TWI810426B - Use and evaluation method of pharmaceutical composition in preparation of medicine for treating cancer - Google Patents
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Abstract
本發明揭示內容是關於用以決定罹癌個體是否適合使用醫藥組合物為癌症用藥的評估方法。藉由標的基因(CHEK1及PIK3CA)的表現量與基本倍數之比較來進行評估，並找出罹癌個體中適合使用醫藥組合物之族群(CHEK1高表現及PIK3CA高表現)。再者，建議使用此醫藥組合物(細胞檢查點激酶(CHK1)抑制劑及磷脂醯肌醇3激酶(PI3K)抑制劑)來抑制族群中癌細胞之存活能力以及腫瘤生長能力，來達到治療癌症之功效。 The disclosure of the present invention relates to an evaluation method for determining whether a cancer-bearing individual is suitable for using a pharmaceutical composition for cancer treatment. The evaluation is performed by comparing the expression levels of the target genes (CHEK1 and PIK3CA) with the basic multiples, and a population group (high expression of CHEK1 and high expression of PIK3CA) suitable for the use of the pharmaceutical composition is found among cancer-bearing individuals. Furthermore, it is suggested to use the pharmaceutical composition (cell checkpoint kinase (CHK1) inhibitor and phosphatidylinositol 3 kinase (PI3K) inhibitor) to inhibit the survival ability and tumor growth ability of cancer cells in the population to achieve the treatment of cancer The effect.
Description
本揭示內容是關於個人化精準醫療之治療癌症的領域。本發明係有關一種醫藥組合物，及其用於製備治療癌症藥物之用途與評估方法。藉由標的基因表現量檢測與基本倍數之評估方法，找出癌症病患中適合使用醫藥組合物之族群，以達到癌症治療的功效。 This disclosure is about the field of personalized precision medicine for treating cancer. The present invention relates to a pharmaceutical composition, and its use and evaluation method for preparing medicines for treating cancer. By means of the detection of target gene expression and the evaluation method of the basic multiple, find out the group of cancer patients who are suitable for using the pharmaceutical composition, so as to achieve the efficacy of cancer treatment.
癌症為身體細胞不正常的生長與快速***導致腫瘤形成，增生的腫瘤細胞侵犯身體的正常細胞與器官，導致個體死亡，癌症是全球死亡原因的第一位。在台灣根據衛生福利部統計，癌症自民國七十一年起已連續37年居國人死因首位。民國一百零七年國人癌症死亡人數統計，有4萬8,784人(男性2萬9,624人，女性1萬9,160人)死於癌症，占總死亡人數之28.2%，較前年上升1.6%(增加747人)。 Cancer is the abnormal growth and rapid division of body cells leading to tumor formation. Proliferating tumor cells invade normal cells and organs of the body, leading to individual death. Cancer is the number one cause of death in the world. In Taiwan, according to statistics from the Ministry of Health and Welfare, cancer has been the number one cause of death for 37 consecutive years since the Republic of China. According to the statistics of the number of cancer deaths in the country in 107, 48,784 people (29,624 males and 19,160 females) died of cancer, accounting for 28.2% of the total deaths, an increase of 1.6% compared to the previous year (an increase of 747 people).
口腔癌是一種全球性好發的癌症，於世界各地均有很多病例報導並為日益嚴重的癌症疾病。於2018年，全球估計約有360,000名新增的口腔癌病患，而約有177,000名病患因口腔癌而死亡，相較於2012年的統計資料可發現罹患口腔癌的個體數亦逐漸攀升，顯示在過去數年間口腔癌病患數目呈現穩定增加的趨勢。在台灣根據衛生福利部民國一百零七年死因統計數據，口腔癌為台灣十大癌症死因第五名，在男性口腔癌死亡率則為第四名順位。所有癌症死亡年齡中位數為69歲，而口腔癌的死亡年齡較早其年齡中位數僅59歲，相較於其他癌症更是提早了十年。根據統計，高達70%以上罹患口腔癌之病患具有抽菸喝酒或嚼食檳榔的習慣。在2010年，台灣政府已將口腔癌篩檢計畫列入國人癌症 篩檢之補助項目，提供高風險族群(30歲以上有咀嚼檳榔或吸菸習慣的個體)每年免費的口腔檢查，可見口腔癌日趨為嚴重的公共健康議題。 Oral cancer is a globally prevalent cancer, with many cases reported all over the world and it is an increasingly serious cancer disease. In 2018, it is estimated that there will be about 360,000 new oral cancer patients worldwide, and about 177,000 patients will die from oral cancer. Compared with the statistics in 2012, it can be found that the number of individuals suffering from oral cancer is gradually increasing , showing a steady increase in the number of oral cancer patients over the past few years. In Taiwan, according to the statistics of the cause of death in 2017 by the Ministry of Health and Welfare, oral cancer is the fifth leading cause of death from cancer in Taiwan, and it ranks fourth in male oral cancer mortality. The median age of death from all cancers is 69 years old, and the median age of death from oral cancer is only 59 years old, ten years earlier than other cancers. According to statistics, up to 70% of patients suffering from oral cancer have the habit of smoking, drinking or chewing betel nuts. In 2010, the Taiwan government has included the oral cancer screening program in the national cancer The screening subsidy program provides high-risk groups (individuals over 30 years old with chewing betel nuts or smoking habits) with annual free oral examinations. It can be seen that oral cancer is becoming an increasingly serious public health issue.
口腔部位可分為唇、夾黏膜(唇及臉頰的內襯)、牙齒、舌頭下方的口腔底部、前三分之二的舌頭、口腔頂部的前面部分(硬顎)、牙齦及臼齒後方的小區域。而口腔癌則為上述區域發生異常的增生，侵犯至周邊正常的組織，甚至轉移至身體其它部位，影響正常功能的運作進而危及生命。造成口腔鱗狀細胞癌的主要致病因素包含吸菸、酗酒、非吸菸性的菸草使用及咀嚼檳榔。 The oral cavity can be divided into lips, mucous membranes (lining of the lips and cheeks), teeth, the floor of the mouth below the tongue, the front two-thirds of the tongue, the front part of the roof of the mouth (hard palate), the gums, and the small area behind the molars. area. Oral cancer is abnormal hyperplasia in the above-mentioned areas, which invades the surrounding normal tissues, and even metastasizes to other parts of the body, affecting normal functioning and endangering life. The main risk factors for oral squamous cell carcinoma include smoking, alcohol abuse, nonsmoking tobacco use, and betel nut chewing.
再者，組織學上可將口腔癌區分為鱗狀細胞癌、疣狀癌、小唾液腺癌、肉瘤及惡性黑色素瘤等，其中以鱗狀細胞癌(oral cavity squamous cell carcinoma,OSCC)為多數，占口腔惡性腫瘤的90%以上，依次為夾黏膜、牙齦、顎、口底部及***部病變。 Moreover, oral cavity cancer can be divided into squamous cell carcinoma, verrucous carcinoma, minor salivary gland carcinoma, sarcoma, and malignant melanoma histologically, among which squamous cell carcinoma (oral cavity squamous cell carcinoma, OSCC) is the majority, It accounts for more than 90% of oral malignant tumors, followed by mucosal, gingiva, jaw, floor of the mouth and lip lesions.
口腔癌為當口腔黏膜長期受到危險因子的刺激，會使得基因調控失衡或突變所造成細胞變異。而與口腔癌相關的危險因子包括：吸菸、喝酒、嚼食檳榔、紫外線照射、蛀牙、不當之假牙、不良之口腔衛生、長期的營養不良、病毒的感染(Human Papillomavirus,HPV16)等。 Oral cancer is when the oral mucosa is stimulated by risk factors for a long time, which will cause cell mutation caused by gene regulation imbalance or mutation. The risk factors related to oral cancer include: smoking, drinking, chewing betel nut, ultraviolet radiation, tooth decay, improper dentures, poor oral hygiene, long-term malnutrition, and viral infection (Human Papillomavirus, HPV16), etc.
