U.S. patent application number 09/427579 was filed with the patent office on 2002-06-27 for prophylactic or therapeutic agents for diseases haivng vascular dysfunction associated with insulin resistance.
Invention is credited to KASHIWAGI, ATSUNORI, KIKKAWA, RYUICHI, NISHIO, YOSHIHIKO, OKAMURA, TOMIO, SHINOZAKI, KAZUYA, TODA, NOBORU.
Application Number | 20020082261 09/427579 |
Document ID | / |
Family ID | 12783164 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082261 |
Kind Code |
A1 |
KASHIWAGI, ATSUNORI ; et
al. |
June 27, 2002 |
PROPHYLACTIC OR THERAPEUTIC AGENTS FOR DISEASES HAIVNG VASCULAR
DYSFUNCTION ASSOCIATED WITH INSULIN RESISTANCE
Abstract
It is an object of the present invention to provide a
pharmaceutical composition for effectively preventing or improving
diseases having vascular dysfunction associated with insulin
resistance. The present invention provides pharmaceutical
compositions for preventing or treating diseases having vascular
dysfunction associated with insulin resistance, comprising as an
active ingredient a compound of the formula (I): 1 wherein R.sup.1
and R.sup.2 each represents a hydrogen atom or taken together with
each other represent a single bond, while R.sup.3 represents
--CH(OH)CH(OH)CH.sub.3, --CH(OCOCH.sub.3)CH(OCOCH.sub.3)CH.sub- .3,
--CH.sub.3, --CH.sub.2OH or a phenyl group when R.sup.1 and R.sup.2
each represents a hydrogen atom, or --COCH(OH)CH.sub.3when R.sup.1
and R.sup.2 together represents a single bond, or a
pharmaceutically acceptable salt thereof.
Inventors: |
KASHIWAGI, ATSUNORI;
(SHIGA-KEN, JP) ; SHINOZAKI, KAZUYA; (SHIGA-KEN,
JP) ; NISHIO, YOSHIHIKO; (SHIGA-KEN, JP) ;
OKAMURA, TOMIO; (SHIGA-KEN, JP) ; TODA, NOBORU;
(OSAKA, JP) ; KIKKAWA, RYUICHI; (OSAKA,
JP) |
Correspondence
Address: |
MANELLI DENISON & SELTER
2000 M STREET NW SUITE 700
WASHINGTON
DC
20036-3307
US
|
Family ID: |
12783164 |
Appl. No.: |
09/427579 |
Filed: |
October 27, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09427579 |
Oct 27, 1999 |
|
|
|
PCT/JP99/00917 |
Feb 25, 1999 |
|
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|
Current U.S.
Class: |
514/249 |
Current CPC
Class: |
A61P 3/06 20180101; A61P
43/00 20180101; A61P 9/10 20180101; A61K 31/505 20130101; A61P 3/00
20180101; A61P 3/08 20180101; A61P 9/00 20180101; A61P 41/00
20180101; A61P 9/12 20180101; A61P 9/08 20180101; A61K 31/519
20130101; A61P 3/14 20180101 |
Class at
Publication: |
514/249 |
International
Class: |
A61K 031/495; A61K
031/47 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 1998 |
JP |
47720/1998 |
Claims
What is claimed is:
1. A pharmaceutical composition for preventing or treating diseases
having vascular dysfunction associated with insulin resistance,
comprising as an active ingredient a compound of the formula (I):
5wherein R.sup.1 and R.sup.2 each represents a hydrogen atom or
taken together with each other represent a single bond, while
R.sup.3 represents --CH(OH)CH(OH)CH.sub.3,
--CH(OCOCH.sub.3)CH(OCOCH.sub.3)CH.sub.3, --CH.sub.3, --CH.sub.2OH
or a phenyl group when R.sup.1 and R.sup.2 each represents a
hydrogen atom, or -COCH(OH)CH.sub.3 when R.sup.1 and R.sup.2
together represents a single bond, or a pharmaceutically acceptable
salt thereof.
2. The pharmaceutical composition of claim 1, wherein the disease
having vascular dysfunction associated with insulin resistance is a
disease with increased production of active oxygen.
