TWI538672B - Use, pharmaceutical composition and kit for applying metformin and sodium butyrate in kras mutation cancer treatment - Google Patents

Use, pharmaceutical composition and kit for applying metformin and sodium butyrate in kras mutation cancer treatment Download PDF

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TWI538672B
TWI538672B TW104100361A TW104100361A TWI538672B TW I538672 B TWI538672 B TW I538672B TW 104100361 A TW104100361 A TW 104100361A TW 104100361 A TW104100361 A TW 104100361A TW I538672 B TWI538672 B TW I538672B
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metformin
sodium butyrate
cancer
pharmaceutical composition
kit
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TW104100361A
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TW201517899A (en
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季匡華
王愈善
張照君
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強普生技股份有限公司
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應用二甲雙胍與丁酸鈉於KRAS基因突變癌症治療之用途、醫藥組合物與醫藥套組 Use of metformin and sodium butyrate in KRAS gene mutation cancer therapy, pharmaceutical composition and medical kit

本發明係關於一種醫藥組合物、醫藥套組以及製造醫藥組合物的用途。 The present invention relates to a pharmaceutical composition, a medical kit, and the use of a pharmaceutical composition.

二甲雙胍(Metformin),或稱甲福明,係由山羊豆(Galega officinalis,或稱山羊草)所萃取出的物質。在過去的幾個世紀,山羊豆一直作為糖尿病的傳統療法而廣為人知,而到了1920年代,科學家更進一步地從山羊豆的萃取物中發現雙胍類的化合物,並經由動物實驗確認了此種化合物係降低血糖濃度的主要活性成份。目前,已知二甲雙胍或其醫療上可接受之鹽類的作用機轉係促進肌肉細胞對葡萄糖的攝取以及醣類的分解,以減少肝臟生成葡萄糖,進而達成抗高血糖的效果,並被廣泛地使用於治療第二型(非胰島素依賴型)糖尿病。 Metformin, or metformin, is a substance extracted from goatpea (Galega officinalis, or goat grass). In the past few centuries, goat beans have been widely known as traditional treatments for diabetes, and in the 1920s, scientists further discovered diterpenoids from goat bean extracts and confirmed them through animal experiments. The main active ingredient that lowers blood glucose levels. At present, it is known that the action mechanism of metformin or its medically acceptable salt promotes the uptake of glucose by muscle cells and the decomposition of sugars, thereby reducing the production of glucose by the liver, thereby achieving an antihyperglycemic effect, and is widely used. Used to treat type 2 (non-insulin dependent) diabetes.

在第二型糖尿病患者中,同時罹患結直腸癌、乳腺癌或胰腺癌此三種癌症其中之一的族群比率異常地高,而目前已有研究指出,服用二甲雙胍或其醫療上可接受之鹽類可以降低第二型糖尿病患者的整體患癌風險。換言之,糖尿病患中,接受二甲雙胍或其醫療上可接受之鹽類治療的患者,其罹患結直腸癌、乳腺癌或胰腺癌的比率較未使用該些化合物治療的患者為低。 In patients with type 2 diabetes, the proportion of one of the three cancers, colorectal cancer, breast cancer or pancreatic cancer, is abnormally high, and studies have now indicated that taking metformin or its medically acceptable salts It can reduce the overall risk of cancer in patients with type 2 diabetes. In other words, in patients with diabetes, patients receiving metformin or its medically acceptable salts have a lower rate of colorectal cancer, breast cancer or pancreatic cancer than patients who are not treated with these compounds.

另一方面,丁酸鈉(Sodium butyrate)是一種組蛋白去乙醯酶抑制劑(Histone deacetylase inhibitor,HDACi)。由於組蛋白去乙醯化酶(HDACs)表現異常上升的現象廣泛地存在於不同類型的癌症之中,因此,丁酸鈉(Sodium butyrate)被視為一種極具治療潛力之物質而應用在許多細 胞或動物實驗的研究中。目前發現,丁酸鈉具有抑制某些特定癌細胞的生長,以及誘導某些特定癌細胞凋亡,進而減緩腫瘤生長速度的功效。另外,丁酸鈉還被認為是一種腸道的保護劑,其可修復腸道黏膜及治療腸道發炎,進而防止結腸炎癌化的機率。另外,丁酸鈉亦被發現可預防乳腺癌的產生。 On the other hand, sodium butyrate is a Histone deacetylase inhibitor (HDACi). Sodium butyrate is widely used as a therapeutic potential because many of the abnormalities in histone deacetylase (HDACs) are widely present in different types of cancer. fine In the study of cellular or animal experiments. It has been found that sodium butyrate has the effect of inhibiting the growth of certain specific cancer cells and inducing apoptosis of certain specific cancer cells, thereby slowing the growth rate of the tumor. In addition, sodium butyrate is also considered to be a protective agent for the intestines, which can repair intestinal mucosa and treat intestinal inflammation, thereby preventing the incidence of colitis cancer. In addition, sodium butyrate has also been found to prevent breast cancer.

歸納而言,目前雖已證實二甲雙胍或其醫藥上可接受之鹽 類、或丁酸鈉分別對於某些特定的癌症或腫瘤有一定程度的療效,但僅單採其各自的治療效果,且在治療上使用二甲雙胍等,也大部分還是取其對糖尿病的療效,至於降低結直腸癌、乳腺癌或胰腺癌之發病機率的效果,則因作用機轉尚未明確,而未被期待。是以,現今對於二甲雙胍及丁酸鈉的後續研究遲未能進一步突破。 In summary, metformin or its pharmaceutically acceptable salt has been confirmed so far. Classes, or sodium butyrate, have a certain degree of efficacy for certain cancers or tumors, but only their respective therapeutic effects, and the use of metformin in treatment, most of them still take its effect on diabetes, As for the effect of reducing the incidence of colorectal cancer, breast cancer or pancreatic cancer, it is not expected that the action is not yet clear. Therefore, the current follow-up study on metformin and sodium butyrate has not been able to break through further.

