KR20240051379A - A pharmaceutical composition for preventing or treating anti-angiogenesis and/or cancer comprising ramucirumab and a HER2 inhibitor - Google Patents

Abstract

One aspect relates to a pharmaceutical composition comprising ramucirumab and a HER2 inhibitor for the prevention or treatment of anti-angiogenesis and/or cancer. According to one aspect, when ramucirumab and a HER2 inhibitor are administered in combination, blood vessel density, length of new blood vessels, and area of PDC spheroids (patient-derived cells spheroids) are significantly higher than when ramucirumab and HER2 inhibitor are administered alone. A decrease was confirmed. In addition, it was confirmed that when ramulirumab and HER2 inhibitor are administered together, the area of PDC spheroids (patient-derived cell spheroids) is reduced, showing excellent cancer prevention or treatment effect through anti-angiogenic effect. and/or may contribute to the market/industry for the treatment of cancer.

Description

A pharmaceutical composition for preventing or treating anti-angiogenesis and/or cancer comprising ramucirumab and a HER2 inhibitor}

It relates to a pharmaceutical composition for preventing or treating anti-angiogenesis and/or cancer, comprising ramucirumab and a HER2 inhibitor.

In 2020, more than 1 million new cases of gastric cancer (GC) occurred and approximately 769,000 deaths. Stomach cancer is the fifth most common cancer and fourth leading cause of death worldwide (1 in 13). Although the survival rate of gastric cancer is increasing with new surgical resection and anticancer drugs, malignant ascites due to peritoneal metastasis in gastric cancer patients is the most common cause of death in advanced gastric cancer, especially diffuse gastric cancer 2, and effective treatment options for cancer are lacking.

Meanwhile, ramucirumab is a fully human monoclonal antibody that targets and binds to vascular endothelial growth factor receptor-2 (VEGFR-2) and blocks the activation of VEGF6. Anti-angiogenic therapy using ramucirumab is useful as monotherapy and in combination with paclitaxel for patients with gastric cancer that progressed after primary chemotherapy, and ramucirumab interferes with paracrine/autocrine signaling. In the second REGARD trial, ramucirumab improved survival compared to best supportive care, whereas in the RAINBOW trial, the combination of ramucirumab and paclitaxel improved survival compared to paclitaxel alone. However, when chemotherapy containing platinum or fluoropyrimidine was administered to gastric cancer patients who had no experience with ramucirumab or bevacizumab (an anti-VEGF monoclonal antibody), It did not improve patient survival.

Accordingly, there was a need to develop an agent that can effectively treat cancer in combination with ramucirumab, and the present inventors found that when ramucirumab and a HER2 inhibitor are administered together instead of paclitaxel, there is a significant inhibition of angiogenesis and a prevention or treatment effect of cancer. By confirming that it can represent, the present invention was developed.

Korean Patent Publication No. 10-2022-0042087

One aspect is to provide a pharmaceutical composition for anti-angiogenesis comprising Ramucirumab and a HER2 inhibitor.

Another aspect is to provide an anti-angiogenic dietary supplement containing Ramucirumab and a HER2 inhibitor.

Another aspect is to provide a pharmaceutical composition for preventing or treating cancer, including Ramucirumab and a HER2 inhibitor.

Another aspect is to provide health functional foods for preventing or improving cancer, including ramucirumab and HER2 inhibitors.

One aspect provides a pharmaceutical composition for anti-angiogenesis comprising Ramucirumab and a HER2 inhibitor.

The term "Ramucirumab" refers to a fully human monoclonal antibody (IgG1) developed for the treatment of solid tumors, PubChem SID 96026051, CAS number 947687-13-0, molecular formula C ₂₈₅ H ₄₃ 4N ₇₄ O It is a human monoclonal antibody with a molecular weight of ₈₈ S ₂ , 6369.07.

The ramucirumab binds to the extracellular domain of VEGFR2 with high affinity and acts as a direct VEGFR2 antagonist that blocks the binding of natural VEGFR ligands (VEGF-A, VEGF-C and VEGF-D).

Meanwhile, the ramucirumab may be a fully human monoclonal antibody (IgG1) developed for the treatment of solid tumors, as well as a fragment of the monoclonal antibody, and the ramucirumab may be an amino acid sequence of the ramucirumab or a fragment thereof. and about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more. It may include proteins or polypeptides having more than % sequence homology.

The term "homology" is intended to indicate the degree of similarity with the wild-type amino acid sequence. Comparison of such homologies can be performed using a comparison program widely known in the art, and homology between two or more sequences is used. It can be calculated as a percentage (%).

The term "HER2 inhibitor (Human epidermal growth factor receptor inhibitor)" refers to a substance that inhibits (inhibits) the expression or activity of the HER2 (ErbB2) gene, and HER2 is human epidermal growth factor receptor 2 (Human epidermal growth factor receptor 2) means.

In one aspect, the HER2 inhibitor is Sapitinib, Trastuzumab, Lapatinib, Margetuximab, Tucatinib, Pertuzumab and Herceptin. It may be one or more selected from the group consisting of (Herceptin), and specifically, it may be one or more selected from the group consisting of Sapitinib, Trastuzumab, Lapatinib and Herceptin, , More specifically, it may be Sapitinib.

