U.S. patent application number 10/228153 was filed with the patent office on 2003-02-13 for smilagenin and its use.
Invention is credited to Brostoff, Jonathan, Gunning, Philip, Hu, Yaer, Rubin, Ian, Wang, Weijun, Whittle, Brian, Xia, Zongqin.
Application Number | 20030032604 10/228153 |
Document ID | / |
Family ID | 26315239 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032604 |
Kind Code |
A1 |
Xia, Zongqin ; et
al. |
February 13, 2003 |
Smilagenin and its use
Abstract
The invention discloses the use of a smilagenin in the treatment
of cognitive dysfunction and similar conditions. Methods of
treatment, and pharmaceutical compositions are also disclosed.
Inventors: |
Xia, Zongqin; (Shanghai,
CN) ; Rubin, Ian; (Leicester, GB) ; Whittle,
Brian; (East Yorkshire, GB) ; Gunning, Philip;
(Essex, GB) ; Hu, Yaer; (Shanghai, CN) ;
Brostoff, Jonathan; (London, GB) ; Wang, Weijun;
(Cambridgeshire, GB) |
Correspondence
Address: |
Attn: Spyros J. Lazaris
OPPENHEIMER WOLFF & DONNELLY LLP
Suite 3800
2029 Century Park East
Los Angeles
CA
90067
US
|
Family ID: |
26315239 |
Appl. No.: |
10/228153 |
Filed: |
August 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10228153 |
Aug 26, 2002 |
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09866234 |
May 25, 2001 |
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09866234 |
May 25, 2001 |
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09362328 |
Jul 28, 1999 |
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6258386 |
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Current U.S.
Class: |
514/26 |
Current CPC
Class: |
A61K 31/704 20130101;
G01N 33/5023 20130101; A61K 31/7048 20130101; A61P 25/28 20180101;
G01N 33/5061 20130101; G01N 33/5008 20130101; G01N 33/502 20130101;
A61K 31/58 20130101; G01N 33/944 20130101; G01N 33/5088 20130101;
G01N 33/5058 20130101 |
Class at
Publication: |
514/26 |
International
Class: |
A61K 031/704 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 1999 |
GB |
GB9905275.5 |
Claims
1. A use of smilagenin in the manufacture of a medicament for the
treatment of a condition characterized by a deficiency in receptor
number, function or turnover.
2. A method of treating a condition characterized by a deficiency
in receptor number, function or turnover, which comprises
administering an effective dose of smilagenin.
3. A method as claimed in claim 2, said method comprising:
providing a composition having receptor number or turnover
enhancing properties, said composition including smilagenin; and
administering said composition to an organism for treating a
condition characterized by said deficiency.
4. The method of claim 2, wherein said condition further comprises
a reduced amount of receptors.
5. The method of claim 2, wherein said composition further
comprises a carrier.
6. The method of claim 2, wherein said condition is found in
postsynaptic neurons.
7. The method of claim 2, wherein said smilagenin is derived from
individual plants belonging to the genera of Smilax, Asparagus,
Anemarrhena, Yucca and Agave, solely or in combinations
thereof.
8. The method of claim 2 which comprises administering a foodstuff
or beverage having an effective amount of smilagenin.
9. The method of claim 2 which comprises administering smilagenin
in the form of a pharmaceutical composition.
10. A use of smilagenin in the manufacture of a medicament for the
treatment of a condition characterized by the presence of
neurofibrillary tangles and/or .beta.-amyloid plaques.
11. A method of treating a condition characterized by the presence
of neurofibrillary tangles and/or .beta.-amyloid plaques, which
comprises administering an effective dose of smilagenin.
12. A method as claimed in claim 11, said method comprising:
providing a composition, said composition including smilagenin; and
administering said composition to an organism for treating a
condition characterized by said presence of neurofibrillary tangles
and/or .beta.-amyloid plaques.
13. The method of claim 12, wherein said composition further
comprises a carrier.
14. The method of claim 11, wherein said smilagenin is derived from
individual plants belonging to the genera of Smilax, Asparagus,
Anemarrhena, Yucca and Agave, solely or in combinations
thereof.
15. The method of claim 11, which comprises administering a
foodstuff or beverage containing an effective amount of
smilagenin.
16. The method of claim 11, which comprises administering
smilagenin in the form of a pharmaceutical composition.
17. A pharmaceutical composition having cognitive function
enhancing properties, said composition comprising a
pharmacologically effective amount of smilagenin.
18. The pharmaceutical composition of claim 17, further comprising
a carrier.
19. The pharmaceutical composition of claim 17, wherein said
smilagenin is in the form of an extract derived from a plant of the
genus Smilax, Asparagus, Anemarrhena, Yucca and Agave, solely or in
combinations thereof.
