AU744799B2 - Improved radiation therapy methods - Google Patents

Improved radiation therapy methods Download PDF

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AU744799B2
AU744799B2 AU29957/99A AU2995799A AU744799B2 AU 744799 B2 AU744799 B2 AU 744799B2 AU 29957/99 A AU29957/99 A AU 29957/99A AU 2995799 A AU2995799 A AU 2995799A AU 744799 B2 AU744799 B2 AU 744799B2
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Gere Dizerega
Kathleen E Rodgers
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University of Southern California USC
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    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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    • C12N2501/32—Angiotensins [AT], angiotensinogen

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Description

1 IMPROVED RADIATION THERAPY METHODS 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, incorporated by reference herein in its entirety). This damage can include fibrosis, remodeling of the extracellular matrix, vascular damage, aberrant angiogenesis, pneumonitis, atherogenesis, osteonecrosis, mucositis, immunosuppression and functional impairment Patent No. 5,616,561, incorporated by reference herein in its entirety). 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 is for radiation dose escalation (Watanabe et al., British J. Haematol. 94:619-627 (1996)).
Repeated or high dose cycles of radiation therapy may be oo• o o IR:\ I BVVj04136.doc:rr WO 99/45945 PCT/US99/05194 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 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 al., 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 WO 99/45945 PCT/US99/05194 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. Patent No. 5,186,931, incorporated by reference herein in its entirety.) 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 al., 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.
4 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 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 15 anti-cancer effects.
Summary of the Invention According to a first embodiment of the present invention there is provided a method for the mitigation of tissue damage due to radiation exposure comprising the administration of an amount effective for the mitigation of tissue damage of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R R8 in the sequence of general formula I
R'-R
2
-R
8 wherein R' is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, 0. Me Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Suc; 25 R 2 is selected from the group consisting of Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;
R
3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;
R
4 is selected from the group consisting of Tyr, Tyr(PO 3 2 Thr, Ser, homoSer, Ala, and azaTyr;
R
5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;
R
6 is His, Arg or 6-NHz-Phe;
R
7 is Pro or Ala; and R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; wherein the active agent does not include R 4 as a terminal Tyr group; IR:\LIBVV]04136.doc:mrr 4a and wherein the compound is not SEQ ID NO: 1.
According to a second embodiment of the present invention there is provided an improved method of radiation therapy for a patient afflicted with a neoplastic disease state, characterised by administering in conjunctive therapy an effective radioprotective amount of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I R -R 2
-R
3
-R
4
-R
5
-R'-R
7
-R
8 wherein R' is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Suc; l0 R 2 is selected from the group consisting of Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;
R
3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;
R
4 is selected from the group consisting of Tyr, Tyr(P0 3 2 Thr, Ser, homoSer, Ala, and azaTyr;
R
5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;
R
6 is His, Arg or 6-NH 2 -Phe;
SR
7 is Pro or Ala; and
R
8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; wherein the active agent does not include R 4 as a terminal Tyr group; and wherein the compound is not SEQ ID NO: 1.
According to a third embodiment of the present invention there is provided an °o'o improved method of treating a patient in need of radiation therapy, characterised by 6 administering to said patient an amount effective for treating a patient in need of radiation 6 S 25 therapy of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I
R'-R
2
-R
3
-R
4
-R
5
-R
6
-R
7
-R
8 wherein R' is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Suc;
R
2 is selected from the group consisting of Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;
R
3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr; RA,, R 4 is selected from the group consisting of Tyr, Tyr(P0 3 2 Thr, Ser, homoSer, S Ala, and azaTyr; Ala, and azaTyr; SR:\lI BVV04136.doc:mrr 4b
R
5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;
R
6 is His, Arg or 6-NH 2 -Phe;
R
7 is Pro or Ala; and
R
8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; wherein the active agent does not include R 4 as a terminal Tyr group; and wherein the compound is not SEQ ID NO: 1.
According to a fourth embodiment of the present invention there is provided an improved method of bone marrow transplantation, characterised by administering an effective amount of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I R R 2
-R
3 R 4
-R
s
R
6
-R
7
-R
8 wherein R' is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Sue;
R
2 is selected from the group consisting of Arg, Lys, Ala, Orn, Ser(Ac), Sar, 15I D-Arg and D-Lys; S 3 .o
R
3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly,
S;
Pro, Aib, Acpc, Lys, and Tyr;
R
4 is selected from the group consisting of Tyr, Tyr(PO 3 2 Thr, Ser, homoSer, Ala, and azaTyr;
R
5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;
R
6 is His, Arg or 6-NH 2 -Phe; a
R
7 is Pro or Ala; and
R
8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; wherein the active agent does not include R 4 as a terminal Tyr group.
25 According to a fifth embodiment of the present invention there is provided a method for increasing megakaryocyte production and mobilization and platelet production in a mammal, which comprises administering to the mammal an amount effective for megkaryocyte production and mobilization and platelet production of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R -R 8 in the sequence of general formula I
R'-R
2
-R
3
-R
4
-R
5
-R
6
-R
7
-R
8 wherein R 1 is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Sue; RA R 2 is selected from the group consisting of Arg, Lys, Ala, Orn, Ser(Ac), Sar, P D-Arg and D-Lys; [R:\LIBVV]04136.doc:mrr 4c
R
3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;
R
4 is selected from the group consisting of Tyr, Tyr(PO 3 2 Thr, Ser, homoSer, Ala, and azaTyr;
R
5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;
R
6 is His, Arg or 6-NH 2 -Phe;
R
7 is Pro or Ala; and R is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; wherein the active agent does not include R 4 as a terminal Tyr group.
According to a sixth embodiment of the present invention there is provided a method for the mitigation of tissue damage due to radiation exposure comprising the administration of an amount effective for the mitigation of tissue damage of at least active agent comprising a sequence of the following general formula: Rl-Arg-R2-R3-R4-His-Pro-R5.
15 wherein R1 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(PO 3 2 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent; and wherein the compound is not SEQ ID NO: 1.
SAccording to a seventh embodiment of the present invention there is provided an improved method of radiation therapy for a patient afflicted with a neoplastic disease state, characterised by administering in conjunctive therapy an effective radioprotective amount of at least one active agent comprising a sequence of the following general 25 formula: R1-Arg-R2-R3-R4-His-Pro-R5 wherein R1 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(PO 3 2 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and is Phe, Ile, or is absent; and wherein the compound is not SEQ ID NO:1.
[R:\LI BVV]041 6.doc:mrr 4d According to an eighth embodiment of the present invention there is provided an improved method of treating a patient in need of radiation therapy, characterised by administering to said patient an amount effective for treating a patient in need of radiation therapy of at least one active agent comprising a sequence of the following general S formula: Rl -Arg-R2-R3-R4-His-Pro-R5 wherein Rl 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 Try and Tyr(PO 3 2 t0 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and is Phe, Ile, or is absent; and wherein the compound is not SEQ ID NO: 1.
According to a ninth embodiment of the present invention there is provided an improved method of bone marrow transplantation, characterised by a sequence of the I following general formula: R 1 -Arg-R2-R3-R4-His-Pro-R5 *o wherein Rl is selected from the group consisting ofH and Asp; R2 is selected from the group consisting of Val and Pro; R3 is selected from the group consisting of Tyr and Tyr(P0 3 2 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and is Phe, Ile, or is absent.
