KR20160111609A - Composition for treating gastric ulcer comprising omeprazole metabolite made by converting enzyme Cytochrome P450 - Google Patents
Composition for treating gastric ulcer comprising omeprazole metabolite made by converting enzyme Cytochrome P450 Download PDFInfo
- Publication number
- KR20160111609A KR20160111609A KR1020150036428A KR20150036428A KR20160111609A KR 20160111609 A KR20160111609 A KR 20160111609A KR 1020150036428 A KR1020150036428 A KR 1020150036428A KR 20150036428 A KR20150036428 A KR 20150036428A KR 20160111609 A KR20160111609 A KR 20160111609A
- Authority
- KR
- South Korea
- Prior art keywords
- omeprazole
- metabolite
- gastric ulcer
- present
- composition
- Prior art date
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
Abstract
Description
본 발명은 시토크롬 P450 효소전환기술을 이용하여 제조한 오메프라졸 대사체를 이용한 위궤양 치료용 조성물에 관한 것이다.
The present invention relates to a composition for treating gastric ulcer using an omeprazole metabolite prepared using cytochrome P450 enzyme conversion technology.
위궤양은 스트레스, 흡연, 알코올, 영양결핍, 감염 및 비스테로이드성 항염제(Nonsteroidal anti-inflammatory drugs:NSAIDs)의 남용에 의해 유발되는 질병이며, 그 원인은 앞서 말한 유발물질들로 인한 직간접적인 위벽 손상으로 인해 위 내부 pH의 감소, 점막 보호물질과 공격인자들의 균형파괴, 헬리코박터에 의한 감염 등으로 알려져 있다. 위궤양은 단순히 부어오르는 등의 경미한 염증반응을 넘어 출혈, 발적, 천공 등의 현상이 일어나 위 내벽이 허물어진 정도가 심할 때로 정의하며 맵고 짠 음식, 뜨거운 음식 등을 섭취할 때 발생빈도가 높아지는 것으로 알려져 있다. 소화성궤양의 발생 빈도에 대하여 국내의 조사는 충분치 않으나 국제적으로 연간 발생률은 총 인구의 0.1 ~ 0.19%이며 연간 유병률이 0.12 ~ 1.5%로 감소하였다. 이는 서양에서 20세기 중반 냉장고가 개발되면서 소화기에 관여하는 균이 상당부분 감소한 영향이 있다고 생각되나 평균수명의 대폭적인 증가와 식습관 문화, 그리고 항생제 남용으로 궤양발생을 증가시킬 수 있는 강한 요인이 여전히 남아있다. The gastric ulcer is a disease caused by the abuse of stress, smoking, alcohol, malnutrition, infection and nonsteroidal anti-inflammatory drugs (NSAIDs), the cause of which is either direct or indirect, Due to a decrease in stomach internal pH, destruction of mucosal protective materials and attack factors, and infection by Helicobacter pylori. The gastric ulcer is defined as a severe inflammatory reaction such as swelling, hemorrhage, eruption, perforation, etc., and the upper internal wall of the stomach is severely broken. It is known that the frequency of ingestion of spicy foods and hot foods is increased have. Domestic surveillance for the incidence of peptic ulcer is not sufficient, but internationally the annual incidence is 0.1 to 0.19% of the total population and the annual prevalence has decreased to 0.12 to 1.5%. This is due to the fact that the development of mid-20th century refrigerators in the West has contributed to a considerable reduction in the number of bacteria involved in the digestive system, but the significant increase in life expectancy, eating habits, and antibiotics abuse are still strong factors have.
위궤양의 경우, 염증이 일어나는 메커니즘이 많이 알려져 있다. 많은 경우, Helicobacter pylori가 관여하고 있으며, 위궤양을 발병시키는 요인은 다양하지만 증상은 비슷하므로, 그에 따른 치료법은 프로톤펌프저해제(proton pump inhibitor) 같은 위 안쪽 pH를 조절해 줄 수 있는 약품들이 많이 개발되어 있다. 그러나 염증에 관한 호르몬과 사이토카인(cytokine)의 작용이 다양한 신호전달체계를 가지고 있어 이를 조절하고 통증을 완화시키는 과정에서 부작용이 따르는 약품들이 많기에 사용에 주의를 필요로 하는 경우가 많다.In the case of gastric ulcers, the mechanisms by which inflammation occurs are well known. In many cases, Helicobacter pylori is involved, and the factors causing gastric ulcer are various, but the symptoms are similar. Therefore, there are many drugs that can regulate the gastric pH such as proton pump inhibitor have. However, many hormones and cytokines have a variety of signal transduction systems, and many of these drugs have side effects in the process of controlling and relieving pain.
본 발명은 이러한 기존의 부작용이 따르는 약품의 한계점을 개선하기 위하여, 자연계에 존재하는 시토크롬 P450 효소를 이용하여 오메프라졸(racemic omeprazole) 및 에소메프라졸(esomeprazole: 오메프라졸의 S-거울상 이성질체) 대사체를 제조하여 기존의 물질들보다 이들의 대사체(hydroxylated metabolite)들이 더 강한 효과를 갖는 오메프라졸 대사체를 도출하여 본 발명을 완성하기에 이르렀다. 현재까지 시토크롬 P450으로 오메프라졸의 대사체를 제조하여 그의 효능에 대한 결과는 전혀 알려진 바 없다.
In order to overcome the limitations of such conventional drugs, the present invention uses a cytochrome P450 enzyme present in nature to produce a racemic omeprazole and an esomeprazole (S-enantiomer) metabolite of omeprazole The present inventors have completed the present invention by deriving an omeprazole metabolite having a stronger effect by hydroxylated metabolites than the existing substances. Until now, the metabolism of omeprazole has been produced with cytochrome P450 and its effect on its efficacy has not been known at all.
본 발명의 목적은 기존의 오메프라졸 대비 효소전환방법으로 제조된 오메프라졸 대사체의 개선된 염증완화 효능을 가지는 위궤양 예방 또는 치료용 조성물을 제공하고자 한다.It is an object of the present invention to provide a composition for preventing or treating gastric ulcer having an improved inflammation-relieving effect of an omeprazole metabolite prepared by an enzyme conversion method compared to conventional omeprazole.
본 발명은 또 다른 목적으로는 기존 오메프라졸(omeprazole의 racemic mixture 또는 S-이성질체) 대비 우수한 염증완화 또는 치료효과를 가지는 오메프라졸 대사체를 포함하는 위궤양 예방 또는 개선용 건강식품을 제공하고자 한다.
Another object of the present invention is to provide a health food for preventing or ameliorating gastric ulcer comprising an omeprazole metabolite having excellent inflammation relieving or therapeutic effect as compared with conventional omeprazole (racemic mixture or S-isomer of omeprazole).