20. The pharmaceutical composition of claim 17, wherein a
pharmacologically effective amount of said composition is
administered to an organism to treat a condition.
21. The pharmaceutical composition of claim 20, wherein said
condition is characterized by cognitive dysfunction and allied
conditions.
22. The pharmaceutical composition of claim 21 wherein said
condition is Alzheimer's disease, senile dementia of the
Alzheimer's type, Parkinson's disease, Lewi body dementia, postural
hypotension, autism, chronic fatigue syndrome, Myasthenia Gravis,
Lambert Eaton disease, diseases and problems associated with Gulf
War Syndrome, occupational exposure to organophosphorus compounds
and problems associated with aging.
23. The pharmaceutical composition of claim 20, wherein a symptom
of said condition includes a deficiency of receptors.
24. The pharmaceutical composition of claim 23, wherein said
deficiency comprises a reduction in receptor number, function or
turnover, or combination thereof.
25. The pharmaceutical composition of claim 20, wherein said
condition is characterized by a presence of neurofibrillary tangles
and/or .beta.-amyloid plaques.
26. A foodstuff or beverage comprising an effective dosage of
smilagenin.
27. The foodstuff or beverage of claim 26, wherein said smilagenin
is in the form of an extract derived from a plant of the genus
Smilax, Asparagus, Anemarrhena, Yucca and Agave, solely or in
combinations thereof.
28. The foodstuff or beverage of claim 26, wherein an effective
amount of said composition is administered to an organism to treat
a condition.
29. The foodstuff or beverage of claim 28, wherein said condition
is characterized by cognitive dysfunction and allied
conditions.
30. The foodstuff or beverage of claim 29, wherein said condition
is Alzheimer's disease, senile dementia of the Alzheimer's type,
Parkinson's disease, Lewi body dementia, postural hypotension,
autism, chronic fatigue syndrome, Myasthenia Gravis, Lambert Eaton
disease, diseases and problems associated with Gulf War Syndrome,
occupational exposure to organophosphorus compounds and problems
associated with aging.
31. The foodstuff or beverage of claim 28, wherein a symptom of
said condition includes a deficiency of receptors.
32. The foodstuff or beverage of claim 31, wherein said deficiency
comprises a reduction in receptor number, function or turnover, or
combination thereof.
33. The foodstuff or beverage of claim 28, wherein said condition
is characterized by the presence of neurofibrillary tangles and/or
.beta.-amyloid plaques.
34. A method of testing a compound for efficacy in improving the
function and/or increasing and/or stabilizing a population of a
specific receptor or type of receptor, the method comprising:
preparing or retrieving suitable cells transfected with DNA for a
specific receptor or type of interest; dividing the transfected
cells into separate portions, a first portion of said separate
portions to serve as a control sample, and a second portion of said
separate portions to serve as a test sample; allowing said control
sample and test sample to grow in the presence of a nutrient
medium; removing the nutrient medium from said control sample and
test sample and then (a) adding the compound dissolved in a
cytologically acceptable carrier to the test sample and (b) adding
an equivalent amount of said carrier to the control samples; and
performing an assay or assays to determine the function, number or
turnover of said specific receptor or type of receptor present in
the control sample and test sample.
35. The method of claim 34, wherein said suitable cells for
transfection are Chinese Hamster Ovary cells.
36. The method of claim 34, wherein said specific receptor is a
muscarinic receptor.
37. The method of claim 34, wherein said compound comprises
smilagenin.
38. A method of testing a compound for efficacy in reducing a
decline in number, over time, of specific receptor or type of
receptor in a cultured cell line, the method comprising: culturing,
in a culture medium, a population of cells expressing a specific
receptor or type of receptor; dividing said population of cells
into a control portion and a test portion; culturing said control
portion and test portions under approximately similar conditions,
wherein said test portion of cells is exposed to a compound;
performing assays to determine the decline of the specific receptor
or type of receptor of interest in said control portion of cells
and in said test portion of cells exposed to said compound over a
time period; and comparing, at a predetermined time, the amounts of
said specific receptor or type of receptor of interest in said
control portion and test portions of cells.
39. The method of claim 38, further comprising determining an
effect of said compound on the decline of said specific receptor or
type of receptor of interest.
40. The method of claim 38, wherein said compound is comprised of
smilagenin.
