U.S. patent application number 10/556309 was filed with the patent office on 2007-10-04 for use of vinca alkaloids and salts thereof.
Invention is credited to Itaru Kojima, Takashi Koyano, Hisako Ohgawara, Kazuo Umezawa.
Application Number | 20070232533 10/556309 |
Document ID | / |
Family ID | 33436426 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070232533 |
Kind Code |
A1 |
Umezawa; Kazuo ; et
al. |
October 4, 2007 |
Use of Vinca Alkaloids and Salts Thereof
Abstract
An agent containing a vinca alkaloid or its pharmacologically
acceptable salt as an active ingredient can induce insulin
production and/or secretion of non-neoplastic cells derived from
the pancreas.
Inventors: |
Umezawa; Kazuo; (Tokyo,
JP) ; Ohgawara; Hisako; (Tokyo, JP) ; Kojima;
Itaru; (Maebashi, JP) ; Koyano; Takashi;
(Tokyo, JP) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Family ID: |
33436426 |
Appl. No.: |
10/556309 |
Filed: |
May 10, 2004 |
PCT Filed: |
May 10, 2004 |
PCT NO: |
PCT/JP04/06567 |
371 Date: |
November 3, 2006 |
Current U.S.
Class: |
514/183 ;
514/283; 514/356; 514/6.7; 514/9.5 |
Current CPC
Class: |
C07D 491/22 20130101;
A61P 13/12 20180101; A61K 31/455 20130101; A61P 3/10 20180101; A61P
27/02 20180101; A61P 25/00 20180101; A61P 9/10 20180101; A61P 43/00
20180101 |
Class at
Publication: |
514/012 ;
514/356; 514/283 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 31/4745 20060101 A61K031/4745; A61K 31/455
20060101 A61K031/455 |
Foreign Application Data
Date |
Code |
Application Number |
May 9, 2003 |
JP |
2003-131256 |
Oct 31, 2003 |
JP |
2003-373665 |
Claims
1. An agent for increasing insulin-producing ability of a
non-neoplastic cell derived from the pancreas, comprising
conophylline or its pharmacologically acceptable salt as an active
ingredient.
2. The agent for increasing insulin-producing ability of a
non-neoplastic cell derived from the pancreas of claim 1, further
comprising nicotinamide.
3. The agent for increasing insulin-producing ability of a
non-neoplastic cell derived from the pancreas of claim 1, further
comprising nicotinamide and hepatocyte growth factor (HGF).
4. An agent for increasing insulin-secreting ability of a
non-neoplastic cell derived from the pancreas, comprising
conophylline or its pharmacologically acceptable salt and
nicotinamide as active ingredients.
5. An agent for increasing insulin-secreting ability of a
non-neoplastic cell derived from the pancreas, comprising
conophylline or its pharmacologically acceptable salt,
nicotinamide, and hepatocyte growth factor (HGF) as active
ingredients.
6. A preventive agent and/or a therapeutic agent for a disease
associated with lack of insulin, comprising conophylline or its
pharmacologically acceptable salt as an active ingredient.
7. The preventive and/or the therapeutic agent for a disease
associated with lack of insulin of claim 6, wherein the disease
associated with lack of insulin is selected from the group
consisting of diabetes, arteriosclerosis, and a complication
resulting from these diseases.
8. The preventive agent and/or the therapeutic agent for a disease
associated with lack of insulin of claim 6, further comprising
nicotinamide.
9. The preventive agent and/or the therapeutic agent for a disease
associated with lack of insulin of claim 6, further comprising
nicotinamide and hepatocyte growth factor (HGF).
10. A blood glucose level-lowering agent comprising conophylline or
its pharmacologically acceptable salt as an active ingredient.
11. The blood glucose level-lowering agent of claim 10, further
comprising nicotinamide.
12. The blood glucose level-lowering agent of claim 10, further
comprising nicotinamide and hepatocyte growth factor (HGF).
13. An agent for inducing differentiation from a non-neoplastic
cell derived from the pancreas into an insulin-producing cell,
comprising conophylline or its pharmacologically acceptable salt as
an active ingredient.
14. The agent for inducing differentiation of claim 13, further
comprising nicotinamide.
15. The agent for inducing differentiation of claim 13, further
comprising nicotinamide and hepatocyte growth factor (HGF).
16. An agent for inducing differentiation from a non-neoplastic
cell derived from the pancreas into an insulin-secreting cell,
comprising conophylline or its pharmacologically acceptable salt
and nicotinamide as active ingredients.
17. An agent for inducing differentiation from a non-neoplastic
cell derived from the pancreas into an insulin-secreting cell,
comprising conophylline or its pharmacologically acceptable salt,
nicotinamide, and hepatocyte growth factor (HGF) as active
ingredients.
18. An agent for promoting induction of differentiation from a
non-neoplastic cell derived from the pancreas into an
insulin-secreting cell, consisting of conophylline or its
pharmacologically acceptable salt.
19. A method for inducing differentiation from a non-neoplastic
cell derived from the pancreas into an insulin-producing cell,
wherein conophylline or its pharmacologically acceptable salt is
added when the non-neoplastic cell derived from the pancreas is
cultured.
20. A method for inducing differentiation from a non-neoplastic
cell derived from the pancreas into an insulin-secreting cell,
wherein conophylline or its pharmacologically acceptable salt and
nicotinamide are added when the non-neoplastic cell derived from
the pancreas is cultured.
21. A method for inducing differentiation from a non-neoplastic
cell derived from the pancreas into a insulin-secreting cell,
wherein conophylline or its pharmacologically acceptable salt,
nicotinamide, and hepatocyte growth factor (HGF) are added when the
non-neoplastic cell derived from the pancreas is cultured.
22. A method for increasing insulin-producing ability of a
non-neoplastic cell derived from the pancreas, wherein conophylline
or its pharmacologically acceptable salt is added when the
non-neoplastic cell derived from the pancreas is cultured.
23. A method for increasing insulin-secreting ability of a
non-neoplastic cell derived from the pancreas, wherein conophylline
or its pharmacologically acceptable salt is added when the
non-neoplastic cell derived from the pancreas is cultured.
24. A method for producing an insulin-producing cell, comprising
culturing a non-neoplastic cells derived from the pancreas in the
presence of conophylline or its pharmacologically acceptable
salt.
25. A method for producing an insulin-secreting cell, comprising
culturing a non-neoplastic cells derived from the pancreas in the
presence of conophylline or its pharmacologically acceptable salt
and nicotinamide.
26. A method for producing an insulin-secreting cell, comprising
culturing a non-neoplastic cells derived from the pancreas in the
presence of conophylline or its pharmacologically acceptable salt,
nicotinamide, and hepatocyte growth factor (HGF).
27. A method for producing insulin, comprising: culturing a
non-neoplastic cell derived from the pancreas in the presence of
conophylline or its pharmacologically acceptable salt and
nicotinamide; and isolating and purifying insulin from the cultured
cell or a medium.
28. A method for producing insulin, comprising: culturing a
non-neoplastic cells derived from the pancreas in the presence of
conophylline or its pharmacologically acceptable salt,
nicotinamide, and hepatocyte growth factor (HGF); and isolating and
purifying insulin from the cultured cell or a medium.
29. An insulin-producing cell that has been induced to
differentiate by the method of claim 19.
