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Methods of inhibiting vascular permeability and apoptosis

A phosphate receptor, vascular endothelium technology, applied in the application field of chemical form in the preparation for treatment and medicine

Inactive Publication Date: 2008-11-12
UNIV OF CONNECTICUT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, persistent or uncontrolled angiogenesis can lead to a variety of disease states or conditions and, for solid tumors, may be necessary to maintain the disease state

Method used

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  • Methods of inhibiting vascular permeability and apoptosis
  • Methods of inhibiting vascular permeability and apoptosis
  • Methods of inhibiting vascular permeability and apoptosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Phosphorylation of FTY720 by Sphingosine Kinase in Vascular Endothelial Cells

[0077] Since S1P is a potent inducer of endothelial cell chemotaxis, the effect of FTY720 and its analogues on endothelial cell migration was tested. HUVEC migration was analyzed using a 96-well chemotaxis microchamber (Neuro Probe Ltd.) (Paik, J.H. et al., J Biol Chem 276: 11830-11837 (2001)). Briefly, polycarbonate filters with a pore size of 8 microns ([mu]m) were coated with 5 micrograms per milliliter ([mu]g / ml) of fibronectin. S1P, FTY720, AAL, FTY720-P or (R)-AFD were diluted to an appropriate concentration with 0.1% ffa-BSA. Add 85 μl of solution or conditioned medium from HUVECs to the lower chamber. Approximately 5 x 10 4 cells were placed in the upper culture chamber. at 5% CO 2 Allow cells to migrate for 4 hr at 37 °C in a humid culture chamber. After the incubation period, the filters were removed, the cells were stained with 0.1% crystal violet in a 96-well pla...

Embodiment 2

[0083] Example 2: Confirmation of phosphorylation of FTY720 and R-AAL

[0084] To confirm that FTY720 and (R)-AAL can be phosphorylated by endothelial cells, in vitro kinase assays were performed using HUVEC whole cell extracts and conditioned medium. Cells were washed 3 times with M199 normal medium and incubated with 0.1% ffa-BAS for the indicated times. After incubation, conditioned medium was removed, centrifuged at 1,000 xg for 10 minutes and used for kinase activity assays. With 400 μl 25mM HEPES pH 7.5, 5mM MgCl 2 , 1X Protease Inhibitor Cocktail Scrape cells at 4°C and disrupt with brief sonication. The cell homogenate was centrifuged at 20,000 xg for 10 minutes at 4°C. In kinase assays, 300 μl of conditioned medium from HUVECs (derived from 5 × 10 5 cells were incubated for 3 hr) or 10 μg whole cell extract as a source of kinase activity. The reaction contained 20 μM sphingosine or 100 μM FTY720 or AAL, [γ- 32 P]ATP (10μCi), 5mM MgCl 2 , 15 mM NaF, 0.5 mM 4-deo...

Embodiment 3

[0086] Example 3: Determination of the role of SK2 in phosphorylation of FTY720 and (R)-AAL

[0087] To determine the role of SK2 in the phosphorylation of FTY720 and (R)-AAL, HEK293T cells were transiently transfected with pcDNA3.1 / Neo expression vector containing SK1 or SK2 cDNA, and FTY720, (R)-AAL and (S)- Phosphorylation of AAL. Figure 4 Shown are TLC-autoradiograms of in vitro kinase assays performed with extracts from control vector 293T cells (293T), SK1-overexpressing 293T cells (SK1-293T), and SK2-overexpressing 293T cells (SK2-293T) . Control vehicle = (C), 100 μM FTY720 = (F), 100 μM (R)-AAL = (R), 100 μM (S)-AAL = (S) or 20 μM sphingosine = (SG) were used as substrates. Contains 50 μM DMS when marked (+). Representative experiments for N=3-5 are shown. The bottom panel shows the fold-specific activity induction of SK1-293T (white bars) and SK2-293T (black bars) vs. vector-transfected 293T. Arrows indicate phosphorylated compounds.

[0088] Moderate amounts ...

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Abstract

Agonist of vascular endothelial sphingosine-1-phosphate receptors are described. Compounds such as FTY720 can be phosphorylated by sphingosine kinase-2 into the phosphorylated froms which serve as sphingosine-1-phosphate receptor agonists. The vascular endothelial sphingosine-1-phosphate receptor agonists are employed in methods of treating a mammal for vascular permeability disorders and unwanted vascular endothelial cell apoptosis, and for the growth of new blood vessels. The sphingosine-1-phosphate receptor agonists can be used for the manufacture of a medicament for treating vascular permeability disorders and unwanted vascular endothelial cell apoptosis, and for the growth of new blood vessels.

Description

[0001] Statement on Federal Funding for Research and Development [0002] The United States Government has certain rights in this invention pursuant to grant numbers HL67330 and HL70694 awarded by the National Institutes of Health. Background technique [0003] The process by which new blood vessels are formed is called "angiogenesis". In angiogenesis, vascular endothelial cells sequentially undergo proliferation, migration and morphogenesis to form a new capillary network. Under normal or non-pathological conditions, angiogenesis occurs in well-established situations, such as in wound healing, ischemic response, and embryonic development. However, persistent or uncontrolled angiogenesis can lead to a variety of disease states or conditions and, for solid tumors, may be necessary to maintain the pathology. [0004] Many extracellular and intracellular signaling molecules interact with cellular receptors and modulate behavior within the vasculature, including processes associ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/133A61P9/00A61K31/00
CPCA61K31/00A61K31/133A61K31/661A61K31/137A61P11/00A61P17/02A61P21/00A61P25/00A61P25/14A61P25/16A61P25/28A61P31/04A61P43/00A61P7/00A61P7/02A61P7/04A61P9/00A61P9/06A61P9/08A61P9/10A61P3/10Y02A50/30
Inventor 蒂莫西·拉特雷莎·桑切斯朴智惠凯文·P·克拉费
Owner UNIV OF CONNECTICUT
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