因此，在臨床上常可見到黏膜表面產生變異或不當增生，形成白斑、紅斑、紅白斑、潰瘍、疣狀上皮增生或是口腔黏膜纖維化，以上皆為口腔癌前期之病變可能情況，而上述病變之情況，又可因為長期發炎刺激，進而進一步演變成口腔癌。 Therefore, clinically, it is often seen that the surface of the mucosa is mutated or inappropriately hyperplasia, forming leukoplakia, erythema, erythema, ulcers, verrucous epithelial hyperplasia, or oral mucosal fibrosis, all of which are possible precancerous lesions of the oral cavity. In the case of lesions, it can further evolve into oral cancer due to long-term inflammation and stimulation.
口腔癌症狀主要為口腔內潰瘍、硬塊、紅斑、白斑或伴有頸部淋巴腫大、不能張口、咀嚼吞食困難等，初期大都沒有疼痛，當腫瘤變大，局部壞死後可能造成細菌感染以致疼痛異常。除此之外，口腔癌常轉移至近處頸部淋巴轉移-顎下區、頦下區、上頸部、中頸部、後頸部；遠處轉移-肺、肝、骨骼、鎖骨下淋巴等。 The symptoms of oral cancer are mainly oral ulcers, hard lumps, erythema, white spots or accompanied by cervical lymphadenopathy, inability to open the mouth, difficulty in chewing and swallowing, etc. Most of them have no pain in the early stage. When the tumor grows larger and local necrosis may cause bacterial infection and pain abnormal. In addition, oral cancer often metastasizes to nearby cervical lymph nodes - submandibular region, submental region, upper neck, middle neck, and posterior neck; distant metastasis - lung, liver, bone, subclavian lymph, etc. .
目前口腔癌治療方式為手術切除或頸部淋巴清除、化學治療、放射治療；一般以手術為主，化學治療、放射治療為輔。而早期第一期與第二期口腔癌治癒率約80~90%。但晚期第三期、第四期口腔癌治癒率只有約30~50%。 又早期癌症症狀不明顯，統計數據顯示超過60%的病患為第III或IV期的病患，因多數病患忽視待發現時多為晚期的口腔癌病患，且晚期口腔癌復發與轉移的病例也呈現高死亡率。由上述可以了解，口腔癌的預防，診斷及治療，有其未滿足的醫療需求。 At present, the treatment methods for oral cancer include surgical resection or cervical lymphadenectomy, chemotherapy, and radiation therapy; generally, surgery is the main method, and chemotherapy and radiation therapy are supplemented. The cure rate of early stage I and II oral cancer is about 80-90%. However, the cure rate of advanced stage III and stage IV oral cancer is only about 30-50%. The symptoms of early cancer are not obvious. Statistics show that more than 60% of the patients are stage III or IV patients, because most patients ignore the patients with advanced oral cancer when they are discovered, and the recurrence and metastasis of advanced oral cancer cases also showed a high mortality rate. From the above, it can be understood that the prevention, diagnosis and treatment of oral cancer have unmet medical needs.
目前口腔癌病患治療僅一種標靶藥物，爾必得舒(ERBITUX)可以使用，此藥物可抑制是針對腫瘤細胞表皮生長因子受體的訊號傳遞，以減緩腫瘤細胞的增生，美國食品藥品監督管理局與台灣衛生福利部均建議，轉移與復發的口腔癌病患，可以使用爾必得舒與化療藥物共同治療，根據文獻研究指出病患同時使用化療藥物與爾必得舒組別，相較於單獨使用化療藥物組別，共同藥物使用組別僅可多延長3個月的存活。又，化療藥物均會造成病患產生不適與強大之副作用。 At present, there is only one targeted drug for the treatment of oral cancer patients. Erbitux (ERBITUX) can be used. This drug can inhibit the signal transmission of the epidermal growth factor receptor of tumor cells to slow down the proliferation of tumor cells. The US Food and Drug Administration Both the Bureau of Health and Welfare of Taiwan suggest that patients with metastatic and recurrent oral cancer can be treated with Erbitux and chemotherapy drugs. With the chemotherapy drug group, the co-drug use group only extended survival by 3 months. In addition, chemotherapy drugs can cause discomfort and powerful side effects to patients.
口腔癌病患具有獨特個人不同的基因調控失衡，不同的基因調控失衡其癌症對應之藥物組合物與化學治療也不盡相同。有鑑於此，由與口腔癌相關之基因檢測與基本倍數評估方法來找尋相對應的藥物治療，以達到個人化精準有效的治療避免無效醫療及提高存活率，為本技術領域人員所欲解決的問題。 Oral cancer patients have unique and different gene regulation imbalances, and different gene regulation imbalances correspond to different drug compositions and chemotherapy for cancer. In view of this, it is a problem that those skilled in the art want to solve by using the genetic detection and basic multiple evaluation methods related to oral cancer to find the corresponding drug treatment to achieve personalized, accurate and effective treatment, avoid ineffective medical treatment and improve survival rate. question.
本發明之主要目的，係提供一種醫藥組合物及於製備治療癌症藥物之用途與評估方法。先藉由適用醫藥組合物之族群之評估方法來找出癌症病患中適合使用醫藥組合物之族群(CHEK1高表現及PIK3CA高表現)。 The main purpose of the present invention is to provide a pharmaceutical composition and its use and evaluation method in the preparation of cancer treatment drugs. Firstly, the group suitable for using the pharmaceutical composition (high expression of CHEK1 and high expression of PIK3CA) among cancer patients is found out by the evaluation method of the group suitable for the pharmaceutical composition.
更進一步，本發明之另一目的係使用醫藥組合物來抑制此族群(CHEK1高表現及PIK3CA高表現)中口腔鱗狀上皮癌細胞之存活能力以及腫瘤生長能力。 Furthermore, another object of the present invention is to use the pharmaceutical composition to inhibit the survival ability and tumor growth ability of oral squamous cell carcinoma cells in this group (CHEK1 high expression and PIK3CA high expression).
為了達到上述之目的，本發明揭示了一種醫藥組合物作為製備治療口腔癌藥物之用途，其中該醫藥組合物包含一細胞檢查點激酶抑制劑及一磷脂醯肌醇3激酶抑制劑，該醫藥組合物係用於抑制一癌細胞。 In order to achieve the above-mentioned purpose, the present invention discloses a pharmaceutical composition used as a medicine for the preparation of oral cancer, wherein the pharmaceutical composition comprises a cell checkpoint kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor, the pharmaceutical composition The substance is used to inhibit a cancer cell.
本發明提供一實施例，其內容在於醫藥組合物作為製備治療癌症藥物之用途，其中該癌細胞係一口腔鱗狀上皮癌細胞。 The present invention provides an embodiment, the content of which is the use of the pharmaceutical composition as a medicine for treating cancer, wherein the cancer cell is oral squamous cell carcinoma.
本發明提供一實施例，其內容在於醫藥組合物作為製備治療癌症藥物之用途，其中該醫藥組合物係抑制該口腔鱗狀上皮癌細胞之存活能力。 The present invention provides an embodiment, which relates to the use of a pharmaceutical composition as a drug for treating cancer, wherein the pharmaceutical composition inhibits the survival ability of the oral squamous cell carcinoma cells.
本發明提供一實施例，其內容在於醫藥組合物作為製備治療癌症藥物之用途，其中該醫藥組合物係抑制該口腔鱗狀上皮癌細胞之腫瘤生長能力。 The present invention provides an embodiment, which is the use of a pharmaceutical composition as a drug for treating cancer, wherein the pharmaceutical composition inhibits the tumor growth ability of the oral squamous cell carcinoma cells.
又，本發明亦揭示了一種醫藥組合物，其包含：一細胞檢查點激酶抑制劑及一磷脂醯肌醇3激酶抑制劑。 Moreover, the present invention also discloses a pharmaceutical composition, which comprises: a cell checkpoint kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor.
