U.S. patent application number 13/296433 was filed with the patent office on 2012-08-02 for use of binding partners for 5-ht5 receptors for the treatment of neurodegenerative and neuropsychiatric disorders.
Invention is credited to Francisco Javier Garcia-Ladona, Hans-Peter Hofmann, Gerd Steiner, Laszlo Szabo.
Application Number | 20120196301 13/296433 |
Document ID | / |
Family ID | 7893918 |
Filed Date | 2012-08-02 |
United States Patent
Application |
20120196301 |
Kind Code |
A1 |
Garcia-Ladona; Francisco Javier ;
et al. |
August 2, 2012 |
USE OF BINDING PARTNERS FOR 5-HT5 RECEPTORS FOR THE TREATMENT OF
NEURODEGENERATIVE AND NEUROPSYCHIATRIC DISORDERS
Abstract
The present invention relates to the use of binding partners for
5-HT5 receptors for the treatment of neuropathological, in
particular neurodegenerative and/or neuropsychiatric, disorders,
which can occur, in particular, in cerebral ischemia, stroke,
epilepsy and seizures in general, chronic schizophrenia, other
psychotic disorders, dementia, in particular Alzheimer's dementia,
demyelinizing disorders, in particular multiple sclerosis, and
brain tumors. The invention also relates to processes for the
identification and characterization of such binding partners, in
particular in the form of screening processes.
Inventors: |
Garcia-Ladona; Francisco
Javier; (Kandel, DE) ; Szabo; Laszlo;
(Dossenheim, DE) ; Steiner; Gerd; (Kirchheim,
DE) ; Hofmann; Hans-Peter; (Limburgerhof,
DE) |
Family ID: |
7893918 |
Appl. No.: |
13/296433 |
Filed: |
November 15, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10836349 |
May 3, 2004 |
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13296433 |
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09889157 |
Jul 11, 2001 |
6750221 |
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PCT/EP2000/000143 |
Jan 11, 2000 |
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10836349 |
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Current U.S.
Class: |
435/7.21 ;
544/250 |
Current CPC
Class: |
G01N 2500/04 20130101;
G01N 33/566 20130101; A61P 9/10 20180101; A61P 25/18 20180101; A61K
31/519 20130101; A61P 25/28 20180101; A61P 25/08 20180101; A61P
25/00 20180101; G01N 2500/02 20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/7.21 ;
544/250 |
International
Class: |
G01N 33/566 20060101
G01N033/566; C07D 495/14 20060101 C07D495/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 11, 1999 |
DE |
19900673.3 |
Claims
1. The use of at least one binding partner for 5-HT5 receptors for
the preparation of an agent for the treatment of neuropathological
disorders and associated indications, symptoms and
dysfunctions.
2. The use as claimed in claim 1 in cerebral ischemia, stroke,
epilepsy and attacks in general, chronic schizophrenia, other
psychotic disorders, dementia, in particular Alzheimer's dementia,
demyelinizing disorders, in particular multiple sclerosis, and
brain tumors.
3. The use as claimed in claim 1 or 2, wherein the K.sub.i values
of the binding partner for its binding to 5-HT5 receptors are less
than 10-.sup.6 M, preferably less than 10-.sup.7 M and in
particular less than 10-.sup.8 M.
4. The use as claimed in one of claims 1 to 3, wherein the binding
affinity of the binding partner for 5-HT5 receptors is greater than
for one or more 5-HT receptors other than 5-HT5.
5. The use as claimed in one of the preceding claims, wherein the
binding affinity of the binding partner for 5-HT5 receptors is
greater than for 5-HT1A, 5-HT1D and/or 5-HT1B receptors.
6. The use as claimed in one of the preceding claims, wherein the
binding partner competitively inhibits the binding of 5-CT to 5-HT5
receptors.
7. A process for the determination of the affinity of binding
partners for 5-HT5 receptors, where the binding partner is brought
into contact with cell systems having 5-HT5 receptors and the
binding affinity is determined.
8. A process for the determination of the activity of binding
partners for 5-HT5 receptors, where the binding partner is brought
into contact with cell systems having 5-HT5 receptors and at least
one binding partner-induced action is determined.
9. A process as claimed in claim 8, where the binding of GTP to G
proteins, intracellular calcium levels, the phospholipase C
activity and/or the cAMP production are determined.
10. A process as claimed in either claim 8 or 9, wherein human
glioma cell lines or h5-HT5-transfected heterologous cell lines are
used.
11. A process as claimed in claim 10, wherein h5-HT5-transfected
CHO cells, h5-HT5-transfected human kidney cells, or
h5-HT5-transfected C-6 glioma cells are used.
12. An in vitro screening process for the identification of a 5-HT5
receptor binding partner, where at least one process as claimed in
claims 7 to 11 is used.
13. A process as claimed in claim 12 for the identification of
binding partners as defined in claims 1 to 6.
Description
[0001] The present invention relates to the use of binding partners
for 5-HT5 receptors for the treatment of neuropathological
disorders and associated indications, symptoms and dysfunctions and
to processes for the identification and characterization of binding
partners of this type.
[0002] At least seven different receptor classes mediate the
manifold physiological activities which are ascribed to an
involvement of the neurotransmitter serotonin (5-hydroxytryptamine,
abbreviated 5-HT). According to an internationally recognized
classification, they are designated by 5-HT1,
5-HT2,5-HT3,5-HT4,5-HT5,5-HT6 and 5-HT7. Most of these classes
moreover include receptor types which can be differentiated
further. Thus the 5-HT1 class includes receptors which can be
divided into at least five subclasses, which are designated by
5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D and 5-HT1E (Boess F. G. and Martin
I. L., Neuropharmacology 33:275-317 (1994)).
[0003] The 5-HT5 class was described for the first time by Plassat
et al., The EMBO Journal Vol. 11 No. 13, pp. 4779-4786 (1992).
5-HT5A and 5-HT5B receptors are differentiated (Erlander et al.,
Proc. Natl. Acad. Sci. USA 90:3452-3456 (1993)). Only small
sequence homologies exist between 5-HT5 and other 5-HT receptors.
The pharmacological profile of these receptors differs markedly.
Using molecular biology techniques, the localization of 5-HT5
receptors was possible in the olfactory bulb, in the hippocampus,
in the cortex, in the cerebral ventricles, in the corpus callosum
and in the cerebellum. By means of immunohistochemical methods, it
was possible to show that 5-HT5 receptors are principally expressed
on astrocytes (Carson et al., GLIA 17:317-326 (1996)). Astrocytes
are directly adjacent to the basal membrane of brain capillaries of
the blood-brain barrier. An abnormal astrocyte endothelium
structure accompanies a loss of the blood-brain barrier. The exact
significance of the astrocytes is unclear. They appear to look
after transport tasks and connective functions. Reactive astrocytes
were observed in connection with reactive gliosis in a number of
pathological brain changes and neuropsychiatric disorders. As a
result of brain injuries, they change their morphologies. The
protein expression pattern changes and growth factors are produced.
In vitro investigations on cultured astrocytes have allowed the
detection of 5-HT5 receptor-mediated responses. It is thus to be
suspected on the one hand that they are involved in recovery
processes of the brain after disorders, but on the other hand it is
also not to be excluded that they contribute to the creation of
damage or even to an increase in damage.
[0004] CNS disorders nowadays concern large sections of the
population. In particular on account of the increase in elderly
people, the numbers of patients are increasing continuously.
Neuropathological conditions such as cerebral ischemia, stroke,
epilepsy and attacks in general, chronic schizophrenia, other
psychotic disorders, dementia, in particular Alzheimer's dementia,
demyelinizing disorders, in particular multiple sclerosis, and
brain tumors lead to damage to the brain and the neuronal deficits
associated therewith.
