U.S. patent application number 17/302143 was filed with the patent office on 2021-08-12 for methods and compositions for protection of cells and tissues from computed tomography radiation.
This patent application is currently assigned to The University of Chicago. The applicant listed for this patent is The University of Chicago. Invention is credited to David J. GRDINA.
Application Number | 20210244751 17/302143 |
Document ID | / |
Family ID | 1000005542512 |
Filed Date | 2021-08-12 |
United States Patent
Application |
20210244751 |
Kind Code |
A1 |
GRDINA; David J. |
August 12, 2021 |
METHODS AND COMPOSITIONS FOR PROTECTION OF CELLS AND TISSUES FROM
COMPUTED TOMOGRAPHY RADIATION
Abstract
Described are methods for preventing or inhibiting genomic
instability and in cells affected by diagnostic radiology
procedures employing ionizing radiation. Embodiments include
methods of preventing or inhibiting genomic instability and in
cells affected by computed tomography (CT) radiation. Subjects
receiving ionizing radiation may be those persons suspected of
having cancer, or cancer patients having received or currently
receiving cancer therapy, and or those patients having received
previous ionizing radiation, including those who are approaching or
have exceeded the recommended total radiation dose for a
person.
Inventors: |
GRDINA; David J.;
(Naperville, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Chicago |
Chicago |
IL |
US |
|
|
Assignee: |
The University of Chicago
Chicago
IL
|
Family ID: |
1000005542512 |
Appl. No.: |
17/302143 |
Filed: |
April 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15862171 |
Jan 4, 2018 |
10987366 |
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17302143 |
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13822223 |
Apr 22, 2013 |
9877976 |
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PCT/US2011/051946 |
Sep 16, 2011 |
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15862171 |
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61383592 |
Sep 16, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/355 20130101;
A61K 33/04 20130101; A61K 45/06 20130101; A61K 31/661 20130101;
A61B 6/032 20130101; A61B 6/508 20130101; A61K 31/145 20130101;
A61K 31/132 20130101; A61K 49/0008 20130101 |
International
Class: |
A61K 31/661 20060101
A61K031/661; A61K 31/132 20060101 A61K031/132; A61K 31/355 20060101
A61K031/355; A61K 33/04 20060101 A61K033/04; A61K 45/06 20060101
A61K045/06; A61B 6/00 20060101 A61B006/00; A61B 6/03 20060101
A61B006/03; A61K 31/145 20060101 A61K031/145; A61K 49/00 20060101
A61K049/00 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made in part with government support
under Grant No. DE-FG02-05ER64086 from the Department of Energy.
The United States Government has certain rights in the invention.
Claims
1. A method of inhibiting genomic instability in a subject caused
by computed tomography (CT) scan radiation comprising administering
to the subject an effective dose of a phosphorothioate compound
prior to and/or following exposure to the CT scan radiation,
wherein the phosphorothioate compound or derivative thereof is
selected from amifostine, WR-1065, WR-638, WR-3689, WR-2822,
WR-2529, WR-77913, WR-255591, WR-2823, WR-33278, WR-255709,
WR-151326, and WR-151327; wherein: the radiation dose is 1-25 cGy;
and inhibiting genomic instability is selected from reducing
micronuclei formation, reducing .gamma.-H2AX formation, reducing
chromosome translocation frequency, reducing HPRT mutant frequency,
and reducing hyper-recombination.
2-32. (canceled)
33. A method of performing a CT scan on a subject, the method
comprising administering to the subject an effective dose of a
phosphorothioate compound or derivative thereof to the subject
prior to and/or following exposure to the CT scan radiation.
34. The method of claim 33, wherein the CT scan radiation comprises
a radiation dose of about 1-25 cGy.
35. The method of claim 33, wherein the phosphorothioate compound
or derivative thereof comprises amifostine, WR-1065, WR-638,
WR-3689, WR-2822, WR-2529, WR-77913, WR-255591, WR-2823, WR-33278,
WR-255709, WR-151326, WR-151327, or any combination thereof.
36. The method of claim 33, wherein the phosphorothioate compound
or derivative thereof is amifostine or WR-1065.
37. The method of claim 33, wherein the effective dose of a
phosphorothioate compound or derivative thereof is administered
prior to and/or up to 1, 2, or 3 hours following exposure to the CT
scan radiation.
38. The method of claim 33, wherein the phosphorothioate compound
or derivative thereof is administered prior to exposure to the CT
scan radiation.
39. The method of claim 33, wherein the phosphorothioate or
derivative thereof is administered at least 3 hours prior to
exposure to the CT scan radiation
40. The method of claim 33, wherein the phosphorothioate compound
or derivative thereof is administered within 5 hours prior to
exposure to the CT scan radiation.
41. The method of claim 33, wherein the phosphorothioate compound
or derivative thereof is administered up to one hour following
exposure to the CT scan radiation.
42. The method of claim 33, wherein the phosphorothioate compound
or derivative thereof is administered prior to and within 30
minutes after exposure to the CT scan radiation.
43. The method of claim 33, wherein the phosphorothioate compound
or derivative thereof is administered subcutaneously,
intravenously, topically, transdermally, orally, or via an
auto-injector device.
44. The method of claim 33, wherein the subject has been diagnosed
with cancer or is suspected of having cancer.
45. The method of claim 33, further comprising diagnosing cancer in
the subject using the CT scan.
46. The method of claim 33, wherein the subject has had 1, 2 or 3
previous CT scans.
47. The method of claim 33, wherein the subject is a human or
non-human animal.
48. The method of claim 33, wherein the CT scan radiation comprises
ionizing radiation.
49. The method of claim 33, further comprising administering to the
subject an antioxidant.
50. The method of claim 49, wherein the antioxidant comprises
ascorbic acid, glutathione, lipoic acid, uric acid,
.beta.-carotene, lycopene, lutein, resveratrol, retinol,
.alpha.-tocopherol, ubiquinol, selenium, catalase, or any
combination thereof.
51. A method of modulating mitochondrial superoxide dismutase 2
(SOD2) levels in a subject exposed to ionizing radiation, the
method comprising administering to the subject an effective dose of
a compound or derivative thereof to the subject prior to and/or
following exposure to the ionizing radiation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/862,171 filed Jan. 4, 2018, which is a
continuation of U.S. patent application Ser. No. 13/822,223 filed
Apr. 22, 2013, which is a national phase application under 35
U.S.C. .sctn. 371 of International Application No.
PCT/US2011/051946 filed Sep. 16, 2011, which claims the benefit of
the filing date of U.S. Provisional Application Ser. No. 61/383,592
filed Sep. 16, 2010. The entire contents of each of the
above-referenced disclosures are specifically incorporated herein
by reference without disclaimer.
BACKGROUND OF THE INVENTION
I. FIELD OF THE INVENTION
[0003] The invention is generally related to biochemistry,
physiology, medicine, and the inhibition of genomic instability
caused by ionizing radiation, such as computed tomography
imaging.
II. BACKGROUND
[0004] Computed tomography (CT) is a powerful medical imaging
method employing tomography created by computer processing.
However, CT scans have been estimated to produce non-negligible
increases in the probability of lifetime cancer mortality, leading
to calls for the use of reduced current settings for CT scans.
Estimated lifetime cancer mortality risks attributable to the
radiation exposure from a CT in a 1-year-old are 0.18% (abdominal)
and 0.07% (head)--an order of magnitude higher than for
adults--although those figures still represent a small increase in
cancer mortality over the background rate. In the United States, of
approximately 600,000 abdominal and head CT examinations annually
performed in children under the age of 15 years, a rough estimate
is that 500 of these individuals might ultimately die from cancer
attributable to the CT radiation. The additional risk is still very
low (0.35%) compared to the background risk of dying from cancer
(23%). However, if these statistics are extrapolated to the current
number of CT scans, the additional rise in cancer mortality could
be 1.5 to 2%. Furthermore, certain conditions can require children
to be exposed to multiple CT scans. Again, these calculations can
be problematic because the assumptions underlying them could
overestimate the risk.
[0005] In 2009 a number of studies appeared that further defined
the risk of cancer that may be caused by CT scans (Brenner et al.,
2001) One study indicated that radiation by CT scans is often
higher and more variable than cited and each of the 19,500 CT scans
that are daily performed in the U.S. is equivalent to 30 to 442
chest x-rays in radiation. It has been estimated that CT radiation
exposure will result in 29,000 new cancer cases just from the CT
scans performed in 2007 (Brenner et al., 2001). The most common
cancers caused by CT are thought to be lung cancer, colon cancer
and leukemia with younger people and women more at risk. Although
CT scans come with an additional risk of cancer (it can be
estimated that the radiation exposure from a full body scan is the
same as standing 2.4 km away from the WWII atomic bomb blasts in
Japan (Nelson, 2009; Semelka et al., 2007), especially in children,
the benefits that stem from their use outweighs the risk in many
cases (Khamsi, 2007). A number of vitamins and nutritional
supplements have been shown to protect the organism when it is
exposed to ionizing radiation. However, improved methods for
consistently and reproducibly reducing the risk of CT associated
radiation are needed.
SUMMARY OF THE INVENTION
[0006] Thus, in accordance with the disclosure, compositions and
methods are provided for protection of cells and tissue from
damaging effects of computer tomorgraphy radiation. In some
embodiments, there are methods of inhibiting genomic instability in
a subject caused by diagnostic radiology procedures that involve
exposure to ionizing radiation. In specific embodiments, methods
comprise administering to the subject an effective dose of a
phosphorothioate compound prior to and/or following exposure to a
diagnostic radiology procedure, including, but not limited to a
lower gastrointestinal (GI) series, which is a medical procedure
that involves taking X-ray pictures and which is also called a
barium enema. In certain embodiments, methods specifically exclude
the use of conventional X-ray imaging that does not involve
computed tomography.
[0007] In certain aspects, methods include inhibiting genomic
instability in a subject caused by computed tomography (CT) scan
radiation comprising administering to the subject an effective dose
of a phosphorothioate compound prior to and/or following exposure
to the CT scan radiation. The dose may provide a blood level of
about 1 to 150 .mu.M. The CT radiation dose may be about 1-25 cGy,
or about 1-10 cGy. In other embodiments, the CT radiation dose may
be about, at least about, or at most about 50, 60, 70, 80, 90, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,
240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,
370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480,
490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,
620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,
750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,
880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000,
1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 (and any
range derivable therein). In certain embodiments, the CT radiation
dosage is not lower than about 20, 30, 40, 50, 60, 70, 80, 90, 100,
110, 120, 130, 140, or 150 millirems. In further embodiments, the
CT radiation dose may be about, at least about, or at most about
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,
2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0,
4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,
5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,
8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,
9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0,
12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5,
18.0, 18.5, 19.0. 19.5, or 20.0 mSv, or any range derivable
therein.
[0008] Inhibiting genomic instability may comprise reducing
micronuclei formation and/or hyper-recombination. The method may
further comprise administering to the subject a second protective
agent, such as vitamin E, selenium or catalase.
[0009] The phosphorothioate may be administered prior to exposure
to the CT scan radiation, or at the time of, or up to one hour
following exposure to the CT scan radiation. The phosphorothioate
may also be provided both before and after the CT scan. The
phosphorothioate may be administered subcutaneously, such as by the
subject using an auto-injector device. The phosphorothioate may be
administered intravenously, topically or orally. The subject may
have cancer or may be suspected of having cancer. The subject may
have had 1, 2 or 3 previous CT scans. The subject may be a human or
a non-human animal.
[0010] Also provided are methods of assessing a subject with
computed tomography (CT) scan radiation comprising (a)
administering to the subject an effective dose of a
phosphorothioate compound prior to and/or immediately or 1 hour
following exposure to the CT scan radiation; and (b) subjecting the
subject to a CT scan.
[0011] Another embodiment comprises a method of diagnosing cancer
in a subject using computed tomography (CT) scan radiation dose
comprising (a) administering to the subject an effective dose of a
phosphorothioate compound prior to and/or immediately or 1 hr
following exposure to the CT scan radiation; and (b) subjecting the
subject to a CT scan.
[0012] A further embodiment involves a method of assessing the
treatment of cancer in a subject using computed tomography (CT)
scan radiation comprising administering to the subject an effective
dose of a phosphorothioate compound prior to and/or immediately
following exposure to the CT scan radiation; (b) treating the
subject with an anti-cancer therapy; and (c) subjecting the subject
to a CT scan. The CT scan may be compared to a pre-treatment CT
scan of the subject.
[0013] Also provided is a method for modulating mitochondrial
superoxide dismutase 2 (SOD2) levels in a subject comprising
administering to the subject an effective dose of a
phosphorothioate compound prior to and/or following exposure to the
CT scan radiation.
[0014] Phosphorothioates used in the inventive methods are
exemplified by, not limited to S-2-(3-aminopropylamino)ethyl
phosphorothioic acid (amifostine, WR-2721),
2-[(aminopropyl)amino]ethanethiol (WR-1065), S-1-(aminoethyl)
phosphorothioc acid (WR-638), S-[2-(3-methylaminopropyl)aminoethyl]
phosphorothioate acid (WR-3689), S-2-(4-aminobutylamino)
ethylphosphorothioic acid (WR-2822), 3-[(2-mercapto ethyl)amino]
propionamide p-toluenesulfonate (WR-2529), S-1-(2-hydroxy-3-amino)
propyl phosphorothioic acid (WR-77913), 2-[3-(methylamino)
propylamino] ethanethiol (WR-255591), S-2-(5-aminopentylamino)
ethyl phosphorothioic acid (WR-2823), [2-[(aminopropyl) amino]
ethanethiol] N,N,'-dithiodi-2,1-(ethanediyl) bis-1,3-propanediamine
(WR-33278),1-[3 -(3 -aminopropyl)
thiazolidin-2-Y1]-D-gluco-1,2,3,4,5 pentane-pentol dihydrochloride
(WR-255709), 3-(3-methylaminopropylamino) propanethiol
dihydrochloride (WR-151326), S-3-(3-methylaminopropylamino) propyl
phosphorothioic acid (WR-151327), a prodrug or salt thereof. In
particular aspects, the phosphorothioate is WR-2721.
[0015] Phosphorothioate can be provided at a dose of at least
about, at most about, or about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 25, 50, 100, 200, 400 mg/kg, or mg/kg/day, including all values
or ranges there between. In certain aspects a phosphorothioate is
administered at a dose of at least about, at most about, or about
20 mg/kg to about 200 mg/kg, and more particularly about 75 mg/kg
or mg/kg/day. Phosphorothioate can be administered 1, 2, 3, 4, 5,
or more times a day, a week, a month, or a year. In certain
aspects, a phosphorothioate is administered about every 24, 48, 72,
96, 120 hours or any range derivable therein. The phosphorothioate
may be provided intravenously, i.v., but also methods may include
administration via other enteral routes--oral, intra-arterial,
subcutaneous, intraperitoneal injection, infusion or perfusion--or
via inhalation routes.
[0016] In certain embodiments, the phosphorothioate may be provided
within, before, or after 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,
170, 180 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or
300 minutes (or any range derivable therein) being exposed to CT
radiation. In certain embodiments, the subject is exposed to CT
radiation
[0017] Methods may further involve administering intravenously to
the subject a contrast agent, which is used to visualize a CT
scan.
[0018] Other embodiments of the invention are discussed throughout
this application. Any embodiment discussed with respect to one
aspect of the invention applies to other aspects of the invention
as well and vice versa. The embodiments in the Example section are
understood to be embodiments of the invention that are applicable
to all aspects of the invention.
[0019] The terms "inhibiting," "reducing," or "prevention," or any
variation of these terms, when used in the claims and/or the
specification includes any measurable decrease or complete
inhibition to achieve a desired result. The use of the word "a" or
"an" when used in conjunction with the term "comprising" in the
claims and/or the specification may mean "one," but it is also
consistent with the meaning of "one or more," "at least one," and
"one or more than one." Throughout this application, the term
"about" is used to indicate that a value includes the standard
deviation of error for the device or method being employed to
determine the value. The use of the term "or" in the claims is used
to mean "and/or" unless explicitly indicated to refer to
alternatives only or the alternatives are mutually exclusive,
although the disclosure supports a definition that refers to only
alternatives and "and/or."
[0020] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0021] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
DESCRIPTION OF THE DRAWINGS
[0022] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0023] FIG. 1--Representative photomicrographs of GFP+/- colonies
displaying delayed hyperrecombination.
[0024] FIG. 2--Percentage of GFP+/- colonies as a function of
treatment.
[0025] FIG. 3--Percentage of GFP+/- colonies as a function of
treatment.
[0026] FIGS. 4A-B--Percentage of cells with micronuclei (A) treated
with WR1065 and irradiated or irradiated 24 h after WR1065 exposure
(B) treated with WR1065 at various times after irradiation.
[0027] FIG. 5--Percentage of cells with micronuclei exposed to 10
cGy x-rays and challenged with 2 Gy various times later.
[0028] FIG. 6--Kinetics of effects of amifostine on MnSOD activity
in normal mouse tissues.
[0029] FIG. 7--Kinetics of effects of amifostine on GPx activity in
normal mouse tissues.
[0030] FIG. 8--Kinetics of effects of amifostine on catalase
activity in normal mouse tissues.
[0031] FIG. 9--Effects of amifostine on miocronuclei formation in
normal mouse tissues.
[0032] FIG. 10A-B--(A) Effects of SOD2-siRNA transfection on
amifostine's ability to prevent radiation induced micronuclei
formation; (B) Correspondence of SOD2-siRNA transfection with
inhibition of elevated SOD2 activity.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Diagnostic radiology currently uses computed tomograpy, or
"CT" scanning technology as a main diagnostic tool. Seventy-two
million CT scans were performed in the USA alone in 2007.
Unfortunately, epidemology studies predict that CT-related
radiation will lead to 29,000 new cancers each year. A typical CT
scan results in organ doses 100 times larger than chest X-rays.
People who have a CT scan for a medical issue generally have on the
average 2 to 3 scans. Children are the most susceptible to the
carcinogenic effect of CT scans. The entire radiation dose range
utilized in CT scans are of the magnitude demonstrated from the
Atomic Bomb Survivors Data to significantly elevate carcinogenic
risk above background levels. Thus, there is a need for a
method/product to reduce these risks thus making CT scans safer for
the general population and specifically children.
[0034] Here, amifostine is shown at radiation doses typical of CT
scans, e.g., 10 cGy, to be effective in protecting against
hyper-recombination cellular processes, an important element in the
development of genomic instability and eventual cancer development
and readily observed in the low radiation dose range utilized by CT
scans. Furthermore, amifostine has been demonstrated to protect
against micronuclei formation in irradiated cells even when
administered up to one hour following irradiation. This is a much
more toxic genomic instability endpoint than mutagenesis since
mutations occur at the gene level while micronuclei formation
represent whole scale chromosomal damage resulting in the loss of
parts of chromosomes.
[0035] It is therefore proposed that amifostine and other
phosphorotioates can be used to reduce the serious side effects of
diagnostic radiology CT scans, namely, the induction of genomic
instability characterized by hyper recombination induced by low
doses of radiation that leads to gross but non-lethal chromosomal
damage as evidenced by micronuclei formation that can culminate in
cancer development. It must be stressed that alteration of the
normal background recombination processes that occur in
unirradiated cells induced by irradiation result in delayed
deleterious effects in which the intregrety of the genome is
negatively affected through hyper, eg, significantly elevated,
recombination processes. Each recombination event carries with it a
probability of incorrect rejoining and hence the introduction of
delayed DNA damage. Hyper recombination is a manifestation of loss
of genomic homeostasis and elevated probability of genomic damage.
Malignancies arise as a consequence of acquired genetic change as
evidenced by genomic instability in cells capable of clonal
expansion. Conventional wisdom holds that the induction of genomic
instability as evidenced by hyper recombination is an early marker
that can be directly observed at very low doses of radiation and
may represent the first critical step in the generation of
radiation-induced cancers. Thus, this method involves the use of
phosphorothioates such as amifostine for the purpose of protecting
against diagnostic radiation level doses of radiation when
administered either 30 min before or 1 h following irradiation. It
is proposed that such agents will advantageously be administered
subcutaneously using a self injector apparatus, optionally with a
second cytoprotective agent.
I. Computed Tomography (CT) Scanning
[0036] Computed tomography (CT) is a medical imaging method
employing tomography created by computer processing. Contrast
enhancing agents are routinely used and these in turn can
exacerbate radiation damage to the cellular DNA by virtue of their
high Z atomic numbers which facilitate the intensity of the
scattered radiation and thus improve the measured image. Such
contrast agents have, therefore, as a side effect the ability to
further sensitize the cells to the genomic damaging properties of
the radiation doses used in acquiring CT images. Digital geometry
processing is used to generate a three-dimensional image of the
inside of an object from a large series of two-dimensional X-ray
images taken around a single axis of rotation.
[0037] CT produces a volume of data which can be manipulated,
through a process known as "windowing," in order to demonstrate
various bodily structures based on their ability to block the X-ray
beam. Although historically the images generated were in the axial
or transverse plane, orthogonal to the long axis of the body,
modern scanners allow this volume of data to be reformatted in
various planes or even as volumetric (3D) representations of
structures. Although most common in medicine, CT is also used in
other fields, such as non-destructive materials testing. Usage of
CT has increased dramatically over the last two decades--an
estimated 72 million scans were performed in the United States in
2007.
[0038] Digital tomosynthesis combines digital image capture and
processing with simple tube/detector motion as used in conventional
radiographic tomography. Although there are some similarities to
CT, it is a separate technique. In CT, the source/detector makes a
complete 360-degree rotation about the subject obtaining a complete
set of data from which images may be reconstructed. In digital
tomosynthesis, only a small rotation angle (e.g., 40 degrees) with
a small number of discrete exposures (e.g., 10) are used. This
incomplete set of data can be digitally processed to yield images
similar to conventional tomography with a limited depth of field.
However, because the image processing is digital, a series of
slices at different depths and with different thicknesses can be
reconstructed from the same acquisition, saving both time and
radiation exposure.
[0039] Because the data acquired is incomplete, tomosynthesis is
unable to offer the extremely narrow slice widths that CT offers.
However, higher resolution detectors can be used, allowing
very-high in-plane resolution, even if the Z-axis resolution is
poor. The primary interest in tomosynthesis is in breast imaging,
as an extension to mammography, where it may offer better detection
rates with little extra increase in radiation exposure.
[0040] Reconstruction algorithms for tomosynthesis are
significantly different from conventional CT, because the
conventional filtered back projection algorithm requires a complete
set of data. Iterative algorithms based upon expectation
maximization are most commonly used, but are extremely
computationally intensive. Some manufacturers have produced
practical systems using off-the-shelf GPUs to perform the
reconstruction.
[0041] Since its introduction in the 1970s, CT has become an
important tool in medical imaging to supplement X-rays and medical
ultrasonography. It has more recently been used for preventive
medicine or screening for disease, for example CT colonography for
patients with a high risk of colon cancer, or full-motion heart
scans for patients with high risk of heart disease. A number of
institutions offer full-body scans for the general population.
However, this is a controversial practice, given its lack of proven
benefit, cost, radiation exposure, and the risk of finding
`incidental` abnormalities that may trigger additional
investigations. The increased use of CT scans has been the greatest
in two fields: screening of adults (screening CT of the lung in
smokers, virtual colonoscopy, CT cardiac screening and whole-body
CT in asymptomatic patients) and CT imaging of children. Shortening
of the scanning time to around 1 second, eliminating the strict
need for subject to remain still or be sedated, is one of the main
reasons for large increase in the pediatric population (especially
for the diagnosis of appendicitis).
TABLE-US-00001 TYPICAL SCAN DOSES Examination Typical effective
dose (mSv) (millirem) Chest X-ray 0.1 10 Head CT 1.5 150 Screening
3 300 mammography Abdomen CT 5.3 530 Chest CT 5.8 580 CT
colonography 3.6-8.8 360-880 Chest, abdomen 9.9 990 and pelvis CT
Cardiac CT angiogram 6.7-13 670-1300 Barium enema 15 1500 Neonatal
abdominal CT 20 2000
For purposes of comparison, the average background exposure in the
UK is 1-3 mSv per year.
[0042] Because contrast CT scans rely on intravenously administered
contrast agents in order to provide superior image quality, there
is a low but non-negligible level of risk associated with the
contrast agents themselves. Many patients report nausea and
discomfort, including warmth in the crotch which mimics the
sensation of wetting oneself. Certain patients may experience
severe and potentially life-threatening allergic reactions to the
contrast dye.
[0043] The contrast agent may also induce kidney damage. The risk
of this is increased with patients who have preexisting renal
insufficiency, preexisting diabetes, or reduced intravascular
volume. In general, if a patient has normal kidney function, then
the risks of contrast nephropathy are negligible. Patients with
mild kidney impairment are usually advised to ensure full hydration
for several hours before and after the injection. For moderate
kidney failure, the use of iodinated contrast should be avoided;
this may mean using an alternative technique instead of CT, e.g.,
MRI. Paradoxically, patients with severe renal failure requiring
dialysis do not require special precautions, as their kidneys have
so little function remaining that any further damage would not be
noticeable and the dialysis will remove the contrast agent.
[0044] An important issue within radiology today is how to reduce
the radiation dose during CT examinations without compromising the
image quality. Generally, higher radiation doses result in
higher-resolution images, while lower doses lead to increased image
noise and unsharp images. Increased dosage raises the risk of
radiation induced cancer--a four-phase abdominal
[0045] CT gives the same radiation dose as 300 chest x-rays.
Several methods exist which can reduce the exposure to ionizing
radiation during a CT scan.
[0046] New software technology can significantly reduce the
required radiation dose. The software works as a filter that
reduces random noise and enhances structures. In this way, it is
possible to get high-quality images and at the same time lower the
dose by as much as 30 to 70%. Other suggestions are to
individualize the examination and adjust the radiation dose to the
body type and body organ examined (different body types and organs
require different amounts of radiation), and, prior to every CT
examination, evaluate the appropriateness of the exam whether it is
motivated or if another type of examination is more suitable
(higher resolution is not always suitable for any given scenario,
such as detection of small pulmonary masses).
[0047] X-ray slice data is generated using an X-ray source that
rotates around the object; X-ray sensors are positioned on the
opposite side of the circle from the X-ray source. The earliest
sensors were scintillation detectors, with photomultiplier tubes
excited by (typically) cesium iodide crystals. Cesium iodide was
replaced during the 1980s by ion chambers containing high pressure
Xenon gas. These systems were in turn replaced by scintillation
systems based on photo diodes instead of photomultipliers and
modern scintillation materials with more desirable characteristics.
Many data scans are progressively taken as the object is gradually
passed through the gantry.
[0048] Newer machines with faster computer systems and newer
software strategies can process not only individual cross sections
but continuously changing cross sections as the gantry, with the
object to be imaged, is slowly and smoothly slid through the X-ray
circle. These are called helical or spiral CT machines. Their
computer systems integrate the data of the moving individual slices
to generate three dimensional volumetric information (3D-CT scan),
in turn viewable from multiple different perspectives on attached
CT workstation monitors. This type of data acquisition requires
enormous processing power, as the data are arriving in a continuous
stream and must be processed in real-time.
[0049] In conventional CT machines, an X-ray tube and detector are
physically rotated behind a circular shroud (see the image above
right); in the electron beam tomography (EBT) the tube is far
larger and higher power to support the high temporal resolution.
The electron beam is deflected in a hollow funnel-shaped vacuum
chamber. X-rays are generated when the beam hits the stationary
target. The detector is also stationary. This arrangement can
result in very fast scans, but is extremely expensive.
[0050] CT is used in medicine as a diagnostic tool and as a guide
for interventional procedures. Sometimes contrast materials such as
intravenous iodinated contrast are used. This is useful to
highlight structures such as blood vessels that otherwise would be
difficult to delineate from their surroundings. Using contrast
material can also help to obtain functional information about
tissues.
[0051] Once the scan data has been acquired, the data must be
processed using a form of tomographic reconstruction, which
produces a series of cross-sectional images. The most common
technique in general use is filtered back projection, which is
straight-forward to implement and can be computed rapidly. However,
this is not the only technique available: the original EMI scanner
solved the tomographic reconstruction problem by linear algebra,
but this approach was limited by its high computational complexity,
especially given the computer technology available at the time.
More recently, manufacturers have developed iterative physical
model-based expectation-maximization techniques. These techniques
are advantageous because they use an internal model of the
scanner's physical properties and of the physical laws of X-ray
interactions. By contrast, earlier methods have assumed a perfect
scanner and highly simplified physics, which leads to a number of
artifacts and reduced resolution--the result is images with
improved resolution, reduced noise and fewer artifacts, as well as
the ability to greatly reduce the radiation dose in certain
circumstances. The disadvantage is a very high computational
requirement, which is at the limits of practicality for current
scan protocols.
[0052] Pixels in an image obtained by CT scanning are displayed in
terms of relative radiodensity. The pixel itself is displayed
according to the mean attenuation of the tissue(s) that it
corresponds to on a scale from +3071 (most attenuating) to -1024
(least attenuating) on the Hounsfield scale. Pixel is a two
dimensional unit based on the matrix size and the field of view.
When the CT slice thickness is also factored in, the unit is known
as a Voxel, which is a three dimensional unit. The phenomenon that
one part of the detector cannot differentiate between different
tissues is called the "Partial Volume Effect." That means that a
big amount of cartilage and a thin layer of compact bone can cause
the same attenuation in a voxel as hyperdense cartilage alone.
Water has an attenuation of 0 Hounsfield units (HU) while air is
-1000 HU, cancellous bone is typically +400 HU, cranial bone can
reach 2000 HU or more (os temporale) and can cause artifacts. The
attenuation of metallic implants depends on atomic number of the
element used: Titanium usually has an amount of +1000 HU, iron
steel can completely extinguish the X-ray and is therefore
responsible for well-known line-artifacts in computed tomograms.
Artifacts are caused by abrupt transitions between low- and
high-density materials, which results in data values that exceed
the dynamic range of the processing electronics.
II. Genomic Instability and Cytoprotection
[0053] A. Genomic Instability
[0054] Genomic instability can be defined as an increased rate of
genetic alterations in the genome of the progeny of irradiated
cells multiple generations after the initial insult such as
chromosomal rearrangements and aberrations, micronuclei formation,
gene mutations, microsatellite instability, changes in ploidy, and
decreased plating efficiency (PE) (Morgan, 2003a; Morgan, 2003b)
with an association between chromosomal instability and delayed
reproductive cell death (Limoli et al., 1997; Marder and Morgan,
1993).
[0055] Micronuclei are small nuclei that forms whenever a
chromosome or a fragment of a chromosome is not incorporated into
one of the daughter nuclei during cell division. In newly formed
red blood cells in humans, these are known as Howell-Jolly bodies.
The can result from genomic toxicity, such as that caused by
radiation. Indeed, micronuclei formation represents a significantly
greater level of chromosomal damage than what is associated with
mutagenesis, which is gene specific.
[0056] Normal recombination processes occur in cells as a means to
preserve genomic integrity and maintain genomic stability. The
frequency of these processes can be negatively affected by stress
inducing agents such as ionizing radiation. The subsequent
elevation of recombinative frequencies in cells leads to a higher
probability of errors and increased DNA damage long after the
initial radiation exposure. In contrast to major deleterious events
induced by high doses of radiation exposure such as loss of cell
viability, mutagenesis, chromosomal and DNA strand breaks, this
deleterious process with its downstream carcinogenic consequence
can be observed and measured at relatively low doses of radiation
in the range used routinely for CT scans. It must be stressed that
the consequences of hyperrecombination are not limited to
mutagenesis but also encompass the potential of gene augmentation
due to hyper duplication and relocation within the genome thus
affecting a myriad of genomic processes including the possibility
of epigenetic processes such as gene silencing.
[0057] Gene conversion can produce a local loss of heterozygosity
(LOH) as well as deletions and inversions at linked repeats and
translocations (Nickoloff, 2002). The inventor has utilized a green
fluorescence protein (GFP)-based assay using human RKO-derived
cells to study the role of delayed hyper-recombination in
radiation-induced genomic instability (Huang et al., 2004). A GFP
direct repeat homologous recombination substrate is incorporated in
RKO cells. One copy is driven by the CMV promoter but is
inactivated by an Xhol linker frame shift mutation, and the second
copy has a wild-type coding capacity but is inactive because it
lacks a promoter.
[0058] Genomic instability can be measured by analysis of delayed
hyperrecombination and mutation/deletion at the GFP direct repeat
substrate. GFP-RKO cells can be GFP+ or GFP+. When these cells are
plated, colonies arising from single cells are either homogenously
colorless (GFP-) or green (GFP+). A GFP- cell can convert to a GFP+
cell directly by radiation-induced delayed hyperrecombination.
Similarly, a GFP+ cell can be converted to a GFP- cell directly by
a radiation-induced point mutation or deletion (see FIG. 1). Mixed
colonies arise by delayed hyperrecombination and can easily be
scored using the criteria of having at least >4 cells per colony
having an altered fluorescent phenotype. The frequency of induced
instability can be calculated as the number of GFP+/- colonies per
total surviving colonies scored.
[0059] Radiation exposure can induce delayed hyper-recombination
with up to 10% of the cells producing mixed GFP+/- colonies. Cells
displaying delayed hyperrecombination show no evidence of delayed
reproductive cell death and there is no correlation between delayed
chromosomal instability and delayed hyperrecombination, indicating
that these forms of genome instability arise by distinct
mechanisms. The inclusion of radiation-induced delayed
hyperrecombination as an important endpoint of genomic instability
and the focus of low dose radiation studies reflects three
important elements thought to be important in the carcinogenic risk
process. First, genome instability associated with delayed
hyperrecombination increases the probability of uncovering
recessive mutations via LOH. Second, delayed hyperrecombination can
be induced by doses of radiation that cause little or no
cytotoxicity, thus increasing the relative fraction of cells at
risk and allowing them to survive and accumulate mutations
necessary for immortalization and cellular transformation. And
third, because delayed hyperrecombination is important for the
repair of DNA double-strand breaks, cells with a hyperrecombination
phenotype may display enhanced resistance to radiation.
[0060] B. Cytoprotection
[0061] Cytoprotection is the use of a chemical agent to prevent
cell killing and/or loss of function in normal tissues exposed to a
deleterious extracellular or intracellular environment, such as
radiation. Amifostine is such a cytoprotective agent, but has been
used in a limited fashion due to early studies with animal models.
The present invention seeks, in one embodiment, to protect cells
from radiation associated with CT scans.
[0062] Evidence of cytoprotection in vivo can also be measured by
analysis of certain biomarkers related to genetic instabilities.
For example, chromosome translocation frequencies have been noted
as the hallmark of exposure to ionizing radiation and have been
utilized to detect chromosomal damage from diagnositic X-rays at
the 50 mGy level. See Tucker and Luckinbill, Radiat Res. 2011,
175(5), 631-7; Bhatti et al., Radiat. Environ. Biophys. 2010,
49(4), 685-692; and Sigurdson et al., Cancer Res. 2008, 68(21),
8825-8831, each of which are hereby incorporated by reference in
their entirety.
[0063] In another example, cytoprotection in vivo can also be
measured by analysis of micronuclei formation. See Kim et al.,
Cancer Res. 2006, 66, 10377-10383, which is hereby incorporated by
reference in its entirety. Micronuclei are formed from either
chromosome fragments or whole chromosomes that lag behind at
anaphase during cell division, and their formation is considered to
be an important biomarker for genotixicity testing. In particular,
micronuclei have been shown to be generated at low radiation dosage
levels (e.g., 10 cGy). See Murley et al., Radiat. Res., 175:57-65,
2011, which is hereby incorporated by reference in its
entirety.
[0064] Evidence of cytoprotection ex vivo can also be measured by
analysis of radiation-induced histone H2AX phosphorylation at
serine 139 (.gamma.-H2AX). It has been shown that quantity of
.gamma.-H2AX-positive cells increase with increasing radiation
dose, with a dose- and time-dependent decay. .gamma.-H2AX formation
in irradiated cells, as a function of relative DNA content, can be
quantified by bivariant flow cytometry analysis with
FITC-conjugated .gamma.-H2AX antibody and nuclear DAPI staining;
measurement can be made after about 1 hour after cellular exposure
to ionizing radiation. See, Kataoka et al., Radiat. Res. 2007, 168,
106-114, which is hereby incorporated by reference in its entirety.
In particular, .gamma.H2AX-based visualization and quantification
of DNA damage induced in peripheral blood mononuclear cells (PBMCs)
can be used to estimate the radiation dose received by adult
patients who undergo multidetector computed tomography (CT), and
has been used to detect dose as low as 6.3 mGy. See Rothkamm et
al., Radiology 2007, 242, 244-251, which is hereby incorporated by
reference in its entirety.
[0065] In another example, evidence of cytoprotection ex vivo can
also be measured by analysis of mutant frequency at the
hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus, for
example, in human G(0) peripheral blood lymphocytes. See Kumar et
al., Radiat Res. 2006, 165(1), 43-50, which is hereby incorporated
by reference in its entirety. HPRT mutant frequency in lymphocytes
has been successfully utilized to estimate average dose of 9.5 cGy
for Russian Chernobyl cleanup workers based on the average increase
in translocation frequency. See Jones et al., Radiat Res. 2002,
158(4), 424-42, which is hereby incorporated by reference in its
entirety.
[0066] In another embodiment, one may combine the use of
phosphorothioate protection with a second cytoprotective strategy.
A description of cytoprotection and methods of assaying for
cytoprotection as it relates to cancer therapies are provided in
U.S. Pat. Nos. 5,567,686; 5,488,042, 5,891,856, and 5,869,338. Any
of the cytoprotective therapies described therein may be combined
with the phosphorothioate treatments of the present invention.
III. PHARMACEUTICAL COMPOSITIONS AND ROUTES OF ADMINISTRATION
[0067] A. Phosphorothioates
[0068] A general description of the class of compounds and their
properties described in this application can be found in Sweeney,
1979 and Giambarresi and Jacobs, 1987, both of which are
incorporated by reference. Compounds and designations exemplary of
the class of phosphorothioates include
S-2-(3-aminopropylamino)ethyl phosphorothioic acid (amifostine,
WR-2721), 2-[(aminopropyl)amino]ethanethiol (WR-1065),
S-1-(aminoethyl) phosphorothioc acid (WR-638),
S-[2-(3-methylaminopropyl)aminoethyl]phosphorothioate acid
(WR-3689), S-2-(4-aminobutylamino)ethylphosphorothioic acid
(WR-2822), 3-[(2-mercapto ethyl)amino]propionamide
p-toluenesulfonate (WR-2529), S-1-(2-hydroxy-3-amino)propyl
phosphorothioic acid (WR-77913),
2-[3-(methylamino)propylamino]ethanethiol (WR-255591),
S-2-(5-aminopentylamino)ethyl phosphorothioic acid (WR-2823),
[2-[(aminopropyl)amino]ethanethiol]N,N,'-dithiodi-2,1-(ethanediyl)bis-1,3-
-propanediamine (WR-33278),
1-[3-(3-aminopropyl)thiazolidin-2-Y1]-D-gluco-1,2,3,4,5
pentane-pentol dihydrochloride (WR-255709),
3-(3-methylaminopropylamino)propanethiol dihydrochloride
(WR-151326), and S-3-(3-methylaminopropylamino)propyl
phosphorothioic acid (WR-151327).
[0069] B. Formulations and Routes of Administration
[0070] The compounds useful in the methods of the invention may be
in the form of free acids, free bases, or pharmaceutically
acceptable addition salts thereof. Such salts can be readily
prepared by treating the compounds with an appropriate acid. Such
acids include, by way of example and not limitation, inorganic
acids such as hydrohalic acids (hydrochloric, hydrobromic,
hydrofluoric, etc.), sulfuric acid, nitric acid, and phosphoric
acid, and organic acids such as acetic acid, propanoic acid,
2-hydroxyacetic acid, 2-hydroxypropanoic acid, 2-oxopropanoic acid,
propandioic acid, and butandioic acid. Conversely, the salt can be
converted into the free base form by treatment with alkali.
[0071] Aqueous compositions of the present invention comprise an
effective amount of the therapeutic compound, further dispersed in
pharmaceutically acceptable carrier or aqueous medium. The phrases
"pharmaceutically or pharmacologically acceptable" refer to
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, or a human, as
appropriate.
[0072] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients also can be
incorporated into the compositions.
[0073] Solutions of therapeutic compositions can be prepared in
water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, mixtures thereof, and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0074] The therapeutic compositions of the present invention are
advantageously administered in the form of injectable compositions
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to inj ection may also
be prepared. These preparations also may be emulsified. A typical
composition for such purpose comprises a pharmaceutically
acceptable carrier. For instance, the composition may contain at
least about, at most about, or about 1, 5, 10, 25, 50 mg or up to
about 100 mg of human serum albumin per milliliter of phosphate
buffered saline. Other pharmaceutically acceptable carriers include
aqueous solutions, non toxic excipients, including salts,
preservatives, buffers and the like.
[0075] A particular form of administration is the use of an
auto-injector that can be pre-loaded with a "unit dose" (see
below), or calibrated to reliably and/or repeatably deliver a unit
dose of phosphorothioate. Most autoinjectors are spring-loaded
syringes. By design, autoinjectors are easy to use and are intended
for self-administration by patients, or administration by untrained
personnel. The site of injection depends on the drug loaded, but it
typically is administered into the thigh or the buttocks. The
injectors were initially designed to overcome the hesitation
associated with self-administration of the needle-based drug
delivery device.
[0076] The autoinjector keeps the needle tip shielded prior to
injection and also has a passive safety mechanism to prevent
accidental firing (injection). Injection depth can be adjustable or
fixed and a function for needle shield removal may be incorporated.
Just by pressing a button, the syringe needle is automatically
inserted and the drug is delivered. Once the injection is completed
some auto injectors have visual indication to confirm that the full
dose has been delivered. Autoinjectors contain glass syringes, this
can make them fragile and contamination can occur. More recently
companies have been looking into making autoinjectors syringes out
of plastic to prevent this issue. Anapen.RTM., EpiPens.RTM., or the
recently introduced Twinject.RTM., are often prescribed to people
who are at risk for anaphylaxis. Rebiject.RTM. and Rebiject.RTM. II
autoinjectors are used for Rebif, the drug for interferon
.beta.-1a, used to treat Multiple Sclerosis. SureClick.RTM.
autoinjector delivers a combination product for drugs Enbrel or
Aranesp to treat arthritis or anemia, respectively. Any of these
technologies could be adapted to deliver the compounds of the
present invention.
[0077] Examples of non aqueous solvents include propylene glycol,
polyethylene glycol, vegetable oil and injectable organic esters
such as ethyloleate. Aqueous carriers include water,
alcoholic/aqueous solutions, saline solutions, parenteral vehicles
such as sodium chloride, Ringer's dextrose, etc. Intravenous
vehicles include fluid and nutrient replenishers. Preservatives
include antimicrobial agents, anti oxidants, chelating agents and
inert gases. The pH and exact concentration of the various
components the pharmaceutical composition are adjusted according to
well known parameters.
[0078] The therapeutic compositions of the present invention may
include classic pharmaceutical preparations. Administration of
therapeutic compositions according to the present invention will be
via any common route so long as the target tissue is available via
that route. This includes oral, nasal, buccal, rectal, vaginal or
topical. Alternatively, administration will be by orthotopic,
intradermal subcutaneous, intramuscular, intraperitoneal or
intravenous injection. Such compositions would normally be
administered as pharmaceutically acceptable compositions that
include physiologically acceptable carriers, buffers or other
excipients. Volume of an aerosol is typically between about 0.01 mL
and 0.5 mL.
[0079] Additional formulations may be suitable for oral
administration. "Oral administration" as used herein refers to any
form of delivery of an agent or composition thereof to a subject
wherein the agent or composition is placed in the mouth of the
subject, whether or not the agent or composition is swallowed.
Thus, `oral administration` includes buccal and sublingual as well
as esophageal administration. Absorption of the agent can occur in
any part or parts of the gastrointestinal tract including the
mouth, esophagus, stomach, duodenum, ileum and colon. Oral
formulations include such typical excipients as, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate and the
like. The compositions take the form of solutions, suspensions,
tablets, pills, capsules, sustained release formulations or
powders.
[0080] In one embodiment, the oral formulation can comprise the
phosphorothioates and one or more bulking agents. Suitable bulking
agents are any such agent that is compatible with the
phosphorothioates including, for example, lactose, microcrystalline
cellulose, and non-reducing sugars, such as mannitol, xylitol, and
sorbitol. One example of a suitable oral formulations includes
spray-dried phosphorothioates-containing polymer nanoparticles
(e.g., spray-dried poly(lactide-co-glycolide)/amifostine
nanoparticles having a mean diameter of between about 150 nm and
450 nm; see, Pamujula, S. et a., J. Pharmacy Pharmacol. 2004, 56,
1119-1125, which is here by incorporated by reference in its
entirety). The nanoparticles can contain between about 20 and 50
w/w % phosphorothioate; for example, between about 25% and 50%.
[0081] When the route is topical, the form may be a cream,
ointment, salve or spray. Topical formulations may include solvents
such as, but not limited to, dimethyl sulfoxide, water,
N,N-dimethylformamide, propylene glycol, 2-pyrrolidone,
methyl-2-pyrrolidone, and/or N-methylforamide. To enhance skin
permeability, if necessary, the skin area to be treated can be
pre-treated with dimethylsulfoxide; see Lamperti et al., Radiation
Res. 1990, 124, 194-200, which is hereby incorporated by reference
in its entirety.
[0082] In other embodiments, the therapeutic compositions may be
for subcutaneous administration (e.g., injection and/or
implantation). For example, implantable forms may be useful for
patients which are expected to undergo multiple CT scans over an
extended period of time (e.g., one week, two weeks, one month,
etc.). In one example, such subcutaneous forms can comprise the
phosphorothioates and a carrier, such as a polymer. The polymers
may be suitable for immediate or extended release depending on the
intended use. In one example, the phosphorothioate can be combined
with a biodegradable polymer (e.g., polylactide, polyglycolide,
and/or a copolymers thereof). In another example, subcutaneous
forms can comprise a microencapsulated form of the
phosphorothioate, see, e.g., Srinivasan et al., Int. J. Radiat.
Biol. 2002, 78, 535-543, which is hereby incorporated by reference
in its entirety. Such microencapsulated forms may comprise the
phosphorothioate and one or more surfactant and other excipients
(e.g., lactose, sellulose, cholesterol, and phosphate- and/or
stearate-based surfactants).
[0083] In a further embodiment, the therapeutic compounds may be
administered transdermally through the use of an adhesive patch
that is placed on the skin to deliver the therapeutic compounds
through the skin and into the bloodstream. An advantage of the
transdermal drug delivery route relative to other delivery systems
such as oral, topical, or intravenous is that the patch provides a
controlled release of the therapeutic compound into the patient,
usually through a porous membrane covering a reservoir of the
therapeutic compound or through body heat melting thin layers of
therapeutic compound embedded in the adhesive. In practicing this
invention, any suitable transdermal patch system may be used
including, without limitation, single-layer drug-in-adhesive,
multi-layer drug-in-adhesive, and reservoir.
[0084] The therapeutic compositions may optionally further comprise
a second protective agent. The second therapeutic agent can be an
antioxidant. Examples of suitable antioxidants include, but are not
limited to ascorbic acid (vitamin C), glutathione, lipoic acid,
uric acid, .beta.-carotene, lycopene, lutein, resveratrol, retinol
(vitamin A), .alpha.-tocopherol (vitamin E), ubiquinol, selenium,
and catalase. In certain embodiments, the second therapeutic agent
is vitamin E, selenium or catalase.
[0085] An effective amount of the therapeutic composition is
determined based on the intended goal, such as enhancing or
extending the lifespan of a beta cell under hyperglycemic
conditions. The term "unit dose" or "dosage" refers to physically
discrete units suitable for use in a subject, each unit containing
a predetermined quantity of the therapeutic composition calculated
to produce the desired responses, discussed above, in association
with its administration, i.e., the appropriate route and treatment
regimen. The quantity to be administered, both according to number
of treatments and unit dose, depends on the protection desired. An
effective dose is understood to refer to an amount necessary to
achieve a particular effect, for example, an increased antioxidant
capability of a cell. In the practice of the present invention, it
is contemplated that doses in the range from 10 mg/kg to 200 mg/kg
can affect the protective capability of these compounds. Thus, it
is contemplated that doses include doses of about 0.1, 0.5, 1, 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,
165, 170, 175, 180, 185, 190, 195, and 200 .mu.g/kg or mg/kg.
Furthermore, such doses can be administered at multiple times
during a day, and/or on multiple days, weeks, or months.
[0086] In certain embodiments, the effective dose is one which can
provide a blood level of about 1 .mu.M to 150 .mu.M. In another
embodiment, the effective dose provides a blood level of about 4
.mu.M to 100 .mu.M.; or about 1 .mu.M to 100 .mu.M; or about 1
.mu.M to 50 .mu.M; or about 1 .mu.M to 40 .mu.M; or about 1 .mu.M
to 30 .mu.M; or about 1 .mu.M to 20 .mu.M; or about 1 .mu.M to 10
.mu.M; or about 10 .mu.M to 150 .mu.M; or about 10 .mu.M to 100
.mu.M; or about 10 .mu.M to 50 .mu.M; or about 25 .mu.M to 150
.mu.M; or about 25 .mu.M to 100 .mu.M; or about 25 .mu.M to 50
.mu.M; or about 50 .mu.M to 150 .mu.M; or about 50 .mu.M to 100
.mu.M. In other embodiments, the dose can provide the following
blood level of the compound that results from a phosphorothioate
compound being administered to a subject: about, at least about, or
at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100 .mu.M or any range derivable therein. In certain embodiments,
the phosphorothioate compound that is administered to a subject is
metabolized in the body to a metabolized phosphorothioate compound,
in which case the blood levels may refer to the amount of that
compound. Alternatively, to the extent the phosphorothioate
compound is not metabolized by a subject, the blood levels
discussed herein may refer to the unmetabolized phosphorothioate
compound.
[0087] In some embodiments, the timing of administration is
relevant. One, 2, 3, 4, or 5 doses may be administered to the
patient before the CT scan is started on the patient. In some
embodiments, the pre-CT scan dose is administered at most 10, 20,
30, 40, 50, or 60 minutes (or any range derivable therein) before
the start of the scan. In further embodiments, the pre-CT scan dose
is administered at least 12, 18 or 24 hours (or any range derivable
therein) before the start of the scan. In some embodiments, a dose
is administered 10, 20, 30, 40, 50, or 60 minutes, or 1, 2, or 3
hours (or any range derivable therein) after the start of the scan
or at the conclusion of the scan. In further embodiments, the
post-CT scan dose is administered at least, up to, or at most 10,
20, 30, 40, 50, or 60 minutes, or up to 1, 2, or 3 hours (or any
range derivable therein) after the start of the scan or at the
conclusion of the scan. In certain embodiments, the dose is given
only within 30 minutes, 1 hour, 1.5, 2, 2.5, or 3 hours (or any
range derivable therein) within the start or end of the scan.
Alternatively, in some embodiments the timing discussed herein may
refer to the time at which the contrast agent is administered.
[0088] Precise amounts of the therapeutic composition also depend
on the judgment of the practitioner and are peculiar to each
individual. Factors affecting dose include physical and clinical
state of the patient, the route of administration, the intended
goal of treatment (alleviation of symptoms versus cure) and the
potency, stability and toxicity of the particular therapeutic
substance or other therapies a subject may be undergoing.
[0089] It will be understood by those skilled in the art and made
aware of this invention that dosage units of .mu.g/kg or mg/kg of
body weight can be converted and expressed in comparable
concentration units of .mu.g/ml or mM (blood levels), such as 4
.mu.M to 100 .mu.M. It is also understood that uptake is species
and organ/tissue dependent. The applicable conversion factors and
physiological assumptions to be made concerning uptake and
concentration measurement are well-known and would permit those of
skill in the art to convert one concentration measurement to
another and make reasonable comparisons and conclusions regarding
the doses, efficacies and results described herein.
[0090] IV. EXAMPLES
[0091] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention
Example 1--Delayed Hyperrecombination Following Thiol Exposure
Prior to Irradiation with 10 cGy
[0092] Genomic instability was evaluated in RKO36 cells by analysis
of delayed hyperrecombination and mutation/deletion at the GFP
direct repeat substrate. All "pure" GFP+ or GFP- colonies reflect
either a prior stable phenotype, or new, stable phenotype directly
induced by radiation but if radiation induces a delayed instability
at the GFP direct repeats, mixed GFP+/- colonies will form (see
FIG. 1). Cells were exposed to 4 mM WR1065 for 30 min and
irradiated 24 h later, the timeframe when MnSOD activity levels
were maximally elevated (Murley et al., 2007). Cells were exposed
to 10 cGy of ionizing radiation using a Philips X-ray generator
operating at 250 kVp and 15 mA at a dose rate of 0.6 cGy/sec. The
frequency of induced instability is calculated as the number of
GFP+/- colonies per total surviving colonies scored (Huang et al.,
2004). Using this system, the inventor assessed whether RKO36 cells
exhibit a thiol-induced adaptive response for the prevention of
radiation-induced delayed genomic instability (delayed
hyperrecombination). The results are shown in FIG. 2. A 10 cGy dose
was not cytotoxic but it induced a significant elevation in the
frequency of GFP+/- colonies over background, e.g., 2.7% vs 0.7%,
P<0.001. The frequencies of GFP+/- colonies in 4 mM WR1065
treated cells or cells exposed to 10 cGy 24 h following treatment
with 4 mM WR1065 were significantly lowered to 1.1% (P=0.02) and
1.6% (P=0.05) as compared to non-WR1065 exposed but irradiated
cells, respectively.
[0093] Investigators have determined that with RKO36 cells, most
GFP+/- mixed colonies are primarily composed of GFP- cells with
varying numbers of GFP+ cells. The frequency of this induced
genomic instability increases with dose, although typical of
non-targeted effects, the dose response is not linear (Smith et
al., 2003).
Example 2--Radiation-Induced Adaptive Responses on Micronuclei
Formation in RKO36 Cells
[0094] The frequency of micronuclei formation following a dose of 2
Gy was significantly reduced if cells were either exposed to WR1065
30 min or 24 h prior to irradiation (see FIG. 4A) or 30 min, 1, 2,
or 3 h following irradiation (see FIG. 4B). Under the treatment
conditions in which cells were exposed to WR1065 either 30 min
before or 3 h following irradiation the protective effect is the
result of a direct interaction between the thiol taken up by the
cells and the damage induced by radiation. This direct protective
effect disappeared when the drug was added 6 h following
irradiation. However, when cells were exposed to the thiol 24 h
prior to irradiation to allow for the induction of elevated MnSOD
enzyme levels, a protective effect was again observed (see FIG. 5).
In contrast to the data described in FIG. 4A, the protection
described in FIG. 4B was mediated not directly by thiol action
(Grdina et al., 1995) but as a result of a thiol-induced adaptive
response mediated through the activation of NF.kappa.B resulting in
the elevation of MnSOD activity (Murley et al., 2002; Murley et
al., 2004; Murley et al., 2006; Murley et al., 2007; Murley et al.,
2008). For comparison, a radiation-induced adaptive response was
investigated by first exposing RKO36 cells to 10 cGy and then
challenging with a 2 Gy dose 0, 4, 8, 12, 16, 20, and 24 h later
(see FIG. 5). Both the thiol- and radiation-induced adaptive
responses measured at 24 h following WR1065 or 10 cGy exposure,
respectively, resulted in the reduction of micronuclei formation to
unirradiated background levels.
Example 3--Thiol-Induced Elevated MnSOD Activity In Vivo
[0095] In collaboration with the Free Radical Biology Core at The
University of Iowa, the inventor characterized the activity of
MnSOD in various normal tissues in C3H mice treated with
amifostine, the prodrug form of WR1065. Animals at 60 days of age,
three per experimental group, were injected i.p. with a single 400
mg/kg dose of amifostine (WR2721).
[0096] Animals were sacrificed 8, 16, 24 and 32 h following
amifostine administration and the small intestine, pancreas, lung,
liver, heart and spleen of each animal was removed, flash frozen
and coded. Coded samples were analyzed for MnSOD, catalase, and
glutathione peroxidase (GPx) activity (Li et al., 1998; Oberley and
Spitz, 1984). Presented in FIG. 6 are composite graphs
demonstrating the effects of a single amifostine injection on the
kinetics of change in MnSOD activity as a function of time.
Pair-wise comparisons of the activities at the 0 h control and the
time interval demonstrating the maximum enhancement or reduction in
MnSOD activity were determined using a two-tailed Student's t test
and are included. MnSOD activity was significantly elevated in
small intestine, pancreas and spleen at either 24 h or 32 h. GPx
activities were elevated in the spleen and pancreas at 24 h and 32
h, respectively (see FIG. 7), and catalase activity was elevated in
the spleen at 32 h (see FIG. 8). These data demonstrate non-protein
thiols can also affect MnSOD enzymatic activity in tissues in an
animal model.
Example 4--In Vivo Inhibition of Micronuclei Formation
[0097] C3H mice at 60 days of age, three per experimental group,
were injected i.p. with a single 400 mg/kg dose of amifostine
(WR2721). After 24 hours (when SOD2 activity was maximally elevated
in the spleens of mice; see, Example 3), the mice were irradiated
with 2 Gy of ionizing radiation, and spleen cells examined for
micronuclei formation. FIG. 9 shows that miocronuclei formation was
significantly inhibited in the amifostine treated animals as
compared to non-amifostine treated control animals. It appears that
amifostine can prevent genomic instability by directly stabilizing
the genome, enhancing the fidelity of repair, slowing down cell
cycle progression thereby facilitating repair before damage is
fixed at cellular division, and by amifostine-induced SOD2
production in normal tissues. Increased intracellular SOD2 levels,
through its endogenous anti-oxidant properties, can extend
protection against genomic instability as evidenced by inhibition
of micronuclei formation.
[0098] As further proof of the proposed mechanism of action, RKO
human colon carcinoma cells (J. S. Murley et al. Radiat. Res. 175,
57-65, 2011; human microvascular endothelial cells (HMEC) and SA-NH
mouse fibrosarcoma tumor cells (J. S. Murley et al. Radiat. Res.
169, 495-505, 2008) were transfected with SOD2-siRNA, (Ambion
(Foster City, Calif.), the sequence of the SOD2-siRNA is 5' AAG GAA
CAA CAG GCC TTA TTC 3')(SEQ ID NO:1) a specific inhibitor of SOD2
synthesis in cells. Control cells were transfected with siRNA that
is not specific to SOD2 (nc-siRNA in FIGS. 10a and 10b). SOD2-siRNA
transfection was found to inhibit amifostine's ability to prevent
radiation induced micronuclei formation (see FIG. 10a). And,
SOD2-siRNA transfection corresponded with inhibition of elevated
SOD2 activity (see FIG. 10b). Thus, amifostine-induced SOD2
elevation can confer a protective property that adds to
amifostine's usefulness as an anti-genomic instability agent.
[0099] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the methods described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
V. REFERENCES
[0100] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
[0101] U.S. Pat. No. 5,488,042 [0102] U.S. Pat. No. 5,567,686
[0103] U.S. Pat. No. 5,869,338 [0104] U.S. Pat. No. 5,891,856
[0105] U.S. Pat. No. 6,984,619 [0106] Bhatti et al., Radiat.
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Sequence CWU 1
1
1121DNAArtificial SequenceSynthetic Primer 1aaggaacaac aggccttatt c
21
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