According to a tenth embodiment of the present invention there is provided a method for increasing megakaryocyte production and mobilization and platelet production in a mammal, which comprises administering to the mammal an amount 25 effective for megakaryocyte production and mobilization and platelet production of at least one active agent comprising a sequence of the following general formula: R 1-Arg-R2-R3-R4-His-Pro-R5 wherein R1 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(PO 3 2 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and is Phe, Ile, or is absent.
[R:\LIBVV]04136.doc:mrr 4e According to an eleventh embodiment of the present invention there is provided at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I of the first embodiment when used to mitigate tissue damage due to radiation exposure.
According to a twelfth embodiment of the present invention there is provided at least one active agent comprising a sequence of at least three contiguous amino acids of groups R -R 8 in the sequence of general formula I of the second embodiment when used in radiation therapy for a patient afflicted with a neoplastic disease state.
According to a thirteenth embodiment of the present invention there is provided at I0 least one active agent comprising a sequence of at least three contiguous amino acids of groups R -R 8 in the sequence of general formula I of the third embodiment when used to treat a patient in need of radiation therapy.
According to a fourteenth embodiment of the present invention there is provided at least one active agent comprising a sequence of at least three contiguous amino acids of groups RI-R in the sequence of general formula I of the fourth embodiment when used in bone marrow transplantation.
According to a fifteenth embodiment of the present invention there is provided at least one active agent comprising a sequence of at least three contiguous amino acids of groups R -R 8 in the sequence of general formula I of the fifth embodiment, when used to increase megakaryocyte production and mobilization and platelet production in a mammal.
According to a sixteenth embodiment of the present invention there is provided at least one active agent comprising a sequence of the general formula R1-Arg-R2-R3-R4- His-Pro-R5 of the sixth embodiment, when used to mitigate tissue damage due to 25 radiation exposure.
According to a seventeenth embodiment of the present invention there is provided at least one active agent comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 of the seventh embodiment when used in radiation therapy for a patient afflicted with a neoplastic disease state.
According to an eighteenth embodiment of the present invention there is provided at least one active agent comprising a sequence of comprising the general formula R1-Arg- R2-R3-R4-His-Pro-R5 of the eighth embodiment, when used to treat a patient in need of radiation therapy.
IR:\LIBVV|04 136.doc:mrr 4f According to a nineteenth embodiment of the present invention there is provided at least one active agent comprising a sequence of comprising the general formula RI-Arg- R2-R3-R4-His-Pro-R5 of the ninth embodiment, when used in bone marrow transplantation.
According to a twentieth embodiment of the present invention there is provided at least one active agent comprising a sequence of comprising the general formula R1-Arg- R2-R3-R4-His-Pro-R5 of the tenth embodiment, when used to increase megakaryocyte production and mobilization and platelet production in a mammal.
According to a twenty-first embodiment of the present invention there is provided use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R -R 8 in the sequence of general formula I of the first embodiment, for the manufacture of a medicament to mitigate tissue damage due to radiation exposure.
According to a twenty-second embodiment of the present invention there is provided use of at least one active agent comprising a sequence of at least three l 15 contiguous amino acids of groups R'-R 8 in the sequence of general formula I of the second embodiment, for the manufacture of a medicament in radiation therapy for a patient afflicted with a neoplastic disease state.
According to a twenty-third embodiment of the present invention there is provided use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R l
-R
8 in the sequence of general formula I of the third embodiment, for the manufacture of a medicament to treat a patient in need of radiation therapy.
9999 :According to a twenty-fourth embodiment of the present invention there is provided use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups RI-R 8 in the sequence of general formula I of the fourth embodiment, for 9 25 the manufacture of a medicament in bone marrow transplantation.
According to a twenty-fifth embodiment of the present invention there is provided use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I of the fifth embodiment, for the manufacture of a medicament to increase megakaryocyte production and mobilization and platelet production in a mammal.
According to a twenty-sixth embodiment of the present invention there is provided use of at least one active agent comprising a sequence of comprising the general formula RI -Arg-R2-R3-R4-His-Pro-R5 of the sixth embodiment, for the manufacture of a j/RAe medicament to mitigate tissue damage due to radiation exposure.
I R:\.IB3V\104136.doc:mrr 4g According to a twenty-seventh embodiment of the present invention there is provided use of at least one active agent comprising a sequence of comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 of the seventh embodiment, for the manufacture of a medicament in radiation therapy for a patient afflicted with a neoplastic disease state.
According to a twenty-eighth embodiment of the present invention there is provided use of at least one active agent comprising a sequence of comprising the general formula R -Arg-R2-R3-R4-His-Pro-R5 of the eighth embodiment, for the manufacture of a medicament to treat a patient in need of radiation therapy.
According to a twenty-ninth embodiment of the present invention there is provided use of at least one active agent comprising a sequence of comprising the general formula RI-Arg-R2-R3-R4-His-Pro-R5 of the ninth embodiment, for the manufacture of a medicament in bone marrow transplantation.
According to a thirtieth embodiment of the present invention there is provided use of at least one active agent comprising a sequence of comprising the general formula R1- 15 Arg-R2-R3-R4-His-Pro-R5 of the tenth embodiment, for the manufacture of a medicament to increase megakaryocyte production and mobilization and platelet production in a mammal.
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 AI analogues, AI fragments and analogues go R\I.I3VVj4136.doc~mrr WO 99/45945 PCT/US99/05194 thereof, angiotensin II (AII), All analogues, All fragments or analogues thereof or All
AT
2 type 2 receptor agonists to a patient in need thereof.
In another aspect of the present invention, an improved cell culture medium and kits are provided for the production of megakaryocytes and platelets wherein the improvement comprises addition to the cell culture medium of an effective amount of angiotensinogen, AI, AL analogues, Al fragments and analogues thereof, All, All analogues, All fragments or analogues thereof or All AT 2 type 2 receptor agonists.
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.
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 All treatment two days following exposure on white blood cell number after irradiation.
WO 99/45945 PCT/US99/05194 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 AII treatment on the day of exposure on megakaryocyte number after irradiation.
Figure 9 is a graph showing the effect of AII treatment two days following exposure on megakaryocyte percentage after irradiation.
Figure 10 is a graph showing the effect of AII 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 monocyte number after irradiation.
Figure 12 is a graph showing the effect 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.
WO 99/45945 PCT/US99/05194 Figure 19 is a graph showing is a graph showing the effect of All analogues and fragments treatment on white blood cell number after irradiation.
Figure 20 is a graph showing is a graph showing the effect of AII analogues and fragments treatment on platelet number after irradiation.
Figure 21 is a graph showing the effect of AII on mouse survival receiving bone marrow transplantation after lethal irradiation.
Figure 22 is a graph showing the effect of All analogues and fragments treatment on white blood cell number after 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 All references patents and patent applications are hereby incorporated by reference in their entirety.
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 megakaryocyte 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 WO 99/45945 PCT/US99/05194 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.
Unless otherwise indicated, the term "active agents" as used herein refers to the group of compounds comprising angiotensinogen, angiotensin I AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof and AII AT 2 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 Enzymology, Vol. 185, edited by D.
Goeddel, 1991, Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology 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, 2 nd 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).
o WO 99/45945 PCT/US99/05194 U.S. Patent No. 5,015,629 to DiZerega (the entire disclosure of which is hereby incorporated by reference) 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-epithelialization and tissue repair. The term AII 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 decapeptide called angiotensin I (AI) which is converted to All by the converting enzyme angiotensinase which removes the C-terminal His-Leu residues from AI (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. Patent No. 5,629,292; U.S. Patent No. 5,716,935; WO 96.;39164; all references herein incorporated by reference in their entirety.) 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. Mol. Cell. Cardiol. 21:S7 (Supp 1II) 1989; Berk et. al., Hypertension 13:305-14 (1989); Kawahara, et al., BBRC 150:52-9 (1988); Naftilan, et al., J. Clin. Invest.
