U.S. patent application number 14/463139 was filed with the patent office on 2015-02-19 for fibrosis causing agent.
The applicant listed for this patent is TERUMO KABUSHIKI KAISHA. Invention is credited to AYAKA AKUTAGAWA, SUGURU HATA, YUICHI TADA.
Application Number | 20150050257 14/463139 |
Document ID | / |
Family ID | 52467000 |
Filed Date | 2015-02-19 |
United States Patent
Application |
20150050257 |
Kind Code |
A1 |
HATA; SUGURU ; et
al. |
February 19, 2015 |
FIBROSIS CAUSING AGENT
Abstract
A fibrosis-causing agent is highly effective in the fibrosis of
tissues. The fibrosis-causing agent contains a fibrosis inducer and
a fibrosis promoter. A fibrosis-causing agent dosage form and a
method of administering the fibrosis-causing agent is also
disclosed.
Inventors: |
HATA; SUGURU; (KANAGAWA,
JP) ; TADA; YUICHI; (KANAGAWA, JP) ;
AKUTAGAWA; AYAKA; (KANAGAWA, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TERUMO KABUSHIKI KAISHA |
TOKYO |
|
JP |
|
|
Family ID: |
52467000 |
Appl. No.: |
14/463139 |
Filed: |
August 19, 2014 |
Current U.S.
Class: |
424/93.72 |
Current CPC
Class: |
A61K 38/4833 20130101;
A61K 31/734 20130101; A61K 35/19 20130101; A61K 35/19 20130101;
A61K 38/363 20130101; A61K 38/4833 20130101; A61K 38/363 20130101;
A61K 45/06 20130101; A61K 35/16 20130101; A61K 9/007 20130101; A61K
31/734 20130101; A61P 11/00 20180101; A61K 35/16 20130101; A61K
47/02 20130101; A61P 43/00 20180101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/93.72 |
International
Class: |
A61K 35/14 20060101
A61K035/14; A61K 47/02 20060101 A61K047/02; A61K 31/734 20060101
A61K031/734 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 19, 2013 |
JP |
2013-169747 |
Claims
1. A fibrosis-causing agent comprising a fibrosis inducer and a
fibrosis promoter.
2. The fibrosis-causing agent according to claim 1, wherein the
fibrosis promoter contains platelets or platelet-rich plasma
(PRP).
3. The fibrosis-causing agent according to claim 2, wherein the
fibrosis promoter contains platelet-rich plasma (PRP) and a PRP
gelling agent.
4. The fibrosis-causing agent according to claim 3, wherein PRP
gelling agent contains at least one selected from the group
consisting of calcium, calcium salt, fibrin, fibrinogen, thrombin,
vitamin K, and factor X.
5. The fibrosis-causing agent according to claim 1, wherein the
fibrosis inducer contains at least one polyanion.
6. The fibrosis-causing agent according to claim 5, wherein the
polyanion is at least one selected from the group consisting of
alginic acid, alginate, and alginic ester.
7. The fibrosis-causing agent according to claim 1, wherein the
fibrosis-causing agent is administered for treatment of pulmonary
emphysema.
8. A method of causing fibrosis by administering to a patient in
thereof, a fibrosis causing agent according to claim 1.
9. A method for causing fibrosis by administering to a subject a
fibrosis causing agent.
10. The method according to claim 9, wherein the fibrosis causing
agent comprises a fibrosis inducer and a fibrosis promoter.
11. The method according to claim 10, wherein the fibrosis promoter
contains platelets or platelet-rich plasma (PRP).
12. The method according to claim 11, wherein the fibrosis promoter
contains platelet-rich plasma (PRP) and a PRP gelling agent.
13. The method according to claim 12, wherein PRP gelling agent
contains at least one selected from the group consisting of
calcium, calcium salt, fibrin, fibrinogen, thrombin, vitamin K, and
factor X.
14. The method according to claim 9, wherein the fibrosis inducer
contains at least one polyanion.
15. The method according to claim 14, wherein the polyanion is at
least one selected from the group consisting of alginic acid,
alginate, and alginic ester.
16. A composition for treating pulmonary emphysema, the composition
consisting of a fibrosis-causing agent, the fibrosis-causing agent
comprising a fibrosis inducer and a fibrosis promoter.
17. A method for treating pulmonary emphysema by administering to a
patient in need thereof, the composition according to claim 16.
18. A fibrosis-causing agent comprising a fibrosis inducer and a
fibrosis promoter, the fibrosis promoter consisting essentially of
platelets or platelet-rich plasma (PRP); and the fibrosis inducer
consisting essentially of at least one polyaninon.
19. The fibrosis-causing agent according to claim 18, wherein the
fibrosis promoter further comprises a platelet-rich plasma (PRP)
gelling agent, the gelling agent is at least one selected from the
group consisting of calcium, calcium salt, fibrin, fibrinogen,
thrombin, vitamin K, and factor X.
20. The fibrosis causing agent according to claim 18, wherein the
polyanion is at least one selected from the group consisting
alginic acid, alginate and alginic ester.
21. A fibrosis-causing agent comprising a fibrosis inducer and a
fibrosis promoter, wherein the fibrosis promoter is platelet-rich
plasma (PRP); and the fibrosis inducer is calcium alginate.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is based on Japanese Application No.
2013-169747, filed on Aug. 19, 2013, the contents of which are
incorporated herein by reference.
BACKGROUND OF THE DISCLOSURE
[0002] The present description relates to a fibrosis-causing
agent.
[0003] Among a large variety of pulmonary diseases which hamper
normal respiration is chronic obstructive pulmonary disease (COPD).
It includes at least one of asthma, pulmonary emphysema, and
chronic bronchitis, which occludes the lung. These diseases often
give rise to their symptoms at one time, thereby making it
difficult to determine which one of them causes lung occlusion in
each case. COPD remains unchanged for several months and hence
chronic bronchitis is clinically identified from the continued
reduction of expiration for two or more years. The most serious
symptoms relating to COPD are chronic bronchitis and pulmonary
emphysema.
[0004] The pulmonary emphysema is characterized by an extraordinary
expansion, accompanied by disorganization, of respiratory
bronchioles, pulmonary alveoli, and alveolar sacs, which are
collectively called alveolar parenchyma for gas exchange. The
alveolar parenchyma in its normal state shrinks at the time of
expiration; however, the enlarged alveolar parenchyma does not
recover after expansion due to breathing. This prevents
satisfactory expiration. Moreover, the pulmonary emphysema
decreases the effective area of pulmonary alveoli and the number of
capillary vessels running in all directions on the surface of
pulmonary alveoli, which reduces the overall ventilating capacity
of the lung. In addition, the lung suffering from pulmonary
emphysema is poor in resilience and unable to keep the airway open
by stretching because it has its elastin and collagen destroyed by
inflammation. This makes the bronchus liable to deformation. The
result is that as the lung shrinks for expiration the bronchus
becomes narrow due to compression by its surrounding air-filled
alveoli and the lung excessively expands, thereby preventing smooth
expiration. This is the reason why patients with pulmonary
emphysema expire breath while keeping their lips pursed up.
[0005] In Japan, there are about 50,000 patients with pulmonary
emphysema, who receive home oxygen therapy. Moreover, those who are
in the incipient or moderate stage of pulmonary emphysema are
estimated to count up to about three million. The present medical
treatment of pulmonary emphysema relies mostly on drug therapy and
oxygen therapy. They are symptomatic therapies intended to
alleviate or eliminate the symptom with the help of a
bronchodilator which expands the bronchus to aid respiration. They
are not necessarily effective. There are other therapies than
mentioned above, such surgical ones as lung implantation, lung
volume reduction surgery (LVRS), and bronchoscopic volume reduction
(BVR). They still have many problems with great burdens on
patients, poor prognosis (in the case of lung implantation), the
possibility of pulmonary emphysema occurring in the remaining lung
(in the case of LVSR), and limited past records proving
effectiveness (in the case of BVR).
[0006] In contrast with the foregoing therapies, a new therapy we
recently designed that reduces the lung capacity in a noninvasive
manner. For example, JP-T-2009-514860 discloses a composition
containing polycations and polyanions such that the ratio of X to Y
is larger than about 1, where X denotes the product of the mass of
polycations and the ratio of the electric charge per mass of
polycations, and Y denotes the product of the mass of polyanions
and the ratio of the electric charge per mass of polyanions.
According to the disclosure, the composition promotes the localized
pulmonary fibrosis in the lung's affected part, thereby achieving
the lung volume reduction (LVR) and curing pulmonary emphysema (or
COPD).
SUMMARY OF THE DISCLOSURE
[0007] The present inventors found that the composition disclosed
in JP-T-2009-514860 does not necessarily achieve fibrosis
sufficiently and hence it does not necessarily produce the effect
of reducing the lung capacity as desired.
Thus, it is an intention of the present disclosure to provide a
fibrosis-causing agent which effectively causes the fibrosis of
tissues. As the result of extensive investigation, the present
inventors found that the above-mentioned problems are solved by a
new fibrosis-causing agent which contains in combination a fibrosis
inducer (which induces fibrosis) and a fibrosis promoter (which
promotes fibrosis). This finding has led to the present disclosure.