Description
CROSS-REFERENCE To RELATED APPLICATION
[0001] This application is a divisional application of copending
U.S. patent application Ser. No. 09/362,328, filed Jul. 28,
1999.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to smilagenin and its use in
treating cognitive dysfunction and allied conditions; and to
compositions for use in such treatments. The invention is also
concerned with the treatment of conditions that are characterized
by a deficiency in the number or function of membrane-bound
receptors. In the following, the present invention will be
described principally with reference to the treatment of
Alzheimer's disease (AD) and senile dementia of the Alzheimer's
type (SDAT), where deficiencies in a number of receptor types have
been demonstrated. However, it is to be understood that the present
invention relates generally to the treatment of conditions
attributable to intrinsic pathological conditions and/or exposure
to adverse environmental conditions these conditions being
characterized by a deficiency in the number or function of
membrane-bound receptors or a deficiency in transmission at the
junctions between neurons or at the junctions of neurons and
effector cells.
[0003] Conditions of the type mentioned above include Parkinson's
disease, Lewi body dementia, postural hypertension, autism, chronic
fatigue syndrome, Myasthenia Gravis, Lambert Eaton disease,
diseases and problems associated with Gulf War Syndrome,
occupational exposure to organophosphorus compounds and problems
associated with aging.
[0004] Alzheimer's disease (AD) and senile dementia of the
Alzheimer's type (SDAT) are grave and growing problems in all
societies where, because of an increase in life expectancy and
control of adventitious disease, the demographic profile is
increasingly extending towards a more aged population. Agents which
can treat, or help in the management of, AD/SDAT are urgently
required.
[0005] Age-associated memory impairment (AAMI) is a characteristic
of older patients who, while being psychologically and physically
normal, complain of memory loss. It is a poorly defined syndrome,
but agents which are effective in treatment of AD/SDAT may also be
of value in these patients.
[0006] Research into AD/SDAT is being carried out by traditional
and conventional medical research methods and disciplines. In
conventional medicine, there are several approaches to the
treatment of AD/SDAT. It is known that the biochemical processes
subserving memory in the cerebral cortex are (at least in part)
cholinergically-mediated. Those skilled in the art will know that
"cholinergically mediated" mechanisms may be directly attributable
to acetylcholine acting on receptors, and these are direct effects.
Other, clinically useful effects may also be caused by modulation
of release of acetylcholine from pre-synaptic nerve endings or
inhibition of enzymes that destroy acetylcholine. These modulating
factors may be exerted through neurones where the mediator is
non-cholinergic; these are referred to as indirect effects. Some
attempts at treatment have focussed on the role of other mediators
such as 5-hydroxytryptamine, which is a mediator in other areas of
brain, such as the mid-brain nuclei. However, since fibres from
these areas are projected forward into the cerebral cortex where
the primary transmitter is acetylcholine, attention has focussed on
the management of this mediator in the search for appropriate
therapeutic agents.
[0007] Cholinergic strategies for the treatment of AD/SDAT have
been directed at several points along the pathway of formation,
synaptic release and removal of released acetylcholine.
[0008] One approach involves treatment with high doses of lecithin
and other precursors of acetylcholine. This is of limited use in
producing sustained improvements in cognitive performance.
[0009] Another approach involves the use of vegetable drugs such as
Polygalae root extract, which has been shown to enhance
choline-acetylcholine transferase (CAT) activity and nerve growth
factor (NGF) secretion in brain. Oral administration of NGF has no
effect on central nervous system neurons because it is a high
molecular weight protein that cannot pass through the blood-brain
barrier. However, agents which can pass through the blood-brain
barrier and have a stimulating effect on NGF synthesis in the
central nervous system have been proposed for the improvement of
memory-related behavior.
[0010] The results of a third clinical approach, which uses
cholinesterase inhibitors such as tacrine hydrochloride, have been
marginally more positive than the above. Substances obtained from
plants used in Chinese and Western medicine, for example huperzine,
galanthamine, and physostigmine have all been shown to be of
some--although limited--benefit in the treatment of AD/SDAT in
clinical studies and also in laboratory models. All of these
substances are inhibitors of acetylcholine esterase (AChE). In
patients with AD/SDAT, there may be reduced synthesis of
acetylcholine (ACh), reduced efficiency in release of ACh from
presynaptic stores, and a decrease in the number or function of
postsynaptic (M.sub.3) receptors. Reductions in pre-synaptic
M.sub.2 receptors have also been shown. The beneficial effect of
AChE inhibitors is attributed to enhancement of acetylcholine
levels at synapses in brain by slowing down the destruction of
released transmitter.
[0011] Compositions which modulate cholinergic function are known
to affect memory and recall. For example, nicotine stimulates
nicotinic acetylcholine receptors, and the short lived memory
enhancing effects of cigarette smoking are thought to be due to the
effect of nicotine. Scopolamine, an antagonist of acetylcholine,
will produce amnesia and impaired cognitive function manifesting in
psychomotor tests as a prolongation of simple reaction times,
possibly as a result of impaired attention, and is used for this
purpose as an adjunctive analgesic treatment. The amnesic effect of
scopolamine can be antagonized by nicotine.