본 발명은 오메프라졸로부터 생산된 하기 화학식 1 또는 2로 표시되는 오메프라졸 대사체를 유효성분으로 포함하는 위궤양 치료용 조성물을 제공한다.The present invention provides a composition for treating gastric ulcer comprising, as an active ingredient, an omeprazole metabolite represented by the following
[화학식 1] [Chemical Formula 1]
[화학식 2] (2)
본 발명의 오메프라졸의 대사체는 상기 거울상 이성질체인 거울상 이성질체(enantiomer)인 5'-히드록실 S-오메프라졸(5’-OH S-omeprazole) 및 5'-히드록실 R-오메프라졸(5'-OH R-omeprazole)을 50:50으로 함유하고 있는 라세미체(racemate) 오메프라졸의 대사체인 것을 특징으로 하는 위궤양 치료용 조성물을 제공할 수 있으나 이에 한정하는 것은 아니다.Metabolites of the present invention are omeprazole enantiomer (enantiomer) of the enantiomers of the 5'-hydroxyl S - omeprazole (5'-OH S -omeprazole) and 5'-hydroxyl R - omeprazole (5'-OH R -omeprazole) at a ratio of 50:50. The present invention is not limited to the composition for treating gastric ulcer.
본 발명의 시토크롬 P450 효소(CYP102A1)를 이용한 효소전환반응으로 제조된 오메프라졸 대사체인 것을 예시할 수 있으나 이에 한정하는 것은 아니다. But the present invention is not limited to the omeprazole metabolism produced by the enzyme conversion reaction using the cytochrome P450 enzyme (CYP102A1) of the present invention.
본 발명의 오메프라졸 대사체는 전체 조성물 총 중량에 대해 0.01 내지 1 중량%를 포함하는 것을 특징으로 하는 위궤양 치료용 조성물을 예시할 수 있으나 이에 한정하는 것은 아니다.The omeprazole metabolite of the present invention may include, but is not limited to, a composition for treating gastric ulcer, which comprises 0.01 to 1% by weight based on the total weight of the total composition.
본 발명은 상기 조성물을 포함하는 위궤양 예방 또는 개선용 건강식품을 제공한다.
The present invention provides a health food for preventing or improving gastric ulcer comprising the composition.
본 발명은 기존물질인 오메프라졸을 시토크롬 P450 효소전환반응으로 제조한 오메프라졸 대사체를 포함하는 위궤양 치료용 조성물에 관한 것으로 상기 위궤양 치료용 조성물을 위궤양을 유발시킨 동물모델에 투여함으로써 기존물질인 오메프라졸 대비 본 발명으로 제조한 오메프라졸 대사체의 경우 보다 개선된 염증완화 효과를 확인하였다. The present invention relates to a composition for treating gastric ulcer comprising an omeprazole metabolite produced by the conversion of omeprazole, which is a conventional substance, by cytochrome P450 enzyme. The composition for treating gastric ulcer is administered to an animal model of gastric ulcer, It was confirmed that the omeprazole metabolite prepared according to the present invention had an improved inflammation mitigating effect.
나아가 본 발명의 효소전환방법으로 제조된 오메프라졸 대사체가 염증반응에 관여하는 인터루킨과 종양괴사인자의 발현에서 기존의 오메프라졸보다 더욱 효과적으로 발현이 조절되고, 활성산소에 관련된 유전자 역시 기존물질보다 발현정도가 크게 나타남을 확인함으로써 기존물질의 부작용을 최소화하면서 보다 우수한 염증완화 효능을 가진 위궤양 치료제로 사용할 수 있으며 보다 획기적인 위궤양 치료제 개발에 기여할 수 있다.
Further, in the expression of the interleukin and tumor necrosis factor involved in the inflammatory reaction, the expression of the omeprazole metabolite produced by the enzyme conversion method of the present invention is more effectively regulated than that of the existing omeprazole, and the expression of the gene related to reactive oxygen species It can be used as a therapeutic agent for gastric ulcer with better anti inflammatory effect while minimizing adverse effects of existing substances and contributing to development of a more innovative gastric ulcer treatment agent.
도 1은 본 발명에 따른 라세믹혼합물인 오메프라졸을 구성하는 S-omeprazole 및 R-omeprazole의 화학구조식 및 시토크롬 P450을 사용하여 제조한 각각 이성질체의 대사체(5'-OH S-omeprazole 및 5'-OH R-omeprazole)의 화학구조를 나타낸 그림이다.
도 2는 본 발명에 따른 오메프라졸 대사체의 HPLC 크로마토그램을 보여주는 것이다(a: 라세미체, b: 5'-OH R-omeprazole, c: 5'-OH S-omeprazole).
도 3은 본 발명에 따른 오메프라졸 대사체의 LC-MS 용출 프로파일을 보여주는 그림이다. (A: CYP102A1 돌연변이체(R47L/F87V/L188Q), B: human CYP2C19 반응에 의한 5'-OH omeprazole 생성을 나타냄).
도 4는 본 발명에 따른 동물모델의 실험스케줄을 나타낸 표(A) 및 위궤양 동물모델의 위 조직을 절편으로 만들어 H&E 염색한 사진(B)의 결과다.
도 5는 본 발명에 따른 위궤양 동물모델의 대변에서 추출한 면역글로불린 A의 레벨을 그래프로 나타낸 결과이다.
도 6은 본 발명에 따른 real-time PCR로 기존 오메프라졸(racemic mixture, 즉 S 및 R-omeprazole의 50:50 혼합물)와 R 및 S-이성질체의 오메프라졸 대사체를 투여하였을 때 염증관련 유전자의 발현 변화를 보여주는 결과이다.
도 7은 acridine orange로 위조직 세포를 염색하여 각 군간 pH의 변화를 보여주는 그래프이다. FIG. 1 is a graph showing the relationship between the isomeric metabolites (5'-OH S -omeprazole and 5'-OH) prepared using the chemical structural formula of S -omeprazole and R -omeprazole and cytochrome P450 constituting the racemic mixture of the present invention, OH R -omeprazole).
FIG. 2 shows HPLC chromatograms of omeprazole metabolites according to the present invention (a: racemate, b: 5'-OH R -omeprazole, c: 5'-OH S -omeprazole).
Figure 3 shows the LC-MS dissolution profile of the omeprazole metabolite according to the invention. (A: CYP102A1 mutant (R47L / F87V / L188Q), B: 5'-OH omeprazole production by human CYP2C19 reaction).
FIG. 4 is a table (A) showing the experimental schedule of the animal model according to the present invention and a result of the H & E stained image (B) of the stomach tissue of the gastric ulcer animal model.
FIG. 5 is a graph showing the levels of immunoglobulin A extracted from the faeces of a gastric ulcer animal model according to the present invention.