30. An insulin-secreting cell that has been induced to
differentiate by the method of claim 20 or 21.
31. A pancreas-derived non-neoplastic cell whose insulin-producing
ability has been increased by the method of claim 22.
32. A pancreas-derived non-neoplastic cell whose insulin-secreting
ability has been increased by the method of claim 23.
33. A preventive and/or a therapeutic method for a disease
associated with lack of insulin, comprising using conophylline or
its pharmacologically acceptable salt.
34. A preventive and/or a therapeutic method for a disease
associated with lack of insulin, comprising: culturing a
non-neoplastic cell derived from the pancreas in the presence of
conophylline or its pharmacologically acceptable salt and
nicotinamide; and using the cultured non-neoplastic cell derived
from the pancreas.
35. A preventive and/or a therapeutic method for a disease
associated with lack of insulin, comprising: culturing a
non-neoplastic cell derived from the pancreas in the presence of
conophylline or its pharmacologically acceptable salt,
nicotinamide, and hepatocyte growth factor (HGF); and using the
cultured non-neoplastic cell derived from the pancreas.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to Japanese
Patent Application No. 2003-13125, filed on May 9, 2003 and
Japanese Patent Application No. 2003-373665, filed on Oct. 31,
2003, both of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to the use of vinca alkaloids
and their salts. More specifically, the present invention relates
to agents that enhance insulin-producing and/or -secreting
abilities of non-neoplastic cells derived from the pancreas,
therapeutic agents for diabetes, blood glucose level-lowering
agents, methods for inducing differentiation of non-neoplastic
cells derived from the pancreas, methods for enhancing
insulin-producing and/or -secreting abilities of non-neoplastic
cells derived from the pancreas, methods for producing
pancreas-derived non-neoplastic cells whose insulin-producing
ability has been enhanced, methods for culturing non-neoplastic
cells derived from the pancreas, methods for producing insulin,
pancreas-derived non-neoplastic cells that have been induced to
differentiate, and pancreas-derived non-neoplastic cells whose
insulin-producing and/or -secreting abilities have been
enhanced.
BACKGROUND ART
[0003] Diabetes is a disease arising from shortage of blood insulin
and impaired insulin function, resulting in increased blood glucose
concentrations, accompanied by complications such as neuropathy,
visual disturbance, renal damage, etc. About 7 million people are
afflicted with diabetes in Japan alone. There are two major types
of diabetes: Type 1 diabetes is caused by an autoimmune destruction
of insulin-producing pancreatic .beta. cells, resulting in an
absolute lack of insulin. On the other hand, type 2 diabetes is
caused by the expression of insulin resistance in target tissues,
such as muscle, fat, and liver, or by a decrease in blood insulin
levels due to a decline in pancreatic .beta. cell function. Thus,
it can be said that both type 1 and type 2 result from impaired
pancreatic .beta.-cell function.
[0004] For treatment of type 1 diabetes, insulin injections are
conventionally used to lower blood glucose levels in most cases. It
is needless to say that, in such cases, four injections a day are
laborious to patients. For treatment of type 2, the PPAR.gamma.
inhibitor, which reduces insulin resistance, is used in some cases.
However, the inhibitor is not very effective and causes obesity as
a side effect, as has been pointed out. In other cases, agents such
as sulfonylurea, which promotes insulin release from .beta. cells,
are used, but they are also not very effective.
[0005] Apart from drug dependent treatment, a new treatment by
transplantation of cells or tissue has been considered promising as
regenerative therapy. If large amounts of cells with
insulin-releasing ability can be transplanted into type I diabetes
patients, four insulin injections per day can be avoided for a long
term. Meanwhile, transplantation of insulin-releasing cells is also
effective for type 2 diabetes because, irrespective of insulin
resistance in target tissues, normal insulin production supresses
the increase of blood glucose level. It is expected that the
efficacy of transplantation is far superior to that of treatment
with agents currently used, e.g., sulfonylurea, that induce insulin
release from .beta. cells.
[0006] Porcine pancreatic cells are expected to be utilized in
regenerative therapy for diabetes because of their ease of
availability, immunological properties, etc. The technique for
collecting large amounts of cells and inducing them to
differentiate into insulin producing and releasing cells to a
sufficient degree has never been known in any cell system.
[0007] Most of pancreatic .beta. cells are generated in the fetal
period and then proliferate and differentiate very slowly (Herrera,
P. L. et al., Development 127: 2317-2322 (2000)). However,
experiments have shown that when the pancreas is damaged .beta.
cells actively differentiate and proliferate. Differentiation and
proliferation of .beta. cells, together with growth of remnant
.beta. cells, occur from pancreatic ductal cells both in adult mice
subjected to a 90% partial pancreatectomy and in mice that have
developed impaired glucose tolerance due to .beta. cell destruction
induced by alloxan. These findings have revealed that stem cells
are also present in the adult pancreas and have regenerative
capacity (Bonner-Wier S. et al., Diabetes 42: 1715-1720 (2000)).
Thus, experiments were performed in which stem cells were isolated
from the pancreatic ducts and induced to differentiate into
insulin-producing cells (Ramiya, V. K. et al., Nature Med. 6:
278-282 (2000) and Bonner-Weir, S. et al., Proc. Nat. Acad. Sci.
USA 97: 7999-800 (2000)). Still, a marker of pancreatic stem cells
was not known. It was reported last year that nestin, a marker
expressed in neural precursor cells, is a marker of pancreatic stem
cells, and nestin was confirmed to be expressed in adult pancreatic
duct. (Hunziker, E. et al., Biochem. Biophys. Res. Commun. 271:
116-119 (2000)). Furthermore, in vitro culture and differentiation
into cells with phenotype including that of insulin-producing cells
was succeeded (Zulewski, H. et al., Diabetes 50: 521-533 (2001)).
In addition, differentiation of ES cells into pancreatic inlet-like
tissues was also succeeded (Lumelysky, N. et al., Science 292:
1389-1394 (2001)). These techniques are expected to be clinically
applied in regenerative medicine of the pancreas.
[0008] While studies are under way on cells that can potentially
serve as materials for pancreatic .beta. cells, physiologically
active substances that induce differentiation of pancreatic .beta.
cells are considered promising for clinical application in the
field of regenerative medicine. Examples of substances that have
thus far been known as inducers of differentiation into .beta.
cells include activin A, which belongs to the TGF-.beta.
superfamily (Demeterco, C. J. et al., Clin. Endo. 85: 3892-3897
(2000)); betacellulin (BTC), which belongs to the EGF family
(Ishiyama, N. et al., Diabetologia 41: 623-628 (1998) and Yamamoto,
K. et al., Diabetes 49: 2021-2027 (2000); hepatocyte growth factor
(HGF) (Ocana, A. G. et al., J. Bio. Chem. 275: 1226-1232(2000));
basic fibroblast growth factor (bFGF) (Assady, S. et al., Diabetes
50: 1691-1697 (2001)); etc. These substances are proteins, which
are not suitable for oral administration. Further, it is difficult
to introduce such substances by means of injection because of their
immunological problem and instability.
[0009] Meanwhile, it has been reported that among
low-molecular-weight compounds, nicotinamide acts as a poly
(ADP-ribose) synthetase inhibitor, promoting regeneration of
pancreatic .beta. cells (Watanabe, T. et al., Proc. Natl. Acad.
