U.S. patent application number 09/929986 was filed with the patent office on 2002-06-27 for method of identifying compounds having antiepileptic, anticonvulsant or anxiolytic activities.
Invention is credited to Civelli, Olivier, Martin, James Richard, Monsma, Frederick, Moreau, Jean-Luc, Nothacker, Hans-Peter, Reinscheid, Rainer.
Application Number | 20020082213 09/929986 |
Document ID | / |
Family ID | 8222893 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082213 |
Kind Code |
A1 |
Civelli, Olivier ; et
al. |
June 27, 2002 |
Method of identifying compounds having antiepileptic,
anticonvulsant or anxiolytic activities
Abstract
The present invention relates to a method of screening for a
therapeutically useful compound which comprises testing an LC132
receptor agonist in a screening assay for psychiatric and/or
neurological disorders. More particularly, the screening method is
based on contacting an LC132 receptor with an agent suspected of
being an agonist of the LC132 receptor function, followed by the
detection of the binding and/or the agonist activity of the
compound and then testing of an agent with LC132 agonist activity
in an antiepileptic, anticonvulsant or anxiolytic screening
assay.
Inventors: |
Civelli, Olivier; (Irvine,
CA) ; Martin, James Richard; (Therwil, CH) ;
Monsma, Frederick; (Riehen, CH) ; Moreau,
Jean-Luc; (Rixheim, FR) ; Nothacker, Hans-Peter;
(Schliengen, DE) ; Reinscheid, Rainer; (Freiburg,
DE) |
Correspondence
Address: |
HOFFMANN-LA ROCHE INC.
PATENT LAW DEPARTMENT
340 KINGSLAND STREET
NUTLEY
NJ
07110
|
Family ID: |
8222893 |
Appl. No.: |
09/929986 |
Filed: |
August 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09929986 |
Aug 14, 2001 |
|
|
|
08868355 |
Jun 3, 1997 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
514/17.5; 514/17.7 |
Current CPC
Class: |
A61P 25/26 20180101;
A61P 25/08 20180101; G01N 33/566 20130101; G01N 2500/00 20130101;
A61P 25/20 20180101; G01N 33/9486 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 1996 |
EP |
96109462.0 |
Claims
What is claimed is:
1. A method for treating a psychiatric or neurological disorder in
a subject comprising administering to the subject Orphanin FQ or a
pharma-cologically active derivative thereof in a daily dosage
amount from about 0.1 nanograms per kilogram body weight to about
0.1 milligrams per kilogram body weight.
2. The method of claim 1 wherein the psychiatric disorder is
anxiety.
3. The method of claim 1 wherein the neurological disorder is
epilepsy or convulsions.
4. A pharmaceutical composition comprising from 0.01 nanograms to
20 milligrams Orphanin FQ or a pharmacologically active derivative
thereof, and a pharmaceutically acceptable carrier.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 08/868,355, filed Jun. 3, 1997.
FIELD OF THE INVENTION
[0002] The present invention relates to a screening method
comprised of testing an LC132 receptor agonist in screening assays
for neurological and/or psychiatric disorders. More specifically,
the screening method is based on bringing an LC132 receptor in
contact with an agent suspected of acting as an agonist of LC132
receptor function followed by the detection of the binding and/or
the agonist activity of the compound and then the testing of an
agent with LC132 agonist activity in an antiepileptic,
anticonvulsant and/or anxiolytic screening test to demonstrate
therapeutically relevant activity in these disorders.
BACKGROUND OF THE INVENTION
[0003] At present, benzodiazepine receptor agonists (e.g.,
alprazolam, diazepam, lorazepam) still represent the predominant
treatment in clinical medicine for anxiety disorders, especially
acute anxiety. Since benzodiazepine receptor agonists have
anticonvulsant properties, some are also used as antiepileptic
drugs. More recently, other drug classes have found clinical use in
the treatment of anxiety disorders, for example selective serotonin
reuptake inhibitors (e.g., fluoxetine) and buspirone. Treatment of
epilepsies is currently dominated by drugs such as carbamazepine,
phenytoin, valproate, ethosuximide, and phenobarbital. As discussed
below, the available drugs used in the pharmacological treatment of
anxiety, epilepsies, and convulsions are not optimal.
