Note: Descriptions are shown in the official language in which they were submitted.
CA 02411192 2002-12-10
WO 02/08178 PCT/SE01/01651
NEW COMBINATION OF SEROTONIN AGONIST (SHT2) AND
ANTAGONIST (SHT6)AS PHARMACEUTICAL FORMULATION
TECHNICAL FIELD
The present invention relates to the prophylaxis or treatment of a 5-HT2~ and
a 5-HT6
receptor-related disease. In addition, the invention provides a pharmaceutical
composition containing a 5-HT2~ receptor agonist and a 5-HT6 receptor
antagonist for
therapeutic use.
BACKGROUND ART
Serotonin (5-hydroxytryptamine or 5-HT) is a key neurotransmitter of the
peripheral
and central nervous system (PNS and CNS) and has been implicated in a variety
of
sensory, motor and behavioral functions such as regulation of eating,
sleeping, body
temperature, blood pressure, emotions and cognition. At least 14 distinct
serotonin
receptor subtypes are expressed in the mammalian PNS and CNS and have been
formally classified; see Glennon, et al., Nezc~osci. Biobehav. Rev. 1990, 14,
35-37; and
D. Hoyer, et al., Pha~macol. Rev. 1994, 46, 157-203. Serotoninergic agonists
and
antagonists have been suggested for the treatment of a wide range of
disorders,
including anxiety, depression, hypertension, migraine, obesity, drug abuse and
addiction, compulsive disorders, schizophrenia, autism, neurodegenerative
disorders
(e.g. Alzheimer's disease, Parkinsonism, and Huntington's chorea), and
chemotherapy-
induced vomiting.
The 5-HT2 subfamily of receptors is composed of three subtypes, the 5-HT2A, 5-
HT2B and 5-HT2c receptors. Serotonin 5-HT2~ receptors are expressed in many
brain
regions and have been implicated in the regulation of food intake (Dourish,
C.T. Obes.
Res. 1995, 3, Suppl. 4, 4495-4625; Bickerdike, M.J., et al. Diabetes, Obes.
Metab.
1999, 1, 207-214). It has been demonstrated that the non-specific 5-HT2C
receptor
agonist arc-chlorophenylpiperazine (nZ-CPP), which has some preference for the
5-HT2C
receptor, reduces food intake in mice that express the normal 5-HT2C receptor
while
the compound lacks activity in mice expressing the mutated inactive form of
the
5-HT2C receptor (Tecott, L.H., et al. Nature 1995, 374, 542-546). .
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WO 02/08178 PCT/SE01/01651
Moreover, it has been reported that m-CPP and the azepinoindole U-22394A, the
latter recently identified to be a 5-HT2~ receptor agonist (unpublished
observation),
reduce body weight in humans following two and nine weeks of treatment,
respectively
(Walsh, A. E. S., Psychopharmacology 1994,116, 120-122; Sargent, P.A., et al.
Psychopha~macology 1997,133, 309-312 and Gallant, D.M., et al. Cu~~.
Thef°. Res.
1967, 9, 579-581).
Recently, a series of pyrrolo[3,2,1-ij]quinoline derivatives was identified to
be
5-HT2~ receptor agonists having selectivity over the 5-HT2A receptor (Isaac
M., et al.,
Bioo~g. Med. Chem. Lett. 2000,10, 919-921). The compounds are said to offer a
novel
approach to the treatment of obesity and epilepsy.
The 5-HT2C receptor subtype has also been suggested to be involved in CNS
disorders, such as depression and anxiety (Jenck, F., et al. Expert Opih.
Invest. Drugs
1998, 7, 1587-1599; Leysen, D.C.M. ID~ugs 1999, 2, 109-120). The 5-HT2C
receptor
subtype has further been suggested to be involved in urinary disorders such as
urinary
incontinence (Leysen, D.C.M. ID~ugs 1999, 2, 109-120).
Also the 5-HT6 receptor (identified in 1993 - Monsma et al., Mol. Pha~macol.
1993, 43, 320-327 and Ruat, M. et al. Biochem. Biophys. Res. Commu~. 1993,193,
269-
276) has been implicated in the regulation of food intake and CNS disorders.
