KR101390059B1 - Pharmaceutical composition for treatment of diabetes mellitus in postmenopausal women - Google Patents

Pharmaceutical composition for treatment of diabetes mellitus in postmenopausal women Download PDF

Info

Publication number
KR101390059B1
KR101390059B1 KR1020120041729A KR20120041729A KR101390059B1 KR 101390059 B1 KR101390059 B1 KR 101390059B1 KR 1020120041729 A KR1020120041729 A KR 1020120041729A KR 20120041729 A KR20120041729 A KR 20120041729A KR 101390059 B1 KR101390059 B1 KR 101390059B1
Authority
KR
South Korea
Prior art keywords
estradiol
pdk4
beta
postmenopausal women
glucose intolerance
Prior art date
Application number
KR1020120041729A
Other languages
Korean (ko)
Other versions
KR20130118658A (en
Inventor
이성규
조경진
Original Assignee
을지대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 을지대학교 산학협력단 filed Critical 을지대학교 산학협력단
Priority to KR1020120041729A priority Critical patent/KR101390059B1/en
Publication of KR20130118658A publication Critical patent/KR20130118658A/en
Application granted granted Critical
Publication of KR101390059B1 publication Critical patent/KR101390059B1/en

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/56Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

본 발명은 폐경 후 여성의 포도당 대사 이상 및 당뇨병 치료에 특이적인 효과를 갖는 약학적 조성물에 관한 것이다. 본 발명은, 장기간 난소적출 상태로 인하여 17베타-에스트라디올이 부족한 마우스에서 발생하는 포도당불내인성을 17베타-에스트라디올을 투여하여 개선할 수 있음을 발견하고, 또한 이렇게 포도당불내인성 개선 효과를 나타내는 타켓분자로서 PDK4(pyruvate dehydrogenase kinase 4) 유전자를 확인하였다. 본 발명은 에스트로겐 및 PDK4 유전자를 타겟으로 하는 약물들에 대해, 폐경 후 여성에 특이적인 당뇨병 치료제라는 새로운 용도를 제공한다.The present invention relates to pharmaceutical compositions having specific effects on the treatment of glucose metabolism abnormalities and diabetes in postmenopausal women. The present invention finds that glucose intolerance, which occurs in mice lacking 17 beta-estradiol due to prolonged ovarian extraction, can be improved by administering 17 beta-estradiol, and thus shows an effect of improving glucose intolerance. As a target molecule, PDK4 (pyruvate dehydrogenase kinase 4) gene was identified. The present invention provides new uses for drugs targeting estrogen and PDK4 genes, which are anti-diabetic agents specific to postmenopausal women.

Description

폐경 후 여성의 당뇨병 치료를 위한 약학적 조성물 {Pharmaceutical composition for treatment of diabetes mellitus in postmenopausal women}Pharmaceutical composition for treatment of diabetes mellitus in postmenopausal women}

본 발명은 포도당 대사 이상 및 당뇨병 치료를 위한 약학적 조성물에 관한 것으로, 특히 폐경 후 여성의 당뇨병 치료에 특이적인 효과를 갖는 약학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for treating glucose metabolic abnormalities and diabetes, and more particularly to a pharmaceutical composition having a specific effect on the treatment of diabetes in postmenopausal women.

에너지 대사 이상으로 발생하는 비만, 인슐린 저항성, 당뇨병, 지질 대사 이상 그리고 이들 복합체인 대사증후군은 전 세계 국가들이 심각한 문제로 인식하고 있다 (국제비만특별조사위원회, 2004년). 미국의 경우는 2020년에 당뇨병 환자가 1천 7백만 명 이상이 될 것으로 추정하고 있으며 (미국 당뇨병학회), 당뇨병과의 전쟁을 선포하고, 국가차원의 체계적인 시스템을 가동하고 있다 (Healthy People 2010, USA). 우리나라의 경우도 당뇨병 환자가 매년 급격히 증가하고 있고, 2020년에는 당뇨병 환자가 전 인구의 10%를 넘을 것으로 추정하고 있다 (대한당뇨병학회 역학소위원회, 건강심사평가원). Obesity, insulin resistance, diabetes, lipid metabolism abnormalities and their complex metabolic syndrome, caused by abnormal energy metabolism, are recognized as a serious problem by countries around the world (International Committee on Obesity, 2004). In the United States, it is estimated that there will be more than 17 million people with diabetes in 2020 (American Diabetes Association), proclaiming the war on diabetes and operating a systematic system at the national level (Healthy People 2010, USA). In Korea, the number of diabetics is increasing rapidly every year, and it is estimated that by 2020, the number of diabetics will exceed 10% of the population (Korean Diabetes Association, Epidemiological Committee of the Korean Diabetes Association).

