KR20220165628A - Composition for treating or preventing colorectal cancer in combination, comprising SYK inhibitor - Google Patents
Composition for treating or preventing colorectal cancer in combination, comprising SYK inhibitor Download PDFInfo
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- KR20220165628A KR20220165628A KR1020210177897A KR20210177897A KR20220165628A KR 20220165628 A KR20220165628 A KR 20220165628A KR 1020210177897 A KR1020210177897 A KR 1020210177897A KR 20210177897 A KR20210177897 A KR 20210177897A KR 20220165628 A KR20220165628 A KR 20220165628A
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
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- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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Abstract
Description
본 발명은 대장암의 MEK 저해제에 대한 내성을 극복하기 위한 병용 치료제 및 MEK 저해제에 내성을 유발할 수 있음을 예측하는 바이오마커로서 비장 티로신 키나아제(spleen tyrosine kinase, SYK) 및 SYK 저해제의 신규한 용도에 관한 것이다. The present invention relates to a novel use of spleen tyrosine kinase (SYK) and SYK inhibitors as a combination therapy for overcoming resistance to MEK inhibitors in colorectal cancer and as a biomarker predicting that resistance to MEK inhibitors may be induced. it's about
암은 대표적인 호발성 난치 질환으로서 전세계적으로 큰 문제가 되고 있다. 그 중 대장암은 2020년 국제암연구센터(IARC)에서 발행된 "Global cancer statistics 2020" 보고서에 따르면 암 사망의 주요 원인 중 2위를 기록하고 있다. 대장암을 치료하기 위한 효과적인 전략 구축을 위해서는 대장암을 더 세분화해서 이해할 필요가 있었고, 이를 위해 대장암을 분자적 수준에서 4개의 하위유형으로 분류한 합의 분자 하위 유형(consensus molecular subtype, CMS)이 제시되었다. 4가지의 CMS 중 CMS4는 암이 진행된 단계에서 발생하는 유형으로 TGF-β 활성화, stromal infiltration, epithelial-mesenchymal transition, angiogenesis의 특징을 갖고 있으면서 임상적 특징으로 다른 유형과 비교했을 때 진행된 암(advanced cancer)에서 주로 발견되며, 전체 생존율이 가장 낮은 유형으로, 효과적인 치료 전략이 필요하다. 현재 암의 주요 치료 전략은 특정 분자를 타겟으로 하여 암 세포만을 사멸시키는 표적 치료 전략으로, 환자 개개인에 맞는 치료를 하는 것으로 발전하고 있다. 대장암의 40% 이상은 KRAS, BRAF 돌연변이를 가지고 있는데, 이는 CMS 각 유형에서도 30-40% 이상으로 나타나는 특징이다. 이런 경우 MAPK 경로의 활성화가 유발되고, 이는 암세포의 생존과 증식을 유도한다. 이 경로의 활성을 억제하기 위해 MAPK 경로의 MEK1/2를 타겟으로 하는 표적 치료제인, MEK 저해제를 적용하는 것을 하나의 치료 전략으로 연구되어왔다. 그러나 MEK 저해제 단일 약물 요법은 내성이 발생하는 경우가 많아 충분한 치료 효과를 보지 못하는 문제가 있다.BACKGROUND OF THE INVENTION [0002] Cancer is a representative incurable disease and has become a major problem worldwide. Among them, colorectal cancer ranks second among the leading causes of cancer death according to the "Global cancer statistics 2020" report published by the International Agency for Research on Cancer (IARC) in 2020. In order to establish an effective strategy for treating colorectal cancer, it was necessary to further subdivide and understand colorectal cancer. To this end, the consensus molecular subtype (CMS), which classified colorectal cancer into four subtypes at the molecular level, presented Among the four types of CMS, CMS4 is a type that occurs at an advanced stage of cancer and has the characteristics of TGF-β activation, stromal infiltration, epithelial-mesenchymal transition, and angiogenesis. ), and is the type with the lowest overall survival rate, requiring an effective treatment strategy. Currently, a major treatment strategy for cancer is a targeted treatment strategy that kills only cancer cells by targeting a specific molecule, and is evolving into a treatment tailored to each patient. More than 40% of colorectal cancers have KRAS and BRAF mutations, which is a characteristic that appears in more than 30-40% of each type of CMS. In this case, the activation of the MAPK pathway is induced, which induces the survival and proliferation of cancer cells. In order to inhibit the activity of this pathway, the application of MEK inhibitors, which are targeted therapeutics targeting MEK1/2 of the MAPK pathway, has been studied as one therapeutic strategy. However, MEK inhibitor single drug therapy has a problem in that it does not show sufficient therapeutic effect in many cases where resistance occurs.
따라서 MEK 저해제에 대한 내성을 극복하여 이의 치료 효과를 증대시킬 수 있는 방법의 개발이 필요하다.Therefore, it is necessary to develop a method capable of overcoming resistance to MEK inhibitors and increasing their therapeutic effect.
이에 본 발명의 발명자는 생존율이 가장 낮은 대장암 합의 분자 하위 유형인 CMS4 대장암의 MEK 저해제에 대한 내성을 극복하기 위하여 연구하던 중, SYK 저해제를 병용 처리시 MEK 저해제의 치료 효과가 증대되는 것을 확인하여 본 발명을 완성하였다. Accordingly, the inventor of the present invention confirmed that the therapeutic effect of the MEK inhibitor is increased when the SYK inhibitor is treated in combination while studying to overcome the resistance to the MEK inhibitor of CMS4 colorectal cancer, which is the molecular subtype of colorectal cancer with the lowest survival rate. Thus, the present invention was completed.
따라서 본 발명의 목적은 항암제 내성 억제 또는 개선용 약학적 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for inhibiting or improving anticancer drug resistance.
본 발명의 다른 목적은 항암 보조제를 제공하는 것이다.Another object of the present invention is to provide an anti-cancer adjuvant.
본 발명의 또 다른 목적은 암의 예방 또는 치료를 위한 병용 투여용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for combined administration for the prevention or treatment of cancer.
본 발명의 또 다른 목적은 암의 예방 또는 치료를 위한 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or treating cancer.
본 발명의 또 다른 목적은 항암제에 대한 내성 억제 또는 개선용 제제의 스크리닝 방법을 제공하는 것이다.Another object of the present invention is to provide a screening method for an agent for inhibiting or improving resistance to an anticancer agent.
본 발명의 또 다른 목적은 항암제에 대한 내성 예측용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for predicting resistance to anticancer drugs.
상기 목적을 달성하기 위하여, 본 발명은 SYK(spleen tyrosine kinase) 저해제를 포함하는 항암제 내성 억제 또는 개선용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for inhibiting or improving anticancer drug resistance comprising a SYK (spleen tyrosine kinase) inhibitor.
상기 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 조성물을 포함하는 항암 보조제를 제공한다.In order to achieve the above other object, the present invention provides an anticancer adjuvant comprising the composition according to the present invention.
상기 또 다른 목적을 달성하기 위하여, 본 발명은 SYK 저해제 및 MAPK(mitogen-activated protein kinase) 신호전달경로 저해제를 포함하는 암의 예방 또는 치료를 위한 병용 투여용 약학적 조성물을 제공한다.In order to achieve the above another object, the present invention provides a pharmaceutical composition for combined administration for the prevention or treatment of cancer containing a SYK inhibitor and a MAPK (mitogen-activated protein kinase) signaling pathway inhibitor.
상기 또 다른 목적을 달성하기 위하여, 본 발명은 SYK 저해제 및 MAPK(mitogen-activated protein kinase) 신호전달경로 저해제를 포함하는 암의 예방 또는 개선용 식품 조성물을 제공한다.In order to achieve the above another object, the present invention provides a food composition for preventing or improving cancer containing a SYK inhibitor and a MAPK (mitogen-activated protein kinase) signaling pathway inhibitor.
상기 또 다른 목적을 달성하기 위하여, 본 발명은 SYK 단백질 또는 SYK 단백질을 코딩하는 유전자를 포함하는 시료에 후보물질을 처리하는 단계; 및 상기 SYK 단백질 또는 유전자의 활성 또는 발현 수준이 후보물질 처리 전보다 감소하는 경우 상기 후보물질은 항암제에 대한 내성 억제 또는 개선 활성이 있는 것으로 판정하는 단계; 를 포함하는 항암제에 대한 내성 억제 또는 개선용 제제의 스크리닝 방법을 제공한다.In order to achieve the above another object, the present invention comprises the steps of processing a candidate substance to a sample containing a SYK protein or a gene encoding the SYK protein; And if the activity or expression level of the SYK protein or gene is reduced than before treatment with the candidate material, determining that the candidate material has an anti-cancer drug resistance suppression or improvement activity; It provides a screening method for an agent for inhibiting or improving resistance to an anticancer agent comprising a.
상기 또 다른 목적을 달성하기 위하여, 본 발명은 SYK(spleen tyrosine kinase)의 발현 또는 활성을 측정하는 제제를 포함하는, 항암제에 대한 내성 예측용 조성물을 제공한다.In order to achieve the above another object, the present invention provides a composition for predicting resistance to anticancer drugs, including an agent for measuring the expression or activity of SYK (spleen tyrosine kinase).