The present disclosure provides a fibrosis-causing agent which
effectively causes the fibrosis of tissues.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1A is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken one week after administration of a
fibrosis-causing agent pertaining to Example 1;
[0009] FIG. 1B is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken four weeks after administration of the
fibrosis-causing agent pertaining to Example 1;
[0010] FIG. 1C is an optical photomicrograph (.times.200,
HE-stained) showing granulomatous inflammation in lung tissues of a
Japanese white rabbit, which was taken one week after
administration of the fibrosis-causing agent pertaining to Example
1;
[0011] FIG. 1D is an optical photomicrograph (.times.100,
HE-stained) showing granulomatous inflammation in lung tissues of a
Japanese white rabbit, which was taken four weeks after
administration of the fibrosis-causing agent pertaining to Example
1;
[0012] FIG. 2A is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken one week after administration of a
fibrosis-causing agent pertaining to Example 2;
[0013] FIG. 2B is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken four weeks after administration of the
fibrosis-causing agent pertaining to Example 2;
[0014] FIG. 2C is an optical photomicrograph (.times.200,
MT-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken one week after administration of the
fibrosis-causing agent pertaining to Example 2;
[0015] FIG. 2D is an optical photomicrograph (.times.200,
MT-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken four weeks after administration of the
fibrosis-causing agent pertaining to Example 2;
[0016] FIG. 2E is an optical photomicrograph (.times.100,
HE-stained) showing granulomatous inflammation in lung tissues of a
Japanese white rabbit, which was taken one week after
administration of the fibrosis-causing agent pertaining to Example
2;
[0017] FIG. 2F is an optical photomicrograph (.times.100,
HE-stained) showing granulomatous inflammation in lung tissues of a
Japanese white rabbit, which was taken four weeks after
administration of the fibrosis-causing agent pertaining to Example
2;
[0018] FIG. 3A is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken one week after administration of a
fibrosis-causing agent pertaining to Comparative Example 1;
[0019] FIG. 3B is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken four weeks after administration of the
fibrosis-causing agent pertaining to Comparative Example 1;
[0020] FIG. 3C is an optical photomicrograph (.times.200,
MT-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken one week after administration of the
fibrosis-causing agent pertaining to Comparative Example 1;
[0021] FIG. 3D is an optical photomicrograph (.times.200,
HE-stained) showing granulomatous inflammation in lung tissues of a
Japanese white rabbit, which was taken one week after
administration of the fibrosis-causing agent pertaining to
Comparative Example 1;
[0022] FIG. 4 is an optical photomicrograph (.times.40, HE-stained)
showing fibrosis in lung tissues of a Japanese white rabbit, which
was taken four weeks after administration of a fibrosis-causing
agent pertaining to Comparative Example 2;
[0023] FIG. 5 is an optical photomicrograph (.times.40, HE-stained)
showing fibrosis in lung tissues of a Japanese white rabbit, which
was taken one week after administration of a fibrosis-causing agent
pertaining to Comparative Example 3;
[0024] FIG. 6A is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken one week after administration of a
fibrosis-causing agent pertaining to Comparative Example 4;
[0025] FIG. 6B is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken four weeks after administration of the
fibrosis-causing agent pertaining to Comparative Example 4;
[0026] FIG. 6C is an optical photomicrograph (.times.200,
MT-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken one week after administration of the
fibrosis-causing agent pertaining to Comparative Example 4;
[0027] FIG. 6D is an optical photomicrograph (.times.200,
MT-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken four weeks after administration of the
fibrosis-causing agent pertaining to Comparative Example 4;
[0028] FIG. 6E is an optical photomicrograph (.times.200,
HE-stained) showing granulomatous inflammation in lung tissues of a
Japanese white rabbit, which was taken one week after
administration of the fibrosis-causing agent pertaining to
Comparative Example 4;
[0029] FIG. 7A is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken one week after administration of a
fibrosis-causing agent pertaining to Example 3;
[0030] FIG. 7B is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken four weeks after administration of the
fibrosis-causing agent pertaining to Example 3;
[0031] FIG. 7C is an optical photomicrograph (.times.200,
MT-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken one week after administration of the
fibrosis-causing agent pertaining to Example 3;
[0032] FIG. 7D is an optical photomicrograph (.times.100,
HE-stained) showing granulomatous inflammation in lung tissues of a
Japanese white rabbit, which was taken one week after
administration of the fibrosis-causing agent pertaining to Example
3;
[0033] FIG. 7E is an optical photomicrograph (.times.100,
HE-stained) showing granulomatous inflammation in lung tissues of a
Japanese white rabbit, which was taken one week after
administration of the fibrosis-causing agent pertaining to Example
3;
[0034] FIG. 8A is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken one week after administration of a
fibrosis-causing agent pertaining to Comparative Example 5;
[0035] FIG. 8B is an optical photomicrograph (.times.200,
HE-stained) showing fibrosis in lung tissues of a Japanese white
rabbit, which was taken four weeks after administration of the
fibrosis-causing agent pertaining to Comparative Example 5; and
[0036] FIG. 8C is an optical photomicrograph (.times.100,
HE-stained) showing granulomatous inflammation in lung tissues of a
Japanese white rabbit, which was taken one week after
administration of the fibrosis-causing agent pertaining to
Comparative Example 5.
DETAILED DESCRIPTION
[0037] The following is a detailed description of an embodiment of
the present disclosure.
[0038] Fibrosis-Causing Agent
[0039] One embodiment of the present disclosure provides a
fibrosis-causing agent containing a fibrosis inducer and a fibrosis
promoter.
[0040] Fibrosis Inducer
[0041] The fibrosis inducer is intended to induce the fibrosis of
tissues. The tissues should preferably be those of trachea or lung
although they are not specifically restricted. More particularly,
they should preferably be those of bronchiole, respiratory
bronchiole, pulmonary alveolus, and alveolar sac, with the last two
being more preferable.
[0042] Examples of the foregoing fibrosis inducer include, without
specific restrictions, polycation, polyanion, composite of
polycation and polyanion, biodegradable material, flexible cured
polymer, adhesive material, and other compounds.
[0043] The foregoing polycation is exemplified by polymers having
amino groups, without specific restrictions. Typical examples of
the polycation include polyamino acid, synthetic polypeptide,
thrombin, polycationic polymer chitosan (such as polyvinylamine and
polyallylamine), partially deacetylated chitin, and basic
polysaccharide (such as aminated cellulose).
[0044] The polyamino acid or synthetic polypeptide is exemplified
by polymers composed of positively charged amino acid such as
lysine, arginine, histidine, ornithine, and 5-hydroxylysine. Their
typical examples include poly-D-lysine, poly-L-lysine,
poly-DL-lysine, polyarginine, polyhistidine, polyornithine,
polyethylamine, and poly-.gamma.-benzyl-L-glutamate. The foregoing
polyamino acid or synthetic polypeptide should preferably be one
which has aminoacid residues as many as 20 to 4000, preferably 50
to 3000, more preferably 100 to 1000, and most desirably 200 to
750. Moreover, the polycation should preferably be one which has a
molecular weight of 10 to 500 kD, preferably 20 to 250 kD, and more
preferably 30 to 200 kD. Incidentally, the foregoing polyamino acid
or synthetic polypeptide may be produced by any known method, such
as chemical synthesis or recombinant DNA technology. The molecular
weight is one which may be determined by any known method, such as
electrophoresis, size exclusion chromatography, and multi-angle
laser beam scattering.
[0045] The foregoing polyanion is exemplified, without specific
restrictions, by polymers having any of carboxyl group, sulfo
group, and phenolic hydroxyl group. Typical examples of the
polyanion include alginic acid, alginate, alginic ester, fibrin,
fibrinogen, heparin, heparan sulfate, glucuronic acid, mannuronic
acid, guluronic acid, dermatan sulfate, condroitin sulfate,
pentosan sulfate, keratan sulfate, mucopolysaccharide polysulfate,
hyaluronic acid, and polymer (such as polyglutamic acid and
polyaspartic acid) composed of negatively charged amino acids (such
as glutamic acid and aspartic acid).
[0046] The foregoing alginic acid is a polymer composed of
.beta.-D-mannuronic acid (M) and .alpha.-L-glucuronic acid (G). It
varies in its properties depending on the molecular weight of M and
G and the ratio between the amounts of M and G. For example, the
one with a high G content easily forms a stable composite material
(in gel form) with divalent cations such as calcium ions. The
resulting gel readily crosslinks to become hard and strong. The
crosslinked gel has a large number of crosslink points which help
reduce the amount of water to be held therein. By contrast, the
alginic acid with a high M content provides a gel which is superior
in flexibility and resilience and capable of holding a large amount
of water. Moreover, the alginic acid is not specifically restricted
in the ratio (M/G) between the contents of .beta.-D-mannuronic acid
(M) and .alpha.-L-glucuronic acid (G). Any adequate ratio should be
selected according to the intention of inducing fibrosis. This
intention may be achieved by carefully selecting raw materials
originating from marine algae.
[0047] The foregoing alginate includes, for example, sodium
alginate, potassium alginate, calcium alginate, iron alginate, and
ammonium alginate.
[0048] The foregoing alginic ester includes those esters of alginic
acid with a C1 to C6 alcohol. The latter is exemplified by
monohydric alcohol (such as methanol, ethanol, propanol, isopropyl
alcohol, and butanol), diol (such as ethylene glycol and propylene
glycol), and triol (such as glycerin). Typical examples of the
alginic ester include propylene glycol alginate, methyl alginate,
ethyl alginate, and ethylene glycol alginate. The preferred alginic
acid is propylene glycol alginate.
[0049] The polyanion capable of forming a composite material with
the polycation includes those listed below and others: heparan
sulfate, heparin/heparan sulfate, dermatan sulfate, condroitin
sulfate, pentosan sulfate, keratan sulfate, keratin sulfate,
mucopolysaccharide polysulfate, carrageenan, sodium alginate,
potassium alginate, hyaluronic acid, polyglutamic acid,
polyaspartic acid, carboxymethylcellulose, randomly structured
nucleic acid; polysaccharides (such as cellulose, xylose,
N-acetyllactosamine, glucuronic acid, mannuronic acid, and
guluronic acid), sulphated products thereof, and carboxymethylated
products thereof; polyamino acid containing a plurality of amino
acids selected from the group consisting of Asp, Glu, Lys, Orn,
Arg, Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp, Asn, Gln, Ser,
Thr, Tyr, Cys, and His, with Asp and/or Glu accounting for no less
than about 25% of the amino acids and Lys, Orn, and Arg accounting
for no more than about 5% of the amino acids; and
[0050] polyamino acid represented by any of the following formulas:
poly(X-Y), poly(X-Y-Y), and poly(X-Y-Y-Y), where X independently
denotes Asp or Glu, and Y independently denotes Gly, Ala, Val, Leu,
Ile, Met, Pro, Phe, Trp, Asn, Gln, Ser, Thr, Tyr, Cys, or His.
[0051] The biodegradable material includes any known ones, such as
thrombin, borate, calcium, magnesium, chondroitin sulfate,
hyaluronic acid, protein (such as gelatin), starch, collagen,
glucosaminoglycan, agarose, dextran, pullulan, polyglycolic acid,
polylactic acid, polyaspartic acid, polycaprolactone,
polyhydroxybutyric acid, polydioxanone, "plastarch," zein,
polydioxane, polylactic acid-glycolic acid copolymer,
polysaccharide, soybean protein, phospho-lipid, cholesterol,
phospholipid-cholesterol copolymer, polymalic acid, sacran,
polyhydroxy butyrate/valerate, polycaprolactam, polybutylene
succinate, polybutylene succinate/adipate, polyethylene succinate,
aliphatic polyester, vinyl acetate, methyl acrylate, vinyl
acetate-methyl acrylate copolymer, biomaterial (such as autologous
blood), and decomposition product resulting from decrosslinking.