The term "Sapitinib (2-[4-[4-(3-chloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl]oxypiperidin-1-yl]-N-methylacetamide)" refers to epithelial growth It refers to a dual tyrosine kinase inhibitor (TKI) of factor receptor (EGFR) HER2 and HER3, and is a HER2 inhibitor with CAS number 848942-61-0, molecular formula C ₂₃ H ₂₅ ClFN ₅ O ₃ , and molecular weight of 473.9. , referred to as AZD8931.

The term “angiogenesis” refers to a process in which vascular sprouts are created from existing microvessels, and the vascular sprouts proliferate to create new capillaries, and when excessive angiogenesis occurs , various diseases can develop.

The term “anti-angiogenesis” means preventing the formation of new blood vessels, and may specifically mean inhibiting the creation of blood vessel sprouts from existing microvessels.

In one aspect, the composition may reduce blood vessel density, the length of new blood vessels, or the PDC spheroid area, and specifically, it may reduce blood vessel density, the length of new blood vessels, and the PDC spheroid area. .

In one aspect, the length of the newly formed blood vessel may refer to the length of an angiogenic sprout.

In one example, an attempt was made to confirm the anti-angiogenic effect when parent blood vessels were formed in endothelial channels, and when ramucirumab and AZD8931 (HER2 inhibitor, Sapitinib) were administered together, blood vessel density and angiogenic sprouts were investigated. It was confirmed that the length and area of PDC spheroids (patient-derived cells spheroids) were decreased.

In addition, it was confirmed that when ramucirumab and AZD8931 (HER2 inhibitor, Sapitinib) were administered in combination, excellent tumor cell toxicity was observed, and tumor induction was lower than when ramucirumab and AZD8931 (HER2 inhibitor, Sapitinib) were administered alone. It was confirmed that angiogenesis was reduced.

In one aspect, the composition may exhibit an excellent anti-angiogenesis effect by reducing blood vessel density, length of generated blood vessels, and area of PDC spheroids (patient-derived cells spheroids).

Additionally, the pharmaceutical composition may contain the active ingredient alone, or may be provided as a pharmaceutical composition including one or more pharmaceutically acceptable carriers, excipients, or diluents.

Specifically, the carrier may be, for example, a colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano-spherical particle. They may form complexes or associate with delivery vehicles and may be used in the art as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers or fatty acids. It can be transported in vivo using known delivery systems.

When the pharmaceutical composition is formulated, it may be prepared using commonly used diluents or excipients such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, disintegrants, and surfactants. You can. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations may contain at least one excipient, such as starch, calcium carbonate, or sucrose. ) or can be prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used, and when manufacturing in the form of eye drops, known diluents or excipients can be used. there is.

In one aspect, the pharmaceutical composition may further include an anti-angiogenic pharmaceutical composition.

The pharmaceutical composition may be provided by mixing with a conventionally known anti-angiogenic pharmaceutical composition or another existing anti-angiogenic pharmaceutical composition, and the anti-angiogenic pharmaceutical composition is a conventionally known anti-angiogenic pharmaceutical composition. It may be a pharmaceutical composition for anti-angiogenesis, an existing pharmaceutical composition for anti-angiogenesis, or a newly developed pharmaceutical composition for anti-angiogenesis.

When the pharmaceutical composition further includes an anti-angiogenic pharmaceutical composition, it is important to mix the mixture in an amount that can obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition may be administered in combination with a known composition having an anti-angiogenic effect or another anti-angiogenic pharmaceutical composition, and may be administered simultaneously, separately, or sequentially, and may be administered singly or multiple times. You can. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition can be administered orally or parenterally, and when administered parenterally, it is administered externally to the skin or intraperitoneally, intrarectally, subcutaneously, intravenously, intramuscularly, intraarterially, intramedullaryly, or intracardially. , intrathecal injection, transdermal injection, intranasal injection, enteral injection, local injection, sublingual injection, intrarectal injection, or intrathoracic injection.

The pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the patient's disease, the activity of the drug, and the drug's effect. It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

In one aspect, the pharmaceutical composition may be administered once a day or may be administered several times. For example, it may be administered every other day, or it may be administered once a week. Specifically, the pharmaceutical composition may be administered at 0.001 to 1000 mg/kg/day, more specifically at 0.1 to 100 mg/kg/day. The administration may be administered once a day, or may be administered several times.

Another aspect provides an anti-angiogenic nutraceutical comprising Ramucirumab and a HER2 inhibitor.

The “ramucirumab”, “HER2 inhibitor”, “anti-angiogenesis”, etc. may be within the above-mentioned range.

In one aspect, the HER2 inhibitor is Sapitinib, Trastuzumab, Lapatinib, Margetuximab, Tucatinib, Pertuzumab and Herceptin. It may be one or more selected from the group consisting of (Herceptin), and specifically, it may be one or more selected from the group consisting of Sapitinib, Trastuzumab, Lapatinib and Herceptin, , More specifically, it may be Sapitinib.

The term "Sapitinib (2-[4-[4-(3-chloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl]oxypiperidin-1-yl]-N-methylacetamide)" refers to epithelial growth It refers to a dual tyrosine kinase inhibitor (TKI) of factor receptor (EGFR) HER2 and HER3, and is a HER2 inhibitor with CAS number 848942-61-0, molecular formula C ₂₃ H ₂₅ ClFN ₅ O ₃ , and molecular weight of 473.9. , referred to as AZD8931.

In one aspect, the health functional food may be one that reduces blood vessel density, the length of new blood vessels, or the PDC spheroid area, and specifically reduces the blood vessel density, the length of new blood vessels, and the PDC spheroid area. You can.