[0005] Therapeutic treatments of the neurodegenerative and
neuropsychiatric disorders outlined were up to now directed at
various membrane receptors with the aim of compensating deficits in
neurotransmission processes. Indeed, it was possible to achieve
neuroprotective effects with serotonogic compounds in animal models
of neuropathological conditions, such as ischemia, cerebral stroke
and excitotoxicity. In some cases, it was also possible to observe
favorable effects on emotional disturbances, such as depression or
anxiety states. Mention may be made here, for example, of 5-HT1A
agonists, such as buspirone or the compound
8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), which is
characterized as a selective 5-HT1A receptor ligand. These active
compounds, however, only decrease neurological deficits to a
limited extent. There is still no effective therapy at present.
[0006] It is therefore an object of the present invention to make
possible the treatment of neuropathological disorders with adequate
efficacy and minor side effects.
[0007] Surprisingly, it has now been found that treatment of the
above disease conditions and associated indications, symptoms and
dysfunctions is made possible by specific use of substances having
binding affinities for 5-HT5 receptors.
[0008] One subject of the present invention is therefore the use of
binding partners for 5-HT5 receptors for the preparation of an
agent for the treatment of neuropathological disorders and
associated indications, symptoms and dysfunctions.
[0009] Neuropathological disorders are understood according to the
invention as meaning disorders which are accompanied by
neurological deficits, i.e. a condition characterized by
neurological deficiency symptoms. The term "disorder" in the sense
according to the invention designates anomalies which, as a rule,
are regarded as pathological conditions and can reveal themselves
in the form of certain signs, symptoms and/or dysfunctions. The
treatment according to the invention can be directed at individual
disorders, viz. anomalies or pathological conditions, but a number
of anomalies which are causally connected to one another can be
combined to give patterns, i.e. syndromes, which can be treated
according to the invention.
[0010] This condition can exist temporarily, progressively or
persistently.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0011] FIG. 1 shows a plot of bound [.sup.3H]-5-CT as a function of
the [.sup.3H]-5-CT concentration.
[0012] FIG. 2 shows 5-HT and 5-CT induced an increase in the
[.sup.35S] GTP.gamma.S binding to cell membranes over basal
values.
[0013] FIG. 3a shows a plot of the percentage of total binding as a
function of the GDP concentration.
[0014] FIG. 3b shows the [.sup.35S] GTP.gamma.S binding
stimulation.
[0015] FIG. 4 shows the [.sup.35S] GTP.gamma.S binding stimulation
for the percentage of the maximal activity as a function of the
5-HT concentration.
[0016] According to the invention, the treatment of
neurodegenerative and/or neuropsychiatric disorders is preferred.
These disorders occur, in particular, in neuropathological
syndromes, as a rule syndromes caused by brain damage, for example
cerebral ischemia, stroke, epilepsy and seizures in general,
chronic schizophrenia, other psychotic disorders, dementia, in
particular Alzheimer dementia, demyelinizing disorders, in
particular multiple sclerosis, and brain tumors. The invention in
particular also relates to the use of 5-HT5 binding partners for
the treatment of those forms of the abovementioned disorders in
whose formation and/or course 5-HT5 receptors are involved, i.e.
disorders which are modulated by a 5-HT5 receptor activity.
[0017] According to a further aspect of the present invention,
neuropathological disorders are treated which accompany a glial
reaction. The use according to the invention relates in particular
to the modulation of a glial reaction.
[0018] An advantageous action of the binding partners is seen in
the preventive or acute treatment of neurological deficits, which
are observed in patients who suffer from psychiatric disorders,
such as epilepsy, psychosis, e.g. psychoses of the acute exogenous
reaction type or concomitant psychoses of organic or exogenous
cause, e.g. after trauma, especially brain lesions and diffuse
brain damage, in metabolic disorders, infections, and
endocrinopathies; endogenous psychoses, such as schizophrenia, and
schizotypic and delusional disorders; effective disorders, such as
depression, mania and manic depressive conditions; and mixed forms
of the psychoses described above; senile dementia and senile
dementia of the Alzheimer type, and in the treatment or prevention
of demyelinization processes.
[0019] The binding partners according to the invention are
efficacious, in particular with respect to the treatment of
ischemic damage, e.g. as a result of brain and spinal cord trauma
and vascular occlusion or heart failure.
[0020] Especially to be mentioned here is stroke (synonym: cerebral
apoplexy, cerebral or apoplectic insult, cerebral stroke).
Transitory ischemic attacks, reversible ischemic neurological
deficits, prolonged reversible ischemic neurological deficits,
partially reversible ischemic neurological symptoms and also
persistent complete cerebral infarcts can be treated according to
the invention. The treatment of acute forms is particularly
advantageous according to the invention.
[0021] One or more of the changes in nerve tissues listed below
underlie the forms of neuropathological disorders which can be
preferably treated according to the invention: degeneration or
death of neurons, in particular of the ganglial cells, e.g.
tigrolysis, indistinctness of the nuclear membrane, plasmolysis,
cytoplasm vacuolization and encrustation, parenchymal necroses of
the brain, cerebral edema, changes to neurons caused by oxygen
deficiency, atrophy, morphological changes, such as
demyelinization, in particular medullary sheath disintegration,
perivascular infiltrates, glial proliferation and/or glial
scarring; degeneration of the Substantia nigra.
[0022] The indication to be treated according to the invention is
often characterized by a progressive development, i.e. the
conditions described above change in the course of time, as a rule
the degree of severity increases and conditions further to already
existing conditions can occur.
[0023] By means of the treatment according to the invention of
neuropathological disorders or of the conditions underlying them, a
number of further signs, symptoms and/or dysfunctions which are
connected with the neuropathological disorders can be treated, i.e.
in particular accompany the disorder conditions described above.
These include, for example, shock lung, brain nerve losses, e.g.
retrobulbar neuritis, eye muscle paralysis, syllabication, spastic
paralysis, cerebella symptoms, sensitivity, bladder and rectal
disorders, euphoria, dementia, hyper- and akinesia, absence
synchynesis, small-step gait, bent posture of trunk and limbs,
pro-, retro- and lateropulsion, tremor, lack of facial expression,
monotonous speech, depression, apathy, labile or rigid affectivity,
impaired spontaneity and resoluteness, slowed thinking, poor
association ability; muscular atrophy.
[0024] A treatment in the sense according to the invention
comprises not only the treatment of acute or chronic signs,
symptoms and/or dysfunctions but also a preventive treatment
(prophylaxis), in particular as a relapse or phase prophylaxis. The
treatment can be achieved symptomatically, for example as symptom
suppression. It can be carried out short-term, be carried our
medium-term, or it can also be a long-term treatment, for example
in the context of a maintenance therapy.
[0025] The term "binding partner for 5-HT5 receptors" describes
substances which bind to 5-HT5 receptors and can therefore also be
designated as 5-HT5 receptor ligands.
[0026] Binding is understood as meaning any molecular interaction
between the binding partner and the receptor, in particular under
physiological conditions. As a rule, these are conventional
interactions, which include electrostatic attraction, hydrogen
bonding, hydrophobic bonds, van-der-Waals forces or metal
complex-like coordinative bonds. In addition to the
above-mentioned, reversible molecular interactions, irreversible
interactions between binding partner and receptor can also be
possible, such as, for example, covalent bonds.
[0027] According to one embodiment, binding partners which can be
used according to the invention competitively inhibit the binding
of comparison binding partners, such as 5-HT (5-hydroxytryptamine)
or 5-CT (5-carboxamidotryptamine), to 5-HT5 receptors.
[0028] Competitive inhibition is understood as meaning that binding
partners which can be used according to the invention compete with
a comparison binding partner, in the present case, for example,
5-HT or 5-CT, for binding to the receptor, i.e. the binding of one
prevents the binding of the other.