Additional examples are biodegradable materials disclosed in
Japanese Patent Laid-open Nos. 2000-160034 and 2002-146219.
[0052] The adhesive material includes, for example, talc,
tetracycline, Picibanil (OK432), anticancer drug, povidone-iodine,
and silver nitrate, which chemically stimulate the pleura, thereby
causing pleuritis. The talc is hydrated magnesium silicate
[Mg.sub.3Si.sub.4O.sub.10(OH).sub.2], which is composed mainly of
SiO.sub.2 (about 60%), MgO (about 30%), and water of
crystallization (4.8%). The Picibanil (OK432) is Streptococcus
pyogenes (Group A, Type 3) strain Su (a species of hemolytic
streptococcus), in the form of penicillin-treated freeze-dried
powder. The anticancer drug includes bleomycin, cisplatin, etc.
[0053] Desirable examples of other compounds as the fibrosis
inducer mentioned above include polyvinyl alcohol, cellulose,
xylose, N-acetyllactosamine, carrageenan, carboxymethylcellulose,
borate, boronate, calcium, magnesium, guluronic acid, heparan
sulfate, dermatan sulfate, pentosan sulfate, keratan sulfate,
mucopolysaccharide polysulfate, hydrogel, acrylamide, agarose,
keratin, chitin, chitosan, partially deacetylated chitin, basic
polysaccharide (such as aminated cellulose), acrylamide,
polyurethane, polyethylene, polyester, fluoroplastics, silica,
silicone, hydroxyapatite, ceramics, bone cement, glass, metal,
silicon compound, siloxane, crosslinked polymer, porous material,
and such material as disclosed in Japanese Patent Laid-open No.
2001-164127.
[0054] The above-mentioned compounds as the fibrosis inducer may be
used alone or in combination with one another.
[0055] The fibrosis inducer mentioned above should preferably
contain a polyanion selected from the group consisting of alginic
acid, alginate, and alginic ester.
[0056] The fibrosis inducer may be in the form of liquid, gel, or
solid (particles).
[0057] In the case where the fibrosis inducer is in the form of
particulate solid, the particles should preferably have an average
particle diameter no larger than twice (more preferably one time)
the entrance diameter of enlarged pulmonary alveolus or alveolar
sac. The average diameter specified above is necessary for the
particles to be retained in the pulmonary alveolus or alveolar sac.
Incidentally, the entrance diameter of the enlarged pulmonary
alveolus or alveolar sac varies depending on the patient's weight
and the degree of seriousness and the position of pulmonary
emphysema. It is usually 1 to 2 mm in the case of human
patient.
[0058] Moreover, the fibrosis inducer should preferably have an
average particle diameter larger than (more preferably no smaller
than 1.1 times) the entrance diameter of the normal pulmonary
alveolus or alveolar sac. The average diameter specified above is
necessary for the fibrosis inducer to be excluded from normal
pulmonary alveoli or alveolar sacs but to enter enlarged pulmonary
alveoli or alveolar sacs, thereby causing fibrosis there only.
Incidentally, the entrance diameter of normal pulmonary alveolus or
alveolar sac varies depending on the patient's weight and other
factors. It is usually 200 to 300 .mu.m in the case of healthy
human.
[0059] The average particle diameter of the fibrosis inducer should
be 200 nm to 2000 .mu.m, preferably 1 .mu.m to 1000 .mu.m. An
average particle diameter no smaller than 200 nm is necessary for
the fibrosis inducer to be protected from phagocytosis by
macrophage or dendritic cell. By contrast, an average particle
diameter no larger than 2000 .mu.m is desirable for the fibrosis
inducer to have a large surface area for sufficient contact with
tissues. Incidentally, the value of "average particle diameter"
used in this specification is measured by observation under a
scanning electron microscope (SEM) or transmission electron
microscope (TEM). It is calculated by averaging particle diameters
of particles observed in several to dozens of visual fields. The
term "particle diameter of particle" means the maximum distance
between any two points on the particle contour at which the
particle contour crosses the line passing through the particle
center.
[0060] In the case where the fibrosis-causing agent is in the form
of solid, the fibrosis inducer mentioned above may have surface
treatment produced by exposure to plasma, addition of polyethylene
glycol, cationization, or anionization. The surface treatment
improves the fibrosis-causing agent in adhesive property and
fibrosis-causing performance and also in protection against
phagocytosis by macrophage or dendric cell.
[0061] Fibrosis Promoter
[0062] The fibrosis promoter is intended to promote the fibrosis of
tissues induced by the fibrosis inducer mentioned above. Usually,
it does not bring about the fibrosis of tissues by itself
[0063] Examples of the fibrosis promoter include the following
without specific restrictions: platelets, erythrocytes, leukocytes,
serum, plasma, platelet-rich plasma (PRP), autologous blood, marrow
fluid, marrow-originating cells, mesenchymal stem cells, fat, fat
stem cells, and other components (such as stem cells) derived from
living bodies; and fibroblast growth factor (FGF), platelet-derived
growth factor (PDGF), vascular endothelial growth factor (VEGF),
nerve growth factor (NGF), epidermal growth factor (EGF),
insulin-like growth factor (IGF), transforming growth factor (TGF),
brain-derived neurotrophic factor (BDNF), granulocyte colony
stimulating factor (G-CSF), granulocyte macrophage colony
stimulating factor (GM-CSF), erythropoietin (EPO), thrombopoietin
(TPO), basic fibroblast growth factor (bFGF or FGF2), hepatocyte
growth factor (HGF), bone morphogenetic protein (BMP), neurotrophin
(neurotrophic factor: NGF, BDNF, NT3, etc.), and other growth
factors belonging to the family of the above-mentioned factors.
[0064] Preferable among the foregoing examples of fibrosis promoter
are platelets and PRP, with the latter being most desirable. They
may be used alone or in combination with one another.
[0065] The foregoing PRP contains platelets and plasma, and may
also contain blood components and anticoagulant.
[0066] The plasma contains water, albumin, protein (such as
immunoglobulin), lipid, carbohydrate, and inorganic salts (such as
sodium ions, potassium ions, and phosphate ions).
[0067] The blood components include leukocytes and
erythrocytes.
[0068] The anticoagulant includes sodium citrate,
ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose
solution (ACD), and sodium fluoride, which serve as a calcium
chelating agent.
[0069] Additional examples of the anticoagulant include vitamin K
dependent blood coagulation factor synthetic inhibitor (such as
warfarin, acenocoumarol, and phenindione); thrombin inhibitor (such
as dabigatran and argatroban); factor Xa inhibitor (such as
rivaroxaban, edoxaban, apixaban, and fondaparinux); and heparin and
low-molecular weight heparin. Preferable among these examples is
calcium chelating agent, particularly sodium citrate. Incidentally,
the foregoing anticoagulants may be used alone or in combination
with one another.
[0070] Platelets and PRP can be obtained usually by centrifuging
blood or with the help of a PRP producing unit (such as Smart PreP2
made by Harvest Technologies Corp. of Terumo group). The ordinary
process for preparation of PRP consists of adding an anticoagulant
to collected blood for prevention of blood coagulation, and
centrifuging the treated blood. The centrifugation separates the
blood into blood cell component containing erythrocytes, buffy coat
containing platelets and leukocytes, and supernatant containing
plasma. The thus obtained supernatant is platelet-rich plasma
(PRP). Incidentally, the resulting PRP may be centrifuged again to
remove erythrocytes remaining in the supernatant. In other words,
the repeated centrifugation of PRP gives rise to more purified
buffy coat and nearly platelet-free plasma or platelet-poor Plasma
(PPP), which are mixed together again to yield purified PRP.
[0071] The fibrosis inducer and the fibrosis promoter should
preferably be mixed together in a ratio of from 1:0.2 to 1:10 (by
mass), more preferably from 1:0.5 to 1:5 (by mass).
[0072] In the case where the fibrosis promoter is platelets or PRP,
the ratio of the fibrosis inducer to the platelets (if the fibrosis
promoter is PRP, platelets contained therein) should preferably be
from 1:0.2 to 1:10 (by mass), more preferably from 1:0.5 to 1:5 (by
mass).
[0073] In the case where the fibrosis promoter is PRP, the content
of platelets in PRP should preferably be 5.times.104 to
1000.times.104 cells/.mu.L, more preferably 10.times.104 to
500.times.104 cells/.mu.L. Incidentally, the content of platelets
in PRP may be determined by measurement with a multi-item automatic
hemocyte counter.
[0074] In the case where the fibrosis promoter is PRP, the content
of anticoagulant should preferably be 0.01 to 20 mg/mL, more
preferably 0.01 to 10 mg/mL, in 1 mL of blood, depending of the
type of anticoagulant.
[0075] The fibrosis-causing agent pertaining to this embodiment is
superior to conventional ones in its fibrosis performance because,
as mentioned above, it is composed of the fibrosis inducer (which
induces fibrosis) and the fibrosis promoter (which promotes
fibrosis) similar to various growth factors released from
platelets. This efficient fibrosis is attributable to the
synergistic action of the fibrosis inducer and the fibrosis
promoter.
[0076] According to one embodiment of the present disclosure, the
combination of the fibrosis inducer and the fibrosis promoter is
such that any one of alginic acid, alginate, and alginic ester is
combined with any one of platelets and PRP. A desirable combination
is alginate and PRP. A more desirable combination is any one of
sodium alginate, calcium alginate, and iron alginate and PRP. The
most desirable combination is calcium alginate and PRP. This
combination is effective for the above-mentioned synergistic
effect.
[0077] Adjuvants
[0078] The fibrosis-causing agent may be incorporated, without
specific restrictions, with a variety of adjuvants selected
according to the type and seriousness of the disease to which it is
applied.
[0079] Examples of the adjuvants include platelet-rich plasma (PRP)
gelling agent, solvent, antibiotic, and steroid. Additional
adjuvants include lipid and surface active agent, which make the
fibrosis inducer stable and multifunctional.
[0080] Platelet-Rich Plasma (PRP) Gelling Agent
[0081] The platelet-rich plasma (PRP) gelling agent causes PRP to
gel. Consequently, it is used in the case where the fibrosis
promoter is PRP. Gelation permits the fibrosis-causing agent to
stay firm at the affected part. This means that the
fibrosis-causing agent even in a small amount can bring about
fibrosis.
[0082] A preferred example of the platelet-rich plasma (PRP)
gelling agent may contain at least one selected from calcium,
calcium salt, fibrin, fibrinogen, thrombin, vitamin K, and blood
coagulation factor X ("factor X").
