a reaction capable of removing the protecting group without giving any influence to the peptide bonding.

The protecting group can be removed by acid treatment, for example with hydrogen chloride, hydrogen bromide, hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or mixtures of these acids. Further, reduction can be performed with metallic sodium in liquid ammonia or catalytic reduction with palladium on carbon.

In the reaction for removing the protecting group by the above acid treatment, an additive such as anisole, phenol or thioanisole may be used.

After completion of the reaction, the peptide prepared by the present invention can be recovered by a conventional process for peptide purification, for example, extraction, partition, reprecipitation, recrystallization or column chromatography.

Further, the peptide of the present invention can be converted into a variant of the peptide as described above, or a pharmaceutically acceptable salt of the peptide, using a conventional method.

The peptides of the invention may be synthesized by an automated peptide synthesizer, or may be generated by genetic engineering techniques. For example, it is possible to prepare a fusion gene encoding a fusion protein composed of a fusion partner and the peptide of the present invention by genetic manipulation, transfecting a host microorganism with the fusion gene, expressing the desired peptide in the form of a fusion protein in the host microorganism, and separating and isolating the peptide of the present invention from the fusion protein with a protease or a compound.

The amino acids used in the present invention are abbreviated according to the IUPAC _ IUB nomenclature as follows:

the dosage of the peptide or variant or pharmaceutically acceptable salt thereof is in the range of 50 μ g to 1mg per day, preferably 0.5mg to 1mg per day for parenteral administration. For oral administration, the dose is 1.2 to 1.5 times the parenteral dose. For rectal administration, the dose is 2 to 5 times the parenteral dose. The peptides of the invention are mainly administered by parenteral routes, such as local injection (intra-discal injection for degenerative disc diseases, and local intralesional injection for fibrosis of other body organs and cancer), intravenous/intramuscular or subcutaneous injection, intracerebroventricular or intraspinal administration or nasal and intrarectal administration. Further, oral administration may be employed if desired.

The peptide or composition of the present invention, in combination with a pharmaceutically acceptable carrier, can be formulated into desired dosage forms, such as injections, suppositories, powders, nasal drops, granules, tablets and the like.

Pharmaceutically acceptable carriers can be prepared according to several factors well known to those skilled in the art, for example, taking into account the following non-limiting factors: the particular physiologically active material to be used and its concentration, stability and intended bioavailability; a disease, disorder or condition being treated; the subject being treated and their age, weight and overall condition; and the intended route of administration of the composition, e.g., nasal, oral, ocular, topical, transdermal and intramuscular. Generally, examples of pharmaceutically acceptable carriers for administration of physiologically active materials other than oral administration routes may include D5W (5% glucose in water), an aqueous solution containing 5% by volume or less of glucose, and physiological saline. In the case of local intralesional injection, various injectable hydrogels can be used to enhance the therapeutic effect and increase the duration of the drug effect. In addition, the pharmaceutically acceptable carrier may contain additional ingredients capable of enhancing the stability of the active ingredient, such as preservatives and antioxidants. Preferably, the peptides or compositions of the invention are formulated into the desired dosage form by any suitable method known in the art, depending on the disease to be treated and the components of the composition, for example as disclosed in "Remington's Pharmaceutical Sciences" (Mack Publishing co., Easton, PA (latest edition)).

The peptides of the invention can be stored in physiological saline solution and, after addition of mannitol and sorbitol, can be freeze-dried in ampoules. The lyophilized peptide may be dissolved in physiological saline or the like for reconstitution before use.

Further, the present invention provides a peptide or a variant or a pharmaceutically acceptable salt thereof as a medicament.

Further, the present invention provides a peptide or a variant or a pharmaceutically acceptable salt thereof for use as a medicament for the treatment and/or prevention of degenerative disc disease, fibrosis of body organs, cancer and/or glomerulosclerosis.

Further, the present invention provides a method for treating and/or preventing degenerative disc disease, body organ fibrosis, cancer and/or glomerulosclerosis, comprising administering to a subject (mammal, including human) a peptide of the present invention or a variant or a pharmaceutically acceptable salt thereof.

Fibrosis of a body organ, cancer, and/or glomerulosclerosis may be treated by inhibiting TGF-beta 1 signaling pathways.

Advantageous effects

The novel peptide or a variant or a pharmaceutically acceptable salt thereof of the present invention is effective in treating and/or preventing degenerative disc disease, body organ fibrosis, cancer and/or glomerulosclerosis, and is effective in inhibiting TGF-beta 1 signaling pathway.