FIG. 6 shows the expression of inflammation-related genes when real-time PCR according to the present invention was administered with the omeprazole metabolites of the existing omeprazole (a 50:50 mixture of S and R -omeprazole) and the R and S -isomer .
FIG. 7 is a graph showing changes in pH between the groups by staining stomach tissue with acridine orange. FIG.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
오메프라졸은 투여 후 사람의 간에서 시토크롬 P450 효소들인 CYP2C19과 CYP3A4에 의해서 대사된다. 간에 주로 존재하는 CYP2C19과 CYP3A4 효소들의 발현량과 활성은 개인에 따라 차이가 있으며, 이에 따라 약물상호작용(drug-drug interaction)의 부작용을 나타내는 원인이 된다. 특히 오메프라졸로부터 5'-히드록시 오메프라졸(5'-OH omeprazole)을 생성하는 CYP2C19은 대표적인 유전적 다형성(genetic polymorphism)을 나타내는 효소이며 CYP3A4는 사람 간에서 많이 발현되면 약물대사에 가장 큰 역할을 수행한다. 대사체를 약물로 사용할 경우, 이러한 약물상호작용 및 개인차를 회피할 수 있는 좋은 수단이 될 수 있다.Omeprazole is metabolized by the cytochrome P450 enzymes CYP2C19 and CYP3A4 in the human liver after administration. The abundance and activity of the CYP2C19 and CYP3A4 enzymes, which are mainly present in the liver, differ from person to person and thus cause side effects of drug-drug interaction. In particular, CYP2C19, which produces 5'-hydroxy omeprazole from omeprazole, is a representative genetic polymorphism, and CYP3A4 plays a major role in drug metabolism when expressed in human liver . When a metabolite is used as a drug, it can be a good means to avoid such drug interactions and individual differences.
본 발명자는 기존 위궤양 치료제의 부작용이 개선된 위궤양 치료제 개발에 유용한 물질을 개발하기 위하여 지속적인 연구를 수행하던 중 기존의 프로톤펌프 저해제(proton pump inhibitor)로 알려져 있는 오메프라졸을 효소전환기법으로 제조한 오메프라졸의 대사체가 기존의 오메프라졸에 비해 염증완화효과가 우수하며 특히, 기존 치료제보다 더 뛰어난 위궤양 치료제로서의 효과가 있음을 최초로 밝혀내어 본 발명을 완성하게 되었다. The inventors of the present invention conducted a continuous study to develop a substance useful in the development of a gastric ulcer treatment drug with improved side effects of existing gastric ulcer treatment drugs, and found that the use of omeprazole, which is known as a proton pump inhibitor, The inventors of the present invention have found that the metabolism is superior to the conventional omeprazole in the inflammation-relieving effect and is more effective as a therapeutic agent for gastric ulcer than the conventional therapeutic agent.
본 발명의 목적을 달성하기 위하여 본 발명은 오메프라졸로부터 생산된 하기 화학식 1 또는 2로 표시되는 오메프라졸 대사체를 유효성분으로 포함하는 위궤양 치료용 조성물을 제공한다.In order to accomplish the object of the present invention, there is provided a composition for treating gastric ulcer comprising, as an active ingredient, an omeprazole metabolite represented by the following
[화학식 1] [Chemical Formula 1]
[화학식 2] (2)
보다 구체적으로는 본 발명의 상기 오메프라졸 대사체는 거울상 이성질체(enantiomer)인 화학식 1로 표시되는 5'-히드록실 S-오메프라졸(5'-OH S-omeprazole) 및 화학식 2로 표시되는 5'-히드록실 R-오메프라졸(5'-OH R-omeprazole)을 50:50으로 함유하고 있는 라세미체(racemate)의 대사체인 것을 특징으로 할 수 있으나 이에 한정하는 것은 아니다. More specifically, the omeprazole metabolite of the present invention is an enantiomer of 5'-hydroxyl S -omeprazole (5'-OH S -omeprazole) represented by the formula (1) and 5'- lock R - omeprazole (5'-OH R -omeprazole) to be characterized in that the metabolite of the racemate (racemate), which contains a 50: 50, but is not limited to this.
상기 오메프라졸 대사체는 상기 S-오메프라졸 및 R-오메프라졸로부터 각각 효소전환기술을 통해 제조할 수 있으나 이에 한정하는 것은 아니다.The omeprazole metabolite may be prepared from the S -omeprazole and R -omeprazole by an enzyme conversion technique, but is not limited thereto.
상기 오메프라졸 대사체는 동일한 기술분야에서 공지된 방법으로 제조하는 것이라면 제한하지 않지만, 본 발명의 일 실시예로는 오메프라졸로부터 시토크롬 P450 효소(CYP102A1)를 이용하여 효소전환기술을 통해 오메프라졸의 대사체를 제조할 수 있으나 이에 한정하는 것은 아니다. Although the omeprazole metabolite is not limited as long as it is produced by a method known in the art, one embodiment of the present invention is a method for producing a metabolite of omeprazole through an enzyme conversion technique using omeprazole using cytochrome P450 enzyme (CYP102A1) But is not limited to this.
구체적으로는, 상기 S-오메프라졸(S-omeprazole)의 5'-carbon 위치가 시토크롬 P450 효소반응에 의해 히드록실화된 5'-OH S-omeprazle(esomeprazole)을 제조할 수 있으나 이에 한정하는 것은 아니다. Specifically, the S - omeprazole is 5'-position carbon of the (S- omeprazole), but can produce a hydroxylated 5'-OH S -omeprazle (esomeprazole) by the cytochrome P450 enzyme reaction is not limited to: .
또한, 상기 R-오메프라졸(R-omeprazole)의 5'-carbon 위치가 시토크롬 P450 효소반응에 의해 히드록실화된 5'-OH R-omeprazole(5'-OH racemic omeprazole) 을 제조할 수 있다(대한민국 특허번호 10-1348984). Furthermore, the R - the 5'-carbon position of omeprazole (R -omeprazole) can be prepared hydroxylated 5'-OH R -omeprazole (5'- OH racemic omeprazole) by the cytochrome P450 enzyme (Republic of Korea Patent No. 10-1348984).
즉, 상기 오메프라졸의 대사체는 상기 P450 효소(CYP102A1 및 CYP102A1 돌연변이체)를 이용한 효소전환기술을 통해 오메프라졸이 5'-히드록실화된 것으로 오메프라졸 대사체를 제조할 수 있으며 그 구조는 상기 화학식 1 또는 2로 표시한다. That is, the metabolite of omeprazole can be prepared by the 5'-hydroxylation of omeprazole through an enzyme conversion technique using the P450 enzyme (CYP102A1 and CYP102A1 mutants) 2.
