U.S. patent application number 13/642639 was filed with the patent office on 2013-08-01 for drug and food/drink for preventing or improving cerebral dysfunction.
This patent application is currently assigned to NIHON UNIVERSITY. The applicant listed for this patent is Shin Aizawa, Hiroyuki Hasegawa. Invention is credited to Shin Aizawa, Hiroyuki Hasegawa.
Application Number | 20130197000 13/642639 |
Document ID | / |
Family ID | 44833976 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130197000 |
Kind Code |
A1 |
Hasegawa; Hiroyuki ; et
al. |
August 1, 2013 |
DRUG AND FOOD/DRINK FOR PREVENTING OR IMPROVING CEREBRAL
DYSFUNCTION
Abstract
[Problem] To provide means for improving symptoms of cerebral
dysfunction. [Solution Means] The inventor has newly found that
peripheral administration of sepiapterin increases the
bioavailability of aromatic monoamines in the brain. Accordingly,
provided are a drug for preventing or improving cerebral
dysfunction, which contains at least one of sepiapterin and its
salt and also provided is a food and or drink for preventing or
improving cerebral dysfunction, which contains at least one of
sepiapterin and its salt. Unlike tetrahydrobiopterin, etc.,
sepiapterin is able to repress the lowering level of aromatic
monoamines (such as serotonin, dopamine and noradrenaline) in
neurons in the brain and also increase the bioavailability.
Therefore, sepiapterin may be effective against cerebral
dysfunction caused by the lowered level of aromatic monoamines in
the brain in neurons in the brain, for example, central mental
disorders such as depression, hyperphagia, autism, impaired
consciousness and concentration, and cognitive disturbance as well
as central motor disorders such as myotonia, rigidity and
tremor.
Inventors: |
Hasegawa; Hiroyuki; (Tokyo,
JP) ; Aizawa; Shin; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hasegawa; Hiroyuki
Aizawa; Shin |
Tokyo
Tokyo |
|
JP
JP |
|
|
Assignee: |
NIHON UNIVERSITY
TOKYO
JP
|
Family ID: |
44833976 |
Appl. No.: |
13/642639 |
Filed: |
April 22, 2011 |
PCT Filed: |
April 22, 2011 |
PCT NO: |
PCT/JP2011/002393 |
371 Date: |
January 1, 2013 |
Current U.S.
Class: |
514/249 ;
544/258 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 25/28 20180101; A23L 2/52 20130101; A61K 31/519 20130101; A61P
25/24 20180101; C07D 475/04 20130101; A23L 33/10 20160801; A61P
25/14 20180101; A61P 25/16 20180101; A61P 25/00 20180101 |
Class at
Publication: |
514/249 ;
544/258 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A23L 1/29 20060101 A23L001/29 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2010 |
JP |
JP2010-098602 |
Claims
1-8. (canceled)
9. A drug for preventing or improving cerebral dysfunction
(excluding cerebral dysfunction of fetuses or newborns), which
contains at least sepiapterin and is peripherally administered
daily at a dose of 0.1 to 100 mg/kg.
10. The drug according to claim 1, which elevates an intracellular
level of tetrahydrobiopterin in neurons in the brain.
11. The drug according to claim 1, which suppresses the decrease in
aromatic monoamines in the brain.
12. The drug according to claim 1 wherein the cerebral dysfunction
is a central mental disorder or a central motor disorder.
13. The drug according to claim 1 wherein the cerebral dysfunction
is a central mental disorder which is any one of depression,
hyperphagia, autism, impaired consciousness and concentration, and
cognitive disturbance, or a central motor disorder which is any one
of myotonia, rigidity and tremor.
14. A food or drink for preventing or improving cerebral
dysfunction (excluding cerebral dysfunction of fetuses or
newborns), which food or drink contains sepiapterin as an active
ingredient and is orally taken daily in an amount of 0.1 to 100
mg/kg.
15. A method for preventing or improving cerebral dysfunction, the
method comprising administering to a patient suffering from
cerebral dysfunction an effective dose of sepiapterin via a
peripheral route of administration.
16. The method of claim 15 wherein sepiapterin is administered
orally to the patient to provide a dose from 0.1 to 100 mg/kg of
body weight per day.
17. The method of claim 15 wherein sepiapterin is administered
intravenously to the patient at a dose of 0.1 to 100 mg/kg of body
weight per day.
18. The method of claim 15 wherein said administration results in
an elevated intracellular level of tetrahydrobiopterin in neurons
of the brain.
19. The method of claim 15 wherein said administration results in
suppression of the decrease in aromatic monoamines in the
brain.
20. Use of sepiapterin in formulation of a drug for preventing or
improving cerebral dysfunction (excluding cerebral dysfunction of
fetuses or newborns), which is peripherally administered daily at a
dose of 0.1 to 100 mg/kg.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a drug and food/drink which
contain sepiapterin for preventing or improving cerebral
dysfunction. More specifically, the present invention relates to a
drug and food/drink for preventing, improving and treating diseases
in which neurotransmitters in the brain are involved, for example,
central mental disorders (such as depression, hyperphagia, autism,
impaired consciousness and concentration, and cognitive
disturbance) or central motor disorders (such as myotonia, rigidity
and tremor).
BACKGROUND ART
[0002] The brain is the highest center of information transmission
via nerves such as motor and consciousness, playing an important
role in human mental activities such as feeling, emotion and reason
as well as optional control of motor. The brain is constructed with
an innumerable number of neurons, and information between the
neurons is transmitted by neurotransmitters in the brain.
[0003] A monoamine neurotransmitter is a generic name for any
non-amino acid neurotransmitter which contains one amino group.
Among these monoamine neurotransmitters, a monoamine
neurotransmitter biosynthesized in the body from tyrosine or
tryptophan of a naturally occurring L-amino acid as a precursor is
referred to as an aromatic monoamine. Representative aromatic
monoamines include serotonin, noradrenaline, dopamine and
adrenaline. Aromatic monoamines are present in the brain and
peripheries as well. It is known that aromatic monoamines present
in the brain play an important role in transmitting information in
the brain and are also deeply involved in control of mental
activities, emotion and motor.
[0004] Serotonin is an aromatic monoamine which is commonly
contained in plants and animals including humans and primarily
present in chromaffin cells of mucous membranes of the small
intestine and in platelets, etc. Serotonin is also partially
present in the central nervous system. This substance functions as
a neurotransmitter in the central nervous system. Serotonin nerves
extend their nerve fibers diversely from nuclei raphes of the
medullary to the brain and spinal cord including the hypothalamus,
basal ganglion and corpus striatum, thereby greatly influencing
mental activities of humans such as emotion, fatigue, pain and
appetite.
[0005] In recent years, a correlation has been found between
serotonin and cerebral dysfunction such as depression, hyperphagia,
autism, impaired consciousness and concentration, and cognitive
disturbance. It is now possible to improve symptoms of cerebral
dysfunction to some extent by means of drugs acting on a serotonin
system. For example, an SSRI (Serotonin Selective Reuptake
Inhibitor) is now commercially available as a drug which inhibits
reabsorption of serotonin released from synapses, thereby improving
symptoms of depression, etc. However, it has been pointed out that
since an SSRI decreases the total amount of serotonin in neurons,
the drug may further exacerbate symptoms of depression for longer
administration.
[0006] Noradrenaline is an aromatic monoamine which is widely
present at sympathetic nerve endings and in the central nervous
system and also a precursor of adrenaline. This substance works as
an adrenocortical hormone and a neurotransmitter at the
peripheries. On the other hand, noradrenaline nerves of the locus
ceruleus project throughout the brain and it is thought that these
are involved in attention, drive impulse, etc. A correlation has
also been found with a change in the noradrenaline system with
depression.
[0007] An SNRI (Serotonin and Norepinephrine Reuptake Inhibitor) is
a drug which inhibits reabsorption of serotonin and noradrenaline
in a synapse, thereby increasing concentration of the
neurotransmitters at perineural cavities to improve symptoms of
depression. It is thought that this drug not only increases the
concentration of serotonin to improve symptoms of depression but
also inhibits reabsorption of noradrenaline to stimulate
sympathetic nerves, thereby exhibiting effects of enhancing
ambition and feeling. However, as in the case of an SSRI, it has
been pointed out that an SNRI also decreases the total amount of
serotonin in neurons and may exacerbate symptoms of depression on
longer administration.
[0008] Dopamine is an aromatic monoamine present in the central
nervous system and also a precursor of adrenaline and
noradrenaline. In the brain, the brainstem ventral tegmental area
and nigral dopamine nerves project on the cerebrum frontal lobe,
corpus striatum, etc., and are involved in control of motor,
regulation of hormones, feelings of pleasure, motivation, learning,
etc.
[0009] In Parkinson's disease, the nigrostriatal dopamine nerves
are decreased to cause motor symptoms such as muscle rigidity,
tremor and akinesia. There is an assumption that links dopamine
with some forms of schizophrenia and depression.
[0010] Of aromatic monoamines, some are present in peripheral
cells, etc., are present in neurons of the central nervous system.
In principle, aromatic monoamines in the brain do not pass through
the blood-brain barrier but they are synthesized and metabolized
independently. That is, no mutual migration or complementation
occurs between the aromatic monoamines present in peripheral cells
and the aromatic monoamines present in neurons of the central
nervous system.
[0011] Aromatic monoamine nerves in the brain release aromatic
monoamines (such as serotonin, noradrenaline and dopamine) stored
in releasing granules in cells. After being released, the aromatic
monoamines are subjected to reuptake by individual neurons, mixed
with newly bio-synthesized aromatic monoamines and taken up again
into the releasing granules. This mechanism is repeated in a
recycling manner, and before being taken up into the releasing
granules, some of the aromatic monoamines are metabolized in the
cells to produce inactive metabolic products. Aromatic monoamines
will not flow into the brain or flow out from the brain due to
functions of the blood-brain barrier. However, their metabolic
products are discharged from the brain into the peripheries.