再者，本發明揭示了一種使用醫藥組合物之評估方法，其步驟包含：使用一即時定量反轉錄聚合酶連鎖反應檢測一腫瘤組織樣本之一CHEK1基因以及一PIK3CA基因，取得一第一CHEK1基因表現量、一第一PIK3CA基因表現量以及檢測一正常組織樣本之該CHEK1基因以及該PIK3CA基因，取得一第二CHEK1基因表現量、一第二PIK3CA基因表現量；比對該第一CHEK1基因表現量與該第二CHEK1基因表現量以及該第一PIK3CA基因表現量與該第二PIK3CA基因表現量，得到一CHEK1差異倍數以及一PIK3CA差異倍數；當該CHEK1差異倍數高於一CHEK1基本倍數以及該PIK3CA差異倍數高於一PIK3CA基本倍數時，形成一比對結果；以及依據該比對結果判斷使用一醫藥組合物，該醫藥組合物係包含一細胞檢查點激酶抑制劑及一磷脂醯肌醇3激酶抑制劑；其中該CHEK1基本倍數係1.7，該PIK3CA基本倍數係2.2。
Furthermore, the present invention discloses an evaluation method using a pharmaceutical composition, the steps of which include: using a real-time quantitative reverse transcription polymerase chain reaction to detect a CHEK1 gene and a PIK3CA gene in a tumor tissue sample to obtain a first CHEK1 gene expression level, a first PIK3CA gene expression level and detecting the CHEK1 gene and the PIK3CA gene in a normal tissue sample to obtain a second CHEK1 gene expression level and a second PIK3CA gene expression level; comparing the first CHEK1 gene expression amount and the second CHEK1 gene expression level and the first PIK3CA gene expression level and the second PIK3CA gene expression level to obtain a CHEK1 difference multiple and a PIK3CA difference multiple; when the CHEK1 difference multiple is higher than a CHEK1 basic multiple and the When the PIK3CA difference multiple is higher than a PIK3CA basic multiple, a comparison result is formed; and a pharmaceutical composition is used according to the comparison result, and the pharmaceutical composition comprises a cell checkpoint kinase inhibitor and a
本發明提供一實施例，其內容在於評估方法，其中於檢測一腫瘤組織樣本之一CHEK1基因以及一PIK3CA基因之步驟中，該腫瘤組織樣本係一癌症腫瘤組織。 The present invention provides an embodiment, which is an evaluation method, wherein in the step of detecting a CHEK1 gene and a PIK3CA gene in a tumor tissue sample, the tumor tissue sample is a cancer tumor tissue.
又，本發明揭示了一種適用醫藥組合物之族群之評估方法，其步驟包括：(a)由一個體取得一腫瘤組織樣本與一正常組織樣本；(b)分別檢測該腫瘤組織樣本與該正常組織樣本之一CHEK1基因之表現量及一PIK3CA基因之表現量；(c)基於步驟(b)計算出一CHEK1基因差異倍數及一PIK3CA基因差異 倍數；以及(d)基於步驟(c)之該CHEK1基因差異倍數及該PIK3CA基因差異倍數來決定該個體是否為適用醫藥組合物之族群；其中，當該CHEK1基因差異倍數高於1.7及該PIK3CA差異倍數高於2.2時，則該個體為適用醫藥組合物之族群。 In addition, the present invention discloses a method for evaluating a group of suitable pharmaceutical compositions, the steps of which include: (a) obtaining a tumor tissue sample and a normal tissue sample from an individual; (b) detecting the tumor tissue sample and the normal tissue sample respectively; The expression level of a CHEK1 gene and the expression level of a PIK3CA gene in the tissue sample; (c) calculate a CHEK1 gene difference multiple and a PIK3CA gene difference based on step (b) multiple; and (d) determine whether the individual is a group suitable for the pharmaceutical composition based on the multiple of the CHEK1 gene difference and the multiple of the PIK3CA gene in step (c); wherein, when the multiple of the CHEK1 gene difference is higher than 1.7 and the PIK3CA gene When the multiple of difference is higher than 2.2, the individual belongs to the group for which the pharmaceutical composition is applicable.
S1~S4:步驟流程 S1~S4: Step process
第1圖：其係本發明之一實施例之步驟流程圖；第2圖：其係本發明之一實施例之CHEK1及PIK3CA之基本倍數圖；第3A圖：其係本發明之一實施例之細胞檢查點激酶抑制劑之濃度結果圖；第3B圖：其係本發明之一實施例之磷脂醯肌醇3激酶抑制劑之濃度結果圖；第4圖：其係本發明之一實施例之同時使用細胞檢查點激酶抑制劑與磷脂醯肌醇3激酶抑制劑(醫藥組合物)之口腔癌細胞之存活能力之實驗結果圖；第5A圖：其係本發明之一實施例之口腔癌小鼠模式之腫瘤生長能力之實驗結果圖；第5B圖：其係本發明之一實施例之口腔癌小鼠模式之腫瘤生長能力之對照實驗結果圖；以及第5C圖：其係本發明之一實施例之口腔癌小鼠模式之體重之實驗結果圖。 Figure 1: It is a flow chart of the steps of an embodiment of the present invention; Figure 2: It is a basic multiple figure of CHEK1 and PIK3CA in an embodiment of the present invention; Figure 3A: It is an embodiment of the present invention The concentration result graph of the cell checkpoint kinase inhibitor; Fig. 3B: it is the concentration result graph of the phosphatidylinositol 3-kinase inhibitor of one embodiment of the present invention; Fig. 4: it is one embodiment of the present invention Figure 5A: It is an oral cancer according to an embodiment of the present invention. Figure 5B: It is a graph of the control experiment results of the tumor growth ability of the oral cancer mouse model of an embodiment of the present invention; and Figure 5C: It is the result of the experiment of the present invention An experimental result diagram of the body weight of the oral cancer mouse model of an embodiment.
為使貴審查委員對本發明之特徵及所達成之功效有更進一步之瞭解與認識，謹佐以實施例及配合說明，說明如後：有鑑於不同的口腔癌病患有不同之口腔癌基因特徵表現，需要與其相對應的藥物治療，為了可以達到更加精準的治療、提高存活率以及減輕習知 化療藥物的副作用。據此，本發明遂提出一種醫藥組合物及於製備治療口腔癌藥物之用途與評估方法，以解決習知技術所造成之問題。 In order to enable your review committee members to have a further understanding and understanding of the characteristics of the present invention and the achieved effects, the examples and accompanying descriptions are provided as follows: In view of the fact that different oral cancer patients have different oral cancer gene characteristics Manifestations, corresponding drug treatment is needed, in order to achieve more precise treatment, improve survival rate and reduce the Side effects of chemotherapy drugs. Accordingly, the present invention proposes a pharmaceutical composition and its application and evaluation method in the preparation of drugs for treating oral cancer, so as to solve the problems caused by the prior art.