3. The pharmaceutical composition of claim 1, wherein the disease
having vascular dysfunction associated with insulin resistance is a
disease with decreased NO production.
4. The pharmaceutical composition of any one of claims 1 to 3,
wherein R.sup.3 is L-erythro-CH(OH)CH(OH)CH.sub.3.
5. The pharmaceutical composition of any one of claims 1 to 4,
which is for preventing or treating coronary vasoconstrictive
angina.
6. The pharmaceutical composition of any one of claim 1 to 4, which
is for preventing or treating effort angina.
7. The pharmaceutical composition of any one of claim 1 to 4, which
is for preventing or treating coronary arteriorestenosis following
percutaneous transluminal coronary angioplasty (PTCA) or coronary
artery bypass grafting (CABG).
8. The pharmaceutical composition of any one of claim 1 to 4, which
is for preventing or treating arteriosclerosis.
9. The pharmaceutical composition of any one of claim 1 to 4, which
is for preventing or treating cerebrovascular constrictive
lesion.
10. Use of a compound of the formula (I): 6wherein R.sup.1 and
R.sup.2 each represents a hydrogen atom or taken together with each
other represent a single bond, while R.sup.3 represents
--CH(OH)CH(OH)CH.sub.3, --CH(OCOCH.sub.3)CH(OCOCH.sub.3 )CH.sub.3,
--CH.sub.3, --CH.sub.2OH or a phenyl group when R.sup.1 and R.sup.2
each represents a hydrogen atom, or --COCH(OH)CH.sub.3 when R.sup.1
and R.sup.2 together represents a single bond, or a
pharmaceutically acceptable salt thereof, for the preparation of a
pharmaceutical composition for preventing or treating diseases
having vascular dysfunction associated with insulin resistance.
11. A method for preventing or treating diseases having vascular
dysfunction associated with insulin resistance, comprising
administering a pharmaceutical composition comprising a compound of
the formula (I): 7wherein R.sup.1 and R.sup.2 each represents a
hydrogen atom or taken together with each other represent a single
bond, while R.sup.3 represents --CH(OH)CH(OH)CH.sub.3,
--CH(OCOCH.sub.3)CH(OCOCH.sub.3 )CH.sub.3, --CH.sub.3, --CH.sub.2OH
or a phenyl group when R.sup.1 and R.sup.2 each represents a
hydrogen atom, or --COCH(OH)CH.sub.3when R.sup.1 and R.sup.2
together represents a single bond, or a pharmaceutically acceptable
salt thereof.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to pharmaceutical compositions
for preventing and/or treating diseases having vascular dysfunction
associated with insulin resistance, comprising as an active
ingredient a compound of the formula (I): 2
[0002] wherein R.sup.1 and R.sup.2 each represents a hydrogen atom
or taken together with each other represent a single bond, while
R.sup.3 represents --CH(OH)CH(OH)CH.sub.3,
--CH(OCOCH.sub.3)CH(OCOCH.sub.3)CH.sub- .3, --CH.sub.3,
--CH.sub.2OH or a phenyl group when R.sup.1 and R.sup.2 each
represents a hydrogen atom, or --COCH(OH)CH.sub.3 when R.sup.1 and
R.sup.2 together represents a single bond, or a pharmaceutically
acceptable salt thereof.
[0003] Insulin resistance is a pathologic state observed in
patients with type II diabetes and typically characterized by a
lack of lowering of blood glucose level even under a high insulin
state. Recently, a pathologic state characterized by a complication
of abnormal glucose tolerance, obesity, hypertension and
hyperlipidemia in one individual was reported and named insulin
resistance syndrome, syndrome X or offal fat syndrome. Large-scale
epidemiological studies showed that these pathologic states are
basically associated with insulin resistance and may be a hazardous
factor in various arteriosclerotic diseases. Consequently, it is
clinically important to explain and prevent these states.
