U.S. patent application number 14/577529 was filed with the patent office on 2015-05-28 for use of il-12 to increase survival.
This patent application is currently assigned to Neumedicines, Inc.. The applicant listed for this patent is Neumedicines, Inc.. Invention is credited to Lena A. Basile.
Application Number | 20150147294 14/577529 |
Document ID | / |
Family ID | 52112445 |
Filed Date | 2015-05-28 |
United States Patent
Application |
20150147294 |
Kind Code |
A1 |
Basile; Lena A. |
May 28, 2015 |
USE OF IL-12 TO INCREASE SURVIVAL
Abstract
The present invention provides methods for increasing survival
in a subject, and/or preserving bone marrow function, and/or
promoting hematopoietic recovery or restoration. The methods
include administering a dose of IL-12 to the subject following an
acute exposure to non-therapeutic whole body ionizing radiation.
Formulations and kits are also provided.
Inventors: |
Basile; Lena A.; (Tujunga,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Neumedicines, Inc. |
Pasadena |
CA |
US |
|
|
Assignee: |
Neumedicines, Inc.
Pasadena
CA
|
Family ID: |
52112445 |
Appl. No.: |
14/577529 |
Filed: |
December 19, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12430016 |
Apr 24, 2009 |
8921315 |
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14577529 |
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61125508 |
Apr 24, 2008 |
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Current U.S.
Class: |
424/85.2 ;
530/351 |
Current CPC
Class: |
A61K 38/18 20130101;
A61K 38/208 20130101; A61K 35/14 20130101; A61P 7/06 20180101; A61K
2300/00 20130101; A61K 45/06 20130101; A61K 38/18 20130101; A61K
2300/00 20130101; C07K 14/5434 20130101; A61K 38/208 20130101; A61K
35/14 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.2 ;
530/351 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61K 45/06 20060101 A61K045/06; C07K 14/54 20060101
C07K014/54 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with US Government support under
contract number BAA-BARDA-08-08 awarded by the Biomedical Advanced
Research and Development Authority, within the Department of Health
and Human Services. The US Government has certain rights in this
invention.
Claims
1-27. (canceled)
28. A kit for increasing survival in a subject, and/or preserving
bone marrow function, and/or promoting hematopoietic recovery or
restoration following non-therapeutic acute exposure to whole body
ionizing radiation, the kit comprising one or more formulations of
a therapeutically effective dose of Interleukin-12 (IL-12).
29. The kit of claim 28, wherein the kit is for increasing survival
in a human subject, wherein the therapeutically effective dose of
IL-12 is between from about 25.66 .mu.g to about 1.71 .mu.g.
30. The kit of claim 28, wherein the kit is for increasing survival
in a human subject, wherein the therapeutically effective dose of
IL-12 is less than about 17.11 .mu.g.
31. The kit of claim 28, wherein the kit is for increasing survival
in a human subject, wherein the therapeutically effective dose of
IL-12 is less than about 13.68 .mu.g.
32. The kit of claim 28, wherein the IL-12 is formulated for
intravenous, intramuscular, intraperitoneal, intracerebrospinal,
subcutaneous, intra-articular, intrasynovial, or intrathecal
administration.
33. The kit of claim 28, wherein the IL-12 is formulated for
injection in solution or as a lyophilized powder.
34. The kit of claim 28, wherein the IL-12 is recombinant
IL-12.
35. The kit of claim 34, wherein the IL-12 is recombinant human
IL-12 (rhIL-12).
36. The kit of claim 38, wherein the rhIL-12 is HemaMax.TM..
37. The kit of claim 28, wherein the IL-12 is modified so as to
reduce the immunogenicity of the protein.
38. The kit of claim 37, wherein the IL-12 protein is modified with
one or more water-soluble polymers.
39. The kit of claim 28, wherein the IL-12 is formulated in a tube,
vial or syringe.
40. The kit of claim 28, wherein the IL-12 is administered with a
carrier molecule or bulking agent.
41. The kit of claim 40, wherein the carrier molecule is an
albumin.
42. The kit of claim 40, wherein the carrier molecule is present at
a concentration of at least about two times the concentration of
IL-12.
43. The kit of claim 28, comprising multiple unit doses of
IL-12.
44. The kit of claim 28, further comprising a compound useful for
supportive care of IL-12 treatment following radiation exposure
selected from the group consisting of antibiotics, hematopoietic
growth factors, G-CSF, IL-3, erythropoietin, erythropoietin-like
molecules, IL-1, IL-4, IL-5, IL-6, IL-7, IL-11, and any combination
thereof.
45. The kit of claim 28, wherein the IL-12 is housed in a radiation
proof or radiation resistant container.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present invention claims priority to U.S. Provisional
Patent Application No. 61/125,508, filed Apr. 24, 2008, the
teachings of which are hereby incorporated by reference in their
entirety for all purposes.
BACKGROUND OF THE INVENTION
[0003] The hematological effects of acute, high dose total body
irradiation (TBI) can lead to death without supportive care, such
as hematopoietic transplant, transfusions and other supportive care
measures. However, such supportive measures cannot be readily
administered to a potentially large number of victims of high dose
radiation exposure following a nuclear accident or direct acts of
terrorism. If such disasters were to occur, military personnel and
civilians alike would be left in great jeopardy. One of the
deleterious effects of high dose radiation is the induction of a
hematopoietic syndrome. To counteract the potentially lethal
effects of hematopoietic syndrome, effective remedial drugs that
can be quickly distributed shortly after the radiation incident are
in great need.
[0004] Currently, there are no available drugs that are effective
in increasing survival and regenerating hematopoiesis. Moreover, in
the face of a radiation-related disaster or act of terrorism, the
immediate distribution of such effective drugs to military
personnel or civilians, if these were to be available, would not be
practically possible. It is anticipated that a lag time of at least
several hours, and perhaps 24 hours or longer, would be necessary
to distribute such drugs to the scene of such a radiation disaster
or act of terrorism. Thus, it is critically important that
effective drugs that can be used to increase survival and
regenerate hematopoiesis exhibit efficacy at protracted time
intervals following acute exposure to ionizing radiation.
[0005] Several studies performed in the mid 80's suggested that
proinflammatory cytokines could confer radioprotection when
administered prior to lethal doses of radiation (Neta R. et al.,
Lymphokine Res. 1986; 5 Suppl 1:S105-10; Neta R. et al., J Immunol.
1986 Apr. 1; 136(7):2483-5; Schwartz G. N. et al., Immunopharmacol
Immunotoxicol. 1987; 9(2-3):371-89). However, it was recognized by
several later studies that the use of IL-12 for prophylactic and
therapeutic treatments of lethal irradiation suffered from
significant drawbacks. One such adverse effect was that IL-12
administered at a high dose of 1000 ng per mouse radiosensitized,
rather than radioprotected, the gastrointestinal tract, resulting
in lethal gastrointestinal syndrome in irradiated mice (Neta R. et
al., J Immunol. 1994 Nov. 1; 153(9):4230-7). This work led to the
conclusion that IL-12 sensitized the intestinal tract at levels
necessary for protection of bone marrow cells (Neta R., Environ
Health Perspect. 1997 Dec.; 105 Suppl 6:1463-5).
[0006] Consistent with these findings, Hixon et al. (Hixon et al.,
Biol Blood Marrow Transplant. 2002; 8(6):316-25) showed that
repeated administration of 500 ng IL-12 to BALBc mice who received
bone marrow transplants following lethal whole body irradiation,
resulted in acute lethal toxicity within 4 to 6 days. In contrast,
Hixon et al. demonstrate that under identical conditions, BALBc
mice that did not receive IL-12 administration recovered 100% of
the time.
[0007] In the event of a nuclear event, whether accidental or
malicious, pre-administration of drugs that promote survival is
simply not possible. For example, in the event of a nuclear
disaster, where large numbers of people and animals may require
therapeutic administration, sufficient time will be required for
the large-scale distribution of radiation treatments. Methods are
needed for treatment that are effective when administered at
protracted times following acute exposure to whole body ionizing
radiation.
[0008] As such, there remains a need in the art for drugs that are
effective in increasing survival and regenerating hematopoiesis
when administrated at protracted times following acute exposure to
whole body ionizing radiation. The present invention satisfies
these and other needs by providing methods of treating a subject
following an acute exposure to non-therapeutic whole body ionizing
radiation via administration of IL-12 at protracted timepoints.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention relates to the use of Interleukin-12
(IL-12) for increasing survival and promoting hematopoietic
recovery following acute exposure to non-therapeutic ionizing
radiation. Use of the methods of the present invention will
increase the number of survivors from a radiation-related disaster,
such as a terrorist attack with a dirty bomb.
[0010] In one aspect, the present invention provides methods for
increasing survival, and/or preserving bone marrow function, and/or
promoting hematopoietic recovery or restoration in a subject
following acute exposure to non-therapeutic, acute whole body
ionizing radiation, comprising the administration of a
therapeutically effective dose of IL-12.
[0011] In a related aspect, the present invention provides
pharmaceutically acceptable formulations of IL-12 for increasing
survival, and/or preserving bone marrow function, and/or promoting
hematopoietic recovery or restoration in a subject following acute
exposure to non-therapeutic whole body ionizing radiation.