然而,尤其對於具有K-ras基因突變之癌症患者而言,目前 藥物治療均不見顯著的治療效果,而臨床上也證實使用二甲雙胍或其醫藥上可接受之鹽類,或使用丁酸鈉對於減緩或改善此種癌症的症狀並無太大幫助。 However, especially for cancer patients with mutations in the K-ras gene, currently No significant therapeutic effects were observed in the medical treatment, and it was clinically confirmed that the use of metformin or a pharmaceutically acceptable salt thereof, or the use of sodium butyrate, did not contribute much to slowing or improving the symptoms of such cancer.

雖單一的二甲雙胍或其醫藥上可接受之鹽類、或丁酸鈉分別對於特定的癌症及腫瘤有某些程度的療效,但並未有研究報導或其他文獻指出將二甲雙胍與丁酸鈉合併使用,然而經發明人研究發現,將二甲雙胍與丁酸鈉合併使用,以用於治療具有K-ras基因突變之癌症患者,具有顯著優於各化合物單獨使用之療效。故本發明據以提出一種應用二甲雙胍與丁酸鈉於具有K-ras基因突變之癌症患者之醫藥組合物、醫藥套組與用途,有效提升對於該種癌症的治療效果。 Although a single metformin or its pharmaceutically acceptable salt, or sodium butyrate, has a certain degree of efficacy for specific cancers and tumors, no studies have reported or other literature has indicated the use of metformin in combination with sodium butyrate. However, the inventors have found that the use of metformin in combination with sodium butyrate for the treatment of cancer patients with mutations in the K-ras gene is significantly superior to the efficacy of each compound alone. Therefore, the present invention has been proposed to use a pharmaceutical composition, a medical kit and a use of metformin and sodium butyrate in a cancer patient having a K-ras gene mutation, thereby effectively improving the therapeutic effect on the cancer.

詳細來說,本發明提供一種二甲雙胍與丁酸鈉用於製造治療一具有K-ras基因突變之癌症患者的醫藥組合物之用途,且當投以醫藥組合物予該癌症患者,二甲雙胍與丁酸鈉提供合併治療之效果。 In particular, the present invention provides a use of metformin and sodium butyrate for the manufacture of a pharmaceutical composition for treating a cancer patient having a K-ras gene mutation, and when administering a pharmaceutical composition to the cancer patient, metformin and butyric acid Sodium provides the effect of combined treatment.

本發明另提供一種治療一具有K-ras基因突變之癌症患者的醫藥組合物,其包括二甲雙胍與丁酸鈉,且當投以醫藥組合物予該癌症患者,二甲雙胍與丁酸鈉提供合併治療之效果。 The present invention further provides a pharmaceutical composition for treating a cancer patient having a K-ras gene mutation, which comprises metformin and sodium butyrate, and when a pharmaceutical composition is administered to the cancer patient, metformin and sodium butyrate are combined. effect.

本發明又提供一種治療一具有K-ras基因突變之癌症患者 的醫藥套組,其包括二甲雙胍與一第一醫藥上可接受之載體、稀釋劑或賦形劑,以及丁酸鈉與一第二醫藥上可接受之載體、稀釋劑或賦形劑,其中,醫藥套組施予該癌症患者後,二甲雙胍與丁酸鈉提供合併治療之效果。 The invention further provides a treatment for a cancer patient having a K-ras gene mutation Medicinal kit comprising metformin and a first pharmaceutically acceptable carrier, diluent or excipient, and sodium butyrate and a second pharmaceutically acceptable carrier, diluent or excipient, wherein After the medical kit is administered to the cancer patient, metformin and sodium butyrate provide a combined treatment effect.

為使之後內容能清楚呈現本發明的技術特徵,以下擬先定 義特定名詞,爾後進一步說明本發明內容。在本發明中,二甲雙胍與丁酸鈉係為合併治療具有K-ras基因突變之癌症患者,而「合併治療」係為同時使用二甲雙胍與丁酸鈉這兩種成分,而使用方式可以製成一種醫藥組合物,其同時包含二甲雙胍與丁酸鈉兩種成分;亦可製成一種醫藥套組(又可稱為組合藥品或組合藥物),其同時包括二種醫藥物,其一具有二甲雙胍成分,另一具有丁酸鈉成分,並同時使用該二種醫藥物。較佳地,醫藥組合物或醫藥套組分別具有一有效劑量之二甲雙胍以及一有效劑量之丁酸鈉,其中,該有效劑量係指兩者可產生協同作用而合併治療之劑量。 In order to make the following content clearly show the technical features of the present invention, the following is intended to be The specific nouns will be further described below. In the present invention, metformin and sodium butyrate are combined to treat a cancer patient having a K-ras gene mutation, and the "combination therapy" is a simultaneous use of two components of metformin and sodium butyrate, and the use can be made into a kind. a pharmaceutical composition comprising both metformin and sodium butyrate; or a medical kit (also referred to as a combination drug or a combination drug), which comprises two kinds of medicines, one of which has a metformin component, The other has a sodium butyrate component and uses both drugs. Preferably, the pharmaceutical composition or the medical kit respectively has an effective dose of metformin and an effective amount of sodium butyrate, wherein the effective dose refers to a dose which synergistically combines the two treatments.

