Composition for preventing or treating side effects caused by anticancer chemotherapy, comprising gv1001 peptide as active ingredient

Abstract

The present invention relates to a composition for use in preventing, treating, or alleviating side effects caused by anticancer chemotherapy using an anticancer chemotherapeutic agent. More specifically, the present invention relates to: a peptide consisting of the amino acid sequence of SEQ ID NO: 1, the peptide having an activity of inhibiting and / or alleviating side effects such as cardiotoxicity, vascular disorders, and inflammation caused by anticancer chemotherapy using an anthracycline-based anticancer agent; and a composition comprising the peptide as an active ingredient. The peptide consisting of the amino acid sequence of SEQ ID NO: 1 and the composition comprising the peptide as an active ingredient according to the present invention can be usefully employed as an anticancer adjuvant capable of alleviating side effects of anticancer chemotherapeutic agents, especially anthracycline-based anticancer agents, and enhancing the effect of anticancer agents.

Description

A composition for the prevention or treatment of side effects caused by anticancer chemotherapy containing GV1001 peptide as an active ingredient

[0001] Cross-reference of related applications

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0123882, provisionally filed on September 11, 2024, and Korean Patent Application No. 10-2025-0128031, filed on September 9, 2025, the full text of which is incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to a composition for preventing, treating, or improving side effects caused by anticancer chemotherapy. More specifically, the present invention relates to a composition for preventing, treating, or improving side effects occurring in patients receiving anticancer chemotherapy using an anthracycline anticancer agent.

[0005] Unlike normal cells, cancer cells exhibit the characteristic of unregulated cell proliferation, leading to continuous growth. Both normal and cancer cells undergo specific stages of cell division and proliferation, during which DNA synthesis is essential. Type II DNA topoisomerase plays a crucial role in maintaining the DNA double helix structure during synthesis; anthracyclines exert an anticancer effect by inhibiting the proliferation of cancer cells through the interference of this enzyme, thereby preventing DNA synthesis. Anthracyclines are a class of anticancer therapies composed of antibiotic compounds and are also referred to as anthracycline anticancer antibiotics or anthracycline anticancer agents. When anthracycline drugs are administered, they produce an anticancer effect by suppressing cancer cells, which proliferate more actively than normal cells, but they also simultaneously inhibit the proliferation of normal cells. Due to this mechanism, systemic side effects such as hair loss, nausea, and vomiting occur during chemotherapy using anthracycline anticancer antibiotics.

[0006] Anthracycline anticancer drugs include daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone. Daunorubicin was the first anthracycline anticancer drug to be developed, followed by doxorubicin as the second. Daunorubicin and doxorubicin or idarubicin share similar basic structural frameworks. Doxorubicin and epirubicin are isomers with the same molecular formula but different structures and physicochemical properties. Epirubicin and idarubicin, which were developed relatively later, tend to be preferred in some chemotherapy regimens due to fewer side effects than daunorubicin or doxorubicin; however, they can all commonly cause side effects such as vomiting, diarrhea, hair loss, stomatitis, and bone marrow suppression, and may also induce cardiac dysfunction. In particular, epirubicin is known to carry a risk of cardiotoxicity, while idarubicin is known to carry a risk of bone marrow suppression and cardiotoxicity.

[0007] In particular, doxorubicin is widely used to treat various malignant tumors, especially resistant and recurrent tumors (Smith, LA; Cornelius, VR; Plummer, CJ; Levitt, G.; Verrill, M.; Canney, P.; Jones, A. Cardiotoxicity of anthracycline agents for the treatment of cancer: Systematic review and meta-analysis of randomised controlled trials. BMC Cancer 2010, 10, 337.). However, vascular disorders such as doxorubicin-induced cardiotoxicity (DIC) and atherosclerosis (Cardinale, D.; Colombo, A.; Bacchiani, G.; Tedeschi, I.; Meroni, CA; Veglia, F.; Civelli, M.; Lamantia, G.; Colombo, N.; Curigliano, G.; et al. Early detection of anthracycline cardiotoxicity and improvement with heart failure therapy. Circulation2015, 131, 1981-.1988.; Armenian, SH; Lacchetti, C.; Barac, A.; Carver, J.; Constine, LS; Denduluri, N.; Dent, S.; Douglas, PS; Durand, JB; Ewer, M.; et al. Prevention and Monitoring of Cardiac Dysfunction in Survivors of Adult Cancers: American Society of Clinical Oncology Clinical Practice Guidelines. J. Clin. Oncol.2017, 35, 893-.911.There are significant difficulties in the clinical application of doxorubicin due to serious side effects, including ). Although the detailed mechanism of DIC is still unclear, mitochondrial dysfunction is considered one of the major causes because doxorubicin preferentially accumulates in mitochondria at concentrations up to 100 times higher than in plasma (Govender, J.; Loos, B.; Marais, E.; Engelbrecht, AM Mitochondrial catastrophe during doxorubicin-induced cardiotoxicity: A review of the protective role of melatonin. J. Pineal Res.2014, 57, 367-380.; Wu, BB; Leung, KT; Poon, EN Mitochondrial-Targeted Therapy for Doxorubicin-Induced Cardiotoxicity. Int. J. Mol. Sci.2022, 23, 1912.). Despite available interventions such as β-blockers and angiotensin II inhibitors, no treatment has demonstrated long-term efficacy in preventing or alleviating DIC (Zamorano, JL; Lancellotti, P.; Rodriguez Munoz, D.; Aboyans, V.; Asteggiano, R.; Galderisi, M.; Habib, G.; Lenihan, DJ; Lip, GYH; Lyon, AR; et al. 2016 ESC Position Paper on cancer treatments and cardiovascular toxicity developed under the auspices of the ESC Committee for Practice Guidelines: The Task Force for cancer treatments and cardiovascular toxicity of the European Society of Cardiology (ESC). Eur.Heart J.2016, 37, 2768-.2801.).

[0008] According to research, doxorubicin induces endothelial-to-mesenchymal transition (EndMT) in vascular endothelial cells, which is characterized by a process in which endothelial characteristics are lost and mesenchymal characteristics are increased (Feng, J.; Wu, Y. Endothelial-to-Mesenchymal Transition: Potential Target of Doxorubicin-Induced Cardiotoxicity. Am. J. Cardiovasc. Drugs2023, 23, 231-246.). EndMT disrupts endothelial barrier function and contributes to both DIC and vascular disorders, such as atherosclerosis. This transition is primarily induced by the TGF-β (transforming growth factor beta) / Smad (suppressor of mothers against decapentaplegic) signaling pathway, proinflammatory cytokines, and the upregulation of transcription factors such as Snail and Twist (Tsai, TH; Lin, CJ; Hang, CL; Chen, WY Calcitriol Attenuates Doxorubicin-Induced Cardiac Dysfunction and Inhibits Endothelial-to-Mesenchymal Transition in Mice. Cells2019, 8, 865.; Grakova, EV; Shilov, SN; Kopeva, KV; Berezikova, EN; Popova, AA; Neupokoeva, MN; Ratushnyak, ET; Teplyakov, AT Anthracycline-Induced Cardiotoxicity: The Role of Endothelial Dysfunction. Cardiology2021, 146, 315-.323.).Oxidative stress and reactive oxygen species (ROS) are known to be major causes of doxorubicin-induced EndMT (Luu, AZ; Chowdhury, B.; Al-Omran, M.; Teoh, H.; Hess, DA; Verma, S. Role of Endothelium in Doxorubicin-Induced Cardiomyopathy. JACC Basic Transl. Sci.2018, 3, 861-.870.; Li, J.; Zhang, Q.; Ren, C.; Wu, X.; Zhang, Y.; Bai, X.; Lin, Y.; Li, M.; Fu, J.; Kopylov, P.; et al. Low-Intensity Pulsed Ultrasound Prevents the Oxidative Stress Induced Endothelial-Mesenchymal Transition in Human Aortic Endothelial Cells. Cell Physiol. Biochem.2018, 45, 1350-.1365.).

[0009] Meanwhile, various anticancer adjuvants are used for purposes such as alleviating the side effects of anticancer drugs, increasing the efficacy of anticancer treatment, and improving the survival rate and quality of life of cancer patients; however, anticancer adjuvants also cause secondary side effects. Interferon and interleukin, which have excellent antitumor and immune-enhancing effects, are protein preparations and have the disadvantage of being expensive. Mesna, used to prevent uremic toxicity, can cause side effects such as nausea, vomiting, decreased appetite, gastrointestinal pain, diarrhea, fever, and dizziness. Aminfostine, a cytoprotective adjuvant used to protect normal cells during anticancer chemotherapy or radiation therapy, has the disadvantage that continuous administration is impossible within 24 hours of administration due to its antihypertensive action.

[0010] Regarding conventionally known anticancer chemotherapy side effect inhibitors and anticancer treatment adjuvants, it has been pointed out as a problem that in the case of natural substances, the active ingredients are often not clearly identified, and in the case of chemical substances, while they reduce the side effects of the target anticancer drug, they cause other toxicities. For example, dexrazoxane, a derivative of EDTA (ethylenediaminetetraacetic acid), is used to prevent cardiotoxicity of anthracycline anticancer drugs including doxorubicin, but transient thrombocytopenia, nausea, vomiting, liver dysfunction, and mutagenicity have been reported (Levine, BS. et al. Cancer Treat Rep 1980, vol.64(12), pp.1211-1215; Von Hoff, DD. et al. Cancer Treat Rep 1981, vol.65(3-4), pp.249-252; Whittaker, P. et al. Environ Mol Mutagen 2001, vol.38(4), pp.347-356; Levine, BS. et al. Cancer Treat Rev 1991, vol.18(4), pp.241-252).

[0011] Therefore, there is a need to develop new substances to alleviate various side effects caused by anticancer chemotherapy using anticancer agents such as anthracycline-based anticancer drugs and / or anticancer treatment adjuvants.

[0012] Meanwhile, the GV1001 peptide, composed of 16 amino acids selected from human telomerase reverse transcriptase (hTERT), has been reported to exhibit anti-inflammatory activity, antioxidant activity, and anticancer activity against some types of cancer, and to be effective in alleviating neurodegenerative diseases, including Alzheimer's disease, and related symptoms. In addition, the toxicity of the GV1001 peptide has been evaluated through numerous clinical trials targeting various diseases, and its safety regarding side effects has been verified.

[0013] However, it is not known at all that GV1001 peptide can improve side effects caused by anticancer chemotherapy agents, particularly anthracycline anticancer agents.

[0014] Surprisingly, the inventors completed the present invention by confirming that the GV1001 peptide can significantly improve side effects caused by anthracycline anticancer drugs, particularly cardiotoxicity, vascular disorders, and inflammation.

[0015]

[0016] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent.

[0017] The objective of the present invention is to provide a health functional food for preventing or improving side effects caused by anticancer chemotherapy using anticancer chemotherapy agents.

[0018] The objective of the present invention is to provide an anticancer adjuvant.

[0019] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising an anticancer adjuvant and an anticancer chemotherapy agent according to the present invention as active ingredients.

[0020] The object of the present invention is to provide a kit for the prevention or treatment of side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent, comprising an anticancer adjuvant and instructions for use according to the present invention.

[0021]

[0022] To achieve the aforementioned objective, the present invention provides a pharmaceutical composition for preventing or treating side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent, comprising as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1 (abbreviated herein as 'GV1001 peptide').

[0023] In addition, to achieve the aforementioned objective, the present invention provides a health functional food for preventing or improving side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent, comprising as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1.

[0024] In addition, to achieve the aforementioned objective, the present invention provides an anticancer adjuvant comprising, as an active ingredient, a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1.

[0025] In addition, to achieve the aforementioned objectives, the present invention provides an anticancer adjuvant comprising an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient, and an anticancer chemotherapy agent comprising an active ingredient, and a pharmaceutical composition for the prevention or treatment of cancer.

[0026] In addition, to achieve the aforementioned objective, the present invention provides a kit for preventing or treating side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent, comprising an anticancer adjuvant containing a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient and instructions for use.

[0027] In addition, to achieve the aforementioned objective, the present invention provides a use of a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1 in the preparation of a composition to be used for preventing, treating, or improving side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent.

