Drug – bio-affecting and body treating compositions – Plant material or plant extract of undetermined constitution... – Containing or obtained from a root – bulb – tuber – corm – or...
Reexamination Certificate
2001-03-12
2003-11-18
Lankford, Jr., Leon B. (Department: 1651)
Drug, bio-affecting and body treating compositions
Plant material or plant extract of undetermined constitution...
Containing or obtained from a root, bulb, tuber, corm, or...
C424S725000
Reexamination Certificate
active
06649196
ABSTRACT:
TECHNICAL FIELD
This invention relates to &bgr;-amyloid (A&bgr;) polypeptides, and more particularly to methods of reducing A&bgr; polypeptides using an extract, or active fraction thereof, of Cimicifuga.
BACKGROUND
Patients with Alzheimer's disease (AD) are typically presented to a clinician by a relative who has observed a decline in memory with or without a change in other cognitive abilities such as declines in executive function, difficulty in word finding or visuo-spatial impairment such as an inability to draw complex geometric structures or becoming lost in familiar places. Neuropathological analysis of brains from patients with AD has revealed extensive neuronal and synaptic loss in select brain regions, neurofibrillary tangles, and the deposition of &bgr;-amyloid (A&bgr;) polypeptides in the form of senile plaques throughout the hippocampus and neocortex.
A&bgr; polypeptides are produced from the amyloid precursor protein (APP) through the combined proteolytic actions of &bgr;- and &ggr;-secretases and are then secreted into the extracellular milieu. Biochemical and immunocytochemical studies have revealed that the A&bgr; polypeptides deposited in brains from patients with AD have substantial amino- and carboxyl-terminal heterogeneity and can contain from 39 to 43 amino acid residues, with &bgr;-amyloid 1-40 (A&bgr;40) and 1-42 (A&bgr;42) being the most predominant. By comparing signals obtained using antibodies directed at the amino terminus of A&bgr; with those obtained by capturing the peptide from an internal epitope, it was found that virtually all A&bgr;42 in AD brain is amino-terminally modified and/or truncated.
SUMMARY
The invention features methods of reducing the level of an A&bgr; polypeptide using a Cimicifuga extract. The invention also features methods of producing an active fraction from an extract of Cimicifuga. An active fraction is one that is able to reduce the level of an A&bgr; polypeptide in or secreted from a cell (in vitro or in vivo). The invention further features a composition containing an active fraction of a Cimicifuga extract and an article of manufacture containing such a composition.
In one aspect, the invention provides methods of reducing the level of a &bgr;-amyloid (A&bgr;) polypeptide in or secreted from a cell. The methods include contacting the cell with an amount of a Cimicifuga extract or of an active fraction thereof in an amount that is effective for reducing the level of the A&bgr; polypeptide and monitoring the level of the A&bgr; polypeptide in or secreted from the cell. Representative cells include H4 cells, M17 cells, 293 cells, Chinese hamster ovary (CHO) cells, primary fibroblasts, C6, primary neuronal, primary mixed brain cultures, Daoy, SK-N-SH, SK-N-AS and SK-N-FI.
In another aspect, the invention provides methods for reducing the level of an A&bgr; polypeptide in a mammal, including administering an amount of a Cimicifuga extract or an active fraction thereof to the mammal in an amount that is effective for reducing the level of the A&bgr; polypeptide and monitoring the level of the A&bgr; polypeptide in the mammal.
In another aspect of the invention, there are provided methods of treating a mammal having AD or at risk to develop AD. The methods include administering an amount of a Cimicifuga extract or an active fraction thereof to the mammal in an amount that is effective for treating or preventing AD. Generally, the extract or active fraction thereof is administered to a mammal orally, intravenously, intracranially, intracerebrally, subcutaneously, intramuscularly, intranasally or intraperitoneally. A representative mammal is a rodent, for example, a mouse. In one embodiment, the mouse expresses an APP carrying a Swedish mutation. An example of such a mouse is a Tg2576 mouse.