Thus, for example, Bentley, J. C., et al., B~. J. Pharmacol. 1999,126, 66P
describes food intake reduction in rats by the administration of a 5-HT6
antagonist.
Also, several antidepressants and atypical antipsychotics display high
affinity for the 5-
HT6 receptor which have suggested the involvement of the 5-HT6 receptor in
schizophrenia (Roth et al. J. Pha~macol. Exp. They. 1994, 268, 1403-1410;
Sleight et al.
Expert Opih. TYcen. Patents 1998, 8, 1217-1224; Bourson et al. B~. J. Pharm.
1998,125,
1562-1566; Boess et al. Mol. Pha~macol. 1998, 54, 577-583; Sleight et al. B~.
J.
Phay~macol. 1998, 124, 556-562). In addition, the 5-HT~ receptor has been
linked to
generalized stress and anxiety states (Yoshioka et al. Life Sci. 1998, 17/18,
1473-1477).
so SUMMARY OF THE INVENTION
According to the present invention it has now unexpectedly been found that the
combined administration of a 5-HT2~ receptor agonist and a 5-HT6 receptor
antagonist
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WO 02/08178 PCT/SE01/01651
reduces food intake by more than the administration of either agonist or
antagonist
alone. Such combined administration of a 5-HT2~ receptor agonist and a 5-HT6
receptor
antagonist may offer therapeutic advantages as compared to treatment with
either
agonist or antagonist alone.
One aspect of the present invention therefore provides a pharmaceutical
composition comprising an effective amount of a combination of a 5-HT2~
receptor
agonist and a 5-HT6 receptor antagonist, and optionally a pharmaceutically
acceptable
carrier.
Another aspect of the invention provides a method of preventing or treating a
disease, in particular obesity, related to the 5-HT2c receptor and the 5-HT6
receptor,
comprising administering to a human or animal subject in need thereof a 5-HT2c
receptor agonist and a 5-HT6 receptor antagonist (simultaneously or
sequentially) in
sufficient amounts to provide a therapeutic effect.
Still another aspect of the invention provides the use of a 5-HT2~ receptor
agonist and a 5-HT6 receptor antagonist for the manufacture of a medicament
for the
treatment of a disease related to the 5-HT2C receptor and the 5-HT6 receptor.
Another aspect of the invention provides a process for preparing a
pharmaceutical composition, wherein a 5-HT2~ receptor agonist and a 5-HT6
receptor
antagonist in a combined therapeutic amount are intimately mixed with a
pharmaceutically acceptable carrier.
Yet another aspect of the invention provides a product containing a 5-HT2C
receptor agonist and a 5-HT6 receptor antagonist as a combined preparation for
simultaneous, separate or sequential use in therapy of a disease, in
particular obesity,
related to the 5-HT2~ receptor and the 5-HT6 receptor.
BRIEF DESCRTPTTON OF THE DRAWINGS
Figure I shows the effect on food intake in ob/ob mice following combined
administration with a 5-HT2~ receptor agonist (PNU-I83933F; 50 mg/kg po) and a
5-
HT6 receptor antagonist (PNU-186053A; 50 mg/kg sc), as well as the effect of
each
agonist and antagonist alone.
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WO 02/08178 PCT/SE01/01651
Figure 2 shows the effect on food intake in ob/ob mice following combined
administration of a 5-HT2~ receptor agonist (BVT.2938F; 5 mg/kg sc) and a 5-
HTg
receptor antagonist (BVT.51~2C; 3 mg/lcg sc), as well as the effect of each
agonist and
antagonist alone.
DETAILED DESCRIPTION OF THE INVENTION
As mentioned above, the present invention is based on the unexpected finding
that
IO combined administration of a 5-HT2~ receptor agonist and a 5-HT6 receptor
antagonist
reduces food intake more than either agonist or antagonist alone. Such
combined
administration of a 5-HT2~ receptor agonist and a 5-HT6 receptor antagonist
may also
offer several benefits, for instance in the treatment of obesity, as compared
to treatment
with either agonist or antagonist alone.
15 Firstly, the combined administration requires lower doses of each compound
to
yield similar or improved reduction of food intake than mono-therapy.