[0028] In addition, to achieve the aforementioned objective, the present invention provides a use of a peptide composed of an amino acid sequence represented by SEQ ID NO. 1 in the manufacture of an anticancer adjuvant for use in preventing, treating, or improving side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent.

[0029] In addition, to achieve the aforementioned objective, the present invention provides a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1 and an anticancer adjuvant comprising the same as an active ingredient, for use in the preparation of a composition to be used in combination with an anticancer chemotherapy agent to prevent, treat, or improve cancer.

[0030] In addition, to achieve the aforementioned objective, the present invention provides a use of a peptide composed of an amino acid sequence represented by SEQ ID NO. 1 in the manufacture of a kit to be used to prevent, treat, or improve side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent.

[0031] In addition, to achieve the aforementioned objective, the present invention provides a method for preventing, alleviating, and / or treating side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent in a subject, comprising the step of administering an anticancer adjuvant containing a peptide having an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient to a subject in need thereof.

[0032] The peptide composed of the amino acid sequence represented by SEQ ID NO. 1 of the present invention can effectively suppress and / or alleviate side effects caused by anticancer chemotherapy using anticancer agents, particularly cardiotoxicity, vascular disorders, and inflammation, and can be usefully used as an anticancer adjuvant to alleviate side effects caused by anticancer chemotherapy using anticancer agents and to enhance the anticancer effect.

[0033] Figures 1a–c illustrate the effects of the GV1001 peptide on doxorubicin-induced EndMT: (a) Representative immunofluorescence staining images showing CD31 (red), α-SMA (green), and DAPI (blue) in HUVECs after 48 hours of exposure to doxorubicin (0.25 μM) and / or GV1001 (10 μg / mL). (b, c) Quantification of fluorescence intensity of CD31 and α-SMA by field of view, analyzed using ImageJ. Statistical significance: Significance levels are as follows: ns = no significant difference; ** p < 0.01, **** p < 0.0001 (n = 5 per group). Scale bar: 20 μm.

[0034] Figures 2a-c illustrate the effects of the GV1001 peptide on EndMT induced by long-term exposure to doxorubicin: (a) Representative immunofluorescence staining images showing CD31 (red), α-SMA (green), and DAPI (blue) in HUVECs after approximately 2 weeks of exposure to doxorubicin (0.125 μM) and / or GV1001 (10 μg / mL). (b, c) Quantification of fluorescence intensity of CD31 and α-SMA by field of view, analyzed using ImageJ. Statistical significance: Significance levels are as follows: ns = no significant difference; ** p < 0.01, **** p < 0.0001 (n = 5 per group). Scale bar: 20 μm.

[0035] Figures 3a-c show the effect of the GV1001 peptide on doxorubicin-induced endothelial cell migration in a scratch wound healing assay. After creating a scratch on a culture dish, the dishes were treated for 2 days with GV1001 (10 μg / mL), TNF-α (10 μg / mL), doxorubicin (0.25 μM), TNF-α (10 μg / mL) + GV1001 (10 μg / mL), or doxorubicin (0.25 μM) + GV1001 (10 μg / mL). Representative images of migrated HUVECs were taken on the 3rd day (Figures 3a and 3b), and the number of migrated cells per field of view was quantified and displayed (Figure 3c). Images were taken at 40x magnification.

[0036] Figures 4a-b show the effect of the GV1001 peptide on the migration (mobility) of HUVECs induced by doxorubicin. (a) Representative image showing changes in HUVEC migration (mobility) due to exposure to GV1001 (10 μg / mL), TNF-α (10 μg / mL), doxorubicin (0.25 μM), or TNF-α (10 μg / mL) + GV1001 (10 μg / mL), or doxorubicin (0.25 μM) + GV1001 (10 μg / mL). Evaluated using the Boyden Chamber Assay. Images are shown at 40x magnification. (b) Results of quantifying the number of migrated cells per field of view. Statistical analysis was performed using one-way ANOVA. Statistical significance: The significance levels are as follows: ns = no significant difference; * p < 0.05; *** p < 0.001; **** p < 0.0001 (n = 5 per group).

[0037] Figures 5a-d show the effects of the GV1001 peptide on factors (TGF-β1, TGF-β2, Smad3, Smad4) associated with the overexpression of doxorubicin-induced inflammatory cytokines in HUVECs. The GV1001 peptide reduces the upregulation of doxorubicin-induced TGF-β1, TGF-β2, Smad3, and Smad4 in HUVECs. Statistical analysis was performed using one-way analysis of variance (ANOVA). Statistical significance: The significance levels are as follows: ns = no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Results are expressed as mean ± standard error (SEM). All experiments were performed in triplicate.

[0038] Figures 6a-c show the reversal effect of the GV1001 peptide on the upregulation of IL-1α, IL-1β, and IL-6 expression induced by doxorubicin in HUVECs. Statistical analysis was performed using one-way analysis of variance (one-way ANOVA). Statistical significance: The significance levels are as follows: ns = no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Results are expressed as mean ± standard error (SEM). All experiments were performed in triplicate.

[0039] Figure 7 shows that the GV1001 peptide inhibits the increase in TGF-β1 and FSP1 induced by long-term exposure to doxorubicin in HUVEC.

[0040] Figures 8a–f show the inhibitory effects of the GV1001 peptide on major pro-inflammatory cytokines overexpressed by doxorubicin in macrophages. (a) IL-1α; (b) IL-1β; (c) IL-6; (d) TNF-α; (e) IL-8; and (f) KC (Keratinocyte Chemoattractant).

[0041] Figure 9 summarizes the schedule of the drug administration experiment conducted on 8-week-old male Apolipoprotein E (ApoE)-deficient mice (C57BL / 6) (30 mice) divided into four groups: PBS, doxorubicin, GV1001, and doxorubicin + GV1001.

[0042] Figures 10a-c show the effects of the GV1001 peptide on the nuclear translocation of NF-κB (p65) induced by doxorubicin in HUVECs. (a) Representative immunofluorescence staining images of NF-κB (p65) (green) in HUVECs exposed for 48 hours to doxorubicin (0.25 μM), GV1001 (10 μg / mL), or doxorubicin (0.25 μM) + GV1001 (10 μg / mL). (b) Calculation of the percentage of cells with nuclear NF-κB (p65) per field of view using ImageJ analysis. Statistical significance: Significance levels are as follows: ns = no significant difference; **** p < 0.001. Scale bar: 20 μm. (c) Western blotting results of phosphorylated p65 (p-p65) and p65 in cells administered with vehicle alone, GV1001 (2 μg / mL or 10 μg / mL), doxorubicin (0.25 μM), or GV1001 + doxorubicin combination. All experiments were performed in triplicate.

[0043] Figures 11a–e show the effects of the GV1001 peptide on doxorubicin-induced reactive oxygen species (ROS) accumulation in the cytoplasm and mitochondria. (a, c) Representative immunofluorescence staining images showing intracellular mitochondrial ROS (red) and cytoplasmic ROS (blue) for 48 hours after administration of doxorubicin (0.25 μM), GV1001 (10 μg / mL), or doxorubicin and GV1001 (10 μg / mL). (b, d) ROS staining intensity quantified using ImageJ software. (e) Relative cellular ATP levels. Statistical significance: Significance levels are as follows: ns = no significant difference; * p < 0.05; ** p < 0.01; **** p < 0.0001. Scale bar: 20 μm. All experiments were performed in triplicate.

[0044] Figures 12a–e show the effect of the GV1001 peptide on structural changes in mitochondria induced by doxorubicin. (a) Representative immunofluorescence staining images of mitochondria (red) in fixed HUVECs after administration of doxorubicin (0.25 μM), GV1001 (10 μg / mL), or the combination of doxorubicin (0.25 μM) and GV1001 (10 μg / mL). (b) Mitochondrial length measured using ImageJ analysis. Statistical significance: Significance levels are as follows: ns = no significant difference; *** p < 0.001; and **** p < 0.0001. Scale bar: 20 μm. (c, e) Mitochondrial ferrous (Mito-Fe) of unfixed HUVECs 2+Representative immunofluorescence staining images of ), lipid peroxides (MitoPeDPP staining), and nuclei (Hoechst nucleic acid staining: Hoechst 33342). (d, f) Concentrations of ferrous iron and lipid peroxides quantified using ImageJ analysis. Statistical significance: Significance levels are as follows: ns = no significant difference; *** p < 0.001; and **** p < 0.0001. Scale bar: 10 μm. All experiments were performed in triplicate.

[0045] Figures 13a-b show the effects of the GV1001 peptide on doxorubicin-induced systemic and vascular inflammation in ApoE-deficient mice. (a) Reversal effect of GV1001 peptide administration (2.0 mg / kg; dissolved in PBS and injected intraperitoneally) on the increase in serum inflammatory cytokine levels induced by doxorubicin administration (5.0 mg / kg; intraperitoneal injection) in ApoE-deficient mice. Cytokine levels were measured using ELISA. Statistical analysis was performed using one-way ANOVA. Statistical significance: The significance levels are as follows: ns = no significant difference; * p < 0.05; ** p < 0.01, **** p < 0.0001. (b) Inhibitory effect of GV1001 peptide administration (2.0 mg / kg) on ​​the increased expression of TNF-α, IL-1β, and IL-6 induced by doxorubicin administration (5.0 mg / kg) in mouse arterial tissue. Gene expression was measured by RT-qPCR using Gapdh as a loading control. Statistical analysis was performed using one-way ANOVA. Statistical significance: The significance levels are as follows: ns = no significant difference; * p < 0.05; ** p < 0.01; **** p < 0.0001. Results are presented as mean ± standard error (SEM) for 7–8 samples.

[0046] Figures 14a–d show that the GV1001 peptide inhibits doxorubicin-induced arterial plaque formation in mice and suppresses lipid deposition in the arterial wall and macrophage / monocyte infiltration. (a) Representative photographs of mouse arteries (8–10 mice per group) stained with Oil Red O after longitudinally cutting and flattening the vessels using the en face preparation method following arterial harvesting. (b) Area of ​​the arteries stained with Oil Red O quantified using ImageJ analysis. Statistical significance: Significance levels are as follows: ns = no significant difference; ** p < 0.01; *** p < 0.001. (c) Representative images of the mouse aortic root stained with Oil Red O. Red: Lipid; L: Lumen; LF: Leaflet; P: Plaque; (D) Representative image of mouse aortic root stained with MOMA-2 (red) by immunofluorescence. Nuclei were stained with DAPI (blue). L: Lumen; AW: Arterial Wall; LF: Leaflet; M: Macrophages / Monocytes. Scale bar: 100 μm.

[0047] The present invention will be described in detail below.

[0048] In one embodiment, the present invention relates to a pharmaceutical composition for preventing or treating side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent, comprising as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1.

[0049] The peptide consisting of the amino acid sequence represented by SEQ ID NO. 1 of the present invention is a peptide consisting of 16 amino acids (EARPALLTSRLRFIPK; SEQ ID NO. 1; 'GV1001 peptide').

[0050] The GV1001 peptide of the present invention effectively inhibits the endothelial-mesenchymal transition (EndMT) induced by doxorubicin in HUVECs (Human Umbilical Vein Endothelial Cells), thereby exhibiting activity that inhibits the downregulation of CD31 and the upregulation of α-SMA (Figs. 1a-1c).

[0051] The GV1001 peptide of the present invention inhibits EndMT induced by long-term exposure to doxorubicin, thereby preserving the normal characteristics of endothelial cells (Fig. 2a-c).

[0052] The GV1001 peptide of the present invention preserves the normal characteristics of endothelial cells by inhibiting doxorubicin-induced EndMT, thereby consequently inhibiting the migration (mobility) of HUVECs (Fig. 3a-c).

[0053] The GV1001 peptide of the present invention inhibits the migration of HUVEC cells induced by doxorubicin, thereby preserving the normal characteristics of endothelial cells and preventing the occurrence of arteriosclerosis (atherosclerosis) (Fig. 4a-b).

[0054] The GV1001 peptide of the present invention inhibits the overexpression of related factors (TGF-β1; TGF-β2; Smad3; Smad4) and inflammatory cytokines (IL-1α; IL-1β; and IL-6) induced by doxorubicin in HUVECs (Figs. 5a-5d; Figs. 6a-c).

[0055] The GV1001 peptide of the present invention inhibits the increase in expression of TGF-β1 and FSP1 induced by long-term exposure to doxorubicin in HUVECs (Fig. 7).