[0012] There are two families of nicotinic receptor subtypes
(.alpha. and .beta.), and each includes four subgroups which differ
in ligand specificity. The role of nicotinic receptors in the CNS
is not well understood at the molecular level. It is possible that
agents binding to nicotinic receptors may modify the rate of
turnover at muscarinic receptor sites in brain. Nicotinic receptors
are ligand-gated ion channels, and their activation causes a rapid
(millisecond) increase in cellular permeability to Na.sup.+ and
Ca.sup.++, depolarisation and excitation.
[0013] Another class of cholinergic receptors can be stimulated by
muscarine. Such muscarinic (M) receptors are G protein-coupled
receptors. Responses of muscarinic receptors are slower; they may
be excitatory or inhibitory. They are not necessarily linked to
changes in ion permeability. Five types of muscarinic receptors
have been detected by cholinergic receptor cloning, and are
designated as m.sub.1-m.sub.5. Pharmacological effects are
associated with four of the cloned receptors and they are
designated as M.sub.1-M.sub.4 based on pharmacological
specificity.
[0014] Using specific receptor proteins and monoclonal antibodies,
it has been possible to further localize muscarinic receptors in
brain as m.sub.1 (postsynaptic) and m.sub.2 (presynaptic). In
heart, M.sub.2 receptors are postsynaptic. Presynaptic muscarinic
receptors are thought to be inhibitory, the binding of ACh to these
receptors attenuating the release of further ACh to provide a
negative feedback mechanism for ACh release. Selective M.sub.2
receptor antagonists which are preferentially distributed to the
brain may therefore be useful in treating Alzheimer's disease.
[0015] It is known that, in disease states such as AD/SDAT, there
is general neuronal loss and deficits in cholinergic nerve
function. It has been speculated that the high affinity nicotinic
binding sites in the remaining cholinergic neurons might be
converted to low affinity binding sites in treating such diseases,
thereby sustaining transmitter release. By lowering the affinity of
the nicotinic binding sites, a quick desensitising process is
avoided.
[0016] Agonist activation at nicotinic receptors in brain has rapid
onset and offset. A decreased affinity of the nicotinic receptors
will reduce the desensitisation process. Schwarz R. D. et al (J.
Neuro Chem 42, (1984), 1495-8) have shown that nicotine binding
sites are presynaptically located on cholinergic (and also
5-hydroxytryptaminergic and catecholaminergic) axon terminals. A
change in high affinity binding sites on AD/SDAT may also induce a
change in the modulatory effect the nicotinic binding sites may
have on other transmitter systems.
[0017] Presynaptic cholinergic mechanisms are also under inhibitory
control by GABAergic neurons and this inhibition is thought to be
intensified in AD/SDAT. Removal or reduction of this inhibition
intensifies presynaptic cortical cholinergic activity and enhances
cognitive processing.
[0018] The interactions of interneuronal fibres innervated by
nicotine (reducing binding affinity), and dis-inhibition of
GABAergic fibres both have a presynaptic locus.
[0019] This is a simplistic model of central transmission, but
provides a framework for understanding the attempts which have been
made to increase the effective concentration of acetylcholine in
central synapses. This further illustrates thc concept of direct
and indirect action. There are disadvantages attaching to the three
conventional therapeutic approaches to AD/SDAT treatment mentioned
above: ACh precursor supplementation, agonist replacement and
acetylcholine esterase inhibition. These treatments may result in a
short-term increase in the availability of ACh which may activate
feedback mechanisms resulting in the desensitisation of
postsynaptic receptors. On theoretical grounds, long term benefits
would not be predicted and when treatment is interrupted, any
benefits in management of AD/SDAT and AAMI disappear and the
condition may even be aggravated.
[0020] It has been shown that a compound with M.sub.1 agonist and
M.sub.2/M.sub.3 antagonist activity improved cognitive performance
in SDAT patients (Sramak et al, Life Sciences vol. 2, No. 3,
195-202, 1997). However, this compound causes unacceptable
cholinergic side effects, such as fatigue, diarrhea and nausea.
[0021] A more radical approach to AD/SDAT and AAMI aims to increase
the number of postsynaptic (M.sub.1) receptors, in brain. It is
known from Chinese Patent No. CN1096031A, that sarsasapogenin (SaG)
can up-regulate M.sub.1 cholinergic receptors and also
down-regulate (i.e. move towards normal levels of)
.beta.-adrenergic receptors, the number of which may be
pathologically-raised in AD/SDAT.
