CYCLIC PEPTIDE SPECIFICALLY BINDING TO APOPTOTIC CELLS AND USE THEREOF

Abstract

Provided is a cyclic peptide (cyclo [Cys-Gln-Arg-Pro-Pro-Arg-Cys] peptide) comprised of the amino acid sequence of SEQ ID NO: 2; and a composition for apoptotic cell detection, drug delivery or imaging, containing the same as an active ingredient. The cyclic peptide (cyclo [Cys-Gln-Arg-Pro-Pro-Arg-Cys] peptide has an excellent effect of binding to apoptotic cells, compared with a linear peptide thereof, thereby greatly facilitating the detection of apoptotic cells and the in vivo imaging of an affected part under apoptosis, while the detection and imaging signal shows a very high correlation in disease prognosis prediction. The cyclic peptide binds to an imaging material, early diagnosing a response of a drug for treating diseases associated with abnormal cell proliferation, and binds to a therapeutic material, selectively delivering a drug to tissues afflicted with Apoptosis-associated diseases.

Claims

1. A cyclic peptide consisting of the amino acid sequence of SEQ ID NO: 2 and specifically binding to apoptotic cells.

2. A composition for detecting apoptotic cells, the composition comprising the peptide of claim 1 as an active ingredient.

3. A composition for imaging an affected part by an apoptosis-related disease, the composition comprising the peptide of claim 1 as an active ingredient.

4. The composition of claim 3, wherein the apoptosis-related disease is any one selected from the group consisting of neoplastic disease, myocardial infarction, arteriosclerosis, neurodegenerative disease, and stroke.

5. The composition of claim 4, wherein the neoplastic disease is any one selected from the group consisting of brain cancer, neuroendocrine cancer, stomach cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, adrenal gland cancer, colorectal cancer, colon cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, and ureteral cancer.

6. The composition of claim 4, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, and Niemann-Pick disease.

7. A composition for screening an initial drug response of a test preparation having apoptosis-inducing activity in a subject afflicted with an abnormal cell proliferation-related disease, the composition comprising the peptide of claim 1 as an active ingredient.

8. The composition of claim 7, wherein the abnormal cell proliferation-related disease is a neoplastic disease or hyperproliferative vascular disease.

9. The composition of claim 2, wherein the peptide is labeled with any one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromophore, a luminescent material, a fluorescer, gadolinium, super paramagnetic particles, and ultrasuper paramagnetic particles.

10. A method for detecting apoptotic cells, the method comprising: (a) mixing peptides of claim 1 with a sample; (b) removing the peptides that are unbound or non-specifically bound; and (c) determining a binding or non-binding of the peptides and a binding position of the peptides.

11. A method for screening an initial drug response of a test preparation in a subject afflicted with an abnormal cell proliferation-related disease, the method comprising: (a) treating a target tissue of an affected part isolated from a subject with a test preparation having apoptosis-inducing activity, wherein the subject is afflicted with an abnormal cell proliferation-related disease; (b) treating the test preparation-treated target tissue of step (a) and a control target tissue treated without a test preparation, with a peptide of claim 1 labeled with a labeling means; and (c) detecting and comparing the labeling means in the peptide-treated target tissues in step (b).

12. The method of claim 11, further comprising (d) determining the target tissue as being responsive to the test preparation if an increased level of the labeling means is detected in the test preparation-treated target tissue in comparison with the control target tissue.

13. The method of claim 11, wherein the labeling means is any one labeling material selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromophore, a luminescent material, a fluorescer, gadolinium, super paramagnetic particles, and ultrasuper paramagnetic particles.

14. A composition for delivering a drug for an apoptosis-related disease, the composition comprising the peptide of claim 1 as an active ingredient.

15. The composition of claim 14, wherein the apoptosis-related disease is any one selected from the group consisting of neoplastic diseases, myocardial infarction, arteriosclerosis, neurodegenerative diseases, and stroke.