[0029] According to a further embodiment, binding partners which
can be used according to the invention inhibit the binding of
comparison binding partners, such as 5-HT (5-hydroxytryptamine) or
5-CT (5-carboxamidotryptamine) to 5-HT5 receptors
noncompetitively.
[0030] Noncompetitive inhibition is understood as meaning that
binding partners which can be used according to the invention
modulate, via their binding to the receptor, the binding of a
comparison binding partner, in the present case, for example, 5-HT
or 5-CT, in particular lower its binding affinity.
[0031] At least in the case of competitive inhibition, i.e. of
reversible binding, the principle applies that the displacement of
one binding partner by another increases with decreasing binding
affinity of the one or increasing binding affinity of the other
with respect to the receptor. More expediently, therefore, binding
partners which can be used according to the invention have a high
binding affinity for 5-HT5 receptors. A binding affinity of this
type allows, on the one hand, an effective displacement of
naturally occurring binding partners for 5-HT5 receptors, such as,
for example, serotonin (5-hydroxytryptamine, 5-HT) itself, where
the necessary concentration of binding partner which can be used
according to the invention for the binding of a certain amount of
this binding partner to 5-HT5 receptors decreases with increasing
binding affinity. With respect to medical use, binding partners are
therefore preferred whose binding affinity is so great that these
can be administered in justifiable amounts in the course of an
effective medical treatment as an active compound. Binding partners
which can be used according to the invention are therefore
preferably administered in daily doses of approximately 0.01 to 100
mg/kg of body weight and in particular of approximately 0.1 to 15
mg/kg of body weight on parenteral administration and 1 to 30 mg/kg
of body weight on oral administration.
[0032] The competition experiments referred to above, with which
that concentration of binding partner which can be used according
to the invention is determined in vitro which displaces 50% of
another comparison binding partner from the receptor binding site
(IC.sub.50 values), offer one possibility of expressing the binding
affinity. Thus the competitive inhibition of the binding of 5-CT to
5-HT5 receptors can also be evaluated to the effect that binding
partners which can preferably be used according to the invention
have half-maximal inhibition constants IC.sub.50 of less than
10.sup.-5 M, preferably of less than 10.sup.-6 M and in particular
of less than 10.sup.-7 M.
[0033] The binding affinity of binding partners which can be used
according to the invention can also be expressed by means of the
inhibition constant K.sub.i, which is in general likewise
determined in vitro using competition experiments. For the binding
of 5-HT5 receptors, binding partners which can be used according to
the invention preferably have K.sub.i values of less than 10.sup.-6
M, advantageously of less than 10.sup.-7 M and particularly
preferably of less than 10.sup.-8 M. K.sub.i values of compounds
which can be used according to the invention lie, for example, in
the range from 110.sup.-7 M to 710.sup.-7 M or in the range from
110.sup.-8 M to 110.sup.-7 M.
[0034] Binding partners which can be used can bind with a lower, an
essentially identical, or a higher affinity to 5-HT5 than to a
specific receptor which is different from 5-HT5.
[0035] Thus binding partners for 5-HT5 receptors with respect to
the use according to the invention in particular include those
whose binding affinity for 5-HT5 receptors compared with the
affinity for 5-HT1 receptors, in particular 5-HT1A, 5-HT1B and/or
5-HT1D, is so high that they are advantageously suitable for the
use according to the invention. This does not necessarily
presuppose a comparatively more selective binding to 5-HT5
receptors, even though selective binding partners for 5-HT5
receptors are a particular embodiment of the present invention.
[0036] For example, binding partners can be used which have high
affinity both for 5-HT5 and for 5-HT1 receptors, in particular for
5-HT1A, 5-HT1B and/or 5-HT1D. In this connection, high affinity
means K.sub.i values as a rule in the range from 110.sup.-9 M to
110.sup.-6 M. According to a particular embodiment, binding
partners which can be used in the high affinity range have a
binding profile for 5-HT receptors which is characterized by a
binding affinity to 5-HT5 which, in comparison to other binding
affinities of this range is essentially identical or only slightly
less. Factors of 10 or less can be advantageous.
[0037] Selective binding partners which can be used according to
the invention have binding affinities for 5-HT5 receptors which are
larger than for one or more 5-HT receptors which are different from
5-HT5, i.e. in particular receptors allocated to the abovementioned
5-HT receptor classes 5-HT1,5-HT2,5-HT3,5-HT4,5-HT6 and 5-HT7. If
the binding affinity for 5-HT5 receptors of a binding partner is
greater than that of a 5-HT receptor which is different from 5-HT5,
we speak of a selective binding of these binding partners to 5-HT5
receptors in relationship to the 5-HT receptor which is different
from 5-HT5. Particular binding partners are those whose binding
affinity for 5-HT5 receptors is greater than for at least one and
in particular all 5-HT1 receptors, in particular for 5-HT1A, 5-HT1D
and/or 5-HT1B receptors. Binding partners whose binding affinity
for 5-HT5 receptors is greater than for all 5-HT receptors which
are different from 5-HT5 constitute a further particular class of
binding partners according to the invention.
[0038] Selectivity is understood as meaning the property of a
binding partner to bind preferably to 5-HT5 receptors.
[0039] It is decisive for the selectivity outlined above that the
binding affinities for 5-HT5 receptors on the one hand and for one
or more of 5-HT receptors which are different from 5-HT5 on the
other hand are adequately different. Affinity differences are
preferred according to which binding affinity ratios of at least 2,
advantageously of at least 5, particularly advantageously of at
least 10, preferably of at least 20, particularly preferably of at
least 50 and in particular of at least 100 are present.
[0040] According to a further embodiment, binding partners which
can be used according to the invention bind selectively to 5-HT5
receptors having the advantageous binding affinities described
above in relation to one or more 5-HT receptors other than
5-HT5.
[0041] According to a further embodiment, binding partners which
can be used according to the invention bind selectively to 5-HT5
receptors having the advantageous binding affinities described
above in relation to all 5-HT receptors other than 5-HT5.
[0042] Binding partners are particularly advantageous which bind to
5-HT5 receptors which are expressed by glia cells and in particular
by astrocytes with the affinities and selectivities described
above.
[0043] According to the invention, the human receptor variant is a
preferred target for the binding partners employed according to the
invention.
[0044] The binding of binding partners according to the invention
to 5-HT5 receptors is coupled to an effector function. Binding
partners can act agonistically or antagonistically and partly
agonistically and/or partly antagonistically.
[0045] Agonists are designated as compounds according to the
invention which completely or partially imitate the activity of
5-HT on 5-HT5 receptors.
[0046] Antagonists are designated as compounds according to the
invention which can block the agonistic activity of 5-HT on 5-HT5
receptors.
[0047] According to a preferred embodiment of the present
invention, binding partners are employed whose binding at least to
5-HT5 receptors of h5-HT5-transfected CHO cells brings about a
change in the agonist-induced stimulation of GTP binding to
membrane-bound G proteins, a change in intracellular calcium
levels, a change in agonist-induced induction of phospholipase C
activity and/or a change in cAMP production. As far as the change
in intracellular calcium levels is concerned, the use of binding
partners which bring about an increase in intracellular calcium
levels represents a particular embodiment of the invention.
[0048] This embodiment also includes binding partners which are
active in known animal models for neurodegenerative and
neuropsychiatric processes.
[0049] Preferred binding partners are those which are also
selective for 5-HT5 receptors in relation to their effector
function in the sense described above.