[0004] We have clinically examined the role of insulin resistance
in vascular endothelial dysfunction and an advanced state thereof,
such as arteriosclerotic process as well as various
arteriosclerotic diseases. We first found that marked
hyperinsulinemia independent from other hazardous factors, i.e.
insulin resistance also occurs in diseases other than diabetes such
as non-diabetic coronary vasoconstrictive angina (Shinozaki, K. et
al., Circulation 1995, 92: 1749-1757). We also showed the presence
of marked insulin resistance in effort angina or cases having
significant constrictive lesion in cerebral angiography (Shinozaki,
K. et al., Diabetes Care 1996, 19: 1-7; Shinozaki, K. et al.,
Stroke 1996, 27:37-43). Furthermore, we also showed the presence of
insulin resistance and initial arteriosclerosis in vivo (Shinozaki,
K. et al., Arterioscler. Thromb. Vasc. Biol., 1997, 17:
3302-3310).
[0005] Vascular endothelium has been known to play an important
role in vascular tonus or thrombopoiesis, and in 1980, the presence
of endothelium-derived relaxing factor (EDRF) was first reported.
The entity of EDRF was proved to be nitric oxide (NO) in 1987. NO
is a gaseous radical and has been shown to readily pass through
cell membranes and exert a wide variety of effects such as
circulation control, neurotransmission, inhibition of platelet
aggregation, antibacterial or anticancer effect. NO not only
controls metabolism by reacting with heme enzyme or SH enzyme
groups, but also has physiological functions and pathological
activity by crosstalking with active oxygen species such as
superoxide (O.sub.2.sup.-), SH compounds, ascorbic acid or the
like. However, its in vivo molecular entity is still unknown in
many respects because all of these molecules are unstable.
[0006] NO having a wide variety of effects as described above is
produced when L-arginine is oxidized from
N.sup.G-hydroxyl-L-arginine into L-citrulline and the reaction is
catalyzed by an enzyme called NO synthase (NOS). NOS widely occurs
in the vascular endothelium, nervous system, kidney, platelets,
cardiac muscles, smooth muscles, etc. and the gene therefor has
already been cloned and structurally analyzed. As a result, the
gene for NOS was found to contain a binding site for
(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (hereinafter referred to
as "BH4") included in compounds of the formula (I) as active
ingredients of the present invention, in addition to those for
coenzymes such as calmodulin (CaM), flavin, NADPH. Moreover, BH4
has been suggested to actually be involved in control of the
function of NOS.
[0007] We examined the in vivo influence of a high insulin state on
superoxide production and endothelium-dependent vasorelaxing
ability, on the hypothesis that vascular tonus abnormality and
vascular endothelial cell disorder might be caused by increased
production of active oxygen species in insulin resistant state. The
result showed that some mechanism hinders activation of NOS in
insulin resistant state while NOS is activated in the vascular
endothelium to maintain the vasorelaxing ability in an exogenous
high insulin state induced by externally administering insulin.
This suggests that the presence of superoxide may be excessive on
vascular walls due to a decrease of NO resulting in an acceleration
of arteriosclerosis and enhancement of vasoconstriction in insulin
resistant states (Shinozaki, K., Kashiwagi, A. et al. : Superoxide
anion impairs endothelium-dependent vascular relaxation in insulin
resistant rat aortas. Jap. J. Pharmacology 75 (1997) suppl. 1, p.
11).
[0008] Thus, the relationship between insulin resistant states and
endothelial dysfunction has been posited. Various studies have been
made on drugs for improving vascular dysfunction caused by insulin
resistance to prevent or treat various diseases associated
therewith and thiazolidine dione derivatives were mentioned as
candidates therefor (Law, R. E. et al., Troglitazone inhibits
vascular smooth muscle cell growth and intimal hyperplasia, J.
Clin. Invest. 98: 1897, 1996). However, no definite conclusion has
yet been reached.
[0009] Recent investigations of various vascular diseases at a
molecular level have led to a therapeutic strategy directed to
blood vessels such as endothelial cells. And it is considered that
one of the most promising therapies is to treat blood vessels with
an agent which controls production of the entity of EDRF, i.e. NO,
or an agent which has an antioxidant action (Gibbons, G. H., Dzau,
V. J., Science. Vol. 272, 689-693, 1996). For example, antioxidant
agents such as vitamins E or probucol are expected to resist
oxidative stress in coronary arteriorestenosis known to be caused
by the metabolite having an oxidant action following percutaneous
transluminal coronary angioplasty (PTCA) or coronary artery bypass
grafting (CABG) (Tardif, J. C. et al. : N. Eng. J. Med. 1997, 337:
365-372), and nitrate agents such as nitroglycerin preparations are
used as exogenous NO donors for therapy of angina. However, no drug
or therapy that satisfies this therapeutic strategy has yet been
established.