Furthermore, aromatic amino acids (such as tryptophan and tyrosine)
which are immediate precursors of aromatic monoamine biosynthesis
will pass through the blood-brain barrier.
[0012] Tetrahydrobiopterin (BH4) is a coenzyme of phenylalanine
hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase and
nitric oxide synthase. This substance is a coenzyme which is
essential for enzymatic reactions such as reactions for synthesis
of tyrosine from phenylalanine, synthesis of serotonin from
tryptophan, synthesis of dopa from tyrosine and synthesis of nitric
oxide and citrulline from arginine.
[0013] The above-described enzymes are incapable of exhibiting
catalytic actions sufficiently, if cells are deficient in
tetrahydrobiopterin. This causes hyperphenylalaninemia and
reduction in bioavailability of monoamine neurotransmitters such as
dopamine, noradrenaline and serotonin.
[0014] Diseases caused by defective production of
tetrahydrobiopterin include malignant hyperphenylalaninemia and
Segawa disease (dopa-responsive dystonia). Furthermore, such a
possibility has been suggested that abnormal metabolism of
tetrahydrobiopterin may be responsible for or exacerbate
depression, hyperphagia, Parkinson's disease, autism,
schizophrenia, etc.
[0015] In cases where tetrahydrobiopterin is transmitted from the
peripheries to the brain, a part of tetrahydrobiopterin is slightly
captured by brain tissues but rapidly discharged outside of brain
tissues at a stage that it does not reach aromatic monoamine
neurons. That is, tetrahydrobiopterin is extremely difficult in
passing through the blood-brain barrier.
[0016] "7,8-Dihydro-6-[(S)-2-hydroxy-1-oxopropyl]-pterin (trivial
name: sepiapterin, hereinafter referred to as sepiapterin)" is an
endogenous compound which widely occurs as an animal pigment in a
variety of animals including humans and also contained in daily
foods in trace amounts. In 1960, Nawa determined a chemical
structure of sepiapterin as one of the pigments contained in the
eyes of a drosophila.
[0017] Nothing is so far known about bioactivity of sepiapterin in
the human body. Sepiapterin is inevitably produced in the human
body by auto-oxidation of tetrahydro-6-lactoyl-tetrahydropterin (an
intermediate in the synthesis of tetrahydrobiopterin from GTP).
However, this substance amounts in trace and is nearly undetectable
in blood or urine.
[0018] It is known that sepiapterin is easily taken up into animal
cells and converted to tetrahydrobiopterin through two-step
enzymatic reactions by SPR (Sepiapterin Reductase) and DHFR
(Dihydrofolate Reductase) (refer to Non-Patent Document 1, for
example).
[0019] In recent years, cell membrane permeation characteristics of
tetrahydrobiopterin, its metabolic product and a prodrug (such as
sepiapterin or dihydrobiopterin) are in the process of being more
clearly understood. For example, Non-Patent Document 2 has
disclosed findings on cell membrane transport of a pterin
compound.
[0020] There have been proposed a variety of drugs for treating
various diseases which contain tetrahydrobiopterin, etc. For
example, Patent Document 1 has disclosed a pterin-derivative
containing drug for treating depression and Parkinson's disease,
Patent Document 2 has disclosed a tetrahydrobiopterin-containing
composition for treating attention deficit hyperactivity disorders
and hyperphenylalaninemia, Patent Document 3 has disclosed a drug
having tetrahydrobiopterin as an active ingredient for treating
spinocerebellar degeneration, and Patent Document 4 has disclosed a
cancer metastasis depressant having a pterin derivative as an
active ingredient. Furthermore, in Non-Patent Document 3,
evaluation has been made for monotherapy with tetrahydrobiopterin
or sepiapterin given to patients with biopterin metabolism
deficiency phenylketonuria. In Non-Patent Document 4, evaluation
has been made for biosynthesis of biopterin in the brain of a rat.
In Non-Patent Document 5, it has been demonstrated that aromatic
monoamines in the brain are increased in concentration only on
peripheral administration of tetrahydrobiopterin at a dose close to
a lethal dose. Furthermore, Non-Patent Document 6 is literature
covering formulation of a prodrug to be described below, Non-Patent
Document 7 is literature covering synthesis of sepiapterin to be
described below, Non-Patent Document 8 is literature covering the
Fukushima-Nixon method to be described below, Non-Patent Document 9
is literature covering a method for measuring amounts of serotonin,
5-hydroxytryptophan and 5-hydroxyindole acetic acid. [0021] [Patent
Document 1] Japanese published Unexamined Patent Application No.
JP59-25323 A1 [0022] [Patent Document 2] Japanese Translation of
International Application (Kohyo) No. JP2008-504295 A1 [0023]
[Patent Document 3] WO96/03989 [0024] [Patent Document 4] Japanese
published Unexamined Patent Application No. JP06-192100 A1 [0025]
[Non-Patent Document 1] K. Sawabe, K. Yamamoto, Y. Harada, A.
Ohashi, Y. Sugawara, H. Matsuoka, and H. Hasegawa, "Cellular uptake
of sepiapterin and push-pull accumulation of tetrahydrobiopterin."
Mol Genet Metab 94 (2008) 410-416. [0026] [Non-Patent Document 2]
H. Hasegawa, K. Sawabe, N. Nakanishi, and O. K. Wakasugi, "Delivery
of exogenous tetrahydrobiopterin (BH4) to cells of target organs:
role of salvage pathway and uptake of its precursor in effective
elevation of tissue BH4." Mol Genet Metab 86 Suppl 1 (2005) S2-10.
[0027] [Non-Patent Document 3] A. Niederwieser, H.-Ch. Curtius, M.
Wang and D. Leupold, "Atypical phenylketonuria with defective
biopterin metabolism. Monotherapy with tetrahydrobiopterin or
sepiapterin, screening and study of biosynthesis in man.": Eur J
Pediatr (1982) 138: 110-112. [0028] [Non-Patent Document 4] G.
Kapatos, S. Katoh and S. Kaufman, "Biosynthesis of biopterin by rat
brain.": Journal of Neurochem. 39, 1152-1162 (1982). [0029]
[Non-Patent Document 5] M. P. Brand, K. Hyland, T. Engle, I. Smith
and S. J. R. Heales, "Neurochemical effects following peripheral
administration of tetrahydropterin derivatives to the hph-1
mouse.": Journal of Neurochem. 66, 1150-1156 (1996). [0030]
[Non-Patent Document 6] K. Beaumont, R. Webster, I. Gardner, K.
Dack, "Design of ester prodrugs to enhance oral absorption of
poorly permeable compounds: challenges to the discovery
scientist.": Current Drug Metabolism (2003), 4(6), 461-485 [0031]
[Non-Patent Document 7] W. Pfleiderer, "Pteridine, LXVIII.
Uberfuhrung von Biopterin in Sepiapterin und absolute Konfiguration
des Sepiapterins (Konfiguration Des Sepiapterins).": Chemische
Berichte 112 (1979) 2750-2755. [0032] [Non-Patent Document 8] T.
Fukushima and J. C. Nixon, "Analysis of reduced forms of biopterin
in biological tissues and fluids": Analytical Biochemistry 102,
176-188 (1980) [0033] [Non-Patent Document 9] F. Inoue, H.
Hasegawa, M. Nishimura, M. Yanagisawa and A. Ichiyama,
"Distribution of 5-hydroxytryptamine (5HT) in tissue of a mutant
mouse deficient in mast cell (W/Wv). Demonstration of the
contribution of mast cells to the 5HT content in various organs":
Agents Actions 16, 2950301 (1985)
DISCLOSURE OF THE INVENTION
Problem to be Solved by the Invention
[0034] As described above, trials have been so far made for
peripherally administering tetrahydrobiopterin to improve symptoms
of diseases caused by defective production of
tetrahydrobiopterin.
[0035] However, peripheral administration of tetrahydrobiopterin
facilitates metabolism of phenylalanine, synthesis of aromatic
monoamines and synthesis of nitric oxide at the peripheries but
hardly facilitates biosynthesis of monoamine neurotransmitters in
the brain. This is assumed to be due to the fact that
tetrahydrobiopterin hardly passes through the blood-brain barrier
and is less likely to pass through cell membranes of aromatic
monoamine neurons, even if a small amount of tetrahydrobiopterin
reaches the brain.
[0036] Therefore, tetrahydrobiopterin is effective in facilitating
metabolism of phenylalanine, synthesis of aromatic monoamines and
synthesis of nitric oxide at the peripheries but not effective in
facilitating synthesis of aromatic monoamines in the brain. That
is, in cerebral dysfunction such as depression, hyperphagia,
autism, impaired consciousness and concentration, and cognitive
disturbance, tetrahydrobiopterin administration hardly improves
their symptoms in reality, and is also not practically viable.
[0037] Under these circumstances, an object of the present
invention is to provide new means for improving symptoms of
cerebral dysfunction, for example, central mental disorders (such
as depression, hyperphagia, autism, impaired consciousness and
concentration, and cognitive disturbance) and central motor
disorders (such as myotonia, rigidity and tremor).
Means for Solving the Problems
[0038] The inventor has newly found that sepiapterin passes through
the blood-brain barrier on peripheral administration of sepiapterin
and is taken up into neurons in the brain and also facilitates
production of aromatic monoamines in the brain, thereby increasing
their bioavailability.
[0039] Under these circumstances, the present invention provides a
drug which contains at least one of sepiapterin and its salt for
preventing or improving cerebral dysfunction, and the invention
further provides a food and or drink which contains at least one of
sepiapterin and its salt for preventing or improving cerebral
dysfunction.
