U.S. patent application number 16/766992 was filed with the patent office on 2021-04-22 for disease-site-specific liposomal formulation.
The applicant listed for this patent is Cardio Incorporated, Osaka University. Invention is credited to Shigeru Miyagawa, Yoshiki Sakai, Yoshiki Sawa, Yasuhiro Yanagi.
Application Number | 20210113464 16/766992 |
Document ID | / |
Family ID | 1000005327783 |
Filed Date | 2021-04-22 |
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United States Patent
Application |
20210113464 |
Kind Code |
A1 |
Sawa; Yoshiki ; et
al. |
April 22, 2021 |
DISEASE-SITE-SPECIFIC LIPOSOMAL FORMULATION
Abstract
The present invention provides a clinically applicable, safe and
convenient, pharmaceutical composition for disease site-specific
treatment. The pharmaceutical composition for disease site-specific
treatment comprises a stealth liposome having a prostaglandin I2
receptor agonist encapsulated therein.
Inventors: |
Sawa; Yoshiki; (Osaka,
JP) ; Miyagawa; Shigeru; (Osaka, JP) ; Sakai;
Yoshiki; (Osaka, JP) ; Yanagi; Yasuhiro;
(Hyogo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Osaka University
Cardio Incorporated |
Osaka
Hyogo |
|
JP
JP |
|
|
Family ID: |
1000005327783 |
Appl. No.: |
16/766992 |
Filed: |
November 27, 2017 |
PCT Filed: |
November 27, 2017 |
PCT NO: |
PCT/JP2017/042350 |
371 Date: |
May 26, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/127 20130101;
A61K 9/1277 20130101; A61K 31/4406 20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/4406 20060101 A61K031/4406 |
Claims
1. A pharmaceutical composition for disease site-specific
treatment, comprising a stealth liposome having a prostaglandin I2
receptor agonist encapsulated therein.
2. The pharmaceutical composition according to claim 1, wherein the
prostaglandin I2 receptor agonist includes at least a compound
represented by formula (I): ##STR00009## wherein ##STR00010##
wherein e represents an integer of 3 to 5, f represents an integer
of 1 to 3, p represents an integer of 1 to 4, q represents 1 or 2,
and r represents an integer of 1 to 3; R.sup.1 represents a
hydrogen atom or a C.sub.1-4 alkyl group; R.sup.2 represents (i) a
hydrogen atom, (ii) a C.sub.1-8 alkyl group, (iii) a phenyl group
or a C.sub.4-7 cycloalkyl group, (iv) a 4- to 7-membered monocyclic
ring containing one nitrogen atom, (v) a C.sub.1-4 alkyl group
substituted with a benzene ring or a C.sub.4-7 cycloalkyl group, or
(vi) a C.sub.1-4 alkyl group substituted with a 4- to 7-membered
monocyclic ring containing one nitrogen atom; and R.sup.3
represents (i) a C.sub.1-8 alkyl group, (ii) a phenyl group or a
C.sub.4-7 cycloalkyl group, (iii) a 4- to 7-membered monocyclic
ring containing one nitrogen atom, (iv) a C.sub.1-4 alkyl group
substituted with a benzene ring or a C.sub.4-7 cycloalkyl group, or
(v) a C.sub.1-4 alkyl group substituted with a 4- to 7-membered
monocyclic ring containing one nitrogen atom; provided that when
##STR00011## is a group represented by (iii) or (iv),
--(C--(CH.sub.2).sub.p-- and .dbd.CH--(CH.sub.2).sub.s-- are bound
to position a or b on the ring, and cyclic structures in R.sup.2
and R.sup.3 are optionally substituted with one to three C.sub.1-4
alkyl groups, C.sub.1-4 alkoxy groups, halogen atoms, nitro groups,
or trihalomethyl groups; or a salt thereof.
3. The pharmaceutical composition according to claim 1, wherein the
prostaglandin I2 receptor agonist includes at least the following
compound (A): (A)
({5-[2-({[(1E)-phenyl(pyridin-3-yl)methylene]amino}oxy)ethyl]-7,8-dihydro-
naphthalen-1-yl}oxy)acetic acid (ONO-1301) represented by formula
(II): ##STR00012## or a salt of compound (A).
4. The pharmaceutical composition according to claim 1, wherein the
prostaglandin I2 receptor agonist includes at least one of the
following compounds (B) to (E): (B) sodium
(.+-.)-(1R,2R,3aS,8bS)-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-h-
ydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta[b]benzofuran-5-butanoate
(beraprost), or a derivative thereof that is a carbacyclic PGI2
derivative, (C)
[4-[(5,6-diphenylpyrazinyl)(1-methylethyl)amino]butoxy]-acetic acid
(MRE-269), (D)
(2E)-7-{(1R,2R,3R)-3-hydroxy-2-[(1E,3S,5S)-3-hydroxy-5-methylnon-1-en-1-y-
l]-5-oxycyclopentyl}hept-2-enoic acid (limaprost), ornoprostil,
enprostil, or misoprostol; or a derivative of any of these
compounds that is a PEF derivative, and (E)
2-{4-[(5,6-diphenylpyrazin-2-yl)(propan-2-yl)amino]butoxy}-N-(methanesulf-
onyl)acetamide (NS-304; selexipag); or a salt of any of compounds
(B) to (E).
5. The pharmaceutical composition according to claim 1, wherein the
prostaglandin I2 receptor agonist includes at least one member
selected from the group consisting of ONO-1301, beraprost,
limaprost, and NS-304.
6. The pharmaceutical composition according to claim 1, wherein the
stealth liposome is obtainable by using at least a prostaglandin I2
receptor agonist and a phospholipid by the Bangham method,
hydration dispersion method, reverse phase evaporation method,
ethanol injection method, ethanol dilution method, homogenization
method, mechanochemical method, direct dispersion method, extruder
method, French press method, remote loading method,
dehydration-rehydration method, freeze-thaw method, ultrasonic
method, or lipid-compound film method; or an improved method of any
of these methods.
7. The pharmaceutical composition according to claim 1, wherein the
stealth liposome has an average particle size of 50 to 200 nm, and
comprises 5 to 50 parts by weight of the phospholipid and 0.05 to 5
parts by weight of PEG-modified phosphoethanolamine, per part by
weight of the prostaglandin I2 receptor agonist.
8. The pharmaceutical composition according to claim 1, wherein the
stealth liposome comprises 0.05 to 5 parts by weight of
MPEG2000-DSPE per part by weight of the prostaglandin I2 receptor
agonist; the prostaglandin I2 receptor agonist includes at least
one member selected from the group consisting of ONO-1301,
beraprost, limaprost, and NS-304; and the stealth liposome releases
the prostaglandin I2 receptor agonist over a period of 3 hours to 4
weeks.
9. The pharmaceutical composition according to claim 1, wherein the
stealth liposome comprises a prostaglandin I2 receptor agonist, a
phospholipid, a PEG-modified phosphoethanolamine, and a
water-miscible organic solvent, and does not comprise a sterol; the
liposome is obtainable by a production method comprising the
following steps (1) to (8): (1) mixing the prostaglandin I2
receptor agonist, the phospholipid, and the PEG-modified
phosphoethanolamine in the solvent in amounts such that at least 5
mg of the phospholipid and at least 0.05 mg of the PEG-modified
phosphoethanolamine are present per mg of the prostaglandin I2
receptor agonist, (2) heating the mixture obtained in step (1) to
prepare a melt, (3) instantly freezing the melt obtained in step
(2), (4) freeze-drying the frozen product obtained in step (3) to
remove the solvent, (5) heating the freeze-dried product obtained
in step (4) to disperse the heated product in an aqueous phosphate
buffer solution, (6) sizing the dispersion obtained in step (5)
with an extruder, (7) ultrafiltrating the dispersion obtained in
step (6) to remove unencapsulated material, and (8) adding a sugar
to the dispersion obtained in step (7) and freeze-drying the
dispersion; and the liposome contains at least 0.001 mg of the
prostaglandin I2 receptor agonist per 1.0 mg of the phospholipid,
and has an average particle size of 50 to 200 nm.
10. The pharmaceutical composition according to claim 1, wherein
the composition is for intravenous administration, intracoronary
administration, inhalation, intramuscular administration,
subcutaneous administration, oral administration, transmucosal
administration, transdermal administration, or an internal organ,
and is in the form of an injectable formulation, an oral
preparation, an inhalant, a nebulizer, an ointment, a patch, or a
spray.
11. The pharmaceutical composition according to claim 1, wherein a
single intravenous dose of the composition is 0.001 to 100 mg in
terms of the prostaglandin I2 receptor agonist.
12. The pharmaceutical composition according to claim 11, wherein a
disease to be treated with the composition is cardiovascular
disease, respiratory disease, urinary disease, gastrointestinal
disease, bone disease, neurodegenerative disease, vascular disease,
dental disease, eye disease, skin disease, other inflammatory
disease, ischemic organ disorder, diabetic complication, tissue
fibrotic disease, tissue degenerative disease, or hair loss; and
the composition comprises a liposome.
13. The pharmaceutical composition according to claim 11, wherein a
disease to be treated with the composition is a cardiovascular
disease such as ischemic and dilated cardiomyopathy,
atherosclerosis obliterans, vasculitis syndrome, valvular disease,
aortic stenosis, chronic heart failure, or diastolic failure; a
respiratory lung disease such as pulmonary hypertension, pulmonary
fibrosis, asthma, or chronic obstructive pulmonary disease; a
gastrointestinal or urinary disease such as chronic kidney disease,
chronic hepatitis, or chronic pancreatitis; or a neurodegenerative
disease such as chronic phase of cerebral infarction, Alzheimer's
disease, diabetic neuropathy, Parkinson's disease, or amyotrophic
lateral sclerosis; and the composition comprises a liposome.
14. The pharmaceutical composition according to claim 2, wherein
the stealth liposome is obtainable by using at least a
prostaglandin I2 receptor agonist and a phospholipid by the Bangham
method, hydration dispersion method, reverse phase evaporation
method, ethanol injection method, ethanol dilution method,
homogenization method, mechanochemical method, direct dispersion
method, extruder method, French press method, remote loading
method, dehydration-rehydration method, freeze-thaw method,
ultrasonic method, or lipid-compound film method; or an improved
method of any of these methods.
15. The pharmaceutical composition according to claim 3, wherein
the stealth liposome is obtainable by using at least a
prostaglandin I2 receptor agonist and a phospholipid by the Bangham
method, hydration dispersion method, reverse phase evaporation
method, ethanol injection method, ethanol dilution method,
homogenization method, mechanochemical method, direct dispersion
method, extruder method, French press method, remote loading
method, dehydration-rehydration method, freeze-thaw method,
ultrasonic method, or lipid-compound film method; or an improved
method of any of these methods.
16. The pharmaceutical composition according to claim 4, wherein
the stealth liposome is obtainable by using at least a
prostaglandin I2 receptor agonist and a phospholipid by the Bangham
method, hydration dispersion method, reverse phase evaporation
method, ethanol injection method, ethanol dilution method,
homogenization method, mechanochemical method, direct dispersion
method, extruder method, French press method, remote loading
method, dehydration-rehydration method, freeze-thaw method,
ultrasonic method, or lipid-compound film method; or an improved
method of any of these methods.
17. The pharmaceutical composition according to claim 5, wherein
the stealth liposome is obtainable by using at least a
prostaglandin I2 receptor agonist and a phospholipid by the Bangham
method, hydration dispersion method, reverse phase evaporation
method, ethanol injection method, ethanol dilution method,
homogenization method, mechanochemical method, direct dispersion
method, extruder method, French press method, remote loading
method, dehydration-rehydration method, freeze-thaw method,
ultrasonic method, or lipid-compound film method; or an improved
method of any of these methods.
18. The pharmaceutical composition according to claim 2, wherein
the stealth liposome has an average particle size of 50 to 200 nm,
and comprises 5 to 50 parts by weight of the phospholipid and 0.05
to 5 parts by weight of PEG-modified phosphoethanolamine, per part
by weight of the prostaglandin I2 receptor agonist.
19. The pharmaceutical composition according to claim 3, wherein
the stealth liposome has an average particle size of 50 to 200 nm,
and comprises 5 to 50 parts by weight of the phospholipid and 0.05
to 5 parts by weight of PEG-modified phosphoethanolamine, per part
by weight of the prostaglandin I2 receptor agonist.
20. The pharmaceutical composition according to claim 4, wherein
the stealth liposome has an average particle size of 50 to 200 nm,
and comprises 5 to 50 parts by weight of the phospholipid and 0.05
to 5 parts by weight of PEG-modified phosphoethanolamine, per part
by weight of the prostaglandin I2 receptor agonist.
Description
TECHNICAL FIELD
[0001] The present invention relates to a disease-site-specific
liposomal formulation.
BACKGROUND ART
[0002] Compound (A) (ONO-1301) is a low-molecular-weight compound
having both PGI2 receptor (IP) agonism and thromboxane (TX) A2
synthase inhibitory activity. Compound (A), which has PGI2
agonistic activity, is known to be useful for prevention and/or
treatment of thrombosis, arteriosclerosis, ischemic heart disease,
gastric ulcer, hypertension, etc. (Patent Literature (PTL) 1).
[0003] On the other hand, prostaglandin (PG) I2 receptor (IP)
agonists, prostaglandin EP2 agonists, and prostaglandin EP4
agonists, such as ONO-1301, can be used as endogenous repair factor
production promoters for many diseases at low doses by inducing
many body regeneration factors, such as a hepatocyte growth factor
(HGF), a vascular endothelial cell growth factor (VEGF), a stromal
cell-derived factor (SDF-1), and a high-mobility group box protein
1 (HMGB1); and these agonists are known to be useful as
regenerative therapies (Patent Literature (PTL) 2).
[0004] However, since there are concerns about side effects of
compound (A), such as diarrhea following oral administration, and
vasodilation and hypotensive effects in intravenous administration,
the development of a dosage form that is capable of preventing
exposure to a high concentration in the gastrointestinal tract or a
rapid increase of the blood concentration, placing less burden on
the patient, and maximizing drug efficacy is strongly desired. In
the development of long-term sustained-release injectable
formulations, many studies have been conducted on methods for
controlling drug release by microspheres (hereinafter sometimes
abbreviated as MS) with an average particle diameter of about 30
.mu.m, containing a drug and using a poorly water-soluble polymer.
A biodegradable polymer is used as the polymer so that the base
does not remain at the site of administration after drug release.
In particular, for example, polylactic acid polymers (hereinafter
sometimes abbreviated as PLA) and lactic acid-glycolic acid
copolymers (hereinafter sometimes abbreviated as PLGA), which have
been used in surgical sutures, bone-fixing bolts, etc., are used.
These biodegradable polymers are used in the LH-RH derivative
injectable formulation Leuplin (sold by Takeda Pharmaceutical Co.,
Ltd.) and the long-acting somatostatin derivative Sandostatin LAR
(sold by Novartis Pharmaceuticals).
[0005] Drugs used in microspheres include bioactive peptides,
various hormones, growth factors, antibodies, peptides such as
genes and various cell growth/differentiation inducing factors,
proteins, nucleic acids, and the like. Compound (A) (Patent
Literature (PTL) 3), and compound (B) and compound (C) (Patent
Literature (PTL) 4) are known as low-molecular compounds.
[0006] These drugs can be administered, for example, by
intramuscular administration, subcutaneous injection, or patch
application to various organs, of an MS formulation; in a dosage
form that can continuously maintain the drug concentration in the
tissue at a disease site, or in a dosage form that can maintain the
blood concentration, such as intravenous infusions. When
administered at a disease site, these formulations for
administration maintain a high drug concentration in the vicinity
of an administration site, and exhibit intravenous infusion-like
blood kinetics; and do not have a drug delivery system (DDS)
effect, which is an effect of accumulating a drug at a disease
site. Specifically, DDS is a technique for delivering a required
amount of a drug to a required place at a required time.
[0007] On the other hand, there is a known lung disease
site-specific therapeutic agent whose mechanism is such that
intravenous injection of a small amount of an MS formulation
accumulates the MS formulation in the lungs and allows gradual
release of a drug in the lungs, thereby maintaining a high
concentration of the drug in the lungs (Patent Literature (PTL) 4).
However, this method has a risk such that mass administration may
cause the development of a pulmonary embolism, and thus has a
safety problem.
[0008] As a method for alleviating these problems and providing DDS
effects in a disease site-specific manner, the production of a
nanosphere (hereinafter sometimes abbreviated as NS) formulation
containing, for example, a PGI2 receptor agonist, such as compound
(A), has been considered. There are many known methods for
producing NS formulations. NS formulations are known as DDS
formulations, which are intravenously administered to utilize
vascular permeability enhancement action at inflammatory sites,
ischemic sites, and/or cancer tissues; and utilize disease
site-specific drug accumulation. However, the production of a
clinically applicable NS formulation comprising a PGI2 receptor
agonist, such as compound (A), has been difficult due to the
stability, content, yield, safety, sustained release rate,
efficacy, etc., of the formulation. In addition, it was extremely
difficult to produce an NS formulation containing compound (A)
capable of accumulation at a disease site and exhibiting the
effect.
[0009] Methods for producing an NS formulation are roughly
classified into breakdown methods and build-up methods. Breakdown
methods are methods of pulverizing particles by spray-drying or a
like method to reduce the particle size to submicron size. Build-up
methods are known to produce, for example, polymer capsule
formulations comprising a lactic acid-glycolic acid copolymer
(PLGA), a lactic acid polymer (PLA), etc.; drug-encapsulating
micelle formulations comprising micellar nanoparticles (polymer
micelles) that have a two-layer structure comprising a block
copolymer (copolymer) formed by combining polyethylene glycol (PEG)
and a polyamino acid; hydrogel formulations produced by
crosslinking gelatin, collagen, or a polymer mixture of hyaluronic
acid, alginic acid, and the like, to form a hydrogel, and
immobilizing a cell growth factor or the like in the hydrogel; and
liposomal formulations having a drug encapsulated in various
phospholipids.
[0010] When fine particles have a size as small as several
nanometers or less, the particles are excreted from the kidney into
urine, and cannot be retained in the body. On the other hand, when
fine particles have a size of 400 nm or more, the fine particles
are quickly eliminated from the body due to the immune mechanism of
eliminating foreign matter by macrophages or the like. Therefore,
as an NS formulation containing a drug, an NS formulation of
several nanometers to 400 nm is recommended due to its enhanced
permeation and retention effect (EPR effect). More specifically,
unlike normal vascular endothelial cells, there is a wide gap of
about 200 nm between vascular endothelial cells in cancer tissue or
at an inflammation or ischemic site; it is known that a
microparticle formulation having a size controlled to about 100 nm,
or a polymer formulation, can be accumulated in tissue of vascular
lesions created by cancer, infectious disease, ischemia,
inflammation, arteriosclerosis, rheumatism, or the like. Thus, as
an NS formulation having a DDS effect, there is known a method of
forming an NS formulation having a particle size adjusted to 50 nm
to 200 nm to allow a drug to reach a lesion site and release the
drug at the lesion site, thus enhancing its therapeutic effect.
Further, as an endocytosis effect, NS formulations are known to
pass through a cell membrane and exhibit effects in cells. It is
known to produce, for example, an oral nanosphere formulation
having calcitonin encapsulated in lactic acid-glycolic acid
copolymer (PLGA) nanoparticles (Non-patent Literature (NPL 1)); a
transpulmonary nanosphere formulation having calcitonin
encapsulated in chitosan nanoparticles (NPL 2); a topical
nanosphere formulation having steroid encapsulated in PLGA
nanoparticles (NPL 3); and a nanosphere formulation having an
anti-inflammatory agent or a mitochondrial injury inhibitor
encapsulated in lactic acid-glycolic acid copolymer (PLGA)
nanoparticles. Such nanosphere formulations are effective for
treating ischemic reperfusion injury (Patent Literature (PTL) 5).
Further, a nanosphere formulation having prostaglandin E1 or a
derivative thereof encapsulated in lactic acid-glycolic acid
copolymer (PLGA) nanoparticles is also known (PTL 6 and NPL 4).
Further, a nanosphere formulation comprising beraprost encapsulated
in lactic acid-glycolic acid copolymer (PLGA) nanoparticles is
known to be effective for pulmonary hypertension (PTL 7).
[0011] Since an encapsulated drug is released from such a PLGA or
PLA nanoparticle formulation by hydrolysis of a lactic
acid-glycolic acid bond with water, a nanoparticle formulation
having a large surface area has a very short drug-release time. In
contrast, the liposomal formulation gradually releases the drug
through enzymatic degradation by lipase etc. in vivo.
[0012] A pharmaceutical composition comprising, as an active
ingredient, a liposome in which an immunosuppressive agent, such as
FK506, FTY720, or cyclosporin A, is encapsulated is also known to
be effective for treating cardiovascular inflammatory diseases,
such as myocardial infarction, myocarditis, and vasculitis syndrome
(PTL 8). Doxil (produced by Janssen Pharmaceutical K.K.) comprising
doxorubicin (an anticancer antibiotic) encapsulated in liposomes
has already been commercially available as an anticancer drug. This
pharmaceutical composition is also commercially available for other
purposes, such as an antifungal agent, a Kaposi's sarcoma
inhibitor, a lymphomatous meningitis inhibitor, an age-related
macular degeneration inhibitor, and a postoperative pain inhibitor.
LipoPGE.sub.1, which is encapsulated in lipid microspheres in the
form of an o/w emulsion of prostaglandin E1 comprising egg yolk
lecithin, oleic acid, olive oil, and glycerin, is already
commercially available (Ripple, sold by Mitsubishi Tanabe Pharma
Corporation) (NPL 4). LipoPGE.sub.1, which has an average particle
size as large as 200 to 300 nm and has no stealth property, is
trapped by the liver and macrophages, and thus has a short blood
retention time.
[0013] On the other hand, no stealth liposomal formulation
comprising a prostaglandin I2 receptor agonist and having an
average particle size of 50 to 200 nm has been reported.
CITATION LIST
Patent Literature
[0014] PTL 1: JPH6-87811A [0015] PTL 2: WO2004/032965 [0016] PTL 3:
WO2008/047863 [0017] PTL 4: WO2014/069401 [0018] PTL 5:
WO2016/006577 [0019] PTL 6: WO2010/058669 [0020] PTL 7:
JP2012-171883A [0021] PTL 8: WO2013/176223
Non-Patent Literature
[0021] [0022] NPL 1: Y. Kawashima, H. Yamamoto, H. Takeuchi and Y.
