KR101306157B1 - Compositions for Preventing or Treating Tumors Comprising Cyclopentapeptides - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of tumor or acromegaly, comprising cyclopentapeptide as an active ingredient.
The inventors found that cyclopentapeptides having the amino acid sequence of Gly1-Tyr2-Arg3-Arg4-Nal5, a CXCR4 antagonist, play an important role in tumor expansion and GH secretion of the pituitary gland. Therefore, the present invention can be usefully used as an active ingredient of a new anticancer agent that targets signaling mechanisms different from the conventional signaling mechanisms and blocks the proliferation of various tumors including the pituitary tumor and induces cell death of tumor cells.
In addition, the present invention can be usefully used as an effective therapeutic composition for acromegaly by inhibiting the production and secretion of growth hormone in pituitary tumor cells that excessively secrete growth hormone.

Description

Composition for Preventing or Treating Tumors Comprising Cyclopentapeptides as Active Ingredients {Compositions for Preventing or Treating Tumors Comprising Cyclopentapeptides}

The present invention relates to a composition for preventing or treating tumors comprising cyclopentapeptide as an active ingredient.

The chemokine SDF-1 (stromal cell-derived factor 1) and its receptor, CXCR4, play an important role in the production and growth of growth hormone (GH) in normal and neoplastic pituitary stimulatory cells. Thus, the chemokine receptor CXCR4 can be an attractive target of anti-tumor agents for acromegaly patients. Synthetic antagonists of cyclic penta-peptide d-Arg3FC131 (c [Gly1- d-Tyr2-d-Arg3-Arg4-Nal5]) of CXCR4 is significantly inhibited the proliferation of GH production and the aqueous phase GH3 cells stimulated in vitro. It also induces apoptosis of GH3 cells by activating the caspase-3 pathway. Systemic administration of d-Arg3FC131 inhibited the growth of tumor cells by inhibiting the growth of GH3 cells transplanted into immunodeficient nude mice through apoptosis induction. These results suggest that d-Arg3FC131 may be a therapeutic agent for pituitary tumors producing excess GH in patients with acromegaly. Pituitary tumors associated with acromegaly are rarely malignant, but high levels of GH and insulin-like growth factor (IGF) can lead to a variety of cardiovascular, respiratory, endocrine and metabolic diseases (1, 2). . Surgical surgery to remove all tumor tissue is the only way to completely treat patients with acromegaly; However, second-line therapies are usually required when managing patients with bulky tumors after surgery or dealing with relapsed patients (3). Three drugs are currently used for the treatment of acromegaly: dopamine agonists, somatostatin receptor ligands (SRLs) and GH receptor antagonists. Treatment with SRL alone induced 60-70% biochemical regulation of serum IGF-1 (4). The combination of SRL and GH receptor antagonists resulted in 95% IGF-1 normalization, but high cost remains a problem (3, 5, 6). If new pharmacological reagents are developed that target different signaling mechanisms in pituitary tumors, they could offer a variety of approaches that combine with current drugs for the treatment of GH-releasing tumors.

Chemokine SDF1 (stromal cell-derived factor-1) and its receptor, CXCR4, are used for the trafficking of hematopoietic precursor cells, infection of human immunodeficiency virus, migration and metastasis of cancer cells, and development of rheumatoid arthritis. It has been reported to be involved in (7-11). SDF1 is known to be expressed in many organs, including the brain, heart, lung, thymus, kidney, spleen, stomach, small intestine and bone marrow of mice. CXCR4 is expressed as broadly as SDF1, especially in nervous tissues (12). SDF1 has been cloned from murine myeloid mesenchymal cells (13), and its amino acid sequence is highly conserved during evolution, suggesting that the site plays an important biological role. SDF1 / CXCR4 interactions include organ-specific growth, survival and breast cancer, kidney cancer, prostate cancer, lung cancer, pancreatic cancer, melanoma, neuroblastoma, non-Hodgkin's lymphoma, multiple myeloma, ovarian cancer and It plays an essential role in the metastasis of malignant tumors, including malignant brain tumors (14-18). Expression of SDF1 and CXCR4 in neurons, astrocytes, microglia and endothelial cells of the central nervous system and anterior pituitary gland suggests that there may be a link between these chemokines and neuroendocrine regulation (12, 20, 21) . We have found that SDF1 and CXCR4 are expressed in the pituitary cells 22 and human pituitary cells 23 of normal and cancerous rats. SDF1 / CXCR4 interactions stimulated Erk1 / 2 phosphorylation and tocaine GH3 cell proliferation stimulated normal and secretion (22), which is consistent with previous studies (19, 24).