[0004] Anxiety is a physiologic phenomenon that acts as a warning
signal for a real or potential danger. Anxiety becomes pathologic
when it occurs in the absence of any real danger or when the
intensity of the emotion is exaggerated. Both physiologic and
pathologic anxiety can be life-threatening when occurring in the
face of pre-existing organic disorders and may create or perpetuate
various physiologic dysfunctions. The diverse anxiety disorders
represent relatively common psychiatric disorders with an estimated
combined prevalence within the general population of about 4-8
percent. The immediate relief provided by benzodiazepine receptor
agonists in treating anxiety disorders is well documented. Although
the efficacy of these drugs appears to be maintained over a long
period, a series of issues arise when treatment is administered for
more than several weeks. Although undesirable side-effects can
largely be avoided by optimising dosage of benzodiazepine receptor
agonists for the individual patient, doses required for severe
cases of anxiety and epilepsies as well as for reducing pathologic
muscle tone frequently depress vigilance to a level that disturbs
intellectual function and reduces attention and precision for
various skills (operation of machines, car driving). The individual
sensitivity to this oversedation varies greatly. Muscle relaxation
may result in blurred speech and disturbed gait, especially in
elderly patients. Behavioral disinhibition may occur at higher
doses and even at normal doses in individuals having minimal
experience with centrally active drugs. Problems can also occur due
to long-lasting exposure to benzodiazepine receptor agonists. One
such is the development of tolerance to a therapeutic effect. Loss
of antiepileptic efficacy occurs in a fair proportion of patients
(manifested as escape phenomena). Physical dependence manifested as
drug discontinuation symptoms following abrupt withdrawal is a
function of duration of drug exposure, dose, duration of action of
the drug, and the personality of patients. In marked contrast to
anxiolytics acting via benzodiazepine receptor agonism (which
exhibit anticonvulsant, muscle relaxant, and sedative/hypnotic
effects), the azaspirodecanedione buspirone presents only
anxiolytic activity. It has been hypothesized that the mechanism of
action of buspirone involves partial agonism at the
serotonin.sub.1A receptor. Advantages of buspirone include less
sedation, less psychomotor impairment in conjunction with ethanol
consumption, reduced physical dependence, and a much lower abuse
liability than for benzodiazepine receptor full agonists. However,
the long latency in the onset of anxiolytic activity is a
pronounced difference to classic benzodiazepine tranquilizers which
act rapidly. In addition, there are possible problems in treatment
compliance for buspirone and questions about efficacy in patients
previously treated with benzodiazepine receptor agonists or
exhibiting severe anxiety. Buspirone is not only a valuable
addition to the medical armamentarium whose place is gradually
becoming more clearly defined, but also very important from the
theoretical standpoint insofar as it is the first anxiolytic to
meet the rigorous clinical efficacy and safety standards of modern.
In view of the mechanism of action of selective serotonin reuptake
inhibitors (SSRIs), it appears that the resulting increased
availability of the neurotransmitter serotonin within the synaptic
cleft is responsible for the pharmacological effects of this drug
class. However, onset of the therapeutic action of SSRIs is slow,
usually requiring at least several weeks. Although originally
developed and predominantly used as antidepressants, SSRIs have
been increasingly used in treating panic disorder, e.g. fluoxetine
or obsessive-compulsive disorder, e.g. fluvoxamine. SSRIs are
generally well tolerated, nonetheless, common adverse effects for
compounds in this class include nervousness, tremor, dizziness,
headache, insomnia, sexual dysfunction, nausea, and diarrhea. In
addition, the tricyclic antidepressant clomipramine, which is a
potent nonselective serotonin reuptake inhibitor, is approved for
treatment of obsessive compulsive disorder [e.g. see: Martin and
Haefely, in "Principles of Pharmacology, Eds. Munson et al.,
Chapman & Hall, N.Y., 1995, pp. 243-277].
[0005] Epilepsy is a neurologic disorder which affects up to about
1 percent of the population. This chronic condition is
characterised by recurrent spontaneous seizures not caused by
active cerebral disease. Seizures are sudden, involuntary,
time-limited alterations in behavior associated with excessive
discharges of cerebral neurons. Today the epilepsies are usually
classified according to the main seizure type presented by the
patient (a given patient may have more than one seizure type but
one is usually more frequent than the others and is used as basis
for classification). A practical advantage of the classification by
seizure type is that it allows, to a certain degree, prediction of
responsiveness to therapeutic alternatives. Generally, only about
half of the patients will have their seizures satisfactorily
controlled with antiepileptic drugs; the remaining half will be
divided into patients having occasional seizures and patients who
have uncontrolled seizures and/or unacceptable adverse effects from
antiepileptic medications. Furthermore, the drugs currently used
frequently cause side effects. There are multiple etiologies for
seizures and the origin often remains obscure, thus, antiepileptics
as well as other drugs exhibiting anticonvulsant effects are
important therapeutically. (e.g. see: Lloyd and Gillenwater, in
"Principles of Pharmacology, Eds. Munson et al., Chapman &
Hall, N.Y., 1995, pp. 363-398).
[0006] Previously, a seventeen amino-acid-long peptide
(F-G-G-F-T-G-A-R-K-S-A-R-K-L-AN-Q) (SEQ ID NO: 1) called orphanin
FQ or nociceptin has been isolated from rat brain (Meunier et al.,
Nature 377: 532-535, 1995) and from porcine hypothalami (Reinscheid
et al., Science 270: 792-794, 1995). The amino acid sequence of
orphanin FQ is identical with that of nociceptin and will be
thereafter referred to as OFQ. Julius (Nature 377: 476, 1995)
discusses the OFQ discovery noting that this peptide shares
greatest sequence similarity with dynorphin A, one of five
established endogenous ligands for opioid receptors. OFQ inhibits
adenylate cyclase in CHO(LC132.sup.+) cells in culture and induces
hyperalgesia when administered intracerebroventricularly to mice
(Meunier et al., loc. cit.). The pattern of results indicate that
this heptadecapeptide is an endogenous agonist of the LC132
receptor and it appears to have pro-nociceptive properties.