[0083] The calcium salt mentioned above includes, for example,
calcium chloride, calcium sulfate, calcium nitrate, calcium
formate, calcium citrate, calcium malate, calcium tartrate, calcium
gluconate, calcium succinate, calcium malonate, calcium glutarate,
calcium maleate, calcium fumarate, calcium glutaconate, and calcium
lactate.
[0084] The PRP gelling agent as exemplified above may be used alone
or in combination with one another.
[0085] An adequate selection of the PRP gelling agent depends on
the anticoagulant used at the time of preparation of PRP. For
example, in the case where the anticoagulant used to prepare PRP is
the calcium chelating agent mentioned above, the desirable PRP
gelling agent is calcium or calcium salt. Moreover, in the case
where preparation of PRP employs any of vitamin K dependent blood
coagulation factor synthetic inhibitor, thrombin inhibitor, factor
Xa inhibitor, and heparin and low-molecular weight heparin, the
preferable PRP gelling agent is any of vitamin K, thrombin, factor
X, fibrin, and fibrinogen.
[0086] Incidentally, calcium, fibrin, fibrinogen, and thrombin
function also as the fibrosis inducer. Therefore, in the case where
the anticoagulant used at the time of preparation of PRP is any of
calcium chelating agent, thrombin inhibitor, and heparin and
low-molecular weight heparin, any of calcium, fibrin, fibrinogen,
and thrombin used as the PRP gelling agent functions also as the
fibrosis inducer and PRP gelling agent.
[0087] Calcium salt, among the numerous PRP gelling agents, may be
used as an adequate adjuvant in the case where the fibrosis inducer
is any of alginic acid, alginate, and alginic ester, even though
PRP is not used as the fibrosis promoter. Alginic acid readily
reacts with calcium salt to form a stable composite material and
thus turns into gel, as mentioned above. Consequently, if the
fibrosis-causing agent is to be administered in the dosage form of
gel, it is possible to add a calcium salt as the gelling agent for
alginic acid. In other words, the calcium salt functions as the
gelling agent for alginic acid and derivatives thereof
[0088] The fibrosis promoter and the PRP gelling agent should be
used in a ratio from 1:0.001 to 1:10 (by mass), preferably from
1:0.003 to 1:5 (by mass).
[0089] Particularly in the case where the fibrosis promoter is PRP
and the PRP contains an anticoagulant, the ratio of the
anticoagulant to the PRP gelling agent should be from 1:0.001 to
1:5 (by mass), preferably 1:0.002 to 1:1 (by mass).
[0090] In the case where the fibrosis inducer is alginic acid or a
derivative thereof and the PRP gelling agent is added for gelation
of alginic acid or derivative thereof, the ratio of the alginic
acid (or a derivative thereof) to the PRP gelling agent should be
from 1:0.001 to 1:10 (by mass), preferably from 1:0.003 to 1:5 (by
mass).
[0091] (Solvent)
[0092] The fibrosis-causing agent may be incorporated with a
solvent as an adjuvant so that it is made fluid. This holds true in
the case where the fibrosis-causing agent lacks fluidity.
[0093] The solvent is not specifically restricted so long as it has
no adverse effects on living bodies. It includes, for example,
water, physiological saline, Ringer's solution, dimethylsulfoxide
(DMSO), dimethylformamide (DMF), HCl, alcohol, glycerol, and any
other aqueous solution (containing water as solvent). These
solvents may be used alone or in combination with one another.
[0094] The fibrosis-causing agent may contain solvents in varied
amounts depending on its dosage form, ranging from approximately 5
to approximately 300% (by mass), preferably approximately 10 to
approximately 200% (by mass), of its mass.
[0095] Antibiotics
[0096] An antibiotic may be added to prevent acute exacerbation due
to infection.
[0097] Examples of the antibiotics include, without specific
restrictions, .beta.-lactam antibiotics (such as penicillin,
cephem, oxacephem, penem, carbapenem, and monobactam);
aminoglycoside antibiotics; tetracycline antibiotics;
chloramphenicol; lincomycin-streptogramine antibiotics; polypeptide
antibiotics; polyene antibiotics; flucytosine; azole antifungal
drugs; terbinafine, butenafine, and amorolfine; and antiviral
agents.
[0098] The foregoing .beta.-lactam antibiotics include, for
example, benzylpenicillin, methicillin, cloxacillin, ampicillin,
amoxicillin, bacampicillin, carbenicillin, sulbenicillin,
cephaloridine, cefotiam, cefoperazone, cefmetazole, latamoxef,
flomoxef, imipenem, panipenem, imipenem-cilastatin mixture,
panipenem-betamipron mixture, aztreonam, and faropenem sodium.
[0099] The foregoing aminoglycoside antibiotics include, for
example, streptomycin, kanamycin, gentamicin, sisomicin, dibekacin,
amikacin, tobramycin, arbekacin, and isepamicin.
[0100] The foregoing tetracycline antibiotics include, for example,
tetracycline, oxytetracycline, minocycline, and
demethylchlortetracycline.
[0101] The foregoing lincomycin-streptogramine antibiotics include,
for example, lincomycin, clindamycin, and quinupristin-dalfopristin
mixture.
[0102] The foregoing polypeptide antibiotics include, for example,
colistin, polymixin B, vancomycin, and teicoplanin.
[0103] The foregoing polyene antibiotics include, for example,
amphotericin B, nystatin, trichomycin, and flucytosine.
[0104] The foregoing azole antifungal drugs include, for example,
econazole, miconazole, fluconazole, and itraconazole.
[0105] The foregoing antiviral agent includes, for example,
aciclovir, vidarabine, ganciclovir, amantadine, rimantadine,
zanamivir, oseltamivir, zidovudine, didanosine, lamivudine,
indinavir ethanolate, ritonavir, saquinavir, interferon
preparations, and ribavirin.
[0106] The antibiotics listed above may be used alone or in
combination with one another.
[0107] The fibrosis-causing agent may contain any of the foregoing
antibiotics in an amount of approximately 0.0005 to approximately
1% (by mass) based on its mass.
[0108] The antibiotics may be administered in a dose of
approximately 5 to approximately 1000 .mu.g/mL.
[0109] Additional Adjuvants
[0110] The fibrosis-causing agent may also contain any of
additional adjuvants listed below:
[0111] radiopaque materials (such as metrizamide, iopamidol, sodium
iothalamate, iodamide sodium, and meglumine, which are
water-soluble, and gold, titanium, silver, stainless steel,
aluminum oxide, and zirconium oxide, which are water-insoluble);
contrast enhancer (such as paramagnetic material, heavy atoms,
transition metal, lanthanide, actinide, dye, and radioactive
nuclear species); steroid; bronchodilator; lipid (such as
acylglycerol, neutral fat, wax, ceramide, phospholipid,
sphingophospholipid, glycerophospholipid, glycolipid,
sphingoglycolipid, glyceroglycolipid, lipoprotein, sulfolipid,
isoprenoid, fatty acid, terpenoid, steroid, and carotenoid);
anionic surface active agent (such as sodium salt of fatty acid,
monoalkyl sulfate, alkylpolyoxyethylene sulfate, alkylbenzene
sulfonate, and monoalkyl phosphate); cationic surface active agent
(such as alkyltrimethyl ammonium salt, dialkyldimethyl ammonium
salt, and alkylbenzyldimethyl ammonium salt); amphoteric surface
active agent (such as alkyldimethylamineoxide and
alkylcarboxybetaine); and nonionic surface active agent (such as
polyoxyethylene alkyl ether, fatty acid sorbitan ester, alkyl
polyglycoside, fatty acid diethanolamide, and alkyl monoglyceryl
ether.
[0112] The additional adjuvants mentioned above may be used alone
or in combination with one another.
[0113] The amount of the additional adjuvants may vary, without
specific restriction, depending on the type and seriousness of the
disease to which the fibrosis-causing agent is applied. The content
of the additional adjuvants is 1 to 200% (by weight) with respect
to the fibrosis-causing agent.
[0114] Dosage Form
[0115] The fibrosis-causing agent may be administered in any form
(such as liquid, gel, particles, and capsules) without specific
restrictions.
[0116] It is only necessary for the fibrosis-causing agent to
assume the desired dosage form at the target part. Consequently, it
is possible to administer the fibrosis inducer and the fibrosis
promoter (as the constituents of the fibrosis-causing agent)
separately in such a way that they produce the desired dosage form
at the target part.
[0117] In addition, the fibrosis-causing agent may be composed of
the fibrosis inducer and the fibrosis promoter which are different
in property. For instance, the former may be a solid and the latter
may be a gel. In this case, the former may be dispersed in the
latter. Alternatively, the fibrosis-causing agent may be in the
form particles or capsules, and in this case the fibrosis inducer
may be contained in the fibrosis-causing agent.
[0118] The fibrosis-causing agent may assume any dosage form which
varies depending on the type of administration to be determined
according to the state of the patient or affected part. For
instance, the fibrosis-causing agent should preferably be in the
dosage form of liquid or particles for administration to a patient
whose bronchial epitheliocytes are poor in resilience due to aging.
Also, the fibrosis-causing agent should preferably be in the dosage
form of viscous liquid, gel, or particles for administration to a
patient who has severe emphysema, in which case the
fibrosis-causing agent securely stays in the affected part.
[0119] Administration of the fibrosis-causing agent in the dosage
form of gel may present difficulties in its delivery to the target
part on account of its high viscosity. This problem may be
addressed by employing a catheter which permits the
fibrosis-causing agent to be delivered efficiently under pressure
or administered immediately after gelation. Another possible way is
to administer the fibrosis inducer and the fibrosis promoter
separately and then administer the PRP gelling agent so that
gelation takes place at the affected part. Incidentally, the
fibrosis-causing agent varies in time required for gelation. An
adequate length of time can be attained by adjusting the structure
of alginic acid (e.g., M/G ratio and molecular weight), the amount
and concentration of the PRP gelling agent, and the concentration
of the anticoagulant.
[0120] In the case where the fibrosis-causing agent is not
administered in its completed form but in the form of combination
of its separate components which turn into the fibrosis-causing
agent at the affected part, it is desirable to use the individual
components (i.e., fibrosis inducer, fibrosis promoter, optional PRP
gelling agent, solvent, and antibiotics) as a kit.
[0121] Use Applications
[0122] The fibrosis-causing agent according to the embodiment of
the present disclosure is capable of causing fibrosis of tissues as
desired. It may be applied to any part without specific
restrictions. Among those parts to which it is properly applied are
trachea and lung tissues, more preferably bronchioles, respiratory
bronchioles, pulmonary alveoli, and alveolar sacs, with the last
two being most desirable. The fibrosis-causing agent brings about
fibrosis in the lung tissues of a patient suffering from pulmonary
emphysema, thereby reducing the lung capacity.