Secondly, the lower doses required by the combined administration may reduce
the risk of adverse events.
Thirdly, the lower doses required by the combined administration may reduce
20 the risk of tolerance development and abuse liability.
Fourthly, therapy based on two targets may increase the individual therapeutic
efficacy relative to therapy based on one target. The risk of non-responsive
efficacy
(non-responders) may be reduced as well.
The beneficial effects of the combined administration of this invention is
useful
25 not only for the modulation of eating behavior, and for treating over-
weight and obesity,
but may also be useful for the treatment of CNS disorders such as, depression,
mania,
schizophreniform disorders, anxiety, memory disorders (such as Alzheimer's
disease)
migraine headache, drug addiction, convulsive disorders, personality
disorders, post-
traumatic stress syndrome, and sleep disorders as well as for treatment of
urinary
30 incontinence (or more generally overactive bladder), sexual dysfunctions,
gastrointestinal disorders and glaucoma.
The term "5-HT2~ receptor agonist" as used herein refers to a compound that
causes activation of the serotonin 5-HT2~ receptor. The 5-HT2~ receptor
agonist
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WO 02/08178 PCT/SE01/01651
preferably has an affinity constant, Ki, of less than 50 nM, preferably less
than 20 nM,
and an in vit~~o intrinsic activity, measured as intracellular Ca2+ levels,
greater than
20%, preferably greater than 50%, relative to 5-HT (1 ~.M).
The term "5-HT6 receptor antagonist" as used herein refers to a compound that
causes blockade of the serotonin 5-HTg receptor mediated responses. The 5-HT6
receptor antagonist preferably has an affinity constant, Ki, of less than 50
nM,
preferably less than 20 nM, and an ih vitoo intrinsic activity, measured as
intracellular
cAMP levels, less than 50%, preferably less than 20%, relative to 5-HT (1 ~M).
I~ vitro assays that may be used for determining the affinity and the
intrinsic
to activity, respectively, of 5-HT2~ receptor agonists and 5-HT6 receptor
antagonists are
known in the art and are also given in the Experimental Part below, as are
assays for
determining affinity to 5-HT2A and 5-HT2B receptors.
Generally, the 5-HT2~ receptor agonists and 5-HT6 receptor antagonists should
be sufficiently selective not to cause any substantial adverse side effects.
The terms
15 "selective" and "substantial" in this context are, however, to be
interpreted broadly, the
meanings thereof being readily apparent to the skilled person.
The 5-HT2~ receptor agonist preferably has a selectivity for the 5-HT2C
receptor
of at least 5, preferably at least 10 and more preferably at least 20,
relative to the 5-
HT2A, 5-HT2g and 5-HT6 receptors, respectively (measured as the affinity
ratios 5-
20 HT2A/5-HT2C, 5-HT2g/5-HT2~ and 5-HT6/5-HT2C)
The 5-HTg receptor antagonist preferably has a selectivity for the 5-HT6
receptor of at least 5, preferably at least 10 and more preferably at least
20, relative to
the 5-HT2A, 5-HT2B and 5-HT2~ receptors, respectively (measured as the
affinity ratios
5-HT2A/5-HT6, 5-HT2B/5-HT6 and 5-HT2~/5-HT6).
25 Relevant tests to determine whether a compound is a selective 5-HT2~
receptor
agonist or a selective 5-HT6 receptor antagonist are known in the art, and
are, as
mentioned above, also outlined in the Experimental Part below.
Compounds known to be 5-HT2~ receptor agonists are, for example, azetidine
and pyrrolidine derivatives of the type described in EP-A-0863136; tricyclic
pyrrole
30 derivatives of the type described in EP-A-0657426; 1-aminoethylindoles of
the type
described in EP-A-0655440; pyrazinoindoles of the type described in EP-A-
0572863;
piperazinylpyrazines of the type described in US 4,081,542; indoline
derivatives of the
type described in WO 00/12475; pyrroloindoles, pyridoindoles and
azepinoindoles of
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WO 02/08178 PCT/SE01/01651
the type described in WO 00/12510; indazole derivatives of the type described
in WO
00/12482; pyrroloquinolines of the type described in WO 00/12502; 2,3,4,4a-
tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6l~ones of the type described in WO
00/35922; indazolylpropylamines of the type described in WO 00/12481;
indazoles of
the type described in WO 00/17170; piperazinylpyrazines of the type described
in WO
00/76984 and in Swedish patent applications Nos. 0004244-0 and 0004245-7,
filed on
20 November 2000; heterocycle fused'y carbolines of the type described in WO
00/77001, WO 00/77002 and WO 00/77010; benzofurylpiperazines of the type
described in WO 01/09111 and WO 01/09123; benzofurans of the type described in
WO
l0 01109122; benzothiophenes of the type described in 01/09126;
pyridinylpiperazines of
the type described in EP 370560; pyrroloquinolines of the type described in
Bioorg.