在本說明書中所使用之「K-ras基因突變」一詞意指K-ras 基因為突變型,其中,K-ras基因係為一種致癌基因,而K-ras基因可分為正常狀態(稱為原生型)及異常狀態(稱為突變型),正常狀態的K-ras基因可抑制腫瘤細胞生長,但異常狀態(突變型)的K-ras基因則會刺激細胞不斷的增生,導致腫瘤的產生。另外,更有研究指出,在肺癌、胰臟癌、大腸直腸癌中,患者之K-ras基因為突變型的機率較其他癌症類型之患者為高,且一般的治療藥物對這些患者的治療效果有限,從而在臨床檢驗上分出非K-ras突變型與K-ras突變型兩種。 The term "K-ras gene mutation" as used in this specification means K-ras The gene is a mutant type, wherein the K-ras gene is an oncogene, and the K-ras gene can be divided into a normal state (called a native type) and an abnormal state (called a mutant type), and a normal state K-ras gene. Can inhibit tumor cell growth, but the abnormal state (mutant) K-ras gene will stimulate the cell proliferation, leading to tumor production. In addition, more studies have pointed out that in lung cancer, pancreatic cancer, and colorectal cancer, the probability of K-ras gene mutation is higher in patients with other cancer types, and the therapeutic effect of general therapeutic drugs on these patients. It is limited, so that non-K-ras mutants and K-ras mutants are classified in clinical tests.

另外,在本說明書中所使用之「協同作用」意指當合併給 予二種或二種以上的物質時,其產生的效應(包括產生相同性質的治療效應,或減少不良反應)等於或大於各單一物質獨自產生的效應之總和。於本發明實驗例中,可知合併給予二甲雙胍與丁酸鈉於K-ras基因突變之癌症患者,其所產生之治療效果,較單一的二甲雙胍或丁酸鈉的治療效果更好。 In addition, the term "synergy" as used in this specification means when combined When two or more substances are administered, the effects (including the therapeutic effects that produce the same properties, or reduce the adverse reactions) are equal to or greater than the sum of the effects of the individual substances alone. In the experimental example of the present invention, it is known that a cancer patient who is administered with metformin and sodium butyrate in a K-ras gene mutation has a better therapeutic effect than a single metformin or sodium butyrate.

在本說明書中所使用之「治療」一詞意指可以減輕、舒緩、改變、改善、改進或影響癌症及其症狀,舉例而言,抑制或治療癌症引發之疼痛或出血、或延緩或反轉腫瘤增生速率。 The term "treatment" as used in this specification means that it can alleviate, soothe, alter, improve, improve or affect cancer and its symptoms, for example, inhibit or treat cancer-induced pain or bleeding, or delay or reverse The rate of tumor proliferation.

在本說明書中所使用之「二甲雙胍」一詞除可指二甲雙胍外,也包括基本二甲雙胍結構經化學或生物學修飾或取代所產生之衍生 物,而且仍保有原基本二甲雙胍結構的性質或具有與原基本二甲雙胍結構相似的性質。當然,「二甲雙胍」一詞亦包括二甲雙胍之醫藥上可接受之鹽類,其係依據二甲雙胍化學結構上之酸或鹼基部份而形成,例如鹽酸二甲雙胍。 The term "metformin" as used in this specification, in addition to metformin, also includes derivatives derived from the chemical or biological modification or substitution of the basic metformin structure. And retains the properties of the original basic metformin structure or has properties similar to those of the original basic metformin structure. Of course, the term "metformin" also includes pharmaceutically acceptable salts of metformin, which are formed based on the acid or base moiety of the chemical structure of metformin, such as metformin hydrochloride.

另外,在本說明書中有提及且與本發明技術特徵實質相關 的化合物或物質,均可涵蓋所稱之化合物或物質以及其任何醫藥上可接受的形式。其中,醫藥上可接受的形式可例如但不限於含非鏡像異構物(diastereomer)與鏡像異構物(enantiomer)的各式異構物(isomer)、鹽類、游離形式、溶劑、前藥(prodrug)、多形體(polymorph)及消旋混合物(racemic mixture)。 In addition, it is mentioned in the present specification and is substantially related to the technical features of the present invention. The compound or substance may encompass the compound or substance referred to and any pharmaceutically acceptable form thereof. Wherein, the pharmaceutically acceptable form can be, for example but not limited to, various isomers, salts, free forms, solvents, prodrugs containing diastereomers and enantiomers. (prodrug), polymorph and racemic mixture.

在本發明中,二甲雙胍與丁酸鈉合併使用時,二甲雙胍的 劑量較佳係介於130至1,000毫克之間,且丁酸鈉的劑量較佳係介於40至300毫克之間。又在本發明中,二甲雙胍與丁酸鈉合併使用時的重量百分比為4:1(體積莫耳濃度比例為2.67:1)。當然,上述有效劑量與重量百分比亦可為醫藥組合物內的成份比例,或是醫藥套組實際使用時的調製比例。 In the present invention, when metformin is combined with sodium butyrate, metformin is used. The dose is preferably between 130 and 1,000 mg, and the dose of sodium butyrate is preferably between 40 and 300 mg. Further, in the present invention, the weight percentage of metformin combined with sodium butyrate is 4:1 (volume molar ratio is 2.67:1). Of course, the above effective dose and weight percentage may also be the proportion of the components in the pharmaceutical composition, or the modulation ratio of the medical kit when actually used.

在本發明中,具有K-ras基因突變之癌症患者可例如但不限 於具有K-ras基因突變之肺癌、胰臟或大腸直腸癌的患者,以治療腫瘤、延緩腫瘤增長或預防腫瘤形成。 In the present invention, a cancer patient having a K-ras gene mutation can be, for example but not limited to For patients with lung cancer, pancreas or colorectal cancer with K-ras gene mutation, to treat tumors, delay tumor growth or prevent tumor formation.