Reinscheid et al. (loc. cit.) describe that when injected
intracerebroventricularly in mice, OFQ decreased locomotor activity
and induced hyperalgesia in the tail-flick test (but not in the
hot-plate test). It was concluded that OFQ may act as a brain
neurotransmitter to modulate nociceptive and locomotor
behavior.
[0007] Vaughan and Christie (Br. J. Pharmacol. 177: 1609-1611,
1996) investigated the actions of OFQ on the membrane properties of
rat dorsal raphe nucleus neurons using whole-cell patch clamp
recording in brain slice. Consistent with the reported presence of
the LC132 receptor mRNA in dorsal raphe neurons (Lachowicz et al.,
J. Neurochem. 64: 34-40, 1995) and G-protein-mediated coupling of
cloned LC132 receptors to the activation of K channels (Zhang and
Yu, J. Biol. Chem. 270: 22772-22776, 1995), it was found that the
LC132 receptor ligand nociceptin potently and efficaciously
increased inwardly rectifying K conductance in dorsal raphe
neurons.
SUMMARY OF THE INVENTION
[0008] It is demonstrated herein that agonists of the LC132
receptor like OFQ have effects in animal models of psychiatric and
neurological disorders (predicting therapeutic efficacy in
patients), especially, but not limited to treatment of anxiety
disorders, epilepsies, and convulsions. This determination has
allowed applicant to develop methods and protocols by which useful
substances can be identified which provide both therapeutically
useful compounds and/or lead compounds to be used in the discovery
of therapeutically useful compounds. Accordingly, the present
invention relates to methods of screening for a therapeutically
useful compound which comprise of testing an LC132 receptor agonist
in a screening assay for psychiatric and/or neurological disorders,
especially in an antiepileptic, anticonvulsant, or anxiolytic
screening test.
[0009] In a preferred embodiment, the method of screening for a
therapeutically useful compound comprises of testing an LC132
receptor agonist in a psychiatric and/or neurological disorder
screening assay. Preferably, the method comprises contacting an
LC132 receptor with an agent suspected of having LC132 receptor
agonist activity and detection of the LC132 receptor-agent binding
and/or the determination of the LC132 receptor agonist activity
followed by testing of an agent having agonist activity in a
psychiatric and/or neurological disorder screening assay, namely an
antiepileptic, anticonvulsant or an anxiolytic screening assay.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The term "LC132 receptor" or "LC132 receptor protein" refers
to the native receptor protein and derivatives thereof. This
receptor or its derivatives from different animal species has had
in the literature several names, the most commonly used being
ORL.sub.1. The LC132 receptor, an orphan receptor whose human and
murine complementary DNAs have recently been characterised,
structurally resembles to opioid receptors (Mollereau et al., FEBS
Lett. 341: 33-38, 1994; Fukuda et al, FEBS Lett. 343: 42-46, 1994;
Chen et al., FEBS Lett. 347: 279-283, 1994; Bunzow et al., FEBS
Lett. 347: 284-288, 1994; Wang et al., FEBS Lett. 348: 75-79, 1994;
Lachowicz et al., loc. cit.; Nishi et al., Biochem. Biophys. Res.
Commun. 205: 1353-1357, 1994 and Wick et al., Molec. Brain Res. 27:
37-44, 1995). The LC132 receptor bioactivity is characterised by
its high-affinity binding of OFQ and is negatively coupled with
adenylate cyclase (Meunier et al., loc. cit.; Reinscheid et al.,
1995, loc. cit.).
[0011] Ordinarily, preferred LC132 receptors derivatives have an
amino acid sequence having at least 80% amino acid sequence
identity with the corresponding human LC132 receptor amino acid
sequence, preferably at least 90% and most preferably at least 95%
and are characterised in that they are capable of binding to OFQ
with high affinity. A particularly advantageous embodiment of the
assay method comprises the use of the native human LC132 receptor
protein.
[0012] The term "LC132 receptor protein" also comprises derivatives
of naturally or non-naturally occurring receptor proteins and
related proteins comprising at least partial protein sequence
capable of binding to the OFQ peptide, i.e. proteins in which one
or more of the amino acids of the natural LC132 receptor or its
fragments have been replaced or deleted without loss of binding
activity. Such derivatives may be produced by known methods of
peptide chemistry or by recombinant DNA technology. The term "LC132
receptor protein" also comprises derivatives which may be prepared
from the functional groups occurring as side chains on the residues
or the N- or C-terminal groups, by means known in the art. These
derivatives may include aliphatic esters of the carboxyl groups,
amides of the carboxyl groups by reaction with ammonia or with
primary or secondary amines, N-acyl derivatives of free amino
groups of the amino acid residues formed with acyl moieties (e.g.
alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free
hydroxyl groups (for example that of seryl- or threonyl residues)
formed with acyl moieties.
[0013] The LC132 (opioid-like) orphan receptor was recently
identified on the basis of close homology with the predicted amino
acid sequence opioid receptors (Mollereau et al, loc. cit.; Bunzow
et al., loc. cit.; Lachowicz et al., loc. cit.). When LC132
receptors were expressed in heterologous systems, e.g. CHO cell
lines, etorphine and dynorphin A weakly inhibited cyclic AMP
formation, but other opioids were inactive. The heptadecapeptide
OFQ has been identified as a potent and efficacious endogenous
agonist of the LC132 receptor (Meunier et al., loc. cit.,
Reinscheid et al. (1995), loc. cit.). The LC132 transcripts are
expressed in several areas of the central nervous system that are
known to be involved in pain regulation, including the
hypothalamus, brainstem and spinal cord dorsal horn (Julius, loc.
cit.).