Sci. USA 91: 3589-3592 (1994) and Sjoholm, A. et al., Endocrinology
135: 1559-1565 (1994)). In addition, nicotinamide has also been
reported to promote the expression of Reg protein (Watanabe, T. et
al., Proc. Natl. Acad. Sci. USA 91: 3589-3592 (1994)), which
promotes the proliferation and differentiation of pancreatic .beta.
cells (Akiyama, T. et al., Proc. Natl. Acad. Sci. USA 98: 48-53
(2001)). In another report, fetal porcine pancreatic islet-like
cell clusters (ICCs) were induced to differentiate into
insulin-producing cells using sodium butyrate and
dexamethasone(Korsgren, O. et al., Ups J. Med. Sci. 98: 39-52
(1993)), but the technology is less likely to be put into practical
use because of the low specificity.
[0010] Meanwhile, the structures of conophylline (Umezawa, K. et
al., Anticancer Res. 14: 2413-2418 (1994)) and conophyllidine (Kam,
T. S. et al., J. Nat. Prod. 56: 1865-1871 (1993)), both of which
are alkaloids isolated from leaves of an Apocynaceae family plant
grown in Malaysia and Thailand are known, as shown in FIG. 1.
Conophylline is known to exhibit anti-tumor activity in animals
(Umezawa, K. et al., Drugs Exptl. Clin. Res. 22: 35-40 (1996)).
[0011] It is also known that conophylline induces insulin
production of pancreatic acinar carcinoma AR42 J-B13 cells. (The
Book of Abstracts (01-21) of the 45th Annual Meeting of the Japan
Diabetes Society held in Tokyo, May 18, 2002). However, these
cancer cells did not release insulin into culture medium.
[0012] The object of the present invention is to provide agents
capable of inducing insulin production and/or secretion of
non-neoplastic cells derived from the pancreas.
DISCLOSURE OF THE INVENTION
[0013] The present inventors have intensively studied to solve the
above-mentioned problems and, as a result, found that vinca
alkaloids markedly induce differentiation of normal pancreatic
cells into insulin-producing and -releasing cells in vitro. Thus,
the present invention has been accomplished.
[0014] The following summarizes the present invention. [0015] 1. An
agent for increasing insulin-producing ability of a non-neoplastic
cell derived from the pancreas, containing a vinca alkaloid or its
pharmacologically acceptable salt as an active ingredient. A
preferred example of the aforementioned vinca alkaloid is
conophylline. The agent preferably further contains nicotinamide,
or nicotinamide and hepatocyte growth factor (HGF). [0016] 2. An
agent for increasing insulin-secreting ability of a non-neoplastic
cell derived from the pancreas, containing a vinca alkaloid or its
pharmacologically acceptable salt and nicotinamide as active
ingredients. A preferred example of the aforementioned vinca
alkaloid is conophylline. The agent preferably further contains
hepatocyte growth factor (HGF). [0017] 3. A preventive and/or a
therapeutic agent for a disease associated with lack of insulin,
containing a vinca alkaloid or its pharmacologically acceptable
salt as an active ingredient. A preferred example of the
aforementioned vinca alkaloid is conophylline. The disease
associated with lack of insulin is selected from the group
consisting of diabetes, arteriosclerosis, and a complication
resulting from these diseases. The preventive and/or the
therapeutic agent for a disease associated with lack of insulin
preferably further contains nicotinamide, or nicotinamide and
hepatocyte growth factor (HGF). [0018] 4. A blood glucose
level-lowering agent containing a vinca alkaloid or its
pharmacologically acceptable salt as an active ingredient. A
preferred example of the aforementioned vinca alkaloid is
conophylline. The blood glucose level-lowering agent preferably
further contains nicotinamide, or nicotinamide and hepatocyte
growth factor (HGF) [0019] 5. An agent for inducing differentiation
from a non-neoplastic cell derived from the pancreas into an
insulin-producing cell, containing a vinca alkaloid or its
pharmacologically acceptable salt as an active ingredient. A
preferred example of the aforementioned vinca alkaloid is
conophylline. The agent for inducing differentiation preferably
further contains nicotinamide, or nicotinamide and hepatocyte
growth factor (HGF). [0020] 6. An agent for inducing
differentiation from a non-neoplastic cell derived from the
pancreas into an insulin-secreting cell, containing a vinca
alkaloid or its pharmacologically acceptable salt and nicotinamide
as active ingredients. A preferred example of the aforementioned
vinca alkaloid is conophylline. The agent for inducing
differentiation preferably further contains nicotinamide, or
nicotinamide and hepatocyte growth factor (HGF). [0021] 7. An agent
for promoting induction of differentiation from a non-neoplastic
cell derived from the pancreas into an insulin-producing cell,
consisting of a vinca alkaloid or its pharmacologically acceptable
salt. A preferred example of the aforementioned vinca alkaloid is
conophylline. [0022] 8. A method for inducing differentiation of a
non-neoplastic cell, in which a vinca alkaloid or its
pharmacologically acceptable salt is added when the non-neoplastic
cell derived from the pancreas is cultured. In this manner, by
culturing non-neoplastic cells derived from the pancreas in the
presence of a vinca alkaloid or its pharmacologically acceptable
salt, the non-neoplastic cell derived from the pancreas
differentiate into an insulin-producing cell or an
insulin-secreting cell. In induction of differentiation of a
non-neoplastic cell derived from the pancreas, nicotinamide, or
nicotinamide and hepatocyte growth factor (HGF) are preferably
used. A preferred example of the aforementioned vinca alkaloid is
conophylline. [0023] 9. A method for increasing insulin-producing
ability of a non-neoplastic cell derived from the pancreas, in
which a vinca alkaloid or its pharmacologically acceptable salt is
added when the non-neoplastic cell derived from the pancreas is
cultured. A preferred example of the aforementioned vinca alkaloid
is conophylline. To increase insulin-producing ability of the
non-neoplastic cell derived from the pancreas, nicotinamide, or
nicotinamide and hepatocyte growth factor (HGF) are preferably
used. [0024] 10. A method for increasing insulin-secreting ability
of a non-neoplastic cell derived from the pancreas, in which a
vinca alkaloid or its pharmacologically acceptable salt is added
when the non-neoplastic cell derived from the pancreas is cultured.
A preferred example of the aforementioned vinca alkaloid is
conophylline. To increase insulin-secreting ability of the
non-neoplastic cell derived from the pancreas, it is preferable to
use nicotinamide, or nicotinamide and hepatocyte growth factor
(HGF). [0025] 11. A method for producing a pancreas-derived
non-neoplastic cell whose insulin-producing and/or secreting
abilities have been increased, which includes culturing the
non-neoplastic cells derived from the pancreas in the presence of a
vinca alkaloid or its pharmacologically acceptable salt. In this
manner, by culturing a non-neoplastic cell derived from the
pancreas in the presence of vinca alkaloid or its pharmacologically
acceptable salt, it is possible to produce a pancreas-derived
non-neoplastic cell whose insulin-producing ability has been
increased (an insulin-producing cell)or a pancreas-derived
non-neoplastic cell whose insulin-secreting ability has been
increased (an insulin-secreting cell) In induction of
differentiation of a non-neoplastic cell derived from the pancreas,
it is preferable to use nicotinamide, or nicotinamide and
hepatocyte growth factor (HGF). A preferred example of the
aforementioned vinca alkaloid is conophylline. [0026] 12. A method
for producing insulin, which includes culturing a non-neoplastic
cell derived from the pancreas in the presence of vinca alkaloid or
its pharmacologically acceptable salt and nicotinamide and
isolating and purifying insulin from a culture (the cultured cell
or a medium). A preferred example of the aforementioned vinca
alkaloid is conophylline. [0027] 13. A method for producing
insulin, which includes culturing a non-neoplastic cell derived
from the pancreas in the presence of conophylline or its
pharmacologically acceptable salt, nicotinamide, and hepatocyte
growth factor (HGF) and isolating and purifying insulin from the
cultured cell or a medium. A preferred example of the
aforementioned vinca alkaloid is conophylline. [0028] 14. A
pancreas-derived non-neoplastic cell (an insulin-producing cell or
an insulin-secreting cell) that has been induced to differentiate
by the method of 8 described above. [0029] 15. A pancreas-derived
non-neoplastic cell whose insulin-producing ability has been
increased by the method of 9 described above. [0030] 16. A
pancreas-derived non-neoplastic cell whose insulin-secreting
ability has been increased by the method of 10 described above.