[0040] Unlike tetrahydrobiopterin, etc., sepiapterin is capable of
preventing decrease in the levels of aromatic monoamines in the
brain (for example, any one of or a plurality of serotonin,
dopamine and noradrenaline) in neurons in the brain on peripheral
administration and also increasing the bioavailability. Therefore,
sepiapterin may be effective against cerebral dysfunction arising
from decreased levels of aromatic monoamines in neurons in the
brain, for example, central mental disorders such as depression,
hyperphagia, autism, impaired consciousness and concentration, and
cognitive disturbance or central motor disorders such as myotonia,
rigidity and tremor.
[0041] A mechanism by which the bioavailability of aromatic
monoamines in the brain increases by sepiapterin is assumed to be
as follows. After peripheral administration, sepiapterin passes
through the blood-brain barrier more easily than
tetrahydrobiopterin and reaches neurons in the brain by a certain
amount. Unlike tetrahydrobiopterin, sepiapterin permeates through
cell membranes of neurons in the brain by facilitated transport and
is taken up into cells. In the neurons in the brain, sepiapterin is
converted to tetrahydrobiopterin through two-step enzymatic
reactions by SPR (Sepiapterin Reductase) and DHFR (Dihydrofolate
Reductase) present in the cells. Thereby, tetrahydrobiopterin in
neurons in the brain is increased in amount, facilitating
biosynthesis of aromatic monoamines in the brain and elevating the
intracellular level of aromatic monoamines in the brain, thus
resulting in an increase in the bioavailability.
[0042] In the case of tetrahydrobiopterin, even if
tetrahydrobiopterin reaches the brain in a small amount through the
blood-brain barrier on peripheral administration, it will not be
easily taken up into neurons. This is fundamentally different in
action mechanism from sepiapterin.
Effects of the Invention
[0043] The present invention may be capable of improving symptoms
of various types of cerebral dysfunction.
BEST MODES FOR CARRYING OUT THE INVENTION
<Action Mechanism of the Present Invention>
[0044] With reference to FIG. 1, an explanation will be made for an
action mechanism of sepiapterin of the present invention in the
brain.
[0045] FIG. 1 is an illustration which depicts a metabolic system
of aromatic monoamines in neurons in the brain. It is noted that
FIG. 1 addresses a metabolic system of serotonin as an example.
However, other aromatic monoamines such as dopamine and
noradrenaline produced from dopamine are also essentially the same
in metabolic system.
[0046] Substances necessary for neurons in the brain such as
nutrients, humoral regulators and physiologically active substances
are supplied from the blood stream (as "blood" in FIG. 1, and the
same shall apply hereinafter) across the blood-brain barrier
("blood-brain barrier" in FIG. 1, and the same shall apply
hereinafter) to neurons (a site described as "monoaminergic neuron"
in FIG. 1, and the same shall apply hereinafter).
[0047] The blood-brain barrier is mainly constituted of (1) a blood
vessel wall in the brain ("blood vessel wall" in FIG. 1, and the
same shall apply hereinafter), (2) a glia cell in the perineural
cavity ("perineural cavity" in FIG. 1, a region between a blood
vessel wall and a neuron, the same shall apply hereinafter), etc.
As described above, migration of substances, drugs, etc., in the
blood stream to the brain is strictly restricted by the blood-brain
barrier and only limited compounds are allowed to migrate to the
brain. Compounds in the brain are also strictly restricted for
discharge by the blood-brain barrier. Only limited metabolites and
metabolic products are principally discharged from the brain.
[0048] A substance whose migration to neurons in the brain is
restricted by the blood-brain barrier is discharged (1) by active
outward transport at blood vessel walls and glia cells after
reaching the vicinity of a neuron by physico-chemical diffusion, or
discharged (2) from the vicinity of the neuron by finally returning
to the blood stream of the brain without being taken up into the
neuron after reaching the vicinity of the neuron.
[0049] On the other hand, a substance taken up into neurons in the
brain reaches the neurons first at the perineural cavity (1) by
physico-chemical permeation and diffusion at cell membranes or
cellular gaps of glia cells, etc., or (2) by cooperative mediation
of a transporter protein group present in the glia cells.
[0050] Next, a substance which has reached a neuron is taken up
into the neuron (1) by physico-chemical permeation and diffusion at
the cell membrane of the neuron or (2) by cooperative mediation of
a transporter protein group present in the neuron. In general,
since the concentration of each substance in a neuron is determined
for each substance, a substance which has been taken up into the
neuron and thereafter rapidly metabolized is further taken up
continuously depending on an amount that has been metabolized.
[0051] Metabolism of aromatic monoamines undergoes in aromatic
monoaminergic neurons in the brain ("monoaminergic neuron" in FIG.
1 and the same shall apply hereinafter).
[0052] For example, serotonin ("Serotonin" in FIG. 1 and the same
shall apply hereinafter) is metabolized by the following mechanism
as shown in FIG. 1. First, tryptophan of L-amino acid ("Tryptophan"
in FIG. 1 and the same shall apply hereinafter) is taken up from
the blood stream via the blood-brain barrier into the monoaminergic
neuron. This tryptophan is converted to 5-hydroxytryptophan ("5HTP"
in FIG. 1 and the same shall apply hereinafter) by actions of
tryptophan hydroxylase ("TPH" in FIG. 1 and the same shall apply
hereinafter) and a coenzyme thereof, that is, tetrahydrobiopterin
("BH4" in FIG. 1 and the same shall apply hereinafter) and further
converted to serotonin by the action of aromatic amino acid
decarboxylase ("AADC" in FIG. 1 and the same shall apply
hereinafter). Serotonin biosynthesis rate-limited at the step of
reaction with tryptophan hydroxylase (TPH).
[0053] It is noted that 5-hydroxytryptophan (5HTP) is a substance
which is now used as the drug of first choice on peripheral
administration to a patient with tetrahydrobiopterin deficiency for
the purpose of increasing the amount of serotonin in the brain. It
is known that, as with tryptophan, 5-hydroxytryptophan is taken up
into an aromatic monoaminergic neuron from the blood stream via the
blood-brain barrier on peripheral administration.
[0054] Biosynthesized serotonin is taken up into
neurotransmitter-releasing granules in an aromatic monoaminergic
neuron and released outside of the cell. However, serotonin does
not migrate to the blood stream but remains in the brain and (1) is
again taken up into the releasing granules in the aromatic
monoaminergic neuron mediated by a serotonin transporter ("SERT" in
FIG. 1 and the same shall apply hereinafter) and (2) flows out to
the blood stream mediated by an organic anion transporter or others
after being subjected to metabolic deactivation by 5HIAA by actions
of monoamine oxidase ("MAO" in FIG. 1 and the same shall apply
hereinafter). It is known that, in the brain, serotonin metabolism
(biosynthesis, release, reuptake, metabolic deactivation and
flowing into the blood stream) is repeated in a cyclic manner and
its metabolic turnover is fast.
[0055] As described earlier, some aromatic monoamines are present
in peripheral cells, etc., are present in neurons of the central
nervous system. Aromatic monoamines in the brain are in principle
do novo synthesized and metabolized therein without passing through
the blood-brain barrier.
[0056] That is, aromatic monoamines in the brain are biosynthesized
and accumulated in neurons in the brain. Release of aromatic
monoamines into perineural cavities, reuptake into the neurons and
metabolic deactivation is also carried out in the brain. Metabolic
deactivation is carried out primarily in glia cells and partially
in neurons.
[0057] Therefore, it is thought that active aromatic monoamines in
the brain will not flow out into periphery as they are after being
released from neurons but will flow out to peripheries after
metabolic deactivation. It is also thought that peripheral aromatic
monoamines will not migrate to the brain or reach neurons in the
brain or perineural cavities. That is, a mere increase in serotonin
at a periphery, for example, in urine, does not necessarily
indicate an increase in serotonin in the brain.
[0058] In general, it is thought that an appropriate level of
aromatic monoamines in the brain is determined by a balance of
various factors such as the rate of biosynthesis in the brain,
accumulation at releasing granules, synaptic release into
pericellular cavities by neurons, reuptake and metabolic
deactivation.
[0059] Tetrahydrobiopterin (BH4) is a coenzyme which is essential
in action of tryptophan hydroxylase (TPH). Tetrahydrobiopterin
(BH4) is capable of passing through a blood vessel wall in the
blood-brain barrier at a rate similar to 5-hydroxytryptophan (5HTP)
but is taken up very little by glia cells, etc., present in a
perineural cavity. Furthermore, since tetrahydrobiopterin (BH4) is
present substantially at a constant level in neurons, it is taken
up very little into the neurons even when elevation of the
concentration of tetrahydrobiopterin (BH4) elevates near the
neurons. Therefore, as shown in FIG. 1, it is thought that the
majority of tetrahydrobiopterin (BH4) is not taken up into glia
cells or neurons after reaching the vicinity of the neurons and
thereafter finally returns to the blood stream in the brain.
[0060] As shown in FIG. 1, sepiapterin ("SP" in FIG. 1 and the same
shall apply hereinafter) is peripherally administered, passes
through the blood-brain barrier after reaching the brain by the
blood stream and is taken up into a monoaminergic neuron. In the
monoaminergic neuron, sepiapterin is converted to
tetrahydrobiopterin (BH4) via dihydrobiopterin ("BH2" in FIG. 1)
and facilitates biosynthesis of serotonin and release thereof as a
coenzyme of tryptophan hydroxylase (TPH) which is the rate limiting
enzyme.
[0061] Sepiapterin (SP) passes through the blood vessel wall in the
blood-brain barrier at a rate substantially similar to
5-hydroxytryptophan (5HTP) and is also taken up by glia cells,
etc., present at a perineural cavity 10 times or more efficiently
than tetrahydrobiopterin (BH4). Therefore, sepiapterin (SP) is
thought to reach neurons in a greater amount than
tetrahydrobiopterin (BH4).