상기 P450 효소(CYP10A1)는 오메프라졸을 산화시켜 5'-히드록실산물로 선택적으로 전환시키는 반응에 보다 안정적이고 효율적으로 수행할 수 있는 효소전환반응에서 가장 효과적인 촉매역할을 할 수 있어 가장 바람직하다. The P450 enzyme (CYP10A1) is most preferable because it can act as a most effective catalyst in the enzyme conversion reaction which can more stably and efficiently perform the reaction of selectively oxidizing omeprazole to 5'-hydroxyl product.
상기 CYP102A1 돌연변이체는 야생형 CYP102A1(표 1)의 아미노산 치환위치와 치환되는 아미노산이 R47L/F87V/L188Q인 것을 사용할 수 있으나 이에 한정하는 것은 아니다. The CYP102A1 mutant may be any of the amino acid substitution positions of wild-type CYP102A1 (Table 1) and substituted amino acids R47L / F87V / L188Q, but the present invention is not limited thereto.
[표 1][Table 1]
본 발명의 일 양태로는 상기 오메프라졸 대사체는 전체 조성물 총 중량에 대해 0.01 내지 1중량%를 포함하는 것을 특징으로 하는 위궤양 치료용 조성물을 제공할 수 있다. 상기 범위의 오메프라졸 대사체로 조성물을 제공할 시 가장 효과적인 위궤양 치료효과를 볼 수 있다.In one embodiment of the present invention, the omeprazole metabolite is 0.01 to 1% by weight based on the total weight of the total composition. When the composition is provided with the above-mentioned range of omeprazole metabolism, the most effective treatment effect of gastric ulcer can be seen.
본 발명의 일 양태로는 오메프라졸 대사체를 포함하는 위궤양 치료용 조성물에 있어서, 본 발명의 위궤양 치료제로서 기존의 위궤양 치료물질인 오메프라졸보다 우수한 효능을 가진 위궤양 치료제 조성물을 제공할 수 있다. 본 발명에 따른 시토크롬 P450 효소전환반응으로 제조된 상기 오메프라졸 대사체를 위궤양을 발병시킨 동물 모델에 사용함으로써, 기존 시판 중인 오메프라졸에 비해 보다 우수한 위궤양 치료물질로 사용될 수 있다.In one embodiment of the present invention, a gastric ulcer therapeutic composition having superior efficacy to omeprazole, which is a conventional gastric ulcer treatment material, can be provided as a gastric ulcer treatment composition of the present invention in a composition for treating gastric ulcer comprising an omeprazole metabolite. The omeprazole metabolite prepared by the cytochrome P450 enzyme conversion reaction according to the present invention can be used as an excellent gastric ulcer treatment material in comparison with existing commercially available omeprazole by using it in an animal model in which a gastric ulcer has developed.
특히, 본 발명의 효소전환반응으로 생산된 오메프라졸 대사체 중 S-오메프라졸 대사체(5'-OH S-omeprazole)의 경우 위궤양 동물모델에서 보다 우수한 항염증 효과를 가진다. 또한, 상기 S-오메프라졸 대사체(5'-OH S-omeprazole)는 S omeprazole에 비해 우수한 약리적 효과를 가지는 것은 물론, 높은 생체이용률에 의한 높은 체내 흡수율을 가짐으로써 기존 치료제에 비해 보다 개선된 위궤양 치료제로써의 효과를 가진다. In particular, the S -omeprazole metabolite (5'-OH S- omeprazole) among the omeprazole metabolites produced by the enzyme conversion reaction of the present invention has a superior anti-inflammatory effect in the gastric ulcer animal model. Further, the S - omeprazole metabolites (5'-OH S- omeprazole) is a peptic ulcer therapeutic agents more improved than the existing therapeutic agents by having high water absorption by the body, as well as, high bioavailability having the excellent pharmacological effect than the S omeprazole .
이에 본 발명에 따른 효소전환기술로 오메프라졸로부터 제조된 오메프라졸 대사체 중 S-오메프라졸 대사체(5'-OH S-omeprazole)을 유효성분을 포함하는 위궤양 치료용 조성물의 경우 기존 오메프라졸 대비 가장 우수한 항염증 효과를 가질 수 있어 바람직하다. Therefore, in the case of a composition for treating gastric ulcer containing an active ingredient, S -omeprazole metabolite (5'-OH S- omeprazole) among the omeprazole metabolites prepared from omeprazole by the enzyme conversion technology according to the present invention, So that it is preferable.
구체적으로는, 본 발명의 일 실시예로서 인도메타신(indomethacin)이라는 비스테로이드성 항염증제(NSAID)로 유도한 위궤양 마우스 모델에서 기존의 오메프라졸과 본 발명의 화학식 1 또는 2로 표시되는 오메프라졸 대사체를 투여하여 기존 오메프라졸 대비 본 발명의 오메프라졸 대사체의 현저한 위궤양 완화효과를 비교실험을 수행하였다. 본 발명의 효소반응으로 제조한 오메프라졸 대사체를 처리함에 따라 발병한 위 조직의 항염증 치료효과를 조직면역학적으로 비교관찰하였다. 그 결과, 본 발명에 따라 제조한 오메프라졸 대사체를 처리함에 따라 유도된 위 조직의 항염증 효과가 기존물질 대비 매우 우수한 항염증 효과를 가지며, 염증으로 인해 변화된 면역글로불린의 회복속도가 기존물질보다 더 우수한 결과를 통해 오메프라졸 대사체의 염증회복능이 보다 우수한 효과를 가진다.Specifically, as an example of the present invention, in a model of gastric ulcer induced by a non-steroidal anti-inflammatory drug (NSAID) called indomethacin, conventional omeprazole and an omeprazole metabolite represented by
나아가, 본 발명의 효소전환방법으로 제조된 오메프라졸 대사체는 기존의 오메프라졸에 비해 염증반응에 관여하는 인터류킨과 종양괴사인자의 발현이 더욱 효과적으로 조절되고, 활성산소에 관련된 유전자 역시 기존보다 발현정도가 크게 나타난다.Furthermore, the metabolism of omeprazole produced by the enzyme conversion method of the present invention is more effective in controlling the expression of interleukin and tumor necrosis factor, which are involved in the inflammatory response, than that of the conventional omeprazole, and the expression of the gene related to reactive oxygen species appear.