[0012] In another aspect, the present invention provides kits
useful for increasing survival, and/or preserving bone marrow
function, and/or promoting hematopoietic recovery or restoration in
a subject following acute exposure to non-therapeutic whole body
ionizing radiation, comprising one or more therapeutically
effective dose of IL-12.
[0013] Further aspects, objects, and advantages of the invention
will become apparent upon consideration of the detailed description
and figures that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 (A-C) shows that IL-12 facilitates multilineage
hematopoietic recovery at various time points of administration and
dosages, and even facilitates significant recovery of all blood
groups at 3 hours post radiation (9 Gy). The blood recovery of
neutrophils (1A), red blood cells (1B) and platelets (1C) are shown
in the graph. The normal threshold count level is depicted for the
different blood cell groups (dotted horizontal line). The depicted
treatment groups are as follows: IL-12 (200 ng; 30 .mu.g/m.sup.2)
administered 3 hrs after radiation (.diamond-solid.), IL-12 (100
ng; 15 .mu.g/m.sup.2) administered 24 hours before radiation ( ),
IL-12 administered 24 hours before and 2 hr after radiation (100 ng
pre/50 ng post; 15 .mu.g/m.sup.2 pre/7.5 .mu.g/m.sup.2 post)
(.box-solid.), and IL-12 administered 24 hours before and 2 hr
after radiation (100 ng pre/100 ng post; 15 .mu.g/m.sup.2 pre/185
.mu.g/m.sup.2 post) (.tangle-solidup.). No control mice remained at
16 days post radiation for blood analysis. Bactrim was removed at
48 days.
[0015] FIG. 2 shows survival effects of IL-12 when administered at
6 hours after a lethal dose of radiation (LD.sub.100/10) and
subcutaneous injection. Female mice C57BL/6 (9 weeks) were used for
this experiment (5-6 mice per group).
[0016] FIG. 3 shows the results of one aspect of the invention as a
Kaplan-Meier plot. Group 1 received vehicle (phosphate buffered
saline (PBS)) and Group 2 received an initial dose of IL-12 (120
ng; 18 .mu.g/m.sup.2) at 6 hours after radiation, followed by one
subsequent dose of IL-12 (100 ng; 15 .mu.g/m.sup.2 at 3 days post
radiation). No antibiotic support was administered. The radiation
dose was 8 Gy (0.9 Gy/min); P=0.001 via Mantel Chi-square analysis.
Mice did not receive antibiotic support.
[0017] FIG. 4 shows remarkable survival effects when administered
at 6 hours after a lethal dose of radiation (LD.sub.100/21) and
subcutaneous injection in male mice. C57BL/6 (14 weeks) were used
for this experiment (5-6 mice per group). Mice were first exposed
to a non-lethal dose of radiation at 7 Gy. At 6 hours after
radiation, mice were either treated with PBS (Group 1) or IL-12
(Group 2 (150 ng; 22.5 .mu.g/m.sup.2). All mice survived the 7 Gy
dose of radiation. On the next round of radiation, all mice
received an 8 Gy radiation dose (0.9 Gy/min). On the second round
of radiation, Group 3 received PBS and Group 2 again received the
same dose of IL-12 (150 ng; 22.5 .mu.g/m.sup.2) that was
administered in the first round of radiation, followed by a
subsequent dose (100 ng; 15 .mu.g/m.sup.2) 48 hrs after the initial
dose at 6 hr post radiation.
[0018] FIG. 5 shows a Kaplan-Meier plot from the second round of
radiation. No antibiotic support was administered; P<0.05 via
Mantel Chi-square analysis.
[0019] FIG. 6 shows survival effects as a result of IL-12
administration at 24 hours after a lethal dose of radiation
(LD.sub.90/27) and subcutaneous injection. Female mice C57BL/6 (9
weeks) were used for this experiment (7-8 mice per group). Group 1
received vehicle (phosphate buffered saline (PBS)) and Group 2
received an initial dose of IL-12 (120 ng; 18 .mu.g/m.sup.2) at 24
hours after radiation, followed by one subsequent dose of IL-12
(100 ng; 15 .mu.g/m.sup.2 at 3 days post radiation). No antibiotic
support was administered. The radiation dose was 8 Gy (0.9 Gy/min);
P=0.001 via Mantel Chi-square analysis.
[0020] FIG. 7 shows a Kaplan-Meier plot at 27 days in the
experimental timeline. No antibiotic support was used. P=0.005 via
Mantel Chi-square analysis.
[0021] FIG. 8 (A-B) shows the results of female C57BL/6 mice (10
per group), which were irradiated with an LD.sub.80 dose of acute
irradiation. The mice were then administered the indicated amounts
of IL-12 as either (A) a double dose at 24 hours and 72 hours post
irradiation, or (B) a single dose at 24 hour post irradiation. The
survival rates for each of the groups are shown.
[0022] FIG. 9 shows the results of female C57BL/6 mice (10 per
group), which were irradiated with an LD.sub.80 dose of acute
irradiation. The mice were then administered the indicated amounts
of IL-12 as either (A) a double dose at 24 hours and 72 hours post
irradiation, or (B) a single dose at 24 hour post irradiation. The
average weight of the surviving mice are plotted as a function of
time in days.
[0023] FIG. 10 illustrates a stratified K-M plot for double dose
experiments performed with female C57BL/6 mice (10 per group).
Briefly, the mice were irradiated with an LD.sub.80 dose of acute
irradiation followed by administration of a double dose of IL-12,
as described in example 6, at 24 and 72 hours post irradiation.
Survival analysis did not indicate a significant overall effect of
IL-12 dose on survival (p<0.59; Tarone-Ware test).
[0024] FIG. 11 illustrates a K-M plot of survival for group 1 (0 ng
IL-12, vehicle control; (.tangle-solidup.)) and group 7 (300 ng
IL-12; 45 .mu.g/m.sup.2; ( )) female C57BL/6 mice irradiated with
an LD.sub.80 dose of acute irradiation followed by administration
of IL-12 at 24 and 72 hours post irradiation. Survival analysis
reveals that administration of 300 ng (45 .mu.g/m.sup.2) IL-12 at
both 24 and 72 hours post irradiation resulted in a significant
increase in survival (p<0.03; Tarone-Ware and Mantel-Cox
tests).
[0025] FIG. 12 illustrates a stratified K-M plot for single dose
experiments performed with female C57BL/6 mice (10 per group).
Briefly, the mice were irradiated with an LD.sub.80 dose of acute
irradiation followed by administration of a single dose of IL-12,
as described in example 6, at 24 hours post irradiation. Survival
analysis indicates a significant overall effect of IL-12 dose on
survival (p<0.02; Tarone-Ware test).
[0026] FIG. 13 illustrates a K-M plot of survival for group 1 (0 ng
IL-12, vehicle control; (.tangle-solidup.)), group 2 (40 ng IL-12;
6 .mu.g/m.sup.2 (.box-solid.)), and group 7 (300 ng IL-12; 45
.mu.g/m.sup.2 ( )) female C57BL/6 mice irradiated with an LD.sub.80
dose of acute irradiation followed by administration of IL-12 at 24
hours post irradiation. Survival analysis reveals that
administration of 40 ng (6 .mu.g/m.sup.2) and 300 ng (45
.mu.g/m.sup.2) IL-12 at 24 hours post irradiation resulted in a
significant increase in survival (p<0.001; Tarone-Ware
test).
[0027] FIG. 14 illustrates a dose response curves for single dose
administration of IL-12 24 hours after irradiation. Female C57BL/6
mice irradiated with an LD.sub.80 dose of acute irradiation
followed by administration of IL-12 at 24 hours post irradiation,
as in example 6. Survival time and percent weight loss are plotted
as a function of administered IL-12 dose.