여성의 경우는 폐경 후에 난소호르몬 결핍 혹은 에스트로겐(17-beta estradiol) 결핍으로 인하여 골다공증 및 골다공증성 골절, 에너지 대사 이상의 표현형인 비만, 이상 지혈증, 당뇨병, 그리고 고혈압, 심혈관 질환 등이 증가하며, 특히 당뇨병과 심혈관 질환의 기저 공통 병태생리에 해당하는 비만, 인슐린 저항성 및 대사증후군이 증가하는 것으로 알려져 있다 [Maturitas 52(Suppl 1):S53-60, 2005; Natl Vital Stat 53:111, 2004; J Korean Med Sci 19:195-201, 2004; Yonsei Med J 46:198-205, 2005; Diabetes Res Clin Pract 65:143-9, 2004]. 지금까지의 국내외 연구에서, 여성들은 폐경 후에 폐경 전보다 비만, 인슐린 저항성 및 대사증후군의 발생이 증가하고, 폐경 후 같은 나이의 남성보다 비만, 인슐린 저항성 및 대사증후군이 더 많이 발생하는 것으로 보고되었다 [J Korean Med Sci 19:195-201, 2004; Yonsei Med J 46:198-205, 2005; Diabetes Res Clin Pract 65:143-9, 2004; Ann Intern Med 85:447-52, 1976; Arterioscler Thromb 13:661-8, 1993; N Engl J Med 321:641-6, 1989; Atherosclerosis 130(1-2):183-9, 1997; Minerva Ginecol 58(1):35-40, 2006; J Hypertens 24:43-5, 2006; J Hypertens 24:131-41, 2006]. 여러가지 기준으로 대사증후군을 평가하였을 때, 국내 폐경 후 여성에서 대사증후군의 빈도는 15%에서 40%에 이르는 것으로 보고되어 있다 [Diabetes Res Clin Pract 65:143-9, 2004]. In women, postmenopausal ovarian hormone deficiency or estrogen (17-beta estradiol) deficiency increases osteoporosis and osteoporotic fractures, obesity, dyslipidemia, diabetes mellitus, hypertension, and cardiovascular disease. It is known that obesity, insulin resistance and metabolic syndrome, which are the underlying common pathophysiology of hypercardiovascular disease, are increased [Maturitas 52 (Suppl 1): S53-60, 2005; Natl Vital Stat 53: 111, 2004; J Korean Med Sci 19: 195-201, 2004; Yonsei Med J 46: 198-205, 2005; Diabetes Res Clin Pract 65: 143-9, 2004]. In domestic and international studies to date, women have been reported to have increased incidence of obesity, insulin resistance and metabolic syndrome after menopause, and more obesity, insulin resistance and metabolic syndrome than men of the same age after menopause [J] Korean Med Sci 19: 195-201, 2004; Yonsei Med J 46: 198-205, 2005; Diabetes Res Clin Pract 65: 143-9, 2004; Ann Intern Med 85: 447-52, 1976; Arterioscler Thromb 13: 661-8, 1993; N Engl J Med 321: 641-6, 1989; Atherosclerosis 130 (1-2): 183-9, 1997; Minerva Ginecol 58 (1): 35-40, 2006; J Hypertens 24: 43-5, 2006; J Hypertens 24: 131-41, 2006]. When evaluating metabolic syndrome based on various criteria, the incidence of metabolic syndrome in postmenopausal women in Korea has been reported to range from 15% to 40% [Diabetes Res Clin Pract 65: 143-9, 2004].

