U.S. patent application number 11/073313 was filed with the patent office on 2005-07-07 for methods of identifying inverse agonists of the serotonin 2a receptor.
Invention is credited to Brann, Mark R., Weiner, David.
Application Number | 20050148018 11/073313 |
Document ID | / |
Family ID | 34709737 |
Filed Date | 2005-07-07 |
United States Patent
Application |
20050148018 |
Kind Code |
A1 |
Weiner, David ; et
al. |
July 7, 2005 |
Methods of identifying inverse agonists of the serotonin 2A
receptor
Abstract
The present invention relates to a method of identifying
compounds which act as inverse agonists of the 5-HT2A receptor, the
method comprising contacting a constitutively active 5-HT2A
receptor with at least one test compound and determining any
decrease in the level of basal activity of the receptor so as to
identify a test compound which is an inverse agonist of the 5-HT2A
receptor. Such inverse agonists may be used in the treatment of
schizophrenia and related psychoses.
Inventors: |
Weiner, David; (San Diego,
CA) ; Brann, Mark R.; (Del Mar, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
34709737 |
Appl. No.: |
11/073313 |
Filed: |
March 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11073313 |
Mar 4, 2005 |
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10130812 |
Nov 6, 2002 |
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10130812 |
Nov 6, 2002 |
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PCT/US99/21439 |
Oct 7, 1999 |
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Current U.S.
Class: |
435/6.14 ;
514/17.5; 514/18.1 |
Current CPC
Class: |
G01N 33/942 20130101;
C12Q 2600/136 20130101; C12Q 1/6883 20130101; G01N 2500/10
20130101; C12Q 2600/156 20130101; G01N 33/566 20130101 |
Class at
Publication: |
435/006 ;
514/002 |
International
Class: |
C12Q 001/68; A61K
038/00 |
Claims
What is claimed is:
1. A method of identifying a compound which is an inverse agonist
of the 5-HT2A receptor, the method comprising: (a) contacting a
constitutively active 5-HT2A receptor with at least one test
compound; and (b) determining any decrease in basal activity level
of the 5-HT2A receptor so as to identify a test compound which is
an inverse agonist of the 5-HT2A receptor.
2. A method of identifying a compound which is an inverse agonist
of the 5-HT2A receptor, the method comprising: (a) culturing cells
which express a constitutively active 5-HT2A receptor; (b)
incubating the cells with at least one test compound; and (c)
determining any decrease in basal activity level of the 5-HT2A
receptor so as to identify a test compound which is an inverse
agonist of the 5-HT2A receptor.
3. The method of claim 2, wherein the cells of step (a) overexpress
said 5-HT2A receptor.
4. The method of claim 2, wherein the identified inverse agonist is
selective for the 5-HT2A receptor.
5. A method of identifying mutation(s) in a gene encoding a
constitutively active 5-HT2A receptor, the method comprising: (a)
extracting nucleic acid from a biological sample obtained from an
individual having a disorder or condition putatively associated
with constitutive activity of the 5-HT2A receptor; (b) preparing
cDNA from the extracted nucleic acid; (c) selecting from the cDNA
in step (b) cDNA encoding the 5-HT2A receptor; (d) transfecting a
cell with an expression vector comprising said selected cDNA; (e)
selecting a cell expressing constitutively active 5-HT2A receptor;
and (f) sequencing the cDNA in said selected cell to detect the
mutation(s).
6. The method of claim 5, wherein said biological sample comprises
blood, platelets or brain tissue.
7. The method of claim 5, wherein the extracted nucleic acid is
RNA.
8. The method of claim 5, further comprising prior to said
transfecting step amplifying the cDNA.
9. The method of claim 5, wherein said cDNA selection is performed
using one or more oligodeoxynucleotide probes specific to the gene
encoding the 5-HT2A receptor.
10. A method of diagnosing a disorder or condition, or a
susceptibility thereto, associated with constitutive activity of
the 5-HT2A receptor, the method comprising: (a) obtaining a
biological sample from an individual putatively affected by or
susceptible to a disorder or condition associated with constitutive
activity of the 5-HT2A receptor; (b) isolating from said biological
sample a nucleic acid sequence encoding said receptor, or a portion
of said nucleic acid sequence corresponding to the portion of the
gene identified to include mutation(s) by the method of claim 5;
and (c) detecting the presence or absence of mutation(s) in said
nucleic acid sequence or said portion thereof.
11. The method of claim 10, wherein said biological sample
comprises blood, platelets or brain tissue.
12. A test kit for detecting mutation(s) in a gene encoding a
constitutively active 5-HT2A receptor, the test kit comprising a
nucleic acid sequence corresponding to a portion of the gene
identified by the method of claim 5 to include at least one
mutation.
13. A method of decreasing the basal activity level of the 5-HT2A
receptor in a subject in need thereof, the method comprising
contacting a 5-HT2A receptor in said subject with an inverse
agonist of the 5-HT2A receptor in an amount effective to
substantially decrease the basal activity level of said
receptor.
14. The method of claim 13, wherein the inverse agonist is
selective for the 5-HT2A receptor.
15. The method of claim 13, wherein the inverse agonist has little
or substantially no anti-dopaminergic activity.
16. A method of ameliorating symptoms of schizophrenia or psychosis
in a subject in need thereof, the method comprising administering
to the subject a therapeutically effective amount of an inverse
agonist of the 5-HT2A receptor.
17. Use of an inverse agonist of the 5-HT2A receptor for the
preparation of a medicament for substantially decreasing the basal
activity level of a constitutively active 5-HT2A receptor.
18. Use according to claim 17, wherein the inverse agonist is
selective for the 5-HT2A receptor.
19. Use according to claim 17, wherein the inverse agonist has
little or substantially no anti-dopaminergic activity.
20. Use of a 5-HT2A receptor to identify a compound acting as an
inverse agonist at said receptor.
21. Use of a 5-HT2A receptor to identify a compound acting as an
inverse agonist at said receptor and useful in the treatment of
schizophrenia or psychosis.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of identifying
compounds which act as inverse agonists of the serotonin (5-HT) 2A
receptor, methods of screening individuals having disorders
putatively associated with constitutively active 5-HT2A receptors,
diagnostic test kits and methods of treatment for such individuals,
methods of decreasing basal activity levels of the 5-HT2A receptor,
and uses of inverse agonists as therapeutic agents for
schizophrenia and psychosis.
BACKGROUND OF THE INVENTION
[0002] Schizophrenia is a devastating neuropsychiatric disorder
that affects approximately 1% of the human population. It is
characterized by a constellation of symptoms: "positive" symptoms
such as hallucinations and delusions; and "negative" symptoms such
as social and emotional withdrawal, apathy, and poverty of speech.
The disorder usually develops early in life and is characterized by
a chronic, often relapsing remitting course. Although the
pathophysiology of this clinically heterogeneous disorder is
unknown, genetic factors play a significant role. It has been
estimated that the total financial cost for the diagnosis,
treatment, and lost societal productivity of individuals affected
by this disease exceeds 2% of the gross national product (GNP) of
the United States. To date, there exist no definitive diagnostic
tests for this disorder. Current treatment options available to
psychiatrists primarily involve pharmacotherapy with a class of
drugs known as antipsychotics. Antipsychotics are effective in
ameliorating positive symptomotology, yet they frequently do not
improve negative symptoms, and significant, treatment-limiting side
effects are common.
[0003] Drugs that possess antipsychotic properties have been in
clinical use since the early 1950's. The first compound shown to
possess this property was chlorpromazine, and many of the
subsequent compounds were derived from this phenothiazine
antipsychotic. Currently, nine major classes of antipsychotics have
been developed and are widely prescribed to treat psychotic
symptoms irrespective of their etiology. Clinical use of these
compounds are limited, however, by their side effect profiles.
Nearly all of the "typical" or older generation compounds have
significant adverse effects on human motor function. These
"extrapyramidal" side effects, so termed due to their effects on
modulatory human motor systems, can be both acute and chronic in
nature. Acute effects include dystonic reactions, and a potentially
life threatening but rare symptom constellation, neuroleptic
malignant syndrome. Chronic side effects include akathisias,
tremors, and tardive dyskinesia, a movement disorder characterized
by involuntary writhing movements of the tongue and oral
musculature seen with long-term administration of these agents. Due
in large part to these disabling side effects, drug development in
this class of compounds has been focused on newer "atypical" agents
free of these adverse effects.