以下將進一步說明本發明之其包含之特性、所搭配之結構及方法：首先，請參閱第1圖，其係本發明之一實施例之步驟流程圖。如圖所示，本發明之一種使用醫藥組合物之評估方法，其步驟包含：S1：使用即時定量反轉錄聚合酶連鎖反應檢測腫瘤組織樣本之CHEK1基因以及PIK3CA基因，取得第一CHEK1基因表現量、第一PIK3CA基因表現量以及檢測正常組織樣本之CHEK1基因以及PIK3CA基因，取得第二CHEK1基因表現量、第二PIK3CA基因表現量；S2：比對第一CHEK1基因表現量與第二CHEK1基因表現量以及第一PIK3CA基因表現量與第二PIK3CA基因表現量，得到CHEK1差異倍數以及PIK3CA差異倍數；S3：當CHEK1差異倍數高於CHEK1基本倍數以及PIK3CA差異倍數高於PIK3CA基本倍數時，形成比對結果；以及S4：依據比對結果判斷使用醫藥組合物，醫藥組合物係包含細胞檢查點激酶抑制劑及磷脂醯肌醇3激酶抑制劑。 The following will further illustrate the features, structures and methods of the present invention: First, please refer to FIG. 1 , which is a flow chart of the steps of an embodiment of the present invention. As shown in the figure, an evaluation method using a pharmaceutical composition of the present invention, the steps include: S1: Use real-time quantitative reverse transcription polymerase chain reaction to detect the CHEK1 gene and PIK3CA gene in tumor tissue samples, and obtain the first CHEK1 gene expression level , the first PIK3CA gene expression level and the detection of CHEK1 gene and PIK3CA gene in normal tissue samples to obtain the second CHEK1 gene expression level and the second PIK3CA gene expression level; S2: compare the first CHEK1 gene expression level with the second CHEK1 gene expression level and the first PIK3CA gene expression level and the second PIK3CA gene expression level to obtain the CHEK1 difference multiple and PIK3CA difference multiple; S3: When the CHEK1 difference multiple is higher than the CHEK1 basic multiple and the PIK3CA differential multiple is higher than the PIK3CA basic multiple, a comparison is made Result; and S4: judging to use the pharmaceutical composition according to the comparison result, the pharmaceutical composition comprises a cell checkpoint kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor.
其中，於本發明中使用之標的基因為CHEK1基因，CHEK1基因轉錄出CHK1蛋白，此蛋白調控細胞週期檢查點訊號，當細胞核酸物質複製發生錯誤時，會激活CHK1蛋白造成細胞週期停滯，並進行修補作用。如在腫瘤細胞中抑制CHK1蛋白之活性，便可以阻礙腫瘤細胞的修復，進而達到殺死腫瘤細胞的功效。 Among them, the target gene used in the present invention is the CHEK1 gene. The CHEK1 gene transcribes the CHK1 protein, which regulates the cell cycle checkpoint signal. When errors occur in the replication of cellular nucleic acid substances, the CHK1 protein will be activated to cause cell cycle arrest, and Repair function. If the activity of CHK1 protein is inhibited in tumor cells, it can hinder the repair of tumor cells, and then achieve the effect of killing tumor cells.
於本發明中使用之另一標的基因為PIK3CA，PIK3CA基因轉錄出PI3K alpha蛋白，此蛋白調控磷脂醯肌醇3-激酶(PI3Ks)訊號路徑，參與細胞增殖與凋亡等多種細胞功能的調節，於腫瘤細胞中此蛋白過度活化導致腫瘤細胞一直快速增生。故，如在腫瘤細胞中抑制PI3K alpha蛋白之活性，可以阻礙腫瘤細胞的生長，進而達到殺死腫瘤細胞的功效。 Another target gene used in the present invention is PIK3CA. The PIK3CA gene transcribes the PI3K alpha protein, which regulates the phosphatidylinositol 3-kinase (PI3Ks) signaling pathway and participates in the regulation of various cell functions such as cell proliferation and apoptosis. The overactivation of this protein in tumor cells leads to the rapid proliferation of tumor cells all the time. Therefore, if the activity of PI3K alpha protein is inhibited in tumor cells, the growth of tumor cells can be hindered, and the effect of killing tumor cells can be achieved.
首先，如步驟S1所示，使用一即時定量反轉錄聚合酶連鎖反應檢測一腫瘤組織樣本之一CHEK1基因以及一PIK3CA基因，取得一第一CHEK1基因表現量、一第一PIK3CA基因表現量。將已依照醫療常規檢查並獲得而置於實驗裝置中之該腫瘤組織樣本(係一癌症腫瘤組織)磨碎，用Trizol試劑來裂解該腫瘤組織樣本中細胞後加入氯仿，使細胞中的物質進行分層，再將含有RNA之水層經由異丙醇沉澱後，再以焦碳酸二乙酯(DEPC)處理過的二次水回溶，並萃取出細胞中的RNA。最後，以該即時定量反轉錄聚合酶連鎖反應的方式來檢測該腫瘤組織樣本之該CHEK1基因以及該PIK3CA基因，並取得該第一CHEK1基因表現量、該第一PIK3CA基因表現量。 First, as shown in step S1, a real-time quantitative reverse transcription polymerase chain reaction is used to detect a CHEK1 gene and a PIK3CA gene in a tumor tissue sample to obtain a first CHEK1 gene expression level and a first PIK3CA gene expression level. Grind the tumor tissue sample (a cancer tumor tissue) that has been inspected and obtained according to medical routine and placed in the experimental device, use Trizol reagent to lyse the cells in the tumor tissue sample, and then add chloroform to make the substances in the cells The layers were separated, and the aqueous layer containing RNA was precipitated by isopropanol, and then redissolved in secondary water treated with diethylpyrocarbonate (DEPC), and the RNA in the cells was extracted. Finally, the CHEK1 gene and the PIK3CA gene in the tumor tissue sample are detected by means of the real-time quantitative reverse transcription polymerase chain reaction, and the first CHEK1 gene expression level and the first PIK3CA gene expression level are obtained.
並以上述同樣的方式，以該即時定量反轉錄聚合酶連鎖反應檢測一正常組織樣本之該CHEK1基因(通常Chk1的激活則會導致細胞週期停滯，並啟動細胞週期檢查點以及DNA修復，來防止受損細胞(包含癌細胞)的死亡)以及該PIK3CA基因(通常PI3K的激活往往與腫瘤發生有關)，並取得一第二CHEK1基因表現量、一第二PIK3CA基因表現量。 And in the same manner as above, detect the CHEK1 gene in a normal tissue sample with the real-time quantitative reverse transcription polymerase chain reaction (usually the activation of Chk1 will lead to cell cycle arrest, and start cell cycle checkpoints and DNA repair, to prevent the death of damaged cells (including cancer cells) and the PIK3CA gene (usually the activation of PI3K is often related to tumorigenesis), and obtain a second CHEK1 gene expression level and a second PIK3CA gene expression level.
接續，如步驟S2所示，比對該第一CHEK1基因表現量(由該腫瘤組織樣本中得到)與該第二CHEK1基因表現量(由該正常組織樣本中得到)，得到一CHEK1差異倍數(由該第一CHEK1基因表現量除以該第二CHEK1基因表現量)。又，比對該第一PIK3CA基因表現量(由該腫瘤組織樣本中得到)與該第二PIK3CA基因表現量(由該正常組織樣本中得到)，得到一PIK3CA差異倍數(由該第一PIK3CA基因表現量除以該第二PIK3CA基因表現量)。 Next, as shown in step S2, compare the first CHEK1 gene expression level (obtained from the tumor tissue sample) with the second CHEK1 gene expression level (obtained from the normal tissue sample), and obtain a CHEK1 difference multiple ( Divide the expression level of the first CHEK1 gene by the expression level of the second CHEK1 gene). In addition, comparing the expression level of the first PIK3CA gene (obtained from the tumor tissue sample) with the expression level of the second PIK3CA gene (obtained from the normal tissue sample), a PIK3CA multiple difference (obtained from the first PIK3CA gene expression level divided by the second PIK3CA gene expression level).
再者，進一步搭配第2圖，其係本發明之一實施例之CHEK1及PIK3CA之基本倍數圖。如步驟S3所示，當該CHEK1差異倍數高於一CHEK1基本倍數以及該PIK3CA差異倍數高於一PIK3CA基本倍數時，形成一比對結果。其中如第2圖可知，該CHEK1基本倍數(CHEK1)係1.7，該PIK3CA基本倍數(PIK3CA)係2.2。該CHEK1基本倍數係1.7係根據口腔癌病患族群(37位)之CHEK1基因表現差異倍數之平均值來訂定出。而該PIK3CA基本倍數係2.2係根據口腔癌病患族群 (31位)之CHEK1基因表現差異倍數之平均值來訂定出。故當該CHEK1差異倍數高於1.7以及該PIK3CA差異倍數高於2.2時，即形成該比對結果。 Furthermore, it is further matched with Fig. 2, which is a basic multiple diagram of CHEK1 and PIK3CA according to an embodiment of the present invention. As shown in step S3, when the CHEK1 difference multiple is higher than a CHEK1 basic multiple and the PIK3CA difference multiple is higher than a PIK3CA basic multiple, a comparison result is formed. As shown in Figure 2, the basic multiple of CHEK1 (CHEK1) is 1.7, and the basic multiple of PIK3CA (PIK3CA) is 2.2. The basic multiple of CHEK1 is 1.7, which is determined based on the average of the differential multiples of CHEK1 gene expression in the oral cancer patient group (37 persons). The basic multiple of PIK3CA is 2.2, which is based on the population of oral cancer patients (31 persons) were determined by the average value of the CHEK1 gene expression fold difference. Therefore, when the CHEK1 difference multiple is higher than 1.7 and the PIK3CA difference multiple is higher than 2.2, the comparison result is formed.
