U.S. patent application number 14/321175 was filed with the patent office on 2014-10-23 for cell preparation for erectile dysfunction or sensory disorders of the lower urinary tract containing adipose tissue derived mesenchymal stem cells.
The applicant listed for this patent is NATIONAL UNIVERSITY CORPORATION NAGOYA UNIVERSITY. Invention is credited to Yasuhito Funahashi, Momokazu Gotoh, Naoshi Koide, Yoshihisa Matsukawa, Yoshifumi Takei, Tokunori Yamamoto.
Application Number | 20140314724 14/321175 |
Document ID | / |
Family ID | 43856626 |
Filed Date | 2014-10-23 |
United States Patent
Application |
20140314724 |
Kind Code |
A1 |
Yamamoto; Tokunori ; et
al. |
October 23, 2014 |
CELL PREPARATION FOR ERECTILE DYSFUNCTION OR SENSORY DISORDERS OF
THE LOWER URINARY TRACT CONTAINING ADIPOSE TISSUE DERIVED
MESENCHYMAL STEM CELLS
Abstract
An object is to provide a novel medical use of adipose
tissue-derived mesenchymal stem cells. It was discovered that
adipose tissue-derived mesenchymal stem cells (ASC) promote
improvement of erectile dysfunction and recovery of the urge to
urinate (improvement of sensory disorders of the lower urinary
tract). On the basis of the discovery, a cell preparation
containing adipose tissue-derived mesenchymal stem cells, which is
effective to erectile dysfunction or sensory disorders of the lower
urinary tract, is provided. In one embodiment, adipose
tissue-derived mesenchymal stem cells and body fat are used in
combination.
Inventors: |
Yamamoto; Tokunori;
(Nagoya-shi, JP) ; Koide; Naoshi; (Nagoya-shi,
JP) ; Takei; Yoshifumi; (Nagoya-shi, JP) ;
Matsukawa; Yoshihisa; (Nagoya-shi, JP) ; Funahashi;
Yasuhito; (Nagoya-shi, JP) ; Gotoh; Momokazu;
(Nagoya-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL UNIVERSITY CORPORATION NAGOYA UNIVERSITY |
Nagoya-shi |
|
JP |
|
|
Family ID: |
43856626 |
Appl. No.: |
14/321175 |
Filed: |
July 1, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13500484 |
Jun 7, 2012 |
8808688 |
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PCT/JP2010/065271 |
Sep 7, 2010 |
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14321175 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61P 13/08 20180101;
A61K 35/28 20130101; A61P 15/00 20180101; A61K 35/28 20130101; A61P
43/00 20180101; A61K 35/35 20130101; A61K 35/30 20130101; C12N
5/0653 20130101; A61P 15/10 20180101; C12N 5/0667 20130101; A61P
13/02 20180101; C12N 5/06 20130101; A61K 35/35 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; C12N 5/0662 20130101; A61P
13/00 20180101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/30 20060101
A61K035/30; A61K 35/12 20060101 A61K035/12 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 6, 2009 |
JP |
2009-232068 |
Mar 12, 2010 |
JP |
2010-056522 |
Claims
1-12. (canceled)
13. A use of adipose tissue-derived mesenchymal stem cells for
producing a cell preparation for sensory disorders of the lower
urinary tract, the use comprising administering a cell preparation
comprising human adipose tissues-derived mesenchymal stem cells to
the external urethral sphincter muscle and/or to the membranous
urethra of external urethral sphincter muscle of a patient
suffering from sensory disorders of the lower urinary tract.
14. A use of adipose tissue-derived mesenchymal stem cells and body
fat, for producing a cell preparation for sensory disorders of the
lower urinary tract, the use comprising administering
therapeutically effective amounts of adipose tissue-derived
mesenchymal stem cells and body fat to the external urethral
sphincter muscle and/or to the membranous urethra of external
urethral sphincter muscle of a patient suffering from sensory
disorders of the lower urinary tract.
Description
TECHNICAL FIELD
[0001] The present invention relates to a cell preparation.
Specifically, the invention relates to a cell preparation effective
to improvement of erectile dysfunction or sensory disorders of the
lower urinary tract. The present application claims priority based
on the Japanese Patent Application No. 2009-232068 filed on Oct.
16, 2009 and priority based on the Japanese Patent Application No.
2010-056522 filed on Mar. 12, 2010, and the contents of the patent
applications are incorporated by reference in their entirety.
BACKGROUND ART
[0002] Stress urinary incontinence refers to a disease of leaking
urine without the urge to urinate in abdominal pressures (exercise,
cough, sneezing, etc.) due to functional disorder of the urethral
sphincter muscle having a function that tightens the urethra in
order not to leak urine from the bladder. Stress urinary
incontinence is a disease shown in both males and females, and
causes in a female are mainly sphincter muscle functional disorders
due to pregnancy and delivery, gynecological surgeries, and aging,
and causes in a male are sphincter muscle disorders due to
surgeries for prostate gland enlargement and prostate gland cancer.
Physical therapy (pelvic floor muscle training) is usually carried
out in an early stage of a treatment for stress urinary
incontinence, but is invalid in a case of having a moderate or
severer symptom. As a surgical treatment, the urethral sling
surgery (that is a vaginal operation, in which an artificial tape
is placed under the urethra to hold the urethra) is generally
broadly performed for female stress urinary incontinence and
favorable results have been attained, but the urethral sling
surgery has a defect such that a foreign material is left inside
the body and, for male stress urinary incontinence, there is no
effective surgical treatment that can be domestically performed.
For a minimally invasive surgical treatment, a treatment of
injecting bovine collagen into the periurethral zone (an endoscope
is transurethrally inserted and collagen is injected to the
membranous urethra through an endoscope) is carried out in some
cases, but a treatment continuing effect is not attained because of
recurrence within 1 to 3 months due to being absorbed within
several weeks after the injection, and also for stress urinary
incontinence after a prostate gland operation, an effectiveness
itself is not more than 20%, which is defective (Non-patent
Document 1). What is more, since collagen is derived from a bovine
tissue, there is a problem that a risk of development of
transferable spongiform encephalopathy cannot be completely
denied.
[0003] The disease has a very high prevalence rate and impairs
quality of life, and there is no minimally invasive, highly
effective therapeutic method, and therefore, there is an urgent
need to develop a minimally invasive, highly effective therapeutic
method. The urethral injection treatment using cultured autologous
skeletal muscle-derived stem cells has been already clinically
applied to a treatment for stress urinary incontinence abroad
(Non-patent Document 2), but because of difficulty of securing
cultured cells to be used in injection (culture method,
environment) and safety problems, development of a new noninvasive
surgical therapeutic method and clinical introduction have been
demanded.
[0004] A stress urinary incontinence treatment using adipose
tissue-derived mesenchymal stem cells (called Adipose-derived stem
cells: ASC, Adipose-derived regeneration cells: ADRC,
Adipose-derived mesenchymal stem cells: AT-MSC, AD-MSC, etc.) has
not been performed in Japan and abroad. It was reported that ASC is
injected into the periurethral zone of a rat to differentiate to a
smooth muscle (Non-patent Document 3), but no functional data
showing improvement of stress urinary incontinence has been shown.
The research group of the present inventors reported in the patent
applications mentioned above that cultured ASC is injected into the
periurethral zone using a stress urinary incontinent rat model to
thus obtain increase of a urethral internal pressure and
improvement of urinary incontinence (Patent Document 1).
PRIOR ART DOCUMENT
Patent Document
[0005] Patent Document 1: WO No. 2008/018450
Non-Patent Document
[0006] Non-patent Document 1: Smith D N, Appell R A, Rackley R R,
Winters JCCOLLAGEN INJECTION THERAPY FOR POST-PROSTATECTOMY
INCONTINENCE J Urol. 1998 August; 160(2): 364-7.
[0007] Non-patent Document 2: Smaldone M C, Chancellor M B. Muscle
derived stem cell therapy for stress urinary incontinence. World J
Urol. 2008 August; 26(4): 327-32. Epub 2008 May 10.