[0056] The GV1001 peptide of the present invention effectively inhibits the overexpression of pro-inflammatory cytokines induced by doxorubicin in macrophages, thereby preventing the generation or exacerbation of systemic or local inflammation, and consequently blocks the occurrence or exacerbation of arteriosclerosis (atherosclerosis) (Figs. 8a-f).

[0057] The GV1001 peptide of the present invention inhibits NF-κB(p65) nuclear translocation induced by doxorubicin in HUVECs (Fig. 10a-c).

[0058] The GV1001 peptide of the present invention inhibits the increase in ROS accumulation in mitochondria and cytoplasm that is significantly increased by doxorubicin (Fig. 11a-d), restores cellular ATP levels that are reduced by doxorubicin (Fig. 11e), and alleviates oxidative stress induced by doxorubicin.

[0059] The GV1001 peptide of the present invention effectively inhibits the accumulation of iron ions and lipid peroxides, including structural damage to mitochondria induced by doxorubicin (Fig. 12a-e).

[0060] The GV1001 peptide of the present invention effectively inhibits systemic and vascular inflammation induced by doxorubicin in ApoE-deficient mice (Fig. 13a-b).

[0061] The GV1001 peptide of the present invention effectively inhibits the development of arteriosclerosis (atherosclerosis) by inhibiting lipid deposition and macrophage / monocyte infiltration into the arterial wall induced by doxorubicin (Fig. 14a-d).

[0062] The GV1001 peptide of the present invention is composed of a total of 16 amino acids and has a relatively short sequence, so it can easily cross tissue barriers in the body and has excellent cell permeability. It has a small molecular weight, so it can be easily synthesized, making mass production easy and relatively low production costs.

[0063] The GV1001 peptide of the present invention does not act as an immunogen itself in the body and does not substantially exhibit toxicity, so it can be safely administered to or ingested by subjects for a long period of time.

[0064] The GV1001 peptide of the present invention should be understood as a concept including structural or functional equivalents. The term "structural or functional equivalent" refers to a peptide that exhibits substantially identical physiological activity while having an amino acid sequence that is completely identical to or has a certain sequence identity with respect to the peptide represented by SEQ ID NO. 1. "Identical physiological activity" means exhibiting at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, or more of the physiological activity of the GV1001 peptide. Furthermore, "structural or functional equivalents" include amino acid sequence variants in which some or all of the amino acids constituting the natural (wild) peptide are substituted, or in which some amino acids are deleted or added. The substitution of amino acids is preferably a conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are as follows: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn), and sulfur-containing amino acids (Cys, Met).

[0065] Structural or functional equivalents of the GV1001 peptide of the present invention may consist of or include the amino acid sequence represented by SEQ ID NO. 1. In an exemplary embodiment, 1 to 20, preferably 1 to 10, more preferably 1 to 5 amino acids may be additionally added to the N-terminus and / or C-terminus of the GV1001 peptide, or an acyl group, an amine group, etc. may be introduced to the side chains of some amino acids constituting the GV1001 peptide, or to the N-terminus and / or C-terminus of the peptide.

[0066] The GV1001 peptide of the present invention or its structural or functional equivalents can be prepared by methods known to those skilled in the art. Such peptides can be produced in prokaryotic or eukaryotic cells by expressing a polynucleotide encoding the peptide sequence of the present invention as part of a larger polypeptide. Alternatively, the GV1001 peptide of the present invention or its structural or functional equivalents can be synthesized by chemical methods. Methods for the expression of heterologous proteins in a recombinant host, chemical synthesis of polypeptides, and in vitro transcription are well known in the field and are described in detail in the literature (Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed, Cold Sprin Harbor, NY; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc, San Diego, Calif; Merrifield, J (1969) J Am Chem Soc 91:501; Chaiken IM (1981) CRC Crit Rev Biochem 11: 255; Kaiser et al (1989) Ann Rev Biochem 57:957; and Offord, RE (1980) Semisynthetic Proteins, Wiley Publishing).

[0067] As used herein, the term "anticancer chemotherapy" refers to the use of chemical substances (anticancer agents) to shrink, suppress, or eliminate cancer, and encompasses monotherapy using a single anticancer agent and combination chemotherapy using two or more anticancer agents. "Anticancer chemotherapy" may be combined with hyperthermia using high frequency, far infrared rays, etc., radiation therapy, hormone therapy, etc., if necessary.

[0068] As used herein, the term "anticancer chemotherapy agent" means a substance (generally, an anticancer agent) used to shrink, inhibit, or eliminate cancer, and encompasses anticancer agents used in monotherapy and combinations of anticancer agents used in combination therapy.

[0069] Examples of anticancer chemotherapy agents include, but are not limited to, cytotoxic anticancer agents, targeted anticancer agents, and immunotherapies.

[0070] Cytotoxic anticancer drugs are drugs that kill rapidly dividing cancer cells and directly inhibit the growth of cancer cells by damaging DNA or interfering with cell replication. Representative cytotoxic anticancer drugs include alkylating agents that bind directly to DNA, antimetabolites that inhibit metabolic processes necessary for DNA replication and cell survival, topoisomerase inhibitors, and microtubule-targeting agents.

[0071] Examples of alkylating agents include, but are not limited to, cyclophosphamide, ifosfamide, bendamustine, melphalan, cisplatin, carboplatin, oxaliplatin, busulfan, dacarbazine, and temozolomide.

[0072] Examples of metabolic antagonists include, but are not limited to, fluorouracil, capecitabine, doxifluridine, tegafur, cytarabine, azacitidine, decitamine, enositabine, methotrexate, pemetrexid, pralatrexate, cladribine, cloparabine, fludarabine, and mercaptopurine.

[0073] Examples of DNA rotase inhibitors include, but are not limited to, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, etoposide, irinotecan, and topotecan.

[0074] Examples of microtubule inhibitors include, but are not limited to, cabazitaxel, paclitaxel, docetaxel, vinblastine, vincristine, and vinorelbine.

[0075] Examples of other cytotoxic anticancer agents include, but are not limited to, bleomycin, hydroxyurea, and mitomycin C.

[0076] Targeted anticancer drugs are drugs that exert an anticancer effect by selectively attacking specific molecular biological targets that have played a leading role in the development or growth of cancer. Representative examples include Iressa (gefitinib), Tarceva (erlotinib), Giotrif (afatinib), and Tagrisso (osimertinib), used for lung cancer; Erbitux (cetuximab) and Avastin (bevacizumab), used for colorectal cancer; and Votrient (pazopanib), Sutent (sunitinib), Inlyta (axitinib), and Cabometyx (cabozantinib), used for kidney cancer, but are not limited to these.

[0077] Immunotherapies are a new class of anticancer drugs that act on the human immune system and exert anticancer effects primarily by enhancing the activity of cytotoxic T cells. Representative immunotherapies include drugs belonging to the immune checkpoint inhibitor class, such as Keytruda (pembrolizumab), Opdivo (nivolumab), Yervoy (ipilimumab), and Tecentriq (atezolizumab), but are not limited to these.

[0078] In this specification, the term "anticancer chemotherapy" may be used interchangeably with "anticancer chemotherapy agent."

[0079] In this specification, the term "anticancer chemotherapy agent" may be used interchangeably with "anticancer agent."

[0080] In one embodiment, the anticancer chemotherapy agent may be a cytotoxic anticancer agent, specifically a DNA rotase inhibitor, more specifically an anthracycline anticancer agent. Preferably, it may be one or more selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin, and most preferably, it may be doxorubicin.

[0081] Side effects caused by anticancer chemotherapy / anticancer chemotherapy agents may have occurred from administering anticancer chemotherapy(agents) alone or in combination of two or more at least once.

[0082] Side effects caused by anticancer chemotherapy / anticancer chemotherapy agents may include memory loss, cognitive decline, bone marrow damage, cytopenia due to bone marrow destruction, hair loss, menstrual irregularities, male infertility, stomatitis, vomiting, dysphagia, digestive disorders, diarrhea, neurological disorders, vascular disorders (including atherosclerosis), inflammation (including systemic or local inflammation, vasculitis), skin or nail discoloration, dyspnea, hearing loss, tinnitus, peripheral neuritis, convulsions, hypersensitivity reactions, cardiovascular reactions, cardiotoxicity (including heart failure, arrhythmia, myocarditis, cardiomyopathy), neuromotor toxicity, neurosensory toxicity, myalgia, arthralgia, nausea, fever, anemia, loss of appetite, lethargy, nausea, constipation, fatigue, infection, hematuria, proteinuria, allergy, abdominal cramps, cell necrosis, or tissue necrosis, but are not limited thereto if they can be prevented or treated by the composition of the present invention.

[0083] In one embodiment, the side effect induced by the anticancer chemotherapy(agent) may be at least one of the side effects induced by anticancer chemotherapy using a cytotoxic anticancer agent, specifically a DNA rotase inhibitor, more specifically an anthracycline-based anticancer chemotherapy agent, specifically cardiotoxicity including heart failure, arrhythmia, myocarditis, cardiomyopathy, etc.; vascular disorders including atherosclerosis, etc.; and inflammation including systemic inflammation, local inflammation, vasculitis, etc.

[0084] In one embodiment, side effects induced by anticancer chemotherapy include side effects that occur or worsen after administration of the chemotherapy agent.

[0085] In one embodiment, if there is no prevention or treatment, side effects induced by anticancer chemotherapy(agent) occur or worsen after about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 1 day, about 2 days, about 4 days, about 7 days, about 2 weeks, about 3 weeks, about 1 month, or about 2 months or more after administration of the chemotherapy agent.

[0086] In one embodiment, the GV1001 peptide of the present invention does not substantially affect the therapeutic effect of anticancer chemotherapy.

[0087] In one embodiment, the GV1001 peptide of the present invention or a pharmaceutical composition comprising the same as an active ingredient may be administered once or more times for a predetermined period about 0.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days or more before administration of an anticancer chemotherapy agent.

[0088] In one embodiment, the GV1001 peptide of the present invention or a pharmaceutical composition containing the same as an active ingredient may be administered one or more times about 1 to about 72 hours, about 1 to about 10 days, or about 2 to about 8 days before administration of anticancer chemotherapy.

[0089] As used herein, the term "prevention" refers to any act of inhibiting, suppressing, delaying, or preventing the occurrence, spread, exacerbation, and recurrence of side effects induced by anticancer chemotherapy(agents) by the administration of the pharmaceutical composition according to the present invention.

[0090] As used herein, the term "treatment" means any act of alleviating, suppressing, blocking, improving, or reversing the side effect (or related symptom) itself and / or its aggravation (development) in a subject who has developed or is at risk of developing a side effect induced by anticancer chemotherapy(agent) through the administration of the pharmaceutical composition, or of altering the relevant cancer and related symptoms, side effects caused by anticancer chemotherapy(agent), overall health status, etc., in a manner favorable to the subject's survival.

[0091] As used herein, the expression "using (or using) an anticancer chemotherapy agent" refers to any act of introducing an anticancer chemotherapy agent into the body of a subject by administering (taking), injecting (injecting), or inhaling it through appropriate means (e.g., syringe, inhaler).

[0092] The pharmaceutical composition of the present invention may be provided in a formulation comprising GV1001 peptide or an equivalent thereof as an active ingredient, and optionally one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0093] The pharmaceutical composition of the present invention may contain GV1001 peptide or an equivalent thereof in an amount of about 0.1 μg / mg to about 1.0 mg / mg, about 1 μg / mg to about 0.5 mg / mg, about 1.5 μg / mg to about 0.25 mg / mg, or about 1.5 μg / mg to about 0.1 mg / mg, based on the total weight of the pharmaceutical composition. The pharmaceutical composition of the present invention may contain GV1001 peptide or an equivalent thereof in an amount of about 0.1 μg / ml to about 95.0 mg / ml, about 1 μg / ml to about 94.0 mg / ml, about 1.5 μg / ml to about 93.0 mg / ml, about 2.0 μg / ml to about 92.0 mg / ml, or about 2.5 μg / ml to about 91.0 mg / ml, based on the total volume of the pharmaceutical composition. When included in the above ranges, it is not only appropriate to exhibit the intended anticancer chemotherapy(agent)-induced side effect of the present invention, but also satisfies both stability and safety of the composition, and it may also be appropriate to include it in the above ranges in terms of cost-effectiveness. In some embodiments, the pharmaceutical composition may comprise a GV1001 peptide or an equivalent thereof and a suitable solvent such as physiological saline, PBS (Phosphate-Buffered Saline), water for injection (WFI), etc.