Med. Chem. Lett. 2000, 10, 919-921; aminoalkylindazoles of the type described
in WO
98/30548; indoles of the type described in WO 01/12603; indolines of the type
described in WO 01/12602; pyrazino(aza)indoles of the type described in WO
00/44753; tricyclic pyrroles or pyrazoles of the type described in WO
98/56768.
Currently preferable 5-HT2c receptor agonists are of the arylpiperazine and
piperazinylpyrazine compound classes, in particular compounds disclosed in WO
00/76984 and in Swedish patent applications Nos. 0004244-0 and 0004245-7,
filed on
November 2000.
2o Compounds known to be 5-HT6 receptor antagonists are, for example,
piperazinylbenzenesulfonamides of the type described in WO 99/37623;
sulfonylbenzene derivatives of the type described in EP-A-0930302; sulfonamide
derivatives of the type described in WO 99/02502; sulfonamide derivatives of
the type
described in WO 99/42465; sulfonamide derivatives of the type described in WO
98/27081; carboxamide derivatives of the type described in WO 98/27058;
sulfonamide
derivatives of the type described in EP-A-0815861; pyrrolidonomethylindole
derivatives of the type described in WO 99/47516; bicyclic piperidine and
piperazine
derivatives of the type described in WO 99/65906; pyrazolopyrimidine and
pyrazolotriazine derivatives of the type described in EP-A-0941994;
arylsulfone-
3o substituted hexahydroazepinoindoles of the type described in WO 01/05793;
oxazinocarbazoles of the type described in WO 01/09142; aminoalkoxycarbazoles
of
the type described in WO 01/17963; diphenylsulfones of the type described in
the
international patent application PCT/US00/30177, filed on June 20, 2000; and
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WO 02/08178 PCT/SE01/01651
arylsulfonylindoles of the type described in the Swedish patent application
No.
0003810-9, filed on October 20, 2000.
Currently preferable 5-HT6 receptor antagonists include the azepinoindole
compound class, such as the class of arylsulfone-substituted
hexahydroazepinoindoles
compounds disclosed in WO 01/05793. Other preferred 5-HT6 receptor antagonists
include the arylsulfonylindole compound class, such as the compound class
described in
the Swedish patent application No. 0003810-9.
The 5-HT2c receptor agonists and the 5-HT6 receptor antagonists may be the
compounds as such or where appropriate the pharmaceutically acceptable salts
(acid or
to base addition salts) thereof or stereochemically isomeric forms thereof
(including
optical isomers, such as enantiomers and racemates).
The pharmaceutically acceptable addition salts as mentioned above are meant to
comprise the therapeutically active non-toxic acid and base addition salt
forms which
the compounds are able to form. Compounds which have basic properties can be
converted to their pharmaceutically acceptable acid addition salts by treating
the base
form with an appropriate acid. Exemplary acids include inorganic acids, such
as
hydrogen chloride, hydrogen bromide, hydrogen iodide, sulphuric acid,
phosphoric
acid; and organic acids such as acetic acid, propanoic acid, hydroxyacetic
acid, lactic
acid, pyruvic acid, glycolic acid, malefic acid, malonic acid, oxalic acid,
benzenesulfonic
2o acid, toluenesulfonic acid, methanesulfonic acid, trifluoroacetic acid,
fumaric acid,
succinic acid, malic acid, tartaric acid, citric acid, salicylic acid, p-
aminosalicylic acid,
pamoic acid, benzoic acid, ascorbic acid and the like. Exemplary base addition
salt
forms are the sodium, potassium, calcium salts, and salts with
pharmaceutically
acceptable amines such as, fox example, ammonia, alkylamines, benzathine, and
amino
acids, such as, e.g. arginine and lysine. The term addition salt as used
herein also
comprises solvates which the compounds and salts thereof are able to form,
such as, for
example, hydrates, alcoholates and the like.
