U.S. patent application number 13/700594 was filed with the patent office on 2013-06-13 for therapeutic agents for pancreatic cancer.
This patent application is currently assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA. The applicant listed for this patent is Masafumi Ikeda, Shuichi Mitsunaga, Atsushi Ochiai. Invention is credited to Masafumi Ikeda, Shuichi Mitsunaga, Atsushi Ochiai.
Application Number | 20130149302 13/700594 |
Document ID | / |
Family ID | 45004029 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130149302 |
Kind Code |
A1 |
Mitsunaga; Shuichi ; et
al. |
June 13, 2013 |
THERAPEUTIC AGENTS FOR PANCREATIC CANCER
Abstract
We achieved the present invention on the basis of the finding
that an excellent therapeutic effect against pancreatic cancer can
be obtained by administering an XL-6 inhibitor and an
antimetabolite to pancreatic cancer patients. We also found that
metastatic lesions from human pancreatic cancer can be reduced and
ascites can be eliminated.
Inventors: |
Mitsunaga; Shuichi;
(Kashiwa-shi, JP) ; Ochiai; Atsushi; (Kashiwa-shi,
JP) ; Ikeda; Masafumi; (Kashiwa-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mitsunaga; Shuichi
Ochiai; Atsushi
Ikeda; Masafumi |
Kashiwa-shi
Kashiwa-shi
Kashiwa-shi |
|
JP
JP
JP |
|
|
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
Tokyo
JP
NATIONAL CANCER CENTER
Tokyo
JP
|
Family ID: |
45004029 |
Appl. No.: |
13/700594 |
Filed: |
May 27, 2011 |
PCT Filed: |
May 27, 2011 |
PCT NO: |
PCT/JP2011/062191 |
371 Date: |
February 7, 2013 |
Current U.S.
Class: |
424/133.1 ;
530/387.3 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 43/00 20180101; A61K 31/7068 20130101; A61K 45/06 20130101;
A61K 39/3955 20130101; A61P 35/00 20180101; C07K 16/2866 20130101;
A61P 1/18 20180101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 39/3955 20130101; A61K 31/7068 20130101; C07K 2317/24
20130101 |
Class at
Publication: |
424/133.1 ;
530/387.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/7068 20060101 A61K031/7068 |
Foreign Application Data
Date |
Code |
Application Number |
May 28, 2010 |
JP |
122838/2010 |
Claims
1. A therapeutic composition for pancreatic cancer comprising an
IL-6 inhibitor, said composition further comprising an
antimetabolite or being administered in combination with an
antimetabolite.
2. The therapeutic composition of claim 1 wherein the IL-6
inhibitor is a substance binding to the IL-6 receptor.
3. The therapeutic composition of claim 2 wherein the substance
binding to the IL-6 receptor is an anti-IL-6 receptor antibody.
4. The therapeutic composition of claim 3 wherein the anti-IL-6
receptor antibody is a chimeric antibody, a humanized antibody or a
human antibody.
5. The therapeutic composition of claim 1 wherein the
antimetabolite is a cytosine analog.
6. The therapeutic composition of claim 5 wherein the cytosine
analog is gemcitabine or a salt thereof.
7. The therapeutic composition of claim 6 wherein gemcitabine or a
salt thereof is gemcitabine hydrochloride.
8. A method for treating pancreatic cancer comprising the step of
administering an IL-6 inhibitor and an antimetabolite to a
subject.
9. The method of claim 8 wherein the IL-6 inhibitor is a substance
binding to the IL-6 receptor.
10. The method of claim 9 wherein the substance binding to the IL-6
receptor is an anti-IL-6 receptor antibody.
11. The method of claim 10 wherein the anti-IL-6 receptor antibody
is a chimeric antibody, a humanized antibody or a human
antibody.
12. The method of claim 8 wherein the antimetabolite is a cytosine
analog.
13. The method of claim 12 wherein the cytosine analog is
gemcitabine or a salt thereof.
14. The method of claim 13 wherein gemcitabine or a salt thereof is
gemcitabine hydrochloride.
15. An IL-6 inhibitor for use in a method for treating pancreatic
cancer by administering it in combination with an
antimetabolite.
16. The inhibitor of claim 15 wherein the IL-6 inhibitor is a
substance binding to the IL-6 receptor.
17. The inhibitor of claim 16 wherein the substance binding to the
IL-6 receptor is an anti-IL-6 receptor antibody.
18. The inhibitor of claim 17 wherein the anti-IL-6 receptor
antibody is a chimeric antibody, a humanized antibody or a human
antibody.
19. The inhibitor of claim 8 wherein the antimetabolite is a
cytosine analog.
20. The inhibitor of claim 19 wherein the cytosine analog is
gemcitabine or a salt thereof.
21. The inhibitor of claim 20 wherein gemcitabine or a salt thereof
is gemcitabine hydrochloride.
Description
TECHNICAL FIELD
[0001] The present invention relates to therapeutic compositions
for pancreatic cancer. More specifically, the present invention
relates to therapeutic compositions for pancreatic cancer
comprising an interleukin 6 (IL-6) inhibitor, said compositions
further comprising or being administered in combination with an
antimetabolite.
BACKGROUND ART
[0002] IL-6 is a cytokine also called B cell stimulating factor 2
(BSF2) or interferon .beta.2. IL-6 was discovered as a
differentiation factor involved in the activation of B lymphocytes
(non-patent document 1), and then demonstrated to be a
multifunctional cytokine that influences functions of various cells
(non-patent document 2). IL-6 has been reported to induce
maturation of T lymphocytes (non-patent document 3).
[0003] IL-6 mediates its biological activities through two proteins
on cells. One is the IL-6 receptor that is a ligand-binding protein
having a molecular weight of about 80 kD to which IL-6 binds
(non-patent document 4, non-patent document 5). The IL-6 receptor
occurs as not only a membrane-bound receptor that traverses the
cell membrane and is expressed on the cell membrane but also
soluble IL-6 receptor mainly consisting of the extracellular
domain.
[0004] The other is gpl30 that is a non-ligand-binding
membrane-bound protein having a molecular weight of about 130 kD
involved in signal transduction. IL-6 and the IL-6 receptor form an
IL-6/IL-6 receptor complex and subsequently bind to gpl30, thereby
mediating biological activities of IL-6 into cells (non-patent
document 6).
[0005] Patent document 1 describes various forms of anti-IL-6 (IL-6
receptor) antibodies such as humanized anti-IL-6 antibodies,
chimeric anti-IL-6 antibodies and the like. Patent document 2
describes therapeutic agents for chronic rheumatoid arthritis and
synovial cell growth inhibitors containing IL-6 antagonists such as
anti-IL-6 antibodies as active ingredients. Patent document 3
describes treatment of diseases caused by IL-6 production such as
plasmacytosis, hyperimmunoglobulinemia, anemia, nephritis,
cachexia, rheumatism, Castleman's disease, mesangium proliferative
nephritis and the like. Patent document 4 describes
prophylactic/therapeutic agents for sensitized T cell-mediated
diseases such as multiple sclerosis, uveitis, chronic thyroiditis,
delayed hypersensitivity, contact dermatitis, atopic dermatitis and
the like, containing anti-IL-6 antibodies as active
ingredients.
[0006] Patent document 5 describes therapeutic agents for systemic
lupus erythematosus, containing anti-IL-6 antibodies as active
ingredients. Patent document 6 describes therapeutic agents for
Crohn's disease, containing anti-IL-6 antibodies as active
ingredients. Patent document 7 describes therapeutic agents for
pancreatitis containing anti-IL-6 antibodies as active ingredients.
Patent document 8 describes therapeutic agents for psoriasis
containing anti-IL-6 antibodies as active ingredients. Further,
patent document 9 describes therapeutic agents for juvenile chronic
arthritis containing anti-IL-6 antibodies as active ingredients.
Patent document 10 describes inhibitors of perineural invasion by
cells containing anti-IL-6 antibodies as active ingredients and
describes that perineural invasion in. human pancreatic cancer can
be suppressed.
[0007] Gemcitabine (Gemzar.RTM.) (HCl) is a cytosine analog that
inhibits DNA synthesis by inhibiting ribonucleotide reductase and
competing with dCTP for incorporation into DNA. Currently, it is
used as a therapeutic agent for some cancers such as pancreatic
cancer. It is also used as a combination therapy with radiotherapy,
but has not achieved significant improvement in extended survival
of pancreatic cancer patients. Some attempts have been made for
other effective combination therapies, but any improvement in
survival rate has not. been reported.
[0008] Pancreatic cancer is diagnosed at unresectable advanced
stages in many cases even today, and associated with rapid progress
and very poor prognosis. Even cases having undergone the only
possible cure resection often lead to early recurrence after
operation. On the other hand, chemotherapy is indicated for
unresectable cases with good performance status (PS) and major
organ function, but even the current standard of care does not have
sufficient therapeutic effect. For example, even gemcitabine
hydrochloride positioned as a first-line therapy has an efficacy in
symptom palliation of 23.8%, a median survival time of 5,7 months,
and a one-year survival rate of 18% (results from foreign phase III
clinical trials). In Japan, 20,000 people are diagnosed with and
22,260 people die of pancreatic cancer annually (The 2004
Demographic Survey by the Ministry of Health, Labour and Welfare of
Japan), and it is the fifth leading cause of cancer-related
death.
[0009] Information about prior art documents related to the
invention of the present application is shown below.
CITATION LIST
Non-Patent Documents
[0010] Non-patent document 1: Hirano, T. et al., Nature (1986) 324,
73-76;
[0011] Non-patent document 2: Akira, S. et al., Adv. in Immunology
(1993) 54, 1-78;
[0012] Non-patent document 3: Lotz, M. et al., J. Exp. Med. (1988)
167, 1253-1258;
[0013] Non-patent document 4: Taga, T. et al., J. Exp. Med. (1987)
166, 967-981:
[0014] Non-patent document 5: Yamasaki, K. et al., Science (1987)
241, 825-828;
[0015] Non-patent document 6: Taga, T. et al., Cell (1989) 58,
573-581.
Patent Documents
[0016] Patent document 1: International Publication WO92/19759;
[0017] Patent document 2: WO96/11020;
[0018] Patent document 3: WO96/12503;
[0019] Patent document 4: WO98/42377;
[0020] Patent document 5: WO98/42377;
[0021] Patent document 6: WO99/47170;
[0022] Patent document 7: WO00/10607;
[0023] Patent document 8: WO02/3492;
[0024] Patent document 9: WO02/080969;
[0025] Patent document 10: WO2009/148148.
SUMMARY OF INVENTION
Technical Problem
[0026] The present invention aims to provide a novel therapeutic
agent for pancreatic cancer.
Solution to Problem
[0027] As a result of careful studies to solve the above problems,
we accomplished the present invention on the basis of the finding
that an excellent therapeutic effect against pancreatic cancer can
be obtained by using an IL-6 inhibitor and an antimetabolite in
combination in pancreatic cancer patients. We also found that
metastatic lesions from human pancreatic cancer can be reduced and
ascites can be eliminated by the combination described above.
[0028] Accordingly, the present invention more specifically
provides [1]-[21] below.
[1] A therapeutic composition for pancreatic cancer comprising an
IL-6 inhibitor, said composition further comprising an
antimetabolite or being administered in combination with an
antimetabolite.
[0029] [2] The therapeutic composition of [1] wherein the IL-6
inhibitor is a substance binding to the IL-6 receptor.
[0030] [3] The therapeutic composition of [2] wherein the substance
binding to the IL-6 receptor is an anti-IL-6 receptor antibody.
[0031] [4] The therapeutic composition of [3] wherein the anti-IL-6
receptor antibody is a chimeric antibody, a humanized antibody or a
human antibody.
[0032] [5] The therapeutic composition of any one of [1]-[4]
wherein the antimetabolite is a cytosine analog.
[0033] [6] The therapeutic composition of [5] wherein the cytosine
analog is gemcitabine or a salt thereof.
[0034] [7] The therapeutic composition of [6] wherein gemcitabine
or a salt thereof is gemcitabine hydrochloride.
[0035] [8] A method for treating pancreatic cancer comprising the
step of administering an IL-6 inhibitor and an antimetabolite to a
subject.
[0036] [9] The method of [8] wherein the IL-6 inhibitor is a
substance binding to the IL-6 receptor.
[0037] [10] The method of [9] wherein the substance binding to the
IL-6 receptor is an anti-IL-6 receptor antibody.
[0038] [11] The method of [10] wherein the anti-IL-6 receptor
antibody is a chimeric antibody, a humanized antibody or a human
antibody.
[0039] [12] The method of any one of [8]-[11] wherein the
antimetabolite is a cytosine analog.
[0040] [13] The method of [12]wherein the cytosine analog is
gemcitabine or a salt thereof.
[0041] [14] The method of [13] wherein gemcitabine or a salt
thereof is gemcitabine hydrochloride.
[0042] [15] An IL-6 inhibitor for use in a method for treating
pancreatic cancer by administering it in combination with an
antimetabolite.
[0043] [16] The inhibitor of [15] wherein the IL-6 inhibitor is a
substance binding to the IL-6 receptor.
[0044] [17] The inhibitor of [16] wherein the substance binding to
the IL-6 receptor is an anti-IL-6 receptor antibody.
[0045] [18] The inhibitor of [17] wherein the anti-IL-6 receptor
antibody is a chimeric antibody, a humanized antibody or a human
antibody.
[0046] [19] The inhibitor of any one of [15]-[18] wherein the
antimetabolite is a cytosine analog.
[0047] [20] The inhibitor of [19] wherein the cytosine analog is
gemcitabine or a salt thereof.
[0048] [21] The inhibitor of [20] wherein gemcitabine or a salt
thereof is gemcitabine hydrochloride.
Advantageous Effects of Invention
[0049] An excellent therapeutic effect against pancreatic cancer
was obtained by using an IL-6 inhibitor and an antimetabolite in
combination. Moreover, it was shown that metastatic lesions from
human pancreatic cancer can be reduced and ascites can be
eliminated by the combination described above. Especially, there
has been no report that ascites is eliminated by monotherapy.
BRIEF DESCRIPTION OF DRAWINGS
[0050] FIG. 1 shows a CT image of ascites of a pancreatic cancer
patient before gemcitabine hydrochloride and tocilizumab are
administered in combination.
[0051] FIG. 2 shows a CT image of ascites of the pancreatic cancer
patient 8 weeks after starting combined administration of
gemcitabine hydrochloride and tocilizumab.
[0052] FIG. 3 shows a CT image of metastatic lesions in the liver
of the pancreatic cancer patient before gemcitabine hydrochloride
and tocilizumab are administered in combination.
[0053] FIG. 4 shows a CT image of metastatic lesions in the liver
of the pancreatic cancer patient 8 weeks after starting combined
administration of gemcitabine hydrochloride and tocilizumab.
DESCRIPTION OF EMBODIMENTS
[0054] As used herein, "IL-6 inhibitor" refers to a substance that
blocks IL-6-mediated signal transduction to inhibit biological
activities of IL-6. Specific examples of IL-6 inhibitors include
substances binding to IL-6, substances binding to the IL-6
receptor, substances binding to gpl30 and the like. IL-6 inhibitors
also include substances inhibiting STAT3 phosphorylation that is a
primary event in IL-6-mediated intracellular signaling, such as
AG490. IL-6 inhibitors include, but not specifically limited to,
anti-IL-6 antibodies, anti-IL-6 receptor antibodies, anti-gpl30
antibodies, IL-6 variants, soluble IL-6 receptor variants, IL-6
peptides, IL-6 receptor peptides, and small molecules showing
similar activities to those of these substances, etc.
[0055] Preferred examples of IL-6 inhibitors include IL-6 receptor
inhibitors, especially anti-IL-6 receptor antibodies.
[0056] The origin from which antibodies used in the present
invention are derived is not specifically limited, but preferably a
mammal, more preferably human.
[0057] Antibodies used in the present invention can be obtained as
polyclonal or monoclonal antibodies by using known means.
Antibodies used in the present invention are preferably monoclonal
antibodies especially derived from mammals. Monoclonal antibodies
derived from mammals include those produced by hybridomas and those
produced by hosts transformed with an expression vector containing
an antibody gene by genetic engineering techniques. Normally, these
antibodies block mediation of biological activities of IL-6 into
cells by binding to IL-6, the IL-6 receptor, gpl30 or the like.
[0058] Hybridomas producing monoclonal antibodies can be
constructed basically using known techniques as follows. That is,
the IL-6 receptor, IL-6, gpl30 or the like is used as an immunizing
antigen to immunize host cells according to a standard immunization
technique, and the resulting immunized cells are fused to known
parent cells by a standard cell fusion technique, and then the
fused ceils are screened for monoclonal antibody-producing cells by
a standard screening method.
[0059] Specifically, monoclonal antibodies can be prepared as
follows. When anti-IL-6 receptor antibodies are to be prepared, for
example, the human IL-6 receptor or mouse IL-6 receptor used as an
immunizing antigen for preparing the antibodies is obtained by
using the nucleotide/amino acid sequence of the IL-6 receptor
disclosed in European Patent Application published as EP 325474 or
Japanese Patent Application published as JPA H03-155795,
respectively.
[0060] The IL-6 receptor protein occurs in two forms, i.e., one is
expressed on the plasma membrane and the other is separated from
the plasma membrane (soluble IL-6 receptor) (Yasukawa, K. et al.,
J. Biochem. (1990) 108, 673-676). The soluble IL-6 receptor differs
from the membrane-bound IL-6 receptor in that it substantially
consists of the extracellular domain of the IL-6 receptor bound to
the plasma membrane and lacks the transmembrane domain or the
transmembrane domain and the intracellular domain. Either IL-6
receptor protein can be used so far as it can be used as an
immunizing antigen for preparing anti-IL-6 receptor antibodies used
in the present invention.
[0061] A suitable host cell is transformed with a known expression
vector system containing the nucleotide sequence of the IL-6
receptor, and then the desired IL-6 receptor protein is purified
from the host cell or the culture supernatant by a known method,
and this purified IL-6 receptor protein can be used as an
immunizing antigen. Alternatively, a cell expressing the IL-6
receptor or a fusion protein of the IL-6 receptor protein with
another protein may also be used as an immunizing antigen.
[0062] Similarly, when IL-6 is used as an immunizing antigen for
preparing antibodies, the human IL-6 is obtained by using the
nucleotide/amino acid sequence of IL-6 disclosed in Eur. J. Biochem
(1987) 168, 543-550, J. Immunol. (1988) 140, 1534-1541 or Agr. Biol
Chem. (1990) 54, 2685-2688. Further, the nucleotide/amino acid
sequence of gpl30 disclosed in European Patent Application
published as EP 411946 can be used as an immunizing antigen for
obtaining anti-gpl30 antibodies.
[0063] Mammals immunized with the immunizing antigens are not
specifically limited, but preferably selected on the basis of the
compatibility with parent cells used for cell fusion, and rodents
such as mice, rats and hamsters are typically used.
[0064] Animals are immunized with the immunizing antigens according
to known methods. For example, a typical method is intraperitoneal
or subcutaneous injection of an immunizing antigen into a mammal.
Specifically, an immunizing antigen is preferably diluted or
suspended in PBS (Phosphate-Buffered Saline) or physiological
saline to an appropriate volume and, if desired, mixed with an
appropriate amount of a conventional adjuvant such as Freund's
complete adjuvant, and emulsified and then administered to a mammal
several times every 4-21 days. A suitable vehicle can be used
during immunization with the immunizing antigen.
[0065] After immunizing the mammal in this manner and confirming an
increase in the serum level of a desired antibody, immunized cells
are collected from the mammal and used for cell fusion. Preferred
immunized cells used for cell fusion include spleen cells among
others.
[0066] As for myeloma cells of the mammal used as parent cells to
which the immunized cells are fused, various previously known cell
lines are appropriately used such as P3X63Ag8.653 (Kearney, J. F.
et al. J. Immunol. (1979) 123, 1548-1550), P3X63Ag8U.1 (Current
Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1
(Kohler, G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519),
MPC-11 (Margulies. D. H. et al., Cell (1976) 8, 405-415), SP2/0
(Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth,
S. F. et al., J. Immunol. Methods (1980) 35, 1-21), S194
(Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323), R210 (Galfre,
G. et al., Nature (1979) 277, 131-133), etc.
[0067] Cell fusion of the immunized cells to myeloma cells can be
performed basically according to known methods such as the method
of Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol,
(1981) 73, 3-46) and the like.
[0068] More specifically, the cell fusion is performed, for
example, in a conventional nutrient culture medium in the presence
of a cell fusion promoter. Fusion promoters that can be used
include, for example, polyethylene glycol (PEG), Sendai virus (HVJ)
and the like, and, if desired, an adjuvant such as dimethyl
sulfoxide can also be added to increase the fusion efficiency.
[0069] Immunized cells and myeloma cells are preferably used in a
ratio of immunized cells to myeloma cells of 1-10, for example.
Culture media that can be used for the cell fusion include, for
example, RPMI1640 and MEM, which are well-suitable for culturing
the myeloma cell lines listed above, and other conventional culture
media used for this type of cell culture, optionally in combination
with serum supplements such as fetal calf serum (FCS).
[0070] Cell fusion is performed by thoroughly mixing given amounts
of the immunized cells and myeloma cells in the culture medium,
adding a PEG solution such as a PEG solution having an average
molecular weight of about 1000-6000 preheated to about 37.degree.
C. typically at a concentration of 30-60% (w/v) and mixing the
suspension to form desired fused cells (hybridomas). Then, cell
fusion promoters and the like that are undesirable for the growth
of hybridomas can be removed by repeating the sequential steps of
adding a suitable culture medium, centrifuging the suspension and
removing the supernatant.
[0071] The hybridomas are selected by incubation in a conventional
selective culture medium such as HAT medium (a culture medium
containing hypoxanthine, aminopterin and thymidine). The incubation
in the HAT medium is continued for a sufficient period to kill
cells other than desired hybridomas (non-fused cells), typically
several days to several weeks. Then, hybridomas producing the
desired antibody are screened by conventional limiting dilution and
are cloned.
[0072] As an alternative to the method for obtaining the hybridomas
by immunizing a non-human animal with an antigen, a desired human
antibody having a binding activity for a desired antigen protein or
antigen-expressing cell can also be obtained by in vitro immunizing
human lymphocytes with the desired antigen or antigen-expressing
cell and fusing the immunized lymphocytes to human myeloma cells
such as U266 (see JPB H01-59878). Alternatively, a desired human
antibody can also be obtained by the method described above except
that an antigen or antigen-expressing cell is administered to a
transgenic animal harboring the human antibody gene repertoire (see
International Patent Applications published as WO 93/12227, WO
92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).
[0073] Hybridomas producing monoclonal antibodies prepared in this
manner can be subcultured in conventional culture media and stored
for a long period in liquid nitrogen.
[0074] Monoclonal antibodies can be obtained from the hybridomas as
culture supernatant after the hybridomas are cultured according to
conventional methods or as ascites fluid after the hybridomas are
grown in a mammal compatible with them. The former method is
suitable for obtaining high purity antibodies while the latter
method is suitable for mass production of antibodies.
[0075] For example, hybridomas producing anti-IL-6 receptor
antibodies can be prepared by the method disclosed in JPA
H03-139293. They can be prepared by injecting PM-1
antibody-producing hybridomas into the peritoneal cavity of a
BALB/c mouse to produce ascites fluid and purifying PM-1 antibodies
from this ascites fluid, or by culturing the hybridomas in a
suitable culture medium such as RPMI1640 containing 10% bovine
fetal serum and 5% BM-Condimed H1 (from Boehringer Mannheim),
Hybridoma SFM (from GIBCO-BRL), PFHM-II (from GIBCO-BRL) or the
like and purifying PM-1 antibodies from the culture
supernatant.
[0076] In the present invention, recombinant antibodies produced
using genetic engineering techniques by transforming a host with a
suitable vector containing an antibody gene cloned from a hybridoma
can be used as monoclonal antibodies (see e.g., Borrebaeck C. A. K.
and Larrick J. W. THERAPEUTIC MONOCLONAL ANTIBODIES, Published in
the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).
[0077] Specifically, mRNA sequences encoding the variable (V)
regions of a desired antibody are isolated from cells producing the
antibody such as hybridomas. Isolation of mRNA is accomplished by
preparing total RNA by known methods such as guanidine
ultracentrifugation (Chirgwin, J. M. et al., Biochemistry (1979)
18, 5294-5299), the AGPC method (Chomczynski, P. et al., Anal.
Biochem. (1987)162, 156-159) or the like and preparing mRNA using
an mRNA Purification Kit (from Pharmacia) or the like. mRNA can be
directly prepared by using QuickPrep mRNA Purification Kit (from
Pharmacia).
[0078] A reverse transcriptase is used to synthesize cDNA sequences
of the antibody V regions from the mRNA sequences obtained.
Synthesis of cDNA can be made by using AMV Reverse Transcriptase
First-strand cDNA Synthesis Kit or the like. Synthesis and
amplification of cDNA can be made by 5'-RACE (Frohman, M. A. et
al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A.
et al., Nucleic Acids Res. (1989) 17, 2919-2932) using 5'-Ampli
FINDER RACE Kit (from Clontech) and PCR. Desired DNA fragments are
purified from the resulting PCR products and linked to vector DNA.
Thus, recombinant vectors are generated and transferred into E.
coli or the like and colonies are selected to prepare desired
recombinant vectors. The nucleotide sequence of the desired DNA is
confirmed by known methods such as dideoxynucleotide chain
termination.
[0079] Once DNA sequences encoding the V regions of the desired
antibody are obtained, they are linked to DNA sequences encoding
the constant regions (C regions) of the desired antibody and
inserted into an expression vector. Alternatively, DNA sequences
encoding the V regions of the antibody can be inserted into an
expression vector containing DNA sequences of the C regions of the
antibody.
[0080] To prepare antibodies used in the present invention, an
antibody gene can be inserted into an expression vector in such a
manner that it can be expressed under the control of expression
regulatory regions such as enhancers and promoters, as described
below. Then, a host cell can be transformed with this expression
vector to express the antibody.
[0081] In the present invention, recombinant antibodies, i.e.
antibodies artificially modified to reduce antigenicity in humans
or for other purposes such as chimeric antibodies and humanized
antibodies can be used. These modified antibodies can be prepared
by known processes.
[0082] Chimeric antibodies can be obtained by linking DNA sequences
encoding the antibody variable regions obtained as described above
to DNA sequences encoding the constant regions of a human antibody
and transforming a host with an expression vector containing the
linked sequences to allow it to produce a recombinant antibody (see
European Patent Application published as EP 125023, International
Patent Application published as WO 92-19759). Chimeric antibodies
useful for the present invention can be obtained by using this
known method.
[0083] Humanized antibodies are also called reshaped human
antibodies, and obtained by grafting the
complementarity-determining regions (CDRs) of an antibody from a
non-human mammal such as a mouse into the
complementarity-determining regions of a human antibody and typical
gene recombination techniques for preparing them are also known
(see European Patent Application published as EP 125023,
International Patent Application published as WO 92-19759).
[0084] Specifically, DNA sequences designed to link the CDRs of a
mouse antibody to the framework regions (FRs) of a human antibody
are synthesized by PCR from several oligonucleotides prepared to
have terminal overlapping regions. The resulting DNA sequences are
linked to DNA sequences encoding the constant regions of the human
antibody and then inserted into an expression vector, which is
transformed into a host to allow it to produce a recombinant
antibody (see European Patent Application published as EP 239400,
International Patent Application published as WO 92-19759).
[0085] The FRs of the human antibody to be linked via the CDRs are
selected so that the complementarity-determining regions form an
appropriate antigen-binding site. If necessary, reshaped human
antibodies may have some amino acid changes in the framework
regions in the variable regions of the antibodies so that the
complementarity-determining regions form an appropriate
antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53,
851-856).
[0086] Chimeric antibodies and humanized antibodies typically use
human antibody constant regions. Human antibody heavy chain
constant regions that can be used include C.gamma. or the like such
as C.gamma.1, C.gamma.2, C.gamma.3 or C.gamma.4. Human antibody
light chain constant regions include, for example, .kappa. or
.lamda.. The human antibody constant regions can be modified to
improve the stability of the antibody or production thereof.
[0087] Chimeric antibodies consist of the variable regions of an
antibody derived from a non-human mammal and the constant regions
derived from a human antibody, while humanized antibodies consist
of the complementarity determining regions of an antibody derived
from a non-human mammal and the framework regions and constant
regions derived from a human antibody so that they are useful as
antibodies used as pharmaceuticals because of their reduced
antigenicity in human bodies.
[0088] Preferred specific examples of humanized antibodies used in
the present invention include humanized PM-1 antibodies (see
International Patent Application published as WO 92-19759).
[0089] In addition to the methods for obtaining human antibodies
described above, methods for obtaining human antibodies by panning
using a human antibody library are also known. For example, phages
that bind to an antigen can be selected by expressing the variable
regions of a human antibody as single chain antibody fragments
(scFvs) on phage surfaces by a phage display method. The DNA
sequences encoding the variable regions of the human antibody that
binds to the antigen can be determined by analyzing the genes of
the phages selected. Once the DNA sequences of the scFvs that bind
to the antigen are known, a suitable expression vector containing
the sequences can be prepared to produce a human antibody. These
methods have already been well known and can be found in WO
92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO
95/01438, and WO 95/15388.
[0090] The antibody gene constructed as described above can be
expressed by known methods. When mammalian cells are used, the
antibody gene can be expressed in a DNA sequence to which
conventional useful promoters, the antibody gene to be expressed
and a polyA signal downstream of or 3' to the gene are operably
linked or a vector containing it. For example, promoters/enhancers
include human cytomegalovirus immediate early
promoter/enhancer.
[0091] Other promoters/enhancers that can be used for expressing
antibodies used in the present invention include viral
promoters/enhancers derived from retroviruses, polyomaviruses,
adenoviruses, simian virus 40 (SV40) and the like or
promoters/enhancers derived from mammalian cells such as human
elongation factor 1.alpha. (HEF1.alpha.).
[0092] For example, expression can be readily accomplished
according to the method of Mulligan et al. (Mulligan, R. C. et al.,
Nature (1979) 277, 108-114) when the SV40 promoter/enhancer is used
or according to the method of Mizushima et al. (Mizushima, S. and
Nagata, 8.Nucleic Acids Res. (1990) 18, 5322) when the HEF1.alpha.
promoter/enhancer is used.
[0093] Hosts using prokaryotic cells include producing systems
using bacterial cells. Known bacterial cells include E. coli and
Bacillus subtilis.
[0094] In E. coli, an antibody gene can be expressed by operably
linking conventional useful promoters, a signal sequence for
antibody secretion and the antibody gene to be expressed. For
example, promoters include the lacZ promoter and araB promoter.
Expression can be accomplished according to the method of Ward et
al. (Ward, E. S. et al., Nature (1989) 341, 544-546; Ward, E. S. et
al. FASEB J. (1992) 6, 2422-2427) when the lacZ promoter is used or
according to the method of Better et al. (Better, M. et al. Science
(1988) 240, 1041-1043) when the araB promoter is used.
[0095] The pelB signal sequence (Lei, S. P. et al J. Bacteriol.
(1987) 169, 4379-4383) can be used as the signal sequence for
antibody secretion when the antibody is to be produced in the
periplasm of E. coli. The antibody produced in the periplasm is
isolated, and then used after the structure of the antibody is
suitably refolded (see e.g., WO96/30394).
[0096] An origin of replication derived from SV40, polyomaviruses,
adenoviruses, bovine papillomavirus (BPV) and the like can be used,
and the expression vector can also contain a selectable marker such
as the aminoglycoside phosphotransferase (APH) gene, thymidine
kinase (TK) gene, E. coli xanthine-guanine phoshoribosyl
transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene or
the like to amplify the gene copy number in the host cell
system.
[0097] Any producing systems can be used to prepare antibodies used
in the present invention. Producing systems for preparing the
antibodies include in vitro and in vivo producing systems. In vitro
producing systems include producing systems using eukaryotic cells
and producing systems using prokaryotic cells.
[0098] Producing systems using eukaryotic cells as hosts include
those using animal cells, plant cells or fungal cells. Known animal
cells include (1) mammalian cells such as CHO, COS, myeloma, BHK
(baby hamster kidney), HeLa and Vero cells; (2) amphibian cells
such as Xenopus oocytes; or (3) insect sells such as sf9, sf21 and
Tn5. Known plant cells include cells derived from Nicotiana
tabacum, which can be grown as callus cultures. Known fungal cells
include yeasts such as Saccharomyces spp., e.g. Saccharomyces
serevisiae and filamentous fungi such as Aspergillus spp., e.g.
Aspergillus niger.
[0099] Antibodies can be obtained by transforming these cells with
a desired antibody gene and culturing the transformed cells in
vitro. Cultivation is performed according to known methods. For
example, DMEM, MEM, RPMI1640 and IMDM can be used as culture media
optionally in combination with serum supplements such as fetal calf
serum (FCS). Alternatively, antibodies may be produced in vivo by
transplanting cells transformed with an antibody gene into the
peritoneal cavity or the like of an animal.
[0100] On the other hand, in vivo producing systems include
producing systems using animals and producing systems using plants.
Producing systems using animals include those using mammals,
insects and the like.
[0101] Mammals that can be used include goat, pig, sheep, mouse,
cow and the like (Vicki Glaser, SPECTRUM Biotechnology
Applications, 1993). Insects that can be used include silkworm.
Plants that can be used include, for example, tobacco.
[0102] An antibody gene is transferred into these animals or plants
and antibodies are produced in vivo in the animals or plants and
recovered. For example, an antibody gene is inserted midway in a
gene encoding a protein produced specifically in milk such as goat
.beta. casein to prepare a fusion gene. A DNA fragment bearing the
fusion gene containing the antibody gene is injected into the
embryo of a goat and this embryo is implanted into a female goat. A
desired antibody is obtained from the milk produced by a transgenic
goat born from the goat impregnated with the embryo or progeny
thereof. To increase the amount of milk containing the desired
antibody produced by the transgenic goat, hormones may be used for
the transgenic goat as appropriate (Ebert, K. M. et al.,
Bio/Technology (1994) 12, 699-702).
[0103] When silkworm is used, a desired antibody is obtained from
the body fluid of silkworm infected with a baculovirus containing
the gene for the desired antibody (Maeda, S. et al., Nature (1985)
315, 592-594). When tobacco is used, the gene for a desired
antibody is inserted into a plant expression vector such as pMON
530, and this vector is transferred into a bacterium such as
Agrobacterium tumefaciens. The desired antibody is obtained from
leaves of tobacco such as Nicotiana tabacum infected with this
bacterium (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24,
131-138).
[0104] When an antibody is to be produced in an in vitro or in vivo
producing system as described above, a host may be cotransformed
with expression vectors separately containing DNA sequences
encoding the heavy chain (H chain) or light chain (L chain) of the
antibody or may be transformed with a single expression vector
containing DNA sequences encoding the heavy and light chains (see
International Patent Application published as WO 94-11523).
[0105] Antibodies used in the present invention may be antibody
fragments or modified products thereof so far as they can be
conveniently used in the present invention. For example, antibody
fragments include Fab, (Fab').sub.2, Fv, or single chain Fv (scFv)
in which heavy and light chain Fv fragments are joined via a
suitable linker.
[0106] Specifically, an antibody is treated with an enzyme such as
papain or pepsin to produce antibody fragments or genes encoding
these antibody fragments are constructed and introduced into an
expression vector and then expressed in a suitable host cell (see
e.g., Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976, Better,
M. & Horwitz, A. H. Methods in Enzymology (1989) 178, 476-496,
Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178,
497-515, Lamoyi, E., Methods in Enzymology (1989) 121, 652-663,
Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-66,
Bird, R. E. et al., TIBTECH (1991) 9, 132-137).
[0107] scFvs are obtained by connecting a heavy chain variable
region and a light chain variable region of an antibody. In the
scFvs, the heavy chain variable region and light chain variable
region are connected via a linker, preferably a peptide linker
(Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,
5879-5883). The heavy chain variable region and light chain
variable region in the scFvs may be derived from any of the
antibodies listed above. Any single-chain peptide composed of 12-19
amino acid residues can be used, for example, as the peptide linker
connecting the variable regions.
[0108] DNA sequences encoding scFvs are obtained by using a DNA
sequence encoding the heavy chain or the heavy chain variable
region and a DNA sequence encoding the light chain or the light
chain variable region of the antibody as templates to amplify a DNA
segment encoding a desired amino acid sequence of the template DNA
sequences by PCR using a primer pair defining both ends of the
segment, and then further amplifying a DNA sequence encoding a
peptide linker segment using a primer pair defining both ends of
the linker segment to be connected to the heavy chain and the light
chain, respectively.
[0109] Once DNA sequences encoding scFvs are prepared, an
expression vector containing them and a host transformed with the
expression vector can be obtained by conventional methods, and
scFvs can be obtained by using the host according to conventional
methods.
[0110] These antibody fragments can be produced by the host after
the genes for them are obtained and expressed in the same manner as
described above. As used herein, "antibody" also means to include
these antibody fragments.
[0111] Modified antibodies including antibodies conjugated with
various molecules such as polyethylene glycol (PEG) can also be
used. These modified antibodies are also included in the "antibody"
as used herein. Such modified antibodies can be obtained by
chemically modifying antibodies produced. These methods have
already been established in this field of art.
[0112] Antibodies produced and expressed as described above can be
isolated from cells or hosts and purified to homogeneity. Isolation
and purification of antibodies used in the present invention can be
perforated by affinity chromatography. Columns used for affinity
chromatography include, for example, protein A columns and protein
G columns. Supports used for protein A columns include, for
example, HyperD, POROS, Sepharose F.F. and the like Any other
isolation/purification methods conventionally used for proteins may
be used without limitation.
[0113] For example, antibodies used in the present invention can be
isolated/purified by appropriately selecting and combining
chromatographies other than affinity chromatography described
above, filtration, ultrafiltration, salting, dialysis and the like.
Chromatographies include, for example, ion exchange chromatography,
hydrophobic chromatography, gel filtration chromatography and the
like. These chromatographies can be applied to HPLC (High
performance liquid chromatography). Reverse phase HPLC can also be
used.
[0114] The concentration of the antibodies obtained above can be
determined by absorbance assays or ELISA or the like. Specifically,
an absorbance assay can be performed by appropriately diluting an
antibody with PBS(-), then measuring the absorbance at 280 nm, and
calculating the concentration on the basis that a 1 mg/ml solution
has 1.35 OD. On the other hand, ELISA assay can be performed as
follows. That is, 100 .mu.l of goat anti-human IgG (from TAG)
diluted to 1 .mu.g/ml with 0.1 M bicarbonate buffer (pH 9.6) is
added to a 96-well plate (from Nunc) and incubated overnight at
4.degree. C. to immobilize the antibody. After blocking, 100 .mu.l
of an appropriately diluted antibody used in the present invention
or a sample containing the antibody or human IgG standard (from
CAPPEL) is added and incubated at room temperature for 1 hour.