[0094] As used herein, the term "pharmaceuticalally acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. Pharmaceutically acceptable carriers, excipients, or diluents may be used without limitation if they are known and commonly used in the pharmaceutical field as buffers, preservatives, analgesics, solubilizers, isotonics, stabilizers, excipients, lubricants, etc.

[0095] Examples of carriers, excipients, or diluents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc.

[0096] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level may be determined based on factors including the subject's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as a single preparation or in combination with other anticancer agents / therapeutic agents, may be administered sequentially or simultaneously with conventional anticancer agents / therapeutic agents, and may be administered as a single or multiple doses. A person skilled in the art can determine, without particular difficulty, the dosage that obtains maximum effect with the minimum amount without adverse effects by considering all of the above factors.

[0097] In one embodiment, the effective dosage of the pharmaceutical composition of the present invention is, based on the amount of active ingredient to be administered to a subject, for example, 0.1 μg / kg / day to 10.0 mg / kg / day, 0.2 μg / kg / day to 9.0 mg / kg / day, 0.3 μg / kg / day to 8.0 mg / kg / day, 0.4 μg / kg / day to 7.0 mg / kg / day, 0.5 μg / kg / day to 6.0 mg / kg / day, 0.6 μg / kg / day to 5.0 mg / kg / day, 0.7 μg / kg / day to 4.0 mg / kg / day, 0.8 μg / kg / day to 3.5 mg / kg / day, 0.9 μg / kg / day to 3.0 mg / body weight kg / day, 1.0 µg / body weight kg / day to 2.5 mg / body weight kg / day, 1.5 µg / body weight kg / day to 2.0 mg / body weight kg / day, 2.0 µg / body weight kg / day to 1.5 mg / body weight kg / day, 2.5 µg / body weight kg / day to 1.0 mg / body weight kg / day, 3.0 µg / body weight kg / day to 900 µg / body weight kg / day, 3.5 µg / body weight kg / day to 850 µg / body weight kg / day, 4.0 µg / body weight kg / day to 800 µg / body weight kg / day, 4.5 µg / body weight kg / day to 750 µg / body weight kg / day, 5.0 µg / body weight kg / day to 700 µg / body weight kg / day, 5.5 μg / body weight kg / day to 650 μg / body weight kg / day, 6.0 μg / body weight kg / day to 600 μg / body weight kg / day, 6.5 μg / body weight kg / day to 550 μg / body weight kg / day, 7.0 μg / body weight kg / day to 500 μg / body weight kg / day, 7.5 μg / body weight kg / day to 450 μg / body weight kg / day, 8.0 μg / body weight kg / day to 400 μg / body weight kg / day, 8.5 μg / body weight kg / day to 350 μg / body weight kg / day, 9.0 μg / body weight kg / day to 300 μg / body weight kg / day, 10.The effective dosage may be 0 μg / kg / day of body weight to 250 μg / kg / day of body weight, but is not limited thereto, and may vary depending on the patient's body weight, age, disease state, etc. In one embodiment, the pharmaceutical composition of the present invention may be administered once to three times a day, but is not limited thereto, and the number of administrations may vary depending on the patient's body weight, age, disease state, etc.

[0098] As used in this specification, the term “subject” means all animals including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs that require improvement of side effects induced by anticancer chemotherapy(agents) or have had or may have had side effects induced by anticancer chemotherapy(agents).

[0099] In one embodiment, the subject may be a human patient who has cancer or is at risk of developing cancer.

[0100] In one embodiment, the subject may be a human patient who is currently receiving, has received, or is scheduled to receive anticancer chemotherapy.

[0101] The route of administration of the pharmaceutical composition of the present invention may be oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal, but the route of administration may be appropriately selected as needed.

[0102] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0103] When the pharmaceutical composition of the present invention is administered parenterally, it may be administered by external application to the skin or by injection (infusion) methods such as intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or thoracic injection, but the method of administration may be appropriately selected as needed.

[0104] The pharmaceutical composition of the present invention may be formulated into a solid dosage form for oral administration, such as a tablet, pill, powder, granule, or capsule.

[0105] The pharmaceutical composition of the present invention may be formulated as a liquid preparation for oral administration, such as a suspension, liquid preparation, emulsion, or syrup.

[0106] The pharmaceutical composition of the present invention may be formulated as a preparation for parenteral administration, such as a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository.

[0107] In one embodiment, the present invention relates to a health functional food for preventing or improving side effects caused by anticancer chemotherapy, comprising as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1.

[0108] GV1001 peptide, anticancer chemotherapy, anticancer chemotherapy agent, anticancer chemotherapy agent-induced side effects, and prevention are as described above.

[0109] As used in this specification, terms such as "improvement" or similar terms such as "alleviation" refer to any action in which side effects induced by anticancer chemotherapy(agents) are improved or the health condition of the subject is beneficially altered by the intake of the GV1001 peptide according to the present invention or a health functional food containing it.

[0110] The health functional food of the present invention refers to a food manufactured and / or processed in various forms to provide a functional benefit to a subject and to prevent or improve side effects induced by anticancer chemotherapy(agents).

[0111] The health functional food of the present invention encompasses not only food in the conventional sense but also functional food.

[0112] There are no specific restrictions on the types of food that may include the health functional food of the present invention, but for example, the health functional food of the present invention may be included in meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc.

[0113] The health functional food of the present invention includes all forms such as functional food, nutritional supplement, health food, and food additives, and these types of food can be manufactured in various forms according to conventional methods known in the art. For example, as a health food, it may be manufactured in the form of a liquid drink for consumption, or consumed by granulation, encapsulation, spherical tablet (pill, etc.), and powdering, or may be manufactured in the form of powder, capsule, soft capsule, tablet, gum, or adhesive type liquid composition for consumption. In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, jarred fruit, jam, marmalade, etc.), fish, meat and its processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, malt syrup, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable protein, retort foods, frozen foods, medicines, various seasonings (e.g., miso, soy sauce, sauces, etc.).

[0114] The health functional food of the present invention may further include ingredients that are ordinarily added during food manufacturing, provided that such addition does not fall outside the scope of the ultimate purpose of the present invention, for example, may further include proteins, carbohydrates, fats, other nutrients, seasonings, and flavorings.

[0115] The health functional food of the present invention may additionally contain various nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The health functional food of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination.

[0116] The content of the active ingredient in the health functional food of the present invention may be 0.01 to 50 w / w%, 0.1 to 45 w / w%, or 0.5 to 35 w / w% based on the total weight of the health functional food finally manufactured, although this is not limited thereto.

[0117] The content of the active ingredient in the health functional food of the present invention may be 0.01 to 50 w / v%, 0.1 to 45 w / v%, or 0.5 to 35 w / v% based on the total volume of the health functional food finally produced, although this is not limited thereto.

[0118] The effective intake amount of the health functional food of the present invention may be, for example, 1 μg / kg / day to 10 mg / kg / day, 10 μg / kg / day to 1 mg / kg / day, or 50 μg / kg / day to 100 μg / kg / day, but is not limited thereto and may vary depending on various factors such as the age, health condition, and complications of the subject.

[0119] In another aspect, the present invention relates to an anticancer adjuvant comprising, as an active ingredient, a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1.

[0120] As used herein, the term "anticancer adjuvant" means a substance that alleviates side effects caused by the administration of an anticancer agent, more specifically, anticancer chemotherapy(agent).

[0121] By administering the anticancer adjuvant of the present invention in combination with anticancer chemotherapy, the occurrence of various side effects caused by anticancer chemotherapy can be prevented or improved. The anticancer adjuvant of the present invention may be administered simultaneously, separately, or sequentially with the anticancer drug. The order of administration of the anticancer adjuvant according to the present invention—that is, which of the anticancer drug and the anticancer adjuvant to administer at what point in time, simultaneously, individually, or sequentially—can be determined by a physician or expert without any particular difficulty.

[0122] In another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising an anticancer adjuvant and an anticancer chemotherapy agent comprising a GV1001 peptide as an active ingredient.

[0123] Since GV1001 peptide, anticancer adjuvant, and anticancer chemotherapy agent are identical to the definitions above, they are omitted from the description in the overlapping scope.

[0124] Examples of cancer include acute leukemia, lymphoma, germ cell tumors, gestational trophoblastic disease, childhood cancers (Ewing sarcoma, rhabdomyosarcoma, Wilms tumor), anal cancer, bladder cancer, breast cancer, chronic leukemia, head and neck tumors, small cell lung cancer, multiple myeloma, ovarian cancer, colorectal cancer, uterine cancer, brain tumor, liver cancer, lung cancer, stomach cancer, melanoma, pancreatic cancer, etc., but are not limited to these.

[0125] In one embodiment, the cancer may be a blood cancer, e.g., acute leukemia, lymphoma, etc., to which an anthracycline anticancer drug is prescribed; or a solid tumor, e.g., breast cancer, lung cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, sarcoma of soft tissue and bone, bladder cancer, etc.

[0126] The pharmaceutical composition of the present invention may include an anticancer adjuvant and an anticancer chemotherapy agent, each in an amount of at least 0.1% by weight and up to 50% by weight, based on the total weight of the composition.

[0127] In another aspect, the present invention relates to a kit for preventing or treating side effects caused by anticancer chemotherapy using an anticancer chemotherapy agent, comprising an anticancer adjuvant containing a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient and instructions for use.

[0128] Since GV1001 peptide and anticancer adjuvants are identical to the definitions above, descriptions are omitted in the overlapping scope.

[0129] In one embodiment, the anticancer chemotherapy agent may be a cytotoxic anticancer agent, specifically a DNA rotase inhibitor, more specifically an anthracycline anticancer agent. Preferably, it may be one or more selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin, and most preferably, it may be doxorubicin.

[0130] The instruction manual may be a means of providing detailed explanations regarding one or more of the dosage, administration route, frequency of administration, and precautions of the anticancer adjuvant, as well as information on the proper use and storage method of the kit.

[0131] In one embodiment, the instruction manual may be provided in a printed form using text, pictures, photographs, diagrams, illustrations, etc., or in an electronic (digitized) form using barcodes or holograms, etc.

[0132] Specific embodiments of the present invention are as follows:

[0133] Example 1. A pharmaceutical composition for preventing or treating side effects caused by anticancer chemotherapy using an anthracycline-based anticancer agent, comprising as an active ingredient a peptide consisting of the amino acid sequence represented by SEQ ID NO. 1.

[0134] Example 2. A pharmaceutical composition for preventing or treating side effects caused by anticancer chemotherapy using an anthracycline anticancer agent, wherein the anthracycline anticancer agent of Example 1 is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin.

[0135] Example 3. A pharmaceutical composition for preventing or treating side effects caused by anticancer chemotherapy using an anthracycline-based anticancer agent, wherein the side effects caused by anticancer chemotherapy using an anthracycline-based anticancer agent in Example 1 are at least one of cardiotoxicity, vascular disorder, and inflammation.

[0136] Embodiment 4. A pharmaceutical composition for the prevention or treatment of side effects induced by anticancer chemotherapy using an anthracycline anticancer agent, wherein in Embodiment 3, cardiotoxicity includes heart failure, arrhythmia, myocarditis, and cardiomyopathy; vascular disorder includes atherosclerosis; and inflammation includes systemic inflammation, local inflammation, and vasculitis.

[0137] Example 5. A pharmaceutical composition for the prevention or treatment of side effects induced by anticancer chemotherapy using an anthracycline anticancer agent, wherein the peptide of Example 1 exhibits at least one activity selected from the group consisting of: an activity that inhibits endothelial-mesenchymal transition (EndMT) in Human Umbilical Vein Endothelial Cells (HUVEC); an activity that inhibits the overexpression of inflammatory cytokines; an activity that inhibits the increase in TGF-β1 and FSP1 expression; an activity that prevents the generation or exacerbation of systemic or local inflammation; an activity that inhibits NF-κB(p65) nuclear transition in HUVEC; an activity that inhibits the increase in ROS accumulation in mitochondria and cytoplasm; an activity that restores reduced cellular ATP levels; an activity that inhibits structural damage to mitochondria; an activity that inhibits the accumulation of iron ions and lipid peroxides; an activity that inhibits lipid deposition into the arterial wall; and an activity that inhibits the infiltration of macrophages and monocytes into the arterial wall.

