KR101804849B1 - Composition for the prevention or treatment of neurodegenerative disease comprising tartrate or pharmaceutically acceptable salts as an active incredient - Google Patents
Composition for the prevention or treatment of neurodegenerative disease comprising tartrate or pharmaceutically acceptable salts as an active incredient Download PDFInfo
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- KR101804849B1 KR101804849B1 KR1020160145705A KR20160145705A KR101804849B1 KR 101804849 B1 KR101804849 B1 KR 101804849B1 KR 1020160145705 A KR1020160145705 A KR 1020160145705A KR 20160145705 A KR20160145705 A KR 20160145705A KR 101804849 B1 KR101804849 B1 KR 101804849B1
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- Prior art keywords
- disease
- tartrate
- tartarate
- present
- pharmaceutically acceptable
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Images
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
- A61K31/225—Polycarboxylic acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- 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
-
- 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
- A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Emergency Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
본 발명은 타르타르산염(tartrate), 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 퇴행성뇌질환 예방 또는 치료용 약학적 조성물에 관한 것이다.
The present invention relates to a pharmaceutical composition for preventing or treating degenerative brain diseases containing tartarate or a pharmaceutically acceptable salt thereof as an active ingredient.
퇴행성뇌질환 환자의 뇌에서 공통적으로 발견되는 병리학적 특징으로는 신경세포의 사멸과 특정단백질의 비정상적 응집이 있다. 아밀로이드 베타(amyloid β), 노인반(senile plaque), 신경섬유농축제(neurofibrillary tangle), 알파 시뉴클린(α-synuclein) 또는 루이소체(lewy body)등의 단백질의 비정상적 응집이 그 자체로 신경세포의 사멸을 유도하거나, 미세아교세포를 활성화해 염증반응을 일으킴으로써 알츠하이머병(Alzheimer's disease) 또는 파킨슨병(Parkinson's disease) 등의 발병에 중요한 역할을 하는 것으로 알려져 있다. Pathologic features commonly found in brain of patients with degenerative brain disease include neuronal death and abnormal aggregation of specific proteins. Abnormal aggregation of proteins such as amyloid beta, senile plaques, neurofibrillary tangles, alpha-synuclein or lewy bodies, itself, (Alzheimer ' s disease or Parkinson ' s disease) by inducing apoptosis or activating microglial cells and causing an inflammatory reaction.
대뇌 흑질(substantia nigra, SN)부위의 도파민성 신경세포(doparminergic neuron)의 사멸과 더불어 흑질의 도파민성 뉴런내에 존재하는 알파 시뉴클린의 응집은 파킨슨병의 주요 병리현상이다. 또한, α-synuclein(SNCA), parkin (PARK 2), leucine-rich repeat kinase 2(PARK 8), PTEN-induced putative kinase 1(PINK 1) 또는 DJ-1(PARK 7)등의 유전자들이 파킨슨병의 원인으로 알려져 있다. Along with the death of dopaminergic neurons in the substantia nigra (SN) region, aggregation of alpha-synuclein present in the black dopant neurons is a major pathology of Parkinson's disease. Genes such as α-synuclein (SNCA), parkin (PARK 2), leucine-rich repeat kinase 2 (PARK 8), PTEN-induced putative kinase 1 (PINK 1) or DJ- .
그중 DJ-1은 20 kDa의 유비퀴틴-세포질성(ubiquitin cytoplasmic) 핵단백질로, 다양한 인간 조직에서 발현되며 Ras와 관련된 신호경로에서 마이토젠 의존적 발암유전자(mitogen-dependent oncogene)로 처음 알려졌다(Nagakubo 등, 1997, Biochem. Biophys. Res. Commun. 231, 509-513). DJ-1은 또한, 산화스트레스가 진행되는 동안 DJ-1은 글루타티온(glutathione)의 합성을 촉진시키고 A53T 시뉴클린의 독성을 억제하기 때문에, DJ-1이 정상적으로 기능할 때는 도파민 세포의 사멸이 방지되지만, DJ-1 유전자에 변이가 생기면 기능이상으로 이어져 도파민 세포의 사멸을 촉발시키는 것이 확인되면서 파킨슨병과의 관련성이 알려졌다(Zhou W., 2005, J. Biol. Chem., 280, 43150-43158).
Among them, DJ-1 is a 20 kDa ubiquitin cytoplasmic nuclear protein expressed in various human tissues and was first known as a mitogen-dependent oncogene in signaling pathways associated with Ras (Nagakubo et al. 1997, Biochem. Biophys. Res. Commun., 231, 509-513). DJ-1 also promotes the synthesis of glutathione and suppresses the toxicity of A53T synuclein during oxidative stress, so that when DJ-1 is functioning normally, dopamine cell death is prevented , A mutation in the DJ-1 gene leads to dysfunction and induces the death of dopamine cells, and thus it is known to be related to Parkinson's disease (Zhou W., 2005, J. Biol. Chem., 280, 43150-43158).
정상적인 DJ-1 기능에 대한 몇몇 보고에 따르면, DJ-1의 시스테인 잔기(Cys106)는 과산화수소(hydrogen peroxid, H2O2)에 의해 쉽게 산화되고(Lee S.J.등, 2003, J. of Biol. Chem., 278, 44552-44559), 산화된 Cys106은 알파 시뉴클린의 응집을 억제하며(Shendelman S.등, 2004, PLoS Biol., 2, e362), 또한 DJ-1은 Daxx/ASK1 세포신호경로를 억제함으로써 활성산소에 의한 도파민성 신경세포의 사멸을 보호한다(Junn E.등, 2005, PNAS, 102, 9691-9696). According to some reports of normal DJ-1 function, cysteine residues (Cys106) in the DJ-1 are easily oxidized by hydrogen peroxide (hydrogen peroxid, H 2 O 2 ) (Lee SJ , etc., 2003, J. of Biol. Chem Oxidized Cys106 inhibits the aggregation of alpha-synuclein (Shendelman S. et al., 2004, PLoS Biol., 2, e362) and DJ-1 inhibits the Daxx / ASK1 cell signaling pathway (Junn E. et al., 2005, PNAS, 102, 9691-9696). ≪ / RTI >
한편, 상기에서 설명한 바와 같이 DJ-1의 기능이상은 파킨슨병의 원인으로 생각되어 지는데, 파킨슨 환자의 뇌에서 불용성의 DJ-1 응집이 증가된 것과(Moore DJ.등, 2005, Hum. Mol. Genet., 14, 71-84; Moore DJ.등, 2005, Annu. Rev. Neurosci., 28, 57-87) DJ-1의 변이체들(L166P, L10P, M26I 및 P158 삭제)의 용해성이 감소하며 봉입체(inclusion)를 형성한다는 보고는(Ramsey CP.등, 2010, Neurosci. Res., 88, 3111-3124; Olzmann JA.등, 2004, J. Biol. Chem., 279, 8506-8515) 이를 뒷받침 한다. 따라서, DJ-1의 변이 및 기능이상을 제어하는 것은 파킨슨병 예방 및 치료에 매우 중요한 타겟이 될 수 있다.