最後，如步驟S4所示，依據該比對結果判斷使用一醫藥組合物，該醫藥組合物係包含一細胞檢查點激酶抑制劑(本發明於後續口腔癌實驗中之較佳實施例為PF477736)及一磷脂醯肌醇3激酶抑制劑(本發明於後續口腔癌實驗中之較佳實施例為BYL719)。該醫藥組合物係用於抑制一癌細胞，其中該癌細胞係一口腔鱗狀上皮癌細胞(為本發明之較佳實施例)。 Finally, as shown in step S4, it is determined to use a pharmaceutical composition according to the comparison result, and the pharmaceutical composition contains a cell checkpoint kinase inhibitor (the preferred embodiment of the present invention in subsequent oral cancer experiments is PF477736) And a phosphatidylinositol 3-kinase inhibitor (the preferred embodiment of the present invention in subsequent oral cancer experiments is BYL719). The pharmaceutical composition is used for inhibiting a cancer cell, wherein the cancer cell is an oral squamous cell carcinoma (a preferred embodiment of the present invention).
接續，請參閱第3A-3B圖，其係分別為本發明之一實施例之細胞檢查點激酶抑制劑之濃度結果圖以及本發明之一實施例之磷脂醯肌醇3激酶抑制劑之濃度結果圖(Y軸為細胞存活率(%)，X軸為Log10濃度(分別有nM以及μM)。由該口腔鱗狀上皮癌細胞(SAS細胞)進行細胞存活率分析法(MTT assay)，測試該細胞檢查點激酶抑制劑(本發明選自由一PF477736(A欄)、一AZD7762(B欄)及一LY2606368(C欄)所組成之其中之一或其組合，但不以上述細胞檢查點激酶抑制劑為限)及該磷脂醯肌醇3激酶抑制劑(一BYL719(A欄)、一GDC0941(B欄)及一GSK1059615(C欄)所組成之其中之一或其組合，但不以上述磷脂醯肌醇3激酶抑制劑為限)，分別觀察對於抑制該口腔鱗狀上皮癌細胞(SAS細胞以及OEC-M1細胞)之生長的效果。 Next, please refer to Figures 3A-3B, which are the concentration results of the cell checkpoint kinase inhibitor in one embodiment of the present invention and the concentration results of the phosphatidylinositol 3-kinase inhibitor in one embodiment of the present invention Graph (Y-axis is the cell survival rate (%), X-axis is the Log 10 concentration (respectively has nM and μ M). Carry out the cell viability analysis method (MTT assay) by this oral squamous epithelial carcinoma cell (SAS cell), test The cell checkpoint kinase inhibitor (the present invention is selected from one or a combination of PF477736 (column A), AZD7762 (column B) and LY2606368 (column C), but does not use the above-mentioned cell checkpoint kinase inhibitors) and the phosphatidylinositol 3-kinase inhibitor (a BYL719 (column A), a GDC0941 (column B) and a GSK1059615 (column C) or a combination thereof, but not Phosphatidylinositol 3-kinase inhibitors are limited), and the effects of inhibiting the growth of the oral squamous cell carcinoma cells (SAS cells and OEC-M1 cells) were observed respectively.
將1×104個該口腔鱗狀上皮癌細胞(SAS細胞以及OEC-M1細胞)種到96孔盤，培養6-8小時細胞貼壁後開始加入不同濃度之不同該細胞檢查點激酶抑制劑(PF477736、LY2606368、AZD7762)或不同之該磷脂醯肌醇3激酶抑制劑(BYL719、GDC0941、GSK1059615)，每個組別做三重複。48小時後，去除含有該細胞檢查點激酶抑制劑(PF477736、LY2606368、AZD7762)或該磷脂醯肌醇3激酶抑制劑(BYL719、GDC0941、GSK1059615)的細胞培養液，並以磷酸鹽緩衝生理鹽水(PBS)清洗兩次後，再加入200μL的10% MTT(20μL MTT和180μL細胞培養液)。在5% CO2，37℃的培養箱培養一小時後，抽掉MTT再加入200μL的DMSO呈色，混和均勻後每個well各取100μL至偵測用96孔盤，以ELISA讀值機(Thermo Fisher Scientific,USA)偵測波長540nm之讀值。該細胞檢查點激酶抑制 劑(PF477736、LY2606368、AZD7762)或該磷脂醯肌醇3激酶抑制劑(BYL719、GDC0941、GSK1059615)處理之各濃度的組別以沒加該細胞檢查點激酶抑制劑(PF477736、LY2606368、AZD7762)或該磷脂醯肌醇3激酶抑制劑(BYL719、GDC0941、GSK1059615)的控制組吸光值相除，再換算成細胞存活率。 Seed 1×10 4 oral squamous cell carcinoma cells (SAS cells and OEC-M1 cells) into 96-well plates, culture for 6-8 hours and start adding different concentrations of different checkpoint kinase inhibitors (PF477736, LY2606368, AZD7762) or different phosphatidylinositol 3-kinase inhibitors (BYL719, GDC0941, GSK1059615), each group was repeated three times. After 48 hours, the cell culture medium containing the cell checkpoint kinase inhibitor (PF477736, LY2606368, AZD7762) or the phosphatidylinositol 3-kinase inhibitor (BYL719, GDC0941, GSK1059615) was removed and washed with phosphate buffered saline ( After washing twice with PBS), 200 μL of 10% MTT (20 μL MTT and 180 μL cell culture medium) was added. After incubating in a 5% CO 2 , 37°C incubator for one hour, remove the MTT and add 200 μL of DMSO for color development. After mixing evenly, take 100 μL from each well to a 96-well plate for detection, and use an ELISA reader ( Thermo Fisher Scientific, USA) detects the reading at a wavelength of 540nm. The groups of each concentration of the cell checkpoint kinase inhibitor (PF477736, LY2606368, AZD7762) or the phosphatidylinositol 3-kinase inhibitor (BYL719, GDC0941, GSK1059615) were treated without the cell checkpoint kinase inhibitor (PF477736 , LY2606368, AZD7762) or the phosphatidylinositol 3-kinase inhibitor (BYL719, GDC0941, GSK1059615) the absorbance value of the control group was divided, and then converted into cell viability.
最後，由第3A及3B圖可知，加入該細胞檢查點激酶抑制劑後(PF477736、LY2606368、AZD7762)會明顯造成該口腔鱗狀上皮癌細胞(SAS細胞以及OEC-M1細胞)死亡，且該PF477736造成SAS細胞死亡至50%時的藥物濃度為120nM(為第3A圖)，該磷脂醯肌醇3激酶抑制劑(BYL719、GDC0941、GSK1059615)亦會造成該口腔鱗狀上皮癌細胞(SAS細胞以及OEC-M1細胞)死亡，且該BYL719造成SAS細胞死亡至50%時的藥物濃度為9μM(為第3B圖)。 Finally, as can be seen from Figures 3A and 3B, the addition of the cell checkpoint kinase inhibitors (PF477736, LY2606368, AZD7762) will obviously cause the death of the oral squamous cell carcinoma cells (SAS cells and OEC-M1 cells), and the PF477736 The drug concentration when causing SAS cell death to 50% is 120nM (Fig. 3A). OEC-M1 cells) died, and the BYL719 caused the SAS cell death to 50% at a drug concentration of 9 μM (Fig. 3B).