83:1419-23 (1989); Taubman et al., 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 WO 99/45945 PCT/US99/05194 (1990). In addition, All 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.
All 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 2 have been identified (Meffert, 1996). The growth-promoting effects of AII have been attributed to mediation by the AT1 receptor, while some evidence suggests that the AT2 receptor may be involved in mediation of the cell differentiation effects of All (Bedecs et al., Biochem. J. 325:449 (1997)).
The effects of AII receptor and AII receptor antagonists have been examined in two experimental models of vascular injury and repair which suggest that both All receptor subtypes (ATI and AT2) play a role in wound healing (Janiak et al., Hypertension 20:737-45 (1992); Prescott, et al., Am. J. Pathol. 139:1291-1296 (1991); Kauffman, et al., Life Sci. 49:223-228 (1991); Viswanathan, et al., Peptides 13:783- 786 (1992); Kimura, et al., BBRC 187:1083-1090 (1992).
Many studies have focused upon AII(1-7) (AII residues 1-7) or other fragments of All to evaluate their activity. AII(1-7) elicits some, but not the full range of effects elicited by All. Pfeilschifter, et al., Eur. J. Pharmacol. 225:57-62 (1992); Jaiswal, et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and Stack, J.
Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol. Exper.
Ther. 265:664-673 (1991); Jaiswal, et al., Hypertension 17:1115-1120 (1991); Portsi, et Br. J. Pharmacol. 111:652-654 (1994).
WO 099/45945 PCT/US99/05194 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 marrow transplantation nephropathy, and acute radiation injury (Moulder et al., Int. J 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. Radiat. Oncol. Biol. Phys. 30:873-878 (1994); Ward et al., Radiation Res. 135:81-87 (1993); Cohen et al., 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 AT1 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 AI analogues, AI fragments and analogues thereof, All, All analogues, All fragments or analogues thereof or AII AT 2 type 2 receptor agonists are potent stimulators of hematopoietic stem cell proliferation Patent Application, Serial No. 09/012,400, hereby incorporated by reference in its entirety). Therefore, it would be expected that the use of these compounds might cause long-term 11 WO 99/45945 PCT/US99/05194 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 AI analogues, Al fragments and analogues thereof, All, All analogues, AII fragments or analogues thereof or All AT 2 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 analogues, Al fragments and analogues thereof, angiotensin II (All), All analogues, All fragments or analogues thereof or All AT 2 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 (All has 100 times higher affinity for AT2 than AT1) is p-aminophenylalanine6-AII ["(p-NH2-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 169:997-10.06 (1990). This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharmacol. 256:93-97 (1994); Bryson, et al., Eur. J.
Pharmacol. 225:119-127 (1992).
The active Al, Al analogues, Al fragments and analogues thereof, All analogues, fragments of AII 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 R'-R 8 in the sequence of general formula I WO W99/45945 PCT/US99/05194
R'-R
2
-R
3
-R
4
-R
5
-R
6
-R
7
-R
8 in which R' and R 2 together form a group of formula X-RA-R wherein X is H or a one to three peptide group,
R
A is suitably selected from Asp, Glu, Asn, Acpc (1aminocyclopentane carboxylic acid), Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Suc,
R
B is suitably selected from Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;
R
3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;
R
4 is selected from the group consisting of Tyr, Tyr(PO 3 2 Thr, Ser, homoSer, Ala, and azaTyr; R' is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;
R
6 is His, Arg or 6-NH2-Phe;
R
7 is Pro or Ala; and
R
8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R 4 as a terminal Tyr group.
Compounds falling within the category of AT2 agonists useful in the practice of the invention include the AII analogues set forth above subject to the restriction that R 6 is p-NH 2 -Phe. In addition to peptide agents, various nonpeptidic agents peptidomimetics) having the requisite AT2 agonist activity are further contemplated for use in accordance with the present invention.
13 *WO 99/45945 PTU9/59 PCT/US99/05194 Particularly preferred combinations for RA and RB' are Asp-Mrg, Asp-Lys, Glu- Mrg and Glu-Lys. Particularly preferred embodiments of this class include the following: All, AIII or AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2); AII(3-8), also known as desi-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AJlIl-7), Asp-Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:4]; AII(2-7). Arg-Val-Tyr-Ile- His-Pro [SEQ ID NO:5]; AII(3-7), Val-Tyr-Ile-His-Pro [SEQ ID NO:6]; AII(5-8), Ile- His-Pro-Phe [SEQ ID NO:7]; AII(1-6), Asp-Arg-Val-Tyr-Ile-His [SEQ ID NO:8]; AII(l Asp-Arg-Val-Tyr-lle [SEQ ID NO:9]; AII(1 Asp-Arg-Val-Tyr [SEQ ID NO:IO0]; and AII(1 Asp-Arg-Val [SEQ ID NO:l 1) Other preferred embodiments include: Arg-norLeu-Tyr-I le-Hi s-Pro-Phe [SEQ ID NO: 121 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:3 AII(6-8), His-Pro-Phe [SEQ ID NO:14] and AII(4- Tyr-le-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: R I -Arg-R2-R3 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(P0 3 2 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and is Phe, Ile, or is absent.
I WO 99/45945 PCT/US99/05194 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 ID 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 R2-R-3R 4
-RS-R
6 -R -R 8 in which R 2 is selected from the group consisting of H, Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;
R
3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;
R
4 is selected from the group consisting of Tyr, Tyr(PO 3 2 Thr, Ser, homoSer and azaTyr; R' is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly;
R
6 is His, Arg or 6-NH 2 -Phe;
R
7 is Pro or Ala; and
R
8 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-RS-His-Pro-Phe [SEQ ID NO:16] wherein R 2
R
3 and R 5 are as previously defined. Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]. Other WO 99/45945 PCT/US99/05194 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 AII(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 Me 2 Gly N,N-dimethylglycyl Bet 1-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt (betaine) Suc Succinyl Phe(Br) p-bromo-L-phenylalanyl azaTyr aza-cx'-homo-L-tyrosyl Acpc 1-aminocyclopentane carboxylic acid Aib 2-aminoisobutyric acid Sar N-methylglycyl (sarcosine) It has been suggested that AII 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 R 3
R
5 and R 7 may be involved in maintaining the appropriate distance between active groups in positions R 4
R
6 and R 8 primarily responsible for binding to receptors and/or intrinsic activity. Hydrophobic 16 WO 099/45945 PCT/US99/05194 side chains in positions R 3
R
5 and R 8 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 R 2 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 R 2 For purposes of the present invention, it is believed that R 3 may be involved in the formation of linear or nonlinear hydrogen bonds with R 5 (in the gamma turn model) or R 6 (in the beta turn model). R 3 would also participate in the first turn 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, R 3 may suitably be selected from Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr. In another preferred embodiment, R 3 is Lys.
With respect to R 4 conformational analyses have suggested that the side chain in this position (as well as in R 3 and R 5 contribute to a hydrophobic cluster believed to be essential for occupation and stimulation of receptors. Thus, R 4 is preferably selected from Tyr, Thr, Tyr (PO 3 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, R 4 is Ala.