[0031] 17. A preventive and/or a therapeutic method for a disease
associated with lack of insulin, which uses a vinca alkaloid or its
pharmacologically acceptable salt. A preferred example of the
aforementioned vinca alkaloid is conophylline. The disease
associated with lack of insulin is selected from the group
consisting of diabetes, arteriosclerosis, and a complication
resulting from these diseases. The preventive and/or the
therapeutic agent for a disease associated with lack of insulin
preferably further contains nicotinamide, or nicotinamide and
hepatocyte growth factor (HGF). [0032] 18. A preventive and/or a
therapeutic method for a disease associated with lack of insulin,
which includes culturing a non-neoplastic cell derived from the
pancreas in the presence of a vinca alkaloid or its
pharmacologically acceptable salt and nicotinamide; and using the
cultured non-neoplastic cell derived from the pancreas.
Alternatively, the method may include culturing a non-neoplastic
cell derived from the pancreas in the presence of a vinca alkaloid
or its pharmacologically acceptable salt, nicotinamide, and
hepatocyte growth factor (HGF); and using the cultured
non-neoplastic cell derived from the pancreas. A preferred example
of the aforementioned vinca alkaloid is conophylline. The disease
associated with lack of insulin is selected from the group
consisting of diabetes, arteriosclerosis, and a complication
resulting from these diseases.
[0033] "Vinca alkaloids" as used herein refer to vinblastine and
vincristine isolated from Vincarosea, an Apocynaceae family plant,
and to alkaloids containing the backbone represented by the
following structural formula: ##STR1## It should be noted that
alkaloids refer to cyclic compounds produced by plants with a
nitrogen atom (N) in the ring. Specific examples of vinca alkaloids
include, but not limited to vinblastine, vincristine, conophylline,
conophyllidine, conofoline, conophyllinine, taberhanine,
pachysiphine etc., as shown in FIG. 1.
[0034] "Non-neoplastic cells derived from the pancreas" as used
herein refer to cells without tumorigenicity that are derived from
the pancreas of individual organisms. Such cells include those
harvested from the pancreas of organisms and those cultivated from
the harvested cells (i.e., cultured cells). Cultured cells include
both primary cultured cells and successively transferred cells.
[0035] "To increase insulin-producing ability and/or -secreting
abilities of cells" is a concept including both causing cells that
do not have insulin-producing and/or -secreting abilities to
acquire insulin-producing and/or -secreting abilities and causing
cells that have insulin-producing and/or -secreting abilities to
enhance their insulin-producing and/or -secretion abilities.
[0036] "To differentiate into .beta. cells" as used herein means
that progenitor cells of .beta. cells come to produce and secrete
insulin.
[0037] As mentioned earlier, it is known that conophylline, a kind
of vinca alkaloid, induces insulin production of pancreatic acinar
carcinoma AR42 J-B13 cells, but the AR42 cells were found to be
incapable of releasing insulin out of cells, though they do produce
insulin. It was unpredictable that, under conditions in which only
such weak effects are known, a vinca alkaloid induces insulin
production, and further, can even release insulin out of cells when
non-neoplastic cells derived from the pancreas were used
instead.
[0038] Activin, which is capable of inducing AR42J cells to produce
insulin, does not exhibit the effect on porcine pancreatic cells.
To induce ES cells to produce insulin, many factors including
leukocyte activating factor (LAF) as well as many processes are
required. The conditions under which cells are induced to
differentiate are different depending on the individual cell type.
For this reason, even those skilled in the art could not predict
that a vinca alkaloid increases insulin production of
non-neoplastic cells derived from pancreas and induces insulin to
be secreted out of the cells.
[0039] Furthermore, since AR42J cells were cancer cells, they could
not be used for regenerative medicine from the viewpoint of safety
even if their insulin-producing ability was increased. The
technique of increasing insulin-producing and -secreting abilities
of non-neoplastic cells derived from the pancreas has been
established by the present invention, which has made it possible to
prepare large amounts of cells available for regenerative
medicine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 shows the chemical structural formulae of several
compounds belonging to the vinca alkaloid family.
[0041] FIG. 2 shows the results of immunostaining of fetal porcine
pancreatic cells cultured in media to which the following were
added for three weeks: vehicle (1) ; only nicotinamide (10 mM) (2);
nicotinamide (10 mM) and HGF (10 ng/ml) (4);only conophylline (0.1
.mu.g/ml) (5); conophylline (0.1 .mu.g/ml) and HGF (10 ng/ml) (7);
conophylline (0.1 .mu.g/ml), nicotinamide (10 mM), and HGF (10
ng/ml) (8). In the figure, N and CNP denote nicotinamide and
conophylline, respectively.
[0042] FIG. 3 shows the results of ELISA measurement of the amount
of insulin produced by fetal porcine pancreatic cells cultured in
media to which the following were added: vehicle (white circle);
nicotinamide and HGF(white triangle); only conophylline (black
circle); conophylline, nicotinamide, and HGF (black triangle). In
the figure, N and CNP denote nicotinamide and conophylline,
respectively.
[0043] FIG. 4 shows the effect of conophylline on blood glucose
levels of streptozotocin-administered rats.
BEST MODE FOR CARRYING OUT THE INVENTION
[0044] Embodiments of the present invention accomplished based on
the above-described findings are hereinafter described in detail by
giving Examples. Unless otherwise explained, methods described in
standard sets of protocols such as J. Sambrook and E. F. Fritsch
& T. Maniatis (Ed.),"Molecular Cloning, a Laboratory Manual
(3rd edition), Cold Spring Harbor Press and Cold Spring Harbor,
N.Y. (2001); and F. M. Ausubel, R. Brent, R. E. Kingston, D. D.
Moore, J. G. Seidman, J. A. Smith, and K. Struhl (Ed.), "Current
Protocols in Molecular Biology," John Wiley & Sons Ltd., or
alternatively, modified/changed methods from these are used. When
using commercial reagent kits and measuring apparatus, unless
otherwise explained, attached protocols to them are used.