In the health functional food, the active ingredient can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement). In general, when manufacturing a food or beverage, the health functional food may be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

The health functional food may be formulated with one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further including one or more of carriers, diluents, excipients, and additives. Foods to which compounds according to one aspect can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, etc.

Specific examples of the carriers, excipients, diluents and additives include lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose. , a group consisting of polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. It may be at least one selected from.

In addition to containing the effective ingredients, the health functional food may contain other ingredients as essential ingredients without any particular restrictions. For example, like regular beverages, it may contain various flavoring agents or natural carbohydrates as additional ingredients. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. You can. The ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, health functional foods according to one aspect include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These components can be used independently or in combination, and the proportions of these additives can also be appropriately selected by those skilled in the art.

In one aspect, the health functional food may further include an anti-angiogenic health functional food.

The health functional food may be provided in combination with a conventionally known anti-angiogenic health functional food or a newly developed anti-angiogenic health functional food.

When the health functional food further includes an anti-angiogenic health functional food, it is important to mix the mixture in an amount that can obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Additionally, in one aspect, the health functional food may be consumed alone or in combination with the anti-angiogenic health functional food.

The health functional food may be consumed in combination with a known composition having an anti-angiogenic effect or other anti-angiogenic health functional food, and may be consumed simultaneously, separately, or sequentially, and may be consumed singly or multiple times. You can. Considering all of the above factors, it is important to consume an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Another aspect provides a pharmaceutical composition for preventing or treating cancer, including Ramucirumab and a HER2 inhibitor.

The “ramucirumab”, “HER2 inhibitor”, “pharmaceutical composition”, etc. may be within the above-mentioned range.

In one aspect, the HER2 inhibitor is Sapitinib, Trastuzumab, Lapatinib, Margetuximab, Tucatinib, Pertuzumab and Herceptin. It may be one or more selected from the group consisting of (Herceptin), and specifically, it may be one or more selected from the group consisting of Sapitinib, Trastuzumab, Lapatinib and Herceptin, , More specifically, it may be Sapitinib.

The term "Sapitinib (2-[4-[4-(3-chloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl]oxypiperidin-1-yl]-N-methylacetamide)" refers to epithelial growth It refers to a dual tyrosine kinase inhibitor (TKI) of factor receptor (EGFR) HER2 and HER3, and is a HER2 inhibitor with CAS number 848942-61-0, molecular formula C ₂₃ H ₂₅ ClFN ₅ O ₃ , and molecular weight of 473.9. , referred to as AZD8931.

In one aspect, the cancer includes lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, ovarian cancer, colon cancer, anal cancer, biliary tract cancer, bladder cancer, gallbladder cancer, head and neck cancer, oral cancer, pharynx cancer, nasopharyngeal cancer, kidney cancer, and muscle cancer. , may be one or more selected from the group consisting of bone cancer, prostate cancer, rectal cancer, small intestine cancer, urethral cancer, thyroid cancer, and breast cancer, and specifically, lung cancer, stomach cancer, liver cancer, pancreas cancer, ovarian cancer, colon cancer, anal cancer, bladder cancer, It may be one or more selected from the group consisting of nasopharyngeal cancer, kidney cancer, and breast cancer, and more specifically, it may be stomach cancer.

In one embodiment, when parent blood vessels were formed in the endothelial channel, when ramucirumab and AZD8931 (HER2 inhibitor, Sapitinib) were administered together, it was confirmed that the area of PDC spheroids (patient-derived cells spheroids) was reduced, thereby demonstrating anticancer activity. It was confirmed that it was effective.

In addition, it was confirmed that when ramucirumab and AZD8931 (HER2 inhibitor, Sapitinib) were administered in combination, excellent tumor cell toxicity was observed, and tumor induction was lower than when ramucirumab and AZD8931 (HER2 inhibitor, Sapitinib) were administered alone. It was confirmed that angiogenesis was reduced.

In one aspect, the composition may exhibit excellent cancer prevention or treatment effects through an anti-angiogenic effect by reducing the area of PDC spheroids (patient-derived cell spheroids).

The term “prevention” may refer to any act of suppressing or delaying the onset of cancer in an individual by administering a pharmaceutical composition according to one aspect.

The term “treatment” may refer to any action in which an individual's cancer symptoms are improved or beneficially changed by administration of a pharmaceutical composition according to one aspect.

The term "administration" refers to introducing a predetermined substance into an individual by an appropriate method, and "individual" refers to all living organisms such as rats, mice, and livestock, including humans, that can harbor cancer. As a specific example, it may be a mammal, including humans.

In one aspect, the pharmaceutical composition may further include a pharmaceutical composition for preventing or treating cancer.

The pharmaceutical composition may be provided by mixing with a conventionally known pharmaceutical composition for the prevention or treatment of cancer or a known pharmaceutical composition for the prevention or treatment of other cancers, and the pharmaceutical composition for the prevention or treatment of cancer It may be a conventionally known pharmaceutical composition for the prevention or treatment of cancer, an existing pharmaceutical composition for the prevention or treatment of cancer, or a newly developed pharmaceutical composition for the prevention or treatment of cancer.

When the pharmaceutical composition further includes a pharmaceutical composition for preventing or treating cancer, it is important to mix the mixture in an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition may be administered in combination with known compositions having a cancer prevention or treatment effect or other pharmaceutical compositions for the prevention or treatment of cancer, and may be administered simultaneously, separately, or sequentially, and may be administered singly or multiple times. may be administered. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Another aspect provides health functional foods for preventing or improving cancer, including ramucirumab and HER2 inhibitors.