[0123] Thus, the fibrosis-causing agent according to one embodiment
of the present disclosure can be properly applied to the lung for
treatment of pulmonary emphysema.
[0124] The dose of the fibrosis-causing agent to be administered
for treatment of pulmonary emphysema varies depending on the state
of patient. It is usually 0.1 to 50 mL/kg, preferably 0.3 to 10
mL/kg.
[0125] Method for Fibrosis
[0126] The fibrosis-causing agent according to one embodiment of
the present disclosure may be applied in various methods as
mentioned below.
[0127] The first method for fibrosis consists of inserting a
catheter into the trachea, bronchus or bronchiole through the
respiratory tract (step (a)) and delivering the fibrosis-causing
agent to the respiration region (including pulmonary alveoli or
alveolar sacs) through the catheter (step (b)).
[0128] The second method for fibrosis consists of inserting a rigid
endoscope and a needle tube (for administration) in the thoracic
cavity (step (c)) and administering the fibrosis-causing agent
below the pleura through the needle tube (step (d)).
[0129] The third method for fibrosis consists of opening the chest
(step (e)) and administering the fibrosis-causing agent to the
desired part (step (f)).
[0130] Fibrosis should preferably be performed on pulmonary alveoli
or alveolar sacs. In addition, fibrosis should preferably be
performed for treatment of pulmonary emphysema.
[0131] The term "respiratory region" used in this specification
generically denotes the respiratory organ beyond the bronchus,
including respiratory bronchioles and two alveoli. To be concrete,
the respiratory region includes bronchi, bronchioles, terminal
bronchioles, respiratory bronchioles, alveolar ducts, pulmonary
alveoli, alveolar sacs, pulmonary veins, and pulmonary arteries. It
should preferably include alveolar ducts, pulmonary alveoli,
alveolar sacs, pulmonary veins. In this specification, the term
"pulmonary alveoli or alveolar sacs" denotes at least either of
pulmonary alveoli or alveolar sacs, and they are collectively
called "alveolar parenchyma."
[0132] The fibrosis-causing agent may be administered to any
object, particularly mammals, without specific restrictions.
Typical examples of mammals include human, pet, household animal,
and farm animal (such as rabbit, dog, cat, horse, sheep, goat,
primate, cow, pig, rat, and mouse). Preferable among them are
human, rabbit, dog, and pig, with human being most desirable.
[0133] The following is a detailed description of the first method
mentioned above.
[0134] Step (a)
[0135] This step involves insertion of a catheter into the trachea,
bronchus, or bronchiole through the respiratory tract. The catheter
may be inserted into any position, however, it should preferably be
inserted such that its forward end extends as far as the eighth
branch or beyond it. The reason for this is that the opening of
enlarged pulmonary alveolus usually exists beyond the eighth to
twelfth branches. The catheter inserted in this manner permits (in
step (b) that follows) the fibrosis-causing agent to be delivered
in a maximum amount selectively to a narrow affected part, i.e.,
the enlarged pulmonary alveolus or alveolar sac (which are simply
referred to as "enlarged alveolar parenchyma" hereinafter). As a
result, the thus administered fibrosis-causing agent effectively
induces fibrosis. In addition, insertion of the catheter up to or
beyond the eighth branch prevents the fibrosis-causing agent from
entering the normal pulmonary alveoli or alveolar sacs (which are
simply referred to as "normal alveolar parenchyma" hereinafter).
This is an effective way of preventing normal pulmonary alveoli or
alveolar sacs from fibrosis while keeping them intact. In view of
the foregoing, the catheter for treatment of a human patient should
preferably be one which has a diameter of 1.5 to 5 mm, more
preferably 2 to 4 mm. Incidentally, the first right-left branch of
the trachea is defined as the first branch in this
specification.
[0136] The catheter is not specifically restricted; it may be
properly selected according to the diameter (or the number of
branches) of the bronchus or bronchiole into which it is inserted.
To be concrete, acceptable catheters include any known medical ones
for the respiratory organ, circulatory organ, and digestive organ.
The catheter may be used according to the method disclosed in U.S.
Patent Application Publication No. 2006/0283462. Moreover, the
catheter is not specifically restricted in its structure; it may or
may not have a balloon. The one having a balloon is preferable from
the standpoint of easy delivery and administration of the
fibrosis-causing agent into the trachea. The catheter is not
restricted either in the number of lumens and the inside diameter.
Adequate values for them should be selected according to the
fibrosis-causing agent to be administered (which varies in dose,
property, shape, and adjuvants) and the presence or absence of a
balloon.
[0137] Insertion of a catheter into the vicinity of an enlarged
alveolar parenchyma may be accomplished with the help of a sheath
inserted into a part close to the enlarged alveolar parenchyma. The
sheath is not specifically restricted in structure, it may or may
not have a balloon. However, it should preferably have a balloon
which closes the bronchus or bronchiole. The balloon fixes the
sheath to the bronchus or bronchiole, thereby allowing the catheter
to be stably inserted into the desired position. The balloon
attached to the sheath and the balloon attached to the catheter may
be placed at any position in the bronchus or bronchiole without
specific restrictions. It is desirable that the balloon attached to
the sheath be placed at the bronchus and the balloon attached to
the catheter be placed at the bronchus near the terminal,
particularly at the bronchiole. Closing the bronchus or bronchiole
with a balloon as mentioned above increases airtightness in the
region beyond the sheath, thereby allowing the fibrosis-causing
agent to be introduced and administered efficiently into the
enlarged alveolar parenchyma through the catheter. It is possible
to cause two balloons attached to the sheath and catheter
respectively to close different parts in the bronchus or
bronchiole, so as to easily control the pressure on the normal
alveolar parenchyma (existing between the two balloons) or the
pressure on the enlarged alveolar parenchyma) beyond the balloon of
the catheter.
[0138] Closing the bronchus or bronchiole with the balloon of the
sheath ensures ventilation with respiration pressure in the near
side from the balloon of the sheath. This leads to efficient and
safe treatment. The balloon of the sheath may be inflated and
deflated in any way without specific restrictions, for example, by
means of a three-way stopcock attached to the base of the
sheath.
[0139] It is possible to stably manipulate the fore-end of the
catheter if the pressure is kept constant in the region beyond the
balloon attached to the sheath. This is accomplished, for example,
by closing the bronchus or bronchiole with the balloon of the
sheath and decompressing the region beyond the sheath. This
procedure permits the balloon of the catheter to closely adhere to
the wall of the bronchus or bronchiole and also prevents air from
entering the region beyond the catheter through the side passage.
The result is easy decompression in the region beyond the catheter.
The reduced pressure (lower than the injection pressure of the
fibrosis-causing agent) in the region beyond the sheath facilitates
the introduction and administration of the fibrosis-causing agent
at a constant pressure into the region beyond the catheter. No
specific restrictions are imposed on the method of controlling the
pressure at the fore-end of the sheath or the fore-end of the
catheter. To be specific, the pressure control may be accomplished
by inserting the catheter into the sheath through a sealing valve
attached to the proximal end of the sheath. The sealing valve
closes the alveolar parenchyma beyond the fore-end of the sheath.
This permits easy pressure control at that part. It is also
possible to control pressure in the alveolar parenchyma beyond the
fore-end of the sheath, if the proximal end of the sheath is
provided with a three-way stopcock through which air is introduced
and discharged. The foregoing method may be applied also to the
pressure control beyond the fore-end of the catheter. The sealing
valve attached to the base of the catheter closes the alveolar
parenchyma beyond the fore-end of the catheter. This permits easy
pressure control at that part. It is also possible to control
pressure in the alveolar parenchyma beyond the fore-end of the
catheter, if the proximal end of the catheter is provided with a
three-way stopcock through which air is introduced and discharged.
Moreover, the inflation and deflation of the catheter's balloon may
be accomplished in any way, without specific restrictions, by means
of the three-way stopcock attached to the proximal end of the
catheter. In addition, the catheter may have a lumen for a guide
wire which facilitates the insertion of the catheter to the desired
position.
[0140] The catheter suitable for the foregoing method is one which
is provided with a balloon to close the bronchus and also with a
lumen which has openings at a far part and a near part and delivers
a liquid to the far part. Another example of the catheter is a
percutaneous transluminal coronary angioplasty (PTCA) catheter of
over-the-wire (OTW) type which is designed for treatment of
cardiovascular stenosis. These catheters may be any commercial ones
listed below. Microcatheter (FINECROSS.RTM. (made by Terumo Corp.)
that permits passage of a guide wire to cardiovascular stenosis.
PTCA catheter (Ryujin Plus OTW.RTM., made by Terumo Corp.).
Occlusion microballoon catheter (ATTENDANT.RTM., made by Terumo
Clinical Supply Co., Ltd.). The foregoing catheter is inserted into
the bronchus through the working lumen of a bronchoscope. Using a
bronchoscope is not essential if the catheter is arranged at any
desired position. The catheter and the catheter's balloon (in its
inflated state) are not specifically restricted in diameter; an
adequate diameter should be selected according to the diameter of
the bronchus and bronchiole. To be concrete, the outside diameter
of the inflated balloon of the catheter should preferably be
slightly larger than the inside diameter of the bronchus or
bronchiole in which the fore-end of the inserted catheter lies. To
be more specific, the outside diameter (Y mm) of the inflated
balloon should be about one to two times larger than the inside
diameter (X mm) of the bronchus or bronchiole. This ratio is
suitable for the catheter or balloon to come into close contact
with the bronchus or bronchiole (which is formed from elastic
smooth muscles) without severe damage.
[0141] This step (a) may be carried out in such a way that, prior
to insertion of the catheter into the bronchus or bronchiole, a
guide wire is inserted into the catheter's lumen (for fluid
delivery). Manipulation in this way permits the fore-end of the
guide wire to be placed beyond the fore-end of the catheter or near
the peripheral position. Thus, the fore-end of the catheter can be
introduced to the vicinity of pulmonary alveoli or alveolar sacs
(air sacs) beyond the bronchus or bronchiole. The guide wire to be
used for this purpose may be any known one designed for
pulmonology, cardiology, and gastroenterology. It should have an
adequate outside diameter which depends on the size of the lumen of
the catheter to be used. Its typical example is Runthrough.RTM. for
cardiology (made by Terumo Corporation), having an outside diameter
of 0.014 inch.