WO 99/45945 PCT/US99/05194 In position R 5 an amino acid with a p aliphatic or alicyclic chain is particularly desirable. Therefore, while Gly is suitable in position R 5 it is preferred that the amino acid in this position be selected from Ile, Ala, Leu, norLeu, Gly and Val.
In the AI, AI analogues, AI fragments and analogues thereof, All, All analogues, fragments and analogues of fragments of particular interest in accordance with the present invention, R' is His, Arg or 6-NH 2 -Phe. The unique properties of the imidazole ring of histidine ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R 6 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 R 7 Similarly, it is presently considered that R 7 should be Pro in order to provide the most desirable orientation of
R
8 In position R 8 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: Analogue 3 Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe SEQ ID NO: 21 Analogue 4 Glu-Arg-Val-Tvr-Ile-His-Pro-Phe SEQ ID NO: 22 Analogue 5 Asp-Lys-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 23 WO 099/45945 PCT/US99/05194 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: 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: Analogue 13 Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe SEQ ID NO: 31 Analogue 14 Asp-Arg-Val-Tyr(PO 3 2 -Ile-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-Phe SEQ ID NO: 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, Ill. (1984) and J. Meienhofer, Hormonal 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. The disclosures of the foregoing treatises are incorporated by reference herein.
In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain Patent No. 5,693,616, herein incorporated by reference in its entirety). 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 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 polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.
19 WO 099/45945 PCT/US99/05194 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 AI analogues, Al fragments and analogues thereof, angiotensin H (AlI), AII analogues, All fragments or analogues thereof or All
AT
2 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.
In another aspect, 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.
WO W99/45945 PCTIUS99/05194 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. Radiationinduced 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. Haematol. 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 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.
WO W99/45945 PCTIUS99/05194 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- 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 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, transdermal patches, topical administrative agents, polyethylene glycol polymers, carboxymethyl cellulose preparations, crystalloid preparations 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, AI, AI analogues, AI fragments and analogues thereof, All, All analogues, All fragments or analogues thereof or AII AT 2 type 2 receptor WO 99/45945 PCT/US99/05194 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, incorporated by reference herein in its entirety. Briefly, marrow is flushed from a subject's femur with Iscove's modification of Dulbecco's medium (IMDM) supplemented with Nutridoma-SP (Boehringer Mannheim, Indianapolis, Ind.), a serum-free medium supplement. For culture studies, a single cell suspension is made by repetitive expulsion through progressively smaller needles. For flow cytometry 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 370 C in a humidified atmosphere in the presence of, preferably, between about 0.1 ng/ml and about 10 mg/ml 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 23 WO 99/45945 PCT/US99/05194 cytometry as described in U.S. Patent No. 5,155,211, incorporated by reference herein in its entirety. Briefly, the appearance of granules and the extensive surfaceconnected open canalicular membrane system as well as a substantial decrease in the nucleus:cytoplasm volume distribution, indicates that the megakaryocyte 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, megakaryocytes and platelets. In a preferred embodiment, subjects are treated with total body irradiation and active agent is administered subcutaneously (10 gg/kg/day or 100 gg/kg/day) at various times before and after irradiation. The number of white blood cells, megakaryocytes 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. Megakaryocyte production is assessed at various time points during culture 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 'rWO 99/45945 PCT/US99/05194 from Becton Dickinson, Sunnyvale, CA, USA) to a streptavidin affinity column (Ceprate SC; CellPro, Bothell, WA, USA) and passing the aspirate through the column, followed 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/ml of the active agents of the invention. The cells are expanded for a period of between 8 and 21 days and megakaryocyte 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 cytokines. Examples of such growth factors and cytokines include, but are not limited to thrombopoietin, lymphokines, 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 10" and 50 x 106 cells per ml in an appropriate medium and reinfused into a subject through intravenous infusions.
WO 99/45945 PCT/US99/05194 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, intracranial, 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, intrasteral, 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 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 WO 99/45945 PCT/US99/05194 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, stabilizers, 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 liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.
WO W99/45945 PCT/US99/05 194 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, 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. 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.
28 'Z WO 99/45945 PCT/US99/05194 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 gg/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, from 1% to 2% by weight of the formulation, although it 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 SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34; SEQ ID SEQ ID NO:36; and SEQ ID NO:37.
WO 99/45945 PCT/US99/05194 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 tissuedamaging 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, Iscove's Modified Dulbecco's Medium, McCoy's Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, Opti- MEM® Reduced-Serum Medium, RPMI 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 WO 99/45945 PCT/US99/05194 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).
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 the claims appended hereto.
Example 1 Effect of AHl 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 p.g/kg/day or 100 gg/kg/day) or saline (placebo) was initiated two days before (-day WO 99/45945 PCT/US99/05194 on the day of (day 0) or 2 days after day 2) irradiation and continued until the animals succumbed to the irradiation or were necropsied. 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 AII starting at two days prior to irradiation did not protect against mortality resulting from irradiation (Figure but that All administration on the day of irradiation (Figure 2) or two days after irradiation (Figure 3) substantially increased survival. Furthermore, All administration at all time periods tested increased the number of circulating white blood cells (Figures Further experiments demonstrated that All administration increased the number of megakaryocytes (Figures 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 Al 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 pg/kg or 100 pg/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 20 and 21 and show that the peptides increase the production of both of these blood elements.
SWO 99/45945 PCT/US99/05194 Table 3: Designation for Analogues/Fragments Name Abbreviation Sequence SEQ ID NO: GSD 28 Ile 8 -AII DRVYIHPI SEQ ID NO: 38 GSD 24B Pro 3 AII DRPYIHPF SEQ ID NO:31 GSD 22A Ala 4 -AIII RVYAHPF SEQ ID NO: 18 AII(1-7) DRVYIHP SEQ ID NO:4 AII DRVYIHPF SEQ ID NO. 1 Example 3. Effect of AII 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/ml angiotensin II (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 106 or 1 x 10" 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 1 x 106 Saline All 1 x 105 AII Saline 1 x 10 6 AII Saline 1x 105 AII All 1 x 106 AII AII 1 x 10 33 SWO 99/45945 PCT/US99/05194 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 22-24, 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 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 oi 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.