On the other hand, as described above, DJ-1 dysfunction is thought to be a cause of Parkinson's disease, and it is known that insulin-like DJ-1 aggregation is increased in the brain of Parkinson's patients (Moore DJ. The solubility of variants (L166P, L10P, M26I, and P158 deletions) of DJ-1 is reduced, Olummann JA, et al., 2004, J. Biol. Chem., 279, 8506-8515) reported that the formation of inclusions (Ramsey CP et al., 2010, Neurosci. Res., 88, 3111-3124; do. Thus, controlling the mutations and dysfunctions of DJ-1 may be a very important target for the prevention and treatment of Parkinson's disease.
무기인(Inorganic phosphate, Pi)은 식품을 통해 섭취되며, 기관(organ)의 발달, 에너지대사 및 세포신호전달과 관련된 다양한 기능을 수행한다. 인간에게서 비정상적인 Pi 섭취에 따른 고인산염혈증(hyperphosphatermia)은 생식저하(Arrata WS.등, 1978, Fertil. Steril., 30, 329-333), 종양생성 증가(Lee S.등, 2015, PLoS One, 10, e0135582; Wulaningsih W.등, 2013, BMC Cancer, 13, 257), 신장기능이상(Alfrey A.C.등, 2004, Kidney Int. Suppl., S13-17), 혈관의 석회화(Jono S.등, 2002, Clin. Calcium, 12, 1067-1072), 세포신호전달 감소, 세포사멸 또는 뼈무기화 저해(Allam O.등, 2011, Br. J. Nutr., 105, 384-392) 등의 병리적 문제를 야기하는 것으로 보고되어 있다. 그러나 파킨슨병 환자에서 Pi가 증가되어 있다는 보고(Barbiroli B.등, 1999, Mov. Disord., 14, 430-435; Brown G.G.등, 1989, Neurology, 39, 1423-1427)와 최근 Pi를 매개로 하여 DJ-1의 응집이 유도된다는 보고(Cha SS.등, 2008, J. Biol. Chem., 283, 34069-34075)는 Pi에 의한 DJ-1의 응집이 파킨슨병의 하나의 원인이 될 수 있음을 시사한다.
Inorganic phosphate (P i ) is ingested through food and performs a variety of functions related to organ development, energy metabolism and cell signaling. Hyperphosphatemia due to abnormal Pi ingestion in humans has been associated with decreased reproduction (Arrata WS. Et al., 1978, Fertil. Steril., 30, 329-333), increased tumorigenesis (Lee S. et al., 2015, PLoS One, 10, e0135582; Wulaningsih W. et al., 2013, BMC Cancer, 13, 257), kidney dysfunction (Alfrey AC et al., 2004, Kidney Int. Suppl., S13-17), vein calcification (Jono S. et al., 2002 , Clin. Calcium, 12, 1067-1072), cell signaling reduction, apoptosis or inhibition of bone mineralization (Allam O. et al., 2011, Br. J. Nutr., 105, 384-392) It is reported to cause. However, it has been reported that Pi is increased in patients with Parkinson's disease (Barbiroli B. et al., 1999, Mov. Disord., 14, 430-435; Brown GG et al., 1989, Neurology, 39, 1423-1427) (Cha SS, et al., 2008, J. Biol. Chem., 283, 34069-34075) suggest that aggregation of DJ-1 by Pi may be one cause of Parkinson's disease .
타르타르산염(tartrate)은 시럽·주스 등에 널리 사용되고, 의약품으로는 청량지갈제(淸凉止渴劑)로서, 또 염색공업·제과·사진·유기합성·금속의 착색 등에 사용되는 화합물이다. 의학적으로 타르타르산염은 Metoprolol tartrate, Rivastigmine tartrate 등과 같이 약효를 나타내는 주 화합물에 부가염으로써 사용될 뿐, 질환에 있어서 타르타르산염 단독 화합물의 효과는 보고된바 없다.
Tartrate is widely used in syrups, juices, etc. It is a compound used as a refreshing agent for cleansing agents, dyeing industry, confectionery, photography, organic synthesis, and coloring of metals. Medically, tartarate is used as an additive salt to the main active compound, such as Metoprolol tartrate, Rivastigmine tartrate, etc., and the effect of tartarate salt alone in the disease has not been reported.
이러한 배경하에 본 발명자들은, DJ-1을 형질주입하여 과발현 시킨 Neuro-2A 또는 SH-SY5Y 신경세포주에 무기인(Inorganic phosphate, Pi)을 처리하였을때 DJ-1이 Pi를 매개로 응집되는 것을 확인하였고, 타르타르산염이 DJ-1과 Pi의 결합부위에 경쟁적으로 결합하여 DJ-1/타르타르산염 복합체를 형성하고, Pi으로 유도된 DJ-1의 응집 및 Pi 또는 활성산소로 유도된 세포사멸을 억제하는 것을 확인함으로써, 타르타르산염을 퇴행성뇌질환 예방 및 치료용 조성물로 유용하게 사용할 수 있음을 규명하여 본 발명을 완성하였다.
Under these circumstances, the inventors of the present invention found that when inorganic phosphorus (P i ) was treated with Neuro-2A or SH-SY5Y neuronal cell line in which DJ-1 was transfected and overexpressed, DJ-1 aggregated via Pi The tartarate was competitively bound to the binding site of DJ-1 and Pi to form a DJ-1 / tartarate complex, and the PI-induced aggregation of DJ-1 and apoptosis induced by Pi or ROS The present inventors have completed the present invention by confirming that tartaric acid salts can be effectively used as a composition for preventing and treating degenerative brain diseases.