[0050] Compounds which can be used according to the invention are
described, for example, in DE 197 24 979.5. These are 3-substituted
3,4,5,6,7,8-hexahydropyrido[3',4':4,5]thieno-[2,3-d]pyrimidine
derivatives of the formula I
##STR00001##
in which R.sup.1 is a hydrogen atom, a C.sub.1-C.sub.4-alkyl group,
an acetyl group, a phenylalkyl C.sub.1-C.sub.4 radical, where the
aromatic is optionally substituted by halogen,
C.sub.1-C.sub.4-alkyl, trifluoromethyl, hydroxyl,
C.sub.1-C.sub.4-alkoxy, amino, cyano or nitro groups or is a
phenylalkanone radical, where the phenyl group can be substituted
by halogen, R.sup.2 is a phenyl, pyridyl, pyrimidinyl or pyrazinyl
group, which is optionally mono- or disubstituted by halogen atoms,
C.sub.1-C.sub.4-alkyl, trifluoromethyl, trifluoromethoxy, hydroxyl,
C.sub.1-C.sub.4-alkoxy, amino, monomethylamino, dimethylamino,
cyano or nitro groups, and which can optionally be fused to a
benzene nucleus, which can optionally be mono- or disubstituted by
halogen atoms, C.sub.1-C.sub.4-alkyl, hydroxyl, trifluoromethyl,
C.sub.1-C.sub.4-alkoxy, amino, cyano or nitro groups and can
optionally contain 1 nitrogen atom, or to a 5- or 6-membered ring
which can contain 1-2 oxygen atoms, A is NH or an oxygen atom,
Y is CH.sub.2, CH.sub.2--CH.sub.2, CH.sub.2--CH.sub.2--CH.sub.2 or
CH.sub.2--CH,
[0051] Z is a nitrogen atom, carbon atom or CH, where the bond
between Y and Z can also be a double bond, and n is the number 2, 3
or 4 and their salts with physiologically tolerable acids.
[0052] Further compounds which can be used according to the
invention are described, for example, in DE 196 36 769.7. These are
3-substituted 3,4,5,6,7,8-hexahydropyrido[4',3':4,5)
thieno-[2,3-d]pyrimidine derivatives of the formula I
##STR00002##
in which R.sup.1 is a hydrogen atom, a C.sub.1-C.sub.4-alkyl group,
an acetyl or benzoyl group, a phenyl C.sub.1-C.sub.4-alkyl radical,
where the aromatic is optionally substituted by halogen,
C.sub.1-C.sub.4-alkyl, trifluoromethyl, hydroxyl,
C.sub.1-C.sub.4-alkoxy, amino, cyano or nitro groups, or is a
naphthyl-C.sub.1-C.sub.3-alkyl radical, a
phenyl-C.sub.2-C.sub.3-alkanone radical or a
phenylcarbamoyl-C.sub.2-alkyl radical, where the phenyl group can
be substituted by halogen, R.sup.2 is a phenyl, pyridyl,
pyrimidinyl or pyrazinyl group, which is optionally mono-, di- or
trisubstituted by halogen atoms, C.sub.1-C.sub.4-alkyl,
trifluoromethyl, trifluoromethoxy, hydroxyl,
C.sub.1-C.sub.4-alkoxy, amino, monomethylamino, dimethylamino,
cyano or nitro groups, and which can optionally be fused to a
benzene nucleus which can optionally be mono- or disubstituted by
halogen atoms, C.sub.1-C.sub.4-alkyl, hydroxyl, trifluoromethyl,
C.sub.1-C.sub.4-alkoxy, amino, cyano or nitro groups and can
optionally contain 1 nitrogen atom, or to a 5- or 6-membered ring
which can contain 1-2 oxygen atoms, or can be substituted by a
phenyl-C.sub.1-C.sub.2-alkyl or -alkoxy group, where the phenyl
radical can be substituted by halogen, or a methyl, trifluoromethyl
or methoxy group, A is NH or an oxygen atom, B is hydrogen or
methyl, C is hydrogen, methyl or hydroxyl, X is a nitrogen
atom,
Y is CH.sub.2, CH.sub.2--CH.sub.2, CH.sub.2--CH.sub.2--CH.sub.2 or
CH.sub.2--CH,
[0053] Z is a nitrogen atom, carbon atom or CH, where the bond,
between Y and Z can also be a double bond, and n is the number 2, 3
or 4, and their salts with physiologically tolerable acids.
[0054] 5-HT5-specific antibodies can also be utilizable as 5-HT5
binding partners. They can be polyclonal antisera, monoclonal
antibodies, antibody fragments, such as F(ab), Fc, etc., chimeric
and recombinant antibodies. Such antibodies can be prepared in a
manner known per se. As an immunogen, 5-HT5 receptor can be used as
such or antigenic fragments thereof, as a rule coupled to customary
carrier proteins.
[0055] Further low molecular weight 5-HT5 binding partners, usually
synthetic compounds, can be used advantageously in many
respects.
[0056] Aptamers, i.e. nucleic acids, as a rule oligonucleotides,
having sufficient affinity for 5-HT5 receptors, can also be used as
binding partners.
[0057] The use according to the invention is not restricted to the
abovementioned binding partners. Rather, any substance which binds
to 5-HT5 receptors in the manner described above, can be used
according to the invention as a 5-HT5 binding partner.
[0058] Assays for the determination of binding affinities of test
substances for 5-HT5 receptors are known in principle. This can be
carried out, for example, by assessing the competitive inhibition
of the binding of a comparison binding partner to 5-HT5 receptors
by the substance to be investigated. Suitable comparison binding
partners are known ligands for 5-HT receptors, such as 5-HT or 5-CT
or LSD. These are expediently labeled such that their binding to
5-HT receptors can be monitored analytically using standard
methods. Radioactive and optical markers are preferred. In binding
studies on 5-HT5 receptors, according to the invention, 5-CT or
LSD, in particular in the form of [.sup.3H]-LSD, is used. The
binding affinities can be expressed as half-maximal inhibition
constants IC.sub.50 or as inhibition constants K.sub.i. This
process is preferably used for primary screening. SPA technology or
FlashPlate technology is preferably used.
[0059] The binding to binding partners to be investigated can also
be determined directly on 5-HT receptors. The inhibition constants
K.sub.i expressing binding affinity can be determined, for example,
calorimetrically, i.e. by measurement of the binding energy
released.
[0060] Effector functions can also be assessed qualitatively or
quantitatively both in vitro and in vivo with the aid of known
functional assays.
[0061] The assessment of an agonistic activity can be based on all
those effects which are produced by the binding of 5-HT to 5-HT5
receptors. It is preferred according to the invention to assess the
effects on the binding of GTP to G proteins, on intercellular
calcium levels, on the phospholipase C activity and/or on the cAMP
production. These processes are preferably used for secondary
screening. Here too, SPA or FlashPlate technology is advantageously
used.
[0062] GTP binding to G proteins can be investigated by using a
nonhydrolyzable analog of GTP, for example [.sup.35S] GTP.gamma.S,
whose binding can be investigated radiologically. This
investigation is preferably carried out on membranes having 5-HT5
receptors.
[0063] For the measurement of intracellular calcium levels, it is
possible to employ suitable calcium probes, as a rule calcium
chelating agents, for example fluorescing compounds, such as Fura
2-acetylmethyl ester or fluo-3-AM. This investigation is preferably
carried out on cell cultures having 5-HT5 receptors, in particular
on individual cells.
[0064] The phospholipase C activity can be determined by means of
the reactions catalyzed by it, for example, the incorporation of
myoinositol, which for detection purposes is preferably
radiolabeled as [.sup.3H)-myoinositol, or the conversion of
PPIP.sub.2 to IP.sub.3, where the PPIP.sub.2 is also preferably
radiolabeled as [.sup.32P)PIP.sub.2]. These investigations are
preferably carried out on individual cells having 5-HT5
receptors.