在本發明中,二甲雙胍與丁酸鈉合併使用時,可以共同調 控癌症之代謝型麩胺酸訊號傳遞途徑以治療具有K-ras基因突變之癌症患者。 In the present invention, when metformin is combined with sodium butyrate, it can be adjusted together. A metabolic glutamate signaling pathway for cancer is treated to treat cancer patients with mutations in the K-ras gene.

綜上所述,依據本發明之用途、醫藥組合物與醫藥套組, 其合併二甲雙胍與丁酸鈉使用,對於改善或增進治療具有K-ras基因突變之癌症或腫瘤患者,提供了顯著的功效。 In summary, the use, the pharmaceutical composition and the medical kit according to the present invention, Its combination with metformin and sodium butyrate provides significant efficacy in improving or enhancing the treatment of cancer or tumor patients with K-ras gene mutations.

圖1為實驗例二之流式細胞分析儀分析經化合物處理後之細胞凋亡結果的數據圖。 Fig. 1 is a graph showing the results of analyzing the apoptosis results of the compound treated by the flow cytometer of Experimental Example 2.

圖2為實驗例三之流式細胞分析儀分析經藥劑處理後相對於對照組之 存活細胞結果的數據圖。 2 is a flow cytometry analyzer of Experimental Example 3, which was analyzed after treatment with a drug and relative to a control group. A data plot of surviving cell results.

以下將配合圖式說明本發明之實施例與實驗例,惟相關文義說明可參照前述,於此不再贅述。 The embodiments and the experimental examples of the present invention will be described below with reference to the drawings, but the related descriptions may refer to the foregoing, and no further details are provided herein.

依據本發明之一種二甲雙胍與丁酸鈉用於製造供合併治療具有K-ras基因突變之癌症患者的醫藥組合物之用途,在一實施例中,可以透過將兩者共同製備成一個醫藥組合物,再給予患者服用來達成。其中,二甲雙胍與丁酸鈉的合成方法均為本發明所屬領域中具有通常知識者所能理解者,於此不再贅述。上述的醫藥組合物可以為固態、液態或半液態的劑型,本發明於此不限。當以固態應用時,較佳是將兩物質均勻混合而製成錠劑,有利於藥效作用。但固態劑型不以二甲雙胍與丁酸鈉兩物質均勻混合調製為限,換言之,於同一劑型的醫藥組合物內,二甲雙胍與丁酸鈉可以具有其他程度的混合,甚至不混合亦可,舉例而言,醫藥組合物可以為一種錠劑或膠囊,其中部分為二甲雙胍,另一部分為丁酸鈉。較佳地,在其他實施例中,可以自販售廠商處直接購得粉狀的二甲雙胍與丁酸鈉原料,而醫藥組合物可藉由混合粉狀之兩者所製成的一散劑。 The use of a metformin and sodium butyrate according to the present invention for the manufacture of a pharmaceutical composition for the combined treatment of a cancer patient having a K-ras gene mutation, in one embodiment, by co-preparing the two into a pharmaceutical composition Then give it to the patient to achieve. Among them, the synthesis methods of metformin and sodium butyrate are all understood by those skilled in the art to which the present invention pertains, and will not be described herein. The above pharmaceutical composition may be in the form of a solid, liquid or semi-liquid, and the present invention is not limited thereto. When applied in a solid state, it is preferred to uniformly mix the two substances to form a tablet, which is advantageous for pharmacodynamic action. However, the solid dosage form is not limited to the uniform mixing of metformin and sodium butyrate. In other words, in the pharmaceutical composition of the same dosage form, metformin and sodium butyrate may have other degrees of mixing, or even no mixing, for example. The pharmaceutical composition may be a tablet or capsule in which part is metformin and the other part is sodium butyrate. Preferably, in other embodiments, powdered metformin and sodium butyrate starting materials are commercially available from a vendor, and the pharmaceutical composition may be a powder prepared by mixing both powders.

當然,為製造、包裝或服藥方便,醫藥組合物較佳可包含一醫藥上可接受之載體、稀釋劑、賦形劑或其組合,以利於製成合用的劑型或配方形式。其中,醫藥上可接受之載體、稀釋劑或賦形劑,可例如公知的碳酸鎂、硬脂酸鎂、滑石、糖、乳糖或其組合。 Of course, for ease of manufacture, packaging or administration, the pharmaceutical compositions preferably comprise a pharmaceutically acceptable carrier, diluent, excipient or combination thereof to facilitate the formulation of a suitable dosage form or formulation. Among them, pharmaceutically acceptable carriers, diluents or excipients may, for example, be known as magnesium carbonate, magnesium stearate, talc, sugar, lactose or a combination thereof.

一般而言,本發明所屬技術領域中具通常知識者可理解二甲雙胍在治療癌症時適於使用的劑量範圍,此同時為二甲雙胍與丁酸鈉合併治療時適用的劑量範圍,舉例而言,具有K-ras基因突變之癌症患者每日每公斤體重可接受的二甲雙胍的劑量範圍在約0.002至約2,000毫克之間;至於合併使用時,則二甲雙胍的劑量範圍控制在約130至約1,000毫克之間,而丁酸鈉的劑量範圍控制在約40至約300毫克之間為佳。在上述範圍內,各劑量間所產生的治療效果可能具有頗大的落差,因此,可參考以下實驗例,以二甲雙胍與丁酸鈉的重量百分比為約4:1進行治療。 In general, those of ordinary skill in the art to which the present invention pertains will appreciate the range of dosages that metformin is suitable for use in the treatment of cancer, while at the same time being a range of dosages suitable for the combined treatment of metformin with sodium butyrate, for example, with K The dose of metformin per kg body weight of cancer patients with -ras gene mutation ranges from about 0.002 to about 2,000 mg per day; for combined use, the dose range of metformin is controlled between about 130 and about 1,000 mg, The dosage range of sodium butyrate is preferably controlled between about 40 and about 300 mg. Within the above range, the therapeutic effect produced between the doses may have a considerable drop. Therefore, reference may be made to the following experimental example in which the weight percentage of metformin and sodium butyrate is about 4:1.