본 발명의 목적은 타르타르산염(tartrate)을 유효성분으로 함유하는 퇴행성뇌질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.
It is an object of the present invention to provide a pharmaceutical composition for preventing or treating degenerative brain diseases containing tartarate as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 하기 화학식 1로 표시되는 화합물, 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 퇴행성뇌질환의 예방 또는 치료용 약학적 조성물을 제공한다:In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases comprising, as an active ingredient, a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
또한, 본 발명은 하기 화학식 1로 표시되는 화합물, 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 퇴행성뇌질환의 개선용 건강기능식품을 제공한다:The present invention also provides a health functional food for improving degenerative brain diseases comprising, as an active ingredient, a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[화학식 1] [ Chemical Formula 1 ]
본 발명에서는, 타르타르산염이 DJ-1 및 Pi(Inorganic phosphate)의 결합부위에 경쟁적으로 결합하여 DJ-1/타르타르산 복합체를 형성함으로써, Pi로 유도된 DJ-1의 응집, 및 Pi 또는 활성산소로 유도된 세포사멸을 억제하는 것을 확인함으로써, 타르타르산염이 퇴행성뇌질환 예방 또는 치료에 유용하게 사용될 수 있음을 확인하였다.
In the present invention, the tartarate is competitively bound to the binding sites of DJ-1 and Pi (Inorganic phosphate) to form a DJ-1 / tartaric acid complex, whereby aggregation of Pi-induced DJ-1, Induced cell death, it was confirmed that the tartarate salt can be effectively used for the prevention or treatment of degenerative brain diseases.
도 1은 Pi(Inorganic phosphate)으로 유도된 DJ-1 응집(aggregation)을 확인한 도이다.
도 2는 DJ-1/tartrate 복합체의 결정구조(crystal structure)를 나타낸 도이다.
도 3a은 Neuro-2A 세포에서 타르타르산염의 Pi로 유도된 DJ-1응집에 대한 억제 효과를 나타낸 도이다.
도 3b는 SH-SY5Y 세포에서 타르타르산염의 Pi로 유도된 DJ-1응집에 대한 저해 효과를 나타낸 도이다.
도 4는 타르타르산염의 산화스트레스로 유도된 DJ-1응집 억제 효과를 나타낸 도이다.
도 5는 타르타르산염의 Pi 및 산화스트레스에 의한 세포사멸에 대한 억제 효과를 나타낸 도이다. FIG. 1 shows DJ-1 aggregation induced by Pi (Inorganic phosphate).
Figure 2 shows the crystal structure of the DJ-1 / tartrate complex.
FIG. 3A shows the inhibitory effect of Pi-induced DJ-1 aggregation of tartarate in Neuro-2A cells. FIG.
FIG. 3B is a graph showing the inhibitory effect on the DJ-1 aggregation induced by Pi of tartarate in SH-SY5Y cells. FIG.
Figure 4 shows the effect of oxidative stress induced DJ-1 aggregation inhibition of tartarate salt .
5 is a graph showing the inhibitory effect of tartarate salt on apoptosis induced by Pi and oxidative stress.
이하 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명은 하기 화학식 1로 표시되는 화합물, 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 퇴행성뇌질환의 예방 또는 치료용 약학적 조성물을 제공한다:The present invention provides a pharmaceutical composition for preventing or treating a degenerative brain disease comprising, as an active ingredient, a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof:
[화학식 1] [ Chemical Formula 1 ]
상기 퇴행성뇌질환은 뇌졸중(Stroke), 파킨슨병(Parkinson's disease), 알츠하이머 치매(Alzheimer's disease), 헌팅톤병(Huntington's disease), 근위축성 측삭 경화증(amyotrophic lateral sclerosis), 알코올성 뇌신경질환, 척수손상(spinal cord injury) 또는 알코올성 치매 및 베르니케-코르사코프 증후군(Wernicke-Korsakoff's syndrome)으로 이루어지는 군으로부터 선택되는 어느 하나인 것을 특징으로 한다The degenerative brain diseases include but are not limited to stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, alcoholic brain disease, spinal cord injury injury or Alcoholic dementia and Wernicke-Korsakoff's syndrome.
또한, 상기 화합물은 Pi(Inorganic phosphate)에 의한 DJ-1 단백질의 응집을 억제하는 것을 특징으로 한다. In addition, the compound is characterized by inhibiting aggregation of DJ-1 protein by Pi (Inorganic phosphate).
또한, 상기 화합물은 Pi 또는 산화스트레스에 의한 세포사멸을 억제하는 것을 특징으로 한다.
In addition, the compound is characterized by inhibiting apoptosis by Pi or oxidative stress.
본 발명의 구체적인 실시예에서, 구체적으로 DJ-1을 과발현하는 SH-SY5Y세포 또는 Neuro-2A 세포에 무기인(Inorganic phosphate, Pi)을 처리하였을때 DJ-1이 응집되는 것을 확인하였고(도 1참조), 상기 화합물 1로 기재되는 본 발명의 타르타르산염이 DJ-1과 Pi의 결합부위에 경쟁적으로 결합하여 DJ-1/타르타르산염 복합체를 형성하며(도 2참조), SH-SY5Y세포 또는 Neuro-2A 세포에서 Pi로 유도한 DJ-1응집이 10 mM 또는 25 mM 타르타르산염 처리에 의해 억제되었고(도 3 내지 도 4참조), Pi 또는 활성산소로 유도된 세포사멸을 억제하는 것을 확인하여(도 5참조), 본 발명의 타르타르산염은 퇴행성뇌질환 예방 및 치료용 조성물로써 유용하게 사용될 수 있다.
