Note: Descriptions are shown in the official language in which they were submitted.
CA 02659664 2009-01-26
76909-151D
IMPROVED RADIATION THERAPY METHODS
This is a divisional application of Canadian Patent Application No. 2,323,237
filed March 8, 1999.
Background of the Invention
Radiation therapy is currently one of the most useful methods of treating
cancerous tumors. However, radiation therapy damages normal tissue surrounding
the
tumor (U.S. Patent No. 5,599,712).
This damage can include fibrosis, remodeling of the extracellular matrix,
vascular
damage, aberrant angiogenesis, pneumonitis, atherogenesis, osteonecrosis,
mucositis,
immunosuppression and functional impairment (U.S. Patent No. 5,616,561).
As a result of these radiation-induced
side effects, techniques have been developed to minimize radiation-induced
damage
to surrounding normal tissues by limiting radiation to the lowest level
effective for
cancer treatment. Since there is a direct relationship between the amount of
radiation
and the effectiveness of the treatment, this method compromises the overall
effectiveness of the treatment.
For some cancer patients, hematopoietic toxicity frequently limits the
opportunity for radiation dose escalation (Watanabe et al., British J
Haematol.
94:619-627 (1996)). Repeated or, high dose cycles of radiation therapy may be
1
CA 02659664 2009-01-26
76909-1511D
responsible for severe stem cell depletion leading to important long-term
hematopoietic sequelea and marrow exhaustion (Masse et al., Blood 91:441-449
(1998). Such stem cell depletion leads to depletion of the full range of
hematopoietic
lineage specific cells, including megakaryocytes, platelets, monocytes,
neutrophils,
and lymphocytes, and the resulting complications of ,such depletion. For
example, in
patients suffering from depressed levels of platelets (thrombocytopenia) the
inability
to form clots is the most immediate and serious consequence, a potentially
fatal
complication of many therapies for cancer. Such cancer patients are generally
treated
for this problem with platelet transfusions. Other patients frequently
requiring platelet
to transfusions are those undergoing bone marrow transplantation or patients
with
aplastic anemia. Platelets for such procedures are obtained by
plateletpheresis from
normal donors. Like most human blood products, platelets for transfusion have
a
relatively short shelf-life and also expose the patients to considerable risk
of exposure
to dangerous viruses, such as the human immunodeficiency virus (HIV).
The administration of hematopoietic growth factors may reduce short-term
side effects induced by radiation, but has been hypothesized to cause long-
term
hematopoietic damage (Masse et al., 1998; Watanabe et al., 1996). Several
studies
have suggested that co-administration of negative hematopoietic regulators can
minimize radiation therapy-induced. myelotoxicity by reducing the number of
progenitor cells that enter the cell cycle. (Watanabe et al., 1996: Dunlop et
al., Blood
79:2221-2225 (1992); Paukovits et al., Blood 81:1755-1761; Bogden et A, Annals
N.Y. Acad. Sci. 628:126-139 (1991); Deeg et al., Ann. Hematol. 74:117-122
(1997);
Masse et al., 1998). This treatment is based on the premise that hematopoietic
stem
CA 02659664 2009-01-26
76909-151D
cells are relatively protected from radiation-related toxicity when quiescent,
particularly when the malignant cells are proliferating (Deeg et al., (1997)).
Bone marrow contains pluripotent stem cells that are capable of reconstituting
the entire hematopoietic system. Bone marrow transplantation has been used to
treat
various intractable hematopoietic diseases including leukemia and severe
aplastic
anemia. (U.S 1 Patent No. 5,186,931).
Typically, a bone marrow transplant patient is subjected to irradiation to
reduce the
leukocyte count to zero, followed by transplantation of bone marrow cells
which
function by producing a sufficient number of normal leukocytes. However,
various
complications, such as death, infectious diseases, graft versus host disease,
radiation
= nephritis, and interstitial pneumonia frequently occur during the time
period between
transplantation and the return to normal white blood cell levels after
transplantation.
As a result of these frequent side effects, no satisfactory methods are
currently
available for supporting bone marrow transplantation which are capable of both
increasing survival of bone marrow transplant patients and also accelerating
the
reconstitution of the hematopoietic system of the patient.
Chronic radiation injuries, such as radiation nephropathy, have been viewed as
inevitable, progressive and untreatable (Moulder et al., Bone Marrow
Transplantation
19:729-735 (1997)). The progressive and untreatable nature of late tissue
damage
follows from the assumption that the injury is due to delayed mitotic cell
death
resulting from genetic injury that is produced and irrevocably fixed in place
at the
time of irradiation (Moulder et at., 1997). Under this view, the only way to
decrease
the probability of injury is by limiting the radiation dose or shielding the
at risk
organs.
3
CA 02659664 2009-01-26
76909-151D
However, recent results indicate that late-onset radiation-induced tissue
injury
involves complex and dynamic interactions among parenchymal and vascular cells
within a particular organ (Moulder et al., 1997). This model of chronic
radiation
injury suggests that pharmacological intervention after radiation exposure
would be
effective.
Thus, despite advances in the field of radiation therapy, prior art methods
have
proven to be of limited utility in minimizing radiation-induced tissue damage,
and
improving the efficacy of tumor radiation therapy and bone marrow
transplantation.
Thus, there is a need for improved therapeutic methods to mitigate radiation
induced
to tissue damage and to improve the effectiveness of radiation therapy.
Furthermore,
the ability to stimulate endogenous platelet formation in thrombocytopenic
patients
with a concomitant reduction in their dependence on platelet transfusion would
be of
great benefit. In addition the ability to correct or prevent thrombocytopenia
in patients
undergoing radiation therapy or chemotherapy for cancer would make such
treatments
safer and possibly permit increases in the intensity of the therapy thereby
yielding
greater anti-cancer effects.
Summary of the Invention
In one aspect, the present invention provides methods and kits for mitigating
radiation induced tissue damage, improving the effectiveness of radiation
therapy, to
support bone marrow transplantation, and promoting megakaryocyte production
and
mobilization and platelet production, each method comprising the
administration of
angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogues
4
CA 02659664 2011-12-06
76909-151D
thereof, angiotensin II (All), All analogues, All fragments or
analogues thereof or All AT2 type 2 receptor agonists to a
patient in need thereof.
Specific aspects of the invention include:
- a use of an effective amount of at least one active
agent for increasing megakaryocyte production and mobilization,
platelet production or a combination thereof, the active agent
comprising a peptide with an amino acid sequence selected_from
the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO:
18, SEQ ID NO: 31, and SEQ ID NO: 38; and
- a megakaryocyte production and mobilization kit, a
platelet production kit or a combination thereof, comprising:
(a) an amount effective to support increasing megakaryocyte
production and mobilization, platelet production or a
combination thereof of at least one active agent comprising a
peptide with an amino acid sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID
NO: 31, and SEQ ID NO: 38, and (b) instructions for using the
effective amount of active agent for increasing megakaryocyte
production and mobilization, platelet production or a
combination thereof.
5
CA 02659664 2011-12-06
76909-151D
In another aspect of the present invention, an improved cell culture medium
and kits are provided for the production of megalcaryocy-tes and platelets
wherein the
improvement comprises addition to the cell culture medium of an effective
amount of
angiotensinogen, Al. Al analogues, Al fragments and analogues thereof; All,
All
analogues, All fragments or analogues thereof or All AT, type 2 receptor
4gonists.
These aspects and other aspects of the invention become apparent in light of
the following detailed description.