[0028] FIG. 15 illustrates a survival curve for female C57BL/6 mice
irradiated with various acute doses of ionizing radiation.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0029] "Interleukin-12 (IL-12)" as used herein includes any
recombinant IL-12 molecule that yields at least one of the
properties disclosed herein, including native IL-12 molecules,
variant IL-12 molecules and covalently modified IL-12 molecules,
now known or to be developed in the future, produced in any manner
known in the art now or to be developed in the future. Generally,
the amino acid sequence of the IL-12 molecule used in embodiments
of the invention is the canonical human sequence related to
IL-12p70. IL-12 comprises two subunits, IL-12A (p35) and IL-12 B
(p40) Polymorphisms, however, are known to exist for IL-12,
especially in the p35 subunit. In particular, a known polymorphism
can exist at amino acid 247 of the p35 human subunit, where
methionine is replaced by threonine. Still other embodiments of the
invention include IL-12 molecules where the native amino acid
sequence of IL-12 is altered from the native sequence, but the
IL-12 molecule functions to yield the properties of IL-12 that are
disclosed herein. Alterations from the native, species-specific
amino acid sequence of IL-12 include changes in the primary
sequence of IL-12 and encompass deletions and additions to the
primary amino acid sequence to yield variant IL-12 molecules. An
example of a highly derivatized IL-12 molecule is the redesigned
IL-12 molecule produced by Maxygen, Inc. (Leong S R, et al., Proc
Natl Acad Sci USA. 2003 Feb. 4; 100(3):1163-8.), where the variant
IL-12 molecule is produced by a DNA shuffling method. Also included
are modified IL-12 molecules included in the methods of invention,
such as covalent modifications to the IL-12 molecule that increase
its shelf life, half-life, potency, solubility, delivery, etc.,
additions of polyethylene glycol groups, polypropylene glycol,
etc., in the manner set forth in U.S. Pat. Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, each of
which is hereby incorporated by reference. One type of covalent
modification of the IL-12 molecule is introduced into the molecule
by reacting targeted amino acid residues of the IL-12 polypeptide
with an organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues of the IL-12
polypeptide. Other IL-12 variants included in the present invention
are those where the canonical sequence has been altered to increase
the glycosylation pattern of the resultant IL-12 molecule, as
compared with the native, non-altered IL-12. This method has been
used to generate second generation molecules of erythropoietin,
referred to as Aranesp. Both native sequence IL-12 and amino acid
sequence variants of IL-12 may be covalently modified. Also as
referred to herein, the IL-12 molecule can be produced by various
methods known in the art, including recombinant methods. Since it
is often difficult to predict in advance the characteristics of a
variant IL-12 polypeptide, it will be appreciated that some
screening of the recovered variant will be needed to select the
optimal variant. A preferred method of assessing a change in the
properties of variant IL-12 molecules is via the lethal irradiation
rescue protocol disclosed below. Other potential modifications of
protein or polypeptide properties such as redox or thermal
stability, hydrophobicity, susceptibility to proteolytic
degradation, or the tendency to aggregate with carriers or into
multimers are assayed by methods well known in the art.
[0030] "Acute Radiation Syndrome" in humans as used herein includes
an acute radiation exposure of 2 Gy or greater.
[0031] "Hematopoietic Syndrome" as used herein includes damage to
the bone marrow compartment which results in pancytopenia, i.e., a
deficiency in peripheral blood cell counts for all blood cell
types, namely white blood cells, red blood cells and platelets.
Hematopoietic Syndrome also refers to loss of hematopoietic
progenitor and stem cells in the bone marrow compartment.
[0032] "Survival" as used herein includes an increase in survival
that can be measured in non-human species as compared to control
groups, such as mice or non-human primates.
[0033] "Hematopoietic Recovery" as used herein includes early
recovery of peripheral blood cell counts for white blood cells, red
blood cells and platelets, as compared to control groups and as
measured in non-human species, such as mice or non-human
primates.
[0034] "Preservation of bone marrow function" as used herein
includes early recovery of cellularity or colony forming units in
the bone marrow compartment, or any other measure of bone marrow
function, as compared to control groups and as measured in
non-human species, such as mice and non-human primates.
[0035] A "therapeutically effective amount or dose" or "sufficient
amount or dose" as used herein includes a dose that produces
effects for which it is administered, for example, a dose
sufficient for increasing survival, and/or preserving bone marrow
function, and/or promoting hematopoietic recovery or restoration in
a subject that has been exposed to an acute dose of whole body
ionizing radiation. The exact dose will depend on the purpose of
the treatment, the timing of the IL-12 administration, certain
characteristics of the subject to be treated, the total amount or
timing of irradiation, and will be ascertainable by one skilled in
the art using known techniques (see, e.g., Lieberman,
Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art,
Science and Technology of Pharmaceutical Compounding (1999);
Pickar, Dosage Calculations (1999); and Remington: The Science and
Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott,
Williams & Wilkins).
[0036] Generally, a dose of a therapeutic agent, according to the
methods and compositions of the present invention, can be expressed
in terms of the total amount of drug to be administered (i.e., ng,
.mu.g, or mg). Preferably, the dose can be expressed as a ratio of
drug to be administered to weight or surface area of subject
receiving the administration (i.e., ng/kg, .mu.g/kg, ng/m.sup.2, or
.mu.g/m.sup.2). When referring to a dose in terms of the mass to be
administered per mass of subject (i.e., ng/kg), it will be
understood that doses are not equivalent between different animals,
and thus conversion factors will need to be used to ensure that one
animal receives the same dose equivalent as another animal.
Suitable factors for the conversion of a mouse "dose equivalent" to
a "dose equivalent" of a different animal are given in the look-up
table below. Thus, in a preferred embodiment, doses are given in
terms of mass to surface area (i.e., ng/m.sup.2, or .mu.g/m.sup.2),
which are equivalent for all animals. The following basic
conversion factors can be used to convert ng/kg to ng/m.sup.2:
mouse=3.0, hamster=4.1, rat=6.0, guinea pig=7.7, human=38.0 (Cancer
Chemother Repts 50(4):219(1966)).
TABLE-US-00001 TABLE 1 Conversion factors and equivalent doses for
several animals. Weight Total Dose Dose Dose Conversion Species
(kg) (ng) (ng/kg) (ng/m.sup.2) Factor Human 65 25655.82 394.7 15000
0.0794 Mouse 0.02 99.47 4973.44 15000 1.0000 Hamster 0.03 130.2
4339.87 15000 0.8726 Rat 0.15 381.12 2540.8 15000 0.5109 Guinea Pig
1.00 1335 1335 15000 0.2684 Rabbit 2 2381.1 1190.55 15000 0.2394
Cat 2.5 2956.44 1182.57 15000 0.2378 Monkey 3 3681.75 1227.25 15000
0.2468 Dog 8 6720 840 15000 0.1689
[0037] As used herein, the term "intermediate dose" includes doses
between a range of about 15 .mu.g/m.sup.2 and about 75
.mu.g/m.sup.2, or between a range of about 20 .mu.g/m.sup.2 and
about 75 j.mu.g/m.sup.2, between a range of about 22.5
.mu.g/m.sup.2 and about 67.5 .mu.g/m.sup.2, between a range of
about 22.5 .mu.g/m.sup.2 and about 52.5 .mu.g/m.sup.2, between a
range of about 30 .mu.g/m.sup.2 and about 60 .mu.g/m.sup.2, between
a range of about 37.5 .mu.g/m.sup.2 and about 52.5 .mu.g/m.sup.2,
and all doses and ranges in-between.
[0038] As used herein, the term "low dose" includes doses less than
about 15 .mu.g/m.sup.2, or less than about 14 .mu.g/m.sup.2, or
less than about 13 .mu.g/m.sup.2, 12 .mu.g/m.sup.2, 11
.mu.g/m.sup.2, 10 .mu.g/m.sup.2, 9 .mu.g/m.sup.2, 8 .mu.g/m.sup.2,
7 .mu.g/m.sup.2, 6 .mu.g/m.sup.2, 5 .mu.g/m.sup.2, 4 .mu.g/m.sup.2,
3 .mu.g/m.sup.2, 2 .mu.g/m.sup.2, 1 .mu.g/m.sup.2, or less than
about 900 ng/m.sup.2, 800 ng/m.sup.2, 700 ng/m.sup.2, 600
ng/m.sup.2, 500 ng/m.sup.2, 400 ng/m.sup.2, 300 ng/m.sup.2, 200
ng/m.sup.2, or 100 ng/m.sup.2. In certain embodiments, a low dose
includes an ultralow dose.
[0039] As used herein, the term "ultralow dose" includes doses less
than about 3 .mu.g/m.sup.2, 2 .mu.g/m.sup.2, 1 .mu.g/m.sup.2, or
less than about 900 ng/m.sup.2, 800 ng/m.sup.2, 700 ng/m.sup.2, 600
ng/m.sup.2, 500 ng/m.sup.2, 400 ng/m.sup.2, 300 ng/m.sup.2, 200
ng/m.sup.2, or 100 ng/m.sup.2.
II. Embodiments
[0040] In one aspect, the present invention is based on the
surprising discovery that Interleukin-12 (IL-12) increases the
survival of subjects exposed to lethal and sub-lethal acute doses
of non-therapeutic whole body ionizing radiation. Significantly, it
was found that administration of IL-12 following acute doses of
ionizing radiation resulted in the regeneration of hematopoietic
activity and peripheral blood cell counts in lethally irradiated
mice, yielding complete hematopoietic recovery without the use of
transplanted cells.
[0041] Advantageously, it was found that IL-12 has the unique and
remarkable property of being able to confer survival on lethally
irradiated mammals when administered at protracted time points
after radiation. Specifically, it was found that IL-12 can rescue
about 90-100% of mammals when administered at 6 hours or 24 hours
after radiation.
[0042] In one aspect, a single dose of IL-12 is sufficient to
confer significant survival, bone marrow preservation, and
promotion of hematopoietic recovery. In other aspects, IL-12 may be
administered in more than one dose such as 2, 3, 4, 5 or more
doses.