In a specific example of the present invention, it was confirmed that when DJ-1 overexpressing SH-SY5Y cells or Neuro-2A cells were treated with inorganic phosphate ( Pi ), DJ-1 aggregated 1), the tartrate of the present invention described as Compound 1 competitively binds to the binding site of DJ-1 and Pi to form a DJ-1 / tartarate complex (see FIG. 2), and SH-SY5Y cells or DJ-1 aggregation induced by Pi in Neuro-2A cells was inhibited by treatment with 10 mM or 25 mM tartarate (see FIGS. 3 to 4), confirming inhibition of Pi or reactive oxygen induced cell death (See FIG. 5), the tartarate of the present invention can be usefully used as a composition for preventing and treating degenerative brain diseases.
본 발명은 화학식 1로 표시되는 화합물뿐만 아니라, 이의 약학적으로 허용되는 염, 이로부터 제조될 수 있는 가능한 용매화물, 수화물, 라세이체 또는 입체이성질체를 모두 포함한다.The present invention includes not only the compounds represented by the formula (1), but also pharmaceutically acceptable salts thereof, possible solvates, hydrates, racemates or stereoisomers thereof which can be prepared therefrom.
본 발명은 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용되는 염의 형태로 사용할 수 있으며, 염으로는 약학적으로 허용 가능한 유리산(free acid)에 의해 형성된 산 부가염이 유용하다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요오드화수소산, 아질산 또는 아인산과 같은 무기산류와 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸이도에이트, 방향족 산류, 지방족 및 방향족 설폰산류와 같은 무독성 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염류로는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트,니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 디하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 숙시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, 하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트 또는 만델레이트를 포함한다.
The present invention can be used in the form of a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Derived from non-toxic organic acids, such as, for example, diesters, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinic acid, succinic acid, succinic acid, succinic acid, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluene sulfonate, chlorobenzene sulfoxide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.
본 발명에 따른 산 부가염은 통상의 방법, 예를 들면 화학식 1로 표시되는 화합물을 과량의 산 수용액 중에 용해시키고, 이 염을 수혼화성 유기 용매, 예를 들면 메탄올, 에탄올, 아세톤 또는 아세토니트릴을 사용하여 침전시켜서 제조할 수 있다. 또한, 동량의 화학식 1로 표시되는 화합물, 및 산 수용액 또는 알코올을 가열하고, 이어서 이 혼합물을 증발시켜서 건조하거나 또는 석출된 염을 흡입 여과시켜 제조할 수도 있다.
The acid addition salt according to the present invention can be produced by a conventional method, for example, by dissolving the compound represented by the formula (1) in an excess amount of an acid aqueous solution, and adding the salt to a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile To precipitate it. It is also possible to prepare the same by heating the compound represented by the general formula (1) and an acid aqueous solution or alcohol, followed by evaporating the mixture or drying the precipitated salt by suction filtration.
또한, 염기를 사용하여 약학적으로 허용가능 한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리 토금속 염은, 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조 시켜 얻는다. 이때, 금속염으로는 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하다. 또한, 이에 대응하는 은 염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 음염(예, 질산은)과 반응시켜 얻는다.
In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).
본 발명의 약학적 조성물은 상기 화합물 1에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 투여를 위해서는 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 산제, 정제, 캡슐제, 환, 과립 또는 주사액제로 제제화 할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The pharmaceutical composition of the present invention may further contain one or more active ingredients which exhibit the same or similar functions in addition to the compound (1). For administration, one or more additional pharmaceutically acceptable carriers may be prepared. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. If necessary, an antioxidant, Other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, powders, tablets, capsules, rings, granules or injection solutions. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.
본 발명의 약학적 조성물은, 통상적인 방법에 의해 정제, 캅셀제, 산제, 과립제, 현탁제, 유제, 시럽제, 기타 액제로 제형화될 수 있다.The pharmaceutical composition of the present invention can be formulated into tablets, capsules, powders, granules, suspensions, emulsions, syrups and other liquid preparations by a conventional method.
구체적으로 본 발명의 약학적 조성물은 경구 투여용 제형, 예를 들면 정체, 트로치제(troches), 로젠지(lozenge), 수용성 또는 우성현탁액, 조제분말 또는 과립, 에멀젼, 하드 또는 소프트 캡슐, 시럽 또는 엘릭시르제(elixirs)로 제제화된다. 정제 및 캡슐 등의 제형으로 제제하기 위해 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴과 같은 결합제, 디칼슘 포스페이트와 같은 부형제, 옥수수 전분 또는 고구마 전분과 같은 붕해제, 스테아르산 마르네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌글리콜 왁스와 같은 윤활유가 함유된다. 캡슐제형의 경우는 상기에서 언급한 물질 이외에도 지방유와 같은 액체 담체를 함유한다.Specifically, the pharmaceutical compositions of the present invention can be formulated for oral administration, for example, as tablets, troches, lozenges, aqueous or aqueous suspensions, prepared powders or granules, emulsions, hard or soft capsules, It is formulated into elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, disintegrants such as starch, , Calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.
또한, 본 발명의 약학적 조성물은 경구 또는 비경구 투여할 수 있으며, 비경구 투여시 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식을 선택하는 것이 바람직하다. 비경구 투여용 제형으로 제제화하기 위해서는 본 발명의 화합물 1과 함께 물에서 혼합하여 현탁액으로 제조하고 이를 앰플 또는 바이알의 단위 투여형으로 제제한다.In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally, and it is preferable to select subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection injection method for parenteral administration. For formulation into parenteral administration formulations,
본 발명에 따른 유효성분의 투여량은 체내에서 활성성분의 흡수도, 불활성화율 및 배설속도, 환자의 연령, 성별 및 상태, 치료할 질병의 중증 정도에 따라 적절히 선택되나, 경구 투여제의 경우 일반적으로 성인에게 1일에 체중 1 kg당 본 발명의 화합물1을 0.0001 ~ 500 mg의 양으로 1회 내지 수회 나누어 투여할 수 있으며, 0.001 ~ 100 mg의 양으로 투여하는 것이 바람직하다.The dosage of the active ingredient according to the present invention is appropriately selected depending on the degree of absorption, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, and severity of the disease to be treated. The
본 발명의 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.
The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.
