보고서 정보
주관연구기관 |
이화여자대학교 Ewha Womans University |
연구책임자 |
김대기
|
참여연구자 |
황성순
,
최선
|
보고서유형 | 1단계보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2017-09 |
과제시작연도 |
2016 |
주관부처 |
(범부처사업) NTIS |
등록번호 |
TRKO202000030050 |
과제고유번호 |
9991006086 |
사업명 |
범부처전주기신약개발 |
DB 구축일자 |
2020-09-26
|
키워드 |
제2형 당뇨병.담즙산 핵 수용체.장내 선택적인 담즙산 핵 수용체 조절물질.혈당조절.체중조절.Type II diabetes.FXR.gut-restricted FXR modulators.glucose homeostasis.body weight control.
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초록
▼
EW-8029 및 FexA1을 기반으로 선도후보물질 142개를 디자인하여 합성완료하고, 이중 123개 화합물의 항당뇨효능을 평가하였음. In vitro GLP-1 secretion test에서 25개의 화합물이 glucose가 없는 상황과 20mM glucose 농도 모두에서 Fexaramine과 FexA1보다 우수한 GLP-1 분비력을 보여주었음. 장내 L 세포인 mGLUTag 세포주에서 8개의 화합물이 FexA1 보다 강력하게 FXR target gene으로 잘 알려진 SHP와 FGF15의 유전자 발현을 동시에 증가시켰음. EW
EW-8029 및 FexA1을 기반으로 선도후보물질 142개를 디자인하여 합성완료하고, 이중 123개 화합물의 항당뇨효능을 평가하였음. In vitro GLP-1 secretion test에서 25개의 화합물이 glucose가 없는 상황과 20mM glucose 농도 모두에서 Fexaramine과 FexA1보다 우수한 GLP-1 분비력을 보여주었음. 장내 L 세포인 mGLUTag 세포주에서 8개의 화합물이 FexA1 보다 강력하게 FXR target gene으로 잘 알려진 SHP와 FGF15의 유전자 발현을 동시에 증가시켰음. EW-8019, EW-8062D, EW-8152, EW-8171H, EW-8196, 혹은 FexA1은 mice에서 최고 용량인 640mg/kg에서도 간에서 SHP 유전자 발현에 영향을 미치지 못한 반면, 장에서는 40mg/kg 용량부터 SHP와 FGF15 유전자 발현을 높혀 이들 화합물 모두가 장내에 선택적으로 작용하는 FXR agonist임을 확인하였음(장/간 선택성은 최소 8배 이상임). Nuclear hormone receptor assay에서 EW-8019와 EW-8027은 FexA1 및 Fexaramine 보다 높은 maximal response를 보였음. EW-8019와 EW-8027은 18종의 nuclear hormone receptor들 중에서 FXR에 대해서만 agonist efficacy를 보여주었음(선택성은 각각 최소 208배, 185배 이상). DIO mice와 ob/ob mice에 EW-8062D와 EW-8152를 0.5% CMC에 현탁액(거의 녹지 않음)으로 투여한 경우, 두 화합물 모두 장내 흡수가 일어나지 않아, 체중증가 억제 효과와 항당뇨효능이 관찰되지 않았음. 그러나, ob/ob mice에 EW-8019, EW-8027, EW-8062D 및 EW-8152를 corn oil에 현탁액(대부분 녹음)으로 투여한 경우, EW-8019와 EW-8152를 투여한 그룹에서 체중증가가 감소하는 경향이 보였으며, 통계상 유의미한 공복 혈당 감소, 통계상 유의미한 공복 혈중 인슐린 수치 감소, 통계상 유의미한 내당능 증가가 관찰되어, 이 두 화합물이 우수한 항당뇨효능를 가진 선도물질임을 확인하였음.
EW-8062D와 EW-8152를 0.5% CMC 현탁액으로, 또는 corn oil 현탁액으로 각각 rat에 경구로 40mg/kg 용량으로 투여 시, 전신 생체이용률은 두 화합물 모두 0%로서 장내에 선택적으로 작용하는 FXR agonist임을 확인하였음. EW-8062D 및 EW-8152를 corn oil에 현탁액 상태로 mice에 경구로 2g/kg 용량으로 1일 1회, 7일간 투여시, EW-8062D 및 EW-8152 투여 그룹은 corn oil을 먹은 대조군에 비하여 혈중 ALT 수치가 통계상 유의미있게 증가하는 것을 확인하였음.
