U.S. patent application number 10/894425 was filed with the patent office on 2006-08-17 for steroidal sapogenins and their derivatives for treating alzheimer's disease.
Invention is credited to Jonathan Brostoff, Phil Gunning, Yaer Hu, Ian Rubin, Weijun Wang, Brian Whittle, Zongqin Xia.
Application Number | 20060182817 10/894425 |
Document ID | / |
Family ID | 26313369 |
Filed Date | 2006-08-17 |
United States Patent
Application |
20060182817 |
Kind Code |
A1 |
Xia; Zongqin ; et
al. |
August 17, 2006 |
Steroidal sapogenins and their derivatives for treating Alzheimer's
disease
Abstract
The invention discloses the use of a number of saponins and
sapogenins, notably those of steroidal structure, in the treatment
of cognitive disfunction and similar conditions. Methods of
treatment, and pharmaceutical compositions are also disclosed.
Inventors: |
Xia; Zongqin; (Shanghai,
CN) ; Hu; Yaer; (Shanghai, CN) ; Rubin;
Ian; (Nottingham, GB) ; Brostoff; Jonathan;
(London, GB) ; Whittle; Brian; (East Yorkshire,
GB) ; Wang; Weijun; (Huntingdon, GB) ;
Gunning; Phil; (Grantchester, GB) |
Correspondence
Address: |
FULWIDER PATTON
6060 CENTER DRIVE
10TH FLOOR
LOS ANGELES
CA
90045
US
|
Family ID: |
26313369 |
Appl. No.: |
10/894425 |
Filed: |
July 17, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10077493 |
Feb 15, 2002 |
6812213 |
|
|
10894425 |
Jul 17, 2004 |
|
|
|
09647110 |
Jan 11, 2001 |
|
|
|
10077493 |
Feb 15, 2002 |
|
|
|
Current U.S.
Class: |
424/725 ;
514/27 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 39/06 20180101; A61K 36/8965 20130101; A61K 36/88 20130101;
A61K 36/896 20130101; A61K 36/8964 20130101; A61P 25/28 20180101;
A61P 25/18 20180101; A61K 31/704 20130101; A61P 39/00 20180101;
A61K 31/7048 20130101; A61P 25/16 20180101; G01N 33/944 20130101;
A61P 39/02 20180101; A61P 9/02 20180101; A61P 43/00 20180101; A61K
36/90 20130101; A61P 9/00 20180101; A61K 31/58 20130101; A61P 21/04
20180101 |
Class at
Publication: |
424/725 ;
514/027 |
International
Class: |
A61K 36/8965 20060101
A61K036/8965; A61K 36/8964 20060101 A61K036/8964; A61K 36/896
20060101 A61K036/896; A61K 31/7048 20060101 A61K031/7048 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 1998 |
GB |
GB9806513.9 |
Mar 8, 1999 |
GB |
GB 9905275.5 |
Claims
1. The use of one or more of smilagenin, prazerigenin, and
astragaloside, tigogenin, ruscogenin, hecoginin and diosgenin in
the manufacture of a medicament for the treatment of a condition
characterised by a deficiency in postsynaptic membrane-bound
receptor number or function.
2-32. (canceled)
Description
[0001] The present invention relates to membrane-bound receptors
and their function; to cognitive disfunction and allied conditions;
to treatments therefor; and to compositions for use in such
treatments. More particularly but not exclusively the invention is
concerned with the treatment of conditions that are characterised
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
characterised by a deficiency in the number or function of
membrane-bound receptors or a deficiency in transmission at the
junctions between neurones or at the junctions of neuroses and
effector cells.
[0002] Conditions of the type mentioned above include 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 ageing.
[0003] Alzheimer's disease (AD) and senile dementia of the
Alzheimer's type (SDAT) are grave and growing problem as 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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 behaviour.
[0009] 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.1) 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.
[0010] 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 antagonised by nicotine.
[0011] 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.
[0012] 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.
[0013] Using specific receptor proteins and monoclonal antibodies,
it has been possible to further localise 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] The interactions of interneuronal fibres innervated by
nicotine (reducing binding affinity), and dis-inhibition of
GABAergic fibres both have a presynaptic locus.
[0018] 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 the 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.
[0019] 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, diarrhoea and
nausea.
[0020] 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.