[0014] The expression of the LC132 receptor protein and derivatives
thereof can be achieved by conventional recombinant DNA technology.
Such techniques are explained in the literature (see e.g. Sambrook,
Fritsch & Maniatis, "Molecular Cloning. A Laboratory Manual",
Cold Spring Harbor Laboratory, N.Y., 1989 and Ausubel et al.,
"Current Protocols in Molecular Biology", Green Publishers
Association & Wiley Interscience, 1987). Further, DNA molecules
or fragments thereof encoding complete or partial LC132 proteins
may be obtained with the polymerase chain reaction (PCR) technique.
Nucleic acid sequences encoding the LC132 receptor may be expressed
using a wide variety of host/vector combinations. Useful expression
vectors may consist of segments of chromosomal, non-chromosomal and
synthetic nucleic acid sequences. Examples of such vectors are
viral vectors, such as the various known derivatives of SV40,
bacterial vectors, such as plasmids from E. coli, phage DNAs, such
as .lambda. phage derivatives, M13 and other filamentous
single-stranded DNA phages, as well as vectors useful in yeasts,
such as derivatives of the 2.mu. plasmid, vectors useful in
eukaryotic cells more preferably vectors useful in animal cells,
such as those containing SV40, adenovirus and/or retrovirus derived
DNA sequences.
[0015] The host cell used for the expression of LC132 receptor
encoding nucleic acid sequences may be selected from a variety of
known hosts. A large number of hosts are available for example from
The American Type Culture Collection (ATCC). Preferred hosts for
the LC132 expression are mammalian cells such as CHO cells. The
preparation of LC132 receptor expressing cell lines is known in the
art and described for example by Reinscheid et al. (1995, loc.
cit.).
[0016] The term "LC132 receptor agonists" refers to compounds
capable of binding to the LC132 receptor and thereby modulating the
LC132 receptor function, i.e. inhibiting forskolin-stimulated
adenylyl cylase activity in a cell transformed with a vector
capable of expressing the LC132 receptor. Exemplary agonists are
high affinity ligands, preferably with IC.sub.50 values of less
than 1 .mu.M , e.g. OFQ or derivatives thereof.
[0017] The term "contacting an LC132 receptor with an agent
suspected of having LC132 receptor agonist activity" may comprise
the following features: An LC132 receptor protein, preferably a
human LC132 receptor protein, is bound in appropriate buffer
conditions to a solid phase. The solid phase is usually
poly(vinylchloride), but may be other polymers such as cellulose,
polyacrylamide, nylon, polystyrene or polypropylene. The solid
supports may be in the form of tubes, beads, discs or micro plates,
or any other surfaces suitable for conducting an assay, and which
on passive binding of the LC132 receptor, exposes the high affinity
receptor binding site. After washing, an agent suspected of binding
to the LC132 receptor and/or of having LC132 receptor agonist
activity may be added in an appropriate buffer solution.
[0018] The preferred way of preparation of the LC132 receptor is
characterised by the transfection of a mammalian cell line, e.g. a
CHO cell line, with a vector containing a nucleic acid sequence
encoding the LC132 receptor followed by the expression of the
receptor protein. Purified membrane containing the receptor protein
may then be bound to the solid surface as described above and
incubated with agents suspected of being an LC132 receptor agonist.
These methods are known in the art and are described e.g. by
Reinscheid et al., loc. cit. In this case the method according to
the invention comprises (a) providing cell membranes containing
LC132 receptors, (b) contracting the membranes with a compound
suspected of being an LC132 receptor agonist and (c) determining
whether the compound binds to the LC132 receptor and exerts agonist
specific activity followed by a psychiatric and/or neurological
disorder screening assay.
[0019] The detection of binding of the agent suspected of being an
agonist to the LC132 receptor may be done by direct labelling of
the agent or by labelling of a competitive agent like OFQ. The
agent or the competitive agent may be marked for example with a
radioactive label. A preferred embodiment is described by
Reinscheid et al. (1995, loc. cit.) and also in EXAMPLE 1.
[0020] The pharmacological characterisation of the LC132 receptor
agonist activity may be determined by the extent of inhibition of
the forskolin-stimulated cAMP accumulation in the host cells (e.g.
CHO cells) transfected with a vector capable of expressing the
LC132 receptor. Concentrations of cAMP may be determined as
described by Reinscheid et al. (1995, loc. cit.) or by other
methods known in the art. A particular preferred embodiment of the
present invention comprising the determination of LC132 receptor
agonist activity is described in EXAMPLE 1.