[0138] Example 6. A health functional food composition for preventing or improving side effects induced by anticancer chemotherapy using an anthracycline-based anticancer agent, comprising as an active ingredient a peptide consisting of the amino acid sequence represented by SEQ ID NO. 1.

[0139] Embodiment 7. A health functional food for preventing or improving side effects caused by anticancer chemotherapy using an anthracycline anticancer agent, wherein in Embodiment 6, the anthracycline anticancer agent is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin.

[0140] Embodiment 8. A health functional food for preventing or improving side effects caused by anticancer chemotherapy using an anthracycline anticancer agent, wherein the side effects caused by anticancer chemotherapy using an anthracycline anticancer agent in Embodiment 6 are at least one of cardiotoxicity, vascular disorder, and inflammation.

[0141] Embodiment 9. A health functional food for preventing or improving side effects induced by anticancer chemotherapy using an anthracycline anticancer agent, wherein in Embodiment 8, cardiotoxicity includes heart failure, arrhythmia, myocarditis, and cardiomyopathy; vascular disorder includes atherosclerosis; and inflammation includes systemic inflammation, local inflammation, and vasculitis.

[0142] Embodiment 10. The health functional food for preventing or improving side effects induced by anticancer chemotherapy using an anthracycline anticancer agent, wherein the peptide of Embodiment 6 exhibits at least one activity selected from the group consisting of: an activity that inhibits endothelial-mesenchymal transition (EndMT) in Human Umbilical Vein Endothelial Cells (HUVEC); an activity that inhibits the overexpression of inflammatory cytokines; an activity that inhibits the increase in TGF-β1 and FSP1 expression; an activity that prevents the generation or exacerbation of systemic or local inflammation; an activity that inhibits NF-κB(p65) nuclear transition in HUVEC; an activity that inhibits the increase in ROS accumulation in mitochondria and cytoplasm; an activity that restores reduced cellular ATP levels; an activity that inhibits structural damage to mitochondria; an activity that inhibits the accumulation of iron ions and lipid peroxides; an activity that inhibits lipid deposition into the artery wall; and an activity that inhibits the infiltration of macrophages and monocytes into the artery wall.

[0143] Example 11. An anticancer adjuvant for alleviating side effects induced by anthracycline-based anticancer drugs, comprising a peptide having the amino acid sequence of SEQ ID NO. 1 as an active ingredient.

[0144] Embodiment 12. An anticancer adjuvant of Embodiment 11, wherein the anticancer adjuvant is administered simultaneously, separately, or sequentially with an anthracycline anticancer agent.

[0145] Example 13. An anticancer adjuvant according to Example 11, wherein the anthracycline anticancer agent is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin.

[0146] Example 14. An anticancer adjuvant of Example 13, wherein the anthracycline-based anticancer agent is doxorubicin.

[0147] Example 15. A pharmaceutical composition for the prevention or treatment of cancer, comprising the anticancer adjuvant of Example 6 and an anthracycline anticancer agent.

[0148] Example 16. A pharmaceutical composition for preventing or treating cancer, wherein the anthracycline anticancer agent of Example 15 is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin.

[0149] Example 17. A pharmaceutical composition for the prevention or treatment of cancer, wherein the anthracycline anticancer agent of Example 16 is doxorubicin.

[0150] Example 18. A kit for preventing or treating side effects caused by anticancer chemotherapy using an anthracycline-based anticancer agent, comprising an anticancer adjuvant containing a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient and instructions for use.

[0151] Embodiment 19. A kit for preventing or treating side effects caused by anticancer chemotherapy using an anthracycline anticancer agent, wherein the anthracycline anticancer agent of Embodiment 18 is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin.

[0152] Embodiment 20. A kit for preventing or treating side effects caused by anticancer chemotherapy using an anthracycline anticancer agent, wherein the side effects caused by anticancer chemotherapy using an anthracycline anticancer agent in Embodiment 18 are at least one of cardiotoxicity, vascular disorder, and inflammation.

[0153] Embodiment 21. A kit for preventing or treating side effects induced by anticancer chemotherapy using an anthracycline anticancer agent, wherein in Embodiment 18, cardiotoxicity includes heart failure, arrhythmia, myocarditis, and cardiomyopathy; vascular disorder includes atherosclerosis; and inflammation includes systemic inflammation, local inflammation, and vasculitis.

[0154] Example 22. A kit for preventing or treating side effects induced by anticancer chemotherapy using an anthracycline anticancer agent, wherein the peptide of Example 18 exhibits at least one activity selected from the group consisting of: an activity that inhibits endothelial-mesenchymal transition (EndMT) in Human Umbilical Vein Endothelial Cells (HUVEC); an activity that inhibits the overexpression of inflammatory cytokines; an activity that inhibits the increase in TGF-β1 and FSP1 expression; an activity that prevents the generation or exacerbation of systemic or local inflammation; an activity that inhibits NF-κB(p65) nuclear transition in HUVEC; an activity that inhibits the increase in ROS accumulation in mitochondria and cytoplasm; an activity that restores reduced cellular ATP levels; an activity that inhibits structural damage to mitochondria; an activity that inhibits the accumulation of iron ions and lipid peroxides; an activity that inhibits lipid deposition into the artery wall; and an activity that inhibits the infiltration of macrophages and monocytes into the artery wall.

[0155] Example 23. In the preparation of a composition for use in preventing, treating, or improving side effects induced by anticancer chemotherapy using an anticancer chemotherapy agent, the use of a peptide comprising the amino acid sequence represented by SEQ ID NO. 1.

[0156] Example 24. Use of a peptide composed of an amino acid sequence represented by SEQ ID NO. 1 in the preparation of an anticancer adjuvant for use in preventing, treating, or improving side effects induced by anticancer chemotherapy using an anticancer chemotherapy agent.

[0157] Example 25. Use of a peptide consisting of the amino acid sequence represented by SEQ ID NO. 1 and an anticancer adjuvant comprising the same as an active ingredient in the preparation of a composition for use in combination with an anticancer chemotherapy agent to prevent, treat, or improve cancer.

[0158] Example 26. Use of a peptide composed of an amino acid sequence represented by SEQ ID NO. 1 in the manufacture of a kit for use in preventing, treating, or improving side effects induced by anticancer chemotherapy using an anticancer chemotherapy agent.

[0159] Embodiment 27. A method for preventing, alleviating, and / or treating side effects caused by anticancer chemotherapy using an anticancer agent in a subject, comprising the step of administering an anticancer adjuvant containing a peptide having an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient to a subject in need thereof.

[0160]

[0161] The terms used in this specification are intended solely for the purpose of describing specific embodiments and are not intended to limit the invention. Terms in which a number is omitted before a noun are not intended to limit the quantity, but rather to indicate that there is one or more objects referred to by the mentioned noun. The terms "comprising," "having," and "containing" are interpreted as open terms.

[0162] In this specification, numeric ranges are merely a convenient alternative to individually referring to each number within the range, and unless otherwise specified, each number should be considered as if it were individually referred to. The end values ​​of all numeric ranges are included within the range and can be combined independently.

[0163] In this specification, the term "approximately" used to describe length, area, volume, time (period), concentration, content, etc., means that there is a tolerance of up to ±20%, and generally ±10%, for the corresponding numerical value or numerical range.

[0164] All methods mentioned herein may be performed in an appropriate order unless otherwise specified or clearly contradictory by the context. The use of any one embodiment and all embodiments or exemplary language (e.g., “like”) is merely to better describe the invention, not to limit the scope of the invention, unless otherwise included in the claims. No language in the specification shall be interpreted as implying that any unclaimed component is essential to the practice of the invention. Unless otherwise defined, technical and scientific terms used herein have the same meaning as generally understood by a person of ordinary skill in the art to which the invention pertains (i.e., a person skilled in the art).

[0165] The preferred embodiment(s) of the present invention include the most optimal mode known to the inventor for carrying out the present invention. Modifications or variations of the preferred embodiment(s) may become apparent to a person skilled in the art upon reading the preceding description. The inventors expect that a person skilled in the art will make appropriate use of such modifications or variations, and the inventors expect the present invention to be practiced in a manner different from that described herein. Accordingly, the present invention includes all modifications and / or variations, including equivalents of the essence of the invention mentioned in the appended claims, as permitted by patent law. Furthermore, any combination of the aforementioned components within all possible modifications and / or variations is included within the scope of the present invention unless otherwise specified herein or clearly contradictory in the context. Although the present invention has been specifically shown and described with reference to exemplary embodiment(s), a person skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the following claims.

[0166]

[0167] Hereinafter, the present invention will be described in detail based on examples and experimental examples to specifically explain the invention. However, the following examples and experimental examples are provided merely to facilitate a better understanding of the invention, and the content of the invention is not limited by the following examples and experimental examples.

[0168] [Example]

[0169] 1. Preparation of GV1001 Peptide

[0170] A peptide composed of 16 amino acids having the structural formula of Chemical Formula 1 below, represented by SEQ ID NO. 1 (GV1001) selected from human telomerase, was prepared.

[0171] <Chemical Formula 1>

[0172] Peptide GV1001 of SEQ ID NO. 1 was prepared according to a conventionally known solid-phase peptide synthesis method. Specifically, the peptides were synthesized by coupling amino acids one by one starting from the C-terminus via Fmoc solid-phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, South Korea). As follows, peptides with the first amino acid of the C-terminus attached to a resin were used. For example, as follows:

[0173] NH2-Lys(Boc)-2-chloro-Trityl Resin

[0174] NH2-Ala-2-chloro-Trityl Resin

[0175] NH2-Arg(Pbf)-2-chloro-Trityl Resin

[0176] All amino acid raw materials used for peptide synthesis were protected at the N-terminus with Fmoc, and the residues were protected with Trt, Boc, t-Bu(t-butylester), Pbf(2,2,4,6,7-pentamethyl dihydro-benzofuran-5-sulfonyl), etc., which are removed by acid. For example, as follows:

[0177] Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ahx-OH, Trt-Mercaptoacetic acid.

[0178] HBTU[2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetamethylaminium hexafluorophosphate] / HOBt[N-Hydroxybenzotriazole] / NMM[4-Methylmorpholine] was used as the coupling reagent. Piperidine in 20% DMF was used for Fmoc removal. A cleavage cocktail [TFA(trifluoroacetic acid) / TIS(triisopropylsilane) / EDT(ethanedithiol) / H2O=92.5 / 2.5 / 2.5 / 2.5] was used to separate the synthesized peptide from the resin and remove the protecting group from the residue.

[0179] Each peptide was synthesized by utilizing a state in which starting amino acids with protecting groups were bound to a solid support, reacting the corresponding amino acids with the support, washing with a solvent, and repeating the deprotection process. The synthesized peptides were cleaved from the resin, purified by HPLC, confirmed to be synthesized by MS, and freeze-dried.

[0180] The specific manufacturing process of the GV1001 peptide is described as follows.

[0181] 1) Coupling step: An amino acid (8 equivalents) protected on NH2-Lys(Boc)-2-chloro-Trityl Resin and coupling reagents HBTU (8 equivalents) / HOBt (8 equivalents) / NMM (16 equivalents) were dissolved in DMF and added, reacted at room temperature for 2 hours, and washed in the order of DMF, MeOH, and DMF.

[0182] 2) Fmoc deprotection step: Piperidine in 20% DMF was added and reacted twice for 5 minutes at room temperature, and washed in the order of DMF, MeOH, and DMF.

[0183] 3) Basic backbone formation step: The reactions of steps 1) and 2) were repeated to form a peptide backbone.

[0184] 4) Cleavage / separation step: A cleavage cocktail was added to the synthesized peptide resin to separate the peptide from the resin.

[0185] 5) Precipitation step: Cooling diethyl ether was added to the obtained mixture, and the peptide obtained by centrifugation was precipitated.

[0186] 6) Purification / Verification / Drying Step: After purification by Prep-HPLC and confirmation of molecular weight by LC / MS, the peptide was finally obtained as a powder by freeze-drying.