[0022] Patent applications have been published which claim the
usefulness of a number of steroid sapogenins having spirostane,
furo-spirostane, spirosolane or solanidine structures in the
treatment of diseases including SDAT. Two patent publications are
of particular relevance here: Chinese patent publication No
CN1096031A claims the use of the spirostane sapogenin,
sarsasapogenin, in the treatment of SDAT. The disclosure in this
document, however, is brief. The other document of relevance is
patent publication DE 4303214A1 which claims the use of a very wide
range of saponins and sapogenins in the treatment of a whole range
of diseases that the inventors consider to be of viral origin. This
disclosure is however of dubious value in that it is well
recognized that there is no infective element to a very large
number of the conditions that are characterized by deficient
synaptic transmission and thus the basic premise of the alleged
invention is flawed. In addition they present no data of any kind
that allows one skilled in the art to be able select a preferred
compound from the large number that are claimed.
[0023] The inventors have found that smilagenin (SMI) exhibits the
ability to regulate receptors. In particular, this compound has
been found to increase the number of M2 receptors in the brain.
Thus, according to one aspect of the invention, there is provided
the use of smilagenin in the manufacture of a medicament for the
treatment of a condition characterized by a deficiency in
postsynaptic membrane-bound receptor number or function.
[0024] Those skilled in the art will be aware of the relationship
between saponins and their sapogenins, and that the latter tend to
be fat-soluble whereas the saponins tend to be water-soluble.
Sapogenins are therefore better able to cross the blood-brain
barrier. The skilled man will also be aware of the epimerisation of
certain sapogenins under conditions of acid hydrolysis.
[0025] The sapogenin of interest in this invention has the
following formula: 1
[0026] With reference to this general formula, smilagenin has the
A/B ring conformation as cis and the stereochernical configuration
at the C25 methyl group is R. The hydroxyl group on the spirostane
ring is in the 3.beta.-OH position.
[0027] Smilagenin occurs naturally in a range of plant species,
notably from the genera Smilax, Asparagus, Anemarrhena, Yucca and
Agave. The species presently of greatest interest include Smilax
regelii Kilip & Morton--commonly known as Honduran
sarsaparilla, Smilax aristolochiaefolia Miller--commonly known as
Mexican sarsaparilla; Smilax ornata Hooker--commonly known as
Jamaican sarsaparilla; Smilax aspera--commonly known as Spanish
sarsaparilla; Smilax glabra Roxburgh; Smilax
febrifuga--Kunth--commonly known as Ecuadorian or Peruvian
sarsaparilla, Anemarrhena asphodeloides Bunge; Yucca schidigera
Roezl ex Ortgies; and Yucca brevifolia Engelm.
[0028] According to a further aspect of the present invention,
there is provided a pharmaceutical composition having cognitive
function enhancing properties which comprises an effective amount
of smilagenin.
[0029] In another aspect, the invention provides a pharmaceutical
composition having cognitive function enhancing properties which
comprises an effective amount of smilagenin in the form of an
extract derived from a plant of the genus Smilax, Asparagus,
Anemarrhena, Yucca or Agave.
[0030] It will be appreciated that the invention embraces within
its scope the use of the compositions defined above. Thus,
according to a fifth aspect, the present invention provides a
method of enhancing cognitive function which comprises
administering to a human or animal an effective dosage of a
composition of the invention.
[0031] The invention also provides a method of enhancing cognitive
function in a human or non-human animal, which comprises
administering an effective dose of smilagenin.
[0032] As used herein, the term "cognitive function" refers to
functions such as thinking, reasoning, remembering, imagining and
learning.
[0033] In identifying compounds that would have use in the
treatment of SDAT and other diseases characterized by reductions in
receptor numbers or synaptic transmission, the inventors have given
consideration to the need to identify compounds that would have the
desired effect but would be devoid of any oestrogenic effects, as
these would be unacceptable, particularly in male patients. A
number of the compounds claimed to have activity in patent
application DE 4303214A1 have marked oestrogenic activity and are
therefore unacceptable. Smilagenin, however, does not display
oestrogenic activity. In addition this compound was tested at other
steroid receptors and was found to have no activity at any of the
following receptors:
[0034] Progesterone
[0035] Glucocorticoid
[0036] Testosterone
[0037] Smilagenin has also been tested for activity in a number of
in-vitro assays. The assays/experiments that Were considered of key
importance in determining possible activity in the elevation of
membrane bound receptor numbers were as follows:
[0038] 1. Chinese hamster ovary (CHO) cells transfected with the a
DNA fragment coding for a muscarinic receptor. The cell line used
for the majority of the experiments was a cell line expressing the
m2 receptor.