To date, inhibition of CXCR4 for the treatment of cancer has shifted from preclinical studies to clinical practice in some malignancies. However, only a few studies have reported the effects on pituitary tumors of CXCR4 antagonists. There are many classes of CXCR4 antagonists including AMD3100, T22 and T140. Each antagonist has a variety of mechanisms by different pharmacokinetics and pharmacodynamics. In T140 [Arg1-Arg2-Nal3-c (Cys4-Tyr5-Arg6-Lys7-d-Lys8-Pro9-Tyr10-Arg11-Cit12-Cys13) -Arg14], one of the potent CXCR4 antagonists, Arg2, Nal3, Tyr5 and Arg14 Residues such as are the most important residues for maintaining antagonistic activity against CXCR4 (25). Since T140 is not stable in mouse serum or rat liver, essential residues (Arg14 in serum; Arg2, Nal3 and Arg14 in liver homogenates) are also inevitably deleted, resulting in a dramatic decrease in efficacy (26, 27). To overcome the structural instability of T 140, we minimized the molecular size and designed a cyclic pentapeptide library. The structure of the cyclo (-Arg1-Arg2-Nal3-Gly4-DTyr5-) was obtained, which showed a potent CXCR4-antagonistic activity similar to T140 (25, 28). Accordingly, the present inventors have found that CXCR4 antagonist cyclic pentafetide FC131 [c (Gly1-d-Tyr2-Arg3-Arg4-Nal5) and its isomeric peptides effectively inhibit the proliferation of pituitary adenoma cells and inhibit the secretion of GH. Assumed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop efficient peptide therapeutics for various tumors, including pituitary tumors. As a result, the present invention was found by the discovery that the cyclopentapeptide consisting of the amino acid sequence of Gly1-Tyr2-Arg3-Arg4-Nal5 inhibits the proliferation, migration and metastasis of cancer cells and induces cell death through antagonism against CXCR4. It was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.

Another object of the present invention to provide a pharmaceutical composition for preventing or treating terminal hypertrophy.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the present invention provides a pharmaceutical composition for the prevention or treatment of tumors comprising a cyclopentapeptide (cyclopentapeptide) consisting of the amino acid sequence of Gly1-Tyr2-Arg3-Arg4-Nal5 as an active ingredient.

The present inventors have made extensive efforts to develop efficient peptide therapeutics for various tumors, including pituitary tumors. As a result, the cyclopentapeptide consisting of the amino acid sequence of Gly1-Tyr2-Arg3-Arg4-Nal5 antagonizes CXCR4. It has been found to inhibit the proliferation, migration and metastasis of tumor cells and induce cell death. CXCR4 is one of the most widely expressed chemokine receptors in human tumor cells, and SDF1 / CXCR4 interactions lead to tumor proliferation, migration, invasion and metastasis as well as tumor angiogenesis (4, 12, 13). ).

According to the present invention, the cyclopentapeptide of the present invention inhibits the production and secretion of growth hormone (GH) in tumor cells, inhibits the proliferation of tumor cells, arrests the cell cycle at G1 checkpoints, and grows tumor cells. In addition to inhibiting the activity, it induces apoptosis of tumor cells. Therefore, the cyclopentapeptide of the present invention can be an effective anticancer composition targeting CXCR4 signaling mechanism.

As used herein, the term “peptide” refers to a linear or cyclic molecule formed by binding amino acid residues to each other by peptide bonds.

As used herein, the term “cyclopentapeptide” refers to a cyclic molecule consisting of five amino acid residues, and Gly1-Tyr2-Arg3-Arg4-Nal5 cyclopentapeptide is Gly, Tyr, Arg, Arg and Nal. It refers to cyclic peptide molecules linked in sequence.

According to a preferred embodiment of the invention, the cyclopentapeptide of the invention is selected from the group consisting of the following formulas (1), (2) and (3):

Formula 1

(2)

(3)

Formula 1 (FC131 [cyclo (Gly1-d-Tyr2-Arg3-Arg4-Nal5)]), 2 (d-Arg3FC131 [cyclo (Gly1-d-Tyr2-d-Arg3-Arg4-Nal5)]) and 3 (d -Arg3-d-Nal5FC131 [cyclo (Gly1-d-Tyr2-d-Arg3-Arg4-d-Nal5)]) is a stereoisomer in which the same five amino acid residues are linked in the same order and differ in their conformation. ).