The “ramucirumab”, “HER2 inhibitor”, “cancer”, “prevention”, “health functional food”, etc. may be within the above-mentioned range.

In one aspect, the HER2 inhibitor is Sapitinib, Trastuzumab, Lapatinib, Margetuximab, Tucatinib, Pertuzumab and Herceptin. It may be one or more selected from the group consisting of (Herceptin), and specifically, it may be one or more selected from the group consisting of Sapitinib, Trastuzumab, Lapatinib and Herceptin, , More specifically, it may be Sapitinib.

The term "Sapitinib (2-[4-[4-(3-chloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl]oxypiperidin-1-yl]-N-methylacetamide)" refers to epithelial growth It refers to a dual tyrosine kinase inhibitor (TKI) of factor receptor (EGFR) HER2 and HER3, and is a HER2 inhibitor with CAS number 848942-61-0, molecular formula C ₂₃ H ₂₅ ClFN ₅ O ₃ , and molecular weight of 473.9. , referred to as AZD8931.

In one aspect, the cancer includes lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, ovarian cancer, colon cancer, anal cancer, biliary tract cancer, bladder cancer, gallbladder cancer, head and neck cancer, oral cancer, pharynx cancer, nasopharyngeal cancer, kidney cancer, and muscle cancer. , may be one or more selected from the group consisting of bone cancer, prostate cancer, rectal cancer, small intestine cancer, urethral cancer, thyroid cancer, and breast cancer, and specifically, lung cancer, stomach cancer, liver cancer, pancreas cancer, ovarian cancer, colon cancer, anal cancer, bladder cancer, It may be one or more selected from the group consisting of nasopharyngeal cancer, kidney cancer, and breast cancer, and more specifically, it may be stomach cancer.

The term “improvement” may refer to any action that at least reduces the severity of a parameter related to the condition being treated, for example, a symptom. At this time, the health functional food can be used simultaneously or separately with drugs for treatment before or after the onset of the disease in order to prevent or improve cancer.

In one aspect, the health functional food may exhibit an excellent cancer prevention or improvement effect through an anti-angiogenic effect by reducing the area of PDC spheroids (patient-derived cell spheroids).

In one aspect, the health functional food may further include a health functional food for preventing or improving cancer.

The health functional food may be provided in combination with a conventionally known health functional food for preventing or improving cancer or a newly developed health functional food for preventing or improving cancer.

When the health functional food further includes a health functional food for preventing or improving cancer, it is important to mix the mixture in an amount that can obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Additionally, in one aspect, the health functional food may be consumed alone or in combination with the health functional food for preventing or improving cancer.

The health functional food can be consumed in combination with known compositions that have a cancer prevention or improvement effect or other health functional foods for the prevention or improvement of cancer, and can be consumed simultaneously, separately, or sequentially, and can be single or multiple. It can be ingested. Considering all of the above factors, it is important to consume an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

According to one aspect, when ramucirumab and a HER2 inhibitor are administered in combination, blood vessel density, length of new blood vessels, and area of PDC spheroids (patient-derived cells spheroids) are significantly higher than when ramucirumab and HER2 inhibitor are administered alone. A decrease was confirmed. In addition, it was confirmed that when ramulirumab and HER2 inhibitor are administered together, the area of PDC spheroids (patient-derived cell spheroids) is reduced, showing excellent cancer prevention or treatment effect through anti-angiogenic effect. and/or may contribute to the market/industry for the treatment of cancer.

Figure 1 is a diagram showing the characteristics of patients registered as stomach cancer patients. Specifically, Figure 1A is a diagram showing the results of classifying samples of registered patients, and Figure 1B is a diagram showing gene mutations in research samples of stomach cancer patients. 1C is a diagram showing the results of unsupervised clustering analysis of patient samples, and FIG. 1D is a diagram showing VEGF-A expression levels according to tissue origin (primary vs. metastases) after the analysis. 1E is a heatmap of gene set variant analysis (GSVA) scores of representative pathways across high and low KDR groups.
Figure 2a is a schematic diagram of establishing an anticancer strategy for HER2, c-MET, and FGFR2, which are major oncogenes found in gastric cancer patients, and Figure 2b is a schematic diagram of establishing an anticancer strategy for VEGFR2 signaling and ERBB, MET, and FGF networks in gastric cancer patients. A diagram showing the results of characterizing , Figure 2c is a diagram showing the results confirming HER2, c-MET, and FGFR2 overexpression through clinicopathological immunohistochemistry indicating positive oncogenic genes, and Figure 2d is a diagram showing the results of confirming overexpression of HER2, c-MET, and FGFR2 through Western blotting. A diagram showing the results of detecting proteins from the mutated gene in this case, and Figure 2e shows the results of analyzing changes in blood vessel morphology according to the concentration of ramucirumab (0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001;ns; not significant), and Figure 2f shows anticancer activity by drug status through quantitative image analysis. This diagram shows the results of analyzing the effects.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

1. Experimental materials and methods

(1) Patient registration and ethical approval

All patients participating in this study agreed to provide biospecimens with approval from the Institutional Review Board (IRB) of Samsung Medical Center (SMC) (IRB#2021-09-052). This study was conducted in accordance with the principles of the Helsinki Declaration and Guidelines for Good Clinical Practice (ClinicalTrial.gov identifier: NCT02589496). Samples were collected from 45 patients diagnosed with advanced gastric cancer, of which 26 samples were primary tumors obtained from surgery and biopsy, and the remaining 19 samples were collected from peritoneal dialysate.