[0065] cAMP production can be determined with the aid of the cAMP
binding protein. This investigation is preferably carried out on
individual cells having 5-HT5 receptors.
[0066] If appropriate, the effector function is also determined,
i.e. the activity of binding partners according to the invention
for other 5-HT receptors. This expediently takes place taking into
account the binding affinities determined for 5-HT5 and other 5-HT
receptors, i.e. in particular taking into account the
selectivity.
[0067] The present invention therefore also relates to processes
for the identification and characterization of binding partners
which can be used according to the invention. These and further
processes which are similarly suitable can form the basis for in
vitro screening processes with which it is possible from a large
number of different compounds to pick out those which, with respect
to future use, appear to be the most promising. For example, by
means of combinatorial chemistry, extensive substance banks can be
prepared which comprise myriads of potential active compounds. The
inspection of combinatorial substance libraries for substances
having desired activity is automatable. Screening robots are used
for the efficient evaluation of the individual assays, which are
preferably arranged on microtiter plates. Thus the present
invention also relates to screening processes, i.e. both primary
and secondary screening processes, in which preferably at least one
of the processes described below is used. If a number of processes
are used, this can be shifted in terms of time or simultaneously
carried out on one and the same sample or on different samples of a
substance to be investigated.
[0068] A particularly effective technology for carrying out
processes of this type is the scintillation proximity assay, called
SPA for short, known in the field of active compound screening.
Kits and components for carrying out this assay can be obtained
commercially, for example from Amersham Pharmacia Biotech. In
principle, solubilized or membrane-bound receptors are immobilized
on small fluoromicrospheres containing scintillation substance. If,
for example, a radioligand binds to the immobilized receptors, the
scintillation substance is stimulated to emit light, since the
spatial vicinity between scintillation substance and radioligand is
specified.
[0069] A further particularly effective technology for carrying out
processes of this type is the FlashPlate.RTM. technology known in
the field of active compound screening. Kits and components for
carrying out this assay can be obtained commercially, for example
from NEN.RTM. Life Science Products. This principle is likewise
based on microtiter plates (96-well or 384-well), which are coated
with scintillation substance.
[0070] The abovementioned assays are known in principle to the
person skilled in the art.
[0071] A first process according to the invention is used for the
determination of the affinity and/or selectivity of binding
partners for 5-HT5 receptors. For this purpose, the binding partner
is brought into contact with 5-HT5 receptors and the binding
affinity is determined.
[0072] For the determination of selectivities, the binding affinity
of the binding partner to be investigated to other 5-HT receptors
is determined in the same manner--if appropriate using the ligands
specific for the respective receptor--and the values obtained are
compared.
[0073] A further process according to the invention relates to the
determination of the activity of binding partners for 5-HT5
receptors, i.e. the determination of agonistic, partly agonistic,
antagonistic and/or partly antagonistic action. For this purpose,
the binding partner is brought into contact with 5-HT5 receptors
and the effects caused by the binding are assessed.
[0074] According to a preferred embodiment, binding partners are
subjected to a primary screening by determining their binding
affinity to 5-HT5 receptors using the [.sup.3H]-5-CT or
[.sup.3H]-LSD competition experiment described above. Those binding
partners which have an inhibition constant IC.sub.50 in the range
of 10.sup.-6M or less are then subjected to a secondary screening
by assessing their effector function in the manner described above,
in particular with respect to GTP binding and/or the intracellular
calcium levels. Finally, the binding partners selected in this way
can be subjected to a counter-screening for selectivity
determination by determining their binding affinity to further 5-HT
receptors essentially in the manner described above--but optionally
using the ligands specific to the respective receptors. For
example, [.sup.3H]-8-hydroxydipropylaminotetralin
([.sup.3H]-8-DPAT) can be used for binding studies to 5-HT1A
receptors, while 5-HT1B and 5-HT1D receptors can be investigated
using [.sup.3H]-5-CT.
[0075] 5-HT5 receptors are preferably made available in the form of
cellular systems, i.e. in the form of membranes, cells, cell
colonies, tissues or organs which carry 5-HT5 receptors. Cellular
systems of this type can express 5-HT5 receptors by nature, but
they can also be induced to express 5-HT5 by suitable genetic
manipulation, e.g. by transfection. In the context of the preferred
embodiment of the present invention concerning h5-HT5, it is
possible to use for this purpose, in particular, the coding
sequence described in Rees S. et al, FEBS Letters 335:242-246
(1994) (accession number X81411). Human glioma cell lines are
preferred as natural cellular systems having 5-HT5 receptors. Of
the h5-HT5-transfected heterologous cell lines, those are preferred
which express the h5-HT5 gene. Mention may be made, for example, of
h5-HT5-transfected CHO cells, h5-HT5-transfected human kidney
cells, in particular h5-HT5-transfected HEK293 cells, or
h5-HT5-transfected C-6 glioma cells.
[0076] For the determination of selectivity, affinity and activity
of binding partners according to the invention, it is also possible
to use brain tissue sections and native membranes from brain
parts.
[0077] If radio labels are employed, the assessment of tissue
sections is preferably carried out autoradiographically.
[0078] The neuroprotective action is preferably determined on
animal models of neurodegenerative and neuropsychiatric
processes.
[0079] Preferred animal models are those for cerebral stroke and
cerebral disorders in the case of multiple infarct, for example
cerebral ischemias as a result of an occlusion of the carotid
artery or the middle cerebral artery (MCA occlusion), fore brain
ischemias and hypoxia tolerance tests, anxiolytic models, for
example active compound-induced convulsions, electroshock- or
isolation-induced aggression, models of antiepileptic activity, for
example electroshock-, active compound- or noise-induced attacks
and genetic models, excitotoxic neurodegeneration models and
demyelinization models.
[0080] In the context of the treatment, the use according to the
invention of 5-HT5 binding partners comprises a process. In this
process, the individual to be treated, preferably a mammal, in
particular a human, agricultural or domestic animal, is
administered an efficacious amount of one or more 5-HT5 binding
partners, as a rule corresponding to pharmaceutical and veterinary
medical practice. Whether such a treatment is indicated and in what
form it has to take place depends on the individual case and is
subject to medical assessment (diagnosis), the signs, symptoms
and/or dysfunctions present, risks of developing certain signs,
symptoms and/or dysfunctions, and additionally includes further
factors.
[0081] As rule, the treatment is carried out by single or repeated
daily administration, if appropriate together or in alternation
with other active compounds or active compound-containing
preparations, such that an individual to be treated is administered
a daily dose of approximately 0.001 g to 10 g, preferably of
approximately 0.001 g to approximately 1 g.
[0082] The invention also relates to the preparation of
pharmaceutical compositions for the treatment of an individual,
preferably a mammal, in particular a human, agricultural or
domestic animal. The binding partners according to the invention
are usually administered in the form of pharmaceutical compositions
which comprise a pharmaceutically tolerable excipient with at least
one inhibitor according to the invention and, if appropriate,
further active compounds. These compositions can be administered,
for example, by the oral, rectal, transdermal, subcutaneous,
intravenous, intramuscular or intranasal route.
[0083] Examples of suitable pharmaceutical formulations are solid
pharmaceutical forms, such as powders, granules, tablets,
pastilles, sachets, cachets, coated tablets, capsules such as hard
and soft gelatin capsules, suppositories or vaginal pharmaceutical
forms, semisolid pharmaceutical forms, such as ointments, creams,
hydrogels, pastes or patches, and also liquid pharmaceutical forms,
such as solutions, emulsions, in particular oil-in-water emulsions,
suspensions, for example lotions, injection and infusion
preparations, eye and ear drops. Implanted delivery devices can
also be used for the administration of binding partners according
to the invention. In addition, liposomes, microspheres or polymer
matrices can also be used.