16. A pharmaceutical composition for preventing and treating a neoplastic disease, the pharmaceutical composition comprising, as active ingredients, the peptide of claim 1 and an anti-tumor substance conjugated thereto.

17. The composition of claim 16, wherein the anti-tumor substance is conjugated to a drug selected from the group consisting of paclitaxel, doxorubicin, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, teniposide, bisantrene, homoharringtonine, Gleevec (STI-571), cisplain, 5-fluouracil, adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, nitrosourea, streptokinase, urokinase, alteplase, angiotensin II inhibitor, aldosterone receptor inhibitor, erythropoietin, NMDA (N-methyl-d-aspartate) receptor inhibitor, lovastatin, rapamycin, Celebrex, Ticlopin, Marimastat, and Trocade.

18. A composition for preventing and treating a neurodegenerative disease, the composition comprising, as active ingredients, the peptide of claim 1 and a neurodegenerative disease therapeutic substance conjugated thereto.

19. A pharmaceutical composition for preventing and treating myocardial infarction, the pharmaceutical composition comprising, as active ingredients, the peptide of claim 1 and a myocardial infarction therapeutic substance conjugated thereto.

20. A pharmaceutical composition for preventing and treating arteriosclerosis, the pharmaceutical composition comprising, as active ingredients, the peptide of claim 1 and an arteriosclerosis therapeutic substance conjugated thereto.

21. A pharmaceutical composition for preventing and treating stroke, the pharmaceutical composition comprising, as active ingredients, the peptide of claim 1 and a stoke therapeutic substance conjugated thereto.

22. The composition of claim 14, wherein the composition further comprises any one labeling material selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromophore, a luminescent material, a fluorescer, gadolinium, super paramagnetic particles, and ultrasuper paramagnetic particles.

23. A method for treating a neoplastic disease, the method comprising administering an effective amount of the peptide of claim 1 and an anti-tumor substance conjugated thereto to a subject in need thereof.

24. A method for preventing or treating a neurodegenerative disease, the method comprising administering an effective amount of the peptide of claim 1 and a neurodegenerative disease therapeutic substance conjugated thereto to a subject in need thereof.

25. A method for preventing or treating myocardial infarction, the method comprising administering an effective amount of the peptide of claim 1 and a myocardial infarction therapeutic substance conjugated thereto to a subject in need thereof.

26. A method for preventing or treating arteriosclerosis, the method comprising administering an effective amount of the peptide of claim 1 and an arteriosclerosis therapeutic substance conjugated thereto to a subject in need thereof.

27. A method for preventing or treating stroke, the method comprising administering an effective amount of the peptide of claim 1 and a stroke therapeutic substance conjugated thereto to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIG. 1 quantitatively shows in vitro detection results of apoptotic cells using linear form of ApoPep-1(A), cyclic form of ApoPep-1(B), and Annexin V(C) after treatment of cells with either cisplatin or cetuximab alone, or in combination thereof (PBS: control group treated without anti-cancer drug, CPT: group treated with cisplatin alone, CET: group treated with cetuximab alone, CPT+CET: group treated with cisplatin and cetuximab in combination).

[0109] FIG. 2 shows fluorescence intensity when mice were treated with either cisplatin or cetuximab alone, or in combination thereof at the 1st and 2nd rounds and then apoptotic cells were subjected to in vivo NIR fluorescence imaging using linear form of ApoPep-1 (A) and cyclic form of ApoPep-1 (B), respectively (PBS: group treated without anti-cancer drug, CPT: group treated with cisplatin alone, CET: group treated with cetuximab alone, CPT+CET: group treated with cisplatin and cetuximab in combination, 1st: group treated with the anti-cancer drug at the 1st round (1 week), 2nd: group treated with the anti-cancer drug at the 2nd round (2 week)).