Kuno, Pharm. Develop. Technol., 5, 77-85, (2000) [0023] NPL 2: Y.
Kawashima, 6th US-Japan Symposium on Drug Delivery Systems,
December 16-21, (2001), Maui [0024] NPL 3: E. Horisawa et al.,
Pharm. Res., 19, 132-139, (2002) [0025] NPL 4: J Pharm Pharmacol.,
2013 August; 65(8): 1187-94
SUMMARY OF INVENTION
Technical Problem
[0026] An object of the present invention is to provide a disease
site-specific stealth liposomal formulation that is effective for
treating a cardiovascular disease, such as ischemic and dilated
cardiomyopathy, obstructive arteriosclerosis, vasculitis syndrome,
valvular disease, aortic stenosis, chronic heart failure, or
diastolic dysfunction; a respiratory disease, such as pulmonary
hypertension, pulmonary fibrosis, asthma, or chronic obstructive
pulmonary disease; a gastrointestinal or urinary disease, such as
chronic kidney disease, chronic hepatitis, or chronic pancreatitis;
and a neurodegenerative disease, such as cerebral infarction
chronic stage, Alzheimer's disease, diabetic neuropathy,
Parkinson's disease, or amyotrophic lateral sclerosis.
[0027] More specifically, an object of the present invention is to
provide a clinically applicable, safe and convenient, stealth
liposomal formulation, which is a liposome (LP) formulation
containing, for example, a PGI2 receptor agonist compound (A), and
which is intermittently administered by intravenous injection,
inhalation, or the like; and thereby specifically accumulated at a
disease site, thus exhibiting a DDS effect.
Solution to Problem
[0028] The present inventors conducted extensive research on the
production of NS formulations having a PGI2 receptor (IP) agonist
encapsulated therein. As a result, the inventors found that a
stealth liposome (hereinafter sometimes abbreviated as LP)
formulation is the optimum formulation to achieve this object.
Through the research of many production methods, the inventors
found an LP formulation that is clinically applicable in terms of
stability, content percentage, yield, safety, efficacy, release,
stealth properties, and the like, from among LP formulations
containing compound (A) or the like. More specifically, the
inventors found that a liposomal formulation that is a
microparticle drug carrier coated with, for example, PEG-modified
phosphoethanolamine and phospholipids can improve drug release
control and stability, as well as exhibit new functions, such as
accumulation at a disease site (targeting) and adhesion to tissue;
thus significantly improving bioavailability (BA) and drug efficacy
and thereby providing an increased effect at a lower dose than each
component used alone, and reducing side effects.
[0029] The present inventors conducted intensive research to solve
the above problems, and found for the first time that in a
liposomal formulation containing a PGI2 receptor (IP) agonist, such
as compound (A), an appropriate combination of the types and
composition ratio of lipids such as a phospholipid component and
PEG-modified phosphoethanolamine having stealth properties; the
average particle size of the liposomal formulation; the weight
ratio of compound (A) or the like to the phospholipid; etc.,
surprisingly allows for the control of the release rate of a PGI2
receptor agonist, such as compound (A), which is a low molecular
compound, and thus improves accumulation at a disease site, thereby
exhibiting a DDS effect.
[0030] The present inventors further found that a specific
combination of lipids allows the liposomal formulation to retain
stealth properties, so that liposomes can escape capture by
macrophages or the like. Further, the inventors found that the
method of the present invention can reliably produce a stealth
liposomal formulation with a high yield. The present invention has
been accomplished through further trial and error based on these
findings, and includes the following inventions.
[0031] Item 1
[0032] A pharmaceutical composition for disease site-specific
treatment, comprising a stealth liposome having a prostaglandin I2
receptor agonist encapsulated therein.
[0033] Item 2
[0034] The pharmaceutical composition according to Item 1, wherein
the prostaglandin I2 receptor agonist includes at least a compound
represented by formula (I):
##STR00001##
wherein
##STR00002##
[0035] (wherein e represents an integer of 3 to 5,
[0036] f represents an integer of 1 to 3,
[0037] p represents an integer of 1 to 4,
[0038] q represents 1 or 2, and
[0039] r represents an integer of 1 to 3);
[0040] R.sup.1 represents a hydrogen atom or a C.sub.1-4 alkyl
group;
[0041] R.sup.2 represents (i) a hydrogen atom, (ii) a C.sub.1-8
alkyl group, (iii) a phenyl group or a C.sub.4-7 cycloalkyl group,
(iv) a 4- to 7-membered monocyclic ring containing one nitrogen
atom, (v) a C.sub.1-4 alkyl group substituted with a benzene ring
or a C.sub.4-7 cycloalkyl group, or (vi) a C.sub.1-4 alkyl group
substituted with a 4- to 7-membered monocyclic ring containing one
nitrogen atom; and
[0042] R.sup.3 represents (i) a C.sub.1-8 alkyl group, (ii) a
phenyl group or a C.sub.4-7 cycloalkyl group, (iii) a 4- to
7-membered monocyclic ring containing one nitrogen atom, (iv) a
C.sub.1-4 alkyl group substituted with a benzene ring or a
C.sub.4-7 cycloalkyl group, or (v) a C.sub.1-4 alkyl group
substituted with a 4- to 7-membered monocyclic ring containing one
nitrogen atom;
[0043] (provided that when
##STR00003##
[0044] is a group represented by (iii) or (iv),
--(C--(CH.sub.2).sub.p-- and --CH--(CH.sub.2).sub.s-- are bound to
position a or b on the ring, and cyclic structures in R.sup.2 and
R.sup.3 are optionally substituted with one to three C.sub.1-4
alkyl groups, C.sub.1-4 alkoxy groups, halogen atoms, nitro groups,
or trihalomethyl groups); or
[0045] a salt thereof
[0046] Item 3
[0047] The pharmaceutical composition according to Item 1, wherein
the prostaglandin I2 receptor agonist includes at least the
following compound (A):
[0048]
(A)({5-[2-({[(1E)-phenyl(pyridin-3-yl)methylene]amino}oxy)ethyl]-7,-
8-dihydronaphthalen-1-yl}oxy)acetic acid (ONO-1301) represented by
formula (II):
##STR00004##
[0049] or a salt of compound (A).
[0050] Item 4
[0051] The pharmaceutical composition according to Item 1, wherein
the prostaglandin I2 receptor agonist includes at least one of the
following compounds (B) to (E):
[0052] (B) sodium
(.+-.)-(1R,2R,3aS,8bS)-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-h-
ydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta[b]benzofran-5-butanoate
(beraprost); or a derivative thereof that is a carbacyclic PGI2
derivative,
[0053] (C) MRE-269,
[0054] (D)
(2E)-7-{(1R,2R,3R)-3-hydroxy-2-[(1E,3S,5S)-3-hydroxy-5-methylno-
n-1-en-1-yl]-5-oxycyclopentyl}hept-2-enoic acid (limaprost),
omoprostil, enprostil, or misoprostol; or a derivative of any of
these compounds that is a PEF derivative, and
[0055] (E) NS-304 (selexipag);
[0056] or
[0057] a salt of any of compounds (B) to (E).
[0058] Item 5
[0059] The pharmaceutical composition according to Item 1, wherein
the prostaglandin I2 receptor agonist includes at least one member
selected from the group consisting of ONO-1301, beraprost,
limaprost, and NS-304.
[0060] Item 6
[0061] The pharmaceutical composition according to any one of Items
1 to 5, wherein the stealth liposome is obtainable by using at
least a prostaglandin I2 receptor agonist and a phospholipid by the
Bangham method, hydration dispersion method, reverse phase
evaporation method, ethanol injection method, ethanol dilution
method, homogenization method, mechanochemical method, direct
dispersion method, extruder method, French press method, remote
loading method, dehydration-rehydration method, freeze-thaw method,
ultrasonic method, or lipid-compound film method; or a modified
method of any of these methods.
[0062] Item 7
[0063] The pharmaceutical composition according to any one of Items
1 to 6, wherein the stealth liposome has an average particle size
of 50 to 200 nm, and comprises 5 to 50 parts by weight of the
phospholipid and 0.05 to 5 parts by weight of PEG-modified
phosphoethanolamine, per part by weight of the prostaglandin I2
receptor agonist.
[0064] Item 8
[0065] The pharmaceutical composition according to any one of Items
1 to 7, wherein the stealth liposome comprises 0.05 to 5 parts by
weight of MPEG2000-DSPE per part by weight of the prostaglandin I2
receptor agonist; the prostaglandin I2 receptor agonist includes at
least one member selected from the group consisting of ONO-1301,
beraprost, limaprost, and NS-304; and the stealth liposome releases
the prostaglandin I2 receptor agonist over a period of 3 hours to 4
weeks.
[0066] Item 9
[0067] The pharmaceutical composition according to any one of Items
1 to 8, wherein the stealth liposome comprises
[0068] a prostaglandin I2 receptor agonist,
[0069] a phospholipid,
[0070] a PEG-modified phosphoethanolamine, and
[0071] a water-miscible organic solvent, and
[0072] does not comprise a sterol;
[0073] the liposome is obtainable by a production method comprising
the following steps (1) to (8):
[0074] (1) mixing the prostaglandin I2 receptor agonist, the
phospholipid, and the PEG-modified phosphoethanolamine in the
solvent in amounts such that at least 5 mg of the phospholipid and
at least one 0.05 mg of the PEG-modified phosphoethanolamine are
present per mg of the prostaglandin I2 receptor agonist,
[0075] (2) heating the mixture obtained in step (1) to prepare a
melt,
[0076] (3) instantly freezing the melt obtained in step (2),
[0077] (4) freeze-drying the frozen product obtained in step (3) to
remove the solvent,
[0078] (5) heating the freeze-dried product obtained in step (4) to
disperse the heated product in an aqueous phosphate buffer
solution,
[0079] (6) sizing the dispersion obtained in step (5) with an
extruder,
[0080] (7) ultrafiltrating the dispersion obtained in step (6) to
remove unencapsulated material, and
[0081] (8) adding a sugar to the dispersion obtained in step (7)
and freeze-dying the dispersion; and the liposome contains at least
0.001 mg of the prostaglandin I2 receptor agonist per 1.0 mg of the
phospholipid and has an average particle size of 50 to 200 nm.
[0082] Item 10
[0083] The pharmaceutical composition according to any one of Items
1 to 9, wherein the composition is for intravenous administration,
intracoronary administration, inhalation, intramuscular injection,
subcutaneous administration, oral administration, transmucosal
administration, transdermal administration, or an internal organ;
and is in the form of an injectable formulation, an oral
preparation, an inhalant, a nebulizer, an ointment, a patch, or a
spray.
[0084] Item 11
[0085] The pharmaceutical composition according to any one of Items
1 to 9, wherein a single intravenous dose of the composition is
0.001 to 100 mg in terms of the prostaglandin I2 receptor
agonist.
[0086] Item 12
[0087] The pharmaceutical composition according to Item 11, wherein
a disease to be treated with the composition is cardiovascular
disease, respiratory disease, urinary disease, gastrointestinal
disease, bone disease, neurodegenerative disease, vascular disease,
dental disease, eye disease, skin disease, other inflammatory
disease, ischemic organ disorder, diabetic complication, tissue
fibrotic disease, tissue degenerative disease, or hair loss;
and
[0088] the composition comprises a liposome.
[0089] Item 13
[0090] The pharmaceutical composition according to Item 11, wherein
the disease to be treated with the composition is a cardiovascular
disease such as ischemic and dilated cardiomyopathy,
atherosclerosis obliterans, vasculitis syndrome, valvular disease,
aortic stenosis, chronic heart failure, or diastolic failure; a
respiratory lung disease such as pulmonary hypertension, pulmonary
fibrosis, asthma, or chronic obstructive pulmonary disease; a
gastrointestinal or urinary disease such as chronic kidney disease,
chronic hepatitis, or chronic pancreatitis; or a neurodegenerative
disease such as chronic phase of cerebral infarction, Alzheimer's
disease, diabetic neuropathy, Parkinson's disease, or amyotrophic
lateral sclerosis; and
[0091] the composition comprises a liposome.
Advantageous Effects of Invention
[0092] According to the present invention, there can be provided a
pharmaceutical composition that is effective for, for example,
cardiovascular diseases, respiratory diseases, gastrointestinal or
urinary diseases, and inflammatory diseases such as
neurodegenerative diseases, ischemic organ disorders, diabetic
complications, tissue fibrotic diseases, tissue degenerative
disease, or hair loss. The pharmaceutical composition of the
present invention can have the effect of enhancing drug efficacy at
a lower dose than a single use of a PGI2 receptor agonist, and also
reducing side effects. Further, the present invention can provide a
stealth liposomal formulation that allows for accumulation of a
PGI2 receptor agonist in a high concentration at a disease site,
and exhibit effects in a sustained manner; and provide a method for
producing the stealth liposomal formulation.
[0093] The liposomal formulation of the present invention
containing compound (A) or the like is effective for circulatory
diseases, respiratory diseases, urinary diseases, gastrointestinal
diseases, and neurodegenerative diseases, when intermittently
administered by intravenous administration, intramuscular
administration, subcutaneous administration, or inhalation
administration. In particular, the liposomal formulation
administered intravenously or by inhalation is accumulated at a
disease site and topically exhibits an endogenous repair factor
production-promoting action, thus being useful as a regenerative
drug. Intravenous administration of the liposomal formulation is
useful for heart diseases, such as myocardial infarction, angina,
dilated cardiomyopathy, aortic stenosis, valvular disease, chronic
heart failure, and diastolic dysfunction, due to its vasodilator
action, angiogenesis action, stem cell differentiation-inducing
action, antifibrotic action, antiapoptotic action, reverse
remodeling action, etc. For pulmonary diseases or the like such as
acute pneumonia, chronic pneumonia, pulmonary hypertension,
pulmonary fibrosis, interstitial pneumonia, COPD, and ARDS,
inhalation administration, in addition to intravenous
administration, is useful. For neurodegenerative diseases such as
amyotrophic lateral sclerosis (ALS), Parkinson's disease,
Alzheimer's disease, and spinal cord injury, intramedullary
administration, in addition to intravenous administration, is
useful.
BRIEF DESCRIPTION OF DRAWINGS
[0094] FIG. 1 is diagrams showing the average particle size
distribution of Formulations 1 to 4.
[0095] FIG. 2 is diagrams showing the average particle size
distribution of Formulations 5 to 8.
[0096] FIG. 3 is a diagram showing the average particle size
distribution of Formulation 9.
[0097] FIG. 4 is a diagram showing the average particle size
distribution of Formulation 10.
[0098] FIG. 5 is diagrams showing the average particle size
distribution of Formulations 11 to 14.
[0099] FIG. 6 is diagrams showing the average particle size
distribution of Formulations 15 to 18.
[0100] FIG. 7 is a diagram showing the average particle size
distribution of Formulation 19.
[0101] FIG. 8 is a diagram showing the average particle size
distribution of Formulation 20.
[0102] FIG. 9 is a graph showing the results of quantification of
compound (A).
[0103] FIG. 10 is a diagram showing the average particle size
distribution of Formulation 21.
[0104] FIG. 11 is transmission electron microscope images of
Formulation 21.
[0105] FIG. 12 is charts of HPLC measurement of Samples 1 to 5.
[0106] FIG. 13 is a diagram showing the average particle size
distribution of Formulation 22.
[0107] FIG. 14 is transmission electron microscope images of
Formulation 22.
[0108] FIG. 15 is an absorption spectrum of a solution of compound
(B).
[0109] FIG. 16 is a diagram showing the average particle size
distribution of Formulation 23.
[0110] FIG. 17 is transmission electron microscope images of
Formulation 23.
[0111] FIG. 18 is an absorption spectrum of compound (C).
[0112] FIG. 19 is diagrams showing the average particle size
distribution of Formulations 24 and 25.
[0113] FIG. 20 shows the results of HPLC analysis.
[0114] FIG. 21 shows the particle size distribution of liposomes
having compound (B) encapsulated therein (Formulation 26).
[0115] FIG. 22 shows UV absorption spectra of compound (B), and
liposomes having compound (B) encapsulated therein.
[0116] FIG. 23 shows the particle size distribution of liposomes
having compound (D) encapsulated therein (Formulation 27).
[0117] FIG. 24 is UV absorption spectra of compound (D), and
liposomes having compound (D) encapsulated therein (Formulation
27).
[0118] FIG. 25 is a diagram showing the average particle size
distribution of liposomes having compound (E) encapsulated therein
(Formulation 28).
[0119] FIG. 26 is absorption spectra of compound (E) and liposomes
having compound (E) encapsulated therein (Formulation 28).
[0120] FIG. 27 shows 42-day survival curves of a group receiving
ONO-1301 by repeated oral administration, and a group receiving
Formulation 21 (ONO-1301LipoNS formulation) by intermittent
intravenous administration.
[0121] FIG. 28 is a graph showing the survival rates of all
groups.
[0122] FIG. 29 is a graph showing a comparison with intermittent
intravenous administration of Formulation 25 (ONO-1301Lipo).
[0123] FIG. 30 is a graph showing a comparison with intermittent
intratracheal administration of Formulation 25 (ONO-1301Lipo).
[0124] FIG. 31 is a drawing showing a method for evaluating a left
ventricle wall thickness and a left ventricle wall area.
[0125] FIG. 32 is photographs showing an infarct area evaluation
method.
DESCRIPTION OF EMBODIMENTS
[0126] The liposomes used in the pharmaceutical composition of the
present invention are not limited, as long as they are closed
vesicles surrounded by a lipid bilayer. The liposomes may be large
unilamellar vesicle (LUV) liposomes, or small unilamellar vesicle
(SUV) liposomes; and may be multilamellar vesicle (MLV) liposomes.
The liposomes can be produced by known production methods.
[0127] There are three types of methods for producing liposomes.
More specifically, the Bangham method is commonly used as a
liposome production method. There are also methods comprising the
Bangham method and some additional operations, which are called the
simple hydration method, ultrasonic treatment method, and extrusion
method. Examples of liposome production methods further include the
direct dispersion method, organic solvent (e.g., ethanol) injection
method, reverse phase evaporation method, calcium fusion method,
surfactant removal method, static hydration method, hexane-span 80
dialysis method, organic solvent globule evaporation method,
mechanochemical method, ultrasonic method, lipid-compound film
method, and the like; and improved methods of these methods.
[0128] The method for adjusting the particle size includes the
extrusion method, extrusion process, French press method, and the
like. Examples of the extruder method or the French press method
includes a method comprising passing particles several times
through a nanopore membrane filter having an appropriate pore size,
which is set in an extruder or a French press to adjust the
liposome size.
[0129] Examples of the method for encapsulating the compound
include the pH gradient (remote loading) method, counter ion
concentration gradient (gelation) method, freeze-thaw method,
supercritical carbon dioxide method, film loading method, and the
like.
[0130] The methods that are superior in encapsulating a
water-soluble drug include the reverse phase evaporation method and
the freeze-thaw method. The methods that are superior in
encapsulating fat-soluble drugs include the Bangham method, the
mechanochemical method, the supercritical carbon dioxide method,
and the film loading method. The methods that are superior in
encapsulating dissociative drugs include the pH gradient (remote
loading) method, the counterionization concentration gradient
method, and the like.
[0131] The Bangham method includes, for example, a method
comprising forming a lipid film; and then applying a mechanical
vibration by vortexing, ultrasonic waves, or the like in an aqueous
buffer to form liposomes (a hydration dispersion method). The
reverse phase evaporation method includes, for example, a method
comprising dissolving a lipid in an organic solvent that is
immiscible with water; then adding an aqueous buffer and performing
ultrasonic treatment to form a reverse micelle (a W/O emulsion),
thereafter removing the organic solvent by vacuum treatment or the
like and achieving a gel state; and then forming liposomes. The
ethanol injection method or the ethanol dilution method includes,
for example, a method comprising dissolving a lipid in ethanol, and
injecting a lipid solution into an aqueous buffer to form
liposomes.
[0132] The homogenization method or the mechanochemical method
includes, for example, a method of forming liposomes by using a
high-pressure emulsifier.
[0133] The direct dispersion method includes a method comprising
directly dispersing a lipid or a mixture of a lipid and a compound
in an aqueous buffer, without preparing a lipid film, to form
liposomes.
[0134] Among the methods for adjusting the particle size, the
extruder method or the French press method includes, for example, a
method comprising passing the liposomes through a nanopore membrane
set in an extruder or a French press to thereby adjust the liposome
size.
[0135] Among the methods of encapsulating the compound, the remote
loading method is an encapsulation method utilizing the difference
in pH solubility of the compound. More specifically, after
liposomes having as an inner aqueous phase a pH solution in which
the compound is water-soluble are formed, the outer aqueous phase
is replaced with a pH solution in which the compound is fat-soluble
by ultrafiltration, dialysis, or the like; and adding a compound
solution to the liposome dispersion to thereby encapsulate the
compound in the aqueous phase of the liposomes. The
dehydration-rehydration method is an encapsulation method in which
liposomes are dehydrated by freeze-drying or the like; and then
rehydrated with an aqueous buffer containing a compound to be
encapsulated, thereby encapsulating the compound.
[0136] The freeze-thawing method includes, for example, a method
comprising mixing a liposome dispersion and a compound solution to
be encapsulated, and repeating freeze-thaw cycles to thereby
encapsulate a compound at a high concentration.
[0137] The method for producing liposomes is not limited to the
production methods described above. The method further includes
improved methods of each of these methods, and the like.
[0138] The lipids that form liposomes are not particularly limited.