[0142] It is desirable that the fore-end of the guide wire and
catheter be provided with a member (agent) capable of radiographic
imaging. This arrangement permits the operator to confirm the
position of the fore-end of the guide wire and catheter (which
projects from the fore-end of the endoscope) at the time of
observation by X-ray radioscopy. In this way the operator can
introduce the guide wire and catheter to the respiratory region
(including enlarged pulmonary alveoli or alveolar sacs) which have
previously been identified by X-ray radioscopy or computed
tomography (CT) scan. In this occasion, the guide wire is pulled
away after it is confirmed by X-ray radioscopy that the fore-end of
the catheter has reached the desired position. The foregoing
operation should preferably be performed in such a way that the
fore-end of the guide wire is placed beyond the fore-end of the
catheter. Moreover, the fore-end of the catheter should preferably
have a network structure or perforated structure so that it will
not adhere to the inner wall of the respiratory region (such as
pulmonary alveoli and alveolar sacs).
[0143] Step (b)
[0144] This step is intended to administer the fibrosis-causing
agent to the respiratory region (including pulmonary alveoli and
alveolar sacs) through the catheter which has been inserted by the
step (a) mentioned above. The operation by this step effectively
places the fibrosis-causing agent in the affected part (enlarged
alveolar parenchyma), thereby inducing and promoting fibrosis in
the affected part and hence reducing the lung capacity.
[0145] Administration in this step may be performed in any manner,
without specific restrictions, because the fibrosis-causing agent
takes on various dosage forms as mentioned above. One method
involves preparation of the fibrosis-causing agent and subsequent
administration of the fibrosis-causing agent. Another method
involves administration of the fibrosis inducer and fibrosis
promoter and subsequent administration of the PRP gelling agent.
Still another method involves administration of the fibrosis
promoter and subsequent sequential administration of the fibrosis
inducer and the PRP gelling agent. The fibrosis inducer, the
fibrosis promoter, and the PRP gelling agent may be administered
together with or separately from the optional solvents and
antibiotics.
[0146] One preferable method involves administration of the
fibrosis inducer and fibrosis promoter and subsequent
administration of the gelling agent for platelet-rich plasma (PRP).
Another preferable method involves simultaneous administration of
the fibrosis inducer, fibrosis promoter, and PRP gelling agent
mixed together.
[0147] The term "administration of the fibrosis-causing agent" used
in this step implies administrating individually the constituents
of the fibrosis-causing agent, because it is only necessary to
create a state in which the fibrosis-causing agent is administered
to the affected part.
[0148] Smooth delivery of the fibrosis-causing agent at the time of
its administration will be ensured if the respiratory region is
pressurized by means of a catheter.
[0149] The specific method for administration of the
fibrosis-causing agent should be established according to the known
technology in comprehensive consideration of the dosage form and
composition of the fibrosis-causing agent to be used and the state
of the patient and effected part.
[0150] For administration of the fibrosis-causing agent in
particulate form, it is desirable to determine the average particle
diameter of the fibrosis-causing agent based on the previously
measured entrance diameter of the enlarged and/or normal pulmonary
alveolus or alveolar sac. In other words, the step (b) of the
procedure according to the present disclosure should be preceded by
a preliminary step to measure the entrance diameter of the enlarged
and/or normal pulmonary alveolus or alveolar sac and then determine
the average particle diameter of the fibrosis-causing agent based
on the thus measured entrance diameter. This preliminary step helps
prevent the fibrosis-causing agent from discharging from enlarged
pulmonary alveoli or alveolar sacs and also prevent the
fibrosis-causing agent from entering normal pulmonary alveoli or
alveolar sacs. Incidentally, it is possible to perform continuously
or intermittently the preliminary steps to "measure the entrance
diameter of the enlarged and/or normal pulmonary alveolus or
alveolar sac" and "determine the average particle diameter of the
fibrosis-causing agent based on the thus measured entrance
diameter."
[0151] There are no specific restrictions on the method of
measuring the entrance diameter of the enlarged and/or normal
pulmonary alveolus or alveolar sac. The measurement may be
accomplished by observation by means of endoscope or CT scan (in
the case where the entrance diameter is as large as 1 mm and
above). An alternative method for measurement is by X-ray
radiography which is preceded by administration of a contrast agent
to the bronchus. Another method involves insertion of a probe into
the vicinity of the entrance of the pulmonary alveolus or alveolar
sac and their imaging with ultrasound or infrared rays. If such
measurements are difficult to carry out, it is possible to adopt,
in place of measured values, statistical values for the entrance
diameter of the enlarged and/or normal pulmonary alveolus or
alveolar sac.
[0152] The fibrosis-causing agent according to this embodiment
effectively induces the fibrosis of tissues; therefore, upon
administration to the respiratory region by the step (b) mentioned
above, it brings about fibrosis in the tissue (specifically
enlarged alveolar parenchyma), thereby reducing the lung capacity.
This produces the effect of relieving and preventing the lung's
overexpansion that weakens the patient due to pulmonary emphysema
and bronchus occlusion. The result is that the enlarged alveolar
parenchyma is made smaller than its original size, and this in turn
produces the effect of relieving and preventing the compression and
occlusion of the bronchus by the surrounding alveolar parenchyma.
In addition, the foregoing method for fibrosis relies on a catheter
and needs no surgical treatment, which leads to a reduced burden on
the patient. Moreover, fibrosis by the foregoing method grows the
connective tissue (particularly fibroblast) on the inner wall of
the enlarged alveolar parenchyma, thereby recovering the resilience
of the alveolar parenchyma and relieving and preventing the lung's
overexpansion.
EXAMPLES
[0153] The present disclosure will be described in more detail with
reference to the following examples, which are not intended to
restrict the scope thereof
Example 1
[0154] Experiments for fibrosis were carried out in which the
fibrosis inducer is sodium alginate and the fibrosis promoter is
platelet-rich plasma (PRP).
[0155] (Preparation of Aqueous Solution of Sodium Alginate)
[0156] An aqueous solution (0.5% w/v) of sodium alginate was
prepared by dissolving 0.15 g of sodium alginate (made by Wako Pure
Chemical Industries, Ltd.) in 30 mL of reverse osmosis water (RO
water), followed by filtration and sterilization through a
sterilizing filter (Millipore 0.22 .mu.m). The resulting solution
was a viscous fluid.
[0157] Preparation of Platelet-Rich Plasma (PRP)
[0158] In the first step, 10 mL each of blood was collected into
two syringes (10 mL) containing 1.0 mL of anticoagulant ACD-A
solution (citric acid glucose solution, made by Terumo Corp.) from
the auricular artery of unanesthetized Japanese white rabbits
(clean, male, 3.0 to 3.49 kg). The collected blood was transferred
to a 15-mL centrifuge tube, which was slowly tumbled for
mixing.
[0159] The blood was centrifuged for ten minutes at 20.degree. C.
and 230.times.g by means of a refrigerated centrifuge (made by
Kubota Corp.) for separation of the supernatant (plasma). The
separated supernatant (plasma) was centrifuged for eight minutes at
20.degree. C. and 840.times.g for precipitation.
[0160] The remaining blood was centrifuged for ten minutes at
20.degree. C. and 1280.times.g for separation of the supernatant
(platelet-poor plasma (PPP)).
[0161] The precipitates obtained as mentioned above were dispersed
again in 1 mL of the platelet-poor plasma (PPP) separated as
mentioned above. Thus, there was obtained the intended
platelet-rich plasma (PRP).
[0162] The resulting PRP was found, by measurement with a
multi-item automatic hemocyte counter (made by Sysmex Corp.), to
contain platelets of 100.times.104 to 130.times.104 cells/.mu.L.
The thus obtained PRP was an ordinary liquid.
[0163] (Preparation of Fibrosis-Causing Agent)
[0164] The fibrosis-causing agent was prepared by mixing the
aqueous solution of sodium alginate and the platelet-rich plasma
(PRP) in a ratio of 1:1 (by volume). The resulting fibrosis-causing
agent was a viscous liquid.
[0165] Administration to Animal
[0166] The test animals were Japanese white rabbits (clean, male,
3.0 to 4.49 kg).
[0167] The test animal was given (by intramuscular injection)
xylazine hydrochloride (diluted four times with physiological
saline) at a dose of 5 mg/kg (1 mL/kg) for preanesthetic
medication.
[0168] The test animal was further given (by intravenous injection
through its auricular vein) somnopentyl (sodium pentobarbiturate,
diluted 3.24 times with physiological saline) at a dose of 20 mg/kg
(1 mL/kg) as an anesthetic agent. The test animal which showed
reflex during operation was given additional injection at a dose of
10 mg/kg (0.5 mL/kg) so that it kept the desired anesthetic
depth.
[0169] After anesthetization, the test animal was given the
fibrosis-causing agent at its bronchus through a catheter.
[0170] To be concrete, this procedure was carried out as follows.
With its sufficient anesthetic depth confirmed, the test animal had
its cervical part dissected at the median part thereof, so that the
trachea was exposed. In the subsequent step, a 0.035-inch guide
wire (made by Terumo Corp.) was inserted into the posterior lobe of
the right lung through the dissected trachea, until the fore-end of
the guide wire reached the position of the seventh rib (or the
upper part of the third branch). The guide wire was passed through
the lumen of a 6-Fr guiding catheter (made by Terumo Corp.) coated
with lidocaine. The catheter was inserted so that the fore-end
thereof reached the seventh rib, and the guide wire was pulled
out.
[0171] By way of the catheter, the fibrosis-causing agent prepared
as mentioned above was infused twice (0.5 mL each), air (10 mL) was
infused, and the fibrosis-causing agent was infused twice again
(0.5 mL each), and finally air (10 mL) was infused. Incidentally,
the infusion of the fibrosis-causing agent and air was coincident
with inspiration.
[0172] After administration of the fibrosis-causing agent, the test
animal had its trachea sutured and then was given viccillin
parenteral solution (0.5 g of ampicillin sodium diluted with 10 mL
of physiological saline) at a dose of 2 mL (100 mg/head) by
intramuscular injection at the paradissected part.
Example 2
[0173] Experiments for fibrosis were carried out in which the
fibrosis inducer is sodium alginate and the fibrosis promoter is
platelet-rich plasma (PRP). These components were used in
combination with calcium chloride as the PRP gelling agent.
[0174] Preparation of aqueous solution of sodium alginate and
preparation of platelet-rich plasma (PRP)
[0175] The same procedure as in Example 1 was repeated to prepare
the aqueous solution of sodium alginate and the platelet-rich
plasma (PRP).