[0021] The inventors have found a number of saponins and sapogenins
which exhibit the ability to regulate receptors. Thus, according to
one aspect of the invention, there is provided the use of one or
more of smilagenin, anzurogenin D, or an astragaloside in the
manufacture of a medicament for the treatment of a condition
characterised by a deficiency in postsynaptic membrane-bound
receptor number or function.
[0022] Those skilled in the art will be aware of the relationship
between saponins and their sapogenins, and that the desired effects
of sapogenins can be exhibited in patients by administration of the
corresponding saponins, or a mixture thereof. Hydrolysis of at
least a proportion of saponin occurs in the gastrointestinal tract.
The skilled man will also be aware of the epimerisation of certain
sapogenins under conditions of acid hydrolysis.
[0023] Not all saponins and/or their aglycones are useful
treatments for AD/SDAT and some, such as the saponins and
sapogenins from digitalis, have potent inotropic actions on the
myocardium. This group of saponins does not appear to have effects
on the central nervous system (CNS) which would predicate
therapeutic use in AD/SDAT; their potency and toxicity at high
doses also rule this out.
[0024] Some of the principal sapogenins are of the following
general formula: ##STR1##
[0025] With reference to this general formula, the structure of
certain sapogenins is as indicated in the Table below:
TABLE-US-00001 A/B ring C25 methyl Cis/Trans/ stereochemistry
Hydroxyl group(s) Compound unsaturation (R or S) on Spirostane ring
Sarasasapogenin Cis S 3.beta.-OH Smilagenin Cis R 3.beta.-OH
Anzurogenin-D Trans R 3.beta.-OH, 5.alpha.-OH, 6.beta.-OH
Sisalgenin Trans S 3.beta.-OH (C.dbd.O at C12) Tigogenin Trans R
3.beta.-OH Diosgenin .DELTA.5 R 3.beta.-OH Ruscogenin .DELTA.5 R
1.beta.-OH, 3.beta.-OH
[0026] The variation in pharmacological properties and
pharmacodynamic actions of various types of sapogenin underlines
the need for selection of those agents which are most useful for
the treatment of AD/SDAT. The discovery of novel facts about the
action of SaG has made it possible to determine which substances
are most useful for the treatment of AD/SDAT and the like.
[0027] The saponins and sapogenins of principal interest in certain
aspects of the present invention occur 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 omata
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. Saponins
and sapogenins which may be of interest also occur naturally in
other genera, for example Dioscorea, Trillium, Solanum,
Strophanthus, Digitalis and Trigonella. As indicated above, some
saponins and sapogenins from these sources possess undesirable
properties and are thus not recommended for use in the
invention.
[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 a saponin or sapogenin. The saponin or sapogenin is preferably a
steroidal saponin or sapogenin. Such a composition preferably
comprises an effective amount of a non-oestrogenic saponin or
sapogenin.
[0029] In another aspect, the invention provides a pharmaceutical
composition having cognitive function enhancing properties which
comprises an effective amount of a saponin or sapogenin (preferably
a non-oestrogenic saponin or sapogenin) derived from a plant of the
genus Smilax, Asparagus, Anemarrhena, Yucca or Agave.
[0030] The invention further provides the use of an extract of a
plant of the genus Smilax, Asparagus, Anemarrhena, Yucca or Agave
in the preparation of a medicament having cognitive function
enhancing properties.
[0031] 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.
[0032] The invention also provides a method of enhancing cognitive
function in a human or non-human animal, which comprises
administering an effective dose of a saponin or sapogenin,
preferably a non-oestrogenic saponin or sapogenin.
[0033] As used herein, the term "cognitive function" refers to
functions such as thinking, reasoning, remembering, imagining and
learning.
[0034] Thus, according to a seventh aspect of the invention, there
is provided the use of one or more of smilagenin, prazerigenin, an
astragaloside, tigogenin, ruscogenin, hecogenin and diosgenin in
the manufacture of a medicament for the treatment of a condition
characterised by a deficiency in postsynaptic membrane-bound
receptor number or function.
[0035] The inventors have also found that when sarsasapogenin is
combined with certain other sapogenins, an unexpected synergistic
effect is obtained.
[0036] Thus, according to an eighth aspect of the invention, there
is provided a composition for the treatment of a condition
characterised by a deficiency in postsynaptic membrane-bound
receptor number or function, the composition comprising at least
two of sarsasapogenin, smilagenin, prazerigenin, an astragaloside,
tigogenin, ruscogenin, hecogenin and diosgenin.