본 발명에 따르면 MEK 저해제에 대해 내성을 가진 CMS4의 대장암 세포주에서 SYK 수준이 높게 발현되며, SYK 저해제는 CMS4 대장암에서 유발된 MEK 저해제에 대한 내성을 극복하여 MEK 저해제의 치료 효과를 증대시킬 수 있음을 확인한 바, 대장암 환자에 있어서, MEK 저해제에 대한 내성 발생 가능성 예측과 더불어 SYK 저해제 및 MEK 병용 처리를 통해 대장암을 효과적으로 치료할 수 있는 새로운 치료 전략으로 유용하게 활용할 수 있다.According to the present invention, SYK levels are highly expressed in CMS4 colorectal cancer cell lines resistant to MEK inhibitors, and SYK inhibitors can overcome the resistance to MEK inhibitors induced in CMS4 colorectal cancer to increase the therapeutic effect of MEK inhibitors. As confirmed, it can be usefully used as a new treatment strategy that can effectively treat colorectal cancer through the combination treatment of SYK inhibitor and MEK, along with prediction of the possibility of resistance to MEK inhibitors in colorectal cancer patients.
도 1은 MEK 저해제에 대해 내성을 보이는 대장암 세포주를 선별한 결과이다.
도 2a 내지 도 2e는 MEK 저해제의 효과 증진을 위한 병용 타겟을 동정한 결과이다.
도 3a 내지 도 3e는 MEK 저해제에 내성을 갖는 대장암 세포주에서 MEK 저해제 및 SYK 저해제 병용처리에 따른 효과를 확인한 결과이다(SYKi: SYK 저해제, HSA: 가장 높은 단일 에이전트(Highest single agent), shScr: sh스크램블(shscramble), Tra: 트라메티닙, Sel: 셀루메티닙.)
도 4는 MEK 저해제에 민감한 대장암 세포주에서 MEK 저해제 및 SYK 저해제 병용처리에 따른 효과를 확인한 결과이다.
도 5는 MEK 저해제와 SYK 저해제 병용 처리에 따른 메커니즘을 확인한 결과이다.
도 6은 MEK 저해제와 SYK 저해제 병용 처리에 있어서, 각 저해제 처리 조건에 따른 신호 전달 네트워크 상태를 나타낸 이미지이다.1 shows the results of screening colon cancer cell lines showing resistance to MEK inhibitors.
2a to 2e are results of identifying combination targets for enhancing the effect of MEK inhibitors.
Figures 3a to 3e are the results of confirming the effect of the combined treatment of the MEK inhibitor and the SYK inhibitor in colorectal cancer cell lines resistant to the MEK inhibitor (SYKi: SYK inhibitor, HSA: Highest single agent, shScr: shscramble, Tra: trametinib, Sel: selumetinib.)
Figure 4 is a result of confirming the effect of the MEK inhibitor and SYK inhibitor combined treatment in colorectal cancer cell lines sensitive to MEK inhibitors.
Figure 5 is a result of confirming the mechanism according to the combined treatment with the MEK inhibitor and the SYK inhibitor.
6 is an image showing the signal transduction network state according to each inhibitor treatment condition in the combined treatment of the MEK inhibitor and the SYK inhibitor.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 항암제에 대해 내성을 가진 CMS4의 대장암에 대한 효과적인 치료 방법에 대한 것으로, SYK(spleen tyrosine kinase) 저해제를 포함하는 항암제 내성 억제 또는 개선용 약학적 조성물을 제공한다.The present invention relates to an effective treatment method for colorectal cancer of CMS4 resistant to anticancer drugs, and provides a pharmaceutical composition for inhibiting or improving anticancer drug resistance including a spleen tyrosine kinase (SYK) inhibitor.
본 발명의 일실시예에 따르면, MEK 저해제 및 SYK 저해제를 CMS4 대장암 세포주에 병용투여시 MEK 저해제에 대한 내성을 극복하여 MEK 저해제의 치료 효과를 증대시킬 수 있음을 확인하였다.According to one embodiment of the present invention, it was confirmed that the therapeutic effect of the MEK inhibitor can be increased by overcoming the resistance to the MEK inhibitor when the MEK inhibitor and the SYK inhibitor are co-administered to the CMS4 colorectal cancer cell line.
본 발명에 있어서, 상기 "MEK(mitogen-activated protein kinase kinase)"는 MEK1과 MEK2로 구성되어 있으며 다양한 성장인자(growth factor), 사이토카인(cytokine), 막 탈분극(membrane depolarization) 또는 칼슘 유입(calcium influx)에 의해 활성화된다. 또한 MEK의 인산화에 의해 ERK(extracellular signal-regulated kinase)가 활성화된다.In the present invention, the "MEK (mitogen-activated protein kinase kinase)" is composed of MEK1 and MEK2, and various growth factors, cytokines, membrane depolarization (membrane depolarization) or calcium influx (calcium activated by influx. In addition, ERK (extracellular signal-regulated kinase) is activated by phosphorylation of MEK.
따라서, 상기 항암제는 이에 제한되는 것은 아니나, 트라메티닙(Trametinib), 셀루메티닙(selumetinib), 비니메티닙(binimetinib), 피마세르팁(Pimasertib), 레파메티닙(Refametinib), 코비메티닙(cobimetinib), U0126-EtOH, PD184352, PD98059, BIX02189, SL-327, BIX02188, AZD8330, TAK-733 및 PD318088으로 이루어진 군으로부터 선택되는 하나 이상의 MEK 저해제일 수 있으며, 보다 바람직하게는 트라메티닙(Trametinib) 또는 셀루메티닙(selumetinib)일 수 있다.Therefore, the anticancer agent is not limited thereto, but is not limited to, trametinib, selumetinib, binimetinib, pimasertib, refametinib, cobimetinib ( cobimetinib), U0126-EtOH, PD184352, PD98059, BIX02189, SL-327, BIX02188, AZD8330, TAK-733, and PD318088. or selumetinib.
본 발명에 있어서, 상기 "SYK(spleen tyrosine kinase)"는 대식세포, 비만세포, B 세포등 각종 염증 관련 세포들에 발현 되어있는 단백질로, SYK가 활성화되면 이러한 염증을 일으키는 면역세포들의 식균 작용 반응을 유도하여, 항체에 의한 염증 증식에 SYK가 중요한 역할을 한다.In the present invention, the "spleen tyrosine kinase (SYK)" is a protein expressed in various inflammation-related cells such as macrophages, mast cells, and B cells, and when SYK is activated, the phagocytosis reaction of immune cells that cause such inflammation , SYK plays an important role in antibody-induced inflammatory proliferation.
본 발명에 있어서, 상기 "SYK 저해제(SYK inhibitor, SYKi)"는 SYK 유전자 또는 SYK 단백질 발현 저해제 또는 SYK 단백질 활성 저해제일 수 있다.In the present invention, the "SYK inhibitor (SYKi)" may be a SYK gene or SYK protein expression inhibitor or a SYK protein activity inhibitor.
상기 SYK 유전자 또는 SYK 단백질 발현 저해제는 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오티드, siRNA(short interfering RNA), shRNA(short hairpin RNA) 및 리보자임으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 제한되지 않는다. 또한, 상기 SYK 단백질 활성 저해제는 상기 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 미메틱스(Peptide Minetics), 앱타머, 항체 및 천연물로 구성된 군으로부터 선택된 하나 이상일 수 있으나, 이에 제한되는 것은 아니다. 상기 항체는 상기 SYK 단백질에 특이적으로 결합할 수 있는 단일클론 항체, 다클론항체, 또는 재조합 항체 등을 포함하며, 당업자에게 알려진 공지의 방법으로 제작하거나 시판되는 것을 구입하여 사용할 수 있다. The SYK gene or SYK protein expression inhibitor may be at least one selected from the group consisting of an antisense nucleotide, short interfering RNA (siRNA), short hairpin RNA (shRNA), and ribozyme that complementarily bind to mRNA of the gene, but is not limited thereto don't In addition, the SYK protein activity inhibitor may be one or more selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the protein, but is not limited thereto. . The antibody includes a monoclonal antibody, a polyclonal antibody, or a recombinant antibody capable of specifically binding to the SYK protein, and may be prepared by a known method known to those skilled in the art or purchased and used commercially.
본 발명에 있어서, 상기 SYK 저해제는 이에 제한되는 것은 아니나, 포스타마티닙(fostamatinib) 및 세비도플레닙(cevidoplenib)으로 구성된 군으로부터 선택된 하나 이상일 수 있으며, 바람직하게는 포스타마티닙일 수 있다. In the present invention, the SYK inhibitor is not limited thereto, but may be at least one selected from the group consisting of fostamatinib and cevidoplenib, preferably fostamatinib.
본 발명의 일 실시예에서는, 상기 SYK 저해제로 포스타마티닙 이나트륨(fostamatinib disodium)을 사용하여 실험을 진행하였다.In one embodiment of the present invention, an experiment was conducted using fostamatinib disodium as the SYK inhibitor.
본 발명은 SYK 저해제와 항암제를 병용 투여하는 경우 항암제에 대하여 유도된 저항성 또는 약제 내성을 극복하고 기존 항암제의 항암 효과를 현저히 개선할 수 있음을 밝힌 것에 그 의의가 있으므로, 상기 SYK 저해제는 본 발명의 기술분야에서 사용되거나 SYK 저해 활성이 있는 것으로 밝혀진 것이라면 그 종류에 상관없이 본 발명에 적용될 수 있고, 구체적인 종류에 한정되는 것이 아님은 당업자에게 자명하다고 할 것이다. Since the present invention is significant in that it is possible to overcome resistance or drug resistance induced to anticancer drugs and significantly improve the anticancer effect of existing anticancer drugs when a SYK inhibitor and anticancer drugs are administered together, the SYK inhibitor is the present invention. Anything used in the art or found to have SYK inhibitory activity can be applied to the present invention regardless of its type, and it will be apparent to those skilled in the art that it is not limited to specific types.