[0187] [Experimental Example]

[0188] Materials and Methods

[0189] 1. Cell culture

[0190] Human umbilical vein endothelial cells (HUVEC; #C2519A, Lonza, Basel, Switzerland) were cultured using endothelial basal medium-2 (EBM-2) and the EGM-2 SingleQuot Kit (#CC-4176, Lonza, Morristown, NJ, USA). The medium was changed every 2 days, and HUVECs were cultured at 37°C in a humid environment with 5% v / v CO2.

[0191] 2. Experimental Animals and Administration Method

[0192] Thirty 8-week-old male apolipoprotein E (ApoE)-deficient mice (C57BL / 6) (body weight 30 g ± 2) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All mice were fed a high-fat diet (HFD; #D12079B, Research Diets, New Brunswick, NJ, USA). The health and behavior of the mice were monitored three times a week during the experiment. The mice were divided into four groups.

[0193] · Group 1 (n=7): PBS (Phosphate-Buffered Saline) intraperitoneally (ip), administered 3 times a week for 9 weeks.

[0194] · Group 2 (n=7): GV1001 peptide (2.0 mg / kg; dissolved in PBS) administered intraperitoneally, 3 times a week for 9 weeks.

[0195] · Group 3 (n=8): Doxorubicin (#25316409, MedChemExpress LLC, Monmouth Junction, NJ, USA) (5.0 mg / kg) intraperitoneally, once a week for 8 weeks, and PBS three times a week for 9 weeks.

[0196] · Group 4 (n=8): GV1001 peptide (2.0 mg / kg; dissolved in PBS) intraperitoneally, 3 times a week, for 9 weeks and doxorubicin (5.0 mg / kg) intraperitoneally, once a week, for 8 weeks.

[0197] Doxorubicin was administered once a week by intraperitoneal injection at a dose of 5.0 mg / kg, as this dose has been proven to be suitable for studies on doxorubicin-induced atherosclerosis in mice in other studies.

[0198] As schematically shown in Fig. 9, PBS or GV1001 peptide was administered three times a week, and doxorubicin administration was started one week after the injection of PBS or GV1001 peptide, with a prescribed dose administered once a week. Intraperitoneal (ip) injection of PBS or GV1001 peptide (2.0 mg / kg) was started at a dose of 0.1 mL one week prior to doxorubicin injection. GV1001 (2.0 mg / kg) was injected intraperitoneally three times a week.

[0199] 3. Sample and Tissue Collection

[0200] Mice were sacrificed after completing injections of PBS, GV1001, or doxorubicin under general anesthesia, and whole blood, whole arteries, hearts, and spleens were collected for analysis as previously reported (Kim, SY; Kim, YJ; Kim, S.; Momeni, M.; Lee, A.; Treanor, A.; Kim, S.; Kim, RH; Park, NH GV1001 Inhibits the Severity of the Ligature-Induced Periodontitis and the Vascular Lipid Deposition Associated with the Periodontitis in Mice. Int. J. Mol. Sci.2023, 24, 12566.). Whole blood was collected from mice via cardiac puncture under isoflurane anesthesia (#G46D22, VetOne, Boise, ID, USA). Then, mice were perfused through the left ventricle for 5 minutes with 4% paraformaldehyde (#158127, Millipore Sigma, Burlington, MA, USA) dissolved in phosphate-buffered saline (PBS). After perfusion, the entire aorta, dilated to the iliac bifurcation, was exposed, and the surrounding tissue was carefully incised and preserved in RNAlater (#AM7020, Thermo Fisher Scientific, Waltham, MA, USA). Additionally, the spleen was resected and the tissue was divided into two parts. One part was fixed in PBS (pH 7.4) containing 4% paraformaldehyde, and the other part was stored in RNAlater at -80°C for analysis of inflammatory cytokine expression.

[0201] 4. Frozen slices

[0202] Cardiac samples were embedded in freezing molds using Tissue-Tek OCT complex (#M71484, Sakura Finetek, Torrence, CA, USA) and stored at -80°C until frozen sections were prepared. The frozen cardiac blocks were sectioned to a thickness of 10 μm at -20°C using a Cryostar NX70 cryostat (Thermo Fisher Scientific, Waltham, MA, USA) according to the previously described procedure (Kim, S.; Bando, Y.; Chang, C.; Kwon, J.; Tarverti, B.; Kim, D.; Lee, SH; Ton-That, H.; Kim, R.; Nara, PL; et al. Topical application of Porphyromonas gingivalis into the gingival pocket in mice leads to chronic-active infection, periodontitis and systemic inflammation. Int. J. Mol. Med.2022, 50, 5159.).

[0203] 5. Histology and Immunofluorescence (IF) Analysis

[0204] For immunofluorescence staining, fixed or live cells, paraffin sections of the spleen, or frozen sections of the aortic roots were stained with primary antibodies, such as CD31 (cluster differentiation 31, #ab28364, Abcam, Cambridge, UK), α-SMA (alpha-smooth muscle actin, #A2547, Millipore Sigma, Burlington, MA, USA), TNF-α (#ab6671, Abcam, Cambridge, UK), p65 (#SC8008, Santa Cruz Biotechnology, Dallas, TX, USA), p-p65 (#CST3036, Cell Signaling, Danvers, MA, USA), Mitosox Red Mitochondrial Superoxide Indicator (#M36008, Invitrogen, Carlsbad, CA, USA), Fluorometric Intracellular ROS probe (#MAK143, Millipore Sigma, Burlington, MA, USA), and MitoTracker Red CMXRos (#CST9082, Cell Signaling Technology, Danvers, MA, USA), BioTracker™ Mitochondrial FerroGreen live cell probe (Mito-FerroGreen) (#SCT262, EMD Millipore Corp.The cells were cultured with the BioTracker Mitochondrial Lipid Peroxide Live Cell Ferroptosis Indicator (#Mct261, Millipore Sigma, Burlington, MA, USA), and MOMA-2 (#MCA519G, Bio-Rad, Hercules, CA, USA), followed by fluorescence detection using Alexa Fluor 488 or 546-conjugated secondary antibodies (#A11029 or #A11010, Thermo Fisher Scientific, Waltham, MA, USA, respectively). Sequentially, sections were mounted on slides using VECTASHIELD™ anti-fade mounting medium and DAPI (4′,6-diamidino-2-phenylindole) (#H1200, Vector Laboratories, Burlington, CA, USA). Immunofluorescence images were taken using a confocal fluorescence microscope (#LSM 700, Carl Zeiss, Oberkochen, Germany). All experiments were performed according to the manufacturer's instructions.

[0205] 6. Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)

[0206] Total RNA was extracted from HUVEC and mouse tissues and reverse transcribed, followed by qPCR. Total RNA was extracted from mouse aorta and spleen tissues using the RNeasy Micro Kit (#74004, Qiagen, Valencia, CA, USA) and reverse transcribed using the SuperScript® III Reverse Transcriptionase Synthesis Kit (#18080044, Thermo Fisher Scientific, Waltham, MA, USA) following the following steps: 5 minutes at 65°C, 2 minutes at 25°C, and 1 hour at 45°C. Then, quantitative PCR (qPCR) was performed using PowerUp™ SYBR-Green Master Mix (#A25742, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as a loading control. Fold induction was calculated using the comparative ΔCq method and relative transcription levels (2 -ΔΔCq It was represented as ). The primer sequences used for RT-qPCR are shown in Table 1.

[0207] GeneForward primer 5'-3'Reverse primer 5'-3'mIL-1βCACAGCAGCACATCAACAAGGTGCTCATGTCCTCATCCTGmTNF-αTCAGGTTGCCTCTGTCTCAGGCTCTGTGAGGAAGGCTGTGmIL-6TGGGACTGATGCTGGTGACAGCCTCCGACTTGTGAAGTGGTmGapdhAGCTTGTCATCAACGGGAAGTTTGATGTTAGTGGGGTCTCGhTGF-β1TACCTGAACCCGTGTTGCTCTCGTTGCTGAGGTATCGCCAGGAAhTGF-β2AAGAAGCGTGCTTTGGATGCGGATGCTCCAGCACAGAAGTTGGChSmad3TGAGGCTGTCTACCAGTTGACCGTGAGGACCTTGTCAAGCCACThSmad4CTACCAGCACTGCCAACTTTCCCCTGATGCTATCTGCAACAGTCChIL-1αTGTGACTGCCCAAGATGAAGCGTGAGTTTCCCAGAAGAAGAGhIL-1βATGGACAAGCTGAGGAAGATGCCCATGTGTCGAAGAAGATAGGhIL-6GGAGACTTGCCTGGTGAAACTGGCTTGTTCCTCACTACTChGapdhAGCCACATCGCTCAGACACGCCCAATACGACCAAATCC

[0208] 7. 엔 페이스(En Face) 분석

[0209] After exposing the entire length of the aorta from the aortic arch to the iliac artery bifurcation along the midline of the abdomen of the mouse, the aorta was dissected from the surrounding tissue under a stereomicroscope (Zeiss, Stemi 305, Oberkochen, Germany). The dissected aorta was stained with Sudan IV (#198102, Sigma-Aldrich, St. Louis, MO, USA) and fixed flat with the intima facing upward. Images of the aorta were captured using a Nikon digital camera (Nikon D7500 DSLR Camera, Tokyo, Japan). The captured images were analyzed using ImageJ Software version 1.48 (NIH, accessed on 11 May 2023, Bethesda, MD, USA).

[0210] 8. Induction of EndMT

[0211] HUVECs were seeded into the chamber wells of a 4-well chamber slide (#C6932, Lab-Tek II, Nunc, Rochester, NY, USA). Cells were treated with doxorubicin (0.25 μM) or 10 μg / mL GV1001 in endothelial growth medium for 2 days. After treatment, cells were fixed in ice-cold 100% methanol (#L13255, Thermo Fisher Scientific, Waltham, MA, USA) for 10 minutes and permeated with 0.1% Tween 20 (#P2287, Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes. Then, cells were blocked with 5% normal goat serum at room temperature for 1 hour. Primary antibodies (CD31, cluster differentiation 31, #ab28364, Abcam, Cambridge, UK; and α-SMA, alpha-smooth muscle actin, A2547, Burlington, MA, USA) were applied overnight at 4°C. Fluorescently labeled secondary antibodies were applied for 1 hour at room temperature (Alexa Fluor 546-conjugated secondary antibody (#A11010, Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 488-conjugated secondary antibody (#A11029, Thermo Fisher Scientific, Waltham, MA, USA). Cells were washed three times with PBS and mounted using VECTASHIELD™ anti-fade mounting medium containing DAPI (#H1200, Vector Laboratories, Burlingame, CA, USA). Immunostaining images were taken using a confocal microscope (Carl Zeiss, LSM 700, Oberkochen, Germany).

[0212] 9. Western Blotting

[0213] Western blot was performed on whole-cell extracts (WCE) of cultured HUVECs. WCEs were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer (#89901, Thermo Fisher Scientific, Waltham, MA, USA). After fractionating the WCEs by SDS-PAGE, they were transferred to Immobilon®-P membranes (#IPVH00010, Millipore, Billerica, MA, USA). The membranes were sequentially incubated with primary and secondary antibodies, and then exposed to a chemiluminescent reagent (#1705061, Bio-Rad, Hercules, CA, USA) for signal detection.

[0214] 10. Enzyme Immunoassay (ELISA)

[0215] Serum concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were measured by enzyme immunoassay (ELISA) using commercially available kits (#BMS607-2HS, #BMS6002, or #BMS603, respectively; Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. The colorimetric reaction was stopped with a stop solution (#423001, BioLegend, San Diego, CA, USA), and absorbance was measured immediately at 450 nm using a plate reader (Bio-Rad Laboratories, PR4100, Hercules, CA, USA). Standard curves were constructed by plotting standard concentrations against absorbance values, and cytokine levels were calculated in pg / mL.