當然,二甲雙胍或丁酸鈉的劑量可能會隨配合的治療上活 性成份、投予路徑或有需要之個體及其生理狀況的不同而有所變化。普遍而言,口服方式需要較高的劑量,反觀治療初期所使用的劑量則相對較低。 Of course, the dose of metformin or sodium butyrate may be alive with the treatment of the combination. Sexual components, routes of administration, or individuals in need and their physiological conditions may vary. In general, higher doses are required for oral administration, whereas the doses used at the beginning of treatment are relatively low.

每一配方中可含有一個劑量單位的醫藥組合物,換言之, 一個配方即含有足可對具有K-ras基因突變之癌症患者產生治療效果的劑量,以方便直接投予。在實施上,可以每一包散劑即含有一個劑量單位的醫藥組合物。當然,在其他實施方式中,一個劑量單位亦可由分散於數個次劑量單位或次包裝中的複數個醫藥組合物組成,例如分散於二至三粒錠劑或膠囊內但包裝在同一吸塑包裝(blister pack)中。 A pharmaceutical composition that can contain one dosage unit per formulation, in other words, One formulation contains a dose sufficient to produce a therapeutic effect on a cancer patient having a K-ras gene mutation to facilitate direct administration. In practice, a powder composition containing one dosage unit per dose may be employed. Of course, in other embodiments, one dosage unit may also consist of a plurality of pharmaceutical compositions dispersed in several sub-doses or sub-packages, for example, dispersed in two to three lozenges or capsules but packaged in the same blister. In the blister pack.

進一步說明,依據本發明之用途,當醫藥組合物投予具有 K-ras基因突變之癌症患者後,其中二甲雙胍與丁酸鈉可發生協同作用,以改善或增進單獨使用二甲雙胍或丁酸鈉對於K-ras基因突變型癌症治療的功效,進而達成消除、抑制、改善、緩解、預防癌症及其症狀、或延緩、阻止、反轉腫瘤增生速率、或達到與上述目的相似的醫療效果。 Further illustrated, in accordance with the use of the present invention, when the pharmaceutical composition is administered After cancer patients with K-ras gene mutation, metformin and sodium butyrate may act synergistically to improve or enhance the efficacy of metformin or sodium butyrate alone in the treatment of K-ras gene mutant cancer, thereby achieving elimination, inhibition, Improves, alleviates, prevents cancer and its symptoms, or delays, prevents, reverses the rate of tumor proliferation, or achieves medical effects similar to those described above.

本發明又提供一種治療一具有K-ras基因突變之癌症患者 的醫藥組合物,其包括二甲雙胍與丁酸鈉。然而,該醫藥組合物與前述說明之醫藥組合物大致相同,可參考前述,於此不再贅述。 The invention further provides a treatment for a cancer patient having a K-ras gene mutation Pharmaceutical composition comprising metformin and sodium butyrate. However, the pharmaceutical composition is substantially the same as the pharmaceutical composition described above, and reference is made to the foregoing, and no further details are provided herein.

本發明又提供一種醫藥套組,其包括二甲雙胍與一第一醫 藥上可接受之載體、稀釋劑或賦形劑,以及丁酸鈉與一第二醫藥上可接受之載體、稀釋劑或賦形劑。其中,二甲雙胍、丁酸鈉與其他有關之說明同樣可參考前述內容,惟此將再針對不足或未說明處加以解釋。 The invention further provides a medical kit comprising metformin and a first medical doctor A pharmaceutically acceptable carrier, diluent or excipient, and sodium butyrate and a second pharmaceutically acceptable carrier, diluent or excipient. Among them, metformin, sodium butyrate and other related descriptions can also refer to the foregoing, but will be explained again for insufficient or unexplained.

第一或第二醫藥上可接受之載體、稀釋劑、賦形劑或其組 合可為本發明所屬技術領域中已被理解的物質或成份,且如何與二甲雙胍或丁酸鈉共同製備的方式與製成的劑型亦為本發明領域具通常知識者所能理解者。而在一實施例中,醫藥套組可具有獨立的包裝或容器,例如吸塑包裝,以分別容置或儲存例如以二甲雙胍與第一醫藥上可接受之賦形劑製成的錠劑以及以丁酸鈉與第二醫藥上可接受之賦形劑製成的錠劑,而於應用時再同時提供給患者服用,或經調製後再投予給患者。惟要說明的是,醫藥套組中二甲雙胍及丁酸鈉仍是要合併使用為限,然則,所稱之合併使用可在兩者其中之一先行投予患者,並未失去作用效果前,另行投予兩者之另一,其中之時間差距可以為一小時至三天;當然較佳係兩者無時間差 地同時投予患者,或其中之一投予後立即投予另一的方式實施。 First or second pharmaceutically acceptable carrier, diluent, excipient or group thereof The materials or ingredients that are understood in the art to which the present invention pertains, and how they are prepared in combination with metformin or sodium butyrate, are also understood by those of ordinary skill in the art. In one embodiment, the kit may have a separate package or container, such as a blister pack, for individually holding or storing, for example, a tablet made with metformin and a first pharmaceutically acceptable excipient, and A tablet made of sodium butyrate and a second pharmaceutically acceptable excipient, which is then administered to the patient at the same time as the application, or modulated and administered to the patient. However, it should be noted that metformin and sodium butyrate in the medical kit are still to be combined. However, the combined use can be referred to one of the two patients before the effect is not lost. Another one of the two, the time difference can be from one hour to three days; of course, there is no time difference between the two The patient is simultaneously administered to the patient, or one of them is administered immediately after administration.