본 발명의 약학적 조성물에 있어서, 상기 SYK 저해제는 항암제와 동시 또는 순차적으로 투여될 수 있다.In the pharmaceutical composition of the present invention, the SYK inhibitor may be administered simultaneously or sequentially with the anticancer agent.
또한, 본 발명은 본 발명에 따른 조성물을 포함하는 항암 보조제를 제공한다.In addition, the present invention provides an anticancer adjuvant comprising the composition according to the present invention.
본 발명에 있어서, 상기 "항암 보조제"는 항암제의 항암효과를 개선, 향상 또는 증대시킬 수 있는 제제를 의미한다.In the present invention, the "anti-cancer adjuvant" refers to an agent capable of improving, improving or enhancing the anti-cancer effect of an anti-cancer agent.
본 발명에 있어서, 상기 항암 보조제는 처리농도에 따라 항암제 또는 항암보조제로서 사용할 수 있으며, 항암제의 감수성을 증진시킬 수 있다. In the present invention, the anticancer adjuvant can be used as an anticancer agent or an anticancer adjuvant depending on the treatment concentration, and can enhance the sensitivity of the anticancer agent.
본 발명에 있어서, 상기 항암 보조제는 암을 예방, 개선 또는 치료하는 효과를 가지는 공지의 화합물과 병용하여 투여될 수 있다.In the present invention, the anticancer adjuvant may be administered in combination with a known compound having an effect of preventing, improving or treating cancer.
또한, 본 발명은 SYK 저해제 및 MAPK(mitogen-activated protein kinase) 신호전달경로 저해제를 포함하는 암의 예방 또는 치료를 위한 병용 투여용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for combined administration for the prevention or treatment of cancer containing a SYK inhibitor and a mitogen-activated protein kinase (MAPK) signaling pathway inhibitor.
본 발명에 있어서, 상기 "MAPK 신호전달경로"는 RAS-RAF-MEK-ERK 캐스케이드(cascade)로 이루어진 신호전달경로로 여러 세포 내 신호전달경로에 공통적으로 존재하는 매우 중요한 신호전달시스템으로 알려져 있다. 특히, 사람 암 조직의 약 30% 정도에서 MAPK 신호전달경로의 비정상적 활성화가 관찰되는 등 MAPK 신호전달경로에서의 이상(aberration)은 암 세포학에서 중요한 역할을 수행한다.In the present invention, the "MAPK signaling pathway" is a signaling pathway consisting of the RAS-RAF-MEK-ERK cascade and is known as a very important signaling system commonly present in several intracellular signaling pathways. In particular, aberrations in the MAPK signaling pathway play an important role in cancer cytology, such as abnormal activation of the MAPK signaling pathway being observed in about 30% of human cancer tissues.
본 발명에 있어서, 상기 MAPK 신호전달경로 저해제(MAPK inhibitor)는 상기 신호전달경로중 MEK를 억제할 수 있는, MEK 저해제를 의미할 수 있으며, MEK 신호전달경로의 활성화 저해를 초래하는 물질이라면 제한 없이 포함된다. In the present invention, the MAPK signaling pathway inhibitor (MAPK inhibitor) may mean a MEK inhibitor capable of inhibiting MEK in the signaling pathway, and any substance that causes activation inhibition of the MEK signaling pathway is not limited. included
본 발명에 있어서, 상기 암은 대장암, 유방암, 폐암, 위암, 전립선암, 갑상선암, 결장직장암, 흑색종, 두경부암, 피부암, 췌장암, 난소암, 방광암, 간암 및 신장암으로 이루어진 군으로부터 선택될 수 있으나, 이에 제한되는 것은 아니다. 바람직하게는 대장암일 수 있으며, 보다 바람직하게는 전체 생존율이 가장 낮은, 분자 하위 유형 4(consensus molecular subtype 4, CMS4)의 대장암일 수 있다. In the present invention, the cancer is selected from the group consisting of colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, thyroid cancer, colorectal cancer, melanoma, head and neck cancer, skin cancer, pancreatic cancer, ovarian cancer, bladder cancer, liver cancer and kidney cancer. It may be, but is not limited thereto. Preferably, it may be colorectal cancer, and more preferably, it may be colorectal cancer of consensus molecular subtype 4 (CMS4), which has the lowest overall survival rate.
본 발명에 있어서, 상기 약학적 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학적 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다. 상기 약학적 조성물은 이들로 한정되는 것은 아니지만, 각각 통상의 방법에 따라 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. In the present invention, the pharmaceutical composition may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be intended for humans. The pharmaceutical compositions are not limited to these, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injection solutions, respectively, according to conventional methods. .
본 발명에 따른 약학적 조성물은 약제학적으로 허용가능한 담체를 포함할 수 있다. 약제학적으로 허용되는 담체는 경구투여시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명에 따른 약학적 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구투여 시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration, and buffers, preservatives, and painless agents for injections. A topical, solubilizing agent, isotonic agent, stabilizer, etc. may be mixed and used, and in the case of topical administration, a base, excipient, lubricant, preservative, etc. may be used. Formulations of the pharmaceutical composition according to the present invention may be variously prepared by mixing with the pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. there is.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may be further included.
본 발명에 따른 약학적 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함된다. 상기 "비경구"는 피하, 피내, 정맥내, 근육내, 관절내, 활액낭내, 흉골내, 경막내, 병소내 및 두개골 내 주사 또는 주입기술을 포함한다. 본 발명에 따른 약학적 조성물은 또한 직장 투여를 위한 좌제의 형태로 투여될 수 있다.The route of administration of the pharmaceutical composition according to the present invention is, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal. The term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition according to the present invention may also be administered in the form of a suppository for rectal administration.
본 발명에 따른 약학적 조성물은 사용된 특정 유효성분의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여시간, 투여경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학적 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있다. 바람직하게는 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여할 수 있으며, 더욱 바람직하게는 1~10000㎍/체중kg/day, 더욱더 바람직하게는 10~1000㎎/체중kg/day의 유효용량으로 하루에 수회 반복 투여될 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The pharmaceutical composition according to the present invention is a variety of factors including the activity of the specific active ingredient used, age, body weight, general health, sex, diet, administration time, route of administration, excretion rate, drug combination, and severity of a specific disease to be prevented or treated. The dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, disease severity, drug type, administration route and duration, but may be appropriately selected by those skilled in the art. Preferably, considering all of the above factors, it is possible to administer an amount that can obtain the maximum effect with the minimum amount without side effects, more preferably 1 to 10000 μg/kg body weight/day, and even more preferably 10 to 1000 mg It can be administered repeatedly several times a day at an effective dose of /kg body weight/day. The dosage is not intended to limit the scope of the present invention in any way.
더불어 본 발명은 SYK 저해제 및 MAPK(mitogen-activated protein kinase) 신호전달경로 저해제를 포함하는 암의 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving cancer containing a SYK inhibitor and a mitogen-activated protein kinase (MAPK) signaling pathway inhibitor.
상기 MAPK 신호전달경로 저해제는 상술한 바와 같이, MEK 저해제일 수 있다.As described above, the MAPK signaling pathway inhibitor may be a MEK inhibitor.
본 발명에 따른 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food), 건강기능식품(health functional food) 및 식품 첨가제(food additives) 등의 모든 형태를 포함한다.The food composition according to the present invention includes all forms such as functional food, nutritional supplement, health food, health functional food and food additives. .
본 발명에 있어서, 용어 "건강기능식품"이란 건강보조의 목적으로 특정성분을 원료로 하거나 식품 원료에 들어있는 특정성분을 추출, 농축, 정제, 혼합 등의 방법으로 제조, 가공한 식품을 말하며, 상기 성분에 의해 생체 방어, 생체리듬의 조절, 질병의 방지와 회복 등 생체조절기능을 생체에 대하여 충분히 발휘할 수 있도록 설계되고 가공된 식품을 말하는 것으로서, 질병의 예방 또는 건강의 회복 등과 관련된 기능을 수행할 수 있는 것을 말한다.In the present invention, the term "health functional food" refers to a food prepared and processed by extracting, concentrating, refining, mixing, etc., a specific ingredient as a raw material or a specific ingredient contained in a food raw material for the purpose of health supplementation, It refers to food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc. by the above components, and performs functions related to disease prevention or health recovery. say what you can do
본 발명에 따른 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다.The food composition according to the present invention can be prepared in various forms according to conventional methods known in the art.
예를 들면, 건강식품으로는 본 발명에 따른 SYK 저해제 및 MAPK 신호전달경로 저해제 혼합 자체를 과립화, 캡슐화 및 분말화하여 섭취하거나 차, 쥬스 및 드링크의 형태로 제조하여 음용하도록 할 수 있다. 또한, 본 발명의 SYK 저해제 및 MAPK 신호전달경로 저해제 혼합을 대장암 치료 또는 MEK 저해제 내성 극복 효과가 알려진 공지의 물질 또는 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다.For example, as a health food, the mixture of the SYK inhibitor and the MAPK signaling pathway inhibitor according to the present invention may be granulated, encapsulated, and powdered to be ingested or prepared in the form of tea, juice, and drink to be consumed. In addition, the mixture of the SYK inhibitor and the MAPK signaling pathway inhibitor of the present invention may be mixed with known substances or active ingredients known to have effects for treating colorectal cancer or overcoming resistance to MEK inhibitors, and preparing a composition in the form of a mixture.