[0004] Various hypotheses have been proposed concerning the
pathophysiology of schizophrenia, including genetic, environmental,
and developmentally based theories. Current neuropharmacological
theories are based, in large part, on the observation that
antipsychotic drugs can improve the symptoms of schizophrenia,
coupled with our current knowledge as to the mechanism of action of
this class of drugs. Antipsychotic drugs have been shown, by both
in vitro and in vivo methods, to interact with a large number of
central monoaminergic neurotransmitter receptors, including
dopaminergic, serotonergic, adrenergic, muscarinic, and
histaminergic receptors. It is likely that the therapeutic and
adverse effects of these drugs are mediated by distinct receptor
subtypes.
[0005] The prevailing theory as to the mechanism of action of
antipsychotic drugs involves antagonism of dopamine D2 receptors.
This is based on the observation that these drugs have high
affinity for this receptor in vitro, and that a correlation exists
between their potency to block D2 receptors and their clinical
efficacy. Unfortunately, it is likely that antagonism of dopamine
D2 receptors also mediates the disabling extrapyramidal side
effects. Interestingly, some antipsychotic drugs have been shown
not to possess high affinity for D2 receptors, and therefore an
alternate mechanism must be responsible for their clinical effects.
The only other consistent receptor interaction that these drugs as
a class display is antagonism of 5-HT2A receptors, suggesting that
antagonism of these receptors is an alternate molecular mechanism
that confers antipsychotic efficacy.
[0006] The observation that many of these drugs are antagonists of
5-HT2A receptors has led investigators to postulate that
schizophrenia might be caused by heightened or exagerrated signal
transduction through serotonergic systems. This theory is bolstered
by a number of basic scientific and clinical observations regarding
serotonergic systems and the 5-HT2A receptor in particular.
Firstly, in addition to the known antipsychotics in widespread
clinical usage, research compounds (e.g. ritanserin) that
selectively block 5-HT2A receptors (with respect to D2 receptors)
have also been shown to possess antipsychotic activity. Secondly,
the 5-HT2A receptor mRNA and protein have been shown to be
expressed in neural systems that mediate higher cognitive and
affective functions, including the cerebral cortex, hippocampus,
and amygdala. Thirdly, some of the positive symptoms that
characterize the disease can be mimicked in normal individuals by
the ingestion of the hallucinogenic indolamine lysergic acid
diethylamide (LSD). It is known that LSD and similar hallucinogens
exert their psychogenic effects, in part, through the activation of
5-HT2A receptors. G-protein coupled neurotransmitter receptors
(GPCR's), including the 5-HT2A receptor, function as transducers of
intercellular communication. Traditionally, these receptors have
been assumed to exist in a quiescent state unless activated by the
binding of an agonist (a drug that activates a receptor). When
activated, receptors interact with G-proteins, resulting in the
generation, or inhibition of, second messenger molecules such as
cyclic AMP, inositol phosphates, and diacylglycerol. These second
messengers then modulate the function of a variety of intracellular
enzymes, including kinases and ion channels, which ultimately
determine neuronal excitability and neurotransmitter release.
[0007] Over the last few years some fundamental observations have
been made relating to ways in which these receptor molecules
function. One of the most important of these has been the
identification and characterization of constitutively active
receptors. It is now appreciated that many, if not most, of the
GPCR monoamine receptors can exist in a partially activated state
in the absence of their agonists. This increased basal activity can
be inhibited by a class of drugs aptly named inverse agonists, in
that they function as the inverse of agonists. Inverse agonists
differ mechanistically from classic (or neutral) antagonists.
Antagonists compete against agonists and inverse agonists for
access to the receptor, but do not possess the intrinsic ability to
inhibit elevated basal or constitutive receptor responses.
[0008] Multiple lines of experimental evidence support the
hypothesis that constitutively active neurotranritter receptors may
exist in the central nervous system and be causative for human
neuropsychiatric disease. Constitutive activity has been observed
with neurotransmitter receptors mutated in vitro. For instance, S.
Cottechia et al. (Proc. Natl. Acad. Sci. USA 87, 1990, pp.
2896-2900) made constitutively active chimeric .alpha.-1 adrenergic
receptor by replacing the third intracytoplasmic loop of the
receptor with that of the .beta.-2 adrenergic receptor. Also, P.
Samama et al. (J. Biol. Chem. 268, 1993, pp. 4625-4636) generated a
constitutively active .beta.-2 receptor by replacing four amino
acid residues in the C-terminal region of the third
intracytoplasmic loop with analogous residues from the .alpha.-1B
receptor. Point mutations have been introduced into the muscarinic
m5 receptor by random saturation mutagenesis (E. S. Burstein et
al., Biochem. Pharmacol. 51, 1996, pp. 539-544; T. A. Spalding et
al., J. Pharm. Exp. Ther. 275, 1995, pp. 1274-1279), resulting in
more than 40 mutants that exhibit varying degrees of constitutive
activity. The relative ease with which these receptors may be
mutated to a constitutively active form suggests that
constitutively active receptors may occur spontaneously in nature
with a high frequency.
[0009] A strong argument for the potential contribution of
constitutively active receptors to human neuropsychiatric disease
would be the finding that similar mutations are causative in other
human diseases. Mutations in the G-protein coupled receptor gene
family are common and are increasingly recognized to cause a number
of human diseases. Most of these mutations are single nucleotide or
point mutations that alter the structure and function of the
receptor molecules. For instance, point mutations in the receptors
rhodopsin and vasopressin (J. Nathans, Cell 78, 1994, pp. 357-360;
W. Rosenthal et al., Nature 359, 1992, pp. 233-235) cause reading
frame shifts, prematurely terminating translation of these
proteins, resulting in non-functioning receptors that subsequently
cause color blindness and nephrogenic diabetes insipidus,
respectively. Robinson and colleagues (P. R. Robinson et al.,
Neuron 9, 1992, pp. 719-725) characterized the first mutation in a
human G-protein coupled receptor that resulted in constitutive
activation of the receptor and caused human disease. They found
that when the amino acid Lys296 was mutated to Glu in the visual
pigment rhodopsin, it was able to activate the G-protein transducin
in the absence of light (its natural "agonist"). This particular
mutation causes a particularly severe phenotype of retinitis
pigmentosa (T. J. Keen et al., Genomics 11, 1991, pp. 199-205).
[0010] The number of constitutively active receptors that cause
human disease is expanding. Multiple endocrinological and
oncological disorders are caused by mutations that give rise to
constitutively active receptors. These mutations have been shown to
occur as a result of both spontaneous somatic events and as
inherited germ line defects. A single point mutation in the
luteinizing hormone receptor (Asp578-Gly), which causes male-linked
precocious puberty, has been shown to be familial in caucasian
populations (A. Shenker et al., Nature 365, 1993, pp. 652-654) and
sporadic in Japanese populations (K. Yano et al., J. Clin.
Endocrin. Metab. 79, 1994, pp. 1818-1823). Two different point
mutations in the parathyroid hormone receptor confer constitutive
activity and cause Jansen's metaphyseal chondroplasia (E. Schipani
et al., New Eng. J. Med. 335, 1996, pp. 708-714; E. Schipani et
al., Science 268, 1995, pp. 98-100). Furthermore, two activating
mutations were found in the thyrotropin receptor, both of which
were found to cause many sporadic thyroid adenomas (J. Parma et
al., Nature 365, 1993, pp. 649-651). Taken together, these data
attest to the widespread biological significance of constitutively
active receptors and their role in human disease. It is, therefore,
highly likely that constitutively active G-protein coupled
receptors exist in the human nervous system and mutations in these
neurotransmitter receptors, including the 5-HT2A receptor, may
cause human neuropsychiatric disease.
[0011] Constitutive activity has been described for a growing
number of G-protein coupled neurotansmitter receptors. The dopamine
D2 receptor has been reported to be constitutively active, and some
antipsychotic compounds have been described as inverse agonists,
although many of these compounds appear to be classical antagonists
(Nilsson, C. L., et al., Neuropsychopharmacology 15, 1996, pp.
53-61; Hall, D. A. and Strange, P. G., Brit. J. Pharm., 121, 1997,
pp. 731-736) Similarly, of the thirteen known serotonin receptor
subtypes, only three have been shown to possess constitutive
activity, the 5-HT1A, 5-HT1D and 5-HT2C receptors. For example, E.
L. Barker et al. (J. Biol. Chem. 269, 1994, pp. 11687-11690)
describe an in vitro assay in which the wild-type 5-HT2C receptor
displays constitutive activity. They further report that certain
classically defined antagonists of the receptor, actually act as
inverse agonists.