[0084] In the production of the compositions, binding partners
according to the invention are usually mixed or diluted with an
excipient. Excipients can be solid, semisolid or liquid materials
which are used as a vehicle or medium for the active compound.
[0085] Suitable excipients include, for example, lactose, dextrose,
sucrose, sorbitol, mannitol, starches, acacia gum, calcium
phosphate, alginates, tragacanth, gelatin, calcium silicate,
microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,
syrup and methylcellulose. In addition, the formulations can
comprise pharmaceutically acceptable vehicles or customary
excipients, such as lubricants, for example tallow, magnesium
stearate and mineral oil; wetting agents; emulsifying and
suspending agents; preservatives, such as methyl and propyl
hydroxybenzoates; antioxidants; antiirritants; chelating agents;
pan-coating auxiliaries; emulsion stabilizers; film-forming agents;
gel-forming agents; flavor-masking agents; flavor corrigents;
resins; hydrocolloids; solvents; solubilizers; neutralizing agents;
permeation accelerators; pigments; quaternary ammonium compounds;
refatting and super fatting agents; ointment, cream or oil bases;
silicone derivatives; spreading auxiliaries; stabilizers;
sterilizing agents; suppository bases; tablet excipients, such as
binders, fillers, lubricants, disintegrants or coatings;
propellants; drying agents; opacifying agents; thickeners; waxes;
plasticizers; white oils. A relevant embodiment is based on expert
knowledge, such as is presented, for example, in Fiedler, H. P.,
Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik and angrenzende
Gebiete (Encyclopedia of excipients for pharmacy, cosmetics and
related areas], 4th Edition, Aulendorf: ECV-Editio-Kantor-Verlag,
1996.
[0086] The present invention is illustrated in greater detail by
means of the following examples, without being restricted
thereto.
REFERENCE EXAMPLE 1
h5-HT5 Receptor-Expressing HEK293 Cells and CHO Cells
[0087] The gene coding for the human 5-HT5 receptor was isolated
from human tissues in a known manner by means of 3'-5'-RT-PCR (RACE
system, Boehringer Mannheim). The gene sequence was then inserted
into a plasmid carrying the neomycin resistance gene (pcDNA3;
Invitrogen, Germany) and amplified in E. coli according to the
manufacturer's instructions. A preparation of the resulting plasmid
was mixed with Lipofectamin.RTM. (Gibco Life-Sciences, Germany),
and HEK293 cells were incubated with a thin layer of this
transfection mixture in petri dishes (2.5 cm). The transfection
mixture was then replaced by neomycin-containing culture medium.
Surviving cells were further cultured in DMEM-F12 medium which was
supplemented with 10% fetal calf serum, 2 mM glutamine and
antibiotics (90 mg of streptomycin, 90 mg of penicillin). The cells
were grown to confluence under 5% CO.sub.2, 95% atmospheric
humidity and 37.degree. C.
[0088] h5-HT5 receptor-expressing CHO cells are obtained
analogously.
REFERENCE EXAMPLE 2
Cell Membrane Preparation
[0089] The method used here essentially follows known methods for
the preparation of cell membranes from cells (Findlay J. B. C. and
Evans W. H. Biological Membranes, Practical Approach (1987)). The
cells cultured according to Reference Example I were carefully
scraped off the surface of the culture vessel and centrifuged in
DMEM-F12 medium at 180.times.g for 10 min. The cell pellets
obtained were resuspended in 5 mM tris HCl buffer containing 5 mM
EDTA, 5 mM EDTA, 0.1 mM PMSF and 3 mM benzamidine (pH: 7.6; buffer
A) and incubated at 4.degree. C. for 15 min. The cell suspension
was homogenized (6.times.3s) in an Ultraturrax.RTM. (15,000 rpm)
and centrifuged at 1000.times.g and 4.degree. C. for 1 min. The
pellet was resuspended in buffer A and, as described above,
homogenized and centrifuged. The supernatants from both steps were
collected and centrifuged at 40,000.times.g and 4.degree. C. for 20
min. The pellet was resuspended and homogenized in buffer A
(1.times.15s). The membrane suspension was centrifuged at
40,000.times.g and 4.degree. C. for 20 min. The resulting pellet
was resuspended in buffer A containing 10% glycerol and 1% bovine
serum albumin. Aliquots were frozen and stored at -80.degree. C.
until use.
REFERENCE EXAMPLE 3
Kinetics of the Saturation Binding of [.sup.3H]-5-CT
[0090] The methodology is essentially known (Ress S. et al., FEBS
Letters 335:242-246 (1994). Membranes obtained according to
Reference Example 2 (200 .mu.l) were incubated in a total volume of
600 .mu.l in 100 mM tri5-HCl containing 1 mM EDTA (pH: 7.7; buffer
B) with increasing concentrations of [.sup.3H]-5-CT (96 Ci/mmol),
10 .mu.m methiothepine being added for the determination of the
specific binding, while methiothepine was not added for the
determination of the total binding. The mixture was incubated at
30.degree. C. for 90 min. The samples were then filtered, a
Skatron.RTM. filtration system and GF/B filters embedded in 0.3%
polyethyleneimide being used. The filters were washed at 4.degree.
C. with 9 ml of buffer B. The radioactivity retained on the filters
was measured by means of liquid scintillation counting, 5 ml of
Ultima-Gold (packard) being used.
REFERENCE EXAMPLE 4
a) [.sup.3H]-5-CT Binding Competition
[0091] The experiments on binding competition were carried out
essentially following known investigations (Rees et al., 1994).
Membranes obtained according to Example 2 (200 .mu.l) were
incubated in the presence of 2 nM [.sup.3H)-5-CT in a total volume
of 600 .mu.l in buffer B with increasing concentrations of selected
compounds. After an incubation time of 75 min at 30.degree. C., the
samples were filtered at 4.degree. C. through GF/B filters embedded
in 0.3% polyethyleneimide using buffer B. The filters were washed
with 9 ml of buffer B. The radioactivity retained on the filters
was determined as in Reference Example 3. The total binding was
defined as that binding of the radioligand which was observed
without addition of further compounds. This nonspecific binding was
defined as that binding of [.sup.3H]-5-CT which was observed in the
presence of 10 .mu.M methiothepine. Similar systems can also be
used which, as a result of use of microtiter plates, allow a high
sample throughput and secondary screening.
[0092] The saturation parameters of the [.sup.3H]-5-CT binding was
determined both by nonlinear regression analysis and from linear
plots using the SigmaPlot software (Jandel Scientific, Germany).
Competition curves were set up in which the radioactive binding is
expressed as a percentage proportion of the total binding. Half
maximal inhibition constants IC.sub.50 and Hill-coefficients
(n.sub.H) were determined by means of nonlinear regression
analysis.
b) Identification of H5-HT5 Receptor Ligands by HTS Using
FlashPlate Technology
[0093] 96-well FlashPlates which are coated with h5-HT5 membranes
can be obtained from Bio Signal Inc. (Canada). [3H)-LSD was diluted
to a suitable concentration in Tris HCl buffer which contains 10 mM
MgCl.sub.2, 0.5 mM EDTA and 0.5% of BSA. The radioligand solution
was added to the wells (25 ml), which either contained or did not
contain test compound. The plates were incubated at room
temperature for 180 minutes and the radioactive signal was measured
using a micro .beta.-counter (Wallac). The nonspecific binding was
determined using methiothepine. [3H]-LSD has an affinity of 12 nM.
With increasing binding affinity of the test compound, the
radioactive signal of [3H]-LSD decreased.