[0037] The substances used in the seventh and eighth aspects of the
invention do not have high overt oestrogenic and/or androgenic
and/or anabolic activity in patients. Nevertheless, in some
embodiments, there is a low level of oestrogenic and/or androgenic
supplementation.
[0038] According to a ninth aspect of the present invention, there
is provided a method for the treatment of a condition which is
characterised by a deficiency in membrane-bound receptor number or
function in a tissue, organ, cell type or organelle, the method
comprising:
[0039] modulating, directly or indirectly, the action of a
cytosolic, nuclear or membrane-bound protein or receptor which,
when it is activated by an agonist binding thereto, or when its
activity is promoted by deactivation of an antagonist thereto,
upregulates and/or normalises the number and/or turnover of
membrane-bound receptors in that tissue, organ, cell type or
organelle.
[0040] Surprisingly, the inventors have found that radiolabelled
SaG is concentrated in the nuclei of brain cells isolated from
rats, and that levels of M receptor mRNA are raised in rats treated
with SaG. Whilst the inventors do not wish to be bound by any
theory, it is believed that SaG exerts the effects described in
Chinese Patent No. CN1096031A by modulating DNA expression.
[0041] One possible explanation in accordance with this invention
is that SaG is an intracellular agonist of a steroid receptor,
possibly the oestrogen receptor, or a transcription factor or
promoter. There are chemical similarities in the structure of
steroids and SaG, and it is therefore possible that the transport
mechanism of SaG from cytoplasm to the nucleus is the same as that
for steroids. Thus, after diffusing across the cell membrane, SaG
binds to a steroid receptor present in the cytoplasm and promotes a
conformational transformation of the receptor so that a
high-affinity nuclear complex is delivered to a response site on
the nuclear DNA protein complex. There, it enhances transcription
of mRNA which migrates from the nucleus to the ribosomes to result
in increased production of muscarinic receptors.
[0042] A second possibility is that SaG is an agonist of an unknown
receptor, which acts to cause an increase in mRNA expression by
binding to the DNA protein complex in the nucleus and acting as a
promoter.
[0043] In either case, the binding of the SaG-receptor complex to
DNA may cause an increase in the expression of mRNA which codes for
cholinergic receptors, dopaminergic receptors, or adrenergic
receptors or other membrane-bound receptors.
[0044] Alternatively, the binding of the SaG receptor complex to
the DNA may cause an increase in the production of linked proteins
such as G protein; or impede their degradation; or later the
linkage between such proteins and associated receptors, thereby
causing secondary changes in receptor number.
[0045] The effects of SaG may be mediated through increases in the
levels of one or more neurotrophic factors, for example nerve
growth factor (NGF).
[0046] It is also recognised that, in addition to the neuronal and
cholinergically mediated synaptic mechanisms, it is possible that
substances such as nitric oxide (NO) and non-cholinergic agonists
can have a modulating effect on cholinergic transmission.
[0047] Whatever the precise nature of the cell component to which
SaG binds in order to exert its effect, this provides a new pathway
on which potential treatments for AD/SDAT, AAMI and the like can be
targeted.
[0048] It has been shown that SaG increases the levels of
membrane-bound receptor mRNA, specifically m.sub.1 receptor mRNA.
It is therefore possible that the cytosolic or nuclear receptor or
promoter, when activated, increases the production of mRNA
molecules in the tissue, organ, cell type or organelle which code
for membrane-bound receptors, or that it decreases the breakdown of
mRNA molecules in the tissue, organ, cell type or organelle which
code for membrane-bound receptors.
[0049] The cytosolic or nuclear receptor, when activated, may also
increase the transcription of mRNA molecules in the tissue, organ,
cell type or organelle which code for membrane-bound receptors.
[0050] As mentioned above, nicotinic receptors may modulate the
number and/or turnover of membrane-bound receptors. Accordingly, in
one embodiment, the action of the cytosolic or nuclear receptor is
modulated by administering a substance which is at least a partial
agonist of nicotinic receptors.
[0051] It is presently preferred that the action of the cytosolic
or nuclear receptor is modulated by administering a substance which
is at least a partial agonist thereof.
[0052] The agonist may be a saponin or a sapogenin, preferably one
or more of sarsasapogenin, smilagenin, prazerigenin, an
astragaloside, tigogenin, ruscogenin, hecogenin and diosgenin.