또한, 기능성 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마멀레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치이즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 본 발명의 SYK 저해제 및 MAPK 신호전달경로 저해제 혼합을 첨가하여 제조할 수 있다.In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled food, jam, marmalade, etc.), fish, meat, and their processed foods (e.g., ham, sausage corned beef, etc.) , breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort It can be prepared by adding a mixture of the SYK inhibitor and the MAPK signaling pathway inhibitor of the present invention to food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.).
본 발명의 식품 조성물 중 상기 본 발명의 SYK 저해제 및 MAPK 신호전달경로 저해제 혼합의 바람직한 함유량으로는 이에 한정되지 않지만 예를 들어 최종적으로 제조된 식품 중 0.01 내지 80 중량%일 수 있으며, 바람직하게는 최종적으로 제조된 식품 중 0.01 내지 50 중량%일 수 있다.The preferable content of the mixture of the SYK inhibitor and the MAPK signaling pathway inhibitor of the present invention in the food composition of the present invention is not limited thereto, but may be, for example, 0.01 to 80% by weight of the final food, preferably the final It may be 0.01 to 50% by weight of food prepared with.
본 발명의 식품 조성물은 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100㎖ 당 일반적으로 약 0.01 내지 0.4g, 바람직하게는 약 0.02 내지 0.03g일 수 있다.The food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional components. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. . The proportion of the natural carbohydrate may be generally about 0.01 to 0.4 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 식품 조성물은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 본 발명의 조성물 100중량부 당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이나, 이에 제한되는 것은 아니다.In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is generally selected from the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto.
또한, 본 발명은 SYK 단백질 또는 SYK 단백질을 코딩하는 유전자를 포함하는 시료에 후보물질을 처리하는 단계; 및 상기 SYK 단백질 또는 유전자의 활성 또는 발현 수준이 후보물질 처리 전보다 감소하는 경우 상기 후보물질은 항암제에 대한 내성 억제 또는 개선 활성이 있는 것으로 판정하는 단계; 를 포함하는 항암제에 대한 내성 억제 또는 개선용 제제의 스크리닝 방법을 제공한다.In addition, the present invention comprises the steps of processing a candidate substance to a sample containing a SYK protein or a gene encoding the SYK protein; And if the activity or expression level of the SYK protein or gene is reduced than before treatment with the candidate material, determining that the candidate material has an anti-cancer drug resistance suppression or improvement activity; It provides a screening method for an agent for inhibiting or improving resistance to an anticancer agent comprising a.
본 발명에 있어서, 유전자의 발현 수준 또는 단백질의 양을 측정하는 방법은 공지의 기술을 이용하여 생물학적 시료로부터 mRNA 또는 단백질을 분리하는 공지의 공정을 포함하여 수행될 수 있다. 상기 생물학적 시료는 상기 유전자의 발현 수준 또는 단백질의 수준이 정상 대조군과는 다른, 생체로부터 채취된 시료를 말하며, 상기 시료로는 예를 들면, 조직, 세포, 혈액, 혈청, 혈장, 타액 및 뇨 등이 포함될 수 있으나, 이에 제한되지 않는다.In the present invention, the method for measuring the expression level of a gene or the amount of a protein may include a known process of isolating mRNA or protein from a biological sample using a known technique. The biological sample refers to a sample collected from a living body in which the expression level or protein level of the gene is different from that of the normal control, and the sample includes, for example, tissue, cell, blood, serum, plasma, saliva, urine, etc. This may be included, but is not limited thereto.
상기 유전자의 발현 수준 측정은 바람직하게는 mRNA의 수준을 측정하는 것이며, mRNA의 수준을 측정하는 방법으로는 역전사 중합효소연쇄반응(RT-PCR), 실시간 역전사 중합효소연쇄반응, RNase 보호 분석법, 노던 블럿 및 DNA 칩 등이 있으나, 이에 제한되지 않는다.The expression level of the gene is preferably measured by measuring the level of mRNA, and methods for measuring the level of mRNA include reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern Blots and DNA chips, etc., but are not limited thereto.
상기 단백질 수준의 측정은 항체를 이용할 수 있는데, 이러한 경우 생물학적 시료 내의 상기 마커 단백질과 이에 특이적인 항체는 결합물, 즉, 항원-항체 복합체를 형성하며, 항원-항체 복합체의 형성량은 검출 라벨(detection label)의 시그널의 크기를 통해서 정량적으로 측정할 수 있다. 이러한 검출 라벨은 효소, 형광물, 리간드, 발광물, 미소입자(microparticle), 레독스 분자 및 방사선 동위원소로 이루어진 그룹 중에서 선택할 수 있으며, 이에 제한되지 않는다. 단백질 수준을 측정하기 위한 분석 방법으로는 웨스턴 블롯, ELISA, 방사선면역분석, 방사선 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 면역침전 분석법, 보체 고정분석법, FACS, 단백질 칩 등이 있으나, 이에 제한되지 않는다.An antibody may be used to measure the protein level. In this case, the marker protein in a biological sample and an antibody specific thereto form a binding product, that is, an antigen-antibody complex, and the amount of the antigen-antibody complex formed is a detection label ( It can be measured quantitatively through the size of the signal of the detection label). These detection labels may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes, but are not limited thereto. Assay methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chips, etc., but are not limited thereto.
본 발명에 있어서, 상기 후보물질은 통상적인 선정방식에 따라 SYK 단백질 또는 SYK 단백질을 코딩하는 유전자의 활성 또는 발현 수준을 저해하는 의약으로서의 가능성을 지닌 것으로 추정되거나 또는 무작위적으로 선정된 개별적인 핵산, 단백질, 기타 추출물 또는 천연물, 화합물 등이 될 수 있다.In the present invention, the candidate substance is an individual nucleic acid or protein presumed to have potential as a drug that inhibits the activity or expression level of SYK protein or a gene encoding SYK protein according to a conventional selection method, or randomly selected. , other extracts or natural products, compounds, and the like.
본 발명의 일 실시예에서는, SYK 저해제를 MEK 저해제와 병용 투여 시 시너지 효과를 통하여 항암제 내성을 극복하고 항암 효과를 증대시킬 수 있음을 확인하였는바, 상기 SYK 단백질 또는 유전자의 활성 또는 발현 수준을 감소시키는 물질은 항암제에 대한 내성 억제 또는 개선용 제제로서 사용될 수 있다.In one embodiment of the present invention, it was confirmed that when a SYK inhibitor is administered in combination with a MEK inhibitor, anticancer drug resistance can be overcome and the anticancer effect can be increased through a synergistic effect, thereby reducing the activity or expression level of the SYK protein or gene. Substances capable of inhibiting can be used as agents for inhibiting or improving resistance to anticancer drugs.
또한, 본 발명은 SYK(spleen tyrosine kinase)의 발현 또는 활성을 측정하는 제제를 포함하는, 항암제에 대한 내성 예측용 조성물을 제공한다.In addition, the present invention provides a composition for predicting resistance to an anticancer agent, including an agent for measuring the expression or activity of SYK (spleen tyrosine kinase).
본 발명의 일실시예에 따르면, MEK 저해제에 대해 내성을 가진 CMS4의 대장암 세포주에서 SYK 수준이 높게 발현됨을 확인하였다.According to one embodiment of the present invention, it was confirmed that SYK levels were highly expressed in CMS4 colorectal cancer cell lines resistant to MEK inhibitors.
따라서 상기 SYK의 유전자, 또는 단백질의 발현 또는 활성 수준이 대조군에 비하여 고발현 또는 고활성화된 경우, 항암제에 대한 내성이 있는 것으로 판정될 수 있다. 이에 있어, 상기 대조군은 MEK 저해제에 대해 내성이 없는 개체로부터 유래된 시료인 것을 특징으로 한다. 본 발명에 있어서, "시료"란 본 발명의 유전자 또는 단백질의 발현이 검출될 수 있는 대상 개체로부터 얻어지는 모든 시료를 의미한다. 상기 시료는 전혈(whole blood), 혈장(plasma), 혈청(serum), 객담(sputum), 눈물(tears), 점액(mucus), 세비액(nasal washes), 비강 흡인물(nasal aspirate), 호흡(breath), 소변(urine), 정액(semen), 침(saliva), 복강 세척액(peritoneal washings), 복수(ascites), 낭종액(cystic fluid), 뇌척수막 액(meningeal fluid), 양수(amniotic fluid), 백혈구(leukocytes), 말초혈액 단핵 세포(peripheral blood mononuclear cells), 백혈구 연층(buffy coat), 선액(glandular fluid), 췌장액(pancreatic fluid), 림프액(lymph fluid), 흉수(pleural fluid), 유두 흡인물(nipple aspirate), 기관지 흡인물(bronchial aspirate), 활액(synovial fluid), 관절 흡인물(joint aspirate), 기관 분비물(organ secretions), 세포(cell), 세포 추출물(cell extract) 및 뇌척수액(cerebrospinal fluid)으로 이루어진 군에서 선택된 어느 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.Therefore, when the expression or activity level of the SYK gene or protein is higher than that of the control group, it can be determined that there is resistance to anticancer drugs. In this regard, the control group is characterized in that a sample derived from a subject not resistant to the MEK inhibitor. In the present invention, "sample" refers to all samples obtained from a subject in which the expression of the gene or protein of the present invention can be detected. The sample is whole blood, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, respiratory (breath), urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid , leukocytes, peripheral blood mononuclear cells, leukocyte buffy coat, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspiration Nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extract and cerebrospinal fluid fluid) may be any one or more selected from the group consisting of, but is not limited thereto.