[0010] The purpose of therapy is to prevent complications of the
vascular system caused by insulin resistance so that patients may
enjoy a prolonged and higher quality of life. This requires
lifelong management by long-term pharmacotherapy. In spite of
various studies on therapeutic agents for insulin resistance as
described above, no drug exists at present that is completely
satisfactory in terms of side effects, safety during long-term use
and improvement in QOL (quality of life). Thus, the development of
therapeutic agents satisfying truly desirable conditions is in
great demand.
[0011] The compounds of the formula (I) as active ingredients in
pharmaceutical compositions of the present invention are known
compounds for use in pharmaceutical compositions against malignant
hyperphenylalaninemia, depression, Parkinson's disease, etc. For
example, see Japanese Patent Public Disclosure (KOKAI) Nos.
25323/84, 76086/84, 277618/86 and 267781/88.
SUMMARY OF THE INVENTION
[0012] It is an object of the present invention to provide a safe
pharmaceutical composition for diseases having vascular dysfunction
associated with insulin resistance without side effects, which
prevents the progress of conditions, prevents the progress of
complications and improves the quality of life of patients.
[0013] We, the inventors, hypothesized about therapy for diseases
having vascular dysfunction associated with insulin resistance that
endothelial dysfunction caused by insulin resistant state might be
improved by controlling both of increased production of active
oxygen species and decreased production of NO to normalize each of
them. As a result of careful studies to improve
endothelium-dependent vasorelaxation, we unexpectedly found that
BH4, which is a coenzyme for NOS, improves decreased production of
endogenous NO and also suppresses increased production of active
oxygen species in insulin resistant state to significantly improve
lowered endothelium-dependent vasorelaxation. Thus, we discovered
the effect of BH4 in improving vascular dysfunction in insulin
resistant states, and as a result accomplished the present
invention.
[0014] The present invention relates to an effective therapy with
BH4 preparations for diseases having vascular dysfunction
associated with insulin resistance.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention relates to a pharmaceutical
composition for preventing or treating diseases having vascular
dysfunction associated with insulin resistance, comprising as an
active ingredient a compound of the formula (I): 3
[0016] wherein R.sup.1 and R.sup.2 each represents a hydrogen atom
or taken together with each other represent a single bond, while
R.sup.3 represents --CH(OH)CH(OH)CH.sub.3,
--CH(OCOCH.sub.3)CH(OCOCH.sub.3)CH.sub- .3, --CH.sub.3,
--CH.sub.2OH or a phenyl group when R.sup.1 and R.sup.2 each
represents a hydrogen atom, or --COCH(OH)CH.sub.3 when R.sup.1 and
R.sup.2 together represents a single bond, or a pharmaceutically
acceptable salt thereof.
[0017] As used herein, the term insulin resistance means a
pathologic state where enhancement of peripheral sugar assimilation
by normally secreted insulin declines, while suppression of the
glucose level released from the liver declines to induce abnormal
glucose tolerance and suppression of lipolysis by insulin also
declines. Characteristically, it often presents with
hyperinsulinemia, but it is not always the case. Insulin resistance
has been reported to occur in not only insulin-sensitive cells but
also vascular wall cells. Diseases having vascular dysfunction
associated with insulin resistance include those caused by insulin
resistance, those aggravated by insulin resistance, those for which
cure is retarded by insulin resistance, etc., such as hypertension,
hyperlipidemia, arteriosclerosis, coronary vasoconstrictive angina,
effort angina, cerebrovascular constrictive lesion, cerebrovascular
insufficiency, cerebral vasospasm, peripheral circulation disorder,
coronary arteriorestenosis following percutaneous transluminal
coronary angioplasty (PTCA) or coronary artery bypass grafting
(CABG), obesity, insulin-independent diabetes, hyperinsulinemia,
lipid metabolism abnormality, coronary arteriosclerotic heart
diseases or the like so far as they are associated with insulin
resistance.