These compounds do not have a high overt oestrogenic and/or
androgenic and/or anabolic activity in patients. A low level of
oestrogenic and/or androgenic supplementation may be beneficial in
the method of the ninth aspect of the present invention.
[0053] The receptor may be located in the cytosol of the cells of
the tissue, organ, cell type or organelle and, when activated by
binding an agonist, migrates to the nucleus of the cells. It is
also possible that the receptor is located in the nucleus of the
cells of the tissue, organ, cell type or organelle, the agonist
diffusing into the nucleus or being transported there by another
mechanism.
[0054] In the method in accordance with the first aspect of the
present invention, it is not essential for an administered
substance to act directly on the cytosolic or nuclear receptor
itself. Instead, action can be taken either upstream or downstream
of the cytosolic or nuclear receptor's or promoter's involvement in
the pathway. Thus, the action of the cytosolic or nuclear receptor
may be modulated by administering a substance which increases
expression of the mRNA molecules in the tissue, organ, cell type or
organelle which code for membrane-bound receptors.
[0055] The role of oestrogen and other related compounds as
possible treatments for SDAT has received considerable interest. In
the studies conducted to look at the effects of a cholinesterase
inhibitor, tacrine, on cognitive function in patients with SDAT a
secondary analysis suggested that all of the improvement was seen
in female patients who were also receiving hormone (oestrogen)
replacement therapy (ERT). Epidemiological data also suggest that
ERT may protect against the development of SDAT. There is extensive
work in the rat that suggests that ovariectomy results in reduced
cognitive function and this effect can be reversed at least in part
by the administration of oestrogen. The effects of oestrogen in
this model may be to increase high affinity choline uptake in
certain areas in the brain, particularly the hippocampus, thereby
improving cholinergic transmission. In the same model,
administration of oestrogen has been shown to increase the levels
of mRNA for brain derived neurotropic factor (BDNF) using suitable
in-situ hybridisation techniques (Singh 1995).
[0056] Possible mechanisms behind the effects of oestrogen have
been investigated in in-vitro experiments. These studies have been
undertaken using a neuroblastoma cell line and the response of the
cells to serum deprivation or the effects of beta amyloid (BA)
fractions. This latter stimulus is thought to be of particular
relevance because of the prominence of amyloid plaques in the late
stages of SDAT. Both serum deprivation and BA induce cell death.
17-.beta. oestradiol has been shown to protect against cell death
induced by serum deprivation and BA. The protective effect was not
abolished when the 17-.beta. oestradiol was tested in the presence
of the oestrogen antagonist, tamoxifen. The non-oestrogenic
enantiomer, 17-.alpha. oestradiol, was as effective in inhibiting
cell death. Subsequent work has suggested that the protective
effects of these compounds depends on the presence of a fully
de-saturated phenolic A ring and an unblocked hydroxyl group at the
three position (Simpkins 1997; Green 1997). In neuroblastoma cell
cultures, oestrogenic compounds were shown to increase the release
of nerve growth factor. The relevance of these findings to the
effects of oestrogen in SDAT remains unclear.
[0057] 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
CN109603A 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
recognised that there is no infective element to a very large
number of the conditions that are characterised 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.
[0058] In identifying compounds that would have use in the
treatment of SDAT and other diseases characterised 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. This data is summarised below in Table 1.
TABLE-US-00002 TABLE 1 Oestrogenic effects of steroid sapogenin
compounds and selected triterpenoid Compound Oestrogenic Activity
Diosgenin Positive Anzurogenin D Negative Ruscogenin Positive
Sarsasapogenin Negative Tigogenin Negative Astragaloside Negative
Smilagenin Negative
[0059] In addition these compounds were tested at other steroid
receptors as it was considered that compounds that would be of
clinical sue should have no effects at the other steroid receptors.
None of the compounds was found to have any activity at any of the
following receptors:
Progesterone
Glucocorticoid
Testosterone
Thus the compounds that were shown not to have activity at the
oestrogen receptor were also inactive at the other important
steroid receptors.