아울러, 본 발명은 하기 화학식 1로 표시되는 화합물, 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 퇴행성뇌질환의 개선용 건강기능식품을 제공한다:In addition, the present invention provides a health functional food for improving degenerative brain diseases comprising, as an active ingredient, a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[화학식 1] [ Chemical Formula 1 ]
상기 퇴행성뇌질환은 뇌졸중(Stroke), 파킨슨병(Parkinson's disease), 알츠하이머 치매(Alzheimer's disease), 헌팅톤병(Huntington's disease), 근위축성 측삭 경화증(amyotrophic lateral sclerosis), 알코올성 뇌신경질환, 척수손상(spinal cord injury) 또는 알코올성 치매 및 베르니케-코르사코프 증후군(Wernicke-Korsakoff's syndrome)으로 이루어지는 군으로부터 선택되는 어느 하나인 것을 특징으로 한다.
The degenerative brain diseases include but are not limited to stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, alcoholic brain disease, spinal cord injury injury or Alcoholic Dementia and Wernicke-Korsakoff's Syndrome.
본 발명의 구체적인 실시예에서, 구체적으로 DJ-1을 과발현하는 SH-SY5Y세포 또는 Neuro-2A 세포에 무기인(Inorganic phosphate, Pi)을 처리하였을때 DJ-1이 응집되는 것을 확인하였고(도 1참조), 상기 화합물 1로 기재되는 본 발명의 타르타르산염이 DJ-1과 Pi의 결합부위에 경쟁적으로 결합하여 DJ-1/타르타르산염 복합체를 형성하며(도 2참조), SH-SY5Y세포 또는 Neuro-2A 세포에서 Pi로 유도한 DJ-1응집이 10 mM 또는 25 mM 타르타르산염 처리에 의해 억제되었고(도 3 내지 도 4참조), Pi 또는 활성산소로 유도된 세포사멸을 억제하는 것을 확인하여(도 5참조), 본 발명의 타르타르산염은 퇴행성뇌질환의 개선용 건강기능식품으로써 유용하게 사용될 수 있다.
In a specific example of the present invention, it was confirmed that when DJ-1 overexpressing SH-SY5Y cells or Neuro-2A cells were treated with inorganic phosphate ( Pi ), DJ-1 aggregated 1), the tartrate of the present invention described as
본 명세서의 "건강기능식품"이란 일상 식사에서 결핍되기 쉬운 영양소나 인체에 유용한 기능을 가진 원료나 성분 (기능성 원료)을 사용하여 제조한 것으로, 인체의 정상적인 기능을 유지하거나 생리기능 활성화를 통하여 건강을 유지하고 개선하는 식품으로 식품의약품안전처장이 정한 것을 의미하나, 이에 한정되지 않으며 통상적인 의미의 건강식품을 배제하는 의미로 사용된 것이 아니다.As used herein, the term "health functional food" is produced by using raw materials or ingredients (functional raw materials) having functions useful for nutrients or human body that are likely to be deficient in daily eating, and is intended to maintain the normal function of the human body, But is not limited to, and is not meant to exclude health food in the usual sense.
본 발명의 건강기능식품은 화합물 1을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다.The health functional food of the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods.
또한, 본 발명의 건강기능식품은 식품학적으로 허용 가능한 식품 보조 첨가제를 더 포함한다. 본 발명에 이용될 수 있는 식품학적으로 허용가능한 식품 보조 첨가제는 포도당, 과당, 말토스, 슈크로스, 덱스트린, 시클로덱스트린과 같은 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알코올과 같은 천연 탄수화물, 타우마틴, 스테비아 추출물 등의 천연 향미제, 사카린, 아스파르탐 등의 합성 향미제, 착색제, 펙트산 또는 그의 염, 알긴산 또는 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산화제 등을 포함하나, 이에 한정되는 것은 아니다.In addition, the health functional food of the present invention further comprises a pharmaceutically acceptable food-aid additive. Food-acceptable food supplementary additives that may be used in the present invention include sugars such as glucose, fructose, maltose, sucrose, dextrin, cyclodextrins, natural carbohydrates such as sugar alcohols such as xylitol, sorbitol, erythritol, Natural flavors such as martin and stevia extract, synthetic flavors such as saccharin and aspartame, colorants, pectic acid or its salts, alginic acid or its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin , Alcohols, carbonating agents, and the like, but are not limited thereto.
상기 외에 본 발명의 건강기능식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등) 등을 함유할 수 있다.
In addition to the above, the health functional food of the present invention may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants, and thickening agents (cheese, chocolate, etc.).
이하, 본 발명을 실시예 및 실험예에 의해서 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.
단 하기 실시예 및 실험예는 본 발명을 예시하기 위한 것일 뿐, 본 발명이 하기 실시예 및 실험예에 의해서 한정되는 것은 아니다.
EXAMPLES The following Examples and Experiments are for the purpose of illustrating the present invention, but the present invention is not limited by the following Examples and Experimental Examples.
<실시예 1> 세포배양 및 DJ-1 형질주입(transfection)≪ Example 1 > Cell culture and DJ-1 transfection
DJ-1의 형질주입세포를 제조하기 위해 하기와 같은 실험을 수행하였다. 구체적으로 사람 및 쥐(mouse) 뉴로블라스토마(neuroblastoma) 세포 SH-SY5Y 또는 Neuro-2A를 10% 소태아혈청(fetal bovine serum)이 포함된 RPMI1640 배지(Gibco)에서 배양하였다(37℃, 5% CO2). DJ-1의 형질주입을 위해서, Lipofectamine 2000 (Life Technologies)을 이용하여 제조사의 지침에 따라 Neuro-2A 또는 SH-SY5Y세포에 DJ-1을 형질주입 하였다.
The following experiment was carried out to prepare transfected cells of DJ-1. Specifically, human and mouse neuroblastoma cells SH-SY5Y or Neuro-2A were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (37 ° C, 5% CO2). For the transfection of DJ-1, use either Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions for Neuro-2A or SH-SY5Y cells DJ-1 was transfected.