Examples of lipids include soy lecithin, hydrogenated soy lecithin,
egg yolk lecithin, phosphatidylcholines, phosphatidylserines,
phosphatidylethanolamines, phosphatidylinositols,
phosphasphingomyelins, phosphatidic acids, long-chain alkyl
phosphates, gangliosides, glycolipids, phosphatidylglycerols,
sterols, and the like. Lipids can be used singly, or in a
combination of two or more. Examples of phosphatidylcholines
include dimyristoylphosphatidylcholine,
dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, and
the like. Examples of phosphatidylserines include dipalmitoyl
phosphatidylserine, sodium dipalmitoylphosphatidylserine, bovine
brain-derived sodium phosphatidylserine, and the like. Examples of
phosphatidylethanolamines include dimyristoyl
phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine,
distearoylphosphatidylethanolamine, and the like. Examples of the
phosphatidylinositols include wheat-derived phosphatidylinositol
sodium, and the like. Examples of phosphasphingomyelins include
bovine-derived sphingomyelin, and the like. Examples of
phosphatidic acids and long-chain alkyl phosphates include
dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid,
distearoyl phosphatidic acid, dicetyl phosphoric acid, and the
like. Examples of gangliosides include ganglioside GM1, ganglioside
GD1a, ganglioside GT1b, and the like. Examples of glycolipids
include galactosylceramide, glucosylceramide, lactosylceramide,
phosphatide, globoside, and the like. Examples of phosphatidyl
glycerol include dimyristoyl phosphatidyl glycerol, dipalmitoyl
phosphatidyl glycerol, distearoyl phosphatidyl glycerol, and the
like. Examples of sterols include cholesterol, dihydrocholesterol,
lanosterol, dihydrolanosterol, sitosterol, campesterol,
stigmasterol, brassicasterol, ergosterol, and the like. When two or
more lipids are used in combination, a phospholipid and cholesterol
are preferably used in combination. The phospholipid is preferably
a phosphatidylcholine. When liposomes are produced by using a
phospholipid and cholesterol, the molar ratio of the phospholipid
to cholesterol is preferably in the range of 1:0.1 to 1:1.5, and
more preferably 1:0.5 to 1:1.25.
[0139] Examples of phospholipids that can be used in the present
invention include the following commercially available products
(sold by Nippon Fine Chemical Co., Ltd.).
[0140] In general, the phospholipid is preferably DOPC or DEPC,
although it may vary depending on the substance to be encapsulated
therein.
TABLE-US-00001 TABLE 1 Abbreviated product name IUPAC name Cas Reg.
No. DPPC 1,2-Dipalmitoyl-sn- 63-89-8 glycero-3-phosphocholine DSPC
1,2-Distoaroyl-sn- 816-94-4 glycero-3-phosphocholine DMPC 1,2-Dimy
is oyl- 18194-24-6 sn-glycerol-3-phosphocholine DOPC
1,2-Dioleoyl-sn-glycero- 4235-96-4 3-phosphocholine DBPC
1,2-Dieracoyl-sn-Glycero- 51779-95-4 3-Phosphocholine POPC
2-Oleoyl-1-palmitoyl-sn- 26853-31-6 glycero-3-phosphocholine PCS
1,2-Diacyl-sn-Glycero- 8002-43-5 3-Phosphocholine (SOY) PCSH
1,2-Diacyl-sn-Glycero- 8002-43-5 3-Phosphocholine (SOY) DPPG
1,2-Dipalmitoyl-sn-Glycero- - 67233-81-9 [Phospho-rac-(1-glycerol)]
(Sodium Salt) DMPG 1,2-Dimy istoyl-sn-Glycero-3- 67232-80-8
[Phospho-rac-(1-glycerol)] (Sodium Salt) DSPG 1,2-Dist
aroyl-sn-Glycero-3- 4537-78-4 [Phospho-rac-(1-glycerol)] (Sodium
Salt) DOPG 1,2-Diole yl-sn-Glycero-3- 62706-09-0
[Phospho-rac-(1-glycerol)] (Sodium Salt) PGE
1,2-Diacyl-sn-Glycero-3- N/A [Phospho-rac-(1-glycerol)] (Sodium
Salt, EGG) PGS 1,2-Diacyl-sn-Glycero-3- N/A
{Phospho-rac-(1-glycerol)] (Sodium Salt, SOY) PGSH
1,2-Diacyl-sn-Glycero-3- N/A [Phospho-rac-(1-glycerol)] (Sodium
Salt, SOY) indicates data missing or illegible when filed
[0141] Conventional liposomal formulations are liposomes comprising
typical phospholipid and cholesterol. There are also stealth
liposomes whose surface is modified with polyethylene glycol (PEG)
or the like to increase the blood retention. These liposomes have
the effect of accumulation specifically at a disease site due to
their EPR effects.
[0142] To increase the stability (stealth properties) of liposomes
in blood, the liposome membrane surface is preferably modified with
a polyethylene glycol (PEG) derivative. The liposomes modified with
a PEG derivative can be produced by using a covalent conjugate of
PEG having a molecular weight of 500 to 20000 and a phospholipid.
The covalent conjugate of PEG and phospholipid is preferably a
PEG-modified phosphoethanolamine, which is a conjugate of PEG
having a molecular weight of 200 to 5000 and
distearoylphosphatidylethanolamine.
[0143] Examples of PEG-modified phosphoethanolamines include
commercially available products, such as DMPE
(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE
(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DSPE
(1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DOPE
(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and the like (all
produced by Nippon Fine Chemical Co., Ltd.), which comprise mPEG
350, mPEG 550, mPEG 750, mPEG 1000, mPEG 2000, mPEG 3000, or mPEG
5000 as a PEG-modifying group.
[0144] A preferable combination is, for example, a combination of
mPEG2000-DSPE: N-(carbonyl-methoxypolyethyleneglycol 5000)-1,2
distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt CAS No.
147867-65-0 (produced by Nippon Fine Chemical Co., Ltd.) and DEPC:
1,2-dierucoyl-sn-glycerol-3-phosphorylcholine CAS No. 51779-95-4
(produced by Nippon Fine Chemical Co., Ltd.).
[0145] The contents of the PEG-modified phosphoethanolamine and
phospholipid are not particularly limited. The content of the
PEG-modified phosphoethanolamine is preferably 0.01% to 10%, and
more preferably 0.01% to 3%, relative to the phospholipid as 1.
[0146] There is also a method in which the liposome surface is
modified with PEG and a targeting molecule, such as an antibody or
a peptide, to increase blood retention and further enhance the
transfer to a target site.
[0147] The present invention provides a stealth liposome
characterized in that the liposome contains a PGI2 receptor agonist
and a phospholipid, and further comprises PEG-modified
phosphoethanolamine. The liposome is preferably formed into a
liposomal formulation by combining a PGI2 receptor agonist and a
phospholipid, and further EG-modified phosphoethanolamine,
according to the purpose; and mixing these components at an
appropriate ratio.
[0148] The stealth liposome comprises a prostaglandin I2 receptor
agonist, a phospholipid, a PEG-modified phosphoethanolamine, and a
water-miscible solvent; and can be produced, for example, by a
production method comprising the following steps:
[0149] mixing a prostaglandin I2 receptor agonist, a phospholipid,
and a PEG-modified phosphoethanolamine in a water-miscible solvent
in amounts such that at least 5 mg of the phospholipid and at least
0.05 mg of the PEG-modified phosphoethanolamine are present per mg
of the prostaglandin I2 receptor agonist, to prepare a mixture;
[0150] heating the mixture to prepare a melt;
[0151] instantly freezing the melt;
[0152] freeze-drying the frozen product to remove the solvent;
[0153] heating the freeze-dried product to disperse the heated
product in an aqueous phosphate buffer solution;
[0154] sizing the dispersion with an extruder;
[0155] ultrafiltrating the dispersion to remove unencapsulated
material; and
[0156] adding a sugar to the dispersion, and freeze-drying the
dispersion.
[0157] The liposomes produced by this method are a stealth
liposomal formulation characterized by containing a PGI2 receptor
agonist, and having an average particle size of 50 to 200 nm.
[0158] The mixture of the PGI2 receptor agonist, the phospholipid,
PEG-modified phosphoethanolamine, and solvent may or may not
contain a sterol, such as cholesterol. Conventional liposomes
preferably comprise a combination of a phospholipid and cholesterol
as constituent lipids. However, the present invention uses no
cholesterol as a constituent lipid, and thereby makes it possible
to produce liposomes in which a PGI2 receptor agonist is stably
encapsulated at a high concentration.
[0159] The size (particle size) of the liposomes is not
particularly limited. The liposomes preferably have an average
particle size of about 10 to 1000 nm, more preferably about 20 to
500 nm, and even more preferably about 50 to 200 nm. The "particle
size" referred to herein means the diameter of a particle
determined by the dynamic light scattering method. A preferable
polydispersity index (PDI) is 0.3 or less. The method for adjusting
the particle size is not particularly limited.
[0160] The present invention provides a prophylactic and/or
therapeutic agent for a cardiovascular disease, a respiratory
disease, a urinary disease, a vascular disease, a gastrointestinal
disease, a neurodegenerative disease, etc., which comprises, as an
active ingredient, liposomes having a PGI2 receptor agonist
encapsulated therein. The PGI2 receptor agonist used in the
pharmaceutical composition of the present invention is not
particularly limited; a known PGI2 receptor agonist can be
preferably used. Examples of known PGI2 receptor agonists include,
for example, pharmaceutical compositions, PGI2 derivatives, and PGE
derivatives, which are compounds represented by the following
formula (I):
##STR00005##
wherein
##STR00006##
[0161] (wherein e represents an integer of 3 to 5,
[0162] f represents an integer of 1 to 3,
[0163] p represents an integer of 1 to 4,
[0164] q represents 1 or 2, and
[0165] r represents an integer of 1 to 3);
[0166] R.sup.1 represents a hydrogen atom or a C.sub.1-4 alkyl
group;
[0167] R.sup.2 represents (i) a hydrogen atom, (ii) a C.sub.1-8
alkyl group, (iii) a phenyl group or a C.sub.4-7 cycloalkyl group,
(iv) a 4- to 7-membered monocyclic ring containing one nitrogen
atom, (v) a C.sub.1-4 alkyl group substituted with a benzene ring
or a C.sub.4-7 cycloalkyl group, (vi) a C.sub.1-4 alkyl group
substituted with a 4- to 7-membered monocyclic ring containing one
nitrogen atom; and
[0168] R.sup.3 represents (i) a C.sub.1-8 alkyl group, (ii) a
phenyl group or a C.sub.4-7 cycloalkyl group, (iii) a 4- to
7-membered monocyclic ring containing one nitrogen atom, (iv) a
C.sub.1-4 alkyl group substituted with a benzene ring or a
C.sub.4-7 cycloalkyl group, (v) a C.sub.1-4 alkyl group substituted
with a 4- to 7-membered monocyclic ring containing one nitrogen
atom;
[0169] (provided that when
##STR00007##
[0170] is a group represented by (iii) or (iv),
[0171] --(C--(CH.sub.2).sub.p-- and .dbd.CH--(CH.sub.2).sub.s-- are
bound to position a or b on the ring, and cyclic structures in
R.sup.2 and R.sup.3 are optionally substituted with one to three
C.sub.1-4 alkyl groups, C.sub.1-4 alkoxy groups, halogen atoms,
nitro groups, or trihalomethyl groups); and salts thereof.
[0172] Preferably, the PGI2 receptor agonist is one of the
following compounds:
[0173] (A)
({5-[2-({[(1E)-phenyl(pyridin-3-yl)methylene]amino}oxy)ethyl]-7-
,8-dihydronaphthalen-1-yl}oxy)acetic acid (CAS 17639141-6; compound
(A)(ONO-1301)) represented by formula (II):
##STR00008##
[0174] (B) carbacyclic PGI2 derivatives such as sodium
(.+-.)-(1R,2R,3aS,8bS)-2,3,3a,8b-tetrahydro-2-hydroxy-1-[(E)-(3S,4RS)-3-h-
ydroxy-4-methyl-1-octene-6-ynyl]-1H-cyclopenta[b]benzofuran-5-butanoate
(CAS: 88475-69-8; beraprost)(compound (B));
[0175] (C)
[4-(5,6-diphenylpyrazinyl)(1-methylethyl)amino]butoxy]-acetic acid
(CAS: 475085-57-5; MRE-269; compound (C));
[0176] (D) PGE derivatives such as
(2E)-7{-(1R,2R,3R)-3-hydroxy-2[-(1E,3S,5S)-3-hydroxy-5-methylnon-1-en-1-y-
l]-5-oxocyclopentyl}-hept-2-enoic acid (CAS: 74397-12-9;
limaprost), omoprostil; 175,20-dimethyl-6-oxo-PGE.sub.1 methyl
ester, emprostil, and misoprostol (compound (D)); and
[0177] (E)
2-{4-[(5,6-diphenylpyrazin-2-)yl)(propan-2-yl)amino]butoxy}-N-(-
methanesulfonyl)acetamide (CAS: 475086-01-2; selexipag; NS-304
(compound (E)).
[0178] The subject to which the pharmaceutical composition of the
present invention is administered is preferably a mammal having an
inflammatory disease, ischemic organ disorder, diabetic
complication, tissue fibrotic disease, tissue degenerative disease,
or the like. Examples of mammals include humans, monkeys, cows,
sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, guinea
pigs, and the like. Humans that have developed an inflammatory
disease, or humans suspected to have an inflammatory disease, are
particularly preferable.
[0179] The method for administering the pharmaceutical composition
of the present invention is not particularly limited, as long as
the active ingredient can reach a disease site. Examples include
injectable formulations, patches, inhalants, nebulizers, sprays,
gels, creams, sprays, ointments, nasal drops, eye drops, and the
like, which are for intravenous administration, intracoronary
administration, drip/infusion, intracoronary administration,
inhalation, intramuscular administration, subcutaneous
administration, oral administration, suppositories, intraperitoneal
administration, transmucosal administration, transdermal
administration, or internal organs. For intraarterial
administration, intracoronary administration is preferable. For
intravenous administration, peripheral intravenous administration
is preferable.
[0180] The injectable formulation may be either an aqueous
injectable formulation or an oily injectable formulation. The
aqueous injectable formulation can be prepared by a known method.
For example, after liposomes having a drug encapsulated therein are
mixed into a solution prepared by appropriately adding
pharmaceutically acceptable additives to an aqueous solvent (e.g.,
water for injectable formulation or purified water), the resulting
mixture is filtered through a filter or the like and sterilized,
and the filtrate is filled into an aseptic container. Examples of
pharmaceutically acceptable additives include isotonic agents such
as sodium chloride, potassium chloride, glycerin, mannitol,
sorbitol, boric acid, borax, glucose, and propylene glycol; buffers
such as phosphoric acid buffer, acetic acid buffer, boric acid
buffer, carbonic acid buffer, citric acid buffer, Tris buffer,
glutamic acid buffer, and epsilon-aminocaproic acid buffer;
preservatives such as methyl paraoxybenzoate, ethyl
paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate,
chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium
dehydroacetate, sodium edetate, boric acid, and borax; thickeners
such as hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinyl
alcohol, and polyethylene glycol; stabilizers such as sodium
bisulfite, sodium thiosulfate, sodium edetate, sodium citrate,
ascorbic acid, and dibutylhydroxytoluene; pH adjusters such as
hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic
acid; and the like. The injectable formulation may further comprise
an appropriate solubilizing agent. Examples of the solubilizing
agent include alcohols such as ethanol; polyalcohols such as
propylene glycol and polyethylene glycol; nonionic surfactants such
as polysorbate 80, polyoxyethylene (50) hydrogenated castor oil,
lysolecithin, and pluronic polyol; and the like. The injectable
preparation may comprise a protein such as bovine serum albumin or
keyhole limpet hemocyanin; a polysaccharide such as aminodextran;
and the like. When an oily injectable formulation is to be
produced, for example, sesame oil or soybean oil can be used as an
oily solvent; and benzyl benzoate, benzyl alcohol, or the like can
be added as a solubilizing agent. The prepared injectable
formulation is usually placed in, for example, an appropriate
ampoule or vial. Liquid formulations such as injectable
formulations can also be preserved after removing water by
cryopreservation, lyophilization, or the like. Lyophilized
formulations are dissolved again at the time of use by adding
distilled water for injectable formulations or the like; and then
used.
[0181] The amount of the drug or the like contained in the
pharmaceutical composition of the present invention varies
depending on the dosage form, administration interval, or
administration route. In the case of the injectable formulation for
intravenous administration, the amount can be appropriately
selected from the range of 0.001 ng/mL to 100 mg/mL. The
administration period and administration method are appropriately
determined according to the disease and the treatment method
therefor, in consideration of safety, convenience, low
invasiveness, patient's burden, compliance, and the like. Any
administration interval may be used as long as the effect can be
expected and the administration interval is convenient. The
administration interval is preferably about twice a day, every day,
once every two days, once every three days, once a week, once every
two weeks, once every three weeks, or once every four weeks; and is
more preferably in the range of once a day to once a week.
[0182] For example, when liposomes having compound (A) encapsulated
therein are intravenously administered to a human that has
developed a heart disease, a single dose in terms of ONO-1301 is
preferably 500 mg or less, and more preferably 100 mg or less. The
lower limit is not particularly limited, and can be any dose that
provides the desired effect.
[0183] When a PGI2 receptor agonist alone is administered at a high
dose, the PGI2 receptor exhibits a hypotensive effect due to its
vasodilatoiy effect; therefore, there is little deviation from the
effective amount. In contrast, the pharmaceutical composition of
the present invention, which comprises, as an active ingredient,
liposomes having a PGI2 receptor agonist encapsulated therein, is
useful in that the liposomal formulation can exhibit effects on
many diseases even at a low dose due to its accumulation at a
disease site by the DDS effect. That is, vascular permeability at a
lesion site, and nano-sized liposomes can be expected to
specifically accumulate in the lesion site (EPR effect).
Furthermore, the liposomal formulation is highly useful in that
since the drug moves into cells due to the endocytosis effect, an
increase in drug efficacy and a reduction in side effects can be
expected. Further, the pharmaceutical composition of the present
invention is useful in that an active ingredient can be delivered
to a target lesion site by administration through a peripheral vein
or the like, without the necessity of using a central venous
catheter or the like. Another advantage is that the pharmaceutical
composition is difficult to be delivered to sites other than the
target site even when administered through a peripheral vein. In
other words, the pharmaceutical composition of the present
invention is highly useful in that the composition can provide an
enhanced tissue repair effect at a low dose; and can reduce side
effects by reducing the dose, suppressing delivery to sites other
than the target site, and eliminating the necessity of using a
central venous catheter or the like.
[0184] When a liposomal formulation is to be produced by the direct
dispersion improvement method, and when a premix of lipids is
produced, the solvent used must meet the following conditions: it
is a solvent in which lipids and the substance to be encapsulated
are soluble; it can be instantly frozen; and it can be removed by
freeze-drying. Any solvent that satisfies the above conditions can
be used.
[0185] In general, the solvent is preferably t-butanol,
cyclohexane+ethanol, hexafluoropropanol, 1-propanol, isopropyl
alcohol, 2-butoxyethanol, and the like. t-Butanol is more
preferable.
[0186] In the freeze-drying of liposomes, it is generally necessary
to add and disperse a sugar, such as maltose, sucrose, or
trehalose, to thereby inhibit cell membrane collapse on freezing
and perform freeze-drying.
Application to Pharmaceutical Products
[0187] PGI2 receptor agonists, such as compound (A), have, for
example, an in vivo regeneration factor production-promoting
action, stem cell differentiation-inducing action, anti-apoptotic
action, reverse remodeling action, anti-fibrotic action, and
angiogenesis-promoting action. Therefore, stealth liposomal
formulations containing such a PGI2 receptor agonist are useful as
therapeutic and/or prophylactic agents for the following various
diseases:
[0188] various organ disorders, inflammatory diseases such as
vascular diseases (e.g., atherosclerosis obliterans (ASO), Berger
disease, Raynaud's disease, arteriosclerosis, vasculitis syndrome,
etc.), cardiovascular diseases (e.g., myocardial infarction,
myocarditis, angina, supraventricular tachyarrhythmia, congestive
heart failure, coronary artery disease, idiopathic cardiomyopathy,
dilated cardiomyopathy, ischemic cardiomyopathy, atrial
fibrillation, chronic heart failure, diastolic dysfunction,
systolic dysfunction, valvular disease, aortic stenosis, etc.),
neurodegenerative diseases (e.g., ischemic encephalopathy,
cerebrovascular disease, stroke, Parkinson's disease, Alzheimer's
disease, diabetic neuropathy, spinal canal stenosis, dementia,
moyamoya disease, spinal cord injury, muscle atrophy lateral
sclerosis (ALS) etc.), respiratory diseases (e.g., acute pneumonia,
pulmonary fibrosis, pulmonary hypertension, chronic obstructive
pulmonary disease (COPD), systemic inflammatory response syndrome
(SIRS), acute lung injury (ALI), acute respiratory distress
syndrome (ARDS), Sarcoidosis, interstitial pneumonia, irritable
pneumonia, asthma, refractory asthma, etc.), bone diseases (e.g.,
osteoarthritis (OA) of, for example, spine or knee, rheumatoid
arthritis (RA), osteoporosis, fracture, spinal cord injury,
periosteal injury, etc.), gastrointestinal liver diseases (e.g.,
fulminant hepatitis, acute hepatitis, cirrhosis, chronic hepatitis,
fatty liver, steatohepatitis, gastric ulcer, gastritis, intestinal
ulcer, etc.), urinary diseases (e.g., acute renal failure, chronic
renal failure, glomerular disease, tubulointerstitial disease,
renal vasculopathy, cystic kidney disease, toxic nephropathy,
tubule transport abnormality, dialysis patient kidney disorders,
nephropathy, nephrotic syndrome, IgA nephropathy, atypical
hemolytic uremic syndrome, acute progressive nephritis syndrome,
renal fibrosis, etc.), gastrointestinal pancreatic diseases (e.g.,
diabetes, chronic pancreatitis, acute pancreatitis, etc.),
gastrointestinal diseases (e.g., esophagitis, gastritis, gastric
ulcer, duodenal ulcer, ulcerative colitis, Crohn's disease, etc.),
diabetic complications (e.g., diabetic neuropathy, skin ulcer,
diabetic nephropathy, diabetic retinopathy, etc.), vascular
endothelial cell damages (e.g., prevention of restenosis after
percutaneous transluminal coronary angioplasty (PTCA)), dental
diseases (e.g., periodontal diseases, tooth extraction wounds, oral
wounds, periodontal tissue disorders, etc.), skin diseases (e.g.,
pressure ulcers, hair loss, etc.), ophthalmic diseases (e.g.,
glaucoma, etc.), organ/cell transplantation (e.g., heart, liver,
kidneys, lungs, pancreas, pancreatic islet cells, bone marrow,
etc.), chronic transplant rejection, and the like. In particular,
the liposomal formulation of the present invention has shown
promise as a prophylactic and/or therapeutic agent for heart
diseases, lung diseases, kidney diseases, bone diseases,
neurodegenerative diseases, liver diseases, pancreatic diseases,
autoimmune diseases, allergic syndromes, and vascular diseases.