[0043] Accordingly, in one aspect, the present invention provides a
method for increasing survival in a subject, and/or preserving bone
marrow function, and/or promoting hematopoietic recovery or
restoration comprising the administration of a dose of IL-12 to the
subject following an acute exposure to non-therapeutic whole body
ionizing radiation. In one embodiment, the dose of IL-12 is less
than about 100 .mu.g/m.sup.2. In one embodiment, the dose of IL-12
is less than about 75 .mu.g/m.sup.2. In another embodiment, the low
dose can be between about 1 .mu.g/m.sup.2 and about 100
.mu.g/m.sup.2, such as 1 .mu.g/m.sup.2, 5 .mu.g/m.sup.2, 10
.mu.g/m.sup.2, 15 .mu.g/m.sup.2, 20 .mu.g/m.sup.2, 25
.mu.g/m.sup.2, 30 .mu.g/m.sup.2, 35 .mu.g/m.sup.2, 40
.mu.g/m.sup.2, 45 .mu.g/m.sup.2, 50 .mu.g/m.sup.2, 55
.mu.g/m.sup.2, 60 .mu.g/m.sup.2, 65 .mu.g/m.sup.2, 70
.mu.g/m.sup.2, 75 .mu.g/m.sup.2, 80 .mu.g/m.sup.2, 85
.mu.g/m.sup.2, 90 .mu.g/m.sup.2, 95 .mu.g/m.sup.2 and 100
.mu.g/m.sup.2 and all doses in-between. In a particular embodiment,
the preferred dose of IL-12 is less than about 75 .mu.g/m.sup.2. In
one embodiment, the dose of IL-12 is either between a range from
about 100 .mu.g/m.sup.2 to about 18 .mu.g/m.sup.2 or between a
range from about 15 .mu.g/m.sup.2 to about 1 .mu.g/m.sup.2.
[0044] In certain embodiments, the IL-12 is a mammalian IL-12,
recombinant mammalian IL-12, murine IL-12 (mIL-12), recombinant
murine IL-12 (rmIL-12), human IL-12 (hIL-12), recombinant human
IL-12 (rhIL-12), canine IL-12 or rIL-12, feline IL-12 or rIL-12,
bovine IL-12 or rIL-12, equine IL-12 or rIL-12, or biologically
active variants or fragments thereof. In one specific embodiment,
the rhIL-12 is HemaMax.TM. (Neumedicines Inc.). In certain
embodiments, the IL-12 can be modified in a fashion so as to reduce
the immunogenicity of the protein after administration to a
subject. Methods of reducing the immunogenicity of a protein are
well known in the art and include, for example, modifying the
protein with one or water soluble polymers, such as a PEG, a PEO, a
carbohydrate, a polysialic acid, and the like.
[0045] In another aspect, the present invention provides methods
for increasing survival in a subject, and/or preserving bone marrow
function, and/or promoting hematopoietic recovery or restoration
comprising the administration of an intermediate dose of IL-12 to
the subject following an acute exposure to non-therapeutic whole
body ionizing radiation. In one embodiment, the intermediate dose
of IL-12 is between a range of about 15 .mu.g/m.sup.2 and about 100
.mu.g/m.sup.2, or between a range of about 20 .mu.g/m.sup.2 and
about 75 .mu.g/m.sup.2, between a range of about 22.5 .mu.g/m.sup.2
and about 67.5 .mu.g/m.sup.2, between a range of about 22.5
.mu.g/m.sup.2 and about 52.5 .mu.g/m.sup.2, between a range of
about 30 .mu.g/m.sup.2 and about 60 .mu.g/m.sup.2, between a range
of about 37.5 .mu.g/m.sup.2 and about 52.5 .mu.g/m.sup.2, and all
doses and ranges in-between. In a particular embodiment, the
preferred intermediate dose of IL-12 is between a range of about
22.5 .mu.g/m.sup.2 and about 52.5 .mu.g/m.sup.2.
[0046] In another aspect, the present invention provides methods
for increasing survival in a subject, and/or preserving bone marrow
function, and/or promoting hematopoietic recovery or restoration
comprising the administration of a low dose of IL-12 to the subject
following an acute exposure to non-therapeutic whole body ionizing
radiation. In one embodiment, the low dose of IL-12 is less than
about 15 .mu.g/m.sup.2. In another embodiment, the low dose can be
between about 3 .mu.g/m.sup.2 and about 12 .mu.g/m.sup.2, such as 3
.mu.g/m.sup.2, 4 .mu.g/m.sup.2, 5 .mu.g/m.sup.2, 6 .mu.g/m.sup.2, 7
.mu.g/m.sup.2, 8 .mu.g/m.sup.2, 9 .mu.g/m.sup.2, 10 .mu.g/m.sup.2,
11 .mu.g/m.sup.2, and 12 .mu.g/m.sup.2 and all doses in-between. In
a particular embodiment, the preferred low dose of IL-12 is about 6
.mu.g/m.sup.2.
[0047] In one particular embodiment, a method is provided for
increasing survival, and/or preserving bone marrow function, and/or
promoting hematopoietic recovery or restoration in a human
comprising the administration of a low dose of IL-12 to said human
following an acute exposure to non-therapeutic whole body ionizing
radiation. In a specific embodiment, the method for treating a
human comprises administering a low dose of rhIL-12 in a range
between about 1 hour and about 24 hours after acute exposure to
whole body ionizing radiation, wherein the low dose is less than
about 400 ng/kg (15 .mu.g/m.sup.2).
[0048] In another aspect, the present invention provides methods
for increasing survival in a subject, and/or preserving bone marrow
function, and/or promoting hematopoietic recovery or restoration
comprising the administration of an ultralow dose of IL-12 to the
subject following an acute exposure to non-therapeutic whole body
ionizing radiation. In one embodiment, an ultralow dose of IL-12
used, such as a dose of less than about 3 .mu.g/m.sup.2. In another
embodiment, the ultralow dose may be between about 300 ng/m.sup.2
and about 2400 ng/m.sup.2, or between about 600 ng/m.sup.2 and
about 1200 ng/m.sup.2. In a particular embodiment, the low dose of
IL-12 is about 900 ng/m.sup.2.
[0049] In still yet another aspect, the present invention provides
a method for increasing survival, and/or preserving bone marrow
function, and/or promoting hematopoietic recovery or restoration in
a human comprising the administration of an ultralow dose of IL-12
to the subject following an acute exposure to non-therapeutic whole
body ionizing radiation. In a specific embodiment, the method for
treating a human comprises administering a low dose of rhIL-12 in a
range between about 1 hour and about 24 hours after acute exposure
to whole body ionizing radiation, wherein the low dose is less than
about 80 ng/kg (3 .mu.g/m.sup.2).
[0050] In certain embodiments of the invention, a low or ultralow
dose of IL-12 suitable for administration may be less than about 14
.mu.g/m.sup.2, or less than about 13 .mu.g/m.sup.2, 12
.mu.g/m.sup.2, 11 .mu.g/m.sup.2, 10 .mu.g/m.sup.2, 9 .mu.g/m.sup.2,
8 .mu.g/m.sup.2, 7 .mu.g/m.sup.2, 6 .mu.g/m.sup.2, 5 .mu.g/m.sup.2,
4 .mu.g/m.sup.2, 3 .mu.g/m.sup.2, 2 .mu.g/m.sup.2, 1 .mu.g/m.sup.2,
or less than about 900 ng/m.sup.2, 800 ng/m.sup.2, 700 ng/m.sup.2,
600 ng/m.sup.2, 500 ng/m.sup.2, 400 ng/m.sup.2, 300 ng/m.sup.2, 200
ng/m.sup.2, or 100 ng/m.sup.2.
[0051] It is well known that solutions of proteins that are
formulated at low concentrations are susceptible to loss of a
significant fraction of the protein prior to administration. One
major cause of this problem is adsorption of the protein on the
sides of tubes, vials, syringes, and the like. Accordingly, in
certain aspects, when administered at low or ultralow doses, it
will be beneficial to administer IL-12 along with a suitable
carrier molecule or bulking agent. In one embodiment, the carrier
agent may be a protein suitable for pharmaceutical administration,
such as albumin. Generally, the carrier molecule or protein will be
present in the formulation in excess of IL-12 in order to minimize
the amount of IL-12 lost prior to administration. In certain
embodiments, the carrier will be present at a concentration of at
least about 2 times the concentration of IL-12, or at a
concentration of at least about 3, 4, 5, 6, 7, 8, 9, 10, 25, 50,
100, or more times the concentration of IL-12 in the
formulation.
[0052] Generally the IL-12 doses used in the methods for increasing
survival in a subject, and/or preserving bone marrow function,
and/or promoting hematopoietic recovery or restoration in a subject
following an acute exposure to non-therapeutic whole body ionizing
radiation will be high enough to be effective for the treatment of
a radiation syndrome, but low enough to mitigate negative side
effects associated with IL-12 administrations, including for
example, radiosensitivity of the GI tract and INF-.gamma.
up-regulation.
[0053] Advantageously, as provided by the methods of the present
invention, administration of IL-12 may occur during any suitable
time period following exposure to acute whole body radiation, up to
and including about a week after exposure. Although the total dose
of acute radiation will factor into the time period in which IL-12
should be administered, according to one embodiment, IL-12 may be
administered at any time up to about 96 hours following exposure to
radiation. In other embodiments, IL-12 can be administered at any
time up to about 72 hours post-irradiation, or at a time up to
about 60 hours, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8
hours, 6 hours, or less following exposure to radiation.