Peptides of the invention can be prepared according to chemical synthesis methods known in the art, in particular solid-phase synthesis techniques (Merrifield, J. Amer . Chem . Soc . 85: 2149-54 (1963) Stewart, et al., Solid Phase Peptide Synthesis , 2nd. ed., Pierce Chem. Co .: Rockford, 111 (1984)).

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19 th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, or the like. Can be.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . A preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.0001-100 mg / kg on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or may be in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

According to a more preferred embodiment of the present invention, the cyclopentapeptide of the present invention is a cyclopentapeptide of formula (2). According to the present invention, the cyclopentapeptide of formula (2) is a cyclic peptide in which the same amino acid residues as in formulas (1) and (3) are bonded in the same order to indicate a compound in which Tyr No. 2 and Arg No. 3 are Form D.

Tumors to which the compositions of the present invention can be applied include breast cancer, kidney cancer, prostate cancer, lung cancer, pancreatic cancer, melanoma, neuroblastoma, non-Hodgkin's lymphoma, multiple myeloma, ovarian cancer and brain tumor . Preferably, the tumor to which the composition of the present invention is applied is a pituitary tumor.

According to a more preferred embodiment of the present invention, the pituitary tumor of the present invention is a tumor that produces and secretes excess growth hormone and the composition of the present invention reduces the production and secretion of growth hormone of the pituitary tumor. According to the present invention, it can be seen that the composition of the present invention significantly inhibits the expression of GH at the transcription level by decreasing the activity of the promoter of the GH gene by 0.02 fold in GH3 pituitary tumor cells that produce and secrete growth hormone (GH). .

According to a preferred embodiment of the present invention, the composition of the present invention induces apoptosis of tumor cells. As shown in the examples of the present invention, the inventors performed a TUNEL analysis to measure apoptosis in tumor tissues, and as a result, in the tumor cells treated with the composition of the present invention, the apoptosis increased three times as compared to the control group. Was observed. Thus, the composition of the present invention can be used as an effective anticancer agent with high cell death efficiency.

According to a preferred embodiment of the present invention, the composition of the present invention inhibits angiogenesis. In order for cancer tissues to grow, angiogenesis is essential as cells of existing blood vessels proliferate and migrate to form new blood vessels. As confirmed in the embodiment of the present invention, as a result of H & E (Hematoxylin and eosin) staining on the tumor tissue, micro-vessels filled with red blood cells in the tumor tissue of the control group is prominent, but almost in the tumor tissue of the mouse treated with the composition of the present invention By observing the fact that it is not visible (Fig. 6), it was confirmed that the composition of the present invention inhibits angiogenesis of tumor tissue and thereby inhibits the growth of tumor tissue.

According to a preferred embodiment of the present invention, the composition of the present invention arrests the cell cycle of tumor cells. According to the present invention, it was confirmed that the G1 cell cycle arrest occurred by observing that the cell ratio of the G0-G1 phase increased due to the treatment of the composition of the present invention through flow cytometry. By blocking the proliferation of tumor cells through such cell cycle arrest, the composition of the present invention can function as an effective anticancer agent composition.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating terminal hypertrophy, comprising a cyclopentapeptide consisting of an amino acid sequence of Gly1-Tyr2-Arg3-Arg4-Nal5 as an active ingredient.

In the present invention, the term "acromegaly" refers to a condition in which an excess growth hormone is produced and secreted from the pituitary gland and the end of the body such as hands, feet or face is enlarged by excessive growth. Extremity hypertrophy, as well as hypertrophy of the end of the body may show symptoms such as excessive, hirsutism, headache, memory loss, weight gain, arthrosis, hypertension, infertility, sequelae, heart hypertrophy, diabetes and perception. Since the composition of the present invention is characterized in that it has a therapeutic effect on the pituitary tumor secreting excess growth hormone, the composition also has an effect on terminal hypertrophy mainly caused by the excessive growth hormone secretion by the pituitary tumor. .

According to a preferred embodiment of the present invention, the cyclopentapeptide of the present invention is selected from the group consisting of the following formula (1), (2) and (3):

Formula 1

(2)

(3)

According to a preferred embodiment of the present invention, the cyclopentapeptide of the present invention is a cyclopentapeptide of formula (2).

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the cyclopentapeptide as an active ingredient.

(b) The present invention can be usefully used as an active ingredient of a new anticancer agent that targets signaling mechanisms different from the conventional signaling mechanisms to block the proliferation of malignant tumors including malignant pituitary tumors and induce cell death of tumor cells.