[0189] As body regeneration factors whose product is induced or
promoted by a PGI2 receptor agonist, such as compound (A), for
example, the following factors are known: a vascular endothelial
cell growth factor (VEGF), a hepatocyte growth factor (HGF),
various fibroblast growth factors (a/bFGF), transforming growth
factor-.beta. (TGF-.beta.), a platelet-derived growth factor
(PDGF), Angiopoietin, a hypoxia-inducible factor (HIF), an
insulin-like growth factor (IGF), a bone morphogenetic protein
(BMP), a connective tissue growth factor (CTGF), an epidermal
growth factor (EGF), a stromal cell-derived factor (SDF-1), a
high-mobility group box 1 (HMGB1), and the like; and growth factors
of their families etc. Examples of other drugs that produce
endogenous repair factors described above include other PGI2
receptor agonists, EP2 and EP4 receptor agonists of PGE2 receptors,
and mixed receptor agonists thereof, and the like. To achieve the
object of the present invention, the drugs mentioned above may be
used in place of compound (A). Examples of the drug that can be
used in place of compound (A) include PGI and PGE derivatives, IP,
EP2 and EP4 receptor agonists, and the like. Specific examples
include compound (B), aeroprost, ornoprostil, compound (C),
compound (D) (limaprost), compound (E), enprostil, misoprostol,
ONO-4232, ONO-8055, and the like.
[0190] These drugs and liposomes containing the drugs exhibit
effects on the same diseases as those on which compound (A) has
effects.
[0191] It is also preferable in the present invention that two or
more drugs selected from compound (A) and drugs described above are
combined according to the purpose, and formed into liposomes. The
drugs may be commercially available, or can be easily produced in
accordance with a known method.
[0192] The dosage form of the liposome of the present invention
includes injectable formulations, ointments, patches, oral
preparations, sprays, and the like, which are for intravenous
administration, coronary artery administration, inhalation,
intramuscular administration, subcutaneous administration, oral
administration, transmucosal administration, and transdermal
administration, or internal organs. In addition to the above, other
examples include implants, transmucosal agents for administration
through the rectum, uterus, oral cavity, or the like, nasal drops,
and intravenous drip injections; or a method for continuous
administration into coronary arteries.
Toxicity
[0193] As compared with the PGI2 receptor agonist alone, the
liposomal formulation of the present invention is less toxic and
fully safe for use as a medicament. Further, the PGI2 receptor
agonist has been confirmed not to have carcinogenesis initiation
and promotion effects in a long-term carcinogenicity test, a
medium-term hepatocarcinogenicity test, etc., using mice and
rats.
EXAMPLES
[0194] The present invention is described in detail below with
reference to Examples; however, the present invention is not
limited to these Examples.
[0195] The following are the reagents used. In the Examples, these
reagents are referred to by the following abbreviations.
[0196] (1) HSPC (hydrogenated soybean phospholipid, hydrogenated
lecithin), product name: COATSOME NC-21 (NOF corporation), CAS:
921228-87-5, 92128-87-5
[0197] (2) DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine),
CAS: 1069-79-0 (Nippon Fine Chemical Co., Ltd.)
[0198] (3) DEPC: 1,2-Dierucoyl-sn-glycerol-3-phosphorylcholine,
CAS: 51779-95-4 (Nippon Fine Chemical Co., Ltd.)
[0199] (4) MPEG2000-DSPE:
N-(carbonyl-methoxypolyethyleneglycol-5000)-1,2
distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt, CAS:
147867-65-0 (Nippon Fine Chemical Co., Ltd.)
[0200] (5) DOPC: 1,2-Dioleoyl-sn-glycero-3-phosphocholine, CAS:
4235-95-4 (Nippon Fine Chemical Co., Ltd.)
[0201] (6) Cholesterol: Cholesterol HP (NOF Corporation)
[0202] (7) PBS(-): Dulbecco's phosphate buffered saline (without Ca
and Mg), filtered and sterilized, tested for mycoplasma and
endotoxin (product code: 14249-95) (Nacalai Tesque Inc.)
[0203] The PGI2 receptor agonists to be encapsulated in liposome
are commercially available from the companies listed below, and can
be purchased generally.
[0204] (i) Compound (A): (ONO-1301), Sigma-Aldrich, CAS:
176391-41-6
[0205] (ii) Compound (B): (Beraprost), Cayman Chemical Company,
CAS: 88475-69-8
[0206] (iii) Compound (C): (MRE-269), Cayman Chemical Company, CAS:
475085-57-5
[0207] (iv) Compound (D): (Limaprost), Cayman Chemical Company,
CAS: 74397-12-9
[0208] (v) Compound (E): (NS-304), Cayman Chemical Company, CAS:
475086-01-2
[0209] Below, examples of the production of liposomes of PGI2
receptor agonists are specifically described in detail.
1. Formulation Example 1 (Remote Loading Method) Preparation of
Liposome
[0210] 1) HSPC (107.4 mg), DSPE (9.0 mg), and cholesterol (35.3 mg)
were weighed and placed in an eggplant flask, and a
chloroform/methanol solution (1/1, v/v) was added and dissolved so
that the lipid concentration was 20 mg/mL.
[0211] 2) The chloroform/methanol was evaporated with a rotary
evaporator, followed by vacuum-drying.
[0212] 3) A 0.1N sodium hydroxide solution (>pH: 12.0) was added
so that the lipid concentration was 10 mg/mL. The mixture was
redispersed by vortexing, and subjected to ultrasonic treatment for
30 minutes in total using a VS-100III produced by AS ONE
Corporation by repeating a cycle of 28 kHz output for 60 seconds,
45 kHz output for 60 seconds, and 100 kHz output for 3 seconds.
After the ultrasonic treatment, the particle size was
confirmed.
[0213] 4) To exchange the liposome external solution, stirred
ultrafiltration (cutoff molecular weight: 300,000 Da) was
performed. The liposome external solution was a 10 mM phosphate
buffer (pH: 8.0) (liposome solution). For ultrafiltration, a
stirred cell (8000 series, a 50-mL cell): Model 8050 5122 produced
by Merck & Co., Inc. and BioMax PBMK 04310 produced by Merck
& Co., Inc. were used. After the preparation of empty
liposomes, lipid quantification was performed.
[0214] 5) Compound (A) (56.5 mg) was weighed, and 1.41 mL of a 0.1N
sodium hydroxide solution was added thereto. The mixture was
dissolved by vortexing, and 5.65 mL of a 10 mM phosphate buffer
(pH: 8.0) was added thereto to thus prepare a solution of 8 mg/mL
(compound (A) solution).
[0215] 6) The compound (A) solution was added to the liposome
solution (three concentration conditions), and the mixtures were
stirred at 60.degree. C. for 1 hour.
[0216] 7) The three concentration conditions (the solution volume:
3 mL) were a 1/10 mixing ratio: 3.29 mg of the compound and 32.9 mg
of the lipids; a 2/10 mixing ratio: 6.58 mg of the compound and
32.9 mg of the lipids; and a 4/10 mixing ratio: 13.16 mg of the
compound and 32.9 mg of the lipids.
[0217] 8) Free compound (A) was removed by ultrafiltration (cutoff
molecular weight: 300,000 Da) using a stirred cell (8000 series, a
10-mL cell): Model 8010 5121 produced by Merck & Co., Inc., and
BioMax PBMK 02510 produced by Merck & Co., Inc. The external
solution was a 10 mM phosphate buffer (pH: 7.4).
[0218] As a result, liposomal formulations having four different
properties shown in Table 2 below were obtained. FIG. 1 is diagrams
showing the average particle size distribution of these
formulations.
TABLE-US-00002 TABLE 2 Average Zeta Lipid Concentration Compound
particle size potential concentration of compound (A) (A)/lipid
Content (Z-Ave, nm) PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu-
Blank 109 0.121 -67 8.44 -- -- -- lation liposome 1 Formu- Mixing
105 0.105 -29 5.40 0.12 0.02 2.2 lation ratio of 2 1/10 Formu-
Mixing 102 0.126 -23 5.46 0.14 0.03 2.6 lation ratio of 3 2/10
Formu- Mixing 103 0.148 -27 5.62 0.16 0.03 2.8 lation ratio of 4
4/10 * Content = calculated from the concentration of compound (A)
per mg of lipid. * Physical property testing was performed after
filtration through a 0.22 .mu.m filter.
2. Formulation Example 2 (Bangham Method) Preparation of
Liposome
[0219] 1) HSPC (102.0 mg), DSPE (8.5 mg), and cholesterol (33.5 mg)
were weighed and placed in an eggplant flask, and a
chloroform/methanol solution (1/1, v/v) was added and dissolved so
that the lipid concentration was 20 mg/mL.
[0220] 2) The chloroform/methanol was evaporated with a rotary
evaporator, followed by vacuum-drying. Four eggplant flasks each
containing 30 mg of the lipids in total were thus obtained.
[0221] 3) Compound (A) (170.6 mg) was weighed, and 4.5 mL of a 0.1N
sodium hydroxide solution was added thereto. The mixture was
dissolved by vortexing, and 2.6 ml of 10 mM phosphate buffer (pH:
8.0) was added thereto to thus prepare a solution of 24 mg/mL
(compound (A) solution).
[0222] 4) The compound (A) solution was added to the vacuum-dried
lipid film (three concentration conditions), followed by stirring
at 37.degree. C. for 1 hour. Since each eggplant flask contained 30
mg of the lipids, the amount of each solution was adjusted with PBS
to 3 mL to achieve a 1/10 mixing ratio: 3.0 mg of compound (A) and
30 mg of the lipids; a 2/10 mixing ratio: 6 mg of compound (A) and
30 mg of the lipids; or a 4/10 mixing ratio: 12 mg of compound (A)
and 30 mg of the lipids.
[0223] 5) Treatment was performed for 60 minutes in total using a
VS-100III, produced by AS ONE Corporation, by repeating a cycle of
28 kHz output for 60 seconds, 45 kHz output for 60 seconds, and 100
kHz output for 3 seconds.
[0224] 6) Ultrasonic treatment was performed for 60 minutes.
[0225] 7) Free compound (A) was removed by ultrafiltration (cutoff
molecular weight: 300,000 Da) using a stirred cell (8000 series, a
10-mL cell): Model 8010 5121 produced by Merck & Co., Inc., and
BioMax PBMK 02510 produced by Merck & Co., Inc.
[0226] The external solution was a 10 mM phosphate buffer (pH:
7.4).
[0227] As a result, liposomal formulations having four different
properties shown in Table 3 below were obtained. FIG. 2 is diagrams
showing the average particle size distribution of these
formulations.
TABLE-US-00003 TABLE 3 Average Zeta Lipid Concentration Compound
particle size potential concentration of compound (A) (A)/lipid
Content (Z-Ave. nm) PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu-
Blank 75 0.172 -23 8.12 -- -- -- lation liposome 5 Formu- Mixing
123 0.162 -30 6.47 0.07 0.011 1.1 lation ratio of 6 1/10 Formu-
Mixing 132 0.146 -32 6.76 0.08 0.012 1.2 lation ratio of 7 2/10
Formu- Mixing 129 0.142 -32 6.66 0.21 0.032 3.2 lation ratio of 8
4/10 * Content = calculated from the concentration of compound (A)
per mg of lipid. * Physical property testing was performed after
filtration through a 0.22 .mu.m filter.
3. Formulation Example 3 (Extruder Method) Preparation of
Liposome
[0228] 1) HSPC (255.0 mg), DSPE (21.3 mg), and cholesterol (83.0
mg) were weighed and placed in an eggplant flask, and a
chloroform/methanol solution (1/1, v/v) was added and dissolved so
that the lipid concentration was 20 mg/mL.
[0229] 2) The chloroform/methanol was evaporated with a rotary
evaporator, followed by vacuum drying.
[0230] 3) Compound (A) (143 mg) was weighed, and 3.76 mL of a 0.1N
sodium hydroxide solution was added thereto. The mixture was
dissolved by vortexing, and 2.2 mL of a 10 mM phosphate buffer (pH:
8.0) was added thereto to thus prepare a solution of 24 mg/mL
(compound (A) solution).
[0231] 4) The compound (A) solution was added to the lipid film,
and a 10 mM phosphate buffer (pH: 8.0) was added to increase the
volume to 36 mL (compound (A): 140 mg, the lipids: 360 mg).
[0232] 5) Ultrasonic treatment was performed at 28 kHz for 1 minute
using a VS-100III produced by AS ONE Corporation so that the lipids
on the wall of the eggplant flask were dispersed.
[0233] 6) At 60.degree. C., heating and stirring were performed for
30 minutes.
[0234] 7) An equivalent amount of 10 mM phosphate buffer (pH: 8.0)
was added, and extruder treatment was performed (60.degree. C., 400
nm, 200 nm). The extruder was performed with a Lipex Thermobarrel
Extruder (100 mL) produced by Northern Lipids, and with Nuclepore
membranes produced by GE Healthcare (400 nm: Product No. 111107;
200 nm: Product No. 111106).
[0235] 8) Free compound (A) was removed by ultrafiltration (cutoff
molecular weight: 300,000 Da) using a stirred cell (8000 series, a
50-mL cell): Model 8050 5122 produced by Merck & Co., Inc., and
BioMax PBMK 04310 produced by Merck & Co., Inc.
[0236] The external solution was a 10 mM phosphate buffer (pH:
7.4).
[0237] As a result, a liposomal formulation having the properties
shown in Table 4 below was obtained. FIG. 3 is a diagram showing
the average particle size distribution of this formulation.
TABLE-US-00004 TABLE 4 Average Zeta Lipid Concentration Compound
particle size potential concentration of compound (A) (A)/Lipid
Content (Z-Ave. nm) PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu-
Mixing 175 0.147 -11 5.12 0.153 0.030 3.0 lation ratio of 9 4/10 *
Content = calculated from the concentration of compound (A) per mg
of lipid. * Physical property testing was performed after
filtration through a 0.22 .mu.m filter.
4. Formulation Example 4 (Lipid-Compound Film Method) Preparation
of Liposome
[0238] 1) Lipids (HSPC: 21.2 mg, DSPE: 1.8 mg, and cholesterol: 7.0
mg) and compound (A) (6.0 mg) were weighed and placed in an
eggplant flask. At this time, the amounts were adjusted so that
compound A) was present in an amount of 2 mg per 10 mg of the
lipids.
[0239] 2) A chloroform/methanol solution (1/1, v/v) was added and
dissolved so that the total weight of the lipids and compound (A)
was 20 mg/mL.
[0240] 3) The chloroform/methanol was evaporated with a rotary
evaporator, followed by vacuum-drying.
[0241] 4) 10 mM phosphate buffer (pH 8.0) was added to the
resulting product so that the lipid concentration was 10 mg/mL.
[0242] 5) Ultrasonic treatment was performed at 28 kHz using a
VS-100III produced by AS ONE Corporation to separate the lipids and
compound (A) from the wall of the eggplant flask, and the mixture
was stirred at 60.degree. C. for 30 minutes.
[0243] 6) An equivalent amount of 10 mM phosphate buffer (pH: 8.0)
was added thereto, and extruder treatment was performed (60.degree.
C., 400 nm, 200 nm). More specifically, the extruder was performed
with a Lipex Thermobarrel Extruder (100 mL) produced by Northern
Lipids, and with Nuclepore membranes produced by GE Healthcare (400
nm: Product No. 111107; and 200 nm: Product No. 111106).
[0244] 7) Free compound (A) was removed by ultrafiltration (cutoff
molecular weight: 300,000 Da) using a stirred cell (8000 series, a
10-mL cell): Model 8010 5121 produced by Merck & Co., Inc., and
BioMax PBMK 02510 produced by Merck & Co., Inc. The external
solution was a 10 mM phosphate buffer (pH: 7.4).
[0245] As a result, a liposomal formulation having the properties
shown in Table 5 below was obtained. FIG. 4 is a diagram showing
the average particle size distribution of this formulation.
TABLE-US-00005 TABLE 5 Average Zeta Lipid Concentration Compound
particle size potential concentration of compound (A) (A)/lipid
Content (Z-Ave. nm) PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu-
Mixing 158 0.132 -13 4.73 0.07 0.015 1.5 lation ratio of 10 2/10 *
Content = calculated from the concentration of compound (A) per mg
of lipid. * Physical property testing was performed after
filtration through a 0.22 .mu.m filter.
5. Production of PEG-Treated Stealth Liposomal Formulation
(1) Formulation Example 5 (Remote Loading Method) Preparation of
Liposome
[0246] 1) HSPC (107.5 mg), MPEG2000-DSPE (35.0 mg), and cholesterol
(35.5 mg) were weighed and placed in an eggplant flask, and a
chloroform/methanol solution (1:1, v/v) was added and dissolved so
that the lipid concentration was 30 mg/mL.
[0247] 2) The chloroform/methanol was evaporated with a rotary
evaporator, followed by vacuum-drying. Then, a 0.1N sodium
hydroxide solution (>pH: 12.0) was added so that the lipid
concentration was 10 mg/mL, and the mixture was redispersed by
vortexing.
[0248] 3) Ultrasonic treatment was performed for 30 minutes in
total using a VS-100III produced by AS ONE Corporation by repeating
a cycle of 28 kHz output for 60 seconds, 45 kHz output for 60
seconds, and 100 kHz output for 3 seconds. Thereafter, the particle
size was confined.
[0249] 4) To exchange the liposome external solution, stirred
ultrafiltration (cutoff molecular weight: 300,000 Da) was
performed. The liposome external solution was 10 mM phosphate
buffer (pH: 8.0) (liposome solution).
[0250] The ultrafiltration was performed using a stirred cell (8000
series, a 50-mL cell): Model 8050 5122 produced by Merck & Co.,
Inc., and BioMax PBMK 04310 produced by Merck & Co., Inc. After
the preparation of empty liposomes, lipid quantification was
performed.
[0251] 5) Compound (A) (56.5 mg) was weighed, and 1.41 mL of a 0.1N
sodium hydroxide solution was added thereto. The mixture was
dissolved by vortexing, and 5.65 mL of 10 mM phosphate buffer (pH:
8.0) was added thereto to thus prepare a solution of 8 mg/mL
(compound (A) solution).
[0252] 6) The compound (A) solution was added to the liposome
solution (three concentration conditions), and the mixture was
stirred at 60.degree. C. for 1 hour. The three concentration
conditions (solution volume: 4 mL) were a 1/10 mixing ratio: 4.0 mg
of compound (A) and 40.0 mg of the lipids; a 2/10 mixing ratio: 8.0
mg of compound (A) and 40.0 mg of the lipids; and a 4/10 mixing
ratio: 16.0 mg of compound (A) and 40.0 mg of the lipids.
[0253] 7) Free compound (A) was removed by stirred ultrafiltration
(cutoff molecular weight: 300,000 Da). Ultrafiltration was
performed by using a stirred cell (8000 series, a 10-mL cell):
Model 8010 5121 produced by Merck & Co., Inc., and BioMax PBMK
02510 produced by Merck & Co., Inc. The external solution was a
10 mM phosphate buffer (pH: 7.4).
[0254] As a result, liposomal formulations having four different
properties shown in Table 6 below were obtained. FIG. 5 is diagrams
showing the average particle size distribution of these
formulations.
TABLE-US-00006 TABLE 6 Average Zeta Lipid Concentration Compound
particle size potential concentration of compound (A) (A)/lipid
Content (Z-Ave. nm) PDI (mV) (mg/mL) (mg/mL) (mg/mL) (%) Formu-
Blank 114 0.139 -23 5.94 -- -- -- lation liposome 11 Formu- Mixing
119 0.113 -32 6.11 0.08 0.01 1.3 lation ratio of 12 1/10 Formu-
Mixing 120 0.096 -33 6.58 0.09 0.01 1.4 lation ratio of 13 2/10
Formu- Mixing 116 0.131 -30 6.58 0.13 0.02 2.0 lation ratio of 14
4/10 * Content = calculated from the concentration of compound (A)
per mg of lipid. * Physical property testing was performed after
filtration through a 0.22 .mu.m filter.
(2) Formulation Example 6 (Bangham Method) Preparation of
Liposome
[0255] 1) HSPC (87.0 mg), MPEG2000-DSPE (28.3 mg), and cholesterol
(29.0 mg) were weighed and placed in an eggplant flask, and a
chloroform/methanol solution (1:1, v/v) was added and dissolved so
that the lipid concentration was 30 mg/mL.
[0256] 2) The chloroform/methanol was evaporated with a rotary
evaporator, followed by vacuum drying. Four eggplant flasks each
containing 10 mg of the lipids in total were thus obtained.
[0257] 3) Compound (A) (164 mg) was weighed, and 4.3 mL of a 0.1N
sodium hydroxide solution was added thereto. The mixture was
dissolved by vortexing, and 2.53 mL of 10 mM phosphate buffer (pH:
8.0) was added thereto to thus prepare a solution of 24 mg/mL
(compound (A) solution).
[0258] 4) The compound (A) solution was added to the vacuum-dried
lipid film (three concentration conditions), followed by stirring
at 37.degree. C. for 1 hour.
[0259] 5) The three concentration conditions (solution volume: 4
mL) were a 1/10 mixing ratio: 1.0 mg of compound (A) and 10.0 mg of
the lipids; a 2/10 mixing ratio: 2.0 mg of compound (A) and 10.0 mg
of the lipids; and a 4/10 mixing ratio: 4.0 mg of compound (A) and
10.0 mg of the lipids.
[0260] 6) Ultrasonic treatment was performed for 60 minutes in
total using a VS-100III produced by AS ONE Corporation by repeating
a cycle of 28 kHz output for 60 seconds, 45 kHz output for 60
seconds, and 100 kHz output for 3 seconds.
[0261] 7) Free compound (A) was removed by ultrafiltration (cutoff
molecular weight: 300,000 Da) using a stirred cell (8000 series, a
10-mL cell): Model 8010 5121 produced by Merck & Co., Inc., and
BioMax PBMK 02510 produced by Merck & Co., Inc. The external
solution was a 10 mM phosphate buffer (pH: 7.4).
[0262] As a result, liposomal formulations having four different
properties shown in Table 7 below were obtained. FIG. 6 is diagrams
showing the average particle size distribution of these
formulations.