[0054] In one specific embodiment, IL-12 is administered to a
subject in need thereof between a range of about 1 hour to about 72
hours after exposure to ionizing radiation. In another embodiment,
IL-12 is administered between a range of about 1 hour and about 24
hours after exposure, or between a range of about 6 hours and about
24 hours following exposure to an acute dose of whole body ionizing
radiation. Of great importance for the usefulness of IL-12 in the
face of a radiation disaster is the fact that IL-12 can be
administered at protracted time points following acute exposure to
ionizing radiation. IL-12 is effective at any time point post
radiation up to one week, but can provide especially high
effectiveness at time points up to 96 hours post radiation.
[0055] In certain other aspects, IL-12 can be administered at any
reasonable time point post radiation event, and be effective in
increasing survival, and/or preserving bone marrow function, and/or
promoting hematopoietic recovery. IL-12 administered at time points
of up to 3 hours, 6 hours, 24 hours, 48 hours and 72 hours are
efficacious in increasing survival, preserving bone marrow
function, and promoting hematopoietic recovery. However, on one
aspect, IL-12 acts on a residual subpopulation of hematopoietic
stem cells, and thus it is expected to be effective at 96 hours,
120 hours and up to one week following acute exposure to ionizing
radiation.
[0056] In another aspect of the invention, methods are provided for
increasing survival in a subject, and/or preserving bone marrow
function, and/or promoting hematopoietic recovery or restoration
comprising the administration of more than one dose of IL-12 to a
subject at protracted times following acute exposure to
non-therapeutic whole body ionizing radiation. For example, in one
embodiment, the method comprises the administration of a first
therapeutically effective dose of IL-12 at a time up to about 24
hours post-irradiation and a second therapeutically effective dose
of IL-12 at a second time up to about 72 hours after said first
dose. In a particular embodiment, the first IL-12 dose can be
administered between a range of about 1 hour and about 24 hours
post-irradiation, or between a range of about 6 hours and about 24
hours post-irradiation, and said second IL-12 dose between a range
of about 48 hours and about 1 week post-irradiation, or between a
range of about 48 hours and about 94 hours post irradiation.
[0057] In one specific embodiment, the methods herein comprise
administering a first dose of less than about 75 .mu.g/m.sup.2
IL-12 to a subject at a time between about 1 hour and about 24
hours after acute exposure to whole body radiation. In other
embodiments, the method comprises administration of less than about
60 .mu.g/m.sup.2 IL-12, or less than about 45 .mu.g/m.sup.2 IL-12,
30 .mu.g/m.sup.2 IL-12, 15 .mu.g/m.sup.2 IL-12, or less IL-12 at an
effective time after acute exposure to radiation.
[0058] In other embodiments, methods of multiple-dose IL-12
administration can comprise the administration of a first low or
ultralow dose of IL-12, for example, a first dose of less than
about 15 .mu.g/m.sup.2, or less than about 14 .mu.g/m.sup.2, or
less than about 13 .mu.g/m.sup.2, 12 .mu.g/m.sup.2, 11
.mu.g/m.sup.2, 10 .mu.g/m.sup.2, 9 .mu.g/m.sup.2, 8 .mu.g/m.sup.2,
7 .mu.g/m.sup.2, 6 .mu.g/m.sup.2, 5 .mu.g/m.sup.2, 4 .mu.g/m.sup.2,
3 .mu.g/m.sup.2, 2 .mu.g/m.sup.2, 1 .mu.g/m.sup.2, or less than
about 900 ng/m.sup.2, 800 ng/m.sup.2, 700 ng/m.sup.2, 600
ng/m.sup.2, 500 ng/m.sup.2, 400 ng/m.sup.2, 300 ng/m.sup.2, 200
ng/m.sup.2, or 100 ng/m.sup.2.
[0059] When administered in multiple doses, i.e. two, three, four,
or more, the first IL-12 dose and subsequent IL-12 dose(s) can be
equivalent doses, or they can be different dose amounts. For
example, in certain embodiments, subsequent dose(s) can be
administered at about 90% of the initial dose, or at about 80%,
75%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 10% or less of the original
dose.
[0060] In one particular embodiment, a method is provided for
increasing survival in a human, and/or preserving bone marrow
function, and/or promoting hematopoictic recovery or restoration,
said method comprising the steps of administering a first dose of
IL-12 to said human within about 24 hours following an acute
exposure to non-therapeutic whole body ionizing radiation, wherein
said first dose is less than about 100 .mu.g/m.sup.2 and
subsequently administering a second dose of IL-12 to said human
within about 96 hours following said acute exposure to
non-therapeutic whole body ionizing radiation, wherein said second
dose is less than about 100 .mu.g/m.sup.2. In a specific
embodiment, said second dose is administered at least about 24
hours after said first dose is administered. In another specific
embodiment, said second dose is less than said first dose.
[0061] In yet other embodiments, methods are provided for
increasing survival in a subject, and/or preserving bone marrow
function, and/or promoting hematopoietic recovery or restoration
following an acute exposure to non-therapeutic whole body ionizing
radiation, comprising repeated administration of IL-12 for at least
about a week, or at least about 2 weeks, or at least about 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, or more weeks. In certain embodiments, the
doses may be administered about once every 12 hours, or about once
every 24 hours, or about 1, 2, 3, 4, 5, 6, or seven times a week.
In other embodiments, IL-12 may be administered about every other
week or about 1, 2, 3, 4, 5, or more times a month. In some
embodiments, each IL-12 doses may be less than about 100
.mu.g/m.sup.2, or alternatively be an intermediate dose, a low
dose, or an ultra low dose. In yet other embodiments, the doses may
be decreased during the course of a repeated administration.
[0062] Without being held to any particular theory, it is believed
that when the hematopoietic system is compromised, as with acute
whole body radiation, the IL-12 mediated pathway leading to the
production of INF-.gamma. may be sensitized. Consistent with this,
Hixon et al. observed that when IL-12 is administered to mice
following whole body irradiation and bone marrow transplant,
INF-.gamma. levels are greatly increased and acute lethal toxicity
of the gut results from the elevated levels of INF-.gamma.. The
observed acute lethal toxicity of the gut was dependant on
INF-.gamma., as INF-.gamma. knockout mice were resistant to IL-12
mediated toxicity. The experiments performed by Hixon et al.
support a model in which IL-12 administration results in an
increase of INF-.gamma. levels, and therefore an increase in Crg-2
and Mig cytokine levels, resulting in anti-angiogenesis effects.
Alternatively, but not mutually exclusive with the above, by
performing bone marrow transplants prior to IL-12 administration,
Hixon et al. provided INF-.gamma. producing lymphocytes to the
mice, further increasing the potential for INF-.gamma.
production.
[0063] Thus, a possible mechanism for the decreased hematopoietic
side effects associated with certain embodiments of the invention
is that when a relatively low dose IL-12 is given to a mammal whose
hematopoietic system is compromised, the dose is insufficient to
upregulate INF-.gamma. production. Since INF-.gamma. inhibits
hematopoiesis and also appears to be the major cytokine responsible
for toxicity, the absence of INF-.gamma. upregulation upon
administration of low and ultralow doses of IL-12, as used in the
methods of the present invention, may be one of the factors
underlying the discovery by the inventors that administration of
low and ultralow doses of IL-12 provides a hematopoietic protective
and recovery effect without apparent toxicity.
[0064] Also of significance is the demonstration that exogenous
administration of IL-12 can expand long-term repopulating (LTR)
hematopoietic stem cells (HSC) in vivo. Thus, without being bound
by theory, HSC expansion by exogenous IL-12 can be the mechanism
responsible for survival from hematopoietic injury resulting from
lethal radiation exposure at later time points, e.g., 24 hours
post-irradiation. Another potential mechanism relates to the
ability of IL-12 to induce DNA repair and reduce apoptosis in
hematopoietic stem cells (HSC) following radiation exposure.
[0065] While most bone marrow progenitor and stem cells are
susceptible to cell death after high dose radiation, subpopulations
of HSC or accessory cells are selectively more radioresistant,
presumably because these cells exist in a largely noncycling
(G.sub.0) state. In humans, these radioresistant cells can play an
important role in recovery of hematopoiesis after exposure to doses
as high as 6 Gy, albeit with a reduced capacity for self-renewal in
the absence of exogenous IL-12.
[0066] Another determinant for hematopoietic reconstitution is
non-homogeneity of the radiation dose, which can spare some marrow
sites that then become the foci of hematopoietic activity. In
either case, i.e., either the residual presence of radioresistant
HSC or inhomogeneity of the radiation dose, the present findings
indicate that a subpopulation of HSC marked by the presence of the
IL-12 receptor (IL-12R+) survives and persists after high dose
radiation, and moreover, that this IL-12R+HSC subpopulation is
activated, expanded, and/or induced to repair itself upon exogenous
administration of IL-12.
[0067] Following radiation exposure, it has been discovered that
IL-12 is effective in mitigating the hematopoietic syndrome
associated with acute radiation syndrome. Specifically, embodiments
of the present invention provide methods for increasing survival,
and/or preserving bone marrow function, and/or promoting
hematopoietic recovery by administering one or more effective
dose(s) of IL-12 to a subject following acute exposure to ionizing
radiation.