[0039] 2. The effects of muscarinic receptor expression in cultured
cell lines of neuronal origin were investigated.
[0040] 3. Cultured cardiac muscle cells obtained from neonatal
Sprague Dawley rats. The cardiac muscle cells express muscarinic
receptors, typically m2. The level of these receptors fails on
prolonged culture and the effects of compounds of interest in
preventing the fall in receptor numbers was investigated.
[0041] The methods and the results of these experiments are now
described in turn.
[0042] 1 CHO Cell Line Experiments
[0043] The effects of various compounds on the expression of m2
receptors on CHO cells transfected with DNA for the m2 receptor
were investigated. Receptor numbers were assayed using tritiated
QNB binding and subtracting non-specific binding. Compounds were
dissolved in DMSO and DMSO was used as a control. Compounds were
tested at a range of final concentrations. Compounds were also
tested in the presence and absence of tamoxifen to try to
distinguish an oestrogen receptor mediated mechanism. The results
are summarized in the Table 2 below, where the compound used in the
invention appears in bold, and data on other sapogenins is given
for comparative purposes:
1TABLE 2 Effects of smilagenin on the expression of m.sub.2
receptors on CHO cells Effect on receptor expression - given as %
increase compared to Molar concentration of control (negative
values in Compound compound brackets) Sarsasapogenin 10.sup.-5 34
10.sup.-6 (14) Anzurogenin D 10.sup.-5 22 10.sup.-6 (26) Sisalgenin
10.sup.-5 NS 10.sup.-6 NS Smilagenin 10.sup.-5 57 10.sup.-6 18
Diosgenin 10.sup.-5 NS 10.sup.-6 NS Ruscogenin 10.sup.-5 (22)
10.sup.-6 NS Tigogenin 10.sup.-5 NS 10.sup.-6 NS NS = No
significant effect
[0044] Thus the experiments indicate that smilagenin was able to
increase the number of muscarinic receptors expressed on the
surface of CHO cells cultured in-vitro. The effect was not
antagonized by tamoxifen, indicating that the mechanism involved
did not involve the oestrogen receptor.
[0045] 2 Effects of Smilagenin on Cell Survival
[0046] Other in vitro assays have been employed to establish the
effects of smilagenin. In particular various neuroblastoma cell
lines including SKN-SN and SH-SY5Y cells as well as
phaechromoacytoma cell lines have been cultured in vitro in the
presence of .beta.-amyloid fragments or serum depletion. A number
of techniques to demonstrate the effectiveness of the compounds in
protecting the cultured cells were investigated. These techniques
included Trypan blue exclusion, chemiluminescence and release of
lactate dehydrogenase. Of most interest was the observation that
incubation of cells, in particular PC12 cells, with .beta.-amyloid
reduced the number of muscarinic receptors measured using
radio-labeled ligand binding techniques. This reduction in receptor
numbers was found to be ameliorated by smilagenin.
[0047] 3 Effects of Smilagenin on Cultured Cardiac Muscle
Cells.
[0048] Cardiac muscle cells were isolated from the ventricular
muscle of neonatal Sprague Dawley rats using standard techniques.
Cells were cultured in vitro and muscarinic receptor numbers
expressed on cell surfaces membrane fragments after homogenization
of cells harvested at various time points were estimated using
specific binding of tritiated QNB. Preliminary experiments
demonstrated that the number of receptors expressed tended to
decline after 10 days of culture. The experiments were therefore
designed to investigate the effects of the various compounds in
inhibiting this decline in receptor numbers.
[0049] The results of these experiments are summarized in Table 3,
where the compound used in the invention appears in bold, and data
on other sapogenins is given for comparative purposes:
2TABLE 3 Effects of various compounds on muscarinic receptor
expression on cultured cardiac muscle cells Concentration of
compound causing a significant increase in number of receptors
expressed on neonatal cardiac muscle after 10 days in vitro
Compound culture Diosgenin NS Anzurogenin D 10.sup.-6M Ruscogenin
NS Sarsasapogenin 10.sup.-5M Tigogenin NS Astragaloside 10.sup.-5M
Smilagenin 10.sup.-6M NS = No significant effect
[0050] It is speculated here that the effect of the active compound
claimed in this patent may operate through an effect on G protein
and that the effects on receptor numbers are secondary to an effect
on G-protein. When a membrane bound G-protein linked receptor is
stimulated two basic sets of events are initiated: the effecter
response; and the internalization of the receptor. The subsequent
processing of the receptor to the state where it is again in a form
on the cell surface or other membrane surface where it can interact
with another receptor ligand appears to be subject to a number of
factors. A number of these factors or mechanisms appear to be
G-protein linked. There is evidence that activation of m.sub.3
receptors may have an effect on G-protein expression or levels. It
is speculated that the actions of the compounds described in this
patent may due to an interaction in the processes of receptor
regeneration, G-protein linkage or G-protein homeostasis.