또한 상기 제제는 SYK에 특이적으로 결합하는 올리고펩타이드, 모노클로날 항체, 폴리클로날 항체, 키메릭(chimeric) 항체, 리간드, PNA(Peptide nucleic acid), 앱타머(aptamer), 안티센스(anti-sense) 뉴클레오티드, 프라이머 또는 프로브일 수 있으나, 이에 제한되는 것은 아니다.In addition, the agent specifically binds to SYK oligopeptide, monoclonal antibody, polyclonal antibody, chimeric antibody, ligand, PNA (peptide nucleic acid), aptamer (aptamer), antisense (anti- sense) nucleotides, primers or probes, but is not limited thereto.
상기 제제를 이용하여 시료내 SYK 단백질을 코딩하는 유전자의 활성 또는 발현을 확인할 수 있으며, 상기 확인은 공지의 어느 방법이든 사용하여 수행할 수 있다. 이를 위하여 역전사효소 중합효소반응(RT-PCR), 경쟁적 역전사효소 중합효소반응(competitive RT-PCR), 실시간 중합효소반응(real time RT-PCR), 실시간 역전사효소 중합효소반응(real time quantitative RT-PCR), RNase 보호 분석법(RNase protection method), 노던 블랏팅(Northern blotting) 및 DNA칩으로 이루어진 군에서 선택된 1종 이상으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The activity or expression of a gene encoding the SYK protein in a sample can be confirmed using the preparation, and the confirmation can be performed using any known method. To this end, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time polymerase reaction (real time RT-PCR), real time reverse transcriptase polymerase reaction (real time quantitative RT-PCR) PCR), RNase protection method (RNase protection method), Northern blotting (Northern blotting) and may be performed by at least one selected from the group consisting of a DNA chip, but is not limited thereto.
또한 상기 제제를 이용하여 시료내 SYK 단백질의 활성 또는 발현을 확인할 수 있으며, 상기 확인은 공지의 어느 방법이든 사용하여 수행할 수 있다. 이를 위하여 웨스턴 블랏팅, ELISA(enzyme linked immunosorbent assay), ELLA (enzyme-linked lectin assay), 방사선면역분석(Radioimmunoassay,RIA), 방사 면역확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케트(rocket) 면역전기영동, 면역조직화학염색, 면역침전 분석법(Immunoprecipitation Assay), 보체고정분석(Complement Fixation Assay), FACS 및 단백질 칩(protein chip)로 이루어진 군에서 선택된 1종 이상으로 수행될 수 있으나, 이에 제한되는 것은 아니다.In addition, the activity or expression of the SYK protein in a sample can be confirmed using the preparation, and the confirmation can be performed using any known method. For this purpose, Western blotting, ELISA (enzyme linked immunosorbent assay), ELLA (enzyme-linked lectin assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, rocket (rocket) It may be performed with one or more selected from the group consisting of immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip. , but is not limited thereto.
상술한 본 발명의 내용은 상호 모순되지 않는 한, 서로 동일하게 적용되며, 당해 기술분야의 통상의 기술자가 적절한 변경을 가해 실시하는 것 또한 본 발명의 범주에 포함된다.The contents of the present invention described above are equally applied to each other unless contradictory to each other, and implementation by adding appropriate changes by a person skilled in the art is also included in the scope of the present invention.
이하 본 발명을 실시예를 통해 상세하게 설명하나 본 발명의 범위가 하기 실시예로만 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through examples, but the scope of the present invention is not limited only to the following examples.
실시예 1. MEK 저해제에 대해 내성을 보이는 대장암 세포주 선별Example 1. Selection of colorectal cancer cell lines resistant to MEK inhibitors
대장암 중 CMS4 세포주에 해당하면서, KRAS 돌연변이를 가지고 있는 HCT116, SW620 및 SW480 세포에 대해서 MEK 저해제(MEK inhibitor)인 트라메티닙(trametinib)과 셀루메티닙(selumetinib)을 처리하였다. HCT116 세포주는 한국 세포주 은행에서 구입했고 SW480 및 SW620는 ATCC에서 구입했다. 모든 세포주는 10% 소태아혈청(fetal bovines serum(FBS), WelGENE)과 항생제(100U/ml의 페니실린(penicillin), 100μg/ml의 스트렙토마이신(streptomycin) 및 0.25μg/ml의 훈기존(Fungizone, Life Technologies))를 넣은 DMEM(WelGENE)에서 37℃, 5% CO2의 조건으로 배양했다. 또한 트라메티닙(GSK1120212, MEK1/2 저해제)와 셀루메티닙(AZD6244, MEK1/2 저해제)은 APExBIO(Boston, USA)에서 구입했다. SYK 저해제인 포스타마티닙 이소듐(Fostamatinib (R788) disodium)은 Chemscene(New Jersey, USA)에서 구입했다. 상기와 같이 준비한 각 세포주에 트라메티닙을 10 nM 또는 100 nM 농도로 처리하였고, 셀루메티닙은 0.1 μM 또는 1.0 μM 농도로 처리하였다. MEK 저해제를 처리하고 141시간까지 세포 성장 분석(cell growth assay)을 통해 모니터링하였고, 크리스탈 바이올렛을 이용한 세포 염색을 수행하였다. 먼저 세포들을 3-10×10³밀도로 96 well plate에 시딩(seeding)하였다. 그 후 18-20시간에 약물을 처리하고 7일 후, 1%(w/v) 크리스탈 바이올렛(Sigma-Aldrich)으로 30분간 상온에서 염색하였다. 그 후 증류수를 이용해 세척하고 건조시킨 후, 각 실험군의 세포의 형태를 확인하였다.HCT116, SW620, and SW480 cells, which correspond to the CMS4 cell line among colorectal cancer and have KRAS mutations, were treated with MEK inhibitors, trametinib and selumetinib. HCT116 cell line was purchased from Korean Cell Line Bank and SW480 and SW620 were purchased from ATCC. All cell lines were treated with 10% fetal bovines serum (FBS), WelGENE) and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml Fungizone, Life Technologies)) in DMEM (WelGENE) and cultured under conditions of 37°C and 5% CO 2 . In addition, trametinib (GSK1120212, MEK1/2 inhibitor) and selumetinib (AZD6244, MEK1/2 inhibitor) were purchased from APExBIO (Boston, USA). Fostamatinib (R788) disodium, a SYK inhibitor, was purchased from Chemscene (New Jersey, USA). Each cell line prepared as above was treated with trametinib at a concentration of 10 nM or 100 nM, and selumetinib at a concentration of 0.1 μM or 1.0 μM. After treatment with the MEK inhibitor, cell growth was monitored through a cell growth assay for up to 141 hours, and cell staining using crystal violet was performed. First, cells were seeded in a 96 well plate at a density of 3-10 × 10³. After that, the drug was treated at 18-20 hours, and after 7 days, it was stained with 1% (w / v) crystal violet (Sigma-Aldrich) for 30 minutes at room temperature. Then, after washing with distilled water and drying, the cell morphology of each experimental group was confirmed.
그 결과 도 1과 같이, SW620과 SW480 세포는 HCT116 세포에 비해 MEK 저해제를 처리하였을 때, 세포 생존율(viability)이 더 높은 것을 확인하였다(도 1 중 A 및 B). 또한 트라메티닙 10 nM, 100 nM을 처리한 세포 사진 상에서도 트라메티닙을 처리하지 않은 세포와 비교했을 때, HCT116보다 SW620과 SW480이 높은 생존율을 보여 MEK 저해제에 대해 내성을 가짐을 확인하였다(도 1 중 C). 따라서 HCT116 세포를 MEK 저해제에 민감한 CMS4 세포주로, SW620, SW480을 MEK 저해제에 대해 내성을 갖는 CMS4 세포주로 분류하였다.As a result, as shown in FIG. 1, it was confirmed that SW620 and SW480 cells had higher cell viability than HCT116 cells when treated with the MEK inhibitor (A and B in FIG. 1). In addition, when compared to cells not treated with trametinib on photographs of cells treated with 10 nM or 100 nM of trametinib, SW620 and SW480 showed higher survival rates than HCT116, confirming resistance to MEK inhibitors (Fig. 1 of C). Accordingly, HCT116 cells were classified as CMS4 cell lines sensitive to MEK inhibitors, and SW620 and SW480 were classified as CMS4 cell lines resistant to MEK inhibitors.
