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Mitochondria protecting agents for treating mitochondria associated diseases

a technology protecting agent, which is applied in the field of mitochondrial membrane potential maintenance and inhibition of the loss of mitochondrial membrane potential

Inactive Publication Date: 2002-05-30
GHOSH SOUMITRA S +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0131] According to this assay, the ability of a mitochondria protecting agent of the invention to inhibit production of ROS intracellularly may be compared to its antioxidant activity in a cell-free environment. Production of ROS may be monitored using, for example by way of illustration and not limitation, 2',7'-dichlorodihydroflurescein diacetate ("dichlorofluorescin diacetate" or DCFC). a sensitive indicator of the presence of oxidizing species. Non-fluorescent DCFC is converted upon oxidation to a fluorophore that can be quantified fluorimetrically. Cell membranes are also permeable to DCFC, but the charged acetate groups of DCFC are removed by intracellular esterase activity, rendering the indicator less able to diffuse back out of the cell.

Problems solved by technology

For example, altered mitochondrial activity may lead to undesirable elevated levels of intracellular reactive oxygen species (ROS) and subsequent intracellular damage or cell death.
Although L-Dopa treatment reduces tremors in most patients for a while, ultimately the tremors become more and more uncontrollable, making it difficult or impossible for patients to even feed themselves or meet their own basic hygiene needs.
Since parkinsonism may be induced by exposure to mitochondrial toxins that affect Complex I activity, it appears likely that defects in Complex I proteins may contribute to the pathogenesis of PD by causing a similar biochemical deficiency in Complex I activity.
Many individuals who have lived normal, productive lives are slowly stricken with AD as they grow older, and the disease gradually robs them of their memory and other mental faculties.
Eventually, they cease to recognize family and loved ones, and they often require continuous care until their eventual death.
This suggests that the COX in AD patients is defective, leading to decreased catalytic activity that in some fashion causes or contributes to the symptoms that are characteristic of AD.

Method used

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  • Mitochondria protecting agents for treating mitochondria associated diseases
  • Mitochondria protecting agents for treating mitochondria associated diseases
  • Mitochondria protecting agents for treating mitochondria associated diseases

Examples

Experimental program
Comparison scheme
Effect test

example 1

DCFC Assay for Inhibition of ROS Production by Mitochondria Protecting Agents

[0146] In the cell-based aspect of the DCFC assay, monolayers of cultured adherent SH-SY5Y human neuroblastoma cells (Biedler et al., Cancer Res. 33:2643, 1973) at or near confluence were rinsed and harvested using trypsin according to standard methods. Single cell suspensions containing 7.5.times.10.sup.4 cells in 200 .mu.l of medium were seeded into 96-well plates for overnight incubation at 37.degree. C. and 5% CO.sub.2 in a humidified cell atmosphere. The following day the wells were gently rinsed once with warm Hanks balanced saline solution (HBSS, Gibco-BRL), 200 .mu.l of 30 .mu.M dichlorofluorescin-diacetate (DCFC-DA, Molecular Probes, Eugene, Oreg.) were added to each well and cultures were incubated for 2 hours at 37.degree. C. / 5% CO.sub.2. The excess DCFC-DA was removed by needle aspiration and each well was gently rinsed twice with HBSS. Each well then received 80 .mu.l of HBSS and 10 .mu.l of mi...

example 2

Assay for Mitochondrial Permeability Transition Using DASPMI

[0151] The fluorescent mitochondria-selective dye 2-,4-dimethylaminostyryl--N-methylpyridinium (DASPMI, Molecular Probes, Inc., Eugene, Oreg.) is dissolved in HBSS at 1 mM and diluted to 25 .mu.M in warm HBSS. In 96-well microculture plates, cultured human cells from an individual known or suspected of having a mitochondria associated disease, or normal (control) cells, are incubated for 0.5-1.5 hrs in 25 .mu.M DASPMI in a humidified 37 C. / 5% CO.sub.2 incubator to permit mitochondrial uptake of the fluorescent dye. Culture supernatants are then removed and candidate mitochondria protecting agents diluted into HBSS from DMF or DMSO stocks, or vehicle controls, are added at various concentrations.