Drawings

Fig. 1 shows pictures taken after staining of normal disc tissue, degenerative disc tissue given DMSO, and degenerative disc tissue given the peptide of example 1.

Fig. 2 shows a graph representing the expression levels of aggrecan genes of a degenerated disc group administered with DMSO and a degenerated disc group administered with the peptide of example 1 in a disc degeneration model compared with a normal disc group as a reference.

FIG. 3 shows the results of Western blot analysis to demonstrate phosphorylated Smad2 expressed in untreated HepG2 cells, in TGF- β 1 treated cells, in TGF- β 1/SB431542 treated cells, in TGF- β 1/peptide treated cells of example 1, and in TGF- β 1/DMSO treated cells.

Detailed Description

Now, the present invention will be described in more detail with reference to the following examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

Example 1: preparation of peptides

Peptron corporation (korea) prepared a peptide having SEQ ID NO:1 (LQVVYLH: SEQ ID NO: 1). Specifically, amino acid units were linked one by one from the C-terminus by Fmoc SPPS (9-fluorenylmethyloxycarbonyl solid-phase polypeptide synthesis method) using an automated peptide synthesizer (ASP48S, Peptron Co.).

By NH₂-His (Trt) -2-chloro-trityl resin, wherein the first amino acid at the C-terminus of the peptide is attached toOn a resin. All amino acids used for synthesizing peptides are protected with trityl (Trt), t-butyloxycarbonyl (Boc), t-butyl (t-Bu), etc., so that the N-terminus is protected with 9-fluorenylmethoxycarbonyl and all residues are removed in acid. 2- (1H-benzotriazole-1-yl) -1, 1, 3 '3' -tetramethylthionurehexafluorophosphate (HBTU)/hydroxybenzotriazole (HOBt)/N-methylmorpholine (NMM) was used as a coupling agent. (1) The protected amino acid (8 eq) and the coupling agent HBTU (8 eq)/HOBt (8 eq)/NMM (16 eq) were dissolved in Dimethylformamide (DMF) and reacted for 2 hours at room temperature. (2) The 9-fluorenylmethyloxycarbonyl group was removed by adding 20% piperidine in DMF followed by a reaction at room temperature for 5 minutes and repeated twice. Reactions (1) and (2) were repeated to prepare the basic peptide backbone and with trifluoroacetic acid (TFA)/1, 2-Ethanedithiol (EDT)/phenylthiomethane/Triisopropylsilane (TIS)/H₂The peptide was separated from the resin at 90/2.5/2.5/2.5/2.5. The peptides were purified by reverse phase high pressure liquid chromatography using a Vydac Everest C18 column (250mm 22mm, 10 μm) and then separated by a linear water-acetonitrile gradient (10-75% (v/v) acetonitrile) containing 0.1% (v/v) trifluoroacetic acid (elution). The molecular weight of the purified peptide was confirmed using LC/MS (Agilent HP1100 series) and then lyophilized.

Example 2: confirmation of the regenerative Effect of intervertebral discs

2-1, preparation of animal model of intervertebral disc degeneration and collection of experimental intervertebral discs

30 rabbits weighing 3 to 3.5kg (New Zealand white rabbits, Orient Bio Inc.) were prepared, both male and female.

Rabbits were anesthetized by intramuscular injection of 5mg/kg xylazine (Rompun, Bayer) and 35mg/kg ketamine hydrochloride (Ketalar, Pfizer). Prior To the start of the procedure, a lateral X-ray film was taken with a fluoroscopy device (model number VPX-200; To shiba, Inc.) To establish the pre-injection baseline height of the disc. Baseline control refers to the standard used for the measurement of the intervertebral disc space. After placing the rabbits on the laboratory table, the intervertebral disc segments L23, L34, L45 and L56 were confirmed with the instrument and the annulus fibrosus was penetrated to the posterior side of the intervertebral disc segments L23, L45 and L56 with an 18G needle. After the animals were resuscitated from anesthesia, they were kept in cages under the following conditions: temperature from 20 to 25 ℃, humidity from 10% to 50%, and light/dark (L/D) cycle: (08:00a.m. to 20:00p.m. are clear). All animals were fed once daily. X-ray films were taken at 2 and 4 weeks after the initial puncture. X-ray films were taken after anesthesia. From the results of the X-ray film, the disc height (IVD height) was measured. From the results of the measurements, the degree of degeneration of the intervertebral disc was quantified by modification of the method disclosed by Lu et al (spine.22: 1828-34, 1997).