EDITORIAL NOTE FOR 29957/99 THE FOLLOWING SEQUENCE LISTING IS PART OF THE DESCRIPTION THE CLAIMS FOLLOW ON PAGE WO 099/45945 PCT/US99/05194 SEQUENCE LISTING <110> Kathleen Rodgers and Gere diZerega <120> Improved Radiation Therapy Methods <130> 97,017-K2 <140> To be assigned <141> To be assigned <160> 38 <170> PatentIn Ver. <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:angiotensin II <400> 1 Page 1 WO099/45945 PCT/US99/051 94 Asp Arg Val Tyr Ile His Pro Phe 1 S <210> 2 <211> 7 <212> PRT <213> Artificial Sequence 2 <223> Description of Artificial Sequence:A112-8 <400> 2 Ary Val Tyr Ile His Pro Phe 1 Page 2 WO 99/45945 "W09945945PCTJUS99/05194 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence 2 <223> Description of Artificial Sequence :AII (3 -8) <400> 3 Val Tyr Ile His Pro Phe 1 <210> 4 <211> 7 <212> PRT Page 3 WO 99/45945 WO 9945945PCTIUS99/05 194 <213> Artificial Sequence 2 <223> Description of Artificial Sequence:AII(l-7) <400> 4 Asp Arg Val Tyr Ilie His Pro 1 <210> S <211> 6 <212> PRT <213> Artificial Sequence 2 Page 4 WO 99/45945 WO 9945945PCT/US99/051 94 <223> Description of Artificial Sequence:AII (2-7) <400> S Arg Val Tyr Ile His Pro 1 <210> 6 <211> <212> PRT <213> Artificial Sequence 2 <223> Description of Artificial Sequence :AII (3- 7 <400> 6 Page WO 99/45945 PCT/US99/051 94 Val Tyr Ile His Pro 1. <210> 7 <211> 4 <212> PRT <213.> Artificial Sequence 220 <223> Description of Artificial Sequence :AII (5-8) <400> 7 Ile His Pro Phe Page 6 WO 99/45945 WO 9945945PCT/US99/05194 .210> 8 '211> 6 <212> PRT <213> Artificial Sequence 2 <223> Description of Artificial Sequence :AII (1-6) <400> 8 Asp Arg Val Tyr Ile His 1 <210> 9 <211> <212> PRT Page 7 WO 99/45945PCUS/O19 PCT/US99/05194 <213> Artificial Sequence 220 <223> Description of Artificial Sequence:AII <4 00 9 Asp Arg Val Tyr Ile 1 '210> '211> 4 <212> PRT <213> Artificial Sequence 2 Page 8 WO 99/45945 WO 9945945PCTIUS99/051 94 <223> Description of Artificial Sequence:AII(1-4) <4 00> Asp Arg Val Tyr 1 <210> 11 <211> 3 <212> PRT <213> Artificial Sequence 2 <223> Description of Artificial Sequence:AII(l- 3 <400> 12.
Asp Arg Val Page 9 WO 99/45945 WO 9945945PCTIUS99/O5I 94 1 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence 220 <221> Xaa at postion 2 is Nie <222> 2 <223> Description of Artificial Sequence:AII analogue <400> 12 Arg Xaa Tyr Ile His Pro Phe 1 Page WO 99/45945 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> Xaa at position 4 is Nle PCT/US99/05194 <222> 4 <223> Description of Artificial Sequence:AII analogue <400> 13 Arg Val Tyr Xaa His Pro Phe Page 11 WO 99/45945 PCTIUS99/051 94 <210> 14 <211> 3 <212> PRT <213> Artificial Sequence 220 <223> Description of Artificial Sequence:AII(6-8) <400> 14 His Pro Phe 1 <210> <211> <212> PRT <213> Artificial Sequence Page 12 WO 99/45945 WO 9945945PCT/US99/051 94 2 <223> Description of Artificial Sequence:AII( 4 -8) <400> Tyr Ile His Pro Phe <210> 16 <211> 7 <212> PRT <213> Artificial Sequence 220 <221> Xaa at position 1 can be Hydrogen,~ Arg, Lys, Ala, Or Ser(Acetylated), MeGly, D-Arg, or D-Lys; Xaa at position Page 13 WO 99/45945 WO 9945945PCT/US99/05194 2 can be Val, Ala, Leu, Nie, Ile, Gly, Pro, Aib, Acp, or Ty Xaa at position 4 can be Ile, Ala, Leu, Nie, Val, or Gly <222> 1-4 <223> Description of Artificial Sequence:AII analogue cla s s <400> 16 Xaa Xaa Tyr Xaa His Pro Phe ~1 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence 2 Page 14 WO 99/45945 W099/5945PCT/US99/051 94 <c223> Description of Artificial Sequence:AII analogue <400> 17 Arg Val Tyr Gly His Pro Phe 1 <210> 18 <211> 7 <212> PRT 213 Artificial Sequence 220 <223> Description of Artificial Sequence:AII analogue <400> 18 Page WO 99/45945 Arg Val Tyr Ala His Pro Phe 1 PCTIUS99/051 94 <210> 19 <211> 8 <212> PRT 213 Artificial Sequence 2 <223> Description of Artificial Sequence:AII analogue i <400> 19 Asp Arg Val Tyr Val His Pro Phe Page 16 WO 99/45945 WO 9945945PCTIUS99/051 94 <210> <211> 8 <212> PRT 213 Artificial Sequence 220 <223> Description of Artificial Sequeflce:AII Analogue 2 <400> Asn Arg Val Tyr Val His Pro Phe 1
S
<210> 21 <211> 11 <212> PRT Page 17 WO 99/45945 PCT/US99/05194 <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 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> Page 18 WO 99/45945 PCT/US99/05194 <223> Description of Artificial Sequence:AII Analogue 4 <400> 22 Glu Arg Val Tyr Ile His Pro Phe 1 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:AII Analogue <400> 23 Page 19 WO99/45945 Asp Lys Val Tyr Ile His Pro Phe 1 PCTIUS99/051 94 <2 10 24 <211> 8 <212> PRT 213 Art if icial Sequence <2 20 <223> Description of Artificial Sequence:AII Analogue 6 <400> 24 Asp Arg Ala Tyr Ile His Pro Phe Page WO 99/45945 WO 9945945PCTIUS99/051 94 <210> <211> 8 <212> PRT <213> Artificial Sequence 2 <223> Description of Artificial Sequence:AII Analoge 7 <400> Asp Arg Val Thr Ilie His Pro Phe 1 <210> 26 <211> 8 <212> PRT Page 21 WO99/45945 <213> Artificial Sequence PCT[US99/05194 2 <223> Description of Artificial Sequence:AII Analogue 8 <400 26 Asp Arg Val Tyr Leu His Pro Phe 1 <2 10> 27 <211> 8 <212> PRT <213> Artificial Sequence 22 0> Page 22 WO 99/45945 PCT[US99/05194 223 Description of Artif icial sequence :AI I Analogue 9 <400> 27 Asp Arg Val Tyr Ile Arg Pro Phe <210> 28 <211> 8 <212> PRT <213> Artificial Sequence 2 <223> Description of Artificial Sequence:AII Analogue <400> 28 Asp Arg Val Tyr Ilie His Ala Phe Page 23 WO 99/45945 WO 9945945PCT/US99/05 194 <2 10> 2 9 <211> 8 <~212> PRT <213> Artificial Sequence 2 <223> Description of Artificial Sequence:AII Analogue 11 <400> 2 9 Asp Arg Val Tyr Ile His Pro Tyr 1 Page 24 WO 99/45945 PCTIUS99/05194 <210> <211> 8 <212> PRT <213> Artificial Sequence 2 <223> Description of Artificial Sequence:AII Analogue 12 <400> Pro Arg Val Tyr Ilie His Pro Phe 1 <210> 31 <211> 8 <212> PRT Page WO 99/45945 PCT/US99/05194 <213> Artificial Sequence <223> Description of Artificial Sequence:AII Analogue 13 .400> 31 Asp Arg Pro Tyr Ile His Pro Phe <210> 32 <211> 8 <212> PRT <213> Artificial Sequence 2 <221> PHOSPHORYLATION Page 26 WO 99/45945 PCT/U5991051 94 <222> 4 <223> Description of Artificial Sequence:AII Analogue 14 <400> 32 Asp Arg Val Tyr Ile His Pro Phe 1 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence 2 <221> Xaa at position 3 is Nle <222> 3 <223> Description of Artificial Sequence:ATI Analogue 1S Page 27 'WO 99/45945 WO 9945945PCT/US99/051 94 <400> 33 Asp Arg Xaa Tyr Ile His Pro Phe 1 '210> 34 <211> 8 '212> PRT <213> Artificial Sequence 220 <221> Xaa at position 5 is Nie <222> <223> Description of Artificial Sequence:AII Analogue 16 <400> 34 Page 28 WO 99/45945 Asp Arg Val Tyr Xaa His Pro Phe 1 <210> 3S PCTIUS99IO5I 94 <211> 9 <212> PRT <213> Artificial Sequence 2 <221> homo Ser <222> 4 <c223> Description of Artificial Sequence:AIT Analogue 17 3 Asp Arg Val Ser Tyr Ile His Pro Phe Page 29 WO 99/45945 WO 9945945PCT[US99/051 94 210 3 6 <211> 8 <212> PRT 213 Artificial Sequence 2 <223> Description of Artificial Sequence:p-amilophenylalanine 6 All <400> 3 6 Asp Arg Val Tyr Ile Xaa Pro Phe <210> 37 Page WO 99/45945PCUS9014 PCT[US99/05194 <211> <212> PRT <213> Artificial Sequence 220 <223> Description of Artificial Sequence:angiotelsi
I
<400> 37 Asp Arg Val Tyr Ile His Pro Phe His Leu 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence Page 3 1 WO099/45945 2 <223> Description of Artificial Sequence:lGD: I1e8-AII PCT/US99/05 194 <400> 38 Asp Arg Val Tyr Ilie His Pro Ilie Page 32

Claims (46)

1. A method for the mitigation of tissue damage due to radiation exposure comprising the administration of an amount effective for the mitigation of tissue damage of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I R-R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8 wherein R' is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Suc; R 2 is selected from the group consisting of Arg, Lys, Ala, Orn, Ser(Ac), Sar, it D-Arg and D-Lys; R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr; R 4 is selected from the group consisting of Tyr, Tyr(PO 3 2 Thr, Ser, homoSer, Ala, and azaTyr; i R 5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly; R 6 is His, Arg or 6-NH 2 -Phe; R 7 is Pro or Ala; and R 8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; wherein the active agent does not include R 4 as a terminal Tyr group; and wherein the compound is not SEQ ID NO:1.
2. The method of claim 1 wherein the active agent is selected from the group consisting of 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, SEQ ID NO:37; and SEQ ID NO:38.
3. An improved method of radiation therapy for a patient afflicted with a neoplastic disease state, characterised by administering in conjunctive therapy an effective radioprotective amount of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I R'- R 2 -R 3 -R 4 -R' R -R -R 8 RA wherein R' is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, S Me 2 Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Suc; [R:\LIBVV\]04136.doc:mrr R 2 is selected from the group consisting of Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys; R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr; R 4 is selected from the group consisting of Tyr, Tyr(PO 3 2 Thr, Ser, homoSer, Ala, and azaTyr; R' is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly; R 6 is His, Arg or 6-NH 2 -Phe; R 7 is Pro or Ala; and i R 8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; wherein the active agent does not include R 4 as a terminal Tyr group; and wherein the compound is not SEQ ID NO:1.
4. The method of claim 3 wherein the active agent is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, 5 SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID SS NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, 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, SEQ ID NO:37 and SEQ ID NO:38.
5. An improved method of treating a patient in need of radiation therapy, *characterised by administering to said patient an amount effective for treating a patient in need of radiation therapy of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I 25 R'-R 2 -R 3 -R 4 -R 5 -R 6 -R 7 R 8 wherein R' is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Suc; R 2 is selected from the group consisting of Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys; R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr; R 4 is selected from the group consisting of Tyr, Tyr(P0 3 2 Thr, Ser, homoSer, Ala, and azaTyr; SR 5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly; R 6 is His, Arg or 6-NH 2 -Phe; [R:\.IB\VV]04 136.doc:mrr R 7 is Pro or Ala; and R 8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; wherein the active agent does not include R 4 as a terminal Tyr group; and wherein the compound is not SEQ ID NO: 1.
6. The method of claim 5 wherein the active agent is selected from the group consisting of 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 i0 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, SEQ ID NO:37 and SEQ ID NO:38.
7. The method of claim 1, 3, or 5 wherein the active agent is SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO: 26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34 and SEQ ID i. 15 NO:38.
8. An improved method of bone marrow transplantation, characterised by administering an effective amount of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I 90* RI R 2 R 3 R 4 R5 R 6 R 7 R 8 R-R2-R-R-R'-R'-R7-R 8 wherein R' is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Suc; R 2 is selected from the group consisting of Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys; R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, 25 Pro, Aib, Acpc, Lys, and Tyr; S R 4 is selected from the group consisting of Tyr, Tyr(PO 3 2 Thr, Ser, homoSer, Ala, and azaTyr; R 3 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly; R 6 is His, Arg or 6-NH 2 -Phe; R 7 is Pro or Ala; and R 8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; wherein the active agent does not include R 4 as a terminal Tyr group.
9. The method of claim 8 wherein the active agent is selected from the group ST4 consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID S SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID [R:\LIB\VV 04136.doc:mrr 38 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, SEQ ID NO:37 and SEQ ID NO:38. The method of claim 8 wherein the active agent is SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38.
11. A method for increasing megakaryocyte production and mobilization and platelet production in a mammal, which comprises administering to the mammal an amount effective for megkaryocyte production and mobilization and platelet production of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R -R8 in the sequence of general formula I *I 2 3 4 5 6 7 8 I5 R-R2-R-R-R -R -R -R wherein R is selected from the group consisting of Asp, Glu, Asn, Acpc, Ala, Me"Gly, Pro, Bet, Glu(NH 2 Gly, Asp(NH 2 and Suc; R 2 is selected from the group consisting of Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys; R 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(PO 3 2 Thr, Ser, homoSer, Ala, and azaTyr; R' is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly; 25 R 6 is His, Arg or 6-NH 2 -Phe; '0 4 R 7 is Pro or Ala; and R is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; wherein the active agent does not include R 4 as a terminal Tyr group.
12. The method of claim 11, wherein 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 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 j NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID IR:\LI BVV104136 doc:mrr 39 NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
13. The method of claim 11 wherein the active agent is SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34 Sand SEQ ID NO:38.
14. A method for the mitigation of tissue damage due to radiation exposure comprising the administration of an amount effective for the mitigation of tissue damage of at least active agent comprising a sequence of the following general formula: R1-Arg-R2-R3-R4-His-Pro-R5. wherein R1 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(PO 3 2 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and is Phe, Ile, or is absent; S 15 and wherein the compound is not SEQ ID NO: 1.
15. The method of claim 14 wherein the active agent is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38.
16. An improved method of radiation therapy for a patient afflicted with a 20 neoplastic disease state, characterised by administering in conjunctive therapy an effective radioprotective amount of at least one active agent comprising a sequence of the following general formula: R 1-Arg-R2-R3-R4-His-Pro-R5 wherein R1 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(PO 3 2 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and is Phe, Ile, or is absent; and wherein the compound is not SEQ ID NO: 1.
17. The method of claim 16 wherein the active agent is selected from the group consisting of SEQ ID NO:4; SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38.
18. An improved method of treating a patient in need of radiation therapy, characterised by administering to said patient an amount effective for treating a patient in IR \LIB\'VV04 136.doc:mrr need of radiation therapy of at least one active agent comprising a sequence of the following general formula: 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 Try and Tyr(PO 3 2 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and is Phe, Ile, or is absent; and wherein the compound is not SEQ ID NO:1.
19. The method of claim 18 wherein the active agent is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38.
20. An improved method of bone marrow transplantation, characterised by a sequence of the following general formula: R1-Arg-R2-R3-R4-His-Pro-R5 wherein R1 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(PO 3 2 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent.