(출처 : 요약서 3p)
Abstract
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Ⅳ. Results
1. Synthesis of 60 lead candidate compounds
a. 86 lead candidate compounds have been designed based on the lead compounds, EW-8029 and FexA1, and successfully synthesized. The structures of the synthesized compounds have been confirmed by 1H NMR, 13C NMR, IR, and HRMS.
b. Docking
Ⅳ. Results
1. Synthesis of 60 lead candidate compounds
a. 86 lead candidate compounds have been designed based on the lead compounds, EW-8029 and FexA1, and successfully synthesized. The structures of the synthesized compounds have been confirmed by 1H NMR, 13C NMR, IR, and HRMS.
b. Docking study of the selected compounds, (R)/(S)-FexA1, EW-8019,(R)/(S)-EW-8027, (R)/(S)-EW-8062D, and (R)/(S)-EW-8152 have been performed by using Glide SP method. All of the compounds showed the same binding mode as co-crystallized Fexaramine, and their docking scores were in the range of –11.274 ~ -12.832, those were the similar to that of Fexaramine’s one, –12.560.
2. Scale-up synthesis of FexA1 and two selected compounds (>25 g) for determination of in vivo efficacy, DMPK, and toxicity
a. Two compounds, EW-8062D and EW-8152 have been selected from 86 lead candidate compounds for in vivo efficacy test, DMPK, and toxicity study.
b. FexA1, EW-8062D, and EW-8152 have been successfully synthesized >25 g.
28.0 g of FexA1 have been synthesized by synthetic route B.
25.1 g of EW-8062D have been synthesized by synthetic route B.
27.3 g of EW-8152 have been synthesized by synthetic route A.
3. Synthesis of additional 30 lead candidate compounds
a. Additional 56 lead candidate compounds have been designed and successfully synthesized.
Some of the lead candidate compounds have methyl or ethyl group in place of deuterium. The structures of the synthesized compounds have been confirmed by 1H NMR, 13C NMR, IR, andHRMS.
b. Docking study of the selected compounds, EW-8171H and (R)/(S)-EW-8196 have been performed by using Glide SP method. All of the compounds showed the same binding mode as co-crystallized Fexaramine, and their docking scores were –12.012, -12.025, -11.821, respectively.
4. In vitro GLP-1 secretion test
a. In GLUTag cells pretreated with DMSO control, Fexaramine, and FexA1 in the absence of glucose, the amount of the secreted GLP-1 were 3.445347±1.162542pM, 3.342203±1.91053pM, and 3.54849±2.533176pM, respectively, showing no difference among the three groups. However, in GLUTag cells pretreated with DMSO control, Fexaramine, and FexA1 in the presence of glucose (20mM), the amount of the secreted GLP-1 were 4.06911±2.668209pM, 30.98462±5.379122pM, and 34.59463±1.960019pM, respectively,showing that Fexaramine and FexA1 enhanced GLP-1 secretion by 2.2-fold and 2.5-fold higher compared to that of DMSO control.
b. Among the tested 123 compounds, 25 compounds showed higher GLP-1 secretion than Fexaramine and FexA1 both in the absence of glucose and in the presence of glucose (20mM).
All of these compounds enhanced GLP-1 secretion more efficiently in response to glucose than in the absence of glucose.
c. Among the tested 123 compounds, EW-8062D and EW-8152 enhanced the highest GLP-1 secretion in the presence of glucose (20mM), 100.6064±7.71074pM and 87.91974±21.71219pM, respectively, showing 7.2-fold and 6.2-fold higher secretion than DMSO control, and 2.9-fold and 2.5-fold higher secretion than FexA1, respectively.
5. FXR target gene expression profiling and promotor assay in cell line
a. In GLUTag cells, among the tested 123 compounds, 8 compounds induced SHP and FGF15 gene expressions more than FexA1 in GLUTag cells.
b. Among the tested 123 compounds, EW-8062D, EW-8171H, and EW-8196 strongly induced classical FXR target genes, thus showing 3.3-fold, 2.7-fold, 2.2-fold higher SHP gene expression, and 1.5-fold, 2.1-fold, 2.1-fold higher FGF15 gene expression than FexA1, respectively.
6. Determination of PD in vivo: FXR target gene expression in ileum and liver in mice
a FXR target gene induction in ileum and liver has been examined in mice after 5-day oral treatment of EW-8019, EW-8062D, EW-8152, EW-8171H, EW-8196, or FexA1 with different doses (vehicle (corn oil), 10mg/kg, 20mg/kg, 40mg/kg, 80mg/kg, 160mg/kg, 320mg/kg,and 640mg/kg). All 6 compounds showed no significant change in SHP expression in liver up to a dose of 640mg/kg, but induced SHP and FGF15 expression in ileum from 40mg/kg, demonstrating that they are all gut-restricted FXR agonists (ileum/liver selectivity indices are>8).
b. EW-8019, EW-8062D, and EW-8152 induced expression of SHP in ileum 3~5-fold higher than FexA1 in tested doses.