실시예 2. MEK 저해제의 효과 증진을 위한 병용 타겟 동정Example 2. Identification of combination targets for enhancing the effect of MEK inhibitors
상기 실시예 1에서 확인한 결과를 기반으로 CMS4에 해당하는 HCT116, SW620 및 SW480 세포주를 MEK 저해제에 민감한 그룹과 내성을 갖는 그룹으로 분류한 후, 단백질체 데이터와 유전자 발현 데이터에 대하여 각각 차이를 분석하였다. 그 중 CD(Characteristic Direction) 값과 DEG(Differentially expressed gene) 결과값 중에서 log2 배수 변화(fold change) 값이 0보다 큰 결과값을 3D 플랏(plot)으로 시각화하여 도 2a 내지 도 2e에 나타내었다. 그 결과, MEK 저해제에 내성을 갖는 세포주 그룹에서 SYK(spleen tyrosine kinase) 수준이 높은 상태를 보이는 것을 확인하였다(도 2a). 또한 HCT116(민감(sensitive))과 SW480(저항(resistant))의 단일세포 유전자 발현 데이터를 기반으로 유전자 공동 발현 네트워크를 구축한 결과, SW480에서 SYK와 MEK이 HCT116 보다 서로 많은 연관성을 가지고 있으며, SYK가 관여하는 많은 다른 노드(node)들이 존재함을 확인하였다(도 2b). 더불어 분석한 생물학적 데이터 결과가 실제 실험에서도 동일한 경향을 보이는지 확인하기 위해 세포실험을 진행하였다. 보다 구체적으로 실시간 정량 중합효소 연쇄반응(Real-time quantitative polymerase chain reaction, qRT-PCR))과 웨스턴블롯(Western blot) 분석을 통해, SYK의 수준을 확인하였다. 먼저 HCT116, SW620 및 SW480 세포주를 각 3×105으로 시딩한 후, 48시간 뒤 세포를 회수하여 RNA를 추출하였다. RNA는 RNA-spin kit(INTRON)을 이용해 추출하였고, cDNA는 제조사의 설명에 따라, DiaStar RT kit(Solgent)와 2X Taq premix(Solgent)를 이용하여 합성했다. 정량적(Quantitative) RT-PCR은 SYBR(GeNet Bio)를 사용하여 QuantStudio5(Applied Biosystem)를 이용하여 수행하였다. 더불어 SYK 프라이머(primer)[정방향(forward): GGAGGAAGGCACACCACTAC, 역방향(reverse): CAGACCAGGCCATCAGACTC]를 사용하였다. 표준화(normalization)는 하우스키핑(housekeeping) 유전자인 GAPDH(Glyceraldehyde 3-phosphate dehydrogenase)를 이용하여 수행하였다. Based on the results confirmed in Example 1, HCT116, SW620, and SW480 cell lines corresponding to CMS4 were classified into a group sensitive to MEK inhibitors and a group resistant to MEK inhibitors, and differences were analyzed for proteomic data and gene expression data, respectively. Among them, the result values having a log2 fold change value greater than 0 among CD (Characteristic Direction) values and DEG (Differentially expressed gene) result values were visualized as 3D plots and shown in FIGS. 2A to 2E. As a result, it was confirmed that a high level of spleen tyrosine kinase (SYK) was observed in the cell line group resistant to the MEK inhibitor (FIG. 2a). In addition, as a result of constructing a gene co-expression network based on single-cell gene expression data of HCT116 (sensitive) and SW480 (resistant), SYK and MEK in SW480 have more correlation with each other than HCT116, and SYK It was confirmed that there are many other nodes involved in (FIG. 2b). In addition, a cell experiment was conducted to confirm that the analyzed biological data showed the same tendency in the actual experiment. More specifically, the level of SYK was confirmed through real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot analysis. First, HCT116, SW620, and SW480 cell lines were each seeded at 3×10 5 , and after 48 hours, the cells were recovered and RNA was extracted. RNA was extracted using RNA-spin kit (INTRON), and cDNA was synthesized using DiaStar RT kit (Solgent) and 2X Taq premix (Solgent) according to the manufacturer's instructions. Quantitative RT-PCR was performed using SYBR (GeNet Bio) and QuantStudio5 (Applied Biosystem). In addition, SYK primers [forward: GGAGGAAGGCACACCACTAC, reverse: CAGACCAGGCCATCAGACTC] were used. Normalization was performed using a housekeeping gene, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).
웨스턴블롯 분석을 위하여, HCT116, SW620 및 SW480 세포주를 각각 3×105으로 시딩한 후, 48시간 뒤 세포를 회수하였다. 이에 프로테아제 억제제(protease inhibitor), 포스파타아제 억제제(phosphatase inhibitor) 칵테일(cocktail)(Thermo-scientific, USA) 0.1%를 넣은 용해 버퍼(20mM HEPES(pH7.2), 150mM NaCl, 0.5% Triton X-100, 10% 글리세롤(glycerol), 0.1% SDS)를 처리하여 단백질을 수득하였다. 안티(anti)-SYK(Santa cruz biotechnology, sc-1240), anti-GAPDH 항체(한국생명공학연구원으로부터 제공받음)를 이용하여 웨스턴블롯을 수행해 수득한 단백질의 활성을 확인하였다.For Western blot analysis, HCT116, SW620 and SW480 cell lines were each seeded at 3×10 5 , and the cells were recovered after 48 hours. Therefore, lysis buffer (20 mM HEPES (pH7.2), 150 mM NaCl, 0.5% Triton X- 100, 10% glycerol, 0.1% SDS) to obtain proteins. Anti-SYK (Santa cruz biotechnology, sc-1240) and anti-GAPDH antibodies (provided from the Korea Research Institute of Bioscience and Biotechnology) were used to confirm the activity of the obtained protein by Western blotting.
그 결과, 앞선 결과와 동일하게 MEK 저해제에 내성을 보이는 CMS4 세포주들에서 높은 SYK 수준을 확인하였다(도 2c). 또한 CMS4 환자의 단백질체 데이터를 이용하여, SYK 수준의 높고, 낮음을 기준으로 카플란-마이어(Kaplan-Meier) 생존 분석 결과, SYK 수준이 높은 환자의 경우 생존율이 낮은 것을 확인한 바, SYK 수준이 환자 생존율에 유의한 영향을 미친다는 사실을 확인하였다(도 2d). 특히 CMS4외의 다른 유형의 환자 데이터에서도 SYK 수준의 유의성을 확인한 결과, 다른 유형에서는 유의성을 보이지 않음을 확인하였다(도 2e). As a result, high SYK levels were confirmed in CMS4 cell lines showing resistance to MEK inhibitors, as in the previous results (FIG. 2c). In addition, as a result of Kaplan-Meier survival analysis based on high and low SYK levels using proteomic data of CMS4 patients, it was confirmed that patients with high SYK levels had low survival rates, It was confirmed that it had a significant effect on (Fig. 2d). In particular, as a result of confirming the significance of the SYK level in other types of patient data other than CMS4, it was confirmed that there was no significance in other types (FIG. 2e).
실시예 3. MEK 저해제에 내성을 갖는 세포주에서 SYK 수준 억제에 따른 효과 확인Example 3. Confirmation of the effect of inhibiting SYK levels in cell lines resistant to MEK inhibitors
상기 실시예에서 확인한 MEK 저해제에 내성을 갖는 SW480, SW620 세포주에 SYK 저해제(SYKi(fostamatinib disodium))와 두 종류의 MEK 저해제 트라메티닙(Trametinib(Tra)), 셀루메티닙(Selumetinib(Sel))을 단독처리, 병용 처리한 후 세포 성장을 확인하였다. 보다 구체적으로, 상기 세포들을 3-10×10³밀도로 96 well plate에 시딩(seeding)하였다. 세포 시딩 후에 20시간이 지난 후 각 실험군에 해당되는 약물을 처리하고 6-7일간 IncuCyte 기기를 이용하여 세포를 촬영했다. 이때 약물은 트라메티닙을 각각 10nM, 100nM 처리하였고, 셀루메티닙을 각각 0.1uM, 1.0uM 처리하였다. 세포 성장을 평가하기 위해 세포의 평균 면적은 Incucyte ZOOM 소프트웨어를 사용하여 설정하였고, 세포 사진은 3시간 간격으로 촬영하였다. 그 결과 도 3a 및 도 3b와 같이, SYK 저해제 및 MEK 저해제를 저농도로 병용 처리했을 때가 고농도로 단독 처리했을 때보다 더 저해제의 효과가 크거나 고농도 단독 처리한 만큼의 효과를 보이는 것을 확인하였다. 또한 MEK 저해제와 SYK 저해제의 병용 약물 처리가 시너지(synergy) 효과를 내는지 확인하기 위해 SynergyFinder을 사용하여 시너지 점수를 계산하였다. 그 결과 도 3c와 같이, SW480 세포주에 MEK 저해제와 SYK 저해제를 병용 처리한 경우, 시너지 점수가 8.33이었고 SW620에 병용 처리한 경우, 시너지 점수가 16.17으로, 병용 투여시 높은 시너지 점수를 가짐을 확인하였다. 더불어 약물을 이용한 방법 외에도 shRNA를 이용해 SYK의 유전자 발현을 낮춘 후, MEK 저해제 두 종류를 처리한 후, MEK 저해제에 대한 내성을 극복하는지 확인하였다. 먼저 HEK 293T 세포에 리포펙타민(lipofectamine, Invitrogen, Waltham, USA)을 이용해서 SYK 유전자를 타겟으로 하는 shRNA(shSYK; SHCLNG-NM_003177, Sigma), 스크램블(shScrambled) 및 유전자 패키징을 위한 혼합물(pLP1, pLP2, pLP/VSVG)을 형질 주입(transfection)하여 렌티바이러스를 생산하였다. 더불어 SW620 세포와 SW480 세포에 4μg/ml의 폴리브렌(polybrene, Sigma-Aldrich)을 함께 넣어 형질 도입(transduction)하였다. 감염된 세포들은 1μg/ml의 퓨로마이신(puromycin, Sigma-Aldrich)를 이용해 선택(selection)하였다. 그 결과 도 3d 및 3e와 같이, shRNA에 의해 SYK 유전자 발현 수준이 감소된 SW620, SW480에 MEK 저해제를 처리했을 때, SYK 유전자 발현 수준을 낮추지 않은 세포에 비해 세포 성장도가 더 떨어지는 것을 확인하였다. 크리스탈 바이올렛 분석 실험에서도 SYK 수준을 낮춘 상태의 MEK 저해제에 저항성을 갖는 세포가 MEK 저해제에 대해 민감한 상태가 되는 것을 확인하였다SYK inhibitor (SYKi (fostamatinib disodium)) and two types of MEK inhibitors, trametinib (Tra) and selumetinib (Selumetinib (Sel)) After single treatment and combined treatment, cell growth was confirmed. More specifically, the cells were seeded in a 96 well plate at a density of 3-10×10³. After 20 hours of cell seeding, the drugs corresponding to each experimental group were treated, and the cells were photographed using an IncuCyte device for 6-7 days. At this time, the drugs were treated with 10 nM and 100 nM of trametinib, respectively, and 0.1 uM and 1.0 uM of selumetinib, respectively. To evaluate cell growth, the average area of cells was set using Incucyte ZOOM software, and pictures of cells were taken at 3-hour intervals. As a result, as shown in FIGS. 3a and 3b, it was confirmed that when the SYK inhibitor and the MEK inhibitor were treated in combination at a low concentration, the effect of the inhibitor was greater than that of the high concentration alone or as high as the high concentration alone. In addition, a synergy score was calculated using SynergyFinder to confirm whether the combined drug treatment of the MEK inhibitor and the SYK inhibitor produced a synergistic effect. As a result, as shown in Figure 3c, when the SW480 cell line was treated in combination with the MEK inhibitor and the SYK inhibitor, the synergy score was 8.33, and in the case of combined treatment with SW620, the synergy score was 16.17, confirming that the combination had a high synergy score. . In addition to the method using drugs, after lowering the gene expression of SYK using shRNA, two types of MEK inhibitors were treated, and then it was confirmed whether resistance to MEK inhibitors was overcome. First, shRNA (shSYK; SHCLNG-NM_003177, Sigma) targeting the SYK gene, scrambled (shScrambled), and a mixture for gene packaging (pLP1, pLP2, pLP/VSVG) were transfected to produce lentiviruses. In addition, 4 μg/ml of polybrene (polybrene, Sigma-Aldrich) was added to SW620 cells and SW480 cells for transduction. Infected cells were selected using 1 μg/ml of puromycin (Sigma-Aldrich). As a result, as shown in Figures 3d and 3e, by shRNA When SW620 and SW480 with reduced SYK gene expression levels were treated with the MEK inhibitor, it was confirmed that the cell growth rate was lower than that of cells without lowering the SYK gene expression level. In crystal violet assay experiments, it was also confirmed that cells resistant to MEK inhibitors with reduced SYK levels became sensitive to MEK inhibitors.