[0068] For humans, as shown in Table 2, the early signs of
hematopoietic syndrome start to occur in the range of radiation
doses of 2 Gy or greater. Similarly, at radiation doses between
about 5.5-7.5 Gy, pancytopenia and moderate GI damage occurs in
humans. Advantageously, when administered according to the methods
of the present invention, IL-12 is effective in alleviating the
pancytopenia at these radiation dose levels, preserving bone marrow
function and will not induce further GI damage. However, the
radiation dose rate can also affect the relative level of radiation
injury. Thus, two radiation doses given at two different dose rates
can show differences in the severity of the relative radiation
injury.
TABLE-US-00002 TABLE 2 Phases of Radiation Injury* Dose
Manifestation of Prognosis Range Gy Prodrome Illness (without
Therapy) 0.5-1.0 Mild Slight decrease in Almost certain blood cell
counts survival 1.0-2.0 Mild to Early signs of bone Highly probable
moderate marrow damage survival (>90% of victims) 2.0-3.5
Moderate Moderate to severe Probable bone marrow damage survival
3.5-5.5 Severe Severe bone marrow Death within damage, slight GI
3.5-6 wk (50% damage of victims) 5.5-7.5 Severe Pancytopenia and
Death probable moderate GI damage within 2-3 wk 7.5-10.0 Severe
Marked GI and bone Death probable marrow damage, hypo- within 1-2.5
wk tension 10.0-20.0 Severe Severe GI damage, Death certain
pneuomonitis, altered within 5-12 d mental status, cogni- tive
dysfunction 20.0-30.0 Severe Cerebrovascular col- Death certain
lapse, fever, shock within 2-5 d *Modified from Walker R I, Cerveny
R J, eds. (21), GI = gastrointestinal
[0069] For other mammals embraced by the methods and compositions
of the present invention, for example mice, rats, guinea pigs,
hamsters, cats, dogs, cattle, horses, sheep, pigs, rabbits, deer,
monkeys, and the like, the radiation dose that can induce
hematopoietic syndrome varies with the species and strain. For
example, for rhesus monkeys, the LD.sub.50 is about 7 Gy. For
certain strains of mice, the LD.sub.50 is also about 7 Gy, for
example Balb-c mice. For other strains of mice, such as C57BL6, the
LD.sub.50 is about 7.5 Gy. The LD.sub.50 can also exhibit
differences based on gender or general health status of the
animal.
[0070] Although it would be difficult to determine the exact extent
of radiation injury in a mammal exposed to acute ionizing radiation
following a radiation-related disaster, IL-12, when used in
accordance with embodiments of the present invention, will increase
survival, and/or preserve bone marrow function, and/or promote
hematopoictic recovery of peripheral blood cell counts.
[0071] Accordingly, in some embodiments of the present invention,
IL-12 is administered to a subject that has been exposed to an
acute dose of ionizing radiation of at least about 1.0 Gy, or an
amount equivalent to an LD.sub.10 in humans. In another embodiment,
IL-12 is administered to a subject that has been exposed to about
3.5 Gy of ionizing radiation, or a dose equivalent to about
LD.sub.50 in humans. In yet other embodiments, IL-12 is useful for
increasing survival, and/or preserving bone marrow function, and/or
promoting hematopoietic recovery of peripheral blood cell counts in
a subject exposed to at least about 2.0 Gy, or at least about 3.0
Gy, 4.0 Gy, 5.0 Gy, 6.0 Gy, 7.0 Gy, 8.0 Gy, 9.0 Gy, 10.0 Gy, 11.0
Gy, 12.0 Gy, 13.0 Gy, 14.0 Gy, 15.0 Gy, 20.0 Gy, 25.0 Gy, 30.0 Gy,
or higher doses of acute ionizing radiation. Similarly, the dose of
ionizing radiation can be expressed in terms of the percent lethal
dose, for example, a dose equivalent of about LD.sub.1, LD.sub.5,
LD.sub.10, LD.sub.20, LD.sub.30, LD.sub.40, LD.sub.50, LD.sub.60,
LD.sub.70, LD.sub.80, LD.sub.90, LD.sub.95, LD.sub.99, or
LD.sub.100.
[0072] A. Supportive Care
[0073] In another aspect of the invention, methods are provided for
increasing survival in a subject, and/or preserving bone marrow
function, and/or promoting hematopoietic recovery or restoration
comprising the administration of one or more dose of IL-12 to a
subject at protracted times following acute exposure to
non-therapeutic whole body ionizing radiation, wherein supportive
care is given to said subject simultaneously or following
administration of IL-12.
[0074] Supportive care modalities useful in conjunction with IL-12
for treatment of a subject who has been exposed to an acute dose of
whole body ionizing radiation include, without limitation,
administration of fluids, one or more antibiotic, blood or blood
component transfusions, administration of one or more growth
factors or hematopoietic growth factors, combination therapies and
the like.
[0075] In one embodiment, supportive care comprises the
administration of one or more antibiotics. Antibiotic support can
be any antibiotic that is useful in preventing infections during
periods of low blood cell counts including, without limitation,
bactrim, ciprofloxacin, moxifloxacin, and the like. Those of skill
in the art will know of other antibiotics useful for supportive
care.
[0076] In another embodiment, supportive care comprises
administration of one or more growth factors, including
hematopoietic growth factors. Many suitable hematopoietic growth
factors are known in the art including, without limitation, colony
stimulating factors (CSF, G-CSF, GM-CSF, M-CSF, IL-3),
erythropoietin, IL-1, IL-4, IL-5, IL-6, IL-7, IL-11, and the like.
Several FDA-approved hematopoietic growth factors are currently
available, and thus may be used in the methods provided herein,
such as G-CSF (Neupogen or Neulasta), IL-11, and erythropoietin
(Epogen, Procrit or Aranesp). In some embodiments, supportive care
comprises the administration of keratinocyte growth factor (KGF or
FGF7).
[0077] In one particular embodiment, erythropoietin administration
can increase survival up to about 50% over and above that of IL-12
alone when super-lethal doses are used with no other supportive
care measures, such as antibiotic support or fluid administration.
Super-lethal doses are defined herein as radiation doses at or
above 5.5 Gy. Erythropoietin is available as a FDA-approved
recombinant protein drug for human use, such as Epogen, Procrit or
Aranesp. Generally, dosing with these erythropoietin drugs will be
simultaneous with or following the administration of IL-12.
Erythropoietin drugs can be repeated as needed, but generally not
administered more than every other day, or every third day.
Preferably erythropoietin is administered about 48 hours after the
last dose of IL-12.
[0078] An effective dose of erythropoietin for a human can be about
20 mg/kg, however, lower doses and higher doses are also effective
in increasing survival when use as an adjuvant to IL-12
administration. Accordingly, in certain embodiments, erythropoietin
is administered at about 1 mg/kg, or at about 2 mg/kg, 3 mg/kg, 4
mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg,
40 mg/kg, 50 mg/kg, or more for treatment in a human, or at a dose
equivalent amount for treatment in an animal other than a
human.
[0079] In another embodiment, supportive care comprises the
administration of a blood transfusion. As used herein, a blood
transfusion may encompass a whole blood transfusion, or
alternatively, transfusion of a blood fraction or blood component,
for example, a red blood cell transfusion, a platelet transfusion,
a white blood cell transfusion. In a related embodiment, supportive
care may comprise the administration of a bone marrow or bone
marrow stem cell transplant.
[0080] B. Formulations
[0081] IL-12 composition provided herein and used according to the
methods of the invention can be formulated for administration via
any known method, such as intravenous administration, e.g., as a
bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, topical, or
inhalation routes. Further, an efficacious dose of IL-12 may differ
with different routes of administration.
[0082] In certain embodiment IL-12 is administered following
radiation exposure by intramuscular (IM) or subcutaneous (SC)
routes. Advantageously, these modes of administration can be
self-administered or administered by a person with little or no
medical training. As such, this aspect of the invention allows for
rapid responses under disaster conditions where attention by those
medical staff may be limited. Other modes of administration,
however, such as intravenous and intraperitoneal, are also
compatible with the present invention.
[0083] The IL-12 compositions can be administered in a variety of
unit dosage forms depending upon the method of administration. For
example, unit dosage forms suitable for oral administration
include, but are not limited to, powder, tablets, pills, capsules
and lozenges. It is recognized that constructs when administered
orally, should be protected from digestion. This is typically
accomplished either by complexing the molecules with a composition
to render them resistant to acidic and enzymatic hydrolysis, or by
packaging the molecules in an appropriately resistant carrier, such
as a liposome or a protection barrier. Means of protecting agents
from digestion are well known in the art.
[0084] In some embodiments, the formulations provided herein
further comprise one or more pharmaceutically acceptable
excipients, carriers, and/or diluents. In addition, the
formulations provided herein may further comprise other medicinal
agents, carriers, adjuvants, diluents, tissue permeation enhancers,
solubilizers, and the like. Methods for preparing compositions and
formulations for pharmaceutical administration are known to those
skilled in the art (see, for example, REMINGTON'S PHARDMACEUTICAL
SCIENCES, 18TH ED., Mack Publishing Co., Easton, Pa. (1990)).