TABLE-US-00007 TABLE 7 Average Lipid particle Zeta concen-
Concentration Compound size potential tration of compound (A)/lipid
Content (Z-Ave. nm) PDI (mV) (mg/mL) (A) (mg/mL) (mg/mL) (%) Formu-
Blank 115 0.205 -48 7.58 -- -- -- lation liposome 15 Formu- Mixing
95 0.213 -45 8.57 0.07 0.009 0.9 lation ratio of 16 1/10 Formu-
Mixing 110 0.243 -48 6.93 0.17 0.024 2.4 lation ratio of 17 2/10
Formu- Mixing 102 0.221 -43 7.77 0.27 0.035 3.5 lation ratio of 18
4/10 * Content = calculated from the concentration of compound (A)
per mg of lipid. * Physical property testing was performed after
filtration through a 0.22 .mu.m filter.
(3) Formulation Example 7 (Extruder Method) Preparation of
Liposome
[0263] 1) HSPC (215.0 mg), MPEG2000-DSPE (70.0 mg), and cholesterol
(71.0 mg) were weighed and placed in an eggplant flask, and a
chloroform/methanol solution (1:1, v/v) was added and dissolved so
that the lipid concentration was 30 mg/mL.
[0264] 2) The chloroform/methanol solution was evaporated with a
rotary evaporator, followed by vacuum-drying.
[0265] 3) Compound (A) (142.7 mg) was weighed, and 3.8 mL of a 0.1N
sodium hydroxide solution was added thereto. The mixture was
dissolved by vortexing, and 2.19 mL of 10 mM phosphate buffer (pH:
8.0) was added thereto to thus prepare a solution of 24 mg/mL
(compound (A) solution).
[0266] 4) The compound (A) solution was added to the lipid film,
and 10 mM phosphate buffer (pH: 8.0) was added thereto to increase
the volume to 35.6 mL (compound (A): 142.4 mg, the lipids: 356.0
mg).
[0267] 5) Ultrasonic treatment was performed for 1 minute and for
30 minutes in total using a VS-1001 produced by AS ONE Corporation
by repeating a cycle of 28 kHz output for 60 seconds, 45 kHz output
for 60 seconds, and 100 kHz output for 3 seconds to separate the
lipids from the wall of the eggplant flask, and the resulting
product was stirred at 60.degree. C. for 30 minutes.
[0268] 6) An equivalent amount of 10 mM phosphate buffer (pH: 8.0)
was added thereto, and extruder treatment was performed (60.degree.
C., 400 nm, 200 nm). The extruder was performed with a Lipex
Thermobarrel Extruder (100 mL) produced by Northern Lipids, and
with Nuclepore membranes produced by GE Healthcare (400 nm: Product
No. 111107; 200 nm: Product No. 111106).
[0269] 7) Free compound (A) was removed by ultrafiltration (cutoff
molecular weight: 300,000 Da) using a stirred cell (8000 series, a
50-mL cell): Model 8050 5122 produced by Merck & Co., Inc., and
BioMax PBMK 04310 produced by Merck & Co., Inc. The external
solution was a 10 mM phosphate buffer (pH: 7.4).
[0270] As a result, a liposomal formulation having the properties
shown in Table 8 below was obtained. FIG. 7 shows is diagrams
showing the average particle size distribution of this
formulation.
TABLE-US-00008 TABLE 8 Average Lipid particle Zeta concen-
Concentration Compound size potential tration of compound (A)/lipid
Content (Z-Ave. nm) PDI (mV) (mg/mL) (A) (mg/mL) (mg/mL) (%) Formu-
Mixing 158 0.114 -35 5.03 0.128 0.025 2.5 lation ratio of 19 4/10 *
Content = calculated from the concentration of compound (A) per mg
of lipid. * Physical property testing was performed after
filtration through a 0.22 .mu.m filter.
(4) Formulation Example 8 (Lipid-Compound Film Method) Preparation
of Liposome
[0271] 1) HSPC (18.0 mg), MPEG2000-DSPE (6.0 mg), cholesterol (6.0
mg), and compound (A) (6.0 mg) were weighed and placed in an
eggplant flask. At this time, the amounts were adjusted so that
compound (A) was present in an amount of 2 mg per 10 mg of the
lipids. Then, a chloroform/methanol solution (1/1, v/v) was added
and dissolved so that the total weight of the lipids and compound
(A) was 30 mg/mL.
[0272] 2) The chloroform/methanol was evaporated with a rotary
evaporator, followed by vacuum-drying.
[0273] 3) 10 mM phosphate buffer (pH 8.0) was added to the
resulting product so that the lipid concentration was 10 mg/mL.
[0274] 4) Ultrasonic treatment was performed at 28 kHz using a
VS-100III produced by AS ONE Corporation to separate the lipids and
compound (A) from the wall of the eggplant flask, and the mixture
was stirred at 60.degree. C. for 30 minutes.
[0275] 5) An equivalent amount of 10 mM phosphate buffer (pH: 8.0)
was added thereto, and extruder treatment was performed (60.degree.
C., 400 nm, 200 nm). The extruder was performed with a Lipex
Thermobarrel Extruder (100 mL) produced by Northern Lipids and with
Nuclepore membranes produced by GE Healthcare (400 nm: Product No.
111107; 200 nm: Product No. 111106).
[0276] 6) Free compound (A) was removed by ultrafiltration (cutoff
molecular weight: 300,000 Da) using a stirred cell (8000 series, a
10-mL cell): Model 8010 5121 produced by Merck & Co., Inc., and
BioMax PBMK 02510 produced by Merck & Co., Inc. The external
solution was a 10 mM phosphate buffer (pH: 7.4).
[0277] As a result, Liposomal Formulation 20 having the properties
shown in Table 9 below was obtained. FIG. 8 is a diagram showing
the average particle size distribution of this formulation.
TABLE-US-00009 TABLE 9 Average Lipid particle Zeta concen-
Concentration Compound size potential tration of compound (A)/lipid
Content (Z-Ave. nm) PDI (mV) (mg/mL) (A) (mg/mL) (mg/mL) (%) Formu-
Mixing 135 0.136 -30 11.30 0.06 0.005 0.5 lation ratio of 20 2/10 *
Content = calculated from the concentration of compound (A) per mg
of lipid. * Physical property testing was performed after
filtration through a 0.22 .mu.m filter.
6. Method for Quantifying Compound (A)
[0278] 1) Compound (A) (5 mg) was weighed and placed in a test
tube, and a 0.1N sodium hydroxide solution (0.125 mL) was added.
The resulting mixture was dissolved by vortexing.
[0279] 2) Purified water (4.875 mL) was added thereto to thus
prepare a compound (A) solution of 1 mg/mL.
[0280] 3) A calibration curve was prepared by measuring the
absorbance at wavelength of 265 nm with respect to 6 different
compound (A) concentrations of 0.0 mg/mL to 0.50 mg/mL.
[0281] 4) Compound (A) solutions of known concentrations (0.1
mg/mL, 0.2 mg/mL, and 0.4 mg/mL) were prepared, and sample
concentrations were obtained from the calibration curve.
[0282] 5) The compound (A) solutions of known concentrations (0.1
mg/mL, 0.2 mg/mL, and 0.4 mg/mL) were each added to blank liposome
(lipid concentration: 4.7 mg/mL), and the compound (A)
concentrations were measured.
[0283] 6) The recovery rates were calculated from the measurement
results of the known concentration solutions, and the measurement
results of the known concentration solutions to which liposome was
added.
[0284] Table 10 and FIG. 9 show the measurement results.
TABLE-US-00010 TABLE 10 Standard Curve Chloroform/methanol solution
Compound A Absorbance 265 nm (mg/mL) {circle around (1)} {circle
around (2)} {circle around (3)} Average 0.000 0.129 0.130 0.127
0.000 0.031 0.293 0.291 0.291 0.163 0.063 0.435 0.450 0.450 0.316
0.125 0.736 0.761 0.761 0.624 0.250 1.360 1.374 1.381 1.243 0.500
2.567 2.587 2.642 2.470 Slope 4.9321 Intercept 0.0064 Recovery
Concentration rate Recovery rate {circle around (1)} {circle around
(2)} {circle around (3)} Average (mg/ml) (%) (-Liposome) Single
drug: 0.588 0.597 0.613 0.503 0.101 101 0.1 mg/ml Single drug:
0.966 0.925 0.998 0.867 0.174 87 0.2 mg/ml Single drug: 1.887 1.911
1.922 1.810 0.366 91 0.4 mg/ml A) Liposome: 0.146 0.146 0.148 0.050
0.009 0.0 mg A) Liposome: 0.591 0.600 0.604 0.502 0.200 100 91 0.1
mg A) Liposome: 1.058 1.056 1.054 0.960 0.193 111 106 0.2 mg A)
Liposome: 1.881 1.922 1.936 1.817 0.367 100 98 0.4 mg B) Liposome:
0.141 0.141 0.144 0.046 0.008 0.0 mg B) Liposome: 0.580 0.591 0.581
0.488 0.098 97 89 0.1 mg B) Liposome: 1.000 1.027 1.011 0.916 0.284
106 101 0.2 mg B) Liposome: 1.845 1.866 1.889 1.770 0.358 98 98 0.4
mg
[0285] The results confirmed that no effect was caused by the
lipids formed into liposomes; thus, compound (A) was quantified by
using an absorbance method.
7. Mass Production of Nanosphere of Stealth Liposome of Compound
(A) by Bangham Method
[0286] (1) A PEG-treated compound (A)-encapsulated liposome was
prepared in large quantities by the Bangham method, and the
properties were tested.
Production Method
[0287] (i) HSPC (5.524 g), cholesterol (1.8419 g), and
MPEG2000-DSPE (1.7978 g) were weighed and placed in an eggplant
flask, a chloroform/methanol (1/1, v/v) solution was added so that
the lipid concentration was 30 mg/mL, and the mixture was dissolved
by stirring at 37.degree. C.
[0288] (ii) The solvent was distilled off with an evaporator in a
nitrogen atmosphere, followed by vacuum-drying. Fifteen 100-ml
eggplant flasks each containing 600 mg of the lipids and one 100-ml
eggplant flask containing 300 mg of the lipids were thus
prepared.
[0289] (iii) Compound (A) was weighed, and a 0.1N sodium hydroxide
solution was added thereto to produce a solution having a compound
(A) concentration of 40 mg/mL. 10 mM phosphate buffer (pH: 8.0) was
added to the solution so that the compound (A) concentration was 24
mg/mL.
[0290] (iv) The solution obtained in (iii) was added to the lipid
film; and 10 mM phosphate buffer (pH: 8.0) was added thereto so
that the lipid concentration was 10 mg/mL, followed by stirring at
37.degree. C. for 1 hour. Then, 22.2 mg of compound (A) was added
to the eggplant flasks each containing 600 mg of the lipids, and
11.1 mg of compound (A) was added to the flask containing 300 mg of
the lipids.
[0291] (V) Ultrasonic treatment was performed for each of the
eggplant flasks for 60 minutes in total using a VS-100III produced
by AS ONE Corporation by repeating a cycle of 28 kHz output for 60
seconds, 45 kHz output for 60 seconds, and 100 kHz output for 3
seconds.
[0292] (vi) A stirred cell (8000 series, a 400-mL cell): Model 8400
5124 produced by Merck & Co., Inc., and BioMax PBMK 07610
produced by Merck & Co., Inc. were used. Since the solution
amount was 930 mL in total, three cells were used for
ultrafiltration (cutoff molecular weight: 300,000 Da) to replace
the outer aqueous phase with 10 mM phosphate buffer (pH 7.4), and
unencapsulated compound (A) was removed.
[0293] As a result, a liposomal formulation having the properties
shown in Table 11 below was obtained. FIG. 10 is a diagram showing
the average particle size distribution of this formulation.
TABLE-US-00011 TABLE 11 Average Lipid particle Zeta concen-
Concentration Compound size potential tration of compound (A)/lipid
Content (Z-Ave. nm) PDI (mV) (mg/mL) (A) (mg/mL) (mg/mL) (%) Formu-
Large-scale 163 0.265 -58 72.2 2.03 0.028 2.8 lation preparation 21
* Content = calculated from the concentration of compound (A) per
mg of lipid. * Physical property testing was performed after
filtration through a 0.22 .mu.m filter.
[0294] The following general test was conducted using part of the
produced PEG-treated compound (A)-encapsulated liposome.
[0295] (2) FIG. 11 is transmission electron microscope images of
Formulation 21.
[0296] Analysis method: Morphological observation
[0297] Photographing device: Hitachi H-7600 at 100 kV
[0298] Sample production method [0299] Dispersion: 400-mesh grid
with carbon support membrane [0300] Staining: Negative staining
(phosphotungstic acid)
[0301] FIG. 11 shows electron micrographs.
[0302] (3) Part of the produced PEG-treated compound
(A)-encapsulated liposome was subjected to HPLC analysis to confirm
the presence or absence of decomposition products. [0303] Analysis
and quantification of compound (A) by HPLC
[0304] Apparatus: Agilent 1290 Infinity LC series
[0305] Column: Shiseido Capcell Pak C18 UG120, 5 .mu.m,
4.6.times.150 mm
[0306] Mobile phase: 1% acetic acid/H.sub.2O: 1% acetic
acid/acetonitrile=60:40
[0307] Flow rate: 1 mL/min
[0308] Detection wavelength: 265 nm
[0309] Column temperature: 25.degree. C.
[0310] Sample Preparation Method
[0311] Sample 1: Compound (A) was dissolved in a 1M sodium
hydroxide solution so that compound A was 1 mg/mL. The resulting
product was allowed to stand at 40.degree. C. overnight (oxide
production sample).
[0312] Sample 2: Compound (A) was added to a 0.1N sodium hydroxide
solution-10 mM phosphate buffer (pH: 8.0) (buffer for
encapsulation) so that compound (A) was 0.5 mg/mL (control
sample)
[0313] Sample 3: Sample 2 was subjected to two-fold dilution with
physiological saline to prepare a solution of 0.25 mg/mL. The
solution was allowed to stand at 37.degree. C. overnight (compound
control sample).
[0314] Sample 4: PEG-treated empty liposomes were diluted 10-fold
with physiological saline. The resulting product was allowed to
stand at 37.degree. C. overnight, followed by ultrafiltration
(lipid control sample).
[0315] Sample 5: The PEG-treated compound (A)-encapsulated liposome
was diluted 10-fold with physiological saline. The resulting
product was allowed to stand at 37.degree. C. overnight, followed
by ultrafiltration (liposome sample).
[0316] The above 5 samples were analyzed by HPLC to confirm the
presence or absence of oxides of compound (A), and the presence or
absence of oxides of compound (A) of the PEG-treated compound
(A)-encapsulated liposome in an environment at a temperature of
37.degree. C.
[0317] FIG. 12 charts of HPLC measurement of Samples 1 to 5.
8. Production of PEG-Treated Compound (B)-Encapsulated Stealth
Liposome by Bangham Method
[0318] (1) Production of Liposome
[0319] (i) HSPC (36.0 mg), cholesterol (12.0 mg), and MPEG2000-DSPE
(12.0 mg) were weighed and placed in an eggplant flask, a
chloroform/methanol (1/1, v/v) solution was added so that the lipid
concentration was 30 mg/mL, and the mixture was dissolved by
stirring at 37.degree. C.
[0320] (ii) The solvent was distilled off with an evaporator in a
nitrogen atmosphere, followed by vacuum-drying.
[0321] (iii) Compound (B) was added to and dissolved in 10 mM
phosphate buffer (pH: 8.0) so that compound (B) was 20 mg/mL.
[0322] (iv) The compound (B) solution was added to the lipid film
to achieve 1/10 (drug/lipid, w/w %), and 10 mM phosphate buffer
(pH: 8.0) was added so that the lipid concentration was 10 mg/mL
(compound (B): 6.0 mg, the lipids: 60.0 mg).
[0323] (v) The resulting product was vortexed for about 20 seconds,
followed by stirring at 37.degree. C. for 1 hour.
[0324] (vi) Ultrasonic treatment was performed for 90 minutes in
total using a VS-100III produced by AS ONE Corporation by repeating
a cycle of 28 kHz output for 60 seconds, 45 kHz output for 60
seconds, and 100 kHz output for 3 seconds.
[0325] (vii) A stirred cell (8000 series, a 10-mL cell): Model 8010
5121 produced by Merck & Co., Inc., and BioMax PBMK 02510
produced by Merck & Co., Inc. were used. The amount was 6 mL at
the time of start and 4 mL at the time of collection, and 1.5-fold
concentration was performed. Since the drug encapsulation amount
was low, ultrafiltration (cutoff molecular weight: 300,000 Da) was
performed to replace the outer aqueous phase with 10 mM phosphate
buffer (pH 7.4), and unencapsulated compound (B) was removed.
[0326] As a result, a liposomal formulation having the properties
shown in Table 12 below was obtained. FIG. 13 is a diagram showing
the average particle size distribution of this formulation.
TABLE-US-00012 TABLE 12 Average Lipid particle Zeta concen-
Concentration Compound size potential tration of compound (B)/lipid
Content (Z-Ave. nm) PDI (mV) (mg/mL) (B) (mg/mL) (mg/mL) (%) Formu-
Mixing 87 0.199 -32 12.19 0.101 0.008 0.8 lation ratio of 22 1/10 *
Content = calculated from the concentration of compound (A) per mg
of lipid. * Physical property testing was performed after
filtration through a 0.22 .mu.m filter.
[0327] Accordingly, a PEG-treated compound (B)-encapsulated
liposome was produced.
[0328] (2) FIG. 14 is transmission electron microscope images of
Formulation 22.
(3) Analysis of Method of Quantifying Compound (B)
[0329] Absorption Spectrum Confirmation Method
[0330] (i) Compound (B) was added to and dissolved in 10 mM
phosphate buffer (pH: 8.0) so that compound (B) was 20 mg/mL.
[0331] (ii) The product obtained in (i) was subjected to two-fold,
four-serial dilutions with purified water.
[0332] (iii) The sample of each concentration was diluted 10-fold
with a chloroform/methanol (1/1, v/v) solution, and the absorption
spectrum was measured at 220 to 700 nm using UV2700.
[0333] The absorption spectrum of compound (B) was measured. As a
result, the maximum absorption wavelength was 285 nm. It was
confirmed that the quantification of compound (B) could be measured
by the absorbance method (absorbance: 285 nm). FIG. 15 is a graph
showing changes in the UV absorption spectrum.
9. Production of PEG-Treated Compound (C)-Encapsulated Stealth
Liposome by Bangham Method
[0334] (1) Liposome Production Method
[0335] (i) HSPC (60.0 mg), cholesterol (12.0 mg), and MPEG2000-DSPE
(12.0 mg) were weighed and placed in an eggplant flask, a
chloroform/methanol (1/1, v/v) solution was added so that the lipid
concentration was 30 mg/mL, and the mixture was dissolved by
stirring at 37.degree. C.
[0336] (ii) The solvent was distilled off with an evaporator in a
nitrogen atmosphere, followed by vacuum-drying.
[0337] (iii) Compound (C) was dissolved in DMSO so that compound
(C) was 20 mg/mL, and 10 mM phosphate buffer (pH: 8.0) was added so
that compound (C) was 10 mg/mL.
[0338] (iv) The compound (C) solution was added to the lipid film
to achieve 1/10 (drug/lipid, w/w %), and the mixture was stirred at
37.degree. C. for 1 hour (compound (C): 6.0 mg, the lipids: 60.0
mg).
[0339] (v) Ultrasonic treatment was performed for 90 minutes in
total using a VS-100III, produced by AS ONE Corporation by
repeating a cycle of 28 kHz output for 60 seconds, 45 kHz output
for 60 seconds, and 100 kHz output for 3 seconds.
[0340] (vi) Ultrafiltration (cutoff molecular weight: 300,000 Da)
was performed using a stirred cell (8000 series, a 10-mL cell):
Model 8010 5121 produced by Merck & Co., Inc., and BioMax PBMK
02510 produced by Merck & Co., Inc. to replace the outer
aqueous phase with 10 mM phosphate buffer (pH 7.4), and
unencapsulated compound (C) was removed.
[0341] (2) As a result, a liposome having the properties shown in
Table 13 was obtained. FIG. 16 is a diagram showing the average
particle size distribution.
TABLE-US-00013 TABLE 13 Average Lipid particle Zeta concen-
Concentration Compound size potential tration of compound (C)/lipid
Content (Z-Ave. nm) PDI (mV) (mg/mL) (C) (mg/mL) (mg/mL) (%) Formu-
Mixing 97 0.158 -34 17.27 1.02 0.059 5.9 lation ratio of 23 1/10 *
Content = calculated from the concentration of compound (A) per mg
of lipid * Physical property testing was performed after filtration
through a 0.22 .mu.m filter.
[0342] Accordingly, a PEG-treated compound (C)-encapsulated
liposome was prepared.
[0343] (3) FIG. 17 is transmission electron microscope images of
Formulation 23.
(4) Analysis of Method of Quantifying Compound (C)
[0344] Absorption Spectrum Confirmation Method
[0345] (i) Compound (C) was added to and dissolved in DMSO so that
compound (C) was 20 mg/mL.
[0346] (ii) An equivalent amount of 10 mM phosphate buffer (pH:
8.0) was added to the product obtained in (i) to produce a 50% DMSO
solution having a compound (C) concentration of 10 mg/mL.
[0347] (iii) The product obtained in (ii) was subjected to
two-fold, three-serial dilution with purified water.
[0348] (vi) The sample of each concentration was diluted 10-fold
with a chloroform/methanol (1/1, v/v) solution, and the absorption
spectrum was measured at 220 to 700 nm using UV2700.
[0349] The absorption spectrum of compound (C) was measured; the
absorption peaks were detected at 300 nm and 368 nm. It was
confirmed that the quantification of compound (C) could be
performed by the absorbance method (wavelength: 300 nm). FIG. 18 is
a graph showing changes in the UV absorption spectrum of compound
(C).
10. Production of Stealth Liposomal Formulation of Compound (A)
(ONO-1301) by Direct Dispersion Improving Method
[0350] 1) Production Method
[0351] (i) The two types of lipids and compound (A) were weighed as
shown in Table 14 below, and dissolved in 20 g of t-butanol at
70.degree. C.
TABLE-US-00014 TABLE 14 Composition 1 Composition 2 Lipid or API
(Formulation 24) (Formulation 25) DEPC 1.88 g 1.80 g MPEG2000-DSPE
0.12 g 0.20 g Compound (A) 100 mg 100 mg
[0352] (ii) The liquid obtained by dissolution in (i) was instantly
frozen in dry ice/acetone.
