U.S. patent application number 12/790626 was filed with the patent office on 2010-12-02 for method of treating anemia by administering il 1ra.
This patent application is currently assigned to Amgen Inc.. Invention is credited to Marco A. Coccia, Jonathan Kay, Dorothy McCabe, Richard Newmark.
Application Number | 20100303815 12/790626 |
Document ID | / |
Family ID | 26936788 |
Filed Date | 2010-12-02 |
United States Patent
Application |
20100303815 |
Kind Code |
A1 |
Kay; Jonathan ; et
al. |
December 2, 2010 |
METHOD OF TREATING ANEMIA BY ADMINISTERING IL 1ra
Abstract
The invention relates to methods of treating a blood disorder in
a mammal with an interleukin-1 (IL-1) inhibitor. The invention also
relates to methods of treating a blood disorder in a mammal with an
IL-1 inhibitor, a TNF inhibitor and an erythropoietin (EPO)
receptor agonist. The invention also relates to compositions of an
IL-1 inhibitor and compositions of an IL-1 inhibitor, a TNF
inhibitor and an EPO receptor agonist.
Inventors: |
Kay; Jonathan; (Newton
Centre, MA) ; McCabe; Dorothy; (Moorpark, CA)
; Newmark; Richard; (Newbury Park, CA) ; Coccia;
Marco A.; (Camarillo, CA) |
Correspondence
Address: |
AMGEN INC.
MAIL STOP 28-2-C, ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Amgen Inc.
Thousand Oaks
CA
|
Family ID: |
26936788 |
Appl. No.: |
12/790626 |
Filed: |
May 28, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11021348 |
Dec 22, 2004 |
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12790626 |
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09969739 |
Oct 2, 2001 |
7087224 |
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11021348 |
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60244792 |
Oct 31, 2000 |
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Current U.S.
Class: |
424/134.1 ;
424/172.1; 514/13.5; 514/7.7 |
Current CPC
Class: |
A61K 47/54 20170801;
A61P 7/00 20180101; A61K 38/1816 20130101; A61P 43/00 20180101;
A61K 47/26 20130101; A61P 7/06 20180101; A61K 9/0019 20130101; A61K
33/26 20130101; A61P 19/02 20180101; A61K 38/20 20130101; A61K
38/1793 20130101; Y02A 50/30 20180101; C07K 2319/30 20130101; A61P
13/12 20180101; A61P 29/00 20180101; A61K 39/395 20130101; A61K
47/55 20170801; A61K 39/395 20130101; A61K 38/1816 20130101; A61K
38/20 20130101; A61K 38/1816 20130101; A61K 38/1816 20130101; A61K
38/17 20130101; A61K 38/1816 20130101; A61K 2300/00 20130101; A61K
38/20 20130101; A61K 2300/00 20130101; A61K 39/395 20130101; A61K
2300/00 20130101; A61K 38/1793 20130101; A61K 2300/00 20130101;
A61K 33/26 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/134.1 ;
424/172.1; 514/13.5; 514/7.7 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 7/06 20060101 A61P007/06; A61K 38/19 20060101
A61K038/19; A61K 38/18 20060101 A61K038/18; A61P 19/02 20060101
A61P019/02; A61P 13/12 20060101 A61P013/12 |
Claims
1. A method of treating anemia in a mammal, comprising
administering a therapeutically effective amount of a
pharmaceutical composition comprising an anti-IL-1 receptor
antibody.
2. The method of claim 1, wherein the treating of anemia raises the
hematocrit in the mammal.
3. The method of claim 2, wherein the target hematocrit is at least
about 30%.
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. The method of claim 2 wherein the mammal suffers from anemia
associated with a decline or loss of kidney function.
15. The method of claim 2, wherein the mammal suffers from anemia
associated with rheumatoid arthritis.
16. The method of claim 2, wherein the mammal suffers from anemia
associated with myelosuppressive therapy.
17. The method of claim 16, wherein the myelosuppressive therapy
comprises chemotherapeutic or anti-viral drugs.
18. The method of claim 2, wherein the mammal suffers from anemia
associated with excessive blood loss.
19. The method of claim 2, further comprising administering a
therapeutically effective amount of iron.
20. The method of claim 2, further comprising administering to the
mammal a therapeutically effective amount of an erythropoietin
(EPO) receptor agonist.
21. (canceled)
22. (canceled)
23. The method of claim 20, wherein the anti-IL-1 receptor antibody
and the EPO receptor agonist are administered in separate
compositions.
24. The method of claim 20, wherein the anti-IL-1 receptor antibody
and the EPO receptor agonist are administered in separate
compositions at different times.
25. The method of claim 20, wherein the anti-IL-1 receptor antibody
and the EPO receptor agonist are administered in separate
compositions at the same time.
26. The method of claim 20, wherein the anti-IL-1 receptor antibody
and the EPO receptor agonist are administered in the same
composition.
27. (canceled)
28. (canceled)
29. The method of claim 20, wherein the EPO receptor agonist is
epoietin alfa.
30. The method of claim 20, wherein the EPO receptor agonist is
darbepoietin alfa.
31. (canceled)
32. (canceled)
33. (canceled)
34. The method of claim 20, wherein the blood disorder treated is
anemia of chronic disease.
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. A composition comprising an anti-IL-1 receptor antibody and an
EPO receptor agonist.
41. (canceled)
42. (canceled)
43. The composition of claim 39, wherein the EPO receptor agonist
is darbepoietin alfa.
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. (canceled)
54. (canceled)
55. (canceled)
56. (canceled)
57. (canceled)
58. (canceled)
59. (canceled)
60. (canceled)
61. (canceled)
62. (canceled)
63. (canceled)
64. (canceled)
65. (canceled)
66. (canceled)
67. (canceled)
68. (canceled)
69. (canceled)
70. (canceled)
71. (canceled)
72. (canceled)
73. (canceled)
74. (canceled)
75. (canceled)
76. (canceled)
77. (canceled)
78. (canceled)
79. (canceled)
Description
[0001] This application is a divisional of U.S. Ser. No.
11/021,348, filed Dec. 22, 2004, now allowed, which is a divisional
of U.S. Ser. No. 09/969,739, U.S. Pat. No. 7,087,224, filed Oct. 2,
2001, which claims the benefit of U.S. Provisional Application No.
60/244,792, filed Oct. 31, 2000, which is hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to treating blood disorders using an
interleukin-1 (IL-1) inhibitor. The invention also relates to
methods of treating blood disorders in a mammal using an IL-1
inhibitor, a tumor necrosis factor (TNF) inhibitor, and an
erythropoietin (EPO) receptor agonist.
BACKGROUND OF THE INVENTION
[0003] Disorders of the blood present many important health
concerns. For example, a decreased hematocrit, e.g. low number of
red blood cells, decreased volume of red blood cells, or reduced
hemoglobin concentration, is indicative of pathological conditions
such as, but not limited to, anemia. The symptoms of anemia may
include fatigue, dizziness, headache, chest pain, shortness of
breath, and depression.
[0004] Treatment of blood disorders such as anemia may involve
increasing the patient's hematocrit. One method of treating anemia
is to give red blood cell transfusions. This procedure, however,
can have risks associated with it. Transfusion reactions may occur
if blood is not correctly and completely matched between the donor
and the patient, and some diseases, especially viral diseases, may
be in the blood being transfused. A theoretical concern, not yet
proven, is that frequent blood transfusions may damage the immune
system, which protects the body from infections.
[0005] Regulatory authorities have approved recombinant human
erythropoietin for treatment of anemia associated with chronic
renal failure (CRF), anemia related to therapy with AZT
(zidovudine) in HIV-infected patients, anemia in patients with
non-myeloid malignancies receiving chemotherapy, and anemia in
patients undergoing surgery to reduce the need of allogenic blood
transfusions.
[0006] Due to the serious health problems arising from blood
disorders, such as those conditions associated with decreased
hematocrit, it is of interest to develop other agents capable of
treating these disorders. These agents include, but are not limited
to, those that increase hematocrit level.
SUMMARY OF THE INVENTION
[0007] In certain embodiments, the invention provides methods of
raising hematocrit in a mammal comprising administering a
therapeutically effective amount of an IL-1 inhibitor. In certain
embodiments, the invention provides methods for maintaining
hematocrit in a mammal. In certain embodiments, the invention
provides methods of treating blood disorders in a mammal comprising
administering a therapeutically effective amount of an IL-1
inhibitor.
[0008] In certain embodiments, the invention provides methods of
treating blood disorders in a mammal comprising administering an
IL-1 inhibitor and an EPO receptor agonist. In certain embodiments,
the invention provides methods of raising and/or maintaining
hematocrit in a mammal comprising administering an IL-1 inhibitor
and an EPO receptor agonist. In certain embodiments, the IL-1
inhibitor and EPO receptor agonists can be administered separately,
at the same time or at different times. In certain embodiments, the
IL-1 inhibitor and EPO receptor agonist can be administered
together at the same time.
[0009] In certain embodiments, the invention provides methods of
treating blood disorders in a mammal comprising administering a TNF
inhibitor and an EPO receptor agonist. In certain embodiments, the
invention provides methods of raising and/or maintaining hematocrit
in a mammal comprising administering a TNF inhibitor and an EPO
receptor agonist. In certain embodiments, the TNF inhibitor and EPO
receptor agonists can be administered separately, at the same time
or at different times. In certain embodiments, the TNF inhibitor
and EPO receptor agonist can be administered together at the same
time.
[0010] In further embodiments, the invention provides methods of
treating blood disorders in a mammal comprising administering an
IL-1 inhibitor, a TNF inhibitor and an EPO receptor agonist. In
certain embodiments, the invention provides methods of raising
and/or maintaining hematocrit in a mammal comprising administering
an IL-1 inhibitor, a TNF inhibitor and an EPO receptor agonist. In
certain embodiments, the IL-1 inhibitor, TNF inhibitor and EPO
receptor agonist can be administered separately, at the same time
or at different times. In certain embodiments, the IL-1 inhibitor,
TNF inhibitor, and EPO receptor agonist can be administered
together at the same time. In further embodiments, the TNF
inhibitor and IL-1 inhibitor can be replaced with a single molecule
(e.g., a P38 inhibitor) that inhibits both TNF and IL-1.
[0011] Also provided are pharmaceutical compositions comprising an
IL-1 inhibitor; an IL-1 inhibitor and an EPO receptor agonist; an
IL-1 inhibitor, a TNF inhibitor, and an EPO receptor agonist; and
an inhibitor of IL-1 and TNF (e.g., a P38 inhibitor) and an EPO
receptor agonist.
DESCRIPTION OF THE FIGURES
[0012] FIG. 1 shows the baseline demographics and disease history
of subjects in the study described in Examples 1, 2, and 3 of this
application. The data is grouped by treatment type and level.
[0013] FIG. 2 compares the distribution of change from baseline in
hematocrit for treatment with anakinra and treatment with placebo
in the study described in Examples 1, 2, and 3 of this
application.
[0014] FIG. 3 shows the baseline demographics and disease history
of subjects in the study described in examples 1, 2, and 3 of this
application who had rheumatoid arthritis and anemia. The data is
grouped by treatment type and level.
[0015] FIG. 4 shows the number and percentage of subjects in the
study described in examples 1, 2, and 3 of this application who had
rheumatoid arthritis and anemia who demonstrated hematocrit
improvement at week 24.
[0016] FIG. 5 shows the number and percentage of subjects in the
study described in Examples 1, 2, and 3 of this application who had
rheumatoid arthritis and anemia and who demonstrated hematocrit
level increases who also met the ACR20 response criteria. The data
is grouped by levels of hematocrit increase.