[0176] Preparation of Mixed Solution
[0177] A mixed solution was prepared from the aqueous solution of
sodium alginate and the platelet-rich plasma (PRP) which were mixed
together in a ratio of 1:1 (by volume). The resulting mixed
solution was a viscous liquid.
[0178] Preparation of Aqueous Solution of Calcium Chloride
[0179] An aqueous solution (40 mM) of calcium chloride was prepared
by dissolving 0.222 g of calcium chloride (made by Wako Pure
Chemical Industries, Ltd.) in reverse osmosis water (RO water) such
that the resulting solution fills a 50-mL volumetric flask. This
aqueous solution was autoclaved at 121.degree. C. for 20 minutes
for sterilization. The thus obtained aqueous solution of calcium
chloride was an ordinary fluid.
[0180] Administration to Animals
[0181] The same procedure as in Example 1 was repeated except that
the mixed solution and the aqueous solution of calcium chloride
were also administered in the following manner
[0182] Administration of Mixed Solution and Aqueous Solution of
Calcium Chloride
[0183] The aqueous solution of calcium chloride was administered
first and then the mixed solution was administered. Administration
in this manner forms in the living organism of a Japanese white
rabbit the fibrosis-causing agent composed of sodium alginate,
platelet-rich plasma (PRP), and calcium chloride. The thus formed
fibrosis-causing agent takes on a gel form.
[0184] By way of the catheter, the aqueous solution of calcium
chloride (0.5 mL), the mixed solution (1.0 mL), and air (10 mL)
were infused sequentially. This step was repeated once again. In
other words, the aqueous solution of calcium chloride (0.5 mL), the
mixed solution (1.0 mL), and air (10 mL) were infused sequentially
in the order listed. Incidentally, the infusion of the aqueous
solution of calcium chloride, mixed solution, and air was
coincident with inspiration.
Comparative Example 1
[0185] Experiments were carried out in which the fibrosis inducer
is sodium alginate alone.
[0186] Preparation of Aqueous Solution of Sodium Alginate
[0187] The same procedure as in Example 1 was repeated to prepare
the aqueous solution of sodium alginate.
[0188] Administration to Animals
[0189] The same procedure as in Example 1 was repeated except that
the aqueous solution of sodium alginate was administered in the
following manner.
[0190] Administration of Aqueous Solution of Sodium Alginate
[0191] By way of the catheter, the aqueous solution of sodium
alginate (0.5 mL each) was infused twice, air (10 mL) was infused,
the aqueous solution of sodium alginate (0.5 mL each) was infused
twice again, and finally air (10 mL) was infused. Incidentally, the
infusion of the aqueous solution of sodium alginate and air was
coincident with inspiration.
Comparative Example 2
[0192] Experiments were carried out in which the fibrosis promoter
is platelet-rich plasma (PRP) alone.
[0193] (Preparation of Platelet-Rich Plasma (PRP))
[0194] The same procedure as in Example 1 was repeated to prepare
platelet-rich plasma (PRP).
[0195] Administration to Animals
[0196] The same procedure as in Example 1 was repeated except that
the platelet-rich plasma (PRP) was administered in the following
manner
[0197] Administration of Platelet-Rich Plasma (PRP)
[0198] By way of the catheter, the platelet-rich plasma (PRP) (0.5
mL each) was infused twice, air (10 mL) was infused, the
platelet-rich plasma (PRP) (0.5 mL each) was infused twice again,
and finally air (10 mL) was infused. Incidentally, the infusion of
the platelet-rich plasma and air was coincident with
inspiration.
Comparative Example 3
[0199] Experiments were carried out in which the fibrosis promoter
is platelet-rich plasma (PRP) and the gelling agent for
platelet-rich plasma (PRP) is calcium chloride in the form of
aqueous solution.
[0200] Preparation of Platelet-Rich Plasma (PRP) and Aqueous
Solution of Calcium Chloride
[0201] The same procedure as in Example 1 was repeated to prepare
platelet-rich plasma (PRP), and the same procedure as in Example 2
was repeated to prepare an aqueous solution of calcium
chloride.
[0202] Preparation of Mixed Solution
[0203] A mixed solution was prepared from the platelet-rich plasma
(PRP) and the aqueous solution of calcium chloride which were mixed
together in a ratio of 9:1 (by volume). The resulting mixed
solution was a viscous liquid.
[0204] Administration to Animals
[0205] The same procedure as in Example 1 was repeated except that
the mixed solution was administered in the following manner.
[0206] Administration of Mixed Solution
[0207] By way of the catheter, the mixed solution (0.5 mL each) was
infused twice, air (10 mL) was infused, the mixed solution (0.5 mL
each) was infused twice again, and finally air (10 mL) was infused.
Incidentally, the infusion of the mixed solution and air was
coincident with inspiration.
Comparative Example 4
[0208] Experiments were carried out in which the fibrosis inducer
is sodium alginate and the gelling agent for platelet-rich plasma
(PRP) is calcium chloride.
[0209] Preparation of Aqueous Solution of Sodium Alginate and
Aqueous Solution of Calcium Chloride
[0210] The same procedure as in Example 1 was repeated to prepare
the aqueous solution of sodium alginate. The same procedure as in
Example 2 was repeated to prepare the aqueous solution of calcium
chloride.
[0211] Administration to Animals
[0212] The same procedure as in Example 1 was repeated except that
the aqueous solution of sodium alginate and the aqueous solution of
calcium chloride were administered in the following manner.
[0213] Administration of Aqueous Solution of Sodium Alginate and
Aqueous Solution of Calcium Chloride
[0214] By way of the catheter, the aqueous solution of calcium
chloride (0.5 mL), the aqueous solution of sodium alginate (1.0
mL), and air (10 mL) were infused sequentially. This step was
repeated once again. In other words, the aqueous solution of
calcium chloride (0.5 mL), the aqueous solution of sodium alginate
(1.0 mL), and air (10 mL) were infused sequentially in the order
listed. Incidentally, the infusion of the aqueous solution of
calcium chloride, aqueous solution of sodium alginate, and air was
coincident with inspiration.
Example 3
[0215] Experiments for fibrosis were carried out in which the
fibrosis inducer is iron alginate and the fibrosis promoter is
platelet-rich plasma (PRP). These components were used in
combination with calcium chloride as the PRP gelling agent.
[0216] Preparation of Aqueous Dispersion of Iron Alginate
[0217] An aqueous solution (1% w/v) of iron chloride was prepared
by dissolving 5.0 g of iron chloride (made by Wako Pure Chemical
Industries, Ltd.) in 500 mL of reverse osmosis water (RO
water).
[0218] Also, an aqueous solution (1% w/v) of sodium alginate was
prepared by dissolving 1.5 g of sodium alginate (made by Wako Pure
Chemical Industries, Ltd.) in 150 mL of reverse osmosis water (RO
water).
[0219] The aqueous solution of sodium alginate (in atomized form)
was added to the aqueous solution of iron chloride being swirled,
so that there was obtained iron alginate in particulate form. The
aqueous dispersion of iron alginate was sifted through a sieve with
an opening of 100 .mu.m and then thoroughly washed with an aqueous
solution of iron chloride. The washed iron alginate was allowed to
stand overnight in an aqueous solution of iron chloride. With the
supernatant removed through an aspirator, the remaining liquid was
centrifuged at 500.times.g for three minutes. The resulting
precipitates were washed with 70% ethanol three times. With the
supernatant discarded, the precipitates were suspended in distilled
water (made by Otsuka Pharmaceutical Co., Ltd.) one half the volume
of precipitates. Thus there was obtained the aqueous dispersion of
iron alginate.
[0220] (Preparation of Platelet-Rich Plasma (PRP) and Aqueous
Solution of Calcium Chloride)
[0221] The same procedure as in Example 1 was repeated to prepare
platelet-rich plasma (PRP), and the same procedure as in Example 2
was repeated to prepare an aqueous solution of calcium
chloride.
[0222] Preparation of Fibrosis-Causing Agent
[0223] The aqueous dispersion of iron alginate and the
platelet-rich plasma (PRP) were mixed together in a ratio of 1:1
(by volume). The resulting mixture was stirred to give a mixed
solution. This mixed solution and the aqueous solution of calcium
chloride were mixed together and stirred in a ratio of 10:1 (by
volume) to give the fibrosis-causing agent. The resulting
fibrosis-causing agent is in the form of gel, with iron alginate
particles dispersed in the gel. Incidentally, the resulting
fibrosis-causing agent was administered to animals before gelation
proceeds completely after mixing with the aqueous solution of
calcium chloride.
[0224] Administration to Animals
[0225] The same procedure as in Example 1 was repeated except that
the fibrosis-causing agent was administered in the following
manner.
[0226] Administration of Fibrosis-Causing Agent
[0227] By way of the catheter, the fibrosis-causing agent (0.6 mL
each) was infused twice, air (10 mL) was infused, the
fibrosis-causing agent (0.5 mL each) was infused twice again, and
finally air (10 mL) was infused. Incidentally, the infusion of the
fibrosis-causing agent and air was coincident with inspiration.
Comparative Example 5
[0228] Experiments were carried out in which the fibrosis inducer
is iron alginate.
[0229] Preparation of Aqueous Dispersion of Iron Alginate
[0230] The same procedure as in Example 3 was repeated to prepare
the aqueous dispersion of iron alginate.
[0231] Administration to Animals
[0232] The same procedure as in Example 1 was repeated except that
the aqueous dispersion of iron alginate was administered in the
following manner.
[0233] Administration of Aqueous Dispersion of Iron Alginate
[0234] By way of the catheter, the aqueous dispersion of iron
alginate (0.5 mL each) was infused twice, air (10 mL) was infused,
the aqueous dispersion of iron alginate (0.5 mL each) was infused
twice again, and finally air (10 mL) was infused. Incidentally, the
infusion of the aqueous dispersion of iron alginate and air was
coincident with inspiration.
Example 4
[0235] Experiments for fibrosis were carried out in which the
fibrosis inducer is calcium alginate and the fibrosis promoter is
platelet-rich plasma (PRP). These components were used in
combination with calcium chloride as the PRP gelling agent.
[0236] Preparation of Aqueous Dispersion of Calcium Alginate
[0237] An aqueous solution (1% w/v) of calcium chloride was
prepared by dissolving 6.0 g of calcium chloride (made by Wako Pure
Chemical Industries, Ltd.) in 600 mL of reverse osmosis water (RO
water).
[0238] Also, an aqueous solution (1% w/v) of sodium alginate was
prepared by dissolving 1.5 g of sodium alginate (made by Wako Pure
Chemical Industries, Ltd.) in 150 mL of reverse osmosis water (RO
water).