[0051] An alternative hypothesis is that the compounds are
increasing the synthesis or release or a decreased rate of
degradation of neurotropic factors such as brain derived growth
factor and/or nerve growth factor. These effects on growth factors
might be due to an effect of the compound on a cytosolic or nuclear
receptor or the binding of a compound to a promoter region with a
consequent effect directly on the rate of production of mRNA for
the growth factor or as a consequence of increasing the production
of another material factor such as G-protein or finally the effects
may be secondary to an effect on receptor or G-protein
procession.
[0052] The increased expression and/or abnormal processing of the
amyloid precursor protein (APP) is associated with the formation of
amyloid plaques and cerebrovascular amyloid deposits which are the
major morphological hallmarks of Alzheimer's disease. Of particular
interest are the processes regulating the proteolytic cleavage of
APP into amyloidogenic and nonamyloidogenic fragments. The cleavage
of APP by the enzyme .alpha.-secretase within the .beta.-amyloid
sequence of the protein results in the formation of a non
amyloidogenic C-Terminal fragment, and the soluble APPs.alpha.
fragment; this latter fragment has been shown to have neurotropic
and neuroprotective activity as well as to enhance memory in mice
when injected intra-cerebro-ventrically (ICV). In contrast,
processing of APP by .beta.-secretase exposes the N-terminus of
.beta.-amyloid.which is released by .gamma.-secretase cleavage at
the variable C-terminus. The resulting .beta.-amyloid peptides,
which contain 39-43 amino acids, have been shown to be neurotoxic
and to accumulate in plaques which interfere with inter-neurone
connections.
[0053] A number of studies have shown that stimulation of the
protein-kinase (PKC) linked muscarinic M.sub.1 and M.sub.3
receptors results in an increase in a-secretase activity. As a
consequence processing of APP to APPs.alpha. with its
neuroprotective effects is increased. In parallel, processing of
APP by .beta.- and .gamma.-secretase is decreased and there is a
consequential reduction of .beta.-amyloid. Other transmitters such
as nerve growth factor (NGF) and brain derived neurotropic factor
(BDNF) as well as bradykinin and vasopressin may have similar
effects in increasing the proportion of APP processed to
APPs.alpha.. There may be a number of factors involved in the
effects of NGF which may include binding of the factor to the
tyrosine kinase receptor (TrkA) and the stimulation of
phospholipase C.gamma. with subsequent phosphorylation and
activation of protein kinase C (PKC) and increase in relative
activity of a-secretase.
[0054] Any treatment which increases activity of protein-kinase C
selectively in brain might therefore be expected to be of use in
the management of Alzheimer's disease. Until recently agonists
selective at the M.sub.1 receptor have not been available.
Non-selective agonists would be expected to stimulate pre-synaptic
M.sub.2 receptors which cause negative feedback and hence would
further severely impair muscarinic transmission. Selective agonists
at the M.sub.1 receptor are now becoming available (talsaclidine)
and such agents are under investigation for the treatment of AD.
There is however, a substantial risk that, as with the chronic
administration of any receptor agonist, the clinical benefits seen
will be severely limited in terms of the size of benefit by
reducing receptor numbers or reducing sensitivity and in terms of
side effects due to lack of receptor specificity. Thus compounds as
described in this invention, which selectively increase muscarinic
M.sub.1 receptor numbers, with little or no effect on muscarinic
M.sub.2 receptor numbers in the brain would be expected to be
devoid of the problems seen with a muscarinic agonist and hence
have particular utility. Indeed the benefits may be seen in three
parts as follows.
[0055] 1. A selective increase in M.sub.1 receptor numbers leading
to increased synaptic transmission. Chronic administration of a
selective agonist will, at best, have no adverse effect on
transmission;
[0056] 2. Secondary to the increased receptor numbers, an increase
stimulation of PKC with a consequential increase in
.alpha.-secretase activity, leading to:
[0057] 2.1 A reduced production of .beta.-amyloid and a consequent
reduction of plaque formation and neuronal loss;
[0058] 2.2 An increase in APPs.alpha. and a consequent improvement
in cerebral function as witnessed by an improvement in short and
long term memory.
[0059] In order to illustrate the invention further by way of
non-limiting example, reference will now be made to the
accompanying drawings and to the Examples which follow; in the
drawings:
[0060] FIG. 1 illustrates a hypothetical mode of action for
smilagenin;
[0061] FIG. 2 illustrates the results obtained in Example 1
below;
[0062] FIG. 3 illustrates the results obtained in Example 2 below;
and
[0063] FIG. 4 illustrates the results obtained in Example 3
below.