<실시예 2> DJ-1 분리, 결정화 및 구조분석<Example 2> DJ-1 separation, crystallization and structural analysis
본 발명자들은 상기 실시예 1의 세포로부터 재조합 DJ-1을 분리 및 분석을 위해 하기와 같은 실험을 수행하였다. 구체적으로, DJ-1 단백질은 상기 <실시예 1>의 DJ-1 유전자가 형질도입된 세포로부터 S.J. Lee et al(J. Biol. Chem., 278, 44552-44559(2003))에 개시된 방법에 의해 분리하고, hanging drop vapor diffusion를 이용하여 결정화 하였다. DJ-1 결정(P3121; a = b = 74.80 Å, c = 75.82 Å; 단량체)은 23℃에서 2 ul의 단백질 용액(~12 mg/ml)을 포함하는 작은 방울(droplets)에서 자랐으며, 1.5 M 타르타르산칼륨나트륨(sodium potassium tartrate), 3.5 M 황화 암모늄(ammonium sulfate) 및 0.1 M 나트륨 카코딜산염(Na-Cacodylate, pH 6.5)을 포함하는 동일한 부피의 침전용액의 회절 해상도(resolution diffraction, A 1.66 Å) 데이터를(표 1) HKL2000을 이용하여 100K의 cryostream 에서 냉각된 결정과 함께 수집하였다. 위상(phasing) 정보는 DJ-1 단량체(PDB 코드 : IJ42)를 검색모델로 하여 PHENIX를 이용한 분자대체(molecular replacement)으로 관찰하였다. 모델 설계 및 정제(refinement)는 COOT, REFMAC5 및 CCP4 suite를 이용하여 실시하였다. The present inventors carried out the following experiments to isolate and analyze recombinant DJ-1 from the cells of Example 1 above. Specifically, the DJ-1 protein was prepared from the cells transduced with the DJ-1 gene of Example 1 described above by the method described in SJ Lee et al (J. Biol. Chem., 278, 44552-44559 (2003) And crystallized using hanging drop vapor diffusion. Monomer) was grown in droplets containing 2 μl of protein solution (~ 12 mg / ml) at 23 ° C and 1.5 μl of protein solution (~ 12 mg / ml) A solution of the same volume of precipitation solution containing sodium potassium tartrate, 3.5 M ammonium sulfate and 0.1 M sodium cacodylate, pH 6.5, A) data were collected (Table 1) along with crystals cooled in a 100K cryostream using HKL2000. The phasing information was observed by molecular replacement using PHENIX using the DJ-1 monomer (PDB code: IJ42) as a retrieval model. Model design and refinement were performed using COOT, REFMAC5 and CCP4 suite.
그 결과, 표 1에 나타난 바와 같이 DJ-1/타르타르산염 혼합체의 최종모델은 14.9%/16.9%의 R work / R free 를 가지며, 3내지 188의 잔기를 포함하고, 한개의 타르타르산염과 229개의 물분자를 포함한다. 입체적 구조는 PROCHEC를 이용하여 입증하였다. As a result, as shown in Table 1, the final model of the DJ-1 / tartarate mixture had R work / R free of 14.9% / 16.9%, containing 3 to 188 residues, and one tartrate salt and 229 Water molecules. The three-dimensional structure was verified using PROCHEC.
α = β = 90°, γ = 120° a = b = 74.80, c = 75.82,
α = β = 90 °, γ = 120 °
<실험예 1> Inorganic phosphate(Pi)에 의한 DJ-1 응집(aggregation)EXPERIMENTAL EXAMPLE 1 DJ-1 aggregation by inorganic phosphate (Pi)
DJ-1 이량체(dimer)는 Pi를 매개로 선형으로 적체되어 프로토필라멘트(protofilament)를 형성하며 이들이 다량 모여 사상형(filamentous) 구조를 형성하는 것으로 알려져 있다. 본 발명자들은 Pi가 DJ-1의 응집을 유발하는지 확인하기 위하여, DJ-1이 형질주입된 Neuro-2A 세포에 2mM의 인산나트륨(sodium phosphate, NaH2PO4)를 처리한 뒤 DJ-1의 세포내 발현을 면역형광염색을 이용하여 확인하였다. The DJ-1 dimer forms a protofilament that is linearly packed via Pi and is known to form a filamentous structure by gathering a large amount of protofilaments. To confirm whether Pi causes aggregation of DJ-1, DJ-1 was transfected with 2 mM sodium phosphate (NaH 2 PO 4 ) to transfected Neuro-2A cells, and DJ-1 Intracellular expression was confirmed by immunofluorescence staining.
구체적으로, 대조군 또는 실험군의 세포를 2% 파라포름알데히드(paraformaldehyde) 및 0.1% 트리톤 X100이 포함된 PBS(phosphate buffered saline)버퍼로 30분 동안 고정시키고, PBS로 3회 세척한 뒤, 10% 염소혈청(goat serum)이 포함된 PBS 버퍼와 반응시켰다. DJ-1 일차항체와 반응시킨 후, 0.1% 트리톤 X100이 포함된 PBS로 3회 세척한 뒤, 이차항체 및 핵을 염색하는 DAPI와 반응시킨 뒤, PBS로 세척하여 immunofluorescence microscope으로 DJ-1 발현을 확인하였다. Specifically, cells in the control or experimental group were fixed with PBS (phosphate buffered saline) buffer containing 2% paraformaldehyde and 0.1% Triton X100 for 30 minutes, washed three times with PBS, And reacted with PBS buffer containing goat serum. After washing with PBS containing 0.1% Triton X100, the cells were reacted with DAPI staining secondary antibody and nuclei, washed with PBS, and analyzed by immunofluorescence microscope for DJ-1 expression. Respectively.
그 결과, 도 1에서 나타난 바와 같이 Neuro-2A세포에서 2 mM 인산나트륨 처리군에서 DJ-1이 반점형태를 형성하는 것을 관찰하였고, DJ-1이 세포내 응집되는 것을 확인하였다(도 1). 또한, SH-SY5Y세포에서 2 mM 인산나트륨 처리에 의해 세포질내에 봉입체(inclusion)가 형성되는 것을 확인함으로써, 상기 결과로부터 Pi에 노출된 DJ-1은 선천적 입체구조를 잃고 응집되는 것을 알 수 있다.
As a result, as shown in Fig. 1, it was observed that DJ-1 formed spot form in 2 mM sodium phosphate-treated group in Neuro-2A cells, and DJ-1 was found to coagulate in the cells (Fig. 1). Furthermore, confirming inclusion formation in the cytoplasm by treatment with 2 mM sodium phosphate in SH-SY5Y cells, it can be seen from the above results that DJ-1 exposed to Pi loses its conformational conformation and coagulates.