[0045] The objective, characteristics, and advantages of the
present invention as well as the idea thereof will be apparent to
those skilled in the art from the descriptions given herein. It is
to be understood that the embodiments and specific examples of the
invention described hereinbelow are to be taken as preferred
examples of the present invention. These descriptions are for
illustrative and explanatory purposes only and are not intended to
limit the invention to these embodiments or examples. It is further
apparent to those skilled in the art that various changes and
modifications may be made based on the descriptions given herein
within the intent and scope of the present invention disclosed
herein.
[0046] One aspect of the present invention is hereinafter explained
in detail.
1. Manufacture of Vinca Alkaloids and Their Salt
[0047] The chemical structural formulae of some compounds belonging
to the vinca alkaloid family are shown in FIG. 1.
[0048] Vinblastine and vincristine can be isolated and purified
from Vinca rosea Linn by the method described in Neuss N, Gorman M,
Hargrove W, et al., & Manning R E (1964) J. Am. Chem. Soc. 86:
1440-1442,
[0049] Conophylline can be isolated and purified from leaves of
Ervatamia microphylla, an Apocynaceae family plant, in the manner
as will be described later in Production example 1 (a method
modified from Umezawa, K. et al. Anticancer Res. 14: 2413-2418
(1994)). About 4 kg of Ervatamia microphylla leaves yields about
500 mg of conophylline crystals.
[0050] Conophyllidine can be prepared from leaves of Ervatamia
microphillae in the same manner as conophylline (Kam, T. S. et al.
J. Nat. Prod. 56: 1865-1871 (1993)).
[0051] Conofoline and pachysiphine can be prepared by the methods
described in Kam T S, Anuradha S. Alkaloids from Tabernaemontana
divaricata, and Phytochemistry (1995) 40: 313-6.
[0052] Conophyllinine and taberhanine can be prepared by the
methods described in Kam T S, Pang H S, Lim T M, Biologically
active indole and bisindole alkaloids from Tabernaemontana
divaricata. Org. Biomol. Chem. (2003) 21; 1(8): 1292-7.
[0053] Examples of salts of vinca alkaloids include hydrochlorides
and sulphates, which are pharmacologically acceptable. These salts
can be produce by known methods.
2. Induction of Insulin Production in Non-Neoplastic Cells Derived
from the Pancreas by Vinca Alkaloids and Their Salts
[0054] First, non-neoplastic cells derived from the pancreas are
prepared. Non-neoplastic cells derived from the pancreas may be
derived from mammals. Such mammals include primates, such as humans
and monkeys, as well as non-primates, such as pigs, cattle, dogs,
and rats. Cells may be derived from healthy animals or from
patients who need treatment. Patients are not limited to humans;
they may be unhealthy non-human animals. When non-neoplastic cells
derived from a healthy animal are used, preferred animals are
fetuses and neonates (for example, in the case of pigs, neonatal
pigs within 3 days of birth) Non-neoplastic cells may be exocrine
cells or endocrine cells, but endocrine cells are preferred.
Further, among endocrine cells, .beta. cells and their progenitor
cells are more preferred.
[0055] Isolation of non-neoplastic cells from the pancreas of
healthy mammalian animals may be performed, for example, as
follows: The pancreas is removed from a mammal and connective
tissue is detached. The pancreas is dissected, buffer is added, and
the mixture is stirred. After stirring, the supernatant is
discarded, an enzyme Liberase is added, and the mixture is stirred
again. Subsequently, a cycle of centrifugation, discarding of the
supernatant, and addition of PBS is repeated. Cells are suspended
with PBS and this cell suspension is overlaid on Histopaque,
followed by centrifugation. Since pancreatic endocrine cells forms
a belt-like white layer at the interface between cell suspension
and Histopaque, this layer is harvested. The sample is centrifuged
and the supernatant is discarded. The cells is suspended in medium
and transferred to the culture chamber to be incubated.
Subsequently, spheroids (loose cell aggregates) are formed by
stirring for an appropriate time, followed by incubation. Cells
detached from the chamber and floating in the medium are used for
the subsequent operations.
[0056] The cells floating in the culture chamber are transferred to
the centrifuge tube, which is let to stand still until spheroids
have sunk to the bottom. Then the supernatant is discarded and
medium was added, followed by light shaking. The tube is let to
stand still and the spheroids are allowed to settle down to the
bottom. After this operation is repeated 2 or 3 times, the cell
lysate is centrifuged and the supernatant is discarded. The
residual cells are cultured under the condition of 5% CO.sub.2 and
37.degree. C. in medium supplemented with a vinca alkaloid or its
pharmacologically acceptable salt. The culture may be stationary
culture. Examples of suitable media include RPMI medium etc. It is
also more preferable to add nicotinamide and hepatocyte growth
factor (HGF) in addition to a vinca alkaloid or its
pharmacologically acceptable salt. The medium may be changed every
4 to 7 days and the culture is incubated for 7 to 35 days. The
morphology of the cells may be examined at suitable time intervals
during the incubation period. Further, when the medium is changed,
the culture solution may be recovered and subjected to the
measurement of the insulin amount in the medium.
[0057] The methods for separating and culturing cells described
above can also be applied when non-neoplastic cells derived from
patients' pancreas are prepared. It should be noted that, to obtain
non-neoplastic cells derived from patients' pancreas, a method is
suggested in which tissue fragments are harvested from patients'
pancreas so that non-neoplastic cells are isolated from these
tissue fragments by the above-described methods.
[0058] As thus far described, by culturing non-neoplastic cells
derived from the pancreas in the presence of a vinca alkaloid or
its salt, differentiation of non-neoplastic cells derived from the
pancreas can be induced. Preferably, non-neoplastic cells derived
from the pancreas can be induced to differentiate into
insulin-producing and -releasing cells (e.g., .beta. cells).
Furthermore, insulin-producing and/or -secreting abilities of
normal pancreatic cells can be increased. Furthermore, by culturing
non-neoplastic cells derived from the pancreas in the presence of a
vinca alkaloid or its salt, insulin can be isolated and purified
from cultures (cultured cells or a medium) by known methods.
[0059] The pancreas-derived non-neoplastic cells whose
insulin-producing and/or -secreting abilities have been increased
by culturing in the presence of a vinca alkaloid or its salt are
capable of producing 10 ng or more, preferably 25 ng or more, and
more preferably 55 ng or more in 1 ml of medium when they were
cultured at the concentration of 2.5.times.10.sup.5 cells per 1 ml
of medium for 7 to 35 days.
[0060] As mentioned earlier, porcine pancreatic cells are expected
to be used in regenerative therapy for diabetes because of their
ease of availability, immunological properties, etc. The method for
collecting large amounts of cells to induce them to differentiate
into insulin-producing and -releasing cells to a sufficient degree
has never been known in any cell system.
[0061] The technique according to the present invention makes it
possible to induce large amounts of cells to differentiate into
insulin-producing and -releasing cells to a sufficient degree.
Accordingly, insulin-producing and -releasing cells obtained by the
methods according to the present invention can be used in
regenerative medicine for diabetes.