REFERENCE EXAMPLE 5
Determination of the Agonist-Induced Stimulation of
[.sup.35S)GTP.gamma.S Binding
[0094] [.sup.35S]GTP.gamma.S binding assays are known. The present
assay was carried out following the previously described method of
Hilf, G. and Jakobs, K. H. (Eur. J. Mol. Pharmacol. 225:245-252
(1992)). Active compound-induced changes in the
[.sup.35S]GTP.gamma.S binding to membranes of HEK293 cells stably
transfected with the h5-HT5 receptor gene were measured (see
Reference Examples 1 and 2). The cell membranes (12 .mu.g) were
incubated with 50 nM triethanolamine HCl buffer (pH: 7.5)
containing 6.75 mM MgCl.sub.2, 150 nM NaCl, 1 mM DTT, 1 mM EDTA, 10
.mu.M GDP and [.sup.35S] GTP.gamma.S. Following a 60-minute
incubation at 30.degree. C. with or without addition of the active
compounds to be tested, the test mixture (100 .mu.l) was rapidly
filtered through GF-B filters using a Skatron.RTM. filtration
device. The filters were rapidly washed with 50 mM tris HCl buffer
(9 ml; pH: 7.5; 4.degree. C.) containing 100 mM NaCl and 5 mM
MgCl.sub.2. The radioactivity retained on the filters was
determined by means of scintillation spectrometry, Ultima Gold
scintillation fluid being used. Similar systems which allow a high
throughput and secondary screening as a result of use of microtiter
plates can likewise be used.
[0095] The active compound activities were expressed as a
percentage proportion of the basic binding measured in the absence
of the active compound. The matching of the curves was carried out
using software for nonlinear regression analysis (SigmaPlot, Jandel
Scientific, Germany) according to the general equation
E=(L.times.E.sub.max)/(L+EC.sub.50), in which E is the action, L is
the ligand concentration, E.sub.max is the maximum action and
EC.sub.50 is that concentration which induces 50% of the maximal
action.
REFERENCE EXAMPLE 6
Determination of Agonist-Induced Changes of Intracellular Calcium
Levels
[0096] The method is known (Kao J. P. Y. Methods in Cell Biology
40:155-181 (1994)). As described in Reference Example 1, h5-HT5
receptor-expressing HEK293 cells were grown in culture vessels. The
cells were carefully scraped off before they were confluent. The
cells were labeled with Fura 2 by incubating at room temperature
with Fura 2-acetylmethyl ester (Sigma). The cells were centrifuged
at 180.times.g for 10 min and resuspended in DMEM-FI2 medium
without serum and incubated at 37.degree. C., 5% CO.sub.2 and 95%
atmospheric humidity for 45 min.
[0097] Intracellular calcium levels were determined with a
fluorescence microscope which was equipped with a suitable filter
exchange system (Olympus/Hamamatsu). The fluorescence ratio (340
nm/380 nm) was determined using the Argus.RTM. software. The
intracellular calcium levels were observed for a short time in
individual cells without the addition of active compounds and then
30 min after addition of the active compound to be tested. Similar
systems which permitted a high throughput and secondary screening
as a result of the use of microtiter plates could likewise be
used.
[0098] The modulation of intracellular Ca.sup.2+ levels can be
assessed analogously in HTS. For this, h5-HT5 receptor-expressing
CHO cells were cultured overnight in 96-well plates (30,0000
[sic]-80,000 cells/well). The cells were labeled for one hour using
HEPES buffer containing 1 mM Fluo-3-AM, 10% pluronic acid and 2.5
mM probencid, and washed. A test compound was added to each well.
For the determination of the calcium levels, the fluorescence
intensity was read off using a fluorometrically operating plate
reader (Fluorometric Imaging Plate Reader; FLIPR).
REFERENCE EXAMPLE 7
Determination of the Agonist-Induced Phospholipase C Activity
[0099] The method is essentially known (Garcia-Ladona F. J. et al.,
Neuroreport 4:691-694 (1993)). The cells were incubated with 0.125
.mu.M [.sup.3H]myoinositol for 24 h. Unincorporated
[.sup.3H]myoinositol was removed from the medium and replaced by
Krebs-Henseleit buffer containing 10 mM LiCl. After incubation for
10 minutes, the active compound to be tested was added. After 45
min, the reaction was stopped by replacing the stimulation medium
by distilled water. If tissue samples are used, a similar procedure
is employed (Garcia-Ladona et al., 1993). The cells were frozen and
stored at -80.degree. C. The production of [.sup.3H]inositol
monophosphate was determined by means of known chromatographic
methods. A similar method can be used with tissue miniprisms. The
determination of the phospholipase C stimulation was likewise
carried out in a similar manner by preparing membrane fractions, as
described in Reference Example 2, and incubating with
[.sup.32P]PIP.sub.2 and active compounds. In this case, the
production of IP3 was determined. Known processes were also
optimized in order to use systems based on microtiter plates.
Commercially obtainable materials allow extension to analyses with
a high throughput and the carrying-out of secondary screening.
REFERENCE EXAMPLE 8
Determination of the Agonist-Induced Change in cAMP Production
[0100] The method used is essentially known (Strada S. S. et al.,
Methods in Neurotransmission receptor analysis: 89-110 (1990)).
Cells were incubated in culture medium without serum and
antibiotics for 10 min. The medium was heated at 95.degree. C. for
15 min in order to stop the reaction. The cell samples were frozen
and stored at -80.degree. C. cAMP levels were determined using
commercially obtainable kits which use the cAMP binding protein.
Known processes were also optimized in order to use systems based
on microtiter plates. Commercially obtainable materials allow
extension to analyses with a high throughput and the carrying-out
of secondary screening.
REFERENCE EXAMPLE 9
Tissue Preparation
[0101] 90 min after administration of the active compound (orally,
intraperitoneally, intravenously or intracerebroventricularly), the
experimental animals were decapitated. The entire brain was rapidly
removed from the skull, frozen on dry ice and stored at -80.degree.
C. Rat brain sections (15 .mu.m) were obtained in a cryostat at
-20.degree. C., applied to gelatin-coated slides and stored at
-30.degree. C. until use.
REFERENCE EXAMPLE 10
Neuroprotective Action
MCA Occlusion
[0102] The neuroprotective activity of the test compounds was
investigated for experimentally caused cerebral stroke in a
standard model. The experiments were performed on male Long Evans
rats. Under nitrous oxide-assisted halothane anesthesia, the middle
cerebral artery (MCA) was severed and permanently ligated distally,
as described in the literature. The resulting infarct volume was
determined 22 hours after MCA occlusion.
REFERENCE EXAMPLE 11
Neuroprotective Action
Experimental Brain Trauma of the Rat
[0103] The lateral fluid percussion method (McIntosh T K,
Neuroscience 28, 233 244, 1989) was used in order to produce an
experimental brain trauma in the rat which is suitable for the
testing of potential neuroprotective substances. By means of this
technique, controlled and consistent tissue damage was produced
underneath the site of the impact, which included the neocortex,
the hippocampus and the thalamus.
[0104] The right parietotemporal region of the skull was exposed in
the anesthetized rat. The skull was trepanned through the parietal
cortex (about 4 mm diameter), the dura being left intact. A Luer
lock attachment was fixed to the skull using dental cement through
the trepanation. After the dental cement had hardened, the rat was
connected to the fluid percussion apparatus according to McIntosh.
This consisted of a cylinder filled with 0.9% NaCl solution, one
end of the cylinder being closed by a piston, while the other end
was connected via a pressure transducer to a Luer lock attachment.
Using this, the counterpart on the rat was tightly closed so that a
liquid column was formed adjacent to the dura. A metal pendulum was
allowed to impact onto the piston from a predetermined height so
that a brief blow of the compressed liquid column on the surface of
the rat brain occurred. High pressures of about 2.5-2.9 bar were
used.