[0110] FIG. 3 shows representative images when mice were treated with either cisplatin or cetuximab alone, or in combination thereof at the 1st and 2nd rounds and then apoptotic cells were subjected to in vivo NIR fluorescence imaging using linear form of ApoPep-1 (C) and cyclic form of ApoPep-1 (D), respectively (PBS: group treated without anti-cancer drug, CPT: group treated with cisplatin alone, CET: group treated with cetuximab alone, CPT+CET: group treated with cisplatin and cetuximab in combination, 1st: group treated with the anti-cancer drug at the 1st round (1 week), 2nd: group treated with the anti-cancer drug at the 2nd round (2 week)).

[0111] FIG. 4 shows changes in tumor volume up to 3 weeks after anti-cancer treatment of mice, which were treated with either cisplatin or cetuximab alone, or in combination thereof at the 1st and 2nd rounds and apoptosis was detected at an early stage using linear form of ApoPep-1 (A) and cyclic form of ApoPep-1 (B), respectively (PBS: group treated without anti-cancer drug, CPT: group treated with cisplatin alone, CET: group treated with cetuximab alone, CPT+CET: group treated with cisplatin and cetuximab in combination, Arrows indicate time points for anti-cancer treatment).

[0112] FIG. 5 shows a tumor weight measured by taking a tumor from mice 3 weeks after anti-cancer treatment when the mice were treated with either cisplatin or cetuximab alone, or in combination thereof at the 1st and 2nd rounds and apoptosis was detected at an early stage using linear form of ApoPep-1 (C) and cyclic form of ApoPep-1 (D), respectively (PBS: group treated without anti-cancer drug, CPT: group treated with cisplatin alone, CET: group treated with cetuximab alone, CPT+CET: group treated with cisplatin and cetuximab in combination, .square-solid., .box-tangle-solidup., .Math., .diamond-solid. indicate measured values for each subject of a subgroup (n=3) and - indicates a mean value).

[0113] FIG. 6 shows TUNEL staining results of tumor tissues taken from mice 3 weeks after anti-cancer treatment when the mice were treated with either cisplatin or cetuximab alone, or in combination thereof at the 1st and 2nd rounds and apoptosis was detected at an early stage using linear form of ApoPep-1 (E) and cyclic form of ApoPep-1 (F), respectively (Green: apoptotic cells; Blue: nucleus, PBS: group treated without anticancer drug, CPT: group treated with cisplatin alone, CET: group treated with cetuximab alone, CPT+CET: group treated with cisplatin and cetuximab in combination, scale bar at the bottom of each image indicates 50 μm).

[0114] Panels A and C of FIG. 7 show linear regression analysis results of the correlation between the in vivo fluorescence intensity and the prognosis of tumor volume when the mice were treated with anticancer drug (either cisplatin or cetuximab alone, or in combination thereof) at the 1st round and subjected to NIR fluorescence imaging using linear form of ApoPep-1 (A) and cyclic form of ApoPep-1 (C), respectively.

[0115] Panels B and D of FIG. 7 show linear regression analysis results of the correlation between the in vivo fluorescence intensity and the prognosis of tumor volume when the mice were treated with anti-cancer drugs (either cisplatin or cetuximab alone, or in combination thereof) at the 2nd round and subjected to NIR fluorescence imaging using linear form of ApoPep-1 (B) and cyclic form of ApoPep-1 (D), respectively.

[0116] FIG. 8 shows C18 reverse-phase FPLC analysis results in the order of time of peptides collected after linear form of ApoPep-1 (A) and cyclic form of ApoPep-1 (B) were incubated in mouse serum for 0-24 hours (Y axis indicates the absorbance unit at 215 nm and X axis indicates the retention time. Arrows indicate the peak of linear or cyclic form of ApoPep-1 peptide).

[0117] FIG. 9A shows MS spectra for peptide peak fractions obtained by incubating linear form of ApoPep-1 in mouse serum for 24 hours, collecting the peptides, and performing C18 reverse-phase FPLC. FIG. 9B shows MS results for the linear form of ApoPep-1 peptide in an initial synthetic state not incubated in serum (Arrows indicate the peak of the linear form of ApoPep-1 peptide). It can be seen through the comparision of FIG. 9A and FIG. 9B that linear form of ApoPep-1 was stably present in serum even after incubation for 24 hours.