[0353] (iii) After freezing, the resulting product was freeze-dried
for about 17 hours with a freeze-dryer.
[0354] (iv) 440 mL of PBS(-) was added to the obtained powder, and
the mixture was placed in a warm bath at 50.degree. C. and
dispersed by a sonicator until no lumps were present (at this time,
the ONO-1301 concentration was 2.5 mg/mL).
[0355] (v) A polycarbonate filter with a pore size of 400 nm and a
drain disc were attached, and sizing was performed at about 50
kg/cm.sup.2 with an extruder in which the jacket was watered with
warm water at 50.degree. C.
[0356] (vi) In the same manner as (v), sizing was performed with a
200-nm filter (3 Pass) to thus obtain a translucent liposome
solution.
[0357] 2) Ultrafiltration
[0358] Ultrafiltration (ultrafiltration membrane: PBMK04310, Merck
Millipore, cutoff molecular weight: 300,000 Da) was performed using
PBS(-) (10-fold dilution of SIGMA D1408) to remove unencapsulated
compounds.
[0359] Ultrafiltration was performed using a stirred cell, 8000
series: Model 8050, product No. 5122, produced by Merck & Co.,
Inc. For the membrane, BioMax PBMK 04310,300 kDa, produced by Merck
& Co., Inc., was used.
[0360] After the filtration, sterilization and filtration with a
0.22-.mu.m filter was performed in a clean bench.
[0361] 3) Confirmation of Properties
[0362] The liposome solution was subjected to ultrafiltration
(cutoff molecular weight: 300,000 Da) with PBS(-): Dulbecco's
phosphate buffered saline (without Ca and Mg), and the resulting
product was used as a sample after ultrafiltration. Additionally,
the presence or absence of oxides of ONO-1301 was confirmed by
HPLC.
[0363] As shown in Table 15, the results showed no significant
change in each physical property before and after the
ultrafiltration. Additionally, as shown in FIGS. 19 and 20, no
peaks of ONO-1301 decomposition products were observed in the
particle size distribution or the HPLC analysis results. The yield
of compound (A) was 88%.
[0364] 4) Stability Test
[0365] After ultrafiltration (Formulation 25), the stability was
analyzed after storage at 4.degree. C. for 9 months using PBS(-):
Dulbecco's phosphate buffered saline (without Ca and Mg); the
results confined no change in the particle size distribution,
content, and other physical properties; thus, the formulation was
confirmed to be stable.
TABLE-US-00015 TABLE 15 Compound Average Lipid Compound of the
particle Zeta concen- of the invention/ size potential tration
invention lipid Content (Z-Ave. nm) PDI (mV) (mg/mL) (mg/mL)
(mg/mL) (%) Formu- Before 91.4 0.16 -3.4 41.8 2.5 0.060 6.0 lation
ultrafil- 24 tration Formu- After 86.8 0.14 -2.1 40.1 2.2 0.055 5.5
lation ultrafil- 25 tration
[0366] 5) Confirmation of Decomposition Product (HPLC Analysis)
[0367] The HPLC measurement results confirmed that no decomposition
products of compound (A) were present under the conditions of this
production.
11. Production of Stealth Liposomal Formulations of Compound (B),
Compound (D), and Compound (E) by Direct Dispersion Improving
Method
[0368] Liposome Production Method
[0369] (i) DEPC (Nippon Fine Chemical Co., Ltd.) (0.188 g) and
MPEG2000-DSPE (Nippon Fine Chemical Co., Ltd.) (0.012 g), and
compound (B, D, or E) (0.01 g) were weighed and placed in a glass
vial.
[0370] (ii) 2.0 g of t-butanol was added to the product obtained in
(i), and the mixture was dissolved by stirring while heating at
37.degree. C.
[0371] (iii) The resulting product was instantly frozen in
ethanol-dry ice.
[0372] (iv) Freeze-drying was performed for 17 hours.
[0373] (v) PBS(-) (Dulbecco's phosphate buffered saline (without Ca
and Mg)) (20 mL) was added to the lipid/compound powder obtained
after freeze-drying.
[0374] (vi) After the resulting product was vortexed lightly,
ultrasonic treatment was performed for 30 minutes while heating at
37.degree. C.
[0375] (vii) Extruder treatment was performed while heating at
37.degree. C. For the apparatus, a LIPEX extruder (100 mL) was
used. After treatment was performed once with a polycarbonate
filter with a pore size of 400 nm, treatment was performed three
times with a polycarbonate filter with a pore size of 200 nm
(treatment pressure: about 5 MPa). The extruder used was a Lipex
Thermobarrel Extruder (100 mL) produced by Northern Lipids, and the
membranes used were Nuclepore membranes produced by GE Healthcare
(400 nm: Product No. 111107; 200 nm: Product No. 111106).
[0376] (viii) Ultrafiltration (ultrafiltration membrane: PBMK04310,
Merck Millipore, cutoff molecular weight: 300,000 Da) was performed
using PBS(-) (10-fold dilution of SIGMA D1408), and unencapsulated
compounds were removed. The ultrafiltration was performed until the
ultrafiltration waste liquid became 140 mL, which was 7 times the
liposome amount (20 mL); and the resulting product in the final
amount of 20 mL was collected.
[0377] The ultrafiltration was performed using a stirred cell, 8000
series: Model 8050, product No. 5122, produced by Merck & Co.,
Inc. For the membrane, BioMax PBMK 04310, 300 kDa, produced by
Merck & Co., Inc., was used. The liposome solution was
introduced into the stirred cell, the apparatus was set, and
feeding of the solution was performed by nitrogen gas
pressurization (0.4 MPa) until 140 mL of waste liquid was
discharged.
[0378] (ix) Sterilization and filtration were performed with a
0.22-.mu.m filter in a clean bench.
[0379] (x) A physical property test was performed. In the physical
property test, the average particle size measurement, zeta
potential measurement, lipid quantification (Wako Pure Chemical
Industries, Phospholipid C-Test Wako), and absorbance measurement
(Abs 680 nm) were performed.
Test Results
(1) Production of Compound (B) (Formulation 26)-Encapsulated
Liposome
[0380] The compound (B)-encapsulated liposome had the physical
properties shown in Table 16 and FIG. 21. As shown in FIG. 22, UV
absorption derived from compound (B) was observed in the UV
absorption spectrum of the compound (B)-encapsulated liposome.
Table 16 below shows the properties (the particle size, PdI value,
and zeta potential) of the obtained liposome. FIG. 21 shows the
liposome particle size distribution of Formulation 26.
[0381] FIG. 22 shows UV absorption spectra of compound (B)
(Beriplast) and liposomes containing the compound (B).
TABLE-US-00016 TABLE 16 Lipid Encapsulated Average particle Zeta
potential (mg/mL) amount (mg/mL) size (nm) PdI (mV) 6.2 0.017 or
less 122 0.190 -19
(2) Production of Compound (D) (Formulation 27)-Encapsulated
Stealth Liposome
[0382] The amount of encapsulated compound (D) was 0.249 mg/mL
according to UV quantification. The obtained liposome had the
properties shown in Table 17 below; i.e., the particle size, PdI
value, and zeta potential. FIG. 23 shows the liposome particle size
distribution of Formulation 27.
[0383] The absorption spectrum of FIG. 24 shows quantitativity at
UV absorption of 233 nm. FIG. 24 is UV absorption spectra of
compound (D) (Limaprost) and liposomes containing Limaprost.
TABLE-US-00017 TABLE 17 Formulation 27: Lipid Encapsulated Average
particle Zeta potential (mg/mL) amount (mg/mL) size (nm) PdI (mV)
6.0 0.249 110 0.200 -19
(3) Production of Compound (E) (Formulation 28)-Encapsulated
Stealth Liposome
[0384] A compound (E)-encapsulated liposome had the physical
properties shown in Table 18 and FIG. 25. The amount of
encapsulated compound (E) was 0.305 mg/mL according to UV
quantification. The table below shows the particle size, PdI value,
and zeta potential. The absorption spectra of FIG. 26 shows
quantitativity at UV absorption of 300 nm.
TABLE-US-00018 TABLE 18 Formulation 28: Lipid Encapsulated Average
particle Zeta potential (mg/mL) amount (mg/mL) size (nm) PdI (mV)
5.9 0.305 131 0.171 -36
11. Various Pharmacodynamic and Pharmacological Tests Using
Compound (A) Stealth Liposomes of Formulations 21 and 25
1) Analysis of Effect of Intermittent Intravenous Administration of
Formulation 21 (ONO-1301Lipo) on Rat Monocrotaline-Induced
Pulmonary Hypertension Model
[0385] The produced Formulation 21 (ONO-1301Lipo formulation) as a
test substance was intermittently administered intravenously once
weekly to a rat monocrotaline (MCT)-induced severe heart failure
(pulmonary hypertension) model from day 7 of the MCT
administration, and the survival rate was compared with a group in
which compound (A) (ONO-1301) was repeatedly orally administered; a
group in which compound (A) was intermittently administered
intravenously once weekly; and, as a positive control, a group in
which an ET-1 antagonist (bosentan) was orally administered. In the
control group, physiological saline (vehicle) was administered once
weekly by intravenous administration.
[0386] For animals, Slc: Wistar male rats, 5 weeks old at the start
of the test, and 66.4 to 90.8 g at arrival (Japan SLC, Inc.) were
used. Monocrotaline (hereinafter referred to as "MCT"), lot No.:
SLBG1999V (Sigma-Aldrich Corporation), was administered once
subcutaneously at the back of the rats at a dose of 60 mg/kg. Six
days after the MCT administration, the animals were divided into
groups according to the weight stratification assignment method
(Table 19).
TABLE-US-00019 TABLE 19 Administered substance/ dose/frequency of
Route of Number of Group administration administration cases 1
Physiological saline/week Intravenous 20 injection 2 Compound (A)
(ONO-1301), Oral 10 3 mg/kg .times. twice/day administration 3
Bosentan, 50 mg/kg .times. twice/day Oral 10 administration 4
Formulation 21.1 mg/kg/week* Intravenous 10 injection 5 Compound
(A) (ONO-1301), Intravenous 10 1 mg/kg/week injection *The dose is
in terms of compound (A) (ONO-1301).
[0387] Group 1: Physiological saline was intravenously administered
7, 14, 21, 28, and 35 days after the MCT administration at weekly
intervals (5 times in total).
[0388] Group 2: Compound (A) was orally administered at 3 mg/kg
twice daily from 7 days to 41 days after the MCT administration
with administration intervals of 8 hours or more.
[0389] Group 3: Bosentan was orally administered at 50 mg/kg twice
daily from 7 days to 41 days after the MCT administration with
administration intervals of 8 hours or more.
[0390] Group 4: Formulation 21 (ONO-1301Lipo) was intravenously
administered at 1 mg/kg 7, 14, 21, 28, and 35 days after the MCT
administration at weekly intervals (5 times in total).
[0391] Group 5: Compound (A) was intravenously administered at 1
mg/kg 7, 14, 21, 28, and 35 days after the MCT administration at
weekly intervals (5 times in total).
[0392] FIG. 27 shows changes in the survival rates of Group 1,
Group 2, and Group 4 until 42 days after the preparation of the
severe heart failure model. Table 20 shows the survival rates of
all of the groups after 42 days.
[0393] In Group 1, one death was observed 15 days after the
preparation of the severe heart failure model by subcutaneous
administration of MCT at 60 mg/kg. Thereafter, 17 deaths were
observed by day 42, and the final survival rate was 10% (number of
animals alive: 2/20).
[0394] In the group in which compound (A) was repeatedly orally
administered at 3 mg/kg twice daily (Group 2), one death was
observed 14 days after the preparation of the severe heart failure
model. Thereafter, 4 more deaths were observed by day 42, and the
final survival rate was 50% (number of animals alive: 5/10),
showing a significant life-prolonging effect compared with the
control group (Group 1) (p<0.05).
[0395] In the group in which bosentan was repeatedly orally
administered at 50 mg/kg twice daily (Group 3), one death was
observed 20 days after the preparation of the severe heart failure
model. Thereafter, another 6 deaths were observed by day 42, and
the final survival rate was 30% (number of animals alive: 3/10),
showing no significant life-prolonging effect compared with the
control group (Group 1).
[0396] Bosentan, which is an ET-1 antagonist, used in the positive
control, has been clinically used as a therapeutic agent for
pulmonary hypertension, and the efficacy thereof has been confirmed
in the same rat MCT-induced heart failure model (Circ. J. 2013; 77:
2127-2133). However, these results are based on repeated oral
administration immediately after the MCT administration. This time,
a significant life-prolonging effect could not be observed in the
administration started 7 days after the MCT administration (Group
3).
[0397] In the group in which Formulation 21 (ONO-1301Lipo) was
intermittently administered intravenously at 1 mg/kg once weekly
(Group 4), one death was observed 27 days after the preparation of
the severe heart failure model. Thereafter, 4 deaths were observed
by day 42, and the final survival rate was 50% (number of animals
alive: 5/10), showing a significant life-prolonging effect compared
with the control group (Group 1) (p<0.05).
[0398] In the group in which compound (A) was intermittently
administered intravenously at 1 mg/kg once weekly (Group 6), two
deaths were observed about 30 minutes after the administration 28
days after the preparation of the severe heart failure model.
Thereafter, 6 deaths were observed by day 42, and the final
survival rate was 20% (number of animals alive: 2/10), showing no
significant life-prolonging effect compared with the control group
(Group 1).
TABLE-US-00020 TABLE 20 Administered substance, Number Survival
dose, frequency of Route of of rate Group administration
administration cases (%) 1 Physiological saline Intravenous 20 10
injection 2 Compound (A) (ONO- Oral 10 50* 1301); 3 mg/kg .times.
twice/day administration 3 Bosentan; 50 mg/kg .times. Oral 10 30
twice/day administration 4 Formulation 21; Intravenous 10 50* 1
mg/kg/week injection 5 Compound (A) (ONO- Intravenous 10 20 1301);
1 mg/kg/week injection
[0399] The total test substance amount of compound (A) administered
from 7 days to 41 days after the MCT administration (35 days in
total) was calculated per animal.
[0400] As a result, the amount was 210 mg/kg/animal (3
mg/kg.times.twice/day.times.35 days) in Group 2, 3500 mg/kg/animal
(50 mg/kg.times.twice/day.times.35 days) in Group 3, and 5
mg/kg/animal (1 mg/kg/week.times.5 times) in Group 4 and Group 5.
Group 4 showed an effect similar to that of Group 2 at a 5/210
(1/42) dose amount of Group 2.
[0401] As a disease site-specific DDS liposomal formulation, a
novel ONO-1301 liposomal formulation (Formulation 21; ONO-1301Lipo)
was produced to analyze the development of a therapeutic method for
developing a more versatile, disease site-specific (DDS)
therapeutic agent for a severe heart failure, by intermittent
intravenous administration.
[0402] The DDS effect was confirmed by comparing the survival rates
in the MCT-induced severe heart failure model by the intermittent
intravenous administration of Formulation 21.
[0403] Bosentan, which is an ET-1 antagonist, and used as the
positive control substance, has been clinically used as a
therapeutic agent for pulmonary hypertension; and the efficacy
thereof has been confirmed in the MCT-induced heart failure model.
However, these results are based on repeated oral administration
immediately after the MCT administration. This time, a significant
life-prolonging effect could not be observed in the administration
treatment 7 days after the MCT administration. In contrast, the
final survival rate of the group in which compound (A) was
repeatedly administered at 3 mg/kg twice daily (Group 2) was 50%,
which showed a significant life-prolonging effect compared with the
control group (Group 1). Additionally, the final survival rate of
the group in which Formulation 21 (ONO-1301Lipo) was intermittently
administered intravenously at 1 mg/kg once weekly (Group 4) was
50%, which also showed a life-prolonging effect comparable with
Compound (A) (Group 2).
[0404] The group in which Formulation 21 was intermittently
administered intravenously at 1 mg/kg/week (Group 4) showed a
life-prolonging effect similar to that of the group in which
compound (A) was repeatedly orally administered at 3 mg/kg twice
daily (Group 2), with the total dose amount of Group 4 being 1/42
of that of Group 2; thus, Group 4 was confirmed to exhibit a DDS
effect as a liposomal formulation.
[0405] In contrast, the group in which the ONO-1301 drug substance
was intermittently administered intravenously at 1 mg/kg/week
(Group 5) showed no effect (Table 20).
[0406] The above results revealed that the repeated oral
administration of compound (A) (ONO-1301) and the intermittent
intravenous administration of Formulation 21 showed a significant
life-prolonging effect by therapeutic administration to the severe
heart failure model after the onset of heart failure (7 days after
the MCT administration). Further, a similar life-prolonging effect
was exhibited at a total dose amount of 1/42, showing a DDS effect
as a stealth liposomal formulation.
2) Analysis of DDS Effect of Formulation 25 (ONO-1301Lipo) on BLM
Pulmonary Fibrosis Model Mice by Various Administration Methods
[0407] 1. Method
[0408] C57BL/6NCr female mice (7 weeks old) were anesthetized with
sodium pentobarbital, and then intratracheally (intrapulmonary)
administered with 20 .mu.L of a bleomycin hydrochloride aqueous
solution (BLM) twice (40 .mu.L in total per animal). In a normal
group (Normal), vehicle (physiological saline) was similarly
intratracheally administered.
[0409] From 6 days to 28 days after the BLM administration, the
test substance was administered to compare the survival rates. As
the test substances, compound (A) (ONO-1301) was repeatedly orally
administration twice daily, compound (A) (ONO-1301) was
intravenously administered once weekly, and compound (A) (ONO-1301)
was intratracheally administered once weekly. Further, Formulation
25 (ONO-1301Lipo) was intravenously administered once weekly, and
Formulation 25 (ONO-1301Lipo) was intratracheally administered once
weekly to analyze the DDS effect of Formulation 25 (ONO-1301Lipo)
by comparing the survival rates.
[0410] 2. Table 21 Shows the Test Group Constitution.
TABLE-US-00021 TABLE 21 Number Route of of Group Test group Dose
administration animals 1 Control 0.5% CMC-Na Oral 10 (vehicle)
aqueous solution .times. administration twice/day 2 Compound (A) 3
mg/kg .times. Oral 10 (ONO-1301) twice/day administration 3
Compound (A) 3 mg/kg .times. Intravenous 10 (ONO-1301) once/week
administration 4 Formulation 25 1 mg/kg .times. Intravenous 10
(ONO- once/week* administration 1301LipoNS) 5 Formulation 25 3
mg/kg .times. Intravenous 10 (ONO- once/week* administration
1301LipoNS) 6 Compound (A) 1 mg/kg .times. Intratracheal 10
(ONO-1301) once/week administration 7 Formulation 25 0.3 mg/kg
.times. Intratracheal 10 (ONO- once/week* administration
1301LipoNS) 8 Formulation 25 1 mg/kg .times. Intratracheal 10 (ONO-
once/week* administration 1301LipoNS) 9 Normal Physiological
Intratracheal 5 saline .times. once/week administration *The dose
is in terms of compound (A) (ONO-1301).
[0411] 3. Test Substance Administration
[0412] 1) Oral Administration: Administration of Vehicle (0.5%
CMC-Na Aqueous Solution) and Compound (A) (Groups 1 and 2)
[0413] From 6 days after the preparation of the lung injury model,
the vehicle and compound (A) (3 mg/kg) were repeatedly orally
administered twice daily for 23 days (the administration interval
between morning and afternoon was 8 hours or more).
[0414] Dose amount: 5 mL/kg.times.twice/day
[0415] Administration method: Gavage oral administration using a
disposable polypropylene syringe and a mouse stomach tube
[0416] 2) Intravenous Administration: Administration of Compound
(A) and Formulation 25 (Groups 3, 4, and 5)
[0417] The administration was performed 6 days after the
preparation of the lung injury model; after that, tail vein
intravenous administration was performed once weekly (4 times in
total, i.e., 6, 13, 20, and 27 or 28 days after the preparation of
the model).
[0418] Dose amount: 1.5 mL/kg
[0419] Administration method: Intravenous administration using a
glass syringe and a disposable injection needle 30G
[0420] 3) Intratracheal Administration: Administration of Compound
(A) and Formulation 25 (Groups 6, 7, 8, and 9)
[0421] The administration was performed 6 days after the
preparation of the lung injury model; after that, intratracheal
administration was performed once weekly (4 times in total, i.e.,
6, 13, 20, and 27 or 28 days after the preparation of the model).
In normal Group 9 (Normal), physiological saline was administered
in a similar manner.
[0422] Dose amount: 0.5 mL/kg
[0423] Administration method: After anesthesia with pentobarbital
(30 to 35 mg/kg, i.p.), intratracheal (intrapulmonary)
administration was performed.
[0424] 4. Results
[0425] 1) FIGS. 28 to 30 Show the Results of the Survival
Rates.
[0426] In the normal group, no deaths were observed. In contrast,
the group in which the vehicle was orally administered to the lung
injury model showed a survival rate of 30%. The group in which
compound (A) was orally administered at 3 mg/kg showed a survival
rate of 60%, thus achieving a life-prolonging effect compared with
the vehicle administration group. The group in which compound (A)
was intravenously administered at 3 mg/kg showed a survival rate of
40%. The groups in which Formulation 25 was intravenously
administered at 1 mg/kg or 3 mg/kg both showed a survival rate of
50%. The group in which compound (A) was intratracheally
administered at 1 mg/kg showed a survival rate of 20%. The groups
in which Formulation 25 was intratracheally administered at 0.3
mg/kg and 1 mg/kg respectively showed survival rates of 50% and
40%, which are higher than that of the vehicle administration group
or the group in which compound (A) was intratracheal administered
at 1 mg/kg.
[0427] 2) Comparison of Survival Rates
[0428] (1) FIG. 29: Comparison with Intermittent Intravenous
Administration of Formulation 25 (ONO-1301Lipo)
[0429] In Group 2, the total dose amount of compound (A) (ONO-1301)
(3 mg/kg.times.twice.times.22 days) was 132 mg/kg.
[0430] Further, the total dose amount of intravenous administration
of compound (A) (ONO-1301) at 3 mg/kg once weekly (Group 3) was 12
mg/kg, and the total dose amount of intravenous administration of
Formulation 25 at 1 mg/kg once weekly (Group 4) was 4 mg/kg. The
total dose amount of intravenous administration of Formulations 25
at 3 mg/kg once weekly (Group 5) was 12 mg/kg.