[0012] The creation of an activated 5-HT2A receptor by mutagenesis
was recently described (Egan, C., T., et., al., J. Pharm. Exp.
Ther. 286(1), 1998, pp. 85-90). Altering amino acid 322 from the
wild type cysteine to lysine, glutamate, or arginine created
activated 5-HT2A receptor mutants. This amino acid was chosen
because it is analogous to the activating mutation produced in the
alb receptor (Kjelsberg, M. A., et al., J. Biol. Chem. 267(3),
1992, pp. 1430-1433). The activated 5-HT2A receptor displayed
measurable constitutive activity, and six antipsychotics were shown
to be inverse agonists (Egan, C. T., ibid.; and Egan, C. T., et
al., Annals N.Y. Acad. Sci., 1999, pp. 136-139). These authors were
unable to measure the constitutive activity of the wild type
receptor in their assay, and an insufficient number of clinically
relevant compounds comprising the various chemical classes of
antipsychotics were tested. This precluded the authors from
recognizing the significance of 5-HT2A receptor inverse agonism and
efficacy as an antipsychotic.
SUMMARY OF THE INVENTION
[0013] Since 5-HT2A receptors may be critical mediators of
antipsychotic drug activity, and as the exact nature of this
interaction (antagonism vs. inverse agonism) is poorly understood,
many antipsychotic compounds have been tested for their functional
activity at this receptor. It has surprisingly been found that the
5-HT2A receptor is constitutively active in the assay described in
the present specification, and that nearly all antipsychotic drugs
are inverse agonists of this receptor. The striking correlation
between antipsychotic efficacy and inverse agonism of the 5-HT2A
receptor argues that inverse agonism of this receptor is a
fundamental molecular mechanism of action of this class of drugs.
This observation has practical applications in the development of
novel antipsychotic agents with more favorable side effect profiles
as well as potentially broader efficacy against the negative
symptomotology of psychotic disorders. This finding also has
important implications for the pathophysiology, diagnosis and
management of schizophrenia and related psychoses.
[0014] Accordingly, the present invention relates in one aspect to
a method of identifying a compound which acts as an inverse agonist
of the 5-HT2A receptor, the method comprising contacting a
constitutively active 5-HT2A receptor with at least one test
compound and determining any decrease in the level of basal
activity of the 5-HT2A receptor so as to identify a test compound
which is an inverse agonist of the 5-HT2A receptor. In a related
aspect, this method is used to identify compounds useful in the
treatment of schizophrenia or psychosis.
[0015] In another aspect, the invention relates to a method of
identifying a mutation in the 5-HT2A receptor gene, the mutation
being suspected of conferring constitutive activity on the
receptor, the method comprising:
[0016] (a) extracting nucleic acid from a biological sample
obtained from an individual having a disorder or condition
putatively associated with constitutive activity of the 5-HT2A
receptor;
[0017] (b) preparing cDNA from the extracted nucleic acid;
[0018] (c) selecting from the cDNA in step (b) cDNA encoding the
5-HT2A receptor;
[0019] (d) transfecting a cell with an expression vector comprising
said selected cDNA;
[0020] (e) selecting a cell expressing constitutively active 5-HT2A
receptor; and
[0021] (f) sequencing the cDNA in said selected cell to detect the
mutation(s).
[0022] In a further aspect, the invention relates to a method of
diagnosing a disorder or condition, or a susceptibility to a
disorder or condition, associated with constitutive activity of the
5-HT2A receptor, the method comprising:
[0023] (a) obtaining a biological sample from an individual
putatively affected by or susceptible to a disorder or condition
associated with constitutive activity of the 5-HT2A receptor,
[0024] (b) isolating from said biological sample a nucleic acid
sequence encoding said receptor, or a portion of said nucleic acid
sequence corresponding to the portion of the gene identified to
include mutation(s) by the mutation identification method described
above; and
[0025] (c) detecting the presence or absence of the mutation(s) in
said nucleic acid sequence or said portion thereof.
[0026] The presence of one or more mutations in the nucleic acid
sequence may, for example, be detected by sequencing the nucleic
acid sequence and comparing it with a sequence known or previously
identified to contain mutation(s).
[0027] In another aspect, the present invention relates to a test
kit for detecting mutation(s) in the gene encoding the 5-HT2A
receptor, said mutations giving rise to constitutive activity of
the 5-HT2A receptor, the test kit comprising a nucleic acid
sequence corresponding to a portion of the gene identified by the
mutation identification method described above to include at least
one mutation.
[0028] Furthermore, the present invention relates to a method of
decreasing the basal activity level of the 5-HT2A receptor in a
subject in need thereof, the method comprising contacting a 5-HT2A
receptor in said subject with an inverse agonist of the 5-HT2A
receptor in an amount effective to substantially decrease the level
of basal activity of said receptor. In a preferred embodiment, the
inverse agonist is selective for the 5-HT2A receptor (i.e., has at
least about ten times greater affinity for the 5-HT2A receptor than
for at least one other neurotransmitter receptor). In another
preferred embodiment, the inverse agonist of the 5-HT2A receptor
has little or substantially no anti-dopaminergic activity. In a
related aspect, the invention relates to a method of decreasing
serotonergic neurotransmission through the 5-HT2A receptor, the
method comprising contacting a 5-HT2A receptor with an inverse
agonist of the 5-HT2A receptor in an amount effective to
substantially decrease the level of basal activity of said
receptor.
[0029] In another aspect, the present invention relates to a method
of ameliorating symptoms of schizophrenia or psychosis in a subject
in need thereof, the method comprising administering to the subject
a therapeutically effective amount of an inverse agonist of the
5-HT2A receptor.
[0030] In yet other aspects, the invention relates to use of an
inverse agonist of the 5-HT2A receptor for the preparation of a
medicament for substantially decreasing the basal activity level of
a constitutively active 5-HT2A receptor. Preferably, in this use,
the inverse 5-HT2A agonist is selective for the 5-HT2A receptor. In
another embodiment relating to such use, the inverse agonist of the
5-HT2A receptor has little or substantially no anti-dopaminergic
activity. The invention also relates in certain aspects to use of a
5-HT2A receptor to identify compounds acting as inverse agonists at
said receptor, as well as use of a 5-HT2A receptor to identify a
compound acting as an inverse agonist at said receptor and useful
in the treatment of schizophrenia or psychosis.
[0031] The present disclosure represents the first reported
measurement of the constitutive activity of the wild type
(non-mutated) human 5-HT2A receptor and correlation of the
molecular property of inverse agonism at this receptor with
antipsychotic efficacy. Since most mutations in GPCR's have been
shown to alter their binding and coupling characteristics, the
ability to measure intrinsic activity at the wild type receptor,
and to use this receptor in assay for drug discovery is
critical.
[0032] Inverse agonists of the 5-HT2A receptor, as identified by
the present methods, may be used to alleviate or treat disorders or
conditions associated with constitutive activity of the 5-HT2A
receptor. It is anticipated that compounds that are inverse
agonists of the 5-HT2A receptor will be less likely to cause
extrapyramidal side effects than many of the typical antipsychotics
in current use. In particular, compounds that are selective for the
5-HT2A receptor, in that they exhibit little or no
anti-dopaminergic activity, are expected to have fewer
extrapyramidal side effects. Furthermore, inverse agonists may be
useful in the alleviation or treatment of the negative symptoms of
schizophrenia. This is supported by the fact that some of the
"atypical" antipsychotics, which are described herein to act as
inverse agonists at the 5-HT2A receptor, have been reported to have
beneficial effects on negative symptoms.
[0033] Definitions
[0034] A "test compound" is intended to include any drug, compound
or molecule with potential biological activity.
[0035] "Constitutive activity" is defined as the elevated basal
activity of a receptor which is independent of the presence of an
agonist. Constitutive activity of a receptor may be measured using
a number of different methods, including cellular (e.g., membrane)
preparations (see, e.g., A. J. Barr and D. R. Manning, J. Biol.
Chem. 27Z, 1997, pp. 32979-32987), purified reconstituted receptors
with or without the associated G-protein in phospholipid vesicles
(R. A. Cerione et al., Biochemistry 23, 1984, pp. 4519-4525), and
functional cellular assays (described herein).
[0036] An "inverse agonist" is defined as a compound which
decreases the basal activity of a receptor (i.e., signal
transduction mediated by the receptor). Such compounds are also
known as negative antagonists.
[0037] An "antagonist" is defined as a compound which competes with
an agonist or inverse agonist for binding to a receptor, thereby
blocking the action of an agonist or inverse agonist on the
receptor. However, an antagonist (also known as a "neutral"
antagonist) has no effect on constitutive receptor activity.