[0118] FIG. 9C shows MS spectra for peptide peak fractions obtained by incubating cyclic form of ApoPep-1 in mouse serum for 24 hours, recovering the peptides, and performing C18 reverse-phase FPLC. FIG. 9D shows MS results for the cyclic form of ApoPep-1 peptide in an initial synthetic state not incubated in serum (Arrows indicate the peak of the cyclic form of ApoPep-1 peptide). It can be seen through the comparision of FIG. 9C and FIG. 9D that cyclic form of ApoPep-1 was stably present in serum even after incubation for 24 hours.

MODE FOR CARRYING OUT THE INVENTION

[0119] Hereinafter, the present invention will be described in detail.

[0120] However, the following examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.

[0121] <Materials and Methods>

[0122] 1. Synthesis and Fluorescence Labeling of Peptides

[0123] Linear form of ApoPep-1 (CQRPPR, SEQ ID NO: 1) and cyclic form of ApoPep-1 (cyclo[CQRPPRC] of the present invention, SEQ ID NO: 2, cyclization via disulfide bonding between amino and carboxy termini) peptides were synthesized by Peptron Inc. (Daejeon, Korea.), and were purified to >95% purity using high performance liquid chromatography (HPLC). Peptides were labeled with FITC (fluorescein isothiocyanate) or FPR675 near-infrared (NIR) fluorescence dye (Bioacts, Inc., Incheon, Korea.).

[0124] 2. In Vitro Binding of Peptides to Apoptotic Cells

[0125] SNU16 human stomach cancer cell line was purchased from KCLB (Seoul, Korea). To induce apoptosis, cells were treated with cisplatin (300 ng/ml), cetuximab (200 ug/ml), or cisplatin (300 ng/ml) plus cetuximab (200 mg/ml) in combination for 24 hours. The concentrations of cisplatin and cetuximab were chosen according to the previous reports (Choi C H, Cha Y J, An C S, Kim K J, Kim K C, et al. (2004) Molecular mechanisms of heptaplatin effective against cisplatin-resistant cancer cell lines: less involvement of metallothionein. Cancer Cell Int 4: 6.; Yun J, Song S H, Park J, Kim H P, Yoon Y K, et al. (2012) Gene silencing of EREG mediated by DNA methylation and histone modification in human gastric cancers. Lab Invest 92: 1033-1044.). After treatment, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated linear or cyclic form of ApoPep-1 peptide at 4□ for 1 hour. As control, cells were stained with Alexa488-conjugated annexin V (Life technologies, Carlsbad, Calif.) for 15 min at room temperature. Percentages of fluorescent (the liner or cyclic form of ApoPep-1 peptide-bound or annexin V-bound fluorescence) cells were calculated by measurement and analysis methods through the selection of the fluorescent signals (the liner or cyclic form of ApoPep-1 peptide-bound or annexin V-bound fluorescence), which were shown in the respective cells when cells in an emulsion state pass through a constant fluorescence detection zone, using flow cytometry (Fluorescence-activated cell sorting (FACS), FACS calibur, BD Biosciences, MA, USA).

[0126] 3. Anti-Tumor Treatment of Mice and Tumor Size Measurement

[0127] All animal experiments were performed in compliance with institutional guidelines and according to the animal protocol approved by the guideline of the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University (permission No. KNU 2012-15).