실시예 4. MEK 저해제에 민감한 세포주에서 SYK 수준 향상에 따른 효과 확인Example 4. Confirmation of the effect of increasing the SYK level in cell lines sensitive to MEK inhibitors
더불어 SYK의 수준이 높은 경우, MEK 저해제에 대해 내성을 갖게 되는지를 확인하기 위하여, MEK 저해제에 민감한 세포인 HCT116 세포주의 SYK 유전자를 과발현(overexpression) 시키고, 세포가 MEK 저해제에 내성을 획득하는지 세포 성장 실험(A)과 상기 실시예 1과 동일한 방법의 크리스탈 바이올렛 분석(B)을 통해 확인하였다. 먼저 SYK 유전자가 과발현된 HCT116세포와 SYK가 과발현되지 않은 HCT116 세포(음성대조군)를 3×10³밀도로 96 웰 플레이트에 시딩하였다. 세포 시딩 후에 20시간이 지난 후 트라메티닙 약물을 2nM 또는 4nM로 처리하고 6-7일간 배양하며 IncuCyte 기기를 이용하여 세포를 촬영했다. 세포 성장을 평가하기 위해 세포의 평균 면적은 Incucyte ZOOM 소프트웨어를 사용하여 설정하였고, 세포 사진은 3시간 간격으로 촬영하였다. 그 결과 도 4와 같이, 음성 대조군(Negative control(NC))과 SYK 과발현된 HCT116 세포에 트라메티닙을 2nM, 4nM 처리했을 때, 음성 대조군 대비 SYK 과발현된 세포에서 세포 성장이 증가하고, 세포 생존율이 증가하는 것을 확인하였다.In addition, in order to confirm whether the level of SYK becomes resistant to MEK inhibitors, the SYK gene of the HCT116 cell line, which is a cell sensitive to MEK inhibitors, was overexpressed, and cell growth was performed to see if the cells acquired resistance to MEK inhibitors. It was confirmed through experiment (A) and crystal violet analysis (B) in the same manner as in Example 1 above . First, HCT116 cells with SYK gene overexpression and HCT116 cells without SYK overexpression (negative control) were seeded in a 96-well plate at a density of 3 × 10³. After 20 hours of seeding, the cells were treated with 2 nM or 4 nM of trametinib, cultured for 6-7 days, and the cells were photographed using an IncuCyte device. To evaluate cell growth, the average area of cells was set using Incucyte ZOOM software, and pictures of cells were taken at 3-hour intervals. As a result, as shown in Figure 4, when the negative control (NC) and SYK overexpressed HCT116 cells were treated with 2nM and 4nM of trametinib, cell growth increased in SYK overexpressed cells compared to the negative control, and cell viability This increase was confirmed.
실시예 5. MEK 저해제와 SYK 병용 처리에 따른 메커니즘 확인Example 5. Confirmation of mechanism according to MEK inhibitor and SYK combined treatment
앞서 확인한 MEK 저해제와 SYK 저해제의 병용 처리 메커니즘을 분석하기 위해, MAPK 경로에 관여하는 단백질인 cRaf, MEK, ERK(extracellular signal-regulated kinase)와 세포 사멸(apoptosis) 마커인 절단된 카스파제-3(cleaved caspase-3)에 대한 웨스턴 블롯을 수행하였다. 내성을 갖는 세포주 중에서 시너지 점수가 더 큰 SW620 세포에 대해서 약물을 처리하지 않은 상태, MEK 저해제인 트라메티닙 10nM를 단독 처리한 상태, SYK 저해제 5μM를 단독 처리한 상태, MEK 저해제와 SYK 저해제를 상기 농도로 병용 처리한 상태의 세포를 48시간 뒤 확보하여 상기 실시예 2와 동일한 방법의 웨스턴블롯을 수행하여 단백질 활성을 확인하였다.To analyze the mechanism of combined treatment of the MEK inhibitor and SYK inhibitor identified above, cRaf, MEK, and ERK (extracellular signal-regulated kinase), which are proteins involved in the MAPK pathway, and cleaved caspase-3 (apoptosis) marker, were analyzed. Western blot for cleaved caspase-3) was performed. Among the resistant cell lines, SW620 cells with a higher synergy score were not treated with the drug, treated with 10 nM of the MEK inhibitor trametinib alone, treated with 5 μM of the SYK inhibitor alone, and the MEK inhibitor and SYK inhibitor as described above. After 48 hours, the cells treated in combination at the same concentration were obtained, and Western blotting was performed in the same manner as in Example 2 to confirm protein activity.
이때 anti-SYK(Santa cruz biotechnology, sc-1240), anti-phosphoSYK(Cell signaling technology, #2710), anti-ERK1/2(Cell signaling technology, #9102), anti-phosphoERK1/2(Cell signaling technology, #4370), anti-phosphoMEK(Cell signaling technology, #9121), anti-phosphocRaf(Cell signaling technology, #9427), anti-caspase3(Santa cruz biotechnology, sc-7272) 항체 및 래빗 폴리클로날(rabbit polyclonal) anti-GAPDH 항체(한국생명공학연구원으로부터 제공받음)를 이용하였다.At this time, anti-SYK (Santa cruz biotechnology, sc-1240), anti-phosphoSYK (Cell signaling technology, #2710), anti-ERK1/2 (Cell signaling technology, #9102), anti-phosphoERK1/2 (Cell signaling technology, #4370), anti-phosphoMEK (Cell signaling technology, #9121), anti-phosphocRaf (Cell signaling technology, #9427), anti-caspase3 (Santa cruz biotechnology, sc-7272) antibody and rabbit polyclonal An anti-GAPDH antibody (provided from Korea Research Institute of Bioscience and Biotechnology) was used.
