Anti-PCSK9 compounds and methods for the treatment and/or prevention of cardiovascular diseases

A compound and heart disease technology, applied in cardiovascular system diseases, active ingredients of heterocyclic compounds, metabolic diseases, etc., can solve problems such as reducing receptor recovery and increasing degradation

Inactive Publication Date: 2016-04-06
实发生物医学公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, Zhang et al. (12) have mapped PCSK9 binding to the EGF-A repeat of LDLR and shown that such binding reduces receptor recycling and increases its degradation

Method used

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  • Anti-PCSK9 compounds and methods for the treatment and/or prevention of cardiovascular diseases
  • Anti-PCSK9 compounds and methods for the treatment and/or prevention of cardiovascular diseases
  • Anti-PCSK9 compounds and methods for the treatment and/or prevention of cardiovascular diseases

Examples

Experimental program
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Effect test

Embodiment 1

[0130] Test for secreted PCSK9

[0131] HEK-293T cells were seeded into 96-well plates in DMEM containing 10% fetal bovine serum medium and incubated overnight at 37 °C. Cells were transfected with the PCSK9 cDNA construct. Compounds were added at different concentrations, followed by an additional 43 hours of incubation. Prior to the PCSK9 assay, cell culture medium was replaced with DMEM serum-free medium containing the same concentration of compound or vehicle and incubated for an additional 5 hours. Cell culture media were analyzed for PCSK9 secretion by immunoblot analysis and imaged and quantified using a LAS-4000 (GE). The results for selected compounds are shown in image 3 .

Embodiment 2

[0133] Test for LDLR upregulation

[0134] A proprietary recombinant assay was used to demonstrate that co-expression of PCSK9 and LDLR DNA in HEK-293 cells resulted in decreased expression levels of LDLR in cells. The present inventors constructed an expression vector of human LDLR under the control of cytomegalovirus promoter-enhancer (pCMV-LDLR). In addition, a construct comprising PCSK9 (pCMV-PCSK9-FLAG) was made. These constructs were used to transfect mammalian cells, and cell lysates and supernatants were subjected to SDS-PAGE and immunoblot analysis using anti-PCSK9 or LDLR antibodies. The blotted data showed that only cells transfected with pCMV-PCSK9-FLAG expressed unprocessed (cells) and processed (medium) PCSK9 ( Figure 4 ). Only cells transfected with pCMV-LDLR showed expression of LDLR in cells ( Figure 4 ). However, cells transfected with both pCMV-PCSK9-FLAG and pCMV-LDLR showed disappearance of the intracellular LDLR band ( Figure 4 ), which provides ...

Embodiment 3

[0137] In situ uptake of Dil-LDL in HepG2 cells

[0138] We also tested the ability of PCSK9 modulator compounds to enhance the uptake of fluorescent DilLDL by HepG2 cells. Briefly, HepG2 cells were plated and grown overnight. Compounds were added to cells followed by the addition of fluorescent Dil-LDL. The cells were washed extensively, and the uptake of fluorescent Di-LDL by the cells was measured using a Synergy2 plate reader ( Figure 7 ).

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Abstract

Disclosed are compounds that modulate the physiological action of the proprotein convertase kexin type 9 (PCSK9), and methods of using these modulators to reduce LDL-cholesterol levels and/or for the treatment and/or prevention of cardiovascular disease (CVD), including treatment of hypercholesterolemia.

Description

[0001] Cross-references to related applications [0002] This application claims the benefit of US Provisional Application No. 61 / 789867, filed March 15, 2013, the entire contents of which are incorporated herein by reference. [0003] Statement Regarding Federally Sponsored Research or Development [0004] This invention was made under SBIR Grant No. HL092712 with support from the National Heart, Lung, and Blood Institute (NHLBI). The US Government has certain rights in this invention. field of invention [0005] The present invention relates to compounds that modulate the physiological effects of proprotein convertase subtilisin Kexin type 9 (PCSK9), including its interaction with the low density lipoprotein receptor (LDLR). More specifically, the invention relates to compositions comprising small molecule modulators of PCSK9 function and methods of using these modulators as pharmaceuticals. Small molecule modulators of PCSK9 function can be used therapeutically to low...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/497C07C69/587A61P9/00
CPCA61K31/167A61K31/17A61K31/18A61K31/235A61K31/245A61K31/397A61K31/40A61K31/401A61K31/415A61K31/4164A61K31/42A61K31/435A61K31/437A61K31/4418A61K31/495A61K31/496A61K31/505A61K31/506A61P3/06A61P9/00A61P9/10A61P43/00C07C235/38C07C237/42C07C271/28C07C275/42C07C311/47C07C2601/02C07C2601/08C07D211/14C07D211/70C07D211/96C07D213/68C07D213/74C07D213/82C07D231/12C07D233/90C07D239/42C07D241/04C07D241/08C07D261/08C07D295/14C07D401/06C07D403/12C07D471/04A61K2300/00C07C275/40C07C2602/02
Inventor 谢林·萨拉赫丁·阿卜杜勒-马吉德马吉德·阿布-阿布扎比本杰明·布拉斯韦恩·奇尔德斯纳比勒·艾尔斯霍尔巴吉维克托·吉杜罗杰利奥·马丁内斯哈罗德·迈耶斯沙克尔·A·穆萨
Owner 实发生物医学公司
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