The 5-HT2~ receptor agonists and the 5-HT6 receptor antagonists may also be
prodrugs or forms that may release the active ingredient in question after
metabolic
3o tranformation i~ vivo. Conventional procedures for the selection and
preparation of
suitable prodrug derivatives are described, for example, in "Design of
Prodrugs" ed. H.
Bundgaard, Elsevier, 1985.
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WO 02/08178 PCT/SE01/01651
The 5-HT2~ receptor agonists and the 5-HT6 receptor antagonists may be
formulated into various pharmaceutical forms for administrative purposes,
either in the
same pharmaceutical dosage form, such as in the same tablet, or in separate
pharmaceutical dosage forms. In the latter case, however, it may be
advantageous to put
the 5-HT2~ receptor agonist unit dosage form and the 5-HT6 receptor antagonist
unit
dosage form in the same package, for example in the same blister.
The 5-HT2~ receptor agonists and the 5-HT6 receptor antagonists, in the form
of
free bases or salt, can be brought into suitable galenic forms, such as
compositions for
oral use, for injection, for nasal spray administration or the like, in
accordance with
1o accepted pharmaceutical procedures. Such pharmaceutical compositions
according to
the invention comprise an effective amount of a 5-HT2~ receptor agonist and a
5-HT6
receptor antagonist in association with compatible pharmaceutically acceptable
carrier
materials, or diluents, as are well known in the art. The carriers may be any
inert
material, organic or inorganic, suitable for oral, enteral, rectal,
percutaneous,
subcutaneous or parenteral administration, such as: water, gelatin, gum
arabicum,
lactose, microcrystalline cellulose, starch, sodium starch glycolate, calcium
hydrogen
phosphate, magnesium stearate, talcum, colloidal silicon dioxide, and the
like. Such
compositions may also contain other pharmacologically active agents, and
conventional
additives, such as stabilizers, wetting agents, emulsifiers, flavoring agents,
buffers, and
the like.
The compositions according to the invention can e.g. be made up in solid or
liquid form for oral administration, such as tablets, pills, capsules,
powders, syrups,
elixirs, dispersable granules, cachets, suppositories and the like, in the
form of sterile
solutions, suspensions or emulsions for parenteral administration, sprays,
e.g. a nasal
spray, transdermal preparations, e.g. patches, and the like.
The dose level of each of the specific 5-HT2~ receptor agonist and 5-HT6
receptor antagonist, and the frequency of dosage of the specific combination
will vary
depending on a variety of factors including the potency of each specific
compound
employed, the metabolic stability and length of action of that compound, the
patient's
3o age, body weight, general health, sex, diet, mode and time of
administration, rate of
excretion, drug combination, the severity of the condition to be treated). The
daily
dosage may, for example, range from about 0.001 mg to about 150 mg per kilo of
body
weight, preferably from about 0.01 mg to about 100 mg per kilo of body weight,
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WO 02/08178 PCT/SE01/01651
especially from about 0.1 to about 50 mg per kilo of body weight of each of
the 5-HT2C
receptor agonise and of the 5-HT6 receptor antagonist, administered singly or
multiply
in doses, e.g. dosages of from about 0.01 mg to about 1 g each. Usually, such
a
combined dosage is given orally but e.g. parenteral or rectal administration
may also be
chosen. An exemplary tablet combination formulation.may be in the form of
either (A)
two separate tablets, i.e. one tablet containing 10 mg, 20 mg or 50 mg of a 5-
HT2C
receptor agonist, and one tablet containing 10 mg, 20 mg or 50 mg of a 5-HT6
receptor
antagonist; or (B) a combined tablet containing 10 mg, 20 mg or 50 mg of a 5-
HT2C
receptor agonist and 10 mg, 20 mg or 50 mg of a 5-HT6 receptor antagonist.