[0038] The "5-HT2A receptor" is defined as the human serotonin
receptor subtype characterized through molecular cloning and
pharmacology as detailed in Saltzman, A G., et al., Biochem.
Biophys. Res. Comm. 181(3), pp. 1469-1478; and Julius, D., et al.,
Proc. Natl. Acad. Sci. 87, pp. 928-932.
[0039] "Transfection" is defined as any method by which a foreign
gene is inserted into a cultured cell.
[0040] A "biological sample" indicates a sample of tissue or body
fluid obtained from a subject. Biological samples relevant to
obtaining 5-HT2A receptors include, but are not limited to, blood,
serum (5-HT2A receptors being present in platelets) and/or brain
tissue, within which the receptor genes are known to be expressed
in identical forms.
[0041] The term "subject" refers to an animal, preferably a mammal,
most preferably a human, who is the object of treatment,
observation or experiment.
[0042] The term "therapeutically effective amount" is used to
indicate an amount of an active compound, or pharmaceutical agent,
that elicits the biological or medicinal response indicated. This
response may occur in a tissue, system, animal or human that is
being sought by a researcher, veterinarian, medical doctor or other
clinician, and includes alleviation of the symptoms of the disease
being treated.
[0043] The terms "selectivity" or "selective," when used in the
context of inverse agonists of 5-HT2A, are used to indicate
compounds having at least approximately 10-fold higher affinity for
the 5-HT2A receptor subtype than towards at least one, and
preferably more than one, other neurotransmitter receptor.
[0044] EC50 for an agonist is intended to denote the concentration
of a compound needed to achieve 50% of a maximal response seen in
R-SAT. For inverse agonists, EC50 is intended to denote the
concentration of a compound needed to achieve 50% inhibition of an
R-SAT response from basal, no compound, levels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 shows the dose response relationship for serotonin at
the 5-HT2A receptor as observed in R-SAT assays. Responses are
plotted as the change in absorbance measured at 420 mm. Ten serial
1:5 dilutions of serotonin starting from 5 M were tested. The
squares depict the response of the 5-HT2A using the PSI.RTM.
expression vector at a DNA concentration of 5 ng per well. The
triangle depicts the response to 1 .mu.M ritanserin. Data are from
duplicate determinations at each drug concentration, where the
error bars denote the standard error of the mean. The EC50 for
serotonin is 7 nM. Note the elevated basal activity of this
receptor as documented by the inhibition below baseline seen with
the inverse agonist ritanserin.
[0046] FIG. 2 shows the dose response relationship at the 5-HT2A
receptor for the inverse agonist ritanserin as determined using
R-SAT analysis. Responses are plotted as the change in absorbance
measured at 420 nm. Ten serial 1:5 dilutions of drug starting from
5 .mu.M were tested. The squares depict the data obtained for
ritanserin, while the triangle denotes the basal, no drug,
response. Data are from duplicate determinations at each drug
concentration, where the error bars denote the standard error of
the mean. The EC50 for ritanserin is 140 pM. Ritanserin displays
high affinity negative intrinsic activity at the 5-HT2A
receptor.
[0047] FIG. 3 shows the dose response relationship for two
representative antipsychotics as inverse agonists of the 5-HT2A
receptor as determined by R-SAT analysis. Responses are plotted as
the change in absorbance measured at 420 nm. Ten serial 1:5
dilutions of drug staring from 5 M were tested. The squares depict
the data obtained for haloperidol in (A), and risperidone in (B),
while the triangles denote the basal, no drug, response. Data are
from duplicate determinations at each drug concentration, where the
error bars denote the standard error of the mean. The EC50 values
are 120 nM for haloperidol and 1 .mu.M for risperidone,
respectively.
[0048] FIG. 4 shows the chemical structures of two representative
compounds identified as inverse agonists of the 5-HT2A receptor
using the screening methods of the present invention. Compound
AC121394, which is haloperidol-like, and compound AC116399, which
is tricyclic-like, were identified out of a library comprising
135,000 structurally diverse organic compounds.
DETAILED DESCRIPTION OF THE INVENTION
[0049] In one embodiment, a method of identifying a compound which
acts as an inverse agonist of the 5-HT2A receptor comprises
contacting a constitutively active 5-HT2A receptor with at least
one test compound and determining any decrease in the level of
basal activity of the 5-HT2A receptor so as to identify the test
compound(s) which act as inverse agonists of the 5-HT2A receptor.
This method may be used to identify compounds useful in the
treatment of schizophrenia or psychosis.
[0050] In a preferred embodiment, a method of identifying a
compound which acts as an inverse agonist of the serotonin 5-HT2A
receptor comprises:
[0051] (a) culturing cells which express a constitutively active
5-HT2A receptor;
[0052] (b) incubating the cells with at least one test compound;
and
[0053] (c) determining any decrease in basal activity level of the
5-HT2A receptor so as to identify a test compound which is an
inverse agonist of the 5-HT2A receptor.
[0054] Where a mutation in the gene encoding the 5-HT2A receptor is
suspected of conferring constitutive activity on the receptor, a
method of identifying a mutation in the 5-HT2A receptor gene
comprises:
[0055] (a) extracting nucleic acid from a biological sample
obtained from an individual having a disorder or condition
putatively associated with constitutive activity of the 5-HT2A
receptor;
[0056] (b) preparing cDNA from the extracted nucleic acid;
[0057] (c) selecting from the cDNA in step (b) cDNA encoding the
5-HT2A receptor;
[0058] (d) transfecting a cell with an expression vector comprising
said selected cDNA;
[0059] (e) selecting a cell expressing constitutively active 5-HT2A
receptor; and
[0060] (f) sequencing the cDNA in said selected cell to detect the
mutation(s).
[0061] The extracted nucleic acid is preferably RNA, from which
cDNA may be prepared by reverse transcription. The cDNA which
encodes the 5-HT2A receptor is preferably amplified using
oligodeoxynucleotide probes specific to the 5-HT2A receptor gene
(i.e., based on the known sequence of the gene).
[0062] The present invention also provides a method of diagnosing a
disorder or condition, or a susceptibility to a disorder or
condition, associated with constitutive activity of the 5-HT2A
receptor. This method comprises:
[0063] (a) obtaining a biological sample from an individual
putatively affected by or susceptible to a disorder or condition
associated with constitutive activity of the 5-HT2A receptor;
[0064] (b) isolating from said biological sample a nucleic acid
sequence encoding said receptor, or a portion of said nucleic acid
sequence corresponding to the portion of the gene identified to
include mutation(s) by the mutation identification method described
above; and
[0065] (c) detecting the presence or absence of the mutation(s) in
said nucleic acid sequence or said portion thereof.
[0066] The presence of such mutations in the nucleic acid sequence
may, for example, be detected by sequencing the nucleic acid
sequence and comparing it with a sequence known or previously
identified to contain mutation(s).
[0067] The present invention also provides a test kit for detecting
mutation(s) in the gene encoding the 5-HT2A receptor, wherein the
mutations give rise to constitutive activity of the 5-HT2A
receptor. The test kit comprises a nucleic acid sequence
corresponding to a portion of the gene identified by the mutation
identification method described above to include at least one
mutation.
[0068] The present invention also provides a method of decreasing
the basal activity level of the 5-HT2A receptor in a subject in
need thereof. This method comprises contacting a 5-HT2A receptor in
said subject with an inverse agonist of the 5-HT2A receptor in an
amount effective to substantially decrease the level of basal
activity of said receptor. In a preferred embodiment, the inverse
agonist is selective for the 5-HT2A receptor. In another preferred
embodiment, the inverse agonist of the 5-HT2A receptor has little
or substantially no anti-dopaminergic activity.
[0069] Transfection of cells in the present invention may be
performed according to any of numerous methods known in the art. In
general, DNA sequences encoding the 5-HT2A receptor may be inserted
into suitable cloning vectors which may conveniently be subjected
to recombinant DNA procedures. These vectors may be autonomously
replicating, i.e., vectors which exist as extrachromosomal
entities, the replication of which are independent of chromosomal
replication (e.g., plasmids). Alternatively, these vectors may be
ones which, when introduced into a host cell, are integrated into
the host cell genome and replicate together with the chromosome(s)
into which they have been integrated.