본 발명의 위궤양 치료용 조성물이 약제로 이용되기 위해서는 약제학적 분야에서 공지된 방법에 의해 제조될 수 있으며, 유효성분으로서의 본 발명의 물질 이외에 약제학적으로 허용 가능한 담체를 포함할 수 있다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용가능한 담체는 재제 시에 통상적으로 이용되는 것으로서 락토스, 덱스트로스, 수크로스, 알기네이트, 젤라틴, 규산 칼슘, 폴리비닐피롤리돈, 셀룰로오스, 물, 메틸 셀룰로오스, 메틸히드록시벤조에이트 등을 포함할 수 있으나 이에 한정하는 것은 아니다. 또한, 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 향미제, 유화제, 현탁제, 보존제 등의 첨가제를 추가로 포함할 수 있다. In order to use the composition for treating gastric ulcer of the present invention as a medicine, it may be prepared by a method known in the pharmaceutical field and may include a pharmaceutically acceptable carrier other than the substance of the present invention as an active ingredient. The pharmaceutically acceptable carrier to be contained in the pharmaceutical composition of the present invention includes those conventionally used in reconstitution such as lactose, dextrose, sucrose, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, Methylcellulose, methylhydroxybenzoate, and the like, but are not limited thereto. In addition, the pharmaceutical composition of the present invention may further include additives such as lubricants, wetting agents, flavoring agents, emulsifying agents, suspending agents, preservatives, etc. in addition to the above components.
본 발명의 약제학적 조성물은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 또는 부형제를 이용하여 제제화 됨으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제, 캅셀제 또는 주사제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. The pharmaceutical composition of the present invention may be prepared in a unit dose form by being formulated using a pharmaceutically acceptable carrier or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs Into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or milks, or they may be in the form of excipients, powders, granules, tablets, capsules or injectables, and may additionally contain dispersing or stabilizing agents.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여방식, 환자의 연령, 체중, 성, 건강상태, 질병 증상의 정도, 음식, 투여시간, 투여방법 및 반응 감응성과 같은 요인들에 의해 적절히 선택될 수 있으며, 바람직하게는 성인기준 1일 당 0.01 ~ 100 mg이 투여될 수 있다. 상기 투여방법으로는 동일한 기술분야에서 사용가능한 방법이라면 모두 제한하지 않고 사용할 수 있으나 경구투여하는 것이 가장 바람직하다. A suitable dosage of the pharmaceutical composition of the present invention is appropriately determined depending on factors such as the formulation method, the administration method, the age, weight, sex, health condition, disease symptom, food, administration time, administration method and responsiveness of the patient And preferably from 0.01 to 100 mg / day on an adult basis. As the above-mentioned administration method, any method which can be used in the same technical field can be used without limitation, but oral administration is most preferable.
본 발명의 또 다른 양태로 상기 조성물을 포함하는 위궤양 예방 또는 개선용 건강식품을 포함한다.Another aspect of the present invention includes a health food for preventing or ameliorating gastric ulcer comprising the composition.
본 발명은 오메프라졸로부터 생산된 하기 화학식 1 또는 2로 표시되는 오메프라졸 대사체를 유효성분으로 포함하는 위궤양 치료용 조성물을 포함하는 위궤양 예방 또는 개선용 건강식품을 제공함으로써, 위궤양을 예방하고 개선에 효과적인 건강식품으로 섭취하여 그 효능을 더욱 발휘할 수 있어 좋다.The present invention provides a health food for preventing or ameliorating gastric ulcer comprising a composition for treating gastric ulcer comprising an omeprazole metabolite represented by the following
[화학식 1] [Chemical Formula 1]
[화학식 2] (2)
상기 위궤양 예방 또는 개선용 건강식품은 다양한 형태의 식품 첨가제 또는 기능성 식품을 제공할 수 있다. 상기 위궤양 예방 또는 개선용 건강식품으로 예를 들면, 침출차, 액상차, 음료, 발효유, 치즈, 요구르트, 주스, 생균제제 및 건강보조식품 등으로 가공될 수 있으며, 그 외 목적에 따라 다양한 식품 첨가제의 형태로 사용될 수 있다. 또한, 건강기능성 식품의 제형은 용액, 유화물, 점성형 혼합물, 타블렛, 분말, 환 등의 다양한 형태일 수 있으며, 이는 단순 음용, 주사투여, 스프레이 방식 또는 스퀴즈 방식 등의 다양한 방법으로 투여될 수 있다.The health food for prevention or improvement of gastric ulcer may provide various types of food additives or functional foods. The health food for prevention or improvement of gastric ulcer can be processed into, for example, an oiled tea, liquid tea, beverage, fermented milk, cheese, yogurt, juice, probiotic agent and health supplement food. Can be used. In addition, the form of the health functional food may be in various forms such as a solution, an emulsion, a viscous mixture, a tablet, a powder, a ring and the like, which can be administered by various methods such as a simple drink, injection, spray or squeeze .
이하, 실시예 및 비교예를 통해 본 발명을 보다 자세하게 설명한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.
Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.
[실시예 1~2] CYP102A1(P450 BM3) 돌연변이체를 이용한 효소전환기술에 의한 오메프라졸 대사체인 5'-OH omeprazole의 제조[Examples 1 to 2] Preparation of 5'-OH omeprazole as an omeprazole metabolite by an enzyme conversion technique using CYP102A1 (P450 BM3) mutant
CYP102A1 돌연변이체(R47L/F87V/L188Q) 효소를 이용하여 오메프라졸을 산화하여 오메프라졸 대사체를 제조하였다(Ryu 등, 2014). 100 mM potassium phosphate 완충액(pH 7.4) 0.25ml에 CYP102A1 50pmol 과 100uM 기질을 넣어 전형적인 steady-state 반응을 시켰다. 반응을 시작하기 위하여 NADPH-생성 시스템(최종 농도: 1 ml 당 10mM glucose 6-phosphate, 0.5mM NADP 및 1IU yeast glucose 6-phosphate dehydrogenase)을 첨가하였다. DMSO로 오메프라졸 20mM 용액을 제조하고, 효소 반응액으로 희석하여 최종 유기용매 농도가 1%(v/v) 이하가 되도록 하였다. Omeprazole metabolites were prepared by oxidizing omeprazole using a CYP102A1 mutant (R47L / F87V / L188Q) enzyme (Ryu et al., 2014). A typical steady-state reaction was performed by adding 50 pmol of CYP102A1 and 100 uM substrate to 0.25 ml of 100 mM potassium phosphate buffer (pH 7.4). To start the reaction, an NADPH-producing system (10 mM glucose 6-phosphate, 0.5 mM NADP and 1 IU yeast glucose 6-phosphate dehydrogenase per 1 ml of final concentration) was added. A 20 mM solution of omeprazole was prepared with DMSO and diluted with the enzyme reaction solution to a final organic solvent concentration of 1% (v / v) or less.
사람 CYP2C19 활성 측정을 위해서는 50pmol CYP2C19, 100pmol NADPH-P450 reductase(CPR), 100pmol 시토크롬 b5 및 45 uM L-알파-디라우로일-sn-글리세롤-3-포스포콜린(L-α-dilauroyl-sn-glycero-3-phosphocholine;DLPC)을 50pmol CYP102A1 대신 사용하였다. 반응액을 37℃에서 30분간 반응시키고 2배의 얼음으로 차갑게 한 디클로로메탄으로 반응을 종결시켰다.