[0021] LC132 receptor agonists may then be further characterised by
any known or coventional psychiatric and/or neurological disorder
screening assay. The term "psychiatric and/or neurological
screening assay" refers to, but is not limited to, anxiolytic,
antiepileptic and/or anticonvulsant screening assays. These assays
are known in the art as described below:
[0022] Anxiety tests (Martin & Haefely, cit. loc.). A large
number of animal models have been developed in the attempt to
predict the anxiolytic activity of novel compounds in man. Many of
these paradigms evaluate animal behavior in a so-called "conflict"
situation, i.e. a behavioral response is simultaneously under the
influence of two opposing motivational states such as approach and
avoidance tendencies. Probably the best known model is the
conditioned punishment conflict paradigm in which animals are
trained to voluntarily exhibit a certain response (e.g. pressing a
lever) in order to receive a reward (e.g. food for a hungry
animal). Once the animals exhibit a constant rate of lever-press
responding, then short periods are introduced (usually signalled by
visual or acoustic signals) during which lever pressing is
simultaneously rewarded by food and punished by mild electrical
foot shock. Animals exhibit a markedly reduced response rate during
these conflict periods, which are also characterised by various
overt signs of emotionality. The characteristic effect of
benzodiazepine receptor agonists, for example the anxiolytic
diazepam, is the disinhibition of punished behavior (resulting in
an increase in the rate of responding under punishment) at doses
that fail to disrupt unpunished responding. Furthermore, these same
active drugs produce an anxiolytic-like effect in the absence of
actual punishment, i.e. when the rate of lever pressing is reduced
by conditioned fear of punishment. The conflict task does not
require conditioned behavioral responses: naive thirsty animals can
be offered the opportunity to drink, with drinking punished via
contact with an electrified spout. Such punishment-suppressed
drinking is disinhibited dose-dependently by benzodiazepine
receptor agonists (e.g., diazepam). Exploratory activity can
likewise be decreased by contingent punishment and released by
treatment with known anxiolytics. Conflict models without
punishment are based on the presence of the natural opposing
motivational states, on the one hand the tendency to explore and,
on the other hand, fear of a novel environment (e.g. dark-light
chamber task, elevated plus-maze, consumption of unfamiliar food or
normal food in an unfamiliar environment, social interaction
between animals unfamiliar with each other). While it is obvious to
ascribe the behavioral disinhibitory effect of benzodiazepine
receptor agonism in these experimental situations to an
anxiolytic-like action, their effect can also be interpreted as a
general reduction of the influence of aversive factors or even to
an impaired ability to withhold innate or conditioned responses. An
anti-frustration effect resulting from benzodiazepine receptor
agonism is suggested by the increase of responding which is
maintained by response-contingent reward in the situation in which
the reward is reduced or omitted. Electrical stimulation of the
periaqueductal gray area of the midbrain via chronically implanted
electrodes in animals is aversive and elicits a number of emotional
reactions; benzodiazepine receptor agonists increase the aversive
threshold. States of acute anxiety characterised by behavioral and
physiological symptoms (cardiovascular, endocrine) can be induced
by chemicals known to be anxiogenic in man, e.g. convulsants such
as pentylenetetrazol, inverse agonists at the benzodiazepine
receptor agonists administered in subconvulsive doses, or even
abrupt drug withdrawal after chronic treatment with high doses of
sedatives. Ultrasonic distress cries by rat pups acutely separated
from their mothers are decreased by benzodiazepine receptor
agonists. The pharmacological specificity of benzodiazepine
receptor agonists in the above paradigms is impressive, whereas
antidepressants, analgesics, and antipsychotics are all relatively
ineffective.
[0023] Antiepilepsy/anticonvulsant tests (Martin & Haefely,
loc. cit.). Benzodiazepine receptor agonists are among the most
potent drugs known for use in preventing or abolishing seizures in
animals induced acutely by chemicals administered systemically
(e.g. pentylenetetrazol, bicuculline, picrotoxin, inhibitors of
GABA biosynthesis, penicillin, local anesthetics, inhibitors of
acetylcholinesterase), applied locally on the cortical surface or
into the ventricular system (cardiac glycosides, the glutamate
receptor stimulant NMDA), or applied chronically into the cortex
(focal aluminium or cobalt epilepsy). They are also effective in
protecting against hyperbaric seizures and from seizures developing
after chronic intermittent electrical stimulation in the limbic
system beginning at a subthreshold intensity (kindled seizures).
Electroconvulsive seizures are also prevented by most (but not all)
benzodiazepine receptor agonists, although at doses considerably
higher than those found to block chemically-induced seizures.
Various genetic models of epilepsy (a petit mal-like phenotype in
rats, seizures induced by acoustic stimulation in genetically-prone
mouse strains or myoclonic seizures induced by photic stimulation
in genetically-prone baboons) respond to benzodiazepine receptor
agonists. Thus, benzodiazepine receptor agonists are effective in
all current animal epilepsy models which are predictive of efficacy
in most forms of human epilepsy or convulsive states. Consistent
with their broad anticonvulsant activity, benzodiazepine receptor
agonists prevent or suppress epileptiform electric activity induced
by various procedures (convulsants, ionic composition) in brain
slices maintained in vitro, particularly hippocampal slices. They
also reduce the neuronal afterdischarges induced in various brain
regions by electrical stimulation. Benzodiazepine receptor agonists
have been found to reduce epileptiform activities in foci, as well
as to inhibit generalisation.