Thereafter, two independent groups, a control group given DMSO and an experimental group given the peptide of example 1, were tested and rabbits were euthanized by injection of ketamine (25mg/kg) and sodium pentobarbital (1.2g/kg, Nembutal, Ovation) according to the planned schedule, and then the discs were removed for histological and biochemical analysis, respectively.

2-2, measuring the regeneration effect of intervertebral discs by staining the tissues of intervertebral discs

Rabbits with degenerative disc in the 2-1 segment were divided into two groups. One group was given dimethyl sulfoxide (DMSO) (0.5 mM/min) by two local intradiscal injections, and one group was given the peptide of example 1 (0.5 mM/min) by two local intradiscal injections. The time point for administration for each group was 4 weeks after induction of disc degeneration and 2 weeks further. After the second dose, animals were housed for 2, 4 and 8 weeks, respectively. At 4, 6 and 10 weeks after the initial administration of the peptide of example 1 and DMSO, respectively, the corresponding individual disc tissues were removed and fixed in formalin. The fixed disc tissue was embedded in the stone 30863 and then serial sections were prepared with a thickness of 4 μm along the sagittal plane. Two of these sections were stained with hematoxylin and eosin (H & E). For comparison with normal disc tissue, discs were removed from rabbits induced by disc degeneration, treated and stained according to the same method as described above.

FIG. 1 is a microscopic result of a single disc tissue removed and stained at week 10. A and B: normal disc tissue, C and D: degenerated disc tissue given DMSO, and E and F: degenerated disc tissue was administered with the peptide of example 1. A. C and E: 40 times magnification, B, D and F: magnification of 400 times. In the 400-fold magnified picture, the arrows indicate the disc nucleus.

As a result, it was observed that nucleus pulposus and annulus fibrosus were clearly distinguished in normal intervertebral disc cells, and that there were a large number of extracellular matrix components (A and B in FIG. 1). In addition, a unique staining of the nucleus was observed in normal disc tissue (B in fig. 1).

On the other hand, DMSO-administered disc tissue showed disc shrinkage, confusion between the annulus fibrosus and nucleus pulposus, and a rarity of extracellular matrix components (C and D in fig. 1). Further, it is difficult to find a stained nucleus in the nucleus pulposus region (D in fig. 1). That is, these results indicate that what was ever present in the nucleus pulposus was dead cells. It is known that cell death is due to disc degeneration and that the lack of cells results in the production of no extracellular matrix components, thus further exacerbating disc degeneration.

The disc tissue administered with the peptide of example 1 showed an enlarged disc, showed distinguishable nucleus pulposus and annulus fibrosus, and had a larger amount of extracellular matrix components (E and F in fig. 1) compared to the disc tissue administered with DMSO. In addition, a vivid staining of the nucleus was observed in the nucleus pulposus region (F in fig. 1).

These results demonstrate that the peptide of example 1 has the effect of treating intervertebral discs by preventing the reduction of extracellular matrix components and cell death due to degeneration of intervertebral discs.

Example 3: confirmation of enhanced Gene expression of aggrecan in intervertebral disc tissue

Real-time PCR was performed to detect the expression level of aggrecan gene, which is a representative extracellular matrix component in the disc tissue.

Animals were prepared in the same manner as in example 2-1 and divided into two groups, one group administered DMSO (0.5 mM/mouse) by local intra-discal injection and one group administered the peptide of example 1 (0.5 mM/mouse) by local intra-discal injection. The time points for administration for each group were 4 weeks after induction of disc degeneration and 2 weeks further, and animals were housed for 2, 4 and 8 weeks after the second administration, respectively. At weeks 4, 6 and 10 after the initial administration of the peptide of example 1 and DMSO, respectively, the corresponding individual disc tissues were removed, the nucleus pulposus and the annulus fibrosus were separated and placed in a test tube, and then flash-frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at-70 ℃.

Total RNA was extracted from the flash frozen and stored disc tissue with Trizol reagent (Invitrogen). cDNA was synthesized using extracted total RNA (2. mu.g), oligodeoxynucleotide (oligo dT), and MMLV-reverse transcriptase (Invitrogen).