(2) Patient-derived cells (PDC) preparation from ascites

Patient-derived cell culture from ascitic fluid was performed as previously reported. Ascites was divided into 50 ml e-tubes and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was removed, the cells were suspended in PBS, and centrifugation was repeated again. To filter the cell suspension, a cell strainer (70 μm; Corning, CAT#CLS431751) was connected to a new 50 ml e-tube and the centrifuged cell suspension was passed through. Then, RBC lysis buffer (Qiagen, CAT#158904) was added to the cell suspension along with PBS (PBS: RBC lysis buffer = 1:3, v/v). The cells were incubated at room temperature for 30 to 60 minutes to allow RBC lysis. This was repeated until the red color disappeared from the cell pellet, then the supernatant was removed and resuspended in PBS. The target cell concentration (usually 10 ⁷ cells in a 150-mm dish) was prepared and the resuspended cells were cultured with PDC medium for passages 2-3. PDC medium was RPMI 1640 (Gibco) supplemented with 10% FBS, 1% antibiotic-antimycotic (GenDEPOT, CAT#CA002), EGF (5 ng/ml, PeproTech), insulin (5 μg/ml, Sigma), and hydrocortisone. (Hydrocortisone) (0.5 μg/ml; Sigma-Aldrich). The absence of mycoplasma contamination was confirmed using a general-purpose mycoplasma detection kit (9 ATCC 30-1012K).

(3) PDC preparation from primary solid tissue

Tissues were transferred to a Petri dish containing fresh, sterilized DPBS supplemented with antibiotics. After rinsing 2-3 times, the tissue pieces were transferred to an e-tube containing 0.5 ml to 1.0 ml of tissue lysis buffer. The composition of tissue lysis buffer was as follows: RPMI 1640 (Gibco) supplemented with collagenase (1.0 mg/ml; Stemcell Technologies, CAT#07912), Dispase (0.5 mg/ml; Stemcell Technologies, CAT# 07923), DNase (0.15 mg/ml; Stemcell Technologies, CAT#07469_C), 5% FBS (Gibco). Pieces of tissue were cut from the e-tube using scissors until the tissue was reduced to very small pieces. The tissue was then transferred to a 15 ml tube containing 1 ml to 2 ml of lysis buffer. Then, the 15 ml tubes were incubated at 37°C for 0.5 to 1.0 hours with vortexing at 70 rpm, and the tubes were centrifuged at 2000 rpm for 5 minutes at room temperature. The supernatant was removed, the pellet was washed with PBS, and then incubated with PDC medium and PDC selection was performed as per the procedure performed by Ascites.

(4) MSI status

Tumor tissue MSI status was determined using PCR analysis of five markers with mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, and NR-27) as previously described. did. Briefly, each sense primer was end-labeled with FAM, HEX, or NED. Pentaplex PCR was performed, and PCR products were run on an Applied Biosystems PRISM 3130 automated genetic analyzer. Allele sizes were estimated using Genescan 2.1 software (Applied Biosystems), and samples with allele size changes in two or more microsatellites were considered MSI-H.

(5) Sample preparation for sequencing

Tumor tissue, ascites malignant cells, and matching peripheral blood samples were obtained. After thorough pathological review, when tumor cellularity was estimated to be greater than 40%, tumor DNA and RNA were extracted from newly obtained tumor tissues using the QIAamp Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNaseA (Qiagen, CAT#19101) was used for RNase digestion during DNA preparation. Concentration and 260/280 and 260/230 nm absorbance were measured using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies LLC, Thermo Fisher Scientific, MA, USA), and DNA and RNA were measured using a Qubit Fluorometer (Life Technologies, CA, USA). was quantified.

(6) Whole exome and transcriptome sequencing

We performed sequencing using genomic DNA (gDNA) obtained from tumor tissue and matched blood samples were isolated using the QIAamp DNA blood kit (Qiagen). A standard exome capture library was generated from 1.0 μg of input gDNA using the Agilent SureSelect Target Enrichment protocol with an Illumina paired-end sequencing library. The SureSelect Human All Exon V6 probe set was used in all cases. We assessed the quantity and quality of DNA using PicoGreen and agarose gel electrophoresis. We diluted 1.0 μg of gDNA in elution buffer and sheared it to a target peak size of 150 bp to 200 bp using a Covaris LE220 focused ultrasonicator (Covaris Inc., Woburn, MA, USA) according to the manufacturer's recommendations. The fragmented DNA was repaired and the A base was ligated to the 3'-end, and the fragments were then ligated with Agilent adapters and amplified by polymerase chain reaction (PCR) analysis. The prepared library was quantified using TapeStation DNA ScreenTape D1000 (Agilent Technologies). For exome capture, 250 ng of DNA library was mixed with hybridization buffer, blocking mix, RNase block, and 5.0 μg of SureSelect All Exon Capture Library according to the standard Agilent SureSelect Target Enrichment protocol. Hybridization to capture baits was performed at 65°C using the thermal cycler cover option heated at 105°C for 24 hours in the PCR machine. Finally, the captured DNA was washed and amplified. The final purified product was quantified by quantitative PCR (qPCR) according to the qPCR quantification protocol guide (KAPA Library Quantification Kit for Illumina Sequencing Platforms) and verified using TapeStation DNA ScreenTape D1000 (Agilent Technologies). Additionally, samples were multiplexed and flow cell clusters were generated using the TruSeq Rapid Cluster kit and TruSeq Rapid SBS kit (Illumina, CA, USA), and the indexed libraries were transferred to an Illumina HiSeq 2500 (Illumina, San Diego, CA, USA). Submitted and paired-end (2 × 100bp) sequencing was performed.