[0062] Moreover, sepiapterin (SP) is readily converted to
tetrahydrobiopterin (BH4) via dihydrobiopterin (BH2) after being
taken up into a neuron. Therefore, sepiapterin in the neuron is
kept relatively low in concentration. As a result, sepiapterin is
continuously further taken up by the amount which has been
converted to tetrahydrobiopterin (BH4). Thus, sepiapterin is
thought to be taken up into the neuron in a greater amount than
tetrahydrobiopterin (BH4).
[0063] When the above findings are comprehensively taken into
account, peripheral supply of sepiapterin (SP) makes it possible to
keep tetrahydrobiopterin (BH4) in a neuron in a greater amount than
peripheral supply of tetrahydrobiopterin (BH4). Therefore, it is
possible to activate tryptophan hydroxylase (TPH) more effectively
and facilitate the biosynthesis of serotonin and release
thereof.
[0064] Drugs such as an SSRI and an SNRI are those which increase
the level of serotonin at a perineural cavity in the brain by
inhibiting reuptake of serotonin by a serotonin transporter (SERT).
Furthermore, a monoamine oxidase inhibitor is a drug which
increases the level of serotonin in the brain by suppressing
metabolic deactivation caused by monoamine oxidase (MAO).
<Drug of the Present Invention>
[0065] The present invention covers a wide variety of drugs which
contain at least one of sepiapterin and its salt for preventing,
improving and treating cerebral dysfunction.
[0066] As described above, sepiapterin means
7,8-dihydro-6-[(S)-2-hydroxy-1-oxopropyl]-pterin. An oxo group
present in a substituent arranged in the 6-position of pterin may
play an important role in allowing sepiapterin to pass through the
blood-brain barrier. Therefore, if such a structure is kept that
the oxo group is retained in the substituent to effect
intracellular conversion to tetrahydrobiopterin, any drug, the
structure of which is partially modified, is also included in
sepiapterin of the present invention.
[0067] For example, isosepiapterin
(7,8-dihydro-6-[(S)-2-oxo-1-hydroxypropyl]-pterin) is different in
position of an oxo group but has an oxo group in the substituent,
as with sepiapterin, and also maintains a structure which can be
converted to tetrahydrobiopterin by actions of enzymes in the body
such as sepiapterin reductase, aldose reductase and dihydrofolate
reductase. Therefore, in the present invention, isosepiapterin is
included in sepiapterin of the present invention as a compound
similar to sepiapterin.
[0068] The drug of the present invention includes not only
sepiapterin in itself and similar products (such as isosepiapterin)
but also pharmaceutically acceptable salts and solvates. The salts
include alkaline metal salts (such as sodium salt, potassium salt
and lithium salt), alkaline earth metal salts (such as calcium
salt, magnesium salt and lithium salt), metal salts (such as
aluminum salt, iron salt, zinc salt, copper salt and nickel salt),
inorganic salts (such as phosphate, sulfate, hydrobromate, ammonium
salt), organic acid salts (such as methanesulfonate,
p-toluenesulfonate, lactate, acetate, trifluoroacetate, citrate,
succinate, fumarate, maleate and salicylate), organic amine salts
(such as methylamine salt, dimethylamine salt, trimethylamine salt,
ethylenediamine salt, diethylamine salt, triethylamine salt,
ethanolamine salt, diethanolamine salt, dibenzylamine salt,
glucosamine salt, dicyclohexylamine salt and tetramethylammonium
salt), amino acid salts (such as glycine salt, lysine salt,
arginine salt, ornithine salt and asparagine salt) and other
organic salts (such as piperidine salt, morpholine salt,
tris-(2-hydroxyl-ethyl) amine salt and choline hydrate).
[0069] The drug of the present invention also includes a prodrug
composed of a compound having at least one of the protective groups
which are pharmacologically acceptable and dissociable under
physiological conditions. The prodrug is made available by a
publicly known method (for example, refer to Non-Patent Document
6). The prodrug is made available by adding free carboxylic acid,
an alkoxy group (for example, ethoxy group), phenalkyloxy group
(for example, benzyloxy group), OCH (R.sup.a) OCOR.sup.b group (for
example, pivaloyloxymethyloxy group), OCH(R.sup.a) OCO.sub.2R.sup.b
group (for example, [[(1-methylethoxy) carbonyl]oxy]ethylester
group and proxetil group), OCH(R.sup.a)OR.sup.b group, 2-alkyl
group, 2-cycloalkyl group, 2-cycloalkyl alkyl group,
oxycarbonyl-2-alkylidene-ethoxy group,
5-alkyl[1,3]dioxyl-2-on-oil-methyloxy group, dialkylamino-alkoxyl
group, and acryloxy group (R.sup.a is a hydrogen atom or
(C.sub.1-C.sub.4) alkyl group, and R.sup.b is any one of a hydrogen
atom, (C.sub.1-C.sub.6) alkyl group, (C.sub.2-C.sub.6) alkenyl
group, (C.sub.1-C.sub.6) alkoxy-(C.sub.1-C.sub.6) alkyl group,
(C.sub.1-C.sub.6) haloalkoxy-(C.sub.1-C.sub.6) alkyl group,
(C.sub.3-C.sub.6) cycloalkyl group, or (C.sub.3-C.sub.6)
cycloalkylmethyl group). Moreover, where a free-form hydroxyl group
is present in the structure, a protective group such as sulphate
(OSO.sub.3H), phosphate (OPO.sub.3H.sub.3), oxymethylene phosphate
(OCH.sub.2OPO.sub.3H.sub.3), succinate ester
(OCOCH.sub.3CH.sub.3COOH), ester of dimethylaminoglycine, a natural
amino acid, an inorganic salt or others is added to make the
prodrug available.
[0070] The drug of the present invention is not in particular
restricted to the dosage form. The drug is available, for example,
in solid preparations (such as tablets, capsules, granules,
powders, and sustained-release tablets) and liquid preparations
(such as syrups and injections).
[0071] A carrier which is pharmacologically acceptable may be used
to formulate a compound of the present invention into a drug. The
carrier includes a variety of organic and inorganic carrier
substances which are commonly used as pharmaceutical
ingredients.
[0072] For example, in solid preparations, a diluting agent, a
smoothing agent, a binder, a disintegrating agent, etc., are
formulated into the drug of the present invention and its carrier.
In liquid preparations, a solvent, a solubilizing agent, a
suspending agent, an isotonic agent, a buffering agent, a soothing
agent, etc., are appropriately formulated into the drug of the
present invention and its carrier. Also, pharmaceutical additives
such as an antiseptic, an antioxidant agent, a coloring agent and a
sweetening agent may be added whenever necessary.
[0073] A preferable diluting agent includes, for example, lactose,
sucrose, D-mannitol, starch, crystalline cellulose and light
silicic anhydride.
[0074] A preferable smoothing agent includes, for example,
magnesium stearate, calcium stearate, talc and colloidal
silica.
[0075] A preferable binder includes, for example, crystalline
cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose,
hydroxypropylmethyl cellulose and polyvinyl pyrrolidone.
[0076] A preferable disintegrating agent includes, for example,
starch, carboxymethyl cellulose, carboxymethyl cellulose calcium,
croscarmellose sodium and sodium carboxylmethyl starch.
[0077] A preferable solvent includes, for example, injection
solvent, alcohol, propylene glycol, macrogol, sesame oil and corn
oil.
[0078] A preferable solubilizing agent includes, for example,
polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate,
ethanol, trisaminomethane, cholesterol, triethanolamine, sodium
carbonate and sodium citrate.
[0079] A preferable suspending agent includes, for example, surface
active agents (such as stearyltriethanolamine, sodium lauryl
sulfate, laurylamino propionate, lecithin, benzalkonium chloride,
benzethonium chloride and glyceryl monostearate) and hydrophilic
high polymers (such as polyvinyl alcohol, polyvinyl pyrrolidone,
sodium carboxymethyl cellulose, methylcellulose, hydroxymethyl
cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose).
[0080] A preferable isotonic agent includes, for example, sodium
chloride, glycerin and D-mannitol.
[0081] A preferable buffering agent includes, for example,
buffering solutions of phosphate, acetate, carbonate, citrate,
etc.
[0082] A preferable soothing agent includes, for example, benzyl
alcohol.
[0083] A preferable antiseptic includes, for example,
p-parahydroxybenzoate esters, chlorobutanol, benzyl alcohol,
phenethyl alcohol, dehydroacetic acid and sorbic acid.
[0084] A preferable antioxidant agent includes, for example,
sulfite and ascorbic acid.
[0085] In addition, the drug of the present invention may contain
auxiliaries, for example, a light absorption pigment helpful in
storage and efficacy retention (such as riboflavin, adenine and
adenosine), a chelating agent/reducing agent for stabilization
(such as vitamin C and citric acid), an amino acid substrate which
enhances effects of sepiapterin in the brain (such as tryptophan)
and analogous substances (such as tetrahydrobiopterin and
dihydrobiopterin).
[0086] Although depending on its dosage form, route of
administration and carrier, the drug of the present invention can
be produced according to common procedures in which sepiapterin is
allowed to be contained usually in a range of 0.1 to 99% (w/w) with
respect to a total amount of formulation.
<Indications and Dosage Regimen of the Drug of the Present
Invention>
[0087] The indications are not in particular restricted and may
include any type of cerebral dysfunction which is decreased in the
intracellular level of aromatic monoamines in the brain.