(c) The present invention can be usefully used as an effective therapeutic composition for acromegaly by inhibiting the production and secretion of growth hormone in pituitary tumor cells that excessively secrete growth hormone.

1 is a diagram showing the structure of a cyclic pentapeptide that functions as a CXCR4 antagonist. FIG. 1A is FC131 [cyclo (Gly1-d-Tyr2-Arg3-Arg4-Nal5)], FIG. 1B is d-Arg3FC131 [cyclo (Gly1-d-Tyr2-d-Arg3-Arg4-Nal5)], and FIG. 1C is d -Arg3-d-Nal5FC131 [cyclo (Gly1-d-Tyr2-d-Arg3-Arg4-d-Nal5)] is shown, respectively.
Figure 2 is a diagram showing the inhibitory effect of GH gene expression of CXCR4 antagonist. Figure 2a is a diagram showing a recombinant adenovirus (Ad-GHp-Luc) comprising a human GH promoter-derived luciferase gene. The E3 site was removed and the GH gene promoter and luciferase gene were inserted into the E1a site of adenovirus type 5. Figure 2b is a diagram showing the results of measuring the transcriptional activity by the GH promoter using a luciferase reporter system after treating GH3 cells infected with Ad-GHp-Luc with various antagonists. * P <0.001 compared with control. † P <0.05 compared to somatostatin-14 (SST) -treated group. Figure 2c is a diagram showing the expression of GH mRNA when treated with CXCR4 antagonist and SST to GH3 cells. Real time RTPCR was performed for 1 hour after treatment with antagonist (100 nM). GAPDH was used as a reference gene to standardize GH mRNA levels. ‡ Compared to P <0.05 control. The results shown in Figures 2b and 2c are averaged ± standard deviation of the average of three independent experiments. FIG. 2D is a diagram showing Western blotting results showing that GH production in GH3 cells was reduced due to d-ArgFC131 (100 nM) treatment. β-actin gene was used as a control. FIG. 2E shows that administration of d-Arg3FC131 and SST reduces the concentration of GH in GH3 cell culture media. Western blotting analysis on the medium was performed continuously for 3 days (100 nM each of d-ArgFC131 and SST).
Figure 3 shows the effect of CXCR4 antagonist on the proliferation of GH3 cells. Proliferation of GH3 cells was measured using nonradioactive cell proliferation assays (FIG. 3A). The number of GH3 cells was expressed as a percentage value relative to the control number. Of the CXCR4 antagonists, d-Arg3FC131 (10, 100 nM) and d-Arg3-d-Nal5FC131 (10, 100 nM) inhibited about 30-60% cell proliferation. The experimental results were expressed as mean ± standard deviation of the average of three independent experimental results. * P <0.001 compared with control. † P <0.001 compared to SST-treated group (100 nM). 3b is a diagram showing that the CXCR4 antagonist d-Arg3FC131 inhibits phosphorylation of Erk1 / 2 and Akt in GH3 cells. After treatment with SDF1 or SDF1 and d-Arg3FC131, phosphorylation of Erk1 / 2 and Akt was examined by Western blotting using specific antibodies.
4 shows the effect of d-Arg3FC131 on cell cycle and apoptosis in GH3 cells. As described in the Examples, GH3 cells were exposed to d-Arg3FC131 (100 nM) for 72 hours and cell cycle distribution was analyzed by flow cytometry to show the proportion of GH3 cells in a specific cell cycle (FIG. 4A). P values compared to the control at 0 h were calculated using linear to linear binding. 4b is a diagram showing that a positive TUNEL response is induced after 72 hours after d-Arg3FC131 (100 nM) treatment. Green fluorescence staining represents TUNEL and red fluorescence staining using PI represents nuclei of GH3 cells for cell density investigation. Figure 3c is a diagram showing the expression of cleaved caspase-3 and BCL-6 in GH3 cells. The apoptosis pathway was investigated by treating cells with d-Arg3FC131 (100 nM) for 72 hours and performing Western blotting with specific antibodies; β-actin gene was used as a control.
5 is a diagram showing the effect of d-Arg3FC131 on the growth of GH3 cells in nude mice. FIG. 5A shows an example of nude mice implanted with GH3 tumors from the control, d-Arg3FC131 (1.23 mg / kg body weight), and octreotide (1.23 mg / kg body weight) treatment groups. Cultured GH3 cells were collected and injected into the flanks of nude mice (1 × 10 ⁶ cells). Red arrows indicate tumor masses on the side. Mice were killed at 1 month and GH3 tumors were isolated. 5B shows tumor formation in xenograft models of nude mice. As described in the Examples, each dot represents the mean ± standard error value for tumor volume of 10 mice in each group. Values for control mice are shown as black squares, values for d-Arg3FC131-treated mice are white squares, and values for octreotide are shown as red circles. * P <0.05 compared with control. There was no statistically significant difference between the d-Arg3FC131- and octreotide treated groups. Figure 5c is a diagram showing the result of comparing the tumor weight of the control mouse, d-Arg3FC131-treated mice and octreotide-treated mice. Black bars represent the average tumor weight of d-Arg3FC131-treated mice and white bars represent the average tumor weight of control nude mice, respectively. * P <0.05 compared with control.
Figure 6 is a diagram showing the histological and immunohistochemical analysis of the tumor tissue of xenograft nude mice. The left column shows tumor tissue from the control and the right column shows tumor tissue from d-Arg3FC131-treated mice. Top panel shows H & E staining results. The red stained part of the tumor tissue of the control group is red blood cells and the adjacent structures are blood vessels, which are rarely observed in d-Arg3FC131-treated mice. Immunostaining of Ki-67 was shown in the middle panel and TUNEL staining in the lower panel (400-fold magnification).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Cell culture and drug treatment