[0062] Insulin-producing and -releasing cells obtained by the
methods according to the present invention may be suspended in a
solution and/or embedded in a support matrix and then administered
to subjects. The solution in which insulin-producing and -releasing
cells are suspended may be a pharmacologically acceptable carrier
and diluent, such as saline, a buffer solution, etc. To the
solution, a preservative (e.g., p-hydroxybenzoic acid ester,
chlorobutanol, thiromesal, etc.) and a stabilizer (e.g., L-arcorbic
acid, etc.) may be added. After insulin-producing and -releasing
cells are suspended in the solution, they may be subjected to a
sterilization treatment. A support matrix in which
insulin-producing and -releasing cells are embedded may be a matrix
that is recipient-compatible and that degrades into a product not
harmful to the recipient. Materials for the matrix may include
natural polymers, synthetic polymers, etc. Examples of natural
polymers include collagen, gelatin, etc. Examples of synthetic
polymers include polyglycolic acid, polylactic acid, etc. The
matrix can be in the form of, but not limited to, film, sheet,
particle, paste, etc.
3. Agents and Drugs Based on Vinca Alkaloids
[0063] Vinca alkaloids and their pharmacologically acceptable salts
can increase insulin-producing and/or -secreting abilities of
non-neoplastic cells derived from the pancreas. Further, vinca
alkaloids and their pharmacologically acceptable salts can also
decrease blood glucose levels. Therefore, these compounds may be
administered to human and other animals as drugs (e.g., therapeutic
agents for diabetes) or may be used as reagents in experiments.
These compounds may be used alone or in combination with other
agents (e.g., other therapeutic agents for diabetes). It should be
noted that, when a vinca alkaloid has been administered to an
animal, its effect is likely to have been enhanced by taking
advantage of endogenous nicotinamide and/or an endogenous
hepatocyte growth factor (HGF); thus, in administering a vinca
alkaloid, nicotinamide and/or hepatocyte growth factor (HGF) may be
simultaneously administered.
[0064] When administered to humans, a vinca alkaloid or its
pharmacologically acceptable salt may be administered orally. The
dosage is, for example, 0.1 to 10 mg/kg bw daily in a single dose
or divided doses. However, the amount of a dose and number of
administration can be suitably changed depending on the symptoms,
age, dosage regimen, etc.
[0065] Vinca alkaloids and their pharmacologically acceptable salts
may be administered orally in preparations, such as tablets,
capsules, granule, powder, syrups, etc. Alternatively, they may be
administered parenterally by intraperitoneal or intravenous
injection in preparations such as injectable formulations,
suppositories, etc. The content of a vinca alkaloid or its
pharmacologically acceptable salt (active ingredient) in a
preparation can vary between 1 to 90% by weight. For example, when
the preparation is in the form of a tablet, a capsule, a granule, a
powder, etc. the content of an active ingredient is preferably 5 to
80% by weight. In the case of a liquid preparation such as syrup,
the content of an active ingredient is preferably 1 to 30% by
weight. In addition, in the case of an injectable preparation for
parenteral administration, the content of an active ingredient is
preferably 1 to 10% by weight.
[0066] Vinca alkaloids and their pharmacologically acceptable salts
are formulated by known methods using the following formulation
additives: excipients (sugars such as lactose, sucrose, glucose,
and mannitol; starches such as potato, wheat, and corn; inorganic
substances such as calcium carbonate, calcium sulfate, and sodium
hydrocarbonate; cellulose crystal; etc.), binders (starch-paste
liquid, gum arabic, gelatin, sodium arginate, methylcellulose,
ethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol,
hydroxypropylcellulose, carmellose, etc.), lubricants (magnesium
stearate, talc, hydrogenerated vegetable oil, macrogol, and
silicone oil), disintegrators (starch, agar, gelatin powder,
cellulose crystal, sodium carboxymethylcellulose, calcium
carboxymethylcellulose, calcium carbonate, sodium hydrocarbonate,
sodium arginate, etc.), correctives (lactose, sucrose, glucose,
mannitol, fragrant essential oils, etc.), solvents (water for
injection, sterile purified water, sesame oil, soybean oil, corn
oil, olive oil, cottonseed oil, etc.), stabilizers (inert gases
such as nitrogen and carbon dioxide; chelating agents such as EDTA
and thioglycolic acid; reducing substances such as sodium
bissulfite, sodium thiosulfate, L-ascorbic acid, and rongalite;
etc.), preservatives (paraoxybenzoic acid, chlorobutanol, benzyl
alcohol, phenol, benzalkonium chloride, etc.),detergents
(hydrogenated castor oil, polysorbates 80 and 20, etc.), buffers
(sodium salts of citric acid, acetic acid, and phosphoric acid;
boric acid; etc.), diluents, etc.
[0067] Vinca alkaloids and their pharmacologically acceptable salts
can be used to prevent and/or treat diseases (e.g., diabetes and
arteriosclerosis) associated with lack of insulin. Vinca alkaloids
and their pharmacologically acceptable salts can also be used in
studies of insulin production and/or secretion of pancreatic cells.
Vinca alkaloids and their pharmacologically acceptable salt can
further be used for blood glucose level-lowering agents as well as
for therapeutic agents for complications resulting from prolonged
high blood glucose levels, such as retinopathy of the eyes,
nephropathy, neuropathy, gangrene, arteriosclerosis, etc.
[0068] Hereinafter, the present invention will be explained in more
detail with reference to Production examples and Experiment
examples. These Production examples and Experiment examples are for
explanatory purposes only and are not intended in any way to limit
the scope of the invention.
[0069] The materials used in the example and their suppliers are as
follows. [0070] Neonatal pigs: Takayama Pig Farm [0071] Roswell
Park Memorial Institute (RPMI) medium: Invitrogen [0072]
Nicotinamide: Sigma Chemical Science (Sigma) [0073] Fetal bovine
serum (FBS): Sigma [0074] Phosphate buffered saline (PBS) [0075]
Histopaque: Sigma [0076] Hepatocyte growth factor (HGF): Sigma
Chemical Science [0077] DMEM: Nissui Pharmaceutical Co., Ltd.
[0078] HEPES: Sigma [0079] Kanamycin: Sigma [0080] Glutamine: Sigma
[0081] Penicillin G: Sigma [0082] Trypsin: Wako [0083] EDTA: Kanto
Chemical Co., Inc. [0084] PBS.sup.- for fluorescent antibodies
[0085] Bovine serum albmine (BSA): Sigma [0086] Anti-insulin
antibody: Biogenesis [0087] Cy3-conjugated anti-guinea pig
antibody: Jackson [0088] ImmunoResearch Laboratories, Inc. (West
Grove, Pa.) [0089] Activin A: R&D Systems,Inc.(Minneapolis,
Mo.) [0090] Hoechst 33258: Polysciences, Inc.
EXAMPLE 1
Production Example 1
Preparation of Conophylline
[0091] Conophylline was isolated and purified from leaves of
Ervatamia microphylla, an Apocynaceae family plant, (harvested in
Khon Khen, Thailand) in the following manner.
[0092] Active substance was extracted from about 4 kg of Ervatamia
micorophylla leaves with 100 L of chloroform to afford about 130 g
of oily substance. This oily substance was chromatographed on a
silica gel column (purification was performed by a total of 5
rounds of column chromatography using about 500 g of silica gel),
eluting sequentially with chloroform:methanol (40:1 and 20:1).
Subsequently, using morphological changes of K-ras-NRK cells as an
activity marker, the fractions exhibiting this biological activity
were recovered. The crude purified product obtained (about 40 g)
was chromatographed on a silica gel column with n-hexane:ethyl
acetate (1:2 and 0:1) (using about 500 g of silica gel; purchased
from Merck Co.) to afford 1.5 g of active fractions. The active
fractions were then chromatographed on a silica gel column (using
150 g of silica gel) with n-hexane:ethyl acetate:chloroform (9:3:1
and 6:3:1), active fractions were recovered, and concentrated to
afford about 500 mg of crystals. The resulting conophylline was
confirmed by mass spectrometry and NMR spectrometry comparing its
data with the literature data (Umezawa, K. et al., Anticancer Res.