[0431] The survival rate in the vehicle group (Group 1) was 30%. In
comparison with the group in which compound (A) (ONO-1301) was
orally administered (Group 2: 60%), the survival rates of Group 4
(dose amount: 1/33 of the total dose amount of Group 2) and Group 5
(dose amount: 1/11 of the total dose amount of Group 2) were
slightly lower than that of Group 2, and both showed the same value
(50%). In contrast, the group in which the compound (A) (ONO-1301)
drug substance was intravenously administered at 3 mg/kg once
weekly (Group 5) showed an even lower survival rate (40%) at a dose
amount 1/11 that of Group 2.
[0432] These results revealed that the intravenous administration
of Formulation 25 once weekly (Groups 4 and 5) to the lung injury
model showed a prolonging effect on the survival rate, compared
with intravenous administration of the compound (A) (ONO-1301) drug
substance once weekly (Group 3). Further, Groups 4 and 5 showed a
slightly lower survival rate than that of the group in which the
compound (A) (ONO-1301) drug substance was repeatedly orally
administered twice daily (Group 2), although the total dose amounts
of Groups 4 and 5 were as low as 1/11 to 1/33 that of Group 2.
These results suggested that the intravenous administration of
Formulation 25 exhibited a DDS effect specific to the lung disease
site.
[0433] (2) FIG. 30: Comparison with Intermittent Intratracheal
Administration of Formulation 25 (ONO-1301Lipo)
[0434] In Group 2, the total dose amount of compound (A) (ONO-1301)
(3 mg/kg.times.twice.times.22 days) was 132 mg/kg.
[0435] The total dose amount of intratracheal administration of
compound (A) (ONO-1301) at 1 mg/kg once weekly (Group 6) was 4
mg/kg, and the total dose amount of intratracheal administration of
Formulation 25 at 0.3 mg/kg once weekly (Group 7) was 1.2 mg/kg.
The total dose amount of intratracheal administration of
Formulation 25 at 1 mg/kg once weekly (Group 8) was 4 mg/kg.
[0436] The survival rate in the vehicle administration group (Group
1) was 30%. In comparison with the group in which compound (A)
(ONO-1301) was orally administered (Group 2:60%), Group 7 (dose
amount: 1/110 of the total dose amount of Group 2) and Group 8
(dose amount: 1/33 of the total dose of Group 2) showed survival
rates of 50% and 40%, respectively.
[0437] In contrast, the group in which the compound (A) (ONO-1301)
drug substance was intravenously administered at 1 mg/kg once
weekly (Group 6) showed an even lower survival rate (20%) at a dose
amount of 1/33 that of Group 2.
[0438] These results revealed that intratracheal administration of
Formulation 25 once weekly (Group 7 and Group 8) to the lung injury
model showed a prolonging effect on the survival rate, compared
with the intratracheal administration of the compound (A)
(ONO-1301) drug substance once weekly (Group 6). Further, Group 7
and Group 8 showed a slightly lower survival rate than that of the
group of repeated oral administration of the compound (A)
(ONO-1301) drug substance twice daily (Group 2), although the total
dose amounts of Group 7 and Group 8 were as low as 1/110 to 1/33
that of Group 2. These results suggested that the intratracheal
administration of Formulation 25 exhibited a DDS effect specific to
the lung disease site. Further, intratracheal administration of
Formulation 25 at 0.3 mg/kg once weekly showed the same survival
rate (50%) as those of the intravenous administration of
Formulation 25 at 1 mg/kg or 3 mg/kg once weekly. This suggested
that intratracheal administration exhibits a DDS effect greater
than that of the intravenous administration.
[0439] Based on the above results, the survival rates of the groups
in which Formulation 25 was intermittently administered
intravenously once weekly (Groups 4 and 5) and the groups in which
Formulation 25 was intermittently administered intratracheally once
weekly (Groups 7 and 8) were compared with that of the vehicle
administration group (Group 1), using the bleomycin-induced lung
injury model mice from day 6 of the bleomycin administration. As a
positive control, a group in which compound (A) (ONO-1301) was
repeatedly orally administered twice daily (Group 2) was created.
Further, a group in which compound (A) (ONO-1301) was
intermittently administered intravenously once weekly (Group 3) and
a group in which compound (A) (ONO-1301) was intermittently
administered intratracheally (Group 6) were created to compare the
DDS effect as ONO-1301 liposomal formulations.
[0440] In the test, the group in which compound (A) (ONO-1301) was
repeatedly orally administered at 3 mg/kg twice daily from day 6 of
the bleomycin administration showed a prolonged survival rate,
suggesting a therapeutic effect.
[0441] The intravenous administration of Formulation 25 once weekly
(Groups 4 and 5) showed a prolonging effect on the survival rate,
compared with intravenous administration of the compound (A)
(ONO-1301) drug substance once weekly (Group 3). Further, Groups 4
and 5 showed a slightly lower survival rate than that of the group
of repeated oral administration of the compound (A) (ONO-1301) drug
substance twice daily (Group 2), although the total dose amounts of
Groups 4 and 5 were as low as 1/11 to 1/33 that of Group 2. These
results suggested that intravenous administration of Formulation 25
showed a DDS effect specific to the lung disease site.
[0442] The group in which Formulation 25 was intratracheally
administered once weekly (Groups 7 and 8) showed a prolonging
effect on the survival rate, compared with the group in which the
compound (A) (ONO-1301) drug substance was intratracheally
administered once weekly (Group 6). Further, Groups 7 and 8 showed
a slightly lower survival rate than that of the group in which the
compound (A) (ONO-1301) drug substance was repeatedly orally
administered twice daily (Group 2), although the total dose amount
was as low as 1/110 to 1/33 that of Group 2. These results
suggested that intratracheal administration of Formulation 25
showed a DDS effect specific to the lung disease site. Further,
intratracheal administration of Formulation 25 at 0.3 mg/kg once
weekly showed the same survival rate (50%) as those of intravenous
administration of Formulation 25 at 1 mg/kg or 3 mg/kg once weekly,
suggesting that the intratracheal administration achieves a greater
DDS effect, i.e., about 10 times that of the intravenous
administration.
3) Analysis of Effect of Formulation 25 (ONO-1301Lipo) on
Spontaneous Dilated Cardiomyopathy (J2N-k) Hamsters
[0443] (1) Test System
[0444] Improvement in the DDS cardiac function in the spontaneous
dilated cardiomyopathy (J2N-k) hamster model by intermittent
intravenous administration of Formulation 25 and compound (A)
(ONO-1301) once every two weeks was evaluated by comparing the left
ventricular ejection fraction (hereinafter referred to as "EF %"),
left ventricular fractional shortening (hereinafter referred to as
"% FS"), and histological evaluation, based on echocardiography,
taken as indices. As a positive control, a group in which compound
(A) (ONO-1301) was repeatedly orally administered twice daily was
created.
[0445] As a test substance, Formulation 25 was used by suspending
and diluting with physiological saline. Compound (A) (ONO-1301) as
a control was used by dissolving in an equivalent amount of aqueous
NaOH, and diluting with physiological saline. For groups of oral
administration of compound (A) (ONO-1301), administration was
performed as a suspension in a 0.5% CMC-Na aqueous solution.
[0446] The spontaneous dilated cardiomyopathy (J2N-k) male
hamsters, 20 weeks old at the start of administration, Japan SLC,
Inc., were administered with the test substances for 8 weeks, and
used for comparison.
[0447] (2) Table 22 Shows the Group Constitution.
TABLE-US-00022 TABLE 22 Number of Group Route of administration
animals Group 1; Control Once/2 weeks Intravenous 4 (physiological
saline) administration Group 2; Formulation 25 *3 Once/2 weeks
Intravenous 5 mg/kg (ONO-1301Lipo) administration Group 3; Compound
(A) Once/2 weeks Intravenous 4 (ONO-1301): 3 mg/kg administration
Group 4; Compound (A) Twice/day Repeated oral 5 (ONO-1301): 3 mg/kg
administration *The dose is in terms of compound (A)
(ONO-1301).
[0448] Liquid dose: 5 mL/kg
[0449] (3) Echocardiography
[0450] Animals arrived at the age of 18 weeks were subjected to a
2-week quarantine/acclimation period, and then to the test at the
time of grouping (test start date) at the age of 20 weeks.
Thereafter, tests were conducted 4 and 8 weeks after the test start
date, and dissection was performed 8 weeks after the test start
date.
[0451] (4) Dissection and Treatment of Removed Tissue
[0452] The final echocardiography was performed 8 weeks after the
start of administration. Thereafter, dissection was performed.
[0453] Dissection was performed as follows. After all of the blood
was collected from the abdominal aorta, and the hamsters were
euthanized under isoflurane anesthesia, the heart and lungs were
removed and weighed. After the weights thereof were measured, the
removed parts, including the left and right ventricles, were
divided into three parts; i.e., apical, middle, and basal parts, at
intervals of about 2 mm along the short axis.
[0454] For the three divided tissues, the short-axis sections
including the right and left ventricles of the middle part were
immersed in 4% paraformaldehyde (for general pathological
examination), and stored.
[0455] (5) Measurement of Left Ventricular Wall Thickness and Area
in Short-Axis Sections of Removed Heart
[0456] When removed, the heart, including the left and right
ventricles, was divided into three parts; i.e., apical, middle, and
basal parts, at intervals of about 2 mm along the short axis. Among
them, one section of the middle part was photographed, and the wall
thickness at the anterior, lateral, posterior, and septum parts,
and the left ventricular area were measured using image-editing
software. For the wall thickness of the left ventricle,
image-editing software was used, the number of pixels in the scale
area of 1 mm.sup.2 on the photograph was obtained, and the outer
diameter and inner diameter of the entire left ventricle wall were
traced to obtain each number of pixels to thus calculate the left
ventricular area by ((the number of pixels of the outer
diameter-the number of pixels of the inner diameter)/1 mm.sup.2).
Thereafter, the wall thickness of each portion was measured.
[0457] FIG. 31 shows the measurement method. The section was
divided into three parts; i.e., apical, middle, and basal areas, at
intervals of about 2 mm. Thereafter, using one section on the
middle side, the wall thickness at the anterior, lateral,
posterior, and septum parts, and the left ventricular area were
measured. For the wall thickness of the left ventricle,
image-editing software was used, the number of pixels in the scale
area of 1 mm.sup.2 on the photograph was obtained, and the outer
diameter and inner diameter of the entire left ventricle wall were
traced to obtain each number of pixels to thus calculate the left
ventricular wall area by ((the number of pixels of the outer
diameter-the number of pixels of the inner diameter)/1 mm.sup.2).
Thereafter, the wall thickness of each portion was measured.
[0458] (6) Test Results
[0459] Table 23 shows the measurement results of EF % values and %
FS values determined by echocardiography.
TABLE-US-00023 TABLE 23 EF % % FS Drugs N Pre 4 W 8 W Pre 4 W 8 W
Group 1; Control 4 42.5 .+-. 3.2 33.6 .+-. 12.3 31.5 .+-. 3.5.sup.a
17.8 .+-. 1.4 13.3 .+-. 6.1 12.6 .+-. 1.6.sup.a Group 4; Compound
(A) 5 43.0 .+-. 7.8 40.4 .+-. 14.9 40.3 .+-. 9.2* 18.2 .+-. 3.9
17.2 .+-. 7.2 .sup. 17.1 .+-. 4.5* (ONO-1301): 3.0 mg/kg, p.o.
Group 3; compound (A) 4 38.8 .+-. 4.3 35.7 .+-. 7.4 30.8 .+-.
2.9.sup.a 16.0 .+-. 2.1 16.1 .+-. 3.1 12.3 .+-. 1.3.sup.a
(ONO-1301): 3.0 mg/kg, intravenous administration Group 2;
formulation 25: 5 40.5 .+-. 5.1 44.5 .+-. 9.9 39.6 .+-. 4.1* 16.9
.+-. 2.6 18.5 .+-. 4.9 .sup. 16.5 .+-. 2.1** 3.0 mg/kg i.v. Data
shown: the mean .+-. standard deviation *p < 0.05, **p < 0.01
(significantly different from the control value by Student's
t-test) .sup.ap < 0.01 (significantly different from the
pre-value by Student's t-test)
[0460] Echocardiography was performed at the time of grouping, and
at week 4 and week 8 after the start of administration. The
measurement was performed 3 times for each individual, and the
average value of the measured data was referred to as the
measurement results.
[0461] In the control group (Group 1), the EF % values at the time
of grouping, and at week 4 and week 8 were 42.5.+-.3.2%,
33.6.+-.12.3%, and 31.5.+-.3.5%, respectively. The EF % value at
week 8 showed a significant decrease (p<0.05) compared with the
EF % value at the time of grouping. The % FS values at the time of
grouping and at week 4 and week 8 were 17.8.+-.1.4%,13.3.+-.6.1%,
and 12.6.+-.1.6%, respectively. Similar to the EF % value, the EF %
value at week 8 showed a significant decrease (p<0.01) compared
with the % FS value at the time of grouping.
[0462] The EF % values of the group of repeat oral administration
of compound (A) (ONO-1301) at 3.0 mg/kg.times.twice/day (Group 4)
at the time of grouping and at week 4 and week 8 were 43.0.+-.7.8%,
40.4.+-.14.9%, and 40.3.+-.9.2%, respectively. Compared with the
value at the time of grouping, the values at week 4 and week 8
showed no significant decrease in cardiac function. The EF % value
at week 8 was higher than that of the control group, showing a
significant difference (p<0.05). The % FS values at the time of
grouping and at week 4 and week 8 were 18.2.+-.3.9%, 17.2.+-.7.2%,
and 17.1.+-.4.5%, respectively, which were similar to the EF %
values.
[0463] The EF % values of the group of intravenous administration
of compound (A) (ONO-1301) at 3.0 mg/kg (Group 3) at the time of
grouping and at week 4 and week 8 were 38.8.+-.4.3%, 35.7.+-.7.4%,
and 30.8.+-.2.9%, respectively. The decrease at week 4 was slower
than that of the control group; however, the decrease at week 8 was
similar to that of the control group. The EF % value at week 8
showed a significant decrease (p<0.01) from the value at the
time of grouping. The % FS values at the time of grouping and at
week 4 and week 8 were 16.0.+-.2.1%, 16.1.+-.3.1%, and
12.3.+-.1.3%, respectively. These results were similar to the EF %
values, showing no efficacy.
[0464] In contrast, the EF % value of the group of intravenous
administration of Formulation 25 (ONO-1301Lipo) at 3.0 mg/kg (Group
2) at the time of grouping and at week 4 and week 8 were
40.5.+-.5.1%, 44.5.+-.9.9%, and 39.6.+-.4.1%, respectively.
Although the difference was not significant, a slight increase was
observed at week 4. At week 8, the decrease was suppressed in a
manner similar to that of the group of oral administration of
compound (A) (ONO-1301) at 3.0 mg/kg, and the value was
significantly higher (p<0.05) compared with that of the control
group. The % FS values at the time of grouping and at week 4 and
week 8 were 16.9.+-.2.6%,18.5.+-.4.9%, and 16.5.+-.2.1%,
respectively; thus, the change was similar to that of the EF %
values. The value at week 8 showed a significant effect (p<0.01)
compared with that of the control group (Group 1), and this effect
was similar to that of the group of repeat oral administration of
compound (A) (ONO-1301) (Group 4).
[0465] 2. Measurement Results of the Wall Thickness of Removed
Heart (Evaluation on the Short-Axis Section of the Middle Part)
[0466] Table 24 shows the wall thickness measurement results.
[0467] As shown in Table 24, the wall thicknesses of the apical,
lateral, posterior, and septum of the middle section in the control
group (Group 1) were 0.8.+-.0.1 mm, 1.1.+-.0.2 mm, 1.1.+-.0.2 mm,
and 1.0.+-.0.2 mm, respectively.
[0468] In the group of oral administration of compound (A)
(ONO-1301) at 3.0 mg/kg (Group 4), the wall thicknesses of the
apical, lateral, posterior, and septum of the middle section were
1.4.+-.0.5 mm, 1.5.+-.0.2 mm, 1.2.+-.0.4 mm, and 1.5.+-.0.3 mm,
respectively. Thus, the thicknesses of the apical, lateral, and
septum were significantly higher (p<0.05) than those of the
control group.
[0469] In the group of intravenous administration of compound (A)
(ONO-1301) at 3.0 mg/kg (Group 3), these values were 1.1.+-.0.6 mm,
1.2.+-.0.3 mm, 1.2.+-.0.2 mm, and 1.0.+-.0.3 mm, respectively.
These measurement results were similar to those of the control
group.
[0470] In contrast, in the group of intravenous administration of
Formulation 25 (ONO-1301Lipo) at 3.0 mg/kg (Group 2), these values
were 1.7.+-.0.4 mm**, 1.5.+-.0.1 mm*, 1.5.+-.0.6 mm.sup..dagger.,
and 1.2.+-.0.3 mm, respectively. These values were significantly
higher and showed a tendency to achieve higher values in the apical
(p<0.01), lateral (p<0.05), and posterior (0.05<p<0.1),
compared with those of the control group.
TABLE-US-00024 TABLE 24 Body weight (g) Before drug Body weight
Left ventricle wall thickness (mm) Drugs N administration at
dissection Anterior Lateral Posterior Septum Group 1: Control 4
120.2 .+-. 7.3 129.4 .+-. 6.7 0.8 .+-. 0.1 1.1 .+-. 0.2 1.1 .+-.
0.2 1.0 .+-. 0.2 Group 4: compound (A) 5 118.8 .+-. 9.6 128.6 .+-.
3.0 1.4 .+-. 0.5* 1.5 .+-. 0.2* 1.2 .+-. 0.4 1.5 .+-. 0.3*
(ONO-1301): 3.0 mg/kg, p.o. Group 3: compound (A) 4 128.7 .+-. 10.4
144.4 .+-. 13.3 1.1 .+-. 0.6 1.2 .+-. 0.3 1.2 .+-. 0.2 1.0 + 0.3
(ONO-1301): 3.0 mg/kg, intravenous administration Group 2;
Formulation 25: 5 119.6 .+-. 10.5 138.8 .+-. 12.4 1.7 .+-. 0.4**
1.5 .+-. 0.1* .sup. 1.5 .+-. 0.6.sup..dagger. 1.2 .+-. 0.3 3.0
mg/kg, intravenous administration Data shown: the mean .+-.
standard deviation .sup..dagger.0.05 < p < 0.1, *p < 0.05,
**p < 0.01 (significantly different from the control value by
Student's t-test)
[0471] 3. Measurement Results of the Left Ventricular Wall Area of
Removed Heart (Evaluation on the Short-Axis Section of the Middle
Part)
[0472] Table 25 shows the left ventricular area measurement
results.
[0473] As shown in Table 25, the measurement results of the left
ventricular wall area of the control group (Group 1), the group of
oral administration of compound (A) (ONO-1301) at 3.0 mg/kg (Group
4), the group of intravenous administration of compound (A)
(ONO-1301) at 3.0 mg/kg (Group 3), and the group of intravenous
administration of Formulation 25 (ONO-1301Lipo) at 3.0 mg/kg (Group
2) were 23.4.+-.5.4 mm.sup.2, 26.4.+-.4.3 mm.sup.2, 23.9.+-.4.5
mm.sup.2, and 29.5.+-.6.4 mm.sup.2, respectively. These measurement
results showed no significant difference; however, reflecting the
wall thickness measurement results, there was a tendency that the
control group (Group 1) and the group of intravenous administration
of compound (A) (ONO-1301) at 3.0 mg/kg (Group 3) showed lower
values, while the group of oral administration of compound (A)
(ONO-1301) at 3.0 mg/kg (Group 4) and the group of intravenous
administration of Formulation 25 (ONO-1301Lipo) at 3.0 mg/kg (Group
4) showed higher values.
TABLE-US-00025 TABLE 25 Left ventricular area (Middle part) Drugs N
(mm.sup.2) Group 1: Control 4 23.4 .+-. 5.4 Group 4: Compound (A)
(ONO- 5 26.4 .+-. 4.3 1301): 3.0 mg/kg, p.o. Group 3: Compound (A)
(ONO- 4 23.9 .+-. 4.5 1301): 3.0 mg/kg, i.v. Group 2; Formulation
25; 5 .sup. 29.5 .+-. 6.4.sup..dagger. 3.0 mg/kg, i.v. Data shown:
the mean .+-. standard deviation 0.05 < p < 0.1
(significantly different from the control value by Student's
t-test)
[0474] As described above, the cardiac function improvement effect
of Formulation 25 (ONO-1301Lipo) was analyzed using spontaneous
dilated cardiomyopathy (J2N-k) hamsters. The test was initiated
after the onset of the pathological condition, and at the age of 20
weeks when the cardiac function considerably decreased. The test
substance was administered until week 28, and the change in the
cardiac function was analyzed.
[0475] In the cardiac function evaluation using EF % and % FS
values determined by echocardiography as indices, the group in
which compound (A) (ONO-1301) was repeatedly orally administered at
3.0 mg/kg.times.twice/day (Group 4) and the group in which
Formulation 25 (ONO-1301Lipo) was intermittently administered
intravenously at 3.0 mg/kg 4 times in total (at the time of
grouping; and at week 2, week 4, and week 6) (Group 2) showed an
effect in terms of suppressing a decrease in the cardiac function,
compared with the control group (Group 1) and the group of
intravenous administration of compound (A) (ONO-1301) at 3.0 mg/kg
(Group 3). In the measurement results of the left ventricle wall
thickness, the control group (Group 1) and the group of intravenous
administration of compound (A) (ONO-1301) at 3.0 mg/kg (Group 3)
showed low values, which suggested the progress of thinning. In
contrast, the group of repeat oral administration of compound (A)
(ONO-1301) at 3.0 mg/kg (Group 4) and the group of Formulation 25
(ONO-1301Lipo) at 3.0 mg/kg (Group 2) showed higher values,
suggesting the suppression of the progress of thinning, compared
with the control group (Group 1) and the group of intravenous
administration of compound (A) (ONO-1301) at 3.0 mg/kg (Group 3).
This is assumed to reflect the results of EF % and % FS values
determined by echocardiography.