Rat pituitary tumor cells, GH3-secreting cells (American Type Culture Collection, Manassas, VA), were treated with 10% FBS (fetal bovine serum, GIBCO-BRL), 100 U / mL penicillin and 100 mg / mL streptomycin (Invitrogen, Carlsbad, Incubated at 37 ° C. under 5% CO ₂ in DMEM (Dulbecco's modified Eagle's medium, GIBCO-BRL, Grand Island, NY). These cells are established models for studying GH gene expression and its molecular mechanisms (22, 29, 30). Cells were either 18 nM SDF1 (CXCL12, R & D Systems, Minneapolis, MN) (22), 100 nM somatostatin-14 (SST, Sigma-Aldrich, St. Louis, MO) or 100 nM FC131, cyclo [Gly1- d-Tyr2-Arg3-Arg4-Nal5]; d-Arg3FC131, cyclo [Gly1-d-Tyr2-d-Arg3-Arg4-Nal5]; And FC131 analogues including d-Arg3-d-Nal5FC131, cyclo [Gly1-d-Tyr2-d-Arg3-Arg4-d-Nal5] (Anygen, Daejeon, Korea, FIG. 1). To determine the concentration of FC131 analogs, GH3 cells were treated with FC131 analogs of 1, 10 and 100 nM. The inhibition of tumor growth was the highest at 100 nM. As such, subsequent experiments were performed using 100 nM FC131 analogs.

Construction of Recombinant Adenovirus Vectors

Cassettes containing the luciferase gene from the human GH promoter (610, +58; cloning in human genome DNA, FIG. 2A) were subcloned into adenovirus delivery plasmids based on pcDNA3 (Invitrogen) (32). To investigate GH gene activation in GH3 cells, recombinant adenovirus (Ad-GHp-Luc) was constructed by known methods (32).

Luciferase Analysis

GH 3 cells were seeded in 12-well plates at a concentration of ⁵ × 10 ⁵ / well and Ad-GHp-Luc was inoculated at 2 plaque forming units (PFU) per cell. Infected cells were treated with CXCR4 antagonist (1 × 10 ⁷ M, Anygen) and somatostatin (1 × 10 ⁷ M, Sigma-Aldrich) (32) for 6 hours in serum-free medium, followed by luciferase assay systems (Promega, Madison, WI). Luciferase activity was measured. Relative light units were measured using a MicroLumat LB96PEG & G luminometer (Berthold Technologies, Bad Wildbad, Germany), and luciferase activity was normalized by known methods (33).

Quantitative Real-Time RT-PCR

Primers for amplifying the GH gene were designed as follows: sense, 5'-gctgcgttctgcttctcag-3 'and antisense, 5'-ccgaggtaccaaacatcagg-3'.

CDNA containing SYBR Green I fluorescent dye and 1 × PCR Master Mix (TaKaRa, Shuzo, Kyoto, Japan) were amplified using an ABI 7300 sequence detection system (Perkin-Elmer Applied Biosystems, Foster City, CA). PCR cycle conditions were as follows: annealing for 5 minutes at 95 ° C., 15 cycles at 95 ° C. for 15 seconds, annealing at 56 ° C. for 15 seconds and extension at 72 ° C. for 1 minute. Relative expression levels were measured using a cycle threshold method (17). Target gene expression levels in all experiments were normalized to endogenous controls and data plotted relative to control mRNA (hormone free).