그 결과 도 5에 나타낸 바와 같이, MEK 저해제를 단독 처리했을 때, cRaf의 활성이 증가하면서 MEK과 ERK의 활성이 완전히 억제되지 않는 것을 확인하였다. 이러한 결과는 기존에 알려진 KRAS 돌연변이가 있는 암세포에 MEK 저해제 처리시 ERK의 음성 되먹임 고리(negative feedback loop)에 의해 내성을 보이는 현상과 일치한다. 반면 SYK 저해제를 단독 처리했을 때에는 오히려 ERK의 활성이 급격히 증가했다. 흥미롭게도, MEK 저해제와 SYK 저해제를 함께 처리했을 때, MEK 저해제를 단독 처리했을 때보다 MEK과 ERK의 활성이 더 강하게 억제되었다. 더불어 세포 사멸 마커인 cleaved caspase3가 두 저해제를 병용 처리했을 때에만 현저히 높게 확인된 바, 이를 통해 단일 저해제 처리는 세포 사멸을 유의미하게 유도하지 못했지만, 두 저해제를 병용 처리했을 때는 세포 사멸을 크게 증가시킬 수 있음을 확인하였다(도 5A). 또한 shRNA를 이용해 SYK 유전자의 발현 수준을 감소시킨 상태에서 트라메티닙 10nM을 처리한 후 48시간 후 웨스턴 블롯으로 ERK의 단백질 활성을 확인한 결과, SYK의 발현이 감소되기만 한 상태에서는 ERK의 단백질 활성이 증가하였으나, SYK의 발현을 감소시키고, 트라메티닙을 함께 처리한 결과에서는 ERK 단백질의 활성이 감소하였음을 확인하였다. 따라서 이를 통해 앞서 확인한 MEK 저해제와 SYK 저해제 병용 처리 결과와 동일한 경향을 보임을 확인하였다(도 5B). 더 나아가 SYK와 MAPK 경로 사이의 상호 조절 관계를 탐색하기 위해, SW620 세포에 SYK 저해제와 MEK 저해제를 각각 저농도 또는 고농도로 처리한 후 48시간 뒤 확보하여 웨스턴 블롯을 수행하였다. As a result, as shown in FIG. 5, when the MEK inhibitor was treated alone, it was confirmed that the activities of MEK and ERK were not completely inhibited while cRaf activity increased. These results are consistent with the phenomenon of showing resistance due to the negative feedback loop of ERK when MEK inhibitors are treated in cancer cells with previously known KRAS mutations. On the other hand, when the SYK inhibitor was treated alone, the activity of ERK increased rapidly. Interestingly, when the MEK inhibitor and the SYK inhibitor were treated together, the activities of MEK and ERK were more strongly inhibited than when the MEK inhibitor was treated alone. In addition, the cell death marker, cleaved caspase3, was found to be significantly high only when the two inhibitors were treated in combination, and thus, single inhibitor treatment did not significantly induce apoptosis, but the combination treatment of the two inhibitors significantly increased apoptosis. It was confirmed that it can be (Fig. 5A). In addition, after treatment with 10 nM of trametinib in a state where the expression level of the SYK gene was reduced using shRNA, the protein activity of ERK was confirmed by
즉, SW620 세포에 SYK 저해제를 0 μM, 0.5μM, 1μM, 5μM, 10μM로 농도를 높여가면서 처리 후 48시간 뒤에 MEK과 ERK에 대한 활성을 확인한 결과, SYK 저해제의 농도가 증가할수록 SYK의 활성도가 떨어지고 ERK의 활성은 증가하는 것을 확인하였다(도 5C). 이를 통해 SYK가 ERK를 억제하는 관계에 있음을 확인하였다. That is, as a result of confirming the activity of MEK and
또한 트라메티닙을 0nM, 1nM, 5nM, 10nM, 50nM로 각각 처리 후 48시간 뒤에 SYK의 활성을 확인한 결과, MEK의 활성이 억제될수록 SYK 활성도 함께 억제되는 것을 확인하였다(도 5D). 이를 통해 MEK이 SYK를 활성화시키는 관계에 있음을 확인하였다. 상기에서 확인한 상호 조절 관계 확인 결과와 단백질 활성화 결과를 종합하여 각 저해제 처리 조건에 따른 신호 전달 네트워크 상태를 추측하여 도 6에 나타내었다.In addition, as a result of confirming
종합적으로, MEK 저해제에 대해 내성을 가진 CMS4의 대장암 세포주에서 SYK 수준이 높게 발현되며, SYK 저해제와 MEK 저해제를 대장암 세포에 병용 처리시 MEK 저해제 단독 처리보다 효과가 현저히 증대됨을 확인한 바, 이를 대장암, 특히 MEK 저해제에 내성을 보이는 대장암의 치료에 유용하게 활용할 수 있으며, 대장암 환자에 있어서, MEK 저해제에 대한 내성 발생 가능성을 예측하는데도 유용하게 활용할 수 있다.Overall, it was confirmed that the level of SYK was highly expressed in CMS4 colorectal cancer cell lines resistant to MEK inhibitors, and that the combined treatment of SYK inhibitors and MEK inhibitors in colorectal cancer cells significantly increased the effect compared to treatment with MEK inhibitors alone. It can be usefully used for the treatment of colorectal cancer, especially colorectal cancer that is resistant to MEK inhibitors, and can also be usefully used to predict the possibility of resistance to MEK inhibitors in colorectal cancer patients.
Claims (15)
A pharmaceutical composition for inhibiting or improving anticancer drug resistance comprising a SYK (spleen tyrosine kinase) inhibitor.
상기 SYK 저해제는 항암제와 동시 또는 순차적으로 투여되는 것을 특징으로 하는, 조성물.
According to claim 1,
The SYK inhibitor is characterized in that administered simultaneously or sequentially with the anticancer agent, the composition.
상기 항암제는 MEK(mitogen-activated protein kinase kinase) 저해제인 것을 특징으로 하는, 조성물.
According to claim 1,
The composition, characterized in that the anticancer agent is a mitogen-activated protein kinase kinase (MEK) inhibitor.
상기 MEK 저해제는 트라메티닙(Trametinib), 셀루메티닙(selumetinib), 비니메티닙(binimetinib), 피마세르팁(Pimasertib), 레파메티닙(Refametinib), 코비메티닙(cobimetinib), U0126-EtOH, PD184352, PD98059, BIX02189, SL-327, BIX02188, AZD8330, TAK-733 및 PD318088으로 이루어진 군으로부터 선택되는 하나 이상인 것을 특징으로 하는, 조성물.
According to claim 3,
The MEK inhibitor is Trametinib, selumetinib, binimetinib, Pimasertib, Refametinib, cobimetinib, U0126-EtOH, Characterized in that at least one selected from the group consisting of PD184352, PD98059, BIX02189, SL-327, BIX02188, AZD8330, TAK-733 and PD318088, the composition.
상기 SYK 저해제는 포스타마티닙(fostamatinib) 및 세비도플레닙(cevidoplenib)으로 구성된 군으로부터 선택된 하나 이상인 것을 특징으로 하는, 조성물.
According to claim 1,
The SYK inhibitor is characterized in that at least one selected from the group consisting of fostamatinib and cevidoplenib, composition.
An anticancer adjuvant comprising the composition according to any one of claims 1 to 5.
A pharmaceutical composition for combined administration for the prevention or treatment of cancer, comprising a SYK inhibitor and a mitogen-activated protein kinase (MAPK) signaling pathway inhibitor.
상기 MAPK 신호전달경로 저해제는 MEK 저해제인 것을 특징으로 하는, 약학적 조성물.
According to claim 7,
The pharmaceutical composition, characterized in that the MAPK signaling pathway inhibitor is a MEK inhibitor.
상기 암은 대장암, 유방암, 폐암, 위암, 전립선암, 갑상선암, 결장직장암, 흑색종, 두경부암, 피부암, 췌장암, 난소암, 방광암, 간암 및 신장암으로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 하는, 약학적 조성물.
According to claim 7,
The cancer is at least one selected from the group consisting of colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, thyroid cancer, colorectal cancer, melanoma, head and neck cancer, skin cancer, pancreatic cancer, ovarian cancer, bladder cancer, liver cancer and kidney cancer To, the pharmaceutical composition.
A food composition for preventing or improving cancer comprising a SYK inhibitor and a mitogen-activated protein kinase (MAPK) signaling pathway inhibitor.
상기 SYK 단백질 또는 유전자의 활성 또는 발현 수준이 후보물질 처리 전보다 감소하는 경우 상기 후보물질은 항암제에 대한 내성 억제 또는 개선 활성이 있는 것으로 판정하는 단계; 를 포함하는 항암제에 대한 내성 억제 또는 개선용 제제의 스크리닝 방법.
processing a candidate substance to a sample containing SYK protein or a gene encoding the SYK protein; and
If the activity or expression level of the SYK protein or gene is reduced than before treatment with the candidate material, determining that the candidate material has resistance suppression or improvement activity against an anticancer agent; A screening method for an agent for inhibiting or improving resistance to an anticancer agent comprising a.
A composition for predicting resistance to an anticancer agent comprising an agent for measuring the expression or activity of SYK (spleen tyrosine kinase).
상기 제제는 SYK에 특이적으로 결합하는 올리고펩타이드, 모노클로날 항체, 폴리클로날 항체, 키메릭(chimeric) 항체, 리간드, PNA(Peptide nucleic acid), 앱타머(aptamer), 안티센스(anti-sense) 뉴클레오티드, 프라이머 또는 프로브인 것을 특징으로 하는, 내성 예측용 조성물.
According to claim 12,
The agent is an oligopeptide that specifically binds to SYK, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), an aptamer, an anti-sense ) A composition for predicting resistance, characterized in that it is a nucleotide, primer or probe.
상기 SYK의 유전자, 또는 단백질의 발현 또는 활성 수준이 대조군에 비하여 고발현 또는 고활성화된 경우, 항암제에 대한 내성이 있는 것으로 판정하는 것을 특징으로 하는, 내성 예측용 조성물.
According to claim 12,
When the expression or activity level of the SYK gene or protein is higher than that of the control group, it is determined that there is resistance to anticancer drugs, the composition for predicting resistance.
상기 대조군은 MEK 저해제에 대해 내성이 없는 개체로부터 유래된 시료인 것을 특징으로 하는, 내성 예측용 조성물.According to claim 14,
The control group is a composition for predicting resistance, characterized in that the sample derived from a subject that is not resistant to the MEK inhibitor.
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| WO2021089791A1 (en) * | 2019-11-08 | 2021-05-14 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for the treatment of cancers that have acquired resistance to kinase inhibitors |
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| WO2021089791A1 (en) * | 2019-11-08 | 2021-05-14 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for the treatment of cancers that have acquired resistance to kinase inhibitors |
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| HUA, BOSTON UNIVERSITY PROQUEST DISSERTATIONS PUBLISHING, 2019, 13887265, 2019.12.31 * |
| YAKTAPOUR ET AL., THE JOURNAL OF CLINICAL INVESTIGATION, 124(11), 5074~5084, 2014.10.20 * |
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