[0018] When administered to patients with these diseases, BH4 can
prevent or treat these diseases by activating the functions of NOS,
increasing NO production and suppressing the production of active
oxygen species to improve disorders of vascular endothelial
cells.
[0019] Accordingly, the treatment or prevention according to the
present invention is directed to insulin resistant diseases
associated with vascular dysfunction having vascular tonus
abnormality or endothelial dysfunction.
[0020] Compounds of the formula (I) as active ingredients of the
present invention include the following ones and pharmaceutically
acceptable salts thereof: 4
[0021] Among aforementioned compounds, 5,6,7,8-tetrahydrobiopterins
or salts thereof are preferable, and BH4 or salts thereof are the
most preferable.
[0022] Compounds of the formula (I) used as active ingredients in
the present invention are known compounds. For example, see
Japanese Patent Public Disclosure (KOKAI) Nos. 25323/84, 76086/84,
277618/86 and 267781/88. These compounds may be used as appropriate
salts with pharmacologically non-toxic acids, including mineral
acids such as hydrochloric acid, phosphoric acid, sulfuric acid,
boric acid; and organic acids such as acetic acid, formic acid,
maleic acid, fumaric acid, mesylic acid.
[0023] Pharmaceutical compositions of the present invention are
effective against the above-mentioned diseases. For example, they
are effective against, but not limited to, hypertension,
hyperlipidemia, arteriosclerosis, coronary vasoconstrictive angina,
effort angina, cerebrovascular constrictive lesion, cerebrovascular
insufficiency, cerebral vasospasm, peripheral circulation disorder,
coronary arteriorestenosis following percutaneous transluminal
coronary angioplasty (PTCA) or coronary artery bypass grafting
(CABG), obesity, insulin-independent diabetes, hyperinsulinemia,
lipid metabolism abnormality, coronary arteriosclerotic heart
diseases or the like so far as they are associated with insulin
resistance.
[0024] Pharmaceutical compositions of the present invention are
prepared by formulating a compound of the formula (I) with a
pharmaceutically common carrier by conventional procedures into a
dosage form suitable for oral, rectal or parenteral administration
(including administration into the vein and cerebrospinal
fluid).
[0025] The carrier used for these pharmaceutical formulations
generally includes excipients, binders, disintegrators, etc.
depending on the dosage form chosen.
[0026] Typical examples of excipients include starch, lactose,
sucrose, glucose, mannitol, cellulose, and examples of binders
include polyvinylpyrrolidone, starch, sucrose,
hydroxypropylcellulose and Arabic gum. Examples of disintegrators
include starch, agar, gelatin powder, cellulose, CMC, but any other
conventional excipients, binders and disintegrators may also be
used.
[0027] In addition to such carriers, pharmaceutical compositions of
the present invention may also contain antioxidants for stabilizing
active ingredients. Antioxidants can be appropriately selected from
those commonly used for pharmaceutical preparations, such as
ascorbic acid, N-acetylcysteine, L-cysteine, dl-.alpha.-tocopherol,
natural tocopherol, etc. They are used in an amount that stabilizes
(one or more) active ingredients, and generally they are preferably
used in the ratio of 0.2 to 2.0 parts by weight to 1 part by weight
of the active ingredient(s).
[0028] Formulations of the present invention suitable for oral
administration may be provided in the form of tablets, sublingual
tablets, capsules, powders, granules or fine granules, or
suspensions in a non-aqueous liquid such as emulsions, potions or
syrups, that contain the prescribed amount of (one or more) active
ingredients.
[0029] For example, granules are prepared by homogeneously mixing
(one or more) active ingredients with one or more auxiliary
ingredients such as carriers and antioxidants as mentioned above,
followed by granulation and sieving to uniform grain size. Tablets
can be prepared by compacting or molding (one or more) active
ingredients optionally with one or more auxiliary ingredients.
Capsules are prepared by filling powder or granules of (one or
more) active ingredients optionally mixed homogenously with one or
more auxiliary ingredients into appropriate capsules using a
capsule filling machine or the like. Formulations for renal
administration can be provided as suppositories using conventional
carriers such as cacao butter. Parenteral formulations can be
provided as dry solids of (one or more) active ingredients sealed
in a nitrogen-filled sterilized container. Such dry solid
preparations can be administered to patients by dispersing or
dissolving them into a determined amount of sterilized water just
prior to administration.