Measurement of cell proliferation

GH3 cells (5 × 10 ³ cells / well) were seeded in 96-well plates in serum-free DMEM and treated with different antagonists twice daily from the next day. On day 6, cell proliferation was measured using a Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega). Optical density was measured at 490 nm and relative data values were expressed as percentages (mean ± standard deviation) (34).

TUNEL

Measurement of cell apoptosis using the Roche Molecular Biochemicals, Indianapolis, IN (TUNEL) assay kit was performed according to the manufacturer's protocol. In summary, GH3 cells were seeded on coverslips and incubated for 16 hours at 37 ° C. in the presence or absence of 100 nM of CXCR4 antagonist. Cells were fixed in 4% formaldehyde and permeated on ice for 2 minutes with 0.1% Triton X-100 dissolved in 0.1% sodium acetate, then exposed to DNA labeling solution at 37 ° C. for 60 minutes and OLYMPUS BX51 microscope (OLYMPUS, Tokyo, Japan), OLYMPUS DP71 camera (OLYMPUS) and DP controller Software (OLYMPUS) were observed.

Flow cell  analysis

We analyzed the effect of CXCR4 antagonist on cell cycle and apoptosis of GH3 cells using fluorescence-activated cell sorting. After treatment with CXCR4 antagonist, GH3 cells (1 × 10 ⁶ cells / mL) were collected and resuspended in 70% ethanol at −20 ° C. for 2 hours. Cells were washed with PBS and repeatedly resuspended in dark at room temperature for 30 minutes in 0.5 mL propidium iodide (Sigma-Aldrich) solution containing 50 mg / mL RNase A, 0.1 mM EDTA and 0.1% Triton X-100. I was. Fluorescence was detected using a PI channel.

Western Blotting

Antagonist-treated GH3 cells were disrupted in RIPA buffer (20 mM Tris pH 7.5, 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 5% phosphatase inhibitor, 0.5% protease inhibitor). Furthermore, GH3 cell culture medium was collected and concentrated for dialysis. The same amount adjusted using the Bradford Protein Assay Kit (Bio-Rad. Richmond, Calif.) Was separated using 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. Phosphorylated extracellular signaling kinase (1: 1000; Cell Signaling, Beverly, MA), phosphorus-protein kinase B / Akt (1: 1000 Cell Signaling), Bcl-2 (1: 1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Bcl-6 (1: 1000; Santa Cruz Biotechnology, Inc.), Caspase-3 (1: 1000; Cell Signaling) or β-actin (1: 1000; Santa Cruz Biotechnology, Inc.) Blots were labeled as internal control using the primary antibody against.

Animal experiment

Animal experiments were performed using 6 week old male non-thymus immunodeficient nude mice (Charles River Japan Inc., Yokohama, Japan). Each group of five animals was placed under a 12 hour light / 12 hour dark condition cycle in an animal room adjusted to 23 ± 2 ° C., 55 ± 5% humidity and fed standard unrestricted food. GH-secreting tumor cells were collected, washed twice in PBS, resuspended in medium, and injected into the flanks of mice on day 0 (2 × 10 ⁶ cells). Thereafter, CXCR4 antagonist (1.23 mg / kg body weight), octreotide (Sandostatin, Norvatis, Stein, Switzerland, 1.23 mg / kg body weight) or PBS was administered twice a day (25). Tumor size (mm ³ ) was measured three-dimensionally using calipers (34) and calculated using the following formula: (3.14 x length x width x depth) / 6. Thirty days after treatment, mice were killed and tumors removed. All animal experiments were approved by the Animal Care and Use Committee of Yonsei University College of Medicine. The collected tumor tissues were fixed with 10% neutral buffered formalin and paraffin blocks were made. 4-μm thick cross sections were stained with H & E, TUNEL and Ki-67. After incubation with fluorescent TUNEL reagent and primary antibody against Ki-67, specifically biotinylated secondary antibody (1: 100, Vector Laboratories, Burlingame, Calif.) And streptavidin-peroxidase (Dako, Kyoto, Japan) were incubated continuously. DAB (Diaminobenzidine, Vector Laboratories) was used as a chromogen and counterstained using hematoxylin.