[0128] Eight-week old female athymic (nu/nu) Balb/c mice were purchased from Orient laboratories (Seongnam, Korea) and were housed under specific-pathogen-free (SPF) conditions with laboratory chow and water ad libitum. Stomach tumor xenografts were established by subcutaneously injecting 1×10.sup.7 SNU-16 cells in 100 ml saline into the right flank. Tumors were allowed to reach 50-60 mm.sup.3 of volume before randomization and initiation of treatment. Treatment of tumor-bearing mice with cisplatin and cetuximab was conducted according to a previously described protocol (Steiner P, Joynes C, Bassi R, Wang S, Tonra J R, et al. (2007) Tumor growth inhibition with cetuximab and chemotherapy in non-small cell lung cancer xenografts expressing wild-type and mutated epidermal growth factor receptor. Clin Cancer Res 13: 1540-1551.). Mice were divided into four treatment groups (n=6 per group) and treated for two weeks: 1) control treated with phosphate buffered saline (PBS, control); 2) cisplatin treatment group (5 mg/kg, intraperitoneal (i.p.) injection, once per week for total two injections); 3) cetuximab treatment group (1.5 mg/kg, i.p., twice per week for total four injections); 4) cisplatin treatment group (5 mg/kg, i.p., once per week for total two injections) plus cetuximab (1.5 mg/kg, i.p., twice per week for total four injections). One round of anticancer drug treatment was conducted in a manner of injection of cisplatin at day 1 per week (the first day of the week on a weekly basis) and cetuximab at day 1 and day 4 per week (the first day and the fourth day of the week on a weekly basis). In the present experiment, a total of two rounds of anticancer drug treatment were conducted for two weeks. Changes in tumor size were measured using an automatic caliper over three weeks. Tumor volumes were calculated using the formula: volume=(length×width×height)/2. Tumor weights were measured after isolation of tumor mass.

[0129] 4. In Vivo NIR Fluorescence Imaging of Tumor Apoptosis

[0130] In vivo NIR fluorescence imaging was performed after the first and second round of treatment. Each treatment group (n=6) was divided into two subgroups (n=3) for imaging with linear and cyclic forms of ApoPep-1, respectively. Linear and cyclic forms of FPR675-labeled ApoPep-1 (1.45 mg/kg and 1.54 mg/kg, respectively; equivalent to 800 nmol/kg for each peptide) was injected through the tail vein into mice. At 90 minutes after injection of the fluorescence-labeled linear and cyclic forms of ApoPep-1 peptide, mice were anesthetized and subjected to imaging. NIR fluorescence (typically, between 650 and 1100 nm) is favored for in vivo optical imaging because of its low tissue absorption and deep tissue penetration properties (Konig K (2000) Multiphoton microscopy in life sciences. J Microsc 200: 83-104). The excitation/emission wavelength of the FPR675 dye used in this study was 675/698 nm. Images were taken using the eXplore Optix optical imaging system (ART Inc., Montreal, Canada), and the acquisition time for a whole-body scanning was 15 minutes per mouse. Fluorescence intensity at region of interest (ROI) was measured using a analysis software provided by the manufacturer (ART Inc.).

[0131] 5. Histologic Analysis of Apoptosis

[0132] After in vivo imaging, mice were euthanized three weeks after the initiation of anticancer drug treatment, and the tumors were removed and frozen quickly in O.C.T. embedding medium (Sakura Finetechnical, Tokyo, Japan). Tissues were cut into 6 um sections, and stained with DAPI (4′,6-diamidino-2-phenylindole) for nucleus counterstaining. Terminal deoxy-nucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was conducted using the Apoptag Red In Situ Apoptosis Detection kit according to guidelines of the manufacturer (Millipore, Billerica, Mass.). The stained tissue sections were observed under a fluorescence microscope (Carl Zeiss, Jena, Germany).

[0133] 6. Correlation Analysis Between Fluorescence Intensity and Tumor Volume

[0134] At three weeks after the initiation of anticancer drug treatment (endpoint of experiments), tumor volumes were measured and tumors were isolated from the mice for the weight measurement. The correlation between NIR fluorescence intensity and tumor volume was evaluated by the linear regression analysis using the Graphpad software.