[0017] FIG. 6 compares the number of subjects in the study
described in Examples 1, 2, and 3 of this application with
rheumatoid arthritis and anemia who showed both hematocrit
improvement and ACR20 response with the total number of subjects
with rheumatoid arthritis and anemia who demonstrated hematocrit
improvement.
[0018] FIG. 7 shows the effect of ARANESP.TM. (darbepoietin alfa)
and Fc-IL-1ra on mean blood hemoglobin concentration in rats
afflicted with anemia of chronic disorders in the study described
in Example 4 of this application. Combination treatment of
Fc-IL-1RA 100 mgs/kg 3.times./wk with ARANESP 6 .mu.g/kg/2 wks was
significantly better at raising blood Hb levels than either single
treatment alone (p<0.02)
[0019] FIG. 8 shows the effect of ARANESP.TM. alone and with
Fc-IL-1ra or a TNF inhibitor (PEG sTNF R1) on paw edema in rats
afflicted with anemia of chronic disorders in the study described
in Example 4 of this application.
[0020] FIG. 9 shows the effect of ARANESP.TM. and Fc-IL-1ra on mean
reticulocyte numbers in rats afflicted with anemia of chronic
disorders in the study described in Example 4 of this application.
Combination treatment of Fc-IL-1RA 100 mgs/kg 3.times./wk with
ARANESP 6 .mu.g/kg/2 wks was significantly better at raising
reticulocyte levels than either single treatment alone
(p<0.004).
[0021] FIG. 10 shows the effect of ARANESP.TM. and Fc-IL-1ra on red
blood cell (RBC) numbers in rats afflicted with anemia of chronic
disorders in the study described in Example 4 of this application.
Combination treatment of Fc-IL-1RA 100 mgs/kg 3.times./wk with
ARANESP 6 .mu.g/kg/2 wks was significantly better at raising RBC
numbers than either single treatment alone (p<0.03).
[0022] FIG. 11 shows the effect of ARANESP.TM. and Fc-IL-1ra on
mean corpuscular volume (MCV) in rats afflicted with anemia of
chronic disorders in the study described in Example 4 of this
application. Combination treatment of Fc-IL-1RA 100 mgs/kg
3.times./wk with ARANESP 6 .mu.g/kg/2 wks was significantly better
at raising MCV than either single treatment alone (p<0.04)
[0023] FIG. 12 shows the effect of ARANESP.TM. and Fc-IL-1ra on
mean corpuscular hemoglobin (MCH) in rats afflicted with anemia of
chronic disorders in the study described in Example 4 of this
application. Combination treatment of Fc-IL-1RA 100 mgs/kg
3.times./wk with ARANESP 6 .mu.g/kg/2 wks was significantly better
at raising MCV than ARANESP treatment alone (p<0.01).
[0024] FIG. 13 shows the effect of ARANESP.TM. and Fc-IL-1ra on
total serum iron concentrations (TSI) in rats afflicted with anemia
of chronic disorders in the study described in Example 4 of this
application. Combination treatment of Fc-IL-1RA 100 mgs/kg
3.times./wk with ARANESP 6 .mu.g/kg/2 wks was significantly better
at raising TSI than either single treatment alone (p<0.02).
[0025] FIG. 14 shows the effect of ARANESP.TM. and Fc-IL-1ra on
total iron binding capacity (TIBC) in rats afflicted with anemia of
chronic disorders in the study described in Example 4 of this
application.
[0026] FIG. 15 shows the effect of ARANESP.TM. and PEG sTNF R1 on
mean blood hemoglobin concentration in rats afflicted with anemia
of chronic disorders in the study described in Example 4 of this
application. Combination treatment of PEG sTNF R1 at 4 mgs/kg
3.times./wk with ARANESP 6 .mu.g/kg/2 wks was significantly better
at raising blood Hb levels than either single treatment alone
(p<0.01)
[0027] FIG. 16 shows the effect of ARANESP.TM. and PEG sTNF R1 on
MCH and MCV in rats afflicted with anemia of chronic disorders in
the study described in Example 4 of this application. Combination
treatment of PEG sTNF R1 4 mgs/kg 3.times./wk with ARANESP 6
.mu.g/kg/2 wks was significantly better at raising MCH
(p<0.0009) and MCV (p<0.01) than either single treatment
alone.
[0028] FIG. 17 shows the effect of ARANESP.TM. and PEG sTNF R1 on
mean reticulocyte numbers and RBC numbers in rats afflicted with
anemia of chronic disorders in the study described in Example 4 of
this application. Combination treatment of PEG sTNF R1 4 mgs/kg
3.times./wk with ARANESP 6 .mu.g/kg/2 wks was significantly better
at raising reticulocyte levels (p<0.002) than either single
treatment alone (p<0.00X).
[0029] FIG. 18 shows the effect of ARANESP.TM. and PEG sTNF R1 on
TSI and the change in TSI in rats afflicted with anemia of chronic
disorders in the study described in Example 4 of this application.
Combination treatment of PEG sTNF R1 4 mgs/kg 3.times./wk with
ARANESP 6 .mu.g/kg/2 wks was significantly better at raising serum
iron levels than either single treatment alone (p<0.02).
[0030] FIG. 19 shows the effect of ARANESP.TM. and PEG sTNF R1 on
the unbound iron binding capacity (UIBC), e.g., serum transferrin
levels, in rats afflicted with anemia of chronic disorders in the
study described in Example 4 of this application. Combination
treatment of PEG sTNF R1 4 mgs/kg 3.times./wk with ARANESP 6
.mu.g/kg/2 wks generated significantly lower UIBC than either
single treatment alone (p<0.015).
[0031] FIG. 20 shows the effect of ARANESP.TM. and PEG sTNF R1 on
transferrin saturation (TSAT) levels (shown as % saturated) in rats
afflicted with anemia of chronic disorders in the study described
in Example 4 of this application. Combination treatment of PEG sTNF
R1 4 mgs/kg 3.times./wk with ARANESP 6 .mu.g/kg/2 wks was
significantly better at raising transferrin saturation than either
single treatment alone (p<0.004).
DEFINITIONS
[0032] The following definitions apply to the terms as used
throughout this specification, unless otherwise limited in specific
instances.
[0033] "Addition variant" refers to a variant that has at least one
more amino acid residue than the parent molecule.
[0034] "Adjuvant" is a substance added to a pharmaceutical
composition that affects the action of the active ingredient of the
composition in a predictable way.
[0035] "Administering" refers to any process of introducing a
composition into or onto the body of a mammal and includes, but is
not limited to, particular methods described in this
specification.
[0036] "Anemia of chronic diseases" (ACD) refers to any mild to
moderately severe anemia that is associated with chronic diseases
such as, but not limited to, trauma, infectious and inflammation
(e.g., rheumatoid arthritis (RA) or inflammatory bowel disease
(IBD)), and neoplastic diseases that persist for greater than 2
months.
[0037] "ARANESP.TM." refers to the super-sialated erythropoietic
protein described in published PCT Application No. WO 00/24893
"Methods and Compositions for the Prevention and Treatment of
Anemia" (incorporated by reference herein for any purpose) and any
derivatives or variants of this protein.
[0038] "Chimeric polypeptide" refers to a polypeptide created by
the fusion between two or more different polypeptides.
[0039] "Deletion variant" refers to a variant that has at least one
less amino acid residue than the parent molecule.
[0040] "Derivative" refers to a chemically modified protein in
which a nonproteinaceous moiety is linked to the protein.
[0041] "Diluent" is a substance used to lessen the concentration of
the other components of a composition.
[0042] "Emulsifier" is an agent such as, but not limited to, a
soap, detergent, steroid or a protein used to stabilize an emulsion
of a hydrophobic phase and an aqueous phase.
[0043] "Erythropoietin (EPO) receptor agonist" refers to molecules
capable of causing activation of the EPO receptor, which may result
from any number of mechanisms. EPO receptor agonists include those
described or referred to in this specification, such as, but not
limited to darbepoietin alfa (ARANESP.TM.), epoietin alfa (EPO,
EPOGEN.RTM.), and anti-EPO receptor agonist antibodies.
[0044] "Fc" refers to all or part of the constant domain of the
heavy or light chain of human immunoglobulin and any variants or
derivatives of this domain.
[0045] "Fc-IL-1ra" refers to a molecule comprising the sequences of
both IL-1ra and the constant domain of the heavy or light chain of
human immunoglobulin and any variants or derivatives of this
domain. Exemplary Fc-IL-1ra is described in PCT Application WO
97/28828, published Jun. 14, 1997 (incorporated by reference) and
any derivatives or variants of this polypeptide.
[0046] "Interleukin-1 (IL-1) inhibitor" refers to molecules capable
of specifically inhibiting activation of cellular receptors by
IL-1, which may result from any one or more number of mechanisms.
Such mechanisms include, but are not limited to, downregulating
IL-1 production, binding free IL-1, interfering with IL-1 binding
to its receptor, interfering with formation of the IL-1 receptor
complex (i.e., association of IL-1 receptor with IL-1 receptor
accessory protein), or interfering with modulation of IL-1
signaling after binding to its receptor. Interleukin-1 inhibitors
include, but are not limited to, those described or referred to in
this specification such as anti-IL-1 receptor antibodies, IL-1
receptor antagonist, and Fc-IL-1ra.
[0047] "Parent molecule" refers to an unmodified molecule or a
variant molecule lacking the particular variation under discussion.
For example, when discussing substitution variants of a parent
molecules, the parent molecule may be a deletion variant.
[0048] "Preservative" is a substance added to a composition to
inhibit chemical change to the composition or contamination of the
composition.
[0049] "Solubilizer" is a substance added to a composition to
increase dissolution of the components of the composition.
[0050] "sTNF-RI" refers to a soluble form of the type I (p55)
receptor for tumor necrosis factor (TNF) and any fusion proteins,
derivatives or variants thereof. Exemplary sTNF-RI is described in
WO 98/01555.
[0051] "Substitution variant" refers to a variant wherein at least
one amino acid residue in a parent molecule is removed and a
different residue inserted in its place.
[0052] "Therapeutically effective amount" is that amount that
results in a measurable improvement of at least one clinical
parameter in a mammal afflicted with the particular disorder.
[0053] "Variant" refers to a polypeptide with at least 75%, or at
least 80%, or at least 90%, or at least 95% or at least 99% amino
acid sequence homology to the reference polypeptide and that
maintains some level, including a reduced level, of relevant
activity of the reference polypeptide. The percentage of homology
as described herein is calculated as the percentage of amino acid
residues found in the smaller of the two sequences which align with
identical amino acid residues in the sequence being compared when
four gaps in a length of 100 amino acids may be introduced to
assist in that alignment, as set forth by Dayhoff (1972), Atlas of
Protein Sequence and Structure, 5:124, National Biochemical
Research Foundation, Washington, D.C., the disclosure of which is
hereby incorporated by reference for any purpose. Variants can be
based on a reference polypeptide in which one or more amino acids
have been deleted from ("deletion variants"), inserted into
("addition variants"), or substituted within ("substitution
variants") the reference molecule.
[0054] Within this application, mention of the singular includes
the plural unless explicitly indicated otherwise.
DETAILED DESCRIPTION OF THE INVENTION
[0055] All documents cited in this application are incorporated by
reference herein for any purpose.
[0056] In certain embodiments, the invention provides for a method
of raising hematocrit in a mammal comprising administering a
therapeutically effective amount of an IL-1 inhibitor. In certain
embodiments, the invention includes maintaining a raised
hematocrit. In certain embodiments, pharmaceutical compositions
comprising an IL-1 inhibitor are provided.