接續，如第4圖所示，其係本發明之一實施例之同時使用細胞檢查點激酶抑制劑與磷脂醯肌醇3激酶抑制劑(醫藥組合物)之口腔癌細胞之存活能力之實驗結果圖。再次進行細胞存活率分析法(MTT assay)，此次除了控制組(符號皆為-)、該細胞檢查點激酶抑制劑(此時本發明選用該PF477736，120nM，符號為+、-)、該磷脂醯肌醇3激酶抑制劑(此時本發明選用該BYL719，9μM，符號為-、+)外，亦有該醫藥組合物(為同時加入該細胞檢查點激酶抑制劑(PF477736，120nM)以及該磷脂醯肌醇3激酶抑制劑(BYL719，9μM)，符號皆為+)於該口腔鱗狀上皮癌細胞(SAS細胞)中。每個組別做三重複，此實驗步驟重複兩次，並將兩次數據合併。其結果如第4圖所示，使用該醫藥組合物(即同時加入該細胞檢查點激酶抑制劑(PF477736，120nM)以及該磷脂醯肌醇3激酶抑制劑(BYL719，9μM)的半抑制劑量(50%)處理該口腔鱗狀上皮癌細胞(SAS細胞)後，其細胞存活率(Y軸)平均值為38.2%，遠低於50%，故表示該醫藥組合物(該細胞檢查點激酶抑制劑加上該磷脂醯肌醇3激酶抑制劑)可以有效抑制該癌細胞(該口腔鱗狀上皮癌細胞(SAS細胞))生長，且比單獨使用各一抑制劑的效果更加顯著。 Next, as shown in Figure 4, it is the experimental result of the viability of oral cancer cells using a cell checkpoint kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor (pharmaceutical composition) at the same time according to an embodiment of the present invention picture. Carry out the cell viability analysis method (MTT assay) again, this time except the control group (the symbols are all -), the cell checkpoint kinase inhibitor (the present invention uses the PF477736, 120nM, the symbols are +, -), the In addition to the phosphatidylinositol 3-kinase inhibitor (this invention chooses the BYL719 at this time, 9 μ M, the symbols are -, +), there is also the pharmaceutical composition (in order to add the cell checkpoint kinase inhibitor (PF477736, 120 nM) and The phosphatidylinositol 3-kinase inhibitor (BYL719, 9 μM), all symbols are +) in the oral squamous epithelial carcinoma cells (SAS cells). Each group was repeated three times, and this experimental procedure was repeated twice, and the two data were combined. The results are shown in Figure 4, using the pharmaceutical composition (i.e. adding the cell checkpoint kinase inhibitor (PF477736, 120nM) and the phosphatidylinositol 3-kinase inhibitor (BYL719, 9μM) half inhibitory dose ( 50%) after treating the oral squamous cell carcinoma cells (SAS cells), the average cell survival rate (Y-axis) is 38.2%, which is far lower than 50%, so it means that the pharmaceutical composition (the cell checkpoint kinase inhibits The drug plus the phosphatidylinositol 3-kinase inhibitor) can effectively inhibit the growth of the cancer cell (the oral squamous cell carcinoma (SAS cell)), and the effect is more significant than that of using each inhibitor alone.
再者，如第5A圖所示，其係本發明之一實施例之口腔癌細胞之腫瘤生長能力之實驗結果圖(X軸為天數)。如圖所示，以該癌症腫瘤組織(為口腔癌腫瘤組織)建立的人源化異種移植小鼠模型進行藥效測試，並檢測腫瘤的生長大 小。其腫瘤大小計算方式：1/2×長×寬×寬，當異種移植動物模型的腫瘤達到300~400mm3時，將小鼠隨機分組並開始各自的治療。治療療程為兩周，第一周以該細胞檢查點激酶抑制劑以腹腔注射(0.5毫克/次，4次/周)，並同時以該磷脂醯肌醇3激酶抑制劑以口腔餵食(1.25毫克，5次/周)，第二周治療只以該磷脂醯肌醇3激酶抑制劑以口腔餵食(1.25毫克，5次/周)，治療期間每周測量腫瘤大小兩次，並於治療後再觀察腫瘤大小一周，腫瘤體積以平均值±平均值標準誤差表示。結果顯示若移植之口腔癌腫瘤組織為CHEK1高表現族群及PIK3CA高表現族群(藉由該評估方法得知該CHEK1差異倍數大於1.7，該PIK3CA差異倍數大於2.2的情況)，在該細胞檢查點激酶抑制劑和該磷脂醯肌醇3激酶抑制劑(本發明之醫藥組合物，為D組，小鼠個數4)合併使用的組別中，腫瘤生長明顯被抑制，與控制組(A組，不給予藥物，小鼠個數4)有顯著差異(*，p<0.05)。並且也比該細胞檢查點激酶抑制劑(B組，小鼠個數3)和該磷脂醯肌醇3激酶抑制劑(C組，小鼠個數4)單獨使用的情況下，能更加有效率的抑制腫瘤生長。 Furthermore, as shown in FIG. 5A , it is an experimental result graph of the tumor growth ability of oral cancer cells according to an embodiment of the present invention (the X axis is the number of days). As shown in the figure, the humanized xenograft mouse model established with the cancer tumor tissue (oral cancer tumor tissue) was used for drug efficacy testing, and the tumor growth and size were detected. The calculation method of the tumor size is: 1/2×length×width×width. When the tumor of the xenograft animal model reaches 300-400mm 3 , the mice are randomly divided into groups and start their respective treatments. The course of treatment was two weeks. In the first week, the cell checkpoint kinase inhibitor was injected intraperitoneally (0.5 mg/time, 4 times/week), and at the same time, the phosphatidylinositol 3-kinase inhibitor was orally fed (1.25 mg , 5 times/week), in the second week of treatment, only the phosphatidylinositol 3-kinase inhibitor was fed orally (1.25 mg, 5 times/week), during the treatment period, the tumor size was measured twice a week, and after treatment The tumor size was observed for one week, and the tumor volume was expressed as mean ± standard error of the mean. The results show that if the transplanted oral cancer tumor tissue belongs to the CHEK1 high-expression group and the PIK3CA high-expression group (by this evaluation method, it is known that the CHEK1 difference is greater than 1.7, and the PIK3CA difference is greater than 2.2), the cell checkpoint kinase In the group where the inhibitor and the phosphatidylinositol 3-kinase inhibitor (the pharmaceutical composition of the present invention is group D, the number of mice is 4) are used in combination, the growth of the tumor is obviously inhibited, compared with the control group (group A, Without drug administration, the number of mice 4) has a significant difference (*, p<0.05). And it can be more efficient than the cell checkpoint kinase inhibitor (group B, number of mice 3) and the phosphatidylinositol 3-kinase inhibitor (group C, number of mice 4) used alone inhibition of tumor growth.
並請一併參閱第5B圖，其係本發明之一實施例之口腔癌小鼠模式之腫瘤生長能力之對照實驗結果圖(X軸為天數)。第5B圖與第5A圖，實驗步驟相同。其差異在於第5B圖實驗中，移植之該癌症腫瘤組織(為口腔癌腫瘤組織)並非CHEK1高表現族群及PIK3CA高表現族群(表示藉由該評估方法得知該CHEK1差異倍數小於1.7，該PIK3CA差異倍數小於2.2)。 Please also refer to FIG. 5B , which is a control experiment result graph of the tumor growth ability of the oral cancer mouse model of an embodiment of the present invention (the X axis is the number of days). Figure 5B is the same as Figure 5A, with the same experimental procedure. The difference is that in the experiment in Figure 5B, the transplanted cancer tumor tissue (oral cancer tumor tissue) is not a CHEK1 high-expression group and a PIK3CA high-expression group (indicating that the CHEK1 difference multiple is less than 1.7, and the PIK3CA The multiple of difference is less than 2.2).
由第5B圖結果可知，不論是控制組(A組，不給予藥物，小鼠個數6)、單純該磷脂醯肌醇3激酶抑制劑(B組，小鼠個數5)還是該細胞檢查點激酶抑制劑和該磷脂醯肌醇3激酶抑制劑(本發明之醫藥組合物，為C組，小鼠個數4)，其治療皆沒有抑制腫瘤生長之功效。顯示本發明之該醫藥組合物僅對CHEK1高表現族群及PIK3CA高表現族群具有良好之治療效果。 As can be seen from the results in Fig. 5B, whether it is the control group (group A, no drug administration, number of mice is 6), the phosphatidylinositol 3-kinase inhibitor alone (group B, number of mice is 5) or the cell check Neither the dot kinase inhibitor nor the phosphatidylinositol 3-kinase inhibitor (the pharmaceutical composition of the present invention, which is group C, the number of mice is 4) has no effect on inhibiting tumor growth. It shows that the pharmaceutical composition of the present invention has a good therapeutic effect only on the CHEK1 high-expression group and the PIK3CA high-expression group.
再者，其係本發明之一實施例之口腔癌小鼠模式之體重之實驗結果圖(Y軸為體重變化倍數，X軸為天數)。如第5C圖所示，每個禮拜測量一次小鼠體重，並以打藥前測量之體重為基準，算出打藥後小鼠體重變化之差異倍數。結果顯示相對於控制組(未加入藥物，為A組)不管是單獨使用該細胞檢查點激酶 抑制劑(B組)和該磷脂醯肌醇3激酶抑制劑(為C組)，或者是使用該醫藥組合物(該細胞檢查點激酶抑制劑及該磷脂醯肌醇3激酶抑制劑，為D組)治療，小鼠體重都沒有顯著降低，顯示該醫藥組合物(該細胞檢查點激酶抑制劑及該磷脂醯肌醇3激酶抑制劑合併)治療不但可以有效抑制腫瘤生長，對於小鼠也不會有脫水及體重減輕之副作用(因為傳統化療藥物會藉由產生DNA交連，導致細胞的死亡，進而使患者有嚴重的脫水及體重減輕等副作用)。 Furthermore, it is an experimental result diagram of the body weight of the oral cancer mouse model in one embodiment of the present invention (the Y axis is the multiple of body weight change, and the X axis is the number of days). As shown in Figure 5C, the body weight of the mice was measured once a week, and the weight difference of the mice after spraying was calculated based on the body weight measured before spraying. The results show that relative to the control group (without adding drugs, it is group A), no matter whether the cell checkpoint kinase is used alone Inhibitor (group B) and the phosphatidylinositol 3-kinase inhibitor (group C), or use the pharmaceutical composition (the cell checkpoint kinase inhibitor and the phosphatidylinositol 3-kinase inhibitor, for D group) treatment, the body weight of the mice did not significantly decrease, showing that the pharmaceutical composition (the cell checkpoint kinase inhibitor and the phosphatidylinositol 3-kinase inhibitor combined) treatment can not only effectively inhibit tumor growth, but also does not affect the mice There will be side effects of dehydration and weight loss (because traditional chemotherapy drugs will cause cell death through DNA cross-linking, which will cause patients to have severe side effects such as dehydration and weight loss).
故本發明之該醫藥組合物(該細胞檢查點激酶抑制劑(PF477736)以及該磷脂醯肌醇3激酶抑制劑(BYL719)之組合為可以取代傳統化療藥物(Cisplatin、鉑帝爾、順鉑俗稱白金，通常以靜脈注射方式來治療，其主要的副作用是嚴重的嘔吐、腎毒性以及神經毒性)之新治療方法，並可減少化療藥物治療所帶來的不適(嚴重的嘔吐、腎毒性以及神經毒性)，可針對CHEK1高表現族群及PIK3CA高表現族群(為該CHEK1差異倍數高於1.7以及該PIK3CA差異倍數高於2.2)之口腔癌病患提供較有效且較為精準的治療以及提高預後存活率。 Therefore, the pharmaceutical composition of the present invention (the combination of the cell checkpoint kinase inhibitor (PF477736) and the phosphatidylinositol 3-kinase inhibitor (BYL719) can replace traditional chemotherapy drugs (Cisplatin, Platinum, Cisplatin, commonly known as Platinum, usually treated by intravenous injection, its main side effects are severe vomiting, nephrotoxicity and neurotoxicity) new treatment method, and can reduce the discomfort caused by chemotherapy drugs (severe vomiting, nephrotoxicity and neurotoxicity) Toxicity), can provide more effective and precise treatment and improve the survival rate of oral cancer patients in the CHEK1 high-expression group and PIK3CA high-expression group (for the CHEK1 multiplicity higher than 1.7 and the PIK3CA multiplier higher than 2.2) .
最後，本發明亦揭示一種適用醫藥組合物之族群之評估方法，步驟包括：(a)由一個體取得一腫瘤組織樣本與一正常組織樣本；(b)分別檢測該腫瘤組織樣本與該正常組織樣本之一CHEK1基因之表現量及一PIK3CA基因之表現量；(c)基於步驟(b)計算出一CHEK1基因差異倍數及一PIK3CA基因差異倍數；以及(d)基於步驟(c)之該CHEK1基因差異倍數及該PIK3CA基因差異倍數來決定該個體是否為適用一醫藥組合物之族群；其中，當該CHEK1基因差異倍數高於1.7及該PIK3CA差異倍數高於2.2時，則該個體為適用該醫藥組合物之族群。 Finally, the present invention also discloses a method for evaluating the population of suitable pharmaceutical compositions, the steps include: (a) obtaining a tumor tissue sample and a normal tissue sample from an individual; (b) detecting the tumor tissue sample and the normal tissue respectively The expression level of a CHEK1 gene and the expression level of a PIK3CA gene in the sample; (c) calculating a CHEK1 gene difference multiple and a PIK3CA gene difference multiple based on step (b); and (d) the CHEK1 gene difference based on step (c) The gene difference multiple and the PIK3CA gene difference multiple are used to determine whether the individual is suitable for a group of pharmaceutical compositions; wherein, when the CHEK1 gene difference multiple is higher than 1.7 and the PIK3CA gene difference multiple is higher than 2.2, then the individual is suitable for the group A family of pharmaceutical compositions.
於步驟(a)之該個體為一哺乳動物，較佳之實施例為一人類。又，該腫瘤組織並不限於口腔癌腫瘤組織，適用於CHEK1基因及PIK3CA基因之活性所媒介之癌症之腫瘤組織皆包括(但不以此限)，其癌症包含：成人與兒童腫瘤、固體腫瘤生長/惡性病、類黏液與圓形細胞癌瘤、局部發展之腫瘤、轉移性癌症、人類軟組織肉瘤，包括歐文氏(Ewing)肉瘤、癌症轉移，包括淋巴性轉移、鱗狀細胞癌瘤，特別指頭與頸部，食道鱗狀細胞癌瘤、口腔癌瘤、血液細胞惡性病(包括多發性脊髓瘤)、白血病(包括急性淋巴細胞白血病、急性抗淋巴細胞白血病、 慢性淋巴細胞白血病、慢性脊髓細胞白血病與毛細胞白血病)、滲漏性淋巴瘤(以體腔為主之淋巴瘤)、胸腺淋巴瘤肺癌(包括小細胞癌瘤、皮膚T細胞淋巴瘤、霍金氏(Hodgkin's)淋巴瘤、非霍金氏淋巴瘤、腎皮質之癌症、產生ACTH之腫瘤、非小細胞癌、乳癌，包括小細胞癌瘤與管癌瘤)、胃腸道癌症(包括胃癌、結腸癌、結腸直腸癌及與結腸直腸新生贅瘤有關之息肉)、胰癌、肝癌、泌尿道癌症(包括膀胱癌，如：原發性表皮膀胱腫瘤、膀胱之侵入性轉換細胞癌瘤及肌肉-侵入性膀胱癌)、攝護腺癌、女性生殖道之惡性病(包括卵巢癌瘤、原發性腹膜上皮瘤、子宮頸癌瘤、子宮內膜癌、***癌、外陰之癌症、子宮癌及卵巢濾泡中之固體腫瘤)、男性生殖道之惡性病(包括睪丸癌與陰莖癌)、腎癌(包括腎細胞癌瘤)、腦癌(包括內因性腦腫瘤、神經母細胞瘤、星形細胞腦腫瘤、神經膠質瘤與侵入中樞神經系統之轉移性腫瘤細胞)、骨癌症(包括骨瘤與骨肉瘤)、皮膚癌症(包括惡性黑色素瘤、人類皮膚角質細胞之腫瘤發展與鱗狀細胞癌)、甲狀腺癌、視網膜母細胞瘤、神經母細胞瘤、腹膜滲漏、惡性胸膜滲漏、間皮瘤、威廉氏(Wilms's)腫瘤、膽囊癌、滋養層瘤、血管外皮細胞瘤與卡波西氏(Kaposi's)肉瘤。 The subject in step (a) is a mammal, preferably a human. In addition, the tumor tissue is not limited to oral cancer tumor tissue, and the tumor tissue applicable to cancer mediated by the activity of CHEK1 gene and PIK3CA gene includes (but not limited to), the cancer includes: adult and children tumors, solid tumors Growth/malignancy, myxoid and round cell carcinomas, locally growing neoplasms, metastatic cancers, human soft tissue sarcomas, including Ewing sarcoma, cancer metastases, including lymphoid metastases, squamous cell carcinomas, especially Fingers and neck, squamous cell carcinoma of the esophagus, oral carcinoma, hematologic malignancies (including multiple myeloma), leukemia (including acute lymphoblastic leukemia, acute lymphoblastic leukemia, Chronic lymphocytic leukemia, chronic myeloid leukemia and hairy cell leukemia), leaky lymphoma (body cavity-based lymphoma), thymic lymphoma lung cancer (including small cell carcinoma, cutaneous T-cell lymphoma, Hawking's ( Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the renal cortex, ACTH-producing tumors, non-small cell carcinoma, breast cancer, including small cell carcinoma and ductal carcinoma), gastrointestinal cancer (including gastric cancer, colon cancer, colon cancer Rectal cancer and polyps associated with colorectal neoplasia), pancreatic cancer, liver cancer, urinary tract cancer (including bladder cancer, such as: primary superficial bladder tumor, invasive transformed cell carcinoma of the bladder and muscle-invasive bladder cancer), prostate cancer, malignant diseases of the female reproductive tract (including ovarian cancer, primary peritoneal epithelial tumor, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, uterine cancer and ovarian follicles solid tumors), malignant diseases of the male reproductive tract (including testicular cancer and penile cancer), kidney cancer (including renal cell carcinoma), brain cancer (including endogenous brain tumors, neuroblastoma, and astrocytic brain tumors) , glioma and metastatic tumor cells invading the central nervous system), bone cancer (including osteoma and osteosarcoma), skin cancer (including malignant melanoma, tumor development of human skin keratinocytes and squamous cell carcinoma), thyroid Carcinoma, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms' tumor, gallbladder carcinoma, trophoblastic tumor, hemangiopericytoma, and Kaposi's )sarcoma.
接續於步驟(b)中使用之檢測方法為即時定量反轉錄聚合酶連鎖反應。於步驟(c)中之計算方式，係將該腫瘤組織樣本之該CHEK1基因之表現量除以該正常組織樣本該CHEK1基因之表現量，同理係將該腫瘤組織樣本之該PIK3CA基因之表現量除以該正常組織樣本該PIK3CA基因之表現量，來分別計算出該CHEK1基因差異倍數及該PIK3CA基因差異倍數。 The subsequent detection method used in step (b) is real-time quantitative reverse transcription polymerase chain reaction. The calculation method in step (c) is to divide the expression level of the CHEK1 gene of the tumor tissue sample by the expression level of the CHEK1 gene of the normal tissue sample, and similarly is the expression of the PIK3CA gene of the tumor tissue sample The expression level of the PIK3CA gene in the normal tissue sample is divided by the expression level of the PIK3CA gene to calculate the multiple of the CHEK1 gene difference and the multiple of the PIK3CA gene difference.
最後如步驟(d)所示，由該CHEK1基因差異倍數及該PIK3CA基因差異倍數來判斷該個體是否為適用醫藥組合物之族群。由上述口腔癌實施例可知，當該CHEK1基因差異倍數高於1.7及該PIK3CA差異倍數高於2.2時，該個體為適用該醫藥組合物(為該細胞檢查點激酶抑制劑以及該磷脂醯肌醇3激酶抑制劑之組合)族群。
Finally, as shown in step (d), it is judged whether the individual belongs to the group suitable for the pharmaceutical composition according to the CHEK1 gene difference multiple and the PIK3CA gene difference multiple. It can be known from the above oral cancer examples that when the CHEK1 gene differential multiple is higher than 1.7 and the PIK3CA differential multiple is higher than 2.2, the individual is suitable for the pharmaceutical composition (the cell checkpoint kinase inhibitor and the
故本發明實為一具有新穎性、進步性及可供產業上利用者，應符合我國專利法專利申請要件無疑，爰依法提出發明專利申請，祈 鈞局早日賜准專利，至感為禱。 Therefore, the present invention is novel, progressive and can be used in the industry. It should meet the patent application requirements of my country's patent law. I file an invention patent application in accordance with the law. I pray that the bureau will grant the patent as soon as possible. I am sincerely praying.
惟以上所述者，僅為本發明之較佳實施例而已，並非用來限定本發明實施之範圍，舉凡依本發明申請專利範圍所述之形狀、構造、特徵及精神所為之均等變化與修飾，均應包括於本發明之申請專利範圍內。 However, the above-mentioned ones are only preferred embodiments of the present invention, and are not used to limit the scope of the present invention. For example, all equal changes and modifications are made according to the shape, structure, characteristics and spirit described in the scope of the patent application of the present invention. , should be included in the patent application scope of the present invention.
S1~S4:步驟流程 S1~S4: Step process
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| TW201622726A (en) * | 2014-04-03 | 2016-07-01 | 艾森塔製藥公司 | Therapeutic combination of a PI3K inhibitor and a BTK inhibitor |
| TWI623533B (en) * | 2016-02-04 | 2018-05-11 | 智擎生技製藥股份有限公司 | 3,5-disubstituted pyrazoles useful as checkpoint kinase 1 (chk1) inhibitors, and their preparations and applications |
| TW201918560A (en) * | 2017-11-07 | 2019-05-16 | 美商南托米克斯有限責任公司 | Circulating RNA for detection, prediction, and monitoring of cancer |
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| TW201622726A (en) * | 2014-04-03 | 2016-07-01 | 艾森塔製藥公司 | Therapeutic combination of a PI3K inhibitor and a BTK inhibitor |
| TWI623533B (en) * | 2016-02-04 | 2018-05-11 | 智擎生技製藥股份有限公司 | 3,5-disubstituted pyrazoles useful as checkpoint kinase 1 (chk1) inhibitors, and their preparations and applications |
| TW201918560A (en) * | 2017-11-07 | 2019-05-16 | 美商南托米克斯有限責任公司 | Circulating RNA for detection, prediction, and monitoring of cancer |
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| Title |
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| 期刊 Wenjuan Wu et al., Combination of the Chk1 inhibitor (prexasertib) with a PI3K/mTOR inhibitor (LY3023414) induces synergistic anti-tumor activity in triple negative breast cancer (TNBC) models. AACR Annual Meeting 2019; [online] https://cancerres.aacrjournals.org/content/79/13_Supplement/3508 March 29-April 3, 2019. Abstract 3508 * |
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