The Cimicifuga extract or active fraction thereof can be from
C. racemosa,
and further can be obtained from the root or rhizome of a
C. racemosa
plant. The
C. racemosa
extract is generally an ethanolic or an aqueous extract. An active component within an active fraction can be soluble in a solvent such as methylene dichloride, ethyl acetate and n-butanol, and active components soluble in such solvents are typically lipophilic. An active component within an active fraction that is capable of reducing the level of an A&bgr; polypeptide can have a molecular weight of less than 10 kD.
A reduction in the level of an A&bgr; polypeptide can be due to decreased production of the A&bgr; polypeptide or increased catabolism of the A&bgr; polypeptide. In an embodiment of the invention, the level of the A&bgr; polypeptide is reduced by at least 10%, at least 25%, at least 50%, or at least 80% compared to the level of the A&bgr; polypeptide in or secreted from a corresponding cell not contacted with the extract or active fraction thereof. A reduction in the level of an A&bgr; polypeptide can be a reduction in the level of A&bgr;40 or A&bgr;42. Further, the reduction in the level of an A&bgr; polypeptide can be a preferential reduction in the level of A&bgr;42. In addition, the extract or active fraction thereof typically should have no significant effect on the level of one or more of APP, CTF&agr;, CTF&bgr;, or sAPP&agr;.
In yet another aspect of the invention, there are provided methods of producing an active fraction of a Cimicifuga extract that reduces the level of an A&bgr; polypeptide upon contact with a cell. The methods include obtaining an extract of Cimicifuga plant material, size-fractionating the extract through a filter to obtain an active fraction, and testing the active fraction to confirm that the active fraction reduces the level of an A&bgr; polypeptide. Generally, an active fraction contains active components having a molecular weight of less than about 10 kD.
The invention also provides methods of producing an active fraction of a Cimicifuga extract that reduces the level of an A&bgr; polypeptide upon contact with a cell, including obtaining an extract of Cimicifuga plant material, extracting the Cimicifuga extract with hexane (thereby producing a hexane-soluble fraction and a hexane-insoluble fraction) and testing the hexane-insoluble fraction to confirm that the hexane-insoluble fraction reduces the level of the A&bgr; polypeptide. A representative hexane is n-hexane. Such methods can additionally include extracting the hexane-insoluble fraction with a dichloroalkane (producing a dichloroalkane-soluble fraction and a dichloroalkane-insoluble fraction) where the dichloroalkane-soluble fraction typically reduces the level of the A&bgr; polypeptide upon contact with a cell. A representative dichloroalkane is methylene dichloride. Methods of producing an active fraction also can include extracting the dichloroalkane-soluble fraction with an alkylacetate (producing an alkylacetate-soluble fraction and an alkylacetate-insoluble fraction) where the alkylacetate-soluble fraction typically reduces the level of an A&bgr; polypeptide. A representative alkylacetate is ethyl acetate. Further, methods of the invention can include extracting the alkylacetate-soluble fraction with an alcohol (producing an alcohol-soluble fraction and an alcohol-insoluble fraction) wherein the alcohol-soluble fraction typically reduces the level of the A&bgr; polypeptide. A representative alcohol is n-butanol. The product of any of the above-described extractions or fractionations can be further concentrated, for example, by lyophilizing.
In another aspect of the invention, there is provided a composition containing an active fraction of a Cimicifuga extract and a pharmaceutically acceptable carrier. Such an active fraction can reduce the level of an A&bgr; polypeptide upon contact with a cell producing the A&bgr; polypeptide.
In still yet another aspect of the invention, there is provided an article of manufacture containing an active fraction of a Cimicifuga extract, a pharmaceutically acceptable carrier, and packaging material. Generally, packaging material contains a label or package insert indicating that the composition is effective for reducing
Eckman Christopher B.
Fauq Abdul
Haugabook Sharie
Yager Debra
Coe Susan D.
Fish & Richardson P.C. P.A.
Lankford , Jr. Leon B.
Mayo Foundation for Medical Education and Research
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