For the measurement of human CYP2C19 activity, 50 pmol CYP2C19, 100 pmol NADPH-P450 reductase (CPR), 100 pmol cytochrome b5 and 45 uM L-alpha-dilauroyl-3-phosphocholine -glycero-3-phosphocholine (DLPC) was used instead of 50 pmol of CYP102A1. The reaction solution was reacted at 37 占 폚 for 30 minutes, and the reaction was terminated with dichloromethane cooled with ice twice.
(1)HPLC 분석(1) HPLC analysis
반응혼합물을 원심분리하여 상등액을 제거하여 버리고 용매를 질소가스 하에서 증발시키고, HPLC로 분석하였다(Ryu 등, 2014). 시료(30ul)를 Gemini C18컬럼(4.6mm×150mm, 5um, Phenomenex, Torrance, CA)에 주입하였다. 이동상은 30% 아세토니트릴을 사용하였다. 이동상 1 ml/min의 속도로 흘려주었고, 용출액을 302nm의 UV로 측정하였다.The reaction mixture was centrifuged to remove supernatant, and the solvent was evaporated under a nitrogen gas and analyzed by HPLC (Ryu et al., 2014). A sample (30 ul) was injected into a Gemini C18 column (4.6 mm x 150 mm, 5 um, Phenomenex, Torrance, Calif.). The mobile phase was 30% acetonitrile. The mobile phase was flowed at a rate of 1 ml / min, and the eluate was measured by UV at 302 nm.
CYP102A1(P450 BM3)이 오메프라졸을 산화할 수 있는지 조사하기 위하여, CYP102A1 효소를 이용하여 오메프라졸의 산화능을 기질의 농도를 100uM로 고정시키고 측정하였다. 그 결과, 도 2의 HPLC 크로마토그램에서도 확인할 수 있듯이, 오메프라졸 대사체(5'-OH omeprazole)를 선택적으로 생산하였음을 나타내고 있다.To investigate the ability of CYP102A1 (P450 BM3) to oxidize omeprazole, the oxidative capacity of omeprazole was measured with CYP102A1 enzyme at a fixed substrate concentration of 100 uM. As a result, as shown in the HPLC chromatogram of FIG. 2, it was shown that omeprazole metabolite (5'-OH omeprazole) was selectively produced.
(a: 라세미체, b: 5'-OH R-omeprazole, c: 5'-OH S-omeprazole)
(a: racemate, b: 5'-OH R -omeprazole, c: 5'-OH S -omeprazole)
(2) LC-MS 분석 및 NMR 분석(2) LC-MS analysis and NMR analysis
CYP102A1 돌연변이체가 생산하는 오메프라졸의 대사산물을 동정하기 위하여, 오메프라졸 및 대사산물의 LC 프로파일과 fragmentation 패턴을 비교하여 LC-MS 분석을 하였다. CYP102A1 돌연변이체 및 사람 CYP2C19를 100uM의 오메프라졸과 NADPH- 생성 시스템의 존재 하에 37℃에서 30분간 반응시켰다. 얼음으로 식힌 2배의 디클로로메탄을 가하여 반응을 종결시켰다. 원심분리 후, 상등액을 제거하여 버리고 유기 용매 층을 질소하에 건조하였다. 반응물을 100ul의 이동상에 볼텍스 믹싱으로 재구성시키고 20초간 소니케이션 하였다. 제조된 용액의 적정량(5ul)을 LC 컬럼에 주입하였다. LC-MS 분석은 LC-MS 소프트웨어를 장착한 쉬마쯔 LCMS-2010 EV 시스템(Shimadzu, Kyoto, Japan)를 사용하여 electro spray ionization(positive) mode에서 실시하였다. 사람 CYP2C19 의해 생성된 대사산물의 총 이온전류(total ion current; TIC) 프로파일 조사하였다. 그 결과, 도 3에서도 확인할 수 있듯이 반응샘플의 mass spectra은 6.200 min(5'-히드록시 오메프라졸), 15.267 min(오메프라졸)에서 피크를 나타내었고, CYP102A1에 의한 5'-히드록실화 산물과 오메프라졸의 mass spectrum은 [M+H]+로 계산하였을 때 각각 362, 346로 관찰되었다. 또한, 상기 반응혼합물의 LC-MS 분석에 의하여 CYP102A1에 의한 5'-히드록실화 오메프라졸(5'-OH omeprazole)의 생산이 확인되었다. CYP102A1 대사산물의 retention time과 fragmentation 패턴이 사람 CYP2C19에 의하여 생산되는 진정 대사산물(authentic metabolites)의 그것과 정확히 일치하는 것을 확인할 수 있었다. In order to identify the metabolites of omeprazole produced by the CYP102A1 mutant, LC-MS analysis was performed by comparing the LC profile and fragmentation pattern of omeprazole and its metabolites. The CYP102A1 mutant and human CYP2C19 were reacted for 30 min at 37 [deg.] C in the presence of 100 [mu] M of omeprazole and an NADPH-producing system. The reaction was quenched by the addition of 2 x dichloromethane cooled to ice. After centrifugation, the supernatant was removed and the organic solvent layer was dried under nitrogen. The reactants were reconstituted in 100 ul mobile phase by vortex mixing and sonicated for 20 seconds. An appropriate amount (5 ul) of the prepared solution was injected into the LC column. LC-MS analysis was performed in an electro spray ionization (positive) mode using a Shimadzu LCMS-2010 EV system (Shimadzu, Kyoto, Japan) equipped with LC-MS software. The total ion current (TIC) profile of metabolites produced by human CYP2C19 was investigated. As a result, as shown in FIG. 3, the mass spectra of the reaction samples showed peaks at 6.200 min (5'-hydroxy-omeprazole) and 15.267 min (omeprazole), and the 5'- hydroxylated products by CYP102A1 and omeprazole The mass spectrum was 362 and 346, respectively, when calculated as [M + H] +. Further, LC-MS analysis of the reaction mixture confirmed the production of 5'-hydroxylated omeprazole (5'-OH omeprazole) by CYP102A1. The retention time and fragmentation patterns of the CYP102A1 metabolites were exactly the same as those of the authentic metabolites produced by human CYP2C19.
상기의 결과들로부터 박테리아 CYP102A1 효소들이 사람 CYP2C19와 동일한 반응을 촉매하여 사람 대사산물인 5'-OH R-omeprazole(실시예 1) 및 5'-OH S-omeprazole(실시예 2)를 생성한다는 것을 확인할 수 있었다(도 2). 사람 P450 기질인 오메프라졸의 산화는 CYP102A1 야생형 및 이의 돌연변이에 의하여 촉매되며, 주요한 대사산물로 히드록실화 산물 즉, 5'-OH 산물이 생성됨을 확인하였고, 상기 생성된 대사산물의 생성은 HPLC 및 LC-MS로 사람 CYP2C19에 의하여 생산되는 산물과 비교함으로써 확인할 수 있었다.