[0088] The above-described diseases include, for example, any one
of the central mental disorders such as depression, hyperphagia,
autism, impaired consciousness and concentration, and cognitive
disturbance, or central motor disorders such as myotonia, rigidity
and tremor. Cerebral dysfunction may be prevented, improved and
treated by, for example, administration of sepiapterin at an
effective dose to patients with cerebral dysfunction.
[0089] The drug of the present invention is applicable to mammals
(for example, humans, horses, cattle, dogs, cats, rats, mice, pigs
and monkeys).
[0090] The drug can be administered orally, for example, as
tablets, capsules (including soft capsules and micro-capsules),
powders and granules, or parenterally as injections, suppositories
and pellets. Parenteral administration includes intravenous,
intramuscular, subcutaneous, intraorgan, intranasal, intradermal,
eye drop, intracerebral, intrarectal, vaginal and intraperitoneal
administrations.
[0091] The drug of the present invention varies in dosage,
depending on an administration route and symptoms. On intravenous
administration to a patient, the drug is administered once daily at
a dose of 0.1 to 100 mg/kg.times.the body weight. For example, the
drug is given at this dose once daily or in one to three divided
doses.
[0092] The drug of the present invention may be administered solely
or concomitantly with other drugs, depending on the aim, usage or
symptoms. For example, an SSRI and an SNRI have certain medicinal
benefits. However, there is a risk that aromatic monoamines may be
decreased in amount on long-term administration. On the other hand,
it may be possible to obtain synergetic effects on concomitant
administration of the drug of the present invention and these
drugs.
[0093] It may also be possible to improve effects of sepiapterin in
combination with, for example, an inhibitor of a retrograde
transporter which prevents intracerebral migration of sepiapterin
or an inhibitor of an extravert transporter which shortens the
retention time of tetrahydrobiopterin in the brain by
administration of sepiapterin.
[0094] Moreover, it is known that probenecid which is a renal
excretion-type inhibitor is capable of prolonging in vivo retention
of tetrahydrobiopterin at the peripheries. Therefore, it may be
possible to increase the effect or prolong the retention time by
using the drug of the present invention together with the renal
excretion-type inhibitor.
[0095] The renal excretion-type inhibitor includes, for example,
probenecid, immunomodulators (such as cyclosporine A, FK506 and
thymosin .alpha.-1), cytokines (such as TNF and TGF-.beta.),
interferons (IFN-.alpha., IFN-.beta., IFN-.gamma.), interleukins
(such as interleukins 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 13),
macrophage/granular cell colony stimulating factors (such as
GM-CSF, G-CSF and M-CSF), erythropoietin, cytokine antagonists
(such as reticulose, ADA, AMD-3100, anti-TNF antibody,
anti-interleukin antibody, soluble interleukin receptor and
proteinkinase C inhibitor), nucleotide transporting inhibitors
(such as dipyridamole, pentoxifylline, N-acetylcysteine (NAC),
procysteine, .alpha.-trichosanthin, phosphonoformic acid, dilazep
and nitrobenzyl thioinosine), non-nucleotide reverse transcriptase
inhibitors (NNRTIs; nevirapine, loviride, delavirdine, calanolide
A, DPC-083, efavirenz, MKC-442 and capravirine), gp120 antagonists
(such as PRO-2000, PRO542 and FP21399), and integrase inhibitors
(such as T-20 and T-1249).
<Food/Drink of the Present Invention>
[0096] The present invention includes any food/drink for
preventing, improving and treating cerebral dysfunction which
contains at least one of sepiapterin and its salt as an active
ingredient.
[0097] Sepiapterin and its salt are allowed to contain in, for
example, health-promoting food (such as specified health-promoting
food and food with nutrient function claims), that is, so-called
health-conscious food/drink and other various types of food/drink.
Furthermore, sepiapterin and its salt can be formulated into
various types of seasoning, etc.
[0098] The food/drink is not in particular restricted to its form
and may be available in liquid, half-solid and solid products.
Specifically, it may be available, for example, as confectionery
such as cookies, senbei (rice cracker), jelly, yokan (sweat jelly
of beans), yogurt and manjyu (bean-jamfilled bun), refreshing
drinks, energy drinks and soups. It may also be available as a tea
by infusion. Furthermore, the above-described drug may be added by
mixture, coating or spraying, for example, in the process of
manufacturing the food/drink of the present invention or to final
products, thereby providing a health-conscious food/drink. Still
furthermore, it may be allowed to be contained in a product that is
temporarily kept in the mouth, for example, toothpaste, breath
fresheners, chewing gum and mouthwash.
<Method for Producing the Drug of the Present Invention>
[0099] Sepiapterin of the present invention can be produced in
accordance with a publicly known method. And, the method thereof is
not restricted in particular.
[0100] For example, it is possible to produce tetrahydrobiopterin
according to procedures in which the 6th-class diastereomer mixture
(6R/6S mixture) is subjected to organic synthesis and 6R is
subjected to chiral separation and purification.
[0101] Sepiapterin is also produced by utilizing the above
synthesis system on the basis of organic synthesis. That is, a
mixture of tetrahydrobiopterin with the 6th-class diastereomer
(6R/6S) is synthesized, thereafter, the mixture is oxidized to
produce a crude sepiapterin sample, and the sample is purified to
produce sepiapterin (as for an example of the sepiapterin
synthesizing method, refer to Non-Patent Document 7, for example).
It is noted that, unlike tetrahydrobiopterin, since sepiapterin
bears achiral in the 6th-class, it is possible to omit the step of
chiral separation in a method for producing sepiapterin.
Example 1
[0102] In Example 1, where PC12 cells and RBL2H3 cells were used as
aromatic monoamine synthesizing cells and sepiapterin was added to
a culture medium, evaluation was made for a total amount of
biopterin in the cells (a total amount of tetrahydrobiopterin, and
its oxidant, dihydrobiopterin and biopterin, the same shall apply
hereinafter).
[0103] The PC12 cells are cultured cells having properties of a
neuron and it is known that they synthesize dopamine, noradrenaline
and adrenaline, with tetrahydrobiopterin used as a coenzyme. The
RBL2H3 cells are cultured cells having properties of a mast cell
and it is known that they synthesize serotonin, with
tetrahydrobiopterin used as a coenzyme.
[0104] The both cells are capable of synthesizing
tetrahydrobiopterin and also retain a certain amount of
tetrahydrobiopterin in the cells but do not retain a saturated
amount thereof. Furthermore, unlike cells which are not capable of
synthesizing aromatic monoamines by themselves but take up aromatic
monoamines extracellularly and capable of secreting aromatic
monoamines in response to stimulation (for example, platelets are
not capable of synthesizing serotonin by themselves but take up
serotonin extracellularly and are capable of secreting serotonin in
response to stimulation), both cells are capable of synthesizing
aromatic monoamines by themselves. In the present example, the
inventor, etc., judged that these cells were suitable as models of
aromatic monoamine synthetic cells and used them accordingly.
[0105] The PC12 cells and the RBL2H3 cells were obtained from the
JCRB Cell Bank (National Institute of Biomedical Innovation,
Japan).
[0106] The PC12 cells were sub-cultured in a DMEM medium
(Dulbecco's Modified Eagle Medium; Dulbecco's medium, and the same
shall apply hereinafter) containing 7% bovine fetal serum and 7%
horse serum. In an experiment, the cells were plated on a 96-well
polylysine-coated plate at 2.times.10.sup.5 per well, the culture
medium was replaced by a serum-free DMEM medium one hour before
start of the experiment on the following day to carry out the
following uptake experiment.
[0107] The RBL2H3 cells were cultured in a DMEM medium containing
10% bovine fetal serum and plated on a 96-well coating-free plate
at 1.times.10.sup.5 per well. Similarly, one hour before start of
the experiment on the following day, the culture medium was
replaced by a serum-free DMEM medium to carry out the following
uptake experiment.
[0108] Sepiapterin or tetrahydrobiopterin was added to the
respective media of the PC12 cell and the RBL2H3 cell so as to give
a final concentration of 100 .mu.M. At 0, 30, 60, 120, and 180
minutes later, the media were washed quickly three times by using
an ice-cold physiological saline (PBS (+): phosphate buffered
saline containing Ca.sup.2+, mg.sup.2+) to measure a total amount
of biopterin in the respective cells (n=6 each).
[0109] A total amount of biopterin in the cells was measured by
using a system of high performance liquid
chromatography/fluorescence detection (HPLC/FD) according to the
Fukusima-Nixon method (refer to Non-Patent Document 8).
[0110] A principle of the Fukushima-Nixon method is as follows.
Upon oxidation with iodine under strong acid or alkaline
conditions, tetrahydrobiopterin is quantitatively oxidized into
oxidized-form biopterin under acid conditions and oxidized into
oxidized-form pterin in which a side chain in the 6th-class
position is removed under alkaline conditions. Dihydrobiopterin is
oxidized into oxidized-form biopterin irrespective of pH
conditions. The oxidized-form biopterin and the oxidized-form
pterin have strong natural fluorescent characteristics (excitation:
350 nm, fluorescence: 450 nm). Therefore, the same sample is
divided into two portions, and one of them and the other are
oxidized with iodine respectively under acid conditions and
alkaline conditions. An amount of the oxidized-form biopterin is
compared with that of the oxidized-form pterin on fluorescence
detection after quantitative determination, thus making it possible
to determine the respective amounts of tetrahydrobiopterin and
dihydrobiopterin in an original sample.
[0111] The thus prepared sample was used in the system of high
performance liquid chromatography/fluorescence detection. And, the
oxidized-form biopterin and the oxidized-form pterin were
quantitatively determined according to an external standard
comparison method. Then, calculated was an amount of
tetrahydrobiopterin and a total amount of biopterin in the cells.
In the high performance liquid chromatography, "Fine-SIL-C18-5T
(made by JASCO Corporation)" was used as a column, and a 7% aqueous
methanol solution was used as an eluent. An FP model made by JASCO
Corporation was used to carry out fluorescence detection.