[0070] The DNA sequences encoding the 5-HT2A receptor may suitably
be derived from sample genomic DNA, or cDNA that has been reverse
transcribed from sample RNA, in accordance with well-established
molecular biological techniques (e.g., as described in Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
[0071] When transfected, the DNA sequence encoding the 5-HT2A
receptor should be operably connected to a suitable promoter
sequence. The promoter may be any DNA sequence which shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell. An example of a suitable promoter is
the SV40 promoter (Subramani et al., Mol. Cell Biol. 1, 1981, pp.
854-864).
[0072] The DNA sequence encoding the 5-HT2A receptor may also be
operably connected to a suitable terminator, such as the human
growth hormone terminator (Palmiter et al.). The vector may further
comprise elements such as polyadenylation signals (e.g., from SV40
or the adenovirus 5 E1b region), transcriptional enhancer sequences
(e.g., the SV40 enhancer) and translational enhancer sequences
(e.g., those encoding adenovirus VA RNAs).
[0073] The vector may further comprise a DNA sequence enabling the
vector to replicate in the host cell in question. An example of
such a sequence (when the host cell is a mammalian cell) is the
SV40 origin of replication.
[0074] The procedures used to ligate the DNA sequences encoding the
5-HT2A receptor, the promoter and the terminator, respectively, and
the procedures used to insert them into suitable vectors containing
the information necessary for replication, are well known to
persons skilled in the art (see, e.g., Sambrook et al., supra).
[0075] Cells which may be used in the present method include any
cells capable of mediating signal transduction via the 5-HT2A
receptor, either via endogenous expression of this receptor (e.g.,
certain types of neuronal cells lines that natively express the
5-HT2A receptor), or following transfection of cells with plasmids
containing the 5-HT2A receptor gene. Such cells are typically
mammalian cells (or other eukaryotic cells, such as insect cells or
Xenopus oocytes), because cells of lower life forms generally lack
the appropriate signal transduction pathways for the present
purpose. Examples of suitable cells include: the mouse fibroblast
cell line NIH 3T3 (ATCC CRL 1658), which responds to transfected
5-HT2A receptors by stimulating growth (described herein); RAT 1
cells (Pace et al., Proc. Natl. Acad. Sci. USA 88, 1991, pp.
7031-7035); and pituitary cells (Vallar et al., Nature 330, 1987,
pp. 556-558). Other useful mammalian cells for the present method
include HEK 293 cells, CHO cells and COS cells.
[0076] Methods of transfecting mammalian cells and expressing DNA
sequences introduced in the cells are described in, e.g., Kaufman
and Sharp, J. Mol. Biol. 159 1982, pp. 601-621; Southern and Berg,
J. Mol. Appl Genet. 1, 1982, pp. 327-341; Loyter et al., Proc.
Natl. Acad. Sci. USA 79, 1982, pp. 422-426; Wigler et al., Cell 14,
1978, p. 725; Corsaro and Pearson, Somatic Cell Genetics 7, 1981,
p. 603; Graham and van der Eb, Virology 52, 1973, p. 456; Neumann
et al., EMBO J. 1, 1982, pp. 841-845; and Wigler et al., Cell 11,
1977, pp. 223-232.
[0077] The screening assay used in the present method may include
any functional assay that would reflect 5-HT2A receptor activity
in, for instance, membrane preparations or living cells, mammalian
and non-mammalian, in response to a ligand (agonist, antagonist
and, inverse agonists) and, in particular, an assay suited for
detecting constitutive activity of receptors. Examples of suitable
assay systems include those using insect cells (such as cells of
Spodoptera frugiperda, Sf9, transfected with baculovirus vector
carrying the receptor gene (e.g., as described in A. J. Barr and D.
R. Manning, J. Biol. Chem. 272, 1997, pp. 32979-32987; J. L.
Hartman and J. K. Northup, J. Biol. Chem. 271, 1996, pp.
22591-22597; J. Labrecque et al., Mol. Pharmacol. 48, 1995, pp.
150-159)), or Xenopus oocytes expressing cloned receptors (e.g., as
described in Y. G. Ni and R Miledi, Proc. Natl. Acad. Sci. USA 94,
1997, pp. 2036-2040), or HEK293 cells transiently expressing cloned
receptors (e.g., as described in M. Tiberi and M. Caron, J. Biol.
Chem. 269, 1994, pp. 27925-27931), or CHO cells (e.g., as described
in A. Newman-Tancredi et al., Neuropsychopharmacology 18, 1998, pp.
396-398). A preferred assay is the Receptor Selection and
Amplification Technology (R-SAT) assay disclosed in U.S. Pat. No.
5,707,798, the disclosure of which is hereby incorporated by
reference in its entirety.
[0078] Although the constitutive activity of the 5-HT2A receptor
may, in certain assays, be detected in itself, it may be more
suitable in other instances to overexpress the receptor to augment
basal signaling and improve the sensitivity of detection of inverse
agonism. Over-expression of receptors in cultured cells, as well as
transgenic animals, has been shown to result in increased
constitutive activity of the receptor (G. Milligan et al., TIPS 16,
pp. 10-13; S. A. Akhter et al. J. Biol. Chem. 272(34), pp.
21253-21259). Over-expression may be experimentally accomplished by
using an excess of plasmid DNA encoding receptors when transfecting
cells as part of functional assays of cloned monoamine receptor
subtypes. The excess of DNA may vary from one assay to the next but
may, in the currently preferred assay, be approximately 10-fold in
excess of that required to provide measurable signaling.
[0079] Attempts have been made to link neurotransmitter receptors
to neuropsychiatric diseases, primarily by identifying
polymorphisms in the receptor genes by methods including
restriction fragment length polymorphism (RFLP), single strand
conformational polymorphism (SSCP) and multipoint, parametric and
non-parametric methods of linkage analysis. For example, the
various dopamine receptors have been shown to possess multiple
polymorphic variants in the human population. (H. H. M. Van Tol et
al., Nature 342, 1992, pp. 149-152; N. Craddock et al., Psychiat.
Genet. 5, 1989, pp. 63-65). However, attempts at associating those
polymorphisms with neuropsychiatric disease are unlikely to succeed
because there is no credible evidence that the polymorphisms have
functional significance. Therefore, the present method of
identifying mutant receptors represents a substantial advantage in
that it identifies only functionally altered mutants. These
phenotypically distinct receptors are much more likely to be
related to human disease.
[0080] Thus, the present diagnostic methods are amenable to
screening human populations for mutant 5-HT2A receptor genes that
create a constitutively active phenotype. As the human 5-HT2A
receptor gene contains introns (A. G Saltzman et al., supra),
amplification of receptor DNA will typically be carried out by
reverse transcriptase-based PCR (RT-PCR; e.g., as described in
Elion, E. A., Current Protocols in Molecular Biology, 1998; F. M.
Ausebel et al., EDS, pp. 3.17.1-3.17.10). This method creates a
representative cDNA pool from an individual's RNA that is extracted
from suitable samples (e.g., serum or brain tissue) and amplifies
the receptor gene using oligonucleotide probes based on the known
sequence of the gene. The resulting PCR products are then subcloned
into mammalian expression vectors, and competent bacteria such as
E. coli are subsequently transformed. Bacterial cultures are
inoculated during transformation, thereby ensuring that the DNA
isolated from this culture represents a mixture of plasmids that
contains copies of both alleles of the amplified 5-HT2A receptor
gene. Phenotypic cellular assays (including R-SAT), select for only
those cells transfected with plasmids that encode functional
receptors, as only these cells will transduce mitogenic signals and
continue to grow. If the transfected receptor cDNA harbors a
mutation that confers a constitutively active phenotype, this is
detectable by the presence of higher levels of basal receptor
activity measured in the assay and verified by incubation of these
transfected cells with a known inverse agonist (e.g. as described
in the Example below).
[0081] After a constitutively active 5-HT2A receptor has been
identified in the assay, a formal characterization of the mutation
responsible for this phenotype is carried out. For example, an
aliquot of the original ligation reaction from all patients in whom
a constitutively active receptor has been identified by screening
is used to re-transform competent bacteria, and individual clones
are selected. The individual clones are then grown in larger
quantities and plasmid DNA is extracted according to any of various
methods known in the art. Restriction enzyme digestions will
identify 5-HT2A gene-containing constructs, and a number of these
are then subjected to automated DNA sequencing.
[0082] Mutant 5-HT2A receptors, identified by the present method,
may be included in a test kit for detecting mutation(s) in the gene
encoding the 5-HT2A receptor. Such a test kit may conveniently
comprise a nucleic acid sequence corresponding to a portion of the
gene encoding the 5-HT2A receptor comprising at least one mutation
identified by the present method to give rise to constitutive
activity of the receptor.