폐경 후에 에너지 대사 이상(당대사 및 지질 대사 이상)의 발생은 노화, 식욕증가의 결과일 수 있지만, 여성호르몬의 영향도 관여됨이 보고되었다 [Atherosclerosis 98:83-90, 1993; The Menopause. Oxford, Elsevier, 2006:p33-40; The Menopause, Oxford, Elsevier, 2006:p41-51]. 장기간 난소적출 상태의 설치류에서 비만이 발생하고(International Journal of Obesity and Related Metabolic Disorders 26:1103-1109, 2002; International Journal of Cardiology 97:485-493, 2004; Maturitas 58:182-190, 2007; Metabolism 58:38-47, 2009), 인슐린 감수성이 저하되며(International Journal of Cardiology 97:485-493, 2004; Endocrinology 150:2109-2117, 2009; Metabolism 58:38-47, 2009), 그 결과로 포도당불내인성이 발생한다 (International Journal of Cardiology 97:485-493, 2004; Endocrinology 150:2109-2117, 2009). 이러한 장기간 난소적출에 의한 대사적 변화는 에스트로겐 결핍의 직접적인 효과와 에스트로겐 결핍에 의한 식욕항진 효과로 발생한 비만으로 의한 이차적인 효과에 의하여 나타나는 것으로 알려져 있다 (International Journal of Obesity and Related Metabolic Disorders 26:1103-1109, 2002; Metabolism 58:38-47, 2009). 장기간 난소적출 상태의 설치류 모델에서, 난소적출 시부터 지속적으로 에스트로겐을 투여하면 비만, 인슐린 저항성 그리고 포도당불내인성이 발생하지 않는 것으로 보고되었다(International Journal of Obesity and Related Metabolic Disorders 26:1103-1109, 2002; International Journal of Cardiology 97:485-493, 2004; Maturitas 58:182-190, 2007; Endocrinology 150:2109-2117, 2009; Metabolism 58:38-47, 2009). 그러나, 장기간 난소적출 상태의 설치류 모델에서 이미 비만, 인슐린 저항성 및 포도당불내인성이 발생한 상태에서 단기간 에스트로겐을 투여하였을 때는 비만을 변화시키지 못하는 것으로 알려져 있다. 그렇지만, 이러한 경우, 즉 장기간 난소적출 상태의 설치류 모델에서 이미 비만, 인슐린 저항성 및 포도당불내인성이 발생한 상태에서 단기간 에스트로겐을 투여하는 것이 포도당불내인성에 어떠한 효과를 미치는 지에 대해서는 알려진 바가 없다.
The development of energy metabolic abnormalities (sugar metabolism and lipid metabolism abnormalities) after menopause may be a result of aging and increased appetite, but the effects of female hormones have also been reported [Atherosclerosis 98: 83-90, 1993; The Menopause. Oxford, Elsevier, 2006: p 33-40; The Menopause, Oxford, Elsevier, 2006: p 41-51. Obesity occurs in rodents with prolonged ovarian extraction (International Journal of Obesity and Related Metabolic Disorders 26: 1103-1109, 2002; International Journal of Cardiology 97: 485-493, 2004; Maturitas 58: 182-190, 2007; Metabolism 58: 38-47, 2009) and decreased insulin sensitivity (International Journal of Cardiology 97: 485-493, 2004; Endocrinology 150: 2109-2117, 2009; Metabolism 58: 38-47, 2009), resulting in glucose intolerance (International Journal of Cardiology 97: 485-493, 2004; Endocrinology 150: 2109-2117, 2009). The metabolic changes caused by long-term ovarian extraction are known to be due to the direct effect of estrogen deficiency and the secondary effect of obesity caused by anorexia efficacy due to estrogen deficiency (International Journal of Obesity and Related Metabolic Disorders 26: 1103- 1109, 2002; Metabolism 58: 38-47, 2009). In long-term ovarian rodent models, continuous estrogen administration from ovarian extraction has not been reported to result in obesity, insulin resistance and glucose intolerance (International Journal of Obesity and Related Metabolic Disorders 26: 1103-1109, 2002 ; International Journal of Cardiology 97: 485-493, 2004; Maturitas 58: 182-190, 2007; Endocrinology 150: 2109-2117, 2009; Metabolism 58: 38-47, 2009). However, long-term ovarian extraction in rodent models has not been known to change obesity after short-term estrogen administration in the presence of obesity, insulin resistance and glucose intolerance. However, there is no known effect of short-term estrogen administration on glucose intolerance in this case, that is, in obesity, insulin resistance and glucose intolerance already occurring in a rodent model with long-term ovarian extraction.

장기간 난소적출 상태에 있는 17베타-에스트라디올 (17beta-estradiol) 이 부족한 마우스에서 발생하는 비만 및 포도당불내인성은 난소적출과 동시에 17베타-에스트라디올을 지속적으로 투여하면 비만 및 포도당불내인성이 발생하지 않는다. 그 이유는 난소적출과 동시에 투여한 17베타-에스트라디올이 난소적출로 유발되는 비만을 억제하기 때문인 것으로 알려져 있다. 그러나, 장기간 난소적출 상태의 설치류 모델에서 이미 비만, 인슐린 저항성 및 포도당불내인성이 발생한 상태에서는 단기간의 에스트로겐 투여로 비만을 변화시키지 못하는 것으로 알려져 있으며, 이러한 단기간의 에스트로겐 투여가 포도당불내인성 및 당뇨병에 어떤 효과를 미치는 지에 대해서는 알려진 바가 없다. Obesity and glucose intolerance in mice lacking 17beta-estradiol after prolonged ovarian extraction do not result in obesity and glucose intolerance following continuous ovarian extraction and continuous administration of 17beta-estradiol. Do not. The reason is that 17beta-estradiol administered at the same time as ovarian extraction suppresses obesity caused by ovarian extraction. However, long-term ovarian extraction in rodent models is known to prevent obesity from short-term estrogen administration when obesity, insulin resistance and glucose intolerance have already occurred. It is not known whether it works.

본 발명에서는, 장기간 난소적출 상태에 있는 17베타-에스트라디올이 부족한 마우스에서 비만 및 포도당불내인성이 이미 발생한 상태에서, 17베타-에스트라디올을 투여하였을 때 비만과 무관하게 직접적으로 포도당 대사 이상으로 인한 포도당불내인성 및 당뇨병에 치료 효과가 있는지를 밝히고, 또한 그 타켓 분자를 발굴하여, 폐경 후 여성에 특이적인 당뇨병 치료를 위한 새로운 약학적 조성물을 제공하고자 한다. In the present invention, obesity and glucose intolerance have already occurred in 17-beta-estradiol-deficient mice in long-term ovarian extraction, and when 17-beta-estradiol was administered, The present invention seeks to find out whether there is a therapeutic effect on glucose intolerance and diabetes, and also to discover its target molecule to provide a new pharmaceutical composition for treating diabetes specific to postmenopausal women.

본 발명에서는, 장기간(10주) 난소적출 상태로 인하여 17베타-에스트라디올이 부족한 마우스에서 발생하는 포도당불내인성을 17베타-에스트라디올을 투여하여 개선할 수 있음을 발견하였다. 또한, 이렇게 포도당불내인성 개선 효과를 나타내는 타켓분자로서 PDK4(pyruvate dehydrogenase kinase 4) 유전자를 확인하였다. 본 발명에서는 PDK4 siRNA 투여 및 PDK4 유전자의 프로모터(Promoter) 부위 중 에스트로겐 수용체 알파가 결합하는 부위를 이용한 Luciferase Assay 로 검증하였다. 사람의 제2형 당뇨병에서 PDK4 발현 증가가 있다는 연구가 있었고(Mol Genet Metab 65:181-186, 1998; Obes Res 11:176-182, 2003; J Appl Physiol 96:2082-2087, 2004), PDK4 억제제가 제2형 당뇨병 치료에 유용할 수 있을 것이라는 제안이 있었으며(Korean Diabetes J 34:274-283, 2010), 17베타-에스트라디올이 에스트로겐 수용체 알파를 통하여 PDK4 프로모터(Promoter) 부위에 결합한다는 연구(Molecular Endocrinology 22(1):10-22, 2008)가 있었으나, 17베타-에스트라디올이 PDK4 를 통하여 17베타-에스트라디올이 부족한 마우스에서 비만과 무관하게 포도당불내인성을 개선시킨다는 것은 본 발명에서 처음 밝혀낸 것이다. 그리고 장기간(10주) 난소적출 상태로 인하여 17베타-에스트라디올이 부족한 마우스에서 에스트로겐의 포도당불내인성 개선 효과와 PDK4 발현 억제에 의한 포도당불내인성 개선 효과를 비교할 때, 에스트로겐의 포도당불내인성 개선에는 PDK4 발현 억제 외에 다른 기전도 더 작용하는 것으로 추정되었다.
In the present invention, it was found that glucose intolerance in mice lacking 17 beta-estradiol due to long-term (10 weeks) ovarian extraction can be improved by administration of 17 beta-estradiol. In addition, PDK4 (pyruvate dehydrogenase kinase 4) gene was identified as a target molecule showing an effect of improving glucose intolerance. In the present invention, PDK4 siRNA administration and Luciferase Assay using the site where the estrogen receptor alpha binds in the promoter region of the PDK4 gene were verified. There has been research showing increased expression of PDK4 in human type 2 diabetes (Mol Genet Metab 65: 181-186, 1998; Obes Res 11: 176-182, 2003; J Appl Physiol 96: 2082-2087, 2004), PDK4 It has been suggested that inhibitors may be useful for the treatment of type 2 diabetes (Korean Diabetes J 34: 274-283, 2010), and studies that 17 beta-estradiol binds to the PDK4 promoter site via estrogen receptor alpha (Molecular Endocrinology 22 (1): 10-22, 2008), but it is the first time in the present invention that 17beta-estradiol improves glucose intolerance in obese or non-obese mice in 17beta-estradiol deficient via PDK4. It is revealed. When comparing the effects of estrogen on glucose intolerance and the effect of improving glucose intolerance by inhibition of PDK4 expression in mice lacking 17beta-estradiol due to long-term (10 weeks) ovarian extraction, PDK4 was effective in improving glucose intolerance. In addition to inhibition of expression, other mechanisms were presumed to act.

이러한 결과를 토대로, 본 발명에서는, Based on these results, in the present invention,

에스트로겐을 유효성분으로 함유하는, 폐경 후 여성의 당뇨병 치료를 위한 약학적 조성물을 제공한다. 본 발명에서 “에스트로겐”은 에스트론, 에스트라디올, 에스트리올 및 이들과 같은 용도로 사용할 수 있는 유사체를 모두 포함하는 의미이다. Provided is a pharmaceutical composition for treating diabetes in postmenopausal women, containing estrogen as an active ingredient. In the present invention, "estrogen" is meant to include all the estrone, estradiol, estriol and the like that can be used for such use.

또한, 본 발명에서는, Further, in the present invention,

PDK4(pyruvate dehydrogenase kinase 4) 유전자의 발현 억제제를 유효성분으로 함유하는, 폐경 후 여성의 당뇨병 치료를 위한 약학적 조성물을 제공한다. 상기 PDK4 유전자의 발현 억제제는 특히 17베타-에스트라디올을 포함할 수 있다. Provided is a pharmaceutical composition for treating diabetes in postmenopausal women, containing an inhibitor of the expression of pyruvate dehydrogenase kinase 4 (PDK4) gene as an active ingredient. Inhibitors of expression of the PDK4 gene may particularly comprise 17beta-estradiol.

본 발명에서는 에스트로겐이 폐경 후 여성의 포도당 대사 이상으로 인한 포도당불내인성의 개선에 직접적인 효과가 있음을 확인함으로써 폐경 후 여성의 당뇨병 치료에 사용할 수 있도록 하고, 그 분자생물학적 기전을 밝혔다. 본 발명에 따라 에스트로겐은 폐경 후 당뇨병이 발생한 여성에 특이적인 당뇨병 치료제로서 새로운 용도로 사용될 수 있으며, PDK4 유전자를 타겟으로 하는 약물들 또한 폐경 후 여성에 특이적인 당뇨병 치료제라는 새로운 용도로 이용될 수 있다. In the present invention, by confirming that estrogen has a direct effect on the improvement of glucose intolerance due to abnormal glucose metabolism in postmenopausal women, it can be used to treat diabetes in postmenopausal women, and revealed its molecular biological mechanism. According to the present invention, estrogen may be used as a new antidiabetic agent specific to postmenopausal women with diabetes, and drugs targeting the PDK4 gene may also be used as a new antimicrobial agent specific to postmenopausal women. .

도 1은 실험군에 대해 10주간의 체중변화를 측정한 결과이다. (*: P<0.05, **: P<0.01, ***: P<0.001)
도 2는 경구당부하 후 혈당을 측정한 결과이다.
도 3은 경구당부하 후 혈당 면적(AUCglucose, Area Under the Curve of Glucose)을 측정한 결과이다 (*: P<0.05, **: P<0.01, ***: P<0.001).
도 4는 중요한 대사 조직(간, 근육 및 지방조직)에서의 PDK4 유전자 발현을 확인한 결과이다 (*: P<0.05, **: P<0.01, ***: P<0.001. GCM (Gastrocnemius muscle, Skeletal muscle), PUF (Periuterine fat, adipose tissue)).
도 5는 17베타-에스트라디올이 PDK4 유전자의 프로모터(Promoter)에 작용하여 이 유전자 발현을 억제하는 Luciferase Assay를 도식화한 것이다.
도 6은 PDK4 유전자의 프로모터(Promoter) Luciferase Assay 결과이다.
Figure 1 is the result of measuring the weight change for 10 weeks for the experimental group. (*: P <0.05, **: P <0.01, ***: P <0.001)
Figure 2 is the result of measuring blood glucose after oral glucose loading.
Figure 3 is the result of measuring the glucose area (AUC glucose , Area Under the Curve of Glucose) after oral glucose load (*: P <0.05, **: P <0.01, ***: P <0.001).
4 shows the results of confirming PDK4 gene expression in important metabolic tissues (liver, muscle and adipose tissue) (*: P <0.05, **: P <0.01, ***: P <0.001.GCM (Gastrocnemius muscle, Skeletal muscle), PUF (Periuterine fat, adipose tissue)).
FIG. 5 is a schematic of Luciferase Assay in which 17 beta-estradiol acts on the promoter of PDK4 gene to inhibit this gene expression.
Figure 6 is a promoter Luciferase Assay results of the PDK4 gene.

이하 구체적인 실시예 및 실험예를 통해 본 발명을 보다 상세히 설명한다. 그러나 이들 실시예 및 실험예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예 및 실험예에 의해 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to specific examples and experimental examples. However, these Examples and Experimental Examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited by these Examples and Experimental Examples.

1. 마우스 난소적출 및 실험 군Mouse ovary extraction and experimental group

C57BL/6 암컷 마우스를 8주령에 가짜수술 및 양측 난소적출술(OV, ovariectomy)을 시행하였다. 난소적출 후 10주간 체중 변화를 측정하였으며, 10주 후에 마우스를 가짜수술 군(SH), 난소적출 군(OV), 난소적출 군에 17베타-에스트라디올을 피하 주사한 군(OVE), 그리고 난소적출 군에 PDK4 siRNA를 복강 주사한 군[(OV + siRNA (PDK4)]의 4개 군으로 나누었다. 체중 변화 측정 결과는 도 1과 같다. OVE 군은 난소적출(OV, ovariectomy) 10주 후에 17베타-에스트라디올 5㎍/마우스 무게(㎏) 피하주사(SC. Injection)를 6회 실시하였으며, [OV+siRNA (PDK4) 군]은 PDK4 siRNA 복강주사(3.3 mg/마우스 무게(kg), IP. Injection)를 2회 실시하였다.
C57BL / 6 female mice underwent sham surgery and bilateral ovary extraction (OV) at 8 weeks of age. Weight change was measured for 10 weeks after ovarian extraction. After 10 weeks, mice were injected subcutaneously with 17 beta-estradiol (SH), ovarian extraction (OV), ovarian extraction group (OVE), and ovary. The abdominal injection of PDK4 siRNA into the extraction group was divided into four groups [(OV + siRNA (PDK4)]. The weight change measurement results are shown in Fig. 1. The OVE group was 17 weeks after 10 weeks of ovariectomy (OV). Beta-estradiol 5 μg / mouse weight (kg) subcutaneous injection (SC. Injection) was performed 6 times, [OV + siRNA (PDK4) group] was PDK4 siRNA intraperitoneal injection (3.3 mg / mouse weight (kg), IP Injection) was performed twice.

2. 2. 경구당부하검사Oral glucose tolerance test

상기 17베타-에스트라디올 피하주사(SC. Injection) 6회 및 PDK4 siRNA 복강주사(IP. Injection) 2회 후 경구당부하검사를 시행하였다. 경구당부하 후 혈당은 도 2와 같으며, 혈당 면적(AUCglucose, Area Under the Curve of Glucose)은 도 3과 같다.The oral glucose tolerance test was performed after sixteen beta-estradiol subcutaneous injections (SC. Injection) and two PDK4 siRNA intraperitoneal injections (IP. Injection). After oral glucose loading, blood glucose is as shown in FIG. 2, and blood glucose area (AUC glucose , Area Under the Curve of Glucose) is as shown in FIG. 3.

도 2, 3에 나타난 바와 같이 SH 군에 비하여 OV 군에서 혈당 면적이 증가하였고, 17베타-에스트라디올 피하주사(SC. Injection)를 6회 실시한 OVE 군은 다시 혈당 면적이 감소하였다. 17베타-에스트라디올을 투여하지 않고 PDK4 siRNA 복강주사(IP. Injection)를 2회 실시한 [OV + siRNA (PDK4) 군]에도 17베타-에스트라디올 투여 시 처럼 다시 혈당 면적이 감소하였다. 즉, 장기간 난소적출로 인해 17베타-에스트라디올 이 부족한 마우스에서 비만 및 포도당불내인성이 발생하였으나(SH), 이렇게 이미 비만 및 포도당불내인성이 발생한 상태에서도 17베타-에스트라디올을 투여하면 포도당불내인성이 개선되었으며, 또한 17베타-에스트라디올 대신 PDK4 siRNA를 투여하여도 17베타-에스트라디올을 투여한 것과 같이 포도당불내인성이 개선되었다. 그러나 17베타-에스트라디올의 포도당불내인성 개선 효과와 PDK4 발현 억제에 의한 포도당불내인성 개선 효과를 비교할 때, 에스트로겐의 포도당불내인성의 개선에는 PDK4 발현 억제 외에 다른 기전도 더 작용하는 것으로 추정되었다. As shown in FIGS. 2 and 3, the blood glucose area was increased in the OV group as compared to the SH group, and the blood glucose area was decreased again in the OVE group which was subjected to 17 beta-estradiol subcutaneous injection 6 times. In the OV + siRNA (PDK4) group, in which PDK4 siRNA ip injection was performed twice without 17 beta-estradiol, blood glucose area was decreased again as in 17 beta-estradiol. In other words, obesity and glucose intolerance occurred in mice lacking 17 beta-estradiol due to prolonged ovarian extraction (SH). However, even when administration of 17 beta-estradiol has already occurred in obesity and glucose intolerance, This improvement was also achieved by improving PDG4 siRNA instead of 17beta-estradiol as well as glucose intolerance as 17beta-estradiol was administered. However, when comparing the effects of improving glucose intolerance of 17 beta-estradiol and improving glucose intolerance by inhibiting PDK4 expression, it was estimated that other mechanisms may be involved in improving glucose intolerance of estrogen.

또한, PDK4 siRNA 투여가 효과적으로 되었는지 알아 보기 위하여, PDK4 siRNA 투여 후 중요한 대사 조직(간, 근육 및 지방조직)에서 PDK4 유전자 발현을 알아 보았다. 그 결과 도 4에서 보는 바와 같이 PDK4 siRNA가 효과적으로 작용하였다. 또한 PDK4 유전자 발현은 OV 군에서 증가하였다가, OVE 군에서 감소하는 것도 알 수 있었다.
In addition, PDK4 gene expression was examined in important metabolic tissues (liver, muscle and adipose tissue) after PDK4 siRNA administration. As a result, as shown in Figure 4 PDK4 siRNA was effective. In addition, PDK4 gene expression was increased in the OV group, but also decreased in the OVE group.

3. 3. PDK4PDK4 유전자의 프로모터( Promoter of gene ( PromoterPromoter ) 부위 중 에스트로겐 수용체 알파가 결합하는 부위를 이용한 Luciferase Luciferase using the site where estrogen receptor alpha binds AssayAssay

마우스에서 17베타-에스트라디올을 투여하여 포도당불내인성이 개선된 것이 17베타-에스트라디올이 PDK4 유전자 발현을 직접 억제하여 나타난 현상인지를 확인하기 위하여, 마우스 간세포주(AML12 cell)에서 PDK4 유전자의 프로모터(Promoter) 부위 중 에스트로겐 수용체 알파가 결합하는 부위를 이용한 Luciferase Assay를 실시하였다 (도 5). 그 결과, 17베타-에스트라디올은 마우스 간세포주(AML12 cell)에서 PDK4 유전자의 프로모터(Promoter) 부위 중 에스트로겐 수용체 알파가 결합하는 부위에서 Luciferase 활성을 감소시켰다 (도 6). 이러한 결과로 17베타-에스트라디올이 PDK4 유전자 발현을 억제함을 확인할 수 있었다.
To determine whether the improved glucose intolerance by administering 17 beta-estradiol in mice is a phenomenon in which 17 beta-estradiol is a result of direct inhibition of PDK4 gene expression, the promoter of PDK4 gene in mouse hepatocytes (AML12 cells) (Promoter) Luciferase assay was performed using a site to which the estrogen receptor alpha binds (FIG. 5). As a result, 17 beta-estradiol reduced Luciferase activity at the site where the estrogen receptor alpha binds in the promoter region of PDK4 gene in mouse liver cell line (AML12 cell) (FIG. 6). These results confirm that 17beta-estradiol inhibits PDK4 gene expression.

이상의 실험 결과로, 장기간 난소적출 상태에서 17베타-에스트라디올이 부족한, 비만해진 마우스에 17베타-에스트라디올을 투여하여 포도당불내인성이 개선됨을 발견하였고, 17베타-에스트라디올을 투여하여 포도당불내인성이 개선된 것이 17베타-에스트라디올이 PDK4 유전자 발현을 직접 억제하여 나타난 현상임을 증명하였다.
As a result of the experiment, it was found that administration of 17 beta-estradiol improved glucose intolerance in obese mice that lacked 17 beta-estradiol under long-term ovarian extraction and improved glucose intolerance by administering 17 beta-estradiol. This improvement proved that 17beta-estradiol was a phenomenon that appeared by directly inhibiting PDK4 gene expression.

Claims (3)

17베타-에스트라디올 또는 PDK4(pyruvate dehydrogenase kinase 4) siRNA를 유효성분으로 함유하는, 폐경 후 당뇨병 여성의 식후 혈당 강하를 위한 약학적 조성물.A pharmaceutical composition for reducing postprandial blood sugar in postmenopausal diabetic women, comprising 17 beta-estradiol or PDK4 (pyruvate dehydrogenase kinase 4) siRNA as an active ingredient. 제1항에 있어서, 상기 폐경 후 당뇨병이 PDK4 유전자의 과발현을 수반하는 것을 특징으로 하는, 폐경 후 당뇨병 여성의 식후 혈당 강하를 위한 약학적 조성물.The pharmaceutical composition of claim 1, wherein the postmenopausal diabetes involves overexpression of the PDK4 gene. 삭제delete
KR1020120041729A 2012-04-20 2012-04-20 Pharmaceutical composition for treatment of diabetes mellitus in postmenopausal women KR101390059B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020120041729A KR101390059B1 (en) 2012-04-20 2012-04-20 Pharmaceutical composition for treatment of diabetes mellitus in postmenopausal women

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020120041729A KR101390059B1 (en) 2012-04-20 2012-04-20 Pharmaceutical composition for treatment of diabetes mellitus in postmenopausal women

Publications (2)

Publication Number Publication Date
KR20130118658A KR20130118658A (en) 2013-10-30
KR101390059B1 true KR101390059B1 (en) 2014-04-29

Family

ID=49636901

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020120041729A KR101390059B1 (en) 2012-04-20 2012-04-20 Pharmaceutical composition for treatment of diabetes mellitus in postmenopausal women