<< 실험예Experimental Example 2> DJ-1/ 2> DJ-1 / 타르타르산염Tartarate (( tartratetartrate ) 복합체의 결정구조(crystal structure)) The crystal structure of the complex
본 발명자들은 DJ-1/타르타르산염 복합체의 결정구조를 확인하기 위하여 하기와 같은 실험을 수행하였다. The present inventors conducted the following experiment to confirm the crystal structure of the DJ-1 / tartarate complex.
구체적으로 상기 <실시예 2>에서 수득한 <실시예 2>의 방법으로 인산버퍼를 이용하여 정제한 재조합 DJ-1(pDJ-1)은 사상형으로 응집되어 있는데(도 2A), 정제된 pDJ-1은 0.4 M 타르타르산칼륨나트륨, 2.0 M 황화 암모늄 및 0.1 M 나트륨 카코딜산염(pH 6.5)을 포함하는 결정화 용액에서 막대형태 결정을 형성하였다. 그러나, 타르타르산염의 농도가 상기용액보다 더 높은 경우 (1.5 M 타르타르산칼륨나트륨, 3.5 M 황화 암모늄 및 0.1 M 나트륨 카코딜산염(pH 6.5))는 사방육면체의(rhombohedron) 결정을 얻지 못하였다. 사방육면체 결정은 DJ-1 이량체의 특징이므로, 그 올리고머 상태를 검사하여 pDJ-1의 사방육면체 구조를 해결했다. 그 결과, 1.66 Å 해상도 결정 구조에 따르면, pDJ-1은 사방육면체 구조에서 이량체로 존재하며(도 2B), 상기 이량체 구조는 DJ-1과 동일하다. 또한, pDJ-1의 주요한 특징은 Pi 결합 부위에 Pi 대신 타르타르산염이 존재한다는 것이다(도 2C). 타르타르산염과 Pi의 결합은 Arg48의 구아니듐(guanidium)기의 곁사슬(side-chain) 및 Asn76의 -NH기의 백본(backbone)과 상호결합 한다는 점에서 유사하나, Asn76의 곁사슬 형태는 리간드 엔티티(ligand entity)에 의해 영향받는다.
Specifically, recombinant DJ-1 (pDJ-1) purified using a phosphate buffer according to the method of Example 2 obtained in Example 2 was coagulated (FIG. 2A), and purified pDJ -1 formed rod-shaped crystals in a crystallization solution containing 0.4 M potassium sodium tartrate, 2.0 M ammonium sulphate and 0.1 M sodium crocodylate (pH 6.5). However, when the concentration of the tartaric acid salt was higher than that of the solution (1.5 M potassium sodium tartrate, 3.5 M ammonium sulphate and 0.1 M sodium crocodylate (pH 6.5)), no rhombohedron crystals were obtained. Since the hexagonal hexagonal crystals are a feature of the DJ-1 dimer, the oligomer state of pDJ-1 was solved by examining its oligomer state. As a result, according to the 1.66 Å resolution crystal structure, pDJ-1 exists as a dimer in the tetragonal hexahedron structure (FIG. 2B), and the dimer structure is the same as DJ-1. In addition, a major feature of pDJ-1 is that tartarate exists instead of Pi at the Pi binding site (Fig. 2C). The bond between tartrate salt and Pi is similar in that it binds with the side chain of the guanidium group of Arg48 and the backbone of the -NH group of Asn76 but the side chain form of Asn76 is a ligand entity (ligand entity).
<실험예 3> 타르타르산염의 Pi로 유도된 DJ-1응집에 대한 저해 효과<Experimental Example 3> Inhibitory effect of tartarate salt on Pi-induced DJ-1 aggregation
<실험예 2>에서 분석한 DJ-1/타르타르산염의 복합체의 존재는 타르타르산염이 Pi가 DJ-1에 결합하여 응집을 유도하는 부위와 동일한 부위에 경쟁적으로 결합하는 것을 의미한다. 따라서, 본 발명자들은 타르타르산염이 Pi에 의한 DJ-1응집을 억제하는지 확인하고자, 하기와 같이 실험하였다. The presence of the DJ-1 / tartarate complex analyzed in Experimental Example 2 means that the tartarate binds competitively to the same site as Pi binds to DJ-1 and induces aggregation. Therefore, the present inventors conducted experiments as follows to confirm whether tartarate inhibits DJ-1 aggregation by Pi.
구체적으로 Neuro-2A 또는 SH-SY5Y 세포에 인산나트륨 및 타르타르산염을 처리한 뒤 면역화학염색을 실시하였다. Specifically, Neuro-2A or SH-SY5Y cells were treated with sodium phosphate and tartarate, followed by immunochemical staining.
그 결과, 도 3에 나타난 바와 같이 2 mM의 인산 나트륨 처리에 의해 DJ-1이 응집되고, 상기 DJ-1 응집이 10 mM 또는 25 mM의 타르타르산염 처리에 의해 감소하는 것을 확인하였다(도 3 A, B). Neuro-2A 세포에서 10 mM 또는 25 mM의 타르타르산염처리군 모두에서 DJ-1응집을 억제하였고, SH-SY5Y 세포에서는 10 mM의 타르타르산염 처리군에서 DJ-1이 응집이 관찰되기는 하지만 그 규모가 대조구(2 mM 인산나트륨)에 비하여 완화되었으며, 25 mM 타르타르산염 처리군에서는 DJ-1응집이 완전히 억제되었다. 상기 결과는 타르타르산염이 Pi에 의해 유도된 DJ-1응집을 억제하는 것을 의미한다.
As a result, it was confirmed that DJ-1 aggregated by treatment with 2 mM sodium phosphate as shown in FIG. 3, and the DJ-1 aggregation was reduced by treatment with 10 mM or 25 mM of tartarate (FIG. 3A , B). DJ-1 aggregation was inhibited in Neuro-2A cells treated with 10 mM or 25 mM of tartarate. In SH-SY5Y cells, DJ-1 aggregation was observed in the 10 mM tartarate-treated group, DJ-1 aggregation was completely inhibited in the 25 mM tartarate-treated group compared to the control (2 mM sodium phosphate). The above results indicate that tartarate inhibits Pi-induced DJ-1 aggregation.