The invention will now be illustrated further by the following non-limiting
Experimental Section.
EXPERIMENTAL SECTION
A. Preparation of test compounds
The free base of the 5-HT2C receptor agonist (2R)-methyl-1-~3-~~-(3
py~idihyloxy)
ethoxyJ-2 py~azinylJpiperazine, fuma~ate ("PNU-183933F") was prepared as
described
in WO 00/76984. The free base was converted to its fumarate salt, m.p. 126-
129°C. MS
2o mlz 315 (M)f. Anal. (C16H2iN502 ~ C~.H40ø) C, H, N.
The 5-HT6 receptor antagonist 6-methyl-9-(phehylsulfohyl)-1, 2, 3, 4, S, 6
hexahydroazepi~o~4,5-bJindole, hydrochloride ("PNU-186053A") was prepared as
described in WO 01/05793.
The 5-HT~~ receptor agonist (2R)-1-(3-~2-((2-ethoxy-3 pyridinyl)oxyJethoxyJ-2-
py~azihyl)-2-methylpiperazi~e, fuma~ate ("BVT.2938F") was prepared as
described in
WO 00/76984.
The 5-HT6 receptor antagonist 1-(phenylsulfo~cyl)-4-(1 piperazi~yl)-1 H
i~dole,
hydf~ochloj°ide ("BVT.5182C") was prepared as described in Swedish
patent application
No. 0003810-9, filed on October 20, 2000. Briefly, BVT.5182C was prepared
according
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WO 02/08178 PCT/SE01/01651
the general procedure depicted in Scheme 1, below,. starting from commercially
available 4-piperazinoindole (compound 1) that undergoes steps (a) to (c) to
afford 1-
(phenylsulfonyl)-4-(1-piperazinyl)-IH indole, hydrochloride (yield 80%). HPLC
purity
>95%; 1H NMR (DMSO-d~ 8 9.64 (br s, 2 H), 8.00-7.85 (m, 3 H), 7.79 (d, J= 3.77
Hz, 1 H), 7.70-7.65 (m, 1 H), 7.63-7.60 (m, 3 H), 7.27-7.22 (m, 1 H), 6.95 (d,
J= 3.76
Hz, 1 H), 6.81-6.77 (m, 1 H), 3.30-3.20 (m, 4 H); 13C NMR (DMSO-d6) 8144.79,
137.02, 135.22, 134.62, 129.82, 126.85, 125.63, 125.54, 123.49, 111.15,
107.87,
107.76, 47.81, 42.86; MS (posES-FIA) m/z 342 (M+H).
Scheme 1
boc
H boc N N HCI
N N CNJ
C ~ CND
N
/ N
/ v
/ N N O=S
H H O ~ /
1 2 3 4
Step (a): TsOC protection of the pipe~azine N4 nitrogen
4-Piperazinoindole (leq), DMAP (0.1 eq) and Et3N (4 eq) were dissolved in
DMF. (BOC)20 (1.1 eq) was added and the reaction mixture was stirred at room
temperature (I2 h). DMF was evaporated and the residue was purified by
chromatography on silica gel using a mixture of chloroform, methanol and
ammonia as
eluent. HPLC: 100 % purity. MS m/z 302.2 (M+H).
Step (b): Preparation of intermediate 3
The intermediate 2 (1.0 eq) was dissolved in DMF and NaH (1.3 eq) was added
and the suspension was stirred for 0.5 h under nitrogen atmosphere.
Benzenesulfonyl
chloride (1.2 eq) was added and the reaction was stirred overnight at room
temperature.
The volatiles were evaporated. The residue was dissolved in DCM, washed with a
saturated solution of NaHC03, dried (MgS04), filtered and concentrated to give
an oily
residue that was purified by chromatography on silica gel using a mixture of
hexane and
ethylacetate (7:3) as eluent to give tent butyl 4-[1-(benzenesulfonyl)-1H
indol-4-yl)]-1-
to
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WO 02/08178 PCT/SE01/01651
piperazinecarboxylate (3). HPLC 100 %. NMR (1H and 13C) and MS analyses
support
the stated structure.