Formulations used according to the methods of the invention may
include, for example, those taught in U.S. Pat. No. 5,744,132,
which is hereby incorporated by reference in its entirety for all
purposes.
[0085] C. Kits
[0086] In another aspect of the invention, kits are provided for
increasing survival in a subject, and/or preserving bone marrow
function, and/or promoting hematopoietic recovery or restoration
following non-therapeutic acute exposure to whole body ionizing
radiation. In one embodiment, a kit of the invention comprises one
or more formulations of a therapeutically effective amount of
IL-12. In certain embodiments, the therapeutically effective amount
of IL-12 comprises a low dose of IL-12, for example, a dose that is
less than about 15 .mu.g/m.sup.2. In other embodiments, the
therapeutically effective amount of IL-12 may comprise an ultralow
dose of IL-12, for example, a dose that is less than about 3
.mu.g/m.sup.2 In yet other embodiments, a kit as provided herein
may comprise multiple doses or formulations suitable for multi-dose
administration of IL-12 following radiation exposure.
[0087] In one specific embodiment, a kit is provided for increasing
survival in a human, and/or preserving bone marrow function, and/or
promoting hematopoietic recovery or restoration following
non-therapeutic acute exposure to whole body ionizing radiation,
comprising a low dose or ultralow dose of human IL-12. In one
embodiment, the human IL-12 is recombinant IL-12 or a recombinant
IL-12 variant. In the kits of the invention, IL-12 can be
formulated so as to be delivered to a patient using a wide variety
of routes or modes of administration. In a particular embodiment,
IL-12 is formulated for injection in solution or as a lyophilized
powder that can be easily reconstituted using sterile water or a
physiologically acceptable buffer.
[0088] In some embodiments, the kits of the invention may also
comprise a formulation of a compound useful for supportive care of
IL-12 treatment following radiation exposure. Suitable compounds
include, without limitation, antibiotics, for example, bactrim,
ciprofloxacin, or moxifloxacin, and hematopoietic growth factors,
such as CSF, G-CSF, GM-CSF, M-CSF, IL-3, erythropoietin,
erythropoietin-like molecules, IL-1, IL-4, IL-5, IL-6, IL-7, IL-11,
and the like.
[0089] In certain embodiments, kits are provided for treating one
or more animal following acute exposure to whole body ionizing
radiation. For example, kits are provided for treating one or more
of a human, dog, cat, guinea pig, hamster, cattle, horse, sheep,
pig, rabbit, and the like. In a particular embodiment, these kits
contain one or more dose of IL-12 specific for the animal to be
treated. For example, a kit for treating cattle may comprise bovine
IL-12, or a kit for treating horses may comprise equine IL-12. In
other embodiments, the kits comprise, for example, murine or human
IL-12 at a dose suitable for administration to the particular
animal to be treated.
[0090] In certain aspects, the kits provided herein are optionally
housed in a radiation proof container (e.g., lead) or
alternatively, the container for the kit is radiation resistant or
proof (e.g., lead).
III. EXAMPLES
Example 1
The IL-12-Facilitated Survival as a Function of Administration
Schedule Following Super-Lethal Radiation (9 Gy)
[0091] This example illustrates the effects of administration time
point of IL-12 in a comparative experiment, where various time
points were directly compared to assess relative survival and
hematopoietic recovery. IL-12 was administered at 3 hours after, 24
hours before, or 24 hours before and 2 hours after (at two
different doses of IL-12) using a 9 Gy radiation dose
(unfractionated dose at a dose rate of 0.9 Gy/min (.gamma.-ray from
cesium 137 irradiator). In this example, female C57BL/6 mice were
used (13 weeks of age). All injections were intravenous, and mice
were started on antibiotics (Bactrim) following radiation.
[0092] As shown in Table 3, survival data demonstrate that there
was not significant differences among the various time points
evaluated. In fact, administration of IL-12 at 3 hours after
radiation or 24 hours before radiation showed only a slight
difference in survival, albeit the two groups were administered a
different dose of IL-12. However, it was determined that 100 ng (15
.mu.g/m.sup.2) is the optimal dose of IL-12 when given 24 hours
before lethal radiation. (See Table 3). Moreover, at the same
overall dose of IL-12 (200 ng/mouse; 30 .mu.g/m.sup.2) given either
in a split dose of given at 24 hour before and 2 hours after
radiation or as a single dose given at 3 hours after radiation,
there was no difference in relative survival. These data suggest
that a residual subpopulation of hematopoietic stem cells persist
after radiation that can be acted upon by IL-12.
TABLE-US-00003 TABLE 3 Survival as a Function of Time of
Administration of IL-12 at an Acute Radiation Dose of 9 Gy Time of
Administration Dose of IL-12 % Survival PBS control 0 0 3 hours
after radiation 200 ng (30 .mu.g/m.sup.2) 60 24 hour before
radiation 100 ng (15 .mu.g/m.sup.2) 50 24 hr before and 2 hr 100
ng, 50 ng 70 after (15 .mu.g/m.sup.2, 7.5 .mu.g/m.sup.2) 24 hr
before and 2 hr 100 ng, 100 ng 60 after (15 .mu.g/m.sup.2, 15
.mu.g/m.sup.2)
Example 2
The IL-12-Facilitated Hematopoietic Recovery as a Function of
Administration Schedule Following Super-Lethal Radiation (9 Gy)
[0093] The peripheral blood recovery for mice that survived the
super-lethal radiation dose is shown in FIG. 1. Although there are
some notable difference in the neutrophil and red blood cell
recovery, overall the recovery of these two blood cell groups was
quite similar, and did not vary significantly as a function of the
time point of IL-12 administration. Platelet recovery, however, was
significantly different for the 3 hour post- and 24 hour
pre-radiation administration of IL-12, as compared to the split
dose (pre-post) administration. For this example, mice were
administered Bactrim in their drinking water, which is known to
induce thrombocytopenia. Mice were taken off antibiotics at about
48 days, and subsequently, all groups showed an increase in
platelet counts. There results show that IL-12 can facilitate
recovery of all blood cell groups, including potent recovery of
platelet counts, at super-lethal radiation dose (9 Gy TBI). Also
notable is the length of the survival time. Mice were monitored for
57 days and were terminated on day 60. All mice appeared in good
health and had regained any losses in body weight.
Example 3
IL-12-Facilitated Survival: Administration of IL-12 at 6 Hours
after Lethal Radiation (LD.sub.100/10) to Female Mice without
Antibiotic Support
[0094] In this example, the effect of IL-12 when administered at 6
hours after an unfractionated (acute), lethal dose of radiation (8
Gy) without the addition of antibiotic support was evaluated.
[0095] A Kaplan-Meier plot of the data is shown in FIG. 2.
Remarkably, at the LD.sub.100/10, 100% of the IL-12-treated mice at
6 hour post radiation survived. Moreover, the health status of the
IL-12-treated mice was remarkably stable during the period in which
control mice experienced the effects of hematopoietic syndrome.
[0096] As shown in FIG. 3, although the IL-12-treated mice did lose
about 14%, on average of their body weight by day 21 post
radiation, they had regained all of their lost body weight, and
were slightly higher in weight than they were at the start of the
experiment, by about day 30 (19.1 g on day 0 vs. 19.6 g on day
30).
[0097] As would be expected, statistical analyses revealed that the
survival effect was highly significant via the Mantel Chi-Square
statistical method (p=0.001), despite the small number of mice (n=5
for Group 1 and n=6 for Group 2). This example demonstrates the
remarkable survival effects of IL-12 at 6 hours post radiation.
Example 4
IL-12-Facilitated Survival: Administration of IL-12 at 6 Hours
after Lethal Radiation (LD.sub.100/21) Using Male Mice and No
Antibiotic Support
[0098] This example evaluated the effect of IL-12 when administered
at 6 hours after an unfractionated (acute), lethal dose of
radiation (8 Gy) in male mice without the addition of antibiotic
support. This example shows a lethal radiation survival study using
male mice.
[0099] Two groups of mice were first exposed to a 7 Gy dose of
radiation and either treated with IL-12 or vehicle (PBS) at about 9
weeks of age. For the first round of radiation, Group 1 received
PBS and Group 2 received IL-12, all mice survived following
radiation. After 5 weeks, when it was clear that all mice were
healthy enough to undergo a second round of radiation, the same
mice were then subjected to a radiation dose of 8 Gy. At this
point, the mice were about 14 weeks of age and weighed 27 grams on
average. The IL-12 was adjusted according to the weight of the
mice. For the second round of radiation, Group 1 again received
only PBS and Group 3 also received PBS, and Group 2 received IL-12
again.
[0100] A Kaplan-Meier plot of the data is shown in FIG. 4 (Groups 1
(PBS) vs. 2 (IL-12)). Remarkably, it was found that after receiving
an accumulated dose of 15 Gy, nearly 70% of the IL-12-treated mice
survived (Group 2) when IL-12 was administered at 6 hours post
radiation. Moreover, the health status of the IL-12-treated mice in
Group 2 was observed to be good during the entire observation
period for the surviving mice. During the second round of
radiation, surviving mice lost about 24% of their body weight by
day 21 post radiation, and on day 30 these mice had regained 10% of
their body weight. These results are shown in FIG. 6. Remarkably,
statistical analyses revealed that the survival effect was
significant via the Chi-Square statistical Mantel method
(p<0.05), despite the small number of mice (n=5 for Group 1 and
n=6 for Group 2).