Brief Description of the Drawings
Figure 1 is a graph showing the effect of All treatment two days prior to
exposure on
post-irradiation mouse mortality.
Figure 2 is a graph showing the effect of All treatment on the day of exposure
on
post-irradiation mouse mortality.
is Figure 3 is a graph showing the effect of All treatment two days following
exposure
on post-irradiation mouse mortality.
Figure 4 is a graph showing the effect of All treatment two days prior to
exposure on
white blood cell number after irradiation.
Figure 5 is a graph showing the effect of All treatment on the day of exposure
on
white blood cell number after irradiation.
Figure 6 is a graph showing the effect of Al! treatment two days following
exposure
on white blood cell number after irradiation.
5a
CA 02659664 2009-01-26
76909-1519
Figure 7 is a graph showing the effect of All treatment two days prior to
exposure on
megakaryocyte number after irradiation.
Figure 8 is a graph showing the effect of All treatment on the day of exposure
on
megakaryocyte number after irradiation.
Figure 9 is a graph showing the effect of All treatment two days following
exposure
on megakaryocyte percentage after irradiation.
Figure 10 is a graph showing the effect of All treatment two days prior to
exposure
on monocyte number after irradiation.
Figure 11 is a graph showing the effect of All treatment on the day of
exposure on
to monocyte number after irradiation.
Figure 12 is a graph showing the cffect of All treatment two days following
exposure
on monocyte number after irradiation.
Figure 13 is a graph showing the effect of All treatment two days prior to
exposure
on neutrophil number after irradiation.
Figure 14 is a graph showing the effect of All treatment on the day of
exposure on
neutrophil number after irradiation.
Figure 15 is a graph showing the effect of All treatment two days following
exposure
on neutrophil number after irradiation.
Figure 16 is a graph showing the effect of All treatment two days prior to
exposure
on lymphocyte number after irradiation.
Figure 17 is a graph showing the effect of All treatment on the day of
exposure on
lymphocyte number after irradiation.
Figure 18 is a graph showing the effect of All treatment two days following
exposure
on lymphocyte number after irradiation. =
6
CA 02659664 2009-01-26
76909-151E)
Figure 19 is a graph showing is a graph showing the effect of AU analogues and
fragments treatment on white blood cell number after irradiation.
Figure 20 is a graph showing is a graph showing the effect of All analogues
and
fragments treatment on platelet number after irradiation.
Figure 21 is a graph showing the effect of All on mouse survival receiving
bone
marrow transplantation after lethal irradiation. =
Figure 22 is a graph showing the effect of All treatment on mouse survival
receiving bone
marrow transplantation after lethal irradiation.
Figure 23 is a graph showing the effect of All on white blood cell number in
the
blood of mice receiving bone marrow transplantation after lethal irradiation.
Figure 24 is a graph showing the effect of All on white blood cell number in
the
blood of mice receiving bone marrow transplantation after lethal irradiation..
Detailed Description of the Preferred Embodiments
The present invention fulfills the needs for improved therapeutic methods to
mitigate radiation induced tissue damage, to improve the effectiveness of
radiation
therapy, to support bone marrow transplantation, and to promote megalcaryocyte
.20 production and mobilization and platelet production.
As defined herein the phrase "mitigation of tissue damage" refers not only to
reduction of damage, but also encompasses recovery of tissue from damage. As
used
herein "tissue" refers to any tissue type, and also includes hematopoietic
stem and
progenitor cells, white blood cells and platelets.
7
CA 02659664 2009-01-26
16909-151D
As defined herein the term "megakaryocyte mobilization" refers to the
movement of a megakaryocyte precursor cell from the bone marrow into the
periphery.
As defined herein, the phrase "improved platelet production" or "improved
megakaryocyte production," means that the number of platelets or
megakaryocytes is
signifiCantly elevated above the normal range of platelets or megakaryocytes
in the
particular mammal involved. The elevation of platelet or megakaryocyte counts
may
occur in a time-dependent manner, and may be cyclical, increasing and then
constant
or decreasing, or constant, etc.
tO Unless otherwise indicated, the term "active agents" as used herein
refers to
the group of compounds comprising angiotensinogen, angiotensin. I (A1), Al
analogues, Al fragments and analogues thereof, angiotensin II (All), All
analogues,
All fragments or analogues thereof and All AT, type 2 receptor agonists.
Within this application, unless otherwise stated, the techniques utilized may
be
found in any of several well-known references such as: Molecular Cloning: A
Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory
Press),
Gene Expression Technology (Methods in Errzymology, Vol. 185, edited by D.
Goeddel, 1991, Academic Press, San Diego, CA), "Guide to Protein Purification"
in
Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR
Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic
Press,
San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, ri Ed.
(R.I.
Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression
Protocols,
pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, NJ), and the
Ambion
1998 Catalog (Ambion, Austin, TX).
8
CA 02659664 2009-01-26
76909-151D
U.S. Patent No. 5,015429 to DiZerega
describes a method for increasing the rate of
healing of wound tissue, comprising the application to such tissue of
angiotensin II
(AII) in an amount which is sufficient for said increase. The application of
All to
wound tissue significantly increases the rate of wound healing, leading to a
more
rapid re-enithelialization and tissue repair. The term AL! refers .to an
octapeptide
present in humans and other species having the sequence Asp-Arg-Val-Tyr-Ile-
His-
= Pro-Phe [SEQ ID NO:1]., The biological formation of angiotensin is initiated
by the
action of renin on the plasma substrate angiotensinogen. The substance so
formed is a
to decapeptide called angiotensin I (Al) which is converted to All by the
converting
enzyme angiotensinase which removes the C-terminal His-Leu residues from Al
(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu [SEQ ID NO:37]). All is a known
pressor agent and is commercially available. The use of All analogues and
fragments,
AT2 agonists, as well as AIII and AIII analogues and fragments in wound
healing has
also. been described. (U.S. Patent No. 5,629,292; U.S. Patent No. 5,716,935;
WO
96.139164).
Studies have shown that All increases mitogenesis and chemotaxis in cultured
cells that are involved in wound repair, and also increases their release of
growth
factors and extracellular matrices (diZerega, U.S. Patent No. 5,015,629; Dzau
et. al.,
= J. Alol. Cell. CardioL 21:S7 (Supp 111) 1989; Berk et. al., Hypertension
13:305-14
(1989); Kawahara, et al., BBRC 150:52-9 (1988); Nafiilan, et al., .1. Clin.
Invest.
83:1419-23 (1989); Taubman et at.. J. BioL Chem 264:526-530 (1989); Nakahara,
et
al., BBRC 184:811-8 (1992); Stouffer and Owens, Circ. Res. 70:820 (1992);
Wolf, et
al., Am. J. Pathol. 140:95-107 (1992); Bell and Madri, Am. J. PathoL 137:7-12
9
CA 02659664 2009-01-26
76909-151D
(1990). In addition, MI was shown to be angiogenic in rabbit corneal eye and
chick
chorioallantoic membrane models (Fernandez, et al., J. Lab. Clin. Med. 105:141
(1985); LeNoble, et al., Eur. J. Pharmacol. 195:305-6 (1991). Therefore, All
may
accelerate wound repair through increased neovascularization, growth factor
release,
reepithelialization and/or production of extracellular matrix.
AII has also been implicated in both cell growth and differentiation (Meffert
et
al., MoL and Cellul. ,Endocrin. 122:59 (1996)). Two main classes of All
receptors,
ATI and AT, have been identified (Meffert, 1996). The growth-promoting effects
of
All have been attributed to mediation by the ATI receptor, while some evidence
suggests that the AT2 receptor may bc involved in mediation of the cell
differentiation effects of All (Bedecs et al., Biochern. J. 325:449 (1997)).
The effects of All receptor and All receptor antagonists have been examined
in two experimental models of vascular injury and repair which suggest that
both All
receptor subtypes (AT! and AT2) play a role in wound healing (Janiak et at.,
Hypertension 20:737-45 (1992); Prescott, etal., Ain. I PathoL 139:1291-1296
(1991);
Kauffman. et al., Life Sci. 49:223-228 (1991); Viswanathan, et al., Peptides
13:783-
786 (1992); Kimura, et at., BBRC 187:1083-1090(1992).
Many studies have focused upon A11(1-7) (All residues 1-7) or other
fragments of All to evaluate their activity. AI1(177) elicits some, but not
the full range
of effects elicited by All. Pfeilschifter, et al., Eur. J. Pharniacol. 225:57-
62 (1992);
Jaiswal, et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and
Stack, J.
Pharrnacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol.
Exper.
Ther. 265:664-673 (1991); Jaiswal, et at., Hypertension 17:1115-1120 (1991);
Portsi,
eta., Br. J. Pharmacol. 111:652-654 (1994).
10
CA 02659664 2009-01-26
76909-1519
While a single pilot study has suggested that All-induced hypertension might
be effective in combination with radiation therapy in the treatment of lung
cancer
patients (Kato et al., Radiation Medicine 11:86-90 (1993)), many studies have
demonstrated that antagonists of angiotensin converting enzyme (ACE), which
mediate the production of All, are effective in reducing radiation
nephropathy, bone
mart;ow transplantation nephropathy, and acute radiation injury (Moulder et
nl., Int. I.
Radiation Onc. Biol. Phys. 27:93-99 (1993); Moulder et al., Bone Marrow
TranspL
19:729-735 (1997); Moulder et al., Radiation Res. 146:106-110 (1996); Cohen et
al.,
J. Lab. Clin. Med. 129:536-547 (1997); Moulder et al., Radiation Res. 136:404-
407
(1993); Yoon et al., Int. J. Radial. Oncol. Biol. Phys. 30:873-878 (1994);
Ward:et al.,
-Radiation Res. 135:81-87 (1993); Cohen et at, Lab. Invest. 75:349-360 (1996);
Cohen
et al., J. Lab. Clin. Med. 124:371-380 (1994); Gerarci et al., Radiation Res.
143:58-68
(1995)). The effect of the ACE inhibitors has been demonstrated, in at least
one case,
to be directly caused by the reduction of activation of the ATI receptor by
All
(Moulder et al.. Radiation Res. 146:106-110 (1996)). These results have led to
the
suggestion that, in the case of radiation nephropathy, the most effective
treatment is
the use of ACE inhibitors (Moulder et al., Bone Marrow Transplantation 19:729-
735
(1997)).
Furthermore, it has recently been demonstrated that angiotensinogen,
angiotensin I (Al). Al analogues, Al fragments and analogues thereof, All, All
analogues, All fragments or analogues thereof or AU AT-, type 2 receptor
agonists are
potent stimulators of hematopoietic stem cell proliferation (j.s. Patent No.
6,335,195).
Therefore, it
would be expected that the use of these compounds might cause long-term
11
CA 02659664 2009-01-26
76909-151D
hematopoietic damage if used in conjunction with radiation therapy (Masse et
al.,
1998; Watanabe et al., 1996).
Based on all of the above, it would be unexpected that the use of
angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogues
thereof, All, All analogues, All fragments or analogues thereof or All AT,
type 2
receptor agonists would be effective in reducing radiation-induced human
tissue
damage or in treating patients in need of radiation therapy.
None of these studies teach or suggest the use of angiotensinogen, angiotensin
I (Al), Al analogues, Al fragments and analogues thereof, angiotensin II
(All), All
analogues, All fragments or analogues thereof or All AT2 type. 2 receptor
agonists to
stimulate the production and mobilization of megakaryocytes, or to stimulate
the
production of platelets.
A peptide agonist selective for the AT2 receptor (MI has 100 times higher
affinity for AT2 than ATI) is p-aminophenylalanine6-AII ["(p-N1-12-Phe)6-
AII)"],
Asp-Arg-Val-Tyr-Ile-Xaa-Pro-Phe [SEQ ID NO.36] wherein Xaa is p-NH2-Phe
(Speth and Kim, BBRC 169:997-1006 (1990). This peptide gave binding
characteristics comparable to AT2 antagonists in the experimental models
tested
(Catalioto, et al., Eur. I. Phartnacol. 256:93-97 (1994); Bryson, et al., Eur.
Pharmacol. 225119-127 (1992).
The active Al, Al analogues, Al fragments and analogues thereof, All
analogues, fragments of All and analogues thereof of particular interest in
accordance
with the present invention are characterized as comprising a sequence
consisting of at
least three contiguous amino acids-of groups RI-R8 in the sequence of general
formula I
CA 02659664 2009-01-26
76909-151D
RI -R2-R3-R4-R5-R6_R7-R8
in which RI and R2 together form a group of formula
X-RA-RB-,
wherein X is H or a one to three peptide group,
RA is - suitably selected from Asp, Glu, Asn, Acpc (1-
aminocyclopentane carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu(NH2), Gly,
Asp(NH2) and Suc,
RB is suitably selected from Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg
and D-Lys;
R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile.
Gly, Pro, Aib, Acpc, Lys, and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(P03)2, Thr, Ser,
homoSer, Ala, and azaTyr;
R5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val
and Gly;
R6 is His, Arg or 6-1\1112-Phe;
R7 is Pro or Ala; and
R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr,
excluding sequences including R4 as a terminal Tyr group.
Compounds falling within the category of AT2 agonists useful in the practice
of the invention include the All analogues set forth above subject to the
restriction
that R6 is p-NH2-Phe. In addition to peptide agents, various nonpeptidic
agents (e.g.,
peptidomimetics) having the requisite AT2 agonist activity are further
contemplated
for use in accordance with the present invention.
13
CA 02659664 2009-01-26
76909-151D
Particularly preferred combinations for RA and RB are Asp-Arg, Asp-Lys, Glu-
Arg and Glu-Lys. Particularly preferred embodiments of this class include the
following: All, AHI or AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:21;
A1I(3-8), also known as desl-AIII or MV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID
NO:31;
AII(1-7), Asp-Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:4]; Arg-Val-Tyr-Ile-
His-Pro [SEQ ID NO:5]; MI(3-7), Val-Tyr-Ile-His-Pro [SEQ ID NO:6]; AII(5-8),
Ile-
His-Pro-Phe [SEQ ID NO:7]; AII(I-6), Asp-Arg-Val-Tyr-Ile-His [SEQ ID NO:8];
A1I(1-5), Asp-Arg-Val-Tyr-Ile [SEQ ID NO:9]; AH(1-4), Asp-Arg-Val-Tyr [SEQ ID
NO:10]; and MI(1-3), Asp-Arg-Val [SEQ ID NO:11]. Other preferred embodiments
to include: Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO:12] and Arg-Val-Tyr-
norLeu-
-His-Pro-Phe [SEQ ID NO:13]. Still another preferred embodiment encompassed
within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-
Tyr-
Ile-His-Pro-Phe [SEQ ID NO:31]. AII(6-8), His-Pro-Phe [SEQ ID NO:14] and AII(4-
8), Tyr-Ile-His-Pro-Phe [SEQ ID NO:15] were also tested and found not to be
effective.
A class of particularly preferred compounds in accordance with the present
invention consists of those with the following general structure:
RI -Arg-R2-R3-R4-His-Pro-R5
wherein RI is selected from the group consisting of H and Asp;
R2 is selected from the group consisting of Val and Pro;
R3 is selected from the group consisting of Tyr and Tyr(P03)2;
R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and
R5 is Phe, Ile, or is absent.
14
CA 02659664 2009-01-26
76909-151D
Particularly preferred embodiment of this class are selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26, SEQ liD
NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38
Another class of compounds of particular interest in accordance with the
present invention are those of the general formula II
in which R2 is selected from the group consisting of H, Arg, Lys, Ma,
Om, Ser(Ac), Sar, D-Arg and D-Lys;
R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile,
Cily, Pro, Aib, Acpc and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(P03)2, Thr, Ser,
homoSer and azaTyr;
R5 is selected from the group consisting of lie, Ala. Leu, norLeu, Val
and Gly;
R6 is His, Aru or 6-NH2-Phe;
R7 is Pro or Ala; and
R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.
A particularly preferred subclass of the compounds of general formula II has
the formula
R2-R3-Tyr-R5-His-Pro-Phe [SEQ ID NO:161
wherein R2, R3 and R5 are as previously defined. Particularly preferred is
angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2].
Other
15
CA 02659664 2009-01-26
76909-151D
preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-
Pro-Phe [SEQ ID NO:17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:18]. The
fragment A11(4-8) was ineffective in repeated tests; this is believed to be
due to the
exposed tyrosine on the N-terminus.
In the above formulas, the standard three-letter abbreviations for amino acid
residues are employed.. In the absence of an indication to the contrary, the L-
form of
the amino acid is intended. Other residues are abbreviated as follows:
TABLE 1
Abbreviation for Amino Acids
Me2Gly N,N-dimethylglycyl
Bet 1-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt
(betaine)
Suc Succinyl
: Phe(Br) p-bromo-L-phenylalanyl
azaTyr aza-a ' -homo-L-tyrosyl
Acpc 1-aminocyclopentane carboglic acid
Alb 2-aminoisobutyric acid
Sar N-methylglycyl (sarcosine) =
It has been suggested that All and its analogues adopt either a gamma or a
beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974). In general,
it is
believed that neutral side chains in position R3, R5 and R7 may be involved in
maintaining the appropriate distance between active groups in positions R4, R6
and R8
primarily responsible for binding to receptors and/or intrinsic activity.
Hydrophobic
16
CA 02659664 2009-01-26
76909-151D
side chains in positions R3, R5 and R8 may also play an important role in the
whole
conformation of the peptide and/or contribute to the formation of a
hypothetical
hydrophobic pocket.
Appropriate side chains on the amino acid in position R2 may contribute to
affinity of the compounds for target receptors and/or play an important role
in the
conformation of the peptide. For. this reason, Arg and Lys are particularly
preferred
as R2.
For purposes of the present invention, it is believed that R3 may be involved
in
the formation of linear or nonlinear hydrogen bonds with R5 (in the gamma turn
to model) or R6 (in the beta turn model). R3 would also participate in the
first tarn in a
beta antiparallel structure (which has also been proposed as a possible
structure). In
contrast to other positions in general formula I. it appears that beta and
gamma
branching are equally effective in this position. Moreover, a single hydrogen
bond
may be sufficient to maintain a relatively stable conformation. Accordingly,
R3 may
suitably be selected from Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and
Tyr. In
another preferred embodiment, R3 is Lys.
With respect to R4, conformational analyses have suggested that the side chain
in this position (as well as in R3 and R5) contribute to a hydrophobic cluster
believed
to be essential for occupation and stimulation of receptors. Thus, R4 is
preferably
selected from Tyr, Thr, Tyr (P03)2, homoSer, Ser and azaTyr. In this position,
Tyr is
particularly preferred as it may form a hydrogen bond with the receptor site
capable of
accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974),
supra). In a
further preferred embodiment, R4 is Ala.
17
CA 02659664 2009-01-26
7 6 9 0 9 - 1 5 1 ID
In position R5, an amino acid with a 13 aliphatic or alicyclic chain is
particularly desirable. Therefore, while Gly is suitable in position Rs, it is
preferred
that the amino acid in this position be selected from Ile, Ala, Leu, norLeu,
Gly and
Val.
In the Al, Al analogues, Al fragments and analogues thereof, All, All
analogues,; fragments and analogues of fragments of particular interest in
accordance
with the present invention, R6 is His, Arg or 6-NH2-Phe. The unique properties
of the
imidazole ring of histidine (e.g., ionization at physiological pH, ability to
act as
proton donor or acceptor, aromatic character) are believed to contribute to
its
particular utility as R6. For example, conformational models suggest that His
may
participate in hydrogen bond formation (in the beta model) or in the second
turn of
the antiparallel structure by influencing the orientation of R7. Similarly, it
is presently
considered that R7 should be Pro in order to provide the most desirable
orientation of
R8. In position R8, both a hydrophobic ring and an anionic carboxyl terminal
appear
to be particularly useful in binding of the analogues of interest to
receptors; therefore,
Tyr and especially Phe are preferred for purposes of the present invention.
Analogues of particular interest include the following:
TABLE 2
Angiotensin II Analogues
All Amino Acid Sequence Sequence
Analogue Identifier
Name
Analogue 1 Asp-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID NO: 19
Analogue 2 Asn-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID NO: 20
Analogue 3 Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe SEQ ID NO: 21
Analogue 4 Glu-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 22
Analogue 5 Asp-Lys-Val-Tyr-Ile-His-No-Phe SEQ ID NO: 23
18
CA 02659664 2009-01-26
6909-15119
Analogue 6 Asp-Arg-Ala-Tyr-Ile-His-Pro-Phe = SEQ ID NO: 24
Analogue 7 Asp-Arg-Val-Thr-Ile-His-Pro-Phe SEQ ID NO: 25
'Analogue 8 _ Asp-Arg-Val-Tyr-Leu-His-Pro-Phe SEQ ID NO: 26 ¨
Analogue 9 Asp-Arg-Val-Tyr-Ile-Arg-Pro-Phe SEQ ID NO: 27 ,
Analogue 10 Asp-Arg-Val-Tyr-Ile-His-Ala-Phe SEQ ID NO: 28
Analogue 11 Asp-Arg-Val-Tyr-Ile-His-Pro-Tyr SEQ ID NO: 29
Analogue 12 Pro-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 30
Analogue 13 _ Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe SEQ ID NO: 31
Analogue 14 Asp-Arg-Val-Ty003)2-11e-His-Pro-Phe SEQ ID NO: 32
Analogue 15 Asp-Arg-norLeu-Tyr-Ile-His-Pro-Phe SEQ ID NO: 33 ,
Analogue 16 _ Asp-Arg-Val-Tyr-norLeu-His-Pro-Phe SEQ ID NO: 34
Analogue 17 Asp-Arg-Val-homoSer-Tyr-Ile-His-Pro-PheSEQID NO: 35
The polypeptides of the instant invention may be synthesized by methods such
as those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide
Synthesis,
2nd ed., Pierce Chemical Co., Rockford, 111. (1984) and J. Meienhofer,
Hormonal
3 Proteins and Peptides. Vol. 2, Academic Press, New York, (1973) for solid
phase
synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press,
New
York, (1965) for solution synthesis.
In general, these methods involve the sequential addition of protected amino
to acids to a growing peptide chain (U.S. Patent No. 5,693,616).
Normally, either the amino or carboxyl group of the first
amino acid and any reactive side chain group are protected. This protected
amino acid
is then either attached to an inert solid support, or utilized in solution,
and the next
amino acid in the sequence,- also suitably protected, is added under
conditions
15 amenable to formation of the amide linkage. After all the desired amino
acids have
been linked in the proper sequence, protecting groups and any solid support
are
removed to afford the crude poTypeptide. The polypeptide is desalted and
purified,
preferably chromatographically, to yield the final product.
19
=
CA 02659664 2009-01-26
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In one aspect, the present invention provides methods and kits for the
mitigation of tissue damage due to radiation exposure comprising the
administration
of angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and
analogues
thereof, angiotensin H (MI), All analogues, All fragments or analogues thereof
or All
AT2 type 2 receptor agonists (the "active agents").
In another aspect, the present invention provides improved methods and kits
for treating a patient .afflicted with a neoplastic disease state that is
being treated with
ionizing or nonionizing radiation, the improvement comprising conjunctive
therapy
whereby an effective radioprotective amount of the active agents is provided.
to In another aspcct, the present invention provides improved methods and
kits
for treating a patient in need of radiation therapy, the improvement
comprising the
administration of the active agents in conjunction with the radiation therapy.
The invention is appropriate for use with any type of ionizing radiation
exposure such as therapeutic or accidental X-ray, gamma ray, or beta particle
exposure. Examples of ionizing radiation exposure suitable for treatment with
the
methods and kits of the present invention include, but are not limited to,
clinical
radiation therapy, medical diagnostics using radioactive tracers, exposure to
naturally
occurring ionizing radiation sources such as uranium and radon, wartime
exposure,
and accidental exposures including occupational exposure at nuclear power
facilities,
and medical and research institutions. Examples of nonionizing radiation
exposure
suitable for treatment with the methods and kits of the present invention
include, but
are not limited to, ultraviolet light, X-rays, microwaves, radio-frequency
waves, and
electromagnetic radiation.
20
CA 02659664 2009-01-26
/6909-151D
Virtually any tissue susceptible to radiation-induced tissue damage can gain
protection by use of the active agents of the invention. For example, breast
tissue is
an excellent candidate for receiving the benefit of the subject invention.
Radiation-
induced tissue damage can be a fatal side effect of over-exposure to radiation
therapy.
Typically, the fibrotic reaction common in normal breast tissue surrounding
the
cancerous tumor being treated with radiation therapy undermines the cosmetic
advantages of radiation therapy over surgical treatment. This disadvantage
will lead
many patients to elect a less effective or more dangerous treatment after
radiation
therapy.The present invention is also particularly suitable for those patients
in need of
repeated or high doses of radiation therapy. For some cancer patients,
hematopoietic
toxicity frequently limits the opportunity for radiation dose escalation
(Watanabe et
al., British J. Haentatol. 94:619-627 (1996)). Repeated or high dose cycles of
radiation therapy may be responsible for severe stem cell depletion leading to
important long-term hematopoietic sequelea and marrow exhaustion. The methods
of
the present invention provide for improved mortality and blood cell count when
used
in conjunction with radiation therapy.
Skin exposure is particularly common in accidental radiation exposure. It is
an
excellent candidate for the inventive therapy, especially as the compounds of
the
.20 invention can be administered topically. Other tissues that are
susceptible to
radiation-induced damage following accidental or therapeutic ionizing or
nonionizing
radiation exposure include, but are not limited to: liver, lung,
gastrointestinal tract,
kidneys, testes, salivary gland, mucosa and brain.
21
CA 02659664 2009-01-26
76909-151D
In another aspect, the present invention provides improved methods and kits
for supporting bone marrow transplantation comprising the administration of
the
active agents to a patient in need thereof. These compounds may be
administered in
combination with auxiliary agents including, but not limited to interleukin
(IL)-3, IL-
1, IL-4,11-5, granulocyte colony stimulating factor (G-CSF), granulocyte-
macrophage
colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-
CSF),
anticancer agents, antiviral agents, and antibiotics.
In a further aspect, the present invention provides kits for mitigating
radiation
induced tissue damage and improving the efficacy of radiation therapy, wherein
the
to kits comprise an effective amount of the active agents of the invention for
mitigating
radiation induced tissue damage or improving the efficacy of radiation
therapy, and
instructions for using the amount effective of active agent as a therapeutic.
In a
preferred embodiment, the kit further comprises a pharmaceutically acceptable
carrier, such as those adjuvants described above. In another preferred
embodiment,
the kit further comprises a means for delivery of the active agent to a
patient. Such
devices include, but are not limited to syringes, matrical or micellar
solutions.
bandages, wound dressings, aerosol sprays, lipid- foams, transderrnal patches,
topical
administrative agents, polyethylene glycol polymers, carboxymethyl cellulose
preparations, crystalloid preparations. (e.g., saline, Ringer's lactate
solution,
phosphate-buffered saline, etc.), viscoelastics, polyethylene glycols. and
polypropylene glycols. The means for delivery may either contain the effective
amount of angiotensinogen, Al, Al analogues, Al fragments and analogues
thereof,
All, All analogues, All fragments or analogues thereof or All AT7 type 2
receptor
11
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76909-15117
agonists, or may be separate from the compounds, which are then applied to the
means for delivery at the time of use.
The methods and kits of the present invention, by mitigating radiation induced
tissue damage and improving the efficacy of radiation therapy and bone marrow
transplantation, significantly enhance the utility of presently available
treatments both
for radiation-induced tissue damage and for clinical radiation therapy.
In a further aspect of the present invention, a method of increasing
megakaryocyte production and mobilization and platelet production by .exposure
to
the active agents of the inventions is disclosed. In one embodiment,
megakaryocytes
are isolated from bone marrow as described in U.S.. Patent No. 5,178,856).
Briefly, marrow is flushed from a
subject's femur with Iscove's modification of Dulbecco's medium (IMDM)
supplemented with Nutridom1SP (Boehringer Mannheim, Indianapolis, hid.), a
serum-free medium supplement. For culture studies, a single cell suspension is
made
by repetitive expulsion through progressively smaller needles. For flow
cytornetry
controls, a monocellular suspension is made by gentle filtration through a 100
micron
nylon mesh. Preferably, adherent cells are -removed to enrich the numbers of
megakaryocytes or their progenitor cells. Up to 2 x 106 cells/ml are placed in
growth
medium at 37 C in a humidified atmosphere in the presence of, preferably,
between
about 0.1 ng/ml and about 10 mg/m1 of the active agents. The cells are
expanded for
a period of between 2 and 21 days and cellular proliferation is assessed at
various
time points during this time period. Subsequent medium changes are performed
as
needed. In a preferred embodiment, megakaryocyte production and mobilization
and
platelet production are assessed by the extent of megakaryocyte ploidization
by flow
*Trade mark 23
CA 02659664 2009-01-26
'6909-1519
cytometry as described in U.S. Patent No. 5,155,211).
Briefly, the appearance of granules and the extensive surface-
connected open canalicular membrane system as well as a substantial decrease
in the
nucleus:cytoplasm volume distribution, indicates that the megalcaryocyte
population
has completed the process of polyploidization but has not yet generated, a
major
portion of their final complement of platelet-specific cytoplasmic components.
In another embodiment, subjects are irradiated as above and active agent is =
injected subcutaneously before, at the time of, and after irradiation. Blood
samples =
are taken at various times after administration of the active agent to monitor
the
number of white blood cells, megalcaryocytes and platelets. In a preferred
embodiment, subjects are treated with total body irradiation and active agent
is
administered subcutaneously (10 jig/kg/day or 100 jig/kg/day) at various times
before
= and after irradiation. The number of white blood cells, megalcaryocytes
and platelets
is preferably determined by counting with a hemacytometer followed by
differential
morphologic analysis.
= In another embodiment of this aspect of the invention; hematopoietic
= precursor cells are isolated from bone marrow, peripheral blood or
umbilical cord
blood and cultured under appropriate growth conditions, in the presence of the
active
agents. Megalcaryocyte production is assessed at various time points during
culture
2Q by differential morphologic analysis.
In a preferred embodiment, hematopoietic precursor cells are isolated from
bone marrow aspirates from the posterior iliac crest (Caplan and Haynesworth,
U.S.
Patent No. 5.486,359). CD34+ hematopoietic precursor cells- are isolated from
the
aspirate by attaching a biotinylated monoclonal antibody specific for CD34
(available
24
CA 02659664 2009-01-26
76909-151D
from Becton Dickinson, Sunnyvale, CA, USA) to a streptavidin affinity column
(Ceprate* SC; CellPio, Bothell, WA, USA) and passing the aspirate through the
column, folldwed by appropriate column washing and stripping, according to
standard
techniques in the art. The isolated cells are suspended in culture medium and
incubated in the presence of, preferably, between about 0.1 ng/ml and about 10
mg/m1
of th'e active agents of the invention. The cells are expanded for a period of
between 8
and 21 days and me_gakaryocyte production is assessed via phase microscopy to
detect
increased size and polyploidization at various points during this time period.
In a further embodiment of the present invention, a method of increasing
megakaryocyte production and mobilization and platelet production by exposure
to
the active agents is disclosed, either in the presence or absence of other
growth factors
and cytoldnes. Examples of such growth factors and cytokines include, but are
not
limited to thrombopoietin, lympholdnes, interleukins - 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11,
granulocyte colony-stimulating factor, granulocyte/macrophage colony
stimulating
factor, macrophage colony-stimulating factor, tumor necrosis factor, epidermal
growth
factor, fibroblast growth factor, platelet derived growth factor, transforming
growth
factor beta, and stem cell factor.
In a further preferred embodiment, megakaryocytes and/or platelets that have
been cultured in the presence of the active agents are used for autologous
transplantation, to reconstitute a depleted hematopoietic system. Prior to
transplantation, the cells are rinsed to remove all traces of culture fluid,
resuspended
in an appropriate medium and then pelleted and rinsed several times. After the
final
rinse, the cells are resuspended at between 0.7 x 106 and 50 x 106 cells per
ml in an
appropriate medium and reinfused into a subject through intravenous infusions.
*Trade mark 25
CA 02659664 2009-01-26
,6909-151D
Following transplantation, subject peripheral blood samples are evaluated for
increased megakaryocyte ploidy and platelet number by standard flow cytometry
and
cell sorting techniques. (Talmadge, et al., supra).
In another aspect of the present invention the active agents are used to
increase
in vivo megakaryocyte production and mobilization and platelet production. For
use
in increasing megakaryocyte production and mobilization and platelet
production, the
active agents may be administered by any suitable route, including orally,
parentally,
by inhalation spray, rectally, or topically in dosage unit formulations
containing
conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
The term
parenteral as used herein includes, subcutaneous, intravenous, intramuscular,
intrasternal, intratendinous, intraspinal, intraeranial, intrathoracic,
infusion techniques
or intraperitoneally. The active agents of all aspects of the present
invention may be administered
by any suitable route, including orally, parentally, by inhalation spray,
rectally, or
topically in dosage unit formulations containing conventional pharmaceutically
acceptable carriers, adjuvants, and vehicles. The term parenteral as used
herein
includes, subcutaneous, intravenous, intraarterial, intramuscular,
intrastemal,
intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques
or
intraperitoneally.
=
The active agents of the invention may be made up in a solid form (including
granules, powders or suppositories) or in a liquid form (e.g., solutions,
suspensions, or
emulsions). The compounds of the invention may be applied in a variety of
solutions.
Suitable solutions for use in accordance with the invention are sterile,
dissolve
sufficient amounts of the peptide, and are not harmful for the proposed
application. In
26
CA 02659664 2009-01-26
76909-151D
this regard, the compounds of the present invention are very stable but are
hydrolyzed
by strong acids and bases. The compounds of the present invention are soluble
in
organic solvents and in aqueous solutions at pH 5-8.
The active agents may be subjected to conventional pharmaceutical operations
such as sterilization and/or may contain conventional adjuvants, such as
preservatives,
stabipzers, wetting agents, emulsifiers, buffers etc. For administration, the
active
agents are ordinarily combined with one or more adjuvants appropriate for the
indicated route of administration. The compounds may be admixed with lactose,
sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid,
talc,
magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric
and
sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine,
and/or
polyvinyl alcohol, and tableted or encapsulated for conventional
administration.
Alternatively, the compounds of this invention may be dissolved in saline,
water,
polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal
solutions,
ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum,
and/or
various buffers. Other adjuvants and modes of administration are well known in
the
pharmaceutical art. The carrier or diluent may include time delay material,
such as
glyceryl monostearate or glyceryl distearate alone or with a wax, or other
materials
well known in the art.Formulations suitable for topical administration include
liquid or semi-liquid
preparations suitable for penetration through the skin (e.g., liniments,
lotions,
ointments, creams, or pastes) and drops suitable for administration to the
eye, ear, or
nose.
27
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/6909-151D
The dosage regimen for mitigating radiation-induced tissue damage and
improving the efficacy of radiation therapy with the active agents is based on
a variety
of factors, including the type of injury, the age, weight, sex, medical
condition of the
individual, the severity of the condition, the route of administration, and
the particular
compound employed. Thus, the dosage regimen may vary widely, but can be
determined routinely by a physician using standard methods. Dosage levels of
the
order of between 0.1 ng/kg and 10 mg/kg body weight of the active agents are
useful
for all methods of use disclosed herein.
The treatment regime will also vary depending, on the disease being treated,
to based on a variety of factors, including the type of injury, thc agc,
weight, sex,
medical condition of the individual, the severity of the condition, the route
of
administration, and the particular compound employed. For example, the active
agents are administered to an oncology patient for up to 30 days prior to a
course of
radiation therapy and for up to 60 days post-radiation exposure. The therapy
is
administered for 1 to 6 times per day at dosages as described above.
In all of these embodiments, the compounds of the invention can be
administered either prior to, simultaneously with; or subsequent to radiation
exposure.
In a preferred embodiment, the active agent is administered subcutaneously.
A suitable subcutaneous dose of active ingredient of active agent is
preferably
between about 0.1 ng/kg and about 10 mg/kg administered twice daily for a time
sufficient to mitigate radiation-induced tissue damage, to provide a
radioprotective
effect for a radiation therapy patient afflicted with a neoplastic disease, to
effectively
treat a patient in need of radiation therapy, to support bone marrow
transplantation
and to promote megakaryocyte production and mobilization and platelet
production.
CA 02659664 2009-01-26
76909-151D
In a more preferred embodiment, the concentration of active agent is between
about
100 ng,/kg body weight and about 10.0 mg/kg body weight. In a most preferred
embodiment, the concentration of active agent is between about 10 jig/kg body
weight
and about 10.0 mg/kg body weight. This dosage regimen maximizes the
therapeutic
benefits of the subject invention while minimizing the amount of agonist or
peptide
needed. Such an application minimizes costs as well as possible deleterious
side
effects.
For subcutaneous administration, the active ingredient may comprise from
0.0001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although
it
to may comprise as much as 10% w/w, but preferably not more than 5% w/w, and
more
preferably from 0.1% to 1% of the formulation.
In another preferred embodiment of the present invention, the active agent is
administered topically. Suitable topical doses and active ingredient
concentration in
the formulation are as described for subcutaneous administration.
In a preferred embodiment of all of the aspects of the invention, the active
agent is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO:2, SEQ
ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,
SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,
SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,
SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34; SEQ ID NO:35,
SEQ ID NO:36; and SEQ ID NO:37.
CA 02659664 2009-01-26
/ 690 9-1 5 1D
In a further preferred embodiment of the above aspects of the invention,
administration of the active agent is localized to the area affected by the
tissue-
damaging radiation.
In another aspect of the present invention, an improved cell culture medium is
provided for megakaryocyte and platelet production, wherein the improvement
comprises addition to the cell culture medium of an effective amount of
between
about 0.1 ng and 10 mg/ml of the active agents of the invention. Any cell
culture
media that can support megakaryocyte and platelet production can be used with
the
present invention. Such cell culture media include, but are not limited to
Basal Media
Eagle, Dulbecco's Modified Eagle Medium, 'wove's Modified Dulbecco's Medium,
McCoy's Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, Opti-
MEM Reduced-Serum Medium, RPM! Medium, and Macrophage-SFM Medium or
combinations thereof.
The improved cell culture medium can be supplied in either a concentrated (ie:
10X) or non-concentrated form, and may be supplied as either a liquid, a
powder, or a
lyophilizate. The cell culture may be either chemically defined, or may
contain a
serum supplement. Culture media is commercially available from many sources,
such
as GIBCO BRL (Gaithersburg, MD) and Sigma (St. Louis, MO)
In a further aspect, the present invention provides kits for megakaryocyte and
platelet production, wherein the kits comprise an amount effective for
megakaryocyte
and platelet production of the active agents of the invention, and
instructions for its
use as a cell culture media supplement.
In a preferred embodiment, the kits further comprise cell culture growth
medium. Any cell culture media that can support megakaryocyte and platelet
30
CA 02659664 2009-01-26
76909-151D
production can be used with the present invention. Examples of such cell
culture
media are described above. The cell culture medium can be supplied in either a
concentrated (ie: 10X) or non-concentrated form, and may be supplied as either
a
liquid, a powder, or a lyophilizate. The cell culture may be either chemically
defined,
or may contain a serum supplement.
In a further preferred embodiment, the kit further comprises a sterile
container,
which can comprise either a sealed container, such as a cell culture flask, a
roller
bottle, or a centrifuge tube, or a non-sealed container, such as a cell
culture plate or
microtiter plate (Nunc; Naperville, IL).
to In another preferred
embodiment, the kit further comprises an antibiotic
supplement for inclusion in the reconstituted cell growth medium. Examples of
appropriate antibiotic supplements include, but are not limited to actimonycin
D,
Fungizone , kanamycin, neomycin, nystatin, penicillin, streptomycin, or
combinations thereof (GIBCO).The present invention may be better understood
with reference to the
accompanying example that is intended for purposes of illustration only and
should
not be construed to limit the scope of the invention, as defined by thc claims
appended
hereto.
=
Example I Effect of All on rat mortality and white blood cell recovery after
irradiation
Female C57B1/6 mice (Jackson Labs, Bar Harbor, Maine) were irradiated
with 600 cGy total body irradiation. Subcutaneous injection with either All
(10
ug/kg/day or 100 ug/kg/day) or saline (placebo) was initiated two days before
(-day
31
CA 02659664 2009-01-26
/ 6909-151 D
2), on the day of (day 0) or 2 days after (+ day 2) irradiation and continued
until the
animals succumbed to the irradiation or were necropsicd. At various times
after
irradiation, the mice were anaesthetized with Metofane* (Pittman-Moore Animal
Health, NZ) and bled via the retro-orbital sinus. Red blood cells were lysed
with
0.3% acetic acid and. the number of white blood cells was determined by
counting
with a hemacytometer. The data in Figures 1-3 show that administration of All
starting at two days prior to irradiation did not protect against mortality
resulting from
irradiation (Figure 1), but that All administration on the day of irradiation
(Figure 2)
or two days after irradiation (Figure 3) substantially increased survivaL
Furthermore,
MI administration at all time periods tested increased the number of
circulating white
blood cells (Figures 4-6). Further experiments demonstrated that All
administration
increased the number of megakaryocytes (Figures 7-9), monocytes (Figures 10-
12),
neutrophils (Figures 13-15), and lymphocytes (Figures 16-18). These. data
demonstrate that in vivo administration of All can improve hematopoietic
recovery
after irradiation.
Example 2. Effect of All and All Analogs/Fragments on WBC and platelet numbers
After Irradiation
The animals were irradiated and treated as in Example 1, however, treatment
started on day 0 only with one subcutaneous injection of either 10. g/lcg or
100 fig/kg
daily until the study was terminated. Analogues and fragments of All (see
Table 3)
were assessed for their effect on WBC recovery and platelet number after
irradiation.
The data are shown in Figures 19 and 20. and show that the peptides increase
the
production of both of these blood ele?nents.
*Trade mark 32
CA 02659664 2009-01-26
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Table 3: Designation for Analogues/Fragments
Name Abbreviation Sequence SEQ ID NO:
GSD 28 I1e8-AII DRVY11HPI SEQ ID NO: 38
GSD 2413 Pro3-AII DRPYIHPF SEQ ID NO:31
GSD 22A A1a4-AIII RVYAHPF SEQ ID NO:18
AII(1-7) DRVYIHP SEQ ID NO:4
All DRVYIHPF SEQ ID NO. 1
Example 3. EffeCt of All on survival of mice receiving bone marrow
transplantation after lethal irradiation
Donor C57B1/6 mice (female, 6-8 weeks old) were irradiated with 600 cGy
total body irradiation. Starting on the day of irradiation, the mice received
either
saline (0.1 ml) or 20 pg/m1 angiotensin 11 (0.1 ml, 100 g/kg) subcutaneously
for
fourteen days. At the end of this period, the bone marrow was harvested from
the
femur by flushing and the number of viable nucleated cells determined by
counting
under a light microscope on a hemacytometer in the presence of trypan blue.
These donor bone marrow cells were then injected intravenously into recipient
mice (female C57B1/6, 6-8 weeks old) that had been lethally irradiated (900
cGy total
body irradiation) at two. concentrations: 1 x 1,06 or I x 105 cells per mouse.
After
injection, the recipient mice received either saline or 100 pg/kg All
subcutaneously
until death or termination. The study design in its entirety is as follows:
Donor Recipient Cell Number
Saline Saline 1 x 106
Saline Saline 1 x 10)
Saline All I X 106
Saline All lx 105
All Saline 1 x 106
All Saline 1 x 105
All All 1 x 106
All All 1 x 105
33
CA 02659664 2009-01-26
76909-151D
The survival of the mice and the number of circulating white blood cells were
measured as a function of time post-bone marrow transplantation. The data are
presented , in Figures 21-24, and demonstrate that All treatment increased
both
survival and white blood cell number in mice receiving bone marrow
transplantation
after irradiation. The greatest benefit was conferred by treatment of both the
donor
bone marrow cells and the recipient mice with All.
The methods and kits of the present invention, by mitigating radiation induced
tissue damage and improving the efficacy of radiation therapy, significantly
enhance
to the utility of presently available treatments both for radiation-induced
tissue damage
and for clinical radiation therapy, as well as bone marrow transplantation by
increasing the survival rate of patients and accelerating the reconstitution
of the
patient's hematopoietic system. Similarly, by providing a method for
megakaryocyte
and platelet production, the present invention will greatly augment clinical
cancer
treatments and bone marrow transplantation and other conditions that lead to
decreased megakaryocyte production and mobilization and platelet production.
The method of the present invention also increases the potential utility of
megakaryocytes as vehicles for gene therapy in hematopoietic disorders, by
providing a more efficient means to rapidly expand transfected megakaryocytes.
It is to be understood that the invention is not to be limited to the exact
details
of operation, or to the exact compounds, compositions, methods, procedures or
embodiments shown and described, as obvious modifications and equivalents will
be
apparent to one skilled in the art, and the invention is therefore to be
limited only by
the full scope of the appended claims.
34
CA 02659664 2009-02-10
SEQUENCE LISTING
<110> Rodgers, Kathleen
diZerega, Gere
<120> Radiation Therapy Methods
<130> 97017K2B
<140> PCT/US99/05194
<141> 1999-03-08
<160> 38
<170> PatentIn Ver. 2.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:ATI
<400> 1
Asp Arg Val Tyr Ile His Pro Phe
1 5
<210> 2
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:ATI (2-8)
<400> 2
Arg Val Tyr Ile His Pro Phe
I 5
<210> 3
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (3-8)
<400> 3
Val Tyr Ile His Pro Phe
1 5
<210> 4
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:ATI (1-7)
<400> 4
Asp Arg Val Tyr Ile His Pro
1 5
1
CA 02659664 2009-02-10
<210> 5
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (2-7)
<400> 5
Arg Val Tyr Ile His Pro
1 5
<210> 6
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (3-7)
<400> 6
Val Tyr Ile His Pro
1 5
<210> 7
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (5-8)
<400> 7
Ile His Pro Phe
1
<210> 8
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-6)
<400> 8
Asp Arg Val Tyr Ile His
1 5
<210> 9
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-5)
<400> 9
Asp Arg Val Tyr Ile
1 5
<210> 10
<211> 4
<212> PRT
<213> Artificial Sequence
2
CA 02659664 2009-02-10
<220>
<223> Description of Artificial Sequence:AII (1-4)
<400> 10
Asp Arg Val Tyr
1
<210> 11
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-3)
<400> 11
Asp Arg Val
1
<210> 12
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
<220>
<221> MOD RES
<222> (2)
<223> Nle
<400> 12
Arg Xaa Tyr Ile His Pro Phe
1 5
<210> 13
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
<220>
<221> MOD_RES
<222> (4)
<223> Nle
<400> 13
Arg Val Tyr Xaa His Pro Phe
1 5
<210> 14
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (6-8)
<400> 14
His Pro Phe
1
3
CA 02659664 2009-02-10
<210> 15
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (4-8)
<400> 15
Tyr Ile His Pro Phe
1 5
<210> 16
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
class
<220>
<221> UNSURE
<222> (1)
<223> Xaa at position 1 can be Arg, Lys, Ala, Orn, Ser,
MeGly, D-Arg, or D-Lys
<220>
<221> UNSURE
<222> (2)
<223> Xaa at position 2 can be Val, Ala, lieu, Nle, Ile,
Gly, Pro, Aib, Acp, or Tyr
<220>
<221> UNSURE
<222> (4)
<223> Xaa at position 4 can be Ile, Ala, lieu, Nle, Val,
or Gly
<400> 16
Xaa Xaa Tyr Xaa His Pro Phe
1 5
<210> 17
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
<400> 17
Arg Val Tyr Gly His Pro Phe
1 5
<210> 18
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
4
CA 02659664 2009-02-10
<400> 18
Arg Val Tyr Ala His Pro Phe
1 5
<210> 19
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 1
<400> 19
Asp Arg Val Tyr Val His Pro Phe
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 2
<400> 20
Asn Arg Val Tyr Val His Pro Phe
1 5
<210> 21
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 3
<400> 21
Ala Pro Gly Asp Arg Ile Tyr Val His Pro Phe
1 5 10
<210> 22
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 4
<400> 22
Glu Arg Val Tyr Ile His Pro Phe
1 5
<210> 23
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 5
<400> 23
Asp Lys Val Tyr Ile His Pro Phe
1 5
5
CA 02659664 2009-02-10
<210> 24
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 6
<400> 24
Asp Arg Ala Tyr Ile His Pro Phe
1 5
<210> 25
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 7
<400> 25
Asp Arg Val Thr Ile His Pro Phe
1 5
<210> 26
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 8
<400> 26
Asp Arg Val Tyr Leu His Pro Phe
1 5
<210> 27
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 9
<400> 27
Asp Arg Val Tyr Ile Arg Pro Phe
1 5
<210> 28
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 10
<400> 28
Asp Arg Val Tyr Ile His Ala Phe
1 5
<210> 29
<211> 8
<212> PRT
<213> Artificial Sequence
6
CA 02659664 2009-02-10
<220>
<223> Description of Artificial Sequence:AII analogue 11
<400> 29
Asp Arg Val Tyr Ile His Pro Tyr
1 5
<210> 30
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 12
<400> 30
Pro Arg Val Tyr Ile His Pro Phe
1 5
<210> 31
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 13
<400> 31
Asp Arg Pro Tyr Ile His Pro Phe
1 5
<210> 32
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 14
<220>
<221> MOD_RES
<222> (4)
<223> PHOSPHORYLATION
<400> 32
Asp Arg Val Tyr Ile His Pro Phe
1 5
<210> 33
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 15
<220>
<221> MOD RES
<222> (3)
<223> Nle
<400> 33
Asp Arg Xaa Tyr Ile His Pro Phe
1 5
7
CA 02659664 2009-02-10
<210> 34
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 16
<220>
<221> MOD RES
<222> (5)
<223> Nle
<400> 34
Asp Arg Val Tyr Xaa His Pro Phe
1 5
<210> 35
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:ATI analogue 17
<220>
<221> MOD_RES
<222> (4)
<223> homo Ser
<400> 35
Asp Arg Val Ser Tyr Ile His Pro Phe
1 5
<210> 36
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial
Sequence:p-aminophenylalanine 6 All
<220>
<221> MOD_RES
<222> (6)
<223> p-aminophenylalanine
<400> 36
Asp Arg Val Tyr Ile Xaa Pro Phe
1 5
<210> 37
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:angiotensin I
<400> 37
Asp Arg Val Tyr Ile His Pro Phe His Leu
1 5 10
8
CA 02659664 2009-02-10
<210> 38
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:1GD: I1e8-AII
<400> 38
Asp Arg Val Tyr Ile His Pro Ile
1 5
9