[0112] The respective cells used in the present example contain by
nature oxidized-form biopterin and oxidized-form pterin only in a
trace amount. The experiment was done on the assumption that they
were not present.
[0113] Sepiapterin is not metabolized in the cells by uptake in a
period of time during which the experiment was performed except for
dihydrobiopterin, irrespective of whether it is exogenous or
endogenous. Dihydrobiopterin in the cells is reduced by
dihydrofolate reductase to tetrahydrobiopterin but some of
dihydrobiopterin remains in the cells. It is found that reactions
other than reduction of dihydrobiopterin to tetrahydrobiopterin and
decomposition of tetrahydrobiopterin hardly take place in a period
of time during which the experiment was performed. It is also clear
that sepiapterin is capable of migrating from inside to outside of
the cells and vice versa, dihydrobiopterin is capable of migrating
in the above-described manner only slightly, and
tetrahydrobiopterin is hardly capable of migrating in the
above-described manner (Non-Patent Document 1). On the basis of the
above findings, a sum of the amount of dihydrobiopterin and the
amount of tetrahydrobiopterin was given a total amount of biopterin
in the cells.
[0114] FIG. 2A and FIG. 2B show the results. FIG. 2A is a graph
which shows the change in the total amount of biopterin in the PC12
cells with the lapse of time, and FIG. 2B is a graph which shows a
change in the total amount of biopterin in the RBL2H3 cells with
the lapse of time. In each of the graphs, a longitudinal axis
indicates the number of moles of a total amount of biopterin per
cell population 1.times.10.sup.6 (total BP, unit: nmol/10.sup.6
cells), while a horizontal axis indicates time after addition of
sepiapterin or tetrahydrobiopterin (Time, unit: min). In each of
the graphs, black circles indicate results obtained when
sepiapterin (SP) was added, and white circles indicate results
obtained when tetrahydrobiopterin (BH4) was added. An error bar
indicates a standard deviation (the same shall apply hereinafter).
Results free of the error bar are smaller in size than a symbol
(the same shall apply hereinafter).
[0115] As shown in FIG. 2A and FIG. 2B, in the respective cells,
there was observed a remarkable increase in the total amount of
biopterin in the cells on addition of sepiapterin. However, there
was observed substantially no change in the total amount of
biopterin in the cells on addition of tetrahydrobiopterin. The
results show a continuous increase in the total amount of biopterin
in aromatic-monoamine secreting cells on addition of sepiapterin.
The results also show essentially no change in the total amount of
biopterin in the aromatic-monoamine secreting cells on addition of
tetrahydrobiopterin.
[0116] There was observed a slight increase in the total amount of
biopterin on addition of tetrahydrobiopterin. This was due to the
fact that tetrahydrobiopterin oxidized in a culture medium under
experiment conditions was turned into dihydrobiopterin and taken up
secondarily (refer to Non-Patent Document 1). Furthermore,
biopterin in the cells was made up of 95% of tetrahydrobiopterin
and remaining percentages of dihydrobiopterin.
[0117] When the above results are comprehensively taken into
account, tetrahydrobiopterin was hardly taken up into aromatic
monoamine synthetic cells on addition of tetrahydrobiopterin, and
there was observed substantially no change in the total amount of
biopterin in the cells. On the other hand, sepiapterin was taken up
into the aromatic monoamine synthetic cells on addition of
sepiapterin and converted to tetrahydrobiopterin in the cells,
therein the total amount of biopterin was increased in the
cells.
[0118] That is, the results of the present example have suggested
that, on peripheral administration of sepiapterin to animals
including humans, unlike tetrahydrobiopterin, sepiapterin passes
through the cell membrane of an aromatic monoamine neuron after
passing through the blood-brain barrier. And, sepiapterin is taken
up into the cells and converted to tetrahydrobiopterin in the
cells, thereby facilitating biosynthesis of aromatic
monoamines.
Example 2
[0119] In Example 2, a cell system of a brain blood vessel wall
model was used to compare passage of sepiapterin with that of
tetrahydrobiopterin across a blood vessel wall.
[0120] As the brain blood vessel wall model, there was used a "BBB
kit, RBT24H (made by PharmaCo-Cell Company Ltd. in Japan)." This
kit was a model system in which rat vascular endothelical cells
were cultured on a porous synthetic resin film having small pores
of 3 .mu.m in inner diameter to form tight intercellular junctions,
thereby forming the blood vessel wall. In this kit, pericytes were
cultured in advance on the back side of the porous synthetic resin
film and astroglia cells were also cultured at the same time in the
well below the film, thereby forming the intercellular tight
junctions of vascular endothelical cells. A cultured area was 0.3
cm.sup.2 per well. In this model, the cell sheet, an upper side of
the cell sheet and a lower side of the cell sheet respectively
correspond to the blood vessel wall, an intravascular cavity
(lumen) and a perineural cavity in the brain (albumen).
[0121] According to an attached manufacture's instruction, each of
tetrahydrobiopterin (BH4), sepiapterin (SP) and 5-hydroxytryptophan
(5HTP) was dissolved in the upper side of the cell sheet
corresponding to the intravascular lumen by using a physiological
balanced salt and added at a concentration of 100 .mu.M. After 30
minutes, each of them was measured for an amount which migrated
downward to the cell sheet.
[0122] An amount of tetrahydrobiopterin (BH4) was calculated
according to the method described in Example 1.
[0123] An amount of sepiapterin (SP) was calculated according to an
external standard comparison method by treating samples by a system
of high performance liquid chromatography/fluorescence detection.
In the high performance liquid chromatography, as with Example 1,
"Fine-SIL-C18-5T (made by JASCO Corporation)" was used as a column,
and a 14% aqueous methanol solution was used as an eluent.
Fluorescence detection was carried out by setting the exciting
wavelength and the fluorescence wavelength to be 412 nm and 527 nm
respectively to make measurement using an FP model made by JASCO
Corporation.
[0124] An amount of 5-hydroxytryptophan (5HTP) was calculated
according to an internal standard comparison method using N-methyl
serotonin by treating samples in the system of high performance
liquid chromatography/fluorescence detection (refer to Non-Patent
Document 9). In the high performance liquid chromatography, as with
Example 1, "Fine-SIL-C18-5T (made by JASCO Corporation)" was used
as a column. Formic acid was added to a 40 mM aqueous sodium
acetate solution, thereby adjusting pH to 3.5, and the aqueous
sodium acetate solution, acetonitrile and methanol were mixed in a
volume ratio of 100:10:5 to prepare a solution, and the solution
was used as an eluent. Fluorescence detection was carried out by
setting the exciting wavelength and the fluorescence wavelength to
be 302 nm and 350 nm respectively to make measurement using an FP
model made by JASCO Corporation.
[0125] As described earlier, 5-hydroxytryptophan (5HTP) is a
substance which is now used as a drug of the first choice on
peripheral administration to patients with tetrahydrobiopterin
deficiency for the purpose of increasing the amount of serotonin in
the brain.
[0126] FIG. 3 shows the results. FIG. 3 is a graph which shows an
amount of downward migration of each of tetrahydrobiopterin (BH4),
sepiapterin (SP) and 5-hydroxytryptophan (5HTP) added over the
upper side of the cell sheet of the brain blood vessel wall model.
In FIG. 3, a horizontal axis indicates the respective results on
addition of tetrahydrobiopterin (BH4), sepiapterin (SP) and
5-hydroxytryptophan (5HTP), and a longitudinal axis indicates the
amount of downward migration to the lower face of the cell sheet
(unit: pmol/well/30 min). Each value was subjected to the Student
t-test (p<0.05).
[0127] As shown in FIG. 3, although a significant difference was
observed, tetrahydrobiopterin (BH4) and sepiapterin (SP) were
similar to 5-hydroxytryptophan (5HTP) in the amount of migration to
the lower side of the cell sheet. That is, the results have
suggested that tetrahydrobiopterin (BH4) and sepiapterin (SP) are
capable of passing through blood vessel walls which constitute the
blood-brain barrier at a rate substantially similar to
5-hydroxytryptophan (5HTP).
Example 3
[0128] In Example 3, comparison was made between sepiapterin and
tetrahydrobiopterin in terms of uptake into astroglia cells.
[0129] The astroglia cells are major glia cells and present tightly
around vessels of the brain. This cell selectively takes up a
substance which has passed through the blood vessel walls of the
brain and supplies the substance to neurons.
[0130] Therefore, CTX TNA2 cells, that is, cultured cells derived
from the astroglia cells, were used to evaluate the uptake of
sepiapterin and that of tetrahydrobiopterin into the astroglia
cells. It is noted that the CTX TNA2 cells used were obtained from
the ATCC in the U.S.A. (American Type Culture Collection).
[0131] On a previous day of the experiment, the CTX TNA2 cells were
inoculated at 1.times.10.sup.5 per well. Thirty minutes after a
culture medium was replaced by Hank's-HEPES (pH 7.4), sepiapterin
was added at 50 .mu.M or tetrahydrobiopterin was added at 100
.mu.M, each of which was cultured for 0, 5, 10, 20, 40 and 60
minutes. After cultivation for the above-described period of time,
the culture medium was removed to quantitatively determine an
amount of each of sepiapterin (SP), dihydrobiopterin (BH2) and
tetrahydrobiopterin (BH4) (n=5 each) accumulated in the cells
according to the method described in Example 1 or Example 2.