[0239] The aqueous solution of sodium alginate (in atomized form)
was added to the aqueous solution of calcium chloride being
swirled, so that there was obtained calcium alginate in particulate
form. The aqueous dispersion of calcium alginate was sifted through
a sieve with an opening of 100 .mu.m and then thoroughly washed
with an aqueous solution of calcium chloride. The washed calcium
alginate was allowed to stand overnight in an aqueous solution of
calcium chloride. With the supernatant removed through an
aspirator, the remaining liquid was centrifuged at 500.times.g for
three minutes. The resulting precipitates were washed with 70%
ethanol three times. With the supernatant discarded, the
precipitates were suspended in distilled water one half the volume
of precipitates. Thus there was obtained the aqueous dispersion of
calcium alginate, having 100 .mu.m or smaller particle diameter
(average particle diameter: 89 .mu.m). Incidentally, the average
particle diameter was measured by using the LS particle size
distribution measuring apparatus (Beckman Coulter).
[0240] Preparation of Platelet-Rich Plasma (PRP) and Aqueous
Solution of Calcium Chloride
[0241] The same procedure as in Example 1 was repeated to prepare
platelet-rich plasma (PRP), and the same procedure as in Example 2
was repeated to prepare an aqueous solution of calcium
chloride.
[0242] Preparation of Fibrosis-Causing Agent
[0243] The aqueous dispersion of calcium alginate and the
platelet-rich plasma (PRP) were mixed together in a ratio of 1:1
(by volume). The resulting mixture was stirred to give a mixed
solution. This mixed solution and the aqueous solution of calcium
chloride were mixed together and stirred in a ratio of 10:1 (by
volume) to give the fibrosis-causing agent. The resulting
fibrosis-causing agent is in the form of gel, with calcium alginate
particles dispersed in the gel. Incidentally, the resulting
fibrosis-causing agent was administered to animals before gelation
proceeds completely after mixing with the aqueous solution of
calcium chloride.
[0244] Administration to Animal
[0245] The same procedure as in Example 1 was repeated except that
the fibrosis-causing agent was administered in the following
manner.
[0246] Administration of Fibrosis-Causing Agent
[0247] By way of the catheter, the fibrosis-causing agent (0.6 mL
each) was infused twice, air (10 mL) was infused, the
fibrosis-causing agent (0.5 mL each) was infused twice again, and
finally air (10 mL) was infused. Incidentally, the infusion of the
fibrosis-causing agent and air was coincident with inspiration.
Table 1 below summarizes the fibrosis-causing agents prepared in
Examples 1 to 4 and Comparative Examples 1 to 5.
TABLE-US-00001 TABLE 1 Fibrosis-causing Agent* Dosage (mL) Form of
Fibrosis Fibrosis Method of Fibrosis Fibrosis Fibrosis- Inducer
Promoter Adjuvant Administration Inducer Promoter Adjuvant causing
Agent Example 1 Alg--Na PRP -- Alg--Na + PRP 1.0 1.0 -- Viscous
liquid Example 2 Alg--Na PRP CaCl.sub.2 CaCl.sub.2 .fwdarw. Alg--Na
+ PRP 1.0 1.0 1.0 Gel Comparative Alg--Na -- -- Alg--Na 2.0 -- --
Viscous liquid Example 1 Comparative -- PRP -- PRP -- 2.0 -- Liquid
Example 2 Comparative -- PRP CaCl.sub.2 PRP + CaCl.sub.2 -- 1.8 0.2
Gel Example 3 Comparative Alg--Na -- CaCl.sub.2 CaCl.sub.2 .fwdarw.
Alg--Na 2.0 -- 1.0 Gel Example 4 Example 3 Alg--Fe PRP CaCl.sub.2
Alg--Fe + PRP + CaCl.sub.2 1.0 1.0 0.2 Gel + Particles Comparative
Alg--Fe -- -- Alg--Fe 2.0 -- -- Particles Example 5 Example 4
Alg--Ca PRP CaCl.sub.2 Alg--Ca + PRP + CaCl.sub.2 1.0 1.0 0.2 Gel +
Particles *Alg--Na: Sodium alginate Alg--Fe: Iron alginate Alg--Ca:
Calcium alginate PRP: Platelet-rich plasma CaCl.sub.2: Calcium
chloride
[0248] Evaluation
[0249] The effect of administrations to animals in Examples 1 to 4
and Comparative Examples 1 to 5 was evaluated in the following
manner.
[0250] One week or four weeks after administration, the Japanese
white rabbit was given an anesthetic by intravenous injection
through its auricular vein. The anesthetic is somnopentyl (sodium
pentobarbiturate) diluted twice with physiological saline, and its
dosage is 45 mg/kg (1 mL/kg).
[0251] The Japanese white rabbit under anesthesia underwent
laparotomy in dorsal position. Then, it underwent perfusion through
the heart with physiological saline (containing heparin, 10
units/mL, 100 mL/head), so that it was killed by bleeding from the
abdominal aorta. Finally, it had its lung extracted. Into the
extracted lung was injected (at a water-gauge pressure of 25 cm)
10% buffered formalin as a preserving and fixing solution for
pathologic tissues (which contains, in 100 mL, 10 mL of formalin
(35 to 38% aqueous solution of formaldehyde), 0.4 g of sodium
dihydrogenphosphate, and 0.65 g of sodium monohydrogenphosphate
anhydride, with the rest being purified water). For immersion
fixation, the lung was allowed to stand for 24 hours in the 10%
buffered formalin. Subsequently, specimens were prepared by
paraffin embedding and staining with hematoxyline-eosin (HE stain)
and masson trichrome (MT stain). The specimens were pathologically
examined under an optical microscope for fibrosis and granulomatous
inflammation in lung tissues. The specimens' optical
photomicrographs taken one week or four weeks after administration
are shown in FIGS. 1A to 8C, which are arranged in the order of
Examples 1 and 2, Comparative Examples 1 to 4, Example 3, and
Comparative Example 5.
[0252] Fibrosis
[0253] Fibrosis can be judged from the presence or absence of the
appearance of fibrocytes and the deposition of extracellular
organs. The rating of fibrosis was given according to the following
criteria.
-: Specimens show no fibrosis. .+-.: Specimens show fibrosis at 1
place. +: Specimens show fibrosis at 2 to 4 places. ++: Specimens
show fibrosis at 5 to 9 places. +++: Specimens show fibrosis at 10
places or more.
[0254] Granulomatous Inflammation
[0255] Granulomatous inflammation can be identified from the
presence or absence of focal lesion due to hyperplasia of
macrophages, multinucleated giant cells, lymphocytes, and fibrous
tissues. The rating of granulomatous inflammation was given
according to the following criteria. Incidentally, granulomatous
inflammation leads to fibrosis.
-: Specimens show no granulomatous inflammation. .+-.: Specimens
show granulomatous inflammation at 1 place. +: Specimens show
granulomatous inflammation at 2 to 4 places. ++: Specimens show
granulomatous inflammation at 5 to 9 places. +++: Specimens show
granulomatous inflammation at 10 places or more.
TABLE-US-00002 TABLE 2 One week Four weeks Fibrosis-causing
Granulomatous Granulomatous Agent* Fibrosis inflammation Fibrosis
inflammation Example 1 Alg-Na + PRP - ++ - + Example 2 CaCl.sub.2
.fwdarw. Alg-Na + PRP ++ ++ ++ ++ Comparative Alg-Na .+-. + - -
Example 1 Comparative PRP - - Example 2 Comparative PRP +
CaCl.sub.2 - - Example 3 Comparative CaCl.sub.2 .fwdarw. Alg-Na +
++ + - Example 4 Example 3 Alg-Fe + PRP + CaCl.sub.2 ++ ++ + +
Comparative Alg-Fe - + + - Example 5 Example 4 Alg-Ca + PRP +
CaCl.sub.2 +++ +++ +++ +++ *Alg-Na: Sodium alginate Alg-Fe: Iron
alginate Alg-Ca: Calcium alginate PRP: Platelet-rich plasma
CaCl.sub.2: Calcium chloride
[0256] The results shown in Table 2 indicate that the
fibrosis-causing agents pertaining to Examples 1 to 4 promote
fibrosis or granulomatous inflammation (prestage thereof) in lungs.
They also indicate that the fibrosis-causing agents pertaining to
Comparative Examples 1 to 5 bring about neither fibrosis nor
granulomatous inflammation in lungs, or they produce only limited
effects.
[0257] To be more specific, it was found that the fibrosis-causing
agent of Example 1 caused almost no fibrosis but remarkably caused
granulomatous inflammation one week and four weeks after
administration (FIGS. 1A to 1D). The noticeable granulomatous
inflammation suggests the possibility of fibrosis occurring in
extended periods. The fact that the effect decreases after one week
or four weeks is probably due to individual differences.
[0258] The fibrosis-causing agent of Example 2 is more effective
than that of Example 1 on account of combination with PRP gelling
agent (calcium chloride). In other words, noticeable fibrosis was
observed after one week and four weeks (FIGS. 2A to 2D). More
noticeable granulomatous inflammation than in Example 1 was also
observed after one week and four weeks (FIGS. 2E and 2F). This
enhanced effect is probably due to the addition of the gelling
agent which makes the fibrosis-causing agent less fluid in the
lung, permitting it to stay longer on cells.
[0259] The results in Comparative Examples 1 and 4 indicate that
sodium alginate has almost no effect on fibrosis (FIGS. 3A to 3C)
although it is slightly effective on granulomatous inflammation
(FIG. 3D). The combination of sodium alginate and PRP gelling agent
merely produces limited effects (FIGS. 6A to 6E).
[0260] The results in Comparative Examples 2 and 3 indicate that
platelet-rich plasma (PRP) induces no fibrosis (FIGS. 4 and 5).
[0261] Moreover, the results in Example 3 and Comparative Example 5
suggest that iron alginate induces fibrosis. It was also found that
iron alginate used alone is not so effective (FIGS. 8A to 8C) but
becomes effective when used in combination with platelet-rich
plasma (PRP) and PRP gelling agent (FIGS. 7A to 7E).
[0262] The results in Example 4 apparently indicate that an
extremely high effect of fibrosis is produced when calcium alginate
is used in combination with platelet-rich plasma (PRP) and PRP
gelling agent.
[0263] It should be understood by those skilled in the art that
various modifications, combinations, sub-combinations and
alterations may occur depending on design requirements and other
factors insofar as they are within the scope of the appended claims
or the equivalents thereof
* * * * *