The amounts of GAPDH and aggrecan mRNA were measured by Prism 7900HT (ABI) using Power SYBR Green PCR Master Mix (Applied Biosystems). 25ng of cDNA, 3. mu.l of 10. mu.M primer and 2 XPowerSYBR Green PCR Master Mix were mixed to make a total volume of 10. mu.l. Real-time PCR was performed under the following reaction conditions: 2 min at 50 ℃ and 10 min at 95 ℃ to induce enzyme activity; then 45 cycles were carried out, each cycle consisting of a reaction at 95 ℃ for 15 seconds and a reaction at 60 ℃ for 1 minute; then each threshold Cycle (CT) value is measured. GAPDH was selected as a control gene and the difference in CT values (Δ CT) between the control gene and aggrecan was calculated. In addition, the difference in CT value (Δ Δ CT)' between the normal disc and the disc to which the peptide of example 1 was administered (or the disc to which DMSO was administered) was also calculated. Then, calculate 2^(-ΔΔCT)And expressed as a percentage relative to normal disc values.

FIG. 2 shows the results of real-time PCR. Fig. 2 is a graph showing the change over time in the expression level of aggrecan gene of the DMSO-administered disc group and the example 1 peptide-administered disc group in the disc degeneration model, compared with the normal disc group as a reference. As shown in the above figure, it can be seen that the disc tissue to which the peptide of example 1 was administered showed an increase in aggrecan gene expression at 4 weeks as compared with the disc tissue to which DMSO was administered. At 6 and 10 weeks, the disc tissue administered the peptide of example 1 showed aggrecan expression levels similar to those of the disc tissue administered DMSO. Since the peptide of example 1 was administered to the animal only at 0 and 2 weeks and then not administered, it can be said that the enhancement of aggrecan gene expression at 4 weeks was due to the potency of the peptide of example 1, whereas the potency of the peptide of example 1 did not maintain aggrecan gene expression for 6 and 10 weeks. From these results, it can be seen that the peptide of the present invention shows a disc regeneration effect by enhancing the gene expression of aggrecan, which is a representative extracellular matrix component of disc tissues that is very important for disc regeneration; and the duration of the effect of the peptide on the enhancement of the expression of the aggrecan gene is not too long, thereby excluding possible side effects due to the excessive enhancement of the expression of the aggrecan gene.

Example 4: confirmation of TGF-beta 1 signaling pathway inhibition

The inhibition of the TGF-. beta.1 signaling pathway by the peptide of example 1 was confirmed by the following experimental method.

It is known that treatment of HepG2 cells with TGF-. beta.1 leads to apoptosis during which Smad2 is first phosphorylated (Park TJ. et al., Mol Carcinog.47: 784-. Using these properties, experiments were performed as follows. Will be 1x10⁶HepG2 cells (ATCC; American Standard Collection of biologicals) were seeded in 60mm dishes, stabilized overnight, and then cultured in serum-free medium (SFM) for 24 hours to deplete nutrients. 5ng/mL of TGF-. beta.1 (PromoKine, Germany) and the above peptides (1, 5 and 25. mu.M) were preincubated at 37 ℃ for 1 hour prior to treating the cells with the peptides of example 1. Furthermore, DMSO (2. mu.l/mL) was also preincubated with TGF-. beta.1 (5ng/mL) at 37 ℃ for 1 hour. Then, the cells were treated with the pre-incubated solution for 30 minAnd then extracting the protein. In addition, cells were pretreated with 10. mu.M SB431542(TOCRIS, USA) only, which is an inhibitor of TGF-beta receptor, followed by incubation for 1 hour, and then treatment with TGF-. beta.1 (5ng/mL) for 30 minutes. The cells were then lysed in Radioimmunoprecipitation (RIPA) buffer (Millipore) {50mM Tris-HCl (pH 7.4), 150mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1mM ethylenediaminetetraacetic acid (EDTA), 1mM phenylmethylsulfonyl fluoride (PMSF), 40mM NaF, 1mM Na₃VO₄1mM Dithiothreitol (DTT) } and mixed on ice. The homogenate was sonicated 5 times with a bran sonofer 450 with output control of 2.56, duty cycle (%) of 20, time set to 6. The cell lysate was centrifuged at 12000rpm for 10 minutes at 4 ℃ and the supernatant was used for Western blot analysis. Protein concentration was determined by the Bradford method. Mu.g of protein was added to SDS sample buffer containing 2-mercaptoethanol. After standing at 95 ℃ for 5 minutes, the resulting mixture was separated by 10% SDS-PAGE, and then subjected to Western blot analysis. For Western blot analysis, the isolated proteins were transferred to nitrocellulose membrane (Bio-Rad Lab) and blocked with PBS-T containing 5% skim milk, followed by reaction with primary antibody diluted 1: 3000 with PBS-T containing 5% skim milk at 4 ℃ overnight. The membrane was then washed three times for 5 minutes each with PBS-T, treated with 5% skim milk in PBS-T, horseradish peroxidase (HRP) -conjugated anti-rabbit secondary antibody (Bio-Rad Lab, 1706515) diluted 1: 5000 at room temperature for 1 hour, and developed with ECL (Amersham pharmacia). Since TGF- β 1 binds to TGF- β receptor while Smad2 is first phosphorylated, phospho-Smad 2(ser465/467) antibody (Cell Signaling, 3101, 8) capable of detecting phosphorylated Smad2 was used as the primary antibody.