[0030] These formulations may preferably be prepared by
incorporating antioxidants as mentioned above optionally with one
or more auxiliary ingredients selected from buffers, flavors,
surfactants, thickeners, lubricants, etc. in addition to active
ingredients and ordinary carriers.
[0031] The dosage of active ingredients, i.e. compounds of the
formula (I) may naturally vary with the administration route, the
symptom to be treated and the particular patient, and may be
ultimately determined by an attendant physician.
[0032] For example, an appropriate dosage for treating diseases
having vascular dysfunction associated with insulin resistance
depends on the purpose of administration, the age, weight,
condition of the patient, etc., but ranges from 0.1 to 50 mg/kg
(body weight)/day, typically 0.5 to 10 mg/kg (body weight)/day for
oral administration.
[0033] A desired dosage of said active ingredients may be
administered once a day or may be administered in divided doses of
two to four times a day at appropriate intervals.
[0034] Active ingredients may be administered alone without being
mixed with other ingredients, or in combination with pharmaceutical
formulations containing other active ingredients suitable for the
disease under treatment to facilitate control of the dosage, for
example.
[0035] In addition to compounds of the formula (I) as active
ingredients, formulations of the present invention may contain at
least one auxiliary active ingredient selected from the group
consisting of substrates or coenzymes or cofactors for NOS such as
L-arginine, flavins (for example, FAD, FMN, etc.) and calcium. More
excellent therapeutic effects can be expected when compounds of the
formula (I) are mixed with these active ingredients than when used
alone. The proportion of each of said auxiliary active ingredients
in formulations of the present invention is not specifically
limited. For example, the weight ratio of at least one selected
from L-arginine, flavins and calcium to 1 part by weight of the
compounds of the formula (I) may be within the range from 0.1 to
10, preferably 0.5 to 2.
[0036] For example, an appropriate dosage of such mixed
formulations for treating diseases having vascular dysfunction
associated with insulin resistance depends on the purpose of
administration, the age, weight, condition of the patient, etc.,
but ranges from 0.1 to 50 mg/kg (body weight)/day, preferably 0.5
to 10 mg/kg (body weight)/day in terms of the total amount of
active ingredients for oral administration.
[0037] A physician may appropriately choose formulations containing
compounds of the formula (I) alone or in combination with other
active ingredients, depending on the age, condition or other
factors of the patient.
[0038] The most preferable active ingredients used in the present
invention are (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and
salts thereof, but their analogues such as
(6R,S)-5,6,7,8-tetrahydrobiopterin,
1',2'-diacetyl-5,6,7,8-tetrahydrobiopterin, sepiapterin,
6-methyl-5,6,7,8-tetrahydropterin,
6-hydroxymethyl-5,6,7,8-tetrahydropter- in or
6-phenyl-5,6,7,8-tetrahydropterin and salts thereof may also be
used. Needless to say, however, BH4 naturally occurring component
in living bodies is preferable. BH4 dihydrochloride exhibits little
toxicity in rats as shown by the acute toxicity of 2 g/kg (body
weight) or more via oral route. An optically inactive analogue,
(6R,S)-5,6,7,8-tetrahydro- biopterin is also only slightly toxic as
reported in Japanese Patent Public Disclosure No. 25323/84 for the
treatment of Parkinson's disease, so that it can also be used for
the therapy according to the present invention. Other compounds of
the formula (I) also exhibit little or no acute toxicity.
[0039] The following examples further illustrate the present
invention in detail without, however, limiting the same
thereto.
EXAMPLES
Example 1
Granules and Fine Granules
[0040] To 1 part (by weight) of polyvinylpyrrolidone (Kollidon 30)
dissolved in sterilized purified water were added 10 parts of
ascorbic acid and 5 parts of L-cysteine hydrochloride to give a
homogeneous solution, and then 10 parts of BH4 dihydrochloride were
added to prepare a homogeneous solution.
[0041] This solution was added to 59 parts of an excipient
(mannitol or lactose) and 15 parts of a disintegrator [corn starch
or hydroxypropylcellulose (LH-22)], and the mixture was kneaded,
granulated, dried, then sieved.