Statistical analysis

All statistical analyzes were performed using Student's t test for comparison between two groups or multiple comparisons between two or more groups, through a linear-by-linear association test. Compared

P <0.05 was considered to be a significant association. Statistical analysis was performed using SPSS for Windows software (version 15.0; SPSS Inc., Chicago IL).

Experiment result

CXCR4  Antagonists GH3  Pituitary Tumor Cells GH Production and secretion Inhibit .

The inventors have found that CXCR4 antagonists have various peptides (somatostatin-14, CXCR4 antagonists FC131 (cyclo [Gly1-d-Tyr2-Arg3-Arg4-Nal5]), d-Arg3 FC131 (cyclo [Gly1-d-Tyr2-d-Arg3-). Arg4-Nal5]) and d-Arg3-d-Nal5 FC131 (cyclo [Gly1-d-Tyr2-d-Arg3-Arg4-d-Nal5])) were investigated to be involved in the production and production of GH. We used GH3 cells transformed with adenovirus containing a GH promoter derived from the luciferase gene (FIG. 1A). Luciferase analysis revealed that d-Arg3 FC131 (100 nM) and d-Arg3-d-Nal5 FC131 (100 nM) significantly reduced the activity of the GH promoter compared to the control group (set 1 to any value) (0.02). By confirming the embryonic activity, it was found that the CXCR4 antagonists d-Arg3FC131 and d-Arg3-d-Nal5FC131 inhibit GH production at the transcription level. Luciferase activity was reduced by 0.3 and 0.4 fold, respectively, after treatment with antagonist FC131 or somatostatin-14 (FIG. 2B).

The inventors then investigated the inhibitory effect of CXCR4 on GH production using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and western blotting. Administration of d-Arg3FC131 and d-Arg3-d-Nal5FC131 100 nM decreased GH transcription levels 0.6 ± 0.1-fold and 0.67 ± 0.2-fold, respectively (FIG. 2C). Western blotting showed that GH protein levels in d-Arg3FC131-treated GH3 cells also decreased time dependently (FIG. 2D). Quantification by Western blotting, GH expression was significantly reduced after 18 hours after treatment with d-Arg3FC131 (100 nM). GH levels in GH3 cell culture medium increased over time. In contrast, treatment with D-Arg3FC131 or SST inhibited the secretion of GH in GH3 (FIG. 2E). These results clearly show that d-Arg-FC131 inhibits GH production in GH3 pituitary tumor cells.

CXCR4  Antagonists GH3  Proliferation of pituitary adenoma cells Inhibit .

To investigate whether CXCR4 antagonist inhibits proliferation of hypothalamic tumor cells, GH3 cells were treated with various doses (1100 nM) of CXCR4 antagonist and somatostatin-14. 6 days after treatment, growth was observed to be inhibited. As shown in Figure 3a, d-Arg3-d-Nal5FC131 is dose-dependent (67.1 ± 13.2% at 10 nM, 59.3 ± 10.6% at 100 nM, P <0.05 compared to both controls) at GH3. Inhibit the growth of cells. The inhibitory effect of d-Arg3FC131 on GH3 cell proliferation was the strongest (100 nM, 30% ± 3.8, p <0.01 compared to the control) and much stronger than that of somatostatin-14 (100 nM, 65.3% ± 11, control Compared to P <0.05) (FIG. 3A). Thus, d-Arg3FC131 was used for the next experiment. There are several reports that SDF1 / CXCR4 interaction activates proliferation and growth of cells by stimulating the Erk1 / 2 and phosphatidylinositol 3-kinase / Akt pathways (25). Therefore, the present inventors investigated whether the cell proliferation inhibitory effect of CXCR4 antagonists was related to the phosphorylation of Erk1 / 2 and Akt. Western blotting results show that Erk1 / 2 and Akt are activated from 5, 10 minutes or 10, 30, 60 minutes after SDF1 treatment (FIG. 3B). However, the antagonist d-Arg3FC131 (100 nM) inhibited this activation.

d- Arg3FC131 The G1  Induce cell cycle inhibition at checkpoint Apoptosis  Cause

To investigate the mechanism of CXCR4 antagonist-induced cell proliferation inhibition, we quantified the number of cells in different cell cycles and the number of cells undergoing apoptosis using flow cytometry. As shown in FIG. 4A, it was confirmed that partial G1 cell cycle arrest occurred by confirming that the cell ratio of G0-G1 phase was increased by d-Arg3FC131 (100 nM) treatment by cell cycle analysis. This arrest lasted 72 hours. In addition, our study revealed that d-Arg3Fc131 induces apoptosis in GH3 cells in a time-dependent manner (FIG. 4A). The percentage of cells that caused apoptosis (8 ± 3.4%, P <0.05) significantly increased after 72 n after 100 nM of d-Arg3Fc131 treatment compared to vehicle-treated GH3 cells (1.35 ± 0.8%).