(7) Variant calling and filtering of the entire exome sequence

Sequenced reads were aligned to the reference human genome GRCh37 (GRCh37) using the Burrows-Wheeler Aligner Maximal Exact Matches (BWA-MEM) algorithm and then marked for duplicates using the Genome Analysis Toolkit (version 4.1.1.0). , preprocessing steps including deletion alignment and base realignment were performed to generate binary alignment map files ready for analysis. Variant calling was initially performed on Mutect255 to establish a set of highly sensitive somatic single nucleotide variants and short indels. Default parameters were applied and both variant callers were run with dbSNP (version 138), 1000G (Phase I) and HapMap (Phase III) data for known polymorphic sites. Variants with a minimum depth <4 or minimum alternative allele <2 were filtered and annotated using Ensembl Variant Effect Predictor (VEP) (release 87) with the GRCh37 database.

(8) Classification into molecular subtypes

EBV detection was performed to define four molecular subtypes. Using Pathseq algorithm, the first group of PDC was detected with high EBV burden samples, and the second group was detected by MSI sensor and showed high mutation rate. The remaining two groups were distinguished by the presence of somatic copy-number aberrations (SCNAs). To distinguish them, the number of chromosomes changed by deletion or insertion was calculated and the genomic instability index (termed CNV GI) was used.

(9) Transcriptome data processing and analysis

The entire transcriptome sequence was annotated with Ensembl (version 98) and aligned to the human reference genome (GRCh38) using STAR (version 2.6.1). We quantified RNA expression in transcripts per million (TPM) using RNA-Seq by Expectation-Maximization (version 1.3.1), applying the parameters recommended by the Genotype-Tissue Expression (GTEx) project. Signal transduction pathways were analyzed using gene set variation analysis.

(10) Primary cell line culture

Human umbilical vein endothelial cells (HUVEC; Lonza, CAT#C2519A) were cultured in EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM (Lonza, CAT#CC-3162) and passages 4 to 5 were used in the experiment. Lung fibroblasts (LF; Lonza, CAT#CC-2512) were cultured in FGMTM-2 Fibroblast Growth Medium-2 BulletKitTM (Lonza, CAT#CC-3132) and passages 5 to 6 were used. HUVEC and LF were cultured at 37°C in 5% CO ₂ for 2-3 days. All cell lines were tested for mycoplasma contamination and authenticated by STR DNA profiling (SMC Basic Research Support Center, Seoul, Korea), and then cells were cultured according to the manufacturer's instructions.

(11) PDC-derived spheroid formation

Cell spheroids were prepared by distributing the PDC suspension in predetermined amounts into U-bottom shaped 96-well plates (Sumitomo Bakelite, Japan, CAT#MS-9096UZ). All spheroids were prepared by adding 1% volume ratio of Matrigel (Corning, USA, CAT#356231) to 150 μL of medium containing 6,000 PDC cells and 500 fibroblast cells, and incubated for two days. Cultured spheroids were used.

(12) Drug treatment and angiogenesis analysis

Ramucirumab was serially diluted in EGM-2 medium to corresponding concentrations of 0.1 μM, 1 μM, 10 μM, and 100 μM. Oncogene-targeting small molecule inhibitors (Sapitinib, AZD8931; Volitinib, AZD6094; AZD4547) (SellekChem, CAT# S2192, S7674, S2801) were dissolved in DMSO to a final concentration of 1 μM according to the manufacturer's instructions. The drugs were injected into the TumorAngioChip along with the medium according to the designed timeline.

(13) Immunocytochemistry and imaging

The sample was fixed in DPBS (WELGENE, Korea) with 4% (w/v) paraformaldehyde (Biosesang, Korea) for 15 minutes, then immersed in 0.20% Triton X-100 (Sigma, X100) for 20 minutes. Permeabilized. Samples were then treated with 3% BSA (Sigma) for 24 h. Conditions for cellular fluorescent staining were as follows: Fluorescein-labeled Ulex Europaeus Agglutinin I (UEA I) (Vector Laboratories, CAT#FL-1061-2; 1:500), Alexa Fluor® 594 anti-human CD31 antibody (Biolegend , CAT# 303126; 1:200), Alexa Fluor® 594 anti-human CD326 (EpCAM) antibody (Biolegend, CAT#324228; 1:300), and Hoechst 33342 (Thermo Scientific™, CAT#62249; 1:1000).

(14) Western blotting analysis

PDC lysates were lysed in lysis buffer (150 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 10% glycerol, 20 mM HEPES [PH 7.4], 1% Triton X-100) with complete protease inhibitors (Invitrogen). Protein concentration was measured using BCA protein reagent (Pierce Biotechnology). PDC lysate (10 μg) was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel (using 12% gel) and transferred to a nitrocellulose membrane with 0.2 μm pore size (Whatman). nitrocellulose membranes) for 60 minutes. The membrane was blocked with 5% skim milk (BD Bioscience) in TBST buffer (Sigma) for 1 hour on a shaker at room temperature and then treated with primary antibody overnight. After washing three times with TBST buffer, the membrane was incubated with the corresponding IgG-horseadish peroxidase (HRP) secondary antibody for 1 h at room temperature. Visualization of the membrane was determined via the ECL detection system (Invitrogen).