[0083] A suitable in vivo experimental system for validation of
both the physiological role of constitutively active 5-HT2A
receptors, and the effects of selective 5-HT2A inverse agonists as
therapeutic agents, is a transgenid animal model in which
constitutive signaling through the 5-HT2A receptor has been
achieved. Transgenic animals, preferably mice, may for instance be
generated by two distinct approaches: 1) brain-specific
over-expression of wild-type human 5-HT2A receptors; and 2)
regulated expression of a constitutively active 5-HT2A receptor
mutant. Both approaches rely upon standard molecular biological
techniques known to those skilled in the art.
[0084] Briefly, the first approach involves subcloning of the wild
type human 5-HT2A receptor gene into an appropriate transgenic
vector, the expression of which is driven by a strong promoter
(e.g., the CMV promoter). Brain-specific expression may be achieved
by incorporating vector constructs comprising the human 5-HT2A
receptor gene into the 5-HT2A genomic promoter region of the host
animal by site-specific homologous recombination (K. Rajewsky et.
al., J. Clin. Invest. 98(3), 1996, pp. 600-603). This is feasible,
as both the human and mouse promoter regions for the 5-HT2A
receptor gene have been cloned and characterized (Zhu, Q., Chen,
K., and Shih, J. C., J Neuroscience 15(7), 1995, pp. 4885-4895.). A
transgenic animal may then be generated by injection of the vector
construct into embryonic stem cells of the selected host animal
(typically, a mouse) in accordance with standard procedures (M. R.
Capecchi, Trends Genet. 5, 1989, pp. 70-76). This approach will
result in regionally specific over-expression of the wild-type
human 5-HT2A receptor in mouse brain.
[0085] The alternative approach requires the generation of a mutant
human receptor which has a significantly higher basal activity than
the wild-type gene. By applying standard PCR-based site-directed
mutagenesis (e.g., as disclosed in E. S. Burstein et al., Biochem.
Pharmacol. 51, 1996, pp. 539-544; and T. A. Spalding et al., J.
Pharm. Exp. Ther. 275, 1995, pp. 1274-1279, for the muscarinic m5
receptor), it is possible to generate a receptor mutant that will
exhibit increased constitutive activity. Using homologous
recombination to incorporate a transgenic expression vector in
which the mutant human gene is expressed from the native mouse
promoter, without overexpression, would result in an animal with
regional specific brain expression of an activated human 5-HT2A
receptor mutant.
[0086] The present disclosure provides a series of human 5-HT2A
receptor mutants that have increased constitutive activity compared
to that observed in the wild type receptor, any of which are
suitable for incorporation into a transgenic mouse model. Inverse
agonists of the 5-HT2A receptor identified by the present methods
may suitably be tested for activity in vivo in the transgenic mouse
models described above, in which the effect of the compounds on
locomotor activity, startle habituation and prepulse inhibition may
conveniently be studied (T. A. Sipes and M. A. Geyer,
Neuropharmacology, 33(3/4), pp. 441-448). Other animal models which
may be used for this purpose include 5-HT agonist induced head
twitches in mice or rats, substantially as disclosed by J. H. Kehne
et al., supra, which may be reduced by administration of inverse
agonists of the 5-HT2A receptor.
[0087] The present invention provides a method of ameliorating
symptoms of schizophrenia or psychosis in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of an inverse agonist of the
5-HT2A receptor.
[0088] Inverse agonists of the 5-HT2A receptor identified by the
methods of the present invention may be formulated in
pharmaceutical compositions comprising one or more inverse agonist
compounds together with a pharmaceutically acceptable diluant or
excipient. Such compositions may be formulated in an appropriate
manner and in accordance with accepted practices such as those
disclosed in Remington's Pharmaceutical Sciences, Gennaro, Ed.,
Mack Publishing Co., Easton Pa., 1990.
[0089] Particularly desirable inverse agonists of the 5-HT2A
receptor will exhibit considerable selectivity for that receptor.
Selectivity may, in the present context, be defined as an at least
10-fold higher affinity for the 5-HT2A receptor subtype than
towards at least one, and preferably more than one, other
neurotransmitter receptor tested. Examples of neurotransmitter
receptors against which potentially selective inverse 5-HT2A
agonists may suitably be tested include histamine, dopamine,
muscarinic and adrenergic receptors, as well as the other existing
serotonin receptor subtypes. 5-HT2A receptor inverse agonists may
be effective in the treatment of a number of neuropsychiatric
diseases and disorders such as psychosis or schizophrenia without
the attendant undesirable extrapyramidal side effects previously
observed with non-selective compounds, notably most classical
antipsychotic drugs. It is currently believed that favorable
therapeutic properties will be found in selective inverse 5-H72A
agonists that have little or substantially no anti-dopaminergic
activity, in particular as antagonists of the dopamine D2 receptor,
as such activity is assumed to give rise to many of these
extrapyramidal side effects. To identify compounds that have the
desired selectivity for 5-HT2A, the present assay method should
also include cells expressing at least one other neurotransmitter
receptor and preferably includes cells expressing a number of
different 20neurotransmitter receptors.
[0090] Advantageously, inverse agonist compounds may be
administered in a single daily dose, or the total daily dosage may
be administered in divided doses two, three or four times daily.
Furthermore, compounds of the present invention may be administered
in intranasal form via topical use of suitable intranasal vehicles,
or via transdermal routes using those forms of transdermal skin
patches well known to persons skilled in the art.
[0091] The dosage regimen for 5-HT2A inverse agonist compounds will
be selected in accordance with a variety of factors. These include
type, species, age, weight, sex and medical condition of the
patient; the severity of the condition to be treated; the route of
administration; the renal and hepatic function of the patient; and
the particular compound employed. A physician of ordinary skill can
readily determine and prescribe the effective amount of the drug
required to prevent, counter or arrest the progress of the disease
or disorder which is being treated.
[0092] The daily dosage may be varied over a wide range from about
0.01 to about 100 mg per adult human per day. An effective amount
is ordinarily supplied at a dosage level of about 0.0001 mg/kg to
about 25 mg/kg body weight per day. Preferably, the range is from
about 0.001 to about 10 mg/kg of body weight per day, and
especially from about 0.001 mg/kg to about 1 mg/kg of body weight
per day. The compounds may be administered on a regimen of 1 to 4
times per day.
[0093] Inverse agonist compounds may be used alone at appropriate
dosages defined by routine testing in order to obtain optimal
pharmacological effect on the serotonin 5-HT2A receptor, while
minimizing any potential toxic or otherwise unwanted effects. In
addition, it is believed that 5-HT2A selective inverse agonists may
be used as adjunctive therapy with known antipsychotic drugs to
reduce the dosage required of these traditional drugs, and thereby
reduce their extrapyramidal side effects.
[0094] The present invention is further disclosed in the following
Example, which is not in any way intended to limit the scope of the
invention as claimed.
EXAMPLE
[0095] The functional receptor assay, Receptor Selection and
Amplification Technology (R-SAT), was used (essentially as
disclosed in U.S. Pat. No. 5,707,798) to investigate the
pharmacological phenotype of the 5-HT2A receptor. The 5-HT2A
receptor gene was amplified by nested PCR from brain cDNA using the
following oligodeoxynucleotides based on published sequences: 5'#1:
5'-agctccgggagaacagcatgta-3'; 5'#2:
5'-gagtgtggatccatcaaggtgaatggtgagcag-3'; 3'#1:
5'-caatgaacagcatagcagcaa-3- '; 3'#2:
5'-ggtttcctctagaaaatagaagttaatttagatt-3 (Saltzman et. al., Biochem.
Biophys. Res. Comm. 181(3), 1991, pp. 1469-1478).
[0096] The cDNA was obtained by reverse transcription of total RNA
isolated from human brain tissue in accordance with standard
techniques (see, Sambrook et al, supra). The human brain tissue was
obtained from a 100-year old female free of neuropsychiatric
disease. The PCR product was subcloned onto the TOPO PCR 2.1.RTM.
vector (Invitrogen, Inc.) in accordance with the manufacturer's
protocol. A Bam-Hl (blunted with T4 polymerase)-Not-1 DNA fragment
containing the gene was subcloned into the mammalian expression
vector PSI.RTM. (Promega, Inc.) for heterologous expression in
R-SAT.