[0135] 7. Stability of Peptides in the Serum

[0136] Peptide stability in the serum was examined by the same method referring to the following documents Yoo S A, Bae D G, Ryoo J W, Kim H R, Park G S, et al. (2005) Arginine-rich antivascular endothelial growth factor (anti-VEGF) hexapeptide inhibits collageninduced arthritis and VEGF-stimulated productions of TNF-alpha and IL-6 by human monocytes. J Immunol 174: 5846-5855. Blood from mice was collected, and then serum was collected by centrifugation at 4□, followed by filtration through 0.22 um-pore filter. Linear and cyclic forms of ApoPep-1 peptides (100 ug of peptide contained in 50 uL of PBS) was incubated with 50 ml of filtered serum at 37□ for 24 hours at time intervals (each sample was composed of serum 50 μL+peptide 50 uL (that is, 100 ug)=100 uL, and reacted in each tube according the time of 0 h, 1 h, 4 h, 8 h, 16 h, 24 h). The samples were diluted to 100-fold, and injected in a volume of 100 μl. The flow rate was 0.3 ml/min, and analyzed by C18 reverse phase FPLC using a linear gradient of 20% from 0 to 100% using acetonitrile through a linear graduation of 20% (Vydac protein and peptide C18, 0.1% trifluoroacetate in water for equilibration, and 0.1% trifluoroacetate in acetonitrile for elution) (Life Technologies, Carlsbad, Calif.). The fraction samples collected according to each peak show as a result of C18 reverse phase FPLC were collected, and frozen dried. To identify the identity of the peptide from the profiles of C18 reverse phase FPLC, each peak was collected, and subjected to mass spectrometry (MS) using an MALDI-TOF mass spectrometer (Life Technologies, Carlsbad, Calif.).

[0137] 8. Statistical Analysis

[0138] The statistical significance of differences between experimental and control groups was analyzed using one-way analysis of variance (ANOVA) (* p<0.05, ** p<0.01, ***p<0.001, statistical significance was shown for each drawing).

Example 1

[0139] In Vitro Detection of Apoptosis of Stomach Tumor Cells Using ApoPep-1 after Treatment with Cisplatin and Cetuximab

[0140] In order to examine the detection of apoptosis according to the structure features of ApoPep-1, stomach tumor cells were treated with cisplatin or cetuximab alone, or cisplatin plus cetuximab in combination, and then incubated with FITC-conjugated linear and cyclic forms of ApoPep-1. Cyclic form of ApoPep-1 (cyclo[CQRPPRC] peptide of the present invention) was prepared by adding cysteine residue at the carboxy terminal of linear form of ApoPep-1 (CQRPPR) and performing cyclization through disulfide bonding. The percentages of apoptotic cells detected by the linear form of ApoPep-1 were approximately 28%, 25%, and 34% in the groups treated with cisplatin alone, cetuximab alone, and cisplatin and cetuximab in combination, respectively (panel A of FIG. 1). The percentages of apoptotic cells detected by the cyclic form of ApoPep-1 were approximately 56%, 49%, and 78% in the groups treated with cisplatin alone, cetuximab alone, and cisplatin and cetuximab in combination, respectively (panel B of FIG. 1). The percentages of apoptotic cells detected by annexin V were approximately 43%, 40%, and 45% in the groups treated with cisplatin alone, cetuximab alone, and cisplatin and cetuximab in combination, respectively (panel C of FIG. 1). These results show that the combined treatment of cisplatin and cetuximab induces apoptosis of stomach tumor cells at higher levels than the treatment of cisplatin or cetuximab alone. Also, these results suggest that the cyclic form of ApoPep-1 more sensitively detects apoptosis of stomach tumor cells than the linear form of ApoPep-1 or annexin V.