承上所述,依據本發明之用途、醫藥組合物與醫藥套組, 其合併二甲雙胍與丁酸鈉使用,對於改善或增進治療具有K-ras基因突變之癌症或腫瘤患者,提供了顯著的功效。 According to the above, the use, the pharmaceutical composition and the medical kit according to the present invention, Its combination with metformin and sodium butyrate provides significant efficacy in improving or enhancing the treatment of cancer or tumor patients with K-ras gene mutations.

實驗例一:製備醫藥組合物 Experimental Example 1: Preparation of a pharmaceutical composition

本發明所使用之二甲雙胍購自中國化學製藥股份有限公司 (China Chemical & Pharmaceutical Co.,Ltd.,Taiwan),丁酸鈉購自台灣默克公司(Merck Ltd.,Taiwan)。取得後,在室溫下分別秤取約331毫克的二甲雙胍(濃度為2mM)與約83毫克(濃度為0.75mM)的丁酸鈉,依照重量百分比約4:1(體積莫耳濃度比約2.67:1)的比例混合粉狀的二甲雙胍與丁酸鈉,即為一單位劑量。接著,包裝為一散劑或扁囊劑之形式,保存於室溫下。 The metformin used in the present invention is purchased from China Chemical Pharmaceutical Co., Ltd. (China Chemical & Pharmaceutical Co., Ltd., Taiwan), sodium butyrate was purchased from Merck Ltd., Taiwan. After obtaining, about 331 mg of metformin (concentration: 2 mM) and about 83 mg (concentration of 0.75 mM) of sodium butyrate were weighed at room temperature, about 4:1 by weight (volume molar ratio of about 2.67). The ratio of :1) is mixed with powdered metformin and sodium butyrate, which is a unit dose. Next, the package is in the form of a powder or cachet and stored at room temperature.

實驗例二:二甲雙胍與丁酸鈉醫藥組合物對癌細胞造成細胞凋亡具有協同作用的效果 Experimental Example 2: The synergistic effect of metformin and sodium butyrate pharmaceutical composition on apoptosis of cancer cells

以組織培養技術培養人類大腸癌(human colon cancer cell)細胞株HCT116(係為具有K-ras基因突變之細胞株)於含有濃度10%之胎牛血清的細胞培養液McCoy’s 5A至適當數量。接著,取含有數量2.5×105之細胞的懸浮液,並接種於6孔(6-wells)培養皿,培養12小時後,分別加入最終濃度為0.75mM之丁酸鈉,最終濃度為2mM之二甲雙胍,以及最終濃度為0.75mM之丁酸鈉及2mM之二甲雙胍的組合物。 The human colon cancer cell line HCT116 (a cell line having a K-ras gene mutation) was cultured by tissue culture technique in a cell culture medium McCoy's 5A containing 10% fetal calf serum to an appropriate amount. Next, a suspension containing cells of 2.5×10 5 was taken and inoculated in a 6-well (6-wells) culture dish. After 12 hours of culture, sodium butyrate at a final concentration of 0.75 mM was added, respectively, and the final concentration was 2 mM. Metformin, and a combination of sodium butyrate and 2 mM metformin at a final concentration of 0.75 mM.

於本實施例中所述之最終濃度,即為加入細胞培養液以進行細胞培養時,該細胞培養液中含有濃度為0.75mM的丁酸鈉,濃度為2mM的二甲雙胍,濃度為0.75mM的丁酸鈉及濃度為2mM的二甲雙胍。 The final concentration described in this example is that when the cell culture medium is added for cell culture, the cell culture solution contains sodium butyrate at a concentration of 0.75 mM, metformin at a concentration of 2 mM, and a concentration of 0.75 mM. Sodium and metformin at a concentration of 2 mM.

細胞株HCT116分別加入上述化合物後,再將培養皿置入37℃的培養箱繼續培養72小時。接著,依序將各孔中的培養液及加入的化合物吸除,另以磷酸鹽緩衝溶液(PBS)清洗,再利用胰蛋白酵素溶液(trypsin-EDTA)收取細胞分析細胞凋亡。 After the cell line HCT116 was separately added to the above compound, the culture dish was placed in an incubator at 37 ° C for further 72 hours. Next, the culture solution and the added compound in each well were sequentially aspirated, and further washed with a phosphate buffer solution (PBS), and then cells were collected by trypsin-EDTA to analyze apoptosis.

細胞凋亡係以磷脂酰絲氨酸外翻分析法並透過使用細胞凋亡分析套組Annexin V(BD Pharmingen)進行,操作流程則依循隨附手冊。概略而言,收取後的HCT116細胞再以PBS清洗3次,接著立即以Annexin V/碘化丙啶(propidium iodide,PI)染色處理。該些處理的細胞先加入濃度1%的胎牛血清蛋白(BSA),其後於加入222.5μl的結合緩衝液(binding buffer)後,直接以10μl的PI以及2.5μl的Annexin V-FITC進行染色,且立即將反應移至低溫且無光的環境下作用10分鐘。以流式細胞分析儀及其分析軟體FACSCalibur計算細胞凋亡百分比。 Apoptosis was performed by phosphatidylserine valgus analysis and by using the apoptosis assay kit Annexin V (BD Pharmingen), and the procedure was followed by the accompanying manual. Roughly, the collected HCT116 cells were washed 3 times with PBS, followed by Annexin immediately. V/propidium iodide (PI) staining treatment. The treated cells were first added with 1% fetal bovine serum albumin (BSA), and then directly stained with 10 μl of PI and 2.5 μl of Annexin V-FITC after addition of 222.5 μl of binding buffer. The reaction was immediately transferred to a low temperature and no light environment for 10 minutes. The percentage of apoptosis was calculated by flow cytometry and its analytical software FACSCalibur.