[0097] Varying doses of 5-HT2A receptor plasmid DNA were
transfected into NIH 3T3 cells (at 70% confluence) using the
transfection reagent Superfect.RTM. (Qiagen, Inc.). 5-HT2A receptor
DNA transfection mixtures (per well of a 96-well cell culture dish)
were composed of from 5 to 50 ng/well of receptor DNA, 25 ng/well
of .beta.-galactosidase plasmid DNA (in the PSI.RTM. vector), 50
.mu.L of DMEM, and 15 .mu.L of Superfect.RTM.. This mixture was
then augmented with additional DMEM (with 10% calf serum and 1%
penicillin/streptomycin/glutamine) sufficient to incubate each well
with 50 .mu.L of the transfection mixture. Cells were transfected
for 12 to 16 hours at 37.degree. C. in a humidified environment
supplemented with 5% CO.sub.2, after which time the media was
replaced by DMEM with 2% cyto-SF3 (Kemp Biotechnologies, Inc.)
containing variable amounts of the compounds being tested.
[0098] Cells were grown in a humidified environment at 37.degree.
C. with 5% CO.sub.2 for five days prior to visualization of
.beta.-galactosidase activity by replacing the media with the
.beta.-galactosidase substrate
o-nitrophenyl-.beta.-D-galactopyranoside (substantially as
described in U.S. Pat. No. 5,707,798). All data were obtained by
measuring the change in absorbance at 420 nm using an automated
plate reader (BioTek EL 310). EC50 values were calculated using the
equation: r=A+B(x/(x+c)), where A=minimum response, B=maximum
response minus minimum response, c=EC50, r=response, and
x=concentration of ligand. Curves were generated by least-squares
fit using the program KaleidaGraph.RTM. (Abelbeck Software).
[0099] Since constitutive activity may be erroneously measured if
experiments are conducted in the presence of minute amounts of
agonist, a number of control experiments were performed to rule out
the possibility that serotonin was present in the media. Firstly,
only synthetic sera were used (cyto-SF3), because bovine calf serum
may contain various monoamines or related receptor agonists.
Second, experiments in which the amount of synthetic sera was about
2-5 times that routinely used did not result in a measurable
increase in constitutive activity (data not shown). In addition,
using other serotonin receptor subtypes in which constitutive
activity has been measured (5-HT2B) revealed compounds that are
neutral antagonists and others that are inverse agonists, arguing
that competition for endogenous serotonin is not occurring
(otherwise, all compounds would appear to be inverse agonists).
[0100] The results of this analysis of the 5-HT2A receptor are
presented in FIG. 1, as a representative pharmacological profile as
determined by R-SAT.
[0101] Based on the results obtained in this analysis, it was
concluded that
[0102] 1) the 5-HT2A receptor is functionally active in R-SAT, and
that the data obtained are in agreement with previously published
binding assays (C. A. Stockmeier et al., J. Pharm. Exper. Ther.
266(3), 1993, pp. 1374-1384).
[0103] 2) expression of 5-HT2A receptors using the PSI.RTM. vector
results in the detection of constitutive activity. Under these
experimental conditions, ritanserin inhibits receptor signaling
below baseline (no drug) values, i.e. it is an inverse agonist
(note ritanserin values in FIG. 1).
[0104] 3) increasing the amount of DNA used for transfection
increased the basal activity of the 5-HT2A receptor (5%
constitutive activity at 5 ng/well vs. 11% constitutive activity at
50 ng/well; data not shown).
[0105] Upon detecting constitutive signaling with the 5-HT2A
receptor, high DNA concentrations were subsequently used to augment
basal responses and facilitate pharmacological analysis of inverse
agonists. All subsequent studies utilized 50 ng/well of 5-HT2A
receptor DNA. FIG. 2 shows the dose response relationship for
ritanserin as a representative 5-HT2A receptor inverse agonist.
[0106] R-SAT was configured to assay simultaneously for compounds
that exhibit both agonism and inverse agonism at this receptor
subtype. Multiple 96-well plates of NIH 3T3 cells were transfected
with 50 ng/well of 5-HT2A receptor DNA and screened against a
640-compound library of medically relevant drugs (RBI Inc, Natick,
Mass.). All compounds were screened at concentrations of 300-500
nM, serotonin (1 .mu.M) was used as a reference agonist, and
ritanserin (1 .mu.M) was used as a reference inverse agonist. The
results of this screen for inverse agonism (for compounds with
greater than 40% inhibition) at the 5-HT2A receptor are shown in
Table 1 below.
1TABLE 1 Screen for Inverse Agonism at 5-HT2A Receptors %
INHIBITION COMPOUND 96 TRIFLUPERIDOL 92 PIRENPIRONE 90 RITANSERIN
87 RISPERIDONE 84 BUTACLAMOL 82 SPIPERONE 82 KETANSERIN 79
MIANSERIN 79 METHIOTHEPIN 77 LOXAPINE 76 OCTOCLOTHEPIN 75 Mdl
26,630 trihcl 75 TRIFLOUPERAZINE 75 CINANSERIN 74 Dag kinase
inhibitor 69 JL-18 (CLOZAPINE) 68 AMOXAPINE 66 CYPROHEPTADINE 65
CHLORPROMAZINE 62 METERGOLINE 61 FLUPHENAZINE 57 FLUSPIRILINE 56
THIORIDAZINE 53 Benztropine 53 5-hydroxy-Ltryptophan 52
Promethazine 52 CLOZAPINE 51 Physostigmine 45 CIS-FLUPENTIXOL 42
PIMOZIDE
[0107] In Table 1, all data are derived from the mean of duplicate
determinations for each test compound, and are presented as a
percentage inhibition referenced to ritanserin (90-100%). The data
include all compounds detected in the screen that displayed a
greater than 40% inhibition from basal, no drug, levels. All
compounds that are known serotonergic drugs are italicized, and all
drugs with known anti-psychotic activity are presented in bold.
[0108] The results of this screen are significant in that:
[0109] 1) The screen identified nearly every antipsychotic drug in
the compound library (18/19 at 30% inhibition or greater),
documenting that these drugs are actually inverse agonists (not
antagonists) at this receptor subtype.
[0110] 2) There is selectivity to this interaction, as multiple
classes of other neuropsychiatric agents (e.g., antidepressants and
anticonvulsants) represented in the library are not inverse
agonists at this receptor subtype.
[0111] 3) The R-SAT technology is amenable to screening compounds
for inverse agonism at the 5-HT2A receptor.
[0112] 4) The R-SAT technology is amenable to screening individuals
for constitutively activating mutations of the 5-HT2A receptor in
an analogous manner to that presented above.
[0113] Having discovered that antipsychotics are inverse agonists
of the 5-HT2A receptor, a detailed pharmacological analysis of many
of these agents was performed to establish their potency and
efficacy. FIG. 3 shows the dose response curves for two known
antipsychotics, the typical agent haloperidol, and the atypical
agent risperidone. Table 2 is a compilation of this detailed
pharmacological analysis presented as negative log EC50 values.
2TABLE 2 Potency of Antipsychotics as Inverse Agonists at the
5-HT2A Receptor DRUG Negative LOG EC50 Sertindole 10.12 +/- 0.18
Tefludazine 9.02 +/- 0.21 Risperidone 8.81 +/- 0.05 Spiperone 8.70
+/- 0.07 Pimozide 8.65 +/- 0.04 Amoxapine 8.64 +/- 0.13 Loxapine
8.49 +/- 0.07 Butaclamol 8.49 +/- 0.19 Fluspirilene 8.49 +/- 0.14
Clozapine 8.17 +/- 0.19 Olanzapine 8.17 +/- 0.07 JL-18 8.11 +/-
0.13 Cis-Flupentixol 8.04 +/- 0.10 Fluphenazine 7.85 +/- 0.10
Chlorpromazine 7.70 +/- 0.11 Triflouperidol 7.59 +/- 0.09
Thioridazine 7.02 +/- 0.18 Triflouperazine 6.76 +/- 0.19
Trans-Flupentixol 6.77 +/- 0.21 Haloperidol 6.79 +/- 0.03
Thiothixene 6.43 +/- 0.11 Sulpiride NO EFFECT Remoxipride AGONIST
Molindone AGONIST
[0114] Table 2 above provides the molar negative log EC50s for
inhibition of constitutive activitty derived from the mean of three
separate dose response experiments (+/-standard error).
Antipsychotics that are generally considered atypical are
highlighted in bold.
[0115] These data allow one to draw the following conclusions
regarding antipsychotics as inverse agonists of the 5-HT2A
receptor:
[0116] 1) Nearly all antipsychotics tested are potent inverse
agonists of the 5-HT2A receptor. No similar activity of these drugs
as inverse agonists at other potentially relevant monoamine
receptors (5-HT1A, 5-HT1B, 5-HT2C, Dopamine D1, D2, D3, and D5,
.alpha.-1B adrenergic, and muscarinic m5 receptors) has been noted
(T. A. Spalding et al., supra; Nilsson, C. L., supra; Hall, D. A.
and Strange, P. G., supra, E. L. Barker et al., supra; A.