Example 2

[0141] In Vivo Imaging of Apoptosis of Stomach Tumor Using ApoPep-1 after Treatment with Cisplatin and Cetuximab

[0142] In order to examine in vivo detection and imaging of apoptosis of apoptosis according to the structural feature of ApoPep-1, the fluorescence intensity at tumor by the accumulation of NIR fluorescence dye (FPR675) labeled-ApoPep-1 to tumor tissue was measured after the first and second round of treatment (equivalent to one week and two weeks after the initiation of treatment, respectively). Quantification of fluorescence intensity at tumor site by linear or cyclic form of ApoPep-1 showed that the intensities were significantly higher in groups treated with cisplatin alone, cetuximab alone, and cisplatin and cetuximab in combination, compared with the control group treated without a drug, after the first or second round of treatment (panel A and panel B of FIG. 2). Fluorescence intensities by linear form of ApoPep-1 were higher in the group treated with cisplatin and cetuximab in combination compared with the group treated with cisplatin alone (p<0.05 and p<0.05 after the first and second round of treatment, respectively, panel A of FIG. 2) or cetuximab alone (p<0.01 after the first round of treatment, not significant after the second round of treatment, respectively, panel A of FIG. 2).

[0143] Particularly, fluorescence intensities by cyclic form of ApoPep-1 were higher in the group treated with cisplatin and cetuximab in combination compared with the group treated with cisplatin alone (p<0.01 and p<0.01 after the first and second round of drug treatment, respectively, panel B of FIG. 2) or cetuximab alone (p<0.001 and p<0.01 after the first and second rounds of treatment, respectively, panel B of FIG. 2).

[0144] Representative whole body fluorescence images by linear and cyclic forms of ApoPep-1 are shown in panel C and panel D of FIG. 3, respectively). As shown in FIG. 3, the difference in fluorescence intensity between experimental groups was confirmed to be definitely differentiated by naked eyes in the fluorescence image by cyclic form of ApoPep-1 compared with linear form of ApoPep-1. Weak background fluorescence signals were observed in other organs, including the liver and lung (panel C and panel D of FIG. 3).

Example 3

[0145] Measurement of Tumor Volumes and Weights after Anti-Tumor Treatment with Cisplatin and Cetuximab

[0146] To examine anti-tumor growth effect by cisplatin or cetuximab alone and in combination, tumor volumes and weights after the drug treatment were measured.

[0147] Treatment with cisplatin and cetuximab alone and in combination reduced tumor volumes compared with control group treated without drug, in the linear form of ApoPep-1 group (p<0.05, p<0.05, and p<0.001, sequentially, panel A of FIG. 4) and in the cyclic form of ApoPep-1 group (p<0.05, p<0.01, and p<0.001, sequentially, panel B of FIG. 4). Combined treatment of cisplatin and cetuximab reduced tumor volumes more efficiently, compared with treatment with cisplatin or cetuximab alone (p<0.05 and p<0.05, respectively, in the linear form of ApoPep-1 group, as shown in panel A of FIGS. 4; and p<0.01 and p<0.01, respectively, in the cyclic ApoPep-1 group, as shown in panel B of FIG. 4).

[0148] Also in changes in tumor weights after treatment with cisplatin and cetuximab alone and in combination, compared with untreated control, the above similar patterns were observed in the linear form of ApoPep-1 group (p<0.01, p<0.01, and p<0.001, sequentially, panel C of FIG. 5) and in the cyclic ApoPep-1 group (p<0.01, p<0.01, and p<0.001, sequentially, panel D of FIG. 5).

[0149] Combined treatment of cisplatin and cetuximab reduced tumor weights more efficiently, compared with treatment with cisplatin or cetuximab alone (p<0.05 and p<0.05, respectively, in the linear form of ApoPep-1 group, as shown in panel C of FIGS. 5; and p<0.01 and p<0.01, respectively, in the cyclic ApoPep-1 group, as shown in panel D of FIG. 5).

[0150] The levels of reduction in tumor volumes and weights after the treatment, between experimental groups injected with linear or cyclic form of ApoPep-1 were similar, and there were no differences in tumor volumes between those two groups at the time of imaging. As shown in FIG. 6, higher levels of apoptosis after treatment with cisplatin and cetuximab in combination, compared with treatment with cisplatin or cetuximab alone, was further demonstrated by the TUNEL staining of the tumor tissues (FIG. 6).