These results suggest that bacterial CYP102A1 enzymes catalyze the same reaction as human CYP2C19 to produce the human metabolites 5'-OH R-omeprazole (Example 1) and 5'-OH S-omeprazole (Example 2) (Fig. 2). The oxidation of omeprazole, a human P450 substrate, was catalyzed by the CYP102A1 wild type and its mutants, confirming the formation of the hydroxylated product, the 5'-OH product, as the major metabolite, and the formation of the resulting metabolite was confirmed by HPLC and LC -MS as compared to the product produced by human CYP2C19.
[실시예 3] Indomethacin을 이용한 위궤양 유발 동물모델 제작[Example 3] Production of gastric-induced animal model using indomethacin
인도메타신(Indomethacin)은 진통제로 알려진 약품이나 궤양을 유발하는 부작용을 가진 약품으로써 indomethacin을 최초 1회 경구투여하여(8mg/kg)을 위궤양을 유발시켰다. Indomethacin 투여 3일전부터 매일 실시예 1~2로 생산된 오메프라졸 대사체를 경구투여(3mg/kg)하여 1주 후 마우스를 경추탈골시켜 희생시킨 후 궤양정도를 조직병리학적으로 측정하였다.
Indomethacin is a drug known to be an analgesic drug or a drug with side effects that causes ulcers. The first oral administration of indomethacin (8 mg / kg) induces gastric ulcers. The omeprazole metabolites produced in Examples 1 and 2 were orally administered (3 mg / kg) daily from
[실시예 4] 위궤양 동물모델에서 R 및 S-오메프라졸과 효소전환 오메프라졸 대사체의 항염증 효능의 조직학적 비교분석[Example 4] Histological comparison analysis of anti-inflammatory effects of R and S -omeprazole and enzyme-converted omeprazole metabolites in an animal model of gastric ulcer
인도메타신으로 유발시킨 위궤양 동물모델에서 실시예 1~2로 제작된 오메프라졸 대사체의 항염증 효능을 조직학적으로 비교하기 위해 실시예 3으로 제작된 위궤양 동물모델의 위 조직을 적출한 후, 10% 파라포름알데하이드로 고정한 후 OCT compound 방법으로 조직절편을 제작하였다. 제작한 조직절편을 H&E 염색법으로 염색하여 조직학적으로 분석하였다. 그 결과는 도 4에 나타내었다.
In order to histologically compare the anti-inflammatory efficacy of the omeprazole metabolites prepared in Examples 1 and 2 in the gastric ulcer animal model induced by indomethacin, the gastric tissues of the gastric ulcer animal model prepared in Example 3 were extracted, After fixation with paraformaldehyde, tissue sections were prepared by OCT compound method. The tissue sections were stained with H & E staining and histologically analyzed. The results are shown in Fig.
[실시예 5] 위궤양 동물모델의 위조직에서 면역글로불린 A 발현의 비교분석[Example 5] Comparative analysis of immunoglobulin A expression in stomach tissue of gastric ulcer animal model
실시예 3에서 제작된 위궤양 동물모델에서 염증이 유발될 때 변화하는 면역글로불린 양을 비교하기 위해 기존 오메프라졸(R 및 S-오메프라졸) 및 실시예 1 및 2에서 제작된 오메프라졸 대사체을 처리한 동물모델의 대변(feces)에서 면역글로불린 A를 ELISA 방법으로 측정하였다. 점막부위에서 감염방어에 관여하는 면역글로불린(IgA)의 그 생성정도를 측정하기 위해 ELISA 방법을 이용하였다. 추출한 대변(feces)에 1:1000으로 희석한 coating buffer를 이용하여 96well plates(Nunc Maxisorb)에 2시간 동안 코팅시켰다. 코팅된 plates를 0.05% Tween-20이 포함된 PBS로 4번 세척하고 5% skin milk 또는 1% bovine serum albumin이 포함된 PBS로 2시간 동안 실온에서 blocking을 수행하였다. 이후 4회 세척한 후 anti-Ig-HRP(1:5000)으로 1시간 동안 반응시켰다. 반응시킨 plates를 30분 이내로 발색시키고 충분히 발색이 진행되면 10% 황산이 포함된 stopping buffer로 발색을 중단시키고 450nm 파장대에서 발색정도를 측정하였다. 그래프로 나타낸 결과, 면역글로불린 A는 인도메타신만은 처리한 군에서 발현이 감소하였다가, 오메프라졸 대사체를 처리한 군에서 회복속도가 가장 빠르게 정상레벨까지 회복된 것을 확인하였다. 오메프라졸 대사체 중 5'-OH S 오메프라졸 대사체의 회복능이 가장 뛰어난 것으로 나타났다. 이는 오메프라졸 대사체가 기존물질보다 면역글로불린 A의 분비로 인한 항염증 효능이 우수함을 나타낸다. 그 결과는 도5에 나타내었다.
To compare the amount of immunoglobulin that changes when inflammation is induced in an animal model of gastric ulcer prepared in Example 3, the effects of conventional omeprazole ( R and S -omeprazole) and animal models treated with the omeprazole metabolites prepared in Examples 1 and 2 Immunoglobulin A was measured by ELISA in feces. ELISA method was used to measure the degree of the production of immunoglobulin (IgA) involved in infection defense at the mucosal site. The coated feces were coated on 96-well plates (Nunc Maxisorb) for 2 hours using a coating buffer diluted 1: 1000. Coated plates were washed 4 times with PBS containing 0.05% Tween-20 and blocked with PBS containing 5% skin milk or 1% bovine serum albumin for 2 hours at room temperature. After washing four times, the cells were reacted with anti-Ig-HRP (1: 5000) for 1 hour. The reaction plates were developed within 30 minutes. When sufficient color development proceeded, the color development was stopped with a stopping buffer containing 10% sulfuric acid and the degree of color development was measured at a wavelength of 450 nm. As a result of the graph, immunoglobulin A showed a decrease in expression in the group treated with indomethacin alone, but the recovery rate in the group treated with the omeprazole metabolite was recovered to the normal level most rapidly. Omeprazole Metabolites of 5'-OH S Omeprazole Metabolites showed the best ability to recover. This indicates that the metabolite of omeprazole is superior to the existing substance in the anti-inflammatory effect due to secretion of immunoglobulin A. The results are shown in Fig.