[0152] Fluorescence of each microculture in the 96-well plate is quantified immediately using a Cytofluor fluorimetric plate reader (model #2350, Millipore Corp., Bedford, Mass.; excitation wavelength=485 nm; emission wavelength=530 ...

example 3

Effect of Mitochondria Protecting Agents on Apoptosis

[0153] In 96-well microculture plates, cultured human cells from an individual known or suspected of having a mitochondria associated disease, or normal (control) cells or cell lines, are cultured for a suitable period in the presence or absence of physiological inducers of apoptosis (e.g., Fas ligand, TNF-.alpha., or other inducers of apoptosis known in the art) and in the presence or absence of candidate mitochondria protecting agents.

[0154] Exteriorization of plasma membrane phosphatidyl serine (PS) is assessed by adding to the 96 well plate annexing-fluorescein isothiocyanate conjugate (annexin-FITC, Oncogene Research Products, Cambridge, Mass.) dissolved in a suitable buffer for binding to cell surfaces at a final concentration of 5 .mu.g / well. (Martin et al., J. Exp. Med. 182:1545, 1995) After 15-30 min in a humidified 37.degree. C. / 5% CO.sub.2 incubator, cells are fixed in situ using 2% formalin, washed to remove non-specif...

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Abstract

The present invention relates generally to mitochondria protecting agents for treating diseases in which mitochondrial dysfunction leads to tissue degeneration and, more specifically, to compounds, compositions and methods related to the same. The methods of this invention involve administration of a pharmaceutically effective amount of a mitochondria protecting agent to a warm-blooded animal in need thereof, and composition of this invention contain a mitochondria protecting agent in combination with a pharmaceutically acceptable carrier or diluent. Mitochondrial associated diseases which may be treated by the present invention include (but are not limited to) Alzheimer's Disease, diabetes mellitus. Parkinson's Disease, neuronal and cardiac ischemia. Huntington's disease and stroke.

Description

[0001] This application claims the benefit of U.S. Provisional Application Nos. 60 / 072,484, 60 / 072,487, 60 / 072,483 and 60 / 072,482, all filed Jan. 26, 1998.THE TECHNICAL FIELD[0002] The present invention relates generally to mitochondria protecting agents for treating diseases in which mitochondrial dysfunction leads to tissue degeneration and, more specifically, to compounds, compositions and methods for treating such diseases.[0003] Mitochondria are the subcellular organelles that manufacture essential adenosine triphosphate (ATP) by oxidative phosphorylation. A number of degenerative diseases may be caused by or associated with either direct or indirect alterations in mitochondrial function. These include Alzheimer's Disease, diabetes mellitus, Parkinson's Disease, neuronal and cardiac ischemia, Huntington's disease and other related polyglutamine diseases (spinalbulbar muscular atrophy, Machado-Joseph disease (SCA-3), dentatorubro-pallidoluysian atrophy (DRPLA) and spinocerebella...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07D215/08A61K31/00A61K31/047A61K31/05A61K31/06A61K31/085A61K31/12A61K31/136A61K31/155A61K31/235A61K31/352A61K31/353A61K31/47A61K47/48A61P3/10A61P9/10A61P25/00A61P25/08A61P25/14A61P25/16A61P25/18A61P25/28A61P27/00A61P43/00C07C39/08C07C39/11C07C39/19C07C69/017C07C215/78C07C215/80C07C217/84C07C279/18C07D215/20C07D311/72
CPCA61K31/00C07D311/72A61K31/06A61K31/136A61K31/155C07C39/08C07C39/11C07C39/19C07C69/017C07C215/78C07C215/80C07C217/84C07C279/18C07D215/20A61K31/05A61P3/10A61P9/10A61P25/00A61P25/08A61P25/14A61P25/16A61P25/18A61P25/28A61P27/00A61P43/00
Inventor GHOSH, SOUMITRA S.MILLER, SCOTT W.DAVIS, ROBERT E.MOOS, WALTER H.
Owner GHOSH SOUMITRA S
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