Example 4

Correlation Between Fluorescence Intensity and Tumor Size

[0151] After the first and second round of treatment, the correlation between the fluorescence intensity of in vivo imaging of apoptosis (measured by the same method at 1 week and 2 weeks after the initiation of treatment, respectively) and later-on tumor volume (at 3 weeks after the initiation of treatment). The fluorescence intensities of images taken by cyclic form of ApoPep-1 after the first round of treatment were inversely correlated with tumor volumes with the strongest agreement (correlation coefficient r.sup.2=0.934, panel C of FIG. 7). The above results showed high correlation, compared with the fluorescence intensities obtained by cyclic form of ApoPep-1 after the second round of treatment (r.sup.2=0.705, panel D of FIG. 7), the fluorescence intensities obtained by linear form of ApoPep-1 after the first round of treatment (r.sup.2=0.631, panel A of FIG. 7), and the fluorescence intensities obtained by linear form of ApoPep-1 after the second round of treatment (r.sup.2=0.402, panel B of FIG. 7). It can be seen through these results that the cyclic form of ApoPep-1 of the present invention can achieve fast diagnosis in tumor response to drugs in an initial stage (even about 1 week) after drug treatment.

Example 5

[0152] Stability of Linear and Cyclic Forms of ApoPep-1 in the Serum

[0153] It was examined whether higher levels of imaging signals by the cyclic form of ApoPep-1 compared with those of the linear form of ApoPep-1 was due to the difference in serum stability of peptides. After incubation of the linear or cyclic form of ApoPep-1 with mouse serum up to 24 hours, the amount of the peptide remaining in the serum was analyzed. The peptide peak was separable from non-specific peaks of serum, and the amount of linear and cyclic forms of peptide remaining in the serum (as calculated by peak area) was not significantly changed up to 24 hours (panel A and panel B of FIG. 8, respectively). MS analysis of each peptide peak confirmed the identity of the linear (FIG. 9A) and cyclic (FIG. 9C) forms of ApoPep-1. These results suggest that both the linear and cyclic forms of ApoPep-1 are stable in the serum up to 24 hours with no difference in stability within the incubation time period, which means that the characteristics in which the cyclic form of ApoPep-1 peptide of the present invention shows significantly improved targeting activity compared with the linear form of peptide are not due to the difference in peptide stability in serum. Therefore, it was suggested that, in cyclic form of ApoPep-1 peptide of the present invention, an artificial structure that binds better to apoptotic cells (histone H1) is generated during the cyclization of the peptide.

INDUSTRIAL APPLICABILITY

[0154] As set forth above, the present invention is directed to a cyclic peptide (cyclo[Cys-Gln-Arg-Pro-Pro-Arg-Cys] peptide) consisting of the amino acid sequence represented by SEQ ID NO: 2 and to a composition comprising the same as an active ingredient for detecting apoptotic cells, delivering a drug, and imaging.

[0155] However, the cyclic peptide (cyclo[Cys-Gln-Arg-Pro-Pro-Arg-Cys] peptide) of the present invention comprising the amino acid sequence represented by SEQ ID NO: 2 has an excellent effect of binding to (or targeting) apoptotic cells as compared with the corresponding linear peptide, thereby facilitating the detection of apoptotic cells and the in vivo imaging of the affected part undergoing apoptosis, while the detection and imaging signals exhibit a very high relevance in view of predicting the prognosis of a disease. Therefore, the cyclic peptide of the present invention can diagnose a response of a therapeutic drug to an abnormal cell proliferation-related disease at an early stage by binding with an imaging material, and can be used for the purpose of selectively delivering a drug to an apoptosis-relating disease tissue by conjugating with a therapeutic material. Accordingly, the present invention is highly industrially applicable.