U.S. patent application number 11/033835 was filed with the patent office on 2005-07-21 for method of pre-sensitizing cancer prior to treatment with radiation and/or chemotherapy and a novel cytokine mixture.
Invention is credited to Talor, Eyal.
Application Number | 20050158275 11/033835 |
Document ID | / |
Family ID | 33552429 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158275 |
Kind Code |
A1 |
Talor, Eyal |
July 21, 2005 |
Method of pre-sensitizing cancer prior to treatment with radiation
and/or chemotherapy and a novel cytokine mixture
Abstract
This invention relates to a breakthrough method for
pre-sensitizing cancer prior to a therapeutic treatment such as
chemotherapy, radiation therapy or immuno-therapy and a novel
cytokine mixture used in the method thereof The cytokine mixture is
a serum-free and mitogen-free mixture comprised of specific ratios
of cytokines such as IL-1.beta., TNF-.alpha., IFN-.gamma. and
GM-CSF to Interleukin 2 (IL-2), which is effective in inducing
cancerous cells to enter a proliferative cell cycle phase thereby
increasing their vulnerability to chemotherapy, radiation therapy
and immuno-therapy. One such novel cytokine mixture is
Multikine.RTM., which can be used alone or in combination with
other drugs for the treatment of cancer thereby increasing the
success of cancer treatment and the disease free survival of cancer
patients.
Inventors: |
Talor, Eyal; (Baltimore,
MD) |
Correspondence
Address: |
Leonard W. Sherman
Sherman & Shalloway
415 N. Alfred Street
Alexandria
VA
22314
US
|
Family ID: |
33552429 |
Appl. No.: |
11/033835 |
Filed: |
January 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11033835 |
Jan 13, 2005 |
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10611914 |
Jul 3, 2003 |
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6896879 |
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Current U.S.
Class: |
424/85.2 |
Current CPC
Class: |
A61K 38/2013 20130101;
A61P 35/00 20180101; A61K 38/193 20130101; A61K 38/191 20130101;
A61K 38/191 20130101; A61K 38/193 20130101; A61K 38/217 20130101;
A61K 38/2006 20130101; A61K 38/217 20130101; A61P 43/00 20180101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
38/2006 20130101; A61K 38/2013 20130101 |
Class at
Publication: |
424/085.2 |
International
Class: |
A61K 038/20 |
Claims
We claim:
1. A method for pre-sensitizing cancer prior to a therapeutic
treatment, comprising the step of: administering a therapeutically
active amount of a serum-free and mitogen-free cytokine mixture to
cancer.
2. The method of claim 1, wherein said therapeutic treatment is
selected from the group consisting of chemotherapy, immuno-therapy
and radiation therapy.
3. The method of claim 1, wherein said serum-free and mitogen-free
cytokine mixture is peritumorally administered three times a week
over a two week period in a range from about 20 IU to 1600 IU
wherein IU represent International Units for Interleukin-2 given in
World Health Organization 1.sup.st International Standard for Human
IL-2, 86/504.
4. The method of claim 1, wherein said serum-free and mitogen-free
cytokine mixture is peritumorally administered three times a week
over a two week period in a range from about 40 to 800 IU wherein
IU represent International Units for Interleukin-2 given in World
Health Organization 1.sup.st International Standard for Human IL-2,
86/504.
5. The method of claim 1, wherein said serum-free and mitogen-free
cytokine mixture is peritumorally administered three times a week
over a two week period in a range from about 35 IU to 75 IU wherein
IU represent International Units for Interleukin-2 given in World
Health Organization 1.sup.st International Standard for Human IL-2,
86/504.
6. The method of claim 1, wherein said serum-free and mitogen-free
cytokine mixture is peritumorally administered three times a week
over a two week period at 55 IU wherein IU represent International
Units for Interleukin-2 given in World Health Organization 1.sup.st
International Standard for Human IL-2, 86/504.
7. The method of claim 1, wherein said serum-free and mitogen-free
cytokine mixture is peritumorally administered three times a week
over a two week period at 400 IU wherein IU represent International
Units for Interleukin-2 given in World Health Organization 1.sup.st
International Standard for Human IL-2, 86/504.
8. The method of claim 1, wherein said serum-free and mitogen-free
cytokine mixture is peritumorally administered three times a week
over a two week period at 800 IU wherein IU represent International
Units for Interleukin-2 given in World Health Organization 1.sup.st
International Standard for Human IL-2, 86/504.
9. The method of claim 1, wherein said serum-free and mitogen-free
cytokine mixture is peritumorally administered five times a week
over a two week period at 800 IU wherein IU represent International
Units for Interleukin-2 given in World Health Organization 1.sup.st
International Standard for Human IL-2, 86/504.
10. The method of claim 1, wherein said serum-free and mitogen-free
cytokine mixture is comprised of specific ratios of cytokines
selected from the group of IL-1.beta., TNF-.alpha., IFN-.gamma. and
GM-CSF to Interleukin-2 (IL-2) as follows: IL-1.beta. to IL-2 at a
ratio range of 0.4-1.5; TNF-.alpha. to IL-2 at a ratio range of
3.2-10.9; IFN-.gamma. to IL-2 at a ratio range of 1.5-10.9; and
GM-CSF to IL-2 at a ratio range of 2.2-4.8.
11. The method of claim 10, wherein said specific ratios of
cytokines are as follows: IL-1.beta. to IL-2 at a ratio range of
0.6 to 0.8; TNF-.alpha. to IL-2 at a ratio range of 7.7 to 11.3;
IFN-.gamma. to IL-2 at a ratio range of 4.9 to 7.1; and GM-CSF to
IL-2 at a ratio range of 3.5 to 4.5.
12. The method of claim 1 wherein the serum-free and mitogen-free
cytokine mixture is Multikine.RTM..
13. A method for inducing tumor cells into a cell cycle selected
from the group of G.sub.1, S, G.sub.2 and M, comprising the step
of: administering a therapeutically active amount of a serum-free
and mitogen-free cytokine mixture to a cancerous cell.
14. The method of claim 13, wherein said serum-free and
mitogen-free cytokine mixture is peritumorally administered three
times a week over a two week period in a range from about 20 IU to
1600 IU wherein IU represent International Units for Interleukin-2
given in World Health Organization 1.sup.st International Standard
for Human IL-2, 86/504.
15. The method of claim 13, wherein said serum-free and
mitogen-free cytokine mixture is peritumorally administered three
times a week over a two week period in a range from about 40 IU to
800 IU wherein IU represent International Units for Interleukin-2
given in World Health Organization 1.sup.st International Standard
for Human IL-2, 86/504.
16. The method of claim 13, wherein said serum-free and
mitogen-free cytokine mixture is peritumorally administered three
times a week over a two week period in a range from about 35 IU to
75 IU wherein IU represent International Units for Interleukin-2
given in World Health Organization 1.sup.st International Standard
for Human IL-2, 86/504.
17. The method of claim 13, wherein said serum-free and
mitogen-free cytokine mixture is peritumorally administered three
times a week over a two week period at 55 IU wherein IU represent
International Units for Interleukin-2 given in World Health
Organization 1.sup.st International Standard for Human IL-2,
86/504.
18. The method of claim 13, wherein said serum-free and
mitogen-free cytokine mixture is peritumorally administered three
times a week over a two week period at 400 IU wherein IU represent
International Units for Interleukin-2 given in World Health
Organization 1.sup.st International Standard for Human IL-2,
86/504.
19. The method of claim 13, wherein said serum-free and
mitogen-free cytokine mixture is peritumorally administered three
times a week over a two week period at 800 IU wherein IU represent
International Units for Interleukin-2 given in World Health
Organization 1.sup.st International Standard for Human IL-2,
86/504.
20. The method of claim 13, wherein said serum-free and
mitogen-free cytokine mixture is peritumorally administered five
times a week over a two week period at 800 IU wherein IU represent
International Units for Interleukin-2 given in World Health
Organization 1.sup.st International Standard for Human IL-2,
86/504.
21. The method of claim 13, wherein said serum-free and
mitogen-free cytokine mixture is comprised of specific ratios of
cytokines selected from the group of IL-1.beta., TNF-.alpha.,
IFN-.gamma. and GM-CSF to Interleukin-2 (IL-2) as follows:
IL-1.beta. to IL-2 at a ratio range of 0.4-1.5; TNF-.alpha. to IL-2
at a ratio range of 3.2-10.9; IFN-.gamma. to IL-2 at a ratio range
of 1.5-10.9; and GM-CSF to IL-2 at a ratio range of 2.2-4.8.
22. The method of claim 21, wherein said specific ratios of
cytokines are as follows: IL-1.beta. to IL-2 at a ratio range of
0.6 to 0.8; TNF-.alpha. to IL-2 at a ratio range of 7.7 to 11.3;
IFN-.gamma. to IL-2 at a ratio range of 4.9 to 7.1; and GM-CSF to
IL-2 at a ratio range of 3.5 to 4.5.
23. The method of claim 13 wherein the serum-free and mitogen-free
cytokine mixture is Multikine.RTM..