[실시예 6] 위궤양 동물모델의 위 조직에서 염증관련 유전자발현 분석[Example 6] Analysis of inflammation-related gene expression in stomach tissue of gastric ulcer animal model
실시예 3에서 제작된 위궤양 동물모델의 위 조직에서 염증관련 유전자발현의 변화를 분석하기 위해 위 조직에서 RNA를 분리하여 cDNA를 합성한 뒤 Real-time PCR로 유전자를 증폭하였다. 그 결과 염증을 일으킬 때 분비되는 사이토카인인 인터루킨-1베타와 인터루킨-6, 인터루킨-7, 및 TNF-α의 레벨이 변화된 것을 확인하였다. 상기 결과로 기존물질인 오메프라졸의 항염증효능보다 효소전환으로 제조된 실시예 1의 오메프라졸의 대사체가 항염증 효능이 더 우수한 효과를 나타내었다. 특히, 5'-OH S-오메프라졸(실시예 2)의 경우, 우수한 항염증 효능으로 인한 염증을 일으킬 때 분비되는 사이토카인 유전자 발현이 가장 작게 나타난 것을 확인하였다. 상기 결과로 보아, 오메프라졸 대사체 중 5'-OH S-오메프라졸(실시예 2)의 경우 항염증 완화효능이 가장 우수한 것을 나타낸다. 그 결과는 도 6에 나타내었다.
In order to analyze the expression of inflammation-related gene expression in the stomach tissue of the gastric ulcer animal model prepared in Example 3, RNA was isolated from the stomach tissue and cDNA was synthesized and amplified by real-time PCR. As a result, it was confirmed that the levels of interleukin-1 beta, interleukin-6, interleukin-7, and TNF-a, which are cytokines secreted in inflammation, were changed. As a result, the metabolite of omeprazole of Example 1, which was prepared by enzyme conversion rather than the anti-inflammatory effect of the existing substance, omeprazole, showed more excellent anti-inflammatory effect. In particular, in the case of 5'-OH S -omeprazole (Example 2), it was confirmed that the expression of cytokine gene secreted when inflammation caused by excellent anti-inflammatory effect was minimized. As a result, the 5'-OH S -omeprazole (Example 2) in the metabolite of omeprazole showed the best anti-inflammatory effect. The results are shown in Fig.
[실시예 7] 위궤양 동물모델의 위 조직 pH의 변화[Example 7] Change in stomach tissue pH of gastric ulcer animal model
위궤양 동물모델에서 추출한 위 조직의 세포샘플을 이용하여 실시예 1~2로 제작된 오메프라졸 대사체를 처리하였을 시의 위 조직의 pH 변화 측정실험을 수행하였다. The measurement of the pH of the stomach tissue was carried out using the cell samples of the stomach tissues extracted from the gastric ulcer animal model when the omeprazole metabolites prepared in Examples 1 and 2 were treated.
우선 위 조직에서 추출한 위 세포샘플과 혼합할 완충용액을 제조하였다. 상기 완충용액은 증류수에 acridine orange(2mg/ml)를 섞은 후 완충용액에 1:1000으로 희석시켜 준비하였다. 위 조직에서 추출한 위 세포샘플을 PBS(10ul)에 세포 수 105 ~ 106개가 되도록 세포샘플을 준비하였다. 실온에서 세포샘플에 완충용액 0.5ml을 더하여 교반시켰다. 교반 후 실온에서 상기 완충용액을 0.5ml 더 첨가한 후 교반시켰다. 교반 후 flow cytometer기를 이용하여 세포샘플의 발광 정도를 측정하였다. R 및 S-오메프라졸을 처리한 군보다는 본 발명에 따른 오메프라졸 대사체를 처리한 군에서 pH 수치가 더 회복된 양상을 보였으며, 특히, S-오메프라졸 대사체(5'-OH S-omeprazole)를 처리한 군의 pH가 다른 군들에 비해 정상대조군의 pH 수치에 더 가깝게 회복된 것으로 나타났다. 그 결과는 도 7에 나타내었다.First, a buffer solution to be mixed with the gastric cell sample extracted from stomach tissue was prepared. The buffer solution was prepared by mixing acridine orange (2 mg / ml) in distilled water and diluting 1: 1000 in a buffer solution. A stomach cell sample extracted from the stomach tissue was prepared in a PBS (10 ul) to a cell number of 10 5 to 10 6 cells. At room temperature, 0.5 ml of buffer solution was added to the cell sample and stirred. After stirring, 0.5 ml of the above buffer solution was further added at room temperature, followed by stirring. After stirring, the degree of luminescence of the cell sample was measured using a flow cytometer. The pH value of the omeprazole metabolite treated group according to the present invention was more restored than the group treated with R and S-omeprazole. In particular, the S-omeprazole metabolite (5'-OH S-omeprazole) The pH of the treated group was recovered closer to the pH value of the normal control group than the other groups. The results are shown in Fig.
Claims (5)
[화학식 1]
[화학식 2]
A composition for treating gastric ulcer comprising, as an active ingredient, an omeprazole metabolite represented by the following formula (1) or (2) produced from omeprazole.
[Chemical Formula 1]
(2)
상기 오메프라졸의 대사체는 거울상 이성질체인 5'-히드록실 S-오메프라졸(5'-OH S-omeprazole) 및 5'-히드록실 R-오메프라졸(5'-OH R-omeprazole)을 50:50으로 함유하고 있는 라세미체(racemate)의 대사체인 것을 특징으로 하는 위궤양 치료용 조성물.
The method according to claim 1,
The metabolites of the omeprazole include enantiomeric 5'-hydroxyl S -omeprazole (5'-OH S -omeprazole) and 5'-hydroxyl R -omeprazole (5'-OH R -omeprazole) Wherein the composition is a metabolite of racemate which is in the form of a racemate.
상기 오메프라졸 대사체는 시토크롬 P450 효소(CYP102A1)를 이용한 효소전환반응으로 생산된 오메프라졸 대사체인 것을 특징으로 하는 위궤양 치료용 조성물.
3. The method of claim 2,
Wherein the omeprazole metabolite is an omeprazole metabolite produced by an enzyme conversion reaction using a cytochrome P450 enzyme (CYP102A1).
상기 오메프라졸 대사체는 전체 조성물 총 중량에 대해 0.01 내지 1중량%을 포함하는 것을 특징으로 하는 위궤양 치료용 조성물.
The method according to claim 1,
Wherein the omeprazole metabolite comprises 0.01 to 1% by weight based on the total weight of the composition.
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| KR20130088209A (en) * | 2012-01-31 | 2013-08-08 | 전남대학교산학협력단 | Novel preparation method of metabolites of omeprazole using bacterial cytochrome p450 and composition therefor |
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| KR20130088209A (en) * | 2012-01-31 | 2013-08-08 | 전남대학교산학협력단 | Novel preparation method of metabolites of omeprazole using bacterial cytochrome p450 and composition therefor |
| KR101348984B1 (en) | 2012-01-31 | 2014-01-10 | 전남대학교산학협력단 | Novel preparation method of metabolites of omeprazole using bacterial cytochrome P450 and composition therefor |
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