Newman-Tancredi et al., Brit. Jour. of Pharm. 120, pp. 737-739; P.
A. Pauwels and F. C. Colpaert, Biochem. Pharm. 50(10), pp.
1651-1658; and D. R. Thomas et al., J. of Receptor and Signal
Transduction Research 15(1-4), pp. 199-211). The correlation
between this singular molecular pharmacological mechanism and
efficacy of a drug as an antipsychotic suggests that this is a
fundamental mechanism of action of this class of drug.
[0117] 2) The atypical antipsychotic agents are amongst the most
potent of 5-HT2A receptor inverse agonists; thus, potent and
selective 5-HT2A inverse agonism should be a property of novel
antipsychotic drugs with improved clinical profiles.
[0118] 3) Since antipsychotics as a class possess the intrinsic
activity to reduce constitutive signal transduction mediated by the
5-HT2A receptors, any condition that favors increased basal
activity of this receptor may be contributory to, or causative of,
psychosis and/or schizophrenia.
[0119] It is apparent that the singular molecular property of
inverse agonism at the 5-HT2A receptor is common to nearly all
compounds with efficacy as an antipsychotic. To further support the
uniqueness of this correlation, a large series of antipsychotics
were pharmacologically profiled against the human 5-HT2C receptor.
This receptor was chosen because: 1) it is genetically and
pharmacologically related to the 5-HT2A receptor, 2) the receptor
RNA and protein are expressed in human brain regions critical to
higher cognitive functioning, and 3) some evidence exists to
support the notion that antagonism of this receptor is relevant to
the mechanism of action of antipsychotic drugs. The wild type human
5-HT2C receptor was PCR-cloned from human cortical cDNA by standard
molecular biological techniques familiar to those skilled in the
art. The receptor construct was subcloned into the PSI.RTM.
mammalian expression vector, and verified by DNA sequencing.
Transfection of 50 ng per well of receptor DNA (identical to the
amount used for 5-HT2A assays) revealed readily measurable
constitutive activity. Thirty-six antipsychotics were
pharmacologically assayed against the 5-HT2C receptor as both
agonists and inverse agonists. Table 3 reports the negative log
EC50 for these compounds as inverse agonists at both the 5-HT2A and
5-HT2C receptors.
3TABLE 3 Potency of Antipsychotics as Inverse Agonists at 5-HT2A
and 5-HT2C Receptors Receptor 5-HT2A Receptor 5-HT2C DRUG EC50
Negative Log EC50 Negative Log Sertindole 10.12 +/- 0.18 7.64 +/-
0.42 Octoclothepin 9.74 +/- 0.98 8.52 +/- 0.56 Tefludazine 9.02 +/-
0.21 8.28 +/- 0.49 Respiridone 8.81 +/- 0.05 <5.0 Tiospirone
8.74 +/- 0.67 6.29 +/- 0.53 Spiperone 8.70 +/- 0.07 No Intrinsic
Activity Pimozide 8.65 +/- 0.04 No Intrinsic Activity Amoxapine
8.64 +/- 0.13 6.92 +/- 0.34 Clothiapine 8.55 +/- 1.09 6.32 +/- 0.57
Butaclamol 8.49 +/- 0.19 No Intrinsic Activity Loxapine 8.49 +/-
0.07 6.30 +/- 0.32 Fluspirilene 8.19 +/- 0.14 No Intrinsic Activity
Clozapine 8.17 +/- 0.19 6.60 +/- 0.64 Olanzapine 8.17 +/- 0.07 6.36
+/- 0.47 JL-18 8.11 +/- 0.13 6.09 +/- 0.45 Cis-Flupentixol 8.04 +/-
0.10 No Intrinsic Activity Fluphenazine 7.85 +/- 0.10 <5.0
Amperozide 7.80 +/- 0.82 No Intrinsic Activity Chlorproethizene
7.70 +/- 0.33 <5.0 Chlorpromazine 7.70 +/- 0.11 No Intrinsic
Activity Triflouperidol 7.59 +/- 0.09 <5.0 Perlapine 7.52 +/-
0.49 5.89 +/- 1.17 Promazine 7.10 +/- 1.27 Agonist Moperone 7.03
+/- 0.59 No Intrinsic Activity Thioridazine 7.02 +/- 0.18 No
Intrinsic Activity Mesioridazine 7.00 +/- 0.30 No Intrinsic
Activity Melperone 6.96 +/- 0.56 No Intrinsic Activity Haloperidol
6.79 +/- 0.03 No Intrinsic Activity Trans-Flupentixol 6.77 +/- 0.21
5.55 +/- 0.37 Triflouperazine 6.76 +/- 0.19 No Intrinsic Activity
Bromperidol 6.66 +/- 0.76 No Intrinsic Activity Prothypendyl 6.60
+/- 0.44 Agonist Quietapine 6.57 +/- 0.80 No Intrinsic Activity
Thiothixene 6.43 +/- 0.11 No Intrinsic Activity Sulpiride No
Intrinsic Activity No Intrinsic Activity Remoxipride AGONIST No
Intrinsic Activity Molindone AGONIST No Intrinsic Activity
[0120] The following conclusions can be drawn from this data:
[0121] 1) The correlation between inverse agonism and efficacy as
an antipsychotic is apparent at the 5-HT2A receptor (33 of 36
compounds), but does not exist at the 5-HT2C receptor (12 of 36
compounds).
[0122] 2) High potency inverse agonism at the 5-HT2A receptor is a
property that many of the "atypical" antipsychotics share, yet no
such correlation between compounds with improved clinical
characteristics ("atypicals") and 5-HT 2C receptor intrinsic
activity can be drawn.
[0123] To identify novel compounds as potential antipsychotic
drugs, the 5-HT2A inverse agonist R-SAT assay was formatted to
conduct high-throughput screening of large libraries of organic
compounds. For these purposes, the constitutive basal response of
the 5-HT2A receptor was augmented by the addition of the alpha
subunit of the heterotrimeric G-protein Gq into the transfection
mixtures. Gq is the signaling molecule utilized by the 5-HT2A
receptor to functionally signal in cells, and coexpressing Gq with
other GPCR's has been previously shown to constitutively activate
receptors in this class (Burstein, E. S., et al., FEBS Lett. 363,
1995, pp. 261-263).
[0124] The 5-HT2A inverse agonist assay was used to screen 135,000
organic compounds for 5-HT2A inverse agonist activity. The
compounds examined were from a library of structurally diverse
organic molecules with an average molecular weight of 350 daltons.
The compounds were dissolved in DMSO and plated onto microtiter
plates with one compound in each well and either 96 or 384
compounds on each plate. The compounds were diluted to a
concentration of 3000 nM, incubated in the presence of transfected
cells for a period of five days, after which time
beta-galactosidase activity was measured to determine the
functional response of potential inverse agonists. These compounds
were also screened against the muscarinic m5 receptor, in an
analogous fashion, to provide a measure of selectivity for the
active compounds.
[0125] Of the 135,000 compounds tested in this manner, 511 were
identified that repressed the 5-HT2A basal activity in replicate
samples greater than 50% of that observed with the control inverse
agonist, 100 nM ritanserin. Of the 511 compounds that repressed
5-HT2A constitutive activity greater than 50% at 3000 nM, 322
compounds repressed significantly at 300 nM as well. Of these, 252
compounds displayed greater than 10-fold selectivity for 5-HT2A
inverse agonism compared to inverse agonism at the muscarinic m5
receptor.
[0126] Of the 252 5-HT2A selective compounds, 111 are related in
structure to the known antipsychotic haloperidol, and 64 compounds
are structurally related to the tricyclic antidepressants compounds
with known antipsychotic activity. Examples of these are the
compound AC121394 in the haloperidol class, and compound AC116399
in the tricyclic class (see FIG. 4). The successful screening of
compounds with 5-HT2A inverse activity that are related in
structure to known antipsychotics is a direct demonstration that
one can identify compounds with potentially improved antipsychotic
activity.
[0127] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed,
since these embodiments are intended as illustrations of several
aspects of the invention. Any equivalent embodiments are intended
to be within the scope of this invention. Indeed, various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims.
[0128] The disclosures of all references cited herein are
incorporated by reference in their entireties.
* * * * *