[0115] After washing, 100 .mu.l of 1:5000 diluted alkaline
phosphatase-conjugated anti-human IgG (from BIO SOURCE) is added
and incubated at room temperature for 1 hour. After washing, a
substrate solution is added and incubated, and then the absorbance
at 405 nm is measured using MICROPLATE READER Model 3550 (from
Bio-Rad) and the concentration of the antibody of interest is
calculated.
[0116] Specific examples of anti-IL-6 antibodies include, but not
specifically limited to, MH166 (Matsuda, T. et al., Eur. J.
Immunol. (1998) 18, 951-956), SK2 antibodies (Sato K et al.,
Academic Conference Proceedings of 21st Annual Meeting of Japanese
Society for Immunology (1991) 21, 166), and the like.
[0117] Specific examples of anti-IL-6 receptor antibodies include,
but not specifically limited to, MR16-1 (Tamura, T. et al. Proc.
Natl. Acad. Sci. USA (1993) 90, 11924-11928), PM-1 antibodies
(Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906), AUK12-20,
AUK64-7 or AUK146-15 (International Patent Application published as
WO 92-19759), etc. Among them, preferred monoclonal antibodies
against the human IL-6 receptor include, but not limited to, PM-1
antibodies, and preferred monoclonal antibodies against the mouse
IL-6 receptor include, but not limited to, MR16-1. A preferred
example of humanized anti-IL-6 receptor antibodies includes the
humanized PM-1 antibody (Tocilizumab, MRA). Other preferred
examples of humanized anti-IL-6 receptor antibodies include the
antibodies described in WO2009/041621. Further, additional
preferred examples of anti-IL-6 receptor antibodies include
anti-IL-6 receptor antibodies recognizing the same epitope as
recognized by the humanized PM-1 antibody (Tocilizumab, MRA).
[0118] Specific examples of anti-gpl30 antibodies include, but not
specifically limited to, AM64 (Japanese Patent Application
published as JPA H03-219894), 4B11, 2H4 (U.S. Pat. No. 5,571,513),
B-P8 (JPA H08-291199), etc.
[0119] IL-6 variants used in the present invention are substances
having binding activity for the IL-6 receptor but not mediating
biological activities of IL-6. In other words, IL-6 variants bind
to the IL-6 receptor competitively with IL-6, but block
IL-6-mediated signal transduction because they do not mediate
biological activities of IL-6.
[0120] IL-6 variants are prepared by introducing variations by
changing amino acid residues of the amino acid sequence of IL-6.
IL-6 variants are derived from IL-6 of any origin, but preferably
human IL-6 in view of antigenicity or the like. Specifically, a
variant is obtained by predicting the secondary structure of IL-6
from its amino acid sequence using a known molecular modeling
program such as WHATIF (Vriend et al., J. Mol. Graphics (1990) 8,
52-56) and evaluating the influence of an amino acid residue to be
changed on the complete amino acid sequence. After a suitable amino
acid residue to be changed is determined, a vector containing a
nucleotide sequence encoding the human IL-6 gene is used as a
template to introduce a variation by conventional PCR so that the
amino acid is changed, whereby a gene encoding an IL-6 variant can
be obtained. This gene can be inserted into a suitable expression
vector as appropriate and manipulated according to the methods for
expressing, producing and purifying recombinant antibodies
described above, whereby the IL-6 variant can be obtained.
[0121] Specific examples of IL-6 variants include the IL-6 variants
disclosed in Brakenhoff et al., J. Biol Chem. (1994) 269, 86-93;
and Savino et al., EMBO J. (1994) 13, 1357-1367, WO 96-18648 and
WO96-17869.
[0122] IL-6 receptor peptides are peptides consisting of the amino
acid sequence of a part or all of the region responsible for
binding of IL-6 to the IL-6 receptor in the amino acid sequence of
the IL-6 receptor. Such peptides typically consist of 10-80,
preferably 20-50, more preferably 20-40 amino acid residues.
[0123] IL-6 receptor peptides can be prepared by commonly known
methods such as genetic engineering techniques or peptide synthesis
on the basis of the amino acid sequence of a part or all of the
region responsible for binding of IL-6 to the IL-6 receptor in the
amino acid sequence of the IL-6 receptor after identifying such a
region.
[0124] To prepare IL-6 receptor peptides by genetic engineering
techniques, the DNA sequence encoding a desired peptide can be
inserted into an expression vector and manipulated according to the
methods for expressing, producing and purifying recombinant
antibodies described above.
[0125] To prepare IL-6 receptor peptides by peptide synthesis,
methods commonly used in peptide synthesis such as solid-phase
synthesis or liquid-phase synthesis can be used.
[0126] Specifically, the method described in "Development of
Pharmaceuticals II", volume 14, Peptide Synthesis, edited by
Haruaki Yajima, Hirokawa Publishing Company 1991 can be followed.
Solid-phase synthesis involves, for example, attaching an amino
acid corresponding to the C-terminus of a peptide to be synthesized
to a support insoluble in organic solvents, and elongating a
peptide chain by alternately repeating a reaction in which amino
acids protected at the .alpha.-amino group and side chain
functional groups by a suitable protecting group are condensed one
by one in the direction from the C-terminus to the N-terminus and a
reaction in which the protecting group of the .alpha.-amino group
of the amino acid or peptide attached to the resin are removed.
Solid-phase peptide synthesis is mainly classified into the Boc
method and the Fmoc method depending on the type of the protecting
group used.
[0127] After the desired peptide is synthesized in this manner, it
is deprotected and cleaved from the support of the peptide chain
take place. For cleavage from the peptide chain, hydrogen fluoride
or trifluoromethanesultonic acid can be typically used in the Boc
method while TFA can be typically used in the Fmoc method. In the
Boc method, the protected peptide bound to the resin described
above is treated in hydrogen fluoride in the presence of anisole,
for example. Then, the protecting group is removed and the peptide
is cleaved from the support and recovered. The recovered peptide is
lyophilized to give a crude peptide. In the Fmoc method, on the
other hand, deprotection and cleavage from the support of the
peptide chain can be performed in TFA, for example, by procedures
similar to those described above.
[0128] The resulting crude peptide can be isolated/purified by HPLC
under optimal conditions eluting with a water/acetonitrile solvent
system commonly used for purification of proteins. Fractions
corresponding to peaks in the resulting chromatographic profile are
collected and lyophilized. The peptide fractions purified in this
matter are identified by mass spectrometry-based molecular weight
analysis, amino acid composition analysis, or amino acid sequence
analysis or the like.
[0129] As used herein, "antimetabolite" is not specifically limited
so far as it is a substance that inhibits cellular reactions by a
structure or function chemically similar to those of physiological
metabolites necessary for nucleic acid synthesis or metabolic
reaction, but especially preferably a nucleotide that inhibits DNA
synthesis. Specifically, examples include thio-6-guanine,
pentostatin, cytosine arabinoside, fluoro-5-uracil (5FU),
fiuorouridine-desoxyribose, capecitabine, gemcitabine, fludarabine
and the like. Especially, cytosine analogs are preferred, among
which gemcitabine is preferred. A preferred example of gemcitabine
includes gemcitabine hydrochloride.
[0130] Therapeutic compositions for pancreatic cancer of the
present invention can be used for treatment and/or prophylaxis of
pancreatic cancer.
[0131] As used herein, "treatment of pancreatic cancer" refers to
inhibition of development of pancreatic cancer, decrease in the
incidence of pancreatic cancer, inhibition of the growth of
pancreatic cancer cells, size reduction of pancreatic cancer
tissue, improvement of symptoms of pancreatic cancer, inhibition of
metastasis of pancreatic cancer, inhibition, reduction and
elimination of pleural effusion or ascites caused by development of
pancreatic cancer and the like.
[0132] As used herein, "combined administration" of an IL-6
inhibitor and an antimetabolite means that these drugs may be
administered simultaneously or continuously or one may be
administered first and then the other at an interval of time. When
an anti-IL-6 inhibitor and an immunorepressor are administered, the
doses can be appropriately adjusted depending on the weight, age,
condition and the like of the subject to be treated, but when the
IL-6 inhibitor is an anti-IL-6 antibody, the dose is, for example,
0.1-100 mg/kg/week or a dose providing an equivalent blood
concentration, preferably 1-50 mg/kg/week or a dose providing an
equivalent blood concentration, more preferably 5-10 mg/kg/week or
a dose providing an equivalent blood concentration. When the
antimetabolite is gemcitabine hydrochloride, the dose is, for
example, 10-10000 mg/m.sup.2/week or a dose providing an equivalent
blood concentration, preferably 100-5000 mg/m.sup.2/week or a dose
providing an equivalent blood concentration, more preferably
500-1500 mg/m.sup.2/week or a dose providing an equivalent blood
concentration.
[0133] The mode of administration, dosing interval and dosage
amount described above can be appropriately selected to provide a
therapeutic effect comparable to the effect of the present
invention. For example, the mode of administration, dosing interval
and dosage amount providing an effect comparable to the examples
above can be selected by measuring the blood concentration of each
drug, and the mode of administration, dosing interval and dosage
amount achieving blood concentrations comparable to the examples
above are also included in the present invention.
[0134] The subject to which compositions or agents of the present
invention are administered is a mammal. The mammal is preferably
human.
[0135] Compositions or agents of the present invention can be
administered orally or parenterally and systemically or topically
in the form of pharmaceuticals. For example, intravenous injection
such as drip injection, intramuscular injection, intraperitoneal
injection, subcutaneous injection, suppositories, rectal
formulations, oral enteric formulations and the like can be
selected and the mode of administration can be appropriately
selected depending on the age and condition of the patient.
[0136] Compositions or agents of the present invention may contain
pharmaceutically acceptable carriers such as preservatives and
stabilizers. Pharmaceutically acceptable carrier refers to a
material that can be administered with the agents described above.
Pharmaceutically acceptable materials include, for example,
sterilized water, physiological saline, stabilizers, excipients,
buffering agents, preservatives, surfactants, chelating agents
(such as EDTA), binders and the like.
[0137] In the present invention, surfactants include nonionic
surfactants , typical examples of which include, for example,
sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan
monolaurate, sorbitan monopalmitate; glycerin fatty acid esters
such as glycerin monocaprylate, glycerin monomyristate, glycerin
monostearate; polyglycerin fatty acid esters such as decaglyceryl
monostearate, decaglyceryl distearate, decaglyceryl monolinoleate;
polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene
sorbitan monolaurate, polyoxyethylene sorbitan monooleate,
polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan
monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene
sorbitan tristearate; polyoxyethylene sorbitol fatty acid esters
such as polyoxyethylene sorbitol tetrastearate, polyoxyethylene
sorbitol tetraoleate; polyoxyethylene glycerin fatty acid esters
such as polyoxyethylene glyceryl monostearate; polyethylene glycol
fatty acid esters such as polyethylene glycol distearate;
polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether;
polyoxyethylene polyoxypropylene alkyl ethers such as
polyoxyethylene polyoxypropylene glycol, polyoxyethylene
polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene
cetyl ether; polyoxyethylene alkyl phenyl ethers such as
polyoxyethylene nonyl phenyl ether; polyoxyethylene hardened castor
oils such as polyoxyethylene castor oil, polyoxyethylene hardened
castor oil (polyoxyethylene hydrogenated castor oil);
polyoxyethylene beeswax derivatives such as polyoxyethylene
sorbitol beeswax; polyoxyethylene lanolin derivatives such as
polyoxyethylene lanolin; polyoxyethylene fatty acid amides such as
polyoxyethylene stearic acid amide, each of which has an HLB of
6-18, and the like.
[0138] Surfactants also include anionic surfactants, typical
examples of which include, for example, alkyl sulfates having a
C10-18 alkyl group such as sodium cetyl sulfate, sodium lauryl
sulfate, sodium oleyl sulfate; polyoxyethylene alkyl ether sulfates
having an average EO mole number of 2-4 and a C10-18 alkyl group
such as sodium polyoxyethylene lauryl sulfate; alkyl sulfosuccinic
acid ester salts having a C8-18 alkyl group such as sodium
laurylsulfosuccinate; natural surfactants including, for example,
lecithin; glycerophospholipids; sphingophospholipids such as
sphingomyelin; sucrose fatty acid esters of C12-18 fatty acids, and
the like.
[0139] Compositions or agents of the present invention can contain
one or more of these surfactants in combination. Preferred
surfactants used in formulations of the present invention include
polyoxyethylene sorbitan fatty acid esters such as Polysorbate 20,
40, 60 or 80, especially Polysorbates 20 and 80. Polyoxyethylene
polyoxypropylene glycols such as poloxamers (e.g. Pluronic
F-68.RTM.) are also preferred.
[0140] The amount of the surfactants added depends on the type of
the surfactants used, but it is typically 0.001-100 mg/mL,
preferably 0.003-50 mg/mL, more preferably 0.005-2 mg/mL in the
case of Polysorbate 20 or Polysorbate 80.
[0141] In the present invention, buffering agents include
phosphoric acid, citrate buffer, acetic acid, malic acid, tartaric
acid, succinic acid, lactic acid, potassium phosphate, gluconic
acid, caprylic acid, deoxycholic acid, salicylic acid,
triethanolamine, fumaric acid and other organic acids and the like,
or carbonate buffer, Tris buffer, histidine buffer, imidazole
buffer and the like.
[0142] Solution formulations may also be prepared by dissolving in
an aqueous buffer known in the field of solution formulations. The
concentration of the buffer is typically 1-500 mM, preferably 5-100
mM, more preferably 10-20 mM.
[0143] Moreover, compositions or agents of the present invention
may contain proteins such as other low molecular mass polypeptides,
serum albumin, gelatin or immunoglobulin; amino acids; sugars such
as polysaccharides and monosaccharides or carbohydrates; and sugar
alcohols.
[0144] In the present invention, amino acids include basic amino
acids such as arginine, lysine, histidine, ornithine, or organic
salts of these amino acids (preferably in the form of
hydrochlorides, phosphates, i.e., amino acid phosphates). When free
amino acids are used, preferred pH values are adjusted by adding a
suitable physiologically acceptable buffering substance such as an
inorganic acid, especially hydrochloric acid, phosphoric acid,
sulfuric acid, acetic acid, formic acid or a salt thereof. Here, it
is especially advantageous to use phosphoric acid salts because
especially stable lyophilized products can be obtained. It is
especially advantageous when preparations contain substantially no
organic acid such as malic acid, tartaric acid, citric acid,
succinic acid or fumaric acid or when no corresponding anion (such
as malate ion, tartrate ion, citrate ion, succinate ion or fumarate
ion) exists. Preferred amino acids are arginine, lysine, histidine,
or ornithine. Moreover, acidic amino acids such as glutamic acid
and aspartic acid and salts thereof (preferably sodium salts), or
neutral amino acids such as isoleucine, leucine, glycine, serine,
threonine, valine, methionine, cysteine or alanine, or aromatic
amino acids such as phenylalanine, tyrosine, tryptophan or its
derivative N-acetyltryptophan can also be used.
[0145] In the present invention, sugars such as polysaccharides and
monosaccharides or carbohydrates include, for example, dextran,
glucose, fructose, lactose, xylose, mannose, maltose, sucrose,
trehalose, raffinose and the like.
[0146] In the present invention, sugar alcohols include, for
example, mannitol, sorbitol, inositol and the like.
[0147] When compositions or agents of the present invention are in
the form of aqueous solutions for injection, they can be mixed with
isotonic solutions containing, for example, physiological saline,
glucose or other adjuvants (e.g., D-sorbitol, D-mannose,
D-mannitol, sodium chloride). The aqueous solutions can also be
used in combination with suitable solubilizers (e.g., alcohol
(ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.),
nonionic surfactants (Polysorbate 80, HCO-50) and the like).
[0148] If desired, they may further contain diluents, solubilizers,
pH modifiers, soothing agents, sulfur-containing reducing agents,
antioxidants and the like.
[0149] In the present invention, sulfur-containing reducing agents
include, for example, sulfhydryl-containing compounds such as
N-acetylcysteine, N-acetylhomocysteine, thioetic acid,
thiodiglycol, thioethanolamine, thioglyceroi, thiosorbitol,
thioglycolic acid and salts thereof, sodium thiosulfate,
glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.
[0150] In the present invention, antioxidants include, for example,
erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole,
.alpha.-tocopherol, tocopherol acetate, L-ascorbic acid and salts
thereof, L-ascorbyl palmitate, L-ascorbyl stearate, sodium
bisulfite, sodium sulfite, triamyl gallate, propyl gallate or
chelating agents such as disodium ethylenediamine tetraacetate
(EDTA), sodium pyrophosphate and sodium metaphosphate.
[0151] If desired, the compositions or agents can be encapsulated
into microcapsules (microcapsules of hydroxymethylcellulose,
gelatin, poly[methylmethacrylic acid] or the like) or formulated
into colloidal drug delivery systems (liposomes, albumin
microspheres, microemulsions, nanoparticles and nanocapsules, etc.)
(see "Remington's Pharmaceutical Science 16.sup.th edition", Oslo
Ed., 1980 or the like). Further, methods for formulating
compositions or agents into sustained release systems are also
known and can be applied to the present invention (Langer et al.,
J. Biomed. Mater. Res. 1981, 15: 167-277; Langer, Chem. Tech. 1982,
12: 98-105; U.S. Pat. No. 3,773,919; European Patent Application
published as (EP) 58,481; Sidman et al., Biopolymers 1983, 22:
547-556; EP 133,988).
[0152] Pharmaceutically acceptable carriers used are selected as
appropriate or in combination from, but not limited to, the above
list depending on the dosage form.
[0153] The subject to which compositions or agents of the present
invention are administered refers to a living body to which the
compositions or agents of the present invention are administered or
a part in the living body. Living bodies include, but not
specifically limited to, animals (e.g., human, domestic animal
species, wild animals).
[0154] A part in the living body is not specifically limited to,
but preferably includes an affected site or the like.
[0155] As used herein, "administration" includes oral or parenteral
administration. Oral administration includes administration in the
form of oral formulations, and dosage forms such as granules,
powders, tablets, capsules, solutions, emulsions, or suspensions
can be selected as oral formulations.
[0156] Parenteral administration includes administration in the
form of injections, and injections include subcutaneous injections,
intramuscular injections, or intraperitoneal injections, etc.
Moreover, agents of the present invention can also be topically
administered to an area to be treated. For example, they can be
administered by topical injection during surgical operation or the
use of a catheter.
[0157] When carrying out methods of the present invention, an agent
of the present invention can be administered as a part of a
pharmaceutical composition with at least one additional drug (for
example, other perineural invasion inhibitors or other therapeutic
agents for pancreatic cancer). In one embodiment, an agent of the
present invention and another agent may be administered
substantially at the same time.
[0158] All of the prior art documents cited herein are incorporated
herein by reference.
EXAMPLES
[0159] The following examples further illustrate the present
invention without, however, limiting the invention thereto. Various
changes and modifications can be made by those skilled in the art,
and such changes and modifications are also included in the present
invention.
Example 1
Effect of Combined Administration of Gemcitabine Hydrochloride and
Tocilizumab on Pancreatic Cancer
[0160] A chemotherapy-naive patient having progressive/metastatic
pancreatic cancer and showing CRP>=2.0 mg/dL (CRP: C-reactive
protein that correlates with the intensity of inflammatory
response) was treated with gemcitabine hydrochloride and a
humanized IL-6 receptor antibody (tocilizumab).
[0161] A male at age 72 with no history of smoking having
pancreatic body cancer of Stage IV as defined by the TNM
classification of UICC.sup.1) had metastatic lesions in the liver,
lymph nodes, ascites and peritoneum and had evident ascites as
shown in FIG. 1. This case had a CRP of 3.28 mg/dL immediately
before administration and also suffered from hyperlipidemia and
chronic atrophic gastritis as complications. This patient was
treated with gemcitabine hydrochloride and tocilizumab in
combination. Gemcitabine hydrochloride was administered by
intravenous drip injection at 1000 mg/m.sup.2 expressed as
gemcitabine over 30 min once a week for 3 weeks followed by the 4th
week of washout. This cycle was repeated. Tocilizumab was
administered by intravenous drip injection at 8 mg/kg over about 60
min every 2 weeks. Depending on the condition of the patient,
gemcitabine or tocilizumab was withdrawn or reduced. For PK
(pharmacokinetics) analysis, medication was started on day 1 for
gemcitabine hydrochloride and on day 2 for tocilizumab. The
subsequent medications were each performed on the same day.
[0162] As for CRP levels that have been reported to be associated
with very poor prognosis when they are 2.0 mg/dL or more.sup.2),
they decreased to 0.66 mg/dL after 1 week and decreased to 0.44
mg/dL falling within the normal range (0.5 mg/dL or less) after 2
weeks. CRP remained in the normal range until the end of treatment.
Ascites was remarkably reduced after 4 weeks, and further reduced
to the extent that it was scarcely observed after 8 weeks as shown
in FIG. 2. Primary and metastatic tumor lesions were reduced by 24%
after 8 weeks, and 31% after 12 weeks. Metastatic lesions in the
liver were also evidently reduced after 8 weeks (FIG. 4) as
compared with before treatment (FIG. 3).
[0163] Tumor markers showed CEA of 112.6 ng/mL and CA19-9 of 47700
U/mL before treatment, but decreased to 20.4 ng/mL and 7900 U/mL
after 8 weeks, and to 9.8 ng/mL and 8820 U/mL after 12 weeks. No
serious side effect was observed.
[0164] We previously closely analyzed clinical performance of
gemcitabine hydrochloride monotherapy in progressive/metastatic
pancreatic cancer patients and found that the average change in
tumor size 3 months after the start of monotherapy (analyzable
cases: 25 cases) was an increase of 36.0% (95% confidence interval:
4.6-67.4%) and that ascites was not eliminated during the
monotherapy of 3 months (analyzable cases: 29 cases). Thus, the
tumor reducing effect and ascites eliminating effect observed in
one case of combination therapy were not observed with
monotherapy.
[0165] These results showed that combined administration of
gemcitabine and tocilizumab had an excellent therapeutic effect as
proved by reduction in pancreatic cancer and metastatic lesions.
Improvements of pancreatic cancer also provided an ascites
eliminating effect that could not be observed with
monotheraphy.
1) Sobin L H, Wittekind C L, editors. TNM classification of
malignant tumors, 6th edition. New York: John Wiley & Sons,
2002. 2) Shuichi Mitsunaga, Masafumi Ikeda, Kohei Nakachi et al.:
Effectiveness of serum CRP levels as an indicator of cachexia in
patients having unresectable pancreatic cancer. The 40th Annual
Academic Meeting of the Japan Pancreas Society, 2009.
INDUSTRIAL APPLICABILITY
[0166] In the present invention, it was shown that an excellent
therapeutic effect against pancreatic cancer can be obtained by
administering an IL-6 inhibitor and an antimetabolite in
combination. Moreover, it was also shown that metastatic lesions
from human pancreatic cancer can be reduced and ascites can be
eliminated.
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