Step (c): Removal of the BOC protecting group
The BOC group on intermediate 3 was removed by dissolving the compound in
methanol followed by addition of ether saturated with HCl gas. The HCl salt
(4) was
filtered and dried.
B. Preparation of a pharmaceutical composition
Tablet
In ~redients m~/tablet
1. 5-HT2~ receptor agonist 10.0
2. 5-HT6 receptor antagonist10.0
3. Cellulose, microcrystalline57.0
4. Calcium hydrogen phosphate15.0
5. Sodium starch glycolate 5.0
6. Silicon dioxide, colloidal0.25
7. Magnesium stearate 0.75
The active ingredients 1 and 2 are mixed with ingredients 3, 4, 5 and 6 for
about 10
minutes. The magnesium stearate (7) is then added, and the resultant mixture
is mixed
for about 5 minutes and compressed into tablet form with or without film-
coating.
C. Receptor affinity and efficacy assays
S HT~~ receptor affi~zity assay
5-HT2~ receptor affinity is determined in competition experiments, where the
ability of a compound in serial dilution to displace 3H-labeled 5-HT, bound to
3o membranes prepared from a transfected HEK293 cell line stably expressing
the human
5-HT2c receptor protein, is monitored by Scintillation Proximity Assay (SPA)
technology. Non-specific binding is defined using 5 ~.M mianserin.
11
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WO 02/08178 PCT/SE01/01651
S HT2~ receptor affinity assay
5-HT2A receptor affinity is determined in competition experiments, where the
ability of a compound in serial dilution to displace 3H-labeled ketanserin or
lysergic
acid diethylamide (LSD), bound to membranes prepared from a transfected CHO
cell
line stably expressing the human 5-HT2A receptor protein, is monitored by
measuring
the radioactivity of filtered membrane homogenates on glass fiber filters in a
scintillation counter. Non-specific binding is defined using 5 ~.M mianserin.
5 HT2B receptor affinity assay
IO 5-HT~B receptor affinity is determined in competition experiments, where
the
ability of a compound in serial dilution to displace 3H-labeled 5-HT, bound to
membranes prepared from a transfected CHO cell line stably expressing the
human 5-
HT~B receptor protein, is monitored by Scintillation Proximity Assay (SPA)
technology. Non-specific binding is defined using 5 ~.M mianserin.
S HT2C receptor efficacy assay
The agonist efficacy at the 5-HT2C receptor is determined by the ability of a
compound to mobilise intracellular calcium in transfected HEI~2.93 cells,
stably
expressing the human 5-HT2~ receptor protein, using the calcium-chelating
fluorescent
dye FLUO-3 (Sigma, St. Louis, MO, U.S.A.). Relative efficacy (%) is measured
relative
to that of serotonin at 1 ELM.
S HT6 receptor affinity assay
The radioligand binding assay uses [3H]-lysergic acid diethylamide (LSD). The
assay is carried out in 96-well sample plates by the addition of 11 ~,I of the
test
compound at the appropriate dilution (the assay employs 11 serial
concentrations of
samples run in duplicate), 11 ~Cl of radioligand, and 178 ~,l of a washed
mixture of
WGA-coated SPA beads and membranes in binding buffer prepared from HEK293-
cells containing cloned human 5-HT6 receptor. The plates are shaken for about
5
3o minutes and then incubated at room temperature for 1 hour. The plates are
then loaded
into counting cassettes and counted in a scintillation counter. The
specifically bound
cpm obtained are fit to a one-site binding model using GraphPad Prism ver.
2Ø
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WO 02/08178 PCT/SE01/01651
Estimated ICso values are converted to Ki (affinity constant) values using the
Cheng-
Prusoff equation (Cheng, Y. C. et al., Biochem. Pha~macol. 1973, ~2, 3099-
3108).
HT6 f~eceptor efficacy assay
5 The antagonist potency at the 5-HT6 receptor is determined by the ability of
a
compound to antagonize the increase in cAMP induced by 5-HT in HEK293 cells,
stably expressing the human 5-HTg receptor protein, using a cAMP SPA direct
screening assay system (RPA559, Amersham Pharmacia Biotech, Uppsala, Sweden).
D. Food intake test
Test compounds
5-HT2~ receptor agonists (2R)-methyl-1-{3-[2-(3-pyridinyloxy)ethoxy]-2-
pyrazinyl}piperazine, fumarate ("PNU-183933F") and (2R)-1-(3-{2-[(2-ethoxy-3-
pyridinyl)oxy]ethoxy}-2-pyrazinyl)-2-methylpiperazine, fumarate ("BVT.2938F")
were
dissolved in saline (0.9% NaCI) and diluted in the same vehicle to the
appropriate
concentration.
5-HT6 receptor antagonists 6-methyl-9-(phenylsulfonyl)-1,2,3,4,5,6-
hexahydroazepino[4,5-b]indole, hydrochloride ("PNU-186053A") and 1-
(phenylsulfonyl)-4-(I-piperazinyl)-1H indole, hydrochloride (5-HT6 receptor
antagonist ("BVT.5182C") were dissolved and diluted in 25% cyclodextrin.
Fresh solutions were prepared on the day of treatment.
Animals
Male mice 8-9 weeks old (C57BL/6JBom-Lep°b (ob/ob), Bomholtsgaard,
Denmark) with an average body weight of 45 g were used. The animals were
housed
singly in cages at 23~1°C, 40-60% humidity and had free access to water
and standard
laboratory chow. The 12112 h light/dark cycle was set to lights off at 5 p.m.
The
animals were conditioned for at least one week before start of study. During
experimental sessions, the animals obtained special chow (BioServ, Frenchtown,
NJ,
USA dust-free precision pellets weighing 20 mg each).
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Experinaehtal section
At the start of the study the animals were transferred to special cages
"operant
test cages" (Habitest Modular Animal Behavior Test System; Colbourn Instr,
Allentown, PA, USA). These cages consist of a feeder trough with sensors for
measurement of food intake, an optic lickometer for registration of water
intake and an
infrared-based monitor for recording overall general motor activity. The
monitors are
coupled to a computer, which controls and monitor events continuously. Food
pellets
were weighed to the amount needed for one whole study and water bottles were
filled
with fresh tap water and weighed. The animals were conditioned to their new
to environment for three days to establish baseline values. The animals were
weighed at 3
p.m. at the start and at the end of the study. The compounds were administered
between
4.20 and 5.00 p.m. before dark onset. Three groups of animals received (i) 5-
HT6
antagonist in 25% cyclodextrin; (ii) 5-HT2~ agonist in saline; and (iii) the
combination .
5-HT2~ agonist/5-HT6 antagonist, respectively. When combined, 5-HT6 antagonist
or
saline was administered 30 min before administration of the 5-HT2~ agonist or
25%
cyclodextrin. A fourth group received respectively vehicle administered in the
same
way. The study ended on the fifth day. Weighing was performed with a computer-
assisted Mettler-Toledo PR5002/PR802 balance.
2o Evaluation of results
Each dose group consisted of 12-16 animals. Data were corrected for food
spillage based on the weighed spillage during 22 hours and assumed to be
proportional
over time. Calculations were performed for the data before and after
treatment. The
values were expressed as % of basal food intake (mean ~ SEM) for the
difference
between food intake before treatment and 3 h (5 pm - 8 pm), 6 h (5 pm - 11
pm), 12 h
(5 pm - 5 am), 21 h (5 pm - 2 pm).
The results shown in Fig. 1 indicate that combined treatment with the 5-HT6
receptor antagonist "PNU-186053A" (50 mg/kg subcutaneously) and the 5-HT2C
receptor agonist "PNLJ-153933F" (50 mg/kg per orally) decreased food
consumption
3o significantly more than the compounds given alone. Correspondingly, the
results shown
in Fig. 2 indicate that combined treatment with the 5-HT2C receptor agonist
"BVT.2938F" (5 mg/kg subcutaneously) and the 5-HT6 receptor antagonist
"BVT.5182C" (3 mg/kg subcutaneously) decreased food consumption, at 12 and 21
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WO 02/08178 PCT/SE01/01651
hours following administration, significantly more than the compounds given
alone.
Thus, it is apparent that combined therapy with a 5-HT~,C receptor agonist and
a 5-HT6
receptor antagonist reduces food intake more efficiently as compared to
treatment with
either agonist or antagonist alone.