(15) Statistical analysis

According to the experimental data, Wilcoxon concordant pairwise signed rank test, unpaired t test, Mann-Whitney U test, and two-way ANOVA were used. p < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; n.s., not significant). Sample numbers and analysis methods are specified in the text and figure legends; no statistics were used to determine sample size, blinding, or randomization methods.

2. Experimental results

(1) Sample preparation and sample classification

Between December 2014 and January 2021, 45 gastric cancer (GC) patients were enrolled (Table 1). We obtained 26 primary tumor tissues or 19 malignant ascitic fluid samples from these patients. Gastric cancer cell lines were purified and established from tumor cells, and all samples were subjected to whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) to decipher the molecular characteristics of the patients. . According to the classification of The Cancer Genome Atlas (TCGA), two samples were classified as Epstein-Barr virus (EBV) and the other was classified as a subgroup with high microsatellite instability (MSI). 35 samples were classified as genomically stable (GS) type and 7 samples were classified as chromosomally unstable (CIN) type (Figure 1a). Consistent with previous reports, we found that mutations in genes including ARID1A, CDH1, MUC6, ERBB2, and RNF43 were frequently found in the subgroup (Figure 1b).

We performed unsupervised clustering of whole transcriptome sequencing (WTS) data using expression levels of the VEGF and VEGFR signaling network (PID VEGF_VEGFR_pathway) to evaluate the efficacy of ramucirumab. was carried out. The cohort was dichotomized into “KDR-high” and “KDR-low” expression groups based on differential expression for KDR proteins (P = 4.481e-08, Wilcoxon signed rank test) (Figure 1c). In the above cohort, it was confirmed that there was no significant difference in the level of VEGF-A expression according to tissue origin (primary vs. metastases) (Figure 1d).

ERBB network and FGF and c-MET signaling pathways were confirmed to be significantly upregulated in the KDR-high group compared to the KDR-low group (Figure 1e, middle). Although further trials are needed, promising combination therapy partners may emerge. We included a set of all previously reported gene pathways to derive all relevant functional abnormalities associated with the KDR-high group. As expected, integrin and syndecan-1-mediated signaling pathway were upregulated in the genomically stable (GS) subtype.

Total (N=45) Age (years) 58 (28 - 80) Sex Male 31 (68.9%) Female 14 (31.1%) Race Asian 45 (100%) Sampling Ascites 19 (42.2%) Primary tumor 26 (57.8%) TCGA GS 35 (77.8%) CIN 7 (15.6%) EBV 2 (4.4%) MSI 1 (2.2%) Tumor grade Tubular adenocarcinoma 15 (33.3%) Metastatic adenocarcinoma 8 (17.8%) Signet-ring cell carcinoma 3 (6.7%) Others 19 (42.2%) HER2 positivity 8 (17.8%) c-MET positivity 7 (15.6%) FGFR2 positivity 3 (6.7%) EBV positivity 1 (2.2%) HER2 & c-MET positivity 1 (2.2%)

(3) Expanding the scale of tumor gene-targeted combination therapy

It is important to optimize treatment strategies for gastric cancer patients according to tumor characteristics. Therefore, we sought to optimize the combination treatment strategy by selecting patient samples with abnormalities in specific genes such as HER2, MET, and FGFR2, and performed large-scale drug screening (Figure 2a).

Our whole transcriptome sequencing (WTS) analysis confirmed the upregulation of ERBB, FGF or c-MET signaling in the KDR-high group (Figure 1e), as demonstrated in Figure 4b. was significantly correlated with the VEGFR2 signal (ERBB network: P < 0.01; c-MET network: P < 0.01; FGF network: P < 0.01, Pearson's correlation test), and at the same time in the KDR-high group (ERBB network: P < 0.01). 0.01; c-MET network: P < 0.01; FGF network: P < 0.01, Wilcoxon rank sum test). We analyzed the pathological examination of patient tissues and confirmed HER2, c-MET, and FGFR2 overexpression by immunohistochemical staining in RAM-23, RAM-24, and RAM-40, respectively (Figure 2c). We also confirmed the presence of HER2, c-MET, and FGFR2 in PDC spheroids established in RAM-23, RAM-24, and RAM-40, respectively (Figure 2d).

This screening system can be used to screen for monotherapy (ramucirumab, AZD8931 (HER2 inhibitor, Sapitinib), AZD6094 (MET inhibitor, Volitinib), AZD4547 (FGFR2 inhibitor)) and combination therapy (ramucirumab + AZD8931 (Sapitinib), ramuciru). The anti-angiogenic effect of Mab + AZD6094 (Volitinib), ramucirumab + AZD4547 (labeled with COMBO) was tested. We developed several indices to accurately reflect changes in angiogenesis following drug treatment.