[0132] FIG. 4A and FIG. 4B show the results. FIG. 4A is a graph
which shows the amounts of sepiapterin (SP), dihydrobiopterin (BH2)
and tetrahydrobiopterin (BH4) accumulated in the cells on addition
of sepiapterin (SP). FIG. 4B is a graph which shows the amounts of
dihydrobiopterin (BH2) and tetrahydrobiopterin (BH4) on addition of
tetrahydrobiopterin (BH4). In each of the graphs, a horizontal axis
indicates cultivation time after addition of sepiapterin or
tetrahydrobiopterin. A longitudinal axis indicates the amounts of
sepiapterin (SP), dihydrobiopterin (BH2) and tetrahydrobiopterin
(BH4) which were quantitatively determined (unit: pmol/10.sup.6
cells).
[0133] As shown in FIG. 4A and FIG. 4B, when addition of
sepiapterin (SP) (FIG. 4A) was compared with addition of
tetrahydrobiopterin (BH4) (FIG. 4B), it was revealed that the
amount of tetrahydrobiopterin (BH4) accumulated in the cultured
cells was at least 10 times greater on addition of sepiapterin than
on addition of tetrahydrobiopterin. And, this amount was at least
20 times greater on conversion of an added amount to the same
concentration.
[0134] The above results have suggested that sepiapterin is taken
up more easily into glia cells 10 times or more than
tetrahydrobiopterin, and tetrahydrobiopterin (BH4) is rapidly
synthesized in the glia cells via dihydrobiopterin (BH2) from
sepiapterin (SP) taken up into the glia cells.
[0135] As described above, the blood-brain barrier is primarily
formed with blood vessel walls and glia cells. Astrocytes, a major
type of glia cell, selectively take up a substance which has passed
through blood vessel walls of the brain and supply the substance to
neurons. Therefore, it is thought that the substance which has
passed through the blood vessel walls and has been taken up by
astroglia cells will reach neurons.
[0136] The results of Example 2 and the present example revealed
that sepiapterin was substantially similar to tetrahydrobiopterin
in amount which has passed through the blood vessel walls further
sepiapterin was at least 10 times greater in amount taken up in
glia cells than tetrahydrobiopterin.
[0137] Therefore, the above results have suggested that sepiapterin
passes through the blood-brain barrier at least 10 times more
easily than tetrahydrobiopterin. That is, when the results of
Example 1 are also taken into account, sepiapterin passes through
the blood-brain barrier more easily than tetrahydrobiopterin on
peripheral administration and is also easily taken up by aromatic
monoamine neurons.
Example 4
[0138] In Example 4, rats were used to measure amounts of
tetrahydrobiopterin, serotonin and 5-hydroxyindoleacetic acid in
the brain on administration of sepiapterin.
[0139] Here, 5-hydroxyindoleacetic acid, which is a metabolic
product of serotonin, is thought to be metabolized and converted
from serotonin mainly in glia cells or serotonin-producing cells.
In this experiment, 5-hydroxyindoleacetic acid was also measured as
an index of the bioavailability of an aromatic monoamine
(serotonin) in the brain.
[0140] Rats used were SD rats (7-8 week old, males) purchased from
Japan SLC, Inc. The rats were kept in a dark place for 12 hours and
in a light place for 12 hours and fed ad libitum with a diet
("MM-3" made by Funabashi Farm Co., Ltd.) and sterilized tap water
as drinking water.
[0141] Sepiapterin or tetrahydrobiopterin was dissolved in 10 mM
hydrochloric acid, the resultant of which was orally administered
to the rats (n=6) under diethylether anesthesia. After 1, 1.5, 2,
3, 4, 6 and 8 hours, the brains were excised under pentobarbital
sodium anesthesia (pentobarbital was intraperitoneally administered
at 40 mg/kg five minutes before), and the brain was divided into
two portions at the median line to obtain left and right brain
samples. Rats not treated with sepiapterin or tetrahydrobiopterin
were used as samples at 0 hours after administration according to
the same procedures.
[0142] An amount of tetrahydrobiopterin in the brain was measured
and calculated according to a method similar to Example 1 by adding
5 times the volume of 100 mM hydrochloric acid to a left brain
sample, homogenizing brain tissues and utilized the supernatant
solution.
[0143] Amounts of serotonin and 5-hydroxyindoleacetic acid in the
brain were calculated by adding 3.5 times the volume of 1.43%
ascorbic acid-containing 100 mM hydrochloric acid which contains
N-methyl serotonin as an internal standard to a right brain sample,
homogenizing brain tissues, adding potassium perchlorate (final
concentration of 5.5%) thereto, ice-cooling the resultant for
removing protein and treating a supernatant thereof in a system of
high performance liquid chromatography/fluorescence detection
(HPLC/FD), (refer to Non-Patent Document 9). In the high
performance liquid chromatography, as with Example 1, etc.,
"Fine-SIL-C18-5T (made by JASCO Corporation)" was used as a column.
As with Example 2, formic acid was added to a 40 mM aqueous sodium
acetate solution, thereby adjusting pH to 3.5. Then, the aqueous
sodium acetate solution, acetonitrile and methanol were mixed in a
volume ratio of 100:10:5 to prepare a solution, and the solution
was used as an eluent. As with Example 1, etc., fluorescence
detection was carried out by setting the exciting wavelength and
the fluorescence wavelength to be 302 nm and 350 nm respectively
and making measurement using an FP model made by JASCO Corporation.
In the system of high performance liquid
chromatography/fluorescence detection (HPLC/FD),
5-hydroxytryptophan (5HTP), serotonin, N-methyl serotonin,
tryptophan and 5-hydroxyindoleacetic acid are eluted in the above
order, thus making it possible to determine quantitatively these
substances at the same time.
[0144] The results are shown in FIG. 5A, FIG. 5B, and FIG. 5C.
[0145] FIG. 5A is a graph which shows change in the amount of
tetrahydrobiopterin in the brain with the lapse of time after
administration of sepiapterin. In the graph, a longitudinal axis
indicates an amount of tetrahydrobiopterin (BH4, unit: nmol/g
brain), and a horizontal axis indicates time after administration
of tetrahydrobiopterin or sepiapterin (time, unit: hour). In the
graph, black circles indicate results on addition of sepiapterin
(SP), and white circles indicate results on addition of
tetrahydrobiopterin (BH4). The two-way analysis of variance
revealed that a value obtained on administration of sepiapterin was
statistically and significantly higher than a value obtained on
administration of tetrahydrobiopterin over 1.5 to 6 hours after
administration (p<0.0001).
[0146] As shown in FIG. 5A, a group treated with
tetrahydrobiopterin did not show an increase in the amount of
tetrahydrobiopterin in the brain, while a group treated with
sepiapterin showed a significant increase in the amount of
tetrahydrobiopterin in the brain.
[0147] The above results have shown that peripheral administration
of sepiapterin increases the amount of tetrahydrobiopterin in the
brain. The results have also shown that peripheral administration
of tetrahydrobiopterin at the same dose does not increase the
amount of tetrahydrobiopterin in the brain.
[0148] Of brain cells, approximately 90% or more are made up of
non-neurons and the remaining part of approximately 10% is made up
of neurons. Aromatic monoamine nerves occupy only a small portion
of the remaining part. Therefore, all tetrahydrobiopterin which has
been elevated in the brain does not necessarily account for an
elevation thereof in aromatic monoamine neurons. However, as shown
in Example 1, sepiapterin easily migrates to aromatic monoamine
synthetic cells and is reduced to tetrahydrobiopterin in the cells.
Furthermore, as shown in Non-Patent Document 1, it is known that
sepiapterin enters into cells in the form of sepiapterin and is
quickly reduced to tetrahydrobiopterin and also the
tetrahydrobiopterin is retained for a relatively long period of
time in the cells. As described above, of continuous elevation of
tetrahydrobiopterin on administration of sepiapterin as shown in
FIG. 5A, a substantial part of the elevation is estimated at a
higher possibility to take place in the aromatic monoamine
synthetic cells. Furthermore, administration of tetrahydrobiopterin
at the same dose does not result in elevation of
tetrahydrobiopterin in the brain, which is in compliance with the
suggestion obtained from the results of Example 1 and content
thereof.
[0149] Then, FIG. 5B is a graph which shows change in the amount of
serotonin in the brain with the lapse of time after administration
of sepiapterin. FIG. 5C is a graph which also shows change in the
amount of 5-hydroxyindoleacetic acid (5HIAA) in the brain with the
lapse of time. In each of the graphs, a horizontal axis indicates
time from administration of tetrahydrobiopterin or sepiapterin
(time, unit: hour) and a longitudinal axis indicates an amount of
serotonin (5HT, unit: nmol/g brain) or that of
5-hydroxyindoleacetic acid (5HIAA, unit: nmol/g brain). In each of
the graphs, black circles indicate results on addition of
sepiapterin (SP) and white circles indicate results on
administration of tetrahydrobiopterin (BH4). The two-way analysis
of variance revealed that, in the above cases, a group treated with
sepiapterin was significantly higher in the amount of serotonin
than a group treated with tetrahydrobiopterin (p<0.0001).
[0150] As shown in FIG. 5B and FIG. 5C, in the group treated with
tetrahydrobiopterin, serotonin and 5-hydroxyindoleacetic acid in
the brain were not increased in amount over a long period of time.
However, in the group treated with sepiapterin, serotonin in the
brain was significantly increased in amount from three to eight
hours and subsequently after administration.
[0151] The above results have suggested that on peripheral
administration of sepiapterin, a part of sepiapterin goes beyond
the blood-brain barrier to reach the brain, then, is taken up into
neurons capable of synthesizing aromatic monoamines in the brain
and converted to tetrahydrobiopterin, thus resulting in an increase
in the amount of serotonin in the brain. Furthermore, peripheral
administration of tetrahydrobiopterin at the same dose does not
significantly increase the amount of serotonin in the brain,
showing that tetrahydrobiopterin which has been peripherally
administered does not substantially reach aromatic monoamine
synthetic cells due to some hindrance. This result is well
complemented by the results of FIG. 5A and not contradictory to the
results of Example 1.