<< 실험예Experimental Example 4> 4> 타르타르산염의Tartarate 산화스트레스로With oxidative stress 유도된 DJ- The induced DJ- 1응집에1 to flocculation 대한 저해 효과 Inhibitory effect on
산화스트레스는 파킨슨병이나 알츠하이머병의 주요한 병인이며 특발성 파킨슨병 환자의 뇌에서 DJ-1이 산화적으로 손상받는 것이 보고되어 있고, 이러한 뇌질환에서 높은 수준의 산화 스트레스가 단백질 응집을 유발하는 기전으로 제시되고 있다. 따라서 본 발명자들은 타르타르산염이 산화 스트레스 및 Pi에 의한 DJ-1의 응집을 억제하는지 확인하고자 하였다. Oxidative stress is a major pathogenesis of Parkinson's disease or Alzheimer's disease and it has been reported that DJ-1 is oxidatively damaged in the brain of patients with idiopathic Parkinson's disease, and a high level of oxidative stress in such brain diseases leads to protein aggregation Is presented. Therefore, the inventors of the present invention sought to determine whether tartaric acid salt inhibits oxidative stress and aggregation of DJ-1 by Pi.
그 결과, 도 4에 나타낸 바와 같이 SH-SY5Y 세포에서 100 uM의 과산화수소(H2O2)에 의해 DJ-1 응집이 유도되고, 2 mM의 인산나트륨과 100 uM의 과산화수소의 혼합처리에 의해 DJ-1의 응집이 더욱 심화되엇으나, 10 mM 및 25 mM의 타르타르산염의 처리에 의하여 상기 과산화수소 또는 과산화수소 및 Pi의 혼합처리에 의한 DJ-1의 응집이 억제되는 것을 확인하였다(도 4). 상기 결과는 타르타르산염이 Pi 및 산화스트레스에 의한 DJ-1 응집을 억제할 수 있음을 의미한다.
As a result, as shown in Fig. 4, DJ-1 aggregation was induced by 100 uM hydrogen peroxide (H2O2) in SH-SY5Y cells and DJ-1 aggregation was induced by mixing 2 mM sodium phosphate and 100 uM hydrogen peroxide However, it was confirmed that the aggregation of DJ-1 by the treatment with hydrogen peroxide or hydrogen peroxide and Pi was inhibited by treatment of 10 mM and 25 mM of tartaric acid salt (FIG. 4). The above results indicate that tartarate salt can inhibit DJ-1 aggregation by Pi and oxidative stress.
<실험예 5> 타르타르산염의 세포사멸 억제 효과Experimental Example 5 Effect of tartaric acid salt on cell death
타르타르산염이 Pi 및 산화스트레스에 의한 세포사멸을 억제하는 효과가 있는지 확인하기 위하여 본 발명자들은 인산화 나트륨 및 과산화수소를 처리하고 타르타르산염을 처리한 뒤 MTT 분석을 이용하여 세포 생존률을 측정하였다. In order to determine whether tartarate has an effect of inhibiting apoptosis caused by Pi and oxidative stress, the present inventors measured cell viability using MTT assay after treatment with sodium phosphate and hydrogen peroxide and treatment with tartarate.
구체적으로 96 well 플레이트에 1 X 104 농도로 SH-SY5Y 세포를 분주하고, 2 mM의 인산화 나트륨, 100 uM의 과산화수소 또는 타르타르산염을 처리하였다. 16시간 후, 0.5 mg/mL의 MTT를 함유하는 RPMI배지 100 ul을 첨가하고, 37℃에서 3시간 동안 배양한 뒤, DMSO(dimethyl sulfoxide)를 첨가하여 진한 파란색의 포마잔(formazan) 결정을 용해시켜 ELISA(enzyme-linked immunosorbent assay) 분석기를 이용하여 570 nm에서 흡광도를 측정하였다. Specifically, SH-SY5Y cells were seeded at a concentration of 1 × 10 4 in a 96-well plate and treated with 2 mM sodium phosphate, 100 μM hydrogen peroxide or tartarate. After 16 hours, 100 μl of RPMI medium containing 0.5 mg / ml of MTT was added and cultured at 37 ° C. for 3 hours. DMSO (dimethyl sulfoxide) was added to dissolve the dark blue formazan crystals And the absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) analyzer.
그 결과, 도 5에 나타낸 바와 같이 SH-SY5Y 세포의 생존율이 대조구에 비하여 인산화나트륨 또는 인산화나트륨 및 과산화수소 처리에 의해 감소하였고, 5, 10 또는 25 mM의 타르타르산염에 의해 세포생존율이 증가하는 것을 확인하였다(도 5). 상기 결과를 통해 타르타르산염이 Pi 또는 산화스트레스에 의한 세포사멸을 억제하는 효과가 있음을 확인하였다.
As a result, as shown in FIG. 5, the survival rate of SH-SY5Y cells was decreased by treatment with sodium phosphate or sodium phosphate and hydrogen peroxide compared to the control, and the cell survival rate was increased by 5, 10 or 25 mM of tartarate (Fig. 5). From the above results, it was confirmed that tartarate inhibits cell apoptosis by Pi or oxidative stress.
Claims (6)
[화학식 1]
A pharmaceutical composition for preventing or treating a degenerative brain disease comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[ Chemical Formula 1 ]
The method according to claim 1, wherein the degenerative brain disease is selected from the group consisting of Stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, , Spinal cord injury or alcoholic dementia, and Wernicke-Korsakoff's syndrome. The pharmaceutical composition for preventing or treating brain diseases according to claim 1,
The pharmaceutical composition according to claim 1, wherein the compound inhibits aggregation of DJ-1 protein by Pi (Inorganic phosphate).
The pharmaceutical composition according to claim 1, wherein the compound inhibits apoptosis by Pi or oxidative stress.
[화학식 1]
A health functional food for improving degenerative brain diseases comprising, as an active ingredient, a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[ Chemical Formula 1 ]
6. The method of claim 5, wherein the degenerative brain disease is selected from the group consisting of Stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, A spinal cord injury or alcoholic dementia, and Wernicke-Korsakoff's syndrome. The health functional food for the improvement of degenerative brain diseases according to claim 1,
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