[0060] The selected compounds have also been tested for their
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: [0061] 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. [0062] 2. The effects of muscarinic
receptor expression in cultured cell lines of neuronal origin were
investigated. [0063] 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
falls on prolonged culture and the effects of compounds of interest
in preventing the fall in receptor numbers was investigated. The
methods and the results of these experiments are now described in
turn. 1 CHO Cell Line Experiments
[0064] 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 summarised in the table 2 below. TABLE-US-00003 TABLE 2 Effects
of compounds on the expression of m.sub.2 receptors on CHO cells
Effect on receptor expression - given as % increase compared to
Molar concentration control (negative values Compound of compound
in 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
[0065] Thus the experiments indicate that several of the compounds
were able to increase the number of muscarinic receptors expressed
on the surface of CHO cells cultured in-vitro. The effect was not
antagonised by tamoxifen, indicating that the mechanism involved
did not involve the oestrogen receptor. Unlike in the work
published by Simpkin et al it was found that there was no need for
an intact phenol A-ring. Equally a number of compounds that are
steroid sapogenins were devoid of activity. Furthermore, additional
experiments indicated that .beta.-oestradiol had a similar effect
in increasing receptor expression when administered at a
concentration of 10.sup.-5M.
2 Effects of Compounds on Cell Survival
[0066] Other in vitro assays have been used to distinguish between
active and non-active compounds. 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-labelled ligand binding techniques. This reduction in
receptor numbers was found to be ameliorated by the active
compounds.
3 Effects of Compounds on Cultured Cardiac Muscle Cells.
[0067] 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 homogenisation
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.
[0068] The results of these experiments are summarised in Table 4:
TABLE-US-00004 TABLE 4 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 Compound neonatal cardiac muscle after 10 days in
vitro 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
[0069] Surprisingly the inventors have found that sapogenins are
preferentially concentrated in the nuclei of cells cultured in
vitro. This is surprising because, as discussed above,
sarasasapogenin (SaG) and some other compounds which have been
shown to increase the number of muscarinic receptors do not bind to
known steroidal receptors. In addition, it is surprising that SaG
is preferentially taken up into the nucleus because the effects of
these compounds can be seen in in-vitro assay systems that express
the muscarinic receptor but where the DNA for the receptor has been
transfected into the cytoplasm and hence is not under the normal
nuclear control mechanism.
[0070] SaG and the other compounds that have been tested and shown
to up-regulate the levels of receptors, have all been shown not to
bind directly to any of the major known classes of membrane bound
receptor. Thus it can be postulated that the observed effects are
probably not due to for instance an effect at the nicotinic
receptor and a consequential increase in the number of muscarinic
receptors. This explanation appears to be even less plausible
(although it cannot be excluded) if one considers that certain of
the compounds have also been shown by the inventors to increase the
number of beta adrenergic receptors expressed on peripheral blood
lymphocytes. Thus the mechanism would appear to be one which has a
more general effect on the regulation of membrane bound
receptors.
[0071] It is speculated here that the effect of the active
compounds 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 internalisation 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.
[0072] 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.
[0073] 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 interneurone
connections.
[0074] 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 .alpha.-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 .alpha.-secretase.
[0075] 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 presynaptic
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.
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;
2. Secondary to the increased receptor numbers, an increase
stimulation of PKC with a consequential increase in
.alpha.-secretase activity, leading to:
2.1 A reduced production of .beta.-amyloid and a consequent
reduction of plaque formation and neuronal loss;
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.
[0076] Finally the effects of the GABA system in modulating
transmission has been discussed above. It is well know that there
is a steroid binding site on the GABA receptor that is distinct
from the benzodiazepine, chloride and GABA binding sites. A number
of therapeutic compounds are know to bind to this site and have
been used to enhance or reduce the level of consciousness. It is
speculated that the chronic administration of a partial agonist at
this site might lead to an enhancement of transmission.
[0077] The invention will be described further in the following
example.
EXAMPLE
Investigation of mRNA Levels Using In Situ Hybridisation
[0078] 20 months old pure-line male SD rats were divided randomly
into 2 groups. One group received an average of 3 mg of
sarsasapogenin per rat per day mixed into the daily feed. The
control group received normal food and water. Four months later,
their brains were used in hybridisation technique experiments, with
4 to 6 months old rats used as a young control group. Other feeding
arrangements for each group were completely identical.
[0079] A cDNA chain which respectively corresponds to the mRNA of
both m.sub.1 and m.sub.2 was synthesised. m.sub.1 corresponds to
the 3-18 amino acid sequence of receptor protein, i.e. TGG TGC CAA
GAC AGT GAT GTT GGG ACT GAC AGC AGG GGG CAC TGA GGT, and M.sub.2 to
the 1-16 amino acid sequence, i.e. ATG AAT AAC TCA ACA AAC TCC TCG
AAC AAT GGC TTG GCT ATT ACC AGT. The cDNA was labelled using a
3'-terminal-label reagent kit with a-.sup.35S-dATP (8.9 TBq/mmol)
as the label material. After the reaction had finished, it was
purified with a nucleotide column. The specific activity of the
batch was estimated (16.67-33.34).times.108 MBq/.mu.g.
a-.sup.35S-dATP, 3'-terminal-label reagent kit and nucleotide
column were obtained from Du Pont Co., USA.