[0476] The above results suggested that the group of repeat oral
administration of compound (A) (ONO-1301) at 3.0
mg/kg.times.twice/day (Group 4) and the group of intermittent
intravenous administration of Formulation 25 (ONO-1301LipoNS) at
3.0 mg/kg once every 2 weeks (Group 2) suppressed a decrease in the
cardiac function in the spontaneous dilated cardiomyopathy (J2N-k)
hamsters.
[0477] The total dose amount of the repeat oral administration of
compound (A) (ONO-1301) at 3 mg/kg.times.twice.times.56 days was
336 mg/kg, while the total dose amount of the intravenous
administration of Formulations 25 (ONO-1301Lipo) at 3 mg/kg.times.4
times was 12 mg/kg; both of these showed a similar efficacy. The
intermittent intravenous administration of compound (A) (ONO-1301)
at 3 mg/kg once every 2 weeks showed no effect. Accordingly, the
intermittent intravenous administration of Formulation 25
(ONO-1301Lipo) once every 2 weeks showed the effect at a dose of
1/28 of that of the repeat oral administration of compound (A)
(ONO-1301). This confirmed the DDS effect of intravenous
administration of Formulation 25 (ONO-1301Lipo).
[0478] Tables 26 and 27 show the body weight change at the time of
grouping and at the time of dissection, and the weights of heart
and lung at the time of dissection. No change was observed in any
of these.
TABLE-US-00026 TABLE 26 Body weight (g) Before drug Body weight at
Drugs N administration dissection Control 4 120.2 .+-. 7.3 129.4
.+-. 6.7 compound (A) (ONO-1301): 5 118.8 .+-. 9.6 128.6 .+-. 3.0
3.0 mg/kg, p.o. Compound (A) (ONO-1301): 4 128.7 .+-. 10.4 144.4
.+-. 13.3 3.0 mg/kg, i.v. Formulation 25: 5 119.6 .+-. 10.5 138.8
.+-. 12.4 3.0 mg/kg, i.v. Data shown: the mean .+-. standard
deviation
TABLE-US-00027 TABLE 27 Body weight (g) Body Tissue weight per 100
g weight at Real tissue weight (g) of the body weight (g) Drugs N
dissection Heart Lung Heart Lung Control 4 129.4 .+-. 6.7 0.4674
.+-. 0.0666 0.5640 .+-. 0.0949 0.3602 .+-. 0.0340 0.4346 .+-.
0.0548 Compound (A) 5 128.6 .+-. 3.0 0.4379 .+-. 0.0136 0.5202 .+-.
0.0336 0.3404 .+-. 0.0032 0.4043 .+-. 0.0203 (ONO-1301): 3.0 mg/kg,
p.o. Compound (A) 4 144.4 .+-. 13.3 0.5028 .+-. 0.0631 0.5740 .+-.
0.0527 0.3475 .+-. 0.0165 0.3998 .+-. 0.0470 (ONO-1301): 3.0 mg/kg,
i.v. Formulation 25: 5 138.8 .+-. 12.4 0.4797 .+-. 0.0673 0.5645
.+-. 0.0625 0.3446 .+-. 0.0222 0.4068 .+-. 0.0276 3.0 mg/kg, i.v.
Data shown: the mean .+-. standard deviation
4) Analysis of Effect of Single Intravenous Administration of
Formulation 25 (ONO-1301LipoNS) on Rat Model of Ischemia by
Complete Coronary Artery Ligation
[0479] An improvement in the cardiac function against ischemic
heart disease and an effect of preventing death from heart failure
by intermittent intravenous administration of Formulation 25
(ONO-1301Lipo) or compound (A) (ONO-1301) were analyzed by using
the left ventricular ejection fraction (hereinafter referred to as
"EF %"), left ventricular fractional shortening (hereinafter
referred to as "% FS"), and histological evaluation, based on
echocardiography, as indices.
[0480] (1) Preparation of Myocardial Ischemia Model
[0481] Male Sprague-Dawley rats (CLEA Japan, Inc., 6 weeks old when
arrived) were anesthetized with a liquid mixture of 0.5 mg/kg of
midazolam (Dormicum Injection 10 mg, Astellas Pharma Inc.) and 2
mg/kg of xylazine (Celactal 2% injection, Bayer Japan Ltd.).
Thereafter, the animal tail veins were secured, and Propofol (1%
Diprivan Injection; AstraZeneca KK) was continuously infused at 6
to 10 mg/kg/hr with a syringe pump (Terufusion TE-3310N; Terumo
Corporation) to maintain deep anesthesia. Thereafter, a tracheal
cannula (a cut-down catheter with an outside diameter of 2.0 mm,
produced by JMS Co., Ltd.) was inserted and indwelled, and a
respirator (for small animals; Shinano Seisakusho) was connected to
maintain breathing at a stroke volume of 1 mL/100 g/stroke (.+-.1
mL) and a stroke rate of 70 times/min (.+-.10 times). Thereafter,
the animals were fixed in a recumbent position from a dorsal fixed
position so that the left chest faced upward, and the epidermis and
muscle layer between the third to fourth or fourth to fifth ribs
were incised with a surgical knife. After confirming that the knife
reached the thoracic cavity, the intercostal space was widened with
a rib spreader, and a state in which the anterior descending branch
(hereinafter referred to as "LAD") from the left atrial appendage
was directly visible at the front was secured.
[0482] Thereafter, the translucent thin pericardium was removed
with tweezers to expose the myocardium. Thereafter, the LAD located
at the margin of the left atrial appendage was pierced and secured
with a nylon thread with a 6-0 or 7-0 needle at a depth of 2 to 3
mm using a microneedle holder, and the LAD was completely ligated.
Thereafter, the muscle layer and epidermis were sutured and closed
with a 4-0 or 5-0 nylon thread. After the chest was closed, 50 mg
of cefamedin was subcutaneously administered to the treatment site,
and the treatment was terminated.
[0483] (2) Table 28 Shows the Group Constitution.
TABLE-US-00028 TABLE 28 Route of Number of Group administration
animals Group 1: Normal -- 3 Group 2: Control Intravenous 5
(Physiological saline) administration Group 3: Formulation 25
Intravenous 5 (ONO-1301Lipo): 0.3 mg/kg administration Group 4:
Formulation 25 Intravenous 5 (ONO-1301Lipo): 1.0 mg/kg
administration Group 5: Formulation 25 Intravenous 5
(ONO-1301Lipo): 3.0 mg/kg administration Group 6: Compound (A)
Intravenous 5 (ONO-1301): 3.0 mg/kg administration 1) Liquid dose:
5 mL/kg 2) Single intravenous administration to the tail 24 hours
after infarction
[0484] (3) Echocardiography
[0485] Echocardiography was performed to confirm the cardiac
function improving effect of the test substances using the left
ventricular ejection fraction (hereinafter referred to as "EF %")
as an index.
[0486] The test was performed 23 hours after the preparation of the
model (test for grouping), the animals whose EF % value decreased
by 25% or more of that of normal animals were selected, grouping
was performed, and a solution for administration of the test
substances was administered by tail vein intravenous administration
24 hours after the preparation of the model. Thereafter,
echocardiography was performed 7 and 14 days after the test
substance administration.
[0487] (4) Dissection and Treatment of Removed Tissue
[0488] Dissection was performed after the completion of the
echocardiography at day 14 after the administration of the test
substance. For dissection, all of the blood was collected from the
abdominal aorta, and the rats were euthanized under isoflurane
anesthesia; thereafter, the heart was removed and weighed. After
measuring the heart weight, the infarct area, including the left
and right ventricles, was divided into three parts along the short
axis.
[0489] (5) Test Results
[0490] (1) Measurement Results of EF % Value Determined by
Echocardiography
[0491] Tables 29 and 30 show the echocardiography results.
[0492] As shown in Table 29, the EF % value in the normal animals
was 85.0.+-.1.9% (n=4). The EF % value of each group 23 hours after
the complete LAD ligation and before the test substance
administration was such that the control group was 52.8.+-.6.8%
(n=5), and the groups of administration of Formulation 25
(ONO-1301Lipo) at 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kg were
55.1.+-.2.4% (n=5), 55.0.+-.4.1% (n=5), and 54.7.+-.5.2% (n=5),
respectively. The group of administration of compound (A)
(ONO-1301) at 3 mg/kg was 56.7.+-.5.5% (n=5).
[0493] The EF % values of all of the groups before the test
substance administration showed a significant decrease (p<0.01)
compared with the EF % value of the normal animals; no significant
difference was observed among the groups.
[0494] As shown in Table 29, the change in the EF % value of each
group at day 7 and day 14 after the administration were as follows.
In the control group, the EF % value was 52.8.+-.6.8% (n=5) before
the administration; and decreased in a time-dependent manner to
48.9.+-.4.0% and 39.0.+-.5.2%, respectively. The EF % value 2 weeks
later showed a significant decrease (p<0.05) compared with that
before the administration.
[0495] In the 0.3 mg/kg administration group of Formulation 25
(ONO-1301LipoNS), the EF % value was 55.1.+-.2.4% (n=5) before the
administration; and the values at day 7 and day 14 were
50.7.+-.6.2% and 42.0.+-.6.2%, respectively, showing similar
changes to those of the control group. Similar to the control
group, the EF % value of this group at day 14 also showed a
significant decrease (p<0.05) compared with that before the
administration.
[0496] In the 1.0 mg/kg administration group, the EF % value was
55.0.+-.4.1% (n=5) before the administration; and the values at day
7 and day 14 were 53.6.+-.7.5% and 54.7.+-.8.2%, respectively,
which were similar to the EF % value before the administration. The
EF % value at day 14 showed a significant increase (p<0.05)
compared with that of the control group.
[0497] In the 3.0 mg/kg administration group, the EF % value was
54.7.+-.5.2% (n=5) before the administration, and increased to
62.4.+-.8.7% and 58.2.+-.13.2%, respectively. The increase in the
EF % value showed a peak on day 7 after the administration compared
with the EF % value before the administration, indicating an
improvement in the cardiac function. The EF % values at day 7 and
day 14 after the test substance administration showed a significant
suppression of a decrease (p<0.05 and p<0.01, respectively)
compared with those of the control group.
[0498] In the group of administration of compound (A) (ONO-1301) at
3.0 mg/kg, the EF % value of was 56.7.+-.5.5% (n=5) before the
administration, and changed to 54.6.+-.8.8% and 53.2.+-.7.1%,
respectively, showing a tendency of a decrease from the EF % value
before the administration, although the difference was not
significant. However, a comparison with those of the control group
at day 7 and day 14 revealed no significant difference in both day
7 and day 14.
[0499] 4. Measurement Results of % FS Value Determined by
Echocardiography
[0500] As shown in Table 29, the % FS value in the normal animals
was 49.4.+-.2.4% (n=4). The % FS value of each group 23 hours after
the complete LAD ligation and before the test substance
administration was such that the control group was 23.9.+-.3.7%
(n=5); and the groups of administration of Formulation 25
(ONO-1301Lipo) at 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kg were
25.0.+-.1.7% (n=5), 25.8.+-.2.4% (n=5), and 25.0.+-.3.0% (n=5),
respectively. The % FS value of the group of administration of
compound (A) (ONO-1301) at 3 mg/kg was 26.5.+-.3.7% (n=5). Usually,
the normal % FS value is clinically said to be 28% or more.
Although the difference was slight, the % FS values of all of the
groups were lower than this value.
[0501] The % FS values of all of the groups before the test
substance administration showed a significant decrease (p<0.01)
compared with the EF % value of the normal animals; no significant
difference was observed among the groups.
[0502] As shown in Table 30, the change in the % FS value of each
group at day 7 and day 14 after the administration were as follows.
In the control group, the % FS value was 23.9.+-.3.7% before the
administration (n=5); and decreased in a time-dependent manner to
21.8.+-.2.2% and 16.7.+-.2.7%, respectively. Similar to the EF %
value, the % FS value 2 weeks later showed a significant decrease
(p<0.05) compared with that before the administration.
[0503] In the 0.3 mg/kg administration group of Formulation 25
(ONO-1301Lipo), the % FS value was 25.0.+-.1.7% (n=5) before the
administration; and the values at day 7 and day 14 were
22.9.+-.3.4% and 18.4.+-.3.2%, respectively, showing similar
changes to those of the control group. Similar to the control
group, the % FS value at day 14 also showed a significant decrease
(p<0.05) compared with that before the administration.
[0504] In the 1.0 mg/kg administration group, the % FS value was
25.8.+-.2.4% (n=5) before the administration; and the values at day
7 and day 14 were 24.8.+-.4.2% and 25.4.+-.5.0%, respectively,
which were similar to the FS % value before the administration.
[0505] In the 3.0 mg/kg administration group, the % FS value was
25.0.+-.3.0% (n=5) before the administration, and increased to
30.1.+-.5.4% and 27.9.+-.8.5%, respectively. The increase in the %
FS value showed a peak on day 7 after the administration compared
with the % FS value before the administration. The % FS values at
day 7 and day 14 after the test substance administration both
showed a significant increase (p<0.05) compared with those of
the control group.
[0506] In the group of administration of compound (A) (ONO-1301) at
3.0 mg/kg, the % FS value was 26.5.+-.3.7% (n=5) before the
administration, and changed to 25.4.+-.5.6% and 24.5.+-.4.4%,
respectively, showing a tendency of a decrease from the % FS value
before the administration, although the difference was not
significant. However, a comparison with those of the control group
at day 7 and day 14 revealed no significant difference in both day
7 and day 14.
[0507] As described above, the measurement results showed
correlation between the EF % and % FS values, which are used as
indices of the evaluation of the cardiac function, in each group;
and the single intravenous administration of Formulation 25
(ONO-1301LipoNS) at 1.0 mg/kg or 3.0 mg/kg showed a cardiac
function improving effect.
TABLE-US-00029 TABLE 29 Drugs N EF % % FS Group 1: Normal 4 85.0
.+-. 1.9 49.4 .+-. 2.4 Group 2: Control 5 52.8 .+-. 6.8** 23.9 .+-.
3.7** Group 3: Formulation 25: 5 55.1 .+-. 2.4** 25.0 .+-. 1.7**
0.3 mg/kg Group 4: Formulation 25: 5 55.0 .+-. 4.1** 25.8 .+-.
2.4** 1.0 mg/kg Group 5: Formulation 25: 5 54.7 .+-. 5.2** 25.0
.+-. 3.0** 3.0 mg/kg Group 6: Compound (A) 5 56.7 .+-. 5.5** 26.5
.+-. 3.7** (ONO-1301): 3.0 mg/kg Data shown: the mean .+-. standard
deviation **p < 0.01 (significantly different from the normal
value by Dunnett's test)
TABLE-US-00030 TABLE 30 EF % % FS Drugs N Pre 1 W 2 W Pre 1 W 2 W
Group 2: Control 5 52.8 .+-. 6.8 48.9 .+-. 4.0 39.0 .+-. 5.2.sup.a
23.9 .+-. 3.7 21.8 .+-. 2.2 16.7 .+-. 2.7.sup.a Group 3:
Formulation 25: 5 55.1 .+-. 2.4 50.7 .+-. 6.2 42.0 .+-. 6.2.sup.a
25.0 .+-. 1.7 22.9 .+-. 3.4 18.4 .+-. 3.2.sup.a 0.3 mg/kg Group 4:
Formulation 25: 5 55.0 .+-. 4.1 53.6 .+-. 7.5 .sup. 54.7 .+-. 8.2*
25.8 .+-. 2.4 24.8 .+-. 4.2 25.4 .+-. 5.0 1.0 mg/kg Group 5:
Formulation 25: 5 54.7 .+-. 5.2 62.4 .+-. 8.7* .sup. 58.2 .+-.
13.2** 25.0 .+-. 3.0 30.1 .+-. 5.4* .sup. 27.9 .+-. 8.5* 3.0 mg/kg
Group 6: Compound (A) 5 56.7 .+-. 5.5 54.6 .+-. 8.8 53.2 .+-. 7.1
26.5 .+-. 3.7 25.4 .+-. 5.6 24.5 .+-. 4.4 (ONO-1301): 3.0 mg/kg
Data shown: the mean .+-. standard deviation *p < 0.05, **p <
0.01 (significantly different from the control value by Dunnett's
test) .sup.ap < 0.05 (significantly different from the pre-value
by t-test)
TABLE-US-00031 TABLE 31 (g) Body weight Heart weight Before Total
weight per 100 g of preparation Before drug At of isolated body
weight Drugs N of AMI administration dissection heart (g) (g) Group
2: Control 5 334.3 .+-. 19.3 315.2 .+-. 15.2 407.6 .+-. 14.8 1.3997
.+-. 0.0876 0.3431 .+-. 0.0219 Group 3: Formulation 25: 5 331.2
.+-. 19.7 317.7 .+-. 16.2 408.2 .+-. 23.2 1.3191 .+-. 0.1493 0.3236
.+-. 0.0362 0.3 mg/kg Group 4: Formulation 25: 5 340.2 .+-. 18.7
320.8 .+-. 21.5 412.4 .+-. 13.4 1.3179 .+-. 0.0655 0.3199 .+-.
0.0206 1.0 mg/kg Group 5: Formulation 25: 5 329.2 .+-. 16.1 315.3
.+-. 14.2 409.8 .+-. 9.1 1.3426 .+-. 0.1025 0.3277 .+-. 0.0255 3.0
mg/kg Group 6: compound (A) 5 339.7 .+-. 13.6 325.1 .+-. 22.3 406.7
.+-. 37.4 1.3312 .+-. 0.1827 0.3267 .+-. 0.0235 (ONO-1301): 3.0
mg/kg
[0508] Infarct Area Evaluation Method
[0509] For the sections, the apical portion was removed, and the
middle area was sectioned into two parts at an interval of about 2
mm. Thereafter, the infarct area of the two sections on the apical
and basal sides was measured. The infarct area was evaluated based
on the ratio of the infarct area to the entire left ventricular
area, and the ratio of the length of the normal area or the infarct
area to the left ventricular outer diameter.
[0510] FIG. 32 shows an infarct area evaluation method. For the
sections, the apical portion was removed, and the middle area was
sectioned into two parts at an interval of about 2 mm. Thereafter,
the infarct area of the two sections on the apical and basal sides
was measured. The infarct area was evaluated based on the ratio of
the infarct area to the entire left ventricular area, and the
ratios of the length of the normal area or the infarct area to the
left ventricular outer diameter.
[0511] The results revealed that the group of administration of
Formulation 25 at 3 mg/kg showed a significant decrease in the
ratio of the infarct area relative to the entire left ventricle
area, and the ratio of the infarct area in the left ventricular
outer diameter area (Table 32 and Table 33).
[0512] The results confirmed that Formulation 25 reduced the
infarct area, indicating that an effect was exhibited. In contrast,
the administration of compound (A) (ONO-1301) at 3 mg/kg showed no
effect. These results suggested that Formulation 25 exhibited a DDS
effect.
TABLE-US-00032 TABLE 32 Left ventricular area (mm.sup.2) Left
ventricular area (mm.sup.2) Normal Infarct Infarct Normal Infarct
Infarct Drugs N region region rate (%) region region rate (%) Group
2: Control 5 35.7 .+-. 6.13 17.8 .+-. 3.22 34 .+-. 7.3 41.1 .+-.
6.80 13.3 .+-. 2.45 25 .+-. 5.2 Group 3: Formulation 25: 5 36.7
.+-. 1.43 23.4 .+-. 8.54 38 .+-. 8.7 41.8 .+-. 10.70 18.9 .+-. 4.56
31 .+-. 5.7 0.3 mg/kg Group 4: Formulation 25: 5 34.6 .+-. 7.73
15.9 .+-. 2.12 32 .+-. 6.4 42.0 .+-. 9.04 17.6 .+-. 2.65 30 .+-.
6.4 1.0 mg/kg Group 5: Formulation 25: 5 41.0 .+-. 4.88 6.5 .+-.
4.83 13 .+-. 9.8** 49.7 .+-. 7.72 8.1 .+-. 3.72 14 .+-. 6.4* 3.0
mg/kg Group 6: Compound A: 5 31.1 .+-. 2.83 18.1 .+-. 5.18 36 .+-.
8.0 41.0 .+-. 8.03 14.6 .+-. 3.23 26 .+-. 2.2 3.0 mg/kg Data shown
in the table: the mean .+-. standard deviation *p < 0.05; **p
< 0.01 (Dunnett's test)
TABLE-US-00033 TABLE 33 Left ventricular Left ventricular outer
diameter (cm) outer diameter (cm) Normal Infarct Infarct Normal
Infarct Infarct Drugs N region region rate (%) region region rate
(%) Group 2: Control 5 1.9 .+-. 0.36 1.4 .+-. 0.18 42 .+-. 4.9 2.5
.+-. 0.23 1.2 .+-. 0.20 32 .+-. 5.2 Group 3: Formulation 25: 5 1.6
.+-. 0.34 1.5 .+-. 0.53 47 .+-. 14.9 2.3 .+-. 0.06 1.3 .+-. 0.19 36
.+-. 3.3 0.3 mg/kg Group 4: Formulation 25: 5 1.9 .+-. 0.53 1.1
.+-. 0.24 37 .+-. 12.2 2.3 .+-. 0.39 1.2 .+-. 0.31 34 .+-. 9.5 1.0
mg/kg Group 5: Formulation 25: 5 2.4 .+-. 0.16 0.6 .+-. 0.28 20
.+-. 7.7** 2.7 .+-. 0.16 0.7 .+-. 0.21 19 .+-. 5.3* 3.0 mg/kg Group
6: Compound A: 5 1.7 .+-. 0.41 1.5 .+-. 0.15 46 .+-. 7.5 2.3 .+-.
0.41 1.2 .+-. 0.37 34 .+-. 10.2 3.0 mg/kg Data shown in the table:
the mean .+-. standard deviation *p < 0.05; **p < 0.01
(Dunnett's test)
[0513] The single intravenous administration of Formulation 25
(ONO-1301Lipo) suppressed the onset of the pathological condition
in the rat heart ischemia model in a dose-correlated manner.
Further, the administration of Formulation 25 at 3 mg/kg showed an
improving effect over the Pre. Although the single intravenous
administration of the compound (A) (ONO-1301) drug substance at 3
mg/kg showed a suppression effect similar to that of Formulation 25
at 1 mg/kg, no effect was observed in terms of reducing the infarct
area.
[0514] These results confirmed that Formulation 25 exhibited a DDS
effect.
* * * * *