21. The method of claim 20 wherein the active agent is selected from the group o. consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26, SEQ ID S NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38.
22. A method for increasing megakaryocyte production and mobilization and platelet production in a mammal, which comprises administering to the mammal an amount effective for megakaryocyte production and mobilization and platelet production of at least one active agent comprising a sequence of the following general formula: R -Arg-R2-R3-R4-His-Pro-R5 wherein R1 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(PO 3 2 R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R R5 is Phe, Ile, or is absent. SR:\LI.3VV]04136.doc:mrr 41
23. The method of claim 22, wherein the active agent is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38.
24. The method of claim 1 wherein the active agent comprises at least four contiguous amino acids of general formula I. The method of claim 1 wherein the active agent comprises at least five contiguous amino acids of general formula I.
26. The method of claim 1 wherein the active agent comprises at least six contiguous amino acids of general formula I. 1 27. The method of claim 1 wherein the active agent comprises at least seven contiguous amino acids of general formula I.
28. The method of claim 1 wherein the active agent consists of at least three i contiguous amino acids of general formula I.
29. The method of claim I wherein the active agent consists of at least four i 5 contiguous amino acids of general formula I. The method of claim 1 wherein the active agent consists of at least five contiguous amino acids of general formula I. S31. The method of claim 1 wherein the active agent consists of at least six contiguous amino acids of general formula I.
32. The method of claim 1 wherein the active agent consists of at least seven .contiguous amino acids of general formula I.
33. The method of claim 3 wherein the active agent comprises at least four contiguous amino acids of general formula I.
34. The method of claim 3 wherein the active agent comprises at least five contiguous amino acids of general formula I. The method of claim 3 wherein the active agent comprises at least six contiguous amino acids of general formula I.
36. The method of claim 3 wherein the active agent comprises at least seven contiguous amino acids of general formula I.
37. The method of claim 3 wherein the active agent consists of at least three contiguous amino acids of general formula I.
38. The method of claim 3 wherein the active agent consists of at least four contiguous amino acids of general formula I. SR4'A, 39. The method of claim 3 wherein the active agent consists of at least five ntiguous amino acids of general formula I. IR:\LIBVV]04 136..doc:mrr 42 The method of claim 3 wherein the active agent consists of at least six contiguous amino acids of general formula I.
41. The method of claim 3 wherein the active agent consists of at least seven contiguous amino acids of general formula I.
42. The method of claim 5 wherein the active agent comprises at least four contiguous amino acids of general formula I.
43. The method of claim 5 wherein the active agent comprises at least five contiguous amino acids of general formula I.
44. The method of claim 5 wherein the active agent comprises at least six Io contiguous amino acids of general formula I. The method of claim 5 wherein the active agent comprises at least seven contiguous amino acids of general formula I.
46. The method of claim 5 wherein the active agent consists of at least three contiguous amino acids of general formula I. S 15 47. The method of claim 5 wherein the active agent consists of at least four contiguous amino acids of general formula I.
48. The method of claim 5 wherein the active agent consists of at least five contiguous amino acids of general formula I.
49. The method of claim 5 wherein the active agent consists of at least six 2o contiguous amino acids of general formula I. The method of claim 5 wherein the active agent consists of at least seven contiguous amino acids of general formula I. 2 51. The method of claim 8 wherein the active agent comprises at least four contiguous amino acids of general formula I.
52. The method of claim 8 wherein the active agent comprises at least five contiguous amino acids of general formula I.
53. The method of claim 8 wherein the active agent comprises at least six contiguous amino acids of general formula I.
54. The method of claim 8 wherein the active agent comprises at least seven o0 contiguous amino acids of general formula I. The method of claim 8 wherein the active agent consists of at least three contiguous amino acids of general formula I.
56. The method of claim 8 wherein the active agent consists of at least four contiguous amino acids of general formula I. I R:\L IIVV]04 136.doc:mrr L 43
57. The method of claim 8 wherein the active agent consists of at least five contiguous amino acids of general formula I.
58. The method of claim 8 wherein the active agent consists of at least six contiguous amino acids of general formula I.
559. The method of claim 8 wherein the active agent consists of at least seven contiguous amino acids of general formula I. The method of claim 11 wherein the active agent comprises at least four contiguous amino acids of general formula I. 61. The method of claim II wherein the active agent comprises at least five 0 contiguous amino acids of general formula I. 62. The method of claim 11 wherein the active agent comprises at least six ocontiguous amino acids of general formula I. .o 63. The method of claim 11 wherein the active agent comprises at least seven contiguous amino acids of general formula I. o• :°o 64. The method of claim 11 wherein the active agent consists of at least three contiguous amino acids of general formula I. The method of claim 11 wherein the active agent consists of at least four contiguous amino acids of general formula I. 66. The method of claim 11 wherein the active agent consists of at least five 20 contiguous amino acids of general formula I. 67. The method of claim 11 wherein the active agent consists of at least six o o.contiguous amino acids of general formula I. 68. The method of claim 11 wherein the active agent consists of at least seven contiguous amino acids of general formula I. 69. The method of claim 1, wherein the active agent is SEQ ID NO:4. The method of claim 3, wherein the active agent is SEQ ID NO:4. 71. The method of claim 5, wherein the active agent is SEQ ID NO:4. 72. The method of claim 8, wherein the active agent is SEQ ID NO:4. 73. The method of claim 11, wherein the active agent is SEQ ID NO:4. 74. A method for the mitigation of tissue damage due to radiation exposure comprising the administration of an amount effective for the mitigation of tissue damage of at least one active agent, which active agent is substantially as hereinbefore described with reference to any one of Examples 1 to 3. An improved method of radiation therapy for a patient afflicted with a neoplastic disease state, characterised by administering in conjuntive therapy an effective IR\LIFIVV04136.doc:tnrr 44 radio protective amount of at least one active agent, which active agent is substantially as herein described with reference to any one of Examples 1 to 3. 76. An improved method of treating a patient in need of radiation therapy, characterised by administering to said patient an amount effective for treating a patient in need of radiation therapy of at least one active agent, which active agent is substantially as herein described with reference to any one of Examples 1 to 3. 77. An improved method of bone marrow transplantation, characterised by administering an effective amount of at least one active agent, which active agent is substantially as herein described with reference to any one of the Examples 1 to 3. 1 78. A method for increasing megakaryocyte production and mobilisation and platelet production in a mammal, which comprises administering to the mammal an amount effective for megakaryocyte production and mobilisation and platelet production •of at least one active agent, which active agent is substantially as herein described with reference to any one of Examples 1 to 3. S 15 79. At least one active agent comprising a sequence of at least three contiguous amino acids of groups R -R8 in the sequence of general formula I as claimed in claim 1 or 2 or 7 when used to mitigate tissue damage due to radiation exposure. 80. At least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in claim 1 or °i 2( 2 or 7 when used to mitigate tissue damage due to radiation exposure, which agent is oo•• substantially as herein described with reference to any one of Examples 1 to 3. At least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in claim 3 or 4 or 7 when used in radiation therapy for a patient afflicted with a neoplastic disease state. 82. At least one active agent comprising a sequence of at least three contiguous amino acids of groups R -R 8 in the sequence of general formula I as claimed in claim 3 or 4 or 7 when used in radiation therapy for a patient afflicted with a neoplastic disease state, which agent is substantially as herein described with reference to any one of Examples 1 to 3. 83. At least one active agent comprising a sequence of at least three contiguous amino acids of groups R -R 8 in the sequence of general formula I as claimed in any one of claims 5 to 7 when used to treat a patient in need of radiation therapy. 