圖1為實驗例二之流式細胞分析儀分析經化合物處理後之 細胞凋亡結果的數據圖。請參考圖1所示,標示「對照組」者為依據相同處理步驟但未加入丁酸鈉或二雙胍之HCT116的凋亡比例,標示「丁酸鈉」或「二甲雙胍」者則分別為依據相同處理步驟但僅加入丁酸鈉或二甲雙胍之HCT116細胞的凋亡比例,而標示「丁酸鈉與二甲雙胍」者則為同時加入兩種化合物。由數據結果明顯可見,在單獨添加丁酸鈉或二甲雙胍時,雖有提高細胞凋亡比例至約20%,相較於對照組提升了約15%,然而,當兩者同時使用時,細胞凋亡比例則提高至約35%,相較於對照組顯著地提升了約30%。因此,由實驗例二可知,含有二甲雙胍與丁酸鈉之醫藥組合物對具有k-Ras基因突變之癌細胞確實有較佳的毒殺效果,造成細胞凋亡的比例上升,且是因為兩者產生協同作用的效果。 Figure 1 is an analysis of the flow cytometer of Experimental Example 2 after compound treatment. A data plot of apoptosis results. Please refer to Figure 1. The "control group" is the percentage of apoptosis of HCT116 based on the same treatment procedure but without sodium butyrate or diterpene. The labeling "sodium butyrate" or "metformin" is the same. The treatment procedure was followed by the addition of sodium butyrate or metformin to the percentage of apoptosis in HCT116 cells, while the labeling "sodium butyrate and metformin" was the simultaneous addition of both compounds. It is obvious from the data results that when sodium butyrate or metformin was added alone, although the percentage of apoptosis was increased to about 20%, it was increased by about 15% compared with the control group. However, when both were used simultaneously, the cells were withered. The proportion of death increased to about 35%, a significant increase of about 30% compared to the control group. Therefore, it can be seen from the experimental example 2 that the pharmaceutical composition containing metformin and sodium butyrate does have a better poisoning effect on cancer cells having a mutation of the k-Ras gene, and the proportion of apoptosis is increased, and both are produced. The effect of synergy.

實驗例三:含有不同最終濃度之二甲雙胍與丁酸鈉的醫藥組合物對癌細胞造成的細胞凋亡效果 Experimental Example 3: Apoptosis effect of cancer cells caused by pharmaceutical compositions containing different final concentrations of metformin and sodium butyrate

依據與實驗例二大致相同的步驟與條件,惟添加不同濃度之丁酸鈉、二甲雙胍、或二甲雙胍與丁酸鈉醫藥組合物處理HCT116細胞。於本實驗例中,為求方便理解,以下將單獨用丁酸鈉處理之HCT116細胞稱為A組、單獨用二甲雙胍處理之HCT116細胞稱為B組、用含有二甲雙胍與丁酸鈉之醫藥組合物處理HCT116細胞稱為C組,其中,A、B及C組又分為七種不同濃度(於本實驗例中,係指最終濃度),整理如下表所示: According to the same steps and conditions as in Experimental Example 2, HCT116 cells were treated with different concentrations of sodium butyrate, metformin, or metformin and sodium butyrate pharmaceutical compositions. In this experimental example, for ease of understanding, the following HCT116 cells treated with sodium butyrate alone are referred to as group A, HCT116 cells treated with metformin alone are referred to as group B, and pharmaceutical compositions containing metformin and sodium butyrate are used. Treatment of HCT116 cells is referred to as group C, in which groups A, B, and C are further divided into seven different concentrations (in this case, the final concentration), as shown in the following table:

圖2為實驗例三之流式細胞分析儀分析經藥劑處理後相對 於對照組之存活細胞結果的數據圖。請參考圖2所示,不同圖案內容的長條圖分別代表A、B、C組,X軸的數字表示添加不同濃度之藥劑(如上表所示),Y軸則表示以流式細胞分析儀及其分析軟體FACSCalibur計算各組及對照組的存活細胞數後,將各組存活細胞數相對於對照組存活細胞數的比值。 Figure 2 is a flow cytometer analysis of the third experimental example. Data plot of surviving cell results in the control group. Please refer to Figure 2, the bar graphs of different pattern contents represent A, B, and C groups respectively. The X-axis numbers indicate the addition of different concentrations of the drug (as shown in the above table), and the Y-axis indicates the flow cell analyzer. And the analysis software FACSCalibur calculated the number of viable cells in each group and the control group, and the ratio of the number of surviving cells in each group to the number of viable cells in the control group.

由數據結果明顯可見,丁酸鈉與二甲雙胍的組合可使具有 k-Ras基因突變之HCT116細胞的存活情形下降,毒殺細胞的效果較強(細胞凋亡的比例較高),優於各別使用的組別,尤其以0.375至3mM丁酸鈉與1至8mM二甲雙胍的組合效果更佳,可見含有二甲雙胍與丁酸鈉之醫藥組合物,可因為協同作用,對於具有K-Ras基因突變之癌細胞產生較佳的治療效果。 It is apparent from the data that the combination of sodium butyrate and metformin can have The survival of HCT116 cells with mutations in the k-Ras gene decreased, and the effect of killing cells was stronger (the proportion of apoptosis was higher), which was superior to the respective groups used, especially 0.375 to 3 mM sodium butyrate and 1 to 8 mM. The combination effect of metformin is better, and a pharmaceutical composition containing metformin and sodium butyrate can be seen to have a better therapeutic effect on cancer cells having a K-Ras gene mutation due to synergistic action.