[0105] The rats were sacrificed 14 days after traumatization for
removal of the brain. After fixation of the brains, sections of
thickness 30 mm were prepared in a freezing microtome and stained
using Toluidine Blue. For the quantification of the neuronal
damage, cell counts were carried out bilaterally in the rostral
hippocampus; the proportion of intact cells in the dentate gyrus
was determined on the traumatized (right) and the (left) opposite
side. The quotient of the cell count in the ipsilateral hippocampus
and the number in the contralateral hippocampus was indicated.
REFERENCE EXAMPLE 12
Synthesis of Tested Binding Partners
a)
3,4,5,6,7,8-Hexahydro-7-methyl-3-[2-(4-(3-trifluoromethylphenyl)piperaz-
in-1-yl)ethyl]pyrido[4',3':4,5]thieno[2,3-d]-pyrimidin-4-one.times.2HCl
(compound A)
[0106] The compound was prepared by heating
2-ethoxymethylenamino-3-cyano-6-methyl-4,5,6,7-tetrahydrothieno-[2,3-c]py-
ridine or
2-ethoxymethylenamino-3-carboethoxy-6-methyl-4,5,6,7-tetrahydrot-
hieno[2,3-c]pyridine under reflux with
1-(2-aminoethyl)-4-(3-trifluoromethylphenyl) piperazine in an inert
solvent such as ethanol. The reaction mixture was worked up, and
the product having a melting point of 309-312.degree. C. was
deposited by precipitating with ethereal hydrochloric acid.
[0107] A further possibility for the preparation consisted in first
heating the abovementioned starting compounds with ethanolamine
under reflux in an inert solvent such as ethanol. After the
reaction mixture had been worked up, the
3,4,5,6,7,8-hexahydro-3-(2-hydroxy)ethyl-7-methylpyrido[4',3':4,5]thieno[-
2,3-d]pyrimidin-4-one obtained was reacted, for example, with
thionyl chloride in order to introduce a suitable leaving group, so
that the reaction of the resulting
3,4,5,6,7,8-hexahydro-3-(2-chloro)-ethyl-7-methylpyrido-[4',3':4,5]thieno-
[2,3-d]pyrimidin-4-one with N-(3-trifluoromethylphenyl)piperazine
in an inert solvent such as xylene under basic conditions (e.g.
potassium carbonate) afforded the desired product after working up
the reaction mixture.
b)
3,4,5,6,7,8-Hexahydro-6-ethyl-3-[2-(4-(1-naphthyl)piperazin-1-yl)ethyl]-
pyrido[3',4':4,5]thieno[2,3-d]pyrimidin-4-one.times.3HCl.times.2H.sub.2O
(compound B)
[0108] Preparation was carried out in principle as described for
compound A.
[0109]
2-Ethoxymethylenamino-3-carboethoxy-5-ethyl-4,5,6,7-tetrahydrothien-
o[3,2-c]pyridine was heated under reflux with
1-(2-aminoethyl)-4-(1-naphthyl)piperazine in an inert solvent such
as ethanol. The reaction mixture was worked up, and the product
converted into the hydrochloride was isolated with a melting point
of 298-300.degree. C.
[0110] Alternatively,
3-(2-chloroethyl)-6-ethyl-3,4,5,6,7,8-hexahydropyrido[3',4':4,5]thieno[2,-
3-d]pyrimidin-4-one was reacted under basic conditions with
N-(1-naphthyl)piperazine in an inert solvent such as xylene.
c)
3,4,5,6,7,8-Hexahydro-7-methyl-3-[2-(4-(7-methoxynaphth-1-yl)piperazin--
1-yl)ethyl]pyrido[4',3':4,5]thieno[2,3-d]pyrimidin-2-one.times.2HCl.times.-
H.sub.2O (compound C)
[0111] Preparation can be carried out analogously to compound
A.
[0112] Compounds A, B and C and also the intermediates needed for
their preparation are known or can be synthesized from analogous
starting materials according to the preparation methods described
in the literature (F. Sauer and P. Stanetty, Monatsh. Chem. (1975),
106(5), 1111-1116; K. Gewald et al., Chem. Ber. 99, 94-100 (1966),
Patent Applications DE 196 36 769.7 and DE 197 24 979.5).
EXAMPLE 1
[0113] According to Reference Example 3, the binding affinity of
[.sup.3H]-5-CT to 5-HT5 receptors was determined. FIG. 1 shows a
plot of bound (.sup.3H]-5-CT as a function of the [.sup.3H]-5-CT
concentration. A dissociation constant of K.sub.d=0.570 nM was
determined. Depending on the clonal cell line, the receptor binding
density (B) varied in a range from 900-28,000 fmol/mg of
protein.
EXAMPLE 2
[0114] According to Reference Example 4, the binding affinities of
serotoninergic compounds were determined by means of [.sup.3H]-5-CT
binding competition. By means of the IC.sub.50 values obtained, the
inhibition constants K.sub.i of the following compounds were
determined (K.sub.i=IC.sub.50/(1+C/K.sub.d)), where C is the
concentration of [.sup.3H]-5-CT and K.sub.d was determined
according to Example 1):
TABLE-US-00001 Compound K.sub.i [M] R(+)-8-OH-DPAT 1.25 10-.sup.7
5-CT 1.44 10-.sup.9 Compound A 6.61 10-.sup.7 Compound B 1.24
10-.sup.7 Compound C 2.37 10-.sup.7
EXAMPLE 3
[0115] According to Reference Example 5, the active
compound-induced binding of GTP to G proteins was investigated. The
coupling of 5-HT5 receptors to G proteins in HEK293 cells was
evident. The typical serotoninergic agonists 5-HT and 5-CT induced
an increase in the [.sup.35S]GTP.gamma.S binding to the cell
membranes of over 40% above the basic value (see FIG. 2). The 5-HT5
receptor needs GDP for the coupling to G proteins, which is
mediated by agonists (see FIG. 3A). The 5-HT effect was
dose-dependent (see FIG. 4) with an EC.sub.50 of 2.6 .mu.M.
EXAMPLE 4
[0116] According to Reference Example 10, the neuroprotective
action of selected 5-HT5 binding partners was tested. The active
compounds were administered intravenously, first as a bolus and
then as a maintenance infusion, 90 minutes after MCA occlusion. The
following results were achieved:
TABLE-US-00002 Infarct volume, % of the control [mean Dose, i.v.
value .+-. SD, (n)] P, Active [mg/kg + Active Student's t compound
mg/kg/h] Placebo compound test, bilateral R(+)-8-OH-D 2 + 1 100
.+-. 11 (12) 72 .+-. 32 (12) 0.0086 PAT Compound A 4 + 2 100 .+-.
25 (12) 62 .+-. 18 (10) 0.0013 Compound B 2 + 1 100 .+-. 20 (12) 66
.+-. 19 (9) 0.0015 Compound C 1 + 0.5 100 67 <0.05
EXAMPLE 5
[0117] According to Reference Example 11, an experimental brain
trauma of the rat was induced using the lateral fluid percussion
method. The protective action of the active compounds tested on the
survival of hippocampal neurons is expressed as the quotient of the
neuron count of the trauma side to the neuron count of the
contralateral side; the mean (m) and the mean error of the mean
(S.sub.m) is in each case indicated
TABLE-US-00003 P, Dose Quotient m .+-. S.sub.m (n) Student's Active
[mg/ Active t test, compound kg i.p.] Placebo compound bilateral
Compound 20 + 20 0.33 .+-. 0.05 (10) 0.56 .+-. 0.05 (10) <0.05 C
[15 + 120 min post] Compound 20 + 20 0.44 .+-. 0.03 (17) 0.64 .+-.
0.03 (12) <0.05 B [15 + 120 min post]
* * * * *