The results are shown in fig. 3. FIG. 3 shows the results of Western blotting (lane 1: untreated HepG2 cells; lane 2: TGF-. beta.1 treated cells; lane 3: TGF-. beta.1/SB 431542 treated cells; lanes 4, 5 and 6: cells treated with 1, 5 and 25. mu.M peptide/TGF-. beta.1, respectively; and lane 7: TGF-. beta.1/DMSO treated cells). In fig. 3, the symbol '+' represents treated with the subject material, and '-' represents untreated with the subject material. The bottom of figure 3 shows the coomassie blue staining results of the membranes used in western blots, indicating that the amount of protein in all lanes is the same.

Referring to fig. 3, lane 1 shows very little phosphorylation of proteins extracted from untreated HepG2 cells, while lane 2 shows significant phosphorylation of proteins due to TGF- β 1. In addition, it was observed that lane 3 showed that SB431542 completely inhibited phosphorylation. When treated with the peptides of example 1 at concentrations of 1. mu.M, 5. mu.M and 25. mu.M, respectively, it was confirmed that the degree of phosphorylation decreased in a dose-dependent manner. Lane 7, which was treated with DMSO, shows the same profile as that of TGF-. beta.1 treatment.

From these results, it can be seen that since the peptides of the present invention show dose-dependent inhibition of the TGF-. beta.1 signaling pathway, diseases curable by the above-mentioned inhibition of the TGF-. beta.1 signaling pathway, such as body organ fibrosis, cancer, and/or glomerulosclerosis (Prud' homme GJ, Lab Invest 87: 1077-. Further, it can be seen that the peptide of example 1, does not completely inhibit the TGF- β 1 signaling pathway as does SB 431542. Since the TGF-. beta.1 signaling pathway is an important regulatory mechanism in humans, complete inhibition of the TGF-. beta.1 signaling pathway, as with SB431542, can lead to side effects. However, the peptides of the invention attenuate the TGF- β 1 signalling pathway in a dose-dependent manner and therefore when the peptides are used to treat a relevant disease, the concentration of the peptides can advantageously be adjusted, thereby reducing possible side effects.

Industrial applications

The novel peptide or a variant or a pharmaceutically acceptable salt thereof of the present invention is effective for the treatment and/or prevention of degenerative disc disease, body organ fibrosis, cancer and/or glomerulosclerosis, and is effective in inhibiting TGF- β 1 signaling pathway, and thus is industrially applicable.

Claims (12)

1. 1, a peptide having the amino acid sequence of SEQ ID NO.

2. A composition for treating and preventing degenerative disc disease, comprising the peptide of claim 1.

3. A composition for treating fibrosis in a body organ, the composition comprising the peptide of claim 1.

4. The composition of claim 3, wherein the treatment of fibrosis in a body organ is by inhibition of the TGF- β 1 signaling pathway.

5. A composition for treating cancer, the composition comprising the peptide of claim 1.

6. The composition of claim 5, wherein the treatment of cancer is by inhibiting TGF- β 1 signaling pathway.

7. A composition for treating glomerulosclerosis comprising the peptide of claim 1.

8. The composition of claim 7, wherein the treatment of glomerulosclerosis is by inhibition of the TGF- β 1 signaling pathway.

9. Use of a peptide according to claim 1 for the preparation of a medicament for the treatment and prevention of degenerative disc disease.

10. Use of a peptide according to claim 1 for the preparation of a medicament for the treatment of fibrosis in a body organ.

11. Use of a peptide according to claim 1 for the manufacture of a medicament for the treatment of cancer.

12. Use of a peptide according to claim 1 for the preparation of a medicament for the treatment of glomerulosclerosis.