Country Status (1)

Country Link
KR (1) KR101390059B1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016125937A1 (en) * 2015-02-06 2016-08-11 경북대학교 산학협력단 Method for screening for therapeutic agent for preventing or treating female menopausal disorders by using pdk2, and pharmaceutical composition for preventing or treating female menopausal disorders, containing therapeutic agent screened for by method as active ingredient

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008127373A (en) 2006-11-24 2008-06-05 Koei Kogyo Kk Estrogen-like composition
JP2010095471A (en) 2008-10-17 2010-04-30 Osaka Industrial Promotion Organization Antiobesic, antilipemic and antidiabetic composition

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008127373A (en) 2006-11-24 2008-06-05 Koei Kogyo Kk Estrogen-like composition
JP2010095471A (en) 2008-10-17 2010-04-30 Osaka Industrial Promotion Organization Antiobesic, antilipemic and antidiabetic composition

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
C.C. Wells et al, Gender Medicine, 2005, Vol. 2, No. 4, pp. 227-237 *
C.C. Wells et al, Gender Medicine, 2005, Vol. 2, No. 4, pp. 227-237*

Also Published As

Publication number Publication date
KR20130118658A (en) 2013-10-30

Similar Documents

Publication Publication Date Title
Bonnet et al. RANKL inhibition improves muscle strength and insulin sensitivity and restores bone mass
Lim et al. Modulation of adiponectin as a potential therapeutic strategy
Staruschenko et al. Role of TRPC6 in progression of diabetic kidney disease
Yamawaki et al. Nrf2 activator for the treatment of kidney diseases
Liu et al. Irbesartan ameliorates diabetic cardiomyopathy by regulating protein kinase D and ER stress activation in a type 2 diabetes rat model
Marcus et al. Angiotensin 1-7 as means to prevent the metabolic syndrome: lessons from the fructose-fed rat model
Yao et al. Natural product vindoline stimulates insulin secretion and efficiently ameliorates glucose homeostasis in diabetic murine models
Lu et al. GIPR antagonist antibodies conjugated to GLP-1 peptide are bispecific molecules that decrease weight in obese mice and monkeys
Farrell Signalling links in the liver: knitting SOCS with fat and inflammation
Zou et al. Fenofibrate ameliorates cardiac hypertrophy by activation of peroxisome proliferator-activated receptor-α partly via preventing p65-NFκB binding to NFATc4
Zhu et al. Insulin regulates titin pre-mRNA splicing through the PI3K-Akt-mTOR kinase axis in a RBM20-dependent manner
Xu et al. Hypoglycemic and hypolipidemic effects of triterpenoid-enriched Jamun (Eugenia jambolana Lam.) fruit extract in streptozotocin-induced type 1 diabetic mice
Devlin et al. Daily leptin blunts marrow fat but does not impact bone mass in calorie-restricted mice
Cha et al. Corn silk extract improves cholesterol metabolism in C57BL/6J mouse fed high-fat diets
Wang et al. Antihyperglycemic effect of ginsenoside Rh2 by inducing islet β-cell regeneration in mice
Hillaert et al. Measuring and targeting aldosterone and renin in atherosclerosis—a review of clinical data
Wei et al. Theobromine ameliorates nonalcoholic fatty liver disease by regulating hepatic lipid metabolism via mTOR signaling pathway in vivo and in vitro
Christensen et al. GIP’s involvement in the pathophysiology of type 2 diabetes
Chen et al. Effects of CB1 receptor blockade on monosodium glutamate induced hypometabolic and hypothalamic obesity in rats
Zha et al. High glucose instigates tubulointerstitial injury by stimulating hetero-dimerization of adiponectin and angiotensin II receptors
Wang et al. Cardioprotection of Klotho against myocardial infarction-induced heart failure through inducing autophagy
Gao et al. PGLP‐1, a novel long‐acting dual‐function GLP‐1 analog, ameliorates streptozotocin‐induced hyperglycemia and inhibits body weight loss
Xu et al. C-peptide ameliorates renal injury in type 2 diabetic rats through protein kinase A-mediated inhibition of fibronectin synthesis
Larsen et al. Does receptor balance matter?–Comparing the efficacies of the dual amylin and calcitonin receptor agonists cagrilintide and KBP-336 on metabolic parameters in preclinical models
Liu et al. The Na/K-ATPase signaling and SGLT2 inhibitor-mediated cardiorenal protection: a crossed road?

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E90F Notification of reason for final refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant
FPAY Annual fee payment

Payment date: 20170208

Year of fee payment: 4

FPAY Annual fee payment

Payment date: 20180418

Year of fee payment: 5

FPAY Annual fee payment

Payment date: 20190322

Year of fee payment: 6