Example 2
Tablets
[0042] The homogeneous solution of an active ingredient prepared in
Example 1 was mixed with 58 parts of lactose and 15 parts of
microcrystalline cellulose, then with 1 part of magnesium stearate
and tableted.
Example 3
Capsules
[0043] The dosage form prepared in Example 1 was filled into
capsules. However, the formulation further contains 0.2% of
magnesium stearate as a lubricant.
Example 4
Injection
[0044]
1 BH4 dihydrochloride 1.5 g Ascorbic acid 1.5 g L-cysteine
hydrochloride 0.5 g Mannitol 6.5 g
[0045] The above ingredients were dissolved into sterilized
purified water to make 100 ml and sterilized, and each of 1 ml or 2
ml aliquot was dispensed into a vial or ampule, then lyophilized
and sealed.
Example 5
Injection
[0046] A solution of 2.0 g of BH4 dihydrochloride dissolved in
sterilized purified water to make 100 ml under an anaerobic
atmosphere was sterilized and sealed in the same way as in Example
4.
Example 6
Suppositories
[0047]
2 BH4 dihydrochloride 150 parts Ascorbic acid 150 parts L-cysteine
hydrochloride 50 parts
[0048] The above ingredients were homogeneously ground and
dispersed into 9950 parts of cacao butter.
Example 7
Granules
[0049]
3 BH4 dihydrochloride 5 parts Ascorbic acid 5 parts L-cysteine
hydrochloride 2 parts
[0050] The above ingredients were used to prepare a homogeneous
solution.
[0051] This solution was added to a homogeneous mixture of 55 parts
of mannitol, 1 part of polyvinylpyrrolidone, 14 parts of
hydroxypropylcellulose and 5 parts of L-arginine or calcium, and
the mixture was kneaded, granulated, dried, then sieved.
Example 8
Granules
[0052]
4 BH4 dihydrochloride 5 parts Ascorbic acid 5 parts L-cysteine
hydrochloride 5 parts Mannitol 52 parts Polyvinylpyrrolidone
(Kollidon 30) 1 part Hydroxypropylcellulose (LH-22) 12 parts
L-arginine or calcium 10 parts
[0053] The above ingredients were granulated and sieved in the same
way as in Example 7.
Example 9
Granules
[0054]
5 BH4 dihydrochloride 5 parts Ascorbic acid 5 parts L-cysteine
hydrochloride 2 parts
[0055] The above ingredients were used to prepare a homogeneous
solution.
[0056] This solution was added to a homogeneous mixture of 10 parts
of L-arginine or calcium, 50 parts of mannitol, 1 part of
polyvinylpyrrolidone (Kollidon 30) and 9 parts of
hydroxypropylcellulose (LH-22), and the mixture was kneaded,
granulated, dried, then sieved.
Example 10
[0057] Male Sprague-Dawley rats (150 g, supplied from Charles
River) were used to prepare a model group with insulin resistance
induced by fructose feed loading (hereinafter referred to as "FR
group") according to the procedure described by Hwang et al.
(Hwang, I-H. et al., Hypertension 10:512, 1987). A control group
grown with a starch feed (hereinafter referred to as "CTR group")
was also prepared (FR group: n=16; CTR group: n=16). FR group
received a feed containing 67% (hereinafter meaning % by weight)
carbohydrate (containing 98% fructose available from Oriental Yeast
Co., Ltd.), 13% fat and 20% protein, while CTR group received a
feed containing 58% carbohydrate, 12% fat and 30% protein. Ten mg
of BH4 dihydrochloride/kg (body weight)/day was orally administered
to 8 animals of each of FR group and CTR group (designated as
"FR+BH4 group" and "CTR+BH4 group", respectively). After 8 weeks,
the thoracic aorta was collected. For each group,
endothelium-separated sections (EC (-)) and non-separated sections
(EC (+)) were prepared and examined for superoxide anion
(O.sub.2.sup.-) production level, endothelial NOS (eNOS) activity
and endothelium-dependent vasorelaxation.
[0058] Statistics
[0059] Group-to-group statistic analyses were made by one-way
ANOVA, with the significance level being p<0.05. Multiple
comparison was made by Bonferronl/Dunn, with the significance level
being p<0.05.