84. At least one active agent comprising a sequence of at least three contiguous Samino acids of groups R1-R in the sequence of general formula I as claimed in any one of [R:\LIJIVV]04 136.doc:mrr claims 5 to 7 when used to treat a patient in need of radiation therapy, which agent is substantially as herein described with reference to any one of Examples 1 to 3. At least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in any one of S claims 8 to 10 when used in bone marrow transplantation. 86. At least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in any one of claims 8 to 10, when used in bone marrow transplantation, which agent is substantially as herein described with reference to any one of Examples 1 to 3. to 87. At least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in any one of claims 11 to 13, when used to increase megakaryocyte production and mobilization and platelet production in a mammal. 88. At least one active agent comprising a sequence of at least three contiguous i5 amino acids of groups R'-R 8 in the sequence of general formula I as claimed in any one of claims 11 to 13, when used to increase megakaryocyte production and mobilization and platelet production in a mammal, which agent is substantially as herein described with .*reference to any one of Examples 1 to 3. 89. At least one active agent comprising a sequence of the general formula R1- 20 Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 14 or 15, when used to mitigate tissue damage due to radiation exposure. 90. At least one active agent comprising a sequence of the general formula R1- Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 14 or 15, when used to mitigate tissue damage due to radiation exposure, which agent is substantially as herein disclosed with reference to any one of Examples 1 to 3. 91. At least one active agent comprising the general formula RI-Arg-R2-R3-R4- as claimed in claim 16 or 17 when used in radiation therapy for a patient afflicted with a neoplastic disease state. 92. At least one active agent comprising the general formula R1-Arg-R2-R3-R4- 1-lis-Pro-R5 as claimed in claim 16 or 17 when used in radiation therapy for a patient afflicted with a neoplastic disease state, which agent is substantially as herein described with reference to any one of Examples 1 to 3. 93. At least one active agent comprising a sequence of comprising the general R~C A I formula RI-Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 18 or 19, when used to treat a patient in need of radiation therapy. ain nne frdainteay I 113VV104136.doc: nrr 94. At least one active agent comprising a sequence of comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 18 or 19, when used to treat a patient in need of radiation therapy which agent is substantially as herein described with reference to any one of Examples 1 to 3. 95. At least one active agent comprising a sequence of comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 20 or 21, when used in bone marrow transplantation. 96. At least one active agent comprising a sequence of comprising the general formula RI-Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 20 or 21, when used in bone o0 marrow transplantation which agent is substantially as herein described with reference to any one of Examples 1 to 3. 97. At least one active agent comprising a sequence of comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 22 or 23, when used to :increase megakaryocyte production and mobilization and platelet production in a 15 mammal. 98. At least one active agent comprising a sequence of comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 22 or 23, when used to increase megakaryocyte production and mobilization and platelet production in a mammal, which agent is substantially as herein described with reference to any one of 20 Examples 1 to 3. 99. Use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in claim 1 or 2 or 7, for the manufacture of a medicament to mitigate tissue damage due to radiation exposure. 100. Use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in claim 1 or 2 or 7, for the manufacture of a medicament to mitigate tissue damage due to radiation exposure which agent is substantially as herein described with reference to any one of Examples 1 to 3. 101. Use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula las claimed in claims 3 or 4 or 7, for the manufacture of a medicament in radiation therapy for a patient afflicted with a neoplastic disease state. SRA;, 102. Use of at least one active agent comprising a sequence of at least three s contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed [R.\LIBVV]04136.doc:mrr 47 in claims 3 or 4 or 7, for the manufacture of a medicament in radiation therapy for a patient afflicted with a neoplastic disease state which agent is substantially as herein described with reference to any one of Examples 1 to 3. 103. Use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R in the sequence of general formula I as claimed in any one of claims 5 to 7, for the manufacture of a medicament to treat a patient in need of radiation therapy. 104. Use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in any one of claims 5 to 7, for the manufacture of a medicament to treat a patient in need of radiation therapy which agent is substantially as herein described with reference to any one of Examples 1 to 3. 105 Use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed S. 5I s in any one of claims 8 to 10, for the manufacture of a medicament in bone marrow transplantation. S..106. Use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in any one of clams 8 to 10, for the manufacture of a medicament in bone marrow 20 transplantation which agent is substantially as herein described with reference to any one of Examples 1 to 3. 107. Use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in any one of claims 11 to 13, for the manufacture of a medicament to increase megakaryocyte production and mobilization and platelet production in a mammal. 108. Use of at least one active agent comprising a sequence of at least three contiguous amino acids of groups R'-R 8 in the sequence of general formula I as claimed in any one of claims 11 to 13, for the manufacture of a medicament to increase megakaryocyte production and mobilization and platelet production in a mammal which agent is substantially as herein described with reference to any one of Examples 1 to 3. 109. Use of at least one active agent comprising a sequence of comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 14 or 15, for the manufacture of a medicament to mitigate tissue damage due to radiation exposure. R 110. Use of at least one active agent comprising a sequence of comprising the eneral formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 14 or 15, for the 35 eieral formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 14 or 15, for the I R:\I.IBV\V]04136.dloc:mrr 48 manufacture of a medicament to mitigate tissue damage due to radiation exposure, which agent is substantially as herein described with reference to any one of Examples 1 to 3. 111. Use of at least one active agent comprising a sequence of comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in claim 16 or 17, for the S manufacture of a medicament in radiation therapy for a patient afflicted with a neoplastic disease state. 112. Use of at least one active agent comprising a sequence of comprising the general formula Rl-Arg-R2-R3-R4-His-Pro-R5 as claimed in claims 16 or 17, for the manufacture of a medicament in radiation therapy for a patient afflicted with a neoplastic disease state which agent is substantially as herein described with reference to any one of Examples I to 3. 113. Use of at least one active agent comprising a sequence of comprising the general formula RI-Arg-R2-R3-R4-His-Pro-R5 as claimed in any one of claims 18 or 19, for the manufacture of a medicament to treat a patient in need of radiation therapy. s: 15 114. Use of at least one active agent comprising a sequence of comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in any one of claims 18 or 19, for the manufacture of a medicament to treat a patient in need of radiation therapy which agent is substantially as herein described with reference to any one of Examples 1 to 3. 115. Use of at least one active agent comprising a sequence of comprising the 20 general formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in any one of claims 20 or 21, for the manufacture of a medicament in bone marrow transplantation. 116. Use of at least one active agent comprising a sequence of comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in any one of claims 20 or 21, for the manufacture of a medicament in bone marrow transplantation which agent is substantially as herein described with reference to any one of Examples 1 to 3. 117. Use of at least one active agent comprising a sequence of comprising the general formula R1-Arg-R2-R3-R4-His-Pro-R5 as claimed in any one of claims 22 or 23, for the manufacture of a medicament to increase megakaryocyte production and mobilization and platelet production in a mammal. [I.\LII LVV'04136.dloc:nmrr 49 118. Use of at least one active agent comprising a sequence of comprising the general formula Rl-Arg-R2-R3-R4-His-Pro-R5 as claimed in any one of claims 22 or 23, for the manufacture of a medicament to increase megakaryocyte production and mobilization and platelet production in a mammal which agent is substantially as herein described with reference to any one of Examples 1 to 3. Dated 22 August, 2001 University of Southern California Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0 4@ S S. *5 0 *6 00 9 0 *0* o° [R:\LIB\'V]04136.doc:mrr
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