[0080] One rat was obtained from each group each time and parallel
experiments were performed. The rats were decapitated and their
brains removed intact. 15 .mu.m thick coronal slices were prepared
in a constantly freezing cryo-microtome (AS-600 cryo-microtome,
Anglia Scientific Co, UK). Slices were taken from different areas
(identical places for each rat) and were mounted on slides smeared
with polylysine, dried in a cool current of air, fixed in a
solution of 4% paraformaldehyde (containing 1.times. phosphate
buffer saline (PBS), pH 7.0) for 5 minutes before being washed
twice in PBS. They were then placed in 0.25% acetic anhydride
solution (containing 0.1 M triethanolamine hydrochloride, Ph 8.0,
and 0.9% sodium chloride) for 10 minutes, dehydrated in 70%, 80%,
95%, and 100% ethyl alcohol for 1 minute, degreased in chloroform
for 5 minutes, and finally treated in 100% and 95% ethyl alcohol
for 1 minute successively.
[0081] Slices used as negative controls were taken and dehydrated
in ethyl alcohol etc. as detailed above, but treated in advance in
100 mg ml RNase and 2.times.SSC solution (salt/sodium citrate
solution containing 300 mmol/L sodium chloride and 45 mmol/L sodium
citrate) for 2 hours at 37.degree. C.
[0082] For hybridisation, the fluid matrix for hybridisation was
compounded with freshly containing 50% deionised formamide,
4.times.SSC, 10% dextran sulphate, 250 .mu.g/.mu.l yeast tRNA,
5.times. Denhard solution, 500 .mu.g/ml denaturation protamine DNA,
10 mmol/L dithiothreitol. Oligonucleotide probe
[(16.67-33.34).times.10 MBq/50 .mu.l] labelled with .sup.35S was
added finally and mixed evenly. 50 .mu.l of the matrix was dripped
onto each slice and a silicate cover glass was placed lightly over,
avoiding airlocks. The slices were then shelved in a hybridisation
box with 2.times.SSC on the bottom to preserve moisture, and
incubated at 37.degree. C. for 18 to 24 hours.
[0083] After hybridisation, the slides were soaked in 1.times.SSC
solution and shaken slightly to rinse the cover glass. They were
washed in 1.times.SSC solution briefly, then vibrated gently in
2.times.SSC containing 50% formamide at 37.degree. C. for 20
minutes with the solution changed four times, and then transferred
into 1.times.SSC solution for vibration at laboratory temperature
for 30 minutes (repeated twice). Finally, the slides were washed
with double distilled water, dehydrated with 70%, then 95% ethyl
alcohol and dried in the air.
[0084] Autoradiographs were prepared in a dark room, the specimen
and the hyperfilm beta max being pressed together using the contact
method and placed in a cassette with a desiccant, exposed at
4.degree. C. for 2 to 3 weeks. They were developed (D196) and fixed
(F5). Finally, the autoradiographs were analysed using a
computerised image analyser (VIDAS imaging analyser, Kontron,
Germany).
[0085] The m.sub.1 probe failed to show any localised area of
activity. The m.sub.1 probe showed activity in dentate nucleus,
cerebral cortex and striatum. Comparison of these three areas for
the different animal groups are shown in Table 5: TABLE-US-00005
TABLE 5 Comparison Area Aged vs young SaG vs Aged Cortex -5.14 .+-.
2.68 (23) 5.77 .+-. 3.82 (20) Hippocampus -3.18 .+-. 2.87 (12) 0.96
.+-. 4.26 (10) Striatum -12.2 .+-. 3.6* 15.71 .+-. 3.27* (10)
Positive means increased compared to the comparator. *p < 0.01.
Numbers in brackets = numbers of slices.
[0086] There was a significant reduction in mRNA expression for
m.sub.1 receptors in the striatum of aged rats compared to young
controls. Administration of SaG resulted in a significant increase
in m.sub.1 receptor mRNA in the same brain area when treated
animals were compared to aged, untreated controls.
* * * * *