[0101] The data presented in FIGS. 3 and 4 were collected at the
same time, i.e., the mice were all irradiated at the same time and
received the same radiation dose. However, there is clearly a
notable difference in the survival curve for control female mice as
compared to control male mice. This may be due to apparent
differences in intrinsic radiation rescue response for males and
females, such as sex differences in endogenous IL-12 production
following radiation or other related factors.
Example 5
IL-12-Facilitated Survival: Administration of IL-12 at 24 Hours
after Lethal Radiation (LD.sub.90/30) Using Female Mice and No
Antibiotic Support
[0102] In this example, IL-12 administration was evaluated when
administered at 24 hours after an unfractionated (acute), lethal
dose of radiation (8 Gy) without the addition of antibiotic
support.
[0103] A Kaplan-Meier plot of the data is shown in FIG. 6.
Remarkably, when IL-12 was administered at 24 hours after an 8 Gy
radiation dose, 90% of the IL-12 treated mice survived, whereas
only 14% of the control mice were alive at 27 days post radiation.
Moreover, the health status of the IL-12-treated mice was
remarkably stable during the period in which control mice
experienced the effects of hematopoietic syndrome.
[0104] As shown in FIG. 7, although the IL-12-treated mice did lose
about 10%, on average, of their body weight by day 21 post
radiation, they had regained all of their lost body weight, and
were slightly higher in weight than they were at the start of the
experiment, by about day 27 (18.5 g on day 0 vs. 19.2 g on day
27).
[0105] Statistical analyses revealed that the survival effect was
significant via the Mantel Chi-Square statistical method (p=0.001),
despite the small number of mice (n=7 for Group 1 and n=8 for Group
2). This example demonstrates the remarkable survival effects of
IL-12 at 24 hours post radiation.
Example 6
Low Dose Administration of IL-12 at 24 Hours and at 24 Hours and 72
Hours after a Lethal Dose of Acute Whole Body Irradiation
(LD.sub.80)
[0106] 160 female C57BL/6 mice in 16 groups with 10 mice per group
were irradiated with a lethal dose of acute whole body irradiation
(LD.sub.80/30) and then treated with IL-12 administration. Both the
dose of IL-12 and the frequency of the dose (single vs. double
dose) were investigated. In the double dose experiment, the first
dose of IL-12 was administered at 24 hours post radiation and the
second dose of IL-12 was administered at 72 hours post radiation.
For the single dose experiment, IL-12 was administered at 24 hours
post radiation only. An outline of the study is shown in Table
4.
TABLE-US-00004 TABLE 4 Experimental design for single dose and
double dose IL-12 administration after acute whole body
irradiation. muIL-12 dose muIL-12 dose Group (ng per mouse)
(.mu.g/m.sup.2) 4A: Double Dose Experiment* 1 0 (vehicle) 0 2 40 6
3 80 12 4 120 18 5 150 22.5 6 200 30 7 300 45 8 500 75 4B: Single
Dose Experiment* 1 0 (vehicle) 0 2 40 6 3 80 12 4 120 18 5 150 22.5
6 200 30 7 300 45 8 500 75 *All injections were subcutaneous.
[0107] Mice treated with a double dose of IL-12 showed a linear
dose response related to administration of mulL-12 at lethal
radiation doses 24 and 72 hours post radiation (FIG. 8A), whereas
studies of single dose treatments demonstrate a biphasic dose
response related to administration of muIL-12 at lethal radiation
doses 24 hours post radiation (FIG. 8B). For the double dose
experiment, higher doses of mulL-12 gave better efficacy, as
measured by survival following a LD.sub.80 radiation dose with the
most efficacious dose being 300 ng (45 .mu.g/m.sup.2) given twice.
Interestingly, for the single dose experiments, the dose response
curve was biphasic with 40 ng (6 .mu.g/m.sup.2) and 200-300 ng (30
.mu.g/m.sup.2-45 .mu.g/m.sup.2)) showing high efficacy at the same
LD.sub.80/30 radiation dose. Average body weights for the surviving
mice are shown in FIG. 9.
[0108] The efficacy at 300 ng (45 .mu.g/m.sup.2) in the double dose
experiment was 70% at the LD.sub.80/30 radiation dose (FIG. 8A).
Notably, in the double dose experiment, there were early deaths in
many of the IL-12 treated groups, as compared to the control.
Without being bound by theory, these early deaths may be due to
protein aggregation resulting in immunogenicity. Consistent with
this notion, there were very few early deaths in the single dose
experiment. As can be seen via the statistical evaluation below, in
the double dose experiment it appears that there may be two
effects: 1) a highly therapeutic effect and 2) an effect that
diminishes the efficacy of the therapeutic effect.
[0109] The results of the single dose experiments show that a
single low dose of IL-12 (40 ng; 6 .mu.g/m.sup.2) administered at
24 hours after acute whole body irradiation resulted in 90%
survival at the LD.sub.80/30 (FIG. 8B). This single dose gave a 70%
increase in survival as compared to the control. Moreover, this
increase in survival was highly statistically significant
(p<0.001; see statistical evaluation below).
[0110] For single dose experiments, The dose response curve was
biphasic with a sharp increase in survival at 40 ng (6
.mu.g/m.sup.2), a dip in survival at intermediate doses and a
second increase in survival time at doses exceeding 200 ng (30
.mu.g/m.sup.2; FIG. 14). Notably, weight loss was a highly
significant predictor of lethality. Mice losing 20% or more of body
weight did indeed subsequently die.
[0111] Without being bound by theory, the biphasic nature of the
dose response curve (FIG. 14) may be explained by the presence of
protein aggregates in the administered IL-12. It is possible that
at the 40 ng (6 .mu.g/m.sup.2) dose, the least amount of
aggregation is occurring, whereas in the middle of the dose range
the protein is more aggregated, and in the higher dose range the
protein is delivered as both an aggregated and non-aggregated form.
Consistently, if there are more aggregates leading to some
immunogenicity in the middle of the dose range, this might explain
presence of a few early deaths, as compared to the control, in the
middle dose range. This type of aggregation and the resultant
biphasic dose response curve has been seen with other
biologics.
[0112] Finally, it should be noted that the concentration actually
delivered to the mice in this example may be as low as 10-20% of
what is indicated. This may be caused by the loss of IL-12 protein
due to adsorption on the sides of tubes and syringes used in the
study, as is common with solutions of very low protein
concentrations. These effects may be mitigated by various measures,
including without limitation, the use of containers with reduced
affinity for the non-specific protein interactions, or the
pre-treatment of containers to reduce adsorption, as well as with
the use of carrier molecules or proteins.
Statistical Analysis
[0113] FIG. 10 shows a stratified K-M analysis of the double dose
experiments. In the double dosing experiments, doses of IL-12,
200-300 ng and above, increased survival in this experiment.
Notably, when group 7 (300 ng; 45 .mu.g/m.sup.2) is compared to
group 1 (0 ng, vehicle control), the effect was statistically
significant (p<0.03; Tarone-Ware and Mantel methods; FIG. 11).
In contrast to the single dose experiments, significant low dose
therapeutic effects were not observed in the double dosing regimes.
Weight loss was not a significant covariate in this experiment
(FIG. 9A), suggesting that the double dose protocol allowed for
considerable weight loss without death, particularly at the highest
IL-12 dose.
[0114] Stratified K-M analysis of the results generated in the
single dose experiments indicated an overall statistically
significant effect of IL-12 dose on survival time (p<0.02;
Tarone-Ware method; FIG. 12). Strikingly, when groups 2 (40 ng; 6
.mu.g/m.sup.2) and 6 (200 ng; 30 .mu.g/m.sup.2) are compared with
group 1 (0 ng, vehicle control) in a three way K-M analysis, the
results are highly statistically significant (p<0.001;
Tarone-Ware Method; FIG. 13).
Example 7
Determination of LD Values for Acute Whole Body Irradiation of
C57BL/6 Female Mice
[0115] Six groups of 10 female C57BL/6 mice each were subjected to
increasing doses of total body radiation using a Gammacell 40
irradiator with Cesium source. The Specific dosage activity
(Adjusted Dose Rate) for the irradiator was 5159 rad/hour. Groups
were irradiated for the time periods shown in Table 5 in order to
achieve the final dose of radiation as delineated in the table.
TABLE-US-00005 TABLE 5 Experimental design for acute exposure to
whole body irradiation. Time Total Body Dose (min:sec) (Rad) 9:00
773.85 9:20 802.22 9:40 831.45 10:00 859.83 10:20 888.21 10:40
917.44
[0116] The irradiated mice were housed for 30 days following
irradiation. The mice were monitored daily for survival with the
day of death recorded for each mouse. The Kaplan-Meier graph of the
results is presented in FIG. 15. Calculated radiation exposures for
LD.sub.30(30), LD.sub.50(30), and LD.sub.70(30) were about 782 rad
(7.82 Gy), 788 rad (7.88 Gy), and 794 rad (7.94 Gy),
respectively.
[0117] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *