U.S. patent application number 13/679656 was filed with the patent office on 2013-03-21 for treatment of parkinson's disease and enhancement of dopamine signal using pde 10 inhibitor.
This patent application is currently assigned to MITSUBISHI TANABE PHARMA COPORATION. The applicant listed for this patent is MITSUBISHI TANABE PHARMA COPORATION. Invention is credited to Taketoshi ISHII, Takeo KITAZAWA, Jun KOTERA, Hiroshi MORIMOTO, Takashi SASAKI, Harutami YAMADA.
Application Number | 20130072477 13/679656 |
Document ID | / |
Family ID | 47359665 |
Filed Date | 2013-03-21 |
United States Patent
Application |
20130072477 |
Kind Code |
A1 |
KOTERA; Jun ; et
al. |
March 21, 2013 |
TREATMENT OF PARKINSON'S DISEASE AND ENHANCEMENT OF DOPAMINE SIGNAL
USING PDE 10 INHIBITOR
Abstract
The present invention relates to a therapeutic or prophylactic
method for treating Parkinson's disease by administering an
effective amount of a compound having a phosphodiesterase 10
inhibitory activity; and also relates to a pharmaceutical
composition for treatment or prophylaxis of Parkinson's disease
comprising as an active ingredient a compound having a
phosphodiesterase 10 inhibitory activity. Moreover, the present
invention relates to a method for enhancing dopamine signal in the
brain, which comprises administering an effective amount of a
compound having a phosphodiesterase 10 inhibitory activity; and
also relates to pharmaceutical composition for enhancing dopamine
signal in brain comprising as an active ingredient a compound
having a phosphodiesterase 10 inhibitory activity.
Inventors: |
KOTERA; Jun; (Hasuda-shi,
JP) ; SASAKI; Takashi; (Kawaguchi-shi, JP) ;
KITAZAWA; Takeo; (Ichikawa-shi, JP) ; ISHII;
Taketoshi; (Tokyo, JP) ; MORIMOTO; Hiroshi;
(Amagasaki-shi, JP) ; YAMADA; Harutami;
(Hasuda-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MITSUBISHI TANABE PHARMA COPORATION; |
Osaka-shi |
|
JP |
|
|
Assignee: |
MITSUBISHI TANABE PHARMA
COPORATION
Osaka-shi
JP
|
Family ID: |
47359665 |
Appl. No.: |
13/679656 |
Filed: |
November 16, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12647880 |
Dec 28, 2009 |
8338420 |
|
|
13679656 |
|
|
|
|
10726634 |
Dec 4, 2003 |
|
|
|
12647880 |
|
|
|
|
Current U.S.
Class: |
514/220 ;
514/221; 514/250; 514/266.23; 514/455; 514/567; 514/614;
514/654 |
Current CPC
Class: |
A61K 31/517 20130101;
A61K 31/55 20130101; A61K 31/137 20130101; A61K 31/4985 20130101;
A61K 31/517 20130101; A61K 31/48 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/198 20130101;
A61K 31/165 20130101; A61K 31/48 20130101; C07D 403/14 20130101;
A61K 31/198 20130101; A61K 31/352 20130101 |
Class at
Publication: |
514/220 ;
514/455; 514/654; 514/221; 514/614; 514/567; 514/250;
514/266.23 |
International
Class: |
A61K 31/198 20060101
A61K031/198; A61K 31/137 20060101 A61K031/137; A61K 31/517 20060101
A61K031/517; A61K 31/165 20060101 A61K031/165; A61K 31/4985
20060101 A61K031/4985; A61K 31/352 20060101 A61K031/352; A61K 31/55
20060101 A61K031/55 |
Claims
1. A method for enhancing dopamine signals in the brain, which
comprises administering to a patient of Parkinson's disease an
effective amount of a compound having a phosphodiesterase 10
inhibitory activity together with inducing dopamine signals by
L-dopa administration or by administration of a dopamine receptor
agonist, and thereby enhancing dopamine signals in the brain of
said patient of Parkinson's disease; wherein the compound having
phosphodiesterase 10 inhibitory activity is not: a non-specific PDE
inhibitor selected from caffeine, IBMX, theophyllamine,
dipyridamole, papaverine, and theophylline; a PDE5 inhibitor
sildenafil; or a PDE4 inhibitor rolipram.
2. The method according to claim 1, wherein the compound having a
phosphodiesterase 10 inhibitory activity has the following
characteristics: 1) IC.sub.50 value is 300 nM or less as measured
under condition of a substrate cAMP concentration of 0.25 .mu.M, or
2) Ki value for phosphodiesterase 10 is 150 nM or less.
3. The method according to claim 1, wherein the compound having a
phosphodiesterase 10 inhibitory activity is characterized by its
inhibitory activity or affinity for phosphodiesterase 10 being 100
times or more as high, as compared to its inhibitory activity or
affinity for phosphodiesterases 1, 3, 4, 5 and 6.
4. The method according to claim 1 or 3, wherein the dopamine
signals are enhanced in a part of the brain, selected from the
group consisting of corpus striatum, nucleus accumbens, olfactory
tubercle and frontal lobe.
5. The method according to claim 1 or 3, wherein the dopamine
signals are enhanced in the corpus striatum.
6. The method according to claim 1 or 3, wherein the dopamine
signals are those induced by L-dopa administration.
7. The method according to claim 1 or 3, wherein the dopamine
signals are those induced by administration of a dopamine receptor
agonist.
8. The method according to claim 1 or 3, wherein the dopamine
signals are those induced by administration of a dopamine type 1
receptor agonist.
9. The method according to claim 1 or 3, wherein the dopamine
signals are those induced by administration of a dopamine type 2
receptor agonist.
10. The method according to claim 1 or 3, wherein the dopamine
signals are those induced by administration of a dopamine type 2
receptor selective agonist.
11. The method according to claim 1 or 3, wherein the dopamine
signals are those induced by administration of bromocriptine.
12. The method according to claim 1 or 3, wherein the compound
having a phosphodiesterase 10 inhibitory activity has the following
characteristics: 1) IC.sub.50 value is 10 nM or less as measured
under condition of a substrate cAMP concentration of 0.25 .mu.M, or
2) Ki value for phosphodiesterase 10 is 5 nM or less.
Description
[0001] The present application is a 37 C.F.R. .sctn.1.53 (b)
divisional of, and claims priority to, U.S. application Ser. No.
12/647, 880 filed Dec. 28, 2009. Application Ser. No. 12/647,880 is
a Continuation of Ser. No. 10/726,634 (now abandoned) filed Dec. 4,
2003, which application claimed the benefit of U.S. provisional
application No. 60/430,641, filed Dec. 4, 2002, U.S. provisional
application No. 60/430,642, filed Dec. 4, 2002, and U.S.
provisional application No. 60/488,375, filed Jul. 21, 2003. The
entire contents of each of these applications is hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a novel method for
treatment or prophylaxis of Parkinson's disease. The present
invention also relates to a novel agent for treatment or
prophylaxis of Parkinson's disease. Further, the present invention
relates to a novel method for enhancing dopamine signals in the
brain.
[0004] 2. Prior Art
[0005] Parkinson's disease (also referred to as PD) is a
progressive degenerate disorder of the central nervous system,
characterized by symptoms such as tremor, rigidity, akinesia,
postural reflex abnormalities (postural disturbance), etc.
[0006] With a partial exception of juvenile Parkinsonism,
development of PD is frequently seen in 40's or 50's or later, and
it is a disease of a high incidence, affecting one out of 2000
people (in the age of 65 or older, one out of 500 people).
[0007] Although neurons in the brain are lost and degenerated with
aging even in a normal case, in a patient of the Parkinson's
disease, significant loss and degeneration of neurons are observed
in the substantia nigra of the midbrain, beyond a rate of normal
aging. Therefore, it is thought that various kinds of symptoms of
Parkinson's disease are caused due to decreasing of dopamine, which
is one of the neurotransmitters produced in the substantia nigra of
the midbrain.
[0008] Dopamine is involved in regulation of postural maintenance
and speed of movement, and when an amount of dopamine produced is
decreased, the symptoms of Parkinson's disease develops. It is
known that dopamine decrease and development of symptoms have a
close relationship. Further, when dopamine is decreased, a balance
is lost between dopamine and acetylcholine, which is another
neurotransmitter, leading to the symptoms of Parkinson's
disease.
[0009] As stated above, Parkinson's disease is a representative
disorder of the central nervous system, whose symptoms is expected
to be alleviated by enhancement of dopamine signals in the brain,
and as a therapeutic agent therefore, L-dopa, a dopamine precursor,
etc. is employed.
[0010] Parkinson's disease and a treatment method thereof are
summarized in review by Marsden et al. and Calne et al. (Marsden,
Lancet, 1990, pp. 948-952; Calne, New England Journal of Medicine,
1993, vol. 329, pp. 1021-1027).
[0011] Among the therapeutic agents for Parkinson's disease, L-dopa
is the most frequently used therapeutic agent. L-dopa is a
precursor of dopamine, and L-dopa therapy is aimed to supplement
dopamine, which is decreased in the substantia nigra-corpus
striatum. Since dopamine does not cross the blood brain barrier
(BBB), it will not reach the brain if it is administered into a
circulating blood. However, L-dopa does cross the blood brain
barrier (BBB), and it is metabolized by dopa decarboxylase in the
brain to give dopamine, which in turn acts on a dopaminergic
receptor.
[0012] However, a long-termed administration of L-dopa causes
complications such as dyskinesia, etc. Further, it has a problem of
instability such as wearing-off phenomenon, on-off phenomenon,
etc., or problem of showing unexpected reaction or involuntary
movements.
[0013] Under such circumstances, there have been needs for
developing a new excellent therapeutic agent for Parkinson's
disease, or a new concomitant agent to avoid a high-dose
administration of L-dopa.
[0014] Meanwhile, the following facts have been known, regarding a
cyclic nucleotide phosphodiesterase of mammals. The cyclic
nucleotide phosphodiesterase (hereinafter simply abbreviated as
phosphodiesterase or PDE) is an enzyme that hydrolyzes a
phosphodiester bond in a cyclic nucleotide such as cAMP (adenosine
3',5'-cyclic monophosphate) or cGMP (guanosine 3',5'-cyclic
monophosphate), etc. as a substrate, to generate nucleotides such
as 5'AMP (adenosine 5'-monophosphate) or 5'GMP (guanosine
5'-monophosphate), etc.
[0015] Cyclic nucleotides such as cAMP, cGMP, etc. are involved in
regulations of many in vivo functions as the second messenger in
the intracellular signal transduction. Intracellular concentrations
of the cAMP and cGMP, changing in response to an extracellular
signal, are regulated by a balance between an enzyme involved in a
synthesis of cAMP and cGMP (adenylate cyclase and guanylate
cyclase), and phosphodiesterase (PDE) involved in a hydrolysis
thereof.
[0016] Until recently, many kinds of the phosphodiesterases have
been isolated and identified in mammals, and they have been
classified into plural types, according to homology of amino acid
sequence, biochemical properties, characterization by an inhibitor,
etc. (Beavo, Physiol. Rev., Vol. 75, pp. 725-748, 1995).
[0017] For example, PDE1 is Ca.sup.2+/calmodulin dependent PDE and
hydrolyses both cAMP and cGMP. PDE2 is activated by cGMP and
hydrolyses both cAMP and cGMP. PDE classified as PDE3 is inhibited
by cGMP. PDE4 specifically recognizes cAMP as a substrate, and is
rolipram-sensitive. PDE5 specifically recognizes cGMP as a
substrate. PDE6 is a photoreceptor cGMP-PDE. PDE7 specifically
recognizes cAMP as a substrate, and is not sensitive to
rolipram.
[0018] Further recently, existences of 4 kinds of novel types of
PDE have been reported. PDE8 specifically recognizes cAMP as a
substrate, and PDE9 specifically recognizes cGMP as a substrate.
Both of PDE8 and PDE9 are reported to be insensitive to IBMX
(3-isobutyl-1-methylxanthine), which is known to be a non-selective
PDE inhibitor. Further, PDE11 recognizes both cAMP and cGMP as a
substrate.
[0019] Regarding PDE10, the followings have been known. Although
PDE10 (PDE10A) recognizes both cAMP and cGMP as a substrate, it has
been reported to have a stronger affinity for cAMP. Further, cDNAs
of human, mouse and rat PDE10A have been isolated and identified.
Still further, existence of the PDE10 protein has been confirmed in
rat (Fujishige et al., J. Biol. Chem., Vol. 274, pp. 18438-18445,
1999; Kotera et al., Biochem. Biophys. Res. Commun., Vol. 261, pp.
551-557, 1999; Soderling, et al., Proc. Natl. Acad. Sci. USA, vol.
96, pp. 7071-7076, 1999; Loughley, et al., Gene, vol. 234, pp.
109-117, 1999)
[0020] As a PDE10 inhibitor, the followings have been known.
WO02/48144 (Bayer) discloses a pyrrolo[2,1-a]dihydro-isoquinoline
compound having a PDE10 inhibitory activity. It is also described
that these compounds with the PDE10 inhibitory activity show
antiproliferative activity, and can be used as an anticancer agent.
Further, it is also described they can be used for treatment of
pains and/or for alleviating a fever in a state of having a fever.
Further, in WO01/24781 (NovaNeuron), application of a modulator of
PDE10 (PDE10A) for Huntington's disease is disclosed.
[0021] The followings have been known regarding a relationship
between Parkinson's disease and the phosphodiesterase inhibitors.
U.S. Pat. No. 4,147,789 (Sandoz) and U.S. Pat. No. 3,961,060 (Astra
Lakemedal) disclose an application of non-specific PDE inhibitors
such as caffeine, theophylline, etc., for treatment of Parkinson's
disease, together with dopaminergic stimulants.
[0022] WO01/78711 (ICOS) discloses an application of a compound
having an inhibitory activity on a cGMP specific PDE (PDE5) for
treatment of Parkinson's disease.
[0023] WO01/32170 (Swope) discloses an application of PDE inhibitor
such as sildenafil for a neurological symptom including Parkinson's
disease, etc.
[0024] Hussain et al. discloses an application of sildenafil, which
is PDE5 inhibitor and a medicament for erectile dysfunction for
sexual dysfunction of patients of Parkinson's disease (Hussain et
al., Journal of Neurology, Nuerosurgery and Psychiatry, 2001, vol.
71, pp. 371-374).
[0025] Swope et al. discloses an application of sildenafil for
treatment of dyskinesia in Parkinson's disease (Swope, et al.,
Neurology, 2000, vol. 54, No. 7, pp. A90-A91).
[0026] In Dicki et al. and Hulley et al., it is described that a
PDE4 inhibitor (Ro20-1724, etc.) is thought to show a protective
activity on cells against neurotoxins (MPP+, MPTP, etc.) (Dicki et
al., Brain Research, 1997, vol. 753, pp. 335-339; and Hulley et
al., Eur. J. Neuroscience, 1995, vol. 7, pp. 2431-2440).
[0027] In Kakkar et al., it is described that deprenyl (MAO-B
inhibitor) and amantadine, known therapeutic agents for Parkinson's
disease are thought to show an inhibitory activity against a
calmodulin dependent PDE (PDE1) (Kakkar et al., Brain Research,
1997, vol. 749, pp. 290-294; and Kakkar et al., Life Sciences,
1996, vol. 59, PL337-341).
[0028] Fredholm et al. describes that the activity of L-dopa is
enhanced by application of PDE inhibitors (caffeine, IBMX,
theophyllamine, dipyridamole, etc.) to an animal model (Fredholm et
al., European Journal of pharmacology, 1976, vol. 38, pp.
31-38).
[0029] Waldeck reports that caffeine affects an activity of
dopamine, and also suggests that this may be based on a PDE
inhibitory activity that caffeine has (Waldeck, Acta Pharmacol.
Toxicol. Suppl., 1975, vol. 36, pp. 1-23).
[0030] However, it has not been known, until today, to apply PDE10
inhibitor for treatment of Parkinson's disease.
[0031] Further, as regards to a dopaminergic receptor, the
following facts have been known. It has been known that several
kinds of dopaminergic receptors exist, and that dopamine type 1
receptor (D1-R) and dopamine type 2 receptor (D2-R) are mainly
expressed in the brain.
[0032] It has been clearly known that Dopamine type 1 receptor
(D1-R) conjugates with Gs (Gs.alpha.) of G protein and conjugates
promotingly with an adenylate cyclase activity. On the other hand,
although dopamine type 2 receptor (D2-R) is said to inhibitingly
conjugate with adenylate cyclase, there is another theory, and
certain points are remained unclear.
[0033] An object of the present invention is to provide a novel
method for treatment or prophylaxis of Parkinson's disease. Another
object is to provide a novel pharmaceutical composition for
treatment or prophylaxis of Parkinson's disease. Still further
object of the present invention is to provide a novel method or
agent for enhancing dopamine signals in the brain in vivo. Objects
other than those above are clear from the following
descriptions.
SUMMARY OF THE INVENTION
[0034] The present inventors have found out that a compound having
an inhibitory activity on phosphodiesterase 10 has an effect of
enhancing an activity caused by L-dopa administration in
Parkinson's disease model animals, that it can be used for
alleviating the symptoms specific for Parkinson's disease, and that
it enhances dopamine signals in the brain, whereby the present
invention has completed.
[0035] The present invention relates to a therapeutic or
prophylactic method for treating Parkinson's disease by
administering to a patient an effective amount of a compound having
a phosphodiesterase 10 inhibitory activity. Further the present
invention relates to a pharmaceutical composition for treatment or
prophylaxis of Parkinson's disease comprising as an active
ingredient a compound having a phosphodiesterase 10 inhibitory
activity. Still further, the present invention relates to a method
for enhancing dopamine signals in the brain, which comprises
administering to a patient an effective amount of a compound having
a phosphodiesterase 10 inhibitory activity. Yet further, the
present invention relates to a pharmaceutical composition for
enhancing dopamine signals in the brain, specifically in vivo,
comprising as an active ingredient a compound having a
phosphodiesterase 10 inhibitory activity. Further, the present
invention relates to the use of a compound having a
phosphodiesterase 10 inhibitory activity for preparation of a
medicament for treating Parkinson's disease, or for enhancing
dopamine signals in vivo.
[0036] (Hereinafter, the compound having a phosphodiesterase 10
inhibitory activity is also referred to as "PDE10 inhibitor".)
[0037] It is thought that action mechanism for the pharmaceutical
or the therapeutic method of the present invention is outlined as
follows. Accordingly, the PDE10 inhibitor as an active ingredient
acts on neurons expressing dopaminergic receptors in the brain,
inhibiting an activity of PDE10, thereby suppressing decomposition
of the second messenger, cAMP. It is thought that, through this
action, signals caused by stimulation of the dopaminergic receptor
(hereinafter referred to as dopamine signals) can be enhanced, and
an effect of alleviating symptoms of Parkinson's disease is
exhibited.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0038] Specific examples of the compound having a phosphodiesterase
10 inhibitory activity include pyrrolo[2,1-a]dihydroisoquinoline
compounds included in the following formula:
##STR00001##
wherein [0039] x and y independently from each other denote zero or
1 with the proviso that x+y=1 or 2; [0040] R.sup.1 and R.sup.2
independently from each other denote hydrogen, C.sub.1-4-alkyl or
CF.sub.3 or [0041] R.sup.1 and R.sup.2 together form a
C.sub.1-4-alkylene bridge; [0042] R.sup.3 and R.sup.4 independently
from each other denote C.sub.1-4-alkyl; [0043] R.sup.5 denotes
C.sub.6-14-aryl, optionally having 1 to 3 further substituents
selected from the group consisting of halogen; [0044]
C.sub.1-6-alkyl which can be further substituted with one or more
radicals selected from the group consisting of OH, halogen,
NH.sub.2 and C.sub.1-6-alkoxy; [0045] C.sub.1-6-alkoxy which can be
further substituted with one or more radicals selected from the
group consisting of OH, halogen, NH.sub.2, C.sub.1-6-alkoxy, and
C.sub.6-10-aryloxy; OH; [0046] NO.sub.2; [0047] CN; [0048]
CF.sub.3; [0049] OCF.sub.3; [0050] NR.sup.6R.sup.7; [0051]
SR.sup.8; [0052] --O--(CH.sub.2).sub.1-4--O-- wherein the oxygen
atoms are bound to the aryl moiety in ortho-position to each other;
[0053] phenyloxy or benzyloxy wherein the phenyl moieties
optionally contain one further substituent selected from the group
consisting of C.sub.1-6-alkyl, C.sub.1-6-alkoxy, halogen and
NO.sub.2; [0054] phenyl, optionally substituted with CN; and 4- to
9-membered aromatic heterocyclic ring containing 1 to 4 heteroatoms
selected from the group consisting of N, O, and S; [0055] R.sup.6
and R.sup.7 independently from each other denote hydrogen,
C.sub.1-6-alkyl, or together with the nitrogen atom to which they
are attached, form a 5- to 7-membered saturated, partially
unsaturated or aromatic ring which can contain up to 3 further
hetero atoms selected from the group consisting of N, O, and S, and
which ring can contain 1 to 3 substituents selected from the group
consisting of C.sub.1-6-alkyl, C.sub.1-6-alkoxy, C.sub.6-10-aryl
and 4- to 9-membered aromatic heterocyclic ring containing 1 to 4
hetero atoms selected from the group consisting of N, O, and S; and
[0056] R.sup.8 denotes hydrogen, C.sub.1-6-alkyl or
C.sub.6-10-aryl-C.sub.1-6-alkyl; with the proviso that
8,9-dimethoxy-3-methyl-2-phenyl-5,6-dihydro-pyrrolo[2,1-a]isoquinoline-1--
carboxylic acid ethyl ester is excluded, [0057] and a
pharmaceutically acceptable salt thereof, etc., specifically
disclosed in WO02/48144 (incorporated herein by reference).
[0058] Other than the above, as the compound having a
phosphodiesterase 10 inhibitory activity, there are mentioned, for
example, Compound Y and Compound Z, which are pyrimidine
derivatives represented by the following formulae, and
pharmaceutically acceptable salts thereof. These compounds also
have a PDE5 (PDEV) inhibitory activity, as well as a PDE10
inhibitory activity.
[0059] Compound Y and Compound Z and pharmaceutically acceptable
salts thereof, and derivatives thereof can be prepared according to
a method disclosed in WO01-83460 (incorporated herein by reference)
(Examples 56 and 128), optionally in combination of conventionally
known methods.
##STR00002##
[0060] Besides the above, as the compounds having a PDE10
inhibitory activity, there are mentioned, for example, Compound A,
a pyrimidine derivative represented by the following formula and a
pharmaceutically acceptable salt thereof. Compound A shows a strong
inhibitory activity on PDE10, and it acts highly specifically on
PDE10, showing 100 times or more as strong inhibitory activity on
PDE10 (IC.sub.50 value of 1/100 or less), as compared to inhibitory
activities on other PDE families (for example, PDE1, 2, 3, 4, 5, 6,
7, 8, 9 and 11) Compound A and a pharmaceutically acceptable salt
thereof can be prepared according to the below-described
Preparation Examples, optionally in combination of the
conventionally known methods.
##STR00003##
[0061] Compounds having a PDE10 inhibitory activity (hereinafter
referred to as PDE10 inhibitors) are not limited to the
above-mentioned specific examples. The compounds having a PDE10
inhibitory activity can be suitably selected and identified from
natural compounds widely existing in the nature and newly
synthesized compounds.
[0062] In the pharmaceutical composition and the therapeutic method
of the present invention, PDE10 inhibitor to be used as an active
ingredient is expected to have a strong inhibitory activity and
affinity for PDE10, for the purpose of obtaining a stronger potency
in terms of medicinal efficacy.
[0063] As such compounds, there may be mentioned, for example, a
compound showing IC.sub.50 value of normally 1 .mu.M or less,
preferably 300 nM or less, more preferably 100 nM or less, and
particularly preferably 30 nM or less, and further preferably 10 nM
or less, when measured in the same manner as in the below-described
Example 1, under the condition of substrate cAMP concentration of
0.25
[0064] Or, there are mentioned a compound having Ki value
(inhibition constant) for PDE10 of normally 500 nM or less,
preferably 150 nM or less, more preferably 50 nM or less,
particularly preferably 15 nM or less, and further preferably 5 nM
or less. Further, in the pharmaceutical composition and the
therapeutic method of the present invention, PDE10 inhibitor to be
used as an active ingredient is expected to have a high selectivity
for PDE10, for the purpose of minimizing side effects.
[0065] As such compound with high selectivity, there may be
mentioned, for example, among the above-mentioned compounds having
a strong inhibitory activity or affinity for PDE10, compounds
having an inhibitory activity or affinity for PDE10 of normally 3
times or more, preferably 10 times or more, more preferably 30
times or more, and particularly preferably 100 times or more as
high, as compared to inhibitory activities or affinities for PDE
families other than PDE10 (ex., PDE 1, 3, 4, 5 and 6, etc. which
are suggested to be involved in physiological activities).
[0066] For example, if IC.sub.50 value (or Ki value) is taken as an
index, the IC.sub.50 value (or Ki value) as measured for PDE10 is
normally 1/3 or less, preferably 1/10 or less, more preferably 1/30
or less, and particularly preferably 1/100 or less, as compared
with the IC.sub.50 value (or Ki value) as measured for the PDE
families other than PDE10 (PDE 1, 3, 4, 5 and 6, etc.).
[0067] Selection and identification of the PDE10 inhibitors and
measurements of inhibitory activities thereof can be carried out in
the similar method as described below in Example 1 or
conventionally known method as disclosed in literatures (for
example, Fujishige et al., Eur. J. Biochem., vol. 266, pp.
1118-1127, 1999, etc.).
[0068] Further, selectivity of the PDE10 inhibitor may be
evaluated, for example, by employing the similar method described
in Example 2 below or the conventionally known method disclosed in
the literatures (for example, Kotera et al., Biochem. Pharmacol.,
vol. 60, pp. 1333-1341, 2000; Sasaki et al., Biochem. Biophys. Res.
Commun., vol. 271, pp. 575-583, 2000; Yuasa et al., Journal of
Biological Chemistry, vol. 275, pp. 31469-31479, 2000, etc.), and
comparing inhibitory activities (or affinities (Ki value)) as
measured for PDE10 and for other PDE families. Through this method,
it is possible to select and identify the compound having selective
inhibitory activity for PDE10.
[0069] Still further, in the pharmaceutical composition and the
therapeutic method of the present invention, PDE10 inhibitor
exhibits its activity in the brain. Therefore, PDE10 inhibitor as
an active ingredient is expected to have good brain uptake and
penetration characteristics and show an intracerebral PDE10 binding
kinetics which enables sustained inhibition of PDE10 in the
brain.
[0070] Different from other tissue, the brain has the blood brain
barrier (BBB) to restrict and regulate the movement of molecules
between the blood and the brain. BBB consists of brain capillary
endothelial cells. The brain capillary endothelial cells are
connected through tight junction, and nutrients and drugs in the
blood are required to pass transcellularly through the brain
capillary endothelial cells. As a mechanism for the drugs to pass
through the BBB, there are mentioned the following five routes; 1)
passive diffusion, 2) carrier-mediated transport, 3)
receptor-mediated transcytosis, 4) absorptive-mediated transcytosis
and 5) active efflux transport by P-glycoprotein. The rate of the
compound to pass through the BBB and enter the brain (PS) via one
or plurals of these mechanisms can be measured by known in vivo
experimental assays such as in situ brain perfusion, integration
plot method, etc., or by the known in vitro experimental assays
such as primary cultured brain capillary endothelial cells, or an
established immortalized brain capillary endothelial cell line,
etc. Further, intracerebral PDE10 binding kinetics of the compound
can be confirmed by pharmacokinetics (PK) analysis and
pharmacodynamics (PD) analysis.
[0071] In the present invention, PDE10 inhibitor to be used as an
active ingredient may be in a free form, or when it can form a
salt, it may be in a pharmaceutically acceptable salt. Examples of
the pharmaceutically acceptable salt include an inorganic acid salt
such as hydrochloride, sulfate, phosphate or hydrobromide, and an
organic acid salt such as acetate, fumarate, oxalate, citrate,
methanesulfonate, benzenesulfonate, tosylate or maleate, etc. In
addition, in case that a compound has a substituent (s) such as
carboxyl group, a salt with a base (for example, an alkali metal
salt such as a sodium salt, a potassium salt, etc., or an
alkaline-earth metal salt such as a calcium salt and the like) may
be mentioned.
[0072] In the pharmaceutical composition or the therapeutic method
of the present invention, administration route is not particularly
limited, and generally employed oral or parenteral administration
method (intravenous, intramuscular, subcutaneous, transdermal,
transnasal, other transmucosal, enteral administration, etc.) can
be applied.
[0073] Further, in case of using a compound which does not have
good brain uptake and penetration characteristics, a method can be
applied in which a pharmaceutical composition is directly or
indirectly introduced into the brain, bypassing the BBB. Examples
for the direct method include a method in which the compound is
administered through catheter placed in the brain of a patient.
More particularly, for example, there may be mentioned methods
including intracerebroventricular (i.c.v.) administration, and a
direct administration in corpus striatum. Such direct
administration methods into the brain are in many case restricted
in terms of invasiveness, when applied to treatment of human being,
however, it is optionally be employed in evaluation for
experimental animals, etc. As the indirect method, there are
mentioned a method of converting a normally water-soluble drug into
an fat-soluble drug or a prodrug (for example, by closing of
hydroxyl, carbonyl, sulfate and primary amine groups, etc.), an
administration method accompanying intravenous injection of
hypertonic solution which enables temporary opening of the BBB
(osmotic opening), etc.
[0074] In the pharmaceutical composition or the therapeutic method
of the present invention, the active ingredient can be prepared
into commonly used pharmaceutical preparations (a tablet, granule,
capsule, powder, injection solution and inhalant, etc.), together
with an inactive carrier, selected depending on an administration
method. Examples of such carriers include a medical additive
acceptable for general pharmaceuticals such as a diluent, binder,
disintegrant, excipient and lubricant, etc.
[0075] In addition, in the pharmaceutical composition or the
therapeutic method of the present invention, dose amount of the
active ingredient may be optionally chosen, depending on potencies
or properties of PDE10 inhibitors as an active ingredient, from a
dose range which is effective enough for exhibiting a drug
efficacy. The dose may vary depending on an administration route,
age, bodyweight, and condition of a patient, and it is suitably
selected from a generally used dose range, for example, a range of
0.01 to 300 mg/kg per day.
[0076] For example, when the PDE inhibitor as specifically
disclosed in the above is used in a high-dose, there is a chance of
lowering a movement of an individual, therefore, it is expected to
select a suitable dose in consideration of such case.
[0077] The pharmaceutical composition or the therapeutic method of
the present invention is applied for alleviating symptoms of
Parkinson's disease.
[0078] For example, the therapeutic method may include the step of
screening a patient of Parkinson's disease, and a step of
administering to the patient an effective amount of a compound
having a phosphodiesterase 10 inhibitory activity.
[0079] As symptoms of Parkinson's disease, for example, there are
mentioned motor-related symptoms as follows:
[0080] Tremor;
[0081] Rigidity;
[0082] Akinesia or bradykinesia; and
[0083] Posture disturbances.
And relating to the above symptoms, there are observed
[0084] Gait disturbances; and
[0085] Speech impairment.
[0086] Further, in treatment of Parkinson's disease, L-dopa (a
precursor of dopamine) is an important medicine to be used as a
primary selected drug. However, as side effects accompanied by a
long-termed use of this L-dopa, the patient often experiences
dyskinesia. Dyskinesia is a functional disorder of voluntary
movement (more specifically, a choreoid symptom which involuntary
appears on face, neck, body, and limbs), and it becomes one of the
big problems in treatment of Parkinson's disease. In addition to
dyskinesia, wearing-off phenomenon, on-off phenomenon are also
symptoms which are troublesome. In many cases, these symptoms can
be alleviated by decreasing dose of L-dopa, however, decreased dose
of L-dopa frequently is accompanied by worsening of Parkinsonian
symptoms, which normally makes it difficult to deal with this
problem.
[0087] The pharmaceutical composition or the therapeutic method of
the present invention can enhance an effect of L-dopa to alleviate
the Parkinsonian symptoms, by applying concomitantly with L-dopa to
Parkinson's disease. Therefore, it is possible to decrease a dose
of L-dopa or number of administration thereof, while maintaining an
effect of L-dopa to alleviate the Parkinsonian symptoms, or it is
possible to prolong a duration of an effect of L-dopa. Further, it
can be expected to alleviate or delay development of unfavorable
symptoms, accompanied by treatment using L-dopa, that is,
dyskinesia, wearing-off phenomenon, on-off phenomenon, etc.
[0088] Further, it is expected that the pharmaceutical composition
or the therapeutic treatment of the present invention is used in
combination with L-dopa in a case where neuronal degeneration is
advanced profoundly, however, in a case where neuronal degeneration
is less advanced, it can be used singly to exhibit an effect.
[0089] The pharmaceutical composition or the therapeutic method of
the present invention can be used in combination with L-dopa
therapy, and it can also be used in combination with drugs for
Parkinson's disease other than L-dopa. Examples of such drugs for
Parkinson's disease other than L-dopa include:
Dopamine receptor agonists [bromocriptine, lisuride, pergolide,
cabergoline, apomorphine, talipexole, ropinirole, pramipexole,
SKF82958, SKF38393, Adrogolide (ABT-431; DAS-431), etc.]; MAO-B
(type B monoamine oxidase) inhibitor [selegiline (deprenyl)]; COMT
(catechol-O-methyl transferase) inhibitor [entacapone, tolcapone,
etc.]; Dopa decarboxylase inhibitor for concomitant use with L-dopa
[carbidopa, benserazide, etc.] Dopamine release accelerator
[amantadine, etc.];
Anticholinergic;
[0090] Adenosine receptor antagonist; and NMDA antagonist, etc.
[0091] Among the above, in case of concomitant use of the dopamine
receptor agonist and the pharmaceutical composition or the
therapeutic method of the present invention, the activity of the
dopamine receptor agonist can be enhanced based on the activity
thereof.
[0092] Further, according to the present invention, the PDE10
inhibitor enhances dopamine signals in the brain. In other words,
the method or the pharmaceutical composition of the present
invention which uses PDE10 inhibitor can be applied to enhance
dopamine signals in the brain in vivo, in living body, including in
patients, by administering an effective amount of the
inhibitor.
[0093] Corpus striatum (putamen and caudate nucleus), nucleus
accumbens, olfactory tubercle and frontal lobe, etc. are known to
be a part in which dopamine signals are involved.
[0094] On the other hand, PDE10 is confirmed to be expressed in
corpus striatum (putamen and caudate nucleus), nucleus accumbens,
olfactory tubercle, frontal lobe, temporal lobe, parietal lobe,
occipital lobe, insular lobe, amygdala, dorsal lateral geniculate
body (apart of thalamus), hippocampus, cerebellum, etc., in the
brain of primates, and especially, strong expressions have been
confirmed in corpus striatum (putamen and caudate nucleus), nucleus
accumbens, and olfactory tubercle, etc. (Example 3 below). Further,
in the brain of rodents, expression thereof has been confirmed in
corpus striatum and nucleus accumbens and olfactory tubercle,
etc.
[0095] Therefore, as a part of the brain in which dopamine signals
can be enhanced by PDE10 inhibitor, there are mentioned corpus
striatum (putamen and caudate nucleus), nucleus accumbens,
olfactory tubercle, frontal lobe, etc. Particularly among them,
preferable effect can be expected in corpus striatum (putamen and
caudate nucleus), nucleus accumbens, olfactory tubercle, etc.
[0096] Further, as the intracerebral dopamine signals to be
enhanced by PDE10 inhibitor, for example, there are mentioned
dopamine signals induced by L-dopa administration, as well as
intrinsic dopamine signals (i.e. dopamine signals based on
endogenous dopamine) in vivo. In addition, dopamine signals induced
by administration of dopamine receptor agonist are also
mentioned.
[0097] Such dopamine signals include dopamine signals induced by
administration of dopamine type 1 receptor agonist (D1 agonist),
and also dopamine signals induced by administration of dopamine
type 2 receptor agonist (D2 agonist).
[0098] Still further, dopamine signals induced by administration of
dopamine type 1 receptor selective agonist (D1 selective agonist)
or dopamine type 2 receptor selective agonist (D2 selective
agonist) are included.
[0099] As described above, since the method or pharmaceutical
composition of the present invention can enhance intracerebral
dopamine signals in vivo, it is expected to exhibit a therapeutic
or prophylactic effect for various central diseases, which are
expected to be alleviated by enhancement of intracerebral dopamine
signals.
[0100] As such central diseases, there are mentioned central
diseases for which L-dopa or dopamine receptor agonist are applied
for treatment or prophylaxis, specifically, for example,
Parkinson's disease, Drug addiction, Cognitive impairment, Restless
legs syndrome, and extrapiramidal syndrome accompanied by treatment
of schizophrenia, etc.
[0101] As the dopamine receptor agonist, the same as the above are
mentioned, among which, as the dopamine type 1 receptor agonist (D1
agonist), there are mentioned pergolide, cabergoline, apomorphine,
SKF82958, SKF38393, Adrogolide (ABT-431; DAS-3), etc.
[0102] As the dopamine type 2 receptor agonist (D2 agonist), there
are mentioned bromocriptine, lisuride, pergolide, cabergoline,
apomorphine, talipexole, ropinirole, pramipexole, etc.
[0103] Among them, pergolide, cabergoline, apomorphine, etc. have
activities as D1 agonist as well as D2 agonist.
[0104] On the other hand, SKF82958, SKF38393, Adrogolide (ABT-431;
DAS-431), etc. are those having less or no activity as a D2
agonist, and are known to be an agonist selective for dopamine type
1 receptor (that is, a dopamine type 1 receptor selective agonist
(D1 selective agonist)).
[0105] Further, bromocriptine, lisuride, talipexole, ropinirole,
pramipexole, etc. are those having less or no activity as a D1
agonist, and are known to be a dopamine type 2 receptor selective
agonist (D2 selective agonist).
[0106] In the present invention, L-dopa includes L-dopa itself and
a prodrug (methyl ester, etc.) thereof, and a pharmaceutically
acceptable salt thereof. Similarly, the dopamine receptor agonists
include the dopamine receptor agonist itself and a prodrug thereof,
and a pharmaceutically acceptable salt thereof.
[0107] Pharmaceutical effect for Parkinson's disease or an effect
for enhancing dopamine signals in the brain of the pharmaceutical
composition and therapeutic method of the present invention, or
PDE10 inhibitor to be used therein as an active ingredient can be
confirmed according to the known method or corresponding methods
thereto.
[0108] For example, as a parkinsonian model animal, a mouse, a rat,
a monkey or the like whose nigrostriatal neurons are impaired by
using neurotoxins, etc. are used, and a pharmaceutical effect can
be evaluated in vivo by administering drugs to these model
animals.
[0109] Such models include, for example, an experimental animal
such as a monkey, a marmoset, etc., which are treated by
administering MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)
as a neurotoxin. In this model, dopaminergic neurons are
degenerated by MPTP, and Parkinson-like symptoms such as motor
disabilities are expressed, in which pharmaceutical effect can be
evaluated by using alleviation of these symptoms as an index
(Examples 6 and 7 below; Burns et al., Proc. Natl. Acad. Sci. USA,
vol. 80, pp. 4546-4550, 1983; and Gerlach, et al., European Journal
of Pharmacology, vol. 208. pp. 273-286, 1991, etc.).
[0110] Alternatively, there are mentioned experimental animals such
as rats which are treated by administration of 6-hydroxydopamine
(6-OHDA). 6-hydroxydopamine is a neurotoxin which mainly acts on
catecholaminergic neurons, and has a dopamine depletion effect. In
this model, by injecting 6-OHDA into the medial forebrain bundle of
the test animal such as a rat, lesion can be formed in the
nigrostriatal neurons on the administration side. Rotational
behavior arise due to dopamine or dopamine agonist, and the
pharmaceutical effect can be evaluated by taking the number of
rotations as an index (Examples 5 and 8 below; and Koga et al.,
European Journal of Pharmacology, vol. 408, pp. 249-255, 2000,
etc.).
[0111] Further, in addition to the above, as a simple method for
evaluating pharmaceutical efficacy, there is mentioned a method in
which an animal such as a mouse, a rat, etc. is treated by
administration of reserpine. Reserpine inhibits transportation
system of catecholamine including dopamine in the brain, causing
depletion of dopamine in neurons. As a result, symptoms similar to
those for Parkinson's disease, such as decrease in spontaneous
movements are observed. The drugs are administered to the animal
and the pharmaceutical effect can be evaluated by measuring the
changes in spontaneous movements, etc. (Ferre et al., European
Journal of Pharmacology, vol. 192, pp. 31-37, 1991).
[0112] Still further, for example, by using a method described in
Example 4 below, or other known method, it is possible to confirm,
in vitro, an enhancing effect for cAMP increase in the neurons
stimulated by dopamine, etc., and to evaluate the strength of the
enhancement.
[0113] The method and the pharmaceutical composition of the present
invention are useful for treatment or prophylaxis of Parkinson's
disease. Particularly, when it is used with L-dopa for enhancing
the effect of L-dopa which is widely employed as a therapeutic
agent of Parkinson's disease, it can decrease the dose amount or
the number of administration of L-dopa and prolong a duration of an
effect of L-dopa. In addition, it is expected to alleviate or delay
development of unfavorable symptoms accompanied by L-dopa
therapy.
[0114] Further, the method and the pharmaceutical composition of
the present invention are useful for enhancing dopamine signals in
the brain in vivo. Therefore, it is expected to show therapeutic or
prophylactic effect for central diseases whose symptoms are
expected to be alleviated by dopamine signal enhancement in the
brain.
[0115] The pharmaceutical composition or the therapeutic method of
the present invention exhibits its pharmaceutical efficacy (a
therapeutic or prophylactic effect for Parkinson's disease, a
dopamine signal enhancing effect in the brain, etc.), based on the
PDE10 inhibitory activity possessed by the compound as an active
ingredient.
[0116] Therefore, pharmaceuticals and therapeutic method expressing
pharmaceutical efficacy, based on the activities other than this
PDE10 inhibitory activity, are not included in the scope of the
present invention.
[0117] For example, there has been a report on an application of
non-specific PDE inhibitors (caffeine, IBMX, theophyllamine,
dipyridamole, papaverine, theophylline, etc.), PDE5 inhibitor such
as sildenafil, etc., or PDE4 inhibitor such as rolipram, etc. for
animal models or for treatment of Parkinson's disease [Fredholm et
al., European Journal of Pharmacology, 1976, vol. 38, pp. 31-38;
Waldeck, Acta Pharmacol. Toxicol. Suppl., 1975, vol. 36, pp. 1-23;
U.S. Pat. No. 4,147,789; U.S. Pat. No. 3,961,060; WO01/78711;
WO01/32170; Swope et al., Neurology, 2000, vol. 54, No. 7, pp.
A90-A91; Dicki et al., Brain Research, 1997, vol. 753, pp. 335-339;
and Hulley et al., Eur. J. Neuroscience, 1995, vol. 7, pp.
2431-2440]. However, effective applications to parkinsonian animal
models or patients of Parkinson's disease, reported therein are not
included in the present invention.
[0118] Hereinafter, the present invention will be explained in more
detail by referring to the following Examples but these Examples do
not intend to limit the present invention.
EXAMPLES
Example 1
Measurement of PDE10 Inhibitory Activity
[0119] (1) Enzyme Preparation of PDE10 (PDE10A)
[0120] According to the methods described in Fujishige et al.
(Fujishige et al., Eur. J. Biochem., vol. 266, pp. 1118-1127, 1999;
and Mukai et al., Br. J. Pharmacol., vol. 111, pp. 389-390, 1994),
the enzyme PDE10A was isolated and prepared as follows.
[0121] At first, corpus striatum was excised from bovine whole
brain (Japanese Black Cattle, purchased from Tokyo Shibaura Zoki,
co., Ltd.), and washed with ice-cold saline (0.9% sodium chloride).
After the tissues were cut in a proper size, it was frozen by
liquid nitrogen, and stored at -80.degree. C.
[0122] To about 1.3 g of this tissue sample was added 8 ml of an
ice-cold homogenization buffer I (20 mM Tris-HCl, pH 7.5, 2 mM
magnesium acetate, 0.3 mM calcium chloride, 1 mM dithiothreitol,
1.3 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride), and the
resultant mixture was homogenized by Polytron homogenizer (PT10/35;
available from KINEMATICA) for about 3 minutes. The homogenate was
centrifuged by a low-speed centrifuge (M160-IV, rotor 50E-12A;
available from Sakuma) at 8000 rpm, at 4.degree. C. for 20 minutes,
and the obtained supernatant was further centrifuged by an
ultracentrifuge (Optima TLX Ultracentrifuge, rotor TLA100.4;
available from Beckman) at 50000 rpm, at 4.degree. C. for 60
minutes. The resultant supernatant was stored at 4.degree. C.
[0123] Successively, this supernatant was applied to
chromatography. Chromatography was performed using FPLC system
(available from Amersham Biosciences). The above supernatant was
applied to a HiTrapQ column (available from Amersham Biosciences)
(5 ml bed volume) equilibrated by elution buffer (20 mM Tris-HCl,
pH7.5, 1 mM calcium chloride, 1 mM dithiothreitol, 5 mM
benzamidine). After washing the column with 20 ml of the elution
buffer (flow rate, 2 ml/ml), and proteins were eluted from the
column by running a sodium chloride gradient (0-400 mM 80 ml,
followed by 400 mM-1000 mM 20 ml). Eluted fractions were collected
by 2 ml each, and fractions eluted at a low salt concentration in
the vicinity of the peak were mixed together.
[0124] To this fraction, glycerol was added to become 50%, and the
mixture was stored at -20.degree. C. This was used for an assay as
PDE10 (PDE10A) enzyme solution.
[0125] Subsequently, as a result of analysis on enzymatic
properties by carrying out the following PDE assay using this
enzyme solution, it was confirmed that Km values for cAMP and cGMP
were 0.17 .mu.M and 3.9 .mu.M, respectively, and almost corresponds
to the Km values of PDE10 (human and rat) which have been reported.
Further, as a result of studies on inhibitions by IBMX, zaprinast
and dipyridamole, IC.sub.50 values were 9.2 .mu.M, 13.7 .mu.M and
1.3 .mu.M, respectively, and it was confirmed that sensitivity for
the inhibitors also corresponded to reported data.
[0126] (2) Measurement of a PDE10 Inhibitory Activity
[0127] The PDE assay was performed using the enzyme solution
obtained in the above (1), and inhibitory activities of the
compounds on PDE10 (PDE10A) were measured.
[0128] PDE assay was performed according to the method described in
Kotera et al. (Kotera et al., Biochem. Pharmacol., vol. 60, pp.
1333-1341, 2000), by the radiolabeled nucleotide method as
follows.
[0129] Accordingly, enzymatic reaction was carried out in 500 .mu.l
of the assay buffer containing as a substrate 4.8 nM [.sup.3H]-cAMP
and 0.25 .mu.M unlabeled cAMP (available from Amersham Biosciences)
[50 mM Tris-HCl, pH8.0, 5 mM MgCl.sub.2, 4 mM 2-mercaptoethanol,
0.33 mg/ml bovine serum albumin].
[0130] After the reaction was carried out while keeping the
temperature at 37.degree. C. for 30 minutes, the reaction was
stopped by boiling the reaction mixture for 2 minutes, and further
added thereto was 100 .mu.l of snake venom (Crotalus atrox snake
venom 1 mg/ml) and the temperature was kept at 37.degree. C. for 30
minutes. Subsequently, 500 .mu.l of methanol was added thereto, and
the reaction mixture was applied to Dowex column (1.times.8-400).
Scintillation cocktail was added to each of the eluates, and the
radioactivity was measured by scintillation counter. From them, PDE
activity taking cAMP as a substrate (an activity to hydrolyze cAMP)
was measured.
[0131] In the measurements of the inhibitory activities of
compounds, a test compound was added to the above-mentioned
reaction mixture, in various concentrations, and PDE activity was
measured in the presence or absence of the test compound. From the
measurements, PDE10 (PDE10A) inhibitory activities were obtained
with respect to the various kinds of compounds.
[0132] IC.sub.50 values were obtained, considering an outline by
changing the concentrations of the inhibitor by order of 10, by
calculating according to the linear equation method, using 2
inhibitory rates obtained from 2 different concentrations, taken
from both sides nearest to 50% inhibition.
[0133] With respect to the compounds which shows the inhibitory
activity, when the affinity to PDE is obtained as Ki values, it can
be obtained, according to the conventional method, through detailed
kinetic analysis of enzyme reactions. For example, PDE activities
(initial reaction rates) are measured by performing an enzymatic
reaction using a substrate (unlabeled AMP) in various
concentrations and a test compound in various concentrations, and
based on the measured values, Ki values are obtained by analysis
such as the secondary plot of Lineweaver-Burk plot and Dixon plot,
etc.
[0134] Alternatively, when the type of the inhibition is
competitive inhibition, Ki values can be obtained based on the
following formula, which represents the relation between Ki values
and IC.sub.50 values:
Ki=IC.sub.50/(1+[S]/Km)
(Cheng-Prusoff equation) (Biochem Pharmacol, 22, 3099, 1973)
Example 2
Measurements of Inhibitory Activities for PDE Families Other than
PDE 10
[0135] (1) Preparation of Enzymes of PDE Families Other than
PDE10
[0136] According to the methods described in the literature, the
enzyme of PDE families other than PDE10 were isolated and prepared
as follows.
Enzyme preparations for PDE1, 2, 3, 4, 5 and 6 were done according
to the method described in Kotera et al. (Biochem. Pharmacol., vol.
60, pp. 1333-1341, 2000).
[0137] Enzyme Preparation of PDE1
[0138] Ventricle was excised from SD rat anesthetized with sodium
pentobarbital (Nihon SLC, about 300 g), and washed with 100 ml of
ice-cold saline 3 times. After the tissues were cut in
approximately 1 g, it was frozen by liquid nitrogen, and stored at
-80.degree. C.
[0139] To about 5 g of this tissue sample was added 16 ml of an
ice-cold homogenization buffer I, and the resultant mixture was
homogenized by Polytron homogenizer for 3 minutes. The homogenate
was centrifuged at 8000 rpm, at 4.degree. C. for 20 minutes, and
the obtained supernatant was further centrifuged by an
ultracentrifuge at 50000 rpm, at 4.degree. C. for 60 minutes. The
resultant supernatant was stored at 4.degree. C.
[0140] Successively, this supernatant was applied to chromatography
under the FPLC system, in the same manner as in Example 1 (1)
above. Eluted fractions were collected by 2 ml each.
[0141] Subsequently, the eluted fractions were used as an enzyme
solution for PDE assay. The reaction was performed in an assay
buffer containing EGTA [ethylene glycol
bis-(.beta.-aminoethylether)-N,N,N',N' tetraacetic acid tetrasodium
salt] (final concentration of 1 mM) or calcium chloride (final
concentration of 2 mM) and calmodulin (40 unit s/ml), and
cGMP-hydrolyzing activity was measured. As a result of PDE assay,
the fraction was selected as a fraction containing PDE1, which
showed higher activity in the presence of calcium chloride and
calmodulin, as compared to the case in the presence of EGTA.
[0142] For these PDE1 containing fractions, cGMP hydrolyzing
activities were analyzed in the presence of E4021 (100 nM), PDE5
specific inhibitor, it was shown that the cGMP-hydrolyzing
activities in these fractions were not inhibited by E4021. From
this fact, it was confirmed that these fractions were not
contaminated by PDE5, which is eluted adjacent to PDE1.
[0143] Further, in the presence or absence of unlabeled cGMP,
cAMP-PDE activities were measured for each of the fractions. PDE2
shows cGMP-hydrolyzing activity, and the presence of cGMP increases
its cAMP hydrolyzing activity. The fractions showing such
properties were excluded for the possibility of contamination of
PDE2.
[0144] Still further, in the presence of rolipram (10 .mu.m), the
PDE4 specific inhibitor, cAMP hydrolyzing activities were measured
for each of the fractions. The fractions showing clear inhibition
by rolipram (10 .mu.M) were excluded due to the possibility of
contamination of PDE4.
[0145] The fractions satisfying the above conditions were selected
and collected, and glycerol was added thereto to become 50%, and
the mixture was kept at -20.degree. C. This was used for PDE assay
as PDE1 enzyme solution.
[0146] (1-2) Enzyme Preparation of PDE2
[0147] One adrenal gland of Japanese Black Cattle, purchased from
Tokyo Shibaura Zoki, co., Ltd. was washed with 100 ml of ice-cold
saline (0.9% sodium chloride) twice. Subsequently, the tissues were
cut into about 1 gram, and it was frozen by liquid nitrogen. To
about 2 g of this tissue sample was added 10 ml of an ice-cold
homogenization buffer II (25 mM Tris-HCl, pH 7.5, 4 mM magnesium
chloride, 0.5 mM EGTA, 2 mM dithiothreitol, 4 mM benzamidine, 0.1
mM phenylmethylsulfonyl fluoride), and the homogenate that was
homogenized by Polytron homogenizer for 3 minutes was centrifuged
at 8000 rpm, at 4.degree. C. for 20 minutes, and the obtained
supernatant was further centrifuged by an ultracentrifuge at 50000
rpm, at 4.degree. C. for 60 minutes. The resultant supernatant was
stored at 4.degree. C.
[0148] This supernatant was applied to chromatography under the
FPLC system, in the same manner as in Example 1 (1) above. Eluted
fractions were collected by 2 ml each [provided that composition of
the elution buffer and gradient of sodium chloride used were as
follows: [0149] Elution buffer composition: 25 mM Tris-HCl, pH 7.5,
0.1 mM EGTA, 1 mM dithiothreitol, 5 mM benzamidine [0150] Gradient
of sodium chloride: 0-500 mM 80 ml and 500 mM-1000 mM 20 ml].
[0151] Subsequently, the eluted fractions were used as an enzyme
solution for PDE assay, and cGMP-hydrolyzing activity was measured.
Or, cAMP-hydrolyzing activity was measured for respective
fractions, in the presence or absence of unlabeled cGMP.
[0152] PDE2 shows an activity for hydrolyzing cGMP and cAMP, and
the presence of cGMP increases its cAMP hydrolyzing activity. On
the other hand, cAMP-hydrolyzing activity of PDE3 is inhibited by
milrinone or cGMP. Further, PDE4 specifically hydrolyzes cAMP, and
its cAMP-hydrolyzing activity is not affected by the presence of
cGMP, and it is inhibited by rolipram, which is a PDE4 specific
inhibitor. The cGMP-hydrolyzing activity of PDE5 is inhibited by
100 nM of E4021. The cGMP-hydrolyzing activity of PDE1 is increased
by addition of 2 mM calcium chloride and 40 units/ml of
calmodulin.
[0153] Using these properties as an index, the fractions containing
PDE2 but not containing other PDE families were selected. These
fractions were collected and glycerol was added thereto to become
50%, and the mixture was stored at -20.degree. C. These were used
as a PDE2 enzyme solution for PDE assay. The PDE activity of this
enzyme solution was inhibited by EHNA
[erythro-9-(2-hydroxy-3-nonyl)adenine], the PDE2 specific
inhibitor, therefore, it was confirmed that this enzyme was
PDE2.
[0154] (1-3) Enzyme Preparation of PDE3
[0155] Apex portion of ventricle of mixed-breed dog (Oriental
Yeast) was excised, and washed with 500 ml of ice-cold saline three
times. Subsequently, the tissues were cut into about 3 g, and it
was frozen by liquid nitrogen, and stored at -80.degree. C. To
about 1 g of this tissue sample was added 10 ml of an ice-cold
homogenization buffer II, and the resultant mixture was homogenized
by Polytron homogenizer for 3 minutes. The homogenate was
centrifuged at 8000 rpm, at 4.degree. C. for 20 minutes, and the
obtained supernatant was further centrifuged by an ultracentrifuge
at 40000 rpm, at 4.degree. C. for 60 minutes. The resultant
supernatant was stored at 4.degree. C.
[0156] This supernatant was applied to chromatography under the
FPLC system, in the same manner as in Example 1 (1) above. Eluted
fractions were collected by 2 ml each [provided that column,
composition of the elution buffer and gradient of sodium chloride
used were as follows: [0157] Column: DEAE Sepharose FF column (20
ml bed volume, XK16/40 column; available from Amersham Biosciences)
[0158] Elution buffer composition: 25 mM Tris-HCl, pH 7.5, 0.1 mM
EGTA, 1 mM dithiothreitol, 5 mM benzamidine [0159] Gradient of
sodium chloride: 0-600 mM 180 ml followed by 600 mM-1000 mM 40
ml].
[0160] Subsequently, the eluted fractions were used as an enzyme
solution for PDE assay, and cGMP-hydrolyzing activity was measured.
Further, cAMP-hydrolyzing activity was measured for respective
fractions, in the presence or absence of unlabeled cGMP.
[0161] PDE3 shows an activity for hydrolyzing cGMP and cAMP, and
the presence of cGMP inhibits its cAMP hydrolyzing activity. In the
same manner as in (1-2) above, using the properties of PDE3 and
other PDE families as indexes, the fractions containing PDE3 but
not containing other PDE families were selected. These fractions
were collected and glycerol was added thereto to become 50%, and
the mixture was stored at -20.degree. C. These were used as a PDE3
enzyme solution for PDE assay. The PDE activity of this enzyme
solution was inhibited by milrinone, the PDE3 specific inhibitor,
therefore, it was confirmed that this enzyme was PDE3.
[0162] (1-4) Enzyme Preparation of PDE4
[0163] The whole lung of mixed-breed dog (Oriental Yeast) was
excised, and washed with 500 ml of ice-cold saline three times.
Subsequently, the tissues were cut into about 3 g, and it was
frozen by liquid nitrogen, and stored at -80.degree. C.
[0164] To about 6 g of this tissue sample was added 15 ml of an
ice-cold homogenization buffer, and the resultant mixture was
homogenized by Polytron homogenizer for 3 minutes. The homogenate
was centrifuged at 8000 rpm, at 4.degree. C. for 20 minutes, and
the obtained supernatant was further centrifuged by an
ultracentrifuge at 50000 rpm, at 4.degree. C. for 60 minutes. The
resultant supernatant was stored at 4.degree. C.
[0165] This supernatant was applied to chromatography under the
FPLC system, in the same manner as in Example 1 (1) above. Eluted
fractions were collected by 2 ml each [provided that column, and
gradient of sodium chloride used were as follows: [0166] Column:
DEAE Sepharose FF column (20 ml bed volume, XK16/40 column;
available from Amersham Biosciences) [0167] Gradient of sodium
chloride: 0-400 mM 180 ml followed by 400 mM-1000 mM 40 ml].
[0168] Subsequently, the eluted fractions were used as an enzyme
solution for PDE assay, and cAMP-hydrolyzing activity was measured
for respective fractions, in the presence or absence of unlabeled
cGMP.
[0169] Activity of PDE4 is inhibited by rolipram, the PDE4 specific
inhibitor. In the same manner as in (1-2) above, using the
properties of PDE4 and other PDE families as indexes, the fractions
containing PDE4 but not containing other PDE families were
selected. These fractions were collected and glycerol was added
thereto to become 50%, and the mixture was stored at -20.degree. C.
These were used as a PDE4 enzyme solution for PDE assay. The PDE
activity of this enzyme solution was inhibited by rolipram, the
PDE4 specific inhibitor, therefore, it was confirmed that this
enzyme was PDE4.
[0170] (1-5) Enzyme Preparation of PDE5
[0171] In the same manner as in (1-4) above, the supernatant
obtained from the homogenate of canine lung tissue was applied to
chromatography under FLPC system, to collect eluted fractions of 2
ml each.
[0172] Subsequently, the eluted fractions were used as an enzyme
solution for PDE assay, and cGMP-hydrolyzing activity was measured
for respective fractions, in the presence or absence of E4021, the
PDE5 specific inhibitor. The fractions which showed activity
inhibition of 80% or more by E4021 were selected as fractions
containing PDE5.
[0173] Further, for examining contamination of PDE1, which is
adjacently eluted, cGMP-hydrolyzing PDE activity of the respective
fractions were measured in the same assay buffer containing EGTA
(final concentration of 1 mM) [or calcium chloride (final
concentration of 2 mM) and calmodulin (40 units/ml)]. As a result
of the assay, the fraction which showed higher activity in the
presence of calcium chloride and calmodulin, as compared to that in
the presence of EGTA were excluded, due to the possibility of
contamination of PDE1.
[0174] These fractions were collected and glycerol was added
thereto to become 50%, and the mixture was stored at -20.degree. C.
These were used as a PDE5 enzyme solution for PDE assay. The PDE
activity of this enzyme solution was inhibited by sildenafil, the
PDE5 specific inhibitor, therefore, it was confirmed that this
enzyme was PDE5.
[0175] (1-6) Enzyme Preparation of PDE6
[0176] From the bovine eyeball, purchased from Tokyo Shibaura Zoki,
co., Ltd., retina (pale red colored) which is present as a membrane
on the pigmented layer was collected and stored at -70.degree.
C.
[0177] To buffer A [16 mM MOPS-NaOH, pH7.5, 1.6 mM dithiothreitol,
8 mM magnesium chloride, 96 mM potassium chloride, 48 mM sodium
chloride, 0.16 mM phenylmethylsulfonyl fluoride, 10 .mu.M pepstatin
A, 10 .mu.M leupeptin] (20 ml), sucrose (6 g) was dissolved. Added
thereto was the above-mentioned retina (about 7 g), and the mixture
was stirred for 30 minutes while cutting the retina by scissors.
The mixture was applied to centrifugation at 2000 rpm, at 4.degree.
C. for 5 minutes and the supernatant was collected. To the
resultant residue was added buffer B[10 mM MOPS, pH7.5, 1 mM
dithiothreitol, 5 mM magnesium chloride, 60 mM potassium chloride,
30 mM sodium chloride, 0.1 mM phenylmethylsulfonyl fluoride, 10
.mu.M pepstatin A, 10 .mu.M leupeptin] (40 ml), and the mixture was
stirred by rolling several times. This was applied to
centrifugation and the resultant supernatant was combined with the
above obtained supernatant. This is further centrifuged at 7000
rpm, at 4.degree. C., for 5 minutes, and the precipitates were
obtained as crude rod outer segment (ROS).
[0178] Subsequently, this was subjected to a stepwise sucrose
density gradient centrifugation. Sucrose solutions with specific
weights of 1.15, 1.13 and 1.11 (8 ml each) were layers from the
bottom in this order, and a sucrose solution with specific weight
of 1.10 (8 ml) in which the above crude ROS was suspended was
layered on top of them, and they were subjected to an
ultracentrifuge at 20000 rpm, 4.degree. C. for 45 minutes. A red
band appearing on the interface of the sucrose solution of specific
weight of 1.11 and that with 1.13 were collected as ROS (rod outer
segment).
[0179] This was diluted with 7 ml of buffer C [100 mM Tris-HCl, pH
7.5, 5 mM dithiothreitol, 5 mM magnesium sulfate, 0.1 mM
phenylmethylsulfonyl fluoride, 10 .mu.M pepstatin A, 10 .mu.M
leupeptin], and then, the mixture was subjected to an
ultracentrifugation at 50000 rpm, at 4.degree. C. for 20 minutes.
The obtained precipitation was suspended in 2 ml of buffer C, and
the suspension was left still on the ice and in room light for 20
minutes. This was further subjected to an ultracentrifugation at
50000 rpm, at 4.degree. C., for 20 minutes, and
suspension-centrifugation process was repeated for the
precipitation, several times, and then, to wash the precipitation.
The obtained precipitation was suspended in 1.5 ml of buffer D [5
mM Tris-HCl, pH 7.5, 5 mM dithiothreitol, 0.5 mM magnesium sulfate,
0.1 mM phenylmethylsulfonyl fluoride, 10 .mu.M pepstatin A, 10
.mu.M leupeptin], and the suspension was centrifuged at 50000 rpm,
at 4.degree. C. for 20 minutes, to give a supernatant. For the
precipitation, suspension and centrifugation were repeated for
several times similarly, and the supernatant was combined with the
above obtained supernatant and they were applied to a gel
filtration.
[0180] Gel filtration was performed using SR25/45 column (available
from Amersham Biosciences) packed with Bio-Gel A-1.5m Gel, under
FPLC system. To the column equilibrated with elution buffer [50 mM
Tris-HCl, pH 7.5, 1 mM dithiothreitol, 150 mM sodium chloride] was
applied a bovine serum albumin solution (1 mg/ml, 1 ml), and the
column was washed with elution buffer. Subsequently, the
above-obtained supernatant (about 2 ml) was applied to the column,
and elution was performed by running elution buffer at a flow-rate
of 0.5 ml/min for about 6 hours, to give eluted fractions of 3 ml
each.
[0181] Further, PDE assay was carried out using the above-eluted
fractions as an enzyme solution, and cGMP-hydrolyzing activity was
measured. The fractions which showed a high cGMP-hydrolyzing
activity was selected and mixed, and to the mixture were added
bovine serum albumin (final concentration of 0.5 mg/ml),
phenylmethylsulfonyl fluoride (0.1 .mu.M), pepstatin A (1
.mu.g/ml), leupeptin (1 .mu.g/ml) and ethylene glycol (40%), and
the mixture was stored at -20.degree. C. Thus, light-activated PDE6
enzyme solution was obtained.
[0182] Subsequently, this light-activated PDE6 was activated by
trypsinization. In other words, to the light-activated PDE6 enzyme
(50-150 .mu.l) were added a buffer solution [20 mM Tris-HCl (pH
7.5), 1 mM magnesium chloride, 0.5 mg/ml bovine serum albumin] to
make the total volume 1 ml. Added thereto was a trypsin solution (7
mg/ml, 10 .mu.l), and the mixture was left to stand at 4.degree. C.
for 30 minutes, and reaction was stopped by addition of a trypsin
inhibitor solution (3 mg/ml, 100 .mu.l). To the reaction mixture,
ethylene glycol (0.7 ml) was added and the mixture was stored at
-20.degree. C. This was used for PDE assay as a trypsin-activated
PDE6 enzyme solution.
[0183] (1-7) Enzyme Preparation of PDE9 (PDE9A)
[0184] Based on the information of cDNA sequence of human PDE9A
(Genbank/EMBL Accession No. AF048837) (Fisher et al., J. Biol.
Chem., 1998, vol. 273, pp. 15559-15564, 1999), a cDNA encoding
human PDE9A was cloned by PCR method. Using this cDNA, a
recombinant protein of human PDE9A was prepared as follows.
[0185] A DNA fragment comprising a cDNA encoding human PDE9A was
linked to pFLAG-CMV-2 (available from Kodak), to give a vector
plasmid for expression of human PDE9A, with a flag tag peptide
added to the N-terminus. COS-7 cells were transfected by these
vector plasmids, and the cells were cultured in Dulbecco's modified
Eagle medium containing 10% bovine fetal serum for about 24 to 48
hours. After incubation, the cells were collected, and washed with
Phosphate buffered saline, and re-suspended in a solution buffer
(20 mM Tris-HCl, pH7.5, 2 mM magnesium acetate, 0.3 mM calcium
chloride, 1 mM dithiothreitol, 1.3 mM benzamidine). The cells were
homogenized by ultrasonic treatment, and subjected to
centrifugation (100,000.times.g, 4.degree. C., 1 hour) to collect a
supernatant.
[0186] This supernatant containing the recombinant human PDE9A was
used for PDE assay as a PDE9 (PDE9A) enzyme solution.
[0187] (1-8) Enzyme Preparations of PDE7 (PDE7A), PDE8 (PDE8A) and
PDE 11 (PDE11A)
[0188] Based on the information of cDNA sequence of human PDE7A
(Genbank/EMBL Accession No. NM002603), a cDNA encoding human PDE7A
was cloned by PCR method, according to a method described in Sasaki
et al. (Sasaki et al., Biochem. Biophys. Res. Commun., vol. 271,
pp. 575-583, 2000). Further, based on the information of cDNA
sequence of human PDE8A (Genbank/EMBL Accession No. AF388183), a
cDNA encoding human PDE8A was cloned by PCR method, according to a
method described in Gamanuma et al. (Gamanuma et al., Cell Signal,
2003, vol. 15, pp. 565-574). Further, a cDNA encoding human PDE11A
was obtained, according to a method reported in Yuasa et al. (Yuasa
et al., J. Biol. Chem., vol. 275, pp. 31469-31479, 2000). Using
these cDNAs, recombinant proteins of human PDE7A, human PDE8A, and
human PDE11A were prepared as follows.
[0189] DNA fragments comprising a cDNA encoding human PDE7A, human
PDE8A or human PDE11A were linked to pcDNA4/HisMax (available from
Invitrogen), to give a vector plasmid for expression of human
proteins, with a histidine tag peptide (hexahistidine), added to
the N-terminus. COS-7 cells were transfected with these vector
plasmids, and the cells were cultured in Dulbecco's modified Eagle
medium containing 10% bovine fetal serum for about 24 hours. The
cultured cells were collected, and washed with phosphate buffered
saline, and re-suspended in a solution buffer (40 mM Tris-HCl,
pH7.5, 15 mM benzamidine, 5 .mu.g/ml pepstatin A, 5 .mu.g/ml
leupeptin). The cells were homogenized by ultrasonic treatment, and
subjected to centrifugation (100,000.times.g, 4.degree. C., 1 hour)
to collect a supernatant. Subsequently, a partial purification was
performed by using a nickel affinity column. In other words, the
above-obtained supernatant was applied to a nickel-nitrotriacetate
resin (available from Qiagen) equilibrated with a solution buffer,
and incubated by shaking at 4.degree. C. for 3 hours. This resin
was filled into a plastic column (0.8.times.5 cm), and the resins
inside the column were washed with a washing buffer (40 mM
Tris-HCl, pH7.5, 15 mM benzamidine, 200 mM sodium chloride, 5 mM
imidazole, 5 .mu.g/ml pepstatin A, 5 .mu.g/ml leupeptin), and
further, the proteins were eluted with an elution buffer (40 mM
Tris-HCl, pH7.5, 15 mM benzamidine, 200 mM sodium chloride, 200 mM
imidazole, 5 .mu.g/ml pepstatin A, 5 .mu.g/ml leupeptin), to
collect fractions containing objective polypeptides. These were
dialyzed with a solution buffer, and stored at -80.degree. C.
[0190] The above-obtained solutions containing recombinant human
PDE7A, recombinant human PDE8A, and recombinant human PDE11A were
used for PDE assays as a PDE7 (PDE7A) enzyme solution, a PDE8
(PDE8A) enzyme solution, and a PDE11 (PDE11A) enzyme solution,
respectively.
[0191] (2) Measurements of Inhibitory Activities on PDE
Families
[0192] The PDE assays were performed using each of the enzyme
solutions obtained in the above (1-1) to (1-8), in the same manner
as in Example 1 (2) above, in the presence of test compounds in
various concentrations or in the absence of the test compounds,
provided that the substrates used in the PDE assays for each of the
PDE families, were as follows:
[0193] PDE1: about 12 nM [.sup.3H]-cGMP and 1 .mu.M cGMP
[0194] PDE2: about 12 nM [.sup.3H]-cGMP and 1 .mu.M cGMP
[0195] PDE3: about 4.8 nM [.sup.3H]-cAMP and 1 .mu.M cAMP
[0196] PDE4: about 4.8 nM [.sup.3H]-cAMP and 1 .mu.M cAMP
[0197] PDE5: about 12 nM [.sup.3H]-cGMP and 1 .mu.M cGMP
[0198] PDE6: about 12 nM [.sup.33H]-cGMP and 10 .mu.M cGMP
[0199] PDE7 (PDE7A): about 4.8 nM [.sup.3H]-cAMP and 0.1 .mu.M
cAMP
[0200] PDE8 (PDE8A): about 4.8 nM [.sup.3H]-cAMP and 0.1 .mu.M
cAMP
[0201] PDE9 (PDE9A): about 12 nM [.sup.3H]-cGMP and 0.1 .mu.M
cGMP
[0202] PDE10 (PDE10A): about 4.8 nM [.sup.31-1]-cAMP and 0.25 .mu.M
cAMP
[0203] PDE11 (PDE11A): about 12 nM [.sup.3H]-cGMP and 1 .mu.M
cGMP
According to the above, various compounds were assayed for
inhibitory activities on each of the PDE families other than
PDE10.
Example 3
Expression of PDE10 in the Brain
[0204] (1) Expression of PDE10 (PDE10A) in the Human Brain
(Detection by PDE Activities)
[0205] Respective parts of the human brain (frontal lobe, temporal
lobe and nucleus accumbens) were examined with respect to the
presence or absence of PDE10 (PDE10A) activities. PDE10 (PDE10A)
activities in the respective parts of the brain were detected by
measuring inhibitory activities using PDE10 (PDE10A) specific
inhibitor, as follows.
[0206] As the PDE10 (PDE10A) specific inhibitor, a compound showing
an excellent selectivity, exhibiting weak or no inhibitory
activities towards PDE families other than PDE10 was used.
[0207] As a reference, to 14.8 mg of corpus striatum of rat brain
was added 1 ml of a homogenation buffer (20 mM Tris-HCl, pH 7.5, 2
mM magnesium acetate, 0.3 mM calcium chloride, 1 mM DTT, 1.3 mM
benzamidine, 0.2 mM PMSF), and the mixture was homogenated by a
sonicator, to give a homogenate. One .mu.L of this homogenate was
used as an enzyme solution, to perform PDE assays in the presence
or absence of PDE10 (PDE10A) specific inhibitor.
[0208] Subsequently, 4 .mu.L each of the homogenates of the human
tissues (respective parts of human brain; frontal lobe, temporal
lobe and nucleus accumbens) purchased from ABS Inc. were used to
perform PDE assays in the presence or absence of the same PDE10
(PDE10A) specific inhibitor.
[0209] In the PDE assay, 0.25 .mu.M cAMP was used as a substrate,
and fractions which showed inhibition by PDE10 (PDE10A) specific
inhibitor were taken as PDE10 (PDE10A) activity. Further, the PDE10
(PDE10A) specific inhibitor was used in a range of compound
concentration of 1.times.10.sup.-5M to 1.times.10.sup.-11 M, and an
inhibition curve was obtained from the results of the assays.
Results:
[0210] According to the PDE 10 (PDE10A) assay using the homogenates
of corpus striatum of rat brain as a reference, it was shown that
based on the total PDE activities in the rat corpus striatum, about
80% thereof was PDE10 (PDE10A) activities. It can be expected that
this is due to a crude condition in the tissues.
[0211] Subsequently, as results of the PDE 10 (PDE10A) assay using
the homogenates of respective parts of the human brain (frontal
lobe, temporal lobe and nucleus accumbens), PDE10 (PDE10A)
activities were detected in any of the frontal lobe, temporal lobe
and nucleus accumbens of the human brain. The ratio of PDE10
(PDE10A) activities to the total PDE activities was shown to be
highest in nucleus accumbens, which was about 50% of the total PDE
activities.
[0212] Expression of PDE10 (PDE10A) in marmoset brain (in situ
hybridization) Localization of mRNA of PDE10A was studied by in
situ hybridization method.
[0213] Probes for hybridization were prepared as follows. Using a
plasmid pFLAG-H10A2 in which a cDNA encoding an entire coding
region of human PDE10A2 was linked top FLAG-CMV-2 (available from
SIGMA), PCR was performed to give a cDNA fragment of human PDE10A
(a fragment corresponding to nucleotides 1112-1449 of Gen-Bank
accession number AB020593). Subsequently, this fragment was
inserted into pGEM-Teasy (available from Promega), to prepare
pGEM-h10A. This plasmid was cut with ApaI for sense probe, and with
PstI for antisense probe, respectively, and DIG labeled by using
DIG-labeling kit (available from Roche) to give sense probes and
antisense probes.
[0214] In situ hybridization of the brain tissue was carried out as
follows. After marmoset (3 years and 3 month old, male) was
anesthetized with diethyl ether, the brain was excised, and
Tissue-Tek optimal cutting temperature compound (Sakura
Finetechnical Co., Ltd.) was dropped thereto. After the tissue was
frozen on dry ice, 16 sections were cut out by a cryostat from the
coronal section through brain and mounted on a polylysine slide
glass. Each section was fixed with 4% formalin, treated with 10
.mu.g/ml proteinase K for 7 minutes, and treated with 0.2 N HCl and
0.1 M triethanolamine/0.25% acetic anhydride. Subsequently,
dehydration was performed by graded ethanols, prehybridization was
done at 50.degree. C. for 30 minutes by hybridization buffer, and
hybridization was done using hybridization buffer containing
probes, at 50.degree. C. for 18 hours. After that, hybridization
mixture was washed with high-stringent buffer at 60.degree. C. for
30 minutes, and treated with RNaseA, and washed again with
high-stringent buffer. Subsequently, signals were detected by
DIG-detection kit (Roche).
Results:
[0215] As a result of the in situ hybridizations of the sections
taken out from 16 points of the coronal section through brain, to
cover the entire region of the marmoset brain, signals were
observed, not only in corpus striatum, nucleus accumbens and
olfactory tubercle, but also in frontal lobe, temporal lobe,
parietal lobe, occipital lobe, insular lobe, amygdala, dorsal
lateral geniculate body (a part of thalamus), hippocampus,
cerebellum, etc. Signals were strongest in corpus striatum, nucleus
accumbens and olfactory tubercle, and they were moderate in frontal
lobe, temporal lobe, parietal lobe, occipital lobe, insular lobe,
amygdala, dorsal lateral geniculate body (apart of thalamus),
hippocampus and cerebellum.
[0216] Test of Coexpression of Dopamine Receptor and PDE10 (I) (In
Situ Hybridization)
[0217] Expression patterns of a dopamine receptor (dopamine type 1
receptor) and PDE10 (PDE10A) in the marmoset brain was studied by
in situ hybridization.
[0218] Probes for hybridization were prepared as follows. For
preparation of probes of dopamine type 1 receptor, PCR was
performed using human brain cDNA contained in Multiple Tissue cDNA
Panels (Clontech) and a primer (5'-GCCTTTGACATCATGTGCTC-3' and
5'-TAGATCCTGGTGTAGGTGAC-3'), to give human D1 dopamine receptor
fragment (Gen-Bank accession number NM000794, nucleotides
1011-1363). The fragment was inserted into pGEM-T easy (Promega),
to prepare pGEM-hD1. The plasmid pGEM-hD1 was cut by SphI for sense
probes, and cut by PstI for antisense probes. Further, the plasmid
pGEM-h10A was cut by ApaI for sense probe, and cut by PstI for
antisense probe. These were DIG labeled with DIG-labeling kit
(Roche), to give sense probes and antisense probes.
[0219] In situ hybridization of the brain tissues were carried out
as follows. Marmoset was systemically perfused with 100 ml of
saline via heart, under etherization, and further perfused with 300
ml of a fixation solution, 4% paraformaldehyde-0.1M phosphate
buffer in the similar manner, and the brain tissue was excised. The
brain was immersed in the same fixation solution (4.degree. C.)
overnight, and sliced. The solution was replaced with 15%, 20% and
30% sucrose-0.1M phosphate buffers in this order, and embedded in
optimal cutting temperature compound (OCT) (Tissue-Tek) in liquid
nitrogen. After the frozen sections were prepared, in situ
hybridizations for mRNAs of dopamine receptor (dopamine type 1
receptor) and PDE10 (PDE10A) were performed by using ISHR Starting
Kit (NIPPON GENE).
Results:
[0220] The results of in situ hybridization were analyzed
particularly paying close attention to the respective parts of
corpus striatum, nucleus accumbens and frontal lobe. From the
observations of any of the parts, it was clear that mRNAs of
dopamine receptor (dopamine type 1 receptor) and PDE10 (PDE10A)
were expressed in about 80% or more of the neurons. This means that
most of the neurons coexpress both of the dopamine receptor
(dopamine type 1 receptor) and PDE10 (PDE10A), and supports the
fact that PDE10 (PDE10A) is involved in regulation of dopamine
signals in the brain (corpus striatum, nucleus accumbens and
frontal lobe).
[0221] Test of Coexpression of Dopamine Receptor and PDE10 (II) (In
Situ Hybridization)
[0222] Coexpression patterns of dopamine receptor (dopamine type 1
receoptor) and PDE10 (PDE10A) in marmoset brain was studied by
double in situ hybridization technique.
[0223] Probes for hybridization were prepared as follows. The
plasmid pGEM-h10A was cut by ApaI for sense probes and cut by PstI
for antisense probes, to prepare sense and antisense probes with
Fluorescein RNA Labeling Mix (Roche)
[0224] In situ hybridization of the brain tissue was performed as
follows. Marmoset was systemically perfused with 100 ml of saline
via heart, under etherization, and further perfused with 300 ml of
a fixation solution, 4% paraformaldehyde-0.1M phosphate buffer in
the similar manner, and the brain tissue was excised. The brain was
immersed in the same fixation solution (4.degree. C.) overnight,
and sliced. The solution was replaced with 15%, 20% and 30%
sucrose-0.1M phosphate buffers in this order, and embedded in
optimal cutting temperature compound (OCT) (Tissue-Tek) in liquid
nitrogen. After the frozen sections were prepared, double staining
of in situ hybridization was performed by using ISHR Starting Kit
(NIPPON GENE). Method and composition of the reagents were based on
the protocol attached to the kit, and only the modified parts will
be described. Proteinase treatment was carried out at a final
concentration of 2 .mu.g/ml for 15 minutes. Prehybridization and
hybridization were done at 50.degree. C. Probes were used by
combining PDE10A-probes and D1 dopamine receptor-probes, and
hybridization was done for 18 hours, with probe's concentration of
15 ng/ml each. Treatment with RNaseA was done for 30 minutes with a
final concentration of 20 .mu.g/ml. For signal detection, DIG
nucleic acid detection kit and HNPP Fluorescent Detection Set
(Roche) were used, and the method and composition of the reagents
were based on the protocol attached to the kit. First, anti-DIG
antibodies and HNPP/Fast Red TR, a fluorescent substrate were used
to detect D1 receptors. The sections were blocked by 1% blocking
buffer for 30 minutes, and then reacted for an hour with anti-DIG
antibodies diluted for 500 folds with the blocking buffer. After
washing, HNPP/Fast Red TR was reacted for 30 minutes, and signals
were detected. Subsequently, each section was treated with 100 mM
glycine-HCl (pH2.2) for 5 minute, twice, to dissociate anti-DIG
antibodies, and subsequently PDE10A signals were detected. The
sections were washed with PBS buffer, and blocked with 1% blocking
buffer for 30 minutes, and were reacted for an hour with anti-FITC
antibodies diluted for 100 folds with the blocking buffer. After
washing, INT/BCIP was reacted in the dark for 18 hours, and PDE10A
signals were detected. In order to confirm if the anti-DIG
antibodies were dissociated, the blocking buffer not containing
anti-FITC antibodies was used, and reaction with INT/BCIP was
carried out, and the signals were not detected. Therefore, it was
confirmed that signals colored by INT/BCIP were derived from
PDE10A.
Results:
[0225] The results of in situ hybridization were analyzed
particularly paying close attention to the respective parts of
corpus striatum and frontal lobe. From the observations of any of
the parts, it was clear that mRNAs of dopamine receptor (dopamine
type-1 receptor) and PDE10 (PDE10A) were coexpressed in the same
neurons. Further, it was shown that most of the neurons coexpress
both of the dopamine receptor (dopamine type-1 receptor) and PDE10
(PDE10A), and this supports the fact that PDE10 (PDE10A) is
involved in regulation of dopamine signals in the brain (corpus
striatum and frontal lobe).
Example 4
Change in Intracellular cAMP in Neurons by PDE10 Inhibitor
[0226] (1) Primary culture of neurons of corpus striatum Female
Wistar rats on day 14 or day 15 of pregnancy were purchased from
Japan SLC, Co. Ltd., and fetuses were taken out on day 18 of
pregnancy. From the fetus, the brain was excised, and stored on
ice-cold Leibovitz's L-15 medium (available from GIBCO) or on
DMEM/F-12 medium (available from GIBCO). In the same medium, dura
matter was removed and corpus striatum was collected. This striatum
part was treated by trypsin to diffuse neurons, and the neurons
were diluted and diffused in DMEM medium containing 10% bovine
fetal serum. These neurons were inoculated in 48-well plate coated
with 0.01% poly-L-lysine (available from Sigma) (2.times.10.sup.5
cells/well) and cultured. On the next day, the medium was changed
to Neurobasal medium (available from GIBCO), containing 50
fold-diluted B-27 supplement (available from GIBCO), and then, half
of the medium was replaced on day 4, 7 and 9 of culture, to
continue culture.
[0227] (2) Measurement of intracellular cAMP
[0228] As in the above (1), neurons were cultured, and on day 10 of
culture, the medium was removed, and then, a test compound (PDE10
inhibitors, etc.) diluted with Hank's balanced salt solution were
added, and incubated at 37.degree. C. for 15 minutes. Following
that, forskolin (final concentration of 1 .mu.M) or dopamine (final
concentration of 100 nM) or D1 agonist (SKF82958 Sigma) (final
concentration of 3 nM, 10 nM, 30 nM) was added thereto, and further
incubated at 37.degree. C. for 15 minutes.
[0229] Intracellular cAMP was measured using cAMP EIA kit available
from Amersham Biosciences. Accordingly, after removing the reaction
mixture, lysis buffer 1B (attached to cAMP EIA kit) was added to
stop the reaction, and to lead cytolysis, and intracellular cAMPs
were extracted. An amount of cAMPs in the extract was measured
according to a protocol attached to the cAMP EIA kit.
[0230] Various kinds of compounds having a PDE10 inhibitory
activity were tested as in the above, and as a result of
measurements of intracellular cAMP in the neurons, it was confirmed
that the various compounds having PDE10 inhibitory activities
enhanced intracellular cAMP increase caused by forskolin stimulus
or dopamine stimulus or D1 agonist stimulus.
Example 5
Evaluation in Rat 6-hydroxydopamine Model (1)
[0231] Preparation of rat 6-hydroxydopamine model According to the
method described in the literature (Koga et al., European Journal
of Pharmacology, vol. 408, pp. 249-255, 2000), 6-hydroxydopamine
(6-OHDA) was administered in the rat's brain, to cause lesion in
the substantia nigra of the brain, to thereby cause motor
disability. As the rats, male SD (Sprague Dawley) rats (about 9 to
10 week old) were used. To these rats was administered desipramine
(available from Sigma) (25 mg/kg, i.p.). Desipramine was used after
being dissolved in distilled water to be 25 mg/ml. After 15
minutes, pentobarbital (trade name: Nembutal Injection, available
from Dainippon Pharmaceutical Co., Ltd.) was administered (50
mg/kg, i.p.) for anesthesia.
[0232] After it was confirmed that the rats were under anesthesia,
hair was cut on the head, and median of scalp was incised for 3-4
cm long. Parietal bone was exposed and a hole was made on the bone
by a hard metal cutter (Miniter Co., Ltd) in a diameter of about 1
mm, in the position of 2 mm to the right and 2.8 mm posterior to
the bregma, to expose dura matter. The rat was placed in a brain
stereotaxic frame, and according to the atlas of Watson et al.
(Paxinos et al., 1986, "The Rat Brain in Stereotaxic Coordinates",
Academic Press, New York), 4 .mu.l of 6-OHDA (available from Sigma)
(2 mg/ml) was administered into the medial forebrain bundle (2 mm
to the right, 2.8 .mu.l posterior and 8.5 mm ventral to the
bregma), in a rate of 1 .mu.l/minute using a microsyringe available
from Hamilton. 6-OHDA was dissolved in ice-cold saline, in advance,
to make the concentration 2 mg/ml, and the solution was frozen and
kept at -20.degree. C. under nitrogen gas. It was thawed on the day
of experiment and cooled on ice until just before the
administration.
[0233] After administration of 6-OHDA, the needle was maintained
for 4 minutes in the same position, and it was removed from the
brain and the scalp was sutured back.
[0234] One week after administration of 6-OHDA, apomorphine was
administered, and rotational behaviors were confirmed to appear due
to the lesion of substantia nigra by 6-OHDA in one hemisphere.
Accordingly, after the rats were acclimated in 45 L cylindrical
plastic bucket in 10 minutes or more, apomorphine (available from
Sigma) was dorsally administered (0.1 mg/kg, s.c.), and the rats
were returned to the plastic bucket, to measure rotational
behaviors. Turnings were counted in every 5-minute interval, until
rotational behaviors disappeared or until an event was repeated
twice in which the number of turnings became 5 or less during the
5-minute interval. The rats showing total rotations of 90 or more
were selected, and used for further experiments.
[0235] (2) Effects of Test Compounds on L-Dopa Induced Rotational
Behaviors
[0236] In the rat 6-OHDA model, the effects of test compounds on
rotational behaviors induced by L-dopa administration was tested as
follows.
[0237] To the model rat prepared in the above (1) (2 weeks after
administration of apomorphine), L-dopa and benserazide (dopa
decarboxylase inhibitor which inhibits degradation of L-dopa) were
administered, and numbers of tunings were measured. Using the
results as an index, grouping was performed, so that the total
numbers of turnings were almost uniform among the groups.
[0238] To these rats, under light etherization, were administered
test compounds (PDE10 inhibitors) or vehicle alone, and further
administered were benserazide (2.5 mg/kg, i.p.) and L-dopa (10
mg/kg, i.p.), and turnings were counted in 5-minute intervals, as
in the above (1).
[0239] L-dopa was administered 30 minutes after benserazide
administration, and turnings were counted after L-dopa
administration. A test compound (PDE10 inhibitor) (or vehicle
alone) was intravenously administered 15 minutes before benserazide
administration, under light etherization.
[0240] Benserazide (available from Sigma) was used after being
dissolved in saline. L-dopa was used by dissolving a prodrug
thereof, methyl L-DOPA (available from Sigma) in saline.
[0241] By comparing numbers of turnings of the group administered
with the test compound (PDE10 inhibitor) and the group administered
with vehicle alone, effects of the test compound (PDE10 inhibitor)
to enhance or to prolong duration of rotational behaviors induced
by L-dopa were evaluated.
Example 6
Evaluation in Marmoset MPTP Model (1)
[0242] Using marmoset MPTP models, effects of test compound on
pharmaceutical efficacy of L-dopa administration were tested as
follows.
[0243] About 16 weeks before carrying out the tests, marmosets were
administered with MPTP
(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) (2.5 mg/kg, i.v.),
according to the method described in the literature (Fukuzaki et
al., Pharmacol. Biochem. Behay., vol. 67 No. 1, pp. 121-129,
September 2000), to degenerate dopaminergic neurons in the brain,
and individuals showing parkinsonian symptoms were used.
[0244] Using these marmosets (age 3-4 years, bodyweight 300-400 g),
the following three kinds of tests were performed. Each test was
performed with respect to the same individual sequentially, and
intervals of 5 days or more were allowed between the tests. [0245]
test 1: vehicle [0246] test 2: vehicle and L-dopa+benserazide
[0247] test 3: test compound (PDE10 inhibitor) and
L-dopa+benserazide
[0248] 10 mg/kg of L-dopa and 2.5 mg/kg of benserazide (dopa
decarboxylase inhibitor to inhibit degradation of L-dopa) were
orally administered at the same time. Further, as test compounds,
compounds confirmed with PDE10 inhibitory activities were
administered via saphenous vein, 90 minutes (or 180 minutes) before
administration of L-dopa+benserazide, under anesthesia by ketamine
and isoflurane (In test 1 and test 2, only vehicle was administered
in place of the test compound in the same manner).
[0249] L-dopa (available from Spectrum Chemical Mfg. Corp.) and
Benserazide hydrochloride (available from Spectrum Chemical Mfg.
Corp.) were suspended in 0.5% methylcellulose solution on the day
of administration and used. A test compound (PDE10 inhibitor) was
dissolved in a solvent 30 minutes before administration and
used.
[0250] After administration of test compound (PDE10 inhibitor) (or
vehicle) and L-dopa+benserazide, each individual was transferred to
an activity cage equipped with four photo cell sensor units, and
observation was continued for 6 hours at longest. For each
individual, locomotor activity and motor disability score were
evaluated in every 10-minute interval.
[0251] Motor disability was assessed according to the following 7
evaluation items, and total score of the 7 items were regarded as
the motor disability score.
<Evaluation Item>(number in [ ] shows a score range, 0 means
normal)
[0252] Alertness [0-2];
[0253] Checking movements [0-2];
[0254] Reaction to stimuli [0-3];
[0255] Attention and eye movements [0-1];
[0256] Posture [0-4];
[0257] Balance/coordination [0-3]; and
[0258] Vocalization [0-2]
[0259] Accordingly, for each of the individuals, results (changes
in locomotor activity and motor disability score) of 3 kinds of
tests were compared. By comparing the results of administration of
L-dopa and vehicle alone (test 2) and the results of administration
of L-dopa and the test compound (PDE10 inhibitor) (test 3), effects
of the test compound (PDE10 inhibitor) for enhancing an activity of
L-dopa or extending a sustained effect of L-dopa to alleviate the
symptoms (that is, improvements in locomotor activity and motor
disability score) were evaluated.
Example 7
Evaluation in Marmoset MPTP Model (2)
[0260] Bromocriptine (bromocriptine mesilate) is known to be a
dopamine receptor agonist, and an agonist which acts on dopamine
type-2 receptor (D2 agonist). As the dopamine type-2 receptor,
subtypes of Dopamine 2, Dopamine 3 and Dopamine 4 exist, and
bromocriptine acts agonistically on any type of them. On the
contrary, as the dopamine type-1 receptor, subtypes of Dopamine 1
and Dopamine 5 exist, and bromocriptine is known to act
antagonistically on Dopamine 1, and to act agonistically on
Dopamine 5. (Japanese clinical medicine, vol. 58, No. 10, pp.
2066-2071).
[0261] Using bromocriptine (bromocriptine mesilate) as a dopamine
receptor agonist, effect of test compound on pharmaceutical effects
caused by administration of dopamine receptor agonist, in marmoset
MPTP model was tested as follows.
[0262] The same method was employed as in Example 6, except for
using bromocriptine (bromocriptine mesilate) in place of L-dopa and
benserazide.
[0263] Each test was performed with respect to the same individual
sequentially, and intervals of 5 days or more were allowed between
the tests. [0264] test 1: vehicle [0265] test 2: vehicle and
bromocriptine mesilate [0266] test 3: test compound (PDE10
inhibitor) and bromocriptine mesilate
[0267] 3 mg/kg of bromocriptine mesilate (available from Sigma)
were orally administered. Further, as test compounds, compounds
confirmed with PDE10 inhibitory activities were administered via
saphenous vein, 90 minutes before administration of bromocriptine
mesilate, under anesthesia by ketamine and isoflurane (In test 1
and test 2, only vehicle was administered in place of the test
compound in the same manner).
[0268] Bromocriptine mesilate was suspended in 0.5% methylcellulose
solution on the day of administration and used. Test compound
(PDE10 inhibitor) was dissolved in a solvent 30 minutes before
administration and used.
[0269] After administration of test compound (PDE10 inhibitor) (or
vehicle) and bromocriptine mesilate, locomotor activity and motor
disability score were evaluated for each individual, in the same
manner as in Example 6.
[0270] Accordingly, for each of the individuals, results (changes
in locomotor activity and motor disability score) of 3 kinds of
tests were compared. By comparing the results of administration of
bromocriptine mesilate and vehicle (test 2) and the results of
administration of bromocriptine mesilate and the test compound
(PDE10 inhibitor) (test 3), effects of the test compound (PDE10
inhibitor) for enhancing an activity of bromocriptine mesilate or
extending a sustained effect of bromocriptine mesilate to alleviate
the symptoms (that is, improvements of locomotor activity and motor
disability score) were evaluated.
Example 8
Evaluation in Rat 6-Hydroxydopamine Model (2)
[0271] SKF38393 is known to be a dopamine receptor agonist which
acts selectively on dopamine 1 receptors. Using SKF38393 as a
dopamine receptor agonist, in rat 6-OHDA model, the effects of test
compounds on rotational behaviors induced by administration of the
dopamine receptor agonist was tested as follows.
[0272] Preparation of model rats and tests were performed in the
same manner as in Example 5, except for using SKF38393 in place of
L-dopa and benserazide.
[0273] However, rotational behaviors of the rats were measured by
an 8-channel rat rotometer (available from Neuroscience Inc.,
Japan). Further, the rats were selected showing total rotations of
100 or more, induced by administration of apomorphine (0.1 mg/kg,
s.c.). The rats were grouped, so that the numbers of turnings at
apomorphine test were almost uniform among the groups, and used for
the following tests.
[0274] To these rats, under light etherization, was administered a
test compound (PDE10 inhibitor)(or vehicle alone), and further
SKF38393 (0.02 mg/kg, s.c.) was administered, and numbers of
turnings were counted (in every 5-minutes interval, over 150
minute).
[0275] SKF38393 (available from Sigma; S-168) was used after being
dissolved in saline. The test compound (PDE10 inhibitor) (or
vehicle alone) was administered intravenously from tail vein, under
light etherization, 30 minutes before administration of
SKF38393.
[0276] By comparing numbers of turnings of the group administered
with the test compound (PDE10 inhibitor) and the reference group
administered with a solvent alone, effects of the test compound
(PDE10 inhibitor) to enhance or to prolong duration of rotational
behaviors induced by dopamine receptor agonist were evaluated.
Example 9
Evaluation of Pharmacological Efficacy of PDE10 Inhibitor
[0277] With respect to Compound A obtained in the preparation
example below, an inhibitory activity on PDE10 was examined in the
same manner as in Example 1. Further, in the same manner as in
Example 2, inhibitory activities on PDE families other than PDE10
were examined. Accordingly, IC.sub.50 values were as shown in Table
1 below, and Compound A of the present invention was shown to have
a specific inhibitory activity on PDE10. Further, from the
estimated value from enzyme kinetic analysis, Ki value of Compound
A of the present invention for PDE10 (PDE1 OA) was approximately
0.9 nM.
TABLE-US-00001 TABLE 1 PDE Family IC.sub.50 (nM) PDE10 (PDE10A) 1.1
PDE1 >1000 PDE2 >1000 PDE3 >1000 PDE4 160 PDE5 >1000
PDE6 290 PDE7 (PDE7A) >1000 PDE8 (PDE8A) >1000 PDE9 (PDE9A)
>1000 PDE11 (PDE11A) >1000
[0278] Further, with respect to Compound A of the present
invention, change in intracellular cAMP in neuron was examined in
the same manner as in Example 4. Accordingly, it was confirmed that
Compound A (concentration 10 .mu.M, 1 .mu.M) enhanced intracellular
cAMP increase caused by forskolin stimulus in neuron. It was also
confirmed that Compound A (concentration 1 .mu.M) enhanced
intracellular cAMP increase caused by dopamine stimulus in neuron.
Further, it was confirmed that Compound A (concentration 10 .mu.M,
1 .mu.M) enhanced intracellular cAMP increase caused by D1 agonist
(SKF82958, available from Sigma) stimulus in neuron.
[0279] Further, Compound A (1 mg/kg, i.v.) was intravenously
administered to rat, and according to integration plot method, a
rate (PS) was measured for Compound A to enter the brain by passing
through the blood brain barrier (BBB), and occupancy of PDE10
binding in the brain was analyzed. It was shown that Compound A had
good brain uptake and penetration characteristics and an excellent
PDE10 binding behaviors inside the brain.
[0280] Still further, in the same manner as in Example 5, effects
of Compound A in the rat 6-hydroxydopamine model were examined.
Accordingly, by administration of Compound A (1 mg/kg, i.v.), it
was confirmed that rotational behaviors induced by L-dopa tended to
increase in terms of total number of turnings, and Compound A
significantly prolonged a duration of rotational behaviors. As
stated above, Compound A enhanced L-dopa induced rotational
behaviors in rat 6-hydroxydopamine model.
[0281] In the same manner as in Example 6, effects of Compound A
were examined in marmoset MPTP model. By comparing the case in
which Compound A (0.3 mg/kg or 1 mg/kg, i.v.) was administered
together with L-dopa and the case in which vehicle alone was
administered with L-dopa, it was shown that Compound A enhanced
and/or prolonged duration of effects of L-dopa to alleviate
symptoms (improvements of locomotor activity and motor disability
score).
[0282] In the same manner as in Example 7, effects of Compound A
were examined in marmoset MPTP model. By comparing the case in
which Compound A was administered together with bromocriptine, a
dopamine receptor agonist (D2 agonist) (0.3 mg/kg) and the case in
which vehicle alone was administered with bromocriptine, it was
shown that Compound A had a tendency to enhance the effects of
bromocriptine to alleviate symptoms (improvements of locomotor
activity and motor disability score).
[0283] [In the above-stated tests using rat and marmoset model
animals, Compound A which was prepared according to preparation
example below was dissolved in a solvent (0.1N--HCl) just before
administration, in accordance with the dose to be used in the
experiments.]
[0284] From these results, Compound A was thought to enhance
dopamine signals in the brain (corpus striatum, etc.), thereby
exhibit a therapeutic effect on Parkinson's disease, based on its
specific inhibitory action on PDE10.
[0285] Further, these results support the fact that compounds
having a specific inhibitory activity on PDE10 enhance dopamine
signals in the brain (corpus striatum, etc.) induced by
administration of L-dopa or dopamine receptor agonist (D2 agonist
such as bromocriptine, etc. and D1 agonist such as SKF82958,
SKF38393, etc.), and the fact that they enhance the pharmaceutical
effect of L-dopa or dopamine receptor agonists (effect to alleviate
symptoms in Parkinson's disease, etc.).
[0286] Preparation Example Preparation of Compound A Compound A,
that is,
2-{(E)-2-[4-(1-imidazolyl)-quinazolin-2-yl]}ethenyl-4-(3,4-dimethoxy)phen-
yl-6-(pyrrolidin-1-yl)pyrimidine was prepared, according to the
method described in the following (1) to (5).
(1) Synthesis of 2,4-dichloro-6-(3,4-dimethoxy)phenylpyrimidine
[0287] 2,4,6-trichloropyrimidine (13.3 g),
3,4-dimethoxyphenylborate (13.2 g), and
dichlorobis(triphenylphosphine)palladium(II) (2.50 g) were added to
1,2-dimethoxyethane (130 mL)-2M sodium carbonate aqueous solution
(90 mL), and the mixture was stirred at 90.degree. C. for 100
minutes. The reaction mixture was cooled down to room temperature,
diluted with ethyl acetate, and washed with brine. The organic
layer was dried over anhydrous sodium sulfate, and the resultant
residue obtained after concentration under reduced pressure was
purified by silica gel column chromatography (hexane:ethyl
acetate=5:1, hexane:ethyl acetate:chloroform=20:20:1, followed by
chloroform:ethyl acetate=10:1), to give 9.23 g of
2,4-dichloro-6-(3,4-dimethoxy)phenylpyrimidine as yellow powder
(yield 45%).
[0288] (Melting point: 165-167.degree. C.)
(2) Synthesis of
2-chloro-4-(3,4-dimethoxy)phenyl-6-(pyrrolidin-1-yl)pyrimidine
[0289] The above obtained
2,4-dichloro-6-(3,4-dimethoxy)phenyl-pyrimidine (3.00 g) and
triethylamine (2.93 mL) were added to N,N-dimethylformamide (42
mL), and to the mixture was added pyrrolidine (966 .mu.L) under ice
cooling. The mixture was stirred at room temperature for 2 hours,
and then, ice-cold water was added to the reaction mixture, and the
mixture was extracted with ethyl acetate. The organic layer was
washed with water and brine, dried over anhydrous sodium sulfate,
and concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (hexane:ethylacetate=1:1), to
give 3.10 g of 2-chloro-4-(3,4-dimethoxy)phenyl-6-(pyrrolidin-1-yl)
pyrimidine as colorless crystals (yield 92%).
[0290] (Melting point: 133-135.degree. C.)
(3) Synthesis of
4-(3,4-dimethoxy)phenyl-6-(pyrrolidin-1-yl)-2-[(E)-2-(tributylstannyl)eth-
enyl]pyrimidine
[0291] According to the method described in literature of Stille et
al. (Organic Synthesis, 1988, vol. 67, pp. 86-97),
(E)-1,2-bis-(tributylstannyl)ethylene was obtained. This
(E)-1,2-bis-(tributylstannyl)ethylene (11.71 g),
2-chloro-4-(3,4-dimethoxy)phenyl-6-(pyrrolidin-1-yl)pyrimidine
(3.09 g) obtained in the above (2),
dichlorobis(triphenylphosphine)-palladium(II) (343 mg),
triphenylphosphine (507 mg), and copper (I) bromide (277 mg) were
mixed in toluene (60 mL), and the mixture was refluxed under
heating for 2 hours. The reaction mixture was cooled down to room
temperature, and purified by silica gel column chromatography
(hexane:ethyl acetate=10:1 to 5:1), to give 2.48 g of
4-(3,4-dimethoxy)phenyl-6-(pyrrollidin-1-yl)-2-[(E)-2-(tributylstannyl)et-
henyl]pyrimidine as pale yellow oil (yield 43%).
[0292] [IR (Neat): 1597, 1575, 1518; APCI-MS m/z 598 (M+H)]
(4) Synthesis of 2-chloro-4-(1-imidazolyl)quinazoline
[0293] To a suspension of 60% sodium hydride (426 mg) in
tetrahydrofuran (4 mL)-N,N-dimethylformamide (24 mL) was added
imidazole (725 mg), and the mixture was stirred at room temperature
for 30 minutes. To the mixture was added dropwise a tetrahydrofuran
solution (35 mL) of 2,4-dichloroquinazoline (2.12 g) synthesized
according to the method of H. C. Scarborough et al. (Journal of
Organic Chemistry, 1962, pp. 957-961), in ice-acetone bath, and the
mixture was stirred for 30 minutes. The reaction mixture was poured
into ice-cold water, and precipitated powder was collected by
filtration, washed with water and dried. The obtained powder was
purified by silica gel column chromatography, (ethyl acetate), to
give 1.88 g of 2-chloro-4-(1-imidazolyl)-quinazoline as colorless
crystals (yield 77%).
[0294] (Melting point: 168-170.degree. C. (decomposition))
(5) Synthesis of
2-{(E)-2-[4-(1-imidazolyl)quinazolin-2-yl]}-ethenyl-4-(3,4-dimethoxy)phen-
yl-6-(pyrrolidin-1-yl)-pyrimidine
[0295] A mixture of 2-chloro-4-(1-imidazolyl)quinazoline (1.44 g)
obtained in the above (4),
4-(3,4-dimethoxy)phenyl-6-(1-pyrrolidinyl)-2-[(E)-2-(tributylstannyl)ethe-
nyl]pyrimidine (2.47 g) obtained in the above (3),
dichlorobis(triphenyl-phosphine)palladium(II) (144 mg),
triphenylphosphine (216 mg), and copper (I) bromide (118 mg) in
toluene (35 mL) was refluxed under heating for 2.5 hours. The
reaction mixture was cooled down to room temperature, diluted with
chloroform (10 mL), and purified by NH-silica gel column
chromatography (hexane:chloroform=7:3, and then, 1:1) and
subsequently by silica gel column chromatography
(chloroform:methanol=50:1). The obtained powder was recrystallized
from dichloromethane-ethyl acetate, to give 1.13 g of
2-{(E)-2-[4-(1-imidazolyl)quinazolin-2-yl]}ethenyl-4-(3,4-dimethoxy)pheny-
l-6-(pyrrolidin-1-yl)pyrimidine (free form) (Compound A) as yellow
crystals (yield 54%).
[0296] (Melting point: 234-239.degree. C.)
* * * * *