[0152] As described above, the results of Example 1 and the present
experiment have strongly suggested a series of action mechanisms in
which, on peripheral administration of sepiapterin, sepiapterin
passes through the blood-brain barrier by a certain amount, reaches
the brain, also passes through cell membranes of aromatic monoamine
neurons, then, is taken up into aromatic monoamine neurons in the
brain, converted to tetrahydrobiopterin in the neurons, and the
tetrahydrobiopterin effectively contributes to the synthesis of
serotonin, thereby increasing the amount of serotonin in the brain.
The results have also suggested that peripheral administration of
tetrahydrobiopterin at the same dose will not enhance the series of
action mechanisms in the brain.
Example 5
[0153] In Example 5, mice were used to measure the amount of
serotonin (5HT) in the brain after administration of
sepiapterin.
[0154] The mice used were those of "hph-1" which were provided by
Dr. K. Hyland (Institute of Metabolic Disease, Baylor University
Medical Center, Dallas, Tex. 75226, USA). The mice are
characterized as being defective in biosynthesis functions of
tetrahydrobiopterin, with the level of tetrahydrobiopterin in the
brain being from 40 to 50% as compared with ordinary mice. The mice
were bred in a dark place for 12 hours and also in a light place
for 12 hours and fed ad libitum with a diet ("MM-3" made by
Funabashi Farm Co., Ltd.) and sterilized tap water as drinking
water.
[0155] An amount of serotonin was measured by dissolving
sepiapterin or tetrahydrobiopterin in 0.01 M hydrochloric acid,
thereafter administering orally each of the resultants at 20 mg/kg
to the mice, giving another oral administration at the same dose
two hours thereafter, and excised the brains from each mouse six
hours after the first administration (n=7 in a group treated with
sepiapterin, and n=8 in a group treated with tetrahydrobiopterin).
Measurement of the amount of serotonin was made by obtaining brain
samples according to a method similar to Example 4, treating the
samples in a system of high-performance liquid
chromatography/fluorescence detection (HPLC/FD) after dissolution
of the samples and protein removal from them, and setting the
excited wavelength and the fluorescence wavelength to be 302 nm and
350 nm respectively. In a control group, measurement was made
similarly by orally administering 0.01M hydrochloric acid.
[0156] FIG. 6 shows the results. FIG. 6 is a graph which shows the
amount of serotonin in the brain after administration of
sepiapterin. In the graph, a longitudinal axis indicates the amount
of serotonin (5HT, unit: nmol/g brain), "v-cont" shows the results
of a control group, "BH4" shows the results on administration of
tetrahydrobiopterin and "SP" shows the result on administration of
sepiapterin, respectively. In the graph, an asterisk shows a
significant difference found by t-test (p<0.05).
[0157] As shown in FIG. 6, a group treated with sepiapterin showed
a significant increase in serotonin in the brain as compared with a
group treated with tetrahydrobiopterin. That is, the results show
that, as with Example 4, a significant increase in the level of an
aromatic monoamine in the brain (serotonin) was observed on
administration of sepiapterin in the experiment with mice as
well.
[0158] As described above, Example 4 and the present example showed
that peripheral administration of sepiapterin increased the amount
of serotonin in the brain but peripheral administration of
tetrahydrobiopterin at the same dose did not increase the
amount.
[0159] It is known that intracerebral biosynthesis, release,
reuptake and metabolism of dopamine whose starting material is
tyrosine of aromatic amino acid are also based on a synthesis
amount of dopamine at dopaminergic neurons and dopamine
biosynthesis is restricted by the amount of intracellular
tetrahydrobiopterin. Therefore, the results of Example 4 and the
present example have suggested that dopamine as well as
noradrenaline and adrenaline synthesized from dopamine are also
increased in intracerebral levels thereof on peripheral
administration of sepiapterin. That is, the above results have
suggested that peripheral administration of sepiapterin may also be
effective in improving central mental disorders and central motor
disorders which are involved in decreased levels of dopamine,
noradrenaline and adrenaline in the brain.
Example 6
[0160] In Example 6, the mice were subjected to a forced swim test
after administration of sepiapterin.
[0161] The forced swim test of the mice is a test method for
evaluating antidepressant effects, and length of "immobile" time is
a criterion of depression. Evaluation is made in such a manner that
the shorter the "immobile" time is, the greater the antidepressant
effects are.
[0162] The mice used were NZB mice purchased from Japan SLC, Inc
(7-week old males). The mice were maintained in a dark place for 12
hours and in a light place for 12 hours and fed ad libitum with a
diet ("MM-3" made by Funabashi Farm Co., Ltd.) and sterilized tap
water as drinking water, except when they were constrained in an
experiment.
[0163] The mice are characterized in that they are more likely to
develop an auto-immune disease with advancement of age. However, in
the present experiment, the mice did not exhibit symptoms of the
disease when being used in the experiment (7-week old). The mice
which had been subjected to 15-minute preliminary swimming without
administration of a drug, etc., on the previous day were used in
the experiment.
[0164] The mice were intraperitoneally administered
tetrahydrobiopterin (n=7) or sepiapterin (n=5) at a single dose of
10 mg/kg and subjected to a forced swim test after 40 minutes to
measure "immobile" time. The test was conducted under dim light by
using a 15 cm-across and 15 cm-deep water tank kept at a
temperature of 22.degree. C. "Immobile" time was measured by
calculating an added value of "immobile" time during
five-minute-swimming. A similar experiment was carried out by
giving a physiological saline to a control group (n=5) and giving
desipramine at 40 mg/kg to a positive control group (n=5).
Desipramine, that is, a tricyclic antidepressant, was used because
it was judged to be used appropriately as a positive control for
demonstrating anti-depressant effects.
[0165] FIG. 7 shows the above results. FIG. 7 is a graph which
shows "immobile" time after administration of sepiapterin in the
mice forced swim test. In the graph, a longitudinal axis indicates
"immobile" time (unit: min. during 5 min.), "control" indicates the
result of the control group, "BH4" indicates the result on
administration of tetrahydrobiopterin, "SP" indicates the result on
administration of sepiapterin, and "Dsp" indicates the result on
administration of desipramine to the positive control group.
Furthermore, the t-test revealed a significant difference of
p=0.005 between "control" and "SP" as well as a significant
difference of p=0.004 between "BH4" and "SP." Furthermore, a
difference between "SP" and "Dsp" was p=0.69, which was estimated
not to be a significant difference in terms of statistics.
[0166] As shown in FIG. 7, a group treated with sepiapterin was
significantly short in "immobile" time as compared with a group
treated with tetrahydrobiopterin and the control group. Thus,
anti-depressant effects were obtained. Furthermore, the group
treated with sepiapterin was also effective in shortening
"immobile" time to an extent similar to the positive control
group.
[0167] As described above, the results of Examples 1 to 6 have
suggested that tetrahydrobiopterin hardly reaches the brain or is
not taken up into neurons on peripheral administration, thus
resulting in no increase in the level of aromatic monoamines in the
brain, and improvement in cerebral dysfunction can be hardly
expected. On the other hand, sepiapterin partially reaches the
brain and is easily taken up into neurons on peripheral
administration, thus resulting in an increased level of aromatic
monoamines in the brain, suggesting effectiveness in improving
cerebral dysfunction.
[0168] Therefore, these results have suggested that the present
invention is effective in preventing, improving and treating
central mental disorders such as depression, hyperphagia, autism,
impaired consciousness and concentration, and cognitive
disturbance. In addition, biosynthesis of dopamine, noradrenaline
and adrenaline depends on the level of tetrahydrobiopterin in the
neurons, as with serotonin. Thus, the results have suggested that
the present invention is effective in preventing, improving and
treating central motor disorders such as myotonia, rigidity and
tremor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0169] FIG. 1 is an illustration which depicts a metabolic system
of aromatic monoamine in neurons in a brain.
[0170] FIG. 2A is a graph which shows change in the total amount of
biopterin taken up into PC12 cells with the lapse of time in
Example 1.
[0171] FIG. 2B is a graph which shows change in the total amount of
biopterin taken up into RBL2H3 cells with the lapse of time in
Example 1.
[0172] FIG. 3 is a graph which shows an amount of downward
migration of each of tetrahydrobiopterin (BH4), sepiapterin (SP)
and 5-hydroxytryptophan (5HTP) added over the upper side of the
cell sheet of the brain blood vessel wall model (RBT24H) in Example
2.
[0173] FIG. 4A is a graph which shows amounts of sepiapterin (SP),
dihydrobiopterin (BH2) and tetrahydrobiopterin (BH4) accumulated in
CTX, TNA2 cells on addition of sepiapterin (SP) in Example 3.
[0174] FIG. 4B is a graph which shows amounts of dihydrobiopterin
(BH2) and tetrahydrobiopterin (BH4) accumulated in the CTX INA 2
cells on addition of tetrahydrobiopterin (BH4) in Example 3.
[0175] FIG. 5A is a graph which shows change in the amount of
tetrahydrobiopterin in the brain with the lapse of time after
administration of sepiapterin in an experiment with rats in Example
4.
[0176] FIG. 5B is a graph which shows change in the amount of
serotonin in the brain with the lapse of time after administration
of sepiapterin in the experiment with rats in Example 4.
[0177] FIG. 5C is a graph which shows change in the amount of
5-hydroxyindoleacetic acid in the brain after administration of
sepiapterin in the experiment with rats in Example 4.
[0178] FIG. 6 is a graph which shows the amount of serotonin in the
brain after administration of sepiapterin in an experiment with
mice (hph-1) in Example 5.
[0179] FIG. 7 is a graph which shows "immobile" time after
administration of sepiapterin in a forced swim test with mice (NZB)
in Example 6.
* * * * *