[0057] The invention, in certain embodiments, provides for treating
blood disorders such as, but not limited to, anemias in a mammal
comprising administering an IL-1 inhibitor, an IL-1 inhibitor and
an EPO receptor agonist, a TNF inhibitor and an EPO receptor
agonist, or an IL-1 inhibitor as well as a TNF inhibitor and an EPO
receptor agonist. In certain embodiments, the invention provides
for raising hematocrit by administering an IL-1 inhibitor and an
EPO receptor agonist, a TNF inhibitor and an EPO receptor agonist,
or an IL-1 inhibitor as well as a TNF inhibitor and an EPO receptor
agonist. In certain embodiments, the invention includes maintaining
a raised hematocrit by administering an IL-1 inhibitor and an EPO
receptor agonist, a TNF inhibitor and an EPO receptor agonist, or
an IL-1 inhibitor as well as a TNF inhibitor and an EPO receptor
agonist. In certain embodiments, pharmaceutical compositions
comprising an IL-1 inhibitor and an EPO receptor agonist, a TNF
inhibitor and an EPO receptor agonist, or an IL-1 inhibitor as well
as a TNF inhibitor and an EPO receptor agonist are provided. In
further embodiments, methods of treatment of blood disorders using
such compositions are provided.
[0058] According to certain embodiments, the invention may be
employed in treatment of blood disorders, such as, but not limited
to, the following: [0059] anemia associated with a decline or loss
of kidney function (chronic renal failure); [0060] anemia
associated with myelosuppressive therapy, such as chemotherapeutic
or anti-viral drugs (such as AZT); [0061] anemia associated with
the progression of non-myeloid cancers, anemia associated with
viral infections (such as HIV); [0062] anemia associated with
relative erythropoietin deficiency; [0063] anemia associated with
congestive heart failure; and [0064] anemia of chronic disease such
as autoimmune disease (e.g., rheumatoid arthritis).
[0065] According to certain embodiments, the invention may be
employed with conditions that may lead to anemia in an otherwise
healthy individual, such as an anticipated loss of blood during
surgery. In certain embodiments, treatment includes once daily
dosing for anemia associated with rheumatoid arthritis (RA). Blood
disorders also include, but are not limited to, disorders of
hematopoiesis, hemachromatosis, and deficiencies in iron
metabolism. Hematopoiesis refers to the formation of blood or blood
cells.
[0066] Anemia can occur as an extra-articular complication of
rheumatoid arthritis (RA) and usually correlates with markers of
disease activity (Peeters H R M et al., Ann. Rheum. Dis., 55:
162-68 1996). The inventors found that treatment with an IL-1
inhibitor of patients with RA increased the hematocrit of these
patients (FIG. 2). In particular, the hematocrit in those RA
patients who were also anemic was increased relative to the placebo
(FIG. 4). Furthermore, the inventors found that the hematocrit
improved even in those patients whose RA response (known as ACR20)
did not meet accepted criteria (FIGS. 5 and 6).
[0067] As noted above, one form of anemia is anemia of chronic
disease (ACD). ACD is a broad term for mild to moderately severe
anemia that is often associated with such conditions as, but not
limited to: [0068] trauma; [0069] infectious inflammation; [0070]
noninfectious inflammation, such as may be associated with
rheumatoid arthritis (RA), inflammatory bowel disease (IBD), lupus
(including systemic lupus erythematosus or SLE) multiple sclerosis
(MS), congestive heart failure (CHF), cardiovascular inflammation,
and neoplastic diseases that persist for greater than two months.
For a review of certain ACD, see Means (1999), International J. of
Hematol. 70(1):7-12. ACD is known to have a multi-factorial
pathogenesis mediated in varying degree by innate and cognate
immune response and pro-inflammatory cytokines such as INF-.gamma.,
TNF-.alpha., and IL-1 (Means (1999), International J. of Hematol.
70(1):7-12; Voulgari, et al., (1999), Clin. Immunol.
92:153-160).
[0071] Immunization of Lewis rats with peptidoglycan-polysaccharide
polymers induces relapsing arthritis and a biphasic ACD that
closely replicates human ACD. The inventors found that
administration of Fc-IL-1ra with ARANESP.TM. to these rats
increased mean Hb level, reticulocyte number, red blood cell
number, corpuscular volume, corpuscular HB level, and total serum
iron concentration to a greater extent than either Fc-IL-1ra or
ARANESP.TM. administered alone (see FIGS. 7 to 15).
[0072] The inventors also administered sTNF-RI alone and in
combination with ARANESP.TM. in the same Lewis rat model of human
ACD. The inventors found that rats treated with PEG sTNF-RI only
trended toward elevated mean Hb levels (FIG. 16) but had
significant reduction in mean paw edema (FIG. 17) compared to
untreated rats. Mean Hb levels in rats receiving ARANESP.TM.
treatment alone were elevated over untreated anemic levels. The
combined therapies significantly enhanced Hb levels compared to
same dose of PEG-sTNF-RI and more than doubled the elevation of Hb
compared to the same doses of ARANESP.TM. alone. The combined
therapies also induced more reticulocytes (FIG. 18) and red blood
cells (FIG. 19) compared to the same doses of ARANESP.TM. and
PEG-sTNF-RI. The combined therapies increased total serum iron
concentrations (FIGS. 20A and 20B), corpuscular volume (FIG. 21)
and corpuscular Hb (FIG. 22) to a greater extent than ARANESP.TM.
alone. The combined therapies also increased total serum iron
concentrations, corpuscular volume, and corpuscular Hb more than
PEG sTNF-RI alone. While not intending to be constrained by theory,
the inventors believe that such results show that the apparent
synergistic effect between ARANESP.TM. and PEG-sTNF-RI is mediated
through enhanced erythropoietic response and by enhancing the
"hemoglobinization" of the increased numbers of RBCs (i.e.,
beneficially affecting RBC phagocytosis, iron sequestration and/or
iron mobilization). In addition, although the data was generated
with PEG-sTNF-RI and ARANESP.TM., other TNF-.alpha. antagonists
(see below) in combination with ARANESP.TM. or other agonists of
epoR (also below) should generate similar results.
[0073] The combination therapies of this invention may also be
useful in treatment of conditions associated with iron storage
disorders. Stored iron is involved in the generation of superoxides
and other reactive species which mediate tissue damage in numerous
diseases or disorders. For such conditions and disorders, the IL-1
inhibitor or TNF inhibitor may aid release of iron and the EPO
receptor agonist may aid consumption of the excess iron by
erythropoiesis. For this or other reasons, the claimed combination
therapies may be useful in treatment of chronic heart disease,
myocardial infarction, stroke, cognitive deficiencies or disorders,
hemachromatosis, inflammatory bowel disease, and the like.
EPO Receptor Agonists
[0074] Erythropoietin (EPO) receptor agonists are molecules capable
of causing activation of the EPO receptor, which may result from
any one or more number of mechanisms. EPO receptor agonists include
those described or referred to in this specification, such as, but
not limited to, ARANESP.TM., EPO and anti-EPO receptor agonist
antibodies.
Interleukin-1 Inhibitors
[0075] Interleukin-1 is a protein produced by numerous cell-types,
including monocytes and some macrophages. This protein has
important physiological effects on a number of different target
cells. Interleukin-1 inhibitors include, but not are limited to,
molecules capable of specifically preventing activation of cellular
receptors to IL-1, which may result from any one or more number of
mechanisms. Such mechanisms include, but are not limited to,
downregulating IL-1 production, binding free IL-1, interfering with
IL-1 binding to its receptor, interfering with formation of the
IL-1 receptor complex (i.e., association of IL-1 receptor with IL-1
receptor accessory protein), or interfering with modulation of IL-1
signaling after binding to its receptor. Classes of interleukin-1
inhibitors include, but are not limited to: [0076] interleukin-1
receptor antagonists such as IL-1ra, discussed below; [0077]
anti-IL-1 receptor monoclonal antibodies (e.g., EP 623674, the
disclosure of which is hereby incorporated by reference for any
purpose); [0078] IL-1 binding proteins such as soluble IL-1
receptors (e.g., U.S. Pat. No. 5,492,888, U.S. Pat. No. 5,488,032,
and U.S. Pat. No. 5,464,937, U.S. Pat. No. 5,319,071, and U.S. Pat.
No. 5,180,812, the disclosures of which are hereby incorporated by
reference for any purpose); [0079] anti-IL-1 monoclonal antibodies
(e.g., WO 9501997, WO 9402627, WO 9006371, U.S. Pat. No. 4,935,343,
EP 364778, EP 267611 and EP 220063, the disclosures of which are
hereby incorporated by reference for any purpose); [0080] IL-1
receptor accessory proteins and antibodies thereto (e.g., WO
96/23067 and WO 99/37773, the disclosures of which are hereby
incorporated by reference for any purpose); [0081] inhibitors of
interleukin-1 beta converting enzyme (ICE) or caspase I (e.g., WO
99/46248, WO 99/47545, and WO 99/47154, the disclosures of which
are hereby incorporated by reference for any purpose), which can be
used to inhibit IL-1 beta production and secretion; [0082]
interleukin-1 beta protease inhibitors; [0083] and other compounds
and proteins which block in vivo synthesis or extracellular release
of IL-1.
[0084] Exemplary IL-1 inhibitors are disclosed in the following
references:
[0085] U.S. Pat. Nos. 5,747,444; 5,359,032; 5,608,035; 5,843,905;
5,359,032; 5,866,576; 5,869,660; 5,869,315; 5,872,095; 5,955,480;
5,965,564;
[0086] International (WO) patent applications 98/21957, 96/09323,
91/17184, 96/40907, 98/32733, 98/42325, 98/44940, 98/47892,
98/56377, 99/03837, 99/06426, 99/06042, 91/17249, 98/32733,
98/17661, 97/08174, 95/34326, 99/36426, 99/36415;
[0087] European (EP) patent applications 534978 and 894795; and
[0088] French patent application FR 2762514.
[0089] The disclosures of all of the aforementioned references are
hereby incorporated by reference for any purpose.
[0090] Certain embodiments of the invention provide variants of
IL-1 inhibitors, which do not substantially adversely affect the
ability to use them to treat anemia. Such additions, deletions, and
substitutions may be at the N-terminal or C-terminal of the
polypeptide, or may be internal to it. In general, relatively small
deletions or additions are less likely to affect structure and/or
function of IL-1 inhibitors. In certain embodiments, deletions,
additions, or substitutions can be from 5-10 amino acid residues,
from 2-5 amino acid residues, or from 1-2 amino acid residues.
[0091] The molecules described in the above references and the
variants and derivatives thereof discussed hereinafter are
collectively termed "IL-1 inhibitors."
Interleukin-1 Receptor Antagonist
[0092] Interleukin-1 receptor antagonist (IL-1ra) is a human
protein that acts as a natural inhibitor of interleukin-1 and that
is a member of the IL-1 family, which includes, but is not limited
to, IL-1.alpha. and IL-1.beta.. Certain receptor antagonists
(including IL-1ra and variants and derivatives, e.g., thereof), as
well as methods of making and using thereof, are described in U.S.
Pat. No. 5,075,222; WO 91/08285; WO 91/17184; AU 9173636; WO
92/16221; WO93/21946; WO 94/06457; WO 94/21275; FR 2706772; WO
94/21235; DE 4219626, WO 94/20517; WO 96/22793; WO 97/28828; and WO
99/36541, the disclosures of which are incorporated herein by
reference for any purpose.