[0064] Smilagenin is represented in the drawings by the
abbreviation SMI.
[0065] Referring to FIG. 1, a diagrammatic representation of the
function of smilagenin is shown. It is believed that smilagenin
acts primarily on cell nuclei; the invention is not, however,
limited to any particular mode of action. The observed increase in
muscarinic receptor number consequential upon administration of
smilagenin is interpreted as leading to increased expression of
muscarinic receptor protein. The possible link between the
secretases and .beta.-amyloid protein formation (discussed above)
is indicated in the drawing.
[0066] The following Examples are provided to illustrate the
invention in a non-limiting manner.
EXAMPLE 1
[0067] In a CHO cell line expressing recombinant human muscarinic
receptors in vitro, the number of muscarinic receptors tends to
decline with time. Smilagenin (1-10 .mu.M) incubated for 72 hours
increases muscarinic receptor density.
[0068] Methods:
[0069] Effect of smilagenin on muscarinic receptor density in CHO
cells expressing recombinant human muscarinic receptors.
[0070] Chinese hamster ovary (CHO) cells expressing high levels of
receptor (.about.2.2 pmoles receptor/mg protein) were cultured in
flasks (150 ml) for 24 hours before the start of the experiment.
Vehicle (DMSO) and smilagenin (at 1 and 10 .mu.M) were added to the
medium for 48 h. The culture medium was discarded, the cells
scraped off and resuspended in Hanks solution, centrifuged and
m-receptor levels determined by incubating with [.sup.3H]-QNB for
30 min followed by liquid scintillation counting. Protein levels
were determined by a micro Lowry method.
[0071] Results:
[0072] These are illustrated in FIG. 2. Over the culturing period
treatment with smilagenin prevents the decrease in muscarinic
receptor number in a concentration-dependent manner.
EXAMPLE 2
[0073] Smilagenin (18 mg/kg/day) administered in the food over 3
months reversed the decline in muscarinic (M) receptor number in
the brain over this period, restoring levels to close to those
observed in young control animals.
[0074] Methods: Single point analysis of brain M receptor
density.
[0075] Sprague-Dawley rats, 23-month-old, (i.e. aged) were divided
into 2 groups, aged-control and aged+smilagenin. Smilagenin (18
mg/kg/day) was mixed in the chow over the 3 month period; Male 4-6
month old rats acted as young controls. At the end of the 3-month
treatment period, pairs of treated and control animals were
sacrificed by cervical dislocation and the intact brain removed.
Single point analysis of brain M receptor density was obtained.
[0076] Results:
[0077] Compared to the young controls there were reductions in M
receptor density in brain of the aged controls (FIG. 3). Compared
to aged controls, smilagenin increased M receptor numbers. Groups
treated with smilagenin were significantly different from untreated
controls (FIG. 3).
EXAMPLE 3
[0078] Smilagenin (18 mg/kg/day) administered in the food over 4
months reversed the decline in cognitive function over this period
compared to that observed in young control animals (FIG. 4).
[0079] Methods: Cognitive function in the Y-maze test
[0080] Sprague-Dawley rats aged 23 months (i.e. aged) were divided
into aged-control or aged+smilagenin treatment group. Rats aged 3
to 4 months acted as young controls. The treatment group received
18 mg/kg/day of smilagenin in chow. Chow without smilagenin was
given to each rat after the chow containing smilagenin had been
consumed. Three months later the animals were tested in the Y-maze
as follows; an equilateral Y-shaped maze was used composed of three
45 cm arms with copper rods in the floor at the end of each arm
which conduct electricity and which were 0.2 cm in diameter, 14 cm
in length and with a 1 cm gap between them. There were 15 W signal
lights at each end. A safe region was indicated by a light signal
in an arm where there was no electricity. If a rat moved to a safe
region, a correct reaction was recorded. If it moved to an arm
where there was no light, an error reaction was recorded. Following
the response, an electric shock was again given after a 5 seconds
interval. Twenty trials in total were conducted and the correct
reaction rate was obtained: number of correct reactions/(20)/mean
response time. Seven such training sessions were completed over
seven days. Memory tests were then repeated 15 and 30 days later
(=4 months) using the same techniques as just described.
[0081] Results:
[0082] Compared to young control rats, aged control animals have a
reduced learning and memory performance in the Y-maze tests. A
regime of 3 months intake of smilagenin (18 mg/kg/day) reversed the
decline in cognitive function (FIG. 4), giving results comparable
to those of young animals.
* * * * *