[0216] 11. Cell migration analysis

[0217] For scratch wound healing analysis, 1.5 × 10⁶ HUVECs were placed in starvation medium containing 0.5% fetal bovine serum (FBS, #10082147, Thermo Fisher Scientific, Waltham, MA, USA). 5Cells were seeded into 12-well plates at a density of cells / mL. Cells were incubated at 37°C and 5% CO2 for 24 hours, followed by pretreatment with 10 μg / mL GV1001 for 3 hours. After treatment, HUVECs were scraped using a sterile 200 μL pipette tip. The cells were then washed with PBS to remove residue. After treatment with 50 ng / mL TNF-α (#210-TA, R&D Systems, Minneapolis, MN, USA) or 0.25 μM doxorubicin at 37°C for 48 hours, the distance between the scraped areas was observed microscopically. Images were captured using an inverted microscope (Olympus, Tokyo, Japan). A Transwell system with a polycarbonate filter (8.0 μm pore size, #3422, Corning, NY, USA) was used for migration analysis. HUVECs were pretreated with 10 μg / mL GV1001 for 3 hours and then transferred to the upper chamber of a Transwell containing medium with 0.5% FBS. The lower chamber was filled with culture medium containing 50 ng / mL TNF-α or 0.25 μM doxorubicin. After 24 hours of incubation, cells that had not migrated from the upper chamber were removed with a cotton swab. Cells that had migrated to the underside of the membrane were fixed, stained with 1% crystal violet (#C6158, Sigma-Aldrich, St. Louis, MO, USA), and photographs were taken. Wound gap areas and the number of migrated cells were analyzed using ImageJ Software version 1.48 (NIH, Bethesda, MD, USA).

[0218] 12. ATP Detection Analysis

[0219]

[0220] 1.5 × 10⁶ HUVEC in 2 mL of EGM-2 medium 5Cells were seeded into 6-well plates at a cell / well density. GV1001 (10 μg / mL) was treated with or without doxorubicin (0.25 μg / mL) for 48 hours. Intracellular ATP content was measured using the ATP Detection Assay Kit (#700410, Cayman Chemical, Ann Arbor) according to the manufacturer's protocol. After mixing the cell lysate with the reaction mixture, ATP concentration was determined by measuring luminescence in a 96-well plate. In summary, cells were washed with pre-chilled PBS, and 1 mL of 1x ATP detection sample buffer was added to each well. The cells were homogenized by pipetting the buffer up and down several times, and the cell lysate was transferred to a pre-chilled tube. The reaction mixture was prepared by mixing D-luciferin and luciferase with 1x ATP detection assay buffer. 100 μL of the freshly prepared reaction mixture and 10 μL of cell lysate were added three times to a white, opaque 96-well plate. The plate was covered and incubated at room temperature for 20 minutes. ATP concentration was determined by measuring luminescence using a Synergy H1 multimode microplate reader (BioTek Instruments, Winooski, VT, USA).

[0221] 13. Statistical Analysis

[0222] All graphs were created using GraphPad Prism Software, and statistical analysis results were calculated using GraphPad Prism 9 (GraphPad Software, Boston, MA, USA). One-way analysis of variance (ANOVA) with the Newman-Keuls test was used for multiple comparisons. A p-value less than 0.05 was considered significant. All in vitro results were verified through at least three independent experiments. Error bars represent the mean ± standard deviation.

[0223] result

[0224] 1. GV1001 peptide inhibits doxorubicin-induced EndMT in HUVECs.

[0225] Numerous studies have demonstrated that atherosclerosis is initiated by vascular endothelial dysfunction, particularly through EndMT (endothelial-to-mesenchymal transition) (Chen, PY; Schwartz, MA; Simons, M. Endothelial-to-Mesenchymal Transition, Vascular Inflammation, and Atherosclerosis. Front. Cardiovasc. Med.2020, 7, 53.). Previous studies have revealed that doxorubicin impairs endothelial barrier function by inducing EndMT in endothelial cells (Clayton, ZS; Brunt, VE; Hutton, DA; VanDongen, NS; D'Alessandro, A.; Reisz, JA; Ziemba, BP; Seals, DR. Doxorubicin-Induced Oxidative Stress and Endothelial Dysfunction in Conduit Arteries Is Prevented by Mitochondrial-Specific Antioxidant Treatment. JACC CardioOncol2020, 2, 475-488.; Sun, Z.; Schriewer, J.; Tang, M.; Marlin, J.; Taylor, F.; Shohet, RV; Konorev, EA. The TGF-beta pathway mediates doxorubicin effects on cardiac endothelial cells. J. Mol. Cell Cardiol.2016, 90, 129-138.). The inventors observed that doxorubicin induces morphological changes in endothelial cells into a mesenchymal phenotype, characterized by the downregulation of CD31 and the upregulation of α-SMA in HUVECs, which is consistent with previous research results.

[0226] The inventors sought to determine whether the GV1001 peptide could inhibit doxorubicin-induced EndMT. When HUVECs were exposed to 0.25 μM doxorubicin and 10 μg / mL GV1001 peptide for 2 days, the GV1001 peptide was found to effectively inhibit doxorubicin-induced mesenchymal transition, thereby suppressing the downregulation of CD31 and the upregulation of α-SMA (Fig. 1a-c). Western blotting analysis revealed that doxorubicin significantly decreased CD31 and increased α-SMA levels in HUVECs (data not shown). In the culture medium, 10 μg / mL of GV1001 peptide completely reversed the changes in CD31 and α-SMA levels induced by doxorubicin. GV1001 peptide can preserve the normal characteristics of endothelial cells by inhibiting doxorubicin-induced EndMT.

[0227] 2. GV1001 peptide significantly inhibits EndMT caused by long-term exposure to doxorubicin.

[0228] It has been proven that atherosclerosis begins with vascular endothelial dysfunction, specifically through EndMT. Previous studies have revealed that doxorubicin impairs endothelial barrier function by inducing EndMT in endothelial cells.

[0229] In this experiment, we aimed to determine whether the GV1001 peptide exhibits an inhibitory effect on EndMT induced by long-term (approx. 2 weeks) exposure to doxorubicin. When HUVEC cells were exposed to doxorubicin (0.125 μM) for approximately 2 weeks, it was found that CD31 expression decreased and α-SMA expression increased, inducing EndMT (Fig. 2a-c; doxorubicin (0.125 μM)). However, when co-treated with the GV1001 peptide (10 μg / mL), CD31 expression was maintained and the increase in α-SMA expression was inhibited. These results suggest that the GV1001 peptide can preserve the normal characteristics of endothelial cells by inhibiting EndMT induced by long-term exposure to doxorubicin.

[0230] 3. The GV1001 peptide inhibited EndMT induced by long-term exposure to doxorubicin, thereby preserving the normal characteristics of endothelial cells; consequently, HUVEC motility was significantly inhibited on the third day after cell removal via scratching.

[0231] The effect of the GV1001 peptide on doxorubicin-induced endothelial cell migration in a scratch wound healing assay was analyzed. After creating scratches in culture dishes, the dishes were treated for 2 days with GV1001 (10 μg / mL), TNF-α (10 μg / mL), doxorubicin (0.25 μM), TNF-α + GV1001, or doxorubicin + GV1001. Representative images of migrated HUVECs were taken on the 3rd day (Figs. 3a and 3b), and the number of migrated cells per field of view was quantified (Fig. 3c). Images were taken at 40x magnification. The inhibition of HUVEC migration suggests that GV1001 inhibited EndMT induced by long-term exposure to doxorubicin, thereby preserving the normal characteristics of endothelial cells.

[0232] 4. Treatment with GV1001 peptide significantly inhibits the migration (mobility) of HUVECs increased by doxorubicin.

[0233] In an in vitro scratch wound healing assay, the migration (mobility) of HUVECs significantly increased in response to TNF-α or doxorubicin. The GV1001 peptide almost completely blocked this increase in migration induced by TNF-α or doxorubicin. Similarly, in the Transwell migration assay, both TNF-α and doxorubicin significantly increased HUVEC migration, while the GV1001 peptide completely inhibited the HUVEC migration induced by TNF-α or doxorubicin (Fig. 4a-b). For the migration assay, HUVECs were cultured in starvation medium (EBM-2 containing 0.5% FBS) to reduce the potential for serum-induced cell proliferation and to maintain the HUVECs, thereby reducing the likelihood of malnutrition. Since TNF-α is known to induce EndMT in HUVECs, it was included as a positive control. These results indicate that the GV1001 peptide plays a protective role in shielding endothelial cells from phenotypic changes induced by doxorubicin, which is associated with the development of atherosclerosis in the body. The GV1001 peptide can prevent the development of atherosclerosis by inhibiting the migration of HUVEC cells induced by doxorubicin, thereby preserving the normal characteristics of endothelial cells.

[0234] 5. GV1001 peptide inhibits the overexpression of doxorubicin-induced inflammatory cytokines and related factors in HUVECs.

[0235] Inflammatory cytokines, including TGF-β, IL-6, IL-1β, and TNF-α, are known to induce EndMT in endothelial cells. To determine whether doxorubicin-induced EndMT is associated with the overexpression of these inflammatory cytokines, HUVECs were exposed to 0.25 μM doxorubicin, and cytokine expression levels were measured using RT-qPCR.

[0236] 5.1. Inhibition of TGF-β and Smad Overexpression by GV1001 Peptide

[0237] As can be seen in Figures 5a-5d, exposure of cells (HUVEC) to 0.25 μM doxorubicin resulted in a significant increase in the expression of TGF-β and its downstream target gene, Smad3, in HUVECs. This effect was completely inhibited by treatment with 2 μM or 10 μM GV1001 peptide.

[0238] 5.2. Inhibition of IL-1α, IL-1β, and IL-6 Overexpression by GV1001 Peptide

[0239] Doxorubicin significantly increased the expression of IL-1α, IL-1β, and IL-6 in HUVECs, and GV1001 peptide was shown to significantly reduce the overexpression of these inflammatory cytokines induced by doxorubicin (Fig. 6a-c). Since inflammatory cytokines induce EndMT in HUVECs, these results suggest that the protective effect of GV1001 peptide against doxorubicin-induced EndMT is associated with the inhibition of inflammatory cytokine overexpression in HUVECs.

[0240] 6. GV1001 peptide inhibits the increase of TGF-β1 and FSP1 induced by long-term exposure to doxorubicin in HUVECs.

[0241] Long-term exposure to doxorubicin induces excessive activation of TGF-β1, which has been reported as a key pathway for Endometrial Transmutation (EndMT). Fibroblast-Specific Protein 1 (FSP1) is a marker that increases during endothelial-to-mesenchymal transition, and its expression increases when the TGF-β1 pathway is activated by doxorubicin. This experiment aimed to determine whether the GV1001 peptide exhibits an inhibitory effect on doxorubicin-induced EndMT. When HUVEC cells were exposed to doxorubicin (0.125 μM) for two weeks, TGF-β1 and FSP1 expression increased, inducing EndMT (Fig. 7; Doxorubicin column in the Merge section). However, when GV1001 peptide (10 μg / mL) was co-treated, the increase in TGF-β1 and FSP1 expression was inhibited (Fig. 7; Doxorubicin + GV1001 column in the Merge section). These results suggest that the GV1001 peptide can preserve the normal characteristics of endothelial cells by inhibiting doxorubicin-induced EndMT.

[0242] 7. GV1001 peptide inhibits the overexpression of major pro-inflammatory cytokines induced by doxorubicin in macrophages (RT-qPCR)

[0243] Atherosclerosis is a chronic inflammatory disease caused by the accumulation of fat and cholesterol in the blood vessel walls, and macrophages play a key role in this process. Macrophages promote the development and progression of atherosclerosis by infiltrating the vascular endothelium, phagocytosing oxidized lipids, and secreting inflammatory substances. Doxorubicin, known to induce systemic inflammation, activates macrophages to increase the secretion of pro-inflammatory cytokines.

[0244] In this experiment, we aimed to investigate the inhibitory effect of the GV1001 peptide on major pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α, and IL-8) overexpressed by doxorubicin in macrophages. When macrophages were exposed to doxorubicin (0.25 μM) for 2 days, the expression of pro-inflammatory cytokines was found to increase (Figs. 8a-f; doxorubicin). When doxorubicin (0.25 μM) and the GV1001 peptide (10 μg / mL) were co-treated, the overexpression of pro-inflammatory cytokines was found to be significantly reduced (Figs. 8a-f; GV1001 + doxorubicin). These results suggest that the GV1001 peptide can effectively inhibit the overexpression of pro-inflammatory cytokines induced by doxorubicin in macrophages, thereby preventing the generation or exacerbation of systemic or local inflammation, and consequently blocking the development or exacerbation of atherosclerosis.

[0245] 8. GV1001 peptide inhibits doxorubicin-induced NF-κB(p65) nuclear translocation in HUVECs.