7. Nuclear hormone receptor assay
a. In nuclear hormone receptor assay, FexA1 and Fexaramine showed 34% and 33% of maximal response to that of GW4064, a FXR full agonist control compound, respectively.
EW-8019 and EW-8027 showed 49% and 55% of maximal response to that of GW4064,respectively, demonstrating that they are more efficient FXR agonists than FexA1 and Fexaramine. EW-8056, EW-8056D, and EW-8150 showed 31%, 33%, and 32% of maximal response to that of GW4064, respectively, exhibiting that their agonist efficacy is similar to that of FexA1 and Fexaramine.
b. EC50 values of GW4064, FexA1, and Fexaramine were 0.15 uM, 0.46 uM, and 0.53 uM,respectively. EC50 values of EW-8019, EW-8027, EW-8056, EW-8056D, and EW-8150 were 0.48 uM, 0.54 uM, 0.35 uM, 0.39 uM, and 0.55 uM, respectively, showing that their values are either lower or equivalent to those of FexA1 and Fexaramine.
8. Nuclear hormone selectivity assay
EW-8019 and EW-8027 showed 46.6% and 52.8% of maximal response to that of GW4064, respectively, at a concentration of 100 uM in FXR receptor assay, however, they did not showed agonist efficacy in a panel of other 17 nuclear hormone receptor assay. EC50 values of EW-8019 and EW-8027 were confirmed to be 0.48 uM and 0.54 uM, respectively, in FXR receptor assay, therefore, their selectivity indices against 17 other nuclear hormone receptor are higher than >208 and >185, respectively.
9. In vivo study (DIO mice: Con (-), EW-8062D, EW-8152; administration vehicle: 0.5% CMC)
a. Either EW-8062D treatment group (40mg/kg, suspension in 0.5% CMC) or EW-8152 treatment group (40mg/kg, suspension in 0.5% CMC) did not show statistically significant weight-reducing effect compared to Con (-) (0.5% CMC) in DIO mice after 6-week oral treatment.
b. There were no differences between drug treatment groups and Con (-) group in non-fasting blood glucose, HbA1c (%), and non-fasting plasma insulin.
c. In OGTT test, there were no differences between drug treatment groups and Con (-) group in fasting blood glucose, blood glucose, AUC of blood glucose, and plasma insulin secretion after 5-week oral treatment.
10. In vivo study (ob/ob mice: control, FexA1, EW-8062D, EW-8152; administration vehicle: 0.5% CMC)
a. Among the 3 drug treatment groups, EW-8152 treatment group (40mg/kg, suspension in 0.5% CMC) showed weight-reducing effect that was not statistically significant compared to Con(-) (0.5% CMC) in ob/ob mice after 4-week oral treatment and then 5-week follow-upperiod.
b. There were no differences between either FexA1 treatment group or EW-8062D treatment group and Con (-) group in fasting blood glucose, HbA1c (%), and fasting plasma insulin. But, EW-8152 treatment group showed elevation of blood glucose due to decreased insulinsecretion.
c. In OGTT test, EW-8152 induced deterioration in glucose tolerance compared to Con (-) treatment due to decreased insulin secretion after 4-week oral treatment.
11. In vivo study (ob/ob mice: control, EW-8019, EW-8027, EW-8062D, EW-8152; administration vehicle: corn oil)
a. Among the 4 drug treatment groups, EW-8019 treatment group (80mg/kg, suspension in corn oil) and EW-8152 treatment group (80mg/kg, suspension in corn oil) showed weight-reducingeffect that was not statistically significant compared to Control (corn oil) in ob/ob mice after4-week oral treatment.
b. EW-8019 treatment group and EW-8152 treatment group reduced fasting glucose that was statistically significant compared to Control in ob/ob mice after 4-week oral treatment.
c. EW-8019 treatment group and EW-8152 treatment group markedly reduced fasting plasma insulin that was statistically significant compared to Control in ob/ob mice after 4-week oral treatment.
d. In OGTT test, EW-8019 and EW-8152 induced improvement in glucose tolerance compared to Control in ob/ob mice after 4-week oral treatment.
e. Blood glucose levels for either EW-8019 treatment group or EW-8152 treatment group were slowly elevated in ob/ob mice after 4-week oral treatment and then 3-week follow-up period(EW-8019 treatment group: from 158mg/dL to 180mg/dL; EW-8152 treatment group: from160mg/dL to 185mg/dL).