In addition, ramucirumab was serially diluted in EGM-2 medium, and morphological quantitative analysis of the three-dimensional vascular network in response to ramucirumab concentration (0 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM) was performed. As a result, it was confirmed that RAM-24 showed significant differences in vessel density, angiogenic sprouting number, and average sprouting length at 1 μM. In the case of RAM-33, it was confirmed that there was a significant difference in the blood vessel density and average angiogenic sprout sprouting length at 1 μM, and in the number of angiogenic sprouts at 10 μM, and in the case of RAM-39, at 1 μM It was confirmed that there was a significant difference in blood vessel density, number of angiogenic sprouts, and average angiogenic sprout sprout length. In the case of RAM-44, there was a significant difference in blood vessel density, average angiogenic sprout sprout length at 0.1 μM and 100 μM. It was confirmed that there was a significant difference in the number of μM angiogenic sprouts (Figure 2e). The arrow shown in FIG. 2E indicates the drug concentration that showed statistical significance according to each analysis standard.

Furthermore, using the above screening system, monotherapy (ramucirumab, AZD8931 (HER2 inhibitor, Sapitinib), AZD6094 (MET inhibitor, Volitinib), AZD4547 (FGFR2 inhibitor)) and combination therapy (ramucirumab + AZD8931 (Sapitinib), The anti-angiogenic effects of ramucirumab + AZD6094 (Volitinib), ramucirumab + AZD4547 (labeled) were tested. We developed several indices to accurately reflect changes in angiogenesis following drug treatment.

For HER2-amplified PDC (RAM-23), vessel density (RAM vs. INH, P = 0.0496; COMBO vs. INH, P = 0.0387) and angiogenic sprout length (RAM vs. INH, P = 0.0036; COMBO vs. INH, P = 0.0003) was significantly reduced in treatment containing ramucirumab, but no significant anti-angiogenic effect was found between ramucirumab alone or in combination, and changes in PDC spheroid area were not observed with monotherapy. was significantly decreased in combination therapy (INH vs. COMBO, P = 0.0013, RAM vs COMBO, P = 0.0005) (Figure 2f). For MET-amplified PDC (RAM-24), changes in blood vessel density did not show significant differences, but changes in sprout length were significantly different from ramucirumab (RAM vs. INH, P = 0.0167; COMBO vs. INH, 0.0211; COMBO vs. RAM, P = 0.9872) was significantly reduced by treatment (Figure 2f). Meanwhile, the changes in PDC spheroid area showed that AZD6094 had anticancer effect and showed more reduction in combination therapy (INH vs. RAM, P = 0.0017; COMBO vs. RAM, P = 0.0006; INH vs. COMBO, P = 0.6908) (Figure 2f).

For FGFR2-amplified PDC, an anti-angiogenic effect was consistently observed across all treatments, taking into account changes in sprout length, which was insufficient to prioritize specific treatment conditions. The change in PDC spheroid area was confirmed to be significantly reduced with the combination treatment (COMBO vs. RAM, P = 0.0236; COMBO vs. INH, P = 0.2861; INH vs. RAM, P = 0.2741) (Figure 2f).

All three models demonstrated that ramucirumab and the targeted agent exhibited excellent tumor cell toxicity. Additionally, the anti-angiogenic effect of the inhibitor was demonstrated using indicators such as blood vessel density (Figure 2f). By combining ramucirumab with a small molecule inhibitor, tumor-induced angiogenesis was substantially reduced in all three PDC models compared to using ramucirumab or the small molecule inhibitor individually.

Claims (12)

A pharmaceutical composition for anti-angiogenesis comprising Ramucirumab and a HER2 inhibitor.
The method of claim 1, wherein the HER2 inhibitor is Sapitinib, Trastuzumab, Lapatinib, Margetuximab, Tucatinib, Pertuzumab and Herceptin. A composition that is at least one selected from the group consisting of (Herceptin).
The composition of claim 1, wherein the composition reduces blood vessel density.
The composition of claim 1, wherein the composition reduces the length of newly formed blood vessels.
The method according to claim 1, wherein the composition reduces the area of PDC spheroids (Patient-derived cells spheroids).
Anti-angiogenic dietary supplement containing Ramucirumab and HER2 inhibitor.
The method of claim 6, wherein the HER2 inhibitor is Sapitinib, Trastuzumab, Lapatinib, Margetuximab, Tucatinib, Pertuzumab and Herceptin. (Herceptin), one or more health functional foods selected from the group consisting of.
A pharmaceutical composition for preventing or treating cancer containing Ramucirumab and a HER2 inhibitor.
The method of claim 8, wherein the HER2 inhibitor is Sapitinib, Trastuzumab, Lapatinib, Margetuximab, Tucatinib, Pertuzumab and Herceptin. A pharmaceutical composition for preventing or treating cancer, which is at least one selected from the group consisting of (Herceptin).
The method of claim 8, wherein the cancer includes lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, ovarian cancer, colon cancer, anal cancer, biliary tract cancer, bladder cancer, gallbladder cancer, head and neck cancer, oral cancer, pharynx cancer, nasopharyngeal cancer, kidney cancer, and muscle cancer. , a pharmaceutical composition for preventing or treating cancer, which is at least one selected from the group consisting of bone cancer, prostate cancer, rectal cancer, small intestine cancer, urethral cancer, thyroid cancer, and breast cancer.
Health functional food for preventing or improving cancer, including Ramucirumab and HER2 inhibitors.
The method of claim 11, wherein the HER2 inhibitor is Sapitinib, Trastuzumab, Lapatinib, Margetuximab, Tucatinib, Pertuzumab and Herceptin. (Herceptin) A health functional food for preventing or improving cancer, which is at least one selected from the group consisting of Herceptin.