[0008] Non-patent Document 3: Jack G S, Almeida F G, Zhang R,
Alfonso Z C, Zuk P A, Rodriguez L V. Processed lipoaspirate cells
for tissue engineering of the lower urinary tract: implications for
the treatment of stress urinary incontinence and bladder
reconstruction. J Urol. 2005 November; 174(5): 2041-5.
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0009] An object of the present invention is to provide a novel
medical use of adipose tissue-derived mesenchymal stem cells
(ASC).
Means for Solving the Problem
[0010] The present inventors conducted a research to develop a new
treatment for stress urinary incontinence using autologous ASC
obtained from subcutaneous adipose tissues as a cell source, that
is, to develop a treatment of ASC periurethral injection through
the urethra under an endoscope, as an initial purpose. Firstly, a
cell injection method into the periurethral zone was studied as a
preliminary experiment. Specifically, a relationship between a
ratio of adipose tissues and ASC separated from adipose tissues in
mixing the both to be injected and the effect of lower urinary
obstruction (bulking effect) was examined. As a result, when the
ratio was set to 1:10 (the case of the maximum ratio of ASC in this
experiment), the largest effect of lower urinary obstruction was
observed and it was revealed that enhancing a mixing ratio of ASC
gives a preferable effect. Subsequently, therapeutic effects of ASC
were examined according to the following procedure on male patients
having stress urinary incontinence due to urethral sphincter muscle
disorder and showing no improvement with conventional treatments
(patients who experienced radical prostate gland extirpation for
prostate gland cancers or transurethral prostate gland excision for
prostate-gland enlargement) as subjects. For each patient, 250-300
g of adipose tissues were collected from the lower abdominal wall
or buttocks with a fat suction device and a thick cell solution
containing a necessary amount of adipose tissue-derived mesenchymal
stem cells was prepared using an adipose tissue-derived mesenchymal
stem cell separation device. Then, a part of the thick cell
solution was injected into the urethral sphincter muscle under
observation with an endoscope inserted through the urethra.
Subsequently, the collected residual thick cell solution and
autologous adipose tissues were mixed and injected to the
membranous urethra transurethrally (injection was continued until
the bladder neck is occluded with swelling). After the operation,
various examinations and questionnaires were periodically
conducted. As a result, increase of blood flow in the periurethral
zone, increase of a single urination amount, and decrease of a
urinary incontinence amount were observed in many patients. That
is, improvement of symptoms of urinary incontinence by ASC
transplantation was confirmed. On the other hand, surprisingly,
appearance of the urge to urinate was observed in one of the
patients after 1 month from the operation, and appearance of the
urge to urinate was observed even in 4 out of 5 patients after 3
months from the operation. Furthermore, as a more interesting
result, improvement of erectile dysfunction (ED) was observed even
in 4 out of 5 patients. Recovery of the urge to urinate and
improvement of erectile dysfunction dramatically enhance quality of
lives of patients having urinary incontinence, and the above
mentioned finding has an extremely important clinical meaning.
[0011] According to the above description, as a result of studies
made by the present inventors, findings which were never expected
at first, that is, improvement of the urge to urinate due to ASC
transplantation and improvement of erectile dysfunction, were
attained. On the other hand, as a result of carrying out an animal
experiment in order to evaluate characteristics of ASC after
transplantation, it was revealed that ASC was differentiated to
smooth muscle cells. In addition, the fact that ASC produces HGF
(stem cell growth factor), which is supposed to relate to urethral
sphincter muscle regrowth and differentiation and have an important
role and the fact that a HGF concentration increases only in an
early stage of regeneration to guarantee safety were confirmed.
What is more, also according to a transplantation experiment using
a large animal (pig) as a subject, effectiveness and safety of a
treatment using ASC were shown. The present inventions shown below
are based on the above mentioned findings.
[0012] [1] A cell preparation for erectile dysfunction or sensory
disorders of the lower urinary tract, containing adipose
tissue-derived mesenchymal stem cells.
[0013] [2] The cell preparation according to [1], further
containing body fat.
[0014] [3] The cell preparation according to [2], which is a kit
constituted with a primary element containing adipose
tissue-derived mesenchymal stem cells and a secondary element
containing body fat separated from a living body.
[0015] [4] The cell preparation according to [2], containing
adipose tissue-derived mesenchymal stem cells, wherein body fat
separated from a living body is administered in combination when
the cell preparation is administered.
[0016] [5] The cell preparation according to any one of [1] to [4],
wherein the adipose tissue-derived mesenchymal stem cells are cell
surface marker CD29 and CD44 positive.
[0017] [6] The cell preparation according to any one of [1] to [4],
wherein the adipose tissue-derived mesenchymal stem cells are
[0018] (1) adhesive cells or their passaged cells, which are
contained in a sedimented cell population sedimented when a cell
population separated from adipose tissues is subjected to
centrifugation under the conditions at 800-1500 rpm for 1-10
minutes,
[0019] (2) cells which are proliferated when the sedimented cell
population is cultured under the low-serum condition,
[0020] (3) cells which are proliferated when a cell population
separated from adipose tissues is cultured under the low-serum
condition,
[0021] (4) a sedimented cell population which is obtained by
treating adipose tissues with protease, thereafter subjecting to a
filtration treatment, and then centrifuging the filtrate to be
recovered as sediments, or
[0022] (5) a sedimented cell population which is obtained by
treating adipose tissues with protease, and then centrifuging the
treated adipose tissues to be recovered as a sediments without
undergoing a filtration treatment.
[0023] [7] The cell preparation according to [6], wherein the
low-serum condition is a condition that a serum concentration in a
culture solution is not more than 5% (V/V).
[0024] [8] The cell preparation according to any one of [1] to [7],
wherein the adipose tissues are human adipose tissues.
[0025] [9] A method for preparing a cell preparation for erectile
dysfunction or sensory disorders of the lower urinary tract,
including the following steps:
[0026] (1) a step of preparing adipose tissue-derived mesenchymal
stem cells and body fat; and
[0027] (2) a step of mixing the adipose tissue-derived mesenchymal
stem cells and the body fat.
[0028] [10] A use of adipose tissue-derived mesenchymal stem cells
for producing a cell preparation for erectile dysfunction or
sensory disorders of the lower urinary tract.
[0029] [11] A use of adipose tissue-derived mesenchymal stem cells
and body fat, for producing a cell preparation for erectile
dysfunction or sensory disorders of the lower urinary tract.
[0030] [12] A method of treating erectile dysfunction or sensory
disorders of the lower urinary tract, including administering the
cell preparation according to [8] to the external urethral
sphincter muscle and/or to the membranous urethra of external
urethral sphincter muscle of a patient suffering from erectile
dysfunction or sensory disorders of the lower urinary tract.
[0031] [13] A method of treating erectile dysfunction or sensory
disorders of the lower urinary tract, including administering a
therapeutically effective amount of adipose tissue-derived
mesenchymal stem cells to the external urethral sphincter muscle
and/or to the membranous urethra of external urethral sphincter
muscle of a patient suffering from erectile dysfunction or sensory
disorders of the lower urinary tract.
[0032] [14] A method of treating erectile dysfunction or sensory
disorders of the lower urinary tract, including administering
therapeutically effective amounts of adipose tissue-derived
mesenchymal stem cells and body fat to the external urethral
sphincter muscle and/or to the membranous urethra of external
urethral sphincter muscle of a patient suffering from erectile
dysfunction or sensory disorders of the lower urinary tract.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows results of measuring cell surface markers of
cells, which were prepared from adipose tissues. It is found to be
CD29 and CD44 positive.
[0034] FIG. 2 shows results of culturing cells which were prepared
from adipose tissues. The cells were cultured in an adipose
differentiation medium to induce differentiation.
[0035] FIG. 3 is a view showing a transurethral fat/ASC injection
procedure. Due to sphincter muscle disorder, the bladder neck and
the urethra were opened (a), but an ASC solution is injected into
the sphincter muscle, followed by injecting a mixed solution of
adipose tissues and ASC to membranous urethra (b), and the urethral
sphincter muscle part is adhered and occluded after the injection
(c).
[0036] FIG. 4 is a table showing a blood flow in the periurethral
zone (A), a single urination amount (B) and a urinary incontinence
amount/day (C) of each patient before and after the treatment.
[0037] FIG. 5 is a graph showing transitions of urinary
incontinence amounts of patients having cases 1 and 2 in a moderate
level of urinary incontinence amounts (cases 1 and 2 in order from
the left). The urinary incontinence amounts were monitored in a
24-hour pad test (evaluated with the average of continuous 3 to 4
days). Urinary incontinence disappears after 24 weeks (6 months) in
the case 1.
[0038] FIG. 6 is a graph showing transitions of urinary
incontinence amounts of patients having cases 3, 4 and 5 in severe
levels of urinary incontinence (cases 3, 4, and 5 in order from the
left). The urinary incontinence amounts were monitored in a 24-hour
pad test (evaluated by the average of continuous 3 to 4 days).
Improvements with time of urinary incontinence amounts continue in
the cases 3 and 4.
[0039] FIG. 7 is a graph showing a transition of QOL (quality of
life) of the patient in the case 1. Variation of ICIQ-SF
(International Consultation on Incontinence Questionnaire-Short
Form), which is an international urinary incontinence symptom/QOL
score, was examined.
[0040] FIG. 8 is a graph showing change of urethral functions in a
urethral internal pressure measurement. Left: graph showing
transitions of maximum urethral closure pressures (cases 1, 2, 3,
4, and 5 in order from the left). Maximum urethral closure
pressures before an operation, and after 2 weeks and 12 weeks from
the operation were compared. Right: graph showing transitions of
functional urethral length (cases 1, 2, 3, 4 and 5 in order from
the left). Urethral sphincter muscle functions in an objective test
are improved in every example. The maximum urethral closure
pressure reflects a closure pressure in the sphincter muscle part,
and the functional urethral length reflects the length of the
sphincter muscle part.
[0041] FIG. 9 shows results of ultrasonic contrast tests in
injection sites (case 1). Increased blood flow with time is
observed after the operation.
[0042] FIG. 10 shows MRI images (case 1) in the injection site. a:
before injection, b: 4 days after injection, c: 4 weeks after
injection, d: 12 weeks after injection. Injected autologous adipose
tissues exist also at the time of passing 12 weeks after
injection.
[0043] FIG. 11 is a table showing presence or absence of appearance
of the urge to urinate before and after a treatment for each
patient.
[0044] FIG. 12 is a table showing EHS scores before and after a
treatment for each patient. 0: The penis is not larger, 1: the
penis is larger but not hard, 2: the penis is hard but not hard
enough for penetration, 3: the penis is hard enough for penetration
but not completely hard, 4: the penis is completely hard and fully
rigid.
[0045] FIG. 13 is a graph showing change of urination states before
and after an operation. The graph shows changes of maximum urine
flow rates (left) and residual urine amounts (right) (cases 1, 2,
3, 4, and 5 in order from the left). In every case, the maximum
urine flow rate does not decrease (that is, the urination state
does not deteriorate), and increase of residual urine is not
observed. That is, the urination (urinary excretion) function is
not damaged.
[0046] FIG. 14 shows HE (Hematoxylin Eosin) stain images of ASC
injection sites (periurethral zone) of a rat. Magnifications are 40
in the left top, 200 in the right top, and 400 in the left
bottom.
[0047] FIG. 15 shows Masson trichrome stain images of ASC injection
sites (periurethral zone) of a rat. Magnifications are 40 in the
left top, 200 in the right top, and 400 in the left bottom.
[0048] FIG. 16 shows .alpha.SMA (smooth muscle) stain images of ASC
injection sites (periurethral zone) of a rat. Magnifications arc 40
in the left top, 200 in the right top, and 400 in the left
bottom.
[0049] FIG. 17 shows calponin type 1 stain images of ASC injection
sites (periurethral zone) of a rat. Magnifications are 40 in the
left top, 200 in the right top, and 400 in the left bottom.
[0050] FIG. 18 is a graph showing an HGF concentration in a culture
supernatant of human ASC.
[0051] FIG. 19 is a graph showing and a HGF concentration (day 3)
of an ASC injection site of a rat. ns: no significant
difference.
[0052] FIG. 20 is a graph showing a VEGF (vascular endothelial
growth factor) producing ability of rat adipose tissue-derived SVF
(stromal vascular fractions).
[0053] FIG. 21 shows tissue images showing cross sections of an ASC
injection site (periurethral zone) of a pig. (1) Macroscopic
observation. (2) Microscopic observation: magnifications are 20 in
the left top, 200 in the right top, and 400 in the left bottom.
[0054] FIG. 22 shows various stain images of ASC injection sites of
a pig. (1) HE stain, (2) Masson trichrome stain image, (3) desmin
stain image, (4) Ki-67 stain, (5) .alpha.SMA stain, (6) calponin
type 1 stain, (7) MHC (myosin heavy chain) stain, (8) S-100 stain,
(9) synaptophysin stain.
[0055] FIG. 23 is a graph showing therapeutic effects by ASC
periurethral injection into an ECM model rat. Urine leakage
pressures of an ASC treatment group, an ECM model group and a sham
operation group (sham group) after 2 weeks and 4 weeks from the
operation were compared for evaluation. The sham operation group,
ECM model group, and ASC treatment group in order from the left.
*p<0.001, #p<0.05
DESCRIPTION OF EMBODIMENTS
[0056] The present invention relates to a cell preparation applied
to a specific disease and a use thereof. The cell preparation of
the present invention contains adipose tissue-derived mesenchymal
stem cells (also may be abbreviated as "ASC" in the specification).
"The adipose tissue-derived mesenchymal stem cells (ASC)" in the
present invention refers to somatic stem cells that are contained
in an adipose tissue, and cells that are obtained by culture of the
somatic stem cells (including subculture) also correspond to "the
adipose tissue-derived mesenchymal stem cells (ASC)" as long as
such cells maintain multipotency. Generally, ASC is obtained from
an adipose tissue separated from a living body as a starting
material, and prepared into "an isolated state" as a cell that
constitutes a cell population (containing cells except for ASC,
which are originated from the adipose tissue). "An isolated state"
herein means that ASC is present in a state of being taken out from
its original environment (that is, a state of constituting a part
of a living body), in other words, a state of being different from
an original state of its existence due to artificial manipulation.
Note that adipose tissue-derived mesenchymal stem cells are also
called ADRC (adipose-derived regeneration cells), AT-MSC
(adipose-derived mesenchymal stem cells), AD-MSC (adipose-derived
mesenchymal stem cells), and so on. In the present specification,
the following terms, that is, adipose tissue-derived mesenchymal
stem cells, ASC, ADRC, AT-MSC, and AD-MSC are used
exchangeably.
(Preparation Method of ASC)
[0057] ASC is prepared through steps such as separation of stem
cells from a fat substrate, washing, concentration, and culture. A
preparation method of ASC is not particularly limited. For example,
ASC can be prepared according to, for example, known methods
(Fraser J K et al. (2006), Fat tissue: an underappreciated source
of stem cells for biotechnology. Trends in Biotechnology; April;
24(4): 150-4. Epub 2006 Feb. 20. Review.; Zuk P A et al. (2002),
Human adipose tissue is a source of multipotcnt stem cells.
Molecular Biology of the Cell; December; 13(12): 4279-95.; Zuk P A
et al. (2001), Multilineage cells from human adipose tissue:
implications for cell-based therapies. Tissue Engineering; April;
7(2): 211-28., and the like are served as references). Further, a
device for preparing ASC from adipose tissues (for example,
Celution (registered trademark) device (Cytori Therapeutics, Inc.,
USA, San Diego)) is also commercially available and ASC may be
prepared using the device. When the device is used, cells that are
cell surface marker CD29 and CD44 positive can be separated from
adipose tissues. Specific examples of a preparation method of ASC
are shown below.
(1) Preparation of Population of Cells from Adipose Tissue
[0058] Adipose tissue can be obtained from an animal by means such
as excision and suck. The term "animal" herein includes human and
non-human mammalians (pet animals, domestic animal, and
experimental animal. Specifically examples include mouse, rat,
guinea pig, hamster, monkey, cow, pig, goat, sheep, dog, cat, and
the like). In order to avoid the problem of immunological
rejection, it is preferable that adipose tissue is collected from
the same individuals as subjects (recipients) to which the cell
preparation of the present invention is to be administered.
However, adipose tissue of the same kinds of animals (other
animals) or adipose tissue heterogeneous animals may be used.
[0059] An example of adipose tissue can include subcutaneous fat,
offal fat, intramuscular fat, and inter-muscular fat. Among them,
subcutaneous fat is a preferable cell source because it can be
collected under local anesthesia in an extremely simple and easy
manner and therefore the burden to a patient in collection is
small. In general, one kind of adipose tissue is used, but two
kinds or more of adipose tissues can be used. Furthermore, adipose
tissues (which may not be the same kind of adipose tissue)
collected in a plurality of times may be mixed and used in the
later operation. The collection amount of adipose tissue can be
determined by considering the kind of donors or kinds of tissue, or
the necessary amount of ASCs. For example, the amount can be from
0.5 g-500 g. When a donor is human, it is preferable that the
collection amount at one time is about 10 g-20 g or less by
considering a burden to the donor. The collected adipose tissue is
subjected to removal of blood components attached thereto and
stripping if necessary and thereafter, subjected to the following
enzyme treatment. Note here that by washing adipose tissue with
appropriate buffer solution or culture solution, blood components
can be removed.
[0060] The enzyme treatment is carried out by digesting adipose
tissue with protease such as collagenase, trypsin and Dispase. Such
an enzyme treatment may be carried out by techniques and conditions
that are known to a person skilled in the art (see, for example, R.
I. Freshney, Culture of Animal Cells: A Manual of Basic Technique,
4th Edition, A John Wiley & Sones Inc., Publication).
Preferably, enzyme treatment is carried out by the bellow-mentioned
techniques and conditions. A cell population obtained by the
above-mentioned enzyme treatment includes multipotent stem cells,
endothelial cells, interstitial cells, blood corpuscle cells,
and/or precursor cells thereof. The kinds or ratios of the cells
constituting the cell population depend upon the origin and kinds
of adipose tissue to be used.
(2) Obtaining of Sedimented Cell Population (SVF Fraction: Stromal
Vascular Fractions)
[0061] The cell population is then subjected to centrifugation.
Sediments obtained by centrifugation are collected as sedimented
cell population (also referred to as "SVF fraction" in this
specification). The conditions of centrifugation are different
depending upon the kinds or amount of cells. The centrifugation is
carried out for example, at 800-1500 rpm for 1-10 minutes. Prior to
the centrifugation, cell population after enzyme treatment can be
subjected to filtration and tissue that has not been digested with
enzyme contained therein can be removed.
[0062] The "SVF fraction" obtained herein includes ASCs. Therefore,
the cell preparation of the present invention can be prepared using
the SVF fraction. That is, the SVF fraction is contained in one
embodiment of the cell preparation of the present invention. The
kinds or ratio of cells constituting the SVF fraction depend upon
the origin and kinds of adipose tissue to be used, conditions of
the enzyme treatment, and the like. The SVF fraction is
characterized by including CD34 positive and CD45 negative cell
population, and that CD34 positive and CD45 negative cell
population (International Publication WO2006/006692A1).
(3) Selective Culture of Adhesive Cells (ASC) and Recovery of
Cells
[0063] Other cell components (such as endothelial cells, stroma
cells, hematopoietic cells, and precursor cells thereof) are
contained in a SVF fraction other than ASC. Thus, in one embodiment
of the present invention, unnecessary cell components are removed
from the SVF fraction by carrying out the following selective
culture. Then, cells that are obtained as a result arc used in the
cell preparation of the present invention as ASC.
[0064] Firstly, a SVF fraction is suspended in an appropriate
medium, and then seeded on a culture dish and cultured overnight.
Floating cells (non-adhesive cells) are removed by replacement of a
medium. Then, culture is continued while suitable replacement of a
medium (for example, once per 3 days). Subculture is carried out
according to necessity. The passage number is not particularly
limited. Note that, for the culture medium, a medium for normal
animal cell culture can be used. Examples such as Dulbecco's
modified Eagle's Medium (DMEM) (NISSUI PHARMACEUTICAL, etc.),
.alpha.-MEM (Dainippon Seiyaku, etc.), DMED: Ham's F12 mixed medium
(1:1) (Dainippon Seiyaku, etc.), Ham's F12 medium (Dainippon
Seiyaku, etc.), and MCDB 201 medium (Research Institute for the
Functional Peptides) can be used. Media added with serums (fetal
bovine serum, human serum, sheep serum, etc.) or serum replacement
s (Knockout serum replacement (KSR), etc.) may also be used. The
adding amount of a serum or serum replacement can be set within the
range from 5% (v/v)-30% (v/v), for example.
[0065] Adhesive cells selectively survive and proliferate according
to the above mentioned operations. Next, the cells proliferated are
collected. The cells may he collected by routine procedures and,
for example, collected easily by enzyme treatment (treatment with
trypsin or Dispase) and then cells are scraped out by using a cell
scraper, a pipette, or the like. Furthermore, when sheet culture is
carried out by using a commercially available temperature sensitive
culture dish, cells may be collected in a sheet shape without
carrying out enzyme treatment. Use of thus collected cells (ASC)
makes it possible to prepare a cell preparation containing ASC at
high purity.
(4) Low-Serum Culture (Selective Culture in a Low-Serum Medium) and
Collection of Cells
[0066] In one embodiment of the present invention, the following
low-serum culture is carried out in place of or after (3) mentioned
above. Then, the cells obtained as a result are used as ASC in the
cell preparation of the present invention.
[0067] In low-serum culture, the SVF fraction (when this step is
carried out after (3), the cells that are collected in (3) are
used) is cultured under the low-serum conditions and a desired
multipotent stem cell (that is, ASC) is selectively proliferated.
Since the amount of serum to be used is small in the low-scrum
culture method, it is possible to use the serum of the subjects
(recipients) themselves to which the cell preparation of the
present invention is administered. That is to say, culture using
autoserum can be carried. By using autoserum, it is possible to
provide a cell preparation capable of excluding heterogeneous
animal materials from manufacturing process and being expected to
have high safety and high therapeutic effect. The "under low-serum
conditions" herein denotes conditions in which a medium contains
not more than 5% serum. Preferably, the sedimented cell population
is cultured in a culture solution containing not more than 2% (V/V)
serum. More preferably, the cells are cultured in a culture
solution containing not more than 2% (V/V) scrum and 1-100 ng/ml of
fibroblast growth factor-2.
[0068] The serum is not limited to fetal bovine serum. Human serum,
sheep serum, and the like, can be used. Preferably, the human
serum, more preferably the serum of a subject to whom the cell
preparation of the present invention is to be administered (that is
to say, autoserum) is used.
[0069] As the medium, a medium for culturing animal cells can be
used on condition that the amount of serum contained in the use is
low. For example, Dulbecco's modified Eagle's Medium (DMEM) (NISSUI
PHARMACEUTICAL, etc.), .alpha.-MEM (Dainippon Seiyaku, etc.), DMED:
Ham's:F12 mixed medium (1:1) (Dainippon Seiyaku etc.), Ham's F12
medium (Dainippon Seiyaku, etc.), MCDB201 medium (Research
Institute for the Functional Peptides), and the like, can be
used.
[0070] By culturing by the above-mentioned method, multipotent stem
cells (ASCs) can be selectively proliferated. Furthermore, since
the multipotent stem cells (ASCs) proliferated in the
above-mentioned culture conditions have a high proliferation
activity, it is possible to easily prepare cells necessary in
number for the cell preparation of the present invention by
subculture. Note here that cells selectively proliferated by
low-serum culture of SVF fraction is CD13, CD90 and CD105 positive
and CD31, CD34, CD45, CD106 and CD117 negative (International
Publication WO2006/006692A1).
[0071] Subsequently, selectively proliferated cells by the
above-mentioned low-serum culture are collected. A collection
operation may be carried out in the same manner as in the case of
(3). Use of thus collected cells (ASC) makes it possible to prepare
a cell preparation containing ASC at high purity.
(5) Formulation
[0072] Cells of a SVF fraction, cells obtained as a result of the
above-mentioned selective culture (3), or cells obtained as a
result of the above-mentioned low-serum culture (4) are suspended
into physiological serine, an appropriate buffer solution (for
example, phosphoric acid buffer solution), or the like and the cell
preparation can be thus obtained. For example,
1.times.10.sup.6-1.times.10.sup.10 cells may be contained as an
amount of single administration so that a pharmaceutically
effective amount of cells is administered. A content of cells can
be suitably adjusted by considering an intended use, objective
disease, and sex, age, body weight, condition of affected area and
condition of cells of a subject to be applied (recipient).
[0073] The cell preparation of the present invention may include,
for example, dimethylsulfoxide (DMSO), serum albumin, and the like,
for protecting the cells; antibiotic and the like for inhibiting
contamination of bacteria; various components (vitamins, cytokines,
growth factors steroids and the like) for activation, proliferation
or differentiation of cells. Examples of cytokines include
interleukin (IL), interferon (IFN), colony stimulating factor
(CSF), granulocyte-colony stimulating factor (G-CSF), and
erythropoietin (EPO), and activin, oncostatin M (OSM). Note that
CSF, G-CSF, EPO, and the like are also growth factors. On the other
hand, examples of the growth factors include hepatocyte growth
factor (HGF), basic fibroblast growth factor (bFGF, FGF2),
epidermal growth factor (EGF), platelet-derived growth factor
(PDGF), insulin-like growth factor (IGF), transforming growth
factor (TGF), nerve growth factor (NGF) and brain-derived
neurotrophic factor (BDNF). Furthermore, the cell preparation of
the present invention may contain pharmaceutically acceptable other
components (for example, carrier, excipient, disintegrating agents,
buffer, emulsifier, suspension, soothing agent, stabilizer,
preservatives, antiseptic, physiologic saline, etc.).
[0074] In the above-mentioned method, the cell preparation is
formed by using cells proliferated by low-serum culture of SVF
fraction. However, the cell preparation may be directly formed by
the low-serum culture of cell population obtained from adipose
tissue (without carrying out centrifugation for obtaining SVF
fraction). That is to say, in one embodiment of the present
invention, cells proliferated by the low-serum culture of cell
population obtained from adipose tissue are used as low-serum
culture ASCs. A cell preparation may also be constituted by
directly using a SVF fraction (containing adipose tissue-derived
mesenchymal stem cells), not using multipotent stem cells that are
obtained according to selective culture ((3) and (4) mentioned
above). The cell preparation in this embodiment contains (a) a
sedimented cell population (SVF fraction) that is collected as
sediments by treating adipose tissues with protease, thereafter
being subjected to a filtration treatment, and then centrifuging
the filtrate, or (b) a sedimented cell population (SVF fraction)
that is collected as sediments by treating adipose tissues with
protease and then centrifuging the treated adipose tissues without
undergoing a filtration treatment. In addition, "directly using"
herein means that a SVF fraction is used as an active ingredient of
the cell preparation without undergoing selective culture.
(Combination Use of ASC and Body Fat)
[0075] In one embodiment, the cell preparation of the present
invention contains body fat in addition to ASC. That is, ASC and
body fat that is separated from a living body are used in
combination in this embodiment. For example, 10-20 g of body fat is
used as an amount of single administration so that a
pharmaceutically effective amount of fat is administered.
[0076] The wording of "adipose tissue-derived mesenchymal stem
cells and body fat are used in combination" or "the cell
preparation is obtained by using adipose tissue-derived mesenchymal
stem cells and body fat in combination" means that adipose
tissue-derived mesenchymal stem cells and body fat are used
concomitantly. Typically, the cell preparation of the present
invention is provided as a compounding agent that is obtained by
mixing a cell population containing ASC and body fat. For example,
the cells of the present invention are suspended into physiological
serine, an appropriate buffer solution (for example, phosphoric
acid buffer solution), or the like to obtain a cell suspension and
body fat may be mixed into the cell suspension. An embodiment of
combination use is not particularly limited to the above-mentioned
example (that is, a compounding agent) and, for example, the cell
preparation of the present invention can be provided in a form of a
kit that is constituted with the primary element containing ASC and
the secondary element containing body fat. In this case, the both
elements are administered to a recipient simultaneously or after
taking a predetermined interval of time. Preferably, the both
elements are administered simultaneously. "Simultaneously" herein
does not demand strict simultaneity. Therefore, the concept of
"simultaneously" includes the case that administration of the both
elements are carried out under the condition of no substantial time
lag, such as administering one element and then administering the
other element quickly, as a matter of course, including the case
that the both elements are administered under the condition of no
time lag, such as mixing the both elements and then administering
the mixed elements to a recipient. On the other hand, in the case
of administering one element and then administering the other
element after taking a predetermined interval of time, it is
preferred that a time lag is set as short as possible so that an
effect of the combination use is favorably exerted. For example,
after administration of one element, the other element is
administered within 15 minutes, preferably within 10 minutes, and
more preferably within 5 minutes.
[0077] A cell preparation containing ASC is prepared and body fat
may be administered in combination at the time of administering the
cell preparation. The timing of administering the cell preparation
and body fat in this case is in the same manner as in the case of
the embodiment of the kit mentioned above. That is, although the
both elements are preferably administered simultaneously, the both
elements may be administered in a predetermined time lag.
Furthermore, opposite to the above-mentioned embodiment, a cell
preparation containing body fat is prepared and ASC may be
administered in combination at the time of administering the cell
preparation. The timing of administration in this case is followed
by the case of the above-mentioned embodiment.
[0078] Examples of body fat include subcutaneous fat, visceral fat,
intramuscular fat, and intermuscular fat. Among them, subcutaneous
fat is particularly preferable because it can be collected under
local anesthesia in an extremely easy manner. A preparation method
of adipose tissues is followed by the description in the section
(1) mentioned above.
(Diseases to be Applied)
[0079] A disease to be treated or prevented with the cell
preparation of the present invention is erectile dysfunction or
sensory disorders of the lower urinary tract. In other words, the
cell preparation of the present invention is used in prevention or
treatment of erectile dysfunction or prevention or treatment of
sensory disorders of the lower urinary tract. Therefore, the cell
preparation of the present invention is generally administered to a
patient suffering from erectile dysfunction (or a potential
patient) or a patient suffering from sensory disorders of the lower
urinary tract (or a potential patient). However, the cell
preparation of the present invention also can be used for the
purpose of experiments and researches to confirm and verify its
effects.
[0080] "Erectile dysfunction (ED)" is one kind of male sexual
functional disorders and refers to "a condition that satisfactory
sexual intercourse cannot be performed since sufficient erection is
not attained during sexual intercourse or sufficient erection
cannot be maintained". Erectile dysfunction (hereinafter also
referred to as "ED") is also called "erectile functional disorder"
or "erectile disorder". ED is classified into the mild type, the
moderate type, and the complete type according to its severity.
Furthermore, ED is divided into the organic factor (caused by
arterial sclerosis, nerve damage, etc.), the psychological factor
(caused by psychological stress), and the mixed factor (generated
by combining the both elements of the organic factor and the
psychological factor) according to causes.
[0081] "Sensory disorders of the lower urinary tract" refers to a
condition of no occurrence of the urge to urinate in need or a
condition of a low degree of the urge to urinate even with
occurrence of the urge to urinate. Sensory disorders of the lower
urinary tract becomes a cause of urinary incontinence, urinary
retention, etc. Sensory disorders of the lower urinary tract is
caused by abnormality of the urinary tract, benign pro static
hypwertrophy (BPH), neurogenic bladder, and the like. Sensory
disorders of the lower urinary tract may occur after an operation
of total extirpation of the prostate gland or due to neurogenic
bladder as aftereffects of diabetes and cerebrovascular disorder.
Amelioration of sensory disorders of the lower urinary tract brings
improvement of quality of life.
(Subjects to be Applied)
[0082] A subject to which the cell preparation of the present
invention is administered is typically a human. However, the cell
preparation can also be constituted for non-human mammalians (pet
animals, domestic animals, and experimental animals, specifically
including, for example, a mouse, rat, guinea pig, hamster, monkey,
cow, pig, goat, sheep, dog, cat, and the like). It has been
observed that ED-like symptoms are rarely shown in dogs and horses
for breeding; for example, the cell preparation of the present
invention can be used also in such cases.
(Administration Method)
[0083] The cell preparation of the present invention is preferably
administered into an affected area by local injection. An injection
site is typically the external urethral sphincter muscle or
membranous urethra. The cell preparation is preferably injected
into both of these two sites, and in this case, the cell
preparation that does not contain adipose tissues (that is, the
cell preparation containing only ASC as the essential component)
may be applied to the external urethral sphincter muscle, and the
cell preparation that contains adipose tissues (that is, the cell
preparation containing ASC and adipose tissues as essential
components) may be applied to membranous urethra. One of intentions
of using the cell preparation that contains adipose tissues lies in
exhibiting an effect of lower urinary obstruction by adipose
tissues. Therefore, injection may be continued until the bladder
neck is occluded by swelling in order to preferably exhibit an
effect of lower urinary obstruction in injection to membranous
urethra. An example of an administration amount (total amount) of
the cell preparation is shown to be, for instance, 0.2 ml-2 ml, and
preferably 1.5 ml-2 ml for application to the external urethral
sphincter muscle, and for example, 2 ml-20 ml, and preferably 15
ml-20 ml (ratio of 1:10) for application to membranous urethra.
Note that the cell preparation may be injected into 2 or more sites
in plural times for both of the application to the external
urethral sphincter muscle and the application to membranous
urethra.
[0084] Administration schedule may be formed by considering a sex,
age, body weight and condition of a subject (patient). The cell
preparation may be administered in plural times serially or
periodically, other than single administration. An administration
interval in plural administrations is not particularly limited and,
for example, 1 day to 1 month. The administration number is not
particularly limited. Examples of the administration number are 2
to 10.
EXAMPLES
1. Study of Cell Injection Method
1-1. Method
(1) Separation of Adipose Tissue-Derived Mesenchymal Stem Cells
(ASC)
[0085] Subcutaneous fat (human) was finely vacuumed with a fat
suction tube after subcutaneous injection of an extracellular
fluid. Then, 260 g out of 300 g of the collected adipose tissues
was injected into an adipose-derived stem cell separation device
(Celution (registered trademark) device) for cell separation, and
then the device was operated according to the instruction for use
to separate stem cells. The separated stem cells were collected
from the device using a syringe. On the other hand, adipose tissues
(about 20 g) for mingling with the separated stem cells were
washed. Note that a disposable set is mounted in the Celution
(registered trademark) device to be able to treat the adipose
tissues in each case. A treatment of detaching stem cells from the
fat substrate was performed by the adipose-derived stem cell
separation device, and in the treatment, the stern cells were
washed and concentrated after being separated. This real time
treatment was performed in a closed environment for minimizing a
risk of contacting with a contaminant and completed within a time
of one surgical treatment.
(2) Study of Mixing Ratio of ASC and Adipose Tissues
[0086] The following test samples (2 cc each) were prepared, and
each test sample was injected subcutaneously into a nude rat with a
18 G needle to compare an effect of lower urinary obstruction
(bulking effect).
[0087] Test sample a: mixture of the separated stem cells and human
adipose tissues in a ratio of 1:0 (that is, only adipose
tissues)
[0088] Test sample b: mixture of the separated stem cells and human
adipose tissues in a ratio of 1:1 (that is, stem cells collected
from 1 g of adipose tissues per 1 g of adipose tissues were
used)
[0089] Test sample c: mixture of the separated stem cells and human
adipose tissues in a ratio of 1:10 (that is, stem cells collected
from 10 g of adipose tissues per 1 g of adipose tissues were
used)
1-2. Results
[0090] ASC was separated from adipose tissues for the purpose of
applications to clinical cases. ASC can be collected as a solution
of a cell component slightly mixed with 5 ml of erythrocytes. An
ischemic ulcer was observed in the center due to decrease of the
total amount of phyma and insufficient vascularization in the test
sample a (only human adipose tissues). A ratio of adipose tissues
and ASC to form phyma in the interior of the urethra and the most
effectively provoke an effect of lower urinary obstruction was
found to be 1:10.
2. Evaluation of Characteristics of Prepared ASC
[0091] Characteristics of cells prepared from human adipose tissues
using the Celution (registered trademark) device were evaluated.
Firstly, a sample immediately after separation of FACS was
hemolyzed with addition of ammonium chloride, and then CD29 and
CD44 that are mesenchymal stem cell markers were measured. As a
result, the sample showed to be CD29 and CD44 positive (FIG.
1).
[0092] On the other hand, cells prepared using the Celution
(registered trademark) device were cultured with an adipose
differentiation medium containing insulin, dexamethasone,
indomethacin, and 3-isobutyl-1-methyl-xanthine (IBMX). Primary
cells showed a fibroblastic form, and on day 4, 70 to 80% of the
cells became confluent. In addition, formation of fat droplet was
observed on day 7 from induction of fat differentiation and stained
with Oil-Red (FIG. 2). As described above, differentiation into
adipose cells was confirmed.
3. Study of Therapeutic Effect of ASC
[0093] The following study was carried out in order to find new
therapeutic uses of ASC collected from adipose tissues.
3-1. Method
(1) Patient (Subject to be Tested)
[0094] Patients suffering from stress urinary incontinence, who
visit the department of urology in the Nagoya university hospital,
were subjects to be tested. All patients experienced radical
prostate gland extirpation for prostate gland cancers, and have
difficult cases of continuing stress urinary incontinence for 1
year or more after the operations. Ages of the patients were 73-83
years old (75.6 years old on average).
(2) Collection of Subcutaneous Adipose Tissues
[0095] A suitable amount of a mixed solution [components:
physiological serine 1000 ml+1% lidocaine (xylocaine) 2 ml+0.1%
adrenaline (bosmin) 1.5 ml+8.4% meylon 10 ml] was injected into
patients' abdominal or gluteal subcutaneous adipose tissues under
general anesthesia, or local and lumbar anesthesia to be
sufficiently distended. A suspension containing adipose tissues was
vacuumed with negative pressure using a special syringe usually
used in the plastic surgery field. In the case of injection under
lumbar anesthesia, when a patient further had algia, intravenous
anesthesia was added for pain relief.
(3) Treatment of Subcutaneous Adipose Tissues (Separation of
ASC)
[0096] ASC was separated and concentrated from about 250-300 g of
the adipose tissues obtained above with an ASC separation device
(Celution (registered trademark) device) (about
1.times.10.sup.6-1.times.10.sup.8/5 ml). Firstly, the collected
adipose tissues were injected into a sterilized disposable set and
washed. Then, a melting treatment was carried out on the adipose
tissues, and an enzyme (Celase.TM.) which separates cells was added
to carry out a digestive treatment and centrifugation of a cell
suspension and washing of the enzyme were automatically performed
in a closed circuit. The collection, adjustment, and
transplantation were performed in an operation room in the Nagoya
university hospital. The cleanliness was in the class of 100-10000,
and clean environment was maintained in all procedures.
(4) Transplantation of ASC into the Periurethral Zone
[0097] Two kinds of injection cell solutions, that is, (a) a cell
solution with 0.5-1.0 ml of ASC and (b) a cell solution obtained by
mixing 20 g of autologous fat and 4.0-4.5 ml of ASC were prepared
using ASC (about 1.times.10.sup.6-1.times.10.sup.8) separated into
5 ml. An endoscope was inserted from the urethra under general
anesthesia, or local and lumbar anesthesia and the above mentioned
2 types of the injection cell solutions were injected into each
patient using a 18 G needle injection device under the endoscope.
That is, 0.5-1.0 ml of the solution (a) was injected into two sites
each of the right and left sides of the external urethral sphincter
muscle and the solution (b) was injected to membranous urethra (4,
8, 6 hours) according to cases in a level that occlusion of the
interior of the urethra due to the effect of lower urinary
obstruction (bulking effect) can be endoscopically observed (FIG.
3).
(5) Test Method and Evaluation Method
[0098] In order to examine therapeutic effects and its duration,
predetermined tests (measurement of a periurethral blood flow,
measurement of single urination amount, test of degree of urinary
incontinence, test of urethra functions, MRI test, ultrasound
examination, etc.) were carried out before a treatment, and after 2
weeks, 1 month, and 3 months from the treatment. Conditions of
urinary incontinence and influence on life were also examined by a
questionnaire (opinionaire). Further, an evaluation (subjective
observation) by ICIQ-SF (urinary incontinence symptom/QOL score:
urinary incontinence frequency, urinary incontinence amounts, and
influence on daily life are evaluated in score with a self
completed questionnaire whose validity has been testified), and as
an objective observation, a 24-hour pad test and a urine flow
dynamic test (the urethral internal pressure measurement; maximum
urethral closure pressure, functional urethral length) were
performed. In the 24-hour pad test, a weight of a pad was measured
every time when urinary incontinence was shown (a weight of a dry
pad is subtracted) and a urinary incontinence amount for 24 hours
was quantitatively measured. The measurement was continued for 4
days and the mean value was calculated as a 1-day urinary
incontinence amount.
3-2. Results
[0099] Results of each test are shown in FIGS. 4 to 12. Increase of
the periurethral blood flow (the periurethral zone/pelvic floor
muscle group contrast effect) is observed in most of the patients
(FIG. 4A). In addition, increase of a single urination amount was
observed in 4 out of 5 patients (FIG. 4B). Similarly, urinary
incontinence amounts were decreased in 4 out of 5 patients (FIG.
4C). Transition of urinary incontinence amounts after operations
was monitored by the 24-hour pad test for a long period of time; as
a result, excellent improvement effects were observed (FIGS. 5 and
6). Further, in evaluation by ICIQ-SF, significant improvements of
urinary incontinence amounts, urinary incontinence frequency, and
QOL were attained (FIG. 7). In the urethral internal pressure
measurement, increase of the maximum urethral closure pressures
(case 1: from 28 cm H.sub.2O to 43 cm H.sub.2O, case 2: from 21 cm
H.sub.2O to 36 cm H.sub.2O) (FIG. 8, left) and increase of
functional urethral lengths (case 1: from 14 mm to 32 mm, case 2:
from 17 mm to 27 mm) (FIG. 8, right) were observed, and improvement
of urethral resistance was objectively confirmed. In the blood flow
evaluation in the ADRC periurethral injection site by the contrast
ultrasound examination, blood flow was increased from the next day
after injection as compared to before injection, and blood flow in
the injection site was increased with time by 56 days after
injection (FIG. 9). Autologous adipose tissues that were injected
to membranous urethra did not disappear due to absorption even at
the time of 3 months after injection and continued to exist in the
injection site (FIG. 10).
[0100] On the other hand, surprisingly, appearance of the urge to
urinate was observed in one of the patients after 1 month from the
operation (FIG. 11), and appearance of the urge to urinate was
observed even in 4 out of 5 patients after 3 months from the
operation. In addition, a more interesting result was obtained with
respect to the index of erectile dysfunction (EHS scores). That is,
improvements of erectile dysfunction were observed even in 4 out of
5 patients (FIG. 12).
[0101] As a result of examining changes of urination states before
and after the operation, disorder of urination functions was not
observed in all cases (FIG. 13).
[0102] As a result of transplantation of ASC, in addition to
improvement of symptoms of urinary incontinence, recovery of the
urge to urinate (improvement of sensory disorders of the lower
urinary tract) and improvement of erectile dysfunction were
observed as described above. The latter two effects are not
predictable at all at first and bear mentioning. Recovery of the
urge to urinate and improvement of erectile dysfunction
dramatically enhance quality of life of a patient suffering from
urinary incontinence. Findings of such effects caused by ASC have
an extremely important clinical meaning. In particular, improvement
of erectile dysfunction can be recognized as an extremely
surprising effect when considering ages of patients (73 to 83 years
old).
[0103] Note that, in both of two cases (cases 1 and 2) performed
this time, improvements of urinary incontinence progressed with
time, and particularly in the case of disappearance of urinary
incontinence, urinary incontinence was progressively improved and
disappeared in 24 weeks (6 months), and a significantly increased
blood flow in the injection site was observed with time by the
contrast ultrasound examination, and thus, improvement of the
urethral sphincter muscle function is not only because of the
bulking effect simply due to injection of adipose tissues, but also
can be considered to deeply relate to the regeneration mechanism.
Collection of subcutaneous adipose tissues can be performed in a
standard minimally invasive method in a plastic surgery that is fat
suction, ASC collection from adipose tissues can be safely carried
out in a short time by using a separation device without requiring
a culture step, and in further serial treatment procedure
processes, collection of subcutaneous fat, collection of ASC,
transurethral injection of collected ADRCs into the periurethral
zone can be performed. Accordingly, an injection treatment of
autologous ASC into the periurethral zone is a highly safe and
useful clinical regeneration therapy as a new non-invasive surgical
treatment for stress urinary incontinence, and is considered to
have high medical significance. Stress urinary incontinence after a
prostate gland operation is very intractable as compared to female
stress urinary incontinence, and by considering that effectiveness
was attained in two cases in this time, effectiveness of ASC to
female stress urinary incontinence is also suggested.
4. Evaluation of ASC Characteristics After Transplantation
[0104] In order to confirm that ASC after transplantation was
differentiated to smooth muscle cells, ASC (ASC prepared from Green
rat subcutaneous fat) was injected into the periurethral zone of a
nude rat and evaluated after 28 days in various stain methods.
Results are shown in FIGS. 14 to 17. In HE stain, a comparatively
differentiated swollen cell clump was observed in the interior of
the urethra (FIG. 14, arrows). In addition, muscular tissues
stained into red with Masson trichrome stain were observed,
corresponding to the swollen sites of the interior of the urethra
that was HE stained (FIG. 15, arrows). On the other hand,
corresponding to the swollen sites of the interior of the urethra
of muscular tissues that were Masson trichrome stained, smooth
muscle tissues were shown with .alpha.SMA stain and calponin type1
stain (FIGS. 16 and 17). As described above, the ASC periurethral
injection site was shown as a swollen cell clump that was
comparatively differentiated in the interior of the urethra. The
injection sites were stained as muscular tissues and further
stained with .alpha.SMA stain with which immature to mature smooth
muscle tissues are stained and which reflects smooth muscle
tissues, and calponin type1 stain with which moderately matured
smooth muscle tissues are stained; on the other hand, the sites
were not stained with MHC (myosin heavy chain) with which matured
smooth muscle tissues are stained (no data is shown), and thus, it
was presumed that the injected cells are differentiated into smooth
muscle tissues and do not reach matured smooth muscle yet.
5. Study of Cytokine Producing Ability of ASC
[0105] Human SVF was collected and subculture was performed
(preparation of human ASC). After culturing 6 passages, a medium
was replaced with a medium containing 10% FBS and the medium was
recovered after 24 hours. A HGF concentration in the medium was
measured as well as calculating the cell number. As a result, it
was confirmed that about 10 ng of HGF per 10.sup.6 cells is
produced (FIG. 18).
[0106] Then, 3.times.10.sup.6 of the above-mentioned cells (human
ASC) after subculture were injected into the interior of the
urethra wall of a 7 week old female nude rat. Then, the nude rat
was sacrificed on day 3, and the HGF (hepatocellular growth factor)
concentration of the injection site was measured. As a result, the
periurethral tissue concentration of HGF produced from the injected
cells reached a peak on day 3 after cell injection (the tissue
concentration was about 300 pg per 1 mg) and decreased to the
detection limit or less from the next day, day 4 (FIG. 19).
[0107] On the other hand, SVF separated from rat adipose tissues
was cultured (using a medium containing 20% FBS and no growth
factor) to measure a VEGF concentration in the culture supernatant.
As a result, the VEGF concentration in the culture supernatant was
increased under both of the normal oxygen condition and the low
oxygen condition (FIG. 20), and it was confirmed that ASC produces
VEGF.
[0108] As described above, it was confirmed that ASC produces HGF
and, at the same time, a HGF concentration in the ASC
transplantation site reaches a peak in a few days after
transplantation and then disappears. That is, the fact that ASC
produces HGF that is supposed to relate to urethral sphincter
muscle regrowth and differentiation and have an important role and
the fact that a HGF concentration increases only in an early stage
of regeneration to guarantee safety were confirmed. In addition, it
was confirmed that ASC also produces VEGF.
6. Preclinical Study Using a Large Animal
[0109] Autologous ASC was extracted from a subject that was a pig
as a large animal with a cell separation device and injected into
the periurethral zone. Effectiveness was evaluated in various stain
methods after 28 days from cell injection. In addition, safety was
also studied.
(1) Injection Operation of Autologous ASC into the Periurethral
Zone
[0110] The pig was in a supine position under general anesthesia.
Bosmin-containing physiological saline (100 ml) was subcutaneously
injected in the same procedure as being performed in a clinical use
and 100 g of autologous adipose tissues was suctioned with a 18 G
subcutaneous fat suction tube. ASC was extracted from the tissues
with an ASC separation device (Celution (registered trademark)
device) (5 ml solution). Simultaneously, entering the pelvic cavity
by lower abdominal median incision (A-1), the bladder neck was
peeled and exposed. The cell-containing solution was injected in
plurality of times into the peritubular zone at a site of about 2
cm distal from the urethra of the bladder neck. No breeding was
confirmed and the surgical site was sutured with a subcutaneous
(absorbable thread; Vicryl 3-0) skin (nonabsorbable thread; silken
thread 1-0) to complete the operation.
(2) Macroscopic and Microscopic Observations in Periurethral
Injection Site on Day 28
[0111] It was observed that the interior of the urethra had a
swollen (FIG. 21 (1)) fusiform cell structure in the palisade
pattern, which corresponded to the injection site (FIG. 21
(2)).
(3) Observations of Immunostaining in Injection Site (Day 28)
[0112] In the HE stain, a swollen, differentiated cell clump is
observed in the interior of the urethra (FIG. 22 (1)). As shown by
the arrow, muscular tissues that are turned red with Masson
trichrome stain were observed, corresponding to a swelling site of
the HE stained interior of the urethra (FIG. 22 (2)). A smooth
muscle or striated muscle-like structure which is stained with
desmin radially from the outside of the swelling site of a Masson
trichrome stained muscular tissue component in the interior of the
urethra is observed (FIG. 22 (3)). Corresponding to the structure,
a cell structure stained with Ki-67 that reflects increase of cell
proliferation is observed (FIG. 22 (4)). The site was stained with
three smooth muscle antibodies, .alpha.SMA (FIG. 22 (5)), calponin
type1 stain (FIG. 22 (6)) and MHC (myosin heavy chain) (FIG. 22
(7)), which specifically stain smooth muscle tissues. The site was
also stained with two antibodies, S-100 (FIG. 22 (8)) and
synaptophysin (FIG. 22 (9)), which reflect nerve cells.
[0113] As described above, the ASC periurethral injection site was
shown as a matured cell clump that is swollen in the interior of
the urethra. The site was stained as a muscular tissue, and further
stained with .alpha.SMA stain which reflects smooth muscle tissues
and stains immature to mature smooth muscle tissues, and calponin
type1 stain which stains moderately matured smooth muscle tissues,
and additionally, MHC which stains matured smooth muscle tissues,
and the smooth muscle component was stained with the three
antibodies. Furthermore, it was presumed that not only smooth
muscles but also nerves that are stained with S-100 and
synaptophysin, which reflect nerve cells, and control functions are
grown in the periphery and the interior. As compared to a small
animal, a bump that was projected in the interior of the urethra of
a large animal was constituted with smooth muscle cells also on 28
days from cell injection.
(4) Postoperative Progress
[0114] Voided urine was shown from the same day of the operation
and, no development of a complication of complete urinary retention
was observed.
[0115] Then, voided urine continued smoothly and there was no need
for a urethral catheterization treatment. Postoperative general
conditions were also preferable and transition of the body weight
was from 33.9 to 48.9 kg and increased by 14.1 kg in 28 days
(nutritious condition after cell injection; body weight
change).
(5) Observations in Blood and Macro and Micro Observations in Major
Organs After 28 Days from Cell Injection
[0116] As a result of a blood test, there were no anemia and
malnutrition and abnormality was not observed in peripheral blood
and biochemical data. On the other hand, macroscopic observations
of extracted organs are as follows and no abnormality was
observed.
[0117] Skin, muscle; no tumor/inflammation/circulatory disorder
[0118] Brain: no abnormality in the color of spinal fluid, no
tumor/inflammation/circulatory disorder in brain surface and cut
surfaces
[0119] Pleural cavity: lung; no pleural
effusion/adherence/tumor/inflammation, no bleeding/stasis
[0120] Heart; normal pericardial fluid, no adherent pericardium, no
bleeding in epicardium
[0121] Abdominal cavity: no ascitic fluid/adherence, no
tumor/inflammation/circulatory disorder
[0122] Digestive tract/liver/gallbladder/pancreas/spleen: no
tumor/inflammation/circulatory disorder
[0123] Retroperitoneum organs; for kidney, ureter, bladder,
prostate gland, seminal vesicle and penis, including the bladder
neck peeled for cell injection and the periurethral zone subjected
to cell injection, an observation of a trace of needle injection,
adherence, tumor, inflammation, and circulatory disorder was not
found.
[0124] In addition, microscopic studies on the above described
organs were made in HE stain, and an observation that suggests
tumor, inflammation, and circulatory disorder was not found.
(6) Summary
[0125] Autologous ASC injection operation into the periurethral
zone was carried out on a large animal as a subject by an operator
who actually performs a treatment in the same method as a clinical
study. Complete urinary retention that is a complication of the
operation was not developed, and for influence that the cell
treatment gave to the entire body, an observation of
differentiation of fat, bone and tumor other than the purpose of
this clinical regeneration and an observation of inflammation and
circulatory disorder were not found in all organs and injection
locals. Further, abnormality was not particularly observed also in
the blood test. In addition, a bump formed in the injection site
was present after 28 days, and effectiveness as a bulking mass was
confirmed. Furthermore, differentiation into smooth muscle was
shown by immunostaining of a bump formed in the injection site.
According to the above description, safety and effectiveness of
periurethral ASC injection were shown also in a pig.
7. Study Using an Animal Model Suffering from Urinary
Incontinence
[0126] Using an ECM model rat (rat having stress urinary
incontinence, whose periurethral zone of the prostate gland was
peeled off with an electric knife; Chermansky C. J. et al.
Neurourology and Urodynamics 23: 166-171 (2004)), which has been
known for an animal model reproducing a condition of intractable
stress urinary incontinence after prostate gland extirpation in a
human, a therapeutic effect of an operation of ASC injection into
the periurethral zone was examined. Firstly, according to a
reported method, the periurethral zone of the prostate gland of a
male wistar rat (250-300 g) was peeled off, and then ASC that has
been previously prepared (prepared from subcutaneous fat of the
same rat in a method described in WO/2008/018450
(PCT/JP2007/065431)) was injected (ASC treated group). For a
control, a solvent (DMEM medium) was injected in place of ASC (ECM
model group). After 2 weeks and 4 weeks from the operations, the
ASC treated group, the ECM model group and the sham operation group
(sham group) were compared for evaluation.
[0127] The evaluation results are shown in FIG. 23. In the ASC
treated group, a urine leakage pressure was significantly high as
compared to the ECM model group. In addition, rise in the urine
leakage pressure with time was also observed. Thus, an effect of
improving urinary incontinence due to adipose tissue derived stem
cell injection into the periurethral zone in intractable stress
urinary incontinence cases after prostate gland cancer operation
was observed also in a basic experiment having constant
conditions.
INDUSTRIAL APPLICABILITY
[0128] The cell preparation of the present invention is used for
improvement of erectile dysfunction or recovery of the urge to
urinate (recovery of sensory disorders of the lower urinary tract).
For a subject (patient) to which the cell preparation of the
present invention is applied, a patient suffering from stress
urinary incontinence, a patient suffering from sensory disorders of
the lower urinary tract after a prostate gland operation, a patient
suffering from urine collection disorder, a patient suffering from
erectile dysfunction, and the like are assumed.
[0129] The invention is not limited by description of the
embodiments and examples of the invention described above at all.
Various modified embodiments are also included in the invention
within the range that a person skilled in the art can easily
conceive of, without departing from the description of the scope of
patent claims.
[0130] Contents of treatises, unexamined patent publications, and
examined patent publications specified in this specification are
all incorporated herein by their references.
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