[0093] Specifically, U.S. Pat. No. 5,075,222 describes several
forms of IL-1ra. One of those, IL-1ra.alpha., called "IL-11" in the
'222 patent, is characterized as a 22-23 kD molecule on SDS-PAGE
with an approximate isoelectric point of 4.8, eluting from a Mono Q
FPLC column at around 52 mM NaCl in Tris buffer, pH 7.6. Another,
IL-1ra.beta., is characterized as a 22-23 kD protein, eluting from
a Mono Q column at 48 mM NaCl. Both IL-1 ra.alpha. and IL-1ra.beta.
are glycosylated. A third, IL-1rax, is characterized as a 20 kD
protein, eluting from a Mono Q column at 48 mM NaCl, and is
non-glycosylated. The U.S. Pat. No. 5,075,222 also discloses
certain methods for isolating genes responsible for coding the
inhibitors, cloning genes in suitable vectors and cell types, and
expressing genes to produce the inhibitors. The proteins include
glycosylated as well as non-glycosylated IL-1 receptor antagonists.
In certain embodiments, glycosylated IL-1 receptor antagonists may
be expressed in human cells, COS cells, or CHO cells. For purposes
of the present invention, IL-1ra and variants and derivatives
thereof as discussed hereinafter are collectively termed "IL-1ra
protein(s)".
TNF-.alpha. Inhibitors
[0094] TNF-.alpha. inhibitors may act by downregulating or
inhibiting TNF production, binding free TNF, interfering with TNF
binding to its receptor, or interfering with modulation of TNF
signaling after binding to its receptor. The term "TNF-.alpha.
inhibitor" thus includes solubilized TNF receptors, antibodies to
TNF, antibodies to TNF receptor, inhibitors of TNF-.alpha.
converting enzyme (TACE), and other molecules that affect TNF
activity. Well known TNF-.alpha. inhibitors within the scope of
this invention include, but are not limited to the following:
[0095] TNF-.alpha. neutralizing antibodies such as infliximab
(REMICADE.RTM.) and D2E7; [0096] antibody fragments such as CDP
870; [0097] TNF-.alpha. receptor antagonist Abs; [0098] soluble
TNF-R1 molecules such as onercept; [0099] soluble TNF-R11 molecules
such as etanercept (ENBREL.RTM.); [0100] TNF-.alpha. production
inhibitors; and [0101] small molecule inhibitors such as P38
inhibitors.
[0102] TNF-.alpha. inhibitors of various kinds are disclosed in the
art, including the following references:
[0103] European patent applications EP 308 378; EP 422 339; GB 2
218 101, EP 393 438; WO 90/13575, EP 398 327, EP; 412 486, WO
91/03553, EP; 418 014, 417 563, JP 127,800/1991, EP 433 900, U.S.
Pat. No. 5,136,021, GB 2 246 569, EP 464 533; WO 92/01002, WO
92/13095, WO 92/16221, EP 512 528; EP 526 905; WO 93/07863, EP 568
928; EP 607 776 (use of leflunomide for inhibition of TNF-.alpha.);
663 210; 542 795; 818 439; 664 128; 542 795; 741 707; 874 819; 882
714; 880 970; 648 783; 731 791; 895 988; 550 376; 882 714; 853 083;
550 376; 943 616; 939 121; 614 984; 853 083
[0104] U.S. Pat. Nos. 5,136,021; 5,929,117; 5,948,638; 5,807,862;
5,695,953; 5,834,435; 5,817,822; 5,830,742; 5,834,435; 5,851,556;
5,853,977; 5,359,037; 5,512,544; 5,695,953; 5,811,261; 5,633,145;
5,863,926; 5,866,616; 5,641,673; 5,869,677; 5,869,511; 5,872,146;
5,854,003; 5,856,161; 5,877,222; 5,877,200; 5,877,151; 5,886,010;
5,869,660; 5,859,207; 5,891,883; 5,877,180; 5,955,480; 5,955,476;
5,955,435; 5,994,351; 5,990,119; 5,952,320; 5,962,481;
[0105] International (WO) patent applications 90/13575, 91/03553,
92/01002, 92/13095, 92/16221, 93/07863, WO 93/21946, WO 93/19777,
EP 417 563, WO 95/34326, WO 96/28546, 98/27298, 98/30541, 96/38150,
96/38150, 97/18207, 97/15561, 97/12902, 96/25861, 96/12735,
96/11209, 98/39326, 98/39316, 98/38859, 98/39315, 98/42659,
98/39329, 98/43959, 98/45268, 98/47863, 96/33172, 96/20926,
97/37974, 97/37973, 97/47599, 96/35711, 98/51665, 98/43946,
95/04045, 98/56377, 97/12244, 99/00364, 99/00363, 98/57936,
99/01449, 99/01139, 98/56788, 98/56756, 98/53842, 98/52948,
98/52937, 99/02510, 97/43250, 99/06410, 99/06042, 99/09022,
99/08688, 99/07679, 99/09965, 99/07704, 99/06041, 99/37818,
99/37625, 97/11668, 99/50238, 99/47672, 99/48491;
[0106] Japanese (JP) patent applications 10147531, 10231285,
10259140, and 10130149, 10316570, 11001481, and 127,800/1991;
[0107] German (DE) application 19731521;
[0108] British (GB) applications 2 218 101, 2 326 881, 2 246 569
and PCT Application No. PCT/US97/12244.
[0109] For purposes of this invention, the molecules disclosed in
these references and the sTNFRs and variants and derivatives of the
sTNFRs and the molecules disclosed in the references below are
collectively termed "TNF-.alpha. inhibitors."
[0110] For example, EP 393 438 and EP 422 339 teach the amino acid
and nucleic acid sequences of a soluble TNF receptor type I (also
known as sTNFR-1 or 30 kDa TNF inhibitor) and a soluble TNF
receptor type II (also known as sTNFR-II or 40 kDa TNF inhibitor),
collectively termed "sTNFRs", as well as modified forms thereof
(e.g., fragments, functional derivatives and variants). EP 393 438
and EP 422 339 also disclose methods for isolating the genes
responsible for coding the inhibitors, cloning the gene in suitable
vectors and cell types, and expressing the gene to produce the
inhibitors.
[0111] sTNFR-I and sTNFR-II are members of the nerve growth
factor/TNF receptor superfamily of receptors which includes the
nerve growth factor receptor (NGF), the B cell antigen CD40, 4-1
BB, the rat T-cell antigen MRC OX40, the fas antigen, and the CD27
and CD30 antigens (Smith et al. (1990), Science, 248:1019-1023).
The most conserved feature amongst this group of cell surface
receptors is the cysteine-rich extracellular ligand binding domain,
which can be divided into four repeating motifs of about forty
amino acids and which contains 4-6 cysteine residues at positions
which are well conserved (Smith et al. (1990), supra).
[0112] EP 393 438 teaches a 40 kDa TNF inhibitor 451 and a 40 kDa
TNF inhibitor 453, which are truncated versions of the full-length
recombinant 40 kDa TNF inhibitor protein wherein 51 or 53 amino
acid residues, respectively, at the carboxyl terminus of the mature
protein are removed.
[0113] PCT Application No. PCT/US97/12244 teaches truncated forms
of sTNFR-I and sTNFR-II which do not contain the fourth domain
(amino acid residues Thr.sup.127-Asn.sup.161 of sTNFR-I and amino
acid residues Pro.sup.141-Thr.sup.179 of sTNFR-II); a portion of
the third domain (amino acid residues Asn.sup.111-Cys.sup.126 of
sTNFR-I and amino acid residues Pro.sup.123-Lys.sup.140 of
sTNFR-II); and, optionally, which do not contain a portion of the
first domain (amino acid residues Asp.sup.1-Cys.sup.19 of sTNFR-I
and amino acid residues Leu.sup.1-Cys.sup.32 of sTNFR-II). The
truncated sTNFRs of the present invention include the proteins
represented by the formula R.sub.1-[Cys.sup.19-Cys.sup.103]-R.sup.2
and R.sub.4-[Cys.sup.32-Cys.sup.115]-R.sub.5. These proteins are
truncated forms of sTNFR-I and sTNFR-II, respectively.
[0114] By "R.sub.1-[Cys.sup.19-Cys.sup.103]-R.sub.2" is meant one
or more proteins wherein [Cys.sup.19-Cys.sup.103] represents
residues 19 through 103 of sTNFR-I, the amino acid residue
numbering scheme of which is provided in FIG. 1 to facilitate the
comparison; wherein R.sub.1 represents a methionylated or
nonmethionylated amine group of Cys.sup.19 or of amino-terminus
amino acid residue(s) selected from any one of Cys.sup.18 to
Asp.sup.1 and wherein R.sub.2 represents a carboxy group of
Cys.sup.103 or of carboxy-terminal amino acid residues selected
from any one of Phe.sup.104 to Leu.sup.110.
[0115] Exemplary truncated sTNFR-I of the present invention include
the following molecules (collectively termed 2.6D sTNFR-I):
NH.sub.2-[Asp.sup.1-Cys.sup.105]-COOH (also referred to as sTNFR-I
2.6D/C105); NH.sub.2-[Asp.sup.1-Leu.sup.108]-COOH (also referred to
as sTNFR-I 2.6D/C106); NH.sub.2-[Asp.sup.1-Asn.sup.105]-COOH (also
referred to as sTNFR-I 2.6D/N105);
NH.sub.2-[Tyr.sup.9-Leu.sup.108]-COOH (also referred to as sTNFR-I
2.3D/d8); NH.sub.2-[Cys.sup.19-Leu.sup.108]-COOH (also referred to
as sTNFR-I 2.3D/d18); and NH.sub.2-[Ser.sup.16-Leu.sup.108]-COOH
(also referred to as sTNFR-I 2.3D/d15), either methionylated or
nonmethionylated, and variants and derivatives thereof.
[0116] By "R.sub.3-[Cys.sup.32-Cys.sup.115]-R.sub.4" is meant one
or more proteins wherein [Cys.sup.32-Cys.sup.115] represents
residues Cys.sup.32 through Cys.sup.115 of sTNFR-II, the amino acid
residue numbering scheme of which is provided in FIG. 2 to
facilitate the comparison; wherein R.sub.3 represents a
methionylated or nonmethionylated amine group of Cys.sup.32 or of
amino-terminus amino acid residue(s) selected from any one of
Cys.sup.31 to Leu.sup.1 and wherein R.sub.4 represents a carboxy
group of Cys.sup.115 or of carboxy-terminal amino acid residue(s)
selected from any one of Ala.sup.116 to Arg.sup.122.
Variants of Proteins
[0117] Variant refers a polypeptide with at least 75%, or at least
80%, or at least 90%, or at least 95% or at least 99% amino acid
sequence homology to the reference polypeptide and that maintains
some level, including a reduced level, of relevant activity of the
reference polypeptide. The percentage of homology as described
herein is calculated as the percentage of amino acid residues found
in the smaller of the two sequences which align with identical
amino acid residues in the sequence being compared when four gaps
in a length of 100 amino acids may be introduced to assist in that
alignment, as set forth by Dayhoff (1972), Atlas of Protein
Sequence and Structure, 5:124, National Biochemical Research
Foundation, Washington, D.C., the disclosure of which is hereby
incorporated by reference for any purpose. Also included within the
term "substantially homologous" are variant(s) of parent molecules
that may be isolated by cross-reactivity with antibodies to the
parent molecule amino acid sequences or whose genes may be isolated
through hybridization with the DNA of parent molecules or segments
thereof under highly stringent or moderately stringent
hybridization conditions.
[0118] Those skilled in the art will understand that one may make
many variants based on IL-1 inhibitors or EPO receptor agonists in
which amino acids have been deleted ("deletion variants"), inserted
("addition variants"), and/or substituted ("substitution
variants"). Such variants should, however, maintain at some level
(including a reduced level) certain relevant activity of the
unmodified or "parent" molecule and thus may be used as IL-1
inhibitors or EPO receptor agonists.
[0119] Variants may be prepared by a variety of mutagenesis
techniques available to one skilled in the art, such as, but not
limited to, site-directed mutagenesis, PCR mutagenesis, and
cassette mutagenesis (Zoller et al. Meth. Enz. 100, 468-500 (1983);
Higuchi, in PCR Protocols pp. 177-183 (Academic Press, 1990); Wells
et al. Gene 34, 315-323 (1985)). Variants may be rapidly screened
to assess their physical properties. It will be appreciated that
such variant(s) will demonstrate similar properties to the
unmodified molecule, but not necessarily all of the same properties
and not necessarily to the same degree as the corresponding parent
molecule.
[0120] There are typically two principal variables in the
construction of amino acid sequence variant(s): the location of the
mutation site and the nature of the mutation. In designing
variant(s), the location of each mutation site and the nature of
each mutation may depend on the biochemical characteristic(s) to be
modified. In certain embodiments, each mutation site can be
modified individually or in series, e.g., by (1) deleting the
target amino acid residue, (2) inserting one or more amino acid
residues adjacent to the located site, or (3) substituting first
with conservative amino acid choices and, depending upon the
results achieved, then with more radical selections.
[0121] In certain embodiments, amino acid sequence deletions range
from about 1 to 30 amino acid residues, from about 1 to 20 amino
acid residues, from about 1 to 10 amino acid residues, or from
about 1 to 5 contiguous amino acid residues. Deletions include, but
are not limited to, amino-terminal, carboxy-terminal and internal
intrasequence. In the case of IL-1ra, in certain embodiments,
deletions may be made in regions of low homology in the IL-1 family
(which comprises IL-1.alpha., IL-1.beta., and IL-1ra). Deletions in
areas of substantial homology with other members of the family
typically will be more likely to significantly modify the
biological activity.
[0122] In certain embodiments, an amino acid sequence addition may
include insertions of an amino- and/or carboxyl-terminal fusion
ranging in length from one residue to one hundred or more residues,
as well as internal intrasequence insertions of single or multiple
amino acid residues. In certain embodiments, internal additions may
range from about 1 to 20 amino acid residues, from about 1 to 10
amino acid residues, from about 1 to 5 amino acid residues, or from
about 1 to 3 amino acid residues.
[0123] An amino-terminus addition is contemplated to include, but
not be limited to, the addition of a methionine (for example, as an
artifact of the direct expression in bacterial recombinant cell
culture). A further example of an amino-terminal addition includes,
but is not limited to, the fusion of a signal sequence to the
amino-terminus of a mature molecule in order to facilitate its
secretion from recombinant host cells. Such signal sequences
typically will be obtained from and thus be homologous to the
intended host cell species. For prokaryotic host cells that do not
recognize and process the native signal sequence of the mature
molecule, the signal sequence may be substituted by a prokaryotic
signal sequence selected in certain embodiments. In certain
embodiments, the native signal sequence may be substituted by, for
example, the alkaline phosphatase, penicillinase or heat-stable
enterotoxin II leader sequences. For expression in yeast cells, in
certain embodiments, the signal sequence may be selected from the
yeast invertase, alpha factor, or acid phosphatase leader
sequences. For mammalian cell expression, in certain embodiments,
the native signal sequences are satisfactory, although other
mammalian signal sequences may be suitable.
[0124] According to certain embodiments, amino- or a
carboxy-terminus additions may include chimeric proteins comprising
the amino-terminal or carboxy-terminal fusion of IL-1 inhibitor
parent molecules or EPO receptor agonist parent molecules with all
or part of the constant domain of the heavy or light chain of human
immunoglobulin (individually or collectively, ("Fc variant(s)"). In
certain embodiments, the immunoglobulin portion of each chimeric
protein comprises all of the domains except the first domain of the
constant region of the heavy chain of human immunoglobulin such as
IgG (e.g., IgG1 or IgG3), IgA, IgM, or IgE. For example, in certain
embodiments, an IL-1 inhibitor is a chimeric protein comprising
IL-1ra and an F.sub.c domain, particularly that of a human IgG1.
See, for example, WO 97/28828, which is hereby incorporated by
reference for any purpose. A skilled artisan will appreciate that,
in certain embodiments, any amino acid of the immunoglobulin
portion can be deleted or substituted with one or more amino acids,
or one or more amino acids can be added as long as the parent
molecule still maintains some level of its relevant activity and
the immunoglobulin portion shows one or more of its characteristic
properties.
[0125] Another group of variants is the amino acid substitution
variants. These are variants wherein at least one amino acid
residue in a parent molecule is removed and a different residue
inserted in its place. Substitution variants include, but are not
limited to, allelic variants which are characterized by
naturally-occurring nucleotide sequence changes in the species
population that may or may not result in an amino acid change. One
skilled in the art can use any information known about the binding
or active site of the polypeptide in the selection of possible
mutation sites.
[0126] In certain embodiments, one method for identifying amino
acid residues or regions for mutagenesis of a protein is called
"alanine scanning mutagenesis", as described by Cunningham and
Wells (1989), Science, 244:1081-1085, the disclosure of which is
hereby incorporated by reference for any purpose. In this method,
an amino acid residue or group of target residues is identified
(e.g., charged residues such as Arg, Asp, H is, Lys and Glu) and
replaced by a neutral or negatively-charged amino acid (most
preferably alanine or polyalanine) to affect the interaction of the
amino acids with the surrounding aqueous environment in or outside
the cell. Those domains/residues demonstrating functional
sensitivity to the substitutions are then refined by introducing
additional or alternate residues at the sites of substitution.
Thus, the site for introducing an amino acid sequence modification
is predetermined. To optimize the performance of a mutation at a
given site, alanine scanning or random mutagenesis may be conducted
and the variant(s) may be screened for the optimal combination of
desired activity and degree of activity in certain embodiments.
[0127] The sites typically of greatest interest for substitutional
mutagenesis include, but are not limited to, sites in which
particular amino acid residues within a parent molecule are
substantially different from other species or other family members
in terms of side-chain bulk, charge and/or hydrophobicity. Other
sites of interest include those in which particular residues of a
parent molecule are identical among other species or other family
members, as such positions are typically important for the
biological activity of a protein.
[0128] In certain embodiments, a skilled artisan will appreciate
that initially sites typically can be modified by substitution in a
relatively conservative manner. Conservative amino acid changes may
involve substitution of one amino acid with another that is similar
in structure and/or function (e.g., amino acids with side chains
similar in size, charge and shape). Examples of such conservative
substitutions include, but are not limited to, those shown in Table
1 under the heading of "Preferred Substitutions". If such
substitutions do not result in a substantial change in biological
activity, then more substantial changes (Exemplary Substitutions)
may be introduced and/or other additions/deletions may be made and
the resulting products screened.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions Original Preferred
Exemplary Residue Substitutions Substitutions Ala (A) Val Val; Leu;
Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Lys; Arg Asp
(D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G)
Pro Pro His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met;
Ala; Phe; norleucine Leu (L) Ile norleucine; Ile; Val; Met; Ala;
Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Leu
Leu; Val; Ile; Ala Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser
Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile;
Leu; Met; Phe; Ala; norleucine
[0129] In making such changes, the hydropathic index of amino acids
may be considered in certain embodiments. The importance of the
hydropathic amino acid index in conferring interactive biological
function on a protein is generally understood in the art (Kyte and
Doolittle (1982), J. Mol. Biol., 157:105-131, the disclosure of
which is incorporated herein by reference for any purpose). It is
known that certain amino acids may be substituted for other amino
acids having a similar hydropathic index or score and still retain
a similar biological activity.
[0130] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the functionally equivalent
protein or peptide thereby created is intended for use in
immunological embodiments. U.S. Pat. No. 4,554,101, the disclosure
of which is incorporated herein by reference for any purpose,
states that the greatest local average hydrophilicity of a protein,
as governed by the hydrophilicity of its adjacent amino acids,
correlates with its immunogenicity and antigenicity, i.e., with a
biological property of the protein.
[0131] In contrast, in certain embodiments, substantial
modifications in the functional and/or chemical characteristics of
a parent molecule may be accomplished by selecting substitutions
that differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the relative charge or hydrophobicity of the protein at the target
site or (c) the bulk of the side chain. Naturally-occurring
residues can be divided into groups based on common side chain
properties:
[0132] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0133] 2) neutral hydrophilic: Cys, Ser, Thr;
[0134] 3) acidic: Asp, Glu;
[0135] 4) basic: Asn, Gln, H is, Lys, Arg;
[0136] 5) aromatic: Trp, Tyr, Phe; and
[0137] 6) residues that influence chain orientation: Gly, Pro.
[0138] Non-conservative substitutions may involve the exchange of a
member of one of these groups for another.
Polypeptide Derivatives
[0139] In certain embodiments, this invention also comprises
chemically modified derivatives of the parent molecules of the IL-1
inhibitor, TNF-.alpha. inhibitor, and EPO receptor agonist, and the
uses thereof, in which the protein is linked to a nonproteinaceous
moiety (e.g., a polymer) in order to modify properties. These
chemically modified parent molecules are referred to herein as
"derivatives". Such derivatives may be prepared by one skilled in
the art given the disclosures herein. In certain embodiments,
conjugates may be prepared using glycosylated, non-glycosylated or
de-glycosylated parent molecule(s) and suitable chemical moieties.
In certain embodiments, non-glycosylated parent molecules and
water-soluble polymers will be used. In certain embodiments,
derivatives encompassed by the invention include post-translational
modifications (e.g., N-linked or O-linked carbohydrate chains,
processing of N-terminal or C-terminal ends), attachment of
chemical moieties to the amino acid backbone, and chemical
modifications of N-linked or O-linked carbohydrate chains. In
certain embodiments, the polypeptides may also be modified with a
detectable label, such as an enzymatic, fluorescent, isotopic or
affinity label to allow for detection and isolation of the protein.
Such polypeptide derivatives should maintain at some level
(including a reduced level) the relevant activity of the parent
molecule and so may be used as IL-1 inhibitors or EPO receptor
agonists.
[0140] In certain embodiments, water-soluble polymers are desirable
because the protein to which each is attached typically will not
precipitate in an aqueous environment, such as a physiological
environment. In certain embodiments, the polymer will be
pharmaceutically acceptable for the preparation of a therapeutic
product or composition. One skilled in the art will be able to
select the desired polymer based on such considerations as, but not
limited to, whether the polymer/protein conjugate will be used
therapeutically and, if so, the therapeutic profile of the protein
(e.g., duration of sustained release; resistance to proteolysis;
effects, if any, on dosage; biological activity; ease of handling;
degree or lack of antigenicity and other known effects of a
water-soluble polymer on a therapeutic proteins).
[0141] Suitable, clinically acceptable, water-soluble polymers
include, but are not limited to, polyethylene glycol (PEG),
polyethylene glycol propionaldehyde, copolymers of ethylene
glycol/propylene glycol, monomethoxy-polyethylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol (PVA), polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, poly (8-amino acids) (either
homopolymers or random copolymers), poly(n-vinyl
pyrrolidone)polyethylene glycol, polypropylene glycol homopolymers
(PPG) and other polyalkylene oxides, polypropylene oxide/ethylene
oxide copolymers, polyoxyethylated polyols (POG) (e.g., glycerol)
and other polyoxyethylated polyols, polyoxyethylated sorbitol, or
polyoxyethylated glucose, colonic acids or other carbohydrate
polymers, Ficoll or dextran and mixtures thereof. As used herein,
polyethylene glycol is meant to encompass any of the forms that
have been used to derivatize other proteins, such as
mono-(C.sub.1-C.sub.10) alkoxy- or aryloxy-polyethylene glycol.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water.
[0142] The water-soluble polymers each may be of any molecular
weight and may be branched or unbranched. Typically, the higher the
molecular weight or the more branches, the higher the
polymer:protein ratio. The water-soluble polymers each typically
have an average molecular weight of between about 2 kDa to about
100 kDa (the term "about" indicating that in preparations of a
water-soluble polymer, some molecules will weigh more, some less,
than the stated molecular weight). In certain embodiments, the
average molecular weight of each water-soluble polymer is between
about 5 kDa and about 40 kDa, between about 10 kDa and about 35
kDa, or between about 15 kDa and about 30 kDa.
[0143] In certain embodiments, the protein has polyethylene glycol
(PEG) attached because PEG typically has very low toxicity in
mammals [Carpenter et al., Toxicol. Appl. Pharmacol., 18, 35-40
(1971)]. A PEG adduct of adenosine deaminase was approved in the
United States for use in humans for the treatment of severe
combined immunodeficiency syndrome. A second advantage that can be
afforded by the conjugation of PEG is that of effectively reducing
the immunogenicity and antigenicity of heterologous proteins. For
example, in certain embodiments, a PEG adduct of a human protein
might be useful for the treatment of disease in other mammalian
species without the risk of triggering a severe immune
response.
[0144] In certain embodiments, polymers such as PEG may be
conveniently attached to one or more reactive amino acid residues
in a protein such as the alpha-amino group of the amino-terminal
amino acid, the epsilon amino groups of lysine side chains, the
sulfhydryl groups of cysteine side chains, the carboxyl groups of
aspartyl and glutamyl side chains, the alpha-carboxyl group of the
carboxyl-terminal amino acid, tyrosine side chains, or to activated
derivatives of glycosyl chains attached to certain asparagine,
serine or threonine residues. Numerous activated forms of PEG
suitable for direct reaction with proteins have been described. In
certain embodiments, PEG reagents for reaction with protein amino
groups include, but are not limited to, active esters of carboxylic
acid or carbonate derivatives, particularly those in which the
leaving groups are N-hydroxysuccinimide, p-nitrophenol, imidazole
or 1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing
maleimido or haloacetyl groups are useful reagents for the
modification of protein free sulfhydryl groups. Likewise, PEG
reagents containing amino, hydrazine or hydrazide groups are useful
for reaction with aldehydes generated by periodate oxidation of
carbohydrate groups in proteins.
[0145] There are a number of attachment methods available to those
skilled in the art, including acylation reactions or alkylation
reactions with a reactive water-soluble molecule. In certain
embodiments, such reactions generate an amino-terminal chemically
modified protein. See, for example, EP 0 401 384; Malik et al.
(1992), Exp. Hematol., 20:1028-1035; Francis (1992), Focus on
Growth Factors, 3(2):4-10, published by Mediscript, Mountain Court,
Friern Barnet Lane, London N20 OLD, UK; EP 0 154 316; EP 0 401 384;
WO 92/16221; WO 95/34326; WO 95/13312; WO 96/11953; WO 96/19459 and
WO 96/19459 and the other publications cited herein that relate to
pegylation, the disclosures of which are hereby incorporated by
reference for any purpose.
[0146] In certain embodiments, pegylation also may be specifically
carried out using water-soluble polymers having at least one
reactive hydroxy group (e.g. polyethylene glycol). In certain
embodiments, the water-soluble polymer can be reacted with an
activating group, thereby forming an "activated linker" useful in
modifying various proteins. The activated linkers can be
monofunctional, bifunctional, or multifunctional.
[0147] Activating groups which can be used to link the
water-soluble polymer to two or more proteins include, but are not
limited to, the following: sulfone, maleimide, sulfhydryl, thiol,
triflate, tresylate, azidirine, oxirane and 5-pyridyl. Useful
reagents having a reactive sulfone group that can be used in the
methods include, without limitation, chlorosulfone, vinylsulfone
and divinylsulfone. These PEG derivatives typically are stable
against hydrolysis for extended periods in aqueous environments at
pHs of about 11 or less, and can form linkages with molecules to
form conjugates which are also hydrolytically stable. Useful
homobifunctional derivatives include, but are not limited to,
PEG-bis-chlorosulfone and PEG-bis-vinylsulfone (see WO
95/13312).
[0148] WO 97/04003, the disclosure of which is hereby incorporated
by reference for any purpose, teaches methods of making
sulfone-activated linkers by obtaining a compound having a reactive
hydroxyl group and converting the hydroxyl group to a reactive
Michael acceptor to form an activated linker, with tetrahydrofuran
as the solvent for the conversion. The application also teaches a
process for purifying the activated linkers, which utilizes
hydrophobic interaction chromatography to separate the linkers
based on size and end-group functionality.
[0149] As an example, chemically modified derivatives of a molecule
may provide such advantages as increased stability, increased time
in circulation, or decreased immunogenicity (see, e.g., U.S. Pat.
No. 4,179,337). The chemical moieties for derivitization may be
selected from water-soluble polymers such as, but are not limited
to, polyethylene glycol, ethylene glycol/propylene glycol
copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and
the like. According to certain embodiments, the polypeptides may be
modified at random positions within the molecule, or at
predetermined positions within the molecule and may include one,
two, three or more attached chemical moieties.
[0150] In certain embodiments, one may specifically desire
N-terminally chemically modified protein. Using polyethylene glycol
as an illustration of the present compositions, in certain
embodiments, one may select from a variety of polyethylene glycol
molecules (by molecular weight, branching, etc.), the proportion of
polyethylene glycol molecules to protein (or peptide) molecules in
the reaction mix, the type of pegylation reaction to be performed,
and the method of obtaining the selected N-terminally pegylated
protein. In certain embodiments, the method of obtaining the
N-terminally pegylated preparation (i.e., separating this moiety
from other monopegylated moieties if necessary) may be by
purification of the N-terminally pegylated material from a
population of pegylated protein molecules. In certain embodiments,
selective N-terminal chemical modification may be accomplished by
reductive alkylation, which exploits differential reactivity of
different types of primary amino groups (lysine versus the
N-terminal) available for derivatization in a particular protein.
Under the appropriate reaction conditions, substantially selective
derivatization of the protein at the N-terminus with a carbonyl
group containing polymer is achieved.
Polyvalent Forms
[0151] Techniques for formation of polyvalent forms include, but
are not limited to, photochemical crosslinking (e.g., exposure to
ultraviolet light), chemical crosslinking (e.g., with bifunctional
linker molecules such as polyethylene glycol), and mutagenesis
(e.g., introduction of additional cysteine residues).
[0152] Polyvalent forms may be constructed by chemically coupling
at least one parent molecule and another moiety with any clinically
accepted linker (e.g., a water-soluble polymer). In principle, the
linker typically should not impart new immunogenicity. In certain
embodiments, the linker also typically should not, by virtue of the
new amino acid residues, alter the hydrophobicity and charge
balance of the structure, which affects its biodistribution and
clearance. A variety of chemical crosslinkers may be used depending
upon which properties of the protein dimer are desired. For
example, in certain embodiments, crosslinkers may be short and
relatively rigid or longer and more flexible, may be biologically
reversible, and may provide reduced immunogenicity or longer
pharmacokinetic half-life.
[0153] In certain embodiments, molecules are linked through the
amino terminus by a two-step synthesis. In the first step, one
molecule is chemically modified at the amino terminus to introduce
a protected thiol, which after purification is deprotected and used
as a point of attachment for site-specific conjugation through a
variety of crosslinkers with a second molecule. Amino-terminal
crosslinks include, but are not limited to, a disulfide bond,
thioether linkages using short-chain, bis-functional aliphatic
crosslinkers, and thioether linkages to variable length,
bifunctional polyethylene glycol crosslinkers (PEG "dumbbells").
Also encompassed by PEG dumbbell synthesis of dimers is a byproduct
of such synthesis, termed a "monobell." A monobell includes, but is
not limited to, a monomer coupled to a linear bifunctional PEG with
a free polymer terminus. Alternatively, in certain embodiments,
molecules may be crosslinked directly through a variety of amine
specific homobifunctional crosslinking techniques which include
reagents such as, but not limited to: diethylenetriaminepentaacetic
dianhydride (DTPA), p-benzoquinone (pBQ), or bis(sulfosuccinimidyl)
suberate (BS.sup.3) as well as others known in the art. In certain
embodiments, it is also possible to thiolate a molecule directly
with reagents such as iminothiolane in the presence of a variety of
bifunctional, thiol specific crosslinkers, such as PEG
bismaleimide, and achieve dimerization and/or dumbbells in a one
step process.
[0154] In certain embodiments, the water-soluble polymers for this
polyvalent form can be, based on the monomers listed herein,
homopolymers, random or block copolymers, terpolymers straight
chain or branched, substituted or unsubstituted. The polymer can be
of any length or molecular weight, but these characteristics can
affect the biological properties. Polymer average molecular weights
particularly useful for decreasing clearance rates in
pharmaceutical applications typically are in the range of 2,000 to
35,000 daltons. In addition, in certain embodiments, the length of
the polymer can be varied to optimize or confer the desired
biological activity.
[0155] In certain embodiments, a bivalent molecule may include two
tandem repeats of parent molecules separated by a polypeptide
linker region. The design of the polypeptide linkers can be similar
in design to the insertion of short loop sequences between domains
in the de novo design of proteins (Mutter (1988), TIBS, 13:260-265
and Regan and DeGrado (1988), Science, 241:976-978, the disclosures
of which are hereby incorporated by reference for any purpose).
Several different linker constructs have been assembled and shown
to be useful for forming single chain antibodies; the most
functional linkers typically vary in size from 12 to 25 amino acids
(amino acids having unreactive side groups, e.g., alanine, serine
and glycine) which together constitute a hydrophilic sequence, have
a few oppositely charged residues to enhance solubility and are
flexible (Whitlow and Filpula (1991), Methods: A Companion to
Methods in Enzymology, 2:97-105; and Brigido et al. (1993), J.
Immunol., 150:469-479, the disclosures of which are hereby
incorporated by reference for any purpose). It has been shown that
a linker suitable for single chain antibodies is effective to
produce a dimeric form of the human sTNFR-II (Neve et al. (1996),
Cytokine, 8(5):365-370, the disclosure of which is hereby
incorporated by reference for any purpose).
[0156] In certain embodiments, polyvalent forms may also be formed
using substitution variants. In certain embodiments, parent
molecules may be modified to form dimers or multimers by
site-directed mutagenesis to create unpaired cysteine residues for
interchain disulfide bond formation.
[0157] Additionally, in certain embodiments, a parent molecule may
be chemically coupled to biotin, and the resulting conjugate may
then be allowed to bind to avidin, resulting in tetravalent
avidin/biotin/parent molecules. In certain embodiments, parent
molecule may also be covalently coupled to dinitrophenol (DNP) or
trinitrophenol (TNP) and the resulting conjugates precipitated with
anti-DNP or anti-TNP-IgM to form decameric conjugates.
[0158] In certain embodiments, recombinant fusion proteins may also
be produced wherein each recombinant chimeric molecule has a parent
molecule(s) sequence amino-terminally or carboxy-terminally fused
to all or part of the constant domains, but to at least one
constant domain, of the heavy or light chain of human
immunoglobulin. In certain embodiments, following transcription and
translation of a heavy-chain chimeric gene, or of a light
chain-containing gene and a heavy-chain chimeric gene, the gene
products may be assembled into a single chimeric molecule having a
parent molecule(s) displayed bivalently. Additional details
relating to the construction of such chimeric molecules are
disclosed, e.g., in U.S. Pat. No. 5,116,964, WO 89/09622, WO
91/16437, WO 97/23614 and EP 315062, the disclosures of which are
hereby incorporated by reference for any purpose.
Pharmaceutical Compositions
[0159] Unless otherwise noted, all statements concerning
pharmaceutical compositions in this specification refer to
compositions comprising IL-1 inhibitor, an EPO receptor agonist, a
TNF-.alpha. inhibitor or a combination of EPO receptor agonist and
IL-1 inhibitor and/or TNF-.alpha. inhibitor.
[0160] In certain embodiments, the invention provides for
pharmaceutical compositions comprising a pharmaceutically
acceptable diluent, carrier, solubilizer, emulsifier, preservative
and/or adjuvant. In certain embodiments, the therapeutic molecules
can be formulated together or packaged together in a kit. In
certain embodiments, the composition may be in a liquid or
lyophilized form and comprises a diluent (Tris, acetate or
phosphate buffers) having various pH values and ionic strengths,
solubilizer such as Tween or Polysorbate, carriers such as human
serum albumin or gelatin, preservatives such as thimerosal or
benzyl alcohol, and antioxidants such as ascrobic acid or sodium
metabisulfite. Also encompassed, in certain embodiments, are
compositions comprising any of the therapeutic molecules modified
with water-soluble polymers to increase solubility or stability. In
certain embodiments, compositions may also comprise incorporation
of any of the therapeutic molecules into liposomes, microemulsions,
micelles or vesicles for controlled delivery over an extended
period of time.
[0161] Specifically, in certain embodiments, compositions herein
may comprise incorporation into polymer matrices such as hydrogels,
silicones, polyethylenes, ethylene-vinyl acetate copolymers, or
biodegradable polymers. Examples of hydrogels include, but are not
limited to, polyhydroxyalkylmethacrylates (p-HEMA), polyacrylamide,
polymethacrylamide, polyvinylpyrrolidone, polyvinyl alcohol and
various polyelectrolyte complexes. Examples of biodegradable
polymers include, but are not limited to, polylactic acid (PLA),
polyglycolic acid (PGA), copolymers of PLA and PGA, polyamides and
copolymers of polyamides and polyesters. Other controlled release
formulations include, but are not limited to, microcapsules,
microspheres, macromolecular complexes and polymeric beads which
may be administered by injection.
[0162] Selection of a particular composition will depend upon a
number of factors, including, but not limited to, the condition
being treated, the route of administration, and the pharmacokinetic
parameters desired. A more extensive survey of component suitable
for pharmaceutical compositions is found in Remington's
Pharmaceutical Sciences, 18th ed. A. R. Gennaro, ed. Mack, Easton,
Pa. (1980), which is hereby incorporated by reference for any
purpose.
[0163] In certain embodiments, an effective amount or amounts of
the therapeutic molecules will depend, for example, upon the
therapeutic objectives, the route of administration, and the
condition of the patient. Accordingly, in certain embodiments, the
therapist may titer the dosage and modify the route of
administration as needed to obtain the optimal therapeutic effect.
A typical daily dosage may range from about 0.1 mg/kg to up to 100
mg/kg or more, depending on the factors mentioned above. In certain
embodiments, a clinician can administer the composition or
compositions until a dosage is reached that achieves the desired
increase in hematocrit or clinical improvement of the blood
disorder.
[0164] In certain embodiments, the composition or compositions may
be administered as a single dose, or as two or more doses of one or
more of the therapeutic molecules. These doses can consist of the
same or different amounts of the therapeutic molecules and can be
administered at the same or different times via the same or
different routes of administration. In certain embodiments, the
composition may be administered as a composition comprising any one
or any combination of the therapeutic molecules. In certain
embodiments, the combination may include the same or different
amounts of the therapeutic molecules. In certain embodiments, the
composition or compositions may be administered as a continuous
infusion via implantation device or catheter. In embodiments in
which the continuous infusion contains more than one therapeutic
molecule, it may contain the same or different concentrations of
the therapeutic molecules.
[0165] As further studies are conducted, information will emerge
regarding appropriate dosage levels for treatment of various
conditions in various patients, and the ordinary skilled worker,
considering the therapeutic context, the type of disorder under
treatment, the age and general health of the recipient, will be
able to ascertain proper dosing.
[0166] The compositions to be used for in vivo administration
typically are sterile. In certain embodiments, this can be readily
accomplished by filtration through sterile filtration membranes.
Where the compositions are lyophilized, sterilization using these
methods may be conducted either prior to, or following,
lyophilization and reconstitution. In certain embodiments, the
compositions for parenteral administration may be stored in
lyophilized form or in solution.
[0167] In certain embodiments, compositions can be placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0168] The route of administration of the compositions are in
accord with known methods including, but not limited to, oral,
nasal, pulmonary, rectal administration or injection or infusion by
subcutaneous, intravenous, intraperitoneal, intracerebral
(intraparenchymal), intracerebroventricular, intramuscular,
intraocular, intraarterial, or intralesional routes, or by
sustained release systems or implantation device which may
optionally involve the use of a catheter. In certain embodiments,
the compositions may be administered continuously by infusion,
bolus injection or by implantation device.
[0169] In certain embodiments, compositions may be administered
locally via implantation into the affected area of a membrane,
sponge, or other appropriate material on to which the therapeutic
molecule or molecules have been absorbed. In certain embodiments,
where an implantation device is used, the device may be implanted
into any suitable tissue or organ, and delivery of the compositions
may be directly through the device via bolus, or via continuous
administration, or via catheter using continuous infusion.
[0170] In certain embodiments, compositions may be administered in
a sustained release formulation or preparation. Suitable examples
of sustained-release preparations include, but are not limited to,
semipermeable polymer matrices in the form of shaped articles, e.g.
films, or microcapsules. Sustained release matrices include, but
are not limited to, polyesters, hydrogels, polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al, Biopolymers, 22: 547-556 [1983]),
poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed.
Mater. Res., 15: 167-277 [1981] and Langer, Chem. Tech., 12: 98-105
[1982]), ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
compositions also may include, but are not limited to, liposomes,
which can be prepared by any of several methods known in the art
(e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692
[1985]; EP 36,676; EP 88,046; EP 143,949).
[0171] In certain embodiments, it may be desirable to use
compositions in an ex vivo manner. Here, cells, tissues, or organs
that have been removed from the patient are exposed to compositions
after which the cells, tissues and/or organs are subsequently
implanted back into the patient.
[0172] In certain embodiments, compositions may be delivered
through implanting into patients certain cells that have been
genetically engineered, using methods known in the art, to express
and secrete the polypeptides, fragments, variants, or derivatives.
In certain embodiments, such cells may be animal or human cells,
and may be derived from the patient's own tissue or from another
source, either human or non-human. In certain embodiments, the
cells may be immortalized. However, in order to decrease the chance
of an immunological response, in certain embodiments, the cells can
be encapsulated to avoid infiltration of surrounding tissues. The
encapsulation materials are typically biocompatible, semi-permeable
polymeric enclosures or membranes that allow release of the protein
product(s) but prevent destruction of the cells by the patient's
immune system or by other detrimental factors from the surrounding
tissues.
[0173] Methods used for membrane encapsulation of cells are
familiar to the skilled artisan, and preparation of encapsulated
cells and their implantation in patients may be accomplished
without undue experimentation. See, e.g., U.S. Pat. Nos. 4,892,538;
5,011,472; and 5,106,627. A system for encapsulating living cells
is described in PCT WO 91/10425 (Aebischer et al.). Techniques for
formulating a variety of other sustained or controlled delivery
products, such as liposome carriers, bio-erodible particles or
beads, are also known to those in the art, and are described, for
example, in U.S. Pat. No. 5,653,975 (Baetge et al.,
CytoTherapeutics, Inc.). In certain embodiments, the cells, with or
without encapsulation, may be implanted into suitable body tissues
or organs of the patient.
[0174] When administered parenterally, proteins may be cleared
rapidly from the circulation and may therefore elicit relatively
short-lived pharmacological activity. Consequently, frequent
injections of relatively large doses of bioactive proteins may be
used to sustain therapeutic efficacy with this method of
administration. Proteins modified by the covalent attachment of
water-soluble polymers such as polyethylene glycol, copolymers of
polyethylene glycol and polypropylene glycol, carboxymethyl
cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or
polyproline are known to exhibit substantially longer half-lives in
blood following intravenous injection than do the corresponding
unmodified proteins [Abuchowski et al., In: "Enzymes as Drugs",
Holcenberg et al., eds. Wiley-Interscience, New York, N.Y., 367-383
(1981), Newmark et al., J. Appl. Biochem. 4:185-189 (1982), and
Katre et al., Proc. Natl. Acad. Sci. USA 84, 1487-1491 (1987),
incorporated by reference for any purpose]. Such modifications may
also increase the protein's solubility in aqueous solution,
eliminate or decrease aggregation, enhance the physical and
chemical stability of the protein, and greatly reduce the
immunogenicity and antigenicity of the protein. As a result, in
certain embodiments, the desired in vivo biological activity may be
achieved by the administration of such polymer-protein adducts less
frequently or in lower doses than with the unmodified protein. In
certain embodiments, IL-1 inhibitors and/or TNF inhibitors can be
used alone or with one or more additional hematopoietic factors or
other therapeutic molecules, such as EPO (Epogen.RTM.), novel
erythropoiesis stimulating protein (NESP, Aranesp.TM.), G-CSF
(Neupogen.RTM.) and derivatives thereof, GM-CSF, CSF-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-18BP, IL-18
inhibitor (e.g., IL-18 antibody), interferon gamma (IFN-.gamma.)
inhibitor (e.g., IFN-.gamma. antibody), IGF-1, or LIF (Leukemic
Inhibitory Factor) in the treatment of hematopoietic disorders.
[0175] There are many diseases which may be treatable with IL-1
and/or TNF inhibitors or such inhibitors and EPO receptor agonists
in certain embodiments. These include, but are not limited to, the
following: myelofibrosis, myelosclerosis, osteopetrosis, metastatic
carcinoma, acute leukemia, multiple myeloma, Hodgkin's disease,
lymphoma, Gaucher's disease, Niemann-Pick disease, Letterer-Siwe
disease, refractory erythroblastic anemia, Di Guglielmo syndrome,
congestive splenomegaly, Hodgkin's disease, Kala azar, sarcoidosis,
primary splenic pancytopenia, miliary tuberculosis, disseminated
fungus disease, fulminant septicemia, malaria, vitamin B12 and
folic acid deficiency, pyridoxine deficiency, Diamond Blackfan
anemia, hypopigmentation disorders such as piebaldism and
vitiligo.
[0176] In certain embodiments, treatment with an IL-1 and/or TNF
inhibitor or such inhibitors and an EPO receptor agonist may also
be used for enhancing hematopoietic recovery after acute blood
loss.
Assays
[0177] To test for suitable compounds for treatment to increase the
hematocrit level, any assays or in vivo protocols that test for
such activity may be employed. Examples of such assays and in vivo
protocols are included in international patent application WO
00/24893, hereby incorporated for reference for any purpose. Those
skilled in the art will be familiar with suitable assays.
[0178] The following examples are offered to illustrate more fully
the invention, but are not construed as limiting the scope
thereof.