d-Arg3FC131-induced apoptosis was measured through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis (FIG. 4B). Apoptotic nuclei increased significantly after 3 days of treatment with 100 nM d-Arg3FC131. When treated with d-Arg3FC131 number of cells that caused apoptosis (33.4 ± 7.1%) the vehicle (vehicle) - it was increased by about three times the processing number of the cells that caused apoptosis in GH3 cells (11.24 ± 4.8%) (P < 0.05). Western blotting analysis for apoptosis-related genes confirmed this. BCL-6, BCL-2, BCL-XL, Bad, Bax, Caspase-3 and cleaved caspase in GH3 cells treated with d-Arg3Fc131 (100 nM) for 72 hours and untreated cells The expression level of -3 was investigated and compared (FIG. 4C). The amount of caspase-3 cleaved form was 0.8% at 6 hours, 1.2% at 12 hours, 1.5% at 18 hours, 1.1% at 24 hours, 48 hours at 0% (control). It increased to 0.8% at day and 0.5% at 72 hours ( P <0.05 in all cases), and an increase in BCL-6 expression compared to the control was observed after 18 hours. However, treatment with d-Arg3FC131 did not induce a significant change in the expression level of the BCL family including BCL-2, BCL-XL, Bad and Bax.

d- Arg3FC131 Nude Mouse Transplant Tumor Model GH3  Inhibits growth in cells Apoptosis  Promote

Based on the discovery that inhibit cell growth and induce apoptosis in vitro CXCR4 antagonist d-Arg3FC131, the present inventors have found that the d-Arg3FC131 would inhibit tumor production by GH- producing pituitary adenoma cells in immunodeficient nude mice Assumed (FIG. 5). GH3 cells were injected subcutaneously into male athymic mice as described in the Examples. Tumors developed within 15 days in mice injected with d-Arg3FC131 or phosphate buffered saline (PBS) as a control, and similar to octreotide-treated mice, tumor formation in d-Arg3FC131-treated mice was more than 10 days compared to the control group. Delayed. In d-Arg3FC131 treated mice, tumor growth rate and size were significantly reduced compared to PBS treated groups. Furthermore, administration of d-Arg3FC131 significantly reduced tumor weight and total body weight (84 ± 1.9 and 28.16 ± 0.5%, respectively, P <0.05). There was no significant difference in tumor growth inhibition between octreotide treated and d-Arg3FC131-treated mice.

We used histology to investigate the effect of CXCR4 antagonist on tumor growth in nude mice. Hematoxylin and eosin (H & E) staining of tumor tissues confirmed that red blood cells-filled microvessels were prominent in the tumor tissues of the control group, but barely visible in the d-Arg3FC131 treated-mouse (Figure 6 top panel). We stained Ki-67 to detect cell proliferation and performed TUNEL analysis to measure apoptosis in tumor tissues (FIG. 6). The number of cells positive for Ki-67 expression and TUNEL were counted using computer image analysis software (Meta Morph, version 4.6, Universal Imaging Co., Downingtown, PA). Tumor tissue of d-Arg3FC131 treated mice showed lower Ki-67 expression level in the cell nucleus compared to the control group (10.3% vs. 4.4%, P <0.05; FIG. 6 middle panel), and the ratio of TUNEL-positive nuclei was d. -Arg3FC131 treated mice increased significantly (5.6% vs. 1.2% (control) P <0.05; Figure 6 bottom panel). Thus, d-Arg3FC131 can reduce transplanted tumor size by inducing apoptosis and inhibiting cell proliferation.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (11)

delete delete delete delete delete delete delete delete A pharmaceutical composition for preventing or treating terminal hypertrophy comprising a cyclopentapeptide consisting of an amino acid sequence of Gly1-Tyr2-Arg3-Arg4-Nal5 as an active ingredient.
The pharmaceutical composition of claim 9, wherein the cyclopentapeptide is selected from the group consisting of Formula 1, Formula 2, and Formula 3 below:
Formula 1

Formula 2

(3)

The pharmaceutical composition according to claim 10, wherein the cyclopentapeptide is a cyclopentapeptide of Formula 2.

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