14: 2413-2418 (1994)). The solvents used were purchased from Kanto
Chemical Co., Inc.
EXAMPLE 2
Experimental Examples I and II
1. Methods
[0093] Neonatal pigs within 72 hours of birth were obtained from
the (Takayama) pig farm. The whole pancreas (ventral and dorsal
pancreas) was removed under general anesthesia immediately after
animals were brought to the operating room. After elimination of
connective tissue covering the removed pancreas and blood adhering
to it, the pancreas was transferred to a 10 ml beaker and dissected
into small pieces with ophthalmic scissors. The dissected pieces
were transferred to a 100 ml conical flask, to which 50 to 60 ml of
phosphate buffered saline (PBS) was added. After a 3 min rotation
at 110 rpm on a low speed stirrer, the mixture was allowed to stand
still and the supernatant was discarded. Then, 40 mL of phosphate
buffered saline containing Liberase PI (Roche) at a concentration
of 2.5 mg/ml was added. After an 8 min rotation at 110 rpm on a low
speed stirrer, the mixture was allowed to stand still and the
supernatant containing the cells was collected. The operation was
repeated 5 times. The supernatant collected was centrifuged at 1200
rpm for 5 min. to collect cells. The cells collected in the
centrifugal sedimentum was suspended in 25 to 50 ml of PBS, 25 ml
of cell suspension was overlaid gently on 10 ml of Histopaque 1077
(Sigma), followed by centrifugation at 1800 rpm for 10 min.
Pancreatic (endocrine) cells form a band-like white layer at the
interface between cell suspension and Histopaque (cell separation
and purification methods)
[0094] Cells present at the interface are recovered with a Pasteur
pipette, suspended in RPMI 1640 supplemented with 10 mM
Nicotinamide and 10% heat-inactivated FBS, collected by
centrifugation at 1200 rpm for 5 min. These cells were resuspended
in the same medium and then subjected to stationary culture in a 75
ml culture flask (5% CO.sub.2 incubator, 37.degree. C.) for a whole
day and night.
Culture Conditions:
[0095] Cells floating in the flask were removed, and cells adhering
to the bottom were detached with EDTA-Trypsin and collected. Cells
were suspended in each of media (groups 1 to 8) and stationary
culture was performed by plating cells (1.25.times.10.sup.3
cells/ml) in culture chambers (2 ml each; 5% CO.sub.2-incubator,
37.degree. C.).
Media Group 1: RPMI 1640 with 10% FBS (Control)
[0096] 2: Control+10 mM nicotinamide
[0097] 3: Control+10 ng/ml HGF
[0098] 4: Control+0.1 .mu.g/ml conophylline
[0099] 5: 2+3
[0100] 6: 2+4
[0101] 7: 3+4
[0102] 8: 2+3+4
[0103] The culture medium was exchanged every four days and the
observation was made for three weeks, during which time the
morphology of cells was observed every week and the appearance of
pancreatic .beta.-cells was confirmed by immunofluorescence using
peroxidase (Experiment example 1). Coloration was performed using
3-amino-9-ethyl carbazole (AEC; 0.75 mg/ml) as a substrate. In
addition, the medium was recovered every time it was exchanged
(every four days) for measurement of the amount of insulin secreted
into the medium by ELISA (Experiment example II).
2. Results
[0104] FIG. 2 shows the results of Example 1 It has been reported
that nicotinamide (Akiyama, T. et al., Proc. Natl. Acad. Sci. USA
98: 48-53 (2001)) and HGF (Ocana, A. G. et al., J. Biol. Chem. 275:
1226-1232 (2000)) promote differentiation of insulin-producing
cells in the limited experimental systems, but their effects are
weak. As shown in FIG. 2, three weeks later, only a few
insulin-producing cells colored in red were seen in vehicle (1),
only nicotinamide (10 mM) (2), nicotinamide and HGF (10 ng/ml) (4),
only conophylline (0.1 .mu.g/ml) (5), and conophylline and HGF (7).
In the triple mixture of nicotinamide, HGF, and conophylline (8),
however, the number of insulin-producing cells colored markedly in
red increased. It should be noted that the mixture of nicotinamide
and conophylline (6) produced a smaller number of insulin-producing
cells than the aforementioned triple mixture the mixture (6) did;
however, nicotinamide-conophylline mixture had a marked increase in
that number, compared with the others (not shown in the
figure).
[0105] FIG. 3 shows the results of Example II. As indicated in FIG.
3, nicotinamide combined with either HGF or conophylline cultured
for 8 to 20 days was able to produce some amount of insulin in the
medium. Furthermore, nicotinamide combined with HGF and
conophylline had a marked increased in the amount of insulin
release, compared with other combinations or alone.
Summary
[0106] To date, there have been no techniques for inducing
differentiation of insulin-producing cells in pancreatic cells of
neonatal pigs. It has been revealed that the combination of
nicotinamide, HGF, and conophylline dramatically increases the
proportion of insulin-producing cells, thereby resulting in a large
amount of insulin release as well. Since the neonatal porcine
pancreas can be supplied in large amounts, it will be possible to
prepare in large amounts implantable cells that release enough
insulin. It is thought that this technique enables regenerative
therapy, which is expected for diabetes in the future.
[0107] Further, conophyllidine, like conophylline, has been
confirmed to induce morphological changes involved in insulin
production of pancreatic acinar carcinoma AR42J cells, suggesting
that conophyllidine, like conophylline, has the effect of
increasing insulin-producing and/or -secreting abilities of
non-neoplastic cells derived from the pancreas.
EXAMPLE 3
Comparative Example 1
Effect in Which Conophylline Induces Differentiation of Rat
Pancreatic Acinar Carcinoma AR42 J-B13 Cells Into Insulin-Producing
Cells
1. Methods
Cell Culture
[0108] DMEM was sterilized by filtration after its pH was adjusted
to 7.4 in the presence of 20 mM HEPES-NaOH. AR42J-B13 cells
(endowed by Dr. Itaru Kojima, Institute for Cellular and Molecular
Regulation, Gunma University) a highly sensitive subclone of AR42J,
were cultured at 37.degree. C. in a 5% CO.sub.2 incubator with 20
ml of culture medium DMEM supplemented with 100 mg/l kanamycin, 0.6
g/l glutamine, 100 unit/ml penicillin G, 5 mM NaHCO.sub.3, and 10%
FBS. To prevent transformation due to a high density of the cells
and to retain differentiation ability of the cells, the cells were
transferred every 2 to 3 days to maintain 2.5% to 5% of confluency.
The cell transfer was performed as follows: After the medium was
removed, the cells were washed twice in PBS.sup.- (Ca.sup.2+,
Mg.sup.2+-free PBS; 8 g/l, NaCl, 0.2 g/l KCl, 0.916
g/Na.sub.2HPO.sub.4, 0.2 g/l KH.sub.2PO.sub.4). Subsequently, the
cells were detached using 2 ml of trypsin-EDTA solution, and then
trypsin was inactivated by addition of 8 ml of the medium.