[0060] Superoxide anion (O.sub.2.sup.-) production level in
thoracic aorta tissue sections
[0061] Measurement of O.sub.2.sup.-production level was made by the
lucigenin-enhanced chemiluminescence method (Ohara et al., J. Clin.
Invest. 91: 2546, 1993). Results in EC (+) of the 4 groups, i.e. FR
group, CTR group, FR+BH4 group and CTR+BH4 group are shown in Table
1 below.
6TABLE 1 O.sub.2 production level (100 cpm/mg dry weight) Number
Standard Group of cases Average deviation CTR 4 123.1 34.5 CTR +
BH4 4 189.3 39.2 FR 4 263.6 35.9 FR + BH4 4 118.0 24.5
[0062] As shown in Table 1, FR group showed a significantly high
O.sub.2.sup.-production level (p<0.001) of twice or more as
compared with CTR group in EC (+) when BH4 was not administered,
but O.sub.2.sup.-production level in FR group significantly
(p<0.001) decreased by BH 4 administration. No significant
difference was found among the 4 groups in EC (-).
[0063] Endothelial NOS activity
[0064] Measurement of eNOS activity was made according to Rees, D.
et al., Hypertension 28: 367, 1996. Results in EC (+) of the four
groups are shown in Table 2 below.
7TABLE 2 eNOS activity (pmol/min/mg protein) Number Standard Group
of cases Average deviation CTR 5 63.4 13.0 CTR + BH4 4 57.0 8.5 FR
5 22.3 10.9 FR + BH4 4 60.2 18.5
[0065] As shown in Table 2, eNOS activity in FR group was
significantly (p<0.001) lowered to 50% or less of CTR group when
BH4 was not administered, but it was significantly (p<0.001)
improved by BH 4 administration.
[0066] Endothelium-dependent vasorelaxation
[0067] Measurement of endothelium-dependent vasorelaxation was made
by the isometric tensiometry (Toda, N. et al., Stroke 24: 1584,
1993) using ink-writing oscillographs (Nippon Kohden Corporation)
and a force-displacement transducer (Nippon Kohden Corporation).
Data were shown as % maximum relaxation vs. the maximum relaxation
induced by 10.sup.-4 M papaverine after aortic sections were
partially precontracted with 1-3.times.10.sup.-7M 1-phenylephrine.
Endothelium-dependent vasorelaxation induced by acetylcholine
(10.sup.31 5M) and calcium ionophore (A23187, Sigma)
(3.times.10.sup.31 7 M) in EC (+) of each of the 4 groups are shown
in Tables 3 and 4, respectively.
8TABLE 3 Endothelium-dependent vasorelaxation induced by
acetylcholine (% maximum relaxation) Number Standard Group of cases
Average deviation CTR 5 89.0 3.9 CTR + BH4 5 92.9 2.9 FR 8 65.9 6.9
FR + BH4 8 82.6 2.9
[0068]
9TABLE 4 Endothelium-dependent vasorelaxation induced by calcium
ionophore (% maximum relaxation) Number Standard Group of cases
Average deviation CTR 8 85.6 7.1 CTR + BH4 8 86.4 8.0 FR 8 66.9 7.9
FR + BH4 8 81.8 2.7
[0069] As shown in Tables 3 and 4, endothelium-dependent
vasorelaxation induced by acethylcholine and calcium ionophore in
FR group was significantly (p<0.001) lowered as compared with
CTR group when BH4 was not administered, but it was significantly
(p<0.001) recovered by BH 4 administration.
[0070] In conclusion, BH4 administration lowered
O.sub.2.sup.-production level, increased eNOS activity and
recovered endothelium-dependent vasorelaxation to improve vascular
dysfunction in FR group. However, any significant change was not
observed in any of these profiles in CTR group even by BH4
administration.
[0071] As has been explained, the present invention provides
pharmaceutical compositions that effectively prevent and/or improve
diseases having vascular dysfunction associated with insulin
resistance. In addition, active ingredients of pharmaceutical
compositions of the present invention have no danger of side
effects or the like even under long-term use because they
inherently occur in living bodies.
* * * * *