上述之實驗例係以二甲雙胍與丁酸鈉的重量百分比為4:1 (體積莫耳濃度比約2.67:1)為例,然,本發明不以此為限。於其他實驗例中,二甲雙胍與丁酸鈉合併使用的劑量可以是,16mM二甲雙胍及其2倍連續稀釋7次的各個濃度,與50mM丁酸鈉及其2倍連續稀釋7次的各個濃度的任意搭配組合使用。詳細可參照下表所示: The above experimental example is exemplified by a weight ratio of metformin to sodium butyrate of 4:1 (volume molar ratio of about 2.67:1), but the invention is not limited thereto. In other experimental examples, the dose of metformin combined with sodium butyrate may be any concentration of 16 mM metformin and its 2-fold serial dilution of 7 times, and each concentration of 50 mM sodium butyrate and its 2-fold serial dilution 7 times. Use in combination with. The details can be seen in the table below:

換言之,二甲雙胍與丁酸鈉合併使用之劑量可為上表所列之各個濃度劑量的任意搭配使用,舉例而言,16mM二甲雙胍可分別與50、25、12.5、6.24、3.12、1.56或0.78mM丁酸鈉合併使用(其餘組合方式,於此不加贅述)。其同樣對於具有K-ras基因突變之癌症或腫瘤患者,提供了改善或增進治療的功效,故本發明不限定二甲雙胍與丁酸鈉的重量百分比需為4:1。 In other words, the dose of metformin combined with sodium butyrate can be used in any combination of the various concentration doses listed above. For example, 16 mM metformin can be combined with 50, 25, 12.5, 6.24, 3.12, 1.56 or 0.78 mM, respectively. Sodium is used in combination (the remaining combinations are not mentioned here). It also provides an effect of improving or enhancing the treatment for a cancer or tumor patient having a K-ras gene mutation, so the present invention does not limit the weight percentage of metformin to sodium butyrate to 4:1.

以上所述僅為舉例性,而非為限制性者。任何未脫離本發明之精神與範疇,而對其進行之等效修改或變更,均應包含於後附之申請專利範圍中。 The above is intended to be illustrative only and not limiting. Any equivalent modifications or alterations to the spirit and scope of the invention are intended to be included in the scope of the appended claims.

Claims (8)

一種治療一具有K-ras基因突變之癌症患者的醫藥組合物,其包括一治療有效量之二甲雙胍與一治療有效量之丁酸鈉,且當投以該醫藥組合物予該癌症患者,該二甲雙胍與該丁酸鈉提供合併治療之效果,其中該二甲雙胍與該丁酸鈉的重量百分比為4:1。 A pharmaceutical composition for treating a cancer patient having a mutation in the K-ras gene, comprising a therapeutically effective amount of metformin and a therapeutically effective amount of sodium butyrate, and when administering the pharmaceutical composition to the cancer patient, the metformin The effect of the combined treatment is provided with the sodium butyrate, wherein the weight percentage of the metformin and the sodium butyrate is 4:1. 如申請專利範圍第1項所述之醫藥組合物,其中該癌症患者包括肺癌、胰臟癌、直腸癌或大腸癌患者。 The pharmaceutical composition according to claim 1, wherein the cancer patient comprises a patient with lung cancer, pancreatic cancer, rectal cancer or colorectal cancer. 如申請專利範圍第1項所述之醫藥組合物,其中該二甲雙胍係與該丁酸鈉協同作用以治療該癌症患者。 The pharmaceutical composition of claim 1, wherein the metformin is synergistic with the sodium butyrate to treat the cancer patient. 如申請專利範圍第1項所述之醫藥組合物,其中該二甲雙胍的劑量係介於130至1,000毫克之間,且該丁酸鈉的劑量係介於40至300毫克之間。 The pharmaceutical composition according to claim 1, wherein the metformin is administered in a dose between 130 and 1,000 mg, and the sodium butyrate is in a dose between 40 and 300 mg. 一種治療一具有K-ras基因突變之癌症患者的醫藥套組,其包括:一治療有效量之二甲雙胍與一第一醫藥上可接受之載體、稀釋劑或賦形劑;以及一治療有效量之丁酸鈉與一第二醫藥上可接受之載體、稀釋劑或賦形劑,其中,該醫藥套組施予該癌症患者後,該二甲雙胍與該丁酸鈉提供合併治療之效果;以及該二甲雙胍與該丁酸鈉的重量百分比為4:1。 A pharmaceutical kit for treating a cancer patient having a mutation in the K-ras gene, comprising: a therapeutically effective amount of metformin and a first pharmaceutically acceptable carrier, diluent or excipient; and a therapeutically effective amount Sodium butyrate and a second pharmaceutically acceptable carrier, diluent or excipient, wherein the medical kit is administered to the cancer patient, the metformin and the sodium butyrate provide a combined treatment effect; and the metformin The weight percentage with sodium butyrate is 4:1. 如申請專利範圍第5項所述之醫藥套組,其中該癌症患者包括肺癌、胰 臟癌、直腸癌或大腸癌患者。 The medical kit of claim 5, wherein the cancer patient comprises lung cancer and pancreas Patients with dirty, rectal or colorectal cancer. 如申請專利範圍第5項所述之醫藥套組,其中該二甲雙胍係與該丁酸鈉協同作用以治療該癌症患者。 The medical kit of claim 5, wherein the metformin is synergistic with the sodium butyrate to treat the cancer patient. 如申請專利範圍第5項所述之醫藥套組,其中該二甲雙胍的劑量係介於130至1,000毫克之間,且該丁酸鈉的劑量係介於40至300毫克之間。 The pharmaceutical kit of claim 5, wherein the metformin has a dosage of between 130 and 1,000 mg and the sodium butyrate is between 40 and 300 mg.
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