Example 1
Administration of an IL-1ra Analog Increases Hematocrit Levels
[0179] In a multi-center, double-blind, dose-ranging study,
subjects with active severe or very severe rheumatoid arthritis
(N=472) received daily subcutaneous injections for 6 months of
placebo, 30, 75, or 150 mg of a recombinant, non-glycosylated
IL-1ra analog ("anakinra"). See FIG. 1 for baseline demographics
and disease history by treatment group. Anakinra is a
non-glycosylated form of human IL-1ra that is recombinantly made in
E. coli.
[0180] Anakinra was injected at concentrations of either 30, 75, or
150 mg/ml of purified anakinra in suspending solution. The
suspending solution contained 140 mM sodium chloride, 10 mM sodium
citrate, 0.1% (w/w) polysorbate 80, 0.5 mM EDTA, and sterile water
for injection. Placebo consisted of the suspending solution.
Anakinra and placebo solutions were stored at temperatures between
20 and 80 C. The pH of all solutions was 6.5.
[0181] A total volume of 1 ml was injected each day. Possible sites
of injection included, but were not limited to, the front of the
thigh, the abdomen above the navel, and the back of the upper arm.
It was recommended that subjects rotate the site of injection.
Subjects were instructed to inject the study solution at the same
time of the day for all 24 weeks, preferably in the evening.
[0182] Hematocrit was assessed at baseline and after 24 weeks of
the anakinra therapy. The difference in change from baseline at
week 24 in hematocrit (volume percentages) between the pooled
anakinra group and the placebo group was assessed via the
Wilcoxon-Mann-Whitney test (E. L. Lehmann, Nonparametrics:
Statistical Methods Based on Ranks, 1975, hereby incorporated by
reference for any purpose) and confirmed with a two sample
t-test.
[0183] The mean hematocrit of subjects treated with anakinra
increased by 0.28 volume-percentages over six months, whereas the
mean hematocrit of subjects treated with placebo decreased by 0.867
volume-percentages over the same time period (FIG. 2).
Example 2
Administration of Anakinra Improves Anemia
[0184] In the study of the effect of anakinra on hematocrit levels
described in Example 1, a subset of subjects with rheumatoid
arthritis were anemic, as defined as hematocrit level less than or
equal to thirty-four percent (Pincus et al., Am. J. Med 89:161,
1990, which is incorporated by reference for any purpose), upon
initiation of the study. Fifty (14.2%) of the anakinra-treated
subjects and thirteen (10.7%) of the placebo-treated subjects were
characterized with this degree of anemia (see FIG. 3 for baseline
demographics and disease history).
[0185] Although the number of anemic subjects in this study was
small, more patients treated with anakinra exhibited improvement in
hematocrit levels after twenty-four weeks of treatment than did
subjects receiving the placebo. Anemia improved in patients taking
each of the three doses of IL-1ra (see FIG. 4 for results).
Example 3
Administration of Anakinra May Improve Anemia Independently of
Articular Disease
[0186] In the study of the effect of the administration of anakinra
on anemia described in Example 2, three of the seven
anakinra-treated patients with greater than or equal to six
volume-percentage improvement in hematocrit levels did not meet the
ACR20 response criteria (FIGS. 5 and 6). The ACR20 defines a
subject as improved if the following criteria are met: >20%
decrease from baseline in the number of tender/painful joints;
>20% decrease from baseline in the number of swollen joints; and
>20% decrease from baseline in 3 of the following 5 criteria: 1)
subject assessment of disease activity; 2) investigator assessment
of disease activity; 3) subject assessment of pain; 4) disability
score from the Health Assessment Questionnaire (HAQ); or 5)
C-reactive protein. Thus, IL-1ra therapy may improve anemia in
rheumatoid arthritis subjects independently of its effect on
articular disease activity.
[0187] While the invention has been described in what is considered
to be its preferred embodiments, it is not to be limited to the
disclosed embodiments, but on the contrary, is intended to cover
various modifications and equivalents included within the spirit
and scope of the appended claims, which scope is to be accorded the
broadest interpretation so as to encompass all such modifications
and equivalents.
Example 4
[0188] On day 0, a group of eight to ten week old female Lewis rats
were immunized with an intraperitoneal (i.p.) injection of
peptidoglycan-polysaccharide polymers (PG-APS) (Lee Laboratories,
Grayson, Ga.) equilibrated to a dose of 15 .mu.g rhamose/kg in
0.85% saline (3 mg/rat in a volume of 0.520 ml, rats injected with
stock PG-APS). Also, on day 0, another group of eight to ten week
old female Lewis rats received 0.5 ml i.p. injections of 0.85%
saline to make carrier-injected rats.
[0189] From day 0 to day 77, blood was collected weekly from tail
arteries into EDTA-coated Microtainer tubes (Becton Dickinson,
Franklin Lakes, N.Y.) and complete blood counts (CBC) were
performed on an ADVIA 120 Hematology System (Bayer Corporation,
Tarrytown, N.Y.) calibrated for rat blood. On day 42, the rats were
divided into two groups. Rats with mean hemoglobin (Hb)
concentrations below 12.0 g/dL were termed anemic. Anemic rats that
possessed similar mean Hb concentrations were placed in the
experimental group.
[0190] The control group consisted of carrier-injected rats that
possessed similar Hb concentrations to each other.
[0191] Beginning on day 43, rats in the experimental group were
subdivided into eight treatment groups. All groups were n=5 except
the carrier group, which had an n=6. Seven groups were treated with
one of the following treatment regimens: a) 100 mg/kg of Fc-IL-1ra
three times per week, b) 3 .mu.g/kg of ARANESP.TM. once every two
weeks c) 6 .mu.g/kg of ARANESP.TM. once every two weeks, d) 100
mg/kg of Fc-IL-1ra three times per week and 3 .mu.g/kg of
ARANESP.TM. once every two weeks, or e) 100 mg/kg of Fc-IL-1ra
three times per week and 6 .mu.g/kg of ARANESP.TM. once every two
weeks f) 4 mg/kg of PEG sTNR-R1 three times per week g) 4 mg/kg of
PEG sTNR-R1 three times per week and 3 .mu.g/kg of ARANESP.TM. once
every two weeks h) 4 mg/kg of PEG sTNR-R1 three times per week and
6 .mu.g/kg of ARANESP.TM. once every two weeks. One group of the
experimental rats was injected with 0.4 mL carrier solution on the
same schedule as the cytokine inhibitors and were housed and bled
the same as all the experimental groups. The treatment period
lasted until day 57.
[0192] Experimental data was obtained blood drawn from tail vein
and analyzed on the ADVIA complete blood count (CBC) analysis
machine (Bayer, Inc) the same day of sampling and according the
manufacture's protocol. Blood for serum was collected into
centrifugal serum separator devices, allowed to clot, spun at 3000
rpm for 5 minutes, serum was pipetted off and put into Eppendorf
tubes, stored in 80 until delivery to LabCorp, Inc. (Research Park,
N.C.). via overnight courier on dry ice. Blood collected for CBC
occurred on days 30, 42, 45, 49, 52, 56, 59, 63, 66, 70, 77. Blood
collected for serum occurred on days 0, 42, 49, 57, 63, 77. In
summary, after therapeutic treatment began, rats were bled twice a
week for CBC analysis and once a week for serum].
[0193] All of these parameters, except iron concentrations, are
part of the data generated by the "complete blood count" (CBC)
analysis which are produced simultaneously by the Advia machine
analysis already described above. Total serum Iron concentrations
(TSI) and transferrin total iron binding capacity were measured by
LabCorp, Inc. (Research Park, N.C.). Unsaturated iron binding
capacity (UIBC) was calculated as follows: TIBC-TSI=UIBC. (See:
"Clinical Biochemistry of Domestic Animals" pages 263-264. Jiro J.
Kaneko, Ed., Academic Press Inc. Harcourt Brace Jovanovich
Publishers). Paw volume was determined as described in Feige U, et
al., Cellular Molecular Life Sciences 57:1457-1470 (2000).
[0194] Rats from the experimental group treated with Fc-IL-1ra
alone trended toward higher mean serum Hb concentrations and
reduced mean paw edema compared to untreated rats from the
experimental group. Administration of 100 mg/kg Fc-IL-1ra increased
mean serum Hb concentrations by 1.8 g/dL over the mean serum Hb
concentrations of untreated rats (p=0.1) (see FIG. 7) and reduced
mean paw volumes by 0.4 ml relative to the mean paw volumes of
untreated rats (p=0.1) (see FIG. 8). Rats from the experimental
group treated with ARANESP.TM. alone at 3 or 6 .mu.g/kg exhibited
elevated mean serum Hb concentrations compared to untreated rats.
Treatment with 3 .mu.g/kg ARANESP.TM. alone increased mean serum Hb
concentrations by 0.4 g/dL over the mean serum Hb concentrations of
untreated rats (p=0.7) (see FIG. 7). Treatment with 6 .mu.g/kg
ARANESP.TM. alone increased mean serum Hb concentrations by 1.3
g/dL over the mean serum Hb concentrations of untreated rats
(p=0.4) (see FIG. 7).
[0195] Treatment of rats from the experimental group with 100 mg/kg
Fc-IL-1ra and 3 .mu.g/kg ARANESP.TM. did not significantly increase
mean serum Hb concentrations compared to treatment by 100 mg/kg
Fc-IL-1ra alone. However, treatment of rats from the experimental
group with 100 mg/kg Fc-IL-1ra and 6 .mu.g/kg ARANESP.TM. more than
doubled the increase in mean serum Hb concentrations compared to
treatment with Fc-IL-1ra or ARANESP.TM. alone. Treatment with 100
mg/kg Fc-IL-1ra and 6 .mu.g/kg ARANESP.TM. increased mean serum Hb
concentrations by 2.2 g/dL more than the increase from treatment
with 100 mg/kg Fc-II-1 ra alone (p=0.04) (see FIG. 7). Treatment
with 100 mg/kg Fc-IL-1ra and 6 .mu.g/kg ARANESP.TM. increased mean
serum Hb concentrations by 2.7 g/dL more than the increase from
treatment with 6 .mu.g/kg ARANESP.TM. alone (p=0.01) (see FIG. 7).
Thus, the total increase rats in mean serum Hb concentrations over
untreated experimental rats resulting from treatment with 100 mg/kg
Fc-IL-1ra and 6 .mu.g/kg ARANESP.TM. was 4.0 g/dL. This is greater
than the sum of the increases in mean serum Hb concentrations over
untreated experimental rats resulting from treatment with 100 mg/kg
Fc-IL-1ra alone (1.8 g/dL) and 6 .mu.g/kg ARANESP.TM. alone (1.3
g/dL).
[0196] Treatment of rats from the experimental group with both 100
mg/kg Fc-IL-1ra and 6 .mu.g/kg ARANESP.TM. increased mean
reticulocyte numbers (p<0.004) compared to the same dose of
either single treatment alone and red blood cell (RBC) numbers
(p<0.03) compared to the same dose of 6 .mu.g/kg ARANESP.TM.
alone (see FIGS. 9 and 10). Treatment of rats from the experimental
group with both 100 mg/kg Fc-IL-1ra and 6 .mu.g/kg ARANESP.TM. also
increased total serum iron concentrations by 44 .mu.g/dL more than
treatment with 6 .mu.g/kg ARANESP.TM. alone (p=0.02) (see FIG. 14).
Treatment with both 100 mg/kg Fc-IL-1ra and 6 .mu.g/kg ARANESP.TM.
also increased mean corpuscular volume to a greater extent than 6
.mu.g/kg ARANESP.TM. alone (p=0.008) or 100 mg/kg Fc-IL-1ra alone
(p=0.04) (see FIG. 11). This treatment also increased mean
corpuscular Hb to a greater extent than 6 .mu.g/kg ARANESP.TM.
alone (p=0.03) or 100 mg/kg Fc-IL-1ra alone (p=0.08) (see FIG.
12).
* * * * *