U.S. patent application number 11/511085 was filed with the patent office on 2007-05-10 for composition and methods for affecting metallocorrinoid uptake.
Invention is credited to Joseph A. Bauer, Daniel J. Lindner.
Application Number | 20070104683 11/511085 |
Document ID | / |
Family ID | 25343974 |
Filed Date | 2007-05-10 |
United States Patent
Application |
20070104683 |
Kind Code |
A1 |
Bauer; Joseph A. ; et
al. |
May 10, 2007 |
Composition and methods for affecting metallocorrinoid uptake
Abstract
The present invention is directed to compositions and methods
for affecting metallocorrinoid uptake. The compositions and methods
of the present invention are particularly useful in enhancing the
uptake or availability of biologically active metallocorrinoids
(e.g. cobalamin and its analogs). The present invention is
particularly useful in the treatment or prevention of conditions
that result from low expression or activity of proteins involved in
the processing of metallocorrinoids, as well as in conditions which
would benefit from enhanced uptake or availability of cobalamin or
its biologically active analogs of cobalamin (e.g. cobalamin drug
conjugates).
Inventors: |
Bauer; Joseph A.; (Akron,
OH) ; Lindner; Daniel J.; (Shaker Heights,
OH) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
25343974 |
Appl. No.: |
11/511085 |
Filed: |
August 28, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10761870 |
Jan 21, 2004 |
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11511085 |
Aug 28, 2006 |
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09864747 |
May 24, 2001 |
6752986 |
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10761870 |
Jan 21, 2004 |
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Current U.S.
Class: |
424/85.6 ;
514/185; 514/52 |
Current CPC
Class: |
A61K 38/215 20130101;
A61K 31/555 20130101; A61K 31/714 20130101; A61K 31/714 20130101;
A61K 2300/00 20130101; A61K 38/215 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/085.6 ;
514/052; 514/185 |
International
Class: |
A61K 38/21 20060101
A61K038/21; A61K 31/555 20060101 A61K031/555; A61K 31/714 20060101
A61K031/714 |
Claims
1. A therapeutic composition comprising a metallocorrinoid and a
cytokine.
2. The therapeutic composition of claim 1, wherein said
metallocorrinoid is vitamin B.sub.12.
3. The therapeutic composition of claim 1, wherein said
metallocorrinoid is a vitamin B.sub.12 analog.
4. The composition of claim 1, wherein said cytokine is
interferon-.beta..
5. The composition of claim 3, wherein said vitamin B.sub.12 analog
is nitrosylcobolamin.
6. The therapeutic composition of claim 3, wherein said vitamin
B.sub.12 analog is selected from the group consisting of
hydroxocobalamin, cyanocobalamin, methylcobalamin, 5'
deoxyadenocobalamin.
7. The therapeutic composition of claim 1 wherein said
metallocorrinoid is a cobalamin drug conjugate and said cytokine is
interferon-.beta..
8. The therapeutic composition of claim 7, further including a
pharmaceutical carrier.
9. A method of enhancing uptake of a metallocorrinoid comprised of
administering a cytokine.
10. The method of claim 9, wherein said metallocorrinoid is vitamin
B.sub.12.
11. The method of claim 9, wherein said metallocorrinoid is a
vitamin B.sub.12 analog.
12. The method of claim 9, wherein said cytokine is an
interferon.
13. The method of claim 12, wherein said interferon is
interferon-.beta..
14. A method of treating a patient comprising the steps of inducing
cytokine production and administering a metallocorrinoid.
15. The method of claim 14, where the step of inducing cytokine
production comprises administering a cytokine.
16. The method of claim 14, wherein said metallocorrinoid is
vitamin B.sub.12.
17. The method of claim 14 wherein said metallocorrinoid is a
vitamin B.sub.12 analog.
18. The method of claim 14, wherein the cytokine is
interferon-.beta..
19. A method for increasing TCII-R activity in a subject to treat a
condition favorably affected by an increase in said TCII-R activity
comprising the step of administering to a subject in need of such
treatment a cytokine in an amount effective to increase TCII-R
activity in the subject.
20. The method of claim 19, further comprising the step of
co-administering a vitamin B.sub.12 analog.
21. The method of claim 19, wherein said cytokine is administered
prior to the vitamin B.sub.12 analog.
22. The method of claim 19, wherein said cytokine is administered
prophylactically.
23. The method of claim 19, wherein said cytokine is administered
acutely.
24. The method of claim 19, wherein said cytokine is an
interferon.
25. The method of claim 24, wherein said interferon is
interferon-.beta..
26. The method of claim 19, wherein said condition is unwanted
cellular proliferation.
27. A method of enhancing bio-availability of a metallocorrinoid
comprising the step of administering interferon-.beta..
28. A method of treating a subject to increase TCII-R activity in a
cell comprising the step of administering to a subject in need of
such treatment a cytokine in an amount effective to increase TCII-R
activity in said cell.
29. The method of claim 28, wherein the subject is cobalamin
deficient.
30. The method of claim 28, wherein the amount is sufficient to
increase TCII-R activity above normal baseline levels.
31. The method of claim 28, wherein the subject has an abnormally
low level of TCII-R activity.
32. The method of claim 28, further comprising the step of
co-administering a ligand of TCII-R.
33. The method of claim 32 herein the ligand of TCII-R is
cobalamin.
34. The method of claim 32, wherein the ligand of TCII-R is a
cobalamin drug conjugate.
35. The method of claim 34, wherein the cobalamin drug conjugate is
nitrosylcobalamin.
Description
RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser.
No. 10/761,870, filed Jan. 21, 2004, which is a continuation of
U.S. application Ser. No. 09/864,747, filed on May 24, 2001 (Now
U.S. Pat. No. 6,752,986, issued on Jun. 22, 2004).
[0002] The entire teachings of the above application(s) are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Metallocorrinoids are corrin rings with a metal-atom center,
such as Co, Fe, Ni, or Mn. A corrin ring is four reduced pyrrole
rings linked together. A subclass of naturally occurring
metallocorrinoids is known as cobalamin, that is, a cobalt-centered
corrin ring. Naturally occurring vitamin B.sub.12, for example, is
a cobalamin.
[0004] Vitamin B.sub.12 compounds are known to have many biological
functions. They are required by the enzyme methionine synthase, for
example, which is involved in the production of DNA. Pregnant women
need increased amounts of vitamin B.sub.12 which is involved in the
production of red blood cells. It is also believed that vitamin
B.sub.12 enhances the effects of other vitamins and nutrients in
tissue repair. Lack of vitamin B.sub.12 leads to megaloblastic
anemia (characterized by large and immature red blood cells) and
neuropathy in man with insidious onset of symptoms. These symptoms
include weakness, tiredness, breathlessness (dyspnea) on exertion,
tingling and numbness (paresthesia), sore tongue (glossitis), loss
of appetite and weight, loss of sense of taste and smell,
impotence, psychiatric disturbances (such as irritability, memory
impairment, mild depression, hallucinations) and sever anemia
(which may lead to signs of cardiac dysfunction). Deficiency of
vitamin B.sub.12 leads to defective DNA synthesis in cells; tissues
most affected are those with the greatest rate of cell turnover,
e.g. the haematopoietic system. In small children Cb1 deficiency
can result in developmental delay, hematological disorders, and
neurological disorders. There may be irreversible damage to the
nervous system with specific demyelination of the spinal cord.
[0005] Increased availability of vitamin B.sub.12, on the other
hand, appears to have a very beneficial effect. Cb1 analogs and
cobalamin drug conjugates have been shown to inhibit the growth of
leukemia cells by possibly deactivating methionine synthase, thus
preventing DNA synthesis. The cobalamins that are analogous to
vitamin B.sub.12 compounds would appear to be potential therapeutic
agents. These include hydroxocobalamin, cyanocobalamin,
nitrocobalamin, mehtylcobalamin, and 5'-deoxyadenocobalamin, as
well as nitrosylcobalamin.
[0006] All forms of vitamin B.sub.12 (adenosyl-, cyano-, hydroxo-,
or methylcobalamin) are bound by the transport proteins intrinsic
factor and transcobalamin II, to be biologically active. Those
transport proteins involved in the uptake of vitamin B.sub.12 are
referred to herein as cobalamin binding proteins. Specifically,
gastrointestinal absorption of vitamin B.sub.12 relies upon the
intrinsic factor-vitamin B.sub.12 complex being bound by the
intrinsic factor receptors in the terminal ileum. Likewise,
intravascular transport and subsequent cellular uptake of vitamin
B.sub.12 throughout the body is dependent upon transcobalamin II
and the cell membrane transcobalamin II receptors, respectively.
After the transcobalamin II-vitamin B.sub.12 complex has been
internalized, the transport protein undergoes lysozymal
degradation, which releases vitamin B.sub.12 into the
cytoplasm.
[0007] Cellular utilization of Cb1 is preceded by two important
receptor-mediated endocytic events. First, the dietary Cb1 bound to
gastric intrinsic factor (IF), a 50-kDa glycoprotein, is
transported across the absorptive enterocyte via an intrinsic
factor-cobalamin receptor that is expressed exclusively in the
apical or the luminal membranes. The plasma transport of cobalamin
to tissues/cells appears to occur via transporter transcobalamin II
(TC II), by receptor-mediated endocytosis via transcobalamin
II-receptor (TC II-R). Intracellularly released Cb1 is then
converted to its biologically active forms, (e.g. methyl-Cb1 and
5'-deoxyadenosyl-Cb1) which are utilized by the cytoplasmic enzyme
methionine synthase (MS) and mitochondrial enzyme
methyl-malonyl-CoA mutase (MMCM), respectively. MS activity is
required for folate metabolism and DNA synthesis and presents a
promising target to block cell proliferation. TCII and serum Cb1
levels are both increased in hepatocarcinomas and leukemias. TCII
has been identified as an acute phase reactant in autoimmune
disorders and infection. Several studies have shown that high
levels of Cb1 inhibited L1210, P388D1, CCRF-CEM, and NCTC929 cell
proliferation. This is likely due to the activation of an
autoimmune response.
[0008] Recent studies have shown that TC II-R is expressed as a
non-covalent homodimer of molecular mass of 124 kDa in tissue
plasma membranes of human, rat, and rabbit. A comprehensive review
of transcobalamin II, the transcobalamin II receptor, and the
uptake of vitamin B.sub.12 is provided in "Transcobalamin II and
Its Cell Surface Receptor Vitamins and Hormones", Vitamins and
Hormones, Vol. 59, pgs. 337-366 (2000) which is incorporated herein
in its entirety by reference thereto. Plasma membrane expression of
TC II-R appears important for the tissue/cellular uptake of Cb1
since its functional inactivation in vivo by its circulatory
antiserum results in intracellular deficiency of Cb1. This
intracellular deficiency in Cb1 results in the development of Cb1
deficiency of the animal as a whole.
[0009] The utilization of vitamin B.sub.12 as a delivery vehicle is
known art. The art describes an oral delivery system that delivers
active substances (hormones, bio-active peptides or therapeutic
agents) by binding these agents to cobalamin or an analog
thereof.
[0010] U.S. Pat. No. 5,936,082, which is hereby incorporated by
reference in its entirety, for example, describes the therapeutic
effectiveness of vitamin B.sub.12 based compounds.
Nitrosylcobalamin (NO-Cb1), in particular, was evaluated for its
chemotherapeutic effect. In five human hematological and eight
solid tumor cell lines, NO-Cb1 exhibited an ID.sub.50 that was
5-100 fold lower in tumor cell lines compared to benign cells
(fibroblasts and endothelial cells). When oxidized from NO-Cb1, the
NO free radical functions in a number of capacities. NO is involved
in vasodilation, and is known to contribute to increased oxidative
stress, inhibition of cellular metabolism and induction of DNA
damage leading to apoptosis and/or necrosis.
[0011] Radiolabelled vitamin B.sub.12 analogs have also been
described in the art as useful in vivo imaging agents. For example,
U.S. Pat. No. 6,096,290, which is hereby incorporated herein in its
entirety by reference thereto, describes the use of radiolabelled
vitamin B.sub.12 analogs as in vivo tumor imaging agents.
[0012] U.S. Pat. No. 6,183,723, which is also incorporated herein
by reference in its entirety, describes certain other
cobalamin-drug conjugates.
SUMMARY OF THE INVENTION
[0013] The multiple components of Cb1 uptake, enzymes, co-factors,
and transport systems present several points of attack for the
therapeutic delivery of cobalamins. As is described herein, the
interrelationship of TCII-R and cytokines make this an attractive
target for the therapeutic delivery of biologically active
metallocorrinoids. Cytokines, in particular interferon .beta., are
shown to enhance the uptake or activity of biologically active
metallocorrinoids, including vitamin B.sub.12 analogs, homologs,
and derivatives.
[0014] Vitamin B.sub.12 analogs can be synthesized in a number of
ways. In addition to conjugation of the side chains of the corrin
ring, conjugation to the Cb1 moiety can also be made, as can
conjugation to the ribose moiety, phosphate moiety, and to the
benzimidazole moiety. The conjugating agent and the drug to be
conjugated depend upon the type of Cb1 group that is modified and
the nature of the drug. One of skill in the art would understand
how to adapt the conjugation method to the particular Cb1 group and
drug to be coupled.
[0015] Preferred methods of attaching the drug to the Cb1 molecule
include conjugation to Cb1 via biotin. Biotin is conjugated to
either the propionamide or the acetamide side chains of the corrin
ring of the Cb1 molecule. The initial biotin-Cb1 complex can be
prepared according to Pathre, et al. (Pathre, P. M., et al.,
"Synthesis of Cobalamin-Biotin conjugates that vary in the position
in cobalamin coupling, Evaluation of cobalamin derivative binding
to transcobalamin II," incorporated by reference). Vitamin B.sub.12
is commercially available in its most stable form as cyanocobalamin
from Sigma Chemical (St. Louis, Mo.).
[0016] One may most easily obtain transcobalamin II in the
following manner: transcobalamin II cDNA is available in the
laboratories of Drs. Seetharam (Medical College of Wisconsin) and
Rothenberg (VA-Hospital, New York) TC II cDNA can be expressed in a
Baculovirus system to make a large amount of functionally active TC
II protein (see Quadors, E. V., et al., Blood 81: 1239-1245, 1993).
One of skill in the art would be able to reproduce the TC II cDNA.
The antibodies to TCII-R were obtained through the laboratory of
Dr. Bellur Seetharam, Med. College of WI.
[0017] One way to make cobalamin drug conjugates is through genetic
engineering. In this method, a DNA sequence encoding TC II and the
peptide drug may be expressed as one chimeric molecule. For
example, it is possible to generate a chimeric construct using the
full-length TC II cDNA and the cDNA for a peptide drug (e.g.
insulin). The chimeric construct can then be expressed to produce a
fusion protein consisting of the TC II-peptide drug. Following
synthesis, the chimeric protein should be tested for both TC II
activity and drug activity. Cobalamin can then be allowed to bind
to this chimeric protein and used for therapy.
[0018] The observation that a cytokine (i.e an interferon such as
interferon-.beta.) upregulates or enhances the activity of the
TCII-R provides a basis for a number of embodiments of the present
invention.
[0019] One embodiment of the present invention is a method for
increasing cobalamin-binding protein activity in a subject in order
to treat a condition favorably affected by an increase in said
cobalamin-binding activity, said method compromising the step of
administering to a subject in need of such treatment a cytokine in
an amount effective to increase cobalamin-binding activity in the
subject. This method may further include the step of administering
a vitamin B.sub.12 analog (which may be a naturally occurring
vitamin B.sub.12 analog), nitrosylcobalamin or other suitable
vitamin B.sub.12 drug conjugate. In this embodiment, the cytokine
may be administered prior, simultaneously, or consecutively with
the vitamin B.sub.12 analog. The cytokine and/or vitamin B.sub.12
analog may be administered prophylactically or acutely. The
increased cobalamin binding protein activity is preferably TCII-R
activity. The cytokine is preferably an interferon such as
interferon-.beta..
[0020] Another embodiment of the present invention is a composition
that is comprised of a metallocorrinoid and a cytokine. It is
preferable that the metallocorrinoid be a vitamin B.sub.12 analog,
homolog, derivative or simply vitamin B.sub.12. This is
particularly useful when there is a deficiency in vitamin B.sub.12
or if the vitamin B.sub.12 analog includes a drug conjugated
thereto. It is particularly preferable that the vitamin B.sub.12
analog be a nitrosylcobalamin, but it may also be others known in
the art, (e.g. hydroxocobalamin, cyanocobalamin, and
methylcobalamin and 5' deoxyadenocobalamin or radiolabelled
cobalamin derivatives). The composition in accordance with this
embodiment of the invention may also include a pharmaceutical
carrier. It is preferable that the cytokine be an interferon, and
more particularly interferon-.beta..
[0021] Another embodiment of the present invention is a therapeutic
composition comprising a cobalamin or a cobalamin drug conjugate
and a cytokine such as interferon-.beta.. In this embodiment, the
therapeutic composition may also further comprise a pharmaceutical
carrier. This is a particular advantageous embodiment when the
cobalamin drug conjugate is designed for a specific aim in mind.
Nitrosylcobalamin is just one cobalamin drug conjugate, and other
drug conjugates may be selected from the group consisting of
hydroxocobalamin, cyanocobalamin, methylcobalamin, and 5'
deoxyadenocobalamin, radiolabelled cobalamin, or other cobalamin
and drug conjugate. This embodiment is particular useful in the
treatment of diseases where the delivery of a therapeutic agent via
a cobalamin delivery mechanism would be beneficial.
[0022] Another embodiment of the present invention is a method of
enhancing uptake or activity of a metallocorrinoid comprised of
administering a cytokine. It is preferable that the
metallocorrinoid be a vitamin B.sub.12 or a vitamin B.sub.12
analog, homolog, or derivative. In this method it is preferable
that the cytokine is an interferon, and more preferably that the
interferon be interferon-.beta..
[0023] Another embodiment of the present invention is a method of
enhancing cellular uptake of a metallocorrinoid comprising the step
of contacting a cell with a cytokine, particularly where the step
of contacting a cell with a cytokine occurs through induction of
cytokine. In this embodiment, it is preferable that the
metallocorrinoid is vitamin B.sub.12 or a vitamin B.sub.12 analog.
As in other embodiments, the vitamin B.sub.12 analog may be any
suitable vitamin B.sub.12 analog, homolog or derivatives such as a
cobalamin drug conjugate. In this embodiment it is preferable that
the cytokine is an interferon, particularly interferon-.beta..
[0024] Another embodiment of the present invention is a method of
treating a patient comprising the steps of inducing cytokine
production; and administering a metallocorrinoid. The step of
inducing cytokine production may include administering a cytokine,
or administering an agent as is known in the art to stimulate
cytokine expression or production. The metallocorrinoid of this
embodiment may be vitamin B.sub.12 or a vitamin B.sub.12 analog,
homolog or derivative such as a cobalamin drug conjugate. The
cytokine is preferably an interferon, more preferably
interferon-.beta..
[0025] Yet another embodiment of the present invention is a method
of enhancing bio-availability of a metallocorrinoid, comprising the
step of administering interferon-.beta. alone or in combination
with a metallocorrinoid.
[0026] Yet another embodiment of the present invention is a method
of treating a subject to increase TCII-R activity in a cell
comprising the step of administering to a subject in need of such
treatment a cytokine to increase TCII-R activity in an amount
effective to increase TCII-R activity in said cell. In this
embodiment, it is preferable that the subject be cobalamin
deficient. Another application of this embodiment is wherein the
amount is sufficient to increase TCII-R activity above normal
baseline levels. Preferably, this method may also be useful when
the subject has an abnormally low level of TCII-R activity. This
method preferably includes the step of co-administering a substrate
(or ligand) of TCII-R, wherein the substrate of TCII-R is a
cobalamin based compound (e.g. cobalamin or a cobalamin drug
conjugate). The cobalamin drug conjugate is preferably
nitrosylcobalamin, but may be any suitable cobalamin drug conjugate
such as those known in the art.
[0027] Yet another embodiment of the present invention is a method
of treating cancer comprised of administering a cytokine (e.g.
interferon-.beta.) to enhance the uptake or increase the
availability of cobalamin analogs, homologs, or derivatives. This
can be done either alone or in combination with the cobalamin
analog, homolog, or derivative.
[0028] Another embodiment of the present invention is a method of
imaging tissue or cells through enhanced uptake of radiolabelled
vitamin B.sub.12 analogs, homologs or derivatives via
administration of a cytokine such as interferon .beta..
[0029] Additional aspects and applications of the present invention
will become apparent to the skilled artisan upon consideration of
the detailed description of the invention, which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0031] FIG. 1 is a bar graph illustrating the anti-proliferative
effect of a cytokine (i.e. an interferon) and NO-Cb1 on NIH-OVCAR-3
ovarian carcinoma;
[0032] FIG. 2 is a graph illustrating a median effect analysis in
accordance with the present invention;
[0033] FIG. 3 is a western blot analysis performed on extracts from
ovarian carcinomas according to the present invention;
[0034] FIG. 4 illustrates a bar graph of a flow cytometric analysis
of Annexin V positive cells;
[0035] FIG. 5 are stained cells illustrating up-regulated TCII-R in
control and IFN-.beta. in NIH-OVCAR-3 treated samples;
[0036] FIG. 6 is a bar graph illustrating the anti-proliferative
effect of a cytokine (i.e. interferon) and NO-Cb1 on WM9
melanoma;
[0037] FIG. 7 is a graph illustrating a median effect analysis on
WM9 human melanoma cells;
[0038] FIG. 8 depicts treated and untreated WM9 tumor cells in
accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0039] While not wishing to be bound by theory, it appears that the
uptake of metallocorrinoids such as vitamin B.sub.12, NO-Cb1 or
other vitamin B.sub.12-based compounds, is dependent upon the TCII
receptor, specific for vitamin B.sub.12. Because the TCII-R plays a
central role in determining metallocorrinoid activity, the
relationship between TCII-R and cytokines (e.g. interferons
("IFNs")) was evaluated. IFNs upregulate the expression of cell
surface markers HLA-I, HLA-II, .beta.2 microglobulin, and tumor
associated antigens such as CEA and CA 125.
[0040] The present invention provides for an increase in receptor
or receptor activity responsible for the uptake of vitamin B.sub.12
derived compounds. The administration of cytokines, particularly
interferons such as IFN-.beta., appears to enhance the activity of
TCII-R. Administering these cytokines prior to or concurrently with
vitamin B.sub.12-based compounds increases the delivery of the
vitamin B.sub.12-based compounds and like metallocorrinoids.
[0041] Increased activity (e.g. TCII-R activity) can be
accomplished in a number of different ways. For example, an
increase in the amount of protein or an increase in the activity of
the protein (while maintaining a constant level of the protein) can
result in increased "activity". An increase in the amount of
protein available can result from increased transcription of the
gene, increased stability of the mRNA or a decrease in protein
degradation.
[0042] The present invention, by causing an increase in Cb1-binding
(e.g. TCII-R) activity, permits not only the re-establishment of
normal base-line levels of Cb1-binding activity, but also allows
increasing such activity above normal base-line levels. Normal
base-line levels are the amounts of activity in a normal control
group, controlled for age and having no symptoms that would
indicate alteration of Cb1-binding activity. The actual base line
level will depend upon the particular age group selected and the
particular measure employed to assay. When using the cytokines of
the present invention not only can normal base-line levels be
restored, but abnormal activity can also be increased desirably far
above normal base-line levels of TCII-R binding activity. Thus,
"increasing activity" means any increase in Cb1-binding protein or
cobalamin uptake in the subject resulting from the treatment,
according to the invention, including, but not limited to, such
activity as would be sufficient to restore normal base-line levels,
and such activity as would be sufficient to elevate the activity
above normal base-line levels.
[0043] In one embodiment of the invention the increase in activity
of the Cb1-binding activity is cytokine induced. Cytokines are
soluble polypeptides produced by a wide variety of cells. Cytokines
control gene activation and cell surface molecule expression. In
what follows, the term "cytokine" incorporates families of
endogenous molecules of various denominations: lymphokines,
monokines, interleukins, interferons, colonization factors and
growth factors and peptides. The known cytokines are in particular
interferon-.alpha. (IFN-.alpha..), interferon-.beta. (IFN-.beta.),
.gamma.-interferon (.gamma.-IFN), interleukin-1 (IL-1) in .alpha.
and .beta. forms, interleukin-2 (IL-2), interleukin-3 (IL-3),
interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6),
interleukin-10 (IL-10), interleukin-12 (IL-12), tumor necrosis
factor (TNF) in .alpha. and .beta. forms, transforming growth
factors (TGF-.beta.), in .beta.1, .beta.2, .beta.3, .beta.1.2
forms, and colony-stimulating factors (CSF) such as the granulocyte
macrophage-stimulating factor (GM-CSF), the granulocyte
colony-stimulating factor (G-CSF) and the macrophage-stimulating
factor (M-CSF) and the epithelial growth factor (EGF),
somatostatin, endorphins, the various "releasing factors" or
"inhibitory factors" such as TRF. There also exist pegilated forms
of interferon. Cytokines play an essential role in the development
of the immune system and thus in the development of an immune
response. However, besides their numerous beneficial properties,
they have also been implicated in the mechanisms for the
development of a variety of inflammatory diseases. For example, the
cytokines TNF-.alpha. and IL-1 are thought to be part of the
disease causing mechanism of atherosclerosis, transplant
arteriosclerosis, rheumatoid arthritis, lupus, scleroderma,
emphysema, etc.
[0044] Important embodiments of the invention involve populations
never before treated with a cytokine such as interferon. Thus, the
invention involves, in certain aspects, treatments of individuals
who are otherwise free of symptoms calling for treatment with
interferons.
[0045] The cytokines and/or cobalamin compounds are preferably
administered in effective amounts. In general, an effective amount
is any amount that can cause an increase in Cb1-binding proteins
activity in a desired cell population or tissue, and preferably in
an amount sufficient to cause a favorable phenotypic change in the
condition such as a lessening, alleviation or elimination of a
symptom or of a condition.
[0046] With regard to the cobalamin or vitamin B.sub.12 derived
compounds, an effective amount is that amount of a preparation that
alone, or together with further doses, produces the desired
response. This may involve only slowing the progression of the
disease temporarily, although more preferably, it involves halting
the progression of the disease permanently or delaying the onset of
or preventing the disease or condition from occurring. This can be
monitored by routine methods. Generally, doses of active compounds
would be from about 0.01 mg/kg per day to 1000 mg/kg per day. It is
expected that doses ranging from 50-500 mg/kg will be suitable,
preferably intravenously, intramuscularly, or intradermally, and in
one or several administrations per day.
[0047] Such amounts will depend, of course, on the particular
condition being treated, the severity of the condition and the
individual patient parameters. Some parameters for consideration
include age, physical condition, size and weight, the duration of
the treatment, the nature of concurrent therapy (if any), the
specific route of administration and like factors within the
knowledge and expertise of the health practitioner. Intravenous
administration and intramuscular administration avoids transport
problems associated with cobalamin when administered orally.
However, if the vitamin B.sub.12 analog, homolog or derivative is
encapsulated, oral delivery may be preferred. In the event that a
response in a subject is insufficient at the initial doses applied,
higher doses (or effectively higher doses by a different, more
localized delivery route) may be employed to the extent that
patient tolerance permits. Multiple doses per day are contemplated
to achieve appropriate systemic levels of compounds. It is
preferred generally that a maximum dose be used, that is, the
highest safe dose according to sound medical judgment. Those of
ordinary skill in the art will understand, however, that a patient
may insist upon a lower does or tolerable does for medical reasons,
psychological reasons or for virtually any other reason.
[0048] The cytokines (e.g. interferons) useful according to the
invention may be combined, optionally, with a
pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptable carrier" as used herein means one or
more compatible solid or liquid fillers, diluents or encapsulating
substances which are suitable for administration into a human. The
term "carrier" denotes an organic or inorganic ingredient, natural
or synthetic, with which the active ingredient is combined to
facilitate the application. The components of the pharmaceutical
compositions also are capable of being co-mingled with the
molecules of the present invention, and with each other, in a
manner such that there is no interaction which would substantially
impair the desired pharmaceutical efficacy.
[0049] The pharmaceutical compositions may contain suitable
buffering agents, including: acetic acid in a salt; citric acid in
a salt; boric acid in a salt; and phosphoric acid in a salt. The
pharmaceutical compositions also may contain, optionally, suitable
preservatives, such as: benzalkonium chloride, chlorobutanol,
parabens and thimerosal.
[0050] A variety of administration routes are available. The
particular mode selected will depend, of course, upon the
particular drug selected, the severity of the condition being
treated and the dosage required for therapeutic efficacy. The
methods of the invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of the active
compounds without causing clinically unacceptable adverse effects.
Such modes of administration include oral, rectal, topical, nasal,
intradermal, inhalation, intra-peritoneal, or parenteral routes.
The term "parenteral" includes subcutaneous, intravenous,
intramuscular, or infusion. Intravenous or intramuscular routes are
particularly suitable for purposes of the present invention.
[0051] The pharmaceutical compositions may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well known in the art of pharmacy. All methods include the
step of bringing the active agent into association with a carrier
that constitutes one or more accessory ingredients. In general, the
compositions are prepared by uniformly and intimately bringing the
active compound into association with a liquid carrier, a finely
divided solid carrier, or both, and then, if necessary, shaping the
product.
[0052] Compositions suitable for parenteral administration
conveniently comprise a sterile aqueous preparation of the
cytokines and/or cobalamins, which is preferably isotonic with the
blood of the recipient. This aqueous preparation may be formulated
according to known methods using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
also may be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example,
as a solution in 1,3-butane diol. 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 a solvent or suspending medium. For
this purpose any bland fixed oil may be employed including
synthetic mono- or di-glycerides. In addition, fatty acids such as
oleic acid may be used in the preparation of injectables. Carrier
formulation suitable for oral, subcutaneous, intravenous,
intramuscular, etc. administrations can be found in Remington's
Pharmaceutical Science, Mack Publishing Co., Easton, Pa. which is
incorporated herein in its entirety by reference thereto.
[0053] Other delivery systems can include time-released, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the active compound, increasing
convenience to the subject and the physician, and may be
particularly suitable for certain cobalamin drug conjugates of the
present invention, particularly the nitrosylcobalamin due to its
activation under acidic conditions found in the early
gastrointestinal tract. Many types of release delivery systems are
available and known to those of ordinary skill in the art. They
include polymer base systems such as poly(lactide-glycolide),
copolyoxalates, polycaprolactones, polyesteramides,
polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.
Microcapsules of the foregoing polymers contains drugs are
described in, for example, U.S. Pat. No. 5,075,109. Delivery
systems also include non-polymer systems that are: lipids including
sterols such as cholesterol, cholesterol esters and fatty acids or
neutral fats such as mono- di- and tri-glycerides; hydrogel release
systems; sylastic systems; peptide based systems; wax coatings;
compressed tablets using conventional binders and excipients;
partially fused implants; and the like. Specific examples include,
but are not limited to: (a) erosional systems in which the active
compound is contained in a form within a matrix such as those
described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and
5,239,660 and (b) diffusional systems in which an active component
permeates at a controlled rate from a polymer such as described in
U.S. Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based
hardware delivery systems can be used, some of which are adapted
for implantation.
[0054] Use of a long-term sustained release implant may be
desirable. Long-term release, are used herein, means that the
implant is constructed and arranged to delivery therapeutic levels
of the active ingredient for at least 30 days, and preferably 60
days. Long-term sustained release implants are well-known to those
of ordinary skill in the art and include some of the release
systems described above.
[0055] In one aspect of the invention, the cytokine is
"co-administered" with a metallocorrinoid which means administered
substantially simultaneously with a metallocorrinoid. By
substantially simultaneously, it is meant that the cytokine (e.g.
interferon-.beta.) is administered to the subject close enough in
time with the administration of the other agent (e.g., vitamin
B.sub.12 or a cobalamin conjugate), whereby the two compounds may
exert an additive or even synergistic effect.
[0056] The following is provided as an illustration of the present
invention as it applies to both in vivo and in vitro. The
materials, methods, examples, results, and discussions should in no
way be viewed as a limitation thereto. For simplicity, the
materials and methods sections is provided after the following
detailed discussion of the present invention.
Examples, Results and Discussion:
[0057] FIG. 1 illustrates NIH-OVCAR-3 ovarian carcinoma evaluated
in accordance with the present invention after 72-hrs growth.
Cytokines, particularly interferons, appear to enhance the activity
or upregulate the cellular receptor for vitamin B.sub.12 (TCII-R),
resulting in enhanced TCII-R activity (in this case demonstrated by
NO-Cb1 uptake). Single agent and combination drug effects were
assessed to determine whether IFN-.beta. enhanced NO-Cb1 activity.
As shown in FIG. 1, NIH-OVCAR-3 cells were treated continuously
with varying concentrations of NO-Cb1 and IFN-.beta.. Consistent
with our hypothesis, we observed synergistic anti-proliferative
activity between IFN-.beta. and NO-Cb1. These matters are shown in
the median effect analysis shown in FIG. 2 (similar to isobologram
analysis) indicated synergy (a combination index <1) between
NO-Cb1 and IFN-.beta. at all 3 doses tested. Cytotoxicity was noted
at the highest combination dose.
[0058] To assess the effect of IFN.beta. on TCII-R expression, a
western blot analysis was performed on extracts from NIH-OVCAR-3
cells (ovarian carcinoma) as shown in FIG. 3. Lane 1 is untreated.
Lanes 2 and 3 are IFN .beta. treated (200 u/ml) at 4 and 16 hrs
respectively. Lanes 4 and 5 are liver and kidney extracts
respectively, and serve as a positive control, since TCII-R is
abundant in these tissues. As shown in FIG. 3, IFN .beta. causes an
increase in the expression of the TCII receptor, identified as the
monomer at 62 kDa with the corresponding dimer at 124 kDa,
consistent with TCII-R. These results correlate with the
anti-proliferative effect of co-treatment of NIH-OVCAR-3 cells with
IFN .beta. and nitrosycobalamin shown in FIG. 1. The increased
expression of the TCII receptor by IFN .beta. treatment results in
the increased uptake of nitrosylcobalamin and thus enhanced
destruction of the cells. The co-delivery of IFN-.beta. and
nitrosylcobalamin appears to result in synergistic destruction of
tumor cells as a result of increased TCII receptor expression or
activity.
[0059] A flow cytometric analysis of Annexin V positive cells was
performed to assess the % apoptosis (programmed cell death) of
NIH-OVCAR-3 cells treated with NO-Cb1, alone and in combination
with IFN-.beta.. This is illustrated in FIG. 4. The ID.sub.25 was
used for both NO-Cb1 (10 uM) and IFN-.beta. (20 U/mL) for 48 hrs.
The effect of IL-2 (250 U/mL) were protective against the effects
of NO-Cb1.
[0060] To further elucidate IFN-.beta. upregulated TCII-R, human
NIH-OVCAR-3 tumors were grown in nude mice to a size of 3 mm in
diameter. The control group received PBS and the treated group
received human IFN-.beta.10.sup.5 units daily for three days.
Tumors were harvested, paraffin embedded, and sections were stained
with rabbit polyclonal anti-TCII-R antibody, (provided by Dr.
Seetharam's lab, Medical College of Wisconsin). FIG. 5 depicts
these treatments. The left panel is an untreated tumor whereas the
right panel is a tumor from a mouse that received IFN-.beta.. The
areas stained brown represent TCII-R. A comparison of the panels
demonstrates increased expression of TCII-R with IFN .beta.
treatment. The increased expression of the TCII receptor allows for
increased uptake of NO-Cb1, consistent with the synergy observed in
the SRB and Annexin V assays upon NO-Cb1 co-treatment with
IFN-.beta..
[0061] WM9 human melanoma was evaluated after 4 days growth. This
is shown in FIG. 6. WM9 cells were treated continuously with
varying concentrations of NO-Cb1 and IFN-.beta.. Similar to the
NIH-OVCAR-3 cells, there was synergistic anti-proliferative
activity between IFN-.beta. and NO-Cb1, as is shown in FIG. 7.
Median effect analysis indicated synergy (a combination index
<1) between NO-Cb1 and IFN-.beta. at all 3 doses tested.
[0062] To further elucidate whether IFN-.beta. upregulated TCII-R,
human WM9 tumors were grown in nude mice to a size of 3 mm in
diameter. The control group received PBS and the treated group
received human IFN-.beta.10.sup.5 units daily for three days.
Tumors were harvested, paraffin embedded, and sections were stained
with rabbit polyclonal anti-TCII-R antibody, (provided by Dr.
Seetharam's lab, Medical College of Wisconsin). FIG. 8 depicts
these treatments. The upper two panels are untreated tumors whereas
the lower panels are tumors from mice that received IFN-.beta.3.
The areas stained brown represent TCII-R. A comparison of the
panels demonstrates increased expression of TCII-R with IFN .beta.
treatment.
[0063] One can see from the basal TCIIr activity in the NIH-OVCAR-3
and WM9 stained sections that when TCII-R expression is lower,
NO-Cb1 uptake is not pronounced. This is reflected by a higher
ID.sub.50 associated with the WM9 cells compared to NIH-OVCAR-3
tumors. Although interferon administration in both NIH-OVCAR-3 and
WM9 resulted in increased effectiveness of NO-Cb1, lower basal
TCIIR expression in WM9 renders these cells less sensitive to the
effect of NO-Cb1 and the combination with IFN-.beta..
[0064] TCII-R is an important component of metallocorrinoid (e.g.
vitamin B.sub.12) metabolism and represents a site-specific target
to regulate vitamin B.sub.12 uptake. Nitrosylcobalamin, a vitamin
B.sub.12 based carrier of nitric oxide (NO), was used to validate
the in vivo functional relevance of increased TCII-R expression.
Intraperitoneal NO-Cb1 treatment of established subcutaneous
NIH-OVCAR-3 tumors resulted in tumor regression. The mean volume of
untreated tumors was 18 fold greater compared to NO-Cb1 treated
tumors at the end of the study. Treated tumors decreased 4-fold in
volume during the treatment period. There was no histologic
evidence of toxicity to normal tissues at NO-Cb1 doses of 170
mg/kg/day after 60 days. IFN-.beta. treatment of NIH-OVCAR-3 cells
in culture resulted in increased expression of the TCII-R, detected
as a monomer (62 kDa) and a dimer (124 kDa). Similarly,
immunohistochemical analysis of NIH-OVCAR-3 xenografts from nude
mice that received human IFN-.beta. showed increased TCII-R
expression compared to controls. Tumors that were resistant to
IFN-.beta. and NO-Cb1 in vivo exhibited minimal to no
immunohistochemical evidence of TCII-R upregulation. In culture,
combination treatment with IFN-.beta. and No-Cb1 resulted in
synergistic anti-proliferative activity in NIH-OVCAR-3 cells and
several different human cells lines including MCF-7 (breast), DU145
and LNCap (prostate), ACHN (renal), A549 (lung), WM9, WM35, WM164,
and WM3211 (melanoma). Treatment of NIH-OVCAR-3 cells with the
combination of NO-Cb1 and IFN-.beta. resulted in a 2-fold increase
in annexin V positive cells compared to NO-Cb1 alone.
Interestingly, a Ribonucleotide Protection Assay revealed a
ten-fold increase in TRAIL and Caspase 7 in NIH-OVCAR-3 cells
treated with the combination of NO-Cb1 and IFN-.beta.. Therefore,
up-regulation and/or increased activity of the TCII-R by IFN-.beta.
results in synergistic anti-tumor effects in vitro and in vivo.
Materials and Methods:
[0065] In-vivo IFN-.beta. treatment of nude mice inoculated with
tumors (e.g. WM9-human melanoma or NIH-OVCAR-3-ovarian carcinoma)
and Immunohistochemical analysis: Nude mice (n=2 each group), were
inoculated with tumors (e.g. WM9-human melanoma or
NIH-OVCAR-3-ovarian carcinoma), subcutaneously (s.c.), one tumor on
each flank. The tumors were grown until 3-5 mm in diameter. Human
IFN-.beta. (10.sup.5 units) was administered s.c. for three days to
the treatment animals. On day four, animals were sacrificed and
tumors were fixed in formalin and paraffin embedded. The sections
were analyzed using standard immunohistochemical techniques.
Anti-TCII-R was used as the primary antibody.
[0066] SRB Anti-Proliferative Cell Survival Assay:
[0067] Cells (2.times.10.sup.3) were seeded in 96-well plates. Data
points represent mean of eight replicates. (n=8). A control plate
was fixed 4 hr after seeding (to allow cells to attach) to
determine the initial seeding density (A.sub.ini). This was defined
as 0% growth. To the wells of the seeded experimental plate,
IFN-.beta. was added and incubation continued for 3-5 days.
Untreated cell controls were included. Growth obtained with this
control was defined as 100% (A.sub.fin). To determine cell number,
cells were fixed with 10% trichloroacetic acid at 4.degree. C. for
1 h. They were stained with 0.4% sulforhodamine B prepared in 1%
acetic acid at 25.degree. C. for 1 h (27). The wells were washed
with 1% acetic acid. Bound dye was eluted with 100 .mu.l of 10 mM
Tris-HCl, pH 10.5 and quantitated in a microplate reader at 570 mm.
Growth in IFN-.beta. treated wells (experimental=exp) was expressed
as a percentage of untreated control growth (mean.+-.SEM). %
Control
growth=100%.times.(A.sub.exp-A.sub.ini)/(A.sub.fin-A.sub.ini) %
STD=100%.times.(STD.sub.exp/(A.sub.fin-A.sub.ini)) %
SEM=100%.times.(SEM.sub.exp/(A.sub.fin-A.sub.ini)) where SEM=STD/
{square root over (n)}
[0068] Western Blot TCII Receptor:
[0069] Cells in culture were treated with vehicle (untreated) or
with IFN-.beta. (500 U/ml) for 4 and 16 hrs, washed twice in PBS,
harvested by scraping, and lysed in buffer containing 100 mM
saline-TRIS. Total cell extracts were homogenized prior to loading.
Protein amounts in clarified cell extracts were determined using
Bio-Rad protein assay reagent. Equivalent amounts of protein (100
.mu.g) were loaded on 10% polyarcylamide SDS separating gels and
electrophoresis was performed using glycine-SDS buffer. Following
electrophoresis, gels were equilibrated in transfer buffer 30 min
at 25.degree. C., and proteins transferred to nitrocellulose
membrane.
[0070] Immunoblot with Electro-Chemiluminescense Detection:
[0071] All steps were performed at 25.degree. C. Following 90 min
electrophoretic wet transfer, the membranes were incubated in
washing buffer TBS-Tween (1.times.TBS, 0.2% X-100,)+4% BSA for 1-2
hr to block non-specific binding. The membrane was washed in
washing buffer. Membranes were then incubated in 25 ml of primary
antibody at 1:500 dilution in the washing buffer overnight at
4.degree. C. Membranes were then washed using the washing buffer
four times, 10 min each. Membranes were incubated in 50 ml
horseradish peroxidase-conjugated secondary antibody (Zymed) at
1:10,000 dilution in washing buffer for 30 minutes. Membranes were
washed in the washing buffer for two hours. Equal volumes of
electro-chemiluminescense (ECL) reagents A and B (Amersham) were
mixed to give enough reagents to develop the blot (0.125
ml/cm.sup.2). Excess buffer was drained from the membrane and it
was placed protein side up on plastic wrap. Detection reagent was
added to the protein side of the membrane. The reaction was allowed
to continue for exactly 1 minute. Excess detection reagent was
drained and the membrane was placed protein side down on plastic
wrap and exposed to film for empirically determined lengths of
time.
[0072] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the composition, methods, and in the
steps or in the sequence of steps of the method described herein,
without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents that are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
[0073] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference:
[0074] McLean G R, William M J, Woodhouse C S, Ziltener H J:
Transcobalamin II and in vitro proliferation of leukemic cells.
Leuk Lymphoma 30: 101-9, 1998. [0075] Tsao C S, Myashita K:
Influence of cobalamin on the survival of mice bearing ascites
tumor. Pathobiology 61: 104-8, 1993. [0076] Jensen H S, Gimsing P,
Pedersen F, Hippe E: Transcobalamin II as an indicator of activity
in metastatic renal adenocarcinoma. Cancer 52: 1700-4, 1983. [0077]
Tsao C S, Miyashita K, Young M: Cytotoxic activity of cobalamin in
cultured malignant and nonmalignant cells, Pathobiology 58: 292-6,
1990. [0078] Shimizu N, Hamazoe R, Kanayama H, Maeta M, Koga S:
Experimental study of antitumor effect of methyl-B12. Oncology 44:
169-73, 1987. [0079] McLean G R, Pathare P M, Wilbur D S, Morgan A
C, Woodhouse C S, Schrader H W, Ziltener H J: Cobalamin analogues
modulate the growth of leukemia cells in vitro. Cancer Res 57:
4015-22, 1997. [0080] Huennekens F M, DiGirolamo P M, Fujii K.
Jacobsen D W, Vitols K S: B12--dependent methionine synthetase as a
potential target for cancer chemotherapy. Adv Enzyme Regul 14:
187-205, 1976. [0081] Bauer, Joseph A., Characterization and nitric
oxide release profile of nitrosylcobalamin: a potential
chemotherapeutic agent. Anti-Cancer Drugs 1998; 9(3): 239-244.
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