Subsequently, the cell were sedimented by centrifugation at 1000
rpm for 5 min, trypsin was removed, 10 ml of fresh medium was
added, and transferred to be 2.5% to 5% of confluency. Cells were
cryopreserved under the condition of 7.5% dimethyl sulfoxide
(DMSO).
[0109] Cells that had been prepared at a density of
4.times.10.sup.5 cells/ml were plated at 500 .mu.l per well on
24-well plates, followed by incubation at 37.degree. C. in a 5%
CO.sub.2 incubator. The next day, either 0.1-0.3 .mu.g/ml
conophylline alone or 0.1 .mu.g/ml conophylline plus 100 pM HGF was
added. After incubation for an appropriate time at 37.degree. C. in
5% CO.sub.2, the medium was removed to a 1.5 ml Eppendorf tube,
cells were washed once in 200 .mu.l of PBS.sup.-, and then 200
.mu.l of fresh PBS.sup.- was added. The morphology of the viable
cells was observed and photographed at 150.times. magnification
with a camera linked to the microscope. Subsequently, cells were
detached with trypsin and all the solution was transferred to the
aforementioned Eppendorf tube for trypan blue exclusion test.
Further, the photograph was enlarged 2.5 times and a morphological
change was defined as having occurred when the full length of a
cell in the diameter became 1.5 times; thus, the rates of
morphological changes were determined.
Immunofluorescence
[0110] Cells that had been prepared at a density of
2.times.10.sup.5 cells/ml were plated at 500 .mu.l per well on
8-well plates, followed by addition of either 0.1 .mu.g/ml
conophylline or 0.1 .mu.g/ml conophylline plus 100 pM HGF.
Subsequently, the cells were incubated at 37.degree. C. in a 5%
CO.sub.2 incubator for 72 hours to differentiate. After the medium
was removed, the cells were washed once with PBS.sup.- for
fluorescent antibodies (8 g/l NaCl, 50.45 g/l
NaH.sub.2PO.sub.4.2H.sub.2O, 1.28 g/l Na.sub.2HPO.sub.4) and fixed
with 3% formaldehyde at 4.degree. C. overnight (or at room
temperature for 30 min). Next, the cells were washed twice with
PBS.sup.- for fluorescent antibodies and blocked by adding 1 ml of
1% BSA solution (a solution of PBS.sup.- for fluorescent
antibodies) per well, followed by incubation at room temperature
for 1 hour. To quench with 50 mM glycine (a solution of PBS.sup.-
for fluorescent antibodies), cells were incubated for 5 min,
followed by washing 3 times. Next, the solution in the plastic
wells was removed, 50 .mu.l per well of alpha-insulin antibody
diluted 100-fold with a 10-fold diluted blocking buffer was placed
taking care not to allow cells to dry, and the plates were allowed
to stand still at room temperature for 1 hour. Here, a well of
cells induced to differentiate with 2 nM activin A and 100 pM HGF
was prepared and preserved without primary antibody for the purpose
of comparing the influence of the nonspecific binding of the
secondary antibody. Next, slides were transferred to the chamber
and washed three times with PBS.sup.- for fluorescent antibodies
for 5 min while shaking on a shaker. Next, Hoechst 33258 was added
to Cy3-conjugated anti-guinea pig antibody diluted 100-fold with a
10-fold diluted blocking solution at a 1:100 dilution, and the
antibody was overlaid as was the primary antibody. The slides were
shielded from light with aluminum foil, allowed to stand still at
room temperature for 1 hour and then placed in the shielded
chamber, followed by washing three times for 10 min with TNT buffer
(0.1 M Tris-HCl, 0.15 MNaCl, 0.05% Tween 20; pH 7.5) under light
shielding on the shaker. The slides were overlaid with 50% glycerol
in PBS.sup.-, covered with coverslips, and photographed under the
microscope.
2. Results
[0111] When conophylline was used alone and in combination with
HGF, process outgrowth such as that seen in nerve cells was
concentration-dependently induced at concentrations of 0.1 to 0.3
.mu.g/ml. Further, combination with HGF slightly enhanced cell
death, thereby increasing the rates of morphological changes.
[0112] The results of the immunofluorescence revealed the
following: After 72 hours, when 100 pM HGF was used alone, no red
coloration indicating insulin was observed in cells. When 0.1
.mu.g/ml conophylline and 100 pM HGF were used in combination,
however, red coloration of insulin was observed in the cytoplasm
excluding the nucleus. Further, the coloration was seen scattered
as if insulin was confined in granules in the cytoplasm. No insulin
coloration was observed in the vicinity of the cell membrane
involved in insulin release. Meanwhile, when 2 nM activin A and 100
pM HGF were used in combination, red coloration of insulin was
observed in granules as in combination of conophylline and HGF, but
its localization was different: the red coloration was observed
along the axis of the process split into two, with some
accumulation at the tips of the neurites.
[0113] In addition, quantification of insulin secretion of the
differentiated cells was attempted using the ELISA method, but the
secretion was found to be below the detection level.
Summary
[0114] In conclusion, it was revealed that conophylline is capable
of inducing morphological changes of ARJ42 cells, thereby inducing
ARJ cells to produce insulin, but that conophylline is incapable of
inducing insulin secretion out of cells.
EXAMPLE 4
Effect of Lowering Blood Glucose Levels in vivo By Conophylline
[0115] Example 2 revealed that conophylline has the effect of
inducing pancreatic cells to produce and release insulin in vitro.
To examine whether the administration of conophylline induces
insulin production in vivo as well, changes in blood glucose levels
caused by the administration of conophylline in vivo were
measured.
[0116] Ten 1-day old Wistar rats (purchased from Japan SLC,
Shizuoka, Japan) were intraperitoneally injected with
streptozotocin (purchased from Wako Pure Chemical Industries, Ltd.,
dissolved in 0.05 mM citrate buffer (pH 4.5)) at 85 .mu.g/g BW per
rat. Streptozotocin decreases insulin production by destroying
pancreatic .beta. cells, thereby inducing diabetes. The day of
streptozotocin injection was defined as day 0. Starting from the
next day (day 1), five rats were injected subcutaneously with a
solution (ethanol) of conophylline at 5 .mu.g/g BW for seven
consecutive days and their random blood glucose levels were
measured daily. Five rats in the control group received the same
volume of solvent (Control). As a result, as shown in FIG. 4, all
the rats showed a remarkable increase in blood glucose levels when
streptozotocin was administered, whereas the rats administered
conophylline (black circle) showed an apparent decrease in blood
glucose levels, compared with the control (white circle),
indicating the effect of lowering blood glucose levels by
conophylline (*: P<0.05, **: P<0.01 vs Control). These
findings revealed that conophylline induces insulin production in
vivo as well.
[0117] In addition, conophylline alone exerted an effect, as
compared with the results obtained in vitro. This suggests that
when conophylline has been administered into the body, endogenous
nicotinamide and/or hepatocyte growth factor (HGF) have been
utilized.
INDUSTRIAL APPLICABILITY
[0118] Agents capable of inducing insulin production and/or
sercretion of non-neoplastic cells derived from the pancreas have
been provided by the present invention.
[0119] The agents according to the present invention can induce
differentiation of non-neoplastic cells derived from the pancreas.
Further, the agents according to the present invention can increase
insulin-producing and/or -secreting abilities of non-neoplastic
cells derived from the pancreas.
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