12. DMPK: analysis of PK profile of 2 selected lead candidate compounds in rats after a single oral administration
a. When an ester compound, EW-8062D in DMAC formulation was administered to rat iv, this compound was rapidly and completely metabolized to an acid compound, EW-8063D in blood.
b. When a solution of EW-8062D in DMAC formulation was administered to rat orally at a dose of 40mg/kg, its metabolite, EW-8063D only was detected in plasma. Oral bioavailability ofEW-8063D was 5.3%.
c. When a suspension of EW-8062D in DMAC formulation was administered to rat orally at a dose of 320mg/kg, its metabolite, EW-8063D only was detected in plasma. Oral bioavailabilityof EW-8063D was 2.2%.
d. When a suspension of EW-8062D in corn oil was administered to rat orally at a dose of 40mg/kg, its metabolite, EW-8063D only was detected in plasma. Oral bioavailability of EW-8063D was 6.6%.
e. When a suspension of EW-8062D in 0.5% CMC was administered to rat orally at a dose of 40mg/kg, neither EW-8062D nor EW-8063D was detected in plasma.
f. When an ester compound, EW-8152 in DMAC formulation was administered to rat iv, this compound was rapidly metabolized to an acid compound, EW-8153 in blood. EW-8152 and EW-8153 were present in plasma in a ratio of 2.3%: 97.7% in terms of AUC0-t..
g. When a solution of EW-8152 in DMAC formulation was administered to rat orally at a dose of 40mg/kg, oral bioavailability of EW-8152 and its metabolite, EW-8153 were 2.0% and 19.0%, respectively.
h. When a suspension of EW-8152 in DMAC formulation was administered to rat orally at a dose of 320mg/kg, oral bioavailability of EW-8152 and its metabolite, EW-8153 were 1.0%and 10.3%, respectively.
i. When a suspension of EW-8152 in corn oil was administered to rat orally at a dose of 40mg/kg, its metabolite, EW-8153 only was detected in plasma. Oral bioavailability of EW-8153 was 17.6%.
j. When a suspension of EW-8152 in 0.5% CMC was administered to rat orally at a dose of 40mg/kg, neither EW-8152 nor EW-8153 was detected in plasma.
13. Toxicity: determination of toxicity of two selected compounds (EW-8062D and EW-8152) after oral administration of a high dose to mice
a. After a suspension of either EW-8062D or EW-8152 in corn oil was administered to mice orally at a daily dose of 2g/kg for 7days, no macroscopic changes in various organs including spleen were observed in sacrificed animals.
b. Treatment of either EW-8062D or EW-8152 elevated blood ALT value that was statistically significant compared to that of corn-oil-treated control group. However, these elevated ALT values were markedly lower compared to the value observed in Acetaminophen-treated positive control group.
(출처 : SUMMARY 10p)
목차 Contents
- 표지 ... 1
- 제출문 ... 2
- 보고서 요약서 ... 3
- 요약문 ... 4
- SUMMARY ... 9
- CONTENTS ... 15
- 목차 ... 16
- 제1장 연구개발과제의 개요 ... 17
- 제1절 타겟 환자군 (Target Patient Population) ... 17
- 제2절 미충족 의학적 수요 (Unmet Medical Needs) ... 19
- 제3절 시장규모와 성장세 (Size and Growth) ... 20
- 제2장 국내외 기술개발 현황 ... 22
- 제1절 전신에 작용하는 FXR full agonist 개발 현황 ... 22
- 제2절 장에 제한적으로 작용하는 partial agonist 개발 현황 ... 22
- 제3장 연구개발수행 내용 및 결과 ... 24
- 제1절 선도물질 합성 ... 24
- 제2절 In vitro 효능 검증 ... 37
- 제3절 In vivo 효능 검증 ... 54
- 제4절 DMPK ... 72
- 제5절 독성 ... 91
- 제4장 목표달성도 및 관련분야에의 기여도 ... 92
- 제1절 선도물질 합성 ... 92
- 제2절 in vitro 효능 검증 ... 92
- 제3절 in vivo 효능 검증 ... 93
- 제4절 DMPK ... 94
- 제5절 독성 ... 94
- 제5장 연구개발결과의 활용계획 ... 95
- 제1절 추가연구의 필요성 ... 95
- 제2절 다른 연구에의 응용 ... 95
- 제3절 기업화 추진방안 ... 95
- 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 96
- 제7장 연구시설ㆍ장비 현황 ... 97
- 제8장 참고문헌 ... 98
- 끝페이지 ... 100
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