[0246] NF-κB(p65) activation is known to induce the expression of inflammatory cytokines. Through this experiment, we aimed to determine whether doxorubicin activates NF-κB(p65) and whether the GV1001 peptide inhibits NF-κB activation by analyzing p65 nuclear translocation and p65 phosphorylation.

[0247] Doxorubicin significantly increased nuclear p65 immunostaining, indicating that NF-κB was activated in HUVECs. Doxorubicin significantly increased p65 phosphorylation, and the GV1001 peptide inhibited doxorubicin-induced p65 phosphorylation in a dose-dependent manner (Fig. 10a-c).

[0248] This suggests that the GV1001 peptide can inhibit NF-κB(p65) nuclear translocation induced by doxorubicin in HUVECs.

[0249] 9. GV1001 peptide prevents the accumulation of reactive oxygen species (ROS) induced by doxorubicin in the cytoplasm and mitochondria.

[0250] Intracellular reactive oxygen species (ROS) are known to induce inflammation and are primarily generated in mitochondria, causing inflammation through the NF-κB (p65) signaling pathway.

[0251] In order to investigate the mechanism by which the GV1001 peptide inhibits doxorubicin-induced NF-κB activation, we aimed to determine the effects of doxorubicin and GV1001 peptide administration on cellular and mitochondrial ROS levels and cellular ATP levels.

[0252] The GV1001 peptide inhibited the increase in mitochondrial and intracellular ROS accumulation significantly increased by doxorubicin (Fig. 11a-d) and restored cellular ATP levels reduced by doxorubicin (Fig. 11e).

[0253] Therefore, GV1001 peptide can alleviate oxidative stress induced by doxorubicin by preventing cytoplasmic and mitochondrial ROS accumulation and restoring cellular ATP levels to near normal levels.

[0254] 10. GV1001 peptide prevents doxorubicin-induced structural damage to mitochondria.

[0255] The accumulation of mitochondrial ROS leads to structural changes in mitochondria and the accumulation of mitochondrial iron ions and lipid peroxides.

[0256] This experiment aimed to investigate the effects of doxorubicin-induced mitochondrial ROS accumulation.

[0257] The effects of doxorubicin on mitochondrial morphology, iron ion levels, and lipid peroxidation were confirmed using immunofluorescence staining. Doxorubicin induced structural damage in mitochondria along with the accumulation of iron ions and lipid peroxidation, and the GV1001 peptide was shown to effectively inhibit such mitochondrial damage (Fig. 12a-e).

[0258] 11. GV1001 peptide exhibits an inhibitory effect on systemic and vascular inflammation induced by doxorubicin in ApoE-deficient mice.

[0259] Systemic and vascular inflammation are major causes of arteriosclerosis, and doxorubicin is known to induce systemic inflammation.

[0260] In this experiment, whether the GV1001 peptide alleviates systemic and vascular inflammation induced by doxorubicin in an ApoE-deficient mouse model was evaluated by measuring the expression levels of inflammatory cytokines (including IL-6, IL-8, TNF-α, IL-1β) in serum and inflammatory cytokines (including IL-6, IL-8, TNF-α, IL-1β) in arterial tissue.

[0261] Administration of doxorubicin (5 mg / kg) significantly increased the levels of inflammatory cytokines such as IL-6, IL-8, TNF-α, and IL-1β in the serum of ApoE-deficient mice. However, administration of GV1001 peptide (2.0 mg / kg) effectively suppressed these increases, thereby alleviating doxorubicin-induced systemic inflammation (Fig. 13a). In arterial tissue, doxorubicin significantly increased the expression of TNF-α, IL-1β, and IL-6, but GV1001 peptide suppressed these increases (Fig. 13b).

[0262] Therefore, GV1001 peptide can effectively reduce systemic inflammation and vascular inflammation (vasculitis) induced by doxorubicin in ApoE-deficient mice.

[0263] 12. GV1001 peptide effectively inhibits doxorubicin-induced arterial wall lipid deposition.

[0264] Vascular inflammation and EndMT are major causes of the development of atherosclerosis, and atherosclerotic plaques are composed of lipids, macrophages, foam cells, etc.

[0265] Through this experiment, we aimed to determine whether the GV1001 peptide improves the increase in arterial wall lipid deposition caused by doxorubicin.

[0266] En face analysis revealed that only minimal lipid deposition was observed in the arterial walls of control mice fed a high-fat diet for 9 weeks. Systemic administration of GV1001 peptide (2.0 mg / kg) alone did not alter lipid deposition in mice. In contrast, administration of doxorubicin (5.0 mg / kg) significantly increased lipid deposition in the arterial walls by more than three times compared to control mice. Surprisingly, co-administration of GV1001 peptide completely suppressed this increase (Fig. 14a-b). Similarly, lipid accumulation in the aortic root induced by doxorubicin was effectively alleviated by GV1001 peptide (Fig. 14c). Furthermore, while doxorubicin treatment significantly increased macrophage and monocyte infiltration within the arterial walls, co-administration of GV1001 significantly reduced this increase (Fig. 14d).

[0267] Therefore, GV1001 peptide can effectively inhibit the development of arteriosclerosis by inhibiting lipid deposition and macrophage / monocyte infiltration in the arterial wall induced by doxorubicin.

[0268]

[0269] The peptide having the amino acid sequence of SEQ ID NO. 1 of the present invention is effective in alleviating at least one of the side effects caused by anticancer chemotherapy, specifically anticancer chemotherapy using an anthracycline-based anticancer agent, particularly cardiotoxicity, vascular disorders, and inflammation. Therefore, it can be usefully utilized as an anticancer adjuvant that can alleviate the side effects of anticancer agents and enhance the effects of anticancer agents, and thus is expected to have great industrial value.

Claims

A pharmaceutical composition for the prevention or treatment of side effects caused by anticancer chemotherapy using an anthracycline-based anticancer agent, comprising as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO.

1. In claim 1, A pharmaceutical composition for preventing or treating side effects caused by anticancer chemotherapy using an anthracycline anticancer agent, wherein the anthracycline anticancer agent is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin. In claim 1, A pharmaceutical composition for the prevention or treatment of side effects caused by anticancer chemotherapy using an anthracycline-based anticancer agent, wherein the side effects caused by anticancer chemotherapy using an anthracycline-based anticancer agent are at least one of cardiotoxicity, vascular disorder, and inflammation. In claim 3, Cardiotoxicity includes heart failure, arrhythmia, myocarditis, and cardiomyopathy; Vascular disorders include atherosclerosis; A pharmaceutical composition for the prevention or treatment of side effects induced by anticancer chemotherapy using anthracycline anticancer agents, the inflammation including systemic inflammation, local inflammation, and vasculitis. In claim 1, The above peptide is a pharmaceutical composition for the prevention or treatment of side effects induced by anticancer chemotherapy using an anthracycline anticancer agent, wherein the peptide exhibits at least one activity selected from the group consisting of: an activity that inhibits endothelial-mesenchymal transition (EndMT) in Human Umbilical Vein Endothelial Cells (HUVEC); an activity that inhibits the overexpression of inflammatory cytokines; an activity that inhibits the increase in TGF-β1 and FSP1 expression; an activity that prevents the generation or exacerbation of systemic or local inflammation; an activity that inhibits NF-κB(p65) nuclear transition in HUVEC; an activity that inhibits the increase in ROS accumulation in mitochondria and cytoplasm; an activity that restores reduced cellular ATP levels; an activity that inhibits structural damage to mitochondria; an activity that inhibits the accumulation of iron ions and lipid peroxides; an activity that inhibits lipid deposition into the arterial wall; and an activity that inhibits the infiltration of macrophages and monocytes into the arterial wall. A health functional food composition for preventing or improving side effects caused by anticancer chemotherapy using an anthracycline-based anticancer agent, comprising as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO.

1. In claim 6, A health functional food for preventing or improving side effects caused by anticancer chemotherapy using an anthracycline anticancer agent, wherein the anthracycline anticancer agent is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin. In claim 6, A health functional food for preventing or improving side effects caused by anticancer chemotherapy using an anthracycline anticancer drug, wherein the side effects caused by anticancer chemotherapy using an anthracycline anticancer drug are at least one of cardiotoxicity, vascular disorder, and inflammation. In claim 8, Cardiotoxicity includes heart failure, arrhythmia, myocarditis, and cardiomyopathy; Vascular disorders include atherosclerosis; A health functional food for the prevention or improvement of side effects induced by anticancer chemotherapy using anthracycline anticancer agents, including systemic inflammation, local inflammation, and vasculitis. In claim 6, The above peptide is a health functional food for preventing or improving side effects induced by anticancer chemotherapy using an anthracycline anticancer agent, wherein the peptide exhibits at least one activity selected from the group consisting of: an activity that inhibits endothelial-mesenchymal transition (EndMT) in Human Umbilical Vein Endothelial Cells (HUVEC); an activity that inhibits the overexpression of inflammatory cytokines; an activity that inhibits the increase in TGF-β1 and FSP1 expression; an activity that prevents the generation or exacerbation of systemic or local inflammation; an activity that inhibits NF-κB(p65) nuclear transition in HUVEC; an activity that inhibits the increase in ROS accumulation in mitochondria and cytoplasm; an activity that restores reduced cellular ATP levels; an activity that inhibits structural damage to mitochondria; an activity that inhibits the accumulation of iron ions and lipid peroxides; an activity that inhibits lipid deposition into the arterial wall; and an activity that inhibits the infiltration of macrophages and monocytes into the arterial wall. An anticancer adjuvant for alleviating side effects induced by anthracycline-based anticancer drugs, comprising as an active ingredient a peptide consisting of the amino acid sequence of SEQ ID NO.

1. In claim 11, An anticancer adjuvant administered simultaneously, separately, or sequentially with an anthracycline anticancer drug. In claim 11, An anticancer adjuvant in which an anthracycline anticancer agent is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin. In claim 13, An anthracycline anticancer agent, an anticancer adjuvant, is doxorubicin. A pharmaceutical composition for the prevention or treatment of cancer, comprising the anticancer adjuvant of claim 6 and an anthracycline anticancer agent. In claim 15, A pharmaceutical composition for the prevention or treatment of cancer, wherein the anthracycline anticancer agent is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin. In claim 16, A pharmaceutical composition for the prevention or treatment of cancer, wherein the anthracycline anticancer agent is doxorubicin. A kit for the prevention or treatment of side effects caused by anticancer chemotherapy using an anthracycline anticancer agent, comprising an anticancer adjuvant containing a peptide consisting of an amino acid sequence represented by SEQ ID NO. 1 as an active ingredient and instructions for use. In claim 18, A kit for the prevention or treatment of side effects caused by anticancer chemotherapy using an anthracycline anticancer agent, wherein the anthracycline anticancer agent is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin. In claim 18, A kit for the prevention or treatment of side effects caused by anticancer chemotherapy using an anthracycline anticancer agent, wherein the side effects caused by anticancer chemotherapy using an anthracycline anticancer agent are at least one of cardiotoxicity, vascular disorder, and inflammation. In claim 18, Cardiotoxicity includes heart failure, arrhythmia, myocarditis, and cardiomyopathy; Vascular disorders include atherosclerosis; A kit for the prevention or treatment of side effects induced by anticancer chemotherapy using anthracycline anticancer agents, including systemic inflammation, local inflammation, and vasculitis. In claim 18, The above peptide is a kit for preventing or treating side effects induced by anticancer chemotherapy using an anthracycline anticancer agent, wherein the peptide exhibits at least one activity selected from the group consisting of: an activity that inhibits endothelial-mesenchymal transition (EndMT) in Human Umbilical Vein Endothelial Cells (HUVEC); an activity that inhibits the overexpression of inflammatory cytokines; an activity that inhibits the increase in TGF-β1 and FSP1 expression; an activity that prevents the generation or exacerbation of systemic or local inflammation; an activity that inhibits NF-κB(p65) nuclear transition in HUVEC; an activity that inhibits the increase in ROS accumulation in mitochondria and cytoplasm; an activity that restores reduced cellular ATP levels; an activity that inhibits structural damage to mitochondria; an activity that inhibits the accumulation of iron ions and lipid peroxides; an activity that inhibits lipid deposition into the arterial wall; and an activity that inhibits the infiltration of macrophages and monocytes into the arterial wall.

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