24. A serum-free and mitogen-free cytokine mixture, comprising
specific ratios of cytokines selected from the group of IL-1.beta.,
TNF-.alpha., IFN-.gamma. and GM-CSF to Interleukin-2 (IL-2) as
follows: IL-1.beta. to IL-2 at a ratio range of 0.4-1.5;
TNF-.alpha. to IL-2 at a ratio range of 3.2-10.9; IFN-.gamma. to
IL-2 at a ratio range of 1.5-10.9; and GM-CSF to IL-2 at a ratio
range of 2.2-4.8.
25. The serum-free and mitogen-free cytokine mixture of claim 24,
wherein said specific ratios of cytokines are as follows:
IL-1.beta. to IL-2 at a ratio range of 0.6 to 0.8; TNF-.alpha. to
IL-2 at a ratio range of 7.7 to 11.3; IFN-.gamma. to IL-2 at a
ratio range of 4.9 to 7.1; and GM-CSF to IL-2 at a ratio range of
3.5 to 4.5.
26. A pharmaceutical composition for use in treating cancer,
comprising specific ratios of cytokines selected from the group of
IL-1, TNF-.alpha., IFN-.gamma. and GM-CSF to Interleukin-2 (IL-2)
as follows: IL-1.beta. to IL-2 at a ratio range of 0.4-1.5;
TNF-.alpha. to IL-2 at a ratio range of 3.2-10.9; IFN-.gamma. to
IL-2 at a ratio range of 1.5-10.9; GM-CSF to IL-2 at a ratio range
of 2.2-4.8, and optionally in combination with a pharmaceutically
acceptable excipient, carrier or additive.
27. The pharmaceutical composition of claim 26, wherein said
specific ratios of cytokines are as follows: IL-1.beta. to IL-2 at
a ratio range of 0.6 to 0.8; TNF-.alpha. to IL-2 at a ratio range
of 7.7 to 11.3; IFN-.gamma. to IL-2 at a ratio range of 4.9 to 7.1;
and GM-CSF to IL-2 at a ratio range of 3.5 to 4.5.
28. The pharmaceutical composition of claim 27, further comprising
an IL-3 to IL-2 ratio in a range from 0.38-0.68, preferably at
0.53.+-.0.15
29. The pharmaceutical composition of claim 27, further comprising
an IL-6 to IL-2 ratio in a range from 37.2-53.8, preferably at
46.+-.5.9.
30. The pharmaceutical composition of claim 27, further comprising
an IL-8 to IL-2 ratio in a range from 261-561.5, preferably at
41.+-.10.6.
31. The pharmaceutical composition of claim 27, further comprising
an IL-1.alpha. to IL-2 ratio in a range from 0.56-0.94, preferably
at 0.75.+-.0.19.
32. The pharmaceutical composition of claim 27, further comprising
an IL-10 to IL-2 ratio in a range from 2.87-3.22, preferably at
3.0.+-.0.18.
33. The pharmaceutical composition of claim 27, further comprising
an IL-16 to IL-2 ratio in a range from 1.24-2.84, preferably at
1.84.+-.0.68.
34. The pharmaceutical composition of claim 27, further comprising
a G-CSF to IL-2 ratio in a range from 2.16-3.78, preferably at
2.97.+-.0.81.
35. The pharmaceutical composition of claim 27, further comprising
a TNF-.beta. to IL-2 ratio in a range from 1.18-2.43, preferably at
1.8.+-.0.63.
36. The pharmaceutical composition of claim 27, further comprising
a MIP-1.alpha. to IL-2 ratio in a range from 16.78-37.16,
preferably at 22.7.+-.7.0.
37. The pharmaceutical composition of claim 27, further comprising
a MIP-1.beta. to IL-2 ratio in a range from 19.2-26.4, preferably
at 22.8.+-.5.7.
38. The pharmaceutical composition of claim 27, further comprising
a RANTES to IL-2 ratio in a range from 2.3-2.7, preferably at
2.5.+-.0.13.
39. The pharmaceutical composition of claim 27, further comprising
a EGF to IL-2 ratio in a range from 0.27-0.28, preferably at
0.275.+-.0.008.
40. The pharmaceutical composition of claim 27, further comprising
a PGE.sub.2 to IL-2 ratio in a range from 3.68-5.42, preferably at
4.5.+-.0.87.
41. The pharmaceutical composition of claim 27, further comprising
a TxB.sub.2 to IL-2 ratio in a range from 23.5-25.1, preferably at
24.3.+-.0.83.
Description
INTRODUCTION
[0001] This invention relates to a breakthrough method for
pre-sensitizing cancer prior to a therapeutic treatment such as
chemotherapy, radiation therapy or immuno-therapy and a novel
cytokine mixture used in the method thereof. The cytokine mixture
is a serum-free and mitogen-free mixture comprised of specific
ratios of cytokines such as IL-1.beta., TNF-.alpha., IFN-.gamma.
and GM-CSF to Interleukin 2 (IL-2), which is effective in inducing
cancerous cells to enter a proliferative cell cycle phase thereby
increasing their vulnerability to chemotherapy, radiation therapy
and immuno-therapy. One such novel cytokine mixture is
Multikine.RTM., which can be used alone or in combination with
other drugs for the treatment of cancer thereby increasing the
success of cancer treatment and the disease free survival of cancer
patients.
BACKGROUND OF THE INVENTION
[0002] Current treatments of cancer and in particular solid tumors,
comprise mainly of surgical intervention followed by radiation
therapy and/or chemotherapy. Dunne-Daly CF, "Principles of
radiotherapy and radiobiology", Semin Oncol Nurs. November 1999;
15(4):250-9; Hensley M L et al., "American Society of Clinical
Oncology clinical practice guidelines for the use of chemotherapy
and radiotherapy protectants.", J Clin. Oncol. October 1999;
17(10):3333-55. In conjunction with such therapies, toxic
chemotherapeutic agents such as Gemcidabin, Vinblastine, Cisplatin,
Fluorouracil, Gleevec, Methotrexate, which are unable to
differentiate between normal and cancerous cells, are used. While
effective, these and other toxic chemotherapeutic agents have done
little to increase overall patient survival. Moreover, current
treatments in general also failed to improve 5-year survival rate
of cancer patients despite synergistic combination of
chemotherapies and radiotherapies. Even anti-epidermal growth
factor receptor agents, anti-angiogenic drugs and immuno and
immuno-adjuvant therapy using drugs such as Rituximab, Erbitux and
Herceptin have failed to significantly increase the 5-year survival
rate of cancer patients. Furthermore, complete remission or disease
free survival of cancer patient irrespective of cancer type have
not been improved upon by any of the aforementioned therapies or
synergistic combinations thereof.
[0003] One treatment modality investigated to improve disease-free
survival rates or lead to complete remission is manipulation of
cell-division cycle of cancerous cells. In particular, cycling
tumor cells are generally more vulnerable to radio- and
chemotherapies than non-cycling tumor cells because complex
biochemical and biomolecular processes such as enzyme-dependent DNA
replication, enzyme-dependent phosphorylation, signal cascades,
association and dissociation of transcriptional activating
molecular complexes, and formation and dissociation of
macromolecular assemblies of cytostructural elements are required
during cell cycling. By inducing tumor cells into a cell cycle
phase, anti-metabolic agents that inhibit any of the complex
biochemical processes such as ribonucleotide reductase (RNR)
inhibitors, dihydrofolate reductase inhibitors or DNA polymerase
inhibitors can be used to stop cell cycling and thereby prevent
tumor proliferation.
[0004] However, known methods taking advantage of cell cycling are
limited to synchronizing cell cycle arrest with sequential
applications of a chemotherapeutic agent. For example, one known
method arrests malignant cells within a S phase of the cell cycle
with pyrimidine analogs followed by exposure to high concentrations
of anti-metabolites. B. Bhutan et al., Cancer Res. 33:888-894
(1973). Few or no cells in the population can proceed beyond this
point of detention after application of the anti-metabolite. W
Vogel et al., Hum. Genet. 45:193-8 (1978).
[0005] Other efforts include methods of manipulating the
cell-division cycle by altering the cell cycle distribution within
the cell population. These protocols stimulate malignant cells from
a dormant phase into a cell cycling phase thereby increasing their
vulnerability to anti-metabolic drugs acting during the vulnerable
DNA replication phase. H H Euler et al., Ann. Med. Interne. (Paris)
145:296-302 (1994); B C Lampkin et al., J. Clin. Invest. 50:2204-14
(1971); Alama et al., Anticancer Res. 10:853-8 (1990). Conversely,
other known methods prohibit normal cells from entering S phase
thereby protecting normal cells from chemotactic drugs.
[0006] Still another known method of synchronizing cell cycle phase
with chemotherapeutics is a so-called pulse dose chemotherapy
described by R E Moran et al., Cancer Treat. Rep. 64:81-6 (1980).
In this approach, leukemic tumor cells in mice were detained in a S
phase of the cell cycle with an infusion of hydroxyurea. After the
infusion, the cells were "released" to continue cell cycling
wherein a "pulse" of a second agent (Ara-C) was given to the mice.
The intent was to maximize impact of the second agent as the
cycling cells were moving through the vulnerable cell cycle
S-phase. However, the results indicated that while mice treated
with Ara-C just after the hydroxyurea infusion showed improved
survival, mice treated with Ara-C at later times after the
hydroxyurea infusion did not show improved survival. Clearly,
simply synchronizing cell cycling with a second agent acting
non-simultaneously did not improve the action of the two
agents.
[0007] Nevertheless, known methods taking advantage of cell cycle
continue to seek an optimal but passive synergy between dosage,
pharmacokinetics, sequence and scheduling.
[0008] It might be expected that confining a cell population to a
vulnerable cell cycle phase where cells are specifically vulnerable
to damage might shift the dynamics of cell killing toward greater
efficiency with a greater reduction in side-effects by diminishing
exposure to toxic drugs. However, actual experiments taking
advantage of cell cycle arrest or static synchronization have been
disappointing because known methods are unable to actually induce
cells into a cell cycle. Rather, all the known methods simply time
the synergistic combination of cell cycle arrest or static
synchronization with the target cell population. Moreover, agents
such as pyrimidine and hydroxyurea used to effect the cell cycle
can cause damage to normal cells.
[0009] Another approach would, of course, be to induce the cells to
enter into a cell cycle phase as opposed to arresting the cell
cycle or synchronizing the cell cycle. However, as would be
otherwise predicted from the art, inducing cells into cell cycling
increases the risk of a rapidly growing and recurring tumor. But
the continued failure of known compositions to improve disease-free
survival rates or lead to complete remission suggests a need for
inducing malignant cells into cell cycling in a manner that does
not proliferate the tumor but increases the susceptibility of the
residual tumor to follow-on treatment with radiation and/or
chemotherapy.
[0010] Therefore, there is a need for methods for inducing tumor
cells into a cell cycle selected from the group of (different
phases of the cell cycle) G.sub.1, S, G.sub.2 and M where the new
methods can be synergistically applied with chemotherapy,
immuno-therapy and radiation therapy. There is also a need for
pre-sensitizing cancer tumors in general along with the need for a
new serum-free and mitogen-free cytokine mixture comprised of
specific ratios of IL-1.beta. to IL-2, TNF-.alpha. to IL-2,
IFN-.gamma. to IL-2 and GM-CSF to IL-2 that unexpectedly
demonstrates far better efficacy over known compositions in
inducing a tumor cells to enter a cell cycle phase or for
pre-sensitizing a cancer.
SUMMARY OF THE INVENTION
[0011] The present invention is based, in part, on methods of
pre-sensitizing cancer in general and a new serum-free and
mitogen-free cytokine mixture having specific ratios of IL-1.beta.
to IL-2, TNF-.alpha. to IL-2, IFN-.gamma. to IL-2 and GM-CSF to
IL-2. Accordingly, the present invention enables the development of
compositions useful as a pharmaceutical or as an adjuvant to be
used in conjunction with therapeutic cancer treatments such as
chemotherapy, immuno-therapy and radiation therapy.
[0012] In embodiments of the invention, a method of improving
conventional chemotherapy or radiotherapy of neoplasms or diseases
of the immune system with a serum-free and mitogen-free cytokine
mixture is disclosed. The methods provide for a pre-sensitizing
step for the treatment of cancer in conjunction with radiotherapies
or other physical modalities of cell killing. A method for inducing
tumor cells into a vulnerable cell cycle phase selected from the
group of (different phases of the cell cycle) G.sub.1, S, G.sub.2
and M is also contemplated. The present invention is not limited to
any one particular type of cancer and can include any type of
cancer. Specific applications include administering a serum-free
and mitogen-free cytokine mixture peritumorally three times a week
over a two week period in a range from about 20 IU to 1600 IU or
specifically at 400 IU or at 800 IU or still further at five times
a week in a range from about 20 IU to 1600 IU or at 400 IU or at
800 IU, wherein IU represent International Units for Interleukin-2
given in World Health Organization 1.sup.st International Standard
for Human IL-2, 86/504.
[0013] Another embodiment includes a serum-free and mitogen-free
cytokine preparation such as Multikine.RTM. in novel and
non-obvious concentrations. The cytokine preparation may further be
part of a pharmaceutical composition. In specific applications, the
new serum-free and mitogen-free cytokine preparation has specific
ratios of cytokine to interleukin 2 (IL-2) as follows: IL-1.beta.
to IL-2 at a ratio range of 0.4-1.5, and preferably at 0.7.+-.0.1
(IL-1.beta./IL-2), TNF-.alpha. to IL-2 at a ratio range of
3.2-10.9, and preferably at 9.5.+-.1.8 (TNF-.alpha./IL-2),
IFN-.gamma. to IL-2 at a ratio range of 1.5-10.9, and preferably at
6.0.+-.1.1 (IFN-.gamma./IL-2), and GM-CSF to IL-2 at a ratio range
of 2.2-4.8, and preferably at 4.0.+-.0.5 (GM-CSF/IL-2).
[0014] In other specific applications, the serum-free and
mitogen-free cytokine preparation or pharmaceutical composition has
further different cytokines and other small biologically active
molecules in Multikine.RTM. wherein the ratio of each of the small
biologically active molecules to Il-2 is as follows: IL-3 to Il-2
in a ratio range of 0.38-0.68, preferably at 0.53.+-.0.15, IL-6 to
Il-2 in a ratio range of 37.2-53.8, preferably at 46.+-.5.9, IL-8
to Il-2 in a ratio range of 261-561.5, preferably at 41.+-.10.6,
IL-1.alpha. to Il-2 in a ratio range of 0.56-0.94, preferably at
0.75.+-.0.19, IL-10 to Il-2 in a ratio range of 2.87-3.22,
preferably at 3.0.+-.0.18, IL-16 to Il-2 in a ratio range of
1.24-2.94, preferably at 1.84.+-.0.68, G-CSF to Il-2 in a ratio
range of 2.16-3.78, preferably at 2.97.+-.0.81, TNF-.beta. to Il-2
in a ratio range of 1.18-2.43, preferably at 1.8.+-.0.63,
MIP-1.alpha. to Il-2 in a ratio range of 16.78-37.16, preferably at
22.7.+-.7.0, MIP-1.beta. to Il-2 in a ratio range of 19.2-26.4,
preferably at 22.8.+-.5.7, a RANTES to Il-2 in a ratio range of
2.3-2.7, preferably at 2.5.+-.0.13, a EGF to Il-2 in a ratio range
of 0.27-0.28, preferably at 0.275.+-.0.008, PGE.sub.2 to Il-2 in a
ratio range of 3.68-5.42, preferably at 4.5.+-.0.87 and TxB.sub.2
to Il-2 in a ratio range of 23.5-25.1, preferably at
24.3.+-.0.83.
[0015] Other objects and advantages of the present invention are
set forth in the following description. The accompanying drawings
and tables, which constitute a part of the disclosure, illustrate
and, together with the description, explain the principle of the
invention. One of ordinary skill in the art will appreciate that
other aspects of this invention will become apparent upon reference
to the attached figures and the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The invention will now be explained in greater detail by the
following description and specific embodiments and with the aid of
the accompanying drawings:
[0017] FIG. 1 represents the mode of action of Multikine.RTM..
[0018] FIG. 2 represents the immuno-histochemistry staining of a
tumor in head and neck cancer squamous cell carcinoma with the
specific cell cycle marker Ki-67.
[0019] FIG. 3 represents morphometric analysis of Ki-67
immuno-histochemistry staining of both tumor stroma and tumor
epithelia where "*" marks a statistically significant differences
where p<0.05 [at .alpha.=0.05].
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0020] The present invention is concerned with methods of
pre-sensitizing cancer in general and a novel serum-free and
mitogen-free cytokine mixture comprised of specific ratios of
IL-1.beta. to IL-2, TNF-.alpha. to IL-2, IFN-.gamma. to IL-2 and
GM-CSF to IL-2. One such novel cytokine mixture is Multikine.RTM.,
which has demonstrated immuno-modulatory capabilities. The clinical
significance of the immuno-suppression in cancer patients
unexpectedly impacts methods of pre-sensitizing cancer prior to
therapeutic treatment and in particular entry of the tumor cell
into a cell cycle phase.
[0021] Immune restoration of head and neck cancer patients is
accomplished by the infusion of cytokines such as IL-2, IFN
.alpha.-.gamma. or IL-12. Whiteside, "Immunobiology and
immunotherapy of head and neck cancer", Curr Oncol Rep 2001;
3:46-55. In head and neck cancer, interleukin-based cytokine
therapy resulted in immuno-augmenting. Cortesina G et al.,
"Interleukin-2 injected around tumor-draining lymph nodes in head
and neck cancer", Head Neck 1991; 13:125-31; De Stefani et al.,
"Treatment of oral cavity and oropharynx squamous cell carcinoma
with perilymphatic interleukin-2: clinical and pathologic
correlations", J Immunother 1996; 19:125-33; Valente et al.,
"Infiltrating leukocyte populations and T-lymphocyte subsets in
head and neck squamous cell carcinomas from patients receiving
perilymphatic injections of recombinant interleukin 2", Mod Pathol
1990; 3:702-8; Whiteside T L et al., "Evidence for local and
systemic activation of immune cells by peritumoral injections of
interleukin 2 in patients with advanced squamous cell carcinoma of
the head and neck", Cancer Res 1990; 53:5654-62; Barrera et al.,
"Combination immunotherapy of squamous cell carcinoma of the head
and neck", Arch Otolaryngol Head Neck Surg 2000; 126:345-51;
Verastegui et al., "A natural cytokine mixture (IRX-2) and
interference with immune suppression induce immune mobilization and
regression of head and neck cancer", Int J Immunopharmacol 1997;
19:619-27; Hadden et al., "Interleukins and contrasuppression
induce immune regression of head and neck cancer", Arch Otolaryngol
Head Neck Surg 1994; 120:395-403. For example, human (r)hIL-2 was
successfully used to improve immune function of head and neck
cancer patients as measured by cytotoxic T lymphocyte [CTL] and
delayed type hypersensitivity [DTH] responses. The decreased
response of T cells was shown by the decreased expression of the T
cell receptor (TCR), its key signaling components, the .zeta. chain
and zap-70, the absence of IL-2 production and increased apoptosis
of T cells. Whiteside T L., "Immunobiology and immunotherapy of
head and neck cancer", Curr Oncol Rep 2001; 3:46-55. Studies
investigating the causes of the impaired T cell function in head
and neck cancer showed that the Fas-FasL system, TGF-.beta. and
PGE.sub.2 are expressed at high levels.
[0022] However, in vivo administration of rIL-2 increased density
of CD25.sup.+ cells as well as natural killer (NK) cells, human
leukocyte antigen (HLA)-DR.sup.+ lymphocytes and T cells. In
another series of studies, positive clinical responses have been
observed when a cytokine mixture was administered perilymphatically
or peritumorally. However, none of these studies correlated an
increased immune response with definitive follow-on treatment such
as surgery, radiation therapy and/or chemotherapy.
[0023] The Technology
[0024] Multikine.RTM., a Leukocyte-Interleukin Injection, is a
serum-free, mitogen-free, antibiotic-free preparation produced from
human peripheral blood mononuclear cells that include T-cells, B
cells and macrophages. There are three "families" of cytokines in
Multikine.RTM. that together impart the unique biological activity
of Multikine.RTM.. They include direct cytotoxic/cytostatic and
virocidal/virostatic cytokines such as TNF-.alpha., and
IFN-.gamma., lympho-proliferative cytokines such as IL-1, and IL-2
and chemotactic cytokines such as IL-6, IL-8 and MIP-1.alpha..
Furthermore, the different cytokine and small biological molecules
that constitute Multikine.RTM. are all derived from the lectin
(PHA) in vitro stimulation of human peripheral blood mononuclear
cells that include T cells, B cells, and macrophages.
Centrifugation on a Ficoll-Paque gradient separates the white blood
cells (including T cells, B cells, and macrophages) from donor
whole blood, and a series of washes (in physiologically buffered
media) facilitates the isolation of lymphocytes, and the removal of
red blood cells, cellular debris and other unwanted cellular
components from the isolated white cell component of the whole
donor blood.
[0025] Multikine.RTM. contains different cytokines present at
specific ratios of each cytokine to Interleukin 2 (IL-2) as
follows: IL-1.beta. to IL-2 at a ratio range of 0.4-1.5, and
preferably at 0.7.+-.0.1 (IL-1.beta./IL-2), TNF-.alpha. to IL-2 at
a ratio range of 3.2-10.9, and preferably at 9.5.+-.1.8
(TNF-.alpha./IL-2), IFN-.gamma. to IL-2 at a ratio range of
1.5-10.9, and preferably at 6.0.+-.1.1 (IFN-.gamma./IL-2), and
GM-CSF to IL-2 at a ratio range of 2.2-4.8, and preferably at
4.0.+-.0.5 (GM-CSF/IL-2).
[0026] The remainder of the different cytokines and other small
biologically active molecules in Multikine.RTM. are also present
within each preparation of the small biologically active molecule
to Il-2 as follows: IL-3 to Il-2 in a ratio range of 0.38-0.68,
preferably at 0.53.+-.0.15, IL-6 to Il-2 in a ratio range of
37.2-53.8, preferably at 46.+-.5.9, IL-8 to Il-2 in a ratio range
of 261-561.5, preferably at 41.+-.10.6, IL-1.alpha. to Il-2 in a
ratio range of 0.56-0.94, preferably at 0.75.+-.0.19, IL-10 to Il-2
in a ratio range of 2.87-3.22, preferably at 3.0.+-.0.18, IL-16 to
Il-2 in a ratio range of 1.24-2.84, preferably at 1.84.+-.0.68,
G-CSF to Il-2 in a ratio range of 2.16-3.78, preferably at
2.97.+-.0.81, TNF-.beta. to Il-2 in a ratio range of 1.18-2.43,
preferably at 1.8.+-.0.63, MIP-1.alpha. to Il-2 in a ratio range of
16.78-37.16, preferably at 22.7.+-.7.0, MIP-1.beta. to Il-2 in a
ratio range of 19.2-26.4, preferably at 22.8.+-.5.7, a RANTES to
Il-2 in a ratio range of 2.3-2.7, preferably at 2.5.+-.0.13, a EGF
to Il-2 in a ratio range of 0.27-0.28, preferably at
0.275.+-.0.008, PGE.sub.2 to Il-2 in a ratio range of 3.68-5.42,
preferably at 4.5.+-.0.87 and TxB.sub.2 to Il-2 in a ratio range of
23.5-25.1, preferably at 24.3.+-.0.83.
[0027] Multikine.RTM. was tested using a characterization protocol
and does not contain the following cytokines and other small
biologically active molecules: IL-4, IL-7, and IL-15, TfR, sICAM,
PDGF-AB, IFN-.alpha., EPO, LTC 4, TGF-.beta.2, FGF basic,
Angiogenin, sE-selectin, SCF, and LIF. Multikine.RTM. contains only
trace quantities (just above the level of detection of the assay)
of IL-12, and LTB 4.
[0028] In the manufacturing process, mononuclear cells are
separated from human donor "buffy coats" by step-gradient
centrifugation and cultured with PHA to enhance production and
secretion of IL-2 and other cytokines from the donor white blood
cells in culture as disclosed in U.S. Pat. Nos. 5,093,479,
4,390,623, 4,388,309, 4,406,830, 4,661,447, 4,681,844 and
4,464,355, all of which are incorporated herein by reference.
Subsequently, the culture supernatant is aseptically harvested,
clarified and subjected to a commercial virus exclusion process.
The supernatant is then further concentrated approximately 10 fold
by ultrafiltration and microfiltration.
[0029] At this point, Human Serum Albumin, Inj. USP is added and
the concentrate is then buffered to a physiological pH and brought
to a target IL-2 concentration per the label claim (example 400
IU/mL). The concentrate is then subjected to a second
micro-filtration (0.22 micron-rated filter) and aseptically
dispensed into sterile serum-type vials and labeled by its IL-2
content. Product potency is measured by the incorporation of
radio-labeled thymidine by a cytotoxic T-lymphoid line (CTLL-2).
The final injectable agent is further tested by ELISA for the
presence of five marker cytokines: IL-2, IL-1.beta., GM-CSF,
IFN-.gamma., and TNF-.alpha..
[0030] Multikine.RTM. is provided frozen in a borosilicate glass
serum vial containing 2.2 mL of drug at the label claim as IL-2
(400 IU/ml) for peritumoral, intratumoral, perilymphatic or
subcutaneous administration. Multikine.RTM. is subjected to quality
control tests for identity, sterility, bacterial endotoxins, pH,
and total protein concentration. Each vial is inspected for
particulate contamination and appearance. The preparation has a
total protein content of 3 mg/mL wherein the material is supplied
sterile and pyrogen free. Multikine.RTM. has an assigned expiration
date of 24 months from date of manufacture when the drug is stored
at -20.degree. C.
[0031] Definitions
[0032] IL-2--Interleukin 2 (IL-2): A 15.5-kD glycoprotein
synthesized by CD4.sup.+ helper T lymphocytes (Formally known as T
cell Growth Factor). IL-2 has an autocrine effect acting on the
CD4+ T lymphocytes that produce it and on other cells of the immune
system (including B lymphocytes, CD8+ T lymphocytes, NK [Natural
Killer] cells and others).
[0033] IL-1.beta. Interleukin 1 beta (IL-1.beta.): A 17-kD cytokine
synthesized by activated mononuclear phagocytes, is found in free
form in the circulation and mediates inflammatory responses. It
acts on CD4+ T lymphocytes to help facilitate their proliferation,
and acts on B-lymphocytes as a growth and differentiation factor.
It also induces the synthesis of IL-6 by mononuclear
phagocytes.
[0034] TNF-.alpha.--Tumor Necrosis Factor alpha (TNF-.alpha.): A
157 amino acid (aa) residues protein, synthesized by stimulated
monocytes, macrophages, B lymphocytes, T lymphocytes, an NK cells
among others, found in a trimmeric form in the circulation. TNF
mediates direct anti-tumor action, causing tumor cell lysis,
facilitates leukocyte recruitment, inducing angiogenesis and
promotes fibroblast proliferation.
[0035] IFN-.gamma.--Interferon Gamma (IFN-.gamma.): A 21-24-kD
glycoprotein homodimer synthesized by activated T lymphocytes and
NK cells, is a powerful activator of monocytes increasing monocytes
ability to destroy intracellular microorganisms and tumor cells. It
has direct anti-viral and anti-proliferative activity, and causes
many cell types to express Class II MHC (Major Histocompatibility
Complex) cell surface molecular complex, as well as increasing the
expression of Class I MHC.
[0036] GM-CSF--Granulocyte Macrophage-Colony Stimulating Factor
(GM-CSF): A 127 aa protein found as a monomer in the circulation,
produced by macrophages and T lymphocytes, fibroblast and
endothelial cells. It is a growth factor for hemopoietic cells, and
stimulates the growth and differentiation of myelomonocytic
lineage.
[0037] IL-3--Interleukin-3 (IL-3): A 20-kD Lymphokine synthesized
by activated CD4+ T helper lymphocytes, acts as a
colony-stimulating factor by facilitating the proliferation of some
hematopoietic cells and promoting the proliferation and
differentiation of T lymphocytes.
[0038] IL-6--Interleukin-6 (IL-6): A 26-kD cytokine produced by
activated T lymphocytes, mononuclear phagocytes, endothelial cells,
and fibroblasts. It acts on many cells but has a special function
in enabling activated B-lymphocytes to differentiate into antibody
secreting plasma cells, and induces hepatocytes to form acute-phase
proteins (implicated in inflammatory responses) as well as
fibrinogen.
[0039] IL-8--Interleukin-8 (IL-8): An 8-kD protein produced by
macrophages and endothelial cells. Is a powerful chemotactic factor
for neutrophils and T lymphocytes, and facilitates neutrophil
adherence to endothelial cells.
[0040] IL-1.alpha.--Interleukin 1 alpha (IL-1.alpha.): A 17-kD
cytokine (like IL-1 is cleaved from a 33-kD precursor molecule,
synthesized by activated mononuclear phagocytes, is rarely found in
free form in the circulation and acts as a membrane-associated
substance.
[0041] It assists IL-1.beta. in mediating inflammatory
responses.
[0042] IL-10--Interleukin-10 (IL-10): An 18-kD polypeptide produced
by CD4+ and CD 8+ T lymphocytes, monocytes, macrophages, activated
B lymphocytes, and keratinocytes. It inhibits macrophages ability
to present antigen particularly to T.sub.H1-type cells, and secrete
IL-6 and TNF.
[0043] IL-16--Interleukin-16 (IL-16): A 14-kD tetrameric protein
produced by CD8+ T lymphocytes, eosinophils, mast cells and
respiratory epithelial cells. It has strong chemoattraction
properties for CD4+ T lymphocytes and monocytes.
[0044] G-CSF--Granulocyte Colony Stimulating Factor (G-CSF): A
22-25-kD homodimer glycoprotein produced by macrophages,
endothelial cells, fibroblasts and stromal cells. It increases
granulocyte progenitor cells in the marrow, and sustains increase
in blood neutrophils. It also enhances the ability of neutrophils
to exhibit enhanced super-oxide production thought to be important
in the destruction of microbially infected cells and tumor
cells.
[0045] TNF-.beta.--Tumor Necrosis Factor beta (TNF---): A 25-kD
protein produced by activated lymphocytes. It can kill tumor cells
in culture, and stimulates proliferation of fibroblasts. In
addition it mimics most of the other actions of TNF-.alpha..
[0046] MIP-1.alpha.--Macrophage Inflamatory Protein-1 alpha
(MIP-1.alpha.): A 66-aa monomeric protein produced by macrophages
and other cells. It is a chemo-attractant for monocytes, T
lymphocytes and eosinophils.
[0047] RANTES--An 8-kD protein produced by T lymphocytes and is a
chemo-attractant to monocytes, T lymphocytes and eosinophils, and
promotes inflammation.
[0048] EGF--Epidermal Growth Factor (EGF): A trisulfated
polypeptide of 53-aa residues. EGF is a member of the tyrosin
kinase family, and has multiple functions including stimulation of
the mitogenic response and assisting in wound healing.
[0049] PGE.sub.2--Prostaglandin E.sub.2 (PGE.sub.2): PGE.sub.2
belong to a family of biologically active lipids derived from
arachidonic acid through the cyclooxygenase enzymatic reaction. It
is released by activated monocytes and blocks MHC Class II
expression on T lymphocytes and macrophages.
[0050] TxB.sub.2--Thromboxane B.sub.2 (TxB.sub.2): TxB.sub.2 is a
member of biologically active compounds derived from
polyunsaturated fatty acids by isomerization of prostaglandin and
endoperoxidase PGH.sub.2 via the enzyme thromboxane synthetase.
TxB.sub.2 has a physiological role in thromboembolic disease, and
anaphylactic reactions.
[0051] CD25.sup.+ Cells--CD25 is a single chain glycoprotein, often
referred to as the .alpha.-chain of the Interleukin-2-receptor
(IL-2R) or the Tac-antigen, that has a mol wt of 55 kDa and is
present on activated T and B cells and activated macrophages. It
functions as a receptor for IL2. Together with the .beta.-chain of
the IL-2R, the CD25 antigen forms a high-affinity receptor complex
for IL-2.
[0052] CTLL-2 (Cell Line)--A line of mouse cytotoxic T lymphocytes
obtained from C57Bl/6 mice. This T cell line is dependent on an
exogenous source of IL-2 for growth and proliferation.
[0053] Fas-FasL--The Fas/Fas Ligand system. The combination of a
Fas antigen, a cell surface transmembrane protein that mediates
apoptosis, and a complementary Fas-activated cytokine on a
neutrophil that transduces an apoptotic signal into cells. Fas is a
type-I membrane protein belonging to the tumor necrosis factor
(TNF) receptor superfamily, and FasL is a member of the TNF family.
FAS ligand is a membrane-bound protein of 31 kDa [kilo Dalton] (278
amino acids). The Fas-Fas ligand system plays important roles in
many biological processes, including the elimination of
autoreactive lymphoid cells. The Fas ligand is predominantly
expressed in activated T lymphocytes and is one of the major
effector molecules of cytotoxic T lymphocytes and natural killer
cells.
[0054] HLA-DR.sup.+Lymphocytes--Lymphocytes containing human
leukocyte antigen (HLA)-DR antigens, a group of polymorphic
glycoproteins determined by a glue sequence found in a leukocyte
loci located on chromosome 6, the major histocompatibility loci in
humans.
[0055] IU (International Units)--A unit of measure of the potency
of biological preparations by comparison to an international
reference standard of a specific weight and strength e.g., WHO
1.sup.st International Standard for Human IL-2, 86/504.
International Units are the only recognized and standardized method
to report biological activity units that are published and are
derived from an international collaborative research effort.
[0056] U (Units as a measure of biological activity)--Shorthand for
a variety of named "units", which each laboratory derives as a
reference, which is further unique to the laboratory where the work
is being performed. Each "unit" is different from one laboratory to
another laboratory and is not a globally recognized standard such
as International Units (IU).
[0057] Mononuclear Infiltrate--Presence of monocytes, plasma cells,
and lymphocytes, in tissue where they "normally" would not be
present; or the presence of these cells in large numbers or
abundance in clusters where they would otherwise be present in only
a small number.
[0058] TCR .zeta. Chain--T-cell receptor-zeta chain. The zeta
subunit is part of the TCR complex and is targeted towards the
interaction of the TCR cell surface receptor with its ligand
(antigen). The zeta subunit extending into the cell cytoplasm
(cytosol) is phosphorylated at its tyrosine residues upon T cell
activation and is implicated in signal transduction after TCR
ligation.
[0059] TIL (Tumor Infiltrating Lymphocytes)--T lymphocytes isolated
from the tumor they are infiltrating. Tumor Infiltrating
lymphocytes have little or no cytotoxicity. TILs include CD4+ CD8+
predominantly T cells, and can be expanded in vitro by culture in
the presence of IL-2. These cells are activated by the treatment
with IL-2 and are frequently more aggressive towards the tumor from
which they were isolated than normal lymphokine activated cells.
The cytotoxic activity of TILs can be enhanced by IFN-.gamma.. The
antitumor activity of TILs in vivo can be blocked by
TGF-.beta..
[0060] ZAP 70--A 70 kD Zeta Associated Protein associated with the
TCR .zeta. Chain that is a tyrosine kinase present in cytosol. ZAP
70 is thought to participate in maintaining T lymphocyte receptor
signaling, mediating the signal transduction which eventually
produces IL-2. The ZAP70 gene is expressed in T-cells and natural
killer cells and maps to human chromosome 2q12.
[0061] .zeta. (Zeta) Chain--See TCR .zeta. Chain--The zeta chain
gene is located on chromosome 1 in humans. The extracellular domain
of this protein is nine amino acids long whereas the transmembrane
domain contains a negatively charged aspartic acid residue and the
cytoplasmic domain is 113 amino acids long. The cytoplasmic tail
contains three of the antigen recognition motifs found in the
cytoplasmic tails of CD3 chains. The zeta chain is also associated
with other receptors such as the Fc (fragment, crystalline)-gamma
receptor of NK cells.
[0062] USP--U.S. Pharmacopeia Monographs.
[0063] P--"p<0.01": A term in mathematical statistics that
denotes the level of probability of an event occurring under
pre-set conditions.
[0064] ANOVA (Analysis of Variances)--A single factor analysis as
described in Statistics and mathematical textbooks e.g., "Handbook
of Statistical Methods for Engineers and Scientists", Harrison M.
Wadsworth, Jr., Ed., McGraw Hill 1990, and "Statistical Operations
Analysis of Health Research Data", Robert P. Hirsch and Richard K.
Riegelman, Eds. Blackwell Science Inc., 1996.
[0065] Mode of action and characterization of Multikine.RTM.
[0066] Multikine.RTM. is a biologically active, minimally toxic,
immunomodulatory mixture of naturally derived and naturally
occurring human cytokines produced under set conditions as
described herein. Multikine.RTM. can be used as a anti-cancer and
anti-viral therapy or as a neo-adjuvant therapy with a
broad-spectrum application for cancer, infectious disease, and
other diseases states responding to immunomodulation.
[0067] Multikine.RTM. was developed based on animal studies, which
demonstrated that "mixed interleukins" have immunomodulatory and
immunostimulatory activity in vitro as shown by Hadden et al.,
"Mixed Interleukins and Thymosin Fraction V Synergistically Induce
T Lymphocyte Development in Hydrocortisone-Treated Aged Mice",
Cell. Immunol. 144:228-236 (1992). Without being limited to any one
theory, it is hypothesized that the local/regional injection of
"mixed interleukins" such as Multikine.RTM. overcomes local
immuno-suppression. Subsequently, a break tolerance to tumor
antigens occurs and allows for an effective local anti-tumor immune
response to occur.
[0068] It has been shown that the local instillation of
interleukins in the region of the tumor or the actual transfection
of Interleukin genes into a tumor markedly augments the anti-tumor
immune response resulting in tumor regression as reported by
Golumbek et al., "Treatment of Established Renal Cancer by Tumor
Cells Engineered to Secrete Interleukin-4", Science 254:713-716
(1991). However, none of these studies discovered the highly
unexpected effect of inducing malignant cells into a cell cycle
phase without causing the active proliferation of the tumor.
[0069] Quite unexpectedly, the administration of Multikine.RTM.
pre-surgery leads to an increase in the number of tumor cells in
the cell cycle phase without increasing the risk of a more rapidly
growing and more rapidly recurring tumor as would otherwise be
predicted from the art. The ability to induce tumor cells into
cell-cycle appears to be unique to Multikine.RTM. and may be due to
the synergistic effect of the different cytokines present in this
investigational drug and the differential effect of these cytokines
on both the host's immune system and the tumor cells.
[0070] Data regarding the recurrence rate of patients treated with
Multikine.RTM. prior to surgery showed no recurrence of cancer at
24 months post treatment with Multikine.RTM.. Remarkably, a small
cohort of 8 sequentially treated patients did not have a single
recurring patient in the 24 months follow-up period. In stark
contrast, the literature teaches the recurrence rate of similar
patients at about 50% at 18-24 months post surgery.
[0071] In particular, Multikine.RTM. treatment did not appear to
induce active proliferation of tumor residing lymphoid cells.
Correspondingly, stromal Ki-67.sup.+ cells decreased while the
frequency of Ki-67.sup.+ cancer cells increased following
Multikine.RTM. treatment. Thus, Multikine.RTM. treatment induced an
increase in the number of cycling tumor cells leading to increased
susceptibility of the residual tumor to follow-on treatment with
radiation and/or chemotherapy. Although other studies conducted
with both natural and recombinant cytokines have shown efficacy in
the treatment of cancer therapy, those studies have failed to teach
the induction of entry into the cell cycling phase or the new
method of synergistically combining Multikine.RTM. with
chemotherapy, immuno-therapy and radiation therapy.
[0072] For example, studies with the use of natural human and
recombinant IL-2 and other cytokines as well as in local-regional
therapy demonstrated the immune augmenting and anti-cancer activity
at various sites. In particular, IL-2 demonstrates activity in the
pleural cavity, liver and the urinary bladder while IFN-.alpha.
demonstrates activity in the ovary while IFN-.beta. demonstrates
activity in the brain as reported by Yasumoto et al., "Induction of
lymphokine-activated killer cells by intrapleural instillations of
recombinant interleukin-2 in patients with, malignant pleurisy due
to lung cancer", Cancer Res 1987; 47:2184-7; Mavilgit et al.,
"Splenic versus hepatic artery infusion of interleukin-2 in
patients with liver metastases", J Clin Oncol 1990; 8:319-24; Pizza
et al., "Tumor regression after intralesional injection of
interleukin-2 (IL-2) in bladder cancer. Preliminary report", Int J
Cancer 1984; 34:359-67; Berek et al., "Intraperitoneal recombinant
.alpha.-interferon for "salvage" immunotherapy in stage III
epithelial ovarian cancer: a gynecologic oncology group study",
Cancer Res 1985; 45:4447-53; and Fettel et al., "Intratumor
administration of beta-interferon in recurrent malignant gliomas-A
phase I clinical and laboratory study", Cancer 1990; 65:78-83. Even
further, IFN-.gamma. has been shown to demonstrate activity in skin
while TNF-.alpha. demonstrates activity in the genitalia while a
mixture of various cytokines demonstrates activity in the head and
neck as reported by Edwards et al., "The effect of intralesional
interferon gamma on basal cell carcinomas", J Am Acad Dermatol
1990; 22:496-500; Irie et al., "A case of vulva cancer responding
to the recombinant human tumor necrosis factor (PT-950) local
injection therapy", Gan No Rinsho 1988; 34:946-50; and Pulley et
al., "Intravenous, intralesional and endolymphatic administration
of lymphokines in human cancer", Lymph Res 1986; 5:S157-63.
Moreover, studies of recombinant IL-2 administrations for 10 days
prior to surgery in the jugular peri-lymphatic or jugular
peri-lymphatic and under the chin showed variable necrosis and
lymphocytic infiltration as reported by Valente et al.,
"Infiltration leukocyte polulations and T-lymphocyte subsets in
head and neck squamous cell carcinomas from patients receiving
perilymphatic injections of recombinant interleukin-2", Mod Pathol
1990; 3:702-8 and DeStefani et al., "Treatment of oral cavity and
oropharynx squamous cell carcinoma with perilymphatic
interleukin-2: clinical and pathologic correlations", J Immunother
1996; 19:125-33. Moreover, microscopic examination of the resected
tumors demonstrated an increase in lymphocytic infiltrate
correlating to the clinical observations of IL-2 as reported by
Saito et al., "Immunohistology of tumor tissue in local
administration of recombinant interleukin-2 in head and neck
cancer", Nip Jibi Gakkai Kaiho 1989; 92:1271-6.
[0073] Nevertheless, none of the aforementioned studies showed any
change in the gross dimensions of resected tumors despite two
remissions at putatively high doses of recombinant IL-2 (800,000 U
for four weeks [U=Units]) in 20 head and neck cancer patients as
reported by Saito et al., "Clinical evaluation of local
administration of rIL-2 in head and neck cancer", Nip Jibi Gakkai
Kaiho. 1989; 921271-6. Furthermore, the aforementioned studies have
been limited by the small number of patients and hampered by the
lack of a pathological comparison to a control group.
[0074] Although a recent randomized multi-center phase III study of
202 OSCC patients by De Stefani et al. indicated that
peri-lymphatic administration of low does (5000 U/day [U=Units]) of
recombinant human IL-2 for 10 days prior to surgery, into the
ipsylateral cervical lymph node chain, resulted in a significant
(p<0.01) increase in disease-free survival, which in turn
resulted in longer overall survival (p<0.03), De Stefani et al.
failed to assess the role of this treatment regimen on cell cycling
and its effect on the improvement of radiation and chemotherapy.
See De Stefani et al., "Improved Survival With Perilymphatic
Interleukin 2 in Patients With Resectable Squamous Cell Carcinoma
of the Oral Cavity and Oropharynx", Cancer 2002; 95: 90-97.
Furthermore, despite teaching 5000 U/day, no comparisons between
the present invention and De Stefani et al. could be made with
regard to De Stefani et al.'s teaching of a "high" and "low" dose
of an administered biologic because the drug potency was measured
by an undefinable U (Units). In contrast, the present invention
validated and completed the full USP analytical methods validation
program for determining the biological activity of Multikine.RTM.
in IU (International Units).
[0075] Methods
[0076] Tumor cell proliferation as measured by the
immunohistochemistry Ki-67 marker, or other equivalent means such
as through the use of PCNA marker, p53 marker were used as a
prognostic parameter. de Vicente et al., "Expression of cyclin D1
and Ki-67 in squamous cell carcinoma of the oral cavity:
clinicopathological and prognostic significance", Oral Oncol 2002;
38:301-8; Bettendorf et al., "Prognostic relevance of Ki-67 antigen
expression in 329 casess of oral squamous cell carcinoma", ORL J
Otorhinolaryngeol Relat Spec 2002; 64:200-5. In conjunction, flow
cytometry or conventional staining methods and the use of
microscopy with clinical, histopathological and tumor staging and
classification (TNM, Tumor, Node, Metastasis) were used with other
to indicate the aggressiveness of the disease process. Kerdpon et
al., "Expression of p53 in oral mucosal hyperplasia, dysplasia and
squamous cell carcinoma", Oral Disease 1997; 3:86-92.
[0077] In particular, a Ki67 cell proliferation marker
differentiates and is specific for only the cells that are in the
cell cycle stages. G.sub.1 is the first growth phase; S is the
second phase marked by the initiation of DNA synthesis by the cell
where cellular DNA replicates, and G.sub.2 the second growth phase
of the cell follows DNA replication in which the cell doubles in
size. M is the last phase in the cell cycle where mitosis occurs
wherein the cell divides into a daughter cell from the original
parent cell. Each resulting cell contains a complete replica of the
DNA of the original parent cell. Ki67 cellular marker being
specific to cells in the cell cycle cannot be found in cells that
are in G.sub.o, which is a resting phase of the cell. During
G.sub.o, the cell does not undergo cellular replication,
proliferation or DNA replication. Notably, the cell cycle phase
phenomena is a property common to all living eukaryotic cells
including tumor cells.
[0078] To detect tumor cell proliferation, the presence of Ki-67 in
residual tumor cell nests following surgical excision are
determined. Raybaud et al., "Nuclear DNA content, an adjunct to p53
and Ki-67 as a marker of resistance to radiation therapy in oral
cavity and pharyngeal squamous cell carcinoma", Int J Oral
Maxillofac Surg 2000; 29:36-41; Koelbl et al., "p53 and Ki-67 as
predictive markers for radiosensitiveity in squamous cell carcinoma
of the oral cavity? An immunohistochemical and clinicopathologic
study", Int J Radiat Oncol Biol Phys 2001; 49:147-54. Generally,
Ki-67 can be found in cells undergoing cell cycle G.sub.1, S,
G.sub.2, and M but not in "resting" tumor cells (G.sub.o). Since
cycling tumor cells are both more radio- and chemo-sensitive, and
non-cycling tumor cells are by-and-large radio- and
chemo-resistant.
[0079] Accordingly, a study was designed that would analyze by
immuno-histopathology tumors from head and neck cancer patients
treated with Multikine.RTM. prior to surgical resection of the
residual tumor followed by radiation therapy. Timr et al., "The
effect of Leukocyte Interleukin, Injection on the peri- and
intratumoral subpopulation of mononuclear cells and on tumor
epithelia--A possible new approach to augmenting sensitivity to
radiation and chemotherapy in oral cancer. A multi-center Phase
I/II clinical trial", The Laryngoscope [accepted for publication
June 2003]. The study was conducted in a blinded manner and
executed by three independent qualified pathologists that were
blinded to the treatment and patient population, treated or
control. Our clinical study, the results of which are reported
here, and are incorporated herein by reference analyzed a cohort of
54 oral squamous cell cancer patients (H&NC) as part of a phase
I-II clinical trial. These patients were investigated for safety of
the therapeutic regimen, tumor and clinical responses, and for the
composition of the mononuclear infiltrate and cell cycling
rates.
[0080] Twenty-seven (27) patients of the 54 patients cohort
received peritumoral administration of Multikine.RTM. in a dose
escalating study. This study resulted in the demonstration that the
pre-treatment of head and neck cancer patients with Multikine.RTM.
tumor induced entry into cell cycle phase, G.sub.1, S, G.sub.2, and
M, but not G.sub.o. This lead to a decrease in recurrence rate and
an increase in disease-free survival of Multikine.RTM. treated
patients.
[0081] In our study, Multikine.RTM. administrations were performed
in the following manner: daily dose was injected peritumorally over
a two-week period (3 times per week) at the following doses for
each of the dose groups tested; low dose, 400 IU (International
Units of IL-2) [IL-2-equivalent] daily (8 patients), medium dose,
800 IU (IL-2-equivalent) daily (12 patients), and 5 times per week
at the high dose, 800 IU (IL-2-equivalent) daily (7 patients). All
Multikine.RTM. injections were administered intra-dermally at the
circumferential margin of the visible/palpable tumor mass. Surgery
aimed at resection of the residual tumor mass was performed between
day 21 and day 28 following the initial administration of
Multikine.RTM.. Local/regional radiation therapy commenced in
post-operative patients following wound healing at variable times
post-surgery and was dependent on the individual patient recovery
from the surgical intervention. Radiotherapy was generally
initiated between two to four weeks post-surgery.
[0082] The administration of Multikine.RTM. was preceded by the
single intravenous infusion of cyclophosphamide, 300 mg/m.sup.2
three-days prior to the first Multikine.RTM. administration.
Indomethacin (25 mg) was self-administered orally (with food),
three times daily, beginning 3 days post cyclophosphamide
administration and until 24 hours prior to surgery. Zinc sulfate
(50 mg) and multivitamin supplement, once daily, was
self-administered beginning 3 days after cyclophosphamide
administration and until 24 hours prior to surgery. The patients
were counseled and encouraged to continue self-administration of
multivitamin and Zinc regimen following surgery. These agents have
no effect whatsoever on tumor cell cycling and were given at doses
that are 3-5 fold bellow the normal cancer therapeutic doses for
these drugs.
[0083] Results
[0084] Detection of cycling cells by Ki-67 expression identified
cancer cells as shown in FIG. 1 and stromal cells (host cells:
mononuclear cells, fibroblasts, endothelial cells etc.).
Morphometric analysis of the density of Ki-67.sup.+ cancer cells
indicated that Multikine.RTM. treatment induced significant
increase (p<0.05) in cycling tumor cells except at the highest
Multikine.RTM. dose administered as shown in FIG. 2. On the other
hand, the incidence of cycling host cells found primarily in the
stromal area of the tumor decreased with an increasing
Multikine.RTM. dose again shown in FIG. 2. Effects were proved to
be significant for the lowest and the highest doses (p<0.05).
These findings support the conclusion that treatment with
Multikine.RTM. treatment causes cancer cells to enter a cell cycle
phase but does not cause the host immune cells or stromal cells to
cycle.
[0085] Accordingly, the present invention contemplates
Multikine.RTM. treatment to induce cell cycle entry of a high
proportion of the tumor cell population based on the expression of
Ki-67 antigen.
[0086] As stated herein, preliminary data regarding the recurrence
rate of patients treated with Multikine.RTM. prior to surgery,
which were either followed by radiation therapy or watchful
waiting, did not exhibit an increase in the recurrence rate at 24
months post treatment with Multikine.RTM.. A small cohort of 8
sequentially Multikine.RTM. treated patients did not have a single
recurring patient in the 24 months follow-up period. In contrast,
the literature pegs the recurrence rate of these patients at about
50% at 18-24 months post surgery.
[0087] Moreover, Multikine.RTM. treatment did not appear to induce
active proliferation of tumor residing lymphoid cells, and
correspondingly stromal Ki-67.sup.+ cells decreased, while the
frequency of Ki-.delta.67.sup.+ cancer cells increased following
Multikine.RTM. treatment. Thus, Multikine.RTM. treatment induced
the increase the number of cycling tumor cells leading to increased
susceptibility of the residual tumor to follow-on treatment with
radiation and/or chemotherapy.
[0088] Treatment Regimen with Multikine.RTM. for Cancer
[0089] The treatment regimen for the pre-sensitization of cancer
with Multikine.RTM. is predicated on treatment protocol developed
for head and neck cancer patients, which has been proven in a
statistically significant manner to significantly increase tumor
cell cycling aimed at rendering these tumor cells more sensitive to
follow on treatment with radiation and/or chemotherapy.
[0090] The treatment will include the administration of
Multikine.RTM. subcutaneously in the area of the submandibular
cervical lymph node chain.
[0091] A two-week course of ten (10) subcutaneous/subdermal daily
injections of Multikine.RTM. at a daily dose ranging from about 20
IU to 1600 IU as IL-2 and will be administered 1/2 peritumorally at
the circumferential margin of the tumor mass, and 1/2 at the
submandibular lymphatic chain ipsylateral to the tumor mass.
Another course of treatment can preferably in the range of 40 IU to
800 IU. Still another range can be in the range of 35 IU to 75
IU.
[0092] One specific non-limiting example of a suggested treatment
contemplates administration of Multikine.RTM. at a daily dose of 55
IU as IL-2 in a two-week course of ten (10) subcutaneous/subdermal
daily injections
[0093] Drug Safety, Pilot Efficacy and Compositions
[0094] Multikine.RTM. has been tested in over 190 Cancer, HIV, and
HIV/HPV infected, patients with no severe adverse events related to
Multikine.RTM. administration as reported by Harris et al.,
"Immunologic approaches to the treatment of prostate cancer", Semin
Oncol. August 1999; 26(4):439-7; Timr et al., "The effect of
Leukocyte Interleukin, Injection on the peri- and intratumoral
subpopulation of mononuclear cells and on tumor epithelia--A
possible new approach to augmenting sensitivity to radiation and
chemotherapy in oral cancer. A multi-center Phase I/II clinical
trial", The Laryngoscope [accepted for publication June 2003];
Brown et al., "A Phase I Open-Label Study of Leukocyte Interleukin,
Injection in HIV-1 infected individuals: preliminary evidence for
improved delayed-type hypersensitivity responses to recall
antigens", Antiviral Therapy 5 (supplement) 18, 2000; Taylor et
al., "Immunotherapy with Leukocyte Interleukin, Injection for human
papilloma virus (HPV) induced cervical dysplasia in HIV patients",
Annual Meeting of the International Society for Interferon and
Cytokine Research, Cleveland, Ohio, October 2001; Taylor et al.,
"Immunotherapy with Leukocyte Interleukin, Injection for human
papilloma virus (HPV) induced cervical dysplasia in HIV patients",
33rd SGO Conference, Miami, Fla., March 2002 Multikine.RTM. was
also shown to be safe in animal toxicological studies in mice,
rats, guinea pigs and dogs. Furthermore, Multikine.RTM. was tested
for and has demonstrated pilot efficacy in head and neck cancer and
cervical dysplasia.
[0095] Multikine.RTM. may further be used as a component of an
immunomodulatory composition together with one or more
pharmaceutically acceptable carriers or adjuvants, either
prophylactically or therapeutically. When provided for use
prophylactically, the immunomodulatory composition is provided in
advance of any evidence of infection or disease. While it is
possible for Multikine.RTM. to be administered in a pure or
substantially pure form, a pharmaceutical composition, formulation
or preparation may also be used.
[0096] The formulations of the present invention, both for clinical
and for human use, comprise Multikine.RTM. as described above
together with one or more pharmaceutically acceptable carriers and,
optionally, other therapeutic ingredients, especially therapeutic
immunological adjuvants. The carrier(s) must be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
[0097] In general, the formulations are prepared by uniformly and
intimately bringing into association the active ingredient with
liquid carriers or finely divided solid carriers or both, and then,
if necessary, bringing the product into the desired formulation.
The term "pharmaceutically acceptable carrier" as used herein
refers to any carrier, diluent, excipient, suspending agent,
lubricating agent, adjuvant, vehicle, delivery system, emulsifier,
disintegrant, absorbant, preservative, surfactant, colorant,
flavorant, or sweetener. The formulations may conveniently be
presented in unit dosage form and may be prepared by any method
well-known in the pharmaceutical art.
[0098] Formulations suitable for intravenous, intramuscular,
subcutaneous, or intraperitoneal, nasal, etc. administration
conveniently comprise sterile aqueous solutions of the active
ingredient(s) with solutions which are preferably isotonic with the
blood of the recipient. The compounds of the present invention may
also be administered orally, parenterally, by inhalation spray,
topically, rectally, buccally, vaginally or via an implanted
reservoir in dosage formulations containing conventional non-toxic
pharmaceutically-acceptable carriers, adjuvants and vehicles. The
term parenteral as used herein includes subcutaneous, intravenous,
intramuscular, intraperitoneally, intrathecally,
intraventricularly, intrasternal and intracranial injection or
infusion techniques.
[0099] Such formulations may be conveniently prepared by dissolving
solid active ingredients in water containing physiologically
compatible substances such as sodium chloride (e.g. 0.1-2.0M),
glycine, and the like, and having a buffered pH compatible with
physiological conditions to produce an aqueous solution and
rendering the solution sterile. These may be present in unit or
multi-dose containers, for example, sealed ampules or vials.
[0100] The compounds of the present invention may also be
administered in the form of sterile injectable preparations, for
example, as sterile injectable aqueous or oleaginous suspensions.
These suspensions may be formulated according to techniques known
in the art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparations may also be
sterile injectable solutions or suspensions in non-toxic
parenterally-acceptable diluents or solvents, for example, as
solutions in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as solvents or suspending mediums. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides. Fatty acids such as oleic acid
and its glyceride derivatives, including olive oil and castor oil,
especially in their polyoxyethylated versions are useful in the
preparation of injectables. These oil solutions or suspensions may
also contain long-chain alcohol diluents or dispersants.
[0101] The compounds of this invention may also be administered
topically, especially when the conditions addressed for treatment
involve areas or organs readily accessible by topical application
including disorders of the eye, the skin, or the lower intestinal
tract. Suitable topical formulations are readily prepared for each
of these areas.
[0102] For topical application to the eye, or ophthalmic use, the
compounds can be formulated as micronized suspensions in isotonic,
pH adjusted sterile saline, or preferably, as solutions in
isotonic, pH adjusted sterile saline, either with or without a
preservative such as benzylalkonium chloride. Alternatively, the
ophthalmic uses of Multikine.RTM. may be formulated in an ointment
such as petrolatum.
[0103] For topical application to the skin, the compounds can be
formulated in a suitable ointment containing the compound suspended
or dissolved in, for example, a mixture with one or more of the
following: mineral oil, liquid petrolatum, white petrolatum,
propylene glycol, polyoxyethylene polyoxypropylene compound,
emulsifying wax and water. Alternatively, the compounds can be
formulated in a suitable lotion or cream containing the active
compound suspended or dissolved in, for example, a mixture of one
or more of the following: mineral oil, sorbitan monostearate,
polysorbate 60, cetyl esters wax, cetearyl alcohol,
2-octyldodecanol, benzyl alcohol and water.
[0104] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. Some factors include the activity of the specific
compound employed, the age, body weight, general health, sex, and
diet of the patients; the time of administration, rate of
excretion, drug combination, and the severity of the particular
disease being treated and form of administration.
[0105] Pharmaceutical methods may also be employed to control the
duration of action. Controlled release preparations may be achieved
through the use of polymer to complex or absorb the peptide. The
controlled delivery may be exercised by selecting appropriate
macromolecules (for example, polyester, polyamino acids, polyvinyl,
pyrrolidone, ethylenevinylacetate, methylcellulose,
carboxymethylcellulose, or protamine sulfate) and the concentration
of macromolecules as well as the methods of incorporation in order
to control release.
[0106] For example, Multikine.RTM. may be incorporated into a
hydrophobic polymer matrix for controlled-release over a period of
days. Such controlled-release films are well known to the art.
Particularly preferred are transdermal delivery systems. Other
examples of polymers commonly employed for this purpose that may be
used in the present invention include non-degradable ethylene-vinyl
acetate copolymer and degradable lactic acid-glycolic acid
copolymers which may be used externally or internally. Certain
hydrogels such as poly(hydroxyethylmethacrylate) or
poly(vinylalcohol) also may be useful, but for shorter release
cycles then the other polymer releases systems, such as those
mentioned above.
[0107] Alternatively, instead of incorporating these agents into
polymeric particles, it is possible to entrap these materials in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly(methylmethacrylate)
microcapsules, respectively, or in colloidal drug delivery systems,
for example, liposomes, albumin microspheres, microemulsions,
nanoparticles, and nanocapsules or in macroemulsions.
[0108] To be effective therapeutically as central nervous system
targets, Multikine.RTM. should also readily penetrate the
blood-brain barrier when peripherally administered. Compounds which
cannot penetrate the blood-brain barrier can be effectively
administered by an intraventricular route or other appropriate
delivery system suitable for administration to the brain.
[0109] Multikine.RTM. may also be supplied in the form of a kit,
alone, or in the form of a pharmaceutical composition as described
above. Administration of Multikine.RTM. can be conducted by
conventional methods. For example, Multikine can be used in a
suitable diluent such as saline or water, or complete or incomplete
adjuvants. Multikine.RTM. can be administered by any route
appropriate for immune system stimulation, such as intravenous,
intraperitoneal, intramuscular, subcutaneous, nasal, oral, rectal,
vaginal, and the like.
[0110] As noted above, Multikine.RTM. may be for either a
prophylactic or therapeutic purpose. When provided
prophylactically, Multikine.RTM. is provided in advance of any
evidence or in advance of any symptom due to disease. When provided
therapeutically, Multikine.RTM. is provided at (or after) the onset
of the disease or at the onset of any symptom of the disease. The
therapeutic administration of Multikine.RTM. serves to attenuate
the disease and improves conventional treatment outcomes.
[0111] The invention being thus described, it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit scope of the invention and
all such modifications are intended to be included within the scope
of the following claims.
* * * * *