[0024] OFQ, an LC132 receptor agonist mentioned above, was injected
intracerebroventricularly in mice and the animals evaluated in a
test used to detect anxiolytic effects (operant conflict test,
light-dark test; EXAMPLES 2 and 3). It was demonstrated in both
test paradigms that OFQ exhibited anxiolytic effects. In addition,
intracerebroventricularly injected OFQ was demonstrated to be
active in an a mouse model of epilepsy (audiogenic seizure model;
EXAMPLE 4) which predicts anticonvulsant effects in general, as
well as antiepileptic action in patients. None of these potential
therapeutic effects of OFQ are described in literature.
[0025] Preferred tests are described in EXAMPLES 1, 2, 3 and 4
below:
[0026] Determination of LC132 receptor agonist activity and
inhibition of forskolin-stimulated cAMP accumulation (EXAMPLE 1)
demonstrates OFQ inhibits forskolin-stimulated cAMP accumulation in
CHO cells transfected with LC132 in comparison to untransfected CHO
cells. OFQ inhibits forskolin-stimulated cAMP accumulation in CHO
cells transfected with the LC132 receptor with a median effective
concentration (EC.sub.50) of 1.05.+-.0.21 nM and a maximal effect
of approximately 80% inhibition at 100 nM. It has no effect on
untransfected cells. For determination of binding kinetics of OFQ
to its receptor the radioligand [.sup.125I]Tyr.sup.14-orphanin was
developed and characterised (Reinscheid et al., J. Biol. Chem. 271:
14163-14168, 1996). [.sup.125I]Tyr.sup.14-orphanin binds to
membranes prepared from CHO cells expressing the LC132 receptor in
a saturable and displaceable manner with a K.sub.d of 56.2.+-.7.3
.mu.M and a B.sub.max of about 200 fmol/mg protein. OFQ inhibits
the binding of [.sup.125I]Tyr.sup.14-orphanin to the LC132 receptor
with an inhibitory constant (K.sub.i) of 0.19.+-.0.02 nM
(Reinscheid et al. (1996), loc. cit.).
[0027] The operant conflict test for anxiolytic effect (EXAMPLE 2)
shows that OFQ (mean .+-.sem=24.6.+-.3.6) produces a nonsignificant
reduction of unpunished responding compared to vehicle injection
(mean .+-.sem=36.3.+-.7.4) indicative of minimal motor impairment.
In contrast, OFQ (mean .+-.sem=16.2.+-.6.0) significantly
(P<0.05) enhances punished responding compared to vehicle
injection (mean .+-.sem=4.3.+-.1.6) indicative of an anxiolytic
effect. This pattern of results is similar to that produced by the
marketed anxiolytic compound diazepam orally administered to these
same two groups of mice, indicating the potential of OFQ as an
anxiolytic drug in patients.
[0028] The light-dark box task for anxiolytic effect (EXAMPLE 3)
shows that OFQ significantly (P's<0.05) increases the time spent
in the illuminated box at the doses 0.3 nmol/mouse (mean
.+-.sem=54.+-.14 sec) and 1 nmol/mouse (mean .+-.sem=58.+-.19 sec)
as compared to vehicle injection (mean .+-.sem=11.+-.5 sec). It
also significantly (P's<0.05) decreases the number of transition
attempts at the doses 0.3 nmol/mouse (mean .+-.sem=1.2.+-.0.6), 1
nmol/mouse (mean .+-.sem=0.7.+-.0.2), and 3 nmol/mouse (mean
.+-.sem=0.4.+-.0.3) as compared to vehicle injection (mean
.+-.sem=3.0.+-.0.5). This pattern of results is indicative of the
anxiolytic activity of OFQ.
[0029] The audiogenic seizure model (EXAMPLE 4) shows that OFQ
exhibited a protective effective against acoustically induced tonic
seizures in genetically sound sensitive mice with an ED.sub.50 dose
of 0.7 nmol/mouse. This result predicts the therapeutic value of
OFQ in epilepsy and in reducing convulsions.
[0030] Accordingly, the present invention also relates to compounds
obtainable by a method described above, e.g. OFQ and its
derivatives and to the pharmaceutical compositions comprising one
or more of these compounds and a therapeutically inert carrier
material. In addition, the invention relates to the use of these
compounds in the manufacture of a composition for the treatment of
psychiatric and/or neurological disorders and to methods for
treatment of psychiatric and/or neurological disorders comprising
administering a therapeutically effective amount of OFQ or a
derivative thereof. Examples for psychiatric and/or neurological
are epilepsy, anxiety or convulsions.
[0031] The term "OFQ and its derivatives" refers to the peptide
F-G-G-F-T-G-A-R-K-S-A-R-K-L-A-N-Q (SEQ ID No: 1), to fragments
and/or to non-naturally occurring peptides or mutants thereof.
These kind of derivatives are peptides in which one or more of the
amino acids of the natural OFQ peptide or its fragments have been
replaced or deleted without loss of the agonist activity. Such
derivatives may be produced by known methods of peptide
chemistry.
[0032] The term "OFQ and its derivatives" also comprises
derivatives which may be prepared from the functional groups
occurring as side chains on the residues or the N- or C-terminal
groups, by means known in the art, and are included in the
invention as long as they remain pharmaceutically acceptable, i.e.
they do not destroy the agonist activity of the peptides and do not
confer toxic properties on compositions containing it. These
derivatives may include, for example, polyethylene glycol
side-chains which may mask antigenic sites and/or extend the
residence of the OFQ peptide derivatives in body fluids. Other
derivatives include aliphatic esters of the carboxyl groups, amides
of the carboxyl groups by reaction with ammonia or with primary or
secondary amines, N-acyl derivatives of free amino groups of the
amino acid residues formed with acyl moieties (e.g. alkanoyl or
carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl
groups (for example that of seryl- or threonyl residues) formed
with acyl moieties.
[0033] The term "OFQ and its derivatives" also includes soluble
forms of the above derivatives. Soluble forms may be prepared by
methods known in the art, e.g. by chemical synthesis.
[0034] The dose ranges for the administration of OFQ and its
derivatives may be determined by those of ordinary skill in the art
without undue experimentation. In general, appropriate dosages are
those which are large enough to produce the desired effect. The
dosage should not be so large as to cause adverse side effects,
such as unwanted cross-reactions, anaphylactic reactions, and the
like. Generally, the dosage will vary with the age, condition, sex
and extent of disease in the patient, counter indications, if any,
immune tolerance and other such variables, to be adjusted by the
individual physician. The expected dose range is about 0. 1
ng/kg/day to about 0.1 mg/kg/day. The OFQ peptide and derivatives
thereof can be administered parenterally by injection or by gradual
perfusion over time. They can be administered intravenously,
intraperitoneally, intramuscularly, or subcutaneously.
[0035] Preparations for parenteral administration include sterile
or aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/ aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers, such as
those based on Ringer's dextrose, and the like. Preservatives and
other additives may also be present, such as, for example,
anti-microbials, anti-oxidants, chelating agents, inert gases and
the like. See, generally, Remington's Pharmaceutical Science, 18th
Ed., Mack Eds., 1990.
[0036] The present invention also relates to compounds obtainable
by a method described above and to the pharmaceutical compositions
comprising one or more of these compound and a therapeutically
inert carrier material.
[0037] Further, the invention comprises the use of the LC132
receptor for the screening of compounds useful for the treatment of
psychiatric and/or neurological disorders, especially for the
treatment of anxiety, epilepsy and convulsions as described
above.
[0038] The following EXAMPLES are intended to illustrate details of
the invention, without thereby limiting it in any manner.
EXAMPLES
Example 1
Determination of LC132 Agonist Activity and Inhibition of
Forskolin-stimulated cAMP Accumulation
[0039] For determination of concentrations of cAMP, LC132 receptor
transfected CHO cells or CHO dhfr.sup.- (wild type) cells were
plated in 24-well plates and grown to confluency. After removal of
the culture medium, test compounds or peptides dissolved in a total
volume of 0.2 ml Dulbecco's Modified Eagle Medium containing 10 mM
HEPES (pH 7.4), 1 mM forskolin and 1 mM
D,L-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (obtainable from
RBI) were added and the cells were incubated for 10 min at
37.degree. C. Reactions were stopped by addition of 0.5 ml ice-cold
ethanol and plates were frozen at -80.degree. C. for 12 hours.
After centrifugation of the plates, portions of the supernatant
were removed and dried for cAMP determination. cAMP assays (Biotrak
SPA, Amersham) were done according to the manufacturer's
instructions. Ligand binding assays were done in 96-well deep
bottom microwell plates. CHO cells stably expressing the LC132
receptor were harvested and resuspended in 10 volumes of binding
buffer containing 50 mM Tris-HCl (pH 7.8), 5 mM MgCl.sub.2, 1 mM
EGTA, 0.1% BSA, and 0.1 mg/ml each of aprotinin, leupeptin and
pepstatin. Membranes were prepared using a tissue homogenizer
(setting 4 for 30 sec; PT20, Kinematica, Lucerne, Switzerland).
Total membrane particulate was obtained after centrifugation at
45,000.times. g for 10 min at 4.degree. C. The resulting pellet was
resuspended and centrifuged twice as described above. Membranes
were finally diluted in an appropriate volume of binding buffer and
stored at -70.degree. C. Protein concentration was determined using
a BCA Protein Assay Kit (Pierce). Incubation of membranes (10-18
.mu.g protein) with [.sup.125I]Tyr.sup.14-orphanin was performed at
room temperature for 1 hour in a total volume of 200 .mu.l binding
buffer. For competition binding experiments 50,000 cpm of
[.sup.125I]Tyr.sup.14-orpha- nin (62.5 pM) were added together with
the indicated concentrations of unlabelled peptides. Non-specific
binding was determined in the presence of 50 nM OFQ. Bound and free
ligand were separated by rapid vacuum filtration through Whatman
GF/C filters using a Brandel multichannel harvester. GF/C filters
had been pretreated with 0.3% polyethylenimine containing 0.1% BSA
for 1 hour at room temperature. Filters were washed six times with
1 ml ice-cold 50 mM Tris-HCl, pH 7.5 and then counted in a gamma
counter.
Example 2
Operant Conflict Test for Anxiolytic Effect
[0040] Female albino mice (Ibm: MORO (SPF); Biological Research
Labs, Fullinsdorf, Switzerland) were used. At the start point of
this experiment these mice were at least 2-3 month old and had
previously been well trained in the operant conflict task. The mice
were individually housed in Macrolon.RTM. type plastic cages (circa
13.times.23.times.13 cm) with sawdust bedding. Room temperature and
humidity were regulated in animal quarters maintained on a natural
light-dark cycle. Tap water was available ad libitum, whereas
access to the standard laboratory chow was restricted. The mice
were maintained at approximately 80-85% of their free feeding body
weight. The training and testing procedures are provided by Martin
et al. (Pharmacol. Biochem. Behav. 46: 905-910, 1993):
Food-deprived mice were well trained in a task in which during the
initial 5 minutes each time a lever was pressed (unpunished
responding) a food reward was automatically delivered and then
during the subsequent 15 minutes each lever-press produced
(punished responding) food reward, but concomitantly a mild
foot-shock was given. Consistent with the published literature,
oral administration of 10 mg/kg diazepam 30 minutes prior to
testing resulted in some reduction of unpunished responding but
enhanced punished lever-pressing in these mice. Two groups (N's=9)
were formed. The volume of intracerebroventricular injection was 2
.mu.l. The vehicle was artificial cerebrospinal fluid. OFQ
(Research Genetics, Inc., Huntsville, Ala.) was given at the dose 3
nmol/mouse. Both unpunished (i.e., the initial 5-min period of a
conflict test) and punished (i.e., the subsequent 15-min period of
a conflict test in which lever pressing resulted in food and
concomitant shock) portions of a conflict test were evaluated. The
technician carrying out the behavioral evaluation was blind
(uninformed) to the treatment conditions for the individual
animals. Statistical analysis was done comparing the two treatment
groups using a Mann-Whitney U test with a p-value less than 0.05
accepted as statistically significant.
Example 3
Light-dark Box Task for Anxiolytic Effect
[0041] Male albino mice [Ibm: MORO (SPF); Biological Research Labs,
4414 Fullingsdorf, Switzerland] were used. The mice were housed in
group cages with sawdust bedding in the laboratory quarters for
several days prior to testing. Room temperature and humidity were
regulated and the animal quarters were maintained on a reversed
light-dark cycle. Tap water and standard laboratory chow was
available ad libitum. Exploratory activity in a light-dark box task
was determined in naive mice. The apparatus consisted of one opaque
and one transparent Plexiglas.RTM. boxes of equal size
(20.times.20.times.14 cm) connected by an opaque plastic tunnel
(5.times.7.times.10 cm). The transparent box was illuminated (4400
lux at the center of this box). Five minutes after
intracerebroventricular injection, a mouse was placed in the tunnel
facing the dark box. The time spent in the illuminated box, the
number of transition attempts (only the head and forepaws entered
into the lighted box), and the number of completed transitions from
the opaque box to the transparent box were recorded for the
5-minute session. A mouse was judged to have changed compartments
when all four paws were placed in the next compartment. The floor
was thoroughly cleaned after each test. Each treatment group was
composed of 10-20 mice. The volume of the intracerebroventricular
injection was 2 .mu.l. OFQ (Research Genetics, Inc., Huntsville,
Ala.) was given at the doses 3, 1, 0.3 and 0.1 nmol/mouse.
Artificial cerebrospinal fluid was used as the vehicle. The
technician carrying out the behavioral evaluation was blind
(uninformed) to the treatment conditions for the individual
animals. Differences between the treatment groups were evaluated
with the Student's t-test with a p-value.ltoreq.0.05 accepted as
statistically significant.
Example 4
Audiogenic Seizure Model for Antiepileptic/Anticonvulsant
Effects
[0042] Young mice [DBA/2J (SPF); Biological Research Labs, 4414
Fullinsdorf, Switzerland] were used. The mice were housed in group
cages with sawdust bedding in the laboratory quarters prior to
testing. Room temperature and humidity were regulated and the
animal quarters were maintained on a natural light-dark cycle. Tap
water and standard laboratory chow was available ad libitum.
Protection from audiogenic seizures was investigated in naive
3-week-old DBA/2J mice (which are known to be genetically seizure
susceptible). Additional experimental details are provided
elsewhere (Martin et al., Psychopharmacology 111: 415-422, 1993) In
short, immediately after intracerebroventricular injection, each
mouse was placed in a separate open transparent Plexiglas.RTM. box
(21.times.44.times.21 cm) containing sawdust bedding. Testing was
done is a sound-isolated chamber and involved exposure to a 14 kHz
sinusoidal tone at 110 dB (zero dB was defined as a pressure level
of 20 .mu.Pa) for 1 minute during which observations were made.
Auditory stimulation was given 5 minutes after treatment. Groups of
8 mice were used to evaluate each dose and the vehicle condition.
The volume of the intracerebroventricular injection was 2 .mu.l.
OFQ (Research Genetics, Inc., Huntsville, Ala.) was given at the
doses 10, 3, 1, 0.1, and 0.01 nmol/mouse. Artificial cerebrospinal
fluid was used as the vehicle. The technician carrying out the
behavioral evaluation was blind (uninformed) to the treatment
conditions for the individual animals. The proportion of the group
failing to exhibit tonic convulsions was recorded. The ED.sub.50
value was calculated with probit analysis.
[0043]
Sequence CWU 1
1
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